Carrot Rhamnogalacturonan-I: Structure–Function Relationship of a Potential Prebiotic and Immunomodulator

Abstract

Carrot Rhamnogalacturonan-I (cRG-I), derived from carrot pomace—a by-product of carrot juice production—is a complex, indigestible dietary glycan with a potential prebiotic and immunomodulatory properties. This study explores the intricate structure-function relationship of cRG-I, focusing on its in vitro fermentation and immune activity. The structural analysis of cRG-I reveals that the RG-I backbone consists of repeating rhamnose (Rha) and galacturonic acid (GalA) units, with side chains of arabinan, galactan, arabinogalactan-I (AG-I), and arabinogalactan-II (AG-II). Six fractions (PF1-6) were separated via size exclusion chromatography, showing variations in Rha substitution, side chain length, and acetylation and methyl esterification levels. The immune activity of the fractions and their saponified derivatives were assessed in vitro. All fractions, except PF2, dose-dependently stimulated the production of TNFα, IL-6, IL-1β, IL-8, and IL-10 in peripheral blood mononuclear cells (PBMCs) from three healthy donors. The cytokine levels were significantly influenced by the specific structural elements, including level and types of side chains, molecular weight (Mw), and degree of esterification of the individual fractions. Notably, the highest Mw fraction (100 kDa) exhibited the most potent activity, which was markedly reduced after the removal of ester residues by saponification. Conversely, the 75 kDa fraction (PF2) was inactive, but its saponified counterpart showed substantial immunomodulatory activity, underscoring the critical role of ester residues in the immune profile of RG-I subpopulations. The fermentation kinetics of cRG-I by fecal microbiota from four donors were studied using an adapted M-SHIME® model. cRG-I was rapidly degraded in the proximal colon, with faster and more complete fermentation over three weeks. The Mw distribution pattern of cRG-I during supplementation revealed two main donor-dependent gut microbial fermentation strategies: the general and preferential pathways. In the general pathway, various cRG-I structures were hydrolyzed concurrently, whereas in the preferential pathway, distinct structures were sequentially fermented. Arabinan side chains were particularly utilized before the RG-I backbone, correlating with an increase in Bifidobacterium longum abundance over the three-week period. MALDI-TOF MS confirmed early degradation of arabinan, galactan, and arabinogalactan side chains. Short-chain fatty acid production, especially acetate and propionate, increased over time. Despite different hydrolysis routes, final RG-I consumption was nearly complete, resulting in similar metabolic profiles after three weeks. The variability in side chains and branching patterns is influenced by the plant source and extraction method. A reproducible protocol was proposed that starts with pre-saponification of acetyl groups and other esters from RG-I samples, followed by solubilization of the dried saponified product as a TBA-salt in DMSO, propyl esterification of all carboxyl groups, and β-elimination to completely degrade the RG-I backbone (DP5-75). The resulting fragments included characteristic RG-I dimers with and without a single side chain. The oligosaccharides released from cRG-I were further fractionated based on size and characterized using HPAEC and MALDI-TOF-MS. The predominant oligosaccharides comprised galactose chains (DP1-5), arabinose chains (DP4-5), or a combination of both arabinose and galactose (DP6). Overall, this research provides an in-depth characterization of the complex structure of cRG-I and underscores its potential as a prebiotic and immunomodulatory dietary fiber.