CN105237543A - Method for purification preparation of N-sulfocarbamoyl toxins - Google Patents
Method for purification preparation of N-sulfocarbamoyl toxins Download PDFInfo
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Abstract
The invention provides a method for purification preparation of N-sulfocarbamoyl toxins. The method comprises the steps of culture of toxin production algae, extraction of toxins, collection and purification of the toxins and the like. The process is simple and effective, meanwhile, the purity of the prepared N-sulfocarbamoyl gonyautoxins-2 (C1) and N-sulfocarbamoyl gonyautoxins-3 (C2) can reach higher than 91%, and the N-sulfocarbamoyl toxins can be used for quality safety detection and scientific research of saxitoxin in aquatic products.
Description
Technical field
The present invention relates to microorganism detection field, relate to a kind of method of purification N-sulphonyl carbamyl toxin specifically.
Background technology
Paralytic shellfish poisoning (PSP) (PSTs) is the water-soluble extremely strong small molecules secondary metabolite of a class that in water body, planktonic algae or microorganism produce.Its basic structure is the how folded six-ring of four aquation purine of two guanidine radicals.According to the difference of substituted radical, mainly be divided into four classes: 1. carbamate toxoid (Carbamatetoxins), comprise saxitoxin (Saxitoxin, STX), N-STX (neoSTX, and GTX1-4 (Gonyautoxins, GTXl-4) NEO); 2. N-sulphonyl carbamyl toxoid (N-sulfocarbamoyltoxins), comprises GTX5 (B1), GTX6 (B2) and C1-C4; 3. deamination formyl radical toxoid (Decarbamoyltoxins), comprises dcSTX, dcneoSTX, dcGTX1-4; 4. deoxidation deamination formyl radical toxoid (Deoxydecarb-amoyltoxins), comprises doSTX, doneoSTX and doGTX1 etc.
PSTs is the efficient sodium-ion channel neurotoxin of a class.After human body has taken in the sea-food polluted by PSTs, clinical initial stage main manifestations has been that mouth, lip, tongue are numb, and paralysis symptom appears in four limbs, and sensation occurs abnormal, and people becomes weak and chaotic; Later stage main manifestations is nauseating, and vomiting, is short of breath, headache fever, dysphagia, dysarteriotony, and severe patient occurs clouding of consciousness even dead.Although itself toxicity of N-sulphonyl carbamyl toxoid is less, very easily sloughs alkylsulfonyl and carbamic acid ester chain by bio-transformation in vivo and generate the very strong STX of toxicity and deamination formyl radical toxoid.In addition, up-to-date Biotransformation experiments also finds and confirms to there is C1/C2 toxin in shellfish fishery products body.
For ensureing the public not by the threat of PSTs, world many countries, area and relevant international organization all carry out stringent regulations and control to shellfish fishery products, and have formulated the PSTs limit standard of corresponding shellfish fishery products and goods thereof.But PSTs standard substance are expensive, therefore associated toxin standard substance preparation and purification technology is shown great attention to.
Due to the unstable of toxin source, PSTs purification technique is the Focal point and difficult point of this toxoid quantitative-qualitative analysis always.The toxin main source of current PSTs purifying is the toxiferous algae strain that in natural marine site, separation and purification obtains.But have greatly algae strain not possess culture condition, have been reported culture condition and the toxiferous algae kind can changing cultivation on a small scale only have A.catenella, A.tamarense (AT, Alexandrium tamarense) and A.minutum etc. a few.But systematic, best method is not still had to the cultivation of toxiferous algae AT.
Therefore be necessary that establishing one can prepare purifying N-sulphonyl carbamyl toxoid method, for relevant fish quality supervision provides technical support.
Summary of the invention
The object of the present invention is to provide a kind of method of purification N-sulphonyl carbamyl toxin, the method comprises the steps:
(1) cultivation of toxiferous algae: the pure algae strain of AT is aseptically accessed in the artificial seawater containing nutritive salt, pure algae strain liquid and artificial seawater volume ratio are 1:2, the cultivation of algae strain is carried out, incubation time 5 days under the condition of f/2 substratum, temperature 23 DEG C, salinity 2.8-3.2% (preferred salinity is 3.0%), pH7.5-8.5 (preferred pH is 8.0), light intensity 16000lux and Light To Dark Ratio 14L:10D;
(2) extraction of toxin: algae liquid is through 0.22 μm of filter membrane suction filtration, the frond got on film is that 1:2 adds extracting solution with volume ratio, room temperature is extracted twice, each 10min, extracted twice liquid is merged after the centrifugal 10min of 4500rpm,-20 DEG C of freezing 4h discard upper strata acetonitrile layer, namely obtain the crude extract of toxin; Wherein extracting solution is that acetonitrile, water and formic acid are formulated according to the volume ratio of 80:19.9:0.1;
(3) collection of toxin and purifying: get crude extract 0.5mL and enter GPC (G-15) purification system, it is wash-out under the isocratic condition of moving phase at 0.1% acetic acid, flow velocity is set to 2.50mL/min, arranges the collection time of effluent liquid, collects with automatic collector; Collect the effluent liquid lyophilize obtained and be settled to 1mL to using up dry doubling 0.1% acetic acid, cross 0.22 μm of filter membrane, enter LC-MS/MS and carry out qualitative to product seed culture of viruses class; HPLC-FLD post-column derivation-peak area normalization method is adopted to calculate toxin purity.
The liquid phase analysis condition wherein used in step (3) is specific as follows:
The hydrophilic post of chromatographic column: TSK-gelAmide-80 (150 × 2mm, 3 μm); Column temperature: 30 DEG C; Flow velocity: 0.3mL/min; Sample size: 5 μ L;
Moving phase: A phase: acetonitrile; B phase: 3.6mM formic acid, the 2mM ammonium formiate aqueous solution (pH3.5);
Gradient elution program: 0min, 15%B; 0-10min, 45%B; 10-11min, 45%B; 11-14min, 15%B, 14-19, min15%B.
The mass spectroscopy condition used in step (3) is specific as follows:
Ion source pattern: positive ion mode (ESI+); Spray voltage: 4.0kv (ESI+); Heat block temperature: 300 DEG C; Ion transfer tube temperature: 350 DEG C; Sheath gas: nitrogen, flow 35arb; Assisted gas: nitrogen, flow 20arb; Collision gas: argon gas, impact pressure 1.5mTorr; The mass spectrometry parameters of toxin is in table 1.
In step (3) use chromatogram and derivatization conditions as follows:
Chromatographic column: AgleaVenusilXBPC8 (4.6 × 150mm, 5 μm); Moving phase (degree of grade): 2mM tetrabutyl ammonium phosphate solution (10% acetic acid or 1% ammoniacal liquor unidirectional adjustment pH to 5.8); Flow velocity: 0.8mL/min; Post-column derivation agent: the 100mM phosphoric acid+5mM Periodic acid aqueous solution (5MNaOH regulates pH to 7.8), flow velocity 0.4mL/min; Souring agent: 0.75M nitric acid, flow velocity 0.4mL/min; Column temperature: 20 DEG C; Derivative pond temperature: 85 DEG C; Fluorimetric detector parameter: excitation wavelength: 330nm; Emission wavelength: 390nm; Sample size: 10 μ L.
Present invention also offers a kind of cultural method of Alexandrium tamarense, the pure algae strain of AT is aseptically accessed in the artificial seawater containing nutritive salt, pure algae strain liquid and artificial seawater volume ratio are 1:2, the cultivation of algae strain is carried out, incubation time 5 days under the condition of f/2 substratum, temperature 23 DEG C, salinity 2.8-3.2% (preferred salinity is 3.0%), pH7.5-8.5 (preferred pH is 8.0), light intensity 16000lux and Light To Dark Ratio 14L:10D.
The present invention is by optimizing the culture condition of Alexandrium tamarense, make it that the highest vigor can be kept to breed, and then produce the PSTs of maximum, this strain algae kind can produce the component of C1/C2 and GTX2/GTX3 tetra-kinds of PSTs, wherein C2 content is the highest, can reach more than 90%; After sephadex G-15 purifying, C1/C2 and GTX2/3 is separated completely; The C1/C2 of purity more than 91% can be obtained by adjustment collection time.Compared with prior art, method is simple, and toxin purity is higher for this technology.
Accompanying drawing explanation
Fig. 1 is that toxin crude extract carries out the toxin Purified rate of recovery through sephadex G-15.
Fig. 2 be toxin Purified after C1/C2HPLC-FLD color atlas.
Embodiment
The present invention is further illustrated for embodiment given below.The present invention is described in conjunction with most preferred embodiment, but after reading example of the present invention, those skilled in the art can understand and in disclosed enforcement, make many changes and also can obtain same or similar result, all belong to the spirit and scope of the present invention.In particular, the alternative reagent disclosed herein of some reagent and obtain identical or similar results.All similar replacements or modification are all considered to the spirit and scope of the present invention, and all above-mentioned equivalent form of values all belong to the scope that claims of the present invention limits.
Embodiment 1
1, materials and methods
1.1 key instruments and reagent
LC-MS/MS: electric spray ion source (ESI) (ThermoFisherScientific, USA);
Fluorescent derivatization system: Watersalliance2690/2475 (Waters, USA) after HPLC column;
Gel-filtration system: J2preplincGPC/SPE/evap combined instrument (J2scientific, USA);
Incubator: PQX-350H growth cabinet, Shanghai Shen Xian thermostatic equipment factory;
Digital P H counts: PHS-3C, Shanghai Lei Ci instrument plant;
Freeze drier: HetoPowerDryLL3000, Shanghai Hui Fen Electronic Science and Technology Co., Ltd.;
Cell disruptor: GA98-IIIDB, excellent bio tech ltd, Wuxi;
Hand refractometer: OTC, Shanghai Tian Lei instrument company limited.
1.2 standard substance and reagent
Toxin standard substance (STX, NEO, GTX1/4, GTX2/3, C1/C2) are purchased from marine life research institute of Canadian NRC; Formic acid, acetonitrile, acetic acid and sephadex G-15 are purchased from traditional Chinese medicines chemical reagent company limited.
Alexandrium tamarense kind: be separated from East China Sea MATTER IN CHANGJIANG ESTUARY, is provided by coastal ocean environmental science National Key Laboratory of Xiamen University;
The preparation reference Guillard of f/2 substratum, R.R.L.1975.Cultureofphytoplanktonforfeedingmarineinverte brates.pp26-60.InSmithW.L.andChanleyM.H. (Eds.) CultureofMarineInvertebrateAnimals.PlenumPress, NewYork, USA
2, experimental technique
2.1 toxin producing algae strain cultural methods
The pure algae strain of AT is aseptically accessed in the artificial seawater containing nutritive salt, pure algae strain liquid and artificial seawater volume ratio are 1:2, the cultivation of algae strain is carried out, incubation time 5 days under the condition of f/2 substratum, temperature 23 DEG C, salinity 3.0%, pH8.0, light intensity 16000lux and Light To Dark Ratio 14L:10D.
2.2 Toxic extraction methods
Algae liquid is through 0.22 μm of filter membrane suction filtration, the frond got on film take volume ratio as the acetonitrile water (volume ratio: acetonitrile+water+formic acid=80:19.9:0.1) that 1:2 adds 80%, room temperature is extracted twice, each 10min, extracted twice liquid is merged after the centrifugal 10min of 4500rpm,-20 DEG C of freezing 4h discard upper strata acetonitrile layer, namely obtain the crude extract of toxin.
2.3 toxin purification process
Get crude extract 0.5mL and enter GPC (G-15) purification system, be wash-out under the isocratic condition of moving phase at 0.1% acetic acid, flow velocity is set to 2.50mL/min, arranges the collection time of effluent liquid, collects with automatic collector, pending; Collect the effluent liquid lyophilize obtained and be settled to 1mL to using up dry doubling 0.1% acetic acid, cross 0.22 μm of filter membrane, enter LC-MS/MS and carry out qualitative to product seed culture of viruses class; HPLC-FLD post-column derivation-peak area normalization method is adopted to calculate toxin purity.
2.4 liquid chromatography mass conditions
(1) liquid-phase condition:
The hydrophilic post of chromatographic column: TSK-gelAmide-80 (150 × 2mm, 3 μm); Column temperature: 30 DEG C; Flow velocity: 0.3mL/min; Sample size: 5 μ L;
Moving phase: A phase: acetonitrile; B phase: 3.6mM formic acid, the 2mM ammonium formiate aqueous solution (pH3.5)
Gradient elution program: 0min, 15%B; 0-10min, 45%B; 10-11min, 45%B; 11-14min, 15%B, 14-19, min15%B.
(2) Mass Spectrometry Conditions:
Ion source pattern: positive ion mode (ESI+); Spray voltage: 4.0kv (ESI+); Heat block temperature: 300 DEG C; Ion transfer tube temperature: 350 DEG C; Sheath gas: nitrogen, flow 35arb; Assisted gas: nitrogen, flow 20arb; Collision gas: argon gas, impact pressure 1.5mTorr.The mass spectrometry parameters of toxin is in table 1.
The mass spectrometry parameters of each toxin of table 1
* quantitative fragmention, what do not mark * is then that the auxiliary alternative ion of qualitative ion excludes in table
2.5HPLC-FLD chromatographic condition
Chromatographic column: AgleaVenusilXBPC8 (4.6 × 150mm, 5 μm); Moving phase (degree of grade): 2mM tetrabutyl ammonium phosphate solution (10% acetic acid or 1% ammoniacal liquor unidirectional adjustment pH to 5.8); Flow velocity: 0.8mL/min; Post-column derivation agent: the 100mM phosphoric acid+5mM Periodic acid aqueous solution (5MNaOH regulates pH to 7.8), flow velocity 0.4mL/min; Souring agent: 0.75M nitric acid, flow velocity 0.4mL/min; Column temperature: 20 DEG C; Derivative pond temperature: 85 DEG C; Fluorimetric detector parameter: excitation wavelength: 330nm; Emission wavelength: 390nm; Sample size: 10 μ L.
3, result and discussion
The optimization of 3.1 toxiferous algae growth conditionss
Daily carry out vitality test to the postvaccinal algae liquid of logarithmic phase, in 5 days, algae liquid is in fast growing period, and the 6th day starts algae cell density substantially constant, determines to become the stagnation point of stable growth phase for logarithmic phase on the 5th day; Under envrionment temperature is 23 DEG C of conditions, AT has the highest cell density, determines that the optimum growth temperature of AT is 23 DEG C; AT almost can not survive in the environment of Low-salinity, but too high salinity (>3.2%) can suppress it to grow equally, and its suitable salinity range, between 2.8 ~ 3.2, selects 3.0% optimum salinity grown as AT; It is 16000lux that incubator can reach light intensity, and close to the AT light saturation point of bibliographical information, under this intensity, cell growth rate obviously slows down, and therefore determines that intensity of illumination is 16000lux; AT is very responsive for the change of pH, and small change (0.1 ~ 0.2) can its growth velocity of remarkably influenced, and it is applicable to pH narrow range of growth, is near 7.5 ~ 8.5, highly consistent with the pH in natural sea-water.During pH regulator artificial seawater acidity to 8.0, obtain the fastest growth velocity.
Finally determine that AT optimal culture condition is: f/2 substratum, temperature 23 DEG C, salinity 3.0%, pH8.0, light intensity 16000lux, Light To Dark Ratio 14L:10D.Under this condition, the maximum cell density obtaining the strain of AT algae is 2.2 × 10
4cell/mL.
The purifying of 3.2 toxin crude extracts
The polar micromolecules material of sephadex G-15 separable 300 ~ 1500.Crude extract after 0.5mL lyophilize being concentrated directly enters GPC (G-15) systematic position, and within every 10 minutes, collect an effluent liquid, collect 10 times altogether, result as shown in Figure 1.
Result shows, and 60 ~ 70min is that PSTs mainly goes out crest segment, and according to the LC-MS/MS detected result display carried out simultaneously, the main ingredient that AT produces PSTs is C1/C2, accounts for more than 97% of toxin total amount, wherein in the majority with C2 again, accounts for more than 90% of toxin total amount.All the other 3% mainly GTX2/3, concentrate on 70 ~ 80min and occur.Experiment shows, C toxoid and GTXs can effectively divide out by G-15, and the elution time of toxin highly stable (<1min).Further separation shows, GTX2/3 mainly occurs at 75 ~ 85min, and C1/C2 mainly occurs at 56 ~ 72min.Because the content of GTX2/3 is less, therefore after purifying, be mainly C1/C2.
Adjust different collection time periods to obtain toxin C 1/C2, cross 0.22 μm of filter membrane after lyophilize is concentrated, HPLC-FLD post-column derivation areas of peak normalization method measures its purity.Post-column derivation C1/C2 color atlas as shown in Figure 2, the rate of recovery and purity result as shown in table 2.
The impact of the table 2 different collection time contratoxin C1/C2 rate of recovery and purity
4, conclusion
The steps such as the cultivation of toxiferous algae that the present invention adopts, the extraction of toxin and purifying; technique is simply effective; N-sulphonyl carbamyl gonyatoxin-2 (C1) simultaneously prepared and N-sulphonyl carbamyl gonyatoxin-3 (C2) purity can reach more than 91%, can be used for saxitoxin quality safety in fishery products and detect and scientific research.
Claims (6)
1. a method for purification N-sulphonyl carbamyl toxin, is characterized in that the method comprises the steps:
(1) cultivation of toxiferous algae: the pure algae strain of AT is aseptically accessed in the artificial seawater containing nutritive salt, pure algae strain liquid and artificial seawater volume ratio are 1:2, the cultivation of algae strain is carried out, incubation time 5 days under the condition of f/2 substratum, temperature 23 DEG C, salinity 2.8-3.2%, pH7.5-8.5, light intensity 16000lux and Light To Dark Ratio 14L:10D;
(2) extraction of toxin: algae liquid is through 0.22 μm of filter membrane suction filtration, the frond got on film is that 1:2 adds extracting solution with volume ratio, room temperature is extracted twice, each 10min, extracted twice liquid is merged after the centrifugal 10min of 4500rpm,-20 DEG C freezing after discard upper strata acetonitrile layer, namely obtain the crude extract of toxin; Wherein extracting solution is that acetonitrile, water and formic acid are formulated according to the volume ratio of 80:19.9:0.1;
(3) collection of toxin and purifying: get crude extract and enter G-15 purification system, be wash-out under the isocratic condition of moving phase at 0.1% acetic acid, flow velocity is set to 2.50mL/min, arranges the collection time of effluent liquid, collects with automatic collector; Collect the effluent liquid lyophilize dry doubling 0.1% acetic acid constant volume extremely to the greatest extent obtained, cross 0.22 μm of filter membrane, enter LC-MS/MS and carry out qualitative to product seed culture of viruses class; HPLC-FLD post-column derivation-peak area normalization method is adopted to calculate toxin purity.
2. the method for purification N-sulphonyl carbamyl toxin according to claim 1, the liquid phase analysis condition wherein used in step (3) is specific as follows:
The hydrophilic post of chromatographic column: TSK-gelAmide-80,150 × 2mm, 3 μm; Column temperature: 30 DEG C; Flow velocity: 0.3mL/min; Sample size: 5 μ L;
Moving phase: A phase: acetonitrile; B phase: 3.6mM formic acid, 2mM ammonium formiate aqueous solution pH3.5;
Gradient elution program: 0min, 15%B; 0-10min, 45%B; 10-11min, 45%B; 11-14min, 15%B, 14-19, min15%B.
3. the method for purification N-sulphonyl carbamyl toxin according to claim 1, the mass spectroscopy condition used in step (3) is specific as follows:
Ion source pattern: positive ion mode ESI+; Spray voltage: 4.0kvESI+; Heat block temperature: 300 DEG C; Ion transfer tube temperature: 350 DEG C; Sheath gas: nitrogen, flow 35arb; Assisted gas: nitrogen, flow 20arb; Collision gas: argon gas, impact pressure 1.5mTorr; The mass spectrometry parameters of toxin is in table 1.
4. the method for purification N-sulphonyl carbamyl toxin according to claim 1, in step (3) use chromatogram and derivatization conditions as follows:
Chromatographic column: AgleaVenusilXBPC8,4.6 × 150mm, 5 μm; Flowing equality: 2mM tetrabutyl ammonium phosphate solution, 10% acetic acid or 1% ammoniacal liquor unidirectional adjustment pH to 5.8; Flow velocity: 0.8mL/min; Post-column derivation agent: the 100mM phosphoric acid+5mM Periodic acid aqueous solution, regulates pH to 7.8, flow velocity 0.4mL/min with 5MNaOH; Souring agent: 0.75M nitric acid, flow velocity 0.4mL/min; Column temperature: 20 DEG C; Derivative pond temperature: 85 DEG C; Fluorimetric detector parameter: excitation wavelength: 330nm; Emission wavelength: 390nm; Sample size: 10 μ L.
5. a cultural method for Alexandrium tamarense, is characterized in that the method comprises the steps:
The pure algae strain of AT is aseptically accessed in the artificial seawater containing nutritive salt, pure algae strain liquid and artificial seawater volume ratio are 1:2, the cultivation of algae strain is carried out, incubation time 5 days under the condition of f/2 substratum, temperature 23 DEG C, salinity 2.8-3.2%, pH7.5-8.5, light intensity 16000lux and Light To Dark Ratio 14L:10D.
6. cultural method according to claim 5, is characterized in that the method comprises the steps:
The pure algae strain of AT is aseptically accessed in the artificial seawater containing nutritive salt, pure algae strain liquid and artificial seawater volume ratio are 1:2, the cultivation of algae strain is carried out, incubation time 5 days under the condition of f/2 substratum, temperature 23 DEG C, salinity 3.0%, pH8.0, light intensity 16000lux and Light To Dark Ratio 14L:10D.
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Application publication date: 20160113 |