The Breeding Of High Glutathione-producing Yeast Strain And Research For Its Optimization Of The Fermentation Conditions

In this study,saccharomyces cerevisiae zf(2-10515) which was preserved in our laboratory was the parent strain.The NTG mutagenesis method was used to breed high glutathione-production strains,and the best NTG concentration was determined which was 0.6 mg/mL.The fatality and positive mutation rate was 54.3%and 5.9%at this concentration respectively.Four high glutathione-producing mutants was breeded by four times NTG mutagenesis contimuously.They were named Yzf(2-10515)-1,Yzf(2-10515)-2, Yzf(2-10515)-3,Yzf(2-10515)-4 respectively.Yzf(2-10515)-1 was the highest glutathione-producing strain. The GSH content of Yzf(2-10515)-1 reached 0.88%in the dried cells and the biomass reached 7.8g/L.The glutathione content in the dry cells was 576.92%higher than the parent stain.A descent of only 0.4%in the glutathione yield and 1.3%in biomass of Yzf(2-10515)-1 was observed after ten times of subculture. Therefore,Yzf(2-10515)-1 was a favourable and stable strain.The medium and cultural conditions of the mutant have been optimized by shake flask.This study chosen malt extract as base medium which concentration was 10°Be′.The optimal medium for Yzf(2-10515)-1 which was determined by single factor experiment was:glucose was the optimal carbon source,(NH₄)₂SO₄ was the optimal nitrogen source,L-cys·HCl was the precursor amino acids salt and the addition of growth factor was no significant effect in glutathione production.The optimal medium which was determined by Box-Benhnken's central composite design and response surface analysis was:glucose 38g, (NH₄)₂SO₄ 6.3g,L-cys.HCl 1.9g,10°Be′malt extract 1000mL,pH natural.Precursor amino acids was added into the above medium.The last optimal medium composition was glucose 38g,(NH₄)₂SO₄ 6.3g,L-cys HCl 1.9g,glutamic acid 5g,10°Be′malt extract 1000mL,pH natural.The optimal fermentation condition of Yzf(2-10515)-1 was determined by single factor was:culture temperature 30℃,initial pH 3.5,rotate speed 140rpm,inoculum age 20h,inoculum size 20%,medium volume 100mL/500mL.In the condition the glutathione content and the weight of dried cells reached to 170mg/L and 9.46g/L which was 148.8%and 21.3%higher than that of non-optimized medium respectively.Glutathione fermentation were studied in a 7.5L scale batch bioreactor on the base of the above experiment.The result showed that the highest glutathione content and the most weight of dried cells weight reached 310mg/L and 17.55g/L after fermented 56h respectively,which was 82%and 86%higher than shake flask respectively.