CN111948311A - Method for extracting microalgae intracellular metabolome product by composite solvent water-adding stimulation method - Google Patents
Method for extracting microalgae intracellular metabolome product by composite solvent water-adding stimulation method Download PDFInfo
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- CN111948311A CN111948311A CN202010800500.9A CN202010800500A CN111948311A CN 111948311 A CN111948311 A CN 111948311A CN 202010800500 A CN202010800500 A CN 202010800500A CN 111948311 A CN111948311 A CN 111948311A
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Abstract
The invention provides a method for dissolving cell membranes and extracting metabolic group products of microalgae by using a composite solvent and a water stimulation process. Firstly, wet algae mud is used as a raw material, and a chloroform and methanol mixed solvent is used for primarily destroying algae cell membranes and extracting metabolites. And secondly, adding deionized water into the extracted algae cells to promote cell membrane disintegration. And thirdly, extracting the intracellular metabolite by using the mixed solvent chloroform and methanol again, combining the chloroform and methanol solutions obtained by two-time extraction, adding a sodium chloride solution, and centrifuging and layering. Wherein, the phosphate sugar such as fructose hexaphosphate and glucose hexaphosphate, organic acid such as pyruvic acid and malic acid, energy substances such as ATP and ADP, and reducing substances such as NA (D) P enter the methanol phase. The cytochrome, fatty acid, etc. enter the chloroform phase. The method has high metabolite dissolution rate, can simultaneously extract various metabolites, and greatly reduces the metabolite oxidative denaturation problem caused by cell drying process.
Description
Technical Field
The invention relates to the technical field of biology, and provides a method for extracting a metabolome product.
Background
Metabolomics research has become an important means for studying cellular metabolism in modern biotechnology. The method is a biotechnology which is developed after genomics and proteomics and can accurately reflect intracellular metabolism level.
Metabonomics dynamically tracks the overall composition of metabolites in body fluids secreted by cells and organisms by using a modern analysis technology with high throughput, high sensitivity and high accuracy, and discriminates and analyzes the physiological and pathological states of a researched object and the relationship between the physiological and pathological states and environmental factors, gene compositions and the like by using a multivariate statistical method.
The preparation of microbial metabolite samples is generally divided into microbial culture, quenching and extraction of metabolites. After the biological sample is collected, biological reaction inactivation and extraction are firstly carried out, and then the type, the content, the state and the change of metabolites in the biological sample are detected by applying nuclear magnetic resonance, mass spectrum or chromatogram and the like to obtain a metabolic profile or a metabolic fingerprint. During sample quenching and metabolite extraction, the principle to be followed is: (1) the quenching process should immediately freeze the cell metabolism so that the metabolome can accurately reflect the metabolic level of the cell at the moment; (2) in the quenching process, the cell membrane is required to be free from obvious damage so as to prevent the leakage of intracellular metabolites; (3) during the extraction process, intracellular metabolites should be extracted as much as possible; (4) the metabolite should not undergo any physical or chemical modification; (5) the resulting sample matrix should be compatible with the chosen analytical method.
Efficient high-quality extraction of microalgae metabolites is always the key to accurate metabonomics determination. Due to the numerous metabolites of microorganisms and the numerous lipid substances, the solubility properties of the components are very different. Therefore, different solvents are generally required to be used for extraction respectively, so that the requirement on the sample amount is high, the extraction workload is high, and the scientific research work is greatly hindered.
In addition, because microalgae cells contain a large amount of oil, the microalgae needs to be dried when oil metabolites are extracted, and the oil in the cells is extracted by utilizing the characteristic that certain solvents can dissolve the oil and entering the cells through osmosis. However, the water content of the microalgae cells exceeds 80%, the drying process consumes time and energy, and the lipid is difficult to be efficiently and thoroughly extracted by utilizing the osmosis of the solvent.
The cell disruption can realize the release of cell contents and can effectively promote the extraction of metabolites, and the conventional methods for disrupting microalgae cells can be divided into mechanical methods and non-mechanical methods. The mechanical method comprises physical methods such as an ultrasonic method, a high-speed high-pressure homogenization method, a repeated freeze-thaw method and the like, and although the crushing efficiency is high, the energy consumption is relatively high, the time consumption of the crushing process is long, and the method is not suitable for metabolic substances which are easy to oxidize and denature; non-mechanical methods include chemical and biological methods such as alkaline thermal methods, acid thermal methods, enzymatic methods, etc., but these methods tend to produce impurities and destroy the quality of the metabolite extraction.
Therefore, the requirement for sampling amount of microalgae is reduced, a single solvent or a solvent combination is used for simultaneously extracting various metabolite components in microalgae cells comprehensively, efficiently and highly, and the compatibility of a sample matrix and a selected analysis method is a key problem in a microalgae metabonomics extraction link.
Disclosure of Invention
The invention aims to provide a method for effectively dissolving microalgae cell membranes by adding water into a composite solvent for stimulation and simultaneously extracting various main metabolites covering cells from wet algae cells so as to overcome the technical defects in the description.
In order to achieve the purpose, the invention provides a method for primarily destroying algae cell membranes by using a chloroform and methanol mixed solution to extract metabolites. Firstly, microalgae cells are mixed with chloroform and methanol, wherein the methanol belongs to a polar solvent and can be combined with polar lipid on microalgae cell membranes to destroy hydrogen bonds between the lipid and proteins. In addition, the addition of chloroform can denature proteins on cell membranes, and the cell membranes have holes under the dual action of the chloroform and the chloroform, so that the mixed solvent can permeate into the cells. And chloroform and methanol belong to nonpolar and polar solvents respectively, are effective solvents of various intracellular metabolites, and can dissolve and extract various intracellular metabolites through pores of cell membranes. And secondly, adding deionized water to further disturb the arrangement of cell membrane phospholipid bilayers and promote the disintegration of cell membranes. Polar water molecules can enter the perforated polar glycerophosphate molecule arrangement layer to further destroy cell membranes, and through observation of a scanning electron microscope at a cell level, water treatment can seriously destroy the cell membranes damaged by the solvent, so that the damage of the mixed solvent to the cell membranes is effectively promoted. The added water treatment step can not generate impurities, can not damage the quality of metabolites, can greatly increase the release of cell contents, and effectively reduce the using amount of a solvent. And thirdly, continuously using a polar and nonpolar mixed solvent (chloroform, methanol) to completely extract the intracellular metabolites. And finally, combining the chloroform methanol solutions of the metabolites extracted twice, adding a sodium chloride solution, centrifuging and layering, wherein phosphate sugars such as fructose hexaphosphate, glucose hexaphosphate and the like, organic acids such as pyruvic acid, malic acid and the like, energy substances such as ATP, ADP and the like, and reducing substances such as NADP, NAD and the like enter a methanol phase, and determining by high performance liquid chromatography. While cytochromes, lipids, fatty acids, etc. were taken into the chloroform phase and measured by gas chromatography.
The invention can directly use the wet algae cells to comprehensively extract the cell metabolites, thereby greatly shortening the sample preparation time. Meanwhile, the requirement for sampling amount is reduced, and all metabolites can be extracted at one time by using one algae sample. In addition, the method of adding water to stimulate the mixed solvent can greatly improve the destruction rate of cell membranes and promote the release of intracellular substances efficiently, quickly and high-quality. Finally, the chloroform and methanol solvents selected by people can be separated by a simple method, the chloroform solution is suitable for gas chromatography analysis after separation, the methanol solution is suitable for high performance liquid chromatography analysis, special adjustment on a detection method is not needed, and the method has the advantages of simple and convenient operation and the like. The invention greatly improves the extraction efficiency of the microalgae metabolome and is beneficial to improving the business flux of the industry.
The method and the process flow of the invention comprise the following steps:
1) adding the wet microalgae thallus 5-10 times of the wet microalgae thallus into a chloroform-methanol solution (the volume ratio of chloroform to methanol is 1:4-4: 1), and uniformly mixing.
2) Ultrasonically treating the mixture in ice water for 10-60 min, primarily destroying algal cell membrane with solvent, and extracting metabolite.
3) Centrifuging the mixture at 4 deg.C and 10000g for 20 min, collecting algae cells, and further crushing and extracting.
4) The chloroform-methanol phase obtained after centrifugation was collected as the first extract.
5) Adding deionized water with the volume 1-3 times of the volume of the sediment cells, and performing vortex oscillation in an ice bath for 30-120 s to further disturb the arrangement of cell membrane phospholipid bilayers and promote the disintegration of cell membranes.
6) Centrifuging at 4 deg.C and 10000g for 5min, collecting supernatant, and adding into the first extract.
7) The first extraction procedure was repeated with the remaining cell particles, and the chloroform methanol phase was then used as the second extract. The two extracts were combined.
8) Adding 1% sodium chloride solution into the extract, centrifuging, and separating to obtain upper methanol phase for detecting sugar phosphate, organic acid, energy and reducing power, and lower chloroform phase for detecting pigment, lipid, fatty acid, etc.
Detailed Description
The present invention will be further clearly and completely described below with reference to specific examples and comparative examples, but the examples and comparative examples described are only a part of the present invention, and not all of it.
Example 1
A method for fully extracting intracellular metabolite of microalgae by using a compound solvent water-adding stimulation method with chlorella as a research object comprises the following steps:
1) 10ml of methanol/chloroform (1: 1) solution is taken and evenly mixed, and 15 percent of microalgae wet thalli are added. The mixture was sonicated in ice water for 30 min.
2) The mixture was centrifuged at 10000g for 20 min at 4 ℃. The upper chloroform methanol solution was collected as the first extract.
3) To the pellet cells, 2mL of distilled water was added and vortexed in an ice bath for 30 s. Centrifuging at 4 deg.C and 10000g for 5min, collecting supernatant, and adding into the first extract.
4) The first extraction procedure was repeated with the remaining cell particles, and the chloroform-methanol phase was collected as a second extract. The two extracts were combined.
5) Adding 1% sodium chloride solution into the extract, centrifuging, and separating to obtain upper methanol phase for detecting sugar phosphate, organic acid, energy and reducing power, and lower chloroform phase for detecting pigment, lipid, fatty acid, etc. The specific results are as follows:
the extraction of representative products was improved by an average of 57.5% and 53.6% over comparative example 1 and comparative example 2, respectively.
Example 2
A method for fully extracting intracellular metabolite of microalgae by using a mixed solvent of chlorella as a research object and adding water for stimulation comprises the following steps:
1) 10ml of methanol/chloroform (2: 3) solution is taken and evenly mixed, and 20 percent of microalgae wet thalli are added. The mixture was sonicated in ice water for 40 min.
2) The mixture was centrifuged at 10000g for 20 min at 4 ℃. The upper chloroform methanol solution was collected as the first extract.
3) To the pellet cells, 3mL of distilled water was added and vortexed in an ice bath for 60 s. Centrifuging at 4 deg.C and 10000g for 5min, collecting supernatant, and adding into the first extract.
4) The first extraction procedure was repeated with the remaining cell particles, and the chloroform-methanol phase was collected as a second extract. The two extracts were combined.
5) Adding 1% sodium chloride solution into the extract, centrifuging, and separating to obtain upper methanol phase for detecting sugar phosphate, organic acid, energy and reducing power, and lower chloroform phase for detecting pigment, lipid, fatty acid, etc. The specific results are as follows:
the extraction of representative products was improved by an average of 60.5% and 56.7% over comparative example 1 and comparative example 2, respectively.
Example 3
A method for fully extracting intracellular metabolite of microalgae by using a mixed solvent of chlorella as a research object and adding water for stimulation comprises the following steps:
1) 10ml of methanol/chloroform (1: 3) solution is taken and evenly mixed, and 10 percent of wet microalgae thalli are added. The mixture was sonicated in ice water for 30 min.
2) The mixture was centrifuged at 10000g for 20 min at 4 ℃. The upper chloroform methanol solution was collected as the first extract.
3) To the pellet cells, 3mL of distilled water was added and vortexed in an ice bath for 120 s. Centrifuging at 4 deg.C and 10000g for 5min, collecting supernatant, and adding into the first extract.
4) The first extraction was repeated with the remaining cell particles, and the chloroform methanol solution was collected as a second extract. The two extracts were combined.
5) Adding 1% sodium chloride solution into the extract, centrifuging, and separating to obtain upper methanol phase for detecting sugar phosphate, organic acid, energy and reducing power, and lower chloroform phase for detecting pigment, lipid, fatty acid, etc. The specific results are as follows:
the extraction of representative products was improved by an average of 63.5% and 59.1% over comparative example 1 and comparative example 2, respectively.
Comparative example 1
Compared with the embodiment, the main difference of the comparative example is that only one organic solvent is used for dissolving the cell membrane and extracting the metabolite, and the extraction process does not have a water-jet step, and the method specifically comprises the following steps:
1) 10ml of methanol solution is taken, and 15% of wet chlorella thallus is added. The mixture was sonicated in ice water for 30 min.
2) The mixture was centrifuged at 10000g for 20 min at 4 ℃. The upper methanol solution was collected as the first extract.
3) The first extraction is repeated with the remaining cell particles, and the methanol solution is collected as a second extract. The two extracts were combined.
4) Adding 10ml chloroform into the extract, shaking and mixing, adding 1% sodium chloride solution, centrifuging, layering, wherein the upper layer methanol phase is used for detecting sugar phosphate, organic acid, energy and reducing power, and the lower layer chloroform phase is used for detecting pigment, lipid, fatty acid, etc. The results are as follows:
comparative example 2
Compared with the embodiment, the main difference of the comparative example is that no water-jet step is adopted in the extraction process, and the method specifically comprises the following steps:
1) 10ml of methanol/chloroform (1: 1) solution is taken and evenly mixed, and 15 percent of microalgae wet thalli are added. The mixture was sonicated in ice water for 30 min.
2) The mixture was centrifuged at 10000g for 20 min at 4 ℃. The upper chloroform methanol solution was collected as the first extract.
3) The first extraction was repeated with the remaining cell particles, and the chloroform methanol solution was collected as a second extract. The two extracts were combined.
4) Adding 1% sodium chloride solution into the extract, centrifuging, and separating to obtain upper methanol phase for detecting sugar phosphate, organic acid, energy and reducing power, and lower chloroform phase for detecting pigment, lipid, fatty acid, etc. The results are as follows:
in the above examples and comparative examples, the above experiments were repeated with the algal species replaced with Phaeodactylum tricornutum, Nannochloropsis, Schizochytrium, etc., and the results were similar.
Claims (5)
1. A method for extracting microalgae intracellular metabolome products by a composite solvent water-adding stimulation method comprises the following steps:
1) adding 5-10 times of microalgae thallus into chloroform and methanol solution (the volume ratio of chloroform to methanol is 1:4-4: 1) and uniformly mixing;
2) carrying out ultrasonic treatment on the mixture in ice water for 10-60 min, and primarily destroying algae cell membranes by using a solvent and extracting metabolites;
3) centrifuging the mixture at 4 deg.C and 10000g for 20 min, collecting algae cells, and further crushing and extracting;
4) collecting the chloroform and methanol phases obtained after centrifugation as a first extract;
5) adding deionized water with the volume 1-3 times that of the sediment cells, and oscillating in an ice bath for 30-120 s to further disturb the arrangement of cell membrane phospholipid bilayers and promote the disintegration of cell membranes;
6) centrifuging at 4 deg.C and 10000g for 5min, collecting supernatant, and adding into the first extract;
7) repeating the first extraction operation on the rest cell particles, taking the chloroform methanol phase as a second extract, and combining the two extracts;
8) adding sodium chloride solution into the extract, centrifuging, and separating to obtain upper methanol phase for detecting phosphate sugar, organic acid, energy and reducing power, and lower chloroform phase for detecting pigment, lipid, fatty acid, etc.
2. The method according to claim 1, wherein the microalgae biomass in step 1) is wet algal mud or dry biomass.
3. The preparation method of claim 1, wherein the shaking method after adding deionized water in step 5) can be vortex or ultrasonic, and is required to be carried out on an ice bath.
4. The method according to claim 1, wherein the chloroform methanol extract is separated into layers by using 1 to 3% sodium chloride solution in step 8).
5. The method of claim 1 or 3, wherein the microalgae comprises chlorella, phaeodactylum tricornutum, nannochloropsis, schizochytrium, thraustochytrium, spirulina, chrysophyceae, or haematococcus pluvialis.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120053357A1 (en) * | 2010-04-06 | 2012-03-01 | Heliae Development, Llc | Extraction of polar lipids by a two solvent method |
| US20140212868A1 (en) * | 2011-09-02 | 2014-07-31 | Centre De Recherche Public - Gabriel Lippmann | Method and Kit For The Isolation Of Genomic DNA, RNA Proteins and Metabolites From A Single Biological Sample |
| CN107044928A (en) * | 2016-02-05 | 2017-08-15 | 中国科学院大连化学物理研究所 | A kind of microalgae intracellular metabolin sample extraction method |
| CN111323494A (en) * | 2018-12-14 | 2020-06-23 | 中国科学院大连化学物理研究所 | Rapid pretreatment method for extracting micromolecular metabolites in adherent cells |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120053357A1 (en) * | 2010-04-06 | 2012-03-01 | Heliae Development, Llc | Extraction of polar lipids by a two solvent method |
| US20140212868A1 (en) * | 2011-09-02 | 2014-07-31 | Centre De Recherche Public - Gabriel Lippmann | Method and Kit For The Isolation Of Genomic DNA, RNA Proteins and Metabolites From A Single Biological Sample |
| CN107044928A (en) * | 2016-02-05 | 2017-08-15 | 中国科学院大连化学物理研究所 | A kind of microalgae intracellular metabolin sample extraction method |
| CN111323494A (en) * | 2018-12-14 | 2020-06-23 | 中国科学院大连化学物理研究所 | Rapid pretreatment method for extracting micromolecular metabolites in adherent cells |
Non-Patent Citations (5)
| Title |
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| ANDRE´ B. CANELAS 等: "Quantitative Evaluation of Intracellular Metabolite Extraction Techniques for Yeast Metabolomics", 《ANAL. CHEM. 2009, 81, 》 * |
| RAQUEL GUIDETTI VENDRUSCOLO 等: "Polar and non-polar intracellular compounds from microalgae: Methods of simultaneous extraction, gas chromatography determination and comparative analysis", 《FOOD RESEARCH INTERNATIONAL 109 (2018) 204–212》 * |
| 何艳丽 等: "基于GC-MS的浒苔代谢产物提取方法研究", 《安徽农业科学》 * |
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| 马建设 等: "裂殖壶菌油脂提取方法的研究", 《温州大学学报(自然科学版)》 * |
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Application publication date: 20201117 |