What are the factors and practices that influence the development of Brettanomyces in wines?

Brettanomyces : Yesterday's Myths and Today's Realities

What are the factors and practices that influence the development of Brettanomyces in wines?

Among the red winemaking techniques, cold pre-fermentation maceration is a practice sometimes used to promote the extraction of color, slow down the extraction of tannins and obtain more regular alcoholic fermentation. This practice is known to be risky. This is not correct if it is conducted properly. First of all, as mentioned earlier, Brettanomyces is not or very little part of the microflora of the grape and even less so if the sulphiting for this practice has been carried out correctly (> 5 g/hl). Then, unless the must is contaminated very early (by the spoiled grapes or by the cellar, the polluted equipment), the population of Brettanomyces remains undetectable or very minor (5 to 10% at most of the yeast microflora) for up to five days of maceration, which is already a lot or excessive if the cold is not sufficiently available [1]. On the other hand, if it settles, this population will continue! Carrying out an initial cold maceration without risk therefore requires using healthy grapes, sufficiently sulphated, cooled to a suitable temperature (< 7-8°C), not handled by pumping (to avoid contamination by the equipment). wine), with a duration less than or equal to eight days at most, to avoid any risk of annoying contamination.

Any accident in the fermentation process then represents an opportunity for the development of Brettanomyces which is an opportunistic germ par excellence. Cases of contamination after stopping fermentation are not rare, but it is especially in the event of a delay in sulfiting after sluggish malolactic fermentation that the risk is highest. In this case, early inoculation with lactic acid bacteria starter is recommended [2]. Unfortunately, in practice, the effectiveness of this technique still remains somewhat uncertain if the inoculation is not early (we then speak of co-inoculation during alcoholic fermentation). Other technical alternatives are possible (see below).

Environmental factors influence the metabolism and growth of Brettanomyces/Dekkera sp. [3]. In terms of the balance of wine composition, the ethanol content of wine negatively affects the ability to Brettanomyces to develop but we must not dream, wines with 15.5% vol of ethanol can also be altered by strains adapted to this type of environment! Brettanomyces is not really sensitive to pH itself but its elevation greatly facilitates the multiplication of Brettanomyces . The naturally more acidic pH of white and rosé wines (pH < 3.5) indirectly protects them from any significant development of Brettanomyces/Dekkera . Below 15°C, growth is greatly slowed down. The increase in temperature favors multiplication up to 32°C; this explains why the frequency of contamination is low in winter and increases sharply in summer or early autumn. We would then tend to think that lowering the temperature of the aging cellars significantly below 15°C would reduce the risk of development of Brettanomyces . This is not inaccurate. However, it must be remembered that it is the ethanol/molecular sulfur dioxide couple which is the most effective in limiting populations by significantly increasing the duration of the latency phase ¹² . However, the content of molecular sulfur dioxide depends, for a given pH, on the quantity of free SO2 and… on the temperature. Thus, significantly reducing the temperature of the cellars quickly finds a limit... In addition, lowering the temperature can have other disadvantages (drying out in the event of artificial air conditioning, higher consumption and oxidation, wood/wood ratio). more important wine, modified speed of evolution of the wine, energy cost, etc.). Consequently, in practice, a temperature between 15 and 17°C is ideal (with 75-80% relative humidity for the barrels). Above 20°C, the risk of contamination increases exponentially and monitoring must follow accordingly!

The residual sugar content of wine represents another sensitive compositional factor. The risk of annoying contamination increases exponentially with the residual sugar content! Brettanomyces is a yeast capable of using a large number of different sugars, including certain disaccharides such as trehalose which can release Saccharomyces at the end of fermentation (especially if the yeasts were stressed) and during aging on lees. A residual quantity of sugars (glucose, fructose, galactose, arabinose, sucrose, trehalose) less than 0.35 g/l may be sufficient to allow the development of a population (1000 cells/ml) capable of synthesizing an ethyl content. -phenols equal to their perception threshold (450 µg/l). However, this quantity of sugars is available in the vast majority of red wines at the end of alcoholic and malolactic fermentation ⁸ . From 1 g/l, the risk of contamination increases dangerously. The quality and speed of completion of fermentation processes, by limiting the energy resources available, represent the first risk control factor. Bettanomyces . Then, due to the increased availability of certain substrates within the lees and their protective effect with respect to sulfur dioxide, it is certain that prolonged aging on the lees of red wines remains a practice that can indirectly promote the development of Brettanomyces ; It must therefore be reserved for the healthiest situations and always limited in time. Racking before summer is recommended. Beyond that, monitoring must be particularly careful if the wine presents favorable compositional factors (pH > 3.7 in particular).

However, many other substrates can be metabolized by this microorganism in wine depending on the availability of oxygen (alcohol, glycerol, etc.). The limiting factor in the development of Brettanomyces is therefore not a priori never the energy resource, but that is not a reason to offer him a stocked pantry. Thus, the quality and speed of the completion of fermentations (alcoholic and malolactic) represent the first prophylactic barrier.

Oak wood is not the cause of the more frequent appearance of the “Brett” character in barrel-aged wines. However, it is obvious that the microporous structure of wood represents an ideal shelter for microorganisms in general. Used barrels therefore represent an obvious source of contamination. Brettanomyces also develops very well as we have already mentioned in new barrels, but also in vats made of inert material. Obviously, it is much easier to clean and disinfect tanks with a smooth surface than barrels or wooden tanks. It is therefore the techniques for controlling the hygiene of wooden containers which make it possible to reduce the risk of development of this micro-organism during the aging of wines (see below).

Brettanomyces can also develop during bottle aging in a more or less random manner (all or part of the bottles from the same batch may be affected). This micro-organism is also systematically detected among the yeasts of old bottled wines, reflecting a remarkable capacity to persist over time. Most often, pre-bottling populations are extremely low. Due to its ability to resist sulfur dioxide, the concentrations used during bottling never allow the residual yeasts to be completely destroyed. As soon as the free sulfur dioxide content has sufficiently decreased in the bottle (by natural oxidation), Brettanomyces can multiply again and produce enough volatile phenols to cause a defect despite a low developed population. The conditions for control and especially preventive elimination of quiescent (and difficult to detect) populations of Brettanomyces therefore represent a crucial issue to guarantee the good development of bottled wines (see below)!

The availability of ethylphenol precursor substrates in wines does not represent a limiting factor either. Cinnamic acids, trans p- coumaric, ferulic and caffeic, respectively precursors of 4-ethyl-phenol, 4-ethyl-guaiacol and 4-ethyl-catechol, exist mainly in grapes in glycolyzed forms and especially esterified with organic acids, mainly acid tartaric, which are not metabolizable by Brettanomyces ¹⁴ . Certain lactic acid bacteria hydrolyze these esters into free acids that are more easily metabolized into volatile phenols later. It was therefore recommended to use lactic acid bacteria selected not to possess the responsible esterase activity in order to reduce the potential for biosynthesis of malodorous ethylphenols by Brettanomyces ¹⁵ . However, the proportion of free cinnamic acids naturally existing in red wines is still naturally largely sufficient to produce quantities of phenols well above the perception thresholds of these compounds. As a result, this approach is completely pointless… except for bacteria sellers.

[1] RENOUF V., PERELLO MC, STREHAIANO P. LONVAUD-FUNEL A., 2006 Global survey of the microbial vasecosystem during alcoholic fermentation in winemaking. J Int Sci Vine Wine,40(2),pp101-116
[2] RENOUF V., LONVAUD-FUNEL A., 2006 Microbiological monitoring of wine. Part 1: From plot to packaging: a tool for sustainable oenology. La Revue des Œnologues, 118, pp27-31
[3] CASTRO-MARTINEZ C. 2007 Brettanomyces bruxellensis : Metabolic kinetic study and modeling. Influence of environmental factors. Thesis Polytechnic Institute of Toulouse N°2487
14 DE LAS RIVAS B. et al . 2009 Molecular screening of wine lactic acid bacteria degrading hydroxycinnamic acids. Journal of Agriculture and Food Chemistry 42, 188-195.
15 https://lallemandwine.com/wp-content/uploads/2014/09/WUP-2-2014-FRANCE.pdf
16 CURTIN C. et al . 2012 Genotype-dependent sulphite tolerance of Australian Dekkera (Brettanomyces) bruxellensis wine isolates. Letters in Applied Microbiology 55, 56-61.
17 ALBERTIN W. et al. 2014 Development of microsatellite markers for the rapid and reliable genotyping of Brettanomyces bruxellensis at strain level. Food Microbiology 42, 188-195.