Application of novel analytical ultracentrifuge analysis to solutions of fungal mannans.

Polysaccharides, the most abundant biopolymers, are required for a host of activities in lower organisms, animals, and plants. Their solution characterization is challenging due to their complex shape, heterogeneity, and size. Here, recently developed data analysis approaches were applied for traditional sedimentation equilibrium and velocity methods in order to investigate the molar mass distribution(s) of a subtype of polysaccharide, namely, mannans from four Candida spp. The molecular weight distributions of these mannans were studied using two recently developed equilibrium approaches: SEDFIT-MSTAR and MULTISIG, resulting in corroboratory distribution profiles. Additionally, sedimentation velocity data for all four mannans, analyzed using ls-g*(s) and Extended Fujita approaches, suggest that two of the fungal mannans (FM-1 and FM-3) have a unimodal distribution of molecular species whereas two others (FM-2 and FM-4) displayed bi-modal and broad distributions, respectively: this demonstrates considerable molecular heterogeneity in these polysaccharides, consistent with previous observations of mannans and polysaccharides in general. These methods not only have applications for the characterization of mannans but for other biopolymers such as polysaccharides, DNA, and proteins (including intrinsically disordered proteins).

Norway spruce galactoglucomannans exhibiting immunomodulating and radical-scavenging activities.

Wood-derived naturally acetylated galactoglucomannans (AcGGM) can be recovered even in ton-scale at mechanical pulp mills using spruce as raw material. These cell wall polysaccharides have a great potential as hydrocolloids and bioactive polymers in food and pharmaceutical applications, or as starting material for production of functional polymers. The immunostimulatory activity of both AcGGM and its deacetylated form (GGM) was now in vitro tested. The biological response of both AcGGM and GGM in the lymphocyte transformation test was dose-dependent. The direct mitogenic as well as comitogenic activities of the AcGGM were comparable to those of the immunogenic corn cob xylan used as control, and GGM showed significantly higher biological responses also at lower doses. In contrast to GGM, AcGGM possessed also DPPH radical-scavenging activity. The results suggested that the spruce AcGGM and GGM are potentially important as additives with immuno-potentiating and antioxidant properties in food products and pharmaceutical formulations.

Effect of microwave irradiation on the molecular and structural properties of hyaluronan.

Hyaluronan (Na(+) salt of hyaluronic acid, HA) was extensively depolymerised by HCl-catalyzed hydrolysis at pH 3 for up to 500min under temperature-controlled microwave irradiation. The effects of microwave heating on the hydrodynamic properties of the polysaccharide were determined by SEC-MALLS and viscometry. The weight-average molecular mass (Mw) of HA decreased from 1.44×10(6) to ∼5000, reaching the region of higher oligosaccharides. The scission of HA chains was found to proceed randomly during the whole degradation process. Treatment of the Mw and intrinsic viscosity data according to the Mark-Houwink equation, [η]=k×Mw(α) suggested three relationships with α1=0.46 for Mw>500,000, α2=0.84 for Mw between 500,000 and 50,000, and α3=1.13 for Mw<50,000. The results revealed that HA with Mw>10,000 adopts a stiffish coil conformation in solution. As monitored by FT-IR and NMR spectroscopic techniques, the primary structure of the HA chains was maintained during the microwave-assisted hydrolysis at pH 3 at 105°C. At reaction times larger than 240min, uv spectroscopy suggested the depolymerisation of HA was accompanied by formation of by-products produced by side reaction.

Almond shell xylo-oligosaccharides exhibiting immunostimulatory activity.

Partially O-acetylated xylo-oligosaccharides (DXO) isolated from almond shells by autohydrolysis as well as their de-acetylated form (DeXO) were subjected to chemical, molecular, and structural analyses. They represent a mixture of neutral and acidic oligomers and low-molecular weight polymers related to (4-O-methyl-D-glucurono)-D-xylan. DXO and DeXO showed direct mitogenic activity and enhancement of the T-mitogen-induced proliferation of rat thymocytes, indicating the immunostimulatory potential of the almond shell xylo-oligosaccharides.

Effect of salt stress on the production and properties of extracellular polysaccharides produced by Cryptococcus laurentii.

The composition, main structural features and molecular properties of exopolysaccharides (EP) produced by Cryptococcus laurentii var. laurentii CCY 17-3-16 under optimal (EPo) and NaCI-stress conditions (EPs) as well as their subfractions isolated by gel chromatography were studied using chemical, FT-IR and NMR spectroscopy methods. The results showed that under stress conditions the yeast produced EP with a lower content of protein and phosphorus. In comparison to EPo, the EPs exhibited a substantially larger proportion of high molecular mass populations. NMR analysis of EPs revealed a higher degree of branching with single xylose side chains of the heteromannan components. The increase of the molecular mass and degree of branching of the macromolecular chains of the heteromannan components might in part be related to the function of EPs to protect the yeast cells from water loss and maintain growth conditions under the salt stress.

Non-cellulosic polysaccharides from the leaves of small balsam (Impatiens parviflora DC.).

The leaves of the annual plant Impatiens parviflora DC., a herbal medicine in Asian countries and invasive in managed forests and natural environments in Central Europe, have the potential as a source of bioactive phenolic compounds and polysaccharides. Extractives accounted for ∼22% of the leaves, whereby, the methanolic extract contains mainly caffeic acid, ferulic acid, kaempferol, and quercetin derivatives, and 1,2,4-trihydroxynaphthalene-1-O-glucoside. From the pre-extracted leaves, non-cellulosic polysaccharides were isolated by a five-step extraction procedure using as extractants cold water, 0.05 M EDTA, and DMSO in the first three steps, and 1% and 5% NaOH in the last two steps. The isolated polysaccharide fractions were characterized by chemical, physicochemical, and spectroscopic analyses (FTIR and (1)H NMR). The first three fractions contained mainly pectin and the alkali-extracted ones methylglucuronoxylan and arabinogalactan. The suggested structural features were confirmed using HSQC NMR and COSY experiments for polysaccharides of the EDTA-fraction and the Pronase-treated 5% NaOH-fraction. The EDTA fraction comprised a pectin with low degree of methyl-esterification (DM, 37%) with few rhamnogalacturonan RG I segments bearing ß-1,4-galactan as side chains. The alkali-extracted fraction comprised a degraded methylglucuronoxylan and type II 3,6-arabinogalactan in about equal amounts.

Bioactive hemicelluloses alkali-extracted from Fallopia sachalinensis leaves.

Fallopia sachalinensis, regarded as an invasive plant in Europe and designated for disposal, is traditionally used in Japan and China as herbal medicine. Attempted for valorization of the leaves, this paper reports on two protein-free polysaccharide fractions, a neutral (FS-5A) and an acidic (FS-5B) one, obtained via alkali extraction and consecutive purification. Both fractions were characterized by chemical, molecular, structural and bioactive properties. FTIR and 1D/2D NMR analyses revealed that FS-5A consisted of a fucogalactoxyloglucan, whereas, glucuronoxylan was the major hemicellulose in FS-5B accompanied with low proportions of fucosylated xyloglucan and pectic RG-I. Both hemicellulose fractions exhibited significant immunostimulating activity in the complement-fixation test and the latter had noticeable DPPH radical-scavenging ability. The results completed information about neutral and acidic bioactive polysaccharide components present in the leaves of F. sachalinensis.

Structural diversity of pectins isolated from the Styrian oil-pumpkin (Cucurbita pepo var. styriaca) fruit.

To evaluate the seeded fruit biomass of the Styrian oil-pumpkin in view of its pectin component, a series of acidic polysaccharides were isolated by a six-step sequential extraction using hot water, EDTA, dilute HCl (twice) and dilute and stronger NaOH solutions. Chemical, physicochemical and spectroscopy analyses revealed that the first four fractions comprised partially methyl-esterified and acetylated pectins with varying proportions of rhamnogalacturonan regions ramified with galactose- and arabinose-containing side chains and showed considerable polymolecularity. The alkali-extracted polysaccharides contained lower amounts of pectins with homogalacturonan and arabinose-rich rhamnogalacturonan regions next to hemicelluloses prevailing in the last polysaccharide. Using (1)H-(13)C HSQC and HMBC spectroscopy, the resonances of free and methylesterified galacturonic acid residues in the purified acid-extracted pectin were unambiguously established and various diads formed by both residues identified. The results might serve as a basis for searching technological conditions to produce pectin from the oil-pumpkin fruit biomass.

Structural features of two heteroxylan polysaccharide fractions from wheat bran with anti-complementary and antioxidant activities.

Wheat bran is a rich source of bioactive substances ascribed to its arabinoxylan component. Two water-soluble arabinoxylans were sequentially extracted from wheat bran. WB1, released during enzymatic digestion of starch and protein, contained medium-branched arabinoxylan (A/X=0.88) consisting of 3-O-substituted (22%), di-substituted (19.8%) and 58% unsubstituted Xylp residues. It was slightly contaminated with (1→3,1→4)-ß-glucan and arabinogalactan, and free of protein. WB2 extracted with 0.5% NaOH contained ∼95% arabinoxylan (A/X=1.09). WB2 and two 5% NaOH-extracted arabinoxylans were rich in protein and phenolic compounds. All radical-scavenging assays indicated a relation with the protein and total phenolics contents. The protein-free WB1 displayed the highest hydroxyl radical scavenging effect indicating the distinct role of phenolic acids. The immunomodulatory activity of WB1 was somewhat lower, whereas, that of WB2 higher in comparison to the immunogenic polysaccharide PMII. The arabinoxylans have the potential as immuno-enhancing and antioxidant additives in functional foods.

Suppressive effect of pectic polysaccharides from Cucurbita pepo L. var. Styriaca on citric acid-induced cough reflex in guinea pigs.

Several water-soluble pectic polysaccharides were isolated from the pumpkin fruit biomass and characterized by composition, structural features and molecular properties. The pectic polysaccharides were tested for antitussive activity by studying the effects of citric acid-induced cough reflex in guinea pigs and reactivity of the airway smooth muscle in vivo conditions in comparison to the narcotic drug codeine. Oral administration of all pectic polysaccharides from pumpkin inhibited the number of coughs induced by citric acid in guinea pigs, but to various extents. The results indicated that the antitussive activity of the pectic polysaccharides is affected by their molecular and structural properties, whereby a synergistic action between the polysaccharide and non-carbohydrate components on the biological response has been suggested as well. The cough depressive efficacy of most of the tested polysaccharides was comparable and even higher than that of codeine. Moreover, the application of these polysaccharides provoked any side effects what is their advantage towards the conventional opioid-derived antitussive agents.

The pharmacological activity of wheat bran polysaccharides.

(Glucurono)arabinoxylans were extracted from the wheat bran with acetate buffer in the first step (WBH1) and with dilute alkali in the second step (WBH2). In both samples xylose and arabinose dominated, accompanied with smaller amounts of galactose, glucose, mannose and uronic acids mainly in WBH1. WBH1 was free of protein and with low content of phenolic compounds. Fraction WBH2 contained relatively low levels of proteins and about 4.5% of the total phenolic. When tested for antitussive activity, the (glucurono)arabinoxylans exhibited comparable cough-suppressing effect with centrally acting codeine. The observed effects of bronchoconstriction are limiting practical application of WBH2.

Production, refining, structural characterization and fermentability of rice husk xylooligosaccharides.

Oligosaccharides produced by hydrothermal processing of rice husks (xylooligosaccharides and glucooligosaccharides) were refined by membrane processing (operating in diafiltration and concentration modes), subjected to xylanase treatment to reduce the average molar mass, and subjected to further purification by ultrafiltration (operating in concentration mode) and ion exchange. The purified products were assayed for composition, molar mass distribution and structural characterization by HPLC, HPAEC-PAD, HPSEC, MALDI-TOF-MS and NMR ((1)H and (13)C). The fermentability of the purified product by fecal inocula was assessed on the basis of the time courses of pH and oligosaccharide concentrations. Succinate, lactate, formiate, acetate, propionate and butyrate were the major products resulting from fermentation experiments.

Weak self-association in a carbohydrate system.

The physiological importance of weak interactions between biological macromolecules (molar dissociation constants >10 microM) is now well recognized, particularly with regard to cell adhesion and immunological phenomena, and many weak interactions have been measured for proteins. The concomitant importance of carbohydrate-carbohydrate interactions has also been identified, although no weak interaction between pure carbohydrate systems has ever been measured. We now demonstrate for the first time to our knowledge using a powerful probe for weak interactions--sedimentation velocity in the analytical ultracentrifuge--that at least some carbohydrates (from the class of polysaccharides known as heteroxylans and demonstrated here to be biologically active) can show well-defined weak self-interactions of the "monomer-dimer" type frequently found in protein systems. The weak interaction between the heteroxylans is shown from a temperature dependence study to be likely to be hydrophobic in nature.