Studies On Fermentation Process Of β-Fructofuranosidase By Arthrobacter

Lactosucrose (O-β-D-galactopyranosyl-(1→2)-β-D-fructofuranoside, LS) is a functional oligosaccharide, which has good processing properties and physiological functions. LS is non-digestible in the human digestive tract and is therefore low in calories and suitable for use in low-calorie foods. Other health benefits such as anti-caries, reduce blood fat, improve fat metabolism, reduce blood cholesterol and triglycerides and so on. In addition, LS is kind of non-reducing oligosaccharides, which does not occur Maillard reaction in the food. So for these reasons, their use as food ingredients has increased rapidly.β-fructofuranosidase (FFase, EC 3.1.2.26) catalyzes transfructosylation in addition to its hydrolutic action. FFase mainly from micro-organisms, but the character of the enzyme from different microbes is different. The enzyme from Arthrobacter sp. catalyzed both transfructosylation and hydrolysis, when it is incubated with sucrose. With lactose as acceptor, the FFase catalyzed preferential transfructosylation of the fructose residue to the acceptor. Despite having high practical potentialities, the use of mannanase is still limited due to low yields and high-production costs.The researches on the FFase from Arthrobacter sp 10138 were very important for the manufacture of LS, which provid an important base for large-scale production and further amplification. The content of this research were as follows:First of all, a high-performance liquid chromatography and evaporative light scattering method (HPLC-ELSD) for analysis LS by FFase synthesis was establishe. The products of enzymatic synthesis were quantitative and qualitative analysis by HPLC-ELSD. The results show that products in addition to containing LS, it also contains glucose, fructose sucrose and lactose. This result was similar with reported in the literature, indicating that FFase catalyzed transfurctosylation in addition to its hydrolytic action.Base on single factor screening test, uniform design and quadratic regression analysis, The optimum medium ingredients for FFase production in flask were as follows:sucrose 25 g/L, beef extract 3.5 g/L, yeast extract 2 g/L, (NH4) 2HPO410 g/ L, KH2PO40.7 g/L, MgSO4·7H2O 0.1 g/L. In the medium with initial pH 7.0. temperature 30℃, shaking speed on 125 r/min, incubated 48h. Under the above optimal conditions, FFase the activity can reach 294.9 U/ml, the fermentation greatly increased the enzyme activity.Using the experimental data of uniform design, artificial neural network (ANN) and genetic algorithm (GA) were applied to optimize the medium components. Artificial intelligence algorithm to optimize the composition as follows:sucrose 33 g/L, beef extract 3 g/L, yeast extract 2 g/L, (NH4)2HPO4 4 g/L, KH2PO4 0.5 g/L, MgSO4·7H2O 0.2 g/L. Maximum enzyme activity of 318.5 U/mL was obtained at the GA-optimized concentrations of medium components. The FFase activity obtained by the ANN-GA was 16% higher than the maximum activity of FFase obtained by UD experiments, and also 8% higher than the quadratic regression model.Finally, two levels fractional factorial experimental design was screened the key factors from various of fermentation conditions,and then use the 5 L fermentor to enlarge. The influences of various agitation speeds and dissolved oxygen (DO) levels onβ-fructofuranosidase production by Arthrobacter sp.10138 were investigated in a 5 L fermentor. The resurts of experiment in 5 L fermentor and shake flask were great different. When control pH 7.0, stirring speed 300 r/min, the ventilation rate 2 L/min, although the Arthrobacter growth was inhibited, FFase yield can reached the maximal value.