Screening And Application Of Guaiacol Functional Bacteria From Huanghelou Wine Ecological Cave

Guaiacols are one of the health factors in liquor.By analyzing the gas chromatographic analysis of the Huanghelou Luzhou-flavored base wine,the guaiacol content is unstable.The project firstly used the diluted coating plate method to separate and purify the bacteria,air,dregs,mud,yellow water and curved bricks in the workshop of the Huanghelou Wine Xianning Winery,and analyze and study the Huanghelou Wine Xianning Winery.The community diversity of bacteria in the workshop air,dross,mud,yellow water,and curved bricks.Through microscopic examination,single-fermentation of the initially isolated Bacillus was carried out,and two strains of guaiacol-producing bacteria 7-06 sediment B-1 and 7-06wall mud B-3 were screened.Molecular biology The identifications were Bacillus amyloliquefaciens and Delftia sp.X-a12,respectively named Y1,Y2,and the total yields were 0.3626 mg/100 mL and 1.4527 mg/100 mL,respectively,and then the tolerance analysis was performed.The results were Y1 and Y2 were resistant to 4%Vol alcohol,pH 4.5,15%sugar.Cell disruption of functional bacteria Y2 was carried out by ultrasonic disruption method,and the effects of ultrasonic output power（W）,each irradiation time（s）,total working time（min）,and bacterial mass concentration（g/mL）on cell disruption were studied.The vanilloid decarboxylase enzyme activity was used as a criterion.The single extraction factor and response surface test were used to obtain better extraction conditions:ultrasonic output power450 W,total treatment time 18 min,and bacterial mass concentration 0.12 g/mL.Under this condition,vanillic acid decarboxylase specific activity was 541.27 U/mL.The crude enzyme solution was subjected to（NH4）2SO4 fractionation precipitation.When the saturation of（NH₄）₂SO₄ reached 60%,the enzyme activity reached the maximum,being 633.34 U/mL.The crude enzyme activity of vanillic acid decarboxylase was studied.The optimal pH of the enzyme was 6.0.When the pH was 5.0⁷.0,the enzyme was relatively stable,indicating that the enzyme has certain acid resistance.The optimal reaction temperature of the enzyme The enzyme was relatively stable at 40℃and between 20 and 40℃.Co²⁺,Mn²⁺,Ba²⁺and Fe³⁺promoted the activity of the enzyme,and the relative enzyme activities were 137.85%,153.08%,123.29%and 131.02%,respectively.Mg²⁺,K⁺,Li⁺and Na⁺had no significant effect on the enzyme,while Ni²⁺and Cu²⁺had strong inhibitory effects on vanillic acid decarboxylase.The response surface test was used to study the preparation process of Y2 bran.Firstly,the single factor test was used to analyze the water,culture temperature,culture time and inoculum size,and then the response surface test was used to optimize according to the single factor test results.The results showed that the main order of factors affecting guaiacol production was culture temperature>inoculation amount>culture time>moisture content.The optimum conditions were 81.5%moisture content,50 h culture time,inoculum volume 11.0%,culture temperature.At 38℃,the guaiacol content was 3.893×10^-4 g/g dry koji.Simulating the solid-state fermentation system of Luzhou-flavor liquor,compared with the traditional medium-high temperature Daqu,the content of guaiacol in the liquor of the experimental group was increased by 45%compared with the blank group,which ensured the content of guaiacol in the ecological liquor.And its stability.