Nutraceutical Beverages - American Chemical Society

Downloaded by UNIV MASSACHUSETTS AMHERST on July 27, 2012 | http://pubs.acs.org Publication Date: December 1, 2003 | doi: 10.1021/bk-2004-0871.ch024

Chapter 24

Powerful Aromatic Volatile Thiols in Wines Made from Several Vitis vinifera Grape Varieties and Their Releasing Mechanism 1

1,2

1

Takatoshi Tominaga , Isabelle Masneuf , and Denis Dub our dieu 1

Faculté d'Œnologie, Université Victor Segalen Bordeaux 2, 351, Cours de la Libération, 33405 Talence Cedex, France Research Associate for Sarco, A Subsidiary of Laffort and Lamothe-Abiet Pinosa, Second to the Faculté d'Œnologie de Bordeaux, France

2

By extracting specific volatile thiols using p-hydroxymercuribenzoate, 4-mercapto-4-methylpentan-2-one (4MMP), 4-mercapto-4-methylpentan-2-ol (4MMPOH), 3mercapto-3-methylbutan-1-ol (3MMB), 3-mercaptohexan-1-ol (3MH), 3-mercaptohexyle acetate (3MHA), and furanmethanethiol (2FM) were identified in wines made from several Vitis vinifera white and red grape varieties. The assay of volatile thiols in certain wines made from Sauvignon blanc, Gewurztraminer, Riesling, Colombard, Petit Manseng and botrytized Semillon confirmed the contribution of 4MMP, A3MH and 3MH to their characteristic aromas reminiscent of box tree, grapefruit, and passion fruit. 2FM, exhibiting a strong roasted coffee aroma, is present in white and red wines elaborated in new oak barrel at much higher concentrations than the perception threshold. 4MMP, 4MMPOH and 3MH can be released during alcoholic fermentation from their S-cysteine conjugate precursors. 2FM is generated in white wines during barrel fermentation. The formation is due to yeast transformation of furfural released from toasted staves.

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© 2004 American Chemical Society In Nutraceutical Beverages; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

315


Introduction Volatile thiols are extremely odoriferous molecules that probably contribute to the aroma of many foods, including fruit and grilled meat (1). Their positive contribution to the aroma of wines made from certain grape varieties has now been proven. However, the fact that these complex natural substances are only present in trace amounts makes them difficult to identify and assay. The analytical methods previously available involved tedious, complicated purification phases that were also rather nonspecific. We concentrated our investigations on the highly characteristic bouquet of Sauvignon blanc wines, with their broad palette of aromas featuring bell peppers, box tree, blackcurrant buds, grapefruit, passion fruit and, in some cases, smoke. This unusual bouquet had long attracted the attention of ampelographers, winemakers, and, of course, aroma chemists. The fact that at least two volatile thiols reminiscent of box tree have been found in the varietal aroma of Sauvignon blanc wines provides a basis for further research into the varietal aromas of non-Muscat grape varieties (2,3). We have spent several years working on the development of a specific method for extracting volatile thiols. We were particularly interested in the property of an organomercuric compound, /?-hydroxymercuribenzoic acid (pHMB), to form specific combinations with volatile thiols, which break down in the presence of excess quantities of other thiols, such as cysteine or glutathion (4). This property was first applied to identify a compound with one of the lowest perception thresholds in Sauvignon blanc wines, 4-mercapto-4methylpentan-2-one (4MMP), which has a odor reminiscent of box tree and broom (3). In the same way, Lavigne et al. (5) later demonstrated that two other volatile thiols, 2-mercaptoethyl acetate and 3-mercaptopropyl acetate, also contributed to the "roasted meat" aromas found in some Semillon and Sauvignon blanc wines. Improvement in our analytical techniques then made it possible to develop a method for extracting specific volatile thiols (6,7). The compounds responsible for the fruity nuances in Sauvignon blanc wines, e.g. passion fruit, grapefruit, etc., had not been identified until we carried out this research. We show that the trace amounts of volatile thiols present in wines contribute to the varietal aroma of certainF/tfj vinifera grape varieties. Furthermore, the volatile thiols that have been identified in wines are almost entirely absent from the corresponding must. The development of varietal aroma in wines made from non-Muscat grape varieties, such as Sauvignon blanc, during alcoholic fermentation remained a largely unexplained phenomenon. Indeed,

In Nutraceutical Beverages; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.


316 although work by Darriet et al, (8) had demonstrated the existence of a nonglycosylated precursor of 4MMP in Sauvignon blanc grapes and must, this compound was difficult to purify and had not yet been identified. Its existence explains the surprising phenomenon of "retour aromatique (aromatic aftertaste)", described by Peynaud (9) and well-known to winemakers. The characteristic aromas are initially barely perceptible when you eat a Sauvignon blanc grape or taste the must whereas, a few seconds or even a minute later, there is a much stronger aroma on the rear palate. The expression "aftertaste" is typically used to describe this delayed perception of the aroma of Sauvignon blanc must. The structure of the precursor in an extract of Sauvignon blanc must was identified thanks to the specific release of volatile thiols by an enzyme capable of cleaving the carbon-sulfur bond (10). Its structure was later confirmed by GC/MS analysis in the form of trimethylsylilated derivatives (77). In this work, we demonstrated the existence of a new chemical family of sulfur-containing aroma precursors in certain fruit. We have also recently demonstrated that furfural, an aldehyde released by toasted barrel wood, is a precursor of the furanmethanethiol found in certain wines (72). This is also the case in coffee (13,14). However, its development in wine depends on the fermentation activity of yeast rather than a transformation due to heat. The formation mechanism of this volatile thiol is discussed in relation to the yeast's sulfur metabolism.

Materials and Methods

Extraction, purification, and assay of the volatile thiols. The volatile thiols were specifically extracted and assayed using the method described by Tominaga et al (6,7).

Assay of hydrogen sulfide in fermenting medium: The hydrogen sulfide assay used in this study is based on the assay for this compound in "headspace" using GC/FPD. A volume of 50 μ ί phydoxypercuribenzoate sulfonium salt (p-HMBS) (1 mM in 0.2 M Tris) was added to 500 μΐ, fermenting must in an Eppendorf tube (2mL), then immediately centrifuged (4000 xg, lmin). The supernatant (275 μ ί ) was recovered in a 2mL vial, which was closed with a capsule. A 20 mM glutathion solution (125 \\L)


317 was introduced into the vial via a syringe to release H S. The vial was then incubated at 30 °C for 30 min. An 125 μΐ. sample was taken from the head-space using a syringe and injected into the GC/FPD, under the conditions described by Lavigne et al (16). Calibration charts were prepared (0-5nmol H S /vial) using a /?-HMBS-H S complex solution as follows: H S gas was put into 1 mL /?-HMBS (1 mM) in 0.2 M Tris for 5min, thenflushedwith nitrogen to remove any excess H S that had not combined with the p-HMBS. In die concentration range (0 to 5 nmol/vial), the standard curve was linear: [H S]nmol/250μL = 0.0058x - 0.1119, r = 0.9901. 2

2

2

2

2

2


2

Assay of S-3-(hexan-l-ol)-L-cysteine in a fermentation medium S-3-(hexan-l-ol)-L-cysteine was assayed in a model medium during fermentation using the method described by Tominaga et al. (11).

Microvinification The model fermentation medium consist of the following solution: 100 g glucose, 100gfructose,3 g tartaric acid, 0.3 g citric acid, 0.3 g malic acid, 2 g potassium phosphate, 0.3 g mesoinositol and 64 mg ammonium sulfate, 127 mg asparagine all in 1L of distilled water adjusted to pH 3.3 with solid potassium hydroxide, and vitamin solution (final concentration d-biotin 40 μg/L, thiamine hydrochloride lmg/L, pyridoxine hydrochloride lmg/L, nicotinic acid 1 mg/L, dpanthothenic acid hemicalcium salt 1 mg/L, /7-aminobenzoic acid 1 mg/L) was added. The fermentation medium for the first microvinification experiment, containing 64mg/L assimilable nitrogen with 15 mg/L added furfural, was supplemented with increasing quantities of nitrogen, by adding either asparagine (90, 140, 190 mg/L) or ammonium sulfate (60, 90, 140 mg/L). The model medium was fermented with Saccharomyces cerevisiae (strain Zymaflore VL3). The fermentation medium for the second microvinification experiment was supplemented with 15 mg/L added furfural, 5 mg/L cysteine, 2g/hL S02 and increasing quantities of nitrogen, by adding ammonium sulfate (21, 32 and 64 mg/L) and asparagine (42, 63 and 127 mg/L). The model medium was fermented with Saccharomyces cerevisiae (strain Zymaflore VL1). For the third microvinification experiment, the fermentation medium with 15 mg/L added furfural was fermented with two yeast strains (Saccharomyces cerevisiae VL3c and VS17). The assimilable nitrogen content was ajusted at 191 mgN/L by adding 64 mg/L of ammonium sulfate and 127 mg/L of asparagine.


318

Results and Discussion


Developing a specific method for extracting volatile thiols using hydroxymercuribenzoate (p-HMB) p-HMB has the property of forming reversible combinations with compounds containing a thiol function (4) (Figure 1). Volatile thiols can be extracted from an organic extract using a p-HMB solution, with which they combine. The original feature of the proposed method (5,7) consists of eliminating impurities by purifying the p-HMB extract using percolation through an anion exchange column (Dowex 1X2-100). The p-HMB, either free or combined with thiols, is fixed on the resin. The column is then rinsed and the volatile thiols are decombined by elution with a cysteine solution. Extraction of the volatile thiols using the method described above was compared to p-HMB extraction only, without percolation through a Dowex column. The peaks for the volatile thiols detected by GC/FPD (figs. 2-a,b) were practically identical, irrespective of the method used. This confirmed that there was no loss of volatile thiols during percolation and rinsing. Contaminant substances were assessed by GC/ FID. Comparison of the two chromatograms (figure 3-a and 3-b) showed that the method using Dowex resin eliminated contaminants (figure 3-a) that could otherwise perturb mass spectrometry identification and assay of the volatile thiols.

Identification of the volatile thiols in Sauvignon blanc wines The volatile thiols were specifically extracted from 0.5 L Sauvignon blanc wine, using the method described above, and analyzed by GC/O, GC/FPD, and GC/MS under the chromatography conditions described by Tominaga et al. (7). The two odoriferous zones with box tree and blackcurrant bud aromas, and a zone reminiscent of the aroma of roasted coffee detected in Sauvignon blanc wine by GC/O were easily perceptible on the gas chromatogram (2,7,17,18). GC/O had already detected these odoriferous zones reminiscent of box tree in Sauvignon blanc wines, with scores of linear relative retention indices, 1459 and 1650 respectively, using a BP-20 (17). One of these two compounds had been identified as 4-mercapto-4-methylpentan-2-one (4MMP) (3). This highlyodoriferous mercaptoketone has also been reported in Scheurebe wines (19) and, more recently, in grapefruit juice (20). The compound responsible for the second odoriferous zone reminiscent of box tree, but with more complex nuances of tropical fruit, was identified as 3-



Figure 2. Comparison of the GC/FPD analyses of the volatile thiols in a Sauvignon blanc wine extracted using two different methods: a) without percolation, b) with percolation.



320

Figure 3. Purity assessment by GC/FID of the volatile thiol extract obtained by two different methods: a) without percolation, b) with percolation.



321 mercaptohexyl acetate in 1996 (21). The new specific extraction method for volatile thiols made it possible to identify three new mercaptoalcohols in Sauvignon blanc wines: 4-mercapto-4-methylpentan-2-ol (4MMPOH), 3mercapto-3-methylbutan-l-ol (3MMB), and 3-mercaptohexan-l-ol (3MH) (22) (Table 1). None of these 4 compounds had previously been found in wine. One of these volatile compounds, 3-methyl-3-mercaptobutan-l-ol, is also found in roasted coffee (23). Engel and Tressl identified 3-mercaptohexan-l-ol and its acetate in passion fruit (24). To our knowledge, 4-mercapto-4-methylpentan-2-ol had never previously been identified in nature. The odoriferous compound corresponding to roasted coffee in certain red and white wines was more recently identified as 2-furanemethanethiol (2FM), using the more specific method for extracting volatile thiols (7). This volatile thiol is well-known in the aroma field for its distinctive roasted coffee odor and extremely low perception threshold. It had previously been identified in several food products: coffee, meat stock, and canned tuna (25-28). We report here that this volatile thiol has been identified for the first time in certain Vitis vinifera wines.

Assay of the volatile thiols identified in wines made from several white and red Vitis vinifera grape varieties The 6 volatile thiols in several Sauvignon blanc wines from Sancerre and Bordeaux were assayed in SIM mode (6,7) (Table 2). The 4MMP and 3MH concentrations were higher than their respective perception thresholds in all wines analyzed. Consequently, the aromatic indices (concentrations found in wine/perception threshold in model solution) (29) for these compounds was considerably higher than 1. 3MHA was no longer present in wines from older vintages. 4MMPOH and 3MMB concentrations were always below the perception threshold, irrespective of the age of the wines analyzed. It is, therefore, undeniable that 4MMP, with its box tree odor, and 3MH, reminiscent of grapefruit and passion fruit, have an impact on the aroma of Sauvignon blanc wines. 3MHA contributes to the box tree aroma in some young wines, but it hydrolyzes rapidly during bottle aging, or even barrel-aging on the lees (75). It is, however, unlikely that 4MMPOH and 3MMB contribute to the aroma of wines madefromthis grape variety. 4MMP is also found in large quantities in the two plants, box tree and broom (30). Thus, the terms "box tree" and "broom", long used to describe these wines, do, in fact, correspond to a chemical reality.


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Table I. Structure of the volatile thiols identified in Sauvignon blanc wines. Compound 4-Mercapto-4-methylpentan-2-one

Structure

(4MMP)

C H — C - CH2-C— ch3 1 II SH Ο

ÇH3 3

4-Mercapto-4-methylpentan-2-ol (4MMPOH)

ÇH3

C H — C - C H 2 - CH—CH 1 1 SH OH 3

3-Mercapto-3-methylbutan-l-ol (3MMB)

3

CH3

CH3—C—CH2 — CH —OH 2

SH 3-Mercaptohexan-l-ol (3MH)

CH3—CH2-CH2-CH-CH2-CH2-OH

3-Mercaptohexyl acetate (3MHA)

SH C H — C H - C H - C H - C H 2 - C H 2 - 0 - C — CH3 3

2

2

SH

2-Furanmethanethiol (2FM)

»

^o^CH SH 2

Table II. Organoleptic incidence of the volatile thiols identified in Sauvignon blanc wines. Compound Olfactory description 4MMP

n

Box tree,

Perception Concentration Aromatic threshold in wines (ng/L) index *

Harriet et al., 1995; Aromatic index = concentration in wine/perception threshold.



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These volatile thiols are present in wines madefromother Vitis vinifera grape varieties (31). For example, 4MMP and 3MH are present in particularly high concentrations in Alsatian Muscat d'Alsace and Gewurztraminer wines, respectively (Figure 4). Concentrations of 3MH are also considerably higher than the perception threshold in Petit Manseng, Gros Manseng, Colombard, and botrytized Semillon wines (31) (Table 3). The presence of 3MH has also been reported in red Cabernet and Merlot wines (32,33). Apparently, however, this volatile thiol has very little impact on the aroma of red wines. On the other hand, 3MH has recently been shown to add a fruity nuance to some Cabernet and Merlot rosé wines (results not shown) (34). 3MHA has been identified in young white Petit Manseng, Gros Manseng, and Colombard wines. White wines fermented in new barrels have a high 2FM content, irrespective of the grape variety, as shown for the Sauvignon blanc wines in figure 6. This volatile thiol is only present in trace amounts in white wines fermented in stainless-steel vats. The red wines analyzed did not systematically contain 2FM. Higher concentrations were found in wines aged in new barrels (Figure 5) (12). The concentration of this volatile thiol in red wines is, however, always lower than in white wines (Figure 6). However the concentration/perception threshold ratio of 2FM in certain red wines is quite high, making it likely that this compound contributes "roasted coffee" nuances to their aroma. Marchand et al. (35) recently reported much higher concentrations of this volatile thiol in certain red Pomerol and St. Emilion wines (70-350 ng/L).

Enzyme release of certain volatile thiols by β-lyase from Eubacterium limosum The surprising "aromatic aftertaste" phenomenon mentioned in the introduction is due to an aroma precursor in Sauvignon blanc. Previous research (8,18) had shown quite clearly that 4MMP could not be released from its precursor by the glycosidases capable of hydrolyzing glycosides, the precursors of monoterpenols. This indicates that the precursor of 4MMP is probably not a glycoside. Among the enzyme activities capable of cleaving a carbon-sulfur bond and releasing a thiol, our attention was attracted by a S-cysteine conjugate β-lyase (EC4.4.1.13). This enzyme, produced by an intestinal bacterium, Eubacterium limosum (36,37), catalyzes the cleaving of the thioether bond in a number of conjugates (S-alkyl- and S-aryl-) of L-cysteine, releasing mercaptan, as well as ammonium and pyruvic acid (Figure 6). The β-lyase activity of Eubacterium limosum is already used in aroma production to synthesize /?-mentha-8-thiol-3one from pulegon (38).



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Figure 4. 3MH and 4MMP concentrations in wines madefromAlsace grape varieties (n=5).

Table 111. Volatile thiol concentrations (ng/L) in wines made from several white grape varieties (n==4) Varieties

4MMP

A3MH

3MH

2FM

Petit manseng

0

5-100

1000-5000

10-60

Gros manseng

0

50-600

5000-10000

0

Colombard

0

20-60

400-1000

0

8-40

0

4000-5000

0-20

0

Nutraceutical Beverages - American Chemical Society

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