Investigation On The Synthesis Of Neo-kestose From Sucrose Catalyzed By Phaffia Rhodozyma

Neokestose was produced by the action of β-fructfuranosidase, in which fructosyl resi-dues were transferred to sucrose by β-2, 6 glycosidic bonds. Neokestose had the ability of re-lief of constipation, decrease of total cholesterol and lipid in the serum, improvement of min-eral adsorption. Previous studies showed that the astaxanthin-producing yeast Phaffia rhodozyma accumulated neokestose as a major transfructosylation product when growing on sucrose, therefore, using Phaffia rhodozyma as biocatalysis had attracted much attention. In order to improve the yield of neokestose, the synthesis of neo-kestose from sucrose catalyzed by Phaffia rhodozyma was investigated.In order to improve the activity of β-fructofuranosidase, the effects of culture conditions on the growth and β-fructofuranosidase activity of Phaffia rhodozyma were investigated in this research using single factor methodology. Xylose could improve the β-fructofuranosidase activity but not be good for the growth. The biomass and β-fructofuranosidase activity were remarkably enhanced by yeast extract. The optimum p H for growth and β-fructofuranosidase activity was different. The results showed that the optimum medium was composed of xylose 10 g/l, yeast extract 10 g/l, malt juice 22 g/l and p H6.0. The β-fructofuranosidase activity was increased from 0.67 U/ml to 2.92 U/ml, which was 3.36 times higher than those without op-timization.The effects of important production parameters on the biotransformation of sucrose by immobilized cells of Phaffia rhodozyma were investigated. Cell concentration could signifi-cantly affect the neokestose yield and the optimal cell concentration was 16 g/l(calculated by dry weight). Sucrose concentration had positive effect on the maximum yield of neokestose within 400 g/l. Elevating the reaction temperature increased the reaction rate but decreased the maximum yield of neokestose. Meanwhile, sugar cane juice could be used as an inexpen-sive substrate for neokestose production.Continuous production of neokestose by immobilized Phaffia rhodozyma with a packed bed reactor was investigated. The fitting curve of yield and time was consistent with quadratic curve using sucrose and sugar cane juice as subsrate, respectively. The highest yield of ne-okestose was achieved at the residence time of 2.0 h for sucrose and 2.2 h for sugarcane juice, respectively. Additionally, a 30-d continuous neokestose production demonstation was veri-fied feasible in a packed bed reactor, indicating that the cell immobilization with chi-tosan-coated alginate had potential for industrial production.The separation and purification of neokestose syrup produced by sucrose biotransfor-mation were investigated. First, Pichia pastoris was used to remove the fructose and glucose of the syrup and the effects of cell concentration of Pichia pastoris and fermentation time on the purity of neokestose were investigated. The results showed that the purity of neokestose increased from 10.92% to 19.39% when the cell concentration and reaction time were 60 g/l and 10 h, respectively. Then, the neokestose syrup was further purified using Bio-Gel P-2 and the effects of sampling amount and flow rate on the purity and recovery of neokestose were researched. Under the optimized conditions of a flow rate of 0.1 ml/min and a sampling amount of 0.5 ml, the purity of neokestose improved from 19.39% to 76.68% and the purity of fructooligosaccharide of 99.22% was obtained.