Breeding Of High-Yield Ethylacetate Yeast And Research Of Application In Fen-Flavour Xiaoqu Liquor

Isolating high-yielding ethyl acetate yeast strain from the xiaoqu liquor starter-making, then through four breeding methods (protoplast ultraviolet mutagenesis, protoplast chemical mutagenesis, and protoplast multipl mutagenesis and protoplast regeneration mutation) to improve the ester-producing yelid and heat-resistant property of the yeast. The ester producing conditions of the mutant were studied. Eventually applied the breeding strain to the brewing of Fen-flavour Xiaoqu liquor to improve the liquor quality. The main research results were as follows:1. The yeast strain Y1 was screened through traditional purification and gas chromatographic analysis, when it was cultured in ester-producing medium for 4 days at 30℃, ethyl acetate quantity could reach to 2.152 g/L, accounting for 90.9% of the total eater. The temperature tolerance of Y1 was 38℃, the optimal growth temperature was 28℃-30℃. Its acidproof performance was good; the optimal initial growth pH was 4. It could resist 10% alcohol. Identificated Y1 by microbial automatic identification equipment and the method of molecular biology, the result showed that Y1 was Hansenula anomala.2. Using the orthogonal experiment to get Y1 protoplast formation and regeneration optimum conditions, the results were as follows: the time of pretreatment was 20 minutes, the concentration of snail enzyme was 1%, the zymolysis time was 30 minutes, in these conditions Y1 protoplast formation rate was 89.7%, regeneration rate was 19.3%. A mutant BY2 was screened through mutation breeding, which yield of ethyl acetate could reach to 2.518 g/L, when compared with the original strain Y1 the yield of ethyl acetate increased by 20.3%, but the tolerability for acid, temperature and alcohol didn't improve. The mutant was stable performance in genetic.3. Study on the effects of diffident culture mediums and diffident culture methods on ester-producing through the single factor test , the results showed that the best ester-producing culture medium for the mutant BY2 was the wheat saccharification mash, the ester yield of static culture was 40% higher when compared with shaking culture. Selected several factors to carry on the orthogonal test once more, the result indicated that the optimization plan was as followed: the brix was 8°, pH was 5, temperature was 25℃, culture time was 4 days, and ethyl alcohol was 2%, total ester production of the BY2 was 4.812g/L in these conditions.4. Strengthened BY2 could also increase content of total ester, total acid of the liquor before and after microbiol culture, but strengthened BY2 before microbiol culture had an greater effect than the after on alcohol yield. Strengthened BY2 after microbiol culture, ethyl acetate content increased 45.9% compared with the control when inoculated quantity was 3%, If inoculation quantity over 3% the ethyl acetate didn't rise but decrease, and alcohol yield decreases with the increase of inoculation quantity; Strengthen BY2 and Saccharomyces cerevisiae at the same time could not increase ester yield and alcohol yield; Strengthen fluid seed was better than strengthen solid seed in terms of the ethyl acetate production .