Resistant starches types 2 and 4 have differential effects on the composition of the fecal microbiota in human subjects

Abstract

Background: To systematically develop dietary strategies based on resistant starch (RS) that modulate the human gut microbiome, detailed in vivo studies that evaluate the effects of different forms of RS on the community structure and population dynamics of the gut microbiota are necessary. The aim of the present study was to gain a community wide perspective of the effects of RS types 2 (RS2) and 4 (RS4) on the fecal microbiota in human individuals.

Methods and findings: Ten human subjects consumed crackers for three weeks each containing either RS2, RS4, or native starch in a double-blind, crossover design. Multiplex sequencing of 16S rRNA tags revealed that both types of RS induced several significant compositional alterations in the fecal microbial populations, with differential effects on community structure. RS4 but not RS2 induced phylum-level changes, significantly increasing Actinobacteria and Bacteroidetes while decreasing Firmicutes. At the species level, the changes evoked by RS4 were increases in Bifidobacterium adolescentis and Parabacteroides distasonis, while RS2 significantly raised the proportions of Ruminococcus bromii and Eubacterium rectale when compared to RS4. The population shifts caused by RS4 were numerically substantial for several taxa, leading for example, to a ten-fold increase in bifidobacteria in three of the subjects, enriching them to 18-30% of the fecal microbial community. The responses to RS and their magnitudes varied between individuals, and they were reversible and tightly associated with the consumption of RS.

Conclusion: Our results demonstrate that RS2 and RS4 show functional differences in their effect on human fecal microbiota composition, indicating that the chemical structure of RS determines its accessibility by groups of colonic bacteria. The findings imply that specific bacterial populations could be selectively targeted by well designed functional carbohydrates, but the inter-subject variations in the response to RS indicates that such strategies might benefit from more personalized approaches.

Conflict of interest statement

Competing Interests: The authors confirm that there has been no interference with an objective assessment of the manuscript, and that they adhere to all the PLoS ONE policies regarding the sharing of data and materials.

Figures

Figure 1. Experimental design used in this study.

Subjects (n = 10) participated in a 17-week double-blind crossover design, in which 3 dietary treatments were assessed: 100 g of crackers containing either native starch or 33 g of RS2 or RS4. An initial baseline period was proceeded by 3-week periods of each dietary treatment in succession interspersed by 2-week washout periods, and a final washout period. Weekly fecal samples were collected throughout the entire study.

Figure 2. Characterization of the fecal microbiota in ten human subjects that consumed a random succession of crackers containing RS2, RS4, and native wheat starch (control) by multiplex pyrosequencing of 16S rRNA tags.

Phylogenetic trees that encompass the phyla (A) Firmicutes (with Clostridiales groups XIVa and IV labeled), (B) Actinobacteria and (C) Bacteroidetes are shown. The trees contain representative sequences of all OTUs detected to be impacted by RS in individual subjects together with sequences of related entries in the database (which included both type strains of known species and sequences from molecular studies of human fecal samples). Sequences were aligned in Muscle 3.6 and the trees were built using the neighbor-joining algorithm with 1,000 bootstrap replicates in MEGA 4.0. Open-black and closed-gray symbols were used to label sequences from individual subjects. OTUs that were not significantly affected in all ten subjects were labeled as ‘not significant’ (NS). The graphs next to the trees show the abundance of OTUs and bacterial groups that were significantly altered in the treatment groups (RS2, RS4, control). These graphs show mean proportions of the three individual samples taken during the treatment periods for each subject. Background refers to samples taken in periods were no crackers were consumed. Repeated measures ANOVA in combination with a Tukey's post-hoc test were performed to indentify differences between treatment groups, and the background was not included in the statistic analysis. *p<0.05; **p<0.01; ***p<0.001.

Figure 3. Bubble plots showing differences in the proportions of bacterial taxa (as per cent of the total microbiota composition) detected between the RS4 (A) and RS2 (B) periods when compared to the control period.

The sizes of the bubbles are proportional to the magnitude of the difference. Black circles represent increases in proportions induced through RS treatment, and white circles show a decrease.

Figure 4. Temporal dynamics of the human fecal microbiota in response to the consumption of crackers containing RS2, RS4, and native wheat starch (control) in five human subjects.

Graphs on the left show proportions of the three main phyla and four representative species (Bifidobacterium adolescentis, Parabacteroides distasonis, Ruminococcus bromii and Clostridium clostridioforme) as determined by pyrosequencing of 16S rRNA tags. Gel images on the right show molecular fingerprints generated by DGGE. Bands that represent Bifidobacterium adolescentis and Parabacteroides distasonis are labeled.