Analytical Investigations of Wine Odorant 3-Mercaptohexan-1-ol and

Chapter 2

Analytical Investigations of Wine Odorant 3-Mercaptohexan-1-ol and Its Precursors

Downloaded by COLUMBIA UNIV on July 17, 2012 | http://pubs.acs.org Publication Date (Web): July 16, 2012 | doi: 10.1021/bk-2012-1104.ch002

Dimitra L. Capone,*,1,2 Mark A. Sefton,2 and David W. Jeffery2 1The

Australian Wine Research Institute, P.O. Box 197, Glen Osmond, South Australia, 5064, Australia 2School of Agriculture, Food and Wine, Waite Research Institute, The University of Adelaide, PMB 1, Glen Osmond, South Australia, 5064, Australia *E-mail [email protected]. Phone +61 8 8303 6689. Fax +61 8 8303 6601

We have developed and applied methods for the analysis of wine odorant 3-mercaptohexan-1-ol (3-MH) and its precursors (including the newly identified cysteinylglycine conjugate) in grape juice and wine. Studies which assessed the effects of grape ripening and processing operations highlighted some important findings. We identified the presence of 3-MH in unfermented juice for the first time and found a dramatic increase in precursor concentrations in the later stages of ripening. We also revealed the effects on precursors from freezing, transportation, fining and inhibiting grape enzymes. Additionally, using labeled (E)-2-hexenal we propose the role of the glutathione-aldehyde adduct as the first intermediate in the formation of 3-MH.

Introduction Among the important grape-derived odorants contained in wine, one group of compounds – polyfunctional thiols – is predominantly associated with Sauvignon Blanc varietal character. The aromas of these “varietal” thiols have been described as “box tree”, “tropical” and “passion fruit” and they are important contributors to wine quality (1). The key thiols for Sauvignon Blanc wine aroma, 4-mercapto-4-methylpentan-2-one (4-MMP), 3-mercaptohexan-1-ol (3-MH) © 2012 American Chemical Society In Flavor Chemistry of Wine and Other Alcoholic Beverages; Qian, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

and 3-mercaptohexyl acetate (3-MHA), have extremely low aroma detection thresholds (Table I). The corresponding odor activity values (OAV) of these thiols, used as a measure of their sensory significance, can number in the hundreds. In particular, 3-MH and 3-MHA have frequently been found in concentrations well above their aroma detection thresholds in Sauvignon Blanc wines (2), especially those from France (3) and New Zealand (NZ) (4). As a result of their abundance and powerful aromas, varietal thiols in wine can influence consumer perception, affecting the level of preference for a particular wine (1, 4).


Table I. Characteristics of varietal thiols found in Sauvignon Blanc wine Aroma detection threshold

Aroma description

Concentration found in wine

Odor activity value

References

4-MMP

3 ng/L

Blackcurrant Box tree Passionfruit

Low ng/L

Up to 30

(2, 3, 5)

3-MH

60 ng/L

Grapefruit Passionfruit

Low ng/L to low μg/L

Up to 210 (310 for NZ wine)

(3, 4, 6)

3-MHA

4 ng/L

Passionfruit Box tree Sweaty

Low ng/L to low μg/L

Up to 195 (625 for NZ wine)

(3, 4, 7)

Since 3-MHA arises from 3-MH during fermentation (8), we focused on factors associated with 3-MH formation. Although often treated as one compound, 3-MH is present in wine as a mixture of enantiomers (Figure 1), each with different aroma detection thresholds and descriptors. (R)-3-MH has an aroma described as “grapefruit” with a threshold of 50 ng/L whereas (S)-3-MH has an aroma described as “passionfruit” and a threshold of 60 ng/L (9). Given their impact, it is essential to understand how these compounds are formed and factors that relate to their stability in wine in order to optimize wine sensory characters as desired.

Figure 1. Structures of the 3-MH enantiomers found in wine.

16 In Flavor Chemistry of Wine and Other Alcoholic Beverages; Qian, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Origins of 3-MH in Wine 3-MH can be generated from odorless precursors which are present in grape juice. The free thiol has not been found in unfermented juice in high concentrations as thiols are released by carbon-sulfur lyase (CSL) activity during vinification (10–14). 3-MH can be further modified by yeast acetyl transferase (ATF) enzymes to generate 3-MHA (8). Precursors to varietal thiol 3-MH, derived from cysteine (Cys-3-MH) (12) and glutathione (Glut-3-MH) (15) have been identified in Sauvignon Blanc juice. These precursors are present as pairs of diastereomers which each release the (R)and (S)-3-MH enantiomers. More recently, the cysteinylglycine conjugate of 3MH (Cysgly-3-MH), an intermediate precursor in the degradation of Glut-3-MH to Cys-3-MH, was identified in Sauvignon Blanc juices (16). As expected, based on its relationship to both Cys- and Glut-3-MH, this compound also exists as two diastereomers (Figure 2).

Figure 2. Structures of the diastereomers of Glut-, Cysgly- and Cys-3-MH found in grape juice. The stereochemical designations relate to the alkyl chain stereocenter.

A range of previous studies of precursors to 3-MH were limited to the cysteine conjugate (3, 11, 17–19) so we further probed the relationships between various 3-MH precursors in juice and 3-MH in wine. We investigated model fermentations of Cys- and Glut-3-MH with VIN13 and modified VIN13 yeast strains, revealing for the first time that yeast can also utilize the glutathione conjugate, leading to the formation of 3-MH (20). This work demonstrated that fermentation of pure (R)-Glut-3-MH resulted in an approximate 3% conversion to (R)-3-MH as a single enantiomer (Figure 3). (R)-Cys-3-MH was also formed during the transformation, presumably through the dipeptide intermediate 17 In Flavor Chemistry of Wine and Other Alcoholic Beverages; Qian, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


(R)-Cysgly-3-MH, but this remained to be confirmed. It appeared that Cys-3-MH was more easily transformed during fermentation compared to its Glut-3-MH counterpart, since the conversion yield of 3-MH from Cys-3-MH was in the order of 14% (20). This observation has since been supported by other studies (21–23).

Figure 3. Fermentation of a single diastereomer of Glut-3-MH ultimately leading to one enantiomer of 3-MH. Other intermediates could include the dipeptide Cysgly-3-MH. The (R)-designation relates to the alkyl chain stereocenter.

Determination of 3-MH Precursors in Juices and Wines Methods for the quantitation of 3-MH precursors in musts or wines had been confined to assessment of the cysteine conjugate (24), most often without resolving the diastereomers. Several methods have utilized GC-MS analysis of Cys-3-MH either indirectly (24) or after derivatization (17, 18, 25) while an HPLC-MS method has also been reported for determination of the unresolved Cys-3-MH diastereomers (26). We recently developed a stable isotope dilution analysis (SIDA) method for 3-MH precursors in juices and wines which resolved both diastereomers of Cysand Glut-3-MH using HPLC-MS/MS (27) and subsequently added Cysgly-3-MH to the method (16). This was the first method where the individual diastereomers of Cys-, Cysgly- and Glut-3-MH were determined in a single analysis. Resolution of diastereomers will be important when studying the evolution of 3-MH enantiomers during winemaking and storage. Cysteine and glutathione conjugates of 3-MH have also been analyzed by Roland et al. (28) using a nanoLC-MS/MS SIDA method (included conjugates of 4-MMP), Kobayashi et al. (21) using HPLC-MS/ MS without internal standard and Allen et al. (29) using SIDA and a modified procedure based on that of Capone et al (27). None of these methods resolved the diastereomers of the 3-MH conjugates. Some concentration ranges for Cys- and Glut-3-MH previously found in juice and wine appear in Table II. While there was good accord with Cys-3-MH concentrations in juice, Glut-3-MH varied considerably between the two reports; this could be due to differences in sample origin or preparation as described below. Analysis of a range of wine samples showed that significant quantities of precursors remained in wine (Table II). This might affect in-mouth release and retronasal perception of 3-MH upon wine consumption (30) or lead to liberation 18 In Flavor Chemistry of Wine and Other Alcoholic Beverages; Qian, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

of free thiols during storage. Furthermore, we found that Pinot Gris, Chardonnay and Riesling juices contained appreciable quantities of 3-MH precursors, but generally Sauvignon Blanc juices were highest (27). Roland et al assessed Melon B., Riesling, and Gewurztraminer juices as well as Sauvignon Blanc, and found that Gewurztraminer typically had the greatest amounts of 3-MH precursors, while Melon B. had the least (28).


Table II. Concentrations of 3-MH precursors determined for commercial Sauvignon Blanc juices

a

Capone et al 2010 Juice (27)a

Roland et al 2010 Juice (28)

Capone et al 2010 Wine (27) a

Cys-3-MH

21 – 55 μg/L

8 – 40 μg/L

1 – 35 μg/L

Glut-3-MH

245 – 696 μg/L

1 – 8 μg/L

138 – 142 μg/L

Sum of individual diastereomers for each precursor type.

Determination of 3-MH in Wines Due to their extremely low concentrations and reactivity, thiol compounds are difficult to measure at near-threshold levels in wine. A common method for extracting these compounds employs p-hydroxymercuribenzoate (p-HMB) solutions to selectively bind the thiols, followed by ion exchange chromatography (5). Although potential problems exist (p-HMB solutions are highly toxic and the methods involve complex extractions), different versions of the p-HMB extraction method have been proposed (31, 32). Other methods have employed derivatizing agents such as 2,3,4,5,6-pentafluorobenzyl bromide (PFBBr), with on-fiber (SPME) or in-cartridge (SPE) derivatization (2, 33, 34). However, routine adoption of these methods has not been forthcoming for various reasons, including problems with linearity, repeatability and sensitivity (34, 35). The methodology involving in-cartridge derivatization with PFBBr followed by SPME has again been improved upon (2, 35), but as with previous methods, the approach requires negative chemical ionisation (NCI) mass spectrometry for sensitivity. GC-MS instruments with NCI capability may not be available in many laboratories and an electron ionization-mass spectrometry (EI-MS) method was considered to be a useful option.

Development of a Quantitative 3-MH Method for Application to Juices and Wines We developed a modified SIDA method for analysing 3-MH in juices and wines for implementation in laboratories containing a GC with conventional EIMS, and eliminated the need for extraction with mercury complexes (36). By combining liquid-liquid extraction and PFBBr derivatization, followed by SPME 19 In Flavor Chemistry of Wine and Other Alcoholic Beverages; Qian, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

sampling of the headspace, we achieved excellent method precision (

Analytical Investigations of Wine Odorant 3-Mercaptohexan-1-ol and

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