Development of a Quantitative GC–MS Method for the Detection of Cyclopropane Fatty Acids in Cheese as New Molecular Markers for Parmigiano Reggiano Authentication

Abstract

Cyclopropane fatty acids (CPFA), as lactobacillic acid and dihydrosterculic acid, are components of bacterial membranes and have been recently detected in milk and in dairy products from cows fed with corn silage. In this paper, a specific quantitative gas chromatography-mass spectrometry (GC-MS) method for the detection of CPFA in cheeses was developed, and the quality parameters of the method (limit of detection, limit of quantitation, and intralaboratory precision) were assessed. Limit of detection and quantitation of CPFA were, respectively, 60 and 200 mg/kg of cheese fat, and the intralaboratory precision, determined on three concentration levels, satisfied the Horwitz equation. This method was applied to 304 samples of PDO cheeses of certified origin, including Parmigiano Reggiano (Italy), Grana Padano (Italy), Fontina (Italy), Comté (France), and Gruyère (Switzerland). Results showed that CPFA were absent in all of the cheeses whose Production Specification Rules expressly forbid the use of silages (Parmigiano Reggiano, Fontina, Comté, and Gruyère). CPFA were instead present in variable concentrations (300-830 mg/kg of fat) in all of the samples of Grana Padano cheese (silages admitted). A mix of grated Parmigiano Reggiano and Grana Padano was also prepared, showing that the method is able to detect the counterfeiting of Parmigiano Reggiano with other cheeses up to 10-20% Grana Padano content. These results support the hypothesis that CPFA can be used as a marker of silage feedings for cheeses, and the data reported can be considered a first attempt to create a database for CPFA presence in PDO cheeses.

Cyclic Fatty Acids in Food: An Under-Investigated Class of Fatty Acids

“…As previously reported, data collected by the authors [10,11,43] on hundreds of milk and dairy samples confirm the strict correlation between the use of silages in the feeding and the presence of CPFA in milk fat. Cyclopropane fatty acids (CPFA) were present only in dairy products from cows fed with silages, and their determination has been demonstrated to be molecular markers of quality for PDO cheeses, as Parmigiano-Reggiano, where the use of silage is forbidden [10].…”

“…Cyclic fatty acids are generally secondary compounds in fatty acid profiles of food; however, due to the recent discovery, some gaps of knowledge must be fulfilled. In some cases, especially cyclopropane fatty acids, they could reach the g/ kg of total fat content in meat and dairy products [12,43] and their dietary intake may be not negligible.…”

“…Finally, the estimated daily, weekly and monthly CPFA dietary intake in the total Italian population (all sex and ages) [45] resulted in the milligrams order, so not negligible in view of a possible physiological action by CPFA on humans. Furthermore, food processing, manufacturing, seasoning steps, and fermentation [10] [43]. SD = standard deviation.…”

“…As reported in the previous paragraphs, no information is present in literature about the fate of CPFA within the human body, and a thorough investigation of how CPFA accumulate and are metabolized in humans is needed. In [13], the rate of CPFA digestibility has been assessed through their lipolysis and resistance to in vitro simulated human gastrointestinal (GI) digestion in Grana Padano cheese, one of the most relevant sources of CPFA [43]. Results showed a high percentage of digestibility of the lipid fraction (more than 90% of free fatty acids and 1-monoglycerides were obtained after digestion).…”

A Novel MOS Nanowire Gas Sensor Device (S3) and GC-MS-Based Approach for the Characterization of Grated Parmigiano Reggiano Cheese

“…It bears a Protected Designation of Origin (PDO) certification adhering to the EC Regulation 2081/92 [1], thus its production is governed by a strict standard following a traditional artisan procedure that takes place exclusively in the provinces of Parma, Reggio Emilia, Modena, Mantova, and Bologna in northern Italy [2]. This semi fat, additive-free, hard cooked cheese produced from raw and semi-skimmed cow’s milk must ripen for at least 12 months and up to 36 months [3,4].…”