Brettanomyces/Dekkera during winemaking

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Brettanomyces/Dekkera during winemaking - Part 2
What the winemaker could do?

Adriaan Oelofse

Adriaan Oelofse and Maret du Toit
PhD Research - An investigation into the role of Brettanomyces/Dekkera in the winemaking process.
Institute for Wine Biotechnology, Stellenbosch University, StellenboschE-mail: oelofse@sun.ac.za

This research is sponsored by

Summary
In order to control the levels of volatile phenol production in wine, Brett populations have to be kept low throughout the winemaking process. This way the volatile phenol contribution may well add to the complexity of the wine, rather than detracting from the favourable aroma profile when at higher concentrations. This section (part 2) follows on a previous section entitled "Brettanomyces during winemaking - What the winemaker should know" (Winelands, Feb 2006) and mentions some strategies for controlling Brett and minimising Brett-associated off-flavours.
Introduction
As with numerous other wine spoilage microbes, it is not a question of prevention, but rather a question of control. Bretts (referring to actual cells) cannot be prevented as it is part of the natural wine microbial flora, but it can be controlled along with the consequent "Brettiness" (referring to the actual off-flavour produced by Brett cells). Good winemaking practices would definitely control Brett along with many other wine spoilage microorganisms, but a holistic approach is required as wine parameters such as, grape quality, sulphur dioxide (SO₂), pH, wine temperature, residual nutrients, O₂, barrel condition and oenological practices are interdependent. For more information on Brettanomyces and its role in winemaking see Part 1 - February edition 2006.
So the question is: What can the winemaker do to limit the growth of Brettanomyces and the potential volatile phenol off-flavours?
What Encourages Brett? - Prevention or Control
If we know what conditions, parameters and/or environments stimulates the growth of Brettanomyces and its enzymes involved in the production of volatile phenols, we have half of the answer. We can use this knowledge to then prevent or control them. The following has been shown to enhance Brett and the levels of the ethyl-derivatives:

From the vineyard
The vineyard does not appear to be a serious contamination source. However, as part of a holistic approach to improve quality, damaged grapes may well contain a higher microbial load than healthy grapes and the removal thereof can be a consideration, along with judicial SO₂ usage at harvest. Winemaking for lighter style wines requires more consideration as these can exhibit phenolic off-flavour due to lower threshold levels.

Poor sanitation (maintain good cellar hygiene)

Longer maceration periods
This can lead to increased amounts of substrates and precursors for Bretty off-flavour (Gerbeaux et al., 2002)

Red wine is more prone to Brett and phenolic off-flavours
"Brettiness" is discouraged by whites, although they do occur in some. This is largely due to higher levels of SO₂, lower pH and fewer pre-cursors.

pH > 3.6 (pH below 3.6 is better)

Temperatures between 25-30°C will encourage their growth (Below 18°C will help with control).

Poor SO₂ usage
There are still a few concerns with regards to the prevention of Brett growth in the wine. Although SO₂ is applied for its antimicrobial properties, the sensitivity of Brett is strain dependant and recently genetic variability among Brett strains has been reported (Godden et al., 2004). The critical additions are at picking and/or crushing and again after MLF before barrel ageing.
Because SO₂ is more effective at lower pH values the potential risk of unwanted microbial populations is much higher with a higher pH as encountered in warmer climate regions (like South Africa). Molecular SO₂ < 0.4 ppm will give poor antimicrobial properties (rather maintain above this level). It has been reported that molecular SO₂ around 0.8 ppm should be sufficient for inhibiting Brett growth in red wine (Australian Wine Research Institute, 2004 annual report). Additionally, research from the AWRI has suggested that it is not just the concentrations of free or bound levels, but rather the ratios thereof. The advised ratio of 0.4 of free to bound/total SO₂ is more ideal to ensure microbial stability (e.g. 30 ppm free to 75 ppm total).

Alcohol below 13%

Residual sugar > 0.2 g/l
It is important to realise that wines classified as dry (<5.0 g/l) still have enough sugar to support the growth of Brett. This is why the residual sugar should be minimised and this can be performed by using a very active starter culture to ferment to dryness.

Additions of amino acids and vitamins, especially biotin and thiamine

Presence of yeasts lees
Yeast lees is a very rich source of nutrients and will encourage many spoilage microbes if the above mentioned points are not controlled.

Delayed racking
During this time the levels of free SO₂ is very low. It has been shown that delayed racking led to increased levels of 4-ethyl phenol (Chatonnet et al., 1995) and therefore winemakers are advised to add SO₂ and clarify the wine as soon as possible after MLF.

Oxidative conditions (Keep containers topped)
The small available amounts of oxygen will support Brett better during conditions of higher ethanol concentrations (Du Toit et al., 2005). In addition, excessive amounts of oxygen will enhance the growth and consequent acetic acid production by Brettanomyces.

Malolactic fermentation
Thus far, it appears as if wine is at its highest risk to Brett spoilage from the onset of MLF and ageing. As MLF is performed when the levels of free available SO₂ are very low, the antimicrobial potential of the wine is also low. The increasing pH (resulting from MLF) and presence of nutrients (resulting from yeast autolysis) can create an environment in which many microbes can proliferate. During early stages of spontaneous MLF, the lactic acid bacterium (LAB) populations are low and Brett populations have more access to substrates and nutrients. Chatonnet et al. (1992) additionally reported that with greater tannic extractions, the LAB exhibit a greater period of latency. Therefore, the use of a strong MLF starter cultures can limit Brett activity. It was observed in certain wines that have high pediococci counts, that they also have high Brett levels or volatile phenol levels (Personal communication, Gafner).

Cross contamination
Use clean wines to top-up and don't use pre-infected barrels.

Other means of Brett elimination that has been mentioned as possibilities are:
Filtration (0.45 um)/Cross flow (0.22 um)
A method believed to be effective for removing Brett is sterile filtration (0.45 mm) (Gafner et al.) This however, along with the other methods mentioned has detrimental effects on the organoleptic quality of the wine and should be carefully considered. Sterile filtration/cross-flow may however be a good option when filling up the barrels. It is crucial that the "top-up" wines and even wines destined for blending are tested or decontaminated before use as this may destroy the entire outcome.
Addition of DMDC (Velcorin)
Dimethyldicarbonate, O.I.V. approved, cost implications.
Photon hydro ionization (Winterthur©) (not investigated for Brett)
Thermal inactivation (Couto et al., 2005) (not investigated for Brett).
Bretts in Barrels
Bretts are also capable of growing in barrels. These yeasts contain á-glucosidase enzyme activity, which cleaves the disaccharide cellobiose (wood sugar) into glucose molecules thus making it readily available for growth. This conversion could however also be the result of the action of other microbial inhabitants of the barrels. With regards to new barrels, the action of fungal cellobiohydrolases on the cellulose could serve as sites for Brett growth. In older barrels the cellobiose might have been previously metabolised (Fugelsang et al., 1993; AWRI). New barrels contain higher amounts of cellobiose and therefore should have a higher potential for supporting Bretts than used barrels. However, when barrels are produced the toasting and firing process should ensure that the wood is microbially clean. With older barrels, research has shown drastic decreases in the amounts of volatile ethyl derivatives produced (up to 85%) after shaving and refiring (Pollnitz et al., 2000). This is ascribed to the destruction of the microbial load in the first few inner layers of barrels. Other things to mention; new barrels pose greater risk if wine is already Brett infected. New barrels can bind more free SO₂ (15 mg/l) than older barrels over 6 months (AWRI) and this should be taken in considereation.
General misconceptions about Brett

Brett only comes in on the grapes
Is in all red wines
Comes in with new barrels
Is not spread by infected barrels
Brett is only found in dirty cellars
Won't grow in dry wine
Won't grow in wine with alcohol above 13%
Brett is a surface film yeast that needs oxygen
Always makes a lot of acetic acid and ethyl acetate
Only Brett gives a mousy taint

Concluding remarks
With regards to Brett, well, only small steps have been taken and there are still many things to be researched. Bretts are but a few of the many organisms that exist in the winemaking environment and because it is a living entity, there will be a lot of diversity amongst its species. Not all Bretts should be deemed bad, although others should. Not all wines are bretty, although in some cases bretty can be good! It is the fluctuation in perception, which is influenced by knowledge that shapes the different opinions. Nevertheless, everybody appreciates and enjoys a good wine within their personal taste!
There is no better control for Brett at this stage than ensuring GOOD WINEMAKING PRACTICES!
References
Barata, A., Laureano, P., D'Antuono, I., Martorell, P., Stender, H., Malfeito-Ferreira, M., Querol, A., Loureiro, V. Enumeration and identification of 4-ethylphenol producing yeasts recovered from the wood of wine ageing barriques after different sanitation treatments (Personal communication).
Chatonnet, P., Dubourdieu, D., Boidron, J.N., 1995. The influence of Brettanomyces/Dekkera spp. yeasts and lactic acid bacteria on the ethyl phenol content of red wines. Am. J. Enol. Vitic. 46, 463-468.
Chatonnet, P., Dubourdieu, D., Boidron, J.N., Pons, M. 1992. The origin and incidence of ethylphenol in red wines. J. Sci. Food Agric. 60, 165.
Couto, J.A., Neves, F., Campos, F., Hogg, T., 2005. Thermal inactivation of the wine spoilage yeasts Dekkera/Brettanomyces. Int. J. Food Microbiol. 104, 337- 344.
Du Toit, W.J., Pretorius, I.S., Lonvaud-Funel A., 2005. The effect of sulphur dioxide and oxygen on the viability and culturability of a strain of Acetobacter pasteurianus and a strain of Brettanomyces bruxellensis isolated from wine. J. Appl. Microbiol. 98, 862-871.
Fugelsang, K. C., Osborn, M.M., Muller, C.J., 1993. Brettanomyces and Dekkera: Implications in winemaking. Beer and production. B.H. Gump (ed). Washington, DC, American Chemical Society
Gafner, J., Porret, N.A., Schneider, K., Hesford, F. 2004. New system for qualitative and quantitative detection of spoilage organisms in wine. Agroscope Research Station, Wädenswil, Switzerland.
Gerbeaux, V., Vincent, B., Bertrand, A. 2002. Influence of maceration, temperature and enzymes on the content of volatile phenols in pinot noir wines. Am. J. Enol. Vitic. 53:2.
Godden, P., Coulter, A., Curtin, C., Cowey, G., Robinson, E., 2004. 12th AWITC, Brettanomyces workshop: Latest research and control strategies, Melbourne, AUS.
Pollnitz, P.A., Pardon, K.H., Sefton, M.A., 2000. Quantitive analysis of 4-ethyl phenol and 4-ethyl guaiacol in red wine. J. Chrom. A. Edt 1, 874, 101-109.

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