We completed development of an analytical procedure using headspace gas chromatography (HS-GC) coupled with sulfur chemiluminescence detection (SCD) which can rapidly and precisely quantify molecular and free sulfur dioxide in wine. The method requires minimal sample preparation and involves no chemical reagents (with the exception of a trace internal standard). At room temperature the method can successfully detect levels of molecular sulfur dioxide at concentrations as low as 0.03 mg/L. The total chromatographic time for the method is 8 minutes and, provided that information on the alcohol concentration and pH is readily available, the molecular and free sulfur dioxide concentrations for the sample can be rapidly calculated using simple formulae. The HS-GC method offers a high degree of precision, with a reported coefficient of variation of 3.72%. Comparisons with standard A/O and Ripper results on a large set of wine samples showed large discrepancies for those wines with high anthocyanin levels, suggesting that SO2 bound to anthocyanins is released during those procedures, inflating the amount of free SO2 that is actually available to protect the wine. The characteristics of rapid analysis, good sensitivity, and high precision, demonstrate that the method could be applicable in a production environment, albeit a large scale operation where a Gas Chromatograph could be utilized and maintained.
Development of volatile sulfur compounds (VSCs) post-fermentation can be a significant issue during both red and white winemaking. Unfortunately our understanding of contributing factors or conditions that impact VSCs is limited due in part to the complexity of their formation. This study focuses on the development of VSCs in Pinot noir during post-fermentation aging. During the first year of the study the impact of lees levels and composition on formation of VSCs was determined. Results showed that although lees levels and yeast strain impacted the amount of sulfur containing amino acids (pre-cursors for the formation of volatile sulfur compounds) in the wine, this did not result in an increase in the formation of volatile sulfur compounds. Wine samples were also provided by collaborating wineries in 2013 and assessed for VSCs so as to determine the cause of early reduction issues in barrel. Wineries were instructed to take juice and wine samples from lots that traditionally had issues with VSCs. Wine samples were taken after pressing and after one, three, and nine months barrel aging. Analysis of these samples indicated that the early formation of reductive smells soon after going to barrel were most likely due to H2S rather than the formation of more complex volatile sulfur compounds such as mercaptens and disulfides. Where this H2S is derived from and what factors impact its formation became the focus of future experiments. Firstly, experiments investigating the role of YAN concentration and content were undertaken. A synthetic grape juice was prepared where the amount and type of YAN (primary amino acids vs. ammonia from diammonium phosphate (DAP)) could be varied. H2S production was measured throughout fermentation and finished wines were assessed for a range of other VSCs. Variation in YAN concentration as well as whether YAN was derived from amino acids or DAP impacted H2S production during fermentation as well as formation of volatile sulfur compounds post-fermentation. In particular, DAP supplementation increased the amount of H2S formed late in fermentation and resulted in the highest amount of methyl thioacetate in the wines post-fermentation. Experiments investigating the role of elemental sulfur in the formation of H2S and other volatile sulfur compounds post-fermentation were also undertaken.
Pinot noir grape fermentations were undertaken where an addition of 0, 5 or 15 ug/g elemental sulfur was made to the grapes. Fermentations were conducted by a high H2S producing yeast strain (UCD522 ) or a no-H2S producing yeast strain (P1Y2). Addition of elemental sulfur to the grapes resulted in H2S formation during the alcoholic fermentation independent of which yeast strain was used. H2S production was higher in fermentations performed by UCD522 with increasing amounts of elemental sulfur resulting in increased production of H2S. In addition, higher elemental sulfur additions also resulted in higher H2S production late in fermentation. This is particularlyimportant as H2S formation late in fermentation is more likely to be retained in the wine due to the reduced production of CO2 by yeast. Higher elemental sulfur also resulted in wines containing higher concentrations of methyl thioacetate post-fermentation. Both of these findings suggest an important role for elemental sulfur in the formation of volatile sulfur compounds during and after fermentation. Overall, this study to date has demonstrated that lees levels impact the concentration of sulfur containing amino acids in the wine but may not directly impact formation of volatile sulfur compounds. Instead, the formation of H2S late in fermentation or early post-fermentation may be the main cause of post-fermentation reduction soon after wine goes to barrel. Current experiments are investigating the impact of YAN, yeast strain, and elemental sulfur on the formation of H2S and other volatile sulfur compounds post-fermentation. This work includes an ongoing effort to measure the amount of elemental sulfur present on grapes at harvest.
The effect of lees contact time during wine barrel aging on volatile sulfur compounds was investigated in this study. Pinot noir wines were made using grapes from the Oregon State University vineyard and fermented with two different commercial yeast strains. One set of wines was produced using the low/no H2S producing yeast strain Saccharomyces cerevisiae P1Y2 , and the other set was produced using Saccharomyces cerevisiae RC212. Fermentations were conducted in triplicate. At the completion of alcoholic fermentation, wines were pressed and split into three different lees treatments based on settling time (0, 24 and 72 hrs) which were named as heavy, medium and light lees treatment respectively in this progress report. Samples were collected at 0, 2 weeks, and 1, 2, 3, 6, 9 months. Volatile sulfur compounds were analyzed by solid phase microextraction-gas chromatography-pulsed flame photometric detection (SPME-GC-PFPD) method. The results showed that hydrogen sulfide (H2S), methylmercaptan (methanethiol) and methyl thioacetate (sulfur containing ester) were the major sulfur compounds of the concern in the wines. Moreover, the concentration of H2S was directly influenced by the type of yeasts in the first month of storage. Wines made from Saccharomyces cerevisiae P1Y2 generally had lower concentration of H2S than those from Saccharomyces cerevisiae RC212. In addition, lower H2S concentration was observed in light lees load than in medium or heavy lees load treatment from Saccharomyces cerevisiae P1Y2 during the first two weeks of aging, whereas more H2S were generated in higher lees loading samples from Saccharomyces cerevisiae RC212. H2S accumulated with time during early aging, and reached to the maximum at one month, then decreased afterwards regardless of types of lees or the amount of lees loaded. Methanethiol also accumulated during aging, and reached its maximum at 2-3 months, then decreased slowly afterwards.
High level of methyl thioacetate was detected in the experimental wines, wines from Saccharomyces cerevisiae RC212 had substantially higher level of methyl thioacetate than those from Saccharomyces cerevisiae P1Y2 regardless of lees level, and the concentration of methyl thioacetate stayed consistent during barrel aging. Heavy lees generally lead to more dimethyl sulfide (DMS) accumulation. During the aging storage, DMS reached to peak level at 2-3 months, decreased at 6 months, and then continued accumulating at 9 months. The levels of other sulfur compounds (carbon disulfide, dimethyl disulfide and dimethyl trisulfide) were very low for flavor contribution. The samples from collaborating wineries were also analyzed, but the results were complicated due to various treatments and remedies performed at winery. H2S was the major volatile sulfur compound in those winery samples, especially at the beginning of the barrel aging. Some winery samples also had high levels of methyl thioacetate and methanethiol. Although various remedies were performed in wineries to remove H2S , the levels of methyl thioacetate were very high in many of the wines after 6 to 9 months of aging. High concentration of methanethiol was also observed in many wines. More winery samples were studied for 2014 harvest and high levels of hydrogen sulfide, DMS and methyl thioacetate were detected in most of those wines. We collaborated with Dr. James Osborne further studied the effect of YAN on volatile sulfur formation. Detailed results were submitted separately as a single report. The study showed that the levels of YAN did not affect the generation of MeSH, CS2, DMDS and DMDS. However, the amount of YAN and the type of YAN did affect the formation of thioactates by Saccharomyces cerevisiae UCD522. DAP addition generated higher thioacetates. We also studied the effect of elemental sulfur on volatile sulfur composition. Although the residual sulfur affected hydrogen sulfide production during fermentation (see the separate report), sulfur addition did not affect the volatile sulfur compounds in the final wine except for methyl thioacetate. Sulfur addition generated more methyl thioacetate by both Saccharomyces cerevisiae P1Y2 and UCD522. High levels of methyl thioacetate could be an important issue for winery. Methyl thioacetate impart sulfur off-flavor in wine, it can also be converted to methanethiol that has a very low sensory threshold. The results suggested the methyl thioacetate and methanethiol could be the major culprits for sulfur off-flavor development during barrel aging. This new finding is significant and needs to have further investigation in future.
To date, the project has completed two goals, and is making progress on two others. We have analyzed the thermodynamics and kinetics for the sulfur dioxide binding of four different aldehydes and ketones, the major binders of SO2. This was carried out using a totally new approach, NMR spectroscopy, which allowed us to analyze the reactions under wine-like conditions. Two-dimensional (1H-1H) homonuclear and heteronuclear (13C-1H) single quantum correlations (COSY and HSQC) nuclear magnetic resonance experiments of wine samples were used for the simultaneous identification and quantification of free and, for the first time, sulfite bound acetaldehyde, pyruvic acid, acetoin, methylglyoxal and α-ketoglutaric acid. This new technological approach opens the door to possible new approaches to measuring free and bound sulfur dioxide. The effect of varying levels of SO2 and glutathione on micro-oxygenation was also investigated and it was surprising to see that both antioxidants suppress oxygen consumption. The cause for this effect may be related to suppression of the free radical formation by the Fenton reaction. When wines were depleted of these antioxidants, it appears that the formation of aldehydes rapidly increased, along with the formation of stabilized pigments. A new method to analyze glutathione, a possible new antioxidant for protecting wine, is also underway.
The first goal of this project was to validate the solid phase microextraction (SPME) method developed in the Ebeler laboratory for the analysis of S-volatiles produced both during fermentation and sur lie aging, and to determine if the method displayed the dynamic range of sensitivity needed to undertake an analysis of the product-precursor relationships for the volatile S-containing compounds that appear during aging on the yeast lees (sur lie). The SPME method has been validated over two years and displays the level of sensitivity needed to undertake the analysis of the chemistry of formation of the S-volatiles under natural or un-spiked conditions.
The second goal of this experiment was to examine the role of several factors on the formation of S-volatiles during sur lie aging. The role of nitrogen, sulfate and vitamin levels and effect of supplementation with methionine, cysteine and glutathione were evaluated and found to not impact the appearance of S-volatiles in synthetic juice media. Likewise the presence of hydrogen sulfide formation during primary fermentation was not correlated with the appearance of S-volatiles sur lie in either synthetic or natural juices. The presence of elemental sulfur impacted H2S formation but to date the presence of elemental sulfur during the primary fermentation has not resulted in the appearance of S-volatiles sur lie. Similarly juice pH has impacted the appearance of negative notes in the wines but these characters do not appear to be associated with S-volatiles. The grape solids level during the primary fermentation did impact the level of S-volatiles formed, but appreciable S-volatiles were found only at the higher solids levels, levels that would not be normally used in production. These wines likewise developed off-characters that could not be correlated with S-volatiles. The off-notes found are reminiscent of S-compounds so it is not surprising that their appearance is blamed on the presence of S-volatiles. However this research suggests that S-volatiles play a more minor role in the appearance of off-characters sur lie than previously reported.
The first goal of this project for the first year of funding was to validate the solid phase
microextraction (SPME) method developed in the Ebeler laboratory and to determine if
the method displayed the dynamic range of sensitivity needed to undertake an analysis of
the product-precursor relationships for the volatile S-containing compounds that appear
during aging on the yeast lees (sur lie). The SPME method has been validated and
displays the level of sensitivity needed to undertake the analysis of the chemistry of
formation of the S-volatiles under natural or un-spiked conditions. The method was used
to profile the appearance of S-volatiles in experimental samples and in wines acquired by
the University for analysis.
The second goal was to examine the impact of nutritional conditions of the primary
fermentation on the appearance of off-odors sur lie. The factors examined were: nitrogen
level, micronutrient level, nitrogen and micronutrient level, yeast strain, hydrogen sulfide
produced during the primary fermentation, supplementation with methionine and cysteine
at levels naturally occurring in juices, and sulfate level. To date none of these conditions
has resulted in the appearance of S-volatiles at the levels expected from the literature over
the five week time course used. Differences in the non-nutritional conditions could
explain the lack of appearance of S-volatiles in our study. A more likely explanation,
however, is the lowered levels of supplements used in our study done deliberately to
more accurately depict actual wine production conditions. The experiments will be repeated
using a longer time course as some of the samples did display low levels of Svolatiles,
detectable chemically but well below the sensory threshold of detection. Other variables
did appear to affect S-volatile formation such as pH and solids content, and these will
be explored more completely in the next year.
The objective of this proposal was to determine the role of cystathionine b-synthase (encoded by the CYS4 gene) in hydrogen sulfide formation in wine strains of Saccharomyces. An analysis of allele diversity in low and high H2S producing yeast strains revealed that one strain, UCD932 (Ba2), carried a specific mutation in CYS4 that may explain the low sulfide production. A series of experiments were performed to define the role of CYS4 allele variation in sulfide production. CYS4 mutant and wild type alleles were cloned and used to transform a series of strains with varying H2S production. Low sulfide production was dominant in UCD932, meaning that transformation of UCD932 with the wild type CYS4 allele did not lead to an increase in sulfide formation. However, transformation of high H2S producing strains with the mutant form of CYS4 did not lead to a reduction in sulfide levels under any condition evaluated. This finding suggests that the mutated allele of CYS4, which leads to stabilized production of cystathionine b-synthase, is not solely responsible for the low H2S producing phenotype of this strain.This proposal was originally requested for two years of research funding. However, better than anticipated progress was made in the first year and the first two objectives have been met or are soon to be completed. The third objective called for a detailed analysis of the impact of truncation mutants of CYS4 on the level of H2S production. Although this is still an interesting area of investigation, we found no evidence that mutation of CYS4 could explain the low level of sulfide produced by UCD932. It is still possible that the altered CYS4 gene of UCD932 in combination with other genetic variations in this strain is responsible for low sulfide production, but it is not sufficient by itself. Therefore, a more global approach to defining the genetic basis of low sulfide production will be proposed in a new grant.
The goal of this proposal was the determination of the genetic basis of the variation in hydrogen sulfide formation observed in commercial and native isolates of Saccharomyces cerevisiae. An initial goal was the characterization of the variability in strain behavior using different synthetic media and natural juices. No correlation was found between the basal level of sulfite reductase activity and hydrogen sulfide production. While strains lacking sulfite reductase did not produce hydrogen sulfide, they did produce copious amounts of sulfite. Methionine and cysteine supplementation had variable effects on volatile sulfur formation. In general these compounds were only of limited effectiveness or lead to the formation of higher sulfur volatiles that are the degradation products of these amino acids. All yeast strains evaluated show an effect of limiting nitrogen in the increased production of hydrogen sulfide, but the level of nitrogen at which this occurs is variable and impacted by medium composition, likely due to relative differences in demand for nitrogen.
There are several major conclusions of this work. First, cytoplasmic cysteine levels are correlated with activity of the sulfate reduction pathway and the production of hydrogen sulfide. Second, cytoplasmic cysteine levels vary up to five fold in different genetic backgrounds in strains grown under identical conditions. Those strains maintaining high internal concentrations of cysteine produce lower amounts of sulfide. Third, methionine and glutathione levels were not found to be correlated with sulfate reduction and H2S production. Fourth, cytoplasmic cysteine levels were a function of the activity of cystathionine b-synthase (CYS4) in most genetic backgrounds. In one genetic background, UCD713, a defective allele of MET17, the step immediately preceding CYS4, carried a mutation reducing cysteine production. Elevation of the activity of this protein restored cysteine levels and reduced sulfide production. Fifth, elevation of CYS4 activity by transformation of strains with a plasmid leading to over-expression of the protein increased cysteine pools and reduced hydrogen sulfide formation in some strains. In other strains over-production of the enzymatic activity did not occur, and in others it was reduced, suggesting that high enzyme levels can catalyze oligomer formation. Sixth, CYS4 allele analysis revealed that one strain that consistently produces little to no H2S carries a mutation in an important regulatory domain. Thus, the differences in hydrogen sulfide formation of different enological strains of Saccharomyces are due to differences in the pool levels of cysteine, which is a function of the relative activity of the enzymes directly involved in cysteine biosynthesis. Finally, alterations of the activity and regulation of Cys4p are associated with greatly diminished hydrogen sulfide production. Allele swap technology should be useful for the generation of low sulfide producing variants of any commercial strain.
In this current grant year, the analysis of the impact of over-expression of two genes involved in consumption of reduced sulfur, CYS4 and MET] 7, on H2S formation in commercial and natural wine strains of Saccharomyces was completed. Interestingly, increasing the level of expression of the CYS4 gene completely eliminated hydrogen sulfide production in four strains, had no effect in others, and in a few resulted in an increase in H2S. Similar results were obtained for MET17. So far, strains that showed reduced volatile sulfur formation with CYS4 did not show any effect with MET 17 and those showing an effect with MET 17 showed no or increased H2S formation with over-expression of CYS4. Strains that were high produces of H2S tended to decrease sulfide release when CYS4 was present, while the moderate producers showed a stronger response with MET 17. Thus, there are multiple underlying genetic causes for the production of hydrogen sulfide. This analysis does indicate that once the cause of H2S release is known for a given strain, it can be corrected genetically. It will also be possible to screen for strains naturally possessing alleles leading to reduced sulfide production to be used in conventional breeding programs. This research has further clarified the basis for the two phases of hydrogen sulfide release observed during fermentation. The early phase of hydrogen sulfide production occurs shortly after maximal cell biomass is attained, within the first few days of active fermentation, and is related to the relative activities of the enzymes generating and consuming reduced sulfur. The later stage, which occurs at the end of fermentation, is related to the nitrogen recycling behavior of the culture. Genomic data indicates that at this point in time numerous pathways have been induced that shunt nitrogen between amino acid components. When this occurs, there is a net shift of nitrogen from the sulfur containing amino acids to the non-sulfur containing amino acids. If nitrogen levels are in ample supply, this is prevented from occurring. Interestingly, analysis of the pattern of production of hydrogen sulfide of the 12 strains used in this study revealed that many of the strains produce hydrogen sulfide continuously during fermentation. Over-expression of MET 17 and CYS4 has the highest impact on the continual producers versus the transient producers.
In this past grant year two significant sub-objectives of this proposal were completed. First, the studies to determine the synthetic juice conditions best mimicking natural grape juice were completed and the twelve yeast strains for the rest of the study were selected from the extensive analysis of the initial 29. The selected strains display a broad array of behaviors with respect to hydrogen sulfide formation under enological conditions. This body of work has been submitted as a manuscript. The second major sub-objective to be completed was the comparison of the Microarray and Proteome technologies for analysis of global gene expression. The microarray analysis profiles the pattern of expression of mRNA, the “transcriptome”, while the proteome analysis directly profiles protein patterns. Both techniques suffer from limitations that may be particularly severe depending upon the relative level of expression of the genes of interest. It was necessary to compare both techniques to determine which one would be most useful for the comparative analysis of expression of proteins of the sulfate reduction pathway. The microarray analysis allows accurate quantitation and comparison of moderately to lowly expressed mRNA species, but is not useful for comparative analyses of highly expressed mRNA species. The mRNAs for the genes of the sulfate reduction pathway were found to be for the most part too highly expressed to be accurately estimated with this technology. However, as described in detail below, the microarray analysis did provide extremely useful information on the physiological status of the cells during nitrogen limitation. The strain used for this study was an isolate of the commercial French White strain. On the other hand, the Proteome analysis was found to be quite suitable for this study. The major proteins of interest are all visible as distinct spots on the proteome gels. In the past year we have fine-tuned this protocol and have achieved better reproducibility than what is typically reported in the literature. The direct comparison of the protein profiles of strains displaying differences in H2S formation has been initiated and will continue next year.