Combined multi-omics approaches reveal the biopreservative potential of lactic acid bacteria against fungal spoilage in dairy products

While fungal spoilage of dairy products remains a significant economic and safety concern, the inherent deficiencies of synthetic chemical additives and growing consumer demand for 'clean label' products have intensified the search for natural biological preservatives. This study employed an integrated multi-omics pipeline combining high-throughput phenotypic screening, LC-MS/MS metabolomics, and whole-genome sequencing (WGS) to identify and mechanistically characterize lactic acid bacteria (LAB) with potent antifungal activity. A total of 84 LAB strains belonging to 17 species, including Lactiplantibacillus plantarum , Enterococcus spp., Lactococcus spp., Leuconostoc spp., and Streptococcus spp., were evaluated against five dairy spoilage fungi ( Aspergillus flavus , Alternaria alternata , Aspergillus niger , Fusarium oxysporum , and Penicillium verrucosum ) via dual-culture and overlay assays, with inhibition rates ranging from 3.0 to 85.0%. E. faecalis T38-3, E. faecalis Tn142-7, and L. plantarum Tn160-3 the three strains with the strongest antifungal activity were selected for in-depth characterization through co-inoculation and cell-free supernatant (CFS) biomass inhibition assays, stability testing (heat treatment, pH neutralization, proteinase K), LC-MS/MS metabolite profiling, and WGS-based genomic analysis. The three selected strains achieved 53–90% biomass inhibition and near-complete suppression of A. flavus , A. alternata , and A. niger in co-culture while maintaining viable counts above 6.0 Log CFU/mL. Stability assays indicated a synergistic mechanism involving pH-dependent organic acids and heat-stable proteinaceous peptides. LC-MS/MS profiling revealed strain-specific organic acid profiles: all three strains produced lactic, acetic, malic, and succinic acids; T38-3 additionally produced propionic, citric, and oxalic acids; while Tn142-7 and Tn160-3 uniquely produced formic acid. Genomic analysis confirmed species identities (ANI >98%, dDDH >89%) and revealed strain-specific biosynthetic gene clusters (BGC): a Type III polyketide synthase (T3PKS) cluster in Tn160-3, an Enterocin AS-48 bacteriocin cluster in T38-3, and sactipeptide clusters in Tn142-7, alongside acid biosynthetic genes ( ldh , ackA/pta , cit operon). Safety screening confirmed Tn160-3 lacked acquired antibiotic resistance genes (ARG), while T38-3 and Tn142-7 carried only intrinsic chromosomal determinants with no critically important resistance genes detected. This integrated multi-omics approach establishes a validated framework for discovering safe and effective LAB-based biopreservatives for sustainable dairy applications.