CN107044928B - A kind of microalgae intracellular metabolite sample extraction method - Google Patents

Abstract

The invention discloses a method for extracting microalgae intracellular metabolite samples, which uses an ice bath to quickly inactivate intracellular enzymes, and ultrasonically breaks through dichloromethane or chloroform, using dichloromethane (or chloroform):methanol:water three-phase The system performs rapid separation of polar and non-polar compounds, insoluble sugars and proteins for subsequent metabolomics analysis. The invention has the characteristics of few operation steps and can simultaneously measure polar and non-polar intracellular components.

Description

A kind of microalgae intracellular metabolite sample extraction method

technical field

The invention relates to a method for extracting microalgae intracellular metabolite samples.

Background technique

Microalgae are autotrophic microorganisms, also known as microalgae plants because of the photosynthesis system in their cells. Microalgae have the ability to efficiently absorb solar energy and fix carbon dioxide to produce biomass (that is, the ability to photosynthesize); at the same time, as a kind of microorganism, microalgae proliferates faster. As single-celled organisms, microalgae are mainly composed of protein, fat, carbohydrates and nucleic acids. At present, microalgae has become an important breeding bait and widely used by farming enterprises. At the same time, microalgae has also become a source of health food such as carotenoids, proteins, and polysaccharides. As the prospect of fossil energy depletion intensifies, the potential application of microalgae in energy products and fine chemicals has also received great attention. In order to effectively utilize the biological resources of microalgae, or use microalgae as a cell factory to regulate microalgae, the analysis of intracellular metabolites in microalgae plays an important role. More and more studies have shown that in the process of metabolite research, the sample extraction method has great nutrition for the later results. On the one hand, the existence of intracellular enzymes may cause endogenous transformation during sample processing. Make the result deviate from expectations, on the other hand, the existence of different polarities and soluble substances, most of the existing methods will lose a part of the sample, reducing the utilization rate of the sample, or the operation is complicated and time-consuming (CN102472741B, CN103713075A, CN102495152B and CN102517349B) .

For endogenous degradation, commonly used methods include low-temperature inactivation, low-temperature solvent inactivation, or high-temperature solvent inactivation. For microalgae, its culture system is a water environment, and the dry-based biomass mass content is usually at a few per thousand levels. Therefore, the existing method is to first centrifuge and then inactivate the treatment. Although the centrifugation time at 4°C was only a few minutes, the cells still had some viability during the process. A better method should first inactivate the intracellular enzymes before harvesting the biomass. The water content of the microalgae biomass collected by centrifugation is between 10-90%.

In conventional extraction methods, freeze drying is used to remove water after sample collection. However, whether it is water extraction of polar substances or extraction using organic solvents, the amount of extractant usually used is much higher than the amount of water collected by centrifugation into the microalgae cell pellet, which does not produce substantial for most subsequent determinations. influences.

At the same time, in conventional extraction methods, only one of the phases is selectively collected for subsequent analysis. If multiple components can be analyzed, more information can be obtained from limited samples.

Therefore, based on the above analysis, the present invention proposes a new intracellular metabolite sample preparation process for the research of microalgae metabolites, especially the characteristics of omics-level research.

Contents of the invention

The invention relates to a method for preparing samples of intracellular metabolites of microalgae metabolism. Firstly, the intracellular enzymes are inactivated in a low-temperature ice bath, and then the cells are collected by centrifugation, and the cells are ultrasonically broken in dichloromethane or chloroform. Separate the metabolites from the aqueous phase to obtain soluble polar (aqueous phase), non-polar (solvent phase) and insoluble polysaccharide and protein components for subsequent metabolomics research. The specific operation is as follows:

(1) Ice bath inactivates intracellular enzymes. Store the microalgae medium sterilized by a 0.22-micron filter membrane or an isotonic solution equivalent to the intracellular osmotic pressure of the microalgae to be treated in a 15mL or 50mL centrifuge tube in a -80°C refrigerator to prepare an ice slant. The medium or isotonic solution used should not exceed half the volume of the centrifuge tube at most. The microalgae culture solution to be analyzed is directly added to the ice slope, and the volume does not exceed the volume of the solution used for the ice slope. Shake vigorously until the ice just melts and disappears.

(2) Collect the cells by centrifugation. Quickly transfer the cell suspension in (1) to a cold centrifuge at 4°C, centrifuge at a speed of 2000-12000g for 5-10 minutes, collect the cells, and discard the supernatant.

(3) Cell washing. After resuspending the collected algal cells with the solution sterilized by 0.22 micron filter membrane in (1) that had been pre-cooled to 4°C and had the same volume of algae liquid at the time of sampling, the cells were collected by centrifugation according to (2) and discarded. clear. Repeat this step 2 to 3 times. The finally obtained cell pellet can be stored at -80°C or directly proceed to step (4).

(4) Ultrasonic crushing. According to the ratio of adding 2 mL of dichloromethane or chloroform for every 2-8 mg of cells initially, add it to the collected cell pellets, and perform ultrasonication in an ice-water mixture. Take 2mL of the above system as an example, the ultrasonic power is 300-500w, follow the ultrasonic 5-8s, stop 5-15s interval cycle operation until there is no visible cell aggregate in the solution.

(5) Extraction. For every 2 mL of dichloromethane or chloroform, add 1 mL of 0-4°C pre-cooled methanol aqueous solution (the volume ratio of methanol to water is about 1:4), mix well, and let stand at 0-4°C for 5-10 minutes until the system becomes clear. Separate the layers and centrifuge at 12000g for 10 minutes at 4°C to obtain three phases. The upper layer is the water phase, mainly polar molecules, the lower layer is the organic phase, mainly non-polar molecules, and the middle solids are polysaccharides and proteins. The above three phases were taken out for subsequent analysis.

In the above method, according to the needs of subsequent analysis, internal standard substance can be added in dichloromethane or chloroform, and methanol aqueous solution used for extraction for quantification.

Experiments show that this method is applicable to both eukaryotic microalgae and prokaryotic cyanobacteria.

The present invention uses an ice bath to quickly inactivate intracellular enzymes, ultrasonically breaks through dichloromethane or chloroform, and uses dichloromethane (or chloroform): methanol: water three-phase system for rapid polar, non-polar compound, insoluble sugar and Protein separation for subsequent metabolomic analysis. The invention has the characteristics of few operation steps and can simultaneously measure polar and non-polar intracellular components.

Detailed ways

The experimental methods described in the following examples, unless otherwise specified, are conventional methods; the reagents and biological materials, unless otherwise specified, can be obtained from commercial sources.

Example 1

1) Isochrysis zhangjiangensis was cultured in seawater supplemented with F/2 medium, and the final concentration of the main components of the F/2 nutrient salt used was NaNO ₃ 75mg/L, NaH ₂ PO ₄ ·2H ₂ O 5mg/L, Vitamin B ₁₂ 0.5μg/L, Biotin 0.5μg/L, vitamin B ₁ 0.1mg/L, FeCl ₃ 6H ₂ O 3.16mg/L, Na ₂ EDTA 2H ₂ O 4.36mg/L, CuSO ₄ 5H ₂ O 9.8 μg/L, Na ₂ MoO ₄ 2H ₂ O 6.3 μg/L, ZnSO ₄ 7H ₂ O 22 μg/L, CoCl ₂ 6H ₂ O 12 μg/L, MnCl ₂ 4H ₂ O 0.18 mg/L. They were cultured in a 500mL photobioreactor for six days, and 7mL samples were taken on days 3, 4, 5 and 6 (corresponding to biomass of 3.4, 5.5, 7.5 and 7.2 mg). One day in advance, take 7mL of seawater sterilized by filtration with a 0.22 micron membrane, put it into a 15mL centrifuge tube with a cover, place it horizontally in a -80°C refrigerator, and prepare an ice slope at 45° to the horizontal plane. After sampling, directly add to the 15mL centrifuge tube prepared with ice slope, close the lid, shake vigorously until the ice visible to the naked eye just dissolves. Quickly transfer to a cold 4°C centrifuge, centrifuge at 10000g for 10 minutes, collect cells, and discard supernatant.

2) Resuspend the collected Chrysophylla cells in 7 mL of seawater pre-cooled to 4°C and sterilized by filtration through a 0.22-micron membrane, and then centrifuge for cleaning according to the aforementioned conditions. Repeat the cleaning operation process once, that is, perform the cleaning operation twice.

3) Add 2 mL of chloroform to the algae cell pellet, and perform ultrasonic crushing under the protection of the ice-water mixture. Ultrasonic power 300-500w, according to the ultrasonic 6s, stop 6s interval cycle operation 3 times, no cell aggregates visible to the naked eye in the solution.

4) Calculated based on the initial volume of chloroform, according to the volume ratio of 2:1, add 1 mL of 4°C pre-cooled methanol aqueous solution (the volume ratio of methanol to water is about 1:4) to the above cell lysate, and mix thoroughly , let stand at 4°C for 5-10 minutes until the system is clearly separated, and centrifuge at 12000g for 10 minutes at 4°C to obtain three phases. The upper layer is the water phase, mainly polar molecules, the lower layer is the organic phase, mainly non-polar molecules, and the middle solids are polysaccharides and proteins. The above three phases were taken out for subsequent analysis.

Embodiment 2: The same operation as in Example 1, except that the difference from Example 1 is that during the above operation, isotope-labeled lactic acid, malic acid and succinic acid with a final concentration of 0.125 μg/mL were added to the aqueous methanol solution, It is used for subsequent quantification of small organic molecules in the aqueous phase by chromatography-mass spectrometry.

Embodiment 3: After the operation process of the above-mentioned embodiment 1, the organic phase is taken out, and weighed after purging the solvent to a constant weight with N2 gas, to obtain the total fat-soluble substance amount determined by gravimetric method. Alternatively, at the same time, the organic phase can be diluted to 1 mg/mL (using acetonitrile:isopropanol solution with a volume ratio of 65:35), and the lipids in it can be determined by mass spectrometry.

Embodiment 4: After the operation process of the above-mentioned embodiment 1, the solid matter was taken out, and the total sugar was determined by using the anthrone sulfate method on the solid matter in embodiment 1.

Example 5: The same operation as in Example 1, the difference from Example 1 is that: after step 1), 5 mg of Tetraspermia subcardiac cells were obtained, and the free amino acid aqueous phase metabolite sample was prepared according to the method in Example 1.

Embodiment 6: The same operation as in Example 1, the difference from Example 1 is that about 4 mg of Chlamydomonas reinhardtii cells cultured using TAP medium are taken, and the seawater in Example 1 is replaced by filter-sterilized TAP medium , to collect samples for protein profile analysis.

Claims (9)

1. a kind of method for being metabolized metabolin sample preparation intracellular for microalgae, which is characterized in that concrete operations are as follows:

(1) ice bath inactivate endocellular enzyme: by microalgae fluid nutrient medium or with the comparable isotonic solution of microalgae to be processed osmotic pressure intracellular, Ice inclined-plane is prepared in centrifuge tube freezing；Used culture medium or isotonic solution volume are to be no more than centrifuge tube body Long-pending half；Centrifuge tube is taken out, the micro algae culturing liquid being analysed to is added directly on ice inclined-plane, and volume is no more than ice inclined-plane The volume of solution used；Acutely oscillation until centrifuge tube in ice just melted disappearance, obtain cell suspension；

(2) cell is collected by centrifugation: the cell suspension in (1) being transferred quickly in -4 to 4 DEG C of centrifuges of pre-cooling, 2000~ It is centrifuged 5~10 minutes under 12000g revolving speed, collects cell, abandon supernatant；

(3) cell cleans: the frustule of collection is prepared ice in the step (1) identical as micro algae culturing liquid volume to be analyzed After the solution on inclined-plane is resuspended, cell is collected according to step (2) pelleted by centrifugation, abandons supernatant, carries out cell cleaning；Wherein, ice is prepared The solution on inclined-plane needs to be cooled to -4 to 4 DEG C in advance；The step is repeated to operate 1~3 time；The cell precipitation finally obtained can be stored in- 80 DEG C or directly carry out step (4) operation；

(4) ultrasonication: according to every 1x10⁸~3x10⁸The ratio of 2mL methylene chloride and/or chloroform is added in a cell, by dichloro Methane and/or chloroform are added in collected cell precipitation, carry out ultrasonication in -4 to 4 DEG C of mixture of ice and water, according to 5~8s of ultrasound stops the interval 5~15s circulate operation and is visible by naked eyes cell aggregation into solution；

(5) extract: the step of corresponding to above-mentioned every addition 2mL methylene chloride and/or chloroform (4) system adds 1mL0~4 DEG C The methanol aqueous solution of pre-cooling, volume ratio 1:3~1:5 of first alcohol and water in methanol aqueous solution, methanol aqueous solution and dichloro in system The volume ratio of methane and/or chloroformic solution is 1:2, is sufficiently mixed, and 0~4 DEG C stands 5~10 minutes and is obviously layered to system, 4 DEG C, 12000g is centrifuged 10 minutes, is obtained three-phase layering system, is at the middle and upper levels water phase, lower layer is organic phase, intermediate solid content Layer；Above-mentioned three-phase is taken out respectively, can be used to subsequent analysis.

2. the method as described in claim 1, it is characterised in that: in Yu Suoshu methylene chloride and/or chloroform and extraction uses One of methanol aqueous solution or two kinds or more solution in, can according to need addition internal standard compound.

3. method according to claim 2, it is characterised in that: water phase internal standard compound can be the amino acid of isotope labelling, core One of acid, glucosan derivative, glycerol derivatives or target molecule to be detected or two kinds or more；Organic phase internal standard compound can To be the fatty acid or one of target molecule to be detected or two kinds or more of isotope labelling.

4. the method as described in claim 1, it is characterised in that: the microalgae is the cyanobacteria of eukaryon microalgae or protokaryon.

5. the method as described in claim 1, it is characterised in that: step (1) will be by the micro- of 0.22 micron membrane filter filtration sterilization Algae culture medium or with the comparable isotonic solution of microalgae to be processed osmotic pressure intracellular, deposited in 15mL or 50mL centrifuge tube- In 80 DEG C of refrigerators, the ice inclined-plane from the horizontal by 15-75 degree is prepared；Used culture medium or isotonic solution volume be from The 1/20 to 1/2 of heart test tube volume；Centrifuge tube is taken out, the micro algae culturing liquid being analysed to is added directly on ice inclined-plane, body The ratio between liquor capacity used in long-pending and ice inclined-plane is 1/20 to 1；Acutely oscillation is until ice has just melted disappearance in centrifuge tube.

6. method as claimed in claim 1 or 5, it is characterised in that:

For algae, isotonic solution is physiological saline or 30~50mM phosphate buffer；

For seawater algae, isotonic solution is that quality volume (g/ml) concentration is 2.5~3.0% saline solutions；

The pH of isotonic solution need to adjust consistent with microdisk electrode.

7. the method as described in claim 1, it is characterised in that: the operation of step (4)；By taking the above-mentioned system of 2mL as an example, ultrasonic function Rate 300-500w stops the interval 5~15s circulate operation and is visible by naked eyes cell aggregation into solution according to 5~8s of ultrasound.

8. the method as described in claim 1, it is characterised in that: step (5) obtains three-phase layering system, is at the middle and upper levels water Phase, wherein mainly polar molecule；Lower layer is organic phase, wherein mainly nonpolar molecule, intermediate solid nitride layer, are polysaccharide And protein.

9. the method as described in claim 1, it is characterised in that:

The subsequent analysis refers to including one of gravimetric detemination each component weight and/or following analysis method or two kinds More than；

Or it is metabolized using thin-layer chromatography, column chromatography, gas-chromatography, liquid chromatogram or chromatograph-mass spectrometer coupling method measurement organic phase Object；

Or pass through gas-chromatography or chromatograph-mass spectrometer coupling side after utilizing liquid chromatogram or chromatograph-mass spectrometer coupling method or derivatization Method measures water phase metabolin；

Or utilize the composition that composition and amino acid or polypeptide sugared in intermediate solid content are measured after enzymatic hydrolysis, digestion method processing.