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Low molecular hyaluronate, preparation method and purpose thereof

A hyaluronic acid salt and hyaluronic acid technology, applied in the fields of enzymology and medicinal chemistry, can solve the problems of low molecular weight HA, which is difficult to achieve, difficult to realize industrial production, low unit enzyme activity, etc., to solve the problems of high cost and low cost Lowering, high enzyme thermostability and pH stability

Active Publication Date: 2013-08-21
BLOOMAGE BIOTECHNOLOGY CORP LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] It is reported that the preparation process of enzymatic hydrolysis to produce low-molecular-weight HA is to first separate and purify HA from the fermentation broth, then add hyaluronidase for enzymolysis, and then obtain low-molecular-weight HA through membrane filtration, ethanol precipitation, dehydration and drying (CN101020724A ), the hyaluronidase used in the above preparation process is a hyaluronidase finished product, the price is very high, and it is difficult to realize industrial production
At present, there are reports in the literature that the unit enzyme activity of hyaluronidase fermentation broth derived from microorganisms is low, and the highest enzyme activity of the fermentation broth is 1.3×10 2 IU / mL (WO2010130810A1), the enzyme activities reported in other literatures are all lower than 10 2 IU / mL, so large-scale production of low molecular weight HA by enzymatic digestion is currently difficult to achieve

Method used

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  • Low molecular hyaluronate, preparation method and purpose thereof
  • Low molecular hyaluronate, preparation method and purpose thereof
  • Low molecular hyaluronate, preparation method and purpose thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0125] Example 1: Obtaining and identification of Bacillus sp. A50

[0126] 1. Acquisition of Bacillus sp. A50

[0127] Open the cover of the plate containing the enriched medium, place it in the air, collect the sedimentation bacteria in the air, cover it after about 1 hour, and place it in an incubator at 25-40°C for aerobic cultivation. After 24 hours of cultivation, Inoculate the isolated single colony in the screening medium, 25-40 ° C, 150 rpm, aerobic culture for 12-16 hours, use the method of Chinese Pharmacopoeia to measure the activity of hyaluronidase, and select the strain with the highest enzyme activity as the present invention. Bacteria, the enzyme activity of the strain can reach 10 5 IU / mL.

[0128] The composition of each medium used above is as follows:

[0129] Enrichment medium (100mL): peptone 0.2-2.0g, yeast powder 0.2-2.0g, K 2 HPO 4 ·3H 2 O 0.05-0.15g, MgSO 4 ·7H 2 O 0.05-0.15g, sodium hyaluronate 0.01-1g, agar powder 2.0g.

[0130] Screenin...

Embodiment 2

[0172] Example 2: Cloning and sequence analysis of hyaluronidase

[0173] The genomic DNA of Bacillus (Bacillus) A50 was extracted with a bacterial genomic DNA extraction kit, and the genomic DNA extraction results were detected by 1% agarose gel electrophoresis. The genome was then sequenced to obtain a whole-genome shotgun sequence of the strain. At the same time, the hyaluronidase isolated and purified from the fermentation broth of Bacillus (Bacillus) A50 was subjected to N-terminal sequencing and trypsin-degraded endopeptide sequencing to obtain a partial amino acid sequence of the hyaluronidase. Use the BLAST tool on NCBI to compare the whole genome shotgun sequence with the partial amino acid sequence, find the gene fragment with 100% similarity to the amino acid sequence, and find the approximate location of the hyaluronidase gene. Then according to the N-terminal sequence of hyaluronidase, the size of the SDS-PAGE electrophoresis band, and the analysis of the open ...

Embodiment 3

[0180] Embodiment 3: Preparation of hyaluronidase (1)

[0181] Slant medium composition (100mL): peptone 0.2g, yeast powder 2.0g, K 2 HPO 4 ·3H 2 O0.05g, MgSO 4 ·7H 2 O 0.05g, glucose 0.5g, agar powder 2.0g, adjust the pH to 6.0 with hydrochloric acid.

[0182] Seed medium composition (100mL): peptone 0.2g, yeast powder 2.0g, K 2 HPO 4 ·3H 2 O0.05g, MgSO 4 ·7H 2 O 0.05g, glucose 0.5g, adjust the pH to 6.0 with hydrochloric acid.

[0183] Fermentation medium composition (100mL): peptone 0.2g, yeast powder 2.0g, K 2 HPO 4 ·3H 2 O0.05g, MgSO 4 ·7H 2 O 0.05g, glucose 0.5g, Tween80 (Tween 80) 0.05mL.

[0184]Take the slant strain (Bacillus sp. A50 CGMCC NO.5744) and inoculate it into the sterilized seed medium, cultivate it at 25°C and 150rpm for 24 hours, then inoculate the seed liquid into the sterilized fermentation medium In the medium, the inoculum size is 10%, 25°C, 200rpm culture for 24 hours, the pH is maintained at 6.0 with sulfuric acid during the fermen...

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Abstract

The invention belongs to the fields of enzymology and pharmaceutical chemistry, and relates to the low molecular hyaluronate, preparation method and purpose thereof. Concretely, the preparation method comprises a degradation step of hyaluronic acid or salt thereof whose molecular weight is greater than 1000 kDa by hyaluronidase prepared by Bacillus spp whose preservation number is CGMCC No. 5744 or SEQ ID No:2 coded hyaluronidase. The preparation of low molecular hyaluronic acid or salts thereof by using hyaluronidase produced by Bacillus spp of the present invention has the advantages of simple operation, mild condition, non-destroy of the product structure and low cost. The product is suitable for large scale industrialized production. The low molecular hyaluronate prepared by the invention has the advantages of good transdermal absorbency, good purity, non-cytotoxicity, good antioxidation, etc., and can be used in the fields of cosmetic, foodstuff and medicine.

Description

technical field [0001] The invention belongs to the fields of enzymology and medicinal chemistry, and relates to a low-molecular-weight hyaluronate, a preparation method and application thereof. Background technique [0002] Hyaluronic acid (HA) is an acidic mucopolysaccharide, an unbranched polymer glycosaminoglycan composed of N-acetylglucosamine and D-glucuronic acid disaccharide repeating units, present in animal tissue cells In the stroma and in 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). [0003] Studies have shown that molecular weight has a great influence on the activity of hyaluronic acid, and hyaluronic acid with different molecular weights even shows diametrically opposite activities (Guo Xueping, Low molecular weight and oligomeric hyaluronic acid, Chinese Journal of Biochemical Medicine, 2003, 24 ( 3): 148-150). [0004] At present,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/26C08B37/08A61K8/73A61K31/728A61P17/02A61P39/06A61P17/18A61P35/00A61P37/04A61Q17/04A61Q19/00A61Q19/08C12R1/07
CPCC12N1/20C12R1/07A61K31/728A61Q17/00A61K8/73C12N9/26C12P19/28C07K14/32A61K8/66A61K8/735A61Q17/04A61Q19/00A61Q19/004A61Q19/08A23L33/17A61P9/00A61P17/00A61P17/02A61P17/16A61P17/18A61P35/00A61P37/04A61P39/06A61P43/00C12N9/2408C12N9/2474C12N1/205C12R2001/07C12P19/04A23V2002/00A61Q19/02C08B37/0072C12P19/26Y02P20/52
Inventor 郭学平石艳丽王冠凤冯宁李海娜乔莉苹毛华栾贻宏刘爱华
Owner BLOOMAGE BIOTECHNOLOGY CORP LTD
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