High purity galacto-oligosaccharides enhance specific Bifidobacterium species and their metabolic activity in the mouse gut microbiome

Abstract

Prebiotics are selectively fermented ingredients that result in specific changes in the composition and/or activity of the gastrointestinal microbiota, thus conferring benefit(s) upon the host health. The aim of this study was to evaluate the influence of a β(1-4)galacto-oligosaccharides (GOS) formulation consisting of 90% pure GOS (GOS90), on the composition and activity of the mouse gut microbiota. Germ-free mice were colonised with microbiota from four pathogen-free wt 129 mice donors (SPF), and stools were collected during a feeding trial in which GOS90 was delivered orally for 14 days. Pyrosequencing of 16S rDNA amplicons showed that Bifidobacterium and specific Lactobacillus, Bacteroides and Clostridiales were more prevalent in GOS90-fed mice after 14 days, although the prebiotic impact on Bifidobacterium varied among individual mice. Prebiotic feeding also resulted in decreased abundance of Bacteroidales, Helicobacter and Clostridium. High-throughput quantitative PCR showed an increased abundance of Bifidobacterium adolescentis, Bifidobacterium pseudocatenulatum, Bifidobacterium lactis and Bifidobacterium gallicum in the prebiotic-fed mice. Control female mice showed a higher diversity (phylogenetic diversity (PD) = 15.1 ± 3.4 in stools and PD = 13.0 ± 0.6 in intestinal contents) than control males (PD = 7.8 ± 1.6 in stool samples and PD = 9.5 ± 1.0 in intestinal contents). GOS90 did not modify inflammatory biomarkers (interleukin (IL)-6, IL-12, IL-1β, interferon gamma and tumour necrosis factor alpha). Decreased butyrate, acetate and lactate concentrations in stools of prebiotic fed mice suggested an increase in colonic absorption and reduced excretion. Overall, our results demonstrate that GOS90 is capable of modulating the intestinal microbiome resulting in expansion of the probiome (autochtonous commensal intestinal bacteria considered to have a beneficial influence on health).

Keywords: Bifidobacterium; mouse gut microbiome; prebiotics.

Figure 1. Study experimental design

Thirteen germ-free mice (GF) were colonized by gavage from a pool of specific pathogen-free (SPF) donors. After 9 days, GOS90 was delivered orally to six mice for 14 days. Fecal samples were collected at three time points (1, 7 and 14 days) and intestinal contents were collected at day 14. Day in negative numbers indicate days before starting the feeding trial.

Figure 2. Microbiome composition of stools and intestinal contents

Phylum and order level distributions of fecal (a,b) and intestinal contents (c,d) microbiota by feeding group (control and GOS90) at day 7 and 14 of the study. The control group included all stool samples from mice not fed the prebiotic. Universal 8F-338R primers were used for stool samples and a combination of the universal and Bifidobacterium-modified primer set were used for the intestinal contents.

Figure 3. Unweighted Principle Coordinate Analysis (PCoA) plot of fecal microbiome from control (n=15) and prebiotic-fed female mice (n=12)

Each dot represents one fecal sample. All time points were included in the analysis. ANOSIM R and P values are indicated in the Figure.

Figure 4. Mean relative abundance of underrepresented genera at days 7 (n=6) and 14 (n=6) of GOS90 feeding compared to control samples (n=15)

Error bars indicate standard deviations (Steel-Dwass All Pairs, * p ≤ 0.05, § p ≤ 0.1).

Figure 5. Mean relative abundances of a) phylum Actinobacteria, b) family Bifidobacteria and c) genus Bifidobacterium by feeding group at days 7 (n= 6) and 14 (n=6)

Data was stratified in responders (n=4) showing increased abundance of Bifidobacterium in response to prebiotic feeding, and non-responders (n=2), showing no change in response to prebiotic feeding. Error bars indicate the 10^th and 90^th percentile. Steel-Dwass All Pairs test *p ≤ 0.05, § ≤ 0.1.

Figure 6. Absolute quantification of Bifidobacterium species by high-throughput qPCR

Changes in abundance between GOS90 (n=12) and controls (n=15) are shown at a) day 7 (n=6) and b) day 14 (n=6). Left panels show data generated using GroEL-specific primers, right panels show data generated using 16S rRNA gene-specific primers.

Figure 7. Relative abundances of specific KEGG pathways and enzymes impacted by GOS90 feeding

a) Galactose metabolism pathway, b) 6-phospho-beta-galactosidase, c) D-lactate dehydrogenase and d) lactate transporter. Control n=15, GOS90 at day 7 n=6, GOS90 at day 14 n=6, Responders to GOS90 at day 14 (n=4) and Non-responders to GOS90 at day 14 (n=2). Boxes represent data between 25^th and 75^th percentile. Error bars indicate the 10^th and 90^th percentile. Steel-Dwass All Pairs tests § p ≤ 0.1, *p ≤ 0.05.