Identification and functionality of bacterial strains in lyophilized probiotic preparations on the Croatian market

Istraživački cilj je uspostaviti pouzdan metodološki pristup za robusnu analizu probiotičkih pripravaka. Prioritetno je definirana ispravnost taksonomske nomenklature i usklađenost koncentracije probiotičkih stanica s preporučenom dnevnom dozom. Mikrobiološka analiza i združena primjena fenotipskih i genotipskih metoda isključila je prisutnost neprobiotičkih bakterija. Probiotički pripravci su sigurni za ljudsku primjenu jer su prema metaboličkim, fiziološkim i fermentacijskim profilima potvrđeni kao vrste iz rodova Lactobacillus ili Bifidobacterium te je ustanovljena niska pojavnost fenotipske rezistencije na antibiotike. Analizom SDS-PAGE proteinskih profila temeljem dendrograma generiranih pomoću Gel Compare II, sojevi su klasificirani u Lactobacillus ili Bifidobacterium rod. Razlikovanje sojeva utemeljeno je na analizi polimorfizma slučajno umnoženih fragmenata DNA, čija je biotipizacija provedena umnažanjem V9 regije 16S rRNA Bifidobacterium ili 16S/23S intergenske rRNA „spacer“ regije Lactobacillus vrste. Sojevi su identificirani sekvencioniranjem 16S rRNA gena. Potencijal kolonizacije i jačanja funkcije intestinalne barijere određeni su karakterizacijom adaptacije na ekstremne uvjete mikrookoliša i gastrointestinalnog trakta, adhezije na mucin i proteinske ligande intestinalnih niša. Pojedini sojevi iskazuju aktivnost hidrolaze žučnih soli, asimilaciju kolesterola ili prebiotičkih supstrata i kompetitivne ekskluzije enteropatogena. Razvijeni integrirani pristup obuhvaća primjenu molekularne metodologije i in vitro eksperimentalnih modela za validaciju probiotičkih bakterija.The research goal was to define a reliable methodological approach for robust analysis of probiotic preparations. The correctness of the taxonomic nomenclature and the compliance of the concentration of probiotic cells with the recommended daily dose was defined as a priority. Microbiological analysis of probiotic products and the combined application of phenotypic and genotypic methods ruled out the presence of contaminating non-probiotic bacteria. The products are safe for use due to the low incidence of phenotypic resistance to antibiotics of strains which are according to metabolic, physiological characteristics and carbohydrate fermentation profiles grouped to Lactobacillus and Bifidobacterium species. SDS-PAGE of soluble cell or surface proteins and computer-aided comparison of the electrophoretic protein patterns generated by Gel Compare II classified bacterial strains into Lactobacillus spp. or Bifidobacterium spp. RAPD-PCR was used to generate unique and identifying DNA profiles of Lactobacillus and Bifidobacterium strains, whose biotyping was performed by amplification of V9 region of 16S rRNA Bifidobacterium or 16S/23S intergenic rRNA "spacer" region of Lactobacillus species. Probiotic strains were identified by 16S rRNA sequencing. Colonization potential and enhancement of intestinal barrier function were determined based on the adaptation to the extreme microenvironment conditions and gastrointestinal tract and adhesion to the mucin and protein ligands of intestinal niches. Strains show bile salt hydrolase activity and cholesterol or prebiotics assimilation and competitive exclusion of enteropathogens. The developed integrated approach includes the application of molecular methodology and in vitro experimental models for the validation of probiotic bacteria