1. The Metabolomics of Brett Flavor

2. Metabolomics Initial analysis done in medium – Greater control of substrate availability Further analysis done in wine – Analyzed internal cell metabolism – Analysis of external metabolites?

3. Brettanomyces Used In These Studies

4. Strain Difference- Head Space Analysis

5. Strain and Substrate Difference -Head Space Analysis

6. Metabolites in Head Space That Account for Differences

7. Fatty Acids 2-methyl butyric acid - pungent acid roquefort cheese, acidic, fruity, dirty, cheesy, acidic with a dairy buttery and cheesy nuance 3-methyl butyric acid (isovaleric acid) – sour, stinky feet, sweaty, cheese, tropical, dairy, acidic, pungent, fruity, stinky, ripe fatty and fruity notes, creamy, fermented, sweet, waxy and berry Octanoic acid (caprylic acid) – fatty, waxy, rancid, oily vegetable, cheesy Isobutyric acid – sharp, acetic, dairy-like, cheesy, buttery with a fruity nuance, sour

8. Fusel Alcohols Phenethyl alcohol – floral, rose, dried rose, flower, rose water, sweet, bready with a rosy honey nuance 2-methyl-1-butanol – roasted, wine, onion, fruity 3-methyl-1-butanol (isoamyl alcohol) - alcoholic, pungent, ethereal, cognac, fruity, banana, molasses, whiskey

9. Esters Ethyl dodecanoate - Oily, fatty, floral with fatty fruity taste Ethyl octanoate – fruity, wine, waxy, sweet, apricot, banana, brandy, pear, musty, pineapple and fruity with a creamy, dairy nuance, mushroom and cognac notes Ethyl-2-methyl butyrate – sharp, sweet, green apple, fruity, berry with fresh tropical nuances, grape, pineapple, mango and cherry notes Phenethyl acetate – floral, sweet, honey, fruity, tropical, rosy with a slight yeasty honey note with a cocoa and balsamic nuance, rosy with a slight green nectar fruity note Ethyl-9-hexadecanoate – brandy, whiskey Ethyl isobutyrate – sweet, ethereal, fruity, alcoholic, rummy, fruity with a rum-and egg nog-like nuance

10. Ethyl Phenols 4-ethyl phenol – phenolic, BandAid, castoreum, smoke, guaiacol, creosote, savory 4-ethyl guaiacol – spicy, smoky, bacon, phenolic, clove, medicinal, woody and sweet vanilla nuances

11. Olfaction SPME head space analysis showed compounds present in media after Brett growth Are these major compounds the ones that are having the most odor impact? SPME GC-Olfactory analysis attempted to answer that question.

12. Greatest Odor Impact by GC-O

13. Compounds Found in SPME Analysis

14. Compounds that Separated Strains and Substrates

15. Compounds Chemically Related to Those Found in the SPME Analysis

16. Metabolomics Analysis of Cellular Metabolites in Wine The chemical complexity of wine makes it difficult to analyze by these techniques Brettanomyces cells grown in wine were analyzed over time to determine which intracellular metabolites were most important during growth in wine (UC Davis Metabolomics Core Facility) Metabolites identified were used to determine metabolic pathways involved

17. Cells in Media Compared to Cells in Wine for 15 Minutes 1 = Media 2 = Wine

18. Cells in Wine at 15 Minutes and 54 Days 1 = 15 minutes 2 = 54 days

19. Media Grown Cells at 8 Days and Wine Grown Cells at 54 Days 3 = Wine 4 = Media

20. Overall Wine Time Course 1 = 15 minutes 2 = 8 days 3 = 54 days

21. Wine Time Course by Strain 208220912807 1 = media 2 = 15 minutes 3 = 8 days 4 = 54 days

22. Most Important Pathways – Wine Time Course Pentose glucuronate Interconversion Alanine, aspartate, glutamate metabolism Nitrogen metabolism Butanoate Metabolism Glycerolipid Metabolism Gly, ser, thr metabolism Glutathione metabolism Arg, pro metabolism Glycerophospholipid

23. Most Important Pathways – First 15 Minutes Ala, asp and glu metabolism Arginine and proline metabolism Terpenoid backbone biosynthesis Glutathione Purine Inositol Phosphate Lysine metabolism

24. What Does This Have to do With Flavor? Metabolites are the final products of the genes and proteins The pathways tell us which products are being made All those pathways are interconnected

25. Pathway to Flavor and Aroma Amino acids/Purine metabolism – L-glutamate and ribonucleotides Butanoate metabolism – short chain fatty acids and alcohols Gultathione metabolism - sulfur and amino acids Glycerolipid/Glycerophospholipid/Inositol Phosphate/Pentose Glucuronate Interconversion - fatty acid Terpenoid backbone synthesis – isoprenoids, monoterpenoids (C10), sesquiterpenoids (C15), and diterpenoids (C20)

27. Flavor? - MSG

28. Aromas Fatty Acid, Alcohol, Aldehyde

29. Aromas -Sulfur Compounds

30. Aromas -Terpenoids

31. Pathway to Flavor and Aroma Amino acids/Purine metabolism – L-glutamate and ribonucleotides Meaty/Savory Butanoate metabolism – short chain fatty acids and alcohols Roasted/Onion/Whiskey/Fruity Glycerolipid/Glycerophospholipid/Inositol Phosphate/Pentose Glucuronate Interconversion - fatty acid Cheesy/Sweaty/Rancid Terpenoid backbone synthesis – isoprenoids, monoterpenoids (C10), sesquiterpenoids (C15), and diterpenoids (C20) Resin/Woody/Floral Ethyl phenols – 4-ethyl phenol, 4-ethyl guaiacol Phenol/Medicinal/Smoke/spice