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Method for preparing oligomeric hyalurate by digestion method, and oligomeric hyalurate and application thereof

A technology of hyaluronate and hyaluronic acid, applied in the biological field, can solve the problems of low degrading enzyme activity, high cost, and limited sources, and achieve broad application prospects, cost reduction, high enzyme thermal stability and pH stability Effect

Active Publication Date: 2013-01-16
BLOOMAGE BIOTECHNOLOGY CORP LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Aiming at the shortcomings of low enzyme activity, limited source and high cost for the biodegradation of hyaluronic acid or its salts, the inventor has isolated a Hyaluronidase-producing Bacillus (Bacillus sp.) A50 has been preserved in the General Microorganism Center (CGMCC) of the China Committee for the Collection of Microorganisms, and the preservation number is CGMCC NO. 5744, deposited on February 8, 2012

Method used

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  • Method for preparing oligomeric hyalurate by digestion method, and oligomeric hyalurate and application thereof
  • Method for preparing oligomeric hyalurate by digestion method, and oligomeric hyalurate and application thereof
  • Method for preparing oligomeric hyalurate by digestion method, and oligomeric hyalurate and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065]Slant medium composition (100mL): peptone 0.2g, yeast powder 2.0g, K 2 HPO 4 ·3H 2 O 0.05g, MgSO 4 ·7H 2 O 0.05g, glucose 0.5g, agar powder 2.0g, adjust the pH to 6.0 with hydrochloric acid.

[0066] Seed medium composition (100mL): peptone 0.2g, yeast powder 2.0g, K 2 HPO 4 ·3H 2 O 0.05g, MgSO 4 ·7H 2 O 0.05g, glucose 0.5g, adjust the pH to 6.0 with hydrochloric acid.

[0067] Fermentation medium composition (100mL): peptone 0.2g, yeast powder 2.0g, K 2 HPO 4 ·3H 2 O 0.05g, MgSO 4 ·7H 2 O 0.05g, glucose 0.5g, Tween80 0.05mL.

[0068] Take the slant bacteria (Bacillus ( Bacillus sp.) A50 CGMCC NO. 5744) inoculated in sterilized seed medium, 25°C, cultured at 150rpm for 24h, and then inoculated the seed liquid into sterilized fermentation medium with an inoculation amount of 10%, at 25°C , cultivated at 200rpm for 24 hours, maintained the pH at 6.0 with sulfuric acid during the fermentation process, and obtained hyaluronidase fermentation broth by fermen...

Embodiment 2

[0070] Slant medium composition (100mL): peptone 1.0g, yeast powder 1.0g, K 2 HPO 4 ·3H 2 O 0.1g, MgSO 4 ·7H 2 O 0.1g, glucose 1.0g, agar powder 2.0g, adjust the pH to 7.0 with phosphoric acid.

[0071] Seed medium composition (100mL): peptone 1.0g, yeast powder 1.0g, K 2 HPO 4 ·3H 2 O 0.1g, MgSO 4 ·7H 2 O 0.1g, glucose 1.0g, adjust the pH to 7.0 with phosphoric acid.

[0072] Fermentation medium composition (100mL): peptone 1.0g, yeast powder 1.0g, K 2 HPO 4 ·3H 2 O 0.1g, MgSO 4 ·7H 2 O 0.1g, glucose 1.0g, Tween80 0.05mL.

[0073] Take the slant bacteria (Bacillus ( Bacillus sp.) A50 CGMCC NO. 5744) inoculated in the sterilized seed medium, 30°C, 100rpm culture for 15h, then inoculated the seed liquid into the sterilized fermentation medium, the inoculation amount was 10%, 35°C , cultured at 300rpm for 16h, during the fermentation process, the pH was maintained at 7.0 with sulfuric acid, and the hyaluronidase fermentation liquid was obtained by fermentation....

Embodiment 3

[0075] Slant medium composition (100mL): peptone 1.5g, yeast powder 1.5g, K 2 HPO 4 ·3H 2 O 0.15g, MgSO 4 ·7H 2 O 0.15g, glucose 1.5g, agar powder 2.0g, adjust the pH to 8.0 with sulfuric acid.

[0076] Seed medium composition (100mL): peptone 1.5g, yeast powder 1.5g, K 2 HPO 4 ·3H 2 O 0.15g, MgSO 4 ·7H 2 O 0.15g, glucose 1.5g, adjust the pH to 8.0 with sulfuric acid.

[0077] Fermentation medium composition (100mL): peptone 0.5g, yeast powder 1.5g, K 2 HPO 4 ·3H 2 O 0.1g, MgSO 4 ·7H 2 O 0.05g, glucose 1.5g, Tween80 0.05mL.

[0078] Take the slant bacteria (Bacillus ( Bacillus sp.) A50 CGMCC NO. 5744) inoculated in sterilized seed medium, 35°C, 200rpm cultured for 13h, then inoculated the seed solution into sterilized fermentation medium, the inoculation amount was 10%, 40°C , cultivated at 100rpm for 12h, maintained the pH at 7.0 with hydrochloric acid during the fermentation process, and fermented to obtain hyaluronidase fermentation broth. The fermentation b...

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Abstract

The invention discloses a method for preparing oligomeric hyalurate by a digestion method, and the oligomeric hyalurate and an application thereof. Bacillus hyaluronidase obtained through fermental cultivation of Bacillus sp. A50 CGMCC NO.5744 is used for degrading hyaluronic acid or a salt thereof, and the method comprises the steps of preparing the hyaluronic acid or the salt thereof, enzymolysis, inactivation, filtering, settling, dewatering and drying. According to the method, the hyaluronidase produced by the Bacillus is used for degrading the hyaluronic acid or the salt thereof, and the oligomeric hyalurate is prepared through dezymotizing, alcohol or ketone settling and dewatering and drying. The method is simple to operate and mild in condition, has no destroy on the product structure and no environmental pollution, the hyaluronidase for fermentation is low in cost and is suitable for large-scale industrial production, the prepared oligomeric hyalurate has the advantages of good percutaneous absorption capability, high purity, no cytotoxicity, strong oxidation resistance and the like, and can be used in fields such as cosmetics, foods and medicines.

Description

technical field [0001] The invention relates to a process for preparing oligomeric hyaluronate by enzymatic cleavage, in particular to a method for preparing oligomeric hyaluronate by using hyaluronidase derived from Bacillus to degrade hyaluronic acid or its salt, and belongs to the field of biotechnology . 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 weight of less than 10 kDa. Studies have shown that molecular weight has a great influence o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/04C12N9/88C08B37/08C12R1/07
Inventor 郭学平石艳丽冯宁王冠凤李海娜乔莉苹王海英栾贻宏刘爱华
Owner BLOOMAGE BIOTECHNOLOGY CORP LTD
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