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Gas chromatography-mass spectrometry detection method for intracellular carbohydrates and organic acids

A gas chromatography and mass spectrometry detection technology, applied in the field of organic acid, gas chromatography-mass spectrometry detection of sugars in cells, can solve the problems of complicated operation, many interference factors of detection technology, low sensitivity, etc., to simplify the operation steps and achieve excellent extraction effect. , the analysis effect is good

Inactive Publication Date: 2014-03-26
LIUZHOU LIANHAI TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a method for detecting intracellular sugars and organic acids by gas chromatography-mass spectrometry, which can make up for the gap in the current detection method for metabolites in microbial fermentation, and overcome the many interference factors, cumbersome operation, and difficult testing of existing detection technologies. Disadvantages such as high cost, low sensitivity, and narrow detection range

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Get the yeast fermentation broth.

[0023] (1) Sample preparation of intracellular metabolites: Take 5ml of fermentation broth, centrifuge to obtain the bacteria, wash the bacteria twice with normal saline, dissolve in cold methanol at -40°C, and the dry weight of the biomass reaches 0.2g / ml, and ultrasonically break down to the cells Cleavage to obtain a lysate, extract the lysate at low temperature, the low temperature condition is: extract at -40°C to -50°C for 4 hours, centrifuge to collect 150ul of the extracted lysate supernatant I, add internal standard ribitol solution, and lyse the supernatant I The ratio of the volume to the weight of the internal standard ribitol is 150ul:20μg, and vacuum-dried at room temperature to obtain the intracellular metabolite sample I.

[0024] (2) Preparation of extracellular fluid samples: take fermentation broth and centrifuge to obtain supernatant II, take 300ul supernatant II, add acetonitrile to supernatant II to remove protei...

Embodiment 2

[0028] Take the lactic acid bacteria fermentation broth.

[0029] (1) Sample preparation of intracellular metabolites: Take 5ml of fermentation broth, centrifuge to obtain the bacteria, wash the bacteria three times with normal saline, dissolve in cold methanol at -40°C, and the dry weight of the biomass reaches 1.0g / ml, and ultrasonically break down to the cells Cleavage to obtain a lysate, extract the lysate at low temperature, the low temperature conditions are: extract at -40°C to -50°C for 5 hours, centrifuge to collect 100ul of the extracted lysate supernatant I, add internal standard ribitol solution, and lyse the supernatant I The ratio of the volume to the weight of the internal standard ribitol is 100ul:20μg, and it is vacuum-dried at room temperature to obtain the intracellular metabolite sample I.

[0030] (2) Preparation of extracellular fluid samples: take the fermentation broth, centrifuge to obtain supernatant II, take 350ul of supernatant II, add acetonitrile ...

Embodiment 3

[0034] Take the Clostridium fermentation broth.

[0035](1) Sample preparation of intracellular metabolites: Take 5ml of fermentation broth, centrifuge to obtain the bacteria, wash the bacteria three times with normal saline, dissolve in cold methanol at -40°C, and the dry weight of the biomass reaches 0.6g / ml, and ultrasonically break down to the cells Cleavage to obtain a lysate, extract the lysate at low temperature, the low temperature condition is: extract at -40°C to -50°C for 3 hours, centrifuge to collect 200ul of the extracted lysate supernatant I, add internal standard ribitol solution, and lyse the supernatant I The ratio of the volume to the weight of the internal standard ribitol is 200ul:20μg, and vacuum-dried at room temperature to obtain the intracellular metabolite sample I.

[0036] (2) Preparation of extracellular fluid samples: take the fermentation broth, centrifuge to obtain supernatant II, take 60ul of supernatant II, add acetonitrile to supernatant II t...

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Abstract

The invention relates to a gas chromatography-mass spectrometry detection method for intracellular carbohydrates and organic acids, and belongs to the technical field of biochemical detection and testing. The gas chromatography-mass spectrometry detection method comprises the following steps: (1) taking a fermentation liquid for high speed centrifugation, and respectively collecting cells and an extracellular fluid; (2) preparing an intracellular metabolite sample, to be more specific, using normal saline to clean collected bacterium, dissolving in cold methyl alcohol for ultrasonication and low temperature extraction, collecting an extraction supernatant by centrifugation, adding an internal standard, and performing vacuum drying; (3) performing sample derivatization, to be more specific, adding a carbohydrate derivatization agent and an amino acid derivatization agent; and (4) performing gas chromatography-mass spectrometry analysis and data acquisition. The gas chromatography-mass spectrometry detection method has the advantages of simple sample treatment, convenient operation, short carbohydrate and organic acid detection time, high sensitivity, high test result repeatability, multiple-sample preparation and the like.

Description

technical field [0001] The invention belongs to the technical field of biochemical detection and determination, in particular to a method for detecting sugar and organic acid in cells by gas chromatography-mass spectrometry. Background technique [0002] Regarding the detection of sugar metabolites by gas chromatography-mass spectrometry, there are related technical reports, such as patent 201210046953.2, which discloses a method for the detection of glucose in urine by gas chromatography-mass spectrometry, including the following steps: (1) Complete the urine sample to be tested Urine enzyme treatment; (2) Add internal standard, use frozen ethanol to precipitate protein, and dry the sample; (3) Perform methyl silylation derivatization on the above sample; (4) Use the above steps 1-3 Process standard sugars to obtain standard sugar samples; (5) Use gas chromatography-mass spectrometry to detect standard sugar samples and urine samples to be tested; (6) Use the detection resu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06
Inventor 陈东梁远雄
Owner LIUZHOU LIANHAI TECH
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