CN104876939A - Method for extracting paralytic shellfish toxins from shell raw materials - Google Patents
Method for extracting paralytic shellfish toxins from shell raw materials Download PDFInfo
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- CN104876939A CN104876939A CN201410072059.1A CN201410072059A CN104876939A CN 104876939 A CN104876939 A CN 104876939A CN 201410072059 A CN201410072059 A CN 201410072059A CN 104876939 A CN104876939 A CN 104876939A
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Abstract
The invention discloses a method for extracting paralytic shellfish toxins from shell tissues. The method comprises the following steps: 1, mixing shell raw materials with acetic acid until the pH value of the obtained mixture is 2-4, carrying out ultrasonic crushing, heating the crushed mixture in boiling water for 5-10min, cooling, filtering, and collecting the obtained filtrate; 2, carrying out reduced pressure concentration on the filtrate, freezing, centrifuging, taking the obtained supernatant, extracting the supernatant by using trichloromethane, uniformly mixing the obtained upper layer extract liquid with ethanol, stirring at room temperature for 20-24h, carrying out secondary centrifuge, collecting the obtained new supernatant, carrying out reduced pressure concentration, drying, dissolving the obtained dried material in acidic methanol, filtering, collecting the obtained new filtrate, drying, dissolving the dried new filtrate in an aqueous acetic acid solution with the pH value of 2-4, adding the obtained sample to a gel column, carrying out column chromatography, and collecting the fraction with the retention time of 160-250min. The method maximally improves the extraction rate of the paralytic shellfish toxins, can effectively remove fats, proteins, carbohydrates and other impurities in the tissues, and maximally reduces the bioactive loss of the toxins.
Description
Technical field
The invention belongs to organic compound and extract field, especially relate to a kind of method extracting paralytic shellfish poisoning (PSP) from shell raw material.
Background technology
Paralytic shellfish poisoning (PSP) (paralytic shellfish poisoning toxins, PSP) be that a class produces the marine biotoxins of paralysis effect to nerve system of human body, produce primarily of toxic dinoflagellate, by food chain shellfish, as: in the organisms such as Perna uiridis (Linnaeus), chlamys farreri, bay scallop, clam, clam, Crassostrea rivularis and basket whelk, accumulation is amplified.When people eat the sea-food of these contaminations by mistake, will occur poisoning or dead.Paralytic shellfish poisoning (PSP) more and more wider in distribution on global, frequency is more and more higher, annual 2000 poisonings occurs, and wherein mortality ratio reaches 15%.PSP toxin is with saxitoxin (Saxitoxin; the derivative of the class tetrahydrochysene purine STX) be derived for skeleton is such as formula shown in I; have now found that this toxoid has more than 50 to plant structure moiety, this toxin can be divided into amino formate, N-sulphonyl carboxamide base class, deamination formyl base class and deoxidation deamination formyl base class according to the difference of R4 group.
Formula I
At present, paralytic shellfish poisoning (PSP) has application in the researchs such as red tide detection, molecular biology, neurobiology, medical diagnosis, drug development, military war agent.At food sanitation and secure context, along with the deterioration of ocean environment, a large amount of generations of red tide, pay attention to more to the inspection of shellfish poison both at home and abroad.The method utilizing cultivation in sea water toxiferous algae class is generally taked in extraction about paralytic shellfish poisoning (PSP), but this geographically has a definite limitation.And from shellfish tissue the toxin of Extraction and enrichment, because this type of content of toxins is very low, in extraction concentration process, be easily subject to the impact of shellfish substrate as materials such as protein, fat, carbohydrates, be difficult to extract the higher sample of purity for toxin correlative study.
Summary of the invention
The object of this invention is to provide a kind of method extracting paralytic shellfish poisoning (PSP) from shell raw material.
The method extracting paralytic shellfish poisoning (PSP) from shell raw material provided by the invention, comprises the steps:
1) pH value shell raw material and aqueous acetic acid mixed to mixture is after 2-4, ultrasonication, then is placed in boiling water and heats 5-10min, cooled and filtered, collects filtrate;
2) by frozen centrifugation after step 1) gained filtrate reduced in volume, get supernatant liquor chloroform extraction, get upper layer of extraction liquid and ethanol mix after after stirring at room temperature 20-24 hour, carry out second time centrifugal, dry after collecting supernatant liquor concentrating under reduced pressure, filter after dissolving with acidified methanol again, after collecting filtrate drying, being dissolved in pH value is after the aqueous acetic acid of 2-4, be splined on gel column and carry out column chromatography, collecting retention time is the flow point of 160min-250min, obtains the extracting solution containing described paralytic shellfish poisoning (PSP);
Described acidified methanol is that the acetic acid of 2-5:95-98 and methyl alcohol form by volume ratio.
In the step 1) of aforesaid method, the concentration of described aqueous acetic acid is 0.05-0.2M, is specially 0.1M;
The pH value of mixture is 2-3;
In ultrasonication step, power is 600-1500W, is specially 600W, and the time is 8-30min, is specially 8-10min.
Step 2) described step 2) in frozen centrifugation step, rotating speed is 4000-6000rpm, is specially 4000-5000rpm; Centrifugal radius is 4-6cm, is specially 5cm;
Time 10-12min, is specially 10min;
Temperature is 3-7 DEG C, is specially 4 DEG C;
In described second time centrifugation step, rotating speed is 8000-10000rpm; Centrifugal radius is 4-6cm, is specially 5cm; Time is 8-10min.
Acidified methanol solubilized toxin, and other residue water-soluble impurity can be removed;
Collect in filtrate drying step, dry object is to remove described acidified methanol.
The method of described drying can be vacuum lyophilization;
Described step 2) in column chromatography steps, applied sample amount is the 2-5% of gel column volume used, is specially 3-4%;
The aqueous acetic acid of eluent to be pH value be 2-4;
Elution speed is 0.4-0.6mL/min, is specially 0.5mL/min;
The consumption of eluent is 3-5 times of gel column volume used, is specially 4 times;
In gel column used, the model of gel is Sephadex G15 or Bio-Gel-p-2;
Length-to-diameter ratio is 30-40:1, is specially 30:1;
The length of gel column is 60-100cm; Diameter is 1-5cm, is specially 2cm.
Described retention time specifically can be 160min-200min or 200min-250min;
Described shell raw material specifically can be Patinopecten yessoensis or Chlamys nobilis.
The present invention improves the extraction yield of paralytic shellfish poisoning (PSP) to greatest extent, effectively can remove the impurity such as tissue interior fat, protein, carbohydrate; And the loss of the biological activity of toxin is reduced to a minimum; And raw material more easily obtains, extract in concentration process, treatment temp is lower, has important using value.
Accompanying drawing explanation
Fig. 1 is that embodiment 1 is separated fluoroscopic examination spectrogram with Sephadex-G15 gel column in 2.
Embodiment
Below in conjunction with specific embodiment, the present invention is further elaborated, but the present invention is not limited to following examples.Described method is ordinary method if no special instructions.Described starting material all can obtain from open commercial sources if no special instructions.Following embodiment step 2) in column chromatography steps, in gel column used, the model of gel is Sephadex-G15, and length-to-diameter ratio is 30:1, and the length of gel column is 60cm, and diameter is 2cm.
Embodiment 1,
1) freezing Patinopecten yessoensis flowing water is thawed, get its sexual gland and drain, take 1 kilogram.Use homogenizer homogeneous, put into the beaker that volume is 3 liters, add 1 liter of 0.1M aqueous acetic acid, after stirring to the pH value of mixture be after 3, utilize Ultrasonic Cell Disruptor under the condition of 600W power broken 10 minutes, be placed in boiling water again and heat 5min, filter with four layers of hospital gauze after cooling, collect filtrate;
2) step 1) gained filtrate vacuum rotary evaporator is carried out concentrating under reduced pressure until in 4 DEG C of frozen centrifugation 10min under 4000rpm, centrifugal radius are the condition of 5cm after 0.3 liter, get supernatant liquor and be placed in separating funnel, use 150mLCHCl3 extracting twice, to remove part lipoid material.Get upper layer of extraction liquid and 300mL ethanol mix after in stirring at room temperature after 24 hours, at 8000rpm, centrifugal radius is the decorating film carrying out second time centrifugal 10min removing precipitation under the condition of 5cm, collect supernatant liquor and be evaporated to the lyophilize of 15mL final vacuum in 50 DEG C, again with being filter after acidified methanol that the acetic acid of 2:98 and methyl alcohol form dissolves by volume ratio, collect filtrate in 34 DEG C of decompression Rotary dryings with after removing acidified methanol, be dissolved in the aqueous acetic acid that pH value is 3, be splined on gel column and carry out column chromatography, applied sample amount is 1ml, for 3% of gel column volume, eluent to be pH value be 3 aqueous acetic acid, the flow velocity of eluent is 0.5ml/min, the consumption of eluent is 4 times of gel column volume used, collecting retention time is the flow point of 160min-200min, namely the extracting solution containing paralytic shellfish poisoning (PSP) is obtained.
Wherein, the determination of different retention time flow point composition is as follows:
From each flow point, respectively get 0.5mL fluoroscopic examination to determine the retention time containing paralytic shellfish poisoning (PSP) flow point, concrete grammar is as follows: being joined by each for 0.5mL flow point in 2mL oxygenant (is buffered soln and the HIO of 9.0 potassium primary phosphates by 50mmol/L, pH value
4composition, wherein HIO
4concentration be 10mmol/L), at 65 DEG C of heating in water bath 3min, it is 0.5mol/L acetic acid that rapid taking-up adds 2.5mL concentration, toxin oxidation solution after acidifying carries out fluoroscopic examination (excitation wavelength: 330nm, emission wavelength: 390nm), measure relative intensity of fluorescence, acquired results as shown in Figure 1.
As seen from the figure, the maximum value of relative intensity of fluorescence is positioned at second peak, is 982.9, and the retention time of its correspondence is 160min-200min, namely obtains the extracting solution containing paralytic shellfish poisoning (PSP), and extraction yield is 76.5%.
According to the mensuration of paralytic shellfish poisoning (PSP) " in the GBT5009.213-2008 shellfish " method, the toxicity measuring this embodiment gained paralytic shellfish poisoning (PSP) extracting solution of gained is 279Mu.
Embodiment 2,
1) Chlamys nobilis is shelled get its sexual gland and drain, take 1 kilogram.Use homogenizer homogeneous, put into the beaker that volume is 3 liters, add 1 liter of 0.1M aqueous acetic acid, after stirring to the pH value of mixture be after 2, utilize Ultrasonic Cell Disruptor under the condition of 600W power broken 8 minutes, be placed in boiling water again and heat 10min, filter with four layers of hospital gauze after cooling, collect filtrate;
2) step 1) gained filtrate vacuum rotary evaporator is carried out concentrating under reduced pressure until in 4 DEG C of frozen centrifugations centrifugal 10min under 5000rpm, centrifugal radius are the condition of 5cm after 0.3 liter, get supernatant liquor and be placed in separating funnel, use 150mLCHCl3 extracting twice, to remove part lipoid material.Get upper layer of extraction liquid and 300mL ethanol mix after in stirring at room temperature after 24 hours, at 10000rpm, centrifugal radius is the decorating film carrying out the decorating film removing precipitation that the centrifugal 8min removing of second time is separated out under the condition of 5cm, collect supernatant liquor and be evaporated to 15mL vacuum lyophilization in 50 DEG C, again with after by volume ratio being the acidified methanol dissolution filter that forms of the acetic acid of 2:98 and methyl alcohol, filtrate is in 40 DEG C of decompression Rotary drying removing acidified methanol, being dissolved in pH value is after the aqueous acetic acid of 3, be splined on gel column and carry out column chromatography, applied sample amount is 4% of gel column volume, eluent to be pH value be 3 aqueous acetic acid, the flow velocity of eluent is 0.4ml/min, the consumption of eluent is 4 times of gel column volume used, collecting retention time is the flow point of 200min-250min, namely the extracting solution containing paralytic shellfish poisoning (PSP) is obtained.
Wherein, the determination of different retention time flow point composition is as follows:
From each flow point, respectively get 0.5mL fluoroscopic examination to determine the retention time of the flow point containing paralytic shellfish poisoning (PSP), concrete grammar is as follows: being joined by each for 0.5mL flow point in 2mL oxygenant (is buffered soln and the HIO of 9.0 potassium primary phosphates by 50mmol/L, pH value
4composition, wherein HIO
4concentration be 10mmol/L), at 65 DEG C of heating in water bath 3min, take out rapidly that to add 2.5mL concentration be 0.5mol/L acetic acid, toxin oxidation solution after acidifying carries out fluoroscopic examination (excitation wavelength: 330nm, emission wavelength: 390nm), measure relative intensity of fluorescence, acquired results is as shown in Figure 1.
As seen from the figure, the maximum value of relative intensity of fluorescence is positioned at second peak, is 660.2, and the retention time of its correspondence is 200-250min, namely obtains the extracting solution containing paralytic shellfish poisoning (PSP), and extraction yield is 72.8%.
According to the mensuration of paralytic shellfish poisoning (PSP) " in the GBT5009.213-2008 shellfish " method, the toxicity measuring this embodiment gained paralytic shellfish poisoning (PSP) extracting solution of gained is 187Mu.
Claims (5)
1. from shell raw material, extract a method for paralytic shellfish poisoning (PSP), comprise the steps:
1) pH value shell raw material and aqueous acetic acid mixed to mixture is after 2-4, ultrasonication, then is placed in boiling water and heats 5-10min, cooled and filtered, collects filtrate;
2) by frozen centrifugation after step 1) gained filtrate reduced in volume, get supernatant liquor chloroform extraction, get upper layer of extraction liquid and ethanol mix after after stirring at room temperature 20-24 hour, carry out second time centrifugal, dry after collecting supernatant liquor concentrating under reduced pressure, filter after dissolving with acidified methanol again, after collecting filtrate drying, being dissolved in pH value is after the aqueous acetic acid of 2-4, be splined on gel column and carry out column chromatography, collecting retention time is the flow point of 160min-250min, obtains the extracting solution containing described paralytic shellfish poisoning (PSP);
Described acidified methanol is that the acetic acid of 2-5:95-98 and methyl alcohol form by volume ratio.
2. method according to claim 1, is characterized in that: in described step 1), and the concentration of described aqueous acetic acid is 0.05-0.2M, is specially 0.1M;
The pH value of mixture is 2-3;
In ultrasonication step, power is 600-1500W, is specially 600W, and the time is 8-30min, is specially 8-10min.
3. method according to claim 1 and 2, is characterized in that: described step 2) in frozen centrifugation step, rotating speed is 4000-6000rpm, is specially 4000-5000rpm; Centrifugal radius is 4-6cm, is specially 5cm;
Time 10-12min, is specially 10min;
Temperature is 3-7 DEG C, is specially 4 DEG C;
In described second time centrifugation step, rotating speed is 8000-10000rpm; Centrifugal radius is 4-6cm, is specially 5cm; Time is 8-10min.
4., according to the arbitrary described method of claim 1-3, it is characterized in that: described step 2) in column chromatography steps, applied sample amount is the 2-5% of gel column volume used, is specially 3-4%;
The aqueous acetic acid of eluent to be pH value be 2-4;
Elution speed is 0.4-0.6mL/min, is specially 0.5mL/min;
The consumption of eluent is 3-5 times of gel column volume used, is specially 4 times;
In gel column used, the model of gel is Sephadex G15 or Bio-Gel-p-2;
Length-to-diameter ratio is 30-40:1, is specially 30:1;
The length of gel column is 60-100cm; Diameter is 1-5cm, is specially 2cm.
5., according to the arbitrary described method of claim 1-4, it is characterized in that: described shell raw material is Patinopecten yessoensis or Chlamys nobilis.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105259292A (en) * | 2015-11-12 | 2016-01-20 | 上海市农业科学院 | Method for measuring paralysis shellfish poison in aquatic products |
| CN108949847A (en) * | 2018-08-01 | 2018-12-07 | 浙江海洋大学 | A method of gonyatoxin is prepared using marine bacteria fermentation |
| CN109142595A (en) * | 2018-08-23 | 2019-01-04 | 中国水产科学研究院黄海水产研究所 | A kind of preparation method of paralytic shellfish poisoning (PSP) standard solution |
| CN114112871A (en) * | 2021-09-27 | 2022-03-01 | 青海大学 | Rapid fluorescence microscopic counting method for salt lake floating viruses |
-
2014
- 2014-02-28 CN CN201410072059.1A patent/CN104876939A/en active Pending
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| 刘晓丽 等: "链状亚历山大藻的培养及麻痹性贝类毒素的提取和检测", 《水产学报》 * |
| 林华娟 等: "扇贝中麻痹性毒素的提取与分离纯化", 《水产学报》 * |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105259292A (en) * | 2015-11-12 | 2016-01-20 | 上海市农业科学院 | Method for measuring paralysis shellfish poison in aquatic products |
| CN108949847A (en) * | 2018-08-01 | 2018-12-07 | 浙江海洋大学 | A method of gonyatoxin is prepared using marine bacteria fermentation |
| CN108949847B (en) * | 2018-08-01 | 2021-06-18 | 浙江海洋大学 | Method for preparing gonyautoxin by fermenting marine bacteria |
| CN109142595A (en) * | 2018-08-23 | 2019-01-04 | 中国水产科学研究院黄海水产研究所 | A kind of preparation method of paralytic shellfish poisoning (PSP) standard solution |
| CN114112871A (en) * | 2021-09-27 | 2022-03-01 | 青海大学 | Rapid fluorescence microscopic counting method for salt lake floating viruses |
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