1. Introduction to the Biology of Spoilage Yeasts and Brettanomyces Linda F. Bisson Department of Viticulture and Enology University of California

2. Presentation Outline  Introduction to Yeast Spoilage  The Biology of Brettanomyces

3. INTRODUCTION TO YEAST SPOILAGE

4. Types of Yeast Spoilage  Film formers  Residual sugar utilizers  Survivalists

5. 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

6. 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

7. Survivalists  Brettanomyces/Dekkera  Pichia guilliermondii  Produce off-characters

8. Types of Yeast Spoilage  Off-character  Turbidity  Films and sediments

9. THE BIOLOGY OF BRETTANOMYCES/DEKKERA

10. 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.

11. 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 CO 2 dominate –Fermentation more rapid in presence of air: Custer ’ s effect

12. Morphology  Cell Morphology –Ogival, bullet shaped, non-uniform –Sometimes arranged in pseudohyphae.  Ascospore Morphology –Conquistador hat- shaped –1 to 4 spores/ascus

13. 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?

14. 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

15. 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

16. 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

17. 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

18. Brettanomyces Spoilage Characters  Vinyl phenols  Ethyl phenols  Biogenic amines –Putrescine –Cadaverine –Spermidine  Acetic acid

19. Vinyl Phenol Formation  Detoxification?  Co-Substrate?

20. 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

21. Production of Vinyl Phenols by Brettanomyces

22. 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

23. 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

24. 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

25. 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