FAQ - Brettanomyces Markers

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

1. Is Brettanomyces really a problem?
   Yes and no.  It all depends on your opinion of the sensory impact of Brettanomyces.  Some winemakers are oblivious to the aromas that result from Brettanomyces or feel that they add to complexity.  Others want no Brettanomyces sensory impact and will do everything possible to control or prevent Brettanomyces growth.
    
2. How are 4-ethylphenol (4-EP) and 4-ethylguaiacol (4-EG) formed?
   4-EP and 4-EG are formed from cinnamic acid precursors in wine.  There are several steps in the synthesis pathway.  The first steps are common to several wine microorganisms.  The last step, the conversion of vinyl phenols to ethyl phenols is unique to Brettanomyces.  As a unique by-product of Brettanomyces, 4-EP is thus an excellent indicator of Brettanomyces presence and activity.
    
3. Is Brettanomyces activity the only source of 4-EP and 4-EG?
   In a word – Yes.  Neither the work done by Chatonnet in France, nor the validation work done here at ETS, has revealed any reason to suspect another source of significant amounts.  Neither 4-EP or 4-EG are normal constituents of red wine.  It is quite common to find three and four year old barrel samples without detectable levels of either 4-EP or 4-EG.
    
4. How do you analyze for 4-EP and 4-EG?
   ETS uses gas chromatography with detection by mass spectroscopy (GC/MS).  The compounds are extracted from the sample along with a deuterated internal standard by solid phase micro extraction (SPME).  This method can detect either compound at 4 ng/mL, low enough to detect minute populations of Brettanomyces.
    
5. What is adequate sample for analysis?
   Samples should consist of a representative sample of at least 30mL.  Ethyl phenols are well distributed within an individual tank or barrel, but sampling must take into consideration the high degree of variation between individual barrels or tanks.
    
6. Are there other analytical techniques that I should consider?
   Plating on selective media can confirm active Brettanomyces populations, but there are several problems inherent in the technique.  Unlike the 4-EP, the Brettanomyces population is not uniformly distributed within barrels.  Sampling is relatively complicated and stirring is often required.  Plating is time consuming (plates are cultured a minimum of 12 days), and often result in false negatives.
    
7. How can I best apply the analysis in my winery?
   There are several applications for 4-EP/4-EG analysis.  Two very beneficial uses are:
   1. Early detection and control of developing Brettanomyces populations.
      
      Analysis of 4-EP allows the winemaker to detect developing populations at very low levels, often less than 2 col/mL.  This allows the winemaker to arrest the development of Brettanomyces at very early stages with only modest SO2 additions.
       
   2. Control of established populations.
      
      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 is determined.  Following treatment, wines with static 4-EP concentrations can be assumed to have inactive populations.  Increases in 4-EP indicate continued Brettanomyces activity and ineffective treatment.
       
8. Can ethyl phenol analysis tell me if there is active Brettanomyces in my wine?
   When applied properly, yes.  In the case of young wines beginning at < 4 ng/mL, any measured 4-EP and 4-EG after time is an indicator of Brettanomyces activity.  For other wines with unknown ethyl phenol history, two data points are required.  An initial number must be first obtained to know the current content of 4-EP and 4-EG.  Once this number is known, any increase in concentration indicates active Brettanomyces.
    
9. What do 4-EP and 4-EG have to do with “Brett Character”?
   An active Brettanomyces population will create several by-products including 4-EP and 4-EG.  These two compounds are responsible for “phenolic” notes associated with the “Brett” character.  4-EP is usually described as “medicinal” or “band aid”, while 4-EG is more often described as “spicy” and “smoky”.
    
10. What are the sensory thresholds of 4-EP and 4-EG?
    The sensory threshold will vary on the taster and the wine matrix, but generally a wine is described as having a Brettanomyces like character when 4-EP reaches between 300 and 600 ng/mL.  4-EG has a lower threshold, somewhere around 50 ng/mL. Contribution of both compounds to the “Brett” character depends on their absolute and relative concentrations.
     
11. Do 4-EP and 4-EG always have a constant ratio in wine?
    Typically, 4-EP is about 8 times higher in wine than 4-EG.  The ratio can vary however. Three hundred red wines recently analyzed at ETS had 4-EP: 4-EG ratios between 3:1 to 22:1.  Variation in the 4-EG level can help to explain why the flavor and intensity of perceived “Brett” character can be very different in wines having similar 4-EP levels.