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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

Introduction to the Biology of Spoilage Yeasts and Brettanomyces

Linda F. Bisson

Department of Viticulture and Enology

University of California

presentation outline
Presentation Outline
  • Introduction to Yeast Spoilage
  • The Biology of Brettanomyces
types of yeast spoilage
Types of Yeast Spoilage
  • Film formers
  • Residual sugar utilizers
  • Survivalists
film formers
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
residual sugar utilizers
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
survivalists
Survivalists
  • Brettanomyces/Dekkera
  • Pichia guilliermondii
  • Produce off-characters
types of yeast spoilage1
Types of Yeast Spoilage
  • Off-character
  • Turbidity
  • Films and sediments
slide9

the Biology of

Brettanomyces/Dekkera

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.
taxonomy
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
morphology
Morphology
  • 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?
brettanomyces characteristics
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
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
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 extreme environments and is found in VNC states
  • Produces diverse metabolic end products from grape components:
      • Volatile Phenols
      • Tetrahydropyrazines
brettanomyces and oxygen
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
brettanomyces spoilage characters
Brettanomyces Spoilage Characters
  • Vinyl phenols
  • Ethyl phenols
  • Biogenic amines
    • Putrescine
    • Cadaverine
    • Spermidine
  • Acetic acid
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
is that smell desirable
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
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

brett signature taints tasting
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
brett alternative substrates tasting
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