Spatial Mass Spectrometry Imaging Combined with Multiomics Reveals the Spatiotemporal Distribution of Monascus -Associated Metabolites during the Ripening of Monascus -Fermented Cheese

Abstract Monascus-fermented cheese (MC) is a dairy product with distinctive red coloration and Monascus-associated metabolites. The spatial distribution patterns and associated metabolic characteristics of metabolites within the cheese matrix remain unclear. In this study, a multiomics strategy integrating matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), metabolomics, metagenomics, and proteomics was applied to investigate the spatiotemporal dynamics of Monascus-associated metabolites during MC ripening. Metabolomic analysis revealed ripening-associated metabolic changes, and the increasing trend of monacolin K during ripening was quantified by targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS). In addition, γ-aminobutyric acid, monascin, and monascorubramine-related ion signals were putatively annotated as Monascus-associated metabolic features and exhibited heterogeneous spatial distributions in MSI analysis. Metagenomics revealed the presence of Monascus and gene potential associated with Monascus-associated metabolite metabolism. Proteomics revealed temporal changes in candidate protein abundances associated with metabolite-related processes. This study provides multiscale insights into spatial metabolic patterns in MC.