Study On Tannase Produced By Aspergillus Niger And Propyl Gallate Biosynthesised By Immobilized Aspergillus Niger Cells In Organic Phase

This research thesis reported that a producing tannase strain, Aspergillus niger TA was improved by mutation screening and optimization of its fermentation conditions; Then after cell immobilization, Aspergillus niger cells were applied to biosynthesize antioxidant propyl gallate (PG) in organic media. Since study on PG biosynthesis by immobilized cell in organic media was first reported home and abroad, this research work was meaningful both in science and application.A new method to determine tannase activity was established, in which PG was selected as substrate and 270nm as measure wavelength. According to the linear relationship between the concentration of PG and the corresponding absorption values, tannase activity was determined from the decreasing amount of substrate. The method exhibited not only simplicity and sensitivity, but also repeatability and accuracy. It was only used to determine the activity of intracellular tannase.A high-yielding tannase mutant strain, Aspergillus niger L2i was screened from its parent strain by combining microwave irradiation with chemical inducing, and its final tannase activity was 130.6 U/ml. Its fermentation parameters in shaking flasks were studied, including concentration of tannic acid, carbon source and nitrogen source with their proportion, initial pH value, agitation speed and incubation temperature, etc. Above seven factors were optimized with L2?(313) orthogonal experiment. Under the optimum conditions, the activity of tannase from Aspergillus niger L2i was 225.8 U/ml.After compare with five kinds of cell immobilization methods, calcium alginate entrapment was adopted to immobilize Aspergillus niger L2i cells. For improving hysteresis effect in mycelium growth and tannase producing, the fermentation parameters about Aspergillus niger L2i immobilization cells were studied, including concentration of sodium alginate, CaCl2 and spores, inoculation amount of gel beads, concentration of tannic acid and medium volume in shaking flasks, etc. Above six factors were further optimized by LIg(37) orthogonal experiment. Under these optimum conditions, immobilized Aspergillus niger L2i cells exhibited not only enzyme-producing earlier and enzyme peak value similar to that of free cells, but also stationary phase longer than that of free cells.At last, immobilized Aspergillus niger L21 cells were applied to biosynthesize PG in organic phase. After reaction system was selected and synthesis parameters preliminary was optimized, about 20% production percent of PG was obtained in the organic media.