Water-insoluble Polysaccharides Research Articles

Structural analysis and antioxidative properties of mutan (water-insoluble glucan) and carboxymethyl mutan from Streptococcus mutans

Streptococcus mutans (MTCC 497) was grown anaerobically in acidic Brain heart infusion (BHI) medium with 15 % sucrose to produce cell-bound and extracellular water-insoluble polysaccharide mutan. Fourier transformed infrared (FTIR) and 13C NMR studies revealed a mixed linkage of α-1−3 and α-1–6 mutan with a production yield of 1.8 g/L. Mutan has a branched structure with a molecular weight (Mw) of 5654 Da. Water-insoluble mutan was carboxymethylated at 0.93 degrees of substitution. FTIR spectra with characteristic peaks at 1603 cm−1 and 1418 cm−1 due to symmetric and asymmetric vibrations of the COO- group confirmed carboxymethylation. Thermal gravimetric analysis showed that native mutan and carboxymethyl mutan exhibited higher thermal stability. Carboxymethylation enhanced solubility and antioxidative radical-scavenging activity. The in-vitro antioxidative radical scavenging analysis revealed 52 % and 47 % inhibition of DPPH and ABTS radicals.

Hindering of Cariogenic Streptococcus mutans Biofilm by Fatty Acid Array Derived from an Endophytic Arthrographis kalrae Strain.

Streptococcus mutans has been considered as the major etiological agent of dental caries, mostly due to its arsenal of virulence factors, including strong biofilm formation, exopolysaccharides production, and high acid production. Here, we present the antivirulence activity of fatty acids derived from the endophytic fungus Arthrographis kalrae isolated from Coriandrum sativum against Streptococcus mutans. The chemical composition of the fatty acids was analyzed by gas chromatography–mass spectrometry GC-MS and revealed nine compounds representing 99.6% of fatty acids, where unsaturated and saturated fatty acids formed 93.8% and 5.8 % respectively. Oleic and linoleic acids were the major unsaturated fatty acids. Noteworthy, the fatty acids at the concentration of 31.3 mg L–1 completely inhibited Streptococcus mutans biofilm, and water insoluble extracellular polysaccharide production in both polystyrene plates, and tooth model assay using saliva-coated hydroxyapatite discs. Inhibition of biofilm correlated significantly and positively with the inhibition of water insoluble extracellular polysaccharide (R = 1, p < 0.0001). Furthermore, Arthrographis kalrae fatty acids at a concentration of 7.8 mg L–1 exhibited acidogenesis-mitigation activity. They did not show bactericidal activity against Streptococcus mutans and cytotoxic activity against human oral fibroblast cells at the concentration used. On the other hand, saliva-coated hydroxyapatite discs treated with sub-minimum biofilm inhibitory concentration of fatty acids showed disturbed biofilm architecture with a few unequally distributed clumped matrices using fluorescence microscopy. Our findings revealed that the intracellular fatty acid arrays derived from endophytic Arthrographis kalrae could contribute to the biofilm-preventing alternatives, specifically Streptococcus mutans biofilms.

Effects of resveratrol on cariogenic virulence properties of Streptococcus mutans

BackgroundStreptococcus mutans is the principal etiological agent of human dental caries. The major virulence factors of S. mutans are acid production, acid tolerance, extracellular polysaccharide (EPS) synthesis and biofilm formation. The aim of this study is to evaluate the effect of resveratrol, a natural compound, on virulence properties of S. mutans.ResultsResveratrol at sub-MIC levels significantly decreased acid production and acid tolerance, inhibited synthesis of water-soluble polysaccharide and water-insoluble polysaccharide, compromised biofilm formation. Related virulence gene expression (ldh, relA, gtfC, comDE) was down-regulated with increasing concentrations of resveratrol.ConclusionsResveratrol has an inhibitory effect on S. mutans cariogenic virulence properties and it represents a promising anticariogenic agent.

Burkholderia cenocepacia H111 Produces a Water-Insoluble Exopolysaccharide in Biofilm: Structural Determination and Molecular Modelling.

Biofilms are a multicellular way of life, where bacterial cells are close together and embedded in a hydrated macromolecular matrix which offers a number of advantages to the cells. Extracellular polysaccharides play an important role in matrix setup and maintenance. A water-insoluble polysaccharide was isolated and purified from the biofilm produced by Burkholderia cenocepacia strain H111, a cystic fibrosis pathogen. Its composition and glycosidic linkages were determined using Gas–Liquid Chromatography–Mass Spectrometry (GLC–MS) on appropriate carbohydrate derivatives while its complete structure was unraveled by 1D and 2D NMR spectroscopy in deuterated sodium hydroxide (NaOD) aqueous solutions. All the collected data demonstrated the following repeating unit for the water-insoluble B. cenocepacia biofilm polysaccharide: [3)-α-d-Galp-(1→3)-α-d-Glcp-(1→3)-α-d-Galp-(1→3)-α-d-Manp-(1→]n Molecular modelling was used, coupled with NMR Nuclear Overhauser Effect (NOE) data, to obtain information about local structural motifs which could give hints about the polysaccharide insolubility. Both modelling and NMR data pointed at restricted dynamics of local conformations which were ascribed to the presence of inter-residue hydrogen bonds and to steric restrictions. In addition, the good correlation between NOE data and calculated interatomic distances by molecular dynamics simulations validated potential energy functions used for calculations.

Isolation and subunit compositions of the xylanosome complexes produced by Cellulosimicrobium species

Cellulosimicrobium cellulans, which is type species of the genus Cellulosimicrobium, produces xylanase predominant nanoscale multienzyme complexes, i.e., xylanosomes, when grown on water-insoluble polysaccharides. Here, we report on the isolation of similar multienzyme complexes (MECs) produced by two other species in genus Cellulosimicrobium (Cellulosimicrobium funkei and Cellulosimicrobium terreum). Functional studies and subunit structure identifications using genomic sequencing and proteomic techniques were also performed. When compared with the xylanosomes produced by C. cellulans F16, the isolated MECs showed a larger particle size and shared at least three conserved multidomain proteins. In addition, they also exhibited different enzymatic activities and subunit compositions, which indicates diverse capability and strategies in degrading hemicelluloses.

Effects of Different Carbon Sources on Enzyme Production and Ultrastructure of Cellulosimicrobium cellulans.

The secretomes of the strain Cellulosimicrobium cellulans F16 grown on different carbon sources were analyzed by zymography, and the subcellular surface structures were extensively studied by electron microscope. The exo-cellulase and xylanase systems were sparse when cells were grown on soluble oligosaccharides, but were significantly increased when grown on complex and water-insoluble polysaccharides, such as Avicel, corn cob, and birchwood xylan. The cellulosome-like protuberant structures were clearly observed on the cell surfaces of strain F16 grown on cellulose, with diameters of 15-20nm. Fibrous structures that connected the adjacent cells can be seen under microscope. Moreover, protuberances that adsorbed the cell to cellulose were also observed. As the adhesion of Cellulosimicrobium cellulans cells onto cellulose surfaces occurs via thick bacterial curdlan-type exopolysaccharides (EPS), such surface layer is potentially important in the digestion of insoluble substrates such as cellulose or hemicellulose, and the previously reported xylanosomes are part of such complex glycocalyx layer on the surface of the bacterial cell.

Identification of DR75-RS11090 Gene Encoding GGDEF Domains, and its Role in Enterococcus faecalis

Background: Enterococcus faecalis is considered the primary pathogenic bacteria in failed root canal therapy due to its excellent biofilm formation ability. The second messenger cyclic dimeric (3’→5’) GMP (c-di-GMP) is involved in bacterial physiological processes, including biofilm formation. C-di-GMP is formed by diguanylate cyclases with the GGDEF domain. Objectives: The current study aimed at investigating the function of DR75-RS11090 in E. faecalis. Methods: In the current experimental study, gene encoding GGDEF domains in E. faecalis were analyzed through National Center for Biotechnology Information (NCBI) database. Enterococcus faecalis strains were cultured in brain-heart-infusion (BHI) broth at 37°C. Growth curves of E. faecalis were measured in an automatic microplate reader. Biofilms were fixed with 2% glutaraldehyde and scanned under the transmission electron microscopy. Acid and water-insoluble polysaccharide productions were also detected by the precise digital acidometer and anthrone method. Results: It was found that DR75-RS11090 gene was predicted to encode GGDEF domain in E. faecalis. The current study observed promoting effects of DR75-RS11090 on E. faecalis biofilm formation, growth, acid production, and water insoluble polysaccharide production. Conclusions: The current study findings improved the understanding of c-di-GMP in E. faecalis and presented a primary study on DR75-RS11090 promoting mechanism.

An insoluble polysaccharide from the sclerotium of Poria cocos improves hyperglycemia, hyperlipidemia and hepatic steatosis in ob/ob mice via modulation of gut microbiota

Metabolic syndrome characterized by obesity, hyperglycemia and liver steatosis is becoming prevalent all over the world. Herein, a water insoluble polysaccharide (WIP) was isolated and identified from the sclerotium of Poria cocos, a widely used Traditional Chinese Medicine. WIP was confirmed to be a (1-3)-β-D-glucan with an average Mw of 4.486 × 106 Da by NMR and SEC-RI-MALLS analyses. Furthermore, oral treatment with WIP from P. cocos significantly improved glucose and lipid metabolism and alleviated hepatic steatosis in ob/ob mice. 16S DNA sequencing analysis of cecum content from WIP-treated mice indicated the increase of butyrate-producing bacteria Lachnospiracea, Clostridium. It was also observed that WIP treatment elevated the level of butyrate in gut, improved the gut mucosal integrity and activated the intestinal PPAR-γ pathway. Fecal transplantation experiments definitely confirmed the causative role of gut microbiota in mediating the benefits of WIP. It is the first report that the water insoluble polysaccharide from the sclerotium of P. cocos modulates gut microbiota to improve hyperglycemia and hyperlipidemia. Thereby, WIP from P. cocos, as a prebiotic, has the potential for the prevention or cure of metabolic diseases and may elucidate new mechanism for the efficacies of this traditional herbal medicine on the regulation of lipid and glucose metabolism.

Effect of three different compositions of topical fluoride varnishes with and without prior oral prophylaxis on Streptococcus mutans count in biofilm samples of children aged 2-8 years: A randomized controlled trial.

Various strategies for controlling caries focus on disrupting the interaction between risk factors. Of these, fluoride varnish has been shown to reduce the colony-forming (CFU) units and water-insoluble extracellular polysaccharide amount. Casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) and xylitol-containing fluoride varnishes have recently gained importance as caries-protective fluoride varnishes. This study aims to assess and compare the reduction in Streptococcus mutans count in biofilm samples after topical application of three different fluoride varnishes and to evaluate the effect of oral prophylaxis prior to fluoride varnish application. Sixty healthy children with no active caries, in the age group of 2-8 years, were randomly divided into Group A = fluoride varnish containing CPP-ACP; Group B = fluoride varnish containing xylitol; and Group C = fluoride varnish with 0.9% difluorosilane; further, the groups were divided into two subgroups, namely A1, B1, and C1 with prior oral prophylaxis and A2, B2, and C2 without oral prophylaxis. Plaque samples were collected at baseline, 1st month, and 3rd month; cultured; and incubated, and CFU/ml was calculated. Data were compiled, and CFU/ml was analyzed by independent t-test, paired t-test, and one-way ANOVA. There was no statistical difference between the fluoride groups. Furthermore, no statistically significant difference was seen between the subgroups. Fluoride varnish containing CPP-ACP showed higher reduction in S. mutans count followed by xylitol-containing fluoride varnish and Fluor Protector®. There was no effect of prior oral prophylaxis on the efficacy of fluoride varnish.

Dietary fibre in legumes, seeds, vegetables, fruits and mushrooms: Comparing traditional and semi-automated filtration techniques

The method AOAC 2011.25 was used to analyze all the dietary fibre (DF) components included in the latest definition by the Codex Alimentarius Commission (2009). The traditional filtration technique, described in the method AOAC 2011.25, was compared with a new semi-automated filtration technique using Fibertec™ 1023 system. For the comparisons, a statistical similarity approach was chosen to evaluate the results of six food matrixes (wheat flour, edible boletus, strawberry, tomato, green pea and horse radish). The total DF contents of the tested matrixes fit within 16% tightest data-induced similarity limit of the manual mean, with one exception (strawberry; 30%). Thus, it was concluded that both techniques are suitable for use with the method AOAC 2011.25, and therefore either technique was used to analyze a wide selection of legumes, seeds, vegetables, fruits and mushrooms (44 foods). Seeds were excellent sources of total DF, as well as water-insoluble (IDF) and water-soluble (SDFP) polysaccharides. A substantial amount of oligosaccharides (SDFS) was found in red onions. Generally, the DF contents were higher in this study than in earlier studies. Higher DF results can be partially explained by the more effective analytical method, and partly by changed varieties.

Extraction and characterization of cellulose and cellulose nanofibers from Citrullus colocynthis seeds

Citrullus colocynthis is a perennial herbaceous plant of the Cucurbitaceae family, cultivated in the Mediterranean basin and tropical countries as a traditional medicinal plant. In this paper, extraction of water-insoluble polysaccharides of Citrullus colocynthis seeds was performed using two chemical extraction processes. The first one involved the deproteinization of grinded Citrullus colocynthis seeds in sodium hydroxide aqueous solution, followed by lipid extraction in the chloroform/methanol solvent. The second process consisted of extracting the lipid material before deproteinization step. Several analytical techniques were used to identify cellulose as the main compound in the water-insoluble extract including ATR-FTIR and IR-Raman spectroscopy, CP/MAS 13CNMR spectroscopy, X-ray diffraction and TEM. Further, almost 75 wt% of cellulose were obtained from the water-insoluble extract after bleaching process which determines the purification efficiency of cellulose and remove lignin and hemicellulose and the final percentages for this extraction are; 23%, 23%, 40% and 13% for lipids, proteins, cellulose and lignin, respectively. Finally, cellulose nanofibers having needle structure and fibrillar network were prepared by acid hydrolysis.

Influence of Tween-80 on the production and structure of water-insoluble curdlan from Agrobacterium sp.

In order to explore the mechanism by which Tween-80 enhances the production of curdlan produced by Agrobacterium sp., the effects of Tween-80 on the production and structure of curdlan and Agrobacterium sp. were evaluated. Maximum curdlan production (51.94g/L) was achieved when 16g/L Tween-80 was added at the beginning of the cell growth stage. The addition of Tween-80 at higher concentration inhibited cell growth. However, the addition of 16g/L Tween-80 enhanced the production of curdlan with a looser ultrastructure, significantly weakened the envelopment of curdlan on Agrobacterium sp., altered the fine structure of cell membrane, and increased the cell membrane permeability. Moreover, the efficiency of oxygen and mass transport, respiration intensity, UTP regeneration, ATP regeneration, activity of curdlan synthetase, capacity of stress response and energy supply of Agrobacterium sp. were all greatly improved by the addition of Tween-80. These findings demonstrate the mechanisms by which Tween-80 enhances curdlan production and provide a cheap and feasible approach to weaken the envelopment of water-insoluble polysaccharides on bacteria.

Dextranase from Arthrobacter oxydans KQ11-1 inhibits biofilm formation by polysaccharide hydrolysis

Dental plaque is a biofilm of water-soluble and water-insoluble polysaccharides, produced primarily by Streptococcus mutans. Dextranase can inhibit biofilm formation. Here, a dextranase gene from the marine microorganism Arthrobacter oxydans KQ11-1 is described, and cloned and expressed using E. coli DH5α competent cells. The recombinant enzyme was then purified and its properties were characterized. The optimal temperature and pH were determined to be 60°C and 6.5, respectively. High-performance liquid chromatography data show that the final hydrolysis products were glucose, maltose, maltotriose, and maltotetraose. Thus, dextranase can inhibit the adhesive ability of S. mutans. The minimum biofilm inhibition and reduction concentrations (MBIC50 and MBRC50) of dextranase were 2 U ml−1 and 5 U ml−1, respectively. Scanning electron microscopy and confocal laser scanning microscope (CLSM) observations confirmed that dextranase inhibited biofilm formation and removed previously formed biofilms.

Differences in the chemical composition of Enterococcus faecalis biofilm under conditions of starvation and alkalinity

ABSTACTThis study aimed to investigate the dynamic changes that occur in the chemical composition of an Enterococcus faecalis (E. faecalis) biofilm under conditions of starvation and in an alkaline environment and to explore the function of chemical composition changes in the resistance of the E. faecalis biofilm to an extreme environment. This study established an in vitro E. faecalis biofilm model under starvation and in an alkaline environment. During the formation of the biofilm, the pH value and nutritional condition of the culture medium were changed, and the changes in chemical composition were observed using biochemical measures. The results showed that, when the pH value of the culture medium was 11, the percentage of water-insoluble polysaccharides in the biofilm was significantly lower than under other conditions. In addition, the percentage of water-soluble polysaccharides in culture medium with pH values of 9 and 11 gradually decreased. The level of the water-soluble polysaccharides in each milligram of dry weight of biofilm at pH 11 increased compared to that under other conditions. The results from this study indicate that the chemical composition of E. faecalis biofilm changed in extreme environments. These changes served as a defensive mechanism for E. faecalis against environmental pressures.

Investigation of CO2 precursors in roasted coffee.

Two CO2 formation pathways (chlorogenic acid (CGA) degradation and Maillard reaction) during coffee roasting were investigated. CGA is shown not a major contributor to CO2 formation, as heating of this compound under typical roasting conditions did not release a large quantity of CO2. However, heating of a CGA moiety, caffeic acid, resulted in high yield of CO2 (>98%), suggesting that CGA hydrolysis could be the rate limiting step for CO2 formation from CGA. A large amount of CO2 was detected from glycine-sucrose model system under coffee roasting conditions, implying the importance of Maillard reactions in CO2 formation. Further studies on the heating of various components isolated from green coffee beans showed that CO2 was generated from various green coffee components, including water insoluble proteins and polysaccharides. Around 50% of CO2 was formed from thermal reactions of lower molecular weight compounds that represent ∼25% by weight in green coffee.

Functional Relationship between Sucrose and a Cariogenic Biofilm Formation.

Sucrose is an important dietary factor in cariogenic biofilm formation and subsequent initiation of dental caries. This study investigated the functional relationships between sucrose concentration and Streptococcus mutans adherence and biofilm formation. Changes in morphological characteristics of the biofilms with increasing sucrose concentration were also evaluated. S. mutans biofilms were formed on saliva-coated hydroxyapatite discs in culture medium containing 0, 0.05, 0.1, 0.5, 1, 2, 5, 10, 20, or 40% (w/v) sucrose. The adherence (in 4-hour biofilms) and biofilm composition (in 46-hour biofilms) of the biofilms were analyzed using microbiological, biochemical, laser scanning confocal fluorescence microscopic, and scanning electron microscopic methods. To determine the relationships, 2nd order polynomial curve fitting was performed. In this study, the influence of sucrose on bacterial adhesion, biofilm composition (dry weight, bacterial counts, and water-insoluble extracellular polysaccharide (EPS) content), and acidogenicity followed a 2nd order polynomial curve with concentration dependence, and the maximum effective concentrations (MECs) of sucrose ranged from 0.45 to 2.4%. The bacterial and EPS bio-volume and thickness in the biofilms also gradually increased and then decreased as sucrose concentration increased. Furthermore, the size and shape of the micro-colonies of the biofilms depended on the sucrose concentration. Around the MECs, the micro-colonies were bigger and more homogeneous than those at 0 and 40%, and were surrounded by enough EPSs to support their structure. These results suggest that the relationship between sucrose concentration and cariogenic biofilm formation in the oral cavity could be described by a functional relationship.

Long-term anti-cariogenic biofilm activity of glass ionomers related to fluoride release

ObjectivesThe aim of this study was to evaluate the difference between anti-cariogenic biofilm activities of glass ionomers (G-Is) during the initial and second fluoride release phases and to define relationships between the anti-biofilm activities and fluoride release. MethodsFluoride release of three commercially available G-Is in a buffer was evaluated for 770h, and then 70-h-old Streptococcus mutans UA159 biofilms were formed on the G-Is that had been immersed in the buffer for 0, 100, 200, or 700h. The dry weight, bacterial cell number, water-insoluble extracellular polysaccharides (EPSs), and accumulated fluoride concentration of the 70-h-old biofilms and fluoride release and acid production rates during biofilm formation were determined. Relationships between the experimental variables and fluoride release rate were also evaluated using linear regression analysis. ResultsIn this study, fluoride release of the tested G-Is did not exhibit a biphasic pattern during biofilm formation. The release was sustained or did not rapidly decrease even over long immersion periods and was strongly correlated with an increase in accumulated fluoride concentration of the biofilms (R=0.99, R2=0.98) and reductions in dry weight, water-insoluble EPSs, and acid production rate of the biofilms (R=−0.99 to −0.96, R2=0.92–0.98). ConclusionsThis study suggests that G-Is can effectively affect acid production, EPS formation, and accumulation of cariogenic biofilms even during the second fluoride release phase, and that the anti-cariogenic biofilm activity is strongly correlated with fluoride release, which may be enhanced by acid production of cariogenic biofilms. Clinical significanceG-Is can affect cariogenic biofilm formation even during the second fluoride release phase.

Fruiting bodies of Hericium erinaceus (Bull.) Pers. – a new source of water-insoluble (1→3)-α-d-glucan

A water-insoluble polysaccharide (WIP) was isolated from the fruiting bodies of &lt;em&gt;Hericium erinaceus&lt;/em&gt; HE01 by an alkaline solution with the yield of 5%. Structural and compositional analyses by total acid hydrolysis, methylation analysis, FT-IR, FT-Raman, and &lt;sup&gt;1&lt;/sup&gt;H NMR spectroscopy as well as other instrumental techniques showed predominantly glucose linked by α-glycosidic bonds and small amounts of mannose, xylose, rhamnose, galactose, and ribose. The methylation analysis showed that (1→3)-linked Glc&lt;em&gt;p&lt;/em&gt; is the major constituent (70.8%) of the polymer, while the 3,4 substituted d-Glc&lt;em&gt;p&lt;/em&gt; represents the main branching residue of the glucan. The presence of (1→3)-α-d-glucan in the hyphae of &lt;em&gt;H. erinaceus&lt;/em&gt; was additionally confirmed by the use of specific fluorophore-labeled antibodies.

Sulfated modification, characterization and property of a water-insoluble polysaccharide from Ganoderma atrum

Sulfated modification was carried out to modify a water-insoluble polysaccharide from Ganoderma atrum (AGAP). The effects of sulfation on structure, physicochemical and functional properties of AGAP were investigated. Three sulfated derivatives were prepared, designated as S-1, S-2 and S-3 with degree of substitution (DS) of 0.35, 0.74 and 1.14, respectively. AGAP was elucidated as an α-(1→3)-glucan with few branches terminated by single mannose or xylose residues. The molecular weight (Mw) and radius of gyration (Rg) were estimated to be 1665kDa and 65.49nm, respectively. After sulfated modification, non-selective sulfation occurred preferably at O-6, partially at O-2 and O-4 positions of the glucosyl residues. The water-solubility of the derivatives was significantly improved in a DS-dependent manner. Mw of the derivatives showed a sharp decrease, and the chain conformation was estimated to be expanded stiff in phosphate buffer. In vitro tests showed that sulfated modification improved its antioxidant activities and anti-proliferative ability against S-180 tumor cells. This study suggested that sulfated modification was an effective approach to improve the water-solubility and functional properties of insoluble polysaccharides.

Effect of brief cetylpyridinium chloride treatments during early and mature cariogenic biofilm formation.

The aim of this study was to investigate the antibiofilm activity of brief cetylpyridinium chloride (CPC) treatments during early and mature Streptococcus mutans biofilm formation. Streptococcus mutans biofilms were formed on saliva-coated hydroxyapatite disks. The biofilms were treated with CPC twice daily (1min/treatment) from 0 to 50h or from 48 to 98h. Acidogenicity, dry weight, viability, and water-insoluble extracellular polysaccharides of the biofilms were analyzed. Confocal laser scanning microscopy (CLSM) images were obtained to confirm the antibiofilm activity during mature biofilm formation and to evaluate the relationship between treatment time and the antibiofilm activity. CPC showed complete antibiofilm activity during early biofilm formation at 0.025% to 0.1%. During mature biofilm formation, CPC inhibited dry weight, viability, and acidogenicity at 0.075% and 0.1%. CLSM images showed an increase in dead cells at 0.075% and 0.1% CPC. The antibiofilm activity during mature biofilm formation increased as the concentration of CPC increased. Images from the CLSM study also showed that antibiofilm activity increased as treatment time increased. Our findings suggest that brief CPC treatments have strong anti-S.mutans biofilm activity. The antibiofilm activity was dependent on the stage of biofilm formation, CPC concentration, and treatment time.