| Acetic acid bacteria is the core microorganisms of vinegar industry, the aim of this study was to screening a group of acetic acid bacteria(AAB) which could resistant to high ethanol, high temperature and high acetic acid, exploring the fermentation characteristics and using it for industrial production. From the aspects of enzymology preliminary reveal the ethanol tolerance mechanism of AAB. Using the excellent AAB to produce watermelon vinegar. The main contents and conclusions of the research are as follows:1) Using the traditional plate screening to screen a batch of ethanol-tolerant AAB from vinegar Pei, further screening out resistance AAB that resistant to high temperature and AAB. Final seven strains were screened out and they could resistant 10 % ethanol, 40 °C temperature and 3 % acetic acid, after physiological and biochemical experiments and 16 s rDNA identification, seven strains were identified as Acetobacter Pasteurianus.2) The culture conditions of AAB was optimized, the optimal fermentation conditions was: 30 °C, 180 r/min, 6 d, pH was 6.0. Comparing to the ethanol, temperature and acid resistance of seven AAB, it can be found that FY-24 could resistant 12 % ethanol, 43 °C temperature and 1.2 % NaCl, and could produce 61.2 g/L acetic acid. Under the condition of compound compared with the fermentation characteristics of AS1.41 and FY-24 in the fermentor, the results shown that under the condition of high temperature and high ethanol at the same time, FY-24 remained high produce acid amount, while the industrial strains AS1.41 almost lost the ability to produce acid. Thus, FY-24 has a potential application for the industrial fermentation.3) The correlation between ethanol resistance of AAB and the activities of ADH and ALDH was studied, the results shown that under the high concentration of ethanol, the activity of ALDH was higher than that of ADH, so the ADH was the restrictive enzyme in high ethanol fermentation. At the same time, the higher ethanol tolerance of AAB and the enzyme activity of ADH and ALDH were significantly positive correlation: under the condition of high ethanol, the higher of the enzyme activity, the bacteria could convert the more ethanol, the growth of AAB was better and more resistant to ethanol. The effect of high temperature and acetic acid on the two enzymes were studied, the results shown that FY-24 and DY-5 could resistant high temperature because of the high activities of their ADH and ALDH; the presence of acetic acid has strong inhibitory effect on ADH and ALDH enzyme activities. The ethanol-tolerant AAB FY-24 and DY-5 could resistant high temperature and acetic acid, these two properties contributed to improve the ethanol tolerance.4) The effect of ethanol induced ADH was studied, it could be found that 8 % ethanol induced the expression of ADH from ethanol-tolerant AAB FY-24 and DY-5, but inhibited the expression of ADH in AS1.41. The purified ADH from FY-24, DY-5 and AS1.41 were carried out polyacrylamide gel electrophoresis, the results shown that their ADH have three subunits, the mass of molecule were 79 kDa(subunit I), 57 kDa(subunit II) and 15 kDa(subunit III). The ethanol, temperature and acetic acid stability of ADH were also studied, the ADH from FY-24 and DY-5 exhibited higher ethanol, temperature and acetic acid stability than that of AS1.41. Thus, we further study the amino acid of ADH, the results shown that the amino acid of the ADH from FY-24, DY-5 and AS1.41 were only a few difference, which may eventually led to the difference of ADH performance.5) By comparing the growth and acid production characteristics of FY-24 and AS1.41 in the process of high temperature watermelon vinegar fermentation, then exploring the feasibility of high temperature fermentation watermelon vinegar. The results shown that FY-24 could grow well and produce much acetic acid at 30-39 °C in the process of watermelon vinegar fermentation, the strain could be used as a brew watermelon vinegar dedicated strains. In order to improve the flavor of watermelon vinegar, brewing watermelon vinegar with high ethanol(8 %), high performance liquid and GC-MS were used to detect the main organic acids and the flavor of watermelon vinegar. Results shown that using the ethanol-tolerant AAB FY-24 to brew watermelon vinegar under the concentration of high ethanol can not only improve the main organic acids(oxalic acid, tartaric acid, formic acid, malic acid, succinic acid) content but also improved the flavor substance especially ester type and content, which has a great potential application in industry.Providing valuable reference for screening AAB, broaden the scope of screening strains. Based on enzymology characteristics of AAB, rovide some theoretical basis for practical production. Finding out the dedicated strains for watermelon vinegar fermentation. |
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