Taxonomic and functional analysis of type I sourdough microbiota after long-term cryopreservation and subsequent reactivation.

Type I sourdough microbiota may be subjected to taxonomic and functional shifts during traditional maintenance by continuous backslopping. This proof-of-concept study evaluated a standardized workflow for long-term cryopreservation (-80 °C for 12 months) and recovery of three artisanal sourdough microbiota differing for their starting microbial compositions (Pediococcus/Fructilactobacillus and Saccharomyces/Kazachstania as dominant taxa). Microbiota stability was assessed through culture-dependent analyses on both purified microbiota pellets and whole sourdough, and through culture-independent (16S/ITS2 metabarcoding) and community-level physiological profiling (OmniLog©, Biolog) on purified microbiota. The storage workflow preserved the main taxonomic and metabolic fingerprints, while viable counts showed sourdough-dependent changes and were mainly affected during the reactivation step. Additionally, post-thaw reactivation resulted in a major taxonomic and functional perturbation, characterized by restructuring of both yeast (increase in Wickerhamomyces and reduction in Saccharomyces) and LAB communities (increased Lacticaseibacillus and Lactiplantibacillus; decreased Pediococcus and Levilactobacillus), with concurrent reduction in carbohydrate utilization and nitrogen-related compound metabolism. Five serial backslopping cycles partially redirected the communities toward sourdough-like taxonomic and metabolic configurations. Although complete taxonomic-metabolic reconstitution was not achieved, essential sourdough functions (acidification, sugar fermentation, and selected amino acid utilization patterns) were restored. These findings support cryopreservation as a viable strategy for microbiota biobanking in Microbial Biological Resource Centers and demonstrate that integrated taxonomic-metabolic profiling provides robust criteria for defining optimal backslopping procedures to optimize sourdough reconstitution performance.