Introductionβ2→1-fructans are dietary fibers. Main objectives of this study were 1) to demonstrate direct signalling of β2→1-fructans on immune cells, 2) to study whether this is mediated by the pattern recognition receptors Toll-like receptors (TLRs) and nucleotide-binding oligomerisation domain-containing proteins (NODs), and 3) to relate the observed effects to the chain length differences in β2→1-fructans.MethodsFour different β2→1-fructan formulations were characterised for their chain length profile. Human peripheral blood mononuclear cells (PBMCs) were stimulated in vitro with β2→1-fructans, and production of IL-1Ra, IL-1β, IL-6, IL-10, IL-12p70, and TNF-α was analysed. Reporter cells for TLRs and NODs were incubated with β2→1-fructans and analysed for NF-κB/AP-1 activation.ResultsCytokine production in human PBMCs was dose- and chain length-dependent. Strikingly, short chain enriched β2→1-fructans induced a regulatory cytokine balance compared to long chain enriched β2→1-fructans as measured by IL-10/IL-12 ratios. Activation of reporter cells showed that signalling was highly dependent on TLRs and their adapter, myeloid differentiation primary response protein 88 (MyD88). In human embryonic kidney reporter cells, TLR2 was prominently activated, while TLR4, 5, 7, 8, and NOD2 were mildly activated.Conclusionsβ2→1-fructans possess direct signalling capacity on human immune cells. By activating primarily TLR2, and to a lesser extent TLR4, 5, 7, 8, and NOD2, β2→1-fructan stimulation results in NF-κB/AP-1 activation. Chain length of β2→1-fructans is important for the induced activation pattern and IL-10/IL-12 ratios.

Beneficial effects of inulin-type fructans are discussed in view of studies that applied the oligosaccharides in colon cancer, chronic inflammatory diseases, vaccination efficacy, and prevention of infection and allergy. In the present paper, we discuss their immunomodulating effects. It is suggested that immunomodulation is elicited through indirect and direct mechanisms. Indirect mechanisms encompass stimulation of growth and activity of lactic acid bacteria, but can also be caused by fermentation products of these bacteria, i.e., short chain fatty acids. Evidence for direct effects on the immune system generally remains to be confirmed. It is suggested that inulin-type fructans can be detected by gut dendritic cells (DCs), through receptor ligation of pathogen recognition receptors (PRRs) such as Toll-like receptors, nucleotide oligomerization domain containing proteins (NODs), C-type lectin receptors, and galectins, eventually inducing pro- and anti-inflammatory cytokines. DCs may also exert antigen presenting capacity toward effector cells, such as B cells, T cells, and natural killer cells locally, or in the spleen. Inulin-type fructans may also ligate PRRs expressed on gut epithelium, which could influence its barrier function. Inulin-type fructans are potent immunomodulating food components that hold many promises for prevention of disease. However, more studies into the mechanisms, dose-effect relations, and structure-function studies are required.

Dietary fiber intake is associated with lower incidence and mortality from disease, but the underlying mechanisms of these protective effects are unclear. We hypothesized that β2→1-fructan dietary fibers confer protection on intestinal epithelial cell barrier function via Toll-like receptor 2 (TLR2), and we studied whether β2→1-fructan chain-length differences affect this process. T84 human intestinal epithelial cell monolayers were incubated with 4 β2→1-fructan formulations of different chain-length compositions and were stimulated with the proinflammatory phorbol 12-myristate 13-acetate (PMA). Transepithelial electrical resistance (TEER) was analyzed by electric cell substrate impedance sensing (ECIS) as a measure for tight junction-mediated barrier function. To confirm TLR2 involvement in barrier modulation by β2→1-fructans, ECIS experiments were repeated using TLR2 blocking antibody. After preincubation of T84 cells with short-chain β2→1-fructans, the decrease in TEER as induced by PMA (62.3 ± 5.2%, P < 0.001) was strongly attenuated (15.2 ± 8.8%, P < 0.01). However, when PMA was applied first, no effect on recovery was observed during addition of the fructans. By blocking TLR2 on the T84 cells, the protective effect of short-chain β2→1-fructans was substantially inhibited. Stimulation of human embryonic kidney human TLR2 reporter cells with β2→1-fructans induced activation of nuclear factor kappa-light-chain-enhancer of activated B cells, confirming that β2→1-fructans are specific ligands for TLR2. To conclude, β2→1-fructans exert time-dependent and chain length-dependent protective effects on the T84 intestinal epithelial cell barrier mediated via TLR2. These results suggest that TLR2 located on intestinal epithelial cells could be a target of β2→1-fructan-mediated health effects.

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Cell wall polysaccharides (CWPs) from chicory root pulp (CRP) and the effect of ensiling on CWP structure to reduce the water-holding capacity (WHC) were studied. Sequential extractions of CRP showed that hot water extraction solubilized arabinan-rich pectin and inulin, each representing 6% of all CRP sugars. A significant amount of pectic sugars (46%) rich in uronic acid from CRP was solubilized by chelating agents. Both dilute alkali extraction, which solubilized branched pectin (14% from CRP), and concentrated alkali extraction, which solubilized hemicellulose dominant in xyloglucans (2.5%) mostly of the XXXG type and mannan (0.9%), from CRP CWPs seemed to influence the WHC of CRP. Alkali-insoluble residue (39% of CRP sugars) mainly comprised cellulose and some branched pectin (17% from CRP). Ensiling reduced the methyl esterification of pectin, caused degradation of homogalacturonan and rhamnogalacturonan, and possibly modified the xyloglucan, mannan, and glucan network, reducing the WHC from 6 mL/g to 3.4 mL/g.

The aim of this research was to study the in vitro fermentation of chicory root pulp (CRP) and ensiled CRP (ECRP) using human fecal inoculum. Analysis of carbohydrate levels in fermentation digests showed that 51% of all CRP carbohydrates were utilized after 24 h of fermentation. For ECRP, having the same cell wall polysaccharide composition as CRP, but with solubilization of 4 times more of CRP pectin due to ensiling, the fermentation was quicker than with CRP as 11% more carbohydrates were utilized within the first 12 h. The level of fiber utilization for ECRP after 24 h was increased by 8% compared to CRP. This effect on fiber utilization from ECRP seemed to arise from (i) increased levels of soluble pectin fibers (arabinan, homogalacturonan, and galactan) and (ii) ahypothesized more open structure of the remaining cell walls in ECRP, which was more accessible to degradation than the CRP cell wall network.

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