Occurrence and Analysis of Sulfur Compounds in Wine

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Chapter 11
Occurrence and Analysis of Sulfur Compounds in Wine

Daniela Fracassetti and Ileana Vigentini
Daniela Fracassetti and Ileana Vigentini
Additional information is available at the end of the chapter
Additional information is available at the end of the chapter
http://dx.doi.org/10.5772/intechopen.72823
Abstract
Sulfur compounds play an important role in the sensory characteristics of wine. These
molecules can derive from the grape, in which the non-volatile forms are usually pres-
ent as glycosylated molecules, the metabolic activities of yeast and bacteria, the chemi-
cal reactions taking place during the wine aging and storage, and the environment. The
sulfur compounds include molecules positively correlated to the aromatic profile of
wine, namely the volatile thiols, and are responsible for certain defects, imparting notes
described as cabbage, onion, rotten egg, garlic, sulfur and rubber. Due to the low con-
centration of these molecules in wine, their high reactivity and the matrix complexity,
the analytical methods which enable their detection and quantification represent a chal-
lenge. The solid phase microextraction (SPME) technique has been developed for sulfur
compounds associated with off-flavors. The analysis of volatile thiols usually requires a
derivatization followed by gas chromatography (GC)-MS or UPLC-MS methods. Besides
the sulfur-containing aromas, another sulfur compound that deserves mention is the
reduced glutathione (GSH) which has been widely studied due to its antioxidant proper-
ties. The analysis of GSH has been proposed using a liquid chromatography technique
(HPLC or UPLC) coupled with fluorescence, MS and UV detectors.
Keywords: sulfur off-flavors, volatile thiols, reduced glutathione, sample preparation,

analysis, wine
1. Introduction
Sulfur-containing compounds strongly affect the sensory properties of wine and must. They
include aromatic molecules, off-flavors and a well-known non-volatile compound with anti-
oxidant properties such as glutathione. The sulfur-containing compounds can be derived from
the grape in which the non-volatile forms are usually present, the metabolic activities of yeast
and bacteria, the chemical reactions taking place during the wine aging and storage, and the
environment [1]. Their presence in wine can be the result of both enzymatic and non-enzymatic
© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons
© 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons
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226 Grapes and Wines - Advances in Production, Processing, Analysis and Valorization
mechanisms. In the first case, the sulfur compounds represent the products of metabolic and
fermentative pathways whose substrates are both amino acids and some sulfur-containing
pesticides. When yeast and bacteria metabolize these thiols, the released sulfur compounds
are generally considered off-flavors [2]. Non-enzymatic processes include photochemical, ther-
mal and other chemical reactions of sulfur compounds during winemaking and storage [3].
The reactions involving the sulfur-containing amino acids, in particular, can bring about the
light-struck taste in case the bottled white wine is exposed to light greatly affecting the sen-
sory properties [4, 5]. The sulfur-containing off-flavors impart negative notes such as cabbage,
onion, rotten egg, garlic, sulfur and rubber [6]. Among them, hydrogen sulfide is probably the
best-known sulfur compound in wine. Hydrogen sulfide is a very reactive species and can
trigger reactions generating compounds such as mercaptans, dimethyl sulfide and polysulfide,
which also have a negative impact on the wine aroma. The long-chain polyfunctional sulfur
compounds, also known as volatile thiols, are one of the most important groups of aroma com-
pounds in wine, which confer pleasant aromatic notes at trace levels; at high concentrations,
these compounds can be objectionable yet [3, 7–9]. Volatile thiols such as 4-mercapto-4-meth-
ylpentan-2-one (4MMP), 3-mercaptohexan-1-ol (3MH) and 3-mercaptohexyl acetate (3MHA)
are particularly important, for example, for characterizing the typical aroma of Sauvignon
Blanc wine [10]. Moreover, in some wines, 4-mercapto-4-methylpentan-2-ol (4MMPOH) is also
detected but its concentration is lower than the thiol-related aromas mentioned above [11].
Besides the sulfur compounds contributing to the sensory characteristics of wine, the gluta-
thione is of particular interest due to its antioxidant properties. This tripeptide can limit the
browning of white must as it reduces back the o-quinones to the correspondent phenols and,
consequently, the formation of the brown polymers are avoided [12].
2. Aromatic compounds containing sulfur
2.1. Sulfur off-flavors
Sulfur-containing compounds are mainly associated with off-odors due to the presence of
hydrogen sulfide and mercaptans whose concentrations are typically low as well as their per-
ception threshold [2]. The most abundant sulfur off-flavors found in wine are represented by
hydrogen sulfide, methanethiol, ethanethiol, dimethylmercaptans (dimethylsulfide, dimeth-
yldisulfide, dimethyltrisulfide) [13] and other sulfur-containing compounds responsible for
off-flavors in wine (Table 1). Hydrogen sulfide is the most recognized sulfur compound asso-
ciated with the rotten egg aroma, it is highly volatile and has a low perception threshold
(10–80 μg/L). Even if hydrogen sulfide could be easily removed by copper treatment [14], it
is very reactive specie participating in the generation of other mercaptans, such as dimeth-
ylsulfide and polysulfide, and ethanthiol, the latter due to its combination with ethanol or
acetaldehyde [7]. Saccharomyces cerevisiae can release this compound through the degradation
of sulfur-containing amino acids and the reduction of elemental sulfur, sulfite or sulfate [15].
However, its synthesis is limited when nitrogen derived from either amino acids (except for
cysteine) or ammonium is added [13]. Variable amounts of hydrogen sulfite can be released
during the alcoholic fermentation. Smith and co-authors [16] suggest that the sulfur-containing
Occurrence and Analysis of Sulfur Compounds in Wine 227
Compound Olfactory description Perception threshold Range in wine

(μg/L) (μg/L)
Hydrogen sulfide Rotten eggs, reduced taste 0.001–150 0–370
Methanethiol Cooked cabbage, reduced taste 0.3 0–16
Ethanthiol Onion, rubber, putrefaction 1.1 0–50
Dimethyl sulfide Cabbage, asparagus, corn, molasses 10–160 0–910
Carbon disulfide Cabbage, rubber >38 0–18
Dimethyl trisulfide Cabbage, onions, cooked vegetable 0.1 0–111
Diethyl sulfide Garlic 0.93–18 0–10
Dimethyl disulfide Cooked cabbage, asparagus, onions 20–45 0–160
Diethyl disulfide Garlic, onion, burnt rubber 4.3–40 0–160
2-Mercaptoethanol Barnyard-like, poultry, farmyard 130 0–400
Methylthioacetate Sulfurous, rotten vegetables 300 0–115
S-Ethylthioacetate Sulfurous 40 0–180
2-(Methylthio)-1-ethanol Cauliflower, French bean 250 0–139
2-(Methylthio)-1-propanol Cauliflower, cooked cabbage 1200 0–5655
2-(Methylthio)-1-butanol Onion, garlic, earthy 100 0–180
Benzothiazole Rubber 50–350 0–30
5-(2-Hydroxyethyl)-4-methylthiazole Green 100–1000 5–50
Table 1. Common fermentative sulfur compounds and off-flavors found in wines [3, 22, 120].
precursors could produce hydrogen sulfite, methanthiol and dimethylsulfide during fermen-
tation and not all these compounds are released in gaseous form. The chemistry behind the
reactions and regulation mechanisms involving hydrogen sulfite and other mercaptans pro-
duced by the yeast is not well understood [17]. Methionine can be metabolized by yeast to
produce fusel alcohol, methionol and 3-methylthio-1-propanol imparting cabbage and cau-
liflower aromas. Cysteine is a precursor of S-containing heterocycle compounds; these com-
pounds can be metabolized by Oenococcus oeni and aroma descriptors such as sulfury, floral,
fruity, toasted and roasted can be imparted to the wine [2, 18]. The microbial degradation of
these amino acids can lead to the presence of dimethyl sulfide which has shown the property
of enhancing the wine aroma due to the interactions with other volatiles, such as esters and
norisoprenoids [19, 20]. Nevertheless, dimethyl sulfide imparts notes such as asparagus, corn
and molasses which are undesired characters in white wine to a certain extent, although the
aroma complexity is increased [21]. The levels of dimethyl sulfide, methionol, diethyl sulfide
and diethyl disulfide increase as the aging time and storage temperature increase and they
can influence the aroma complexity of aged wine [13, 22]. Mercaptans are responsible for
reduced aroma in wine. Different strategies can be used for the removal of sulfur off-flavors.
The addition of diammonium phosphate or other yeast-based nutrients leads to an increase
of the readily assimilable nitrogen enabling the yeast to convert sulfur-containing amino acids
into hydrogen sulfide [23]. The aeration of wine during the alcoholic fermentation as well as
wine racking in aerated conditions showed a protective effect against the formation of sulfur
off-flavors. The oxygen positively contributes to the fermentative capacity of the yeast [24, 25].
Thereafter, the oxygen presence maintains lower levels of sulfur off-flavors during storage
[26] probably due to either the formation molecular mass sulfur compounds or the quinone
ability for trapping the sulfur compounds [16]. The aging of the lees can have a positive effect
since the mercaptans can link to the cysteinyl residues of the yeast cell wall to give disulfides
[27]. Wine treatment with copper or silver showed to be affective for the removal of hydrogen
sulfide as copper or silver sulfide precipitates were formed [28]. However, recent studies have
indicated that copper addition post-bottling increases the level of sulfur off-flavors during
aging [24, 29, 30]. The effect of copper still needs to be clarified as well as its dosages.
The sulfur-containing compounds are associated with the occurrence of light-struck taste, a
defect affecting white wine bottled in clear bottles and exposed to light. The photochemical
oxidations may affect different wine components including phenols, acids and alcohols [31,
32]. The formation of the light-struck taste is related to the presence of riboflavin, a vitamin
highly sensitive to light and methionine. The pathway proposed by Maujean and Seguin [4]
involves the photoxidative degradation of methionine to give methional and reduced ribofla-
vin. Methional is unstable when exposed to light and decomposes to acrolein and methanethiol
(Figure 1). Two molecules of the latter eventually yield dimethyl disulfide (DMDS). The light-
struck taste is described as cooked cabbage mainly due to the formation of methanethiol and
DMDS though hydrogen sulfide may have a role [33–35]. Light exposure can also lead to the
formation of other undesirable compounds, such as furfural [36] which has been positively cor-
related to the “cooked vegetable” aroma of white wines stored under oxidative conditions [37,
38]. Not all wines are susceptible to developing this defect. The concentration of riboflavin plays
the major role [5, 39] and when it is lower than 80–100 μg/L, the risk of the light-struck taste
decreases. The riboflavin content in the grape hardly exceeds a few tens of micrograms per liter
and the metabolic activities of S. cerevisiae affect its levels in wine [40, 41]. The levels of riboflavin
can be reduced by means of wine treatment with bentonite or charcoal as well as by using a low
producer yeast strain of this vitamin. Finally, nutrients commonly employed for favoring yeast
growth could further increase the amount of riboflavin in wine [41]. Moreover, the amounts of
methionine could influence the occurrence of light-struck taste as well as the presence of oxygen
[42]. The light-struck taste can be limited by the addition of antioxidant compounds. The use of
gallic tannins seems to be particularly promising in protecting wine possibly due to the binding
of sulfur off-flavors and the quinones present in the polyphenol-based mixture [42].
2.2. Varietal thiols
The varietal thiols including 3-mercaptohexan-1-ol (3MH), 3-mercaptohexylacetate (3MHA)

and 4-mercapto-4-methylpentan-2-one (4MMP) are sulfur-containing aromas associated with
the typical flavor of Sauvignon Blanc wines [11, 43, 44]. Moreover, in some wine, 4-mercapto-
4-methylpentan-2-ol (4MMPOH) was also detected, but its level was lower than the volatile
thiols mentioned above [44] (Table 2). They are characterized by some fruity aromas, like cassis
[45], grapefruit [46], passion fruit [47] and guava [48]. The impact of sulfur compounds on wine
aroma has been updated in literature and the varietal character is affected by several of these
Figure 1. Reaction scheme of methional formation due to light exposure [4].
molecules if their concentration is close to the perception threshold [22, 49, 50]. 3MH and 3MHA
have olfactory perception thresholds of 60 and 4 ng/L, respectively and they are responsible for
passion fruit-like and grapefruit-like olfactory notes [11]. These compounds were also found in
red wines [51]. The 4MMP has an olfactory perception threshold of 0.8 ng/L, and its aroma is
described as box tree-like, black currant-like, or even cat urine-like when occurring at high con-
centration [10]. The 4MMPOH has a perception threshold of 55 ng/L in aqueous alcoholic solu-
tion and it is reminiscent of citrus zest and grapefruit [44]. 3MH is the most abundant in wine,
in concentrations generally higher than its perception threshold, while 4MMPOH in wine is
generally lower than its perception threshold. The level of 4MMP is dependent on the grape
cultivar and it changes between different samples in the same cultivar [52]. The concentrations
of 3MH and 3MHA in Sauvignon Blanc wines from different countries have been reported to be
in the range 688–18,681 and 10–2507 ng/L, respectively [53–55]. Besides Sauvignon Blanc wines,
the volatile thiols were detected in white wine made from different Vitis vinifera grape varieties,
such as Gewürtztraminer, Muscat, Riesling, Sylvaner, Pinot Gris, Pinot Blanc, Colombard, Petit
Manseng, botrytised Semillon and Grenache [44, 56]. Moreover, these compounds were also
found in certain Italian autochthonous varieties including Verdicchio Bianco (also known as
Trebbiano di Lugana) [57], Arneis [58], Grillo and Catarratto Bianco Comune [59].
The content of volatile thiols decreases during wine aging, according to the oxidative conditions.
Glutathione, sulfur dioxide and anthocyanin content exert a protective effect. In contrast,
Compound Structure Olfactory Perception Range in

description threshold wine (ng/L)b
(ng/L)a
3-mercaptohexan-1-ol Passion fruit, 60 26–18,000
(3MH) grape fruit,
guavac
3-mercaptohexylacetate Passion fruit, 4 0–2500

(3MHA) grape fruit, box
tree, guavad
4-mercapto-4- Box tree, black 0.8 0–40

methylpentan-2-one currant, passion
(4MMP) fruite
4-mercapto-4- Citrus zestf 0–90

methylpentan-2-ol
(4MMPOH)
a
In model wine solution [8, 10, 11, 147].
b
Range Vitis vinifera wines [11, 28, 53–55, 92, 148].
c
[43].
d
[8, 11].
e
[8, 147].
Table 2. Contribution of volatile thiols in Vitis vinifera wines.
increased contact with oxygen, particularly in the presence of catechin derivatives, promotes
their degradation [52, 60, 61]. Three different mechanisms proposed lead to their decay with
a different degradation kinetics. The volatile thiols can be easily oxidized when oxygen and
iron are present, forming disulfides [62, 63]. Since the volatile thiols act as electrophiles,
they can react with phenolic compounds [64]. Moreover, the presence of oxygen and cata-
lyst metals, namely iron and copper, lead the oxidation of phenols into the corresponding
o-quinones which can bind the volatile thiols [65, 66]. A rapid decline of 3MHA is observed
after 3 months of bottle storage, while a much slower decline in 3MH can be noticed. After 1
year of storage, the ester has completely disappeared, while 3MH is still present but its con-
tent is halved. The chemical structure of the volatile thiols affects their degradation: 4MMP,
a tertiary thiol, results less able to react with o-quinones and decreases slower in comparison
to 3MH, a secondary thiol, maybe due to its steric hindrance [63]. The wine composition
and oxygen exposure are the major reasons for loss of volatile thiols both during winemak-
ing and post-bottling [29, 67]. The presence of antioxidants, such as sulfur dioxide, ascorbic
acid and reduced glutathione, limit the polyphenol oxidation either by removing oxygen
from wine or by reversing and altering the oxidation process. In particular, the addition of
glutathione, up to 20 mg/L before bottling, led to higher 3MH level after 6 months of storage
in bottle [29].
2.2.1. Varietal thiol precursors
The varietal thiols occur in grape berry as non-volatile cysteinyl- and glutathionyl-conjugated
precursors [29, 68]. 3MH bound as cysteinyl-conjugate (Cys-3MH), glutathionyl-conjugate
(GSH-3MH) and also cysteinylglycin-conjugate (CysGly-3MH) have been reported [70, 71].
4MMP occurs in the grape and must as cysteine conjugate (Cys-4MMP) and glutathione con-
jugate (GSH-4MMP) [72, 73]. Moreover, 3MH can be derived from either (E)-2-hexen-1-ol or
(E)-2-hexenal [74, 75]. Through a lipoxygenase/lyase sequence active in the presence of oxygen,
(E)-2-hexenal can be obtained from linolenic acid and converted to GSH-3MH by coupling with
glutathione [75–77]. (E)-2-hexenal may act as a precursor when hydrogen sulfide is released in
the early part of the fermentation [73, 74, 78]. Under aerobic conditions, S. cerevisiae is able to oxi-
dize (E)-2-hexen-1-ol into (E)-2-hexenal, and the reverse process can occur under anaerobic con-
ditions [77]. Recently, S-3-(hexan-1-al)-glutathione (GSH-3MHAl) was identified in Sauvignon
Blanc juice [79] and it can be considered a precursor of thiol aromas. These compounds are split
by the S. cerevisiae through the enzymatic activity of carbon-sulfur lyase [10, 80], the volatile
thiols are liberated. 3MHA is obtained by yeast activity from 3MH esterified with acetic acid as
precursor [7]. This finding correlated the ester and volatile thiol metabolism in yeast for the first
time [2]. The capability of S. cerevisiae to liberate volatile thiols from their precursors is genetically
determined; the yeast selection can represent a useful tool to favor the 3MH and 4MMP release
[8, 50]. The fermentation temperature affects the varietal thiol concentrations in the resulting
wines: in the range of alcoholic fermentation for white wines, at higher fermentation temperature
and higher concentrations of varietal thiols is released. Among the S. cerevisiae strains commonly
used, VL3 and EG8 release more volatile thiols, in comparison to VL1 and 522 [80]. S. bayanus var.
uvarum strains and hybrids of the latter yeast and S. cerevisiae showed a higher ability to release
both 3MH and 4MMP when compared to S. cerevisiae strain [81]. Furthermore, non-Saccharo
myces yeasts, like Torulaspora delbrueckii and Pichia kluyveri, have been proposed as innovative
tools to improve wine quality, being able to release varietal thiols [82, 83]. The 3MHA formation
from 3MH is also dependent on the strain genetic characteristics [84]. S. cerevisiae strains showed
the enhanced ability to hydrolyze the S-cysteinyl link rather than the ester synthetic activity.
The combined use of different yeast strains, one of them having hydrolyzing ability, with stron-
ger esterification ability, can represent a useful tool to affect the volatile thiol composition [50].
However, several researches have indicated that no clear correlation between precursor concen-
trations in must and free thiol concentration in wine exist [8, 16, 59, 73, 80, 85–89].
The harvest has a significant effect on the content of thiol precursors in juice. Capone and
Jeffery [90] showed that machine harvest and transportation for 12 h of the grapes led to
higher levels of thiol precursors in comparison to hand harvest grapes immediately processed.
The authors suggested the minimal berry damage results in less formation of the precur-
sors. More than half of the total cysteine conjugates is located in the grape skin; as a conse-
quence, an increased skin contact time augments Cys-3MH in grape musts, while it has little
effect on Cys-4MMP and Cys-4MMPOH concentrations [91–93]. Stronger pressing conditions
allow a major extraction of thiol precursors in comparison to free run juice. Roland and co-
authors [94] demonstrated an easier extraction of Cys-3MH in comparison to GSH-3MH due
to their distribution in different parts of the skin. The produced wines contained 3MH at
higher concentrations when the juice was collected at the end of the pressing cycle. On the
contrary, Patel et al. [95] found lower amounts of 3MH and 3MHA in wine produced from
juice obtained from stronger pressing conditions in which the thiol precursors were present
in higher amounts. This can be due to the higher extraction of oxidizable phenols inducing
the rapid decrease of glutathione and the oxidation of varietal thiols. The pressing performed
on an industrial scale caused a strong decrease of thiol precursors. The factors affecting the
GSH-portion degradation of the precursors are not completely clear and mechanisms, other
than oxidation or proteolysis, could induce the loss of the thiol precursors [59].
3. The reduced glutathione
The reduced glutathione (GSH) is a tripeptide constituted by l-cysteine, γ-glutamic acid and
glycine exerting antioxidant and detoxifying activities in the cell [96, 97]. This compound exerts
several activities in must and wine. The antioxidant property of GSH is well known: it can reduce
the o-quinone deriving from the enzymatic oxidation carried out by the polyphenoloxidase
enzymes (PPO) on the tartaric esters of hydroxycinnamic acids. This hinders the formation poly-
mers causing the browning of must [12]. During aging, the o-quinones are produced as a result
of the non-enzymatic oxidation (also known as chemical oxidation) of o-diphenols [98]. The level
of o-diphenols in wine is correlated to the browning of white wines [28, 98, 99]. Caffeoyl-tartaric
acid (caftaric acid) and coumaric-tartaric acid (coutaric acid) are some of the most abundant
hydroxycinnamic acids in must representing the substrate mainly oxidized by the enzymatic
action. The GSH can reduce the oxidized caftaric acid, generating 2-S-glutathionyl caftaric acid,
also known as Grape Reaction Product (GRP) [100]. The GRP is not a substrate of the PPO and it
can trap the o-quinone, limiting the formation of brown polymers which are responsible for color
changes of white must and wine. GRP can oxide enzymatically by the Botrytis cinerea laccase and
chemically by the caffeoyl-tartaric acid quinone. In this way, the GRP quinone is originated; it
can be a substrate of condensation reaction with the phenols. This molecule is responsible for
brown compound formation. The high GSH concentration allows a second nucleophile attack,
in position 5 of the benzyl ring. The 2,5-diglutathionyl caftaric acid (GRP2) is formed and is a
substrate of the laccase action [12]. With low GSH concentration, the GRP can be oxidized by the
excess caftaric acid quinones which can cause an intense browning (Figure 2) [101, 102].
GSH is able to limit the loss of the flavoring volatile thiols acting as a competitor for the reduc-
tion of the quinones [103]. In fact, GSH concentration is about a thousand times higher than that
of volatile thiols which are protected against oxidation. Lavigne and Dubourdieu [103] reported
that when the GSH concentration ranged from 6 to 10 mg/L it slowed down the decrease of
volatile thiols. Additionally, other aromatic compounds, such as isoamyl acetate (3-methyl-1-bu-
tyl acetate), ethyl hexanoate and linalool (3,7-dimethylocta-1,6-dien-3-ol), are better protected
during bottle storage [104]. GSH can limit the formation of sotolon (3-hydroxy-4,5-dimethyl-
2(5H)-furanone), a compound responsible for the atypical aging character of white wine [103].
It confers aroma descriptors such as dried fig and rancid and its perception threshold is 7 μg/L
[105]. Besides sotolon, 2-aminoacetophenone (1-(2-aminophenyl)-ethanone) is also responsible
for the atypical aging and it has a lower perception threshold than sotolon, corresponding to
1 μg/L. Both sotolon and 2-aminoacetophenone concentrations increase due to the exposition
of wine to oxygen during bottling [106]. GSH can have a protective action on the wine aroma
during the oxidative aging. GSH reduces the formation of both these off-flavors during storage.
Moreover, GSH can have a positive effect on white wine color which appears to be more stable
during aging [103, 107]. The first source of GSH is the grape in which it can exceed 200 mg/L of
grape juice according to grape cultivar, environmental conditions and viticultural practices [108]
Figure 2. Scheme of GRP and browning compound formation [100–102].

and the amounts of readily assimilable nitrogen in the soil [103]. Lower levels were found in must
up to 100 mg/L [108] and it can be affected by exposure to oxygen, tyrosinase activity and pre-
fermentative grape skin maceration [93, 109]. The loss of GSH in must production can negatively
affect the formation of precursors of the varietal thiol compounds [73] as well as the residual con-
tent of GSH during wine aging. Glutathione was reported to be consumed by S. cerevisiae at the
beginning of the alcoholic fermentation and then to be released by the yeast cell lysis [103, 110].
At the beginning of the alcoholic fermentation GSH almost disappears and then its concentration
increases as an effect of the yeast cell synthesis and lysis. However, winemaking on an industrial
scale did not preserve GSH in must and the level of this antioxidant increased during the alco-
holic fermentation due to the yeast metabolism [111]. Lavigne and Dubourdieu [103] reported
the GSH level becomes stable 1 month after the alcoholic fermentation is started. On the contrary,
in recent researches, the highest levels of GSH were observed after the racking [111, 112]. The con-
centration of GSH in wine is lower than in the juice and grapes and it ranges from 3 to 35.5 mg/L
[109, 111–113] and can be increased through the choice of an adequate yeast strain [15]. No GSH
is released from yeast under nitrogen starvation during the alcoholic fermentation [103, 111]. The
GSH concentration decreased after the racking as well as during wine aging on lees [57, 95, 111]
maybe due to the adsorption of GSH on the yeast lees as occurs for other low-molecular weight
thiols during wine aging [114].
4. Analytical methods
The analytical methods developed for the analysis of sulfur compounds in wine need to over-
come the low concentrations and the high reactivity of these molecules as well as the com-
plexity of the matrix. Sulfur compounds in wine are frequently divided into “light” (boiling
point < 90°C) and “heavy” (boiling point > 90°C) compounds [3] indicating difficulty in using
a relevant common sampling/enrichment technique.
4.1. Sulfur off-flavors
The most common technique employed for the analysis of sulfur compounds associated
with off-flavors in wine is static headspace analysis by means of solid phase micro extraction
(SPME) combined with gas chromatography (GC) coupled with different detectors (Table 3).
Methods in dynamic headspace by using Purge and Trap equipment was also described as an
alternative to the static headspace. In static headspace technique, the analytes reach the equi-
librium state between the liquid and gas phases and then are adsorbed in a fiber. In dynamic
space, the analytes in the gas phase into the headspace and the atmosphere around the sample
is constantly swept away by a flow of carrier gas, taking volatile analytes with it. Through
this technique, the equilibrium state is not reached and, thus, more of the volatile dispersed
in the matrix will pass into the headspace whose size results in increased sampling phase. As
a consequence, the trapping stage of the analysis offers good sensitivity [43, 115]. The proper
combination of sorbent and temperature may permit collection and concentration of specific
analytes while venting others. Despite this, the instrumentation requires more complexity and
it is more expensive than other sampling techniques, such as the static headspace. Moreover,
Analytical technique Advantages Disadvantages

Dynamic Purge and trap—gas Increased sampling Many sources of errors
headspace chromatography phase; good sensitivity
Static headspace Solid phase micro extraction Cheaper and simpler Compromise between sensitivity and
(SPME)—gas chromatography instrumentation extraction/temperature conditions
needs to be properly set
SPME fiber
Carboxen-polydimethylsiloxane Suggested for sulfur Less versatile than CAR-PDMS-DVB

(CAR-PDMS)a compounds with low fiber
boiling point
Carboxen-polydimethylsiloxane- Best extraction yield for Suitable analytical method setting for
divinylbenzene the analytical conditions increasing the extraction yield (sample
(CAR-PDMS-DVB)b applied temperature, ionic strength, extraction
time)
a
[117, 118].
b
[22, 119, 120].
Table 3. Analytical methods for the determination of sulfur off-flavors.
many sources of error in Purge and Trap instruments have been reviewed [116]. As for the
dynamic headspace, the choices of the proper fiber as well as sample temperature and the
presence of salt increasing the ionic strength can improve extraction yield in static headspace.
In particular, the use of carboxen-polydimethylsiloxane fiber (CAR-PDMS) [117, 118] or car-
boxen-polydimethylsiloxane-divinylbenzene (CAR-PDMS-DVB) [22, 119] has been proposed
for the analysis of fermentative sulfur compounds. The use of the latter fiber has been shown
to produce good results in terms of repeatability and reproducibility [22]. Moreover, the best
modified ionic strength has been obtained by using magnesium sulfate. Due to the different
boiling temperature of the fermentative sulfur compounds, a good compromise needs to be
set to allow for the adsorption of these compounds through the SPME technique. As reported
by Nguyen and co-authors [120], the incubation of the sample was carried out at 45°C for
5 min and the extraction at 45°C for 30 min under agitation. The GC equipment was coupled
with MS detector and the compounds of interest were detected in single ion monitoring (SIM)
mode and quantified by means of different internal standards properly chosen. The analytical
methods described above are suitable for the analysis of the volatiles characterized by low
boiling point (lower than 90°C) that is not the case for 3MH, 3MHA and 4MMP.
4.2. Varietal thiols
Among headspace sampling techniques, only purge and trap has been used to analyze 3MH
and 3MHA in wines, reaching detection limits closer to their perception threshold [121]. Despite
this, the other heavy volatile sulfur aromas were not identified at perception threshold, thus leav-
ing derivatization procedures as the most promising technique for the extraction and analysis of
volatile thiols. Due to the low concentration of these compounds, in the order of magnitude of
ng/L, the sample preparation has provided a liquid/liquid extraction followed by the evapora-
tion of the organic solvent. As the varietal thiols are present in wine in low concentration as well
as being highly reactive, the deuterated analogues are commonly used as internal standards
compensating for the eventual loss taking place during the sample preparation [122].
Among the derivatizing compounds, the use of p-hydroxymercuribenzoate (pHMB) was first
proposed by Tominaga and co-authors [10]. The volatile thiols were derivatized with pHMB,
isolated with the use of strong basic anion exchange column followed by the liquid/liquid
extraction with dichloromethane. The sample was concentrated and analyzed by GC-MS. As
a result of the analytical method improvement, varietal thiols were quantified in several
wines produced with Vitis vinifera grape varieties [44], suggesting these sulfur compounds
play a key role in the aroma of different white wines. Besides 3MH, 3MHA and 4MMP, the
method allows the identification and quantification of two other sulfur-containing aromas,
such as 2-furanmethanethiol [11] and benzenemethanethiol [123] in wines. The identifica-
tion of these two latter molecules, together with the identification of ethyl 3-mercaptopropio-
nate, established the role of certain volatile thiols in the bouquet of aged champagne wines
[124]. Although very efficient, this procedure is time consuming. In an effort to reduce the
time required for the sample preparation, a covalent chromatography was employed for
the enrichment of the thiols from the wine extract. Specifically, a cross-linked agarose gel
containing phenylmercuric chloride was used where the volatile thiols were trapped after
their liquid/liquid extraction with dichlomethane [122]. The ability of some common solid
phase extraction sorbents to retain organomercuric salts for selective concentration of thiols in
wines was also proposed using styrene-divinylbenzene copolymer sorbent. Nevertheless, the
organomercury salt that is formed for the detection of varietal thiols by the analytical meth-
ods described (considered as hazardous poison) still remains the key point of this method.
Other analytical approaches employ pentafluorobenzyl bromide as a derivatizing agent,

which transforms thiols into their corresponding pentafluorobenzyl derivatives [125–128].
The derivatizing reaction is usually carried out in a purified extract (i.e. water) [128], organic
solvent [126], in-cartridge [127], or in-fiber [125]. Another very promising derivatizing agent
in the gas chromatography analysis of thiols is ethyl propiolate, which is able to derivatize
thiols directly in wine matrix and is a suitable derivatizing reagent for the electron impact
mass spectrometry detection system [129].
Moreover, Piano and co-authors [55] proposed an analytical method in which the varietal
thiols are detected by liquid chromatography (UPLC) coupled with MS/MS. The sample
preparation required several steps in order to protect the thiol aromas from oxidation and the
liquid/liquid extraction in order to achieve the concentration of the analytes. The o-phthaldi-
aldehyde (OPA) is used as derivatizing agent. The method allows the quantification of 3MH
and 3MHA, but the derivatization of 4MMP does not occur. The formation of the OPA deriva-
tive of 4MMP was probably prevented by the hydrogen bonding between the thiol group and
the carbonyl moiety within the compound itself, and its steric hindrance. Derivatization of
4MMP was an issue when other derivatizing reagents were used [127].
4.2.1. Varietal thiol precursors
The analytical methods proposed for the determination of volatile thiol precursors include
both indirect and direct methods [71]. In the first case, the precursors are transformed in
volatile compounds and determined by both GC and HPLC coupled with MS detector. In the
second case, the precursors are quantified after their proper purification.
In the first method developed, the Cys-4MMP was detected by GC coupled with flame pho-
tometric detectors (FDP) by synthetizing it and 4MMP was then detected [69]. Tominaga and
co-authors [130] quantified the cysteinylated precursors by GC-MS after their derivatization.
The addition of labeled thiol precursors allowed the quantification of those contained in the
must by GC-FDP and GC-MS [131]. The GC coupled either with atomic emission detection
(AED) [132] or detection-capture mass spectrometry (DCMS) detector [86] were also proposed
employing propyl thioacetate as internal standard and derivatizing the released volatile thi-
ols with ethyl chloroformate, respectively. The precursors of 3MH were quantified using the
labeled isotopic standard d10-3MH by GC-MS.
Both GC-MS and liquid chromatography techniques were used for the direct determination of
thiol precursors. In the case of GC-MS, their derivatization is necessary and different deriva-
tizing agents were proposed [133–135]. In the case of liquid chromatography, both HPLC-MS
and HPLC-MS/MS were applied. The use of these types of equipment did not require a
derivatization of thiol precursors, but a solid phase extraction (SPE) was applied achieving
the sample purification. The quantification was carried out by means of the patterns of labeled
compounds [73, 87, 136–139] as well as without them [59, 72]. The quantification by liquid
secondary ionization mass spectrometry (LSIMS) was also reported [140].
4.3. The reduced glutathione
Several analytical methods have been proposed for GSH quantification in the grape, must
and wine using different analytical techniques [141]. GSH was quantified after a treatment
with glutathione reductase enzyme in white wine [113]. In grapevine tissues, the GSH was
derivatized with 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) after the enzymatic treatment and
the detection was performed spectrophotometrically at 412 nm [142]. The determination of
total GSH could be performed after the enzymatic treatment which allowed the breakdown
of disulfide bridges. The GSH could be derivatized pre-column with o-phthalaldehyde (OPA)
and quantified by HPLC; the detection was than conducted by fluorescence [143, 144]. The
use of the fluorescence detector allowed the GSH quantification after derivatization with
2,3-naphthalenedialdehyde (NDA), as described by Marchand and de Revel [145]. The tech-
nique involved the pre-column derivatization and the separation by HPLC. The determination
of oxidized glutathione was also allowed by enzymatic treatment with glutathione reductase.
The fluorescence detector was employed for the GSH determination after its derivatization
with monobromobimane (mBB); the reaction adduct was identified by capillary electropho-
resis [110]. As described above, the mBB is photodegradable and a GSH content underesti-
mation could occur [146]. The method proposed by Du Toit et al. [109] was based on liquid
chromatography coupled with a mass spectrometry detector (LC-MS/MS). The determination
of both reduced and oxidized glutathione was allowed in the same run. The ethanol present
in wine had to be removed prior to the analysis and this step could cause an underestimation
of the GSH content. Another analytical method for the GSH quantification was developed
using the atomic adsorption spectrometry [147]. The method is based on the reactivity of the
mercury toward the thiols. The liquid chromatography (both HPLC and UPLC) coupled with
UV detector allows the quantification of GSH after its derivatization with p-benzoquinone
[148]. The sample preparation is not required for must and wine, while the juice needs to be
obtained from grape under reductive condition prior to the derivatization [111]. Suitable pre-
parative conditions are mandatory for monitoring GSH concentration during must extraction
and clarification since such wine making steps are responsible for the chemical and enzymatic
oxidations.
5. Conclusions
Several sulfur compounds occur in wine with a strong influence on its aromatic profile. On
one hand, some of these molecules impart negative notes and their presence in wine should
be counteracted and limited. The formation mechanisms are not completely clear and fur-
ther researches are needed also to better understand the treatments and/or the winemak-
ing practices potentially decreasing the appearance of these undesired compounds. On the
other hand, the varietal thiols are responsible for positive characteristics of wine aroma up
to a certain extent. Their protection is essential for the maintenance of the aromatic profile
throughout both the winemaking and storage of wine by means of antioxidants of which
reduce glutathione can represent a good natural candidate. Further studies will be necessary
to investigate the fate of their precursors during winemaking on an industrial scale as well as
to increase the aromatic potential of the produced wine. In the last decades, improvements
of the analytical methods have been carried out in terms of sensitivity and identification of
new odorants has been achieved. The goal for the researches has being the set up more sensi-
tive and less-time consuming methods with a reduced impact on the environment in order to
minimize defects and optimize the aromatic profile during winemaking.
Author details
Daniela Fracassetti* and Ileana Vigentini

*Address all correspondence to: daniela.fracassetti@unimi.it
Department of Food, Environmental and Nutritional Sciences (DeFENS), Università degli
Studi di Milano, Milan, Italy
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Occurrence and Analysis of Sulfur Compounds in Wine

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Keywords: sulfur off-flavors, volatile thiols, reduced glutathione, sample preparation,

2. Aromatic compounds containing sulfur

2.1. Sulfur off-flavors

Compound Olfactory description Perception threshold Range in wine

Methanethiol Cooked cabbage, reduced taste 0.3 0–16

Ethanthiol Onion, rubber, putrefaction 1.1 0–50

Dimethyl sulfide Cabbage, asparagus, corn, molasses 10–160 0–910

Carbon disulfide Cabbage, rubber >38 0–18

Dimethyl trisulfide Cabbage, onions, cooked vegetable 0.1 0–111

Diethyl sulfide Garlic 0.93–18 0–10

Dimethyl disulfide Cooked cabbage, asparagus, onions 20–45 0–160

Diethyl disulfide Garlic, onion, burnt rubber 4.3–40 0–160

2-Mercaptoethanol Barnyard-like, poultry, farmyard 130 0–400

Methylthioacetate Sulfurous, rotten vegetables 300 0–115

S-Ethylthioacetate Sulfurous 40 0–180

2-(Methylthio)-1-ethanol Cauliflower, French bean 250 0–139

2-(Methylthio)-1-propanol Cauliflower, cooked cabbage 1200 0–5655

2-(Methylthio)-1-butanol Onion, garlic, earthy 100 0–180

Benzothiazole Rubber 50–350 0–30

5-(2-Hydroxyethyl)-4-methylthiazole Green 100–1000 5–50

2.2. Varietal thiols

The varietal thiols including 3-mercaptohexan-1-ol (3MH), 3-mercaptohexylacetate (3MHA)

Figure 1. Reaction scheme of methional formation due to light exposure [4].

Compound Structure Olfactory Perception Range in

3-mercaptohexylacetate Passion fruit, 4 0–2500

4-mercapto-4- Box tree, black 0.8 0–40

4-mercapto-4- Citrus zestf 0–90

Table 2. Contribution of volatile thiols in Vitis vinifera wines.

2.2.1. Varietal thiol precursors

3. The reduced glutathione

Figure 2. Scheme of GRP and browning compound formation [100–102].

4.1. Sulfur off-flavors

Analytical technique Advantages Disadvantages

Carboxen-polydimethylsiloxane Suggested for sulfur Less versatile than CAR-PDMS-DVB

Table 3. Analytical methods for the determination of sulfur off-flavors.

4.2. Varietal thiols

Other analytical approaches employ pentafluorobenzyl bromide as a derivatizing agent,

4.2.1. Varietal thiol precursors

4.3. The reduced glutathione

Daniela Fracassetti* and Ileana Vigentini

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