CAUSES OF HYDROGEN SULFIDE FORMATION IN WINEMAKING

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1 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 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 H 2 S 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 H 2 S 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 H 2 S formation in winemaking. More specifically the role of the micro- and macro-nutritional make-up will be discussed. What are the chemical mechanisms for H 2 S 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 H 2 S (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 H 2 S 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 H 2 S formation. An additional proposed mechanism for the chemical formation of H 2 S involves metals or their cations. Accordingly, wine acids attacking metal fixtures are proposed to produce nascent hydrogen that directly reduces bisulfite to H 2 S (Rankine, 1963). Alternatively, Cu 2+, Zn 2+ or Sn 2+ cations in particular have been suggested to split disulfide bridges in proteins to yield products that include H 2 S. Clearly, with the widespread use of stainless steel or nonmetallic fermentation vessels and fittings, either mechanism is of reduced significance. Even so, these chemical routes to H 2 S liberation will only remain of minor importance if the conditions upon which they are dependent are avoided. What are the biological origins of H 2 S? H 2 S 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 H 2 S liberation to the insignificant. Therefore the more important biological origin of this off-flavour compound undoubtedly involves yeast. In this case H 2 S 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.

2 JIRANEK CAUSES OF HYDROGEN SULFIDE FORMATION IN WNEMAKING, PAGE 2 What are the precursors of the H 2 S formed? In order to understand the causes of H 2 S 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 H 2 S 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 H 2 S (Aida et al., 1969, Tokuyama et al., 1973). Methionine is a similarly effective inducer of H 2 S 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 H 2 S 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 H 2 S 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 H 2 S 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 H 2 S 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, H 2 S 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 H 2 S. 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 H 2 S 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 H 2 S liberation could be modest. On the other hand, the intracellular organic sulfur reserved compound, glutathione (γ-l-glutamyl-l-cysteinylglycine), does contribute to H 2 S liberation by wine yeast. Our work has shown that when yeast are precultured with an inhibitor of glutathione accumulation, the amount of H 2 S 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

3 JIRANEK CAUSES OF HYDROGEN SULFIDE FORMATION IN WNEMAKING, PAGE 3 liberation of H 2 S. But the majority of H 2 S 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 H 2 S (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 in insufficient amounts in grape juices, as is often the case (Amerine et al., 1980, Henschke and Jiranek, 1993), yeast meets the requirement for sulfur by exploiting inorganic forms present in grape juice. Sulfate (SO 4 2- ) is the predominant naturally occurring form of sulfur in grape juice (up to 1200 mg/l; Leske et al., 1997) whereas sulfite (SO 3 2- ) can arise through additions during winemaking or as a by-product from the growth of several yeasts, including wine strains of Saccharomyces cerevisiae (Eschenbruch, 1974b). The assimilation of sulfate occurs via the Sulfate Reduction Sequence (SRS, Fig 1). In summary, sulfate enters the cell via an active transport process and is sequentially reduced to sulfide via sulfite, the terminal step being mediated by the sulfite reductase enzyme. Whether entering the cell by diffusion (Stratford and Rose, 1986) or, active transport (Macris and Markakis, 1974), sulfite is also able to channel into the SRS via sulfite reductase. The organic sulfur compound, homocysteine, is subsequently formed through the condensation of a nitrogenous precursor, O-acetylhomoserine with sulfide. The ability of O-acetylserine to also condense with sulfide to yield cysteine directly has been shown in vitro but is of uncertain importance in vivo. Only when sulfide is incorporated into organic sulfur compounds in this way is it prevented from being lost from the cell to the fermentation medium. Therefore, the nitrogenous precursor compounds, whether derived from assimilable nitrogen accumulated from the must (largely) or from cellular nitrogen reserves, must be available for this synthesis and the retention of H 2 S to occur. Several crucial steps in this incorporation and hence retention of H 2 S are therefore immediately apparent. These include i) those ultimately leading to formation of O-acetylhomoserine and ii) the condensation of O-acetylhomoserine with sulfide to yield homocysteine and downstream derivatives. Successful operation of these steps is dependent on there being sufficient supply of macro-nutrients such as assimilable nitrogen and/or micro nutrients such as the vitamins pantothenate and pyridoxine. What are the key biological mechanisms for H 2 S formation? Studies with defined media and near-real-time determination of H 2 S liberation have been instrumental in delineating the principal precursors and mechanisms for H 2 S liberation during winemaking. Thus H 2 S liberation from model cider and wine fermentations containing sulfate as sole sulfur source demonstrates that this inorganic compound can act as a precursor (Stratford and Rose, 1985, Jiranek et al., 1995b). Moreover, the use of radiolabelled precursors allowed the origin of the sulfur atom in liberated H 2 S to be traced back specifically to the sulfate in the medium (Stratford and Rose, 1985, Jiranek et al., 1995b). The inclusion of sulfite in this experiment lead to the liberation of largely non-radioactive H 2 S, thereby demonstrating that not only can sulfite act as a precursor, it is actually preferred when both sulfur sources are present. As explained above, these inorganic sulfur compounds are reduced to H 2 S by way of the SRS, as part of sulfur assimilation by this organism. The fact that these inorganic sulfur sources are

4 JIRANEK CAUSES OF HYDROGEN SULFIDE FORMATION IN WNEMAKING, PAGE 4 playing such a dominant role in H 2 S liberation highlights the central role of the SRS and specifically the sulfite reductase enzyme in this process. The actual cause of H 2 S liberation via this process is shown to be a depletion of assimilable nitrogen. Specifically, H 2 S liberation occurs within 15 min of nitrogen depletion, can be quickly suppressed by nitrogen supplementation and resumes once added nitrogen is depleted (Stratford and Rose, 1985, Jiranek et al., 1995b). This mechanism is in keeping with the frequently insufficient amounts of assimilable nitrogen that are found in grape juices (Amerine et al., 1980, Henschke and Jiranek, 1993) as well as the typical responsiveness of H 2 S liberation during wine ferments to nitrogen supplements. These key findings suggest that H 2 S liberation as caused by the depletion of assimilable nitrogen occurs because the H 2 S that is formed under these conditions is in excess of cellular needs because the nitrogenous precursors of the SAA with which sulfide ordinarily combines are also depleted. Therefore, sulfide that has been formed from sulfate and/or sulfite by sulfite reductase is not incorporated into organic sulfur compounds and is lost from the cell into the fermentation where it becomes apparent to the winemaker. To know a little bit more... The model of H 2 S formation by yeast presented in this paper predicts that rates of H 2 S liberation should be related to levels of sulfite reductase activity of the culture. This is confirmed experimentally. Rates of H 2 S liberation occurring from sulfite in response to nitrogen depletion at different times during growth correspond to the amount of sulfite reductase activity detectable in crude cell extracts prepared from the culture at the same time points (Jiranek et al., 1995b, 1996). Accordingly, highest rates of H 2 S liberation occurred during the active growth phase when the growth-related demand for SAA and hence sulfite reductase activity would be expected and in fact was shown to be highest. The extent of synthesis of the sulfite reductase enzyme results, in part, from the degree of expression of the genes encoding this enzyme. This expression is in turn controlled by the cellular availability of various organic sulfur compounds (Cherest et al., 1969, 1971, 1973). Thus when these compounds are present, the genes encoding sulfite reductase are turned off since a reduced synthesis of organic sulfur is clearly required. During nitrogen deficiency, such regulatory organic sulfur compounds will be depleted. This has given rise to suggestions that the basis for an increased liberation of H 2 S following nitrogen depletion is actually a response to the reduced cellular content of organic sulfur compounds and hence an increased gene expression and synthesis of sulfite reductase. Whether such a synthesis could occur is put in doubt by the fact that the onset of H 2 S liberation occurs within 15 min of nitrogen being depleted from the medium (Jiranek et al., 1995b). Even if time permitted such a synthesis, it would appear unsustainable given that the cell would be starved of the very amino acids that would be required by this process. Further experimental data argue against this mechanism. For example, the addition of cycloheximide induces H 2 S liberation at rates similar to those seen during nitrogen starvation, even though this antibiotic specifically inhibits protein synthesis (Stratford and Rose, 1985). Also, we have shown that the amounts of sulfite reductase activity seen across extremes of nitrogen availability are relatively constant, unlike the wide changes seen in rates of H 2 S liberation (Jiranek et al., 1996). Therefore, H 2 S liberation induced by nitrogen starvation, at least that attributable to inorganic sulfur reduction, is the result of an existing sulfite reductase activity and sulfite reductase activities are not markedly increased following nitrogen depletion.

5 JIRANEK CAUSES OF HYDROGEN SULFIDE FORMATION IN WNEMAKING, PAGE 5 The close association between rates of H 2 S liberation by cultures and crude extracts is, nonetheless, somewhat at odds with reports of H 2 S acting as an inhibitor of this enzyme (Yoshimoto and Sato, 1968a, Dott and Trüper, 1976). The implication is that any such inhibition is ineffective either in the wine strains studied or under wine conditions. Perhaps this is so because sulfide never accumulates to inhibitory concentrations intracellularly. Any sulfide will predominantly take the form of H 2 S, especially at wine ph (ca 3.5), and being highly volatile and reactive will be quickly lost from the system. Therefore this inhibitory mechanism is of little significance to existing sulfite reductase enzyme under these conditions. This in turn appears to leave action of sulfite reductase largely without control, with the formation of H 2 S being dependent on the supply of a substrate (sulfite), reducing power (NADPH) and the longevity of the enzyme. The liberation of H 2 S can be shown to decay with similar kinetics to those determined by the half-life of sulfite reductase in the absence of enzyme replacement by protein synthesis (Jiranek et al., 1996). Consequently, such H 2 S liberation can continue for many hours or even days in some strains. Apparently, the availability of reducing power is not severely limited in these nitrogen-starved cultures. The key factor that allows a rapid cessation of H 2 S liberation after nitrogen depletion is the nature of the sulfur substrate for sulfite reductase. Thus when sulfate is the sole source of sulfur, H 2 S liberation does not achieve the same maximal rate as that from sulfite and it is quickly inhibited (Hallinan et al., 1999). Importantly, cultures that have ceased H 2 S liberation from sulfate do not, however, loose the ability to reduce sulfite to H 2 S. Supplementation of such sulfate-grown cultures with sulfite rapidly restores high rates and extended H 2 S liberation (Jiranek et al., 1995b). Clearly the basis for the differing ability of the cell to limit H 2 S liberation from sulfite vs sulfate is independent of sulfite reductase and is effected through a control point further upstream in the SRS. How is sulfate reduction controlled in yeast? From the point of view of minimising the energetic burden of the unnecessary reduction of sulfate during nitrogen starvation, a logically control point is that of sulfate transport. Such control could be mediated in a manner analogous to that by which sugar transporters are quickly lost upon nitrogen starvation (Busturia and Lagunas, 1986; Salmon, 1989). Sulfate transport has been shown to be lost in laboratory strains with a half-life of 10 mins in a nitrogenand glucose-free medium (Horák et al., 1981). However, starvation for nitrogen alone did not have the same impact on sulfate transport in wine yeasts (Hallinan et al., 1999). Rates of sulfate transport remained stable for at least 7 h, albeit after an artefact of the experimental system brought about an initial protein-synthesis dependent increase in sulfate transport capacity. As such it would appear that the point for controlling the futile reduction of sulfate during nitrogen starvation is downstream of sulfate transport. Given that a period of sulfate starvation can temporarily remove the block on continued sulfate reduction, a build-up of inhibitory intermediates of the SRS is implicated as the process responsible (Hallinan et al., 1999). Further work is required to determine the nature of the inhibitor as well as which of the remaining SRS enzymes, ATP sulfurylase, APS kinase or PAPS reductase (Fig. 1), are the targets. Which elements of must composition are important? The discussion so far has highlighted several key points with regard to the formation of H 2 S by yeast. A deficiency of the micro-nutrients, pantothenate and pyridoxine, will lead to H 2 S liberation by different mechanisms. Pantothenate shortages will deprive the yeast of the precursors of sulfur amino acid synthesis thereby leading to a relative excess of H 2 S. On the other hand, pyridoxine deprivation will prevent the condensation of such precursors with H 2 S,

6 JIRANEK CAUSES OF HYDROGEN SULFIDE FORMATION IN WNEMAKING, PAGE 6 thereby leading to a shortage of homocysteine and the compounds that regulate the SRS. A derepression of the SRS and over-production of H 2 S is expected to follow (see Fig. 1). Given their role in the assimilation of inorganic nitrogen, other vitamins are vital, such that their absence will effectively lead to nitrogen starvation when organic nitrogen sources are unavailable (C. Edwards, pers. comm.). Such shortages will prevent the sequestration of H 2 S into organic sulfur. Other causes of an apparent nitrogen starvation might include the accumulation of ethanol later in fermentation or else the use of an inoculum cultivated with insufficient exposure to oxygen (Calderbank et al., 1984; van Uden 1989). In both cases the function of transporters, including those for assimilable nitrogen, may be compromised thereby giving rise to an intracellular deficiency of nitrogen. What are the real implications of assimilable nitrogen depletion in musts? Where the various scenarios described above, are not an issue, the unavailability of assimilable nitrogen per se will be the key factor allowing for H 2 S liberation. Whether such liberation occurs depends on: i) the nature of the inorganic sulfur source present (sulfate vs sulfite, though both are typically present) and ii) the amount of sulfite reductase activity (determined genetically and being dependent on the point within the growth curve when nitrogen has become depleted). Recent reports suggest that qualitative aspects of nitrogen availability are important (Spiropoulos et al., 2000). Specifically the ratio of methionine to assimilable nitrogen determines whether the enzymes of the SRS are repressed. However, Bisson s group misquoted the amounts, and hence importance, of methionine in the media used in earlier work (Jiranek et al., 1995b) and also extrapolated from gene expression patterns for laboratory yeast and conditions. While we too have yet to quantify SRS gene expression in wine yeasts, our investigations reveal that even with the inclusion of high concentrations (8.3 mm, 1200 mg/l) of the SRS repressor, methionine, sulfite reductase activity was only reduced by ca 70% of the unsupplemented culture (Jiranek et al., 1996). It therefore seems unlikely that methionine will ever occur in grape juice in concentrations sufficient to effect significant repression of the SRS and sulfite reductase. How to control H 2 S formation in winemaking? Accordingly, the relationship between assimilable nitrogen and H 2 S appears to be one limited to the ability of nitrogen to provide precursors of sulfur amino acid synthesis, thereby sequestering H 2 S away into organic sulfur rather than allowing it to be lost from the cell. As such the most important strategy for dealing with the majority of H 2 S problems will be the maintenance of intracellular nitrogen supplies. This will be achieved through the accurate determination of juice nitrogen content combined with supplementation as required to meet yeast nitrogen requirements of an effectively propagated strain of selected genetic makeup. Selection criteria for appropriate strains would incorporate their relative nitrogen requirement (Jiranek et al., 1995a; Julien et al., 2000), propensity to produce H 2 S (Jiranek et al., 1995c) and compatibility with wine-like ethanol concentrations and extended anaerobic growth. This group is undertaking several projects based on these criteria, which aim to provide improved wine strains for industry, ultimately by non-recombinant means.

7 JIRANEK CAUSES OF HYDROGEN SULFIDE FORMATION IN WNEMAKING, PAGE 7 NH 4 + amino acids SO 4 2- amino acids Intracellular nitrogen homoserine NH adenylylsulfate 3 -phospho-5 -adenylylsulfate Acetyl CoA CoA O-acetylhomoserine INSIDE CELL sulfite sulfide ATP PPi ATP PPi NADPH PAP + NADP + 3 NADPH 3 NADP + ATP sulfurylase. APS kinase. SO 4 2- PAPS reductase. sulfite reductase. homocysteine synthase. OUTSIDE CELL SO 2 H 2 S adenosine homocysteine cystathionine S-adenosylhomocysteine methionine ATP PPi + Pi S-adenosylmethionine cysteine glutathione Figure 1. Sulfur amino acid biosynthesis (including the Sulfate Reduction Sequence [SRS]) in wine yeast. Substrates move into the yeast cell via various transporters (depicted as white or shaded circles) or else via diffusion as in the case of molecular sulfur dioxide (SO 2 ). Only selected enzymes and cofactors have been included. The vitamin pantothenate is involved in sulfur amino acid synthesis in being a component of Acetyl CoA, which is required for the formation of O-acetylhomoserine. Pyridoxine is a component of pyridoxal phosphate, a co-factor required by homocysteine synthase. Abbreviations: ATP, adenosine triphosphate; CoA, coenzyme A; NADPH, nicotinamide adenine dinucleotide phosphate (reduced); NADP +, nicotinamide adenine dinucleotide phosphate (oxidised); NH 4 +, ammonium; PAP, phosphoadenosylphosphate; PPi, inorganic pyrophosphate; Pi, inorganic orthophosphate.

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