Sulfur Compounds in Wine

1. Sulfur Compounds in Wine Linda Bisson Department of Viticulture and Enology

2. Introduction to S-Containing Faults

3. Why Are Sulfur Compounds a Problem? • Low thresholds of detection • Negatively-associated aromas • Chemical reactivity • Difficulty in removal • Difficulty in masking

4. The Classic Sulfur Fault Descriptors • Rotten egg • Fecal • Rubber/Plastic tubing • Burnt match • Burnt molasses • Burnt rubber • Rotten vegetable: cauliflower, cabbage, potato, • asparagus, corn • Onion/Garlic • Clam/Tide pool • Butane/Fuel/Chemical

5. The Sulfur Taints • Hydrogen sulfide • Higher sulfides • Dimethyl (Diethyl) sulfide • Dimethyl disulfide • Mercaptans • Methyl (Ethyl) mercaptan • Thioesters • Methyl (ethyl) thioacetate • Other S-amino acid metabolites • Thioethers • Cyclic and heterocyclic compounds

6. Sources of Sulfur Compounds • Non-biological • Elemental sulfur • S-containing pesticides • Biological • Sulfate/Sulfite reduction and reduced sulfide reactions • S-containing amino acid metabolism • S-containing vitamins and cofactors degradation • Glutathione metabolism and degradation • S-containing pesticides degradation • Elemental sulfur

7. Timing of Sulfur Fault Formation • Primary Fermentation Early: Hydrogen Sulfide • Primary Fermentation Late: Hydrogen Sulfide • Post Fermentation: Hydrogen Sulfide or Sur Lie Faults • Bottling: S-fault development

8. Hypotheses to Explain S-Taint Formation • Correlated with H2S formation during the primary fermentation • Correlated with late H2S formation (peak 2) but not with H2S formation during primary fermentation • Associated with S-containing amino acid levels during primary fermentation • Due to degradation of S-containing metabolites during yeast lees aging, but not related to levels of these compounds present in the initial juice • Yeast strain most important • Juice composition most important

9. Problems with Previous Studies • Lack of control of all variables • Invalid comparisons (too many variables) • Confounding factors not considered to be important • Differences in strains and conditions used • Driving reactions by having an excess of precursors, beyond anything found in juices or wines

10. Hydrogen Sulfide

11. Why is H2S formed? • Off-shoot of metabolism • Reductive environment • Signaling molecule

12. Hydrogen Sulfide Formation: Off-Shoot of Metabolism • 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 • Also a stress response • Strain variation

13. Stress Response: Reduction Pathway Remains Operational • Need cysteine for glutathione (tripeptide cytoplasmic redox (electron) buffer • Need methionine for S-adenosylmethionine and one carbon transfers needed for ethanol tolerance

14. Sulfate Reduction Pathway SO4 SUL1, SUL2 SO4 MET3 Adenylylsulfate MET14 H2S Phosphoadenylylsulfate MET16 (1,8,20,22) Sulfite MET10 (1,5?,8,20) Sulfide MET17/25/15 Cysteine Cystathionine Homocysteine Methionine CYS3 CYS4 MET6

15. Hydrogen Sulfide Formation: Reductive Environment • Biological energy is obtained from recapture of light (carbon bond) energy, from proton movements and from electron movements • Cell is dealing with an excess of electrons that exceeds buffering capacity • Many electrons can be used to reduce a single sulfate molecule restoring the proper balance of cytoplasmic electrons

16. Hydrogen Sulfide Formation: Reductive Environment • Tank dimensions leading to stratification of electron gradients • Settling of yeast cells • Chemical composition of juice • Oxygen level and content of juice

17. Hydrogen Sulfide Formation: Signaling Molecule • Hydrogen sulfide coordinates population metabolic activities: shuts down respiration in favor of fermentation, coordinating population of cells in fermentation • Hydrogen sulfide inhibits respiration of a variety of organisms: allows more rapid domination of fermentation • Explains selective pressure for high sulfide producers in the wild

18. Current Understanding of H2S Formation • Nitrogen levels not well-correlated with H2S formation, but generally see increased H2S at lower nitrogen • Tremendous strain variation in H2S production • Can get H2S with high nitrogen • Get more H2S with higher solids content • Get more H2S with unsound fruit

19. 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

20. Timing of Formation of H2S H2S Brix Time

21. Timing of Formation of H2S • Early (first 2-4 days): due to N/vitamin shortage, electron imbalance, signaling • Late (end of fermentation): due to degradation of S-containing compounds • Sur lie (post-fermentation aging): due to autolysis • In Bottle: screw cap closures: return from an altered chemical form

22. Higher SulfideS

23. 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 amino acids • Biological • Chemical • From reaction of reduced sulfur intermediates with other cellular metabolites? • Formed chemically due to reduced conditions? • Degradation of cellular components: autolysis

24. 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

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

26. Defining Metabolic Behaviors Resulting in Taint Formation • S-amino acid catabolism • Vitamin/Co-factor interactions and metabolism • Glutathione turnover and reactions • Metabolic roles of sulfate reduction

27. Defining Metabolic Behaviors Resulting in Taint Formation • S-amino acid catabolism • Degradation of methionine and cysteine: methional and methionol • Chemical reaction products of methionine and cysteine: stress resistance • Influence of wine composition and chemistry on yeast behavior • Vitamin/Co-factor interactions and metabolism • Glutathione turnover and reactions • Metabolic roles of sulfate reduction

28. Defining Metabolic Behaviors Resulting in Taint Formation • S-amino acid catabolism • Vitamin/Co-factor interactions and metabolism • Role of thiamin • Role of S-adenosylmethionine • Glutathione turnover and reactions • Metabolic roles of sulfate reduction

29. Defining Metabolic Behaviors Resulting in Taint Formation • S-amino acid catabolism • Vitamin/Co-factor interactions and metabolism • Glutathione turnover and reactions • Role in stress response: prevention of oxidative damage • Impact of nitrogen level on metabolism • Biological turnover of ‘reacted’ glutathione • Metabolic roles of sulfate reduction

30. Defining Metabolic Behaviors Resulting in Taint Formation • S-amino acid catabolism • Vitamin/Co-factor interactions and metabolism • Glutathione turnover and reactions • Metabolic roles of sulfate reduction • Stress response: • Prevention of oxidative damage • Role in ethanol tolerance • Environmental/metabolic detoxification • Banking on reactivity to inactivate a toxic substance • Metabolic demands

31. Understanding the Interface between Metabolite Production and Wine Chemistry and Composition • What environmental conditions impact S-compound metabolic activities? • Separating a biological response from a chemical one • Control the metabolites • Control the chemistry

32. Sulfur Compound Flight #1Spiked Compounds • Glass 1: Control Wine (Cabernet Sauvignon) • Glass 2: Hydrogen sulfide H2S • Glass 3: Dimethyl sulfide CH3-S-CH3 • Glass 4: Dimethyl trisulfide: CH3-S-S-CH3 • Glass 5: Diethyl sulfide: C2H5-S-C2H5 • Glass 6: Diethyl disulfide: C2H5-S-S-C2H5

33. Sulfur Compound Flight #1Spiked Compounds • G 1: Control Wine (Cabernet Sauvignon) • G2: Hydrogen sulfide: rotten egg • 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

34. Sulfur Compound Flight #2:Taints produced late in fermentation • Glass 1: Control Wine (Cabernet Sauvignon) • Glass 2: Ethanethiol • Glass 3: Mercapto -2- methyl propanol (Methionol) • Glass 4: Methyl thiopropionaldehyde (Methional) • Glass 5: Mercapto-3-methyl butanol • Glass 6: BM45 French Colombard

35. Sulfur Compound Flight #2Spiked Compounds • G 1: Control Wine (Cabernet Sauvignon) • G 2: Ethanethiol: onion, rubber, natural gas • G 3: Methionol: cauliflower, cabbage, potato • G 4: Methional: musty, potato, onion, meaty • G 5: Mercapto-3-methyl butanol: meaty • G 6: French Colombard: reduced

36. BM 45: • Isolated in Montalcino • Produces high polyphenol reactive polysaccharides = mouth feel • Has high nitrogen requirements and can produce H2S • Aroma characteristics: fruit jam, rose, cherry, spice, anise, cedar and earthy