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In this study, the mucilage polysaccharide (MP) from Amanita hemibapha subspecies javanica was prepared by hot water extraction and ethanol precipitation and then fractionated using anion-exchange chromatography equipped with a DEAE Sepharose fast flow column. The most immune-enhancing polysaccharide fraction 2 (MPF2) was subjected to a structural modification such as hydrolysis or over-sulphation. The sulphate and molecular weight (Mw) of over-sulphated (OS1-3) and hydrolysed (HS1-3) derivatives of MPF2 differed between 9.85% and 14.2% and 32.8 and 88.1 × 103 g/mol, respectively. Further, the immune-enhancing properties of MPF2 and its derivatives were tested on RAW264.7 and NK cells through various in vitro assays. Interestingly, a low molecular weight of HS1-3 significantly increased the nitric oxide (NO) production (p &lt, 0.05) more than MPF2, indicating that Mw is a major factor in RAW264.7 cell stimulation. In addition, RAW264.7 cells produced various cytokines by up-regulating mRNA expression levels and the activation of nuclear factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. On the other hand, OS1-3-treated natural killer (NK) cells induced cytotoxicity in HepG2 cells through the expression of IFN-γ, Grandzyme-B, perforin, NKp30, and FasL. These results demonstrated that sulphate derivatives play an important role in NK cell activation. Further, this study also explores how polysaccharide binds to RAW264.7 and NK cells. MPF2 and HS3 may activate RAW264.7 cells via binding to TLR4 receptors, and OS2 could be activated through the CR3 signalling pathways.

Soaking grains in water during the preparation of germinated brown rice (GBR) can result in the multiplication of bacterial contaminants. To address this problem, a system was designed to automatically change the soaking water every 6 h. A significant reduction of the contaminants was observed. GBR was saccharified by mold bran Amylomyces rouxii. The 5 day saccharified GBR that contained the highest -amylase and glucoamylase activities was further used for alcohol fermentation by yeast Saccharomyces cerevisiae. Then, acetic acid was produced by Acetobacter aceti through a semi-continuous process. In the evaluation of the nutritional quality during processing, significant increments in the levels of -aminobutyric acid (GABA), antioxidant activities (DPPH), and total phenolic contents were found during soaking, but these levels progressively decreased in wine and were lowest in vinegar. Moreover, the soaking period from 36 h to 48 h had no significant effect on these substances. GC-MS analysis indicated that four important volatile organic compounds in the produced wine and vinegar were reported for their antimicrobial activity. Furthermore, no change in GABA was found in the vinegar over 6 months of storage. Thus, using this method could successfully produce brewed vinegar with health benefits from GBR.

In this study, Cetraria islandica polysaccharide (CIPs) was extracted by hot water extraction method and its effect on structural modification of immunomodulatory activities was investigated. This polysaccharide mainly consisted of carbohydrates (97.0%), Sulfate (1.2%) with one type of glucose. The average molecular weight (328.7 × 103 g/mol) was determined by size exclusion chromatography. The structure of the polysaccharide was found to be composed of (1→3) and (1→4)-β-D-glucopyranosyl units. The immunomodulatory activities of the crude polysaccharide and its derivatives, over-sulfated (OS1,2,3) and hydrolyzed (H1,2,3), were tested against NK-92 cells and RAW264.7 cells. The results obtained clearly demonstrated that over-sulfated (OS1,2,3)-treated NK-92 cells induced cytotoxicity in HeLa cells through the expressions of IFN-γ, NKp44, NKp30, and FasL. On the other hand, the hydrolyzed derivatives (H1,2,3) activated RAW264.7 cells through production of nitric oxide (NO) and mRNA expression of iNOS, IL-1, TNF-α, IL-6, IL-10 and IL-12 through the nuclear factor kappa-B (NF-κB) and mitogen activated protein kinases (MAPK) pathways. Our findings suggest that the presence of sulfate in the polysaccharide played a pivotal role in NK-92 cell however the Mw was a determinant factor in RAW264.7 cell activation, in which both cells were activated through the CR3 and TLR-4 signaling pathways.

The aqueous polysaccharide from Polygonatum sibiricum was extracted and fractionated using anion-exchange chromatography to obtain F1 fraction. The F1 was chemically sulfated and partially acid-hydrolyzed for the production of its over-sulfated (OS1,2,3) and hydrolyzed (HP1,2,3) derivatives, in which the sulfate content of OS1,2,3 was 7.5–17.1%, and the Mw of HP1,2,3 ranged from 18.2 × 103 to 57.3 × 103 g/mol. Considerable RAW264.7 cell activation was observed by HP1,2,3 with NO production of 34.9, 44.3 and 42.7 μM, respectively, as well as the mRNA expression of cytokines (IL-1β, IL-6, IL-10 and IL-12). NK cell cytotoxicity against HT-29 cell was facilitated by OS1,2,3 treatment with the increased gene expressions of INF-γ, Granzyme-B, perforin, NKG2D, and FasL. RAW264.7 cells appeared to be activated via MR and TLR4 mediated signaling pathway, but CR3 and TRL2 might play a main role in stimulating NK cells. Overall, the present study suggests the potential application of polysaccharides from P. sibiricum in functional foods and pharmacological industries.

Water-soluble sulfated polysaccharides extracted from Ulva intestinalis and fractionated using DEAE Sepharose fast flow column to identify their molecular properties and macrophage cells stimulating activities. Crude and fractions (F1 and F2) were formed of neutral sugars (58.7–74.7%), sulfates (6.2–24.5%), uronic acids (4.9–5.9%) and proteins (3.2–10.4%). Different levels of sugar constituents including rhamnose (30.1–39.1%), glucose (39.0–48.4%), galactose (0.0–15.8%), xylose (8.5–11.3) and arabinose (0.0–5.1%). The molecular weight (Mw) of crude and fractionated polysaccharides ranged from 87.1 × 103 to 194.1 × 103 (g/mol). Crude polysaccharides were not toxic to RAW264.7 cells and fractions induced cell proliferation. Fraction F1 stimulated RAW264.7 cells to release considerable amounts of nitric oxide, IL-1β, TNF-α, IL-6, IL-10 and IL-12 cytokines. The main backbone of the most immunostimulating polysaccharide (F1) was consisted of mixed linkages of (1 → 2)-linked rhamnose and (1 → 2)-linked glucose residues. Keywords: Ulva intestinalis, Sulfated polysaccharides, Ulvan, Molecular weight, Immunostimulating activity

The aqueous crude sulfated fucan (SF) from Stichopus japonicus was extracted and fractionated using anion-exchange chromatography to obtain four fractions (F1, F2, F3 and F4) and to investigate their NK cell activation and cytotoxicity. The most potent NK cell cytotoxicity (45% at 250 μg/mL) against HeLa cells was observed by F1 treatment, on the other hand, F3 and F4 treatment exhibited strong NK cell cytotoxicity (31–34% at 250 μg/mL) against HepG2 and HT-29 cells. The SF treatment enhanced the activation of NK cells through the mRNA expression of IFN-γ, an activating receptor (NKp30), lysing proteins (perforin and granzyme-B) as well as a death ligand (FasL). However, the treatment of the SF derivatives, deproteinated-F1 and desulfated-F3 (DP-F1 and DS-F3), markedly lowered the levels of NK cell cytotoxicity and mRNA expression of the activating factors, suggesting that the protein and sulfate were pivotal for the interaction between the SF and NK cells. The antibody neutralization test revealed that complement receptor-3 (CR3) may be a critical receptor involved in NK cell activation by the SF.

Summary This research aimed to extract polysaccharides and improve the property of sulphate-modified purple glutinous rice bran, cv. Kum Doi Saket. The effects of temperature, ratio of sulphur trioxide–trimethylamine (STMA) to sample and reaction times were studied. The results showed that the degree of substitution (DS) of sulphated polysaccharides was 0.01–0.53. Low molecular weight sulphated polysaccharides were obtained, and the functional groups of the sulphated polysaccharides were confirmed using FT-IR. No enhancement of the antioxidant activity of the sulphated polysaccharides was observed. Interestingly, immunomodulatory activity, including inducing cytokine production (iNOS, TNF-α, IL-1β and IL-10) via up-regulated mRNA expression, was significantly increased by 10–86% when compared to the crude polysaccharides.

The water soluble protein-sulfated fucan (PSF) complex isolated from the body wall of Stichopus japonicus was fractionated using an anion-exchange chromatography to obtain four purified fractions (F1, F2, F3, and F4) and to investigate their structural characteristics and immuno-enhancing activities. The crude PSF and fractions mostly consisted of neutral sugars, proteins and sulfates in various proportions. Fucose was their major monosaccharide unit with small portion of mannose, glucose and galactose. The molecular weight (Mw) of the crude PSF and fractions ranged from 323.6×103 to 3630.0×103g/mol. The crude PSF, F2, and F3 were potent stimulator of RAW264.7 cells inducing marked nitric oxide (NO) and cytokines production. The glycosidic linkage of polysaccharides was evaluated using GC-MS and confirmed by 2D-NMR. The main backbone of highly immunostimulating F3 fraction was (1→3)-α-l-linked fucosyl residue with sulfation at C-2 and/or C-4.

Polysaccharides were extracted from Schizophyllum commune (a common mushroom) and their structural and immune-enhancing properties were investigated. Crude and fractions (F1 and F2) were composed of sugars (50.3-82.8%), proteins (1.46-20.1%), and sulfates (1.33-7.01%). Monosaccharide compositions of Cr and F1 were mainly composed of glucose (75.5% and 88.2%) with small amounts of mannose, galactose and xylose whereas the F2 was mainly composed of manose (55.2%) with minor amounts of galactose, glucose, and xylose. Their immune-enhancing activities were tested using RAW264.7 cells. Proliferation activity of RAW264.7 cells was over 100% after treatment with these polysaccharides. In addition, RAW264.7 cells produced large amounts of nitric oxide and various cytokines by up-regulating mRNA expression levels and the activation of nuclear factor-kappa (NF-κB) and mitogen-activated protein kinase (MAPK) after treatment with these polysaccharides. In addition, RAW264.7 cells were activated mainly through CR-3 and TLR-4 receptors. The backbone of F2 with excellent immune-enhancing activity was mainly linked by (1→3)-linked-mannopyranosyl and (1→2,3)-linked-mannopyranosyl residues.

Water-soluble polysaccharides isolated from the rhizome of Polygonatum sibiricum and fractionated using ion-exchange chromatography were investigated to determine their structure and immunostimulating activity. Crude and fractions (F1 and F2) consisted of carbohydrates (85.1~88.3%) with proteins (4.51~11.9%) and uronic acid (1.79~7.47%), and included different levels of mannose (62.3~76.3%), glucose (15.2~20.3%), galactose (4.35~15.3%), and arabinose (4.00~7.65%). The crude contained two peaks with molecular weights (Mw) of 151×103 and 31.8×103, but F1 and F2 exhibited one major peak with Mw of 103×103 and 628×103, respectively. Little immunostimulatory activity was observed by the crude; however, F1 and F2 significantly activated RAW264.7 cells to release nitric oxide and various cytokines, suggesting they were potent immunostimulators. The backbone of the most immunostimulating fraction (F1) was (1→4)-manno- and (1→4)-gluco-pyranosyl residues with galactose and glucose attached to O-6 of manno-pyranoside.

Water-soluble sulfated heteropolysaccharides were extracted from Cladophora glomerata Kützing and fractionated by ion-exchange chromatography, which yielded two subfractions, F1 and F2. The crude and fractionated polysaccharides (F1 and F2) mostly consisted of carbohydrates (62.8–74.5%) with various amounts of proteins (9.00–17.3%) and sulfates (16.5–23.5%), including different levels of arabinose (41.7–54.4%), galactose (13.5–39.0%), glucose (0.80–10.6%), xylose (6.84–13.4%), and rhamnose (0.20–2.83%). Based on the size exclusion chromatography (SEC) profiles, the crude and fractions mainly contained one peak with shoulders having molecular weight (Mw) ranges of 358–1,501 × 103. The F1 fraction stimulated RAW264.7 cells to produce considerable amounts of nitric oxide and cytokines compared to the crude and F2 fraction. The backbone of the most potent immunostimulating fraction (F1) was α-(1→4)-L-arabinopyranoside with galactose and xylose residues as branches at O-2 position, and sulfates mainly at O-2 position as well.

The effect of molecular structures of sulfated polysaccharide (NF1) isolated from Cladophora glomerata Kutzing on the NO releasing capacity was investigated. It was possible to obtain the NF1 derivatives having various amounts of proteins (2.55-8.50%) and sulfates (6.88-13.1%) as well as different Mw (141-610 × 103 g/mol), which enabled to investigate the effects of various proteins, sulfates and Mw of NF1 on the NO releasing capacity from RAW 264.7 cells. The activity was lost after desulfation of the polysaccharides and moreover slightly increased NO releasing capacity after oversulfation of NF1 polysaccharides with the degree of sulfation (DS, 3.70). Suggesting that the sulfate groups were essential to activate NF1 polysaccharides are key factors to regulate the immunomodulatory activities.

Water-extractable polysaccharides obtained from Chlorella vulgaris were fractionated using anion-exchange chromatography to investigate molecular characteristics and biological activities. Crude and fractionated polysaccharides F1, F2, and F3 mostly consisted of carbohydrates (40.0-64.7%), proteins (18.3-31.0%), ash (4.10-16.6%), and uronic acids (1.02-7.30%). Monosaccharide constituents were predominantly glucose (83.9-100%) with small amounts of xylose (10.8-16.1%). Crude polysaccharide and fractions contained 1 or 2 sub-fractions with average Mw values ranging from 19.7 to 1,145×103 g/mol. Fractions F1, F2, and F3 stimulated RAW264.7 cells, inducing release of NO, PGE2, and the inflammatory cytokines TNF-α, IL-6, and IL-10. The biologically active part of the immune-enhancing fraction (F3) was mainly connected via 1,6-linked glucose with branches at C-3 positions.

The water-soluble crude polysaccharides, extracted from the rhizome of Smilax glabra, were fractionated using an anion exchange chromatography, yielding two fractions, F1 and F2. The crude and fractions (F1 and F2) mainly consisted of carbohydrates (66.7%–91.1%), proteins (7.30%–23.9%) and minor amount of sulfates (1.60%–9.40%). Glucose was the major monosaccharide unit of the polysaccharides with different levels of sugar constituents including galactose, arabinose, rhamnose and mannose. The molecular weight (Mw) of crude and fractions ranged from 32,102–6.3 × 103 g/mol. The crude and fractions could stimulate RAW264.7 cells to release nitric oxide and cytokines via up-regulation of their mRNA expression by the activation of NF-κB and MAPKs pathways. The related pattern recognition receptors (PRRs) on the surface of the cells appeared to be TLR2 and CR3. The GC–MS analysis revealed that the main backbone of the most immune-enhancing F2 was (1 → 4)-linked glucose and galactose chain with minor linkages of (1 → 6)-galactose, (1 → 3)-mannose, (1 → 2)-rhamnose and (1 → 5)-arabinose with some branches at C-3 and C-4 rhamnose, or C-6 galactose.

Molecular characteristics, structural properties and immunostimulatory activities of polysaccharides from pomegranate peel were evaluated after 0.1 M HCl, Cellic CTec2 and buffer extractions. The isolated polysaccharides were mainly formed of neutral sugars (32.1%-51.1%) and uronic acids (19.9%-30.8%) as well as varying levels of proteins (15.0%-39.5%). Different levels of sugars including glucose (44.9%-68.1%), galactose (14.6%-19.4%), mannose (3.4%-18.1%), arabinose (3.1%-18.1%) and rhamnose (3.5-6.0%) constructed the structure of isolated polysaccharides. The average molecular weight (Mw) of polysaccharides differed notably and ranged from 422.5 × 103 to 18,631.8 × 103 g/mol. Polysaccharide molecules obtained using Cellic CTec2 enzyme induced RAW264.7 murine macrophage cells to release considerable amount of inflammatory mediators including nitric oxide, IL-1β, TNF-α, IL-6 and IL-10. The polysaccharide stimulation of macrophage cells initiated through NF-κB and MAPKs signaling pathways and activation of p-NF-κB, p-JNK, p-ERK and p-p38 proteins. The most potent immunostimulatory polysaccharides were consisted of (1 → 3)-linked glucose, (1 → 6)-linked galactose, (1 → 4)-linked mannose and (1 → 4)-linked arabinose residues with branching points at C6 and C3. These results indicated that the lower molecular weight of polysaccharides isolated with Cellic CTec2 enzyme could be one of the major determinant structural characteristics in stimulating macrophage cells.

Sulfated polysaccharides (SP) isolated from freshwater green algae, Spirogyra neglecta (Hassall) Kützing, and fractionated SPs were examined to investigate their molecular characteristics and immunomodulatory activity. The crude and fractionated SPs (F1, F2, and F3) consisted mostly of carbohydrates (68.5–85.3%), uronic acids (3.2–4.9%), and sulfates (2.2–12.2%) with various amounts of proteins (2.6–17.1%). d-galactose (23.5–27.3%), d-glucose (11.5–24.8%), l-fucose (19.0–26.7%), and l-rhamnose (16.4–18.3%) were the major monosaccharide units of these SPs with different levels of l-arabinose (3.0–9.4%), d-xylose (4.6–9.8%), and d-mannose (0.4–2.3%). The SPs contained two sub-fractions with molecular weights (Mw) ranging from 164 × 103 to 1460 × 103 g/mol. The crude and fractionated SPs strongly stimulated murine macrophages, producing considerable amounts of nitric oxide and various cytokines via up-regulation of their mRNA expression by activation of nuclear factor-kappa B and mitogen-activated protein kinases pathways. The main backbone of the most immunoenhancing SP was (1→3)-l-Fucopyranoside, (1→4,6)-d-Glucopyranoside, and (1→4)-d-Galactopyranoside.

Exopolysaccharides (EPS) obtained from the culture medium of Lactobacillus confusus TISTR 1498 were investigated to determine their molecular characteristics and the effect of molecular weight (Mw) on immunomodulatory activity. The EPS mainly consisted of carbohydrates (81.9±2.4%) with only one type of monosaccharide, D-glucose, which was mostly connected by α-(1→6) glycosidic linkages. The EPS itself was unable to stimulate RAW264.7 cells to produce pro-inflammatory mediator nitric oxide (NO) and cytokines. However, considerable stimulation of RAW264.7 cells was observed by the low Mw of EPSs having Mw values≤70×10(3)g/mol. The partially hydrolyzed EPS stimulated RAW264.7 cells to induce considerable NO and various cytokine production such as TNF-α, IL-1β, IL-6 and IL-10 via up-regulation of their mRNA expression. In addition, the degradation Iκ-B and the phosphorylation of c-Jun NH2-terminal kinase (JNK) were facilitated by BW-30 and MW-40, suggesting that the partially hydrolyzed EPS stimulated RAW264.7 cells through the activation of NF-κB and JNK pathways.

The effects of sulfates and proteins of the sulfated polysaccharide-F2 (SP-F2) from Codium fragile on the NK cell activation and cytotoxicity were systematically investigated. The SP-F2 treatment significantly increased both NK cell proliferation (129%/100μg/mL) and their potent cytotoxic effects against HeLa cells (46%). The SP-F2 treatment appeared to enhance NK cell activation through the expression of the activating receptor, NKp30; the secretion of the cytokine, IFN-γ and the release of the lysing proteins, perforin and granzyme-B. However, the treatment of the SP-F2 derivatives, deproteinated and desulfated-F2 (DP-F2 and DS-F2), markedly lowered the mRNA expression levels of IFN-γ, granzyme-B, NKp30 and FasL, suggesting that the proteins and sulfates were essential for the interaction between the SP-F2 and NK cells. The antibody neutralization test revealed that CR3 might be a critical receptor involved in SP-F2 NK cell activation.

Food and Applied Bioscience, 1, 1, 49-58