Native Exopolysaccharide Research Articles

A DFT study of the gallium ion-binding capacity of mature Pseudomonas aeruginosa biofilm extracellular polysaccharide.

Intravenous gallium therapy is a non-antibiotic approach to limit Pseudomonas aeruginosa biofilm proliferation, by outcompeting iron for siderophore binding. Gallium therapy represents a viable therapeutic strategy for cystic fibrosis (CF) patients harbouring mucoid P. aeruginosa biofilm lung infections. Siderophore deficient P. aeruginosa isolates still demonstrate a hindered biofilm proliferation when exposed to gallium but it is currently unknown whether exogenous gallium has any disruptive influence on the exopolysaccharide (EPS), the major mucoid P. aeruginosa CF lung biofilm matrix component. To that end, Density-Functional Theory (DFT) was deployed to assess whether gallium (Ga3+) could be substituted into the mature mucoid EPS scaffold in preference of calcium (Ca2+)-the native EPS cross-linking ion. Removal of the stable, bound native calcium ions offers a large enthalpic barrier to the substitution and the mature EPS fails to accommodate exogenous gallium. This suggests that gallium, perhaps, is utilising a novel, possibly unknown, ferric uptake system to gain entry to siderophore deficient cells.

Effect of Polysaccharides on the Physical, Rheological and Thermal Properties of a Balanced Canine Food


 The structuring of food is mainly done through a complex set of various food materials, such as proteins, polysaccharides, lipids, minerals, and water. Among these components, proteins and polysaccharides are the most used materials in food processing. In extrusion processing, the molecular interactions between its main components play a synergistic role in the achieved structure by performing specific functions in food that affect the sensory, physicochemical, and functional properties. In this research, food for pet animals based on beans (Phaseolus vulgaris L.), fodder corn (Zea mays L.), polished rice (Oryza Sativa), and polysaccharides as inulin, sodium alginate, and/or a native microbial exopolysaccharide from mining waste, was designed and evaluated. To produce the pellet, a single-screw extruder at 140 rpm and 140°C at the exit zone and the effects of the moisture content [13-18%] and concentration of each polysaccharide [0.3-0.9%] on the physicochemical properties of the food were evaluated. The obtained responses were: 1.16-1.4 for expansion index, 0.8-1.7 g/cm3 for bulk density, 1.5-7.4 g/g for water absorption index, 8.8-12.6% for water solubility index, and 8.8-35 N for hardness. According to the recorded values, native exopolysaccharides can provide better physicochemical characteristics than commercial polysaccharides.

Polyguluronate simulations shed light onto the therapeutic action of OligoG CF-5/20

Chronic mucoid P. aeruginosa cystic fibrosis (CF) lung infections are associated with the development of a biofilm composed of anionic acetylated exopolysaccharide (EPS) alginate, electrostatically stabilised by extracellular Ca2+ ions. OligoG CF-5/20, a low molecular weight guluronate rich oligomer, is emerging as a novel therapeutic capable of disrupting mature P. aeruginosa biofilms. However, its method of therapeutic action on the mucoid biofilm EPS is not definitively known at a molecular level. This work, utilising molecular dynamics (MD) and Density-Functional Theory (DFT), has revealed that OligoG CF-5/20 interaction with the EPS is facilitated solely through bridging Ca2+ ions, which are not liberated from their native EPS binding sites upon OligoG CF-5/20 dispersal, suggesting that OligoG CF-5/20 does not cause disruptions to mature P. aeruginosa biofilms through breaking EPS-Ca2+-EPS ionic cross-links. Rather it is likely that the therapeutic activity arises from sequestering free Ca2+ ions and preventing further Ca2+ induced EPS aggregation.

Lyophilization induces physicochemical alterations in cryptococcal exopolysaccharide

Microbial polysaccharide characterization requires purification that often involves detergent precipitation and lyophilization. Here we examined physicochemical changes following lyophilization of Cryptococcus neoformans exopolysaccharide (EPS). Solution 1H Nuclear Magnetic Resonance (NMR) reveals significant anomeric signal attenuation following lyophilization of native EPS while 1H solid-state Nuclear Magnetic Resonance (ssNMR) shows few changes, suggesting diminished molecular motion and consequent broadening of 1H NMR polysaccharide resonances. 13C ssNMR, dynamic light scattering, and transmission electron microscopy show that, while native EPS has rigid molecular characteristics and contains small, loosely packed polysaccharide assemblies, lyophilized and resuspended EPS is disordered and contains larger dense aggregates, suggesting that structural water molecules in the interior of the polysaccharide assemblies are removed during extensive lyophilization. Importantly, mAbs to C. neoformans polysaccharide bind native EPS more strongly than lyophilized EPS. Together, these observations argue for caution when interpreting the biological and immunological attributes of polysaccharides that have been lyophilized to dryness.

Effect of Gamma Irradiation on Enhanced Biological Activities of Exopolysaccharide from Halomonas desertis G11: Biochemical and Genomic Insights.

In this work, a native exopolysaccharide (nEPS) produced by Halomonas desertis G11 isolated from a Tunisian extreme environment was modified by gamma irradiation. Characterization as well as the antioxidant and antitumor activities of nEPS and its gamma-irradiated derivatives (iEPSs) were comparatively evaluated. In vitro and in vivo antioxidant potentials were determined by using different methods and through different antioxidant enzymes. The antitumor activity was checked against a human colon cancer cell line. Analyses of the complete genome sequence were carried out to identify genes implicated in the production of nEPS. Thus, the genomic biosynthesis pathway and the export mechanism of nEPS were proposed. Analyses of irradiation data showed that iEPSs acquired new functional groups, lower molecular weights, and gained significantly (p < 0.05) higher antioxidant and antitumor abilities compared with nEPS. These findings provide a basis for using iEPSs as novel pharmaceutical agents for human therapies.

Potential of Exopolysaccharide from Porphyridium marinum to Contend with Bacterial Proliferation, Biofilm Formation, and Breast Cancer.

Exopolysaccharide (EPS) from marine microalgae are promising sources of a new generation of drugs. However, lot of them remain to be discovered and tested. In this study, EPS produced by Porphyridium marinum and its oligomers prepared by High Pressure Homogenizer have been tested for different biological activities, i.e., antibacterial, anti-fungal and antibiofilm activities on Candida albicans, as well as for their effects on the viability of murine breast cancer cells. Results have shown that all EPS samples present some biological activity. For antibacterial and antibiofilm activities, the native EPS exhibited a better efficiency with Minimum Inhibitory Concentration (MIC) from 62.5 µg/mL to 1000 µg/mL depending on the bacterial strain. For Candida albicans, the biofilm formation was reduced by about 90% by using only a 31.3 µg/mL concentration. Concerning breast cancer cells, lower molar masses fractions appeared to be more efficient, with a reduction of viability of up to 55%. Finally, analyses of polymers composition and viscosity measurements were conducted on all samples, in order to propose hypotheses involving the activities caused by the intrinsic properties of polymers.

Isolation and characterization of a novel exopolysaccharide secreted by Lactobacillus mucosae VG1

A novel strain of Lactobacillus mucosae was isolated from a faecal sample of an individual who had adhered to a strict vegetarian diet for nine years. The strain displayed a ropy character when grown on plates and generated a relatively small amount (62 mg/L) of an exopolysaccharide (EPS) when grown in broth culture.The EPS eluted from a size exclusion chromatography column as a single band with a weight average molecular mass of 1.51 × 104 g/mol. Monomer analysis and sugar absolute configuration analysis confirmed that the EPS was a D-galactan. Using linkage analysis in combination with 1D and 2D-NMR spectroscopy, with spectra being recorded for both the native EPS and for the products generated by Smith degradation of the EPS, the following structure was determined for the repeat unit of the polysaccharide: [Display omitted] This is a novel D-galactan and represents the first structure for an EPS produced by a strain of Lactobacillus mucosae to be reported.

Bifidogenic effects of Cordyceps sinensis fungal exopolysaccharide and konjac glucomannan after ultrasound and acid degradation

The bifidogenic effects of exopolysaccharide (EPS) of a medicinal fungus (Cordyceps sinensis) and a well-known food polysaccharide konjac glucomannan (KGM) with different molecular weight (MW) ranges were evaluated through in vitro experiments in liquid cultures of Bifidobacteria. Native EPS and KGM were partially degraded with power ultrasound (US) to improve the water solubility, and further hydrolysed with trifluoroacetic acid to much lower MW. The acid-hydrolysed fractions (EPS-AH and KGM-AH) supported the growth of all five tested bifidobacterial species, while the US-degraded high MW fractions, EPS-US and KGM-US, could only slightly support the growth of some species. All EPS fractions increased the acetic acid production of most bifidobacterial species. Most remarkably, the high MW EPS-US, EPS-AH and KGM-US fractions significantly enhanced the cell viability with much higher colony forming unit (CFU) counts, suggesting a protective effect of these high MW polysaccharides for the bacterial survival. The results have shown that MW was a significant factor on the bifidogenic properties of partially degraded EPS and KGM.

Construction of a Cordyceps sinensis exopolysaccharide-conjugated selenium nanoparticles and enhancement of their antioxidant activities

A Cordyceps sinensis exopolysaccharide (EPS)-conjugated selenium nanoparticles (SeNPs) were successfully constructed through the reduction of SeO32−. The EPS-SeNPs were characterized in terms of formation, morphology, size, Se distribution and phase by UV–vis, FT-IR, transmission electron microscopy (TEM), dynamic light scattering (DLS), energy dispersive X-ray (EDX) and wide angle X-ray diffraction (WAXD) measurements. Results revealed that the SeNPs conjugated to EPS were amorphous and could be well dispersed at a size range of 80–125nm. The interactions between the OH groups of EPS and SeNPs substituted for intermolecular interaction in native EPS to form new CO⋯Se bonds, resulting in good dispersion of SeNPs in the EPS matrix. Besides, the EPS-SeNPs at different Se/P ratios exhibited significant scavenging ability on superoxide anion radical (O2−) and ABTS radical cation (ABTS+) when compared to pure EPS, indicating that the conjugated SeNPs reinforced antioxidant effect of EPS. This work not only provides a simple and efficient way to construct well-dispersed SeNPs in aqueous system, and demonstrates the vital role of the EPS as a biopolymer template for dispersion, stabilization and size control of SeNPs, but also finds the EPS-SeNPs can potentially serve as a good antioxidant towards O2− and ABTS+.

Depolymerization of Pseudomonas stutzeri exopolysaccharide upon fermentation as a promising production process of antibacterial compounds.

Many researchers have focused on high molecular weight (Mw) exopolysaccharides (EPS) as a source of potentially bioactive lower Mw derivatives. Therefore, it is of interest to find means for efficient and safe production of depolymerized-polymer derivatives. Exopolysaccharide-depolymerization products (EDP) varying in molecular weight were recovered from fermentative depolymerization of a native EPS produced by Pseudomonas stutzeri AS22. Following their purification and physicochemical characterization, the antibacterial activity of EDP on food spoilage and food poisoning microorganisms was evaluated through the measurement of the inhibition zone diameter, the half maximal (IC50) and the minimal (MIC) inhibitory concentrations. Our results indicate that the lower the Mw, the higher will be the effectiveness of EDP on reducing Gram-negative bacteria growth and the opposite trend was observed in the case of Gram-positive bacteria. EDP bioactivities may provide novel insights into the potentiality of P. stutzeri EPS and its derivatives to be used as functional-food components.

ANTIMICROBIAL, ANTICOAGULATION, FIBRINOLYTIC AND PREBIOTIC ACTIVITIES OF EXOPOLYSACCHARIDE PRODUCED BY MARINE KLEBSIELLA SP

The authors previously, achieved a high yield of exopolysaccharide production from a marine Klebsiella sp. The aim of the present study is determination of this exopolysaccharide composition and evaluation of its biological activities. The monosaccharide moieties of this exopolysaccharide were determined by complete hydrolysis, qualitative and quantitative paper chromatography of the hydrolysis product. The monosaccharide composition of this exopolysaccharide was galactose 16%, fructose 20%, glucose 32%, fucose 22%, and uronic acid 10%. The biological activities of the native and modified sulfated bacterial exopolysaccharide were investigated. No anticoagulation or fibrinolytic activities were recorded for the sulfated exopolysaccharide, but the native form of exopolysaccharide showed both activities. The native exopolysaccharide also showed higher antibacterial activity against S. aureus and E. coli but both the native and sulfated form had no antifungal activity against Candida albicans. MIC of the native and sulfated exopolysaccharide that sustain the highest bacterial inhibition zone was 15 mg/dl. No prebiotic activities were recorded for both. In conclusion, this biopolymer has significant biological activities and it has two rare monosaccharide moieties with very significant value namely fucose and uronic acid that require further adaptation for practical and industrial application.

Structural studies of the exopolysaccharide from Lactobacillus plantarum C88 using NMR spectroscopy and the program CASPER

Some lactic acid bacteria, such as those of the Lactobacillus genus, have the ability to produce exopolysaccharides (EPSs) that confer favorable physicochemical properties to food and/or beneficial physiological effects on human health. In particular, the EPS of Lactobacillus plantarum C88 has recently demonstrated in vitro antioxidant activity and, herein, its structure has been investigated using NMR spectroscopy and the computer program CASPER (Computer Assisted Spectrum Evaluation of Regular polysaccharides). The pentasaccharide repeating unit of the O-deacetylated EPS consists of a trisaccharide backbone, →4)-α-d-Galp-(1→2)-α-d-Glcp-(1→3)-β-d-Glcp-(1→, with terminal d-Glc and d-Gal residues (1.0 and 0.8equiv per repeating unit, respectively) extending from O3 and O6, respectively, of the →4)-α-d-Galp-(1→ residue. In the native EPS an O-acetyl group is present, 0.85equiv per repeating unit, at O2 of the α-linked galactose residue; thus the repeating unit of the EPS has the following structure: →4)[β-d-Glcp-(1→3)][β-d-Galp-(1→6)]α-d-Galp2Ac-(1→2)-α-d-Glcp-(1→3)-β-d-Glcp-(1→. These structural features, and the chain length (∼103 repeating units on average, determined in a previous study), are expected to play an important role in defining the physicochemical properties of the polymer.

The Disassociation of Cordyceps Exopolysaccharide in Aqueous Solution Induced by Ultrasound

The self-aggregation of polysaccharides (PS) in solution has led to high viscosity, unstable biological activity and poor resolution in instrumental characterization as well. In this work, ultrasound with low power level was applied to disrupt the PS aggregation of an exopolysaccharide(EPS) from Cordyceps sinensis mycelial culture. Ultrasonic irradiation with 20% amplitude (about 4 W) for 0.5 min decreased the specific viscosity of EPS by 59%. Dynamic light scattering(DLS) experiments on native EPS showed four distinct population with diameter range of 10~500 nm, 500~2500 nm, 3500~12300 nm and 22150~115400 nm respectively. The peaks at large size range in the intensity-weighted size distribution disappeared or shifted to small size range, indicating a gradual disassociation of EPS with the prolongation of ultrasonic duration. AFM observation proved the disassociation of EPS from a highly across-linked gel into single strand under ultrasonic treatment. According to DLS analysis, ultrasonic treated EPS did not show any sign of aggregation over 8 days in contrast to an immediate aggregation growth with native EPS. Ultrasonic irradiation control PS association by means of weakening the hydrogen bonds through the hydrodynamic shear forces arising from acoustic cavitation in the irradiated liquid, which has importance in the applications and structural characterization of PS.

Direct visualization of the interaction between pilin and exopolysaccharides of Myxococcus xanthus with eGFP-fused PilA protein

Type IV pili (TFP) and exopolysaccharides (EPS) are important components for social behaviors in Myxococcus xanthus, including gliding motility and fruiting body formation. Although specific interactions between TFP and EPS have been proposed, there have as yet been no direct observations of these interactions under native conditions. In this study, we found that a truncated PilA protein (PilACt) containing only the C-terminal domain (amino acids 32-208) is sufficient for EPS binding in vitro. Furthermore, an enhanced green fluorescent protein (eGFP) and PilACt fusion protein were constructed and used to label the native EPS in M. xanthus. Under confocal laser scanning microscope, the eGFP-PilACt-bound fruiting bodies, trail structures and biofilms exhibited similar patterns as the wheat germ agglutinin lectin-labeled EPS structures. This study showed that eGFP-PilACt fusion protein was able efficiently to label the EPS of M. xanthus, providing evidence for the first time of the direct interaction between the PilA protein and EPS under native conditions.

Ultrasonic depolymerization of an exopolysaccharide produced by a bacterium isolated from a deep-sea hydrothermal vent polychaete annelid

Low frequency ultrasound was used to depolymerize a high-molecular-weight exopolysaccharide (EPS) produced by a deep-sea hydrothermal bacterium Alteromonas macleodii subsp. fijiensis biovar deepsane. The influence of several parameters was examined including the duration of ultrasonic irradiation, EPS concentration, reaction temperature and volume of the sonicated solution. With the aim of optimizing the depolymerization, the native EPS was simultaneously treated with hydrogen peroxide and ultrasound. This study identified the sonication conditions that produce low-molecular-weight derivatives from the native EPS (>10 6 Da) with good reproducibility.

Structural characterisation of a perdeuteriomethylated exopolysaccharide by NMR spectroscopy: characterisation of the novel exopolysaccharide produced by Lactobacillus delbrueckii subsp. bulgaricus EU23

The exopolysaccharide (EPS) from Lactobacillus delbrueckii subsp. bulgaricus EU23 was perdeuteriomethylated and the perdeuteriomethylated EPS (pdm-EPS) purified by elution from a C 18 Sep-Pak cartridge. Both 1D and 2D NMR spectra were recorded for the pdm-EPS and these were interpreted to provide assignments for the individual 1H and 13C resonances of the sugar residues of the repeating unit. Using a combination of the results from monomer analysis and linkage analysis of the native EPS and the ROESY and HMBC NMR spectra of the pdm-EPS the following structure has been determined for the repeating unit: A process for characterising polysaccharides having low solubility in aqueous solution is reported.

Structure determination of the exopolysaccharide produced by Lactobacillus rhamnosus strains RW-9595M and R.

Exopolysaccharides (EPSs) were isolated and purified from Lactobacillus rhamnosus strains RW-9595M, which has been shown to possess cytokine-stimulating activity, and R grown under various fermentation conditions (carbon source, incubation temperature and duration). Identical (1)H NMR spectra were obtained in all cases. Molecular masses were determined by gel permeation chromatography. The primary structure was elucidated using chemical and spectroscopic techniques. Organic acid, monosaccharide and absolute configuration analyses gave the following composition: pyruvate, 1; D-glucose, 2; D-galactose, 1; and l-rhamnose, 4. Methylation analysis indicated the presence of three residues of 3-linked rhamnose, and one residue each of 2,3-linked rhamnose, 2-linked glucose, 3-linked glucose and 4,6-linked galactose. The EPS was submitted to periodate oxidation followed by borohydride reduction. Monosaccharide analysis of the resulting polysaccharide gave the new composition: rhamnose, 4; and glucose, 1. Methylation analysis confirmed the loss of the 2-linked glucose and 4,6-linked galactose residues. On the basis of one- and two-dimensional (1)H and (13)C NMR data, the structure of the native EPS was consistent with the following heptasaccharide repeating unit: [3Rha alpha-3Glc beta-3[Gal4,6(R)Py alpha-2]Rha alpha-3Rha alpha-3Rha alpha-2Glc alpha-](n) where Rha corresponds to rhamnose (6-deoxymannose) and Py corresponds to pyruvate acetal. Complete (1)H and (13)C assignments are reported for the native and the corresponding pyruvate-hydrolysed polysaccharide. Electrospray MS and MS/MS data are given for the oligosaccharide produced by Smith degradation.

Structure determination of the exopolysaccharide produced by Lactobacillus rhamnosus strains RW-9595M and R

Exopolysaccharides (EPSs) were isolated and purified from Lactobacillusrhamnosus strains RW-9595M, which has been shown to possess cytokine-stimulating activity, and R grown under various fermentation conditions (carbon source, incubation temperature and duration). Identical 1H NMR spectra were obtained in all cases. Molecular masses were determined by gel permeation chromatography. The primary structure was elucidated using chemical and spectroscopic techniques. Organic acid, monosaccharide and absolute configuration analyses gave the following composition: pyruvate, 1; d-glucose, 2; d-galactose, 1; and l-rhamnose, 4. Methylation analysis indicated the presence of three residues of 3-linked rhamnose, and one residue each of 2,3-linked rhamnose, 2-linked glucose, 3-linked glucose and 4,6-linked galactose. The EPS was submitted to periodate oxidation followed by borohydride reduction. Monosaccharide analysis of the resulting polysaccharide gave the new composition: rhamnose, 4; and glucose, 1. Methylation analysis confirmed the loss of the 2-linked glucose and 4,6-linked galactose residues. On the basis of one- and two-dimensional 1H and 13C NMR data, the structure of the native EPS was consistent with the following heptasaccharide repeating unit: {3Rhaα-3Glcβ-3[Gal4,6(R)Pyα-2]Rhaα-3Rhaα-3Rhaα-2Glcα-}n where Rha corresponds to rhamnose (6-deoxymannose) and Py corresponds to pyruvate acetal. Complete 1H and 13C assignments are reported for the native and the corresponding pyruvate-hydrolysed polysaccharide. Electrospray MS and MS/MS data are given for the oligosaccharide produced by Smith degradation.

Characterization, chemical modifications and in vitro anticoagulant properties of an exopolysaccharide produced by Alteromonas infernus

A new low-molecular-weight ‘heparin-like’ component was obtained from an exopolysaccharide produced by a mesophilic strain found in deep-sea hydrothermal vents. Data concerning the structure of the native high-molecular-weight exopolysaccharide (10 6 g/mol, 10% sulfate content) are reported for the first time. Two depolymerization processes were used to obtain low-molecular-weight (24–35×10 3 g/mol) oversulfated fractions (sulfate content 20 or 40%). Nuclear magnetic resonance studies indicated that after sulfation (40%), the low-molecular-weight fraction obtained by free radical depolymerization was less sulfated in the 6- O-position than the fraction depolymerized by acid hydrolysis. The free radical depolymerized product also had sulfated residues in the 4- O-position and disulfated ones in the 2,3- O-positions. Moreover, the compounds generated by the free radical process were more homogeneous with respect to molecular mass. Also for the first time, the anticoagulant activity of the low-molecular-weight exopolysaccharide fractions is reported. When the fractions obtained after sulfation and depolymerization were compared with heparins, anticoagulant activity was detected in oversulfated fractions, but not in native exopolysaccharide. The free radical depolymerized fraction inhibited thrombin generation in both contact-activated and thromboplastin-activated plasma, showing a prolonged lag phase only in the contact-activated assay. Affinity co-electrophoresis studies suggested that a single population of polysaccharide chains binds to antithrombin and that only a subpopulation strongly interacts with heparin cofactor II.

Effects of structural modifications on some physical characteristics of exopolysaccharides from Lactococcus lactis.

The relationship between the primary structure and the chain stiffness of exopolysaccharides (EPSs) and modified EPSs produced by two strains of Lactococcus lactis subsp. cremoris was investigated. The molar mass and radius of gyration of these exopolysaccharides were analyzed by multiangle static light scattering after size-exclusion chromatography. From these results and the chemical structure of the repeating units of the investigated EPSs, the Kuhn lengths could be calculated. We found that the initial Kuhn lengths of the two native EPSs are similar. Modification of the EPSs by removing parts of the side groups resulted in a decrease in both the absolute value and the normalized value of the Kuhn length. It is therefore concluded that partial removal of the side groups of these polysaccharides could make them less efficient as thickeners if no specific interaction with other components occurs.