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One of the most difficult issues in the juice industry is to manufacture juices where processing processes minimise the impact on the native characteristics of the fruits. In this study, high-pressure technology was used on whole apple fruits in order to evaluate the effect on the juice production. Two varieties, cv. Limoncella and cv. Pink Lady, were considered. Preliminarily, the fruits were subjected to different pressures, and histological as well as pomological measurements were taken in order to identify the best treatment condition, which was established to be 600 MPa for 3 min. Juice samples were then characterised by measuring the colour, viscosity, total antioxidant capacity (TAC), and total phenolic content (TPC). The storage colour stability of the juices for both varieties showed not significant L* values between the untreated and pre-treated fruits. Juices obtained from pre-treated fruits had a viscosity significantly higher than that obtained from untreated ones. Interestingly, the TPC of high-pressure processing (HPP) pre-treated juice resulted in being significantly higher compared to the untreated ones. The HPP pre-treatment can be considered as a commercial application to modulate some quality standards for apple juice production. [ABSTRACT FROM AUTHOR]

Microalgae show potential as renewable and environmentally friendly fuel resources. Wastewaters (WWs) can be utilized as growth media, reducing the associated cultivation costs. Therefore, the aim of this study was to investigate how organic-rich WWs affect the growth and fatty acid methyl esters (FAME) profile of Chlorella vulgaris. This particular strain exhibits high biomass productivity and can thrive in a wide range of WWs. It also has the ability to shift its metabolism from autotrophic to hetero/mixotrophic. Glycerol can serve as a means to direct metabolism towards lipids production. Consequently, C. vulgaris was cultivated in brewery wastewater (BWW) containing varying concentrations of glycerol under both metabolic conditions. When C. vulgaris was cultivated in a mixotrophic environment, it achieved a notably higher biomass yield compared to heterotrophic cultivation. The highest biomass yield, reaching 1.33 g L-1, was achieved utilizing 2 mL of glycerol in BWW, surpassing the control with 1.08 g L-1. However, when a two-phase metabolism was applied, comprising the ten days of mixotrophy followed by the final five days in heterotrophy (MHB), the biomass yield was slightly lower than that obtained under continuous mixotrophic conditions. Nevertheless, it was still double the biomass obtained in a strictly heterotrophic environment. The FAME profile analysis revealed that, within the considered trophic conditions, the highest content of saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), and polyunsaturated fatty acids (PUFA) was observed during mixotrophic cultivation with 10 mL of glycerol, mixotrophic cultivation with 4 mL of glycerol, and MHB with 4 mL of glycerol (35.36%wt, 46.89%wt, and 31.60%wt, respectively). An initial examination of the saturated and unsaturated components of the FAME suggests that lipids extracted from C. vulgaris biomass cultivated mixotrophically and heterorophically in BWW could potentially serve as a valuable feedstock for biodiesel production. [ABSTRACT FROM AUTHOR]

An untargeted Nuclear Magnetic Resonance (NMR) spectroscopy-based metabolomics approach was applied as a first attempt to explore the metabolome of pigs treated with antibiotics. The final goal was to investigate the possibility of discriminating between antibiotic-treated (TX group) and untreated pigs (CTRL group), with the further perspective of identifying the authentication tools for antibiotic-free pork supply chains. In particular, 41 samples of pig liver were subjected to a biphasic extraction to recover both the polar and the non-polar metabolites, and the 1H NMR spectroscopy analysis was performed on the two separate extracts. Unsupervised (principal component analysis) and supervised (orthogonal partial least squares discriminant analysis) multivariate statistical analysis of 1H NMR spectra data in the range 0–9 ppm provided metabolomic fingerprinting useful for the discrimination of pig livers based on the antibiotic treatment to which they were exposed. Moreover, within the signature patterns, significant discriminating metabolites were identified among carbohydrates, choline and derivatives, amino acids and some lipid-class molecules. The encouraging findings of this exploratory study showed the feasibility of the untargeted metabolomic approach as a novel strategy in the authentication framework of pork supply chains and open a new horizon for a more in-depth investigation. [ABSTRACT FROM AUTHOR]

Perennial grains provide various ecosystem services compared to the annual counterparts thanks to their extensive root system and permanent soil cover. However, little is known about the evolution and diversification of perennial grains rhizosphere and its ecological functions over time. In this study, a suite of -OMICSs - metagenomics, enzymomics, metabolomics and lipidomics - was used to compare the rhizosphere environment of four perennial wheat lines at the first and fourth year of growth in comparison with an annual durum wheat cultivar and the parental species Thinopyrum intermedium. We hypothesized that wheat perenniality has a greater role in shaping the rhizobiome composition, biomass, diversity, and activity than plant genotypes because perenniality affects the quality and quantity of C input - mainly root exudates - hence modulating the plantmicrobes crosstalk. In support of this hypothesis, the continuous supply of sugars in the rhizosphere along the years created a favorable environment for microbial growth which is reflected in a higher microbial biomass and enzymatic activity. Moreover, modification in the rhizosphere metabolome and lipidome over the years led to changes in the microbial community composition favoring the coexistence of more diverse microbial taxa, increasing plant tolerance to biotic and abiotic stresses. Despite the dominance of the perenniality effect, our data underlined that the OK72 line rhizobiome distinguished from the others by the increase in abundance of Pseudomonas spp., most of which are known as potential beneficial microorganisms, identifying this line as a suitable candidate for the study and selection of new perennial wheat lines. [ABSTRACT FROM AUTHOR]

By-products from the fruit supply chain, especially seeds/kernels, have shown great potential to be valorised, due to their high content of macronutrients, such as lipids, protein, and fibre. A mild enzymatic assisted extraction (EAE) involving the use of a protease was tested to evaluate the feasibility of a cascade approach to fractionate the main fruit by-products components. Protease from Bacillus licheniformis (the enzyme used in the AOAC 991.43 official method for dietary fibre quantification) was used, and besides protein, the conditions of hydrolysis (60 °C, neutral pH, overnight) allowed us to dissolve a portion of soluble fibres, which was then separated from the solubilized peptide fraction through ethanol precipitation. Good protein extraction yields, in the range 35–93%, were obtained. The soluble fibre extraction yield ranged from 1.6% to 71% depending on the by-product, suggesting its applicability only for certain substrates, and it was found to be negatively correlated with the molecular weight of the fibre. The monosaccharide composition of the soluble fibres extracted was also diverse. Galacturonic acid was present in a low amount, indicating that pectin was not efficiently extracted. However, a predominance of arabinose and galactose monomers was detected in many fractions, indicating the isolation of a fruit soluble fibre portion with potential similarity with arabinogalactans and gum arabic, opening up perspectives for technological applications. The residual solid pellet obtained after protease assisted extraction was found to be an excellent fibre-rich substrate, suitable for being subjected to more "hard" processing (e.g., sequential pectin and hemicellulose extraction) with the objective to derive other fractions with potential great added economic value. [ABSTRACT FROM AUTHOR]

The increase of greenhouse gases into the atmosphere represents today one of the most global concern. The inevitable depletion of fossil fuels and the adverse climate changes push the scientific community to seek renewable and sustainable sources of fuel. In this scenario microalgae can be potentially exploited as renewable and environmentally friendly fuel resources. Wastewaters (WW) can be used as culture media reducing the costs associated to their cultivation. Hence, the goal of this study was to examine the effect of an organic rich WW on Chlorella vulgaris growth and fatty acid methyl esters (FAME) profile. This strain shows high biomass productivity thriving in a wide range of WWs and is able to shift its metabolism from autotrophic to hetero/mixotrophic one. Glycerol can be used to convey metabolism towards lipids production. Therefore, C. vulgaris was cultivated in dairy waste (DWW) with different concentrations of glycerol under both metabolisms. When C. vulgaris was cultivated under mixotrophy attained a high biomass yield compared to heterotrophy. The highest biomass yield (1.72 g L-1) was obtained with 10 mL of glycerol in DWW compared to the control (1.08 g L-1). When a two-phase metabolism was adopted, that is the first ten days under mixotrophy followed by the last five days in heterotrophy (MHD), the biomass was almost doubled with 2 mL of glycerol in DWW. FAME profile reveled that compared to the control the highest saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), and polyunsaturated fatty acids (PUFA) content were obtained under heterotrophy with 10 mL of glycerol, under MHD with 2 mL and with 4 mL of glycerol (46.73%wt, 41.79%wt, and 30.34%wt, respectively). A preliminary analysis on the saturated and unsaturated components of the FAME suggests that lipids extracted from C. vulgaris biomass cultivated mixotrophically and heterorophically in DWW could represents a feedstock to be exploited for biodiesel production. [ABSTRACT FROM AUTHOR]

Plasma Activated Water (PAW) has recently emerged as a promising non-chemical and non-thermal technology for the microbial decontamination of food. However, its use as a replacement for conventional disinfection solutions needs further investigation, as the impact of reactive species generated by PAW on nutritional food quality, toxicology, and safety is still unclear. The purpose of this study is to investigate how treatment with PAW affects the health-promoting properties of fresh-cut rocket salad (Eruca sativa). Therefore, the polyphenolic profile and antioxidant activity were evaluated by a combination of UHPLC-MS/MS and in vitro assays. Moreover, the effects of polyphenolic extracts on cell viability and oxidative status in Caco2 cells were assessed. PAW caused a slight reduction in the radical scavenging activity of the amphiphilic fraction over time but produced a positive effect on the total phenolic content, of about 70% in PAW-20, and an increase in the relative percentage (about 44–50%) of glucosinolate. Interestingly, the PAW polyphenol extract did not cause any cytotoxic effect and caused a lower imbalance in the redox status compared to an untreated sample. The obtained results support the use of PAW technology for fresh-cut vegetables to preserve their nutritional properties. [ABSTRACT FROM AUTHOR]

Pomegranate produces fruit with high nutritional value. Of particular interest is the woody part of the seed, which influences consumer acceptability and is rich in bioactive components. The aim of this study was to morphoanatomically and biochemically characterize the tegmen of local and commercial pomegranates grown in Italy. SEM and a texturometer were used for morphoanatomical and hardness analysis, respectively, and gravimetric and Soxhlet extraction, as well as a GS-MS instrument, were used for chemical analysis. In addition to the classic morphometric parameters, we described, for the first time, the bundles on pomegranate tegmen, identifying four forms (FAN, TREE, COMB and RAMIFIED); local cultivars showed a higher variability compared to the commercial cultivars. Lignin content showed no correlation with seed strength. We developed a new hierarchical model in which geometric parameters and the resistant volume of the lignified tissue can explain the variability in hardness observed in the tests. Quantitative differences were observed in total lipids and unsaponifiable fractions; in particular, all oils were rich in unsaturated fatty acids. We propose that bundles form as a novel trait for characterization and a new hierarchical model to evaluate the hardness of seeds. The content of fatty acids and other biocompounds in pomegranate seeds can promote their valorization as a resource rather than a waste product. [ABSTRACT FROM AUTHOR]

The increase of greenhouse gases into the atmosphere, mainly due to industrialization, has affected all the ecosystems. Current worldwide living standards are still heavily dependent on non-renewable fuels. The inevitable depletion of fossil fuels and the adverse climate changes push the scientific community to seek renewable and sustainable sources of fuel. In this scenario microalgae can be potentially exploited as renewable and environmentally friendly fuel resources. Wastewaters (WW) can be used as culture media minimizing the costs associated to their cultivation. Hence, the goal of this study was to examine the effect of agro-industrial WWs rich in organic nutrients on algal lipid content and fatty acid methyl esters (FAME) profile. For this purpose, the fresh water green algae Chlorella vulgaris was selected. This strain is able to thrive in a wide range of WWs with high biomass productivity and to shift its metabolism from autotrophic to hetero/mixotrophic one. C. vulgaris was cultivated in brewery (BWW), dairy (DWW), oil mill WWs and media supplemented with sugarcane molasses. High biomass yields were obtained when C. vulgaris was cultivated in BWW and DWW (1.76 g L-1 and 1.56 g L-1, respectively) compared to the control and the other WWs. The assessment of FAMEs composition (i.e. level of unsaturation) of algae cultivated under all the investigated conditions demonstrated that the former ones can be viably used as sources for producing biofuels. [ABSTRACT FROM AUTHOR]

Cyclopropane fatty acids (CPFAs) are an investigated class of secondary fatty acids of microbial origin recently identified in foods. Even though the dietary daily intake of this class of compounds it has been recently estimated as not negligible, to date, no studies specifically have investigated their presence in human plasma after consumption of CPFA-rich sources. Therefore, the aims of this study were (i) to test CPFAs concentration in human plasma, thus demonstrating their in vivo bioaccessibility and potential bioavailability, (ii) to investigate a dose-response relationship between medium term chronic intake of CPFAs-rich foods and both CPFAs and plasma total fatty acid profiles in healthy subjects. Ten healthy normal weight adults were enrolled for conducting an in vivo study. Participants were asked to follow a CPFA-controlled diet for 3 weeks, consuming 50 g of Grana Padano cheese (GP) and 250 mL of whole cow milk, which correspond to a total of 22.1 mg of CPFAs. Fasting CPFAs concentration were monitored for eight timepoints during the whole study and plasma total fatty acids composition was determined by GC-MS. CPFAs, mainly dihydrosterculic acid (DHSA), were identified in plasma total fatty acids profile at the beginning of the study and after dietary treatment. A significant (p < 0.05) increase of CPFAs mean plasma concentration (n = 10) were observed at the end of the dietary intervention. Contrarily, the total fatty acids composition of the general plasma fatty acids profile did not significantly change (p ≥ 0.05) during the dietary intervention period. This is the first investigation demonstrating that CPFAs are bioaccessible in vivo and, as expected, their plasmatic concentration may be affected by consumption of CPFAs-rich foods. This research will open the door to further detailed research, which may better elucidate the role of these compounds in human health. [ABSTRACT FROM AUTHOR]

Cyclopropane fatty acids (CPFAs) are the most abundant cyclic fatty acids in microorganisms with unknown role(s) regarding their dietary relevance and biological effects in humans. This work was aimed to draw up a list of CPFAs-containing foods for estimating their dietary intake in the Italian population to provide a basis for evaluating their nutritional relevance and potential health-related effects. The CPFAs content of more than 500 food items was investigated and a preliminary dietary intake was assessed (12.0 ± 6.0 mg/day), based on the data reported by the Italian National Food Consumption Survey INRAN-SCAI 2005-06. CPFAs should be considered of dietary relevance in view of their potential physiological activity in humans and their presence in significant amounts in dairy products, as Grana Padano cheese (9.0-30.0 mg/100 g), and in bovine meat (0.7-4.0 mg/100 g). Future studies should elucidate whether this uncommon class of fatty acids has a biological role in human health. [ABSTRACT FROM AUTHOR]

Cyclopropane fatty acids (CPFAs) are unusual fatty acids of microbial origin, recently detected in milk and dairy products. CPFAs have been demonstrated to be interesting molecular markers for authentication of dairy products obtained without ensiled feeds. Moreover, they can also be recognized as a new secondary component of human diet. Information is lacking on the presence of cyclic fatty acids in other food sources. Cyclopropane fatty acids have been detected by GC-MS analysis in cheese and other animal fats in concentration ranging from 200 to 1000 mg/kg fat, but in some cases, the complex fatty acid profile and the possible presence of interfering peaks make the separation not straightforward and the quantification uneasy. Therefore, a new reliable 1H NMR method was developed to detect and measure CPFA content in different foods of animal origin, based on the detection of the characteristic signals of cyclopropane ring. The 1H NMR (600 MHz) method showed detection limits comparable with those of full scan GC-MS, and it allowed the identification and quantitation of the cyclopropane fatty acids in different foods. [ABSTRACT FROM AUTHOR]

A survey was conducted to determine the content of cyclopropane fatty acids (CPFAs) and ω-cyclohexyl fatty acids (CHFAs) by using gas chromatography- mass spectrometry (GC-MS) and proton nuclear magnetic resonance (1H NMR) techniques in various meat samples from different species, including commercial samples and complex and thermally processed products (i.e., Bolognese sauce). The CPFAs concentration (as the sum of two isomers, namely dihydrosterculic acid and lactobacillic acid) in bovine meat fat (ranging between 70 and 465 mg/kg fat) was positively related to a silage-based diet, and therefore, they are potential biomarkers for monitoring the feeding system of cattle. CHFAs, such as 11-cyclohexylundecanoic and 13-cyclohexyltridecanoic acids, were only found in lipid profiles from ruminant species, and a linear trend was observed in their content, together with iso-branched fatty acids (iso-BCFAs) deriving from ruminal fermentation, as a function of bovine meat percentage in both raw and cooked minced meat. Thus, CHFAs are potential biomarkers for the assurance of the meat species and, combined with iso-BCFAs, of the beef/pork ratio even in complex meat matrices. The proposed approaches are valuable novel tools for meat authentication, which is pivotal in the management of meat quality, safety, and traceability. [ABSTRACT FROM AUTHOR]

Cyclopropenoid fatty acids (CPEFA), found in oilseeds from Malvaceae and Sterculiaceae, have been shown to interfere with the endogenous synthesis of several bioactive lipids of dairy fat, such as cis-9, trans-11 18:2 and cis-9 18:1, by inhibiting Δ9-desaturase. No previous study has reported the presence of sterculic acid in animal fat and its incorporation in tissues after its administration, due to the lack of a proper methodology. In the present research, a GC-MS method based on cold base derivatization to fatty acids methylesters was developed to determine CPEFA in ewe milk triglycerides, after infusing sterculic acid (0.5 g/day) to six lactating ewes. An alternative derivatization based on silanyzation followed by GC-MS analysis was also tested, showing its possible applicability when CPEFA are present in the form of free fatty acids. Sterculic acid was detected in ewe milk triglycerides, demonstrating its incorporation from the bloodstream into milk by the mammary gland. The mean transfer rate represented 8.0 ± 1.0% of the daily dose. This study provides, for the first time, the presence of sterculic acid in milk fat, supporting the importance of understanding its occurrence in vivo and encouraging further research to determine whether it can be present in foods, such as dairy products, obtained under practical farming conditions. [ABSTRACT FROM AUTHOR]

Coffee capsules market is on the rise as it allows access to a wide selection of coffee, differing in taste and brand. However, few data about the chemical characterization of the capsule-brewed coffee aroma are available. In this work, an untargeted approach using headspace solid-phase microextraction (HS-SPME) coupled to gas chromatography–mass spectrometry (GC-MS) and combined to chemometrics was performed to study and compare aroma profile from 65 capsule-brewed espresso coffees (ECs) commercialized by five of the most representative brands in Italy. Volatile profiles obtained from ECs were subjected to multivariate statistical analysis, which generally did not show a significant variability among coffees belonging to the same brand, except for those modified after the addition of specific flavor additives or aromatic substances (such as caramel, chocolate, etc.). Similarities may be related to the starting coffee brew or the processing method, which is likely the same for each individual brand. Additionally, partial least squares discriminant analysis (PLS-DA) showed that capsules from a specific brand contain the highest concentration of pyrazines, thus characterized by an intense and characteristic aroma, and a stronger note than those from the other brands. This study supports that the chemical analysis in conjunction with chemometric tools is a useful approach for assessing flavor quality, even if the need remains to identify volatile markers of high-quality beverages. [ABSTRACT FROM AUTHOR]

In this study we investigated the oligopeptide pattern in fermented cocoa beans and derived products after simulated gastrointestinal digestion. Peptides in digested cocoa samples were identified based on the mass fragmentation and on the software analysis of vicilin and 21 KDa cocoa seed protein sequences, the most abundant cocoa proteins. Quantification was carried out by liquid chromatography/electrospray ionisation mass spectrometry (LC/ESI-MS) using an internal standard. Sixty five peptides were identified in the digested samples, including three pyroglutamyl derivatives. The in vitro angiotensin-converting enzyme (ACE)-inhibitory activity of cocoa digests were tested, demonstrating a high inhibition activity, especially for digestates of cocoa beans. The peptides identified were screened for their potential ACE inhibitory activity through an in silico approach, and about 20 di-, three- and tetra-peptides actually present in our samples were predicted as active. Two of the potentially active peptides were chemically synthesized and then assessed for their inhibitory activity by using the ACE in vitro assay. These peptides demonstrated an ACE inhibitory activity, however, that was too weak to explain alone the high activity of cocoa digestates, suggesting a synergic effect of all cocoa peptides. As a whole, results showed that an average chocolate portion (30 g) ensures an amount of peptides after digestion that, assuming complete absorption, could reach almost a complete inhibition of ACE. [ABSTRACT FROM AUTHOR]

By-products from the fruit supply chain, especially seeds/kernels, have shown great potential to be valorised, due to their high content of macronutrients, such as lipids, protein, and fibre. A mild enzymatic assisted extraction (EAE) involving the use of a protease was tested to evaluate the feasibility of a cascade approach to fractionate the main fruit by-products components. Protease from Bacillus licheniformis (the enzyme used in the AOAC 991.43 official method for dietary fibre quantification) was used, and besides protein, the conditions of hydrolysis (60 °C, neutral pH, overnight) allowed us to dissolve a portion of soluble fibres, which was then separated from the solubilized peptide fraction through ethanol precipitation. Good protein extraction yields, in the range 35-93%, were obtained. The soluble fibre extraction yield ranged from 1.6% to 71% depending on the by-product, suggesting its applicability only for certain substrates, and it was found to be negatively correlated with the molecular weight of the fibre. The monosaccharide composition of the soluble fibres extracted was also diverse. Galacturonic acid was present in a low amount, indicating that pectin was not efficiently extracted. However, a predominance of arabinose and galactose monomers was detected in many fractions, indicating the isolation of a fruit soluble fibre portion with potential similarity with arabinogalactans and gum arabic, opening up perspectives for technological applications. The residual solid pellet obtained after protease assisted extraction was found to be an excellent fibre-rich substrate, suitable for being subjected to more "hard" processing (e.g., sequential pectin and hemicellulose extraction) with the objective to derive other fractions with potential great added economic value.

Black soldier fly (BSF) is being increasingly used for agro-food by-products valorisation. Adult flies, puparia, and excess of prepupae are the by-products of this process, which could be further valorised. Lactic fermentation of BSF biomasses with two different strains ( L. rhamnosus and L. plantarum ) has been used for this purpose. Deep changes in the molecular composition were observed, without significant differences related to the different strains used. The lipid and protein fractions were the most impacted. Fermentation enriched the biomass in monounsaturated and polyunsaturated fatty acids and essential amino acids, significantly improving the nutritional properties of the substrates. Although not particularly marked, a proteolytic activity of lactobacilli was observed on the BSF muscular and cuticular proteins, especially in the samples of adult flies and puparia, where fermentation resulted more effective. Conversely, there was no evidence of chitinolytic activity., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Author(s).)

Cyclopropane fatty acids (CPFA) were found in milk fat from cows fed maize silage and suggested to be synthesized by lactic acid bacteria during ensiling. This study aimed to elucidate some gaps of knowledge about the microbial synthesis of CPFA, to strengthen the current authentication method based on their detection in cheese fat and performed for Parmigiano Reggiano (UNI11650), whose Specifications forbid the use of silage. CPFA were screened in different ensiled cows' feeding by gas chromatography-mass spectrometry, and the effect of feed ingredients and ruminal digestion on CPFA microbial production were further examined by in vitro tests. Results showed that solely the environmental conditions developed in silos for specific plant materials (e.g., maize) are essential for the bacterial synthesis of CPFA, whereas rumen activity did not affect CPFA levels in feeds. This supports the suitability of using CPFA as biomarkers of a crop silage-based diet forbidden by certain PDO feedstock regulations.

This study aimed to investigate cyclopropane fatty acids (CPFAs) as quality biomarkers of forage feedings in cheese fat obtained from ewe's milk, based on two different dietary treatments (hay and silage). The gas chromatography-mass spectrometry (GC-MS) analysis detected CPFAs in most cheese samples, both from hay and silage-based diets. CPFA levels in cheese fat from hay feeding were positively correlated to the total trans -monounsaturated fatty acids (MUFAs) and n-6 polyunsaturated fatty acids (PUFAs), whereas they were negatively correlated to cis -MUFAs, odd- and branched-chain fatty acids (i.e., C13:0 anteiso , C16:0 iso , and C17:1), and C22:5n-3, which are mainly associated with a low starch intake and grass pasture. Overall, the presence of CPFAs in ovine cheese fat suggests the use of silage, but it can also be an indicator of poor-quality hay forages. This approach confirmed the reliability of CPFAs as biomarkers of forage quality, especially in relation to the use of conserved forages and good livestock practices.

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.