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Microbial limit detection method of cross-linking or high molecular hyaluronic acid type substances

A microbial limit and high molecular weight technology, applied in the field of hyaluronic acid microbial detection, can solve the problems of unsuitable microbial reproduction, microbial proliferation, etc., to solve the problem of inability to accurately measure microbial limit, inhibit proliferation, and reduce the difficulty of formulation and coating. Effect

Pending Publication Date: 2020-07-03
BLOOMAGE BIOTECHNOLOGY CORP LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Aiming at the problem that the enzymatic hydrolysis temperature and pH routinely used in hyaluronic acid products are likely to cause a large number of microorganisms to proliferate, the inventors conducted research and found that although the enzymatic hydrolysis time will be prolonged in a low temperature and weak acid environment, the hyaluronic acid can still be reduced. The viscosity of acid products, and more importantly, the low temperature and weak acid environment is not suitable for most of the microorganisms in the environment to reproduce, so the original microbial content in the sample to be tested can be better maintained during the test, and the amount of microorganisms to be tested can be greatly improved. Detection accuracy of sample microbial limit index

Method used

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  • Microbial limit detection method of cross-linking or high molecular hyaluronic acid type substances

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] (1) Preparation of sterile hyaluronidase solution

[0037] will contain 100,000 IU of Bacillus ( Bacillus sp.) A50 CGMCC NO.5744 producing hyaluronidase, sterilized and filtered, added to 5 mM sterile phosphate buffer (pH5.0) containing appropriate amount of glass dispersed beads, to prepare sterile hyaluronidase containing Phosphate solution 90 mL.

[0038] (2) Preparation of samples to be tested

[0039] Weigh 10 g of high molecular weight sodium hyaluronate, the molecular weight of the high molecular weight sodium hyaluronate is 2800 kDa, and the dynamic viscosity of its 0.1 wt% aqueous solution is 280 mPa·s. Add 10 g of high molecular weight sodium hyaluronate to the above 90 mL of sterile phosphate solution containing hyaluronidase, oscillate and mix well to disperse and suspend, shake in a water bath at 15°C for 40 minutes, and the obtained enzyme The hydrolysis solution is uniform and transparent, with no obvious viscosity visible to the naked eye, then take ...

Embodiment 2

[0049] (1) Preparation of sterile hyaluronidase solution

[0050] will contain 150,000 IU of Bacillus ( Bacillus sp.) A50 CGMCC NO.5744 hyaluronidase producing hyaluronidase was sterilized and filtered, added to 50 mM sterile phosphate buffer (pH4.5) containing appropriate amount of glass dispersion beads to prepare sterile hyaluronidase containing Phosphate solution 90 mL.

[0051] (2) Preparation of samples to be tested

[0052] Weigh 10 g of cross-linked sodium hyaluronate, the dynamic viscosity of the cross-linked sodium hyaluronate 0.1wt% aqueous solution is 450 mPa·s. Add 10 g of cross-linked sodium hyaluronate to the above 90 mL of sterile phosphate solution containing hyaluronidase, shake and mix well to make it dispersed and suspended, shake in a water bath at 18°C ​​for 60 minutes, and the obtained enzyme The hydrolysis solution is uniform and transparent, with no obvious viscosity visible to the naked eye, then take 10 mL of the enzymatic hydrolysis solution, ad...

Embodiment 3

[0055] (1) Preparation of sterile hyaluronidase solution

[0056] will contain 3,000,000 IU of Bacillus ( Bacillus sp.) A50 CGMCC NO.5744 producing hyaluronidase, sterilized and filtered, added to sterile phosphate buffer (pH4.8) containing appropriate amount of glass dispersion beads 100 mM, to prepare sterile phosphoric acid containing hyaluronidase Saline solution 90 mL.

[0057] (2) Preparation of samples to be tested

[0058] Weigh 10 g of cross-linked magnesium hyaluronate, the dynamic viscosity of the cross-linked magnesium hyaluronate 0.1wt% aqueous solution is 1500mPa·s. Add 10 g of cross-linked magnesium hyaluronate to the above-mentioned 90 mL of sterile phosphate solution containing hyaluronidase, shake and mix well to disperse and suspend, shake in a water bath at 10°C for 120 minutes, and the obtained enzyme The hydrolysis solution is uniform and transparent, with no obvious viscosity visible to the naked eye. Then take 10 mL of the enzymatic hydrolysis solut...

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Abstract

The invention discloses a microbial limit detection method of cross-linking or high molecular hyaluronic acid type substances. The method comprises the steps of performing enzymolysis treatment on thecross-linking or high molecular hyaluronic acid type substances to be detected under the condition of low temperature and weak acid, so as to reduce the stickiness of the solution of the cross-linking or high molecular hyaluronic acid type substances; and preparing a detection solution from enzymatic hydrolysate with reduced stickiness, so as to realize microbial limit detection of samples to bedetected. The samples to be detected under the condition of low temperature and weak acid are treated, so that the stickiness of the detection solution can be reduced, the difficult problems that thecross-linking or high molecular hyaluronic acid type substances are low in solubility of solid powder and difficult to uniformly coat on the surface of a plating medium in a detection course, and thetrue data of microbial limit cannot be obtained, are solved, and a convenient and effective detection method is provided for microbial limit detection of the cross-linking or high molecular hyaluronicacid type substances.

Description

technical field [0001] The invention relates to a microbial limit detection method for cross-linked or high-molecular-weight hyaluronic acid substances, and belongs to the technical field of hyaluronic acid microbial detection. Background technique [0002] Hyaluronic acid is a natural mucopolysaccharide widely present in animals and humans. It is composed of N-acetylglucosamine and D-glucuronic acid disaccharide repeating units through β-(1→4) glycosidic bonds and β-( 1→3) Unbranched polymer glycosaminoglycans composed of glycosidic bonds. Due to its unique physical properties such as viscoelasticity and lubricity, it is widely used in the fields of medicine, cosmetics and food. [0003] Cross-linked hyaluronic acid is a polymer gel modified by chemical cross-linking of hyaluronic acid. After cross-linking, the molecular chain of hyaluronic acid is longer and has a more complex three-dimensional structure. Compared with natural hyaluronic acid, it is A liquid with high vi...

Claims

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Application Information

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IPC IPC(8): C12Q1/06C12Q1/04
CPCC12Q1/06C12Q1/045Y02A50/30
Inventor 乔莉苹石艳丽王冠凤穆淑娥曲建全周宁郭学平
Owner BLOOMAGE BIOTECHNOLOGY CORP LTD
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