Yielding Glutathione Yeast Strain Selection And Culture Conditions

Ⅰthe Aim of the Study:Glutathione was one kind of the three peptide compounds which contain L-glutamate, L-cysteine and glycine. There were restore state (GSH) and oxidation state (GSSG) in the body. GSH had a variety of important physiological function, particularly played a vital role in the maintenance of appropriate redox state. Glutathione was widely used in food, medicine, healthy products and cosmetics industry.The glutathione of our country depend on importation, so it is urgent to study on glutathione production. As a result of the fermentation by bacteria or yeast culture easily, production methods and process of continuous improvement and perfection, the use of microbial fermentation GSH industrialization had become the most common method of production. The past 30 years, yeast had been the choice of GSH production of micro-organisms fermentation. The breeding of high glutathione-producing yeast strains by UV, 60Co mutagenesis coupled protoplast fusion and optimizations of cultivation conditions were studied to get high content GSH and provide data for industrial production.ⅡMethods of Study1·Glutathione presence in the cells was often used to fermentation produce and a broken cell wall, including chemistry, physics, enzyme methods, etc. The ethanol direct extraction method was study, in order to provide the date support for the GSH industrial production, including the concentration of alcohol, extract time and temperature to identify the most economic extract conditions.2·The effect of medium composition on the production of glutathione by the purchase of glutathione-producing strains CⅠwas studied in shaking-flask culture. The effect of factors such as carbon source, nitrogen source, inorganic salt, that affected the GSH productivity of CⅠwas tested.3·The strains were mutagenized by UV, 60Co and UV- 60Co, selecting ZnCl2 resistance and L-Ethionine resistance mutants, respectively.4·Comparison of the two breeding methods, the better strain ZC1 was selected. The effects of culture condition on the production of glutathione were studied in shaking-flask culture.ⅢResults of Study1. The optimization condition of extraction by the orthogonal analysis was: temperature 50℃, reaction time 90min, alcohol concentration 40%. The total GSH in broth was 75.1 mg/L that increased 37.3%.2. The optimal fermentation conditions for CⅠwhich were determined by direct cross experiment was: 25g/L glucose, l0g/L yeast extract, (NH4)2SO4 3g/L, K2HP04 1.2g/L, KH2P04 1.8 g/L and MgS04 0.3 g/L were also added to the medium. The total GSH in broth was 96 mg/L that increased 27.8%.3. The mutant UC1 with high glutathione-producing was selected, finally. The total GSH in broth was 169.4 mg/L that increased 76.5%. Diploid marker strain Z5 (produced transparent circle in high ZnCl2, ZnCl2r) and C4 (yellow, Ethr ) with high GSH content were chosen. The total GSH of the mutant Z5 in broth was 121mg/L that increased 26%. The total GSH of the mutant C4 in broth was 127 mg/L that increased 32.3%. Diploid marker strain with high GSH content were chosen as parental stains. The fusant R54 was constructed by inactived protoplast fusion, selection and identification. The condition of production, regeneration of protoplast and protoplast fusion were studied. The biomass of R54 was 10.4g/L; the accumulation of glutathione was 157.2mg/L. The accumulation of glutathione of the mutant R54 increased 29.8%and 23.6% in comparison with the two original strains Z5 and C4.4. The optimum ferment condition was culture under 30℃for 33h, the original PH of the medium was 6.5, and the inoculating Concentration was 10%, the liquid volume of 30 mL in 250 mL.In short, GSH content of the starting strain CⅠ, were increased 215.7% by 54.7mg / L to 172.7mg / L.