Screening And Application Of Acid-producing Microorganisms In Pit Mud

Butyric acid bacteria and caproic acid bacteria play an important role in the brewing of liquor.The organic acids produced by their metabolism are the precursors for the synthesis of the main ester flavors in liquor,and they are also important constituents of the acids in liquor.In this paper,the separation and screening of butyric acid bacteria and caproic acid bacteria,the acid-producing characteristics of growth,the co-cultivation of caproic acid bacteria with butyric acid bacteria,Saccharomyces cerevisiae,and the acid-generating properties of compound caproic acid bacteria solution.Relevant research has been carried out on the application of acid bacteria liquid to simulate the solidification of liquor solid brewing.The main research results are as follows:(1)Separation and screening from high-quality cellar mud of a well-known winery Using ES and RCM.After observing the color development of CuSO4,detecting the composition of the fermentation broth and identifying the molecular biology,a hexanoic acid-producing strain E-6(Clostridium celerecrescens)was obtained and a butyric acid-producing strain R-2(Clostridium tyrobutyricum).(2)Cultivation of R-2(Clostridium tyrobutyricum)and E-6(Clostridium celerecrescens)in different culture methods showed that: the test tube liquid deep culture and the flask(fermentation plug)culture method is conducive to the metabolic acid production of R-2 and E-6.A study on the growth and acid production characteristics of R-2 showed that the biomass of R-2 was the largest when cultured to 60 h,and the yield of butyric acid could reach 6.9 g/L when the glucose content was 20 g/L,When the pH is less than 5,the growth and metabolism of the bacteria will be inhibited.A study on the acid production characteristics of strain E-6 showed that the metabolic acid production cycle of strain E-6 was longer,and the hexanoic acid production reached the highest value of 9.42 g/L when cultured to the 12 th day.After single factor cultivation and orthogonal experiment,The optimal culture conditions for the production of hexanoic acid from E-6 are 12% inoculation amount,Culture temperature 35 ?,2 mL/100 mL ethanol addition and 80% volime.(3)The acid production test of mixed fermentation of R-2(Clostridium tyrobutyricum)and E-6(Clostridium celerecrescens)showed that the fermentation broth with the compounding ratio of 2:1 and 1:1 had been cultivated until the 11 th day.The acid yields were 9.75 g/L and 9.45 g/L,respectively,which were higher than those of caproic acid produced by E-6 single-bacteria fermentation.The mixed fermentation of E-6 and Saccharomyces cerevisiae mixed fermentation acid production test showed that: E-6 and Saccharomyces cerevisiae co-cultivation with the appropriate inoculation ratio can effectively increase the yield of hexanoic acid,the content of Saccharomyces cerevisiae in the compounding ratio is too high is not conducive to the formation of hexanoic acid,the compounding ratio of the content of hexanoic acid in the co-culture fermentation broth is 3:1>2:1>1:1>1:0>1:2.The mixed fermentation of E-6,R-2 and Saccharomyces cerevisiae produced acid.The test results showed that the content of hexanoic acid was the highest under the mixed fermentation condition with an inoculation ratio of 2:1:1,which was 10.4 g/L.Proportionally mixed into the compound hexanoic acid bacterial solution,and then the response surface experiment was used to optimize the acid-producing culture conditions of the compound hexanoic acid bacterial solution.The optimized hexanoic acid production was 13.73 g/L,an increase of about 32% compared with that before optimization.(4)The compound hexanoic acid bacterial liquid was added to Xiaotan to simulate the solid-state brewing of strong-flavor liquor in different proportions.After testing,the content of esters and acids in the experimental group increased greatly.The content of ethyl hexanoate in the distilled wine of the experimental group L-40 was 698.12 mg/L,which was 2.77 times higher than that of the blank experimental group.Heat map analysis and principal component analysis showed that there was little difference between the groups of L-0 and L-20,L-60 and L-80.