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j.foodchem.2017.10.123

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Food Chemistry 245 (2018) 676–686
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Evaluation of putative precursors of key ‘reductive’ compounds in wines
post-bottling
MARK
⁎
Marlize Z. Bekker , Eric N. Wilkes, Paul A. Smith
The Australian Wine Research Institute, P.O. Box 197, Glen Osmond, SA 5064, Australia
A R T I C L E I N F O
A B S T R A C T
Keywords:
Volatile sulfur compounds
Hydrogen sulfide
Methanethiol
Ethanethiol
Precursors
Wine
Copper
pH
Precursors to hydrogen sulfide (H2S), methanethiol (MeSH), ethanethiol (EtSH), and dimethyl sulfide (DMS)
were assessed in wines post-bottling, and the percent yield of VSCs from each precursor determined. Cysteine
(Cys) and glutathione (GSH) were associated with small increases in H2S concentrations, with a maximum yield
of 0.18% and 1.3%, respectively. Greater yields of H2S were obtained from the combined Cys/GSH and copper
treatments in white wine. Copper, acting on unknown precursors, was associated with large increases in H2S in
Shiraz wines. Dimethyl disulfide and methyl thioacetate were important precursors to MeSH, and produced
maximum yields of 72% and 33%, respectively. Ethyl thioacetate was a key precursor to EtSH, with a maximum
yield of 39% obtained. Copper and pH were important in modulating MeSH and EtSH accumulation in wines. A
maximum yield of 23% of DMS from S-methylmethionine was obtained, with dimethyl sulfoxide producing
significantly less DMS with a maximum yield of only 9.4%.
1. Introduction
The origin and management of volatile sulfur compounds (VSCs) in
wine concerns many winemakers, as these compounds can have a significant impact on wine aroma attributes and wine quality. The compounds associated with typical ‘reduced’ characters are well defined;
however, precursors to these ‘reductive’ aroma compounds have yet to
be validated in real wines (Ugliano, 2013; Waterhouse, Sacks, & Jeffery,
2016a).
Hydrogen sulfide (H2S) and methanethiol (MeSH) are assumed to be
primarily responsible for the formation of ‘reductive’ aromas post-bottling (Ugliano, 2013; Ugliano et al., 2011, 2012). These two compounds
are characterized by aromas of rotten egg, sewage, and rubber when
present in concentrations of greater than their odor thresholds (OT) of
1.1–1.6 µg/L and 1.8–3.1 µg/L, respectively (Siebert, Solomon, Pollnitz,
& Jeffery, 2010). It has been suggested that elevated concentrations of
cysteine (Cys) may lead to increased H2S accumulation in wine postbottling, possibly through desulfurization of Cys (Gruenwedel &
Patnaik, 1971). However, the ability of Cys to act as a precursor to H2S
is yet to be confirmed in real wine. The observation that the presence of
glutathione (GSH) increased H2S accumulation in white wine during
bottle maturation suggests that GSH may play a role in modulating free
H2S concentrations in wines post-bottling (Ugliano et al., 2011). The
formation of H2S catalyzed by metal ions acting on yet to be identified
sulfur sources has been identified as an important pathway for H2S
⁎
formation in wines post bottling (Bekker, Mierczynska-Vasilev, Smith,
& Wilkes, 2016; Bekker, Smith, Smith, & Wilkes, 2016; Ugliano et al.,
2011; Viviers, Smith, Wilkes, & Smith, 2013).
Methanethiol (MeSH) and ethanethiol (EtSH) can play important
roles in the perception of ‘reductive’ aromas in wine, as both compounds have low OT values (Siebert et al., 2010) as well as the ability to
act as strong suppressors of fruit and floral attributes in wine (FrancoLuesma et al., 2016). Ethanethiol imparts onion, rubbery, and sulfidiclike aromas when present above its OT of 1.1 µg/L (Goniak & Noble,
1987). However, EtSH is rarely observed in wine, even in wine rated as
reduced (Rauhut, Kurbel, MacNamara, & Grossmann, 1998; Siebert
et al., 2010). Precursors to MeSH have been suggested to include methionine (Met), methyl thioacetate (MeSAc), and dimethyl disulfide
(DMDS). The role of these compounds as precursors to MeSH remains to
be established in real wine, noting that previous studies showed no
correlation between MeSAc, DMDS, and MeSH concentrations (Fedrizzi
et al., 2011; Ugliano et al., 2012). The ability of Met to act as a precursor to MeSH has similarly been reported in model wine matrices
(Pripis-Nicolau, De Revel, Bertrand, & Maujean, 2000) but not yet in
real wine.
It is known that Cys and Met are the main sources of H2S and MeSH
during fermentation (Jiranek, Langridge, & Henschke, 1995; Perpete
et al., 2006). Juice containing high concentrations of Cys and Met will
produce high concentrations of H2S and MeSH during fermentation that
will remain in the wine, either as free, loosely bound, or complexed
Corresponding author.
E-mail address: marlize.bekker@awri.com.au (M.Z. Bekker).
http://dx.doi.org/10.1016/j.foodchem.2017.10.123
Received 20 June 2017; Received in revised form 23 October 2017; Accepted 25 October 2017
Available online 05 November 2017
0308-8146/ © 2017 Elsevier Ltd. All rights reserved.
Food Chemistry 245 (2018) 676–686
M.Z. Bekker et al.
(99%), dimethyl sulfide (99%), dimethyl sulfoxide (DMSO, 99.9%),
ethanethiol (99.7%), ethyl methyl sulfide (EMS, 96%), L-glutathione
reduced (98%), iron (III) sulfate hydrate (97%), L-methionine (99.5%),
L-methionine-S-methylsulfonium iodide (99%), potassium metabisulfite
(PMS, 98%), sodium hydrosulfide hydrate (NaSH3·xH2O, 71%), and
sodium thiomethoxide (NaSMe, 95%), were obtained from SigmaAldrich (Castle Hill, NSW, Australia). Methyl thioacetate (98.8%), ethyl
thioacetate (99.5%), and propyl thioacetate (PrSAc, 99.7%) were obtained from Lancaster Synthesis (Jomar Bioscience, Kensington, SA,
Australia). Potassium hydrogen tartrate (Fluka) was supplied by SigmaAldrich. Tartaric acid (99.5%) and sodium chloride (99.5%) were obtained from Merck (Frenchs Forest, NSW, Australia). Aluminum (III)
potassium sulfate dodecahydrate and copper (II) sulfate pentahydrate
(99%) were purchased from Ajax Chemicals (Sydney, NSW, Australia).
Zinc (II) sulfate heptahydrate (99.5%) was obtained from Standard
Laboratories, (Melbourne, VIC, Australia). Ethanol (99.5%, Rowe
Scientific) was redistilled in-house prior to use. All reference standards
for VSC analyses were prepared as described by Siebert et al. (2010).
thiol-compounds in wine post-bottling. However, the desulfurization of
Cys and Met to produce H2S and MeSH in wines post-bottling without
the assistance of yeast may be more challenging. The correlation of high
concentrations of Cys and Met in wines post-bottling with high concentrations of H2S and MeSH post-bottling may be the result of remnants of fermentation chemistry and not necessarily the products of
post-bottling desulfurization of amino acids.
It has been suggested that disulfides and thioacetates that are produced during fermentation, such as diethyl disulfide (DEDS) and ethyl
thioacetate (EtSAc), can release thiols such as EtSH in wine (Rankine,
1963). However, there is little evidence for these thiol formation reactions in wines post-bottling (Ugliano, 2013). Thioacetates, such as
MeSAc and EtSAc, are regularly identified in red wines rated as ‘reductive’ and thioacetates also occur more regularly in ‘reductive’ wines
than disulfides, such as DMDS and DEDS (Fang & Qian, 2005;
Leppanen, Denslow, & Ronkainen, 1980; Rauhut et al., 1998; Siebert
et al., 2010). The OTs of MeSAc, EtSAc, DMDS, and DEDS are significantly higher than those of H2S, MeSH, and EtSH, at 50 µg/L, 10 µg/
L, 29 µg/L, and 4.3 µg/L, respectively (Siebert et al., 2010). Interestingly, it has been shown that a relationship exists between the presence
of MeSAc and EtSAc and the presence of MeSH and EtSH in fermenting
Shiraz wines prepared under reductive conditions (Bekker, Day, Holt,
Wilkes, & Smith, 2016).
Dimethyl sulfide (DMS) is usually measurable in both red and white
wines, and is associated with aromas of canned corn, asparagus, or
vegetal aromas when present in high concentrations (Mestres, Busto, &
Guasch, 2000). It has been shown that S-methylmethionine (SMM) is an
important precursor to DMS in wines (Loscos et al., 2008; Segurel,
Razungles, Riou, Salles, & Baumes, 2004; Segurel, Razungles, Riou,
Trigueiro, & Baumes, 2005). Factors affecting DMS formation in wines
post-bottling include grape variety, viticultural and vinification processes, and wine pH. These factors most likely impact on the precursor
compounds to DMS (Bekker et al., 2016; Escudero, Campo, Farina,
Cacho, & Ferreira, 2007; Segurel et al., 2005; Ugliano, 2013). Vinification variables, such as oxygen exposure during fermentation, as well
as the presence of metal ions, such as copper, iron, or manganese, do
not impact DMS concentrations post-bottling (Bekker et al., 2016;
Viviers et al., 2013).
In this study, the roles of precursor compounds in determining final
concentrations of compounds associated with ‘reductive’ aromas in
wines post-bottling were investigated. Some of these compounds, such
as Cys, GSH, Met, disulfides, and thioacetates, have been identified as
possible precursors to H2S, MeSH, and EtSH; however, these hypotheses
have not been tested in real wine (Ugliano, 2013; Waterhouse, Sacks, &
Jeffery, 2016b). These putative precursors to H2S, MeSH, EtSH, and
DMS were assessed for their ability to release their corresponding
smaller molecular mass thiol compounds under normal wine conditions. In addition, the molar % yield of VSC from each precursor was
determined, and the accumulation of each VSC in white and red wine
compared, to gain insight into the potential of each precursor compound to modulate wine aroma. Factors modulating the formation of
VSCs were also assessed. Factors investigated were the presence of
elevated metal concentrations and varying pH levels. Overall, the main
aim of this study was to evaluate the ability of the precursors to release
their corresponding ‘reductive’ aroma compound, to assess the factors
that modulated the release of VSCs, and to determine the expected %
yield of H2S, MeSH, EtSH, and DMS from their individual precursor
compounds.
2.2. Wine samples
2.2.1. Precursors to hydrogen sulfide
Certified organic Verdelho wine from the 2012 vintage and Shiraz
wine from the 2010 vintage, produced in South Eastern Australia, were
obtained from a local winery. Both wines had not been treated with
either copper or SO2 during winemaking. Analyses of the chemical
compositions of the two base wines were conducted by The Australian
Wine Research Institute (AWRI) Analytical Service (Adelaide, Australia)
and are as follows: pH 3.3, 5.8 g/L residual sugars, 13% (v/v) alcohol,
0.85 g/L volatile acidity (as acetic acid), 6.1 g/L titratable acidity (as
tartaric acid), 1.0 mg/L free SO2 and 4.0 mg/L total SO2 for the
Verdelho wine; and pH 3.5, 0.70 g/L residual sugars, 14% (v/v) alcohol,
0.37 g/L volatile acidity (as acetic acid), 6.6 g/L titratable acidity (as
tartaric acid), < 4.0 mg/L free SO2 and 4.0 mg/L total SO2 for the
Shiraz wine.
2.2.2. Precursors to methanethiol
A dry white wine (2014 vintage) and a Cabernet Merlot (2014
vintage) produced in South Australia, were obtained from a local
winery. Analyses of the chemical compositions of the two base wines
were conducted by The AWRI Analytical Service (Adelaide, Australia)
and are as follows: pH 3.3, 12% (v/v) alcohol, 0.24 g/L volatile acidity
(as acetic acid), 5.7 g/L titratable acidity (as tartaric acid), 57 mg/L free
SO2 and 170 mg/L total SO2 for the dry white wine; and pH 3.6, 0.20 g/
L residual sugars, 14% (v/v) alcohol, 0.53 g/L volatile acidity (as acetic
acid), 5.9 g/L titratable acidity (as tartaric acid), 42 mg/L free SO2 and
61 mg/L total SO2 for the Shiraz.
A Chardonnay wine (vintage 2014) and a Shiraz wine (vintage
2014), produced in South Australia, were obtained from a local winery
to evaluate the effects of pH on MeSH formation from MeSAc. Analyses
of the chemical compositions of the two base wines were conducted by
The AWRI Analytical Service (Adelaide, Australia) and are as follows:
pH 3.5, 3.5 g/L residual sugars, 15% (v/v) alcohol, 0.35 g/L volatile
acidity (as acetic acid), 4.9 g/L titratable acidity (as tartaric acid),
18 mg/L free SO2 and 63 mg/L total SO2 for the Chardonnay; and pH
3.7, 0.30 g/L residual sugars, 14% (v/v) alcohol, 0.40 g/L volatile
acidity (as acetic acid), 6.2 g/L titratable acidity (as tartaric acid),
19 mg/L free SO2 and 67 mg/L total SO2 for the Shiraz.
2. Materials and methods
2.2.3. Precursors to ethanethiol
The same Chardonnay and Shiraz wines (vintage 2014) used for the
pH study on MeSH formation from MeSAc (Section 2.2.2) were used to
investigate the ability of EtSAc to act as a precursor to EtSH.
2.1. Chemicals
All chemicals were of analytical reagent grade, and water was
prepared from a Milli-Q purification system (Millipore, North Ryde,
NSW, Australia). L-Cysteine hydrochloride (99%), dimethyl disulfide
2.2.4. Precursors to dimethyl sulfide
A Sauvignon Blanc wine (vintage 2012) and a Cabernet Sauvignon
677
Food Chemistry 245 (2018) 676–686
M.Z. Bekker et al.
as internal standard (250 µM). The LC-MSMS conditions were as follows: 1 µL of sample was injected at a flow rate of 0.8 mL/min. Solvent
A was 0.1% formic acid in Milli-Q-Water, and solvent B was 0.1%
formic acid in acetonitrile. Separation was achieved using an Agilent
Zorbax Eclipse Plus C18 RRHD (2.1 × 100 mm 1.8 µm, Forest Hill, VIC,
Australia) column, held at 60 °C. The solvent ramp was as follows:
0 min to 0.5 min (1% B), 3.5 min (10% B), 6 min (15% B), 6.5 min (20%
B), 6.6 min to 7.5 min (75% B), 7.6 min to 10 min (1% B). The MS
conditions were as follows: gas temperature 315 °C, gas flow 14 L/min,
nebulizer pressure 40 psi, sheath gas heater 400 °C, sheath gas flow 11
L/min, capillary voltage 3800 V, nozzle voltage 1500 V, start time
0 min, with dynamic multiple reaction monitoring scan type. The concentrations of the Cys, Met, SMM, and GSH that were spiked into the
base wines are given in Table 1.
wine (vintage 2011), produced in South Australia, were obtained from
a local winery. Analyses of the chemical compositions of the two base
wines were conducted by The AWRI Analytical Service (Adelaide,
Australia) and are as follows: pH 3.2, 2.6 g/L residual sugars, 13% (v/v)
alcohol, < 0.25 g/L volatile acidity (as acetic acid), 6.6 g/L titratable
acidity (as tartaric acid), 15 mg/L free SO2 and 92 mg/L total SO2 for
the Chardonnay; and pH 3.7, 3.4 g/L residual sugars, 14% (v/v) alcohol,
0.44 g/L volatile acidity (as acetic acid), 5.9 g/L titratable acidity (as
tartaric acid), 8.0 mg/L free SO2 and 51 mg/L total SO2 for the Cabernet
Sauvignon.
2.2.5. Model wine
For the MeSH and EtSH precursor studies, model wine was prepared
that consisted of 12% ethanol and 10 g/L potassium hydrogen tartrate,
using degassed water obtained from a Milli-Q purification system
(oxygen concentration < 5 ppb). The pH of the model wine was adjusted using 40% tartaric acid (w/v) to pH 3.4. Additional model wine
samples with pH adjusted to pH 3.0 were prepared, to assess the effect
of pH on the hydrolysis of MeSAc and EtSAc and the subsequent release
of MeSH and EtSH from their corresponding thioacetates.
2.3.3. Metal Analyses
Base wines and stock solutions were analyzed for their metal concentrations by Flinders Analytical, Flinders University (Adelaide,
Australia) using an Agilent 7500 cx inductively coupled plasma mass
spectrometer (Agilent Technologies, Tokyo, Japan) as described in
Thiel, Geisler, Blechschmidt, and Danzer (2004). The concentrations of
metals spiked into the base wines are in Table 1.
2.3. Chemical analyses
2.3.4. Oxygen measurement
Colorless vials identical to the ones used for each precursor experiment were fitted with PreSens Pst6 oxygen sensors (Presens,
Regensburg, Germany) and were prepared, in triplicate, with the base
wine (white, red, or model) or with no treatments (control). Wine
samples were stored in kegs under a positive nitrogen (N2) pressure.
Oxygen sensors were used to confirm that the samples were not exposed
to oxygen and to ensure against dissolved oxygen uptake during storage. Oxygen measurements were carried out using a PreSens Fibox 3
trace v3 oxygen meter (Presens, Regensburg, Germany).
2.3.1. Gas chromatography coupled to sulfur chemiluminescence detection
The base wines were analyzed for their VSC profiles before the start
of each experiment 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). Instrument control and data analysis were performed
with Agilent GC ChemStation software, Rev. B.04.03-SP2 [105] and
Maestro software integrated version 1.4.40.1/3.5. The gas chromatograph was fitted with a 15 m × 0.25 mm Varian Wax FactorFour
VFWAXms fused silica capillary column, 0.50 μm film thickness
(Varian, Mulgrave, VIC, Australia) connected with a fused silica universal straight connector (Grace Davison Discovery Sciences) to a
60 m × 0.25 mm Varian VB-5 fused silica capillary column, 0.50 μm
film thickness (Varian, Mulgrave, VIC, Australia) with a 2 m × 0.53 mm
retention gap. Helium (Air Liquide ultrahigh purity) was used as a
carrier gas. The method used to analyze the VSCs is the method described by Siebert et al. (2010) without modification. Using this
method, the base concentrations of H2S, MeSH, EtSH, and DMS were
determined in the wines. Wine samples were again analyzed for MeSAc,
EtSAc, and DMDS after the addition of these putative precursor compounds to the wines, and the concentrations of MeSAc, EtSAc, DMDS, as
well as H2S, MeSH, EtSH, and DMS were followed for the 12-month
duration of each experiment. The concentrations of the MeSAc, EtSAc,
and DMDS present in the wines after addition are given in Table 1.
2.4. Sample preparation and analyses
The experiments used to evaluate the putative precursors to H2S,
MeSH, EtSH, and DMS were treated as separate investigations and as
such there were inter-experimental variations regarding wine variety,
sample size, and variables such as pH and metal additions. However,
there were no intra-experimental differences in these variables. These
parameters were consistent for each experimental set. For each experiment, modulating factors were selected that have been proposed as
possible contributors to specific VSC formation. As such copper was
used for the H2S, MeSH, and EtSH precursor studies (Bekker et al.,
2016; Nedjma & Hoffmann, 1996; Ugliano et al., 2011; Viviers et al.,
2013), aluminum and zinc were used for the DMS precursor study
(Viviers et al., 2013), and wine pH was included as a variable for the
thioacetate precursor studies (Bekker et al., 2016).
Given that oxygen exposure plays a fundamental role in VSC formation (Ugliano, 2013), all samples were prepared in an anaerobic
hood using wines (red, white, and model) with < 5 ppb dissolved
oxygen. The wines were subsampled inside an anaerobic hood into the
following vials: H2S and DMS precursors studies – 20 mL amber vials
with crimp cap lids (Chromacol, Part of Thermo Fisher Scientific Inc.,
Scoresby, VIC, Australia); MeSH precursors study – 210-mL green piccolo wine bottles sealed (AG 31185NNPB sparkling piccolo, Best Bottlers PTY LTD, Mildura, Australia) with Zavent 200-mL screwcaps (Best
Bottlers PTY LTD, Mildura, Australia); and thioacetates’ role as precursors – 40-mL clear vials with solid caps with Teflon liners (Grace
Davison Discovery Sciences, Rowville, VIC, Australia).
Stock solutions of precursors and metals were prepared by weighing
out individual compounds into volumetric flasks under atmospheric
oxygen conditions, then transferring the volumetric flasks to an anaerobic hood where the stock solutions were prepared volumetrically
using degassed ethanol (oxygen concentration < 5 ppb) for DMDS,
2.3.2. Determination of L-cysteine, L-methionine, S-methylmethionine, and
L-glutathione
The concentrations of Cys, Met, SMM, and GSH were determined
immediately after spiking into the wine samples. The concentrations of
Cys, Met, GSH, and SMM were determined by Metabolomics Australia
(The Australian Wine Research Institute, Adelaide, Australia) using the
method described by Boughton et al. (2011) with slight adaptation.
Quantification of amino acids was performed using a derivatization
technique with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate
(AQC) followed by liquid chromatography (LC) tandem mass spectrometry (MS/MS) in ESI positive ionization mode. Samples were analyzed
using an Agilent 1290 Infinity LC system coupled with Agilent 6490
triple quadrupole LC/MS system with iFunnel technology (Forest Hill,
VIC, Australia). In short, wine samples were diluted 1:100 with Milli-Q
water to be in the appropriate concentration range. Calibration standards for Cys, Met, GSH, and SMM were prepared with 12 calibrant
levels, ranging from 0.0005 µM to 200 µM. L-Valine-13C5, 15N was used
678
Food Chemistry 245 (2018) 676–686
M.Z. Bekker et al.
Table 1
Concentrations of precursor compounds and metals that were added to the base wines for each experiment.
Precursor
Cysteine
Glutathione
L-Methionine
Methyl thioacetateh
Methyl thioacetate
(exp.2)h
Dimethyl disulfide
Ethyl thioacetate
S-Methylmethionine
Dimethyl sulfoxide
Base wine precursor
concentration (µg/L)a
Total precursor concentration
µg/L)b
White
wine
White
wine
Red
wine
Model
wine
d
4293
9635
5365
ndi
nd
550
2777
765
nd
nd
n/a
n/a
0
0
0
nd
nd
nd
n/al
nd
nd
nd
n/al
0
0
0
n/al
Red wine
Model
wine
Concentration
range in wine
(µg/l)
(min–max)
e
Base wine metal concentrations (µg/L)
Total metal
concentrations (µg/L)
White wine
White wine, red wine,
model wine
Red wine
Cuc
Alc
Znc
Cu
Al
Zn
Cu
Al
Zn
8000
18,000
8000
50
50
8000
18,000
8000
50
50
n/a
n/a
8000
50
50
240–6000
1300–34,700f
0–37,000g
0–12.5j
26
26
190
190
250h
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
15
15
100
100
180h
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
1000
1000
500
500
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
50
60
10,000
2500m
50
60
10,000
2500m
50
60
n/a
n/a
nd–1.5j
ndj
0–4460k
< 0.097–1230n
190
190
n/a
n/a
n/a
n/a
440
440
n/a
n/a
930
930
100
100
n/a
n/a
n/a
n/a
235
235
n/a
n/a
715
715
500
500
n/a
n/a
n/a
n/a
4000
4000
n/a
n/a
4000
4000
a
Concentrations (µg/L) of precursor compounds in base wine.
Total concentrations of precursors after spiking into wines.
c
Concentration ranges of Al, Cu, and Zn in Australian wines are 0–1.79 mg/L, 0–1.89 mg/L, and 0.01–2.27 mg/L, respectively (Martin, Watling, & Lee, 2012). The legal limits for Al,
Cu, and Zn for export of Australian wine to the European Union are 8.0 mg/L, 1.0 mg/L, and 5 mg/L, respectively (Analytical Requirements for the Export of Australian Wine, 2016).
d
n/a: precursor experiment not performed in model wine; metals not included as experimental variable.
e
Concentration range of Cys in French wines (Pripis-Nicolau, De Revel, Marchand, Beloqui, & Bertrand, 2001).
f
Concentration range of GSH in Sauvignon Blanc wines (Janes, Lisjak, & Vanzo, 2010).
g
Concentration range reported for Met in wine (Ough & Amerine, 1988).
h
Two experiments were performed that investigated MeSAc as precursor to MeSH. For the first experiment, the concentration of copper was 190 µg/L in the base white wine and
100 µg/L in the base red wine; in the second experiment the concentration of copper was 250 µg/L in the base white wine, and 180 µg/L in the base red wine.
i
nd: not detected.
j
Concentration range of MeSAc, DMDS, and EtSAc in Australian wines (Siebert et al., 2010).
k
Average “potential DMS” concentrations in Grenache, Syrah, Gros Manseng, and Petit Manseng (Loscos et al., 2008; Segurel et al., 2004).
l
Dimethyl sulfoxide concentrations not measured in base wines.
m
Dimethyl sulfoxide concetrations added to Sauvignon Blanc and Cabernet Sauvignon base wines.
n
Average dimethyl sulfoxide concentrations in Australian and New Zealand red and white wines (De Mora, Lee, Shooter, & Eschenbruch, 1993).
b
3. Results and discussion
MeSAc, EtSAc, and PrSAc stock solution preparations. The stock solutions of copper, iron, aluminum, zinc, Cys, Met, SMM, GSH, and DMSO
were prepared as described above; however, these stock solutions were
prepared volumetrically using degassed water obtained from a Milli-Q
purification system (oxygen concentration < 5 ppb). Each treatment in
each wine (red, white, or model) for each experiment was prepared in
triplicate.
All samples were stored at room temperature (22 °C) in 19-L post
mix Cornelius kegs (Ambar technology, Alexandria, NSW, Australia)
where they were protected from light. All the samples were also protected from oxygen exposure during aging as each keg was flushed with
N2 (g) until they reached < 1 ppb measured oxygen and maintained at
a slight positive pressure of 1 psi N2 (g). The lid of each keg was fitted
with a PreSens Pst6 oxygen sensor (Presens, Regensburg, Germany) to
monitor and prevent oxygen ingress into the kegs. Samples of each
experiment were analyzed at five time points over the course of
12 months to determine the long-term effects of the precursors in real
wine systems. Samples were only analyzed once and then discarded.
The formation of VSCs in wines has been proposed to occur by two
pathways, the first being the de novo formation from unidentified precursor compounds, and the second being the release from loosely bound
metal complexes (Franco-Luesma & Ferreira, 2016a, 2016b). In this
study, the focus was on the formation of VSCs from precursor compounds (de novo formation) and not the release of VSCs from loosely
bound metal complexes. To correct for the effect of the metals on other
precursor compounds and loosely-bound thiol-metal complexes, control
samples were prepared for each experiment that only contained the
base wine with added metals. When evaluating the role of the added
precursor compounds (Cys, Met, GSH, DMDS, MeSAc, EtSAc, SMM, and
DMSO), the yields obtained from the added precursors were interpreted
with respect to the amount of VSCs naturally produced in the control
wines; and the amount of VSCs produced in the control wines with
added metals.
The abilities of compounds to act as precursors of H2S, MeSH, EtSH,
or DMS were evaluated and the percentage yield of either H2S, MeSH,
EtSH, or DMS (% yield) was calculated, to obtain an objective measure
of the contribution of each precursor compound to final concentrations
of H2S, MeSH, EtSH, and DMS in wines post-bottling. The following
equation was used to calculate the % yield:
2.5. Statistical
All significance tests (Student’s t-test, ANOVAs and Tukey analyses)
were conducted using GraphPad Prism statistics software (v7.03;
GraphPad Software Inc., La Jolla, CA). The significance of the compounds as precursors to H2S, MeSH, EtSH, and DMS was determined
through two-way repeated measures (RM) ANOVA, and Dunnetts
multiple comparison test was used to correct the p-value for multiple
comparisons. Statistical significance was assigned if p < .05 (95%
confidence interval). All values are represented as mean ± standard
deviation (Stdev).
%yield
⎡ (mol VSC produced in wines with added precursor compound) ⎤
⎢
⎥
−(mol VSC already present in control wines)
⎥
=⎢
⎢ theoretical mol VSC that should be produced from added ⎥
⎢
⎥
⎢
⎥
⎣ precursor compound
⎦
(1)
× 100
The mol VSCs produced from the added precursor compounds were
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Scheme 1. The summary of the effects of cysteine
(Cys), glutathione (GSH), methionine (Met), methyl
thioacetate (MeSAc), dimethyl disulfide (DMDS),
ethyl thioacetate (EtSAc), dimethyl sulfoxide
(DMSO), and S-methylmethionine (SMM) as precursors to hydrogen sulfide (H2S), methanethiol
(MeSH), ethanethiol (EtSH), and dimethyl sulfide
(DMS), respectively. The maximum percentage (%)
accumulation for H2S, MeSH, EtSH, and DMS over
12 months is reported for each wine evaluated. The
factors modulating VSC formation that were evaluated (copper, aluminum, zinc, pH) are shown next to
the respective reaction. (i)Maximum concentration of
H2S, MeSH, EtSH, DMS, DMDS, or DEDS measured in
a specific wine matrix over the course of 12 months.
(ii)
Modulating factor that was evaluated for its ability
to modulate VSC formation from each precursor.
(iii)
The increase in H2S concentration cannot be
quantified as a molar percentage as the unidentified
precursors responsible for the H2S accumulation associated with added copper are not yet identified. As
such the increases in H2S concentrations are discussed in relation to the amount of H2S produced in
the control wines without added copper. (iv)Normal
pH levels for the wines were as follows: Chardonnay
pH 3.5, Shiraz pH 3.7, model wine pH 3.4. Lower pH
levels for all the wines were pH 3.0. (v)Maximum
accumulation of the VSC in wines with or without
copper are indicated by ‘ ± Cu2+’. (vi)Maximum accumulation for the VSCs in wines with or without
aluminum
and
zinc
are
indicated
by
‘ ± Al3+ ± Zn2+’.
the respective precursor compound (theoretical yield), and multiplying
this value by 100 to give a percentage value [Eq. (1)]. For samples with
added metals, the mol H2S, MeSH, EtSH, or DMS produced in the
control wines with added metals were used to correct for the actual
calculated by subtracting the mol H2S, MeSH, EtSH, or DMS produced
in the control sample from the mol H2S, MeSH, EtSH, or DMS produced
in the treated samples (actual yield), and dividing this value by the
theoretical mol H2S, MeSH, EtSH, or DMS that should be produced from
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M.Z. Bekker et al.
Fig. 1. Hydrogen sulfide (H2S) produced from cysteine (Cys) and glutathione (GSH) in Verdelho and Shiraz wines, as modulated by copper, Cu(II), addition.
original H2S concentrations in the Shiraz wines.
These results in real wines support observations made in a previous
study in model wine that showed that only small increases in H2S
concentrations were associated with elevated Cys and GSH (Bekker
et al., 2016).
yield.
Variables considered to modulate the formation of H2S, MeSH,
EtSH, or DMS in wines were also investigated (modulating factors). The
modulating factors investigated in this study were metal ions (copper,
aluminum, and zinc) and wine pH. The differences in accumulation of
H2S, MeSH, EtSH, or DMS were studied in white wines and red wines, to
investigate the manner of VSC accumulation in white and red wines. A
summary of all precursor and modulating factor effects are given in
Scheme 1.
3.1.2. Effects of copper on H2S formation
Significantly increased H2S concentrations were measured in
Verdelho and Shiraz wines with added copper. A maximum of 8.4 µg/L
H2S and 28 µg/L H2S was produced in Verdelho and Shiraz wines, respectively, treated with only copper after 12 months (Fig. 1c, f; Table
S1). This is a 4.2-fold and 11-fold increase in H2S concentration in
copper-treated wines, respectively, when compared to H2S concentration in the control Verdelho and Shiraz wines (Fig. 1c, f; Table S1).
These results show that the formation of H2S through copper catalyzed
reactions, from unidentified precursor compounds already present in
the wine, is another major contributing factor to H2S concentrations in
wines post-bottling.
In the Verdelho wines, the % yield of H2S from Cys or GSH increased
when either Cys or GSH were added in combination with copper
(Fig. 1b; Tables S1 and S2). In the presence of copper, the maximum %
yield of H2S increased from 0.02% to 0.18% for Cys, and from 0.02% to
1.3% for GSH in the Verdelho wines (Fig. 1b, Table S2). This translated
to an increase from a maximum 8.4 µg/L H2S (control wines with
copper) to 11.6 µg/L H2S and 35.9 µg/L H2S for Cys and GSH-treated
Verdelho wines, respectively. The treatment of these Verdelho wines
with GSH and copper resulted in a 4-fold increase in H2S concentrations.
In the Shiraz wines with added copper, the maximum concentration
of H2S produced from copper alone was 28.5 µg/L compared to the
maximum concentration of H2S produced from “Cys + Cu(II)” of
17.6 µg/L and from “GSH + Cu(II)” of 7.8 µg/L. It appeared that Cys
and GSH, in combination with copper, suppressed H2S accumulation
(Fig. 1e, f; Tables S1 and S2). The inhibitory effects of Cys and GSH
were only measured in the Shiraz wines, suggesting that this effect was
related to red wine compounds.
3.1. Hydrogen sulfide
3.1.1. Precursors to H2S
Cysteine and GSH were evaluated as precursors to H2S in wines
post-bottling. Over the 12 months of the experiment, H2S concentrations did not increase significantly in wines with added Cys in the
Verdelho or the Shiraz wines (Fig. 1a, d; Table S1). A maximum % yield
of H2S from Cys of only 0.02% and 0.05% was measured over the
course of 12 months post-bottling in Verdelho and Shiraz wines, respectively (Fig. 1a, d; Table S2). The highest concentrations of H2S
produced from Cys over 12 months were 2.5 µg/L and 3.7 µg/L for the
Verdelho and Shiraz wines, respectively (Table S1), compared to the
control wines that produced maximum H2S concentrations of 2.1 µg/L
and 2.6 µg/L (Table S1) over the course of the experiment.
The presence of GSH was not associated with significantly increased
H2S concentrations in the Verdelho wines (Fig. 1a, Table S1). In the
Shiraz wines a small but significant increase in H2S concentration associated with GSH was measured at three and six months of storage
(Fig. 1d; Table S1). The maximum % yield of H2S associated with GSH
addition was only 0.02% in Verdelho and 0.18% in Shiraz wines over
the 12 months (Fig. 1a, d; Table S2). The highest concentrations of H2S
produced from GSH over 12 months were 2.1 µg/L and 6.1 µg/L for the
Verdelho and Shiraz wines, respectively (Table S1), compared to the
control wines that produced maximum H2S concentrations of 2.1 µg/L
and 2.6 µg/L (Table S1). Although the % yield of H2S from GSH in the
Shiraz wine appears small at 0.18%, the impact of GSH on H2S concentrations was significant and produced greater than double the
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3.1.3. Accumulation of H2S in white and red wine
There were significant differences in H2S accumulation in Verdelho
and Shiraz wines. In Verdelho wines ‘GSH + copper’ produced larger
concentrations of H2S than in the same treatment in Shiraz wines
(Fig. 1b, c vs e, f; Table S1). In Shiraz wines ‘Cys + copper’ produced
larger concentrations of H2S than in the same treatment in Verdelho
wines (Fig. 1f vs c), however, H2S accumulation was greater in samples
with only copper, than in samples with ‘Cys + copper’ in the Shiraz
wines (Fig. 1f vs c; Table S1). As discussed in Section 3.1.2, the accumulation of H2S associated with copper addition was suppressed by Cys
or GSH addition in Shiraz wines (Fig. 1e, f). This decrease in H2S accumulation was only measured in the Shiraz wines and must therefore
be related to red wine compounds that are not present in white wine.
This may be explained through the interaction of copper, H2S, Cys,
GSH, and other wine compounds, such as polyphenols, quinones, and
tannins. The decrease in H2S concentrations in the red wines may be
explained through the formation of disulfide complexes of Cys and/or
GSH with H2S, or the inclusion of H2S into larger polysulfanes. The role
of polysulfanes in modulating the concentrations of certain VSCs postbottling has recently been proposed by Kreitman, Danilewicz, Jeffery,
and Elias (2017).
The abilities of Cys and GSH to act as precursors to H2S, and the
modulating effects of copper in determining Cys and GSH roles as
precursors, are summarized in Scheme 1(A).
Ethyl thioacetate played a significant role in modulating free EtSH
concentrations, with a maximum yield of 24% EtSH (8.1 µg/L) obtained
in a Chardonnay wine (pH 3.5), 9.0% EtSH (3.2 µg/L) in a Shiraz wine
(pH 3.7), and 18% EtSH (6.2 µg/L) in a model wine (pH 3.4)
(Fig. 2j, k, l; Tables S7 and S8). It is clear that elevated concentrations of
thioacetates, such as EtSAc or MeSAc, are a substantial risk for increased EtSH or MeSH formation in wines post-bottling.
The impact of the conversion of thioacetates or disulfides to their
corresponding thiols will profoundly impact wine aroma, considering
that the OTs of the thioacetates and disulfides are significantly higher
than those of the smaller thiol molecules. The relationship between the
formation of thioacetates and disulfides during fermentation from thiols
such as MeSH, EtSH, and the subsequent release of these thiols from the
thioacetates and disulfides in wines post-bottling is important in modulating ‘reductive’ aromas in wines post-bottling.
The contribution of Met to MeSH concentrations in wines postbottling was negligible. The maximum % MeSH yield from Met was
0.12% (5.5 µg/L vs 5.1 µg/L in controls for dry white Month 1; 2.9 µg/L
vs 0 µg/L in controls for Cabernet Merlot Month 6; and 0 µg/L produced
from Met in model wine) as measured in dry white wine, Cabernet
Merlot, and model wine (Fig. 2a, b, c; Tables S3 and S4). This yield was
similar to the % H2S yield obtained from Cys as discussed in Section 3.1.
This suggests that sulfur-containing amino acids (Cys, Met) are not as
readily converted to produce H2S or MeSH in wines post-bottling.
3.2. Methanethiol and ethanethiol
3.2.2. Effects of copper and pH on MeSH and EtSH formation
The role of copper and pH in modulating the release of VSCs from
precursors was also investigated. Copper played an important role in
modulating MeSH formation from DMDS; and wine pH played an important role in modulating MeSH and EtSH formation from MeSAc and
EtSAc in wines post-bottling. The accumulation of MeSH and EtSH after
their release from acid-catalyzed hydrolysis of MeSAc or EtSAc, was
significantly impacted by presence of copper. Significantly less MeSH
and EtSH accumulated in wines when copper was present, and copper
addition was also associated with the formation of the oxidation products of MeSH and EtSH, DMDS and DEDS.
When copper was added to wines with added DMDS, large increases
in MeSH yields were measured, with a maximum MeSH yield of 72%
(44 µg/L) obtained in dry white wine and a yield of 13% MeSH (8.5 µg/
L) obtained in Cabernet Merlot wines (Fig. 2d, e; Tables S3 and S4). In
contrast, no free MeSH was measured in the model wines treated with
DMDS and copper (Fig. 2f; Table S4). This lack of accumulation of
MeSH in the model wine (Fig. 2c, f) was not expected, and could be the
result of an equilibrium between the formation of MeSH from DMDS
and the oxidation of MeSH to DMDS in the simple matrix of model
wines. This experiment demonstrated that the disulfide DMDS has the
potential to release up to 70% of its corresponding thiol compound
when in the presence of copper (Fig. 2d). The maximum amount of
MeSH produced from DMDS increased from 44% to 72% in the dry
white wine when DMDS was in the presence of copper (Fig. 2a vs d;
Table S4). As such, disulfides represent an important source of thiols in
wines, and the presence of copper adds an increased risk for disulfide
reduction to produce the corresponding smaller thiol molecules.
The presence of copper in combination with Met treatment had no
significant effects on MeSH formation in the three wines evaluated
(Fig.2d, e, f, Tables S3 and S4), reinforcing the lack of contribution
made by Met alone as previously discussed (Fig. 2a, b, c).
Lowering the pH of the wines increased MeSH and EtSH formation
from MeSAc and EtSAc, respectively, in all wines tested
(Fig. 2g, h, i, j, k, l; Tables S5 and S7), which is in agreement with
previous studies (Rauhut et al., 1998). At the unadjusted and higher pH
levels (Chardonnay pH 3.5, Shiraz pH 3.7, model wine pH 3.4) the
concentrations of MeSAc and EtSAc decreased to give the following
maximum % yields of MeSH and EtSH over 16 months (Fig. 2g to l;
Tables S5, S6, S7, and S8): 18% MeSH (11 µg/L, Chardonnay) > 13%
MeSH (4.1 µg/L, model wine) > 3% MeSH (4.1 µg/L, Shiraz); and 24%
3.2.1. Precursors to MeSH and EtSH
Of the three evaluated precursors of MeSH (DMDS, MeSAc, and
Met), only DMDS and MeSAc contributed significantly to free MeSH
concentrations in the wines post-bottling. Similarly, EtSAc contributed
significantly to free EtSH concentrations in wines post-bottling.
In the dry white wine, DMDS produced a maximum yield of 44%
MeSH (31 µg/L) after six months of storage (Fig. 2a; Tables S3 and S4).
For the Cabernet Merlot wines the maximum % yield of MeSH from
DMDS was considerably less at 10% MeSH (5.3 µg/L) after six months
of storage (Fig. 2b; Tables S3 and S4). No MeSH was measurable in the
model wines that were treated with DMDS (Fig. 2c; Table S3). A molar
conversion of DMDS to MeSH of either 44% or 10% in the white and red
wines, respectively, remains substantial. Furthermore, the concentrations of produced MeSH were 17-times and 3-times greater than the OT
of MeSH (1.8–3.1 µg/L). These results indicate that DMDS has the potential to contribute significantly to ‘reductive aromas’ in wines postbottling, either through the direct effects that DMDS has on wine aroma
when present above its OT of 29 µg/L (Siebert et al., 2010), or through
the reduction of the disulfide over time to produce MeSH that has a
much lower OT value. This experiment emphasizes the importance of
other disulfides, and possibly polysulfanes, in modulating thiol concentrations in wines post-bottling, as DMDS can be considered as a
precursor that is likely to be representative of a wider range of disulfides and polysulfanes.
The maximum amount of MeSH produced from MeSAc was considerably lower than the amount of MeSH produced from DMDS in the
dry white wines (Fig. 2a; Table S4) and similar % yields of MeSH were
produced in the Cabernet Merlot from MeSAc as from DMDS (Fig. 2b;
Table S4). The % MeSH yield produced from MeSAc reached a maximum of 33% MeSH (13 µg/L) in the dry white wine, 13% (3.4 µg/L) in
the Cabernet Merlot, and 18% in the model wines (4.7 µg/L)
(Fig. 2a, b, c; Tables S3 and S4). These results showed that thioacetates
have the potential to significantly impact wine aroma when present in
wines post-bottling, as it they produce a molar yield of up to 33% of the
corresponding thiol, depending on the whether it is a white wine or a
red wine. The concentration of MeSH did not remain elevated in the
Cabernet Merlot wines (Fig. 2b), which suggests that the released MeSH
may have reacted with other wine compounds, resulting in decreased
MeSH accumulation over time.
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Fig. 2. The % yields of methanethiol (MeSH) produced from methyl thioacetate (MeSAc), methionine (Met), and dimethyl disulfide (DMDS) in dry white wine, Cabernet Merlot, and
model wine, as a function of copper, Cu(II), addition are shown in 2a to 2f. The % yields of MeSH produced from MeSAc and ethanethiol (EtSH) produced from ethyl thioacetate (EtSAc)
in Chardonnay, Shiraz wines and model wine, as modulated by pH are shown in 2g to 2l.
remained in the wine are displayed; as well as in Fig. 3c to h where the
% MeSH or % EtSH produced, % MeSAc or EtSAc remaining, and the %
DMDS or DEDS are displayed.
When DMDS was added to wines, similar trends in DMDS consumption were observed in both dry white wine and Cabernet Merlot
wine (Fig. S1a vs S1b); however, the concentration of DMDS remained
relatively stable in the model wines (Fig. S1c). The decrease in DMDS
concentration in the real wines did coincide with increased MeSH
concentrations in the dry white wine (Fig. S1a), but not in the Cabernet
Merlot wines (Fig. S1b). Furthermore, the final concentration of MeSH
always decreased in the white and red wines towards the end of the 12month storage period (Fig. S1a, S1b), suggesting that the highly reactive MeSH reacted with other wine compounds that subsequently
decreased the total concentration of free MeSH present in these wines
(Fig. S1a, S1b). The fact that MeSH did not accumulate in the model
wines treated with DMDS over the 12-month storage period (Fig. S1c,
S1d) suggests that an equilibrium between MeSH oxidation to DMDS,
and DMDS reduction to MeSH may have been reached.
Copper significantly increased the loss of DMDS in the dry white
wines after one month of storage (Fig. S1a vs a), and in the Cabernet
Merlot wines after three months of storage (Fig. S1b vs b). In the dry
white wine only 20% of DMDS remained after one month of storage
when copper was present (Fig. 3a), whereas similar decreases in DMDS
EtSH (8.1 µg/L, Chardonnay) > 18% EtSH (6.2 µg/L, model wine) >
9% EtSH (3.2 µg/L, Shiraz).
When the pH of the Chardonnay, Shiraz, and model wines was
lowered to pH 3.0 from pH 3.5, 3.7, and 3.4, respectively, the amount
of thiols produced from thioacetates increased significantly and produced substantially higher % yields of MeSH and EtSH over 16 months
relative to the higher pH’s (Fig. 2g, h, i, j, k, l; Tables S5, S6, S7, and
S8): 28% MeSH (14 µg/L, Chardonnay) > 14% MeSH (4.4 µg/L, model
wine) > 6% MeSH (4.8 µg/L, Shiraz); and 39% EtSH (14 µg/L, Chardonnay) > 21% EtSH (7.4 µg/L, model wine) > 11% EtSH (3.9 µg/L,
Shiraz).
Thioacetates clearly present a risk to ‘reductive aroma’ formation in
wines post-bottling, and managing wine pH will have significant effects
on the magnitude of thiol release from the corresponding thioacetates.
3.2.3. Accumulation of MeSH and EtSH in white and red wine
The broader compositional differences of the white, red, and model
wine matrices had significant effects on the accumulation of MeSH and
EtSH. The accumulation effects were strikingly demonstrated when
considering the increasing complexity of the wines: model wine <
white wine < red wine. The variable response of MeSH accumulation
in the different wine matrices is shown in Fig. 3a and b, in which the
sum of the molar % MeSH produced and the molar % DMDS that
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Fig. 3. Total percentage (%) of methanethiol (MeSH) accumulation and the% dimethyl disulfide (DMDS) remaining in dry white (a) and Cabernet Merlot (b). The total
percentage (%) of methanethiol (MeSH) and ethanethiol
(EtSH) accumulation, dimethyl disulfide (DMDS) and diethyl disulfide (DEDS) formation, and remaining methyl
thioacetate (MeSAc) and ethyl thioacetate (EtSAc) in
Chardonnay (c, d), Shiraz (e, f), and model wines (g, h) as
modulated by pH and copper, Cu(II), addition.
d; e vs f). When comparing the white wines to the red wines, significant
increases in MeSH and EtSH accumulation were once again only measured in the white wines (Fig. 3d vs f).
The symmetrical disulfides, DMDS and DEDS, were only produced
in the simple model wine matrices, where the probability of identical
thiols oxidizing to produce symmetrical disulfides was greater than in
complex matrices where there were many other compounds that the
thiols could react with. Irrespective of the pH or copper addition, in all
model wines with added MeSAc and EtSAc, varying concentrations of
DMDS and DEDS were measured (Fig. 3g, h, S1e, S1f, S1g, S1h).
Copper significantly impacted the oxidation of MeSH and EtSH to
DMDS and DEDS, respectively. The oxidation of DMDS and DEDS in
model wines with added copper from the MeSH and EtSH (produced
from MeSAc and EtSAc) (Fig. 3g, h) was greater than the oxidation of
DMDS and DEDS from MeSH and EtSH (produced from MeSAc and
EtSAc) in model wines without copper (Fig. S1e, S1f, S1g, S1h). Approximately 25% more DMDS and DEDS was produced in model wines
treated with added copper than without. It has been suggested that the
concentrations were only reached after 12 months of storage without
added copper (Fig. S1a). Similarly, in the Cabernet Merlot wines DMDS
decreased to less than 10% after six months of storage when copper was
added (Fig. 3b), whereas approximately 15% of DMDS remained even
after 12 months of storage when no copper was present (Fig. S1b). A
corresponding increase in %MeSH was not measured in the Cabernet
Merlot wines (Fig. 3b). This suggests that as MeSH was formed in the
red wines, MeSH reacted with wine compounds, such as tannins,
polyphenols, quinones, metal ions, that suppressed the accumulation of
free MeSH.
There were differences in accumulation of MeSH and EtSH from
MeSAc and EtSAc in the white wines and the red wines (Fig. 3c, d vs e,
f). The decrease in MeSAc and EtSAc proceeded slowly over 16 months
at similar rates in the Chardonnay at pH 3.5 and Shiraz at pH 3.7
(Fig. 3c, e). The MeSAc and EtSAc concentrations decreased at a faster
rate in the model wines at pH 3.4 (Fig. S1c, S1d) compared to the
Chardonnay and Shiraz wines (Fig. 3c, d). When the pH was lowered to
pH 3.0, the rate of MeSAc and EtSAc consumption increased (Fig. 3c vs
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molar % yield of approximately 1% did not produce as much H2S in
wines post-bottling compared to the amount of H2S produced from Cys
and GSH during fermentation. However, Cys and GSH were associated
with increased H2S concentrations of up to 3-times the concentration of
H2S in the control wines. The small % yield of H2S from Cys and GSH
may significantly impact wine aroma and quality. Verdelho and Shiraz
wines treated with only copper produced large concentrations of H2S.
This suggests that a pool of yet to be identified precursor compounds
are also involved in modulating final H2S concentrations in wine
through copper-catalyzed reactions.
The disulfide, DMDS (as a representative for disulfide and possibly
higher polysulfanes compounds), is likely to be an important precursor
that determines free MeSH concentrations in wine post-bottling. In this
experiment, DMDS produced a % yield of up to 70% of MeSH in dry
white wine when in the presence of copper. A wide range of disulfides
may release large amounts of their corresponding smaller thiol compounds, especially when in the presence of copper.
The thioacetates, MeSAc and EtSAc, act as important precursors to
MeSH and EtSH, and for these compounds wine pH played an important
role in modulating the magnitude of thiol release from the corresponding thioacetate. The maximum amount of MeSH and EtSH produced from MeSAc and EtSAc, respectively, in the Chardonnay wines
was consistently between 18% and 39%, and between 13 and 21% in
the model wines. The fact that smaller concentrations of MeSH and
EtSH accumulated in the red wines, suggest that these thiols interact
with wine compounds that inhibit their accumulation in red wine.
Finally, some risk remains that under changing wine conditions any RSH incorporated into other molecular forms may again be released. The
impacts of the formation of MeSH or EtSH from thioacetates or disulfides will have significant impacts on wine aroma and quality, seeing
that the OTs of MeSH and EtSH are much lower than those of the
thioacetates or disulfides.
Taken together, our results demonstrate that understanding the
consequences of the presence of thioacetates and disulfides in finished
wine is important, as these compounds can be byproducts of yeast
metabolism. Furthermore, winemaking processes may contribute to the
formation of disulfides such as DMDS. Hence managing thioacetate and
DMDS formation, through yeast selection and through the management
of copper additions to wine, may help winemakers protect their wines
from MeSH and EtSH formation later during bottle storage and maturation.
formation of the symmetrical disulfides (such as DMDS and DEDS) in
wine is a possible pathway that explains the decrease in MeSH and EtSH
concentrations in wine over time (Limmer, 2005). However, this study
has demonstrated that DMDS and DEDS are only produced from MeSH
and EtSH, respectively, in the absence of other matrix compounds and
only after an extended storage period, with higher concentrations of
DMDS produced in the presence of copper. In complex wine matrices
the formation of unsymmetrical disulfides may be preferred to the
formation of symmetrical disulfides.
The ability of DMDS, MeSAc, and Met to act as precursors to MeSH,
and the modulating effects of copper and pH in determining the roles of
these compounds as precursors to MeSH, are summarized in
Scheme 1(B). The ability of EtSAc to act as a precursor to EtSH, and the
modulating effects of copper and pH in determining the roles of EtSAc
as precursor to EtSH, are summarized in Scheme 1(C).
3.3. Dimethyl sulfide
3.3.1. Precursors to DMS
Previous studies have shown that SMM is a significant precursor to
DMS, but that DMSO can also contribute to DMS concentrations in
wines post-bottling (Loscos et al., 2008; Segurel et al., 2005). The aim
of this experiment was to determine the expected % accumulation of
DMS from SMM or DMSO when these compounds are present in wines
post-bottling.
On evaluation of the expected % conversion of SMM and DMSO to
DMS, it was found that SMM produced a yield approximately 20% of
DMS (854 µg/L and 917 µg/L in Sauvignon blanc and Shiraz, respectively), with DMS production from SMM not plateauing after even
12 months of storage (Fig. S2c and S2d; Tables S9 and S10). Similarly,
DMSO continued to produce significant concentrations of DMS to give a
maximum yield of 9% (258 µg/L in Sauvignon blanc) after 12 months of
storage, with the rate of DMS formation not plateauing even after
12 months of storage (Fig. S2c, S2d; Tables S9 and S10).
3.3.2. Effects of aluminum and zinc on DMS formation
Additional factors that have been shown to impact DMS formation
post-bottling were the presence of aluminum and/or zinc, with both
metals associated with lower DMS formation previously (Viviers et al.,
2013), and as such these metals were included in this experiment.
However, on this occasion aluminum and zinc did not significantly
impact DMS formation (Fig. S2a, S2b, Table S9). The only obvious
difference was observed between ‘SMM’ vs ‘SMM + Zn(II)’ treatments
after 10 months of storage in the Shiraz wines, with 1.3% more DMS
produced from SMM with added zinc than from SMM alone (687 µg/L
vs 654 µg/L, Table S10). The effects of aluminum and zinc may be less
defined in this study, as the concentrations of the metals were approximate 2.5 times lower than the concentrations of these metals in
the previous study (Viviers et al., 2013).
Acknowledgments
We thank Pamela Solomon and Tracey Siebert from the Australian
Wine Research Institute (AWRI, Adelaide, Australia) for technical support and Jason Young at Flinders University Analytical Department
(Adelaide, Australia) for ICPMS analyzes. The AWRI is a member of the
Wine Innovation Cluster at the Waite Precinct 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.
3.3.3. The accumulation of DMS in white and red wines
There were no differences in the accumulation of DMS. The % DMS
yield produced from either SMM or DMSO was similar in both
Sauvignon Blanc and Shiraz wines (Fig. S2c, S2d; Table S10). The
abilities of SMM and DMSO to act as precursors to DMS, and the
modulating effects of aluminum and zinc in determining the roles of
these compounds as precursors to DMS, are summarized in
Scheme 1(D).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.foodchem.2017.10.123.
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