CN113999330B - Separation and co-production process of low-salt-concentration heparin sodium and active intestinal protein peptide - Google Patents
Separation and co-production process of low-salt-concentration heparin sodium and active intestinal protein peptide Download PDFInfo
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- 229920000669 heparin Polymers 0.000 title claims abstract description 86
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 84
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 79
- ZFGMDIBRIDKWMY-PASTXAENSA-N heparin Chemical compound CC(O)=N[C@@H]1[C@@H](O)[C@H](O)[C@@H](COS(O)(=O)=O)O[C@@H]1O[C@@H]1[C@@H](C(O)=O)O[C@@H](O[C@H]2[C@@H]([C@@H](OS(O)(=O)=O)[C@@H](O[C@@H]3[C@@H](OC(O)[C@H](OS(O)(=O)=O)[C@H]3O)C(O)=O)O[C@@H]2O)CS(O)(=O)=O)[C@H](O)[C@H]1O ZFGMDIBRIDKWMY-PASTXAENSA-N 0.000 title claims abstract description 65
- 229960001008 heparin sodium Drugs 0.000 title claims abstract description 60
- 230000000968 intestinal effect Effects 0.000 title claims abstract description 51
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 27
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 24
- 238000000926 separation method Methods 0.000 title abstract description 8
- 239000011347 resin Substances 0.000 claims abstract description 62
- 229920005989 resin Polymers 0.000 claims abstract description 62
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 54
- 210000004347 intestinal mucosa Anatomy 0.000 claims abstract description 46
- 238000001179 sorption measurement Methods 0.000 claims abstract description 42
- 238000001035 drying Methods 0.000 claims abstract description 26
- 210000000936 intestine Anatomy 0.000 claims abstract description 23
- 239000003963 antioxidant agent Substances 0.000 claims abstract description 18
- 230000003078 antioxidant effect Effects 0.000 claims abstract description 18
- 238000010828 elution Methods 0.000 claims abstract description 16
- 230000007935 neutral effect Effects 0.000 claims abstract description 11
- 238000001556 precipitation Methods 0.000 claims abstract description 10
- 239000002994 raw material Substances 0.000 claims abstract description 10
- 108091005804 Peptidases Proteins 0.000 claims abstract description 9
- 150000001875 compounds Chemical class 0.000 claims abstract description 9
- 102000035195 Peptidases Human genes 0.000 claims abstract description 7
- 210000000813 small intestine Anatomy 0.000 claims abstract description 7
- 239000003480 eluent Substances 0.000 claims description 100
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 72
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 57
- 239000000243 solution Substances 0.000 claims description 45
- 238000003756 stirring Methods 0.000 claims description 44
- 239000007788 liquid Substances 0.000 claims description 41
- 238000000034 method Methods 0.000 claims description 38
- 239000011780 sodium chloride Substances 0.000 claims description 38
- 238000010438 heat treatment Methods 0.000 claims description 36
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 claims description 21
- 229960002897 heparin Drugs 0.000 claims description 21
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 20
- 238000005406 washing Methods 0.000 claims description 18
- 239000011259 mixed solution Substances 0.000 claims description 15
- 239000000047 product Substances 0.000 claims description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 14
- 230000002255 enzymatic effect Effects 0.000 claims description 12
- 238000002360 preparation method Methods 0.000 claims description 12
- 239000008213 purified water Substances 0.000 claims description 12
- 239000000413 hydrolysate Substances 0.000 claims description 11
- 238000001291 vacuum drying Methods 0.000 claims description 11
- 239000002244 precipitate Substances 0.000 claims description 10
- 238000005086 pumping Methods 0.000 claims description 10
- 239000006228 supernatant Substances 0.000 claims description 9
- 239000012043 crude product Substances 0.000 claims description 7
- 238000000605 extraction Methods 0.000 claims description 7
- 238000001914 filtration Methods 0.000 claims description 7
- 239000003055 low molecular weight heparin Substances 0.000 claims description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 6
- 238000001816 cooling Methods 0.000 claims description 6
- 229940127215 low-molecular weight heparin Drugs 0.000 claims description 6
- 241000282887 Suidae Species 0.000 claims description 5
- 239000003957 anion exchange resin Substances 0.000 claims description 5
- PGWTYMLATMNCCZ-UHFFFAOYSA-M azure A Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 PGWTYMLATMNCCZ-UHFFFAOYSA-M 0.000 claims description 5
- 230000018044 dehydration Effects 0.000 claims description 5
- 238000006297 dehydration reaction Methods 0.000 claims description 5
- 239000000843 powder Substances 0.000 claims description 5
- DWAQJAXMDSEUJJ-UHFFFAOYSA-M Sodium bisulfite Chemical compound [Na+].OS([O-])=O DWAQJAXMDSEUJJ-UHFFFAOYSA-M 0.000 claims description 4
- 239000007864 aqueous solution Substances 0.000 claims description 4
- 235000010267 sodium hydrogen sulphite Nutrition 0.000 claims description 4
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 claims description 4
- 238000006243 chemical reaction Methods 0.000 claims description 3
- 239000000463 material Substances 0.000 claims description 3
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- 239000012153 distilled water Substances 0.000 claims description 2
- 230000001376 precipitating effect Effects 0.000 claims description 2
- 235000010288 sodium nitrite Nutrition 0.000 claims description 2
- 150000003839 salts Chemical class 0.000 abstract description 16
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- 229920002683 Glycosaminoglycan Polymers 0.000 abstract description 9
- 230000007062 hydrolysis Effects 0.000 abstract description 8
- 238000006460 hydrolysis reaction Methods 0.000 abstract description 8
- 238000005904 alkaline hydrolysis reaction Methods 0.000 abstract description 2
- 230000009286 beneficial effect Effects 0.000 abstract description 2
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- 230000033228 biological regulation Effects 0.000 description 16
- 238000001514 detection method Methods 0.000 description 13
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- 102000015728 Mucins Human genes 0.000 description 5
- 108010063954 Mucins Proteins 0.000 description 5
- 150000001805 chlorine compounds Chemical class 0.000 description 5
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- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 2
- XKMRRTOUMJRJIA-UHFFFAOYSA-N ammonia nh3 Chemical compound N.N XKMRRTOUMJRJIA-UHFFFAOYSA-N 0.000 description 2
- 239000003146 anticoagulant agent Substances 0.000 description 2
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- SQDAZGGFXASXDW-UHFFFAOYSA-N 5-bromo-2-(trifluoromethoxy)pyridine Chemical class FC(F)(F)OC1=CC=C(Br)C=N1 SQDAZGGFXASXDW-UHFFFAOYSA-N 0.000 description 1
- 229920001287 Chondroitin sulfate Polymers 0.000 description 1
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- 108090000790 Enzymes Proteins 0.000 description 1
- 206010062713 Haemorrhagic diathesis Diseases 0.000 description 1
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- 239000004367 Lipase Substances 0.000 description 1
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- 108090001060 Lipase Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
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- 238000004925 denaturation Methods 0.000 description 1
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- 230000000415 inactivating effect Effects 0.000 description 1
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- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
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- HRZFUMHJMZEROT-UHFFFAOYSA-L sodium disulfite Chemical compound [Na+].[Na+].[O-]S(=O)S([O-])(=O)=O HRZFUMHJMZEROT-UHFFFAOYSA-L 0.000 description 1
- 229940001584 sodium metabisulfite Drugs 0.000 description 1
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- 206010043554 thrombocytopenia Diseases 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
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- 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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- 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
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
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Abstract
The invention relates to the field of biological pharmacy, in particular to a separation and co-production process of low-salt-concentration heparin sodium and active intestinal protein peptide. Fresh pig small intestine mucosa is used as an initial raw material, a sausage casing is peeled by a intestine scraper to obtain the intestinal mucosa, and an antioxidant protective agent is added to protect mucosal protein so as to improve the additional value of the intestinal mucosal protein peptide. Under the condition of low salt concentration, the protein and the glycosaminoglycan are efficiently dissociated by combining neutral compound proteolytic enzyme hydrolysis and high-temperature alkaline hydrolysis, and then the crude heparin sodium and the active intestinal protein are co-produced by adopting physical operations such as resin adsorption, gradient salinity elution, alcohol precipitation, drying, crushing and the like. The co-production process is beneficial to less discharge of waste water containing salt and the like, improves the added value of products and has important economic value.
Description
Technical Field
The invention relates to the field of biological pharmacy, in particular to a separation and co-production process of low-salt-concentration heparin sodium and active intestinal protein peptide.
Background
Heparin sodium belongs to glycosaminoglycan molecules with complex structures and is the most effective clinical anticoagulant and antithrombotic drug. The Chinese pharmacopoeia 2020 edition stipulates that raw materials for producing the heparin sodium are from healthy porcine small intestinal mucosa. China is a big pig slaughtering country and controls about 70% of the upstream industrial chain of heparin raw materials worldwide. The porcine small intestine mucosa mainly contains protein, glycosaminoglycan, nucleic acid, lipid substances and the like, wherein glycosaminoglycan and protein are combined by covalent bonds, and the glycosaminoglycan is also called proteoglycan. The production process of the crude heparin sodium requires that protein and glycosaminoglycan are dissociated firstly, and then the crude heparin sodium is prepared through the steps of resin adsorption elution, alcohol precipitation, drying and the like. Two methods are commonly used for extracting and dissociating crude heparin sodium: enzymolysis and salting out.
The technical route of the enzymolysis method is as follows: (1) scraping intestines and extracting intestinal mucosa (2) for enzymolysis: adding 2 degree saline into the mixed solution of intestinal mucosa, adjusting pH to 8.0-9.0 with sodium hydroxide solution, controlling the temperature of the mixed solution at 35-40 deg.C, adding trypsin, keeping the temperature for 4-6h, and heating for inactivating. (3) Separation: separating denatured protein, oil and fat from the enzymatic hydrolysate. (4) Cooling to about 50 ℃. (5) Adsorption: adjusting the pH value of the enzymolysis liquid to be more than 8.0, and the salinity is lower than 2.7 percent. Resin stirring adsorption is carried out at 50 ℃. (6) Collecting resin: collecting the adsorbed resin with a resin collecting pipe, and washing with purified water to remove impurities such as protein and oil. (7) And (3) elution: washed with 15% saline and the eluate was collected. (8) And (5) carrying out alcohol precipitation. (9) Drying and crushing.
The technical route of the salt decomposition method comprises the following steps: (1) intestinal mucosa extraction by scraping intestine (2) salt lysis: adding 4 degree saline into the mixed solution of intestinal mucosa, adjusting pH to 8.0-9.0 with sodium hydroxide solution, controlling the temperature of the mixed solution at 58 deg.C, and maintaining the temperature for 4-6 h. (3) Adsorption: adjusting the pH value of the enzymolysis liquid to be more than 8.0, and the salinity is lower than 2.7 percent. Resin stirring adsorption is carried out at 50 ℃. (4) Collecting resin: collecting the adsorbed resin with a resin collecting pipe, and washing with purified water to remove impurities such as protein and oil. (5) And (3) elution: washed with 15% saline and the eluate was collected. (6) And (5) carrying out alcohol precipitation. (7) Drying and crushing.
The production processes of the enzymolysis method and the salt hydrolysis method are two production processes commonly used in the industry at present, and the process adopts higher salinity and temperature to cause denaturation damage to intestinal mucosa protein, and the denatured intestinal protein is mainly used as animal husbandry feed. The molecular weight of the crude heparin sodium produced by the salt hydrolysis method is higher, and the subsequent production of heparin sodium raw material medicines and low-molecular heparin raw material medicines is easily influenced by partial batches. The yield of the heparin sodium produced by the enzymolysis method is low, so that the production cost is high. The salinity of the production wastewater generated by the salt decomposition method and the enzymolysis method is higher (higher than 10000ppm) and can reach more than 5 times of that of the domestic sewage, thus causing larger environmental protection pressure.
CN103724456 discloses a normal temperature salt-free extraction process of heparin sodium, however, the process has the following disadvantages: the components such as nuclease, lipase and the like in the complex enzyme have overhigh cost, and the proteoglycan is slowly dissociated under the normal temperature condition and the reaction is not complete.
Disclosure of Invention
In order to solve the technical problems, the invention provides a separation and coproduction process of low-salt-concentration heparin sodium and active intestinal protein peptide. Under the condition of low salt concentration, the protein and the glycosaminoglycan are efficiently dissociated by combining neutral compound proteolytic enzyme hydrolysis and high-temperature alkaline hydrolysis, and then physical operations such as resin adsorption, gradient salinity elution, alcohol precipitation, drying, crushing and the like are adopted, so that the co-production of crude heparin sodium and active intestinal protein is finally realized.
The technical scheme for solving the problems is as follows:
a process for separating and coproducing low-salt-concentration heparin sodium and active intestinal protein peptide comprises the following process steps: fresh small pig intestines are taken as raw materials, intestine casings are peeled off through an intestine scraper to obtain intestinal mucosa, antioxidant is added to protect mucosal protein, and the joint production of crude heparin sodium and active intestinal protein is realized through operations of low salinity enzymolysis, resin adsorption, gradient salinity elution, alcohol precipitation, drying, crushing and the like.
The invention has the following beneficial effects:
(1) the invention combines the neutral compound protease hydrolysis and alkaline condition hydrolysis to effectively dissociate glycosaminoglycan and protein, thereby avoiding the generation of high salinity wastewater and ensuring that the salinity of the wastewater is lower than 2000 ppm.
(2) According to the invention, the antioxidant is added into the intestinal mucosa breaking solution to perform antioxidant protection on the intestinal protein, so that the intestinal mucosa protein is effectively protected, and the commercial value of the byproduct intestinal mucosa protein peptide is improved.
(3) In the separation step, the resin adsorption and gradient salinity elution processes are adopted, so that the joint production of the crude heparin product and the intestinal mucosa protein peptide is realized, the production cost is reduced, and the product value of the whole upstream industrial chain of the crude heparin product is improved.
Drawings
FIG. 1 shows a process for separating and co-producing crude heparin sodium and active intestinal protein under the condition of low salt concentration.
Detailed Description
The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
Detailed Description
Example 1
A process for separating and coproducing crude heparin sodium and active intestinal protein under the condition of low salt concentration comprises the following process steps: fresh small pig intestines are taken as raw materials, intestine casings are peeled off through an intestine scraper to obtain intestinal mucosa, antioxidant is added to protect mucosal protein, and the joint production of crude heparin sodium and active intestinal protein is realized through operations of low salinity enzymolysis, resin adsorption, gradient salinity elution, alcohol precipitation, drying, crushing and the like.
The specific process steps are as follows:
s1, intestinal mucosa extraction: taking 100 fresh small intestines of pigs, peeling off casings by using a intestine scraper to obtain intestinal mucosa, simultaneously washing with purified water, collecting intestinal mucosa mixed liquor 224L, and adding 224g of sodium metabisulfite into the intestinal mucosa mixed liquor to perform antioxidation protection on intestinal proteins.
S2, low-salinity enzymolysis: pumping the intestinal mucosa mixed solution obtained in the step S1 into an enzymolysis tank, adding 6mol/L sodium hydroxide solution to adjust the pH value range of the mixed solution to 6.0, heating and stirring, controlling the temperature to 45 ℃, adding 112g of Novoxil neutral compound proteolytic enzyme, stirring and carrying out enzymolysis for 4 hours. Adding sodium hydroxide solution to adjust the pH value to 9.0, raising the temperature of the feed liquid to 85 ℃, and continuing stirring for 1 h. Adding concentrated hydrochloric acid into the feed liquid to adjust the pH value to 7.5, and cooling to 50 ℃.
S3, resin adsorption: pumping the enzymatic hydrolysate obtained in the step S2 into an adsorption tank by a pump, adding treated A98 type anion exchange resin, heating to maintain the temperature of the feed liquid at 50 ℃, stirring for 1h until azure A test paper detects that the enzymatic hydrolysate does not show red, opening a filter valve below an adsorption tube for filtration, collecting the enzymatic hydrolysate, further washing the resin by purified water with 3 times of the volume of the resin, collecting washing liquid, and combining the washing liquid with the enzymatic hydrolysate.
S4, gradient salinity elution: firstly, preparing 2.0% sodium chloride eluent with 3 times of resin volume and 15% sodium chloride eluent with 3 times of resin volume, heating to 50 ℃, firstly adding 2.0% sodium chloride eluent with 1.5 times of resin volume into an adsorption tank, heating to 55 ℃, stirring for 1h, collecting eluent 1, adding 2.0% sodium chloride eluent with 1.5 times of resin volume into the adsorption tank again, heating to 55 ℃, stirring for 1h, collecting eluent 2, merging eluent 1 and eluent 2, and merging eluent 1, eluent 2 and the enzymolysis liquid obtained in the step S3 to obtain total protein eluent; then adding 15% sodium chloride eluent with the volume 1.5 times of that of the resin into the adsorption tank, heating to 55 ℃, stirring for 1h, and collecting eluent 3. Then adding 15% sodium chloride eluent with the volume 1.5 times of the resin volume again, heating to 55 ℃, stirring for 1h, and collecting eluent 4. And combining the eluent 3 and the eluent 4 to obtain the crude heparin sodium eluent.
S5, preparation of active intestinal protein peptide: and (4) adding ethanol with the volume of 1.5 times into the total protein eluent obtained in the step (S4), centrifugally separating by a continuous flow centrifuge to obtain an active intestinal protein wet product, then transferring the active intestinal protein wet product into a reduced pressure vacuum drying oven for drying for 3 hours, and crushing to obtain 1210g of active intestinal protein peptide powder.
S6, preparation of crude heparin sodium: and (4) adding 1.5 times of ethanol by volume into the crude heparin eluent obtained in the step (S4), standing for 4 hours, and removing the supernatant. And (3) taking the thick heparin crude product at the bottom, adding ethanol for dehydration, collecting powdery crude heparin sodium, transferring the powdery crude heparin sodium into a reduced-pressure vacuum drying oven for drying for 3 hours, and crushing to obtain 98g of crude heparin sodium.
The process flow diagram for this example is shown in figure 1.
The crude heparin, intestinal mucosa protein peptide and the salt content of the production wastewater prepared in the embodiment are detected, and the detection results are shown in tables 1 to 3:
TABLE 1 crude heparin sodium assay results
TABLE 2 intestinal mucin peptide assay results
| Inspection item | Internal control standard | The result of the detection |
| Nitrogen content | 15-20％ | 16％ |
| Loss on drying | ≤10％ | 2.4％ |
| Chloride compound | ≤50ppm | Compliance with regulations |
| Heavy metals | ≤30ppm | Compliance with regulations |
| pH value | 5.0-7.0 | 6.5 |
| Rumination gene | Negative of | Compliance with regulations |
| Oversulfated chondroitin sulfate | Is lower than the detection limit | Compliance with regulations |
TABLE 3 detection results of production wastewater
| Inspection item | Internal control standard | The result of the detection |
| Sodium chloride content | ≤2000ppm | 1820ppm |
| Ammonia nitrogen | ≤8 | 6.2 |
| COD | ≤60 | 52.3 |
Example 2
A process for separating and coproducing crude heparin sodium and active intestinal protein under the condition of low salt concentration comprises the following process steps: fresh small pig intestines are taken as raw materials, intestine casings are peeled off by an intestine scraper to obtain intestinal mucosa, antioxidant is added to protect mucosal protein, and the joint production of crude heparin sodium and active intestinal protein is realized by low-salinity enzymolysis, resin adsorption, gradient salinity elution, alcohol precipitation, drying, crushing and other operations.
The specific process steps are as follows:
s1, intestinal mucosa extraction: taking 100 fresh small intestines of pigs, peeling off the casings by using a intestine scraper to obtain intestinal mucosa, simultaneously washing with purified water, collecting an intestinal mucosa mixed solution 231L, and adding 462g of antioxidant sodium bisulfite into the intestinal mucosa mixed solution to perform antioxidant protection on intestinal proteins.
S2, low-salinity enzymolysis: pumping the intestinal mucosa mixed solution obtained in the step S1 into an enzymolysis tank by a pump, adding 6mol/L sodium hydroxide solution to adjust the pH value range to 7.5, heating and stirring, controlling the temperature to 55 ℃, adding 184g of Novoxil neutral compound type proteolytic enzyme, stirring and carrying out enzymolysis for 5 hours. Adding sodium hydroxide solution to adjust the pH value to 9.5, raising the temperature of the feed liquid to 90 ℃, and continuing stirring for 1 h. Adding concentrated hydrochloric acid into the feed liquid to adjust the pH value to 8.0, and cooling to 55 ℃.
S3, resin adsorption: pumping the enzymatic hydrolysate obtained in the step S2 into an adsorption tank by a pump, adding treated A98 type anion exchange resin, heating to maintain the temperature of the feed liquid at 55 ℃, stirring for 2 hours until azure A test paper detects that the enzymatic hydrolysate does not show red, opening a filter valve below an adsorption tube, filtering, and collecting the enzymatic hydrolysate. The resin is further washed with purified water in an amount of 3 times the volume of the resin, and the washing solution is collected and combined with the enzymatic hydrolysate.
S4, gradient salinity elution: firstly, preparing 2.0 percent sodium chloride eluent with 3 times of resin volume and 15 percent sodium chloride eluent with 3 times of resin volume, and heating to 55 ℃; firstly, adding 2.0% sodium chloride eluent with the volume 1.5 times that of the resin into an adsorption tank, heating to 55 ℃, stirring for 1h, collecting eluent 1, adding 2.0% sodium chloride eluent with the volume 1.5 times that of the resin into the adsorption tank again, heating to 55 ℃, stirring for 1h, collecting eluent 2, combining eluent 1 and eluent 2, and combining eluent 1, eluent 2 and the enzymolysis liquid obtained in the step S3 to obtain total protein eluent; adding 15% sodium chloride eluent with the volume 1.5 times of that of the resin, heating to 55 ℃, stirring for 1h, and collecting eluent 3. Then adding 15% sodium chloride eluent with the volume 1.5 times of the resin volume again, heating to 55 ℃, stirring for 1h, and collecting eluent 4. And combining the eluent 3 and the eluent 4 to obtain the crude heparin sodium eluent.
S5, preparation of active intestinal protein peptide: and (4) adding 2.0 volume of ethanol into the total protein eluent obtained in the step (S4), performing centrifugal separation by a continuous flow centrifuge to obtain an active intestinal protein wet product, then transferring the active intestinal protein wet product into a vacuum reduced pressure drying oven for drying for 3 hours, and crushing to obtain 1330g of active intestinal protein peptide powder.
S6, preparation of crude heparin sodium: and (5) adding 2.0 times of volume of ethanol into the crude heparin eluent obtained in the step S4, standing for 5 hours, and removing supernatant. And (3) adding ethanol into the thick heparin crude product at the bottom for dehydration, collecting powdery crude heparin sodium, transferring the powdery crude heparin sodium into a reduced-pressure vacuum drying oven for drying for 3 hours, and crushing to obtain 103g of crude heparin sodium.
The process flow diagram for this example is shown in figure 1.
The crude heparin, intestinal mucin peptide and production wastewater prepared in this example were tested for salt content, and the test results are shown in tables 4 to 6:
TABLE 4 crude heparin sodium detection results
| Inspection item | Internal control standard | The result of the detection |
| Potency of the drug | 80-120IU/mg | 93IU/mg |
| Loss on drying | ≤10％ | 4.2％ |
| Chloride compound | ≤50ppm | Compliance with regulations |
| Heavy metals | ≤30ppm | Compliance with regulations |
| pH value | 5.0-7.0 | 6.1 |
| Rumination gene | Negative of | Compliance with regulations |
TABLE 5 intestinal mucin peptide assay results
| Inspection item | Internal control standard | The result of the detection |
| Nitrogen content | 15-20％ | 17％ |
| Loss on drying | ≤10％ | 3.4％ |
| Chloride compound | ≤50ppm | Compliance with regulations |
| Heavy metals | ≤30ppm | Compliance with regulations |
| pH value | 5.0-7.0 | 6.6 |
| Rumination gene | Negative of | Compliance with regulations |
TABLE 6 test results of wastewater
| Inspection item | Internal control standard | The result of the detection |
| Sodium chloride content | ≤2000ppm | 1620ppm |
| Ammonia nitrogen | ≤8 | 6.5 |
| COD | ≤60 | 51.6 |
Example 3
A process for separating and coproducing crude heparin sodium and active intestinal protein under the condition of low salt concentration comprises the following process steps: fresh small pig intestines are taken as raw materials, intestine casings are peeled off through an intestine scraper to obtain intestinal mucosa, antioxidant is added to protect mucosal protein, and the joint production of crude heparin sodium and active intestinal protein is realized through operations of low salinity enzymolysis, resin adsorption, gradient salinity elution, alcohol precipitation, drying, crushing and the like.
The specific process steps are as follows:
s1, intestinal mucosa extraction: taking 100 fresh small intestines of pigs, peeling off the casings by using a intestine scraper to obtain intestinal mucosa, simultaneously washing with purified water, collecting an intestinal mucosa mixed solution 228L, and adding 342g of antioxidant sodium bisulfite into the intestinal mucosa crushed solution to perform antioxidant protection on intestinal proteins.
S2, low-salinity enzymolysis: pumping the intestinal mucosa mixed solution obtained in the step S1 into an enzymolysis tank by a pump, adding 6mol/L sodium hydroxide solution to adjust the pH value range to 6.8, heating and stirring, controlling the temperature to 48 ℃, adding 160g of Novoxil neutral compound type proteolytic enzyme, stirring and carrying out enzymolysis for 4.5 h. Adding sodium hydroxide solution to adjust the pH value to 8.0, raising the temperature of the feed liquid to 87 ℃, and continuing stirring for 3 hours. Adding concentrated hydrochloric acid into the feed liquid to adjust the pH value to 7.0, and cooling to 53 ℃.
S3, resin adsorption: pumping the enzymolysis liquid obtained in the step S2 into an adsorption tank by a pump, adding treated A98 type anion exchange resin, heating to maintain the temperature of the material liquid at 52 ℃, stirring for 1.5h until azure A test paper detects that the enzymolysis liquid does not show red, opening a filtering valve below an adsorption tube, filtering, and collecting the enzymolysis liquid. Further washing the resin with purified water 3 times the volume of the resin, collecting the washing liquid and combining with the enzymolysis liquid.
S4, gradient salinity elution: firstly, preparing 2.0% sodium chloride eluent with 3 times of resin volume and 15% sodium chloride eluent with 3 times of resin volume, heating to 52 ℃, firstly adding 2.0% sodium chloride eluent with 1.5 times of resin volume into an adsorption tank, heating to 55 ℃, stirring for 1 hour, collecting eluent 1, adding 2.0% sodium chloride eluent with 1.5 times of resin volume into the adsorption tank again, heating to 55 ℃, stirring for 1 hour, collecting eluent 2, merging eluent 1 and eluent 2, and merging eluent 1, eluent 2 and the enzymolysis liquid obtained in the step S3 to obtain total protein eluent; then adding 15% sodium chloride eluent with the volume 1.5 times that of the resin into the adsorption tank, heating to 55 ℃, stirring for 1h, and collecting eluent 3. Then adding 15% sodium chloride eluent with the volume of 1.5 times of the resin volume again, heating to 55 ℃, stirring for 1h, and collecting eluent 4. And combining the eluent 3 and the eluent 4 to obtain the crude heparin sodium eluent.
S5, preparation of active intestinal protein peptide: and (4) adding ethanol with the volume being 1.8 times that of the feed liquid into the total protein eluent obtained in the step S4, separating by a continuous flow centrifuge to obtain an active intestinal protein wet product, then transferring the active intestinal protein wet product into a reduced pressure vacuum drying oven for drying for 5 hours, and crushing to obtain 1410g of active intestinal protein peptide powder.
S6, preparation of crude heparin sodium: adding ethanol with the volume 1.8 times that of the crude heparin eluent, standing for 6h, and removing the supernatant. And (3) adding ethanol into the thick heparin crude product at the bottom for dehydration, collecting powdery crude heparin sodium, transferring the powdery crude heparin sodium into a reduced-pressure vacuum drying oven for drying for 5 hours, and crushing to obtain 97g of crude heparin sodium.
The process flow diagram for this example is shown in figure 1.
The crude heparin, intestinal mucin peptide and production wastewater prepared in this example were tested for salt content, and the test results are shown in tables 7 to 9:
TABLE 7 crude heparin sodium assay results
| Inspection item | Internal control standard | The result of the detection |
| Potency of the drug | 80-120IU/mg | 99IU/mg |
| Loss on drying | ≤10％ | 3.4％ |
| Chloride compound | ≤50ppm | Compliance with regulations |
| Heavy metals | ≤30ppm | Compliance with regulations |
| pH value | 5.0-7.0 | 6.8 |
| Rumination gene | Negative of | Compliance with regulations |
TABLE 8 detection results of intestinal mucin peptides
| Inspection item | Internal control standard | The result of the detection |
| Nitrogen content | 15-20％ | 16％ |
| Loss on drying | ≤10％ | 3.9％ |
| Chloride compound | ≤50ppm | Compliance with regulations |
| Heavy metals | ≤30ppm | Compliance with regulations |
| pH value | 5.0-7.0 | 6.4 |
| Rumination gene | Negative of | Compliance with regulations |
TABLE 9 test results of wastewater
Example 4
Heparin has many negative effects such as bleeding and the induction of thrombocytopenia in long-term use, and low molecular weight heparin is a fraction obtained by separating normal heparin or a fraction produced after lysis. The substance has low molecular weight and single structure, the antithrombotic effect of the substance is better than that of heparin, the anticoagulant effect of the substance is lower than that of heparin, and the substance has the characteristics of high bioavailability, long half-life period in vivo, small bleeding tendency, easy absorption by oral administration and the like.
The specific process steps are as follows:
s1, intestinal mucosa extraction: taking 100 fresh small intestines of pigs, peeling off casings by using a intestine scraper to obtain intestinal mucosa, simultaneously washing with purified water, collecting an intestinal mucosa mixed solution 228L, and adding 342g of antioxidant sodium bisulfite into the intestinal mucosa crushing solution to perform antioxidant protection on intestinal proteins.
S2, low-salinity enzymolysis: pumping the intestinal mucosa mixed solution obtained in the step S1 into an enzymolysis tank by a pump, adding 6mol/L sodium hydroxide solution to adjust the pH value range to 6.8, heating and stirring, controlling the temperature to 48 ℃, adding 160g of Novoxil neutral compound type proteolytic enzyme, stirring and carrying out enzymolysis for 4.5 h. Adding sodium hydroxide solution to adjust the pH value to 8.0, raising the temperature of the feed liquid to 87 ℃, and continuing stirring for 3 hours. Adding concentrated hydrochloric acid into the feed liquid to adjust the pH value to 7.0, and cooling to 53 ℃.
S3, resin adsorption: pumping the enzymolysis liquid obtained in the step S2 into an adsorption tank by a pump, adding treated A98 type anion exchange resin, heating to maintain the temperature of the material liquid at 52 ℃, stirring for 1.5h until azure A test paper detects that the enzymolysis liquid does not show red, opening a filtering valve below an adsorption tube, filtering, and collecting the enzymolysis liquid. Further washing the resin with purified water 3 times the volume of the resin, collecting the washing liquid and combining with the enzymolysis liquid.
S4, gradient salinity elution: firstly, preparing 2.0% sodium chloride eluent with 3 times of resin volume and 15% sodium chloride eluent with 3 times of resin volume, heating to 52 ℃, firstly adding 2.0% sodium chloride eluent with 1.5 times of resin volume into an adsorption tank, heating to 55 ℃, stirring for 1 hour, collecting eluent 1, adding 2.0% sodium chloride eluent with 1.5 times of resin volume into the adsorption tank again, heating to 55 ℃, stirring for 1 hour, collecting eluent 2, merging eluent 1 and eluent 2, and merging eluent 1, eluent 2 and the enzymolysis liquid obtained in the step S3 to obtain total protein eluent; then adding 15% sodium chloride eluent with the volume 1.5 times of that of the resin into the adsorption tank, heating to 55 ℃, stirring for 1h, and collecting eluent 3. Then adding 15% sodium chloride eluent with the volume of 1.5 times of the resin volume again, heating to 55 ℃, stirring for 1h, and collecting eluent 4. And combining the eluent 3 and the eluent 4 to obtain the crude heparin sodium eluent.
S5, preparation of active intestinal protein peptide: and (4) adding ethanol with the volume being 1.8 times that of the feed liquid into the total protein eluent obtained in the step S4, separating by a continuous flow centrifuge to obtain an active intestinal protein wet product, then transferring the active intestinal protein wet product into a reduced pressure vacuum drying oven for drying for 5 hours, and crushing to obtain 1410g of active intestinal protein peptide powder.
S6, preparation of crude heparin sodium: adding ethanol with the volume 1.8 times that of the crude heparin eluent, standing for 6h, and removing the supernatant. And (3) taking the thick heparin crude product at the bottom, adding ethanol for dehydration, collecting powdery crude heparin sodium, transferring the powdery crude heparin sodium into a reduced-pressure vacuum drying oven for drying for 5 hours, and crushing to obtain 97g of crude heparin sodium.
S7, preparation of low molecular weight heparin sodium: dissolving the crude product heparin sodium prepared in the step S6 in 1.5% acetic acid solution, adding sodium nitrite aqueous solution, adjusting the pH value of the solution to 2.5, controlling the temperature to be 25 ℃, stirring for 4h, then adding 4mol/L sodium hydroxide solution to adjust the pH value of the solution to be neutral, raising the temperature to 40 ℃, adding sodium borohydride, reducing the heparin sodium, then adding 6mol/L hydrochloric acid to adjust the pH value of the solution to be 3.5, keeping for 30min, then adding 4mol/L sodium hydroxide solution to adjust the pH value of the solution to be 6.5, adding ethanol with the volume 2.5 times of that of the reaction solution, standing for 12h, removing supernatant, centrifuging and collecting precipitate; dissolving the precipitate in sodium chloride aqueous solution, adjusting the pH value of the solution to 2.5 by using 6mol/L hydrochloric acid, keeping the solution for 20min, adding 4mol/L sodium hydroxide solution to adjust the pH value of the solution to 6.5, adding 2.5 times of ethanol, standing for 10h, removing supernatant, centrifuging and collecting precipitate; dissolving the precipitate in distilled water, separating with chromatographic column, collecting fractions, mixing the fractions with high concentration, ultrafiltering, concentrating, precipitating with ethanol, standing, centrifuging, collecting precipitate, and vacuum drying to obtain low molecular weight heparin sodium.
The detection results of the crude heparin and the intestinal mucosa protein peptide obtained by the embodiment of the invention all accord with the proposed internal control standard, and the product has reliable quality, controllable process and lower production cost; according to the invention, neutral compound protease hydrolysis and alkaline condition hydrolysis are combined to effectively dissociate glycosaminoglycan and protein, so that high-salinity wastewater is avoided, and the discharge of salt-containing nitrogen-containing wastewater is greatly reduced; meanwhile, the antioxidant is added into the mixed liquor of the intestinal mucosa to carry out antioxidant protection on the intestinal mucosa, thus effectively protecting the intestinal mucosa protein and leading the product to have higher economic value. When the low molecular weight heparin sodium is prepared, sodium borohydride is used as a reducing agent to reduce the heparin sodium, hydrochloric acid is added into the solution, then the solution is neutralized by sodium hydroxide solution, and ethanol is precipitated.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Although embodiments of the present application have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the application, the scope of which is defined in the appended claims and their equivalents.
Claims (1)
1. A process for separating and coproducing low-salt-concentration heparin sodium and active intestinal protein peptide is characterized by comprising the following process steps: fresh small pig intestines are taken as raw materials, intestine casings are peeled off by an intestine scraper to obtain intestinal mucosa, antioxidant is added to protect mucosal protein, and the joint production of crude heparin sodium and active intestinal protein is realized by low-salinity enzymolysis, resin adsorption, gradient salinity elution, alcohol precipitation, drying and crushing;
the method comprises the following process steps:
s1, intestinal mucosa extraction: taking 100 fresh small intestines of pigs, peeling off casings by using a intestine scraper to obtain intestinal mucosa, simultaneously washing with purified water, collecting an intestinal mucosa mixed solution 228L, and adding 342g of antioxidant sodium bisulfite into the intestinal mucosa crushed solution to carry out antioxidant protection on intestinal protein;
s2, low-salinity enzymolysis: pumping the intestinal mucosa mixed solution obtained in the step S1 into an enzymolysis tank by a pump, adding 6mol/L sodium hydroxide solution to adjust the pH value range to 6.8, heating and stirring, controlling the temperature to 48 ℃, adding 160g of Novoxil neutral compound proteolytic enzyme, stirring and carrying out enzymolysis for 4.5 hours; adding sodium hydroxide solution to adjust the pH value range to 8.0, raising the temperature of the feed liquid to 87 ℃, and continuing stirring for 3 hours; adding concentrated hydrochloric acid into the feed liquid to adjust the pH value to 7.0, and cooling to 53 ℃;
s3, resin adsorption: pumping the enzymatic hydrolysate obtained in the step S2 into an adsorption tank by a pump, adding treated A98 type anion exchange resin, heating to maintain the temperature of the feed liquid at 52 ℃, stirring for 1.5h until azure A test paper detects that the enzymatic hydrolysate does not show red, opening a filter valve below an adsorption tube, filtering, and collecting the enzymatic hydrolysate; further washing the resin with purified water in an amount which is 3 times the volume of the resin, and collecting washing liquid and combining the washing liquid with the enzymolysis liquid;
s4, gradient salinity elution: firstly, preparing 2.0% sodium chloride eluent with 3 times of resin volume and 15% sodium chloride eluent with 3 times of resin volume, heating to 52 ℃, firstly adding 2.0% sodium chloride eluent with 1.5 times of resin volume into an adsorption tank, heating to 55 ℃, stirring for 1 hour, collecting eluent 1, adding 2.0% sodium chloride eluent with 1.5 times of resin volume into the adsorption tank again, heating to 55 ℃, stirring for 1 hour, collecting eluent 2, merging eluent 1 and eluent 2, and merging eluent 1, eluent 2 and the enzymolysis liquid obtained in the step S3 to obtain total protein eluent; then adding 15% sodium chloride eluent with the volume 1.5 times that of the resin into the adsorption tank, heating to 55 ℃, stirring for 1h, and collecting eluent 3; then adding 15% sodium chloride eluent with the volume 1.5 times of that of the resin again, heating to 55 ℃, stirring for 1h, and collecting eluent 4; mixing the eluent 3 and the eluent 4 to obtain a crude heparin sodium eluent;
s5, preparation of active intestinal protein peptide: adding ethanol with the volume 1.8 times that of the feed liquid into the total protein eluent obtained in the step S4, separating by a continuous flow centrifuge to obtain an active intestinal protein wet product, then transferring the active intestinal protein wet product into a reduced pressure vacuum drying oven for drying for 5 hours, and crushing to obtain 1410g of active intestinal protein peptide powder;
s6, preparation of crude heparin sodium: adding ethanol with the volume 1.8 times that of the material liquid into the crude heparin eluent, standing for 6 hours, and removing supernatant; taking a thick heparin crude product at the bottom, adding ethanol for dehydration, collecting powdery crude heparin sodium, transferring the powdery crude heparin sodium into a reduced-pressure vacuum drying oven for drying for 5 hours, and crushing to obtain 97g of crude heparin sodium;
s7, preparation of low molecular weight heparin sodium: dissolving the crude product heparin sodium prepared in the step S6 in 1.5% acetic acid solution, adding sodium nitrite aqueous solution, adjusting the pH value of the solution to 2.5, controlling the temperature to be 25 ℃, stirring for 4h, then adding 4mol/L sodium hydroxide solution to adjust the pH value of the solution to be neutral, raising the temperature to 40 ℃, adding sodium borohydride, reducing the heparin sodium, then adding 6mol/L hydrochloric acid to adjust the pH value of the solution to be 3.5, keeping for 30min, then adding 4mol/L sodium hydroxide solution to adjust the pH value of the solution to be 6.5, adding ethanol with the volume 2.5 times of that of the reaction solution, standing for 12h, removing supernatant, centrifuging and collecting precipitate; dissolving the precipitate in sodium chloride aqueous solution, adjusting the pH value of the solution to 2.5 by using 6mol/L hydrochloric acid, keeping the solution for 20min, adding 4mol/L sodium hydroxide solution to adjust the pH value of the solution to 6.5, adding 2.5 times of ethanol, standing for 10h, removing supernatant, centrifuging and collecting the precipitate; dissolving the precipitate in distilled water, separating with chromatographic column, collecting fractions, mixing the fractions with high concentration, ultrafiltering, concentrating, precipitating with ethanol, standing, centrifuging, collecting precipitate, and vacuum drying to obtain low molecular weight heparin sodium.
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Denomination of invention: A low salt concentration heparin sodium and active intestinal peptide separation and co production process Effective date of registration: 20231129 Granted publication date: 20220819 Pledgee: Bank of China Limited Huangchuan sub branch Pledgor: HUANGCHUAN PENGSHENG LIVESTOCK PRODUCTS CO.,LTD. Registration number: Y2023980068138 |