2.3.1. Pure cultures
Pure cultures were carried out in 1 l sterile Erlenmeyer flasks, closed with dense cotton plugs, containing 400 ml of pasteurised (10 min at 100 °C) Sauvignon Blanc grape must (110 g l−1 glucose, 109 g l−1 fructose, 2.77 g l−1 l-malic acid, 588 mg l−1 total nitrogen, pH 3.45) supplemented with sulphur dioxide (30 mg l−1) (vintage 2010). Each experiment was performed in triplicate. Yeasts were pre-cultured in a YPD liquid medium at 30 °C for 48 h. Then pasteurised Sauvignon Blanc grape must was inoculated with 106 cells ml−1 and incubated at 20 °C, without agitation (semi-anaerobic conditions). Fermentation progress and yeast growth were monitored throughout the fermentation process, by assessing sugar concentration and by viable cell counts. Cell viability was determined by flow cytometry using fluorescein diacetate (FDA) as a marker of viability (Malacrinò et al., 2001).