Antioxidant Vitamins and Prebiotic FOS and XOS Differentially Shift Microbiota Composition and Function and Improve Intestinal Epithelial Barrier In Vitro

Abstract

Human gut microbiota (HGM) play a significant role in health and disease. Dietary components, including fiber, fat, proteins and micronutrients, can modulate HGM. Much research has been performed on conventional prebiotics such as fructooligosaccharides (FOS) and galactooligosaccharides (GOS), however, novel prebiotics or micronutrients still require further validation. We assessed the effect of FOS, xylooligosaccharides (XOS) and a mixture of an antioxidant vitamin blend (AOB) on gut microbiota composition and activity, and intestinal barrier in vitro. We used batch fermentations and tested the short-term effect of different products on microbial activity in six donors. Next, fecal inocula from two donors were used to inoculate the simulator of the human microbial ecosystem (SHIME) and after long-term exposure of FOS, XOS and AOB, microbial activity (short- and branched-chain fatty acids and lactate) and HGM composition were evaluated. Finally, in vitro assessment of intestinal barrier was performed in a Transwell setup of differentiated Caco-2 and HT29-MTX-E12 cells exposed to fermentation supernatants. Despite some donor-dependent differences, all three tested products showed beneficial modulatory effects on microbial activity represented by an increase in lactate and SCFA levels (acetate, butyrate and to a lesser extent also propionate), while decreasing proteolytic markers. Bifidogenic effect of XOS was consistent, while AOB supplementation appears to exert a specific impact on reducing F. nucleatum and increasing butyrate-producing B. wexlerae. Functional and compositional microbial changes were translated to an in vitro host response by increases of the intestinal barrier integrity by all the products and a decrease of the redox potential by AOB supplementation.

Keywords: SCFA; fructooligosaccharides; intestinal barrier; microbiota; vitamins; xylooligosaccharides.

Figure 1

Schematic representation of the experimental set up: (A) Short-term incubations (part 1) to assess interindividual variability, using fecal samples from six donors D1–D6 (48 h); (B) Long-term SHIME^® study (part 2) using the selected fecal samples (D2 and D4) from the screening of two donors (4 weeks). AOB = antioxidant blend; XOS = xylooligosaccharides; FOS = fructo-oligosaccharides; SFCA = short-chain fatty acid; ST/SI = stomach/small intestine; PC = proximal colon; DC = distal colon; SHIME^® = simulator of the human intestinal microbial ecosystem.

Figure 2

Changes in (A) total SCFA, (B) acetate, (C) propionate, (D) butyrate, (E) BCFA, and (F) PCA of metabolic markers after short-term fecal batch incubations (48 h) for six donors, upon treatment with AOB, XOS and FOS versus a blank control. In the PCA, ellipses indicate a confidence interval of 95%. The selected donors are marked with thick, black lines. CTRL= control; AOB = antioxidant blend; XOS = xylooligosaccharides; FOS = fructooligosaccharides; D = donor; BCFA = branched-chain fatty acids; SCFA = short-chain fatty acids. Significant differences are indicated with different letters (a, b, c, d; p < 0.05).

Figure 3

Principle component analysis plots for (left) PC and (right) DC long-term SHIME samples, representing metabolic data (acetate, propionate, butyrate, BCFA, total SCFA and lactate) obtained on four different time points (days 3/4/10/11 of treatment) for blank control, AOB, XOS and FOS for two donors (D2 and D4). Ellipses indicate a confidence interval of 95%. PC = proximal colon; DC = distal colon; CTRL = control; AOB = antioxidant blend; XOS = xylooligosaccharides; FOS = fructooligosaccharides; D = donor; BCFA = branched-chain fatty acids; SCFA = short-chain fatty acids.

Figure 4

Changes in (A) acetate, (B) propionate, (C) butyrate, (D) BCFA and (E) total SCFA, and (F) lactate for donor 2, upon treatment with AOB, XOS and FOS versus a blank control for PC and DC. Bars represent the average and standard deviation of four treatment days (D3/4/10/11) (n = 4). CTRL = control; AOB = antioxidant blend; XOS = xylooligosaccharides; FOS = fructooligosaccharides; PC = proximal colon; DC = distal colon; BCFA = branched-chain fatty acids; SCFA = short-chain fatty acids. Significant differences are indicated with different letters (a, b, c, A, B, C, D; p < 0.05).

Figure 5

Principle component analysis plots for (left) PC and (right) DC long-term SHIME samples of donor 2, representing microbial community composition (significantly affected OTUs) obtained on four different time points (days 3/4/10/11 of treatment) for blank control, AOB, XOS and FOS. Ellipses indicate a confidence interval of 95%. PC = proximal colon; DC = distal colon; AOB = antioxidant blend; XOS = xylooligosaccharides; FOS = fructooligosaccharides.

Figure 6

Effect of AOB, XOS and FOS on microbial community composition of donor 2 at phylum level during 2 weeks of treatment versus the control for PC (A) and DC (B). Average of 4 time points (day 3/4/10/11) as absolute abundance (16 S gene copies/mL)of the different phyla for the two colon regions (PC and DC). PC = proximal colon; DC = distal colon; CTRL = control; AOB = antioxidant blend; XOS = xylooligosaccharides; FOS = fructooligosaccharides.

Figure 7

Changes in TEER (transepithelial electric resistance) for donor 2, upon treatment with AOB, XOS and FOS versus a blank control for PC and DC. Data are averages of six technical replicates with standard deviation STDEV (n = 6). CTRL = control; AOB = antioxidant blend; XOS = xylooligosaccharides; FOS = fructooligosaccharides; PC = proximal colon; DC = distal colon. Significant differences (p < 0.05) are indicated with small letters (a, b, c) for the proximal colon and capital letters (A, B, C) for the distal colon. Bars with different letters are significantly different.

Figure 8

Changes in redox potential for donor 2 and 4, upon treatment with AOB, XOS and FOS versus a blank control for PC and DC. Data are averages of two technical replicates for both donor 2 and 4 with standard deviation STDEV (n = 4). CTRL = control; AOB = antioxidant blend; XOS = xylooligosaccharides; FOS = fructooligosaccharides; PC = proximal colon; DC = distal colon. Significant differences (p < 0.05) are indicated with small letters (a, b, c) for the proximal colon and capital letters (A, B) for the distal colon. Bars with different letters are significantly different.