Cryopreservation of fermented table olives microbiomes: an integrative case study on viability, functional stability, and biobanking applications

Microorganisms and their assembly in composite microbiomes play a crucial role in food fermentation, significantly influencing product quality, safety, and nutritional value. Preserving these microbiomes in culture collections is essential for maintaining biodiversity and supporting bio-based innovation in sustainable food systems. However, the long-term conservation of complex microbiomes remains challenging due to microbial heterogeneity and sensitivity to preservation methods. This study evaluated the effectiveness of two cryopreservation protocols for the long-term storage of microbiomes from naturally fermented table olives (cv. Leccino), a traditional Mediterranean diet staple food. Glycerol and dimethyl sulfoxide (DMSO), both at 15% (v/v), were used as cryoprotectants and microbiome samples were stored at -140 °C. Microbial viability, taxonomic composition, and metabolic functionality were assessed over one year using culture-dependent techniques, RNA-based metabarcoding, and Biolog® EcoPlate profiling. Moreover, the oleuropein degradation was evaluated in microbiome samples recovered after one year of cryopreservation. Results indicated that the viability of the microbial consortium slightly decreased, regardless of the cryoprotectant used, and no significant changes in the metabolic profile were observed. In addition, the metabarcoding analysis revealed no significant differences in relative abundances after the storage period. The study confirmed the effectiveness of the cryopreservation method, proper maintenance of the microbial consortium viability and functionality after long-term storage. These findings support the feasibility of microbiome biobanking for fermented foods and highlight the importance of tailored preservation strategies to ensure the stability and functionality of microbial consortia. This approach contributes to the conservation of microbial resources, reproducibility in microbiome science, and the development of high-quality standardisation in fermented products.