A Mechanistic Study of the Fermentation of beta-glucans from Different Sources by Bifidobacteria.

摘要

beta-Glucans are a kind of non-digestible carbohydrate (NDC) that are known for their benefits for human gut health, but there are very few studies on their fermentability by human colon microbiota. In this study four beta-glucans were selected for in vitro fermentation by three bifidobacteria. The beta-glucans included those from a seaweed called Laminaria digitata (laminarin), barley, a bacterium called Alcaligenes faecalis (curdlan), and a mushroom sclerotia from Pleurotus tuber-regium. Inulin from Dahlia tubers was used as control.;The content of beta-glucan in the NDCs prepared from the mushroom sclerotium of Pleurotus tuber-regium was 80.8 % with proteins less than 1.0 %, while that of curdlan, barley and laminarin all have more than 95% beta-glucan. All the beta -glucans contained almost purely glucose as their sugar components with only trace amount of mannose ( 2%) being found in laminarin. beta-glucan from barley had a MW of 590 kDa and a linear chain with mixed 1→3 and 1→4 beta-linkages in the ratio of 1:3. Curdlan had a beta-(1→3) linked unbranched linear chain with a MW of 10 to 30 kDa. Laminarin had a beta-(1→3) linked backbone with beta-(1→6) branches, having a MW of 6 kDa. beta-Glucan from mushroom sclerotia had a highly branched main chain with mixed glycosidic 1→3, 1→4 and 1→6 beta-linkages with a MW of 96 kDa.;Batch systems of in vitro fermentation of individual NDCs by B.longum subsp. infantis ( B. infantis), B. longum and B. adolescentis were carried out for 24 h under anaerobic condition. All the systems showed a significant drop (p 0.05) of at least 0.5 units in their pH values. The populations of B. infantis increased by 3 log10 CFU on all the NDCs while those of B. longum and B. adolescentis increased by about 1 to 1.5 log10 CFU and 2 to 2.3 log10 CFU, respectively. Utilization of the NDCs by the bifidobacteria evaluated by organic matter disappearance ranged from 4.52% in barley to 41.3% in inulin. The total short chain fatty acid (SCFA) produced by B. infantis was higher than that in B. longum and B. adolescentis for all the beta -glucans. The SCFA profile of inulin and all beta-glucans produced by all the bifidobacteria was dominated by acetate (96%). The ratio of acetic : propionic : butyric acid in the SCFA profile of the fermentation of all the beta-glucans by B. infantis was 8: 1: 1, which was very different from that of B. longum and B. adolescentis.;Based on the in vitro fermentation results, B. infantis was selected for a mechanistic study on the fermentation of the Pbeta-glucans from different sources by proteomic and molecular biology approaches. In the proteomic study, the gels of the two-dimensional difference gel electrophoresis (2D-DIGE) containing the total proteins from the B. infantis cells fermented with beta-glucans from barley, seaweed and mushroom sclerotia were compared with each other to isolate the differentially expressed protein spots. In all the comparisons, a total number of 198 protein spots were identified based on their mass spectra. These proteins were classified according to their functional annotation, including ABC transporters, phosphotransferase system (PTS), transketolase and others. Several genes encoding the proteins that probably play a role in the transport and degradation of beta-glucans including the ABC transporter gene, PTS gene and membrane protein gene underwent real time RT-PCR for transcriptional analysis. Hydrolytic enzyme activity assay showed that intracellular beta-1, 3 glucanase activity was present when B. infantis was incubated with beta-glucans from seaweed and mushroom.;Based on the above results, a model for beta-glucan catabolism in B. infantis was proposed. The fbeta -glucan molecules might be captured and imported inside the bacterial cells either by ABC transporters or PTS. They were then subjected to hydrolysis by glucan beta-1, 3 glucosidase. The released glucose molecules were readily incorporated into the central fermentation pathway, the 'bifid shunt', in which the hydrolyzed residues were further degraded or exported. This study has deepened our understanding on the fermentation of beta-glucans by bifidobacteria and demonstrated the potential of beta-glucans to be used as a novel prebiotic.