Study On Fermentation Conditions Optimization For High-Yielding Strains And Preparation Of Thermostable Lactase

Lactase is a kind of hydrolase that mainly applied in fields such as food, medicine, analyzing, etc. at present. Optimization of fermentation conditions in shaking fask level of producing lactase and fermentation in 10L fermentor are investigsted by the Aspergillus niger DL116 which was stored in our lab in this thesis. The preparation processing of solid and liquid enzyme was studied primarily, the enzyme properties which will be a foundation for its industrial production. The Aspergillus niger DL116 lactase which has a broad application prospects because of its low production costs, fine stability, effective prevention on other strains in the production is a extracelluar and high temperature enzyme. The main results were as following:On the process of optimizing fermentation condition, impact of inoculation amount, temperature, initial pH, dissolution oxygen on fermentation by mycelium inoculation have been studied in shake flask level. The fermentation conditions was optimized by single-factor and orthogonal design experiments. The optimum fermentation conditions on producing lactase were as follows: 28°C, initial pH6.0, inoculation amount 6%, flask liquid volume 20mL/300mL. After optimization of fermentation condition, the lactase activity can reach 116.54U/mL, which increased 22.85% before optimization and the fermentation periods shortened 2 days.Based on the results at shake flask level, scale up studies were carried out in 10L fermentor. The metabolizing parameters including dissolution oxygen, pH, total sugar, deoxidize sugar, ammonia nitrogen and lactase activity were tested. It laid a foundation for large scale fermentation.In order to increase the lactase thermal stability, the effects of different substances were investigated. The results showed that the thermal stability of lactase could be increased for adding glycerol, lactose, maltodextrin, soy protein etc. into the lactase solution. Based on the results of single factor experiments, an orthogonal experiment design was used to optimize the best compounding of combined protective agent which including 4% glycerol, 3% lactose, 4% maltodextrin, 4% soy protein. Under the protection of the combined protective agent, the enzyme activity remained 85.28% after incubation of 2h at 70°C. So the combined protective agent had a significant effect on the enhancement of the lactase thermal stability. The concentrated enzyme liquid was gained by filtration and ultrafiltraion. The combined protective agent and 0.1% sodium benzoate were added to the concentrated enzyme liquid, the liquid lactase preparation showed good storage stability, The lactase activity was 88.77% stored on room temperature for 60 days. It was increased by 20.63% comparing with the control. The lactase activity is 424.84U/mL of the liquid lactase preparation, the optimum temperature is 70°C and pH is 4.0.After ultrafiltraion, the condensated lactase was been sedimentated by ethanol. Finally the solid enzyme was gotten by vacuum drying. The lactase activity is 14286.19U/g of the solid lactase preparation, the optimum temperature is 70°C and pH is 4.0. The lactase activity was 96.46% stored on room temperature for 30 da>s.