Vairation Of The Dominant Microbes During Acetic Acid Fermentation Process Of Zhenjiang Aromatic Vinegar Through Molecule Ecology Techniques

Vinegar is well received by Chinese people as an acidulous condiment, and Zhenjiangaromatic vinegar （ZAV） is one of the most famous Chinese traditional vinegars. It is producedthrough traditional solid-state fermentation and unique cycle-inoculation style, and featuringfull flavor and soft sour with the metabolism of various kinds of microorganisms. Thefermentation of ZAV is composed of alcoholic fermentation and acetic acid fermentation.Alcohol is produced through the metabolism of yeast and mould with sticky rice as the mainraw material during the alcoholic fermentation, and alcohol is converted to acetic acid byvarious kinds of bacteria and fungi during the acetic acid fermentation, which is thecommitted step to form the vinegar flavor. In this study, several molecule ecology techniqueswere applied to investigate the dynamic variation of some high abundance microorganismsduring the solid-state acetic acid fermentation process of ZAV, and the results had establishedresearch foundation for the mechanism analysis and fermentation process control of vinegarbrewing.Denaturing Gradient Gel Electrophoresis （DGGE） and Multiplex PCR （MPCR） wereapplied to investigate the dynamics of some high abundance bacteria during the acetic acidfermentation process of ZAV. Firstly, the dominant bacteria during the acetic acidfermentation process of ZAV were initially identified through PCR-DGGE, and a pure-culturemethod was applied to isolate the bacteria in the vinegar fermentation culture. MPCRapproach was established to investigate the variation of the dominant microorganismsincluding Acetobacter pasteurianus, Lactobacillus helveticus, Lactobacillus crispatus andBacillus cereus during the acetic acid fermentation process of ZAV according to the results of16S rDNA sequencing and DGGE analysis.Species-specific primer sets for Real-time quantitative PCR （RT-qPCR） were applied toresearch on the biomass variation of some high abundance microorganism includingAcetobacter, Lactobacillus, Saccharomyces and A. pasteurianus, L. helveticus, L. crispatus, B.cereus during the acetic acid fermentation process of ZAV. Results showed that the biomass oftotal bacteria varied similarly with a tendency of increasing in the first a few days followed bya gradually decent with the fermentation performed, while Acetobacter and Lactobacillusreached the maximum biomass of1.14×10¹⁰and3.37×10¹¹copies/g dry grains on the7th and4th day respectively, and the maximum biomass of A. pasteurianus, L. helveticus and L.crispatus were1.01×10¹⁰,1.95×10¹¹and2.74×10⁹copies/g dry grains which was reached onthe7th,4th and5th day repetitively. The biomass of B. cereus decreased rapidly to zero onday6-9while it increased slowly in the first five days. As to Saccharomyces, the variationtrend was different from bacteria, which changed in the initial stage of fermentation, whilebiomass of Saccharomyces decreased rapidly to0on day8-12.