Efficiency of population-dependent sulfite against Brettanomyces bruxellensis in red wine

Brettanomyces bruxellensis is considered as a spoilage yeast encountered mainly in red wine. It is able to reduce vinylphenols from phenolic acids to ethylphenols. These volatiles are responsible for the phenolic "Brett character" described as animal, farm, horse sweat and animal leather odors. Other molecules are responsible for organoleptic deviations described as "mousiness taint". SO₂ is the product most often used by winemakers to prevent B. bruxellensis growth. Usually, the recommended molecular dose of SO₂ (active SO₂, mSO₂) is highly variable, from 0.3 to 0.8mg/L. But these doses do not take into account differences of strain resistance to sulfites or population levels. Moreover, SO₂ is known as a chemical stressor inducing a viable but nonculturable (VBNC) state of B. bruxellensis. These cells, which are non-detectable by plate counting, can lead to new contamination when the amount of sulfite decreases over time. Consequently, we first assessed the effect of SO₂ levels in red wine on two strains with phenotypically different sulfite resistances. Then, we studied the relationship between amounts of SO₂ (0, 0.5, 0.9 and 1.1mg/L active SO₂) and population levels (10³, 10⁴ and 10⁵cells/mL) in red wine. Yeasts were enumerated by both plate counting and flow cytometry over time using viability dye. Our results showed different SO₂ resistances according to the strain used. A relationship between yeast population level and SO₂ resistance was demonstrated: the higher the yeast concentration, the lower the efficiency of SO₂. Under certain conditions, the VBNC state of B. bruxellensis was highlighted in red wine. Yeasts in this VBNC state did not produce 4-EP. Moreover, cells became culturable again over time. All these results provide new information enabling better management of sulfite addition during wine aging.