Transcript 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
 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