Utilizing Malolactic Fermentation as a Tool to Prevent Brettanomyces Bruxellensis Wine Spoilage
Brettanomyces bruxellensis is considered one of the most problematic wine spoilage yeasts due to the difficulty of controlling it, the potential significant financial losses due to loss of wine quality, and the cost of prevention and remediation measures. Wine is particularly vulnerable to B. bruxellensis infection during and shortly after the malolactic fermentation (MLF) as SO2 cannot be added until this process is complete. It has been suggested that conducting a rapid MLF initiated by inoculation of Oenococcus oeni is a useful strategy to prevent B. bruxellensis spoilage as this minimizes the length of time the wine is not protected by SO2. This project investigates an additional benefit of conducting a rapid MLF, the prevention of B. bruxellensis growth due to inhibitory interactions with O. oeni. Pinot noir wine (no SO2 additions, no MLF) was produced and used to test the ability of a large number of commercial O. oeni strains to inhibit B. bruxellensis growth at the end of MLF. Sterile filtered wine was inoculated with one of eleven commercial O. oeni strains and growth and malic acid monitored. When MLF was complete, wines were inoculated with a select strain of B. bruxellensis and growth and volatile phenol production monitored.
All O. oeni strains tested inhibited the growth of B. bruxellensis UCD2049 in Pinot noir wine with O. oeni strain variation observed. O. oeni strains Alpha, 350, VP41, MBR31 and PN4 most strongly inhibited growth of B. bruxellensis UCD2049, while strains CH11, Omega, Beta, and VFO 2.0 inhibited B. bruxellensis to a lesser extent. The potential mechanism of this inhibition was investigated by using a dialysis membrane to physically separate O. oeni and B. bruxellensis cells but allow free movement of nutrients and other potential inhibitory compounds. The physical separation of O. oeni from B. bruxellensis relieved the inhibition of B. bruxellensis by O. oeni that occurred when the two microorganism were in present together. These results indicate that inhibition is not due to nutrient depletion by O. oeni as nutrients could flow freely across the dialysis membrane. It is also unlikely that B. bruxellensis inhibition was due to the production of an inhibitory compound by O. oeni as any potential inhibitory compound would also have passed through the dialysis membrane. Instead, these results provide strong evidence that the inhibition of B. bruxellensis by O. oeni is due to cell-cell contact.
The sensitivity of additional B. bruxellensis strains to O. oeni was also determined. While B. bruxellensis UCD2049 populations declined rapidly when inoculated into Pinot noir wine that had just completed MLF with O. oeni Alpha, growth of the other B. bruxellensis strains tested was not impacted. Why B. bruxellensis strain UCD2049 was inhibited by O. oeni while the other B. bruxellensis strains were not was subsequently investigated. Initial experiments considered whether ethanol tolerance between B. bruxellensis strains impacted inhibition by O. oeni. Given that earlier experiments had been conducted in 13% (v/v) wine, wines were instead adjusted to 12.5% or 14% (v/v) ethanol. In low (12.5%) ethanol wine that had undergone MLF, B. bruxellensis UCD2049 grew well, in contrast to what was observed in 13% wine where growth was inhibited. B. bruxellensis strains AWRI-1499 and Copper Mountain also grew well in low ethanol wine with no difference between treatments. In higher ethanol wine, B. bruxellensis UCD2049 struggled to grow whether the wine had undergone MLF or not. In contrast, B. bruxellensis strains AWRI-1499 and Cooper Mountain grew well in the higher ethanol wine. B. bruxellensis strains AWRI-1499 populations recovered slower in wine that had undergone MLF while the opposite occurred for strain Copper Mountain. These results demonstrate that ethanol tolerance differences between B. bruxellensis strains impact their inhibition by O. oeni. For example, strain UCD2049 was not inhibited by O. oeni in wine at 12.5% ethanol but was inhibited in 13% and 14% ethanol wine. Additional experiments will be conducted where pH will also be considered as tolerance to this factor is known to differ between B. bruxellensis strains. Experiments are also underway exploring how long MLF induced B. bruxellensis inhibition last as well as whether B. bruxellensis inhibition occurs if infection happens at the beginning or mid-point of MLF.