Specificity of polysaccharide use in intestinal bacteroides species determines diet-induced microbiota alterations.

The intestinal microbiota impacts many facets of human health and is associated with human diseases. Diet impacts microbiota composition, yet mechanisms that link dietary changes to microbiota alterations remain ill-defined. Here we elucidate the basis of Bacteroides proliferation in response to fructans, a class of fructose-based dietary polysaccharides. Structural and genetic analysis disclosed a fructose-binding, hybrid two-component signaling sensor that controls the fructan utilization locus in Bacteroides thetaiotaomicron. Gene content of this locus differs among Bacteroides species and dictates the specificity and breadth of utilizable fructans. BT1760, an extracellular beta2-6 endo-fructanase, distinguishes B. thetaiotaomicron genetically and functionally, and enables the use of the beta2-6-linked fructan levan. The genetic and functional differences between Bacteroides species are predictive of in vivo competitiveness in the presence of dietary fructans. Gene sequences that distinguish species' metabolic capacity serve as potential biomarkers in microbiomic datasets to enable rational manipulation of the microbiota via diet.

The effect of in vitro simulated colonic pH gradients on microbial activity and metabolite production using common prebiotics as substrates.

BACKGROUND: The interplay between gut microbiota (GM) and the metabolization of dietary components leading to the production of short-chain fatty acids (SCFAs) is affected by a range of factors including colonic pH and carbohydrate source. However, there is still only limited knowledge on how the GM activity and metabolite production in the gastrointestinal tract could be influenced by pH and the pH gradient increases along the colon. RESULTS: Here we investigate the effect of pH gradients corresponding to levels typically found in the colon on GM composition and metabolite production using substrates inulin, lactose, galactooligosaccharides (GOS), and fructooligosaccharide (FOS) in an in vitro colon setup. We investigated 3 different pH regimes (low, 5.2 increasing to 6.4; medium, 5.6 increasing to 6.8 and high, 6.0 increasing to 7.2) for each fecal inoculum and found that colonic pH gradients significantly influenced in vitro simulated GM structure, but the influence of fecal donor and substrate was more pronounced. Low pH regimes strongly influenced GM with the decreased relative abundance of Bacteroides spp. and increased Bifidobacterium spp. Higher in vitro simulated colonic pH promoted the production of SCFAs in a donor- and substrate-dependent manner. The butyrate producer Butyricimonas was enriched at higher pH conditions, where also butyrate production was increased for inulin. The relative abundance of Phascolarctobacterium, Bacteroides, and Rikenellaceae also increased at higher colonic pH, which was accompanied by increased production of propionate with GOS and FOS as substrates. CONCLUSIONS: Together, our results show that colonic substrates such as dietary fibres influence GM composition and metabolite production, not only by being selectively utilized by specific microbes, but also because of their SCFA production, which in turn also influences colonic pH and overall GM composition and activity. Our work provides details about the effect of the gradients of rising pH from the proximal to distal colon on fermenting dietary substrates in vitro and highlights the importance of considering pH in GM research.

Identification of carbohydrate gene clusters obtained from in vitro fermentations as predictive biomarkers of prebiotic responses.

BACKGROUND: Prebiotic fibers are non-digestible substrates that modulate the gut microbiome by promoting expansion of microbes having the genetic and physiological potential to utilize those molecules. Although several prebiotic substrates have been consistently shown to provide health benefits in human clinical trials, responder and non-responder phenotypes are often reported. These observations had led to interest in identifying, a priori, prebiotic responders and non-responders as a basis for personalized nutrition. In this study, we conducted in vitro fecal enrichments and applied shotgun metagenomics and machine learning tools to identify microbial gene signatures from adult subjects that could be used to predict prebiotic responders and non-responders. RESULTS: Using short chain fatty acids as a targeted response, we identified genetic features, consisting of carbohydrate active enzymes, transcription factors and sugar transporters, from metagenomic sequencing of in vitro fermentations for three prebiotic substrates: xylooligosacharides, fructooligosacharides, and inulin. A machine learning approach was then used to select substrate-specific gene signatures as predictive features. These features were found to be predictive for XOS responders with respect to SCFA production in an in vivo trial. CONCLUSIONS: Our results confirm the bifidogenic effect of commonly used prebiotic substrates along with inter-individual microbial responses towards these substrates. We successfully trained classifiers for the prediction of prebiotic responders towards XOS and inulin with robust accuracy (≥ AUC 0.9) and demonstrated its utility in a human feeding trial. Overall, the findings from this study highlight the practical implementation of pre-intervention targeted profiling of individual microbiomes to stratify responders and non-responders.

The emergence of inflammatory microglia during gut inflammation is not affected by FFAR2 expression in intestinal epithelial cells or peripheral myeloid cells.

Gut inflammation can trigger neuroinflammation and is linked to mood disorders. Microbiota-derived short-chain fatty acids (SCFAs) can modulate microglia, yet the mechanism remains elusive. Since microglia do not express free-fatty acid receptor (FFAR)2, but intestinal epithelial cells (IEC) and peripheral myeloid cells do, we hypothesized that SCFA-mediated FFAR2 activation within the gut or peripheral myeloid cells may impact microglia inflammation. To test this hypothesis, we developed a tamoxifen-inducible conditional knockout mouse model targeting FFAR2 exclusively on IEC and induced intestinal inflammation with dextran sodium sulfate (DSS), a well-established colitis model. Given FFAR2's high expression in myeloid cells, we also investigated its role by selectively deleting it in these populations of cells. In an initial study, male and female wild-type mice received 0 or 2% DSS for 5d and microglia were isolated 3d later to assess inflammatory status. DSS induced intestinal inflammation and upregulated inflammatory gene expression in microglia, indicating inflammatory signaling via the gut-brain axis. Despite the lack of significant effects of sex in the intestinal phenotype, male mice showed higher microglial inflammatory response than females. Subsequent studies using FFAR2 knockout models revealed that FFAR2 expression in IECs or immune myeloid cells did not affect DSS-induced colonic pathology (i.e. clinical and histological scores and colon length), or colonic expression of inflammatory genes. However, FFAR2 knockout led to an upregulation of several microglial inflammatory genes in control mice and downregulation in DSS-treated mice, suggesting that FFAR2 may constrain neuroinflammatory gene expression under healthy homeostatic conditions but may permit it during intestinal inflammation. No interactions with sex were observed, suggesting sex does not play a role on FFAR2 potential function in gut-brain communication in the context of colitis. To evaluate the role of FFAR2 activated by microbiota-derived SCFAs, we employed the same knockout and DSS models adding fermentable dietary fiber (0 or 2.5% inulin for 8 wks). Despite no genotype or fiber main effects, contrary to our hypothesis, inulin feeding augmented DSS-induced inflammation and signs of colitis, suggesting context-dependent effects of fiber. These findings highlight microglial involvement in colitis-associated neuroinflammation and advance our understanding of FFAR2's role in the gut-brain axis. Although not integral, we observed that the role of FFAR2 differs between homeostatic and inflammatory conditions, underscoring the need to consider different inflammatory conditions and disease contexts when investigating the role of FFAR2 and SCFAs in the gut-brain axis.

Production of polyhydroxybutyrate by coupled saccharification-fermentation of inulin.

Inulin is a fructose-based polysaccharide that can be found in several plant species, from grass and onions to chicory roots; thus, it has the potential to be an excellent renewable source of fructose for several industrial applications. Among them, inulin hydrolysis can be coupled to a fermentation operation to produce polyhydroxybutyrate (PHB) using Cupriavidus necator H16. This work reports the PHB production process involving chicory root inulin hydrolysis using inulinase Novozym 960 followed by a C. necator fermentation. It was found that the maximum saccharification (95% wt.) was reached at 269 U/ginulin after 90 min. The hydrolysates obtained were then inoculated with C. necator, leading to a biomass concentration of 4 g/L with 30% (w/w) polymer accumulation. Although PHB production was low, during the first hours, the cell growth and polymer accumulation detected did not coincide with a fructose concentration decrease, suggesting a simultaneous saccharification and fermentation process, potentially alleviating the product inhibition inherent to the inulinase-fructose system. The characterization of the obtained PHB showed a polymer with more homogeneous values of Mw, and better thermal stability than PHB produced using pure fructose as a fermentation substrate. The results obtained demonstrate a viable alternative carbon substrate for PHB production, opening the possibility for inulin-rich renewable feedstock valorization.

Prebiotic inulin as a treatment of obesity related nonalcoholic fatty liver disease through gut microbiota: a critical review.

The microbial-derived products, including short chain fatty acids, lipopolysaccharide and secondary bile acids, have been shown to participate in the regulation of hepatic lipid metabolism. Previous studies have demonstrated that prebiotics, such as oligosaccharide and inulin, have abilities to change the concentration of microbial-derived products through modulating the microbial community structure, thus controlling body weight and alleviating hepatic fat accumulation. However, recent evidence indicates that there are individual differences in host response upon inulin treatment due to the differences in host microbial composition before dietary intervention. Probably it is because of the multiple relationships among bacterial species (e.g., competition and mutualism), which play key roles in the degradation of inulin and the regulation of microbial structure. Thereby, analyzing the composition and function of initial gut microbiota is essential for improving the efficacy of prebiotics supplementation. Furthermore, considering that different structures of polysaccharides can be used by different microorganisms, the chemical structure of processed inulin should be tested before using prebiotic inulin to treat obesity related nonalcoholic fatty liver disease.

Properties of the extracellular alkaline inulinase produced by haloalkaliphilic phototrophic bacteria Ectothiorodospirea mobilis.

The studies have revealed alkaline exoinulinase produced by haloalkaliphilic phototrophic bacteria Ectothiorhodospirea mobilis Al-2 for the first time. A new method for the isolation of a homogeneous exoinulinase from the culture broth was developed and the properties of this enzyme have been investigated. It was shown that specified exoinulinase in contrast to the studied exoinulinases produced by microorganisms exhibits catalytic activity at the wide range of pH (7.0-10) and a temperature (20-60 °C) with a maximum of the inulolitic activity at pH 9.0 and 50 °C. The studied exoinulinase possessing also invertase activity (I/S1.4) is a monomeric protein with molecular mass 57Kda, as well as Km and Vmax for inulin 3.8 mM/ml and 10 µmol/ml/min-1, respectively. The studies of the influence of different metal ions on enzyme activity have shown that Mn+2, Cu+2, Co+2, Mg+2, NaCl 5-7% promote relatively higher catalytic activity while Zn+2, Cu+2 and Fe+2 partially suppress the enzyme activity and Hg2+completely inactivates the enzyme.The formation of only fructose and glucose at the enzymatic hydrolysis of inulin confirms that the studied exoinulinase belongs to the exo-type of enzymes. The obtained results supplement our fundamental knowledge in biochemistry-enzymology, as well as the biodiversity of microorganisms expressing exoinulinase. The studied exoinulinase exhibits activity at salinity of the medium and can potentially be used in the biotechnology of inulin bioconversion into bioproducts under alkaline conditions.

Dietary Galactooligosaccharides Supplementation as a Gut Microbiota-Regulating Approach to Lower Early Life Arsenic Exposure.

Prebiotics may stimulate beneficial gut microorganisms. However, it remains unclear whether they can lower the oral bioavailability of early life arsenic (As) exposure via regulating gut microbiota and altering As biotransformation along the gastrointestinal (GI) tract. In this study, weanling mice were exposed to arsenate (iAsV) via diet (7.5 µg As g-1) amended with fructooligosaccharides (FOS), galactooligosaccharides (GOS), and inulin individually at 1% and 5% (w/w). Compared to As exposure control mice, As concentrations in mouse blood, liver, and kidneys and As urinary excretion factor (UEF) were reduced by 43.7%-74.1% when treated with 5% GOS. The decrease corresponded to a significant proliferation of Akkermansia and Psychrobacter, reduced percentage of inorganic arsenite (iAsIII) and iAsV by 47.4% and 65.4%, and increased proportion of DMAV in intestinal contents by 101% in the guts of mice treated with 5% GOS compared to the As control group. In contrast, FOS and inulin either at l% or 5% did not reduce As concentration in mouse blood, liver, and kidneys or As UEF. These results suggest that GOS supplementation may be a gut microbiota-regulating approach to lower early life As exposure via stimulating the growth of Akkermansia and Psychrobacter and enhancing As methylation in the GI tract.

Phenotypic and genomic analysis of inulin consumption by Lactiplantibacillus plantarum strains from Sichuan pickle.

AIMS: To investigate the capability, properties, and molecular mechanism of inulin fermentation by lactic acid bacteria (LAB) from Sichuan pickle. METHODS AND RESULTS: A total of 79 LAB strains were purified from 30 aged Sichuan pickle brine samples, and only 21 Lactiplantibacillus plantarum strains (26.58%, 21/79) derived from 15 samples grew well through utilizing inulin as a carbon source. The fermentation tests through using long-chain inulin (lc-inulin) as a carbon source showed that only 6 L. plantarum strains grew well, while other 15 strains could only utilize short-chain oligofructose (FOS), and thin-layer chromatography analysis evidenced a strain specificity of inulin consumption patterns. Lactiplantibacillus plantarum YT041 is a vigorous inulin fermenter, and whole genome sequencing data revealed that sacPTS1 and fosRABCDXE operons might be associated with the fermentation of FOS and lc-inulin, respectively. CONCLUSIONS: The phenotype of inulin consumption is commonly present in LAB from Sichuan pickle, which is strain-specific and largely depends on their specific ecological niche and degree of polymerization.

Oligofructose, 2'fucosyllactose and ß-glucan in combination induce specific changes in microbial composition and short-chain fatty acid production compared to sole supplementation.

AIMS: In this study, we explored the effects that the prebiotic inulin-type fructans, and prebiotic candidates: 2'fucosyllactose and ß-glucan from barley, singular and in combination had on microbial load, microbiome profile, and short-chain fatty acid production. This was carried out as a prescreening tool to determine combinations that could be taken forward for use in a human intervention trial. METHODS AND RESULTS: Effects of inulin-type fructans, 2'fucosyllactose and ß-glucan from barley in singular and combination on microbial load and profile and short-chain fatty acid production (SCFA) was conducted using in vitro batch culture fermentation over 48 h. Changes in microbial load and profile were assessed by fluorescence in situ hybridization flow cytometry (FISH-FLOW) and 16S rRNA sequencing, and changes in SCFA via gas chromatography. All substrates generated changes in microbial load and profile, achieving peak microbial load at 8 h fermentation with the largest changes in profile across all substrates in Bifidobacterium (Q < 0.05). This coincided with significant increases in acetate observed throughout fermentation (Q < 0.05). In comparison to sole supplementation combinations of oligofructose, ß-glucan and 2'fuscosyllactose induced significant increases in both propionate and butyrate producing bacteria (Roseburia and Faecalibacterium praunitzii), and concentrations of propionate and butyrate, the latter being maintained until the end of fermentation (all Q < 0.05). CONCLUSIONS: Combinations of oligofructose, with ß-glucan and 2'fucosyllactose induced selective changes in microbial combination and SCFA namely Roseburia, F. praunitzii, propionate and butyrate compared to sole supplementation.

Effect of resistant starch types as a prebiotic.

Since the role of intestinal microbiota in metabolism was understood, the importance of dietary components such as fibres and prebiotics, which affect the modulation of microbiota, has been increasing day by day. While all prebiotic components are considered dietary fibre, not every dietary fibre is considered a prebiotic. While fructooligosaccharides, galactooligosaccharides, inulin, and galactans are considered prebiotics, other fermentable carbohydrates are considered candidate prebiotic components based on in vitro and preclinical studies. Resistant starch, one of such carbohydrates, is considered a potential prebiotic component when it is made resistant to digestion naturally or chemically. In this review, both in vitro and in vivo studies in which the prebiotic capacity of type II, type III, and type IV resistant starch isolated from food and produced commercially was assessed were analyzed. According to the results of current studies, certain types of resistant starch are thought to have a high prebiotic capacity, and they may be candidate prebiotic components although positive results have not been achieved in all studies. KEY POINTS: • Resistant starch is undigested in the small intestine and is fermented in the large intestine. • Resistant starch fermentation positively affects the growth of Bifidobacterium and Lactobacillus. • Resistant starch can be considered a prebiotic ingredient.

Inulin increases the EPS biosynthesis of Lactobacillus delbrueckii ssp. bulgaricus LDB-C1.

OBJECTIVE: Its eps gene cluster, the antioxidant activity and monosaccharide composition of exopolysaccharides, the expression levels of related genes at different fermentations were analyzed for clarifying the exopolysaccharide biosynthesis mechanism of Lactobacillus delbrueckii subsp. bulgaricus LDB-C1. RESULTS: The comparison analysis of eps gene clusters indicated that the gene clusters present diversity and strain specificity. The crude exopolysaccharides from LDB-C1 exhibited a good antioxidant activity. Compared with glucose, fructose, galactose, and fructooligosaccharide, inulin significantly improved the exopolysaccharide biosynthesis. The structures of EPSs were significantly different under different carbohydrate fermentation conditions. Inulin obviously increased the expressions of most EPS biosynthesis related genes at fermentation 4 h. CONCLUSION: Inulin accelerated the beginning of the exopolysaccharide production in LDB-C1, and the enzymes promoted by inulin was beneficial for the accumulation of exopolysaccharide at the whole fermentation process.

Production, purification, and characterization of inulinase from Streptomyces anulatus.

Inulinase is an enzyme that catalyzes inulin to d-fructose. This enzyme can be extracted from plants, but it is difficult to obtain it in large quantities, so its production cost is high. Therefore, microbial inulinase has great potential for industrial needs. In the last decade, there have been very few reports on actinobacterial inulinases, especially on purification and characterization of inulinase process extraction. This study aims to select actinomycetes that possess high inulinase activity from the soil. To screen inulinase-producing bacteria, modified Czapex-Dox agar supplemented with 1% inulin powder was used. The most effective isolate was Streptomyces sp. EFBO8, morphological and genotypic identification methods, confirmed that the strain is Streptomyces anulatus and that its nucleotide sequence has been deposited in GenBank under accession number OQ073700. To optimize inulinase production, kinetics were performed by using S. anulatus strain, which proved to be most productive with a value of 24,024 EU/mL. The enzyme was purified from the culture filtrate by precipitation with ammonium sulfate (NH4 )2 SO4 , followed by column chromatography Sephadex (G-50) separation. Purified protein has a molecular mass of 3331.83 Da.

Influence of pH on Inulin Conversion to 2,3-Butanediol by Bacillus licheniformis 24: A Gene Expression Assay.

2,3-Butanediol (2,3-BD) is an alcohol highly demanded in the chemical, pharmaceutical, and food industries. Its microbial production, safe non-pathogenic producer strains, and suitable substrates have been avidly sought in recent years. The present study investigated 2,3-BD synthesis by the GRAS Bacillus licheniformis 24 using chicory inulin as a cheap and renewable substrate. The process appears to be pH-dependent. At pH 5.25, the synthesis of 2,3-BD was barely detectable due to the lack of inulin hydrolysis. At pH 6.25, 2,3-BD concentration reached 67.5 g/L with rapid hydrolysis of the substrate but was accompanied by exopolysaccharide (EPS) synthesis. Since inulin conversion by bacteria is a complex process and begins with its hydrolysis, the question of the acting enzymes arose. Genome mining revealed that several glycoside hydrolase (GH) enzymes from different CAZy families are involved. Five genes encoding such enzymes in B. licheniformis 24 were amplified and sequenced: sacA, sacB, sacC, levB, and fruA. Real-time RT-PCR experiments showed that the process of inulin hydrolysis is regulated at the level of gene expression, as four genes were significantly overexpressed at pH 6.25. In contrast, the expression of levB remained at the same level at the different pH values at all-time points. It was concluded that the sacC and sacA/fruA genes are crucial for inulin hydrolysis. They encode exoinulinase (EC 3.2.1.80) and sucrases (EC 3.2.1.26), respectively. The striking overexpression of sacB under these conditions led to increased synthesis of EPS; therefore, the simultaneous production of 2,3-BD and EPS cannot be avoided.

Dietary Supplementation of Inulin Contributes to the Prevention of Estrogen Receptor-Negative Mammary Cancer by Alteration of Gut Microbial Communities and Epigenetic Regulations.

Breast cancer (BC) is among the most frequently diagnosed malignant cancers in women in the United States. Diet and nutrition supplementation are closely related to BC onset and progression, and inulin is commercially available as a health supplement to improve gut health. However, little is known with respect to inulin intake for BC prevention. We investigated the effect of an inulin-supplemented diet on the prevention of estrogen receptor-negative mammary carcinoma in a transgenic mouse model. Plasma short-chain fatty acids were measured, the gut microbial composition was analyzed, and the expression of proteins related to cell cycle and epigenetics-related genes was measured. Inulin supplementation greatly inhibited tumor growth and significantly delayed tumor latency. The mice that consumed inulin had a distinct microbiome and higher diversity of gut microbial composition compared to the control. The concentration of propionic acid in plasma was significantly higher in the inulin-supplemented group. The protein expression of epigenetic-modulating histone deacetylase 2 (Hdac2), Hdac8, and DNA methyltransferase 3b decreased. The protein expression of factors related to tumor cell proliferation and survival, such as Akt, phospho-PI3K, and NF-kB, also decreased with inulin administration. Furthermore, sodium propionate showed BC prevention effect in vivo through epigenetic regulations. These studies suggest that modulating microbial composition through inulin consumption may be a promising strategy for BC prevention.

Phytochemical Characterization of Purple Coneflower Roots (Echinacea purpurea (L.) Moench.) and Their Extracts.

Echinacea purpurea is a perennial plant that belongs to the Asteraceae family. It has a wide range of applications mainly in the treatment and prevention of inflammations in the respiratory system. The current study aimed to perform a phytochemical characterization of purple coneflower (Echinacea purpurea) roots and their extracts (water, 40%, 50%, 60% ethanol, and 60% glycerol). Phytochemical characterization was carried out by gravimetric, spectrophotometric, and chromatographic methods. Echinacea roots were characterized by a low lipid (0.8%) content. In contrast, carbohydrates (45%) and proteins (20%) occupied a large part of the dry matter. Amongst the extracts, the highest yield was obtained using water as a solvent (53%). Water extract was rich in protein and carbohydrates as fructans (inulin) were the most abundant carbohydrate constituent. The most exhaustive recovery of the phenolic components was conducted by extraction with 40% ethanol and 60% glycerol. It was found that water is the most suitable extractant for obtaining a polysaccharide-containing complex (PSC) (8.87%). PSC was composed mainly of fructans (inulin) and proteins with different molecular weight distributions. The yield of PSC decreased with an increasing ethanol concentration (40% > 50% > 60%) but the lowest yield was obtained from 60% glycerol extract. The obtained results showed that Echinacea roots contained a large amount of biologically active substances-phenolic components and polysaccharides and that glycerol was equally efficient to ethanol in extracting caffeic acid derivatives from purple coneflower roots. The data can be used for the preparation of extracts having different compositions and thus easily be incorporated into commercial products.

Film Coatings Based on Aqueous Shellac Ammonium Salt "Swanlac® ASL 10" and Inulin for Colon Targeting.

Over the past decades, increasing interests took place in the realm of drug delivery systems. Beyond treating intestinal diseases such as inflammatory bowel disease, colon targeting can provide possible applications for oral administration of proteins as well as vaccines due to the lower enzymatic activity in the distal part of GIT. To date, many strategies are employed to reach the colon. This article encompasses different biomaterials tested as film coatings and highlights appropriate formulations for colonic drug delivery. A comparison of different films was made to display the most interesting drug release profiles. These films contained ethylcellulose, as a thermoplastic polymer, blended with an aqueous shellac ammonium salt solution. Different blend ratios were selected as well for thin films as for coated mini-tablets, mainly varying as follows: (80:20); (75:25); (60:40). The impact of blend ratio and coating level was examined as well as the addition of natural polysaccharide "inulin" to target the colon. In vitro drug release was measured in 0.1 M HCl for 2 h followed by phosphate buffer saline pH 6.8 to simulate gastric and intestinal fluids, respectively. Coated mini-tablets were exposed to fresh fecal samples of humans in order to simulate roughly colonic content. Several formulations were able to fully protect theophylline as a model drug up to 8 h in the upper GIT, but allowing for prolonged release kinetics in the colon. These very interesting colonic release profiles were related to the amount of the natural polysaccharide added into the system.

Psyllium reduces inulin-induced colonic gas production in IBS: MRI and in vitro fermentation studies.

OBJECTIVE: Health-promoting dietary fibre including inulin often triggers gastrointestinal symptoms in patients with IBS, limiting their intake. Our aim was to test if coadministering psyllium with inulin would reduce gas production. DESIGN: A randomised, four-period, four-treatment, placebo-controlled, crossover trial in 19 patients with IBS. Subjects ingested a 500 mL test drink containing either inulin 20 g, psyllium 20 g, inulin 20 g+ psyllium 20 g or dextrose 20 g (placebo). Breath hydrogen was measured every 30 min with MRI scans hourly for 6 hours. Faecal samples from a subset of the patients with IBS were tested using an in vitro fermentation model. Primary endpoint was colonic gas assessed by MRI. RESULTS: Colonic gas rose steadily from 0 to 6 hours, with inulin causing the greatest rise, median (IQR) AUC(0-360 min) 3145 (848-6502) mL·min. This was significantly reduced with inulin and psyllium coadministration to 618 (62-2345) mL·min (p=0.02), not significantly different from placebo. Colonic volumes AUC(0-360 min) were significantly larger than placebo for both inulin (p=0.002) and inulin and psyllium coadministration (p=0.005). Breath hydrogen rose significantly from 120 min after inulin but not psyllium; coadministration of psyllium with inulin delayed and reduced the maximum increase, AUC(0-360 min) from 7230 (3255-17910) ppm·hour to 1035 (360-4320) ppm·hour, p=0.007.Fermentation in vitro produced more gas with inulin than psyllium. Combining psyllium with inulin did not reduce gas production. CONCLUSIONS: Psyllium reduced inulin-related gas production in patients with IBS but does not directly inhibit fermentation. Whether coadministration with psyllium increases the tolerability of prebiotics in IBS warrants further study. TRIAL REGISTRATION NUMBER: NCT03265002.

Non-Ionic Inulin-Based Polymer Nanodiscs Enable Functional Reconstitution of a Redox Complex Composed of Oppositely Charged CYP450 and CPR in a Lipid Bilayer Membrane.

Although polymer-based lipid nanodiscs are increasingly used in the structural studies of membrane proteins, the charge of the belt-forming polymer is a major limitation for functional reconstitution of membrane proteins possessing an opposite net charge to that of the polymer. This limitation also rules out the reconstitution of a protein-protein complex composed of oppositely charged membrane proteins. In this study, we report the first successful functional reconstitution of a membrane-bound redox complex constituting a cationic cytochrome P450 (CYP450) and an anionic cytochrome P450 reductase (CPR) in non-ionic inulin-based lipid nanodiscs. The gel-to-liquid-crystalline phase-transition temperature (Tm) of DMPC:DMPG (7:3 w/w) lipids in polymer nanodiscs was determined by differential scanning calorimetry (DSC) and 31P NMR experiments. The CYP450-CPR redox complex reconstitution in polymer nanodiscs was characterized by size-exclusion chromatography (SEC), and the electron transfer kinetics was carried out using the stopped-flow technique under anaerobic conditions. The Tm of DMPC:DMPG (7:3 w/w) in polymer nanodiscs measured from 31P NMR agrees with that obtained from DSC and was found to be higher than that for liposomes due to the decreased cooperativity of lipids present in the nanodiscs. The stopped-flow measurements revealed the CYP450-CPR redox complex reconstituted in nanodiscs to be functional, and the electron transfer kinetics was found to be temperature-dependent. Based on the successful demonstration of the use of non-ionic inulin-based polymer nanodiscs reported in this study, we expect them to be useful in studying the function and structures of a variety of membrane proteins/complexes irrespective of the charge of the molecular components. Since the polymer nanodiscs were shown to align in an externally applied magnetic field, they can also be used to measure residual dipolar couplings (RDCs) and residual quadrupolar couplings (RQCs) for various molecules ranging from small molecules to soluble proteins and nucleic acids.

Inulin activates FXR-FGF15 signaling and further increases bile acids excretion in non-alcoholic fatty liver disease mice.

Non-alcoholic fatty liver disease (NAFLD) is a metabolic disorder defined as the presence of intrahepatic lipid deposition and steatosis as well as chronic inflammation without excessive alcohol consumption. Our previous studies found that inulin could dramatically improve lipid metabolism disorders in NAFLD murine models. In recent years, mounting evidence has approved that there are disproportionately increased bile acids (BAs) in patients with NAFLD while the hepatic bile acids signaling is suppressed. Meanwhile the primary function of bile acids is to promote the excretion of cholesterol and therefore keep the cholesterol metabolism balance. Hence, we investigate whether inulin exerts beneficial effects on lipid metabolism disorders by modulating bile acids signaling in our present study. And we found that inulin treatment significantly reversed the abnormal accumulation of bile acids in high-fat-induced NAFLD mice. Furthermore, our data confirmed that inulin supplementation attenuates NAFLD via restoring the activity of FXR accompanied by increasing hepatic bile acids de novo synthesis and further enhancing bile acids excretion in mice.