Izolacija, karakterizacija i ispitivanje probiotskih osobina autohtonih sojeva laktobacila

Abstract

Cilj ove doktorske disertacije je bio ispitivanje probiotskih osobina novih, autohtonih sojeva laktobacila izolovanih iz gastrointestinalnog trakta zdravih beba i uzoraka domaćeg kravljeg sira sa područja Gornjeg Milanovca. Ukupno je obrađeno 46 uzoraka (43 humanog i 3 poreklom iz sira) iz kojih su izolovana 143 bakterijska soja. Od ovog broja 120 izolata su bili Gram pozitivne koke, 15 izolata Gram pozitivni bacili i 8 izolata Gram negativni bacili. Pripadnost rodu Lactobacillus je potvrđena kod 8 izolata (7 humanog i 1 poreklom iz sira) za koje je pokazano da su katalaza negativni, nesporulativni Gram pozitivni bacili, sposobni da rastu na MRS podlozi u anaerobnim uslovima. Genotipska identifikacija je pokazala da izolati Lac1, Lac2, Lac6 i Lac7 pripadaju vrsti Lactobacillus plantarum, izolati Lac3 i 5s vrsti Lactobacillus casei, izolat Lac4 vrsti Lactobacillus paracasei i izolat Lac5 vrsti Lactobacillus gasseri. Iz daljih ispitivanja je isključen izolat Lac5 zbog slabog rasta na čvrstoj podlozi. Fiziološkom i biohemijskom karakterizacijom ispitivanih izolata pokazano je da se radi o nehemolitičnim, homofermentativnim vrstama koje rastu u širokom opsegu temperature (10-40ºC), koncentracije NaCl (2,0-6,5%) i pH vrednosti (2,0-8,0). Takođe, utvrđeno je da izolat 5s raste mnogo sporije u odnosu na ostale izolate. Izolati su pokazali visok stepen otpornosti na lizozim, kisele uslove sredine i prisustvo žučnih soli. Proteolitička aktivnost je detektovana samo kod izolata Lac1. Svi ispitivani izolati rastu u prisustvu fenola od 0,4%, a izolat Lac2 i pri koncentraciji od 1%. Ispitivanje osetljivosti na najčešće upotrebljavane antibiotike pokazalo je da su svi izolati rezistentni prema vankomicinu, amikacinu, gentamicinu i norflokscinu, a osetljivi prema ampicilinu i rifampicinu. Ni jedan od ispitivanih izolata nije pokazao osetljivost prema -amilazi, tripsinu, himotripsinu, pronazi E i katalazi. Ispitivanje osnovnih probiotskih osobina pokazalo je visok procenat (preko 79%) preživljavnja izolata u simuliranim uslovima gastrointestinalnog trakta. Osim toga, bezćelijski supernatanti svih izolata su pokazali antimikrobno dejstvo prema pojedinim Gram pozitivnim i Gram negativnim bakterijama, dok je ono izostalo prema kvascima i plesnima, kao i prema vrstama iz roda Lactobacillus...

The aim of this Doctoral thesis was to investigate the probiotic properties of new, indigenous lactobacilli strains isolated from the gastrointestinal tract of healthy babies and homemade cheese samples originating from the area of Gornji Milanovac. We collected 46 samples (43 of human and 3 of dairy origin), from which 143 bacterial strains were isolated. Among them, 120 isolates were identified as Gram positive cocci, 15 isolates as Gram positive bacilli and 8 isolates as Gram negative bacilli. We showed that 8 isolates (7 of human and 1 of dairy origin) were catalase-negative, Gram positive bacilli that do not form spores and are able to grow on MRS medium in anaerobic conditions, and confirmed that they belong to the genus Lactobacillus. Genotypic identification showed that isolates Lac1, Lac2, Lac6 and Lac7 belong to the species Lactobacillus plantarum, isolates Lac3 and 5s to the Lactobacillus casei, Lac 4 to the Lactobacillus paracasei and isolate Lac5 to the Lactobacillus gasseri. Isolate Lac5 was excluded from further studies due to its poor growth on agar medium. Physiological and biochemical characterization showed that all isolates were nonhemolytic, homofermentative species, growing in a wide range of temperature (10 to 40ºC), NaCl concentration (2.0 to 6.5%) and pH (2.0 to 8.0). Comparing to the other isolates, the 5s grew slower. The isolates showed high degree of resistance to lysozyme, acidic environmental conditions and the presence of bile salts. Only Lac1 isolate possessed proteolytic activity. All isolates grew in the presence of 0.4% phenol and Lac2 even at its concentration of 1%. Investigation of isolates sensitivity to antibiotics showed that all isolates were resistant to vancomycin, amikacin, gentamicin and norfloxacin, but sensitive to ampicillin and rifampicin. All tested isolates showed no sensitivity to α-amylase, trypsin, chymotrypsin, pronase E and catalase. Investigation of probiotic properties showed that isolated strains survived in high percentage (over 79%) in simulated conditions of gastrointestinal tract. Additionally, all isolates showed antimicrobial effect against certain Gram positive and Gram negative bacteria, but not against yeasts and molds. No antimicrobial effect was detected against other species of the Lactobacillus genus. This last feature is important because it gives the possibility of combining two or more species in the potential probiotic product...