Enes Dertli

Impact of the exopolysaccharide layer on biofilms, adhesion and resistance to stress in Lactobacillus johnsonii FI9785.

BackgroundThe bacterial cell surface is a crucial factor in cell-cell and cell-host interactions. Lactobacillus johnsonii FI9785 produces an exopolysaccharide (EPS) layer whose quantity and composition is altered in mutants that harbour genetic changes in their eps gene clusters. We have assessed the effect of changes in EPS production on cell surface characteristics that may affect the ability of L. johnsonii to colonise the poultry host and exclude pathogens.ResultsAnalysis of physicochemical cell surface characteristics reflected by Zeta potential and adhesion to hexadecane showed that an increase in EPS gave a less negative, more hydrophilic surface and reduced autoaggregation. Autoaggregation was significantly higher in mutants that have reduced EPS, indicating that EPS can mask surface structures responsible for cell-cell interactions. EPS also affected biofilm formation, but here the quantity of EPS produced was not the only determinant. A reduction in EPS production increased bacterial adhesion to chicken gut explants, but made the bacteria less able to survive some stresses.ConclusionsThis study showed that manipulation of EPS production in L. johnsonii FI9785 can affect properties which may improve its performance as a competitive exclusion agent, but that positive changes in adhesion may be compromised by a reduction in the ability to survive stress.

Spontaneous Mutation Reveals Influence of Exopolysaccharide on Lactobacillus johnsonii Surface Characteristics

As a competitive exclusion agent, Lactobacillus johnsonii FI9785 has been shown to prevent the colonization of selected pathogenic bacteria from the chicken gastrointestinal tract. During growth of the bacterium a rare but consistent emergence of an altered phenotype was noted, generating smooth colonies in contrast to the wild type rough form. A smooth colony variant was isolated and two-dimensional gel analysis of both strains revealed a protein spot with different migration properties in the two phenotypes. The spot in both gels was identified as a putative tyrosine kinase (EpsC), associated with a predicted exopolysaccharide gene cluster. Sequencing of the epsC gene from the smooth mutant revealed a single substitution (G to A) in the coding strand, resulting in the amino acid change D88N in the corresponding gene product. A native plasmid of L. johnsonii was engineered to produce a novel vector for constitutive expression and this was used to demonstrate that expression of the wild type epsC gene in the smooth mutant produced a reversion to the rough colony phenotype. Both the mutant and epsC complemented strains had increased levels of exopolysaccharides compared to the wild type strain, indicating that the rough phenotype is not solely associated with the quantity of exopolysaccharide. Another gene in the cluster, epsE, that encoded a putative undecaprenyl-phosphate galactosephosphotransferase, was deleted in order to investigate its role in exopolysaccharide biosynthesis. The ΔepsE strain exhibited a large increase in cell aggregation and a reduction in exopolysaccharide content, while plasmid complementation of epsE restored the wild type phenotype. Flow cytometry showed that the wild type and derivative strains exhibited clear differences in their adhesive ability to HT29 monolayers in tissue culture, demonstrating an impact of EPS on surface properties and bacteria-host interactions.

Structure and biosynthesis of two exopolysaccharides produced by Lactobacillus johnsonii FI9785.

Background: Bacterial cell surface polysaccharides are important in pathogenesis, cell adhesion, and protection against harsh environments. Results: Two novel exopolysaccharide (EPS) structures were identified in Lactobacillus johnsonii. Conclusion: The eps cluster is essential for production of both EPS, but epsE is required only for the heteropolymer. Significance: This study will guide functional analysis of EPS in survival and colonization of gut commensals. Exopolysaccharides were isolated and purified from Lactobacillus johnsonii FI9785, which has previously been shown to act as a competitive exclusion agent to control Clostridium perfringens in poultry. Structural analysis by NMR spectroscopy revealed that L. johnsonii FI9785 can produce two types of exopolysaccharide: EPS-1 is a branched dextran with the unusual feature that every backbone residue is substituted with a 2-linked glucose unit, and EPS-2 was shown to have a repeating unit with the following structure: -6)-α-Glcp-(1–3)-β-Glcp-(1–5)-β-Galf-(1–6)-α-Glcp-(1–4)-β-Galp-(1–4)-β-Glcp-(1-. Sites on both polysaccharides were partially occupied by substituent groups: 1-phosphoglycerol and O-acetyl groups in EPS-1 and a single O-acetyl group in EPS-2. Analysis of a deletion mutant (ΔepsE) lacking the putative priming glycosyltransferase gene located within a predicted eps gene cluster revealed that the mutant could produce EPS-1 but not EPS-2, indicating that epsE is essential for the biosynthesis of EPS-2. Atomic force microscopy confirmed the localization of galactose residues on the exterior of wild type cells and their absence in the ΔepsE mutant. EPS2 was found to adopt a random coil structural conformation. Deletion of the entire 14-kb eps cluster resulted in an acapsular mutant phenotype that was not able to produce either EPS-2 or EPS-1. Alterations in the cell surface properties of the EPS-specific mutants were demonstrated by differences in binding of an anti-wild type L. johnsonii antibody. These findings provide insights into the biosynthesis and structures of novel exopolysaccharides produced by L. johnsonii FI9785, which are likely to play an important role in biofilm formation, protection against harsh environment of the gut, and colonization of the host.

Development of a fermented ice-cream as influenced by in situ exopolysaccharide production: Rheological, molecular, microstructural and sensory characterization

This study aimed to investigate the role of in situ exopolysaccharide (EPS) production by EPS(+)Streptococcus thermophilus strains on physicochemical, rheological, molecular, microstructural and sensory properties of ice cream in order to develop a fermented and consequently functional ice-cream in which no stabilizers would be required in ice-cream production. For this purpose, the effect of EPS producing strains (control, strain 1, strain 2 and mixture) and fermentation conditions (fermentation temperature; 32, 37 and 42 °C and time; 2, 3 and 4h) on pH, S. thermophilus count, EPS amount, consistency coefficient (K), and apparent viscosity (η50) were investigated and optimized using single and multiple response optimization tools of response surface methodology. Optimization analyses indicated that functional ice-cream should be fermented with strain 1 or strain mixture at 40-42 °C for 4h in order to produce the most viscous ice-cream with maximum EPS content. Optimization analysis results also revealed that strain specific conditions appeared to be more effective factor on in situ EPS production amount, K and η50 parameters than did fermentation temperature and time. The rheological analysis of the ice-cream produced by EPS(+) strains revealed its high viscous and pseudoplastic non-Newtonian fluid behavior, which demonstrates potential of S. thermophilus EPS as thickening and gelling agent in dairy industry. FTIR analysis proved that the EPS in ice-cream corresponded to a typical EPS, as revealed by the presence of carboxyl, hydroxyl and amide groups with additional α-glycosidic linkages. SEM studies demonstrated that it had a web-like compact microstructure with pores in ice-cream, revealing its application possibility in dairy products to improve their rheological properties.

Effect of in situ exopolysaccharide production on physicochemical, rheological, sensory, and microstructural properties of the yogurt drink ayran: an optimization study based on fermentation kinetics.

Exopolysaccharide (EPS)-producing starter cultures are preferred for the manufacture of fermented milk products to improve rheological and technological properties. However, no clear correlation exists between EPS production and the rheological and technological properties of fermented milk products such as the yogurt drink ayran. In this study, 4 different strain conditions (EPS- and EPS+ Streptococcus thermophilus strains) were tested as a function of incubation temperature (32, 37, or 42°C) and time (2, 3, or 4 h) to determine the effect of culture type and in situ EPS production on physicochemical, rheological, sensory, and microstructural properties of ayran. Furthermore, we assessed the effect of fermentation conditions on amounts of EPS production by different EPS-producing strains during ayran production. A multifactorial design of response surface methodology was used to model linear, interaction, and quadratic effects of these variables on steady shear rheological properties of ayran samples and in situ EPS production levels. The physicochemical and microbiological characteristics of ayran samples altered depending on incubation conditions and strain selection. Steady shear tests showed that ayran samples inoculated with EPS+ strains exhibited pseudoplastic flow behavior. Production of ayran with EPS- strain (control sample) resulted in the lowest apparent viscosity values (η50), whereas those produced with the combination of 2 EPS+ strains yielded ayran with notably increased η50 values. We concluded that incubation time was the variable with the greatest effect on η50, consistency coefficient (K), and flow behavior index (n) values. In situ EPS production was also affected by these conditions during ayran fermentation in which strain-specific metabolism conditions were found to be the most important factor for EPS production. In addition, these findings correlated the amount of in situ EPS produced with the rheological properties of ayran. Scanning electron microscopy images of the samples showed differences in structural features, revealing a prominent network strand structure in the ayran samples inoculated with the admixture of 2 EPS-producing strains incubated at 37°C for 3 h. These results provide useful information for large-scale production of ayran by the dairy industry.

EpsA is an essential gene in exopolysaccharide production in Lactobacillus johnsonii FI9785

Lactobacillus johnsonii FI9785 has an eps gene cluster which is required for the biosynthesis of homopolymeric exopolysaccharides (EPS)‐1 and heteropolymeric EPS‐2 as a capsular layer. The first gene of the cluster, epsA, is the putative transcriptional regulator. In this study we showed the crucial role of epsA in EPS biosynthesis by demonstrating that deletion of epsA resulted in complete loss of both EPS‐1 and EPS‐2 on the cell surface. Plasmid complementation of the epsA gene fully restored EPS production, as confirmed by transmission electron microscopy and nuclear magnetic resonance (NMR) analysis. Furthermore, this complementation resulted in a twofold increase in the expression levels of this gene, which almost doubled amounts of EPS production in comparison with the wild‐type strain. Analysis of EPS by NMR showed an increased ratio of the heteropolysaccharide to homopolysaccharide in the complemented strain and allowed identification of the acetylated residue in EPS‐2 as the (1,4)‐linked βGlcp unit, with the acetyl group located at O‐6. These findings indicate that epsA is a positive regulator of EPS production and that EPS production can be manipulated by altering its expression.

Highly Enantioselective Production of Chiral Secondary Alcohols Using Lactobacillus paracasei BD101 as a New Whole Cell Biocatalyst and Evaluation of Their Antimicrobial Effects

Chiral secondary alcohols are valuable intermediates for many important enantiopure pharmaceuticals and biologically active molecules. In this work, we studied asymmetric reduction of aromatic ketones to produce the corresponding chiral secondary alcohols using lactic acid bacteria (LAB) as new biocatalysts. Seven LAB strains were screened for their ability to reduce acetophenones to their corresponding alcohols. Among these strains, Lactobacillus paracasei BD101 was found to be the most successful at reducing the ketones to the corresponding alcohols. The reaction conditions were further systematically optimized for this strain and high enantioselectivity (99%) and very good yields were obtained. These secondary alcohols were further tested for their antimicrobial activities against important pathogens and significant levels of antimicrobial activities were observed although these activities were altered depending on the secondary alcohols as well as their enantiomeric properties. The current methodology demonstrates a promising and alternative green approach for the synthesis of chiral secondary alcohols of biological importance in a cheap, mild, and environmentally useful process.

Characterization of functional properties of Enterococcus faecium strains isolated from human gut

The aim of this work was to characterize the functional properties of Enterococcus faecium strains identified after isolation from human faeces. Of these isolates, strain R13 showed the best resistance to low pH, bile salts, and survival in the simulated in vitro digestion assay, and demonstrated an important level of adhesion to hexadecane as a potential probiotic candidate. Analysis of the antibiotic resistance of E. faecium strains indicated that in general these isolates were sensitive to the tested antibiotics and no strain appeared to be resistant to vancomycin. Examination of the virulence determinants for E. faecium strains demonstrated that all strains contained the virulence genes common in gut- and food-originated enterococci, and strain R13 harboured the lowest number of virulence genes. Additionally, no strain contained the genes related to cytolysin metabolism and showed hemolytic activity. The antimicrobial role of E. faecium strains was tested against several pathogens, in which different levels of inhibitory effects were observed, and strain R13 was inhibitory to all tested pathogens. PCR screening of genes encoding enterocin A and B indicated the presence of these genes in E. faecium strains. Preliminary characterization of bacteriocins revealed that their activity was lost after proteolytic enzyme treatments, but no alteration in antimicrobial activity was observed at different pHs (3.5 to 9.5) and after heat treatments. In conclusion, this study revealed the functional characteristics of E. faecium R13 as a gut isolate, and this strain could be developed as a new probiotic after further tests.

Highly Enantioselective Production of Chiral Secondary Alcohols with Candida zeylanoides as a New Whole Cell Biocatalyst

The increasing demand for biocatalysts in synthesizing enantiomerically pure chiral alcohols results from the outstanding characteristics of biocatalysts in reaction, economic, and ecological issues. Herein, fifteen yeast strains belonging to three food originated yeast species Candida zeylanoides, Pichia fermentans, and Saccharomyces uvarum were tested for their capability for asymmetric reduction of acetophenone to 1‐phenylethanol as biocatalysts. Of these strains, C. zeylanoides P1 showed an effective asymmetric reduction ability. Under optimized conditions, substituted acetophenones were converted to corresponding optically active secondary alcohols in up to 99% enantiomeric excess and at high yields. The preparative scale asymmetric bioreduction of 4‐nitroacetophenone (1m) by C. zeylanoides P1 gave (S)‐1‐(4‐nitrophenyl)ethanol (2m) with 89% yield and > 99% enantiomeric excess. Compound 2m has been obtained in an enantiomerically pure and inexpensive form. Additionally, these results indicate that C. zeylanoides P1 is a promising biocatalyst for the synthesis of chiral alcohols in industry.

Impact of exopolysaccharide production on functional properties of some Lactobacillus salivarius strains

AbstractThe aim of this work was to characterize functional properties of Lactobacillus salivarius strains isolated from chicken feces. Detection of genes responsible for exopolysaccharide (EPS) production revealed that all strains harbored a dextransucrase gene, but p-gtf gene was only detected in strain E4. Analysis of EPS production levels showed significant alterations among strains tested. Biofilm formation was found to be medium composition dependant, and there was a negative correlation with biofilm formation and EPS production. Autoaggregation properties and coaggregation of L. salivarius strains with chicken pathogens were appeared to be specific at strain level. An increment in bacterial adhesion to chicken gut explants was observed in L. salivarius strains with the reduction in EPS production levels. This study showed that strain-specific properties can determine the functional properties of L. salivarius strains, and the interference of these properties might be crucial for final selection of these strains for technological purposes.