Breeding And Immobilization Of High-yield Ethyl Acetate And Tolerant Saccharomyces Cerevisiae

Qingxiang liquor is one of the representative aromatic flavor of Chinese liquor.It has a long history and profound cultural background.The main aroma components are ethyl acetate and ethyl lactate.Saccharomyces cerevisiae is the main microbial group in the process of wine-making,which can produce alcohol and ethyl acetate by metabolism.It is difficult to detect the deficiency of the common ester-producing yeast in the fermented grains at the late stage of fermentation,which affects the synthesis of ethyl acetate,the main aroma component.Therefore,the selection of yeast with high yield of ethyl acetate plays an important role in increasing the yield of ethyl acetate and improving the quality of base liquor.In this paper,22 strains of S.cerevisiae isolated from Daqu and fermented grains of Qingxiang Liquor were screened,and S.cerevisiae,with high yield of ethyl acetate was compared with W.anomalus isolated from the same source for tolerance to acid and ethanol.Then,S.cerevisiae and W.anomalus,were used to optimize the immobilization conditions of the mutants.Finally,the yield of ester was analyzed in the wine-making experiment.The main results are as follows:1)S.cerevisiae is the main wine-producing and ethyl acetate-producing microorganism in the brewing of white spirit,Screening S.cerevisiae with high yield of ethyl acetate is of great significance for the study and improvement of liquor quality.In this experiment,22strains of S.cerevisiae were selected for high-yield ethyl acetate.The results showed that ethyl acetate?ethyl acetate?was a fragrant and easy-to-diffuse aroma component.Therefore,after48 h culture at 28?of YPD solid culture medium,the primary screen was carried out using a sniffer method.The 14 strains had a fruit flavor and 8 S.cerevisiae had the alcohol taste.After48 h of culture at 28?,in a liquid fermentation medium,14 strains of S.cerevisiae with fruit flavor were screened according to the yield of ethyl acetate,and the yield of S.cerevisiae J12-1 was the strongest.The ester content was 0.68 g/L,followed by S.cerevisiae J12-6,S.cerevisiae J10-4,and the ester content was 0.59 g/L,0.57 g/L,respectively.2)W.anomalus is a common ester-producing microorganism.After a short period of fermentation in the brewing process,the growth was inhibited and gradually replaced by Saccharomyces cerevisiae.In order to analyze the reasons why the growth of W.anomalus was inhibited in the late stage of liquor fermentation,S.cerevisiae J12-1 and W.anomalus J2-5 were inoculated into the fermentation medium of different alcohol concentration and different pH for 6 days at 28?.The cell concentration of OD₆₀₀00 was detected regularly,and the tolerance of W.anomalus J2-5 and S.cerevisiae J12-1 to alcohol and acid was compared.The results of acid tolerance test showed that with the increase of fermentation time and the decrease of pH in fermentation broth,The cell concentration of S.cerevisiae J12-1 decreased as a whole,but the OD value of W.anomalus J2-5 was 4.81 when pH was 3.6 for 6 days,which indicated that the effect of pH on the growth was less.However,in the alcohol tolerance test,the alcohol tolerance of,W.anomalus J2-5 was poor.After 6 days of cultivation with 12%alcohol concentration,the cell concentration was the lowest compared with other alcohol concentration conditions at the same time,the OD value was 3.09.3)In the late stage of liquor fermentation,the growth of yeast was inhibited with the increase of metabolite such as ethanol and total acid in fermented grains,which made it difficult to increase the amount of ethyl acetate in liquor samples.In order to obtain the yeast with high yield and tolerance,in this experiment,the strain W.anomalus J2-5 was irradiated150s by UV irradiation of 254 nm,then mutated by microwave for 35 s,and S.cerevisiae J12-1 strain was irradiated by UV light of 254 nm for 240 s,then mutated by microwave for35 s.And after the ultraviolet microwave combined mutagenesis,the cells were cultured in the medium supplemented with ethyl acetate and bromcresol violet indicator for 2d,and were screened according to the ratio of transparent circle D/d.The results showed that the D/d value of W.anomalus Y5-5 mutant strain was 1.96 times that of the original strain J2-5,while the D/d value of the S.cerevisiae Z3-3 mutant strain was 8.67,which was the original.3.85times of strain J12-1.The ester content rescreen was determined by the saponification method and the tolerance was measured in YPD liquid culture medium inoculated to different alcohol concentrations.The mutant with high yield,high alcohol resistance,strong acid resistance and good genetic stability was W.anomalus Y5-5 and S.cerevisiae Z3-3,and the yield of ester reached 3.75 g/L and 2.68 g/L,respectively.4)W.anomalus Y5-5 was superior to S.cerevisiae Z3-3 in ester yield and alcohol tolerance,while ethanol significantly inhibited the growth and fermentation performance of W.anomalus Y5-5.In order to increase the activity of lipid-producing yeast and finally increase the total ester content in brewing process,the immobilization conditions of W.anomalus Y5-5 were optimized by single factor and orthogonal experiment.Then the effects of immobilized W.anomalus Y5-5 on the wine-making process were analyzed and compared.The results showed that the immobilization of W.anomalus Y5-5 was the best when the concentration of alginate was 3%,the concentration of calcium chloride was 2.0 mol/L,the temperature of immobilization was 25?,and the amount of entrapped bacteria was 10⁹/mL.It was applied to the simulated wine-making experiment,and the sample was steamed in the total acid.Under the condition of no significant difference in alcohol precision and pH between the control group and the control group,the content of ethyl acetate was up to 3.4g/L,which was about twice as high as that of the control group.