Reprinted with Permission from: Proceedings of the 5th International Symposium on Cool Climate Viticulture and Oenology, 16-20 January 2000, Melbourne, Australia Brettanomyces in Wine Thomas Henick-Kling1, Christoph Egli, Jonathan Licker, Craig Mitrakul, Terry E. Acree 1 Associate Professor for Enology, Cornell University, Department of Food Science and Technology, Geneva, NY 14456-0462, USA. The Brettanomyces / Dekkera yeasts can be found in fermenting must and in wine. Typically they grow after alcoholic and malolactic fermentation during storage of wine in tank, barrel, or bottle. They contribute characteristic ‘bretty’ flavors which are described as smoky, barnyard, plastic, burnt plastic, vinyl, Bandaid ™, and creosote. They can also contribute a ‘metallic’ bitterness. Compounds which are responsible for brett flavor in wine include 4-ethyl phenol, 4-ethyl guaiacol, isovaleric acid, and unidentified "burnt plastic" compound. Descriptive sensory evaluations show an inverse relationship between fruity and bretty flavor perception. The brett aromas in some wines are considered a positive attribute, especially when present at low concentration. Often these flavors are considered a defect. The wine’s varietal and regional flavor characteristics might be completely masked by these flavors and the wine can be unpleasantly bitter. 4-ethyl phenol is used by some wineries as an indicator compound for the activity of Brettanomyces. Yet some wines having a strong brett flavors do contain none or very little 4-ethyl phenol. Preliminary studies show that 4-ethyl phenol is formed all through growth of Brettanomyces. Thus this compound can be used to confirm the presence of bretty flavors. The search for indicator compounds formed during early stages of growth of B. continues. The Brettanomyces yeasts can produce the characteristic brett flavors when growing at low cell density of several hundred to several thousand cells per mL. The fact that these yeasts are often present at low cell numbers and that they are slow growing makes detection difficult. Genetic analyses of wine isolates have shown that only strains of B. bruxellensis grow in wine. Semi-selective plating is performed prior to genetic analysis on media including cycloheximide. The development of DNA based probes has made it possible to reliably identify the yeast once it is plated on a nutrient agar plate. Further developments of specific probes and of probe application techniques will improve the specificity and speed of detection. What are of Brettanomyces / Dekkera yeasts? The management of Brettanomyces is fast becoming one of the most important winemaking issues. Brettanomyces/Dekkera yeasts are reported to be involved in spoilage by off-flavor production in beer and wine (Anonymous 1998, Chatonnet et al. 1992, Chatonnet et al. 1995, Edlin et al. 1995, Kuniyuki et al. 1984, Spaepen & Verachtert 1982). In wine these yeasts typically grow in low cell numbers after completion of the alcoholic and malolactic fermentation during aging of wine in barrels and bottles. The aroma characteristics of their spoilage-causing metabolites were described as burnt plastic, smoky, barnyard, horse sweat, leather, and wet wool (Boulton et al. 1996, Chatonnet et al. 1992, Fugelsang 1997, Heimoff 1996, Heresztyn 1986, Ibeas et al. 1996, Licker et al. 1998). To some people the presence of some of these aroma characteristics can be a positive aroma attribute. To others, even the slightest hint of Brettanomyces character is cause for a wine’s rejection. Despite the traditional negative connotations of 'brett' character, in some cases, the yeasts may add positive characters and complexity to red wines (Heimoff 1996, Fugelsang 1997). These flavors though can become so dominant that they completely mask the varietal and regional flavor characteristics of a wine. These flavors which we call bretty here and which we will describe further in this paper should not to be confused with mousiness (see discussion below). To be able to protect the wines against spoilage by these yeasts and to possibly be able to positively integrate some of their flavor into some wines it is important that winemakers are familiar with the Brettanomyces flavor characteristics and with its physiology. Winemakers need new tools for reliable and early detection of Brettanomyces in their wines before the wine quality is altered. In 1960, van der Walt and van Kerken (1960) reported the ascospore formation in yeast strains previously classified as the non-sporeforming yeasts Brettanomyces and proposed that a new genus be created to accommodate the sporogenous forms. Van der Walt proposed the name Dekkera for the new genus and reported two species, D. bruxellensis and D. intermedia (1964). Several taxonomic changes have occurred since van der Walt's introduction of the new genus with additions and deletions to both genera as the demonstration of spore formation and the absence of spore formation occurred. Brettanomyces, the anamorph form of the genus Dekkera includes five species. B. nanus was added to the four recognized species B. bruxellensis, B. anomalus, B. custersianus and B. naardenensis following the renaming of Eeniella nana on the basis of rDNA sequence homology (Anonymous 1998, Boekhout et al. 1994, Hoeben and Clark-Walker 1986, Yamada et al. 1995). Molecular studies of species within this genus were initiated based on the mitochondrial genome (Hoeben and Clark-Walker 1986) and the rRNA genes (Molina et al. 1993). Molina et al. (1993) carried out restriction analysis of the 18S rRNA from various Brettanomyces yeasts and found good correlation with isoenzyme electrophoresis and DNA homology analysis. Further investigation of the ribosomal RNA genes on the partial sequence level of the 18S and 26S rRNA was done by Yamada et al. (1994) and Cai et al. (1996), who determined the sequence of various Brettanomyces and Dekkera yeast strains by PCR and direct sequencing. Ibeas et al. (1996) used nested PCR to track one single strain in sherry wine. Mitrakul et al. (1999) used RAPD-PCR to discriminate yeast strains within the species B. bruxellensis. Although strain discrimination worked out well by RAPD-PCR in that study, results can be difficult to reproduce using this method. Alternatively, ribosomal genes have been used extensively to give information about the phylogenetic relationship of taxa. In many eukaryotes ribosomal genes in yeast are organized as 18S-5.8S-26S operons which are tandemly repeated 50 to 200 times per haploid genome (Planta and Raué 1989). The two internal transcribed spacers (ITS1 and ITS2) separate the 18S and the 26S from the 5.8S rRNA gene. Conserved portions of rRNA genes are useful for inferring phylogenetic relationships between strains. ITS regions are less conserved due to less evolutionary constraints (Musters at el. 1990) and therefore can be used to discriminate species within some genera. Ribosomal DNA restriction fragments length polymorphism and comparative sequence analysis of the two internal transcribed spacer (ITS) regions located between the ribosomal RNA genes was carried out using Brettanomyces/Dekkera yeast reference strains and wine isolates (Egli et al. 2000). Both ITS1 and ITS2 were found to contain distinct regions with sufficient sequence divergence to make them suitable as specific identification target sites. Specific oligonucleotides were designed for each Brettanomyces/Dekkera species and evaluated for specificity and reliability using reference strains and wine isolates. Use of these specific primers allowed for species-specific discrimination. Brettanomyces/Dekkera yeast isolates from wine were shown to uniquely belong to the species B. bruxellensis. The traditional methods of identification as described by van der Walt and Yarrow (1984) and Barnett et al. (1990) are laborious and time consuming. Identification takes 3 to 4 weeks and often results are ambiguous and can be unreliable (Mitrakul et al. 1999). The development of molecular techniques for yeast identification has substantially reduced the time for identification with improved reliability through the direct analysis of DNA, thus identification will not vary with the physiological state (Dowhanick, T. M. 1995, Ness et al. 1993). Included among the molecular techniques for identification are DNA fingerprinting methods. DNA fingerprinting describes any procedure which provides a unique profile of DNA for a given organism (Meaden 1990). Mitrakul et al. (1998) and Egli et al. (2000) isolated and described Brettanomyces from 3 Cabernet Sauvignon wines which were characterized by tasting panels as having no, some, and strong bretty flavor characteristics (Licker et al. 1999). Using the molecular methods of karotyping, restriction fragment length polymorphism (RFLP), and RAPD-PCR analysis the isolates were indentified as Brettanomyces / Dekkera bruxellensis (Fig. 1). To test the specificity of the OPA-03 primer in the RAPD-PCR assay for Brettanomyces yeast, several other yeasts which are abundant in the must or wine were taken as controls. Included were Candida guilliermondii (lane 13), Pichia anomala (lane 14), Hanseniaspora uvarum (lane 15), and Saccharomyces cerevisiae (lane 16). Of these yeasts Hanseniaspora uvarum has the ability to grow in the presence of cycloheximide as do Brettanomyces/Dekkera yeasts (Barnett et al. 1990; Fugelsang 1997). None of the fingerprints of these yeasts was similar to those obtained for Brettanomyces/Dekkera yeasts (Fig. 1B) confirming the usefulness of primer OPA-03 for detection and discrimination purposes. Similar patterns were found for all isolates of the three vintages 1989, 1992, and 1994 matching with D. bruxellensis reference strains (Fig. 1C). Further investigations showed that over the years, different strains of Brettanomyces evolved in the wines of this winery (Egli, unpublished). Each wine typically contained one strain of Brettanomyces (Mitrakul et al. 1999). A M 1 2 3 4 5 6 7 8 9 10 11 12 c bp 26 45 16 05 11 98 67 6 51 7 46 0 39 6 35 0 B bp 26 45 16 05 11 98 67 6 51 7 46 0 39 6 35 0 M 13 14 15 16 c C M 89 92 94 c bp 26 45 16 05 11 98 67 6 51 7 46 0 39 6 35 0 Figure 1. Species determination of Brettanomyces/Dekkera strains and wine isolated yeasts using RAPD-PCR fingerprints generated with primer OPA-03. (A) Lanes 1-7 represent strains of the species D. bruxellensis (lane 1, D. bruxellensis; lane 2, B. custersii; lane 3, D. abstinens; lane 4, B. bruxellensis; lane 5, B. intermedius; lane 6, B. lambicus; lane 7, D. intermedia), lanes 8-10 represent strains of the species D. anomala (lane 8, D. anomala; lane 9, B. claussenii; lane 10, B. anomalus), and lanes 11 and 12 represent single strains of the species D. custersiana (B. custersianus) and D. naardenensis (D. naardenensis), respectively. (B) Lane 13 represents Candida guilliermondii; lane 14, Pichia anomala; lane 15, Hanseniaspora uvarum; and lane 16, S. cerevisiae. (C) Lanes 89, 92, and 94 represent single isolated colonies from the 1989, 1992, and 1994 wine samples, respectively. The patterns were identical for all ten isolated strains in each vintage. Only one strain is shown for that reason. Lanes M show the D-15 DNA marker, and lanes c the negative control containing no DNA. (Mitrakul et al. 1998) Occurrence of Brettanomyces In 1930 Krumbholz and Tauschanoff (1933) isolated the yeast Mycotorula intermedia from a French grape must; Custer (1940) reclassified it as Brettanomyces bruxellensis. In 1911 from Wädenswil, Osterwalder (1912) isolated and identified the yeast Monilia vini in Swiss apple wine; Schanderl (1950) and Schanderl & Draczynski (1952) presumed it to be a Brettanomyces species; based on the physiological evidence of Osterwalder, van der Walt & van Kerken (1958b) characterized it as Brettanomyces intermedius. In the early 1950's Schanderl (1950) and Schanderl & Draczynski (1952) of Geisenheim reported the first uncontested occurrence of Brettanomyces in bottled wine (van der Walt and van Kerken, 1958a) ; the isolation was from a German sparkling wine. Since then Brettanomyces yeast have been reported in almost every winemaking region of the world (Licker et al. 1998). Although Brettanomyces/Dekkera yeasts are typically not included in the genera of yeasts found on the surface of grapes (Fleet & Heard 1993), it is quite common for these yeasts to be found in the winery (Chatonnet et al. 1995, Fugelsang 1997, Licker 1999). We have indications that Brettanomyces can already occur on some grapes. Studies currently underway will be testing this hypothesis. Researchers isolated Brettanomyces in fermenting grape must from around the world: France (Krumbholz and Tauschanoff, 1933; Domercq, 1956); Germany (Schanderl, 1950);Italy (Florenzano,1951; Trioli, 1996), South Africa (van der Walt and van Kerken, 1961); Uzbekistan (Mavlani, 1968); New Zealand (Wright and Parle, 1974); and Spain (Mateo et al., 1991; Querol et al., 1990). Brettanomyces populations were found to be a minority in the microflora of fermenting musts. Domercq (1956) detected two Brettanomyces cultures out of 80 red; no cultures were isolated out of 38 white grape musts sampled from French wineries. With a refinement of isolation procedures Brettanomyces were isolated consistently from fermentations in 6 of 15 New Zealand wineries, and occasionally from 4 of the 15 (Wright and Parle, 1974). In total they were isolated in 33 of 124 fermenting white and red musts in New Zealand. The alteration of wines by Brettanomyces/ Dekkera yeasts typically occurs during barrel aging of wine prior to bottling (Chatonnet 1995). These yeasts have caused wine spoilage in wine producing areas around the world (Fugelsang 1997, Boulton et al. 1996, Chatonnet et al. 1992, Heimoff 1996, Heresztyn 1986, Ibeas et al. 1996). However, little is known about these yeasts and rapid and reliable detection of these yeasts is needed before positive or negative aromas and flavors can be attributed to particular strains. Kunkee and Amerine (1970) wrote of the problems associated with yeast detection: "The major problem of studies on yeast ecology results from the methods used for isolating the microflora. So many different techniques have been used that comparisons of frequency of occurrence must be made with caution". Van der Walt & van Kerken (1961) were initially unable to isolate Brettanomyces from musts and winery equipment using customary media and techniques. It can take a week or longer before colonies are visible, a far longer incubation time compared to Saccharomyces and other yeast. Custer (1940) was first to note the characteristically slow growth of the genera. Saccharomyces, Kloeckera, Metschnikowia, Pichia, Candida, or "wild" yeast develop earlier in culture thereby hindering detection of Brettanomyces. Early mold development inhibits detection by covering the media surface. Brettanomyces may go undetected; plates may be discarded before colonies develop. Addition of 100 ppm cycloheximide (actidione), 10 ppm thiamin, and 0.5% calcium carbonate to media aids in the selective detection of Brettanomyces yeast (Phaff and Starmer, 1980). Another problem of Brettanomyces detection in the winery is large variations in barrel to barrel populations. In a 45 week barrel sampling study of stored Cabernet Sauvignon wine, Blazer and Schleußner (1995) determined it was necessary to stir barrels before plating. Measured populations increased after stirring - in some cases by 10-fold or more; in others, detection depended on stirring. Fugelsang (1993) stated that wood cooperage is the most frequently cited source of Brettanomyces within the winery. It must be remembered though that Brettanomyces can enter a winery with the grapes. Wineries are encouraged by some enologists in the United States to destroy Brettanomycesinfected barrels to avoid further contamination within the winery (Boulton et al., 1996; Heimoff, 1996). Another problem is barrel to barrel variations within the same lot. In a recent two-year Californian winery investigation (Frey et al., 1996; Heimoff, 1996), new oak, stainless steel, and previously Brettanomyces infected barrels were filled with Cabernet Sauvignon inoculated with 4 cells/mL of a Brettanomyces culture. Similar barrels were filled with sterilized wine. All of the wooden barrels (new & old containing inoculated & sterile wine) developed Brett. populations. Brettanomyces populations were undetectable in all of the stainless steel barrels. Flavors associated with Brettanomyces yeasts Brettanomyces yeasts and their characteristic flavors were first described in English beers (Licker et al. 1998). To obtain the "English character" flavor production in beer Claussen (1904) stressed "a general rule cannot be given for all cases, but the quality of Brettanomyces to be added must be regulated by local circumstances, more especially by the time the beer has to be stored and by the temperature of the storing room." A Brettanomyces inoculation with a wort of 1.055 specific gravity and a room temperature of 24-27 °C would achieve the "English" character. Shimwell confirmed these conditions: a 1.060 specific gravity was essential to achieve a "vinous" wine-like flavor (Shimwell, 1947); in contrast, a beer under 1.050 would produce an unpalatable and turbid beer with an objectionable, insipid flavor and aroma (Shimwell, 1938) . As Shimwell (1947) noted, Brettanomyces can behave "as a desirable organism in one beer and an undesirable one at one and the same brewery". Clearly, in winemaking practice we are not yet at the stage where we could utilize the flavor characteristics of Brettanomyces as deliberately as was discribed by these two brewing researchers. We still lack sufficient understanding to the different flavor characteristics, of the different strains of Brettanomyces, of the conditions under which they grow in wine, and the conditions which affect the type of flavors they produce. We started our investigations of the flavor contribution of Brettanomyces yeasts by obtaining different wines which were described by winemakers as being bretty. Licker et al. (1999) studied two groups of commercial wines with suspected “Brett” character using two trained panels of judges. One group of judges (A) were winemakers from New York State and experienced tasters from the New York Agricultural Experiment Station. The second panel (B) was an international group of enologists including members from Australia, France, Germany, Switzerland, and the USA. The first group of wines included four Cabernet Sauvignon and two Pinot Noir wines; the second included four Cabernet Sauvignon wines. All were evaluated by sensory descriptive analysis and GC/MS 4-ethyl phenol analysis. Characteristic “Brett” aromas such as plastic, burnt plastic, Band-aid™, cow manure, barnyard, and horse sweat were summarized by panel A as ‘plastic’. For panel B ‘plastic’ included only plastic, burnt plastic, and Band-aid™ odors. Dry manure and sweaty/animal were separate descriptors. In both groups, the wines were differentiated by univariate analysis of variance (ANOVA) and multivariate discriminant analysis (DA) by two descriptors: plastic and fruity. Figure 2 shows the clear separation of the 3 Cabernet Sauvignon wines, the youngest wine being the most fruity and least plastic (bretty), the oldest being the most plastic (bretty) and least fruity. The 1992 wine was characterized by both some fruit and some plastic, the brett aromas appeared to give it some spicy aroma character. The a priori “Brett” observations from the winemakers proved to be a consistent predictor of Brett character for all wines. In these wines the intensity of perceived brett character agreed with the 4-ethyl phenol concentrations. The “strong Brett” wines were the older vintage wines with higher 4-ethyl phenol concentrations, higher plastic and lower fruity mean scores. Wines with “maybe some” and “no Brett” had the lower 4-ethyl phenol concentrations and more fruit with less plastic scores. This investigation shows that aroma modifications by Brettanomyces yeasts can be reliably detected and quantified with trained tasters. Further investigations into the chemical basis of the ‘Brett’ aromas will allow us to use chemical indicators to detect activity of these yeasts early. 1 Odor activ ity 100 % 2 4 . 9 8 5 6 3 12 Odor activ ity Odor activ ity 92CS 15 14 b 2 3 7 5 9 8 10 14 1 4 6 15 1 1 100 % 89 CS 7 11 10 13 1 100 % 0 a 12 13 3 2 4 5 7 11 15 700 10 14 8 6 912 1 3 Retentio n Ind ex (OV101) 1500 ~ 94CS plastic ~ smoky ~ ~ ~ ~~~ ~ ~ ~~ ~ c fruity DF1 = 87% ~ spicy Figure 2. Scatterplot of the canonical scores for the two discriminant functions based on data from panel A (1989, 1992, and 1994 Cabernet Sauvignon produced in the same winery from the same vineyard sources). Figure 3. Odor spectrum gas chromatograms on an OV101 column - the top 15 odor active compounds: (a) "no Brett", (b) "medium Brett", (c) "high Brett". Numbers on the chromatogram refer to the chemical structures in figure 4. an OV101 column - the top 15 odor active compounds: (a) "no Brett", (b) "medium Brett", (c) "high Brett". Numbers on the chromatogram refer to the chemical names in Table 1. Comparing the odor spectrum gas chromatograms of the three wines (Fig. 3 and Table 1), in a general effect was observed. 'Floral', 'fruity' compounds were the dominant odorants in the "no Brett" wine while 'rancid', 'plastic' odors accounted for 1/3 or less of the odor activity; in the "medium Brett" wine, the 'floral', 'fruity' compounds decrease to 1/2 or less of the odor activity while the 'rancid', 'plastic' compounds increase to 2/3; in the "high Brett" wine, the 'rancid', 'plastic' compounds were the dominant odorants while the 'floral', 'fruity' compounds were far less dominant. The 'floral' odorant identified as 2-phenyl ethanol was the dominant compound in "no Brett" and "medium Brett" wines; in the "high Brett" wine, it was equally as dominant as isovaleric acid and the unknown compound. The 'fruity' odorant ß-damascenone was equally dominant among the three wines; for this reason, it should not be considered as a contributor to "Brett" aroma. Odor/Compound 0 Odor activity +/- S. E. 50 100 rancid, isovaleric acid burning tires, cis-2-nonenal burning tires, trans-2-nonenal plastic, guaiacol plastic, 4-ethyl phenol plastic, ethyl decanoate plastic, unknown C94CS C92CS C89CS Figure 4. Comparison of the odor activity of 7 dominant bretty odor components in Cabernet Sauvignon wines from 3 different vintages from the same winery (GCO analysis, Licker 1999). The odor active compounds were ranked from 1-15, 1 being the most odor active compound. The numbers above each peak in the chromatogram correspond to the compound and associated descriptor in the table. Isovaleric acid (3-methyl butanoic acid) was found to be the dominant odorant in the "high Brett" wine as detected by CharmAnalysis. The odor described by the GCO sniffer was 'rancid'; the chemical identity of the odorant was confirmed by GC-MS. This acid is produced in wine by yeast as a metabolic byproduct of protein (99). Volatile phenolic compounds, such as 4-ethyl guaiacol, guaiacol, and 4-ethyl phenol, were also among the dominate odor active compounds in this wine; however, the individual contribution by each of the three phenolics was half or less than the odor activity of isovaleric acid. The chemical identity of the second most dominate odorant in the "high Brett" wine, identified as 'plastic' by CharmAnalysis, remains 'unknown'. Bretty and mousy flavors Bretty flavors should not be confused with mousy flavors. When we refer to bretty flavors we refer to aromas such as burnt plastic, bandaid™, barnyard, manure, creosote, wet wool, sweaty leather, and a bitter, matallic-like taste. These are the terms with which California and New York winemakers described bretty flavors (Licker et al. 1998, Licker et al. 1999). In tasting many wines with different intensities of brett flavors only once have we tasted a wine that was bretty and mousy. On the other hand we have tasted many wines which were mousy but not bretty. Mousiness is a distinct flavor which is perceived retronasally, it is almost never smelled. It is described as the aroma of mouse or rodent urine, in some mild cases it has been described as black walnut like. While the smoky, barnyardy, sweaty aromas are found attractive by some people, the mousy flavor is always strongly rejected by people who perceive it. Heresztyn (1986) described tetrahydropyridines formed by some isolates of Brettanomyces and by some lactobacilli as the chemicals responsible for the mousy taint. The taint was produced by each class of microorganisms only in the presence of lysine and ethanol. Researchers at the Australian Wine Research Institute (Henschke 1996a) have since identified two additional compounds associated with mousiness and produced by both Brettanomyces and Lactobacillus species: 2-acetyl-1-pyrroline (ACPY) and ethyltetrahydropyridine (ETPY). In tainted wines ETPY was found at concentrations below its high odor threshold, thereby contributing little to the sensory character of mousiness. The two acetyl-tetrahydropyridines and ACPY were reported to be the main mousy taint compounds. It is still unclear how the Brettanomyces implicated in the mousy taint relate to the many Brettanomyces strains isolated from bretty wines which were not mousy. Clearly more research is necessary to compare wine descriptions from different countries and to understand which strains of Brettanomyces, under what conditions, can form the mousy flavors in wine. Physiology of Brettanomyces yeasts – conditions which favor their growth and ways in which winemakers can limit their growth Even if some infection by Brettanomyces and some amount of bretty flavors is attractive in some wines in other wines this contribution of Brettanomyces to the wine flavor profile is not desired at all. Therefore the winemaker must constantly look out for signs of infection (presence of Brettanomyces or appearance of characteristic bretty aromas). Then infected and non-infected wines can be kept separate and treated as necessary. We still lack very good ecological studies which show the path of infection with Brettanomyces yeasts in the winery. Now that we have the molecular biological tools to identify individual strains of Brettanomyces (Mitrakul et al. 1998, Egli et al. 1999) we can design studies which tell us where exactly a particular strain in a particular wine came from. Brettanomyces can be brought in with the grapes particularly damaged grapes contain a large number of microorgansims. Also,apparently more commonly, Brettanomyces is spread to new batches of wine from other batches of infected wine or from contaminated barrels and other winery equipment. It appears that Brettanomyces yeast are not very competitive with the main fermentative yeast Saccharomyes. We don’t know the nutrient requirements or the range of pH, temperature, and alcohol concentration over which Brettanomyces can grow. Why is Brettanomyces found almost always in red wines and almost never in white? We do know that these yeasts are sensitive to SO2, they can be removed by appropriately tight filters (1 micrometer and smaller) and they are killed by ozone and dimethyl dicarbamate (DMDC, Velcorin™). Good cellar hygiene is the first step in controlling infection of Brettanomyces. Equipment, tanks and barrels, the cellar environment should be kept clean at all times. New and used barrels can bring Brettanomyces into a winery. Batches of wine infected with Brettanomyces should be kept separate and monitored carefully (add SO2 or filter if yeast population rises to high cell density, more than 10 2 cells per mL). Before blending an infected batch with another wine remove the yeast by filtration or kill the yeast with Velcorin™. Carefully sanitize the container in which the infected wine was stored. Several studies indicate that Brettanomyces can grow in the grape must. This is important to remember when skin contact or cold soaking is used. To reduce the risk of Brettanomyces growth before alcoholic fermentation, damaged grapes should be avoided, SO2 should be added (20-50 mg/L). After cold soak the must should be warmed quickly to suitable fermentation temperature (for most wine yeast 16-20°C) and a yeast starter culture should be added once the must has been warmed. A good active fermentation with Saccharomyces cerevisiae apparently suppresses growth of Brettanomyces. After completion of alcoholic fermentation the wine again is under increased risk of infection by Brettanomyces. To limit this risk, malolactic fermentation should be induced quickly, best with the addition of a starter culture. After completion of malolactic fermentation the wine should be racked and sulfited or a least sulfited (to maintain 0.8 mg/L molecular SO2). Maintaining adequate free SO2 during barrel or tank aging of wine will prevent in most cases Brett growth. According to Chatonnet et al., (1993), the only way to limit the growth of Brettanomyces in red wines aged in oak barrels is to maintain a sufficient concentration of free sulfur dioxide (SO 2) throughout the aging process. At least 7 g/barrel of SO2 gas should be used to disinfect empty barrels. Filled wine barrels should receive 20 to 25 mg/L of free SO2, 30 to 35 mg/L in the hot summer. These concentrations should be maintained throughout the aging process to limit Brettanomyces development (Chatonnet et al., 1993). Scott (1995) showed yeast and bacteria could penetrate the porous cellular structure of oak barrels and establish active permanent sub-surface mixed cultures even after cleaning. Variations in wine composition (pH, anthocyanin concentration, nutrient content, and temperature) can affect SO 2 treatment in wine (Rose, 1987; Smith, 1996). Brettanomyces can endure SO2 treatment in barrels (Hock, 1990). The yeast survives treatment in areas of limited SO 2 contact: around bung holes, in the oak, and in the yeast sediment (lees). Of course in the case of red wines it is often not possible or desirable to maintain high free SO2 concentrations during wine aging. In this case the wines must be monitored regularly for presence of Brettanomyces yeasts and for flavor change. Today, monitoring the concentration of 4-ethylphenol is still the most convenient method to monitor many batches of wine. In most cases an increase of 4-ethylphenol indicates presence and activity of Brettanomyces. Not always though is the presence of Brettanomyces indicated by 4-ethylphenol and not always when 4-ethylphenol is found can live Brettanomyces be found. The main disadvantage is that once we find increased concentrations of 4-ethylphenol in a wine the wine already is noticeably bretty. Thus the 4-ethylphenol content only confirms what a trained winemaker can taste. Preliminary data from Zoecklein, Whiton, and Fugelsang (personal communication 1999) indicate that 4ethylphenols is formed continually while live cells of Brettanomyces are present in wine (Fig. 5). The 4-ethylphenol concentration increases with cell number and as long as live yeast are present (including decline phase of growth, in this case after). We should have other indicator chemicals which are formed by Brettanomyces during early stages of infection, before the typical brett aromas are developed. Also we need microbiological detection tests which are easier, quicker, and more reliable than the plating tests and identification tests. A direct assay for Brettanomyces cells or DNA in the wine is needed. This test should be quick (minutes) so that it can be used during wine transfers.