Provider: - Institution: - Data provided by Europeana Collections- The probiotic potential of bacteria depends on the surface characteristics of bacterial cells. Keeping this in mind, in this work the surface components of the cells responsible for the aggregation ability and the production of exopolysaccharides (EPS) were analyzed from the natural isolates of lactobacilli. Lactobacilli strains used in this work were isolated from autochthonous cheeses produced in households according to the traditional technology. Selected lactobacilli showing the autoaggregation ability (BGAR75, BGGR2-68, BGGR2- 82, BGDP9-85, BGDP1-84, BGNJ1-3, BGNJ1-61, BGNJ1-70) as well as two selected strains which do not form aggregates (BGAR76 and BGGR2-20) according to the comparison of their 16S rRNA gene sequence to the NCBI database were classified in the group Lactobacillus casei. The strain BGDU4-71 using the 16S rRNA gene sequence was determined as Lactobacillus delbrueckii subsp. bulgaricus and the strain BGCG11, the producer of the exopolysaccharide (EPS-CG11), was determined by AFLP methodology as Lactobacillus paraplantarum. In order to characterize factors involved in the aggregation process the kinetics and the type of autoaggregation were analyzed. The kinetics (spectrofotometrically determined) as well as the shape of the aggregates was variable among the strains. Strains with fastest and the largest aggregates were BGSJ2-8, BGDP1- 84 and BGNJ1-6. The characterization of the nature of factors involved in autoaggregation of selected strains of lactobacilli was performed by exhaustive washing of the strains in distilled water and in PBS solution. It was noticed that the autoaggregation ability was lost after exhaustive washing in distilled water in all tested strains except BGDP1-84, which led to the conclusion that the presence of some ions was necessary for the formation of the aggregates. Besides, it was shown that some of the factors promoting autoaggregation were of proteinaceous nature, since the ability was lost after the proteinase K treatment. Lb. paracasei subsp. paracasei BGSJ2-8 was able to form coaggregates with Listeria innocua ATCC33090, Escherichia coli ATCC25922 or with Salmonella enterica ser. Typhimurium TR251, while its derivative BGSJ2-81 that was not able to autoaggregate, did not show coaggregation. Spectrofotometrical measurements of the interaction showed that the coaggregation was disturbed after proteinase K treatment, which indicated that coaggregation, as well as the autoaggregation, involve some factors with proteinaceous nature. The analysis of the surface characteristics of strains showing autoaggregation by hexadecane adhesion method showed that all strains with the autoaggregation ability were highly hydrophobic, while the ones that do not aggregate (as well as for the derivative BGSJ2-81) have hydrophilic cell surface . The other part of the work was including the analysis of the probiotic potential of the strain Lb. paraplantarum BGCG11, which produces exopolysaccharide EPS-CG11, in in vitro model systems. In this part of the work, the Muc derivatives from the strain BGCG11 (NB1, NB4, NB16), obtained by novobiocin plasmid curing, were included in analyses. The derivatives produce significantly lower amount of EPS that differs from EPS-CG11 in composition. The analysis of survival in simulated transit trough gastrointestinal tract (GIT), as well as the adhesion to three cell lines (Caco-2, HT29, HT29-MTX) were performed by counting of bacterial colonies before and after the treatment/interaction and the percentage of survival/adhesion was calculated referring to the number of bacteria before the incubation with cell lines. BGCG11 and Muc derivatives (NB1, NB4 and NB16), resuspended in 10% skimmed milk, survived (1- 2%) the simulated GIT transit. The EPS-CG11 was isolated and purified from the supernatant of the liquid BGCG11 culture, cultivated in minimal media with glucose as the only sugar source. HPLC method with MALLS detector was used to monitor the change in molar mass and the amount of EPS-CG11 polymer after simulated transit trough gastric and intestinal digestion. Obtained results showed that the molar mass and the amount of EPSCG11 remained unchanged, indicating that the enzymes in GIT did not affect the stability of the purified EPS-CG11. Testing of the adhesion ability of BGCG11, as well as the control probiotic strain Lb. rhamnosus GG, showed similar percentage of adhesion for these two lactobacilli, while Mucderivatives NB1, NB4 and NB16 expressed statistically significant higher percentage of adhesion to all three cell lines. All tested strains showed very low percentage of adhesion to HT29-MTX cell line, what is the most likely due to the presence of the mucus barrier (HT29-MTX cell line, produces mucins, while Caco-2 cells do not). The probiotic potential of the strain BGCG11 was also monitored by in vitro analysis of the induction of the immune response in the presence of UV-inactivated bacteria (BGCG11, Mucderivatives) and purified EPS-CG11, in human peripheral blood lymphocytes (PBLs) isolated from healthy volunteers. The immune response was monitored by the proliferation of PBLs (by using kit for the proliferation measurement) and the cytokine production (IFNγ, TNFα, IL-12, IL-10, IL-1β and IL-17) by flow cytometry. It was noticed that PBLs proliferated in the presence of lactobacilli (BGCG11 and Mucderivatives NB1, NB4 and NB16), while EPS-CG11 did not affect to the proliferation of PBLs, which led to the conclusion that some other molecules, rather than EPS were involved in inducing of the proliferation. Generally analyzing the type of the immune response, the presence of BGCG11 and the EPS-CG11 (in concentration of 100 g/ml) induced anti-inflammatory and a pro-Th17 response, while Muc derivatives NB1, NB4 and NB16 induced inflammatory response in PBLs. Furthermore, the detailed analysis of the influence of potential pathogenic microorganisms, alone or in coincubation with BGCG11, derivative NB1 and EPS-CG11, on HT29-MTX cell line was performed. The cytotoxic effect in HT29-MTX cell line (performed by measurement of the lactate dehydrogenase activity) and the analysis of the production IL-8 (by ELISA method) was performed. Obtained results were compared to the effects of Lb. rhamnosus GG and its isolated and purified EPS-GG in the same experimental conditions. From all tested pathogens, only Listeria monocytogenes LMG13305 induced significant cell lyses in HT29-MTX cells, which was reduced after coincubation of L. monocytogenes LMG13305 with EPS-CG11. The level of the IL-8 production in HT29-MTX cells after coincubation of pathogens with lactobacilli (BGCG11, Mucderivative NB1 and Lb. rhamnosus GG) or with EPS molecules (EPS-CG11 or EPS-GG) was different in comparison to levels obtained after incubation only with pathogenic strains. BGCG11 and NB1 induced significantly higher level of IL-8 in the presence of Salmonella enterica ser. Thyphymurium LMG15660, Shigella sonnei LMG10473 and Yersinia enterocolitica LMG7899. In coincubation with L. monocytogenes LMG13305, only BGCG11 increased the level of IL-8 production. EPS-CG11 decreased the level of this cytokine in coincubation with L. monocytogenes LMG13305, while both type of EPS molecules (EPS-CG11 and EPS-GG) significantly decreased the IL-8 production in coincubation with Clostridium difficile LMG21717. The conclusion is that different molecules from the cell surface of pathogens, and lactobacilli modify nonspecific immune response of HT29-MTX cell line, while the presence of purified EPS molecules showed silencing of this response. Hence, it is important to see whether the effects of EPS-CG11 and EPS-GG are different, since the difference was seen when Lb. rhamnosus GG and Lb. paraplantarum BGCG11 strains were. At the end of this work, the potential EPS-CG11 operon was localized and characterized. Using techniques of molecular genetics (cloning, sequencing, DNA-DNA hybridization) 26463 bp of the sequence of large plasmid pCG1, lacking in Muc derivatives, were obtained. In this plasmid the potential operon EPS-CG11 was localized: the region of 15 kb with 15 open reading frames (ORFs). Based on the amino acid sequence homology these ORFs were identified as: the priming glycosyltransferase (ORF 1), glycosyltransferases (ORF 2, 3, 4, 5 and ORF 8), polysaccharide polymerases (ORF 6 and 7), chain length determinators (ORF 9, 10 and 11) and the EPS biosynthesis regulators (ORF 10 and 11). Downstream from these ORFs was the transposase, while ORF 13, 14, 15 and 16 showed homology at the amino acid level with proteins involved in synthesis of dTDP-rhamnose (rfbACBD genes) that is a part of the EPS-CG11 composition. In this way, the new and original structure of the EPS-CG11 gene operon was for the first time characterized in Lactobacillus paraplantarum species. Moreover, this operon is also specific by its plasmid localization.- Probiotički potencijal bakterija u velikoj meri zavisi od površinskih karakteristika bakterijske ćelije. Stoga su u ovom radu analizirane površinske komponente ćelija odgovorne za agregacione sposobnosti i produkciju egzopolisaharida (EPS) prirodnih izolata laktobacila. Sojevi laktobacila korišćeni u ovom radu su izolovani iz autohtonih sireva proizvedenih u domaćinstvima prema tradicionalnoj tehnologiji. Odabrani laktobacili koji su ispoljavali autoagregaciju (BGAR75, BGGR2-68, BGGR2-82, BGDP9-85, BGDP1-84, BGNJ1-3, BGNJ1-61, BGNJ1-70), kao i dva odabrana soja koja ne agregiraju (BGAR76 i BGGR2-20), su klasifikovani na osnovu poređenja nukleotidnih sekvenci gena za 16S rRNK sa NCBI bazom podataka i svrstani u grupu Lactobacillus casei. Soj BGDU4-71 je determinisan sekvenciranjem 16S rDNK kao Lactobacillus delbrueckii subsp. bulgaricus, a soj BGCG11, proizvođač egzopolisaharida (EPS-CG11), je determinisan AFLP metodom kao Lactobacillus paraplantarum. U cilju karakterizacije fakto