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Method for rapidly detecting glucosamine

A technology of glucosamine and glucose solution, applied in the field of rapid detection of glucosamine, can solve problems such as complexity and affect efficiency, and achieve the effect of improving experimental efficiency

Active Publication Date: 2021-07-23
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

When testing a large amount of fermentation broth, the detection method will inevitably affect the efficiency of the experiment due to the complexity of its own operation.

Method used

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  • Method for rapidly detecting glucosamine
  • Method for rapidly detecting glucosamine

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Effect test

Embodiment 1

[0025] The detection reagent is a gallic blue detection reagent, and its specific configuration method is: preparation pH is 8.0, and concentration is the N-acetylglucosamine solution of 100g / L, adds 1% concentration in solution and is the gall blue of 10mmol / L (final concentration is 100μmol / L).

[0026] The detection method is as follows: 1. Take 50 μL of fermentation broth in a 96-well plate;

[0027] 2. Add 50 μL detection reagent and incubate at 37°C for 10 minutes;

[0028] 3. Fluorescence detection was performed with a microplate reader, the excitation wavelength was 560nm, and the emission wavelength was 640nm.

Embodiment 2

[0030] In order to verify the accuracy of this detection method, in this embodiment, we cultivated two bacterial strains, a high-expression strain and a non-expression strain (control strain) of hexobutadiose deacetylase, and cultivated 24h, 48h, 72h and 96h, adopt the method of the present invention and existing phthalaldehyde detection method to detect the total enzyme activity in the fermented liquid. The result is as figure 1 , figure 2 shown. The results showed that the enzyme-producing strain showed higher total enzyme activity after 72 hours of culture, and continued to increase after 96 hours of culture. However, the detection reagent proposed by the present invention can also effectively detect the level of enzyme production, and the fluorescence intensity decreases with the increase of enzyme activity. This example proves that the method is easy to operate and can effectively distinguish high and low enzyme activities, and is applicable to high-throughput screeni...

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Abstract

The invention discloses a method for rapidly detecting glucosamine. The method comprises the following steps: preparing an N-acetylglucosamine solution, and adding gallocyanine into the N-acetylglucosamine solution to obtain a gallocyanine detection reagent; adding a gallocyanine detection reagent into fermentation liquor containing a glucosamine production strain, performing culturing, detecting the fluorescence intensity, and judging the content of glucosamine according to the reduction of the fluorescence intensity. The method for rapidly detecting the glucosamine is mainly applied to measurement of the total catalytic activity of the hexachlorodisaccharide deacetylase capable of converting the substrate N-acetylglucosamine into the glucosamine. Different from an existing phthalic dicarboxaldehyde detection method, the detection method provided by the invention only needs one-step reagent adding and mixing operation, and detection can be carried out by a microplate reader after culture. Compared with an existing method, the method is simple and efficient, and the experiment efficiency can be obviously improved.

Description

technical field [0001] The invention relates to a method for rapidly detecting glucosamine, which belongs to the technical field of biological detection. Background technique [0002] Glucosamine is a small molecular monosaccharide that widely exists in nature and is widely used in the fields of medicine and health care products. At present, the industrial production of glucosamine mainly adopts chemical hydrolysis. This method uses chitin in shrimp and crab shells as a substrate and uses strong acid for hydrolysis. The reaction conditions are harsh, it is easy to cause environmental pollution, and the production efficiency is limited. With the promotion of green chemistry and environmentally friendly manufacturing, more research has begun to focus on green and environmentally friendly microbial fermentation technology, and use cheap products such as chitin and glucose as substrates to produce glucosamine through hydrolysis or synthesis. Among them, hexuccinate deacetylase ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
CPCG01N21/6428
Inventor 刘龙陈坚吕雪芹堵国成李江华刘延峰孙国运
Owner JIANGNAN UNIV
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