Sulfur Compounds in the Whisky Production Process: History
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Akira Wanikawa

Whisky is produced through malting, mashing, fermentation, distillation, and maturation. During these processes, several sulfur compounds are formed and/or reduced in quantity. To date, alkyl sulfides, thiols, methylthio group compounds, thiophenes, thiazoles, dithiapentane derivatives, and furfuryl compounds have been detected in the final spirit. Distillers might be able to control quality better if the pathways and contributions of these chemicals were known; currently, however, only some of them have been identified, while others remain to be revealed.

  • malting
  • fermentation
  • distillation
  • maturation

1. Formation of Dimethyl Sulfide in Malting

In the malting process, barley is initially steeped in water and then rested in air for 36–48 h at 16 °C. Subsequently, the water is drained off and the barley is allowed to germinate for around 5–6 days. The germinated barley is then kilned at 60–70 °C for 24 h [1].
There have been several reports on the formation of DMS during malting. DMS is described as “cooked sweet corn”, “cooked vegetables”, and “cooked tomato” [2][3], and its threshold is reported as 5 μg/L in 20% ethanol solution [4]. According to Bamforth [5], S-methyl methionine is formed from methionine during germination and converted to DMS during kilning, and at higher kilning temperatures, the amount of DMS will be increased. Bathgate also claimed that both the kilning temperature and the design of the equipment affect the character of the malt, including sulfur and vegetable-like aromas [6]. Therefore, temperature might be a key target for decreasing the amount of DMS that forms. In addition, during kilning, a portion of DMS might oxidize into odorless methylsulfonylmethane (dimethyl sulfoxide), which would then be converted back to DMS by yeast during fermentation [2].

2. Formation of Sulfur Donors during Fermentation

After mashing using malt or commercial enzymes, yeast is added to the cooled wort or mash. During fermentation, yeast converts sugars into ethanol and carbon dioxide, but is also involved in the formation of sulfur compounds. Miller summarized the sulfur compounds found in wort and whisky as follows: three alkyl sulfides, DMS, DMDS and DMTS; three thiols, sulfane, methanethiol and ethanethiol [7] (p171); five methylthio derivatives, such as 3-methylsulfanylpropane-1-ol (3-(methylthio) propanol, MTP); and seven thiophene derivatives, including thiophene, 2-methylthiolan-3-one, 2-methylthiophene, benzothiophene, benzothiazole, and 2-methyl-3-(methyldisulfanyl)furan (methyl-(2-methyl-3-furyl) disulfide, MMFDS).
Masuda and Komura also reviewed the formation of flavors including sulfur compounds during fermentation [8]. According to them, MTP, 3-methylsulfanylpropyl acetate (3-(methylthio) propyl acetate, MTPA), and 2-methylthiolan-3-one were formed from methionine during fermentation in malt whisky production. Schreier et al. examined metabolites formed from methionine as the sole nitrogen source, reporting the formation of MTP at 50–60% and trace amounts of MTPA, 3-methylsulfanyl propanal, and 2-methylthiolan-3-one [9]. More recently, Etschmann et al. studied the formation of MTP and MTPA from methionine by S. cerevisiae [10]. While the wild-type yeast produced large amounts of MTP and trace MTPA in a synthetic medium containing methionine as the sole nitrogen source, considerable amounts of both MTP and MTPA were produced by a genetically modified yeast. Thus, MTP and MTPA seem to be formed from methionine by yeast during fermentation; these compounds have onion-/potato-like, and sulfurous characteristics, respectively. Deed et al. also studied the formation of MTA from methionine by S. cerevisiae [11]. They examined three Ehrlich pathway genes, which suggested that alternative pathways may be involved in the formation.
Hydrogen sulfide is known to be unpleasant, with a rotten egg aroma, and can act as a sulfur donor due to its high reactivity. Stewart and Ryder discussed the sulfur cascade during fermentation, especially from sulfate to sulfur-containing amino acids [2]. It is well established that S. cerevisiae can assimilate sulfate into cells, incorporating sulfur dioxide and hydrogen sulfide into the amino acids cysteine and serine via the MET17 gene in the methionine biosynthesis pathway. A portion of sulfur dioxide and hydrogen sulfide may also leak out of yeast cells. In biochemical and molecular biological studies using the stable isotope 34S and a mutant yeast, Kinzurik et al. showed that hydrogen sulfide could be converted into ethanethiol, S-ethyl thioacetate, and diethyl disulfide [12]. Because the yeast strain with MET17 gene deletion ultimately accumulated hydrogen sulfide, it might be possible to identify metabolites of hydrogen sulfide. Moreover, the same research group proposed the conversion of hydrogen sulfide [13], ethanethiol, and methanethiol both through biological transformation by S. cerevisiae and through chemical conversion. Thus, hydrogen sulfide might be converted to ethanethiol by yeast, and ethanethiol might be converted to ethylsulfanylethane via S-ethyl thioacetate. In addition, hydrogen sulfide and ethanethiol might be turned into methanethiol chemically. From the methanethiol, three alky sulfides (DMS, DMDS, and DMTS) might be produced by chemical reaction, while S-methyl thioacetate might be formed by yeast. It seems, therefore, that hydrogen sulfide, ethanethiol, and methanethiol may act as sulfur donors, forming several sulfur volatiles during the fermentation process, although the kinetics and equilibria of the reaction remain to be elucidated. Ethanethiol and ethylsulfanylethane have garlic-like aromas, while methanethiol has a rotten cabbage character. The threshold of ethanethiol has been reported as 0.03 μg/L in 20% ethanol [4]. As a side note, hydrogen sulfide has also been reported to act as a sulfur donor in the formation of an onion-like off-flavor in beer, namely 2-methyl-3-sulfanylbutan-1-ol [14][15]. Hence, because the formation of hydrogen sulfide is associated with fermentation speed and sulfur-containing amino acids, fermentation conditions might be a key target for controlling unpleasant sulfur volatiles. Investigations into yeast strain, fermentation temperature, and yeast inoculation, among other factors, might lead to better control of these compounds in the future.
Hydrogen sulfide and other sulfur volatiles that are formed from hydrogen sulfide might be associated with the sulfurous character reported in the following two studies. First, Daute et al. examined the influence of pretreatment of wort [16], including boiling, autoclaving, and filtration, on sensory and chemical analysis, demonstrating that larger amounts of volatiles, such as esters, fusel alcohols and sulfides, were formed as compared with control wort, although no significant differences were detected between pretreated and control wort. However, pretreated wort had more feinty characteristics, such as meaty, cereal, and sulfury notes. It was suggested that these changes might be due to differences in the components of wort, because there was a decrease in fermentation speed and ethanol yield. Second, Waymark and Hill investigated the influence of 24 yeast strains [17], including distilling yeasts, wine yeasts, and brewer’s yeasts, in malt whisky fermentation, carrying out sensory analysis on new-make spirits prepared on the laboratory scale. They demonstrated a difference in sensory intensity among the new-make spirits; in particular, brewer’s yeasts produced whisky with higher scores of cereal, sulfury, and feinty notes.
Sulfur volatiles produced by brewer’s yeast have also been reported. Yomo et al. studied the effect of brewer’s yeast [18], which was originally used in malt whisky making many years ago. Indeed, some distilleries use a mixture of distilling and brewer’s yeast even today, although most distilleries use only distilling yeast. The researchers demonstrated that new-make spirits produced with the mixed yeasts had a full-body character and increased amounts of sulfur compounds, including DMTS, 2-(methyldisulfanyl) ethanol, and 2-(methyldisulfanyl) ethyl acetate. The dithiapentyl derivative was reported to have a mushroom character. The researchers also compared fresh brewer’s yeast with starved yeast, showing that the latter produces spirits with a more complex and full-body character in sensory analysis due to the formation of larger amounts of these sulfur compounds. They suggested that physiological changes in the starved yeast lead to desirable product qualities after maturation. Overall, their findings are identical to those of Waymark and Hill [17], who showed that brewer’s yeast tends to produce a more intense feinty note, although there is a difference between fresh and spent yeast.

3. Formation and Removal of Sulfur Volatiles during Distillation

The behaviors of sulfur volatile compounds during distillation have been previously explored in relation to the copper still. Normally, malt whisky is distilled twice: a first distillation from wash to low wine, and a second distillation from low wine to spirits. During distillation, water is supplied to the condenser, which condenses the vapor back to liquid. There are two types of condenser: a traditional worm tub, composed of a coil in a tub; and a shell-and-tube, composed of numerous small tubes in big shells [19]. It is generally believed that the type of condenser affects the product quality, especially sulfur characters, with the worm tub producing whisky with greater sulfury and heavier notes due to the surface area and duration of contact with copper, as Bathgate has pointed out [6].
The level of copper in the distillate is an indicator of the consistency of spirits, and higher levels are identified in those produced by a shell-and-tube condenser [20], which is why this type of condenser provides a lighter quality. For differentiation and authentication, Hopfer et al. determined 53 elements in multiple whiskies from different countries [21], reporting higher levels of copper in Scotch and Japanese whisky than in Irish, bourbon, and Tennessee whiskeys, although they did not discuss the reasons. Webster et al. evaluated the influence of the two types of condenser on the amounts of alkyl sulfides, MMFDS, and copper in spirits from two commercial distilleries [22]. They observed that the amount of MMFDS in whisky from the distillery with the worm tub condenser was lower than that from the distillery with the shell-and-tube condenser. However, the operation and shape of the pot still also differed between the two distilleries, and the researchers concluded that other factors, in addition to condenser type, might affect the behavior of sulfur volatiles during distillation.
Distillation is reported to cause both the formation of sulfur volatiles and a decrease in sulfur levels, leading Miller to suggest that it might be necessary to integrate the two theories and propose new mechanisms [7] (p. 172). Based on their experience, distillers believe that copper removes sulfur compounds, as mentioned above; thus, a decrease in levels might be considered to occur during whisky production. On the contrary, however, hydrogen sulfide and alkyl thiols—the proposed precursors of DMDS and DMTS—may react easily with other compounds in the presence of copper, as described in the fermentation section. Therefore, it seems reasonable that both reactions might occur during distillation, although the mass balance of the two will vary. The threshold of DMDS and DMTS has been reported as 30 and 0.05 μg/L in 20% ethanol solution, respectively [4].
Regarding the formation of sulfur compounds, Masuda and Nishimura compared amounts of DMDS in whisky prepared using copper and glass stills of the same shape [23], demonstrating that the amount in the distillate from the glass still was about one-tenth that of the copper still. Nedjma and Hoffmann studied the formation of alkyl sulfides from hydrogen sulfide, methanethiol, and ethanethiol in a buffer solution in the presence of Cu2+ [24]. They proposed that DMDS was formed from methanethiol, while DMTS was formed from methanethiol and hydrogen sulfide. Furusawa proposed that DMDS and DMTS were formed from MTP and from MTP with hydrogen sulfide during distillation owing to copper salts [25]. All in all, while the last two studies were performed in model solutions, it will be clearly difficult to prove these reactions in distillation.
In terms of a decrease in the amounts of sulfur compounds, Jack et al. evaluated the effects of copper and non-copper stills on sulfury sensory scores, such as cereal, feinty, sulfury, and meaty notes [26]. They carried out three distillation tests using all copper, non-copper in the first and copper in the second distillation, and the reverse combination (copper and non-copper). They observed that all-copper and copper/non-copper distillation led to lower sensory scores, while non-copper/copper had significantly higher scores, suggesting that the contact wash with copper led to a decrease in sulfur compounds. Subsequently, Harrison et al. examined the importance of copper in more detail [27]. They designed a still that could be interchanged with copper or stainless steel in three parts: the pot, lyne arm, and condenser. Distillation experiments were carried out on a laboratory scale, the amount of DMTS in the distillates was determined by using a headspace GC–sulfur chemiluminescence detector (SCD), and the score in terms of meaty and sulfury notes in sensory analysis was evaluated. In short, the authors attempted to identify crucial contact points—namely, liquid in the pot, vapor in the lyne arm, or liquid in the condenser. They demonstrated that both the amount of DMTS and the sensory score were lower in all-copper distillation. In terms of the three still parts, the pot had the most effect, although not all 64 combinations were examined. This indicated that contact with copper for both liquid in the pot and vapor in the lyne arm is the most important for reducing levels of sulfur compounds, while copper salt was involved in their formation. In addition, two unknown peaks were observed in GC-SCD analysis, especially in distillate from the stainless steel still, suggesting that these compounds might contribute to meaty and sulfury characteristics.

4. Decrease in Alkyl Sulfides during Maturation

Distilled spirits are matured in oak casks. In regard to sulfur volatiles, there have been a few novel findings in recent years. It is known that the rate of decrease in sulfur compounds during maturation depends upon the sulfur compounds. Masuda and Nishimura measured sulfur volatiles, including three alkyl sulfides [23], three thiophenes, four MTP derivatives, and benzothiazole, in malt whisky that was unaged and aged for 1–10 years. There was observed three types of behavior: DMTS, 5-methylthiophene-2-carbaldehyde, benzothiophene, and benzothiazole did not change; DMDS gradually decreased; and other compounds decreased markedly within a few years. In another study, Leppänen et al. reported that DMTS decreased slowly, consistent with currently held views [28][29]. Thus, DMS decreases markedly, DMDS decreases gradually, and DMTS slowly decreases due to the charcoal layer inside the oak. As pointed out by Masuda and Komura [8], these alkyl sulfides contribute to typical immature characteristics and their presence indicates the lack of a substantial maturation period.

This entry is adapted from the peer-reviewed paper 10.3390/molecules27051672

References

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