Preparation Technology Of Reduced Glutathione By Microbial Fermentation

Glutathione (reduced glutathione, GSH) is a mercapto tripeptide, connected with L-glutamic acid, L-cysteine and glycine. GSH is an important metabolic substence in human cells with multiple physiological functions, such as scavenging free radical, detoxification and promoting the liver enzymatic activity. In food industry, GSH also can enhance the meat aroma, prevent pigmentation of tin and fruit brown stain. GSH has been researched in Japan and other developed countries, and GSH is dominated in Chinese market. In China, GSH is produced by fermentation technology. So the technology needs to be improved to increase the yield of GSH urgently.In this article, GSH high-yield yeast was selected from the IAE culture preservation library, with the production of 9.6 mg GSH per gram dry cells. The medium was adjusted to accelerate the cells growth and increase the GSH production. The intracellular GSH content was reached 12.94 mg per gram dry cells through adding L-cysteine and glucose, and cell concentration increased by 12.4 times.Hot water and ethanol extraction methods were used to extract the intracellular GSH. Four factors of pH value (A), extraction temperature (B), extraction time (C) and solid to liquid ratio (D) were optimized through the single factor and orthogonal test. According to the GSH extraction efficiency and the conditions, hot water method was to be proved the better, and the optimal conditions were: solid to liquid ration was 1:3, extract pH 2.0, in 90 ℃ water bath to extract 10 min, GSH extraction content was 14.27 mg per gram dry cells.Ion exchange and Sephadex technology were used to separate and purify GSH from the yeast extraction. Through the static test, the optimal separation conditions were determined: D0017 cation exchange resin, the optimal extraction solution pH 4.5 and the elution buffer was disodium hydrogen phosphate-citrate buffer+0.3mol/L NaCl with pH 7.0. The dynamic separation test was performed under the optimal conditions, which was the column height was 15 cm and the flow rate was 2.0 milliliter per min, then the recovery rate of GSH was 91.46%. The effluent liquid containing GSH concentration over 250 μmol per litre were freeze-dried. The Sephadex G-10 gel chromatography column was used to purify the concentrated solution, the optimal conditions were that column height was 45 cm, loading quantities was 10% the column volume, eluent was deionized water, flow rate was 1.0 milliliter per min, then the recovery rate of GSH was 86.18%, miscellaneous removal rate was 87.6%.According to the results, hot water extraction method had many advantages, such as saving time, no pollution, extracting completely. The ion exchange and Sephadex gel chromatography were used to separate and purify GSH and it was high effective to remove the impurity proteins and easy to operate. The processes were suitable for the industrial application.