New Zealand winegrowers use mechanical harvesting as a norm. This is probably out of necessity, however it is by far the harvesting method delivering the highest concentration of thiols. This is contradictory to what has been believed for years to be best practice when producing top quality wines.
     Research studies comparing harvesting methods such as hand-picked with whole bunch pressing, hand-picked with crushing and destemming and machine harvesting showed an increase in thiol concentration as the "roughness" of the method increased.11 This observation falls in line with the scientific explanation involving oxygen, enzymes and C6 compounds together with a sulfur donor.
     An actual increase of some C6 compounds when comparing hand-picked grapes to machine-harvested grapes has been observed. With greater maceration (longer skin contact) of the fruit and greater enzyme activity, the levels of the C6 compounds increased. The addition of maceration enzyme and sufficient time given for the reaction to occur can increase the thiols formed during the process.
     Wine producers can mimic the effect that mechanical harvesting has on grape berries. Some of these tools are listed below, but the fundamental aspect would be to cause some berry damage with sufficient protection to avoid too much oxidation.

Presence of antioxidants
When it comes to volatile thiols the importance of the presence of antioxidants cannot be overstated. The -SH group of the volatile thiols makes these compounds extremely susceptible to oxidation. It is therefore important to protect the wine from oxygen exposure and ensure sufficient presence of antioxidants such as sulfur dioxide, which will help preserve the aromatic compounds.12
     The earlier the addition of antioxidants after harvest, the better. Studies show that increasing the amount of antioxidants also increased the amount of volatile thiols formed and maximum thiol potential seemed to be reached so long as a moderate (30 - 50 ppm) level of SO2 was present prior to fermentation.2 This does have some limits due to fermentation difficulties in the presence of too much SO2 and inhibition of the enzyme responsible for increasing C6 compounds (lipoxygenase) during harvesting.
     However this effect has not been conclusively demonstrated. Excessive SO2 concentrations (300 mg/L) led to lower 3MHA concentration probably due to the acetylation pathway converting 3MH to 3MHA being interrupted.
     Timing of SO2 additions had an influence on 3MH and 3MHA production. A two-hour delay of the SO2 additions resulted in wines containing thiol concentrations of about half compared to wines where SO2 was added as early as 10-15 minutes after mechanical harvesting.2
     Finding practical methods for early SO2 additions can be challenging. New Zealand wine companies have used methods such as drip-feed of a concentrated SO2 solution, however, care needs to be taken over corrosion issues with equipment.
     In another New Zealand study, the addition of increasing amounts (0, 60, 120 mg/L) of SO2 led to increases in volatile thiol production obtaining levels of up to 12,000 ng/L which would be considered a high thiol wine.2 This wine was not Sauvignon Blanc, but rather Pinot Gris. These results show the necessity to investigate the contribution of volatile thiols to other varieties and highlights the importance of the use of antioxidants.
     Other antioxidants that have been under investigation are ascorbic acid and glutathione. Ascorbic acid can be added to juice and wine as a supplement to SO2 in order to increase antioxidant capacity. Glutathione, on the other hand, is not (yet) registered as a permitted additive, however glutathione levels can possibly be increased by adding some commercially available inactive dry yeast products.13
     In a study where 30 mg/L SO2 was added to grape must together with either 100 mg/L ascorbic acid or glutathione, the levels of all three important volatile thiols increased with 4MMP increasing significantly with glutathione addition.14 A 30 mg/L SO2 addition together with 100 mg/L glutathione showed 3MH and 3MHA concentrations of at least double the value compared to treatments where glutathione was added alone. It would thus seem that the combined protective effect could be more effective than the individual antioxidants.
     After fermentation, the presence of SO2 is of utmost importance as the instability of volatile thiols leads to drastic decreases if not protected.53 The thiol most susceptible to degradation and oxidation is 3MHA. During oxidation, very reactive quinones are formed which will readily bind to wine constituents including the volatile thiols rendering them odorless. Sulfur dioxide binds these quinones rapidly and thus prevents further oxidation reactions from occurring.
     The methods used for antioxidant additions need to be controlled for efficiency. Concentrated SO2 mixes quickly with the must in the bins as grapes are transferred into trucks or winery receiving hopper. The efficiency of powder should be investigated and it is important to check the uniformity and distribution of the antioxidant by taking samples from the juice arriving at the winery. Pockets of juice with low free SO2 are likely to have lower antioxidant potential.

Cold soaking/freezing of grapes
In a study where Sauvignon Blanc grapes were frozen to -20° C (-4 F) using dry ice and then thawed over 24 hours the results showed an increase in 3MH and 3MHA content.14 Wines made from hand-harvested grapes that underwent this cryogenic maceration contained about 300% more 3MH and 3MHA compared to wines that did not undergo cryogenic maceration.
     The level of thiols obtained were comparable with the same grapes that were harvested using the mechanical harvester. The explanation for this could be the increased leaching of precursors and enzymes into the grape must due to berry damage from ice crystal formation. Ice formation thus not only increases contact between reactants, but also concentrates the reactants in the available liquid thereby facilitating the reaction. The expense involved in using this freeze/thaw cycle may not be economical when processing large volumes of must, however, this technique could be applied in small batches to obtain larger diversity in wine styles.

Pressing and oxidation of juice
Studies have shown that wines made from juice obtained from the press (1 bar) contained either the same or a lesser amount of thiols when compared to wines made from free run juice.17,18 This could be due to the higher potential for oxidation due to larger phenolic extraction as the pressing pressure increases.
     Sufficient SO2 should be present prior to pressing to achieve a reductive atmosphere (using carbon dioxide or nitrogen gas) during the pressing process. Higher pressing fractions might contain higher concentrations of thiol precursors (including C6 compounds). However you run the risk of increasing the potential for oxidation (due to higher phenolic content). This could be risky seeing that an increase in the conjugated precursors would not necessarily lead to an increase in free thiols in the corresponding wine.
     Juice oxidation (measured by absorbance at 420 nm) can influence the volatile thiol concentration of the corresponding wines. Higher concentrations of 3MH were obtained from juice with lower 420 nm measurements.17 However, a low 420 nm measurement did not guarantee high thiol concentrations as other important factors could have a greater effect on the formation of precursors and volatile thiols. The oxidation of juice is an important factor to consider, however the addition of sufficient SO2 at this stage can minimize negative effects occurring due to oxygen addition delivering volatile thiol levels equivalent of juice that was not exposed to oxygen.18
     It is advisable to keep juice fractions that might be in advanced stages of oxidation, separate due to lower potential for volatile thiol formation. The wine can then be bottled or blended if proven to have sufficient volatile thiols present after fermentation. Another option would be to eliminate phenolic compounds through the use of a specialized fining agent on the juice. This prevents formation of quinones at a later stage and preserves thiol-containing compounds.21

Fermentation conditions
The yeast strain is extremely important that could determine the amount of precursors converted during fermentation. However, the yeast strain will only have a limited effect. Juice composition (such as the presence of volatile thiol precursors), needs to be of a certain standard and composition for the yeast to be able to convert and form volatile thiols. Other than precursors, the exact composition needed is unknown. The effect of the same yeast strain was investigated on different musts and the results showed that juice diversity was the primary thiol determinant,20 with yeast strain selection having a secondary effect. The strains can, however have an important effect and multiple fold increases have been seen when comparing yeast strain and volatile thiol production.
     3MHA is not formed directly from a precursor, but rather due to an esterification reaction that occurs during the fermentation process. Not all strains of Saccharomyces cerevisiae have the same capacity to express these compounds. Some strains are good thiol producers in that they release 3MH and 4MMP from corresponding precursors or it can create the volatile thiols from other compounds. Other yeast strains can be good converters in that they can efficiently convert 3MH to 3MHA. A mixture of these yeasts can be inoculated to maximize both production and conversion.
     S. cerevisiae is not the only yeast strain capable of releasing/producing volatile thiols from the precursors and other yeast strains, such as Pichia kluyveri, that have proven to be effective in increasing volatile thiol production.23
     Higher fermentation temperatures (irrespective of yeast strain used) resulted in increased volatile thiol concentration when compared to lower temperatures. However, in some cases the extended higher temperature during fermentation led to a decrease towards the end.24 It is therefore advised to commence fermentation at 62.5° or 64.5° F (17° or 18° C) for about 30 grams of sugar fermented and then, depending on the yeast added, whether it is cold-tolerant or not, gradually lower the fermentation temperature to about 59° to 61° F (15° to 16° C) in order to preserve the released volatiles.

Storage temperature
It is absolutely vital to keep the wine at a low temperature.25 Higher temperatures will not only accelerate the oxidation reaction, but will encourage hydrolyses of 3MHA to form 3MH (in some cases even leading to an increase of 3MH concentration). This way you will lose some of the aroma potency due to the higher perception threshold reported for 3MH, and a change in aroma quality.
     3MHA is the volatile thiol most affected during storage of wine, while much smaller losses were seen for 3MH. Some studies have shown the effect of temperature to be even more important than oxygen exposure during wine storage. It would thus be advised to keep wine at a temperature as low as possible not only until bottling but for consumption.
     Differences in storage temperature of only 5.5° F (3? C) such as 15? C vs 18? C or 59° F compared to 64.5° F could have a massive impact on thiol preservation. The cost of refrigeration should be considered and a workable compromise of 50° to 53.5° F (10-12? C) has been identified.2 Temperature logging during exports should be considered to ensure preservation of all aroma compounds and especially the thiols. It might be worth the cost and effort to ensure the transport temperature and condition is of a certain standard to guarantee better preservation.

Interaction with other wine components
The Top 10 Sauvignon Blanc competition, presented by the Sauvignon Blanc Interest Group of South Africa and sponsored by First National Bank, is the country's foremost platform for Sauvignon Blanc producers to showcase and benchmark their wines. The ten selected 2015 wines were subjected to various chemical analyses including volatile thiol and methoxypyrazine, while the sensory profile of each wine was determined using projective mapping.
     Results from the top 10 2015 winners showed great diversity in wine styles: from fresh and fruity to green and even wooded wines and volatile thiol concentration in these wines ranged from less than 10 ng/L to 547 ng/L for 3MHA; while 3MH concentrations ranged from 328 ng/L to 1,638 ng/L. These values are much lower than the maximum values determined by Vinlab for 2015 which ranged from less than 10 ng/L to 2,440 ng/L for 3MHA and 29 ng/L to 4,140 ng/L for 3MH.
     The diversity of wine styles chosen as competition winners, shows great complexity in quality South African Sauvignon Blanc wines. The contribution of other aroma compounds in these wines should not be underestimated as compounds such as esters and monoterpenes can significantly influence the aromatic composition of the wine delivering great complexity and desirable profiles. The sensory results of the selected wines did not always correspond to the chemical profile highlighting the importance of other aroma compounds impacting the wines and interactions occurring between volatile compounds.
     Analysis of the 2016 Top 10 Sauvignon Blanc wines showed very interesting results for 4MMP. The values ranged from 0 to 122 ng/L, which are huge amounts of this thiol especially considering, in general, that this thiol is mostly absent in wines from other countries. Even to such an extent that some researchers have stopped analyzing for it. From the selection of top 10 wines, the wines containing copious amounts of 4MMP had a distinct black currant character and it added a different dimension to the wines' aromatic characteristics. It would seem as if the presence of 4MMP in South African wines could give a needed edge to distinguish the wines in the international market.
     The above mentioned techniques can be used to tailor-make a wine to fit the aroma profile desired, however, it is not an easy task. Not only is there no guarantee for success, there is the added hurdle of wine complexity where sensory interactions can play a bigger role than anticipated.
     Sauvignon Blanc needs to be considered holistically by the way the compounds work together to create wine aroma. The presence of other molecules in the wine, whether it be aromatic or non-aromatic, can influence the perception of volatile thiols. This is a complex relationship within the wine medium and there is no real way to control these type of interactions as the effects of many have not been investigated.
     Some relationships have been studied. The interaction of 3MH with oxidation-related compounds (especially methional, reminiscent of cooked potato) showed a strong suppressive effect with methional reducing the intensity of the fruity aroma significantly.1 Conversely, acetaldehyde actually enhanced the perception of fruitiness brought by 3MH with moderate concentrations.26 As soon as elevated concentrations of acetaldehyde were present the interaction changed from enhancing to suppressing.
     A mutual suppressive effect was seen between IBMP and 3MH at specific concentrations.3 The contribution of the volatile thiols to the green aroma in wine has been observed in various studies. In a pressing study, the decrease in volatile thiol concentration in press fractions of higher inflation led to a decrease in the "fresh green capsicum" aroma even though there was no change in the IBMP concentration.27

Tailor your thiols to your desire
Volatile thiols plays an integral role in the aroma of Sauvignon Blanc wines and potentially other cultivars. Various techniques, some explained in this article, are available for the winemaker to optimize the formation of these odorous compounds, however a greater understanding of the formation of the precursors and the reactivity of the compounds is needed to be able to fully take advantage of these tools. This article might be able to equip you with the necessary knowledge to tailor the thiols to your desire.
     Even though volatile thiols can potentially be an overpowering attribute in a wine, in most cases it participates in complex aromatic interactions. These interactions lead to overall impressions of the wine aroma and perhaps can bring complexity to an otherwise one-
dimensional wine. Thus, the presence of certain aroma compounds does not guarantee the clear perception of the accompanied attributes. Assuming a certain aromatic profile based on a few chemical compounds would thus lead to incorrect conclusions.
     Another mistake would be to assume that all Sauvignon Blanc grapes behave the same. For example, harvesting studies from different sites showed some agreement in results, however occasionally the treatment did not lead to enhanced thiols.19 This should be kept in consideration to conduct small scale studies before opting for larger volumes to save time and money.
     A great wine starts in the vineyard. If the potential is not there to start with, the chances of achieving significant volatile thiol levels are slim. Therefore, it is wise to keep track of high thiol producing blocks between vintages and primarily use those grapes for production of high thiol wines.
     Some of this information is hard to comprehend as it will go against what has been considered for years to be good winemaking practice (especially for Sauvignon Blanc). South African Sauvignon Blanc is considered to be the mid-way point between New Zealand and the Loire (France) and innovative winemakers should push the envelope in order to stand out.

Dr. Carien Coetzee completed her Ph.D. at the University of Stellenbosch in South Africa. Her studies evolved around the effect of oxidation on Sauvignon Blanc wines with a central theme of aromatic compounds and their stability. She is currently employed at Vinlab, an accredited laboratory supporting the South African wine industry.

1. Coetzee, C., J. Brand, G. Emerton, D. Jacobson, A.C. Silva Ferreira and W.J. du Toit. 2015 "Sensory interaction between 3-mercaptohexan-1-ol, 3-isobutyl-2-methoxypyrazine and oxidation-related compounds." Aust. J. Grape Wine Res. 21 (2), 179-188.
2. Kilmartin, P. 2016 "Thiols found in Sauvignon blanc and their significance." In New Zealand Society for Viticulture & Oenology Sauvignon blanc Workshop; 24-80.
3. van Wyngaard, E., J. Brand, D. Jacobson and W.J.; du Toit. 2014 "Sensory interaction between 3-mercaptohexan-1-ol and 2-isobutyl-3-methoxypyrazine in dearomatised Sauvignon Blanc wine." Aust. J. Grape Wine Res. 20 (2), 178-185.
4. Swiegers, J.H., R. Willmott, A. Hill-Ling, D.L. Capone, K.H. Pardon, G.M. Elsey, K.S. Howell, M.A. de Barros Lopes, M.A. Sefton, and M. Lilly. et al. 2006 "Modulation of volatile thiol and ester aromas by modified wine yeast." Dev. Food Sci. 43 (C), 113-116.
5. Coetzee, C. and W.J. du Toit. 2012 "A comprehensive review on Sauvignon blanc aroma with a focus on certain positive volatile thiols. Food Res. Int. 45 (1), 287-298.
6. Subileau, M., R. Schneider, J.M. Salmon and E. Degryse. 2008 "New insights on 3-mercaptohexanol (3MH) biogenesis in Sauvignon Blanc wines: Cys-3MH and (E)-hexen-2-al are not the major precursors." J. Agric. Food Chem. 56 (19), 9230-9235.
7. Thibon, C., C. Böcker, S. Shinkaruk, V. Moine, P. Darriet and D. Dubourdieu. 2016 "Identification of S-3-(hexanal)-glutathione and its bisulfite adduct in grape juice from Vitis vinifera L. cv. Sauvignon blanc as new potential precursors of 3SH." Food Chem. 199, 711-719.
8. Pinu, F.R., S. Jouanneau, L. Nicolau, R.C. Gardner and S.G. Villas-Boas. 2012 "Concentrations of the Volatile Thiol 3-Mercaptohexanol in Sauvignon blanc Wines: No Correlation with Juice Precursors." Am. J. Enol. Vitic. 63 (3), 407-412.
9. Harsch, M.J., F. Benkwitz, A, Frost, B. Colonna-Ceccaldi, R.C. Gardner and J.M. Salmon. 2013 "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. 61 (15), 3703-3713.
10. Kubo, I., K.I. Fujita, A. Kubo, K.I. Nihei and C.S. Lunde. 2003 "Modes of antifungal action of (2E)-alkenals against Saccharomyces cerevisiae." J. Agric. Food Chem. 51, 3951−3957.

11. Jouanneau, S. 2011 "Survey of aroma compounds in Marlborough Sauvignon blanc wines. Regionality and small scale winemaking," University of Auckland.
12. Coetzee, C. and W.J. Du Toit. 2015 "Sauvignon blanc wine: Contribution of aging and oxygen on aromatic and non-aromatic compounds and sensory composition - a review." South African J. Enol. Vitic.
13. Kritzingera, E.C., M.A. Standerb and W.J. Du Toita. 2013 "Assessment of glutathione levels in model solution and grape ferments supplemented with glutathione-enriched inactive dry yeast preparations using a novel UPLC-MS/MS method." Food Additives & Contaminants: Part A, Vol. 30, No. 1, 80-92,
14. Rosales del Prado, D. 2015 "Effect of glutathione and inactive yeast additions to Sauvignon blanc at harvest on wine aroma." University of Auckland.

15. Herbst-Johnstone, M., l. Nicolau and P.A. Kilmartin. 2011 "Stability of varietal thiols in commercial sauvignon blanc wines. Am. J. Enol. Vitic. 62 (4), 495-502.
16. Olejar, K.J., B. Fedrizzi and P.A. Kilmartin. 2015 "Influence of harvesting technique and maceration process on aroma and phenolic attributes of Sauvignon blanc wine." Food Chem. 183, 181-189.
17. Maggu, M., R. Winz, P.A. Kilmartin, M.C.T. Trought and L. Nicolau. 2007 " Effect of Skin Contact and Pressure on the Composition of Sauvignon Blanc Must." J. Agric. Food Chem. 55 (25), 10281-10288.
18. Parish, K.J. M. Herbst-Johnstone, F. Bouda, S. Klaere and B. Fedrizzi. 2016 "Pre-fermentation fining effects on the aroma chemistry of Marlborough Sauvignon blanc press fractions." Food Chem. 208, 326-335.
19. Allen, T., M. Herbst-Johnstone, M. Girault, P. Butler, G. Logan, S. Jouanneau, L. Nicolau and P.A. Kilmartin. 2011 "Influence of grape-harvesting steps on varietal thiol aromas in Sauvignon blanc wines." J. Agric. Food Chem. 59 (19), 10641-10650.
20. Coetzee, C., K. Lisjak, L. Nicolau, P. Kilmartin and W.J. du Toit. 2013 "Oxygen and sulfur dioxide additions to Sauvignon blanc must: Effect on must and wine composition." Flavour Fragr. J. 28 (3), 155-167.
21. Moine, V., M.L. Murat, C. Arfeuillère and C. Thibon. 2011 "Collage des jus de presse blanc. Influence sur leurs teneurs en composes phénoliques, en glutathion et précurseur d'arômes." Rev. des œnologues 138, 45-47.
22. Lee, S.A., f.E. Rick, J. Dobson, M. Reeves, H. Clark, M. Thomson and R.C. Gardner. 2008 "Grape juice is the major influence on volatile thiol aromas in Sauvignon blanc." Aust. N.Z. Grapegrower & Winemaker 533 (6), 78-86.
23. Anfang, N., M. Brajkovich and M.R. Goddard. 2009 "Co-fermentation with Pichia kluyveri increases varietal thiol concentrations in sauvignon blanc." Aust. J. Grape Wine Res. 15 (1), 1-8.
24. Masneuf-Pomarède, I., C. Mansour, M. Murat, T. Tominaga and D. Dubourdieu. 2006 "Influence of fermentation temperature on volatile thiols concentrations in Sauvignon blanc wines." Int. J. Food Microbiol. 108 (3), 385-390.
23. Makhotkina, O., B. Pineau and P.A. Kilmartin. 2012 "Effect of storage temperature on the chemical composition and sensory profile of Sauvignon Blanc wines." Aust. J. Grape Wine Res. 18 (1), 91-99.
26. Coetzee, C., J. Brand, D. Jacobson and W.J. du Toit. 2016 "Sensory effect of acetaldehyde on the perception of 3-mercaptohexan-1-ol and 3-isobutyl-2-methoxypyrazine." Aust. J. Grape Wine Res.
27. Parish, K. 2012 "The significance of pressing conditions on key aroma volatiles in Marlborough Sauvginon blanc," University of Auckland.