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Method for rapidly determining content of hyaluronic acid in fermentation liquor

A hyaluronic acid and rapid determination technology, applied in the fields of enzymology and medicinal chemistry, can solve the problems of complex operation, influence judgment, and many interference factors, and achieve the effect of large number of samples, high specificity, and simple operation

Active Publication Date: 2020-08-18
BLOOMAGE BIOTECHNOLOGY CORP LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The classic detection method for hyaluronic acid content is the carbazole sulfate method, and the carbazole chromogenic method is considered to be the most widely used method for hyaluronic acid detection. Poor specificity, the components in the hyaluronic acid fermentation broth are relatively complex, and there are too many interference factors for carbazole color detection directly using the fermentation broth, especially in the early stage of fermentation process development, the properties of the hyaluronic acid fermentation broth fermented by different media The difference is relatively large, which is bound to affect the judgment
Therefore, it is necessary to pre-alcoholize and redissolve the hyaluronic acid fermentation broth before further using the carbazole method to determine the hyaluronic acid content, which is complicated to operate.

Method used

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  • Method for rapidly determining content of hyaluronic acid in fermentation liquor
  • Method for rapidly determining content of hyaluronic acid in fermentation liquor
  • Method for rapidly determining content of hyaluronic acid in fermentation liquor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] 1. Preparation of standard curve

[0045] 1. Accurately weigh 0.1 g (WS) of hyaluronic acid unsaturated disaccharide (4,5-unsaturated disaccharide) standard (use phosphorus pentoxide as desiccant and vacuum-dry to constant weight, content> 99.5%), Put it in a 100 ml volumetric flask, add purified water to dissolve and dilute to the mark, shake well, and use it as a stock solution. Accurately measure 5.0 ml of the stock solution before use, put it in a 100 ml measuring bottle, add water to make a solution containing 50 μg of hyaluronic acid unsaturated disaccharide per 1 ml, shake well, and obtain the hyaluronic acid unsaturated disaccharide standard solution.

[0046] 2. Take 0ml, 1ml, 2ml, 3ml, 4ml, and 5 ml of standard solutions in test tubes, respectively, add 5ml, 4ml, 3ml, 2ml, 1ml, and 0 ml of purified water, mix well, and adjust to zero with purified water. Determination of UV absorbance value A at 232 nm 232 .

[0047] 3. Take the hyaluronic acid unsaturated...

Embodiment 2

[0058] Take 100 ml of hyaluronic acid fermentation broth in 3 parallel batches, add 10 g of activated carbon respectively, stir for 50 min, centrifuge to get the supernatant, and use enzymatic hydrolysis buffer (80 mM / L Na 2 HPO 4 -NaH 2 PO 4 Buffer, pH6.0) Dilute each fermentation broth 40 times to prepare the solution to be tested; take 1 ml of the solution to be tested, add 150 IU of hyaluronidase, and dilute the enzymatic hydrolysis buffer to 10 ml, at a temperature of 25 Complete enzymatic hydrolysis at ℃ for 25 minutes, and boil at 100℃ for 2 minutes. Measure the ultraviolet absorption value of the enzymolysis solution at 232nm to obtain the A 232 .

[0059] Except for replacing the hyaluronidase with an equal volume of purified water, the fermentation broth was processed synchronously according to the same steps above to obtain the A before enzymolysis of the fermentation broth dilution. 232 .

[0060] A before and after enzymolysis according to the fermentation...

Embodiment 3

[0063] Take 100 ml of the hyaluronic acid fermentation broth fermented for 16 h, 20 h, 24 h, 28 h, 32 h, 36 h, 40 h, and 44 h, and add LX-60 adsorption resin from Xi’an Lanxiao Technology Co., Ltd. 20 g, shake for 1 h, and centrifuge to get the supernatant. Enzyme digestion buffer (50 mM / L Na 2 HPO 4 -NaH 2 PO 4buffer, pH6.0) to dilute each fermentation broth 20 times to prepare the solution to be tested; take 1 ml of the solution to be tested, add 900 IU of hyaluronidase, and dilute the enzymatic hydrolysis buffer to 10 ml, at a temperature of 30 Complete enzymatic hydrolysis at ℃ for 20 minutes, and boil at 100℃ for 2 minutes. Measure the ultraviolet absorbance value of the enzymolysis solution at 232 nm to obtain the A 232 .

[0064] Except for replacing the hyaluronidase with an equal volume of purified water, the fermentation broth was processed synchronously according to the same steps above to obtain the A before enzymolysis of the fermentation broth dilution. 2...

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Abstract

The invention discloses a method for rapidly determining the content of hyaluronic acid in fermentation liquor. The method comprises the following steps: adsorbing and purifying fermentation liquor; diluting the adsorbed and purified supernatant and then carrying out enzymolysis; thoroughly performing enzymolysis on hyaluronic acid in fermentation liquor to obtain 4, 5-unsaturated disaccharide, detecting ultraviolet absorption values of the fermentation liquor before and after enzymolysis, substituting a difference value of the ultraviolet absorption values of the fermentation liquor before and after enzymolysis into a standard curve of the concentration of the 4, 5-unsaturated disaccharide and the ultraviolet absorption values, and calculating and obtaining the content of the hyaluronic acid in the fermentation liquor. The method is simple and convenient to operate and high in specificity, hyaluronic acid in the fermentation liquor does not need to be purified, the number of samples detected in batches is large, the method can be widely applied to detection of the content of hyaluronic acid in the field of hyaluronic acid fermentation, and a convenient and efficient tool is provided for optimization and process monitoring of a hyaluronic acid fermentation process.

Description

technical field [0001] The invention relates to a method for quickly measuring the content of hyaluronic acid in a fermented liquid, in particular to a method for thoroughly enzymatically hydrolyzing hyaluronic acid fermented liquid by using hyaluronidase or chondroitin sulfate, and using the fermented liquid before and after enzymatic hydrolysis The invention relates to a detection method for calibrating the content of hyaluronic acid in a fermented liquid by absorbing difference at 232nm, and belongs to the technical fields of enzymology and medicinal chemistry. 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. Almost all animal tissues contain HA, and cockscombs and human umbilical cords are mainly used as ...

Claims

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Application Information

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IPC IPC(8): G01N21/33G01N1/28G01N1/38
CPCG01N21/33G01N1/28G01N1/38
Inventor 乔莉苹石艳丽陈玉娟穆淑娥郭学平栾贻宏周宁魏玉洁孙元军
Owner BLOOMAGE BIOTECHNOLOGY CORP LTD
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