Study On The Degradation Of Phytosterol Into Androstenone By Mycobacterium

In this experiment, phytosterols were cleaved successfully into ADD and AD through cleaving the C17side chain of sterols by Mycobacterium Ml and M2. The mainly researching contents are blow:1. The methods of analysis and determination: The method of Layer Chromatography which employed silica gel and Ether-ethyl acetate-Petroleum(6 : 4) was mainly used to qualitatively analyze the substrates and the products. The Ultraviolet Absorption Spectrometry which worked at two kinds of wave length (254nm and 298nm) was mainly used to quantitatively analyze ADD and AD. According to the similar work theory of the Layer Chromatography and the High Performance Liquid Chromatography, the method of High Performance Liquid Chromatography with silica gel column was established. Through the methods of Calibration curve and Internal standard, the products ADD and AD could be quantitatively determined. Using FID detector and large calibre capillary column, the Gas Chromatography could be used to analyze the four kinds of phytosterols in the substrate. And according to the work theory of FID detector, the relative emendation factors were determined. So the Gas Chromatography could be used to analyze the sterols and AD(D) in the fermentation sample.2. The quality of strains used in production: During the consuming of nutrition, the cell morph changed regularly from long bacilli to short bacilli and last to cocci. The strain capacity of cleaving sterols had some relationship to the cell morph. The transformation capacity was high when the cell was long bacilli, but the transformation capacity became low when the cell became to short bacilli, and last when the cell morph turn to cocci, the transformation disappeared. There were no difference in cell morph between Mycobacterium Ml and M2, but their production capability was different. According to their transformation capability, strain M2 was selected as the mainly researching object.3. The parameters of processes: In this experiment one step process and two steps process were studied in detail. And the parameters of processes were determined, for instance: in one step process, the slant culture should be cultured in culture medium MYC/S2 under 29℃ and its preservative time should be no more than 20 days, the inoculation quantum to MYC/02 should be 7%, and in the two steps process, the liquid culture should be added to the fermentation medium in the period of 35h-40h by 7% of MYC/02. The optimal culture conditions of MYC/02 were also determined, for instance : pH=7.0, culture temperature was 29℃ andthe dissolved oxygen should be ample.4. The methods of adding substrate : Among the methods of adding alcohol, cyclodextrin, Tween80 and ultrasonic, employing 0.3%-1% Tween80 to disperse substrate is the best method, In this method phytosterols were suspended in 0.5%-1.0% Tween80, and the suspending liquid was emulsified in 120℃, then it was used to prepare fermentation culture medium MYC/02.5. The optimization of transformation process in 5L fermentation reactor : Based on the experiments of flask, the operation parameters of 5L fermentation reactor were established. During the course of cultivation, the temperature was controlled in 29℃, the pH=7 was adjusted through adding 10% NaOH and 2M hydrochloric acid, and the abundant oxygen could be acquired through adjusting the air flux and the stir rotate speed. The transformation effects (transformation 168h) of 5L fermentation reactor were below: ①When the adding rate was 0.3%, the substrate transformation rate was 100%, and AD(D) yield rate was above 90%. ②When the adding rate was 0.5%, the substrate transformation rate was about 95%, and AD(D) yield rate was about 85%. ③When the adding rate was 1.0%, the substrate transformation rate was 50%~60%, and AD(D) yield rate was about 50%.