Wheat bran particle size strongly influenced the amounts and proportions of SCFAs produced

We tested whether wheat bran particle size would impact the metabolism and community structure of fecal microbiota through in vitro batch fermentations of wheat bran size fractions (separated by sieving from an identical initial source), which had been previously digested enzymatically in vitro to mimic transit through the stomach and small intestine and inoculated with fecal microbiota from three healthy donors. To evaluate metabolic outcomes, we measured the production of the terminal SCFAs most abundantly found in fecal samples (acetate, propionate, and butyrate) at 0, 6, 12, 24, and 48 h time points (post-inoculation with fecal microbiota). The particle size fractions tested were (1) 180–300 μm (the finest bran), (2) 300–500 μm, (3) 500–800 μm, (4) 850–1,000 μm, and (5) >1,700 μm (the coarsest bran). The rapidly-fermentable soluble fiber, fructooligosaccharide (FOS)35, was also included as a positive control for fermentation. As we hypothesized, different wheat bran size fractions not only impacted the rate and absolute amounts of SCFAs produced by fecal microbiota (Fig. 1) but also significantly (p < 0.05) influenced the molar ratios of the SCFAs (Fig. 2). At all time points, fermentation of FOS generated the highest amount of total SCFA, acetate, and propionate (Fig. 1). Among bran fractions, the finest bran produced the highest amount of total SCFA, acetate, and propionate at all the time points, and gradual decreases in the amounts of these microbial byproducts were observed as the particle size increased. The same trend was also observed for butyrate production up to 24 h post-inoculation within the bran fractions. After this time point, fermentation of the finest bran fraction generated butyrate concentrations indistinguishable from FOS, which is widely-regarded as a highly-butyrogenic fiber35. Interestingly, although the coarsest bran generated the lowest amount of butyrate in the first 24 h post-inoculation, by 48 h post-inoculation butyrate concentrations in coarse bran fermentations were also indistinguishable from those of FOS (p < 0.05) (Fig. 1).

Figure 1 Short-chain fatty acid (SCFA) production by fecal microbiota in in vitro fermentations over time. FOS (fructooligosaccharide) was used as a fast-fermenting, butyrate-producing positive control. The blank did not contain any carbon substrate. Total SCFA is the sum of acetate, propionate, and butyrate. Error bars represent the standard error of the mean of three separate replicates. Mean values with the same letter are not significantly different (Tukey’s multiple comparisons test, p < 0.05). Full size image

Figure 2 Relative abundances of acetate, propionate and butyrate (relative to total SCFA) produced by fecal microbiota in in vitro fermentations over time. FOS (fructooligosaccharide) was used as a fast fermenting-butyrate producing comparator. The blank did not contain any substrate. Error bars represent the standard error of the mean of three separate replicates. Mean values with the same letter are not significantly different (Tukey’s multiple comparisons test, p < 0.05). Total SCFA is the sum of acetate, propionate, and butyrate. Full size image

In terms of SCFA molar ratios, at early time points (6 and 12 h post-inoculation) there was a direct relationship between size fraction and the proportion of acetate with respect to the total of all SCFAs (R2 = 0.94, and R2 = 0.79, respectively; Fig. S1), with the coarsest wheat bran producing the highest proportion of acetate and the finest one generating the lowest proportion (Fig. 2). However, this relationship was not maintained over increasing incubation times (Fig. S1). For propionate generation no significant differences were observed at the 6 h time point between treatments, except that fermentation of 300–500 μm wheat bran particles resulted in the highest propionate production. At later time points (24 h post-inoculation and later), propionate proportions of the two smallest size fractions were significantly higher than the other bran treatment groups but not statistically distinguishable from each other (p < 0.05) (Fig. 2). The relationship between particle size fraction and butyrogenesis was temporally complex; we observed an inverse relationship between butyrate molar ratio and wheat bran particle size fraction at early time points (6 and 12 h post-inoculation) with the coarsest wheat bran generating the lowest proportion of butyrate and the finest one the highest (Figs 2 and S1). Surprisingly, this inverse relationship reversed at later time points (48 h), at which a direct relationship between relative abundance of butyrate and wheat bran particle size fraction emerged (R2 = 0.84), with the highest proportion of butyrate produced from the coarsest bran (Figs 2 and S1). The relative abundance of butyrate produced from the coarsest bran at 48 h post-inoculation reached 20%, which was almost double the relative abundance of butyrate generated from FOS (10.24%) (Fig. 2). These data clearly indicated that particle size fraction dramatically influenced the metabolic outcome of wheat bran fermentation by fecal microbiota.

Wheat bran particle size fraction significantly impacted the fecal microbiota community structure

To determine whether the observed alterations in metabolism of wheat bran size fractions were accompanied by shifts in the microbiota, we assessed the effects of wheat bran size fraction on colonic microbiota composition by amplicon sequencing targeting the V4 and V5 region of the bacterial 16S rRNA gene, using genomic DNA extracted at 24 and 48 h post-inoculation. Sequences were clustered into OTUs defined at the 97% identity level, from which α- and β-diversity metrics were calculated. As expected, fermentation of FOS and bran fractions resulted in very different microbial community structures over time (Fig. 3). Within bran-consuming cultures, the microbiota associated with distinct size fractions were significantly different across both time points (AMOVA, p < 0.001), although we observed no significant differences (p < 0.05) in α-diversity metrics (Fig. S2). Although size fraction clusters were not clearly resolved after 24 h post-inoculation, (Fig. 3a), clear separations between microbiota consuming different bran size fractions were evident 48 h post-inoculation. Especially evident were demarcations of microbiota growing on the finest wheat bran treatment and those consuming the coarsest bran (Fig. 3b). Taken together, these data suggest that divergent communities arise as colonic microbiota ferment wheat bran particles of differing size fractions.

Figure 3 Principal component analysis of community structures associated with wheat bran size fractions, as determined by 16S rRNA gene amplicon sequencing. Bray-Curtis dissimilarity of fecal microbiota was based on the relative abundances of OTUs at a 97% identity level after in vitro fermentation for (a) 24 h and (b) 48 h. FOS (fructooligosaccharide) was used as a fast-fermenting, butyrate-producing positive control. The blank did not contain any substrate. Dissimilarity was also calculated using ThetaYC; the result was not substantially different from that visualized by Bray-Curtis dissimilarity. Full size image

The selective effect of bran particle size fraction operates at fine taxonomic resolutions

Though particle size fraction resulted in significant differences in abundances of taxa at the family level and higher (Fig. S3), the selective effects of particle size also occurred at the genus or species levels. In general, overrepresentation of phylum Firmicutes in association with the coarsest brans was driven by increases in members of Lachnospiraceae, whereas increases in the relative abundances of members of Bacteroidaceae drove increased representation of phylum Bacteroidetes associated with the finest brans. However, within genus Bacteroides, distinct OTUs increased in abundance in response to differential bran size fractions. The most obvious change within bran treatments, compared to the inoculum, was a 20-fold increase in the relative abundance of OTU6 Bacteroides (of which 59% of reads within the cluster could be classified as B. intestinalis with a bootstrap value ≥95%) with the coarsest bran treatment. Similarly, we observed 4-, 8-, 20-, and 23-fold increases over the initial microbiota in the relative abundances of OTU8 Coprococcus eutactus, OTU5 Roseburia, OTU12 Lachnospiraceae and OTU15 Lachnospiraceae, respectively, in fermentations of the coarsest bran. In contrast, growth on wheat bran caused dramatic decreases in the relative abundances of Bifidobacterium-related OTUs; the coarsest wheat bran treatment resulted in 5-, and 3-fold decreases in OTU11, and OTU7, respectively, compared to the inoculum (Figs 4 and S4).

Figure 4 Relative abundances (percentage of sequences) based on the top 50 OTUs in each sample. The top 50 OTUs account for more than 90% of the total sequences of all wheat bran treatment groups at all time points (Fig. S7). Error bars represent the standard error of the mean of three separate replicates. Mean values with the same letter are not significantly different (Tukey’s multiple comparisons test, p < 0.05). Full size image

More surprisingly, we observed either direct or inverse relationships between individual OTU abundances and wheat bran size fraction. For example, there was an inverse relationship between OTU3 Bacteroides (of which 67% of reads could be classified within B. dorei) and wheat bran particle size fraction (R2 = 0.775) (Fig. S5), such that increasing particle size led to decreasing relative abundance of this OTU (Figs 4, S4 and S5). Conversely, at the 48 h post-inoculation time point, OTU6 Bacteroides (of which 59% of the reads could be classified within B. intestinalis), OTU8 Coprococcus eutactus, OTU12 Lachnospiraceae and OTU15 Lachnospiraceae displayed a direct relationship with wheat bran size fraction (R2 = 0.95, R2 = 0.89, R2 = 0.76, R2 = 0.76, respectively) such that the relative abundances of these species gradually increased with increasing wheat bran particle size (Figs 4, S4 and S5).

To identify the specific bacterial taxa representative of the extremes in wheat bran size fractions, we compared the microbial compositions of the coarsest and the finest wheat bran treatments at 48 h post-inoculation using the linear discriminant analysis effect size (LEfSe) method (Fig. 5). Members of Lachnospiraceae (specifically, Coprococcus eutactus and Roseburia hominis) were shown to be discriminators for the coarsest bran treatment, whereas members of Bacteroides and its parent taxa were differentiators for the finest bran treatment (Fig. 5a, LDA > 4). However, within genus Bacteroides, OTU6 (attributed as Bacteroides intestinalis) and OTU4 (attributed as Bacteroides stercoris) were differentiators for the coarsest bran particles. These relationships suggest that stable relationships exist between bran size fraction and individual species (especially, members of Bacteroides) within the colonic microbiota.

Figure 5 Linear discriminant analysis of taxa differentiating the finest and coarsest wheat bran size fractions. Initial and blank communities were also included in the analysis to prevent misattribution of taxa to size fractions that were more highly represented in controls. (a) Taxa with LDA scores >4.0 in the finest (180–300 μm) and coarsest (>1700 μm) size fractions at 48 h post-inoculation; linear discriminants of initial and blank conditions not shown. (b) Cladogram depicting taxa that are overrepresented in the finest and coarsest bran fractions compared with abundances in the initial inoculum and substrate-free blank incubations. Full size image

Different wheat bran fractions possess distinct monosaccharide compositions

To identify potential mechanisms driving differences in microbial metabolism of wheat bran size fractions and its consequent impact upon community structure, we next investigated whether, in addition to differences in physical size, the variously-sized wheat bran fractions were chemically distinct. Accordingly, we measured the neutral sugar contents of wheat bran fractions after they had been digested with salivary α-amylase, pepsin, and pancreatin enzymes to mimic upper gastrointestinal (GI) tract transit; this treatment removes the vast majority of accessible starch (Fig. 6). No significant differences in the proportions of rhamnose, mannose, and galactose were observed among the samples (p < 0.05) (Fig. 6a). The finest wheat bran particles contained the highest amount of glucose (47.92%), and the glucose proportion of the brans decreased as the particle size increased (Fig. 6a). In wheat, the two main sources of glucose are starch and cellulose, so the majority of the glucose measured in the neutral sugar analysis likely belonged to one of these two polysaccharides. We then measured the total starch content of the samples to determine whether the observed glucose proportions correlated with particle starch content (Fig. 6b). Indeed, the finest bran particles displayed the greatest amount of starch (11.72%) and, as particle size increased, starch content decreased (with the coarsest bran displaying less than 1% starch). These data strongly suggested that the distinctions in the glucose proportions measured by neutral sugar analysis arose from differences in the starch, rather than the cellulose, contents of the wheat bran fractions. However, these starches were not removed by treatment with upper-GI-simulating amylase treatment; thus, the starches measured are likely to be resistant.

Figure 6 (a) Neutral monosaccharide compositions of the wheat bran samples (%, mole basis). Mean values of each constituents were compared across the samples, and those with the same letter are not significantly different (Tukey’s test, p < 0.05). No letter was included where the mean values are not statistically different (Tukey’s test, p < 0.05). (b) Total starch contents of the samples. Error bars represent the standard error of the mean of three separate replicates. Mean values with the same letter are not significantly different (Tukey’s multiple comparisons test, p < 0.05). (c) Arabinose:xylose ratio of the samples. Error bars represent the standard error of the mean of three separate replicates. No statistical differences were observed between the mean values (Tukey’s multiple comparisons test, p < 0.05). Full size image

We also found significant differences in the proportions of xylose and arabinose among the size fractions (p < 0.05). In contrast to glucose, we observed direct relationships between wheat bran size fraction and arabinose (R2 = 0.83) and xylose contents (R2 = 0.77), with the finest bran fraction displaying the lowest proportions of xylose (22.66%) and arabinose (24.06%) (Figs 6a and S6). The relative proportions of these sugars increased with bran size fraction, with the coarsest bran possessing the highest proportions (37.39%, and 38.68% xylose and arabinose, respectively) (Figs 6a and S6). Xylose and arabinose are the building blocks of arabinoxylan polymers, with the former composing the backbone of the molecule and the latter forming the branching points36. Therefore, the arabinose-to-xylose ratio is useful in estimating the branch density of the molecule. The arabinose to xylose ratio among bran size fractions were not significantly different (p < 0.05) (Fig. 6c), suggesting similar arabinoxylan structure among size fractions.