Metabolic profiling of Eurotium cristatum during liquid fermentation using untargeted UHPLC-MS/MS metabolomics

Untargeted metabolomics based on ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was used to systematically characterize metabolites in the liquid fermentation broth of Eurotium cristatum at 0, 24, and 40 h. Principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), differential metabolite screening, metabolite classification, and KEGG pathway enrichment analysis were applied to investigate dynamic metabolic changes and key regulatory pathways during fermentation. PCA explained 82.20% of the total variance, clearly separating samples from different fermentation stages and revealing phased metabolic patterns. Amino acids, peptides and their analogues (29.03%), lipids and lipid-like molecules (17.06%), and organic acids (8.40%) were the predominant metabolite classes showing significant stage-dependent changes. The metabolite profiles suggested that early fermentation was mainly associated with primary metabolic processes supporting fungal growth, whereas the later fermentation stage was characterized by the increased abundance of metabolites associated with secondary metabolism, with substantial accumulation of putative bioactive metabolites, including TCA cycle-related organic acids, terpenoids, and lipids. Pathway analysis using the KEGG database showed that differentially expressed metabolites were primarily linked to metabolic pathways, amino acid metabolism, biosynthesis of secondary metabolites, nucleotide metabolism, and ABC transporter pathways. These results provide a foundation for industrial fermentation of E. cristatum and targeted production of high-value metabolites.