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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. The Old Problem . . . . Brettanomyces. Historical Background.

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Overview of the biology of brettanomyces a new look at an old problem

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

Historical background
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 background1
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


  • 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


  • Cell Morphology

    • Ogival, bullet shaped, non-uniform

    • Sometimes arranged in pseudohyphae.

  • Ascospore Morphology

    • Conquistador hat-shaped

    • 1 to 4 spores/ascus

Brettanomyces genomics
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
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
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
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
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 saccharomyces1
Brettanomyces vs. Saccharomyces

  • Saccharomyces

    • Whole genome duplication

    • Domestication events

    • Reversible adaptation

  • Brettanomyces

    • Intensified local adaptive evolution

    • Terminal, non-reversible adaptation

Brettanomyces vs saccharomyces2
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
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
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


Role as a Spoilage Organism


Brettanomyces spoilage characters
Brettanomyces Spoilage Characters

  • Vinyl phenols

  • Ethyl phenols

  • Isovaleric Acid

  • Biogenic amines

    • Putrescine

    • Cadaverine

    • Spermidine

  • Acetic acid

  • Host of other compounds

The main spoilage characters
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
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
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
Vinyl Phenol Formation

  • Detoxification?

  • Co-Substrate?

Vinyl phenol formation1
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
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
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)







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)

The Standards


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

Black: CA

Pink: Canada

Lavender: NY

Blue: MO

Red: France

Green: Germany

Orange: Chile

Dark Blue: NZ

Brown: Belgium

Light Green: Thailand

What does this mean
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
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
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


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
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
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
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 strain1
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
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
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