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

Abstract

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

Surviving in the presence of sulphur dioxide: strategies developed by wine yeasts

“…In these conditions, entry and exit from the VBNC state can be experimentally appreciated by visualising the difference in cell counts obtained using a culture-independent technique (e.g. epifluorescence (Millet and Lonvaud-Funel 2000;Divol and Lonvaud-Funel 2005;du Toit et al 2005) or flow cytometry (Serpaggi et al 2010a)) and plating (Fig. 4).…”

Detection and identification of microorganisms in wine: a review of molecular techniques

“…The primary use of flow cytometry in the wine industry, however, has been to detect spoilage organisms [66,103]. Most often, methods use the 16S or 26S rDNA genes as a target for FISH analysis, as discussed above.…”

“…However, the lack of commercial antibodies for many of the yeasts and bacteria present in wine means that probes need to be designed for detection of most spoilage microbes. Serpaggi et al [103] were able to detect Dekkera grown in Pinot noir at a concentration of 10 2 cfu ml -1 using FISH-based flow cytometry. However, phenolic compounds interfered with initial methods for permeabilizing the cells and the method developed took over 24 h to complete.…”

Isolation, identification and control of osmophilic spoilage yeasts in sweetened condensed milk

“…The spoilage caused by yeasts may alter both the physical and sensorial properties of foods as a result of their activity (Loureiro and Querol, 1999), which has caused major economic loss in many sectors of food and beverage industries, especially in baking and beverages industry. Current losses to the food industry caused by yeast spoilage are estimated at tens of millions pounds annually in the world (Querol, 2006;Gerez et al, 2010;Serpaggi et al, 2010).…”