DEAE Anion Exchange Chromatography Research Articles

Expression, Purification, and Anti-UV Irradiation Effect of RsSOD on HCE-T Human Corneal Epithelial Cells.

Superoxide dismutase (SOD) is a class of enzymes that catalyze the disproportionation of superoxide anion radicals into hydrogen peroxide and oxygen. It can remove excessive free radicals in organisms and acts as a potent antioxidant, cleaning free radicals generated by radiation and protecting cells from oxidative damage. In this study, we obtained a MnSOD gene from the radiation-resistant bacterium Radiobacillus sp. (RsSOD) and constructed its recombinant expression vector through gene synthesis. The recombinant RsSOD protein was efficiently expressed using IPTG induction, and purified via repeated freezing and thawing, heating, and DEAE anion-exchange chromatography. The purified RsSOD exhibited an enzyme activity of 2072.5 U/mg. Furthermore, RsSOD was demonstrated to have robust resistance to high temperatures, acid, alkali, and artificial intestinal fluid. Further studies were performed to investigate the radiation resistance of RsSOD against ultraviolet (UV) irradiation in human corneal epithelial (HCE-T) cells. The results indicated that a low concentration of RsSOD (6.25 U/mL) could promote HCE-T cell proliferation and protect these cells from damage caused by both long-term and short-term UV exposure, effectively reducing apoptosis induced by short-term UV irradiation. These findings suggest that the RsSOD protein possesses significant anti-UV irradiation property and is expected to be a candidate for treating ocular radiation-related diseases.

Effects of Solid Fermentation on Polygonatum cyrtonema Polysaccharides: Isolation, Characterization and Bioactivities.

Polygonati Rhizoma is a widely used traditional Chinese medicine (TCM) with complex pre-processing steps. Fermentation is a common method for processing TCM to reduce herb toxicity and enhance their properties and/or produce new effects. Here, in this study, using Bacillus subtilis and Saccharomyces cerevisiae, we aimed to evaluate the potential application of solid fermentation in isolating different functional polysaccharides from Polygonatum cyrtonema Hua. With hot water extraction, ethanol precipitation, DEAE anion exchange chromatography and gel filtration, multiple neutral and acidic polysaccharides were obtained, showing different yields, content, compositions and functional groups after fermentation. Combining in vitro experiments and in vivo aging and immunosuppressed mouse models, we further compared the antioxidant and immunomodulating bioactivities of these polysaccharides and found a prominent role of a natural polysaccharide (BNP) from fermented P. cyrtonema via Bacillus subtilis in regulating intestinal antioxidant defense and immune function, which may be a consequence of the ability of BNP to modulate the homeostasis of gut microbiota. Thus, this work provides evidence for the further development and utilization of P. cyrtonema with fermentation, and reveals the potential values of BNP in the treatment of intestinal disorders.

Polysaccharide from aerial part of Chuanminshen violaceum alleviates oxidative stress and inflammatory response in aging mice through modulating intestinal microbiota.

Aging is a biological process of progressive deterioration of physiological functions, which poses a serious threat to individual health and a heavy burden on public health systems. As population aging continues, research into anti-aging drugs that prolong life and improve health is of particular importance. In this study, the polysaccharide from stems and leaves of Chuanminshen violaceum was obtained with water extraction and alcohol precipitation, and then separated and purified with DEAE anion exchange chromatography and gel filtration to obtain CVP-AP-I. We gavaged natural aging mice with CVP-AP-I and performed serum biochemical analysis, histological staining, quantitative real-time PCR (qRT-PCR) and ELISA kit assays to analyze inflammation and oxidative stress-related gene and protein expression in tissues, and 16SrRNA to analyze intestinal flora. We found that CVP-AP-I significantly improved oxidative stress and inflammatory responses of the intestine and liver, restored the intestinal immune barrier, and balanced the dysbiosis of intestinal flora. In addition, we revealed the potential mechanism behind CVP-AP-I to improve intestinal and liver function by regulating intestinal flora balance and repairing the intestinal immune barrier to regulate the intestinal-liver axis. Our results indicated that C. violaceum polysaccharides possessed favorable antioxidant, anti-inflammatory and potentially anti-aging effects in vivo.

Preparation and catalytic properties of catalase-inorganic hybrid nanoflowers

Catalase is widely used in the food, medical, and textile industries. It possesses exceptional properties including high catalytic efficiency, high specificity, and environmental friendliness. Free catalase cannot be recycled and reused in industry, resulting in a costly industrial biotransformation process if catalase is used as a core ingredient. Developing a simple, mild, cost-effective, and environmentally friendly approach to immobilize catalase is anticipated to improve its utilization efficiency and enzymatic performance. In this study, the catalase KatA derived from Bacillus subtilis 168 was expressed in Escherichia coli. Following separation and purification, the purified enzyme was prepared as an immobilized enzyme in the form of enzyme-inorganic hybrid nanoflowers, and the enzymatic properties were investigated. The results indicated that the purified KatA was obtained through a three-step procedure that included ethanol precipitation, DEAE anion exchange chromatography, and hydrophobic chromatography. Then, by optimizing the process parameters, a novel KatA/Ca3(PO4)2 hybrid nanoflower was developed. The optimum reaction temperature of the free KatA was determined to be 35 ℃, the optimum reaction temperature of KatA/Ca3(PO4)2 hybrid nanoflowers was 30-35 ℃, and the optimum reaction pH of both was 11.0. The free KatA and KatA/Ca3(PO4)2 hybrid nanoflowers exhibited excellent stability at pH 4.0-11.0 and 25-50 ℃. The KatA/Ca3(PO4)2 hybrid nanoflowers demonstrated increased storage stability than that of the free KatA, maintaining 82% of the original enzymatic activity after 14 d of storage at 4 ℃, whereas the free KatA has only 50% of the original enzymatic activity. In addition, after 5 catalytic reactions, the nanoflower still maintained 55% of its initial enzymatic activity, indicating that it has good operational stability. The Km of the free KatA to the substrate hydrogen peroxide was (8.80±0.42) mmol/L, and the kcat/Km was (13 151.53± 299.19) L/(mmol·s). The Km of the KatA/Ca3(PO4)2 hybrid nanoflowers was (32.75±2.96) mmol/L, and the kcat/Km was (4 550.67±107.51) L/(mmol·s). Compared to the free KatA, the affinity of KatA/Ca3(PO4)2 hybrid nanoflowers to the substrate hydrogen peroxide was decreased, and the catalytic efficiency was also decreased. In summary, this study developed KatA/Ca3(PO4)2 hybrid nanoflowers using Ca2+ as a self-assembly inducer, which enhanced the enzymatic properties and will facilitate the environmentally friendly preparation and widespread application of immobilized catalase.

Polysaccharide from Salviae miltiorrhizae Radix et Rhizoma Attenuates the Progress of Obesity-Induced Non-Alcoholic Fatty Liver Disease through Modulating Intestinal Microbiota-Related Gut-Liver Axis.

Non-alcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease worldwide, thus treatments for it have attracted lots of interest. In this study, the Salviae miltiorrhizae Radix et Rhizoma (SMRR) polysaccharide was isolated by hot water extraction and ethanol precipitation, and then purified by DEAE anion exchange chromatography and gel filtration. With a high-fat-diet-induced obesity/NAFLD mouse model, we found that consumption of the SMRR polysaccharide could remarkably reverse obesity and its related progress of NAFLD, including attenuated hepatocellular steatosis, hepatic fibrosis and inflammation. In addition, we also reveal the potential mechanism behind these is that the SMRR polysaccharide could regulate the gut–liver axis by modulating the homeostasis of gut microbiota and thereby improving intestinal function.

Purification and Biochemical Characterization of Two Anionic Peroxidase Isoenzymes from Raphanus sativus L. var niger Roots.

Peroxidase is a commonly used enzyme with a wide range of applications. Horseradish (Armoracia rusticana) is the most well-known source of peroxidase enzyme. Peroxidases extracted from other plant sources have also been proved as useful, sometimes even superior, comparing to traditional horseradish peroxidase (HRP). In the present study, two novel peroxidase isoenzymes were purified and characterized from Raphanus sativus L. var niger roots. Two anionic peroxidase isoenzymes were purified using diafiltration, ammonium sulfate precipitation, DEAE anion-exchange chromatography, and concanavalin A affinity chromatography. The heaviest anionic isoenzyme (isoenzyme A) has a MW of about 110 KD, and the other anionic isoenzyme (isoenzyme B) has a MW of 97 KD. Both isoenzymes have an optimum temperature of 40°C, but the activity of isoenzyme B is much more dependent on temperature with a Q10 of 3.5, while isoenzyme A has a Q10 of 1.7. These isoenzymes showed great thermal stability at 37°C and 4°C. Isoenzyme A showed the highest activity at pH 5 and it was found to be more stable at pH 6, whereas isoenzyme B showed the highest activity at pH 6 and is more stable at pH 7. Isoenzyme A has a Km value of 10.63mM and 0.043mM, and isoenzyme B has a Km of 15.38mM and 0.067mM for 4-aminoantipyrine and H2O2, respectively. The isoenzymes purified from Raphanus sativus L. var niger offer excellent chemical and thermal stability, which encourages further studies on their suitability for biotechnological applications.

Extraction, Purification, Characterization and In Vitro Antibacterial properties of Sulfated Polysaccharide Extracted using Sargassum sp.

In the present study, Sulfated polysaccharides were extracted from macroalga Sargassum whitey collected from the Gulf of Mannar, Tamil Nadu, India. The maximum yield of 1.95g sulfated polysaccharide was extracted from Sargassum whitey and 0.81mg/ml of purified sulfated polysaccharide was obtained through DEAE anion exchange chromatography. The purified form was characterized using Fourier Transform Infrared Spectroscopy (FTIR), Energy Dispersive X-ray spectroscopy (EDX), and Field Emission Scanning Electron Microscopy (FESEM) analyses. The in vitro biological properties such as antibacterial, antioxidants activity (DPPH, Hydroxyl & Superoxide anion) of sulfated polysaccharides were evaluated and the results showed that 45μg mL-1 concentration of sulfated polysaccharide exhibited good antibacterial inhibitory activity against all the tested pathogenic bacteria strains, Escherichia coli, Klebsiella pneumoniae and Staphylococcus aureus. The in vitro antioxidant activity also found to be significant, where the exhibited IC50 concentrations by the purified sulfated polysaccharide was 183.2µg/mL for DPPH, 204.2µg/mL for superoxide anion, and 163.8µg/mL for Hydroxyl scavenge free radicals activities.

Fermentation, Purification, and Tumor Inhibition of a Disulfide-Stabilized Diabody Against Fibroblast Growth Factor-2.

Angiogenesis is considered one of the hallmarks of cancer and plays a critical role in the development of tumor. Fibroblast growth factor 2 (FGF-2) is a member of the FGF family and participates in excessive cancer cell proliferation and tumor angiogenesis. Thus, targeting FGF-2 was considered to be a promising anti-tumor strategy. A disulfide-stabilized diabody (ds-Diabody) against FGF-2 was produced in Pichia pastoris (GS115) by fermentation and the anti-tumor activity was analyzed. The novel 10-L fed batch fermentation with newly designed media was established, and the maximum production of the ds-Diabody against FGF-2 reached 210.4 mg/L. The ds-Diabody against FGF-2 was purified by Ni2+ affinity chromatography and DEAE anion exchange chromatography. The recombinant ds-Diabody against FGF-2 could effectively inhibit proliferation, migration, and invasion of melanoma and glioma tumor cells stimulated by FGF-2. Furthermore, xenograft tumor model assays showed that the ds-Diabody against FGF-2 had potent antitumor activity in nude mice by inhibiting tumor growth and angiogenesis. The tumor growth inhibition rate of melanoma and glioma was about 70 and 45%, respectively. The tumor angiogenesis inhibition rate of melanoma and glioma was about 64 and 51%, respectively. The results revealed that the recombinant ds-Diabody against FGF-2 may be a promising anti-tumor drug for cancer therapy.

Expression, Purification, and Bioactivity of a Soluble Recombinant Ovine Interferon-tau in Escherichia Coli.

IntroductionOvine interferon-tau (oIFN-τ) is a newly discovered type I interferon. This study used biochemical techniques to transform the oIFN-τ gene into Escherichia coli to obtain the mass and soluble expression of the recombinant protein.Material and MethodsFirst, total RNA was extracted from fresh sheep embryonic tissues with TRIzol reagent and then used as a template to reverse transcribe and amplify the mature oIFN-τ gene with RT-PCR. The amplified product was next digested with the HindIII and XhoI restriction enzymes and inserted into the pET-32a(+) vector to construct the prokaryotic expression plasmid. The corrected in-frame recombinant plasmid, pET-32a(+)-oIFN-τ, was transformed into E. coli Rosetta (DE3) competent cells. After induction with isopropyl-beta-D-thiogalactopyranoside (IPTG), the recombinant protein was detected in bacteria. Finally, the bacteria were lysed by sonication, and the recombinant protein was purified by nickel affinity chromatography and DEAE anion exchange chromatography.ResultsThe protein was confirmed to be oIFN-τ, which mainly existed in the soluble lysate fraction, as proven by SDS-PAGE and Western blot assays.ConclusionPurified IFN-τ exists mostly in a soluble form, and its anti-vesicular stomatitis virus (VSV) activity reached 7.08×10(6)IU/mL.

Pectic polysaccharide from Nelumbo nucifera leaves promotes intestinal antioxidant defense in vitro and in vivo.

In this study, the Nelumbo nucifera leaf polysaccharide (NNLP) was isolated by hot water extraction and ethanol precipitation. DEAE anion exchange chromatography and gel filtration were further performed to obtained the purified fraction NNLP-I-I, the molecular weight of which was 16.4 kDa. The monosaccharide composition analysis and linkage units determination showed that the fraction NNLP-I-I was a pectic polysaccharide. In addition, the NMR spectra analysis revealed that NNLP-I-I mainly consisted of a homogalacturonan backbone and rhamnogalacturonan I, containing a long HG region and short RG-I region, with AG-II and 1-3 linked rhamnose as side chains. The biological studies demonstrated that NNLP-I-I displayed antioxidant properties through mediating the Nrf2-regulated intestinal cellular antioxidant defense, which could protect cultured intestinal cells from oxidative stress and improve the intestinal function of aged mice.

Isolation and Characterization of Glutaminase-free L-asparaginase Produced by Staphylococcus sp. MGM1

L-asparaginase is an important therapeutic enzyme used in combination with other drugs for therapy of Acute Lymphoblastic Leukemia (ALL). L-asparaginase catalyzes the conversion of asparagine to aspartic acid and ammonia. In recent years, this enzyme gained applications in many fields of science such as clinical research, pharmacological, and food industries. This study was aimed at isolation and identification of a strain with the ability to producing extracellular glutaminase free L-asparaginase from soil and determination of enzyme stability. The isolation was done on M9 medium. Biochemical tests and 16S rDNA sequence was used for strain identification. L-asparaginase was partially purified using ammonium sulfate precipitation, dialysis, and DEAE-anion exchange chromatography. The effect of pH and temperature on enzyme activity was investigated. The isolated bacteria were identified as Staphylococcus sp. The optimum pH and temperature for maximum L-asparaginase activity were found at 8 and 35 °C. The enzyme purification showed a single band around 115 kDa on SDS-Page. The optimal activity for the enzyme produced by MGM1 was similar to the physiological conditions of the human body, therefore, further studies on this enzyme would be of great value in finding a new efficient asparaginase enzyme.

Soluble Expression, Rapid Purification and Antiviral Activity of Recimbinant Bovine Interferon-α in Escherichia coli

In this study, the cDNA of mature bovine interferon-α (BoIFN-α) gene was cloned and expressed in the Escherichia coli expression system. Total RNA was extracted from bovine liver cells and reverse transcribed into cDNA, and then BoIFN-α gene fragment was amplified with specific primers by PCR method before being ligated into the pET-32a (+) expression vector. Since the pET-32a (+) plasmid expresses the thioredoxin A (TrxA) tag protein, it can increase the solubility of the target protein. In order to express the target protein, the pET-32a-BoIFN-α recombinant plasmid was transformed into E. coli Rosetta (DE3) strain followed by IPTG induction. It was found that the recombinant fusion protein Trx-rBoIFN-α was finally expressed in a soluble form in the host cell, accounting for about 33.45% of the total cellular protein. The protein was purified by two-step chromatography, including Ni2+-chelating Sepharose affinity chromatography and DEAE anion exchange chromatography. The purity of the purified product reached 93.0%, and the cytopathic effects (CPE) inhibition method was adopted to determine the antiviral activity of the expressed fusion protein in vesicular stomatitis virus/ Madin-Darby bovine kidney cells titration system. The specific activity of the recombinant fusion protein Trx-rBoIFN-α was measured as (3.6 ± 0.25) × 106 U/mg. After cutting of the Trx tag protein by enterokinase digestion, the remaining solo protein was confirmed as rBoIFN-α protein by Western blot assay. These findings will enable us to produce a large number of bioactive rBoIFN-α protein for future application.

Design of a chimeric antigen containing multiple immunodominant epitopes and its use in detection of IgM antibodies against Rubella virus

A chimeric antigen designated B103 containing six immunodominant regions derived from three structural proteins of Rubella virus (RV) was designed and its utility in serological diagnosis was assessed. Protein B103 is comprised of aa 1-30 & aa 96-123 of C protein, aa 31-105 of E2 protein, as well as aa 11-39, aa 154-277 & aa 389-412 of E1 protein. In addition, it contains thioredoxin (TRX) at the N-terminal and His tag at the C-terminal. B103 was expressed in Escherichia coli BL21(DE3) and purified by Streamline Chelating affinity and DEAE anion exchange chromatography. Based on the antigenicity of B103 as verified by Western blotting analysis, we constructed and evaluated a novel capture ELISA for RV-IgM detection. B103 was expressed in a soluble form, accounting for 18.57% of the total bacterial proteins. After purification, the concentration and purity of protein B103 were 3.026 mg/mL and 95.35%, respectively. Western blotting analysis demonstrated that protein B103 could react with acute-phase serum of RV. By ELISA, 40 negative sera and 40 RV-acute phase sera were detected. The sensitivity, specificity, positive predictive value, negative predictive value and coincidence rate of the ELISA were 92.50%, 95.00%, 94.87%, 92.68% and 93.75%, respectively. The McNemer analysis suggested that there was no statistical difference between the 'Gold standard' and the novel ELISA with a kappa coefficient of 0.900, indicating excellent consistency. B103 chimeric protein with excellent antigenicity obtained from prokaryotic expression followed by chromatography purification could prove useful for early diagnosis of RV infection.

Preparation of multi-epitope recombinant diagnostic antigen of Mycobacterium tuberculosis

Multi-epitope recombinant diagnostic antigen (designated 'B102') of Mycobacterium tuberculosis (Mtb) was prepared and evaluated as a serological diagnostic antigen. With TRX at the N-terminal and His tag at the C-terminal, the multi-epitope Mtb recombinant diagnostic antigen including 11 predicted B-cell epitopes from 6 Mtb antigens (PstS1, ESAT6, CFP10, Ag85B, Ag85A and PPE54) was expressed in Escherichia coli BL21 (DE3) and purified by Ni²⁺-Chelating affinity and DEAE anion exchange chromatography. Based on the antigenicity of B102 confirmed in Western blotting analysis, we constructed and evaluated a double-antigen sandwich ELISA for diagnosis of Mtb infection. The protein B102 exists in the form of inclusion bodies, accounting for 31.25% of the total proteins of the bacteria. After purification and renaturation, protein B102 exists in soluble form with the concentration 3.124 mg/mL and the homogeneity 96.71%. WB analysis demonstrated that protein B102 could react with antibodies in Mtb positive serum. Using the novel antigen in ELISA, we tested 60 Mtb-related positive and negative serum; The results showed the sensitivity, specificity, positive and negative predictive values and coincidence rate of the detection method is 90.00%, 93.33%, 93.10%, 90.32% and 91.67%, respectively. The McNemer analysis suggested there was no statistical difference between the 'Gold standard' and the novel ELISA with kappa 0.833, which suggested the excellent consistency. By prokaryotic expression and chromatography purification, the multi-epitope recombinant antigen B102 was obtained with excellent antigenicity, which could be applied for Mtb-related serological detection.

Characterization of Inulin-Type Fructan from Platycodon grandiflorus and Study on Its Prebiotic and Immunomodulating Activity.

Platycodon grandiflorus is a plant widely used in traditional Chinese medicine, of which polysaccharides are reported to be the main components responsible for its bio-functions. In this work, the inulin-type fructan (PGF) was obtained by DEAE anion exchange chromatography from the water extracted from P. grandifloras. Characterization was performed with methanolysis, methylation, and NMR and the results showed that PGF is a β-(2-1) linked fructan, with terminal glucose and with a degree of polymerization of 2–10. In order to study its biofunctions, the prebiotic and immunomodulation properties were assayed. We found that PGF exhibited good prebiotic activity, as shown by a promotion on six strains of lactobacillus proliferation. Additionally, the PGF also displayed direct immunomodulation on intestinal epithelial cells and stimulated the expressions of anti-inflammatory factors. These results indicated that the inulin from P. grandiflorus is a potential natural source of prebiotics as well as a potential intestinal immunomodulator, which will be valuable for further studies and new applications.

Soluble expression, rapid purification, biological identification of chicken interferon-alpha using a thioredoxin fusion system in E. coli and its antiviral effects to H9N2 avian influenza virus

In this paper, we report a soluble expression based on Escherichia coli and two-step purification of a novel thioredoxin-tagged chicken interferon-α fusion protein (Trx-rChIFN-α) by using pET32a(+) expression system. The mature ChIFN-α gene was amplified by Reverse transcriptase-polymerase chain reaction (RT-PCR) and subcloned into pET-32a (+) vector prior to transformation into Rosetta (DE3) competent cells. After IPTG induction, the recombinant fusion protein was expressed efficiently in the soluble fraction. The protein purification was performed by nickel affinity chromatography and DEAE anion exchange chromatography. The purified product has a purity of 95% with a yield of 47.3 mg/L of culture. The specific activity of the fusion protein reaches to 2.0 × 107 IU/mg as determined in the CEF/VSV titration system. After excision of the Trx tag by enterokinase, the remaining solo protein was confirmed as rChIFN-α protein by SDS-PAGE, N-terminal sequencing and mass spectrometry. The effects of this Trx-rChIFN-α fusion protein against H9N2 influenza virus infection were also evaluated in ovo. The results showed that the Trx-rChIFN-α protein could significantly reduce the hemagglutination titer of H9N2 virus, and the H9N2 viruses HA gene copy numbers. These findings will enable us to produce large amount and bio-active rChIFN-α protein for future applications.

Identification of a small antimycotic peptide produced by Bacillus amyloliquefaciens 6256

Bacillus sp. 6256 is a good biocontrol agent against Botrytis cinerea which caused tomato gray mold disease. Strain 6256 was identified as B. amyloliquefaciens by analysis of its partial gyrB gene sequence. To identify and characterize the antimycotic peptides from the culture broth of the bacterium, the antimicrobial substances produced by B. amyloliquefaciens 6256 were isolated by ammonium sulfate precipitation, Superdex 200 gel filtration chromatography and DEAE anion exchange chromatography. The purified compound was designated as P657. The biological activity of P657 was stable at as high as 100 °C for 20 min and in pH value ranged from 5 to 10. The antimycotic compound was resistant to trypsin and proteinase K, and could completely inhibit spore germination of Botrytis cinerea in vitro. MALDI-TOF-MS analysis results showed the presence of fengycins A (C16–C17) and fengycins B (C15–C17) isoforms in P657.

Effects of Cu2+ on alkaline phosphatase from Macrobrachium rosenbergii

To gain insight into the effect of Cu2+ on the activity and structure of alkaline phosphatase (ALP) from Macrobrachium rosenbergii, the enzyme was purified using ammonium sulfate fractionation, Sephacryl S-200, and DEAE anion exchange chromatography. We studied Cu2+-mediated inhibition and aggregation of ALP, and found that Cu2+ significantly inactivated ALP activity with an IC50 of 1.47 ± 0.02 mM. We further revealed that Cu2+ reversibly inhibited ALP in a mixed-type manner with Ki = 0.41 ± 0.02 mM. Time-interval kinetics showed that the inhibition followed first-order reaction kinetics. This process was associated with conformational changes and significant transient free-energy change. Spectrofluorometry results showed that Cu2+ induced ALP tertiary structural changes, including the exposure of hydrophobic surfaces that directly induced ALP aggregation. The results provide new information regarding ALP from M. rosenbergii.

Characterization and prebiotic activity in vitro of inulin-type fructan from Codonopsis pilosula roots

The inulin-type fructan was obtained by DEAE anion exchange chromatography from C. pilosula Nannf. var. modesta (Nannf.) L. T. Shen, after optimized extract condition, which was established by response surface methodology, designed using Box-Behnken factorial design and the optimum condition were: extracting 2.5 h with ratio of solvent to material 40 mL/g at 100 °C, twice. The maximum extraction yield was 20.6 ± 0.2%. It was confirmed as β-(2-1) linkage fructan, with terminal glucose, and with a degree of polymerization of 2–17 (DPav = 6), shown by the results of methanolysis, methylation, nuclear magnetic resonance and molecular weight determination. The prebiotic activity was proven on account of stimulation effect on Lactobacillus and pH reduction of medium in vitro. The results indicated that the inulin from C. pilosula could be used as a potential natural source of prebiotics.

Macrophage-stimulating activities of newly isolated complex polysaccharides from Parachlorella kessleri strain KNK-A001

Our previous studies demonstrated that the microalga Parachlorella kessleri (KNK-A001) has immunostimulatory activities, which were observed as an increase in natural killer (NK) cell activity in mice after intraperitoneal injection or as a protective effect on a virus-infected model shrimp after oral administration. In this study, we attempted to gain insight into the constituent substances of KNK-A001 that are responsible for the immunostimulatory activity. First, we obtained five polysaccharide fractions from KNK-A001 by DEAE anion exchange chromatography. Among the fractions, F5 showed the most potent induction of nitric oxide (NO) secretion in RAW264.7 cells, and both mRNA and protein expression levels of inducible NO synthase (iNOS) were increased in F5-treated RAW264.7 cells. A significant increase in the nuclear translocation of the p65 subunit of nuclear factor-kappa B (NF-κB) was observed in F5-treated RAW264.7 cells. F5 also induced the secretion of tumor necrosis factor (TNF)-α in RAW264.7 cells. Analysis using mitogen-activated protein (MAP) kinase inhibitors suggested that c-Jun N-terminal kinase (JNK) and p38 MAP kinase were mainly involved in F5-induced NO and TNF-α productions. The compositional analysis of F5 identified the main constituents as galactose, glucose, galacturonic acid, and mannose. Gel-filtration analysis suggested that molecular mass of F5 was approximately 400kDa.