Brettanomyces Unfold

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Charné Biermann – 27 July 2018

Brettanomyces (commonly known as just Brett) is a wine spoilage yeast and often the cause for the unfavorable change in aromatic-profiles of a final wine. The compounds responsible for these aromatic changes are 4-ethylphenol (4-EP), 4-ethylguaiacol, (4EG) and isovaleric acid. The reason why some winemakers fear Brett in their wine is because of the associated undesirable sensory characteristics such as band-aid, medicinal and plastic for 4-EP; smokey, spicy and sweaty for 4-EG and rancid and goaty for isovaleric acid. The combination of these attributes is commonly described as “Brett character” and delivers a band-aid, banyard and leather aroma to the wine. Brett produces these volatile compounds through the enzymatic proses of decarboxylation of phenolic (hydroxycinnamic) acids that are naturally present during the winemaking process. B. bruxellensis is reported to be the most common species of Brett in wine and most likely the culprit spoiling your wine.

 

The origin of Brett includes the following; the vineyard, grape wine processing and handling equipment, contaminated old barrels, wine brought in from other wineries and possibly through fruit flies.

 

The detection of Brett-cells is normally associated with stages after alcoholic fermentation, during the process of malolactic fermentation (MLF) and barrel aging. Conditions during malolactic fermentation is favorable for the growth and activity of Brett cells due to the low concentration of free sulfur dioxide at this stage, the presence of residual sugars and the absence of other microorganisms which can compete for available nutrients. During barrel aging, the presence of cellobiose (sugar resource) and oxygen can promote the growth and activity of Brett cells. Low sulfur dioxide levels and poor filtration prior to bottling can also lead to the presence of Brett cells in the bottled wine and subsequent spoilage of the wine. Early detection of spoilage can be used to prevent and control the formation of Brett associated aroma. Detecting Brett spoilage in your wine can be done by either measuring 4EP and 4EG concentrations or testing for Brett cell presence and activity in the wine. Filtering your wine to get rid of Brett, can be helpful, but 4-EG and 4-EP is not affected by filtration. Thus, resulting in no Brett-growth and high volatile phenol compound results. Therefore, it is of utmost importance to do a microscopic and chromatography analysis to accurately determine the presence of Brett in you wine.

 

The incubation and growth of Brett cell colonies take up to 7 days. Cell colony detection of Brett is a significant tool for determining the level of contamination. Vinlab makes use of polimerase chain reaction (PCR) for accurate identification of the Brett cells. PCR is a highly sensitive DNA amplification method with the advantage of producing results rapidly.

 

Taking a representative sample

Brett yeast cells are normally concentrated at the bottom of a tank, so either stir the barrel if a cell count is required or take a sample from the bottom if just a positive/negative answer is required. The wine can be racked from the lees. Cross-contamination should be avoided when sampling takes place, especially in batches with known infections.

 

Cell morphology of Brett

Microscopic analysis of Brettanomyces done by Vinlab

The vegetative cells in the microscopic analysis above, are ogival in shape; which is a result of repeated polar budding.

 

The sensory threshold for 4-EP is between 0.4 and 0.67 mg/L and 0.15 and 0.35mg/l respectably. 4EP and 4EG concentrations in wines differ significantly with 4EP usually being about 10 times higher than 4 EG (broadly speaking). This ratio is not static and can differ quite dramatically between wines. The ratio of the two compounds will determine the aromatic contribution and attributes of the faulty aroma in the wine. At levels below the sensory threshold, they are thought to add smokiness and positive aromatic characteristics to the wine. At levels above the sensory threshold, complexity and breakdown of fruity esters will occur and the wine is considered spoilt.

 

Conditions favoring Brett growth

  • Brett is capable for growing in wine by using glucose, fructose (at levels as small as <0.2g/L), non-fermentable sugars, glycerol, ethanol and malic acid as carbon source.
  • Nitrates and amino-acids can be used as a nitrogen source.
  • Vitamins (biotin and thiamine) available as left-overs after fermentation.
  • Optimal temperature for Brett growth ranges between 25-30 ̊
  • Although Brett will grow in an anaerobic environment, the availability of oxygen will enhance growth.
  • Fermentation lees and high pH levels encourages Brett growth, thus normally but not exclusively associating Brett activity with red wines.

 

Brett management program                                                                                        

When to analyse:

Analysis High level of Brett Some Brett present Brett not detected
Initially Ongoing Initially Ongoing Initially Ongoing
Microbiological analysis pH<3.6 Monthly Monthly 3-Monthly Monthly if infected 3-Monthly 3-6 Monthly
Microbiological analysis pH>3.6 Monthly Monthly Monthly Monthly if infected 3-Monthly 3-6 Monthly
4-EP, 4-EG analysis First establish a baseline/starting values for all batches of wine
3-Monthly 3-Monthly 3-Monthly 3-Monthly 3-6 Monthly Only if microbiological analysis is positive

 

 Guidelines for taking representative and meaningful barrel samples after baselines have been established.

High level of Brett Some Brett present Brett not detected
Microbiological analysis

Culture individual barrels

25-30% of barrels per batch

Use same barrels each time

Culture individual barrels 15-25% of barrels per batch

Use same barrels each time

 

Composite sample per batch

10-15% of barrels per batch

Use same barrels each time

4-EP, 4-EG analysis

Composite sample per batch

25-30% of barrels per batch

Use same barrels each time

Composite sample per batch

15-25% of barrels per batch

Use same barrels each time

Composite sample per batch

10-15% of barrels per batch

Use same barrels each time

 

Interpreting results

Interpretation of results should be taken into account of the following;

  • 50-200 cells/ml are potentially undesirable.
  • Recent SO2 additions may give false results.
  • If sample becomes to hot (>38 ̊C) on its way to the laboratory, the cells may die, giving false results.
  • 4-EP production can sometimes lag behind cell growth by as much as 2 months.
  • There can be situations where the cells are viable but not culturable.

 

Managing Brett populations in your wine

Fermentation management

–  Reduce carbon, nitrogen    and vitamin sources

Avoid stuck fermentations through efficient inoculation and balanced yeast nutrition.

Dry wines are less conducive to Brett growth.

Measure YAN levels before fermentation to avoid over-additions of nitrogen.

Add vitamins at appropriate points during fermentation to avoid excess residual levels.

pH management

Where possible pH should be kept <3.60 to ensure SO2 is most effective.

Adjust pH down using tartaric acid during wine maturation.

Readjust before bottling using potassium carbonate (calcium carbonate can result in CaT precipitation after cold stabilization if there is excess Ca in the wine).

SO2 management

Free molecular SO2 should be >0.4mg/L.

Molecular SO2 is pH dependent and will be easier to achieve at lower pH levels.

Test SO2 levels regularly (monthly).

Monitor SO2 levels more frequently in new barrels where SO2 levels drop more quickly.

Free SO2 is lower in turbid wines (SO2 is more rapidly bound) than in clearer wines, thus turbid wines should be avoided where possible.

Temperature management Where possible keep cellar temperatures below 16 ̊C
Barrel management

Where possible keep Brett-infected wine out of new barrels which have high levels of cellobiose (carbon source for Brett) and in which SO2 levels drop more quickly.

Wash barrels at each racking with a high pressure, hot water treatment or ozone.

Keep empty barrels sanitized through the use of sulphur.

Avoid initial contamination

Keep wines with residual sugar out of old or infected barrels

Keep infected wines out of new barrels.

Reduce cellar populations by sanitizing cellar equipment in between cellar operations.

Don’t buy in used barrels.

Avoid cross contamination

Keep infected batches sperate from uninfected batches.

Sampling devises and sharing barrels bungs are often the causes of cross-contamination.

Sanitize cellar equipment in between processing each batch.

Monitor and reduce population levels

An egg white fining followed by a racking can significantly reduce known population levels.

Filtration can reduce population levels, the extent of which is dependent on filter pore size. 0.65µm is sufficient to remove Brett cells.

 

The filtration of your wine through a membrane filter is an important aspect to get rid of unwanted microorganisms, as well as sanitizing of the wine-barrels. Wine barrels can be sanitized by using hot water and steam (60 ̊C). Sanitizing agents containing sulfite, chlorine dioxide and phosphoric acid is used to kill micro-organisms through cell membrane degradation and metabolic disruption.