Application of flow cytometry to wine microorganisms.

Flow cytometry (FCM) is a powerful technique allowing detection and enumeration of microbial populations in food and during food process. Thanks to the fluorescent dyes used and specific probes, FCM provides information about cell physiological state and allows enumeration of a microorganism in a mixed culture. Thus, this technique is increasingly used to quantify pathogen, spoilage microorganisms and microorganisms of interest. Since one decade, FCM applications to the wine field increase greatly to determine population and physiological state of microorganisms performing alcoholic and malolactic fermentations. Wine spoilage microorganisms were also studied. In this review we briefly describe FCM principles. Next, a deep revision concerning enumeration of wine microorganisms by FCM is presented including the fluorescent dyes used and techniques allowing a yeast and bacteria species specific enumeration. Then, the last chapter is dedicated to fluorescent dyes which are used to date in fluorescent microscopy but applicable in FCM. This chapter also describes other interesting "future" techniques which could be applied to study the wine microorganisms. Thus, this review seeks to highlight the main advantages of the flow cytometry applied to wine microbiology.

Design and Performance Testing of a DNA Extraction Assay for Sensitive and Reliable Quantification of Acetic Acid Bacteria Directly in Red Wine Using Real Time PCR.

Although strategies exist to prevent AAB contamination, the increased interest for wines with low sulfite addition leads to greater AAB spoilage. Hence, there is a real need for a rapid, specific, sensitive, and reliable method for detecting these spoilage bacteria. All these requirements are met by real time Polymerase Chain Reaction (or quantitative PCR; qPCR). Here, we compare existing methods of isolating DNA and their adaptation to a red wine matrix. Two different protocols for isolating DNA and three PCR mix compositions were tested to select the best method. The addition of insoluble polyvinylpolypyrrolidone (PVPP) at 1% (v/v) during DNA extraction using a protocol succeeded in eliminating PCR inhibitors from red wine. We developed a bacterial internal control which was efficient in avoiding false negative results due to decreases in the efficiency of DNA isolation and/or amplification. The specificity, linearity, repeatability, and reproducibility of the method were evaluated. A standard curve was established for the enumeration of AAB inoculated into red wines. The limit of quantification in red wine was 3.7 log AAB/mL and about 2.8 log AAB/mL when the volume of the samples was increased from 1 to 10 mL. Thus, the DNA extraction method developed in this paper allows sensitive and reliable AAB quantification without underestimation thanks to the presence of an internal control. Moreover, monitoring of both the AAB population and the amount of acetic acid in ethanol medium and red wine highlighted that a minimum about 6.0 log cells/mL of AAB is needed to significantly increase the production of acetic acid leading to spoilage.

Efficiency of population-dependent sulfite against Brettanomyces bruxellensis in red wine.

Brettanomyces bruxellensis is considered as a spoilage yeast encountered mainly in red wine. It is able to reduce vinylphenols from phenolic acids to ethylphenols. These volatiles are responsible for the phenolic "Brett character" described as animal, farm, horse sweat and animal leather odors. Other molecules are responsible for organoleptic deviations described as "mousiness taint". SO2 is the product most often used by winemakers to prevent B. bruxellensis growth. Usually, the recommended molecular dose of SO2 (active SO2, mSO2) is highly variable, from 0.3 to 0.8mg/L. But these doses do not take into account differences of strain resistance to sulfites or population levels. Moreover, SO2 is known as a chemical stressor inducing a viable but nonculturable (VBNC) state of B. bruxellensis. These cells, which are non-detectable by plate counting, can lead to new contamination when the amount of sulfite decreases over time. Consequently, we first assessed the effect of SO2 levels in red wine on two strains with phenotypically different sulfite resistances. Then, we studied the relationship between amounts of SO2 (0, 0.5, 0.9 and 1.1mg/L active SO2) and population levels (103, 104 and 105cells/mL) in red wine. Yeasts were enumerated by both plate counting and flow cytometry over time using viability dye. Our results showed different SO2 resistances according to the strain used. A relationship between yeast population level and SO2 resistance was demonstrated: the higher the yeast concentration, the lower the efficiency of SO2. Under certain conditions, the VBNC state of B. bruxellensis was highlighted in red wine. Yeasts in this VBNC state did not produce 4-EP. Moreover, cells became culturable again over time. All these results provide new information enabling better management of sulfite addition during wine aging.