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Wine Flavor 101 January 11, 2013
Overview of the Biology of
Brettanomyces: A New Look at
an Old Problem
Linda F. Bisson
Department of Viticulture and Enology
University of California
Brettanomyces
The Old Problem . . .
Historical Background
 Brettanomyces
is a budding yeast found
widely distributed in nature
 Discovered in beer in 1904 (Claussen), in
wine (Krumbholz & Tauschanoff,1930) and
again in 1940 (Custers)
 Results in a variety of aromas
– English Character or Lambic Beers
– Spoilage/Regional Character in Wines
Historical Background
 Brettanomyces
produces a wide array of
aromatic compounds
 Brettanomyces cellar contamination was
widespread
 Brettanomyces characters became synonymous
with “terroir” and regional signature
 Brettanomyces characters can compete with
varietal characters for dominance of wine profile
Taxonomy
 Anamorphic/non-sexual
form: Brettanomyces
Teleomorphic/sexual form: Dekkera
 Several species are found: B. bruxellensis, B.
anomala, B. custerianus
 Characteristic traits:
–
–
–
–
–
Ascomycete yeast
Reproduce by budding
Observation of sporulation is rare
Pseudohyphae formed
Fermentation end products: acetic acid and CO2
dominate
– Fermentation more rapid in presence of air: Custer’s
effect
Morphology
Cell Morphology
– Ogival, bullet shaped,
non-uniform
– Sometimes arranged in
pseudohyphae.
Ascospore Morphology
– Conquistador hatshaped
– 1 to 4 spores/ascus
Brettanomyces Genomics
 Chromosomal
number varies by strain
 Chromosome configuration not well preserved
 Not a simple haploid or diploid
– Hybrid between two strains with similar but different
genomes?
– Diploid progenitor that lost the ability to engage in
sexual reproduction (genome renewal)
 Accumulation
of allelic differences and
polymorphisms
– Hyper-mutagenic?
– Defective in repair?
Metabolism of Brettanomyces
 Can
use numerous sugars, ethanol, other
carbon compounds, and even amino acids as
carbon sources
 Can survive in very nutrient poor condition
 Can survive and metabolize in extreme
environments and is found in VNC states
 Produces diverse metabolic end products
from grape components:
» Volatile Phenols
» Tetrahydropyrazines
Brettanomyces Characteristics
 Highly
metabolically versatile
 Capable of ethanol production from sugars
anaerobically
 Produce acetic acid from sugars aerobically
 Can produce viable petite (non-fermenting)
off-spring
Brettanomyces and Oxygen
 Oxygen
stimulates growth, acetic acid
formation and glycolysis
 Oxidation of acetaldehyde to acetic acid is
favored over reduction to alcohol
 Leads to depletion of NAD+
 Requires co-substrates or oxygen for acetic
acid production
 Redox state of cytoplasm has a strong
impact on metabolites produced
Brettanomyces vs. Saccharomyces
 Saccharomyces:
grows 5 times faster
 Brettanomyces has slightly higher ethanol yields (1015%)
 Saccharomyces produces more glycerol (6 fold higher)
 Brettanomyces produces more biomass (20 to 30%
more)
 Brettanomyces more tolerant of large changes in pH
and temperature
 Brettanomyces has a more energy-efficient
metabolism: can do more with less
Brettanomyces vs. Saccharomyces
 Saccharomyces
– Whole genome duplication
– Domestication events
– Reversible adaptation
 Brettanomyces
– Intensified local adaptive evolution
– Terminal, non-reversible adaptation
Brettanomyces vs. Saccharomyces
Whole genome duplication (Saccharomyces: buy
all the gear I need and carry it with me) vs.
Intensified local adaptive evolution
(Brettanomyces: live off the land)
 Both strategies allow successful adaptation to
challenging environments and enable switching
between metabolic modes.
 The WGD is more confining of subsequent
strain integrity; strains are more similar than in
the case of intensified local adaptive evolution
What Does This Mean for
Winemakers and Consumers?
 Significant
diversity in compounds
produced by Brettanomyces strains
 Regional specificity of compounds
produced due to highly adapted local
populations
Spoilage Organism or Agent of
Regional Character?
 Brettanomyces
makes a host of aromatic
compounds
 Compounds made differ by strain
 Compounds made differ by winery
 Compounds made differ by vineyard
Brettanomyces
Role as a Spoilage Organism
Brettanomyces Spoilage Characters
 Vinyl
phenols
 Ethyl phenols
 Isovaleric Acid
 Biogenic amines
– Putrescine
– Cadaverine
– Spermidine
 Acetic
acid
 Host of other compounds
Production of Vinyl Phenols by Brettanomyces
OH
OH
H
H
Cinnamate
CH
OH
decarboxylase
CH
CH
H
Vinyl phenol
reductase
CH2
CH2
CH2
COOH
H
= coumaric
OH
= caffeic
OMe
= ferulic
The Main Spoilage Characters
 Three
main phenolic spoilage compounds:
– 4-Ethylphenol (band aid)
– 4-Ethylguaiacol (smoky medicinal)
– 4-Ethylcatechol (horsy)
 Isovaleric
acid (rancid, sour, vomit and
sometimes barnyard note)
Is That Character Desirable?
 Detection
threshold varies with varietal from
126 to 420 ppb of 4-EP depending upon matrix
 Recovery Thresholds:
– 50% of tasters can detect 605 ppb in wine or 440 ppb
in water of 4-EP
 Chatonnet
has defined spoilage as:
– >426 ppb of 4-EP and 4-EG
– >620 ppb of 4-EP
Incidence of Spoilage
Country
>426ppb
>620ppb
France
36%
28%
Italy
49%
19%
Australia
59%
46%
Portugal
42%
27%
Wines may contain up to 50 ppm (!) of 4-EP
Vinyl Phenol Formation
 Detoxification?
 Co-Substrate?
Vinyl Phenol Formation
 4-EP
formation is growth associated
 4-EP formation not correlated with acetic
acid formation
 High 4-EP producers tolerate higher
environmental levels of p-coumaric acid
When Is It Spoilage?
 High
concentration, dominating wine
profile
 Conflict with wine matrix characters
 Suppression of varietal character
 Enhancement of off-notes
 Lactic acid bacteria often found in wines
with Brettanomyces
The Wirz Strain Trial
 Take
a large collection of 35
Brettanomyces strains: 17 Strains from
CA, 1 from NY, 1 from MO, 4 from France,
2 from Germany, 2 from New Zealand, 2
from Chile, 2 from Malta, 2 from Belgium,
1 from Canada, 1 from Thailand
 Perform descriptive analysis with trained
panelists following growth in Cabernet
Sauvignon wine
 14 panelists participated
The Main Aromas Found:
 Band-Aid
(4-Ethyl Phenol)
 Earthy (Geosmin)
 Horsy
 Leather
 Putrid
 Soy
 Tobacco
The Standards
8 aroma standards were selected by the
panelists:
– Soy (Soy Sauce)
– Band-Aid (4-Ethyl Phenol)
– Horsy (Horse Sweat-soaked Towel)
– Putrid (Burnt Fava Beans)
– Tobacco (Shredded Cigarette)
– Leather (Leather Shoelace)
– Earthy (Geosmin)
ANOVA
Analysis of Variance (ANOVA) showed that
the variance in the data for 5 of the 7
attributes could be explained by the wines:
– Band-Aid, horsey, earthy, putrid, soy. (p<0.06)
– Leather and tobacco  judge interaction was
too high
3
PC2 (27.9%)
Black: CA
Pink: Canada
Lavender: NY
Blue: MO
Red: France
Green: Germany
Orange: Chile
Dark Blue: NZ
Brown: Belgium
Light Green: Thailand
-3
2091
2076
2092
1
Bandaid
2054
2093
2059
2052
2065
2060
2079
2080
738
-2
2
2049
2067
2041
752
-1 2050
2063
0
1
Control
2066
Earthy
2062
2051
2053
2085
Soy
2058 Horsey 2081
2083
0
2030
2046
2077
2078
2
3
Putrid
2082
-1
615
2075
2047
-2
2048
-3
PC1 (41.4%)
What Does This Mean?
 There
is a group of strains that showed no effect
on the wine: grew but no off-characters were
produced
 Other strains showed differing impacts on the
wine
 Aroma groupings were observed:
– Band-Aid & Soy vs. Earthy & Putrid; Horsey vs.
nothing. All vs. nothing.
 Correlation
of descriptors
– Earthy and Putrid are very highly correlated, Band-Aid
and Soy, Soy and Horsey also correlated but less
highly
Lessons Learned
 Strain
diversity evident in same wine:
strains use different metabolic strategies
 Not much correlation with geographical
location of origin
 Many strains were on the “positive” side:
not just absence of off-characters
 Some panelists seemed to be “blind” to
some characters as the consensus
descriptor
Subsequent Studies
 Lucy
Joseph: GC-Olfactory
 Brad Kitson: Role of specific precursors in
wine
 Beth Albino: Survey of strains, precursors
and diversity of aromatic impressions:
Birth of the Brett Aroma Wheel
 Lucy Joseph: Metabolomic analyses,
refinement of Brett Aroma Wheel
Brettanomyces
The New Look
Brettanomyces Aroma Impact
 The
sensory analysis demonstrated that
different strains impact the same wine in
different ways
 Some of the Brettanomyces-infected wines
were “preferred” over the control in judge
comments
 Judges disagreed on the nature of the
positive descriptors
 New goal: better understanding of the
positive impacts of Brettanomyces on wines
Brettanomyces and Regional
Character
 Local
Brettanomyces strains contribute to
the expected aromatic profile of wines
when allowed to bloom during aging
 Some people, cultures, population
segments are attracted to those characters
 Many more people are attracted to the
positive Brettanomyces characters if the
vinyl phenols and isovaleric acid
contributions are minimized
Brettanomyces Characters
 Are
described differently by different people
 Strongly trigger complex memory responses
 See the perception phenomenon of “filling in
the aroma gap”
 Show strong matrix effects
 Can add to complexity
The Brett Aroma Impact Wheel
 Not
a typical descriptive analysis
 Wanted the consensus terms but also
breadth of descriptors
The Search for a Neutral/
Positive Strain
 Neutral
Strains: may be useful in making
wines stable against further Brettanomyces
infection via consumption of residual nutrients
 Positive Strains: may impart some of the
spicy, complex characters Brettanomyces is
known for minus the negatives
 Better understanding of metabolism may lead
to a better understanding of the negative
impacts of wine and allow better prediction of
which wines to use for this “style”
The Search for a Neutral/
Positive Strain
 Variations
in vinyl phenol production
 Not consistently stable
 Strong matrix influence that is not well
understood
 Bottom Line: Brettanomyces cannot be
trusted metabolically, but if historical
winery experience is positive and there is
no subsequent adaptive pressure, the
positive influence may recur
Today’s Program: AM
 The
Brettanomyces Signature Spoilage
Characters
– Spiked Cabernet Wine
 Influence
of Matrix:
– Characters in different wines
» Smoky Red Fruit Grenache
» Gamy Pinot noir
» Barbera
Today’s Program: PM
 Brettanomyces
Aroma Impact Wheel
– Tasting of Merlot Wine with Different Strains
– Tasting of Brett “suppressed and salvaged”
wine

Brettanomyces in Commercial Wines:
– Winemaker Panel
– Tasting of Commercially wines with Brett
contributions: selected by use of Aroma
Impact Wheel descriptors