Molecular Detection and Identification ofBrettanomyces/Dekkera bruxellensisandBrettanomyces/Dekkera anomalusin Spoiled Wines

Abstract

In this paper we describe the development of a PCR protocol to specifically detect Brettanomyces bruxellensis and B. anomalus. Primers DB90F and DB394R, targeting the D1-D2 loop of the 26S rRNA gene, were able to produce amplicons only when the DNA from these two species were used. No amplification product was obtained when DNA from other Brettanomyces spp. or wine yeasts were used as the templates. The 305-bp product was subjected to restriction enzyme analysis with DdeI to differentiate between B. bruxellensis and B. anomalus, and each species could be identified on the basis of the different restriction profiles. After optimization of the method by using strains from international collections, wine isolates were tested with the method proposed. Total agreement between traditional identification and molecular identification was observed. The protocol developed was also used for direct detection of B. bruxellensis and B. anomalus in wines suspected to be spoiled by Brettanomyces spp. Application of culture-based and molecular methods led us to the conclusion that 8 of 12 samples were spoiled by B. bruxellensis. Results based on the application of molecular methods suggested that two of the eight positive samples had been infected more recently, since specific signals were obtained at both the DNA and RNA levels.A wide variety of yeast species have been implicated in wine spoilage. Of the spoilage yeasts, species of Brettanomyces (imperfect state, Dekkera) are probably the most serious (21) and controversial. These organisms are most frequently found in red wine 6 to 10 months after barrelling (4), but they have also been isolated from other fermented beverages, such as beer and cider. Brettanomyces species can survive, multiply, and contaminate wines from transfer piping or cooperage that has been insufficiently cleaned and disinfected after use. These potential spoilage yeasts have been identified in almost every wine-producing area of the world (12). Wines infected with Brettanomyces/Dekkera yeasts develop off-flavors, which are described as animal, stable, barnyard, horse blanket, and burnt plastic (5,12,13,16,18). Brettanomyces can produce a distinct haziness when the concentration of cells present in the wine is Ͻ3 ϫ 10 3 cells/ml (4) or higher (13). Brettanomyces species can synthesize volatile phenolic compounds, including phenol, syringol (16), and several ethylphenols (6). Control of these organisms is difficult due to their relative resistance to normally used concentrations of sulfur dioxide (15). The physiology and ecology of this spoilage genus are still unclear, and little is known about it (14).Current methods for identification and enumeration of Brettanomyces contamination take 1 to 2 weeks and rely on growth on semiselective culture media or selective culture media, followed by final identification by biochemical and physiological analysis and morphology as determined by microscopic examination (19). Newer techniques for rapid detection and identification of Brettanomyces, such as a nested PCR...

Significance of Brettanomyces and Dekkera during Winemaking: A Synoptic Review

“…The genus Dekkera was introduced to the taxonomy in 1964 after the production of ascospores (sporulating-form) was observed (Van der Walt, 1984). Currently, the five species jointly belonging to the genera Brettanomyces and Dekkera are: Brettanomyces custersianus, Brettanomyces naardenensis, Brettanomyces nanus, B. anomalus and B. bruxellensis (Kurtzman & Fell, 2000;Cocolin et al, 2004). Teleomorphs (perfect state) are known for the last two species, Dekkera anomala and Dekkera bruxellensis, respectively (Kurtzman & Fell, 2000).…”

“…The details regarding the morphological, biochemical and physiological characteristics of these species are well described in recent classification manuals (Barnett et al, 2000;Boekhout et al, 2002;Kurtzman & Fell, 2000). From the five species currently known, the species primarily associated with winemaking is B. bruxellensis (D. bruxellensis) (Egli & HenickKling, 2001;Stender et al, 2001;Cocolin et al, 2004), although B. anomalus (D. anomala) and B. custersianus isolations from must fermentations have been reported in two instances (Querol et al, 1990;Esteve-Zarzoso et al, 2001). With advances in DNAbased methods, recent wine-related investigations often include D. anomala along with the predominant species D. bruxellensis as conventional methods had showed difficulty in differentiating between these two species .…”

Assessing Genetic Diversity among Brettanomyces Yeasts by DNA Fingerprinting and Whole-Genome Sequencing

“…Apart from isolations from beer and wine, Brettanomyces species have been detected and isolated in other foods, such as cider, soft drinks, dairy products, and olives (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). Despite its economic importance either as a spoilage contaminant in wine and nonalcoholic beverages or as a vital component of the fermentation biota in the production of certain beers, the physiology and ecology of Brettanomyces yeasts have only recently been the subject of intensive research (1,(21)(22)(23)(24)(25). However, little is still known about the level of genomic interstrain variation within B. bruxellensis or within the genus Brettanomyces.…”

“…So far, most genetic studies on Brettanomyces have focused on rapid fingerprinting using rRNA sequencing (26,27), PCRrestriction fragment length polymorphism (RFLP) analysis (28), random amplified polymorphic DNA (RAPD) analysis (29), amplified fragment length polymorphism (AFLP) analysis, arbitrarily primed PCR (AP-PCR), and microsatellite fingerprinting (25,30). However, so far these studies have mostly been performed with strains belonging to the same species, most often B. bruxellensis (17,20,29).…”

Volatile phenols in wine: Control measures of Brettanomyces/Dekkera yeasts

“…Brettanomyces/Dekkera yeasts exist in two forms: Brettanomyces, the asexual, non-sporulating form, and Dekkera, the sexual, sporulating form. These genera are particularly known as spoiling agents in beer, wine, cider and soft drinks industries (Deak and Beuchat, 1996 (Cocolin et al 2004;Oelofse et al, 2008). Different strains of Brettanomyces can show great differences in their production of volatile phenols (Joseph and Bisson, 2004).…”