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Method for large-scale preparation of high-purity unsaturated hyaluronic acid disaccharide

A technology for large-scale preparation and hyaluronic acid, which is applied in the field of oligomeric hyaluronic acid preparation, can solve problems such as unfavorable industrial production, low product purity and reduction, and achieve the effects of less impurities, high product purity and good precipitation effect.

Active Publication Date: 2020-04-10
BLOOMAGE BIOTECHNOLOGY CORP LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] Aiming at the problems that the purification method for preparing unsaturated hyaluronic acid disaccharide in the prior art is not conducive to industrial production, and the purity of the obtained product is low, the present invention provides a method for large-scale preparation of high-purity unsaturated hyaluronic acid disaccharide, The method uses high-molecular hyaluronic acid or its salt as a raw material, firstly through the first enzymatic hydrolysis to reduce the molecular weight of hyaluronic acid or its salt, and after enzymatic hydrolysis, it is preliminarily purified by organic solvent precipitation to obtain high-purity hyaluronic acid. Low-molecular-weight hyaluronic acid or its salts, and then low-molecular-weight hyaluronic acid or its salts are subjected to a second enzymatic hydrolysis at a higher concentration. After enzymatic hydrolysis, high-fold organic solvent precipitation is used for purification, and finally high-purity Unsaturated hyaluronic acid disaccharide

Method used

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  • Method for large-scale preparation of high-purity unsaturated hyaluronic acid disaccharide
  • Method for large-scale preparation of high-purity unsaturated hyaluronic acid disaccharide
  • Method for large-scale preparation of high-purity unsaturated hyaluronic acid disaccharide

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] 1. To 1.5 m 3 Add 1 m to the stainless steel dissolution tank 3 Purify water, raise the temperature to 30°C, and adjust the pH to 5.0 with hydrochloric acid.

[0047] 2. Set the stirring speed to 20 rpm, and add 1.8×10 8 IU of Bacillus hyaluronidase, after stirring for 1 min, slowly add 100 kg of sodium hyaluronate with a molecular weight of 2000 kDa for 1 h. Add 50 kg of sodium hyaluronate for 1 hour, continue enzymatic hydrolysis for 1 hour after adding, then continue to slowly add 30 kg of sodium hyaluronate with a molecular weight of 2000 kDa for 1 hour, continue enzymatic hydrolysis until the molecular weight is reached When it is less than 50 kDa, raise the temperature to 50°C and maintain it for 60 min.

[0048] 3. Filter the enzymatic hydrolyzate after deactivation with a 0.45 µm mixed cellulose filter, and then filter it with a 3 m 3 Precipitate with ethanol to obtain sodium hyaluronate precipitate, dehydrate the precipitate with ethanol, and then vacuum dr...

Embodiment 2

[0052] 1. To 1.5 m 3 Add 1 m to the stainless steel dissolution tank 3 Purify water, raise the temperature to 35°C, and adjust the pH to 6.0 with hydrochloric acid.

[0053] 2. Set the stirring speed to 30 rpm, and add 2.4×10 7 IU of Bacillus hyaluronidase, after stirring for 1 min, slowly add 80 kg of potassium hyaluronate with a molecular weight of 1300 kDa for 1 h, enzymatic hydrolysis for 1 h after adding, and then continue to slowly add potassium hyaluronate with a molecular weight of 1300 kDa Add 40 kg of potassium hyaluronate for 1 hour, continue enzymatic hydrolysis for 1 hour after adding, then continue to slowly add 20 kg of potassium hyaluronate with a molecular weight of 1300 kDa for 1 hour, continue enzymatic hydrolysis until When the molecular weight is less than 80 kDa, raise the temperature to 55°C and maintain it for 40 min.

[0054] 3. Filter the enzymatic hydrolyzate after deactivation with a 0.45 µm mixed cellulose filter, and then filter it with a 5 m ...

Embodiment 3

[0058] 1. To 1.5 m 3 Add 1 m to the stainless steel dissolution tank 3 Purify water, raise the temperature to 40°C, and adjust the pH to 6.5 with hydrochloric acid.

[0059] 2. Set the stirring speed to 50 rpm, and add 1.0×10 7 IU of Bacillus hyaluronidase, after stirring for 1 min, slowly add 40 kg of calcium hyaluronate with a molecular weight of 1800 kDa for 1 h, enzymatic hydrolysis for 1 h after adding, and then continue to slowly add calcium hyaluronate with a molecular weight of 1800 kDa Add 35 kg of calcium hyaluronate for 1 hour, continue enzymatic hydrolysis for 1 hour after adding, then continue to slowly add 25 kg of calcium hyaluronate with a molecular weight of 1800 kDa for 1 hour, continue enzymatic hydrolysis until the molecular weight is reached When it is less than 55 kDa, raise the temperature to 60°C and maintain it for 20 min.

[0060] 3. Filter the enzymatic hydrolysis solution after deactivation with a 0.45 µm mixed cellulose filter, and then filter i...

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Abstract

The invention discloses a method for large-scale preparation of high-purity unsaturated hyaluronic acid disaccharide. The method adopts two-step enzymolysis and two-step purification, and specificallyincludes the steps of: firstly, carrying out enzymolysis on macromolecular hyaluronic acid or salt thereof by hyaluronidase, purifying the obtained product with a low organic solvent multiple to obtain low-molecular-weight hyaluronic acid or salt thereof with high purity, further performing thorough enzymolysis on the low-molecular-weight hyaluronic acid or salt thereof, and purifying the obtained product with a high organic solvent multiple to obtain the high-purity unsaturated hyaluronic acid disaccharide. The method has simple technological process operation, mild conditions, high production efficiency, low energy consumption, and is suitable for large-scale industrial production. The obtained product has high purity and low cytotoxicity, has the characteristic of promoting the proliferation of human umbilical vein endothelial cells and human corneal epithelial cells, can be used as a standard substance for content and purity detection of hyaluronic acid and related products thereof, and also has wide application prospect in the field of medicines.

Description

technical field [0001] The present invention relates to a preparation method of unsaturated hyaluronic acid disaccharide, in particular to a preparation method of unsaturated hyaluronic acid disaccharide with high purity, large yield and suitable for large-scale production, which belongs to the preparation technology of oligomeric hyaluronic acid field. Background technique [0002] Hyaluronic acid (HA) is an acidic mucopolysaccharide widely present in humans and mammals. It is composed of N-acetylglucosamine and D-glucuronic acid disaccharide repeating units through β-(1→ 4) Unbranched single-chain polymer glycosaminoglycan composed of glycosidic bonds and β-(1→3) glycosidic bonds. Due to the unique physical properties of hyaluronic acid, high moisture retention, viscoelasticity, and lubricity, it is widely used in medicine, cosmetics, food and other fields, and its molecular weight is generally 10. 5 ~10 7 Da (Dalton). [0003] Oligomeric hyaluronic acid refers to hyal...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/26C12P19/14C07H3/04C07H1/06
CPCC12P19/26C12P19/14C07H3/04C07H1/06
Inventor 乔莉苹石艳丽陈玉娟李强冯宁周宁刘福全郭学平刘爱华
Owner BLOOMAGE BIOTECHNOLOGY CORP LTD
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