Multi-omic approach for assessing microbial viability, taxonomic composition, and metabolic activity after freeze-drying of fermented olives microbiomes

Background: The development of food microbiome biobanks within research infrastructures aims to preserve viable, well-characterized microbial consortia for both scientific and industrial applications. Preservation strategies support positioning culture collections as key infrastructure for enabling safe, resilient, and sustainable fermentations. In this study, we evaluated an integrated workflow that combines culture-dependent and culture-independent approaches to assess the microbial viability, taxonomic composition, and metabolic activity of the microbiome in fermented table olive cv. Bella di Cerignola during freeze-drying. Methods: An integrated multi-omic workflow combining viability assays, MALDI-TOF-MS identification, metabarcoding analysis, and phenomic profiling was applied before and after the freeze-drying procedure. Microbial viability was assessed by plate counting on selective culture media. The taxonomic profile was evaluated by a metabarcoding approach, and representative colonies were randomly selected for identification based on MALDI-TOF-MS analyses. Functional diversity and metabolic potential were assessed using the Omnilog EcoPlate™ system. Results and Discussion: Culturomics enabled quantification of viable populations after freeze-drying and reliable taxonomic identification. Culture-dependent results were consistent with metabarcoding data and MALDI-TOF-MS identification, confirming workflow robustness. Functional profiling indicated that the microbial consortium retained substantial metabolic versatility after freeze-drying, supporting preservation of community functionality. Conclusions: The integrated multi-omic workflow provides a high-resolution framework for assessing microbiome preservation. These findings also support the development of standardized freeze-drying protocols for preserving microbial consortia, alongside cryopreservation methods. We acknowledge the contribution and support from the Italian national Node (MIRRI-IT) of the European Research Infrastructure MIRRI-ERIC.