Shotgun Metagenomic Sequencing Revealed the Prebiotic Potential of a Fruit Juice Drink with Fermentable Fibres in Healthy Humans

Abstract

Fibre-based dietary interventions are at the forefront of gut microbiome modulation research, with a wealth of 16S rRNA information to demonstrate the prebiotic effects of isolated fibres. However, there is a distinct lack of data relating to the effect of a combination of soluble and insoluble fibres in a convenient-to-consume fruit juice food matrix on gut microbiota structure, diversity, and function. Here, we aimed to determine the impact of the MOJU Prebiotic Shot, an apple, lemon, ginger, and raspberry fruit juice drink blend containing chicory inulin, baobab, golden kiwi, and green banana powders, on gut microbiota structure and function. Healthy adults (n = 20) were included in a randomised, double-blind, placebo-controlled, cross-over study, receiving 60 mL MOJU Prebiotic Shot or placebo (without the fibre mix) for 3 weeks with a 3-week washout period between interventions. Shotgun metagenomics revealed significant between-group differences in alpha and beta diversity. In addition, the relative abundance of the phyla Actinobacteria and Desulfobacteria was significantly increased as a result of the prebiotic intervention. Nine species were observed to be differentially abundant (uncorrected p-value of <0.05) as a result of the prebiotic treatment. Of these, Bifidobacterium adolescentis and CAG-81 sp900066785 (Lachnospiraceae) were present at increased abundance relative to baseline. Additionally, KEGG analysis showed an increased abundance in pathways associated with arginine biosynthesis and phenylacetate degradation during the prebiotic treatment. Our results show the effects of the daily consumption of 60 mL MOJU Prebiotic Shot for 3 weeks and provide insight into the functional potential of B. adolescentis.

Keywords: Bifidobacterium adolescentis; Lachnospiraceae; arginine; fibre; gut microbiota; prebiotics; shotgun metagenomics.

Figure 1

Study design.

Figure 2

Effects of 3-week supplementation of the MOJU Prebiotic Shot or placebo (without fibres) on gut microbiota in healthy adults. Boxplot showing the (A) Shannon and (B) Simpson alpha diversity, grouped by treatment, coloured by timepoint, and faceted by phase. (C) Boxplot showing the percent abundance of B. adolescentis, coloured by treatment, grouped by time point, and faceted by treatment phase.

Figure 3

Effects of 3-week supplementation of the MOJU Prebiotic Shot or placebo (without fibres) on gut microbiota beta diversity in healthy adults. Principal component analysis (PCA) is based on Aitchison distance matrixes. PCA plots of PCA dimensions 1, 2, and 3 of beta diversity analysis. The ellipses represent 80% confidence intervals. Lines connect the same samples during each time point, with shapes indicating the time point, coloured by treatment and grouping. Both graphs are bordered by violin plots showing projections onto the principal component axes. Violin plots with significantly different groupings are marked, with *** = p-value < 0.01.

Figure 4

Effects of 3-week supplementation of the MOJU Prebiotic Shot or placebo (without fibres) on gut microbiota in healthy adults. Bar chart showing the percentage composition of the 20 most abundant species, coloured by genus, grouped by phylum, faceted by subject, and ordered by treatment and timepoint. Note: Firmicutes_A is a placeholder phylum which has been classified as Firmicutes in the NCBI database but does not meet the clustering requirements for GTDB. Unused space represents other species not in the top-20 most abundant.

Figure 5

Effects of 3-week supplementation of the MOJU Prebiotic Shot or placebo (without fibres) on gut microbiota composition in healthy adults. Data represent a volcano plot of differential abundant gut microbiota species showing effect size and −log10 of the uncorrected p-value taxa between time points under (A) prebiotic fibres and (B) placebo (without fibres). Coloured by change relative to baseline and species with an uncorrected p-value < 0.05 labelled. The horizontal line represents the unadjusted p-value cut-off at 0.05. N = 14.

Figure 6

Effects of 3-week supplementation of the MOJU Prebiotic Shot or placebo (without fibres) on gut microbiota functional metagenome in healthy adults. Data represent a volcano plot of differential abundant KEGG modules (A) with prebiotic fibres and (C) placebo, and KEGG Pathways (B) with prebiotic fibres and (D) placebo, between groups. The X-axis position of each point represents effect size differences at the end of the intervention, coloured by change relative to baseline. The horizontal line represents the unadjusted p-value cut-off at 0.05; the vertical lines represent effect sizes of −0.1 and 0.1. N = 14.

Figure 6

Effects of 3-week supplementation of the MOJU Prebiotic Shot or placebo (without fibres) on gut microbiota functional metagenome in healthy adults. Data represent a volcano plot of differential abundant KEGG modules (A) with prebiotic fibres and (C) placebo, and KEGG Pathways (B) with prebiotic fibres and (D) placebo, between groups. The X-axis position of each point represents effect size differences at the end of the intervention, coloured by change relative to baseline. The horizontal line represents the unadjusted p-value cut-off at 0.05; the vertical lines represent effect sizes of −0.1 and 0.1. N = 14.

Figure 7

Effects of 3-week supplementation of the MOJU Prebiotic Shot or placebo (without fibres) on gut microbiota functional metagenome in healthy adults. Principal component analysis (PCA) is based on Aitchison distance matrixes. PCA plots of PCA dimensions 1, 2, and 3 of (A) KEGG modules and (B) KEGG pathways. Ellipses represent 80% confidence intervals. Lines connect the same samples during each time point, with shapes indicating the time point, coloured by treatment and grouping. Both graphs are bordered by violin plots. Violin plots with significantly different groupings are marked, with * = p-value < 0.1, ** = p-value < 0.05, and *** = p-value < 0.01.

Figure 7

Effects of 3-week supplementation of the MOJU Prebiotic Shot or placebo (without fibres) on gut microbiota functional metagenome in healthy adults. Principal component analysis (PCA) is based on Aitchison distance matrixes. PCA plots of PCA dimensions 1, 2, and 3 of (A) KEGG modules and (B) KEGG pathways. Ellipses represent 80% confidence intervals. Lines connect the same samples during each time point, with shapes indicating the time point, coloured by treatment and grouping. Both graphs are bordered by violin plots. Violin plots with significantly different groupings are marked, with * = p-value < 0.1, ** = p-value < 0.05, and *** = p-value < 0.01.

Figure 8

Effect of 3-week supplementation of the MOJU Prebiotic Shot or placebo (without fibres) on gut microbiota functional metagenome in healthy adults, showing upregulated arginine biosynthesis by Bifidobacterium adolescentis KEGG modules M00844, M00845, and the urea cycle M00029 during the prebiotic phase.