Application of culture culture-independent molecular biology based methods to evaluate acetic acid bacteria diversity during vinegar processing

Abstract

Acetic acid bacteria (AAB) are considered fastidious microorganisms because they are difficult to isolate and cultivate. Different molecular approaches were taken to detect AAB diversity, independently of their capacity to grow in culture media. Those methods were tested in samples that originated during traditional vinegar production. Bacterial diversity was assessed by analysis of 16S rRNA gene, obtained by PCR amplifications of DNA extracted directly from the acetification container. Bacterial composition was analyzed by RFLP-PCR of 16S rRNA gene, Temporal Temperature Gradient Gel Electrophoresis (TTGE) separation of amplicons containing region V3–V5 of 16S rRNA gene and cloning of those amplicons. TTGE bands and clones were grouped based on their electrophoretic pattern similarity and sequenced to be compared with reference strains. The main microorganism identified in vinegar was Acetobacter pasteurianus, which at the end of the acetification process was considered to be the only microorganism present. The diversity was the highest at 2% acetic acid, where indefinite species of Gluconacetobacter xylinus/ europaeus/ intermedius were also present.