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Grifola frondosa mycelium anti-tumor glycoprotein and preparation method

A technology of Grifola frondosa mycelium and glycoprotein, which is applied in the field of bioengineering, can solve the problems of damaging product activity, restricting universality, and affecting product safety, etc., and achieves short production cycle, obvious technical advantages, broad application prospects and The effect of potential economic benefits

Active Publication Date: 2014-01-15
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the patents disclosed above, in the process of extracting polysaccharides and their complexes, reagents such as acetic acid and strong alkali are used, and even steps such as deproteinization that may damage the activity of the product are involved, which limits the use of this type of product to a certain extent. The extensiveness of the product also affects the safety of the product
[0006] However, the research on the purification method, structural analysis and biological activity of glycoprotein or glycopeptide in the submerged fermentation mycelium of Grifola frondosa has not been reported or technically protected

Method used

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  • Grifola frondosa mycelium anti-tumor glycoprotein and preparation method

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Experimental program
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Effect test

Embodiment 1

[0038] Grifola frondosa strain ( Grifola frondosa 50072), purchased from China Microorganism Culture Collection Center. The strains were first cultured on slant media. The test tubes and tampons used for culture need to be kept at 121 o C sterilized for 30 min, and then filled with medium 121 o C sterilization for 30 min, placed on a slant to cool to make it solidify into a slant. Then the ultra-clean bench was inoculated. The slant was cultured at a constant temperature of 25 o C or so, for a period of 7 days. Cut the slant bacteria into small pieces and inoculate them into the seed culture medium. After the medium is divided, put a cotton plug on the conical flask to prevent external microorganisms from entering the medium and cause pollution, and ensure good ventilation performance. . The composition of the seed culture solution is: glucose 20 g / L, peptone 2 g / L, KH 2 PO 4 2 g / L, MgSO 4 ·7H 2 O 1 g / L, corn steep liquor 15 g / L, the rest is water, pH is natura...

Embodiment 2

[0040] 100 g of Grifola frondosa mycelium was added to 1 times the volume of water and the homogenate was crushed, 4 o C extraction, centrifugation, discard insoluble matter, collect supernatant, add ammonium sulfate to 80% saturation after supernatant concentration, let it stand for 24 hours, high-speed centrifugation to get the precipitate, add a small amount of water to redissolve the above precipitate, and reconstitute The solution was placed in a dialysis bag of 8000-14000 Da, dialyzed with deionized water, the dialysate was collected, concentrated, and subjected to DEAE-Sepharose Fast Flow column chromatography, followed by gradient elution with distilled water and different concentrations of NaCl as the eluent, and collected Glycoprotein complex fractions eluted with different concentrations of NaCl solutions, and each collection was freeze-dried.

[0041] OD 280 On-line detection showed 3 peaks in total; the anti-tumor activity was detected, and the anti-tumor activit...

Embodiment 3

[0043] 100 g of Grifola frondosa mycelium was added to 1 times the volume of water and the homogenate was crushed, 4 o C extraction, centrifugation, discard the insoluble matter, collect the supernatant, add ammonium sulfate to 70% saturation after concentrating the supernatant, let it stand for 24 hours, centrifuge at high speed to get the precipitate, add a small amount of water to redissolve the above precipitate, and reconstitute The solution was placed in a dialysis bag of 8000-14000 Da, dialyzed with deionized water, the dialysate was collected, concentrated, and subjected to DEAE-Sepharose Fast Flow column chromatography, followed by gradient elution with distilled water and different concentrations of NaCl as the eluent, and collected Glycoprotein complex fractions eluted with different concentrations of NaCl solutions, and each collection was freeze-dried.

[0044] OD 280 On-line detection yielded 3 peaks; the anti-tumor activity detection showed that the D2 componen...

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Abstract

The invention discloses a grifola frondosa mycelium anti-tumor glycoprotein and a preparation method, and belongs to the technical field of bioengineering. The glycoprotein is prepared by the following steps: using submerged fermented grifola frondosa mycelium as a raw material, homogenating and crushing, extracting with cold water, centrifuging, taking supernatant, precipitating with ammonium sulfate, dialyzing, and carrying out DEAE (diethyl-aminoethanol) -sepharose Fast Flow and superdexTM 75 prepgrad column chromatography and other steps to systematically separate and purify. The glycoprotein is a compound of polysaccharide and protein, wherein polysaccharide is 2-10% in content and composed of four monosaccharides including arabinose, fructose, mannose and glucose; the protein is 30-90% in content and composed of 17 amino acids including aspartic acid, methionine, glutamic acid and so on; the molecular weight of the glycoprotein is 30-90 KDa. The glycoprotein can inhibit growth of human gastric carcinoma cell SGC-7901 and human breast cancer cell MCF-7, and can be used for preparing a possible anti-cancer drug. Besides, the grifola frondosa mycelium anti-tumor glycoprotein can be generally applied to separation and purification of glycoprotein obtained from mycelium of various officinal and edible fungi through submerged fermentation.

Description

technical field [0001] The invention relates to a Grifola frondosa submerged fermentation mycelia glycoprotein with antitumor and other activities and a preparation method thereof, belonging to the technical field of bioengineering. Background technique [0002] The separation and structure determination of the effective components of edible and medicinal fungi is one of the current research hotspots. The types and contents of active ingredients contained in edible and medicinal fungi directly determine different physiological activities and functions, so different fungal metabolites have their unique chemical composition and physiological activity. Fungal polysaccharides and their complexes, together with proteins and nucleic acids, are the three most important biological macromolecules. With the further improvement of methods such as separation, purification, composition determination and structural analysis of polysaccharides, the biological functions of polysaccharides ...

Claims

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Application Information

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IPC IPC(8): C07K14/375C07K1/36C07K1/30C07K1/16A61P35/00
CPCC07K14/375
Inventor 崔凤杰昝新艺李云虹孙文敬张志才钱静亚黄达明董英
Owner JIANGSU UNIV
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