Impact of sulfur dioxide and temperature on culturability and viability of Brettanomyces bruxellensis in Wine.

Brettanomyces is a major threat to red wine quality, causing off-odors such as "medicinal," "barnyard," or even "sewage" during aging. Although sulfites (SO2) are used to limit spoilage by these yeast cells, reduced storage temperatures may lessen SO2 requirements. To test this hypothesis, a 4 | 4 factorial experimental design with molecular SO2(mSO2) concentration (0.0, 0.2, 0.5, or 1.1 mg/liter) and storage temperature (22, 18, 15, or 10°C) was devised. Of three strains evaluated, B5 was the lone strain to regain culturability following exposure to 0.5 mg/liter mSO2 (18°C), whereas only F3 remained culturable in the absence of mSO2 at 10°C. Application of fluorescence microscopy using two different probes and quantitative PCR assays revealed only a 2-log reduction in metabolically active cells from wines with SO2 that were not culturable on nonselective media. Culturability in these wines eventually returned regardless of the concentration of mSO2 present. In addition, 4-ethylphenol production ceased upon addition of SO2. These findings provide additional support that Brettanomyces can enter a "viable-but-not-culturable" state upon exposure to sulfites. Given the diversity among strains, maintaining conditions of ≤15°C and ≥0.4 mg/liter mSO2 will help limit spoilage by Brettanomyces but will not lead to its complete eradication.