Impact of dietary resistant starch type 4 on human gut microbiota and immunometabolic functions

Abstract

Dietary modulation of the gut microbiota impacts human health. Here we investigated the hitherto unknown effects of resistant starch type 4 (RS4) enriched diet on gut microbiota composition and short-chain fatty acid (SCFA) concentrations in parallel with host immunometabolic functions in twenty individuals with signs of metabolic syndrome (MetS). Cholesterols, fasting glucose, glycosylated haemoglobin, and proinflammatory markers in the blood as well as waist circumference and % body fat were lower post intervention in the RS4 group compared with the control group. 16S-rRNA gene sequencing revealed a differential abundance of 71 bacterial operational taxonomic units, including the enrichment of three Bacteroides species and one each of Parabacteroides, Oscillospira, Blautia, Ruminococcus, Eubacterium, and Christensenella species in the RS4 group. Gas chromatography-mass spectrometry revealed higher faecal SCFAs, including butyrate, propionate, valerate, isovalerate, and hexanoate after RS4-intake. Bivariate analyses showed RS4-specific associations of the gut microbiota with the host metabolic functions and SCFA levels. Here we show that dietary RS4 induced changes in the gut microbiota are linked to its biological activity in individuals with signs of MetS. These findings have potential implications for dietary guidelines in metabolic health management.

Figure 1. Study design.

(a) Time line of the placebo-controlled, crossover, dietary intervention with resistant starch (RS4) and control flour (CF). Stool and blood samples were collected before and after each treatment period (indicated by arrows). (b) Trial profile and numbers of participants in the study.

Figure 2. Separation of the microbiome post intervention in RS4 and CF groups.

Two-dimensional principal coordinate analyses (PCoA) based on the weighted UniFrac distance between samples, given the abundance of 5,831 taxa present in at least one sample (n = 19). Axes 1 and 2 explain 26% and 13% of the variation, respectively (p = 0.01).

Figure 3. Differential gut microbial composition after RS4 intervention at the species level.

(a) Relative abundance of bacterial species (log fold change) in the RS4 group compared with the CF group post intervention (n = 19). Significant compositional variation between the two groups before the intervention was previously ruled out. (b) The Firmicutes/Bacteroidetes ratio after intervention (n = 14). The dotted line represents this ratio at baseline. (c) Abundance of major bacterial species (log fold change) before and after RS4 treatment. #, the closest hit from the NCBI 16S rRNA database cross referenced with the OTU from the Greengenes database. *q ≤ 0.05, **q ≤ 0.01, ***q ≤ 0.001, ^§q ≤ 0.09 (trend/approaching significance), n = 19.

Figure 4. Effects of RS4 on faecal SCFAs.

(a) SCFA abundance before and after RS4 intervention (*p ≤ 0.05, n = 19). (b) Positive correlation of six bacterial species with increased SCFA levels in an RS4-specific manner (all, p < 0.05). Pearson coefficients are shown on heat map. #, the closest hit from the NCBI 16S rRNA database cross referenced with the OTU from the Greengenes database. †, species either significantly enriched or approached significance in the RS4 group.

Figure 5. Associations between gut microbiota and host biological parameters after RS4 and CF interventions.

(a) Heat map showing Pearson’s r values (all, p < 0.05). Black rectangular borders indicate an association present only post RS4 intervention. #, the closest hit from the NCBI 16S rRNA database cross referenced with the OTU from the Greengenes database. †, species either significantly enriched or approached significance in the RS4 group, n = 15.

Figure 6. Intra-associations within bacterial species that were correlated with metabolic functions or SCFAs in an RS4-specific manner.

Heat map showing Pearson’s r values, corresponding to the size of the circle (n = 19). The black border indicates clustering of species (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, shown only in the upper triangle). #, the closest hit from the NCBI 16S rRNA database cross-referenced with the OTU from the Greengenes database. †, species either significantly enriched or approached significance in the RS4 group.