Improved detection and enumeration of yeasts in wine by Cells-qPCR

Quantitative PCR by directly sampling (Cells-qPCR) has been adapted to detect and quantify total yeasts, and B. bruxellensis, S. cerevisiae and Z. bailii species, in grape musts and wines. To increase assay sensitivity, the effects of a previous cell wall lysis, by both enzymatic and mechanical methods, were evaluated. Cell wall disruption by mechanical methods showed the best results to enhance assay sensitivity. Numerous standard curves were constructed by mechanically lysed cells in culture medium, and in white and red grape musts and wines. Good regression values (>0.99) and efficiencies (>0.99) were obtained, and it was possible to detect one single cell per reaction in all the matrices. Moreover, the population evolution of yeasts during the winemaking process showed that the method provides an effective tool to detect and enumerate yeasts during industrial wine fermentation, and to rapidly control wine spoilage risks.