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Method for preparing ultra-low molecular weight hyaluronic acid oligosaccharides and salt thereof through combination of solid-liquid biphasic enzymolysis and ultrafiltration

A technology of hyaluronic acid oligosaccharides and hyaluronic acid, applied in the direction of fermentation, etc., can solve the problems of hyaluronic acid time-consuming, labor-intensive, rising, high price of hyaluronidase, etc., and achieve low production costs and reduced production costs , high thermal stability and pH stability

Active Publication Date: 2018-06-29
BLOOMAGE BIOTECHNOLOGY CORP LTD +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, as the molecular weight of hyaluronic acid obtained by degradation decreases, the amount of enzyme required for the degradation process will increase, and the amount of enzyme required for the preparation of hyaluronic acid oligosaccharides by enzymatic hydrolysis is far greater than that required for the preparation of low molecular weight hyaluronic acid. The amount of enzyme needed, and commercial hyaluronidase is expensive, so it has not been possible to carry out large-scale industrial production to prepare hyaluronic acid oligosaccharides
[0006] In addition, hyaluronic acid is a high-molecular polysaccharide, and its solution viscosity increases with the increase of molecular weight and concentration. During the enzymolysis process, adding hyaluronic acid to the enzymolysis solution is easy to form lumps of external liquid and internal solid, transparent Time-consuming and labor-intensive process of urethane dispersion

Method used

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  • Method for preparing ultra-low molecular weight hyaluronic acid oligosaccharides and salt thereof through combination of solid-liquid biphasic enzymolysis and ultrafiltration
  • Method for preparing ultra-low molecular weight hyaluronic acid oligosaccharides and salt thereof through combination of solid-liquid biphasic enzymolysis and ultrafiltration
  • Method for preparing ultra-low molecular weight hyaluronic acid oligosaccharides and salt thereof through combination of solid-liquid biphasic enzymolysis and ultrafiltration

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] to 1m 3 Add 1m to the stainless steel dissolution tank 3 Purified water, warming up to 20°C, adjusting the pH to 4.5, adding 4.8×10 7 IU of Bacillus hyaluronidase, after stirring for 5 minutes, add 1kg of zirconia dispersion beads with a diameter of 1.8-2.0mm, adjust the stirring speed to 30-40rmp, then quickly add 60kg of potassium hyaluronate with a molecular weight of 20kDa, and the addition time is 2min, continue to stir at this speed for 3-5min after adding, then adjust the stirring speed to 15-20rpm, stir evenly, and obtain a hyaluronic acid-enzymolysis liquid-solid-liquid two-phase enzymatic hydrolysis system; enzymolysis at 15-20rpm After 0.5h, adjust the rotation speed to 30-40rmp again, add 60kg of potassium hyaluronate with a molecular weight of 20kDa, the addition time is 3min, stir for 3-5min after adding, adjust the rotation speed to 15-20rpm, and stir evenly to obtain hyaluronic acid Solid-liquid two-phase enzymatic hydrolysis system; after enzymolysis ...

Embodiment 2

[0045] to 1m 3 Add 1m to the stainless steel dissolution tank 3 Purified water, warming up to 30°C, adjusting the pH to 4.0, adding 5.6×10 7 IU of Bacillus hyaluronidase, after stirring for 5 minutes, add 1kg of zirconia dispersion beads with a diameter of 1.8-2.0mm, adjust the stirring speed to 30-40rmp, then add 70kg of calcium hyaluronate with a molecular weight of 10kDa, and the addition time is 1 -2min, continue to stir at this speed for 3-5min after adding, then adjust the stirring speed to 15-20rpm, stir evenly, and obtain a hyaluronic acid-enzymolysis liquid-solid-liquid two-phase enzymatic hydrolysis system; After solution for 0.5h, adjust the rotation speed to 30-40rmp again, add 70kg of calcium hyaluronate with a molecular weight of 10kDa, the addition time is 2-3min, stir for 3-5min after adding, adjust the stirring speed to 15-20rpm, stir evenly, The hyaluronic acid solid-liquid two-phase enzymatic hydrolysis system was obtained; after enzymatic hydrolysis at 15...

Embodiment 3

[0048] to 1m 3 Add 1m to the stainless steel dissolution tank 3 Purified water, warming up to 35°C, adjusting the pH to 5.0, adding 1.2×10 8 IU of bacillus hyaluronidase, after stirring for 5min, add 1.4kg of zirconia dispersion beads with a diameter of 1.8-2.0mm, adjust the stirring speed to 30-40rmp, then add 100kg of sodium hyaluronate with a molecular weight of 300kDa, and the addition time is 2-3min, continue to stir at this speed for 3-5min after adding, then adjust the stirring speed to 15-20rpm, stir evenly, and get hyaluronic acid-enzymolysis liquid solid-liquid two-phase enzymatic hydrolysis system; at 15-20rpm After 1 hour of enzymatic hydrolysis, adjust the rotation speed to 30-40rmp again, add 100kg of sodium hyaluronate with a molecular weight of 300kDa, the addition time is 3-4min, stir for 3-5min after adding, adjust the stirring speed to 15-20rpm, and stir evenly. The hyaluronic acid solid-liquid two-phase enzymatic hydrolysis system was obtained; after enzy...

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Abstract

The invention discloses a method for preparing ultra-low molecular weight hyaluronic acid oligosaccharides and a salt thereof through combination of solid-liquid biphasic enzymolysis and ultrafiltration. The method comprises the following steps: degrading hyaluronic acid and a salt thereof by utilizing hyaluronidase produced by bacilli; preparing high-concentration hyaluronic acid enzymatic hydrolysate by adopting the method of combining a solid-liquid biphasic enzymolysis system and an ultrafiltration system, and then performing inactivating, activated carbon adsorption and impurity removal,filtering and spray-drying to obtain the ultra-low molecular weight hyaluronic acid oligosaccharides and the salt thereof with the molecular weight of less than or equal to 3 kDa. The product preparedby the method is high in stability, ultra-low in molecular weight, good in inflammation resistance and percutaneous absorptivity, high in purity, strong in oxidation resistance, does not have cytotoxicity, has the advantages of repairing skin cell damage and the like, and can be widely applied to the fields of cosmetics, food and medicines. According to the method, the process flow is easy to operate; conditions are mild; various alcohols are not used; the energy consumption is relatively low; the method does not have damage to a product structure, does not have environmental pollution, and is suitable for large-scale industrial production.

Description

technical field [0001] The invention relates to a process for preparing hyaluronic acid oligosaccharides and salts thereof by enzymatic cleavage, in particular to a method for preparing ultra-low molecular weight hyaluronic acid oligosaccharides and salts thereof by combining solid-liquid two-phase enzymatic hydrolysis and ultrafiltration The method belongs to the technical field of hyaluronic acid. Background technique [0002] Hyaluronic acid (HA) is an acidic mucopolysaccharide composed of N-acetylglucosamine and D-glucuronic acid disaccharide repeating units through β-(1→4) glycosidic bonds and β-(1 →3) Unbranched high-molecular glycosaminoglycans composed of glycosidic bonds exist in the intercellular matrix of animal tissues and the capsule of some bacteria. Hyaluronic acid is widely used in medicine, cosmetics, food and other fields, and its molecular weight is generally 10 5 ~10 7 Da (Dalton). Oligomeric hyaluronic acid refers to hyaluronic acid with a molecular ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/26C12P19/14C08B37/08
CPCC08B37/0072C12P19/14C12P19/26
Inventor 乔莉苹申凤同陆震薛蔚吕旸范馨仪石艳丽郭学平
Owner BLOOMAGE BIOTECHNOLOGY CORP LTD
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