Recognizing Wine Off-Characters of Microbial Origin

1. Recognizing Wine Off-Characters of Microbial Origin Wine Flavor 101 January 16, 2014 Linda F. Bisson Department of Viticulture and Enology

2. Wine Off-Characters of Microbial Origin • Off-colors • Off-flavors • Hazes/cloudiness • Sediment/precipitates

3. Source of Microbial Off-Characters • Vineyard flora • Winery flora • Fermentation microbes • Saccharomyces • Lactic acid bacteria • Spoilage microbes • Acetic acid bacteria • Lactic acid bacteria • Flor yeasts • Brettanomyces • Non-Brett Spoilage Yeasts

4. Off-Characters • Same off-character may come from different sources (acetaldehyde) • Some off-characters arise only in specific chemical/microbial environments • Compound(s) responsible for some taints are unknown • Best course of action: not getting them in the first place!

5. Microbial Off-Characters • Pre-fermentation • During fermentation arising from normal flora • Post-fermentation

6. Pre-Fermentation Off-Characters • Derive from the metabolic activities of non-Saccharomyces yeasts and bacteria • Increased impact with rot • Increased impact with hang time • Juice and must processing can encourage pre-fermentation character formation

7. Pre-Fermentation Processing • Lower temperatures: favor non-Saccharomyces yeasts • Cold soak/maceration (reds) • Cold settling (whites) • See bloom of Hanseniaspora/Kloeckera • Warm temperatures: favor bacteria • Cap management strategies • Refrigeration capacity

8. Pre-Fermentation Processing • Oxygen exposure: favors both non-Saccharomyces yeasts and bacteria • All organisms benefit from presence of oxygen • Obligate aerobes not inhibited • pH adjustment: high pH values (over 3.8) favor growth of bacteria over yeasts • Low, no or late sulfite additions: favor both non-Saccharomyces yeasts and bacteria • Favors growth of both wild lactics and acetic acid bacteria • Favors growth of non-Saccharomyces yeasts

9. Pre-Fermentation Spoilage Characters • Ethyl acetate • Acetic acid • Amplification of unripe or green characters • Sour taint

10. Off-Characters Produced by Yeast During Fermentation • Common • Sulfur volatiles • Esters ___________________________________ • Rare • Higher alcohols • Acetaldehyde • Higher aldehydes • Volatile acids

11. Fermentation Off-Characters Influenced by yeast strain • Different strains vary ten-fold or more in compound production within dynamic range of odor detection • Strains dominate fermentation with differing efficiencies

12. Fermentation Off-Characters Impacted by growth conditions • Volatilization of compounds • Temperature • Head space • Nutrient availability • Nitrogen • Micronutrients • Oxygen • Microbial competition • Sulfite use • Inoculation practices

13. Fermentation Off-Characters Affected by juice composition • Availability of precursors • pH • Presence of stressors • High sugar/high ethanol • Previous microbial history

14. The Common Yeast Off-Odors • Sulfur volatiles • Fermentation esters

15. Yeast Sulfur Volatiles • Hydrogen sulfide • Complex sulfides

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

17. 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

18. Hydrogen Sulfide

19. Hydrogen Sulfide Issues in Wine • Confers a distinctive rotten egg character • Compound can be hidden in an oxidized form and return as redox conditions of the wine change • Character interacts with other characters to give a more complex off-character: fecal, burnt rubber • Wine aroma is attenuated at levels below recognition threshold

20. Hydrogen Sulfide Formation: • Due to release of reduced sulfide from the enzyme complex sulfite reductase or 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 from catabolism of sulfur-containing amino acids if present in excess

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

22. Hydrogen Sulfide Formation:Explaining Yeast Variation • Hydrogen sulfide plays an important population signaling role • Inhibits respiration: coordinated population fermentation • Inhibits respiration: inactivation of bacteria and other yeasts • Hydrogen sulfide inhibits aerobic bacteria • Hydrogen sulfide formation is protective against oxidative stress • Strain variation due to exposure to different environmental conditions in combination with the multiplicity of roles of H2S

23. Timing of Formation of H2S H2S Brix Time

24. 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 • Surlie(post-fermentation aging): due to autolysis

25. 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

26. Complex Sulfides

27. 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

28. The Classic Yeast Sulfur Fault Descriptors • 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

29. The Complex Sulfur Taints • 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

30. 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

31. 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

32. Sulfur CompoundAroma Descriptors • Dimethyl sulfide: cabbage, cooked corn, asparagus, canned bean/vegetable • Dimethyl trisulfide: meaty, fishy, clams, green onion, garlic, cabbage • Diethyl sulfide:garlic, onion • Diethyl disulfide: overripe onion, greasy, garlic, burnt rubber, manure • Ethanethiol: onion, rubber, natural gas • Methionol: cauliflower, cabbage, potato • Methional: musty, potato, onion, meaty • Mercapto-3-methyl butanol: meaty

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

34. 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)

35. Fermentation Esters as Off-Characters

36. What Is an Ester? • Volatile molecule • Formed from the reaction of an alcohol and a keto acid • Formed enzymatically from an alcohol and a keto acid bound to the cofactor, Coenzyme A • Characteristic fruity and floral aromas

37. Ester Formation O R1-OH + R2-CCoA O R1-O-C-R2

38. How Are Esters Formed? • Can be formed by the chemical reaction of an alcohol and a keto acid • Can be formed enzymatically by the plant • Can be formed enzymatically by microbes

39. Where do Esters Come from in Wine? • Can be formed by the chemical reaction of an alcohol and a keto acid • Can be formed enzymatically by the plant • Can be formed enzymatically by microbes • Non-Saccharomyces yeasts • Saccharomyces • Lactic acid bacteria • Acetic acid bacteria

40. Ester Classes • Ethyl esters of acids • Acetate esters of alcohols

41. Common Esters Found in Wine • Ethyl Propanoate • Ethyl -2-Methylpropanoate • Ethyl-2 -Methylbutanoate • Ethyl-3-Methylbutanoate • Isobutyl Acetate • 2-Methylpropyl Acetate • 2-Methylbutyl Acetate • 3-Methylbutyl Acetate (Isoamyl acetate) • Hexyl Acetate • Requires grape precursor • Ethyl Lactate • Bacterial in origin

42. Positive Wine Characters Associated with Esters • Fruit • Apple • Apricot • Fig • Melon • Peach • Pear • Prune • Raspberry • Strawberry • Honey • Tropical fruit • Banana • Coconut • Mango • Pineapple • Floral • Rose • Butter • Spice • vanilla • Yeast (bread)

43. Esters Associated with Apple • Amyl acetate • Ethyl acetate • Ethyl butyrate • Isobutyl acetate • Phenethyl acetate

44. Esters Associated with Pineapple • Ethyl acetate • Ethyl butanoate (Ethyl butyrate) • Ethyl hexanoate

45. Negative Wine Characteristics Associated with Esters • Foxy • Nail polish • Bubble gum/cotton candy • Soapy • Candle wax • Perfume • Intense fruit • Intense floral

46. Ester Expression • Dependent upon chemical species present • Dependent upon concentrations: relative and absolute • Dependent upon matrix factors • Dependent upon yeast strain and substrates

47. In General . . . • The higher the concentration the more negative the impression is of the character • Longer chain esters fall into soapy, perfume range • Combinations of esters can confer a stronger aroma than the sum of the individual compounds

48. Negative Ester Characters • Nail polish/glue: ethyl acetate • Soap: ethyl octanoate, ethyl decanoate • Perfume: hexyl acetate • Rose: phenethylacetate, phenethyl alcohol