CN102961403B - Composition extracted from animal organs - Google Patents
Composition extracted from animal organs Download PDFInfo
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- CN102961403B CN102961403B CN201210532287.3A CN201210532287A CN102961403B CN 102961403 B CN102961403 B CN 102961403B CN 201210532287 A CN201210532287 A CN 201210532287A CN 102961403 B CN102961403 B CN 102961403B
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Abstract
The invention relates to a composition extracted from animal organs, wherein the animal organs are selected from livers, small intestines, large intestines and lungs, and animals are selected from cows, pigs, sheep and dogs.
Description
Technical field
The present invention relates to a kind of composition extracting from animal organ, the composition and method of making the same especially extracting from animal organ.
Background technology
The composition extracting from animal organ is the important sources of medicine, food, daily necessities, makeup always.
The mastocyte of animal connective tissue can produce acidic mucopolysaccharide material, wherein a kind of heparin sodium that cries.Heparin sodium has strong anticoagulation, energy anti-freezing, antithrombotic form and adjust blood fat, be used as the choice drug of the thrombotic diseases such as control deep-vein thrombosis formation, heparin sodium also has anti-inflammatory, antianaphylaxis, the various biological function such as antiviral, anticancer, can be used for product, food, daily necessities, makeup.Though heparin sodium, for the history of clinical existing more than 60 years, can only extract from the portion of tissue of animal, can not synthetic.
Liver, small intestine, large intestine, lung are the raw materials that extracts heparin sodium, and current extracts heparin sodium by salting-out process or enzyme salt binding method conventionally.The main program of enzyme salt binding method is: first intestinal mucosa thing carried out to enzymolysis, filtration, then uses resin absorption, wash-out, and redeposition, purifying post-drying.
Summary of the invention
The object of this invention is to provide a kind of composition extracting from animal organ, the composition and method of making the same especially extracting from animal organ, its productive rate is high, the height of tiring, energy consumption is low, cost is low.
Technical scheme of the present invention relates to a kind of composition.
Technical scheme of the present invention relates to a kind of composition extracting from animal organ.
The present invention relates to a kind of preparation method of the composition extracting from animal organ.
The preparation method who the present invention relates to a kind of composition, comprises the steps:
(1) liver, mucous membrane of small intestine, large intestine or, lung adds quartz sand, pulverize;
(2) hydrolysis crushed material;
(3) absorption, comprises ion-exchange fiber absorption, macroporous resin adsorption;
(4) wash-out, comprises ion-exchange fiber absorption, macroporous resin wash-out;
(5) precipitation;
(6) purifying;
(7) be drying to obtain sterling.
It is ox, pig, sheep, dog that animal comes from.
The present invention adopts following technical scheme for the extracting method of heparin sodium,
The preparation method who the present invention relates to a kind of composition, comprises the steps:
(1) liver, mucous membrane of small intestine, large intestine or, lung adds quartz sand, pulverize;
(2) use enzymic hydrolysis crushed material;
(3) ion-exchange fiber absorption;
(4) ion-exchange fiber wash-out;
(5) heparin sodium precipitation;
(6) heparin sodium purification;
(7) be drying to obtain sterling heparin sodium.
The present invention adopts following technical scheme for the extracting method of heparin sodium,
An extracting method for heparin sodium, is characterized in that comprising the steps:
(1) liver, mucous membrane of small intestine, large intestine or, lung adds quartz sand, micronizing;
(2) use enzymic hydrolysis crushed material, the zymin that hydrolysis adopts is: cellulase: amylase: the mixture that lipase mixes;
(3) ion-exchange fiber absorption;
(4) ion-exchange fiber wash-out;
(5) heparin sodium precipitation;
(6) heparin sodium purification;
(7) be drying to obtain sterling heparin sodium.
The present invention adopts following technical scheme for the extracting method of heparin sodium,
An extracting method for heparin sodium, is characterized in that comprising the steps:
(1) liver, mucous membrane of small intestine, large intestine or, lung adds quartz sand, micronizing;
(2) use enzymic hydrolysis crushed material, the zymin that hydrolysis adopts is: cellulase: amylase: lipase is with (1～3): (1～2): the mixture that the mass parts ratio of (1～3) mixes;
(3) ion-exchange fiber absorption;
(4) ion-exchange fiber wash-out;
(5) heparin sodium precipitation;
(6) heparin sodium purification;
(7) be drying to obtain sterling heparin sodium.
The present invention adopts following technical scheme for the extracting method of heparin sodium,
An extracting method for heparin sodium, comprises the following steps:
(1) 100～200 part of liver, mucous membrane of small intestine, large intestine or, lung adds 10～20 parts of quartz sands, micronizing;
(2) use enzymic hydrolysis ground mixt, the zymin that hydrolysis adopts is: cellulase: amylase: lipase is with (1～3): (1～2): the mixture that the mass parts ratio of (1～3) mixes;
In reactor, add 100～200 parts of ground mixts, be warming up to 40～50 ℃, insulation 10～20min, adds water 50～80 parts, adds 1～2 part of NaCL, adds MgCL
20.01～0.02 part, control 30～40 ℃ of temperature, with lye pH adjustment be 7～9, add 1～2 part of zymin, stir 1～2h and add precipitation agent, described precipitation agent is to select to 2～5 parts of at least one materials in polymerize aluminum chloride, aluminium chlorohydroxide, Tai-Ace S 150, leaves standstill 2～4h, by 100～200 order filter-cloth filterings for supernatant liquid, collect filtrate;
(3) ion-exchange fiber absorption:
In the container with whipping appts, add 100～200 parts of filtrates that obtained by step (2), add 4～8 parts of ion-exchange fibers, stir 4～8h, ion-exchange fiber is leached, add again 4～8 parts of ion-exchange fibers, stir 4～8h, ion-exchange fiber is leached, the ion-exchange fiber leaching is merged to water and clean 1～2 time;
(4) ion-exchange fiber wash-out:
The ion-exchange fiber that step (3) is reclaimed, adding quality is in 3～4mol/L NaCL solution of 1～2 times of ion-exchange fiber quality, stirs 2～4h, filters, collects elutriant; Adding quality is 2～3mol/LNaCL solution of 1～2 times of ion-exchange fiber quality again, stirs 2～4h, filters, and collects elutriant; Then use with 2～3mol/L NaCL solution stirring of ion-exchange fiber quality equal mass and wash once, collect washing lotion, for the wash-out of lower batch of ion-exchange fiber;
(5) precipitation of heparin sodium:
The elutriant of the 1st time and the 2nd time in step (4) is merged, and adding concentration is that 85～95% ethanol stir, and makes elutriant alcohol concn reach 45～50%, leaves standstill 6～12h, and supernatant liquor that will precipitation heparin sodium reclaims, and collects heparin sodium precipitation;
(6) purifying of heparin sodium:
The heparin sodium precipitation that step (5) is reclaimed with appropriate 1～2%NaCL solution or/and, appropriate 0.1～0.2%Na
2sO
4, or/and, NaHSO
4solution dissolves, insolubles is removed in centrifugation, adding concentration is that 85～95% ethanol stir, make heparin sodium aqua alcohol concn reach 45～50%, leaving standstill 7～14h, filter, collect heparin sodium precipitation, is that 85～95% washing with alcohol heparin sodiums precipitate 1～2 time by concentration, filter, collect heparin sodium precipitation;
(7) dry: heparin sodium is dry under vacuum condition, obtain product.
Ion-exchange fiber is selected from: strong-acid cation exchange fibre, Subacidity cation exchange fiber, strongly basic anion ion exchange fibre, weakly basic anion exchange fibre
Ion-exchange fiber is preferentially selected from: ZB-2 strongly basic anion ion exchange fibre.
Ion-exchange fiber also can be replaced with D208 wide aperture resin.
Advantage of the present invention:
The present invention first by substrate micronizing, breaks its cellularstructure before enzymolysis, then heating heating, adopt again new formula zymin, utilize multi-enzyme system that heparin and albumen composition are dissociated, dissociation effect is good, under the condition relaxing, makes cell walls sex change complete enzymic hydrolysis process.
Before filtering hydrolysed mix, add precipitation agent, add precipitation agent the protein in hydrolyzed solution, intestines slag etc. effectively can be separated, reclaim, can reduce the absorption of absorption process to them, reduce the cost of ion-exchange fiber.
Ion-exchange fiber is the function novel material that a kind of chemical property is comparatively stable, physical strength is higher of developing on ion exchange resin principle basis.The present invention is the extraction for heparin sodium by ion-exchange fiber first, has improved traditional enzymolysis process, and technique is simple, with short production cycle, easy and simple to handle, less investment, being applicable to technical scale produces, heparin purity 300～450IU/mg, extraction efficiency can reach 100,000,000 IU/500～1000 chitterlings, improves 30～40%, joint salt 40～50%, water saving 40～50%, energy-conservation 40～50%, the crude protein rate of recovery reaches 60%.
The present invention can summarize by the specific form without prejudice to spirit of the present invention or principal character with other; in the situation that not departing from the above-mentioned subject area of the present invention; various changes and the replacement made according to ordinary skill knowledge and customary means; all fall into protection scope of the present invention, be also not limited in the specific embodiment of the present invention.
Embodiment
embodiment 1
An extracting method for heparin sodium, is characterized in that comprising the steps:
(1) pig intestinal mucosa adds quartz sand, micronizing;
(2) use enzymic hydrolysis crushed material, the zymin that hydrolysis adopts is: cellulase: amylase: lipase is with (1): (1): the mixture that the mass parts ratio of (1) mixes;
(3) ion-exchange fiber absorption;
(4) ion-exchange fiber wash-out;
(5) heparin sodium precipitation;
(6) heparin sodium purification;
(7) be drying to obtain sterling heparin sodium.
embodiment 2
An extracting method for heparin sodium, is characterized in that comprising the steps:
(1) calf intestinal mucous membrane adds quartz sand, micronizing;
(2) use enzymic hydrolysis crushed material, the zymin that hydrolysis adopts is: cellulase: amylase: lipase is with (1): (1): the mixture that the mass parts ratio of (1) mixes;
(3) ion-exchange fiber absorption;
(4) ion-exchange fiber wash-out;
(5) heparin sodium precipitation;
(6) heparin sodium purification;
(7) be drying to obtain sterling heparin sodium.
embodiment 3
An extracting method for heparin sodium, comprises the following steps:
(1) 100 part of pig intestinal mucosa adds 10 parts of quartz sands, micronizing,
(2) use enzymic hydrolysis ground mixt, the zymin that hydrolysis adopts is: Trichodermareesei cellulase-producing: alkaline β～amylase: rabbit steapsase, with (1): (1): the mixture that the mass parts ratio of (1) mixes;
In reactor, add 100 parts of ground mixts, be warming up to 40 ℃, insulation 10min, adds water 50 parts, adds 1 part of NaCL, adds MgCL
20.01 part, control 30 ℃ of temperature, with lye pH adjustment be 8, add 1 part of zymin, stir 1h and add precipitation agent, described precipitation agent is to select to 2 parts of polymerize aluminum chlorides, leaves standstill 2h, by 100 order filter-cloth filterings for supernatant liquid, collection filtrate;
(3) ion-exchange fiber absorption:
In the container with whipping appts, add 100 parts of filtrates that obtained by step (2), add 4 parts of ion-exchange fibers, stir 4h, ion-exchange fiber is leached, add again 4 parts of ion-exchange fibers, stir 4h, ion-exchange fiber is leached, the ion-exchange fiber leaching is merged to water and clean 1 time;
(4) ion-exchange fiber wash-out:
The ion-exchange fiber that step (3) is reclaimed, adding quality is in the 3mol/L NaCL solution of 1 times of ion-exchange fiber quality, stirs 2h, filters, collects elutriant; Adding quality is the 2mol/LNaCL solution of 1 times of ion-exchange fiber quality again, stirs 2h, filters, and collects elutriant; Then use with the 2mol/L NaCL solution stirring of ion-exchange fiber quality equal mass and wash once, collect washing lotion, for the wash-out of lower batch of ion-exchange fiber;
(5) precipitation of heparin sodium:
The elutriant of the 1st time and the 2nd time in step (4) is merged, and adding concentration is that 85% ethanol stirs, and makes elutriant alcohol concn reach 45%, leaves standstill 6h, and supernatant liquor that will precipitation heparin sodium reclaims, and collects heparin sodium precipitation;
(6) purifying of heparin sodium:
The heparin sodium appropriate 1%NaCL solution of precipitation that step (5) is reclaimed and appropriate 0.1%Na
2sO
4solution dissolves, and insolubles is removed in centrifugation, and adding concentration is that 85% ethanol stirs, make heparin sodium aqua alcohol concn reach 45%, leave standstill 7h, filter, collect heparin sodium precipitation, be that 85% washing with alcohol heparin sodium precipitates 1 time by concentration, filter, collect heparin sodium precipitation;
(7) dry: heparin sodium is dry under vacuum condition, obtain product.
Ion-exchange fiber is preferentially selected from: ZB-2 strongly basic anion ion exchange fibre.
Heparin purity 350IU/mg, extraction efficiency can reach 100,000,000 IU/1000 root chitterlings, compared with conventional heparin sodium extraction method (in < pig intestinal mucosa, heparin sodium extracts and process for refining research >), joint salt 45%, water saving 45%, energy-conservation 50%, the crude protein rate of recovery reaches 60%.
embodiment 4
An extracting method for heparin sodium, comprises the following steps:
(1) 100 part of pig intestinal mucosa adds 10 parts of quartz sands, micronizing,
(2) use enzymic hydrolysis ground mixt, the zymin that hydrolysis adopts is: Trichodermareesei cellulase-producing: alkaline β～amylase: rabbit steapsase is with (1): (1): the mixture that the mass parts ratio of (1) mixes;
In reactor, add 100 parts of ground mixts, be warming up to 40 ℃, insulation 10min, adds water 50 parts, adds 1 part of NaCL, adds MgCL
20.01 part, control 30 ℃ of temperature, with lye pH adjustment be 8, add 1 part of zymin, stir 1h and add precipitation agent, described precipitation agent is to select to 2 parts of polymerize aluminum chlorides, leaves standstill 2h, by 100 order filter-cloth filterings for supernatant liquid, collection filtrate;
(3) ion-exchange fiber absorption:
In the container with whipping appts, add 100 parts of filtrates that obtained by step (2), add 4 parts of ion-exchange fibers, stir 4h, ion-exchange fiber is leached, add again 4 parts of ion-exchange fibers, stir 4h, ion-exchange fiber is leached, the ion-exchange fiber leaching is merged to water and clean 1 time;
(4) ion-exchange fiber wash-out:
The ion-exchange fiber that step (3) is reclaimed, adding quality is in the 3mol/L NaCL solution of 1 times of ion-exchange fiber quality, stirs 2h, filters, collects elutriant; Adding quality is the 2mol/LNaCL solution of 1 times of ion-exchange fiber quality again, stirs 2h, filters, and collects elutriant; Then use with the 2mol/L NaCL solution stirring of ion-exchange fiber quality equal mass and wash once, collect washing lotion, for the wash-out of lower batch of ion-exchange fiber;
(5) precipitation of heparin sodium:
The elutriant of the 1st time and the 2nd time in step (4) is merged, and adding concentration is that 85% ethanol stirs, and makes elutriant alcohol concn reach 45%, leaves standstill 6h, and supernatant liquor that will precipitation heparin sodium reclaims, and collects heparin sodium precipitation;
(6) purifying of heparin sodium:
The appropriate 1%NaCL solution of heparin sodium precipitation that step (5) is reclaimed dissolves, insolubles is removed in centrifugation, adding concentration is that 85% ethanol stirs, make heparin sodium aqua alcohol concn reach 45%, leaving standstill 7h, filter, collect heparin sodium precipitation, is that 85% washing with alcohol heparin sodium precipitates 1 time by concentration, filter, collect heparin sodium precipitation;
(7) dry: heparin sodium is dry under vacuum condition, obtain product.
Ion-exchange fiber is preferentially selected from: ZB-2 strongly basic anion ion exchange fibre.
Heparin purity 450IU/mg, extraction efficiency can reach 100,000,000 IU/500 root chitterlings, compared with conventional heparin sodium extraction method (in < pig intestinal mucosa, heparin sodium extracts and process for refining research >), joint salt 50%, water saving 40%, energy-conservation 40%, the crude protein rate of recovery reaches 60%.
embodiment 5
An extracting method for heparin sodium, comprises the following steps:
(1) 200 part of calf intestinal mucous membrane adds 20 parts of quartz sands, micronizing,
(2) use enzymic hydrolysis ground mixt, the zymin that hydrolysis adopts is: filamentous fungal strains Gibberella fujikuroi cellulase-producing: alkaline β～amylase: rabbit steapsase is with (3): (2): the mixture that the mass parts ratio of (3) mixes;
In reactor, add 200 parts of ground mixts, be warming up to 50 ℃, insulation 20min, adds water 80 parts, adds 2 parts of NaCL, adds MgCL
20.02 part, control 37 ℃ of temperature, with lye pH adjustment be 9, add 2 parts of zymins, stir 2h and add precipitation agent, described precipitation agent is 5 parts, Tai-Ace S 150, leaves standstill 4h, by 200 order filter-cloth filterings for supernatant liquid, collection filtrate;
(3) ion-exchange fiber absorption:
In the container with whipping appts, add 200 parts of filtrates that obtained by step (2), add 8 parts of ion-exchange fibers, stir 8h, ion-exchange fiber is leached, add again 8 parts of ion-exchange fibers, stir 8h, ion-exchange fiber is leached, the ion-exchange fiber leaching is merged to water and clean 1～2 time;
(4) ion-exchange fiber wash-out:
The ion-exchange fiber that step (3) is reclaimed, adding quality is in the 4mol/L NaCL solution of 2 times of ion-exchange fiber quality, stirs 4h, filters, collects elutriant; Adding quality is the 3mol/LNaCL solution of 2 times of ion-exchange fiber quality again, stirs 4h, filters, and collects elutriant; Then use with the 3mol/L NaCL solution stirring of ion-exchange fiber quality equal mass and wash once, collect washing lotion, for the wash-out of lower batch of ion-exchange fiber;
(5) precipitation of heparin sodium:
The elutriant of the 1st time and the 2nd time in step (4) is merged, and adding concentration is that 95% ethanol stirs, and makes elutriant alcohol concn reach 50%, leaves standstill 12h, and supernatant liquor that will precipitation heparin sodium reclaims, and collects heparin sodium precipitation;
(6) purifying of heparin sodium:
The heparin sodium appropriate 2%NaCL solution of precipitation that step (5) is reclaimed and appropriate 0.2%NaHSO
4solution dissolves, and insolubles is removed in centrifugation, and adding concentration is that 95% ethanol stirs, make heparin sodium aqua alcohol concn reach 50%, leave standstill 14h, filter, collect heparin sodium precipitation, be that 95% washing with alcohol heparin sodium precipitates 2 times by concentration, filter, collect heparin sodium precipitation;
(7) dry: heparin sodium is dry under vacuum condition, obtain product.
Ion-exchange fiber is preferentially selected from: ZB-2 strongly basic anion ion exchange fibre.
Heparin purity 400IU/mg, extraction efficiency can reach 100,000,000 IU/1000 root chitterlings, compared with conventional heparin sodium extraction method (in < pig intestinal mucosa, heparin sodium extracts and process for refining research >), joint salt 40%, water saving 40%, energy-conservation 40%, the crude protein rate of recovery reaches 60%.
embodiment 6
An extracting method for heparin sodium, comprises the following steps:
(1) 200 part of calf intestinal mucous membrane adds 20 parts of quartz sands, micronizing,
(2) use enzymic hydrolysis ground mixt, the zymin that hydrolysis adopts is: viride F-U V cellulase-producing: alkaline beta-amylase: the mould lipase in ground is with (3): (2): the mixture that the mass parts ratio of (3) mixes;
In reactor, add 200 parts of ground mixts, be warming up to 50 ℃, insulation 20min, adds water 80 parts, adds 2 parts of NaCL, adds MgCL
20.02 part, control 37 ℃ of temperature, with lye pH adjustment be 9, add 2 parts of zymins, stir 2h and add precipitation agent, described precipitation agent is 5 parts, Tai-Ace S 150, leaves standstill 4h, by 200 order filter-cloth filterings for supernatant liquid, collection filtrate;
(3) ion-exchange fiber absorption:
In the container with whipping appts, add 200 parts of filtrates that obtained by step (2), add 8 parts of ion-exchange fibers, stir 8h, ion-exchange fiber is leached, add again 8 parts of ion-exchange fibers, stir 8h, ion-exchange fiber is leached, the ion-exchange fiber leaching is merged to water and clean 1-2 time;
(4) ion-exchange fiber wash-out:
The ion-exchange fiber that step (3) is reclaimed, adding quality is in the 4mol/L NaCL solution of 2 times of ion-exchange fiber quality, stirs 4h, filters, collects elutriant; Adding quality is the 3mol/LNaCL solution of 2 times of ion-exchange fiber quality again, stirs 4h, filters, and collects elutriant; Then use with the 3mol/L NaCL solution stirring of ion-exchange fiber quality equal mass and wash once, collect washing lotion, for the wash-out of lower batch of ion-exchange fiber;
(5) precipitation of heparin sodium:
The elutriant of the 1st time and the 2nd time in step (4) is merged, and adding concentration is that 95% ethanol stirs, and makes elutriant alcohol concn reach 50%, leaves standstill 12h, and supernatant liquor that will precipitation heparin sodium reclaims, and collects heparin sodium precipitation;
(6) purifying of heparin sodium:
The appropriate 2%NaCL solution of heparin sodium precipitation that step (5) is reclaimed dissolves, insolubles is removed in centrifugation, adding concentration is that 95% ethanol stirs, make heparin sodium aqua alcohol concn reach 50%, leaving standstill 14h, filter, collect heparin sodium precipitation, is that 95% washing with alcohol heparin sodium precipitates 2 times by concentration, filter, collect heparin sodium precipitation;
(7) dry: heparin sodium is dry under vacuum condition, obtain product.
Ion-exchange fiber is preferentially selected from: ZB-2 strongly basic anion ion exchange fibre.
Heparin purity 350IU/mg, extraction efficiency can reach 100,000,000 IU/1000 root chitterlings, compared with conventional heparin sodium extraction method (in < pig intestinal mucosa, heparin sodium extracts and process for refining research >), joint salt 40%, water saving 40%, energy-conservation 40%, the crude protein rate of recovery reaches 60%.
embodiment 7
An extracting method for heparin sodium, comprises the following steps:
(1) 100 part of pig intestinal mucosa adds 10 parts of quartz sands, micronizing,
(2) use enzymic hydrolysis ground mixt, the zymin that hydrolysis adopts is: viride F-U V cellulase-producing: alkaline beta-amylase: the mould lipase in ground is with (1): (1): the mixture that the mass parts ratio of (1) mixes
In reactor, add 100 parts of ground mixts, be warming up to 40 ℃, insulation 10min, adds water 50 parts, adds 1 part of NaCL, adds MgCL
20.01 part, control 30 ℃ of temperature, with lye pH adjustment be 8, add 1 part of zymin, stir 1h and add precipitation agent, described precipitation agent is to select to 2 parts, polymerize aluminum chloride material, leaves standstill 2h, by 100 order filter-cloth filterings for supernatant liquid, collection filtrate;
(3) D208 wide aperture resin absorption:
In the container with whipping appts, add 100 parts of filtrates that obtained by step (2), add 4 parts of D208 wide aperture resins, stir 4h, D208 wide aperture resin is leached, add again 4 parts of D208 wide aperture resins, stir 4h, D208 wide aperture resin is leached, the D208 wide aperture resin leaching is merged to water and clean 1 time;
(4) D208 wide aperture resin elution:
The D208 wide aperture resin that step (3) is reclaimed, adding quality is in the 3mol/L NaCL solution of 1 times of D208 wide aperture resin quality, stirs 2h, filters, collects elutriant; Adding quality is the 2mol/LNaCL solution of 1 times of D208 wide aperture resin quality again, stirs 2h, filters, and collects elutriant; Then use with the 2mol/L NaCL solution stirring of D208 wide aperture resin quality equal mass and wash once, collect washing lotion, for the wash-out of lower batch of D208 wide aperture resin;
(5) precipitation of heparin sodium:
The elutriant of the 1st time and the 2nd time in step (4) is merged, and adding concentration is that 85% ethanol stirs, and makes elutriant alcohol concn reach 45%, leaves standstill 6h, and supernatant liquor that will precipitation heparin sodium reclaims, and collects heparin sodium precipitation;
(6) purifying of heparin sodium:
The heparin sodium appropriate 1%NaCL solution of precipitation that step (5) is reclaimed and appropriate 0.1%Na
2sO
4solution dissolves, and insolubles is removed in centrifugation, and adding concentration is that 85% ethanol stirs, make heparin sodium aqua alcohol concn reach 45%, leave standstill 7h, filter, collect heparin sodium precipitation, be that 85% washing with alcohol heparin sodium precipitates 1 time by concentration, filter, collect heparin sodium precipitation;
(7) dry: heparin sodium is dry under vacuum condition, obtain product.
Heparin purity 420IU/mg, extraction efficiency can reach 100,000,000 IU/1000 root chitterlings, compared with conventional heparin sodium extraction method (in < pig intestinal mucosa, heparin sodium extracts and process for refining research >), joint salt 45%, water saving 50%, energy-conservation 40%, the crude protein rate of recovery reaches 60%.
embodiment 7
An extracting method for heparin sodium, comprises the following steps:
(1) 100 part of pig intestinal mucosa adds 10 parts of quartz sands, micronizing;
(2) use enzymic hydrolysis ground mixt, the zymin that hydrolysis adopts is: filamentous fungal strains Gibberella fujikuroi cellulase-producing: alkali alpha amylase: Candida lipalytica Lipase is with (1): (1): the mixture that the mass parts ratio of (1) mixes;
In reactor, add 100 parts of ground mixts, be warming up to 40 ℃, insulation 10min, adds water 50 parts, adds 1 part of NaCL, adds MgCL
20.01 part, control 30 ℃ of temperature, with lye pH adjustment be 8, add 1 part of zymin, stir 1h and add precipitation agent, described precipitation agent is to select to 2 parts of polymerize aluminum chlorides, leaves standstill 2h, by 100 order filter-cloth filterings for supernatant liquid, collection filtrate;
(3) D208 wide aperture resin absorption:
In the container with whipping appts, add 100 parts of filtrates that obtained by step (2), add 4 parts of D208 wide aperture resins, stir 4h, D208 wide aperture resin is leached, add again 4 parts of D208 wide aperture resins, stir 4h, D208 wide aperture resin is leached, the D208 wide aperture resin leaching is merged to water and clean 1 time;
(4) D208 wide aperture resin elution:
The D208 wide aperture resin that step (3) is reclaimed, adding quality is in the 3mol/L NaCL solution of 1 times of D208 wide aperture resin quality, stirs 2h, filters, collects elutriant; Adding quality is the 2mol/LNaCL solution of 1 times of D208 wide aperture resin quality again, stirs 2h, filters, and collects elutriant; Then use with the 2mol/L NaCL solution stirring of D208 wide aperture resin quality equal mass and wash once, collect washing lotion, for the wash-out of lower batch of D208 wide aperture resin;
(5) precipitation of heparin sodium:
The elutriant of the 1st time and the 2nd time in step (4) is merged, and adding concentration is that 85% ethanol stirs, and makes elutriant alcohol concn reach 45%, leaves standstill 6h, and supernatant liquor that will precipitation heparin sodium reclaims, and collects heparin sodium precipitation;
(6) purifying of heparin sodium:
The appropriate 1%NaCL solution of heparin sodium precipitation that step (5) is reclaimed dissolves, insolubles is removed in centrifugation, adding concentration is that 85% ethanol stirs, make heparin sodium aqua alcohol concn reach 45%, leaving standstill 7h, filter, collect heparin sodium precipitation, is that 85% washing with alcohol heparin sodium precipitates 1 time by concentration, filter, collect heparin sodium precipitation;
(7) dry: heparin sodium is dry under vacuum condition, obtain product.
Heparin purity 400IU/mg, extraction efficiency can reach 100,000,000 IU/01000 root chitterlings, compared with conventional heparin sodium extraction method (in < pig intestinal mucosa, heparin sodium extracts and process for refining research >), joint salt 40%, water saving 40%, energy-conservation 40%, the crude protein rate of recovery reaches 60%.
embodiment 9
An extracting method for heparin sodium, comprises the following steps:
(1) 200 part of calf intestinal mucous membrane adds 20 parts of quartz sands, micronizing;
(2) use enzymic hydrolysis ground mixt, the zymin that hydrolysis adopts is: Trichodermareesei WX112 fermentative production cellulase: alkali alpha amylase: Candida lipalytica Lipase is with (3): (2): the mixture that the mass parts ratio of (3) mixes
In reactor, add 200 parts of ground mixts, be warming up to 50 ℃, insulation 20min, adds water 80 parts, adds 2 parts of NaCL, adds MgCL
20.02 part, control 37 ℃ of temperature, with lye pH adjustment be 9, add 2 parts of zymins, stir 2h and add precipitation agent, described precipitation agent is 5 parts, Tai-Ace S 150, leaves standstill 4h, by 200 order filter-cloth filterings for supernatant liquid, collection filtrate;
(3) D208 wide aperture resin absorption:
In the container with whipping appts, add 200 parts of filtrates that obtained by step (2), add 8 parts of D208 wide aperture resins, stir 8h, D208 wide aperture resin is leached, add again 8 parts of D208 wide aperture resins, stir 8h, D208 wide aperture resin is leached, the D208 wide aperture resin leaching is merged to water and clean 1-2 time;
(4) D208 wide aperture resin elution:
The D208 wide aperture resin that step (3) is reclaimed, adding quality is in the 4mol/L NaCL solution of 2 times of D208 wide aperture resin qualities, stirs 4h, filters, collects elutriant; Adding quality is the 3mol/LNaCL solution of 2 times of D208 wide aperture resin qualities again, stirs 4h, filters, and collects elutriant; Then use with the 3mol/L NaCL solution stirring of D208 wide aperture resin quality equal mass and wash once, collect washing lotion, for the wash-out of lower batch of D208 wide aperture resin;
(5) precipitation of heparin sodium:
The elutriant of the 1st time and the 2nd time in step (4) is merged, and adding concentration is that 95% ethanol stirs, and makes elutriant alcohol concn reach 50%, leaves standstill 12h, and supernatant liquor that will precipitation heparin sodium reclaims, and collects heparin sodium precipitation;
(6) purifying of heparin sodium:
The heparin sodium appropriate 2%NaCL solution of precipitation that step (5) is reclaimed and appropriate 0.2%NaHSO
4solution dissolves, and insolubles is removed in centrifugation, and adding concentration is that 95% ethanol stirs, make heparin sodium aqua alcohol concn reach 50%, leave standstill 14h, filter, collect heparin sodium precipitation, be that 95% washing with alcohol heparin sodium precipitates 2 times by concentration, filter, collect heparin sodium precipitation;
(7) dry: heparin sodium is dry under vacuum condition, obtain product.
Heparin purity 300IU/mg, extraction efficiency can reach 100,000,000 IU/1000 root chitterlings, compared with conventional heparin sodium extraction method (in < pig intestinal mucosa, heparin sodium extracts and process for refining research >), joint salt 40%, water saving 40%, energy-conservation 40%, the crude protein rate of recovery reaches 60%.
embodiment 10
An extracting method for heparin sodium, comprises the following steps:
(1) 200 part of calf intestinal mucous membrane adds 20 parts of quartz sands, micronizing;
(2) use enzymic hydrolysis ground mixt, the zymin that hydrolysis adopts is: Trichodermareesei WX112 fermentative production cellulase: alkali alpha amylase: Candida lipalytica Lipase is with (3): (2): the mixture that the mass parts ratio of (3) mixes;
In reactor, add 200 parts of ground mixts, be warming up to 50 ℃, insulation 20min, adds water 80 parts, adds 2 parts of NaCL, adds MgCL
20.02 part, control 37 ℃ of temperature, with lye pH adjustment be 9, add 2 parts of zymins, stir 2h and add precipitation agent, described precipitation agent is 5 parts, Tai-Ace S 150, leaves standstill 4h, by 200 order filter-cloth filterings for supernatant liquid, collection filtrate;
(3) D208 wide aperture resin absorption:
In the container with whipping appts, add 200 parts of filtrates that obtained by step (2), add 8 parts of D208 wide aperture resins, stir 8h, D208 wide aperture resin is leached, add again 8 parts of D208 wide aperture resins, stir 8h, D208 wide aperture resin is leached, the D208 wide aperture resin leaching is merged to water and clean 1-2 time;
(4) D208 wide aperture resin elution:
The D208 wide aperture resin that step (3) is reclaimed, adding quality is in the 4mol/L NaCL solution of 2 times of D208 wide aperture resin qualities, stirs 4h, filters, collects elutriant; Adding quality is the 3mol/LNaCL solution of 2 times of D208 wide aperture resin qualities again, stirs 4h, filters, and collects elutriant; Then use with the 3mol/L NaCL solution stirring of D208 wide aperture resin quality equal mass and wash once, collect washing lotion, for the wash-out of lower batch of D208 wide aperture resin;
(5) precipitation of heparin sodium:
The elutriant of the 1st time and the 2nd time in step (4) is merged, and adding concentration is that 95% ethanol stirs, and makes elutriant alcohol concn reach 50%, leaves standstill 12h, and supernatant liquor that will precipitation heparin sodium reclaims, and collects heparin sodium precipitation;
(6) purifying of heparin sodium:
The heparin sodium appropriate 2%NaCL solution of precipitation that step (5) is reclaimed and appropriate 0.2%NaHSO
4solution dissolves, and insolubles is removed in centrifugation, and adding concentration is that 95% ethanol stirs, make heparin sodium aqua alcohol concn reach 50%, leave standstill 14h, filter, collect heparin sodium precipitation, be that 95% washing with alcohol heparin sodium precipitates 2 times by concentration, filter, collect heparin sodium precipitation;
(7) dry: heparin sodium is dry under vacuum condition, obtain product.
Heparin purity 300IU/mg, extraction efficiency can reach 100,000,000 IU/1000 root chitterlings, compared with conventional heparin sodium extraction method (in < pig intestinal mucosa, heparin sodium extracts and process for refining research >), joint salt 40%, water saving 40%, energy-conservation 40%, the crude protein rate of recovery reaches 60%.
Claims (1)
1. an extracting method for heparin sodium, comprises the following steps:
(1) 100 part of pig intestinal mucosa adds 10 parts of quartz sands, micronizing,
(2) use enzymic hydrolysis ground mixt, the zymin that hydrolysis adopts is: Trichodermareesei cellulase-producing: alkaline beta-amylase: rabbit steapsase is with (1): (1): the mixture that the mass parts ratio of (1) mixes
In reactor, add 100 parts of ground mixts, be warming up to 40 ℃, insulation 10min, adds water 50 parts, adds 1 part of NaCl, adds MgCl
20.01 part, control 30 ℃ of temperature, with lye pH adjustment be 8, add 1 part of zymin, stir 1h and add precipitation agent, described precipitation agent is to be selected from 2 parts of polymerize aluminum chlorides, leaves standstill 2h, by 100 order filter-cloth filterings for supernatant liquid, collection filtrate;
(3) ion-exchange fiber absorption:
In the container with whipping appts, add 100 parts of filtrates that obtained by step (2), add 4 parts of ion-exchange fibers, stir 4h, ion-exchange fiber is leached, add again 4 parts of ion-exchange fibers, stir 4h, ion-exchange fiber is leached, the ion-exchange fiber leaching is merged to water and clean 1 time;
(4) ion-exchange fiber wash-out:
The ion-exchange fiber that step (3) is reclaimed, adding quality is in the 3mol/L NaCl solution of 1 times of ion-exchange fiber quality, stirs 2h, filters, collects elutriant; Adding quality is the 2mol/L NaCl solution of 1 times of ion-exchange fiber quality again, stirs 2h, filters, and collects elutriant; Then use with the 2mol/L NaCl solution stirring of ion-exchange fiber quality equal mass and wash once, collect washing lotion, for the wash-out of lower batch of ion-exchange fiber;
(5) precipitation of heparin sodium:
The elutriant of the 1st time and the 2nd time in step (4) is merged, and adding concentration is that 85% ethanol stirs, and makes elutriant alcohol concn reach 45%, leaves standstill 6h, and supernatant liquor that will precipitation heparin sodium reclaims, and collects heparin sodium precipitation;
(6) purifying of heparin sodium:
The appropriate 1%NaCl solution of heparin sodium precipitation that step (5) is reclaimed dissolves, insolubles is removed in centrifugation, adding concentration is that 85% ethanol stirs, make heparin sodium aqua alcohol concn reach 45%, leaving standstill 7h, filter, collect heparin sodium precipitation, is that 85% washing with alcohol heparin sodium precipitates 1 time by concentration, filter, collect heparin sodium precipitation;
(7) dry: heparin sodium is dry under vacuum condition, obtain product;
Ion-exchange fiber is preferentially selected from: ZB-2 strongly basic anion ion exchange fibre.
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