RECOGNIZING WINE
OFF-CHARACTERS OF
MICROBIAL ORIGIN
Wine Flavor 101
January 16, 2014
Linda F. Bisson
Department of Viticulture and Enology
Wine Off-Characters of Microbial Origin
• Off-colors
• Off-flavors
• Hazes/cloudiness
• Sediment/precipitates
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
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!
Microbial Off-Characters
• Pre-fermentation
• During fermentation arising from normal flora
• Post-fermentation
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 prefermentation character formation
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
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
Pre-Fermentation Spoilage Characters
• Ethyl acetate
• Acetic acid
• Amplification of unripe or green characters
• Sour taint
Off-Characters Produced by Yeast During
Fermentation
• Common
• Sulfur volatiles
• Esters
___________________________________
• Rare
• Higher alcohols
• Acetaldehyde
• Higher aldehydes
• Volatile acids
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
Fermentation Off-Characters
Impacted by growth conditions
• Volatilization of compounds
• Temperature
• Head space
• Nutrient availability
• Nitrogen
• Micronutrients
• Oxygen
• Microbial competition
• Sulfite use
• Inoculation practices
Fermentation Off-Characters
Affected by juice composition
• Availability of precursors
• pH
• Presence of stressors
• High sugar/high ethanol
• Previous microbial history
The Common Yeast Off-Odors
• Sulfur volatiles
• Fermentation esters
Yeast Sulfur Volatiles
• Hydrogen sulfide
• Complex sulfides
Why Are Sulfur Taints a Problem?
• Low thresholds of detection
• Chemical reactivity
• Difficulty in removal
• Difficulty in masking
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
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
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
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
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
Timing of Formation of H2S
Brix
H2S
Time
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
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
COMPLEX SULFIDES
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
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
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
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
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
Sulfur Compound Aroma 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
Sources of Higher Sulfides
• S-Containing Amino Acids
• S-Containing Vitamins and Co-factors
• Glutathione (Cysteine-containing tripeptide involved in
redox buffering)
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)
FERMENTATION ESTERS
AS OFF-CHARACTERS
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
Ester Formation
O
R1-OH
+
R2-CCoA
O
R1-O-C-R2
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
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
Ester Classes
• Ethyl esters of acids
• Acetate esters of alcohols
Common Esters Found in Wine
• Ethyl Propanoate
• 2-Methylpropyl Acetate
• Ethyl -2-
• 2-Methylbutyl Acetate
Methylpropanoate
• 3-Methylbutyl Acetate
• Ethyl-2 (Isoamyl acetate)
Methylbutanoate
• Hexyl Acetate
• Ethyl-3-Methylbutanoate • Requires grape
precursor
• Isobutyl Acetate
• Ethyl Lactate
• Bacterial in origin
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)
Esters Associated with Apple
• Amyl acetate
• Ethyl acetate
• Ethyl butyrate
• Isobutyl acetate
• Phenethyl acetate
Esters Associated with Pineapple
• Ethyl acetate
• Ethyl butanoate (Ethyl butyrate)
• Ethyl hexanoate
Negative Wine Characteristics Associated
with Esters
• Foxy
• Nail polish
• Bubble gum/cotton candy
• Soapy
• Candle wax
• Perfume
• Intense fruit
• Intense floral
Ester Expression
• Dependent upon chemical species present
• Dependent upon concentrations: relative and absolute
• Dependent upon matrix factors
• Dependent upon yeast strain and substrates
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
Negative Ester Characters
• Nail polish/glue: ethyl acetate
• Soap: ethyl octanoate, ethyl decanoate
• Perfume: hexyl acetate
• Rose: phenethylacetate, phenethyl alcohol