Isolation And Characterization Of High Acetic Acid-yielding Acetic Acid Bacteria With Tolerance And Its Application In Plum Vinegar

At present,the fruit vinegar industry in China is mainly based on blending beverage vinegar,and the fruit vinegar produced by brewing method with broad market prospect abroad is less in China.The core of fruit vinegar industry is strain breeding and process optimization.The purpose of this study is to screen high acetic acid-yielding acetic acid bacteria with thermotolerant and ethanol-resistance,to explore their fermentation and enzymatic properties at high temperature and high ethanol,and to apply them to ferment plum vinegar at high temperature and high ethanol.To provide a reference for the applying of this glorious acetate strain in soured fruit vinegar business underneath non-strict management conditions.The main research contents and conclusions are as follows:1)Isolation,characterization and preliminary study on tolerance of high ethanoic acid-yielding acid acid microorganism.Various types of fruit samples(plum,peach,mango,banana,loquat)were used for the study,and 40 strains of acetic acid-producing bacteria were screened by enrichment culture,calcium plate,and acetic acid qualitative test.The acid production of only 9 acetic acid bacteria was higher than that of AS1.41,compared with the acid production of 40 and Acetobacter pasteurianus AS1.41 strains.The results showed that LO2 not only produced more acid than AS1.41,but also grew well at 37?or 11%(v/v)ethanol(culture medium),with its OD₆₀₀ up to 0.56 and 0.61,respectively.The LO2 strains were morphologically and 16S r DNA molecular and finally identified as Acetobacter sp.LO2(nos.MN602472).2)Effect of initial ethanol concentration and temperature on the growth of 9 high-yielding acetic acid bacteria.At 30?,when ethanol concentration exceeds 13%,only LO2and AS1.41 grow.At 30?,when ethanol concentration was 15%,only LO2 grew.At 37?,when ethanol concentration exceeds 11%,only LO2 and AS1.41 grow.At 37?,when ethanol concentration exceeds 13%,only LO2 grew.At 40?,only LO2 strain could grow under 13%ethanol concentration.Therefore,the LO2 has good ethanol and temperature tolerance.3)Effects of initial ethanol concentration and temperature on industrial strains and the best resistant strains.The acid production and growth of LO2 and AS1.41 tended to be stable after 5 days,and LO2 showed the best growth and acetic acid production(about 41.85 g/L)at 30?and 5%ethanol concentration.Within the initial ethanol concentration of 3%?10%,with the increase of ethanol concentration,the acid production and growth of LO2 and AS1.41 strains increased,and gradually decreased after exceeding 5%ethanol concentration.In the fermentation temperature of 30??40?,with the increase of fermentation temperature,the delay period increased,and the acid production and growth of strain AS1.41gradually decreased to no growth,while the acid production and growth of strain LO2decreased slowly.Under the combined conditions of 30??40?fermentation temperature and 3%?10%initial ethanol concentration,the growth and acid production of LO2 strain were better than AS1.41,and it could tolerate the combination of 37?and 10%ethanol or40?and 8%ethanol.4)Fermentation temperature and initial ethanol concentration influenced the key acid-producing enzymes of Acetobacter.And the study of temperature and alcohol tolerance of acid-producing key enzymes.With the increase of fermentation temperature,the acetate dehydrogenase(ADH)and aldehyde dehydrogenase(ALDH)of LO2 and AS1.41 strains reached the peak at 35?and 30?,respectively.The enzyme activity of ADH was on top of that of ALDH,and therefore the enzyme activity of LO2 strain was on top of that of AS1.41.With the increase of initial ethanol concentration,the ADH and ALDH activities of LO2 and AS1.41 strains reached the peak in 5%ethanol and 3%ethanol,respectively.ADH enzyme activity was higher than ALDH enzyme activity,and the enzyme activity of LO2strain was higher than as1.41.The ADH and ALDH of LO2 strain had the best temperature tolerance of 37?and alcohol tolerance of 10%,while that of AS1.41 strain was 30?and5%ethanol,respectively.5)Monitoring the fermentation process of plum vinegar by 4)LO2 strain and AS1.41strain under high temperature and high ethanol.The growth and acid production of the two strains were similar under suitable conditions.The maximum acid production of plum vinegar produced by LO2 strain(53.88 g/L)was higher than that of AS1.41 strain(51.64g/L).Under the condition of high ethanol(8%ethanol),the lag period of acetic acid bacteria was prolonged,and the maximum acid yield of plum vinegar produced by LO2 strain(59.80g/L)was higher than that of AS1.41(47.78 g/L);/L).At high temperature(37?),the acid yield of plum vinegar produced by LO2 strain(46.64 g/L)is 1.79 times that of AS1.41(25.92g/L).Under the conditions of high temperature and high ethanol,AS1.41 hardly grows,and LO2 strain still keeps fermenting.6)Quality analysis of plum vinegar fermented by 5)LO2 strain and AS1.41 strain under high temperature and high ethanol.Under three fermentation conditions[high temperature(37?,5%ethanol),high ethanol(30?,8%ethanol)and high temperature and high ethanol(37?,8%ethanol),the organic acid content of plum vinegar fermented by strain LO2(AS1.41)decreased by 8.37%(1.37%)Under stress condition,compared with AS1.41,the number of volatile components in plum vinegar fermented by LO2 strain is more(2?10).The most important contributors to the overall flavor of plum vinegar are esters and alcohols,and their relative content is higher(1.47%?14.16%).The characteristic volatile flavor components are more abundant,and the highest ROAV value of plum vinegar is isoamyl acetate Meanwhile,principal component analysis can effectively distinguish 8 kinds of plum vinegar.The sensory evaluation of plum vinegar fermented by LO2 strain is better than that of AS1.41 strain,and it has unique plum color,strong plum fruit aroma and vinegar aroma,and has typical characteristics of fermented vinegar.