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The Metabolomics of Brett Flavor
Metabolomics
• Initial analysis done in medium
– Greater control of substrate availability
• Further analysis done in wine
– Analyzed internal cell metabolism
– Analysis of external metabolites?
Brettanomyces Used In These Studies
Strain Difference-Head Space Analysis
Strain and Substrate Difference-Head Space
Analysis
Metabolites in Head Space That Account
for Differences
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
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
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
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
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.
Greatest Odor Impact by GC-O
Compounds Found in SPME Analysis
Compounds that Separated Strains and Substrates
Compounds Chemically Related to Those
Found in the SPME Analysis
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
Cells in Media Compared to Cells in Wine for 15
Minutes
1 = Media
2 = Wine
Cells in Wine at 15 Minutes and 54
Days
1 = 15 minutes
2 = 54 days
Media Grown Cells at 8 Days and Wine
Grown Cells at 54 Days
3 = Wine
4 = Media
Overall Wine Time Course
1 = 15 minutes
2 = 8 days
3 = 54 days
Wine Time Course by Strain
2082
2091
2807
1 = media
2 = 15 minutes
3 = 8 days
4 = 54 days
Most Important Pathways – Wine Time
Course
Nitrogen metabolism
Alanine, aspartate, glutamate metabolism
Pentose glucuronate
Interconversion
Butanoate Metabolism
Glutathione metabolism
Arg, pro metabolism
Glycerolipid Metabolism
Glycerophospholipid
Gly, ser, thr metabolism
Most Important Pathways – First 15
Minutes
Arginine and proline metabolism
Ala, asp and glu metabolism
Purine
Glutathione
Terpenoid backbone biosynthesis
Lysine
Inositol Phosphate
metabolism
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
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)
Flavor? - MSG
Aromas
Fatty Acid, Alcohol, Aldehyde
Aromas -Sulfur Compounds
Aromas -Terpenoids
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