Introduction to the Biology of Spoilage Yeasts and Brettanomyces

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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
 Film
formers
 Residual sugar utilizers
 Survivalists
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
 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
 Brettanomyces/Dekkera
 Pichia
guilliermondii
 Produce off-characters
Types of Yeast Spoilage
 Off-character
 Turbidity
 Films
and sediments
THE BIOLOGY OF
BRETTANOMYCES/DEKKERA
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
 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
 Cell
Morphology
– Ogival, bullet shaped,
non-uniform
– Sometimes arranged in
pseudohyphae.
 Ascospore
Morphology
– Conquistador hatshaped
– 1 to 4 spores/ascus
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
 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
 Saccharomyces:
grows 5 times faster
 Brettanomyces has slightly higher ethanol yields (1015%)
 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
 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
 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
 Vinyl
phenols
 Ethyl phenols
 Biogenic amines
– Putrescine
– Cadaverine
– Spermidine
 Acetic
acid
Vinyl Phenol Formation
 Detoxification?
 Co-Substrate?
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
Production of Vinyl Phenols by Brettanomyces
OH
OH
H
H
Cinnamate
CH
OH
decarboxylase
CH
CH
H
Vinyl phenol
reductase
CH2
CH2
CH2
COOH
H
= coumaric
OH
= caffeic
OMe
= ferulic
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
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
 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
 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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