Preparative Separation Of Homogeneous Xylooligosaccharides And Their Fermentation By Bifidobacteria In Vitro

The discipline of enzymatic hydrolysate of xylooligosacchardes(XOS) separation with polyacrylamide gel was investigated, and high purity of homogeneous XOS were obtained by using a established preparative gel permeation chromatography system. The mechanism of Bifidobacterium adolescentis metabolizing XOS mixture and homogeneous XOS was studied by anaerobic batch cultivation in vitro. The components and structures of XOS mixture were analyzed, and the results showed that the content of arabinosyl groups in XOS mixture was 14.75 %, which composed of neutral xylobiose to xylohexaose, a little of acid XOS, xylan, enzyme protein and lignin. Xylobiose and xylotriose were not substituted with arabinose or 4-O-methylglucuronic acid proved by high performance liquid chromatography and electrospray ionization mass spectrometry. Xylopentaose and xylohexaose contained corresponding isomeric compounds with arabinose sidechains. High purity of compounds from xylobiose to xylohexaose were obtained in the order of molecular weight when XOS were separated with polyacrylamide gel Bio-Gel P-2 with degassed and purified water as eluant. In the course of separation, the molecular size of XOS was the dominant factor governing the separation in this system, and there was no specific absorption between polyacrylamide gel and XOS. The resolution range between two adjacent peaks was 0.60~1.11, and selective factor a was less than 1.2. Considering the separation rate and yield, the best flow rate was 12 ml/h. The system for preparative separation of XOS had been established, and this system was controlled by ?KTA FPLC separate XOS by Bio-Gel P-2 with column XK 50/60, with degassed high-purified water as eluant at 120 ml/h at 48℃, and detected by differential refractometer and 280 nm ultraviolet. A large amount of xylobiose and xylotriose with purity of 97.08 % and 94.19 % were obtained by twice separation with this system, and they can be used as standard samples of XOS. The biological activity of XOS mixture was investigated, and the results showed that the total sugar concentration decreased from 5.0 to 3.21 g/L when it was used as carbon sources for B. adolescentis after 48 h fermentation in vitro, the pH value decreased from 7.0 to 4.70, and the maximal cell-dry-weight was 0.37 g/L during 48 h fermentation. Its ability to proliferation of B. adolescentis was stronger than xylose, less than glucose. The components with low degree of polymerization had stronger ability of proliferation than XOS mixture with high DP. Arabinose was released from arabinosyl groups in XOS mixture, the macromolecules and the components with low degree of polymerization by α-L-arabinofuranosidases from B. adolescentis during the fermentation. Free xylose accumulated during the fermentation. The concentration of lactate, acetate, propionate and butyrate was 2.34, 1.60, 0.36 and 4.07 g/L, respectively, after XOS mixture was metabolized by B. adolescentis for 24 h. The biological activity of xylobiose and the discipline of B. adolescentis metabolizing xylobiose were investigated, and the results showed that the total sugar concentration decreased from 5.0 to 2.93 g/L when it was used as carbon sources for B. adolescentis after 48 h fermentation in vitro, the pH value decreased from 7.0 to 4.38, and the maximal dry weight cells was 0.33 g/L during 48 h fermentation. Its ability to proliferation of B. adolescentis was similar with XOS mixture, stronger than xylose and XOS mixture with high DP, and less than glucose. There was no arabinose produced and xylose accumulated during the fermentation. The main metabolites were 4.38 g/L lactate and 2.62 g/L acetate after 24 h fermentation, and the concentration of propionate was 0.47 g/L and no butyrate was detected. The biological activity of xylotriose and the discipline of B. adolescentis metabolizing xylotriose were investigated, and the results showed that the total sugar concentration decreased from 5.0 to 1.65 g/L when it was used as carbon sources for B. adolescentis after 48 h fermentation in vitro, the pH value decreased from 7.0 to 4.44, and the maximal cell-dry-weight was 0.47 g/L during 48 h fermentation. Its ability to proliferation of B. adolescentis was stronger than xylobiose and XOS mixture, and less than glucose. There was no arabinose produced and xylose accumulated during the fermentation. The main metabolite was 3.98 g/L lactate, and the concentration of acetate and propionate was 1.64 and 0.36 g/L, respectively, but no butyrate was detected. The biological activity of xylopentaose and the discipline of B. adolescentis metabolizing xylopenatose were investigated, and the results showed that the total sugar concentration decreased from 5.0 to 2.36 g/L when it was used as carbon sources for B. adolescentis after 48 h fermentation in vitro, the pH value decreased from 7.0 to 4.44, and the maximal dry weight cells was 0.41 g/L during 48 h fermentation. Its ability to proliferation of B. adolescentis was stronger than xylobiose and XOS mixture, and less than xylotriose and glucose. B. adolescentis decomposed xylopentaose into xylobiose and xylotriose during the fermentation. A large amount of arabinose was released from arabinose sidechains in xylopentaose by α-L-arabinofuranosidases from B. adolescentis, and free xylose accumulated during the fermentation. The concentration of lactate, acetate, propionate and butyrate was 4.77, 2.62, 0.40 and 3.65 g/L, respectively, after xylopenatose was metabolized for 24 h.The biological activity of xylohexaose and the discipline of B. adolescentis metabolizing xylohexaose were also investigated, and the results showed that the total sugar concentration decreased from 5.0 to 2.09 g/L when it was used as carbon sources for B. adolescentis after 48 h fermentation in vitro, the pH value decreased from 7.0 to 4.64, and the maximal dry weight cells was 0.40 g/L during 48 h fermentation. Its ability to proliferation of B. adolescentis was similar with xylopentaose, stronger than xylobiose and XOS mixture, but less than xylotriose and glucose. B. adolescentis decomposed xylohexaose into xylotriose during the fermentation. A large amount of arabinose was released from arabinose sidechain in xylohexaose by α-L-arabinofuranosidases from B. adolescentis and was used up at 48 h, and free xylose accumulated during the fermentation. The concentration of lactate, acetate and propionate was 3.88, 2.39 and 0.47 g/L, respectively, but no butyrate was detected. In conclusion, high purity of xylobiose and xylotriose that can be used as standard samples of xyloolgosacchairdes had been obtained by second separation with Bio-Gel P-2; the relative proliferation ability of homogeneous xylooligosaccharides had been found out by anaerobic batch cultivation in vitro; the discipline and mechanism of Bifidobacterium adolescentis metabolizing xylooligosaccharides had been investgated. This dissertation provided substance guarantee for the research and development of xylooligosacchrides, provided the theoretical guidance for the R&D of xylooligosaccharides with high biological activity, and also provided the theoretical foundation for the mechanism of bifidobacteria metabolizing functional oligosaccharides.