| Summary: | Phenyllactic acid (PLA), a promising food preservative, is safe and effective against a broad spectrum of food-borne pathogens. However, its mechanisms against toxigenic fungi are still poorly understood. In this study, we applied physicochemical, morphological, metabolomics, and transcriptomics analyses to investigate the activity and mechanism of PLA inhibition of a typical food-contaminating mold, <i>Aspergillus flavus</i>. The results showed that PLA effectively inhibited the growth of <i>A. flavus</i> spores and reduced aflatoxin B<sub>1</sub> (AFB<sub>1</sub>) production by downregulating key genes associated with AFB<sub>1</sub> biosynthesis. Propidium iodide staining and transmission electron microscopy analysis demonstrated a dose-dependent disruption of the integrity and morphology of the <i>A. flavus</i> spore cell membrane by PLA. Multi-omics analyses showed that subinhibitory concentrations of PLA induced significant changes in <i>A. flavus</i> spores at the transcriptional and metabolic levels, as 980 genes and 30 metabolites were differentially expressed. Moreover, KEGG pathway enrichment analysis indicated PLA-induced cell membrane damage, energy-metabolism disruption, and central-dogma abnormality in <i>A. flavus</i> spores. The results provided new insights into the anti-<i>A. flavus</i> and -AFB<sub>1</sub> mechanisms of PLA.
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