Metabolomics-based screening identifies Aspergillus strains that accumulate bioactive leucine catabolites and fatty acid ethyl esters in koji mold-ripened cheese.

Aspergillus koji molds, which play a principal role in manufacturing fermented foods, have been applied to the production of koji cheese. In this study, Aspergillus oryzae strains and their metabolic attributes were studied using nontargeted metabolomics and identified as starter cultures that can introduce novel chemical characteristics. In volatile compounds, A. oryzae Q was recognized as an uncommon strain following the previously selected strain KC43, and its cheese scarcely increased the levels of straight-chain fatty acids and methyl ketones, sharp cheesy odorants. Strain O can accumulate substantial levels of fatty acid ethyl esters (FAEEs) in koji cheese, indicating its potential contribution to the floral note and masking of sharpness in cheese, as reported in blue cheese. In water-soluble compounds, the tested strains as well as the previously selected strains were commonly characterized by a marked increase in amino acid levels. By contrast, 3-hydroxy-3-methylbutyric acid (HMB) and 3-hydroxy-3-methylglutaric acid (HMG), which were detected as unannotated compounds, were highly responsible for strain Q. Metabolic tracing using a 13C-labeled substrate identified that HMB and HMG were extracellular metabolites converted from l-leucine, along with the formation of α-ketoisocaproic acid and isovaleric acid. The production quantity of these four compounds was strongly correlated with the initial concentration of l-leucine (R > 0.975). The findings proposed the hypothesis that these compounds derived from their CoA-thioesters in l-leucine degradation pathway required for HMG-CoA and mevalonate synthesis. This study is the first to report HMB and HMG as metabolites produced by A. oryzae and as major compounds in Aspergillus-fermented foods.