II 1 iI n d Control of hydrogen sulfide formation during fermentation

INTRODUCTION Hydrogen sulfide and related sulfur compounds produced internally by yeast during fermentation are considered highly undesirable if they are excreted and exceed threshold concentrations in the finished wine, as they cause an unpleasant "reduced character". The threshold for hydrogen sulfide is on the order of parts per billion and that for some of the other reduced sulfur compounds derived from hydrogen sulfide ranges from parts per million to parts per billion. Factors which affect production of these compounds include, but are not limited to, the nutritional status of the grapes, yeast strain, and fermentation conditions (Rauhut, 1993). Formation of excessive reduced character in table wines produced from sound grapes using good winemaking practices is a perplexing problem because it can occur unpredictably, and spoil affected wines. Yeast produces this class of compounds internally as a byproduct of the normal and necessary synthesis of S-adenosyl-methionine and the sulfur-containing amino acids, methionine and cysteine (Thomas and Surdin-Kerjan, 1997). Excessive synthesis leads to excretion of hydrogen sulfide into the wine. Although excess hydrogen sulfide formation has generally been correlated with nitrogen deficiency (Jiranek et al., 1995a, 1995b), deficiencies in vitamin B6 and pantothenic acid, and residual sulfur on grapes from the application of fungicides have also been implicated. In one study, formation of hydrogen sulfide was found not to correlate with variation in activity of the formative enzyme, sulfite reductase, which reduces sulfite to hydrogen sulfide (Jiranek et al., 1996). This suggests that the amount of extracellular hydrogen sulfide formed is controlled by other rate-limiting steps: its utilization in sulfur amino acid biosynthesis, its active or passive transport from the cell, or consumption through other biochemical pathways. Formation still occurs unexpectedly, and often enough to be considered a persistent problem (Boulton et al. 1996; Park et al. 2000). One complicating factor in the study of hydrogen sulfide production is that formation during the early stages of fermentation is not always problematic, either because the yeast eventually takes up and reuses the excreted sulfide or because the volatile sulfide is removed by entrainment in the stream of carbon dioxide that evolves during the course of fermentation. At the latter stages of fermentation, when excretion of the hydrogen sulfide is believed to be most troublesome, the less vigorous yeast may no longer take it up, or evolution of carbon dioxide may be so slowed as to no longer be an efficient means of removal. As a consequence, the total amount of hydrogen sulfide formed in a fermentation is likely to be a poorer predictor of eventual reduced character than measurement at the end of fermentation. Because hydrogen sulfide is produced within the yeast cell directly from sulfite, its immediate biochemical precursor, we are testing the hypothesis that excretion of sulfite by yeast will minimize hydrogen sulfide production. Excretion of sulfite occurs due to the activity of the yeast protein, Ssu1 (Avram and Bakalinsky, 1997; Avram et al. 1999; Park et al. 1999; Park and Bakalinsky, 2000; Goto-Yamamoto et al. 1998), which has also been implicated in selenium efflux (Pinson et al. 2000). If the hypothesis is found to be true, it will provide a simple means of identifying wine yeast strains that tend to produce low levels of hydrogen sulfide during fermentation.