One of the greatest yet least understood variable influencing hydrogen sulfide formation by yeast during wine production is differences in genetic background. The overall goal of this research program is to define and categorize the pattern of strain differences in mRNA and protein profiles correlated with low and high H2 S production. Hydrogen sulfide formation was evaluated in 15 different yeast strains, 6 commercial yeasts and 9 natural winery isolates, in three different media at several different nitrogen concentrations. Two media were synthetic grape juice formulations and the third was a Pinot noir juice. All 15 strains increased hydrogen sulfide production upon H2S limitation, but the magnitude of the effect varied greatly (from 100 fold to a less than 50%increase with a 10 fold decrease in nitrogen concentration). Other juice parameters thought to impact hydrogen sulfide formation were also evaluated at levels at which they are normally found in wines: metal ions, sulfur dioxide, glutathione. None of these compounds had a significant effect on H2S formation in any of the 15 strains at these concentrations. The effect of the amino acids threonine, methionine and cysteine were also evaluated. Cysteine at concentrations found in juice had no effect on H2S production in any of the strains evaluated. Threonine, at the high end of the concentration range at which it is found in juice generally increased hydrogen sulfide production in all strains tested, although the magnitude of the effect varied. The effect of methionine was quite variable. Methionine inhibited hydrogen sulfide formation in several strains, but had no effect in the others. In general, strains showed similar responses but differed dramatically in the magnitude of the response to juice conditions. In order to further clarify the genetic basis of this difference in response, mRNA and protein profiling is being conducted. Strains display dramatic changes in both mRNA and protein patterns upon nitrogen limitation, however, there are great differences in protein profiles between different strains grown under nutrient rich conditions. The genome-wide analytical technologies will allow a detailed description of strain differences.

To date we have confirmed the high degree of variation in H2S formation among commercial yeast strains and natural isolates. In general, all display an increase in H2S formation upon nitrogen and pantothenate limitation, but the magnitude of the effect varies widely among the strains. Some are far more sensitive to limiting nutrients than are others. The degree of response is not well-correlated with the intrinsic sulfite reduction activity as determined from color on BiGGY agar suggesting that factors other than the activity of this pathway are critical in hydrogen sulfide production. Glutathione addition to the medium did not appear to impact H2S formation. Internal glutathione levels were manipulated using two metabolic inhibitors, which seemed to have an effect on lag phase but not total amount of H2S formed once cells had adapted to the inhibitor. This adaptation would be prevented in the presence of both inhibitors, which is currently being investigated. This work has provided an important foundation for comparison of the protein and gene expression profiles of strains grown under conditions of low and high hydrogen sulfide formation.

Sixteen samples of grapes from commercial vineyards were fermented during the 1996 harvest studies. These included 1 each of Chardonnay, Cabernet Franc, Sangiovese and Syrah, 4 Merlot and 8 Cabernet Sauvignon samples. Musts were supplemented with diammonium phosphate and a vitamin mixture to avoid deficiencies in these being a cause of sulfide formation. The levels of hydrogen sulfide produced during fermentation were measured by GC using direct headspace sampling and flame photometric detection. The values obtained ranged from 0 to 155.8 ug (mean of 23.6, sd of 25.9). Yeast strain comparisons on 3 musts gave the following sulfide formation results (in ug): Fermevin (mean of 26.1, sd of 27.2), Lavin D254 (mean of 30.2, sd of 37.3), PDM (32.8,sd of 42.3) and Montrachet (mean of 35.6, sd of 44.1) and are not significantly different from each other when tested on multiple juices. The juices were analyzed for individual amino acids, glutathione and sulfate concentrations and these were correlated with the formation of hydrogen sulfide using principal component analysis (PC A). The levels of sulfate, glutathione and free amino nitrogen (FAN) were all positively correlated with higher sulfide production. Studies with one of the juices show the effects of addition of glutathione and separately, sulfate on the increased levels of hydrogen sulfide production.

We have used an HPLC method to assay reduced and oxidized glutathione and cysteine in musts and wines. This method is based on carboxymethylation with iodoacetic acid followed by derivatization of the amino groups with 2,4-dinitro-l-fluorobenzene. During the 1995 season we implemented the procedure and have accomplished the following. We can derivatize and separate reduced glutathione and cysteine in grape juices. Using this method we have determined the level of glutathione and cysteine in Chardonnay berries. In berries harvested at 22.5 Brix we determined that the amount of glutathione present before crushing was 214 nmoles/g fresh weight and the amount of cysteine was 12.16 nmoles/g fresh weight. This gives a ratio of glutathione to cysteine of 17.6 showing that glutathione is more than 17 times as abundant as cysteine in Chardonnay berries prior to crushing. In wines only oxidized glutathione was detected. Reduced glutathione and cysteine, and oxidized cysteine were not found in wines. Wines to which oxidized glutathione had been added were less brown than reference wine. Aroma difference testing of wines to which oxidized glutathione was added to give 30 times the normal were not different from reference wines at the 95%level. We initiated experiments to determine the sensory effects of adding glutathione to musts prior to fermentation. Preliminary results indicate that musts to which glutathione has been added ferment more quickly than the control fermentations without glutathione.

The GC used for analysis of sulfur volatiles has been recalibrated to permit evaluation of the split effluent of the GC by sensory (sniff) evaluation and by flame photometry. In all surveys of headspace of spoiled wines, no new volatiles were detected other than those previously reported (Park et al, 1994). The natural wine yeast strain, Saccharomyces cerevisiae UCD934, was investigated to determine the number of genes responsible for H2S production during yeast fermentation. The strain is heterozygous for the MET5 gene which encodes for a component of sulfite reduaase. The parental strains were sporulated, tetrads dissected, and crossbreeding was conducted. Compared to the parents, the progeny strains showed dramatic variation in H2S production capacity. By the tetrad analysis of these yeast and further crosses made since the submission of the progress report in March, 1995, it was confirmed that five genes including MET 5 are involved in H2S production.