Investigation of ‘stone fruit’ aroma in Chardonnay, Viognier and botrytis Semillon wines

Accepted Manuscript Investigation of ‘stone fruit’ aroma in Chardonnay, Viognier and botrytis Semillon wines Tracey E. Siebert, Sheridan R. Barter, Miguel A. de Barros Lopes, Markus J. Herderich, I. Leigh Francis PII: DOI: Reference:

S0308-8146(18)30353-4 https://doi.org/10.1016/j.foodchem.2018.02.115 FOCH 22499

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

25 October 2017 19 February 2018 21 February 2018

Please cite this article as: Siebert, T.E., Barter, S.R., de Barros Lopes, M.A., Herderich, M.J., Leigh Francis, I., Investigation of ‘stone fruit’ aroma in Chardonnay, Viognier and botrytis Semillon wines, Food Chemistry (2018), doi: https://doi.org/10.1016/j.foodchem.2018.02.115

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Investigation of ‘stone fruit’ aroma in Chardonnay, Viognier and botrytis Semillon wines Tracey E. Sieberta,b, , Sheridan R. Bartera, Miguel A. de Barros Lopesb, Markus J. Herdericha and I. Leigh Francisa,b a

The Australian Wine Research Institute, PO Box 197, Glen Osmond (Adelaide) SA 5064, Australia

b

University of South Australia, School of Pharmacy and Medical Science, City East Campus, Adelaide, SA 5000, Australia

Corresponding author’s email: [email protected]

Abstract Despite numerous studies, the identity of the compounds that are responsible for ‘stone fruit’ aroma in wine has not been conclusively established. This study focussed on wine varieties that often display peach and apricot characters, such as Chardonnay, Viognier and botrytis-affected sweet Semillon wines. Wines with high and low ‘stone fruit’ aroma were evaluated by gas chromatography-olfactometry-mass spectrometry (GC-O-MS) using extracts representative of the aroma of the wine in a glass. No aromaactive zone was described as ‘stone fruit’ aroma across all three wine varietals. However, for the individual varieties, terpenes, such as linalool and geraniol, in the Viognier wines, several esters in the Chardonnay wines, and γ-nonalactone in the botrytis Semillon were associated with ‘stone fruit’ aroma. Notably, this is the first study assessing the aroma profile of Viognier wine by GC-O. In addition, an extension study of Viognier grape monoterpene profiles clarified its classification as an aromatic variety.

Keywords  Corresponding author at: The Australian Wine Research Institute, P.O. Box 197, Glen Osmond (Adelaide), SA 5064, Australia. E-mail address: [email protected] (T. E. Siebert).

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Wine; aroma; flavour; GC-O-MS; stone fruit; Viognier; botrytis Semillon; Chardonnay

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1.

Introduction Different wine varieties have diverse flavours, and there can be large sensory differences within varieties, resulting from vineyard origin and winemaking or

viticultural practices (Ferreira & San Juan, 2011; Polášková, Herszage, & Ebeler, 2008). As wine is a high value beverage, understanding flavours that give distinct varietal characters or add to the appeal of a wine is beneficial to wine producers. The wine aroma and flavour descriptive term ‘stone fruit’ is often used by winemakers, wine judges and wine writers when describing specific wine varieties, especially for Chardonnay, Viognier and botrytis wines (Iland, Gago, Caillard, & Dry, 2009). Synonyms for ‘stone fruit’ include apricot, yellow or white peach, nectarine, and dried or canned apricot. Despite numerous studies on wine aroma, the identity of the volatile compound or compounds that might be responsible for ‘stone fruit’ aromas in wine has not been convincingly established. Consumers also appreciate the ‘stone fruit’ character in wines and a study of Chardonnay wines linked high consumer preference to the wines displaying ‘peach’ together with ‘tropical’, ‘melon’, and ‘confectionary’ characters (Saliba, Heymann, Blackman, & MacDonald, 2013). Another study comprising Chardonnay, Riesling and Sauvignon blanc wines found the majority of consumers preferred wines with ‘sweetness’, ‘pineapple’, ‘apricot’ and ‘pear’ flavours (Lesschaeve, Bowen, & Bruwer, 2012). Volatile aroma compounds make a large contribution to wine flavour (Polášková et al., 2008). Sometimes individual aroma compounds can have a strong contribution to wine flavour as so-called impact compounds, but more often the volatile composition of different wine varieties will only differ in the proportions of aroma compounds (Ferreira et al., 2011; Polášková et al., 2008). Some impact aroma compounds found in fruits and vegetables have also been found to be the impact aroma compounds that give the same aroma attributes in wines, for example 2-isobutyl-3-methoxypyrazine (IBMP) for ‘capsicum/bell pepper’ (Harris, Lacey, Brown, & Allen, 1987) and (S)-3mercapto-1-hexanol for ‘passion fruit’ (Tominaga, Darriet, & Dubourdieu, 1996). In fresh stone fruit, the n-alkyl γ-lactones (octa-, deca- and dodeca-) and δ-decalactone are considered to be impact aroma compounds (Belitz, Grosch, & Schieberle, 2009). Guth (1997a) conducted a pioneering study to determine and compare the aroma profiles of two white wines, the varieties Scheurebe and Gewürztraminer, using aroma extract dilution analysis (AEDA) and static headspace-gas chromatography-olfactometry (SHS-GC-O). In the study, 44 aroma compounds were identified. The main differences between the two wine varieties were two specific aroma compounds, cis-rose oxide, ‘rose water-like’, for Gewürztraminer and 4-mercapto-4-methyl-2-pentanone,

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‘box tree/blackcurrant-like’ for Scheurebe. Many studies have since used GC-O to investigate the aroma profile of numerous wine varieties and wine styles, with the more recent studies using mass spectrometry (MS) concurrently with GC-O (Ferreira et al., 2011). Some studies of different dry white wine varieties, including Chardonnay, Riesling, Albariño, and Sauvignon blanc, have reported ‘stone fruit’ attributes determined by GC-O and/or by sensory analyses (Benkwitz, Nicolau, Lund, Beresford, Wohlers, & Kilmartin, 2012; Lee & Noble, 2003; Schüttler, Friedel, Jung, Rauhut, & Darriet, 2015; Vilanova, Zamuz, Tardáguila, & Masa, 2008). Similar studies of botrytisaffected sweet white wines and sweet ice wines have also reported ‘stone fruit’ attributes (Genovese, Gambuti, Piombino, & Moio, 2007; Ma, Tang, Xu, & Li, 2017; Sarrazin, Dubourdieu, & Darriet, 2007). Little has been published regarding Viognier wine aroma composition (Zoecklein, Wolf, Pélanne, Miller, & Birkenmaier, 2008). Furthermore, no studies could be found that investigated ‘stone fruit’ or the more specific ‘apricot’ aroma attribute in Viognier wines, even though this variety can often have particularly strong ‘apricot’ flavour (Iland et al., 2009). Some of the wine studies involving GC-O-MS suggested that ‘stone fruit’ aromas in wine might be due to n-alkyl lactones (Ma et al., 2017; Sarrazin et al., 2007). However, the sample preparation methods for the GC-O for these studies involved large volumes of wine, with liquid- or solid-phase extraction followed by a marked concentration step and this might have led to a biased profile of volatile compounds in the extracts compared to that in the headspace of the wine (Abbott, Etievant, Langlois, Lesschaeve, & Issanchou, 1993; Le Fur, Mercurio, Moio, Blanquet, & Meunier, 2003). Quantitative analytical data has shown that generally sub-aroma detection threshold concentrations of the n-alkyl lactones are present in dry white wines (Cooke, Capone, Van Leeuwen, Elsey, & Sefton, 2009; Genovese et al., 2007; Langen, Wang, Slabizki, Wall, & Schmarr, 2013). However, the wines in these previous studies had not been specifically selected for their ‘stone fruit’ aroma attributes. The aim of this study was the identification of volatiles in Chardonnay, Viognier and botrytis Semillon wines that contribute to ‘stone fruit’ character. 2.

Materials and methods

2.1.

Reagents and standards Aroma compounds (≥ 93% purity) used as reference standards during GC-O-MS were supplied by Sigma-Aldrich (Castle Hill, NSW, Australia), 4-ethylguaiacol was

supplied by Apin Chemicals (Abingdon, UK), and 4-methylguaiacol was supplied by TCI (Chem-Supply, Gilman, SA, Australia). Compounds used as standards for

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quantitative GC-MS analyses, namely 1-hexan-d13-ol, monoterpenes and γ-lactones (C8–C12) were supplied by Sigma-Aldrich (≥97% purity), or synthesised previously inhouse, namely the d7-γ-lactones (C8–C12) (Cooke et al., 2009), d7-geraniol (Pedersen, Capone, Skouroumounis, Pollnitz, & Sefton, 2003), d6-linalool and d6-α-terpineol. Sodium dodecyl sulfate was supplied by BDH (VWR, Murrarie, QLD, Australia), ethanol was supplied by VWR, tartaric acid and sodium chloride were supplied by Merck (Bayswater, VIC, Australia), potassium hydrogen tartrate was supplied by Sigma-Aldrich. Model wine was prepared using saturated potassium hydrogen tartrate in 13.0% v/v aqueous ethanol, adjusted to pH 3.40 with tartaric acid (20% w/v). All chemicals were of analytical reagent grade unless otherwise stated; water was obtained from a Milli-Q purification system (Millipore, North Ryde, NSW, Australia). 2.2.

Wine selection Sixty-five commercially available wines, described as having ‘stone fruit’ aroma either on their back label or on the winery’s tasting notes, were assessed under blind

conditions by a group of experienced wine tasters (n = 6), with up to 12 wines evaluated per session. The wines were assessed in a dedicated open-plan sensory laboratory, and following independent assessment using free choice notes, the samples were discussed. Subsequently, a set of 10 wines was selected: four Australian Chardonnay; three Australian Viognier; one French Viognier; and two Australian botrytis Semillon. Half the wines chosen had high ‘stone fruit’ aroma intensity and the other half had negligible ‘stone fruit’ aroma. The selected wines were up to four years old. Six of the wines were donated by the wineries and four were purchased from wine retail outlets in Adelaide, South Australia. 2.3.

Grape samples

Eleven clones of Viognier (Vitis vinifera L.) were obtained from a single commercial vineyard block in the Eden Valley growing region of South Australia, Australia (34° 36' S, 139° 01' E), altitude 540 m. The grapevines were planted in 2005: average yield 6.25 T/ha; 2.75 m (row) and 1.6 m (vine) spacing; on own roots; cane pruned, 2 canes per vine; and vertical shoot-positioning wires with seven to nine rows of each clone, except one, which consisted of three rows. The grapevines were consistently managed across the block throughout each growing season. All clones were sampled immediately prior to harvest for three vintages (2014, 2015 and 2016), except for one clone (Yalumba 1)

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in 2015, as it had already been harvested earlier for commercial reasons. For each clone, ten bunches were taken from three separate zones within the rows (3 samples × 11 clones). The samples were held at 4 °C until the following day. The berries from each ten-bunch sample were removed by hand and randomised before sub-sampling for analysis of standard grape composition parameters (200 berries). The remaining berries were stored at ‒20 °C until required for GC-MS analysis. All 33 samples were analysed. 2.4.

Basic Wine and Grape Composition

The basic chemical composition of all wines and grape samples were determined by the Australian Wine Research Institute Commercial Services, using methods as detailed in Iland, Bruer, Edwards, Caloghiris, and Wilkes (2013) (Supporting information, Tables S1 and S2). For dry wines, pH, titratable acidity (TA), volatile acidity (VA), residual sugar (glucose + fructose), specific gravity and alcohol were measured using FTIR WineScan (FOSS, Hillerød, Denmark) and free and total sulfur dioxide (F/T SO2) using flow injection analysis (FIA; QuikChem 8000 Series; Lachat Instruments, Loveland , CO). For botrytised wines, pH and TA were measured with a combined pH meter and auto-titrator (TIM840; Radiometer Analytical, Dusseldorf, Germany), Free and total SO2 were measured by aspiration, residual sugar by enzymatic test kit (Randox, Paramatta, NSW, Australia), specific gravity by density meter, and VA and alcohol by distillation. For grape berry samples, pH and TA were measured with a combined pH meter and auto-titrator and total soluble solids (°Brix) was determined using a digital refractometer. 2.5. Gas Chromatography- Olfactometry-Mass Spectrometry (GC-O-MS) 2.5.1. GC-O Method Development To determine the most appropriate sample introduction, different enrichment and injection techniques were compared using an Agilent 7890A gas chromatograph (GC) (Agilent Technologies Australia Pty Ltd, Mulgrave, VIC, Australia), fitted with a Gerstel CIS 4 cooled injection system, and equipped with a Gerstel MPS2 XL multipurpose sampler, a Gerstel cryotrap system (CTS 2) and a Gerstel ODP 3 olfactory detection port (Lasersan Australasia Pty Ltd, Robina, QLD, Australia). Deactivated fused silica tubing (Agilent, dimensions of 2 m × 0.25 mm i.d.) was installed from the CIS 4 inlet, through the CTS 2 and then to the ODP. The carrier gas was helium (ultra-

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high purity, BOC, Adelaide, SA, Australia) at a constant flow rate of 6 mL/min for 1.05 min, decreased to 1 mL/min for 0.3 min, then increased to 12 mL/min and held at that flow. The oven and ODP transfer line were held at 220 °C. The CTS 2 was held at ‒100 °C for 1.6 min, then increased to 220 °C at 20 °C/s and held for 1 min. For static headspace (SHS) injection, the CIS 4 inlet was used in solvent vent mode with a Tenax TA filled liner installed (Gerstel, Lasersan Australasia Pty Ltd). A 10-mL aliquot of wine was added to a 20-mL glass, crimp cap, headspace vial (Agilent) and the sample was equilibrated at 40 °C for 10 min with agitation. The HS syringe was held at 60 °C and, after pressurising the vial, 1000 μL of the vial headspace was injected. The inlet temperature was held at 20 °C during injection, then increased to 300 °C at 10 °C/s and held for 2 min. For headspace solid phase micro-extraction (HS-SPME) injection, the CIS 4 inlet was used with an SPME liner (1.0 mm i.d.) installed. Four SPME fibre phases (1 cm) were compared: polyacrylate (PA), 85 μm, white; divinylbenzene/Carboxen/polydimethylsiloxane (DVB/CAR/PDMS), 50/30 μm, grey; PDMS, 100 μm, red; and DVB/PDMS, 65 μm, blue (Agilent). SPME fibre selection is discussed in Section 3.1. A 10-mL aliquot of wine was added to a 20-mL glass, screw-cap, SPME vial and the sample was equilibrated at 40 °C for 0.5 min. The fibre was exposed to the headspace for 10 min with agitation. The SPME fibre was desorbed in splitless mode and left in the inlet for 2 min. The splitter, at 10:1, was opened after 1 min. The inlet temperature was held at 250 °C. For post-chromatography assessment, a DB-5ms column (Agilent; dimensions of 60 m × 0.25 mm i.d. with 0.25 μm film thickness) was installed from the CIS 4 inlet and connected to deactivated fused silica tubing (Agilent, dimensions of 1 m × 0.25 mm i.d.). The deactivated fused silica section was threaded through the CTS 2 and then connected to the OPD. The carrier gas was helium (ultra-high purity, BOC, Adelaide, SA, Australia) at a constant flow rate of 2 mL/min for 14.5 min, decreased to 0.5 mL/min and held for 1 min, then increased to 4 mL/min and held for 2.5 min. The CTS 2 was held at ‒100 °C for 1.6 min, then increased to 220 °C at 20 °C/s and held for 1 min. The oven temperature started at 40 °C, held for 1 min, then raised to 250 °C at 20 °C/min, and held at that temperature for 7 min. The ODP transfer line was held at 250 °C. The instrument was controlled with Agilent G1701EA ChemStation software in conjunction with Gerstel Maestro software (Version 1.4.8.14). 2.5.2. GC-O-MS Analysis

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Gas chromatography-olfactometry-mass spectrometry (GC-O-MS) was performed utilising two separate instruments. Instrument 1 was an Agilent 6890 GC, fitted with a standard split/splitless inlet, coupled to an Agilent 5973N mass selective detector and equipped with a Gerstel MPS2 multipurpose sampler and a Gerstel ODP 2 olfactory detection port (Lasersan Australasia Pty Ltd). The instrument was controlled with Agilent G1701DA ChemStation software in conjunction with Gerstel Maestro software (Version 1.3.3.51/3.3). Instrument 2 was an Agilent 7890A GC, fitted with a standard split/splitless inlet, coupled to an Agilent 5975C mass selective detector and equipped with a Gerstel MPS2 XL multipurpose sampler, a Gerstel cryotrap system (CTS 2) and a Gerstel ODP 3 olfactory detection port (Lasersan Australasia Pty Ltd). The instrument was controlled with Agilent G1701EA ChemStation software in conjunction with Gerstel Maestro software (Version 1.4.8.14). For GC-O-MS analysis, both DB-Wax and DB-5ms columns (Agilent, dimensions of 60 m × 0.25 mm i.d. with 0.25 μm film thickness) were used. A deactivated glass Y connector (Agilent) was installed at the end of the analytical column, approximately 1.2 m of 0.22 mm i.d. deactivated fused silica tubing (Agilent) was connected from the Y connector to the ODP, and approximately 2 m of 0.11 mm i.d. deactivated fused silica tubing (SGE Analytical Science Pty Ltd, Ringwood, VIC, Australia) was connected from the Y connector to the MS. The carrier gas was helium (ultra-high purity, BOC, Adelaide, SA, Australia) and constant pressure mode set to 183 kPa (nominal initial flow 1.8 mL/min) was used. This resulted in the flow being split between MS and ODP in a 1:1 ratio. The mass spectrometer quadrupole temperature was set at 150 °C, and the source was set at 230 °C. The ODP and MS transfer lines were held at 250 °C. Positive ion electron impact spectra at 70 eV were recorded in the range of m/z 35−350. For HS-SPME injection, a DVB/CAR/PDMS (2 cm) 50/30 μm SPME fibre (Agilent) was exposed to the headspace of the sample for 30 min at 40 °C, with agitation. The SPME fibre was desorbed in the splitless mode and left in the injector for 10 min. The splitter, at 29:1, was opened after 1 min. The injector temperature was held at 270 °C. The oven temperature started at 40 °C, held for 5 min, then raised to 250 °C at 5 °C/min, and held at that temperature for 10 min. The GC-O-MS parameters were based on those used by Mayr, Geue, Holt, Pearson, Jeffery, and Francis (2014). For individual aroma compounds, different sensitivities between people have been observed, resulting in a range of aroma detection thresholds across a group from very sensitive to anosmia effects (Langen, Wegmann-Herr, & Schmarr, 2016). Thus, a GC-O sensory panel of four assessors was used. The assessors (n = 4) were staff that had extensive previous experience with sensory evaluation of wine, being members of the descriptive analysis panel, and with GC-O assessments. Assessors initially evaluated wines from the study, presented in wine glasses (ISO, 30 mL), to familiarise themselves with ‘stone fruit’ attributes. The assessors evaluated two additional wines

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by GC-O for training purposes, a wine spiked with a mixture of wine aroma compounds and a wine considered to have strong ‘apricot’ character. The panel members were given free choice in their use of aroma descriptors to allow for potential unknown compounds or unexpected aromas. To minimise fatigue, two panellists alternately ‘sniffed’ in 5-minute segments of the run and then repeated the next GC run in reverse order to ensure each panellist evaluated the entire GC run. The pairing of panellists was switched around for each wine. When each panellist detected an aroma, they described the aroma and rated its intensity as very low, low, medium-low, medium, mediumhigh or high intensity. Subsequently, the intensity ratings were ascribed a numerical score of 0.5, 1, 1.5, 2, 2.5 and 3 respectively. If a particular aroma was not detected by an assessor, then it was given a score of zero. As an indicator of the averaged aroma strength across the panel for each aroma, the modified frequency percentage (MF (%)) was calculated (Campo, Ferreira, Escudero, Marqués, & Cacho, 2006): MF (%) = (F (%) × I (%))1/2 where F (%) = the detection frequency percentage of the assessors, and I (%) = the average intensity expressed as percentage of the maximum intensity. Data analysis was performed using MassHunter Qualitative Analysis software (Agilent, Version B.07.00). Aroma compound identity was achieved by chromatogram deconvolution and comparison to mass spectral libraries (NIST11, Wiley275) then comparing each compound’s calculated linear retention index (LRI) to reference compounds or to that found in the literature. 2.5.3. Semi-quantitative analysis of aroma compounds GC-MS semi-quantitative analysis was performed using Instrument 2 as described above, except the analytical column was connected directly to the MS. Sample preparation, extraction conditions and GC–MS method were the same as above (Section 2.5.2) except 100 μL of 1-hexan-d13-ol (491 mg/L) were added as internal standard to each vial, with 10 mL of wine. Both splitless and split (1:10) injection modes were employed for each column type. The MS was used in simultaneous scan and selected ion monitoring (SIM) modes to allow for more sensitive screening of the samples for γ-lactones. MS data were recorded for scan mode in the range of m/z 35−350 and for SIM the ions monitored were m/z 85 and 128. 2.5.4. Targeted analysis of γ-lactones in wines

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The n-alkyl γ-lactones (C8–C12) were quantified in the wines using GC-MS stable isotope dilution analysis (SIDA) in selected ion monitoring mode after sample clean-up and concentration by solid-phase extraction (SPE) (Cooke et al., 2009). Briefly, 50 mL of wine were spiked with d7-n-alkyl γ-lactone analogues as internal standards and the sample was loaded onto a pre-conditioned Bond Elut-ENV cartridge. After washing and drying the cartridge, the analytes were eluted with dichloromethane (2 mL), dried with MgSO4 and concentrated under N2. Additionally, the MS was used in simultaneous SIM/scan mode and extra ions were monitored (m/z 71, 99, 114) to determine if any δ-lactones were present in the wines. Instrument control and data analysis were performed with Agilent ChemStation software (E.02.02.1431). 2.5.5. Targeted analysis of free monoterpenes The free monoterpenes, linalool, geraniol and α-terpineol, were quantified in the Viognier wines using a previously published method (Pedersen et al., 2003). The Viognier clone berry samples were analysed using HS-SPME-GC-MS with SIDA in SIM mode, incorporating elements from previously published methods (Pedersen et al., 2003; Perestrelo, Barros, Rocha, & Câmara, 2011). A GC-MS instrument equivalent to Instrument 1, fitted with a Peltier-cooled tray holder (Gerstel), was utilised. Destemmed berries (100 g), partially thawed, were homogenised using a hand-held stick blender (Breville Wizz Stick). Grape berry homogenate (7 g) was added to a 20-mL glass, screw-cap, SPME vial (Agilent) together with sodium dodecyl sulfate solution (20% w/v, 100 µL), sodium chloride solution (saturated, 3 mL), and IS solution (d6linalool; d7-geraniol; and d6-α-terpineol, 10 mg/L, 35 µL). After vortexing the vial, the sample vials were placed in the Peltier cooler tray and held at 4 °C. Immediately prior to injection, the sample was equilibrated at 80 °C for 1 min. The SPME fibre (DVB/CAR/PDMS, grey, 2 cm) was exposed to the headspace for 20 min with agitation, desorbed in pulsed splitless mode (110 kPa) and left in the inlet for 10 min. The splitter, at 42:1, was opened after 2.1 min. The injector temperature was held at 200 °C. A DB-5ms column (Agilent, dimensions of 30 m × 0.25 mm i.d. with 0.25 μm film thickness) was installed with the carrier gas at a constant flow rate of 1.2 mL/min. The oven temperature started at 40 °C, held for 2 min, raised to 200 °C at 5 °C/min, then raised to 280 °C at 100 °C/min and held at that temperature for 7 min. The ions monitored were: m/z 93, 124 and 142 for d6-linalool; 93, 121 and 136 for linalool; 65, 124 and 142 for d6-α-terpineol; 121, 136 and 139 for α-terpineol; 69, 99 and 161 for d7-geraniol; 93,121, and 154 for geraniol. The underlined ion for each compound was used for quantitation and the other ions were used as qualifiers. Calibration functions were obtained

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by a series of duplicate standard additions of all compounds (0 to 200 μg/L, n = 8 × 2) to glucose and fructose solution (100 g/L each) and were linear throughout the concentration range. Data analyses were performed using MassHunter Quantitative Analysis software (Agilent, Version B.07.01). 2.6. Qualitative sensory evaluation of γ-lactones A sensory evaluation session using model wine was conducted by combining comparable amounts of the highest concentrations of the n-alkyl γ-lactones (C8–C12) quantified in the selected Chardonnay and Viognier wines. γ-Nonalactone was also spiked into model wine individually at three concentrations (5, 25, 50 µg/L). The spiked samples were assessed by a group of experienced wine tasters (n = 5) in comparison to the base model wine medium under conditions as described above. 2.7. Statistical analysis Chemical data were analysed using the statistical package Minitab 18 (Version 18.1, Minitab Inc., State College, PA). Analysis of variance (ANOVA) and Tukey HSD test were used to interpret the differences in means (p = 0.05). 3.

Results and discussion

3.1.

GC-O method development GC-O is a powerful technique for investigating the aroma profile of wine and identifying the aroma-active components therein (Ferreira et al., 2011). In this study, it

was imperative that a representative sample of the whole wine aroma, that which can be smelled in the wine glass, complete with its ‘stone fruit’ attribute, survived the sample preparation and the GC analysis (Abbott et al., 1993; Le Fur et al., 2003). Rega, Fournier, and Guichard (2003) compared the aromas of total orange juice SPME extracts to the aroma of orange juice to assess which best represented the juice aroma. The entire volatilised injected sample was released to the olfactometry port all at once and, thus, sniffed altogether. For this current study, GC parameters were chosen to minimise the width of the eluting band of aroma compounds to the ODP nose cone with a short, fused silica column fitted rather than an analytical capillary column. A

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high He flow rate (6 mL/min) was set on the GC because the actual He flow, measured at the end of the fused silica tubing, was only 2 mL/min. The wine aroma compounds volatilised in the GC inlet were cryo-trapped and then quickly re-released from the CTS 2. Decreasing the He flow (to 1 mL/min) while the CTS 2 was heated and then increasing the He flow (to 12 mL/min) allowed for a narrow aroma band width (15 sec). Two experienced wine tasters with GC-O expertise assessed two wines, both with high levels of ‘stone fruit’ character, in the glass and compared their aromas to that of the entire GC-O aroma band for each of the different sample preparation techniques. The aroma band released was considered to represent well the aroma from a glass of wine as determined by the two assessors. When comparing the four SPME fibre phases (1 cm): DVB/CAR/PDMS, 50/30 μm; PDMS, 100 μm; DVB/PDMS,65μm; and PA, 85μm, the aroma produced from the SPME injection using a DVB/CAR/PDMS fibre most closely resembled the wine in the glass. The aroma produced from the SHS injection also resembled the wine aroma in the glass but was less intense and had a burnt note. An added benefit of using a DVB/CAR/PDMS fibre was that it is available in a 2-cm option and that enhances the amount of volatiles extracted and overcomes potential sensitivity issues due to the loss of half of the effluent when the carrier gas flow is split (1:1) post GC column between the ODP and the MS. Therefore, HS-SPME using a DVB/CAR/PDMS fibre (2 cm) was the chosen technique for the GC-O-MS study. The initial volume of wine sample and vial (10 and 20 mL respectively) as well as temperature of incubation and fibre exposure time were chosen based on previous studies in-house and by other researchers (Martí, Mestres, Sala, Busto, & Guasch, 2003). Different fibre exposure times were compared (15, 30 and 45 min) and 30 min was the most suitable. After the most suitable HS- SPME parameters had been established, a post-chromatography sensory assessment of the total effluent from the DB-5ms analytical column was conducted. The aroma was perceived as slightly less fruity but was still representative of the wine in the glass and retained clear ‘stone fruit’ character. 3.2.

GC-O identification For this study, each GC-O panellist assessed each of the 10 wines on two different types of GC capillary column phases, non-polar and polar, to assist in identifying

and overcoming any masking or mixing of aromas due to co-elution of peaks. Excellent repeatability between the chromatography runs was found by overlaying the chromatograms of repeated samples. From the semi-quantitative GC-MS scan data, the peak area of the IS was consistent between the dry wines (< 10% RSD). The IS peak

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areas for the botrytis Semillon samples were higher than in the dry wines due to lower ethanol and higher residual sugar content affecting the headspace partitioning of volatiles but were consistent between the two samples. Aroma profiles of the wines were evaluated by GC-O-MS. More than 100 discrete aromas were detected by two or more assessors in any wine. Generally, aroma compounds were identified based on their calculated linear retention index (LRI), mass spectral library match and aroma match compared to reference compounds or literature. Table 1 highlights the 56 aroma compounds considered important to this study because they were detected in more than one wine, except for botrytis Semillon, and lists each compound’s LRI (wax and DB-5), aroma descriptor and averaged aroma strength, calculated as MF (%). A further 15 compounds could not be identified. Unfortunately, no ‘stone fruit’ aromas were consistently described at any aroma-active region by the panellists across all the wines considered to be high in ‘stone fruit’ character. This could mean that, in some of the wines, the aroma for ‘stone fruit’ was masked by another aroma or that a combination of compounds was required to make up ‘stone fruit’ aroma. Interestingly though, both the high and low ‘stone fruit’ botrytis Semillon wines had an aroma-active region described as medium intensity ‘peach’, ‘nectarine’. The corresponding GC-MS peak was identified as γ-nonalactone and was only evident in the high ‘stone fruit’ botrytis Semillon sample. Numerous volatile aroma compounds, such as fermentation derived aroma compounds, methional, β-damascenone, α-terpineol, limonene and vitispirane, were detected in all the wines by GC-O but with differences in aroma intensities. Differences between the GC-O profiles of the wine varieties were observed: geraniol aroma was only smelled in the Viognier wines; 3-isobutyl-2-methoxypyrazine and γ-nonalactone were only recognised in botrytis Semillon; ethyl heptanoate and furfural were only perceived in the high ‘stone fruit’ botrytis Semillon; no aromas were unique to Chardonnay wines but linaloyl oxide, linalool and 2-phenylacetate were not smelled in the Chardonnay wines.

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Table 1 Aroma compounds detected by GC-O-MS in Chardonnay, Viognier and botrytis Semillon wines with high and low ‘stone fruit’ attributes; GC retention indices, aroma descriptors, compound identity and modified frequency (%) LRIa DB-wax

Aroma Descriptorsb

Identityc

Important to Viognier 1110 971 1549 1102 1869 1256 1288 1090 1530 969 1638 812 1652 1392 2165 1175 1530

Viognier

Compound

DB-5

Chardonnay

B. Semillon

H-1

H-2

L-1

L-2

H-1

H-2

L-1

L-2

H

L

A, MS, RI A, MS, RI A, MS, RI A, MS, RI A, MS, RI A, MS, RI A, MS, RI

Linaloyl oxide Linalool Geraniol δ-Terpinene Benzaldehyde Butanoic acid Ethyl decanoate ni ni ni

41 71 50 29 58 58 50 54 41 41

41 74 29 65 41 29 54 54 50 58

29 68 29 50 41 61 68 58 29 41

65 29 − 41 29 58 50 50 58 29

− − − 58 41 50 − 32 41 −

− − − − − − 46 58 − −

− − − 84 41 − − 61 29 41

− − − 67 − 29 54 − − −

46 − − 71 58 50 − − 29 35

46 50 − − 50 61 − − 50 29

A, MS, RI A, MS, RI

Ethyl pentanoate cis-Rose oxide ni ni

35 58 50 76

29 74 29 46

− − − −

− − − −

− 54 41 −

50 − 41 −

50 41 58 −

41 − 29 −

74 41 41 −

50 41 − −

Not detected in Viognier 2256 1990 Honey, toast

A, MS, RI

Ethyl hexadecanoate

−

−

−

−

41

50

41

29

0

29

Only detected in botrytis Semillon 1333 1098 Tropical juice, honey 1477 839 Savoury, tomato dust 1539 1181 Green capsicum

A, MS, RI A, MS, RI A, RI

Ethyl heptanoate Furfural 3-Isobutyl-2methoxypyrazine γ-Nonalactone ni ni

− −

− −

− −

− −

− −

− −

− −

− −

58 65

− −

−

−

−

−

−

−

−

−

41

54

− − −

− − −

− − −

− − −

− − −

− − −

− − −

− − −

50 71 65

58 − 29

2-Phenylethyl acetate

50

−

74

41

−

−

−

−

50

29

ni ni

− −

− −

− −

29 −

41 43

29 41

− −

− −

− −

58 −

Floral, latex Floral, citrus leaf Floral - rose Chemical (sweet), confectionary Dried apple Cheese Tropical juice, lemonade Confectionery - raspberry, bubblegum Cedar, citrus, green leaf Leather

Present in high 'stone fruit' Viognier 1135 900 Confectionery - raspberry 1362 1113 Floral - rose water 1141 Floral, confectionary 1481 Waxy, citrus, strawberry

2064

1368 931 1468

Peach, nectarine, tobacco Talc, Geranium Green - leaf, grass

Not detected in Chardonnay 1830 1260 Floral - rose petal Present in high 'stone fruit' Chardonnay 2223 Floral, confectionary 2341 Savoury, smoke

A, MS, RI

A, MS, RI

14

Table 1 (continued) LRIa DB-wax

Aroma Descriptorsb

Identityc

Viognier

DB-5

Present in low 'stone fruit' Chardonnay 1180 Floral, confectionary 1186 Cheese, sweat 1735 985 Savoury, boiled potato 2057 1279 Spice, fruit cake, toffee Detected in all wines (n = 9 or 10) 650 450 Putrid, cabbage, egg 973 765 Confectionery - raspberry, apple 1042 800 Confectionery - raspberry, banana 1060 854 Confectionery -banana, apple 1074 861 Confectionery - musk, apple 1124 881 Confectionery - banana, bubblegum 1218 745 Cheese, sweat 1235 998 Tinned pineapple 1188 1030 Confectionery - pineapple, citrus 1446 1201 Fruit 1456 633 Vinegar 1467 908 Cooked potato 1551 1293 Floral, hot metal, green leaf 1680 862 Cheese, sweat 1711 1211 Floral, pine, minty 1788 1282 Confectionery - raspberry, apple 1848 1386 Stewed apple, iced-tea 1884 1089 Smoky bacon, smoky wood 1935 1122 Floral, rose, green 2198 1360 Smoky bacon, sweet spice 2227 1119 Curry, dusty rose, cinnamon 2284 1384 Leather, dusty 1908 Confectionery - strawberry, raspberry 825 Solvent 1417 Hot metal, green, geranium Detected in many wines (4 ≤ n ≤ 8) 716 427 Bruised apple 896 616 Fruit, ether 919 Smoke, savoury 990 596 Caramel, butter 968 714 Plastic 1167 844 Fruit - tropical 1275 1013 Floral, pineapple

Chardonnay

B. Semillon

Compound H-1

H-2

L-1

L-2

H-1

H-2

L-1

L-2

H

L

ni ni Methionol 4-Ethylguaiacol

29 − 50 74

− − − 58

− 29 50 46

29 − − 61

− − − −

− − − −

29 29 71 87

41 29 71 53

41 35 50 74

41 41 29 50

A, MS, RI A, MS, RI A, MS, RI A, MS, RI A, MS, RI A, MS, RI A, MS, RI A, MS, RI A, MS, RI A, MS, RI A, MS, RI A, RI A, MS, RI A, MS, RI A, MS, RI A, MS, RI A, MS, RI A, MS, RI A, MS, RI A, MS, RI A, RI A, MS, RI

Methanethiol Ethyl 2-methylpropanoate Ethyl butanoate Ethyl 2-methylbutanoate Ethyl 3-methylbutanoate 3-Methylbutyl acetate 3-Methylbutanol Ethyl hexanoate Limonene Ethyl octanoate Acetic acid Methional Vitispiranes 3-Methylbutanoic acid α-Terpineol Diethyl pentanedioate β-Damascenone Guaiacol 2-Phenylethanol Eugenol Sotolon Decanoic acid ni ni ni

68 74 74 68 71 41 79 87 79 71 54 74 76 82 74 54 96 46 87 61 50 68 61 68 79

65 58 76 50 71 41 79 87 46 41 58 54 65 61 71 54 68 41 61 58 41 74 68 58 50

76 71 82 54 50 65 89 91 58 65 29 71 58 74 74 71 82 41 74 50 84 87 65 61 −

58 65 79 68 61 41 87 96 50 65 41 58 71 74 79 41 91 − 68 54 − 84 41 89 65

− 82 84 68 79 76 74 84 54 82 50 87 58 65 65 58 61 58 68 58 29 46 54 41 46

50 58 61 50 50 74 58 87 29 54 58 76 76 58 76 50 74 41 61 29 29 71 41 50 76

61 84 74 79 61 41 79 89 65 − − 87 65 58 74 − 94 58 74 − 58 50 58 71 84

41 76 65 61 65 58 76 82 41 50 58 82 71 79 71 41 73 50 58 41 41 71 41 58 68

58 65 79 71 65 41 67 89 50 68 82 68 68 82 65 29 84 58 71 71 58 61 71 79 61

46 78 76 76 50 46 78 91 50 58 58 65 87 89 84 41 74 41 71 71 61 76 58 65 79

A, MS, RI A, MS, RI

Acetaldehyde Ethyl acetate ni Diacetyl Ethyl propanoate Ethyl 2-butenoate Hexyl acetate

50 41 29 − − 29 −

29 50 29 58 − 41 −

− − − − − − 65

41 50 41 41 41 41 −

29 41 41 − 29 − 71

− − − 58 29 29 58

41 50 29 − 29 − −

− − 29 − − − −

− − 50 41 58 29 −

29 41 − 41 − 29 29

A, MS, RI A, RI

A, MS, RI A, MS, RI A, MS, RI A, MS, RI

15

Table 1 (continued) LRIa

Aroma Descriptorsb

DB-wax

DB-5

1304 1315 1380 1400 1411 1515 1776

980 890 852

1859 1993 2085 2206 2369

984 1284 1370 1597 1326 1155 1168 1353 1475

Mushroom (fresh) Chicken (cooked), savoury Citrus, green, stalky Floral, musk, watermelon Green, woody, mushroom Floral, confectionary - musk Floor polish, lime (cooked)

A, RI A, RI A, MS, RI

Green tea, pineapple (tinned) Coconut, spice, wood Toffee, honeycomb Leather, barnyard Spice, cedar, smoky Confectionery - raspberry, bubblegum Confectionery - musk, strawberry

A, MS, RI A, MS, RI A, RI A, MS, RI

a

Calculated linear retention index (LRI)

b

Summary of the comments from panellists

Viognier

Identityc

A, MS, RI A, MS, RI A, MS, RI

Chardonnay

B. Semillon

Compound Octene-3-one 2-Methyl-3-furanthiol (E)-3-Hexen-1-ol ni 1-Octen-3-ol Geranyl ethyl ether 1,1,6-Trimethyl-1,2dihydronaphthalene Ethyl dodecanoate cis-Oak lactone Homofuraneol 4-Ethylphenol ni ni ni

H-1

H-2

L-1

L-2

H-1

H-2

L-1

L-2

H

L

50 41 46 65 − 50 87

− − 29 29 58 − 50

35 58 − 41 − 29 −

29 − 41 − − − 68

50 61 41 41 29 41 −

− 76 − − − − −

− 61 − − 29 − 41

− 74 − − − − −

82 − 71 41 76 50 54

50 50 78 29 65 29 58

− − − 50 50 68 −

29 65 − − 50 65 −

− − − 41 41 50 −

− − 35 − − − 50

46 41 50 35 − − 54

65 − − 41 29 71 61

61 76 41 74 61 58 58

− 65 − 50 29 41 50

− 54 50 − 54 29 41

− 84 54 61 50 61 54

c

Method of identification: A, aroma match with literature; MS, data in agreement with those of authentic compound or NIST11/Wiley 275 libraries; RI, data in agreement with those of authentic compound and/or literature. In italics are compounds identified only on GC Wax phase column, in bold are those identified on both GC Wax and DB5 phases. n, number of wines. ni, not identified. H, high ‘stone fruit’ aroma in wine; L, low ‘stone fruit’ aroma in wine.

16

3.3.

GC-MS analyses

3.3.1. Quantitative analysis of volatile aroma compounds The compounds of the wines were quantified using an accurate and precise SIDA method for the monoterpenes and γ-lactones. A single internal standard was used for relative quantification of the other compounds identified from GC-O analysis. Table 2 shows that the Viognier wines contained higher levels of limonene, δ-terpinene, geranyl ethyl ether, linalool, α-terpineol and geraniol than those made from Chardonnay. Chardonnay wines were higher than the Viognier wines only in 2-phenylethanol. Botrytis Semillon was higher than the other wine varietals in ethyl acetate and acetic acid, as expected, but also in ethyl heptanoate and ethyl pentanoate. Most other fermentation-derived aroma compounds in the botrytis Semillon wines were at relatively low levels. cis-Rose oxide was only detected in the high ‘stone fruit’ botrytis Semillon. When comparing the high to low ‘stone fruit’ wines for the separate varieties, there were some differences observed. For Chardonnay, ethyl butanoate, 3-methylbutyl acetate, hexyl acetate, butanoic acid, 2phenylethyl acetate and ethyl hexadecanoate were higher in the high ‘stone fruit’ samples, while the branchedchain esters ethyl 2-methylpropanoate, ethyl 2-methylbutanoate, the vitispiranes, methionol, diethyl pentanedioate and 2-phenylethanol were higher in the low ‘stone fruit’ samples. For Viognier, limonene, δterpinene, ethyl heptanoate, geranyl ethyl ether, benzaldehyde, linalool, butanoic acid, α-terpineol, methionol and geraniol were higher in the high ‘stone fruit’ samples. For the two botrytis Semillon wines, there were many differences between the high and low samples. The four aroma compounds that were at high concentration (more than three-fold higher ratio) in the high ‘stone fruit’ sample were ethyl 3-methylbutanoate, 1,1,6trimethyl-1,2-dihydronaphthalene (TDN), vitispiranes, cis-oak lactone and there were four compounds at relatively high concentration in the low ‘stone fruit’ sample: hexyl acetate, 1-octen-3-ol, 2-phenylethyl acetate and β-damascenone. The averaged aroma intensity, as MF (%), often matched the relative peak area ratio for individual aroma compounds but several compounds could only be detected by GC-O and not by MS, such as methional, sotolon, 1-octen-3-one, 4-ethylguaiacol and 3-isobutyl-2-methoxypyrazine. As HS-SPME-GC-O-MS is a solvent-free technique, another noted advantage was that it allowed for the assessors to evaluate the GC effluent immediately after injection. This enabled highly volatile aroma compounds to be smelled, such as methanethiol, acetaldehyde, diacetyl and dimethyl sulfide. However, these highly volatile compounds could not be accurately quantified, due to poor peak shape and coelution with other compounds, notably carbon dioxide, ethyl acetate and ethanol.

17

Table 2 Relative concentration (Peak Area analyte/Peak AreaIS) of aroma compounds identified by GC-O in Chardonnay, Viognier and botrytis Semillon wines with high and low ‘stone fruit’ attributes; GC retention indices; and CAS numbers. LRI DB-wax 650 716 896 968 973 990 1042 1060 1074 1110 1124 1135 1167 1188 1218 1235 1275 1288 1304 1315 1333 1362 1380 1446 1446 1456 1467 1477 1515 1530

DB-5 450 427 616 714 765 596 800 854 861 971 881 900 844 1030 745 998 1013 1090 980 890 1098 1113 852 1201 984 633 908 839 1284 969

CAS registry number 74-93-1 75-07-0 141-78-6 105-37-3 97-62-1 431-03-8 105-54-4 7452-79-1 108-64-5 7392-19-0 123-92-2 539-82-2 623-70-1 138-86-3 123-51-3 123-66-0 142-92-7 586-62-9 4312-99-6 28588-74-1 106-30-9 876-17-5 928-97-2 106-32-1 3391-86-4 64-19-7 3268-49-3 98-01-1 40267-72-9 100-52-7

Viognier

Chardonnay

B. Semillon

Compound H-1

H-2

L-1

L-2

H-1

H-2

L-1

L-2

H

L

2.09 0.030 0.034

3.16 0.018 0.026

2.72 0.022 nd

1.81 0.029 0.031

1.97 0.017 0.024

1.98 0.017 0.002

3.01 0.022 0.077

1.19 0.023 0.037

6.36 0.056 0.064

4.03 0.026 0.027

0.512 0.020 0.043 0.162 0.87 nd 0.060 0.583 6.78 10.0 0.13 0.450

0.294 0.014 0.020 0.012 1.27 nd 0.013 0.187 4.80 15.4 0.29 0.117

0.431 0.011 0.011 nd 10.81 nd 0.023 0.091 5.08 17.0 3.66 0.061

0.279 0.029 0.057 0.063 0.75 nd 0.013 0.101 4.87 15.3 0.21 0.085

0.355 0.010 0.032 nd 5.20 nd 0.039 0.022 6.41 12.5 1.84 nd

0.331 nd 0.005 nd 8.04 nd 0.015 0.021 6.30 14.6 2.55 0.016

0.156 0.050 0.098 nd 0.39 nd 0.016 0.007 6.53 8.6 0.02 nd

0.287 0.019 0.027 nd 2.80 nd 0.014 0.019 5.15 14.6 1.12 0.013

0.107 0.044 0.050 0.013 0.44 0.025 0.006 0.018 2.25 3.0 0.04 0.028

0.090 0.023 0.014 nd 1.19 0.008 0.003 0.018 3.02 5.4 0.30 0.040

0.027 nd nd 42.7 nd 0.35

0.016 nd 0.023 82.9 nd 0.84

0.008 nd 0.018 94.4 nd 0.15

0.008 nd 0.033 66.0 nd 0.38

0.016 nd 0.003 66.2 nd 0.34

0.043 nd nd 67.9 nd 0.25

0.014 nd 0.009 52.4 nd 0.67

0.012 nd 0.009 84.5 nd 0.19

0.425 0.006 nd 7.3 0.067 1.72

0.177 0.003 0.003 18.6 0.250 1.74

0.24 0.337 0.072

0.72 0.205 0.712

0.07 0.054 0.032

0.52 0.071 0.028

0.74 nd 0.063

0.09 0.015 nd

0.73 nd 0.057

0.17 nd 0.000

0.97 0.032 0.885

0.63 0.029 0.307

a

Methanethiol Acetaldehydea Ethyl acetate Ethyl propanoate Ethyl 2-methylpropanoate Diacetyla Ethyl butanoate Ethyl 2-methylbutanoate Ethyl 3-methylbutanoate Linaloyl oxide 3-Methylbutyl acetate Ethyl pentanoate Ethyl 2-butenoate Limonene 3-Methylbutanol Ethyl hexanoate Hexyl acetate δ-Terpinene 1-Octen-3-oneb 2-Methyl-3-furanthiolb Ethyl heptanoate cis-Rose oxidec (E)-3-Hexen-1-ol Ethyl octanoate 1-Octen-3-olc Acetic acid Methionalb Furfural Geranyl ethyl ether Benzaldehydec

18

Table 2 (continued)

DB-wax

DB-5

1539

1181

CAS Registry Number 24683-00-9

1549 1551 1582 1638 1652 1680 1711 1735 1776

1102 1293 790 812 1392 862 1211 985 1370

78-70-6 65416-59-3 513-85-9 107-92-6 110-38-3 503-74-2 98-55-5 505-10-2 30364-38-6

LRI

1788 1282 818-38-2 1830 1260 103-45-7 1848 1386 23726-93-4 1859 1597 106-33-2 1869 1256 106-24-1 1884 1089 90-05-1 1935 1122 60-12-8 1993 1326 39212-23-2 2057 1279 2785-89-9 2064 1368 104-61-0 2085 1155 27538-10-9 2198 1360 97-53-0 2206 1168 123-07-9 2227 1119 28664-35-9 2256 1990 628-97-7 2284 1384 334-48-5 LRI, calculated linear retention index a

Detectable but not quantifiable.

b

Not detected by MS.

c

Quantified on DB-5 GC column.

Viognier

Chardonnay

B. Semillon

Compound 3-Isobutyl-2methoxypyrazineb Linaloolc Vitispiranesc 2,3-Butanediol Butanoic acid Ethyl decanoate 3-Methylbutanoic acid α-Terpineol Methionol 1,1,6-Trimethyl-1,2dihydronaphthalene Diethyl pentanedioate 2-Phenylethyl acetate β-Damascenone Ethyl dodecanoate Geraniolc Guaiacolc 2-Phenylethanol cis-Oak lactone 4-Ethylguiacolb γ-Nonalactonec Homofuraneolb Eugenol 4-Ethylphenol Sotolonb Ethyl hexadecanoate Decanoic acid

H-1

H-2

L-1

L-2

H-1

H-2

L-1

L-2

H

L

0.244 0.505 0.327 0.040 21.9 nd 0.184 0.009 2.72

0.239 0.458 0.234 0.037 29.1 0.017 0.219 0.036 0.41

0.149 0.024 0.352 0.020 40.3 nd 0.039 nd 0.09

0.021 0.575 0.304 0.017 20.6 0.025 0.116 nd 1.61

nd 0.144 0.180 0.041 32.5 nd 0.021 0.012 0.21

0.017 0.065 0.266 0.042 27.5 nd 0.014 0.011 0.14

nd 0.778 0.221 0.035 28.0 nd 0.018 0.112 0.66

nd 0.222 0.287 0.024 28.9 nd nd 0.033 0.20

nd 3.728 0.527 nd 2.1 nd 0.028 nd 3.55

nd 0.550 0.503 nd 4.5 nd 0.020 nd 0.47

0.015 0.08 nd 1.62 0.337 nd 2.05 nd

0.005 0.20 0.078 1.08 0.144 0.014 2.36 0.022

nd 1.94 nd 4.37 0.045 nd 2.47 nd

0.012 0.15 nd 1.15 0.056 nd 1.85 nd

0.008 0.80 nd 2.73 nd nd 2.91 0.014

nd 0.93 0.043 2.08 nd nd 3.23 nd

0.034 0.11 nd 3.10 nd nd 4.66 0.065

0.018 0.70 nd 1.63 nd nd 3.31 nd

0.023 0.06 0.023 0.13 nd 0.005 1.12 0.055

0.007 0.24 0.070 0.15 nd nd 1.76 0.004

nd

nd

nd

nd

nd

nd

nd

nd

0.031

nd

nd nd

0.010 nd

nd nd

nd nd

nd nd

nd nd

nd 0.128

nd nd

0.009 nd

nd nd

0.248 1.52

0.451 3.09

0.516 4.71

0.339 2.86

0.530 4.99

0.436 3.62

0.352 2.97

0.401 4.56

0.141 0.31

0.209 0.84

19

3.3.2. γ-Lactones As previously published quantitative n-alkyl γ-lactone studies in wine had not focused on wines with high ‘stone fruit’ aroma, assessing the involvement of γ-lactones was an important aim of this study. However, γ-nonalactone was only detectable by GC-MS in scan mode in one of the botrytis Semillon wines. Therefore, targeted analyses of the n-alkyl γ-lactones was performed, while also monitoring for δ-lactones. Table 3 shows the range of concentrations of the n-alkyl γ-lactones found in the wines (also Supporting Information, Table S3). The concentrations found were quite low for the dry white wines (not detected to 7.2 µg/L), in agreement with the average levels reported in other studies (Cooke et al., 2009; Langen et al., 2013). Nonetheless, significantly higher concentrations of γ-octa- (p < 0.05) and γ-nonalactone (p < 0.01) were found in the high ‘stone fruit’ dry white wines. No difference was found between the dry white wines in the concentrations of γ-deca- or γdodecalactone. γ-Undecalactone was not detected in any of the wines. Low concentrations of δ-nona-, δ-decaand δ-dodecalactones (0.4–28 µg/L) have been reported previously in dry white wines (Langen et al., 2013). However, no peaks corresponding to expected LRIs and ions of the n-alkyl δ-lactones were detected in this set of wines. Table 3. Concentration of the n-alkyl γ-lactones in the white wines studied. γ-Lactones (µg/L) Wine Octa-

Nona-

Deca-

Dodeca-

High ‘stone fruit’ (n = 4)

1.0 ± 0.2a

5.5 ± 1.7

0.8 ± 0.2

0.4 ± 0.3

Low ‘stone fruit’ (n = 4)

0.5 ± 0.3

2.0 ± 0.5

0.8 ± 0.2

0.5 ± 0.1

High ‘stone fruit’ (n = 1)

6.9

62.6

2.9

0.7

Low ‘stone fruit’ (n = 1)

2.6

20.5

1.8

0.6

Aroma detection thresholdb

700c

76c

10d

29c

Chardonnay and Viognier

Botrytis Semillon

a

Values are shown as the mean ± SD (standard deviation)

b

In model wine: 10‒14% v/v ethanol in water + tartaric acid to pH 3.2‒3.5

c

Jarauta, Ferreira, and Cacho (2006)

d

Moyano, Zea, Moreno, and Medina (2010)

When the individual aroma detection thresholds of the n-alkyl γ-lactones, and especially γ-octa- and γnonalactone, are compared to the concentrations found in the wines (Table 3), then it becomes apparent that they

20

were not likely to be major contributors to ‘stone fruit’ aroma in the Chardonnay and Viognier wines studied. In contrast, the botrytis Semillon wines contained ten-fold more γ-nonalactone and five-fold more γ-octalactone than the dry white wines. As the concentration of γ-nonalactone in the high ‘stone fruit’ botrytis Semillon wine is near its aroma detection threshold, it might have contributed to the perceived ‘stone fruit’ aroma. The higher concentrations in the sweet wines could be due in part to the botrytis-driven concentration factor of grape berry shrivel as well as chemical transformations (Magyar & Soós, 2016). Other studies have reported higher concentrations of γ-nona- and γ-decalactone in botrytized wines compared to dry wines (Genovese et al., 2007; Sarrazin et al., 2007). Jarauta et al. (2006) reported that sub-threshold levels of alkyl γ-lactones strongly interact synergistically to produce an aroma higher than that expected based on their individual thresholds. To determine if additive interactions were occurring between the n-alkyl γ-lactones at the concentrations found in the dry white wines in this study, a sensory addition experiment was conducted. No ‘stone fruit’ aroma was noted in a model wine spiked with a mixture of C8‒C12 alkyl γ-lactones (1.0, 7.0, 1.0, 0.0 and 1.0 µg/L respectively). Hence, a more complex mixture including wine aroma compounds from other chemical families might be needed for the reconstituted wine aroma to be perceived with a ‘stone fruit’ character. A mixture base of several lactones, vanillins, cinnamates and terpenes produced ‘sweet fruit’ and ‘peach’ aromas in model wine, but was only ‘floral’ and ‘sweet’ when added to a neutral white wine (Loscos, Hernandez-Orte, Cacho, & Ferreira, 2007). As γ-nonalactone was the most prevalent γ-lactone in the wines, it was also evaluated. Four out of the five assessors noted the 25 µg/L sample was different, with some comments of ‘slightly fruity’, compared to the base model wine, which suggests that γ-nonalactone was likely to be important in the aroma of botrytis Semillon. 3.3.3. Monoterpenes The monoterpenes linalool, α-terpineol and geraniol were indicated as important to Viognier wines based on GC-O data (Table 1) and were associated with high ‘stone fruit’ aroma in Viognier wines with relatively high concentrations determined in these wines (Table 2). Therefore, these aroma compounds were also accurately quantified in the Viognier wines with a targeted analysis (Table 4). The monoterpenes were detected in all Viognier wines and the targeted results agree with the trend seen with the relative ratio data (Table 2). The relatively high concentrations were noteworthy, considering Viognier has been reported previously as a neutral variety that does not rely on monoterpenes for varietal flavour (Mateo & Jiménez, 2000). On the contrary, a number of authors have stated that Viognier is an aromatic variety, i.e. having floral, Muscat-like flavour (Iland, Dry, Proffitt, & Tyerman, 2011; Ribéreau-Gayon, Glories, Maujean, & Dubourdieu, 2006), but no quantitative

21

monoterpene data have been reported to support this, to our knowledge. One study indicated a Viognier grape sample had a similar volatile profile to a Riesling sample with relatively low monoterpene concentration, but concentrations were not presented (Chacón, García, Martínez, Mena, & Izquierdo, 2012). The mean concentrations of linalool, α-terpineol and geraniol (58, 91 and 16 µg/L) found in the high ‘stone fruit’ Viognier wines were substantially higher than the median reported concentrations of these compounds in wines of other ‘neutral’ varieties, such as Chardonnay (7, 7, 2 µg/L) and Sauvignon blanc (10, 12, 1 µg/L), and were higher than commonly observed in Riesling wines (12, 54, 18 µg/L), an aromatic variety where monoterpenes are generally considered to be key contributors to flavour (Black, Parker, Siebert, Capone, & Francis, 2015). The potential involvement of monoterpenes in ‘stone fruit’ aroma of wine was an unexpected outcome because much research has been conducted regarding monoterpenes and wine aroma as recently reviewed by Black et al. (2015), and they are considered to imbue ‘floral’, ‘citrus’ and ‘pine-like’ characters. These same aromas were described during the GC-O for the linalool, α-terpineol and geraniol peaks (Table 1). These observations add further evidence to the hypothesis that a number of compounds, in a particular ratio, are required for a Viognier wine to be perceived as having a ’stone fruit’ character, similar to what has been shown for ‘overripe orange’ flavour in Bordeaux dessert wines (Stamatopoulos, Fr rot, Temp re, Pons, & Darriet, 2014) and key aroma attributes in red wine (Ferreira, Sáenz-Navajas, Campo, Herrero, de la Fuente, & Fernández-Zurbano, 2016). Table 4 Concentrations of free monoterpenes (µg/L) in the Viognier wines with high and low ‘stone fruit’ attributes. Compound Linalool α-Terpineol Geraniol

Aroma detection thresholda 25b

VH-1

VH-2

VL-1

VL-2

56

60

35

2

250b

96

86

27

29

30c

13

19

7

1

VH, Viognier wine high in ‘stone fruit’ aroma; VL, Viognier wine low in ‘stone fruit’ aroma. a

in model wine: 10‒11% v/v ethanol in water ± tartaric acid to pH 3.4, glycerine (7 g/L).

b

Ferreira, López, and Cacho (2000).

c

Guth (1997b).

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As monoterpenes are grape-derived aroma compounds, it was of interest to determine the concentration of linalool, α-terpineol and geraniol in Viognier grapes. A set of 11 Viognier berry samples, collected from different grapevine clones over three vintages ‒ from a vineyard considered to regularly produce wines with varietal ‘stone fruit’ character ‒ was analysed for these compounds. Fig. 1 shows the varied concentrations between the clones for linalool, α-terpineol and geraniol. A generally consistent profile for each monoterpene across the clones and from vintage to vintage was found even though the concentrations were quite different across vintages. The ‘Tablas Creek’ clone had the lowest concentrations of linalool, α-terpineol and geraniol (Fig. 1). The ‘Yalumba 3’ clone had relatively higher concentrations of linalool and α-terpineol in 2014 compared to 2015 and 2016, and the ‘Yarra Yering’ clone had a higher concentration of geraniol in 2016 than expected by comparison to the other clones’ profiles. The concentrations of linalool and α-terpineol previously reported in Viognier juices, from a vine training systems study over two vintages (2002, 2003), ranged from 34‒90 µg/L and 10‒ 56 µg/L respectively (Zoecklein et al., 2008), which were similar to the concentrations found in 2016 for the current study. In summary, the relatively high concentrations of monoterpenes found support the wine data in the conclusion that Viognier is an aromatic terpene-rich variety. To confirm if the differences in monoterpene concentration between the clones was driven by sugar ripeness (TSS), an ANOVA with TSS as a co-variate was conducted for each vintage. For the 2014 and 2016 vintages, there were significant differences among clones for linalool, α-terpineol and geraniol (p < 0.01), but TSS was not a significant covariate. In 2015, TSS was a significant covariate in the model (p < 0.05), but there were nonetheless significant differences among the clones for each monoterpene. Therefore, the observed clonal differences in grape monoterpenes appear not to be driven by the differences in TSS and maturity. 4.

Conclusions This paper presents an investigation into the aroma compounds responsible for ‘stone fruit’ aroma in

Chardonnay, Viognier and botrytis Semillon wines. To our knowledge, this is the first detailed study regarding the aroma profile of Viognier wine by GC-O. As an important first step, the wine extract used for GC-O was demonstrated to represent the overall aroma of the wine in a glass. Even though the overall aroma compound compositions of the three wine varieties were similar, several notable differences were observed. The impact aroma compounds of apricots and peaches, γ-octa-, γ-deca-, γ-dodeca- and δ-decalactone, were not found to be major contributors to ‘stone fruit’ aroma in Chardonnay and Viognier wines. However, γ-nonalactone might influence ‘stone fruit’ aroma in botrytis Semillon. Interestingly, other aroma compounds which can be important to the flavour of stone fruit, such as linalool, α-terpineol and geraniol (Belitz et al., 2009) but are noted more

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generally as having ‘floral’ characters, might be of major importance for ‘stone fruit’ aroma in Viognier wine. The data obtained on both wine and grape samples provide new insight into this scarcely studied grape variety, highlighting the high concentration of monoterpenes. The differing concentrations of monoterpenes between Viognier clones could provide winemakers with options for desired wine style or blending. In Chardonnay wine, where stone fruit aroma can be more subtle, a specific mixture of esters was identified which could contribute to ‘stone fruit’ aroma. The wine aroma descriptive term ‘stone fruit’ encompasses more specific descriptors, including ‘apricot’, ‘peach’ (yellow and white), ‘nectarine’ and they can each be further separated into fresh, cooked or dried terms. Therefore, an even more comprehensive sensory study encompassing a large number of Chardonnay and Viognier wines with a range of different ‘stone fruit’ descriptors and intensities is needed to determine if synergistic and/or masking effects in specific mixtures of aroma compounds are responsible for different ‘stone fruit’ aromas in wine, and also to confirm the sensory contribution of monoterpenes to Viognier flavour.

Conflict of interest The authors declare no competing financial interest.

Acknowledgements We thank Alice Barker and Wes Pearson and our colleagues from the AWRI for assisting with the wine selection, the participants in the GC-O study and Natoiya Lloyd and Kevin Pardon for the synthesis of d6linalool and d6-α-terpineol. We also thank Yalumba for their donation of wine and grape samples, and Louisa Rose and Brooke Howell for their assistance and valuable discussions. We are grateful to members of the Australian wine industry for their assistance and provision of numerous wine samples, especially Chapel Hill Winery, De Bortoli Family Winemakers, Henschke Cellars, Trentham Estates. This work was supported by Australian grapegrowers and winemakers through their investment body Wine Australia, with matching funds from the Australian government. The AWRI is a member of the Wine Innovation Cluster located at the Waite precinct in Adelaide, South Australia. This work was also supported by the Australian Government Research Training Program Scholarship through the University of South Australia (Adelaide, Australia).

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The Human Research Ethics Committee of the University of South Australia approved the methods of sensory testing and data collection used in this study.

Appendix Tables S1–3

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Figure Caption Fig. 1. Concentration of free monoterpenes (µg/kg) in the grape berry homogenate of 11 Viognier clones over three consecutive vintages (2014‒2016): (a) linalool, (b) geraniol, (c) α-terpineol. Bars are means of triplicate vineyard samples; error bar is half HSD. No sample was available for the Yalumba 1 clone in 2015.

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Highlights 

Detailed analysis of aroma-active compounds in white wines with ‘stone fruit’ aroma.



First aroma profile of Viognier wine by GC-O.



Monoterpenes might be of major importance for ‘stone fruit’ aroma in Viognier wine.



Clarification of Viognier being classified as an aromatic terpene-rich variety.

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