Formation and fate of positive and negative sulfur compounds
Abstract
Low molecular weight sulfur compounds (LMWSCs) can contribute both positive and negative sensory attributes to wines. Positive aromas include ‘passionfruit’, ‘tropical’ and ‘blackcurrant’ and negative characters include ‘rotten egg’, ‘rubber’, ‘sewage’ and ‘canned corn’. This project focused on increasing understanding of the formation and fate of sulfur compounds responsible for these sensory attributes and applying this knowledge to develop practical strategies to manipulate and modulate LMWSCs during winemaking. A further aspect was to ensure that positive and negative compounds do not undergo any further unwanted changes in tank or bottle, once a wine has a desirable LMWSC profile.
As part of this project, new advanced analytical methods were developed to enable characterisation of the very challenging and reactive LMWSCs at part per billion (ppb) concentrations and below. Another very significant breakthrough was the identification of precursors to several key LMWSCs. The work also showed that the addition of copper significantly influenced the evolution of LMWSCs. Importantly, the effects of copper differed depending on dissolved oxygen concentration post-bottling and also as a function of the residual copper concentration. In addition, copper effects were further influenced by pH and SO2 and it was demonstrated that post-bottling formation of H2S, MeSH, DMS, and CS2 is significantly impacted by both copper additions and wine pH. Beyond copper, the project showed that the formation of LMWSCs from their precursors in wine is influenced by the presence of other metal ions that naturally occur in wine, especially when present in high concentrations. Data on factors modulating the chemical formation of LMWSCs in wine was augmented by the discovery of significant differences among wine yeast in their ability to release both positive and negative LMWSCs. Taken together, this information will help winemakers to make informed choices regarding yeast strain and winemaking conditions to suit their wine style, potentially reducing the formation of negative LMWSCs in a winery environment.
Summary
Low molecular weight sulfur compounds (LMWSCs) can contribute both positive and negative sensory attributes to wines. Positive characteristics can include ‘passionfruit’, ‘tropical’ and ‘blackcurrant’ but the negative characteristics include ‘rotten egg’, ‘rubber’, ‘sewage’ and ‘canned corn’. Their control in a winery environment is an important avenue to increasing the value of wine by either increasing positive sensory attributes or decreasing those deemed to be negative. Their occurrence can be influenced by factors including yeast selection and fermentation conditions; the nature and quantity of precursor compounds; the availability or absence of oxygen at different points of the winemaking process; and availability and speciation of transition metal ions such as copper. Broadly, this project focused on increased understanding of these factors; this knowledge was then used to develop practical strategies to manipulate and modulate LMWSCs during winemaking and to ensure that once a wine has produced the expressed LMWSCs, positive or negative, they do not undergo any further unwanted changes in tank or bottle.
More specifically, the project aimed to develop an in-depth understanding of the role of compounds suspected to be the main precursors of the sensorially important LMWSCs, with a focus on negative sensory characters deriving from hydrogen sulfide (H2S), methanethiol (MeSH), ethanethiol (EtSH), dimethylsulfide (DMS), phenylmethanethiol (BnSH) and the generally positive varietal thiol sensory characteristics of 3-sulfanylhexan-1-ol (3-MH) and 3-sulfanylhexyl acetate (3-MHA). The project sought to understand the metabolic and chemical pathways that lead to formation of these thiol compounds and the chemical and environmental switches that lead to otherwise innocuous sulfur-based compounds being converted to those that have a significant sensory impact.
As part of the enabling technology required for this project, new advanced analytical methods were developed to enable characterisation of these very challenging and reactive LMWSCs. A new HPLC-MS method for the analysis of thiols and disulfides in wine was developed and validated which quantifies a wider range of LMWSCs than previous methods. In addition, to support investigations of sulfur-containing amino acids as LMWSC precursors, an amino acid assay method was developed in collaboration with the AWRI Metabolomics group for cysteine (Cys), methionine (Met) and glutathione (GSH).
A very significant breakthrough from this project was the identification of precursors to several key LMWSCs. At the outset of this project, it was suspected that the presence of certain compounds such as Cys, GSH, Met, disulfides, and thioacetates such as methyl thioacetate (MeSAc) and ethyl thioacetate (EtSAc) were contributing factors in determining H2S, MeSH, EtSH, and DMS concentrations in wines post-bottling. However, these hypotheses had yet to be tested in real wines. This study demonstrated that the direct desulfurisation of Cys, Met, or GSH did not pose an obvious risk of H2S or MeSH formation in wine post-bottling. However, the presence of MeSAc, or disulfides such as DMDS, significantly increased the risk of MeSH formation, with up to a 20% MeSH yield and a 70% MeSH yield obtained from MeSAc and DMDS, respectively, as measured in wines over a 12-month storage period. Additionally, model studies do not support the theory that BnSH forms from benzaldehyde reacting with H2S, leaving unanswered the question about how BnSH and the associated ‘struck flint’ character are formed. Based on these findings it is recommended that thioacetates and disulfides are monitored to assess potential risk of post-bottling LMWSC release, in addition to the ‘total packaged oxygen’ (TPO) concentration of wine.
It was shown that the addition of copper significantly influenced the evolution of LMWSCs, but the effects of copper differed depending on dissolved oxygen concentration post-bottling and with the residual copper concentration. As expected, at higher oxygen concentrations, some metals such as copper, significantly reduced the concentration of the thiols in the wine tested. During wine maturation, once the oxygen concentration decreased to non-detectable, the effect of copper was reversed, with the presence of copper now being associated with a significant increase in MeSH concentration, regardless of the presence or absence of other metals. Higher residual copper concentrations also resulted in significantly higher H2S concentrations in wine, but interestingly, these differences did not become evident until 12 months post-bottling. This work highlights the copper concentration dependency of H2S formation and the weakness of traditional benchtop trials used to determine copper additions, where only the immediate effects are assessed and not the long-term impacts in-bottle resulting from residual copper.
In addition, copper effects can be influenced by pH and SO2 and this study has demonstrated that the post-bottling formation of H2S, MeSH, DMS, and CS2 is significantly impacted by both copper additions and wine pH. For some ‘reductive’ aroma compounds, the interaction between pH and copper treatment was an important factor in determining their final concentrations in Chardonnay and Shiraz wines post-bottling. The current results established that both wine pH and copper additions have significant impacts on H2S, MeSH, and DMS concentrations in wines post-bottling. Specifically, less H2S and MeSH
were produced through copper-catalysed reactions in wines at a lower pH than in wines at a higher pH level. It should be noted also that different effects were observed in red wines and white wines, likely due to the differing nature of the wines’ matrix components. Investigations also showed that the presence of SO2 plays a fundamental role in the modulation of H2S profiles through copper interactions in both red and white wine post-bottling.
One possibility for managing copper effects is to bind copper to chelating compounds that completely remove it from the wine matrix or chemically isolate it so that it can no longer participate in the formation of reductive characters. The results from this project suggest that differing metal chelation environments can be established using additives and that these can have a significant effect on H2S generation. It is possible to optimise copper’s beneficial effects through the timing of its addition and in terms of limiting residual copper concentrations it is more desirable to add copper at 0° Brix (during the final phase of active fermentation) rather than at the end (post-active ferment) which is a common industry practise. This timing ensures that yeast and solids adsorb copper and limit the residual copper concentration in a final wine.
The project has shown that the formation of LMWSCs from their precursors in wine is influenced by the presence of not only copper, but also by other metal ions that naturally occur in wine when present in sufficiently high concentrations. These metals include manganese, aluminium, iron and zinc, and it is recommended that producers monitor these and keep metal concentrations as low as possible in wine, to minimise the post-bottling evolution of wine LMWSCs.
As part of this project the AWRI collaborated with a project led by Dr Andrew Clark at Charles Sturt University (CSU). The results from the collaboration demonstrate that major non-volatile matrix components (red wine tannin, white wine protein, white wine polysaccharide, red wine polyphenol, white wine polyphenol) had limited impact on the form of copper present in wine, most likely because the major form of copper in most wines is as copper(I) sulfide (Cu2S), rather than as complexes with matrix components as suggested by previous studies.
In further research to understand the chemical forms of copper in wine, differences in particle size and concentration of copper-tartrate complexes suggested that the various types of copper-tartrate complexes produced at varying pH levels may affect the binding sites of copper that are available to either catalyse the formation of LMWSCs such as H2S, or quench the thiols produced to form copper sulfide complexes. This may explain some of the pH-related effects observed in these experiments.
This project has also discovered significant differences among wine yeast in their ability to release both positive and negative LMWSCs. Small-scale fermentation experiments with a large number of yeast strains have shown the great diversity in the production of LMWSC by yeast. This analytical information, combined with genomic data on these yeast strains, has allowed the identification of several yeast markers associated with the formation of important LMWSCs, such as the ‘tropical’ thiols 3-MH and 4-MMP and H2S, from their precursors. This information will help winemakers to make informed yeast strain choices to suit their wine style, and potentially reduce the formation of negative LMWSCs in a winery environment.