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Extraction and analysis method for metabolites in tribonema cells

An analytical method and technology of Xanthomonas algae, applied in the direction of analyzing materials, sampling, material separation, etc., to achieve the effects of high sensitivity, simple operation and high yield

Inactive Publication Date: 2018-03-30
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the total lipid content under heterotrophic conditions can only reach about 20% of the dry weight

Method used

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  • Extraction and analysis method for metabolites in tribonema cells
  • Extraction and analysis method for metabolites in tribonema cells
  • Extraction and analysis method for metabolites in tribonema cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] (1) Collect the fresh Xanthophyllum cells cultivated to the logarithmic growth phase, wash them repeatedly with double distilled water for 3 times, place the obtained clean cells in liquid nitrogen, quench for 5 minutes, terminate the metabolic reaction, and freeze-dry;

[0025] (2) Weigh 20 mg of the above-mentioned freeze-dried cells and place them in a mortar, add quartz sand and grind them to a fine powder (see Figure 1A );

[0026] (3) Add 1 mL of cold methanol aqueous solution (methanol: water = 1:1, v / v, -40°C) to the above powdered cells, mix well and place in a centrifuge tube, close the cap tightly and place in liquid nitrogen, Repeated freezing and thawing 5 times (freeze in liquid nitrogen for 3 minutes, and thaw in a refrigerator at -20°C), then centrifuge in a low-temperature refrigerated centrifuge at 5000 rpm for 5 minutes, and collect the supernatant; add 0.5 mL of cold methanol aqueous solution (methanol: water = 1:1, v / v, -40℃), the supernatant colle...

Embodiment 2

[0033] The difference from Example 1 is to change the grinding method, using liquid nitrogen grinding (see Figure 1B ).

Embodiment 3

[0035] The difference from Example 1 is that the extraction solvent is changed and the hot ethanol method is used for extraction: 75% ethanol is placed in a 95°C water bath until the temperature is constant, and it is ready for use;

[0036] Weigh 20 mg of freeze-dried cells, add quartz sand and grind to fine powder, add 1.5 mL of ethanol solution prepared in the above water bath, mix well and place in a centrifuge tube, put in a 95°C water bath for 3 minutes, take it out and put it in an ice bath at 0°C 3min (see Figure 2B ).

[0037] by the above Figure 1A The total ion chromatograms obtained by the two methods of B and B can more intuitively compare the influence of the two grinding methods on the extraction of metabolites. The number of chromatographic peaks ground with quartz sand is more than that ground with liquid nitrogen, indicating that quartz sand The crushing effect on the cell wall of yellow hair algae is better.

[0038] After using quartz sand to grind the ...

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Abstract

The invention belongs to the technical field of analysis of microalgae metabolite components, and particularly relates to an extraction and analysis method for metabolites in tribonema cells. The extraction and analysis method comprises the following steps: extracting pretreated tribonema cells in an excessive solvent, centrifugally collecting supernatant after extraction, adding a pyridine solution of methoxylamine hydrochloride into the supernatant, uniformly mixing the supernatant and the pyridine solution, and putting the mixture into a thermostatic water bath at the temperature of 30 to 40 DEG C for 80 to 90 minutes; after the water bath reaction is finished, adding N-methyl-N-trimethylsilyl trifluoroacetamide (MSTFA) into a reaction system for uniform mixing, and then incubating themixture in a thermostatic water bath at the temperature of 37 to 40 DEG C for 30 to 80 minutes, thus obtaining a tribonema metabolite derived sample; carrying out metabolite detection on the sample byusing a gas chromatograph-mass spectrometer so as to qualitatively determine effective components. The method disclosed by the invention has the advantages that small molecule intracellular metabolites can be extracted from the tribonema cells, the yield is high, and a reference can be provided for extracting active substances from the tribonema cells; in addition, the extraction and analysis operation is simple.

Description

technical field [0001] The invention belongs to the technical field of metabolome analysis of microalgae, and in particular relates to a method for extracting and analyzing intracellular metabolites of Xanthophyllum algae. Background technique [0002] Xanthophyllum is a class of eukaryotic microalgae whose plants are unbranched filaments [1] , with good industrial properties of high oil yield, anti-pollution and easy harvesting, it is an ideal material for biodiesel production [2,3] . [0003] Previous studies have found that Xanthophyllum can use some organic carbon sources for heterotrophic fermentation, and the growth rate is more than 10 times higher when glucose is used as a carbon source than under autotrophic conditions. [4] . However, the total lipid content under heterotrophic conditions can only reach around 20% of dry weight. How to improve the lipid content of Xanthophyllum under heterotrophic conditions, in addition to optimizing the culture process, it is ...

Claims

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Application Information

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IPC IPC(8): G01N30/88G01N30/06G01N1/28
CPCG01N30/88G01N1/286G01N30/06G01N2001/2866
Inventor 汪辉邵慧敏刘天中张妍
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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