Wine is particularly vulnerable to infection by Brettanomyces bruxellensis during or shortly after malolactic fermentation (MLF). While different methods and techniques enable winemakers to prevent wine spoilage due to this organism, no single intervention is universally sufficient. Moreover application of sulfur dioxide, the most commonly used of these methods, is unavailable to winemakers during MLF due to the sensitivity of Oenococcus oeni to that substance. It is therefore generally suggested that winemakers carry out a rapid and healthy MLF in order to reduce the time a wine is without the protection of sulfur dioxide. In recent years however, some studies have suggested that MLF may also act to inhibit wine spoilage by Brettanomyces bruxellensis. This study investigated this additional benefit of MLF by studying the influence of MLF on the growth and volatile phenol production of Brettanomyces bruxellensis. Additionally, the impacts of microbial strain and wine ethanol concentration were also investigated with respect to that influence. Potential causes for the inhibitory interactions between O. oeni and B. bruxellensis were also explored. Experiments were conducted in Pinot noir wine produced in the 2018 and 2019 winegrowing seasons. A strain of Brettanomyces bruxellensis that had previously shown potential sensitivity to MLF, strain UCD-2049, was inoculated into wine at the end of a 14 day MLF conducted by ten different strains of Oenococcus oeni. UCD-2049 populations declined after inoculation after MLF for all strains of O. oeni tested. When inoculated into a control wine that had not undergone MLF with O. oeni, no suppression of growth was observed. Significantly higher concentrations of 4-ethyl phenol and 4-ethyl guaiacol were measured in wine that had not undergone MLF when compared to wine that had undergone MLF using any of the ten strains tested.
To determine possible mechanisms of inhibition an experiment was conducted where B. bruxellensis was inoculated into wine that had just completed MLF but O. oeni cells were separated from B. bruxellensis by a dialysis membrane. The dialysis membrane allowed physical separation of the microorganisms but free flow of wine. While B. bruxellensis populations declined rapidly and remained repressed for many weeks when in direct contact with O. oeni, populations only declined slightly and quickly recovered if O. oeni was separated from B. bruxellensis by a dialysis membrane. This finding suggests that inhibition by O. oeni is related to cell to cell contact rather than depletion of nutrients or production of an inhibitory compound.
Strain variation in Brettanomyces bruxellensis was tested by selecting a highly “suppressive” strain of Oenococcus oeni, strain Alpha, and determining the effect of MLF by this strain on the growth and volatile phenol production of a number of strains of Brettanomyces bruxellensis. The impact of Oenococcus oeni strain Alpha was tested by inoculating each different Brettanomyces bruxellensis strain into a wine at the end of a 14 day MLF. B. bruxellensis populations in both the control and MLF treated wines recovered to a similar level by the end of the experiment. The possibility that ethanol tolerance differences between the B. bruxellensis strains contributed to their variable susceptibility to inhibition by O. oeni was investigated. B. bruxellensis strains were inoculated into wines that had been adjusted to a high or low ethanol content and that had or had not just completed MLF. While B. bruxellensis populations behaved similarly to the control groups in the low ethanol wines, in the high ethanol wines two of the three strains of B. bruxellensis tested were inhibited in the MLF treated wines with respect to the control. For the third B. bruxellensis strain, the reverse was true. B. bruxellensis strain was a major factor in volatile phenol production, though some suppression of volatile phenol production by MLF was observed in cases where there was no suppression of growth.
This study demonstrated that Brettanomyces bruxellensis response to MLF is dependent on Brettanomyces bruxellensis strain, and that this response may be impacted by the ethanol concentration of the wine. It was also determined that sensitivity to MLF with respect to culturable cell growth is likely related to cell-cell contact between Brettanomyces bruxellensis and Oenococcus oeni.