Microbiota associated to fermented table olive of the cultivar Carolea assessed by molecular methods

Natural fermentation is one of the most used methods to process the table olives; final product characteristics are determined by the specific microbiota of the olive cultivars. Aim of the work was to assess the microbiota of the cultivar Carolea olives that were naturally fermented using the following brines: 1) 8% NaCl (w/v), 2) 8% NaCl acidified to pH 4.30, 3) 5% NaCl for 20 days and then brought to 8% NaCl, 4) 5% NaCl for 20 days, then brought to 8% NaCl and acidified to pH 4.30. Lactic acid bacteria (LAB) and yeasts were analysed at the 240 days using both culture-dependent and culture-independent approach. LAB and yeasts were isolated, according to the colonies features, and stored at -80°C in the Microbial Collection of the Laboratory of Microbiology. The LAB identification was performed by multiplex PCR assay of the recA gene while the yeasts were identified by PCR-ITS and RFLP analysis of ITS-5.8S rRNA region. Total DNA was extracted from the brines and subjected to PCR-DGGE. LAB and yeasts representative strains and amplicons of bands excised from polyacrylamide gels were sequenced. The microbial load ranged from 5.18 to 5.41 Log CFU/ml and from 5.18 to 5.80 Log CFU/ml for LAB and yeasts, respectively. Lactobacillus plantarum was predominant in all the trials while among the yeasts Pichia kudriavzevii, Candida boidinii, and Wickerhamomyces anomalus were detected. The DGGE analysis confirmed the presence of L. plantarum for the LAB population in all the trials and of C. boidinii in the trials 1), 3), 4), and W. anomalus in the trial 2) for the yeasts.