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80 WINES&VINES April 2018 GRAPEGROWING PRACTICAL WINERY & VINEYARD glutathione (Glut-3MH) bound precursors came to the fore. 5 A substantial amount of work has been done to understand these precursors. In follow-up fermenta- tions, it has been conclusively proven that these conjugated pre- cursors are not the main precur- sors and only contribute to a fraction of the formed volatile thiols. 6 Recently, other similar pre- cursors such as Glut-3MH-Al and Glut-3MH-SO 3 have been identi- fied, 7 but these still only account for a small percentage of volatile thiols formed during fermenta- tion. Other conjugated com- pounds are being investigated as potential precursors. The conjugated precursors are not released linearly, and a low (0.1%-12%) conversion rate when using commercial wine yeast was observed, as well as an almost complete lack of correla- tion between the bound precur- sor concentrations and the final thiol concentrations in wine. 8 E v e n w i t h t h i s i n f o r m a t i o n widely known and accepted, these conjugated precursors are still commonly referred to as being the main source of volatile thiols in wine, though the state- ment remains unsupported. The presence of these precursors does, however, excite the scien- tific community. The fact that the conversion is so low suggests a lot of potential in the juice. A method to draw this through to the wine would be key. Other pathways for the forma- tion of volatile thiols are being investigated that may be more significant sources than conju- gated precursors. These pathways include the contribution of H 2 S (or another –SH moiety) as a sul- fur donor in combination with a C6 compound such as (E)-2-hex- enal or (E)-2-hexenol, during the first few hours of fermentation. 9 This is a more direct pathway for the formation of 3MH. However, this reaction is not a straightfor- ward chemical reaction and re- quires the presence of yeast activity (possibly enzymatic), as tests have shown that the reaction does not take place in un-inocu- lated media. These observations imply that the formation of 3MH and /or 3MHA via the H2S pathway re- quires yeast activity and suggest that it is not driven by straightfor- ward chemical reactions. C6 compounds such as hexenal and hexenol are formed from C18 polyunsaturated fatty acids in plant cell membranes. This reac- tion occurs rapidly during the damaging of plant cells due to the oxidation of the polyunsaturated fatty acids in the presence of oxy- gen and lipoxygenase enzymes. The grape damage occurs during harvesting and processing. Why does the plant/berry pro- duce C6 compounds in "stress" situations? Evolutionarily speak- ing, the reaction makes great sense: The C6 compounds have anti-microbial properties, and this reaction (which occurs in berry- damaging situations) will lead to production of toxic-protecting compounds (in this case the C6 compounds) to minimize the po- tential damage caused by the spe- cific injury or infection. As a result, the (somewhat damaged) grapes have elevated concentra- tions of these protecting C6 com- pounds. What is known is that (E)-2-hexenal has a proven inhibi- tory and fungicidal activity against Saccharomyces cerevisiae, 10 which is probably one of the reasons that yeast metabolizes it, together with its related alcohol (E)-2-hexen- 1-ol, into the less toxic and less reactive hexan-1-ol. The tripeptide glutathione al- ready naturally present in the grape tissue can react with C6 compounds to yield the non-toxic Glut-3MH precursor, in this way protecting the berry from elevated levels of C6 compounds. A thiol precursor is formed during the detoxification process! In a similar manner, it is hypoth- esized that yeast inoculated into the grape must will disable the toxic compounds in its own way by con- verting, for example, hexenol to hexanol and by facilitating the bind- ing of antioxidants with these C6 compounds, thereby resulting in precursor/volatile thiol formation. The plant and/or yeast will use mechanisms to convert the toxic substances formed during berry damage to an inactive form resulting in an odorous product. H 2 S has been considered to possibly be a main antioxidant binding compound for the binding of these C6 compounds. However, production of H 2 S by the yeast does not coincide with the uptake of these C6 compounds (within 24 hours after inoculation during the yeast lag phase) and other sources can be considered. This suggests the availability of another sulfur donor in the early fermentation. However, again a huge untapped potential remains in that large amounts of C6 com- pounds are present during early fermentation stages that can be converted to volatile thiols if the correct sulfur donor is available. A patent is currently pending to allow the bubbling of H 2 S through the grape must during early stages of fermentation to facilitate the reaction of C6 compounds with H 2 S. This addition is currently il- legal except for research wines produced using this process. In a study where H 2 S was added to the must before fermen- tation, an unprecedented amount of 257 µg/L of 3MH and 35 µg/L 3MHA were present in the fin- ished wines. These concentrations are up to 38 times the amount found in "high thiol" wines. Hex- enol and hexenal formation is not unique to Sauvignon Blanc—or other "thiol-containing cultivars," for that matter—they can occur in musts from all grape varieties. If the bubbling of H 2 S is ap- proved, the application should be done with great caution due to the unpleasant sensory effect unre- acted H 2 S can have on wine aroma and the toxicity of H 2 S. Using a more natural approach by encour- aging the earlier formation of H 2 S by yeast or delaying the disappear- ance of C6 compounds could have the same effect. The extent to which this occurs in commercial winemaking needs further study. The presence of elemental sulfur can have an effect on the thiol con- centration in wines. Where in- creased amounts of elemental sulfur were added to Sauvignon Blanc grape must, an increase in the volatile thiols was also observed. However, these increases were ac- companied with increases in reduc- tive compounds such as H 2 S and methyl-thiol and corresponding reductive aroma attributes. 9 These processes seem promis- ing, however the exact origin of the volatile thiols in wine contin- ues to elude the scientific com- munity at present. The race to expose the pathway involved in volatile thiol formation is a con- stant pressure point in the re- search community due to the implications it will have on pro- duction of all wine varieties, espe- cially Sauvignon Blanc. The secret to this mechanism will unlock huge potential and a wealth of possibilities. Other than control- ling thiol production in the win- ery, it will provide viticulturists and winemakers with the tools needed to predict the thiol poten- tial of grapes in vineyards. Until then, the procedures known to increase volatile thiol levels in wine should be exploited in order to ensure maximum thiol produc- tion if that is what the winemaker is going for. Dr. Carien Coetzee completed her Ph.D. at the University of Stellenbosch. Her studies evolved around the effect of oxi- dation on Sauvignon Blanc wines with a central theme of aromatic compounds and their stability. She is currently em- ployed at Vinlab, an accredited laboratory supporting the South African wine indus- try. Contact her at carien@vinlab.com. The references for this article are available online at winesandvines.com/features. VOLATILE THIOLS IN NEW ZEALAND AND SOUTH AFRICAN SAUVIGNON BLANC 3MH (ng/L) 3MHA (ng/L) 4MMP (ng/L) New Zealand 100 – 20,000 5 – 2,500 2 - 50 South Africa* 29 – 6,700 1 – 3,100 2 - 122 While levels of 3MH are significantly higher in New Zealand Sauvignon Blanc, South African Sauvignon Blanc may contain higher levels of 4MMP. * Based on results from VinLab (Pty.) Ltd. and the University of Stellenbosch for 2014 to 2016 Sauvignon Blanc wines.