Screening,Characteristics And Application Of High-yielding Ethyl Acetate Yeast

High-yielding ethyl acetate yeast is in great demand in the production of food and condiment industries,but there are few strains that can meet practical production applications.For this reason,in this thesis,wine and vinegar spirits collected in different distilleries and vinegar factories were used as isolation sources,and the yeast strains were initially screened using traditional strain screening methods combined with morphological observation and molecular biology identification.Then,the yeast strains with high ethyl acetate production performance were screened using the initially screened strains for liquid fermentation and the ethyl acetate content in the fermentation broth measured by gas chromatography as an evaluation index.At the same time,the strains were comprehensively evaluated by studying the functional properties of the strains in combination with tolerance experiments.The factors influencing the production of ethyl acetate by the strains were optimized by using single-factor and response surface experiments.Finally,high yielding ethyl acetate yeast powder was prepared using vacuum freeze-drying method and the formulation of freeze-dried protectant was optimized to provide experimental basis and experimental data for the application of direct-pitch fermenters.The main conclusions drawn from this study were as follows:(1)Thirty-three yeast strains were initially screened out from industrial distilled spirit and vinegar brewing mass samples,and two of which,Y10 and Y12,with high ethyl acetate production capacity were re-screened by ethyl acetate production ability assay,which could reach 6.68 g/L and 4.73 g/L respectively.And both strains were identified by molecular biology as Wickerhamomyces anomalus.The results of tolerance experiments showed that the highest tolerated glucose and ethanol volume fractions of yeast strains Y10 and Y12 were 30%and 10%respectively.Moreover,the lowest tolerated pH value was 2.5 and the highest tolerated temperature was 36 ℃,which indicating that these two yeast strains had good adaptability to the changes of fermentation environment.(2)Optimization of ester production conditions of yeast strain.Based on the results of single-factor experiments,three factors with more obvious effects on yeast ethyl acetate production of yeast were selected from five different factors,namely,medium brix,fermentation temperature,ethanol addition,pH and fermentation time.These factors were used to conduct response surface for the optimization of the experiments.The results showed that the ethyl acetate yield of strain Y10 reached 13.15 g/L,which increased by 49.2%compared with that before optimization under the conditions of fermentation temperature 28℃,ethanol volume fraction 1.5%and medium pH6.Meanwhile,for strain Y12 the ethyl acetate yield reached 12.96 g/L under the conditions of fermentation temperature 28℃,ethanol volume fraction 3%and medium pH6,which increased by 68.5%.Combined with the performance of the strain and the stability of ethyl acetate yield,strain Y10 was selected as the object of study in the subsequent experiments.(3)Preparation of yeast lyophilized powder and optimization of protective agent.In the process of lyophilized powder preparation,the optimal conditions for collecting yeast slurry were 6000 rpm,10 min.Based on the results of single-factor experiments,the protectant was optimized by response surface design experiments.With the formulation of 8%skim milk powder,12%sucrose and 5%alginate sugar,and the mixing ratio of 1:2 between bacterial slurry and lyophilized protectant,the cell survival rate of lyophilized yeast strain Y10 reached 81.5%.Both strains of Wickerhamomyces anomalus,Y10 and Y12,had good fermentation properties and high ethyl acetate production capacity,while the lyophilized powder made from strain Y10 was also able to maintain high cell viability after lyophilization.Therefore,both yeast strains have good pre-application potential in food fermentation industries.