CN102603890A - Method for extracting hirudin - Google Patents
Method for extracting hirudin Download PDFInfo
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- CN102603890A CN102603890A CN2012100797819A CN201210079781A CN102603890A CN 102603890 A CN102603890 A CN 102603890A CN 2012100797819 A CN2012100797819 A CN 2012100797819A CN 201210079781 A CN201210079781 A CN 201210079781A CN 102603890 A CN102603890 A CN 102603890A
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- hirudin
- leech
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Abstract
The invention relates to a method for extracting hirudin. The method comprises the following steps: washing and freezing Hirudo and grinding as a whole into a slurry, heating the Hirudo to 60-80 DEG C and keeping the temperature for 2-3min, regulating the pH value to 4-4.4, then cooling to 25-30 DEG C, adding NaCO3 with the mass concentration of 10%, regulating the pH value to 6-7, adding purified water and purifying for 3 times, carrying out net collection on all leach liquor, centrifugalizing with a tube centrifuge, dialyzing the mother liquor with dialysis membrane for 36h, replacing the dialysis membrane once every 12h, preparing the crude product of hirudin by using a freeze drying method, and finally preparing the refined product of hirudin through freeze drying. The extraction method is reasonable in process, convenient to operate and practical. The finished product of hirudin has the characteristics of good quality stability and high yield. The yield of the hirudin extracted by the method of the invention is up to more than 99%.
Description
Technical field
The invention belongs to preparing method's technical field of r-hirudin, relate to a kind of product yield and reach the r-hirudin process for extracting more than 99%.
Background technology
R-hirudin is the intravital a kind of protein of leech, contains 65 amino-acid residues and 3 pairs of disulfide linkage, has the antithrombin activity of high special, and the Trombin inhibiting bound substrates is so there is blood coagulation resisting function.
At present in preparation during r-hirudin, normally with smashing to pieces after the leech cooling, and leech difficulty when smashing to pieces of living is very big, because the leech health is soft being difficult for and smashs to pieces, and leech constantly runs away in the course of processing, climb around be very to feel sick; Also have plenty of with boiling water leech is boiled, it is after death smashed to pieces again, but r-hirudin secretory product runs off from the leech oral cavity when crossing water; Usually to the muddy leech with electric furnace or manual boiling, like this can not industriallization, again can not thermostatically heating; Adopt the whizzer spinning incomplete to leech liquid, the r-hirudin product yield is low, because there is the irrational shortcoming of above-mentioned technology in existing r-hirudin process for extracting, thereby the quality stability of r-hirudin finished product is poor, and yield has only about 80% usually.
Summary of the invention
In order to overcome the above-mentioned shortcoming of prior art
,The present invention provides the good and yield of a kind of quality stability with reasonable, the easy to operate practicality of technology, r-hirudin finished product to bring up to the process for extracting of the r-hirudin more than 99%.
The technical solution adopted for the present invention to solve the technical problems is: a kind of process for extracting of r-hirudin, and its process step is:
The first step: leech clean removed carry out behind the earth impurity freezingly, leech is carried out integral body after freezing and smash to pieces, the leech after then integral body being smashed to pieces is worn into muddy with industrial soybean milk maker;
Second step: will be heated to 60-80 ℃ through the muddy leech that the first step makes, and keep 2-3 minute;
The 3rd step: to will in the leech slurry after second step heated, adding mass percent concentration is the Glacial acetic acid min. 99.5 of 10 ﹪, and adjustment pH value is cooled to 25-30 ℃ immediately at 4-4.4, adds the NaCO that mass percent concentration is 10 ﹪ again
3, adjustment pH value 6-7;
The 4th step: the leech slurry after the 3rd step handled is collected the mother liquor residue with the cloth bag filtration, adds pure water then and successively carries only 3 times, collects whole vat liquors only, carries out spinning with tubular-bowl centrifuge again;
The 5th step: will be through the 4th mother liquor that obtain of step with the dialysis membrane 36h that dialyses, every 12h changes a dialysis membrane;
The 6th step: will make the r-hirudin bullion with freeze-drying through the dialyzate that the 5th step obtained;
The 7th step: will carry out water dissolution with suitable zero(ppm) water, the 16h that dialyses again, gained solution obtained by freeze drying r-hirudin elaboration through the bullion that the 6th step obtained.
When in the said the first step, smashing to pieces, also smash to pieces together with the ice that leech is external.
In said second step, adopt the interlayer heating and stirring pot to heat, put the water in the interlayer after stopping to heat, in interlayer, cool off again when to be cooled through tap water.
Said pH value adjustment in the 3rd step; Be that the purpose of acid adjustment is that the leech oxyphorase is solidified, and solidifies the most complete during pH value 4 after the second step heat treated; Therefore the pH value of acid adjustment must put in place; Cooling back adjust pH to 7, the acidity that neutralized like this is to liquid colour-changeable and till separating out clarifying water.
In the 4th step, adopt tubular-bowl centrifuge to carry out spinning, will filtrate and carry out three spinnings repeatedly, collect liquid and remove the residue in the pipe.
Be banded at dialysis membrane described in the 5th step, path length is greater than 30mm, and the disposable dialysis of ability is complete like this.
The invention has the beneficial effects as follows: adopt freezing processing to make r-hirudin not run off, when smashing to pieces, also smash to pieces simultaneously,, wear into muddy to it through the soybean milk maker of industry then so there is not the loss of r-hirudin together with the external ice of leech; Adopt the heating and stirring pot of interlayer to heat, can industriallization operate like this, can carry out thermostatically heating again, stop to put interbedded water after the heating, in interlayer, cool off again when to be cooled through tap water; After heating, the purpose of acid adjustment is that the oxyphorase in the r-hirudin is solidified, and solidifies the most fully during pH value 4, and the pH value of acid adjustment settles at one go, cooling back adjust pH to 7, and the acidity that neutralized like this is to liquid colour-changeable and till separating out clarifying water; Use tubular-bowl centrifuge, the residue in the pipe of liquid and place to go is collected in three spinnings repeatedly of will filtrating, and improves yield; The dialysis desert is banded, and path length is more than 30mm, and disposable like this dialysis is complete, and dialyzate adopts pure water to make low molecular material to discharge fully; Have the good and yield of the quality stability of reasonable, the easy to operate practicality of technology, r-hirudin finished product and bring up to the characteristics more than 99%.
Embodiment
Below in conjunction with embodiment the present invention is further specified.
A kind of process for extracting of r-hirudin; Its process step is: the first step: freezing with carrying out behind the clean removal of the leech earth impurity; Adopt freezing processing to make r-hirudin not run off, leech is carried out integral body after freezing to smash to pieces, the leech after then integral body being smashed to pieces is worn into muddy with industrial soybean milk maker; When smashing to pieces, also smash to pieces together with the ice that leech is external, so there is not the loss of r-hirudin.Second step: will be heated to 60-80 ℃ through the muddy leech that the first step makes, and keep 2-3 minute; Adopt the interlayer heating and stirring pot to heat, can industriallization operate like this, can carry out thermostatically heating again, put the water in the interlayer after stopping to heat, in interlayer, cool off again when to be cooled through tap water.The 3rd step: to will in the leech slurry after second step heated, adding mass percent concentration is the Glacial acetic acid min. 99.5 of 10 ﹪, and adjustment pH value is cooled to 25-30 ℃ immediately at 4-4.4, adds the NaCO that mass percent concentration is 10 ﹪ again
3, adjustment pH value 6-7; The purpose of acid adjustment is that the leech oxyphorase is solidified, and solidifies the most fully during pH value 4, so the pH value of acid adjustment must put in place, cooling back adjust pH to 7, and the acidity that neutralized like this is to liquid colour-changeable and till separating out clarifying water.The 4th step: the leech slurry after the 3rd step handled is collected the mother liquor residue with the cloth bag filtration, adds pure water then and successively carries only 3 times, collects whole vat liquors only, carries out spinning with tubular-bowl centrifuge again; Adopt tubular-bowl centrifuge to carry out spinning, will filtrate and carry out three spinnings repeatedly, collect liquid and remove the residue in the pipe, improve yield.The 5th step: will be through the 4th mother liquor that obtain of step with the dialysis membrane 36h that dialyses, every 12h changes a dialysis membrane; It is banded that said dialysis membrane is, and path length is greater than 30mm, and the disposable dialysis of ability is complete like this, and dialyzate adopts pure water to make low molecular material to discharge fully.The 6th step: will make the r-hirudin bullion with freeze-drying through the dialyzate that the 5th step obtained.The 7th step: will carry out water dissolution with suitable zero(ppm) water, the 16h that dialyses again, gained solution obtained by freeze drying r-hirudin elaboration through the bullion that the 6th step obtained.
The present invention has the characteristics that quality stability is good and yield is high of reasonable, the easy to operate practicality of technology, r-hirudin finished product.The yield of handling the gained r-hirudin through the present invention reaches more than 99%.
Claims (6)
1. the process for extracting of a r-hirudin is characterized in that process step is:
The first step: leech clean removed carry out behind the earth impurity freezingly, leech is carried out integral body after freezing and smash to pieces, the leech after then integral body being smashed to pieces is worn into muddy with industrial soybean milk maker;
Second step: will be heated to 60-80 ℃ through the muddy leech that the first step makes, and keep 2-3 minute;
The 3rd step: to will in the leech slurry after second step heated, adding mass percent concentration is the Glacial acetic acid min. 99.5 of 10 ﹪, and adjustment pH value is cooled to 25-30 ℃ immediately at 4-4.4, adds the NaCO that mass percent concentration is 10 ﹪ again
3, adjustment pH value 6-7;
The 4th step: the leech slurry after the 3rd step handled is collected the mother liquor residue with the cloth bag filtration, adds pure water then and successively carries only 3 times, collects whole vat liquors only, carries out spinning with tubular-bowl centrifuge again;
The 5th step: will be through the 4th mother liquor that obtain of step with the dialysis membrane 36h that dialyses, every 12h changes a dialysis membrane;
The 6th step: will make the r-hirudin bullion with freeze-drying through the dialyzate that the 5th step obtained;
The 7th step: will carry out water dissolution with suitable zero(ppm) water, the 16h that dialyses again, gained solution obtained by freeze drying r-hirudin elaboration through the bullion that the 6th step obtained.
2. the process for extracting of r-hirudin according to claim 1 is characterized in that: when in the said the first step, smashing to pieces, also smash to pieces together with the ice that leech is external.
3. the process for extracting of r-hirudin according to claim 1 is characterized in that: in said second step, adopt the interlayer heating and stirring pot to heat, put the water in the interlayer after stopping to heat, in interlayer, cool off through tap water when to be cooled again.
4. the process for extracting of r-hirudin according to claim 1 is characterized in that: the said pH value adjustment in the 3rd step, be through second go on foot heat treated after; The purpose of acid adjustment is that the leech oxyphorase is solidified; Solidify the most fully during pH value 4, so the pH value of acid adjustment must put in place, cooling back adjust pH to 7; The acidity that neutralized like this is to liquid colour-changeable and till separating out clarifying water.
5. the process for extracting of r-hirudin according to claim 1 is characterized in that: in the 4th step, adopt tubular-bowl centrifuge to carry out spinning, will filtrate and carry out three spinnings repeatedly, collect liquid and remove the residue in managing.
6. the process for extracting of r-hirudin according to claim 1, it is characterized in that: be banded at dialysis membrane described in the 5th step, path length is greater than 30mm, and the disposable dialysis of ability is complete like this.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012100797819A CN102603890A (en) | 2012-03-23 | 2012-03-23 | Method for extracting hirudin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012100797819A CN102603890A (en) | 2012-03-23 | 2012-03-23 | Method for extracting hirudin |
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| Publication Number | Publication Date |
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| CN102603890A true CN102603890A (en) | 2012-07-25 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN2012100797819A Pending CN102603890A (en) | 2012-03-23 | 2012-03-23 | Method for extracting hirudin |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107365381A (en) * | 2017-09-01 | 2017-11-21 | 荆州市民康生物科技有限公司 | A kind of method had children outside the state plan ripple auxiliary and improve Hirudo japonica element extract yield |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1082409A (en) * | 1992-07-28 | 1994-02-23 | 胡维明 | The extracting method of medical natural hirudiu |
| WO2004113386A2 (en) * | 2003-06-26 | 2004-12-29 | Merck Patent Gmbh | Modified hirudin proteins and t-cell epitopes in hirudin |
| CN101230087A (en) * | 2007-01-22 | 2008-07-30 | 广东海洋大学 | Technique for producing hirudin by using poecilobdella manillensis frozen fresh body as raw material |
| CN102286098A (en) * | 2011-08-18 | 2011-12-21 | 周维官 | Production method of natural hirudin |
-
2012
- 2012-03-23 CN CN2012100797819A patent/CN102603890A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1082409A (en) * | 1992-07-28 | 1994-02-23 | 胡维明 | The extracting method of medical natural hirudiu |
| WO2004113386A2 (en) * | 2003-06-26 | 2004-12-29 | Merck Patent Gmbh | Modified hirudin proteins and t-cell epitopes in hirudin |
| CN101230087A (en) * | 2007-01-22 | 2008-07-30 | 广东海洋大学 | Technique for producing hirudin by using poecilobdella manillensis frozen fresh body as raw material |
| CN102286098A (en) * | 2011-08-18 | 2011-12-21 | 周维官 | Production method of natural hirudin |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107365381A (en) * | 2017-09-01 | 2017-11-21 | 荆州市民康生物科技有限公司 | A kind of method had children outside the state plan ripple auxiliary and improve Hirudo japonica element extract yield |
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Application publication date: 20120725 |