RESUMO
To map qualitative and quantitative metabolome alterations when Penicillium roqueforti is grown in an environment where l-tyrosine levels are perturbed, the recently established differential off-line LC-NMR (DOLC-NMR) approach was successfully applied in connection with an absolute metabolite quantitation using a quantitative 1H NMR protocol following the ERETIC 2 (Electronic REference To access In vivo Concentrations) methodology. Among the 23 influenced metabolites, amino acid degradation products like 2-(4-hydroxyphenyl)acetic acid and 2-(3,4-dihydroxyphenyl)acetic acid underwent a tremendous upregulation in the amino acid perturbed approach. Moreover, the output of secondary metabolites like andrastin A, eremofortin B, and the tetrapeptide d-Phe-l-Val-d-Val-l-Tyr was affected in the case of the presence or absence of the added aromatic amino acid. Furthermore, the isolated secondary metabolites of P. roqueforti have been quantified for the first time in five divergent Penicillium isolates by means of a validated LC-ECHO-MS/MS method. This technique is used to compensate the effect of co-extracted matrix compounds during the analysis and to utilize quasi-internal standards to quantify all metabolites of interest accurately. This screening outlined the great variety between the different fungi of the same species. The metabolite spectra of wild-type fungi included more toxic intermediates compared to a selected fungi used as a starter culture for blue-mold cheese production. In addition, these secondary metabolites were quantified in commercially available white- and blue-mold cheese samples. The main differences between the analyte profiles of white and blue cheeses were linked to the impact of the used starter culture. Specific metabolites detected from P. roqueforti like andrastin A and B or roquefortine C could not be detected in white cheese. Among the blue cheese samples, different metabolite pattern could be observed regarding various P. roqueforti starter cultures.