Unveiling the Microbial Community of Naturally Fermented Bella di Cerignola PDO Table Olives Integrating Culture-Dependent, MALDI-TOF MS Identification and Long-Read Metabarcoding Approaches

Spontaneous fermentation of table olives is driven by complex microbial communities that influence product quality and safety. This study investigated the microbial community during the intermediate and late stages (from 3 to 6 months) of spontaneous fermentation of Bella di Cerignola table olives in brine using an integrated culture-dependent and culture-independent approach. Bacterial and fungal communities were monitored using viable counts, isolation and identification by MALDI-TOF MS, and long-read metabarcoding targeting the 16S rRNA gene and ITS region. Lactic acid bacteria remained dominant throughout fermentation, whereas the yeast population progressively declined during the last period. MALDI-TOF MS identified Lactiplantibacillus pentosus as the predominant culturable bacterial species and Pichia membranifaciens, Pichia manshurica, and Wickerhamomyces anomalus as the dominant yeasts. Metabarcoding confirmed the ecological relevance of these yeasts and revealed a bacterial community dominated by the Secundilactobacillus collinoides/paracollinoides group, which was not recovered by cultivation, and the Lactiplantibacillus plantarum group. The integration of MALDI-TOF MS and long-read metabarcoding analysis provided a comprehensive overview of the microbial community of Bella di Cerignola olives, demonstrating the complementary value of the two approaches for the microbiota characterisation of spontaneous olive fermentations and for the identification of autochthonous candidate strains to further investigate as starter cultures.