Carrot Rhamnogalacturonan-I Supplementation Shapes Gut Microbiota and Immune Responses: A Randomised Trial in Healthy Adults

Abstract

Background: Rhamnogalacturonan-I (RG-I) is a pectic polysaccharide with emerging prebiotic and immunomodulatory potential. This randomised, double-blind, placebo-controlled trial (ID: NCT06081972) evaluated the effects of carrot-derived RG-I (cRG-I) supplementation, compared to placebo (maltodextrin), on gut microbiota composition and immune cell activation in healthy adults. Methods: A total of 54 participants (18-70 years old) were randomised in a double-blind manner to receive either 500 mg/day of cRG-I or placebo for four weeks. Pre-screening ensured balanced randomisation based on habitual fibre intake and faecal Bifidobacterium counts. Questionnaires assessed potential gut health and well-being effects, while in vitro and ex vivo models were used to evaluate effects on intestinal permeability. Results: cRG-I was well tolerated with excellent compliance. Faecal Bifidobacterium counts increased significantly, peaking at week 3. Isobutyric acid levels rose, though no other SCFAs differed. Immunologically, cRG-I enhanced the percentage of circulating myeloid dendritic cells expressing activation markers (CD86, HLA-DR) on. Stool consistency improved slightly. Preclinical models further showed that cRG-I and its fermentation products protected intestinal barrier integrity under stress. Conclusion: These results support cRG-I as a safe, low-dose dietary intervention capable of beneficially modulating gut microbiota, immune responses, and barrier function in healthy adults within a short supplementation period.

Keywords: DC activation; bifidobacteria; cRG-I; diarrhoea score; healthy adults; prebiotic; rhamnogalacturonan-I.

Figure 1

(A) Study design and (B) subjects’ disposition. * The number of individuals included in the analysis corresponds to those actively participating in the study at each specific timepoint. At baseline, the total number of participants was n = 26. Created in BioRender. https://BioRender.com/b97z315 (accessed on 30 July 2025); https://BioRender.com/m49u742 (accessed on 30 July 2025).

Figure 1

(A) Study design and (B) subjects’ disposition. * The number of individuals included in the analysis corresponds to those actively participating in the study at each specific timepoint. At baseline, the total number of participants was n = 26. Created in BioRender. https://BioRender.com/b97z315 (accessed on 30 July 2025); https://BioRender.com/m49u742 (accessed on 30 July 2025).

Figure 2

The change in the bifidobacteria counts during 4 weeks after the start of supplementation. Each symbol depicts the mean ± SEM of at least 22 observations. Significance is not indicated on the figure, as changes over time were analysed using GEE and are described in the results.

Figure 3

The change in diarrhoea score between baseline and week 4 in healthy subjects was larger in the cRG-I group compared to the placebo. *: statistically significant compared to placebo (p < 0.05).

Figure 4

(A) Change in the proportion of cells expressing activation markers and (B) change in expression of cell surface activation markers (MFI) on human blood dendritic cells (mDCs) after 4 weeks intervention. Data are baseline-corrected and presented as mean ± SEM (%) at week 4. *: statistically significant compared to placebo over time (p < 0.05).

Figure 5

Protective effect of fermented cRG-I on gut barrier integrity assessed using an in vitro Caco-2/THP-1 co-culture model of epithelial inflammation. TEER values (% of initial) were measured 24 h after PMA stimulation. CM: cell culture medium; F_NC: faecal fermentation supernatant without carbon source; F_cRG-I: faecal fermentation supernatant with cRG-I. Bars represent mean ± SD of three technical replicates. * p < 0.05 compared to baseline of each treatment; † p < 0.05 compared to F_NC; (Wilcoxon matched-pairs signed-rank test).

Figure 6

The effect of cRG-I on gut barrier integrity on colon biopsies. (A) Effects on colonic paracellular permeability (absolute concentration values) in biopsies mounted in Ussing chambers for SDC responders (control n = 7), SDC (n = 6), cRG-I (n = 7), cRG-I + SDC (n = 7), F_cRG-I (n = 7), F_cRG-I + SDC (n = 7). (B) Effects on transcellular permeability (absolute concentration values) in biopsies mounted in Ussing chambers for SDC responders (control n = 10), SDC (n = 9), cRG-I (n = 10), cRG-I + SDC (n = 10), F_cRG-I (n = 10), F_cRG-I + SDC (n = 10). † p < 0.05 statistically significant compared to control; * p < 0.05 statistically significant compared to SDC; Wilcoxon matched-pairs signed rank test.