## 3 Results and Discussion
### Acetification Process Parameters  
DCM, a cheap, readily available, and inert biomaterial, was used as an immobilization carrier for the microorganisms involved and as a promoter of the FSS wine acidification process [15]. The composition of FSS and its aqueous extract, as well as its use for dry winemaking using _S. cerevisiae_AXAZ-1 immobilised on DCM, were previously reported by [10]. One of the novelties of the methods presented in this study is that the alcoholic fermentation was partial until a sweet FSS wine was obtained, therefore the subsequent acidification was carried out in the presence of sugars in the substrates. In Table 1, parameters of the sweet wine acidification process using the wild and the selected AAB cultures, free and immobilized on DCM and after 2 months of cold storage, are presented, as means of 3 repeated acidification batches. Analysis of variance (One-way ANOVA at the \(p=0.05\) level) between the data populations of the 3 batches, showed that the means of sugar, ethanol, and acidity concentrations were not significantly different  
Figure 2: SEM images of the immobilized biocatalysts on DCM during the acidification of sweet FSS wine. (**a**): wild culture (ICW). (**b**,**c**): _A. aceti_ and _K. europeus_ culture (ICM). (\(p>0.05\)), with the exception of the ethanol levels for FCM, FCW-2M, FCM-2M, and ICW-2M (\(p<0.05\)). For FCM-2M the means of all parameters of the 3 batch fermentations were significantly different. Generally, it can be observed that all cultures, free or immobilised, can produce vinegar with 46.65 \(\pm\) 5.43 g/L levels of acidity (as g acetic acid/L) from sweet wine with 5.08 \(\pm\) 1.19% \(v/v\) alcohol concentration. The differences observed in the initial sugar and ethanol concentration of the substrates are due to the production method used, i.e., an amount of the produced vinegar was decanted and the remaining liquid served as the starter of the next batch, as in the case of the common industrial SmF processes. This is a technological feature that needs further investigation. However, this is a low-volume bench-scale experiment and these variations are not easy to control compared to the commonly used 8-10 tn, or higher capacity, industrial acetate vessels. Moreover, the final industrial vinegar products are commonly diluted with water to the desired acidity before packaging. The most important requirement in the vinegar industry is to keep the acetators functioning because acidification cease is usually detrimental for the AAB and the whole process must restart. Figures 3 and 4, present the kinetics of the acidification of the sweet wine at 30 \({}^{\circ}\)C using the wild or selected cultures, free, immobilized, and after 2 months of cold storage. The average values of 3 batches plus standard deviations are illustrated for the evolution of sugar, ethanol, and acidity levels during the process. Best curve fitting of the data was done using [PERSON]'s sigmoidal function, on Microcal Origin 6.0 software, achieving very high coefficients of determination (R\({}^{2}>0.99\)) for the majority of curve fits (Tables S1 and S2, Figures S1 and S2). Specifically, Figure 3 illustrates the acidification kinetics of the FSS sweet wine using the fresh, free and immobilised, wild AAB cultures (FCW, ICW) and the mixed _A. aceti_ and _K. europaeus_ cultures (FCM, ICM). Comparing the data from Table 1 and Figure 3, it can be observed that it is possible to produce vinegar from sweet FSS wine in repeated acidification batches using the same culture, by all 4 treatments. The acidification times ranged from 3-5 days to 2-9 days for the fresh wild and mixed cultures, respectively, free and immobilised. The effect of cell immobilization on the acidification kinetics was more obvious in the case of the selected, mixed culture.