Carrot-Derived Rhamnogalacturonan-I Consistently Increases the Microbial Production of Health-Promoting Indole-3-Propionic Acid Ex Vivo

Abstract

Background: Using dietary interventions to steer the metabolic output of the gut microbiota towards specific health-promoting metabolites is often challenging due to interpersonal variation in treatment responses.

Methods: In this study, we combined the ex vivo SIFR^® (Systemic Intestinal Fermentation Research) technology with untargeted metabolite profiling to investigate the impact of carrot-derived rhamnogalacturonan-I (cRG-I) on ex vivo metabolite production by the gut microbiota of 24 human adults.

Results: The findings reveal that at a dose equivalent to 1.5 g/d, cRG-I consistently promoted indole-3-propionic acid (IPA) production (+45.8% increase) across all subjects. At a dose equivalent to 0.3 g/d, increased IPA production was also observed (+14.6%), which was comparable to the effect seen for 1.5 g/d inulin (10.6%). IPA has been shown to provide protection against diseases affecting the gut and multiple organs. The Pearson correlation analysis revealed a strong correlation (R = 0.65, p_adjusted = 6.1 × 10^-16) between the increases in IPA levels and the absolute levels of Bifidobacterium longum, a producer of indole-3-lactic acid (ILA), an intermediate in IPA production. Finally, the community modulation score, a novel diversity index, demonstrated that cRG-I maintained a high α-diversity which has previously been linked to elevated IPA production.

Conclusions: The results from the ex vivo SIFR^® experiment mirrored clinical outcomes and provided novel insights into the impact of cRG-I on the gut microbiome function. Importantly, we demonstrated that cRG-I promotes tryptophan conversion into IPA via gut microbiome modulation, thus conferring benefits via amino acid derived metabolites extending beyond those previously reported for short chain fatty acids (SCFA) resulting from carbohydrate fermentation.

Keywords: Bifidobacterium longum; carrot rhamnogalacturonan-I (cRG-I); dietary fiber; ex vivo; indole-3-propionic acid; prebiotic; tryptophan metabolism.

Figure 1

Study design using the ex vivo SIFR^® technology to assess the impact of cRG-I, IN and XA on the human gut microbiota. (A) Reactor design using the ex vivo SIFR^® technology to test the impact of the fibers with different specificities at a dose equivalent to 0.3 g/d (cRG-I_L) or 1.5 g/d (cRG-I_H, IN and XA), compared to a no-substrate control (NSC) in fecal samples of 24 human adults in parallel. (B) Timeline and analyses at 0 h and 48 h. Analysis of key fermentation parameters and microbial composition was reported earlier by Van den Abbeele et al., 2023 [15].

Figure 2

cRG-I, XA and IN stimulated the microbial production of different metabolites. The heat map displays the impact of a dose equivalent to 0.3 g/d carrot-derived rhamnogalacturonan-I (cRG-I_L) or 1.5 g/d carrot-derived rhamnogalacturonan-I (cRG-I_H), inulin (IN) and xanthan (XA) on a selection of metabolites identified at level 1 and 2a, as quantified Via untargeted LC-MS after 48 h of incubation. Colonic fermentation was simulated using SIFR^® technology for healthy adults (n = 24). The reported metabolites were significantly affected by the treatments (FDR < 0.20). Significant differences are indicated in bold of the log₂-transformed average fold change (abundance treatment/abundance NSC). Metabolite classes and subclasses (based on the precursor amino acids or nucleobases) are indicated on the left side of the heat map. cRG-I: carrot-derived rhamnogalacturonan-I, IN: inulin, XA: xanthan.

Figure 3

cRG-I enhanced the microbial production of health-related metabolites and reduced the production of harmful linoleic acid derivatives. (a) The bar chart showing level 1/2a metabolites that were significantly affected (highlighted by asterisks) by an equivalent dose of 0.3 and 1.5 g/d carrot-derived rhamnogalacturonan-I (cRG-I_L and cRG_H, respectively), after 48 h of SIFR^® colonic incubation for healthy adults (n = 24). The data are presented as log₂-transformed average fold change (abundance treatment/abundance NSC). Potentially beneficial and harmful metabolites are highlighted in green and yellow, respectively, while metabolites in gray are not discussed with respect to health benefits. (b) log2-transformed fold change versus NSC for a selection of health-related metabolites promoted by cRG-I. (c) Disease-associated linoleic acid derivatives that were reduced by cRG-I.

Figure 4

The fermentation of cRG-I promoted indole-3-propionic acid (IPA) production consistently across 24 donors, correlating with the consistent increase in Bifidobacterium longum (OTU10). (a) Absolute IPA levels (area under curve, AUC) and (b) log₂-transformed fold change versus NSC, as quantified Via LC-MS after 48 h SIFR^® colonic fermentation of carrot-derived rhamnogalacturonan-I (cRG-I), inulin (IN) and xanthan (XA) by the gut microbiota of 24 healthy adults. (c) Absolute levels (cells/mL) and (d) log₂-transformed fold change in B. longum (OTU10). (e) The Pearson correlation analysis between B. longum (OTU3) and IPA across all study arms. The Pearson correlation coefficient (R) and corrected p-value indicating the significance of the correlation are presented. (f) Schematic presentation of reductive conversion of tryptophan into IPA Via indole-3-pyruvic acid (IPyA) and indole-3-lactic acid (ILA). cRG-I likely promotes IPA Via stimulation of ILA-producing B. longum. Competing pathways that convert tryptophan to indole-3-acetic acid (IAA) and indole are shown in gray. Interactions between B. thetaiotaomicron and E. coli that suppress indole biosynthesis upon pectin supplementation are also shown [19].