Exploring Microbial Communities of Spanish-style Green Table Olives of Conservolea and Halkidiki Cultivars During Modified Atmosphere Packaging in Multi-Layered Pouches Through Culture-Dependent Techniques and Metataxonomic Analysis

The aim of this study was to investigate the microbiological and physicochemical changes of pitted green olives (cvs. Conservolea and Halkidiki) elaborated in the Spanish style method and packaged in two types of multilayered pouches under nitrogen atmosphere (100% N2) for a period of 12 months. Moreover, the evolution of microbial consortia at the beginning (0 days), middle (180 days) and final (360 days) time points of storage was elucidated by plating, genotyped and identified through RAPD-PCR and ITS region amplicon sequencing, respectively, and subsequently metataxonomic analysis (for fungal communities only). Results showed that no enterobacteria could be detected on olive drupes, whereas the dominant microbiota from the onset of storage in both pouches consisted of LAB in populations ranging between ca. 4.2-6.6 log CFU/g. Although yeasts were initially enumerated at ca. 5.5 log CFU/g, they declined rapidly and could not be detected by plate counting after 30 days. The pH values increased from 4.11 to 4.24 and 4.03 to 4.12 at the beginning and end of storage for cvs. Halkidiki and Conservolea green olives, respectively. The total color difference index (Delta E*) presented a perceivable change in visual color (Delta E* > 2.0) only in cv. Conservolea olives during storage. Finally, molecular fingerprinting RAPD-PCR and 16S amplicon based identification revealed the dominance of five LAB species, namely Pediococcus ethanolidurans, Lactiplantibacillus pentosus, Lentilactobacillus rapi, Lentilactobacillus parafarraginis and Lentilactobacillus buchneri. Regarding the identification of yeasts, the metagenetic amplicon sequencing approach revealed fungal complexity in the olive samples. Pichia manshurica and Pichia membranifaciens prevailed during the first and middle stages of storage, whereas at the final stage higher complexity was noticeable.