Preliminary results for application of culture-independent molecular methods to olive brines bacterial communities.

Transformation of green table olives requires a brining stage during which olives ferment. In naturally transformed olives, both flavour improvement and debittering are associated to fermentation. Because of brine features, naturally occurring bacteria and yeasts are progressively selected by the high salt concentration, low pH and phenolic compounds with antimicrobial activity. The establishment of an adequate fermenting microflora is essential to obtain a good and safe product; then, during transformation, control of chemical and microbiological parameters is crucial. Currently, microflora is periodically monitored by plate-counting of main groups. These routine analyses, however, don’t show neither the complexity of population nor possible differences between various samples, because the most used cultural media are often not selective enough, and fail to differentiate single species. We carried out preliminary trials to evaluate the possibility of application of molecular analyses to describe microflora dynamic and complexity in fermenting green olive brines. We used culture-independent methods, based on analysis of total DNA directly extracted from the whole microbial and blastomycetic community in naturally fermented olive brines. Olives (cv. “Nocellara del Belice”) were transformed with both traditional (NaCl only) and modified brines (with low NaCl content, where the total ionic strength was ensure by KCl and/or CaCl2, to obtain “hyposodic” olives ) which allow regular fermentation. In particular, we carried out analyses on PCR amplified fragments of polymorphic rDNA regions with length or sequence variability among different species, fractionating them by denaturing gel electrophoresis (RISA, Ribosomal Intergenic Sequences Analysis) and by automated capillary electrophoresis (ARISA, Automated-RISA), or by DGGE (Denaturing Gradient Gel Electrophoresis) when analyzing variable regions (but with similar length) of 16S rDNA. Resulting specific profiles can be compared between different samples, allowing to show the dynamic of microbial populations. By DNA cloning and sequencing, we were able to identify dominant species, assigning them to specific bands. Although these are preliminary data, we think these techniques might be rapid and useful tools, complementary to traditional microbiological analyses, to study brine microflora and to follow simultaneously, comparing patterns, the proper growth of a number of species in complex populations (with known profiles) used as natural starters, when more specific markers are not available.