## 2 Materials and Methods  
### Yeast Strains and Culture Conditions  
Commercial- and wild-type yeast strains of the species _Saccharomyces cerevisiae_ were selected and studied in synthetic media targeting application of selected yeast strains as starter cultures for wine-making of _Vitis vinifera_ Greek grape varieties from the Santorini region. Specifically, the new isolated strains LMBF Y-10, Y-25, Y-35 and Y-54, originated from the culture collection of the Laboratory of Microbiology and Biotechnology of Foods (Department of Food Science and Human Nutrition, Agricultural University of Athens, Greece), originated from various types of food-stuffs (_viz._ commercial wine, grape musts and grapes) and not having been previously systematically studied regarding their fermentation potential, were used as cell factories in the present study. The commercial strains: Symphony, Cross X and Passion Fruit, were also used for comparison reasons. Strains were regenerated in YPDA slants (20 g/L glucose, 10 g/L yeast extract, 10 g/L peptone and 25 g/L agar) every 4 months to maintain the yeast viability [26]. Pre-cultures were performed in 250 mL non-baffled conical flasks filled with 50 mL of medium (YPD medium: 20 g/L glucose, 10 g/L yeast extract, 10 g/L peptone, pH \(\approx\) 3.5) previously autoclaved at \(T\) = 115 \({}^{\circ}\)C/1.5 atm for 15 min. Aerobic experiments were performed in 250 mL (filled with 50 \(\pm\) 1 mL medium) agitated flasks (use of a ZHWY-211B Rocking Incubator) at 180 \(\pm\) 5 rpm in which commercial glucose (Hellenic Industry of Sugar SA, Orestiada, Greece) was used as carbon source. Glucose-based media presented the following salt composition in g/L: KH\({}_{2}\)PO\({}_{4}\) 7.0; Na\({}_{2}\)HPO\({}_{4}\) 2.5; MgSO\({}_{4}\)-7H\({}_{2}\)O 1.5; CaCl\({}_{2}\)-2H\({}_{2}\)O 0.15; FeCl\({}_{3}\)-6H\({}_{2}\)O 0.15; ZnSO\({}_{4}\)-7H\({}_{2}\)O 0.02; MnSO\({}_{4}\)-H\({}_{2}\)O 0.06 [26]. The nitrogen sources used were peptone and yeast extract (at concentrations 3.0 and 3.0 g/L respectively), while initial glucose (Glc\({}_{0}\)) was added to _ca_ 100 g/L and _ca_ 200 g/L. The pH value was adjusted to 3.5 \(\pm\) 0.2, while incubation temperature \(T\) = 30 \(\pm\) 1 \({}^{\circ}\)C was employed. Flasks were aseptically inoculated with 1 mL of yeast preculture (thus, a 2% \(v/v\) inoculation occurred). In the elaborated kinetics, each flask constituted the experimental point for the relevant experiments. Microaerophilic/anaerobic trials were performed during wine-making experiments in static cultures [14]; grape must from two different _Vitis vinifera_ Greek grape varieties of Santorini indigenous, Assyrtiko and Mavrottragano, were used for white and red wine production, respectively. The initial concentration of total reducing sugars (TS) for Assyrtiko must was 221.5 g/L, pH was =3.3 and total acidity was =6.0 g/L (expressed as tartaric acid) while for Mavrottragano must the respective values were 213.5 g/L, 3.4 and 5.8 g/L. In addition, SpringFerm(tm) (Fermentis, France) nutrients (0.2 g/L) were applied in each fermentation batch targeting enhanced fermentation rates [27]. Subsequently, alcoholic fermentations were carried out by applying the strains LMBF Y-54 and Passion Fruit under static cultures, ensuring microaerophilic (initially) and nearly anaerobic (after the initial steps of the fermentation and the CO\({}_{2}\) accumulation in the must and the bottle) conditions [12,14] in 1.0 L Duran bottles containing 500 mL of grape must. Alcoholic fermentations were performed at \(T\) = 18 \({}^{\circ}\)C constant temperature incubating an initial yseat population of 5 \(\times\) 10\({}^{6}\) cfu per 500 mL of must (_viz._ 5 mL of exponential pre-culture in 500 mL of must). During fermentation, samples were obtained aseptically at 24-h intervals for further analysis, while sugar depletion signified the end of the wine-making process [7].