Cryopreservation of fermented olives microbiomes: Viability, functional and taxonomic stability after long-term storage

Microbial communities are essential to the functioning of ecosystems, playing a central role in biological processes across the planet. In food ecosystems, the microbiomes of fermented products not only enhance food safety and shelf life but also contribute to human health and well-being. Within this framework, Microbial Culture Collections serve as vital infrastructures also for ensuring food safety and quality, as well as fostering the development of bio-based innovations. As the demand for sustainable food systems grows, both industry and researchers are increasingly focused on harnessing the potential of complex microbial consortia in fermentation and developing nature-inspired solutions. However, a significant challenge remains: the absence of standardised, effective methods for the long-term preservation of these microbiomes, along with reliable assessments of their post-preservation viability and functionality. Recent investigations conducted under the EU-funded SUS-MIRRI.IT project (N° IR0000005) have advanced our understanding of microbiome preservation, particularly in fermented food systems. This study presents novel findings on the cryopreservation of microbiomes derived from table olives and their brine. The protocols tested involved storage at ultra-low temperatures using a -150°C freezer, combined with the use of glycerol and dimethyl sulfoxide (DMSO) as cryoprotective agents. The microbial consortia were analysed before and after a one-year storage period by using culture-based techniques and RNA-based metabarcoding, as well as metabolic profiling by Biolog Ecoplate assays. Moreover, the oleuropein degradation ability was evaluated in microbiome samples recovered and propagated after 1 year of cryopreservation. Results indicated a slight reduction in microbial viability after one year, independent of the cryoprotectant used. Metabolic activity and functional performance, specifically the ability to degrade oleuropein, remained stable. Furthermore, metabarcoding analysis revealed no significant alterations in the composition of the microbial community. This integrated methodological approach offers a valuable model for preserving microbiomes from other fermented foods, reinforcing the crucial role of culture collections in maintaining viable and functionally robust microbial resources for future applications.