Isolation and cultivation of acetic acid bacteria from high selective sources

Cultivable acetic acid bacteria (AAB) are used as oxidising bacteria in several biotechnological applications (e.g vitamin C, dihydroxyacetone, gluconate, ketogluconates synthesis) as well as in fermentative processes (vinegar). Troubles about their isolation and cultivation in laboratory media has been well reported. The viable but not cultivable state seems mainly influenced by environmental stress conditions and current isolation methods allow to access only a small subset of AAB population. Also the handling and preservation of strains isolated from high selective sources is still a hard practice. Among vinegars, traditional balsamic vinegar is made through cooking the grape must, in which spontaneous alcoholic fermentation and acetic oxidation take place, then it is aged to be bottled. Cooked grape must is characterized by very low pH and high sugars content. In previous works we observed high selective pressure of cooked grape must on AAB growth and the loss of strains viability under laboratory conditions. Moreover culture independent studies by PCR/DGGE, highlighted that only easier cultivable strains are isolated from cooked grape must by conventional culture-dependent methods. In this study AAB strains were isolated from cooked grape musts by plating system using three isolation media, molecular characterized by PCR/RFLP analysis and partial sequencing of rDNA region. To test the ability to carry out the oxidation process in high selective substrates, cooked grape must, wine and “Passmore and Carr” medium, with different pH, titrable acidity, soluble solids and ethanol content, were used as substrates. The results showed the high soluble solids parameter as the main limiting factor of AAB growth and activity in cooked grape must. Improvement of isolation and cultivation methods could provide recognition of new species, a better preservation system of strains, allowing an AAB strains selection of biotechnological interest.