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 hat-

shaped– 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 (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 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

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

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