Selection of autochthonous lactic acid bacteria for preparation of probiotic starter cultures

Autohtoni izolati bakterija mliječne kiseline (BMK) identificirani su sekvencioniranjem 16S rRNA gena te su provedena istraživanja u okviru probiotičkog koncepta s ciljem in vitro odabira potencijalnih probiotičkih kultura. Kako bi se isključila mogućnost širenja antibiotičke rezistencije, ispitivana je osjetljivost autohtonih izolata BMK na različite skupine antibiotika. Autohtoni izolati BMK su pokazali osjetljivost na svih 12 ispitanih antibiotika, a jedino su predstavnici Lactobacillus vrsta bili rezistentni na vankomicin, zbog uroñene rezistencije na taj antibiotik. Kako je glavni zahtjev pri izboru probiotičkih sojeva njihovo preživljavanje u gastrointestinalnom traktu (GIT), ispitano je preživljavanje autohtonih izolata BMK u simuliranim uvjetima GIT. Osobito visok stupanj preživljavanja pri pH vrijednosti simuliranog želučanog soka 2 i u simulaciji soka tankog crijeva, pokazali su bakterijski sojevi: ZG2-25, ZG3-17, ZG3-18, ZG4-14, ZG1-54, BGGO5-3 , BGLE1-6, BGAL1-1 i ZG5-9. Za postizanje probiotičkog učinka potrebno je u organizam unijeti visok broj živih probiotičkih bakterija u koncentriranom, praškastom pripravku koji se može dobiti liofilizacijom. Stoga je u ovom radu ispitano preživljavanje autohtonih izolata BMK, odabranih na temelju in vitro testa simuliranih gastrointestinalnih uvjeta, tijekom liofilizacije, uz dodatak različitih lioprotektora. Autohtoni izolati BMK preživljavaju proces liofilizacije u većem broju kad su prisutni lioprotektori obrano mlijeko, inulin i oligosaharidi. Lactococcus lactis subsp. lactis BGLE1-6 najbolje preživljava u prisutnosti inulina, Lactobacillus pentosus/paraplantarum/plantarum BGGO5-3 uz dodatak prebiotika oligosaharida, dok Lactococcus lactis BGAL1-1 i Lactobacillus pentosus ZG3-17 najbolje preživljavaju proces liofilizacije u obranom mlijeku. Liofilizacija odabranih sojeva BMK s različitim lioprotektorima, doprinosi povećanju broja živih bakterijskih stanica u probiotičkim pripravcima što doprinosi iskazivanju njihovog probiotičkog učinka kad se primjene kao bioterapeutici.Research was conducted in probiotic concept with the main goal of in vitro selection of the potential probiotic cultures. Autochthonous lactic acid bacteria (LAB) isolates were identified by sequencing of 16S rRNA genes. Sensitivity of LAB isolates was tested to various groups of antibiotics to exclude the possibility of the spread of antibiotic resistance. LAB isolates showed sensitivity to all tested antibiotics, with exception of Lactobacillus species which were resistant to vankomycin due to their intrinsic resistance. The survival of LAB isolates in simulated gastrointestinal tract (GIT) was examined since it is the main requirement in the selection of probiotic strains. High rate of survival in simulated gastric juice at pH 2.0 and simulated small intestine juice was exhibited by the following bacterial strains: ZG2-25, ZG3-17, ZG3-18, ZG4-14, ZG1-54, BGGO5-3, BGLE1-6, BGAL1-1 and ZG5-9. To achieve the probiotic effect, it is necessary to ingest high numbers of live probiotic bacteria in concentrated, powdered form. LAB can be obtained in such form by lyophilization. LAB isolates (selected on the basis of in vitro test) was tested for survivability during lyophilization. LAB isolates survived the process of lyophilization in higher numbers with lyoprotectants such as skimmed milk, inulin and oligosaharides. Lactococcus lactis subsp. lactis BGLE1-6 survives in highest numbers with inulin present in the system, Lactobacillus pentosus/paraplantarum/plantarum BGGO5-3 survives in highest numbers with oligosaharides as lyoprotectonts while Lactococcus lactis BGAL1-1 and Lactobacillus pentosus ZG3-17 survives the process of lyophilization in highest numbers with skimmed milk. Lyophilization of selected LAB strains with different lyoprotectants contributes to increase in number of live bacterial cells in probiotic formulations thus contributing to the demonstration of the probiotic effect when used as biotherapeutics