| Trehalose is one of the most primal oligosaccharide which is developed recently. It possesses characters including low-sweet, non-distress with tooth, avirulent, stable chemical property and non-caramelization. In the creature body, trehalose not only is a structure component which provides energy, but also is a metabolized substance which can protect the protein, the fat, the glucide and the nucleic acid in creature cell at dry, low temperature, high pervasive condition against demolishing. These unique protective properties of trehalose make it an interesting compound for several applications, for example as cryoprotectant for cells in medicine and microbiology, as an effective component in cosmetics, as a stabilizer for clinical reagents and bioproducts, or even as a preservative for fresh foodstuff. Because of these distinct properties, many technology workers carring out research and developing in production technology of trehalose.This paper studied on breeding and screening trehalose-enriched yeast strain H-16 by protoplast mutagenesis and violet mutagenesis, high trehalose-producing activity strain was breeded.Through carring out three-factors and three-level orthogonal experiment, the factors including enzyme concentration,enzymolysis temperature and enzymolysis time, the optimized conditions were: enzyme concentration 1.5%, enzymolysis temperature 30℃, enzymolysis time 1.5h. In single factor experiment, the amount ofβ-mercaptoethanol was determined asβ-mercaptoethanol 0.2%, and action time was 10min, homeo-osmosis stabilizer was KCl. Under the optimized condition, the formation rate of protoplast was 89.5%, regeneration rate was 16.2%.The relationship between ultra-violet irradiated dose and lethality of trehalose-producting yeast protoplast was also examined in this study. We obtained the lethality curve of ultra-violet to protoplast, irradiate under ultra-violet by 20W viltalight lamp to apart from 30cm, yeast fungus mutagenic agent was 50s, the lethality rate was 81.3%. Protoplast was ultra-violet irradiated by optimum, mutation strain was conservated after mutagenesis, and conservation was to end until ideal strain was screened. Eight high trehalose-producting activity strains were screened from 203 mutation strains. Among the 8 strains, trehalose yield of strain UV-6 was 1971.8μg/mL.In genetic stability experiment, UV-3 genetic stability was better. Trehalose yield of strain UV-6 was 1867.4μg/mL, increased 1.16 times comparing with the yield 862.8μg/mL of primitive strain.It can be realized to increase the yield of trehalose fermentation by optimization the fermentation condition and medium constitution. Firstly, based on single factor experiment, to produce trehalose of the strain UV-3the optimized conditions were: temperature 30℃, pH6.5, rotation speed 180rpm, volume of medium 40mL in 250 triangular flask, inoculating amount 8%, cultivation time 24h, trehalose production was maxmum. Beacause of the effect of medium moity to strain production was comparatively large, four-factor second order rotation combination experiment was applied in systematic optimize of medium trehalose-producting. The results of single factor experiment were made as zero level of two revolving orthogonal experiment, regression equation were deteced by F-analysis. Result discripted regression equation fitted better, and the optimized conditions were: cane sugar 2.61%, beef extract 0.53%, yeast extract 0.53%, MgSO4·7H2O0.03%. Under the best fermentation condition, the highest trehalose yield of fermentation was 3098.6μg/mL appeared after 24h, which was 0.71 times compared with prevenient strain 1231.2μg/mL.
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