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 Substrates

• • Grown 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

Caffeic Acid

Phenolic Acids

Amino Acids

Lysine Tryptophan Phenylalanine Tyrosine

Vinyl and Ethyl Phenols

OH H OH H OH H CH CH COOH Cinnamate decarboxylase CH CH 2 Vinyl phenol reductase CH 2 CH 2 H = coumaric OH = caffeic OMe = ferulic

Proposed Pathway for Mousy Aromas from Lysine in Brettanomyces

ETHP = 2-ethyltetrahydropyridine ATHP = 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?

10 million olfactory sensory neurons on the olfactory epithelium 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.

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 occur on 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 Substrates Substrate

Coumaric Acid Ferulic Acid Phenylalanine

Time (min)

16 to 18 22 3 to 4 6 7 to 8 14 to 15 18 to 22 3 to 4 6 to 8 14 to 15 19 to 20

Aroma

Chemical, asphalt, irritating Clove, animal Fruity, butterscotch Orange, sweet, floral Cheesy, dirty sock, sweaty Floral, almond oil, ink Spicy, smoky, tumeric, medicinal Fruity, rotting flowers, plastic Plant, sweaty, stinky Floral, sweet, medicinal, rose Metallic, sulfur Tyrosine/Tryptophan 3 to 4 6 to 8 14 Sweet, fruity, chemical, sharp Rotten, cheesy, sweaty, rancid Perfume, rose, pepper, unpleasant floral

Types of Chemical Compounds Produced

Type of Compound

Ethyl/Vinyl Phenols

Substrates

Phenolic acids

Panel’s Aroma Descriptors

Chemical, smoke, spicy, plastic Fatty Acids Fatty Acid Esters Long Chain Alcohols Pyridines Terpenes Amino Acids Amino Acids Rotten, rancid, sweaty, barnyard Artificial fruit and floral Phenolic Acids Amino Acids Amino Acids Chemical, solvent, floral, fruit Animal, mousy Phenolic Acids Amino Acids Spicy, floral, tropical, toasty

Genetics of Fatty Acid Metabolism in

Brettanomyces

Genes found in

Brettanomyces

that are not found in Saccharomyces

Species found in

Saccharomyces kluyveri Pichia pastoris Pichia pastoris Ashbya gossypii Pichia pastoris

Gene Name Function

FAD3 omega-3 fatty acid desaturase delta 8-(E)-sphingolipid desaturase delta 4-(E)-sphingolipid desaturase Sphingolipid C9-methyltransferase Ceramide glucosyltransferase

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