In its 2020-21 fiscal year, the Oregon Wine Board of Directors granted $350,000 to researchers for eight projects with the potential to advance quality grape growing and winemaking in Oregon. The update below is part of a series to let you know about the status of these projects.

Dr. James Osborne is an associate professor and enology extension specialist in the Department of Food Science and Technology and the Oregon Wine Research Institute at Oregon State University. His current research focuses on the impact of wine microorganisms such as lactic acid bacteria, Brettanomyces and non-Saccharomyces yeast on wine quality. He has prepared the update below.

Utilizing malolactic fermentation as a tool to prevent Brettanomyces bruxellensis wine spoilage

Project objectives:
The overall objective of this study is to investigate interactions between the wine spoilage yeast Brettanomyces bruxellensis (“Brett”) and the malolactic bacteria Oenococcus oeni (O. oeni) in order to reduce the risk of wine spoilage.

The specific project objectives are to:

  • Investigate how O. oeni inhibits Brett growth and volatile phenol production and determine if strain variability exists;
  • Determine the mechanism by which Brett is inhibited by O. oeni; and
  • Investigate the impact of timing of Brett infection relative to malolactic fermentation on Brett growth inhibition and the persistence of Brett inhibition.

Importance to the Oregon wine community:
Brettanomyces bruxellensis is considered the most problematic wine spoilage yeast 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. Winemakers are limited in the tools available to prevent the infection and growth of Brett in wine, with SO2 and sanitation being the main options. However, SO2 cannot be added to a wine until malolactic fermentation (MLF) has been completed, making this time period a critical point where Brett spoilage can occur.

It has been suggested that conducting a rapid MLF initiated by inoculation of O. oeni is a useful strategy to prevent Brett 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 Brett growth due to inhibitory interactions with O. oeni.

Progress so far:
Process
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 Brett growth at the end of MLF. Sterile filtered wine was inoculated with one of 10 commercial O. oeni strains and growth and malic acid levels were monitored. When MLF was complete, wines were inoculated with a select strain of Brett, and growth and volatile phenol production were monitored.

The potential mechanism of Brett inhibition by O. oeni was investigated by using a dialysis membrane to physically separate O. oeni and B. bruxellensis cells in wine but allow free movement of nutrients and other potential inhibitory compounds. This allowed us to determine if inhibition occurs via cell-to-cell contact, nutrient depletion, or production of an inhibitory compound by O. oeni.

The impact of timing of Brett infection relative to MLF was explored by inoculating Brett into wine at the beginning, middle, or end of MLF conducted by O. oeni. In addition, the sensitivity of additional B. bruxellensis strains to O. oeni was also determined in a similar manner to the O. oeni screening.

Finally, the interactive impact of ethanol concentration and MLF on Brett inhibition was investigated by adjusting Pinot noir wine to three different ethanol concentrations. A portion of these wines underwent MLF while a portion did not. Brett was then inoculated into these wines and monitored for growth and volatile phenol production.

Findings
All 10 O. oeni strains tested inhibited B. bruxellensis UCD-2049 growth and volatile phenol production in Pinot noir wine when Brett was inoculated into wine at the end of MLF. Some O. oeni strain variation was observed. The potential mechanism of this inhibition was determined to most likely be due to cell-cell contact as physical separation of O. oeni cells from B. bruxellensis cells by a dialysis membrane relieved the inhibition of B. bruxellensis by O. oeni that occurred when the two microorganism were present together. This suggests that inhibition was not due to nutrient depletion by O. oeni and was also unlikely to be caused by an inhibitory compound.

The sensitivity of additional B. bruxellensis strains to O. oeni was also determined. While B. bruxellensis UCD-2049 populations declined rapidly when inoculated into Pinot noir wine that had just completed MLF with O. oeni Alpha, growth of other B. bruxellensis strains tested was not impacted.

Why strain UCD-2049 is more sensitive to O. oeni than the other Brett strains tested is unknown at this point. One possibility is that the inhibition of Brett by O. oeni was influenced by wine conditions such as pH and ethanol as Brett strains vary in their sensitivity to these wine parameters. Subsequent experiments showed that ethanol tolerance differences between B. bruxellensis strains may have played a role in their sensitivity to repression by O. oeni. For example, strain UCD-2049 was inhibited by O. oeni in wine at 14% and 13% (v/v) ethanol but not in wine adjusted to 12.5% (v/v) ethanol. In contrast, the more ethanol tolerant Brett strain AWRI-1499 was only inhibited by MLF in the high ethanol wine (14%) and not in wine containing 12.5% or 13% (v/v) ethanol.

Next steps:
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 currently 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.