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Development of a Quantitative GC-MS Method for the Detection of Cyclopropane Fatty Acids in Cheese as New Molecular Markers for Parmigiano Reggiano Authentication Augusta Caligiani, Marco Nocetti, Veronica Lolli, Angela Marseglia, and Gerardo Palla J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b00913 • Publication Date (Web): 02 May 2016 Downloaded from http://pubs.acs.org on May 9, 2016

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Journal of Agricultural and Food Chemistry

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

Augusta Caligiani1*, Marco Nocetti2, Veronica Lolli1, Angela Marseglia1, Gerardo Palla1 Dipartimento di Scienze degli alimenti, Università degli Studi di Parma, Parco Area delle Scienze 17/A - 43124 Parma (Italy) Servizio Tecnico Consorzio del Formaggio Parmigiano Reggiano, Via J. F. Kennedy, 18 42124 Reggio Emilia (Italy)

*Corresponding author: Augusta Caligiani Dip. Scienze degli alimenti Università degli Studi di Parma Parco Area delle Scienze 17/A 43124-Parma, Italy Tel: +39 0521 905407; fax: +39 0521 905472; E-mail: [email protected]

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ACS Paragon Plus Environment


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Abstract

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Cyclopropane fatty acids (CPFA), as lactobacillic acid and dihydrosterculic acid, are components of

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bacterial membranes and have been recently detected in milk and in dairy products from cows fed

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with corn silage. In this paper, a specific quantitative GC-MS method for the detection of CPFA in

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cheeses was developed and the quality parameters of the method (LOD, LOQ, linearity,

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intralaboratory precision) were assessed. Limit of detection and quantitation of CPFA were

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respectively 60 and 200 mg/kg of cheese fat, and the intralaboratory precision, determined on

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three concentration levels, satisfied the Horwitz equation. Method was applied to 304 samples of

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PDO cheeses of certified origin, comprising Parmigiano Reggiano (Italy), Grana Padano (Italy),

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Fontina (Italy), Comté (France), Gruyère (Switzerland). Results showed that CPFA were absent in

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all the cheeses whose Production Specification Rules expressly forbid the use of silages

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(Parmigiano Reggiano, Fontina, Comté and Gruyère). CPFA were instead present, in variable

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concentrations (300-830 mg/kg of fat), in all the samples of Grana Padano cheese (silages

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admitted). Mix of grated Parmigiano Reggiano and Grana Padano were also prepared, showing

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that the method is able to detect the counterfeiting of Parmigiano Reggiano with other cheeses

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until 10-20 %. These results comfort the hypothesis that CPFA can be used as a marker of silage

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feedings for cheeses, and the data reported can be considered a first attempt to create a database

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for CPFA presence in PDO cheeses.

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Key words:

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Cyclopropane fatty acid, dihydrosterculic acid, GC-MS, PDO cheese, Parmigiano Reggiano,

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authentication

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INTRODUCTION

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Food authentication represents an important strategic issue for the food industry because

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consumers are becoming increasingly interested in quality and origin of foods. This is especially

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true when consumers purchase expensive certified and high-added-value products, such as

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organic, protected denomination of origin (PDO), or protected geographical indication (PGI)

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products.1 The higher prices of PDO products encourage more frequent counterfeiting. In dairy

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sector the most important issue in authentication is related to PDO cheeses which are high

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commercial value products confined according to legislative and proper labelling rules. Their

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authenticity is associated with several factors such as the geographical area of production,

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materials and technology used. In fact, cheese production can differ according to the feeding

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system of the animals providing the milk, the starters used, the heating temperature, the salting,

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the use or not of preservatives and the ripening time, as these parameters generate defined

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characteristics, which in turn can be detected by several analytical techniques, providing indication

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of the origin of the cheeses.2, 3

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Among PDO cheeses, Parmigiano Reggiano (PRRE) cheese represents one of the most important

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cheese in terms of quality and from an economical point of view: it costs more than double than

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generic similar cheeses, so it is important to protect it against mislabeling. The production is

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regulated by the Parmigiano-Reggiano Cheese Consortium (CFPR). In compliance with the

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European Regulation 510/2006 and following 1151/12, the designation PDO Parmigiano Reggiano

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can be exclusively awarded to the cheese produced following traditional artisan methods and

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produced in a restricted area of Italy (Provinces of Parma, Reggio Emilia, Modena, Mantova and

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Bologna) from milk produced in the same area. It is an additive-free, hard and long aged (at least

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twelve months) cheese made in copper vats from partly skimmed, unpasteurised milk.4 The

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production specification of Parmigiano Reggiano reports specific rules for cow diets, and the use of

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ensiled feeds is expressly forbidden (see ‘Single document of the PDO Parmigiano Reggiano’:

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http://www.parmigianoreggiano.com/consortium/rules_regulation_2/default.aspx).

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The traditional analysis applied by the CFPR to differentiate PRRE cheese from the major

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commercial hard type cheese brands are the determination of soluble nitrogen and free amino

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acids (evaluation of ripening time), the presence of the added lysozyme and the amount of

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copper. In order to enforce the protection of PRRE from the mislabelling fraud, it is desirable to

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develop new objective and robust methods capable of verifying the authenticity of the marketed 3



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products, especially for grated or shredded cheese, for those the direct visual check of the

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trademark logo fire-marked on the cheese rind is not available. Grated PRRE can be easily mixed

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with other low quality cheeses.

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Therefore, more recently spectroscopic techniques have been tested to determine the degree of

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ripening and the geographical origins of PRRE cheese, for example nuclear magnetic resonance,5,6

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stable isotope analysis combined with multielemental analysis7,8 and middle and near infrared

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spectroscopy coupled with statistical techniques.9 Regarding degree of ripening, other analytical

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methods employed were the determination of free amino acids, oligopeptides and D-amino

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acids.10-15

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Fatty acids in food authentication were mainly used for the determination of the cow feeding

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system, in fact the complex fatty acid profile of milk fat furnishes several information related to

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diet composition and to ruminal fermentation pattern and many papers were published on these

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topics.16,17 Fatty acid composition of milk is also affected by the zone of origin and cow feeds, and

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has been proposed to predict cow diet composition and altitude of bulk milk.18,19

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Fatty acids profile were previously determined in PRRE to understand its nutritional properties20

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and free fatty acids were explored as possible indicators of ripening.21 However, no specific

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applications of fatty acids in PRRE authentication were reported. Recently, we reported the

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presence of cyclopropane fatty acids (CPFA, mainly dihydrosterculic acid and lactobacillic acid,

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Figure 1) in milk and dairy products.22 CPFA are produced by some bacterial strains in stress

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conditions.23-26

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Data collected by our research group on hundreds of milk and dairy samples22,27,28 showed that

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CPFA were present in milk only when cows were fed with ensiled feeds (mainly corn silage). On

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the contrary, products obtained without the use of silages (as PRRE and other PDO cheeses, milk,

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butter and cheeses from alpine regions) were found always negative to cyclopropane fatty acid.

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Therefore, CPFA can be proposed as markers in dairy products of the use of silages in cow feeding

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and can represent a useful tool for the quality control of PDO cheeses, whose specifications of

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production does not allow the use of silages: among these PRRE but also Fontina (Italy), Gruyere,

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Comte and so on. It appears clear that to have an analytical method able to discriminate the origin

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of milk used for PRRE production, specifically linked to the type of cow feed used, it is extremely

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important to verify the authenticity of this cheese. In order to enforce the correlation between 4


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CPFA absence and PRRE authenticity in this paper we classify 304 cheese samples based on the

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presence/absence of CPFA. The samples cover four typologies of PDO cheeses of three different

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European countries whose specification rules forbid the use of silages in cow feeding, and one

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PDO cheese that permits the use of silages. Moreover, based on the determination of CPFA

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concentration, we try to define the threshold of adulteration of grated Parmigiano Reggiano with

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other less valuable cheeses that are commonly produced using milk from cows fed with silages.

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MATERIALS AND METHODS

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Chemicals. Dihydrosterculic acid methyl ester (DHSA, purity > 98%) was from Abcam, Cambridge.

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Tetracosane (99%), methanol, potassium hydroxide, n-pentane, n-hexane, acetone, sodium

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bisulphate were from Sigma Aldrich, Saint Louis, MO, USA.

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Sampling. 304 PDO cheese samples of certified origin were provided by Consorzio del Formaggio

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Parmigiano-Reggiano (Reggio Emilia, Italy): Parmigiano Reggiano (200 samples), Grana Padano (68

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samples), Fontina (16 samples), Comté (10 samples), Gruyère (10 samples). Additionally, mixtures

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of grated Grana Padano and Parmigiano Reggiano in different proportions (Figure 4) were

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prepared.

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Whole cheeses are subjected to analysis procedure after removing the surface layer (rind); grated

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cheeses are directly subjected to analysis.

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Preparation of dihydrosterculic acid (DHSA) and tetracosane standard solutions. Appropriate

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amounts of tetracosane (internal standard) and DHSA were weighed and added to hexane (100

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ml), in order to yield final stock solution concentrations of 50 mg/l. The stock solution of DHSA was

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used to obtain diluted solutions of 20, 10, 4, 2 mg/l. 1 ml of each DHSA solution was mixed in vials

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with 1 ml of tetracosane solution (50 mg/l), in order to obtain working solutions at 25, 10, 5, 2 and

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1 mg/l of DHSA, all containing 25 mg/l of tetracosane. These solutions were used to determinate

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response factor, linearity, limit of detection and limit of quantitation.

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Cheese fat extraction. Cheese fat was extracted with n-pentane, according to ISO14156/IDF 172,

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utilizing an automatic extraction apparatus (SER148 Extractor unit, VELP Scientifica) that permits

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the reduction of extraction times to 120 minutes (90 minutes immersion and 30 minutes washing).

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The extract was taken to dryness in a rotary evaporator at 50 °C.

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As comparison, manual fat extractions were also performed: 2 g of grated cheese sample were

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stirred with hexane/acetone mixture (20 ml, 4/1 v/v) or with hexane only heating to about 50 °C to

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melt fat. Extraction was performed twice, the two extracts collected. The extract was taken to

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dryness in a rotary evaporator at 50 °C.

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Cheese fat methylation. The fatty acid methyl esters were prepared according to ISO 15884,

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utilizing n-hexane for fatty acids methyl esters dissolution. 100 mg of fat were dissolved in 4 ml

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hexane and added to 1 ml of tetracosane (50 mg/l in hexane) and mixed for few minutes with 0.2

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ml of KOH 10% in methanol. The upper layer was neutralized with sodium bisulphate, centrifuged

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and 1 µl was used for the GC-MS injection (split mode, 1:20).

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GC-MS analysis. GC-MS analysis was carried out on an Agilent Technologies 6890N gas-

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chromatograph (Agilent Technologies, Palo Alto, CA, USA) coupled to an Agilent Technologies 5973

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mass spectrometer (Agilent Technologies, Palo Alto, CA, USA). A low-polarity capillary column

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(SLB-5ms, Supelco, Bellafonte, USA) was used. The chromatogram was recorded in the scan mode

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(40-500 m/z) with a programmed temperature from 40°C to 280°C. Peak identity of CPFA was

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confirmed by comparison with DHSA pure standard.

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Preparation of cheese spiked samples. 100 mg of cheese fat negative to CPFA (authentic

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Parmigiano Reggiano) was spiked with the appropriate amount of DHSA solutions, in order to

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obtain concentrations of DHSA of 1000, 500, 250, 100 and 50 mg/kg fat. Spiked fat was treated

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according to the methylation protocol. These samples were used for the determination of

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recovery and of the eventual matrix effect on LOD, LOQ, and linearity.

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Determination of CPFA response factor. 1 µl of the solution containing 25 mg/l of DHSA and

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tetracosane was injected in the GC-MS system. The peaks were manually integrated and used to

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determinate response factors utilizing the following equation:

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fCPFA=

where mCPFA is the mass, in milligrams of CPFA in the calibration solution wCPFA is the purity, expressed aa mass fraction in milligrams per milligrams of CPFA Ais is the peak area of the internal standard (tetracosane) mis is the mass, in milligrams of internal standard in the calibration solution wis is the purity, expressed aa mass fraction in milligrams per milligrams of tetracosane ACPFA is the peak area of CPFA

Limit of detection (LOD) and limit of quantitation (LOQ). The limit of detection (LOD) and the limit

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of quantitation (LOQ) were calculated utilizing the S/N ratio methods, based on the determination

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of the peak to peak noise.29 LOD and LOQ were therefore calculated as the concentrations added 6


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to cheese fat sample negative to CPFA producing a recognizable peak with a signal-to-noise ratio

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of, respectively, 3.3 and 10.

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LOD and LOQ were determined both in pure standard solution and in a sample of cheese fat

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negative to CPFA spiked with different concentrations of DHSA.

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Linearity. Linearity of the method was checked at a range between 5 mg/kg fat to 1000 mg/kg fat

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for DHSA and five concentration levels (50, 100, 250, 500, 1000 mg/kg fat) were considered. Three

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replicates were performed for each concentration. Each DHSA solution (25, 10, 5, 2 and 1 mg/l of

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CPFA, all containing 25 mg/l of tetracosane) was added to a ‘blank’ cheese fat sample, and

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subjected to the extraction and derivatization protocol. Regression was performed on the ratio of

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peak areas of analyte and internal standard referred to the fat (mg/kg fat) versus analyte

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concentrations added. The goodness of fit was determined by means of Mandel test at the 99%

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significance.

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Repeatability. Repeatability were calculated for three concentration levels ranging from the limit

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of quantitation 200 mg/kg fat to 1000 mg/kg fat. Results are reported in Table 2. The results of

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precision were compared with Horwitz predicted intralaboratory precision (PRSD), by calculating

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the Horwitz ratio (HORRAT).30

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Data Analysis. Data are obtained as duplicate analysis of two independent sample extractions and

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are presented as mean ± SD. Positivity or negativity of samples to CPFA was defined based on the

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threshold of 60 mg/kg fat; this value corresponds to the experimental detection limit, calculated at

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a signal to noise ratio > 3.

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Content of CPFA was reported as absolute amount in milligrams per kilogram of fat calculated

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respect to the internal standard, according to the following equation:

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wCPFA =

*1000

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where

182 183

mis is the mass, in milligrams of internal standard in the calibration solution

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wis is the purity, expressed as mass fraction in milligrams per milligrams of tetracosane

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ACPFA is the peak area of CPFA

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fCPFA is the mean response factor for CPFA 7



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Ais is the peak area of the internal standard (tetracosane)

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ms is the mass, in grams of the cheese fat sample

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RESULTS AND DISCUSSION

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Development and validation of analytical method for the quantitative determination of CPFA in

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cheese

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Choice of the chromatographic conditions. Chromatographic separation of CPFA was initially based

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on the method published by Montanari et al.,31 using relatively long chromatographic run to

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obtain the separation of the two CPFA isomers (Figure 1). However, we verified that in dairy

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samples lactobacillic acid is in lower amount respect to dehydrosterculic acid, and generally

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behind the limit of quantitation. Therefore, for the purpose of routine quality control of cheeses, it

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resulted more convenient to use shorter run (25 min) and give the results as the sum of

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dihydrosterculic and lactobacillic acid (Figure 2).

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Choice of the internal standard. CPFA were previously determined in milk and dairy samples22 and

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expressed in a semi-quantitative way as percentage on total chromatographic area of fatty acids

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methyl esters. However, this method has some drawbacks, mainly due to the possible saturation

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of the detector for the main fatty acids (as palmitic and oleic acids) that has as outcome the

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overestimation of the percentage of CPFA. Because the determination of CPFA has becoming an

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important criterion for the authenticity of PRRE cheese, a validated quantitative method is

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needed.

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The quantitative analysis of CPFA needed an internal standard. CPFA are minor fatty acids, present

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in low concentration in milk fat, and the internal standard has to be added in comparable amount.

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Milk fat is known to contain the highest number of fatty acids of all edible fats and recently more

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than four hundred different fatty acids have been identified in milk fat.17 In this context, it resulted

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very difficult to find a commercial fatty acid absent in cheese fat. The choice of an internal

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standard easy commercially available was considered of priority, in order to make the method

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easy applicable as a routine control. Therefore, a hydrocarbon (tetracosane) was chosen.

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Tetracosane, on low polarity capillary columns, eluted in a chromatographic zone close to the

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CPFA signal free from other fatty acids methylesters, immediately after docosanoic acid (FIGURE

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2b). The use of an internal standard of different molecular structure needs the accurate

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determination of response factor. It was demonstrated that response factor of CPFA respect to 8


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tetracosane (mean value 1.06 ± 0.23) is not constant during time as it depends by the tuning

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conditions of the instrument. Therefore, to obtain reliable quantitative results, determination of

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response factor has to be performed in duplicate every day before each series of analysis. Another

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point considered was the eventual different recovery of tetracosane and dihydrosterculic acid

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after transesterification. Response factors were determined for solutions of tetracosane and

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dihydrosterculic acid before and after transesterification, and results were not significantly

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different (data not shown).

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Effect of Fat extraction methods. Cheese fat was extracted according to ISO14156/IDF 172, Milk

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and milk products –Extraction methods for lipids and liposoluble compounds. As alternative, the

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following methods, Soxhlet with different solvents and manual extractions, have proven to give

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the same results in terms of dihydrosterculic acid extraction (Table 1). Results of the different

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extractions were compared with one-way ANOVA and the differences were not statistically

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significant (p-level 0.05). The invariance of analytical results in different extraction conditions can

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be considered a prove of the ruggedness of the method.

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Method validation. Optimized GC-MS method was subjected to validation in terms of precision,

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accuracy, linearity, detection and quantitation limits, following recommendations of the

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International Conference on Harmonization (ICH (2005). Validation of analytical procedures: text

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and methodology. Harmonized tripartite guideline, Q2(R1)). All the validation tests were

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performed on cheese sample negative to CPFA spiked with standard of CPFA (dihydrosterculic

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acid) at different concentration levels.

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Limit of detection (LOD), limit of quantitation (LOQ), linearity. The limit of detection (LOD) and

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the limit of quantitation (LOQ) were calculated utilizing the S/N ratio methods, based on the

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determination of the peak to peak noise.29 LOD and LOQ were therefore calculated as the

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concentrations added to cheese fat sample negative to CPFA producing a recognizable peak with a

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signal-to-noise ratio of, respectively, 3.3 and 10. The limit of detection (LOD) were obtained at 60

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mg/kg fat (S/N ratio 3.3), while limits of quantitation (LOQ) was found to be respectively 200

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mg/kg fat (S/N ratio 10). These values were also confirmed by the blank signal determined by the

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intercept of the regression line.

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The linearity was demonstrated in the range 60-1000 mg /kg fat (R2 = 0.9942; slope 0.0044 ±

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0.0005; intercept 0.002 ± 0.002). Mandel test resulted not significant and the linearity within the 9



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range of the sample concentration also demonstrates the constancy of the response factors in the

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same interval.

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Repeatability. Repeatability was calculated on three concentration levels as intra-day precision

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(intermediate precision), obtained using the same method on identical test material in the same

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laboratory by the same operator using the same equipment within a short interval of time. The

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results of precision were compared with Horwitz predicted intralaboratory precision (PRSD),

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calculated as 0.66*2*c-0.1505, where c is the concentration level expressed as mass fraction.

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Considering that a commonly used criterion is that satisfactory precision produce results with a

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HORRAT value less than 2, the repeatability obtained (Table 2) satisfy the Horwitz equation.

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Presence of CPFA in PDO cheese samples

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The quantitative method was applied to the determination of CPFA in authentic PDO cheeses

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obtained with different technologies. Cheeses were chosen based on the possibility of using or not

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ensiled products for cow feeding, as reported in the respective Production Specification Rules. In

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particular, three important Italian PDO cheeses as Parmigiano Reggiano, Fontina and Grana

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Padano were sampled, together with a Switzerland Cheese (Gruyère) and a French cheese

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(Comtè). Except for Grana Padano, Production Specification Rules of the other cheeses expressly

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forbid the use of ensiled feeds in cow feeding. Results obtained for CPFA quantity are reported in

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Table 3.

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CPFA were always absent in PDO cheeses for which the use of silages is forbidden as Parmigiano-

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Reggiano, Fontina (Italy, http://www.fontina-dop.it/pdf/DISCIPLINARE%20FONTINA_inglese.pdf),

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Gruyère (Switzerland, http://gruyere.com/content/ressources/cahier-des-charges-gruyere-aop-

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anglais-2016.pdf)

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http://www.comte.com/fileadmin/upload/mediatheque/documents_pdf/cahier_des_charges_co

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mte_6_mars_2015-.pdf). Grana Padano samples were instead always positive to the CPFA,

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reflecting that silages are not forbidden for their production (Figure 2c). The amount of CPFA

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found in Grana Padano is variable and it ranges from 300 to 830 mg/kg fat, with a global mean

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value of 540 mg/kg, as reported in Table 3. The most frequent values of CPFA in GP are comprised

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between 400-600 mg/kg fat, as evidence by the graph reported in Figure 3, where samples of GP

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are divided in classes according to the range of CPFA.

and

Comté

279 10


(France,

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We previously showed that 80 samples of milk from cows feed with corn silage, were found

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positive for the presence of CPFA and 140 samples of milk from cows fed with forage without corn

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silages were all negative.22 The results reported here for PDO cheeses confirm the strong

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correlation between CPFA presence and use of silage in cow feeding. The results are of particular

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significance for Parmigiano Reggiano, due to the large sampling, comprising cheeses sampled in

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more than half of the Parmigiano Reggiano producing factories (200 in total, spread in all the

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geographical area defined in the Production Specification Rules).

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The presence of CPFA in milk and dairy products probably derives from their presence in ensiled

288

feeds, where CPFA can be released by bacteria during fermentation. The environmental conditions

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developed in silos seem to be essential for the production and release of CPFA from bacteria. In

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fact, we recently demonstrated that any production of CPFA by lactic acid bacteria occurs during

291

milk fermentation, and fermented milks and yogurt maintained the percentages of CPFA present

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in the starting milk. Moreover, CPFA content does not change during the shelf life of the

293

products.28

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Presence of CPFA in mixtures of grated cheeses. We also evaluated the minimum level of

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adulteration of Parmigiano Reggiano that can be evidenced with the proposed method, artificially

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preparing mixtures of Parmigiano Reggiano and other grated hard cheese (Grana Padano). Results

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showed that the CPFA content is proportional to the increase of GP percentage and a linear

298

correlation was observed between the percentage of Grana Padano added to Parmigiano

299

Reggiano and the CPFA content (Figure 4). The dispersion of the values are due to the different

300

samples of GP and PR utilized to prepare the mix, which were chosen different to achieve a more

301

real system. The more important aspect is that the addition of ten percent of Grana Padano to

302

Parmigiano Reggiano can be still detected. Adulteration behind this value has scarce commercial

303

significance. These results demonstrate that CPFA amount can give also information on the entity

304

of adulteration of Parmigiano Reggiano with other cheeses.

305 306

As a whole, CPFA can be considered interesting molecular markers, able to distinguish cheeses

307

obtained from milk of dairy cows fed with silage-based diet from milk of cows fed with hay-based

308

diets. The analysis of CPFA can be a useful tool for the quality control of PDO cheeses, such as

309

Parmigiano Reggiano, whose specifications of production does not allow the use of silages. We

310

found no exception regarding the absence of CPFA in authentic PRRE cheese, so the data collected 11



311

suggests that the presence of CPFA could be used as one of the marker of Parmigiano Reggiano

312

authenticity. Moreover, the quantitative method proposed is relatively simple, assures a quick

313

sample preparation and relies on available instrumentation, thus making it suitable for the

314

screening of a large number of samples with a good cost/analysis ratio.

315 316

References

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Mariani, P. Dairy maturation of milk used in the manufacture of Parmigiano-Reggiano cheese:

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Effects on physico-chemical characteristics, rennet-coagulation aptitude and rheological

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properties. J. Dairy Res. 2008, 75, 218–224.

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5) Consonni, R.; Cagliani, L. R. Ripening and geographical characterization of Parmigiano Reggiano

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cheese by 1H NMR spectroscopy. Talanta. 2008, 76, 200–205.

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493-502.

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434

Legends to figures

435

Figure 1: Structures and chromatographic separation of CPFA according to the method of

436

Montanari et al. 31

437

Figure 2: a) Total chromatogram (gas chromatography-mass spectrometry) of FAMEs of a Grana

438

Padano cheese fat, b) enlarged view of CPFA peak elution zone and c) comparison between a

439

sample positive and a sample negative to CPFA.

440

Figure 3. Distribution of Grana Padano samples in classes according to ranges of CPFA values

441

Figure 4. CPFA content (mg/kg fat) in known mix of Parmigiano Reggiano and Grana Padano grated

442

cheeses

16


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Table 1. Comparison of different extraction methods on the quantitative amount (mg/kg fat) of CPFA in a Grana Padano cheese Method of extraction and solvent utilized Soxhlet - pentane (ISO14156/IDF 172) Soxhlet - hexane Soxhlet hexane/acetone Manual hexane Manual hexane/acetone

CPFA mean (n=4) 490 500 460 510 480

standard deviation 10 20 10 30 10

17


RSD % 2,3 4,6 3,0 4,8 2,3


Table 2: intra-day repeatability of the method, calculated on a blank cheese fat sample spiked with different CPFA levels

CPFA level

Intra-day repeatability (RSD %)

Horwitz ratio for intra-day repeatability

200

2.30

0.11

500

1.55

0.10

1000

1.99

0.19

(mg/kg fat)

18


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Table 3: Presence of CPFA in cheeses of known origin Sample Parmigiano Reggiano Fontina Comtè Gruyere

Use of No. of No. of samples silages samples positive to CPFA* forbidden 200 0

Mean (mg/kg fat) ± Range SD (min-max) Nd nd

forbidden 16 forbidden 10 forbidden 10

0 0 0

nd nd nd

nd nd nd

68

540 ± 110

300-830

Grana Padano permitted 68

*positivity for CPFA > 60 mg/kg fat (LOD)

19



Figure 1

Figure 2

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Figure 3

Figure 4

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TOC graphic

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