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Transcript
Lecture 10: Problem Fermentations Reading Assignment: Text, Chapter 4, pages 168181 This lecture will cover the principle types of fermentation problems that can arise during the alcoholic fermentation: Stuck fermentations and offcharacter production Problem Fermentations • Slow (sluggish) fermentation • Stuck (incomplete, arrested) fermentation • Off-character production – Hydrogen sulfide – Sulfur volatiles – Acetic acid – Undesired Esters Stuck and Sluggish Fermentations Stuck and Sluggish Fermentations • Characterized by failure of yeast to consume sugar • Multiple causes • Difficult to treat • Leads to reduced wine quality 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. GOAL: To be able to distinguish a normal from a problem fermentation as soon as possible. Fermentation Profile 5 1 Brix 2 3 4 Time 1: lag time; 2: max fermentation rate; 3: transition point; 4: post-transition fermentation rate; 5: overall time to dryness 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 Fermentation Capacity Is a Function of: • Yeast Biomass Concentration • Fermentative Ability of Individual Cells Causes of Stuck/Sluggish Fermentations • Nutrient limitation 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 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. Amination NH4 + -ketoglutarate glutamate NH4 + glutamate glutamine 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? Amino Acid Transport Amino acid H+ H+ Transporter Amino acid ATPase Pump ATP H+ ADP 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 Sources of Nutrients • Grape • Nutrient additions (winemaker) – Diammonium phosphate – Yeast extracts – Yeast “ghosts” – Proprietary yeast nutrient mix • Yeast autolysis Causes of Stuck/Sluggish Fermentations • Nutrient limitation • Ionic imbalance 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 Causes of Stuck/Sluggish Fermentations • Nutrient limitation • Nutrient imbalance • Substrate inhibition Substrate Inhibition Transporters with a high substrate affinity can get “jammed” at high substrate concentrations F G G FG F Causes of Stuck/Sluggish Fermentations • • • • Nutrient limitation Nutrient imbalance Substrate inhibition Ethanol toxicity Ethanol Toxicity Plasma membrane is the most ethanol-sensitive cell structure: Composition: Protein Lipid Other 50% 40% 10% Functions: Permeability barrier Regulation of uptake Mediates response to environment Maintains electrochemical gradients Mediates cell-cell interactions 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” Membrane Fluidity Is Required for Transport G G G 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? Ergosterol HO Fatty Acid Saturation Saturated Unsaturated Phospholipid Head Groups FA PO4 HO OH HO OH FA FA FA PO4 PO4 PO4 CH2 CH2 CH2 CH CH2 CH2 NH2 OOC NH2 OH H3C- N -CH3 CH + 3 PhoshpatidylInositol Serine Ethanolamine Choline 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-adenosylmethionine) be available Causes of Stuck/Sluggish Fermentations • • • • • Nutrient limitation Nutrient imbalance Substrate inhibition Ethanol toxicity Presence of toxic substances 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 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 Causes of Stuck/Sluggish Fermentations • • • • • • Nutrient limitation Nutrient imbalance Substrate inhibition Ethanol toxicity Presence of toxic substances Poor adaptation of strain 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 Causes of Stuck/Sluggish Fermentations • • • • • • • Nutrient limitation Nutrient imbalance Substrate inhibition Ethanol toxicity Presence of toxic substances Poor adaptation of strain Low pH 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 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 Temperature Shock • Super-cooling/heating of tank due to equipment failure • High temperature fermentations becoming too warm due to yeast metabolism 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. Off-Character Production The Saccharomyces Off-Characters • Volatile sulfur compounds 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 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 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 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? 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 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 Timing of Formation of H2S Brix H2S Time Timing of Formation of H2S Early (first 2-4 days): due to N imbalance Late (end of fermentation): due to autolysis, degradation of S-containing compounds H2S produced early can be driven off by carbon dioxide during active phase of fermentation Sulfate Reduction Pathway SO4 SUL1, SUL2 SO4 MET3 Adenylylsulfate MET14 Phosphoadenylylsulfate MET16 (1,8,20,22) Sulfite MET10 (1,5?,8,20) Sulfide Cysteine Cystathionine CYS3 MET17/25/15 Homocysteine Methionine CYS4 MET6 Regulation of the Sulfate Reduction Pathway • Methionine (SAM) Repression • Cysteine Inhibition of Inducer Production (O-acetyl serine) • General Amino Acid Control • Sub-Pathway Controls Homocysteine Methionine S-Adenosylhomocysteine Met-tRNA S-Adenosylmethionine Cysteine Cys-tRNA γ-Glutamylcysteine GlutathioneRD GlutathioneOX Methionine Repression Antagonized by: • • • • • • • Threonine Serine Aspartate Glycine Glutamate Histidine Lysine Methionine Repression is Augmented by: • Leucine The Amino Acids of General Amino Acid Control • • • • • • • • • • Lysine Histidine Arginine Leucine Valine Serine Phenylalanine Tryptophan Proline Methionine The Saccharomyces Off-Characters • Volatile sulfur compounds • Acetic Acid Acetic Acid Production by Saccharomyces • Levels made by Saccharomyces are generally low, below threshold of detection • Strain differences in amount formed • Derived from: – Fatty acid biosynthesis/degradation – Amino acid degradation The Saccharomyces Off-Characters • Volatile sulfur compounds • Acetic Acid • Higher Alcohols – Fusel oils – Phenethyl alcohol Higher Alcohols ( C2) • Also called “fusel oils” • Formed during amino acid degradation R Deamination R Decarboxylation R HCNH C=O HC=O COOH COOH Amino Acid RCHOH Alcohol Reduction RCOOH Acid Oxidation Phenethyl Alcohol OH H2C CH2 Generic “floral” May be too intense for some wines The Saccharomyces Off-Characters • • • • Volatile sulfur compounds Acetic Acid Higher Alcohols Acetaldehyde/Higher Aldehydes Aldehyde Production • Acetaldehyde from glycolysis – Released when conversion to ethanol is blocked – Released as SO2 adjunct • Higher aldehydes from amino acid degradation – Released when formation of higher alcohols is blocked The Saccharomyces Off-Characters • • • • • Volatile sulfur compounds Acetic Acid Higher Alcohols Acetaldehyde/Higher Aldehydes Unwanted Esters – Fatty acid metabolism – Amino acid metabolism • Phenethyl Acetate Unwanted Esters Esters form from the reaction of an alcohol and an acyl-CoA molecule O R1-OH + R2-CSCoA O R1-O-C-R2 Source of Esters • Most common ester is ethyl acetate made from the reaction of ethanol with acetyl-CoA • Esters can derive from amino acid degradation and reaction of acids with ethanol or of alcohols with acetyl-CoA • Esters can derive from fatty acid metabolism Phenethyl Acetate • Degradation product of phenyalanine • Characteristic “rose oil” odor • May be too pungent O CH2-CH2O-C-COOH The Saccharomyces Off-Characters • • • • • • Volatile sulfur compounds Acetic Acid Higher Alcohols Acetaldehyde/Higher Aldehydes Unwanted Esters Vinyl Phenols Vinyl Phenols OH H OH H OH H CH CH CH COOH CH2 CH2 Decarboxylase CH3 Vinyl Phenol Reductase Vinyl Phenols • Responsible for sweaty, horsy, stable,pharmaceutical off aromas • Usually formed by Brettanomyces • Saccharomyces possesses the enzymes needed to make vinyl phenols and there are reports that it will make them under certain conditions Moral: Yeast needs are simple, but it can be challenging to keep them happy.