1. Linda F. Bisson Department of Viticulture and Enology University of California, Davis Impact of Vineyard Residues on Wine Quality

2. Types of Vineyard Residues Fungicide treatments Fungicide treatments Pesticides Pesticides Foliar sprays Foliar sprays

3. Possible Impacts of Vineyard Residues Primary Impacts: Primary Impacts: –Inhibition of yeast fermentation –Inhibition of malolactic conversion –Off-character formation  Direct  Due to yeast metabolism  Due to bacterial metabolism Secondary Impacts: Secondary Impacts: –Alteration of berry microbiota

4. Common Impacts on Wine Aroma Direct detection of residue: rare Direct detection of residue: rare S 0 or S-containing compounds: S-volatile formation S 0 or S-containing compounds: S-volatile formation Antifungal agents: can impact fermentation aroma in addition to fermentation progression Antifungal agents: can impact fermentation aroma in addition to fermentation progression Antibacterial agents: can impact fermentation aroma in addition to MLF progression Antibacterial agents: can impact fermentation aroma in addition to MLF progression Sometimes the impact is a depression of aroma Sometimes the impact is a depression of aroma

5. Why Is There an Impact on Aroma? Microbes must detoxify component Microbes must detoxify component Component may simply be metabolizable by microbial enzymes Component may simply be metabolizable by microbial enzymes May inhibit a primary pathway forcing a secondary pathway to be used instead May inhibit a primary pathway forcing a secondary pathway to be used instead May simply be altered chemically due to the reductive conditions established by yeast metabolism May simply be altered chemically due to the reductive conditions established by yeast metabolism

6. Is There a Problem with Vineyard Residues? Generally all products should have been tested thoroughly for impacts on microbes and wine aroma Generally all products should have been tested thoroughly for impacts on microbes and wine aroma Timing of use can prevent any residuals from entering the winery Timing of use can prevent any residuals from entering the winery More growers are adopting organic and sustainable practices More growers are adopting organic and sustainable practices

7. Elemental Sulfur Used to control mildew Used to control mildew No resistances have been developed No resistances have been developed Is a natural fungicide (not synthetic) Is a natural fungicide (not synthetic) Residues can pose a problem: Residues can pose a problem: –High levels inhibitory to yeast –Low levels lead to H 2 S formation

8. Elemental Sulfur Residues Depends upon how S 0 was applied Depends upon how S 0 was applied Depends upon timing of application versus harvest date Depends upon timing of application versus harvest date References: References: –Thomas, CS, et al (1993) AJEV 44:205 (residue levels with different S 0 formulations) –Thomas, CA et al (1993) AJEV 44:211 (impact of S 0 spikes on H 2 S formation)

9. Hydrogen Sulfide (H 2 S) in Winemaking Hydrogen sulfide production by Saccharomyces occurs as a part of yeast metabolism during fermentation. Hydrogen sulfide production by Saccharomyces occurs as a part of yeast metabolism during fermentation. Rotten egg character Rotten egg character Range of production is 0 – 300 μg/L. Range of production is 0 – 300 μg/L. Threshold of human odor detection is 0.1- 1 μg/L in wine. Threshold of human odor detection is 0.1- 1 μg/L in wine. Varies by genetic background of yeast Varies by genetic background of yeast

10. H 2 S is Formed in Three Ways during Wine Fermentation Sulfur MET/CYS Sulfate Sulfur MET/CYS Sulfate Chemical conversion Sulfate reduction pathway in yeast H 2 S Sulfide H 2 S Sulfide H 2 S in Wine H 2 S in Wine

11. Elemental Sulfur and H 2 S Several studies suggest H 2 S increases with increasing S 0 Several studies suggest H 2 S increases with increasing S 0 –Not strain dependent –Due to reductive environmental conditions Strain effects dominate that of S 0 addition Strain effects dominate that of S 0 addition Beliefs: Beliefs: –S 0 residues converted to H 2 S during fermentation –Nitrogen addition does not prevent H 2 S formation from S 0

12. Problems with Research to Date Yeast strains make H 2 S from sulfate reduction and other sources: high background levels Yeast strains make H 2 S from sulfate reduction and other sources: high background levels Yeast strain sulfide levels often exceed those of S 0 Yeast strain sulfide levels often exceed those of S 0 Not all of the S 0 can be accounted for as H 2 S Not all of the S 0 can be accounted for as H 2 S

13. Research Approach Take advantage of MET10-932 (H 2 S-less) yeast Take advantage of MET10-932 (H 2 S-less) yeast MET10-932 allele: MET10-932 allele: –Yeast do not make H 2 S –Can change just this one gene and compare impact of increasing S 0 levels –Can assess wine for the appearance of other off-odors or traits that are associated with S 0

14. Goals Evaluate the impact of elemental sulfur addition on H 2 S production by using strains with differing abilities to reduce sulfate to sulfide Evaluate the impact of elemental sulfur addition on H 2 S production by using strains with differing abilities to reduce sulfate to sulfide Can yeasts still produce H 2 S from elemental sulfur when sulfite reductase is not functional? Can yeasts still produce H 2 S from elemental sulfur when sulfite reductase is not functional? How much impact ? How much impact ?

15. Genotypes and Phenotypes of Modified Yeast Strains H 2 S Production H 2 S Production UCD522 (wild type) UCD522 (wild type) UCD932 (wild type) UCD932 (wild type) S288C (wild type) S288C (wild type) 522Δmet10 522Δmet10 522MET10 932 522MET10 932 522MET10 522 522MET10 522 522MET10 S288C 522MET10 S288C --++--++ +-++-+

16. Growth on Selective Medium YPD YNB BiGGY media UCD522 UCD932 522 MET10 522 522 Δmet10 522 MET10 932 522 MET10 S288C H 2 S Producers / H 2 S Producers

17. Fermentation Media Synthetic grape juice (Minimal Must Media) Synthetic grape juice (Minimal Must Media) 433, 208, 123 mg/L of nitrogen 433, 208, 123 mg/L of nitrogen 20 mg/L of methionine 20 mg/L of methionine Chardonnay juice (’08 Woodbridge, filtered, 23.5 Brix) Chardonnay juice (’08 Woodbridge, filtered, 23.5 Brix) 450.3 mg/L of nitrogen 450.3 mg/L of nitrogen 12.02 mg/L of methionine 12.02 mg/L of methionine Directly spiked with elemental sulfur (Thiosperse) Directly spiked with elemental sulfur (Thiosperse) 0, 1.7, 3.4, 6.8 mg/L of sulfur 0, 1.7, 3.4, 6.8 mg/L of sulfur

18. Fermentation Conditions 150mL media / 250mL flask 150mL media / 250mL flask Silicon stopper Silicon stopper FIGASA ® H 2 S detecting tube FIGASA ® H 2 S detecting tube 25°C, 120 rpm 25°C, 120 rpm Until reach dryness (<0.25% sugar) Until reach dryness (<0.25% sugar) Triplicates Triplicates 250

19. H 2 S Measurement on Tube Blackened length in mm on scaled lead acetate tube Blackened length in mm on scaled lead acetate tube 1 mm equals to 1.2 µg of H 2 S 1 mm equals to 1.2 µg of H 2 S

20. H 2 S Production in 433 mg/L N

21. H 2 S Production in 208 mg/L N

22. H 2 S Production in 123 mg/L N

23. H 2 S Production in Chardonnay

24. Conclusions From This Study High S 0 does lead to increased H 2 S High S 0 does lead to increased H 2 S Production of H 2 S both de novo and from S 0 depended upon the allele at MET10 Production of H 2 S both de novo and from S 0 depended upon the allele at MET10 Production of H 2 S both de novo and from S 0 depended upon nitrogen level Production of H 2 S both de novo and from S 0 depended upon nitrogen level –Moderate N gave highest levels of H 2 S with and without S 0 –Impact of S 0 more pronounced at high N levels

25. H 2 S Production in Red Wine with S 0 Spikes Assess impact of S 0 residues in presence and absence of ability to make H 2 S Assess impact of S 0 residues in presence and absence of ability to make H 2 S Evaluate changes in wine during aging Evaluate changes in wine during aging

26. The Experiment for Today’s Wines 2010 UC Davis Grenache noir The Students: Constantin Heitkamp Shaunt Oungoulian Alex Stauffer Aaron Whitlatch

27. Experimental Design Crush / Destem50 Gal TJsAdditions/Inoc Racked to Barrel Press and Return to TJ Monitor Brix/Temp/Odor Pumpover 10 min per 12 hr Cold StorageBottling Sensory Evaluation

28. Elemental Sulfur Spiking Elemental Sulfur was added to juices prior to inoculation and mixed in Elemental Sulfur was added to juices prior to inoculation and mixed in Sulfur residue levels were: O, 2, 4 mg/L Thiolux Sulfur residue levels were: O, 2, 4 mg/L Thiolux Two yeast strains: Montrachet (UCD522) and P1Y0-2 (Phyterra H 2 S-less yeast) Two yeast strains: Montrachet (UCD522) and P1Y0-2 (Phyterra H 2 S-less yeast)

29. Montrachet Fermentation

30. P1Y0-2 Fermentation

31. Wine Sensory Analysis Hedonic Ranking - December 2010 P0 > M0, M2, M4 P2, P4 > M0, M2 M4 > M0, M2 All > M2 Higher elemental sulfur did not lead to a decrease in aroma preference Indicates a clear preference to Phyterra Yeast During fermentation M2 and M4 had the greatest sulfur aroma, but M4 did not score as poorly as M2 when assessing the wine

32. Difference Testing Triangle Test P0 > M0 M0 > M2 M0 > M4 This reaffirms the hedonic test ranking that Phyterra yeast is different from Montrachet The triangle test supports the hypothesis that increased elemental sulfur added to the fermentation leads to less preference in the wine (assumed to be sulfur aroma defect)

33. Conclusions Phyterra yeast preferred immediately post fermentation: wines described as fresher and fruitier Phyterra yeast preferred immediately post fermentation: wines described as fresher and fruitier M2 fermentation was considered to be the least preferred and most defective in aroma M2 fermentation was considered to be the least preferred and most defective in aroma Impact of aging? Impact of aging?

34. Acknowledgements American Vineyard Foundation American Vineyard Foundation California Competitive Grant Program for Research in Viticulture and Enology California Competitive Grant Program for Research in Viticulture and Enology Maynard A. Amerine Endowment Maynard A. Amerine Endowment

35. Sulfur Residues Flight Glass 1: Montrachet, 0 ug/L S 0 Glass 1: Montrachet, 0 ug/L S 0 Glass 2: Montrachet, 2 mg/L S 0 Glass 2: Montrachet, 2 mg/L S 0 Glass 3: Montrachet, 4 mg/L S 0 Glass 3: Montrachet, 4 mg/L S 0 Glass 4: P1Y0-2, 0 ug/L S 0 Glass 4: P1Y0-2, 0 ug/L S 0 Glass 5: P1Y0-2, 2 mg/L S 0 Glass 5: P1Y0-2, 2 mg/L S 0 Glass 6: P1Y0-2, 4 mg/L S 0 Glass 6: P1Y0-2, 4 mg/L S 0