| The conditions that affected the protoplast generation quotiety and regeneration quotiety of K.Marxianus were studied in this paper. Five-factor and four- level orthogonal experiments were designed to study the single factor, including hypha age, enzymes, temperature and osmotants, analyze the range of each factor and assess affecting degree of each factor. Finally the optimum conditions for KMarxianus protoplast generation and regeneration were determined: hypha age was 12h, enzyme concentration was 1.5%, enzymatic hydrolysis time was In, enzymatic temperature was 28C and potassium chloride was selected as osmotants. Under the optimum condition determined, the generation quotiety and regeneration quotiety were 92.3% and 29.6% respectively.The relationship between UV-light irradiated dose and lethality of K.Marxianus protoplast was also examined in this study. We got the UV-light to lethality curve. The results suggested that when UV-light irradiated dose was 20 watt, distance was 30cm and irradiated time was 50 second, the lethality of protoplast was 80.2%. The regenerated protoplast was primarily screened under the condition above.After K.Marxianus protoplast mutagenization with UV, TTC double-plate method was chosen for primary screening. The thick degree of mutant strain color was selected as criterion to evaluated the mutant strain's ability of producing alcohol. 24 mutant strains of thick color from plates were selected for secondary screening.Mutant strains from primary screening were incubated in whey medium and fermented in small tubes for 48h. The rate and amount of producing gas were chose as criterions for secondary screening. Finally we got 8 mutant strains of higher gas-producing rate and amount.8 mutant strains of milk koumiss from secondary screening were fermented in 250ml shaking flakes for third screening. We got one strain of high alcohol yield and ferment rate. The alcohol concentration of this strain was 3.8%, which was increased by 52% compared with original strain, and the residue of sugar was 0.1%. We named the strain obtained ZR-20.The effect of serial passage on the genetic stability was studied in this paper and the results demonstrated that compared with original strain, the ferment ability of ZR-20 from 1st to 10 th passage did not vary significantly.In addition, the growth and ferment characteristics of ZR-20 were examined in this paper. We obtained series of curves including temperature, pH, ferment temperature and ferment pH for ZR-20 and studied their effection on growth and ferment. The optimum combination for ZR-20 growth was 34C, 4.9( pH), which was as same as that of original strain. The effect of grow curve and ferment curve was studied by differenttemperature ,pH, incubation amount .The results indicate: growth curve was effected significantly by incubation amount ,more incubation amount ,shorter time to reach stability phase .But the final product amount was not effected by incubation amount .Growth vary but not significantly at different pH. Time to reach stability phase at pH5.9 was the shortest. Incubation amount did not effected ferment significantly to ferment curve .Ferment curve was not effected significantly by pH(3.9~5.9)A three-factor and three-level orthogonal experiment was designed to study the effection of ferment temperature, pH and incubation amount on final ferment results, analyze the range of each factors and optimize the ferment condition of ZR-20. The optimum ferment condition for ZR-20 was determined: 28 C, pH 4.9, incubation amount 2 X 107. Under the optimum ferment condition, the alcohol concentration post final ferment of ZR-20 was 4.2%, which was increased by 10.5% and 68% respectovely compared with those of preoptimized mutant strain and original strain.
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