Endo-1,3(4)-β-Glucanase-Treatment of Oat β-Glucan Enhances Fermentability by Infant Fecal Microbiota, Stimulates Dectin-1 Activation and Attenuates Inflammatory Responses in Immature Dendritic Cells

Abstract

Background: Non-digestible carbohydrates are added to infant formula to mimic the effects of human milk oligosaccharide by acting as prebiotics and stimulating the immune system. Although not yet used in infant formulas, β-glucans are known to have beneficial health effects, and are therefore of potential interest for supplementation. Methods and results: We investigated the in vitro fermentation of native and endo-1,3(4)-β-glucanase-treated oat β-glucan using pooled fecal inocula of 2- and 8-week-old infants. While native oat β-glucan was not utilized, both inocula specifically utilized oat β-glucan oligomers containing β(1→4)-linkages formed upon enzyme treatment. The fermentation rate was highest in the fecal microbiota of 2-week-old infants, and correlated with a high lactate production. Fermentation of media supplemented with native and enzyme-treated oat β-glucans increased the relative abundance of Enterococcus and attenuated pro-inflammatory cytokine production (IL-1β, IL-6, TNFα) in immature dendritic cells. This attenuating effect was more pronounced after enzyme treatment. This attenuation might result from the enhanced ability of fermented oat β-glucan to stimulate Dectin-1 receptors. Conclusion: Our findings demonstrate that endo-1,3(4)-β-glucanase treatment enhances the fermentability of oat β-glucan and attenuates pro-inflammatory responses. Hence, this study shows that especially enzyme-treated oat β-glucans have a high potential for supplementation of infant formula.

Keywords: cytokine production; dendritic cells; in vitro fermentation; infant formula; microbiota; oat β–glucan.

Figure 1

HPSEC profiles of native oat β-glucan at the start and after 14, 20 and 26 h of fermentation using pooled fecal inoculum of 2- (A) and 8- (B) week-old infants. Calibration of the system using pullulan standards is indicated.

Figure 2

UHPLC-PGC-MS profiles of enzyme-treated oat β-glucan (eOBG) at the start and after 14, 20 and 26 h of fermentation using pooled fecal inoculum of 2- (A) and 8- (B) week-old infants. Peaks are labelled with 1a: 1 = degree of polymerization, a = letter qualifier. Corresponding structures are placed above label with —: β(1–4)-linkage and /: β(1–3)-linkage.

Figure 3

Relative abundance of bacteria at the highest classified taxonomy in duplicate fermentation digesta collected at the start and after 14 and 26 h from in vitro fermentation of media supplemented with native oat β-glucan (OBG) and enzyme-treated oat β-glucan (eOBG) using pooled fecal inoculum of 2- and 8-week-old infants.

Figure 4

Production of SCFAs, lactic acid and succinic acid upon fermentation of media supplemented with native oat β-glucan (A) and enzyme-treated oat β-glucan (B) using pooled fecal inoculum of 2- and 8-week-old infants.

Figure 5

Induced cytokines (fold change of induction by 0 h sample) by digesta of control fermentation, native oat β-glucan (OBG) and enzyme-treated oat β-glucan (eOBG) fermentation with pooled fecal inocula of 2- and 8-week-old infants. Statistical differences were tested using a mixed-effects model (REML) test followed by a Tukey’s multiple comparison test. Stars above bars represent statistical differences compared to the time-matched control samples (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Statistical differences between samples of OBG and eOBG fermentations are indicated above graphs (# p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001) (n = 6).

Figure 6

Relative NF-κB release by (A) HEK-Dectin1a and (B) HEK-Dectin1b reporter cell stimulation with (A) samples of native oat β-glucan (OBG) and enzyme-treated oat β-glucan (eOBG) fermented with 2-week old fecal inoculum and (B) OBG and eOBG fermented with 8-week old fecal inoculum. For all groups, a medium control and Zymosan were included. Statistical differences were tested using a OneWay ANOVA test followed by a Tukey’s multiple comparison test. Stars above bars represent statistical differences compared to the medium control (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Statistical differences between t = 14 or t = 26 and the matched t = 0 are indicated above graphs (# p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001). Experiments are repeated three times in triplicate.