Synthesis of oligosaccharides corresponding to the polysaccharides of Lactobacillus and Thermophlilus strains

Synthesis of a branched trisaccharide and a tetrasaccharide repeating units corresponding to the polysaccharides of Lactobacillus spp. G-77 and Thermus thermophilus Samu-SA1 as their methyl glycosides has been achieved in excellent yield. Most of the glycosyl linkages are 1,2-cis in these oligosaccharide fragments

Further characterization of the epa gene cluster and Epa polysaccharides of Enterococcus faecalis.

We previously identified a gene cluster, epa (for enterocococcal polysaccharide antigen), involved in polysaccharide biosynthesis of Enterococcus faecalis and showed that disruption of epaB and epaE resulted in attenuation in translocation, biofilm formation, resistance to polymorphonuclear leukocyte (PMN) killing, and virulence in a mouse peritonitis model. Using five additional mutant disruptions in the 26-kb region between orfde2 and OG1RF_0163, we defined the epa locus as the area from epaA to epaR. Disruption of epaA, epaM, and epaN, like prior disruption of epaB and epaE, resulted in alteration in Epa polysaccharide content, more round cells versus oval cells with OG1RF, decreased biofilm formation, attenuation in a mouse peritonitis model, and resistance to lysis by the phage NPV-1 (known to lyse OG1RF), while mutants disrupted in orfde2 and OG1RF_163 (the epa locus flanking genes) behaved like OG1RF in those assays. Analysis of the purified Epa polysaccharide from OG1RF revealed the presence of rhamnose, glucose, galactose, GalNAc, and GlcNAc in this polysaccharide, while carbohydrate preparation from the epaB mutant did not contain rhamnose, suggesting that one or more of the glycosyl transferases encoded by the epaBCD operon are necessary to transfer rhamnose to the polysaccharide. In conclusion, the epa genes, uniformly present in E. faecalis strains and involved in biosynthesis of polysaccharide in OG1RF, are also important for OG1RF shape determination, biofilm formation, and NPV-1 replication/lysis, as well as for E. faecalis virulence in a mouse peritonitis model.

Ozonolysis for selectively depolymerizing polysaccharides containing β-d-aldosidic linkages

The depolymerization of polysaccharides, particularly those containing acid-sensitive components, into intact constituent repeating units can be very difficult. We describe a method using ozonolysis for depolymerizing polysaccharides containing β-d-aldosidic linkages into short-chain polysaccharides and oligosaccharides. This method is carried out on polysaccharides that have been fully acetylated whereby β-d-aldosidic linkages are selectively oxidized by ozone to form esters, from which the polysaccharides are subsequently cleaved with a nucleophile. Ozone oxidation of aldosidic linkages proceeds under strong stereoelectronic control, and reaction rates depend on the conformations of glycosidic linkages. Thus, β-d-aldosidic linkages with different conformations can have very different reaction rates even in the absence of substantial chemical differences. These rate differences allowed for very high selectivity in cleaving β-d-linkages of polysaccharides. Several polysaccharides from group B Streptococcus and other bacterial species were selectively depolymerized with this method. The repeating units of the group B Streptococcus polysaccharides all contain an acid-sensitive sialic acid residue in a terminal position on a side chain and several β-d-residues including galactose, glucose, and N-acetylglucosamine; however, with each polysaccharide, one type of linkage was more reactive than others. Selective cleavage of the most sensitive linkage occurs randomly throughout the polymer chain, yielding fragments of controllable and narrowly distributed sizes and the same repeating-unit structure. The average size of the molecules decreases exponentially, and desired sizes can be obtained by stopping the reaction at appropriate time points. With this method the labile sialic acid residue was not affected.

A Cross-Polarization, Magic-Angle-Spinning, 13C-Nuclear-Magnetic-Resonance Study of Polysaccharides in Sugar Beet Cell Walls1

Solid-state nuclear magnetic resonance relaxation experiments were used to study the rigidity and spatial proximity of polymers in sugar beet (Beta vulgaris) cell walls. Proton T1ρ decay and cross-polarization patterns were consistent with the presence of rigid, crystalline cellulose microfibrils with a diameter of approximately 3 nm, mobile pectic galacturonans, and highly mobile arabinans. A direct-polarization, magic-angle-spinning spectrum recorded under conditions adapted to mobile polymers showed only the arabinans, which had a conformation similar to that of beet arabinans in solution. These cell walls contained very small amounts of hemicellulosic polymers such as xyloglucan, xylan, and mannan, and no arabinan or galacturonan fraction closely associated with cellulose microfibrils, as would be expected of hemicelluloses. Cellulose microfibrils in the beet cell walls were stable in the absence of any polysaccharide coating.

Borate-Rhamnogalacturonan II Bonding Reinforced by Ca2+ Retains Pectic Polysaccharides in Higher-Plant Cell Walls1

The extent of in vitro formation of the borate-dimeric-rhamnogalacturonan II (RG-II) complex was stimulated by Ca2+. The complex formed in the presence of Ca2+ was more stable than that without Ca2+. A naturally occurring boron (B)-RG-II complex isolated from radish (Raphanus sativus L. cv Aokubi-daikon) root contained equimolar amounts of Ca2+ and B. Removal of the Ca2+ by trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid induced cleavage of the complex into monomeric RG-II. These data suggest that Ca2+ is a normal component of the B-RG-II complex. Washing the crude cell walls of radish roots with a 1.5% (w/v) sodium dodecyl sulfate solution, pH 6.5, released 98% of the tissue Ca2+ but only 13% of the B and 22% of the pectic polysaccharides. The remaining Ca2+ was associated with RG-II. Extraction of the sodium dodecyl sulfate-washed cell walls with 50 mm trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid, pH 6.5, removed the remaining Ca2+, 78% of B, and 49% of pectic polysaccharides. These results suggest that not only Ca2+ but also borate and Ca2+ cross-linking in the RG-II region retain so-called chelator-soluble pectic polysaccharides in cell walls.

Novel, Starch-Like Polysaccharides Are Synthesized by an Unbound Form of Granule-Bound Starch Synthase in Glycogen-Accumulating Mutants of Chlamydomonas reinhardtii1

In vascular plants, mutations leading to a defect in debranching enzyme lead to the simultaneous synthesis of glycogen-like material and normal starch. In Chlamydomonas reinhardtii comparable defects lead to the replacement of starch by phytoglycogen. Therefore, debranching was proposed to define a mandatory step for starch biosynthesis. We now report the characterization of small amounts of an insoluble, amylose-like material found in the mutant algae. This novel, starch-like material was shown to be entirely dependent on the presence of granule-bound starch synthase (GBSSI), the enzyme responsible for amylose synthesis in plants. However, enzyme activity assays, solubilization of proteins from the granule, and western blots all failed to detect GBSSI within the insoluble polysaccharide matrix. The glycogen-like polysaccharides produced in the absence of GBSSI were proved to be qualitatively and quantitatively identical to those produced in its presence. Therefore, we propose that GBSSI requires the presence of crystalline amylopectin for granule binding and that the synthesis of amylose-like material can proceed at low levels without the binding of GBSSI to the polysaccharide matrix. Our results confirm that amylopectin synthesis is completely blocked in debranching-enzyme-defective mutants of C. reinhardtii.

Natural Pectin Polysaccharides as Edible Coatings

The most fashionable trends in food packaging research are targeted towards improvements in food quality and safety by increasing the use of environmentally-friendly materials, ideally those able to be obtained from bio-based resources and presenting biodegradable characteristics. Edible films represent a key area of development in new multifunctional materials by their character and properties to effectively protect food with no waste production. The use of edible films should be considered as a clean and elegant solution to problems related with waste disposal in packaging materials. In particular, pectin has been reported as one of the main raw materials to obtain edible films by its natural abundance, low cost and renewable character. The latest innovations in food packaging by the use of pectin-based edible films are reviewed in this paper, with special focus on the use of pectin as base material for edible coatings. The structure, properties related to the intended use in food packaging and main applications of pectins are herein reported.

The thermal performance of the polysaccharides extracted from hardwood:cellulose and hemicellulose

The pyrolytic behaviour of individual component in biomass needs to be understood to gain insight into the mechanism of biomass pyrolysis. A comparative study on the pyrolysis of cellulose (hexose-based polysaccharides) and hemicallulose (pentose-based polysaccharides) is performed by two sets of experiments including TG analysis and Py-GC-MS/FTIR. The samples of these two polysaccharide components are thermally decomposed in TGA at the heating rate of 5 and 60 K/min to demonstrate the different characteristics of mass loss stage(s) between them. The yield of pyrolytic products is examined by a fluidized-bed fast pyrolysis unit. The experiment confirms that cellulose mainly contributes to bio-oil production (reaching the maximum of 72% at 580 °C), while hemicellulose works as an important precursor for the char production (∼25%). The compounds in the gaseous mixture (CO and CO2) and bio-oil (levoglucosan, furfural, aldehyde, acetone and acetic acid) are further characterized by GC-MS for cellulose and GC-FTIR for hemicellulose, and their formations are investigated thoroughly. © 2010 Elsevier Ltd. All rights reserved.

Silanisation de polysaccharides en milieu liquide ionique et réactivité d’un organoalkoxysilane vis-à-vis de nucléophiles simples : vers des hydrogels injectables pour l’ingénierie tissulaire du cartilage

Tissue engineering is a real challenge for the treatment of cartilage pathologies. In this field, biomimetic hydrogels based on natural polymers are among the most commonly used matrices. A hydrogel made of silanized hydroxypropylmethylcellulose (HPMC-Si) is especially promising because it can be injected in cartilaginous lesions by minimally invasive surgery. However, the current synthesis of HPMC-Si is limited by the insolubility of hydroxypropylmethylcellulose (HPMC). This thesis work was focused on finding new synthesis conditions for the design of HPMC-Si hydrogel. In order to obtain a complete solubilization of HPMC and to improve its functionalization by the (3-glycidyloxypropyl) trimethoxysilane (GPTMS), the use of ionic liquids (IL), which are excellent solvents for polysaccharides, was undertaken. The beginning of this study was first devoted to the selection of an IL and then to the development of new reaction conditions. With these new conditions, higher silicon rates were obtained for HPMC modified in ionic liquid medium, however no hydrogel could be formed. The second part was therefore devoted to the synthesis of GPTMS 13C. Indeed, thanks to this radiolabeling, a structural characterization by 13C NMR of the HPMC-Si could be achieved. Finally, the reactivity in organic solvents of three organosilanes, including the GPTMS, was investigated toward nucleophiles representing the common functions found in natural polymers (e.g. -NH2, -OH, -SH). The results of this thesis have provided insights into the GPTMS reactivity in organic medium and thus paves the way to new conditions for the silanization of polysaccharides.

Valorization of the Macroalgae Sargassum Muticum by Enzymatic Hydrolysis, Interest of Surfactants to Improve the Extraction of Phlorotannins and Polysaccharides

The use of surfactants to improve enzymatic hydrolysis of the macroalgae Sargassum muticum has been investigated. Visible absorption spectroscopy has been used to quantify the solubilization of both polysaccharides and phlorotannins in the hydrolysates.   After total extraction, results showed that Sargassum muticum contained 2.74% (expressed in percent of the dry weight of the algae) of phlorotannins whose 32 % were in the cell wall. This result shows that it is important to access to the parietal phlorotannins. To reach this objective, we chose the enzymatic approach for destructurating the cell wall of the algae. The use of 5% dry weight (DW - 5% by weight of hydrolyzed algae) of an enzymatic mix containing a commercial beta-glucanase, a commercial protease and an alginate lyase extracted from Pseudomonas alginovora led after 3 hours of hydrolysis to the solubilization of 2.43% DW polysaccharides and 0.52% DW phlorotannins. The use of 0.5% volume of the surfactant Triton® X-100 with 10% DW of the enzymatic mix has allowed to reaching the value of 2.63% DW of solubilized phlorotannins, that is 96% of the total phenolic content.   The use of non-ionic surfactant, combined to enzymatic hydrolysis, showed an increased efficiency in disrupting cell wall and solubilizing phlorotannins in Sargassum muticum.

Sulphated polysaccharides from Ulva clathrata and Cladosiphon okamuranus seaweeds both inhibit viral attachment/entry and cell-cell fusion, in NDV infection

Sulphated polysaccharides (SP) extracted from seaweeds have antiviral properties and are much less cytotoxic than conventional drugs, but little is known about their mode of action. Combination antiviral chemotherapy may offer advantages over single agent therapy, increasing efficiency, potency and delaying the emergence of resistant virus. The paramyxoviridae family includes pathogens causing morbidity and mortality worldwide in humans and animals, such as the Newcastle Disease Virus (NDV) in poultry. This study aims at determining the antiviral activity and mechanism of action in vitro of an ulvan (SP from the green seaweed Ulva clathrata), and of its mixture with a fucoidan (SP from Cladosiphon okamuranus), against La Sota NDV strain. The ulvan antiviral activity was tested using syncytia formation, exhibiting an IC50 of 0.1 μg/mL; ulvan had a better anti cell-cell spread effect than that previously shown for fucoidan, and inhibited cell-cell fusion via a direct effect on the F0 protein, but did not show any virucidal effect. The mixture of ulvan and fucoidan showed a greater anti-spread effect than SPs alone, but ulvan antagonizes the effect of fucoidan on the viral attachment/entry. Both SPs may be promising antivirals against paramyxovirus infection but their mixture has no clear synergistic advantage

Effect of Lycii fructus polysaccharides on ovulation failure in rats

Purpose: To investigate the effect of Lycii fructus polysaccharides (LFPS) on ovulation failure. Methods: A rat model of ovulation failure was established by intragastric administration of hydroxyurea (300 mg/kg). Rats with ovulation failure then received LFPS via oral administration at doses of 100, 200, or 400 mg/kg. The body, uterus and ovary of each rat were weighed using electronic scales. The hypothalamic-pituitary-ovarian (HPO) axis hormones, including estradiol (E2) level, follicle-stimulating hormone (FSH) activity, and luteinizing hormone (LH) activity in the serum of each rat were determined by enzyme-linked immunosorbent assay (ELISA). The levels of pro-apoptotic proteins (Fas, FasL, FADD, c-caspase-8, c-caspase-10, c-caspase-3, c-caspase-6, and c-caspase-7) in the ovarian tissue of each rat were detected by western blot. Results: Hydroxyurea reduced significantly (p < 0.01) uterus and ovary indices (uterus or ovary weight/body weight) (0.119 and 0.026 %), E2 level (3.42 pmol/L), and FSH and LH activities (2.28 and 2.76 U/L), compared with those in the normal group (0.169 and 0.039 %; 6.72 pmol/L; 2.76 and 3.75 U/L). Hydroxyurea increased significantly (p < 0.01) the levels of the above-mentioned pro-apoptotic proteins relative to those in the normal group. LFPS (100, 200, or 400 mg/kg) reversed significantly (p < 0.05 or 0.01) the effect of hydroxyurea on all of the above indices. Conclusion: LFPS exhibits a protective effect on hydroxyurea-induced ovulation failure by regulating the HPO axis hormones and death receptor-mediated apoptotic pathway.

Antitumor effect and mechanism of action of polysaccharides extracted from Polygonum perfoliatum L whole plant in human lung carcinoma A549 cell line

Purpose: To optimize the extraction conditions of polysaccharides from Polygonum perfoliatum L. (PSDP) and to evaluate their anti-tumor activities on A549 cell line. Methods: Extraction of PSDP was optimized using Box-Behnken design (BBD). Three factors of response surface methodology (RSM) including extraction time, ratio of water to raw material and number of extractions were employed to optimize the yield of PSDP. The cytotoxic effect of PSDP on human lung carcinoma A549 cell line was evaluated in vivo, while its effects on expressions of caspase3, caspase-9, Bcl-2 and Bax were determined by western blot assay. Result: BBD was significant and applicable to PSDP extraction. Based on the contour plots, response surface plots and variance analysis, it predicted that the optimum conditions for PSDP extraction were: 1.58 h (extraction time); 30.18 mL/g (ratio of water to raw material); and 2.02 (number of extractions). PSDP had significant inhibitory effect on the growth of A549 cells in a concentration- and timedependent manner (p < 0.05). After treatment with PSDP, caspase-3, caspase-9 and Bax were significantly up-regulated (p < 0.05), whereas Bcl-2 was down-regulated, all concentration-dependently. Conclusion: RSM analysis is an appropriate method to optimize PSDP extraction. The results also indicate that PSDP has significant anti-tumor effect against A549 cells, most likely via inducing mitochondria-mediated apoptosis.

Metabolism of plant polysaccharides by Leucoagaricus gongylophorus, the symbiotic fungus of the leaf-cutting ant Atta sexdens L.

Atta sexdens L, ante feed on the Fungus they cultivate on cut leaves inside their nests. The fungus, Leucoagaricus gongylophorus, metabolizes plant polysaccharides, such as xylan, starch, pectin, and cellulose, mediating assimilation of these compounds lay the ants, This metabolic integration may be an important part of the ant-fungus symbiosis, and it involves primarily xylan and starch, both of which support rapid fungal growth. Cellulose seems to be less important for symbiont nutrition, since it is poorly degraded and assimilated by the fungus. Pectin is rapidly degraded but slowly assimilated by L. gongylophorus, and its degradation may occur so that the fungus can more easily access other polysaccharides in the leaves.