Fluorescence in situ hybridisation detection of Lactobacillus plantarum group on olives to be used in natural fermentations

Abstract

At present there are very few studies on the bacterial diversity of olives and on the importance of the microbial species for the fermentation of olives aimed to table olives production. Most of the authors report on the occurrence of Lactobacillus plantarum as principal member of these communities or at least as the species responsible for the fermentation. In this study, fluorescence in situ hybridisation (FISH) with 16S rRNA probes was used to evaluate the occurrence of L. plantarum in olives. A 18-bp oligonucleotide probe was used in FISH experiments to evaluate the specificity of detection among Lactobacillus species. The probe was tested against 30 Lactobacillus species and appeared to be specific for L. plantarum, L. paraplantarum and L. pentosus. The probe was then used to investigate the occurrence of these species in 25 samples of olives (cultivar “Leccino”) collected in Campania region (Southern Italy). The olives were washed in a saline solution and the suspensions were then analysed by FISH and observed by fluorescence microscopy. No hybridisation signal was detected in at least 30 fields of observation when the L. plantarum-specific probe was used, probably due to the low sensitivity of the FISH method. Olive samples were plated on Rogosa agar and about 40% of the samples did not give growth after 5 days. When colony growth was observed, bulk cells from Rogosa agar plates were collected and analysed by DNA extraction followed by 16S rDNA Polymerase chain reaction–denaturing gradient gel electrophoresis (PCR–DGGE). The different microbial species were identified by direct sequencing of DGGE bands. Leuconostoc pseudomesenteroides was the most frequently found species, occurring in more than 50% of the samples that had shown growth on Rogosa agar. The closest relatives of the species of the genera: Leuconostoc, Pediococcus, Pseudomonas and Raoultella were also identified suggesting that guided fermentation by using selected LAB starters is advisable for a safe and desired table olives production.