Shotgun metagenomics of Castelvetrano olives

Table olives processed in the Castelvetrano style are rapidly debittered with sodium hydroxide, then treated with sea salt and stored in cold rooms to preserve sensory properties and the proliferation of pathogenic microorganisms. Before commercialisation, olives undergo multiple rinses to remove residual soda, immersion in acidified brine, and, in some cases, pasteurisation, followed by packaging. At the industrial level, even when stored at low temperatures, olives sometimes spoil and become unsuitable for sale. To identify the microbial communities involved in the spoilage of table olives during storage prior to commercialisation, a shotgun metagenomic investigation of both suitable and unsuitable olives was conducted. DNA extracted from olives and brine was quantified and submitted for library preparation following Illumina DNA Prep instructions. The prepared libraries were sequenced using P3 reagents (2 × 150 cycles) on the Illumina Nextseq 2000 platform. Raw sequencing data were analysed using open-access bioinformatics tools, including quality control (Falco, version 1.2.4), trimming (FastP, version 1.0.1), depletion of olive DNA (Bowtie 2, version 2.5.3), taxonomic classification (Kraken 2, version 2.1.3), and core-metagenome visualisation (Krona pie chart, version 2.7.1). Alpha- and beta- diversity were calculated using Shannon and Bray-Curtis indices, respectively. The main results demonstrated that, on an industrial scale, in preserved olives suitable for further processing according to the Castelvetrano style, Leuconostoc mesenteroides, Loigolactobacillus coryniformis, and Paucilactobacillus nenjiangensis are the most represented species. In olives, these microorganisms primarily participate in oleuropein hydrolysis and secondary debittering. In particular, L. coryniformis and P. nenjiangensis help reduce softening by regulating pectinolytic activity and contribute to the sensory profile. Conversely, in olives unsuitable for Castelvetrano-style processing, the most abundant species is Pichia membranifaciens, followed by Kluyveromyces lactis and Debaryomyces hansenii. P. membranifaciens is a yeast with high osmotic tolerance, which gives it a competitive advantage over lactic acid bacteria. It can produce compounds that contribute to off-flavours and biotin, which can promote the growth of undesirable and sometimes pathogenic microorganisms.