Aromatic diversity of
Brettanomyces
C.M. Lucy Joseph Department of Viticulture and
Enology
U.C. Davis
Brettanomyces Aromas
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Horse sweat - Leather
Earthy
Medicinal
Band Aid
Smoky
Tobacco
Barnyard
Putrid
Lilac
Brettanomyces Substrates
Grown in a defined medium with the
following substrates:
• Cinnamic acids:
– Coumaric Acid
– Ferulic Acid
– Caffeic Acid
• Amino Acids
– Lysine
– Phenylalanine
– Tryptophan
– Tyrosine
Bench Test Indicated Differences in Odor
Sensitivities
• Some individuals did not feel that any of the odors
were typical of what they thought of as “Brett
aroma”
• Others did not detect typical Brettanomyces
aroma compounds (i.e. 4-ethylphenol): “specific
anosmia”
• Samples were described quite differently by
different people making it necessary to determine
if they were detecting different compounds in the
same sample
• Samples without known precursors gave typical
Brettanomyces odors
Phenolic Acids
Caffeic Acid
Amino Acids
Lysine
Tryptophan
Phenylalanine
Tyrosine
Vinyl and Ethyl Phenols
OH
OH
H
H
Cinnamate
CH
OH
decarboxylase
CH
CH
H
Vinyl phenol
reductase
CH2
CH2
CH2
COOH
H
= coumaric
OH
= caffeic
OMe
= ferulic
Proposed Pathway for Mousy Aromas from
Lysine in Brettanomyces
ETHP = 2-ethyltetrahydropyridine
ATHP = 2-acetyltetrahydropyridine
Amino Acid to Alcohols in
Saccharomyces
• Phenylalanine
Amino Acid to Alcohols in Saccharomyces
• Tryptophan
Sensory Analysis
The analysis of the Brettanomyces
samples suggested that people were
either detecting different
compounds or multiple compounds
produced from the precursor.
Review of Human Olfactory Sensory
Detection
How Do We Perceive Aroma?
10 million olfactory
sensory neurons on the olfactory
epithelium
Every olfactory receptor
binds many similar odorants
with different affinities.
Olfactory sensory neurons are
directly connected to the olfactory
bulb which is connected directly to
the primitive brain or the limbic
system. This system is involved
in processing memory and emotion.
Genetics of Olfaction
• Humans have 388 genes that code for olfactory
receptors (OR) and about 414 pseudogenes
• These genes have different alleles but only one
allele is expressed at a time
• OR genes are divided into 17 families and 127
subfamilies based on sequence and protein
structure
• Each receptor reacts with one type of chemical or
chemical constituent
• “Aroma” often consists of a mixture of these
signals to receptors that we learn to associate with
a given object like bacon or coffee
Olfactory Receptor Gene Distribution
Olfactory receptor genes occur
on all chromosomes except 20
and Y.
Analysis of Brettanomyces Produced Compounds by
Solid Phase Micro-extraction with Gas
Chromatography and a Mass Spectrometer Detector
with an Olfactory Port
(SPME-GCMS-O)
• Tested cultures in defined medium not wine
• Analyzed samples with substrates added at levels
normally found in wine to determine which
compounds were odor active
• Tested standards to determine if the chemicals
identified had the same retention times and
aroma descriptors as those in the samples
Solid Phase Micro-extraction with Gas Chromatography and
a Mass Spectrometer Detector with an Olfactory Port
• The fiber was exposed to the
head space of 10 ml samples of
media, with cells removed, for
30 minutes.
• The fiber was desorbed into the
GC column injector.
• The sample could be split for
olfactory detection and mass
spec. analysis or un-split for
mass spec. analysis alone.
Olfactory Port
• Half of the sample goes to
the Mass Spec. detector and
the other half is carried with
humidified air to the glass
nose cone for olfactory
detection by a human.
• Panelist responds to aroma
by pushing button to
indicate time and duration
of aroma
• Panelist also describes
aroma and notes time
during the run
Antech Solutions
Difference in Panelists Perception of Standards
Difference in Panelists Perception of the Same Sample
Difference in Odor Perception of One Panel
Member on Different Days
Summary of Panel Members Perceptions of
Standards
• Only the aroma compounds that were detected by a
majority of panelists were identified chemically
• Some standard compounds were detected by all of
the panelists while others were only detected by half
of the panelists
• Responses to the compounds varied among
panelists, from 100% detection to 75% detection of
all standards
Aromas Associated with Substrates
Substrate
Time (min)
Aroma
Coumaric Acid
16 to 18
Chemical, asphalt, irritating
22
Clove, animal
3 to 4
Fruity, butterscotch
6
Orange, sweet, floral
7 to 8
Cheesy, dirty sock, sweaty
14 to 15
Floral, almond oil, ink
18 to 22
Spicy, smoky, tumeric, medicinal
3 to 4
Fruity, rotting flowers, plastic
6 to 8
Plant, sweaty, stinky
14 to 15
Floral, sweet, medicinal, rose
19 to 20
Metallic, sulfur
3 to 4
Sweet, fruity, chemical, sharp
6 to 8
Rotten, cheesy, sweaty, rancid
14
Perfume, rose, pepper, unpleasant
floral
Ferulic Acid
Phenylalanine
Tyrosine/Tryptophan
Types of Chemical Compounds Produced
Type of Compound
Substrates Panel’s Aroma Descriptors
Ethyl/Vinyl Phenols
Phenolic acids
Chemical, smoke, spicy, plastic
Fatty Acids
Amino Acids
Rotten, rancid, sweaty, barnyard
Fatty Acid Esters
Amino Acids
Artificial fruit and floral
Long Chain Alcohols
Phenolic Acids Chemical, solvent, floral, fruit
Amino Acids
Pyridines
Amino Acids
Terpenes
Phenolic Acids Spicy, floral, tropical, toasty
Amino Acids
Animal, mousy
Genetics of Fatty Acid Metabolism in
Brettanomyces
Genes found in Brettanomyces that are not found in Saccharomyces
Species found in
Saccharomyces
kluyveri
Gene Name Function
Pichia pastoris
omega-3 fatty acid desaturase
delta 8-(E)-sphingolipid
desaturase
delta 4-(E)-sphingolipid
desaturase
Ashbya gossypii
Sphingolipid C9-methyltransferase
Pichia pastoris
Ceramide glucosyltransferase
Pichia pastoris
FAD3
Conclusions
• Brettanomyces produces a variety of odor active
compounds
• The production of odor active compounds by
Brettanomyces is controlled by substrate
availability and metabolic state of the cell
• The metabolic state of the cell is dependent upon
its environment
• Interactions between aroma compounds and
individual ability to perceive odor active
compounds will affect perception of overall aroma