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Lecture 10: Problem Fermentations

Lecture 10: Problem Fermentations. Reading Assignment: Text, Chapter 4, pages 168- 181. This lecture will cover the principle types of fermentation problems that can arise during the alcoholic fermentation: Stuck fermentations and off-character production. Problem Fermentations.

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Lecture 10: Problem Fermentations

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  1. Lecture 10: Problem Fermentations

  2. Reading Assignment: Text, Chapter 4, pages 168- 181

  3. This lecture will cover the principle types of fermentation problems that can arise during the alcoholic fermentation: Stuck fermentations and off-character production

  4. Problem Fermentations • Slow (sluggish) fermentation • Stuck (incomplete, arrested) fermentation • Off-character production • Hydrogen sulfide • Sulfur volatiles • Acetic acid • Undesired Esters

  5. Stuck and Sluggish Fermentations

  6. Stuck and Sluggish Fermentations • Characterized by failure of yeast to consume sugar • Multiple causes • Difficult to treat • Leads to reduced wine quality

  7. The main challenge of slow and arrested fermentations is that they are not recognizable until after fermentation rate has changed. At this point it may be too late to change the adaptive response of the yeast.

  8. GOAL: To be able to distinguish a normal from a problem fermentation as soon as possible.

  9. Fermentation Profile 5 1 2 Brix 3 4 Time 1: lag time; 2: max fermentation rate; 3: transition point; 4: post-transition fermentation rate; 5: overall time to dryness

  10. Fermentation Profile • Lag time • Duration? • Maximum fermentation rate • Rate value? • Duration? • Transition point • At what Brix level? • How sharp? • Post-transition fermentation rate • Value relative to max fermentation rate? • Length of time? • Brix/ethanol/nitrogen level at which it occurs? • Overall time to dryness

  11. Fermentation Capacity Is a Function of: • Yeast Biomass Concentration • Fermentative Ability of Individual Cells

  12. Causes of Stuck/Sluggish Fermentations • Nutrient limitation

  13. Nutrient Limitation: Nitrogen • Nitrogen: most often limiting • Amino acids • Can be degraded as N source via transamination • Can be interconverted with related amino acids • Can be used as that amino acid • Ammonia • Mobilized by direct amination

  14. Transamination Glutamate + X -ketoglutarate + N-X Glutamine + X’ glutamate + N-X’ Alanine + X’’ pyruvate + N-X’’ Where “X” is an intermediate in amino acid/ nucleotide biosynthesis, and “N-X” is an amino acid or nucleotide base.

  15. Amination NH4 + -ketoglutarate glutamate NH4 + glutamate glutamine

  16. Preference for Nitrogen Sources • How readily can it be converted to NH4, glutamate or glutamine? • Expense of utilization (ATP, cofactor, oxygen requirement) • Toxicity of C-skeleton • What else is available?

  17. Amino Acid Transport H+ H+ Amino acid Transporter ATPase Pump ATP ADP Amino acid H+

  18. Factors Affecting Nitrogen Compound Utilization and Preference • pH • Transport is coupled to H+ ion movements • Ethanol • Inhibits amino acid transporter function (80% at 5% ethanol for the general amino acid permease) • Increases passive proton flux • Other N compounds • Competition for uptake • Nitrogen repression • Induction • Yeast strain differences

  19. Sources of Nutrients • Grape • Nutrient additions (winemaker) • Diammonium phosphate • Yeast extracts • Yeast “ghosts” • Proprietary yeast nutrient mix • Yeast autolysis

  20. Causes of Stuck/Sluggish Fermentations • Nutrient limitation • Ionic imbalance

  21. Ionic Imbalance Ratio of K+:H+ Must be at least 25:1 Needs to be adjusted early in fermentation Probably important in building an ethanol tolerant membrane

  22. Causes of Stuck/Sluggish Fermentations • Nutrient limitation • Nutrient imbalance • Substrate inhibition

  23. Substrate Inhibition Transporters with a high substrate affinity can get “jammed” at high substrate concentrations G G F F F G

  24. Causes of Stuck/Sluggish Fermentations • Nutrient limitation • Nutrient imbalance • Substrate inhibition • Ethanol toxicity

  25. Ethanol Toxicity Plasma membrane is the most ethanol-sensitive cell structure: Composition: Protein 50% Lipid 40% Other 10% Functions: Permeability barrier Regulation of uptake Mediates response to environment Maintains electrochemical gradients Mediates cell-cell interactions

  26. Ethanol Toxicity Impact of ethanol Perturbs membrane structure at protein:lipid interface Leads to increased “passive proton flux” and acidification of cytoplasm Inhibits protein activity Affects membrane “fluidity”

  27. Membrane Fluidity Is Required for Transport G G G

  28. Ethanol Toxicity Adaptation of membrane requires: Increasing content of sterols Increasing relative content of proteins Increasing level of desaturation (number of double bonds) in fatty acid side chains Modification of phospholipid head groups?

  29. Ergosterol HO

  30. Fatty Acid Saturation Saturated Unsaturated

  31. Phospholipid Head Groups FA PO4 FA PO4 CH2 CH FA PO4 CH2 CH2 NH2 FA PO4 CH2 CH2 N -CH3 HO OH HO OH H3C- OOC NH2 + CH3 OH Phoshpatidyl- Inositol Serine Ethanolamine Choline

  32. Ethanol Toxicity Sterol and fatty acid desaturation are Oxygen-requiring processes New protein synthesis requires nitrogen be available Phospholipid head group synthesis requires cofactors (S-adenosyl- methionine) be available

  33. Causes of Stuck/Sluggish Fermentations • Nutrient limitation • Nutrient imbalance • Substrate inhibition • Ethanol toxicity • Presence of toxic substances

  34. Presence of Toxic Substances • Toxins may arise from the metabolic activity of other microbes • Toxins may arise from metabolic activity of Saccharomyces • Toxins may have arisen in vineyard, but are not inhibitory until ethanol has accumulated

  35. The Most Common Toxins • Acetic acid • Higher organic acids (C2 – C4) • Medium chain fatty acids/fatty acid esters • Acetaldehyde • Fungicide/Pesticide residues • Higher alcohols • Higher aldehydes • Killer factors • Sulfur dioxide

  36. Causes of Stuck/Sluggish Fermentations • Nutrient limitation • Nutrient imbalance • Substrate inhibition • Ethanol toxicity • Presence of toxic substances • Poor adaptation of strain

  37. Poor Adaptation of Strain • Strain may not display ethanol tolerance • Strain may have high nitrogen/vitamin requirements • Strain may be a poor fermentor, but capable of dominating the fermentation • Temperature effects

  38. Causes of Stuck/Sluggish Fermentations • Nutrient limitation • Nutrient imbalance • Substrate inhibition • Ethanol toxicity • Presence of toxic substances • Poor adaptation of strain • Low pH

  39. pH • pH is reduced by metabolism of Saccharomyces • Low pH musts (below pH 3.0) may drop to an inhibitory level (pH 2.7) • Dependent upon K+ concentration

  40. Causes of Stuck/Sluggish Fermentations • Nutrient limitation • Nutrient imbalance • Substrate inhibition • Ethanol toxicity • Presence of toxic substances • Poor adaptation of strain • Low pH • Temperature shock

  41. Temperature Shock • Super-cooling/heating of tank due to equipment failure • High temperature fermentations becoming too warm due to yeast metabolism

  42. The factors leading to arrest of fermentation are interacting. Limitation for nutrients enhances the toxicity of ethanol as does high temperature and the presence of other toxic substances.

  43. Off-Character Production

  44. The Saccharomyces Off-Characters • Volatile sulfur compounds

  45. Volatile Sulfur Compounds • Hydrogen Sulfide: H2S • Methanethiol: CH3-SH • Ethanethiol: C2H5-SH • Dimethyl sulfide: CH3-S-CH3 • Dimethyl disulfide: CH3-S-S-CH3 • Diethyl sulfide: C2H5-S-C2H5 • Diethyl disulfide: C2H5-S-S-C2H5

  46. 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

  47. Hydrogen Sulfide Formation • Due to nitrogen limitation • Sulfate reduction regulated by nitrogen availability • Lack of nitrogenous reduced sulfur acceptors leads to excessive production of reduced sulfate and release as H2S • Strain variation

  48. Higher Sulfides • Come from degradation of sulfur containing amino acids • From reaction of reduced sulfur intermediates with other cellular metabolites? • Formed chemically due to reduced conditions?

  49. 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

  50. 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

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