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