Study On Overproduction And Stability Of Glutathione With Yeast

Glutathione(GSH) was a kind of important tripeptide with biology activity,widely used in food and medicine industries.Using yeast fermentation to synthesise GSH was the main way of GSH industrialized production.So far,the study on improving the synthesis of GSH were more, but the research on the stability of intracellular and extracellular GSH was less,while the stability of GSH directly affected the efficiency of cell enzyme catalytic synthesis of GSH.The mainly research contents and results of this thesis were as follows:(1) In the study of amino acid added strategy optimization, using the three precursor, and methionine which had synergy with cysteine, study the influence of the different time or different concentration added on the GSH content and cell dry weight.The results showed that amino acids added had a greater effect on the GSH content and cell dry weight.Through horizontal comparison,at stagnation(2 h) adding10 mM glutamic acid, logarithmic phase(15 h) 7 mM glycine,stability(24 h) 10 mM cysteine and 8 mM methionine could effectively increase the GSH content, and had less effect on the cell dry weight.Selecting these points, combined with a variety of adding methods, further designed the amino acid added strategy.According to the results of in fermentation 2 h adding 10 mmol/L glutamic acid,15 h added 7 mM glycine,24 h added 10 mM cysteine and 8mM methionine,the GSH content reached maximum,3.89 mg/g wet cells, when using the added strategy of single point, two points and three points.In the ofverification experiment.The result was 1.04 times higher than control, and cell dry weight was only slightly smaller than controls.(2) The glutathione synthesis conditions optimized.With the GSH content as the response value,GSH synthesis fermentation conditions were optimized using the single factor experiment and the Box-Behnken response surface analysis method, and the GSH synthesis optimum fermentation conditions were determined through the RSM method: fermented liquid pH 4.86, amount of carbohydrate supplement 14.09g/L, L-cysteine tendency for 13.00 mM, methionine tendency for 8.24 mM.The GSH content in predictive value could reach 4.02 mg/g wet cells,and in the verification experiment was the measured with a mean of 3.99 mg/g wet cells, very close to the model prediction, and the relative error was 0.75%, increased by about 108.9% than that of before optimization.These results showed that the experimental results were coincide with the predicted value of the regression equation, and the model could wellpredict the response value results of the actual fermentation.(3)The stability study of intracellular and extracellular GSH.At yeast ferme ntation cultivation of 24 h,the 200 mg/Lof GSH refinement was added to fer mentation liquor, then examined the stability of GSH in the fermented liquid u nder the conditions of normal environment.The results showed that extracellular GSH existed unstable phenomenon, and GSH oxidation were the important f actors that affect the stability of GSH, while intracellular GSH change was not obvious.By regulating pH, temperature and oxygen environment factors, and o ptimizing the yeast fermentation conditions,finally,the optimal fermentation condi tions were determined for pH=3.5-4.5, the temperature of 25 ? or less and an aerobic culture, then the stability of GSH was increased than the control group obviously.(4) The influence of antioxidants and metal ions on the stability of GSH.Antioxidants DTT or Vc added had significant protective effects on the stabilit y of GSH,which could effectively reduce the oxidation of GSH;Under the proper pH, when cultivating 6 h after adding 0.4% DTT or Vc,the extracellular GSH contentcould keep over 87.61%,improved more than 30% compared with the control group;While the three kinds of metal ions(Zn2+?Mn2+?Cd2+) had little effects on the stability of extracellular GSH,when treated with Zn2+ and Mn2+,the extracellular GSH content was slightly higher than that of the control and Cd2+ was similar tothe control, but the Zn2+ and Cd2+ could increase the c ontent of intracellular GSH, and improve the synthesis of GSH.