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Specific identification and quantification of the spoilage microorganism Brettanomyces in wine by flow cytometry: A useful tool for winemakers

THE yeast Brettanomyces is the major microbial cause for wine spoilage worldwide and causes significant economic loss. This yeast produces from grape acid phenols, 4-ethylphenol and 4-ethylguaiacol, which confer characteristic aroma described as mousy, smoky, burnt plastic, barnyard, horse sweat, leather, and wet wool. Moreover, these yeasts are high acetic acid producers and are capable of producing a mousy taint caused by 2-acetyl-1,4,5,6-tetrahydropyridine and 2-acetyl-3,4,5,6-tetrahydropyridine. Thus, this peculiar yeast metabolism is deleterious to wine quality. The monitoring of Brettanomyces remains a problem for most wine cellars. Recently, flow cytometry (FCM) has been proposed to quantify Brettanomyces in wine (1). However, cells are dyed with fluorescein diacetate, which stained all viable wine microorganisms and thus is not specific to Brettanomyces. By contrast, methods with some specificity, which are available to identify Brettanomyces, present other disadvantages. Most of them take 1 or 2 weeks and spoilage may occur before it is noticed by the winemaker. Detection of Brettanomyces by FCM using antibodies coupled to fluorochrome could be another strategy as it has been used successfully for other microorganisms (2). However, Brettanomyces antibodies are not available. Among the many molecular methods for analyzing microorganisms, fluorescence in situ hybridization (FISH) is a widely used method for monitoring microorganisms. Indeed, the FISH method presents advantages comparing with other molecular methods. It can target DNA or RNA regions and combines a counting technique with an identification technique and FISH analyses with rRNA targeted probes are applied for the identification and quantification of microorganisms. FISH coupled with microscopy detection has been the most commonly applied technique for analysis of specific microorganisms (3). However, visual counting is difficult and time consuming. The accuracy of this quantification approach is relatively low (4) taking into account the bias associated with interobserver variability. In recent years, the combination of FISH with FCM detection (FISH-FCM) appeared to be a very high throughput method for identification and quantification of specific microorganisms (5–7). In this study, we propose a FCM-FISH method adapted to detect and enumerate Brettanomyces cells grown in red wine using specific probes modified with the fluorescent dye Alexa Fluor 488 (Molecular Probes, Eugene, OR, USA) at the 50end. Probes 26S-D.brux.1.1 (TACCGGAACGACCGCAGT), 26S-D.brux.1.2 (TGTCACAGGCCTCTACATTG), and 26SD.brux.5.1 (CTTACTCAAATCCCTCCGGT), previously designedAdvancement of Cytometry (8) and targeting the 26S rRNA gene, were used. For experiments conducted in contaminated red wine, 1.10 cells from a 72 h YPD culture were added in an adaptation medium composed of 50% YPD and 50% red wine (Burgundy Pinot noir). After 5 days at 28 C, red wine was inoculated at 10 cells/mL and incubated at 28 C for 2 days. An aliquot of 10 cells from red wine was centrifuged (9000 g, 10 min, 25 C) and rinsed twice in PBS (130 mM NaCl, 5 mM NaH2PO4, and 5 mM Na2HPO4; pH 7.4). Permeabilization treatments were conducted to increase the percentage of fluorescent Brettanomyces cells. Indeed, first