CN102516367A - Method for extracting high-purity microcystins - Google Patents
Method for extracting high-purity microcystins Download PDFInfo
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- CN102516367A CN102516367A CN2011104551315A CN201110455131A CN102516367A CN 102516367 A CN102516367 A CN 102516367A CN 2011104551315 A CN2011104551315 A CN 2011104551315A CN 201110455131 A CN201110455131 A CN 201110455131A CN 102516367 A CN102516367 A CN 102516367A
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Abstract
The invention relates to a method for extracting high-purity microcystins, which includes steps of repeatedly freezing and thawing water samples of microcystis aeruginosa by means of liquid nitrogen, extracting supernate from the frozen and thawed water sample centrifugally for reserving, pretreating C18 solid-phase extraction columns, enriching and purifying MC(microcystin)-LR in the water samples by the C18 solid-phase extraction columns, and finally dissolving and concentrating the MC-LR to obtain the high-purity microcystins. By the method, MC-LR in milligram level, which is low in impurity and high in purity, can be extracted. The operation of realizing wall breaking of MC cells by freezing and thawing through liquid nitrogen is simple and convenient and intracellular and extracellular MC-LR can be released completely. In addition, the method is environment-friendly, safe and economical.
Description
Technical field
The present invention relates to a kind of process for extracting of Microcystin, be specially a kind of method of from produce malicious microcystic aeruginosa, extracting the high purity Microcystin, belong to environmental technology field.
Background technology
In recent years, along with the aggravation of global water body eutrophication degree, (Harmful Algal Blooms, HABs) frequency and the amplitude of incident generation increase the harmful algae wawter bloom day by day all over the world.Algal bloom has become the global problem that has a strong impact on quality of water environment and water ecology safety, and the healthy and other biological safety of not only great harm humans also causes enormous economic loss to the whole world.Some wawter bloom algae in metabolic process or the frond back of breaking in water body, discharge the algae toxin, bring very big harm for human health, hydrocoles, poultry etc.Like the Microcystin (Microsystins that microcystic aeruginosa produced; Be called for short MC) be ring-type seven peptide compounds of one type of biologically active; In recent decades, animal experiment, crowd's epidemiology survey both at home and abroad shows that Microcystin has effects such as body liver toxicity, short cancer, fetal toxicity, genetoxic, immunity infringement.
Microcystin is seven peptide monocycle hepatotoxin of one type of biologically active, and relative molecular weight is between 900-1100.Up to the present, the isomer of the Microcystin of discovery reaches kind more than 60, wherein exists more and toxicity is bigger is microcapsule phycotoxin MC-LR, MC-YR, MC-RR.The algae that can produce Microcystin mainly contains Microcystis aeruginosa (Microcystis), anabena (Anabaena), the algae that quivers (Oscillatoria) and beads algae (Nostoc) etc.Because it is comparatively general that MC-LR exists, toxicity is stronger, and separation and Culture goes out the pure article of standard, so present algae toxin study Chang Yiqi is representative index, WHO is that benchmark recommendation drinking-water algae toxin standard is 1.0 μ g/L with MC-LR content.
Along with to extensively the carrying out of Microcystin Study on degradation, the scientific research personnel is also increasing to the demand of Microcystin sample, the research content that the detection and the Study on degradation of Microcystin in the water body become the water surrounding field.
Have only offshore company of several family just can buy the standard substance such as MC-LR of high purity (purity is more than 95%) at present; (like about 3000 yuan of the U.S. 500mg of sigma company standard substance price) costs an arm and a leg; Factors such as the purchase formality is numerous and diverse, and waiting time is long are brought great inconvenience to research.All accomplish if test required Microcystin sample, also will increase experimental cost greatly by the import standard substance.Therefore, exploitation have an independent intellectual property right high purity Microcystin process for extracting for launching more significant about the research of Microcystin in water surrounding and health field.
Chinese patent 200910033693 discloses the method for a kind of separation, purifying microcystin; The blue-green algae of producing microcystic toxins is with 1: 20～80 solvent extraction; Extracting solution is through the SPE column purification; The column chromatography system that utilizes constant flow pump, C18 chromatography column, automatic Fraction Collector to constitute then carries out separation and purification to Microcystin, and moving phase is made up of 35～50% water, 50～65% methyl alcohol and 0.05～0.1% acid.Because methanol concentration is on the low side, also have the existence of acid can make the degraded of algae toxin moiety, plastics or Glass Containers can have absorption to MC; The ultrasonication frustule makes algae toxin wherein discharge simultaneously with other materials, is unfavorable for the acquisition of high purity algae toxin.
Summary of the invention
The purpose of this invention is to provide a kind of method of extracting the high purity Microcystin, this method is efficient, economy, purity are high, has solved present Microcystin standard substance and has cost an arm and a leg, obtains problems such as formality is numerous and diverse.
The technical scheme that the present invention takes is:
A kind of method of extracting the high purity Microcystin comprises that step is following:
(1) water sample pre-treatment: the microcystic aeruginosa water sample through liquid nitrogen multigelation 2-4 time, with broken frustule, is discharged microcapsule phycotoxin MC-LR in the born of the same parents, then that the water sample centrifuging and taking supernatant after the freeze thawing is subsequent use;
(2) enrichment of the pre-treatment of SPE solid-phase extraction column, MC-LR and purifying:, clean 2-4 time with ultrapure water again with washed with methanol solid-phase extraction column 2-4 time; The supernatant that feeds step (1) in the solid-phase extraction column after cleaning is crossed post, crosses the intact back of post and has made the leacheate solid-phase extraction column of MC-LR that cleaned enrichment with methanol aqueous solution, removes impurity;
(3) stripping of MC-LR with concentrate: with 100% methyl alcohol as elutriant cleaning enrichment the solid-phase extraction column of MC-LR with wash-out MC-LR; Make the MC-LR stripping; The MC-LR-methanol mixed solution of stripping is placed Rotary Evaporators, 40-60 ℃, rotary evaporation 10-20min.
Microcystic aeruginosa water sample frustule concentration about 10 described in the above-mentioned steps (1)
7Individual/ml, described centrifugal under 8000-10000rpm speed centrifugal 8-12 minute.
Solid-phase extraction column described in the step (2) is the C18 solid-phase extraction column; The described post speed of crossing is 5ml/min; Described methanol aqueous solution concentration is 10%-20% (v/v), washs 1-2 time.
The invention has the beneficial effects as follows:
(1) this patent is used is that 100% chromatographically pure methyl alcohol is made moving phase, and extraction effect is better, and sample keeps more stable, and after concentrating through Rotary Evaporators, the purity of algae toxin sample is higher, can extract to obtain a milligram level MC-LR, and impurity is few, and purity is high;
(2) MC in the Microcystis aeruginosa sample comprises MC in outer MC of born of the same parents and the born of the same parents; Therefore, total MC is a MC sum in outer MC of born of the same parents and the born of the same parents, and this research adopts the method for frozen-thawed to make the frustule broken wall; Can obtain in the born of the same parents simultaneously and the outer MC of born of the same parents; Saved the step of holding back and extract the filter membrane residue in the traditional filtering method, easy, the environmental protection of working method, and safety, economy;
(3) be easy to suitability for industrialized production;
(4) sample source is in the product of laboratory culture poison microcystic aeruginosa, and starting material are inexpensive, be easy to get; In the research Microcystin, can also study the product poison Changing Pattern of microcystic aeruginosa etc.
Description of drawings
Fig. 1 is the liquid chromatogram of embodiment 1 sample;
Fig. 2 is embodiment 1 sample and the reacted color atlas of F5.
Embodiment
Further specify the present invention below in conjunction with accompanying drawing and embodiment.
But the microcystic aeruginosa water sample can be gathered also laboratory enlarged culturing.
A kind of method of extracting the high purity Microcystin comprises that step is following:
(1) (the algae kind numbers 905 available from aquatic institute of the Chinese Academy of Sciences, and frustule concentration is about 10 after the activation to get the microcystic aeruginosa water sample
7Individual/ml) 100ml, centrifugal behind twice of frozen-thawed (effendorf, 8000r/min 10min), insert the blank glass bottle with supernatant.
(2) clean the C18 solid-phase extraction column 2 times with 10ml methyl alcohol (chromatographically pure) earlier, cross post speed 5ml/min, clean the C18 enriching column 2 times with the 20ml ultrapure water then, crossing post speed still is 5ml/min;
Supernatant is fed the C18 solid-phase extraction column (crossing post speed is 5ml/min) that cleaned through solid-phase extraction device, and the methyl alcohol of using 20ml 10% then is as 2 C18 enriching columns of leacheate continuous wash (cross post speed and still be 5ml/min).
(3) clean the C18 enriching column 2 times with 10ml methyl alcohol as elutriant, with the MC-LR stripping in the blank glass bottle.The 20ml MC-LR-methanol mixed solution of stripping is placed Rotary Evaporators, and 60 ℃ of rotary evaporation 20min are concentrated into 1ml.
2 one kinds of methods of extracting the high purity Microcystin of embodiment comprise that step is following:
(1) (the algae kind numbers 905 available from aquatic institute of the Chinese Academy of Sciences, and frustule concentration is about 10 after the activation to get the microcystic aeruginosa water sample
7Individual/ml) 100ml, centrifugal behind twice of frozen-thawed (effendorf, 8000r/min 10min), insert the blank glass bottle with supernatant.
(2) clean the C18 solid-phase extraction column 2 times with 10ml methyl alcohol (chromatographically pure) earlier, cross post speed 5ml/min, clean the C18 enriching column 2 times with the 20ml ultrapure water then, crossing post speed still is 5ml/min;
Supernatant is fed the C18 solid-phase extraction column (crossing post speed is 5ml/min) that cleaned through solid-phase extraction device, and the methyl alcohol of using 20ml 10% then is as 2 C18 enriching columns of leacheate continuous wash (cross post speed and still be 5ml/min).
(3) clean the C18 enriching column 2 times with 10ml methyl alcohol as elutriant, with the MC-LR stripping in the blank glass bottle.The 20ml MC-LR-methanol mixed solution of stripping is placed Rotary Evaporators, and 60 ℃ of rotary evaporation 20min are concentrated into 1ml.
The glass fiber filter of 1ml sample behind the embodiment 1 concentrated constant volume with 0.22 μ m filtered, and the liquid phase bottle of packing into of filtrating advances performance liquid appearance analyzing and testing.Qualitative according to RT, according to peak area with the external standard standard measure.
Testing used HPLC is the Agilent1100 high performance liquid chromatograph, and chromatographic column is the reverse analytical column of SB-C18; Moving phase is methyl alcohol: water (containing 0.1% trifluoroacetic acid)=70: 30 (V/V); Flow velocity is 0.7ml/min; The ultraviolet detection wavelength is 238nm; Sample IR: 5 μ l; Column temperature: 40 ℃; Through detecting, the concentration of this sample MC-LR is 98.5mg/L.The liquid chromatogram that detects this sample is as shown in Figure 1.Can find out, the almost not assorted peak of color atlas, main peak clearly is MC-LR, and interpret sample purity is higher, and impurity is few.
Test 2
(frustule concentration is about 10 to get the malicious microcystic aeruginosa liquid 100ml of product
7Individual/ml), centrifugal behind twice of frozen-thawed (effendorf, 8000r/min 10min), insert the blank glass bottle with supernatant.Bacterium liquid (the bacterial concentration about 10 that adds 20ml Microcystin degradation bacteria F5 in the bottle
9Individual/ml), put into the shaking culture case (25 ℃, 220r/min), the reaction 5d after detected result.
Detection to the MC-LR concentration in the vial is operated according to the step of test 1.Detected result shows that after Microcystin degradation bacteria F5 reaction, the concentration of MC-LR is 262mg/L in the bottle, and warp calculating F5 is 68.1% to the degradation rate of MC-LR, and this result is with very nearly the same with the result of MC-LR standard substance test.Thereby explain, when research Microcystin degradation experiment, can cultivate Microcystis aeruginosa fully voluntarily and then extract Microcystin; And the algae toxin concentration that extracts is the milligram level, and purity is higher, and general colleges and universities or scientific research institutions laboratory can be realized; To practice thrift experimental cost greatly, improve conventional efficient.As shown in Figure 2 in the test 2 with the reacted sample chromatogram figure of F5.
As stated, embodiments of the invention have been carried out explanation at length, but as long as not breaking away from inventive point of the present invention and effect in fact can have a lot of distortion, this will be readily apparent to persons skilled in the art.Therefore, such variation also all is included within protection scope of the present invention.
Claims (4)
1. a method of extracting the high purity Microcystin is characterized in that, comprises that step is following:
(1) water sample pre-treatment: the microcystic aeruginosa water sample through liquid nitrogen multigelation 2-4 time, with broken frustule, is discharged microcapsule phycotoxin MC-LR in the born of the same parents, then that the water sample centrifuging and taking supernatant after the freeze thawing is subsequent use;
(2) enrichment of the pre-treatment of SPE solid-phase extraction column, MC-LR and purifying:, clean 2-4 time with ultrapure water again with washed with methanol solid-phase extraction column 2-4 time; The supernatant that feeds step (1) in the solid-phase extraction column after cleaning is crossed post, crosses the intact back of post and has made the leacheate solid-phase extraction column of MC-LR that cleaned enrichment with methanol aqueous solution, removes impurity;
(3) stripping of MC-LR with concentrate: with 100% methyl alcohol as elutriant cleaning enrichment the solid-phase extraction column of MC-LR with wash-out MC-LR; Make the MC-LR stripping; The MC-LR-methanol mixed solution of stripping is placed Rotary Evaporators, 40-60 ℃, rotary evaporation 10-20min.
2. a kind of method of extracting the high purity Microcystin according to claim 1 is characterized in that, centrifugal described in the step (1) is under 8000-10000rpm speed centrifugal 8-12 minute.
3. a kind of method of extracting the high purity Microcystin according to claim 1 is characterized in that the solid-phase extraction column described in the step (2) is the C18 solid-phase extraction column; The described post speed of crossing is 5ml/min.
4. a kind of method of extracting the high purity Microcystin according to claim 1 is characterized in that the methanol aqueous solution concentration described in the step (2) is 10%-20%, washs 1-2 time.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103484394A (en) * | 2013-07-04 | 2014-01-01 | 河南师范大学 | Toxic microcystis bacterial strain and purification method of toxins produced by toxic microcystis bacterial strain |
| CN108752440A (en) * | 2018-06-21 | 2018-11-06 | 水利部中国科学院水工程生态研究所 | A kind of other extensive method for extraction and purification of the gram-grade of Microcystin |
| CN109060478A (en) * | 2018-08-27 | 2018-12-21 | 温州大学苍南研究院 | A kind of extracting method of microcystic aeruginosa extracellular polymeric |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101585867A (en) * | 2009-06-25 | 2009-11-25 | 江苏省农业科学院 | A kind of method of separation and purification Microcystin |
| CN102153635A (en) * | 2011-01-10 | 2011-08-17 | 常州大学 | Method for extracting microcystin-LR (Laboratory reagent) from blue-green algae in Taihu Lake |
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- 2011-12-30 CN CN2011104551315A patent/CN102516367A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101585867A (en) * | 2009-06-25 | 2009-11-25 | 江苏省农业科学院 | A kind of method of separation and purification Microcystin |
| CN102153635A (en) * | 2011-01-10 | 2011-08-17 | 常州大学 | Method for extracting microcystin-LR (Laboratory reagent) from blue-green algae in Taihu Lake |
Non-Patent Citations (1)
| Title |
|---|
| 任晶等: "应用HPLC优化铜绿微囊藻胞内毒素提取方法的研究", 《中国科技论文在线》, 3 March 2010 (2010-03-03) * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103484394A (en) * | 2013-07-04 | 2014-01-01 | 河南师范大学 | Toxic microcystis bacterial strain and purification method of toxins produced by toxic microcystis bacterial strain |
| CN103484394B (en) * | 2013-07-04 | 2015-02-18 | 河南师范大学 | Toxic microcystis bacterial strain and purification method of toxins produced by toxic microcystis bacterial strain |
| CN108752440A (en) * | 2018-06-21 | 2018-11-06 | 水利部中国科学院水工程生态研究所 | A kind of other extensive method for extraction and purification of the gram-grade of Microcystin |
| CN108752440B (en) * | 2018-06-21 | 2022-08-26 | 水利部中国科学院水工程生态研究所 | Gram-level large-scale extraction and purification method of microcystin |
| CN109060478A (en) * | 2018-08-27 | 2018-12-21 | 温州大学苍南研究院 | A kind of extracting method of microcystic aeruginosa extracellular polymeric |
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Application publication date: 20120627 |