High Molecular Weight Exopolysaccharide Research Articles

The activity of hydrolytic enzymes and antibiotics against biofilms of bacteria isolated from industrial-scale cooling towers

BackgroundCooling towers (CTs) are crucial to myriad industrial processes, supporting thermal exchange between fluids in heat exchangers using water from lakes and rivers as coolant. However, CT water can sometimes introduce microbial contaminants that adhere to and colonize various surfaces within the CT system. These microorganisms can form biofilms, significantly hindering the system’s thermal exchange efficiency. Current treatment strategies employ oxidizing biocides to prevent microbial growth. However, despite their affordability, they do not eliminate biofilms effectively and can lead to corrosive damage within the system. Herein, we aim to devise an anti-biofilm strategy utilizing hydrolytic enzymes (such as α-amylase, glucoamylase, pectin-lyase, cellulase, protease, and DNase) alongside antibiotics (including meropenem, ciprofloxacin, gentamicin, erythromycin, chloramphenicol, and ceftriaxone) to combat microbial growth and biofilm formation in cooling systems.ResultsAll enzymes reduced the development of the biofilms significantly compared to controls (p < 0.05). The polysaccharidases exhibited biomass reduction of 90%, except for pectin-lyase (80%), followed by DNAse and protease at 43% and 49%, respectively. The antibiotics reduced the biofilms of 70% of isolates in concentration of > 2 mg/mL. The minimal biofilm eradication concentration (MBEC) lower than 1 mg/mL was detected for some 7-day-old sessile isolates. The enzymes and antibiotics were also used in combination against biofilms using the modified Chequerboard method. We found six synergistic combinations, with Fractional inhibitory concentrations (FIC) < 0.5, out of the ten tested. In the presence of the enzymatic mixture, MBECs presented a significant decrease (p < 0.05), at least 4-fold for antibiotics and 32-fold for enzymes. Moreover, we characterized high molecular weight (> 12 kDa) exopolysaccharides (EPS) from biofilms of ten isolates, and glycosyl composition analysis indicated a high frequency of glucose, mannose, erythrose, arabinose, and idose across isolates EPS contrasting with rhamnose, allose, and those carbohydrates, which were detected in only one isolate.ConclusionThe synergistic approach of combining enzymes with antibiotics emerges as a highly effective and innovative strategy for anti-biofilm intervention, highlighting its potential to enhance biofilm management practices.

Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1 eps gene knockout mutants reduced exopolysaccharide synthesis and immunomodulatory activities

In this study, eps genes in Cluster I of Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1 were deleted via conjugal transformation to understand the role of key eps genes in exopolysaccharide (EPS) synthesis and their immunomodulatory effects. Eps gene knockout mutants of OLL1073R-1, ΔepsIB, ΔepsIC, ΔepsID, ΔepsIE, and ΔepsIF were isolated by homologous recombination with the conjugative plasmid pGMβ1. Significant reductions in high molecular weight EPS (H-EPS) production were observed in ΔepsIB, ΔepsIC, and ΔepsIE and partly in ΔepsIF, but not in ΔepsID fermented milk. H-EPS purified from OLL1073R-1 fermented milk showed significant induction of IL-6, IL-10 and IL-12, whereas low molecular weight EPS (L-EPS) showed significant induction of IL-10 and IL-12 in THP-1 dendritic cells. ΔepsIE with less production of H-EPS exhibited significant reductions in IL-6, IL-10 and IL-12 productions. The present study firstly reported that H-EPS in OLL1073R-1 contributes to IL-6, IL-10 and IL-12 production in THP-1 dendritic cells.

Rheological properties and thickening effect of high molecular weight exopolysaccharide and intracellular polysaccharide from Schizophyllum commune

Edible and medicinal fungal polysaccharides with excellent biological activity and high viscosity are potential thickeners for food applications. In this study, the rheological properties of aqueous solutions of exopolysaccharide or fermentation broth polysaccharide (SCFP) and intracellular polysaccharide or mycelial polysaccharide (SCMP) obtained from liquid submerged fermentation of the fungus Schizophyllum commune (S. commune), and their thickening effects on coffee and fruit tea were investigated. The polysaccharide aqueous solutions behaved as pseudoplastic fluids, and their viscosity was concentration-dependent but not significantly affected by temperature (10–70 °C). The critical concentrations (c*) of SCFP and SCMP were 0.275 g/L and 0.356 g/L, respectively. The intensity of the thixotropic properties of the polysaccharide aqueous solutions was positively correlated with polysaccharide concentration. Both polysaccharide solutions exhibited solid-like viscoelastic behavior and formed weak gels, with enhanced gel strength at high concentrations and low temperatures. The viscosity at a shear rate of 50 s−1 (η50) of the 5 g/L SCFP- and SCMP-thickened coffee was 0.07 Pa·s, while that of SCFP- and SCMP-thickened fruit tea was 0.1 Pa·s and 0.08Pa·s, respectively, which were significantly higher than that of polysaccharide aqueous solutions (0.05 Pa·s for SCFP and SCMP). The yield stress values increased with increasing polysaccharide concentration, suggesting a potential role of for these polysaccharides in dysphagia management. Overall, SCFP and SCMP have significant potential for application in the food industry.

Rheological and emulsifying properties of an exopolysaccharide produced by potential probiotic Leuconostoc citreum-BMS strain

EPS-BMS, is to our knowledge, the first high molecular weight exopolysaccharide from potential probiotic Leuconostoc citreum-BMS strain that consists on a mixture of α-(1,6)-dextran branched at the third position and β-(2,6)-levan. This sample exhibited interesting rheological and emulsifying properties under different conditions. Steady shear experiments proved that EPS-BMS had a pseudoplastic behavior without thixotropic properties. Interestingly, pseudoplasticity was maintained even under stress conditions of temperature, pH and salts, which could provide some sensory properties for food products such as mouth feel. Dynamic oscillatory measurements reflected a liquid-like behavior of the sample regardless of the studied EPS concentration, pH, temperature and ionic force. Results related to the emulsifying as well as interfacial properties showed that EPS-BMS had great potential to be applied as emulsifier and/or emulsion stabilizer in both neutral and acidic conditions. Based on the properties reported in this work, EPS-BMS could be potentially applied in the food industry.

Exopolysaccharide Producing Bifidobacterium animalis subsp. lactis Strains Modify the Intestinal Microbiota and the Plasmatic Cytokine Levels of BALB/c Mice According to the Type of Polymer Synthesized.

Bacteria-host interactions are mediated by different microbial associated molecular patterns which are most often surface structures such as, among others, exopolysaccharides (EPSs). In this work, the capability of two isogenic EPS-producing Bifidobacterium animalis subsp. lactis strains to modulate the gut microbiota of healthy mice, was assessed. Each strain produces a different type of polymer; the ropy strain S89L synthesized a rhamnose-rich, high-molecular weight EPS in highest abundance than the non-ropy DMS10140 one. BALB/c mice were orally fed for 10 days with milk-bifidobacterial suspensions and followed afterward for 7 post-intervention days (wash-out period). The colonic content of mice was collected in several sampling points to perform a metataxonomic analysis. In addition, the influence of specific microbial clades, apparently stimulated by the ropy and non-ropy strains, on mouse plasmatic cytokine levels was investigated through hierarchical association testing. Analysis of 16S rRNA gene sequences showed that the abundance of Firmicutes phylum significantly increased 7 days after cessing the treatment with both strains. The relative abundance of Alloprevotella genus also rose, but after shorter post-treatment times (3 days for both DMS10140 and S89L strains). Some bacterial clades were specifically modulated by one or another strain. As such, the non-ropy DMS10140 strain exerted a significant influence on Intestinomonas genus, which increased after 4 post-administration days. On the other hand, feeding with the ropy strain S89L led to an increase in sequences of Faecalibaculum genus at 4 post-treatment days, while the abundance of Erysipelotrichaceae and Lactobacillaceae families increased for prolonged times. Association testing revealed that several lactobacilli and bifidobacterial significantly stimulated by ropy S89L strain were positively associated with the levels of certain cytokines, including IL-5 and IL-27. These results highlight relevant changes in mice gut microbiota produced after administration of the ropy S89L strain that were associated to a potential immune modulation effect.

A high-molecular weight exopolysaccharide from the Cs-HK1 fungus: Ultrasonic degradation, characterization and in vitro fecal fermentation

This work was to examine the impact of power ultrasound (US) on the molecular properties of a high-molecular weight (MW) exopolysaccharide (EPS) from the Cs-HK1 medicinal fungus and the utilization, and prebiotic function of the US-treated EPS fractions in human fecal microflora in vitro. The US treatment caused notable reduction of intrinsic viscosity, average MW and aggregate size of EPS in water but no significant changes in the molecular structure. The US-treated EPS fractions were consumed more rapidly by the fecal microflora, resulting in a higher total level of short chain fatty acids. They also affected the relative abundance in the microflora more beneficially than the original EPS. The results suggest that power US is effective for modifying and improving the prebiotic properties of high-MW polysaccharides.

Protective effects of exopolysaccharide of a medicinal fungus on probiotic bacteria during cold storage and simulated gastrointestinal conditions

The efficacy of probiotic health products depends on the capability of the constituent probiotic bacteria to survive through long period of cold storage and the gastrointestinal tract. This study was to evaluate the protective effects of a high-molecular weight (MW) exopolysaccharide (EPS) from a medicinal fungus Cs-HK1 on three different bifidobacteria. The EPS had a total dietary fiber content about 70% (w/w), which was close to its total carbohydrate content. It was resistant to artificial gastric acid (pH 2) with no more than 4% (w/w) hydrolysis in 6 h. EPS at 5 g/L significantly increased the survival rate of the probiotic bacteria during cold storage (4 °C) and in simulated gastric acid, reducing the death rate of different bacterial strains by 50% to 70%. The protective effect of EPS was weaker when the concentration was decreased to 3 g/L or when the MW of EPS was reduced by partial degradation with power ultrasound. EPS also showed significantly protective effect on the all bacterial strains in bile juice. The results have demonstrated the potential value of Cs-HK1 EPS as a novel prebiotic fiber for the formulation of synbiotic products with probiotic bacteria.

Cross-reactivity of latex agglutination assay complicates the identification of Burkholderia pseudomallei from soil.

The monoclonal antibody-based latex agglutination tests targeting a high molecular weight exopolysaccharide antigen of Burkholderia pseudomallei are commercially available. The tests are primarily used in routine diagnosis of melioidosis in major hospitals in Thailand and some endemic countries. Being a rapid test, this technique was employed as a presumptive test to identify colonies of B. pseudomallei among many others grown from soil specimens collected from southern Thailand. Cross-reactivity of these tests with other soil bacteria was a concern since it complicated the identification of B. pseudomallei. Here, we describe the cross-reactivity of two commercial latex agglutination tests for melioidosis with B. territorii, B. pseudomultivorans, B. multivorans and B. cenocepacia isolates from soil.

Protection of Bifidobacterial cells against antibiotics by a high molecular weight exopolysaccharide of a medicinal fungus Cs-HK1 through physical interactions

This study was to assess the protective effect of exopolysaccharide (EPS) produced by a medicinal fungus Cordyceps sinensis Cs-HK1 on Bifidobacteria against antibiotic inhibition. The high-molecular weight EPS fractions showed significant protective effect on all five bifidobacterial strains against four common antibiotics, leading to a dramatic increase in the minimal inhibitory and minimal bactericidal concentrations. The protective effect of EPS on the bacteria was probably attributed to the formation of a viscous layer around the bacterial cell resisting the access by the antibiotics. The EPS layer surrounding the bacteria cell also promoted the aggregation of bacteria and formation of biofilm as observed by microscopy. EPS also enhanced the bifidobacterial adhesion to Caco-2 cell monolayer. In general, the protective effect as well as biofilm formation due to EPS was significantly correlated with the molecular weight of EPS fractions.

Exopolysaccharide Produced by Probiotic Strain Lactobacillus paraplantarum BGCG11 Reduces Inflammatory Hyperalgesia in Rats.

The aim of this study was to test the potential of high molecular weight exopolysaccharide (EPS) produced by the putative probiotic strain Lactobacillus paraplantarum BGCG11 (EPS CG11) to alleviate inflammatory pain in Wistar rats. The EPS CG11 was isolated from bacterial surface and was subjected to Fourier-transform infrared spectroscopy (FTIR) and thermal analysis. FTIR spectra confirmed the polysaccharide structure of isolated sample, while the thermal methods revealed good thermal properties of the polymer. The antihyperalgesic and antiedematous effects of the EPS CG11 were examined in the rat model of inflammation induced by carrageenan injection in hind paw. The results showed that the intraperitoneal administration of EPS CG11 produced a significant decrease in pain sensations (mechanical hyperalgesia) and a paw swelling in a dose-dependent manner as it was measured using Von Frey anesthesiometer and plethysmometer, respectively. These effects were followed by a decreased expression of IL-1β and iNOS mRNAs in rat’s paw tissue suggesting that the antihyperalgesic and antiedematous effects of the EPS CG11 are related to the suppression of inflammatory response. Additionally, we demonstrated that EPS CG11 exhibits immunosuppressive properties in the peritonitis model induced by carrageenan. Expression levels of pro-inflammatory mediators IL-1β, TNF-α and iNOS were decreased, together with the enhanced secretion of anti-inflammatory IL-10 and IL-6 cytokines, while neutrophil infiltration was not changed. To the best of our knowledge, this is the first study which reports an antihyperalgesic effect as the novel property of bacterial EPSs. Given the high demands of pharmaceutical industry for the replacement of commonly used analgesics due to numerous side effects, this study describes a promising natural compound for the future pharmacological testing in the area.

Purification of the exopolysaccharide produced by Alteromonas infernus: identification of endotoxins and effective process to remove them.

Alteromonas infernus bacterium isolated from deep-sea hydrothermal vents can produce by fermentation a high molecular weight exopolysaccharide (EPS) called GY785. This EPS described as a new source of glycosaminoglycan-like molecule presents a great potential for pharmaceutical and biotechnological applications. However, this unusual EPS is secreted by a Gram-negative bacterium and can be therefore contaminated by endotoxins, in particular the lipopolysaccharides (LPS). Biochemical and chemical analyses of the LPS extracted from A. infernus membranes have shown the lack of the typical LPS architecture since 3-deoxy-D-manno-oct-2-ulopyranosonic acid (Kdo), glucosamine (GlcN), and phosphorylated monosaccharides were not present. Unlike for other Gram-negative bacteria, the results revealed that the outer membrane of A. infernus bacterium is most likely composed of peculiar glycolipids. Furthermore, the presence of these glycolipids was also detected in the EPS batches produced by fermentation. Different purification and chemical detoxification methods were evaluated to efficiently purify the EPS. Only the method based on a differential solubility of EPS and glycolipids in deoxycholate detergent showed the highest decrease in the endotoxin content. In contrast to the other tested methods, this new protocol can provide an effective method for obtaining endotoxin-free EPS without any important modification of its molecular weight, monosaccharide composition, and sulfate content.

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.

Characterization of new exopolysaccharide production by Rhizobium tropici during growth on hydrocarbon substrate

Exopolysaccharide (EPS) are produced by a diverse of rhizobia species and has been demonstrated to be a bioemulsifier with potential applications in the degradation of hydrocarbons. In the present study, attempts were made to obtain the new exopolysaccharide production by Rhizobium tropici (SEMIA 4080 and MUTZC3) strains during growth on hydrocarbon substrate. Under the different cultivation conditions, the high molecular weight exopolysaccharides from Rhizobium tropici strains cultivated for 96h mainly consisted of carbohydrates (79–85%) and a low percentage of protein. The EPSC3-D differed from the others, with only 60% of carbohydrate. However, all strains produced polymers with distinct rheology properties, such as viscosity of each EPS sample, suitable for different applications. In addition, RP-HPLC, FTIR and NMR studies revealed EPS produced by rhizobia strains were similar indicating minimal difference between EPS compositions.

Selection of EPS-producing Lactobacillus strains isolated from kefir grains and rheological characterization of the fermented milks

In this study the ability to produce exopolysaccharides during growth in milk of 28 Lactobacillus, previously isolated from kefir grain, was investigated and the rheological properties of the obtained fermented milks were also studied. During fermentation all microorganisms were able to produce polysaccharide with final concentration ranging from 20 mg L−1 to 370 mg L−1. Lactobacillus kefiranofaciens and Lactobacillus plantarum strains produced oligosaccharide with low degree of polymerization. The exopolysaccharides produced by the five Lactobacillus paracasei strains presented also a high molecular weight fraction. In consequence the acid milk gels obtained by fermentation with all the L. paracasei strains were the ones that presented the highest viscosities. Nevertheless the viscolestic characteristics of the resulting acid gels were different. L. paracasei CIDCA 83123 produces a milk having a viscous behavior whereas fermented milks with L. paracasei CIDCA 8339, CIDCA 83120, CIDCA 83121 and CIDCA 83124 has gel structure.The five L. paracasei strains isolated from kefir studied in the present work were the first described that produce high molecular weight exopolysaccharides. Since production of high molecular weight exopolysaccharides affects the viscosity of fermented milk these strains could have a successful application improving texture of fermented dairy product.

Characterization and antibacterial activity of silver nanoparticles prepared with a fungal exopolysaccharide in water

This work demonstrates a simple and feasible approach for the synthesis of silver nanoparticles (AgNPs) in water with a high molecular weight (MW) exopolysaccharide fraction, EPS1 produced by a medicinal fungus Cs-HK1. The formation and properties of AgNPs were evaluated at various temperatures, time periods, and silver nitrate/EPS1 concentrations in water. At suitable conditions (100 °C, 60 min and 10 mM AgNO3 with 1.0 mg/mL EPS1), AgNPs were formed with an average diameter of 50 nm and a narrow size distribution, remaining as a stable dispersion for at least 2 months. EPS1 may be acting as a reducing and stabilizing agent for the formation of AgNPs, which were attached to the hydroxyl groups of EPS1. The AgNPs formed in EPS1 solution exhibited a concentration-dependent inhibition of both Gram-negative and -positive bacteria but a very low cytotoxicity on the RAW264.7 murine macrophage cells. The results demonstrated the feasibility for green synthesis of AgNPs as potential antibacterial agents using natural polysaccharides dissolved in water.

Purification and Characterization of Biofilm-Associated EPS Exopolysaccharides from ESKAPE Organisms and Other Pathogens

In bacterial biofilms, high molecular weight, secreted exopolysaccharides can serve as a scaffold to which additional carbohydrates, proteins, lipids, and nucleic acids adhere, forming the matrix of the developing biofilm. Here we report methods to extract and purify high molecular weight (>15 kDa) exopolysaccharides from biofilms of eight human pathogens, including species of Staphylcococcus, Klebsiella, Acinetobacter, Pseudomonas, and a toxigenic strain of Escherichia coli O157:H7. Glycosyl composition analysis indicated a high total mannose content across all strains with P. aeruginosa and A. baumannii exopolysaccharides comprised of 80–90% mannose, K. pneumoniae and S. epidermidis strains containing 40–50% mannose, and E. coli with ∼10% mannose. Galactose and glucose were also present in all eight strains, usually as the second and third most abundant carbohydrates. N-acetyl-glucosamine and galacturonic acid were found in 6 of 8 strains, while arabinose, fucose, rhamnose, and xylose were found in 5 of 8 strains. For linkage analysis, 33 distinct residue-linkage combinations were detected with the most abundant being mannose-linked moieties, in line with the composition analysis. The exopolysaccharides of two P. aeruginosa strains analyzed were consistent with the Psl carbohydrate, but not Pel or alginate. The S. epidermidis strain had a composition rich in mannose and glucose, which is consistent with the previously described slime associated antigen (SAA) and the extracellular slime substance (ESS), respectively, but no polysaccharide intracellular adhesion (PIA) was detected. The high molecular weight exopolysaccharides from E. coli, K. pneumoniae, and A. baumannii appear to be novel, based on composition and/or ratio analysis of carbohydrates.

Thermal inactivation reaction rates for ricin are influenced by pH and carbohydrates

Ricin is a lethal protein toxin produced by the castor bean plant. Ricin is known to possess significant heat resistance. Therefore, we placed it in a variety of foods to study the influence of the food matrix on behavior of a thermally stable protein toxin. First order rate constants for the thermal inactivation of ricin in foods and simple buffers were measured using cytotoxicity assays. We observed greater thermal stability at 75 °C for the cytotoxic activity of ricin when it was placed in a yogurt-containing fruit drink compared to its stability when placed in the other foods tested. We found that galactose and high molecular weight exopolysaccharides present in various dairy products contributed to the thermal stability of ricin. Differential scanning calorimetry also showed enhanced thermal stability for ricin at pH 4.5. Our results demonstrate the importance of considering pH and the presence of stabilizing ligands in the thermal inactivation of protein toxins in foods.

Conditional Requirement for Exopolysaccharide in the Mesorhizobium–Lotus Symbiosis

Rhizobial surface polysaccharides are required for nodule formation on the roots of at least some legumes but the mechanisms by which they act are yet to be determined. As a first step to investigate the function of exopolysaccharide (EPS) in the formation of determinate nodules, we isolated Mesorhizobium loti mutants affected in various steps of EPS biosynthesis and characterized their symbiotic phenotypes on two Lotus spp. The wild-type M. loti R7A produced both high molecular weight EPS and lower molecular weight (LMW) polysaccharide fractions whereas most mutant strains produced only LMW fractions. Mutants affected in predicted early biosynthetic steps (e.g., exoB) formed nitrogen-fixing nodules on Lotus corniculatus and L. japonicus 'Gifu', whereas mutants affected in mid or late biosynthetic steps (e.g., exoU) induced uninfected nodule primordia and, occasionally, a few infected nodules following a lengthy delay. These mutants were disrupted at the stage of infection thread (IT) development. Symbiotically defective EPS and Nod factor mutants functionally complemented each other in co-inoculation experiments. The majority of full-length IT observed harbored only the EPS mutant strain and did not show bacterial release, whereas the nitrogen-fixing nodules contained both mutants. Examination of the symbiotic proficiency of the exoU mutant on various L. japonicus ecotypes revealed that both host and environmental factors were linked to the requirement for EPS. These results reveal a complex function for M. loti EPS in determinate nodule formation and suggest that EPS plays a signaling role at the stages of both IT initiation and bacterial release.

Structure characterization of a fucose-containing exopolysaccharide produced by Enterobacter cloacae Z0206

A novel high molecular weight (1.1×106Da) exopolysaccharide (EPS) produced by Enterobacter cloacae Z0206 strain was isolated by column chromatography. Complete hydrolysis of the EPS followed by gas chromatography mass spectrometry (GC–MS) and high performance liquid chromatography (HPLC) analyses showed that the EPS is composed of l-fucose, d-glucose, d-galactose, d-glucuronic acid and pyruvic acid in the approximate molar ratio of 2:1:3:1:1. Partial acid hydrolysis of the purified EPS followed by gel permeation chromatography (GPC) yielded a hexasaccharide. A combination of chemical analysis coupled with mass spectrometry and 1D and 2D NMR spectroscopy applied to the oligosaccharide showed that the EPS comprises a heptasaccharide repeating unit.

Membrane Topology and Functional Analysis ofMethylobacillussp. 12S GenesepsFandepsG, Encoding Polysaccharide Chain-Length Determining Proteins

The EpsF and EpsG of the methanol-assimilating bacterium Methylobacillus sp. 12S are involved in the synthesis of a high molecular weight exopolysaccharide, methanolan. These proteins share homology with chain-length determiners in other polysaccharide-producing bacteria. The N- and C-termini of EpsF were found to locate to the cytoplasm, and EpsF was predicted to have two transmembrane regions. EpsG showed both ATPase and autophosphorylation activities.