| The Lactosucrose which is a functional oligosaccharide, can promote the proliferation of bifidobacteria and regulation of gut microflora, improve intestinal immunity and other physiological functions. Lactosucrose also has good processing properties, which is expected to become another functional food additive after the fructooligosaccharides and galactooligosaccharides being functional food additives. The industrialization of Lactosucrose in our country has not been reported, and major difficulties are as follows:the yield of beta-fructofuranosidase is low; Arthrobacter fermentation conditions are limited to the shake flask level; the enzymatic synthesis yield of Lactosucrose is low, which will cause high cost of raw materials and post-purification.To slove these two issues, in this paper, first, batch fermentation of beta-fructofuranosidase produced by Arthrobacter in5L fermentor was studied; the optimized conditions were selected for kinetic modeling and providing data for further optimization of fermentation conditions or technology amplification. Second, the immobilization of beta-fructofuranosidase by glutaraldehyde-sodium alginate embedding-crosslinking was applied; response surface method was used to optimize the immobilization process. Last, yeast ferment was used to remove the byproduct of glucose dynamically in the reaction of synthesis Lactosucrose in our country for the first time to increase the yield of Lactosucrose. The main results in this paper are summarized as follows:1. Batch fermentation technology of Arthrobacter production FFase in5L fermentor was studied, and the best conditions are rotate speed of200r/min, controlled pH6.5, temperature30℃. The final FFase activity was249.81U/mL Equation Logistic, Leudeking-Pire and Leudeking-Piret-Like were applied to describe the growth of Arthrobacter, synthetization of FFase and dynamic regularity of substrate consumption. The dynamic equations are mentioned as follows: Three equations above indicated that changed regularity among growth of Arthrobacter, substrate consumption and synthetization of FFase was elucidated well.2. FFD screening was applied to obtain process conditions of immobilizing FFase by alginate. The concentration of sodium alginate, fixed time and crosslinking temperature are key factors. Three-factor-three-level Box-Behnken design response surface analysis was used to obtain the optimal immobilization conditions:the concentration of sodium alginate of4g/L, fixed time of42min, cross-linking temperature of26℃, and FFase enzyme activity recovery was59.33%. In addition, the stability of immobilized FFase was studied. Results showed that the immobilized FFase enzyme has greatly improved the heat, pH and storage stability compared with free enzyme; even if immobilized FFase was recycled five times, the relative activity recovery was still51.71%.3. Rate changes of Lactosucrose synthetic reaction were researched by the Boltzman equation; different concentrations of substrate and glucose were tested, which demonstrated that glucose could inhibit Lactosucrose synthesis, especially when the glucose concentration was high, the substrate concentration was low, and the reaction was conducted to later period, suppression inhibition was particularly evident. ANN-GA model was employed to optimize conditions of the invertase deletion yeast removing byproduct glucose dynamically:initial pH6.65, yeast amount of1.4%, yeast adding time of11.66h, and substrate concentration of550mmol/L. The final Lactosucrose concentration was158.75g/L. The results were very satisfactory, and compared with reaction without yeast:Lactosucrose concentration increased from102.98g/L to158.75g/L, lactose conversion rate grew28.35%, final Lactosucrose content increased to50.67%from27.37%, and purified degree of the product was ascended at the same time. |
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