Odorant Screening and Quantitation of Thiols in Carmenere Red Wine

Article pubs.acs.org/JAFC

Odorant Screening and Quantitation of Thiols in Carmenere Red Wine by Gas Chromatography−Olfactometry and Stable Isotope Dilution Assays Carolina Pavez,*,†,‡ Eduardo Agosin,†,‡ and Martin Steinhaus§ †

Department of Chemical and Bioprocess Engineering, School of Engineering, Pontificia Universidad Católica de Chile, Avenida Vicuña Mackenna, 4860 Macul, Santiago, Chile ‡ Centro de Aromas y Sabores, DICTUC, Avenida Vicuña Mackenna, 4860 Macul, Santiago, Chile § Deutsche Forschungsanstalt für Lebensmittelchemie (German Research Center for Food Chemistry), Lise-Meitner-Straße 34, 85354 Freising, Germany ABSTRACT: The sensory impact of thiols in Vitis vinifera ‘Carmenere’ red wines was evaluated. For this purpose, aroma extract dilution analysis was applied to the thiols isolated from a Carmenere red wine by affinity chromatography with a mercurated agarose gel. Results revealed the presence of four odorants, identified as 2-furanylmethanethiol, 3-sulfanylhexyl acetate, 3-sulfanyl1-hexanol, and 2-methyl-3-sulfanyl-1-butanol, with the latter being described here for the first time in Carmenere red wines. Quantitation of the four thiols in the Carmenere wine screened by aroma extract dilution analysis and in three additional Carmenere wines by stable isotope dilution assays resulted in concentrations above the respective orthonasal odor detection threshold values. Triangle tests applied to wine model solutions with and without the addition of the four thiols showed significant differences, thus suggesting that the compounds do have the potential to influence the overall aroma of red wine. KEYWORDS: AEDA, Carmenere red wine, GC−olfactometry, odor-active thiols, SIDA, thiol quantitation

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INTRODUCTION The human nose is able to detect some volatile thiols at a very low odor threshold (nanograms per liter). Such odorous thiols are among the key aroma compounds of many kinds of food, including onion,1 grapefruit,2 guava,3 coffee,4 and mango.5 Thiols are also the crucial odorants responsible for the passion fruit, grapefruit, and box tree aroma notes in white wine.6,7 The most extensively studied odorous thiols in wine are 3-sulfanyl1-hexanol (3SH), 3-sulfanylhexyl acetate (3SHA), and 4methyl-4-sulfanyl-2-pentanone (4MSP). In the wine grapes, 3SH and 4MSP are linked to cysteine or glutathione. The odorless adducts are cleaved during the fermentation process by means of a yeast β-lyase to yield the free thiols.8−10 A yeast alcohol acetyltransferase in parts converts 3SH to 3SHA.10 Further important odor-active thiols found in wine include 2furanylmethanethiol (FMT) and phenylmethanethiol (PhMT). FMT is responsible for roasted coffee and toasty aroma notes and can be formed from furfural extracted from toasted oak barrels.11 PhMT accounts for smoky aroma notes; however, its origin in wine is yet unclear.12 Whereas the contribution of thiols to white wine aroma is well-established and of particular importance in Sauvignon Blanc wines,13,14 only few studies have addressed the influence of thiols on red wine aroma thus far. The presence of 2-methyl3-sulfanyl-1-propanol, 3SH, 3SHA, FMT, 4MSP, and 2-methyl3-furanthiol was reported in Bordeaux red wines.15−17 Aroma extract dilution analysis (AEDA) demonstrated that notably 3SH is a potent odorant in Bordeaux red wines.18 Recently, the concentrations of the thiols 4MSP, 3SH, and 3SHA in red wines from Languedoc, France, were compared to sensory data. High concentrations of 4MSP were correlated with a strong © XXXX American Chemical Society

blackcurrant aroma, which may further be enhanced by 3SH and 3SHA.18 Vitis vinifera L. ‘Carmenere’ is a red grape cultivar originating from Bordeaux, France. Carmenere was thought to be extinct after the grape phylloxera plague in Europe in the 19th century. In 1994, however, it was rediscovered in Chile, where it had previously been misidentified as Merlot. Currently, with an approximate acreage of 11 000 ha, Chile is by far the top producer of Carmenere grapes, although minor acreages can also be found in other countries.19 Sensory studies have characterized the aroma of Carmenere red wine as vegetablelike, spicy, fruity, and cassis-like. Vegetable-like aroma notes are clearly attributed to methoxypyrazines, such as 3-isobutyl-2methoxypyrazine and 3-isopropyl-2-methoxypyrazine.20 However, the odorants responsible for the fruity and cassis-like aroma notes in Carmenere wine have not yet been fully elucidated. In this work, our aim was to isolate the volatile thiols from a Carmenere red wine, screen them for potent odorants by AEDA, quantitate the resulting odorous thiols, and evaluate their sensory impact on red wine aroma by sensory experiments.

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MATERIALS AND METHODS

Wine. The four Carmenere red wines employed in this study are described in Table 1. For the screening experiments, sample CMR 1

Received: January 26, 2016 Revised: April 11, 2016 Accepted: April 12, 2016

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DOI: 10.1021/acs.jafc.6b00411 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry was used. Quantitation of thiols was accomplished with all four samples.

dilutions of 1:10, 1:100, and 1:1000. Diluted samples were analyzed by HRGC−O. Each odorant was assigned a flavor dilution (FD) factor representing the dilution factor of the highest diluted sample in which the odorant was still detected by HRGC−O. FD factors obtained from four sniffers were averaged.26 Quantitation of Thiols by Stable Isotope Dilution Assays (SIDAs). Sodium chloride (10 g) and aliquots (500 μL) of solutions of the internal standards (2H5)-FMT, (2H2)-3SHA, (2H2)-2M3SB, and (2H2)-3SHA in dichloromethane (all at concentrations of 0.2 μg/mL) were added to the wine sample (100 mL). Isolation of thiols was performed by affinity chromatography with a mercurated agarose gel, as detailed above. 23 The eluate obtained from the affinity chromatography was purified by SAFE at 40 °C.24 The distillate was concentrated to 200 μL using a Vigreux column (0.5 cm inner diameter) and a microdistillation device.26 Calibration curves were prepared for each target compound from the analysis of defined analyte/standard mixtures covering ratios from 1:5 to 5:1. Calibration curves were built by plotting the concentration ratio between the unlabeled and labeled compound against the corresponding area ratio. The limit of detection (LOD) and limit of quantitation (LOQ) were approximated as the concentration at which the signal-to-noise ratios (S/N) were 3 (LOD) and 10 (LOQ). HRGC−MS measurements were performed by means of twodimensional heart-cut gas chromatography−mass spectrometry (GC− MS). The system consisted of a CombiPal autosampler (CTC Analytics, Zwingen, Switzerland), a Trace Ultra gas chromatograph (Thermo Scientific, Dreieich, Germany), a heated (250 °C) transfer line, a GC 3800 gas chromatograph (Varian, Darmstadt, Germany), and a Saturn 2200 ion trap mass spectrometer (Varian). The first gas chromatograph was equipped with a cold on-column injector (Thermo), a column DB-FFAP (30 m × 0.32 mm inner diameter, 0.25 μm film thickness, J&W, Agilent), and a moving column stream switching system (MCSS, Thermo). A FID (Thermo) and a tailormade sniffing port served as monitor detectors. The temperature program for the first GC oven started at 40 °C for 2 min, followed by a gradient of 6 °C/min until the temperature reached 230 °C. The MCSS was connected to a DB-1701 column (30 m × 0.25 mm inner diameter, 0.25 μm film thickness, J&W, Agilent) in the second chromatograph via a heated (250 °C) transfer line consisting of an uncoated but deactivated fused silica capillary (0.32 mm inner diameter). The oven temperature for the second column started at 40 °C for 2 min, followed by an initial gradient of 8 °C/min until reaching 170 °C and a subsequent gradient of 40 °C/min until reaching 230 °C. Volatiles conveyed to the second oven by time control of the MCSS were refocused by a jet of cold nitrogen gas (−196 °C) applied to the end of the transfer line until the second oven program commenced. GC−MS chromatograms were recorded in the chemical ionization (CI) mode with methanol as the reagent gas. Odor Threshold Determination. The odor threshold of 2M3SB was determined orthonasally in a synthetic wine matrix solution (11% ethanol, v/v, and 5 g/L tartaric acid, pH adjusted to 3.4 with 1 M NaOH). Results were obtained following the ASTM E679-04 procedure for the determination of odor and taste thresholds by a forced-choice ascending concentration series method of limits.27 Odor threshold values of FMT, 3SHA, and 3SH in synthetic wine matrix solution were taken from the literature.10,17 Sensory Experiments. Experiments on the sensory impact of FMT, 3SHA, 2M3SB, and 3SH were based on a red wine aroma model consisting of the synthetic wine matrix solution detailed above to which 28 major red wine odorants had been added in their natural concentrations.28 Triangle tests according to ISO 4120:200429 were applied to red wine model solutions with and without the addition of thiols. Five triangle tests were prepared using the following thiol concentrations: test 1 (T1), 4 ng/L FMT; test 2 (T2), 5 ng/L 3SHA; test 3 (T3), 40 ng/L 2M3SB; test 4 (T4), 100 ng/L 3SH; and test 5 (T5), 4 ng/L FMT, 5 ng/L 3SHA, 40 ng/L 2M3SB, and 100 ng/L 3SH. Solutions were presented in covered polytetrafluoroethylene (PTFE) vessels and coded with three-digit numbers. A total of 18 panelists from the German Research Center for Food Chemistry, previously trained to identify and detect the target thiols in synthetic

Table 1. Carmenere Red Wine Samples Used in This Study sample CMR 1a

CMR 2

CMR 3

CMR 4

origin Cachapoal Valley, Chile Cachapoal Valley, Chile Cachapoal Valley, Chile Maipo Valley, Chile

harvest

sample description

2011

sampled directly after fermentation (no filtration and bottling)

2011

sampled from the same batch as CMR 1 after fermentation, filtration, and bottling

2012

sampled directly after fermentation (no filtration and bottling)

2011

commercial bottled sample

a

Carmenere red wine sample employed for the AEDA screening experiments.

Reference Odorants and their Labeled Analogues. FMT was purchased from Sigma-Aldrich, Taufkirchen, Germany, and 3SH was purchased from Alfa Aesar, Karlsruhe, Germany. 3SHA was synthesized from 3SH and acetyl chloride.21 (2H5)-FMT, (2H2)-3SH, and (2H2)-3SHA were synthesized according to published procedures.3,22 2M3SB and (2H2)-2M3SB were synthesized from 2-methyl2-butenal (Sigma-Aldrich) using the method recently detailed for the synthesis of 2-methyl-3-sulfanyl-1-pentanol.1 The concentrations of the synthesized compounds in the stock solutions were determined by gas chromatography−flame ionization detector (GC−FID) using methyl octanoate as an internal standard. Detector signal correction was executed by application of response factors, as described previously.22 Isolation of Volatile Thiols from Carmenere Red Wine. Sodium chloride was added to each 100 mL Carmenere red wine sample until saturation. Each mixture was extracted 3 times with diethyl ether (200, 100, and 100 mL). The combined organic phases were dried over anhydrous sodium sulfate and concentrated to 5 mL by means of a Vigreux column (50 × 1 cm). The concentrated extract was applied to 1 g of mercurated agarose gel in a glass column (0.5 cm inner diameter) previously conditioned with 5 mL of isopropanol and then rinsed with 50 mL of pentane/dichloromethane (2:1, v/v). Thiols were eluted with 10 mM dithiothreitol in 50 mL of pentane/ dichloromethane (2:1, v/v).23 The eluate was purified by solventassisted flavor evaporation (SAFE) at 40 °C.24 The SAFE distillate was concentrated to 1 mL using a Vigreux column (0.5 cm inner diameter). High-Resolution Gas Chromatography Olfactometry (HRGC−O) and High-Resolution Gas Chromatography Mass Spectrometry (HRGC−MS). HRGC−O was performed using a 5160 Mega Series gas chromatograph (Carlo Erba Instruments, Milan, Italy) and fused silica capillaries (30 m × 0.32 mm inner diameter, 0.25 μm film thickness, J&W Scientific, Agilent Technologies, Waldbronn, Germany) equipped with either a FFAP or DB-5 separation phase. Samples were introduced into the chromatograph by cold on-column injection at 40 °C using helium gas as a carrier. Oven programs for both columns included 2 min at 40 °C, followed by a gradient of 6 °C/ min until reaching 230 °C. Helium flow was maintained at 2 mL/min. The column effluent was split 1:1 (v/v) using a Y-shaped glass splitter and two deactivated fused silica capillaries (50 cm × 0.20 mm inner diameter). One part of the eluate was directed into a flame ionization detector (FID), while the other part was directed into a tailor-made sniffing port21 kept at 220 °C. Linear retention index (RI) calculations were performed after co-injection with a series of n-alkanes, as described previously.25 HRGC−MS was performed with a HP 5890 gas chromatograph (Hewlett-Packard, Heilbronn, Germany) connected to a Finnigan-type MAT 95 S mass spectrometer (Finnigan, Bremen, Germany) run in the electron impact (EI) mode at 70 eV. AEDA. The thiol fraction isolated from Carmenere red wine, as detailed above, was diluted stepwise in dichloromethane to obtain B

DOI: 10.1021/acs.jafc.6b00411 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry wine matrix solution, were selected for the tests. The panelists performed each test in three replicates during three different sessions. The data were subjected to statistical analysis using the sensory evaluation software Compusense Five 5.4 (Compusense, Guelph, Ontario, Canada).

Results of the quantitation experiments (Table 4) showed concentrations of 33.1 ng/L (FMT), 10.0 ng/L (3SHA), 530 ng/L (2M3SB), and 617 ng/L (3SH) in the sample CMR 1, the Carmenere red wine screened by AEDA. Thus, all four compounds were present in concentrations exceeding their respective threshold values determined in a synthetic wine matrix solution (cf. Table 3). The highest odor activity value (OAV), i.e., the ratio of the concentration of an odorant to its odor threshold value, was calculated for 2M3SB (2700), followed by FMT (83), 3SH (10), and 3SHA (2). CMR 2 was produced from the same batch of grape berries and under the same fermentation conditions as CMR 1, including malolactic fermentation in oak barrels. However, CMR 1 was sampled directly after fermentation, whereas CMR 2 additionally underwent filtration and bottling. Results showed losses associated with filtration and bottling that ranged between 20 and 40% for 3SHA, 2M3SB, and 3SH but a complete loss of FMT. These changes might be due to oxidative degradation. It has been shown that thiols are prone to disulfide formation under oxidative conditions and that, among different thiols, particularly FMT is highly susceptible to this reaction.33 CMR 3 was produced at the same winery and sampled in the same way as CMR 1 but in 2012, whereas CMR 1 was collected in 2011. Concentrations of FMT, 3SHA, and 3SH were in the same range for both samples, indicating that their concentration is influenced only a little by the vintage. A huge difference, however, was found for 2M3SB. 2M3SB has been identified as an impact odorant in Sauternes wine (Figure 1).34 This special type of wine is produced in the Sauternes region of France from Sémillon, Sauvignon Blanc, and Muscat grapes colonized by noble rot (Botrytis cinerea).35 Therefore, the presence of 2M3SB in the Carmenere red wines analyzed here could be associated with this fungus, and the differences in the 2M3SB concentrations between CMR 1 and CMR 3 may simply reflect different degrees of infestation. Actually, there is some probability that Carmenere grapes become infested with this rot, because the chances of B. cinerea development increase with the degree of ripening19 and Carmenere grapes are harvested rather late to reduce the concentration of undesirable methoxypyrazines, which account for strong and undesirable vegetable-like aroma notes.20 The late harvest of Carmenere grapes matches with the beginning of the rain season in Chile, which further promotes the occurrence of Botrytis as a result of high humidity.19 On the other hand, in CMR 4, a commercial bottled sample originating from a different location, no 2M3SB was detected. However, the concentrations of FMT, 3SHA, and 3SH were in the same range as determined in CMR 1 and CMR 3, suggesting that the

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RESULTS AND DISCUSSION Thiol Screening. HRGC−O applied to the volatile thiol fraction obtained from the Carmenere red wine CMR 1 revealed four odor-active compounds. A comparison of the retention indices, odor qualities, and mass spectra of the compounds with respective data of reference compounds analyzed under the same conditions allowed for their identification as FMT, 3SHA, 2M3SB, and 3SH (Table 2). Table 2. Thiols Identified in a Carmenere Red Wine by GC− Olfactometry LRIa odorant

b

FMT 3SHA 2M3SB 3SH

c

FFAP

DB-5

FDd

roasted coffee box tree sweet onion grapefruit

1430 1716 1726 1839

907 1244 1016 1121

1 10 10 100

odor quality

a LRI = linear retention index. bThe compound was identified by comparing its mass spectrum (MS−EI), retention indices on capillary columns FFAP and DB-5, and its odor quality perceived by GC−O to data obtained from reference compounds. cOdor quality perceived at the sniffing port. dFD = flavor dilution factor as determined by AEDA.

Application of an AEDA revealed the highest FD factor (100) for 3SH, suggesting that this compound was the major odoractive thiol in the Carmenere red wine. This finding was consistent with results previously reported for other red wine varieties (Cabernet Sauvignon and Merlot) from Bordeaux, France.16 Quantitation of Thiols. Detailed information is available on the concentrations of a variety of thiols in white wines, particularly in Sauvignon Blanc, and the role of 4MSP, 3SH, and 3SHA is well-described for this type of wine.30−32 In contrast, data on the role of thiols in red wine were limited,18 and no data were available on the concentrations of thiols in Carmenere red wine. Therefore, we quantitated the thiols identified above in the Carmenere red wine used for the screening and in the three additional Carmenere red wines detailed in Table 1. Quantitation was accomplished by stable isotope dilution assays. Calibration parameters (Table 3) showed good linearities for all four compounds. The LOQ was well below the odor threshold values for compounds 3SHA and 3SH and only slightly above the respective odor threshold values for FMT and 2M3SB. Table 3. Calibration Parameters of the Quantitation Method quantifier ions (m/z)

qualifier ions (m/z)

odorant

labeled standard

analyte

standard

analyte

standard

R2

slope

LODa (ng/L)

LOQb (ng/L)

odor thresholdc (ng/L)

FMT 3SHA 2M3SB 3SH

(2H5)-FMT (2H2)-3SHA (2H2)-2M3SB (2H2)-3SH

115 177 121 135

120 179 123 137

81 83−117 103 83−101

86 85−119 105 85−103

0.9964 0.9990 0.9995 0.9989

0.9690 0.9844 0.6798 0.8531

0.150 0.0200 0.110 0.150

1.35 0.170 1.03 1.36

0.40 4.2 0.19 60

a LOD = limit of detection, approximated as the concentration at which the S/N was 3. bLOQ = limit of quantitation, approximated as the concentration at which the S/N was 10. cDetermined in a synthetic wine matrix solution.

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Table 4. Concentrations and Odor Activity Values Obtained for FMT, 3SHA, 2M3SB, and 3SH in Four Different Carmenere Red Wines FMT

3SHA b

2M3SB

sample

concentration (ng/L)

OAV

concentration (ng/L)

OAV

concentration (ng/L)

OAV

concentration (ng/L)

OAVb

CMR CMR CMR CMR

1 2 3 4

33.1 ndc 31.9 38.7

83

10.0 8.00 21.0 22.0

2 2 5 5

530 306 35.2 1 in the four Carmenere wine samples analyzed, their contribution to the overall aroma was not yet unequivocally established. The OAVs were based on thresholds in a synthetic wine matrix without further aromaactive compounds, whereas in real wine, a complex mixture of aroma-active compounds is present28 and interactions during the olfactory perception, therefore, may lead to the suppression of certain aroma compounds.36 To approach this problem, the impact of an addition of FMT, 3SHA, 2M3SB, and 3SH to a red wine model solution, including 28 major red wine odorants in their natural concentrations,28 was studied by triangle tests. The concentrations tested were 4 ng/L for FMT, 5 ng/L for 3SHA, 40 ng/L for 2M3SB, and 100 ng/L for 3SH. These values were chosen because they were in between the threshold values in the synthetic wine matrix solution (cf. Table 3) and the typical concentrations found in the Carmenere wine samples (cf. Table 4). Compounds were added individually as well as in combination of all four. In all five cases, the triangle test resulted in a significant difference (α = 0.05) between the test sample with thiol addition and the reference sample without thiol addition, indicating that FMT, 3SHA, 2M3SB, and 3SH do have the potential to influence the overall aroma of red wines in the concentrations observed.

AUTHOR INFORMATION

Corresponding Author

*Telephone: +56-2-2354-7259. Fax: +56-2-2354-4076. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Johannes Polster and Jörg Stein for their helpful advice on synthetic approaches, Kathrin Röver and Julia Bock for their skillful technical assistance, Romina Montealegre for her assistance with the statistical analysis, and Anakena winery for providing the Carmenere wine samples.

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DOI: 10.1021/acs.jafc.6b00411 J. Agric. Food Chem. XXXX, XXX, XXX−XXX