## 4 Discussion  
Climate change is a threat to grapevine growers and the wine industry. Indeed, a variety of bioclimatic indices have been developed in order to scrutinize productivity in the field, accompanied by possible adaptation strategies [50; 51; 52; 53; 54; 55; 56; 57; 58; 59; 60]. At the winemaking level, the proposed strategies include chemical and microbiological acidification [61]. Chemical acidification through the use of citric, tartaric, or malic acids is not a feasible option due to their instability and the effect on sensorial properties. On the contrary, lactic acid is more stable and organoletically softer than the aforementioned acids [62]. Therefore, biological acidification through the use of _L. thermotolerans_, with its unique metabolic attributes, in combination with _S. cerevisiae_, is promoted as a feasible option [63]. In our study, we would like to go further and examine this acidification effect in an already acidic variety, in terms of microbial dominance and sensory attributes. Time of inoculation, inoculum ratio, and fermentation temperature have been reported to affect the growth and persistence of _L. thermotolerans_ and _S. cerevisiae_[64; 65; 66]. Sequential fermentation seems to be the strategy of choice in order to avoid co-existence with _S. cerevisiae_. According to this scheme, _L. thermotolerans_ is inoculated first, and when the acidity reaches the desired level, depending on the capacity of the strain, inoculation with _S. cerevisiae_ takes place. This way, acidification takes place and _L. thermotolerans_ experiences no antagonism by _S. cerevisiae_. On the contrary, co-inoculation will result in antagonism for nutrients, especially nitrogen, and redirection of the _L. thermotolerans_ carbon catabolism from glycolysis to the pentose phosphate pathway; both will result in the reduction of lactic acid production [28; 67]. Concerning _S. cerevisiae_ implantation control, besides the  
Figure 3: Sensory description of the wines produced in the present study. W1: _L. thermotolerans_ strain Lt1—_S. cerevisiae_ strain A&Y10 (dark blue); W2: _L. thermotolerans_ strain Lt1—_S. cerevisiae_ strain A26Y23 (red); W3: _L. thermotolerans_ strain Lt1—_S. cerevisiae_ strain Sc1 (gray); W4: _S. cerevisiae_ strain A&Y10 (yellow); W5: _S. cerevisiae_ strain A26Y23 (light blue); W6: _S. cerevisiae_ strain Sc1 (green). The asterisk next to each attribute indicates statistically significant differences between the different wines. These are presented by the box and whisker charts. Different letters above each box designate statistically significant differences (_p_ < 0.05) of the specific attribute between the different wines. strain A26Y23, which could dominate at 100% (W5), all the other inoculated strains were present at approximately 60%. As Assyrtiko must is known for its high acidity and low pH, the indigenous strains are well adapted and are able to survive until the end of the fermentation process [47]. This statement is in accordance with the implantation control results, where the indigenous strains remained in an increased population until the end of alcoholic fermentation. In general, low fermentation temperatures (\(\leq\)20 \({}^{\circ}\)C) enhance the competition potential of _L. thermotolerans_, especially when _S. cerevisiae_ is sequentially and not simultaneously inoculated, leading to protracted persistence in high populations [68]. However, this is observed in laboratory fermentations using pasteurized grape juice and not under industrial fermentation conditions. In the latter case, _L. thermotolerans_ seems to lack effective competitiveness and therefore its final biomass is rather restricted [68]. In the present study, in which fermentation took place under industrial conditions, _S. cerevisiae_ strains were added 48 h after _L. thermotolerans_. Based on the oenological analysis, this period was adequate for the activation of lactic acid production. Regarding the duration of fermentation, it has been stated that incorporation of _L.