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 CO 2 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

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

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?  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