The Mutation Breeding Of High Yield Glutathione Strain And Optimization Of Its Fermentation Conditions

Glutathione is a nonprotein tripeptide of glutamic acid,L-Cysteine,and glycine found in most eukaryotes and some prokaryotes,reduced Type(GSH)accounts for the majority in living organisms.Glutathione is widely used in food,pharmaceutical and cosmetic industries due to its antioxidant,free radical scavenging,detoxifying and whitening properties.As a result,demand for Glutathione at home and abroad has shown an upward trend in recent years.In this study,Pichia pastoris was used to produce Glutathione.Firstly,the original strain BY-1 was mutagenized by ARTP combined with gradient plate screening,the strain BY-2-24 was obtained by adaptive evolution of MMC,and the fermentation medium was optimized by response surface analysis(RSM),and the yield of shake flask was increased successfully Finally,the fermentation process was further optimized by 1.5 L quadruple fermenter culture.The results of the study are as follows:(1)Mutation breeding and adaptive evolution of high yield Glutathione strains.The mutation of Pichia pastoris BY-1 was induced by atmospheric pressure and room temperature plasma,and MMC adaptive evolution of the mutant was carried out by using microbial droplet culture apparatus.The results showed that the mutant with high yield could be obtained by the treatment of ARTP,the screening of 1,2,4-triazole gradient plate,the evolution of MMC adaptation and the verification of shaking flask.The results showed that the mutant with high yield of Glutathione could be obtained by the treatment of ARTP,the screening of 1,2,4-triazole gradient plate,the evolution of MMC adaptation and so on.The yield of BY-2-24 was 312.1 mg/L,which was 2.34 times of that of BY-2-24,and it still had good genetic stability after 7 subcultures.(2)Optimization of fermentation medium for Glutathione production by Pichia pastoris.First,the PB experiment was designed to screen the significant factors affecting the fermentation process of Glutathione,and then the BBD experiment was used to optimize the significant factors to determine the optimal medium.The results showed that yeast extract,anhydrous potassium dihydrogen phosphate and magnesium sulfate heptahydrate had the most significant effect on the fermentation process of Glutathione.The optimum medium for Glutathione fermentation was obtained by response surface analysis: glucose 30 g/L,yeast extract 26 g/L,ammonium sulfate 7.5 g/L,anhydrous potassium dihydrogen phosphate2.6 g/L,magnesium sulfate heptahydrate 0.3 g/L,sodium chloride 4.5 g/L,calcium chloride 1.5 g/L,cysteine 0.75 g/L.Under these conditions,the yield of BY-2-24 strain was 418.3 mg/L,which was much higher than that of BY-2-24 before optimization(312.1 mg/L).(3)Preliminary study on the fermentation of high-yield strain in fermentor.By-2-24,a high-yield strain of Glutathione,was cultured in a1.5 L 4-fermenter,and the mycelium concentration,p H value,residual sugar content,amino nitrogen and Glutathione production were measured.The results showed that when the initial sugar concentration was 25 g/L and DO was controlled at 35%,Pichia pastoris could grow best and the yield of Glutathione could reach up to 429.9 mg/L in the batch fermentation of Glutathione.Finally,the fermentation level of Glutathione was further improved by adding glucose.After 48 hours of fermentation,the yield of Glutathione was up to 436.3 mg/L.