The viable but not culturable (VBNC) state has been studied in detail in bacteria. It has been suggested that the VBNC state also exists in eukaryote cells, such as wine yeasts, including Brettanomyces in particular. We investigated the VBNC state in this yeast, focusing on the conditions for entry and exit, and the morphological and metabolic modifications associated with this state. We added sulfite (0.8 mg.L-1 molecular SO2) to induce the VBNC state. Increasing the pH of the medium inactivated the sulfite, allowing the cells to exit from the VBNC state and to become culturable again. In these conditions, we found that Brettanomyces VBNC cells were smaller than culturable cells, and that spoilage by volatile phenols could persist during VBNC state. Furthermore, according to our proteome comparison, it seems that the blockade of ATP synthesis was compensated by an increase in energy-producing metabolism pathway. This study provides the first insight into the VBNC state in eukaryote cells, showing common trend to the VBNC state of prokaryotic cells. The existence of VBNC state in Brettanomyces cells can also provoke errors of detection. A new tool of detection by fluorescence in situ hybridization and reading by flow cyometry was thus set up. This method allows the revealing of Brettanomyces cells presence in wine in an efficient and fast way.
