Enzymatic Hydrolysis Cello-oligosaccharide Of Medicago Sativa L. By β-glucanase

Cello-oligosaccharide as a significant component of functional oligosaccharides is mainly used on food &feed additive and biopesticide. It not only enhances immunity effectiveness of animals and plants, but can also be friendly to the environment since it is non-toxic and non-persistent. Plant cellulose is a renewable resource which abounds in the nature what makes it the desirable raw material of cello-oligosaccharide. Medicago sativa is chose as the raw material in this experiment to explore the method of cello-oligosaccharide preparation by hydrolyzing natural plant cellulose. This research is carried on preparation of cello-oligosaccharide through medicago sativa enzymolysis, purification and analysis from following items:The obtainment of standard substance of single-component oligosaccharide and research on the method of analysis was required at the very beginning, for the supply of reference standard and qualitative & quantitative analysis in the further experiment. The cello-oligosaccharide which was separated and purified by preparative silica column chromatography was prepared by mix acid degradation and molecular weights from DP two to DP five which were detected by ESI-TOF-MS.TLC,FACE and HPLC were adopted in the qualitative & quantitative analysis of cello-oligosaccharide. The results showed that the separation degrees of cello-oligosaccharide with different polymerization degrees appeared clear bandings with different Rf Values in the TLC& FACE analysis.Single chromatographic peak was appeared in the HPLC analysis of cellobiose, but there were two chromatographic peaks in the same analysis of cellotriose, either in the analysis of cellotetraose and cellopentaose. We concluded that the structural isomers exist in the cellotriose, cellotetraose and cellopentaose, which based on the results of UPLC-MS/MS analysis that the molecular weights of two chromatographic peak in the same cello-oligosaccharide were the same.The medicago sativa was pretreated by high shear emulsification and sig water, then bleached by hydrogen peroxide. NaOH solutions with five different concentration (0.5%,1%,2%,3%,4%) were selected in this experiment. Through comparative fiber filter bag rapid analysis, we found 1% NaOH solutions is the optimal choice. The HPLC analysis results of pretreated medicago sativa showed that the cellulose was 56.54% and hemicellulose was 9.22%.Medicago sativa crude powder was used for the substrate and for preparing cello-oligosaccharides byβ-glucosan. Firstly, through the single-factor experiments, the four main factors in the enzyme hydrolysis were determined, time (x1), temperature (x2), pH (x3) and enzyme-substrate ratio (E/S, x4). While, the optimal enzyme hydrolysis conditions were predicted as 8h, 50.0℃, pH 5.5 and E/S 0.5. Under this zymolysing conditions , the cellobiose yield was 6.75% and the cellotrioseyield was28.22%relevantly.