Studies On The Preparation Technology Of Gentio-oligsaccharides With Immobilized β-glucosidase

Gentio-oligosaccharides are a new type of functional oligosaccharides,which have many physiological functions, such as proliferating intestinalbeneficial bacteria apparently, adjusting the balance of intestinal flora andimproving the laxative effect. In order to reduce the production cost ofgentio-oligosaccharides and provide technical parameters for their industrialproduction, the preparing technology of gentio-oligosaccharides by immobilizedβ-glucosidase (IBGL) were studied, the objective was obtained after separationby Sephadex gel chromatography, and ESI-MS and~1H-NMR were chosen toidentify the objective in this paper. The main results were as follows:1. Preparation process of BGL: according to the ratio of1:10, buffersolution (HAc-NaAc) was added into the solid fermentation medium ofAspergillus niger, and the solid medium was extracted three times under thenatural temperature. The extraction rate could reach93.82%. Crude BGL waspurified by the method of two-step salting-out, and it could be purified1.6times.2. Preparation process of IBGL:3.5%(w/v) sodium alginate solution10mLwas mixed adequately with moderate diluted BGL2mL (according to the ratio of105U BGL:1g dry carrier), and then1%（v/v）glutaraldehyde was added in andtreated at4℃for3h. Finally, dripped the solution into2%calcium chloride andhardened for12h. Activity and hardness of IBGL gel ball was17.78U/g wet gelball,658.32g respectively, and recovery ratio of the process was66.34%,3. Nature of IBGL: the optimum pH and temperature of IBGL was4.8,65℃respectively. The lost activity of IBGL was30%when it was stored at4℃for60d, K_m of IBGL was1.07mmol/L. The glucose conversion rate of BGL andIBGL was47%,43%respectively. Compared with BGL, pH tolerance range, theoptimum temperature and stability of IBGL all increased. 4. Preparation process of gentio-oligosaccharides by IBGL in the fixed bed:according to the ratio of30U IBGL:1g glucose, IBGL was added into the60%（w/v）glucose buffer solution, reacted and transformed for36h at65℃, pH4.8.When IBGL reacted for4times, the average glucose conversion rate was41.07%, and its residual activity was82.23%. The production ability of IBGLwas about3.5times as much as that of BGL.5. Isolation and identification of the product gentio-oligosaccharides:1) Separation conditions: Sephadex G-10gel column Φ1cm×40cm,10%ethanol as eluent, elution velocity10mL/h, column temperature50℃.Twocomponents were obtained after separation. TLC results indicated thatcomponent Ⅰ was the glucose, and component Ⅱ might be gentiobiose.2) Identification of component Ⅱ: ESI-MS results identified its molecularweight was the same with gentiobiose.~1H-NMR (400MHz, D2O) δ:5.06,5.05,3.85~3.65,3.60~3.50,3.38~3.25,3.20~3.00. Combined with the results of massspectrometry and1H-NMR, it could be determined that component Ⅱ wasgentiobiose.6. Research on methodology of residual substrate glucose in system ofgentiooligsaccharide prepared by enzyme: In order to determine yield ofgentiooligsaccharide quantitatively when it was prepared by enzyme, theGlucose Assay Kit Method was used to determine content of residual substrateglucose, and to measure yield of gentiooligsaccharide indirectly. It showed theresults of determining content of residual substrate glucose by the method werestable in two hours (RSD=0.81%), with good repeatability (RSD=0.97%),high precision (RSD=1.55%), and slight inference (the average recovery=101.68%).7. Cost analysis: total cost of gentio-oligosaccharides prepared by BGL was2.8times as much as that of gentio-oligosaccharides prepared by IBGL. Theproduction cost of gentio-oligosaccharides could be reduced remarkably whenthey were prepared by IBGL.