1. Aromatic diversity of Brettanomyces C.M. Lucy Joseph Department of Viticulture and Enology U.C. Davis

2. Brettanomyces Aromas Horse sweat - Leather Earthy Medicinal Band Aid Smoky Tobacco Barnyard Putrid Lilac

3. Brettanomyces Substrates Grown in a defined medium with the following substrates: Cinnamic acids: – Coumaric Acid – Ferulic Acid – Caffeic Acid Amino Acids – Lysine – Phenylalanine – Tryptophan – Tyrosine

4. Bench Test Indicated Differences in Odor Sensitivities Some individuals did not feel that any of the odors were typical of what they thought of as “Brett aroma” Others did not detect typical Brettanomyces aroma compounds (i.e. 4-ethylphenol): “specific anosmia” Samples were described quite differently by different people making it necessary to determine if they were detecting different compounds in the same sample Samples without known precursors gave typical Brettanomyces odors

5. Phenolic Acids Caffeic Acid

6. Amino Acids Tyrosine Phenylalanine Tryptophan Lysine

7. Vinyl and Ethyl Phenols

8. Proposed Pathway for Mousy Aromas from Lysine in Brettanomyces ETHP = 2-ethyltetrahydropyridine ATHP = 2-acetyltetrahydropyridine

9. Amino Acid to Alcohols in Saccharomyces Phenylalanine

10. Amino Acid to Alcohols in Saccharomyces Tryptophan

11. Sensory Analysis The analysis of the Brettanomyces samples suggested that people were either detecting different compounds or multiple compounds produced from the precursor.

12. Review of Human Olfactory Sensory Detection

13. How Do We Perceive Aroma? Every olfactory receptor binds many similar odorants with different affinities. Olfactory sensory neurons are directly connected to the olfactory bulb which is connected directly to the primitive brain or the limbic system. This system is involved in processing memory and emotion. 10 million olfactory sensory neurons on the olfactory epithelium

14. Genetics of Olfaction Humans have 388 genes that code for olfactory receptors (OR) and about 414 pseudogenes These genes have different alleles but only one allele is expressed at a time OR genes are divided into 17 families and 127 subfamilies based on sequence and protein structure Each receptor reacts with one type of chemical or chemical constituent “Aroma” often consists of a mixture of these signals to receptors that we learn to associate with a given object like bacon or coffee

15. Olfactory Receptor Gene Distribution Olfactory receptor genes occur on all chromosomes except 20 and Y.

16. Analysis of Brettanomyces Produced Compounds by Solid Phase Micro-extraction with Gas Chromatography and a Mass Spectrometer Detector with an Olfactory Port (SPME-GCMS-O) Tested cultures in defined medium not wine Analyzed samples with substrates added at levels normally found in wine to determine which compounds were odor active Tested standards to determine if the chemicals identified had the same retention times and aroma descriptors as those in the samples

17. Solid Phase Micro-extraction with Gas Chromatography and a Mass Spectrometer Detector with an Olfactory Port The fiber was exposed to the head space of 10 ml samples of media, with cells removed, for 30 minutes. The fiber was desorbed into the GC column injector. The sample could be split for olfactory detection and mass spec. analysis or un-split for mass spec. analysis alone.

18. Olfactory Port Half of the sample goes to the Mass Spec. detector and the other half is carried with humidified air to the glass nose cone for olfactory detection by a human. Panelist responds to aroma by pushing button to indicate time and duration of aroma Panelist also describes aroma and notes time during the run Antech Solutions

19. Difference in Panelists Perception of Standards

20. Difference in Panelists Perception of the Same Sample

21. Difference in Odor Perception of One Panel Member on Different Days

22. Summary of Panel Members Perceptions of Standards Only the aroma compounds that were detected by a majority of panelists were identified chemically Some standard compounds were detected by all of the panelists while others were only detected by half of the panelists Responses to the compounds varied among panelists, from 100% detection to 75% detection of all standards

23. Aromas Associated with Substrates SubstrateTime (min)Aroma Coumaric Acid16 to 18Chemical, asphalt, irritating 22Clove, animal Ferulic Acid3 to 4Fruity, butterscotch 6Orange, sweet, floral 7 to 8Cheesy, dirty sock, sweaty 14 to 15Floral, almond oil, ink 18 to 22Spicy, smoky, tumeric, medicinal Phenylalanine3 to 4Fruity, rotting flowers, plastic 6 to 8Plant, sweaty, stinky 14 to 15Floral, sweet, medicinal, rose 19 to 20Metallic, sulfur Tyrosine/Tryptophan3 to 4Sweet, fruity, chemical, sharp 6 to 8Rotten, cheesy, sweaty, rancid 14Perfume, rose, pepper, unpleasant floral

24. Types of Chemical Compounds Produced Type of CompoundSubstratesPanel’s Aroma Descriptors Ethyl/Vinyl PhenolsPhenolic acidsChemical, smoke, spicy, plastic Fatty AcidsAmino AcidsRotten, rancid, sweaty, barnyard Fatty Acid EstersAmino AcidsArtificial fruit and floral Long Chain AlcoholsPhenolic Acids Amino Acids Chemical, solvent, floral, fruit Pyridines Amino Acids Animal, mousy TerpenesPhenolic Acids Amino Acids Spicy, floral, tropical, toasty

25. Genetics of Fatty Acid Metabolism in Brettanomyces Species found inGene NameFunction Saccharomyces kluyveriFAD3omega-3 fatty acid desaturase Pichia pastoris delta 8-(E)-sphingolipid desaturase Pichia pastoris delta 4-(E)-sphingolipid desaturase Ashbya gossypiiSphingolipid C9-methyltransferase Pichia pastorisCeramide glucosyltransferase Genes found in Brettanomyces that are not found in Saccharomyces

26. Conclusions Brettanomyces produces a variety of odor active compounds The production of odor active compounds by Brettanomyces is controlled by substrate availability and metabolic state of the cell The metabolic state of the cell is dependent upon its environment Interactions between aroma compounds and individual ability to perceive odor active compounds will affect perception of overall aroma