Liberation of Hydrogen Sulfide from Dicysteinyl Polysulfanes in Model

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Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Liberation of Hydrogen Sulfide from Dicysteinyl Polysulfanes in Model Wine Marlize Z. Bekker,*,† Gal Y. Kreitman,‡ David W. Jeffery,§ and John C. Danilewicz⊥ †

The Australian Wine Research Institute, P.O. Box 197, Glen Osmond, South Australia 5064, Australia 134 Western Avenue, Morristown, New Jersey 07960, United States § School of Agriculture, Food and Wine, Waite Research Institute, The University of Adelaide, PMB 1, Glen Osmond, South Australia 5064, Australia ⊥ 44 Sandwich Road, Ash, Canterbury, Kent CT3 2AF, United Kingdom J. Agric. Food Chem. Downloaded from pubs.acs.org by UNIV OF SOUTH DAKOTA on 12/12/18. For personal use only.

‡

S Supporting Information *

ABSTRACT: Diorganopolysulfanes can be generated when hydrogen sulfide (H2S) and thiols are oxidized in the presence of Cu(II) under conditions usually aimed at removing H2S from wine. This work sought to understand if polysulfanes could act as latent sources of H2S during postbottling storage. The stability of the polysulfanes formed in situ in model wine containing cysteine, H2S, and transition metals was dependent both on the number of sulfur linking atoms (Sn) and on the presence of a reducing agent, such as sulfur dioxide or ascorbic acid. A polysulfane containing three linking sulfur atoms was the most stable, with 84% of the relative initial amount remaining in solution after six months, compared to polysulfanes containing four or more linking sulfur atoms that decomposed rapidly, with 26% remaining after six months. Importantly, sulfur dioxide was associated with the rapid degradation of polysulfanes and subsequent liberation of H2S. Three cysteine-S-sulfonates were also tentatively identified, which gives insight into the possible release mechanisms involved with H2S reappearance. KEYWORDS: polysulfanes, thiosulfates, hydrogen sulfide, cysteine, reductive aromas, wine, antioxidants

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which are not readily removed by filtration.10 As outlined previously, two thiols likely coordinate to Cu(II) to form an initial complex (1) as depicted in Figure 1A,10,11 whereupon it is proposed that electron transfer from one sulfur atom produces the Cu(I) complex, which dimerizes (2), allowing S− S bond formation to give the disulfide. The Cu(I) complex then aggregates and Cu(II) is regenerated in the presence of oxygen and Fe(III) to continue the process of thiol oxidation. Importantly, only recently have a number of nonvolatile thiols (e.g., cysteine and glutathione, RSH) been considered.10−12 These are present in large molar excess to H2S (R = H) so it is envisaged that an organodisulfane (RSSH) would result. When RSSH is incorporated into the cycle as in (3), a diorganotrisulfane (RSSSR) (4) would be generated and other diorganopolysulfanes (referred to simply as “polysulfanes” from here on), including mixed analogues (i.e., RSnR′), could form as H2S is further incorporated into an organopolysulfane. Highlighting these reactions, it has been demonstrated in model wine that the oxidation of a mixture of H2S and cysteine in the presence of Cu(II) produced putative polysulfanes with up to six-linked sulfur atoms, and with glutathione (GSH) in place of cysteine, putative polysulfanes with up to seven linked sulfur atoms were found.11 Polysulfanes were also observed upon oxidation of a commercial white wine spiked with GSH and H2S (and

INTRODUCTION The origin and management of volatile sulfur compounds (VSCs) in wine concerns many winemakers, as these compounds have a significant impact on wine aroma attributes and wine quality. Whereas some VSCs can be associated with varietal characters and viewed positively, hydrogen sulfide (H2S) is one of the primary compounds responsible for negative “reductive” aroma characters in wine,1,2 due to imparting aromas of rotten egg and sewage when present in concentrations above its odor threshold (OT) of 1.1 to 1.6 μg/ L.3 H2S is produced during fermentation, and although much is lost due to the purging action of CO2 that is also produced, H2S may remain above its OT and be problematic in the resulting wine.4 Commonly, H2S is removed by aeration and/ or Cu(II) addition (in a process known as copper fining),5 but this treatment may be temporary as H2S (and other VSCs such as methanethiol) can often reappear postbottling, when reductive conditions are re-established.6,7 Hypotheses invoke that the above oxidative treatments produce substances, which, though temporarily stable, are vulnerable to reductive cleavage. As such, likely latent sources of VSCs involve metal-bound sulfides and S−S compounds (i.e., disulfides and diorganopolysulfanes).8 It had been assumed that upon Cu(II) fining, H2S formed the highly insoluble Cu(II)S that is easily removed by filtration, but this is not the case.9 It is important to take into account that the H2S is accompanied by a large molar excess of endogenous nonvolatile thiols such as cysteine, N-acetylcysteine, and homocysteine, as well as glutathione. Thiols and H2S rapidly reduce Cu(II), and the resulting Cu(I) complex apparently aggregates to produce polynuclear nanoclusters, © XXXX American Chemical Society

Received: August 29, 2018 Revised: November 2, 2018 Accepted: November 10, 2018

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DOI: 10.1021/acs.jafc.8b04690 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Once produced, polysulfanes are proposed to act as latent VSC sources in wine that may release sulfhydryls by nucleophilic S−S bond cleavage by thiolate ions, which may be further assisted by coordination of a linking sulfur to a metal.8 The resulting hydrosulfide would then undergo further degradation [Figure 1C].8 However, thiolate ions will be present at very low concentrations at wine pH. More likely is that polysulfanes might act as latent H2S sources through a sulfitolysis mechanism, as shown for the cleavage of a disulfide [Figure 1D],24 or possibly through the reduction of the disulfide bonds by ascorbic acid.25 The potential degradation of polysulfanes as a result of sulfitolysis or ascorbic acid reduction is of particular interest as both sulfur dioxide and ascorbic acid are routinely used as wine additives. The sulfitolysis of disulfides or polysulfanes proceeds through an SN2 reaction.8,24 Arapitsas et al.26 reported evidence of the sulfitolysis of GSSG produced by the oxidation of GSH, resulting in the formation of GSSO3H and demonstrating in real wines that GSSG formed through the oxidation of GSH is rapidly converted into GSSO3H (with minor concentrations of GSH) when sulfur dioxide was added to wines. This outcome explains why GSSG is not usually reported in wines. Therefore, it seems plausible that similar cleavage reactions could ultimately result in the release of H2S from polysulfanes in wine, potentially via sulfitolysis of RSSxH or another unknown mechanism. Based on the notion that mono- and diorganopolysulfanes can form when H2S is oxidized in the presence of nonvolatile thiols, the aim of this study was to determine conditions under which putative polysulfanes could rerelease H2S postbottling. In particular, we investigated the ability of dicysteinyl polysulfanes to liberate H2S over time under reductive conditions established by treating polysulfanes with sulfite, ascorbic acid, or catechin (alone or in combination) in model wine stored anoxically.

Figure 1. Proposed mechanism for the Cu-mediated reaction of H2S with thiols to produce polysulfanes, showing only the thiol ligands (A,B); SN2 reaction mechanism of thiol-diorganopolysulfane exchange (C) and sulfitolysis of a disulfide and acid-catalyzed cleavage of organic thiosulfate (D).8 The R groups may or may not be the same organic group, such as cysteine or GSH, for example, leading to the formation of symmetric or asymmetric polysulfanes. Adapted with permission from ref 11.

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methanethiol) to facilitate compound identification.11 The significance of polysulfanes to the regeneration of VSCs during storage still needs to be investigated, however. In contrast, bulk metal sulfides (e.g., CuS, FeS) arising through reaction of sulfhydryls and transition metals present in wine (either naturally or added as in the case of copper fining) have been implicated in playing an important role in the liberation of H 2 S in wine postbottling under anoxic storage;13−15 however, a portion of liberated H2S remains unexplained. One suggestion involves desulfurization of cysteine or GSH to liberate H2S in wine postbottling,16 but cleavage of a sulfur−carbon bond should not proceed readily in wine without high temperature catalysis17−19 or microbial action.20 Although direct desulfurization of cysteine (and analogues) and GSH to liberate H2S appears unlikely as a way of directly accounting for much of the H2S that arises postbottling, it has been shown that when a Sauvignon Blanc wine was treated with both GSH and copper and stored under anoxic conditions, a positive correlation existed between increased GSH and increased copper concentrations and H2S accumulation;21 it seems possible that these relatively abundant thiols could be involved in regenerating H2S via their role in polysulfane formation. Alternatively, they may displace H2S from a metal complex. Polysulfanes may potentially be generated in the presence of Cu as outlined earlier,10,11 as well as from the degradation of elemental sulfur pesticide residues,22 or possibly through yeast-mediated polysulfane production during fermentation.23

MATERIALS AND METHODS

Chemicals. All chemicals were of analytical reagent grade, and water was obtained from a Milli-Q purification system (Millipore, North Ryde, NSW, Australia). All chromatographic solvents were of high pressure liquid chromatography (HPLC) grade and purchased from Rowe Scientific (Lonsdale, SA, Australia). L-Ascorbic acid (99%), (+)-catechin hydrate (98%), L-cysteine hydrochloride (99%), ethylmethyl sulfide (EMS, 96%), formic acid (98%), potassium metabisulfite (PMS, 98%), and sodium sulfide nonahydrate (Na2S· 9H2O, 98%) were obtained from Sigma-Aldrich (Castle Hill, NSW, Australia). Iron(III) sulfate hydrate (97%) was purchased from Acros Organics (Thermo Fisher, Scoresby, VIC, Australia). Ethanol (HPLC grade), copper(II) sulfate (99%), tartaric acid (99.5%), and sodium chloride (99.5%) were obtained from Merck (Frenchs Forest, NSW, Australia). Preparation of Cysteine Polysulfanes. Model wine (3 L) was prepared in a 5 L Schott bottle with a Teflon-lined cap fitted with a sampling port and valve, by dissolving tartaric acid (5 g/L) in water, followed by the addition of ethanol to yield a final concentration of 12% v/v. The solution was adjusted to pH 3.6 with sodium hydroxide (10 M) and brought to volume with water. Dicysteinyl polysulfanes were generated by adding L-cysteine (500 μM) and Na2S·9H2O (250 μM) to the model wine, followed by Fe(III) (100 μM) and Cu(II) (50 μM), and the solutions were thoroughly mixed. The model wine solution was stored in the dark at room temperature under air. After 24 h the headspace of the model wine was analyzed for H2S by fitting a H2S detector tube (Kitagawa, Japan), loaded with lead acetate/ copper sulfate to the sampling port of the Schott bottle cap, opening the valve, and aspirating 100 mL of headspace through the detector tube using the aspirating pump, as per the manufacturer instructions. B

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Figure 2. Structures of the dicysteinyl polysulfanes determined by LC−MS. The measurement was repeated every 24 h. No H2S was measurable 48 h after the model wine solution was prepared. The solution was then degassed by bubbling helium though a 2 μm sinter for 40 min until the oxygen concentration was 50 μg/L according to Presens oxygen measurement (Presens, Regensburg, Germany) and transferred to an anaerobic hood. Preparation of Polysulfane Solutions to Study the Release of H2S. Stock solutions of ascorbic acid, sulfur dioxide (as PMS), and (+)-catechin were prepared with complete oxygen exclusion. Degassed Milli-Q water (oxygen concentration ≤ 20 μg/L) and ethanol (oxygen concentration ≤ 0.5 μg/L) were placed inside an anaerobic hood. Stock solutions of ascorbic acid (120 mM) and sulfur dioxide (144 mM) were prepared in an anaerobic hood using degassed Milli-Q water, and the stock solution of (+)-catechin (37 mM) was prepared using degassed ethanol. Five sets of triplicate samples consisting of five treatments were prepared. For each sample, vials were almost completely filled with 24 mL of the degassed model wine solution containing the prepared dicysteinyl polysulfanes and sealed with Teflon lined solid caps (Sigma-Aldrich, Castle Hill, NSW, Australia). The treatments were prepared by consecutively adding 100 μL of the 120 mM ascorbic acid stock solution, 100 μL of the 144 mM sulfur dioxide stock solution, and 200 μL of the 37 mM (+)-catechin stock solution as appropriate to give the following treatments: (1) Control (no addition); (2) AA (500 μM of ascorbic acid); (3) SO2 (600 μM sulfur dioxide); (4) SO2 + AA (600 μM sulfur dioxide, 500 μM ascorbic acid); and (5) SO2 + Cat [600 μM sulfur dioxide, 310 μM (+)-catechin]. Samples were stored in boxes to protect from light exposure inside the anaerobic hood at 20 °C until required for analysis. Each set of samples was used per time point [Day 1 (24 h after treatment), Day 3, Day 6, Month 1, and Month 6], with 10 mL subsampled for SCD analysis of VSCs and 2 mL subsampled for LC− MS analysis of dicysteinyl polysulfanes. The remainders of the samples for each time point were stored at −20 °C. Preparation of Cysteine Control Samples. Model wine was prepared inside an anaerobic hood using degassed ethanol (oxygen concentration ≤0.5 μg/L) and degassed Milli-Q water (oxygen concentration ≤ 20 μg/L), as previously described. Stock solutions of L-cysteine (120 mM), Cu(II) (12 mM), and Fe(III) (24 mM) using preweighed neat compounds and degassed Milli-Q water were prepared inside an anaerobic hood. The control samples were prepared by making appropriate additions of 100 μL of the 120 mM L-cysteine solution, 100 μL of the 12 mM Cu(II) solution, 100 μL of the 24 mM Fe(III) solution, 100 μL of the 120 mM ascorbic acid solution, and 100 μL of the 144 mM sulfur dioxide solution to 24 mL of model wine to give the following treatments (and nominal concentrations): (1) Cys (500 μM L-cysteine); (2) Cys + metals [500 μM L-cysteine, 50 μM Cu(II), 100 μM Fe(III)]; (3) Cys + SO2 + AA (500 μM L-cysteine, 600 μM sulfur dioxide, 500 μM ascorbic acid). Chemical Analyses. Metals Analysis. The concentrations of the Cu(II) and Fe(III) stock solutions were confirmed after preparation

using an inductively coupled plasma mass spectrometer (ICPMS, Nexion 350D PerkinElmer, United States) by The Australian Wine Research Institute (AWRI) Commercial Services. The following settings were used: RF power 1400 W, plasma argon flow rate 18 L/ min, nebulizer flow rate 0.75−0.80 L/min. Solutions were aspirated through a PFA MicroFlow 400 μL/min nebulizer into a quartz cyclonic spray chamber cooled to 2 °C. Samples were introduced to the system via an S10 autosampler connected to an ESI-FAST switching valve, with a 500 μL polytetrafluoroethylene sample loop. Online (postsampling) dilution of approximately 1:5 was achieved by adding internal standard solution containing 50 μg/L of yttrium in 2% v/v HNO3 (Suprapur grade, Merck Millipore) and 2% v/v ethanol (RCI Chemicals) to the sample inflow via a T-piece and appropriate diameter peristaltic pump tubing, with a pump rate of 5 rpm. The ICPMS was calibrated using solutions of known concentration (1− 1200 μg Cu/L, 50−5000 μg Fe/L) in a matrix of 2% v/v HNO3 and 12% v/v ethanol in Milli-Q water. Oxygen Measurement. Oxygen concentration of the degassed model wine solution, as well as of degassed Milli-Q water and degassed ethanol was determined using a PreSens PSt6 oxygen dip-in probe connected to a PreSens Fibox 3 trace v3 oxygen meter (Presens, Regensburg, Germany), with a detection limit of the PreSens PSt6 dip-in probe of 0.5 μg/L. Gas Chromatography Coupled to Sulfur Chemiluminescence Detection. The treatment and control samples were analyzed for H2S at each time point using an Agilent 355 sulfur chemiluminescence detector (SCD) coupled to an Agilent 6890A gas chromatograph (GC) (Forest Hill, VIC, Australia). The GC-SCD system was equipped with a Gerstel multipurpose sampler (MPS 2XL, Lasersan Australasia, Robina, QLD, Australia) controlled by Maestro software, integrated version 1.3.9.13/3. Instrument control and data analysis were performed with Agilent GC ChemStation software, rev. B.04.02 [96]. The method described by Siebert et al. was used without modification.27 Liquid Chromatography−Mass Spectrometry Analysis of Polysulfanes. Analysis of polysulfanes was carried out with an Agilent 1200 HPLC system apart from a binary pump (Agilent Infinity 1290) (Agilent Technologies, Santa Clara, CA, USA) coupled with a Sciex 4500 triple quadrupole mass spectrometer (AB Sciex, Framingham, MA, USA). Chromatographic separation was performed with an Altima C18 column (250 × 2.1 mm, 5 μm, 100 Å, Grace Davison) under gradient conditions with mobile phase A composed of 0.5% formic acid in water and mobile phase B composed of 0.5% formic acid in acetonitrile. The mobile phase was delivered at a flow rate of 0.2 mL/min using the following gradient program: 1 to 10% of B in 10 min, 10 to 80% B in 10 min, then 80% B for 5 min. The column was re-equilibrated for 10 min before the next injection (35 min analysis time). The column oven temperature was set to 25 °C, and the injection volume was 10 μL. The mass spectrometer was equipped with a Turbo V ion source and operated in positive electrospray ionization (ESI) mode. The ESI capillary voltage was set at 5500 V in positive ion mode with a declustering potential of 60 V. The mass C

DOI: 10.1021/acs.jafc.8b04690 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry spectrometer was operated in selected ion monitoring (SIM) mode with a 100 ms survey scan, and tandem mass spectrometry (MS/MS) product ion scans (100 ms) of the SIM ions were recorded per duty cycle using a collision energy of 20 eV. Dicysteinyl polysulfanes SIM of [M + H]+ ions: m/z 121, 241, 273, 305, 337, 369; L-cysteine sulfonates SIM of [M + H]+ ions: m/z 202, 234, 266, 298, 330, 362; polythionate SIM of [M + H]+ ions: m/z 195, 227, 259, 291, 323. Statistical Analyses. Values obtained from replicates are represented as mean ± standard deviation (SD). Significant treatment effects were determined through two-way repeated measures analysis of variance (ANOVA), with Tukey multiple comparison testing of means, using GraphPad Prism statistics software (v7.03 GraphPad Software Inc., La Jolla, CA). Statistical significance was assigned if P < 0.05 (95% confidence interval). Principal component analysis (PCA) was carried out with The Unscrambler X (CAMO Software AS, Oslo, Norway).

H2S is more rapidly incorporated into polysulfanes through the S−S bond forming mechanism specified in Figure 1, whereas cysteine, in molar excess, exists as the main sulfhydryl bound to Cu near the end of the oxidation process, thus forming cystine at that stage. Polysulfane Degradation and H2S Liberation. The degradation of polysulfanes and the liberation of H2S were followed over six-months under anoxic conditions for the various treatments, with both polysulfane and H2S concentrations being measured at each time point. In the control samples, in which no antioxidant compound additions were made, dicysteinyl trisulfane concentrations decreased slowly over the six-month period, with a loss of 3% over the first 6 days (P value = 0.0345) and 16% in total over six months (P value < 0.0001) (Figure 4, Tables S1, S3). The tetrasulfane remained stable over the first month, with a loss of only 11% (P value < 0.0001); however, after six months 73% of the tetrasulfane decomposed (P value < 0.0001) (Figure 4, Tables S1, S3). The relative amount of the longer chain polysulfanes (pentasulfanes and hexasulfanes) also decreased on average by 70% after six months of storage (P values < 0.0001) (Figure 4, Tables S1, S3). The small but significant decreases in polysulfane concentrations in the control samples after the first days of storage (Table S3) were seemingly reflected in the small amounts of H2S that were liberated in these samples (Figure 5, Tables S5, S6). The concentrations of H2S in the control samples slowly increased to slightly greater than its odor threshold of 1.1−1.6 μg/L3 after one month of storage, followed by a decrease to slightly below its odor threshold after six months (average of 0.7 μg/L; Figure 5, Table S5). Generally, over the course of six months, dicysteinyl polysulfanes containing three sulfur atoms were relatively stable (or were replenished), whereas polysulfanes containing more than three sulfur atoms slowly decomposed over that time (Figure 4). Addition of ascorbic acid had a significant effect on polysulfane decomposition (P value < 0.0001) (Figure 4, Tables S2, S4). The trisulfane was again found to decompose slowly with a 42% decrease after six months (P value < 0.0001) compared to an average of 91% for tetrasulfanes to hexasulfanes (P values < 0.0001) (Table S2). Significantly greater concentrations of H2S were liberated in samples containing ascorbic acid compared to control samples after six months (Figure 5, Table S6). Initially, treatment samples followed a similar trend to the controls, liberating small amounts of H2S after 6 days of storage, but after six months, the amount of H2S increased to an average of 5 μg/L of H2S (almost an order of magnitude greater than control samples) (Figure 5, Table S5). It is interesting to note that ascorbic acid had no effect on cystine concentration, whereas sulfur dioxide decreased its concentration very significantly, presumably by sulfitolysis26 (Figure 5). It can be concluded that ascorbic acid appears to have the ability to cleave S−S bonds of polysulfanes to liberate H2S through an unknown mechanism, but the reaction kinetics appear to be slow, as discussed previously.8 All polysulfanes were significantly depleted by the sulfur dioxide treatments (P < 0.0001), whether alone (SO2) or in combination with ascorbic acid (SO2 + AA) or (+)-catechin (SO2 + Cat) (Figure 4, Tables S1−S4). It is immediately apparent that the relative concentrations of tetrasulfanes to hexasulfanes were decreased to very low levels compared to the control sample (Figure 4, Tables S1−S4). Evidently, these polysulfanes reacted rapidly with bisulfite, and on average, 91%

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RESULTS AND DISCUSSION Formation of Polysulfanes in Model Wine. When H2S and cysteine were oxidized in air together in the presence of Cu(II) and Fe(III) in model wine, cystine and four polysulfanes containing three to six sulfur atoms were observed (Figure 2). These products were tentatively identified using liquid chromatography (LC) coupled with mass spectrometry (MS) by comparing their mass spectra and elution order with published data.11 Additional peaks observed in the chromatograms (Figure S3) may be explained by the formation of diastereoisomers due to racemization of the cysteine fragment or possibly from on-column degradation of higher polysulfanes, as discussed by Kreitman et al. (2017). 11 Representative chromatograms of the treatments and MS/ MS spectra of the polysulfanes are provided in the Supporting Information (Figures S3−S13). There were only small amounts of cysteine remaining in the model wines at Day 1 after the initial rapid oxidation phase. The majority of the cysteine (>99.9%) was either oxidized with incorporation of H2S to produce the polysulfanes or oxidized with itself to produce cystine (Figure 3, Figure S1, Table S1).

Figure 3. Relative distribution of dicysteinyl polysulfanes produced in model wine as measured immediately after all the oxygen was removed from the samples.

The number of sulfur linking atoms of the polysulfane species was indirectly proportional to their concentration distribution (as reported in peak area) showing trisulfane (65.6%) ≫> tetrasulfane (22.5%) ≫ pentasulfane (10.1%) ≫ hexasulfane (1.4%). The longer chain polysulfanes were either not stable or their formation was less favorable, but they were still formed in greater relative amounts than cystine (0.3%).28 This situation was somewhat reminiscent of the preferential consumption of H2S when present with cysteine and Cu/Fe,12 and implies that D

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Figure 4. Relative abundances of (a) cysteine, (b) cystine, and (c−f) polysulfanes (S = 3−6) determined at different time intervals over six months in model wine without any treatment (control) and treated with 500 μM ascorbic acid (AA), 600 μM sulfur dioxide (SO2), sulfur dioxide (600 μM) and ascorbic acid (500 μM) (SO2 + AA), and sulfur dioxide (600 μM) and (+)-catechin (310 μM) (SO2 + Cat).

of the tetrasulfane and 96% of pentasulfane and hexasulfane counterparts had decomposed after 24 h compared to the controls. In contrast, the trisulfane was more stable in the presence of sulfur dioxide with a 59% decrease after 24 h relative to the control. The decomposition rate of all polysulfanes treated with SO2 + AA was consistently lower than the decomposition rate of the polysulfanes treated with either SO2 or SO2 + Cat, with 79%, 91%, and 94% of the tetrasulfanes, pentasulfanes, and hexasulfanes decomposed after 24 h, respectively. It appears that the presence of ascorbic acid inhibited the rate of decomposition of the polysulfanes. All polysulfane samples treated with sulfur dioxide (alone or in combination) liberated between 2.1−212 μg/L of H2S, and the increase in H2S concentrations were significantly greater (except for those measured on Day 6) than the amount of H2S produced in the control samples or those treated with ascorbic acid alone (P values ranging from 0.0256 to 3 implied that their stability decreased as sulfur chain length increased. Liberation of H2S over the course of the experiment correlated with a decrease in overall relative polysulfane concentration, with the greatest decline in polysulfane abundance and largest release of H2S (up to 212 μg/L) being associated with the presence of reducing agents, whereas the controls barely yielded more than 1 μg/L. Sulfur dioxide had a much greater effect on polysulfane degradation than ascorbic acid and the larger polysulfanes (tetrasulfanes to hexasulfanes) decomposed rapidly when treated with sulfur dioxide, with less than 10% of the original amount remaining after 24 h. Desulfurization of cysteine did not appear to contribute greatly to H2S formation, but sulfitolysis was implicated in its release from polysulfanes, and several cysteine-S-sulfonates were detected by LC−MS. Even though the results are encouraging and expand our understanding of the potential causes of VSC reappearance postbottling, further work needs to be done to confirm the presence and reactivity of polysulfanes in real wines. As an initial step, representative dicysteinyl polysulfanes could be prepared and fully characterized and their cleavage reactions and ability to form H2S (or other thiols) under reductive conditions could be examined in greater detail. Having such reference compounds available would facilitate confirmation of identity and quantification of these polysulfanes in real wines and determination of conditions that favor their production. A considerable complication in real

Figures showing relative concentrations of polysulfanes presented as stacked bar graphs; H2S concentrations liberated in the anoxically prepared cysteine control samples; figures showing mass spectra and chromatograms of dicysteinyl polysulfanes, cysteine control samples, and cysteine-S-sulfonates. Tables containing data for means and standard deviations of relative polysulfane and H2S concentrations; and significant differences in polysulfane aand H2S concentrations as a function of time and treatments (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Telephone: +61 883136600. Fax +61 883136601. E-mail [email protected]. ORCID

Marlize Z. Bekker: 0000-0002-9378-438X David W. Jeffery: 0000-0002-7054-0374 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank Yoji Hayasaka, Allie Kulcsar, and Matthew Wheal (AWRI) for technical support and Paul Smith (AWRI) for helpful discussions and encouragement. The AWRI is a member of the Wine Innovation Cluster in Adelaide. The work was supported by Australia’s grape growers and winemakers through their investment body, Wine Australia, with matching funds from the Australian Government.

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ABBREVIATIONS USED AA, ascorbic acid; Cat, (+)-catechin; EMS, ethylmethyl sulfide; GSH, glutathione; GSSG, oxidized glutathione; H2S, hydrogen sulfide; Na2S·9H2O, sodium sulfide nonahydrate; PCA, principal component analysis; PMS, potassium metabisulfite; OT, odor threshold; SCD, sulfur chemiluminescence detector; SD, standard deviation; SIM, selected ion monitoring; VSCs, volatile sulfur compounds H

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DOI: 10.1021/acs.jafc.8b04690 J. Agric. Food Chem. XXXX, XXX, XXX−XXX