## 3 Results and Discussion
### Cell Growth Dynamics of the Strains and Evolution of Total Soluble Solids during Mixed Fermentation  
The P4 _S. cerevisiae_ strain was tested in pure and mixed fermentations with four non-_Saccharomyces_ strains, which were CR1 (_Z. bailii_), LC2-1 (_T. delbrueckii_), TSF and CZ3 (_C. zemplinina_). These four non-_Saccharomyces_ strains were chosen for their inability to ferment maltose (data not shown), resulting in suitable candidates for production of low  
Figure 1: The apparent (AA) and real (RA) degree of attenuation (%) of the beers obtained by P4 and the three commercial starters (Pilsen starter (SP), Weizen starter (SW) and Amber starter (SA)) in wort obtained from malt extract for Pilsen (PW), Weizen (WW) and Amber (AW) beers. Data are the mean of two independent experiments. alcohol beers. Among the non-_Saccharomyces_ yeasts used in this work, _T. delbrueckii_ was first suggested as a potential brewing yeast by [PERSON] and [PERSON] [37]. After that, different studies evaluated the use of this yeast species for brewing [19; 22], whereas it should be emphasised that, currently, few data have been reported on the use of _Z. bailii_ and _C. zemplinina_ strains for brewing [38]. The fermentation trials were performed in duplicate and monitored by evaluation of the cell growth dynamics of each strain and evolution of total soluble solids (TSS). The results are reported in Figure 2.  
The overall duration of the fermentations was different, depending on the word and the starter culture used. In fact, for Pilsner and Weizen beers, the primary fermentation performed with P4, P4-CR1 and P4-LC2-1 finished in 10 days, whereas more days were necessary to complete the primary fermentation by P4-TSF and P4-CZ3. The primary fermentation in AW was faster than in the other two ports, although differences were found among the starter cultures. As already reported for PW and WW fermentations, P4, P4-CR1 and P4-LC2-1 starter cultures consumed the sugars before P4-TSF and P4-CZ3. Figure 2: Fermentation kinetics of single and mixed starter cultures composed of _S. cerevisiae_ (P4) and non-_Saccharomyces_ strains (_Z. bailii_ CR1, _T. delbrueckii_ LC2-1, _C. zemplinina_ TSF and CZ3) during fermentation in three different ports (PW, WW, AW), reported as cell counts on the left _y_-axis and reduction of total soluble solids (TSS) on the right _y_-axis. Continuous lines, viable cells; dotted lines, TSS. Data are the mean values \(\pm\) standard deviations of two independent experiments. The duration of the refermentation in bottle was 14 days for all beers, independently of the word used. The initial amount of TSS was 9 degrees Plato (\({}^{\circ}\)P) in PW and WW, and 9.5 \({}^{\circ}\)P in AW, whereas the final content of TSS was around 5 \({}^{\circ}\)P in all the beers produced. As regards the reduction of TSS during the time, the mixed cultures including _Z. bailii_ and _T. delbrueckii_ (P4-CR1 and P4-LC2-1, respectively) showed a similar trend to the pure P4 culture in PW and WW, with a progressive decrease from 9 to 5 \({}^{\circ}\)P until the sixth day of fermentation and successive stability until the end of the process. Conversely, in AW, these two mixed starter cultures showed a slower sugar reduction than the P4 strain. As regards _T. delbrueckii_, most previous studies reporting the use of this yeast in beer production found that the overall speed of fermentation seemed to be slower than that of the usual _S. cerevisiae_ brewing strains. The results obtained in our study were different, as in PW and WW fermentation, the rate of TSS reduction in mixed fermentation including the _T. delbrueckii_ strain was similar to those exhibited by a pure culture of _S. cerevisiae_, whereas a slightly slower reduction was observed in AW fermentation, confirming that the fermentation performance of _T. delbrueckii_ strongly depends on the strain [39]. The reduction of TSS in the fermentations performed by the two mixed cultures P4-TSF and P4-CZ3, both consisting of _S. cerevisiae_ and _C.