Causes of Hydrogen Sulfide Formation in Winemaking

JIRANEK – CAUSES OF HYDROGEN SULFIDE FORMATION IN WNEMAKING, PAGE 1 CAUSES OF HYDROGEN SULFIDE FORMATION IN WINEMAKING Vladimir JIRANEK Department of Horticulture, Viticulture and Oenology, The University of Adelaide, PMB 1 Glen Osmond, SA 5064. The application of Saccharomyces cerevisiae yeast to the wine fermentation accomplishes more than the mere catabolism of sugar to ethanol and carbon dioxide. A myriad of flavour compounds are also formed. While the desirability of some of these compounds is a function of their concentration and wine style, others are generally regarded as contributors to off-flavour. One such off-flavour compound is hydrogen sulfide. The occurrence of H2S is widespread and frequent in the beverage fermentation industries. Accordingly, much has been and continues to be published on this topic, yet uncertainty regarding its origin remains. What is clear is that H2S can arise via numerous mechanisms and that the more important of these for contemporary winemaking are those that are biological in nature. This paper will focus on the causes of H2S formation in winemaking. More specifically the role of the micro- and macro-nutritional make-up will be discussed. What are the chemical mechanisms for H2S formation in wine? Different chemical mechanisms for this phenomenon have been identified. As an example of the former, elemental sulfur residues have long been recognised to be a potent precursor of H2S (reviewed by Rankine, 1963). This reductive process is inversely correlated with sulfur particle size and pH, increases with temperature, reductive conditions, ethanol concentration and the presence of metal ions (Acree et al., 1972, Schütz and Kunkee, 1977). While direct yeast-sulfur contact appears necessary, the process is nevertheless a chemical reduction. The most effective strategy for dealing with this cause of H2S formation has been adoption of vineyard and winery practices that avoid introduction of elemental sulfur residues into the wine. Thus adherence to withholding periods following the use of sulfur-containing fungicides or else their complete avoidance in the vineyard ensures no significant transfer of elemental sulfur to grape juice or wine (Thomas et al., 1993a, 1993b). Similarly, in the context of the somewhat historic use of sulfur candles for the sterilisation of cooperage and winery equipment, either judicious washing of equipment before its use or else alternate sterilisation/sanitization methods will minimise H2S formation. An additional proposed mechanism for the chemical formation of H2S involves metals or their cations. Accordingly, wine acids attacking metal fixtures are proposed to produce nascent hydrogen that directly reduces bisulfite to H2S (Rankine, 1963). Alternatively, Cu2+, Zn2+ or Sn2+ cations in particular have been suggested to split disulfide bridges in proteins to yield products that include H2S. Clearly, with the widespread use of stainless steel or non- metallic fermentation vessels and fittings, either mechanism is of reduced significance. Even so, these chemical routes to H2S liberation will only remain of minor importance if the conditions upon which they are dependent are avoided. What are the biological origins of H2S? H2S has been shown to be formed by both yeast and bacteria. However, suppression of bacteria such as Oenococcus, Lactobacillus and Acetobacter spp. by the appropriate use of sulfur dioxide to is likely to relegate such sources of H2S liberation to the insignificant. Therefore the more important biological origin of this off-flavour compound undoubtedly involves yeast. In this case H2S liberation is strongly influenced by the nutritional make-up of the medium. Links have been reported to the deficiency of nutrients such as assimilable nitrogen, vitamins or else rapid changes in growth conditions or kinetics. VINIDEA.NET – WINE INTERNET TECHNICAL JOURNAL, 2002, N°3 JIRANEK – CAUSES OF HYDROGEN SULFIDE FORMATION IN WNEMAKING, PAGE 2 What are the precursors of the H2S formed? In order to understand the causes of H2S liberation, early studies focussed on the identification of possible precursor compounds. Brewing researchers demonstrated very effectively that organic sulfur compounds such as cysteine or methionine were potent inducers of H2S when these amino acids were added to a yeast culture (reviewed by Lawrence and Cole, 1968). Such additions are also effective in oenological fermentations (Rankine, 1963, Eschenbruch et al., 1973, Jiranek et al., 1995b). The mechanism is suggested to involve an enzymic degradation of cysteine as part of the utilisation of this amino acid as a nitrogen source. Thereby, in addition to yielding nitrogenous derivatives of cysteine, the cysteine-degrading enzymes also bring about the release of the sulfur component of this amino acid as H2S (Aida et al., 1969, Tokuyama et al., 1973). Methionine is a similarly effective inducer of H2S liberation under these conditions, presumably due to its ready interchangeability with cysteine. However, despite the efficacy of these organic precursors in supplementation trials, the significance of this mechanism for H2S liberation during winemaking is limited by the fact that only trace amounts of small organic sulfur compounds occur in grape juices (Eschenbruch, 1974a, Gallander et al., 1969, Amerine et al., 1980, Jiranek and Henschke, 1993). Researchers subsequently sought to identify alternate sources of the sulfur amino acids (SAA) in order to account for the amounts of H2S that were occurring in affected fermentations. Proteolytic release of these amino acids from grape or yeast proteins was therefore suggested (Eschenbruch, 1974b, Eschenbruch et al., 1978, Vos and Gray, 1979). Importantly, as an explanation for the inverse correlation between juice assimilable nitrogen content and H2S liberation, such proteolysis was suggested to specifically arise as a nitrogen-scavenging mechanism in response to a nitrogen deficiency (Vos and Gray, 1979). The search for proteolytic activities has been extensive and continues because of the possible benefits of these enzymes in preventing protein hazes in wine. Nevertheless, no extracellular activities of consequence under oenological conditions have been identified (Rosi et al., 1987, Sturley and Young, 1988, Lagace and Bisson, 1990, Dizy and Bisson, 2000). Even if such activities were to exist, the SAA content of target proteins would typically be low relative to other amino acids. Thus the amounts of SAA released before other amino acids inhibit/repress transport of the SAA or else satisfy the nitrogen needs of the cell could well be low and so too the H2S that might be produced from their degradation. At any rate, the ability of a culture to synthesise and export proteolytic enzymes when experiencing nitrogen starvation should be seriously limited. Certainly, H2S can be liberated upon the addition of cycloheximide, an inhibitor of protein synthesis (Stratford and Rose, 1985), thereby arguing against a need for synthesis of new enzymes such as proteases to bring about the observed H2S. For these reasons, degradation of extracellular proteins and in turn the hydrolysis of any resulting methionine and cysteine is unlikely to be of importance to H2S liberation during winemaking. As for the role of turnover of intracellular proteins and peptides particularly during nitrogen limitation, the situation is probably different. Saccharomyces has an extensively studied assortment of enzymes for such processes (e.g. see Van Den Hazel et al., 1996), especially under conditions of nitrogen limitation (Large, 1986). Given that degradation of intracellular proteins might also be expected to liberate non-sulfur amino acids as well as methionine and cysteine, the impact of the latter on H2S liberation could be modest. On the other hand, the intracellular organic sulfur reserved compound, glutathione (γ-L-glutamyl-L-cysteinylglycine), does contribute to H2S liberation by wine yeast. Our work has shown that when yeast are pre- cultured with an inhibitor of glutathione accumulation, the amount of H2S liberated by such cultures under conducive conditions is initially reduced (Hallinan et al., 1999). This finding suggests that as part of the process for dealing with nitrogen deficiency, intracellular glutathione reserves are mobilised, and the cysteine component of this tri-peptide is degraded with the VINIDEA.NET – WINE INTERNET TECHNICAL JOURNAL, 2002, N°3 JIRANEK – CAUSES OF HYDROGEN SULFIDE FORMATION IN WNEMAKING, PAGE 3 liberation of H2S. But the majority of H2S liberated by such cultures was still derived from other sulfur compounds (Hallinan et al., 1999). While the full significance of glutathione degradation needs to be determined, a role for this compound in the so-called “end-of fermentation” H2S (Henschke and Jiranek, 1991), is a possibility worthy of investigation. Several possible candidate precursors arise through the course of sulfur assimilation and metabolism. How does yeast metabolize sulfur? Of the total dry weight of yeast cells, approximately 0.2 and 0.9% is represented by sulfur (Lawrence and Cole, 1968). Most is found in the organic sulfur compounds, primarily the amino acids, methionine and cysteine (60%), the reserve compound, glutathione (20%) as well as enzyme cofactors such as acetyl coenzyme A, the methyl group donor, S-adenosylmethionine and the vitamins, thiamine and biotin. Sulfur is therefore an essential requirement for growth, but one that is typically not limiting in grape juice. Thus when sulfur-containing amino acids occur

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