Karakterizacija površinskih molekula bakterijskih ćelija odgovornih za potencijalnu probiotičku aktivnost prirodnih izolata laktobacila

Abstract

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 faktora uključenih u proces agregacije u ovom radu su analizirani brzina i tip autoagregacije. Brzina autoagregacije (utvrđivana spektrofotometrijski) kao i oblik agregata su varirali kod sojeva. Najbrže su agregirali i formirali najkrupnije agregate sojevi BGSJ2- 8, BGDP1-84 i BGNJ1-6. Karakterizacija prirode faktora koji utiču na sposobnost autoagregacije izabranih sojeva laktobacila je rađena intenzivnim pranjem sojeva koji agregiraju u bidestilovanoj vodi i u PBS rastvoru. Uočeno je da se autoagregacija gubila intenzivnim pranjem u bidestilovanoj vodi kod svih sojeva, osim kod BGDP1-84, na osnovu čega je zaključeno da je prisustvo jona neophodno za formiranje agregata. Osim toga, pokazano je da su neki od faktora autoagregacije proteinske prirode s obzirom da se ova karakteristika gubila nakon tretmana proteinazom K. Lb. paracasei subsp. paracasei BGSJ2-8 je formirao koagregate sa Listeria innocua ATCC33090, Escherichia coli ATCC25922 ili sa Salmonella enterica ser. Typhimurium TR251, dok njegov derivat BGSJ2-81 koji je izgubio sposobnost autoagregacije nije koagregirao...

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...