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The challenge of Brettanomyces in wine
LWT - Food Science and Technology 43 (2010) 1474e1479
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The challenge of Brettanomyces in wine Danielle Wedral, Robert Shewfelt, Joseph Frank* Department of Food Science and Technology, University of Georgia, Athens, GA 30602, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 February 2010 Received in revised form 1 June 2010 Accepted 8 June 2010
Brettanomyces spp. is an indigenous yeast that can grow during wine fermentation and impact its flavor. The characteristic flavor is often described as phenolic/medicinal, spices, cloves, earthy, barnyard, and horsy. The acceptability of Brettanoymces-induced flavors depends on the flavor intensity, personal preference, and consumer expectations. The yeast has been isolated from grapes, barrels, and winery equipment. It impacts wines from all the top producing regions in the world, with associated flavors most often found in red wines. Prevention of growth of this yeast in wine involves attention to fruit quality and winery sanitation, control of sulfite and oxygen levels, and the use uncontaminated barrels. Greater understanding of how Brettanomyces and its metabolites contribute to wine flavor is required to recognize its impact on consumer acceptability. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Brettanomyces Wine
1. Introduction Brettanomyces bruxellensis is a yeast found on the surfaces of grapes as well as in barrels, but the greatest concern is its presence in wine. It characteristically produces 4EP and 4EG, two easily measured compounds that are indicative of Brett activity, as well as a variety of other compounds that can contribute to wine aroma complexity. At low concentrations, these and other compounds that the yeast produces can contribute to wine aroma complexity. Higher levels of metabolites impart intense flavors that overwhelm the wine aroma and create an unpleasant experience (Chatonnet & Pons, 1990). Therefore Brett-associated metabolites in wine can be either negative or positive depending on concentration and consumer expectation. 2. Characteristics of Brettanomyces spp. Brettanomyces spp. are the non-spore forming counterparts of the genus Dekkera. Brettanomyces and Dekkera are often used interchangeably and due to Brettanomyces being the more common name found in the literature, it will be used in this review. There are five species belonging to the genus Brettanomyces/Dekkera, including Brettanomyces custersianus, Brettanomyces naardenensis, Brettanomyces nanus, Brettanomyces anomalus, and B. bruxellensis (Kurtzman & Fell, 2000). B. bruxellensis most commonly affects wine, and it has been found on the surfaces of grapes as well as in
* Corresponding author. Tel.: þ1 7065420994. E-mail address: [email protected] (J. Frank). 0023-6438/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2010.06.010
barrels. B. bruxellensis has an oval to an ellipsoidal shape and reproduces by budding. Cell morphology changes from elliptic to branched shape after a few months of incubation (Fig. 1). The physiology of B. bruxellensis has been well studied because of its unusual aromatic bi-products and because it is an example of the Custer effect, which is the inhibition of alcoholic fermentation under anaerobic conditions due to high production of acetic acid and redox imbalance (Henschke, Curtin, & Grbin, 2007; Van Dijken & Scheffers, 1986; Vigentini et al., 2008).
3. Historical aspects Brettanomyces spp. can be found in traditional wine and beer produced using indigenous cultures. Classic English stock beers went through a secondary fermentation in a cask where Brettanomyces was naturally present or was added. This imparted the typical English stock ale character, much of which is believed due to growth of Brettanomyces spp. The name Brettanomyces was first introduced by N Hjelte Claussen of New Carlsberg Brewery to specifically describe the yeast required to produce English stock beers, and became a recognized genus in the 1920s when a similar yeast was isolated from Lambic ales of Belgium (Henschke et al., 2007). In the 1950s and 1960s the yeast was identified as Brettanomyces ssp. and was isolated from wine in France, Italy and South Africa, but it wasn’t until the 1980s and 1990s that the yeast was characterized for its ability to impart characteristic aroma to wine (Henschke et al., 2007). Loureiro and Malfeito-Ferreira (2006, pp. 364e398) reviewed information on the presence of Brettanomyces in various products.
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(Arvik, Conterno, & Henick-Kling, 2002). Research on B. bruxellensis has been conducted in many parts of the world and the organism has been isolated in wines from all major wine producing regions for wine production (Conterno, Joseph, Arvik, Henick-Kling, & Bisson, 2006). A Portuguese study that analyzed wine volatiles from different countries found that more than 25% of red wines had 4-EP levels higher than the preference threshold of 620 mg/L, above which some consumers reject product (Chatonnet, Dubourdieu, Boidron, & Pons, 1992; Lesschaeve, 2007; Loureiro & MalfeitoFerreira, 2003). These results are not based on random sampling, but indicate that volatile phenols related to Brettanomyces are a worldwide issue. Another study performed in Burgundy on Pinot noir wine demonstrated that these yeasts are present in about 50% of the wines undergoing maturation and about 25% of bottles (Gerbaux, Naudin, Meurgues, & Monamy, 2000). Various authors have concluded that controlling growth of Brettanomyces is the most important microbiological challenge of modern winemaking, and is responsible for significant economic losses worldwide (Boulton, Singleton, Bisson, & Kunkee, 1996; Fugelsang, 1997). 5. Chemistry of Brettanomyces-induced flavor
Fig. 1. Photomicrograph of B. bruxellensis. Photo by D. Wedral.
Brett has been isolated from a variety of products and places ranging from wines and winery equipment, beer (in particular lambic beers), cider, apple cider factories, tequila, dairy equipment and kombucha tea. Brett is often found in these products due to its ability to survive for long periods and initiate growth in stored or maturing products. For example, Brettanomyces is commonly isolated from lambic beers after one year of maturation when Saccharomyces spp. are no longer found (Loureiro & MalfeitoFerreira, 2006). At present, Brettanomyces spoilage of wines usually occurs when they are fermented or aged in oak barrels (Rayne & Eggers, 2008). The yeast grows slowly so it usually imparts flavors only when the wine is aged. Since Brettanomyces is present at low numbers early in the fermentation, it is outnumbered by other indigenous yeast and may go undetected. Modern production practices for beer including sanitation practices, high carbon dioxide levels, and low nitrogen contents have made growth of Brettanomyces in this product infrequent. 4. The current issue of Brettanomyces in wine The main factor affecting the sensory properties imparted by B. bruxellensis is the production of 4-ethylphenol (4-EP) and 4-ethylguaiacol (4-EG) (Vigentini et al., 2008). Small differences in 4-EP and 4-EG concentrations can be detected by consumers and quantifed by trained tasters (Licker, Acree, & Henick-Kling, 1999). The presence of Brett character can be considered either negative or positive depending on concentration and expectation of a particular wine. At low concentrations, these compounds can contribute to wine aroma complexity, but high concentrations can overwhelm the wine aroma and create an unpleasant experience (Chatonnet & Pons, 1990). The quality of wines containing Brettanomyces metabolites is controversial in that this judgment depends on individual and cultural preferences. For example in 4-EP sensory analysis, a New York State group of experts described the flavor as plastic, burnt plastic, phenolic/medicinal, cow manure, barnyard and horse sweat, whereas an international group only included the more neutral terms burnt plastic and phenolic/medicinal in their descriptors. In general, growth of Brettanomyces in wine is considered undesirable in the United States, some consider this a global issue
The initial detection of Brettanomyces-induced aromas in wine generally occurs during the barrel maturation (Godoy, Martínez, Carrasco, & Ganga, 2008). These aromas result primarily from the enzymatic conversion of ferulic and p-coumaric acid into 4-vinylguaiacol and 4-vinylphenol by hydroxycinnamate decarboxylase. These compounds are then reduced to 4-EG and 4-EP by vinylphenol reductase (Suárez, Suárez-Lepe, Morata, & Calderón, 2007). Other yeasts can reduce p-coumaric acid to form 4-vinylphenol, but B. bruxellensis appears unique in its ability to reduce vinylphenols to sensory detectable amounts of 4-EP and 4-EG (Chatonnet, Dubourdieu, & Boidron, 1995). The aroma of 4-EP is often described as phenolic/medicinal and that of 4-EG is often described as clove/holiday spice. Cinnamate decarboxylase activity of B. bruxellensis, unlike that in S. cerevisiae, is not inhibited by phenolic compounds in grapes, so it can produce several milligrams of ethylphenols per liter of wine, the amount produced being directly proportional to its population (Chatonnet et al., 1995). A high polyphenol content of the wine will drive the enzymatic reaction to produce more end products. A low correlation between amount of 4-EP and number of yeast colony forming units present in the wine is often observed, suggesting that the metabolite (or enzyme producing it) is released upon yeast cell death and autolysis (Fugelsang & Zoecklein, 2003). Another Brettanomyces -produced flavor compound is 4-ethylcatechol (4-EC), which is noted for its medicinal aroma. Caffeic acid is the precursor compound for this metabolite. Due to the requirement to be derivatize to be detected by gas chromatography 4-EC has not been studied to the extent of the other volatile phenols, but it does have a lower detection threshold than other ethyl phenols, so may be of importance (Loureiro & Malfeito-Ferreira, 2006). In addition to flavor changes, wines contaminated with Brettanomyces may have an undesirable color. This may result from glycosidic activities or formation of vinylphenolic pyranoanthocyanins (Oelofse, Pretorius, & Du Toit, 2008). 5.1. Chemistry Brettanomyces-induced flavor in wine The average concentrations where 4-EP and 4-EG may influence wine flavor are 0.62 mg/l and 0.14 mg/l with recognition thresholds of 770 and 436 mg/l, respectively (Chatonnet et al., 1992). 4-EP and 4-EG appear in wines at various amounts and ratios depending on the variety of grape used and wine style. On average they appear at a 10:1 ratio respectively, which corresponds to the respective
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precursor ratio of p-coumaric acid and ferulic acid (Chatonnet et al., 1992; Romano, Perello, Revel, & Lonvaud-Funel, 2008). Another study showed average ratios of 4-EP to 4-EG differed with different wine types and was approximately 10:1 for Cabernet Sauvignon, 9:1 for Shiraz, 8:1 for Merlot and 3.5:1 for Pinot Noir. Little work has been done on determining thresholds of these compounds in different varieties, but some indicate thresholds to be higher in Cabernet Sauvignon and lower in Tempranillo wines (Suárez et al., 2007). Studies have also shown no difference in ethylphenol concentrations produced in Cabernet Franc, Cabernet Sauvignon, Merlot, Pinot Noir, and Syrah wines (Rayne & Eggers, 2008). Recent studies have demonstrated that B. bruxellensis is not the only organism that can produce 4-EP and 4-EG, and that not all strains of B. bruxellensis produce these compounds. One study found that 20% of Brettanomyces strains did not produce these compounds (Conterno et al., 2006). Winemaking strains of S. cerevisiae differ in their ability to decarboxylate hydroxycinnamic acids to vinylphenols, therefore the idea that Brettanomyces/Dekkera yeasts differ in this characteristic as well seems plausible (Curtin, Bellon, Henschke, Godden, & De la Lopes, 2007; Shinohara, Kubodera, & Yanagida, 2000). Some microorganisms including Lactobacillus brevis and Pediococcus pentosaceus can decarboxylate p-coumaric acid to form 4-vinylphenol, but few, including Brettanomyces and Lactobacillus plantarum, are capable of producing ethylphenols (Chatonnet et al., 1995). Thirty seven percent of lactic acid bacteria tested could produce volatile phenols from p-coumaric acid and 9% could produce 4-ethylphenol (Couto, Campos, Figueiredo, & Hogg, 2006). What differentiates Brettanomyces from other microorganisms is the relative high production of ethylphenols. For example, Pichia gulliermondii is capable of converting p-coumaric acid to 4-ethylphenol in grape juice, grapes and winery equipment, but has shown low to no conversion rates in wine (Dias, Dias, et al., 2003), making Brettanomyces/Dekkera yeasts the sole known cause for ethylphenol-associated off flavors (Barata, Nobre, Correia, Malfeito-Ferreira, & Loureiro, 2006; Vigentini et al., 2008). The formation of Brett character is influenced by the microbial strain, wine pH, nutrient availability in the must and stage of winemaking at which contamination occurs (Romano et al., 2008). Nitrogen availability is more important in limiting flavor production than is the presence of residual sugars (Conterno et al., 2007). P-coumaric acid appears to induce off-flavor production more than does ferulic acid, but strain variation is significant in this regard (Harris, Ford, Jiranek, & Grbin, 2009).
5.2. Genetic diversity and flavor character Genetic-associated strain variation is behind much of the variability in sensory descriptions of Brettanomyces-induced flavors and variations in its growth ability (Vigentini et al., 2008). For example, B. bruxellensis strains exhibit significant variation in their ability to grow in Pinot Noir wines (Fugelsang & Zoecklein, 2003), and individual strains differ in their impact on phenolic profiles of the wine (Silva et al., 2005). Similar genetic subtypes of B. bruxellensis have been found worldwide, an indication that the yeast has spread geographically, perhaps due to international trade in grape juice, barrels, and winery equipment. It is also possible that similar genetic adaptations have arisen independently due to similar stresses in the wine making environment (Curtin et al., 2007). Because of inherent strain variation, indicators of the capacity of strains to induce off-flavors are important. The ability to produce off-flavors in wine is related to a strain’s production of volatile phenolic compounds. Therefore, measurement of ethyl phenol production can be used as a simple indicator of strain spoilage capacity (Vigentini et al., 2008).
6. Growth of Brettanomyces during winemaking B. bruxellensis can be present at any stage in wine making, but due to its high ethanol and low sugar tolerances it is most often associated with aging in oak barrels, stuck or slow alcoholic fermentations and malolactic fermentation (Dias, Pereira-da-Silva, Tavares, Malfeito-Ferreira, & Loureiro, 2003; Renouf et al., 2006). Brett can tolerate 14e14.5% ethanol in red wine (Loureiro & Malfeito-Ferreira, 2006), and may grow in high alcohol wines and during stuck fermentations (Silva, Cardoso, & Gerós, 2004). Brettanomyces can also slow the growth of Saccharomyces by producing acetic acid (Uscanga, Delia, & Strehaiano, 2003). Red wines are particularly susceptible to Brettanomyces bruxenellis infection due to their lower acidity, higher polyphenol content and barrel aging. Therefore Vitis vinifera red varietals with higher precursor polyphenol content are the most susceptible to the Brettanomyces defect. For example, Pinot Noir generally has low levels of p-coumaric acid providing B. bruxenellis less substrate for 4-EP production. B. bruxellensis’s slow growth makes wines that are stored for long periods in wooden barrels particularly susceptible to its influence (Suárez et al., 2007). Wines that are aged longer in barrels are more susceptible to the defect if B. bruxenellis is in the barrel even though such wines generally start barrel aging as a premium product. High quality red wines often are barrel aged for longer periods, putting them at greater risk for spoilage. B. bruxenellis has been found at a penetration depth of 8 mm within the wood of barrels (Malfeito-Ferreira, 2005). Once B. bruxellensis has infected a barrel, the organism cannot be removed by cleaning, shaving or other techniques, although precautions can be taken to limit its growth (Garde-Cerdán et al., 2008). Malolactic fermentation is another vulnerable period for contamination because it is associated with low levels of free sulfur dioxide in the presence of residual sugar (Oelofse et al., 2008). Brettanomyces is most commonly identified in reds and less frequently isolated in white wines (Dias Pereira-da-Silva, et al., 2003b). The loss of viability in white wines is largely due to the efficacy of sulfur dioxide at low pH (Loureiro & Malfeito-Ferreira, 2006). White wines do not seem to have the Brettanomyces aroma character due to absence of precursor compounds (Chatonnet et al., 1992). 7. Controlling Brettanomyces-induced flavor defects Because B.bruxellensis is the predominant spoilage organism in bottled wine much effort is spent in the wine industry on control measures (Renouf, Perello, de Revel, & Lonvaud-Funell, 2007). Control steps are instituted from the vineyard to bottling. Control measures may center on controlling the presence and growth of B. bruxellensis as well as controlling the formation of flavor precursors. B. bruxellensis is found on damaged grapes and on winery equipment, so effective general sanitation is the first step in prevention. Adequate amounts of sulfur dioxide should be added to fermentation vessels and maintained during fermentation to inhibit growth of the organism. Brettanomyces can enter a viablebut-not-culturable state after sulfur dioxide addition, so the yeast may still be present in the wine after the depletion of free sulfur dioxide (Umiker & Edwards, 2007). Free sulfur dioxide should be maintained above 30 mg/L throughout the wine-making process (Barata et al., 2008), although the effectiveness of this level is reduced in higher pH wines. Control factors to consider while fermenting a wine are using a starter culture, alcohol and acid levels, temperature and oxygen exposure. Using a starter culture can decrease indigenous fermentation, decreasing the opportunity for Brettanomyces to grow. Though fermenting wines to a higher alcohol level may often inhibit development of flavor precursors,
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ethanol tolerance is also a strain-dependent character (Vigentini et al., 2008). Either using grapes high in acid content or adding weak acids during the wine making process may decrease the growth of Brettanomyces, although some research has shown that pH values are not correlated with Brett spoilage (Oelofse, LonvaudFunel, & du Toit, 2009). Sorbic acid can act as an inhibitor in conversion of precursor compounds to 4-EP, but Brettanomyces appears to be one of the more resistant yeast species in wine to sorbic acid as an inhibitor, so winemakers must be aware of appropriate doses (Benito, Palomero, Morata, Calderón, & SuárezLepe, 2009). Fermentation can provide an opportunity for growth of B. bruxellensis. Oxygen has a positive effect on the growth of B. bruxellensis, so micro-oxygenation should be used with caution. Topping off barrels limits surface area and oxygen contact with the wine/must. 4-EP and 4-EG concentrations in red wines were found to positively correlate with dissolved oxygen levels and negatively correlate with cellar humidity (Rayne & Eggers, 2008). Negative correlations between contamination and humidity may be due to the lack of evaporation of wine in barrels, which creates less surface area for oxygen exposure. Reduction in contamination levels and prevention of growth during fermentation are essential because control measures after this stage can only reduce, not eliminate the Brettanomyces influence. For example, ozone treatment of barrels can delay but not eliminate the onset of growth of B. bruxellensis (Oelofse et al., 2008). Using proteins to fine wines before introducing them to barrels can significantly reduce levels of B. bruxellensis (Suárez et al., 2007). Filtering can reduce the yeast and bacteria populations by 1000-fold, but yeast in a viable-but-nonculturable state are small in size and may pass through a 0.45 mm filtration membrane (Millet & Lonvaud-Funel, 2000). Though filtering and fining is often (and controversially) perceived as undesirable for premium quality wines, these processes physically reduce levels of Brettanomyces (Arvik & Henick-Kling, 2002). Treating wine with dimethyl dicarbonate (VelcorinÔ) inactivates B.bruxellensis and other yeasts that may initiate refermentation in the bottle. It is used to preserve wines that have residual sugar or are unfiltered. Ethyphenols can be adsorbed out of wine using absorbent resins (Oelofse et al., 2008). Controlling the occurrence of Brettanomyces-associated flavors is most effectively accomplished by applying simple cellar practices including effective cleaning and hygiene, using appropriate amounts of sulfur dioxide, topping off barrels general oxygen reduction in the must, and most importantly not using contaminated barrels. Some Brettanomyce-strains have been found to be rapid and early flavor producers, while others are slow and late producers, making early detection of the microorganism a useful quality control tool (Agnolucci et al., 2009). The control strategies discussed in this section will not always eliminate Brettanomyces-induced flavors from wine, but they can reduce its effect to tolerable levels. 8. Detection Brettanomyces in wine can be detected either directly or indirectly by analysis of metabolites. Direct determinations of the microorganism utilize culturing and polymerase chain reaction (PCR) techniques. Recently, a fluorescence in situ hybridization method using peptide nucleic acid probes has been developed. Neither morphological features nor other classical methods are adequate to confirm the identity of yeast cultures as Brettanomyces spp. Therefore, classical microbiological culturing methods are often combined with other techniques such as fluorescence microscopy, PCR, gas chromatography mass spectrometry or sensory analysis with trained tasters (Suárez et al., 2007). B. bruxellensis can be present at any stage during wine making, but can remain in a viable-but-not-culturable (VNC) or dormant
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state in wine for long periods of time, meaning cells that cannot be cultured by conventional methods may continue to have the potential to revive and resume growth (Arvik & Henick-Kling, 2002). As a result of cell dormancy and slow growth B. bruxellensis spoilage in dry wines can take place over several months (Romano et al., 2008). The VNC population in a dry wine may be 10 times greater than the culturable population (Millet & LonvaudFunel, 2000). Brettanomyces is also unique in that it can produce characteristic flavor when growing at low cell density of several hundred to several thousand cells per mL, therefore physical detection is very difficult due to slow growth and low cell numbers (Arvik et al., 2002). Volatile metabolites can be present even when the yeast cells are undetectable or minimally detectable. 8.1. Culturing Selective culturing of B. bruxellensis often employs cycloheximide to inhibit bacteria. S. cerevisiae, and competing indigenous yeasts (Couto, Barbosa, & Hogg, 2005). Ethanol is another commonly used selective agent (Renouf et al., 2006; Silva et al., 2004). P-coumaric acid is often added to culture media as a precursor for 4-EP production (Couto et al., 2005). When p-coumaric acid is used in culturing, trained investigators can identify the resulting characteristic odor. Sugar may be added to selective media to decrease production of acetic acid, which can interfere with the olfactory detection of the 4-EP (Couto et al., 2005). Bromocresol green is often added to selective media as a differential agent to provide evidence of acid production (Rodrigues, Gonçalves, Pereira, Malfeito, & Loureiro, 2001). Selective media for detection of B. bruxellensis are often incubated for up to two weeks to account for slow growth (Rodrigues et al., 2001). Although plating methods are of questionable accuracy due to VNC and slow growing organisms, they are still widely used, generally in conjunction with physiological, chemical or sensory analysis. Once isolates of B. bruxellensis are obtained they can be subtyped by using amplified fragment length polymorphism analysis (Henschke et al., 2007). PCR is a reliable, sensitive, and rapid method for detecting various microorganisms in wine, however it does not provide information about the level of contamination (Delaherche, Claisse, & Lonvaud-Funel, 2004). There is concern about PCR inhibitors such as polyphenols and tannins preventing the detection of B. bruxellensis in wine when it is present in low numbers (Loureiro & Malfeito-Ferreira, 2006). Even so, PCR is widely used because of its convenience and reliability. VNC cells contain sufficient intact DNA to produce a positive PCR result. 8.2. Chemical analysis of Brett metabolites Chemical and sensory analysis can only determine if growth of Brettanomyces has already affected the wine, too late for preventative actions. For the past two decades, the presence of B. bruxellensis early in fermentation has been determined by analysis for 4-EP and 4-EG. However, recent literature questions the validity of this approach. In a genetic and physiological study of B. bruxellensis, approximately 80% of strains produced 4-EP and 4-EG, but only 50% produced them at high levels (Conterno et al., 2006). Testing for 4-EP and 4-EG and other metabolites may still provide useful information on the potential for off-flavor development, and will likely continue to be used because of a lack of viable alternative methods. B. bruxellensis yeasts can be identified by gas chromatography (GC) by the analysis of extracted cell membrane fatty. This method, although accurate, can be time consuming. When GC is used for identification, headspace analysis is often preferred due to less
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preparation time. Static headspace through purge and trap or a solid phase microextraction are predominantly used. GC is often combined with mass spectrometry (GCMS) or olfactory methods for volatile identification (Whiton & Zoecklein, 2000). Head space mass spectrometry electronic nose (MS e_nose) also has potential use (Cynkar, Dambergs, Janik, & Gishen, 2006). 8.3. Sensory analysis for B. bruxenellis metabolites For sensory profiling of red V. vinifera wines for Brett character, the SpectrumÔ Method may be the most applicable due to its use of customized descriptors. This method uses lexicon development determined by a panel after tasting a wine with known Bretannomyces-induced off flavor. Flavor attributes are agreed on by the panel. The panel leader can create references to define terms and intensities (Meilgaard, Civille, & Carr, 1999). Potential flavor compounds produced by B. bruxenellis in addition to 4-EP and 4-EG include medium and short chain fatty acids including dedecanoic, isobutryic, isovaleric, and 2-methylbutryic acids, 2-phenolethanol, isoamyl alcohol, cis-2-nonenal, trans-2-nonenal, B- damascenone, and ethyl decanoate (Fugelsang & Zoecklein, 2003). Some studies indicate that volatile phenols, isovaleric acid, acetic acid, carboxylic acids, short chain fatty acids and ethyl-decanoate are also of sensory significance (Romano et al., 2008). Recent studies have shown that isobutryic and isovaleric acid may be more important than previously thought in defining the sensory character of wines effected by B. bruxenellis, and these compounds may produce a masking effect on sensory detection of ethylphenol (Romano, Perello, Lonvaud-Funel, Sicard, & de Revel, 2009). Isobutryic and isovaleric acids are associated with flavors described as sweaty, dirty socks and cheesy. 8.4. Sensory thresholds for 4-EP and 4-EG Chemical analysis of Brettanomyces metabolites provides objective data, however, the implications of finding such metabolites for sensory quality of the wine is often uncertain. The sensory perception thresholds for 4-EP and 4-EG in red wine aree605 mg/L and 100 mg/L, respectively (Chatonnet et al., 1992). When both compounds are present in red wine, the sensory perception threshold of 4-EP is reduced. Sensory perception of these compounds may be affected by other compounds. For example, in a light-bodied red wine with little oak influence, the sensory perception threshold of 4-EP may be e350 mg/L as opposed to a 1000 mg/L threshold in a full-bodied red wine with more oak influence (Coulter et al., 2003). The concept that Brettanomyces can be associated with a consistent flavor profile in wine has been challenged by studies examining the production ethylphenols and other metabolites by different strains. These studies have demonstrated that experts often cannot accurately identify contaminated wines. This could be due to chemical interactions that reduce perception thresholds for 4-EP and 4-EG (Conterno et al., 2006; Romano et al., 2009). 9. Conclusions Growth of Brettanomyces spp. in wine can influence the quality of the product by contributing to flavor complexity when at low levels and causing undesirable flavors at high levels. The flavor characteristic imparted to the wine also differs between strains and the ability of wine components to modify sensory impact of the metabolites. Individuals differ significantly in the ability to detect Brettanomyces-induced flavors in wine and in their acceptance of these flavors. The presence of Brettanomyces spp. in wine is difficult to prevent, as the yeast is found on grapes, in the winery and in
barrels. Controlling its growth in wine involves making the wine a less friendly growth environment by producing it with at least moderate acidity, low sugar content, the application of sulfite, as well as employing good sanitation and fining and filtration before bottling. Acknowledgements The authors gratefully acknowledge the critical review and comments on the finished manuscript by Jessica Just. References Agnolucci, M., Vigentini, I., Capurso, G., Merico, A., Tirelli, A., Compagno, C., et al. (2009). Genetic diversity and physiological traits of Brettanomyces bruxellensis strains isolated from Tuscan Sangiovese wines. International Journal of Food Microbiology, 130(3), 238e244. Arvik, T., Conterno, L., & Henick-Kling, T. (2002). Brettanomyces bruxellensis in New York State wines: A global issue. In Proceedings from the 31st Annual New York Wine Industry Workshop. Geneva, New York. Arvik, T., & Henick-Kling, T. (2002). 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F., & Kunkee, R. E. (1996). Principles and practices of winemaking. New York: Chapman and Hall. Chatonnet, P., Dubourdieu, D., & Boidron, J. (1995). The influence of Brettanomyces/ Dekkera sp. yeasts and lactic acid bacteria on the ethylphenol content of red wines. American Journal for Enology and Viticulture, 46(4), 463e468. Chatonnet, P., Dubourdieu, D., Boidron, J., & Pons, M. (1992). The origin of ethylphenols in wines. Journal of the Science of Food and Agriculture, 60, 165e178. Chatonnet, P., & Pons, M. (1990). Elevage des vins rouges futs de chene: evoluton de certains composes volatiles et de lur impact aromatique. Sciences des Aliments, 10, 565e587. Conterno, L., Joseph, L. C., Arvik, T., Henick-Kling, T., & Bisson, L. (2006). Genetic and physiological characterization of Brettanomyces bruxellensis strains isolated from wines. American Journal for Enology and Viticulture, 57(2), 139e147. Conterno, L., Lasik, G., Tomasino, E., Schneider, K., Hesford, F., & Henick-Kling, T. (2007). Influence of sugar and nitrogen sources on growth and phenolic offflavor production by Brettanomyces bruxellensis isolated from wine. American Journal of Enology and Viticulture, 58(3), 411A. Coulter, A., Robinson, E., Cowey, G., Francis, I., Lattey, K., Capone, D., et al. (2003). Dekkera/Brettanomyces yeast: An overview of recent AWRI investigations and some recommendations for its control. In proceedings of the 2002 ASVO Seminar (pp. 41e50). Tanunda, Australia. Couto, J. A., Barbosa, A., & Hogg, T. (2005). A simple cultural method for the presumptive detection of the yeasts Brettanomyces/Dekkera in wines. Letters in Applied Microbiology, 41(6), 505e510. Couto, J. A., Campos, F. M., Figueiredo, A. R., & Hogg, T. (2006). Ability of lactic acid bacteria to produce volatile phenols. American Journal for Enology and Viticulture, 57(2), 166e171. Curtin, C. D., Bellon, J. R., Henschke, P. A., Godden, P. W., & De la Lopes, M. A. (2007). Genetic diversity of Dekkera bruxellensis yeasts isolated from Australian wineries. FEMS Yeast Research, 7(3), 471e481. Cynkar, W., Dambergs, B., Janik, L., & Gishen, M. (2006). Feasibility study on the use of head space mass spectrometry electronic nose (MS e_nose) to monitor red wine spoilage induced by Brettanomyces yeast. Research done at the Cooperative Research Center for Viticulture. Adelaide: Australia. Delaherche, A., Claisse, O., & Lonvaud-Funel, A. (2004). Detection and quantification of Brettanomyces bruxellensis and ‘ropy’ Pediococcus damnosusstrains in wine by real-time polymerase chain reaction. Journal of Applied Microbiology, 97(5), 910e915. Dias, L., Dias, S., Sancho, T., Stender, H., Querol, A., Malfeito-Ferreira, M., & Loureiro, V. (2003). Identification of yeasts isolated from wine-related environments and capable of producing 4-ethylphenol. Food Microbiology, 20(5), 567. Dias, L., Pereira-da-Silva, S., Tavares, M., Malfeito-Ferreira, M., & Loureiro, V. (2003). Factors affecting the production of 4-ethylphenol by the yeast Dekkera bruxellensis in enological conditions. Food Microbiology, 20(4), 377e384. Fugelsang, K. (1997). Wine microbiology. New York: The Chapman and Hall Enology Library.
D. Wedral et al. / LWT - Food Science and Technology 43 (2010) 1474e1479 Fugelsang, K., & Zoecklein, B. (2003). Population Dynamics and effects of Brettanomyces bruxellensis strains on Pinot noir (Vitis vinifera L.) wines. American Journal for Enology and Viticulture, 54(4), 294e300. Garde-Cerdán, T., Lorenzo, C., Carot, J. M., Jabaloyes, J. M., Esteve, M. D., & Salinas, M. R. (2008). Statistical differentiation of wines of different geographic origin and aged in barrel according to some volatile components and ethylphenols. Food Chemistry, 111(4), 1025e1031. Gerbaux, V., Naudin, R., Meurgues, O., & Monamy, C. (2000). Etude des phénols volatils dans les vins de Pinot noir en Bourgogne. Bulletin de l’O.I.V., 73 (835e836), 581e599. Godoy, L., Martínez, C., Carrasco, N., & Ganga, M. A. (2008). Purification and characterization of a p-coumarate decarboxylase and a vinylphenol reductase from Brettanomyces bruxellensis. International Journal of Food Microbiology, 127(1/2), 6e11. Harris, V., Ford, C., Jiranek, V., & Grbin, P. (2009). Survey of enzyme activity responsible for phenolic off-flavour production by Dekkera and Brettanomyces yeast. Applied Microbiology & Biotechnology, 81(6), 1117e1127. Henschke, P., Curtin, C., & Grbin, P. (2007). Molecular characterization of the wine spoilage yeast e Dekkera (Brettanomyces) bruxellensis. Microbiology Australia, 28, 76e78. Kurtzman, C., & Fell, J. W. (2000). The yeasts, a taxonomic study (4th ed.). Amsterdam: Elsevier Science Publisher BV. Lesschaeve, I. (2007). Sensory evaluation of wine and commercial realities: review of current practices and perspectives. American Journal for Enology and Viticulture, 58(2), 252e258. Licker, L., Acree, T., & Henick-Kling, T. (1999). Impact of Brettanomyces yeast on wine flavor: Sensory description of wines with different ‘Brett’ aroma character. Geneva, New York: New York State Agricultural Experiment Station, Department of Food Science and Technology. Loureiro, V., & Malfeito-Ferreira, M. (2003). Spoilage yeasts in the wine industry. International Journal of Food Microbiology, 86(1e2), 23e50. Loureiro, V., & Malfeito-Ferreira, M. (2006). Food spoilage microorganisms. Boca Raton: Woodhead Publishing. Malfeito-Ferreira, M. (2005). Avances recients en el control de Brettanomyces/Dekkera bruxellensis en vinos. Zaragoza, Spain: Enotour Agrovin. Meilgaard, M., Civille, G. V., & Carr, T. B. (1999). Sensory evaluation techniques (3rd ed.). Boca Raton, FL: CRC Press. Millet, V., & Lonvaud-Funel, A. (2000). The viable but non-culturable state of wine micro-organisms during storage. Letters in Applied Microbiology, 30, 136e141. Oelofse, A., Lonvaud-Funel, A., & du Toit, M. (2009). Molecular identification of Brettanomyces bruxellensis strains isolated from red wines and volatile phenol production. Food Microbiology, 26(4), 377e385. Oelofse, A., Pretorius, I. S., & Du Toit, M. (2008). Significance of Brettanomyces and Dekkera during winemaking: a synoptic review. South African Journal of Enology & Viticulture, 29(2), 128e144.
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Rayne, S., & Eggers, N. (2008). 4-Ethylphenol and 4-ethylguaiacol concentrations in barreled red wines from Okanagan Valley Appellation British Columbia. American Journal for Enology and Viticulture, 51(1), 92e97. Renouf, V., Falcou, M., Miot-Sertier, C., Perello, M. C., De Revel, G., & LonvaudFunel, A. (2006). Interactions between Brettanomyces bruxellensis and other yeast species during the initial stages of winemaking. Journal of Applied Microbiology, 100(6), 1208e1219. Renouf, V., Perello, M. C., de Revel, G., & Lonvaud-Funell, A. (2007). Survival of wine microorganisms in the bottle during storage. American Journal of Enology and Viticulture, 58(3), 379e386. Rodrigues, Gonçalves, Pereira, S., Malfeito, F., & Loureiro, V. (2001). Development and use of a new medium to detect yeasts of the genera Dekkera/Brettanomyces. Journal of Applied Microbiology, 90(4), 588e599. Romano, A., Perello, M., Lonvaud-Funel, A., Sicard, G., & de Revel, G. (2009). Sensory and analytical re-evaluation of “Brett character”. Food Chemistry, 114(1), 15e19. Romano, A., Perello, M., Revel, G., & Lonvaud-Funel, A. (2008). Growth and volatile compound production by Brettanomyces/Dekkera bruxellensis in red wine. Journal of Applied Microbiology, 104(6), 1577e1585. Shinohara, T., Kubodera, S., & Yanagida, F. (2000). Distribution of phenolic yeasts and production of phenolic off-flavors in wine fermentation. Journal of Bioscience and Bioengineering, 90(1), 90e97. Silva, L. R., Andrade, P. B., Valentão, P., Seabra, R. M., Trujillo, M. E., & Velázquez, E. (2005). Analysis of non-coloured phenolics in red wine: effect of Dekkera bruxellensis yeast. Food Chemistry, 89(2), 185e189. Silva, P., Cardoso, H., & Gerós, H. (2004). Studies on the wine spoilage capacity of Brettanomyces/Dekkera spp. American Journal for Enology and Viticulture, 55(1), 65e72. Suárez, R., Suárez-Lepe, J. A., Morata, A., & Calderón, F. (2007). The production of ethylphenols in wine by yeasts of the genera Brettanomyces and Dekkera: a review. Food Chemistry, 102(1), 10e21. Umiker, N. L., & Edwards, C. G. (2007). Impact of sulfur dioxide on culturability and viability of Brettanomyces in wine. American Journal for Enology and Viticulture, 58(3), 417A. Uscanga, M. G., Delia, M. L., & Strehaiano, P. (2003). Brettanomyces bruxellensis effect of oxygen on growth and acetic acid production. Applied Microbiology and Biotechnology, 61(2), 157e162. Van Dijken, P. J., & Scheffers, W. A. (1986). Redox balances in the metabolism of sugars by yeasts. FEMS Microbiol Rev, 32, 199e224. Vigentini, I., Romano, A., Compagno, C., Merico, A., Molinari, F., Tirelli, A., et al. (2008). Physiological and oenological traits of different Dekkera/Brettanomyces bruxellensis strains under wine-model conditions. FEMS Yeast Research, 8(7), 1087e1096. Whiton, R., & Zoecklein, B. (2000). Optimization of headspace solid-phase microextraction for analysis of wine aroma compounds. American Journal for Enology and Viticulture, 51(4), 379e382.
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LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt
The challenge of Brettanomyces in wine Danielle Wedral, Robert Shewfelt, Joseph Frank* Department of Food Science and Technology, University of Georgia, Athens, GA 30602, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 February 2010 Received in revised form 1 June 2010 Accepted 8 June 2010
Brettanomyces spp. is an indigenous yeast that can grow during wine fermentation and impact its flavor. The characteristic flavor is often described as phenolic/medicinal, spices, cloves, earthy, barnyard, and horsy. The acceptability of Brettanoymces-induced flavors depends on the flavor intensity, personal preference, and consumer expectations. The yeast has been isolated from grapes, barrels, and winery equipment. It impacts wines from all the top producing regions in the world, with associated flavors most often found in red wines. Prevention of growth of this yeast in wine involves attention to fruit quality and winery sanitation, control of sulfite and oxygen levels, and the use uncontaminated barrels. Greater understanding of how Brettanomyces and its metabolites contribute to wine flavor is required to recognize its impact on consumer acceptability. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Brettanomyces Wine
1. Introduction Brettanomyces bruxellensis is a yeast found on the surfaces of grapes as well as in barrels, but the greatest concern is its presence in wine. It characteristically produces 4EP and 4EG, two easily measured compounds that are indicative of Brett activity, as well as a variety of other compounds that can contribute to wine aroma complexity. At low concentrations, these and other compounds that the yeast produces can contribute to wine aroma complexity. Higher levels of metabolites impart intense flavors that overwhelm the wine aroma and create an unpleasant experience (Chatonnet & Pons, 1990). Therefore Brett-associated metabolites in wine can be either negative or positive depending on concentration and consumer expectation. 2. Characteristics of Brettanomyces spp. Brettanomyces spp. are the non-spore forming counterparts of the genus Dekkera. Brettanomyces and Dekkera are often used interchangeably and due to Brettanomyces being the more common name found in the literature, it will be used in this review. There are five species belonging to the genus Brettanomyces/Dekkera, including Brettanomyces custersianus, Brettanomyces naardenensis, Brettanomyces nanus, Brettanomyces anomalus, and B. bruxellensis (Kurtzman & Fell, 2000). B. bruxellensis most commonly affects wine, and it has been found on the surfaces of grapes as well as in
* Corresponding author. Tel.: þ1 7065420994. E-mail address: [email protected] (J. Frank). 0023-6438/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2010.06.010
barrels. B. bruxellensis has an oval to an ellipsoidal shape and reproduces by budding. Cell morphology changes from elliptic to branched shape after a few months of incubation (Fig. 1). The physiology of B. bruxellensis has been well studied because of its unusual aromatic bi-products and because it is an example of the Custer effect, which is the inhibition of alcoholic fermentation under anaerobic conditions due to high production of acetic acid and redox imbalance (Henschke, Curtin, & Grbin, 2007; Van Dijken & Scheffers, 1986; Vigentini et al., 2008).
3. Historical aspects Brettanomyces spp. can be found in traditional wine and beer produced using indigenous cultures. Classic English stock beers went through a secondary fermentation in a cask where Brettanomyces was naturally present or was added. This imparted the typical English stock ale character, much of which is believed due to growth of Brettanomyces spp. The name Brettanomyces was first introduced by N Hjelte Claussen of New Carlsberg Brewery to specifically describe the yeast required to produce English stock beers, and became a recognized genus in the 1920s when a similar yeast was isolated from Lambic ales of Belgium (Henschke et al., 2007). In the 1950s and 1960s the yeast was identified as Brettanomyces ssp. and was isolated from wine in France, Italy and South Africa, but it wasn’t until the 1980s and 1990s that the yeast was characterized for its ability to impart characteristic aroma to wine (Henschke et al., 2007). Loureiro and Malfeito-Ferreira (2006, pp. 364e398) reviewed information on the presence of Brettanomyces in various products.
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(Arvik, Conterno, & Henick-Kling, 2002). Research on B. bruxellensis has been conducted in many parts of the world and the organism has been isolated in wines from all major wine producing regions for wine production (Conterno, Joseph, Arvik, Henick-Kling, & Bisson, 2006). A Portuguese study that analyzed wine volatiles from different countries found that more than 25% of red wines had 4-EP levels higher than the preference threshold of 620 mg/L, above which some consumers reject product (Chatonnet, Dubourdieu, Boidron, & Pons, 1992; Lesschaeve, 2007; Loureiro & MalfeitoFerreira, 2003). These results are not based on random sampling, but indicate that volatile phenols related to Brettanomyces are a worldwide issue. Another study performed in Burgundy on Pinot noir wine demonstrated that these yeasts are present in about 50% of the wines undergoing maturation and about 25% of bottles (Gerbaux, Naudin, Meurgues, & Monamy, 2000). Various authors have concluded that controlling growth of Brettanomyces is the most important microbiological challenge of modern winemaking, and is responsible for significant economic losses worldwide (Boulton, Singleton, Bisson, & Kunkee, 1996; Fugelsang, 1997). 5. Chemistry of Brettanomyces-induced flavor
Fig. 1. Photomicrograph of B. bruxellensis. Photo by D. Wedral.
Brett has been isolated from a variety of products and places ranging from wines and winery equipment, beer (in particular lambic beers), cider, apple cider factories, tequila, dairy equipment and kombucha tea. Brett is often found in these products due to its ability to survive for long periods and initiate growth in stored or maturing products. For example, Brettanomyces is commonly isolated from lambic beers after one year of maturation when Saccharomyces spp. are no longer found (Loureiro & MalfeitoFerreira, 2006). At present, Brettanomyces spoilage of wines usually occurs when they are fermented or aged in oak barrels (Rayne & Eggers, 2008). The yeast grows slowly so it usually imparts flavors only when the wine is aged. Since Brettanomyces is present at low numbers early in the fermentation, it is outnumbered by other indigenous yeast and may go undetected. Modern production practices for beer including sanitation practices, high carbon dioxide levels, and low nitrogen contents have made growth of Brettanomyces in this product infrequent. 4. The current issue of Brettanomyces in wine The main factor affecting the sensory properties imparted by B. bruxellensis is the production of 4-ethylphenol (4-EP) and 4-ethylguaiacol (4-EG) (Vigentini et al., 2008). Small differences in 4-EP and 4-EG concentrations can be detected by consumers and quantifed by trained tasters (Licker, Acree, & Henick-Kling, 1999). The presence of Brett character can be considered either negative or positive depending on concentration and expectation of a particular wine. At low concentrations, these compounds can contribute to wine aroma complexity, but high concentrations can overwhelm the wine aroma and create an unpleasant experience (Chatonnet & Pons, 1990). The quality of wines containing Brettanomyces metabolites is controversial in that this judgment depends on individual and cultural preferences. For example in 4-EP sensory analysis, a New York State group of experts described the flavor as plastic, burnt plastic, phenolic/medicinal, cow manure, barnyard and horse sweat, whereas an international group only included the more neutral terms burnt plastic and phenolic/medicinal in their descriptors. In general, growth of Brettanomyces in wine is considered undesirable in the United States, some consider this a global issue
The initial detection of Brettanomyces-induced aromas in wine generally occurs during the barrel maturation (Godoy, Martínez, Carrasco, & Ganga, 2008). These aromas result primarily from the enzymatic conversion of ferulic and p-coumaric acid into 4-vinylguaiacol and 4-vinylphenol by hydroxycinnamate decarboxylase. These compounds are then reduced to 4-EG and 4-EP by vinylphenol reductase (Suárez, Suárez-Lepe, Morata, & Calderón, 2007). Other yeasts can reduce p-coumaric acid to form 4-vinylphenol, but B. bruxellensis appears unique in its ability to reduce vinylphenols to sensory detectable amounts of 4-EP and 4-EG (Chatonnet, Dubourdieu, & Boidron, 1995). The aroma of 4-EP is often described as phenolic/medicinal and that of 4-EG is often described as clove/holiday spice. Cinnamate decarboxylase activity of B. bruxellensis, unlike that in S. cerevisiae, is not inhibited by phenolic compounds in grapes, so it can produce several milligrams of ethylphenols per liter of wine, the amount produced being directly proportional to its population (Chatonnet et al., 1995). A high polyphenol content of the wine will drive the enzymatic reaction to produce more end products. A low correlation between amount of 4-EP and number of yeast colony forming units present in the wine is often observed, suggesting that the metabolite (or enzyme producing it) is released upon yeast cell death and autolysis (Fugelsang & Zoecklein, 2003). Another Brettanomyces -produced flavor compound is 4-ethylcatechol (4-EC), which is noted for its medicinal aroma. Caffeic acid is the precursor compound for this metabolite. Due to the requirement to be derivatize to be detected by gas chromatography 4-EC has not been studied to the extent of the other volatile phenols, but it does have a lower detection threshold than other ethyl phenols, so may be of importance (Loureiro & Malfeito-Ferreira, 2006). In addition to flavor changes, wines contaminated with Brettanomyces may have an undesirable color. This may result from glycosidic activities or formation of vinylphenolic pyranoanthocyanins (Oelofse, Pretorius, & Du Toit, 2008). 5.1. Chemistry Brettanomyces-induced flavor in wine The average concentrations where 4-EP and 4-EG may influence wine flavor are 0.62 mg/l and 0.14 mg/l with recognition thresholds of 770 and 436 mg/l, respectively (Chatonnet et al., 1992). 4-EP and 4-EG appear in wines at various amounts and ratios depending on the variety of grape used and wine style. On average they appear at a 10:1 ratio respectively, which corresponds to the respective
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precursor ratio of p-coumaric acid and ferulic acid (Chatonnet et al., 1992; Romano, Perello, Revel, & Lonvaud-Funel, 2008). Another study showed average ratios of 4-EP to 4-EG differed with different wine types and was approximately 10:1 for Cabernet Sauvignon, 9:1 for Shiraz, 8:1 for Merlot and 3.5:1 for Pinot Noir. Little work has been done on determining thresholds of these compounds in different varieties, but some indicate thresholds to be higher in Cabernet Sauvignon and lower in Tempranillo wines (Suárez et al., 2007). Studies have also shown no difference in ethylphenol concentrations produced in Cabernet Franc, Cabernet Sauvignon, Merlot, Pinot Noir, and Syrah wines (Rayne & Eggers, 2008). Recent studies have demonstrated that B. bruxellensis is not the only organism that can produce 4-EP and 4-EG, and that not all strains of B. bruxellensis produce these compounds. One study found that 20% of Brettanomyces strains did not produce these compounds (Conterno et al., 2006). Winemaking strains of S. cerevisiae differ in their ability to decarboxylate hydroxycinnamic acids to vinylphenols, therefore the idea that Brettanomyces/Dekkera yeasts differ in this characteristic as well seems plausible (Curtin, Bellon, Henschke, Godden, & De la Lopes, 2007; Shinohara, Kubodera, & Yanagida, 2000). Some microorganisms including Lactobacillus brevis and Pediococcus pentosaceus can decarboxylate p-coumaric acid to form 4-vinylphenol, but few, including Brettanomyces and Lactobacillus plantarum, are capable of producing ethylphenols (Chatonnet et al., 1995). Thirty seven percent of lactic acid bacteria tested could produce volatile phenols from p-coumaric acid and 9% could produce 4-ethylphenol (Couto, Campos, Figueiredo, & Hogg, 2006). What differentiates Brettanomyces from other microorganisms is the relative high production of ethylphenols. For example, Pichia gulliermondii is capable of converting p-coumaric acid to 4-ethylphenol in grape juice, grapes and winery equipment, but has shown low to no conversion rates in wine (Dias, Dias, et al., 2003), making Brettanomyces/Dekkera yeasts the sole known cause for ethylphenol-associated off flavors (Barata, Nobre, Correia, Malfeito-Ferreira, & Loureiro, 2006; Vigentini et al., 2008). The formation of Brett character is influenced by the microbial strain, wine pH, nutrient availability in the must and stage of winemaking at which contamination occurs (Romano et al., 2008). Nitrogen availability is more important in limiting flavor production than is the presence of residual sugars (Conterno et al., 2007). P-coumaric acid appears to induce off-flavor production more than does ferulic acid, but strain variation is significant in this regard (Harris, Ford, Jiranek, & Grbin, 2009).
5.2. Genetic diversity and flavor character Genetic-associated strain variation is behind much of the variability in sensory descriptions of Brettanomyces-induced flavors and variations in its growth ability (Vigentini et al., 2008). For example, B. bruxellensis strains exhibit significant variation in their ability to grow in Pinot Noir wines (Fugelsang & Zoecklein, 2003), and individual strains differ in their impact on phenolic profiles of the wine (Silva et al., 2005). Similar genetic subtypes of B. bruxellensis have been found worldwide, an indication that the yeast has spread geographically, perhaps due to international trade in grape juice, barrels, and winery equipment. It is also possible that similar genetic adaptations have arisen independently due to similar stresses in the wine making environment (Curtin et al., 2007). Because of inherent strain variation, indicators of the capacity of strains to induce off-flavors are important. The ability to produce off-flavors in wine is related to a strain’s production of volatile phenolic compounds. Therefore, measurement of ethyl phenol production can be used as a simple indicator of strain spoilage capacity (Vigentini et al., 2008).
6. Growth of Brettanomyces during winemaking B. bruxellensis can be present at any stage in wine making, but due to its high ethanol and low sugar tolerances it is most often associated with aging in oak barrels, stuck or slow alcoholic fermentations and malolactic fermentation (Dias, Pereira-da-Silva, Tavares, Malfeito-Ferreira, & Loureiro, 2003; Renouf et al., 2006). Brett can tolerate 14e14.5% ethanol in red wine (Loureiro & Malfeito-Ferreira, 2006), and may grow in high alcohol wines and during stuck fermentations (Silva, Cardoso, & Gerós, 2004). Brettanomyces can also slow the growth of Saccharomyces by producing acetic acid (Uscanga, Delia, & Strehaiano, 2003). Red wines are particularly susceptible to Brettanomyces bruxenellis infection due to their lower acidity, higher polyphenol content and barrel aging. Therefore Vitis vinifera red varietals with higher precursor polyphenol content are the most susceptible to the Brettanomyces defect. For example, Pinot Noir generally has low levels of p-coumaric acid providing B. bruxenellis less substrate for 4-EP production. B. bruxellensis’s slow growth makes wines that are stored for long periods in wooden barrels particularly susceptible to its influence (Suárez et al., 2007). Wines that are aged longer in barrels are more susceptible to the defect if B. bruxenellis is in the barrel even though such wines generally start barrel aging as a premium product. High quality red wines often are barrel aged for longer periods, putting them at greater risk for spoilage. B. bruxenellis has been found at a penetration depth of 8 mm within the wood of barrels (Malfeito-Ferreira, 2005). Once B. bruxellensis has infected a barrel, the organism cannot be removed by cleaning, shaving or other techniques, although precautions can be taken to limit its growth (Garde-Cerdán et al., 2008). Malolactic fermentation is another vulnerable period for contamination because it is associated with low levels of free sulfur dioxide in the presence of residual sugar (Oelofse et al., 2008). Brettanomyces is most commonly identified in reds and less frequently isolated in white wines (Dias Pereira-da-Silva, et al., 2003b). The loss of viability in white wines is largely due to the efficacy of sulfur dioxide at low pH (Loureiro & Malfeito-Ferreira, 2006). White wines do not seem to have the Brettanomyces aroma character due to absence of precursor compounds (Chatonnet et al., 1992). 7. Controlling Brettanomyces-induced flavor defects Because B.bruxellensis is the predominant spoilage organism in bottled wine much effort is spent in the wine industry on control measures (Renouf, Perello, de Revel, & Lonvaud-Funell, 2007). Control steps are instituted from the vineyard to bottling. Control measures may center on controlling the presence and growth of B. bruxellensis as well as controlling the formation of flavor precursors. B. bruxellensis is found on damaged grapes and on winery equipment, so effective general sanitation is the first step in prevention. Adequate amounts of sulfur dioxide should be added to fermentation vessels and maintained during fermentation to inhibit growth of the organism. Brettanomyces can enter a viablebut-not-culturable state after sulfur dioxide addition, so the yeast may still be present in the wine after the depletion of free sulfur dioxide (Umiker & Edwards, 2007). Free sulfur dioxide should be maintained above 30 mg/L throughout the wine-making process (Barata et al., 2008), although the effectiveness of this level is reduced in higher pH wines. Control factors to consider while fermenting a wine are using a starter culture, alcohol and acid levels, temperature and oxygen exposure. Using a starter culture can decrease indigenous fermentation, decreasing the opportunity for Brettanomyces to grow. Though fermenting wines to a higher alcohol level may often inhibit development of flavor precursors,
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ethanol tolerance is also a strain-dependent character (Vigentini et al., 2008). Either using grapes high in acid content or adding weak acids during the wine making process may decrease the growth of Brettanomyces, although some research has shown that pH values are not correlated with Brett spoilage (Oelofse, LonvaudFunel, & du Toit, 2009). Sorbic acid can act as an inhibitor in conversion of precursor compounds to 4-EP, but Brettanomyces appears to be one of the more resistant yeast species in wine to sorbic acid as an inhibitor, so winemakers must be aware of appropriate doses (Benito, Palomero, Morata, Calderón, & SuárezLepe, 2009). Fermentation can provide an opportunity for growth of B. bruxellensis. Oxygen has a positive effect on the growth of B. bruxellensis, so micro-oxygenation should be used with caution. Topping off barrels limits surface area and oxygen contact with the wine/must. 4-EP and 4-EG concentrations in red wines were found to positively correlate with dissolved oxygen levels and negatively correlate with cellar humidity (Rayne & Eggers, 2008). Negative correlations between contamination and humidity may be due to the lack of evaporation of wine in barrels, which creates less surface area for oxygen exposure. Reduction in contamination levels and prevention of growth during fermentation are essential because control measures after this stage can only reduce, not eliminate the Brettanomyces influence. For example, ozone treatment of barrels can delay but not eliminate the onset of growth of B. bruxellensis (Oelofse et al., 2008). Using proteins to fine wines before introducing them to barrels can significantly reduce levels of B. bruxellensis (Suárez et al., 2007). Filtering can reduce the yeast and bacteria populations by 1000-fold, but yeast in a viable-but-nonculturable state are small in size and may pass through a 0.45 mm filtration membrane (Millet & Lonvaud-Funel, 2000). Though filtering and fining is often (and controversially) perceived as undesirable for premium quality wines, these processes physically reduce levels of Brettanomyces (Arvik & Henick-Kling, 2002). Treating wine with dimethyl dicarbonate (VelcorinÔ) inactivates B.bruxellensis and other yeasts that may initiate refermentation in the bottle. It is used to preserve wines that have residual sugar or are unfiltered. Ethyphenols can be adsorbed out of wine using absorbent resins (Oelofse et al., 2008). Controlling the occurrence of Brettanomyces-associated flavors is most effectively accomplished by applying simple cellar practices including effective cleaning and hygiene, using appropriate amounts of sulfur dioxide, topping off barrels general oxygen reduction in the must, and most importantly not using contaminated barrels. Some Brettanomyce-strains have been found to be rapid and early flavor producers, while others are slow and late producers, making early detection of the microorganism a useful quality control tool (Agnolucci et al., 2009). The control strategies discussed in this section will not always eliminate Brettanomyces-induced flavors from wine, but they can reduce its effect to tolerable levels. 8. Detection Brettanomyces in wine can be detected either directly or indirectly by analysis of metabolites. Direct determinations of the microorganism utilize culturing and polymerase chain reaction (PCR) techniques. Recently, a fluorescence in situ hybridization method using peptide nucleic acid probes has been developed. Neither morphological features nor other classical methods are adequate to confirm the identity of yeast cultures as Brettanomyces spp. Therefore, classical microbiological culturing methods are often combined with other techniques such as fluorescence microscopy, PCR, gas chromatography mass spectrometry or sensory analysis with trained tasters (Suárez et al., 2007). B. bruxellensis can be present at any stage during wine making, but can remain in a viable-but-not-culturable (VNC) or dormant
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state in wine for long periods of time, meaning cells that cannot be cultured by conventional methods may continue to have the potential to revive and resume growth (Arvik & Henick-Kling, 2002). As a result of cell dormancy and slow growth B. bruxellensis spoilage in dry wines can take place over several months (Romano et al., 2008). The VNC population in a dry wine may be 10 times greater than the culturable population (Millet & LonvaudFunel, 2000). Brettanomyces is also unique in that it can produce characteristic flavor when growing at low cell density of several hundred to several thousand cells per mL, therefore physical detection is very difficult due to slow growth and low cell numbers (Arvik et al., 2002). Volatile metabolites can be present even when the yeast cells are undetectable or minimally detectable. 8.1. Culturing Selective culturing of B. bruxellensis often employs cycloheximide to inhibit bacteria. S. cerevisiae, and competing indigenous yeasts (Couto, Barbosa, & Hogg, 2005). Ethanol is another commonly used selective agent (Renouf et al., 2006; Silva et al., 2004). P-coumaric acid is often added to culture media as a precursor for 4-EP production (Couto et al., 2005). When p-coumaric acid is used in culturing, trained investigators can identify the resulting characteristic odor. Sugar may be added to selective media to decrease production of acetic acid, which can interfere with the olfactory detection of the 4-EP (Couto et al., 2005). Bromocresol green is often added to selective media as a differential agent to provide evidence of acid production (Rodrigues, Gonçalves, Pereira, Malfeito, & Loureiro, 2001). Selective media for detection of B. bruxellensis are often incubated for up to two weeks to account for slow growth (Rodrigues et al., 2001). Although plating methods are of questionable accuracy due to VNC and slow growing organisms, they are still widely used, generally in conjunction with physiological, chemical or sensory analysis. Once isolates of B. bruxellensis are obtained they can be subtyped by using amplified fragment length polymorphism analysis (Henschke et al., 2007). PCR is a reliable, sensitive, and rapid method for detecting various microorganisms in wine, however it does not provide information about the level of contamination (Delaherche, Claisse, & Lonvaud-Funel, 2004). There is concern about PCR inhibitors such as polyphenols and tannins preventing the detection of B. bruxellensis in wine when it is present in low numbers (Loureiro & Malfeito-Ferreira, 2006). Even so, PCR is widely used because of its convenience and reliability. VNC cells contain sufficient intact DNA to produce a positive PCR result. 8.2. Chemical analysis of Brett metabolites Chemical and sensory analysis can only determine if growth of Brettanomyces has already affected the wine, too late for preventative actions. For the past two decades, the presence of B. bruxellensis early in fermentation has been determined by analysis for 4-EP and 4-EG. However, recent literature questions the validity of this approach. In a genetic and physiological study of B. bruxellensis, approximately 80% of strains produced 4-EP and 4-EG, but only 50% produced them at high levels (Conterno et al., 2006). Testing for 4-EP and 4-EG and other metabolites may still provide useful information on the potential for off-flavor development, and will likely continue to be used because of a lack of viable alternative methods. B. bruxellensis yeasts can be identified by gas chromatography (GC) by the analysis of extracted cell membrane fatty. This method, although accurate, can be time consuming. When GC is used for identification, headspace analysis is often preferred due to less
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preparation time. Static headspace through purge and trap or a solid phase microextraction are predominantly used. GC is often combined with mass spectrometry (GCMS) or olfactory methods for volatile identification (Whiton & Zoecklein, 2000). Head space mass spectrometry electronic nose (MS e_nose) also has potential use (Cynkar, Dambergs, Janik, & Gishen, 2006). 8.3. Sensory analysis for B. bruxenellis metabolites For sensory profiling of red V. vinifera wines for Brett character, the SpectrumÔ Method may be the most applicable due to its use of customized descriptors. This method uses lexicon development determined by a panel after tasting a wine with known Bretannomyces-induced off flavor. Flavor attributes are agreed on by the panel. The panel leader can create references to define terms and intensities (Meilgaard, Civille, & Carr, 1999). Potential flavor compounds produced by B. bruxenellis in addition to 4-EP and 4-EG include medium and short chain fatty acids including dedecanoic, isobutryic, isovaleric, and 2-methylbutryic acids, 2-phenolethanol, isoamyl alcohol, cis-2-nonenal, trans-2-nonenal, B- damascenone, and ethyl decanoate (Fugelsang & Zoecklein, 2003). Some studies indicate that volatile phenols, isovaleric acid, acetic acid, carboxylic acids, short chain fatty acids and ethyl-decanoate are also of sensory significance (Romano et al., 2008). Recent studies have shown that isobutryic and isovaleric acid may be more important than previously thought in defining the sensory character of wines effected by B. bruxenellis, and these compounds may produce a masking effect on sensory detection of ethylphenol (Romano, Perello, Lonvaud-Funel, Sicard, & de Revel, 2009). Isobutryic and isovaleric acids are associated with flavors described as sweaty, dirty socks and cheesy. 8.4. Sensory thresholds for 4-EP and 4-EG Chemical analysis of Brettanomyces metabolites provides objective data, however, the implications of finding such metabolites for sensory quality of the wine is often uncertain. The sensory perception thresholds for 4-EP and 4-EG in red wine aree605 mg/L and 100 mg/L, respectively (Chatonnet et al., 1992). When both compounds are present in red wine, the sensory perception threshold of 4-EP is reduced. Sensory perception of these compounds may be affected by other compounds. For example, in a light-bodied red wine with little oak influence, the sensory perception threshold of 4-EP may be e350 mg/L as opposed to a 1000 mg/L threshold in a full-bodied red wine with more oak influence (Coulter et al., 2003). The concept that Brettanomyces can be associated with a consistent flavor profile in wine has been challenged by studies examining the production ethylphenols and other metabolites by different strains. These studies have demonstrated that experts often cannot accurately identify contaminated wines. This could be due to chemical interactions that reduce perception thresholds for 4-EP and 4-EG (Conterno et al., 2006; Romano et al., 2009). 9. Conclusions Growth of Brettanomyces spp. in wine can influence the quality of the product by contributing to flavor complexity when at low levels and causing undesirable flavors at high levels. The flavor characteristic imparted to the wine also differs between strains and the ability of wine components to modify sensory impact of the metabolites. Individuals differ significantly in the ability to detect Brettanomyces-induced flavors in wine and in their acceptance of these flavors. The presence of Brettanomyces spp. in wine is difficult to prevent, as the yeast is found on grapes, in the winery and in
barrels. Controlling its growth in wine involves making the wine a less friendly growth environment by producing it with at least moderate acidity, low sugar content, the application of sulfite, as well as employing good sanitation and fining and filtration before bottling. Acknowledgements The authors gratefully acknowledge the critical review and comments on the finished manuscript by Jessica Just. References Agnolucci, M., Vigentini, I., Capurso, G., Merico, A., Tirelli, A., Compagno, C., et al. (2009). Genetic diversity and physiological traits of Brettanomyces bruxellensis strains isolated from Tuscan Sangiovese wines. International Journal of Food Microbiology, 130(3), 238e244. Arvik, T., Conterno, L., & Henick-Kling, T. (2002). Brettanomyces bruxellensis in New York State wines: A global issue. In Proceedings from the 31st Annual New York Wine Industry Workshop. Geneva, New York. Arvik, T., & Henick-Kling, T. (2002). 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F., & Kunkee, R. E. (1996). Principles and practices of winemaking. New York: Chapman and Hall. Chatonnet, P., Dubourdieu, D., & Boidron, J. (1995). The influence of Brettanomyces/ Dekkera sp. yeasts and lactic acid bacteria on the ethylphenol content of red wines. American Journal for Enology and Viticulture, 46(4), 463e468. Chatonnet, P., Dubourdieu, D., Boidron, J., & Pons, M. (1992). The origin of ethylphenols in wines. Journal of the Science of Food and Agriculture, 60, 165e178. Chatonnet, P., & Pons, M. (1990). Elevage des vins rouges futs de chene: evoluton de certains composes volatiles et de lur impact aromatique. Sciences des Aliments, 10, 565e587. Conterno, L., Joseph, L. C., Arvik, T., Henick-Kling, T., & Bisson, L. (2006). Genetic and physiological characterization of Brettanomyces bruxellensis strains isolated from wines. American Journal for Enology and Viticulture, 57(2), 139e147. Conterno, L., Lasik, G., Tomasino, E., Schneider, K., Hesford, F., & Henick-Kling, T. 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