Analysis of acetic acid bacteria by different culture-independent techniques in a controlled superficial acetification
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Three molecular techniques [denaturing gradient gel electrophoresis (DGGE-PCR), restriction fragment length polymorphism of the 16S rRNA gene amplicon (RFLP-PCR 16S rRNA) and real-time PCR (RT-PCR) with SybrGreen and with specific TaqMan-Minor Groove Binder (MGB) probes] were used to identify and monitor acetic acid bacteria (AAB) species during a controlled acetification. This process was initiated by seeding a starter culture comprising a mixture of one strain each of Acetobacter pasteurianus, Gluconacetobacter europaeus and Gluconacetobacter hansenii. Analysis at the species level indicated that the population of A. pasteurianus increased quickly, subsequently acquiring a dominant position, whereas the other two species gradually disappeared. All three methods confirmed this result. When the total AAB population was estimated, the results obtained based on summing the three species by TaqMan-MGB RT-PCR, total AAB RT-PCR and the direct microscopic count method were similar. Using TaqMan-MGB RT-PCR we were able to detect species with populations 3 log units lower than that of the major species and which could not be detected by other methods.
KeywordsReal time-PCR DGGE-PCR Surface culture Traditional vinegar Wine
The authors are grateful for financial assistance from the Spanish Government (Project AGL2007-66417–C02-02) and the European Commission (Project WINEGAR, Cooperative Research under the Sixth Framework Programme of the European Community, 2005-2007). We thank the Language Service of the Rovira i Virgili University for revising the manuscript.
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