CN111285944A - Casing heparin sodium combined extraction process - Google Patents
Casing heparin sodium combined extraction process Download PDFInfo
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- CN111285944A CN111285944A CN201811483543.8A CN201811483543A CN111285944A CN 111285944 A CN111285944 A CN 111285944A CN 201811483543 A CN201811483543 A CN 201811483543A CN 111285944 A CN111285944 A CN 111285944A
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- heparin 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
- C08B37/006—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
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
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- Proteomics, Peptides & Aminoacids (AREA)
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Abstract
The invention discloses a combined technology for extracting heparin sodium and intestinal membrane protein by using small intestines of pigs, and a finished product casing is obtained at the same time. The extraction raw material resin and the ethanol are recycled in the extraction process. Scraping the upper intestinal mucosa of the small intestine of the pig, adsorbing heparin sodium by resin after enzymolysis of pig pancreas, and performing salt dissolution and ethanol precipitation with different concentrations to obtain a purified heparin sodium product; carrying out continuous enzymolysis on the supernatant, then carrying out membrane concentration, and drying to obtain high-purity intestinal membrane protein; and (5) carrying out leak detection and pickling on the casing to obtain a finished product casing. The pig small intestine has the advantages of easily available raw materials in the Chinese slaughtering industry, high operation mechanization degree, capability of recycling the raw materials, high utilization rate of casing, suitability for large-scale industrial production, high purity of the obtained heparin sodium and the obtained intestinal membrane protein, good economic benefit and wide prospect.
Description
Technical Field
The invention relates to an extraction technology of casing heparin sodium, and a combination technology for simultaneously obtaining casing protein and finished casings.
Background
Heparin sodium is extracted from intestinal mucosa of animals such as pig or cattle, and is a highly effective anticoagulant. The molecular structure is complex and cannot be artificially synthesized in a short time. The heparin sodium raw material is derived from small intestinal mucosa of healthy live pigs, the intestinal mucosa also contains a large amount of protein, nucleic acid, microorganism and other components, and high-purity heparin sodium and intestinal membrane protein products can be obtained after separation and purification.
At present, the annual slaughtering amount of domestic animals such as pigs, cattle and sheep is billions, at present, the utilization rate of animal casings of domestic slaughtering and processing enterprises is low, and high value-added products are not fully utilized.
Disclosure of Invention
The invention aims to provide a casing heparin sodium combined extraction process, which is a combined efficient and rapid extraction technology, can effectively improve the utilization efficiency of domestic small intestines of pigs, and obtains high-purity heparin sodium and intestinal membrane protein products.
As conceived above, the technical scheme of the invention is as follows: a casing heparin sodium combined extraction process is characterized in that: the method comprises the following steps:
① regulating the pH value of the collected intestinal mucosa to 9.0-10.0, adding pig pancreas, and performing enzymolysis at 50-55 deg.C to obtain enzymolysis solution;
② cooling the enzymolysis solution to room temperature, heating to 50-55 deg.C, adding resin and stirring to adsorb heparin sodium component in the solution, filtering to obtain resin and filtrate, standing, and filtering;
③ filtering the filtrate with resin, adjusting pH to 9.0-10.0 at 50-55 deg.C, adding pig pancreas, performing enzymolysis to obtain enzymolysis solution, membrane concentrating to obtain concentrated solution, and spray drying to obtain intestinal membrane protein;
③ rinsing the resin adsorbed with heparin sodium component with clear water, adjusting pH to 9.0-10.0, eluting for 2 times, mixing the eluates, adjusting pH to 9.5, stirring, standing, separating out supernatant, mixing the supernatant and filtrate, fine adjusting pH to 6.0, precipitating with ethanol overnight, recovering the supernatant, collecting the lower precipitate, and vacuum drying to obtain crude heparin sodium product;
④ and eluting the precipitate obtained by salt dissolving and ethanol precipitating the crude heparin sodium product, and vacuum drying to obtain refined heparin sodium product.
The enzymolysis time in the step ① is 30 min.
The amount of the resin used in the step ② is 3% of the enzymolysis solution.
Fully rinsing the resin adsorbed with the heparin sodium component in the step ③ with clear water, eluting the solution with 1.2mol/L sodium chloride solution for 2 times, wherein the first time of elution is 1.5 times of the volume of the resin, the second time of elution is 0.8 times of the volume of the resin, the elution time is 2 hours, the eluates are combined, the obtained eluent is adjusted to pH 9.5, the mixture is stirred for 30 minutes and then stands for 12 hours, then supernatant is carefully separated out, the clear solution and filtrate are combined, the pH is finely adjusted to 6.0, 1.5 times of 95% ethanol is added for precipitation overnight, the supernatant is recycled, the lower precipitate is collected, and the crude heparin sodium product is obtained by vacuum drying.
The specific operation steps of the step ④ are that the obtained crude heparin sodium is completely dissolved by 2% sodium chloride solution to prepare 6% solution, when the temperature of the filtrate is reduced to 36 ℃, the pH value is adjusted to 10.5 by saturated sodium hydroxide solution, the feed liquid is filtered by a 'sterilizing filter', the filtrate is adjusted to 6.0 by hydrochloric acid, 1 time of 95% ethanol is added, precipitation treatment is carried out for 24 hours at the temperature of 8 ℃, the precipitate is collected and dissolved by 10% sodium chloride solution, 3 times of 95% ethanol is added for precipitation, the precipitate is collected and then eluted once by 95% ethanol, and refined heparin sodium is obtained by vacuum drying.
The invention obtains the intestinal mucosa, the sausage casing and the intestinal skin by the pig small intestine raw material. The intestinal mucosa is subjected to enzymolysis, resin adsorption, filtration, elution, drying and other technologies to obtain heparin sodium and intestinal membrane protein products, and can also obtain casings for production. The invention realizes the maximum utilization rate of small intestine products by a combined extraction technology, and meanwhile, the resin and ethanol solution used in the purification process can be recycled after being recovered, thereby reducing waste.
Detailed description of the preferred embodiments
The following is a further detailed description of the present invention.
The embodiment 1 is a casing heparin sodium combined extraction process, fresh pig intestines are carefully cleaned by clear water to remove internal and external dirt and surface fat, and then the heparin sodium on the casing surface is scraped by a intestine scraper and is cleaned and collected by the clear water. Adjusting pH to 9.5, adding refined pancreas Sus Domestica, and performing enzymolysis at 0 deg.C for 30min to obtain enzymolysis solution. Cooling the enzymolysis solution to room temperature, carefully removing the oil flake layer floating on the liquid surface, controlling the temperature to be 50-55 ℃, adding the pretreated resin and continuously stirring. The resin is 3% of the feed liquid, and can adsorb heparin sodium component in the feed liquid, and the intestinal membrane protein is in the solution. Filtering to obtain resin adsorbed heparin sodium component and filtrate. Then standing and filtering. The resin adsorbed with heparin component obtained by filtration was rinsed thoroughly with clear water, the pH was adjusted to 9.5, and the solution was eluted 2 times with 1.2mol/L NaCl solution. The first elution is 1.5 times of the resin volume and is eluted for 5h, the second elution is 0.8 times of the resin volume and is eluted for 2h, and the eluates are combined. Adjusting the pH of the obtained eluent to 9.5, stirring for 30min, standing for 12h, carefully separating out a supernatant, combining the supernatant and a filtrate, finely adjusting the pH to 6.0, adding 1.5 times of 95% ethanol for precipitating overnight, recycling the supernatant ethanol, collecting a lower precipitate, and drying in vacuum to obtain a crude product of heparin sodium. Filtering the filtrate with resin, adjusting pH to 9.5 at 55 deg.C, adding porcine pancreas, performing enzymolysis for 1 hr to obtain enzymolysis solution, concentrating with membrane to obtain concentrated solution, and spray drying at 180 deg.C to obtain intestinal membrane protein. And completely dissolving the obtained heparin sodium crude product by using a 2% sodium chloride solution to prepare a solution with the concentration of 6%. When the temperature of the filtrate is reduced to 36 ℃, the pH of the filtrate is adjusted to 10.0 by saturated sodium hydroxide solution, the feed liquid is filtered by a sterilizing filter, the filtrate is adjusted to 6.0 by hydrochloric acid, 1 time of 95% ethanol is added, and the precipitate treatment is carried out for 24 hours at the temperature of 8 ℃. And (3) dissolving the collected precipitate with 10% sodium chloride solution, adding 3 times of 95% ethanol for precipitation, collecting the precipitate, eluting once with 95% ethanol, and vacuum-drying to obtain refined heparin sodium product.
Example 2: a casing heparin sodium combined extraction process comprises carefully cleaning fresh pig intestine with clear water to remove internal and external dirt and surface fat, scraping off heparin sodium on the casing surface with a intestine scraper, and cleaning with clear water. Adjusting pH to 10, adding refined pancreas Sus Domestica, and performing enzymolysis at 0 deg.C for 40min to obtain enzymolysis solution. Cooling the enzymolysis solution to room temperature, carefully removing the oil flake layer floating on the liquid surface, controlling the temperature to be 50-55 ℃, adding the pretreated resin and continuously stirring. The resin is 3% of the feed liquid, and can adsorb heparin sodium component in the feed liquid, and the intestinal membrane protein is in the solution. Filtering to obtain resin adsorbed heparin sodium component and filtrate. Then standing and filtering. The resin adsorbed with heparin component obtained by filtration was rinsed thoroughly with clear water, the pH was adjusted to 10, and the solution was eluted 2 times with 1.2mol/L NaCl solution. The first elution is 1.5 times of the resin volume and is eluted for 5h, the second elution is 0.8 times of the resin volume and is eluted for 2h, and the eluates are combined. Adjusting the pH of the obtained eluent to 9.5, stirring for 30min, standing for 12h, carefully separating out a supernatant, combining the supernatant and a filtrate, finely adjusting the pH to 6.0, adding 1.5 times of 95% ethanol for precipitating overnight, recycling the supernatant ethanol, collecting a lower precipitate, and drying in vacuum to obtain a crude product of heparin sodium. Filtering the filtrate with resin, adjusting pH to 9.5 at 55 deg.C, adding porcine pancreas, performing enzymolysis for 1 hr to obtain enzymolysis solution, concentrating with membrane to obtain concentrated solution, and spray drying at 180 deg.C to obtain intestinal membrane protein. And completely dissolving the obtained heparin sodium crude product by using a 2% sodium chloride solution to prepare a solution with the concentration of 6%. When the temperature of the filtrate is reduced to 36 ℃, the pH of the filtrate is adjusted to 10 by saturated sodium hydroxide solution, the feed liquid is filtered by a sterilizing filter, the filtrate is adjusted to 6.0 by hydrochloric acid, 1 time of 95% ethanol is added, and the precipitate treatment is carried out for 24 hours at the temperature of 8 ℃. And (3) dissolving the collected precipitate by using a 10% sodium chloride solution, adding 95% ethanol with the volume being 3 times of that of the precipitate for precipitation, collecting the precipitate, eluting the precipitate by using the 95% ethanol once, and drying in vacuum to obtain a refined heparin sodium product.
Example 3: a casing heparin sodium combined extraction process comprises carefully cleaning fresh pig intestine with clear water to remove internal and external dirt and surface fat, scraping off heparin sodium on the casing surface with a intestine scraper, and cleaning with clear water. Adjusting pH to 9, adding refined pancreas Sus Domestica, and performing enzymolysis at 55 deg.C for 30min to obtain enzymolysis solution. Cooling the enzymolysis solution to room temperature, carefully removing the oil flake layer floating on the liquid surface, controlling the temperature to be 50-55 ℃, adding the pretreated resin and continuously stirring. The resin is 3% of the feed liquid, and can adsorb heparin sodium component in the feed liquid, and the intestinal membrane protein is in the solution. Filtering to obtain resin adsorbed heparin sodium component and filtrate. Then standing and filtering. The resin adsorbed with heparin component obtained by filtration was rinsed thoroughly with clear water, the pH was adjusted to 9, and the solution was eluted 2 times with 1.2mol/L NaCl solution. The first elution is 1.5 times of the resin volume and is eluted for 5h, the second elution is 0.8 times of the resin volume and is eluted for 2h, and the eluates are combined. Adjusting the pH of the obtained eluent to 9, stirring for 30min, standing for 12h, carefully separating out a supernatant, combining the supernatant and a filtrate, finely adjusting the pH to 6.0, adding 1.5 times of 95% ethanol for precipitating overnight, recycling the supernatant ethanol clear solution, collecting a lower precipitate, and drying in vacuum to obtain a crude product of heparin sodium. Filtering the filtrate with resin, adjusting pH to 9.5 at 55 deg.C, adding porcine pancreas, performing enzymolysis for 1 hr to obtain enzymolysis solution, concentrating with membrane to obtain concentrated solution, and spray drying at 180 deg.C to obtain intestinal membrane protein. And completely dissolving the obtained heparin sodium crude product by using a 2% sodium chloride solution to prepare a solution with the concentration of 6%. When the temperature of the filtrate was reduced to 36 ℃, the PH was adjusted to 10 with saturated sodium hydroxide solution, the feed was filtered through a "sterilizing filter", the filtrate was adjusted to PH 6.0 with hydrochloric acid, 1 time of 95% ethanol was added, and the mixture was precipitated at 8 ℃ for 24 hours. And (3) dissolving the collected precipitate by using a 10% sodium chloride solution, adding 3 times of 95% ethanol for precipitation, collecting the precipitate, eluting the precipitate by using 95% ethanol once, and performing vacuum drying to obtain a refined heparin sodium product.
The above examples are merely illustrative of the embodiments of the present invention, but the present invention is not limited to the above embodiments, and other embodiments and modifications of the present invention are possible. In addition, any modifications, equivalent substitutions of ingredients, improvements and equivalents made by those skilled in the art to the present invention are within the scope of the present invention as defined by the appended claims.
Claims (5)
1. A casing heparin sodium combined extraction process is characterized in that: the method comprises the following steps:
① regulating the pH value of the collected intestinal mucosa to 9.0-10.0, adding pig pancreas, and performing enzymolysis at 50-55 deg.C to obtain enzymolysis solution;
② cooling the enzymolysis solution to room temperature, heating to 50-55 deg.C, adding resin and stirring to adsorb heparin sodium component in the solution, filtering to obtain resin and filtrate, standing, and filtering;
③ filtering the filtrate with resin, adjusting pH to 9.0-10.0 at 50-55 deg.C, adding pig pancreas, performing enzymolysis to obtain enzymolysis solution, membrane concentrating to obtain concentrated solution, and spray drying to obtain intestinal membrane protein;
③ rinsing the resin adsorbed with heparin sodium component with clear water, adjusting pH to 9.0-10.0, eluting for 2 times, mixing the eluates, adjusting pH to 9.5, stirring, standing, separating out supernatant, mixing the supernatant and filtrate, fine adjusting pH to 6.0, precipitating with ethanol overnight, recovering the supernatant, collecting the lower precipitate, and vacuum drying to obtain crude heparin sodium product;
④ and eluting the precipitate obtained by salt dissolving and ethanol precipitating the crude heparin sodium product, and vacuum drying to obtain refined heparin sodium product.
2. The process of claim 1, wherein the enzymolysis time in step ① is 30 min.
3. The process of claim 1, wherein the amount of resin used in step ② is 3% of the amount of the enzymatic hydrolysate.
4. The process of claim 1, wherein the resin adsorbed with heparin sodium component in step ③ is rinsed thoroughly with clear water, the solution is eluted with 1.2mol/L NaCl solution 2 times, the first eluent is 1.5 times the resin volume and 5 hours and the second eluent is 0.8 times the resin volume and 2 hours, the eluates are combined, the obtained eluates are adjusted to pH 9.5, stirred for 30min and then kept stand for 12 hours, then the supernatant is carefully separated out, the supernatant and the filtrate are combined, the pH 6.0 is finely adjusted, 1.5 times of 95% ethanol is added for precipitation overnight, the supernatant is recycled, the lower precipitate is collected and vacuum dried to obtain the crude heparin sodium product.
5. The process of claim 1, wherein the step ④ comprises dissolving the crude heparin sodium product completely in 2% sodium chloride solution to obtain 6% solution, adjusting pH to 10.5 with saturated sodium hydroxide solution when the temperature of the filtrate is reduced to 36 ℃, filtering the feed solution with a "sterilizing filter", adjusting pH to 6.0 with hydrochloric acid, adding 1 time of 95% ethanol, precipitating at 8 ℃ for 24h, collecting the precipitate, dissolving the precipitate in 10% sodium chloride solution, precipitating with 3 times of 95% ethanol, collecting the precipitate, eluting with 95% ethanol once, and vacuum drying to obtain refined heparin sodium product.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811483543.8A CN111285944A (en) | 2018-12-06 | 2018-12-06 | Casing heparin sodium combined extraction process |
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| CN201811483543.8A CN111285944A (en) | 2018-12-06 | 2018-12-06 | Casing heparin sodium combined extraction process |
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| CN111285944A true CN111285944A (en) | 2020-06-16 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113999330A (en) * | 2021-12-07 | 2022-02-01 | 潢川县鹏升畜产品有限公司 | Low-salt-concentration heparin sodium and active intestinal protein peptide separation and co-production process |
| 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 |
-
2018
- 2018-12-06 CN CN201811483543.8A patent/CN111285944A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113999330A (en) * | 2021-12-07 | 2022-02-01 | 潢川县鹏升畜产品有限公司 | Low-salt-concentration heparin sodium and active intestinal protein peptide separation and co-production process |
| 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: 20200616 |
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