CN104530262A - Production method for extracting heparin from pig lung - Google Patents
Production method for extracting heparin from pig lung Download PDFInfo
- Publication number
- CN104530262A CN104530262A CN201510036218.7A CN201510036218A CN104530262A CN 104530262 A CN104530262 A CN 104530262A CN 201510036218 A CN201510036218 A CN 201510036218A CN 104530262 A CN104530262 A CN 104530262A
- Authority
- CN
- China
- Prior art keywords
- pig lung
- heparin
- production method
- enzymolysis
- pig
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Polysaccharides And Polysaccharide Derivatives (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to a production method for extracting heparin from a pig lung. The production method comprises the following steps: mincing a fresh pig lung into a slurry; carrying out heat preservation and enzymolysis on the slurry; filtering enzymolysis liquid, and collecting filter liquor; and carrying out deproteinization treatment, ultrafiltration, oxidation precipitation and freeze drying on the filter liquor to obtain a crude heparin product. The production method disclosed by the invention has the beneficial effects that the pig lung slurry can be more thoroughly and comprehensively dissolved by adopting an enzymolysis method, and the enzymolysis is terminated by using trypsin; by virtue of a vacuum freeze drying technology, the product quality is stabilized, the yield is enhanced, and the obtained crude product is few in impurity and high in valence; according to the production method, clarifying agent is adopted to replace a traditional process of removing protein through repeated ethanol precipitation, so that the removal rate is high, the usage amount of a large amount of ethanol is saved and the production cost is lowered; the production method is reasonable in process design, non-toxic, harmless and biologically green and environmental-friendly in production, easily available in pig lung raw material sources, low in production cost and suitable for large-scale industrial production.
Description
Technical field
The present invention relates to biomedicine field, particularly relate to a kind of production method extracting heparin from pig lung.
Background technology
Heparin is acidic mucopolysaccharide sulfuric acid ester anticoagulant.Recent study proves that heparin also has reducing blood lipid, and for many years, world market is always very vigorous to the demand of heparin.Obtain because heparin extracts processing from pluck, output is subject to a definite limitation, often can not meet the market requirement.From the later stage nineties 20th century, world market heparin demand significantly increases, and China's heparin is produced and exported year after year all to rise by a relatively large margin, and export volume and export amount of money are quick growing trend.China has become premier heparin bulk drug big export country in the world.Calendar year 2001, national heparin product export amount of money is 4,460 ten thousand dollars.2002, export amount of money reached 5,894 ten thousand dollars, increases 32.17 % than last year.Before heparin product ranks among in China's pharmaceutical products export total money sequence 5, be only second to vitamins C, citric acid, vitamin-E and sulfanilamide (SN).
The heparin product that China produces, except meeting domestic market, also exports in a large number.For a long time, China's products export is smooth and easy, as long as quality reaches requirement, usually has how many just outlet how many.In contrast thereto, due to the Economic Development Status of China and the restriction of living standards of the people, to this costliness (injection fine work 8 ~ 10 dollars/mega, for oral use is 7 ~ 8 dollars/mega) anticoagulant current home consumption amount minimum, mainly earn foreign exchange, sell to the developed countries and regions such as Japan, the U.S., Europe.
At present, the method of domestic production heparin, mainly salt solution and acid hydrolyzation, such as publication number is CN101735340A, CN101649008A, the Chinese invention application of CN101519459A etc., these two kinds of methods are all adopt pig intestinal mucosa to be raw material, in the basic conditions by salt solution, enzymolysis, go out after partial protein, by Trimethylamine 99 quaternary ammonium strong basicity cationic exchange resin adsorption agent wash-out, in elutriant in addition alcohol settling both heparin sodium primary raw materials is sold, its shortcoming reaches 48h at the production cycle, temperature of reaction is 95-100 DEG C, energy consumption is large, hydrolysis not exclusively, yield is low, produce 100,000,000 international unit (IU) heparin crude product and need 2800-3000 root pig intestinal mucosa, products obtained therefrom purity is low, crude product is at most 80 containing heparin and tires/milligram, and produce a large amount of large intestines slags and waste water, contaminate environment, and resin regeneration difficulty in production process, this preparation method is long due to the production cycle, is hydrolyzed not exclusively, not thoroughly, obtained heparin sodium purity is low, yield is low, it is low to tire, and is difficult to carry out Produce on a large scale, and can not meets the heavy demand in market.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art part, there is provided that a kind of life cycle of the product is short, purity is high, height of tiring, impurity are few, the pig lung that utilizes of environmental protection extracts the production method of heparin crude product, this technique adopts biologic enzymolysis method to extract heparin crude product from the pig lung that starting material are easy to get.
The technical scheme that the present invention realizes object is as follows:
A kind of production method extracting heparin from pig lung, it is characterized in that: step is as follows: (1) pre-treatment: pig lung is cleaned, after removing inside and outside dirt and fat, rub into rotten shape, and under fully stirring, add water stirring and obtain pig lung mixed solution, wherein the mass ratio of pig lung and water is 1:3-4, (2) enzymolysis: adjust pig lung mixed solution pH value to be 6.5 ~ 8.0, add the chondroitin sulfate prozyme of 1.5% ~ 2.5% of pig lung weight, stir, be warming up to 30 ~ 45 DEG C, after insulation reaction 30 ~ 60min, the pH value keeping reaction solution is 7.0 ~ 7.5, add 0.25% ~ 0.5% trypsinase of pig lung weight, stir, insulation 2 ~ 4h, continue to be warmed up to 90 ~ 100 DEG C, keep 5 ~ 10min, cooling, obtain pig lung enzymolysis solution (3) removing protein: filter pig lung enzymolysis solution, remove surface oil lipid layer, filtrate pH value is adjusted to be 8.5 ~ 10.5, stir the finings adding 0.1% ~ 0.4% of pig lung weight when being warmed up to 55 ~ 65 DEG C, continue to be warming up to 90 ~ 95 DEG C, insulation reaction 2 ~ 4h, be cooled to normal temperature, (4) ultrafiltration: aforesaid liquid 0.2 ~ 0.4 μm of filter membrane is filtered, obtains filtered liquid, (5) oxidation precipitation: add hydrogen peroxide, make the content of hydrogen peroxide in filtered liquid be 10 ~ 30g/kg, normal-temperature reaction 8 ~ 12h filters, obtain filtrate, filtrate is adjusted pH to 4.5 ~ 5.5, after adding isopyknic dehydrated alcohol precipitates overnight, removing supernatant liquid, throw out is crude product heparin, (6) lyophilize: crude product heparin is put into vacuum freezing drying oven, design temperature-20 ~ 10 DEG C, vacuum tightness 0.05 ~ 0.08Mpa, time of drying 2 ~ 4h.
Described a kind of production method extracting heparin from pig lung, is characterized in that: described step (2) chondroitin sulfate prozyme is the combination of in chondrosulphatase A, B and C two kinds or more.
Described a kind of production method extracting heparin from pig lung, is characterized in that: described step (3) finings is natural 1+1 finings, is made into the solution of 1% before use with Glacial acetic acid.
Described a kind of production method extracting heparin from pig lung, is characterized in that: the acid used of described step (5) adjust pH is one in Glacial acetic acid, oxalic acid, citric acid or two kinds of mixing.
Advantage of the present invention and beneficial effect are: the enzyme solution that 1, the present invention adopts more thoroughly can dissolve pig lung slurries all sidedly, enzymolysis is stopped with trypsinase, Vacuum Freezing & Drying Technology stabilizes the quality of product and improves yield, and the crude product impurity simultaneously obtained is few, height of tiring; 2, adopt in production method of the present invention finings come alternative ethanol repeatedly alcohol precipitation remove the traditional technology of protein, there is clearance high, saved the usage quantity of a large amount of ethanol, reduced production cost; 3, present invention process is reasonable in design, produces nontoxic, harmless, biological green environmental protection, and pig lung raw material sources are easy to get, and operation cost is low, is applicable to large-scale industrial production.
Embodiment
Embodiment 1: a kind of production method extracting heparin from pig lung, it is characterized in that: step is as follows: (1) pre-treatment: pig lung 5kg is cleaned, after removing inside and outside dirt and fat, rub into rotten shape, and under fully stirring, add the stirring of 15kg water and obtain pig lung mixed solution, (2) enzymolysis: adjust pig lung mixed solution pH value to be 6.5 with sodium hydroxide, add chondroitin sulfate A (CSA) and the C of 75g, stir, be warming up to 30 DEG C, after insulation reaction 30min, the pH value keeping reaction solution is 7.0, add 12.5g trypsinase, stir, insulation 2h, continue to be warmed up to 90 DEG C, keep 5min, cooling, obtain pig lung enzymolysis solution (3) removing protein: filter pig lung enzymolysis solution, remove surface oil lipid layer, filtrate pH value is adjusted to be 8.5 with sodium hydroxide, stir the finings adding 5g when being warmed up to 55 DEG C, continue to be warming up to 90 DEG C, insulation reaction 2h, be cooled to normal temperature, (4) ultrafiltration: aforesaid liquid 0.2 μm of filter membrane is filtered, obtains filtered liquid, (5) oxidation precipitation: add hydrogen peroxide, makes the content of hydrogen peroxide in filtered liquid be that 10/kg, normal-temperature reaction 8h filter, obtain filtrate, filtrate is adjusted pH to 4.5, after adding the dehydrated alcohol precipitates overnight of 20kg with Glacial acetic acid, removing supernatant liquid, throw out is crude product heparin, (6) lyophilize: crude product heparin is put into vacuum freezing drying oven, design temperature-20 DEG C, vacuum tightness 0.05Mpa, time of drying 2h, both heparin crude product 20 g, it is tired as every milligram of 100 units.
Embodiment 2: a kind of production method extracting heparin from pig lung, it is characterized in that: step is as follows: (1) pre-treatment: 100kg pig lung is cleaned, after removing inside and outside dirt and fat, rub into rotten shape, and under fully stirring, add the stirring of 350kg water and obtain pig lung mixed solution (2) enzymolysis: adjust pig lung mixed solution pH value to be 7.0 with sodium hydroxide, add chondroitin sulfate A (CSA) and the B of the 2kg of pig lung weight, stir, be warming up to 40 DEG C, after insulation reaction 45min, the pH value keeping reaction solution is 7.0, add 0.3kg trypsinase, stir, insulation 3h, continue to be warmed up to 95 DEG C, keep 7min, cooling, obtain pig lung enzymolysis solution (3) removing protein: filter pig lung enzymolysis solution, remove surface oil lipid layer, filtrate pH value is adjusted to be 9.0 with sodium hydroxide, stir the finings adding 0.2kg when being warmed up to 60 DEG C, continue to be warming up to 93 DEG C, insulation reaction 3h, be cooled to normal temperature, (4) ultrafiltration: aforesaid liquid 0.2 μm of filter membrane is filtered, obtains filtered liquid, (5) oxidation precipitation: add hydrogen peroxide, makes the content of hydrogen peroxide in filtered liquid be that 20g/kg, normal-temperature reaction 10h filter, obtain filtrate, filtrate is adjusted pH to 5.0, after adding 350kg dehydrated alcohol precipitates overnight with oxalic acid, removing supernatant liquid, throw out is crude product heparin, (6) lyophilize: crude product heparin is put into vacuum freezing drying oven, design temperature-10 DEG C, vacuum tightness 0.07Mpa, time of drying 3h, both heparin crude product 0.5kg, it is tired as every milligram of 90 units.
Embodiment 3: a kind of production method extracting heparin from pig lung, it is characterized in that: step is as follows: (1) pre-treatment: 1000kg pig lung is cleaned, after removing inside and outside dirt and fat, rub into rotten shape, and under fully stirring, add the stirring of 4000kg water and obtain pig lung mixed solution (2) enzymolysis: adjust pig lung mixed solution pH value to be 8.0, add chondroitin sulfate B and the C of the 25kg of pig lung weight, stir, be warming up to 45 DEG C, after insulation reaction 60min, the pH value keeping reaction solution is 7.5, add 5kg trypsinase, stir, insulation 4h, continue to be warmed up to 100 DEG C, keep 10min, cooling, obtain pig lung enzymolysis solution (3) removing protein: filter pig lung enzymolysis solution, remove surface oil lipid layer, filtrate pH value is adjusted to be 10.5 with sodium hydroxide, stir the finings adding 4kg when being warmed up to 65 DEG C, continue to be warming up to 95 DEG C, insulation reaction 4h, be cooled to normal temperature, (4) ultrafiltration: aforesaid liquid 0.4 μm of filter membrane is filtered, obtains filtered liquid, (5) oxidation precipitation: add hydrogen peroxide, makes the content of hydrogen peroxide in filtered liquid be that 30g/kg, normal-temperature reaction 12h filter, obtain filtrate, filtrate is adjusted pH to 5.5, after adding the dehydrated alcohol precipitates overnight of 4000kg with citric acid, removing supernatant liquid, throw out is crude product heparin, (6) lyophilize: crude product heparin is put into vacuum freezing drying oven, design temperature 0 DEG C, vacuum tightness 0.08Mpa, time of drying 4h, both heparin crude product 4.8kg, it is tired as every milligram of 100 units.
Claims (4)
1. one kind is extracted the production method of heparin from pig lung, it is characterized in that: step is as follows: (1) pre-treatment: pig lung is cleaned, after removing inside and outside dirt and fat, rub into rotten shape, and under fully stirring, add water stirring and obtain pig lung mixed solution, wherein the mass ratio of pig lung and water is 1:3-4, (2) enzymolysis: adjust pig lung mixed solution pH value to be 6.5 ~ 8.0, add the chondroitin sulfate prozyme of 1.5% ~ 2.5% of pig lung weight, stir, be warming up to 30 ~ 45 DEG C, after insulation reaction 30 ~ 60min, the pH value keeping reaction solution is 7.0 ~ 7.5, add 0.25% ~ 0.5% trypsinase of pig lung weight, stir, insulation 2 ~ 4h, continue to be warmed up to 90 ~ 100 DEG C, keep 5 ~ 10min, cooling, obtain pig lung enzymolysis solution (3) removing protein: filter pig lung enzymolysis solution, remove surface oil lipid layer, filtrate pH value is adjusted to be 8.5 ~ 10.5, stir the finings adding 0.1% ~ 0.4% of pig lung weight when being warmed up to 55 ~ 65 DEG C, continue to be warming up to 90 ~ 95 DEG C, insulation reaction 2 ~ 4h, be cooled to normal temperature, (4) ultrafiltration: aforesaid liquid 0.2 ~ 0.4 μm of filter membrane is filtered, obtains filtered liquid, (5) oxidation precipitation: add hydrogen peroxide, make the content of hydrogen peroxide in filtered liquid be 10 ~ 30g/kg, normal-temperature reaction 8 ~ 12h filters, obtain filtrate, filtrate is adjusted pH to 4.5 ~ 5.5, after adding isopyknic dehydrated alcohol precipitates overnight, removing supernatant liquid, throw out is crude product heparin, (6) lyophilize: crude product heparin is put into vacuum freezing drying oven, design temperature-20 ~ 10 DEG C, vacuum tightness 0.05 ~ 0.08Mpa, time of drying 2 ~ 4h.
2. a kind of production method extracting heparin from pig lung according to claim 1, is characterized in that: described step (2) chondroitin sulfate prozyme is the combination of in chondrosulphatase A, B and C two kinds or more.
3. a kind of production method extracting heparin from pig lung according to claim 1, is characterized in that: described step (3) finings is natural 1+1 finings, is made into the solution of 1% before use with Glacial acetic acid.
4. a kind of production method extracting heparin from pig lung according to claim 1, is characterized in that: the acid used of described step (5) adjust pH is one in Glacial acetic acid, oxalic acid, citric acid or two kinds of mixing.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510036218.7A CN104530262B (en) | 2015-01-26 | 2015-01-26 | A kind of production method extracting heparin from Pulmonis Sus domestica |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510036218.7A CN104530262B (en) | 2015-01-26 | 2015-01-26 | A kind of production method extracting heparin from Pulmonis Sus domestica |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104530262A true CN104530262A (en) | 2015-04-22 |
| CN104530262B CN104530262B (en) | 2016-08-24 |
Family
ID=52845919
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510036218.7A Active CN104530262B (en) | 2015-01-26 | 2015-01-26 | A kind of production method extracting heparin from Pulmonis Sus domestica |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104530262B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111269334A (en) * | 2020-03-11 | 2020-06-12 | 无棣县兴亚生物科技有限公司 | Production process of chondroitin and bone paste |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5849722A (en) * | 1991-04-16 | 1998-12-15 | Seikagaku Kogyo Kabushiki Kaisha | Oligosaccharide having affinity for fibroblast growth factor and process for producing same |
| CN102212149A (en) * | 2011-06-15 | 2011-10-12 | 天津宝迪农业科技股份有限公司 | Preparation process for extracting crude sodium heparin from pig lungs |
| CN103755836A (en) * | 2013-11-25 | 2014-04-30 | 青岛九龙生物医药有限公司 | Preparation technology for extracting heparin sodium crude product by utilizing animal lung |
| CN103980387A (en) * | 2014-05-26 | 2014-08-13 | 安徽科宝生物工程有限公司 | Method for preparing heparin sodium by biological enzyme process |
-
2015
- 2015-01-26 CN CN201510036218.7A patent/CN104530262B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5849722A (en) * | 1991-04-16 | 1998-12-15 | Seikagaku Kogyo Kabushiki Kaisha | Oligosaccharide having affinity for fibroblast growth factor and process for producing same |
| CN102212149A (en) * | 2011-06-15 | 2011-10-12 | 天津宝迪农业科技股份有限公司 | Preparation process for extracting crude sodium heparin from pig lungs |
| CN103755836A (en) * | 2013-11-25 | 2014-04-30 | 青岛九龙生物医药有限公司 | Preparation technology for extracting heparin sodium crude product by utilizing animal lung |
| CN103980387A (en) * | 2014-05-26 | 2014-08-13 | 安徽科宝生物工程有限公司 | Method for preparing heparin sodium by biological enzyme process |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111269334A (en) * | 2020-03-11 | 2020-06-12 | 无棣县兴亚生物科技有限公司 | Production process of chondroitin and bone paste |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104530262B (en) | 2016-08-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103724442B (en) | A kind of preparation method of low albumen high-purity Stichopus japonicus polysaccharide | |
| CN103665192B (en) | A kind of method extracting sodium heparin and co-producing protein powder from chitterlings | |
| CN104072544A (en) | Method for extracting sialic acid from bird nests by means of crystallization | |
| CN103773828A (en) | Method for extraction of collagen from fish scale | |
| CN103342669A (en) | Simple method for extracting natural taurine | |
| CN103333240A (en) | Method for reclaiming human albumin from component IV precipitate | |
| CN101829501A (en) | Combined membrane filtering method for Chinese traditional medicine extraction, separation and concentration | |
| CN104489488A (en) | Method for preparing Kvas bread concentrate | |
| CN103493968A (en) | Preparation method of high-purity peony seed protein powder for oil | |
| CN106490608A (en) | A kind of Herba Dendrobii enhancing immunity oral liquid, electuary, capsule, tablet | |
| CN103130904A (en) | High-valued utilization method for patinopecten yessoensis offal | |
| CN102382203B (en) | High-efficiency extraction process for polysaccharide of lotus seeds | |
| CN102558377A (en) | Preparation method of soybean polysaccharide gum | |
| CN109957045A (en) | A kind of joint production process extracting heparin sodium and protein peptides from animal's liver | |
| CN102327314A (en) | Preparation method of phyllemblic tannin | |
| CN103923050B (en) | Utilize rutin to prepare the method for Quercetin | |
| CN104387504A (en) | Method for preparing heparin sodium by using small intestines of pigs | |
| CN103848929A (en) | Process for high-efficiently extracting sodium heparin | |
| CN104480090A (en) | Preparation method of porcine pepsin and gastric mucin | |
| CN104530262A (en) | Production method for extracting heparin from pig lung | |
| CN105399795A (en) | Method for extracting astragaloside from radix astragali | |
| CN106317259A (en) | Joint production technology for extracting heparin sodium and protein peptide powder from pork lung | |
| CN104448027A (en) | Method for extracting fucoidan and fucoxanthin from kelp | |
| CN106279345A (en) | A kind of industrialized preparing process of thick glycyrrhizic acid | |
| CN107156864B (en) | Method for preparing water-soluble fiber from pueraria lobata waste |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: Production method for extracting heparin from pig lung Effective date of registration: 20190925 Granted publication date: 20160824 Pledgee: Huaibei Tongchuan financing guarantee Group Co., Ltd. Pledgor: Anhui Chen-Bright Bioengineering Co., Ltd. Registration number: Y2019340000083 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20210719 Granted publication date: 20160824 Pledgee: Huaibei Tongchuan financing guarantee Group Co.,Ltd. Pledgor: ANHUI CHEM BRINGHT BIOENGINEERING Co.,Ltd. Registration number: Y2019340000083 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right |