Cheeses

Chapter 19

Cheeses Tullia Tedeschi, Gianni Galaverna, Arnaldo Dossena and Stefano Sforza Department of Organic and Industrial Chemistry, University of Parma, Parco Area delle Scienze 17A, Parma, Italy

Chapter Outline 1. PDO Cheeses 479 1.1. The Protected Designation of Origin for Cheeses 479 1.2. PDO Cheeses in the EU481 1.3. Characteristics of Some of the Most Famous PDO Cheeses 481 2. Traditional Techniques for the Determination of the Authenticity of Cheeses 494 2.1. Physico-Chemical Analyses 494 2.2. ELISA Assays 495 2.3. HPLC 495 2.4. GC 496

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2.5. Sensory Analyses 3. Advanced Techniques for the Determination of the Authenticity of Cheeses 3.1. NIR and MIR Spectroscopy 3.2. Nuclear Magnetic Resonance Spectroscopy 3.3. Stable Isotope Analysis 3.4. DNA Analysis 3.5. LC/MS 4. Conclusions and Future Trends References

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498 498

500 502 503 504 505 506

PDO CHEESES

1.1 The Protected Designation of Origin for Cheeses The designation of origin is a system that protects a cultural and gastronomic heritage. The system guarantees the product’s origin and uniqueness, and it ensures that its production follows strict specifications established through traditional and ancestral know-how. This approach is typical of the European Countries and PDO is part of the protected geographical status system in the EU. It is mainly focussed on the careful legislation of the labelling to protect European food and drink products from potential fraudulent reproduction. Comprehensive Analytical Chemistry, Vol. 60. http://dx.doi.org/10.1016/B978-0-444-59562-1.00019-0 © 2013 Elsevier B.V. All rights reserved.

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For a dairy product to be acknowledged as a designation of origin, it must (1) be produced in a determined region or zone, (2) follow specific production regulations, (3) have a historically established reputation, and (4) apply for and obtain a PDO recognition from the European Union. In addition to the PDO label, within the European Union, other labels are also used, such as the protected geographical indicator (PGI) label and the traditional specialty guaranteed label, based on the legal framework provided by the Council Regulation (EC) No 510/2006 of 20 March 2006 (Official Journal of the European Union, L93/12, 31 March 2006). These labels are used slightly differently from protected designation of origin (PDO), but all are designed to protect foods that are unique to specific regions of Europe, especially the rural areas. The general aim is to ensure the preservation of traditional methods of food production, which can also encourage people to stay settled in rural areas by providing an economic incentive to produce traditional foods, and which can increase consumer confidence by certifying that foods with a PDO label are produced according to a basic standard. The law is aiming at protecting the reputation of regional foods, promoting rural and agricultural activity, helping producers to obtain a premium price for their products and eliminate unfair competition and misleading of consumers by non-genuine products, which may be of inferior quality or of a different flavour. Among the EU countries, France, Italy, and Spain are owners of the highest number of foods with a PDO label. Indeed, they all have their own version of the PDO system: ‘Appellation d’origine controlee’ in France, ‘Denominazione di origine controllata’ in Italy, and ‘Denominacion de Origen’ in Spain. In many cases, the EU PDO/PGI system works parallel with the system used in the specific country, and in some cases, it is subordinated to the appellation system that was already applied, particularly with wine, for example, and in France with cheese. For example, many French cheeses have both PDO (AOP in French) and AOC classifications, but generally only the AOC classification is shown. As far as the specification related to the PDO label is concerned, in the case of cheese, it may indicate several peculiar characteristics such as that the cheese must be produced in a particular place from unpasteurized milk produced by a specific breed of cattle and that it must be cured in moulds of a certain size and shape. The procedure to obtain a PDO or PGI label is as follows: first an application is made to the authorities of the specific Member State; then, if it is judged consistent with the criteria in the regulation and thus acceptable, it is forwarded to the European Commission for final approval. In many cases, very specific names have been applied, in order to protect a specific product, without harming more general business. As an example, Cheddar cheese is a generic name used for the production of this type of cheese in different countries (e.g., the USA, Australia and New Zealand) and thus it is not protected, but the PDO ‘West Country farmhouse Cheddar cheese’ was applied

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to the specific product from the United Kingdom. On the contrary, Feta was registered as a PDO, thus disappointing cheese makers outside Greece. In some cases, products are protected in Europe but not elsewhere: as an example, Buffalo Mozzarella is protected in Europe, but the name is used without restrictions by U.S. dairy companies. Another peculiar characteristic is related to geographical limitations, which can be quite strict. As an example, Stilton cheese can only be produced in the three English counties of Derbyshire, Leicestershire, and Nottinghamshire. This tells a peculiar story, as Stilton village is in the traditional county of Huntingdonshire, now a district of Cambridgeshire, which results in the impossibility to produce Stilton cheese in Stilton. The protection for names on products both made and sold outside the EU is not unconditional outside the European Union; however, there are a number of bilateral agreements with the EU and other countries. As examples, agreements exist between the EU and Australia (wine, 1994) (but not cheese), Canada (wine and spirits, 2003), Chile (wine and spirits, 2002), Colombia (coffee, 2007), Mexico (spirit drinks, 1997), and South Africa (wine and spirits, 2002). Within the EU, many different kinds of cheeses are produced, which are the expression of ancient tradition and food production capacities, thus resulting in a large number of typical cheese products which successfully applied for the PDO logo. In the following paragraphs, the PDO cheeses produced in the EU are listed and described.

1.2 PDO Cheeses in the EU Each region in the EU has some ancient tradition of cheese making related also to the history and costumes of the territory and its population. In Tables 1–7, the complete list of PDO cheeses approved within the EU is given: the milk-type and the specific country or region of production are indicated (http://www.fromages-aop.com/index.php/en/european-pdo). There is also a PDO product from Cyprus, Halloumi, which is produced from eweand goat milk. A very large variety of characteristics of production (e.g., milk-type, type of starter cultures or inoculum, type of clotting enzymes and type of processing), which give rise to very different textural and organoleptic features, is one of the most striking examples of diversification in food production systems. In the following paragraph, some of the most famous PDO cheeses produced within the EU are described.

1.3 Characteristics of Some of the Most Famous PDO Cheeses 1.3.1 Gorgonzola (Italy) Gorgonzola (www.gorgonzola.com) is a veined Italian blue cheese whose origins were related to the town of Gorgonzola (Lombardy) where it has been

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TABLE 1 PDO Cheeses of Austria and Germany Name of the Cheese

Milk Type

Country of Origin

Gaitaler Almka¨se

Cow milk

Austria

Tiroler Almka¨se/Tiroler Alpka¨se

Cow milk

Austria

Tiroler Bergka¨se

Cow milk

Austria

Tiroler Grauka¨se

Cow milk

Austria

Vorarlberger Alpka¨se

Cow milk

Austria

Vorarlberger Bergka¨se

Cow milk

Austria

Allga¨uer Bergka¨se

Cow milk

Germany

Allga¨uer Emmentaler

Cow milk

Germany

Altenburger Ziegenka¨se

Cow milk and goat milk

Germany

Odenwa¨lder Fru¨hstu¨ckska¨se

Cow milk

Germany

TABLE 2 PDO Cheeses of GREECE Name of the Cheese

Milk Type

Anevato

Ewe and/or goat milk

Batzos

Ewe and/or goat milk

Feta

Ewe and/or goat milk

Formaella Arachovas Parnassou

Ewe and/or goat milk

Galotyri

Ewe and/or goat milk

Graviera Agrafon

Ewe and/or goat milk

Graviera Kritis

Ewe and/or goat milk

Graviera Naxou

Cow milk

Kalathaki Limnou

Ewe and/or goat milk

Kasseri

Ewe and/or goat milk

Katiki Domokou

Goat and/or ewe milk

Kefalograviera

Ewe and/or goat milk

Kopanisti

Cow, ewe and goat milk

Ladotyri Mytilinis

Ewe and/or goat milk

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TABLE 2 PDO Cheeses of GREECE—Cont’d Name of the Cheese

Milk Type

Manouri

Ewe and/or goat milk

Metsovone

Cow, ewe and goat milk

Pichtogalo Chanion

Ewe and/or goat milk

San Michali

Cow milk

Sfela

Ewe and/or goat milk

Xynomyzithra Kritis

Ewe and/or goat milk

TABLE 3 PDO Cheeses of Northern Europe Name of the Cheese

Milk Type

Country of Origin

Imokilly Regato

Cow milk

Ireland

Bryndza Podhalan˜ska

Ewe and cow milk

Poland

Oscypek

Ewe and cow milk

Poland

Fromage de Herve

Cow milk

Belgium

Boeren-Leidse met sleutels

Cow milk

The Netherlands

Kanterkaas, Kanternagelkaas, Kanterkomijnekaas

Cow milk

The Netherlands

Noord-Hollandse Edammer

Cow milk

The Netherlands

Noord-Hollandse Gouda

Cow milk

The Netherlands

Beacon Fell traditionnal Lancashire cheese

Cow milk

The United Kingdom

Bonchester cheese

Cow milk

The United Kingdom

Buxton blue

Cow milk

The United Kingdom

Dovedale cheese

Cow milk

The United Kingdom

Single Gloucester

Cow milk

The United Kingdom

Staffordshire Cheese

Cow milk

The United Kingdom

Swaledale cheese/ swaledale ewes’ cheese

Ewe milk

The United Kingdom

Continued

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TABLE 3 PDO Cheeses of Northern Europe—Cont’d Name of the Cheese

Milk Type

Country of Origin

West Country Farmhouse Cheddar Cheese

Cow milk

The United Kingdom

White Stilton Cheese/Blue Stilton Cheese

Cow milk

The United Kingdom

TABLE 4 PDO Cheeses of Portugal Name of the Cheese

Milk Type

Queijo de Azeitao

Ewe milk

Queijo de cabra Transmontano

Goat milk

Queijo de Evora

Ewe milk

Queijo de Nisa

Ewe milk

Queijo de Pico

Ewe milk

Queijo Rabac¸al

Ewe and goat milk

Queijo S. Jorge

Cow milk

Queijo Serpa

Ewe milk

Queijo Serra da Estrela

Ewe milk

Queijo Terrincho

Ewe milk

Queijos da Beira Baixa

Ewe and goat milk

TABLE 5 PDO Cheeses of Spain Name of the Cheese

Milk-Type

Cabrales

Goat and/or ewe milk

Idiazabal

Ewe milk

Mahon–Menorca

Cow milk

Picon Bejes-Tresviso

Cow and goat and ewe milk

Queso de l’Alt Urgell y la Cerdanya

Cow milk

Queso de la Serena

Ewe milk

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TABLE 5 PDO Cheeses of Spain—Cont’d Name of the Cheese

Milk-Type

Queso de Murcia

Goat milk

Queso de Murcia al vino

Goat milk

Queso Ibores

Goat milk

Queso Majorero

Goat milk

Queso Manchego

Ewe milk

Queso Nata de Cantabria

Cow milk

Queso Palmero ou Queso de La Palma

Goat milk

Queso Tetilla

Cow milk

Queso Zamorano

Ewe milk

Quesucos de Lie´bana

Cow and ewe and goat milk

Roncal

Ewe milk

Torta del Casar

Ewe milk

TABLE 6 PDO Cheeses of Italy Name of the Cheese

Milk Type

Asiago

Cow milk

Bitto

Cow milk

Bra

Cow milk

Caciocavallo Silano

Cow milk

Canestrato Pugliese

Ewe milk

Casciotta d’Urbino

Ewe milk

Castelmagno

Cow milk

Fiore Sardo

Ewe milk

Fontina

Cow milk

Formai de Mut dell’Alta Valle Brembana

Cow milk

Gorgonzola

Cow milk

Grana Padano

Cow milk Continued

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TABLE 6 PDO Cheeses of Italy—Cont’d Name of the Cheese

Milk Type

Montasio

Cow milk

Monte Veronese

Cow milk

Mozzarella di Bufala Campana

Buffalo milk

Murazzano

Ewe and cow milk

Parmigiano-Reggiano

Cow milk

Pecorino di Filiano

Ewe milk

Pecorino Romano

Ewe milk

Pecorino Sardo

Ewe milk

Pecorino Siciliano

Ewe milk

Pecorino Toscano

Ewe milk

Provolone Valpadana

Cow milk

Quartirolo Lombardo

Cow milk

Ragusano

Cow milk

Raschera

Cow milk

Ricotta Romana

Cow milk

Robiola di Roccaverano

Cow milk

Spressa delle Giudicarie

Cow milk

Stelvio ou Stilfser

Cow milk

Taleggio

Cow milk

Toma Piemontese

Cow milk

Valle d’Aosta Fromadzo

Cow milk

Valtellina Casera

Cow milk

produced since 879 B.C. It was matured in natural caves to acquire the characteristic veining due to the presence of specific strains of mould. Today, it is mainly produced in the northern Italian regions of Piedmont and Lombardy, using whole cow milk to which rennet and starter bacteria are added, along with spores of Penicillium roqueforti. The whey is removed during curdling, and the cheese is dry-salted and aged at low temperatures (ageing time typically 3–4 months). Its texture changes from buttery to firm, crumbly and quite salty, with a ‘bite’ from its blue veining. The cheese’s characteristic veining is

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TABLE 7 PDO Cheeses of France Name of the Cheese

Milk Type

Region

Munster

Cow milk

Alsace-Lorraine

Bleu d’Auvergne

Cow milk

Auvergne

Cantal

Cow milk

Auvergne

Fourme d’Ambert

Cow milk

Auvergne

Saint-Nectaire

Cow milk

Auvergne

Salers

Cow milk

Auvergne

Epoisses

Cow milk

Burgundy

Chaource

Cow milk

Champagne

Langres

Cow milk

Champagne

Brocciu

Ewe milk

Corsica

Bleu de Gex Haut-Jura

Cow milk

Franche-Comte´

Comte´

Cow milk

Franche-Comte´

Mont d’Or

Cow milk

Franche-Comte´

Morbier

Cow milk

Franche-Comte´

Camembert de Normandie

Cow milk

Normandy

Isigny Butter and Cream

Cow milk

Normandy

Livarot

Cow milk

Normandy

Neufchaˆtel

Cow milk

Normandy

Pont-l’Eveˆque

Cow milk

Normandy

Chabichou du Poitou

Goat milk

Poitou-Charentes

Charentes-Poitou Butter

Cow milk

Poitou-Charentes

Abondance

Cow milk

Savoy

Beaufort

Cow milk

Savoy

Chevrotin

Goat milk

Savoy

Reblochon

Cow milk

Savoy

Tome des Bauges

Cow milk

Savoy

Chavignol

Goat milk

The Loire Valley

Pouligny-St-Pierre

Goat milk

The Loire Valley

Sainte Maure de Touraine

Goat milk

The Loire Valley Continued

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TABLE 7 PDO Cheeses of France—Cont’d Name of the Cheese

Milk Type

Region

Selles-sur-Cher

Goat milk

The Loire Valley

Valenc¸ay

Goat milk

The Loire Valley

Brie de Meaux

Cow milk

The North-East (Brie and Thierache)

Brie de Melun

Cow milk

The North-East (Brie and Thierache)

Maroilles

Cow milk

The North-East (Brie and Thierache)

Banon

Goat milk

The South (the Mediterranean)

Pe´lardon

Goat milk

The South (the Mediterranean)

Ossau-Iraty

Ewe milk

The South-West (Aquitaine)

Bleu des Causses

Cow milk

The South-West (Midi-Pyre´ne´es)

Laguiole

Cow milk

The South-West (Midi-Pyre´ne´es)

Rocamadour

Goat milk

The South-West (Midi-Pyre´ne´es)

Roquefort

Ewe milk

The South-West (Midi-Pyre´ne´es)

Bleu du Vercors-Sassenage

Cow milk

The South-East (Rhoˆne-Alpes)

Fourme de Montbrison

Cow milk

The South-East (Rhoˆne-Alpes)

Picodon

Goat milk

The South-East (Rhoˆne-Alpes)

obtained by inserting and removing metal rods during ageing, thus allowing the development of the mould spores into hyphae. Two main varieties are produced: Gorgonzola Dolce (sweet) and Gorgonzola Piccante (piquant), which differ in ageing time. The PDO is referred to the product from the provinces of Novara, Bergamo, Brescia, Como, Cremona, Cuneo, Lecco, Lodi, Milan, Pavia, Varese, Verbano-Cusio-Ossola, and Vercelli, as well as some municipalities in the area of Casale Monferrato (province of Alessandria).

1.3.2 Pecorino Sardo (Italy) Pecorino Sardo (www.pecorinosardo.it) is a hard cheese made from fresh whole sheep milk in the island of Sardinia (Italy). It is curdled using lamb or kid rennet, poured into moulds to obtain the characteristic shape; it has a firm consistency and a rich flavour. After a brief period in brine, the moulds are lightly smoked and left to ripen in cool cellars in central Sardinia (average weight about 3.5 kg), resulting in a rind varying from deep yellow to dark brown in colour and with a white to straw-yellow paste.

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1.3.3 Asiago (Italy) Asiago (www.asiagocheese.it) is produced from cow milk in two main types according to the ageing time: fresh Asiago Pressato, with a smooth texture and aged Asiago d’Allevo, with a crumbly texture and a stronger flavour. It is produced in a restricted area between the Po valley and the Asiago plateau (Vicenza, Veneto region): the area extends to the entire Vicenza and Trento provinces and part of the provinces of Padua and Treviso. The cheese produced and aged in dairies located more than 600 m above sea level has the additional label ‘Product of the Mountains’. Its origins date back to 1000 B.C., although the use of bovine milk instead of sheep milk became common during the nineteenth century. Pressed Asiago cheese is made from fresh whole milk, curdled with rennet and cooked; it gives its name to the squeezing of the mould by hydraulic pressing. After brining, the maturing period is about 20–40 days, giving a finished cheese with a cylindrical shape with a diameter of 30–40 cm and height of about 15 cm (average weight 11–15 kg). The crust is thin and elastic with an inner soft, buttery, white or slightly yellowish paste with a creamy and milky flavoured sweet and delicate taste. Asiago d’Allevo is produced by using a mixture of whole milk and skimmed milk, curdled with rennet and cooked. The paste is formed in moulds with cheesecloths, drained and turned several times, and then salted by spreading salt over the surface or by soaking in brine. The ageing process lasts at least 60 days in warehouses with storage temperature and relative humidity around 10–15  C and 80–85%. According to the ageing time, we have Asiago Mezzano (3–8 months ageing), Asiago Vecchio (9–18 months ageing), and Asiago Stravecchio (more than 18 months of ageing), changing from a compact paste, straw-coloured and with a sweetish taste to a very hard and grainy paste, amber-coloured, with a bitter and spicy taste. 1.3.4 Parmigiano-Reggiano (Italy) Parmigiano-Reggiano (www.parmigiano-reggiano.it) is a hard, granular cheese, cooked but not pressed, named after the producing areas near Parma, Reggio Emilia, Modena, and Bologna (all in Emilia-Romagna), and Mantova (in Lombardia), produced from raw cow milk. Traditionally, cows have to be fed only on grass or hay. Only natural whey culture is allowed as a starter, together with calf rennet. The whole milk of the morning milking is mixed with the naturally skimmed milk (it is left in large shallow tanks to allow the cream to separate) of the previous evening’s milking, resulting in a partially skimmed mixture. Copper-lined vats are used for curdling (with calf rennet) and cooking (55  C). The curd is then collected in a piece of muslin before being divided into two parts and placed in moulds. Salting is performed for 20–25 days by dipping into brine, after that ageing lasts an average of 2 years (final product averages about 38-kg weight). Parmigiano-Reggiano cheese has a sharp, complex fruity/nutty, and umami taste (it is characterized by a high amount of free glutamate, 1.2 g ca. per 100 g) with a strong savoury

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flavour and a slightly gritty texture. According to legend, Parmigiano-Reggiano was created in the course of the Middle Ages in Bibbiano, in the province of Reggio Emilia. It was cited as early as 1348 in the writings of Boccaccio, who, in the Decameron, describes ‘a mountain, all of grated Parmesan cheese’, on which ‘dwell folk that do nought else but make macaroni and ravioli, and boil them in capon’s broth, and then throw them down to be scrambled for; and hard by flows a rivulet of Vernaccia, the best that ever was drunk, and never a drop of water therein’. (Giovanni Boccaccio, Decamerone, VIII 3).

1.3.5 Taleggio (Italy) Taleggio (www.taleggio.it) is a washed rind and smear-ripened Italian cheese that is named after Val Taleggio (Bergamo province). The cheese has a strong aroma but its flavour is comparatively mild, with an unusual fruity tang. Its crust is thin. The name Taleggio has been used before the tenth century in the caves of Val Taleggio: it might be one of the oldest soft cheeses. The production takes place every autumn and winter. First, the acidified milk from milk calves is brought to the laboratory. The cheese is set on wooden shelves in chambers, sometimes traditionally in caves, and matures within 6–10 weeks. It is washed once a week with a seawater sponge, in order to prevent mould infestation, and to prevent the cheese from forming an orange or rose crust. Today, the area of production is Lombardy (provinces of Bergamo, Brescia, Como, Cremona, Lecco, Lodi, Milano, Pavia), Piedmont (province of Novara), and Veneto (province of Treviso). 1.3.6 Mozzarella di Bufala (Italy) Mozzarella di bufala is a mozzarella made from the milk of the domestic water buffalo. In Italy, the cheese is produced in areas ranging from Rome in Lazio to Paestum (near Salerno) in Campania, and there is a production area near Foggia, Puglia. Buffalo mozzarella from Campania has the PDO label ‘Mozzarella di Bufala Campana’ (www.mozzarelladop.it). Different theories are present for the origin of the domestic water buffalo in Italy, probably introduced by strangers during migration. Buffalo mozzarella became widespread throughout the south of Italy, especially from the second half of the eighteenth century. For the production, after milk is heated and curdled with natural whey, hot water is poured on the curd to obtain ‘pasta filata’ (soft). After cooling, the cheese is pickled with the original whey and packaged in special bags or plastic. 1.3.7 Feta (Greece) Feta is a brined curd cheese traditionally made in Greece (first record from the Byzantine Empire). Feta is a crumbly aged cheese, commonly produced in blocks, and has a slightly grainy texture. Since 2002, feta has been a PDO product in the EU. According to the relevant EU legislation, only those

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cheeses produced in a traditional way in some areas of Greece (mainland and the island of Lesbos), and made from sheep milk or from a mixture of sheep and goat milk (up to 30%) of the same area, may bear the name ‘feta’. This has resulted after a long legal battle with Denmark, which produced an artificially blanched cow milk cheese with the same name. Feta is salted and cured in a brine solution (with water or whey) for several months; when removed from the brine, it dries rapidly.

1.3.8 Manchego (Spain) Queso Manchego (www.quesomanchego.es) is a cheese made in the La Mancha region of Spain from the milk of sheep of the Manchega breed. Official Manchego cheese is aged for a period of 60 days to 2 years. Manchego has a firm and compact consistency and a buttery texture and often contains small, unevenly distributed air pockets. The colour of the cheese varies from white to ivory-yellow and the inedible rind from yellow to brownish beige. The cheese has a distinctive flavour, well developed but not too strong, creamy with a slight piquancy, and leaves an aftertaste that is characteristic of sheep milk. To be designated as Queso Manchego, the cheese must be produced in a restricted area of the provinces of Albacete, Ciudad Real, Cuenca, and Toledo (La Mancha region). The cheese is produced by pressing in a cylindrical mould that has a maximum height of 12 cm and a maximum diameter of 22 cm. Manchego cheese can be made from pasteurized or raw milk (labelled as Artesano). The only permitted additives are natural rennet, or an approved coagulating enzyme, and sodium chloride (salt). The moulds in which the cheese is pressed are barrel shaped and leave a distinctive zigzag pattern (known as pleita) on the rind. There are three versions of the cheese: (1) Fresco, fresh cheese aged for only 2 weeks, with a rich but mild flavour; (2) Curado, a semi-firm cheese aged for 3–6 months, with a sweet and nutty flavour; and (3) Viejo, aged for 1 year, firm with a sharper flavour and a rich deep pepperiness. 1.3.9 Cheddar (United Kingdom) Cheddar cheese is a relatively hard, pale yellow to off-white (unless artificially coloured), and sometimes sharp-tasting, cheese. Originating in the English village of Cheddar in Somerset, cheeses of this style are produced also in several countries around the world. Indeed, the name ‘Cheddar cheese’ is widely used (in the United States, Australia, and New Zealand) and has no PDO within the European Union, but only Cheddar produced from local milk within four counties of South West England (Somerset, Devon, Dorset, and Cornwall) may use the name ‘West Country Farmhouse Cheddar’ (www. ukprotectedfoods.com/west-country-farmhouse-cheddar-pdo). The cheese originates from the village of Cheddar in Somerset, South- West England. Cheddar Gorge on the edge of the village contains a number of caves, which

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provided the ideal humidity and constant temperature for maturing the cheese, which has been produced since at least the twelfth century. Cheddar is usually a deep to pale yellow (off-white) colour with a sharp, pungent flavour, often slightly earthy. The term cheddaring refers to an additional step in the production of Cheddar-style cheese where, after heating, the curd is kneaded with salt, cut into cubes to drain the whey, then stacked and turned. Strong, extra-mature Cheddar, sometimes called vintage, needs to be matured for up to 15 months.

1.3.10 Stilton (United Kingdom) Stilton is a type of English cheese, known for its characteristic strong smell and taste. It is produced in two varieties: the well-known blue and the less-known white. This PDO status means that only cheese produced in the three counties of Derbyshire, Leicestershire, and Nottinghamshire and made according to a strict code may be called ‘Stilton’ (www.stiltoncheese.co.uk). Blue Stilton’s distinctive blue veins are created by piercing the crust of the cheese with stainless steel needles, allowing air into the core. The manufacturing and ripening process takes approximately 9–12 weeks. 1.3.11 Edam (The Netherlands) Edam (Dutch: Edammer) is a Dutch cheese traditionally sold in spheres with a pale yellow interior and a coat of red paraffin wax. It is named after the town of Edam in the province of North Holland, where the cheese is coated for export sale and for the tourist high season. After ageing for at least 17 weeks, the cheese sphere is coated with black wax, rather than the usual red or yellow. Most ‘young’ Edam cheese sold in stores has a very mild flavour and is slightly salty or nutty. As the cheese ages, its flavour sharpens, and it becomes firmer (www.edam.com/edam_cheese.htm). 1.3.12 Roquefort (France) Roquefort (www.roquefort.fr) is a sheep-milk blue cheese from the south of France, and together with Bleu d’Auvergne, Stilton, and Gorgonzola is one of the world’s best known blue cheeses. Only those cheeses aged in the natural Combalou caves of Roquefort-sur-Soulzon may bear the name Roquefort. Roquefort is made entirely from the milk of the Lacaune, Manech, and Basco-Be´arnaise breeds of sheep. The cheese is produced throughout the de´partement of Aveyron and part of the nearby de´partements of Aude, Loze`re, Gard, He´rault, and Tarn. The cheese is white, tangy, crumbly and slightly moist, with distinctive veins of green mould and no rind. It has a characteristic odour and flavour, with a notable taste of butyric acid. The overall flavour sensation is mild, sweet, smoky and salty. A typical wheel of Roquefort weighs between 2.5 and 3 kg and is about 10-cm thick. Roquefort, or similar style cheese, is mentioned in literature as far back as AD 79, when Pliny the Elder

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remarked upon its rich flavour. The mould that gives Roquefort its distinctive character (P. roqueforti) is found in the soil of the local caves. Traditionally, the cheesemakers extracted it by leaving bread in the caves for 6–8 weeks until it was consumed by the mould. The interior of the bread was then dried to produce a powder. In modern times, the mould can be grown in a laboratory, which allows for greater consistency. The mould may either be added to the curd, or introduced as an aerosol, through holes poked in the rind.

1.3.13 Camembert (France) Camembert is a soft, creamy, surface-ripened cow milk cheese. It was first made in the late eighteenth century at Camembert, Normandy, in northern France (www.france.fr/en/gastronomy/fiche-synthetique/camembert-normandy). The AOC variety ‘Camembert de Normandie’ is required by law to be made only with unpasteurized milk. The cheese is made by inoculating warm milk with mesophilic bacteria, then adding rennet and allowing the mixture to coagulate. The curd is then cut into roughly 1 cm (1/2 in.) cubes, salted, and transferred to low cylindrical Camembert moulds. The moulds are turned every 6–12 h to allow the whey to drain; after 48 h, each mould contains a flat, cylindrical, solid cheese mass weighing approximately 350 g. The surface of each cheese is then sprayed with an aqueous suspension of the mould Penicillium camemberti and the cheeses are left to ripen for at least 3 weeks. The ripening process produces the distinctive powdery rind and creamy interior texture characteristic of the cheese. Once the cheeses are sufficiently ripe, they are wrapped in paper and placed in wooden boxes for transport. Camembert cheese gets its characteristic flavour from many naturally occurring chemical substances, including ammonia, succinic acid, and salt. 1.3.14 Comte´ (France) Comte´ (also called Gruye`re de Comte´) (www.comte.com) is a French cheese made from unpasteurized cow milk in the Franche-Comte´ region of eastern France since the twelfth century. The cheese has the shape of circular discs (40–70 cm in diameter, 10 cm in height, 50 kg). The rind is usually a dustybrown colour, and inside it is a pale creamy yellow. The texture is relatively hard and flexible, and the taste is strong and slightly sweet. Fresh milk is poured into large copper vats where it is gently warmed and coagulated with rennet. After placing into moulds and pressing out of the whey, the salted cheese is matured for several months (up to 24 months and more). 1.3.15 Cabrales (Spain) Cabrales (www.quesocabrales.org) is a cheese made in the artisan tradition by rural dairy farmers in the north of Spain. This cheese can be made from pure, unpasteurized cow milk or blended in the traditional manner with goat and/or sheep milk, conferring a stronger, spicy flavour.

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Cabrales must be produced with milk from cows in a small zone of production in Asturias, in the mountains of the Picos de Europa. The milk is first heated and curdled by the addition of rennet. After removing the whey, the curd is packed into cylindrical moulds called arnios and salted. After the initial curing period of around 2 weeks, the Cabrales is then aged a further 2–5 months in natural caves in the mountains of the area. The cheeses are placed on wooden shelves known as talameras, where they are periodically turned and cleaned: during this period, penicillium moulds develop, producing blue–green veins throughout the cheese. Cabrales has a strong flavour, sometimes quite acidic, and along with Gorgonzola and Roquefort it is one of the most famous blue cheeses. Traditionally, Cabrales was sold wrapped in the moist leaves of Acer pseudoplatanus (Sycamore maple), but nowadays it is sold in a dark green-coloured aluminium foil.

2 TRADITIONAL TECHNIQUES FOR THE DETERMINATION OF THE AUTHENTICITY OF CHEESES In order to detect adulteration and to guarantee authenticity, a classical approach is to determine the amount of one or more marker compounds in a suspect cheese and to compare this amount with a ‘reference’ cheese of certain origin. Actually, cheese production can differ according to the feeding system of the animals providing the milk, the starters used, the heating temperature, the salting, and the ripening time, and all these parameters generate defined chemical, physical, or microbial differences, which in turn can be detected, providing indication of the origin of the cheeses [1]. This approach might be hampered by several problems, of which the most important is certainly the often limited knowledge on the correct lower and upper boundaries for reference compounds (usually requiring a large number of ‘known’ samples to build up a reliable reference dataset), in turn linked to the intrinsic variability expected to be present in cheeses produced at different times, especially for artisanal-produced cheeses. As a consequence, it is not always possible to make a definitive statement, even by measuring a large number of compounds, on the authenticity of all products. Moreover, the large number and diversity of analyses required, and the complexity of their execution and interpretation often make this approach feasible only for advanced research centers and universities.

2.1 Physico-Chemical Analyses In general, simple physico-chemical analyses (total and soluble nitrogen content, salt, moisture, pH) are the simplest way to gather data on cheese composition. They are, in general, quite easy to perform, and thus affordable for many small laboratories, but have the drawback of providing a very ‘bulk’ view of the compounds contained in a cheese, with no or scarce molecular

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details. Most of the time, the use of these data for a clear-cut cheese authentication is at best difficult, even if some promising examples are reported in the literature. In a striking example of analysis performed on 80 Spanish cheeses belonging to 10 different varieties, it was shown that those cheeses could be correctly classified by measuring very simple chemical parameters easily obtainable through relatively simple analyses (nitrogen, non-protein nitrogen, moisture, salt, pH) and combining them in a multivariate analysis [2]. In a very similar approach, total nitrogen, water-soluble nitrogen, 12% TCAsoluble nitrogen and pH were used to discriminate Emmental cheeses produced in different European countries at different times of the year. The result was again obtained by the use of advanced multivariate statistical analysis and neural networks [3]. In a different approach, performed on a traditional semihard French cheese, cheeses produced in the mountains had higher proteolysis values than cheeses produced on the valleys [4].

2.2 ELISA Assays Enzyme Immunoassays such as ELISA are also quite widespread as authentication methods, being easy to use, rapid and eventually readily automatable [5]. Many examples of enzyme immunoassays have been reported for species authentication in milk and cheese. Indirect competitive ELISA has been developed to detect low percentages of undeclared bovine milk in goat, ewe, and buffalo milk [6]. Quite analogously, an indirect ELISA using monoclonal antibodies against bovine beta-casein was developed for the detection of adulteration in non-declared cow cheeses [7]. In the market, there are many immunoassay kits for species authentication in milk and cheeses. These kits require a minimal amount of training and equipment and can be used for the rapid identification and quantification of the milk species used in cheeses [8].

2.3 HPLC Beside these simple analyses, more sophisticated ones, but also quite widespread, such as chromatography, can also be used for getting more information on the molecular composition of the cheeses. Reversed-phase HPLC was used for analyzing the retentate and permeate of the water soluble fraction of 60 Cheddar cheeses, varying in age and flavour quality. Rather poor classification was obtained in these cases, mostly based on amino acid content, the most abundant molecules in these fractions [9]. Chemometric tools (principal component analysis (PCA) and hierarchical cluster analysis) were applied to RP-HPLC chromatograms of ethanol-soluble and ethanol-insoluble fractions and free amino acids, to evaluate proteolysis in model cheeses (Cheddar-type) made with different single strain starters, succeeding in differentiating the samples based on the composition of the starters [10]. RP-HPLC was used to detect and quantify different percentages of bovine, ovine and

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caprine milk used to manufacture mixed cheeses, through the chromatographic profiles of whey proteins extracted from these cheeses themselves, which turned out to be very similar to the profiles obtained from the corresponding milk mixtures [11]. Similar approaches were used for analyzing the ethanol-soluble and the ethanol-insoluble fractions extracted from Italian PDO ewe cheeses, showing differences in the peptide profiles [12]. Similarly, different proteolytic profiles obtained by RP-HPLC and capillary electrophoresis were analysed by PCA, allowing the discrimination of three different Norwegian cheese varieties. The difference in the profiles appeared to be due to the different composition of the starter cultures [13]. Quite analogously, HPLC profiles were used to analyse the products of proteolysis in 40 Hungarian cheeses of different ripening time, allowing, by applying PCA procedures on those profiles, discrimination of the ageing time [14]. Mahon cheeses made with raw milk or pasteurized milk, and having different ageing times, were also analysed by HPLC, applying PCA to amino acids and other compounds identified, allowing the discrimination of cheeses according to their ripening times and the different treatment of milk [15]. All these results demonstrate that traditional HPLC techniques can be very useful for authentication of cheeses but also that several problems are present. Besides the fact that the sample preparation is usually quite long and cumbersome and also that HPLC instrumentation, albeit common, is still not widespread in all production sites and requires trained personnel, the main problem is that the mixture of amino acids and peptides obtained often yield complex profiles in HPLC, which require advanced statistical procedures for discriminating the samples. The traditional UV detection of HPLC in these cases offers no insights into the actual molecular composition of these mixtures, hampering the identification of suitable markers.

2.4 GC Besides HPLC, GC can also be used to identify in the volatile fraction of cheeses (or milk) compounds that might be useful for authenticating the products, most of the times analyzing the headspace. As GC/MS instruments are also quite common and quite easy to use, direct structural information on the marker compounds is generally more easily obtained than in the case of HPLC. One of the most striking examples is the detection of terpenes: terpene content and profile in cheeses is affected by the feeding system of the cows (or other animals providing the milk), especially the grazing or non-grazing system, so a GC analysis of terpenes might help to discriminate between those two systems. Studies done on milk have demonstrated that changing the species composition of the plants used to feed the cows has an immediate influence on the amount and the types of terpenes found in the lipidic fraction of milk [16] and that terpene compounds can provide useful fingerprints for the characterization of dairy products, not only according to the feeding

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system (grazing or stable) but also their geographical origin [17]. The terpenes in the volatile fraction of two French cheeses were studied by dynamic headspace GC/MS, in both cases analyzing cheeses produced by using raw or pasteurized milk; the two varieties were both manufactured with the same milk, obtained from cows grazing in the same place. Quite interestingly, it was found that milk pasteurization did not change the terpene profiles, whereas much bigger differences were found between the two cheese varieties, even if the milk origin was the same. Evidently, the differences in the terpene profiles could be ascribed to the technological differences applied in cheese manufacturing, which were suggested to be due to the different microbial population or ageing time [18]. Also, Emmental cheeses produced in Switzerland could be well discriminated from similar cheeses produced in different European countries by analyzing the volatiles present in the head space with GC, equipped with both FID and MS detectors [19]. Fatty acid composition of milk is also affected by the zone of origin, where cows feed: milk produced at higher altitudes was found to have a smaller amount of saturated short- and medium chain fatty acids, and more polyunsaturated fatty acids, than milk produced at lower latitudes [20]. GC was also found to be very useful to detect molecules, such as hexanal, related to the state of conservation of milk, allowing discrimination of fresh milk from that stored at room temperature [21]. In general, though headspace analysis by GC, and mostly by GC/MS, can be considered a very promising tool for differentiating volatiles from various cheeses, several problems prevent its widespread use for robust authentication. Sample preparation might be affected by the variability in the systems used to sample head space, and again GC instrumentation, albeit common, is still not widespread in all production sites and requires trained personnel. Finally, even though it is clear that volatiles in the headspace are strictly linked to the geographical origin of the milk and to the technological parameters used for cheese manufacturing, and mostly to the composition of bacterial population, it is also true that small changes in these parameters might lead to large differences in the volatiles, in turn, hampering or making impossible a clear-cut authentication of the cheeses.

2.5 Sensory Analyses Finally, a very traditional way to authenticate cheeses is the use of sensory analysis. Sensory analyses can be used to authenticate textural parameters of the cheeses [22] or gross composition, such as fat content [23]. Although sensory analyses can be a very rapid way to obtain answers on cheese authenticity without the use of expensive instruments or laboratory facilities, again several problems have to be considered. First, sensory analyses are obviously much more complicated than ‘tasting’ the cheese and require trained and experienced personnel, who are not always available in all the production

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facilities. Also, these methods can hardly be implemented for practical purposes when many samples need to be analysed online in a food industry. In conclusion, the traditional methods widely employed nowadays for assessing cheese authenticity only partially help in this process. The complexity of a matrix-like cheese, the natural variability observed in many artisanal products making it difficult to firmly fix limits for the authentic products, and the problem of performing analyses often requiring expensive and complex instruments and skilled and trained personnel make these analyses at most an indication of a likely or not likely authenticity. Advanced chemometric methods can be of tremendous help in many difficult cases, but again advanced statistics require trained personnel, and it is not always easy to defend cases related to authentication of food products in a court. For these reasons, more and more advanced techniques are appearing in the literature, aimed at providing more robust tools for cheese authentication.

3 ADVANCED TECHNIQUES FOR THE DETERMINATION OF THE AUTHENTICITY OF CHEESES Discrimination between different dairy products and confirmation of the authenticity are basically the same analytical problem. The basic assumption behind the application of advanced spectroscopic techniques to solve this problem is the generation of the ‘fingerprint’ of foods. An individual dairy product with a given chemical composition exposed to a light source will have a characteristic spectrum, which is a result of the absorption by various chemical constituents. Because the exact composition of any natural material is dependent on factors such as variety, season and location, it is possible to collect a range of typical spectra for any food matrix. Therefore, what is needed is a library of representative spectra, to which the spectrum of a test food material may be compared, in order to establish its quality or authenticity. Given the nature of the data sets involved, multivariate chemometric techniques are required and a number of commercial software packages can be used to better elucidate and process all the data.

3.1 NIR and MIR Spectroscopy IR spectroscopy is a rapid and non-destructive technique for the authentication of food samples. Analysis of a food sample using the mid-infrared (MIR) spectrum (4000– 400 cm 1) reveals information about the molecular bonds present and can therefore give details of the types of molecules present in the food. Nearinfrared (NIR) spectroscopy utilizes the spectral range from 14,000 to 4000 cm 1 and provides much more complex structural information related to the vibrational behaviour of combinations of bonds. These techniques are

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suited for application in an industrial setting due to their ease of use and the relatively low financial cost of obtaining and running the equipment. Considerable work has been carried out in the area of cheese quality and authenticity determination using NIR and MIR spectroscopic techniques on a laboratory scale. Recent technical developments in NIR and MIR spectroscopies and chemometrics will facilitate the transfer of this technology from laboratory to online application, which, in addition to the rapid, non-destructive, and relatively low-cost nature of infrared spectroscopy, make it an ideal analytical tool. Although for most of the applications mentioned in this field thus far, the use of NIR spectroscopy is more prevalent than MIR spectroscopy, in the area of cheese authenticity, which encompasses geographical origin and adulteration detection, there is more scope for other technologies found in the literature. Karoui et al. [24] outlined the feasibility of discriminating the manufacturing process and sampling zone in ripened soft cheeses using MIR (3000–900 cm 1) and reflectance NIR (315–1700 nm) spectroscopy. This chapter gives a good comparison of the two spectroscopic techniques used on the same cheese samples. Regarding the MIR spectra, the percentage of samples correctly classified into six groups (three for external and three for central zones) by factorial discriminant analysis (DA) was 64.8% and 33.3%, respectively, for the calibration and validation sets, respectively. Better classification was obtained from the NIR spectra where the corresponding results were 85.2% and 63.2%. This chapter would suggest that although many papers have been published in the area of cheese authenticity using MIR spectroscopic techniques, NIR spectroscopy is still a more accurate tool in this area. The same authors [25] overcame some of these difficulties of the MIR technique by concentrating on a particular wavelength range. In assessing the potential of infrared spectroscopies for the determination of the geographical origin of Emmental cheeses, NIR spectroscopy was found to give 89% and 86.8% correct classification for the calibration and validation spectral data sets, respectively. The MIR results were comparable to the NIR results, giving corresponding correct classification figures of 84.1% and 85.7% within the 3000- to 2800-cm 1 region. NIR results were still superior to the MIR results, but by concentrating on this section of the wavelength range, a closer comparison was achieved. In a third work, 12 samples of cheeses (three types of Saint-Nectaire PDO cheeses and Savaron cheeses) differing in manufacturing and ripening conditions, from 12 different producers, were characterized by MIR [26]. The results obtained in this study demonstrated that this technique is useful for recognizing the manufacturing conditions of Saint-Nectaire cheeses. While physicochemical and rheology data allowed only 72.2% and 91.7% of correct classification of the investigated cheeses, MIR classified all the investigated cheeses correctly. It appears that MIR spectra recorded directly on cheeses are fingerprints allowing the identification of cheeses, and moreover, the analysis of

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spectral collections by multidimensional statistical methods makes it possible to derive molecular information on the structure of cheese components. Very recently, NIR spectroscopy has also been used for the authentication of Asiago d’allevo [27], a PDO cheese from northern Italy. Latent variable models applied on spectral data were developed and used to estimate several chemical properties and to classify the available dataset according to the location and management of the cheese-making factory (lowland and alpine), the ripening age (6, 12, 18, and 36 months), the altitude of milk production (low, medium, medium–high, and high), and the period of the year of the cheese production (May, July, and September). The variable importance in projection index was used to identify the most informative spectral regions for discrimination. Results showed that NIR spectra can be used both to accurately estimate several chemical properties and to classify the samples according to the different experimental conditions under investigation with the same discrimination capacity provided by traditional chemical analysis.

3.2 Nuclear Magnetic Resonance Spectroscopy Nuclear Magnetic Resonance (NMR) spectroscopy involves the analysis of the energy absorption by atomic nuclei with non-zero spins in the presence of a magnetic field. The energy absorptions of the atomic nuclei are affected by the nuclei of surrounding molecules, which cause small local modifications to the external magnetic field. NMR spectroscopy can therefore provide detailed information about the molecular structure of a food sample, given that the observed interactions of an individual atomic nucleus are dependent on the atoms surrounding it. High-resolution NMR (HR-NMR; utilizes frequencies above 100 MHz) has been applied in many more food authenticity studies than low-resolution NMR (LR-NMR; uses frequencies of 10–40 MHz). The advantage of HRNMR over LR-NMR is that it is possible to obtain much more detailed information regarding the molecular structure of a food sample. The major disadvantage of HR-NMR is that it is one of the most expensive analytical techniques to employ, both in terms of the initial capital outlay and running costs. Some examples of NMR studies have been reported focusing on the authenticity and geographical origin determination. NMR studies on aqueous extracts of both PDO Italian cheeses, Grana Padano [28] and ParmigianoReggiano [29], the two most known Italian hard ripened cheeses, allowed the identification of several low molecular compounds, in particular amino acids, and their distribution along the ripening time. The work on Grana Padano also allowed for the quantification of the amino acid content, which was in agreement with previous studies on amino acid production during the proteolysis steps in hard ripened cheeses, which was substantial only in the first months of ripening.

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Foreign ‘Grana-type’ cheeses from eastern countries were found to be well differentiated from Italian Parmigiano-Reggiano cheeses; however, the work was limited to a few samples and typologies. Even if the results were too preliminary for drawing firm conclusions on the use of NMR analysis on amino acids as a way to define cheese typology, the data demonstrated the feasibility of the NMR technique for characterizing typical cheeses. Shintu and Caldarelli [30] also tested the potential of HR-MAS-NMR, performed on solid samples, to study 20 samples of Emmental cheeses from seven different geographical regions. PCA and DA were used to analyze the data set NMR spectra and succeeded in grouping the studied samples according to their geographical origins. In this case, the molecular markers identified were both amino acids and fatty acids. Another Italian traditional cheese that bears the PDO trademark is the already mentioned Mozzarella. Some studies have been performed in the last years also by HR-NMR on this kind of cheese. In particular, Brescia et al. [31] used HR-NMR combined with other analytical techniques with the goal of determining the geographical origin of the milk and of the finished mozzarella product obtained from it. Spectral data were processed by chemometric methods in order to obtain the geographical characterization of buffalo milk mozzarella cheeses originating from two areas of Southern Italy. Mozzarella aqueous extracts were analysed by HR-NMR and many molecular components were identified. From the multivariate analysis, it resulted that the amino acid tyrosine was a possible marker to discriminate Campaniamozzarellas from Apulia ones. Anyway, the work was limited to 14 samples, and the same authors stated that this analysis should be repeated in following years and on a higher number of samples in order to analyse the annual variability of the milk. Recently, the same authors reported a more comprehensive study (39 samples) in which the HR-NMR technique was used to discriminate mozzarella cheeses obtained from Italian milk from those obtained from other milk [32]. In particular, protonic NMR spectra of the aqueous extracts indicate differences in amino acid composition between the other and Italian samples. The authors stated that the smaller proportion of amino acids in the latter is probably due to the different feeding regime of the animals. A rapid and simple NMR method has also been proposed, by Mammi et al. [33], to discriminate Asiago d’Allevo cheese samples from different production chains. In this case, a fast and reproducible extraction of the organic fraction was employed to characterize the fatty acid content. By applying chemometric analysis to NMR data, it is possible to differentiate PDO Asiago cheese produced in alpine farms from those produced in lowland and mountain industrialized factories. PCA of both 1H and 13C NMR spectra showed a good separation of alpine farm products from the other ones, whereas the lowland and mountain industrialized cheeses are undistinguishable. In this study, the samples were differentiated on the basis of a higher content of unsaturated fatty acids, principally oleic, linoleic, linolenic, and conjugated

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linoleic acids for the alpine farm cheeses and a higher content of saturated fatty acids for the industrialized products. As shown in these examples, NMR can provide information concerning different specific molecular markers which cannot be gathered anywhere else. In addition, NMR might be a better choice for heterogeneous samples, as a larger quantity of a sample is probed with NMR than with, for example, NIR reflectance or transmittance. The main disadvantage of this technique is that the equipment is too complex to optimize compared with, for example, the NIR instrument, and that measurements are time dependent. Indeed, the pulse sequence used is crucial to obtain valuable results. The use of wrong pulse sequence or parameter settings might result in a negative outcome of the experiments. Therefore, NIR might be preferred for fast, non-invasive quantitative measurements on samples which are relatively homogeneous.

3.3 Stable Isotope Analysis The ratios of stable isotopes provide an interesting analytical tool to confirm quality and/or identity of dairy products as there are sometimes region-specific patterns in environmental isotopic ratios (soil, water). Like trace elements, isotopes are incorporated into local feeds and the body of the animals. Therefore, these ratios may be specific for those areas. The ratios of hydrogen (H/D) and oxygen (16O/18O) isotopes in body tissues are primarily influenced by water. Isotopic ratios of H, C, N, S, and Sr (12C/13C, 14N/15N, 32S/34S, 86Sr/87Sr) are more indicative of soil and feed origin. Analyses utilizing the isotopic ratios of H, O, N, and S have been applied to determine the identity of dairy products. Manca et al. [34] showed the stable isotope ratios (13C/12C and 15N/14N) of casein measured by isotope ratio mass spectrometry, and some free amino acid ratios (His/Pro, Ile/Pro, Met/Pro, and Thr/Pro) determined by HPLC in samples of ewe milk cheese from Sardinia, Sicily, and Apulia were found to be parameters independent of ripening time. Multivariate data treatments performed by applying both unsupervised (PCA and cluster analysis) and supervised linear discriminant analysis (LDA) methods revealed good discrimination possibilities for the cheeses according to place of origin. In this respect, particularly significant were the variables Ile/Pro, Thr/Pro, 13C/12C, and 15N/14N ratios on which basis 100% discrimination and classification of the samples by LDA were obtained. Together with the isotopic ratio of other bioelements (S and H in casein and C and O in glycerol), the stable isotope analysis permitted the separation of different European cheeses from France, Italy, and Spain [35]; Emmental cheese from Finland, Bretagne, and Savoy [36]; and Peretta cheese of Sardinia from competitors produced in Northern Europe [37]. Recently, a study of H, C, N, and S stable isotopes and mineral profiles has also been developed to guarantee the authenticity of grated hard cheeses and, in particular, of Italian PDO Parmigiano-Reggiano [38].

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This study presents two statistical models, based on isotopic and elemental composition, able to trace the origin of cheese also in grated and shredded forms, for which it is not possible to check the logo firemarked on the rind. One model is able to predict the origin of seven types of European hard cheeses (in a validation step, 236 samples out of 240 were correctly recognized) and the other can discriminate the PDO Parmigiano-Reggiano cheese from nine European and two nonEuropean imitators (260 out of 264 correct classifications). The most significant variables for cheese traceability common in both models are 13C, 2H, 15N, 34S and Sr, Cu, Mo, Re, Na, U, Bi, Ni, Fe, Mn, Ga, Se, and Li. These variables are linked not only to geography but also to cow diet and cheese-making processes. However, the stable isotope approach also has some important constraints. Conclusions made from results using the stable isotopes must be based on uniform environment features (e.g. climate, altitude, distance from oceans) allowing few or no differences in isotopic ratios of the dairy products. Therefore, dairy products from animals originating from different, but climatically or geologically similar, areas might have an identical isotopic pattern. Another disadvantage of analysing stable isotopes is the time-consuming and expensive preparation of samples for some elements and the high costs of the analytical equipment.

3.4 DNA Analysis In recent years, growth in research into food authentication methods based on the analysis of the DNA has been observed to grow. The majority of the work related to exploiting DNA analysis has focused on using polymerase chain reaction (PCR) to amplify the specific areas of DNA of interest. The principle of PCR is that specific lengths of DNA can be copied enough times to provide a sufficient amount of that area of DNA to be analysed using a variety of methods with electrophoretic techniques being the most frequently used. Species-specific PCR has proved to be a suitable method to control food authenticity because a specific target sequence can be detected even in matrices containing a pool of heterogeneous genomic DNA, such as milk or other dairy products. In these food products, molecular markers can be found in the DNA of different grass species and forages commonly fed to animals or DNA coming from the microbial environment. These improvements open the possibility that, in the future, many types of cheeses requiring the PDO certification could be validated by the the plastidial DNA fragments present in the milk used for their production. Instead, milk bacterial composition has been studied by using intergenic transcribed spacer–PCR fingerprinting to provide analytical support to the important issue represented by the authentication of the geographical origin of alpine milk products [39]. This work showed for the first time that the

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composition of these communities is related to the altitude of pastures strongly enough to allow the distinction of milk origin in terms of altitude by means of the adopted internal transcribed spacers (ITS)-PCR fingerprinting protocol followed by multivariate statistical analysis of the resulting band patterns. Although further research is required to better elucidate this relatedness and to study its behaviour over time in depth, this new analytical approach and its findings can be of significant interest for the manufacture, protection, traceability and promotion of the PDO Fontina cheese and other alpine dairy products. The same methodology has also been applied to distinguish Italian PDO water buffalo Mozzarella from different producers on a molecular basis in relation to the place of manufacturing within the production district [40]. Microbial DNA was isolated from Mozzarella’s governing liquid to amplify the whole microflora’s ribosomal 16S–23S ITS-PCR fingerprinting by means of an original primer pair. Although further analyses are required to better investigate the method’s capabilities and limitations, and, in particular, the possibility to univocally identify samples and their places of production, different PDO water buffalo Mozzarellas were distinguished using the described protocol. This simple technique provided an information level sufficient to observe a relationship between the genetic diversity of the cheeses’ microbial communities and the dairies’ geographical position; in addition, the electrophoretic profiles obtained by this technique did not show major changes over 1 year, suggesting that Mozzarella bacterial populations are substantially stable over time. Concerning genetic traceability, the use of DNA markers as diagnostic tools for food authenticity, provenance, and traceability of variety/type composition of complex food matrices has been investigated in an increasing number of projects worldwide. However, some processed food contains highly degraded DNA and/or PCR inhibitors, both of which may affect the subsequent PCRs used for the amplification of diagnostic DNA sequences. These effects may be overcome by modification of the DNA extraction process and PCR assay design and conditions. It is sometimes possible to overcome these inhibitory effects by extensive dilution of the DNA extract; however, this may not be an option when the amount of DNA in the sample is limited. In these cases, a sensitive method for the detection of small amounts of highly degraded DNA is necessary. Therefore, as a first step, it is important to be able to optimize the DNA extraction method, which should be adapted to the nature of the food matrices.

3.5 LC/MS Liquid chromatography combined with mass spectrometry (LC–MS) can be a very efficient advanced analytical technique in the agrifood field, in particular, to assess food authenticity.

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In a recent study, Czerwenka et al. [41] proposed an LC/ESI-MS method aimed at analyzing b-lactoglobulin as a way to detect the presence of cow milk in water buffalo milk and mozzarella cheese, based on the fact that the molecular mass of bovine and bubaline b-lactoglobulins are different, due to several aminoacidic substitutions. Therefore, although having very similar chromatographic retention times, they can be easily discriminated by mass spectrometry and indeed, after having set up a simple extraction method, the lactoglobulins could be easily detected by LC/ESI-MS. Although the qualitative determination of the presence of bovine milk was rather straightforward, the correct quantitation proved to be more difficult, due to the varying lactoglobulin content in the two types of milk, required for a nonlinear calibration curve in order to perform a correct quantitation of the amount of bovine milk used in fraudulent samples. Thus, in general, it can be assumed that all chromatographic methods aimed at protein detection require a very careful calibration in order to avoid inaccurate quantitative results in terms of milk percentage. Besides proteins, the chromatographic determination of the peptides generated by the proteolytic events, either taking place in cheeses or performed in cheese samples for analytical purposes, can be used for assessing the species providing the milk used for cheese production. The underlying principle is that, deriving from specific proteins, peptide sequences can be species-specific as well. Given the complexity of the peptide mixtures in cheeses, and the very similar chromatographic retention time of homologous peptides derived from similar proteins, a precise detection of these marker peptides can be accomplished only by means of mass spectrometric detection. In a method proposed by our group and aimed at determining the presence of cows milk in sheep milk cheeses, the full peptide fraction was extracted by cheeses and isolated through subsequent filtration and ultrafiltration steps, and finally the peptides were analyzed by LC/ESI-MS in order to identify, among all the different proteolytic peptides, the species-specific ones. Two different marker peptides arising from the N-terminal part of a-S1 casein, the fragments 1–23 and the fragment 1–14, very common proteolytic peptides usually present in all cheeses, were identified as suitable, differing for two amino acids between the two species. The selective detection of these peptides allowed the identification of the presence of cow milk in sheep milk down to 1% [42].

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CONCLUSIONS AND FUTURE TRENDS

Several PDO cheeses are nowadays on the market and probably an increased number will be available in the future, on the basis of the several regional and traditional peculiarities of the different regions. Certainly, PDO labels have met both the interest of the producers for obtaining a premium price for their valuable products as well as the interest of the consumers to obtain a guarantee for the authenticity of the product itself. There is thus no doubt that

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authentication of cheeses is a problem of extraordinary economic importance, with many quality and sometimes safety implications, and a problem which still, in many cases, has not found a general solution yet. Simple physicochemical analyses can provide few answers, and they need to be integrated by more sophisticated analyses (NIR, NMR, stable isotopes, DNA analysis, LC/MS), which cannot always be performed in all laboratories, and their implementation in production sites is very far in the future. Chemometric tools are almost always essential to provide the desired answers related to authenticity tools, also because most of the time those answers rely on a smart combination of very diverse data collected by diverse analyses. It is quite clear that the authenticity problems are also diverse, and thus a fit-to-all method does not exist, but many good methods rely on a combination of several different data. As a striking prototypical example, we can consider the actual approach used by the Consortium of Parmigiano-Reggiano to determine the authenticity of Parmigiano-Reggiano cheeses, particularly in the case of grated samples, where traceability might be problematic due to the absence of the full wheel with the labels on the rind. The only official analysis reported in the production is the level of free amino acids, but this parameter is suited only to determine ageing time (and even then, with some limitations) and totally not suitable for discriminating authentic from fake Parmigiano-Reggiano grated cheeses. Thus, lately, the Consortium implemented screening actions, by internally measuring the lysozyme content (which should be absent in authentic Parmigiano-Reggiano cheeses), making sensory analyses, and using NIR to determine moisture, ageing, soluble proteins, and percentage of rind. Moreover, through external laboratories, it also measures the amount of copper content and the activity of alkaline phosphatase. Only on samples which are ‘suspicious’, it implements, again through external laboratories, isotope and rare earth analyses, aimed at assessing the authentic origin of the samples. All these parameters are integrated in a single statistical approach that, through a Bayesian method, provides an answer to the question of authenticity of the analyzed samples. Such a multifactorial approach, which requires careful calibration and validation, is becoming more and more common for providing solutions to authenticity-related problems in cheeses, in turn allowing the use of efficient tool to actually enforce the rules associated with PDO cheeses.

REFERENCES [1] Karoui R, De Baedemaker J. A review of the analytical methods coupled with chemometric tools for the determination of the quality and identity of dairy products. Food Chem 2007;102:621. [2] Millan R, Saavedra P, Sanjuan E, Castelo M. Application of discriminant analysis to physicochemical variables for characterizing Spanish cheeses. Food Chem 1996;55:189. [3] Pillonel L, Butikofer U, Schlichtherle-Cerny H, Tabacchi R, Bosset JO. Geographic origin of European Emmental. Use of discriminant analysis and artificial neural network for classification purposes. Int Dairy J 2005;15:557.

Chapter

19

Cheeses

507

[4] Bugaud C, Buchin C, Coulon JB, Hauwuy A, Dupont D. Influence of the nature of alpine pastures on plasmin activity, fatty acid and volatile compound composition of milk. Lait 2001;81:401. [5] Hurley IP, Coleman RC, Irealand HE, Williams JH. Measurement of bovine IgG by indirect competitive ELISA as a means of detecting milk adulterations. J Dairy Sci 2004;87:215. [6] Hurley IP, Coleman RC, Ireland HE, Williams JHH. Use of sandwich IgG ELISA for the detection and quantification of adulteration in milk and cheese. Int Dairy J 2006;16:805. [7] Lopez-Calleja IM, Gonzalez I, Fajardo V, Hernandez PE, Garcia T, Martin R. Application of an indirect ELISA and a PCR technique for detection of cows’ milk in sheep’s and goats’ milk cheeses. Int Dairy J 2006;17:87. [8] Asensio L, Gonzalez I, Garcia T, Martin R. Determination of food authenticity by enzymelinked immunosorbent assay (ELISA). Food Control 2008;19:1. [9] O’Shea BA, Uniacke-Lowe T, Fox PF. Objective assessment of Cheddar cheese quality. Int Dairy J 1996;6:1135. [10] Pripp AH, Kieronczyk A, Stepaniak L, Srhaug T. Comparison of the biochemical characteristics of three Norwegian cheese varieties using multivariate statistical analysis. Milchwissenschaft 1999;54:558. [11] Ferreira IMPLVO, Cacote H. Detection and quantification of bovine, ovine and caprine milk percentages in protected denomination of origin cheeses by reversed-phase high performance liquid chromatography of beta-lactoglobulins. J Chromatogr 2003;1015:111. [12] Di Cagno R, Banks J, Sheehan L, Fox PF, Brechany EY, Corsetti A, et al. Comparison of the microbiological, compositional, biochemical, volatile profile and sensory characteristics of three Italian PDO ewes’ milk cheeses. Int Dairy J 2003;13:961. [13] Pripp AH, Rehman S-U, McSweeney PLH, Fox PF. Multivariate statistical analysis of peptide profiles and free amino acids to evaluate effects of single-strain starters on proteolysis in miniature Cheddar-type cheeses. Int Dairy J 1999;9:473. [14] Bara-Herczegh O, Horvath-Almassy K, Orsi F. Application of multivariate methods to identify the indices of secondary proteolysis for Trappist cheese maturity and quality. Eur Food Res Technol 2002;214:516. [15] Frau M, Massanet J, Rossello C, Simal S, Canellas J. Evolution of free amino acid content during ripening of Mahon cheese. Food Chem 1997;60:651. [16] Viallon C, Martin B, Verdier-Metz I, Pradel P, Garel JP, Coulon JB, et al. Transfer of monoterpenes and sesquiterpenes from forages into milk fat. Lait 2000;80:635. [17] Fernandez C, Astier C, Rock E, Coulon JB, Berdague´ JL. Characterization of milk by analysis of its terpene fractions. Int J Food Sci Technol 2003;38:445. [18] Cornu A, Kondjoyan N, Martin B, Verdier-Metz I, Pradel P, Berdague´ JL, et al. Terpene profiles in Cantal and Saint-Nectaire-type cheese made from raw or pasteurised milk. J Sci Food Agric 2005;85:2040. [19] Pillonel L, Ampuero S, Tabacchi R, Bosset JO. Analytical methods for the determination of the geographic origin of Emmental cheese: volatile compounds by GC/MS-FID and electronic nose. Eur Food Res Technol 2003;216:179. [20] Collomb M, Butikofer U, Sieber R, Jeangros B, Bosset JO. Composition of fatty acids in cow’s milk fat produced in the lowlands, mountains and highlands of Switzerland using high-resolution gas chromatography. Int Dairy J 2002;12:649. [21] Ulberth F, Roubicek D. Monitoring of oxidative deterioration of milk powder by headspace gas chromatography. Int Dairy J 1995;5:523. [22] Lebecque A, Laguet A, Devaux MF, Dufour E. Delineation of the texture of Salers cheese by sensory analysis and physical methods. Lait 2001;81:609.

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[23] Ritvanen T, Lampolahti S, Lilleberg L, Tupasela T, Isoniemi M, Appelbye U, et al. Sensory evaluation, chemical composition and consumer acceptance of full fat and reduced fat cheeses in the Finnish market. Food Qual Prefer 2005;16:479. [24] Karoui R, Mouazen AM, Ramon H, Schoonheydt R, De Baerdemaeker J. Feasibility study of discriminating the manufacturing process and sampling zone in ripened soft cheeses using attenuated total reflectance MIR and fiber optic diffuse reflectance VIS–NIR spectroscopy. Food Res Int 2006;39:588. [25] Karoui R, Dufour E, Pillonel L, Schallerc E, Picque D, Cattenoz T, et al. The potential of combined infrared and fluorescence spectroscopies as a method of determination of the geographic origin of Emmental cheeses. Int Dairy J 2005;15:287. [26] Boubellouta T, Karoui R, Lebecque A, Dufour E´. Utilisation of attenuated total reXectance MIR and front-face fluorescence spectroscopies for the identification of Saint-Nectaire cheeses varying by manufacturing conditions. Eur Food Res Technol 2010;231:873. [27] Ottavian M, Facco P, Barolo M, Berzaghi P, Segato S, Novelli E, et al. Near-infrared spectroscopy to assist authentication and labeling of Asiago d’allevo cheese. J Food Eng 2012;113:289. [28] de Angelis Curtis S, Curini R, Delfini M, Brosio E, D’Ascenzo F, Bocca B. Amino acid profile in the ripening of Grana Padano cheese: a NMR study. Food Chem 2000;71:495. [29] (a) Consonni R, Cagliani LR. Ripening and geographical characterization of Parmigiano Reggiano cheese by 1H NMR spectroscopy. Talanta 2008;76:200(b) Shintu L, Caldarelli S. High-resolution MAS NMR and chemometrics: characterization of the ripening of Parmigiano Reggiano cheese. J Agric Food Chem 2005;53:4026. [30] Shintu L, Caldarelli S. Toward the determination of the geographical origin of Emmental(er) cheese via high resolution MAS NMR: a preliminary investigation. J Agric Food Chem 2006;54:4148. [31] Brescia MA, Monfreda M, Buccolieri A, Carrino C. Characterisation of the geographical origin of buffalo milk and mozzarella cheese by means of analytical and spectroscopic determinations. Food Chem 2005;89:139. [32] Sacco D, Brescia MA, Sgaramella A, Casiello G, Buccolieri A, Ogrinc N, et al. Discrimination between Southern Italy and foreign milk samples using spectroscopic and analytical data. Food Chem 2009;114:1559. [33] Schievano E, Pasini G, Cozzi G, Mammi S. Identification of the production chain of Asiago d’allevo cheese by nuclear magnetic resonance spectroscopy and principal component analysis. J Agric Food Chem 2008;56:7208. [34] Manca G, Camin F, Coloru GC, Del Caro A, Depentori D, Franco MA, et al. Characterization of the geographical origin of Pecorino Sardo cheese by casein stable isotope (13C/12C and 15N/14N) ratios and free amino acid ratios. J Agric Food Chem 2001;49:1404. [35] Camin F, Wietzerbin K, Blanch Cortes A, Haberhauer G, Lees M, Versini G. Application of multielement stable isotope ratio analysis to the characterization of French, Italian, and Spanish cheeses. J Agric Food Chem 2004;52:6592. [36] Pillonel L, Badertscher R, Froidevaux P, Haberhauer G, Ho¨lzl S, Horn P, et al. Stable isotope ratios, major, trace and radioactive elements in Emmental cheeses of different origins. Lebensm Wiss Technol 2003;36:615. [37] Manca G, Franco MA, Versini G, Camin F, Rossmann A, Toia A. Correlation between multielement stable isotope ratio and geographical origin in Peretta cows’ milk cheese. J Dairy Sci 2006;89:831. [38] Camin F, Wehrensa R, Bertoldi D, Bontempo L, Ziller L, Perini M, et al. H, C, N and S stable isotopes and mineral profiles to objectively guarantee the authenticity of grated hard cheeses. Anal Chim Acta 2012;711:54.

Chapter

19

Cheeses

509

[39] Bonizzi I, Buffoni JN, Feligini M, Enne G. Investigating the relationship between raw milk bacterial composition, as described by intergenic transcribed spacer–PCR fingerprinting, and pasture altitude. J Appl Microbiol 2009;107:1319. [40] Bonizzi I, Feligini M, Aleandri R, Enne G. Genetic traceability of the geographical origin of typical Italian water buffalo Mozzarella cheese: a preliminary approach. J Appl Microbiol 2006;102:667. [41] Czerwenka C, Muller L, Lindner W. Detection of the adulteration of water buffalo milk and mozzarella with cow’s milk by liquid chromatography–mass spectrometry analysis of b-lactoglobulin variants. Food Chem 2010;122:901. [42] Sforza S, Aquino G, Cavatorta V, Galaverna G, Mucchetti G, Dossena A, et al. Proteolytic oligopeptides as molecular markers for the presence of cows’ milk in fresh cheeses derived from sheep milk. Int Dairy J 2008;18:1072.