- Email: [email protected]
Aromatic potential of botrytized white wine grapes: Identification and quantification of new cysteine-S-conjugate flavor precursors
Analytica Chimica Acta 660 (2010) 190–196
Contents lists available at ScienceDirect
Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
Aromatic potential of botrytized white wine grapes: Identification and quantification of new cysteine-S-conjugate flavor precursors Cécile Thibon a,∗ , Svitlana Shinkaruk b , Michael Jourdes a , Bernard Bennetau c , Denis Dubourdieu a , Takatoshi Tominaga a a b c
Université de Bordeaux, UMR Œnologie, INRA, ISVV, Bordeaux, F-33000, France Université de Bordeaux, ENITAB, CS 40201, Bordeaux, F-33000, France Université de Bordeaux, CNRS, UMR 5255 ISM, F-33405, France
a r t i c l e
i n f o
Article history: Received 25 July 2009 Received in revised form 8 October 2009 Accepted 10 October 2009 Available online 17 October 2009 Keywords: Sweet wine Aroma precursor Cysteine-S-conjugate GC derivatization Vitis vinifera Botrytis cinerea
a b s t r a c t Sweet wines made from botrytized grapes contain much higher concentrations of volatile thiols, especially 3-sulfanylhexan-1-ol (3SH), than dry white wines. Three new specific volatile thiols (3sulfanylpentan-1-ol (3SP), 3-sulfanylheptan-1-ol (3SHp), and 2-methyl-3-sulfanylbutan-1-ol (2M3SB) were recently identified in Sauternes wines. Like most volatile thiols, these compounds were almost totally absent from must, mainly being formed during alcoholic fermentation. In this work, we describe the identification and quantification of three new cysteine-S-conjugate precursors in must made from Botrytis-infected grapes. S-3-(pentan-1-ol)-l-cysteine (P-3SP), S-3-(heptan-1-ol)-l-cysteine (P-3SHp), and S-3-(2-methylbutan-1-ol)-l-cysteine (P-2M3SB) were identified by direct GC–MS analysis of their derivative forms obtained by silylation of an enriched fraction, isolated from must by affinity chromatography. Concentrations were considerably higher when Botrytis cinerea had developed on the grapes. In botrytized must, the mean levels of P-3SP, P-3SHp, and P-2M3SB were in the vicinity of 700, 50, and 500 nM, respectively, whereas concentrations in healthy must ranged from 0 to 50 nM. This indicated that these three new sulfanyl alcohols, responsible for the characteristic aroma of botrytized wines, were formed by the yeast metabolism during alcoholic fermentation from the corresponding non-volatile cysteine-S-conjugate precursors. Moreover, these results highlighted the predominant role of botrytization in developing grape aroma potential. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Sulfur compounds, especially volatile thiols, are extremely odoriferous molecules, which contribute to the aroma of many fruits, plants, and foods [1]. For example, certain thiols contribute to the characteristic aroma of blackcurrant [2], grapefruit [3,4], passion fruit [5], guava [6], fringed rue (Ruta chalepensis) [7], and box tree (Buxus sempervirens) [8]. The key role played by thiols in the odor of roasted coffee [9], popcorn, and grilled meat is also well known [10–12]. Finally, the contribution of compounds in this family to the aroma of beer was recently reported [13]. Since the early 1990s, several highly odoriferous volatile thiols have been identified in wines made from Vitis vinifera L. cv. Sauvignon blanc. These cover a broad aromatic palette, including such key descriptors as: green pepper, boxwood, broom, eucalyptus, blackcurrant buds, rhubarb, tomato leaves, nettles, grapefruit, passion fruit, white peaches, gooseberries, and asparagus broth,
∗ Corresponding author. Tel.: +33 5 57 57 58 61; fax: +33 5 57 57 58 13. E-mail address: [email protected] (C. Thibon). 0003-2670/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2009.10.018
as well as acacia flowers and wood. The first molecule identified as a characteristic component of Sauvignon blanc wine aroma was 4-methyl-4-sulfanylpentan-2-one (4MSP) [14]. Assays of this sulfanylketone in certain Sauvignon blanc wines revealed concentrations as high as 40 ng L−1 , considerably higher than the perception threshold (0.8 ng L−1 in model solution) [15], confirming its decisive organoleptic role in wines characteristic of this grape variety. Several other odoriferous volatile thiols were later identified in Sauvignon blanc wines: 3-sulfanylhexyl acetate (3SHA) [16], 4-methyl-4-sulfanylpentan-2-ol (4MSPOH), and 3-sulfanylhexan1-ol (3SH) [17], smelling of boxwood and zest, citrus zest, and grapefruit and passion fruit, respectively. More recently, 3-sulfanylpentan-1-ol (3SP), 3-sulfanylheptan1-ol (3SHp), 2-methyl-3-sulfanylbutan-1-ol (2M3SB), and 2-methyl-3-sulfanylpentan-1-ol (2M3SP) were identified in Sauternes wines [18]. These thiols, together with 3SH, significantly enhance the varietal aroma of botrytized sweet white wines. 3SP and 3SHp have pronounced citrus zest aromas, while 3SH smells fruity. Concentrations of 3SP in botrytized wines are always well below the perception threshold (900 ng L−1 ), while 3SHp rarely exceeds its perception threshold (35 ng L−1 ). However, an additive
C. Thibon et al. / Analytica Chimica Acta 660 (2010) 190–196
191
Fig. 1. Structures of 3-sulfanylpentan-1-ol (3SP), 3-sulfanylhexan-1-ol (3SH), 3-sulfanylheptan-1-ol (3SHp), 2-methyl-3-sulfanylbutan-1-ol (2M3SB), and their corresponding precursors: S-3-(pentan-1-ol)-l-cysteine (P-3SP), S-3-(hexan-1-ol)-l-cysteine (P-3SH), S-3-(heptan-1-ol)-l-cysteine (P-3SHp), and S-3-(2-methylbutan-1-ol)-l-cysteine (P2M3SB).
effect of these volatile thiols, combined with 3SH, has been clearly demonstrated [18]. Therefore, 3SP and 3SHp have a considerable impact on overall aromatic complexity in the presence of other volatile thiols, such as 3SH, although concentrations remain below the perception threshold. Concentrations of 3SH, 3SP, and 3SHp in botrytized wines are strongly affected by the development of Botrytis cinerea. Wines made from healthy grapes contain 3SH but only traces of the other two thiols, while those in the “pourri plein” stage (entirely botrytized but not desiccated) of noble rot have much higher thiol concentrations [19,20]. Like most volatile thiols, these compounds are almost totally absent from must, mainly being formed during alcoholic fermentation. Tominaga et al. showed that 3SH, present in a cysteinylated conjugate precursor form in must, was released by yeast metabolism during alcoholic fermentation [21]. The putative cysteinylated conjugates of 3SP, 3SHp, and 2M3SB, similar in chemical structure to 3SH, were studied in botrytized must to determine whether they were formed by the same pathway (Fig. 1). In this work, we describe the identification of three new cysteine-S-conjugate precursors in must made from Botrytisinfected grapes (Fig. 1). S-3-(pentan-1-ol)-l-cysteine (P-3SP), S-3-(heptan-1-ol)-l-cysteine (P-3SHp), and S-3-(2-methylbutan-1ol)-l-cysteine (P-2M3SB) were identified by direct GC–MS analysis of their derivative forms, obtained by silylation of an enriched fraction, isolated from must by affinity chromatography, as previously described for the precursor of 3SH (P-3SH) [22]. This GC–MS method was simple, accurate, effective for identifying new cysteine-S-conjugates (aroma precursors), as well as determining concentrations in must during grape botrytization.
2. Experimental 2.1. Chemicals and reagents trans-2-Pentenal (95%), trans-2-hexenal (95%), trans-2-heptenal (97%), trans-2-methyl-2-butenal (98%), S-benzyl-l-cysteine (97%), N-(tert-butoxycarbonyl)-l-cysteine (Boc-cysteine) (99.5%), cesium carbonate (99%), and sodium borohydride (98%) were purchased from Sigma–Aldrich (L’Isle d’Abeau, France). Nmethyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide with 1% tert-butyldimethychlorosilane (MTBSTFA:t-BDMCS) and anhydrous pyridine were obtained from Perbio Science (Brebières, France). Chelating Sepharose Fast Flow was purchased from GE Healthcare (Buckinghamshire, England). 2.2. Nuclear magnetic resonance (NMR) spectroscopy 1 H, and 13 C NMR spectra were recorded on a Bruker AC-300 FT (1 H: 300.13 MHz, 13 C: 75.4 MHz). No internal standard was present in D2 O samples and spectra were referenced using the frequency of the deuterated solvent (lock frequency). Chemical shifts (ı) and coupling constants (J) are expressed in ppm and Hz, respectively.
2.3. Synthesizing cysteine-S-conjugates New cysteine-S-conjugates were synthesized in three steps by the Michael addition of Boc-protected cysteine to corresponding ␣,-unsaturated aldehydes, followed by reducing the carbonyl group to alcohol and deprotecting the amino group, as previously reported for P-3SH synthesis (Scheme 1) [22,23].
Scheme 1. Three-step synthesis of new cysteine-S-conjugate precursors: (a) Cs2 CO3 , CH3 CN, r.t.; (b) 1. NaBH4 , MetOH, r.t., 2. 10 M HCl, r.t.
192
C. Thibon et al. / Analytica Chimica Acta 660 (2010) 190–196
Table 1 MS/MS conditions for the acquisition of cysteine-S-conjugate derivatives. Compound
Retention time (min)
Parent ion (window)
Resonance excitation voltage (V)
Recorded mass range
Product ions for quantification
t-BDMS-P-2M3SB t-BDMS-P-3SP t-BDMS-P-3SH t-BDMS-P-3SHp t-BDMS-S-benzylcysteine
21.0–21.3 21.4 22.0 23.1 20.8
492 (3) 492 (3) 506 (3) 520 (3) 354 (3)
1.50 1.50 1.50 1.50 1.50
454–502 454–502 468–516 482–530 134–240
464 464 478 492 147
2.3.1. S-3-(pentan-1-ol)-l-cysteine (P-3SP) Beige solid (purity determined by GC–MS after trimethylsilylation, 96%). 1 H NMR (D2 O) ı 0.84 (t, J = 7.3 Hz, 3H, CH3 -5); 1.51 (m, 2H, CH2 -4); 1.60 (m, 2H, CH2 -2a); 1.73 (m, 1H, CH2 -2b); 2.71 (m, 1H, CH-3); 2.96 (m, 2H, CH2 -); 3.60 (m, 2H, CH2 -1); 3.78 (m, 1H, CH-␣). 13 C NMR (D2 O) ı 10.03 and 10.14 (CH3 -5); 27.00 and 27.06 (CH2 -4); 30.44 and 30.47 (CH2 -); 35.43 and 35.67 (CH2 -2); 44.01 and 44.29 (CH-3); 53.97 and 53.99 (CH-␣); 59.11 and 59.15 (CH2 1); 172.69 and 172.71 (COOH). All 13 C peak values were slightly different, depending on the stereospecific features of the chemical structure of each diastereoisomer. 2.3.2. S-3-(heptan-1-ol)-l-cysteine (P-3SHp) Beige solid (purity determined by GC–MS after trimethylsilylation, 95%). 1 H NMR (D2 O) ı 0.77 (t, J = 7.2 Hz, 3H, CH3 -7); 1.11–1.34 (m, 4H, CH2 -6 and CH2 -5); 1.49 (m, 2H, CH2 -4); 1.63 (m, 2H, CH2 2a); 1.75 (m, 1H, CH2 -2b); 2.78 (m, 1H, CH-3); 2.85–2.94 (m, 2H, CH2 -); 3.63 (m, 2H, CH2 -1); 3.80 (dd, J = 6.8, 4.5 Hz, 1H, CH-␣). 13 C NMR (D O) ı 13.26 (CH -7); 21.90 (CH -6); 27.97 and 28.05 2 3 2 (CH2 -5); 30.39 and 30.45 (CH2 -); 33.88 (CH2 -4); 35.88 and 36.12 (CH2 -2); 42.53 and 42.76 (CH-3); 53.99 (CH-␣); 59.14 and 59.18 (CH2 -1); 172.6 (COOH). Some 13 C peak values were slightly different, depending on the stereospecific features of the chemical structure of each diastereoisomer. 2.3.3. S-3-(2-methylbutan-1-ol)-l-cysteine (P-2M3SB) Beige solid (purity determined by GC–MS after trimethylsilylation, 90%). As trans-2-methyl-2-butenal contains two stereogenic centers and the configuration of cysteine C-␣ is constant (R configuration), the four synthesized stereoisomers formed two pairs of diastereomers. They differed by syn or anti orientation of substituents at the C-2 and C-3 chiral centers formed during the Michael addition reaction. NMR spectra and 2D correlations (COSY, HMQC, and HMBC) confirmed the presence of two diastereomer pairs in a ratio of 1:2. Although full stereochemical determination was not possible, due to signal complexity and overlap, 2,3-anti addition was apparently preferred, according to previously published data [18,24]. 1 H NMR (D O) ı 0.63–0.73 and 0.77–0.84 (two massifs with a 2 relative integration ratio of 2:1, 3H, CH3 -2 ); 0.95–1.14 (four overlapping doublets, J = 6.8 Hz, 3H, CH3 -4); 1.63–1.75 and 1.93–2.02 (two massifs with a relative integration ratio of 2:1, 1H, CH-2); 2.78–2.90 (m, 1H, CH-3); 2.91–3.06 (m, 2H, CH2 -); 3.18–3.48 (m, 2H, CH2 -1); 4.10–4.18 and 4.21–4.32 (two massifs with a relative integration ratio of 2:1, 1H, CH-␣). 13 C NMR (D2 O) ı 11.48, 11.67, 12.13, and 12.44 (CH3 -2 ); 15.56, 15.60, and 18.85 (CH3 -4); 30.06, 30.24, and 30.28 (CH2 -); 39.08, 39.22, 39.83, and 39.99 (CH-2); 42.09, 42.24, 43.41, and 44.02 (CH-3); 52.34, 52.42, 52.50, and 52.62 (CH-␣); 64.28, 64.36, and 64.41 (CH2 -1); 168.36, 168.49, 170.06, and 170.13 (COOH). 2.4. Must preparation and purification of cysteine-S-conjugate Sauvignon blanc and Semillon grapes were picked from the same plot in the 2008 vintage (Château d’Yquem, Appellation Sauternes, France). Semillon musts were also studied in the 2007
vintage. Two 1000-grape samples were harvested at four stages in botrytization: healthy (not infected by B. cinerea), pourri plein (entirely botrytized but not desiccated), pourri rôti (botrytized and desiccated), and late pourri rôti (shriveled grapes left for an additional week before picking). In the laboratory, samples were manually destemmed, sulfured (10 g hL−1 ), and crushed in a blender (2 min) under inert gas (CO2 ). Must obtained from each grape sample was stored at −20 ◦ C prior to analysis. The method for purifying cysteine-S-conjugates from must was based on the protocol described by Thibon et al. [22]. Briefly, samples (500 L) containing 200 pg S-benzyl-l-cysteine as an internal standard were adjusted to pH 10 and loaded directly onto a Chelating Sepharose column, containing immobilized copper. The retained fraction was washed with 2 mL potassium phosphate buffer (50 mM, pH 7), eluted by percolating it with 3 mL hydrochloric acid solution (25 mM), and evaporated dry under vacuum. The dry residue was dispersed in 500 L ethanol–acetone mix (50/50). The supernatant was evaporated dry under vacuum in a 2-mL vial. 2.5. Derivatization and GC–MS analysis The tert-butyldimethylsilyl (t-BDMS) derivatives were prepared as described by Thibon et al. [25]. Briefly, MTBSTFA:t-BDMCS (25 L) and anhydrous pyridine (40 L) were added in vial containing the dry residue The mixture was shaken briefly and heated to 70 ◦ C for 15 min. Electronic impact (EI) mass spectra were recorded at 70 eV on a Polaris Q mass spectrometer, equipped with an ion trap analyzer (Thermo Electron Corporation, Milan, Italy), connected to a Trace GC gas chromatograph (Thermo Electron) equipped with a type BPX35 capillary column (SGE: 50 m, internal diameter: 0.25 mm, film thickness: 0.22 m), and a splitless injector (200 ◦ C). A total of 2-L of the derivatized sample was injected. The temperature program was as follows: 80 ◦ C for 1 min, then increasing to 240 ◦ C at 15 ◦ C min−1 , 310 ◦ C at 4 ◦ C min−1 , and 320 ◦ C at 10 ◦ C min−1 , followed by a 5 min isotherm. Derivatives were quantified in MS/MS mode. Ions were selected for quantification on the basis of peak intensity and ion specificity. The MS/MS acquisition conditions for cysteine-S-conjugate derivatives are presented in Table 1. 2.6. Method validation For calibration, must samples were spiked prior to purification by adding the appropriate volume of a mixture of standard stock solution of all cysteine-S-conjugates to achieve concentrations of 10, 25, 100, 250, and 1000 nM for P-3SP, P-2M3SB, and P-3SHp and of 100, 500, 2500, 10,000, and 25,000 nM for P-3SH, then analyzed by GC–MS, as described above. Each point of calibration was carried out in triplicate. Calibration curves plotted relative area of the total sum of diastereoisomers versus concentration of standard solutions. Repeatability was assessed over a series of five replicates. The limit of detection (LOD) was defined as the concentration that gave a signal-to-noise ratio (S/N) of three. The limit of quantification (LOQ) was defined as the concentration that gave an S/N ratio of 10. These parameters were experimentally calculated from the S/N obtained in must sample analyses.
C. Thibon et al. / Analytica Chimica Acta 660 (2010) 190–196
193
Table 2 Repeatability, calibration line parameters, limits of quantification (LOQ), limits of detection (LOD), and recovery (spiking level = 100 nM) from healthy and botrytized grape juice (n = 5) of S-3-(pentan-1-ol)-l-cysteine (P-3SP), S-3-(hexan-1-ol)-l-cysteine (P-3SH), S-3-(heptan-1-ol)-l-cysteine (P-3SHp), and S-3-(2-methylbutan-1-ol)-l-cysteine (P-2M3SB). Compound
P-3SP P-3SH P-3SHp P-2M3SB a b c
RSDa (%)
5.6 2.8 5.9 6.8
Calibration line
y = 1.65x y = 1.37x y = 4.02x y = 1.24x
R2
0.9868 0.9915 0.9873 0.9988
LOQb
0.6 0.4 0.7 1.0
LODc
0.2 0.1 0.2 0.3
Average recovery (%) Healthy
Botrytized
96 102 108 105
103 97 113 98
RSD: relative standard deviation (n = 5). Limits of quantification (nM). Limits of detection (nM).
2.7. Thiol production assay in fermented synthetic medium Volatile thiol release was measured at the end of fermentation of synthetic grape juice, previously described as KP medium, in 350mL bottles at 24 ◦ C [26]. This medium was buffered to pH 3.3 and contained 80 g L−1 glucose, 80 g L−1 fructose, and 190 mg L−1 available nitrogen. Each aroma precursor (P-3SP, P-3SH, P-3SHp, and P-2M3SB) was added to the medium before yeast inoculation at 1000 nM. Two yeasts strains were used in this experiment: VL3-1D (wildtype) and ure2. The first was derived from a commercial starter VL3c (Laffort Enologie, Bordeaux, France) described previously for its strong capacity to reveal volatile thiols [27–29]. ure2 is an homozygous spore clones of VL3-1D carrying two deleted URE2 gene copies. This mutant released three times more 3SH than the wild-type strain [30]. Yeast strains were inoculated at 1 × 106 cells mL−1 from an overnight pre-culture in KP medium diluted with water (1:1). Cell numbers were measured using an electronic particle counter (ZII; Coulter-Counter, Coultronics, Margency, France). Volatile thiol release was measured after complete fermentation (measured by weight loss). All experiments were carried out in triplicate. At the end of fermentation, the volatile thiols were extracted from 250 mL synthetic medium, using the method described by Tominaga et al. (1998), as modified by Tominaga et al. (2000) [15,31]. Amounts of 3SP, 3SH, 3SHp, and 2M3SB were quantified by GC–MS, as described by Sarrazin et al. [18]. Briefly, volatile thiol extracts were analyzed on a 6890N gas chromatograph (Agilent technologies) using a type BP20 capillary column [SGE, 50 m, internal diameter: 0.22 mm, film thickness: 0.25 m]. The detector was a mass spectrometer (MS 5973, Agilent Technologies), functioning in EI mode. SIM mode was used to quantify 3SP, 3SH, 3SHp, and 2M3SB, selecting the followings ions: m/z 120 and 86 for 3SP and 2M3SB, m/z 134 and 100 for 3SH, and m/z 148 and 114 for 3SHp.
3. Results and discussion 3.1. Identification and quantification of new cysteine-S-conjugates in silyl derivative form Each derivatized synthetic reference compound was initially analyzed by GC–MS in scan mode to obtain the retention time and fragmentation pattern. Fig. 2 shows the total ion chromatogram and mass spectra of the cysteine-S-conjugate mixture in t-BDMS derivative form. Adequate resolution was observed in the chromatogram, i.e. no overlapping peaks. As the derivatization step increased steric hindrance and maximized the dissimilarity between diastereoisomers, a partial differentiation of derivatives was observed. The presence of three stereogenic centers in P-2M3SB explains the
massif form of the peak ranging from 21.0 to 21.3 min. A slight separation of diastereoisomeric forms was observed for P-3SP and P-3SH at 21.4 and 22.0 min, respectively. In contrast, the P-3SHp derivative presented a Gaussian peak at 23.1 min. The mass spectrum of pure P-3SP, P-3SH, P-3SHp, and P-2M3SB, obtained by chemical synthesis, was determined by GC–MS in full scan mode (Fig. 2). All the compounds revealed similar fragmentation patterns, with several identical ions, such as m/z 189, 218, 258, 302, and 316, all resulting from the fragmentation of the derivatized cysteine moiety of P-3SP, P-3SH, P-3SHp, and P-2M3SB. Moreover, the other ions, such as [M–15]+ , [M–57]+ , [M–85]+ , [M–159]+ , [M–189]+ , [M–217]+ and [M–350]+ , differed by 14 mass units, e.g.: P-2M3SB and P-3SP compared to P-3SH or 28 mass units for P-3SHp. This difference is due to the upper alkyl-alcohol moiety of P-3SP, P-3SH, P-3SHp, and P-2M3SB (Fig. 2, see supporting information). The intense fragment, [M–57]+ at m/z 492 for P-2M3SB and P-3SP, m/z 506 for P-3SH and m/z 520 for P-3SHp, resulted from the cleavage of a tert-butyl group from the molecular ion of P-3SP, P-3SH, P-3SHp, and P-2M3SB. Due to this very easy cleavage, the molecular ion of each compound was not detected in our GC–MS condition. Moreover, the [M–85]+ ion at m/z 464 for P-2M3SB and P-3SP, m/z 478 for P-3SH, and m/z 492 for P-3SHp was also a very intense ion, corresponding to the main fragment in MS/MS analysis of the [M–57]+ ion for each compound (see supporting information). Thus the [M–85]+ ion resulted from the loss of 57 mass units (tert-butyl group), followed by the loss of 28 mass units from its parent ion [M–57]+ . The loss of 28 mass units was due to the loss of the CO group from the silylated acid function of the cysteine moiety of P-3SP, P-3SH, P-3SHp, and P-2M3SB. Ions were selected for quantification on the basis of peak intensity and ion specificity, as well as potential interference from other compounds. Consideration was primarily given to peak intensity, to obtain the highest sensitivity. The [M–57]+ peaks of compound derivatives (EI mode) were chosen as the parent ion. As the MS/MS spectrum of the precursor ion [M–57]+ had one major peak at [M–85]+ , m/z 464, m/z 478, m/z 492, and m/z 464 were used as the quantitative ions for assessing P-3SP, P-3SH, P-3SHp, and P-2M3SB (see supporting information). Mass spectra and retention times for synthesized cysteine-Sconjugates were used to assay the analytes in the Sauvignon blanc or Semillon juice, obtained from healthy or botrytized grapes. Must extract was obtained by the assaying method based on the protocol described in Section 2. The first stage consisted of purifying the cysteine-S-conjugates by percolating the must directly onto a Chelating Sepharose column, containing immobilized copper. In the second stage, the precursors were derivatized to increase their volatility prior to GC–MS/MS analysis. GC–MS/MS analysis of the botrytized grape juice extract revealed the presence of P-3SP, P3SH, P-3SHp, and P-2M3SB, by comparison with the retention times and MS/MS spectra of the synthetic reference compounds (data not shown).
194
C. Thibon et al. / Analytica Chimica Acta 660 (2010) 190–196
Fig. 2. Chromatograms, mass spectra, and structures of the main fragments obtained from the t-BDMS derivatives of the reference cysteine-S-conjugates: P-2M3SB, P-3SP, P-3SH, and P-3SHp. (The structure of the other ions is shown in supporting information.)
3.2. Validating the method for quantifying cysteine-S-conjugates in must The peak areas of the total sum of diastereoisomers on the MS/MS chromatograms were calculated for [M–85]+ ions: m/z 464 (t-BDMS derivatives of P-3SP and P-2M3SB), m/z 478 (t-BDMS derivative of P-3SH), m/z 492 (t-BDMS derivative of P-3SHp), and m/z 147 (t-BDMS derivative of S-benzylcysteine, IS). Table 2 shows the calibration lines and R2 obtained for each of the four com-
pounds. Calibration curves were plotted for five concentrations, from 10 to 1000 nM (for P-3SP, P-2M3SB, and P-3SHp) and from 100 to 25,000 nM for P-3SH. Each point on the curve represents the average value of three replicated measurements. The calibration curves of the ratio of the [M–85]+ peak areas to those at m/z 147 (IS) were linear for GC-injections with a correlation coefficient of >0.986 and a relative standard deviation (RSD) of <6.8%. Repeatability was determined using healthy and botrytized grape juice. Recovery trials were performed at 100 nM. The average recov-
C. Thibon et al. / Analytica Chimica Acta 660 (2010) 190–196
195
Table 3 Quantitative assays (nanomoles per liter) of P-3SP, P-3SH, P-3SHp, and P-2M3SB in must at different stages in botrytization, from two grape varieties in different vintages. Vintage 2008
2007
* a b
Variety
Botrytis stage
P-3SP
P-3SH
P-3SHp
P-2M3SB
Sauvignon
Healthy Pourri plein Pourri rôti Late pourri rôti
21 774* 803* 750*
± ± ± ±
6 33 8 25
453 9902* 20301* 15606*
± ± ± ±
50 856 1656 1380
13 30 36* 20
± ± ± ±
3 14 12 5
87 645* 643* 739*
± ± ± ±
28 21 7 17
Semillon
Healthy Pourri plein Pourri rôti Late pourri rôti
11 420* 460* 320*
± ± ± ±
8 112 92 59
98 8383* 11782* 5120*
± ± ± ±
24 1351 871 489
17 13 34* 39*
± ± ± ±
2 9 1 6
52 252* 338* 299*
± ± ± ±
2 34 10 18
Semillon
Healthy Pourri plein Pourri rôti Late pourri rôti
21 474* 614* 404*
± ± ± ±
8 5 114 66
158 19721* 23407* 19254*
± ± ± ±
24 1430 4202 2185
15 31* 105* 62*
± ± ± ±
2 5 16 9
42 312* 417* 909*
± ± ± ±
4 11 109 205
a
b
Statistically different from healthy stage, ANOVA, P = 0.01. Mean value (n = 2). Standard deviations s (n = 2).
ery ranged from 96% to 113%. Detection and quantification limits were determined by analyzing several botrytized musts, naturally containing cysteine-S-conjugates, and calculating the concentration required to give a signal-to-noise ratio of 3 or 10, respectively. Detection and quantification limits ranged from 0.1 to 0.3 and from 0.4 to 1 nM, respectively. 3.3. Impact of noble rot development on grapes on S-conjugate content The concentrations of 3SH, 3SP, and 3SHp in botrytized wines are strongly affected by the development of B. cinerea. Wines produced from grapes affected by the “pourri plein” stage (entirely botrytized but not desiccated) of noble rot have much higher sulfanyl alcohol concentrations. Like most volatile thiols, these compounds were almost totally absent from must, mainly being formed during alcoholic fermentation [18]. Several grape juice samples were analyzed to assess the impact of B. cinerea development on cysteine-S-conjugate formation. Grape samples were harvested at four stages in botrytization: healthy, pourri plein, pourri rôti, and late pourri rôti. For each grape sample, the cysteinylated precursors were extracted, as described above, and this process was repeated twice. Table 3 lists S-conjugate concentrations in nanomoles per liter during the botrytization of Sauvignon blanc and Semillon grapes in the 2007 and 2008 vintages. Healthy grape juice contained only trace levels of P-3SP, P-3SHp, and P-2M3SB (ranging from 11 to 87 nM). The P-3SH content of healthy Semillon and Sauvignon blanc musts was approximately 100 and 500 nM, respectively. The enrichment previously described for P-3SH during botrytization [22,25,32] was also observed for the other S-conjugates. In both varieties and vintages, a considerable increase in S-conjugate concentrations was observed, starting in the first stages of botrytization. Indeed, P-3SP levels increased rapidly, reaching an average of 30-fold between the healthy and pourri rôti stages. The increase in P-3SHp and P-2M3SB was more moderate, ranging from 2.5- to 7-
fold and 6- to 17-fold, respectively. The decrease in grape volume by desiccation ranged from 40% to 60% at the pourri rôti and late pourri rôti stages (data not shown). Therefore, this rate of increase in four aroma precursors cannot be explained merely by grape desiccation. Indeed, irrespective of the S-conjugate observed, concentrations were considerably higher when B. cinerea had developed on the grapes. These results confirm the predominant role of the B. cinerea metabolism in developing grape aroma potential and explain the higher volatile thiol concentrations in sweet compared to dry white wines. 3.4. Bioconversion rate of S-conjugates into volatile thiols by yeast To investigate the capacity of Saccharomyces cerevisiae to release volatile thiols from cysteine-S-conjugates during alcoholic fermentation, reference compounds were added to a synthetic medium prior to fermentation. Two yeasts strains were used in this experiment. VL3-1D strain was selected for its strong capacity to reveal volatile thiols [27–29]. In previous work, we demonstrated the predominance role of nitrogen catabolic repression (NCR) in volatile thiol release [30]. NCR consists of the specific inhibition of transcriptional activators by the cytoplasmic protein Ure2p. This inactivation occurred when optimal sources of nitrogen are available, reducing the transcription level of many target genes involved in poor nitrogen source utilization. Therefore, the effect of NCR was equally tested on the release of 3SP, 3SHp, and 2M3SB by measuring the production of volatile thiols after fermentation by the NCR derepressed strain (ure2) in relation to the wild-type strain (wt). Fermentations were carried out in 350-mL batch cultures using KP synthetic grape juice (see Section 2). Each synthetic precursor was added to the medium at 1000 nM. All the fermentation processes were completed in comparable times. Table 4 shows the
Table 4 Bioconversion of cysteine-S-conjugates into volatile thiols by VL3-1D and ure2 strains. Synthetic precursors (P-3SP, P-3SH, P-3SHp, and P-2M3SB) were added to the medium at 1000 nM. Volatile thiol release was measured after complete fermentation (n = 3), expressed in nanograms per liter and conversion rates were calculated (expressed in %). ure2
VL3-1D (wt) Mean (ng L−1 ) 3SP 3SH 3SHp 2M3SB a b
2273a 1809 283 1990
Mean value (n = 3). Standard deviations s (n = 3).
± ± ± ±
156b 210 29 130
Conversion rate (%)
Mean (ng L−1 )
2.0 1.6 0.3 1.8
7707 6897 1290 8171
± ± ± ±
569 422 184 625
Conversion rate (%) 6.8 6.1 1.1 7.2
196
C. Thibon et al. / Analytica Chimica Acta 660 (2010) 190–196
volatile thiol levels after fermentation and the conversion rate of each aroma precursor by the two strains. First, bioconversion of cysteine-S-conjugates by yeast was strongly affected by the general mechanism of nitrogen catabolic repression similarly to 3SH release. Indeed the mutant (ure2) released three times more 3SH, 3SP, 3SHp, and 2M3SB than the wild-type strain (VL3-1D). This may indicate that all volatile thiols studied were released by the same metabolic pathway of yeast controlled by nitrogen sensing. Bioconversion rates were similar for P-3SP, P-3SH, and P-2M3SB, i.e. 2% with VL3-1D approximately, and about 7% with ure2. The P-3SHp conversion rate was 6-fold lower that those of the other cysteine-S-conjugates, irrespective of the strain. Indeed, with the same initial precursor content, only 283 ng L−1 3SHp were released by VL3-1D (wt), but 2273 ng L−1 3SP. This result apparently indicates that the yeast bioconversion rate is higher when the carbon chain is shorter. The 3SHp content of young botrytized wines is partly due to the low level of P-3SHp in must and its poor bioconversion rate by yeast. Indeed, Sarrazin et al. observed that this volatile thiol was found in sweet wines at mean levels 2- and 4-fold lower than those observed for 2M3SB and 3SP, respectively [18]. As the bioconversion rates of 3SH, 3SP, 2M3SB are almost identical, the precursor levels in must correlate directly with the volatile thiol concentrations found in botrytized wines [18–20]. Finally, it was noted that cysteine-Sconjugates with long carbon chains (highly hydrophobic) were not assimilated as well by yeast as those with short carbon chains. This may indicate that cysteine-S-conjugates are not assimilated passively via the membrane but by a specific transport. As they are similar in structure to amino acids, the uptake of aroma precursors from must into the yeast cytoplasm may be driven by amino acid transporters [30,33], thus explaining why a long carbon chain inhibits their uptake and subsequent bioconversion. 4. Conclusion This article reports the identification and quantification of new cysteine-S-conjugate precursors in botrytized must. Concentrations were considerably higher when B. cinerea had developed on the grapes. The increase in precursor content observed here is in agreement with the volatile thiol concentrations previously found in sweet wines [3]. This indicates that the sulfanyl alcohols responsible for the characteristic aroma of botrytized wines are formed by the yeast metabolism from the corresponding cysteine-S-conjugate precursors. Further work is now required to enhance our understanding of aromatic potential and, more particularly, to elucidate the metabolism of cysteine-S-conjugate production by V. vinifera and B. cinerea in the vineyard. Acknowledgments We would like to thank Sandrine Garbay at Château d’Yquem (Sauternes, France) for her help, as well as Aquitaine Traduction
for reviewing the English manuscript. This work was supported by grants from the Bordeaux Wine Council (CIVB), Laffort Oenologie, and Château d’Yquem. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.aca.2009.10.018. References [1] M.L. Shankaranarayana, B. Raghavan, K.O. Abraham, C.P. Natarajan, in: I.D. Morton, A.J. Macleod (Eds.), Food Flavors, Part A, Elsevier Science Publishers, 1982, p. 169. [2] J. Rigaud, P. Etievant, R. Henry, A. Latrasse, Sci. Aliments 6 (1986) 213. [3] A. Buettner, P. Schieberle, J. Agric. Food Chem. 47 (1999) 5189. [4] E. Demole, P. Enggist, G. Ohloff, Helv. Chim. Acta 65 (1982) 1785. [5] K.H. Engel, R. Tressl, J. Agric. Food Chem. 39 (1991) 2249. [6] H. Idstein, P. Schreier, J. Agric. Food Chem. 33 (1985) 138. [7] S. Escher, Y. Niclass, M. van de Waal, C. Starkenmann, Chem. Biodivers. 3 (2006) 943. [8] T. Tominaga, D. Dubourdieu, Flavour Frag. J. 12 (1997) 373. [9] R. Tressl, R. Silwar, J. Agric. Food Chem. 29 (1981) 1078. [10] U. Gasser, W. Grosch, Z. Lebensm, Unters. Forsch. 186 (1988) 489. [11] H. Guth, W. Grosch, J. Agric. Food Chem. 42 (1994) 2862. [12] P. Schieberle, J. Agric. Food Chem. 39 (1991) 1141. [13] C. Vermeulen, I. Lejeune, T.T. Tran, S. Collin, J. Agric. Food Chem. 54 (2006) 5061. [14] P. Darriet, T. Tominaga, V. Lavigne, J.N. Boidron, D. Dubourdieu, Flavour Frag. J. 10 (1995) 385. [15] T. Tominaga, M.L. Murat, D. Dubourdieu, J. Agric. Food Chem. 46 (1998) 1044. [16] T. Tominaga, P. Darriet, D. Dubourdieu, Vitis 35 (1996) 207. [17] T. Tominaga, A. Furrer, R. Henry, D. Dubourdieu, Flavour Frag. J. 13 (1998) 159. [18] E. Sarrazin, S. Shinkaruk, T. Tominaga, B. Bennetau, E. Frerot, D. Dubourdieu, J. Agric. Food Chem. 55 (2007) 1437. [19] T. Tominaga, R. Baltenweck-Guyot, C. Peyrot des Gachons, D. Dubourdieu, Am. J. Enol. Vitic. 51 (2000) 178. [20] T. Tominaga, N. Niclass, E. Frérot, D. Dubourdieu, J. Agric. Food Chem. 54 (2006) 7251. [21] T. Tominaga, C. Peyrot Des Gachons, D. Dubourdieu, J. Agric. Food Chem. 46 (1998) 5215. [22] C. Thibon, S. Shinkaruk, T. Tominaga, B. Bennetau, D. Dubourdieu, J. Chromatogr. A 1183 (2008) 150. [23] S. Shinkaruk, C. Thibon, J. Schmitter, P. Babin, T. Tominaga, M. Degueil, B. Desbat, C. Jussier, B. Bennetau, D. Dubourdieu, C. Bennetau-Pelissero, Chem. Biodivers. 5 (2008) 793. [24] G. Acuna, M. Gautschi, F. Kumli, J. Schmid, J. Zsindely, Mercapto-alkanol flavor compounds, UNITED STATES PATENT AND TRADEMARK OFFICE GRANTED PATENT, 2003. [25] C. Thibon, D. Dubourdieu, P. Darriet, T. Tominaga, Food Chem. 114 (2009) 1359. [26] P. Marullo, M. Bely, I. Masneuf-Pomarede, M. Pons, M. Aigle, D. Dubourdieu, FEMS Yeast Res. 6 (2006) 268. [27] K.S. Howell, J.H. Swiegers, G.M. Elsey, T.E. Siebert, E.J. Bartowsky, G.H. Fleet, I.S. Pretorius, M.A. de Barros Lopes, FEMS Microbiol. Lett. 240 (2004) 125. [28] I. Masneuf-Pomarede, C. Mansour, M.L. Murat, T. Tominaga, D. Dubourdieu, Int. J. Food Microbiol. 108 (2006) 385. [29] M.L. Murat, I. Masneuf, P. Darriet, V. Lavigne, T. Tominaga, D. Dubourdieu, Am. J. Enol. Vitic. 52 (2001) 136. [30] C. Thibon, P. Marullo, O. Claisse, C. Cullin, D. Dubourdieu, T. Tominaga, FEMS Yeast Res. 8 (2008) 1076. [31] T. Tominaga, L. Blanchard, P. Darriet, D. Dubourdieu, J. Agric. Food Chem. 48 (2000) 1799. [32] J.L. Luisier, H. Buettner, S. Volker, T. Rausis, U. Frey, J. Agric. Food Chem. 56 (2008) 2883. [33] M. Subileau, R. Schneider, J.M. Salmon, E. Degryse, FEMS Yeast Res. 8 (2008) 771.
Contents lists available at ScienceDirect
Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
Aromatic potential of botrytized white wine grapes: Identification and quantification of new cysteine-S-conjugate flavor precursors Cécile Thibon a,∗ , Svitlana Shinkaruk b , Michael Jourdes a , Bernard Bennetau c , Denis Dubourdieu a , Takatoshi Tominaga a a b c
Université de Bordeaux, UMR Œnologie, INRA, ISVV, Bordeaux, F-33000, France Université de Bordeaux, ENITAB, CS 40201, Bordeaux, F-33000, France Université de Bordeaux, CNRS, UMR 5255 ISM, F-33405, France
a r t i c l e
i n f o
Article history: Received 25 July 2009 Received in revised form 8 October 2009 Accepted 10 October 2009 Available online 17 October 2009 Keywords: Sweet wine Aroma precursor Cysteine-S-conjugate GC derivatization Vitis vinifera Botrytis cinerea
a b s t r a c t Sweet wines made from botrytized grapes contain much higher concentrations of volatile thiols, especially 3-sulfanylhexan-1-ol (3SH), than dry white wines. Three new specific volatile thiols (3sulfanylpentan-1-ol (3SP), 3-sulfanylheptan-1-ol (3SHp), and 2-methyl-3-sulfanylbutan-1-ol (2M3SB) were recently identified in Sauternes wines. Like most volatile thiols, these compounds were almost totally absent from must, mainly being formed during alcoholic fermentation. In this work, we describe the identification and quantification of three new cysteine-S-conjugate precursors in must made from Botrytis-infected grapes. S-3-(pentan-1-ol)-l-cysteine (P-3SP), S-3-(heptan-1-ol)-l-cysteine (P-3SHp), and S-3-(2-methylbutan-1-ol)-l-cysteine (P-2M3SB) were identified by direct GC–MS analysis of their derivative forms obtained by silylation of an enriched fraction, isolated from must by affinity chromatography. Concentrations were considerably higher when Botrytis cinerea had developed on the grapes. In botrytized must, the mean levels of P-3SP, P-3SHp, and P-2M3SB were in the vicinity of 700, 50, and 500 nM, respectively, whereas concentrations in healthy must ranged from 0 to 50 nM. This indicated that these three new sulfanyl alcohols, responsible for the characteristic aroma of botrytized wines, were formed by the yeast metabolism during alcoholic fermentation from the corresponding non-volatile cysteine-S-conjugate precursors. Moreover, these results highlighted the predominant role of botrytization in developing grape aroma potential. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Sulfur compounds, especially volatile thiols, are extremely odoriferous molecules, which contribute to the aroma of many fruits, plants, and foods [1]. For example, certain thiols contribute to the characteristic aroma of blackcurrant [2], grapefruit [3,4], passion fruit [5], guava [6], fringed rue (Ruta chalepensis) [7], and box tree (Buxus sempervirens) [8]. The key role played by thiols in the odor of roasted coffee [9], popcorn, and grilled meat is also well known [10–12]. Finally, the contribution of compounds in this family to the aroma of beer was recently reported [13]. Since the early 1990s, several highly odoriferous volatile thiols have been identified in wines made from Vitis vinifera L. cv. Sauvignon blanc. These cover a broad aromatic palette, including such key descriptors as: green pepper, boxwood, broom, eucalyptus, blackcurrant buds, rhubarb, tomato leaves, nettles, grapefruit, passion fruit, white peaches, gooseberries, and asparagus broth,
∗ Corresponding author. Tel.: +33 5 57 57 58 61; fax: +33 5 57 57 58 13. E-mail address: [email protected] (C. Thibon). 0003-2670/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2009.10.018
as well as acacia flowers and wood. The first molecule identified as a characteristic component of Sauvignon blanc wine aroma was 4-methyl-4-sulfanylpentan-2-one (4MSP) [14]. Assays of this sulfanylketone in certain Sauvignon blanc wines revealed concentrations as high as 40 ng L−1 , considerably higher than the perception threshold (0.8 ng L−1 in model solution) [15], confirming its decisive organoleptic role in wines characteristic of this grape variety. Several other odoriferous volatile thiols were later identified in Sauvignon blanc wines: 3-sulfanylhexyl acetate (3SHA) [16], 4-methyl-4-sulfanylpentan-2-ol (4MSPOH), and 3-sulfanylhexan1-ol (3SH) [17], smelling of boxwood and zest, citrus zest, and grapefruit and passion fruit, respectively. More recently, 3-sulfanylpentan-1-ol (3SP), 3-sulfanylheptan1-ol (3SHp), 2-methyl-3-sulfanylbutan-1-ol (2M3SB), and 2-methyl-3-sulfanylpentan-1-ol (2M3SP) were identified in Sauternes wines [18]. These thiols, together with 3SH, significantly enhance the varietal aroma of botrytized sweet white wines. 3SP and 3SHp have pronounced citrus zest aromas, while 3SH smells fruity. Concentrations of 3SP in botrytized wines are always well below the perception threshold (900 ng L−1 ), while 3SHp rarely exceeds its perception threshold (35 ng L−1 ). However, an additive
C. Thibon et al. / Analytica Chimica Acta 660 (2010) 190–196
191
Fig. 1. Structures of 3-sulfanylpentan-1-ol (3SP), 3-sulfanylhexan-1-ol (3SH), 3-sulfanylheptan-1-ol (3SHp), 2-methyl-3-sulfanylbutan-1-ol (2M3SB), and their corresponding precursors: S-3-(pentan-1-ol)-l-cysteine (P-3SP), S-3-(hexan-1-ol)-l-cysteine (P-3SH), S-3-(heptan-1-ol)-l-cysteine (P-3SHp), and S-3-(2-methylbutan-1-ol)-l-cysteine (P2M3SB).
effect of these volatile thiols, combined with 3SH, has been clearly demonstrated [18]. Therefore, 3SP and 3SHp have a considerable impact on overall aromatic complexity in the presence of other volatile thiols, such as 3SH, although concentrations remain below the perception threshold. Concentrations of 3SH, 3SP, and 3SHp in botrytized wines are strongly affected by the development of Botrytis cinerea. Wines made from healthy grapes contain 3SH but only traces of the other two thiols, while those in the “pourri plein” stage (entirely botrytized but not desiccated) of noble rot have much higher thiol concentrations [19,20]. Like most volatile thiols, these compounds are almost totally absent from must, mainly being formed during alcoholic fermentation. Tominaga et al. showed that 3SH, present in a cysteinylated conjugate precursor form in must, was released by yeast metabolism during alcoholic fermentation [21]. The putative cysteinylated conjugates of 3SP, 3SHp, and 2M3SB, similar in chemical structure to 3SH, were studied in botrytized must to determine whether they were formed by the same pathway (Fig. 1). In this work, we describe the identification of three new cysteine-S-conjugate precursors in must made from Botrytisinfected grapes (Fig. 1). S-3-(pentan-1-ol)-l-cysteine (P-3SP), S-3-(heptan-1-ol)-l-cysteine (P-3SHp), and S-3-(2-methylbutan-1ol)-l-cysteine (P-2M3SB) were identified by direct GC–MS analysis of their derivative forms, obtained by silylation of an enriched fraction, isolated from must by affinity chromatography, as previously described for the precursor of 3SH (P-3SH) [22]. This GC–MS method was simple, accurate, effective for identifying new cysteine-S-conjugates (aroma precursors), as well as determining concentrations in must during grape botrytization.
2. Experimental 2.1. Chemicals and reagents trans-2-Pentenal (95%), trans-2-hexenal (95%), trans-2-heptenal (97%), trans-2-methyl-2-butenal (98%), S-benzyl-l-cysteine (97%), N-(tert-butoxycarbonyl)-l-cysteine (Boc-cysteine) (99.5%), cesium carbonate (99%), and sodium borohydride (98%) were purchased from Sigma–Aldrich (L’Isle d’Abeau, France). Nmethyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide with 1% tert-butyldimethychlorosilane (MTBSTFA:t-BDMCS) and anhydrous pyridine were obtained from Perbio Science (Brebières, France). Chelating Sepharose Fast Flow was purchased from GE Healthcare (Buckinghamshire, England). 2.2. Nuclear magnetic resonance (NMR) spectroscopy 1 H, and 13 C NMR spectra were recorded on a Bruker AC-300 FT (1 H: 300.13 MHz, 13 C: 75.4 MHz). No internal standard was present in D2 O samples and spectra were referenced using the frequency of the deuterated solvent (lock frequency). Chemical shifts (ı) and coupling constants (J) are expressed in ppm and Hz, respectively.
2.3. Synthesizing cysteine-S-conjugates New cysteine-S-conjugates were synthesized in three steps by the Michael addition of Boc-protected cysteine to corresponding ␣,-unsaturated aldehydes, followed by reducing the carbonyl group to alcohol and deprotecting the amino group, as previously reported for P-3SH synthesis (Scheme 1) [22,23].
Scheme 1. Three-step synthesis of new cysteine-S-conjugate precursors: (a) Cs2 CO3 , CH3 CN, r.t.; (b) 1. NaBH4 , MetOH, r.t., 2. 10 M HCl, r.t.
192
C. Thibon et al. / Analytica Chimica Acta 660 (2010) 190–196
Table 1 MS/MS conditions for the acquisition of cysteine-S-conjugate derivatives. Compound
Retention time (min)
Parent ion (window)
Resonance excitation voltage (V)
Recorded mass range
Product ions for quantification
t-BDMS-P-2M3SB t-BDMS-P-3SP t-BDMS-P-3SH t-BDMS-P-3SHp t-BDMS-S-benzylcysteine
21.0–21.3 21.4 22.0 23.1 20.8
492 (3) 492 (3) 506 (3) 520 (3) 354 (3)
1.50 1.50 1.50 1.50 1.50
454–502 454–502 468–516 482–530 134–240
464 464 478 492 147
2.3.1. S-3-(pentan-1-ol)-l-cysteine (P-3SP) Beige solid (purity determined by GC–MS after trimethylsilylation, 96%). 1 H NMR (D2 O) ı 0.84 (t, J = 7.3 Hz, 3H, CH3 -5); 1.51 (m, 2H, CH2 -4); 1.60 (m, 2H, CH2 -2a); 1.73 (m, 1H, CH2 -2b); 2.71 (m, 1H, CH-3); 2.96 (m, 2H, CH2 -); 3.60 (m, 2H, CH2 -1); 3.78 (m, 1H, CH-␣). 13 C NMR (D2 O) ı 10.03 and 10.14 (CH3 -5); 27.00 and 27.06 (CH2 -4); 30.44 and 30.47 (CH2 -); 35.43 and 35.67 (CH2 -2); 44.01 and 44.29 (CH-3); 53.97 and 53.99 (CH-␣); 59.11 and 59.15 (CH2 1); 172.69 and 172.71 (COOH). All 13 C peak values were slightly different, depending on the stereospecific features of the chemical structure of each diastereoisomer. 2.3.2. S-3-(heptan-1-ol)-l-cysteine (P-3SHp) Beige solid (purity determined by GC–MS after trimethylsilylation, 95%). 1 H NMR (D2 O) ı 0.77 (t, J = 7.2 Hz, 3H, CH3 -7); 1.11–1.34 (m, 4H, CH2 -6 and CH2 -5); 1.49 (m, 2H, CH2 -4); 1.63 (m, 2H, CH2 2a); 1.75 (m, 1H, CH2 -2b); 2.78 (m, 1H, CH-3); 2.85–2.94 (m, 2H, CH2 -); 3.63 (m, 2H, CH2 -1); 3.80 (dd, J = 6.8, 4.5 Hz, 1H, CH-␣). 13 C NMR (D O) ı 13.26 (CH -7); 21.90 (CH -6); 27.97 and 28.05 2 3 2 (CH2 -5); 30.39 and 30.45 (CH2 -); 33.88 (CH2 -4); 35.88 and 36.12 (CH2 -2); 42.53 and 42.76 (CH-3); 53.99 (CH-␣); 59.14 and 59.18 (CH2 -1); 172.6 (COOH). Some 13 C peak values were slightly different, depending on the stereospecific features of the chemical structure of each diastereoisomer. 2.3.3. S-3-(2-methylbutan-1-ol)-l-cysteine (P-2M3SB) Beige solid (purity determined by GC–MS after trimethylsilylation, 90%). As trans-2-methyl-2-butenal contains two stereogenic centers and the configuration of cysteine C-␣ is constant (R configuration), the four synthesized stereoisomers formed two pairs of diastereomers. They differed by syn or anti orientation of substituents at the C-2 and C-3 chiral centers formed during the Michael addition reaction. NMR spectra and 2D correlations (COSY, HMQC, and HMBC) confirmed the presence of two diastereomer pairs in a ratio of 1:2. Although full stereochemical determination was not possible, due to signal complexity and overlap, 2,3-anti addition was apparently preferred, according to previously published data [18,24]. 1 H NMR (D O) ı 0.63–0.73 and 0.77–0.84 (two massifs with a 2 relative integration ratio of 2:1, 3H, CH3 -2 ); 0.95–1.14 (four overlapping doublets, J = 6.8 Hz, 3H, CH3 -4); 1.63–1.75 and 1.93–2.02 (two massifs with a relative integration ratio of 2:1, 1H, CH-2); 2.78–2.90 (m, 1H, CH-3); 2.91–3.06 (m, 2H, CH2 -); 3.18–3.48 (m, 2H, CH2 -1); 4.10–4.18 and 4.21–4.32 (two massifs with a relative integration ratio of 2:1, 1H, CH-␣). 13 C NMR (D2 O) ı 11.48, 11.67, 12.13, and 12.44 (CH3 -2 ); 15.56, 15.60, and 18.85 (CH3 -4); 30.06, 30.24, and 30.28 (CH2 -); 39.08, 39.22, 39.83, and 39.99 (CH-2); 42.09, 42.24, 43.41, and 44.02 (CH-3); 52.34, 52.42, 52.50, and 52.62 (CH-␣); 64.28, 64.36, and 64.41 (CH2 -1); 168.36, 168.49, 170.06, and 170.13 (COOH). 2.4. Must preparation and purification of cysteine-S-conjugate Sauvignon blanc and Semillon grapes were picked from the same plot in the 2008 vintage (Château d’Yquem, Appellation Sauternes, France). Semillon musts were also studied in the 2007
vintage. Two 1000-grape samples were harvested at four stages in botrytization: healthy (not infected by B. cinerea), pourri plein (entirely botrytized but not desiccated), pourri rôti (botrytized and desiccated), and late pourri rôti (shriveled grapes left for an additional week before picking). In the laboratory, samples were manually destemmed, sulfured (10 g hL−1 ), and crushed in a blender (2 min) under inert gas (CO2 ). Must obtained from each grape sample was stored at −20 ◦ C prior to analysis. The method for purifying cysteine-S-conjugates from must was based on the protocol described by Thibon et al. [22]. Briefly, samples (500 L) containing 200 pg S-benzyl-l-cysteine as an internal standard were adjusted to pH 10 and loaded directly onto a Chelating Sepharose column, containing immobilized copper. The retained fraction was washed with 2 mL potassium phosphate buffer (50 mM, pH 7), eluted by percolating it with 3 mL hydrochloric acid solution (25 mM), and evaporated dry under vacuum. The dry residue was dispersed in 500 L ethanol–acetone mix (50/50). The supernatant was evaporated dry under vacuum in a 2-mL vial. 2.5. Derivatization and GC–MS analysis The tert-butyldimethylsilyl (t-BDMS) derivatives were prepared as described by Thibon et al. [25]. Briefly, MTBSTFA:t-BDMCS (25 L) and anhydrous pyridine (40 L) were added in vial containing the dry residue The mixture was shaken briefly and heated to 70 ◦ C for 15 min. Electronic impact (EI) mass spectra were recorded at 70 eV on a Polaris Q mass spectrometer, equipped with an ion trap analyzer (Thermo Electron Corporation, Milan, Italy), connected to a Trace GC gas chromatograph (Thermo Electron) equipped with a type BPX35 capillary column (SGE: 50 m, internal diameter: 0.25 mm, film thickness: 0.22 m), and a splitless injector (200 ◦ C). A total of 2-L of the derivatized sample was injected. The temperature program was as follows: 80 ◦ C for 1 min, then increasing to 240 ◦ C at 15 ◦ C min−1 , 310 ◦ C at 4 ◦ C min−1 , and 320 ◦ C at 10 ◦ C min−1 , followed by a 5 min isotherm. Derivatives were quantified in MS/MS mode. Ions were selected for quantification on the basis of peak intensity and ion specificity. The MS/MS acquisition conditions for cysteine-S-conjugate derivatives are presented in Table 1. 2.6. Method validation For calibration, must samples were spiked prior to purification by adding the appropriate volume of a mixture of standard stock solution of all cysteine-S-conjugates to achieve concentrations of 10, 25, 100, 250, and 1000 nM for P-3SP, P-2M3SB, and P-3SHp and of 100, 500, 2500, 10,000, and 25,000 nM for P-3SH, then analyzed by GC–MS, as described above. Each point of calibration was carried out in triplicate. Calibration curves plotted relative area of the total sum of diastereoisomers versus concentration of standard solutions. Repeatability was assessed over a series of five replicates. The limit of detection (LOD) was defined as the concentration that gave a signal-to-noise ratio (S/N) of three. The limit of quantification (LOQ) was defined as the concentration that gave an S/N ratio of 10. These parameters were experimentally calculated from the S/N obtained in must sample analyses.
C. Thibon et al. / Analytica Chimica Acta 660 (2010) 190–196
193
Table 2 Repeatability, calibration line parameters, limits of quantification (LOQ), limits of detection (LOD), and recovery (spiking level = 100 nM) from healthy and botrytized grape juice (n = 5) of S-3-(pentan-1-ol)-l-cysteine (P-3SP), S-3-(hexan-1-ol)-l-cysteine (P-3SH), S-3-(heptan-1-ol)-l-cysteine (P-3SHp), and S-3-(2-methylbutan-1-ol)-l-cysteine (P-2M3SB). Compound
P-3SP P-3SH P-3SHp P-2M3SB a b c
RSDa (%)
5.6 2.8 5.9 6.8
Calibration line
y = 1.65x y = 1.37x y = 4.02x y = 1.24x
R2
0.9868 0.9915 0.9873 0.9988
LOQb
0.6 0.4 0.7 1.0
LODc
0.2 0.1 0.2 0.3
Average recovery (%) Healthy
Botrytized
96 102 108 105
103 97 113 98
RSD: relative standard deviation (n = 5). Limits of quantification (nM). Limits of detection (nM).
2.7. Thiol production assay in fermented synthetic medium Volatile thiol release was measured at the end of fermentation of synthetic grape juice, previously described as KP medium, in 350mL bottles at 24 ◦ C [26]. This medium was buffered to pH 3.3 and contained 80 g L−1 glucose, 80 g L−1 fructose, and 190 mg L−1 available nitrogen. Each aroma precursor (P-3SP, P-3SH, P-3SHp, and P-2M3SB) was added to the medium before yeast inoculation at 1000 nM. Two yeasts strains were used in this experiment: VL3-1D (wildtype) and ure2. The first was derived from a commercial starter VL3c (Laffort Enologie, Bordeaux, France) described previously for its strong capacity to reveal volatile thiols [27–29]. ure2 is an homozygous spore clones of VL3-1D carrying two deleted URE2 gene copies. This mutant released three times more 3SH than the wild-type strain [30]. Yeast strains were inoculated at 1 × 106 cells mL−1 from an overnight pre-culture in KP medium diluted with water (1:1). Cell numbers were measured using an electronic particle counter (ZII; Coulter-Counter, Coultronics, Margency, France). Volatile thiol release was measured after complete fermentation (measured by weight loss). All experiments were carried out in triplicate. At the end of fermentation, the volatile thiols were extracted from 250 mL synthetic medium, using the method described by Tominaga et al. (1998), as modified by Tominaga et al. (2000) [15,31]. Amounts of 3SP, 3SH, 3SHp, and 2M3SB were quantified by GC–MS, as described by Sarrazin et al. [18]. Briefly, volatile thiol extracts were analyzed on a 6890N gas chromatograph (Agilent technologies) using a type BP20 capillary column [SGE, 50 m, internal diameter: 0.22 mm, film thickness: 0.25 m]. The detector was a mass spectrometer (MS 5973, Agilent Technologies), functioning in EI mode. SIM mode was used to quantify 3SP, 3SH, 3SHp, and 2M3SB, selecting the followings ions: m/z 120 and 86 for 3SP and 2M3SB, m/z 134 and 100 for 3SH, and m/z 148 and 114 for 3SHp.
3. Results and discussion 3.1. Identification and quantification of new cysteine-S-conjugates in silyl derivative form Each derivatized synthetic reference compound was initially analyzed by GC–MS in scan mode to obtain the retention time and fragmentation pattern. Fig. 2 shows the total ion chromatogram and mass spectra of the cysteine-S-conjugate mixture in t-BDMS derivative form. Adequate resolution was observed in the chromatogram, i.e. no overlapping peaks. As the derivatization step increased steric hindrance and maximized the dissimilarity between diastereoisomers, a partial differentiation of derivatives was observed. The presence of three stereogenic centers in P-2M3SB explains the
massif form of the peak ranging from 21.0 to 21.3 min. A slight separation of diastereoisomeric forms was observed for P-3SP and P-3SH at 21.4 and 22.0 min, respectively. In contrast, the P-3SHp derivative presented a Gaussian peak at 23.1 min. The mass spectrum of pure P-3SP, P-3SH, P-3SHp, and P-2M3SB, obtained by chemical synthesis, was determined by GC–MS in full scan mode (Fig. 2). All the compounds revealed similar fragmentation patterns, with several identical ions, such as m/z 189, 218, 258, 302, and 316, all resulting from the fragmentation of the derivatized cysteine moiety of P-3SP, P-3SH, P-3SHp, and P-2M3SB. Moreover, the other ions, such as [M–15]+ , [M–57]+ , [M–85]+ , [M–159]+ , [M–189]+ , [M–217]+ and [M–350]+ , differed by 14 mass units, e.g.: P-2M3SB and P-3SP compared to P-3SH or 28 mass units for P-3SHp. This difference is due to the upper alkyl-alcohol moiety of P-3SP, P-3SH, P-3SHp, and P-2M3SB (Fig. 2, see supporting information). The intense fragment, [M–57]+ at m/z 492 for P-2M3SB and P-3SP, m/z 506 for P-3SH and m/z 520 for P-3SHp, resulted from the cleavage of a tert-butyl group from the molecular ion of P-3SP, P-3SH, P-3SHp, and P-2M3SB. Due to this very easy cleavage, the molecular ion of each compound was not detected in our GC–MS condition. Moreover, the [M–85]+ ion at m/z 464 for P-2M3SB and P-3SP, m/z 478 for P-3SH, and m/z 492 for P-3SHp was also a very intense ion, corresponding to the main fragment in MS/MS analysis of the [M–57]+ ion for each compound (see supporting information). Thus the [M–85]+ ion resulted from the loss of 57 mass units (tert-butyl group), followed by the loss of 28 mass units from its parent ion [M–57]+ . The loss of 28 mass units was due to the loss of the CO group from the silylated acid function of the cysteine moiety of P-3SP, P-3SH, P-3SHp, and P-2M3SB. Ions were selected for quantification on the basis of peak intensity and ion specificity, as well as potential interference from other compounds. Consideration was primarily given to peak intensity, to obtain the highest sensitivity. The [M–57]+ peaks of compound derivatives (EI mode) were chosen as the parent ion. As the MS/MS spectrum of the precursor ion [M–57]+ had one major peak at [M–85]+ , m/z 464, m/z 478, m/z 492, and m/z 464 were used as the quantitative ions for assessing P-3SP, P-3SH, P-3SHp, and P-2M3SB (see supporting information). Mass spectra and retention times for synthesized cysteine-Sconjugates were used to assay the analytes in the Sauvignon blanc or Semillon juice, obtained from healthy or botrytized grapes. Must extract was obtained by the assaying method based on the protocol described in Section 2. The first stage consisted of purifying the cysteine-S-conjugates by percolating the must directly onto a Chelating Sepharose column, containing immobilized copper. In the second stage, the precursors were derivatized to increase their volatility prior to GC–MS/MS analysis. GC–MS/MS analysis of the botrytized grape juice extract revealed the presence of P-3SP, P3SH, P-3SHp, and P-2M3SB, by comparison with the retention times and MS/MS spectra of the synthetic reference compounds (data not shown).
194
C. Thibon et al. / Analytica Chimica Acta 660 (2010) 190–196
Fig. 2. Chromatograms, mass spectra, and structures of the main fragments obtained from the t-BDMS derivatives of the reference cysteine-S-conjugates: P-2M3SB, P-3SP, P-3SH, and P-3SHp. (The structure of the other ions is shown in supporting information.)
3.2. Validating the method for quantifying cysteine-S-conjugates in must The peak areas of the total sum of diastereoisomers on the MS/MS chromatograms were calculated for [M–85]+ ions: m/z 464 (t-BDMS derivatives of P-3SP and P-2M3SB), m/z 478 (t-BDMS derivative of P-3SH), m/z 492 (t-BDMS derivative of P-3SHp), and m/z 147 (t-BDMS derivative of S-benzylcysteine, IS). Table 2 shows the calibration lines and R2 obtained for each of the four com-
pounds. Calibration curves were plotted for five concentrations, from 10 to 1000 nM (for P-3SP, P-2M3SB, and P-3SHp) and from 100 to 25,000 nM for P-3SH. Each point on the curve represents the average value of three replicated measurements. The calibration curves of the ratio of the [M–85]+ peak areas to those at m/z 147 (IS) were linear for GC-injections with a correlation coefficient of >0.986 and a relative standard deviation (RSD) of <6.8%. Repeatability was determined using healthy and botrytized grape juice. Recovery trials were performed at 100 nM. The average recov-
C. Thibon et al. / Analytica Chimica Acta 660 (2010) 190–196
195
Table 3 Quantitative assays (nanomoles per liter) of P-3SP, P-3SH, P-3SHp, and P-2M3SB in must at different stages in botrytization, from two grape varieties in different vintages. Vintage 2008
2007
* a b
Variety
Botrytis stage
P-3SP
P-3SH
P-3SHp
P-2M3SB
Sauvignon
Healthy Pourri plein Pourri rôti Late pourri rôti
21 774* 803* 750*
± ± ± ±
6 33 8 25
453 9902* 20301* 15606*
± ± ± ±
50 856 1656 1380
13 30 36* 20
± ± ± ±
3 14 12 5
87 645* 643* 739*
± ± ± ±
28 21 7 17
Semillon
Healthy Pourri plein Pourri rôti Late pourri rôti
11 420* 460* 320*
± ± ± ±
8 112 92 59
98 8383* 11782* 5120*
± ± ± ±
24 1351 871 489
17 13 34* 39*
± ± ± ±
2 9 1 6
52 252* 338* 299*
± ± ± ±
2 34 10 18
Semillon
Healthy Pourri plein Pourri rôti Late pourri rôti
21 474* 614* 404*
± ± ± ±
8 5 114 66
158 19721* 23407* 19254*
± ± ± ±
24 1430 4202 2185
15 31* 105* 62*
± ± ± ±
2 5 16 9
42 312* 417* 909*
± ± ± ±
4 11 109 205
a
b
Statistically different from healthy stage, ANOVA, P = 0.01. Mean value (n = 2). Standard deviations s (n = 2).
ery ranged from 96% to 113%. Detection and quantification limits were determined by analyzing several botrytized musts, naturally containing cysteine-S-conjugates, and calculating the concentration required to give a signal-to-noise ratio of 3 or 10, respectively. Detection and quantification limits ranged from 0.1 to 0.3 and from 0.4 to 1 nM, respectively. 3.3. Impact of noble rot development on grapes on S-conjugate content The concentrations of 3SH, 3SP, and 3SHp in botrytized wines are strongly affected by the development of B. cinerea. Wines produced from grapes affected by the “pourri plein” stage (entirely botrytized but not desiccated) of noble rot have much higher sulfanyl alcohol concentrations. Like most volatile thiols, these compounds were almost totally absent from must, mainly being formed during alcoholic fermentation [18]. Several grape juice samples were analyzed to assess the impact of B. cinerea development on cysteine-S-conjugate formation. Grape samples were harvested at four stages in botrytization: healthy, pourri plein, pourri rôti, and late pourri rôti. For each grape sample, the cysteinylated precursors were extracted, as described above, and this process was repeated twice. Table 3 lists S-conjugate concentrations in nanomoles per liter during the botrytization of Sauvignon blanc and Semillon grapes in the 2007 and 2008 vintages. Healthy grape juice contained only trace levels of P-3SP, P-3SHp, and P-2M3SB (ranging from 11 to 87 nM). The P-3SH content of healthy Semillon and Sauvignon blanc musts was approximately 100 and 500 nM, respectively. The enrichment previously described for P-3SH during botrytization [22,25,32] was also observed for the other S-conjugates. In both varieties and vintages, a considerable increase in S-conjugate concentrations was observed, starting in the first stages of botrytization. Indeed, P-3SP levels increased rapidly, reaching an average of 30-fold between the healthy and pourri rôti stages. The increase in P-3SHp and P-2M3SB was more moderate, ranging from 2.5- to 7-
fold and 6- to 17-fold, respectively. The decrease in grape volume by desiccation ranged from 40% to 60% at the pourri rôti and late pourri rôti stages (data not shown). Therefore, this rate of increase in four aroma precursors cannot be explained merely by grape desiccation. Indeed, irrespective of the S-conjugate observed, concentrations were considerably higher when B. cinerea had developed on the grapes. These results confirm the predominant role of the B. cinerea metabolism in developing grape aroma potential and explain the higher volatile thiol concentrations in sweet compared to dry white wines. 3.4. Bioconversion rate of S-conjugates into volatile thiols by yeast To investigate the capacity of Saccharomyces cerevisiae to release volatile thiols from cysteine-S-conjugates during alcoholic fermentation, reference compounds were added to a synthetic medium prior to fermentation. Two yeasts strains were used in this experiment. VL3-1D strain was selected for its strong capacity to reveal volatile thiols [27–29]. In previous work, we demonstrated the predominance role of nitrogen catabolic repression (NCR) in volatile thiol release [30]. NCR consists of the specific inhibition of transcriptional activators by the cytoplasmic protein Ure2p. This inactivation occurred when optimal sources of nitrogen are available, reducing the transcription level of many target genes involved in poor nitrogen source utilization. Therefore, the effect of NCR was equally tested on the release of 3SP, 3SHp, and 2M3SB by measuring the production of volatile thiols after fermentation by the NCR derepressed strain (ure2) in relation to the wild-type strain (wt). Fermentations were carried out in 350-mL batch cultures using KP synthetic grape juice (see Section 2). Each synthetic precursor was added to the medium at 1000 nM. All the fermentation processes were completed in comparable times. Table 4 shows the
Table 4 Bioconversion of cysteine-S-conjugates into volatile thiols by VL3-1D and ure2 strains. Synthetic precursors (P-3SP, P-3SH, P-3SHp, and P-2M3SB) were added to the medium at 1000 nM. Volatile thiol release was measured after complete fermentation (n = 3), expressed in nanograms per liter and conversion rates were calculated (expressed in %). ure2
VL3-1D (wt) Mean (ng L−1 ) 3SP 3SH 3SHp 2M3SB a b
2273a 1809 283 1990
Mean value (n = 3). Standard deviations s (n = 3).
± ± ± ±
156b 210 29 130
Conversion rate (%)
Mean (ng L−1 )
2.0 1.6 0.3 1.8
7707 6897 1290 8171
± ± ± ±
569 422 184 625
Conversion rate (%) 6.8 6.1 1.1 7.2
196
C. Thibon et al. / Analytica Chimica Acta 660 (2010) 190–196
volatile thiol levels after fermentation and the conversion rate of each aroma precursor by the two strains. First, bioconversion of cysteine-S-conjugates by yeast was strongly affected by the general mechanism of nitrogen catabolic repression similarly to 3SH release. Indeed the mutant (ure2) released three times more 3SH, 3SP, 3SHp, and 2M3SB than the wild-type strain (VL3-1D). This may indicate that all volatile thiols studied were released by the same metabolic pathway of yeast controlled by nitrogen sensing. Bioconversion rates were similar for P-3SP, P-3SH, and P-2M3SB, i.e. 2% with VL3-1D approximately, and about 7% with ure2. The P-3SHp conversion rate was 6-fold lower that those of the other cysteine-S-conjugates, irrespective of the strain. Indeed, with the same initial precursor content, only 283 ng L−1 3SHp were released by VL3-1D (wt), but 2273 ng L−1 3SP. This result apparently indicates that the yeast bioconversion rate is higher when the carbon chain is shorter. The 3SHp content of young botrytized wines is partly due to the low level of P-3SHp in must and its poor bioconversion rate by yeast. Indeed, Sarrazin et al. observed that this volatile thiol was found in sweet wines at mean levels 2- and 4-fold lower than those observed for 2M3SB and 3SP, respectively [18]. As the bioconversion rates of 3SH, 3SP, 2M3SB are almost identical, the precursor levels in must correlate directly with the volatile thiol concentrations found in botrytized wines [18–20]. Finally, it was noted that cysteine-Sconjugates with long carbon chains (highly hydrophobic) were not assimilated as well by yeast as those with short carbon chains. This may indicate that cysteine-S-conjugates are not assimilated passively via the membrane but by a specific transport. As they are similar in structure to amino acids, the uptake of aroma precursors from must into the yeast cytoplasm may be driven by amino acid transporters [30,33], thus explaining why a long carbon chain inhibits their uptake and subsequent bioconversion. 4. Conclusion This article reports the identification and quantification of new cysteine-S-conjugate precursors in botrytized must. Concentrations were considerably higher when B. cinerea had developed on the grapes. The increase in precursor content observed here is in agreement with the volatile thiol concentrations previously found in sweet wines [3]. This indicates that the sulfanyl alcohols responsible for the characteristic aroma of botrytized wines are formed by the yeast metabolism from the corresponding cysteine-S-conjugate precursors. Further work is now required to enhance our understanding of aromatic potential and, more particularly, to elucidate the metabolism of cysteine-S-conjugate production by V. vinifera and B. cinerea in the vineyard. Acknowledgments We would like to thank Sandrine Garbay at Château d’Yquem (Sauternes, France) for her help, as well as Aquitaine Traduction
for reviewing the English manuscript. This work was supported by grants from the Bordeaux Wine Council (CIVB), Laffort Oenologie, and Château d’Yquem. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.aca.2009.10.018. References [1] M.L. Shankaranarayana, B. Raghavan, K.O. Abraham, C.P. Natarajan, in: I.D. Morton, A.J. Macleod (Eds.), Food Flavors, Part A, Elsevier Science Publishers, 1982, p. 169. [2] J. Rigaud, P. Etievant, R. Henry, A. Latrasse, Sci. Aliments 6 (1986) 213. [3] A. Buettner, P. Schieberle, J. Agric. Food Chem. 47 (1999) 5189. [4] E. Demole, P. Enggist, G. Ohloff, Helv. Chim. Acta 65 (1982) 1785. [5] K.H. Engel, R. Tressl, J. Agric. Food Chem. 39 (1991) 2249. [6] H. Idstein, P. Schreier, J. Agric. Food Chem. 33 (1985) 138. [7] S. Escher, Y. Niclass, M. van de Waal, C. Starkenmann, Chem. Biodivers. 3 (2006) 943. [8] T. Tominaga, D. Dubourdieu, Flavour Frag. J. 12 (1997) 373. [9] R. Tressl, R. Silwar, J. Agric. Food Chem. 29 (1981) 1078. [10] U. Gasser, W. Grosch, Z. Lebensm, Unters. Forsch. 186 (1988) 489. [11] H. Guth, W. Grosch, J. Agric. Food Chem. 42 (1994) 2862. [12] P. Schieberle, J. Agric. Food Chem. 39 (1991) 1141. [13] C. Vermeulen, I. Lejeune, T.T. Tran, S. Collin, J. Agric. Food Chem. 54 (2006) 5061. [14] P. Darriet, T. Tominaga, V. Lavigne, J.N. Boidron, D. Dubourdieu, Flavour Frag. J. 10 (1995) 385. [15] T. Tominaga, M.L. Murat, D. Dubourdieu, J. Agric. Food Chem. 46 (1998) 1044. [16] T. Tominaga, P. Darriet, D. Dubourdieu, Vitis 35 (1996) 207. [17] T. Tominaga, A. Furrer, R. Henry, D. Dubourdieu, Flavour Frag. J. 13 (1998) 159. [18] E. Sarrazin, S. Shinkaruk, T. Tominaga, B. Bennetau, E. Frerot, D. Dubourdieu, J. Agric. Food Chem. 55 (2007) 1437. [19] T. Tominaga, R. Baltenweck-Guyot, C. Peyrot des Gachons, D. Dubourdieu, Am. J. Enol. Vitic. 51 (2000) 178. [20] T. Tominaga, N. Niclass, E. Frérot, D. Dubourdieu, J. Agric. Food Chem. 54 (2006) 7251. [21] T. Tominaga, C. Peyrot Des Gachons, D. Dubourdieu, J. Agric. Food Chem. 46 (1998) 5215. [22] C. Thibon, S. Shinkaruk, T. Tominaga, B. Bennetau, D. Dubourdieu, J. Chromatogr. A 1183 (2008) 150. [23] S. Shinkaruk, C. Thibon, J. Schmitter, P. Babin, T. Tominaga, M. Degueil, B. Desbat, C. Jussier, B. Bennetau, D. Dubourdieu, C. Bennetau-Pelissero, Chem. Biodivers. 5 (2008) 793. [24] G. Acuna, M. Gautschi, F. Kumli, J. Schmid, J. Zsindely, Mercapto-alkanol flavor compounds, UNITED STATES PATENT AND TRADEMARK OFFICE GRANTED PATENT, 2003. [25] C. Thibon, D. Dubourdieu, P. Darriet, T. Tominaga, Food Chem. 114 (2009) 1359. [26] P. Marullo, M. Bely, I. Masneuf-Pomarede, M. Pons, M. Aigle, D. Dubourdieu, FEMS Yeast Res. 6 (2006) 268. [27] K.S. Howell, J.H. Swiegers, G.M. Elsey, T.E. Siebert, E.J. Bartowsky, G.H. Fleet, I.S. Pretorius, M.A. de Barros Lopes, FEMS Microbiol. Lett. 240 (2004) 125. [28] I. Masneuf-Pomarede, C. Mansour, M.L. Murat, T. Tominaga, D. Dubourdieu, Int. J. Food Microbiol. 108 (2006) 385. [29] M.L. Murat, I. Masneuf, P. Darriet, V. Lavigne, T. Tominaga, D. Dubourdieu, Am. J. Enol. Vitic. 52 (2001) 136. [30] C. Thibon, P. Marullo, O. Claisse, C. Cullin, D. Dubourdieu, T. Tominaga, FEMS Yeast Res. 8 (2008) 1076. [31] T. Tominaga, L. Blanchard, P. Darriet, D. Dubourdieu, J. Agric. Food Chem. 48 (2000) 1799. [32] J.L. Luisier, H. Buettner, S. Volker, T. Rausis, U. Frey, J. Agric. Food Chem. 56 (2008) 2883. [33] M. Subileau, R. Schneider, J.M. Salmon, E. Degryse, FEMS Yeast Res. 8 (2008) 771.