Breeding Of High-Yielding Trehalose Strain And Studies On Production Technology

Trehalose is a nonreducing disaccharide with two glucose residues. Because trehalose exhibit protective action against damage of high molecular substances , it achieve widespread application in a variety of fields including foods, pharmaceutics and biochemistry etc . Baker's yeast (Saccharomyces cerevisiae) is a major strain of trehalose production. Trehalose is accumulated at the end of the reproductive stages and under extreme environmental conditions. Because the trehalose is intracellular product, its productivity is limited. This paper examines the breeding of the mutant with high-yielding trehalose and the production of extracellular trehalose via the immobilized trehalose synthase. The main research contents include the methods for trehalose determination, the breeding of trehalose high-producing strain, the optimization of medium and culture conditions, the production of extracellular trehalose and the purification of the intra- and extracellular trehalose.The methods for trehalose determination were studied in this paper. The methods of paper chromatography-mottle elution cnromometry and enzyme-DNS colorimetric were defined. These methods were simple, cheap and accuate .The results showed that the positive correlation was obtained between trehalose concentration and acetic acid or osmotic tolerant. Through the test, the strain of Y7, which had the highest content of trehalose and strong tolerance to sodium chloride and acetic acid, was defined as a parental strain. In order to enhance the trehalose production, Y7 was used for mutation with EMS. Through primary screening and compound screening, strain L61 with high-yielding trehalose and deep neutral trehalase activity was screened. Comparing with the Y7, the specific growth rate of mutant increased by 22.7%, the activities of neutral and acid trehalase droped by 19.2%and 6.7%, respectively. Under the non-optimized condition, the production of trehalose increased by 57.2% compared with that of parental strain Y7.The factors affecting the trehalose production were studied in this paper. The Placket-Burman design method and Respond Surface analysis method were used to optimize the medium and the reaction conditions were optimized too. Under the optimized condition strain L61 could produce 0.82g/L trehalose after reaction for 3 hours by flask- shaking batch culture. The production of trehalose increased by 46.4% compared with that of the initial condition (0.56g/L). An investigation has been carried out of fed batch culture according to the optimum condition of batch culture. The intermittent feeding glucose was helpful to the trehalose production in the condition of 20g/L initial glucose concentration. The trehalose production of the fed batch cultureincreased by 50.4% compared with that of flask- shaking batch culture.In the yeast, the heat shock of 50 癈 can be helpful to the increase of the activity of trehalose synthase, on the other hand it can destroy yeast's cell wall. The intracellular trehalose produced could excreted to the culture medium. Keeping the pH at 6.0, 3% of the initial glucose concentration , 5% of seed volume, 1% of glucose concentration, heat shock at 50 癈 for 6 hours resulted in 8.94g/L trehalose.Based on the flask-shaking fed-batch culture, the test of the fed-batch reaction which synthesized the extracellular trehalose was performed with strain L61 in 5-liter bioreactor. The optimized factors were defined. Keeping the pH of the culture broth at 6.0,the initial glucose concentration of l%,the strain could produce 14.5g/L trehalose after reaction at 50 for 6 hours, the production of trehalose increased by 62.2% than that of flask-shaking culture. Meanwhile, the results showed that althougt the trehalose was produced successively , the intracellular trehalose concentration was 0 at the end of the reaction. Trehalose was excreted to culture medium completely. Yeast's cell was only as a carrier of the trehalose synthase. Trehalose could be synthesized successively with the feeding of glucose.Extraction...