A combined metabolomics and peptidomics approach to discriminate anomalous rind inclusion levels in Parmigiano Reggiano PDO grated hard cheese from different ripening stages

Parmigiano Reggiano is a hard cheese with a Protected Designation of Origin (PDO) certification that also applies to the grated product. The percentage of rind in grated Parmigiano Reggiano is regulated by the PDO production Specification and must not exceed the limit of 18% (w/w). The present study evaluates the potential of an untargeted foodomics approach to detect anomalous inclusions of rind in grated Parmigiano Reggiano cheese. In particular, a combined metabolomics and peptidomics approach was used to detect potential markers of counterfeits (rind > 18%). In the framework of realistic food integrity purposes, non-Parmigiano Reggiano grated samples and different ripening times were also considered. Untargeted metabolomics allowed detecting 347 compounds, with a prevalence of amino acids and peptide derivatives, followed by fatty acyls and other compounds (such as lactones, ketones, and aldehydes) typically related to proteolysis and lipolysis events. Overall, the unsupervised multivariate statistics showed that the ripening time plays a hierarchically higher impact than rind inclusion in determining the main differences in the chemical profiles detected. Interestingly, supervised statistics highlighted distinctive markers for ripening time and rind inclusion, with only 16 common discriminant compounds being shared between the two conditions. The best markers of rind inclusion > 18% were 2-hydroxyadenine (VIP score = 1.937; AUC value = 0.83) and the amino acid derivatives argininic acid (VIP score = 1.462; AUC value = 0.75) and 5-hydroxyindole acetaldehyde (VIP score = 1.710; AUC value = 0.86). Interestingly, the medium-chain aldehyde 4-hydroperoxy-2-nonenal was a common marker of both ripening time and anomalous rind inclusion (>18%), likely arising from the lipid oxidation processes. Finally, among potential marker peptides of rind inclusion, the alpha-S1 casein proteolytic product (F)FVAPFPEVFGK(E) could be identified.

Automated summary

The study utilized an untargeted foodomics approach to detect anomalies in grated Parmigiano Reggiano cheese, identifying that ripening time has a hierarchically higher impact than rind inclusion in determining chemical profiles. Distinctive markers for rind inclusion over 18% were discovered, such as 2-hydroxyadenine, argininic acid, and 5-hydroxyindole acetaldehyde. Common markers for both ripening time and anomalous rind inclusion included the medium-chain aldehyde 4-hydroperoxy-2-nonenal.