4-EP/4-EG Technical Bulletin
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Brettanomyces Monitoring by Analysis

Brettanomyces Monitoring by Analysis of 4-ethylphenol and 4-ethylguaiacol

Volatile Phenols 

Brettanomyces yeast are unique in their ability to readily synthesize the volatile phenols 4-ethylphenol (4-EP) and 4-ethylguaiacol (4-EG) in standard wine conditions. These compounds are useful for monitoring Brettanomyces and are an important component of Brettanomyces wine character.

ETS Laboratories has offered 4-ethylphenol as a tool for monitoring Brettanomyces in wine since 1993. New methods and equipment now allow detection and measurement of 4-ethylguaiacol as a standard part of the 4-ethylphenol report.

Brettanomyces and Ethyl Phenols
Heresztyn and Chatonnet demonstrated the strong link between 4-EP in wine and Brettanomyces. Subsequent research has shown that Brettanomyces is the primary wine organism capable of producing 4-ethylphenol. As a by-product of infection, 4-EP is an excellent indicator of Brettanomyces presence and activity.

The biosynthesis of ethyl phenols by Brettanomyces is based on the enzymatic conversion of vinyl phenols derived from cinnamic acids.

Early Detection
Analysis of wines for 4-EP enables wineries to identify the presence of Brettanomyces populations large enough to impact wine sensory.  Once Brettanomyces is detected, SO2 can be used to slow development and the wine can be targeted for ongoing monitoring.

Population Control

4-EP can be used to monitor the effectiveness of Brettanomyces control programs in a wine where Brettanomyces is known to exist. First, a baseline concentration of 4-EP needs to be established. Following treatment, wines with static 4-EP concentrations can be assumed to have inactive populations. Increases indicate continued Brettanomyces activity and ineffective treatment.

Significance of 4-Ethylguaiacol (4-EG)

4-EG is also a valuable indicator of active Brettanomyces populations. As with 4-EP, 4-EG is synthesized from cinnamic acid precursors, but at lower concentrations. The average production ratio is 8 parts 4-EP for every 1 part 4-EG. The aroma profiles of 4-EG and 4-EP are distinctly different. Their combined concentration can be used to estimate the sensory impact of Brettanomyces. Relative concentrations of 4-EG and 4-EP influence the character of Brettanomyces sensory effects.  relative concentration.

Graph #1 displays measured concentrations of 4-EP and 4-EG in three wines. In each case, the concentration of 4-EP is fairly consistent. The major difference is the relative concentration of 4-EG. Displayed ratios of 4-EP: 4-EG range from 3:1 to 18:1

Graph #2
presents the same data in terms of the relative sensory impact. “Olfactory Units” are calculated by dividing the concentration by the respective sensory threshold. The results clearly illustrate the significance of 4-EG to the overall sensory impact of Brettanomyces.

Sensory Impact

Both 4-ethyl phonel (4-EP) and 4-ethyl guaiacol (4-EG) are often described as “phenolic”. Other descriptors often used for 4-EP are  “medicinal” and “band aid”, while 4-EG is more often described as “spicy” and “smoky”.

Typically, 4-EG is present in much lower quantities in red wine than 4-EP, usually about 8 times less. However, 4-EG is a more volatile compound with a sensory threshold much lower than 4-EP. In a wine with Brettanomyces, both compounds may be well above sensory thresholds.

Perceived "Brett" character in red wine is influenced by the concentration of both compounds. Variation in the concentration of 4-EG helps to explain why the flavor and intensity of perceived Brett character can be very different with wines having similar 4-EP concentrations.

Analysis for 4-EG in conjunction with 4-EP offers a more complete and superior analytical tool than testing for a single compound. It also offers several advantages over simple colony counts derived from plating wine samples on specialized media.

Plating vs. Ethyl Phenol Analysis
Plating on specialized media followed by microscopic evaluation can be used to monitor Brettanomyces presence and populations. However, there are several limitations to plating which make chemical analysis an attractive adjunct or alternative.
  • Brettanomyces yeast are not uniformly distributed in tanks or barrels so representative samples are often difficult to obtain.
  • Incubation groth and presumptive identification can take up to 14 days.
  • Other yeasts with similar growth characteristics can result in false positives.
  • Low cell viability due to SO2 additions often results in false negatives.
  • Plating will not detect viable but non-culturable cells (VNC).

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