CN107044928A - A kind of microalgae intracellular metabolin sample extraction method - Google Patents
A kind of microalgae intracellular metabolin sample extraction method Download PDFInfo
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Abstract
The invention discloses a kind of microalgae intracellular metabolin sample extraction method, using ice bath mode fast deactivation endocellular enzyme, by dichloromethane or chloroform ultrasonication, with dichloromethane (or chloroform)：Methanol：Water three-phase system carries out the separation of fast polarity, non-polar compound, insoluble sugar and albumen, for follow-up metabonomic analysis.The present invention has operating procedure few, the characteristics of polarity and nonpolar intracellular component can be determined simultaneously.
Description
Technical field
The present invention relates to a kind of microalgae intracellular metabolin sample extraction method.
Background technology
Microalgae is autotrophic type microorganism, there is photosynthesizer because its is intracellular and is also referred to as microalgae plant.
There is microalgae efficient absorption solar energy, fixed carbon dioxide to produce ability (the i.e. photosynthesis of biomass
Ability)；While the one kind of microalgae as microorganism, its growth rate is very fast.As unicellular organism,
The main composition of microalgae is albumen, fat, carbohydrate and nucleic acid etc..At present, microalgae has been
Important breeding bait by breeding enterprise extensive use, while microalgae also have become carotenoid,
The source of the health foods such as protein, polysaccharide.With the expected aggravation that fossil energy is exhausted, microalgae exists
The potential application of energy product and fine chemicals has also obtained very big concern.In order to effectively utilize microalgae
Living resources, or microalgae as cell factory is oriented into regulation and control microalgae, microalgae intracellular metabolite analysis
Important function has all been played wherein.Increasing research shows, in metabolin research process, sample
Product extracting method has very big nutrition to anaphase, on the one hand, the presence of intracellular enzyme system, it is possible to
Endogenous conversion is produced in sample handling processes so that result, which deviates, is expected, on the other hand, different
The presence of polarity and dissolved matter, most existing method can lose a part of sample, reduce sample
Utilization rate, or complex operation, time-consuming (CN102472741B, CN103713075A,
CN102495152B and CN102517349B).
For endogenous degradation, it is molten that conventional method includes low-temperature inactive, low-temperature solvent inactivation or high temperature
Agent inactivation is several.For microalgae, its cultivating system is water environment, butt biomass mass content
Generally in some thousandths of level, therefore existing method is first to centrifuge to inactivate processing again.Although 4 DEG C of centrifugations
Time only has a few minutes, but cell still has certain activity during being somebody's turn to do.Relatively good method should
Endocellular enzyme inactivation is first set to carry out biomass collection again.The microalgae biomass water content being collected by centrifugation exists
Between 10-90%.
In general extraction methods, the removal of moisture can be carried out after sample collection using freeze-drying.But
Either to the water extraction of polar substances, or it is stripped using organic solvent, the use of usual extractant
The water in microalgae cell precipitation can be significantly larger than collected by centrifugation in amount, and the latter is for most of follow-up surveys
It is fixed not produce materially affect.
Meanwhile, in general extraction methods, only selective collection wherein one mutually carries out subsequent analysis more,
If can analyze multiple components, more information can be obtained from limited sample.
Therefore, the present invention is based on above-mentioned analysis, is studied for microalgae metabolin, and especially group learns level
A kind of Research Characteristics, it is proposed that new intracellular metabolin sample preparation flow.
The content of the invention
The present invention relates to a kind of method that intracellular metabolin sample preparation is metabolized for microalgae, pass through first
Low temperature ice bath inactivates endocellular enzyme, then cell is collected by centrifugation, and ultrasound is broken in dichloromethane or chloroform
Broken cell, metabolin separation is carried out using the aqueous phase containing methanol, obtain solvable polarity (aqueous phase),
Nonpolar (solvent phase) and insoluble polysaccharide and protein component, for follow-up metabolism group research.
Concrete operations are as follows：
(1) ice bath inactivation endocellular enzyme.By the micro-algae culture medium Jing Guo 0.22 micron membrane filter filtration sterilization
Or the isotonic solution suitable with pending microalgae intracellular osmotic pressure, in 15mL or 50mL centrifuge tubes
Deposit in -80 DEG C of refrigerators, prepare ice inclined-plane.Used culture medium or isotonic solution do not surpass at most
Cross the half of centrifuge tube volume.The micro algae culturing liquid being analysed to is added directly on ice inclined-plane, body
Volume of the product no more than solution used in ice inclined-plane.Acutely vibration is until ice has just melted disappearance.
(2) cell is collected by centrifugation.Cell suspension in (1) is transferred quickly to the cold 4 DEG C
In centrifuge, centrifuged 5~10 minutes under 2000~12000g rotating speeds, collect cell, abandon supernatant.
(3) cell is cleaned.By the frustule of collection with by be cooled in advance 4 DEG C, sampling when algae
After the solution of 0.22 micron membrane filter filtration sterilization is resuspended in liquid product identical (1), according to (2) bar
Cell is collected by centrifugation in part, abandons supernatant.The step is repeated to operate 2~3 times.The cell precipitation finally obtained can
With the operation for being stored in -80 DEG C or directly carrying out step (4).
(4) ultrasonication.2mL dichloromethane or chloroform are added according to initial every 2~8mg cells
Ratio, be added in collected cell precipitation, ultrasonication carried out in mixture of ice and water.With
Exemplified by the above-mentioned systems of 2mL, ultrasonic power 300-500w, according to 5~8s of ultrasound, stops 5~15s intervals
Circulate operation is visible by naked eyes cell aggregation into solution.
(5) extract.For every 2mL dichloromethane or chloroform, the first of 0~4 DEG C of precooling of 1mL is added
Alcohol solution (the wherein volume ratio of first alcohol and water about 1:4), it is sufficiently mixed, 0~4 DEG C stands 5~10 points
Clock is substantially layered to system, 4 DEG C, and 12000g is centrifuged 10 minutes, obtains three-phase.It is at the middle and upper levels
Aqueous phase, mainly polar molecule, lower floor are organic phase, mainly nonpolar molecule, middle solid content
For polysaccharide and protein etc..Above-mentioned three-phase is taken out for subsequent analysis respectively.
In the above-mentioned methods, the need for can be according to subsequent analysis, in dichloromethane or chloroform, with
And extract in the methanol aqueous solution used, internal standard compound is added, is quantified.
Experiment shows that this method is applied to eucaryon microalgae, is also applied for the blue-green algae of protokaryon.
The present invention uses ice bath mode fast deactivation endocellular enzyme, broken by dichloromethane or chloroform ultrasound
It is broken, with dichloromethane (or chloroform)：Methanol：Water three-phase system carries out fast polarity, nonpolar chemical combination
The separation of thing, insoluble sugar and albumen, for follow-up metabonomic analysis.The present invention has operation step
It is rapid few, the characteristics of polarity and nonpolar intracellular component can be determined simultaneously.
Embodiment
Experimental method described in following embodiments, is conventional method unless otherwise specified；The examination
Agent and biomaterial, unless otherwise specified, are commercially obtained.
Embodiment 1
1) lsochrysis zhanjiangensis (Isochrysis zhangjiangensis) is incubated at addition F/2 culture mediums
Seawater in, the final concentration of NaNO of used F/2 nutritive salt main component375mg/L,
NaH2PO4·2H2O 5mg/L, VitaminB120.5 0.5 μ g/L, vitamin B of μ g/L, Biotin10.1
Mg/L, FeCl3·6H2O 3.16mg/L,Na2EDTA·2H2O 4.36mg/L, CuSO4·5H2O
9.8 μ g/L, Na2MoO4·2H2O 6.3 μ g/L, ZnSO4·7H2O 22 μ g/L, CoCl2·6H2O 12
μ g/L, MnCl2·4H2O 0.18mg/L.Cultivated six days in 500mL bioreactors, point
Not in 3, sample within 4,5 and 6 days each 7mL (correspondence biomass is 3.4,5.5,7.5 and 7.2mg).Carry
The previous day, the seawater 7mL for 0.22 micron membrane filter filtration sterilization of learning from else's experience loads 15mL centrifugation examinations with cover
Guan Zhong, is placed horizontally in -80 DEG C of refrigerators, is prepared into the ice inclined-plane with the horizontal 45 degree.Sampling
Afterwards, it is added directly into the centrifugal pipes of 15mL for preparing ice inclined-plane, covers lid, acutely vibration is straight
Just dissolved to macroscopic ice.It is transferred quickly in 4 DEG C of centrifuges to the cold, in 10000g
Centrifuged 10 minutes under rotating speed, collect cell, abandon supernatant.
2) by the chrysophyceae cell of collection be cooled in advance 4 DEG C, it is through 0.22 micron membrane filter filtration sterilization
Seawater 7mL is resuspended, and cleaning operation is carried out according still further to aforementioned condition centrifugation.Repeated washing operating process 1
Secondary, i.e., cleaning operation is carried out 2 times.
3) 2mL chloroforms are added in frustule precipitation, under the protection of mixture of ice and water, is surpassed
Sound is crushed.Ultrasonic power 300-500w, according to ultrasonic 6s, stops 6s intervals circulate operation 3 times, molten
Cell aggregation is visible by naked eyes in liquid.
4) calculated with initial chloroform volume, according to 2:1 volume ratio, in above-mentioned cell pyrolysis liquid
Middle methanol aqueous solution (the wherein volume ratio of first alcohol and water about 1 for adding 4 DEG C of precoolings of 1mL:4),
After being sufficiently mixed, 4 DEG C of standings are substantially layered for 5~10 minutes to system, 4 DEG C, 12000g centrifugations 10
Minute, obtain three-phase.It is at the middle and upper levels aqueous phase, mainly polar molecule, and lower floor is organic phase, main
If nonpolar molecule, middle solid content is polysaccharide and protein etc..Above-mentioned three-phase is taken out into use respectively
In subsequent analysis.
Embodiment 2：The operation of be the same as Example 1, difference from Example 1 is：In above-mentioned behaviour
During work, the breast of final concentration of 0.125 μ g/mL isotope marks is added in methanol aqueous solution
Acid, malic acid and butanedioic acid, for being determined subsequently through application of gas chromatorgraphy/mass organic molecule in aqueous phase
Amount.
Embodiment 3：After the operating process of above-described embodiment 1, organic phase is taken out, swept with N2 air-blowings
Weighed after solvent to constant weight, obtain total liposoluble substance amount of gravimetric detemination.Or, simultaneously can be by
It (is 65 using volume ratio that organic phase, which is diluted to 1mg/mL,:35 acetonitrile：Aqueous isopropanol), to it
In lipid carry out carry out mass spectroscopy.
Embodiment 4：After the operating process of above-described embodiment 1, solid content is taken out, by embodiment 1
In solid content using sulfuric acid anthrone method determine total reducing sugar.
Embodiment 5：The operation of be the same as Example 1, difference from Example 1 is：In step 1)
The sub- slit bamboo or chopped wood frustules of cardioid four of 5mg are obtained afterwards, and free amino acid aqueous phase generation is prepared according to the method for embodiment 1
Thank to thing sample.
Embodiment 6：The operation of be the same as Example 1, difference from Example 1 is：Take about 4mg
Using the chlamydonomas reinhardtii cells of TAP medium cultures, replaced using the TAP culture mediums Jing Guo filtration sterilization
For the seawater in embodiment 1, sample is collected, for protein spectrum parsing.
Claims (9)
1. a kind of method that intracellular metabolin sample preparation is metabolized for microalgae, it is characterised in that specific
Operation is as follows：
(1) ice bath inactivation endocellular enzyme：By microalgae fluid nutrient medium or with pending microalgae intracellular osmotic pressure
Suitable isotonic solution, ice inclined-plane is prepared in centrifuge tube freezing；Used culture medium is isotonic
Liquor capacity is the half of no more than centrifuge tube volume；Centrifuge tube is taken out, is analysed to
Micro algae culturing liquid is added directly on ice inclined-plane, and volume is no more than the volume of solution used in ice inclined-plane；It is acute
It is strong to vibrate up to ice has just melted disappearance in centrifuge tube, obtain cell suspension；
(2) cell is collected by centrifugation：Cell suspension in (1) is transferred quickly to -4 to 4 DEG C of precooling
In centrifuge, centrifuged 5~10 minutes under 2000~12000g rotating speeds, collect cell, abandon supernatant；
(3) cell is cleaned：By the frustule of collection with micro algae culturing liquid volume identical to be analyzed
After the solution resuspension that ice inclined-plane is prepared in step (1), cell is collected according to step (2) pelleted by centrifugation,
Supernatant is abandoned, cell cleaning is carried out；Wherein, preparing the solution on ice inclined-plane needs to be cooled to -4 to 4 DEG C in advance；Weight
The multiple step is operated 1~3 time；The cell precipitation finally obtained can be stored in -80 DEG C or directly be walked
Suddenly the operation of (4)；
(4) ultrasonication：According to every 1x108~3x108Individual cell add 2mL dichloromethane and/or
The ratio of chloroform, dichloromethane and/or chloroform is added in collected cell precipitation, at -4 to 4 DEG C
Ultrasonication is carried out in mixture of ice and water, according to 5~8s of ultrasound, stops 5~15s intervals circulate operation extremely
Cell aggregation is visible by naked eyes in solution；
(5) extract：The step of corresponding to above-mentioned often addition 2mL dichloromethane and/or chloroform (4)
System, adds methanol aqueous solution (the wherein volume ratio of first alcohol and water of 0~4 DEG C of precooling of 1mL
1:3~1:5) it is that the volume ratio of methanol aqueous solution and dichloromethane and/or chloroformic solution in system is 1:2,
It is sufficiently mixed, 0~4 DEG C of standing is substantially layered for 5~10 minutes to system, 4 DEG C, 12000g centrifuges 10 points
Clock, obtains three-phase layering system, and it is at the middle and upper levels aqueous phase, and lower floor is organic phase, middle solid nitride layer；
Above-mentioned three-phase is taken out respectively, you can for subsequent analysis.
2. the method as described in claim 1, it is characterised in that：In the dichloromethane and/or chloroform
In and the methanol aqueous solution that uses of extraction in one or two or more kinds of solution in, can be according to need
Add internal standard compound.
3. method as claimed in claim 2, it is characterised in that：Aqueous phase internal standard compound can be isotope
In amino acid, nucleic acid, glucosan derivative, glycerol derivatives or the target molecule to be detected of mark
One or two or more kinds；Organic phase internal standard compound can be the aliphatic acid or to be detected of isotope marks
One or two or more kinds in target molecule.
4. the method as described in claim 1, it is characterised in that：The microalgae be eucaryon microalgae or
The blue-green algae of protokaryon.
5. the method as described in claim 1, it is characterised in that：Step (1) will be micro- by 0.22
The degerming micro-algae culture medium of rice membrane filtration or the vadose solution such as suitable with pending microalgae intracellular osmotic pressure
Liquid, is deposited in 15mL or 50mL centrifuge tubes in -80 DEG C of refrigerators, prepare from the horizontal by
The ice inclined-plane of 15-75 degree；Used culture medium or isotonic solution volume is centrifuge tube volume
1/20 to 1/2；Centrifuge tube is taken out, the micro algae culturing liquid being analysed to is added directly on ice inclined-plane,
Volume is 1/20 to 1 with the ratio between liquor capacity used in ice inclined-plane；Acutely vibration is up to ice in centrifuge tube
Disappearance is just melted.
6. the method as described in claim 1 or 5, it is characterised in that：
For algae, its isotonic solution is physiological saline or 30~50mM phosphate buffers；
For seawater algae, its 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)；With 2mL
Exemplified by above-mentioned system, ultrasonic power 300-500w, according to 5~8s of ultrasound, stops the circulation of 5~15s intervals
Operation is visible by naked eyes cell aggregation into solution.
8. the method as described in claim 1, it is characterised in that：Step (5) obtains three-phase layering
System, it is at the middle and upper levels aqueous phase, wherein mainly polar molecule；Lower floor is organic phase, wherein mainly
It is nonpolar molecule, middle solid nitride layer, it is polysaccharide and protein.
9. the method as described in claim 1, it is characterised in that：
The subsequent analysis refers to include gravimetric detemination each component weight, and/or in following analysis method
One or two or more kinds；
Or utilize thin-layer chromatography, column chromatography, gas-chromatography, liquid chromatogram or chromatograph-mass spectrometer coupling method
Determine organic phase metabolin；
Or pass through gas-chromatography or color after utilizing liquid chromatogram or chromatograph-mass spectrometer coupling method or derivatization
Spectrum-mass spectrometry combination method determines aqueous phase metabolin；
Or utilize composition and amino acid sugared in solid content in the middle of being determined after enzymolysis, digestion method processing
Or the composition of polypeptide.
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Cited By (2)
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| CN111948311A (en) * | 2020-08-11 | 2020-11-17 | 山东理工大学 | Method for extracting microalgae intracellular metabolome product by composite solvent water-adding stimulation method |
| CN112540133A (en) * | 2020-11-02 | 2021-03-23 | 暨南大学 | Detection and analysis method for marine microalgae intracellular metabolome |
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| CN112540133A (en) * | 2020-11-02 | 2021-03-23 | 暨南大学 | Detection and analysis method for marine microalgae intracellular metabolome |
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