Method for detecting organic acid, amino acid and sugar in fermented liquid by gas chromatography-mass spectrometry

A gas chromatography and mass spectrometry detection technology, which is applied to measurement devices, instruments, scientific instruments, etc., can solve the problems of narrow detection range, high test cost, low sensitivity, etc., and achieve the effect of saving time, excellent extraction effect and simplifying operation steps.

Inactive Publication Date: 2014-05-21
广州恩康生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The purpose of the present invention is to provide a method for gas chromatography-mass spectrometry detection of organic acids, amino acids, and sugars in fermentation broth, to make up for the blanks in the current detection method for metabolites in microbial fermentation, and to overcome the many interference factors of existing detection technologies. Disadvantages such as tediousness, high test cost, low sensitivity, and narrow detection range

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  • Method for detecting organic acid, amino acid and sugar in fermented liquid by gas chromatography-mass spectrometry

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Take the Clostridium fermentation broth.

[0024] (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 0.8g / 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 2 hours, centrifuge to collect 50ul 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 50ul:20μg, and vacuum-dried at room temperature to obtain the intracellular metabolite sample I.

[0025] (2) Preparation of extracellular fluid samples: take fermentation broth and centrifuge to obtain supernatant II, take 200ul supernatant II, add acetonitrile to supernatant II to remove p...

Embodiment 2

[0030] Get the lactic acid bacteria fermentation liquid.

[0031] (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 vacuum-dried at room temperature to obtain the intracellular metabolite sample I.

[0032](2) Preparation of extracellular fluid samples: take the fermentation broth, centrifuge to obtain supernatant II, take 350ul of supernatant II, add acetonitrile to supe...

Embodiment 3

[0036] Take the mold fermentation broth.

[0037] (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.

[0038] (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 to remo...

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Abstract

The invention relates to a method for detecting an organic acid, an amino acid and sugar in fermented liquid by gas chromatography-mass spectrometry, belonging to the technical field of biochemical examination and determination. The detection method comprises the following steps: (1) centrifuging taken fermentation liquid at a high speed and respectively collecting cells and extracellular fluid; (2) preparing a fermentation liquid sample: adding acetonitrile in a liquid supernatant prepared in the step (1) to remove protein; carrying out vortex shaking, and centrifuging and collecting liquid supernatant III; adding an internal standard substance ribitol solution and drying at a room temperature in vacuum to obtain an extracellular fluid sample II; (3) deriving the sample adding a sugar deriving agent and an amino acid deriving agent; and (4) carrying out the gas chromatography-mass spectrometry and data acquisition. The method provided by the invention has the advantages of simplicity in sample treatment, simplicity and convenience in operation, short detection time of organic acid, amino acid and sugar, high sensitivity, high repeatability of a detection result, capability of preparing a plurality of samples 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 organic acids, amino acids and sugars in fermentation broth by gas chromatography-mass spectrometry. Background technique [0002] In terms of using gas chromatography-mass spectrometry to detect amino acids and sugar metabolites, there are related technical reports, such as patent 201210046953.2, which discloses a method for gas chromatography-mass spectrometry detection of urinary organic acids, including the following steps: (1) the urine to be tested (2) add internal standard, use frozen ethanol to process protein precipitation, and dry the sample; (3) carry out methyl silylation derivatization treatment on the above sample; (4) use the above 1- Step 3 to process the standard organic acid to obtain the standard organic acid sample; (5) use gas chromatography-mass spectrometry to detect the standard organic acid sample and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/88G01N30/06
Inventor 陈东
Owner 广州恩康生物科技有限公司
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