## 3 Results and Discussion
### Fermentations  
#### 3.2.1 Fermentation Efficiency  
Thermally-dried free or immobilized kefir cells were initially evaluated regarding their fermentation efficiency in simultaneous alcoholic and ML repeated batch fermentations at 30, 20, and 5 \({}^{\circ}\)C for a period greater than 6 months. Although low alcohol winemaking using both wet and freeze-dried immobilized kefir culture on DCM, apple pieces, and grape skins was recently proposed [17], to the best of the authors' knowledge this is the first time the use of thermally-dried immobilized kefir cells is investigated. Fermentation kinetic data and other important parameters are presented in Table 2.  
Figure 1: Survival rate (%) of thermally-dried free and immobilized kefir cells. (**a**) Free kefir culture, (**b**) immobilized kefir cells on apple pieces, (**c**) immobilized kefir cells on delignified cellulosic material (DCM), (**d**) immobilized kefir cells on grape skins. Ethanol content, ethanol productivity, ethanol production yield, and acetic acid concentration were significantly (\(p<0.05\)) affected only by the fermentation temperature. On the other hand, total acidity values were significantly (\(p<0.05\)) affected by the nature of the culture (free or immobilized cells). Fermentation time, residual sugars content and sugar conversion, malic acid concentration and malic acid conversion, lactic acid and glycerol concentration, volatile acidity and pH were affected significantly (\(p<0.05\)) by both factors, while strong interactions (\(p<0.05\)) were noted. No significant differences were observed in citric acid content. As expected, significantly (\(p<0.05\)) higher fermentation times and significantly (\(p<0.05\)) lower ethanol productivity values were recorded as the fermentation temperature was lowered, in all cases [14; 20; 30; 31; 32; 33; 34]. Ethanol concentration ranged from 4.4 to 10.0% (\(\
icefrac{{v}}{{v}}\)) in fermentations performed with immobilized kefir cells, depending on fermentation temperature, as previously reported [17]. Higher ethanol productivities (up to 15.3 g/L d) were noted in fermentations with thermally-dried immobilized cells at all fermentation temperatures in comparison to free cells [35], although not significantly in all cases, and in levels greater than usually noticed in traditional practice or similar to industrial fermentations [35; 36]. Notably, high ethanol yield values (up to 0.47 g/g, accounting for over 90% of the total theoretical yield) that could be of high interest for the wine-making industry [37] were noted at 20 \({}^{\circ}\)C for both thermally-dried free and immobilized cells. Lower ethanol yield values were recorded at the first batches (30 \({}^{\circ}\)C), in all cases, but similar to yields recently associated with parallel alcohol and yeast biomass production (known as the \"Crabtree effect\") in grape must fermentation [38]. As stated above, thermal drying may result in diminished cell populations, mainly due to cell membrane damage and oxidative stress (caused by free radical formation) [39], thus the recovery of cell biomass at the initial stages of fermentations with thermally-dried cells is not uncommon [26]. However, the continuous improvement of fermentation kinetics as the repeated batches proceeded, indicated adaptation of the thermally-dried cells [25], an attribute that has also been noticed when other drying methods have been applied [40; 41; 42]. On the other hand, at 5 \({}^{\circ}\)C, ethanol yield values exhibited a substantial drop (although not significant in the case of immobilized cells), which may be attributed to a variety of causes like greater yeast susceptibility to ethanol toxicity in low temperatures [43], oxidative stress [44], changes to yeast metabolism due to cold stress [45; 46], or even cell exhaustion after an increased number of repeated batches [47]. As a result, significantly lower sugar conversion values (\(p<0.05\)) and significantly (\(p<0.05\)) higher residual sugars were documented at 5 \({}^{\circ}\)C, in all cases.