The Sulfur Taints

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

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

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

4. Sources of Sulfur Compounds • Sulfate reduction pathway • Degradation of sulfur containing amino acids • Inorganic sulfur • Non-enzymatic • Requires reducing conditions established by yeast • 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 • Reduction of sulfate decoupled from amino acid synthesis • Sulfate reduction regulated by nitrogen availability • Lack of nitrogenous reduced sulfur acceptors leads to excessive production of reduced sulfate and release as H2S • See strain variation

7. Hydrogen Sulfide Formation:The New Story • 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 • Strain variation due to exposure to different environmental conditions in combination with the multiplicity of roles of H2S

8. Sulfate Reduction Pathway SO4 SUL1, SUL2 SO4 MET3 Adenylylsulfate MET14 Phosphoadenylylsulfate MET16(1,8,20,22) Sulfite MET10 (1,5?,8,20) Sulfide MET17/25/15 Cysteine Cystathionine Homocysteine Methionine CYS3 CYS4 MET6

9. Hydrogen Sulfide Formation • 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 • S-compounds needed for 1C transfers and adaptation to ethanol and other stressors • Strain variation

10. Current Understanding of H2S Formation • Nitrogen levels not well-correlated with H2S formation, but generally see increased H2S at lower nitrogen • Under complex genetic control • Tremendous strain variation in H2S production

11. Factors Impacting H2S Formation • Level of total nitrogen • Level of methionine relative to total nitrogen • Fermentation rate • Use of SO2 • Vitamin deficiency • Presence of metal ions • Inorganic sulfur in vineyard • Use of pesticides/fungicides • Strain genetic background

12. Timing of Formation of H2S H2S Brix Time

13. Timing of Formation of H2S • 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 • H2S 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 • Need to make sure it is gone and not just converted to a non-volatile form • Use of volatiles stripping technologies • Precipitation via copper • Emerging issue: health and environmental concerns about copper • Use of fining agents • Use of strains not producing sulfides

15. Hydrogen Sulfide • Screen of native isolates to define non-producers • Screen of mutant library to define genes involved in sulfide production • Genetic crosses to identify the genes altered in the native populations • 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 • Does not affect protein structure • Does not affect activity • Prevents sulfide release • Eliminates sulfide production in several strains including UCD522 • Can be crossed into any commercial strain background

17. Sulfate Reduction Pathway SO4 SUL1, SUL2 SO4 MET3 Adenylylsulfate MET14 Phosphoadenylylsulfate MET16(1,8,20,22) Sulfite MET10 (1,5?,8,20) Sulfide MET17/25/15 Cysteine Cystathionine Homocysteine Methionine CYS3 CYS4 MET6

18. Higher Sulfides

19. Higher Sulfides • Emerge late in fermentation and during sur lie aging • Release of compounds during entry into stationary phase by metabolically active yeast • 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 • Enzymatic • Chemical

20. Common Volatile Sulfur Compounds • Methanethiol: CH3-SH • Ethanethiol: C2H5-SH • Dimethyl sulfide: CH3-S-CH3 • Dimethyl disulfide: CH3-S-S-CH3 • Dimethyl trisulfide: CH3-S-S-S-CH3 • Diethyl sulfide: C2H5-S-C2H5 • Diethyl disulfide: C2H5-S-S-C2H5

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

22. Ehrlich Pathway S-Compounds • Ehrlich Pathway: source of fusel oils • Removal of N from amino acid compounds • Generates aldehyde • 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)

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

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

25. DIAGNOSIS OF SULFUR TAINTS

26. Correct Diagnosis of Fault Is Important • Is it an S-containing compound? • When did taint first appear? • 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 • Other classes of off-odors can be reminiscent of S-compounds • 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 • 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? • Associated with unsound fruit? • Associated with specific processing? • Inert gas blanketing • Type of vessel • Associated with specific fermentation conditions?

30. Taint Prevention

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

32. Preventing S-Taint Formation • 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 • Yeast strain selection • Fermentation management

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

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

35. Preventing S-Taint Formation • Vineyard • Wine chemistry • Yeast strain selection • 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 • Need to make sure it is gone and not just converted to a non-volatile form • Use of volatiles stripping technologies • Precipitation via copper • Emerging issue: health and environmental concerns about copper • Use of fining agents • Use of strains not producing sulfides

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

39. Conclusions • Sulfur taints can be controlled • Need to use correct strain • 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

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

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

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