CN110437351A - A kind of process for extracting heparin sodium from intestinal mucosa - Google Patents
A kind of process for extracting heparin sodium from intestinal mucosa Download PDFInfo
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- CN110437351A CN110437351A CN201810423373.8A CN201810423373A CN110437351A CN 110437351 A CN110437351 A CN 110437351A CN 201810423373 A CN201810423373 A CN 201810423373A CN 110437351 A CN110437351 A CN 110437351A
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- resin
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- sodium chloride
- salinity
- sodium
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
Abstract
The present invention relates to technical field of heparin sodium production, provide a kind of process that heparin sodium is extracted from intestinal mucosa.The present invention looks for another way, and uses fresh pig pancreas for waste trypsase enzyme solution, is used further to extract heparin sodium, greatly reduces the use of synthetic material in this way, improve the natural sex of product；Moreover, extracting heparin sodium using trypsase enzyme solution of the invention, it can significantly reduce peculiar smell in production process and generate, be conducive to environmental protection.The present invention uses fresh pig pancreas to extract heparin sodium, high income for waste trypsase enzyme solution, and 100,000,000 international unit heparin sodium crudes of production only need 1600 or so chitterlings, and product purity is high, the potency > 100U/mg of heparin sodium.
Description
Technical field
It is the present invention relates to technical field of heparin sodium production, in particular to a kind of from the technique side that intestinal mucosa extracts heparin sodium
Method.
Background technique
Heparin sodium is also known as heparin, is a kind of natural anticoagulative substance of the acid mucopolysaccharides containing sulfate.Heparin sodium is
White or off-white powder, odorless, tasteless, have draw it is moist, it is soluble easily in water, it is organic molten insoluble in ethyl alcohol, acetone, dioxane etc.
Agent.
Heparin sodium is widely present in mammal liver, lung, in intestinal mucosa, and complex presence is mostly combined into protein.Enzyme
It solves albumen and separates heparin sodium, heparin sodium is the mucopolysaccharide of sulfur acid, amino, glycuronic acid.It is negatively charged in pH8-9, it can be with
Anionite carries out ion exchange, carries out crude separation, it is consummate that polysaccharide liquid precipitates progress in high concentration ethanol.Heparin sodium is
Best anti-coagulants in blood chemistry measurement.Heparin sodium is a kind of glutinous polysaccharide of sulfur-bearing acid groups, and molecular weight is 1.5 ten thousand,
Its anticoagulant mechanism be with anticoagulant enzyme II together, the effect between low concentration energy inhibiting factor Ⅸ a, VIII and PF3, and capable of reinforcing anti-
Fibrin ferment III inactivates serine protease, so that fibrin ferment be prevented to be formed；There are also inhibit fibrin ferment self catalysis and inhibit because
The effect of sub- X.
The defect of the enzymatic hydrolysis production method of traditional heparin sodium is: the production cycle is long, and energy consumption is high, and yield is low, production 100,000,000
International unit heparin sodium crude needs the mucous membrane of 2800-3000 root chitterlings, and product purity is low, and the potency of heparin sodium is at most 80U/
Mg, and a large amount of intestines slag and waste water are generated, pollute environment.The disclosure of the invention of CN1308088A is a kind of to utilize bio-fermentation process
The method for producing heparin sodium.This method is using 2709 protease as catalyst, D204 strong-base anion-exchange resin conduct
Ion exchange resin, and be filtered using 150-180 mesh multilayer cloth screen.2709 protease are synthetic material, reduce liver
The natural sex of plain sodium product, and 2709 protease hydrolyzeds are used, the peculiar smell generated in production process is larger, pollutes environment.Together
When the product yield of this method, purity it is relatively low.
Summary of the invention
It is an object of the invention to overcome drawbacks described above of the existing technology, provide a kind of from intestinal mucosa extraction heparin sodium
Process, with short production cycle, product yield, purity are higher, can significantly reduce in production process peculiar smell and generate,
Conducive to environmental protection, the use of synthetic material is greatly reduced in extraction, improves the natural sex of product.
The technical solution adopted by the present invention to solve the technical problems is:
A kind of process for extracting heparin sodium from intestinal mucosa, the processing step are as follows:
(1) prepared by trypsase enzyme solution: fresh pig pancreas is taken, remained on surface grease is removed, is rubbed with meat grinder, it will
The pig pancreas of rubbing: saturated limewater=1:9-12 weight ratio is uniformly mixed to obtain trypsase enzyme solution.
(2) it digests: enzymatic vessel is added in pig intestinal mucosa slurries, pig intestinal mucosa slurries are pig intestinal mucosa: water=1:
21 ° -24 ° of salinity of sodium chloride solution is added into enzymatic vessel and adjusts feed liquid in enzymatic vessel for the mixture of the weight ratio of 2.5-4
Salinity controls the pH of feed liquid 8.5 ± 0.5 to 5 ± 0.5 °；Then add the enzyme amount of 15 ± 5kg to enzymatic vessel by every 1000L feed liquid
Interior addition trypsase enzyme solution, for control feed liquid temperature at 53 ± 3 DEG C, 4.2 ± 0.2 ° of salinity, the holding 3 ± 0.5 of pH7.0 ± 0.5 is small
When；It is finally warming up to 85-88 DEG C, keeps 20-25min to obtain enzymolysis liquid, filters off residue.The last enzymolysis liquid of this step filters off residual
When slag, residue is often gone using Bag filter, such efficiency is often lower, and inventor has been invented by practical exploration using vibration
It is dynamic to be sieved through the mode for filtering off residue, the rate of filtration is substantially increased, production efficiency is improved, is the good side instead of Bag filter
Method.
(3) it adsorbs: enzymolysis liquid being inputted into adsorption tanks, is cooled to 57 ± 3 DEG C, add water to adjust salinity to 3.0 ± 0.5 °, control
PH7.0-7.5 is added the amount of resin of 3-8kg by every 1000L enzymolysis liquid, and ROHM AND HAAS FPA98CL type resin is added into adsorption tanks,
After stirring and adsorbing 6-8 hours, resin is collected.
(4) it washs: the resin of collection being washed with water to pH and is put into 4 ± 1 ° of salinity, temperature 55- for neutrality, then by resin
60 DEG C of sodium chloride solution, stirring are washed at least 1 hour, wherein resin: sodium chloride solution=1:1.3-1.7 (w/v).
Sodium chloride solution temperature is controlled at 55-60 DEG C, convenient for cleaning up the attachment of resin surface grease type, convenient for elution.
(5) it elutes: the resin after step (4) washing being poured into elution tank and is eluted twice, wherein is eluted for the first time
Are as follows: resin is added to salinity and is 21 ± 1 °, stirs at least 3 hours in 55-60 DEG C of temperature of sodium chloride solution, resin: chlorination
Sodium solution=1:1-1.4 (w/v)；Second of elution are as follows: it is 21 ± 1 °, temperature that the resin after eluting first time, which is added to salinity,
At least 3 hours are stirred in 55-60 DEG C of sodium chloride solution, resin: sodium chloride solution=1:0.8-1.2 (w/v)；Merging is received twice
The eluent of collection.Control twice elution elution when sodium chloride solution temperature at 55-60 DEG C, convenient for by the heparin sodium of grease-like from
It is eluted on resin, increases yield.
(6) it precipitates: the eluent that step (5) is collected into being poured into settling tank, is added under stirring condition into settling tank
The alcohol that 85 ± 5 ° of alcoholic strength, alcohol dosage are subject to the alcoholic strength for surveying mixed liquor in settling tank to 45 ° -60 °, are staticly settled
15 hours or more, collect sediment.
(7) dry: the alcohol that the sediment that step (6) obtains is added 85 ° of alcoholic strength or more carries out dehydration 1-3 hours, filter
Drying and processing is done after dry up to heparin sodium product.W/v in the present invention is mass volume ratio specifically: kg:L.
The present invention looks for another way, and uses fresh pig pancreas for waste trypsase enzyme solution, is used further to extract heparin sodium,
The use for greatly reducing synthetic material in this way improves the natural sex of product；Moreover, using trypsase enzyme of the invention
Liquid extracts heparin sodium, can significantly reduce peculiar smell in production process and generate, be conducive to environmental protection.
The present invention uses fresh pig pancreas to extract heparin sodium, high income, production 100,000,000 for waste trypsase enzyme solution
International unit heparin sodium crude only needs 1600 or so chitterlings, and product purity is high, the potency > 100U/mg of heparin sodium.
Preferably, step (2) handles obtained enzymolysis liquid under 6000-8000 revs/min of revolving speed, centrifugal treating 20-
After 40min, the absorption of next step is entered back into.
Milkiness shape liquid is obtained after enzymatic hydrolysis, enters centrifuge after filtering off residue, starts centrifuge, enzymolysis liquid is in centrifugation masterpiece
Under, after so that milkiness shape enzymolysis liquid is entered bowl, solid particle or the biggish liquid of density (containing albumen) are to rotatory drum wall
Sedimentation forms sediment, and the lesser heparin sodium decomposed solution of density is assembled to rotary drum center position, flow to overflow port discharge, becomes point
Chaotropic.Sediment squeezes out sediment from slag-drip opening by worm-drive, and separating liquid is discharged by centrifuge from discharge port, conveying
Next step resin adsorption process is carried out in adsorption tanks.
Partial impurities are directly removed in decomposing workshop section after increasing this step, the resin in absorption process is enable sufficiently to inhale
Attached heparin sodium improves resin adsorption validity.The separation that impurity is directly carried out in decomposition process, improves the pure of final products
Degree.Centrifugal speed and time are controlled, guarantees the effect of centrifuge separation.
Preferably, remaining feed liquid is delivered to adsorption tanks again after step (3) are collected resin, control temperature 57 ±
3 DEG C, pH7.0-7.5, add the amount of resin of 2-3kg that ROHM AND HAAS FPA98CL type resin is added into adsorption tanks by every 1000L feed liquid
After adsorbing 6-8 hours again, resin is collected, after then merging with the resin that step (3) are collected, into the purge step of next step
Suddenly.
Feed liquid after resin adsorption is re-started absorption by this step, and the yield of heparin sodium can be improved, and reduces waste, together
When can be reduced the content of albumen in waste liquid, be conducive to wastewater treatment.
Preferably, being reused after the resin activated processing of ROHM AND HAAS FPA98CL type, it is activated are as follows: resin is used
50-60 DEG C of warm water impregnate 20 hours it is above after rinsed well and drained with clear water, add mass concentration as 10% hydroxide
It is more than hour to stir 1 for sodium solution, and it is neutral for finally being rinsed with clear water to washing lotion pH, and the additional amount of the sodium hydroxide solution is
The meter of 1L sodium hydroxide solution is added in every kilogram resin；Resin after activation is under 4000-5000 revs/min of revolving speed, centrifugation
10-30min is handled, is used further to adsorb.
Resin ready for use after activation processing is entered into centrifuge by being centrifuged machine inlet.Start centrifuge, resin exists
Under 4000-5000 revs/min of revolving speed effect, after resin enters bowl, solid particle resin is settled to rotatory drum wall, is glued
It is attached in bulging wall.The lesser substance of liquid or density that centrifugal action is thrown away is assembled to rotary drum center position, flow to overflow port row
Out, become waste.The resin for staying in bulging wall is collected, prepares to be used for absorption process.Moisture is sufficiently got rid of in this step resin hole
It is dry, to increase the adsorption space of resin, improve the adsorption capacity of resin.
Preferably, the eluent that step (5) is collected enters back into step (6) precipitating, ultrafilter after entering ultrafilter ultrafiltration
Ultrafiltration membrane aperture at 0.45 μm -0.6 μm.
By ultrafiltration, can by eluent waste water and impurity exclude, effectively improve the purity of product, and can be reduced next
Walk the alcohol use amount in the heavy technique of wine, save the cost.
Preferably, step (5) collect eluent under 8000-10000 revs/min of revolving speed, centrifugal treating 10-
After 20min, step (6) precipitating is entered back into.
Eluent is entered centrifuge by being centrifuged machine inlet, starts centrifuge, eluent is in decomposed solution in centrifugal force
Under (8000-10000 revs/min) effect, after so that milkiness shape eluent is entered bowl, solid particle or the biggish liquid of density
Body (containing albumen) is settled to rotatory drum wall, forms sediment, and the lesser heparin sodium eluent of density is assembled to rotary drum center position, flow to
Overflow port discharge, becomes separating liquid.Sediment squeezes out sediment from slag-drip opening by worm-drive, and separating liquid passes through centrifuge
It is discharged from discharge port, is transported in settling tank and carries out next step alcohol precipitation process.This step increases the work being centrifuged to eluent
Sequence to improve the purity of final products, and can be reduced next to directly remove partial impurities and albumen in elution procedure
Walk the alcohol use amount in the heavy technique of wine, save the cost.
Preferably, remaining supernatant inputs in another empty settling tank after step (6) collects sediment, it is heavy to another sky
Saturated sodium chloride solution is added in the tank of shallow lake to stir evenly, and adjusting pH is 9-10, the volume of supernatant and saturated sodium chloride solution
Than after staticly settling 8-12 hours, collecting precipitating, entering after then merging with the sediment that step (6) obtains for 1:0.5-0.7
The drying of next step.
This step carries out wine to supernatant of the first time wine after heavy again and sinks, and can effectively improve the yield of heparin sodium, reduce
Waste is conducive to wastewater treatment.
Preferably, the temperature of step (7) drying and processing is 60-80 DEG C.
Preferably, the residue that the filtering of collection step (2) enzymolysis liquid obtains, 5 ± 0.5 ° of salinity are added into enzymatic vessel
Then sodium chloride solution rejoins residue in enzymatic vessel, the weight ratio of residue and 5 ± 0.5 ° of salinity of sodium chloride solution is
1:5-6 controls the pH of feed liquid 8.5 ± 0.5, then adds the enzyme amount of 10-12kg that pancreas is added into enzymatic vessel by every 1000L feed liquid
Protease enzyme solution controls feed liquid temperature at 55 ± 1 DEG C, keeps the temperature 6-7 hours；It is finally warming up to 88-90 DEG C, keeps 20-25min,
Enzymolysis liquid is obtained after standing 20-30min again, enzymolysis liquid is under 6000-8000 revs/min of revolving speed, after centrifugal treating 20-40min, into
Enter the absorption of next step.
In prior art, digests the i.e. intestines slag of remaining residue and often give it up, relatively waste in this way and increase environment
Burden.And the present inventor passes through many years practical exploration, has invented the processing step for effectively recycling residue, passes through
To the enzymolysis processing again of residue, the further residue decomposition of energy extracts heparin sodium, increases the yield of heparin sodium, reduce
Waste discharge is conducive to environmental protection.
The beneficial effects of the present invention are:
1, it uses fresh pig pancreas for waste trypsase enzyme solution, is used further to extract heparin sodium, greatly reduce in this way
The use of synthetic material, improves the natural sex of product；Moreover, extracting heparin using trypsase enzyme solution of the invention
Sodium can significantly reduce peculiar smell in production process and generate, be conducive to environmental protection.
2, high income, 100,000,000 international unit heparin sodium crudes of production only need 1600 or so chitterlings, and product purity is high, liver
The potency > 100U/mg of plain sodium.
Specific embodiment
Below by specific embodiment, technical scheme of the present invention will be further explained in detail.
In the present invention, if not refering in particular to, used raw material and equipment etc. are commercially available or commonly used in the art.
Method in following embodiments is unless otherwise instructed the conventional method of this field.
Resin activated processing:
By new ROHM AND HAAS FPA98CL type resin, (commercially available import resin, dealer Beijing Bo Saisi biotechnology are limited
Company) impregnated 20 hours with 50 DEG C of warm water after rinsed well and drained with clear water, add the hydroxide that mass concentration is 10%
It is more than hour to stir 1 for sodium solution, and it is neutral for finally being rinsed with clear water to washing lotion pH, and the additional amount of the sodium hydroxide solution is
The meter of 1L sodium hydroxide solution is added in every kilogram resin.Studies have shown that the warm water temperature used in resin activated processing is in 50-
Between 60 DEG C, soaking time between 16-24 hours, the activation processing effect to resin be it is equivalent, do not do go to live in the household of one's in-laws on getting married one by one herein
It states.Under 4000-5000 revs/min of revolving speed, centrifugal treating 10-30min is used further to adsorb resin after activation.
Embodiment 1:
(1) prepared by trypsase enzyme solution: fresh pig pancreas is taken, remained on surface grease is removed, is rubbed with meat grinder, it will
The pig pancreas of rubbing: saturated limewater=1:10 weight ratio is uniformly mixed to obtain trypsase enzyme solution.
(2) it digests: enzymatic vessel, pig is added in 1000L pig intestinal mucosa slurries (handled through archenteron-scrapping machine by chitterlings and obtained)
Mucous membrane of small intestine slurries are pig intestinal mucosa: the mixture of water=1:3 weight ratio, and 22 ° of salinity of chlorination is added into enzymatic vessel
Sodium solution adjusts the salinity of feed liquid in enzymatic vessel to 5 ± 0.5 °, controls the pH of feed liquid 8.5 or so；Then every 1000L feed liquid is pressed
The enzyme amount of 15kg is added to be added trypsase enzyme solution into enzymatic vessel, control feed liquid temperature is at 53 DEG C, 4.2 ± 0.2 ° of salinity, pH7.0
Left and right is kept for 3 hours；86 DEG C are finally warming up to, keeps 23min to obtain enzymolysis liquid, filters off residue.Then enzymolysis liquid 7000 turns/
Under the revolving speed divided, after centrifugal treating 30min, the absorption of next step is entered back into.
The residue that enzymolysis liquid filtering obtains is collected, 5 ± 0.5 ° of salinity of sodium chloride solution is added into enzymatic vessel, then will
Residue rejoins in enzymatic vessel, and the weight ratio of residue and 5 ± 0.5 ° of salinity of sodium chloride solution is 1:5, controls the pH of feed liquid
8.5 or so, then add the enzyme amount of 10kg that trypsase enzyme solution is added into enzymatic vessel by every 1000L feed liquid, controls feed liquid temperature
Degree keeps the temperature 6 hours at 55 DEG C；90 DEG C are finally warming up to, 22min is kept, then obtains enzymolysis liquid after standing 25min, enzymolysis liquid exists
Under 7000 revs/min of revolving speed, after centrifugal treating 30min, into the absorption (enzymatic hydrolysis digested with pig intestinal mucosa of next step
Liquid all inputs adsorption tanks).
(3) it adsorbs: enzymolysis liquid being inputted into adsorption tanks, is cooled to 57 DEG C, add water to adjust salinity to 3.0 ± 0.5 °, control
PH7.0 is added the amount of resin of 5kg by every 1000L enzymolysis liquid, and ROHM AND HAAS FPA98CL type resin is added into adsorption tanks, and stirring is inhaled
After attached 7 hours, resin is collected；Remaining feed liquid is delivered to adsorption tanks again after resin being collected, and controls temperature at 57 DEG C,
PH7.0 adds the amount of resin of 2.5kg that ROHM AND HAAS FPA98CL type resin is added into adsorption tanks and adsorbs again by every 1000L feed liquid
After 7 hours, resin is collected, after then merging with the resin that absorption enzymolysis liquid is collected, into the washing step of next step.
(4) it washs: the resin of collection being washed with water to pH and is put into 4 ± 1 ° of salinity, 58 DEG C of temperature for neutrality, then by resin
Sodium chloride solution, stirring washed for 2 hours, wherein resin: sodium chloride solution=1:1.5 (w/v).
(5) it elutes: the resin after step (4) washing being poured into elution tank and is eluted twice, wherein is eluted for the first time
Are as follows: resin is added to salinity and is 21 ± 1 °, stirred 4 hours in 58 DEG C of temperature of sodium chloride solution, resin: sodium chloride solution=
1:1.2(w/v)；Second of elution are as follows: the resin after eluting first time is added to the chlorination that salinity is 21 ± 1 °, 58 DEG C of temperature
It is stirred 4 hours in sodium solution, resin: sodium chloride solution=1:1.0 (w/v)；Merge the eluent collected twice.
The eluent of collection enters back into precipitating in next step after entering ultrafilter ultrafiltration, and the ultrafiltration membrane aperture of ultrafilter is 0.45
μm-0.6μm。
(6) it precipitates: the eluent being collected into being poured into settling tank, alcoholic strength is added under stirring condition into settling tank
85 ° of alcohol, alcohol dosage are subject to the alcoholic strength for surveying mixed liquor in settling tank to 50 °, are staticly settled 18 hours, and it is heavy to collect
Starch.
Remaining supernatant inputs in another empty settling tank after collecting sediment, and saturation chlorine is added into another empty settling tank
Change sodium solution to stir evenly, and adjusting pH is 9, the volume ratio of supernatant and saturated sodium chloride solution is 1:0.6, staticly settles 10
After hour, precipitating is collected, the drying of next step is entered after then merging with the sediment that elution liquid precipitate obtains.
(7) dry: the alcohol for being added 90 ° of alcoholic strength to sediment carries out dehydration 1 hour, 80 DEG C of livers obtained by drying after being filtered dry
Plain sodium product.
It is detected, 100,000,000 international unit heparin sodium crudes of production only need 1600 or so chitterlings, and product purity is high, heparin
The potency > 100U/mg of sodium.
Embodiment 2:
(1) prepared by trypsase enzyme solution: fresh pig pancreas is taken, remained on surface grease is removed, is rubbed with meat grinder, it will
The pig pancreas of rubbing: saturated limewater=1:9 weight ratio is uniformly mixed to obtain trypsase enzyme solution.
(2) it digests: enzymatic vessel is added in 2000L pig intestinal mucosa slurries, pig intestinal mucosa slurries are pig intestinal mucosa: water
The salt of feed liquid in 21 ° of salinity of sodium chloride solution adjusting enzymatic vessel is added into enzymatic vessel for the mixture of the weight ratio of=1:2.5
Degree controls the pH of feed liquid 8.0 to 5 ± 0.5 °；Then add the enzyme amount of 10kg that pancreas egg is added into enzymatic vessel by every 1000L feed liquid
White enzyme enzyme solution, at 50 DEG C, 4.2 ± 0.2 ° of salinity, pH6.5 is kept for 3.5 hours control feed liquid temperature；85 DEG C are finally warming up to, is protected
It holds 25min and obtains enzymolysis liquid, filter off residue.Then enzymolysis liquid is under 6000 revs/min of revolving speed, after centrifugal treating 40min, then into
Enter the absorption of next step.
The residue that enzymolysis liquid filtering obtains is collected, 5 ± 0.5 ° of salinity of sodium chloride solution is added into enzymatic vessel, then will
Residue rejoins in enzymatic vessel, and the weight ratio of residue and 5 ± 0.5 ° of salinity of sodium chloride solution is 1:6, controls the pH of feed liquid
8 or so, then add the enzyme amount of 12kg that trypsase enzyme solution is added into enzymatic vessel by every 1000L feed liquid, controls feed liquid temperature
At 54 DEG C, 7 hours are kept the temperature；88 DEG C are finally warming up to, 25min is kept, then obtains enzymolysis liquid after standing 30min, enzymolysis liquid is 6000
Rev/min revolving speed under, after centrifugal treating 40min, into the absorption of next step.
(3) it adsorbs: enzymolysis liquid being inputted into adsorption tanks, is cooled to 54 DEG C, add water to adjust salinity to 3.0 ± 0.5 °, control
PH7.5 is added the amount of resin of 3kg by every 1000L enzymolysis liquid, and ROHM AND HAAS FPA98CL type resin is added into adsorption tanks, and stirring is inhaled
After attached 8 hours, resin is collected；Remaining feed liquid is delivered to adsorption tanks again after resin being collected, and controls temperature at 54 DEG C,
PH7.5 adds the amount of resin of 2kg that ROHM AND HAAS FPA98CL type resin is added into adsorption tanks and adsorbs 8 again by every 1000L feed liquid
After hour, resin is collected, the washing step of next step is entered after then merging with the resin of enzymolysis liquid absorptive collection.
(4) it washs: the resin of collection being washed with water to pH and is put into 4 ± 1 ° of salinity, temperature 60 C for neutrality, then by resin
Sodium chloride solution, stirring washed for 1 hour, wherein resin: sodium chloride solution=1:1.3 (w/v).
(5) it elutes: the resin after step (4) washing being poured into elution tank and is eluted twice, wherein is eluted for the first time
Are as follows: resin is added to salinity and is 21 ± 1 °, stirred 5 hours in 55 DEG C of temperature of sodium chloride solution, resin: sodium chloride solution=
1:1(w/v)；Second of elution are as follows: the resin after eluting first time is added to the sodium chloride that salinity is 21 ± 1 °, 55 DEG C of temperature
It is stirred 5 hours in solution, resin: sodium chloride solution=1:0.8 (w/v)；Merge the eluent collected twice.
The eluent of collection is under 8000 revs/min of revolving speed, after centrifugal treating 20min, enters back into and precipitates in next step.
(6) it precipitates: eluent is poured into settling tank, 80 ° of alcoholic strength of alcohol is added under stirring condition into settling tank,
Alcohol dosage is subject to the alcoholic strength for surveying mixed liquor in settling tank to 45 °, staticly settles 20 hours, collects sediment.
Remaining supernatant inputs in another empty settling tank after collecting sediment, and saturation chlorine is added into another empty settling tank
Change sodium solution to stir evenly, and adjusting pH is 10, the volume ratio of supernatant and saturated sodium chloride solution is 1:0.5, is staticly settled
After 12 hours, precipitating is collected, the drying of next step is entered after then merging with the sediment that elution liquid precipitate obtains.
(7) dry: the alcohol for being added 85 ° of alcoholic strength to sediment carries out dehydration 3 hours, 60 DEG C of livers obtained by drying after being filtered dry
Plain sodium product.
It is detected, 100,000,000 international unit heparin sodium crudes of production only need 1600 or so chitterlings, and product purity is high, heparin
The potency > 100U/mg of sodium
Embodiment 3:
(1) prepared by trypsase enzyme solution: fresh pig pancreas is taken, remained on surface grease is removed, is rubbed with meat grinder, it will
The pig pancreas of rubbing: saturated limewater=1:12 weight ratio is uniformly mixed to obtain trypsase enzyme solution.
(2) it digests: enzymatic vessel is added in pig intestinal mucosa slurries, pig intestinal mucosa slurries are pig intestinal mucosa: water=1:4
Weight ratio mixture, be added into enzymatic vessel 24 ° of salinity of sodium chloride solution adjust the salinity of feed liquid in enzymatic vessel to 5 ±
0.5 °, the pH of feed liquid is controlled 9.0；Then add the enzyme amount of 20kg that trypsase enzyme is added into enzymatic vessel by every 1000L feed liquid
Liquid, at 56 DEG C, 4.2 ± 0.2 ° of salinity, pH7.5 is kept for 2.5 hours control feed liquid temperature；88 DEG C are finally warming up to, 20min is kept
Enzymolysis liquid is obtained, residue is filtered off.Enzymolysis liquid after centrifugal treating 20min, enters back into next step under 8000 revs/min of revolving speed
Absorption.
The residue that enzymolysis liquid filtering obtains is collected, 5 ± 0.5 ° of salinity of sodium chloride solution is added into enzymatic vessel, then will
Residue rejoins in enzymatic vessel, and the weight ratio of residue and 5 ± 0.5 ° of salinity of sodium chloride solution is 1:5, controls the pH of feed liquid
9, then add the enzyme amount of 12kg that trypsase enzyme solution is added into enzymatic vessel by every 1000L feed liquid, controls feed liquid temperature 56
DEG C, keep the temperature 6 hours；90 DEG C are finally warming up to, 20min is kept, then obtains enzymolysis liquid after standing 30min, enzymolysis liquid is at 8000 revs/min
Revolving speed under, after centrifugal treating 20min, into the absorption of next step.
(3) it adsorbs: enzymolysis liquid being inputted into adsorption tanks, is cooled to 60 DEG C, add water to adjust salinity to 3.0 ± 0.5 °, control
PH7.0 is added the amount of resin of 8kg by every 1000L enzymolysis liquid, and ROHM AND HAAS FPA98CL type resin is added into adsorption tanks, and stirring is inhaled
After attached 6 hours, resin is collected；Remaining feed liquid is delivered to adsorption tanks again after resin being collected, and controls temperature at 60 DEG C,
PH7.0 adds the amount of resin of 3kg that ROHM AND HAAS FPA98CL type resin is added into adsorption tanks and adsorbs 6 again by every 1000L feed liquid
After hour, resin is collected, after then merging with the resin of enzymolysis liquid absorptive collection, into the washing step of next step.
(4) it washs: the resin of collection being washed with water to pH and is put into 4 ± 1 ° of salinity, temperature 60 C for neutrality, then by resin
Sodium chloride solution, stirring washed for 1 hour, wherein resin: sodium chloride solution=1:1.7 (w/v).
(5) it elutes: the resin after step (4) washing being poured into elution tank and is eluted twice, wherein is eluted for the first time
Are as follows: resin is added to salinity and is 21 ± 1 °, stirred 3 hours in the sodium chloride solution of temperature 60 C, resin: sodium chloride solution=
1:1.4(w/v)；Second of elution are as follows: the resin after eluting first time is added to the chlorination that salinity is 21 ± 1 °, temperature 60 C
It is stirred 3 hours in sodium solution, resin: sodium chloride solution=1:1.2 (w/v)；Merge the eluent collected twice.
The eluent of collection is under 10000 revs/min of revolving speed, after centrifugal treating 10min, enters back into and precipitates in next step.
(6) it precipitates: eluent is poured into settling tank, 90 ° of alcoholic strength of alcohol is added under stirring condition into settling tank,
Alcohol dosage is subject to the alcoholic strength for surveying mixed liquor in settling tank to 60 °, staticly settles 15 hours, collects sediment.
Remaining supernatant inputs in another empty settling tank after collecting sediment, and saturation chlorine is added into another empty settling tank
Change sodium solution to stir evenly, and adjusting pH is 10, the volume ratio of supernatant and saturated sodium chloride solution is 1:0.7, staticly settles 8
After hour, precipitating is collected, the drying of next step is entered after then merging with the sediment that elution liquid precipitate obtains.
(7) dry: the alcohol for being added 90 ° of alcoholic strength to sediment carries out dehydration 2 hours, 70 DEG C of livers obtained by drying after being filtered dry
Plain sodium product.
It is detected, 100,000,000 international unit heparin sodium crudes of production only need 1600 or so chitterlings, and product purity is high, heparin
The potency > 100U/mg of sodium
Present invention substantially reduces the uses of synthetic material, improve the natural sex of product；Moreover, using of the invention
Trypsase enzyme solution extracts heparin sodium, can significantly reduce peculiar smell in production process and generate, be conducive to environmental protection.
Above-mentioned embodiment is only a preferred solution of the present invention, not the present invention is made in any form
Limitation, there are also other variations and modifications on the premise of not exceeding the technical scheme recorded in the claims.
Claims (5)
1. a kind of process for extracting heparin sodium from intestinal mucosa, it is characterised in that: the processing step is as follows:
(1) prepared by trypsase enzyme solution: taking fresh pig pancreas, removes remained on surface grease, rubbed, will be rubbed with meat grinder
Pig pancreas: saturated limewater=1:9-12 weight ratio be uniformly mixed to obtain trypsase enzyme solution；
(2) it digests: enzymatic vessel is added in pig intestinal mucosa slurries, pig intestinal mucosa slurries are pig intestinal mucosa: water=1:2.5-4
Weight ratio mixture, into enzymatic vessel be added 21 ° -24 ° of salinity sodium chloride solution adjust enzymatic vessel in feed liquid salinity
To 5 ± 0.5 °, the pH of feed liquid is controlled 8.5 ± 0.5；Then the enzyme amount of 15 ± 5kg is added to add into enzymatic vessel by every 1000L feed liquid
Enter trypsase enzyme solution, at 53 ± 3 DEG C, 4.2 ± 0.2 ° of salinity, pH7.0 ± 0.5 is kept for 3 ± 0.5 hours control feed liquid temperature；
It is finally warming up to 85-88 DEG C, keeps 20-25min to obtain enzymolysis liquid, filters off residue；
(3) it adsorbs: enzymolysis liquid being inputted into adsorption tanks, is cooled to 57 ± 3 DEG C, add water to adjust salinity to 3.0 ± 0.5 °, control
PH7.0-7.5 is added the amount of resin of 3-8kg by every 1000L enzymolysis liquid, and ROHM AND HAAS FPA98CL type resin is added into adsorption tanks,
After stirring and adsorbing 6-8 hours, resin is collected；
(4) it washs: the resin of collection being washed with water to pH and is put into 4 ± 1 °, 55-60 DEG C of temperature of salinity for neutrality, then by resin
Sodium chloride solution, stirring washed at least 1 hour, wherein resin: sodium chloride solution=1:1.3-1.7 (w/v)；
(5) it elutes: the resin after step (4) washing being poured into elution tank and is eluted twice, wherein is eluted for the first time are as follows:
Resin is added to salinity and is 21 ± 1 °, stirs at least 3 hours in 55-60 DEG C of temperature of sodium chloride solution that resin: sodium chloride is molten
Liquid=1:1-1.4 (w/v)；Second of elution are as follows: it is 21 ± 1 °, temperature 55- that the resin after eluting first time, which is added to salinity,
At least 3 hours are stirred in 60 DEG C of sodium chloride solution, resin: sodium chloride solution=1:0.8-1.2 (w/v)；Merging is collected twice
Eluent；
(6) it precipitates: the eluent that step (5) is collected into being poured into settling tank, alcohol is added under stirring condition into settling tank
The alcohol of 85 ± 5 ° of degree, alcohol dosage are subject to the alcoholic strength for surveying mixed liquor in settling tank to 45 ° -60 °, it is small to staticly settle 15
When more than, collect sediment；
(7) dry: the alcohol that the sediment that step (6) obtains is added 85 ° of alcoholic strength or more carries out dehydration 1-3 hours, after being filtered dry
Drying and processing is done up to heparin sodium product；
The enzymolysis liquid that step (2) processing obtains after centrifugal treating 20-40min, enters back under 6000-8000 revs/min of revolving speed
The absorption of next step；
Remaining feed liquid is delivered to adsorption tanks again after step (3) are collected resin, and control temperature is at 57 ± 3 DEG C, pH7.0-
7.5, add the amount of resin of 2-3kg that ROHM AND HAAS FPA98CL type resin is added into adsorption tanks by every 1000L feed liquid and adsorbs 6- again
After 8 hours, resin is collected, after then merging with the resin that step (3) are collected, into the washing step of next step；
Remaining supernatant inputs in another empty settling tank after step (6) collects sediment, is added into another empty settling tank full
It is stirred evenly with sodium chloride solution, and adjusts pH as 9-10, the volume ratio of supernatant and saturated sodium chloride solution is 1:0.5-
0.7, after staticly settling 8-12 hours, precipitating is collected, into the dry of next step after then merging with the sediment that step (6) obtains
It is dry；
The residue that the filtering of collection step (2) enzymolysis liquid obtains 5 ± 0.5 ° of salinity of sodium chloride solution is added into enzymatic vessel, so
Residue is rejoined in enzymatic vessel afterwards, the weight ratio of residue and 5 ± 0.5 ° of salinity of sodium chloride solution is 1:5-6, control material
The pH of liquid then adds the enzyme amount of 10-12kg that trypsase enzyme solution is added into enzymatic vessel 8.5 ± 0.5 by every 1000L feed liquid,
Feed liquid temperature is controlled at 55 ± 1 DEG C, keeps the temperature 6-7 hours；It is finally warming up to 88-90 DEG C, keeps 20-25min, then stand 20-
Enzymolysis liquid is obtained after 30min, enzymolysis liquid is under 6000-8000 revs/min of revolving speed, after centrifugal treating 20-40min, into next step
Absorption.
2. the process according to claim 1 for extracting heparin sodium from intestinal mucosa, it is characterised in that: the ROHM AND HAAS
It reuses, is activated after the resin activated processing of FPA98CL type are as follows: use after 50-60 DEG C of warm water of resin impregnates 20 hours or more
Clear water is rinsed well and is drained, and adds the sodium hydroxide solution that mass concentration is 10% and it is more than hour to stir 1, finally with clear
It is neutrality that water, which is rinsed to washing lotion pH, and the additional amount of the sodium hydroxide solution is that 1L sodium hydroxide solution is added in every kilogram resin
Meter；Under 4000-5000 revs/min of revolving speed, centrifugal treating 10-30min is used further to adsorb resin after activation.
3. the process according to claim 1 for extracting heparin sodium from intestinal mucosa, it is characterised in that: step (5) is collected
Eluent enter after ultrafilter ultrafiltration and enter back into step (6) precipitating, the ultrafiltration membrane aperture of ultrafilter is at 0.45 μm -0.6 μm.
4. the process according to claim 1 for extracting heparin sodium from intestinal mucosa, it is characterised in that: step (5) is collected
Eluent under 8000-10000 revs/min of revolving speed, after centrifugal treating 10-20min, enter back into step (6) precipitating.
5. the process according to claim 1 for extracting heparin sodium from intestinal mucosa, it is characterised in that: step (7) drying
The temperature of processing is 60-80 DEG C.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115490784A (en) * | 2022-08-03 | 2022-12-20 | 江苏派瑞克生物科技有限公司 | Extraction process of heparin sodium |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103183747A (en) * | 2012-09-19 | 2013-07-03 | 杭州龙扬生物科技有限公司 | Technology for extracting high-purity heparin sodium from intestinal mucosa by trypsin method |
| CN103183748A (en) * | 2012-09-19 | 2013-07-03 | 杭州龙扬生物科技有限公司 | Technology for extracting heparin sodium from intestinal mucosa with trypsin method |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103183747A (en) * | 2012-09-19 | 2013-07-03 | 杭州龙扬生物科技有限公司 | Technology for extracting high-purity heparin sodium from intestinal mucosa by trypsin method |
| CN103183748A (en) * | 2012-09-19 | 2013-07-03 | 杭州龙扬生物科技有限公司 | Technology for extracting heparin sodium from intestinal mucosa with trypsin method |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115490784A (en) * | 2022-08-03 | 2022-12-20 | 江苏派瑞克生物科技有限公司 | Extraction process of heparin sodium |
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Application publication date: 20191112 |