## 3 Results and Discussion
### Standard Quality Parameter of Wine  
Basic quality parameters for produced wines clearly indicated that yeast strain and fermentation protocol significantly affect the Tamjanika grape wine characteristics (Table 1). Compared to the control wine sample, the fermentation with native yeast strains, in both fermentation protocols, significantly reduced the concentration of ethanol. Precisely, the concentration of ethanol was lower in the samples produced in sequential fermentations compared to the control, from 0.36% \(v/v\) to 0.53% \(v/v\), for the _H. uvarum_ S-2 and _C. famata_ WB-1, respectively, while the highest ethanol reduction (1.06% \(v/v\) less compared to the control) was found in the wine sample produced with monoculture of _C. famata_ WB-1 strain. Such results indicated that selected strains can be used to reduce the content of ethanol in large-scale wine production, which is in line with the interests of modern winemaking and consumer demands for products with a lower content of ethanol. The ethanol reduction was earlier confirmed for non-_Saccharomyces_ species, and this ability highly depends on the yeast strain or/and fermentation conditions [35; 36; 37]. Although statistically significant differences in residual sugar content are observed between produced wines, all the fermentations reached dryness (reducing sugar lower than 4 g/L). Lower ethanol concentration and similar sugar consumption for used yeast strains compared to the commercial _S. cerevisiae_ QA23 could be explained by the ability of certain yeast strains to produce different metabolic byproducts (glycerol, succinic acid, lactic acid) or to accumulate greater yeast biomass [38]. Figure 1: Kinetics of pure and sequential fermentations inoculated with two non-_Saccharomyces_ isolates (_C. famata_ WB-1 and _H. uvarum_ S-2) and _S. cerevisiae_ QA23 (SC). Data were the mean \(\pm\) SD of two biological replicates of fermentation. Although in our previous papers we found that C. _famata_ WB-1 and \(H\). _warm_ S-2 isolates were not able to ferment sugar to dryness in the sterile must, we highlighted that both isolates can produce dry wines from fresh must in a non-sterile environment that is more similar to the real conditions during wine fermentation. Completed fermentation of fresh Prokupac grape, or Tamjanika grape must in this work, confirmed the presence of grape or winery indigenous microbiota, which is able to prevail and finish the fermentation. It is worth mentioning that in our previous experiment we found that the inoculation with 6 log CFU/mL should be considered sufficient to ensure the growth and proliferation of inoculated isolates and their predominance at least during the early stages of fermentation, which will result in a substantial impact on aroma and sensory quality of produced wines. It has been reported that although non-_Saccharomyces_ yeasts are mainly present in the first stage of fermentation, they significantly affect the characteristics of the wine [39]. This is not just based on the direct production of secondary metabolites, but related to the enzyme production responsible for the bioconversion of nonvolatile and odorless flavor precursors into their active version [40]. The total and volatile acidity of wines produced by different yeast isolates or fermentation protocols reveal slight differences. A higher level of total acidity was found in the wines produced by non-_Saccharomyces_ yeast strains in monoculture. These findings are in line with our previous results for _C. famata_ WB-1 strain [19]. This ability should be considered a positive characteristic, especially in warm climate areas where the trend of the total acidity reduction in wines is observed [41; 42]. Independently of the fermentation protocol, volatile acids were slightly higher in wines produced with _C. famata_ WB-1 and \(H\). _uavrum_ S-2, but still, in all produced wines, this level was far below the limits proposed by the OIV (1.2 g/L of acetic acid).