The branching ratio of enzymatically synthesized α-glucans impacts microbiome and metabolic outcomes of in vitro fecal fermentation.

The aim of this study was to evaluate the impacts of enzymatically synthesized α-glucans possessing α-1,4- and α-1,6-glucose linkages, and varying in branching ratio, on colonic microbiota composition and metabolic function. Four different α-glucans varying in branching ratio were synthesized by amylosucrase from Neisseria polysaccharea and glycogen branching enzyme from Rhodothermus obamensis. The branching ratios were found to range from 0 % to 2.8 % using GC/MS. In vitro fecal fermentation analyses (n = 8) revealed that the branching ratio dictates the short-chain fatty acid (SCFA) generation by fecal microbiota. Specifically, slightly branched (0.49 %) α-glucan resulted in generation of significantly (P < 0.05) higher amounts of propionate, compared to more-branched counterparts. In addition, the amount of butyrate generated from this α-glucan was statistically (P > 0.05) indistinguishable than those observed in resistant starches. 16S rRNA sequencing revealed that enzymatically synthesized α-glucans stimulated Lachnospiraceae and Ruminococcus related OTUs. Overall, the results demonstrated metabolic function of colonic microbiota can be manipulated by altering the branching ratio of enzymatically synthesized α-glucans, providing insights into specific structure-function relationships between dietary fibers and the colonic microbiome. Furthermore, the slightly branched α-glucans could be used as functional carbohydrates to stimulate the beneficial microbiota and SCFAs in the colon.

When simplicity triumphs: niche specialization of gut bacteria exists even for simple fiber structures.

Structurally complex corn bran arabinoxylan (CAX) was used as a model glycan to investigate gut bacteria growth and competition on different AX-based fine structures. Nine hydrolyzate segments of the CAX polymer varying in chemical structure (sugars and linkages), CAX, five less complex non-corn arabinoxylans, and xylose and glucose were ranked from structurally complex to simple. The substrate panel promoted different overall growth and rates of growth of eight Bacteroides xylan-degrading strains. For example, Bacteroides cellulosilyticus DSM 14838 (Bacteroides cellulosilyticus) grew well on an array of complex and simple structures, while Bacteroides ovatus 3-1-23 grew well only on the simple structures. In a competition experiment, B. cellulosilyticus growth was favored over B. ovatus on the complex AX-based structure. On the other hand, on the simple structure, B. ovatus strongly outcompeted B. cellulosilyticus, which was eliminated from the competitive environment by Day 11. This adaptation to fine structure and resulting competition dynamics indicate that dietary fiber chemical structures, whether complex or simple, favor certain gut bacteria. Overall, this work supports a concept that fiber degraders diversify their competitive abilities to access substrates across the spectrum of heterogeneity of fine structural features of dietary fibers.

Dietary Fibers of Tree Nuts Differ in Composition and Distinctly Impact the Fecal Microbiota and Metabolic Outcomes In Vitro.

This study aimed to evaluate and compare the effects of dietary fibers (DFs) of commercially important tree nuts (almond, cashew, hazelnut, pistachio, and walnut) on gut microbiota in vitro. Microbial compositions and short-chain fatty acids were determined using 16S rRNA sequencing and gas chromatography (GC), respectively. Neutral and acidic monosaccharides were analyzed using GC/MS and spectrophotometry, respectively. Our results revealed that cashew fibers exhibit higher butyrate formation compared to others. Accordingly, cashew fiber promoted butyric acid-producing bacteria-related operational taxonomic units (OTUs; Butyricimonas and Collinsella) at higher relative abundances. The higher butyrogenic capacity of cashew fiber is mainly attributed to its higher soluble/total DF ratio and remarkably distinct monosaccharide composition. Additionally, nut fibers stimulated family Lachnospiraceae- and Ruminococcaceae-related OTUs. These findings show that although the degree of promotion is nut type-dependent, nut fibers are generally capable of promoting beneficial microbes in the colon, further suggesting that DFs of tree nuts are contributing factors to their health-promoting effects.

Sex-dependent colonic microbiota modulation by hazelnut (Corylus avellana L.) dietary fiber.

Although many efforts have been made to characterize the functional properties of hazelnut constituents (mainly its oil, protein, and phenolics), those of its dietary fiber (DF) have not been elucidated yet. Here, we aimed to investigate the impact of DF of natural and roasted hazelnuts, and hazelnut skin on the colonic microbiota in vivo (C57BL/6J mouse models) by determining their composition through 16S rRNA sequencing and microbial short-chain fatty acids (SCFAs) using gas chromatography. Our results revealed that hazelnut DF generally showed an acetogenic effect in male mice, whereas the same trend was not observed in the female counterparts. The 16S rRNA sequencing results showed that hazelnut DF, especially that of natural hazelnuts, increased the relative abundances of Lactobacillus-related OTUs that have probiotic potential. LEfSe analysis indicated that, for female mice, Lachnospiraceae, Prevotella, Ruminococcaceae, and Lactobacillus were found to be discriminators for DF of natural hazelnuts, roasted hazelnuts, hazelnut skin, and control, respectively, whereas Bacteroides, Lactobacillus, Prevotella, and Lactococcus were the discriminators for the male counterparts, respectively. This study clearly indicates that, although the roasting process slightly alters the functionalities, hazelnut DF favors beneficial microbes and stimulates beneficial microbial metabolites in the colon in a sex-dependent way, which could be a contributing factor to the health-promoting effects of hazelnuts. Furthermore, hazelnut skin, a byproduct of the hazelnut industry, was found to have potential to be utilized to produce functional DF targeting colonic health.

Corn arabinoxylan has a repeating structure of subunits of high branch complexity with slow gut microbiota fermentation.

Corn arabinoxylan (CAX), a cell wall-derived dietary fiber, was extracted with alkali, partially purified, and treated with hydrolytic enzymes in order to investigate the relationship of fine structure and fermentability by the human gut microbiota. Glycosyl composition and linkage analysis of CAX and two hydrolysates, coupled with molecular size analysis, indicated an organized structural feature of the native polymer, which consists of a repeating structural subunit containing complex branching patterns along the xylan backbone and flanked by regions of less complexity. The two lengths of the highly branched subunit were isolated and were shown to have enhanced slow fermentation property compared to the native structure (3.3 vs. 5.9 mL gas, 4 h), that was related to increasing complexity of the branched structures. Lower molecular size structures with higher branch complexity fermented slower, contrary to a conventional view that small fiber structures approaching the oligosaccharide level are necessarily more rapidly fermented.

Gut microbiota modulation with long-chain corn bran arabinoxylan in adults with overweight and obesity is linked to an individualized temporal increase in fecal propionate.

BACKGROUND: Variability in the health effects of dietary fiber might arise from inter-individual differences in the gut microbiota's ability to ferment these substrates into beneficial metabolites. Our understanding of what drives this individuality is vastly incomplete and will require an ecological perspective as microbiomes function as complex inter-connected communities. Here, we performed a parallel two-arm, exploratory randomized controlled trial in 31 adults with overweight and class-I obesity to characterize the effects of long-chain, complex arabinoxylan (n = 15) at high supplementation doses (female: 25 g/day; male: 35 g/day) on gut microbiota composition and short-chain fatty acid production as compared to microcrystalline cellulose (n = 16, non-fermentable control), and integrated the findings using an ecological framework. RESULTS: Arabinoxylan resulted in a global shift in fecal bacterial community composition, reduced α-diversity, and the promotion of specific taxa, including operational taxonomic units related to Bifidobacterium longum, Blautia obeum, and Prevotella copri. Arabinoxylan further increased fecal propionate concentrations (p = 0.012, Friedman's test), an effect that showed two distinct groupings of temporal responses in participants. The two groups showed differences in compositional shifts of the microbiota (p ≤ 0.025, PERMANOVA), and multiple linear regression (MLR) analyses revealed that the propionate response was predictable through shifts and, to a lesser degree, baseline composition of the microbiota. Principal components (PCs) derived from community data were better predictors in MLR models as compared to single taxa, indicating that arabinoxylan fermentation is the result of multi-species interactions within microbiomes. CONCLUSION: This study showed that long-chain arabinoxylan modulates both microbiota composition and the output of health-relevant SCFAs, providing information for a more targeted application of this fiber. Variation in propionate production was linked to both compositional shifts and baseline composition, with PCs derived from shifts of the global microbial community showing the strongest associations. These findings constitute a proof-of-concept for the merit of an ecological framework that considers features of the wider gut microbial community for the prediction of metabolic outcomes of dietary fiber fermentation. This provides a basis to personalize the use of dietary fiber in nutritional application and to stratify human populations by relevant gut microbiota features to account for the inconsistent health effects in human intervention studies. TRIAL REGISTRATION: Clinicaltrials.gov, NCT02322112 , registered on July 3, 2015. Video Abstract.

Maize Bran Particle Size Governs the Community Composition and Metabolic Output of Human Gut Microbiota in in vitro Fermentations.

Differences in the chemical and physical properties of dietary fibers are increasingly known to exert effects on their fermentation by gut microbiota. Here, we demonstrate that maize bran particle size fractions show metabolic output and microbial community differences similar to those we previously observed for wheat brans. As for wheat brans, maize bran particles varied in starch and protein content and in sugar composition with respect to size. We fermented maize bran particles varying in size in vitro with human fecal microbiota as inocula, measuring their metabolic fate [i.e., short-chain fatty acids (SCFAs)] and resulting community structure (via 16S rRNA gene amplicon sequencing). Metabolically, acetate, propionate and butyrate productions were size-dependent. 16S rRNA sequencing revealed that the size-dependent SCFA production was linked to divergent microbial community structures, which exerted effects at fine taxonomic resolution (the genus and species level). These results further suggest that the physical properties of bran particles, such as size, are important variables governing microbial community compositional and metabolic responses.

Subtle Variations in Dietary-Fiber Fine Structure Differentially Influence the Composition and Metabolic Function of Gut Microbiota.

The chemical structures of soluble fiber carbohydrates vary from source to source due to numerous possible linkage configurations among monomers. However, it has not been elucidated whether subtle structural variations might impact soluble fiber fermentation by colonic microbiota. In this study, we tested the hypothesis that subtle structural variations in a soluble polysaccharide govern the community structure and metabolic output of fermenting microbiota. We performed in vitro fecal fermentation studies using arabinoxylans (AXs) from different classes of wheat (hard red spring [AXHRS], hard red winter [AXHRW], and spring red winter [AXSRW]) with identical initial microbiota. Carbohydrate analyses revealed that AXSRW was characterized by a significantly shorter backbone and increased branching compared with those of the hard varieties. Amplicon sequencing demonstrated that fermentation of AXSRW resulted in a distinct community structure of significantly higher richness and evenness than those of hard-AX-fermenting cultures. AXSRW favored OTUs within Bacteroides, whereas AXHRW and AXHRS favored Prevotella Accordingly, metabolic output varied between hard and soft varieties; higher propionate production was observed with AXSRW and higher butyrate and acetate with AXHRW and AXHRS This study showed that subtle changes in the structure of a dietary fiber may strongly influence the composition and function of colonic microbiota, further suggesting that physiological functions of dietary fibers are highly structure dependent. Thus, studies focusing on interactions among dietary fiber, gut microbiota, and health outcomes should better characterize the structures of the carbohydrates employed.IMPORTANCE Diet, especially with respect to consumption of dietary fibers, is well recognized as one of the most important factors shaping the colonic microbiota composition. Accordingly, many studies have been conducted to explore dietary fiber types that could predictably manipulate the colonic microbiota for improved health. However, the majority of these studies underappreciate the vastness of fiber structures in terms of their microbial utilization and omit detailed carbohydrate structural analysis. In some cases, this causes conflicting results to arise between studies using (theoretically) the same fibers. In this investigation, by performing in vitro fecal fermentation studies using bran arabinoxylans obtained from different classes of wheat, we showed that even subtle changes in the structure of a dietary fiber result in divergent microbial communities and metabolic outputs. This underscores the need for much higher structural resolution in studies investigating interactions of dietary fibers with gut microbiota, both in vitro and in vivo.

Dietary fibre profiles of Turkish Tombul hazelnut (Corylus avellana L.) and hazelnut skin.

Dietary fibre (DF) profiles of natural hazelnut, roasted hazelnut and hazelnut skin were analyzed. Insoluble (IDF) and soluble (SDF) DFs were examined for monosaccharide and glycosyl-linkage compositions using gas chromatography-mass spectrometry (GC-MS). Total DF contents of natural hazelnut, roasted hazelnut, and hazelnut skin were 17.8, 15.4, and 69.8%, respectively; majority of which (>96%) were water-insoluble. IDFs of natural and roasted hazelnuts were composed of cellulose (~49%), pectic polysaccharides (~30%), and xyloglucans (~15%), whereas that of hazelnut skin made up lignin (~55%) and fibre polysaccharides (cellulose, pectic polysaccharides, and xyloglucans, ~45%). Unlike the ones from other sources, pectic polysaccharides in IDFs had lower proportion of smooth region and higher proportion of hairy region that is heavily branched with arabinan and galactan side chains. Xyloglucans were also densely branched with monomeric and/or dimeric side chains. SDFs of the samples were composed of heavily branched heteromannans (~60%), slightly branched pectic polysaccharides (~25%), and xyloglucans possessing monomeric side chains (~5%). These results suggest that hazelnut is rich in DFs that have potential to improve large bowel function and hazelnut skin, a byproduct of hazelnut roasting process, could be utilized for the production of functional carbohydrates having prebiotic capacities.

Arabinose biosynthesis is critical for salt stress tolerance in Arabidopsis.

The capability to maintain cell wall integrity is critical for plants to adapt to unfavourable conditions. l-Arabinose (Ara) is a constituent of several cell wall polysaccharides and many cell wall-localised glycoproteins, but so far the contribution of Ara metabolism to abiotic stress tolerance is still poorly understood. Here, we report that mutations in the MUR4 (also known as HSR8) gene, which is required for the biosynthesis of UDP-Arap in Arabidopsis, led to reduced root elongation under high concentrations of NaCl, KCl, NaNO3 , or KNO3 . The short root phenotype of the mur4/hsr8 mutants under high salinity is rescued by exogenous Ara or gum arabic, a commercial product of arabinogalactan proteins (AGPs) from Acacia senegal. Mutation of the MUR4 gene led to abnormal cell-cell adhesion under salt stress. MUR4 forms either a homodimer or heterodimers with its isoforms. Analysis of the higher order mutants of MUR4 with its three paralogues, MURL, DUR, MEE25, reveals that the paralogues of MUR4 also contribute to the biosynthesis of UDP-Ara and are critical for root elongation. Taken together, our work revealed the importance of the Ara metabolism in salt stress tolerance and also provides new insights into the enzymes involved in the UDP-Ara biosynthesis in plants.

Among older adults, age-related changes in the stool microbiome differ by HIV-1 serostatus.

BACKGROUND: HIV-1 infection and physiological aging are independently linked to elevated systemic inflammation and changes in enteric microbial communities (dysbiosis). However, knowledge of the direct effect of HIV infection on the aging microbiome and potential links to systemic inflammation is lacking. METHODS: In a cross-sectional study of older people living with HIV (PLWH) (median age 61.5 years, N = 14) and uninfected controls (median 58 years, n = 22) we compared stool microbiota, levels of microbial metabolites (short-chain fatty acid levels, SCFA) and systemic inflammatory biomarkers by HIV serostatus and age. FINDINGS: HIV and age were independently associated with distinct changes in the stool microbiome. For example, abundances of Enterobacter and Paraprevotella were higher and Eggerthella and Roseburia lower among PLWH compared to uninfected controls. Age-related microbiome changes also differed by HIV serostatus. Some bacteria with inflammatory potential (e.g. Escherichia) increased with age among PLWH, but not controls. Stool SCFA levels were similar between the two groups yet patterns of associations between individual microbial taxa and SCFA levels differed. Abundance of various genera including Escherichia and Bifidobacterium positively associated with inflammatory biomarkers (e.g. soluble Tumor Necrosis Factor Receptors) among PLWH, but not among controls. INTERPRETATION: The age effect on the gut microbiome and associations between microbiota and microbial metabolites or systemic inflammation differed based on HIV serostatus, raising important implications for the impact of therapeutic interventions, dependent on HIV serostatus or age.

Shear-thickening behavior of gelatinized waxy starch dispersions promoted by the starch molecular characteristics.

An unusual shear-thickening behavior was observed in 10% w/w gelatinized waxy potato and waxy corn starch dispersions at shear rates around 20 s-1. However, the phenomenon was not found in gelatinized dispersions of waxy rice. The aim of this study was to investigate reasons for the observed shear-thickening behavior as well as its relationship with the amylopectin molecular structure. This unique phenomenon only appears at a shear rate of about 20 s-1 during the increasing shear rate stage of the first cycle of a two-cycle steady shear flow test. After 7 d storage, the shear-thickening behavior of waxy potato starch dispersions disappeared, while it remained in waxy corn starch dispersions. A small strain temperature sweep test applied to waxy potato starch dispersions stored for 7 d showed a significant increase in the elastic behavior of dispersions at temperatures lower than 60 °C. This behavior was not observed on fresh and 7 d stored waxy corn and waxy rice dispersions. The study provides valuable information on a unique rheological behavior of waxy starch dispersions and its relationship to the amylopectin structure, thus opening opportunities for the design of novel foods with desired nutritional and textural properties.

Fecal Microbiota Responses to Bran Particles Are Specific to Cereal Type and In Vitro Digestion Methods That Mimic Upper Gastrointestinal Tract Passage.

Although in vitro studies to identify interactions between food components and the colonic microbiota employ distinct methods to mimic upper gastrointestinal (GI) tract digestion, the effects of differences in protocols on fermentation have not been rigorously addressed. Here, we compared two widely used upper GI tract digestion methods on four different cereal brans in fermentations by fecal microbiota to test the hypotheses that (1) different methods are varyingly efficient in removing accessible starches and proteins from dietary components and (2) these result in cereal-specific differences in fermentation by fecal microbiota. Our results supported both hypotheses, in that the methods differed significantly in bran starch and protein retention and that the effects were cereal-specific. Furthermore, these differences impacted fermentation by the fecal microbiota of healthy donors, altering both short-chain fatty acid production and microbial community composition. These data suggest that digestion methods should be standardized across laboratories for in vitro fiber fermentation studies.

Divergent short-chain fatty acid production and succession of colonic microbiota arise in fermentation of variously-sized wheat bran fractions.

Though the physical structuring of insoluble dietary fiber sources may strongly impact their processing by microbiota in the colon, relatively little mechanistic information exists to explain how these aspects affect microbial fiber fermentation. Here, we hypothesized that wheat bran fractions varying in size would be fermented differently by gut microbiota, which would lead to size-dependent differences in metabolic fate (as short-chain fatty acids; SCFAs) and community structure. To test this hypothesis, we performed an in vitro fermentation assay in which wheat bran particles from a single source were separated by sieving into five size fractions and inoculated with fecal microbiota from three healthy donors. SCFA production, measured by gas chromatography, uncovered size fraction-dependent relationships between total SCFAs produced as well as the molar ratios of acetate, propionate, and butyrate. 16S rRNA sequencing revealed that these size-dependent metabolic outcomes were accompanied by the development of divergent microbial community structures. We further linked these distinct results to subtle, size-dependent differences in chemical composition. These results suggest that physical context can drive differences in microbiota composition and function, that fiber-microbiota interaction studies should consider size as a variable, and that manipulating the size of insoluble fiber-containing particles might be used to control gut microbiome composition and metabolic output.

Dietary Fiber Treatment Corrects the Composition of Gut Microbiota, Promotes SCFA Production, and Suppresses Colon Carcinogenesis.

Epidemiological studies propose a protective role for dietary fiber in colon cancer (CRC). One possible mechanism of fiber is its fermentation property in the gut and ability to change microbiota composition and function. Here, we investigate the role of a dietary fiber mixture in polyposis and elucidate potential mechanisms using TS4Cre×cAPCl°x468 mice. Stool microbiota profiling was performed, while functional prediction was done using PICRUSt. Stool short-chain fatty acid (SCFA) metabolites were measured. Histone acetylation and expression of SCFA butyrate receptor were assessed. We found that SCFA-producing bacteria were lower in the polyposis mice, suggesting a decline in the fermentation product of dietary fibers with polyposis. Next, a high fiber diet was given to polyposis mice, which significantly increased SCFA-producing bacteria as well as SCFA levels. This was associated with an increase in SCFA butyrate receptor and a significant decrease in polyposis. In conclusion, we found polyposis to be associated with dysbiotic microbiota characterized by a decline in SCFA-producing bacteria, which was targetable by high fiber treatment, leading to an increase in SCFA levels and amelioration of polyposis. The prebiotic activity of fiber, promoting beneficial bacteria, could be the key mechanism for the protective effects of fiber on colon carcinogenesis. SCFA-promoting fermentable fibers are a promising dietary intervention to prevent CRC.

Reciprocal Prioritization to Dietary Glycans by Gut Bacteria in a Competitive Environment Promotes Stable Coexistence.

When presented with nutrient mixtures, several human gut Bacteroides species exhibit hierarchical utilization of glycans through a phenomenon that resembles catabolite repression. However, it is unclear how closely these observed physiological changes, often measured by altered transcription of glycan utilization genes, mirror actual glycan depletion. To understand the glycan prioritization strategies of two closely related human gut symbionts, Bacteroides ovatus and Bacteroides thetaiotaomicron, we performed a series of time course assays in which both species were individually grown in a medium with six different glycans that both species can degrade. Disappearance of the substrates and transcription of the corresponding polysaccharide utilization loci (PULs) were measured. Each species utilized some glycans before others, but with different priorities per species, providing insight into species-specific hierarchical preferences. In general, the presence of highly prioritized glycans repressed transcription of genes involved in utilizing lower-priority nutrients. However, transcriptional sensitivity to some glycans varied relative to the residual concentration in the medium, with some PULs that target high-priority substrates remaining highly expressed even after their target glycan had been mostly depleted. Coculturing of these organisms in the same mixture showed that the hierarchical orders generally remained the same, promoting stable coexistence. Polymer length was found to be a contributing factor for glycan utilization, thereby affecting its place in the hierarchy. Our findings not only elucidate how B. ovatus and B. thetaiotaomicron strategically access glycans to maintain coexistence but also support the prioritization of carbohydrate utilization based on carbohydrate structure, advancing our understanding of the relationships between diet and the gut microbiome.IMPORTANCE The microorganisms that reside in the human colon fulfill their energy requirements mainly from diet- and host-derived complex carbohydrates. Members of this ecosystem possess poorly understood strategies to prioritize and compete for these nutrients. Based on direct carbohydrate measurements and corresponding transcriptional analyses, our findings showed that individual bacterial species exhibit different preferences for the same set of glycans and that this prioritization is maintained in a competitive environment, which may promote stable coexistence. Such understanding of gut bacterial glycan utilization will be essential to eliciting predictable changes in the gut microbiota to improve health through the diet.

A perspective on the complexity of dietary fiber structures and their potential effect on the gut microbiota.

Even though there are many factors that determine the human colon microbiota composition, diet is an important one because most microorganisms in the colon obtain energy for their growth by degrading complex dietary compounds, particularly dietary fibers. While fiber carbohydrates that escape digestion in the upper gastrointestinal tract are recognized to have a range of structures, the vastness in number of chemical structures from the perspective of the bacteria is not well appreciated. In this article, we introduce the concept of "discrete structure" that is defined as a unique chemical structure, often within a fiber molecule, which aligns with encoded gene clusters in bacterial genomes. The multitude of discrete structures originates from the array of different fiber types coupled with structural variations within types due to genotype and growing environment, anatomical parts of the grain or plant, discrete regions within polymers, and size of oligosaccharides and small polysaccharides. These thousands of discrete structures conceivably could be used to favor bacteria in the competitive colon environment. A global framework needs to be developed to better understand how dietary fibers can be used to obtain predicted changes in microbiota composition for improved health. This will require a multi-disciplinary effort that includes biological scientists, clinicians, and carbohydrate specialists.