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Introduction to the Biology of Spoilage Yeasts and
Brettanomyces
Linda F. Bisson
Department of Viticulture and Enology
University of California
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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: Custer’s
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 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
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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 (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
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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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CH
CH
COOH
H
OH
CH
CH2
CH2
CH2
OH OH
H H
Cinnamate
decarboxylaseVinyl phenol
reductase
H OH OMe= coumaric = caffeic = ferulic
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:
– >426 ppb of 4-EP and 4-EG– >620 ppb of 4-EP
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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
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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