## 3 Results and Discussion
### Adaptive Evolution-Mediated Changes in Resveratrol Concentrations
Some of these approaches focus on developing genetically modified wine yeasts that will be able to produce resveratrol during fermentation. For example, [PERSON] et al. [54] made an attempt to reconstruct a biochemical pathway in a heterologous host to produce resveratrol [54]. Other strategies are aimed at selecting the best \(\beta\)-glucosidase-producer yeasts that are able to hydrolyze glycosylated forms of resveratrol  
Figure 4: Concentrations of resveratrol [mg/L] after the end of fermentation. Grape juice containing 18% sugars (10.2 g/L glucose and 7.7 g/L fructose) or 30% sugars (15.82 g/L glucose; 13.55 g/L fructose) were used as fermentation media. Data are presented as the means \(\pm\) SD; \(n=3\). Significant differences are marked by * (at \(p\) < 0.05). 1 gen—lst generation (ancestral strains); YPD–YPD-evolved derivatives (control); KCl_a–1.25 M KCl-evolved derivatives; KCl_b–1.5 M KCl-evolved derivatives. under enological conditions [52; 53]. For example, in one study, an extracellular glucan \(\beta\)-glusosidase precursor was identified and isolated from _Dekera bruxellensis_ wine yeast [55]. This enzyme consists of two subunits, SCW4p and glucan \(\beta\)-glucosidase precursor, although only the latter subunit has been shown to possess extracellular \(\beta\)-glucosidase activity that was responsible for the conversion of picoid from _Polygonum cuspidatum_ into resveratrol [55]. Nevertheless, _D. bruxellensis_ is generally considered the main spoilage yeast during winemaking due to the production of specific compounds like various pungent volatile phenols, associated with unpleasant aromas, which are collectively known as the phenolic taint or \"brett\" flavor [1; 2]. Therefore, in another study, 308 autochthonous yeast strains isolated from _Vitis labrusca_ Bordo grapes were screened for \(\beta\)-glucosidase production; 33 out of the 308 analyzed strains were reported to be able to hydrolyze picoid into free resveratrol in synthetic media (along with the ability to hydrolyze other substrates, such as cellobiose and arbutin and/or esculin) [52]. Among them were strains from the following yeast species: _Hanseniapson warum_ (\(n=23\)), _Hanseniapson oputiae_ (\(n=4\)), _Candida zemplinina_ (\(n=3\)), _S. cerevisiae_ (\(n=1\)), _Zygoascus meyerea_ (\(n=1\)), and _Zygoasccharomyces beili_ (\(n=1\)). With regard to _S. cerevisiae_, it also should be mentioned that only one strain out of 11 was able to hydrolyze picoid into resveratrol due to \(\beta\)-glucosidase activity [52]. Here, we showed that adaptive evolution under osmotic stress can be a useful approach to develop yeast strains towards the production of wines with an increased content of resveratrol. Of course, there are some strain-dependent limitations. For example, the evolved derivatives of strain 3 failed to produce resveratrol-enriched wines. In fact, \(\beta\)-glucosidase activity, which is responsible for cleaving the glycosylated precursors of resveratrol, can be significantly decreased and/or inhibited under winemaking conditions, such as, i.e., low pH value, high initial glucose concentration, and high ethanol concentration [53]. On the other hand, there are yeasts that produce \(\beta\)-glucosidase whose enzymatic activity and stability are not affected by enological conditions. These are not repressed by a high concentration of glucose and are resistant to ethanol and a low pH [21]. Perhaps, strains 1 and 2 are producers of such \(\beta\)-glucosidase. The findings showing that KCl-evolved strains 1 and 2 are more efficient in producing resveratrol-enriched wines in comparison to the corresponding ancestral strains, which might result from adaptive evolution-mediated changes in protein structures [3; 4], including \(\beta\)-glucosidase, that affect their activity and/or stability under enological conditions. As subtle changes in protein structures might result from adaptive evolution-driven mutations, one can assume that our suggestions are in line with the results obtained by [PERSON] et al. [55]. [PERSON] et al.