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Keywords:

  • 4-methylguaiacol;
  • bushfire;
  • gas chromatography-mass spectrometry;
  • glycoside;
  • grapes;
  • guaiacol;
  • smoke;
  • wine

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

Background and Aims:  Taint in smoke-exposed grapes have been associated with elevated levels of guaiacol and 4-methylguaiacol. Previous research has reported guaiacol and 4-methylguaiacol in both fruits and wines. In some cases, these compounds were not detected, or were detected at low levels in the fruit while high levels were subsequently identified during or after winemaking. Later research indicated that this was due to the presence of glycosidic conjugates. Here we report a method for the routine analysis of guaiacol and 4-methylguaiacol released after acid hydrolysis of glycoside precursors.

Methods and Results:  Chardonnay, Merlot, Shiraz, Sangiovese and Cabernet Sauvignon fruits were collected following bushfire events in 2006–2007 in the King Valley wine region of NE Victoria, Australia. Gas chromatography-mass spectrometry (GC-MS) was used to detect free guaiacol and 4-methylguaiacol in both fruits and wines. Low levels of free and bound forms were present in fruit not exposed to smoke. Substantial levels of free guaiacol and 4-methylguaiacol were detected in the wines made from the smoke-affected fruits. These compounds increased during bottle storage. Acid hydrolysis of wines and berries resulted in a several-fold increase in free guaiacol and 4-methylguaiacol.

Conclusions:  The validated GC-MS method is suitable for monitoring free and glycosidically bound guaiacol and 4-methylguaiacol after acid hydrolysis in both fruits and wines. Acid hydrolysis of wines provided evidence that bound volatiles, most probably glycosidically, act as reserve for guaiacol and 4-methylguaiacol, which are released during ageing of wines.

Significance of the Study:  This is the first study published in a refereed journal to demonstrate that smoke taint-associated volatiles increase during ageing of wine and bound forms of guaiacol and 4-methylguaiacol represent an aroma reserve for smoke taint in ageing/bottled wines.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

Bushfires are a natural feature of the Australian environment (Gill 1979). While fire events are a necessary element in the life cycle of some native plants (Bell et al. 1993, Dixon et al. 1995), they can have a devastating effect on communities and industries in fire-prone regions (http://www.bom.gov.au/climate). In the grape and wine industry, the negative impacts of bushfires can also occur in areas not directly threatened by fire because of the effects of bushfire smoke (Whiting and Krstic 2007).

Smoke has been shown to directly impact grape composition and subsequent wine quality (Kennison et al. 2007). Wines made from smoke-affected grapes have been described as having smoky, dirty, earthy, burnt, smoked meat, damp fire and ashtray characteristics (Kennison et al. 2007, Whiting and Krstic 2007).

In grapes and wine, a number of compounds have been identified that have sensory characteristics similar to those used to describe wines made from smoke-affected fruit (Maga 1988, Kennison et al. 2007). Two of these, guaiacol and 4-methylguaiacol, have been widely used as indicator compounds in assessing the degree to which fruit and wines have been affected by smoke (Kennison et al. 2007, Whiting and Krstic 2007, Sheppard et al. 2009).

Guaiacol and 4-methylguaiacol are lignin degradation products (Maga 1984, Wittkowski et al. 1992). These are commonly found in wines that have been aged in oak barrels (Towey and Waterhouse 1996, Pollnitz et al. 2000). Guaiacol and 4-methylguaiacol may also be derived from corks (Simpson et al. 1986) and occur naturally in the fruit and leaves of some grape varieties, for example Shiraz, Merlot and Muscat of Alexandria (Sefton 1998, Wirth et al. 2001). At low levels, these compounds add complexity to wine flavour and aroma (Francis and Newton 2005), but at higher levels are undesirable and considered as a taint or fault in the wine (Boidron et al. 1988, Kennison et al. 2007). Boidron et al. (1988) reported that the detection thresholds for guaiacol and 4-methylguaiacol in red wines were 75 µg/L and 65 µg/L, respectively, and in white wines were 95 µg/L and 65 µg/L. Whereas, Simpson et al. (1986) reported a much lower detection threshold of 20 µg/L for guaiacol.

Smoke taint in wine occurs as a result of incorporation of smoke components into the fruit and their subsequent extraction into wine (Kennison et al. 2007, Sheppard et al. 2009). To avoid the loss of quality associated with smoke taint, fruits from smoke-affected vineyards tends not to be harvested; thus, these fruits represent a financial loss to growers and winemakers. For example, in 2006–2007, an estimated AUD$20 million worth of winegrapes, or approximately AUD$75 million worth of wine were lost in the north east of Victoria because of smoke taint (Whiting and Krstic 2007).

Following bushfire events, grape samples are routinely analysed for the presence of guaiacol and 4-methylguaiacol as indicators of smoke taint in the fruits as well as in the fermenting musts and finished wines (Jones 2009). It has been proposed previously that the concentration of these compounds increases during vinification because of the hydrolytic release of guaiacol and 4-methylguaiacol from their glycosylated forms (Sefton 1998, Wirth et al. 2001, Kennison et al. 2008). The hypothesis is supported by the recent finding that juice from smoke-affected grapes contained higher levels of glucosides of guaiacol and 4-methylguaiacol compared with unsmoked grapes (Hayasaka et al. 2010) and proposed that enzymatic hydrolysis is the major source of the release of smoke taint volatiles during fermentation. Analysis of the free guaiacol and 4-methylguaiacol in fruit and wines may substantially underestimate the level of potential smoke taint compounds in grapes and wine (Kennison et al. 2008).

While this initial work demonstrated an increase in guaiacol and 4-methylguaiacol during fermentation and anticipated increasing levels of these compounds in wines made from smoke-affected fruit during bottle ageing, no evidence of this has been published to date. Here, we report levels of guaiacol and 4-methylguaiacol in wines made from fruits exposed to smoke in the north east of Victoria during bushfires in 2006–2007 analysed immediately post-bottling and after 2 years of bottle ageing. In addition, we have adapted an existing analytical methodology (Zoecklein et al. 2000, Whiton and Zoecklein 2002) to enable routine analysis of the smoke taint potential represented by bound forms of guaiacol and 4-methylguaiacol in both fruits and wines.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

Chemicals

High-performance liquid chromatography grade acetonitrile, methanol, ethanol, sulphuric acid (H2SO4) and sodium hydroxide (NaOH) was purchased from Merck and Co. Inc. (Darmstadt, Germany). Guaiacol, 4-methylguaiacol standards and n-hexane (gas chromatography (GC) grade) were obtained from Sigma–Aldrich (St. Louis MO, USA). 4-ethyl-d5-2-methoxyphenol (d5-4-EG) was purchased from CDN isotopes (Pointe-Claire, QB, Canada). Purity of all standards was verified by GC-mass spectrometry (MS) before preparation of stock solutions. Deionised water was obtained through a MilliQ system (Milli-RX Analytical-Grade Water Purification System, Millipore, Billerica MA, USA).

Wine Samples 2006–2007 and 2008–2009

Wines were made from Vitis vinifera L. cv. Chardonnay, Merlot, Shiraz, Sangiovese and Cabernet Sauvignon fruits collected from the King Valley wine region of north eastern Victoria (36°42″ South, 146°25″ East), Australia. Fruits were collected in March 2007 following bushfire events in December 2006 and January 2007 (Table 1), and again in the March 2009 following bushfires events in February 2009. Because the region is incorporated within a phylloxera inclusion zone, fruits were frozen at –20°C for at least 7 days prior to shipping to a small-scale winery for winemaking or to the research laboratory for analysis of guaiacol and 4-methylguaiacol. Wines were made according to a standardised methodology (Walker et al. 1998). For the purposes of comparison, non-smoked fruits of Shiraz and Cabernet Sauvignon varieties (Table 1) from the 2006 vintage and wines prepared from these fruits were analysed for both free and bound forms of guaiacol and 4-methylguaiacol. In 2005–2006, there was no bushfire activity in the major Australian viticultural production areas.

Table 1.  Sites (or vineyards) and harvest °Brix of winegrape varieties, exposed to smoke as a result of bushfires in the 2006–2007 season in north eastern Victoria.
VarietyVineyard and location°Brix at Harvest
Cabernet Sauvignon (non smoked, control)Mildura21.4
34°42″ South 142°28″ East
Shiraz (non smoked, control)Mildura23.6
34°42″ South 142°28″ East
ChardonnayKing Valley23.9
36°41″ South 146°25″ East
ChardonnayWhitfield24.1
36°45″ South 146°24″ East
MerlotMoyhu24.7
36°34″ South 146°22″ East
MerlotKing Valley25.3
36°41″ South 146°25″ East
MerlotChestnut24.0
36°47″ South 146°25″ East
ShirazMyrrhee24.7
36°43″ South 146°19″ East
ShirazChestnut25.5
36°47″ South 146°25″ East
ShirazKing Valley26.7
36°41″ South 146°25″ East
ShirazChestnut South24.8
36°55″ South 146°23″ East
ShirazWhitfield27.4
36°45″ South 146°24″ East
SangioveseMyrrhee 36°43″ South 146°19″ East24.1
SangioveseKing Valley23.7
36°41″ South 146°25″ East
SangioveseWhitfield25.9
36°45″ South 146°24″ East
Cabernet SauvignonEdi Upper23.6
36°41″ South 146°29″ East
Cabernet SauvignonChestnut South21.8
36°55″ South 146°23″ East
Cabernet SauvignonKing Valley23.0
36°41″ South 146°25″ East

Wines prepared from 2006–2007 growth season were analysed for free guaiacol and 4-methylguaiacol by the Australian Wine Research Institute (AWRI; Adelaide, South Australia) as described by Pollnitz et al. (2004). This method has been reported to have a limit of detection (LOD) of 1 µg/L in both grapes and wines, with an uncertainty of ±1.0 µg/L or ±10% (whichever is greater). AWRI analysed wines on two occasions: 2 weeks post-bottling in June 2007 and in May 2009. These wines were also analysed in March 2010 by the method described below. Fruits collected at harvest in March 2007 were also analysed by the AWRI laboratory for free guaiacol and 4-methylguaiacol in April 2007 (Pollnitz et al. 2004).

Grape and wine samples (2008–2009 growth season) were analysed for both free and bound forms of guaiacol and 4-methylguaiacol according to the methodology described below.

Sample preparation

Whole berries.  Frozen whole berries were homogenised to a fine powder using a Retsch grinder (GM 200, Retsch, Haan, Germany) in the presence of liquid nitrogen. Ground berry material was transferred to a 50-mL polypropylene tube and stored at −80°C until analysed. For analysis, frozen berry material was thawed and centrifuged for 10 min at 2469 × g. Ten millilitres of the supernatant were transferred to a clean 15-mL tube to which was added 1.5 mL of 10 M NaOH before vortex mixing and filtration through a 0.45-µm syringe filter.

Wine.  A 20-mL aliquot of the wine to be analysed was frozen in liquid nitrogen and dried using a freeze dryer (Freezone, Labconco Corporation, Kansas City MO, USA) at −75°C. The dried sample was redissolved in 10 mL of deionised water, 1.5 mL of 10 M NaOH was added and then the samples passed through a 0.45 µm syringe filter.

Hydrolysis of guaiacol and 4-methylguaiacol bound precursors.  For the hydrolysis of bound forms of guaiacol and 4-methylguaiacol, the whole berry supernatant, or the freeze dried wine samples, were purified utilising solid phase extraction (SPE) adapted to a 2-mL 96-well plate system (Oasis® HLB Plate, Waters Corporation, Milford MA, USA) and vacuum manifold (96 Well Plate Manifold, Waters Corporation, Milford MA, USA). Solid phase columns were conditioned with 0.5 mL methanol followed by a rinse of 0.5 mL deionised water. For whole berries, 0.5 mL of sample, or 1 mL of wine sample was loaded in triplicate into wells and the liquid phase removed. Samples were rinsed three times with 1 mL aliquots of deionised water.

Solid phase plate columns were eluted with 0.17 mL ethanol (99.9%) and were rinsed with 0.33 mL water into a clean 2-mL 96-well plate. One millilitre of each of the three replicates of each sample was then transferred to 20-mL GC-MS headspace autosampler vials. To these was added 4 mL of H2SO4 (pH1.0). Autosampler vials were then sealed and incubated for 1 h at 100°C. Samples were cooled on ice and transferred to Kimble tubes (PYREX® Corning, New York, USA) containing 1.05 g NaCl. These samples were spiked with 10 µL of internal standard (4 mg/L d5-4-EG in ethanol) and vortexed for 1 min followed by incubation at room temperature for 30 min. Immediately after incubation 2 mL of n-hexane was added to the tubes and vortexed for 1 min. Samples were stored at room temperature for 2–3 h before spinning at 2469 × g for 5 min. A 1-mL portion of the organic phase was transferred to a 2-mL GC autosampler vial, capped and analysed for guaiacol and 4-methylguaiacol using GC-MS method described below.

Free forms of guaiacol and 4-methylguaiacol were measured by extracting 5 mL of the wine to be analysed in triplicate with 2 mL of n-hexane after spiking with 10 µL of d5-4-EG and adding 1.05 g NaCl similar to the sample procedure described above. d5-4-EG was used as an internal standard because purchase of suitable stable isotope labelled analogues of guaiacol and 4-methylguaiacol were not commercially available.

Gas chromatography mass spectral analysis of guaiacol and 4-methylguaiacol.  Grape and wine samples were analysed for guaiacol and 4-methylguaiacol using an Agilent 7890A gas chromatograph and 5975 mass spectrometer (Agilent Technologies, Palo Alto CA, USA). The column used was a fused silica capillary (DB-1701P, 0.25 mm I.D. × 30 m length × 0.25 µm film thickness, J&W Scientific, Folsom CA, USA). High purity helium (BOC Gases, Adelaide SA, Australia) was used as a carrier gas with an average linear velocity of 37 cm/s and a flow rate of 1 mL/min. Liquid samples (1 µL) were injected using a CTC-PAL autosampler (CTC Analytics AG, Zwingen, Switzerland) into front GC inlet injector at 240°C by using a 4-mm i.d. liner (Agilent Technologies, Palo Alto CA, USA). GC injector (inlet 1) was operated in the split/splitless mode with a splitless time of 1.5 min followed by a split flow of 50 mL/min. Oven temperature started at 40°C and held for 3 min before increasing to 50–100°C/min. Temperature was then increased by 4°C/min until it reached 180°C and then increased to 220°C at 80°C/min and held for 2.33 min. Under this temperature programme the analyte retention times (RT) were RT = 9.066 min for guaiacol, RT = 11.268 min for 4-methylguaiacol and RT = 13.228 min for d5-4-EG.

The MS ion source temperature was 230°C and the GC-MS transfer line temperature was 220°C. A solvent delay of 5 min was set up and data acquisition mode was set to selective ion monitoring mode. The ions monitored were m/z 81, 109 and 124 for guaiacol; m/z 95, 123 and 138 for 4-methylguaiacol; and m/z 124, 139 and 157 for d5-4EG (National Institute of Standards and Technology virtual library). The selected ions were monitored for 50 ms each.

Calibration standards and method validation.  Solutions containing 500, 250, 100, 50, 25, 10, 5, 2.5 and 1.0 µg/L guaiacol and 4-methylguaiacol were prepared in n-hexane. The calibration standard curves were prepared by transferring 1.0 mL of the mixed guaiacol/4-methylguaiacol solution to a 2-mL vial and adding 10 µL of d5-4-EG internal standard solution.

The specificity, precision and validation of the analytical method were determined by spiking a series of standards to red wines (Shiraz and Cabernet Sauvignon). The wines were spiked in triplicate with 0, 10, 20, 40, 80, 160 and 200 µg/L of mixed guaiacol and 4-methylguaiacol standards to determine recovery of these chemicals in n-hexane phase.

The values of LOD and limit of quantification (LOQ) of guaiacol and 4-methylguaiacol were determined by analysing n-hexane spiked with 1 µg/L of mixed guaiacol/4-methylguaiacol standards. Standard deviation (SD) from ten independent analyses was determined to calculate LOD (3 × SD) and LOQ (10 × SD) (Clesceri et al. 1995).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

Method validation

The parameters of the method were optimised using standards of guaiacol and 4-methylguaiacol in n-hexane. It was verified that these analytes presented rates of recovery and levels of detection compatible with their thresholds of perception and the concentrations expected in non-smoked fruit and wine (Table 2). The recovery of smoke taint compounds was >100% with relative SD (RSD) < 10% (Figure 1).

Table 2.  Concentration of G and 4-MG in grapes and wines exposed to smoke as result of bushfires during the 2008–2009 season in north eastern Victoria.
VarietySiteGrapesWine
Free G (µg/Kg)Free 4-MG (µg/Kg)Bound G (µg/L)Bound 4-MG (µg/L)Free G (µg/L)Free 4-MG (µg/L)Bound G (µg/L)Bound 4-MG (µg/L)
  1. The samples were analysed for free and bound G and 4-MG by the method described in materials and methods. Values represent mean ± standard deviation (n = 3); the limits of detection determined for G and 4-MG were between 0.32 and 0.38 µg/L. Control grapes and wines were not exposed to smoke. ND, not detected; G, guaiacol; 4-MG, 4-methylguaiacol.

Shiraz (control)MilduraNDND29.8 ± 2.0ND12.7 ± 1.2ND46.0 ± 6.3ND
Cabernet Sauvignon (control)MilduraNDND4.1 ± 0.2ND10.9 ± 2.7ND8.9 ± 1.1ND
MerlotKing ValleyNDND35.8 ± 1.8ND2.8 ± 1.1ND21.9 ± 0.8ND
ShirazMoyhu4.7 ± 0.8ND105.5 ± 3.75.0 ± 0.819.7 ± 0.6ND73.0 ± 0.2ND
SangioveseKing Valley0.9 ± 0.1ND38.2 ± 4.15.3 ± 2.16.8 ± 0.1ND27.9 ± 2.4ND
image

Figure 1. Recovery of guaiacol (G) and 4-methylguaiacol (4-MG) in n-hexane extracts after spiking of Shiraz wine with known amount of these analytes for each recovery level. The bars indicate standard deviation (n = 3).

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The detection limits (LOD) determined for guaiacol and 4-methylguaiacol were between 0.32–0.38 µg/L. The detection limits of the method allowed us to use it in routine analysis of guaiacol and 4-methylguaiacol. For guaiacol and 4-methylguaiacol, the LOQ was 1.27 µg/L and 1.06 µg/L, respectively. These values were close to the lowest concentration level of the working range.

The reproducibility and reliability of the method was evaluated by measuring free guaiacol and 4-methylguaiacol after acid hydrolysis of SPE-extracted bound forms of these compounds over 2 days by two operators (Table 3). The RSD obtained by the method described was less than 10% for all the analytes and no significant differences were observed between the sets of data produced by the two operators.

Table 3.  Validation of analytical method showing the concentration of guaiacol (µg/L) released after acid hydrolysis of purified glycosides of guaiacol in wine produced from smoke-affected Merlot winegrapes in 2008–2009 season in King Valley (North Eastern Victoria).
VarietySourceDayOperator 1Operator 2RSD (%)
  1. The samples were analysed in triplicate by two operators on two different days. Values represent mean ± standard deviation (n = 3). RSD, relative standard deviation.

MerlotKing Valley123.8 ± 0.223.3 ± 0.81.5
221.9 ± 1.523.0 ± 1.33.5

It was apparent that guaiacol and 4-methylguaiacol levels increased significantly after hydrolysis with H2SO4 at 100°C for 1 h (Figure 2). A time course experiment revealed that acid hydrolysis at 100°C for 30 min would be sufficient to release all of the bound form of guaiacol and 4-methylguaiacol into free forms for analysis by GC-MS.

image

Figure 2. Time course acid hydrolysis at 100°C to determine optimum time required to release bound forms of guaiacol and 4-methylguaiacol of Shiraz wine prepared from grapes exposed to bushfire smoke during the 2006–2007 growing season (n = 3).

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Analysis of free guaiacol and 4-methylguaiacol in new and aged wines

Wines were made from smoke-affected fruits collected after bushfires in Victoria (Australia), which occurred during the 2006–2007 growing season. Following bottling of the wines, made in a small-scale experimental winery, the wines were analysed for the presence of guaiacol and 4-methylguaiacol and then wines were re-analysed 2 years later (Figure 3). Guaiacol concentration in the fruits ranged from 6.9 µg/kg to 183 µg/kg of fruits, while 4-methylguaiacol concentration in the fruits ranged from 1.3 µg/kg to 28.7 µg/kg. No guaiacol or 4-methylguaiacol were detected in unsmoked control Shiraz and Cabernet Sauvignon grapes.

image

Figure 3. Concentration of guaiacol and 4-methylguaiacol in grapes (a), and guaiacol (b) and 4-methylguaiacol (c) in wines prepared from these grapes exposed to smoke as a result of bushfires in the 2006–2007 season in north eastern Victoria (King Valley (KV), Whitfield (W), Moyhu (M), Chestnut (C), Myrrhee (My), Chestnut South (CS), Edi Upper (EU)). Grapes were analysed following harvest in 2007 for free guaiacol and 4-methylguaiacol by Pollnitz et al. (2004) method. (n = 1). Wines were analysed for free guaiacol and 4-methylguaiacol immediately post-bottling in 2007 and 2 years later in 2009 (n = 1) by Pollnitz et al. (2004) method. Wines were subsequently analysed by the method described here for both free and bound forms of guaiacol and 4-methylguaiacol in 2010, values represent mean of analytical replicates (n = 3). Totals represent the sum of free and bound guaiacol analysed in 2010.

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In the wines measured 2 weeks post-bottling, the concentration of guaiacol ranged from 13 µg/L to 377.0 µg/L, and 4-methylguaiacol ranged from 5 µg/L to 82 µg/L. As with the fruits, the highest concentrations of both guaiacol and 4-methylguaiacol were observed in the Sangiovese wines, while the lowest concentrations were observed in wines made from Chardonnay grapes.

After 2 years of bottle ageing, wines were re-analysed and the concentrations of both guaiacol and 4-methylguaiacol in most of the wines had increased (Figure 3). The highest concentration of guaiacol was still observed in the Sangiovese wines and had increased by 10–15% during 2 years of bottle ageing. The highest concentration of 4-methylguaiacol at this time was observed in a wine made from Cabernet Sauvignon grapes which had an increase of around 45% in the concentration of 4-methylguaiacol. Indeed, two of the three highest concentrations of 4-methylguaiacol were in Cabernet Sauvignon wines; the third highest level was observed in Cabernet Sauvignon wine from the Edi Upper vineyard, which had increased by 67% during 2 years of bottle ageing. The biggest increase in guaiacol and 4-methylguaiacol concentrations during ageing was observed in the Chardonnay wines with an increase of over 200% in guaiacol and ∼300% in 4-methylguaiacol.

These wines were analysed in March 2010 for free guaiacol and 4-methylguaiacol by the GC-MS method described here; for most wines guaiacol and 4-methylguiacol had continued to increase. The Chardonnay and two of the Merlot wines remained below the sensory threshold for perception, with one of these Merlot wines also now close to that threshold (73.2 µg/L).

Analysis of bound precursors in grapes and wine

The method utilised to determine the concentrations of bound guaiacol and 4-methylguaiacol in grapes and wines was adapted from an assay previously used to determine the levels of flavour and aroma precursors in grapes and wine known as the glycosyl-glucose assay (Zoecklein et al. 2000). Here, we have applied that methodology to the purification of the glycosylated precursors of guaiacol and 4-methylguaiacol, the acid-catalysed hydrolytic release of guaiacol and 4-methylguaiacol and identification of the aglycones by GC-MS.

The methodology was applied to wines made from fruit exposed to smoke during bushfires in 2006–2007 and to wines made from fruits that had not been exposed to smoke (2005–2006).

Wines made from fruits exposed to smoke during bushfire events in 2006–2007 had detectable levels of guaiacol and 4-methylguaiacol when measured immediately post-bottling. It was subsequently observed that the concentration of guaiacol and 4-methylguaiacol had increased when these wines were analysed again in 2009 and 2010, indicating release of these compounds from a pool of conjugated precursors. To examine the pool of potential smoke taint compounds these wines were hydrolysed with acid to release the remaining guaiacol and 4-methylguaiacol. Concentrations of free guaiacol and 4-methylguaiacol detected after acid hydrolysis of purified glycosidic forms of guaiacol and 4-methylguaiacol for unsmoked (control) and smoked grape varieties are shown (Figure 3).

Following acid hydrolysis of the wines from 2006–2007, high concentrations of both guaiacol and 4-methylguaiacol were observed in all of the wines. In all cases, the concentrations were above the sensory thresholds for detection and in many cases the concentration was greater than ten times the threshold for sensory detection.

Different levels of guaiacol and 4-methylguaiacol were observed in the wines made from grapes, collected from various sites (Table 1), exposed to bushfire smoke during the 2006–2007 growing season. For example, in Chardonnay wines, the guaiacol and 4-methylguaiacol concentrations ranged from 192 µg/L (King Valley) to 262 µg/L (Whitfield) and 86 µg/L (King Valley) to 152 µg/L (Whitfield), respectively (Figure 3). These values include both the free form and that released by acid hydrolysis at the time of analysis in 2010 (Figure 3). Highest levels of guaiacol were found in Merlot (1196.8 µg/L) and Shiraz wines (1158.7 µg/L) made from fruits collected at Cheshunt site.

By comparison, wines made from fruits that had not been exposed to smoke had relatively low levels of the bound precursors of guaiacol. In Shiraz, the detected level of guaiacol was 46.0 ± 6.3 µg/L and in Cabernet Sauvignon, 8.9 ± 1.1 µg/L (Table 2). Furthermore, bound precursors for 4-methylguaiacol were not detected through acid hydrolysis of the wines.

To explore the levels of free and bound smoke taint precursors in fruits, the methodology was refined using grapes and the wines made from those grapes following bushfire events in 2008–2009, and also from fruits that had not been exposed to smoke (Table 2). No free or bound precursors of 4-methylguaiacol were detected in control fruits. But wine made from control fruit had low levels of free and bound forms of guaiacol. In contrast, smoke-exposed fruit and wines showed elevated levels of both free and bound precursors of smoke taint. Among the three varieties studied, the Shiraz fruits and wines had highest levels of free and bound guaiacol and 4-methylguaiacol (Table 2).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

A GC-MS based liquid injection method was developed to determine free analytical levels of guaiacol and 4-methylguaiacol after acid hydrolysis of purified precursors of taint compounds. The optimised conditions for releasing the free form of guaiacol and 4-methylguaiacol were hydrolysis with 2.5 M H2SO4 (pH 1.0) at 100°C for 30 min (Figure 2). It is still remained to be seen whether acid hydrolysis completely hydrolysed the precursors under experimental conditions used in this study. Nevertheless, these findings are consistent with recently published data (Hayasaka et al. 2010), where glycoside precursors were found to be the source of free taint compounds in smoke-exposed winegrapes. They reported that both acid and enzymatic hydrolyses released free guaiacol and 4-methylguaicol from glycosidic precursors.

The red wine matrix (Shiraz and Cabernet Sauvignon) did not have any significant impact on recovery, precision and specificity of the method for the detection of guaiacol and 4-methylguaiacol (Figure 1 and data not shown). The recovery of smoke taint compounds was better than 100% with RSD < 10% (Figure 1). This could be due to hydrolysis of soluble guaiacol and 4-methylguaiacol precursors at higher injector block temperatures as has also been reported previously especially when polar solvents were used at high injector temperatures (Pollnitz et al. 2004). Another reason for this discrepancy could be matrix enhancement of the GC response; usually from the active sites in the liner and column being shielded by compounds in the matrix resulting in a larger response for the target compounds. The RSD for the lowest concentration (i.e. 1.0 µg/L) of guaiacol and 4-methylguaiacol was 9–10% indicating high repeatability of the method. The LOD and LOQ values enabled us to identify and quantify guaiacol and 4-methylguaiacol routinely with 99% confidence. Furthermore, under analyses conditions (described in Materials and Methods), the expected values of controls and unknown samples determined by two operators were within <10% RSD.

Using this method, we have examined the levels of free and bound guaiacol and 4-methylguaiacol in a range of both smoke-affected and non-smoked grape and wine samples. Previous research has shown that smoke exposure alters the chemical composition and aroma of winegrapes and the wine products. Guaiacol and 4-methylguaiacol were the main compounds implicated in taint in smoke-affected fruit and wines. In this study we have demonstrated that the smoked grapes and resultant wines had enhanced levels of guaiacol and 4-methylguaiacol compared with wines and fruit that had not been exposed to smoke. In addition, we have shown that during 2 years of storage, the concentration of guaiacol and 4-methylguaiacol increased in bottled wines, providing evidence that the wines contained high levels of guaiacol and 4-methylguaiacol precursors, most likely as glycosides, acting as a pool of taint precursors. To our knowledge this is first demonstration of an increase in smoke taint during bottle ageing.

Grapes smoked, as a result of bushfires during 2006–2007 in the King Valley wine region of north eastern Victoria, had elevated levels of guaiacol and 4-methylguaiacol (Figure 3). These compounds were not detected in control non-smoked fruits. The highest concentrations of guaiacol were observed in Sangiovese and the lowest in Merlot. Although duration and intensity of smoke for all these sites needs to be determined, it is tempting to speculate that there is differential accumulation of smoke taint compounds based on variety. This hypothesis is further supported by high levels of guaiacol observed in Sangiovese (1000 µg/L) and Shiraz (1090 µg/L) wines, prepared from fruit collected at King Valley site, compared with Cabernet Sauvignon (522 µg/L), whereas, Merlot wines had intermediary levels of guaiacol i.e. 823 µg/L (Figure 3) at the same site. It would be interesting to compare smoke intensity and duration for all these sites and correlate it to the responsive phenological stage of each variety. Recently, 7days post-veraison was identified as a peak period of Merlot vine sensitivity to smoke (Kennison et al. 2009).

The 4-methylguaiacol concentrations were also highest in the Sangiovese fruits, while both Shiraz and Merlot had similarly low levels. The ratio of guaiacol and 4-methylguaiacol concentrations in fruits varied between 2.8–8.1, suggesting a differential absorption of guaiacol and 4-methylguaiacol into grape skin. The ratio resulting from combustion of different wood sources such as Ponderosa pine and Quercus sp. (oak) have been reported to range between 1.6 to 1.85 (Edye and Richards 1991, Guillén and Manzanos 2002).

Of the wines prepared from smoke-affected fruit, only the Chardonnay, Merlot and one of the Shiraz wines had guaiacol and 4-methylguaiacol concentrations below the sensory detection threshold (Figure 3). Rapp and Versini (1996) reported guaiacol to have a negative impact on wine aroma at concentrations exceeding 80 µg/L. Simpson et al. (1986) reported guaiacol to be responsible for an off-flavour in wine; the taint, originating from contaminated corks, was attributed to guaiacol levels ranging from 70 µg/L to 2630 µg/L with a detection threshold of 20 µg/L being reported in the study.

Wines produced from Shiraz and Cabernet Sauvignon winegrapes not exposed to smoke had low levels of guaiacol and 4-methylguaiacol (Table 2). It is possible that these levels could originate from other environmental factors or that guaiacol and 4-methylguaiacol are synthesised or accumulate to detectable levels in these cultivars. These compounds may exist in all varieties below the current level of detection. Previous studies have reported guaiacol as a glycoside in the berries of Tempranillo, Grenache (Lopez et al. 2004), Shiraz (Wirth et al. 2001), Merlot (Sefton 1998) and in Chardonnay juice (Hayasaka et al. 2010).

Analysis of 2006–2007 season wines in 2009 and 2010, showed significant increases in free guaiacol and 4-methylguaiacol (Figure 3) during ageing in bottles. Because these wines were never in contact with wood at any stage of winemaking or storage, the most likely source of additional free smoke taint analytes is the hydrolysis of bound forms of guaiacol and 4-methylguaiacol present in the wines at bottling. If the taint- and wood-derived precursors are the same, then data presented in this paper are consistent with previously published results where concentrations of free guaiacol and 4-methylguaiacol had been shown to increase in wines during cooperage in oak barrels (Sefton 1998, Wirth et al. 2001, Moreno and Azpilicueta 2007, Ortega-Heras et al. 2007). This increase in free guaiacol and 4-methylguaiacol had been suggested to be due to hydrolysis of glycosides of compounds present in the wood (Wittkowski et al. 1992, Kennison et al. 2008). Release of free guaiacol and 4-methylguaiacol from their precursors after acid hydrolysis in this study, supports this hypothesis. Concentrations of free 4-methylguaiacol increased in all the bottled wines during 3 years of bottle storage but remained lower than the sensory detection threshold and were always significantly lower than concentrations of free guaiacol (Figure 3). Because guaiacol levels increased substantially during storage and were above the sensory detection threshold, we suggest that for routine analysis, it would probably be sufficient for the wine industry to use only free and bound guaiacol concentrations as indicators of smoke taint and smoke taint potential in grapes and wine.

Concentrations of hydrolytically released guaiacol from its bound forms ranged from 141.3 µg/L to 1123.6 µg/L and 4-methylguaiacol ranged from 62.9 µg/L to 760.8 µg/L in the wines measured 2 years post-bottling (Figure 3). The highest concentrations of hydrolytically released guaiacol and 4-methylguaiacol were observed in Merlot and Shiraz, while the lowest concentrations were observed in wines from Chardonnay grapes. This observation was consistent with previous reports that wines made from white grapes tended to have lower levels of guaiacol and 4-methylguaiacol and that this was because of the absence of skin contact during winemaking with the wines being made from free-run juice (Kennison et al. 2008). This suggests that guaiacol and 4-methylguaiacol are concentrated in the skin (or seeds) of the berry.

The thickness of grape berry skin can vary between 3–8 µm and depending on vine variety; skins constitute 10–12% by weight of a mature grape berry. Recently, Sheppard et al. (2009) observed that thicker-skinned berries absorbed less guaiacol when exposed to smoke. Earlier observations of low levels of guaiacol and 4-methylguaiacol in white wines made without skin contact suggested that smoke taint compounds were confined to the skins of the fruit, possibly adhering to the cuticular wax of the berry (Høj et al. 2003, Whiting and Krstic 2007, Kennison et al. 2008, Sheppard et al. 2009). While this may be the case for the free analytes, it is possible that the skin is not the sole source of guaiacol and 4-methylguaiacol extracted from the berries and that high levels of the glycosylated precursors may be present especially in the mesocarp (flesh) of berries exposed to high smoke levels. This probably is the case in the samples analysed here as indicated by the significant increases in guaiacol and 4-methylguaiacol in Chardonnay wines upon acid hydrolysis (Figure 3). This is consistent with previous work where higher levels of these compounds were detected in the flesh of the berry because of the very high smoke exposure of grapes (Kennison et al. 2007). It would be interesting to analyse these compounds in each of the berry tissues (skin, seeds and flesh) separately to localise their distribution in the berry.

As discussed above, hydrolysis of the glycosylated precursors resulted in an increase in free smoke taint compounds in wines during bottle ageing (Figure 3). The presence of smoke taint precursors in wine has negative implications for the shelf life of bottled wines, although the full implications of this are uncertain because the rate of release is unknown. It would be necessary to study the kinetics of hydrolysis of these precursors in different wine matrices and under different storage conditions to develop a predictive model for release of guaiacol and 4-methylguaiacol and to determine whether an equilibrium between free and bound forms is reached. This information could then be used to determine shelf life of wines containing levels of free smoke taint compounds below the sensory threshold but with a significant pool of bound compounds. A further gap in our current knowledge is the extraction of both free and bound forms into the wine during fermentation or maceration. Together, these data could be used to evaluate the potential risk posed by a range of guaiacol levels in fruits at harvest.

As a corollary to this work, it is important to understand the smoke intensity and its duration in the field, resulting in levels of guaiacol that represent a significant risk. This risk will vary with variety and with wine style and with the expected shelf (or cellar) life of the wine. While there has been some research into the dose-response of grapes to smoke, this needs to be extended to include both bound and free forms of guaiacol in all of the varieties currently in production in smoke prone regions. Different winemaking styles will also need to be considered. This investigation must also include a network of smoke detectors in these regions that will both generate valuable research data and a risk management tool for the industry.

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

In this study, we demonstrated that guaiacol and 4-methylguaiacol accumulate in bottled wines, produced from smoke-exposed grapes, during ageing. GC-MS data strongly suggest that hydrolysis of precursors of guaiacol and 4-methylguaiacol is the most likely source of increase in smoke taint content during storage of bottled wines. Guaiacol was always present at concentrations considerably in excess of 4-methylguaiacol. The optimised and validated GC-MS method was demonstrated to be useful for quantitative analysis of smoke taint compounds in grape berries and wines. In an event of bushfire, this technique could be used to verify the results of sensory analysis, and to certify the criteria for acceptance or rejection of fruit by wine makers.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

The authors gratefully acknowledge the assistance of Mr Stephen Lowe of the King Valley Vignerons in the collection and transport of samples from bushfire-affected regions of NE Victoria. The authors also thank Mr Peter NanKervis (Agilent Technologies Australia) for his tremendous help during method development and Mrs Marica Mazza (DPI Vic.) for providing non-smoked grapes and wines for comparison. Analysis of free guaiacol and 4-methylguaiacol, in the 2006–2007 season grapes and wines, was conducted by the Australian Wine Research Institute (AWRI). The authors also recognise the invaluable support of Mrs Joanne Butterworth-Gray of the Victorian Wine Industry Association and Mr Liam Fogarty of the Victorian Department of Sustainability and Environment. This research was supported by E & J Gallo Winery, California (USA), the Victorian Department of Primary Industries and the Victorian Department of Sustainability and Environment.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References
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