The Study On High GSH And Selenium-rich Technology By Candida Utilis

Glutathione is an important physiological active compound, plays significant rolein treatment of tumor, cancer, aging and coordination of endocrine, and so on.Glutathione is widely used in medical, food, feed and other industries. Selenium is anessential nutritional trace element, which is used to the normal physiology of human.Selenium deficiency can lead to many diseases, such as keshan disease, hepaticnecrosis, cancer and other disease. In nature, the selenium in the form of inorganicselenium and organic selenium, inorganic selenium has high toxicity, organicselenium has low toxicity, strong biological activity, high absorption rate,environmental pollution, etc. The microbial fermentation is the most economic, safeand practical method.The contents and the conclusion of this experimental study are as follows:（1）Study on Neocuproine method for determining the content of GSH in theyeast extract, used HPLC method and Neocuprine method determination ofglutathione. A comparative study on the results found by the2methods was made,and it was shown that best accuracy and selectivety was achieved by the HPLCmethod. For HPLC method, accuracy, precision, recovery, and the measured valuecan reach a higher level. For Neocuproine method, accuracy, precision, recovery, andthe measured value close with HPLC method, but it would influenced by thecoexistence of other thiol compounds, its precision significantly lower than that ofHPLC. Neocuproine method applied to the determination of GSH in yeast extract,due to the yeast extract the coexistence of less other thiol compounds. Neocuproinemethod has advantages as follows, simple operation, low detection limit, the testingcost is low, and measurement for large-scale production.（2）The fermentation conditions of GSH produced by Candida utilis werestudied, the effects of extraction methods, medium component and the glucosefeeding on production of GSH were investigated. The optimal conditions wereobtained as follows: ethanol method was chosen as the extraction method ofintracellular GSH by screening. The yeast extract powder and ammonium sulfate had the greatest effect, especially, the yield reached25.48mg/g by addition of ammoniumsulfate as nitrogen source. Addition volume of glucose was16mL and the optimalfeeding time was10h. Under such conditions, Dry weight of biomass was8.14g/L,the production of glutathione was113.45mg/L. By shake flask culture, which was2.89fold improvement over that of the initial condition.（3）The effect of concentration of sodium selenite in plating medium toselenium-rich by candida utilis effect was studied. The color of the strain would turnred, when the plate adding sodium selenite medium, and the concentration of sodiumselenite higher, the color deeper. The rich selenium content reached the maximum,when sodium selenite concentration exceeded1500mg/L, through mutagenesis strainrich selenium content increased5.6times. Using breeding strains for experiments，inorder to observe relationship between the concentration of sodium selenite and strainquantity, The tolerance of candida utilis in sodium selenite is limited, When theconcentration of more than50mg/L will inhibit the growth of bacteria.（4）The fermentation conditions of selenium enrichment by candida utilis werestudied. Based on the results of the sole experiment，three factors was selected: Theconcentration of sodium selenite, inital pH and incubation temperature, the responsesurface method was used to optimize the various factors. The optimal conditions areobtained as follows: incubation time of30h, time of adding selenium ismid-logarithmic, the sodium selenite concentration is35mg/L,inital pH of6.6,incubation concentration of10%, incubation temperature of27℃, themediumvolume of150mL/500mL. Under the optimal conditions，the biomass is6.87g/L, Thetotal selenium content of candida utilis is12639.7μg/L, the selenium content of thecells is1839.8μg/g, in which sodium selenite conversion rate of79.1%and theorganic Se is higher than90%. The actual value of selenium content which aresubstantially consistent with the theoretical value, response surface methodology isapplicable for the fermentation conditions of Selenium enriched by Candida utilis.