Acetic acid bacteria from biofilm of strawberry vinegar visualized by microscopy and detected by complementing culture-dependent and culture-independent techniques

Maria José Valera, Maria Jesús Torija, Albert Mas, Estibaliz Mateo

Biotecnologia Enológica, Dept. Bioquímica i Biotecnologia, Facultat d'Enologia, Universitat Rovira i Virgili, C/Marcellí Domingo s/n, 43007 Tarragona, Spain.

Abstract

Acetic acid bacteria (AAB) usually develop biofilm on the air–liquid interface of the vinegar elaborated by traditional method. This is the first study in which the AAB microbiota present in a biofilm of vinegar obtained by traditional method was detected by pyrosequencing. Direct genomic DNA extraction from biofilm was set up to obtain suitable quality of DNA to apply in culture-independent molecular techniques. The set of primers and TaqMan – MGB probe designed in this study to enumerate the total AAB population by Real Time – PCR detected between 8 × 10⁵ and 1.2 × 10⁶ cells/g in the biofilm. Pyrosequencing approach reached up to 10 AAB genera identification. The combination of culture-dependent and culture-independent molecular techniques provided a broader view of AAB microbiota from the strawberry biofilm, which was dominated by Ameyamaea, Gluconacetobacter, and Komagataeibacter genera. Culture-dependent techniques allowed isolating only one genotype, which was assigned into the Ameyamaea genus and which required more analysis for a correct species identification. Furthermore, biofilm visualization by laser confocal microscope and scanning electronic microscope showed different dispositions and cell morphologies in the strawberry vinegar biofilm compared with a grape vinegar biofilm.

Keywords: Wine; Vinegar; Next-generation sequencing; PCR-DGGE; 16S rRNA gene.