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Method for extracting microalgae intracellular metabolite by composite solvent and water stimulation method

A compound solvent and metabolomics technology, applied in the biological field, can solve the problems of time-consuming crushing process, damage to the quality of metabolites, and easy generation of impurities, etc., and achieve the effects of shortening sample preparation time, improving extraction efficiency, and increasing destruction rate

Inactive Publication Date: 2020-11-17
SHANDONG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Mechanical methods include ultrasonic method, high-speed high-pressure homogenization method, repeated freeze-thaw method and other physical methods. Although these methods have high crushing efficiency, the energy consumption is relatively high, and the crushing process takes a long time. extraction; non-mechanical methods include chemical and biological methods such as alkaline heat method, acid heat method, and enzymatic hydrolysis, but these methods are prone to produce impurities and damage the quality of metabolite extraction

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  • Method for extracting microalgae intracellular metabolite by composite solvent and water stimulation method

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Embodiment 1

[0023] A method for fully extracting microalgae intracellular metabolome products by a composite solvent plus water stimulation method using chlorella as the research object, comprising the following steps:

[0024] 1) Take 10ml of methanol / chloroform (1:1) solution and mix well, add 15% microalgae wet cells. The mixture was sonicated in ice water for 30 min.

[0025] 2) Centrifuge the mixture at 4°C, 10000g for 20 min. The upper chloroform-methanol solution was collected as the first extract.

[0026] 3) Add 2 mL of distilled water to the precipitated cells, and vortex for 30 s in an ice bath. Centrifuge at 4°C and 10000g for 5min, collect the supernatant and add it to the first extract.

[0027] 4) Repeat the first step of extraction for the remaining cell particles, and then collect the chloroform-methanol phase as the second extraction. The two extracts were combined.

[0028] 5) Add 1% sodium chloride solution to the extract for centrifugation and layering. The upper...

Embodiment 2

[0032] A method for fully extracting microalgae intracellular metabolome products by a mixed solvent plus water stimulation method using chlorella as the research object, comprising the following steps:

[0033] 1) Take 10ml of methanol / chloroform (2:3) solution and mix well, add 20% microalgae wet cells. The mixture was sonicated in ice water for 40 min.

[0034] 2) Centrifuge the mixture at 4°C, 10000g for 20 min. The upper chloroform-methanol solution was collected as the first extract.

[0035] 3) Add 3 mL of distilled water to the precipitated cells, and vortex in an ice bath for 60 s. Centrifuge at 4°C and 10000g for 5min, collect the supernatant and add it to the first extract.

[0036] 4) Repeat the first step of extraction for the remaining cell particles, and then collect the chloroform-methanol phase as the second extraction. The two extracts were combined.

[0037] 5) Add 1% sodium chloride solution to the extract for centrifugation and layering. The upper met...

Embodiment 3

[0041] A method for fully extracting microalgae intracellular metabolome products by a mixed solvent plus water stimulation method using chlorella as the research object, comprising the following steps:

[0042] 1) Take 10ml of methanol / chloroform (1:3) solution and mix well, add 10% microalgae wet cells. The mixture was sonicated in ice water for 30 min.

[0043] 2) Centrifuge the mixture at 4°C, 10000g for 20 min. The upper chloroform-methanol solution was collected as the first extract.

[0044] 3) Add 3 mL of distilled water to the precipitated cells, and vortex for 120 s in an ice bath. Centrifuge at 4°C and 10000g for 5min, collect the supernatant and add it to the first extract.

[0045] 4) Repeat the first step of extraction for the remaining cell particles, and then collect the chloroform-methanol solution as the second extraction. The two extracts were combined.

[0046] 5) Add 1% sodium chloride solution to the extract for centrifugation and layering. The upper m...

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Abstract

The invention provides a method for carrying out cell membrane dissolution and metabolite product extraction on microalgae by using a composite solvent and water stimulation process by taking the microalgae as an object. The method comprises the following steps: step 1, taking wet algae mud as a raw material, preliminarily destroying algae cell membranes through a chloroform and methanol mixed solvent, and extracting metabolites; 2, adding deionized water into the extracted algae cells, and promoting cell membrane disintegration, and 3, extracting intracellular metabolites by using the mixed solvent chloroform and methanol again, combining the chloroform and methanol solutions obtained by two times of extraction, adding a sodium chloride solution, and carrying out centrifugal layering. Phosphosaccharides such as fructose hexaphosphate and glucose hexaphosphate, organic acids such as pyruvic acid and malic acid, energy substances such as ATP and ADP, and reducing power substances such as NA (D) P enter a methanol phase. Cytochrome, fatty acid and the like enter a chloroform phase. According to the method, the metabolite dissolution rate is high, multiple metabolites can be extractedat the same time, and the metabolite oxidative denaturation problem caused in the cell drying process is greatly reduced.

Description

technical field [0001] The invention relates to the field of biotechnology and provides a method for extracting metabolome products. Background technique [0002] Metabolomics research has become an important means of studying cell metabolism in modern biotechnology. It is a biotechnology developed after genomics and proteomics that can accurately reflect the level of intracellular metabolism. [0003] Metabolomics uses high-throughput, high-sensitivity and high-precision modern analysis techniques to dynamically track the overall composition of metabolites in body fluids secreted by cells and organisms, and to identify and analyze the researched objects with the help of multivariate statistical methods Physiological and pathological states and their relationship with environmental factors, genetic composition, etc. [0004] The preparation of microbial metabolite samples is generally divided into microbial cultivation, quenching and extraction of metabolites. After biolo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06
CPCG01N30/02G01N30/06G01N2030/062
Inventor 任晓洁赵新河宋元达
Owner SHANDONG UNIV OF TECH
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