Karakterizacija površinskih molekula bakterijskih ćelija odgovornih za potencijalnu probiotičku aktivnost prirodnih izolata laktobacila
Characterization of surface molecules from bacterial cells involved in the potential probiotic activity of natural lactobacilli isolates
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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 ukl...jučenih 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 det...ermined 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...