Trichoderma pseudokoningii exocellular polysaccharide, preparation method and application thereof

A technology of Trichoderma cells and polysaccharides, applied in the field of microorganisms, can solve problems such as increasing the spleen index, and achieve the effect of enhancing the phagocytic activity of macrophages

Inactive Publication Date: 2008-07-16
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there are no reports or patent publications about the immune activity of the exopolysaccharide of Trichoderma pseudoconii CGMCCNo.1443. The polysaccharide we isolated and purified from the fermentation broth of Trichoderma pseudoconii has strong immune activation ability and can significantly induce Apoptosis of fibroma cells prolongs the lifespan of S180 ascites tumor mice, reduces tumor weight and increases spleen index of S180 solid tumor mice

Method used

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  • Trichoderma pseudokoningii exocellular polysaccharide, preparation method and application thereof
  • Trichoderma pseudokoningii exocellular polysaccharide, preparation method and application thereof
  • Trichoderma pseudokoningii exocellular polysaccharide, preparation method and application thereof

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Embodiment 1

[0037] The preparation of embodiment 1 Trichoderma pseudoconii exopolysaccharide

[0038](1) Preparation of crude polysaccharide: Trichoderma pseudoconii CGMCC No.1443 was inoculated on PDA medium, activated at 28°C for 2 days, inserted into liquid medium from the plate, and cultured with shaking at 28°C and 130rpm for 6 days. Filter with four layers of gauze to remove hyphae and spores, centrifuge at 4000rpm for 15min, decolorize the supernatant through a 3520 resin column, collect the eluate, and concentrate under reduced pressure at 48°C to 1 / 4 of the original volume. The concentrate was deproteinized by the Sevag method (chloroform:n-butanol=4:1, V / V). Add 3 times the volume of 95% ethanol and let it stand overnight at 4°C, centrifuge at 5000rpm for 15 minutes, collect the precipitate, wash with ether, acetone, and 95% ethanol in sequence, dissolve the precipitate in distilled water, dialyze for 48 hours, concentrate to 1 / 4 of the original volume, and freeze Dried to obta...

Embodiment 2

[0041] Example 2 Purification of Trichoderma pseudoconii exopolysaccharide

[0042] Sephadex G-75 Sephadex column chromatography was used. Weigh 20g of Sephadex G-75 and suspend it in a large volume of water, let it settle naturally for 4 hours, remove suspended fine particles by pouring method, after repeated several times, add enough water to gradually raise the temperature to boiling, and keep it warm for 3 hours. Cool, vacuumize, and continuously load into a chromatographic column (2.5×80 cm) under gentle stirring, and pass 2-3 times of column bed volume of water to stabilize the column head. Accurately weigh 1.0 g of Trichoderma extracellular crude polysaccharide, dissolve it in 5 ml of water, put it on the column, and carry out elution with distilled water at room temperature at a flow rate of 0.4 ml / min, 15 min / tube. Use the phenol-sulfuric acid chromogenic method to track and detect tube by tube, and measure the light absorption value at 490nm. The elution volume was...

Embodiment 3

[0043] Example 3 Purity identification of Trichoderma pseudoconii exopolysaccharide

[0044] The purity and molecular weight of the polysaccharide samples were measured by high-performance gel filtration (HPGPC). According to the relationship between the molecular weight of polysaccharides on the gel column and the elution volume, a standard curve was first prepared with a series of standard dextran (Dextran) with different molecular weights. , and then compare it with the standard curve according to the elution volume of the sample (because the flow rate is constant, so the retention time is actually used). The purity of the sample was judged according to its peak shape, and the molecular weight of the sample was obtained from the standard curve.

[0045] The HPLC chromatograph used is composed of Waters 515 HPLC pump and Waters 2410 differential detector; the chromatographic column is two columns of Waters Ultrahydrogel 2000 and Ultrahydrogel 500 connected in series. The mo...

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Abstract

The invention discloses a polysaccharide from Trichoderma pseudokoningii, which is characterized in that (1) the high performance gel filtration chromatography test shows that the polysaccharide has a symmetrical peak with 18325 molecular weight; (2) an improved sulfuric acid-phenol method and a 3-aminophenol method test show that the neutral polysaccharide content is 65.2 percent, while the uronic acid content is 32.6 percent; (3) the GC analysis of the alditol acetate shows that the monosaccharide components are rhamnose, glucose and galactose, the molar ratio is Rha:Glc:Gal relates to 5.6:2.7:1.0, while when the polysaccharide is completely reduced, the molar ratio is Rha:Glc:Gal relates to 14.5:9.3: 1.0, having 26.6 molar percent glucuronic acid. The preparation method of the polysaccharide from Trichoderma pseudokoningii consists of the crude polysaccharide preparation and the polysaccharide purification. The polysaccharide from the Trichoderma pseudokoningii has broad applications in preparing the drugs or functional food for improving the mammal immunocompetence and for treating or adjuvant treating the tumors.

Description

technical field [0001] The invention belongs to the field of microorganisms, and relates to a polysaccharide capable of enhancing immunity and inhibiting tumor growth, in particular to an exopolysaccharide with immune activity produced by Trichoderma pseudokoningii CGMCC No.1443 (Trichoderma pseudokoningii) (abbreviated as Trichoderma polysaccharide, EPS) and its preparation method and use. Background technique [0002] According to the traditional classification system, Trichoderma belongs to Deuteromycotina, Hyphomycetes, Hyphomycetales, and Hyphomycetaceae. Trichoderma is a widely distributed fungus. Except for a few species which are weak pathogenic bacteria, most of them have antagonistic effect on plant pathogenic bacteria. It is the most researched and applied type of biocontrol fungus. It has inhibitory effect on various pathogenic bacteria. It is an ideal biocontrol fungus for preventing and controlling soil-borne diseases. [0003] Fungal polysaccharides are isol...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C08B37/00C12P19/04A61K31/715A61P35/00C12R1/885
Inventor 张鹏英陈靠山郝林华魏广金
Owner SHANDONG UNIV
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