Introduction
‘Brett’ is still a hushed topic for many a winemaker. It causes noticeable spoilage which can detract from the wine quality both in terms of aroma and taste. Often winemakers don’t realise they have a problem in their wine until it is too late, costing them entire batches and vintages. In most cases, oak – often old, but can be new as well, is the source and vector of the spoilage.
But the simple fact is, everyone has Brettanomyces – but whether it is allowed to grow is the question.
A number of basic chemical parameters can be used to identify the risk of any wine developing Brettanomyces. This article will seek to outline some of those key parameters and a best practice monitoring strategy to minimise microorganism growth and provide ongoing monitoring.
What is Brettanomyces bruxellensis?
‘Brett’ or Brettanomyces bruxellensis(B.bruxellensis)is a spoilage yeast commonly found in wineries, in part due to its tolerance for high alcohol, low pH and low oxygen levels. B. bruxellensis has the potential to cause off flavours in wines, through production of the volatile phenolic compounds 4-ethylphenol (4-EP) and 4-ethylguaiacol (4-EG).This is often associate with a reduction in fruit and other desirable aromatic profiles.
Where does B.bruxellensis come from?
Whilst B.bruxellensis may be found anywhere including vineyards and cellar equipment, B.bruxellensis tends to become evident during the maturation process in oak. They grow very slowly in comparison to other yeast but are essentially scavengers and can metabolise a number of analytes that many other yeast can’t in order to survive. The porous nature of the wood allows the influx of small amounts of oxygen (Swaffield & Scott, 1995; Loureiro & Malfeito-Ferreira, 2006) and the presence of cellobiose can serve as a sugar resource in new oak (Boulton et al., 1996).
What are the risk factors I can control in my wine?
Temperature
At lower temperatures, yeast will grow slower. The warmer the temperature, the quicker they will grow. A wine with a high population of B.bruxellensis was monitored at 3°C, 10°C, 15°C and 20°C over 50 days. The wine stored at 3°C and 10°C did not present any significant increase in 4EP/4EG levels, however the wines stored at 15°C and 20°C both presented with over 1500 ug/L 4-EG at the end of the 50-day period (Malfeito-Ferreira, M., 2011). The time of year and storage conditions can play a role in spoilage.
Residual Sugar
Like many yeast, whilst B.bruxellensis can live off many substrates, they do have a preference for simple sugars like glucose and fructose. Incrementing amounts of glucose and fructose were demonstrated to stimulate the growth of B.bruxellensis. (Chatonnet et al 1995). Ensuring your wine has completely finished fermentation is critical as anything over 0.2g/L of GF was found to stimulate B.bruxellensis growth. Whilst a wine with 4 g/L glucose/fructose may not taste like a sweet wine, this presents a huge risk for the growth of B.bruxellensis.
Alcohol and Sulfite tolerance
Multiple studies have found that B.bruxellensis are not able to grow well over 15% alcohol whilst Saccharomyces cerevisiae can grow up to 18% alcohol. Despite this, they are able to remain viable for long periods of time. This is attributed to their exceptional resistance to minimal nutrient conditions.
B. bruxellensis can be extremely sulfite resistant. There are different genetic variants which are more or less Sulfite resistant (Varela et al 2019). Levels of 0.8 mg/L molecular SO2 had varying levels of effect on 3 different strains over a 1-year storage period. Levels up to 0.6 mg/L had very little effect on any of the strains. This means a red wine stored in oak at 15°C, with a pH of 3.65 with a free SO2 amount of 35 mg/L would only amount to 0.5 mg/L of molecular SO2. (Coulon et al 2011). Extended malolactic fermentation where a wine may be left without SO2 for prolonged periods is a high-risk scenario where B.bruxellensis often start growing.
I’m worried I added too much DAP and now there are excess levels of Nitrogen – will this promote Brett?
Interestingly, no significant impact on the growth of B.bruxellensis was found in wines with incrementing levels of yeast assimilable nitrogen at the end of fermentation in ranges expected post autolysis.
How can I monitor my wines?
Taint
There are a number of different ways to monitor wines for the presence of B.bruxellensis. Many people choose to monitor the taint that is produced rather than checking for the cells themselves. 4EP and 4EG may be quantified employing a GC-MS approach (Table 1). This methodology ensures precise detection and quantification of these volatile compounds, often linked to sensory descriptors such as "band-aid" or "wet leather." The proportion of 4EP to 4EG differs across various wines, with a 3.5:1 ratio for Pinot, 8:1 for Merlot, 9:1 for Shiraz and a 10:1 ratio for Cabernet Sauvignon.
Thresholds for this vary depending on the source, however generally anything over 400 ug/L for 4-EP and 150 ug/L for 4-EG are able to be perceived. A number of different compounds from oak and varietal characters from the wine itself may mask the taint.
Yeast detection
Yeast may be cultured or detected by molecular methods, the most common being some variation of PCR (Table 1). Culturing or plating, assumes that the yeast is able to grow in ideal conditions and can take up to 14 days. Molecular methods tend to be quicker, and involve looking at the DNA itself.
It is common to see wines that have recently been sulfured to not have B.bruxellensis present via plating, literature suggesting that the cells may be temporarily viable non culturable (VBNC). Most molecular methods including qPCR will generally detect the cells whether or not they are VBNC. Often a combination of both tests over time will contribute to a comprehensive data set allowing the winemaker to identify high risk wines and practices.
B. bruxellensis testing methods
| Culturable cells | qPCR/PCR | Flow cytometry | 4EP/4EG |
|---|---|---|---|
| (PLATING) This tells us how many cells can grow in a lab environment.
An inexpensive monthly follow up during wine maturation. |
Measures viable and non-viable B. bruxellensis DNA. A fast, highly specific test for the detection and the quantification of B. bruxellensis. | Differentiates between viable and non-viable based on either cell wall integrity or cell function. A fast and highly specific test. | 4-Ethylphenol (4EP) and 4-Ethylguaiacol (4EG).
Chemical taint produced by ‘Brett’ once it reaches critical population. |
My wine has Brett – what can I do?
Absolute filtration can remove any yeast, including B. bruxellensis, prior to bottling. It is always best to keep the population low to reduce the load on the filters and maximise their efficiency. However constantly sterile filtering a wine in storage may not be the best practice. B. bruxellensis grows slowly fighting against many inhibiting factors as it proliferates, including other microorganisms. By sterile filtering the wine constantly and then returning it to an infected vessel, this can promote even quicker growth. It is important to treat both the wine and the source of the infection, including the oak.
How can I kill Brett in barrel?
B. bruxellensis cells are able to be rendered non-viable with the use of heat. One study pointed to 55°C for 2 minutes being enough to reduce the Brettanomyces population to zero (Couto et al 2005). However microorganisms are not just present in the wine, they are able to permeate the oak itself, meaning that any heat treatment must reach the outside of the barrel to be effective. The time and temperature required to achieve this will vary depending on the capability of the steamer, temperature of the water and size of the barrel.
Chitosan
Curative application
Application of chitosan/ β-glucanase based treatments is often more common once a problem has been detected. Its application is able to effectively kill the yeast cell, literally destroying the cell structure. The population required to commence producing 4-EP and 4-EG will vary depending on the wine in question, but often produces detectable levels in the 1x102 to 1x103 cells/mL range (Chatonnet et al 1992). At this population level, SO2 will be consumed very quickly – both free and total SO2 will be greatly reduced here and unavailable from an antimicrobial point of view if added. At this cell level it is ultra-critical to reduce the microbial load in order to ensure a portion of molecular SO2 in order to prevent further growth.
Analytical checkpoint – Wines which are tested for Brettanomyces with molecular methods after the addition of Chitosan often present with positive results for a period of time as the DNA is still present, despite the cells being completely denatured.
Preventative application
Chitosan/beta-glucanase combinations may be used successfully as a preventative form of treatment before B.bruxellensis has the opportunity to proliferate. Because the combination affects the cell viability, it will inhibit any growth before the cell mass is able to grow. Often, the presence of B.bruxellensis is only determined by the winemaker when taint is detected sensorially. By this stage, the B.bruxellensis population has reached high cell numbers, in the order of 50-100 cells/mL. By preventing the growth in the first place, a winemaker is able to maintain a higher proportion of molecular SO2 by ensuring there is a growth prevention strategy such as OENOBRETT® in place. This method is also much less dependent on personnel to monitor the problem and becomes part of a standard operating procedure. This preventative treatment can be used:
- On topping wine in case of barrel ageing
- On wine lees
- On red pressings (these contain a higher microbial load than the free run fraction)
- On wine with high pH (less available molecular SO2)
- On wine with high micro-organisms population
If my wine comes up positive for B. bruxellensis via PCR, but negative for plating, what does that mean?
qPCR measures DNA which is present in both live and dead B. bruxellensis cells. SO2 additions may cause B. bruxellensis cells not to show up positive via plating. qPCR can pick up cells that are not necessarily culturable via plating which are sometimes called Viable but non-culturable (VBNC).
Picking up VBNC by qPCR may give you a positive result that you would not have otherwise gotten with plating. If B. bruxellensis is not picked up due to a recent SO2 addition, it may falsely lead to the conclusion that B.bruxellensis is not present. It is also possible that the cells are no longer alive, and only dead cells have been detected, but they may have still been alive long enough to produce some 4EP/4EG. This may also be the case when chitosan-based products such as OENOBRETT® are used, as the cell membrane is compromised creating a dead cell, whereby the DNA is still able to be detected via qPCR. Ongoing routine checks are recommended to detect any growth before the cells start producing ethyl phenols.
What if my wine comes up positive via PCR and plating, but there is no 4EP/4EG being produced?
B. bruxellensis cells need to reach a critical population before they can start producing off-flavours (4EP/4EG). This population level may be different for each wine depending on the chemical factors and other microorganisms present. It is likely that if there is a population in the wine that it will start producing 4EP/4EG over time.
My wines are at 50 ppm free SO2 – why am I getting a viable result via plating?
B. bruxellensis are highly SO2 tolerant and can survive significant levels of SO2. The amount of available SO2 will be dependent on pH. Any residual sugar available can stimulate their growth. Each wine matrix is different and will have factors working with and against B.bruxellensis growth. Brand new barrels contain carbohydrates which can also be metabolised by Brett, further supporting population growth.
Will B.bruxellensis keep growing after it goes to bottle?
B. bruxellensis will keep growing in bottle if the environment allows. They are able to grow without any air (anaerobically) as well as with air (aerobically), so if the product is not sterile filtered to bottle via membrane filtration, there is a risk that a single cell could turn into a large population over a number of years and produce significant levels of 4EP/4EG.
Concluding notes
B. bruxellensis is ever present in wineries and can cause significant financial and reputational damage. The gold standard of B.bruxellensis management is preventative, with ongoing routine monitoring, however often this only gets put in place after a major infection. Some basic parameters to monitor may assist in controlling or preventing the proliferation of B.bruxellensis:
- Ensuring all residual sugar has been consumed during primary alcoholic fermentation
- Avoiding long periods of time where the wine is left unsulfured – the shortest possible timeframe from alcoholic fermentation to the completion of MLF is desirable
- Understand that lower alcohol wines present a higher risk of infection ie. a 12% v/v red wine will be more at risk than a 16% red wine
- Oak, often old but sometimes new, can promote the growth of B. bruxellensis. Understanding that this maturation phase is high risk may assist in management of the barrels.
- Sulfur additions by themselves may not be enough to control B.bruxellensis growth – additional methods may be required
- Treat the wine and the vessel, not just the wine, otherwise re-infection is possible.
- Use of preventative treatments such as chitosan-based products may assist with the management of high-risk wines with low alcohol, high sugar levels and warmer temperatures in addition to SO2
- B. bruxellensis can grow over time with a molecular SO2 level of 0.6 mg/L
References
Agnolucci, M., Rea, F., Sbrana, C., Cristani, C., Fracassetti, D., Tirelli, A. and Nuti, M., 2010. Sulphur dioxide affects culturability and volatile phenol production by Brettanomyces/Dekkera bruxellensis. International journal of food microbiology, 143(1-2), pp.76-80.
Boulton, R.B., Singleton, V., Bisson, L.F. & Kunkee, R., 1996. Principles and Practices of Winemaking. Chapman & Hall Publishers New York, NY.
Chatonnet, P., Dubourdieu, D. & Boidron, J.N., 1995. The influence of Brettanomyces/Dekkera sp. yeasts and lactic acid bacteria on the ethylphenol content of red wines. Am. J. Enol. Vitic. 46, 463-468.
Childs BC, Bohlscheid JC, Edwards CG. Impact of available nitrogen and sugar concentration in musts on alcoholic fermentation and subsequent wine spoilage by Brettanomyces bruxellensis. Food microbiology. 2015 Apr 1;46:604-9.
Coulon, J., Raffestin, V., Bellan, M. and Lonvaud-Funel, A., 2011. Molecular SO2 effect on Brettanomyces bruxellensis populations during a one-year storage. In 9th International Symposium of Oenology, Bordeaux, France.
Couto JA, Neves F, Campos F, Hogg T. Thermal inactivation of the wine spoilage yeasts Dekkera/Brettanomyces. International journal of food microbiology. 2005 Oct 25;104(3):337-44
Freer SN. Acetic acid production by Dekkera/Brettanomyces yeasts. World Journal of Microbiology and Biotechnology. 2002 Apr;18(3):271-5.
Loureiro, V. & Malfeito-Ferreira, M., 2006. Dekkera/Brettanomyces spp. Chapter 13. In: Blackburn, C. de W. (ed). Food spoilage microorganisms. Woodhead Publishing Ltd, Abington, Cambridge, UK. pp. 353-398.
Malfeito-Ferreira M. Yeasts and wine off-flavours: a technological perspective. Annals of Microbiology. 2011 Mar;61(1):95-102.
Serpaggi, V., Remize, F., Recorbet, G., Gaudot-Dumas, E., Sequeira-Le Grand, A. and Alexandre, H., 2012. Characterization of the “viable but nonculturable” (VBNC) state in the wine spoilage yeast Brettanomyces. Food microbiology, 30(2), pp.438-447.
Willenburg, E. and Divol, B., 2012. Quantitative PCR: an appropriate tool to detect viable but not culturable Brettanomyces bruxellensis in wine. International journal of food microbiology, 160(2), pp.131-136.
Avramova, M., Vallet-Courbin, A., Maupeu, J., Masneuf-Pomarède, I. and Albertin, W., 2018. Molecular diagnosis of Brettanomyces bruxellensis’ sulfur dioxide sensitivity through genotype specific method. Frontiers in Microbiology, 9, p.1260.
Copyright© 2023 Winechek Laboratories. No part of this publication may be reproduced in any form or by any means without the permission in writing from the copyright owner.
Varela C, Bartel C, Roach M, Borneman A, Curtin C. Brettanomyces bruxellensis SSU1 haplotypes confer different levels of sulfite tolerance when expressed in a Saccharomyces cerevisiae SSU1 null mutant. Applied and Environmental Microbiology. 2019 Feb 15;85(4):e02429-18.
Tubia I, Prasad K, Pérez-Lorenzo E, Abadín C, Zumárraga M, Oyanguren I, Barbero F, Paredes J, Arana S. Beverage spoilage yeast detection methods and control technologies: A review of Brettanomyces. International journal of food microbiology. 2018 Oct 20;283:65-76.
Swaffield, C.H., Scott, J.A. & Jarvis, B., 1997. Observations on the microbial
ecology of traditional alcoholic cider storage vats. Food Microbiol. 14, 353-361.
