Winemaking appears to be a relatively simple process; the conversion of grape sugar into ethanol by wine yeasts. However, the microbiology of wine production is much more complex. Wine quality is an intrinsic compilation of unique characteristics, not only from the wine yeast (Saccharomyces cerevisiae), but also from the bacteria and wild yeasts present from the onset of fermentation. These yeasts include the genera Candida, Debaryomyces, Hanseniaspora, Kloeckera, Kluyveromyces, Pichia, Toruloaspora, Zygosaccharomyces and Brettanomyces. However, from the onset of alcoholic fermentation the influence of the wild yeasts diminishes quickly as the increase of ethanol concentrations drastically influences their growth (Loureiro and Malfeito-Ferreira, 2003).

One of the yeasts that can withstand the toxicity of ethanol levels and which has become the latest concern for most winemakers as a result of phenolic off-flavours, is Brettanomyces/Dekkera (Licker et al., 1999; Loureiro and Malfeito-Ferreira, 2003).

The name Dekkera, which is often mentioned together with Brettanomyces in literature, refers to the interchangeable name for Brettanomyces (Barnett et al., 1990; Heresztyn, 1986). Dekkera is the sporogenous form (ascospore-forming) or sexual anamorph of Brettanomyces.

Wines typically associated with a "Bretty character" is commonly recognised by aromatic defects ranging from medicinal smells (elastoplast, band-aid) to farmyard-like odours (horse sweat, wet animal, wet leather) and even spicy clove-like aromas (Licker et al., 1999). However, the true significance of Brett during winemaking is vague and therefore the "Brett problem" has spawned an interest of international magnitude.

Brett in my wine?

What are we talking about? From a microbiologist's viewpoint, are there actual Brett cells? Or are we talking about a flavour characteristic? The latter being more common amongst winemakers and consumers. If we talk about actual cells, then there's a very specific problem. If we are talking about an already formed off-flavour, the question remains; was it produced by Brett? Research is ongoing in an attempt to answer this question.

Origin and occurrence of Brett

What's in a name? It was shown in 1904 that a slower secondary fermentation followed the primary fermentation in the production of an English stock beer (Claussen, 1904). This fermentation was induced by a non-spore forming non-Saccharomyces yeast. This yeast or ''British brewing fungus'' was designated Brettanomyces, seeing that ''Brettano'' means British brewer and ''myces'' means fungus (Licker et al., 1999). The flavours that were produced by Brettanomyces during the secondary fermentation were very characteristic of the British beer of that time.

With regards to wine, Brettanomyces have been isolated from wineries all around the world and it is therefore unlikely that winemaking regions exist that are free of potential Brettanomyces growth (Arvik and Henick-Kling, 2002). There is evidence that Brett comes from the grapes and survives in the vineyard but difficulties are experienced with the detection and isolation of Brett from grapes (personal communication). However, preliminary results indicate very low Brett numbers which are not to be seen as reason for concern, but rather as caution, as with all the other wine microbes coming from the grapes (unpublished data). The detection levels are very low and this is typical of organisms renowned for having a VBNC (viable but non-culturable) state (Millet and Lonvaud-Funel, 2000). It is also interesting to note that Bretts have been isolated from fruit flies (Drosophila) and bees, and this may additionally serve as a source of contamination (Henick-Kling, 1998). It appears however, as if these yeasts are more established in wineries (Chatonnet et al., 1995; Fugelsang, 1997) and this can be ascribed to more suitable niches that exist within a winery as a result of ineffective cleaning of the crushing equipment, transfer lines and the barrels (wooden cooperage). Chatonnet et al. (1995) states that used barrels are the most notorious source of Brett contamination.

Origin of Brett flavour

The substrates from which "Bretty aromas" are produced are called hydroxycinnamic acids (also generally described as phenolic acids). Phenolic acids are abundant in plants (e.g. grapes) and form part of the structure of plant cell walls (Barthelmebs et al., 2000a). The action of enzymes (hemicellulases) from fungi and bacteria can however release these weak acids. Phenolic acids serve as natural toxins for the plant to inhibit the growth of unwanted microbes (Barthelmebs et al., 2000b). Some microbes can however degrade these acids, specifically the hydroxycinnamic acids p-coumaric (PCA), ferulic (FA) and caffeic acid (CA), as a method of survival. However, the consequence of this action is the production of phenolic off-flavours. The organisms involved include wine bacteria (lactic acid bacteria), wild yeasts (e.g. Brett) and the wine yeast S. cerevisiae. Some contribute indirectly to the so-called "Bretty flavour" (production of precursors, vinyl derivatives) and others more directly (production of ethyl derivatives). Nevertheless, the significance of the flavours produced by these yeasts is argued both ways. Some consider the slightest hint of Brett character as an indication of spoilage. Others see the contribution of these contentious yeasts as an integral part of a red wine's complexity.

Figure 1. Brettanomyces/Dekkera under a light microscope (1000x microscopic). Figure 2. Production of volatile phenols by wine micro-organisms.

Morphology and physiology

With regards to their taxonomy, Brettanomyces/Dekkera have seen numerous reclassifications in the last few years and there are currently five recognised species: D. bruxellensis, D. anomala, D. custersianus, D. naardenensis and D. nanus (Egli and Henick-Kling, 2001). From the research that has been done, D. bruxellensis have thus far been associated with wine, and more recently D. anomala have also been added after the molecular techniques improved for the identification of isolates (Cocolin et al., 2004). Amongst these two species of Brett, there are many strains that exist and it is still unclear if all strains are bad (some may be harmless). This is being investigated as part of my research and other international research groups.

Factors you should know (Metabolism)

Like most yeasts, Brettanomyces can utilise a large number of substrates for their sugar requirement. Any available amounts of glucose, fructose, arabinose and trehalose contributing to the residual sugar of the wine will greatly suit their growth or carbon source needs along with alcohols already present such as ethanol and glycerol. Wine yeasts (Saccharomyces) often do not metabolise the last bit of sugar because of the gradual rise in ethanol levels and consequently small amounts of residual sugar are remaining (<2.0 g/l) that can be utilised by some spoilage wild yeasts (e.g. Brettanomyces, Schizosaccharomyces) (Loureiro and Malfeito-Ferreira, 2003). In addition, instances of stuck/sluggish fermentation may create sufficient time for a Brett bloom and any residual sugar would be beneficial to Brett for metabolising ethanol as a carbon source. Brett however, do not need sugar, but it gives them energy to survive. With regards to nitrogen sources, care should be taken with the addition of diammonium phosphate (DAP). Analysis for yeast assimible nitrogen (YAP) should be performed so that the winemaker can avoid unnecessary additions. Excessive usage in an attempt to improve slow fermentations could lead to increased levels of nitrogen. This could further encourage the growth of Bretts and other spoilage microbes during later stages, such as post MLF (Godden et al., 2004).

Spoilage products

The growth of Brettanomyces/Dekkera in wine has been associated with various forms of spoilage including cloudiness, pellicle formation (less common) and a range of off-flavours (Chatonnet et al., 1995; Henick-Kling, 1998). Depending on the conditions and precursors available, these yeasts can produce acetic acid, contributing to wine's volatile acidity (Arvik and Henick-Kling, 2002; Guadalupe Aguilar Uscanga et al., 2003); tetrahydropyridines that are reminiscent of mouse-urine (mousiness) (Heresztyn, 1986); isovaleric acid, known for a rancid cheesy aroma and volatile phenols (Edlin et al., 1995; Dias et al., 2003; Coghe et al., 2004). The latter is determined by a few factors, including the substrate and enzyme activity. E.g. if the substrate is p-coumaric acid, it will be converted by an enzyme to form the vinyl derivative, 4-vinyl phenol, which will be further reduced by another enzyme to form the ethyl form or 4-ethyl phenol (See Fig. 2). It is the ethyl derivatives that are typically associated with "Bretty character", as the vinyl derivatives (precursor of ethyl derivatives) can be produced by many other wine microbes as already mentioned (Cavin et al., 1993). Excluding the influence of grape variety, the enzymatic activity or capability of the various organisms indicated differs, depending on the wine conditions, and therefore the production levels, precursors and concentrations of these volatile phenols will also vary in different wines.

Important: What many should realise is that the precursors of the ethyl derivatives, namely the vinyl derivatives (e.g. 4-vinyl phenol and 4-vinyl guaiacol) can also be produced by the wine yeast, by other non-Saccharomyces yeasts (other than Brett) and by lactic acid bacteria (LAB) (van Wyk and Rogers, 2000; Cerd n et al., 2001; Cavin et al., 1997). Therefore, all these organisms contribute to the phenolic off-flavours and form part of the so-called "Bretty character" during some stage. Focusing only on the ethyl derivatives, there have been a few questions: (1) Is it really only Brett that can produce these in wine? (2) Are all strains of Brett equally bad? In my opinion one of the most important issues, (3) What are the concentrations of the substrates PCA and FA (free form) in the grape juice as these will have the most influence on the potential formation of volatile off-flavours? (4) Why aren't the substrates measured? (5) Shouldn't it become a standard measurement of grape juice? How do these levels differ between cultivars? These questions are some of the aspects investigated and will be answered in time.

Table 1. Typical "Bretty" aromas.

Concluding remarks

The microbiology of wine is very complex and this makes it often difficult to pin-point the exact problem and its origin in a habitat such as wine. Nonetheless, large-scale investigations on controlling wine spoilage in attempt to improve wine quality have been undergone for many years with great results and improvements originating. In order to build on this it is very important that the winemaking community, winemakers, researchers, and consumers communicate and keep each other informed.

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