Ethlyphenols analysis is an excellent indicator of Brettanomyces presence and activity and is a good complement to other types of microbiological testing, like Scorpions analysis.
Is Brettanomyces Really A Problem?
Yes and no. It all depends on your opinion of the sensory impact of Brettanomyces. Some winemakers accept or encourage the aromas that result from various concentrations of Brettanomyces. Others want no Brettanomyces sensory impact and will do everything possible to control or prevent Brettanomyces growth.
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 is the conversion of vinyl phenols to ethyl phenols. As a common byproduct of Brettanomyces, 4-EP is an excellent indicator of Brettanomyces presence and activity.
Is Brettanomyces Activity The Only Source Of 4-EP And 4-EG?
No, other organisms, for example, Pichia guillermondi, can produce low levels of 4-EP. However, the work done by Chatonnet in France, and validation work done at ETS, has revealed no reason to suspect another significant source of 4-EP in standard wine. Neither 4-EP nor 4-EG are normal constituents of red wine. It is not unusual to find three- and four-year-old barrel samples without detectable levels of either 4-EP or 4-EG.
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 an internal standard by solid phase microextraction (SPME). This method can detect either compound at 4 ng/mL.
How large of a sample do I need for analysis?
Proper analysis requires a representative sample of at least 50mL. 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.
Are There Other Analytical Techniques That I Should Consider?
Plating on selective media can identify presumptive Brettanomyces populations, but will not detect yeast in a viable but nonculturable state. Plating provides information on the number of actively growing Brettanomyces in a wine.
The ETS Scorpions™ Yeast Panel positively identifies all viable Brettanomyces in the sample. The Scorpions™ Yeast Panel complements the plating analysis because it provides information on the percentage of Brettanomyces that are actively growing versus viable but non-culturable.
These analyses both complement the 4-EP and 4-EG analyses because they provide information on total viable and actively growing cell populations, which can be used to determine if 4-EP and 4-EG levels are at risk to increase.
Can Ethylphenol 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 usually an indicator of Brettanomyces activity. For other wines with unknown ethyl phenol history, two data points are required. An initial number must first be obtained to measure the current concentration of 4-EP and 4-EG. Once this number is known, any increase in concentration usually indicates active Brettanomyces.
How Can I Best Apply The Analysis In My Winery?
There are several applications for 4-EP/4-EG analysis:
Early detection and control of spoilage produced by Brettanomyces populations.
Analysis of 4-EP allows the winemaker to detect populations when they first start to impact the wine. This allows the winemaker to arrest the development of Brettanomyces at very early stages.
4-EP can be used to monitor the effectiveness of 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.
What Do 4-EP / 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”
What Are The Sensory Thresholds Of 4-EP And 4-EG?
The sensory threshold will vary with 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. The contribution of both compounds to the “Brett” character depends on their absolute and relative concentrations.
Can I Use 4EP / 4EG Analysis To See If My Sterile Filtration Was Successful?
No, the presence of 4-EP and 4-EG will remain in the wine even if all active Brettanomyces have been removed from the wine.
Do 4-EP And 4-EG Always Have A Constant Ratio In Wine?
No, the ratio of 4-EP to 4-EG varies from wine to wine. This variation helps explain why the flavor and intensity of perceived “Brett” character can be different in wines having similar 4-EP levels but different levels of 4-EG. Typically, 4-EP is about 8 times higher in wine than 4-EG. However, this ratio can vary. In a recent study at ETS three hundred red wines were analyzed and 4-EP: 4-EG ratios ranged between 3:1 to 22:1.