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Introduction to the Biology of Spoilage Yeasts and Brettanomyces

Introduction to the Biology of Spoilage Yeasts and Brettanomyces. Linda F. Bisson Department of Viticulture and Enology University of California. Presentation Outline. Introduction to Yeast Spoilage The Biology of Brettanomyces. Introduction to Yeast Spoilage. Types of Yeast Spoilage.

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Introduction to the Biology of Spoilage Yeasts and Brettanomyces

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  1. Introduction to the Biology of Spoilage Yeasts and Brettanomyces Linda F. Bisson Department of Viticulture and Enology University of California

  2. Presentation Outline • Introduction to Yeast Spoilage • The Biology of Brettanomyces

  3. Introduction to Yeast Spoilage

  4. Types of Yeast Spoilage • Film formers • Residual sugar utilizers • Survivalists

  5. Film Formers • Candida, Pichia • Candida spp • P. anomala • P. membranifaciens • Torulaspora • Hansenula • Dependent upon oxygen exposure and head space • May be aromatically neutral or sources of off-aromas

  6. Residual Sugar Utilizers • Saccharomyces • Zygosaccharomyces • Z. bailii • Z. bisporous • Z. rouxii • Saccharomycodes ludwigii • Can grow in bottle post-bottling • Can form turbidity and be aromatically neutral • Can form off-characters

  7. Survivalists • Brettanomyces/Dekkera • Pichia guilliermondii • Produce off-characters

  8. Types of Yeast Spoilage • Off-character • Turbidity • Films and sediments

  9. the Biology of Brettanomyces/Dekkera

  10. 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.

  11. 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

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

  13. 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?

  14. Brettanomyces Characteristics • Custer’s effect: oxygen stimulates glycolysis • Capable of ethanol production from sugars anaerobially • Produce acetic acid from sugars aerobically • Can produce viable petite (non-fermenting) off-spring

  15. 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

  16. 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 extreme environments and is found in VNC states • Produces diverse metabolic end products from grape components: • Volatile Phenols • Tetrahydropyrazines

  17. Brettanomycesand Oxygen • Oxygen stimulates growth, acetic acid formation and glycolysis (Custer’s effect) • 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

  18. Brettanomyces Spoilage Characters • Vinyl phenols • Ethyl phenols • Biogenic amines • Putrescine • Cadaverine • Spermidine • Acetic acid

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

  20. 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

  21. Production of Vinyl Phenols by Brettanomyces

  22. Is That Smell Desirable? • Three main spoilage compounds: • 4-Ethylphenol (band aid) • 4-Ethylguaiacol (smoky medicinal) • 4-Ethylcatechol (horsy) • 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

  23. 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

  24. Brett Signature Taints Tasting • Glass 1: Control (Merlot) • Glass 2: 1000ppb 4-EP • Glass 3: 620 ppb 4-EP • Glass 4: 400ppb 4-EG • Glass 5: 430: 350ppb 4-EP + 80 4-EG • Glass 6: 2200: 1800 4-EP + 400 4-EG

  25. Brett Alternative Substrates Tasting • Glass 1: Control: Brett in media minus supplements • Glass 2: Ferulic acid • Glass 3: Coumaric acid • Glass 4: Phenylalanine • Glass 5: Tryptophan and Tyrosine • Glass 6: Lysine

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