## 3 Results and Discussion
### Prebiotic Potential
Generally, bacteria synthesize degradative enzymes only when substrates for these enzymes are present in their environment. Moreover, the concentration of the produced enzymes in bacterial cells could be in relevance with the amount of the available substrate [60]. The investigation of the type and concentration of the produced glycolhydrolases plays a crucial role in the detection of substrates nature used by bacteria during their fermentation [61]. The utilization of _P. major_ poly- and oligosaccharides were evaluated by measuring the activity of \(\alpha\)-galactosidase, \(\alpha\)-glucosidase and \(\beta\)-xylosidase produced by probiotic bacteria during the fermentation process. Glucose was used as a control for glycohydrolase synthesis. All _Lactobacillus_ strains showed highest values for \(\alpha\)-galactosidase activity (up to 0.64 U/mg), followed by \(\alpha\)-glucosidase activity (up to 0.38 U/mg), and about 5-10 times lower values of \(\beta\)-xylosidase activity (up to 0.06 U/mg) (Figure 5). Regarding the dynamics of glycohydrolase secretion, all strains showed higher rates of \(\alpha\)-galactosidase activity at the 20 th hour (0.14-0.68 U/mg) compared to the 6 th hour (0.08-0.24 U/mg). Except _L. acidophilus_ N, which showed higher \(\alpha\)-glucosidase activity at the end fermentation hour, all other _Lactobacillus_ strains demonstrated similar values of \(\alpha\)-glucosidase activity at the 6 th (0.21-0.38 U/mg) and 20 th hour (0.22-0.33 U/mg). As expected, no enzyme activity was detected in all investigated strains using glucose in the culture media (data not shown). Mainly, bacteria do not need the production of glycohydrolases when they use monosaccharides as carbon sources [60]. Similar results had [PERSON] et al. [52] which reported for 17 _Bifidobacterium_ strains low or no enzyme activity when using glucose as energy source. the glycosidic bonds in the structure of the polysaccharide fragments hydrolyzed by the particular enzymes. On the other hand, there is a strain specificity regarding the possibility of secretion of the studied enzymes. For example, the strain \(L\). _sakei_ S16 showed the highest \(\alpha\)-galactosidase activity, reaching two to three times higher values than the other investigated strains. Except for \(L\). _acidophilulus_, all other strains showed almost identical values of \(\alpha\)-glucosidase activity. The obtained results indicated, on the one hand, the presence of accessible \(\alpha\)-glycosidic linkages in the polysaccharide fractions, and on the other hand, that the tested strains had affinity towards more easily digestible monosaccharide components for their subsequent utilization. The established \(\beta\)-xylosidase activity in most of the tested strains is a proof of the branched structure of _P. major_ heteropolsaccharide fractions and the ability of the strains to adapt to growth media with different carbohydrate substrates. \(\beta\)-xylosidase activity of \(L\). _sakei_ S16 and \(L\). _brevis_ S27 was higher compared to the other tested strains but it was 8-10 times lower than the values of \(\alpha\)-glucosidase activity. A three-way ANOVA test was performed three times in order to evaluate the statistical significance of the individual factors-carbon source (glucose, PWPs and PLM), bacterial strains (\(L\). _A._ N, \(L\). _P._ S30, \(L\). _S._ S16 and \(L\). _B._ S27) and time (6 h and 20 h) on the dependant variables (the enzyme activities: \(\alpha\)-galactosidase, \(\alpha\)-glucosidase and \(\beta\)-xylosidase activity). The results (Supplementary Materials: Table S3) showed that there was a statistically significant difference between all of the individual factors and their combination interactions (\(p<0.05\)), with R\({}^{2}\) value close to 1 (0.996-0.997), indicating a considerable linear relationship between the factors and the dependant variable. [PERSON]'s HSD tests for post-hoc analysis were also carried out in each of the three different ANOVA tests.