Overview of the Biology of Brettanomyces: A New Look at an Old Problem

1. 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

2. The Old Problem . . . Brettanomyces

3. 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

4. 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

5. 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

6. Morphology • Cell Morphology • Ogival, bullet shaped, non-uniform • Sometimes arranged in pseudohyphae. • Ascospore Morphology • Conquistador hat-shaped • 1 to 4 spores/ascus

7. 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?

8. 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

9. 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

10. 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

11. 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

12. Brettanomyces vs. Saccharomyces • Saccharomyces • Whole genome duplication • Domestication events • Reversible adaptation • Brettanomyces • Intensified local adaptive evolution • Terminal, non-reversible adaptation

13. 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

14. 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

15. 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

16. Role as a Spoilage Organism Brettanomyces

17. Brettanomyces Spoilage Characters • Vinyl phenols • Ethyl phenols • Isovaleric Acid • Biogenic amines • Putrescine • Cadaverine • Spermidine • Acetic acid • Host of other compounds

18. Production of Vinyl Phenols by Brettanomyces

19. 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)

20. 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

21. 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

22. Vinyl Phenol Formation • Detoxification? • Co-Substrate?

23. 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

24. 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

25. 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

26. Band-Aid (4-Ethyl Phenol) Earthy (Geosmin) Horsy Leather Putrid Soy Tobacco TheMainAromasFound:

27. 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

28. 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

29. Black: CA Pink: Canada Lavender: NY Blue: MO Red: France Green: Germany Orange: Chile Dark Blue: NZ Brown: Belgium Light Green: Thailand

30. 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

31. 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

32. 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

33. Brettanomyces The New Look

34. BrettanomycesAromaImpact • 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

35. 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

36. BrettanomycesCharacters • 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

37. The Brett Aroma Impact Wheel • Not a typical descriptive analysis • Wanted the consensus terms but also breadth of descriptors

38. 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”

39. 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

40. 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

41. 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