Wines Enology Grape Chemistry Lab at Virginia Tech

Enology Notes

Enology Notes # 92 July 29, 2004

To: Regional Wine Producers

From: Bruce Zoecklein, Head, Wine/Enology-Grape Chemistry Group, Virginia Tech

Subjects: Brettanomyces; Laboratory Services; New Books

Brettanomyces. Brettanomyces continues to be a problem for premium wine producers. In a study funded by the Virginia Winegrowers Advisory Board (Fugelsang and Zoecklein, 2002), replicated, sterile Pinot noir (Vitis vinifera L.) wines were individually inoculated with one of eight strains of Brettanomyces bruxellensis at initial viable cell numbers < 50 cfu/mL. In two separate studies, population changes were monitored for 23 months, or until cell densities peaked and subsequently declined to < 30 cfu/mL. Significant variation was noted in both growth rate and population densities among strains. Significant increases in the concentration of 4-ethylphenol occurred after accumulated cell populations reached 2.5 x 105 cfu/mL.

This study demonstrated that strains of Brett could have very different growth patterns (see Figure 1). As can be noted, several strains appeared to decline in cell population, and then bloom again. This may have resulted from a phenomenon now known as “active but not culturable” (Phister and Mills, 2004). This calls into serious question the validity of the traditional culture plate method used by industry to determine the concentration of viable Brettanomyces.

Brettanomyces has the ability to produce a number of organic compounds which can impact wines. However, the sensory attributes of Brett wines relate not simply to concentration of certain metabolites, but to the ratio of these components and their interactions with the wine matrix. For example, it is possible to have wines with high concentrations of metabolites, including the traditional troublemaker, 4-ethylphenol, and not have a Brett character.

Therefore, wine composition can greatly influence the sensory threshold of volatile compounds produced by Brett. Wine composition is a function of the fruit and processing. Wines like Cabernet Sauvignon have a high detection threshold, while Temprenillo and others have low detection thresholds (Phister and Mills, 2004).

Tests conducted at the Wine/Enology-Grape Chemistry Group lab, and elsewhere, have demonstrated that volatile phenols can bind with higher molecular weight phenols during thermal processing and microoxygenation. This lowers volatility and makes some Brett metabolites less perceptible. On the other hand, oxygenation in the presence of viable Brett (splash racking, microoxygenation) can increase the population and may increase the production of taint metabolites.

Regardless, the analysis of 4-ethylphenol does not always correlate to the intensity of Brett character, or to the concentration of viable Brettanomyces species present.

There seems to be a significant strain variation. A survey conducted in Australia demonstrated that the ratio of two important metabolites, 4-ethylphenol to 4-ethylguaiacol, was lower in cooler than in warmer climates. This may a function of malvidin-3-coumaroyl glucoside production. This grape skin phenol is believed to be a precursor to 4-ethylphenol, and is found in higher concentrations in shaded fruit. Thus, there may be a link between vineyard management and Brettanomyces.

Variation among Brett strains was recently highlighted by Joseph and Bisson (2004).

16% of the Brett strains surveyed produced no 4-ethylphenol or 4-ethylguaiacol.

Some strains can metabolize ethanol. It had been assumed that Brett used sugars derived from barrels, micro-molar concentrations of glucose remaining in dry wines, or possibly the glucose derived by glycoside hydrolysis. Mansfield, Zoecklein, and Whiton (2002) demonstrated that Brett has the ability, in model solutions, to break down phenolic glycosides, like anthocyanins, liberating glucose. This breakdown can provide a carbon source and renders the anthocyanin molecule unstable, a reason why Brett wines frequently lack desirable color.

Joseph and Bisson (2004) reported that 30% of the Brett strains surveyed grew at 10C, but some grew at elevated temperatures (37C).

50% of the Brett strains reviewed grew in 30 mg/L free sulfur dioxide at pH 3.4. Brett strains demonstrate a large diversity in sulfur dioxide tolerance, from 14-56 mL molecular free SO2. This would suggest that it is much better to attempt control with a few large doses of sulfur dioxide, rather than a number of smaller doses.

Brettanomyces may be able to utilize DAP (diammonium phosphate) and nutrients in some commercial nitrogen supplements. This would suggest that the addition of excess N is counter-indicative. Too much N increases the fermentation rate, and changes the ratio of esters to long-chain alcohols produced by Saccharomyces. Additionally, excess nitrogen may stimulate the growth of undesirable organisms such as Brettanomyces. For a simple test to determine the nitrogen status of the juice, see our website,; go to Extension, then On-Line Publications & Current Topics, then Reduced Volume Formol Titration.

50% of the strains surveyed by Joseph and Bisson (2004) formed biofilms. An example of a biofilm is the plaque which forms on teeth. At this liquid-solid interface, microorganisms such as Brettanomyces can slough off into the environment. How well a sanitation procedure breaks down the biofilm determines, to a large degree, its effectiveness.

Laboratory Services. Members of the Virginia Wineries Association are working with Virginia Tech to upgrade the extension lab service. We are in the process of obtaining bids for an automated system to provide complete and rapid analytical support to the Virginia wine industry. This would be a fee-based service, the proceeds of which would be used for industry extension activities.

New Books. Two new books are available which I believe are highly useful. Growing Quality Grapes to Winery Specifications (Krstic et al., 2003) discusses quality measurement and management options for winegrowers. This is an excellent resource that every grower should have (published by Winetitles, ISBN # 1 875130 39 X).

Soil, Irrigation and Nutrition (Nicholas, 2004) discusses the physical and biological characteristics of soils, soil management, including cover crops and herbicide application, soil nutrition, etc., in a very easy-to-read format (published by South Australian Research and Development Institute, ISBN # 1 875130 40 3).

References for Brettanomyces.

Fugelsang, K.C., and B.W. Zoecklein. 2003. Population dynamics and effects of Brettanomyces bruxellensis strains on Pinot noir (Vitis vinifera L.) wines. Am. J. Enol. Vitic. 54:294-300.

Joseph, C.M.L., and L. Bisson. 2004. Physiological diversity of Brettanomyces/Dekkera isolated from wine. In Technical Abstracts, 55th Annual Meeting, San Diego, California, p. 28. American Society for Enology and Viticulture, Davis, CA.

Mansfield, A.K., B.W. Zoecklein, and R.S. Whiton. 2002. Quantification of glycosidase activity in selected strains of Brettanomyces bruxellensis and Oenococcus oeni. Am. J. Enol. Vitic. 53:303-307.

Phister, T.G., and D.A. Mills. 2004. Novel methods to detect Brettanomyces (Dekkera) in wine. In Technical Abstracts, 55th Annual Meeting, San Diego, California, p. 30. American Society for Enology and Viticulture, Davis, CA.

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Dr. Bruce Zoecklein
Professor and Enology Specialist Head Enology-Grape Chemistry Group
Department of Food Science and Technology, Virginia Tech
Blacksburg VA 24061
Enology-Grape Chemistry Group Web address:
Phone: (540) 231-5325
Fax: (540) 231-9293