Variable responses of human microbiomes to dietary supplementation with resistant starch
© The Author(s). 2016
Received: 16 February 2016
Accepted: 10 June 2016
Published: 29 June 2016
The fermentation of dietary fiber to various organic acids is a beneficial function provided by the microbiota in the human large intestine. In particular, butyric acid contributes to host health by facilitating maintenance of epithelial integrity, regulating inflammation, and influencing gene expression in colonocytes. We sought to increase the concentration of butyrate in 20 healthy young adults through dietary supplementation with resistant starch (unmodified potato starch—resistant starch (RS) type 2).
Fecal samples were collected from individuals to characterize butyrate concentration via liquid chromatography and composition of the microbiota via surveys of 16S rRNA-encoding gene sequences from the Illumina MiSeq platform. Random Forest and LEfSe analyses were used to associate responses in butyrate production to features of the microbiota.
RS supplementation increased fecal butyrate concentrations in this cohort from 8 to 12 mmol/kg wet feces, but responses varied widely between individuals. Individuals could be categorized into three groups based upon butyrate concentrations before and during RS: enhanced, high, and low (n = 11, 3, and 6, respectively). Fecal butyrate increased by 67 % in the enhanced group (from 9 to 15 mmol/kg), while it remained ≥11 mmol/kg in the high group and ≤8 mmol/kg in the low group. Microbiota analyses revealed that the relative abundance of RS-degrading organisms—Bifidobacterium adolescentis or Ruminococcus bromii—increased from ~2 to 9 % in the enhanced and high groups, but remained at ~1.5 % in the low group. The lack of increase in RS-degrading bacteria in the low group may explain why there was no increase in fecal butyrate in response to RS. The microbiota of individuals in the high group were characterized by an elevated abundance of the butyrogenic microbe Eubacterium rectale (~6 % in high vs. 3 % in enhanced and low groups) throughout the study.
We document the heterogeneous responses in butyrate concentrations upon RS supplementation and identify characteristic of the microbiota that appear to underlie this variation. This study complements and extends other studies that call for personalized approaches to manage beneficial functions provided by gut microbiomes.
The microbiota in the large intestine provides several functions that are beneficial to human health such as producing short-chain fatty acids, modifying primary to secondary bile acids, and providing colonization resistance to some enteric pathogens [1, 2]. Managing this community of microbes to maintain and improve these beneficial functions could promote health and reduce the incidence of preventable diseases including obesity and type 2 diabetes , colon cancer , chronic and acute undernutrition , and infections by Clostridium difficile .
One of the beneficial functions derived from the colonic microbiota is the production of butyric acid that is generated from the fermentation of dietary fiber. The conjugate base of the acid—butyrate—is the preferred energy source for colonocytes . Butyrate improves the intestinal barrier by facilitating tight-junction assembly , suppresses inflammatory and allergic responses by inducing differentiation of colonic regulatory T cells , regulates cell apoptosis , and stimulates production of anorectic hormones . Indeed, reduced concentrations of butyrate have been associated with the incidence of graft-versus-host disease [12, 13], kwashiorkor , colon cancer , and obesity . Especially in these circumstances, increased butyrate production in the large intestine may be beneficial to human health.
One approach to increasing butyrate production is to deliver more fermentable carbohydrates to gut microbiomes. Here, we report on the impact of dietary supplementation with resistant starch (unmodified potato starch; RS type 2) on fecal butyrate concentrations and composition of the gut microbiota in 20 healthy young adults. RS consists of starch that is resistant to hydrolysis by human enzymes and passes through the small intestine unabsorbed. In the large intestine, RS can be metabolized and then fermented by microbes to a variety of products, including butyric acid . In this study, we find that the response to RS supplementation varies between individuals in ways that can be explained, at least in part, by variation in the composition of their microbiota. Recognizing inter-individual variability in responses to fiber supplementation and determining the microbiota characteristics that underlie it are essential first steps towards personalized plans for managing gut microbiomes for desirable functionality, including the production of butyrate.
Prospective volunteers were students in research-based sections of Introductory Biology 173 at the University of Michigan. Individuals with a self-reported history of bowel disorders such as irritable bowel syndrome, inflammatory bowel disease, or colorectal cancer were excluded from the study. Twenty individuals (10 males, 10 females) participated in the 3-week study. The age range was 19–20 years, and the BMI range was from 19–63. Each participant gave his or her written, informed consent before participating in the study. This research was carried out in compliance with the Helsinki Declaration and was approved by the Institutional Review Board of the University of Michigan Medical School (HUM00094242).
Participants were provided waxed tissue paper (Epitope Diagnostics, San Diego, CA), which was laid down on the water in the toilet bowl prior to defecation. This paper sticks to the sides of the toilet bowl so that the fecal sample is not readily contaminated by water in the toilet. Fecal samples were collected using the deoxyribose nucleic acid (DNA) Genotek Omnigene Gut Collection kits (DNA Genotek, Ontario, Canada) following the manufacturer’s instructions. The DNA Genotek tube contains 2 mL of a stabilization buffer and a steel ball to facilitate mixing of the fecal material with the buffer. Approximately, 0.67 g of fecal material was collected per tube. The kits were returned to a −20 °C freezer within 24 h of collection. Frozen samples were thawed and aliquots were withdrawn for DNA extraction, measurement of fermentation products, and to create sample archives.
DNA extraction, PCR, sequencing, and sequence processing
For DNA extraction, 0.25 mL of the fecal suspension in the DNA Genotek tube was deposited into a MoBio PowerMag Soil DNA Isolation Bead Plate. DNA was extracted following MoBio’s instructions on an epMotion 5075 liquid handling workstation (Eppendorf, Hauppauge, NY). The V4 region of the 16S-ribosomal RNA (rRNA) encoding genes was amplified from each sample using the dual-indexing sequencing strategy . Sequencing was performed on the Illumina MiSeq platform, using a MiSeq Reagent Kit V2 500 cycles (Cat# MS-102-2003), according to the manufacturer’s instructions. Sequences were curated using mothur v1.31.2  and clustered into operational taxonomic units at ≥ 97 % sequence identity using the average neighbor algorithm.
Measurement of fermentation products
One millimeter of fecal suspension was aliquoted from each DNA Genotek OmniGut tube and centrifuged at 4 °C for 10 min (4500×g). The supernatant fraction was withdrawn and passed sequentially through 1.2, 0.65, and 0.22-μm hydrophilic low protein-binding Durapore membrane filters (EMD Millipore, Darmstadt, Germany). Filtered samples were stored at −20 °C until analysis. Once thawed, samples were maintained at 4 °C in an autosampler. A Shimadzu HPLC (Shimadzu Scientific Instruments, Columbia, MD) equipped with a dual UV (214 nm for short-chain fatty acids (SCFAs))/refractive index detector (for ethanol) and an Aminex HPX-87H column (Bio-Rad Laboratories, Hercules, CA) heated to 50 °C was used to measure acetic, propionic, and butyric acids and ethanol in the filtrate. The mobile phase was 0.01 N H2SO4 at a flow rate of 0.6 ml/min, and the injection volume was 10 μl. Samples were randomized in regard to injection order, and the average value from two technical replicates was used in all subsequent analyses. Ethanol, which was a component of the stabilization buffer, served to monitor injection volumes. Samples in which the peak height for ethanol was less than 70 % of the average peak height for ethanol in all samples were excluded from further analysis. Eight external standards (0.1–20 mM) for acetic, propionic, and butyric acids were used to generate the standard curve. These standards were run after every 100 samples. LC Solutions Software (Shimadzu Scientific Instruments, Columbia, MD) was used to curate the data and calculate concentrations based on the standard curve generated during the run. The concentrations were then normalized by the average weight of the fecal samples (0.67 g) collected in the DNA Genotek tubes.
All statistical analyses were performed using RStudio version 0.99.489  and the software PAST . To evaluate the impact of RS supplementation on acetate, propionate, and butyrate concentrations in the study population, a nested repeated measures analysis of variance (ANOVA) was used. This revealed that fecal butyrate and acetate concentrations increased with RS intake in the study population (p < 0.05). For all subsequent analyses, the median value of butyrate for each person before and during RS consumption was used so that the number of samples compared was not artificially inflated. Paired or unpaired t tests were employed as appropriate. To determine if RS intake altered microbial community composition, a permutational analysis of variance (PERMANOVA) analysis was conducted with the Bray-Curtis similarity index. Each individual was used as the blocking factor to account for repeated measures of microbiota composition from each individual. This analysis revealed that RS intake altered the composition of gut microbiota in the study population (p < 0.05). For further analyses, we used the median abundance of each operational taxonomic unit (OTU) in each individual before and during RS consumption. To identify specific OTUs that had changed with RS consumption, LEfSe and Random Forest analyses were performed for each individual separately (comparing their four samples before and four samples during RS consumption). LEfSe  was implemented within mothur using the correction for multiple comparisons. PERMANOVA, analysis of similarity (ANOSIM), and permutational analyses of dispersions (PERMDISP) analyses were performed with the package vegan in R. Random Forest was implemented using the package randomForest in R.
The impact of RS on the composition of the gut microbiota and the concentration of three short-chain fatty acids (acetic, propionic, and butyric) in feces was determined in 20 healthy young adults. RS was gradually introduced into their regular diets during an acclimatization period of 3 days (Fig. 1). Thereafter, study participants included 48 g of unmodified potato starch in their daily diet (~24 g as RS) for 7 days. Four fecal samples were collected before the introduction of RS and another four during the period of maximum RS supplementation (Fig. 1).
Effect of dietary supplementation with RS on concentration of select fermentation products (mmol/kg wet feces)
p value (repeated measures ANOVA)
Median ± IQR
Median ± IQR
8 ± 6
12 ± 7
27 ± 6
34 ± 10
13 ± 6
12 ± 5
Like the fecal SCFA concentrations, there was also some intra-individual variability in the composition of microbiota. However, at least 75 % of OTUs were consistently detected in all samples from an individual with the coefficient of variation in their relative abundances ranging from 39 to 65 %. Unlike the SCFAs, where statistical tests did not detect differences in response to RS for an individual, ANOSIM tests showed that RS supplementation altered the composition of microbiota in most individuals in the study (ANOSIM with Bray-Curtis similarity; four samples before vs. four during RS for each individual; p < 0.10 for 16 out of 20 individuals). This conclusion was corroborated by results from a PERMANOVA analysis to determine if RS supplementation altered the composition of microbiota in the overall study population. In order to incorporate intra-individual variability, each individual was considered as the blocking factor in the PERMANOVA. This analysis revealed that the composition of the gut microbiota was altered with RS supplementation in our study population (PERMANOVA using Bray-Curtis similarity blocking for each individual; p = 0.001). A PERMDISP analysis further revealed that the PERMANOVA was not affected by differences in the dispersion of communities before and during RS (PERMDISP p = 0.44).
Effect of dietary supplementation with RS on relative abundance of four dominant bacterial phyla
During RS (%)
Median ± IQR
Median ± IQR
paired t test
1.3 ± 0.6
6.2 ± 0.6
37.1 ± 10.7
33.2 ± 5.2
53.3 ± 13.2
51.5 ± 9
4.7 ± 4.1
4.8 ± 4.1
To identify characteristics of the microbiota that may underlie the variable responses to RS, we first used Random Forest regression to identify relationships between the abundances of OTUs and butyrate concentrations before and during consumption of RS. No OTUs were particularly strong predictors of butyrate concentrations either before or during RS consumption. Butyrate concentrations before RS were weakly related to baseline abundances of OTU 4 (Eubacterium rectale) (R 2 = 0.14; p = 0.10). Unexpectedly, this relationship was not detectable during RS supplementation.
Results of Random Forest regression and LEfSe to identify OTUs that distinguish the three response groups
Distinguishing microbiota features
Low vs. enhanced
[OTU 7] during RS
High vs. low
[OTU 4] before and during RS
[OTU 4] before and during RS
[OTU 3] before RS ‡
High vs. enhanced
[OTU 4] before and during RS
[OTU 4] before and during RS
Cultivars of B. adolescentis are capable of breaking down RS . OTU #7 in which sequences were identical to the 16S rRNA encoding gene of B. adolescentis was detected in 14 out of 20 total individuals and increased in abundance in 12 individuals. This OTU did not increase in individuals U026 and U024 (LEfSe p > 0.05 correcting for multiple comparisons). However, OTU #7 [B. adolescentis] was not detected in six individuals. In these individuals, LEfSe was used to find other OTUs that increased in abundance during RS supplementation, since these could be RS-degrading bacteria. In three individuals in whom B. adolescentis sequences did not increase, OTU 19 increased in abundance (LEfSe p < 0.05 correcting for multiple comparisons). Sequences in that OTU are identical to Ruminococcus bromii, another group of RS-degrading bacteria . In individual U005, neither B. adolescentis nor R. bromii increased in abundance with RS. A potential candidate for a RS-degrading organism in this individual is OTU 50, whose average abundance increased from 2.6 to 7.1 % albeit not statistically significant (p = 0.29). Sequences in this OTU are most closely related to Ruminiclostridium [Eubacterium] siraeum. This organism has not been reported to degrade RS, but it is in the same taxonomic family as R. bromii.
Since B. adolescentis and R. bromii are the strongest candidates for RS-degrading organisms in this study, we summed the abundance of these two organisms for each individual (Fig. 4b). Individuals in all three groups start with a similar abundance of these RS-degrading microbes. RS elicits dramatic increases in their abundance in high and enhanced groups, but not in the low group (Fig. 4b).
The impact of dietary supplementation with resistant starch on fecal butyrate concentrations and the composition of the microbiota was examined in 20 healthy young adults. In this cohort, the average ratios of acetate:propionate:butyrate before and during RS supplementation were 58:25:15 and 58:22:20, respectively. These ratios agree well with previously documented ratios of ~60:20:20 in human feces [26–29]. In response to RS supplementation, the concentration of fecal butyrate increased from 8 to 12 mmol/kg wet feces in the overall study population (repeated measures ANOVA p = 0.02). The inter-individual variation in butyrate concentrations before and during RS was striking (Fig. 2). Most previous studies with RS have documented only population-wide response in butyrate concentrations , while one other study reported considerable heterogeneity between individuals in regard to fecal butyrate concentrations . This pronounced inter-individual variability suggests that a single approach to improving beneficial functions from the microbiome is unlikely to be universally successful. Rather, personalized approaches may be needed to manage microbiomes for health. In line with this endeavor, we attempted to identify features in the microbiota that could explain different responses to dietary supplementation with RS.
Random Forest was used to identify relationships between butyrate concentrations and OTUs in the study population. Weak relationships were identified between butyrate concentrations before RS and the abundances of OTU 4 (E. rectale). Random Forest failed to reveal any relationships between OTU abundances and butyrate concentrations during RS supplementation. It is possible that the number of individuals in this study (n = 20) may have been too small to detect robust correlations between butyrate and the abundance of OTUs at the population level. Alternatively, it is not necessary that the abundance of a single organism be correlated with butyrate concentrations. After all, the potential for producing butyrate is fairly widespread within the phylum Firmicutes . Rather, we expect that the abundance of genes encoding butyryl-CoA:acetate CoA transferase (but) and butyrate kinase (buk) would be correlated to butyrate concentrations.
Our next step was to create groupings in the data based upon similar responses to RS. This clustering approach should constrain the variability within each group and increase the probability of identifying microbiota features that characterize each type of response. K-means clustering using butyrate concentrations before and during RS identified three groups: enhanced, high, and low. Butyrate increased on average from 9 to 15 mmol/kg wet feces in the enhanced group, whereas butyrate concentrations remained consistently high or low in the other two groups (≥11 and ≤8 mmol/kg wet feces, respectively; Fig. 3). With these clusters, we were able to identify features of the microbial communities that differentiated the three groups.
The relative abundance of proposed RS-degrading bacteria—B. adolescentis or R. bromii—increased with RS supplementation in the enhanced and high groups (from 2 to 9 %), but not in the low group (~1.5 % throughout; Fig. 4). This finding complements and extends previously published reports on the effects of RS supplementation on the composition of the human gut microbiota. In a study with overweight adult males, only individuals with detectable abundances of R. bromii in their gut microbiota were able to degrade RS (type 3—Novelose 330; Walker et al. ). In another study with ten human subjects (five males and females; 28–38 years old), dietary supplementation with either type 4 RS (FiberSym RW) or type 2 RS (HiMaize260) increased the relative abundance of B. adolescentis and R. bromii, respectively (Martinez et al. ). These in vivo studies are nicely complemented by in vitro microcosm studies, which showed that isolates of R. bromii and B. adolescentis are capable of degrading several forms of RS . Thus, different types of resistant starches are likely to promote the growth of B. adolescentis or R. bromii, with both organisms exhibiting substrate specificity depending upon the type and source of RS.
The relative abundance of RS-degrading organisms and butyrate concentrations did not increase in the low group. This suggests that their microbiota did not break down RS and so could not lead to increased butyrate production. This suggestion is supported by the fact that the concentrations of neither acetate nor propionate increased in the low group. So why does the abundance of RS-degrading organisms remain at 1.5 % in the low group? Possible explanations include limitation by antagonistic microbes or lack of synergistic microbes. Our attempts to identify antagonistic and synergistic interactions by constructing correlation networks  between RS-degrading organisms and other OTUs did not yield any compelling relationships.
In addition to RS-degrading organisms, another OTU (OTU #4) was identified as distinguishing the high group from the enhanced and low groups (Table 3, Fig. 5). Its V4 sequence is identical to that of E. rectale, well established as a prominent butyrogenic bacterium in the human gut microbiome [24, 35]. Its relative abundance in the high group was consistently about 6 %, compared to 3 % in the other two groups. Surprisingly, the abundance of E. rectale did not change in any of the groups in response to RS—even though butyrate concentrations increased appreciably in the enhanced group (from 9 to 15 mmol/kg wet feces). E. rectale generates butyrate from acetate, and there is a net gain of ATP in that process . So if E. rectale was responsible for increased butyrate production in the enhanced group with RS, one might expect an increase in its relative abundance. We offer two possible explanations for the increased butyrate production in the enhanced group without a measurable increase in the abundance of E. rectale. First, changes in the relative abundance of E. rectale may be subtle and masked by the dramatic increase in the abundance of RS-degrading organisms (~from 2 to 10 % in the enhanced group). It is also possible that populations of E. rectale take longer to respond to RS supplementation. We used an acclimatization period of only 3 days during which the amounts of RS in diet were gradually increased, and after this period, four fecal samples were collected. It remains to be seen whether there is a detectable increase in the abundance of E. rectale following a longer duration of RS consumption. Also, as mentioned earlier, it is well documented that butyrate production is widespread within the phylum Firmicutes in human gut microbiomes. Therefore, rather than a single organism, if more acetate is being converted to butyrate, the genes encoding butyryl-CoA:acetate CoA transferase (but) and butyrate kinase (buk) should increase in abundance with RS supplementation.
Our data show that dietary supplementation with RS type 2 as unmodified potato starch increases fecal butyrate concentration, but with remarkable inter-individual variation. We were able to infer potential explanations for some of these differential effects by investigating the composition of microbiota. Fecal butyrate concentrations increased by an average of 67 % in a subset of the study population (n = 11 of 20; 9 to 15 mmol/kg wet feces). Most individuals in this group showed a dramatic increase in the relative abundance of the RS-degrading organisms—B. adolescentis or R. bromii. In five of these individuals, the prominent butyrogenic microbe E. rectale also increased in abundance. Another subset of the population (3 of 20) consistently maintained high butyrate concentrations both before and with RS (≥12 mmol/kg wet feces). In this subset, RS-degrading organisms increased in abundance, suggesting that RS is being degraded. But there was no concomitant increase in fecal butyrate concentrations. These individuals may be experiencing a plateau effect  in butyrate production even before RS is administered. In fact, the microbiota structures of these individuals also did not change as much with RS (ANOSIM R = 0.17) as compared to changes in the microbiota in the enhanced group (ANOSIM R = 0.64). It is tempting to suggest that the microbiota of these individuals are performing well with regard to butyrate production, and they do not benefit from additional dietary input of fermentable carbohydrates. A third subset of our study population (6 of 20) had consistently low concentrations of butyrate even when consuming RS (≤8 mmol/kg wet feces). In this group, RS-degrading organisms did not increase in abundance, suggesting that their microbiota did not break down RS. Based upon this result, we propose that increasing butyrate in these individuals will require either (i) testing another form of dietary fiber such as inulin or arabinoxylan that their microbiota might degrade, (ii) a synbiotic approach that combines a dietary fiber with the appropriate fiber-degrading bacteria, or (iii) targeted removal of microbes if any are antagonistic to RS-degrading organisms. The findings of this study illustrate the importance of studying individual responses to dietary modifications. This will uncover the mechanisms that underlie these responses and in time provide actionable insights towards precision management of microbiomes.
ANOVA, analysis of variance; DNA, deoxyribose nucleic acid; g, gram(s); h, hour(s); HPLC, high-pressure liquid chromatography; LC, liquid chromatography; ml, milliliter(s); mM, millimolar; °C, degrees centigrade; OTU, operational taxonomic unit; rRNA, ribosomal RNA; RS, resistant starch; μl, microliters
The authors thank Drs. Frank Gerberick and JoseCarlos Garcia-Garcia (Procter & Gamble Company, Cincinnati, OH) for providing feedback on data analysis and interpretation.
This research was supported by grants from the Procter and Gamble Company and the Howard Hughes Medical Institute (52008119).
Availability of data and materials
The sequence and metadata have been deposited in the GenBank database [accession number: SRP067761].
AV, JRS, AS, CW, KRT, and TMS conceived of the study, participated in its design and coordination, and helped draft the manuscript. AV, AS, and CW conducted the microbiota analyses. JRS conducted the analyses of fermentation products. KRT participated and advised in statistical analyses. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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