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Method for simultaneously analyzing glycerin and dihydroxy acetone in microorganism fermentation liquor

A technology of microbial fermentation broth and dihydroxyacetone, applied in the field of chemical analysis, can solve the problems of low detection accuracy, difficult to implement enzymatic detection, weak response signal, etc. Effect

Inactive Publication Date: 2012-03-07
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] However, the above methods have their own shortcomings, such as: the detection accuracy of thin layer chromatography and spectrophotometer method is not high; the enzymatic detection is not easy to realize; the detection of high performance liquid chromatography requires a differential detector and a special Sugar column, while the response signal is weak; the detection by silane derivatization gas chromatography requires derivatization in anhydrous environment and high temperature; the detection by online cracking gas chromatography requires a special cracker

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  • Method for simultaneously analyzing glycerin and dihydroxy acetone in microorganism fermentation liquor
  • Method for simultaneously analyzing glycerin and dihydroxy acetone in microorganism fermentation liquor
  • Method for simultaneously analyzing glycerin and dihydroxy acetone in microorganism fermentation liquor

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Example 1 , method validation

[0026] 1.1. Linear analysis

[0027] Prepare standard solutions of glycerol and dihydroxyacetone with concentrations of 1, 10, 25, 50, and 100 g / l respectively, and prepare 10 g / l n-hexadecane solution as an internal standard, and use the internal standard method for quantification to correct operation and instrument errors.

[0028] Then, take 10ul of standard solutions of different concentrations, add 10ul 1-methylimidazole and 0.5ml acetic anhydride respectively, and react at room temperature for 1-5min, add 100ul deionized water to degrade excess acetic anhydride, then add 10ul normal Hexadecane internal standard solution, then extract the reaction solution with 100ul dichloromethane, the organic phase of extraction is dried with anhydrous sodium sulfate, the extract after drying is directly carried out gas chromatography analysis, obtains the chromatogram of the standard solution of different concentrations, Among them, the chrom...

Embodiment 2

[0049] Example 2 , Biological fermentation broth sample preparation and pretreatment

[0050] G. oxydans DSM2003 of the genus Gluconobacter (Gluconobacter) is used to ferment glycerol to produce dihydroxyacetone, wherein,

[0051] DSM2003 strain seed medium: 8.0% sorbitol, 2.0% yeast powder, 0.1% K 2 HPO 4 , 0.05% MgSO 4 ·7H 2 O.

[0052] DSM2003 strain fermentation medium: 8.0% glycerol, 2.0% yeast powder, 0.1% K 2 HPO 4 , 0.05% MgSO 4 ·7H 2 O.

[0053] Insert the Gluconobacter oxydans DSM2003 strain into the seed medium, and at 30° C., 200 rpm shaking culture for 24 hours to obtain the seed culture solution, then insert 2L fermentation medium with 10% inoculum size, in a 3.7L fermenter (KLF2000, Switzerland ) to produce dihydroxyacetone by fermentation.

[0054] Sampling was taken at intervals during the fermentation process, and the microbial fermentation broth was centrifuged at 10,000 rpm for 5 minutes to remove bacterial precipitates, and the supernatant was ...

Embodiment 3

[0055] Example 3 , Fermentation broth supernatant derivatization treatment and chromatographic detection

[0056] Take 10 μl of the centrifuged supernatant obtained in step 2, derivatize the fermentation broth according to the method described in step 1.1, and carry out gas chromatography analysis and detection of the acetylated product, wherein, the detection results of the 16-h sample are as follows: figure 2 As shown, the chromatograms of the remaining samples are consistent with figure 2 resemblance.

[0057] figure 2 The results show that the method of the present invention is not affected by other impurities in the microbial fermentation broth in practical application, therefore, it is feasible to use the above-mentioned method to detect the content of glycerol and dihydroxyacetone in the microbial fermentation broth. Realize the control of microbial fermentation process.

[0058] To sum up, the present invention can directly carry out acetylation treatment on mi...

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Abstract

The invention aims at providing a method for simultaneously analyzing glycerin and dihydroxy acetone in microorganism fermentation liquor, which comprises the following steps of: A) processing the supernatant of the microorganism fermentation liquor by acetylizing; and B) carrying out gas chromatography detection on the samples after being processed by acetylizing. The method can be used for directly carrying out acetylizing treatment on the fermentation liquor at normal temperature and can avoid dihydroxy acetone from being decomposed under the high temperature needed by common acetylizing. The acetylizing of the glycerin and dihydroxy acetone in the microorganism fermentation liquor has quick reaction speed, is simple and convenient. Meanwhile, the method needs less sample amount, has high accuracy and good precision, can realize simultaneous quantifying on the glycerin and dihydroxy acetone in the microorganism fermentation liquor with complex components in a short time, solves theproblems existed in the existing method and can completely meet the detection, regulation and control on the process of producing dihydroxy acetone by fermenting microorganism glycerin.

Description

technical field [0001] The invention belongs to the field of chemical analysis, in particular to a method for simultaneously analyzing glycerin and dihydroxyacetone in microbial fermentation broth. Background technique [0002] Dihydroxyacetone is not only widely used in the cosmetic industry, but also a useful synthetic module in fine chemicals. Dihydroxyacetone is an oxidation product of glycerol, which is mainly produced industrially by microorganisms of the genera Acetobacter and Gluconobacter oxidans (Cassandra, D.M., et al. Crit Rev in Biotechnol. 2007, 27:147-171). However, due to the complex composition of the biological fermentation broth, glycerin and dihydroxyacetone have similar chemical structures and physical and chemical properties, it is difficult to detect at the same time. Therefore, how to accurately and quantitatively detect the content of glycerol and dihydroxyacetone, so as to optimize and regulate the biotransformation process , is an important aspect...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/02G01N30/06G01N30/60C12P7/28C12R1/01
Inventor 林金萍魏东芝吴建
Owner EAST CHINA UNIV OF SCI & TECH