| Glutathione (the reduced form of GSH), consisted of L-glutamate, L-cysteine and glycine, is oneof the major non-protein thiol compounds. GSH is widely distributed in nature and plays manyimportant physiological roles in living cells. As an antioxidant, GSH can enhance the immunity ofthe body promptly and maintain the normal redox environment of cells. Since GSH could be widelyused in the many fields, i.e. clinical medicine, food industry, athletic sports and biological research,the demand of GSH has been expanding. Fermentative production of GSH is one of the mostpromising methods for GSH production at present.In this study, Candida utilis WSH 02-08 which could accumulate high concentration of GSHintracellularly was selected for GSH production.According to the principles of fermentation optimization a series of feasible approaches orstrategies were carried out to achieve high concentration, high yield and high productivity of GSHby the optimization of C. utilis WSH 02-08 cultivation. The main results were showed as follows:(1) As the key amino acid for GSH synthesis, L-cysteine showed important influence on GSHproduction. The addition of L-cysteine at the late stage of cultivation greatly improved GSHconcentration and intracellular GSH content. GSH concentration reached 347 mg·L-1 with theaddition of 10 mmol·L-1 L-cysteine and increased by 56% than the control.(2) During the batch or fed-batch culture of C. utilis WSH 02-08 in a 7 L stirred fermentor, theaddition of L-cysteine at the end of cell growth also had positive effects on both GSH productionand intracellular GSH content. By the addition of L-cysteine, the ultimate GSH concentrationreached 1150 mg·L-1 in fed-batch culture of glucose feeding. It is indicated that L-cysteine additioncombined with glucose feeding was no doubt one of the most efficient approaches tosimultaneously improve both GSH production and intracellular GSH content.(3) Based on the previous results on GSH fermentation in flask and 7 L fermentor, the scale up ofGSH production from 7 L to 30 L fermentor was investigated. Both GSH concentration (1027 mg/L)and GSH content (3.53%) in 30 L fermentor were very close to those in 7 L fermentor.(4) When Candida utilis WSH 02-08 was cultivated in a glucose-ammonium sulfate mediumwithout pH control, glutathione leakage occurred when the pH of the culture broth dropped to 1.5.Further experiments showed most of intracellular glutathione could be leaked into the medium bykeeping the pH at 1.2 for 3 h in which the cells were still alive. Thereby, a low pH-stress strategywas developed and applied in fed-batch productions of glutathione. Total glucose concentrationreached 737.1 mg/l and increased by 24.9% and 20.0% than the controls. 197.3 mg/l of glutathionecould be secreted into the culture broth during the process. Glutathione content was 2.67 % (w·w -1)which were only 2.28% (w·w -1) and 2.11% (w·w -1) in the controls. This demonstrates theimportance of a physiology-based fermentation strategy in the production of useful compounds.(5) The effects of DMSO on the cell growth of C. utilis WSH 02-08 were investigated. Additionof DMSO did not show negative effect on yeast cell growth at its lower concentration. Only slightinhibition to cell growth was observed in the higher DMSO concentration. However, when theconcentration of DMSO was between 20-40 g·L-1, intracellular GSH leakage occurred. When theconcentration of DMSO was 32 g·L-1, half of the total GSH (129 mg/L) accumulatedextracellularly and the total GSH concentration reached 278 mg/L. Therefore, the addition ofDMSO would be in favor of GSH excretion, which also helped to simplify the process of GSHrecovery in the following downstream operation.(6) The intracellular or extracellular distribution of GSH was quite different under the differentaddition time of DMSO during GSH fermentation. Although the earlier addition of DMSOincreased the extracellular GSH, the total GSH concentration was in a low level. The addition ofDMSO at the proper time (late stage of exponential phase) would be in favor of both GSH excretionand total GSH concentration.(7) As to the cultivation of C. utilis WSH 02-08 by batch or fed-batch process in a 7 L stirredfermentor, the addition of DMSO at 12 h increased the extracellular GSH and the further feeding of30 g·L-1 glucose at 24 h increased the intercellular GSH which reached the equivalent level of thecontrol. On significant difference was observed by the addition of DMSO. However, the maximumGSH concentration reached 784.5 mg·L-1, increased by 27.6% than the control, and the GSHcontent reached 3.04%. 187.3 mg/l of GSH was secreted into medium and the GSH productivityreached 18.68 mg·L-1·h-1 which was 27.7% higher than the control. |
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