Determination of the Importance of In-Mouth Release of Volatile

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Determination of the Importance of In-Mouth Release of Volatile Phenol Glycoconjugates to the Flavor of Smoke-Tainted Wines Christine M. Mayr,† Mango Parker,† Gayle A. Baldock,† Cory A. Black,† Kevin H. Pardon,† Patricia O. Williamson,† Markus J. Herderich,† and I. Leigh Francis†,* †

The Australian Wine Research Institute, P.O. Box 197, Glen Osmond (Adelaide), South Australia 5064, Australia S Supporting Information *

ABSTRACT: The volatile phenols guaiacol, 4-methylguaiacol, syringol, 4-methylsyringol, o-, m-, and p-cresol, as well as their glycoconjugates, have previously been shown to be present in elevated concentrations in smoke-tainted wine. Sensory descriptive analysis experiments, with addition of free volatile phenols in combination with their glycosidically bound forms, were used to mimic smoke taint in red wines. The addition of volatile phenols together with glycoconjugates gave the strongest off-flavor. The hydrolysis of glycosidically bound flavor compounds in-mouth was further investigated by in vitro and in vivo experiments. The results indicate that enzymes present in human saliva are able to release the volatile aglycones from their glycoconjugates even under low pH and elevated ethanol conditions, confirming that in-mouth breakdown of monosaccharide and disaccharide glycosides is an important mechanism for smoke flavor from smoke affected wines, and that this mechanism may play an important general role in the flavor and aftertaste of wine. KEYWORDS: bushfire, glycosides, flavor reconstitution, smoke taint, sensory descriptive analysis, PLS, in-mouth release, wine, volatile phenols

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INTRODUCTION The effect of bushfire and controlled forest burn events in recent years has resulted in the production of wines described as “smoke tainted”. Such wines are characterized by an undesirable sensory character described as “smoky”, “burnt”, “ash”, and “ashtray”.1 This latter feature of smoke-tainted wines is notable as a lingering, unpleasant ashy aftertaste. It is of particular concern to many wine producers and is a sensory property that is generally not present in wines that have been exposed to heavily toasted or charred oak wood during production, which can otherwise give a smoky flavor.2 This undesirable taint has resulted in large financial losses for the Australian wine industry, and several research groups have investigated the effect of smoke on the quality of wine and the compounds causing the taint. Several volatile phenols were identified at elevated levels in wine made from smoke-affected grapes,3 with guaiacol being the most abundant, and therefore used, together with 4methylguaiacol, as an indicator for the smoke taint. Other phenolic compounds, notably syringol, 4-methylsyringol, and o-, p-, and m-cresol also have been found in increased concentrations in smoke-tainted grapes and wines.1,4 It has been demonstrated that the concentration of guaiacol in wine increases during fermentation,5,6 pointing toward the presence of precursors of guaiacol in smoke-affected fruit. This was confirmed7−9 with glycosidically bound volatiles of guaiacol and other phenolic compounds, such as syringol and m-cresol, identified following grapevine smoke exposure, including numerous disaccharide glycosides, and also through demonstrating the in vivo conjugation of guaiacol in grapes following application of guaiacol. The relationships between the concentration of volatile phenol compounds, both free and bound, and the sensory panel © 2014 American Chemical Society

ratings of smoke attributes in affected wines have recently been studied.4 This correlative study indicated that guaiacol, o-cresol, m-cresol, and p-cresol were most important to smoky sensory attributes of wines. Moreover, some monosaccharide glycosides of phenols were shown to have a flavor effect when presented to sensory panelists in model wine, ascribed to in-mouth breakdown of the glycosides. In this work, the sensory contribution of glycosylated phenols relative to free volatile phenols was investigated through an examination of smoke-affected wines and through reconstitution studies. The primary aim was to assess the inmouth flavor effect of the glycoconjugates relative to the volatiles, as this is characteristic of smoke-tainted wines. The direct release of glycosidically bound volatiles through the influence of human saliva was also investigated in vivo and in vitro.

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MATERIALS AND METHODS

Wine. Commercially produced smoke-affected and reference, unaffected wines from several wine regions from the 2007 and 2009 vintages were used (see Supporting Information, SI, Table S1) for the first sensory descriptive study, together with one wine made during 2011 under small-scale research winemaking conditions from frozen smoke-affected 2009 vintage Shiraz grapes, according to the small-scale winemaking protocol previously described.7 Two of these wines were used in the subsequent reconstitution study (Samples 18 and 19, see SI Table S1). Smoke-affected grapes for the small-scale research wine were sourced from a vineyard in Yarra Valley, Victoria (Australia) in March 2009 that had been exposed to smoke generated by a series of Received: Revised: Accepted: Published: 2327

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Figure 1. Synthesis of (A) guaiacyl β-D-primeveroside (6) and (B) tri-O-isobutyryl-α-D-xylopyranosyl bromide. bushfires that occurred in the period 7 February−14 March 2009. These fires, particularly those in the period of 7 February, were of extremely high intensity, causing extensive loss of vineyards and winery buildings in the Yarra Valley, and can be considered one of the worst fire events in Australia’s history. Smoke was present in the area for an extended period as a result. Smoke-exposed grapes and wines made from smoke-exposed grapes were obtained from producers who identified these as showing clear smoke flavor. Materials. Guaiacol, 4-methylguaiacol, o-cresol, m-cresol, p-cresol, syringol and 4-methylsyringol, and d7-p-cresol were purchased from Sigma-Aldrich (Castle Hill, Australia). d3-Guaiacol and d3-4-methylguaiacol had been previously synthesized in-house.9,10 d3-Syringol was prepared according to the method of Aihara et al.,11 and syringol β-Dglucoside and syringol β-D-gentiobioside were synthesized in house, as previously reported.7 All chemicals were analytical-reagent-grade unless otherwise stated. Water was obtained from a Milli-Q purification system (Millipore, North Ryde, Australia). Guaiacol-β-D-Primeveroside (2-methoxyphenyl-6-O-(β-D-xylopyranosyl)-β-D-glucopyranoside) Synthesis. All chemicals were analytical-reagent grade. Guaiacyl β-D-primeveroside (6) was synthesized using a procedure (Figure 1A) based on a method employed previously by our group to prepare d3-syringyl β-D-gentiobioside.12 Briefly, guaiacyl-2′,3′,4′-tri-O-acetyl-β-D-glucopyranoside (4) was prepared in four steps by coupling guaiacol and tetra-O-acetyl-α-Dglucopyranosyl bromide, followed successively by glucose deprotection, tritylation, and acetylation, and finally detritylation. The protected disaccharide was prepared using synthesized isobutyrylprotected xylopyranosyl bromide as the glycosyl donor (Figure 1B) to avoid excessive orthoester byproduct formation during coupling. Deprotection with Amberlite(OH) resin gave the disaccharide 6. Instrumentation. Proton (1H) and carbon (13C) Nuclear Magnetic Resonance (NMR) spectra were recorded with a Bruker spectrometer operating at 400 or 600 MHz for proton and 100 or 150 MHz for carbon nuclei, respectively. Chemical shifts were recorded as δ values in parts per million (ppm). Spectra were acquired in chloroform-d or

deuterium oxide (D2O) at ambient temperature, and resonances were assigned by routine 2D correlation experiments. For 1H NMR spectra, the peak as a result of residual CHCl3 (δ 7.26) or the CH3 peak of acetonitrile (δ 2.06), added when D2O was the solvent, was used as the internal reference. For 13C NMR spectra, the central peak of the CDCl3 triplet (δ 77.16) or the CH3 peak of acetonitrile (δ 1.47), added when D2O was the solvent, was used as the internal reference. High-Resolution Mass Spectrometry (HRMS) spectra were obtained on a Bruker microTOF-Q II with electrospray ionization (ESI) or atmospheric-pressure chemical ionization (APCI) in positive mode. Samples dissolved in water or methanol at concentrations of approximately 1−2 mg/L were analyzed by flow injection. Specific optical rotations were recorded with a PolAAr 21 polarimeter, referenced to the sodium D line (589 nm) at 20 °C, using the spectroscopic-grade solvents specified and at the concentrations (c, g/ 100 mL) indicated. The measurements were carried out in a cell with a 1 dm path length. Melting points were measured with a Buchi Melting Point B-540 unit and were uncorrected. Synthesis of Tri-O-isobutyryl-D-xylopyranose. Isobutyryl chloride (51 g, 480 mmol) in CHCl3 (100 mL) was added dropwise to a stirred solution of D-(+)-xylose (10 g, 68 mmol) in anhydrous pyridine (49 mL, 610 mmol) at 0 °C under N2. The yellow mixture was stirred at room temperature for 18 h. The resultant orange mixture was diluted with a further 200 mL CHCl3 and washed with 2 M HCl (3 × 150 mL), saturated NaHCO3 (3 × 150 mL), water (2 × 50 mL), and brine (3 × 50 mL). The organic layer was dried (Na2SO4), filtered, and concentrated in vacuo. The oily reaction product was purified by silica gel column chromatography eluted with 15% EtOAc/hexane to give a colorless oil as a 97:3 mixture of α/β epimers (29 g, 99%). Rf 0.33 (15% EtOAc/hexane). [α]D + 65.4 (c 0.67, CH2Cl2). 1H NMR for α epimer (600 MHz, CDCl3): δ 6.27 (1H, d, J = 3.7 Hz, H1); 5.52 (1H, t, J = 9.9 Hz, H3); 5.06 (1H, dd, J = 9.9, 3.7 Hz, H2); 5.05 (1H, dd, J = 11.1, 9.9 Hz, H4); 3.91 (1H, dd, J = 11.0, 5.8 Hz, H5eq); 3.68 (1H, app t, J = 11.0 Hz, H5ax); 2.70−2.41 (4H, m, CHMe2); 1.24−1.07 (24H, m, Me). 13C NMR (150 MHz, CDCl3): δ 176.10−175.10 (CO); 89.16 2328

dx.doi.org/10.1021/jf405327s | J. Agric. Food Chem. 2014, 62, 2327−2336

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7.9, 1.4 Hz, H3); 6.90 (1H,td, J = 7.9, 1.4 Hz, H5); 5.36 (1H, dd, J = 9.3, 8.0 Hz, H2′); 5.24 (1H, app t, J = 9.3 Hz, H3′); 5.20 (1H, app t, J = 9.5 Hz, H4′); 4.97 (1H, d, J = 8.0 Hz, H1′); 3.84 (3H, s, H7); 3.62 (1H, ddd, J = 9.5, 5.4, 2.2 Hz, H5′); 3.22 (1H, dd, J = 10.4, 2.2 Hz, H6b′); 3.15 (1 H, dd, J = 10.4, 5.4 Hz, H6a′); 2.08 (3H, s, COCH3); 2.02 (3H, s, COCH3); 1.74 (3H, s, COCH3). 13C NMR (150 MHz, CDCl3): δ 170.56 (CO); 169.69 (CO); 169.14 (CO); 150.81 (C2); 146.42 (C1); 143.67 (ipso CPh3); 128.78 (ortho CPh3); 127.94 (meta CPh3); 127.13 (para CPh3); 124.68 (C4); 121.04 (C5); 120.49 (C6); 112.71 (C3); 101.07 (C1′); 86.73 (CPh3); 73.65 (C5′); 73.00 (C3′); 71.53 (C2′); 68.82 (C4′); 61.88 (C6′); 56.12 (C7); 20.86 (2 × COCH 3 ); 20.51 (COCH 3 ). APCI-HRMS (m/z): Calcd for C38H42NO10+ ([M + NH4]+), 672.2803; Found, 672.2791. Compound 3 (390 mg, 0.59 mmol) was dissolved in HOAc/H2O (4:1, 3.9 mL), and the solution was stirred under N2 at 60 °C for 4 h. The reaction mixture was cooled, toluene (3 mL) was added, and the solution was concentrated in vacuo. This step was repeated twice, and the crude material was purified by silica gel column chromatography eluted with 20% Et2O/CH2Cl2 to give 4 as a white solid (200 mg, 83%). Rf 0.18 (15% Et2O/CH2Cl2). mp 160.6−160.8 °C. [α]D = −30.8 (c 0.55, CH2Cl2). 1H NMR (600 MHz, CDCl3): δ 7.08 (1H, dd, J = 7.8, 1.2 Hz, H6); 7.06 (1H, td, J = 7.8, 1.2 Hz, H4); 6.90 (1H, dd, J = 7.8, 1.2 Hz, H3); 6.87 (1H, td, J = 7.8, 1.2 Hz, H5); 5.32 (1H, app t, J = 9.5 Hz, H3′); 5.26 (1H, dd, J = 9.5, 7.8 Hz, H2′); 5.10 (1H, app t, J = 9.5 Hz, H4′); 5.02 (1H, d, J = 7.8 Hz, H1′); 3.81 (3H, s, H7); 3.74 (1H, ddd, J = 11.9, 8.2, 2.0 Hz, H6b′); 3.63 (1H, dd, J = 11.9, 5.9 Hz, H6a′); 3.59 (1H, ddd, J = 9.5, 5.0, 2.0 Hz, H5′); 2.35 (1H, dd, J = 7.8, 5.9 Hz, OH); 2.07 (3H, s, COCH3); 2.05 (3H, s, COCH3); 2.03 (3H, s, COCH3). 13C NMR (150 MHz, CDCl3): δ 170.45 (CO); 170.13 (CO); 169.54 (CO); 150.71 (C2); 146.12 (C1); 124.80 (C4); 121.02 (C5); 119.96 (C6); 112.82 (C3); 100.84 (C1′); 74.46 (C5′); 72.63 (C3′); 71.48 (C2′); 68.74 (C4′); 61.45 (C6′); 56.07 (C7); 20.79− 20.77 (3 × COCH3). APCI-HRMS (m/z): [M + NH4]+ Calcd for C19H28NO10+, 430.1708; Found, 430.1682. Synthesis of Guaiacyl-2′,3′,4′-tri-O-acetyl-2″,3″,4″-tri-O-isobutyryl-β Primeveroside (5). Under a N2 atmosphere at −15 °C, anhydrous 1,2-dichloroethane (2.6 mL) was added to AgCF3SO3 (0.12 g, 0.48 mmol), activated 4 Å molecular sieves (0.23 g), tri-O-isobutyryl-α-Dxylopyranosyl bromide (190 mg, 0.45 mmol) and 4·(93 mg, 0.23 mmol). The slurry was stirred for 10 min, then 2,6-di-tert-butyl-4methylpyridine (93 mg, 0.45 mmol) was added, and the mixture was stirred overnight at room temperature. The reaction mixture was diluted with CH2Cl2, filtered through diatomaceous earth, and concentrated to dryness. The resulting crude material was purified by silica gel column chromatography, eluting with 95:4:1 petroleum ether/Et2O/CH2Cl2 to give 5 as a white gummy solid (160 mg, 91%). Rf = 0.22 (95:4:1 petroleum ether/Et2O/CH2Cl2). [α]D = −51.8 (c 0.55, CHCl3). 1H NMR (400 MHz, CDCl3): δ 7.11 (1H, d, J = 7.8 Hz, H6); 7.10 (1H, td, J = 7.8, 1.6 Hz, H4); 6.98 (1H, td, J = 7.8, 1.6 Hz, H5); 6.93 (1H, dd, J = 7.8, 1.6 Hz, H3); 5.26 (1H, app t, J = 9.4 Hz, H3′); 5.22 (1H, dd, J = 9.4, 7.6 Hz, H2′); 5.13 (1H, app t, J = 9.0 Hz, H4″); 4.97 (1H, d, J = 7.6 Hz, H1′); 4.96 (1H, app t, J = 9.4 Hz, H4′); 4.92 (1H, app t, J = 9.0 Hz, H3″); 4.89 (1H, dd, J = 9.0, 7.2 Hz, H2″); 4.53 (1H, d, J = 7.2 Hz, H1″); 4.05 (1H, dd, J = 11.6, 5.4 Hz, H5eq″); 3.83 (3H, s, H7); 3.73 (2H, m, H6′); 3.70 (1H, ddd, J = 9.3, 5.4, 2.8 Hz, H5′); 3.22 (1H, dd, J = 11.6, 9.3 Hz, H5ax″); 2.50 (1H, sept, J = 7.0 Hz, CHMe2); 2.48 (1H, sept, J = 7.0 Hz, CHMe2); 2.33 (1H, sept, J = 7.0 Hz, CHMe2); 2.07 (3H, s, COCH3); 2.03 (3H, s, COCH3); 2.01 (3H, s, COCH3); 1.12 (6H, d, J = 7.0 Hz, (CH3)2CH); 1.11 (6H, d, J = 7.0 Hz, (CH3)2CH); 1.09 (6H, d, J = 7.0 Hz, (CH3)2CH); 1.08 (6H, d, J = 7.0 Hz, (CH3)2CH); 1.01 (6H, d, J = 7.0 Hz, (CH3)2CH). 13C NMR (150 MHz, CDCl3): δ 175.99 (Me2CHCO); 175.96 (Me2CHCO); 175.45 (Me2CHCO); 170.39 (CH3CO); 169.73 (CH3CO); 169.51 (CH3CO); 150.81 (C2); 146.18 (C1); 125.02 (C4); 121.54 (C5); 120.41 (C6); 113.05 (C3); 100.83 (C1′); 100.76 (C1″); 74.53 (C5′); 72.70 (C3′); 71.43 (C2′); 71.36 (C4″); 70.47 (C2″); 69.07 (C4′); 68.84 (C3″); 67.23 (C6′); 62.47 (C5″); 56.20 (C7); 34.01 (CHMe2); 33.99 (CHMe2); 33.91 (CHMe2); 20.77 (3 × COCH3); 19.01 (2 × ((CH3)2CH); 18.96 (2 × ((CH3)2CH);

(C1); 69.49 (C2); 69.09 (C3); 68.40 (C4); 60.89 (C5); 34.16−33.89 (4 × CHMe2); 19.13−18.76 (8 × (CH3)2CH). APCI-HRMS (m/z): [M + NH4]+ Calcd for C21H38NO9+ 448.2541; Found, 448.2511. Synthesis of Tri-O-isobutyryl-α-D-xylopyranosyl Bromide. Under an N2 atmosphere with NaOH pellets in a trap, 33% HBr/HOAc (12 mL, 66 mmol) was added to a stirred solution of tetra-O-isobutryl-Dxylose (97:3 α:β, 6.2 g, 14 mmol) in CH2Cl2 (30 mL) at 0 °C. After 20 h, CH2Cl2 (200 mL) was added, and the reaction mixture was successively washed with sat. aq. NaHCO3 solution (2 × 100 mL), H2O (100 mL), and brine (100 mL). The organic layer was dried (Na2SO4) and concentrated in vacuo (5.8 g, 95%). A portion was recrystallized from EtOH for characterization purposes to afford tri-Oisobutyryl-α-D-glucopyranosyl bromide as white crystals. Rf 0.73 (1:4:95 petroleum ether/Et2O/CH2Cl2), mp 54.1−54.7 °C. [α]D + 179.2 (c 0.94, CHCl3). 1H NMR (400 MHz, CDCl3): δ 6.57 (1H, d, J = 4.1 Hz, H1); 5.62 (1H, t, J = 9.8 Hz, H3); 5.06 (1H, td, J = 10.3, 5.9 Hz, H4); 4.78 (1H, dd, J = 9.8, 4.1 Hz, H2); 4.03 (1H, dd, J = 11.2, 5.9 Hz, H5eq); 3.87 (1H, app t, J = 11.2 Hz, H5ax); 2.56 (1H, sept, J = 7.0 Hz, CHMe2); 2.53 (1H, sept, J = 7.0 Hz, CHMe2); 2.50 (1H, sept, J = 7.0 Hz, CHMe2); 1.17−1.10 (18H, m, (CH3)2CH). 13C NMR (100 MHz, CDCl3): δ 176.04−175.73 (CO); 87.97 (C1); 70.81 (C2); 69.22 (C3); 67.84 (C4); 62.78 (C5); 34.04−33.93 (CHMe2); 19.04−18.78 (6 × (CH3)2CH). APCI-HRMS (m/z): [M−Br]+ Calcd for C17H27O7+ 343.1751; Found, 343.1757. Synthesis of Guaiacyl 2′,3′,4′,6′-tetra-O-acetyl-β-D-glucopyranoside (1). Under N2, a solution of tetra-O-acetyl-α-D-glucopyranosyl bromide (3.9 g, 9.4 mmol) in acetone (20 mL) was added via cannula into a stirred solution of guaiacol (1.2 g, 9.5 mmol) in 0.3 M NaOH (31.5 mL, 9.5 mmol). The resultant orange reaction mixture was stirred under N2. The pH was monitored and maintained at 12 over a 5 h period using 1 M NaOH. The solution was acetylated with Ac2O (8.5 mL, 90 mmol) and 4-dimethylaminopyridine (DMAP) (10 mg, 0.08 mmol) in pyridine (9 mL, 110 mmol) and stirred overnight at room temperature. The flask was concentrated in vacuo to give a syrup. Et2O/CH2Cl2 (4:1, 150 mL) was added, and the suspension was filtered then washed successively with 0.2 M HCl (2 × 75 mL), water (3 × 50 mL), and brine (3 × 50 mL). The organic layer was dried (Na2SO4), filtered and concentrated in vacuo. The crude product was purified by silica gel column chromatography using 0→5% Et2O/ CH2Cl2 giving a white solid. This was recrystallized from EtOH to give 1.4 g (34%) of 1 as white crystals. Rf 0.33 (10% Et2O/CH2Cl2). 1H NMR data agreed with the literature.13 Synthesis of Guaiacyl β-D-glucopyranoside (2). A suspension of 1 (1.4 g, 3.2 mmol) was stirred in a mixture of MeOH/Et3N/H2O (8:1:1, 25 mL) overnight at room temperature. Additional MeOH/ Et3N/H2O (8:1:1, 5 mL) was added, and the solution was stirred for a further 5 h. The resultant clear solution was concentrated in vacuo, H2O was added, and the solution was concentrated again. This procedure was repeated several times until the solid reached a constant weight. This crude solid was recrystallized from EtOH to give 2 as a white crystalline solid (820 mg, 90%). 1H NMR data agreed with the literature.8 Synthesis of Guaiacyl-2′,3′,4′-tri-O-acetyl-6′-O-trityl-β-D-glucopyranoside (3). Anhydrous pyridine (2.2 mL, 27 mmol) was added via syringe to 2 (0.63 g, 2.2 mmol), DMAP (8 mg, 0.07 mmol), and Ph3CCl (1.1 g, 3.8 mmol) under a N2 atmosphere. The mixture was stirred at 50 °C for 9 h, and then stirred overnight at room temperature. Ac2O (2.2 mL, 23 mmol) was added, and the reaction was stirred for a further 90 min. The flask was placed on ice, MeOH (2.5 mL) was added, and the resultant mixture was stirred for 30 min. The solvent was removed in vacuo, and the crude solid was dissolved in CH2Cl2. This solution was washed sequentially with 0.2 M HCl, water, and brine. The organic layer was dried (Na2SO4), filtered, and concentrated in vacuo. The crude material was purified by silica gel column chromatography eluted with 3→10% Et2O/CH2Cl2 to give a white solid (800 mg, 56%). Rf 0.24 (3% Et2O/CH2Cl2). mp 90−95 °C. [α]D +13.8 (c 0.44, CHCl3). 1H NMR (600 MHz, CDCl3): δ 7.45 (6H, dt, J = 7.7, 1.6 Hz, ortho CPh3); 7.36 (1H,, dd, J = 7.9, 1.4 Hz, H6); 7.28 (6H, tt, J = 7.7, 1.6 Hz, meta CPh3); 7.22 (3H, tt, J = 7.2, 1.6 Hz, para CPh3); 7.09 (1H, td, J = 7.9, 1.4 Hz, H4); 6.93 (1H, dd, J = 2329

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Samples were assessed at 22−24 °C in three or five formal sessions, lasting a maximum of 2 h, under sodium lighting in isolated ventilated tasting booths. Assessors were presented with 15 (smoke affected wines) or 16 (reconstitution study) samples per session, in sets of three or four for the two studies, respectively, with 90 s rest breaks between each sample. Assessors were instructed to rinse their mouths after each evaluation with aqueous citrus pectin solution (1 g/L, Fluka), followed by two rinses of water. There was a forced rest of at least 10 min between each of the sets of three or four samples. Samples (30 mL) were presented in a random and balanced order across the assessors, in coded, covered International Organization for Standardization tasting glasses. Two replications of each of the 20 samples for the smoke-affected wine study and three replications of the 20 reconstitution samples were assessed. FIZZ software (Version 2.46, Biosystemes, France) was used for the collection of all data. Experimental Design of the Reconstitution Study. Different volatiles, singly and in combination; an isolated glycoside fraction from smoke-affected grapes; and synthesized glycosides were added at different concentrations to a red base wine, as specified in Table 1. The

18.84 ((CH3)2CH), 18.81 ((CH3)2CH). ESI−HRMS (m/z): [M + Na]+ Calcd for C36H50NaO17+, 777.2940; Found, 777.2934. Synthesis of Guaiacyl β-Primeveroside (6). A solution of 5 (66 mg, 0.09 mmol) in MeOH (10 mL) containing Amberlite IRA-400(OH) resin (0.5 g) was stirred gently overnight. The reaction mixture was filtered and washed successively with MeOH and water. The combined filtrate was concentrated in vacuo to give the product as a white solid (27 mg, 73%). mp 209.9−210.9 °C. [α]D = −63.9 (c 0.204, H2O). 1H NMR (400 MHz, D2O): δ 7.23 (1H, dd, J = 8.2, 1.3 Hz, H3); 7.15 (1H, td, J = 8.2, 1.3 Hz, H4); 7.12 (1H, dd, J = 8.2, 2.6 Hz, H6); 7.02 (1H, ddd J = 8.2, 6.4, 2.6 Hz, H5); 5.14 (1H, m, H1′); 4.39 (1H, d, J = 7.8 Hz, H1″); 4.12 (1H, dd, J = 12.0, 1.8 Hz H6b′); 3.91 (1 H, dd, J = 11.6, 5.4 Hz, H5eq″); 3.88 (3H, s, H7); 3.86 (1H, dd, J = 12.0, 5.8 Hz, H6a′); 3.75 (1H, ddd, J = 9.6, 5.8, 1.8 Hz, H5′); 3.63 (1H, dd, J = 8.8, 7.6 Hz, H2′); 3.61 (1H, dd, J = 9.6, 8.8 Hz, H3′); 3.59−3.51 (2H, m, H4″, 4′); 3.35 (1H, app t, J = 9.2 Hz, H3″); 3.25 (1H, dd, J = 9.4, 7.8 Hz, H2″); 3.18 (1H, dd, J = 11.6, 10.5 Hz, H5ax″). 13C NMR (100 MHz, D2O): δ 149.47 (C2); 145.85 (C1); 124.60 (C4); 122.19 (C5); 117.07 (C6); 113.71 (C3); 103.80 (C1″); 100.94 (C1′); 76.15 (C3″); 76.08 (C3′); 76.02 (C5′); 73.59 (C2″); 73.44 (C2′); 69.88 (C4′); 69.80 (C4″); 68.78 (C6′); 65.68 (C5″); 56.48 (C7). ESI−HRMS (m/z): [M + Na]+ Calcd for C18H26NaO11+, 441.1367; Found, 441.1364. Isolation of Glycosides from Smoke Affected Grapes. Ten kg of smoke-tainted grapes were homogenized using a knife mill (Grindomix GM 200, Retsch GmbH, Germany) centrifuged at 4000 rpm for 5 min, and the supernatant was slowly (over 20 h) passed through a low-pressure column (capacity 600 mL) of an absorbent polymer resin (Amberlite FPX66, The Dow Chemical Company, Camberwell, Victoria, Australia). After removal of nonabsorbed material with water (5 L) and removal of volatiles with pentane (2.5 L), the glycosidic fraction was eluted from the column with food grade ethanol (2 L), concentrated on a rotary evaporator to dryness, then redissolved in 50 mL of ethanol, and stored at −20 °C until required. The glycosides were quantified as previously reported.14 For the reconstitution study, an aliquot of the ethanolic solution was added directly to the base wine. Chemical Analyses. Standard chemical analysis and quantification of free volatile phenols for all wines were conducted by Commercial Services at the Australian Wine Research Institute (AWRI, Adelaide, Australia), as reported elsewhere.7 Sensory Analysis. The reconstitution and the smoke affected wine sensory studies were conducted several months apart in mid-2011, with the sensory protocols being very similar. Ten panelists were used for the smoke affected wine study (six female), while 11 were used for the reconstitution study (seven female), all were part of the AWRI trained descriptive analysis panel with experience in wine descriptive analysis studies (age range, 32−60), and recruitment and training criteria as previously described.4 All data were collected in accordance with standard institutional procedures, including risk assessment and informed consent, and all samples were expectorated. For both studies, the assessors had three training sessions: two 2 h discussion sessions to generate and define attributes by consensus though assessment of pairs of wines, followed by one practice rating session in isolated booths, where a subset of the samples was presented in duplicate and performance was assessed. For the smoke affected wine study, the assessors rated six aroma (overall f ruit, vanilla, oaky, earthy/dirty, smoky/burnt, and band-aid) and 11 palate attributes (overall f ruit f lavor, vanilla f lavor, oaky f lavor, burnt/charred f lavor, bandaid flavor, bitterness, astringency, hotness, ashy af tertaste, acid af tertaste, and burning af tertaste). For the reconstitution study, ten aroma (overall f ruit, red f ruit, dark f ruit, f resh green, musty/dusty, woody, earthy, smoky, medicinal, and alcohol) and eight palate attributes (overall f ruit f lavor, woody f lavor, smoky f lavor, burnt rubber f lavor, medicinal flavor, bitterness, astringency, and burnt/ashy af tertaste) were rated. For attribute definitions and reference standards used, see SI Tables S4 and S5. The intensity of each attribute was rated using an unstructured 15 cm line scale from 0 to 10, with indented anchor points of “low” and “high” placed at 10% and 90%, respectively.

Table 1. Samples for Sensory Analysis with Details of Concentrations of Each Compound Added to a Base Winea sample code

details

a

base wine all Ph Gu all Cr m-Cr o-Cr p-Cr Syr Gu+4-Me Gu+all Cr low Glyc high Gly

high Gly + all Ph low Gly + all Ph Gu + Gu-Glu Gu-Glu m-Cr-Glu Syr-Glu-Glu wine 18d wine 19

no addition all volatile phenolsb guaiacol 79 μg/L o-cresol 20 μg/L, p-cresol 10 μg/L, m-cresol 16 μg/L m-cresol 16 μg/L o-cresol 20 μg/L p-cresol 10 μg/L syringol 148 μg/L guaiacol 79 μg/L, 4-methylguaiacol 43 μg/L guaiacol 79 μg/L, o-cresol 20 μg/L, p-cresol 10 μg/L, m-cresol 16 μg/L low glycoside-isolate addition (equivalent to 500 μg/L SyrGG) high glycoside-isolate addition (equivalent to 1500 μg/L SyrGG) high glycoside-isolate and all volatile phenols in combinationb low glycoside-isolate and all volatile phenols in combinationb guaiacol 79 μg/L and guaiacol glucoside 500 μg/L guaiacol glucoside 500 μg/L m-cresol glucoside 500 μg/L syringol gentiobioside 500 μg/L smoke-tainted wine 2007, Pinot Noir, Yarra Valley smoke-tainted wine 2009, Shiraz, Yarra Valley

a Base wine: 2010 Merlot bag-in-box wine (2L). bVolatile phenols matching the concentration of phenols of smoke tainted wine (Shiraz, 2007): Guaiacol 79 μg/L, 4-Methylguaiacol 43 μg/L, Syringol 148 μg/ L, Methylsyringol 38 μg/L, o-Cresol 20 μg/L, p-Cresol 10 μg/L, mCresol 16 μg/L. cGlycoside fraction isolated from smoke affected grapes. dFor details of wines 18 and 19, see SI Table S1.

red base wine was a relatively low flavor bag-in-box commercial Shiraz wine with negligible or not detected concentrations of volatile phenols. The concentrations of the compounds added were chosen with regard to measured concentrations in a smoke-tainted wine (Wine 15, SI Table S2). Two smoke-affected wines were also included. Wine 15 was not included due to volume limitations. Stock solutions of all compounds were prepared using 96% v/v food grade ethanol (Tarac Technologies, South Australia, Australia) and stored at 4 °C during the experiment, with appropriate volumes added directly to the base red wine 1 h prior to testing. Sample Preparation. Saliva Collection and Incubation. Saliva was collected from healthy nonsmoking volunteers, with normal 2330

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Figure 2. Partial least-squares regression analysis of volatile phenols, their glycoconjugates and sensory attributes (a) x and y loading plot; x-loading: chemical composition; y-loadings: sensory attributes; Ph: Phenol; Cr: Cresol; Gu: Guaiacol; Mg: 4-Methylguaiacol; Sy: Syringol; Msy: 4Methylsyringol; GG: β-D-glucosyl-β-D-glucosides; PG: Pentosylglucosides; RG: α-L-rhamnosyl-β-D-glucoside (b) scores plot for the 20 wine samples, sample codes as per SI Table S1. salivary flow and olfactory and gustatory function from within the staff of the AWRI, and selected from prior knowledge that they could consistently perceive smoke flavor from tasting glycosides. Samples were taken in duplicate at different days at different times. Note that these subjects were not members of the sensory panels. The salivary flow was increased by chewing Parafilm (Pechiney Plastic Packaging) for approximately 10 min and collected in a test tube through expectoration. At each collection period, saliva (3.0 mL) was transferred to a 22 mL amber vial with screw cap Teflon seal. A standard condition was used with guaiacol monoglucoside added (6.32 μg in 1 mL water) incubated in a water bath (37 °C) and stirred for 30 min on a magnetic stirrer. The reaction was stopped by adding saturated aqueous CaCl2 (2 mL), and the free volatiles quantified. In a single study using a single subject different conditions were examined with standard conditions involving 3 mL saliva, 6.32 μg guaiacol monoglucoside in 2 mL saturated tartaric acid pH 3.5, 0% v/v alcohol, 0 g/L glucose, and no mouthwash, with variables studied involving pH

(adjusted using 2 M NaOH or 2 M tartaric acid); temperature; ethanol concentration; glucose concentration; and chlorhexidine mouthwash rinsing (0.2% w/v chlorhexidine gluconate, GlaxoSmithKline Consumer Healthcare GmbH&Co., Stuttgart, Germany). The latter was investigated following thorough rinsing for one minute with saliva collected immediately after rinsing and after five minutes. Volatile Phenol Analysis. For analysis of the released volatiles by solid-phase microextraction (SPME), deuterated standards (d3guaiacol, d3-4-methylguaiacol, d7-p-cresol, and d3-syringol) were added to the saliva samples in the 22 mL amber vials, after the reaction was stopped. A Gerstel autosampler (MPS) was fitted with a 85 μm PA (polyacrylate) fiber assembly (Supelco) to sample the headspace above the stirred wine sample for 30 min at 50 °C, immediately prior to instrumental analysis. Instrumental analyses were carried out with a Hewlett-Packard (HP) 6890 gas chromatograph and a HP 5973 mass selective detector (Agilent Technologies, Forest Hill, Australia) fitted with a Gerstel autosampler (MPS). 2331

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The gas chromatograph was fitted with a 60 m × 0.25 mm J&W fused silica capillary column DB-WAX, 0.25 μm film thickness. The carrier gas was helium, linear velocity was 33 cm/sec, and flow rate was 1.4 mL/min. The oven temperature was started at 40 °C, held at this temperature for 2 min, increased to 240 °C at a 10 °C/min rate, and held at this temperature for 10 min. The injector temperature was 220 °C, and the transfer line was held at 280 °C. For quantification, mass spectra were recorded in Selective Ion Monitoring (SIM) mode. The ions used for quantification in SIM runs were m/z 127 for d3-guaiacol; m/z 124 for guaiacol; m/z 141 for d3-4-methylguaiacol; m/z 138 for 4methylguaiacol; m/z 115 for d7-cresol; m/z 108 for cresol; m/z 157 for d3-syringol; and m/z 154 for syringol. In Mouth Measurements with SBSE (Stir Bar Sorptive Extraction). This method was modified from the BOSS System, developed by Buettner et al.15,16 A SBSE bar (film thickness 1.0 mm, 20 mm length, Gerstel, Germany) was held in the mouth for 5 min, by attaching it to a very thin spatula and rotating it by the subject. The lips and velum were kept closed, and the spatula was moved carefully within the oral cavity. After equilibration, the SBSE bar was removed from the oral cavity, dipped into water, briefly dried with lint-free tissue, and placed in individually sealed glass liners in the autosampler tray for analysis. Thermodesorption of the samples was performed by inserting the glass liners in a thermodesorption unit (TDU, Gerstel GmbH) in combination with a CIS-4 PTV injector (Gerstel GmbH), which allows cryofocusing of the analytes. The sampling parameters were as follows: splitless thermal desorption was performed with the TDU set at 40 °C for 0.2 min and then heated with a rate of 100 to 240 °C for 5 min. Cryofocusing was performed with liquid nitrogen at −100 °C. Injection was performed with a ramp of 12 °C/s from −100 to 240 °C (5 min). The gas chromatographic conditions were as for the SPME analysis. Gas Chromatography-Olfactometry (GC-O). GC-O was performed using an Agilent 6890 gas chromatograph, equipped with a Gerstel MPS2 multipurpose sampler and coupled to an Agilent 5973N mass selective detector. The GC was also fitted with a Gerstel olfactory detection port (ODP) and the flow was split between MS and ODP in a 1:1 ratio. The instrument was controlled with Agilent G1701DA ChemStation software with the Maestro software integrated version 1.3.3.51/3.3. The GC column and oven program was as described for GC-SPME analysis. Data Analysis. Sensory descriptive data for each attribute were analyzed using an analysis of variance (ANOVA) testing for the effects of sample, judge and replicate and their two-way interactions, treating judge as a random effect. Tukey’s HSD was applied as a means comparison test. Principal component analysis (PCA) and partial leastsquares regression (PLS) were carried out using The Unscrambler 10.2 (CAMO Technologies Inc., Woodbridge, NJ). All PLSR analyses were carried out using standardized data, using the PLS2 algorithm with full cross-validation, with the y data set being the sensory attribute scores, and the x data being the chemical compositional data. Following ANOVA of the reconstitution study, contrast tests were performed to assess significant differences between particular pairs of samples.

comparable concentrations of volatile phenols and glycosides of phenols to the control wines (see SI Tables S2 and S3), there were many wines with concentrations substantially higher, with differences much greater than the analytical error (coefficient of variation of less than 15%7,12). Generally, there was a high degree of correlation among the phenolic compounds, with Pearson coefficients above approximately 0.8 for almost all-pair wise correlations, and all being statistically significant (P < 0.05, r > 0.44, n = 20), with the exception of the gentiobioside glycoside of phenol (Ph-GG), which was only weakly and insignificantly related to the other analytes. Wine 15 was highest in almost all the compounds measured. This indicates that smoke exposure gives consistent concentrations of the range of volatile phenols. From the ANOVA of the sensory descriptive analysis data, there were significant differences among the wines for all sensory attributes rated. Considering the fact that these were commercially produced wines with varied extent of oak treatment and of different vintages, and because it was of greatest interest to assess the relationship of the smoke related sensory attributes with the phenol compounds measured for each wine, the fruit and oak related attributes were not considered in subsequent analysis. A partial least-squares regression (PLS) analysis (Figure 2) showed that the smoke related attributes bandaid and smoky burnt aroma, and bandaid, burnt/charred, and ashy af tertaste (palate) were associated strongly with most of the volatile phenols and their glycosides, being rated highest in the wines 15 and 17. The burning af tertaste attribute was strongly associated with the alcohol concentration of the wines, and was not related to the smokerelated attributes. Three factors were considered important to the model, with the third component showing a separation of the bandaid attributes from the other smoke related attributes, and bandaid was more associated with the three cresols (data not shown). There was a good ability to model the smoke related sensory attributes with the chemical data (76% of the variance of the sensory data explained), with the coefficients of determination for predicted versus measured (calibration) being from 0.71 to 0.86 for each of the smoke-related palate attributes. The validation values were also similarly high (0.61 to 0.66), indicating the predictive ability of the model for new samples, not included in the model, was good. Regarding the key in-mouth attribute ashy aftertaste, using a model including only the volatiles (two-factor model, 72% of the variance of the sensory data explained) gave slightly poorer R2 values for this attribute compared to the complete model (R2 predicted vs measured: calibration, x, validation y), while a model including glycosides only (one-factor model, 59% of the variance of the sensory data explained) also gave slightly poorer R2 values for the ashy aftertaste (R2 predicted vs measured: calibration, 0.63, validation 0.59), indicating that both volatile phenols and nonvolatile precursors are contributing to this attribute. While almost all volatile and glycoside conjugates of phenols were important to the model, the analytes with greatest positive contribution were 4-methyl guaiacol, guaiacol, the three volatile cresols, as well as phenol rutinoside (Ph-RG), methylguaiacol rutinoside (MGu-RG), guaiacol rutinoside (GuRG), and cresol rutinoside (Cr-RG). The free volatile compounds syringol and 4-methyl syringol had small negative or negligibly positive regression coefficients. The results of this study confirmed the results of the previous work,4 that multiple volatile phenols and nonvolatile glycoconjugates were together associated with smoky off-flavor. The

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RESULTS AND DISCUSSION Sensory Investigations. The importance of nonvolatile glycoconjugates of phenols relative to volatile phenols to the inmouth characteristics of smoke-affected wines was assessed in two ways, first with an examination of wines made from grapes affected by bushfire events, and second by a reconstitution study with volatiles and nonvolatiles added to a base wine. An initial study was aimed at confirming the importance of volatile phenols and their glycosides on the sensory of smokeaffected wines, which was shown in an earlier investigation,4 using wines with a wider range of phenol concentrations, and to allow selection of a wine with known composition as point of reference for the subsequent reconstitution study. While some wines made from potentially smoke-affected grapes had 2332

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Figure 3. Principal component analysis biplot of the flavor sensory attribute mean values for wines with added volatiles (△), glycoconjugates (■), or both (●).

From the ANOVA, the attributes medicinal f lavor, smoky f lavor, and burnt ashy af tertaste were rated significantly different across the samples. The attributes smoky f lavor and burnt ashy af tertaste were highly correlated (r = 0.74, n = 18). Figure 3 shows the PCA for the significant flavor attributes. The major separation along PC1, which accounted for close to 70% of the variance in the sensory data, related to differences in ratings for burnt ashy af tertaste and smoky flavor. Separation along PC2 was related mainly to differences in medicinal flavor. The base wine, plotted to the lower left quadrant of the figure, was rated low in each of these attributes. The addition of syringol and the individual cresols (o-, m-, and p-cresol) had only a small effect on the flavor properties of the base wine, while the base wine with the three cresols added together (All Cr) was shifted strongly to the right and to the upper half of the figure, demonstrating an additive effect of the three compounds together, giving higher medicinal and smoky flavor. The samples with guaiacol alone (Gu) and guaiacol with 4-methyl guaiacol (Gu+4-Me) were rated higher again in burnt/ashy and smoky flavor, with the Gu+4-Me wine being lower in the medicinal flavor score. The combination of guaiacol and cresols (Gu + All Cr) had a very high rating in the smoky attributes. Interestingly, the All Ph sample, was rated lower in the burnt/ashy and smoky f lavor attributes than the Gu and all Cr samples, indicating a suppressing effect of the other phenols in the mixture. Assessing statistical significance for these two attributes, there was a trend (P = 0.097) for the All Ph sample to be rated lower in burnt/ashy af tertaste than the Gu sample, while other pairwise comparisons for All Ph, Gu, and All Cr were not significant. Regarding the glycosides, the wine with added syringol gentiobioside (Syr-Glu-Glu) was rated very similarly to the base wine, while the Gu-Glu sample was rated significantly higher only for the medicinal attribute. The m-cresol glucoside was rated substantially higher in burnt/ashy and smoky, at a similar intensity to the free guaiacol alone. Importantly, for the glycoside fraction isolated from the smoke affected grapes,

composition of the wine 17 was used as the basis for a confirmatory study using a reconstitution approach to further assess the sensory significance of the volatile phenols and the nonvolatile glycosides. Reconstitution Sensory Study. Descriptive sensory analysis was used to characterize differences in the flavor attributes of a red wine, after addition of volatiles, synthetic glycoconjugates, fractions containing glycoconjugates isolated from smoke-affected grapes, or combinations thereof. The combinations used were based on hypotheses based on results of the initial sensory correlative study and a previous study,4 so that the relative influence of individual volatile phenols and combinations, notably guaiacol, 4-methyl guaiacol and the cresols, were investigated at concentrations that were found in the smoke-affected wine 17 (see SI Table S1). Also included in the sensory descriptive analysis study were two smoke-affected wines (wines 18 and 19, see SI Table S1) as reference samples. Table 1 provides details of the 20 samples included. From an inspection of the data for the 20 samples, it was noted that the smoke-affected wines (Wines 18 and 19) were rated highly in several attributes, including “woody” and “fresh green”, which were not evident in the reconstitution samples or the base wine, and these two wines (Wines 18 and 19) were rated substantially higher for the smoke related attributes. To assess the various additions, the two wines made from smokeaffected grapes were excluded, and ANOVA was conducted for the remaining 18 samples listed in Table 1. Aroma attributes were rated for the samples as well as in-mouth attributes, and from the ANOVA there were no significant differences between any of the glycoside samples with no addition of volatiles (that is Low Gly, High Gly and m-Cr-Glu, and Syr-Glu-Glu and GuGlu) and the base wine in the smoke and medicinal aroma attributes, as determined by contrast tests. The concentration of free volatile phenols in the glycoside samples was also found to be negligible, and below the detection limit of the analytical method. Accordingly, only the palate attributes were investigated further. 2333

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there was a perceptible flavor effect for the low Gly sample, being rated significantly higher in medicinal (P = 0.05) compared to the base wine, while the high Gly was rated significantly higher in smoky flavor compared to the base wine (P = 0.02). As expected, the glycoside samples with volatile phenols added were rated higher in the smoky attributes than their counterparts with no volatiles added, so that the (Gu+Gu-Glu) wine was rated moderately highly in the smoke related attributes, at a concentration similar to that of the guaiacol addition alone, and the high Gly+ All Ph sample was overall rated as most different from the base wine, being situated to the far right of Figure 3, and substantially and significantly higher in scores for these attributes than the (All Ph) wine, confirming that the glycoside fraction contributed flavor. Breakdown of Glycoconjugates with Incubation of Saliva. Results from the reconstitution study confirmed a direct sensory effect of glycosidically bound volatiles on smoke related flavor attributes. In addition, previous studies had demostrated that monoglucosidically bound volatiles in a model wine system or water could give rise to a smoky flavor in-mouth and that the respective volatiles were released.4 These investigations from Parker et al.4 indicated that the release of volatiles is highly variable across individuals. A series of investigations were conducted to gain a better understanding of the variability between individuals and to assess factors responsible for differences in release of phenols through of glycoside hydrolysis. Table 2 presents a summary of a set of experiments aiming to assess release of guaiacol from incubation with saliva from four

was lower when incubated at room temperature and nearly totally inhibited when incubated at 0 °C in comparison to body temperature (37 °C). Table 3. Percentage Release of Guaiacol from Guaiacol Monoglucoside Following Incubation with Saliva in Vitro From One Subjecta with Each Value Obtained As a Single Replicate As a Function of Temperature, pH, Alcohol Concentration, Glucose Concentration, and Time after Rinsing with Chlorhexidine Antibacterial Mouthwashb condition temperature (°C) 37 20 0 pH 7.0 5.6 3.5 ethanol (% v/v) 0 10 15 glucose (g/L) 0 3 45 time after chlorhexidine mouthwash (min) 0 5

A B C D a

mean release (standard deviation) % 17 31 12 63

(6) (13) (8) (5)

minimum release (%)

maximum release (%)

number of replicates

9.7 21.0 3.8 57.0

25 52 20 68

6 9 4 4

18 6 n.d.c 22 17 n.d. 39 18 6 55 5 4 0.2 0.1

a

Subject A from Table 1. bUnless otherwise stated, conditions were 30 min incubation, 3 mL saliva, 6.32 μg guaiacol monoglucoside in 2 mL saturated tartaric acid pH 3.5, 37 °C, 0% v/v alcohol, 0 g/L glucose, no mouthwash. cNot detected.

Table 2. Release of Guaiacol from Guaiacol Monoglucoside Following Incubation with Saliva Obtained from Four Subjects in Vitroa subject

release (%)

The alcohol concentration was found to have a strong effect on the release of guaiacol (Table 3), with the release decreasing from 39% to 6.3% with increasing ethanol concentration. However, the presence of alcohol did not lead to a complete inhibition of the guaiacol release. The same effect could be seen for a high glucose concentration, with 3 g/L of glucose leading to a strong decrease in the guaiacol release from 55% to 5%. Another inhibiting effect of the release could be seen by using antibacterial mouthwash. The use of chlorhexidine mouthwash completely inhibited the release of guaiacol from the glucoside for up to 2 h after application. These results are in agreement with the findings that bacterial microorganisms in saliva are the agents for release.17 Beside guaiacol, monoglucosides of syringol and m-cresol were also investigated. The mean release of six measurements for each glycoside was determined (Table 4). The proportion of both compounds released was high, similar to that of guaiacol glucoside. Most volatile phenols in grapes and wine are found as disaccharide glycosides, and some in relatively high concentrations.7 Therefore, the interaction of these glycoconjugates with saliva is of great interest. The degradation of syringol gentiobioside (2,6-dimethoxyphenyl-1-O-β-D-glucopyranosyl(1→6)-β-D-glucopyranoside) and guaiacol primeveroside (2methoxyphenyl-6-O-(β-D-xylopyranosyl)-β-D-glucopyranoside) was studied, and the results are shown in Table 4. The syringol gentiobioside reacted with the saliva in a similar way to its

30 min incubation, 37 °C.

individuals. Subject A had a mean release of guaiacol from its monoglucoside of 17% (release range of 9.7−25%). A comparison across the four different subjects showed that person D had a greater, and more consistent, activity to release guaiacol than the other subjects. The wide variation among individuals may be due to differing mouth biota and will be investigated in future studies. For this study, the glucosides, dissolved in water, were added directly to human saliva. When the glycosides were added to a model wine (pH 3.5, 12% v/v alcohol) and added to the saliva, the release was strongly reduced, so that there was a complete inhibition if saliva was mixed with model wine in a 3:2 ratio. A series of samples were examined to assess the influence of the model wine system on the release, in a single replicate for each condition from a single subject. Replication was not possible due to limitations in quantity of the guaiacol glucoside, but the results are comparable across treatments to provide screening data of the most important variables influencing release. As expected, the release decreased with lowering of the pH, with a total inhibition at a pH of 3.5 (Table 3). The release of guaiacol 2334

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Table 4. Release of Guaiacol, Syringol and m-Cresol from Mono- and Disaccharide Glycosides Following Incubation with Saliva in Vitroa glycoside syringol glucoside m-cresol glucoside syringol gentiobioside guaiacol primeveraside

samples, and Con Simos and Adrian Coulter for arranging the samples. The Australian Wine Research Institute (AWRI), a member of the Wine Innovation Cluster on the Waite precinct in Adelaide, is supported by Australian grapegrowers and winemakers through their investment body the Grape and Wine Research and Development Corporation (GWRDC), with matching funds from the Australian Government.

release (standard deviation) % 40 50 27 3

(8) (9) (3.7) (2.4)

■

30 min incubation, two subjects, 37 °C, mean of three replicates per subject.

a

(1) Høj, P.; Pretorius, I. S.; Blair, R. The Australian Wine Research Institute, Annual Report, 2003, p 37. (2) Prida, A.; Chatonnet, P. Impact of Oak-Derived Compounds on the Olfactory Perception of Barrel-Aged Wines. Am. J. Enol. Vitic. 2010, 3, 408−413. (3) Kennison, K. R.; Wilkinson, K. L.; Williams, H. G.; Smith, J. H.; Gibberd, M. R. Smoke-Derived Taint in Wine: Effect of Postharvest Smoke Exposure of Grapes on the Chemical Composition and Sensory Characteristics of Wine. J. Agric. Food Chem. 2007, 55, 10897−10901. (4) Parker, M.; Osidacz, P.; Baldock, G.; Hayasaka, Y.; Black, C.; Pardon, K. H.; Jeffery, D. W.; Geue, J. P.; Herderich, M. J.; Francis, I. L. Contribution of Several Volatile Phenols and Their Glycoconjugates to Smoke-Related Sensory Properties of Red Wine. J. Agric. Food Chem. 2012, 60, 2629−2637. (5) Kennison, K. R.; Gibberd, M. R.; Pollnitz, A. P.; Wilkinson, K. L. Smoke-Derived Taint in Wine: The Release of Smoke-Derived Volatile Phenols During Fermentation of Merlot Juice Following Grapevine Exposure to Smoke. J. Agric. Food Chem. 2008, 56, 7379− 7383. (6) Singh, D. P.; Chong, H. H.; Pitt, K. M.; Cleary, M.; Dokoozlian, N. K.; Downey, M. O. Guaiacol and 4-Methylguaiacol Accumulate in Wines Made from Smoke-Affected Fruit Because of Hydrolysis of Their Conjugates. Aust. J. Grape Wine Res. 2011, 17, S13−S21. (7) Hayasaka, Y.; Baldock, G. A.; Parker, M.; Pardon, K. H.; Black, C. A.; Herderich, M. J.; Jeffery, D. W. Glycosylation of Smoke-Derived Volatile Phenols in Grapes as a Consequence of Grapevine Exposure to Bushfire Smoke. J. Agric. Food Chem. 2010, 10989−10998. (8) Hayasaka, Y.; Dungey, K. A.; Baldock, G. A.; Kennison, K. R.; Wilkinson, K. L. Identification of a Beta-D-Glucopyranoside Precursor to Guaiacol in Grape Juice Following Grapevine Exposure to Smoke. Anal. Chim. Acta 2010, 660, 143−148. (9) Hayasaka, Y.; Baldock, G. A.; Pardon, K. H.; Jeffery, D. W.; Herderich, M. J. Investigation into the Formation of Guaiacol Conjugates in Berries and Leaves of Grapevine Vitis vinifera L. Cv. Cabernet Sauvignon Using Stable Isotope Tracers Combined with HPLC-MS and MS/MS Analysis. J. Agric. Food Chem. 2010, 58, 2076− 2081. (10) Pollnitz, A. P.; Pardon, K. H.; Sykes, M.; Sefton, M. A. The Effects of Sample Preparation and Gas Chromatograph Injection Techniques on the Accuracy of Measuring Guaiacol, 4-Methylguaiacol and Other Volatile Oak Compounds in Oak Extracts by Stable Isotope Dilution Analyses. J. Agric. Food Chem. 2004, 52, 3244−3252. (11) Aihara, K.; Urano, Y.; Higuchi, T.; Hirobe, M. Mechanistic Studies of Selective Catechol Formation from o-Methoxyphenols Using a Copper (II)-Ascorbic Acid-Dioxygen System. J. Chem. Soc., Perkin Trans. 2 1993, 2165−2170. (12) Hayasaka, Y.; Parker, M.; Baldock, G. A.; Pardon, K. H.; Black, C. A.; Jeffery, D. W.; Herderich, M. J. Assessing the Impact of Smoke Exposure in Grapes: Development and Validation of a HPLC-MS/MS Method for the Quantitative Analysis of Smoke-Derived Phenolic Glycosides in Grapes and Wine. J. Agric. Food Chem. 2013, 61, 25−33. (13) Zhong, F.-Y.; Zhou, J.-H. 6-Acetoxymethyl-2-(2Methoxyphenoxy)tetrahydropyran-3,4,5-Triyl Triacetate. Acta Crystallogr. 2005, E61, o2701−o2703. (14) Dungey, K. A.; Hayasaka, Y.; Wilkinson, K. L. Quantitative Analysis of Glycoconjugate Precursors of Guaiacol in Smoke-Affected Grapes Using Liquid Chromatography−Tandem Mass Spectrometry

monoglucoside, with a mean of six measurements giving 27% release. A much lower mean release of 3% resulted for the guaiacol primeveroside, indicating that the active principle in saliva, presumably bacterial enzyme activity, is able to hydrolyze both mono- and disaccharides, with evidence for variability in activity according to the nature of the sugar and the aglycone. In addition, the release of free volatiles was confirmed in vivo, by placing SBSE bars in mouth after holding the glycoside solution in mouth. Surprisingly, free guaiacol and syringol could be detected, by means of GC-olfactometry, up to 15 min after spitting out the glycoside solution In conclusion, monoglucosides and disaccharide glycosides can be released by the activity of saliva, with a great dependency on the glycoside and the individual. The release is greatly affected by a low pH, alcohol, and glucose concentration. Considering the buffering properties and alcohol dilution of saliva, as well as rapidly elevated temperature following ingestion, hydrolysis of glycosides is likely to continue as long as there is residual wine in the oral cavity. The decrease in release following chlorhexadine mouth wash, as observed previously in studies on flavonoid glucosides,7 provides further evidence that it is in-mouth bacteria that play a role in the hydrolysis, with previous studies indicating the presence of gram-positive cocci in tetrads and gram-negative diplococci,7 although oral epithelial cells may also be involved.7 The reconstitution study clearly showed that the glycosidically bound volatiles have a direct impact on perceived smoke flavor, including aftertaste, and together with the data from the in vitro experiments, it is apparent that the breakdown of glycosides in mouth, including disaccharide glycosides, is an important mechanism of flavor perception in wine.

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ASSOCIATED CONTENT

S Supporting Information *

Basic wine chemical composition and vintage and variety details; free volatile phenol and glycoconjugate concentration of the red wines; and sensory attributes and their definitions used in the two sensory descriptive analysis studies. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Phone: +61 883036600; fax: +61 883036601; e-mail: leigh. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the sensory panelists, Dr. David Jeffery for advice with the synthesis, Belinda Bramley for assistance with the sensory panel, the Victorian wine industry for donating 2335

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Based Stable Isotope Dilution Analysis. Food Chem. 2011, 126, 801− 806. (15) Buettner, A. Investigation of Potent Odorants and Afterodor Development in Two Chardonnay Wines Using the Buccal Odor Screening System (BOSS). J. Agric. Food Chem. 2004, 52, 2339−2346. (16) Buettner, A.; Otto, S.; Beer, A.; Mestres, M.; Schieberle, P.; Hummel, T. Dynamics of Retronasal Aroma Perception During Consumption: Cross-Linking On-Line Breath Analysis with MedicoAnalytical Tools to Elucidate a Complex Process. Food Chem. 2008, 108, 1234−1246. (17) Hemingway, K. M.; Alston, M. J.; Chappell, C. G.; Taylor, A. J. Carbohydrate-Flavor Conjugates in Wine. Carbohydr. Polym. 1999, 38, 283−286.

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