Application of molecular methods for the differentiation of acetic acid bacteria in a red wine fermentation

Aims: To apply rapid and reliable molecular techniques for typing acetic acid bacteria and studying their population dynamics during wine-making processes. Methods and Results: We tested the usefulness of the Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-PCR) and Repetitive Extragenic Palindromic-PCR (REP-PCR) techniques with reference strains of most of the species of acetic acid bacteria. We obtained exclusive patterns for each strain with the ERIC-PCR technique, proving the utility for characterizing below species level. REP-PCR technique was not as adequate for this purpose because some strains yielded identical fingerprint. One hundred twenty isolates from a commercial red wine fermentation were fingerprinted using both techniques. We detected a high degree of strain diversity in the first stage of fermentation that decreased throughout the process. However, several strains and species were dominant in the alcoholic fermentation phases. The identification of different strains or genotypes at the species level was carried out by restriction analysis of the 16S ribosomal DNA gene. Gluconobacter oxydans dominated the fresh must, while Acetobacter aceti was the only isolated species at the end of the process. Gluconacetobacter hansenii and G. liquefaciens were also isolated in significant numbers at the beginning of fermentation. Conclusions: ERIC-PCR and REP-PCR techniques proved useful for characterizing strains of acetic acid bacteria. Significance and Impact of the study: The availability of molecular techniques for a fast and reliable genotypic characterization should increase our knowledge of the ecology of acetic acid bacteria and determine more accurately their growth behaviour during various stages of vinification.