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Proteases of Lactobacillus and Lactococcus strains catalyze casein degradation in fermented dairy products, which can result in the production of bioactive peptides. Proteolytic properties of a selection of lactic acid bacteria (LAB) strains previously isolated in Croatia, including Lactococcus lactis and Lactobacillus strains, are described. All strains of Lactobacillus and Lactococcus showed an Fmc+ phenotype that can be associated with efficient growth in milk. The significant caseinolytic effect, after incubation of culture supernatant or concentrated cell biomass, was observed for Levilactobacillus brevis D6 and Lactiplantilactobacillus plantarum D13 after growth in the optimal growth medium, while for Lc. lactis ZGBP5-32 and Levilactobacillus brevis SF9B strains after growth in skimmed milk. To assess the LAB growth in skimmed milk, the acidification rate was monitored. Statistically, significant acidification capacity was determined for L. plantarum D13 in the optimal medium and by the proteolytic strain L. helveticus M92 in skimmed milk. After extraction of proteinases from the strains with caseinolytic activity, protein samples were analysed by the SDS-PAGE method. The protein extract of the strains Lc. lactis ZGBP5-32 and ZGZA7-10, retained proteolytic activity even at very low concentrations. The ultrafiltration improved protein extraction which possibly contained putative protease, as a decrease in contaminating proteins was confirmed by SDS-PAGE in samples of L. brevis D6 and SF9B, L. fermentum D12 and L. plantarum D13., Proteinaze Lactobacillus i Lactococcus sojeva kataliziraju reakcije razgradnje kazeina iz fermentiranih mliječnih proizvoda pri čemu se mogu sintetizirati peptidi s bioaktivnim učincima. Određena su proteolitička svojstva Lactococcus lactis sojeva izoliranih iz tradicionalno proizvedenih hrvatskih svježih sireva i probiotičkih te funkcionalnih starter kultura iz roda Lactobacillus. Odabrani su sojevi Lactobacillus i Lactococcus vrsta kod kojih je ustanovljen Fmc+ fenotip, a koji se povezuje s prisutnošću proteinaza s kazeinolitičkim djelovanjem. Analizom fenotipa hidrolize kazeina ustanovljeno je značajnije kazeinolitičko djelovanje sojeva Levilactobacillus brevis D6 i Lactiplantilactobacillus plantarum D13 nakon kultivacije u optimalnoj hranjivoj podlozi za rast, a sojeva Lactococcus lactis ZGBP5-32 i Levilactobacillus brevis SF9B nakon rasta u obranom mlijeku. U svrhu procjene rasta sojeva u obranom mlijeku praćeno je zakiseljavanje mlijeka. Statistički značajna acidifikacija određena je nakon rasta L. plantarum D13 preko noći u optimalnom mediju odnosno nakon rasta proteolitičkog soja L. helveticus M92 u obranom mlijeku. Nakon pripreme ekstrakta proteina sojeva koji iskazuju kazeinolitičku aktivnost, proteinski uzorci su analizirani SDS-PAGE metodom. Djelomično pročišćene proteinaze sojeva Lc. lactis ZGBP5-32 i ZGZA7-10 i u vrlo niskim koncentracijama su pokazale kazeinolitičku aktivnost. Implementacija ultrafiltracije u postupku ekstrakcije doprinijela je pročišćavanju proteinaza što je potvrđeno SDS-PAGE metodom za proteinske ekstrakte sojeva L. brevis D6 i SF9B, L. fermentum D12 i L. plantarum D13. Daljnja istraživanja bit će usmjerena na karakterizaciju bioaktivnih peptida uslijed proteazne aktivnosti odabranih kazeinolitičkih bakterija mliječne kiseline.

This study aimed to select the Lactobacillus strains, from human milk microbiota, with specific properties of synthesis of exopolysaccharides (EPS), S-proteins and bacteriocins with the potential to be characterised as therapeutic biomolecules. Detection of the „ropy“ phenotype was used for screening of EPS producers, while bacteriocinogenic activity was analysed by the determination of antagonistic activities in vitro. S-proteins of Lactobacillus brevis strains MB1, MB2, MB13 and MB20, were detected by SDS-PAGE analysis of GHCl extracted surfaceome and further analysed by LC-MS/MS using database search by Mascot. For the identification of the genes of interest, responsible for the expression of targeted biomolecules, PCR with specific primers was applied. 1H-NMR analysis confirmed that Lactobacillus fermentum MC1 strain produces EPS. PCR method with eps primers confirmed the genes of the eps cluster. Dendrograms of SDS-PAGE electrophoretic profiles, obtained using GelCompar II, showed characteristic bands of approximately 45 kDa in L. brevis strains identified by mass spectrometry. The spectrum of Lactobacillus strains showed bacteriocinogenic activity, however, the presence of plnJ, plnA and plnEF genes involved in the bacteriocin biosynthetic pathway was confirmed in the genome of six Lactobacillus plantarum strains. The comparative genomic analysis provided insights into the clusters related to the expression of potential biomolecules. The analysis of breast milk microbiota revealed the presence of Lactobacillus strains with the potential to express specific biomolecules. Further investigations will be directed towards deciphering mechanisms of probiotic actions triggered by these biomolecules in order to develop next-generation probiotics.

U svrhu odabira potencijalnih probiotika nove generacije, iz prikupljenih uzoraka majčinog mlijeka izolirano je 100 bakterijskih sojeva. Elektroforezom RAPD-PCR produkata, dobiveni su jedinstveni genetički profili svakog bakterijskog soja, koji su hijerarhijskom klaster analizom svrstani u 5 dendrograma. Analizom dendrograma, odabrano je 28 genetički različitih bakterijskih sojeva koji su fenotipski okarakterizirani nizom metoda i identificirani sekvenciranjem 16S rRNA gena. Usporedbom rezultata sekvenciranja s rezultatima hijerarhijske klaster analize, ustanovljeno je da 46 % sojeva pripada rodu Enterococcus, 27 % Staphylococcus, 22 % Lactobacillus i 5 % Streptococcus. SDS-PAGE analizom je utvrđeno da čak 4 Levilactobacillus brevis sojeva (MB1, MB2, MB13 i MB20) sadrže površinske (engl. Surface, S) proteine, po prvi put identificirane u sojevima izoliranim iz majčinog mlijeka. PCR analizom je ustanovljeno da 6 Lactiplantibacillus plantarum sojeva sadrže gene koji kodiraju za bakteriocine (plnJ, plnA i plnEF). Ispitivanjem antimikrobne aktivnosti, preživljavanja u nepovoljnim uvjetima te agregacije i adhezije na epitelne i subepitelne strukture intestinalnog trakta, ustanovljeno je da najveći probiotički potencijal među sojevima izoliranim iz majčinog mlijeka, imaju producenti potencijalnih terapijskih biomolekula S-proteina i bakteriocina.

Competition of commensal and probiotic bacteria with pathogens for colonization of colon is one of the protective mechanisms of human gastrointestinal tract. To be able to successfully compete with pathogens for binding sites, probiotic bacteria must have advanced adhesion properties. Since in our previous work we have shown that specific surface proteins, named S-layer proteins, have important role in adhesion of bacterial cells, in this study, we examined the ability of five S-layer-expressing Lactobacillus strains to inhibit the adhesion of Salmonella enterica serovar Typhimurim FP1 and Escherichia coli 3014 to human colon adenocarcinoma Caco-2 cells. Two types of experiments were done: preincubation (lactobacilli were incubated with Caco-2 cells first, after which potentially pathogenic bacteria were added) and coincubation (both bacteria were added to Caco-2 cells simultaneously). The number of adherred potentially pathogenic bacteria decreased in lesser extent in coincubation experiment than in the case of preincubation of Lactobacillus strains, when they were given advantage in creating interaction with Caco-2 cells. It is also important to emphasise that all of the five Lactobacillus strains showed antimicrobial activity against both of the potentially pathogenic strains, which is aditional mechanism of prevention of pathogen colonisation in human gastrointestinal tract.

The autochthonous strain D6, which was identified as Lactobacillus brevis, carries a 45-kDa S-layer protein. This strain displayed the highest percentage of autoaggregation (70.37% ± 5.79 in 5h) among lactic acid bacteria (LAB) strains tested, and the autoaggregation was affected by the removal of S-layer proteins by 5M guanidine hydrochloride (GHCl) (54.87±7.13 in 5h). Since S-layer proteins have been identified within lactic acid bacteria (LAB), only among representatives of Lactobacillus species, it was of special interest to study its functional role in Lb. brevis D6. Subsequently, the capacities of the Lb. brevis D6 to adhere to extracellular matrix proteins (ECM) and intestinal epithelial cell lines in vitro were assayed. The removal of the S-layer proteins reduced the adhesive ability of the strain D6 to Caco-2 cell lines and ECM components, while its adhesiveness to the IPEC-1 cell line was low. Pathogen exclusion through coaggregation ability and survival in simulated conditions of the gastrointestinal tract (GIT) and during freeze drying of Lb. brevis D6, before and after GHCl extraction of S-layer proteins from the cell surface, were followed. Results implied on potential S-layer protective functions on Lb. brevis D6 cells under aforementioned stress conditions. Additionally, cytokine production in monocyte-derived dendritic cells (moDC) and moDC maturation in response to Lb. brevis D6 bacterial cells and its purified S-layer proteins were determined by ELISA and FACS, respectively. However, no immunodulatory effects were observed, besides a slight induction of TNF-alpha, IL-12 and IP-10 production. The results suggest that the S-layer protein enhances certain probiotic activities of Lb. brevis D6, particularly protection in rigorous GIT conditions and adhesion to intestinal cell lines.

Biološka uloga S-proteina (engl. surface layer (S-layer) proteins) prisutnih na površini stanične stijenke nekih vrsta bakterija iz roda Lactobacillus, još nije u potpunosti razjašnjena. Između dvjestotinjak autohtonih sojeva bakterija mliječne kiseline (BMK), koje su izolirane iz tradicionalno proizvedenih fermentiranih proizvoda, a zatim identificirane i okarakterizirane kao probiotičke i/ili starter kulture, te pohranjene u Zbirci bakterija mliječne kiseline (ZBMK), Laboratorija za tehnologiju antibiotika, enzima, probiotika i starter kultura Prehrambeno-biotehnološkog fakulteta, odabrani su sojevi BMK iz roda Lactobacillus koji sadrže S-proteine na površini stanice. Nakon karakterizacije S-proteina odabranih bakterijskih sojeva, provedena su istraživanja u okviru probiotičkog koncepta. Ispitana je uloga S-proteina u auto- i koagregaciji bakterijskih stanica, adheziji na crijevne epitelne stanice, kompetitivnoj ekskluziji patogena, kao i uloga S-proteina, tijekom proizvodnje i primjene suhih aktivnih probiotičkih pripravaka. Posebno je istražena uloga S-proteina u imunomodulacijskom djelovanju odabranih sojeva BMK s ciljem istraživanja mogućnosti njihove primjene kao mukoznih vakcina. Ispitan je imunomodulacijski učinak, s naglaskom na ulogu S-proteina odabranih sojeva BMK u poticanju sazrijevanja odnosno proizvodnje citokina u humanim dendritičnim stanicama. Proizvodnja citokina i sazrijevanje dendritičnih stanica, kao rezultat imunostimulacije, bilo pomoću bakterijskih stanica ili njihovih izoliranih i pročišćenih S-proteina, su određeni ELISA metodom, te metodom protočne citometrije. Odabrani sojevi bakterija iz roda Lactobacillus koji sadrže S-proteine su inducirali nastajanje citokina i sazrijevanje dendritičnih stanica, a dobiveni rezultati su bili različiti u odnosu na različite sojeve.

Bacterial surface (S-) S-layers are macromolecular paracrystalline arrays covering cell envelope which are composed of identical protein or glycoprotein subunits. So far, among numerous lactic acid bacteria (LAB) species these proteins have been identified only between several Lactobacillus members. The S-layer proteins of Lactobacillus strains have specific features and their biological roles have not yet been fully elucidated. Although these surface proteins are yet to be characterised, several functional roles in Lactobacillus cells have been proposed. In fact, some probiotic properties of Lactobacillus bacteria, such as adhesion, pathogens exclusion and immunomodulation have been related with the occurrence of S-layers. Hence, in this manuscript the most recent studies together with the results obtained in PhD. Jagoda Šušković, Prof. research group, concerning S-layer protein characterisation are summarised. Especially the functional role of S-layer protein in important probiotic traits, adhesion to intestinal epithelial cells, immunomodulation activity and in competitive exclusion of pathogens by Lactobacillus strains, is discussed.