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Method for preparing hyaluronate oligomer according to digestion method, prepared hyaluronate oligomer and application thereof

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

Inactive Publication Date: 2012-09-26
BLOOMAGE BIOTECHNOLOGY CORP LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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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 sp. A50 has been preserved in the General Microorganism Center (CGMCC) of the China Committee for the Collection of Microorganisms, with the preservation number CGMCC NO. 5744, and the preservation time is 2012 February 8

Method used

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  • Method for preparing hyaluronate oligomer according to digestion method, prepared hyaluronate oligomer and application thereof
  • Method for preparing hyaluronate oligomer according to digestion method, prepared hyaluronate oligomer and application thereof
  • Method for preparing hyaluronate oligomer according to digestion method, prepared hyaluronate oligomer and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0065] Inclined 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 2g, adjust the pH to 6.0 with hydrochloric acid.

[0066] Composition of seed culture medium (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] Composition of fermentation medium (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 (Tween 80) 0.05mL.

[0068] Take the slant strain (Bacillus sp. A50 CGMCC NO. 5744) and inoculate it in the sterilized seed culture medium, incubate at 25°C and 150 rpm for 24 hours, and then inoculate the seed liquid into the sterilized fermentation medium The inoculum was 10%, cultured at 25°C, 200 rpm for 24 hours. During the fermentation, the pH was maintained at 6.0 with sulfuric acid. The fermentation broth was produced to obtain hyaluronidas...

Embodiment 2

[0070] Inclined 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 2g, pH was adjusted to 7.0 with phosphoric acid.

[0071] Composition of seed culture medium (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, pH was adjusted 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] Inoculate the slant strain (Bacillus sp. A50 CGMCC NO. 5744) into the sterilized seed culture medium, cultivate at 30°C and 100 rpm for 15 hours, and then inoculate the seed liquid into the sterilized fermentation medium The inoculum was 10%, 35℃, 300rpm culture for 16 hours, the pH was maintained at 7.0 with sulfuric acid during the fermentation process, and the hyaluronidase fermentation broth was obtained by fermentation. The fermentatio...

Embodiment 3

[0075] Inclined 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 2g, adjust the pH to 8.0 with sulfuric acid.

[0076] Composition of seed culture medium (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] The slant strain (Bacillus sp. A50 CGMCC NO. 5744) was inoculated into sterilized seed culture medium, cultivated at 35°C and 200 rpm for 13 hours, and then the seed liquid was inoculated into sterilized fermentation medium The inoculum was 10%, 40°C, 100rpm culture for 12 hours, the pH was maintained at 7.0 with hydrochloric acid during the fermentation process, and the hyaluronidase fermentation broth was obtained by fermentation. The fermen...

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Abstract

The invention discloses a method for preparing a hyaluronate oligomer according to a digestion method, a prepared hyaluronate oligomer and an application thereof. Bacillussp. A50 CGMCC NO.5744 is fermented and cultured, thereby obtaining Bacillussp hyaluronidase for degrading hyaluronic acid or salt solution thereof. The method comprises the following steps of: preparing a solution of the hyaluronic acid or salt solution thereof; performing enzymolysis; inactivating; filtering; depositing; and dewatering and drying. According to the invention, the Bacillussp hyaluronidase generated by the Bacillussp is utilized to degrade the hyaluronic acid or salt thereof; the hyaluronate oligomer is prepared according to the steps of dezymotizing, ethanol precipitation, dewatering and drying; the method is simple in operation, mild in condition, free from damage to product structure and free from environmental pollution; the cost of the hyaluronidase as a fermentation source is low; the method is suitable for large-scale industrial production; the prepared hyaluronate oligomer has the advantages of excellent transdermal absorption property, high purity, zero cytotoxicity, strong oxidation resistance, and the like; and the hyaluronate oligomer can be applied to the fields of cosmetics, food 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 polymeric glycosaminoglycans composed of glycosidic bonds exist in the intercellular matrix of animal tissues and in the membrane 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 on...

Claims

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Application Information

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IPC IPC(8): C12P19/04C12N1/20C12N9/88C12R1/07
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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