A kind of preparation method of sea cucumber fucoidan sulfate ester and chondroitin sulfate oligosaccharide

A technology of fucoidan sulfate and chondroitin sulfate, which is applied in the field of small molecule compound preparation, can solve the problems of complicated desalination steps and high cost of ion exchange chromatography column, and achieves simplified separation and degradation process, low cost and short heating time. Effect

Active Publication Date: 2021-08-31
OCEAN UNIV OF CHINA
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  • Application Information

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Problems solved by technology

However, the cost of ion-exchange chromatographic columns is high, and the product desalting steps are cumbersome

Method used

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  • A kind of preparation method of sea cucumber fucoidan sulfate ester and chondroitin sulfate oligosaccharide
  • A kind of preparation method of sea cucumber fucoidan sulfate ester and chondroitin sulfate oligosaccharide
  • A kind of preparation method of sea cucumber fucoidan sulfate ester and chondroitin sulfate oligosaccharide

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Experimental program
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Effect test

Embodiment 1

[0026] 1) Dried Apostichopus japonicus Dried sea cucumber is crushed into powder state. Weigh 10g powder, add 15mmol / L EDTA-Na2, 5mmol / L cysteine, 300mL 0.1mol / L pH 8.0 phosphate buffer solution, 500mg alkaline protease and 150mg trypsin, and stir in a water bath at 50°C for enzymolysis 24h. Afterwards, the reactant was boiled in a water bath for 5 min to inactivate the enzyme, and cooled to room temperature. Centrifuge at 5000×g for 10 min, and take the supernatant.

[0027] Add 16 mL of 10% cetylpyridinium chloride aqueous solution to the obtained supernatant (i.e. sea cucumber enzymatic hydrolysis solution), let stand at room temperature for 5 hours, centrifuge at 2000×g for 15 minutes at 20°C, discard the supernatant to obtain a precipitate .

[0028] The obtained precipitate was dissolved in 150 mL of NaCl ethanol solution (3 mol / L sodium chloride, 86% ethanol). Then add 300mL of 95% ethanol, place it at 4°C for 5h, centrifuge (4000×g, 15min, 20°C), and take the preci...

Embodiment 2

[0042] In order to specifically verify the influence of sample volume and degradation time on the prepared fucoidan sulfate and fucosyl sulfate chondroitin oligosaccharides in steps 3) and 5), change the sample volume and degradation time in steps 3) and 5). Time, all the other are with embodiment 1. The specific sample volume, degradation time and corresponding yield, molecular weight and sulfate content of fucoidan sulfate ester and fucosyl sulfate chondroitin oligosaccharide are shown in Table 2.

[0043] 2.1: The volume of the sea cucumber sulfated polysaccharide solution was increased to 100 mL for thermal degradation, and the yield of the obtained fucoidan sulfated oligosaccharides was 21.2%, and the yield of fucosyl chondroitin sulfated oligosaccharides was 43.2%. The weight average molecular weight of the fucoidan sulfate oligosaccharide is 4563.64Da, the number average molecular weight is 3213.83Da, and the dispersion coefficient is 1.42. The weight average of fucosy...

Embodiment 3

[0048] In order to verify the purity of the prepared fucoidan sulfate oligosaccharides and fucosyl chondroitin sulfate oligosaccharides, the liquid chromatography-mass spectrometry technique was used to analyze the fucoidan sulfate oligosaccharides and rock Composition of Cocosyl Chondroitin Sulfate Oligosaccharides.

[0049] 1) The method of liquid chromatography-mass spectrometry analysis is as follows: 10 mg / mL sulfated polysaccharide degradation products were mixed with acetonitrile in equal volumes, and then centrifuged at 10 000 r / min for 10 min. The supernatant was taken, and the composition and structure of oligosaccharides in the product were analyzed by high performance liquid chromatography-mass spectrometry. Chromatographic instrument: UltiMate 3000 high performance liquid chromatograph, liquid condition: chromatographic column is Luna 3μHILIC (2.0mm×150mm, ), mobile phase A is 5mmol / L ammonium acetate aqueous solution 10mL sea cucumber sulfate polysaccharide pH5...

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Abstract

The invention provides a preparation and separation and purification method of sea cucumber fucoidan sulfate oligosaccharides and chondroitin sulfate oligosaccharides, that is, the sea cucumber sulfate polysaccharide mixture extracted from sea cucumbers is used as raw material, and the pH value of the sea cucumber sulfate polysaccharide solution is adjusted 5.0-6.0, use 110-115°C high-pressure steam to heat and degrade for 60-90 minutes, so that the fucoidan sulfate is first degraded into oligosaccharides, and the fucoidan sulfate oligosaccharides are separated by ultrafiltration. Then the ultrafiltration retentate with a molecular weight greater than 5kDa was adjusted to a pH of 3.0-4.0, thermally degraded at 120-130°C for 90-120 minutes, concentrated and freeze-dried to obtain sea cucumber fucosyl chondroitin sulfate oligosaccharides. The method of the invention saves steps such as ion chromatography separation, gel chromatography purification and desalination of fucoidan sulfate and chondroitin sulfate in sea cucumbers, and realizes the degradation and separation and purification of sea cucumber fucoidan sulfate and chondroitin sulfate in one step , which greatly simplifies the preparation process.

Description

technical field [0001] The invention belongs to the technical field of preparation of small molecular compounds, and in particular relates to a preparation method of sea cucumber fucoidan sulfate ester and chondroitin sulfate oligosaccharide. Background technique [0002] Sea cucumber belongs to the Echinodermata genus Holothuria, and is a sea treasure with important nutritional and economic value. There are two sulfated polysaccharides, fucoidan sulfate and fucosyl chondroitin sulfate, in the body wall of sea cucumbers. Fucoidan sulfate is a straight-chain sulfuric acid formed by linking fucose with α1→3 or α1→4 bonds A polysaccharide with a small amount of fucose side chains. Sea cucumber fucosyl chondroitin sulfate is a glycosaminoglycan with glucuronic acid and acetylgalactosamine as repeating disaccharide units, connected by β-1,4- and β-1,3 glycosidic bonds, chondroitin sulfate There are sulfated fucose branched chains on the acetylgalactosamine and glucuronic acid o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C08B37/00C08B37/08
CPCC08B37/0003C08B37/006C08B37/0069
Inventor 赵雪杨毅张鸿伟卢海燕石德玲薛长湖
Owner OCEAN UNIV OF CHINA
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