The Preparation Of Glucomanno-Oligosaccharides By Whole-cell Catalysis With Recombinant Engineered Bacteria

Glucomannan-oligosaccharides is a water-soluble polysaccharide polymer, as a functional oligosaccharides, can improve animal intestinal microflora, improve immune function and oxidation resistance, widely used in food, medicine and feed industry. Their special physiological functions and broad application prospects greatly aroused the interest of scholars. The glucomanno-oligosaccharides were prepared with recombinant engineering bacterium containing beta mannase gene by whole-cell catalysis;These enzymatic hydrolysates were detected by Thin layer chromatography,bleached and processed to remove monosaccharides.The glucomanno-oligosaccharides were prepared with the Recombinant engineering bacterium Rossate-pET32a-CH3 containing beta mannase gene by whole-cell catalysis. By single factor optimization analysis, the influences of different digestion temperature, digestion time, pH, substrate concentration and enzyme amount on the rate of hydrolysis were explored.The results showed that enzymatic hydrolysis reaction of optimum temperature of 50?, the best time of 12 h, the best pH value of 7.0, the best substrate concentration was 3%, the best enzyme amount was 1000 U.Because the influence of time and enzyme on hydrolysis rate was not obvious,reaction temperature (A),substrate concentration (B), pH (C) were selected as the three variables, hydrolysis rate Y as response value.According to the principle of Box-Behnken, test plan were designed by the Design-Expert software. Finally,test results determines the optimum condition:reaction temperature was 50.6?, the substrate concentration was 3.4%, pH value of 6.8,making an eventual konjac powder hydrolysis rate reached 41.34%, in conformity with the predictive value of 41.541%.Because of the thin layer chromatography (TLC) with low cost, fast detection, the advantages of simple operation, therefore, glucomanno-oligosaccharides were prepared and enzyme products were detected by thin layer chromatography. Enzymolysis liquid and oligosaccharides standard containing mannose, mannobiose manninotriose, mannotetrose were spot on Sillca 60 silica gel plate,to analyze by thin layer chromatography.The analysis results showed the main components of enzymatic hydrolysis product were mannobiose manninotriose, mannotetrose,did not contain or contain a small amount of monosaccharides.Using yeast fermentation way to remove the monosaccharides in enzymolysis products, and through the single factor and, the best condition of yeast to remove monosaccharides were studied.With single factor optimization,results showed that the best conditions of yeast to remove monosaccharides:fermentation time for 22 h, the substrate concentration was 2%, yeast addition amount of 2%.Through orthogonal test optimization, the sequence of effects of three factors on removal rate of monosaccharides was:substrate concentration (C)>fermentation time (A)> yeast acount(B) and that the best conditions was fermentation time of 24 h, the substrate concentration 1%, yeast 2%.Under the best conditions, monosaccharides removal rate reached 69.50%.With the powerful and efficient adsorption of activated carbon powder,the effects of activated carbon dosage,decolorization time,decolorization temperature on the decolonzation were studied.Results showed that the optimal conditions were activated carbon dosage of 0.4%, decolorization time 1 h,decolorization temperature 70?.Under the best conditions, the decolorization of active carbon rate was 88.53%, the retention rate was 85.43%.