Overview of the Biology of Brettanomyces: A New Look at an Old Problem Linda F. Bisson Department of Viticulture and Enology University of California 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
Wine Flavor 101 January 11, 2013
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 hat-
shaped– 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 (10-15%) 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
CH
CH
COOH
H
OH
CH
CH2
CH2
CH2
OH OH
H H
Cinnamate
decarboxylaseVinyl phenol
reductase
H OH OMe= coumaric = caffeic = ferulic
Production of Vinyl Phenols by Brettanomyces
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
Bandaid
Horsey
Earthy
Soy
PutridControl
615
738
752 2030
2041
2046
2047
2048
2049
2050
2051
2052
2053
2054
2058
2059
2060
2062
2063
2065
2066
2067
2075
2076
2077
2078
2079
2080
2081
2082
2083
2085
2091
2092
2093
-3
-2
-1
0
1
2
3
-3 -2 -1 0 1 2 3
PC1 (41.4%)
PC
2 (
27
.9%
)Black: CAPink: CanadaLavender: NYBlue: MORed: FranceGreen: GermanyOrange: ChileDark Blue: NZBrown: BelgiumLight Green: Thailand
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