Gas Chromatography−Mass Spectrometry (GC−MS) Characterization

The volatile composition of 26 premium quality vinegars belonging to three different protected geographical indications (traditional balsamic vinegar ...
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J. Agric. Food Chem. 2009, 57, 4784–4792 DOI:10.1021/jf804005w

Gas Chromatography-Mass Spectrometry (GC-MS) Characterization of Volatile Compounds in Quality Vinegars with Protected European Geographical Indication FABIO CHINNICI,*,† ENRIQUE DURA´N GUERRERO,‡ FRANCESCA SONNI,† NADIA NATALI,† RAMO´N NATERA MARI´N,‡ AND CLAUDIO RIPONI† ‡

† Food Science Department, University of Bologna, Viale Fanin, 40, Bologna 40127, Italy, and Analytical Chemistry Department, Faculty of Sciences, University of Ca´diz, Post Office Box 40, Pol. Rı´ o San Pedro, Puerto Real 11510, Ca´diz, Spain

The volatile composition of 26 premium quality vinegars belonging to three different protected geographical indications (traditional balsamic vinegar of Modena, balsamic vinegar of Modena, and sherry vinegar) has been characterized by means of a solid-phase extraction (SPE) gas chromatography-mass spectrometry GC-MS method. Among the about 90 quantified compounds, shortchain fatty acids, furanic compounds, enolic derivatives, and some esters were found to discriminate the samples as a consequence of differences in the extent of Maillard reactions, presence of alcoholic fermentation, or duration of wood aging. KEYWORDS: Traditional balsamic vinegar of Modena; balsamic vinegar of Modena; sherry vinegar; vinegar aroma; GC-MS; SPE; volatile compounds; furanic compounds

INTRODUCTION

In the European Union, the promotion of foods that have been recognized to be traditionally linked to a certain geographical area is based on a legislative system known as “protected denomination” (1 ). Nowadays, most of the member states possess a number of food specialties with a protected designation of origin (PDO) or a protected geographical indication (PGI), with each of them obtained following approved specifications related to their chemical-sensory features, their production process, and certification. For what concern vinegars, at the European level, two different PDOs exist: (i) traditional balsamic vinegar of Modena (TBVM) and (ii) traditional balsamic vinegar of Reggio Emilia (TBVRE) (2 ). Two further specialties are the sherry vinegar (JVs), a product which has its own denomination of origin and has recently been accepted by the European Commission as PDO (3 ), and balsamic vinegar of Modena (BVM), which is under consideration of the European Commission, with its geographical indication now temporarily protected (4 ). TBVM and TBVRE are Italian vinegars coming from the Emilia-Romagna region, differing almost exclusively from the production area and the grape cultivars used to obtain the starting must. They consist of a dark, syrupy sausage, in which the sour notes are well-balanced by a sweet background deriving from the initial unfermented must. Briefly, for TBVM, the process starts with the cooking of the grape must in open boilers to reduce the initial volume of about 30%. During this phase, conditions are propitious for the development of caramelization and Maillard reactions (5 ) that generate compounds, some of which with a *To whom the correspondence should be addressed. Telephone: +39512096015. Fax: +39512096017. E-mail: [email protected].

pubs.acs.org/JAFC

remarkable aromatic impact. Once cooked, the must is transferred to a cask, where a partial alcoholic fermentation takes place (up to 70 g/L of ethanol). After 5 or 6 months, the acetic fermentation process is promoted and the product is divided into some smaller casks (typically five) made of different kinds of wood (oak, chestnut, mulberry, or juniper) and decreasing volume. During this period, acetic acid bacteria grow on the media surface, where the oxygen concentration is higher. Vinegar oxidation is produced mainly by strains belonging to Acetobacter, Gluconobacter, and Gluconacetobacter species, which oxidize ethanol to acetic acid (6 ). A long aging period follows during which, because of evaporation, casks are refilled using the method consisting of taking the finished product from the smaller cask (the fifth cask) and refilling it with the vinegar coming from the fourth cask. Then, the fourth cask is subsequently refilled from the third cask and so on until the larger cask (the first), which is filled with fresh cooked must. Only after at least 12 years of aging, the vinegar could be withdrawn to be marketed as TBVM, while for the older “Extravecchio” vinegar, the aging should be at least 25 years. Balsamic vinegar of Modena (BVM) differs from the abovecited TBVM in the fact that it substantially comes from a wine vinegar, to which a minor portion of cooked must and caramel have been added, and that it has undergone a shorter aging period (from 6 months to 2 years) usually in one single oak cask. Finally, sherry vinegar (JVs) is a Spanish D.O. produced in the JerezXeres-Sherry wine region (Andalucı´ a) and derives from a wine vinegar aged in casks often following the traditional “solera” method, where a volume of older vinegar from the “solera” (placed on the ground) line of casks is bottled. This volume is refilled with younger vinegar from another line of casks and so on until the line of casks with the youngest vinegar, which is refilled

Published on Web 04/22/2009

© 2009 American Chemical Society

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Table 1. Principal Characteristics of the Studied Samples vinegar

code

studied samples

raw material

aging type

aging time

caramel addition

traditional balsamic vinegar of Modena “extravecchio” traditional balsamic vinegar of Modena “affinato” sherry vinegar sherry vinegar reserva balsamic vinegar of Modena

TBVM-ST TBVM-AF VJ VR BVM

5 5 4 6 6

cooked must cooked must sherry wine sherry wine wine

dynamic dynamic dynamic dynamic static

>25 years >12 years >6 months >2 years 12 years old “affinato” and 5 >25 years old “extravecchio”) supplied by different Italian producers, 6 BVM ( 6 months old “vinagre de Jerez” and 6 >2 years old “vinagre de Jerez Reserva”) purchased from a Spanish market were analyzed. The acidity of the samples (expressed in grams of acetic acid/100 g of vinegar) varied from 5 to 7 for TBVM and from 7 to 10 for JVs. The acidity of BVM was 6% for all of the samples. With this limited number of studied vinegars (26 samples), the different types of products were covered. Furthermore,

The volatile compounds identified in vinegars are reported in Table 2. Although the used method is not suitable to determine major volatile compounds (acetaldehyde, ethanol, methanol, and ethyl acetate), a total of 93 compounds were identified, 57 of which were confirmed by comparing their rT and mass spectra with authentic standards. For the remaining volatiles, identification was accomplished by matching their mass spectra with Nist 2.0 and Wiley 7 libraries and further confirmed by the comparison to linear retention indexes (RIs) found in the literature (7-12). Identified volatiles were grouped in function of the chemical class,

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Table 2. Quantification and Identification Criteria for Volatile Compounds Identified in at Least One Vinegar Sample number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68

RIa 1142 1208 1240 1246 1265 1279 1286 1348 1360 1379 1389 1407 1443 1469 1472 1497 1507 1521 1524 1534 1547 1555 1567 1577 1594 1597 1601 1611 1617 1627 1654 1661 1667 1670 1673 1673 1687 1725 1742 1748 1768 1774 1780 1786 1791 1818 1839 1870 1875 1898 1899 1903 1925 1948 1967 1975 1996 2019 2027 2044 2047 2051 2061 2064 2082 2092 2103 2110

compound 1-butanol 3-methyl-1-butanol ethyl hexanoate 3-hydroxy-3-methyl-2-butanone dihydro-2-methyl-3(2H)-furanone hexyl acetate 3-hydroxy-2-butanone ethyl lactate 4-hydroxy-4-methyl-2-pentanone 3-ethoxy-1-propanol 3-acetiloxy-2-butanone tetradecane linalool oxide isomer 2-furancarboxaldehyde linalool oxide isomer butanediol diacetate Isomer 2-acetylfuran benzaldehyde ethyl 3-hydroxybutirate butanediol diacetate isomer propanoic acid 1,2-etanediol diacetate 5-methyl-2-furancarboxaldehyde isobutyric acid δ-valerolactone dihydro-4-methyl-2(3H)-furanone 2-acetyl-5-methylfurane dihydro-2(3H)-furanone 2-acetoxy-1-propanol butanoic acid propanediol diacetate Isomer 2-furanmethanol 4-hydroxy-3-pentanoic acid γ-lactone 3-ethyl-2-hydroxy-2-cyclopenten-1-one isopentanoic acid diethyl succinate γ-hexalactone benzyl acetate propanediol diacetate isomer pentanoic acid methyl salicilate 1-(5-methyl-2-furyl)-2-propanone ethyl 2-phenylacetate 5-valerolactone butoxyethoxyethanol 2-phenylethyl acetate 2-hydroxy-3-methyl-2-cyclopenten-1-one hexanoic acid 2-methoxyphenol trans-4-methyl-5-butyldihydro-2(3H)-furanone benzyl alcohol 1,4-butanediol diacetate 2-phenylethanol 5-ethoxymethyl-2-furaldehyde trans-4-methyl-5-butyldihydro-2(3H)-furanone 3-hydroxy-2-methyl-4H-pyran-4-one 2,5-furandicarboxaldehyde 1-(2-furanyl)-2-hydroxyethanone 4-methyl-5,6-dihydropyran-2-one 1H-pyrrole-2-carboxaldehyde 4-ethyl-2-methoxyphenol dihydro-3-hydroxy-4,4-dimethyl- 2(3H)-furanone 3-acetoxypropene diethyl malate 5-ethoxydihydro-2(3H)-furanone octanoic acid 1,2,3-propanetriyl triacetate 4-methylphenol

other name

isoamyl alcohol

acetoin diacetone alcohol

furfural

2-furylmethyl ketone

5-methyl furfural

γ-butirolactone

furfuryl alcohol angelicalactone isovaleric acid caprolactone

valeric acid

cyclotene caproic acid guaiacol trans-whiskeylactone

phenylethyl alcohol cis-whiskeylactone maltol furylhydroxymethyl ketone dehydromevalonic lactone p-ethylguaiacol pantalactone allyl acetate solerone triacetin

quantificationb

identificationc

supplierd

56 55 + 70 88 59 NQ 43 43 + 45 45 59 59 + 71 NQ 85 + 71 59 95 + 96 59 TIC 95 + 110 105 + 106 43 TIC 73 + 74 43 + 86 109 + 110 TIC TIC NQ TIC 42 + 86 TIC 60 TIC 82 + 97 98 + 55 TIC 60 101 + 129 85 TIC TIC 60 + 73 120 NQ 164 TIC 75 104 TIC 60 + 73 109 + 124 99 79 + 108 TIC 91 + 122 TIC 99 126 TIC TIC TIC TIC 137 + 152 71 TIC 117 TIC 60 + 73 43 TIC

S, MS S, MS S, MS S, MS MS S, MS S, MS S, MS S, MS S, MS MS S, MS S, MS S, MS S, MS MS S, MS S, MS S, MS MS S, MS S, MS S, MS MS MS MS MS S, MS MS S, MS MS S, MS S, MS MS S, MS S, MS S, MS MS MS S, MS S, MS MS S, MS MS S, MS S, MS MS S, MS S, MS S, MS S, MS MS S, MS MS S, MS S, MS MS MS MS MS S, MS S, MS MS S, MS MS S, MS S, MS MS

Aldrich Merck Sigma Sigma Aldrich Sigma Sigma Sigma Aldrich Aldrich Fluka Sigma Fluka Sigma Fluka Aldrich Sigma Fluka Sigma

Fluka Fluka Fluka Aldrich Sigma Sigma Aldrich

Aldrich Sigma fluka Aldrich Aldrich Aldrich Sigma Aldrich Aldrich Fluka Fluka Aldrich

Sigma Fluka Sigma Sigma Aldrich

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Table 2. Continued number 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93

RIa

compound

2185 2198 2215 2244 2271 2272 2275 2283 2295 2328 2404 2455 2467 2509 2519 2567 2574 2658 2667 2722 2786 2827 2854 2864 2925

2-methoxy-4-(2-propenyl)phenol 4-ethylphenol 5-acetoxymethyl-2-carboxaldehyde 1,2,3-propanetriol diacetate 1,2,3-propanetriol monoacetate 2,6-dimethoxyphenol 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one decanoic acid 3,5-dihydroxy-2-methyl-4H-pyran-4-one 4-(1-hydroxyethyl)γ-butanolactone ethyl hydrogensuccinate benzoic acid 2-furancarboxylic acid 5-hydroxymethyl-2-carboxaldehyde 5-acetyl-2-furanmethanol 4-hydroxy-3-methoxybenzaldehyde phenylacetic acid 4-hydroxy-3-methoxybenzoic acid ethyl ester 1-(4-hydroxy-3-methoxyphenyl)ethanone tetradecanoic acid 4-hydroxy-3-methoxyphenethanol hexadecanoic acid 4-hydroxy-3,5-dimethoxybenzaldehyde 4-hydroxybenzaldehyde 4-hydroxyphenyl ethanol

other name eugenol acetoxymethyl furfural

siringol ddmp 5-hydroxymaltol

2-furoic acid HMF vanillin ethyl vanillate acetovanillone homovanillyl alcohol siringaldehyde tyrosol

quantificationb

identificationc

supplierd

164 107 + 122 126 TIC TIC 139 + 154 TIC 60 + 73 TIC NQ 101 105 + 122 95 + 112 97 + 126 NQ 151 + 152 91 + 136 196 151 TIC TIC 73 + 129 181 + 182 TIC 107

S, MS S, MS S, MS MS MS S, MS MS S, MS MS MS S, MS S, MS S, MS S, MS MS S, MS S, MS S, MS S, MS MS MS S, MS S, MS MS S, MS

Sigma Sigma Aldrich

Fluka Sigma

Sigma Sigma Sigma Sigma Sigma Aldrich Aldrich Aldrich

Sigma Aldrich Sigma

a

Retention index. b Mass fragments used for quantification purposes (TIC denotes the use of the peak area as acquired in the TIC mode; NQ stands for not quantified). c Identification obtained by means of standard compounds (S) and/or mass spectra (MS). d Supplier of the standard compound.

and their amount was compared between samples, also taking into account the starting material and the production process specific for each vinegar. Overall, the amounts of alcohols found in our samples were substantially in accordance with other works on TBV (7, 8), BVM (8, 9), and JVs (8, 10-12) (Table 3). If compared to BVM and JVs, a general lower amount of these compounds could be seen in TBVM, likely because of the almost absent alcoholic fermentation during the production phases of this latter. This is particularly evident for 3-methyl-1-butanol and, to a lesser extent, for 2-phenyl ethanol and tyrosol, which typically derive from amino acid metabolism of yeast cells during alcoholic fermentation (14 ). As already found by Zeppa et al. (7 ), furthermore, 3-methyl-1-butanol decreases as the aging phase progresses, being the highest in BVM (where the mean aging period is shorter) and the lowest in both the JVs commercial classes. A similar trend was shown for benzyl and homovanillyl alcohols, which were lower in TBVM than in BVM and JVs. Aldehydes may derive from alcohol oxidation or wood leakage. Significant differences between vinegars were found for benzaldehyde, syringaldehyde, and vanillin, with the latter being the highest in long wood aged TBVM and JVs. Similar amounts of vanillin were found by Callejo´n et al. (9 ), who did not consider TBVM in their work. Quite surprisingly, Zeppa et al. (7 ) did not find vanillin or syringaldehyde in three different batteries of TBVRE, maybe because of the extraction method employed by these authors, which used a different type of cartridge (C18 instead of LiChrolut-EN) and an higher volume of washing water (30 mL) before the elution of volatile compounds. In vinegars, acids mainly come from alcohol oxidation acted by acetic bacteria. Their presence, hence, is expected to be somehow related to the amount of alcohols in the raw material. On the basis of our data, acids were significantly higher in VJ and VR, while both the TBVM and BVM had the lowest concentration. This finding is particularly evident for short-chain acids (C3-C6),

which were statistically the highest in VR (may be because of the longer acetification process with respect to VJ). Excluding the nonstudied acetic acid, isovaleric acid represents by far the main acid in vinegars (up to about 80% of the total in JVs), and its concentration in VJ and VR is slightly higher than that reported by Dura´n Guerrero et al. (12 ) or other authors (9, 10). In BVM and TBVM, however, our data largely agree with Callejo´n et al. (9 ) and Zeppa et al. (7 ), respectively. Acetates are formed by esterification between acetic acid and mono- or polyalcohols and are expected to increase according to aging duration (7 ). However, only slight differences were found between vinegars in the sum of acetates (Table 3). On the contrary, taking into account each single ester, it can be noticed that VR had the highest amount of propanediol and benzyl acetates, while TBVM contained the highest quantities of butanediol diacetate and triacetin. The latter appeared to be characteristic of TBVM, and its amount discriminated the “younger” TBVM “affinato” from the “older” TBVM “extravecchio”. Quantitatively, similar amounts of benzyl and 2-phenylethyl acetates were reported by Natera Marı´ n et al. (11 ), Dura´n Guerrero et al. (12 ), and Callejo´n et al. (9 ), in JVs and BVM, respectively. Acetates coming from di- or triols were only reported by Zeppa et al. (7 ) in TBVM, at lower concentration ranges. Ethylic esters are another family of compounds already reported in vinegars. Their amount has been significantly correlated with the ethanol content of the raw material (10 ). As a sum, in fact, vinegars coming from an alcoholic matrix (BVM, VJ, and VR) had the highest ethyl ester amounts, while both of the TBVMs (affinato and extravecchio) had the lowest. A major part of this difference is due to ethyl hydrogen succinate, which is the main ethyl ester in all of the vinegars. However, significant differences between TBVM and all of the other vinegars were also found for ethyl lactate, diethyl succinate, methyl salicilate, and ethyl-2-phenylacetate. The latter seems to

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Table 3. Concentrations (μg/kg) of Different Groups of Volatile Compounds in European Protected Denomination Vinegar Samples

a

TBV affinato compound

median min

TBV extravecchio

max

median min

BVM

max

median

VJ

min

max

nd 25.61 141 16.1 22.1 443 13.3 90.2 7.19 48.1

7.45 77.46 1084 59.7 47.3 1106 28.7 232 27.1 132

9.77 43.0 nd 9.88 0.06

median

VR

min

max

median

loq 27.01 239 31.4 a 25.9 b 656 ab 17.9 ab 345 ab 45.2 b 90.6

nd 15.62 177 23.5 21.1 524 14.3 286 25.0 65.3

1.39 37.08 307 39.2 46.5 849 21.2 486 54.7 107

23.4 625 792 32.3 1.44

a a a

61.2 1039 3133 55.8 4.25

17.5 700 2984 48.1 3.83

110 3317 8849 292 12.6

ab b b

1283 816 535 11.0 44.6 862 518 7.26 15.1 227 583 246 16.3

4107 2442 2372 36.7 142 2452 2434 129 133 922 2999 1183 52.3

b a a a a b b a

3164 2775 1400 110 132 2336 1748 156 127 1835 3897 3311 134

2028 2243 1264 75.2 115 1871 1258 56.7 86.0 871 3172 2376 94.3

4296 3105 4263 154 161 2768 2745 409 217 10886 8627 11795 192

bc b ab b ab b b ab

9.21 nd nd nd 103 6.63 1139 1169 11.1 nd nd 282 loq 3.62

109.94 nd nd nd 799 29.5 4766 2241 80.4 19.1 nd 1273 117 6.99

25.97 nd nd nd a 368 a 22.5 b 2033 1642 a 29.4 a 3.93 a nd 882 65.8 ab 5.08

12.35 nd nd nd 243 16.3 1761 1123 19.0 loq nd 733 48.6 4.47

76.38 nd nd nd 431 28.7 2270 1892 67.6 12.7 nd 1155 88.8 5.49

nd 770 89.1 100 loq 14.3 nd 42.5 1.39 50.9

nd 11925 145 1915 34.4 67.2 nd 193 130 199

loq 1698 147 b 823 a 88.3 ab 32.8 loq b 145 117 b 157

loq 979 65.9 181 63.5 19.6 loq 60.0 96.4 60.6

6.35 19427 168 1366 236 37.7 78.5 182 147 185

min

max

loq 25.83 276 42.2 41.3 1079 21.8 253 30.2 90.8

nd 14.73 163 40.3 20.0 848 15.9 112 12.2 44.0

10.65 48.44 1600 86.8 93.0 1532 29.8 830 85.2 145

71.5 1537 3409 89.4 5.23

63.4 1165 1585 64.2 3.03

198 2821 6684 226 9.80

b b b

4573 3749 3459 151 367 3255 2065 165 98.4 715 6381 8804 195

3425 3560 1593 139 188 2766 1577 97.4 30.6 173 3607 5530 168

6337 4775 8069 218 1156 3966 2420 386 166 1890 8733 13642 260

c c c c b c b b

50.37 nd 8.38 nd 995 59.3 3232 2364 62.4 18.2 loq 1323 113 8.14

19.34 nd loq nd 582 46.3 2349 2111 32.3 2.49 nd 1040 75.3 6.79

75.13 3.55 31.6 nd 1589 68.8 6335 5086 87.6 61.9 0.03 2264 203 13.42

6.43 3681 188 194 207 57.7 nd 99.0 96.6 103

loq 427 138 61.6 108 27.8 nd 16.1 47.0 16

20.7 8166 283 689 368 76.8 nd 131 228 140

Alcohols 1-butanol 3-methyl-1-butanol (mg/kg) 3-ethoxy-1-propanol 2-acetoxy-1-propanolb butoxyethoxyethanol benzyl alcohol 2-phenylethanol (mg/kg) homovanillyl alcoholc tyrosol (mg/kg) sum alcohols (mg/kg)

0.53 68.5 30.8 141 177 8.65 75.9 8.17 18.4

nd 0.02 55.4 17.6 72.4 81.6 3.55 41.4 5.71 13.2

nd 16.6 391 83.6 276 292 9.89 80.6 16.9 26.0

a

b a a a a a

0.10 103 53.8 120 342 14.12 87.4 18.0 30.8

nd 0.00 26.4 47.7 18.8 231 8.14 84.2 12.0 23.7

nd 0.79 438 148 373 510 35.3 110 32.9 70.2

loq 56.21 242 38.6 b 29.1 a 576 ab 18.2 a 138 a 11.6 a 88.1 a

c

b

a b ab b b b

b

ab c b b b b

Aldehydes benzaldehyde vanillin siringaldehyde 4-hydroxybenzaldehyded sum aldehydes (mg/kg)

9.47 967 887 45.1 1.94

4.82 399 233 20.9 0.70

11.2 1161 1982 80.5 3.05

a 8.87 b 1476 ab 1568 82.7 ab 3.13

4.86 1248 1440 54.2 2.82

15.3 4810 6313 119 11.2

a 15.8 b 192 ab 185 18.5 b 0.42

a

b

b

Acids propanoic acid isobutyric acide butyric acid isovaleric acid (mg/kg) pentanoic acid hexanoic acid octanoic acid decanoic acid tetradecanoic acidf hexadecanoic acid phenylacetic acid benzoic acid sum acids (mg/kg)

502 600 196 8.42 24.7 350 63.1 loq 52.6 395 2506 1757 15.4

128 272 67.2 0.98 4.50 117 loq loq 6.48 85.9 955 479 8.13

837 1606 353 35.5 54.3 990 382 10.3 77.9 669 2713 3646 44.2

a a a a a a a a

397 737 200 16.0 31.5 561 210 10.7 67.0 547 ab 3537 a 4673 a 30.5

309 638 129 4.01 26.1 307 101 loq 51.6 286 2795 1650 13.6

636 1748 489 24.9 77.0 988 1197 136 265 1497 5332 5411 33.4

a a a a a a a a

2143 1358 724 17.4 74.4 1522 1325 79.3 58.4 492 ab 913 a 476 a 27.7

a a a

bc ab b

c b b

Acetates hexyl acetate butanediol diacetate isomerg butanediol diacetate isomerg 1,2-etanediol diacetate propanediol diacetate isomerg benzyl acetate propanediol diacetate isomerg 2-phenylethyl acetate 1,4-butanediol diacetateg allyl acetate triacetin 1,2,3-propanetriol diacetateg 1,2,3-propanetriol monoacetateg sum acetates (mg/kg)

6.87 loq loq loq 76.6 6.86 349 693 40.6 27.8 15.2 670 43.1 2.61

loq loq loq loq 30.6 loq 158 279 30.8 9.21 loq 485 19.2 1.14

33.65 36.6 72.5 5.75 368 16.3 1673 2189 67.0 63.1 84.8 1891 169 4.83

2.25 2.73 22.7 loq a 341 a 11.7 a 811 1599 ab 136 ab 63.2 b 112 2465 240 a 5.97

loq nd 4.16 nd 229 8.94 462 746 111 61.2 79.4 1232 97.2 3.38

25.12 69.5 149 33.5 458 31.4 1160 4999 231 187 351 4867 519 12.3

16.01 nd nd nd a 574 a 18.9 a 3291 1812 b 31.3 b loq c nd 681 37.5 ab 6.82

a a ab a ab a

ab

b b b a ab a

b

Esters ethyl hexanoate ethyl lactate ethyl 3-hydroxybutirate diethyl succinate ethyl 2-phenylacetate methyl salicilate diethyl malate ethyl hydrogensuccinate (mg/kg) ethyl vanillate sum esters (mg/kg)

loq 126 51.9 loq 2.09 5.02 nd 0.14 16.5 0.71

nd loq 10.2 loq loq 1.34 nd loq 3.57 0.04

18.2 366 368 21.4 24.0 15.7 nd 6.94 28.8 7.02

a a a a a a

nd 10.0 168 nd loq 5.06 loq loq 37.2 0.33

nd 1.86 113 nd loq 1.41 loq loq 19.9 0.15

loq 379 661 loq 29.2 30.5 249 18.6 119 20.0

a a a a a a

nd 3209 99.4 635 3.82 20.4 nd 85.7 56.0 90.8

b

b b b ab b b

b ab b b b b

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Table 3. Continued TBV affinato compound

median min

TBV extravecchio

max

median min

BVM

max

median

VJ

min

max

median

nd loq 73.8 169 0.25

9.6 514 275 1533 1.96

a b a

716 2.74 238 nd 90.8 loq 153 loq 216 nd 259 262 427 430

3081 41.7 842 13.1 713 165 680 7985 555 9.29 1052 2328 931 1001

107 19.6 1786 66 1.98

157 186 14997 b 335 15.6 b

23.0 14.9 1.22 loq 181 1189 101 63.4 1086 612 4.49

16.9 loq loq loq 97.8 637 80.6 45.4 569 89.2 3.13

34.7 26.6 12.6 0.1 367 2433 178 463 2043 1100 7.27

a a a a a

24.6 7.50 92.6 5.53 2.67 190 321

22.6 4.86 30.8 5.02 1.44 17.0 116

12.7 6.45 19.2

VR

min

max

median

min

max

nd loq 83.9 96.6 0.18

nd loq 45.4 20.9 0.07

nd 76.9 123 1025 1.22

a a a

nd 6.86 178 234 0.40

nd loq 99.1 175 0.31

nd 36.5 311 2050 2.35

a a a

ab loq a 1.82 ab 60.1 nd b 44.3 a nd b nd ab nd b nd a nd b 10.5 a 1504 ab 11.5 b 15.1

loq 1.38 13.4 nd 7.78 nd nd nd nd nd 10.5 1228 4.46 7.51

962 5.88 254 nd 88.2 nd nd nd nd nd 43.2 2154 51.8 60.2

a a a

51.6 5.12 164 nd 1.65 nd nd nd nd nd 10.5 2112 12.9 19.9

loq 3.66 110 nd 1.65 nd nd nd nd nd 10.5 1766 0.69 6.86

487 21.6 519 3.38 198 nd nd nd nd nd 39.1 2907 46.3 70.9

a a a

263 30.8 6992 181 7.66

247 25.0 1789 167 2.32

607 91.1 11042 b 249 11.5 b

336 75.3 5039 244 6.150

180 26.6 2399 130 2.972

765 270 9654 b 527 10.00 b

18.4 18.1 139 22.3 193 592 a 97.7 62.8 666 bc 160 a

13.8 7.41 124 6.88 109 397 65.0 60.6 269 64.3

32.7 31.6 167 26.5 382 1418 155 76.1 713 424 3.37

33.3 64.1 115 27.2 470 1122 a 122 107 616 ab 69.1 3.18 4.04

18.5 30.6 22.6 loq 320 740 91.0 79.4 462 4.7 4.29

76.7 99.3 157 33.5 828 2297 171 234 1188 107 6.50

27.6 9.85 137 7.34 3.70 283 452

20.9 6.37 b 149 12.9 6.49 b 237 ab 430

18.3 5.47 75.2 11.0 3.77 143 266

21.5 9.38 237 21.0 7.22 249 542

b b

18.8 9.84 141 14.5 6.30 199 401

18.0 6.31 105 11.7 5.17 142 318

24.7 14.1 176 82.0 7.40 249 471

b b

50.3 23.4 73.7

b b b

10.9 5.09 16.2

18.9 9.30 28.3

a a a

28.3 19.1 47.4

25.1 14.3 39.4

40.5 22.3 62.9

ab b b

Enolic Derivatives 3-ethyl-2-hydroxy-2-cyclopenten-1-one cycloteneh maltol 5-hydroxymaltolh sum enolic derivatives (mg/kg)

h

21.6 198 462 585 1.36

15.3 100 317 174 0.69

97.3 269 611 1989 2.72

ab 42.3 bc 300 ab 1003 1003 ab 2.55

21.1 249 791 743 2.19

126 457 1417 2654 3.85

b c b b

6.7 181 111 360 0.61

a

a

a

Furanic and Pyranic Derivatives 5-methyl-2-furaldehyde furfural (mg/kg) 2-acetylfuran 2-acetyl-5-methylfuranei 2-furanmethanol 5-ethoxymethyl-2-furaldehydei 2,5-furandicarboxaldehydej furylhydroxymethyl ketonei 1H-pyrrole-2-carboxaldehydej 5-acetoxymethyl-2-furaldehyde (mg/kg) ddmph 2-furancarboxylic acid 5-hydroxymethyl-2-furaldehyde (mg/kg) sum furanic compounds (mg/kg)

4436 75.2 802 loq 118 loq 1778 2544 283 89.6 273 1167 1614 1773

2499 40.0 396 loq 94.4 nd 680 loq 155 54.8 125 901 728 886

5224 76.7 1790 1092 149 122 2439 6362 518 97.4 292 2339 2308 2466

bc 3894 b 54.3 ab 767 33.8 ab 147 ab 153 c 1934 ab 4178 b 433 b 215 b 298 ab 3854 b 2055 c 2388

2150 38.5 428 12.1 122 loq 1404 2406 180 103 131 2889 1456 1689

6664 90.2 2422 246 248 2647 3586 9504 567 271 331 4679 2782 3117

c b b

1229 9.12 631 loq ab 264 b loq c 227 b 1888 b 351 b loq b 392 b 858 b 646 c 659

a a a a a a a ab a a

a a a a a a a ab a a

Ketones acetovanillone 3-hydroxy-3-methyl-2-butanone 3-hydroxy-2-butanone (acetoin) 4-hydrohy-4-methyl-2-pentanone sum ketones (mg/kg)

102 19.8 322 241 0.77

63 10.3 loq 38 0.43

173 28.0 1702 a 522 2.06 a

267 18.0 102 217 0.66

161 12.0 loq 196 0.59

431 32.2 623 304 1.25

a a

124 38.3 5960 198 6.42

Lactones 5-valerolactonek caprolactone trans-whiskeylactone cis-whiskeylactone dehydromevalonic lactonek pantalactone soleronek δ-valerolactonek γ-butirolactone angelicalactonek sum lactones (mg/kg)

43.7 14.9 loq 17.0 412 1461 234 127 713 567 4.50

34.4 4.72 loq 7.04 291 420 105 85.5 422 391 2.91

61.7 33.0 7.12 43.0 1033 1911 286 257 858 635 5.22

ab b a ab ab

70.2 54.8 2.23 46.7 1588 2569 ab 403 268 1368 bc 924 a 8.35

41.5 29.2 loq 35.1 1356 2454 305 168 946 702 7.67

99.3 172 428 551 3406 2895 622 711 2325 1377 12.8

b c a b b b

c b

a b b ab a

ab c b ab a a

a a

Phenols siringol guaiacol p-ethylguaiacol 4-methylphenoll eugenol 4-ethylphenol sum phenols

22.5 6.1 8.31 12.0 5.02 47.1 105

19.5 3.2 loq 8.55 2.67 38.9 82.3

26.5 6.5 22.8 14.6 8.13 89.7 160

a

a a

25.7 6.8 8.93 21.5 8.09 89.0 162

24.8 5.7 3.82 15.1 2.41 57.4 127

30.4 12.43 37.1 a 38.6 23.5 206.3 ab 320 ab

b

b

Terpenes linalool oxide isomer 1 linalool oxide isomer 2 sum terpenes

5.35 4.90 10.01

loq loq loq

7.49 a 8.60 a 17.21 a

7.20 5.98 13.18

loq 21.0 1.89 9.54 2.01 30.5

a a a

35.1 18.1 55.3

12.8 7.56 20.4

a

In the same row, different letters indicate significant difference (p < 0.01). Median, median value; min, minimun value; max, maximum value; nd, below the detection limit; loq, below the quantification limit. Flagged compounds are expressed as follows. b 3-Ethoxy-1-propanol. c Vanillin. d Benzaldehyde. e Butyric acid. f Decanoic acid. g Triacetin. h Maltol. i 2-Acetyl furan. j Furoic acid. k γ-Butyrolactone. l Guaiacol.

be characteristic of Spanish vinegars (Table 3). Our data confirm the finding of other authors (15 ), who, in a stepwise linear discriminant analysis (SLDA) study aimed at differentiating the

aroma of balsamic, Jerez, wine, and cider vinegars, reported that ethyl-2-phenylacetate and methyl salicilate are among the most discriminating variables. In quantitative terms, again, our data

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J. Agric. Food Chem., Vol. 57, No. 11, 2009

Chinnici et al.

Figure 1. MS spectrum and formula of cyclotene.

agree with previous reports (7, 9-12) for TBVM, BVM, and JVs, respectively, and confirm that those esters strongly related to alcoholic fermentation, such as diethyl succinate and ethylmalate, are almost exclusively present in BVM, VJ, and VR. It has been suggested that alkyl-substituted 5- or 6-membered cyclic R-diketones, such as maltol and related compounds, exhibit a typical burnt sugar, sweet maple flavor and that this feature could be due to their stable keto-enolic form (16, 17). These compounds typically arise from hexose thermal or acid-catalyzed degradation (17 ), and some of them have been already reported in foods or beverages, such as coffee (18 ) or sherry wines (19 ). Furthermore, their presence was also noticed in charred woods used in wine and vinegar aging (20 ) as a consequence of pyrolytic degradation of cellulose. For what concern vinegars, Zeppa et al. (7 ) reported the presence of some enolic derivatives (maltol, cyclotene, and 5-hydroxymaltol) in TBVRE, while a furanone with an enol-carbonyl structure (sotolon) was identified in long aged JVs by Callejo´n et al. (9, 10). As shown in Table 3, our data suggest that TBVM possesses the highest amounts of enolic derivatives in comparison to BVM and VJ. 5-Hydroxymaltol was the main contributor to this class of compounds followed by maltol and cyclotene. To the best of our knowledge, this is the first report on the presence of cyclotene in BVM and JVs. Figure 1 shows its formula and MS spectrum. It is worth mentioning that, for this latter compound, the odor threshold in water is around 0.3 mg/L (21 ) and may well-contribute to the aromatic complexity of TBVM. None of our samples revealed the presence of sotolon. This can be explained by the relatively low recovery shown by this compound during the extraction process. This low recovery could come from the extremely high solubility of sotolon in water, and therefore, its amount could be reduced during the washing step of the extraction process. A further enolic compound (3-ethyl-2-hydroxycyclopenten2-one) was identified exclusively in Italian balsamic vinegars. This volatile, which could be considered the ethyl homologue of cyclotene, possesses a strong burnt-sugar sweet aromatic impact, with an odor threshold notably lower than this latter

compound (17 ). The larger amounts of enolic derivatives in TBVM with respect to JVs and BVM may derive from the initial cooking of the must and the prolonged wood aging of this sugarrich product. On the other side, the weak amounts of 3-ethyl-2hydroxycyclopenten-2-one in BVM could be due to the admitted practice of caramel addition. Other classes of volatiles, which originate from sugar thermal degradation, are furanic and pyranic compounds. A wide number of them, in fact, were reported after the heating of glucose solutions, mostly with the presence of amino acids (22, 23). It is known, on the other hand, that TBVM shows a relevant amount of HMF and furfural, together with other furanic derivatives, such as 2-acetylfuran and furoic acid (24 ). As expected, both the TBVM type of vinegars had the highest total amount of furanic compounds (Table 3), independent from the age of the vinegar. BVM revealed a minor, even if relevant, content of total furans (likely coming from caramel addition), while JVs had the lowest content of these sugar derivatives. HMF was, by far, the major representative (up to 93.5% in TBVM “affinato”), followed by furfural and 5-methylfurfural. In TBVM, 5-acetoxymethylfurfural is a further major furanic compound, which comes from the esterification between acetic acid and HMF. This volatile, already reported in TBVM at a concentration varying from 7.28 mg/kg (7 ) to 188 mg/kg (24 ) seems to be characteristic of Italian TBVM and could represent a discriminant parameter between “affinato” and “extravecchio” TBVMs, as already suggested by Giacco et al. (25 ). Three further volatiles were found exclusively in Italian TBVM and BVM. They are 2,5-furandicarboxaldehyde, furylhydroxymethyl ketone, and 1H-pyrrole-2-carboxaldehyde. All of them may derive from Maillard reactions produced by heating proline or phenylalanine mixtures (26 ) and have already been reported in toasted oak extracts (20, 27). Their sensory impact has been described as “honey” and “toasty caramel” (27 ) and, in TBVM and BVM, may derive from the cooking of the must and the caramel addition, respectively. Other furanic derivatives somewhat relevant are 2-acetyl-5-methylfuran, with a nutty

Article reminiscence, and DDMP (2,3-dihydro-2,5-dihydroxy-6-methyl4H-pyran-4-one), the role of which in the Maillard pathway has been described as pivotal for the generation of enolic derivatives (5-hydroxymaltol, maltol, and cyclotene in particular) (22 ). Acetoin is a wine constituent initially produced during alcoholic fermentation. When wines are submitted to acetic fermentation, the acetoin amount increases because of the transformation of R-acetolactate and 2,3-butanediol acted by acetic bacteria (28 ). Despite the low recovery that this compound showed by the extraction method that we used (13 ), our data suggest that vinegars coming from wine tend to have a higher amount of this compound, which is characterized by a buttery odor. Considerable amounts of lactones have been found in our samples, which may come from cyclicization of hydroxyacids during fermentation or from sugar degradation (7 ). On the other hand, the presence of whiskey lactones is in relationship with wood aging, and the ratio between the two isomers is thought to depend upon the oak species used (29 ). Quite surprisingly and diversely from the finding of other authors (9 ), the cis/trans ratio of JVs was found to be