Solid phase microextraction (SPME) combined with gas-chromatography and olfactometry-mass spectrometry for characterization of cheese aroma compounds

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Lebensm.-Wiss. u.-Technol. 37 (2004) 139–154

Solid phase microextraction (SPME) combined with gas-chromatography and olfactometry-mass spectrometry for characterization of cheese aroma compounds Damian Conrad Frank, Caroline Mary Owen, John Patterson* Sensory Neuroscience Laboratory, School of Biophysical Sciences and Electrical Engineering, Swinburne University of Technology, P.O. Box 218, Hawthorn 3122, Australia Received 26 August 2002; accepted 17 June 2003

Abstract The applicability of solid phase microextraction (SPME) as a technique for the concentration of cheese aroma for analysis by gas chromatography-mass spectrometry (GC-MS) and gas chromatography-olfactometry (GC-O) was assessed in this preliminary study. Three each of the following cheese varieties were examined: cheddar, hard grating and mold-ripened blue. Volatile components were concentrated by Carboxen-PDMS SPME fibres for 16 h (overnight) and analysed by GC-MS and GC-O. Odor compounds, which could be perceived at the olfactory port (OP), were matched with electron impact (EI) and methanol chemical ionization (CI) mass spectra. The volatile compounds identified were compared to previously reported cheese aroma compounds. Of the components identified via olfactometry, methanethiol, methional, dimethyl trisulfide and butanoic acid were present in all of the cheeses implying their essential role in the formation of basic cheese aroma. A number of alkyl-pyrazines were also found to impart roasted nutty, raw potato and savoury broth-like notes in some of the cheeses. In all cases, the aroma active compounds identified via olfactometry were in agreement with those reported in the literature. In a separate study, it was demonstrated on a number of cheeses that the adsorption of most important aroma volatiles increased consistently up to 16 h; i.e. sulfur compounds, lactones, pyrazines, phenolic compounds and benzene derivatives. For the strong-smelling pecorino and blue cheeses, however, some analyte displacement effects were observed. Data indicated that a sampling time between 9 and 16 h was appropriate and displacement/ competition reactions tended to occur in strong cheeses, i.e. those which have undergone extensive lipolysis e.g. pecorino, very high concentration of butanoic and hexanoic acid, and/or b-oxidation e.g. blue cheese, with high concentrations of 2-heptanone and 2nonanone. r 2003 Published by Elsevier Ltd. on behalf of Swiss Society of Food Science and Technology. Keywords: Cheese aroma; GC-MS; Olfactometry; SPME

1. Introduction Analysis of cheese aroma compounds by traditional methods typically involves the use of concentration– extraction equipment such as vacuum distillation, liquid–liquid extraction and more recently, purge and trap techniques (Bosset & Gauch, 1993; Engels, Dekker, de Jong, Neeter, & Visser, 1997; Thierry, Maillard, & Le Que! re, 1999). Vacuum distillation, whilst effective, involves delicate equipment, use of organic solvents and can be prohibitively time consuming for general *Corresponding author. Tel.: +62-3-9214-8862; fax: +62-3-98190856. E-mail address: [email protected] (J. Patterson).

application. Microscale liquid–liquid extraction apparatus offers a less costly alternative, but suffers the drawback of requiring elevated temperatures, leading to the generation of chemical artefacts and loss of highly volatile components. A number of purge and trap approaches have been successfully applied to cheese aroma analysis, however these require specific equipment. In addition these techniques are not easily automated, precluding their wide-scale use. In contrast, SPME flavour analyses can be performed at low cost with relatively simple equipment. SPME sampling can be easily automated and provides a rapid, solvent-free method for the concentration of cheese aroma compounds for GC-MS and olfactory analyses. Regardless of the method of volatile

0023-6438/$30.00 r 2003 Published by Elsevier Ltd. on behalf of Swiss Society of Food Science and Technology. doi:10.1016/S0023-6438(03)00144-0

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concentration, many important aroma compounds such as some of the sulfur volatiles may be present at concentrations below the limits of conventional methods of detection. In such cases olfactometry may be the only reliable means of detection. SPME has received much attention in the literature for the analysis of food volatile compounds including dairy products (Marsili, 1999; Shooter, Jayatissa, & Renner, 1999; Pe! re" s, Viallon, & Berdague! , 2001), wine (Mestres, Sala, Marit, Busto, & Guasch, 1999; Rocha, Ramalheira, Barros, Delgadillo, & Coimbra, 2001) and other fermented products (Marsili & Miller, 2000). The use of SPME as a concentration technique for the analysis of a range of cheese odor compounds was evaluated in the following study. The primary aims of this study were to: (1) ascertain how many previously identified cheese aroma compounds could be detected in a wide range of cheese styles using SPME-GC-MS and (2) to evaluate the efficacy of Carboxen-PDMS fibres as a sampling method for olfactory profiling of cheese headspace.

2. Materials and methods 2.1. Source of cheeses The nine cheeses (three each of cheddar, blue-mold and hard-grating styles) used in this study were representative of popular cheeses widely available in local (Melbourne) supermarkets. Three blue cheeses were selected: two strong blue-mold varieties (SBlue1 and SBlue2) and one mild blue cheese (MBlue). The Cheddars were all ‘vintage’ i.e. matured for 12–20 months (Ched1, Ched2 and Ched3). The hard cheeses used were: parmesan (HardParm1), pecorino (HardPec2) and Grana Padano (HardGP3); all in block form. The strong blue cheeses had typical blue cheese aroma and the mild blue cheese had a distinctive roast nuttymushroom type of aroma. The typically sweet fruity aroma of the Grana Padano was in contrast to the stronger savoury aroma of the parmesan and pecorino. The matured cheddars had comparatively mild but typical cheddar aroma. For the fibre equilibrium studies, a young pecorino and parmesan were sourced from a local cheese maker and the blue cheese was purchased at the local supermarket. 2.2. Sample preparation and SPME sampling Cheese samples were refrigerated at 4 C until use. For the hard and cheddar cheeses, the outer 2 cm was removed and the sample was grated using a conventional grater. For the blue cheeses, which were packaged as wedges, samples were taken from the apex, which was generally the most prolifically mold-affected area. A 7 gcheese sample was weighed into a 20 ml headspace vial,

and a teflon-lined septum was immediately sealed with an aluminium crimp seal. The headspace depth was approximately 4 cm. 75 mm Carboxen-PDMS fibres (Supelco, Bellafonte, USA) in manual SPME fibre holders were used to concentrate the cheese headspace. The fibres were exposed to the headspace at ambient temperature (22 C) overnight (16 h). For each of the cheeses reported at least three olfactory and three GCMS analyses were performed. For the equilibrium studies, the parmesan and pecorino were finely grated and thoroughly mixed before being transferred to headspace vials and sealed. The blue cheese was cut into small cubes and ground to a homogeneous powder with a mortar and pestle in liquid nitrogen and added to headspace vials. 2.3. GC-olfactometry and GC-MS analyses A Varian Chrompack 3800 (Walnut Creek, CA) gas chromatograph with electronic flow control (EFC) was used for all analyses. Ultra-high purity helium (BOC Gases, Australia) was used as the carrier gas. Separation was achieved on a BP21-polyethylene glycol terephthalic acid-treated column (30 m, 0.32 mm ID, 0.25 mm film thickness; SGE Australia). Mass spectral analyses were conducted with the analytical column directly connected to an ion-trap mass spectrometer (Varian Saturn 2000) and the EFC was set to a constant flow of 1.5 ml/min. The injector (Varian 1098) was held at 250 C (splitless to 3 min then 1:10 split). The oven was temperature programmed as follows: held at 35 C for 8 min, then the temperature was raised to 190 C (4 C/min, held 5 min) to a final temperature of 210 C (5 C/min, held 5 min). The ion trap conditions were as follows: transfer line temperature 160 C, trap temperature 180 C, scan range 40–250 amu (3 microscans/scan). EI mass spectra were identified with the NIST-98 (version 1.7) mass spectral library. In addition to EI mass spectra, methanol chemical ionization (CI) was also employed to obtain molecular mass, where applicable. Methanol CI gives a prominent M+1+ ion, with little or no fragmentation, and is especially good for determination of the molecular-mass of ketones, esters and carboxylic acids. In the case of the later, methanol CI gives a prominent methylated M+14+ ion, with a smaller M+ ion. Methanol CI is not generally effective for long-chain, saturated hydrocarbons or long-chain, saturated alcohols. Quantification of compounds was achieved using the Saturn-Star software (Varian) using characteristic ions or in some cases, the total ion current for particular compounds. For the GC-O work the analytical column was connected to a capillary outlet splitter with a 1:10 ratio (SGE Australia). The higher flow column was connected to the olfactory port (OP; SGE Australia) and the lesser flow was connected to a flame ionization detector (FID). The flow into the olfactory port was set

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at 20 ml/min. A retention index mixture of C5-C22 n-alkanes was used to set the column flow to match the retention times with the column directly connected to the mass spectrometer. 2.4. Sniffing Olfactory analyses were performed by a single sniffer; Caucasian male, 34 years old, occasional social smoker. The sniffer’s olfactory performance was first assessed using the ‘Sniffin’ Sticks’olfactory performance test (Burghart, Wedel, Germany). This clinical test of olfactory performance consists of a basic screening test and an advanced test. The screening test includes a basic smell identification task. The advanced test is composed of three sub-tests: odor identification, odor discrimination and a butanol threshold test (Hummel, Sekinger, Wolf, Pauli, & Kobal, 1997). The scores from the threshold, discrimination and identification tests are summed to form the TDI score, with a total maximum score out of 48. The TDI score of the sniffer of 38 indicated that the participant was within the range for normal olfactory function (Kobal et al., 2000). The sniffer has had wide exposure to dairy and non-dairy aroma references and suffers from no known anosmias. When odors were perceived at the OP their elution time, aroma quality and intensity were noted. The retention times of the odors were consistent for each of the three overnight runs for each cheese. A number of standard compounds were run through the GC-MS to confirm identification. All chemicals were of analytical grade purity (Sigma-Aldrich, Australia). The retention times of the odors at the OP were calculated and compared with retention times of components on total ion chromatograms (TICs). Descriptions of the odors were based on the single sniffer’s subjective response.

3. Experimental data In the first part of the study, the range of odor compounds picked up by Carboxen-PDMS fibres were compared with those reported for particular varieties in the literature (cheddar, hard-grating and blue-mold). The potential for the fibres to pick up characteristic chemical differences between the three cheese varieties was also considered. Headspace profiles of gross differences between the hard-grating, blue and cheddar cheeses are shown in Fig. 1. For each cheese around 300–350 separate peaks were resolved, the majority of which were present at low concentration. More than 200 compounds were identified on the basis of a combination of some or all of the following criteria: (1) EI full scan mass spectra, (2) CI parent ion mass (where applicable), (3) agreement of retention time with authentic standards, and (4) odor quality at the OP.

141

Some gross differences were observed in the profiles of the cheeses. Butanoic and hexanoic acids dominated the profiles of all cheeses, with relatively high concentrations of both compounds measured in the headspaces of the strong blue cheeses and the pecorino. High concentrations of 2-heptanone, 2-nonanone, 2-octenone, phenyl ethyl alcohol and higher-alcohols were measured in the blue cheeses. These components are well-defined products of the extensive b-oxidation of free fatty acids (FFA) brought about by Penicillium roqueforti (Kinsella & Hwang, 1976). The profile for Grana Padano-style indicated a higher concentration of ethyl hexanoate, which was found in sniffing experiments to be an impact compound. In the Parmesan and pecorino cheeses trimethyl- and tetramethyl-pyrazine peaks were present on profiles. Most of the differences in the headspace chemical composition of the cheeses involved trace components, many of which were aroma active, as determined by olfactometry analysis. Table 1 lists odor-active compounds perceived at the OP together with their odor quality and relative intensity. Odor intensity was rated on a scale of 1–5; just perceived (1), mild, but clearly definable (2), strong (3), very strong (4) and extremely strong (5). The various different classes of aroma compounds are discussed in the following. 3.1. Sulfur compounds At least 14 sulfur compounds were identified in the cheese headspace on the basis of full-scan EI mass spectra, including methanethiol, methional (3-methylthiopropionaldehyde), dimethyl sulfide, dimethyl disulfide and dimethyl trisulfide. Sulfur compounds were semi-quantitatively analysed using the characteristic ions listed in Table 2. Methanethiol, with its distinctive fermented cabbage smell, was readily perceived in all the cheeses ranging from mild to very strong (HardParm1). The quantitative data (Table 2) for the HardParm1, Ched3 and the two strong blue cheeses was reflected in odor intensity at the OP. The distinctive savoury, roast potato aroma of methional was also present as a major impact aroma in all the cheeses surveyed (strong to extremely strong). In the blue cheeses, methional coeluted with a larger peak, tentatively identified as isopentyl hexanoate making it difficult to quantify despite the characteristic aroma at the OP. Dimethyl sulfide and dimethyl disulfide were present in all of the cheeses, but had only very low odor intensity at the OP in a few of the cheeses. In contrast, dimethyl trisulfide, detected in all of the cheeses, was readily identified by its characteristic garlic-savoury smell and mass spectrum, ranging from mild to extremely strong. Dimethyl tetrasulfide and methionol (methyl thiopropanol) had similar retention times and alliaceous savoury and cooked cauliflower aromas, respectively. Dimethyl

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Fig. 1. Gross differences in the headspace profiles of the nine different cheeses after sampling with Carboxen-PDMS fibres for 16 h at 22 C.

tetrasulfide was present in the headspace of the three hard cheeses and in two of the cheddar varieties. This compound was most concentrated in the HardParm1, which had the strongest odor at the OP. Methionol was

detected in all of the cheeses (except one cheddar) and registered as a garlicy savoury note at the OP. This compound was stronger in all of the blue cheeses, which was reflected in the quantitative data (Table 2). 2-methyl

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143

Table 1 Relative intensities of odor-active compounds in the headspace of the nine cheeses (16 h sampling with Carboxen-PDMS fibres at ambient temperature of 22 C) Compound name

RI

Odor description

Ched1 Ched2 Ched3 MBlue Sblue1 Sblue2 Hard Hard Hard Parm1 Pec2 GP3

Methanethiolb, c, d, f, i Dimethyl sulfidea, c, d, f, i, y,  2-Butanonea, b, c, d, e, f, i, y,  Methyl butanoatea, b, c, d, e, k, y,  Diacetyla, b, c, d, e, f, i, l, k, y,  Ethyl butanoateb, c, d, e, f, k, l, i, n, y,  Dimethyl disulfidec, d, e, f,  Methyl-butanala, d, e, f,  Unidentified Unidentified Ethyl pentanoatea, d,  2-Heptanonea, b, c, d, f, h, l, i, y,  Unidentified Ethyl hexanoatea, c, d, e, i, l, n, y,  Methyl pyrazine Methylbutyl butanoate 2-Octanonea, d, h, i,  1-Octene-3-onef, l, m, y 2,5-Dimethyl pyrazineh, n, y,  2,6-Dimethyl pyrazineh, n, y,  Dimethyl trisulfidea, b, c, d, e, f, m, n, y,  Methyl propyl pyrazine 2-Ethyl-5-methyl pyrazinen,  2-Nonanonea, b, c, d, e, f, i,  Trimethyl-pyrazinen, y,  Sesquiterpene (MW 204) Methoxy-2-methylbenzeney,  Dimethylethyl pyrazinen,  Methionalb, c, d, l, m, n, y,  Dimethylethyl pyrazinen,  Tetramethyl pyrazinen, y,  Acetic acidb, e, i, k, m, n, y,  2-Nonenaln, y 3,5-Diethyl 2-methyl pyrazineh,  2,3,5-Trimethyl-6 ethyl pyrazinen, Sesquiterpene (MW 204) 2-methyl thioethanol Sesquiterpene (MW 204) Unidentified Unidentified Sesquiterpene (MW 204) Unidentified Butanoic acidb, e, f, h, i, m, n, y Unidentified Isovaleric acidi, h, n, y Unidentified Dimethyl tetrasulfideb,  Naphthalene/unknownh,  Methylthio proponolb, i, y,  1,4-Dimethoxybenzene Allylmethyl sulfidey Pentanoic acid (valeric)b, c, e, h, n, y,  g-Hexalactoneb, h,  Unidentified 2-Methoxy phenolh,  Hexanoic acidb, c, e, h, i, n, y,  g-Octalactoneh, i, n,  2-Phenylethyl alcoholh, i, y,  g-Cyanotoluene Heptanoic acidh, y

o900 o900 o900 o900 923 954 979 995 1002 1007 1016 1128 1183 1202 1252 1253 1263 1299 1317 1322 1358 1361 1381 1390 1413 1424 1439 1460 1463 1480 1499 1485 1508 1516 1517 1531 1531 1565 1573 1587 1588 1632 1683 1654 1678 1688 1697 1698 1702 1716 1729 1736 1760 1787 1815 1817 1846 1850 1882 1905

Fermented cabbage Garlic-rotten Sap-acetone Fruity apple Buttery-sweet Fruity-melon, sweet Garlic, onion Grainy malty Peppermint/sweet Rubber-like Melon, fruity, sweet Musty, varnish, sweet Fatty, sweet, hay Fruity, grape melon Nutty, grainy Sweet, fruity Fruity, green Fungus, woody Nutty, roast grain Nutty roast grain Sweet, garlic, spicy Nutty, savoury Peanut, green Floral, fruity, peachy Nutty, musty, beans Dirt, fungus Disinfectant, aniseed Raw potato, beans, Roast potato Savoury brothy Raw potato, beans Vinegar sour, sharp Fatty, cucumber Savoury nutty Savoury nutty Wet wood, fungus Roast chicken garlicy Strong dirt Green exotic Burnt matches, nutty Strong fungus Garlic, savoury cheesy, rotten, sharp Milky, custard Rotten cheesy Burnt rubber, plant- like Sulphur, garlic Green, fresh Garlic sulphur Woody, earthy, phenol Garlic, savoury Cheesy, meaty, rotten Coconut, sweet Garlic, onion Smoked, phenolic Sharp, goaty Coconut, melon sweet Rose, floral Smoked, chemical Goaty, cheesy

2 1 2

2 1 3

3 1 2

2

3

3

3

2 1

2

1 3 1 1

3 3 1 1

3 3 1

3 3 1

1

4 1 3

2

2

1

3 2

3 4 1

2

2 2

2

4 1 1 2 2

1

1 1 3

1

2 1

1

3

2 4

2

3

3 1

2 1

3

4

2 1

5

3 1

4 4 4 3 4 3 1 3 3 3

2 1

2 2 3 1 1 2

2 1 2 3 2

2 4

3

2

3 3

3

4

4

3

4 3

4 3

3

3

3 1

1 3

1

3 3

2 1

2 1 3

4 3 1

1 2 2

1 3 2

2 3 3 3 1 3 2

1 3 1 1 4 3

1

3

4

4

3

3

2 3

2 3

2 3

3 2 2

1

1

1

3 2

1 5 3 2 2 2 3 2 2 3 2 2 2 4 3 3 3

1 1 1

2 2 2 5

2 2 3 3

3 3

1 1 3

2

3

2

1 5

3 3 2 2 3

1 2 2 3 1

3

2

1 2 3 3 3 3

2 3 4 2

3 3 2

3 1 2 1 2

1

5 4 4 1 3

2 4 3 3 2 3 2

1 2 1

2

1

3 1

3

1

2

1

1

1

3 4 3 2

1

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144 Table 1 (continued)

RI

Compound name Diphenyl ether Furaneold, l, m, y,  p-Cresolb, c, h, i, y,  Unidentified Homofuraneold, l, m, n, y,  d-Decalactoneb, h, i, l, m, n, y,  Ethyl phenoly, i Sotoloney,  Unidentified 3-Propyl phenol Unidentified n-Decanoic acidh, n,  d-Dodecalactoneh, i, m, 

Odor description

1920 1951 1981 1990 1984 2036 2047 2058 2086 2104 2112 2113 2187

Ched1 Ched2 Ched3 MBlue Sblue1 Sblue2 Hard Hard Hard Parm1 Pec2 GP3

Melon, geranium milky Caramel burnt sugar Barnyard, phenolic Honey floral Caramel burnt sugar Coconut, sweet Disinfectant Coriander, curry Wet-dog,-cardboard Disinfectant Grass, plant Waxy-sweet Fruity-apricots

2

2 2 1 3 3 2

2

3

1 1

3

3 1 1

1

1

1 2 3

2 1 3 4 1 2

1

2 3

2 3 3

3 2 4 3

4 2 3 1

3 5 4 5 2

1 1 3 2 1

3 1

3

1

4 2

2

1

2

2 2

w The odor profiles were determined in triplicate by the one sniffer for each cheese. Odor descriptions are the sniffer’s own. The intensity of the compounds was rated from 1 to 5; just perceived at the olfactory port (1), mild aroma, clearly perceived (2), strong aroma (3), very strong (4) and extremely strong (5). RI, retention index. Identified on the basis of EI and CI mass spectral data. yAn authentic sample was used to confirm identity. Previously identified aroma component: aBosset and Gauch (1993), bUrbach (1995), cUrbach (1993), dThierry et al. (1999), eEngels et al. (1997), f P!er"es et al. (2001), hMeinhart and Schreier (1986), iMolimard and Spinnler (1996), kHa and Lindsay (1991a, b), lPreiniger and Grosch (1994), mMilo and Reineccius (1997), nQian and Reineccius (2002).

Table 2 Relative concentration (integrated rea counts) of sulfur compounds in the nine cheeses Compound

Ion

Ched1

Ched2

Ched3

Mblue1

SBlue1

SBlue3

Hard Parm1

Hard Pec2

Hard PG3

Methanethiol Dimethyl sulfide Dimethyl disulfide Dimethyl trisulfide Methional Dimethyl tetrasulfide Methyl thio-propanol Allyl methyl sulfide

47 62 94 126 104 158 106 88

9205 18 596 86 382 45 519 1139 1200 7327 —

38 475 25 489 180 799 40 096 5199 — 1861 —

61 807 12 803 243 152 101 769 5799 1271 1836 —

22 874 653 31 197 10 961 1000 — 22 240 —

53 911 1775 29 214 5756 — — 393 618 62 720

67 262 1014 6288 1011 1020 — 218 431 41 423

182 213 69 122 8510 259 036 43 772 7914 6357 —

34 463 5088 11 663 22 694 7008 1500 1379 —

39 886 17 079 126 887 46 513 8020 2064 14 138

Numbers are the mean area counts for three determinations on 7 g samples of cheese sampled for 16 h. A dash indicates the absence of the compound on TICS. Ion was the mass spectral ion used for quantitation. Standard errors for the three determinations were within 20%. Highest concentrations of a particular compound over the nine cheeses are indicated in bold.

thioethanol (methyl b-mercaptoethanol) was present in a number of the cheeses and perceived as a mild savoury chicken-broth aroma. In the strong blue cheeses, allylmethyl sulfide (3-methylthio-1-propene) was found to be responsible for a mild sulfurous aroma at the OP. Although other sulfur compounds, such as benzthiazole, were detected in cheese headspace, their contribution to the aroma profile was negligible. Most of these sulfur compounds have been identified previously in cheeses (Forss, 1979; Urbach, 1993; Molimard & Spinnler, 1996; McSweeny & Sousa, 2000). 3.2. Esters and fatty acids At least 35 combinations and permutations of possible esters were picked up by the fibres and readily identified from the complimentary of EI-MS and CI-MS data. The ester compounds gave clear M+1+ ions facilitating unambiguous identification together with EImass spectra. Despite the diverse number of ester-

compounds picked up by the SPME fibre, only a small number actually contributed to a definable odour at the OP. These aroma-impact esters were: ethyl butanoate (fruity, apple-like), ethyl pentanoate (melon, fruity), ethyl hexanoate (fruity grape, melon-like). Isoamyl butanoate (3-methyl butyl butanoate) contributed a mild fruity note to the SBlue1. Other ester compounds present may have contributed to a very mild slightly grainy-sweet background aroma: in this study only readily perceived compounds were reported. All of the esters reported by Bosset and Gauch (1993) were identified using the Carboxen fibres. The series of straight chain carboxylic acids, ethanoic (acetic) through to tetradecanoic were present as well as the branched chain isovaleric acid (3-methyl butanoic acid). Acetic acid contributed a mild to strong sharpvinegar note to many of the cheeses. Butanoic acid was generally a major aroma impact compound with its characteristic cheesy sharp aroma. Hexanoic acid was perceived as a very mild to strong sharp, goat-like smell.

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Pentanoic acid was detected as a mild to strong savoury cheesy aroma in some cheeses. Isovaleric acid also imparted a rotten cheesy aroma, especially in the two strong blue cheeses. 3.3. Aldehydes, ketones, diketones and alcohols Although more than 27 ketones and diketones were present in the cheeses, only a small number of these compounds contributed to cheese aroma. Butan-2-one was present in all of the cheeses, imparting a brothy, sap-like aroma. Diacetyl (butane-2,3-dione) was an important impact compound in the headspace of some of the cheeses, with a sweet buttery aroma perceived at the OP. A number of ketones were important in the blue cheese aroma: 2-heptanone (musty, sweet, moldy, varnish) and 2-nonanone (floral fruity, peach). 1octene-3-one was found to be responsible for the mild to very strong fungus mushroom aroma, and was especially strong in the SBlue2 and MBlue1. Although the weak EI mass spectrum was consistent with this compound, the methanol CI mass spectrum yielded a significant increase in sensitivity, with a strong ion at m=z 127. 3.4. Pyrazines and nitrogen compounds A number of nitrogen heterocycles were identified in the cheeses (Tables 1 and 3). Pyridine was present in all of the cheeses as was pyrazine and a number of alkyl pyrazines. All of the isomers of dimethyl pyrazine (2,3-, 2,5-, and 2,6-) were resolved by the column. The 2,6isomer was present in most cheeses as the most abundant of the three (Table 3). A mild to intense roast nut aroma was perceived at the olfactory port at this retention time. Overall, the whole MBlue1 cheese had a dominant roast nutty aroma; two separate intense nutty peaks were detected corresponding with 2,5- and 2,6-

145

dimethyl pyrazine in this cheese; the high odour intensities at the OP were also reflected in the quantitative data (Table 3). Trimethyl- and tetramethyl-pyrazine were present in a number of the cheeses, particularly the hard samples, and had strong savoury, musty potato-like and milder bean-like aromas, respectively. Two closely eluting peaks had mass spectra consistent with isomers of dimethyl ethyl pyrazine, with a prominent ion present at m=z 135. Brothy, savoury aromas were perceived at the OP. Diethyl methyl pyrazine, also identified in the headspace of the hard cheeses (strong peak at m=z 149), was perceived as a savoury-brothy aroma at the OP. A number of other nitrogen heterocycles were tentatively identified on the basis of complimentary EI and CI mass spectral data, but were not found to make a contribution to aroma profiles (Table 1). 3.5. Benzene, phenol and naphthalene derivatives By far the largest chemical group was that represented by aromatic benzene, phenol and naphthalene derivatives. Alkyl benzene derivatives predominated, many of which have been previously identified in cheeses. Apart from naphthalene (previously identified), there were a number of alkyl naphthalenes detected; however, they were not perceived at the OP. A major characteristic of all the blue cheese varieties was a high concentration of 2-methyl anisole (methoxy-2-methyl benzene), with an aniseed disinfectant odor. 4-methoxy anisole (1,4dimethoxybenzene) also present in the headspace of some of the blue cheeses had a strong resin-like aroma. 2-Phenylethyl alcohol (benzene ethanol), with a strong floral rose aroma was a character impact compound in all of the blue cheeses as was p-cresol (4-methyl phenol), which was strong in all of the blue cheeses. This compound was also detected as a mild aroma in the cheddars and the hard-cheeses except ParmHard1, in

Table 3 Relative concentrations (integrated area counts) of alkyl-pyrazines in the nine cheeses Alkyl group

Ion

Ched1

Ched2

Ched3

MBlue1

SBlue2

SBlue3

Hard Parm1

Hard Pec2

Hard GP3

Methyl2,5-dimethyl2,6-Dimethyl2,3-Dimethyl2-Methylpropyl 2-Ethyl-5-methyl Trimethyl 2,6-Diethyl 3-Ethyl-2,5-dimethyl Tetramethyl 3,5-Diethyl-2-methyl

94 108 108 108 136 121 122 135 135 136 149

1000 o1000 — — — o1000 6471 — — — —

— 4927 39 864 1827 — — 13 658 — — — —

o1000 2559 68 691 o1000 — o1000 5265 — — — —

28 620 125 752 729 689 2687 18 370 4717 38 365 — 1347 5669 o1000

28 203 — 16 537 4860 — o1000 — — — — —

o1000 13 402 1934 — — o1000 906 — — — —

1248 49 736 56 494 10 513 — 2669 211 727 3447 9042 287 176 112 614

9145 24 573 19 217 3779 — 1345 271 145 974 4957 55 902 128 765

4627 65 239 86 367 16 812 — 1613 11 874 6070 6450 2723 3197

Numbers are the mean area counts for three determinations on 7 g samples of cheese sampled for 16 h. A dash indicates the absence of the compound. Ion was the mass spectral ion used for quantitation. Standard errors for the three determinations were within 20%. Highest concentrations of a particular compound over the nine cheeses are indicated in bold.

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which it was strong. In addition to the methyl phenols, ethyl phenol and 3-propyl phenol contributed strong medicinal/disinfectant aromas to the blue cheeses. A strong chemical-smoked aroma was perceived in all of the blue cheeses; the compound responsible was tentatively identified as 2-cyanotoluene (2-methy benzonitrile) on the basis of EI and CI mass spectral data. Another separate smoked aroma in two of the blue cheeses was found to be due to 2-methoxy phenol. Diphenyl ether contributed a mild sweet geranium odor mainly to the three blue cheeses. Many alkyl benzenes were present including toluene, ethylbenzene, xylenes, styrene and others, which did not contribute to the aroma. 3.6. Lactones and furanones Although many of the lactones, which have been reported previously in cheese, were present on SPMEGC-MS profiles, few of them were at a high enough concentration to be perceived at the OP. g-hexalactone contributed a mild sweet coconut aroma to some of the cheeses and g-octalactone was very strong in only the SBlue2 blue cheese. d-decalactone was perceived as a mild sweet coconut note in all of the cheddars and was particularly strong in HardParm1. d-dodecalactone was perceived as a weak apricot sweet aroma in a number of cheeses. All of these lactones have been previously reported in cheeses. Two separate intense sweet caramel, burnt sugar aromas were present in many of the cheeses. The compounds responsible were identified as homofuraneol (5-ethyl-4-hydroxy-2-methyl-3(2H)furanone) and furaneol (2,5-dimethyl-4-hydroxy-3(2H)furanone). Both compounds have low odor thresholds: furaneol (25 mg/kg) and homofuraneol (6 mg/kg) (Preiniger & Grosch, 1994). Furaneol yielded a weak but clear signal at m=z 128 (signal to noise ratio; S=N 129–245) in EImode and a clear signal at m=z 129 in methanol CImode. Similarly, despite the often-strong aroma, a weak but clear signal at m=z 142 was present in EI-mode (S=N 142–189) for homofuraneol. In CI mode a clear signal at m=z 143 was also obtained. The presence of these compounds was further confirmed by running authentic standards. An intense curry powder-coriander aroma was also present in a number of the cheeses, which was extreme in HardParm1. Despite the intense aroma in this sample, there was not a characteristic EI mass spectrum at this retention time on TICs. Using CI ionization a clearly discernible peak at m=z 129 (S=N 52) was present. Running an authentic sample confirmed that this compound was sotolone- (4,5-dimethyl-3hydroxy-2(5 H)-furanone), which had an identical retention time and CI mass spectrum. Sotolone has an exceedingly low odor threshold, in the region of around 1 mg/kg in water (Kobayashi, 1989). Sotolone has been previously identified in dairy products (Karagul-Yuceer, Drake, & Cadwallader, 2001).

3.7. Non-aromatic hydrocarbons More than 20 hydrocarbon compounds were readily identified by their EI mass spectra and, in some cases, also from complementary CI data. Although 1-limonene and a-pinene were identified in cheese headspace, no perceivable odor was detected at the OP. At least 12 sesquiterpene-type compounds (MW 204) were identified in the blue cheeses. Two closely spaced sesquiterpene peaks eluting at RI 1565 and RI 1588 had strong woody, musty, fungus aromas in the three blue cheeses. The mass spectra of these compounds were similar to that of 5-epi-aristolochene and germacrene A (Cane, Wu, Proctor, & Hohn, 1993) 5-epi-aristolochene has been identified as the main sesquiterpene product synthesized by a fungal terpinoid cyclase isolated from the blue cheese mold Penicillium roqueforti. Sesquiterpene compounds have previously been identified as fungal volatile metabolites (Viallon et al., 1999, 2000). There were further milder mold-like notes perceived for some of the blue cheeses at RI 1424 and 1531.The fungus odour perceived at RI 1531 was present in the HardParm1, HardPec2 and MBlue1 indicating that this sesquiterpene may have originated from the milk rather than through synthesis by bacterial enzymes, such as Aristolochene synthase. Sesquiterpenes have been previously measured in raw milk (Christensen & Reineccius, 1995; Dirinck & De Winne, 1999). 3.8. Equilibrium study Young pecorino, parmesan and a strong typicalflavoured blue cheese were used to examine the time dependent adsorption of various volatile components onto the fibres. Three new Carboxen-PDMS fibres were conditioned prior to the study and used throughout. Measurements were performed in triplicate and fibres were left in the cheese headspace for a nominal time, 1, 2, 4, 8 and 16 h. The integrated areas for dominant and trace analytes for each cheese variety were calculated and the average values were plotted against time (Figs. 2–4). The time dependent change in concentration of headspace components of the parmesan cheese is shown in Fig. 2. All volatile components increased in concentration over time up to 16 h, with no displacement/ competition effects measured. The fibre concentration of major volatile components, e.g. acetic, butanoic and hexanoic acids and acetoin (3-hydroxy-2-butanone) and important trace aroma compounds, such as methanethiol, dimethyl trisulfide, diacetyl, ethyl hexanoate, trimethyl and tetramethyl pyrazine and others increased over time. Some compounds reached maximum concentration after 8 h, however most increased up to 16 h. Fig. 3 shows changes in the on-fibre concentration for headspace components of a strong smelling blue cheese. Generally the on-fibre concentration of all low and

ARTICLE IN PRESS D.C. Frank et al. / Lebensm.-Wiss. u.-Technol. 37 (2004) 139–154 dimethyl sulfone pyridine 2-hydroxy propanoate trimethylpyrazine methyl sulphide 2-nonanone 4-penten-1-ol 2,6-dimethyl pyrazine trimethylethylpyrazine propanoic acid pentanoic acid

350000 300000 250000 200000

147

methanethiol ethyl octanoate octanoic Acid benzaldehyde pentyl butanoate styrene methyl pentanol hydroxy pentanone

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methylethylthiazole methyl-2(5H)-furanone 2,3-dimethyl pyrazine, 3-methyl-2-buten-1-ol xylene methyl-1-hexene phenylethyl alcohol

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ethyldimethyl pyrazine 3-methyl 2-butenoic acid isocyanomethylbenzene heptanoic acid

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2-furanmethanol phenylmethylacetate d-octalactone phenol methional 3,4-diethyl-thiophene, p-cresol d-decalactone methionol

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acetic acid butanoic acid hexanoic acid acetoin

160000000 140000000 120000000 100000000 80000000

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dimethyl disulfide dimethyl trisulfide 3-methyl heptene ethyl butanoate ethyl hexanoate hexyl isovalerate 2-heptanone tetramethylpyrazine diacetyl

6000000

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Fig. 2. Change in the on-fibre concentration or volatile analytes (integrate area counts) over time (16 h) for the parmesan-style cheese.

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148 16000

12000 10000

methanethiol 2,6-dimethylpyrazine 2,3-dimethylpyrazine ethylmethoxybenzene methyl acetophenone benzyl nitrile d-octalactone

50000 40000 integrated area

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dimethyl sulphide dimethyl trisulphide methionol t-decalactone d-decalactone ethyl phenol propyl phenol

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time (hours) phenylethyl acetate heptanoic acid sesquiterpene2 methionol pentyl nonanoate ethyl dodecanoate dimethyl sulfone phenol

50000 45000

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toluene 2-butanone styrene isopentyl isobutyrate 2-octanone 2-decanone benzaldehyde

1600000 1400000 1200000 integrated area

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2-dodecanol sequiterpene 1 isopentyl octanoate methylphenol dehydromevalonic lactone ? -octalactone decanoic acid 1-nonanol

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butanoic acid hexanoic acid ethyl octanoate ethyl hexanoate 2-pentanone 2-heptanone 2-nonanone

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methoxymethylbenzene isopentyl hexanoate

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long chain alcohol

300000000

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octanoic acid

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Fig. 3. Change in the on-fibre concentration (integrate area counts) over time (16 h) for the blue-mold cheese.

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3000000 integrated area

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acetoin styrene 2-nonanone dimethyl hexane pentanone ethyl butanoate pyridine

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time (hours) methional benzoic acid ethyldimethyl pyrazine ethylmethyl furan furan methanol methyl butenoate ?-octalacone d-octalactone

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methylbutyl butanoate ethyl nonanoate undecanone d-hexalactone d-decalactone phenol

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800000000 700000000 600000000 500000000 400000000 300000000 200000000 100000000 0 0

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Fig. 4. Change in the on-fibre concentration (integrate area counts) over time (16 h) for the pecorino-style cheese.

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medium concentration headspace compounds increased up to 16 h, however some displacement phenomena were observed mainly for the macro-concentration components. Increasing concentrations of 2-nonanone and ethyl octanoate displaced 2-pentanone, 2-hexanone and 2-heptanone. Both butanoic and hexanoic acids were displaced over time, as were 2-heptanol, methyl butanol and acetic acid. In contrast, little or no displacement was observed for trace and medium concentration headspace components, which generally increased up to 16 h. The fibre concentration of sulfur aroma compounds such as, methanethiol, dimethyl trisulfide, allyl methylsulfide and methionol increased or remained constant over time. 2,6-diethylpyrazine, and aroma impact benzene and phenol derivatives also increased over time: methoxy methylbenzene, 4-methyl phenol, ethyl and propyl phenol and phenylethyl alcohol. The fibre concentration of other important trace aroma compounds increased up to 16 h e.g. lactones and sesquiterpene-type compounds. In addition a very strong smelling pecorino was examined. Butanoic and hexanoic acids dominated the headspace. As the on-fibre concentration of hexanoic acid increased over time, other components were displaced; butanoic acid, acetic acid, acetoin, 2-pentanone, and ethyl butanoate. Other components, including the sulfur compounds, either increased or remained constant over time without any significant displacement. The equilibrium data indicated that certain classes of compounds were apparently not affected by displacement phenomena, such as, sulfur compounds, pyrazines, lactones or phenolic compounds.

4. Discussion The Carboxen-PDMS fibres demonstrated an ability to concentrate a wide range of cheese volatile components for GC-MS and GC-O analyses. In addition to well-characterized cheese components, the fibres successfully adsorbed many other types of aroma compounds, such as trace sulfur compounds, pyrazines, furanones and sesquiterpene compounds. 4.1. Pyrazines in cheese aroma The contribution of dimethyl pyrazines to a nutty dimension in cheese aroma has been reported in Emmental (Preiniger & Grosch, 1994), Parmigiano Reggiano (Ha & Lindsay, 1991a, b) and in Gruye" re (Liardon, Bosset, & Blanc, 1982). Alkyl pyrazines are produced in cheese via the condensation of aminoketones, which are formed through Maillard and Strecker degradation reactions (Forss, 1979; Aston & Dulley, 1982; Liardon et al., 1982; Griffith & Hammond, 1989; Barbieri et al., 1994; Shu, 1998). Bosset and Gauch (1993) reported the tentative presence of ethyl

dimethyl pyrazines in Apenzeller cheeses. Liardon and co-workers (1982) reported the presence of all of the pyrazines identified in the present study in the outer layer and smear of Swiss Gruye" re. These alkyl-pyrazines were also identified in both Parmesan (Meinhart & Schreier, 1986) and Swiss Emmental (Qian & Reiniccius, 2002). Of the dimethyl pyrazines, the 2,6-dimethyl isomer was generally found to be the most abundant (Table 3), which is in agreement with the findings of Meinhart and Schreier (1986). In the present study it was demonstrated that not only were alkyl-pyrazines present in cheese headspace, but also that they were perceived at the OP, implying that they may make a contribution to the nutty, savoury dimension of cheese aroma, especially the 2,6-dimethyl pyrazine and trimethyl pyrazine. 4.2. Sulfur compounds in cheese aroma Many researchers have indicated the essential role of sulfur compounds in cheese volatile flavour (Forss, 1979; Urbach, 1993; Molimard & Spinnler, 1996; McSweeny & Sousa, 2000). Both methanethiol and methional have been implicated as important components of cheeses aroma (Urbach, 1993). Methional is produced from both enzymatic and non-enzymatic breakdown of methionine, which is the most abundant sulfur amino acid present in caseins (McSweeny & Sousa, 2000). Methanethiol and dimethyl disulfide are decomposition products of methional. Dimos, Urbach, and Miller (1996) demonstrated a strong correlation between methanethiol and cheddar flavour. All of the cheeses examined in the current study had mild to very strong fermented cabbage aroma associated with methanethiol. Although both dimethyl sulfide and dimethyl disulfide were clearly present on TICs of all cheeses, they contributed only marginally to the aroma perceived at the OP. In contrast, the aroma intensity at the OP of both methional and dimethyl trisulfide were consistently high. Interestingly, dimethyl trisulfide with its pungent savoury, garlic aroma is barely discussed as a major component of cheddar flavour (Urbach, 1993), although it has been previously detected in cheddar headspace (Engels et al., 1997). Dimethyl trisulfide was not reported in an extract of aged cheddar (Christensen & Reineccius, 1995), however the authors conceded in their report that their extraction technique may have led to losses of the most volatile components. In a more recent study (Zehentbauer & Reineccius, 2002), dimethyl trisulfide was reported as a high impact aroma compound in a mild Cheddar extract; none of the other sulfide compounds were found to make a contribution to odor. In another recent study of Parmigiano Regianno (Qian & Reiniccius, 2002) dimethyl trisulphide was found to make a high contribution to volatile flavour:

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once again the other sulfides did not. There is much evidence that the aroma profile of cheese is highly dependent on the concentration methodology employed. Arora, Cormier, and Lee (1995) used a purge and trap method to concentrate cheddar odor and performed olfactory profiling; however neither dimethyl trisulfide nor methional were reported. Engels et al. (1997) reported the presence of dimethyl disulfide and dimethyl trisulfide in the water-soluble fraction of a number of ripened cheeses (including cheddar) using a purge and trap technique. Methional was found to be the compound with the highest odor activity in Emmentaler cheese (Preiniger & Grosch, 1994) and a key flavour component of Cheddar (Zehentbauer & Reineccius, 2002) and Parmigiano Regianno (Qian & Reineccius, 2002). Meinhart and Schreier (1986) did not report the presence of any of the sulfur compounds in Table 3 in extracts of Parmigiano Regianno. The reported sensory thresholds for these methyl sulfides vary considerably (Guadagni, Buttery, & Okano, 1963; Buttery, Guadagni, Ling, Seifert, & Lipton, 1976): dimethyl sulfide (0.3 mg/kg), dimethyl disulfide (0.33–12 mg/kg) and dimethyl trisulfide (0.01 mg/kg). The latter has by far the lowest detection threshold, which may explain why dimethyl trisulfide was invariably detected as the strongest of the four methyl sulfides in all of the cheeses. Threshold data for dimethyl tetrasulfide does not appear to be reported in the literature. Shooter et al. (1999) suggested that the concentration of both methanethiol and dimethyl disulfide in butter were influenced by grass-type and season; if this is true then this would also presumably apply to other important sulfur compounds such as methional and dimethyl trisulfide. The high aroma value of both homofuraneol and furaneol in cheese aroma extracts has been demonstrated by a number of researchers (Rychlik, Warmke, & Grosch, 1997; Qian & Reiniccius, 2002). Furaneol was found to have a high aroma dilution factor in dynamic headspace dilution assay extract of a mild Cheddar cheese (Zehentbauer & Reineccius, 2002) and both furaneol and homofuraneol were found to be potent aroma in the acidic fraction of Cheddar using aroma extract dilution assay. The contribution of sotolone to the aroma of dairy products has been cited recently (Karagul-Yuceer et al., 2001).

the blue cheeses examined. 1-Octen-3-one, which has a low odor threshold of around 10 mg/kg (Preiniger & Grosch, 1994) has been detected previously in Emmental (Preiniger & Grosch, 1994) and other varieties (Molimard & Spinnler, 1996; Kub!ıckova! & Grosch, 1997; Pe! re" s et al., 2001). 2-Octanone was present as a mild fruity-green aroma in the two strong blue cheeses. A number of low concentration high impact compounds were present in blue cheese profiles: 1-methyl anisole (aniseed, disinfectant, floral), b-phenyl ethyl alcohol (rose, floral), 2-methyl cyanotoluene (burnt rubber, smoked ham), p-cresol (barnyard, manure-like, medicinal), ethyl and n-propyl phenol (mild disinfectant, chemical). The characteristic barnyard aroma of pcresol and has been linked to an ‘unclean’ dimension when out of balance in Cheddar (Dunn & Lindsay, 1985). Conversely p-cresol has been shown to be an important character impact compound in smoked cheeses (Ha & Lindsay, 1991a, b). Moio et al. (2000) identified methyl anisole as an odor-impact compound in Gorganzola aroma. This compound was shown to contribute to the odor profiles in the current study, especially for the blue-mold cheeses. 2-phenylethyl alcohol has been identified as a desirable aroma impact compound in mold-ripened Camembert (Molimard & Spinnler, 1996) and as a ‘fault’ in some Cheddars (Dunn & Lindsay, 1985). A number of unidentified sesquiterpene compounds were also found to contribute to the aroma of the blue cheeses, which has not been reported in previous studies of mold-ripened cheeses (Kinsella & Hwang, 1976; Larroche et al., 1989; Tomasini et al., 1995; Moio et al., 2000).

4.3. Blue cheese aroma

4.5. SPME and aroma analysis

The methyl ketones, 2-heptanone and 2-nonanone, produced via the b-decarboxylation of free fatty acids by P. roqueforti, are considered to be important components in blue-cheese aroma (Kinsella & Hwang, 1976; Larroche, Artpah, & Gros, 1989; Tomasini, Bustillo, & Lebeault, 1995; Moio, Piombino, & Addeo, 2000). 2heptanone (moldy, musty, varnish) and 2-nonanone (floral, fruity, peachy) dominated both TICs and OPs of

Olfactometry experiments can only provide insights into the complexity of cheese aroma and dominant ‘flavour notes’. Clear perception of an aroma at the OP does not necessarily signify that this particular component will be clearly perceived in a complex mixture. Human perception of individual components in a complex aroma mixture is surprisingly limited (Laing & Jinks, 2001). Full interpretation of the role of a single

4.4. Grana Padano aroma Moio and Addeio (1998) reported the impact compounds in Grana Padano, via GC-OP: ethyl butanoate, 2-heptanol, methional, 1-octen-3-ol, ethyl hexanoate and 2-nonanal were the most potent odorants. From SPME-GC-O profiles, the esters ethyl butanoate and butyl hexanoate were present in relatively high concentrations compared to the other cheeses and were very strong at the olfactory port. Methional was also perceived as very strong at the OP.

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aroma component needs to be assessed via a technique such as aroma extract dilution analysis in conjunction with thorough sensory testing with a trained panel. Carboxen-PDMS fibres concentrate analytes primarily through the process of adsorption of molecules into micro-pores, which is distinctly different to the absorption processes of other phases, and particularly effective in the concentration of odorous low molecular weight compounds. SPME concentration occurs through the process of partitioning analytes between the solid-phase coating and the sample headspace. The theoretical basis of SPME has been outlined extensively in the literature ! (Zhang, Yang, & Pawliszyn, 1994; Gorecki & Yu, 1999; Pawliszyn, 2000; Pillonel, Bosset, & Tabacchi, 2002). The time required to reach equilibrium for different analytes varies, often requiring many hours to occur ! (Gorecki et al., 1999). In the present study it was demonstrated that many important trace cheese aroma compounds required up to 16 h or more to reach maximum on-fibre concentration at ambient (22 C) sampling. Although no analyte displacement was observed for the mild parmesan, the strong pecorino and blue-mold cheese exhibited selected displacement, implying that the available micro-pores had reached capacity. Although the Carboxen-phase is theoretically chemically unbiased, the PDMS absorbent phase may introduce some chemical specificity, i.e. some compounds may be absorbed more strongly than others (Bartelt, 1997; Shirey, 2000). Such bias effects should be considered when making quantitative comparisons between components within a cheese and across cheeses. When certain analytes are present at relatively high concentration in a sample, e.g. 2-heptanone or hexanoic acid, the distribution coefficients of trace analytes between the solid/gas phase may be affected. Headspace composition is strongly affected by the partitioning of volatiles between the solid/liquid phase and gas phase. The fat, pH, protein and moisture content of a particular sample matrix, i.e. cheese, has a significant effect on the degree of release of particular volatiles as does the polarity of the analytes themselves (Jo & Ahn, 1999; Gijs, Piraprez, Perpe! te, Spinnler, & Collin, 2000; Haahr, Bredie, Stahnke, Jensen, & Refsgaard, 2000; Uichard & Langourieux, 2000). Headspace measurement of analyte concentration may be quite different to results obtained via solvent extraction or other techniques. Hence SPME analysis can only be considered valid for volatile flavour analysis and not for total flavour analysis i.e. SPME does not measure components partitioned in fat, protein, water fractions. As initial smell perception plays such an important role in the overall desirability of cheese, SPME volatile profiling can, however, provide an important complementary tool in defining the odorous part of cheese flavour. In the current study a number of high-impact odor compounds were close to or below the

limits of instrumental detection, underlining the importance of GC-O as a tool for aroma analysis. The semi-quantitative comparisons of sulfur and pyrazine volatile compounds in the cheese headspaces (Table 3 and Table 4) were considered valid in the light of the equilibrium study data, showing the lack of displacement observed for these classes of compounds. On the basis of the small number of representative cheeses i.e. three, examined for each of the broad cheese classes i.e. cheddar, hard-grating, and blue, it would not be possible to make definitive statements regarding what is ‘typical’ with respect to sulfur compound- or pyrazine compound-profiles. As the primary purpose of this study was to assess the performance of CarboxenPDMS fibres for concentrating and characterizing chemical differences between cheese varieties only one experienced sniffer was used. For any thorough sensory evaluation of the aroma of a particular cheese a larger number of trained sniffers would be required to perform olfactory profiling experiments.

5. Conclusion Although hundreds of volatile compounds have been identified in the headspace of cheeses, (Bosset & Gauch, 1993; Urbach, 1993) only a small number contribute to actual cheese aroma. Carboxen-PDMS fibres have been shown to be the fibre of choice for the analysis of a wide range of aroma compounds (Carlata & Ebeler, 1998; Marsili, 1999; Mestres et al., 1999; Shooter et al., 1999; Shirey, 2000; Pe! re" s et al., 2001; Rocha et al., 2001). In the current study a broad range of cheese aroma compounds were concentrated by Carboxen-PDMS fibres. It was shown that many important low concentration odorous compounds were not displaced over time by other components, implying SPME-GC-MS may be a suitable analytical technique for the reliable measurement of these important trace-concentration flavour compounds. Generally it was indicated that dimethyl trisulfide might play a greater role generally in cheese aroma than previously supposed and additionally, sulfur compounds such as methionol (methylthio propanol) may be more important in blue cheese aroma. The data also indicated that the concentration of higher substituted pyrazines, such as trimethyl pyrazine, tetramethyl pyrazine and diethylmethyl pyrazines, was higher in hard grating cheeses, possibly contributing their overall aroma. For routine quantitative analyses of cheeses, preequilibrium i.e. shorter sampling times would be employed, depending on the particular analytes of interest. Overall, the data indicated that CarboxenPDMS SPME fibres and 16 h equilibration times were appropriate for the concentration of most volatile cheese flavour compounds, however the caveat applies that

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displacement phenomena need to be considered, especially in strong smelling cheeses.

Acknowledgements The authors gratefully acknowledge the financial support of the Australian Dairy Research and Development Corporation.

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