Determination of flavour compounds in a mountain cheese by headspace sorptive extraction-thermal desorption-capillary gas chromatography-mass spectrometry

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LWT 41 (2008) 185–192 www.elsevier.com/locate/lwt

Determination of flavour compounds in a mountain cheese by headspace sorptive extraction-thermal desorption-capillary gas chromatography-mass spectrometry S. Panseri, I. Giani, T. Mentasti, F. Bellagamba, F. Caprino, V.M. Moretti Department of Veterinary Science and Technology for Food Safety, Faculty of veterinary Medicine, University of Milan, Via Trentacoste 2, 20134 Milan, Italy Received 31 January 2007; accepted 9 February 2007

Abstract Headspace sorptive extraction (HSSE) combined with thermal desorption (TD), gas chromatography (GC) and mass spectrometric detection (MSD) was applied to determine the volatile compounds of an Italian mountain cheese (Bitto) with protected designation of origin. Samples were taken from local dairy farms in Valtellina (Northern Italy), in which goats and cows were grazing on pastures during summer. A total of 84 compounds that belong to the following chemical families were detected and identified: aldehydes (17), ketones (10), esters (15), fatty acids (12), alcohols (11), terpenes (11) and hydrocarbons (8). The impact of each volatile compound on the flavour profile of Bitto cheese was discussed. The flavour profile of this cheese is important for the producer who wants to claim the right of naming cheese manufactured in a specified restricted area. Actually, this approach might become mainly useful for products, like Bitto cheese, with a PDO registration. HSSE coupled with thermal desorption and GC–MS analysis provided a versatile tool for the analysis of volatiles in cheese. r 2007 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. Keywords: Headspace sorptive extraction; Mountain cheese; Volatile compounds

1. Introduction The Bitto cheese is an Italian traditional dairy product with protected designation of origin (PDO), produced in Northern Italy, since 1996. The milk utilized for Bitto production mainly comes from Bruna breed local cows, with a possible addition of goat’s milk. This artisanal cheese is produced only from June until September, when cows and goats are maintained on alpine pasture on the mountain area that is bounded by province of Sondrio (Valtellina). The cheese is made with whole raw cow milk without the use of starter cultures, to which no more than 10% of goat milk can be added. Immediately after collection, the raw milk is heated to 35–37 1C; throughout the process it is stirred by a rotary blade to ensure the Corresponding author. Tel.: +39 02 50315760; fax: +39 02 50315746.

E-mail address: [email protected] (V.M. Moretti).

distribution of the heat. Calf rennet is added to the heated milk and, once the curdling has been completed, the curd is broken into slivers with a size like wheat grains. The curd is then separated from the whey by using a special cloth and put it in a traditional cheese mould, which gives it its characteristic shape. The cheese is pressed for 24 h to extract all remaining whey and then is dry-salted on both faces and left for ripening in traditional room at temperature of 12–16 1C, for a minimum of 70 days. The ageing stage ranges, in general, from 1 to 8 months. The cheese rind has a yellow-straw colour, which tends to become more intense during ripening. The Bitto cheese manufacturing is unchanged during centuries and it represents a core product in the agricultural economy of the production area at Valtellina. For these reasons, the preservation of the peculiarity of this cheese is needed and it requires the identification of those properties that much better describe the characteristics of the product.

0023-6438/$30.00 r 2007 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2007.02.011

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It is well known that each type of cheese is distinguished not only by its physicochemical characteristics but also by its flavour, which is dependent on the milk, the starter addition, the technological parameters and the ripening condition (Curioni & Bosset, 2002; Dirinck & Winne, 1999; Gomez-Ruiz, Ballesteros, Gonzales-Vinas, Cabezas, & Martinez-Castro, 2002; Partitario, Barbosa, & Vilas Boas, 1998). The analysis of flavour compounds in dairy products is a complex problem due to the complexity of the matrix and usually involves the use of concentration-extraction equipments such as vacuum distillation, liquid–liquid extraction or purge and trap techniques. (De Frutos, Sanz, & Martinez-Castro, 1991; Lubbers, Landy, & Voilley, 1998; Mariaca & Bosset, 1997; Sides, Robards, & Helliwell, 2000). Extraction methods are continually revised and improved with new technologies in order to reduce the time, the solvents use and the laboratory staff resources, and to increase the sensitivity and application versatility. More recently, a novel approach for sample enrichment, stir bar sorptive extraction (SBSE) has been introduced as a solventless sample preparation method for the extraction of organic compounds from aqueous matrices (Demyttenaere, Martı´ nez, Verhe´, Sandra, & De Kimpe, 2003; Baltussen, Sandra, David, & Cramers, 1999; Baltussen, Cramers, & Sandra, 2002; Juan Dı´ ez, Domı´ nguez, Guille´n, Veas, & Barroso, 2004; Larra`yoz, Addis, Gauch, & Bosset, 2001). The SBSE method has found acceptance for a wide range of application approaches (Guerrero, Natera Marı´ n, Mejı´ as, & Garcı´ a Barroso, 2006; Marı´ n, Zalacain, De Miguel, Alonso, & Salinas, 2005). The SBSE consists of a bar that is coated with a layer of polydimethylsiloxane (PDMS) gum (TwisterTM, Gerstel) stirring in a liquid samples for a fixed time that results in sorptive extraction of the analytes into the PDMS coating. After stirring the bar is placed in a glass tube, which is transferred into a thermal desorption unit (TDU), where the analytes are thermally recovered and analysed with a GC–MS system. In addition to the extraction of organic analytes from aqueous samples, the PDMS stir bar is also suitable as the extraction medium for the enrichment of analytes from gaseous (headspace) samples (headspace sorptive extraction, HSSE). In the HSSE, the stir bar is suspended in the headspace of the sample by means of a stainless steel stem or by a suited headspace vial. The HSSE has been already used to characterize the volatile fraction of wiskey, coffee and sage leaves (Bicchi, Iori, Rubiolo, & Sandra, 2002; Bicchi, Cordero, Liberto, Rubiolo, Sgorbini, David et al., 2005), essential oils (Bicchi, Cordero, Liberto, Rubiolo, Sgorbini, & Sandra, 2005), medicinal plants (Bicchi, Cordero, Iori, Rubiolo, & Sandra, 2000), olive oil (Cavalli, Fernandez, LizzaniCuvelier, & Loiseau, 2003) and fungi metabolites (Demyttenaere, Morina, De Kimpe, & Sandra, 2004). To our knowledge, no articles have been published on the determination of flavour compounds in dairy products using this extraction technique.

Considering the observation mentioned above and the lack of information about the flavour compounds of Bitto cheese, the aim of this preliminary work was to apply this new technique, HSSE, for the evaluation of aroma compounds of this Italian mountain cheese. 2. Materials and methods 2.1. Cheese samples Fourteen samples of Bitto cheese samples from Valtellina at 75 days of ripening, weighting around 250 g, were analysed. They were purchased from five dairy farms located in Valtellina, in which goats and cows were grazing on pastures during summer. Representative samples of cheese were obtained by discarding about 1 cm of rind and smear and all samples were kept frozen at 20 1C, until analysis. 2.2. Headspace sampling procedure A 1 cm long stir bar with 0.5 mm PDMS coating (for a total volume of 24 ml) was used. The stir bars are manufactured by Gerstel (Mullheim, Rhur, Germany) under the name of Twisters. The outer 2 cm of the cheese samples were removed and the samples were grated using a conventional grater. A stir bar was suspended in 20 ml headspace vial containing 2 g of finely grated cheese and then the adsorption was carried out for 60 min at 50 1C, in a water bath. After the extraction, the bar was placed into a glass tube and thermodesorption was carried out into a Gerstel thermodesorption unit (TDU). New stir bars were thermally conditioned for 3 h at 300 1C, while between sampling, they were reconditioned for 30 min at 250 1C in the GC injection port to avoid carry-over effects. 2.3. Analysis by thermodesorption-gas chromatography–mass spectrometry Analyses were performed with an Agilent 6890N gas chromatograph (Agilent Technologies, DE, USA) coupled to a quadrupole mass spectrometer MSD 5973N (Agilent Technologies, DE, USA). The TDU was installed on the top of Agilent 6890 GC equipped with a CIS-4 programmable temperature vaporizing (PTV) injector (Gerstel, Mullheim, Ruhr, Germany). The stir bar was thermally desorbed and volatiles were cryofocused in the liner of the PTV and then injected into the GC–MS system. Splitless thermal desorption was performed by programming the TDU from 25 to 280 1C at a rate of 20 1C min1. The analytes were cryofocused in the PTV at 150 1C with liquid nitrogen and the PTV was ramped from 150 to 250 1C at 12 1C s1, with a split ratio of 30:1. The GC system was equipped with a fused-silica capillary column (HP-5 ms, 30 m  0.25 mm i.d., film thickness 0.25 mm) (Agilent Technologies, DE, USA).

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Oven temperature was programmed as follow: from 35 to 120 1C at 2 1C min1, then from 120 to 280 1C at 5 1C min1, and held for 5 min. The carrier gas was helium at a flow rate of 1 ml min1. The mass spectra of volatile compounds in cheese samples were obtained by electron ionization (EI) at 70 eV, and the detector operated in scan mode from 35 to 350 amu, with scanning velocity of 2.48 scan s1 and using solvent delay of 1 min. The terpenes and sesquiterpenes in cheese samples were also detected using the MSD in the selected ion monitoring (SIM) mode, after extraction of m/z 93 and 136 for terpenes, and m/z 204 and 161 for sesquiterpenes. The dwell time was set to 100 ms. Identification of all volatile compounds was carried out by comparing GC retention time with those of standard compounds, and by Nist 98 and Wiley 275 mass spectral libraries. In order to identify unknown spectra, the linear retention indices (LRI) were also calculated for each peak using as a reference the series of hydrocarbons C6–C25 and compared with literature LRI values. The data were recorded and analysed with HP Chemstation Software. All analyses were done in duplicate.

2.4. Statistical analyses Data are reported as mean values7standard error of mean (SEM). Homogeneity of variance was confirmed and comparison between means was by ANOVA. StudentNewman–Keuls was used as post hoc test for comparison of the means among different farms. Significance was accepted at probabilities of 0.05. All the statistical analyses were performed by SPSS 12.0 (SPSS, Chicago, IL, USA).

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3. Results and discussion The volatile substances extracted by HSSE and identified were 84. The largest group of volatiles in the headspace of cheese samples were represented by aldehydes, ketones, fatty acids and esters, followed by hydrocarbons, terpenes and alcohols. 3.1. Aldehydes and ketones The abundance of aldehydes and ketones in the volatile fraction of Bitto cheese is shown in Tables 1 and 2. Hexanal, heptanal, nonanal, 2,4-decadienal, represented the most abundant aldehydes in all cheese samples, followed by 2-nonenal, 1,4-hexadienal, 2,4-heptadienal and benzenacetaldehyde. Benzaldehyde was found only in cheese samples from farms 1, 2, 3, and 4. These aldehydes are quite common in cheese and they are characterized by green-grass and herbaceous aromas. Nonanal is important for aroma of some cheese, like Grana Padano and water buffalo Mozzarella and 2-nonenal confers a pleasant green flavour to Pecorino and Ragusano cheese types (Moio & Addeo, 1998). Aldehydes, together with ketones, are the major secondary products of autoxydation of unsaturated fatty acids (Molimard & Spinnler, 1996). Hexanal, heptanal and 2-nonenal, in fact, probably arise from autoxydation of a-linolenic acid. Aldehydes may also derive from a-ketoacids produced by transamination of amino acids during ripening (Barbieri et al., 1994; Helinck, Le Bars, Moreau, & Yvon, 2004; Yvon & Rijnen, 2001). They are considered unstable and transitory compounds in cheese, because they are reduced to alcohols or oxidized to corresponding acid. The large amount of these compounds

Table 1 Relative abundance1 (mean7SEM) of aldehydes detected in the headspace of Bitto cheese samples made in different farms Compound

Farm1 (n ¼ 4) Farm2 (n ¼ 3) Farm3 (n ¼ 3) Farm4 (n ¼ 2) Farm5 (n ¼ 2) Odour description2

3-Methyl-1-butanal 0.1970.11 pentanal n.d. 2-Pentenal 0.3170.05 n-Hexanal 4.1470.42 2-Hexenal 0.3770.17 n-Heptanal 3.06a70.65 Benzaldehyde 1.0670.25 1,4 Hexadienal 1.48a,b70.01 2,4 Heptadienal 1.4370.23 Benzenacetaldehyde 0.93a,b70.03 n-Octanal 0.28a70.04 2-Nonenal 1.88a70.87 n-Nonanal 1.3770.13 2-Decanal 0.7070.34 2,4-Decadienal 1.62a70.47 n-Decanal 0.4770.12 n-Hexadecanal 0.5070.26

0.4970.03 n.d. 0.2770.05 1.8670.04 n.d. 2.11a70.15 1.1370.06 0.64a70.05 0.65770.06 1.26b70.04 0.19a70.02 0.52a70.02 1.1770.05 n.d. 0.54a70.03 n.d. 0.4570.07

n.d. n.d. 0.3570.13 1.6270.68 1.5270.74 2.15a70.21 0.5570.12 2.61b70.22 2.1670.20 0.46a70.00 0.35a70.04 0.93a70.15 0.7470.27 0.1770.06 1.63a70.23 n.d. 0.4570.05

n.d. n.d. 0.5170.22 12.3570.69 0.5270.09 9.48b72.36 0.7570.01 1.43a,b70.12 1.0970.54 1.18b70.03 0.90b70.04 8.97b70.04 2.1070.70 1.2070.02 1.77a70.12 0.1370.08 n.d.

1.9270.14 6.6470.28 1.1870.01 19.4079.77 0.5470.12 13.18b70.05 n.d. 0.94a70.23 2.0870.20 0.83a,b70.17 1.52c70.01 8.22b70.45 3.0170.80 2.1670.11 5.76b70.19 0.0970.05 0.0670.04

Means within rows with different superscript are significantly (Po0.05). NS, not significant. 1 Relative abundance expressed as percentage on total volatile compounds detected. 2 Odour description from Flavors & Fragrances, Aldrich International Edition 2003–2004.

Mild, Oil, Butter — Green, Vegetable Herbaceous, Woody Fatty, Grassy Sour milk, Dairy, Bitter Almond Sweet Fresh, Green, Floral, Cinnamon like Nutty Almond like, Nutty Green, Herbaceous Penetrant, Fatty, Waxy Floral, Citrus, Green Orange, Fatty, Fried Powerful, Fatty Penetrant, Sweet, Waxy, Floral, Citrus Waxy, Floral

P NS NS NS NS NS o0.05 NS o0.05 NS o0.05 o0.05 o0.05 NS NS o0.05 NS NS

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Table 2 Relative abundance1 (mean7SEM) of ketones detected in the headspace of Bitto cheese samples made in different farms Compound

Farm1 (n ¼ 4)

Farm2 (n ¼ 3)

Farm3 (n ¼ 3)

Farm4 (n ¼ 2)

Farm5 (n ¼ 2)

Odour description2

P

2-Pentanone 3-Hydroxy-2-butanone 2-Heptanone 8-Nonen-2-one 2-Nonanone 2-Undecanone 2-Tridecanone Diphenylmetanone 2-Pentadecanone 5,9-Undecadien-2-one

2.2970.37 n.d. 12.3372.61 0.5870.18 9.8872.67 5.3470.28 3.0370.36 0.28a70.04 1.1670.28 0.2470.01

1.6770.03 n.d. 18.2674.92 0.6870.27 15.9973.46 6.4170.27 4.9070.73 0.74b70.02 1.7670.30 n.d.

2.0170.27 n.d. 13.2773.44 0.7470.15 10.5473.36 3.9871.18 4.8570.21 0.38ab70.09 2.0170.12 0.3370.06

0.1770.10 n.d. 6.0172.15 0.2470.08 6.2072.34 4.1770.42 2.2670.16 0.12a70.07 0.7970.02 0.0970.05

n.d. 3.1070.39 6.4471.47 0.5970.13 6.3171.71 5.9670.29 3.7470.18 0.15a70.09 2.0070.14 n.d.

Sweet, Floral, Ethereal Buttery Blue cheese, Spicy — Fruity, Floral Citrus, Rose, Iris Warm, Herbaceous — Delicate musk —

NS NS NS NS NS NS NS o0.05 NS NS

Means within rows with different superscript are significantly (Po0.05). NS, not significant. 1 Relative abundance expressed as percentage on total volatile compounds detected. 2 Odour description from Flavors & Fragrances, Aldrich International Edition 2003–2004. Table 3 Relative abundance1 (mean7SEM) of free fatty acids detected in the headspace of Bitto cheese samples made in different farms Compound

Farm1 (n ¼ 4)

Farm2 (n ¼ 3)

Farm3 (n ¼ 3)

Farm4 (n ¼ 2)

Farm5 (n ¼ 2)

Odour description2

P

Butanoic acid Pentanoic acid Hexanoic acid Octanoic acid Decanoic acid Dodecanoic acid Tetradecanoic acid Tetradecenoic acid Hexadecanoic acid Octadecanoic acid 3-Methyl butanoic acid 2-Methyl butanoic acid

6.1672.49 1.8970.20 4.3770.54 1.2270.29 3.2170.26 0.9270.34 0.0570.03 1.43a70.83 3.8370.74 0.6670.38 3.0570.31 1.5970.13

7.0470.05 0.2370.06 3.2470.32 2.2670.06 2.5271.46 0.2570.14 n.d. 5.36b70.07 0.6770.19 1.1670.18 n.d. 0.9170.13

5.3271.55 1.8470.53 5.2071.31 2.4971.05 2.1671.16 0.8270.03 0.1670.03 1.30a70.08 2.9670.04 0.9270.11 1.3070.02 0.3570.20

5.3270.33 0.0770.04 2.1870.03 0.8270.23 1.4270.19 0.3970.12 n.d. 2.64a70.38 4.3470.21 21.7670.08 1.2470.02 n.d.

1.2470.19 n.d. 3.8770.80 1.8770.53 3.6670.24 n.d. 0.0570.03 0.17a70.04 n.d. n.d. 0.3270.01 0.0970.05

Sharp, Cheesy Putrid, Sweety, Rancid Sickening, Sour Unpleasant, Oily Fatty Fatty Faint, Waxy, Oily — Virtually, Odourless Odourless — —

NS NS NS NS NS NS NS o0.05 NS NS NS NS

Means within rows with different superscript are significantly (Po0.05). NS, not significant. 1 Relative abundance expressed as percentage on total volatile compounds detected. 2 Odour description from Flavors & Fragrances, Aldrich International Edition 2003–2004.

found in all samples, is probably due to the early stage of ripening of analysed cheese. It is interesting to observe that these compounds are able to bestow a characteristic floral note to cheese; hexanal, one of the most abundant aldehydes found in all samples, for example, gives the green note of immature fruit, while nonanal is described having an aromatic note reassembly rose (Izco & Torre, 2000). The aldehydes can play a very important role in Bitto cheese, also because their low perception threshold (Carbonell, Nun˜ez, & Ferna´ndez-Garcı´ a, 2002). Also many methyl ketones are intermediate compounds, which may be reduced to alcohols. The major representative of 2-alkanones with odd number of carbon atoms were: 2-heptanone, 2-nonanone, 2-undecanone, 2-pentadecanone and 2-tridecanone, while there were only small amounts of 8-nonen-2-one, pentadecanone and diphenylmetanone and they are presented in Table 2. The 2-heptanone and 2-nonanone were the most abundant compounds isolated in the headspace of Bitto cheese samples and they are considered as impact

compounds in natural Gorgonzola cheese (Moio, Piombino, & Addeo, 2000). Due to their typical odours and their low perception threshold, ketones, and especially methylketones, are primarily known for their contribution to the aroma of cheese and they are more abundant in the cheeses manufactured by artisan dairies (Karahadian, Josephon, & Lindsay, 1985). These odour-active compounds are also considered an important part of the aroma in bleu cheese (Dumont & Adda, 1987), and in other varieties as Swiss Gruye`re, Gruye`re de Comte` and Camembert (Dirinck & Winne, 1999; Rychlik & Bosset, 2001). Moreover, 2heptanone, together with 2-nonanone and diacetyl, is considered the main component of butter aroma (Belitz & Grosch, 1999). 3.2. Fatty acids and esters Free fatty acids and esters were another series of important flavour constituents found in Bitto cheese (Tables 3 and 4).

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Table 4 Relative abundance1 (mean7SEM) of esters detected in the headspace of Bitto cheese samples made in different farms Compound

Farm1 (n ¼ 4) Farm2 (n ¼ 3) Farm3 (n ¼ 3) Farm4 (n ¼ 2) Farm5 (n ¼ 2) Odour description2

Ethyl acetate 7.0074.04 Methyl butyrate 1.9970.15 Ethyl butanoate 10.6574.33 Propyl butanoate 0.1370.08 Methyl pentanoate 0.3070.08 Methyl hexanoate 2.49a70.29 Ethyl hexanoate 7.7271.23 Propyl hexanoate 0.1770.10 Methyl octanoate 0.4370.01 Ethyl octanoate 2.8970.59 Ethyl decanoate 1.6570.53 Ethyl dodecanoate 1.5270.57 Ethyl tetradecanoate 0.3370.08 Methyl hexadecanoate n.d. Methyl octadecanoate n.d.

5.4570.06 0.6970.40 4.3570.55 0.4470.09 0.5970.29 2.05a71.18 5.3370.92 0.1670.09 0.3770.21 1.3670.05 0.6970.19 0.1170.06 0.1170.07 1.7370.63 0.2870.16

24.6970.43 1.3670.01 4.5272.05 0.3270.00 0.3570.05 2.13a70.20 4.9670.59 0.1070.06 0.1770.10 1.9270.37 1.4570.02 1.1470.28 0.2770.09 1.8370.42 0.7570.34

n.d. 0.2770.04 12.4075.59 n.d. 0.1070.06 17.08b71.07 2.8670.08 0.1870.04 0.2570.00 1.1270.25 0.1070.04 n.d. 0.0770.04 0.0770.04 0.0470.02

n.d. n.d. n.d. n.d. 0.2470.06 1.00a70.13 1.7070.11 0.00 0.3770.09 0.4170.06 0.2170.05 0.0670.03 0.0370.02 n.d. n.d.

Fruity, Fragrant, Banana–pineapple Fragrant, Ethereal, Sweet Sharp, Cheesy Fruity, Sour Cheese, Parmesan Pineapple, Ethereal Sickening, Sour Wine-like, Cheese Green, Fruity Unpleasant, Oily Fatty Fatty Mild waxy, Soapy Virtually, Odourless Virtually, Odourless

P NS NS NS NS NS o0.05 NS NS NS NS NS NS NS NS NS

Means within rows with different superscript are significantly (Po0.05). NS, not significant. 1 Relative abundance expressed as percentage on total volatile compounds detected. 2 Odour description from Flavors & Fragrances, Aldrich International Edition 2003–2004.

Thirteen fatty acids and sixteen esters were isolated from Bitto cheese; the importance of these compounds in term of aroma depends among products and, in the great majority of cheese, they play a positive contribution to the typical flavour (Parliment, Kolor, & Rizzo, 1982). Fatty acids originate mainly from the hydrolysis of triacylglycerols (Careri, Manini, Spagnoli, Barbieri, & Bolzoni, 1994). Fatty acids are not only aroma compounds by themselves, but also serve as precursors of methyl-ketones, alcohols, lactones and esters. In general, long-chain fatty acids (412 carbon atoms) play a minor role in the flavour, because of their relatively high perception threshold (Preininger, Warmke, & Grosh, 1996). Higher level of medium chain fatty acids, (C6–C10), with even number of carbon atoms were present in Bitto cheese. Butanoic acid, hexanoic acid and octanoic acid were the most abundant free fatty acids extracted in all samples analysed. Butanoic acid was isolated in all samples of cheese and has a rancid cheese-like odour which plays a role in the flavour of some cheese such as Camembert and Grana Padano. Branched-chain fatty acids like 2- and 3-methylbutanoic acids were isolated in Bitto samples, according to results reported by Yvon and Rijnen (2001). These compounds probably derive from isoleucine and leucine and they impart ‘‘sweaty’’ and ‘‘ripened cheese’’ aroma. Among esters, more ethyl esters were contained in the volatile fraction of all samples, in accordance with results found in other varieties of cheese (Buchin et al., 1998). Ethyl butanoate, ethyl hexanoate and ethyl octanoate were the most abundant esters isolated in the headspaces of the samples. These compounds have been identified as ones of the most potent odorants of Cheddar, Grana Padano and

Ragusano (Bellesia et al., 2003; Moio & Addeo, 1998). Esterification reactions may occur between short to medium chain fatty acids and alcohols. Nevertheless, it has been recently shown that cheese esters might also be synthesized directly from triglycerides and alcohols via an alcoholysis reaction (Thierry, Maillard, Richoux, & Lortal, 2006). These compounds are probably the result of microbial metabolism of fatty acids. They play an important role in the formation of the fruity character in cheese and they are able to give this main fruity note to the flavour of some Italian cheeses (Viallon et al., 2000). They also contribute to the balance of the flavour by minimizing the sharpness imparted by free fatty acids (Gallois & Langlois, 1990). 3.3. Alcohols and hydrocarbons Eleven alcohols were identified in the volatile fraction of Bitto cheese sample (Table 5). These compounds may be rapidly produced from aldehydes under the strong reducing condition present in cheese, or from other metabolic pathways, such as lactose metabolism, and amino acid catabolism (Molimard & Spinnler, 1996). The most abundant compounds present in the cheese samples were: 1-butanol, 2-butanol, phenylethylalcohol and 3-methyl-1-butanol. The last one was characterized in cheese samples from farms 1, 2 and 3, and reasonably derived from amino acid catabolism (Urbach, 1997). It is responsible for the pleasant aroma of some soft cheeses, giving an alcoholic floral note and it is interesting to observe that alcohols, when present at high levels, are responsible for defects in some cheese, such as Cheddar and Gouda (Molimard & Spinnler, 1996). Among the hydrocarbons, xylenes, were present in great quantity in all

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Table 5 Relative abundance1 (mean7SEM) of alcohols detected in the headspace of Bitto cheese samples made in different farms Compound

Farm1 (n ¼ 4) Farm2 (n ¼ 3) Farm3 (n ¼ 3) Farm4 (n ¼ 2) Farm5 (n ¼ 2) Odour description2

Ethanol 1.5770.91 2-Butanol 5.0472.91 1-Butanol 1.6070.52 2-Pentanol 1.2370.71 3-Methyl-1-butanol 2.3470.40 2-Methyl-1-butanol 0.8870.21 2-Furanmethanol 0.7970.02 1-Hexanol 0.31ab70.01 2-Heptanol n.d. Phenylethylalcohol 1.59a70.27 Benzenethanol 0.3470.02

n.d. 5.0270.03 3.2370.88 n.d. 2.3570.01 0.9570.16 1.2670.05 0.04a70.02 n.d. 0.45b70.08 2.5870.14

n.d. n.d. 1.8070.56 n.d. 1.7270.03 0.2770.16 0.00 0.41ab70.05 n.d. 0.33b70.01 n.d.

n.d. n.d. 0.9970.07 n.d. n.d. n.d. 0.00 0.77b70.13 0.0770.04 0.12b70.07 0.1370.08

n.d. n.d. 1.6170.66 n.d. n.d. n.d. 1.2170.01 0.51ab70.04 n.d. 0.09b70.05 n.d.

P

— NS Medicinal NS Medicinal NS Mild green, Fusel oil NS Herbaceous, Hearthy, Oily NS — NS — NS Herbaceous, Fragrant, Green, Woody o0.05 Earthy, Oily NS — o0.05 — NS

Means within rows with different superscript are significantly (Po0.05). NS, not significant. 1 Relative abundance expressed as percentage on total volatile compounds detected. 2 Odour description from Flavors & Fragrances, Aldrich International Edition 2003–2004.

Table 6 Relative abundance1 (mean7SEM) of hydrocarbons detected in the headspace of Bitto cheese samples made in different farms Compound

Farm1 (n ¼ 4)

Farm2 (n ¼ 3)

Farm3 (n ¼ 3)

Farm4 (n ¼ 2)

Farm5 (n ¼ 2)

Odour description2

P

Toluene Methylbenzene Ethylbenzene p-Xylene Benzene1,4 dimethyl Tetradecane 2-Hexadecene Naphtalene

2.3070.41 1.8170.45 0.8170.23 1.6870.33 0.2470.09 0.5870.24 1.1670.46 0.9970.18

0.9370.54 2.9470.37 1.2970.09 1.1470.57 0.3970.22 0.8270.32 1.7570.62 0.8870.04

n.d. 1.0270.58 0.8270.21 1.3870.14 0.4670.26 0.7770.07 2.0370.44 3.4671.23

0.7170.10 1.3770.30 0.3570.05 0.9870.24 n.d. 9.9275.62 0.0470.02 0.4870.00

n.d. 1.1570.11 0.4070.06 1.3670.42 0.0270.01 0.2770.08 0.6570.06 0.4570.13

— Fruity, Fragrant Heavy, Floral — Sweet, Almond, Cherry, Spicy — — —

NS NS NS NS NS NS NS NS

Means within rows with different superscript are significantly (Po0.05). NS, not significant. 1 Relative abundance expressed as percentage on total volatile compounds detected. 2 Odour description from Flavors & Fragrances, Aldrich International Edition 2003–2004.

samples of Bitto; these compounds seem arise from the degradation of plant material (Table 6).

grazing animals and ultimately into the cheese (Bosset, Butikofer, Gauch, & Sieber, 1994).

3.4. Terpenes

4. Conclusions

Eleven terpenes has been isolated in the volatile fraction of cheese samples, and they are presented in Table 7. The most abundant terpenes isolated in cheese samples were a-pinene, b-myrcene, limonene followed by sabinene, trans-b-caryophyllene, d-3-carene and g-terpinene. Terpenes are important in those cheeses which are manufactured in alpine regions by artisan farmers. They originate from the plants that constitute the forage mixture of the pasture (Bugaud, Buchin, Hauwuy, & Coulon, 2001; Dumont & Adda, 1978). Their occurrence in cheese, in fact, is not related to the ripening process, but to the diet of cows grazing in high mountain pastures, although some volatile terpenes, like apinene, may be biosynthesize by microrganisms (Larsen, 1997). These molecules are transferred into the milk of the

As evidenced by this initial study and from the results obtained, it may be concluded that Bitto cheese has a complex volatile profile that belongs to the following families: aldehydes, ketones, free fatty acids, esters and alcohols. It can be also speculated that the flavour of this cheese seems to depend, not only on particular key components, but rather on a ‘‘balance’’ of all components present. The present work provides new data to characterize Bitto cheese and to increase knowledge about the compounds responsible for the formation of its characteristic aroma. The SBSE extraction technique in Bitto samples represents a novel approached application for cheese matrix and the broad range of aroma compounds isolated and characterized allow to consider this method suitable to resolve this analysis.

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191

Table 7 Relative abundance1 (mean7SEM) of terpenes detected in the headspace of Bitto cheese samples made in different farms Compound

Farm1 (n ¼ 4)

Farm2 (n ¼ 3)

Farm3 (n ¼ 3)

Farm4 (n ¼ 2)

Farm5 (n ¼ 2)

Odour description2

P

a-Pinene Camphene Sabinene b-Pinene b-Myrcene Cumene d-3-Carene Limonene g-Terpinene Verbenol Trans-b-caryophyllene

1.0270.24 0.4370.25 0.4770.27 0.7070.40 0.9870.41 0.1770.10 0.5670.09 1.2670.32 0.3470.04 0.3370.19 0.20a70.01

1.3170.76 0.1770.10 0.9070.52 0.6270.36 1.0970.63 0.4670.26 0.2170.12 1.5670.26 0.1970.11 0.2370.13 0.06a70.03

1.5570.25 n.d. 0.7370.42 1.9470.05 1.2470.06 0.5170.04 0.4270.06 1.1370.21 0.2070.11 n.d. 0.46b70.02

0.1470.08 0.2970.10 0.6170.03 1.0870.03 0.3070.02 0.2570.02 0.3970.09 0.8370.13 0.0570.03 n.d. 0.15a70.01

0.4770.08 0.8470.05 1.2670.08 n.d. 1.5570.19 n.d. 0.2970.09 0.7770.17 0.3770.22 1.2170.13 0.11a70.06

Sharp, Pine Camphoraceous — Woody, Pine Sweet, Balsamic, Plastic Pungent, Acid — Warm, Spearmint Herbaceous, Citrus Spicy, Minty, Champhoraceous Terpene Odour, Woody, Spicy

NS NS NS NS NS NS NS NS NS NS o0.05

Means within rows with different superscript are significantly (Po0.05). NS, not significant. 1 Relative abundance expressed as percentage on total volatile compounds detected. 2 Odour description from Flavors & Fragrances, Aldrich International Edition 2003–2004.

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