The Metabolomics of Brett Flavor Metabolomics Initial analysis

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, Band. Aid, 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 (C 10), sesquiterpenoids (C 15), and diterpenoids (C 20)

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 (C 10), sesquiterpenoids (C 15), and diterpenoids (C 20) Resin/Woody/Floral • Ethyl phenols – 4 -ethyl phenol, 4 -ethyl guaiacol Phenol/Medicinal/Smoke/spice