Preserving fermented-foods microbial diversity through systematic culturomics for the discovery of multi-strain probiotic candidates

Fermented foods (FFs) represent complex living ecosystems that deliver viable microbes and bioactive metabolites linked to human health benefits. However, many probiotic strains isolated from FFs fail to reproduce these effects in vivo , likely due to the disruption of their natural ecological synergy during isolation. Here, we employed a systematic, ecology-aware culturomics framework to transform the Kimchi microbiome into genome-vetted, multi-species probiotic candidates while preserving ecological fidelity. Specifically, 56 distinct enrichment culture conditions were established using six liquid media (In-situ, MRS, NB, TSB, BHI, BB) across varied redox states (aerobic, anaerobic, microaerophilic), incubation periods (12 h, 66 h), and selective suppressants (CHIR-090, nalidixic acid). Results indicated that In-situ and MRS media under microaerophilic conditions effectively preserved the lactobacilli core, whereas generalist media and aeration expanded taxonomic breadth to include rare taxa. Furthermore, extended incubation (66h) successfully unlocked 107 unique taxa compared to the limited diversity of short incubation (12h). Shotgun metagenomic mining further revealed promising functional properties, including acid tolerance, adhesion modules, and diverse bacteriocin-skewed biosynthetic gene clusters. Crucially, the collection exhibited a strong safety profile: only 1% of identified risk factors were antibiotic resistance genes (ARGs) on mobile genetic elements (MGEs), and only 4% represented colocalized ARGs, virulence factors, and MGEs. Systematic-culturomic isolation later yielded over 90 strains, including Weissella , Bacillus , and Lactococcus , significantly expanding beyond standard lactobacilli-centric portfolios. Overall, this study confirms that ecology-aware culturomics captures the functional diversity of the Kimchi microbiome, providing a scalable model for realizing the full therapeutic potential of FFs. • Ecology-guided culturomics can transform kimchi microbiomes into NGPs. • In situ media preserve kimchi consortia, retaining intra-/inter-species diversity. • Genome-vetted MAGs reveal glycan use, EPS/adhesion, and bacteriocin BGCs. • Low mobile ARG/VF burden nominates safer Leuconostoc – Weissella – Lactococcus. • Generalist media and extended incubation time unlock taxa beyond core-LAB.