The significance of the H2S-C6 pathway for volatile thiol production

The significance of the H₂S-C6 pathway was tested by monitoring the initial stages of fermentation using a defined synthetic grape medium and various yeast strains.

Introduction

Volatile thiols (3MH, 3MHA and 4MMP) are sulphur-containing compounds responsible for potent tropical fruit aroma in especially Sauvignon blanc wine, where levels can reach far beyond the sensory perception threshold. In the grapes, 3MH, 3MHA and 4MMP are not present in their free aromatic form and the aromatic compounds are formed during fermentation. For many years, researchers have been trying to establish the source of volatile thiols and the mechanisms involved in the formation thereof.

Conjugated precursors

Certain non-aromatic, non-volatile conjugated precursors are present in the grape juice.^1,2 The yeast transforms these precursors during alcoholic fermentation to release the free and aromatic volatile thiols from the conjugated compound. 3MHA does not have a conjugated precursor in the grape. It is formed during fermentation through the esterification of 3MH with acetic acid, a reaction regulated by specific enzymes produced by the yeast.

Although the conjugated precursor’s involvement in the formation of volatile thiols has been established,³ several studies determined that these precursors only account for a small fraction of the total amount of thiols present in wine.^1,4-7 Moreover, no direct correlation between precursor concentration in the juice and the amount of free volatile thiols in the resulting wines has been observed.^8,9

H₂S-C6 pathway

Another possible source of volatile thiols is the formation through a mechanism where a sulphur-containing compound reacts with a six-carbon (C6) compound to produce the volatile thiols. Previous studies have shown that early H₂S production by the yeast in the presence of unsaturated C6 compounds (such as hexenal) induces direct yeast-mediated formation of volatile thiols.¹⁰ This specific pathway of thiol production is disputed, mainly because the C6 precursors are rapidly converted to less reactive compounds within a few hours after yeast inoculation¹⁰ (before H₂S formation), likely rendering it unavailable for the addition reaction.

However, the early production of H₂S by the yeast, together with the decline in C6 compounds and the possible formation and release of volatile thiols during these initial stages of fermentation, has only recently been scientifically investigated by New Zealand researchers. In this study,¹¹ the significance of the H₂S-C6 pathway was tested by monitoring the initial stages of fermentation using a synthetic grape medium with known composition and various laboratory and commercial yeast strains.

Materials and methods

Laboratory scale fermentation was performed by inoculating 100 mL of synthetic grape medium (supplemented with 1.5 mg/L of hexenal) with 11 selected yeast strains. After 12 hours of incubation at 25°C and agitation, the fermentation was stopped by removing the yeast cells through centrifugation, and the remaining media (slightly fermented juice) was retrieved for testing.

Results

Natural H₂S production by the yeast during the initial 12 hours

Results showed the early production of H₂S by the yeast, as well as significant variability in the H₂S-producing potential of the selected yeast strains, with some yeast producing more H₂S than others during these initial stages of fermentation.

C6 decrease during the initial 12 hours

The decrease of unsaturated hexenal and hexenol during the initial 12 hours after initiation of fermentation was monitored. Results showed that all the yeast strains tested could metabolise hexenal, and the concentration of the C6 compounds significantly decreased in the presence of yeast.

The production of volatile thiols during the initial 12 hours

Despite the natural formation of H₂S by the yeast and the consumption of the C6 compound, no 3MH or 3MHA was detected during the initial 12 hours after the initiation of fermentation. It is unsure if the concentration of H₂S formation under the conditions of the study was sufficient to initiate thiol production and/or if the amount of volatile thiols produced was too low to be able to be detected analytically. Other than that, the volatile thiol concentration in the slightly fermented synthetic must was measured, therefore excluding intracellular volatile thiol content. It is possible that the yeast still needs to release the volatile thiols from the cell. The measurement of intracellular volatile thiols poses many challenges and might not be possible to conduct at this stage.

The production of volatile thiols during the initial 12 hours when an exogenous source of H₂S was provided

The early addition of exogenous H₂S (in the form of NaSH) resulted in the production of 3MH (but not 3MHA), suggesting that sufficient H₂S concentrations must be present to obtain thiols through the H₂S-C6 pathway.

Conclusion

Even though the yeast naturally produced H₂S and rapidly consumed the C6 compounds during the early stages of fermentation, no volatile thiols were detected in the medium. It could be that the concentration of endogenously formed H₂S is not high enough to contribute substantially to free volatile thiols in a synthetic grape medium, especially considering that the addition of NaSH (which provided an exogenous source of H₂S) did lead to increases in 3MH. Other than that, it is unclear if the yeast would have started producing and excreting the thiols if the fermentation continued. Challenges regarding the measurement of low concentrations of thiols could also contribute to further difficulties in determining the significance of this mechanism in volatile thiol formation. Therefore, the contribution of the H₂S-C6 mechanism to the final concentration of volatile thiols in wines remains unclear, and further work is needed.

Abstract

The study aimed to test the significance of the H₂S-C6 pathway in yeast for volatile thiol production. The study found that even though the yeast naturally produced H₂S and rapidly consumed the C6 compounds during the early stages of fermentation, no volatile thiols were detected in the medium. However, the addition of NaSH (which provided an exogenous source of H₂S) led to measurable increases in 3MH. The contribution and significance of the H₂S-C6 pathway in the formation of volatile thiols (when no exogenous source of H₂S was provided) remains unclear and needs further investigation.

Abbreviations

4MMP: 4-mercapto-4-methylpentan-2-one; 3MH: 3-mercaptohexan-1-ol; 3MHA: 3-mercaptohexyl acetate; hexenal: (E)-hex-2-enal; hexenol: (E)-hex-2-en-1-ol.

References

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2. Tominaga, T.; Darriet, P.; Dubourdieu, D. Identification of 3-Mercaptohexyl Acetate in Sauvignon Wine, a Powerful Aromatic Compound Exhibiting Box-Tree Odor. Vitis 1996, 35, 207–210.
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7. Fedrizzi, B.; Pardon, K. H.; Sefton, M. A.; Elsey, G. M.; Jeffery, D. W. First Identification of 4-S-Glutathionyl-4-Methylpentan-2-One, a Potential Precursor of 4-Mercapto-4-Methylpentan-2-One, in Sauvignon Blanc Juice. Journal of Agricultural and Food Chemistry 2009, 57 (3), 991–995.
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9. Roland, A.; Cavelier, F.; Schneider, R. New Insights in Varietal Thiols Biogenesis in Wine. In International conference series on wine active compounds; Oenoplurimedia: Beaune, France, 2011; pp 21–23.
10. Harsch, M. J.; Benkwitz, F.; Frost, A.; Colonna-Ceccaldi, B.; Gardner, R. C.; Salmon, J. M. New Precursor of 3-Mercaptohexan-1-Ol in Grape Juice: Thiol-Forming Potential and Kinetics during Early Stages of Must Fermentation. J Agric Food Chem 2013, 61 (15), 3703–3713. https://doi.org/10.1021/jf3048753.
11. Hou, R.; Jelley, R. E.; van Leeuwen, K. A.; Pinu, F. R.; Fedrizzi, B.; Deed, R. C. Hydrogen Sulfide Production during Early Yeast Fermentation Correlates with Volatile Sulfur Compound Biogenesis but Not Thiol Release. FEMS Yeast Res 2023, 23. https://doi.org/10.1093/femsyr/foad031.

For more information, contact Carien Coetzee at carien@basicwine.co.za.

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