Comparison and Optimization of Quantification Methods for Shigella flexneri Serotype 6 O-antigen Containing Galacturonic Acid and Methyl-Pentose.

Shigella is a leading diarrheal cause of morbidity and mortality worldwide, especially in low- and middle-income countries and in children under five years of age. Increasing levels of antimicrobial resistance make vaccine development an even higher global health priority. S. flexneri serotype 6 is one of the targets of many multicomponent vaccines in development to ensure broad protection against Shigella. The O-antigen (OAg) is a key active ingredient and its content is a critical quality attribute for vaccine release in order to monitor their stability and to ensure appropriate immune response. Here, the optimization of two methods to quantify S. flexneri 6 OAg is reported together with the characterization of their performances. The optimized Dische colorimetric method allows a tenfold increment of the sensitivity with respect to the original method and is useful for fast analysis detecting selectively methyl-pentoses, as rhamnose in S. flexneri 6 OAg. Also, a more specific HPAEC-PAD method was developed, detecting the dimer galacturonic acid-galactosamine (GalA-GalN) coming from S. flexneri 6 OAg acid hydrolysis. These methods will facilitate characterization of S. flexneri 6 OAg based vaccines. The colorimetric method can be used for quantification of other polysaccharide containing methyl-pentoses, and the HPAEC-PAD could be extended to other polysaccharides containing uronic acids.

Study on a novel spherical polysaccharide from Fructus Mori with good antioxidant activity.

A novel polysaccharide (MFP1P) was isolated from Fructus Mori, followed by purification via DEAE-52 cellulose and 27 % ethanol fraction. The MFP1P had the molecular weight of 56.78 kDa and the total sugar content of 93.32±0.54 %. And the MFP1P is mainly composed of glucose, galactose, galacturonic acid and mannose with molar ratio of 66.62 %, 13.94 %, 18.24 % and 1.20 %, respectively. MFP1P was mainly composed of →3)-α-D-Gal (1→, ß-D-Man-(1→ and →6)-α-D-Glc (1→ glycosidic bond and showed a spherical chain conformation with uniform distribution in solution. The MFP1P exhibited great antioxidant activity with oxygen-free radical absorption capacity (ORAC) values of 291.63±6.81 µmol TE/g and MDA IC50 of 0.289±0.022 mg/mL.

Microwave-assisted extraction of arabinan-rich pectic polysaccharides from melon peels: Optimization, purification, bioactivity, and techno-functionality.

The effects of water to solids ratio (WSR, 10-30 mL/g), power (180-540 W), and irradiation time (IT, 5-15 min) in microwave-assisted extraction (MAE) were optimized to extract polysaccharides from melon peels (PMP). The maximum extraction yield (32.81 %) was obtained under 20.94 mL/g WSR, 414.4 W power, and 12.75 min IT. The main monosaccharide composition of purified PMP with an average molecular weight of 5.71 × 104 kDa were d-galacturonic acid, arabinose, glucose, and galactose. An ascending dose-dependent antiradical and antioxidant behavior for PMP (0-5.0 mg/mL) was found. The initial foaming capacity (38.6-110.3 %) and foaming stability (5.2-65.2 %) were significantly increased as a function of PMP concentration (1.0-5.0 %), while they reduced by increasing the mixing time (p < 0.05). The highest emulsifying activity index (44.1 m2/g) and emulsifying stability (69.3 %) at 5.0 % PMPs were determined. PMP gels with FTIR-identified functional groups can be formulated in new gluten-free functional products.

Optimization of polysaccharides extraction from quince peels: partial characterization, antioxidant and antiproliferative properties.

In this study, Box-Behnken Design was used to optimize the ultrasonic extraction of polysaccharides from quince peels (QPPs) by ascorbic acid and the effect of extraction temperature, extraction time and pH was evaluated. Under optimized conditions of temperature 90 °C, 60 min sonication time and pH = 3.26, the extraction yield, the galacturonic acid yield and the concentration of sample required to scavenge 50% of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic) acid (ABTS) values of QPPs were respectively 10.25%, 3.86% and 1.35 mg/mL. The QPPs extracted under optimum conditions was characterized by Fourier transform infrared spectroscopy (FTIR), Nuclear magnetic resonance (1 H NMR) and Size exclusion chromatography (SEC/MALS/VD/DRI). The monosaccharide analysis revealed that arabinose was the most abundant, followed by galactose, glucose, mannose and xylose. Moreover, QPPs showed significant antioxidant activities (2,2-diphenyl-1-picrylhydrazyl (DPPH) and Ferric- reducing antioxidant power (FRAP)) and reduced viability of human Caco-2 and murine B-16 cell lines in a dose-dependent manner. Hence QPPs could be used as antitumor agent in functional foods andpharmaceutical industries.

Structural characterization and anticomplement activities of three acidic homogeneous polysaccharides from Artemisia annua.

ETHNOPHARMACOLOGICAL RELEVANCE: Artemisia annua L. is a heat-clearing Chinese medicine and well-known for its antimalarial constituent, artemisinin. It has gained increasing attention for its anti-inflammatory and immunoregulatory activities. Interestingly, the crude polysaccahrides of A. annua exhibited potent anticomplement activity. This study was to isolate and characterize its anticomplement homogeneous polysaccharides from A. annua, and reveal the relationship between structures and anticomplement activities of the isolated polysaccharides. MATERIALS AND METHODS: Water-soluble crude polysaccharides from the aerial parts of A. annua were extracted and fractionated by DEAE-cellulose and Sephacryl S-300 gel permeation chromatography. Homogeneity, molecular weight, monosaccharide composition, methylation and NMR analysis were performed to characterize the structures of homogeneous polysaccharides. Their anticomplement activities and targeting components in the complement activation cascade were evaluated by hemolytic assays. RESULTS: Three homogeneous polysaccharides (AAP01-1, AAP01-2 and AAP01-3) were obtained from A. annua. AAP01-1 was composed of seven monosaccharides, including mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose. AAP01-2 and AAP01-3 had similar monosaccharides with AAP01-1, except the absence of glucuronic acid. They were all branched acidic heteropolysaccharides with different contents of galacturonic acid (8%, 28% and 15% for AAP01-1, AAP01-2 and AAP01-3, respectively). AAP01-2 showed potent anticomplement activity with CH50 value of 0.360 ±â€¯0.020 mg/mL through the classical pathway and AP50 value of 0.547 ±â€¯0.033 mg/mL through the alternative pathway. AAP01-3 exhibited slightly weaker activity (CH50: 1.120 ±â€¯0.052 mg/mL, AP50: 1.283 ±â€¯0.061 mg/mL), while AAP01-1 was inactive. Moreover, AAP01-2 acted on C1q, C3, C4, C5 and C9 components and AAP01-3 interacted with C3, C4 and C5 components in the activation cascade of complement system. CONCLUSION: These results indicated that the relatively high contents of galacturonic acid were important for anticomplement activities of the polysaccharides from A. annua. The anticomplement polysaccharides are another kind of bioactive constituents conferring heat-clearing effects of A. annua.

Structural characterization of a galacturonic acid-rich polysaccharide from Ziziphus Jujuba cv. Muzao.

Jujube (Zizyphus jujuba Mill.) have been widely used as a health food and medicinal herb in oriental medicine. In the present study, a novel galacturonic acid-rich polysaccharide (PZMP3-2) was isolated from Zizyphus Jujuba cv. Muzao through hot water extraction, ethanol precipitation, and purification with DEAE-Sepharose Fast Flow and Sephacryl S-300 column chromatography. The chemical structures of PZMP3-2 were elucidated by methylation analysis, along with HPGPC, FT-IR spectroscopy, GC-MS, 1D NMR spectroscopy, and 2D NMR spectroscopy. Its morphological properties were further characterized by SEM, AFM, and XRD. Monosaccharide compositional analysis of PZMP3-2 revealed the presence of rhamnose, arabinose, galactose, and galacturonic acid at a molar ratio of 1.74:2.00: 1.00:18.69, and the HPGPC data demonstrated an average molecular weight of 58.21 kDa. Structural and linkage analysis by GC-MS and NMR spectroscopy suggested that PZMP3-2 was a homogalacturonan (HG)-like pectic polysaccharide branched at C-2 with some Araf and Rhap residues. Such unique structure of PZMP3-2 may indicate distinct bioactivities and further application in food and even clinic.

Extraction and characterization of an alginate from the Iranian brown seaweed Nizimuddinia zanardini.

Sodium alginate from Nizimuddinia zanardini (an Iranian brown algae) was extracted with acid and alkaline solutions, partially and totally hydrolyzed and analyzed for its biochemical composition. 1H NMR spectroscopy, SEC-MALLS, HPAEC and FT-IR were performed to determine its structure and its physico-chemical properties. This alginate has a M/G ratio of 1.1, a molecular weight of 103 kDa, a polydispersity index of 1.22, and an intrinsic viscosity of 342 mL/g. Its antioxidant activity was tested by DPPH radical scavenging showing its potential for food preservation. Rheological properties of solutions of this alginate with concentrations between 1 and 5% (w/v) in water and 0.5 M NaCl were investigated indicating a Newtonian fluid type behaviour in water and a shear thinning fluid type behaviour in NaCl solutions.

Impact of Prosopis alba exudate gum on sorption properties and physical stability of fish oil alginate beads prepared by ionic gelation.

This study was conducted to evaluate the effect of Prosopis alba exudate gum (G) as encapsulating matrix component on water-solid interactions, physical state, oxidative damage and appearance properties of alginate-chitosan encapsulates containing fish oil. With this purpose, water sorption isotherms were obtained at 25 °C. G increased the hygroscopicity of encapsulates, showing a higher monolayer water content (7.87 ±â€¯0.47% db.) than control (1.07 ±â€¯0.04% db.). G introduction reduced the plasticizing effect of water, increasing the aw range (aw < 0.45) at which samples were in amorphous state and providing the highest protection against lipid oxidation. Appearance properties (chromatic and optical) were affected by hydration and were better maintained in samples containing G at aw > 0.52. These results allow considering Prosopis alba exudate gum, as a novel excipient to protect fish oil encapsulated in low moisture polyelectrolyte systems, with the added benefits of employing an undervalued natural resource.

Reduction of heavy metal (Pb2+) biosorption in zebrafish model using alginic acid purified from Ecklonia cava and two of its synthetic derivatives.

Heavy metal contamination has become a major problem that causes severe environmental and health issues due to their biosorption, bioaccumulation, and toxicity. This study was designed to evaluate heavy metal chelating abilities of alginic acid (AA) extracted from the brown seaweed Ecklonia cava and two of its derivatives prepared by the partial oxidation of the 2° OH groups (OAA) and partial carboxylation of the monomeric units (CAA) upon reducing the heavy metal biosorption in zebrafish (Danio rerio) modal. Metal ions were quantified using ICP-OES and biopolymers were characterized by FTIR and XRD analysis. All investigated biopolymers indicated potential ability for chelating Pb2+, Cu2+, Cd2+, As3+, and Ag+. The sorption capacities were in the order of CAA>OAA>AA. All biopolymers indicated a comparatively higher chelation towards Pb2+. AA, OAA, and CAA could effectively reduce Pb2+ induced toxicity and Pb2+ stress-induced ROS production in zebrafish embryos. Besides, they could reduce the biosorption of Pb2+ in adult zebrafish which could lead to bioaccumulation. Since alginic acid purified from E. cava and its derivatives could be utilized as seaweed derived biopolymers to purify heavy metals contaminated water and as a dietary supplement to reduce heavy metal biosorption in organisms.

Magnetic molecularly imprinted polymers for recognition and enrichment of polysaccharides from seaweed.

New magnetic molecularly imprinted polymers with two templates were fabricated for the recognition of polysaccharides (fucoidan and alginic acid) from seaweed by magnetic solid-phase extraction, and the materials were modified by seven types of deep eutectic solvents. It was found that the deep eutectic solvents magnetic molecularly imprinted polymers showed stronger recognition and higher recoveries for fucoidan and alginic acid than magnetic molecularly imprinted polymers, and the deep eutectic solvents-4-magnetic molecularly imprinted polymers had the best effects. The practical recovery of the two polysaccharides (fucoidan and alginic acid) purified with deep eutectic solvents-4-magnetic molecular imprinted polymers in seaweed under the optimal conditions were 89.87, and 92.0%, respectively, and the actual amounts extracted were 20.6 and 18.7 µg/g, respectively. To sum up, the developed method proved to be a novel and promising method for the recognition of complex polysaccharide samples from seaweed.

Centrifugal partition chromatography in a biorefinery context: Optimisation and scale-up of monosaccharide fractionation from hydrolysed sugar beet pulp.

The isolation of component sugars from biomass represents an important step in the bioprocessing of sustainable feedstocks such as sugar beet pulp. Centrifugal partition chromatography (CPC) is used here, as an alternative to multiple resin chromatography steps, to fractionate component monosaccharides from crude hydrolysed sugar beet pulp pectin. CPC separation of samples, prepared in the stationary phase, was carried out using an ethanol: ammonium sulphate (300gL-1) phase system (0.8:1.8v:v) in ascending mode. This enabled removal of crude feedstream impurities and separation of monosaccharides into three fractions (l-rhamnose, l-arabinose and d-galactose, and d-galacturonic acid) in a single step. Throughput was improved three-fold by increasing sample injection volume, from 4 to 16% of column volume, with similar separation performance maintained in all cases. Extrusion of the final galacturonic acid fraction increased the eluted solute concentration, reduced the total separation time by 24% and removed the need for further column regeneration. Reproducibility of the separation after extrusion was validated by using multiple stacked injections. Scale-up was performed linearly from a semi-preparative 250mL column to a preparative 950mL column with a scale-up ratio of 3.8 applied to mobile phase flow rate and sample injection volume. Throughputs of 9.4gL-1h-1 of total dissolved solids were achieved at the preparative scale with a throughput of 1.9gL-1h-1 of component monosaccharides. These results demonstrate the potential of CPC for both impurity removal and target fractionation within biorefinery separations.

Effects of extraction methods on molecular characteristics, antioxidant properties and immunomodulation of alginates from Sargassum angustifolium.

The relationship between molecular structure and bioactivity was evaluated for alginates obtained under different extraction methods (water, acid, alcalase and cellulase) from Sargassum angustifolium. The use of enzymes considerably reduced protein (from 14.58% to <0.4%) and polyphenol (from 16.0% to <1.7mg GA/g sample) contaminations of alginates compared to those of water and acid. The FT-IR spectrum revealed that extraction method did not affect the structure of the recovered alginates. The highest molecular weight (Mw) (557.1×103g/mol) was found in acid treated alginate while the Mw of cellulase assistant alginate (356.2×103g/mol) was the minimum. The SVg values varied from 2.79-5.17cm3/g revealing the loosed conformational structures of alcalase and cellulase assistant alginates. Alcalase assistant alginate stimulated RAW264.7 cells to release nitric oxide and inflammatory cytokines TNF-α, IL-1, IL-6, IL-10 and IL-12. Enzyme treated alginates showed maximum DPPH radical scavenging activity and reducing power. Therefore, the present results showed the determinant effect of pretreatment during the extraction process of alginate and the beneficial influence of enzymatic process when biological functions of alginates are of high interest in the industry.

Plant gum identification in historic artworks.

We describe an integrated and straightforward new analytical protocol that identifies plant gums from various sample sources including cultural heritage. Our approach is based on the identification of saccharidic fingerprints using mass spectrometry following controlled enzymatic hydrolysis. We developed an enzyme cocktail suitable for plant gums of unknown composition. Distinctive MS profiles of gums such as arabic, cherry and locust-bean gums were successfully identified. A wide range of oligosaccharidic combinations of pentose, hexose, deoxyhexose and hexuronic acid were accurately identified in gum arabic whereas cherry and locust bean gums showed respectively PentxHexy and Hexn profiles. Optimized for low sample quantities, the analytical protocol was successfully applied to contemporary and historic samples including 'Colour Box Charles Roberson &Co' dating 1870s and drawings from the American painter Arthur Dove (1880-1946). This is the first time that a gum is accurately identified in a cultural heritage sample using structural information. Furthermore, this methodology is applicable to other domains (food, cosmetic, pharmaceutical, biomedical).

Microbial alginate dressings show improved binding capacity for pathophysiological factors in chronic wounds compared to commercial alginate dressings of marine origin.

Marine alginates are well established in wound management. Compared with different modern wound dressings, marine alginates cannot prove superior effects on wound healing. Alginates from bacteria have never been studied for medical applications so far, although the microbial polymer raises expectations for improved binding of wound factors because of its unique O-acetylation. Due to its possible positive effects on wound healing, alginates from bacteria might be a superior future medical product for clinical use. To prove the binding capacity of microbial alginates to pathophysiological factors in chronic wounds, we processed microbial alginate fibres, produced from fermentation of the soil bacterium Azotobacter vinelandii ATCC 9046, into needle web dressings and compared them with commercial dressings made of marine alginate. Four dressings were assessed: Marine alginate dressings containing either ionic silver or zinc/manganese/calcium, and microbial alginate dressings with and without nanosilver. All dressings were tested in an in vitro approach for influence on chronic wound parameters such as elastase, matrix metalloproteases-2, tumour necrosis factor-α, interleukin-8, and free radical formation. Despite the alginate origin or addition of antimicrobials, all dressings were able to reduce the concentration of the proinflammatory cytokines TNF-α and IL-8. However, microbial alginate was found to bind considerable larger amounts of elastase and matrix metalloproteases-2 in contrast to the marine alginate dressings. The incorporation of zinc, silver or nanosilver into alginate fibres did not improve their binding capacity for proteases or cytokines. The addition of nanosilver slightly enhanced the antioxidant capacity of microbial alginate dressings, whereas the marine alginate dressing containing zinc/manganese/calcium was unable to inhibit the formation of free radicals. The enhanced binding affinity by microbial alginate of Azotobacter vinelandii to pathophysiological factors may be interesting to support optimal conditions for wound healing.

Combined De-Algination Process as a Fractionation Strategy for Valorization of Brown Macroalga Saccharina japonica.

A combined process, de-algination followed by enzymatic saccharification, was designed to produce alginate and glucose from Saccharina japonica consecutively. The process conditions of de-algination were optimized separately for each stage of acidification and alkaline extraction. Collectively, the de-algination yield was 70.1% under the following optimized conditions: 2.4 wt% of Na2CO3, 70 °C, and 100 min with the acidified S. japonica immersed in a 0.5 wt% H2SO4 solution for 2 h at room temperature. The glucan content in the de-alginated S. japonica increased to 38.0%, which was approximately fivefold higher than that of the raw S. japonica. The enzymatic hydrolysis of the de-alginated S. japonica almost completed in 9 h, affording 5.2 g (96.8% of glucan digestibility) of glucose at a de-alginated S. japonica loading of 14.2 g.

Optimization of alginate alkaline extraction technology from Sargassum latifolium and its potential antioxidant and emulsifying properties.

Alginate was recovered from Sargassum latifolium biomass using different conditions of alkali treatment. Box-Behnken experimental design was evaluated to study the influence of alkali:alga ratio, temperature and time on alginate yield, and its molecular weight (MW) and mannuronic/guluronic acid ratio (M/G). The second-order polynomial equations were analyzed by appropriate statistical methods. Extraction temperature and time were the most important factors during alginate alkaline extraction. MW and M/G ratio played an important role in controlling the reducing power of alginate. Increasing pH of the alginate solutions enhanced its reducing capacity, while thermal treatment showed a negative effect. Additionally, alginate exhibited good emulsion stabilizing capacities with diverse hydrophobic compounds. Emulsifying activity was less sensitive to temperature, ionic strength and more stable at acidic pH.

Optimization Extraction, Preliminary Characterization and Antioxidant Activities of Polysaccharides from Semen Juglandis.

The optimization extraction process, preliminary characterization and antioxidant activities of polysaccharides from Semen Juglandis (SJP) were studied in this paper. Based on the Box-Behnken experimental design and response surface methodology, the optimal extraction conditions for the SJP extraction were obtained as follows: temperature 88 °C, extraction time 125 min and ratio of liquid to solid 31 mL/g. Under these conditions, experimental extraction yield of SJP was (5.73 ± 0.014)% (n = 5), similar to the predicted value of 5.78%. Furtherly, the purified SJP obtained from SJP extract by DEAE-52 and Sephacryl S-100 chromatography was analyzed to be rhamnose, galacturonic acid, galactose, arabinose and fucose in the molar ratio of 1:6.34:1.38:3.21:1.56. And the weight-average molecular weight and radius of gyration of the purified SJP in 0.1 M NaCl were determined to be 2.76 × 104 g/mol and 122 nm by SEC-MALLS, respectively. More importantly, it exhibited appreciable antioxidant activities compared to the standard Vc, such as DPPH radical scavenging activity (IC50 0.21 mg/mL), strong reducing power, ABTS radical scavenging activity (IC50 0.29 mg/mL), and hydroxyl radical scavenging activity (IC50 0.38 mg/mL). These results indicate that SJP may be useful for developing functional health products or natural antioxidant.

Comparison of the properties of membranes produced with alginate and chitosan from mushroom and from shrimp.

Dense and porous chitosan-alginate membranes (1:1 in mass) useful as coverages of skin wounds treated through cell therapy were produced using chitosan of different chain sizes from fungal (white mushrooms) and animal (shrimp shells) sources. Porous materials were obtained by adding the surfactant Poloxamer 188 to the formulations. The influence of chitosan type on membranes physicochemical properties and toxicity to fibroblasts was evaluated. Porosity was noticed to be more pronounced in membranes obtained with fungal chitosan and increased with its molecular mass. These formulations showed the highest values of thickness, roughness, opacity, liquid uptake and water vapor permeability. The membranes were not toxic to fibroblasts, but the lowest cytotoxicity values (0.16-0.21%) were observed for membranes prepared with fungal chitosan in the presence of surfactant. In conclusion, it is possible to replace chitosan from animal sources by chitosan of fungal origin to produce membranes with negligible cytotoxicity while maintaining appropriate physicochemical properties.

Structural and immunological feature of rhamnogalacturonan I-rich polysaccharide from Korean persimmon vinegar.

The crude polysaccharide (KPV-0) isolated from Korean persimmon vinegar was fractionated using gel filtration chromatography to enhance the immunostimulatory activity and to identify the structural features of active fraction. Among three fractions, KPV-I obtained in a void volume, demonstrated the potent production of macrophage-stimulating mediators, including tumor necrosis factor-α, interleukin (IL)-6, IL-12, and nitric oxide. KPV-I showed a combined single peak with high molecular weight of 55,000Da by high performance size exclusion chromatography. Component sugar analysis revealed that KPV-I contained mainly of arabinose, mannose, galactose, rhamnose and galacturonic acid. Single radial gel diffusion assay using ß-glucosyl Yariv reagent showed that KPV-I contained arabinogalactan protein with 13.7%. Methylation analysis indicated that KPV-I contained 21 kinds of neutral glycosidic linkages, which seemed to be composed three kinds of polysaccharide; that is a rhamnogalacturonan-I (65-70%) derived from persimmon as a raw material, a mannan (20-25%) derived from fermentation-associated microorganisms, and a linear glucans (less than 10%). In conclusion, polysaccharide isolated from persimmon vinegar could augment the macrophage stimulation, and a large amounts of RG-I polysaccharide derived from persimmon is likely a crucial role in expression of the activity in persimmon vinegar.

Molecular structure, chemical properties and biological activities of Pinto bean pod polysaccharide.

Pinto bean pod polysaccharide (PBPP) was successfully extracted with yield of 38.5g/100g and the PBPP gave total carbohydrate and uronic acid contents of 286.2mg maltose equivalent/g and 374.3mgGal/g, respectively. The Mw of PBPP was 270.6kDa with intrinsic viscosity of 0.262dm(3)/g, which composed of mannose (2.5%), galacturonic acid (15.0%), rhamnose (4.0%), glucose (9.0%), galactose (62.2%), xylose (2.9%) and arabinose (4.3%) with trace amount of ribose and fucose. The result suggested that PBPP has a spherical conformation with a highly branched structure. Fourier Transform Infrared analysis showed that PBPP has a similar structure as commercial pectin with an esterification degree of 59.9%, whereas scanning electron microscopy study showed that the crude polysaccharide formed a thin layer of film that was made of multiple micro strands of fibre. PBPP exhibited substantial free radical scavenging activity (7.7%), metal reducing capability (2.04mmol/dm(3)) and α-amylase inhibitory activity (97.6%) at a total amount of 1mg. PBPP also exhibited high water- and oil-holding capacities (3.6g/g and 2.8g/g, respectively). At a low concentration, PBPP exhibited emulsifying activity of 39.6% with stability of 38.6%. Apart from that, PBPP was able to show thickening capability at low concentration (0.005kg/dm(3)).