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A multicenter study to evaluate a targeted proteomics method for the qualitative and quantitative analysis of the different types of milk used in mixed-milk cheeses

EuPA initiatives - Full Day Session

Abstract #158 Posted on26 March 202512 May 2025 12 May 2025 13h08

Abstract

Most mixed milk cheeses are made from a binary or ternary mixture of cow, sheep and goat milk and represent an important market share in Europe (in Spain 40-45%). The percentage of milk of each species must be clearly indicated on the label of the product. However, the large (2-3 times) price differences between different types of milk represent a powerful incentive to commit composition fraud. Until now, an analytical method capable of monitoring milk proteins in a complex and highly altered matrix, such as cheese, was not available. We have developed a PRM-based targeted proteomics method to monitor specific tryptic peptides generated from cow, sheep and goat milk reporter proteins. It allows for the accurate determination of the percentage of protein from each of the species present in a mixed milk cheese sample. We evaluated our method with reference cheeses made with different but known percentages of milk from each species following standard cheese making procedures. Analytical parameters such as the working range, limit of detection and quantification, repeatability, reproducibility, accuracy and precision have been evaluated. Finally, we demonstrated the applicability of the method for mixed-milk commercial cheeses with variable (1-11 months) ripening periods. A multicenter study (n=10 labs) is currently active to determine the universal applicability of the method under different analytical conditions. The conclusions of the study will be published by mid 2026.

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Most mixed milk cheeses are made from a binary or ternary mixture of cow, sheep and goat milk and represent an important market share in Europe (in Spain 40-45%). The percentage of milk of each species must be clearly indicated on the label of the product. However, the large (2-3 times) price differences between different types of milk  represent a powerful incentive to commit composition fraud. Until now, an analytical method capable of monitoring milk proteins in a complex and highly altered matrix, such as cheese, was not available. We have developed a PRM-based targeted proteomics method to monitor specific tryptic peptides generated from cow, sheep and goat milk reporter proteins. It allows for the accurate determination of the percentage of protein from each of the species present in a mixed milk cheese sample. We evaluated our method with reference cheeses made with different but known percentages of milk from each species following standard cheese making procedures. Analytical parameters such as the working range, limit of detection and quantification, repeatability, reproducibility, accuracy and precision have been evaluated. Finally, we demonstrated the applicability of the method for mixed-milk commercial cheeses with variable (1-11 months) ripening periods. A multicenter study (n=10 labs) is currently active to determine the universal applicability of the method under different analytical conditions. The conclusions of the study will be published by mid 2026.

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