Evidence for viable but nonculturable yeasts in botrytis-affected wine

Aims: In Botrytis-affected wine, high concentrations of SO2 are added to stop the alcoholic fermentation and to stabilize the wine. During maturing in barrels or bottle-ageing, an unwanted refermentation can sometimes occur. However, results of the usual plate count of wine samples at the beginning of maturing suggest wine microbial stability. The aim of this study was to investigate the mode of yeasts survival after the addition of SO2 and to identify surviving yeasts. Methods and Results: Using direct epifluorescence technique, we observed the behaviour of cells after SO2 addition and compared the cell number determined by this method with the result of plate counts. The persistent yeast species were identified using two methods: polymerase chain reaction (PCR)-restriction fragment length polymorphism and PCR-denaturing gradient gel electrophoresis. They were identified as Saccharomyces cerevisiae and Candida stellata, and after few months of maturing, other spoiling yeasts appeared, like Rhodotorula mucilaginosa or Zygosaccharomyces bailii. Conclusions: All characteristics of the cells lead to the conclusion that yeast persisted in wine in a viable but nonculturable-like state (VBNC). Suppression of the effect of free-SO2 did not lead to the resuscitation of the cells; however, another method proved the capacity of the cells to exit from the VBNC-like state. Significance and Impact of the Study: This study permits the characterization of the presence of VBNC-like yeasts in wine. The 'refermentation' phenomenon is probably due to the exit of the VBNC state.