Introduction to the Biology of Spoilage Yeasts and Brettanomyces

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

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Introduction to Yeast Spoilage

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Types of Yeast Spoilage

• Film formers
• Residual sugar utilizers
• Survivalists

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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

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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

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Survivalists

• Brettanomyces/Dekkera
• Pichia guilliermondii
• Produce off-characters

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Types of Yeast Spoilage

• Off-character
• Turbidity
• Films and sediments

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the Biology of Brettanomyces/Dekkera

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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.

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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
  Custers effect

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Morphology

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

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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?

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Brettanomyces Characteristics

• Custers 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

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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

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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

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Brettanomyces and Oxygen

• Oxygen stimulates growth, acetic acid formation
  and glycolysis (Custers 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

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Brettanomyces Spoilage Characters

• Vinyl phenols
• Ethyl phenols
• Biogenic amines
• Putrescine
• Cadaverine
• Spermidine
• Acetic acid

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Vinyl Phenol Formation

• Detoxification?
• Co-Substrate?

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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

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Production of Vinyl Phenols by Brettanomyces
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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
• gt426 ppb of 4-EP and 4-EG
• gt620 ppb of 4-EP

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Incidence of Spoilage
Country gt426ppb gt620ppb France
36 28 Italy 49 19 Australia
59 46 Portugal 42 27
Wines may contain up to 50 ppm (!) of 4-EP
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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

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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