Defining sulfur spray recommendations based upon the re-emergence of hydrogen sulfide off-aromas in wine post-bottling
Elemental sulfur (S0) is an environmentally and economically sustainable control against powdery mildew, a disease common to humid grape-growing regions; however, S0 residues are shown to increase hydrogen sulfide (H2S) production during fermentation. My recent work has shown that H2S formation can continue under post-bottling, reductive conditions, even if all free H2S is removed prior to bottling. We have hypothesized that this is due to the formation of a water-soluble precursor that can later be reduced to create the characteristic “rotten egg” aroma of H2S. The goal of this research is to elucidate the mechanism for post-bottling H2S production, in order that we might define more appropriate S0 spraying limitations, develop methods to predict post-bottling H2S formation, as well as develop methods for prophylactic treatment. Grape juice spiked with high levels of S0 was fermented, racked, sparged, and bottled under reductive conditions. The resulting wine was aged reductively for 3 months and tested for both free and TCEP-releasable H2S, according to an assay developed by our group. Spiked samples were fractionated by HPLC and the fractions analyzed for potential precursor compounds.
The objectives as described in the original plan of work are:
- Define new limits for use of S0 fungicides on the basis of H2S formed during storage rather than H2S formed during fermentation.
- Identify the wine soluble precursor compound responsible for H2S formed during storage.
Grape juice was fermented in the presence of S0 as described in the original proposal. S0 was spiked at 0 mg/L, 20 mg/L, and 100 mg/L into grape juice samples, in triplicate. Yeast nutrient in the form of diammonium phosphate was added prophylactically to all samples. Wines were fermented to dryness, racked off of the yeast lees, and the H2S removed by means of aeration. Wines were tested via the previously described method to quantify the free H2S – which was verified to be undetectable in all samples – as well as “latent” H2S, according to our previously described assay using the reducing compound tris(2-carboxyethyl)phosphine (TCEP). Wines were bottled reductively and tested periodically for free and “latent” H2S. The free H2S produced post-bottling showed a linear correlation to the level of S0 initially spiked into the wine, which indicates that post-bottling H2S is predictable given the pre-fermentation quantification of S0 residues. Variability in weather and pre-fermentation practices have been shown to cause a wide variation in S0 persistence (2), and it is recommended that S0 residues be quantified prior to fermentation.
Initial attempts to isolate the unknown water-soluble precursor compound were performed using liquid-liquid extraction techniques. Samples of wine that had been spiked with the highest concentration of S0, and therefore contained the highest concentration of “latent” H2S at the beginning of storage, were used. Liquid-liquid extraction was performed using a variety of solvents including ethyl acetate, dichloromethane, hexane, and chloroform. Following the extraction, both the aqueous and organic fraction were analyzed according to the TCEP-releasable H2S assay. In all cases, the organic fraction showed no detectable H2S, but the aqueous portion did show detectable H2S. This indicated that the unknown H2S precursor compound always remained in the aqueous portion, and is probably a polar compound. In another attempt to isolate the unknown compound, cation exchange solid phase extraction was performed. The eluant, the wash, and the SPE loaded sample were all tested using the TCEP-releasable H2S assay in order to determine which portion contained the unknown compound. The only portion that gave a signal for “latent” H2S in this case was the SPE-loaded sample.
In another attempt to elucidate the identity of the unknown compound, it was hypothesized that the precursor to H2S was a glutathione polysulfide. For example, previous work has shown that the enzymatic reduction of glutathione trisulfide produces H2S (3). Although, this has not yet been shown non-enzymatically, it is possible that a similar reaction could be occurring in the reductively aged wine samples. To test this, glutathione trisulfide was synthesized according to previously described methods (3). Oxidized (glutathione disulfide, GSSG) was reacted with S0 at pH10 and acidified to stabilize the glutathione trisulfide product compound. The excess elemental sulfur was removed. HPLC was performed on the glutathione trisulfide reaction mixture, as well as the S0-spiked wine sample. The spectra were overlaid for comparison. Fractions were collected from both the trisulfide synthesis and the spiked wine sample. Fractions will be analyzed via direct injection electrospray mass spectrometry to confirm the presence or absence of glutathione trisulfide in the wine sample.
Conferences and Presentations:
- 2014 CRAVE (Cornell Recent Advances in Viticulture and Enology) Conference
- The Northern Grapes Project Webinar Series
The CRAVE conference is an annual event that takes place at Cornell University, which includes faculty, extension staff, and viticulture and enology students. Departments include horticulture, PPPMB, Entomology, Food Science, and Applied Economics; extension programs include 5 different regions in New York. A 15 minute presentation regarding the ongoing work of this project was given.
A 45-60 minute Webinar was given as part of the Northern Grapes Project Webinar Series on December 16, 2014. The title of the presentation was “Stuck on You – Sulfur Spray Residues in the Vineyard and Winery”. Included was a presentation of prior related work on the persistence of sulfur spray residues in the vineyard and the winery, as well as recent results from this project having to do with the post-bottling formation of H2S.
Work has gone according to plan. The hypothesis made in the original proposal, that the unknown precursor is a glutathione polysulfide, is currently being tested.
Impacts and Contributions/Outcomes
Our recommendations to growers and winemakers based upon our current results are to measure the sulfur residues on grapes prior to processing. Several variables other than spraying frequency and intervals contribute to the persistence of S0 residues on grapes, including weather and pre-fermentation practices (2). Previous work by our group involved the development of an inexpensive, simple assay for the quantification of sulfur residues on grapes (1). Knowing that excess S0 residues are present prior to fermentation can allow growers and winemakers to adjust their plans accordingly. For example, growers can delay harvest to minimize S0 residues, and winemakers can clarify juice to rid it of excess S0 residues. These were the main recommendations recently presented during the Northern Grapes Project Webinar Series (see above).
- M. T. Kwasniewski, W. F. Wilcox, G. L. Sacks. Am. J. Enol. Vitic. 63(3), 462A-463A 2012.
- Kwasniewski, Misha T. Diss. Cornell University, 2013.
- Moutiez, M.; Aumercier, M.; Teissier, E.; Parmentier, B.; Tartar, A.; Sergheraert, C. Biochemical and Biophysical Research Communications 1994, 202 (3), 1380-1386.
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