Breeding Of Ergosterol-Producing Strain And Study On Its Fermentation Condition

The parameters of High Performance Liquid Chromatography method to determine ergosterol content were studied. Saccharomyces cerevisiae TK15-94-33 (a lys" phe") and Saccharomyces kluyveri TK63-41-72 (a ade" leu") were screened as parent strains. Using protoplast fusion technique, a fusant TKF37 with high erogsterol content and biomass was obtained. At the same time, the optimum medium and fermentation process of TKF37 were studied.The parameters of HPLC were determined. The operating conditions included chromatographic column Nucleosil ODS(250mm X 4.6mm ID,5 u m),methanol: water(97:3,v/v) as mobile phase with a flow rate of 1.5mL/min and UV detection at 282nm. The relative standard deviation of detection results was no more than l%,and the recovery rate was over 98%. The accuracy and precision of the method were good.The yeast (S. cerevisiae) with high biomass and the yeast (S. kluyveri) with high ergosterol content in dry cell were screened form ten different species and genuses in yeast. Two haploids TK15-94 and TK63-41 were separated from the two yeast. The mutants with two genetic markers, TK15-94-33(a lys- phe-)and TK63-41-72(a ade- leu-), were selected after strains TK15-94 and TK63-41 were treated by EMS.The optimum conditions of protoplast formation on the yeast TK 15-94 and TK63-41 were studied. Using O.lmg/mL zymolyase to treat cells of TK15-94 for 50min,the protoplast formation rate was 98.5% and the regeneration rate was 26.3%. Using 0.12mg/mL zymolyase to treat cells of TK63-41 for 60 min, the protoplast formation rate was 97.7% and the regeneration rate was 32.1%.A fusant with good characters of two parent strains TK15-94-33 and TK63-41-72 was obtained by protoplast fusion. By identifying cell morpha and genetic stabilization test, the strain TKF37 was stable fusant. The ergosterol content of the yeast TKF37 reached 2.1%, and biomass was 3.71g/100mL.The optimal seed and fermentation mediums were obtained by orthogonal design test,and the optimal fermentation conditions were also studied. The optimum medium consisted of(%):glucose4, tryptonel, MgSO4 - 7H2O 0.1, KH2PO4-3H20 0.2. A 500mL flask containing 50mL medium was cultivated at 30 癈 for 32h in pH5.5 and the rotating speed was 220r/min.The ergosterol content reached 2.4%,the biomass reached 4.3g/100mL.