We used glutathione reaction product (GRP) and glutathione (GSH) labeled with radioactive 35S to study changes in these compounds during crushing and aging. During crushing GSH is converted to GRP as expected, but a large portion is oxidized to GSH disulfide (GSSG). After only 5 days incubation in a white wine, glutathione is largely oxidized to GSSG and also converted to a much less polar metabolite having an Rf of 0.62 on paper chromatography in 1-butanol/acetic acid/water (4:1:1 (V/V). During a 55 day aging study GRP appeared unchanged but GSH was converted to at least 4 other compounds, one of which was identified as GSSG. Since GRP might give rise to thiophenols during fermentation or aging we studied the sensory properties of two commercially available thiophenols. The sensory threshold for 4-hydroxythiophenol in a white wine was found to be lppm. The threshold for 4-mercapto toluene was estimated to be 0.06ppm. These results underline the importance of identifying the metabolic products of glutathione disulfide (GSSG) and glutathione reaction product (GRP) during fermentation and wine aging.
In previous years, we examined the effect of carbon monoxide (CO) as a substitute for sulfur dioxide in the treatment of must and wines for prevention of the proliferation of spoilage organisms. Initial results showed that CO was effective in controlling juice organisms such as Hansenula at 90 ppm. Wine spoilage organisms, with the exception of Brettanomyces were reticent to control at this level. This years’ studies, using the same methodology as in previous years, focused on the control of organisms whose presence is becoming more apparent as wineries limit the amount of sulfur dioxide used. Furthermore, we examined the effect of CO on organisms such as Zygosaccharomyces, which are refractive to control by sulfur dioxide and sorbate. These organisms are of high economic significance inasmuch as they are becoming more apparent as spoilage organisms in concentrates and wines sweetened with them. This years’ work focused on controlling the above-mentioned organisms amd on corroborating results obtained previously. Results obtained therewith, show that indeed Brettanomyces and Hansenula can be fully controlled at 90 ppm in juice, Dekkera at 120 ppm and Kloeckera at 240 ppm. Zygosaccharomyces was delayed for 96 hours in juice at 240 ppm and controlled at 360 ppm. Zygosaccharomyces was fully controlled in wine at 24 0 ppm. Saccharomyces cerevisiae (Montrachet) was unaffected at levels as high as 72 0 ppm CO in juice.
The formation of volatile sulfur compounds during fermentation was studied using juice from grapes with a history of production of sulfide odors. Juice was supplemented with different levels of glutathione (GSH) and glutamic acid to investigate the role of these compounds in production of sulfur vblatiles. Juices supplemented with high levels of GSH produced the highest levels of H2S during fermentation. For Cabernet, the control produced no ethanethiol (EtSH), whereas other supplemented juices produced about 20 ppb EtSH. In contrast for Merlot musts, all produced ethanethiol. The low level GSH must fermented faster than the control (17 days vs. 22 days), whereas supplementation with a nitrogen equivalent amount of glutamic acid had no effect on fermentation rate. This Finding may be important since the control juice had not only a sulfide odor problem but also slow fermentation. The 300 pprn GSH supplemented juice produced eight different type of thiol compounds with high concentrations whereas lower addition of GSH and GLU produced only three thiol compounds at much lower concentrations. Model media with different nitrogen levels were investigated for their pattern of H2S production and the preliminary result showed the production of H2S was directly related to the levels of nitrogen content with the highest levels when yeast actively growing. In no case was H2S present at the end of fermentation.Further experimental fermentation will be conducted based on this preliminary result. To compare the effect of the size of fermentation on the formation of sulfur volatiles, wines from lab scale fermentations were compared with commercial wine produced in large scale fermentation of the same grapes. The commercial wine had a strong sulfide odor and an extremely high level (about 1 ppm) of H2S and lesser amounts of methyl and ethyl mercaptan, whereas wines from our small fermentor did not have any off-odor and lower concentrations of sulfur volatiles, suggesting the importance of redox potential and dissolved oxygen differences between the two systems.
Research Accomplishments: As previously suggested, nitrogen-deficient grape juices produced higher concentration of hydrogen sulfide. However, despite the examination of musts reported to have produced serious sulfur spoilage problems and which were shown to be nitrogen deficient, no wines were produced which contained serious volatile sulfur spoilage problems as shown in these two studies by the absence of mercaptans. The role of nitrogen deficiency in musts or of different yeast strains in the production of sulfur compounds, despite very rigorous analytical analyses of precisely controlled fermentations is still unclear. As demonstrated by the results of the last two years, no simple relationship between must composition and production of sulfur off-flavors exists. Complex factors which require painstaking and systematic study appear to regulate the production of these compounds.