Rapid Identification and Enumeration of Saccharomyces cerevisiae Cells in Wine by Real-Time PCR

Abstract

Despite the beneficial role of Saccharomyces cerevisiae in the food industry for food and beverage production, it is able to cause spoilage in wines. We have developed a real-time PCR method to directly detect and quantify this yeast species in wine samples to provide winemakers with a rapid and sensitive method to detect and prevent wine spoilage. Specific primers were designed for S. cerevisiae using the sequence information obtained from a cloned random amplified polymorphic DNA band that differentiated S. cerevisiae from its sibling species Saccharomyces bayanus, Saccharomyces pastorianus, and Saccharomyces paradoxus. The specificity of the primers was demonstrated for typical wine spoilage yeast species. The method was useful for estimating the level of S. cerevisiae directly in sweet wines and red wines without preenrichment when yeast is present in concentrations as low as 3.8 and 5 CFU per ml. This detection limit is in the same order as that obtained from glucose-peptone-yeast growth medium (GPY). Moreover, it was possible to quantify S. cerevisiae in artificially contaminated samples accurately. Limits for accurate quantification in wine were established, from 3.8 ؋ 10 5 to 3.8 CFU/ml in sweet wine and from 5 ؋ 10 6 to 50 CFU/ml in red wine.Wine can become a growth substrate for a range of undesirable yeast species, both during and after fermentation. Uncontrolled yeast growth at either of these two stages can alter the chemical composition of wine, detracting from its sensory properties of appearance, aroma, and flavor. If these faults are severe, the wine is rejected by consumers. Thus, wine spoilage constitutes an important concern to wine producers.It is well known that Saccharomyces cerevisiae plays a beneficial role in wine fermentation, in which it is the predominant species. Nevertheless, S. cerevisiae is able to spoil wine after fermentation if the yeast is not properly eliminated or if the bottle is contaminated by yeast cells present in the wine bottling line or in the cork. S. cerevisiae is an important spoilage yeast because it resists high ethanol concentrations. It has been found mainly in sweet wines, where fermentable sugar can support growth, and also in semidry bottled wines.It is important to detect spoilage yeasts quickly to enable wineries to intervene rapidly and effectively. Methods based on PCR appear to be the best alternative. In the case of S. cerevisiae, several methods have been shown suitable for the detection of this species. This is the case of the restriction analysis of the rRNA region spanning the 5.8S gene and the two internal transcribed spacers (ITSs) (5.8S-ITS region). The amplification patterns of S. cerevisiae when its DNA is digested with the endonuclease CfoI, HaeIII, or HinfI identify this species accurately. These data are summarized in references 10 and 13 and are available online (http://www.yeast-id.com). Masneuf et al. (18) developed a similar system based on PCR amplification and subsequent restriction analysis using the nuclear gene MET2. These author...

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