1. Linda Bisson Department of Viticulture and Enology The Sulfur Taints

2. Why Are Sulfur Taints a Problem? Low thresholds of detection Low thresholds of detection Chemical reactivity Chemical reactivity Difficulty in removal Difficulty in removal Difficulty in masking Difficulty in masking

3. S-Volatiles: Negative Impacts on Flavor Hydrogen Sulfide: Rotten egg Hydrogen Sulfide: Rotten egg Post-fermentation S-taints Post-fermentation S-taints Sur lie Sulfide Taints Sur lie Sulfide Taints

4. Sources of Sulfur Compounds Sulfate reduction pathway Sulfate reduction pathway Degradation of sulfur containing amino acids Degradation of sulfur containing amino acids Inorganic sulfur Inorganic sulfur Non-enzymaticNon-enzymatic Requires reducing conditions established by yeastRequires reducing conditions established by yeast Degradation of S-containing pesticides/fungicides Degradation of S-containing pesticides/fungicides

5. HYDROGEN SULFIDE

6. Hydrogen Sulfide Formation: The Old Story Due to release of reduced sulfide from the enzyme complex sulfite reductase Due to release of reduced sulfide from the enzyme complex sulfite reductase Reduction of sulfate decoupled from amino acid synthesis Reduction of sulfate decoupled from amino acid synthesis Sulfate reduction regulated by nitrogen availability Sulfate reduction regulated by nitrogen availability Lack of nitrogenous reduced sulfur acceptors leads to excessive production of reduced sulfate and release as H 2 S Lack of nitrogenous reduced sulfur acceptors leads to excessive production of reduced sulfate and release as H 2 S See strain variation See strain variation

7. Hydrogen Sulfide Formation: The New Story Hydrogen sulfide plays an important population signaling role Hydrogen sulfide plays an important population signaling role –Inhibits respiration: coordinated population fermentation –Inhibits respiration: inactivation of bacteria and other yeasts Hydrogen sulfide formation is protective against stress Hydrogen sulfide formation is protective against stress Strain variation due to exposure to different environmental conditions in combination with the multiplicity of roles of H 2 S Strain variation due to exposure to different environmental conditions in combination with the multiplicity of roles of H 2 S

8. Sulfate Reduction Pathway SUL1, SUL2 SO 4 Adenylylsulfate Phosphoadenylylsulfate Sulfite Sulfide Cysteine Cystathionine Homocysteine Methionine MET3 MET14 MET16 (1,8,20,22) MET10 (1,5?,8,20) MET17/25/15 MET6CYS4CYS3

9. Hydrogen Sulfide Formation From degradation of S-containing amino acids From degradation of S-containing amino acids –When present in excess to harvest nitrogen –As Redox needs change: S-containing amino acids needed to maintain redox status of cells Also a stress response Also a stress response –S-compounds needed for 1C transfers and adaptation to ethanol and other stressors Strain variation Strain variation

10. Current Understanding of H 2 S Formation Nitrogen levels not well-correlated with H 2 S formation, but generally see increased H 2 S at lower nitrogen Nitrogen levels not well-correlated with H 2 S formation, but generally see increased H 2 S at lower nitrogen Under complex genetic control Under complex genetic control Tremendous strain variation in H 2 S production Tremendous strain variation in H 2 S production

11. Factors Impacting H 2 S Formation Level of total nitrogen Level of total nitrogen Level of methionine relative to total nitrogen Level of methionine relative to total nitrogen Fermentation rate Fermentation rate Use of SO 2 Use of SO 2 Vitamin deficiency Vitamin deficiency Presence of metal ions Presence of metal ions Inorganic sulfur in vineyard Inorganic sulfur in vineyard Use of pesticides/fungicides Use of pesticides/fungicides Strain genetic background Strain genetic background

12. Timing of Formation of H 2 S Brix Time H2SH2S

13. Timing of Formation of H 2 S Early (first 2-4 days): due to N imbalance? Or signaling? Late (end of fermentation): due to degradation of S-containing compounds Sur lie (post-fermentation aging): due to autolysis H 2 S produced early can be driven off by carbon dioxide during active phase of fermentation

14. Elimination of Hydrogen Sulfide Rely on volatility and fermentation gas or inert gas sparging to remove Rely on volatility and fermentation gas or inert gas sparging to remove –Need to make sure it is gone and not just converted to a non-volatile form Use of volatiles stripping technologies Use of volatiles stripping technologies Precipitation via copper Precipitation via copper –Emerging issue: health and environmental concerns about copper Use of fining agents Use of fining agents Use of strains not producing sulfides Use of strains not producing sulfides

15. Hydrogen Sulfide Screen of native isolates to define non- producers Screen of native isolates to define non- producers Screen of mutant library to define genes involved in sulfide production Screen of mutant library to define genes involved in sulfide production Genetic crosses to identify the genes altered in the native populations Genetic crosses to identify the genes altered in the native populations Assessment of allele swap in transferring the non-producer phenotype to producer strains Assessment of allele swap in transferring the non-producer phenotype to producer strains

16. MET10-932 Contains a change of amino acid 662 from threonine to lysine Contains a change of amino acid 662 from threonine to lysine Does not affect protein structure Does not affect protein structure Does not affect activity Does not affect activity Prevents sulfide release Prevents sulfide release Eliminates sulfide production in several strains including UCD522 Eliminates sulfide production in several strains including UCD522 Can be crossed into any commercial strain background Can be crossed into any commercial strain background

17. Sulfate Reduction Pathway SUL1, SUL2 SO 4 Adenylylsulfate Phosphoadenylylsulfate Sulfite Sulfide Cysteine Cystathionine Homocysteine Methionine MET3 MET14 MET16 (1,8,20,22) MET10 (1,5?,8,20) MET17/25/15 MET6CYS4CYS3

18. HIGHER SULFIDES

19. Higher Sulfides Emerge late in fermentation and during sur lie aging Emerge late in fermentation and during sur lie aging Release of compounds during entry into stationary phase by metabolically active yeast Release of compounds during entry into stationary phase by metabolically active yeast Come from degradation of sulfur containing compounds by viable cells Come from degradation of sulfur containing compounds by viable cells –Biological –Chemical  From reaction of reduced sulfur intermediates with other cellular metabolites?  Formed chemically due to reduced conditions? Degradation of cellular components: autolysis Degradation of cellular components: autolysis –Enzymatic –Chemical

20. Common Volatile Sulfur Compounds Methanethiol: CH 3 -SH Methanethiol: CH 3 -SH Ethanethiol: C 2 H 5 -SH Ethanethiol: C 2 H 5 -SH Dimethyl sulfide: CH 3 -S-CH 3 Dimethyl sulfide: CH 3 -S-CH 3 Dimethyl disulfide: CH 3 -S-S-CH 3 Dimethyl disulfide: CH 3 -S-S-CH 3 Dimethyl trisulfide: CH 3 -S-S-S-CH 3 Dimethyl trisulfide: CH 3 -S-S-S-CH 3 Diethyl sulfide: C 2 H 5 -S-C 2 H 5 Diethyl sulfide: C 2 H 5 -S-C 2 H 5 Diethyl disulfide: C 2 H 5 -S-S-C 2 H 5 Diethyl disulfide: C 2 H 5 -S-S-C 2 H 5

21. Common Volatile Sulfur Compound Ranges in Wine Hydrogen sulfide: Trace to 80 ug/L Hydrogen sulfide: Trace to 80 ug/L Methanethiol: Trace Methanethiol: Trace Ethanethiol: 1.9 -18.7 ug/L Ethanethiol: 1.9 -18.7 ug/L Dimethyl sulfide: 1.4 - 474 ug/L Dimethyl sulfide: 1.4 - 474 ug/L Dimethyl disulfide: Trace to 1.6 ug/L Dimethyl disulfide: Trace to 1.6 ug/L Dimethyl trisulfide: 0.09 - 0.25 ug/L Dimethyl trisulfide: 0.09 - 0.25 ug/L Diethyl sulfide: 4.1 - 31.8 ug/L Diethyl sulfide: 4.1 - 31.8 ug/L Diethyl disulfide: Trace - 85 ug/L Diethyl disulfide: Trace - 85 ug/L

22. Ehrlich Pathway S-Compounds Ehrlich Pathway: source of fusel oils Ehrlich Pathway: source of fusel oils Removal of N from amino acid compounds Removal of N from amino acid compounds Generates aldehyde Generates aldehyde Aldehyde reduced to alcohol Aldehyde reduced to alcohol In fermentation see high concentrations of methionine-derived “fusel” compounds: Methionol (100-6,300 ug/L) and Methional (generally trace, but reaction products are more aromatic) In fermentation see high concentrations of methionine-derived “fusel” compounds: Methionol (100-6,300 ug/L) and Methional (generally trace, but reaction products are more aromatic)

23. Sources of Higher Sulfides S-Containing Amino Acids S-Containing Amino Acids S-Containing Vitamins and Co-factors S-Containing Vitamins and Co-factors Glutathione (Cysteine-containing tripeptide involved in redox buffering) Glutathione (Cysteine-containing tripeptide involved in redox buffering)

24. Management of S-Taints Diagnosis of Taint Diagnosis of Taint Taint Prevention Taint Prevention Taint Mitigation Taint Mitigation

25. DIAGNOSIS OF SULFUR TAINTS

26. Correct Diagnosis of Fault Is Important Is it an S-containing compound? Is it an S-containing compound? When did taint first appear? When did taint first appear? What factors are associated with appearance of the taint? What factors are associated with appearance of the taint?

27. Is It a sulfur-containing compound? Be familiar with the characteristic off- odors of sulfur compounds Be familiar with the characteristic off- odors of sulfur compounds Other classes of off-odors can be reminiscent of S-compounds Other classes of off-odors can be reminiscent of S-compounds Thresholds of detection are so low compound may be difficult to detect chemically Thresholds of detection are so low compound may be difficult to detect chemically

28. When did taint first appear? Provides important clues as to the reason taint is occurring Provides important clues as to the reason taint is occurring If know why it is being made can take steps to prevent formation If know why it is being made can take steps to prevent formation

29. What factors are associated with appearance of the taint? Always found with a specific vineyard? Always found with a specific vineyard? Associated with unsound fruit? Associated with unsound fruit? Associated with specific processing? Associated with specific processing? –Inert gas blanketing –Type of vessel Associated with specific fermentation conditions? Associated with specific fermentation conditions?

30. TAINT PREVENTION

31. Preventing S-Taint Formation Vineyard Vineyard Wine chemistry Wine chemistry Yeast strain selection Yeast strain selection Fermentation management Fermentation management

32. Preventing S-Taint Formation Vineyard Vineyard –Judicious use of elemental sulfur –Eliminate reliance on S-containing pesticides –Address excesses of metal ions –Address vine stress: nutritional or otherwise Wine chemistry Wine chemistry Yeast strain selection Yeast strain selection Fermentation management Fermentation management

33. Preventing S-Taint Formation Vineyard Vineyard Wine chemistry Wine chemistry –Minimize use of ‘enabling’ practices  Temperature  Solids content  “Reductive” aging conditions  Role of the ‘lie’ in ‘sur lie’ Yeast strain selection Yeast strain selection Fermentation management Fermentation management

34. Preventing S-Taint Formation Vineyard Vineyard Wine chemistry Wine chemistry Yeast strain selection Yeast strain selection –Match strain to winemaking conditions –Meet the nutritional needs of the specific strain Fermentation management Fermentation management

35. Preventing S-Taint Formation Vineyard Vineyard Wine chemistry Wine chemistry Yeast strain selection Yeast strain selection Fermentation management Fermentation management –Provide adequate nutrition –Keep cells suspended –Mix to prevent reductive stratification of tanks –Remove from lees/yeast residue at first sign of trouble post-fermentation

36. TAINT MITIGATION

37. Elimination of Hydrogen Sulfide Rely on volatility and fermentation gas or inert gas sparging to remove Rely on volatility and fermentation gas or inert gas sparging to remove – Need to make sure it is gone and not just converted to a non-volatile form Use of volatiles stripping technologies Use of volatiles stripping technologies Precipitation via copper Precipitation via copper – Emerging issue: health and environmental concerns about copper Use of fining agents Use of fining agents Use of strains not producing sulfides Use of strains not producing sulfides

38. Treatments for Higher Sulfides Removal by reduction of disulfide bond with ascorbate and copper binding Removal by reduction of disulfide bond with ascorbate and copper binding Sulfide trapping by quinones Sulfide trapping by quinones O OH O OH O + RSH OH O + RSH OH SR SR Removal by charcoal fining Removal by charcoal fining

39. Conclusions Sulfur taints can be controlled Sulfur taints can be controlled Need to use correct strain Need to use correct strain Need to minimize use of compounds in vineyard and winemaking techniques that amplify S-compound formation Need to minimize use of compounds in vineyard and winemaking techniques that amplify S-compound formation Many factors leading to S-taint appearance are still not well understood Many factors leading to S-taint appearance are still not well understood

40. Sulfur Compound Flight #1: Taints produced late in fermentation Glass 1: Control wine Glass 1: Control wine Glass 2: Hydrogen Sulfide Glass 2: Hydrogen Sulfide Glass 3: Dimethyl sulfide (2 ug/L) Glass 3: Dimethyl sulfide (2 ug/L) Glass 4: Dimethyl sulfide (20 ug/L) Glass 4: Dimethyl sulfide (20 ug/L) Glass 5: Dimethyl sulfide (60 ug/L) Glass 5: Dimethyl sulfide (60 ug/L) Glass 6: Diethyl sulfide (20 ug/L) Glass 6: Diethyl sulfide (20 ug/L)

41. Sulfur Compound Flight #2 Spiked Compounds Glass 1: Control Wine (Cabernet Sauvignon) Glass 1: Control Wine (Cabernet Sauvignon) Glass 2: Diethyl disulfide (40 ug/L) Glass 2: Diethyl disulfide (40 ug/L) Glass 3: Dimethyl trisulfide (0.2 ug/L) Glass 3: Dimethyl trisulfide (0.2 ug/L) Glass 4: Ethanthiol (15 ug/L) Glass 4: Ethanthiol (15 ug/L) Glass 5: Methionol (500 ug/L) Glass 5: Methionol (500 ug/L) Glass 6: Methional (50 ug/L) Glass 6: Methional (50 ug/L)

42. Sulfur Compound Flight #2 Spiked Compounds G 1: Control Wine (Cabernet Sauvignon) G 1: Control Wine (Cabernet Sauvignon) G 2: Dimethyl sulfide: low concentrations enhance varietal character G 2: Dimethyl sulfide: low concentrations enhance varietal character G 3: Dimethyl sulfide: cabbage, cooked corn, asparagus, canned vegetable G 3: Dimethyl sulfide: cabbage, cooked corn, asparagus, canned vegetable G 4: Dimethyl trisulfide: meaty, fishy, clams, green, onion, garlic, cabbage G 4: Dimethyl trisulfide: meaty, fishy, clams, green, onion, garlic, cabbage G 5: Diethyl sulfide: garlic, onion G 5: Diethyl sulfide: garlic, onion G 6: Diethyl disulfide: overripe onion, greasy, garlic, burnt rubber, manure G 6: Diethyl disulfide: overripe onion, greasy, garlic, burnt rubber, manure