Preparation method for k-carrageenan oligosaccharide with low polymerization degree

A technology of carrageenan oligosaccharides and low degree of polymerization, which is applied in the field of microorganism and enzyme engineering, and can solve the problems of poor solubility and absorbability

Inactive Publication Date: 2013-05-22
WEIHAI KANGBOER BIOLOGICAL PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the large molecular weight of carrageenan, its solubility and absorbability are poor, which limits its application.

Method used

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  • Preparation method for k-carrageenan oligosaccharide with low polymerization degree
  • Preparation method for k-carrageenan oligosaccharide with low polymerization degree
  • Preparation method for k-carrageenan oligosaccharide with low polymerization degree

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Thin-layer chromatography (TLC) detection of κ-carrageenan oligosaccharides

[0024] Mix TLC silica gel powder and sodium carboxymethyl cellulose in a ratio of 1:3, grind thoroughly, spread evenly on a glass plate, activate at 100°C for 2 hours, and store in a desiccator for later use. Use a capillary pipette to spot the sample on the prepared silica gel chromatography plate. The distance between the point of sample point and the bottom of the plate is 1cm, and the distance between the point of point of sample point is 1cm. Use a hair dryer to slowly blow over the point of sample point to make the solvent evaporate quickly. Put the chromatographic plate in the chromatographic tank and develop it at room temperature, wait until the developing agent (n-butanol: glacial acetic acid: water is 2:1:1) is developed to a distance of 1 cm from the top of the plate, take it out and dry it, and use the chromogenic agent ( Aniline-diphenylamine) for color development, an...

Embodiment 2

[0026] Embodiment 2: HPLC and ESI-TOF-MS detection carrageenan enzymolysis product

[0027] Agilent1100 high-performance liquid chromatography was used to separate the preliminary purified samples. Separation conditions: mobile phase A: ACN (acetonitrile), mobile phase B: 100 mM ammonium formate (pH 3.0), gradient: 80% to 60% A, time 0 to 20 min. The molecular weight of κ-carrageenan oligosaccharides was detected by ESI-TOF-MS in positive ion mode (G1969A, Agilent, UN).

[0028] Results: After preliminary separation and purification of enzymatically hydrolyzed κ-carrageenan oligosaccharides, the results obtained by high performance liquid chromatography were as follows: figure 2 , as shown in the figure, three sample peaks b, c, and d are obtained, indicating that the degradation products of κ-carrageenan degrading enzyme are three components, and peak a is the solvent peak.

[0029] The ESI-TOF-MS spectrum shows that the basic unit of κ-carrageenan oligosaccharide is disac...

Embodiment 3

[0030] Example 3: 13 C-NMR chromatography to detect the structure of oligosaccharides

[0031] Use German Bruker AVANCE500MHz NMR instrument to carry out oligosaccharide 13 C-NMR spectrum analysis, 10mg sample was dissolved in 0.5mL 2 h 2 In O, the detection process was carried out at 30°C.

[0032] result: 13 C-NMR chromatography is used to detect the structure of the enzymatic hydrolysis product, such as image 3 , only one characteristic signal was detected at 102.64ppm, and the carbon reducing end of the A-terminus of the oligosaccharide was detected at 96.79ppm and 95.08ppm.

[0033] NMR spectroscopy can provide valuable information for the structural analysis of polysaccharides, especially polysaccharide molecules composed of the same or similar block units. pass 13 C-NMR analysis can determine the action site and enzymatic hydrolysis mode of κ-carrageenan degrading enzyme cleavage. Such as image 3The spectrum of κ-carahexaose sulfate shown shows two characteri...

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Abstract

The invention discloses a preparation method for k-carrageenan oligosaccharide with a low-polymerization degree. The preparation method comprises the following steps of: carrying out enzymolysis preparation on the k-carrageenan; and separating and purifying the degradation products to obtain pure k-carrageenan oligosaccharide mixing component. The preparation method is characterized in that carrageenan degradative enzyme is obtained from a marine microorganism Cellulophaga lytica strain: N5-2(NCBI (National Center of Biotechnology Information), GENBANKD registry number being GU129978) and used for degrading the k-carrageenan to obtain the k-carrageenan oligosaccharide mixture with the low polymerization degree. The enzyme is an extracellular enzyme, can be acted to beta-1,4 glucosidic bond of the k-carrageenan, wherein the acting sites are respectively octasaccharide and hexaose; and the product is low-polymerization degree oligosaccharide consisting of disaccharide repeating units. The preparation method disclosed by the invention is low in cost and simple. Compared with the methods such as chemical method and a physical method, the obtained product is single, the side products are less and the large-scale production is convenient.

Description

technical field [0001] The invention relates to the fields of microorganisms, enzyme engineering and the like. The present invention degrades κ-carrageenan with extracellular enzymes produced by isolating natural marine microbial strains to obtain κ-carrageenan oligosaccharides with a low degree of polymerization, the main product of which is a mixture with a degree of polymerization of 2, 6, and 8. The sugar component is relatively simple, and has various activities such as anti-tumor, anti-oxidation, anti-virus and immune regulation. Background technique [0002] Kappa-carrageenan is an acidic sulfated polysaccharide composed of repeating units of 3,6-endether-α-D-galactose and 4-sulfate-β-D-galactose. In recent years, studies have found that carrageenan has various biological activities such as lowering blood lipids, anticoagulation, antithrombosis, immune regulation, and stimulating connective tissue growth, showing attractive application prospects in the field of biome...

Claims

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Application Information

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IPC IPC(8): C12P19/14C12R1/01
Inventor 吴海歌姚子昂于国友于丽萍
Owner WEIHAI KANGBOER BIOLOGICAL PHARMA
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