# _Forentation_
## 3 Results  
### Fermentation Kinetics  
Laboratory-scale fermentations (400 mL) were conducted by inoculating pure or mixed (simultaneous and sequential) cultures of two regional strains belonging to the species _P. kudriavzevii_ and _S. cerevisiae_ into sterile Granny Smith apple must (Table 1). In simultaneous fermentations, both _S. cerevisiae_ and _P. kudriavzevii_ species were inoculated at ratios 1:1 (Sim 1:1) or 1:10 (Sim 1:10), respectively. On the other hand, sequential fermentations were inoculated with _P. kudriavzevii_ (from the beginning) and _S. cerevisiae_ (after 48 h of fermentation) (Seq P-S) or vice versa (Seq S-P). The progression of fermentations was monitored by measuring \({}^{\circ}\)Brix (and total reducing sugars after \({}^{\circ}\)Brix stabilization) and cfu/mL counts (Figure 1). All fermentations except for those inoculated with the pure _P. kudriavzevii_ culture showed complete sugar consumption (less than 2 g/L of total reducing sugars) after 16 days of inoculation. In contrast, fermentations carried out by the pure _P. kudriavzevii_ culture took a total of 23 days to complete sugar consumption. The evolution data of \({}^{\circ}\)Brix obtained from fermentations with _P. kudriavzevii_ alone (P) and with sequential inoculation initiated with _P. kudriavzevii_ (Seq P-S) fitted the sigmoid or modified Gompertz decay model, whereas the data for the remaining fermentation trials (S, Sim 1:1, Sim 1:10, Seq S-P) fitted the exponential decay model (Table 2). The most notable difference among fermentations was the substrate consumption rate (parameter _K_). This parameter can be compared only among fermentations that fit the same model. For fermentations fitted to the sigmoid model, those conducted with the pure culture of _P. kudriavzevii_ exhibited a significantly lower rate of sugar consumption (lower \(K\) value) compared to the sequential fermentation initiated with this strain (Seq P-S). Among the fermentations fitted to the exponential model, those simultaneously inoculated with the two strains (both Sim 1:1 and Sim 1:10) exhibited the highest \(K\) values (Table 2), possibly due to the highest yeast inoculum concentration (Table 1). Figure 1: Yeast viable count and \({}^{\circ}\)Brix evolution in pure and mixed fermentations with _S. cerevisiae_ _NIF8 and P. kudriavzevii_ NPCC1651. Bars represent yeast viable count (log cfu/mL) for _S. cerevisiae_ (gray) and _P. kudriavzevii_ (white). Lines indicate modeled \({}^{\circ}\)Brix values, and points indicate observed \({}^{\circ}\)Brix values. Different letters indicate significant differences between _P. kudriavzevii_ and _S. cerevisiae_ cfu/mL values on a given fermentation day (Tukey test, n = 2). P: _P. kudriavzevii_ NPCC1651; S: _S. cerevisiae_ NIF8; Sim 1:1: Simultaneous fermentation by S:P ratio 1:10; Seq P-S: Sequential fermentation by _S. cerevisiae_ NIF8 48 h after _P. kudriavzevii_ NPCC1651; Seq S-P: Sequential fermentation by _P. kudriavzevii_ NPCC1651 48 h after _S. cerevisiae_ NIF8. In order to evaluate the sugar consumption kinetics of the different fermentations, glucose and fructose concentrations were measured at various stages of the alcoholic fermentation (Figure 2). As in the case of \({}^{\circ}\)Brix, the observed data were fitted to the modified sigmoid decay model and to the exponential decay model, and the model that showed the best fit was selected to obtain the kinetic parameters (Table 3).