Aromatic diversity of Brettanomyces

C.M. Lucy Joseph Department of Viticulture and Enology

U.C. Davis

Brettanomyces Aromas

• Horse sweat - Leather• Earthy• Medicinal• Band Aid• Smoky• Tobacco• Barnyard• Putrid• Lilac

Brettanomyces SubstratesGrown in a defined medium with the

following substrates:• Cinnamic acids:– Coumaric Acid– Ferulic Acid– Caffeic Acid

• Amino Acids– Lysine– Phenylalanine– Tryptophan– Tyrosine

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

Phenolic Acids

Caffeic Acid

Amino Acids

Tyrosine

Phenylalanine

Tryptophan

Lysine

Vinyl and Ethyl Phenols

CH

CH

COOH

H

OH

CH

CH2

CH2

CH2

OH OH

H H

Cinnamate

decarboxylaseVinyl phenol

reductase

H OH OMe= coumaric = caffeic = ferulic

Proposed Pathway for Mousy Aromas from Lysine in Brettanomyces

ETHP = 2-ethyltetrahydropyridineATHP = 2-acetyltetrahydropyridine

Amino Acid to Alcohols in Saccharomyces

• Phenylalanine

Amino Acid to Alcohols in Saccharomyces

• Tryptophan

Sensory Analysis

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

Review of Human Olfactory Sensory Detection

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

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

Olfactory Receptor Gene Distribution

Olfactory receptor genes occuron all chromosomes except 20 and Y.

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

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.

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

Difference in Panelists Perception of Standards

Difference in Panelists Perception of the Same Sample

Difference in Odor Perception of One Panel Member on Different Days

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

Aromas Associated with SubstratesSubstrate Time (min) Aroma

Coumaric Acid 16 to 18 Chemical, asphalt, irritating

22 Clove, animal

Ferulic Acid 3 to 4 Fruity, butterscotch

6 Orange, sweet, floral

7 to 8 Cheesy, dirty sock, sweaty

14 to 15 Floral, almond oil, ink

18 to 22 Spicy, smoky, tumeric, medicinal

Phenylalanine 3 to 4 Fruity, rotting flowers, plastic

6 to 8 Plant, sweaty, stinky

14 to 15 Floral, sweet, medicinal, rose

19 to 20 Metallic, sulfur

Tyrosine/Tryptophan

3 to 4 Sweet, fruity, chemical, sharp

6 to 8 Rotten, cheesy, sweaty, rancid

14 Perfume, rose, pepper, unpleasant floral

Types of Chemical Compounds Produced

Type of Compound Substrates Panel’s Aroma DescriptorsEthyl/Vinyl Phenols Phenolic acids Chemical, smoke, spicy, plastic

Fatty Acids Amino Acids Rotten, rancid, sweaty, barnyard

Fatty Acid Esters Amino Acids Artificial fruit and floral

Long Chain Alcohols Phenolic Acids

Amino Acids

Chemical, solvent, floral, fruit

PyridinesPyridines Amino AcidsAmino Acids Animal, mousyAnimal, mousy

Terpenes Phenolic Acids

Amino Acids

Spicy, floral, tropical, toasty

Genetics of Fatty Acid Metabolism in Brettanomyces

Species found in Gene Name FunctionSaccharomyces kluyveri FAD3 omega-3 fatty acid desaturase

Pichia pastoris delta 8-(E)-sphingolipid desaturase

Pichia pastoris delta 4-(E)-sphingolipid desaturase

Ashbya gossypii Sphingolipid C9-methyltransferase

Pichia pastoris Ceramide glucosyltransferase

Genes found in Brettanomyces that are not found in Saccharomyces

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