The functional role of exopolysaccharides and bacteriocins in the probiotic activity of autochthonous lactic acid bacteria strain

Funkcionalne biomolekule posreduju u funkcionalnosti probiotičkih bakterija in vivo. Ciljanom selekcijom autohtonih bakterija mliječne kiseline (BMK) iz znanstveno etablirane bakterijske kolekcije probiotičkih sojeva i funkcionalnih starter kultura odabrani su sojevi sa specifičnim svojstvima biosinteze bakteriocina i egzopolisaharida (EPS-a) te sojevi koji eksprimiraju proteaze za razgradnju kazeina do biopeptida. Glavni istraživački cilj je strukturna i funkcionalna karakterizacija ovih biomolekula kao postbiotika za definiranje molekularnh mehanizama probiotičke aktivnosti sojeva producenata. Sojevi koji su pokazali fenotip sinteze biomolekula, identificirani su potpunim sekvencioniranjem genoma. Producent bakteriocina, Lactiplantibacillus plantarum D13, koji inhibira Listeria monocytogenes ATCC®19111™ i Staphylococcus aureus 3048, u genomu sadrži pln genski klaster za biosintezu plantaricina. Trodimenzionalne strukture plantaricina PlnJK i PlnEF određene su in silico. Limosilactobacillus fermentum D12, sintetizira EPS-e, glikogen i razgranati heteropolisaharid koji sadrži t-Glcp i 2,6-vezane Galf, u omjeru 1:12. eps klaster sadrži gene potrebne za biosintezu EPS-a gtf, epsA, B, C, C’, D i E, gene odgovorne za polimerizaciju i transport wzx i wzy te gene za aktivaciju prekursora molekula galE, galT, galU, rfbB, pgm i glf. Dokazano je da EPS-i posreduju u adhezijskim i autoagregacijskim svojstvima soja producenta D12 te imaju zaštitnu ulogu tijekom liofilizacije i mikroinkapsulacije. Lactobacillus i Enterococcus faecium sojevi imaju kazeinolitičko djelovanje. Primjena konzorcija BMK kao funkcionalne starter kulture u kontroliranoj fermentaciji, rezultirala je biosintezom 15 biopeptida iz kazeina. In vitro i in vivo ispitivanjem funkcionalnosti konzorcija BMK utvrđen je kolonizacijski potencijal i učinak na modulaciju sastava intestinalnog mikrobioma. Optimirana je biotehnološka proizvodnja funkcionalnih mikroformulacija konzorcija BMK. Primjena definiranog konzorcija BMK doprinjela je proizvodnji fermentiranog produkta s dodanom funkcionalnom vrijednošću i omogućila je proizvodnju inovativnog napitka na bazi nusproizvoda fermentacije, sirutke s povećanim sadržajem biopeptida. Zaključno, plantaricin D13, odnosno EPS D12, imaju potencijal probiotičkih molekula nove generacije te uz biokatalitički potencijal sustava proteaza obećavajući su postbiotici koji djeluju kao okidačke molekule za specifične funkcionalnosti sojeva producenata.Functional biomolecules mediate the functionality of probiotic bacteria in vivo. Targeted selection of autochthonous lactic acid bacteria (LAB) from the scientifically established bacterial collection of probiotic strains and functional starter cultures was undertaken to identify strains with specific properties of bacteriocin and exopolysaccharide (EPS) biosynthesis, and those expressing proteases, that degrade casein to biopeptides. The main research goal was the structural and functional characterization of these biomolecules as postbiotics for defining the molecular mechanisms of probiotic activity of the producer strain. Strains with the desired phenotype of biomolecule synthesis were identified by whole-genome sequencing. The bacteriocin producer, Lactiplantibacillus plantarum D13, inhibits Listeria monocytogenes ATCC®19111™ and Staphylococcus aureus 3048, contains the pln gene cluster for the biosynthesis of plantaricin. The three-dimensional structures of plantaricin PlnJK and PlnEF were determined in silico. Limosilactobacillus fermentum D12, synthesizes EPSs, glycogen and branched heteropolysaccharide containing t-Glcp and 2,6-linked Galf, in a ratio of 1:12. eps cluster contains genes required for the biosynthesis of EPSs gtf, epsA, B, C, C', D and E, genes responsible for the polymerization and transport wzx and wzy, and genes for the activation of precursor molecules galE, galT, galU, rfbB, pgm and glf. EPSs mediate the adhesion and autoaggregation ability of D12-producer strain and has a protective role during exposure to freeze-drying and microencapsulation. Lactobacillus and Enterococcus faecium strains have caseinolytic activity. When LAB consortium was applied as a functional starter culture in fermentation, an increase in the biosynthesis of 15 biopeptides from casein was detected. Colonization potential and the biomodulation capacity, through monitoring the changes in the intestinal microbiome composition, were determined by in vitro and in vivo functionality studies of the LAB consortium. The biotechnological production of functional microformulations of the LAB consortium has been optimized. Designed LAB consortium has contributed to the production of a fermented product with added functional value and enabled the production of an innovative beverage based on fermentation by-product, whey with increased content of biopeptides. Plantaricin D13 and EPS D12, have the potential for next generation of probiotic molecules, and with the biocatalytic potential of protease system, promise as postbiotics that act as trigger molecules for the specific functionalities of producer strains