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Serum-derived bovine immunoglobulin (SBI) prevents translocation and inflammation via direct binding of microbial components. Recently, SBI also displayed potential benefits through gut microbiome modulation. To confirm and expand upon these preliminary findings, SBI digestion and colonic fermentation were investigated using the clinically predictive ex vivo SIFR ® technology (for 24 human adults) that was, for the first time, combined with host cells (epithelial/immune (Caco-2/THP-1) cells). SBI (human equivalent dose (HED) = 2 and 5 g/day) and the reference prebiotic inulin (IN; HED = 2 g/day) significantly promoted gut barrier integrity and did so more profoundly than a dietary protein (DP), especially upon LPS-induced inflammation. SBI also specifically lowered inflammatory markers (TNF-α and CXCL10). SBI and IN both enhanced SCFA (acetate/propionate/butyrate) via specific gut microbes, while SBI specifically stimulated valerate/bCFA and indole-3-propionic acid (health-promoting tryptophan metabolite). Finally, owing to the high-powered cohort (n = 24), treatment effects could be stratified based on initial microbiota composition: IN exclusively stimulated (acetate/non-gas producing) Bifidobacteriaceae for subjects classifying as Bacteroides / Firmicutes -enterotype donors, coinciding with high acetate/low gas production and thus likely better tolerability of IN. Altogether, this study strongly suggests gut microbiome modulation as a mechanism by which SBI promotes health. Moreover, the SIFR ® technology was shown to be a powerful tool to stratify treatment responses and support future personalized nutrition approaches.

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., Competing Interests: No competing interest., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

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., Competing Interests: The authors S.D., A.B., and P.V.d.A. are employees of Cryptobiotix SA.

Long-chain dextrans are α-glucans that can be produced by lactic acid bacteria. NextDext TM , 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.

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., Competing Interests: Authors PV, AB, SD are employed by Cryptobiotix. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Lemons, Narrowe, Liu, Firrman, Mahalak, Van den Abbeele, Baudot, Deyaert, Li and Yu.)

Research on gut microbiota has generally focused on fecal samples, representing luminal content of the large intestine. However, nutrient uptake is restricted to the small intestine. Abundant immune cell populations at this anatomical site combined with diminished mucus secretion and looser junctions (partly to allow for more efficient fluid and nutrient absorption) also results in intimate host-microbe interactions despite more rapid transit. It is thus crucial to dissect key differences in both ecology and physiology between small and large intestine to better leverage the immense potential of human gut microbiota imprinting, including probiotic engraftment at biological sensible niches. Here, we provide a detailed review unfolding how the physiological and anatomical differences between the small and large intestine affect gut microbiota composition, function, and plasticity. This information is key to understanding how gut microbiota manipulation, including probiotic administration, may strain-dependently transform host-microbe interactions at defined locations., Competing Interests: Declaration of interests A.C.O., M.N., B.P., D.S., L.G.W.S., and P.V.D.A. work full time for IFF, ADM, Yakult, Novozymes, Caelus Health, and Cryptobiotix respectively. These companies were not involved in carrying out this research., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

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.

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., Competing Interests: PVdA, SD and AB are employees of Cryptobiotix. CT and CP are employees of Roquette, while EW and DB are employees of DSM. DW is employed by CosmosID. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Van den Abbeele, Deyaert, Thabuis, Perreau, Bajic, Wintergerst, Joossens, Firrman, Walsh and Baudot.)

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.

The human gut microbiome contributes crucial bioactive metabolites that support human health and is sensitive to perturbations from the ingestion of alcohol and antibiotics. We interrogated the response and recovery of human gut microbes after acute alcohol or broad-spectrum antibiotic administration in a gut model simulating the luminal and mucosal colonic environment with an inoculated human microbiome. Both alcohol and antibiotic treatments reduced the production of major short-chain fatty acids (SCFAs) (acetate, propionate, and butyrate), which are established modulators of human health. Treatment with a microbial synbiotic restored and enhanced gut function. Butyrate and acetate production increased by up to 29.7% and 18.6%, respectively, relative to untreated, dysbiotic samples. In parallel, treatment led to increases in the relative abundances of beneficial commensal organisms not found in the synbiotic (e.g., Faecalibacterium prausnitzii and the urolithin-producing organism Gordonibacter pamelaeae) as well as species present in the synbiotic (e.g., Bifidobacterium infantis), suggesting synergistic interactions between supplemented and native microorganisms. These results lead us to conclude that functional shifts in the microbiome, evaluated by both metabolite production and specific taxonomic compositional changes, are an appropriate metric to assess microbiome "recovery" following a dysbiosis-inducing disruption. Overall, these findings support the execution of randomized clinical studies to determine whether a microbial synbiotic can help restore microbiome function after a disruption. IMPORTANCE The human gut microbiome is sensitive to disruptions by common stressors such as alcohol consumption and antibiotic treatment. In this study, we used an in vitro system modeling the gut microbiome to investigate whether treatment with a microbial synbiotic can help restore microbiome function after stress. We find that a complex gut community treated with alcohol or antibiotics showed reduced levels of production of short-chain fatty acids, which are critical beneficial molecules produced by a healthy gut microbiota. Treatment of stressed communities with a microbial synbiotic resulted in the recovery of SCFA production as well as an increase in the abundance of beneficial commensal organisms. Our results suggest that treatment with a microbial synbiotic has the potential to restore healthy gut microbiome function after stress and merits further investigation in clinical studies.

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.

The human gastrointestinal tract consists of different regions, each characterized by a distinct physiology, anatomy, and microbial community. While the colonic microbiota has received a lot of attention in recent research projects, little is known about the small intestinal microbiota and its interactions with ingested compounds, primarily due to the inaccessibility of this region in vivo . This study therefore aimed to develop and validate a dynamic, long-term simulation of the ileal microbiota using the SHIME ® -technology. Essential parameters were identified and optimized from a screening experiment testing different inoculation strategies, nutritional media, and environmental parameters over an 18-day period. Subjecting a synthetic bacterial consortium to the selected conditions resulted in a stable microbiota that was representative in terms of abundance [8.81 ± 0.12 log (cells/ml)], composition and function. Indeed, the observed community mainly consisted of the genera Streptococcus, Veillonella, Enterococcus, Lactobacillus , and Clostridium (qPCR and 16S rRNA gene targeted Illumina sequencing), while nutrient administration boosted lactate production followed by cross-feeding interactions towards acetate and propionate. Furthermore, similarly as in vivo , bile salts were only partially deconjugated and only marginally converted into secondary bile salts. After confirming reproducibility of the small intestinal microbiota model, it was integrated into the established M-SHIME® where it further increased the compositional relevance of the colonic community. This long-term in vitro model provides a representative simulation of the ileal bacterial community, facilitating research of the ileum microbiota dynamics and activity when, for example, supplemented with microbial or diet components. Furthermore, integration of this present in vitro simulation increases the biological relevance of the current M-SHIME® technology., Competing Interests: FM, MM, and TW are employees of ProDigest, that provides services to other companies using the model developed in this study. SD and PA were employees of ProDigest when this project was conducted. During the current project WP, Bv, and EK were employed by BaseClear, the company providing NGS services throughout the study. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Deyaert, Moens, Pirovano, van den Bogert, Klaassens, Marzorati, Van de Wiele, Kleerebezem and Van den Abbeele.)

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.

The gut microbiota plays a pivotal role in health and disease. The use of probiotics as microbiota-targeted therapies is a promising strategy to improve host health. However, the molecular mechanisms involved in such therapies are often not well understood, particularly when targeting the small intestinal microbiota. In this study, we investigated the effects of a probiotic formula (Ecologic®825) on the adult human small intestinal ileostoma microbiota. The results showed that supplementation with the probiotic formula led to a reduction in the growth of pathobionts, such as Enterococcaceae and Enterobacteriaceae , and a decrease in ethanol production. These changes were associated with significant alterations in nutrient utilization and resistance to perturbations. These probiotic mediated alterations which coincided with an initial increase in lactate production and decrease in pH were followed by a sharp increase in the levels of butyrate and propionate. Moreover, the probiotic formula increased the production of multiple N-acyl amino acids in the stoma samples. The study demonstrates the utility of network theory in identifying novel microbiota-targeted therapies and improving existing ones. Overall, the findings provide insights into the dynamic molecular mechanisms underlying probiotic therapies, which can aid in the development of more effective treatments for a range of conditions.

Clostridioides difficile infection (CDI) is the leading cause of antibiotic-associated diarrhea and an important nosocomial infection with different severity degrees. Disruption of the gut microbiota by broad-spectrum antibiotics creates a proper environment for C. difficile colonization, proliferation, and clinical disease onset. Restoration of the gut microbial ecosystem through prebiotic interventions can constitute an effective complementary treatment of CDI. Using an adapted simulator of the human gut microbial ecosystem, the PathoGut TM SHIME, the effect of different long-term and repeated dose lactulose treatments was tested on C. difficile germination and growth in antibiotic-induced dysbiotic gut microbiota environments. The results showed that lactulose reduced the growth of viable C. difficile cells following clindamycin treatment, shifted the antibiotic-induced dysbiotic microbial community, and stimulated the production of health-promoting metabolites (especially butyrate). Recovery of the gut microenvironment by long-term lactulose administration following CDI was also linked to lactate production, decrease in pH and modulation of bile salt metabolism. At a structural level, lactulose showed a significant bifidogenic potential and restored key commensal members of the gut ecosystem such as Lactobacillaceae , Veillonellaceae and Lachnospiraceae . These results support further human intervention studies aiming to validate the in vitro beneficial effects of lactulose on gut microbiome recovery during antibiotic exposure and CDI., Competing Interests: D.F., and A.K.K. are employees of Fresenius-Kabi.

Akkermansia muciniphila is a champion of mucin degradation in the human gastrointestinal tract. Here, we report the isolation of six novel strains from healthy human donors and their genomic, proteomic and physiological characterization in comparison to the type-strains A. muciniphila MucT and A. glycaniphila PytT. Complete genome sequencing revealed that, despite their large genomic similarity (>97.6%), the novel isolates clustered into two distinct subspecies of A. muciniphila: Amuc1, which includes the type-strain MucT, and AmucU, a cluster of unassigned strains that have not yet been well characterized. CRISPR analysis showed all strains to be unique and confirmed that single healthy subjects can carry more than one A. muciniphila strain. Mucin degradation pathways were strongly conserved amongst all isolates, illustrating the exemplary niche adaptation of A. muciniphila to the mucin interface. This was confirmed by analysis of the predicted glycoside hydrolase profiles and supported by comparing the proteomes of A. muciniphila strain H2, belonging to the AmucU cluster, to MucT and A. glycaniphila PytT (including 610 and 727 proteins, respectively). While some intrinsic resistance was observed among the A. muciniphila straind, none of these seem to pose strain-specific risks in terms of their antibiotic resistance patterns nor a significant risk for the horizontal transfer of antibiotic resistance determinants, opening the way to apply the type-strain MucT or these new A. muciniphila strains as next generation beneficial microbes.

The validated SHIME model was used to assess the effect of repeated administration of two different lactulose dosages (5 g/d and 10 g/d) on the human gut microbiome during and following amoxicillin-clavulanic acid treatment. First, antibiotic treatment strongly decreased Bifidobacteriaceae levels from 54.4% to 0.6% and from 23.8% to 2.3% in the simulated proximal and distal colon, respectively, coinciding with a marked reduction in butyrate concentrations. Treatment with lactulose enhanced acetate and lactate levels during antibiotic treatment, likely through lactulose fermentation by Lachnospiraceae and Lactobacillaceae . One week after cessation of antibiotic treatment, Bifidobacteriaceae levels re-increased to 20.4% and 7.6% in the proximal and distal colon of the 5 g lactulose/d co-administered unit, as compared with 1.0% and 2.2% in the antibiotic-treated unit, and were even further stimulated upon extension of lactulose administration. Marked butyrogenic effects were observed upon prolonged lactulose supplementation, suggesting the establishment of cross-feeding interactions between Bifidobacteriaceae and butyrate producers. Furthermore, a limited Enterobacteriaceae outgrowth following antibiotic treatment was observed upon dosing with 10 g lactulose/d, indicating inhibition of pathogenic colonization by lactulose following antibiotic therapy. Overall, lactulose seems to be an interesting candidate for limiting the detrimental effects of amoxicillin-clavulanic acid on the human gut microbiome, though further studies are warranted to confirm these findings.

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.

Environmental pH is a critical parameter for maintenance of the gut microbiota. Here, the impact of pH on the gut microbiota luminal and mucosal community structure and short chain fatty acid (SCFA) production was evaluated in vitro, and data compiled to reveal a donor-independent response to an increase or decrease in environmental pH. The results found that raising environmental pH significantly increased luminal community richness and decreased mucosal community evenness. This corresponded with an increased abundance of Ruminococcaceae Ruminococcus and Erysipelotrichaceae Erysipelatoclostridium, and a decreased abundance of Coriobacteriaceae Collinsella and Enterobacteriaceae Shigella for both the luminal and mucosal communities. Total SCFA levels were significantly higher, primarily due to an increase in acetic and 2-methylbutanoic acids. Lowering pH decreased luminal community evenness and decreased mucosal community evenness and richness. This corresponded with an increased abundance of Lachnospiraceae Enterocloster, Veillonellaceae Megasphaera, Veillonellaceae Sporomusa, Erysipelotrichaceae Eubacterium, and Alcaligenaceae Sutterella, and decreased abundance of Odoribacteraceae Butyricimonas, Fusobacteriaceae Fusobacterium, Veillonellaceae Phascolarctobacterium, and multiple Enterobacteriaceae species for both the luminal and mucosal communities. Total SCFA levels were significantly lower, with an observed drop in acetic and propionic acids, and increased butyric and valeric acids. Taken together, these results indicate that alterations to environmental pH can modulate the gut microbiota community structure and function, and some changes may occur in a donor-independent manner., (Published by Oxford University Press on behalf of FEMS 2022.)

The American cranberry, Vaccinium macrocarpon , contains fibers and (poly)phenols that could exert health-promoting effects through modulation of gut microbiota. This study aimed to investigate how a freeze-dried whole cranberry powder (FCP) modulated metabolite production and microbial composition using both a 48-h incubation strategy and a long-term human gut simulator study with the M-SHIME (Mucosal Simulator of the Human Intestinal Microbial Ecosystem). FCP was repeatedly administered over three weeks. The studies included five and three study subjects, respectively. In both models, FCP significantly increased levels of health-related short-chain fatty acids (SCFA: acetate, propionate and butyrate), while decreased levels of branched-chain fatty acids (markers of proteolytic fermentation). Interestingly, FCP consistently increased luminal Bacteroidetes abundances in the proximal colon of the M-SHIME (+17.5 ± 9.3%) at the expense of Proteobacteria (-10.2 ± 1.5%). At family level, this was due to the stimulation of Bacteroidaceae and Prevotellaceae and a decrease of Pseudomonodaceae and Enterobacteriaceae . Despite of interpersonal differences, FCP also increased the abundance of families of known butyrate producers. Overall, FCP displayed an interesting prebiotic potential in vitro given its selective utilization by host microorganisms and potential health-related effects on inhibition of pathogens and selective stimulation of beneficial metabolites.

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.

Purpose: Cereboost®, an American ginseng extract, has shown improved short-term memory and attention/alertness in healthy young and middle-aged individuals, potentially via modulation of the gut microbiome and upregulation of neurotransmitters such as acetylcholine. Here, we explored the effects of Cereboost® on cognition and mood in the first 6 h post intervention (acute), after 2 weeks daily supplementation (chronic), and whether 2 weeks daily supplementation altered the response to a single acute dose (acute-on-chronic). A concurrent in vitro study evaluated effects of repeated Cereboost® administration on human gut microbiota., Methods: Cognitive effects of Cereboost® were assessed using a double-blind, randomized, placebo-controlled clinical trial, with 61 healthy young adults. Modulation of the gut microbiome was concurrently modelled using the Simulator of the Human Microbial Ecosystem (SHIME®), using a young adult donor., Results: Consistent with previous findings, Cereboost® improved working memory and attention during the immediate postprandial period; effects that were amplified following two weeks' treatment (acute-on-chronic) compared to acute testing alone. Chronic supplementation improved cognition on an acetylcholine-sensitive attention task and improved mental fatigue and self-assurance aspects of mood. The parallel in vitro study revealed significantly increased acetate, propionate, and butyrate levels in simulated proximal and distal colon regions, linked with observed increases in Akkermansia muciniphila and Lactobacillus., Conclusion: This study confirmed the promising effects of Cereboost® on cognitive function and mood, while suggesting a possible link to alterations of the gut microbiome and modulation of acetylcholine. Further studies will be required to unravel the underlying mechanisms that are involved., Registration: The study was pre-registered at ClinicalTrials.gov on 6th July 2018 (Identifier: NCT03579095)., (© 2021. The Author(s).)

A short-chain fructo-oligosaccharide (sc-FOS) was tested in a simulator of the human gut microbial ecosystem (SHIME) in vitro model to quantify its prebiotic effects according to Prebiotic Index (PI) and Measure of prebiotic effect (MPE) equations. FossenceTM, (sc-FOS, 0.5%) was fermented in a simulated human proximal colonic condition, using a fecal inoculum from a healthy individual. We analysed the pH reduction, substrate utilization, lactate and short-chain fatty acid (SCFA) production and microbial community modulation. Microbial fermentation of sc-FOS strongly reduced the media pH indicating the production of lactate and SCFA with accumulation of lactate and enhanced levels of acetate (34.38 ± 0.38 mM), propionate (20.93 ± 0.56 mM) and butyrate (4.93 ± 0.03 mM) compared to 18.46 ± 0.20 mM, 6.24 ± 0.10 mM and 3.3 ± 0.06 mM in the blank, respectively. Total SCFA production in test media was 61.91 ± 0.87 mM compared to 33.65 ± 0.36 mM in blank and the contribution of free-sugars present in sc-FOS to SCFAs was negligible. Modulation of the microbial community was analysed through 16S rRNA sequencing and we found that sc-FOS greatly stimulated the beneficial bacteria such as Bifidobacteria and Lactobacillus. We report the PI and MPE values for FossenceTM, as 14.9 and 0.01 respectively at the end of 24 h, which is an indicator of a strong prebiotic effect., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

MegaSporeBiotic™ is an oral, spore-based probiotic comprised of five Bacillus spp. (Bacillus indicus HU36, Bacillus subtilis HU58, Bacillus coagulans SC208, Bacillus licheniformis SL307, and Bacillus clausii SC109). The effects of MegaSporeBiotic™ on gut microbiota activity and community composition were evaluated for the first time using an in vitro model of the human gastrointestinal tract, the simulator of the human intestinal microbial ecosystem (SHIME®), under healthy conditions. Following a stabilization period and a control period (2 weeks each), the reactor feed was supplemented with daily MegaSporeBiotic™ for 3 weeks (treatment period). Changes in microbial community activity and composition between the control and treatment periods were evaluated for each colon compartment (ascending [AC], transverse [TC], and descending colon [DC]). Propionate levels increased significantly in the TC (week 2, P = 0.02; week 3, P = 0.0019) and DC (week 2, P = 0.03) with treatment while lactate levels significantly decreased in the TC (week 3, P = 0.03). Ammonium levels were significantly decreased during the final week of treatment (TC, P = 0.02; DC, P = 0.03). Overall, Akkermansia muciniphila, Bifidobacteria spp., and Firmicutes increased with treatment while Lactobacillus spp. and Bacteroidetes decreased. The Firmicutes:Bacteroidetes ratio increased with treatment in the AC compartment. MegaSporeProbiotic™ treatment resulted in changes in metabolism and increased bacterial diversity., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

While a large set of in vitro models are available to study the effects of specific food ingredients (e.g. pre- and probiotics) on the human gut microbiome, the availability of such models for companion animals is limited. Since improving gut health of such animals is an emerging research field, the Simulator of the Canine Intestinal Microbial Ecosystem (SCIME™) was recently developed and validated with in vivo data. The current study presents a further improvement of this model by using an alternative method for feed preparation, i.e. by administering digestive enzymes to mimic upper gastro-intestinal digestion followed by a dialysis approach to mimic small intestinal absorption. As opposed to the previously implemented method, this resulted in a more optimal simulation of protein digestion and absorption. Further, upon entrance in the colon, increased production of the health-promoting butyrate and lower levels of Lactobacillus spp. and Bifidobacterium spp. were observed, which corresponded better with obtained in vivo data. A second model improvement consisted of the implementation of a mucosal environment to not only simulate luminal but also mucosal microbiota. In consistency with the human model for which this technology was previously validated, it was found that for all canine microbiota mucin beads were enriched with members of the Lachnospiraceae (~ Clostridium cluster XIVa), a family containing multiple well-known butyrate producers. The SCIME™ was thus upgraded to a so-called Mucosal SCIME™ (M-SCIME™). In conclusion, the current study presents improvements of the SCIME™, further increasing the relevance of obtained data with this in vitro model for dogs., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The human gut microbiome is currently recognized to play a vital role in human biology and development, with diet as a major modulator. Therefore, novel indigestible polysaccharides that confer a health benefit upon their fermentation by the microbiome are under investigation. Based on the recently demonstrated prebiotic potential of a carrot-derived pectin extract enriched for rhamnogalacturonan I (cRG-I), the current study aimed to assess the impact of cRG-I upon repeated administration using the M-SHIME technology (3 weeks at 3g cRG-I/d). Consistent effects across four simulated adult donors included enhanced levels of acetate (+21.1 mM), propionate (+17.6 mM), and to a lesser extent butyrate (+4.1 mM), coinciding with a marked increase of OTUs related to Bacteroides dorei and Prevotella species with versatile enzymatic potential likely allowing them to serve as primary degraders of cRG-I. These Bacteroidetes members are able to produce succinate, explaining the consistent increase of an OTU related to the succinate-converting Phascolarctobacterium faecium (+0.47 log 10 (cells/mL)). While the Bifidobacteriaceae family remained unaffected, a specific OTU related to Bifidobacterium longum increased significantly upon cRG-I treatment (+1.32 log 10 (cells/mL)). Additional monoculture experiments suggested that Bifidobacterium species are unable to ferment cRG-I structures as such and that B. longum probably feeds on arabinan and galactan side chains of cRG-I, released by aforementioned Bacteroidetes members. Overall, this study confirms the prebiotic potential of cRG-I and additionally highlights the marked consistency of the microbial changes observed across simulated subjects, suggesting the involvement of a specialized consortium in cRG-I fermentation by the human gut microbiome., Competing Interests: A.M. and R.A. are employees of Nutrileads, the funder of the study. Nutrileads holds patents for the use cRG-I. A.M. participated in the design of the study and the writing of the manuscript. R.A. conceptualized the work and participated in the writing of the manuscript.

The human gut microbiota has been linked to the health status of the host. Modulation of human gut microbiota through pro- and prebiotic interventions has yielded promising results; however, the effect of novel prebiotics, such as chitin-glucan, on gut microbiota-host interplay is still not fully characterized. We assessed the effect of chitin-glucan (CG) and chitin-glucan plus Bifidobacterium breve (CGB) on human gut microbiota from the luminal and mucosal environments in vitro. Further, we tested the effect of filter-sterilized fecal supernatants from CG and CGB fermentation for protective effects on inflammation-induced barrier disruption and cytokine production using a co-culture of enterocytes and macrophage-like cells. Overall, CG and CGB promote health-beneficial short-chain fatty acid production and shift human gut microbiota composition, with a consistent effect increasing Roseburia spp. and butyrate producing-bacteria. In two of three donors, CG and CGB also stimulated Faecalibacterium prausniitzi . Specific colonization of B. breve was observed in the lumen and mucosal compartment; however, no synergy was detected for different endpoints when comparing CGB and CG. Both treatments included a significant improvement of inflammation-disrupted epithelial barrier and shifts on cytokine production, especially by consistent increase in the immunomodulatory cytokines IL10 and IL6.

While many beneficial host-microbiota interactions have been described, imbalanced microbiota in the gut is speculated to contribute to the progression and recurrence of chronic inflammatory diseases such as Crohn's disease (CD). This in vitro study evaluated the impact of a cranberry concentrate Type M (CTM) on adherent-invasive Escherichia coli (AIEC) LF82, a pathobiont associated with CD. Different stages of pathogenic infection were investigated: (i) colonization of the mucus layer, and (ii) adhesion to and (iii) invasion of the epithelial cells. Following 48 h of fecal batch incubation, 0.5 and 1 mM of CTM significantly altered AIEC LF82 levels in a simulated mucus layer, resulting in a decrease of 50.5% in the untreated blank, down to 43.0% and 11.4%, respectively. At 1 mM of CTM, the significant decrease in the levels of AIEC LF82 coincided with a stimulation of the metabolic activity of the background microbiota. The increased levels of health-associated acetate (+7.9 mM) and propionate levels (+3.5 mM) suggested selective utilization of CTM by host microorganisms. Furthermore, 1 mM of both fermented and unfermented CTM decreased the adhesion and invasion of human-derived epithelial Caco-2 cells by AIEC LF82. Altogether, this exploratory in vitro study demonstrates the prebiotic potential of CTM and supports its antipathogenic effects through direct and/or indirect modulation of the gut microbiome.

Increasing insight into the impact of the gut microbiota on human health has sustained the development of novel prebiotic ingredients. This exploratory study evaluated the prebiotic potential of baobab fruit pulp powder, which consists of pectic polysaccharides with unique composition as compared to other dietary sources, given that it is rich in low methoxylated homogalacturonan (HG). After applying dialysis procedures to remove simple sugars from the product (simulating their absorption along the upper gastrointestinal tract), 48 h fecal batch incubations were performed. Baobab fruit pulp powder boosted colonic acidification across three simulated human adult donors due to the significant stimulation of health-related metabolites acetate (+18.4 mM at 48 h), propionate (+5.5 mM at 48 h), and to a lesser extent butyrate (0.9 mM at 48 h). Further, there was a trend of increased lactate levels (+2.7 mM at 6h) and reduced branched chain fatty acid (bCFA) levels (-0.4 mM at 48 h). While Bacteroidetes levels increased for all donors, donor-dependent increases in Bifidobacteria , Lactobacilli , and Firmicutes were observed, stressing the potential interindividual differences in microbial composition modulation upon Baobab fruit pulp powder treatment. Overall, Baobab fruit pulp powder fermentation displayed features of selective utilization by host microorganisms and, thus, has promising prebiotic potential (also in comparison with the 'gold standard' prebiotic inulin). Further research will be required to better characterize this prebiotic potential, accounting for the interindividual differences, while aiming to unravel the potential resulting health benefits.

Clostridioides difficile (formerly Clostridium difficile ) infection (CDI) is one of the most common hospital-acquired infections, which is often triggered by a dysbiosed indigenous gut microbiota (e.g., upon antibiotic therapy). Symptoms can be as severe as life-threatening colitis. The current study assessed the antipathogenic potential of human milk oligosaccharides (HMOs), i.e., 2'-O-fucosyllactose (2'FL), lacto-N-neotetraose (LNnT), and a combination thereof (MIX), against C. difficile ATCC 9689 using in vitro gut models that allowed the evaluation of both direct and, upon microbiota modulation, indirect effects. During a first 48 h fecal batch study, dysbiosis and CDI were induced by dilution of the fecal inoculum. For each of the three donors tested, C. difficile levels strongly decreased (with >4 log CFU/mL) upon treatment with 2'FL, LNnT and MIX versus untreated blanks, coinciding with increased acetate/ Bifidobacteriaceae levels. Interindividual differences among donors at an intermediate time point suggested that the antimicrobial effect was microbiota-mediated rather than being a direct effect of the HMOs. During a subsequent 11 week study with the Pathogut TM model (specific application of the Simulator of the Human Intestinal Microbial Ecosystem (SHIME ® )), dysbiosis and CDI were induced by clindamycin (CLI) treatment. Vancomycin (VNC) treatment cured CDI, but the further dysbiosis of the indigenous microbiota likely contributed to CDI recurrence. Upon co-supplementation with VNC, both 2'FL and MIX boosted microbial activity (acetate and to lesser extent propionate/butyrate). Moreover, 2'FL avoided CDI recurrence, potentially because of increased secondary bile acid production. Overall, while not elucidating the exact antipathogenic mechanisms-of-action, the current study highlights the potential of HMOs to combat CDI recurrence, help the gut microbial community recover after antibiotic treatment, and hence counteract the adverse effects of antibiotic therapies.

Human gut microbiota has a fundamental role in human health, and diet is one of the most relevant factors modulating the gut microbial ecosystem. Fiber, fat, proteins, and micronutrients can shape microbial activity and structure. Much information is available on the role of defined prebiotic fibers on gut microbiota, but less known are the effects of intact dietary fiber sources on healthy gut ecosystems. This research investigated in vitro the short-term effect of 22 commercially available food sources of dietary fiber on gut microbiota activity [pH, gas, short-chain fatty acids (SCFA), branched fatty acids (BCFA), lactate] and specific composition of Firmicutes, Bacteroidetes, bifidobacteria, and lactobacilli populations. More than 80% (19 of 22) of the products were highly fermentable and induced SCFAs production, with specific product differences. In general, all the whole grain cereals had a similar effect on gut microbiota modulation, inducing acetate and butyrate production and increasing bifidobacteria levels. Incorporating and comparing a large variety of products, including "non-conventional" fiber sources, like konjac, bamboo fiber, or seeds fiber, about which there is little information, contributes to our knowledge on the modulatory activity of diverse food fiber sources on human gut microbiota, and therefore potential health promotion through dietary fiber diversification., Competing Interests: AB and ER are employees of the Kellogg Company. PV was employed by ProDigest. MC and JG are employed by ProDigest. MM is CEO of ProDigest. The authors declare that this study received funding from Kellogg Company. The funder had the following involvement in the study: conceptualization, funding acquisition, data curation, utilizing resources, writing and editing., (Copyright © 2021 Calatayud, Van den Abbeele, Ghyselinck, Marzorati, Rohs and Birkett.)

We report here the potential role of a 4-strain probiotic suspension for use with patients with Parkinson's disease (PD). Stool samples from a group of three patients with diagnosed PD were used to create microbiotas in an in-vitro gut model. The effects of dosing with an oral probiotic suspension (Symprove) on bacterial composition and metabolic activity in the microbiotas was evaluated over 48 h and compared with healthy controls. Additionally, the effect of probiotic dosing on epithelial tight-junction integrity, production of inflammatory markers and wound healing were evaluated in cell culture models. In general, the relative proportions of the main bacterial phyla in the microbiotas of PD patients differed from those of healthy subjects, with levels of Firmicutes raised and levels of Bacteroidetes reduced. Dosing with probiotic resulted in a change in bacterial composition in the microbiotas over a 48 h period. Several other indicators of gut health changed upon dosing with the probiotic; production of short chain fatty acids (SCFAs) and lactate was stimulated, levels of anti-inflammatory cytokines (IL-6, IL-10) increased and levels of pro-inflammatory cytokines and chemokines (MCP-1 and IL-8) decreased. Tight junction integrity was seen to improve with probiotic dosing and wound healing was seen to occur faster than a control. The data suggest that if development and/or progression of PD is influenced by gut microbiota dysbiosis then supplementation of the diet with a properly formulated probiotic may be a useful adjunct to standard treatment in clinic., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors.)

Candida albicans is a major fungal pathogen of humans. It exists as a commensal in the oral cavity, gut or genital tract of most individuals, constrained by the local microbiota, epithelial barriers and immune defences. Their perturbation can lead to fungal outgrowth and the development of mucosal infections such as oropharyngeal or vulvovaginal candidiasis, and patients with compromised immunity are susceptible to life-threatening systemic infections. The importance of the interplay between fungus, host and microbiota in driving the transition from C. albicans commensalism to pathogenicity is widely appreciated. However, the complexity of these interactions, and the significant impact of fungal, host and microbiota variability upon disease severity and outcome, are less well understood. Therefore, we summarise the features of the fungus that promote infection, and how genetic variation between clinical isolates influences pathogenicity. We discuss antifungal immunity, how this differs between mucosae, and how individual variation influences a person's susceptibility to infection. Also, we describe factors that influence the composition of gut, oral and vaginal microbiotas, and how these affect fungal colonisation and antifungal immunity. We argue that a detailed understanding of these variables, which underlie fungal-host-microbiota interactions, will present opportunities for directed antifungal therapies that benefit vulnerable patients., (© The Author(s) 2020. Published by Oxford University Press on behalf of FEMS.)

Background:   Okoubaka aubrevillei is used in traditional West African medicine and in homeopathy for treatment and prevention of several gastrointestinal problems. The aim of this in vitro study was to evaluate the effect of repeated doses of two Okoubaka products (10 % ethanolic tincture, mother tincture (MT); 3 rd decimal potency, 3X) on the microbial activity of physiological human colon microbiota using a Simulator of the Human Intestinal Microbial Ecosystem (SHIME ® ) and to investigate any preventive effect against infections with diarrhea-causing pathogens., Methods:  Upon inoculation with fecal microbiota from a healthy donor, 4 parallel proximal colon compartments of the SHIME were treated either with Okoubaka MT, Okoubaka 3X, ethanol control or blank control for 7 days. Using the Okoubaka -adapted microbial community from SHIME, 48 h challenge tests were performed with enterotoxigenic Escherichia coli (ETEC) and Salmonella enteritidis in 4 different doses (10 3 -10 8 colony forming units as typical in vivo infectious doses). Pathogen concentrations, short-chain fatty acids (SCFAs) and branched SCFA production were measured in triplicate at 0, 24 and 48 h., Results:  In the challenge tests, both Okoubaka products were able to restrict the colonization of ETEC and Salmonella at 3 of the 4 pathogen doses (except the highest doses), with a stronger anti-pathogenic effect for MT, which included a reduction of 2.0 log-units of ETEC (p < 0.0001) and 1.1 log-units of Salmonella (p < 0.0001). Total SCFA levels remained unaffected, but butyrate increased during the first 24 h (p < 0.0001 for ETEC), accompanied by decreased acetate production., Conclusion:  We observed in vitro a systemic activating effect of Okoubaka on intestinal microbiome resistance, which resulted in an anti-pathogenic effect, especially against ETEC. We hypothesize that the mode of action in vivo is also based on systemic regulative effects., Competing Interests: The research presented in this publication was financed by DHU-Arzneimittel GmbH & Co KG, Karlsruhe, Germany (DHU).Susann Buchheim-Schmidt and Petra Klement are employees of DHU.Uwe Peters received an advisory fee from DHU.Cindy Duysburgh, Pieter Van den Abbeele and Massimo Marzorati are employees of Prodigest. ProDigest received payment from DHU for conducting the research and analysing the data.Thomas Keller is owner of ACOMED statistik. ACOMED statistik received payment from DHU for statistical analysis of the study.David Martin and Stephan Baumgartner report no conflict of interest., (Thieme. All rights reserved.)

The importance of the gut microbiota in human health and disease progression makes it a target for research in both the biomedical and nutritional fields. To date, a number of in vitro systems have been designed to recapitulate the gut microbiota of the colon ranging in complexity from the application of a single vessel to cultivate the community in its entirety, to multi-stage systems that mimic the distinct regional microbial communities that reside longitudinally through the colon. While these disparate types of in vitro designs have been employed previously, information regarding similarities and differences between the communities that develop within was less defined. Here, a comparative analysis of the population dynamics and functional production of short-chain fatty acids (SCFAs) was performed using the gut microbiota of the same donor cultured using a single vessel and a 3-stage colon system. The results found that the single vessel communities maintained alpha diversity at a level comparable to the distal regions of the 3-stage colon system. Yet, there was a marked difference in the type and abundance of taxa, particularly between families Enterobacteriaceae, Bacteroidaceae, Synergistaceae, and Fusobacteriaceae. Functionally, the single vessel community produced significantly less SCFAs compared to the 3-stage colon system. These results provide valuable information on how culturing technique effects gut microbial composition and function, which may impact studies relying on the application of an in vitro strategy. This data can be used to justify experimental strategy and provides insight on the application of a simplified versus complex study design. KEY POINTS : • A mature gut microbiota community can be developed in vitro using different methods. • Beta diversity metrics are affected by the in vitro culturing method applied. • The type and amount of short-chain fatty acids differed between culturing methods.

Human gut microbiota (HGM) play a significant role in health and disease. Dietary components, including fiber, fat, proteins and micronutrients, can modulate HGM. Much research has been performed on conventional prebiotics such as fructooligosaccharides (FOS) and galactooligosaccharides (GOS), however, novel prebiotics or micronutrients still require further validation. We assessed the effect of FOS, xylooligosaccharides (XOS) and a mixture of an antioxidant vitamin blend (AOB) on gut microbiota composition and activity, and intestinal barrier in vitro. We used batch fermentations and tested the short-term effect of different products on microbial activity in six donors. Next, fecal inocula from two donors were used to inoculate the simulator of the human microbial ecosystem (SHIME) and after long-term exposure of FOS, XOS and AOB, microbial activity (short- and branched-chain fatty acids and lactate) and HGM composition were evaluated. Finally, in vitro assessment of intestinal barrier was performed in a Transwell setup of differentiated Caco-2 and HT29-MTX-E12 cells exposed to fermentation supernatants. Despite some donor-dependent differences, all three tested products showed beneficial modulatory effects on microbial activity represented by an increase in lactate and SCFA levels (acetate, butyrate and to a lesser extent also propionate), while decreasing proteolytic markers. Bifidogenic effect of XOS was consistent, while AOB supplementation appears to exert a specific impact on reducing F. nucleatum and increasing butyrate-producing B. wexlerae . Functional and compositional microbial changes were translated to an in vitro host response by increases of the intestinal barrier integrity by all the products and a decrease of the redox potential by AOB supplementation.

The prevalence of acute respiratory infections and their impact on quality of life underlies the need for efficacious solutions that are safe, sustainable and economically viable. Polysaccharides in several (traditional) plant extracts have been shown to be immunostimulatory, and some studies suggest beneficial effects against respiratory infections. The aim of this study was to (i) identify the active polysaccharide constituents from affordable and renewable crops (bell pepper and carrot) using activity-guided fractionation, (ii) evaluate in vitro effects on innate immune responses (phagocytosis and cytokine secretion), microbiota modulation and production of short chain fatty acids, followed by (iii) the evaluation of effects of a bell pepper extract enriched for the active component in a human proof of concept study. We identified rhamnogalacturonan-I (RG-I) as the nutricophore responsible for the immunostimulatory activity with substantial structural and functional equivalence between bell pepper (bp) and carrot (c). The in vitro studies showed that bpRG-I and cRG-I comprise similar immune- and microbiota modulatory potential and the human study demonstrated that bpRG-I was well tolerated and enhanced innate immune responsiveness in vivo. This is an important step towards testing the efficacy of RG-I from bpRG-I or cRG-I in an infection trial in humans.

Because of the recognized health benefits of breast milk, it is recommended as the sole nutrition source during the first 6 months of life. Among the bioactive components are human milk oligosaccharides (HMOs) that exert part of their activity via the gut microbiota. Here, we investigated the gut microbiota fermentation of HMO 2'fucosyllactose (2'-FL), using two in vitro models (48 h fecal incubations and the long-term mucosal simulator of the human intestinal microbial ecosystem [M-SHIME ® ]) with fecal samples from 3-month-old breastfed (BF) infants as well as 2-3 year old toddlers. The short-term model allowed the screening of five donors for each group and provided supportive data for the M-SHIME ® study. A key finding was the strong and immediate increase in the relative abundance of Bifidobacteriaceae following 2'-FL fermentation by both the BF infant and toddler microbiota in the M-SHIME ® . At the metabolic level, while decreasing branched-chain fatty acids, 2'-FL strongly increased acetate production together with increases in the health-related propionate and butyrate whilst gas production only mildly increased. Notably, consistently lower gas production was observed with 2'-FL fermentation as compared to lactose, suggesting that reduced discomfort during the dynamic microbiome establishment in early life may be an advantage along with the bifidogenic effect observed.

The prebiotic activity of a commercially available oat product and a novel oat ingredient, at similar β-glucan loads, was tested using a validated in vitro gut model (M-SHIME ® ). The novel oat ingredient was tested further at lower β-glucan loads in vitro, while the commercially available oat product was assessed in a randomised, single-blind, placebo-controlled, and cross-over human study. Both approaches focused on healthy individuals with mild hypercholesterolemia. In vitro analysis revealed that both oat products strongly stimulated Lactobacillaceae and Bifidobacteriaceae in the intestinal lumen and the simulated mucus layer, and corresponded with enhanced levels of acetate and lactate with cross-feeding interactions leading to an associated increase in propionate and butyrate production. The in vitro prebiotic activity of the novel oat ingredient remained at lower β-glucan levels, indicating the prebiotic potential of the novel oat product. Finally, the stimulation of Lactobacillus spp. was confirmed during the in vivo trial, where lactobacilli abundance significantly increased in the overall population at the end of the intervention period with the commercially available oat product relative to the control product, indicating the power of in vitro gut models in predicting in vivo response of the microbial community to dietary modulation.

Human milk contains prebiotic components, such as human milk oligosaccharides (HMOs), which stimulate the growth of specific members of the infant gut microbiota (e.g., Bifidobacteria ). Plant-based or synthetic oligosaccharides are often added to infant formulas to simulate the bifidogenic effect of HMOs. Cow milk, the most common source of protein in infant formula, and goat milk, used increasingly in the manufacture of infant formula, contain naturally-occurring prebiotics. This study compared the upper gastrointestinal digestion and subsequent colonic fermentation of human milk vs. goat and cow milk-based infant formulas (goat IF and cow IF, respectively), without additional oligosaccharides using an in vitro model for 3-month-old infants based on the Simulator of the Human Intestinal Microbial Ecosystem (SHIME®). First, a dialysis approach using 3.5 kDa membranes was demonstrated to simulate small intestinal absorption of carbohydrates in conditions similar to those in vivo . During the in vitro digestion experiment, oligosaccharides were detected in human milk and goat IF but barely detected in the cow IF. Further, all three milk matrices decreased colonic pH by boosting acetate, lactate, and propionate production, which related to increased abundances of acetate/lactate-producing Bifidobacteriaceae for human milk (+25.7%) and especially goat IF (33.8%) and cow IF (37.7%). Only cow IF stimulated butyrate production which correlated with an increase in Lachnospiraceae and Clostridiaceae . Finally, Enterobacteriaceae and Acidaminococcaceae also increased with all three milk matrices, while production of proteolytic metabolites (branched-chain fatty acids) was only detected for the cow IF. Overall, goat and cow milk-based formulas without added oligosaccharides impacted gut microbial activity and composition similarly to human milk. This suggests that even without supplementation of formula with oligosaccharides, whole goat milk, whole cow milk and cow milk ingredients already supply compounds in formulas that exert beneficial bifidogenic effects. Further clinical research is warranted to elucidate the effect of whole goat milk-based formulas on the infant gut microbiome., Competing Interests: SG and CP are employees of Dairy Goat Co-operative (NZ) Ltd, that financially supported the study and manufactured the formulas for use in this study. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Gallier, Van den Abbeele and Prosser.)

The current study evaluated the effect of five yeast-derived formulations (T1-T5) on microbial metabolism and composition of the canine and feline gut microbiota using a novel in vitro colonic incubation approach. This novel in vitro model allowed for growth of the entire spectrum of dog- and cat-derived bacteria from the inoculum, thus offering an excellent platform to evaluate effects of nutritional interventions on the gut microbiota. Further, yeast-derived ingredients differentially increased production of acetate, propionate, butyrate, ammonium, and branched short-chain fatty acids, with T5 and T1 consistently stimulating propionate and butyrate, respectively. 16S-targeted Illumina sequencing coupled with flow cytometry provided unprecedented high-resolution quantitative insights in canine and feline microbiota modulation by yeast-derived ingredients, revealing that effects on propionate production were related to Prevotellaceae, Tannerellaceae, Bacteroidaceae, and Veillonellaceae members, while effects on butyrate production were related to Erysipelotrichaceae, Lachnospiraceae, Ruminococcaceae, and Fusobacteriaceae. Overall, these findings strengthen the health-promoting potential of yeast-derived ingredients.

The colon gut microbiota is responsible for complex chemical conversions of nutrients and subsequent release of metabolites that have diverse biological consequences. However, information on the metabolic dynamics that occur longitudinally through the colon is limited. Here, gas and liquid chromatographies coupled with mass spectrometry were applied to generate metabolic profiles of the region-specific microbial communities cultured using an in vitro platform simulating the ascending (AC), transverse (TC), and descending (DC) colon regions. Comparative analysis revealed a large divergence between metabolic profiles of the AC and the TC and DC regions in terms of short-chain fatty acid production, metabolic spectrum, and conversion of bile acids. Metagenomic evaluation revealed that the regionally derived metabolic profiles had strong correlation to community composition and genetic potential. Together, the results provide key insights regarding the metabolic divergence of the regional communities that are integral to understand the structure-function relationship of the gut microbiota.

Dietary fibers are essential components of a balanced diet and have beneficial effects on metabolic functions. To gain insight into their impact on host physiology and gut microbiota, we performed a direct comparison of two specific prebiotic fibers in mice. During an 8-wk follow up, mice fed a high-fat diet (HFD) were compared with mice on a normal diet (basal condition, controls) and to mice fed the HFD but treated with one of the following prebiotics: fructooligosaccharides (FOS) or soluble corn fiber (SCF). Both prebiotic fibers led to a similar reduction of body weight and fat mass, lower inflammation and improved metabolic parameters. However, these health benefits were the result of different actions of the fibers, as SCF impacted energy excretion, whereas FOS did not. Interestingly, both fibers had very distinct gut microbial signatures with different short-chain fatty acid profiles, indicating that they do not favor the growth of the same bacterial communities. Although the prebiotic potential of different fibers may seem physiologically equivalent, our data show that the underlying mechanisms of action are different, and this by targeting different gut microbes. Altogether, our data provide evidence that beneficial health effects of specific dietary fibers must be documented to be considered a prebiotic and that studies devoted to understanding how structures relate to specific microbiota modulation and metabolic effects are warranted.

Symprove, a multi-strain probiotic, has been shown to exert a mild anti-inflammatory effect in patients with ulcerative colitis (UC). We examined stool samples from 3 patients with UC in order to create microbiotas in an in-vitro gut model. The effects of Symprove on bacterial diversity and metabolic activity in the microbiotas was evaluated over 48 h. In addition, the influence of probiotic dosing on epithelial tight-junction integrity, production of inflammatory markers and wound healing were evaluated in cell culture models. The relative proportions of the main bacterial phyla in UC patients differed from those of healthy subjects studied previously; levels of Firmicutes were lowered and levels of Bacteroidetes were raised. Addition of Symprove changed the bacterial composition in the microbiotas over a 48 h period. Several other factors generally implicated in good gut health changed after dosing with probiotic; production of short chain fatty acids (SCFAs) and lactate was stimulated, levels of anti-inflammatory cytokines (IL-6, IL-10) increased, levels of pro-inflammatory cytokines and chemokines (MCP-1 and IL-8) decreased, epithelial tight junction integrity improved and wound healing occurred faster than a control. The results imply it is not the simple addition of probiotic bacteria that improves gut health. Rather, the probiotic bacteria generate lactate, which then stimulates growth of commensal gut bacteria, raising SCFA levels (particularly butyrate). The increased butyrate concentration positively influences inflammation response and time of wound healing., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: This study evaluated the impact of Bifidobacterium animalis ssp. lactis CNCM I-3446, Bovine Milk-derived OligoSaccharides (BMOS) and their combination on infant gut microbiota in vitro. In addition, a novel strategy consisting of preculturing B. lactis with BMOS to further enhance their potential synbiotic effects was assessed., Method: Short-term fecal batch fermentations (48 h) were used to assess the microbial composition and activity modulated by BMOS alone, B. lactis grown on BMOS or dextrose alone, or their combinations on different three-month-old infant microbiota., Results: BMOS alone significantly induced acetate and lactate production (leading to pH decrease) and stimulated bifidobacterial growth in 10 donors. A further in-depth study on two different donors proved B. lactis ability to colonize the infant microbiota, regardless of the competitiveness of the environment. BMOS further enhanced this engraftment, suggesting a strong synbiotic effect. This was also observed at the microbiota activity level, especially in a donor containing low initial levels of bifidobacteria. In this donor, preculturing B. lactis with BMOS strengthened further the early modulation of microbiota activity observed after 6 h., Conclusion: This study demonstrated the strong synbiotic effect of BMOS and B. lactis on the infant gut microbiota, and suggests a strategy to improve its effectiveness in an otherwise low- Bifidobacterium microbiota.

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.

The outer cell wall of yeast is characterized by high levels of β-glucans and mannan-oligosaccharides (MOS), which have been linked with beneficial effects on intestinal health and immune status in dogs. In this study, a standardized in vitro simulation of the canine gastrointestinal tract (Simulator of the Canine Intestinal Microbial Ecosystem; SCIME) was used to evaluate the effect of a Saccharomyces cerevisiae-based product, consisting of 27.5% β-glucans and 22.5% MOS, on the activity (as assessed by measurement of fermentative metabolites) and composition (as assessed by 16S-targeted Illumina sequencing) of canine intestinal microbiota. The S. cerevisiae-based product was tested at three different dosages, i.e., 0.5, 1.0, and 2.0 g/d. A dose-dependent fermentation pattern was observed along the entire length of the colon, as shown by the increased production of the health-related acetate, propionate, and butyrate for the three concentrations tested (0.5, 1.0, and 2.0 g/d). A consistent finding for all three tested concentrations was the increased propionate production (P < 0.05) in the simulated proximal and distal colon. These changes in terms of fermentative metabolites could be linked to specific microbial alterations at the family level, such as the specific stimulation of the propionate-producing families Porphyromonadaceae and Prevotellaceae upon in vitro exposure to the S. cerevisiae-based product. Other consistent changes in community composition upon repeated exposure included the decrease in the Enterobacteriaceae and the Fusobacteriaceae families, which both contain several potentially opportunistic pathogens. Altogether, the generated data support a possible health-promoting role of a product high in β-glucans and MOS when supplemented to the dogs' diet., (© The Author(s) 2020. Published by Oxford University Press on behalf of the American Society of Animal Science.)

: Background: Prebiotics used as a dietary supplement, stimulate health-related gut microbiota (e.g., bifidobacteria, lactobacilli, etc.). This study evaluated potential prebiotic effects of an artichoke aqueous dry extract (AADE) using in vitro gut model based on the Simulator of Human Intestinal Microbial Ecosystem (SHIME ® )., Methods: Short-term colonic fermentations (48 h) of AADE, fructo-oligosaccharides (FOS), and a blank were performed. Microbial metabolites were assessed at 0, 6, 24, and 48 h of colonic incubation via measuring pH, gas pressure, lactate, ammonium, and short-chain fatty acids (SCFAs) levels. Community composition was assessed via targeted qPCRs., Results: After 24 and 48 h of incubation, bifidobacteria levels increased 25-fold with AADE ( p < 0.05) and >100-fold with FOS ( p < 0.05) compared to blank. Lactobacillus spp. levels only tended to increase with AADE, whereas they increased 10-fold with FOS. At 6 h, pH decreased with AADE and FOS and remained stable until 48 h; however, gas pressure increased significantly till the end of study. Acetate, propionate, and total SCFA production increased significantly with both at all time-points. Lactate levels initially increased but branched SCFA and ammonium levels remained low till 48 h., Conclusion: AADE displayed prebiotic potential by exerting bifidogenic effects that stimulated production of health-related microbial metabolites, which is potentially due to inulin in AADE., Competing Interests: A.V., A.Z., and E.R. are employed by Euromed S.A. The study funders (Euromed S.A.) had no role in the design, collection, or analysis of the data.

Enrichment of food crops with iodine is an option to alleviate dietary deficiencies. Therefore, foliar iodine fertilizer was applied on wheat and rice, in the presence and absence of the other micronutrients zinc and selenium. This treatment increased the concentration of iodine, as well as zinc and selenium, in the staple grains. Subsequently, potential iodine losses during preparation of foodstuffs with the enriched grains were studied. Oven-heating did not affect the iodine content in bread. Extraction of bran from flour lowered the iodine in white bread compared to wholegrain bread, but it was still markedly higher compared to the control. During subsequent in vitro gastrointestinal digestion, a higher percentage of iodine was released from foods based on extracted flour (82-92%) compared to wholegrain foods (50-76%). The foliar fertilization of wheat was found to be adequate to alleviate iodine deficiency in a population with a moderate to high intake of bread.