CN117402234A - Collagen extraction process for removing pepsin residues - Google Patents
Collagen extraction process for removing pepsin residues Download PDFInfo
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- 229940111202 pepsin Drugs 0.000 title claims abstract description 140
- 102000057297 Pepsin A Human genes 0.000 title claims abstract description 122
- 108090000284 Pepsin A Proteins 0.000 title claims abstract description 122
- 108010035532 Collagen Proteins 0.000 title claims abstract description 94
- 102000008186 Collagen Human genes 0.000 title claims abstract description 94
- 229920001436 collagen Polymers 0.000 title claims abstract description 94
- 238000000605 extraction Methods 0.000 title claims abstract description 27
- 239000004005 microsphere Substances 0.000 claims abstract description 64
- 235000010410 calcium alginate Nutrition 0.000 claims abstract description 55
- 239000000648 calcium alginate Substances 0.000 claims abstract description 55
- 229960002681 calcium alginate Drugs 0.000 claims abstract description 55
- OKHHGHGGPDJQHR-YMOPUZKJSA-L calcium;(2s,3s,4s,5s,6r)-6-[(2r,3s,4r,5s,6r)-2-carboxy-6-[(2r,3s,4r,5s,6r)-2-carboxylato-4,5,6-trihydroxyoxan-3-yl]oxy-4,5-dihydroxyoxan-3-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylate Chemical compound [Ca+2].O[C@@H]1[C@H](O)[C@H](O)O[C@@H](C([O-])=O)[C@H]1O[C@H]1[C@@H](O)[C@@H](O)[C@H](O[C@H]2[C@H]([C@@H](O)[C@H](O)[C@H](O2)C([O-])=O)O)[C@H](C(O)=O)O1 OKHHGHGGPDJQHR-YMOPUZKJSA-L 0.000 claims abstract description 55
- 238000000034 method Methods 0.000 claims abstract description 17
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- 239000000243 solution Substances 0.000 claims description 71
- 238000005406 washing Methods 0.000 claims description 35
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 33
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims description 31
- 239000007788 liquid Substances 0.000 claims description 31
- 235000010413 sodium alginate Nutrition 0.000 claims description 31
- 239000000661 sodium alginate Substances 0.000 claims description 31
- 229940005550 sodium alginate Drugs 0.000 claims description 31
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 30
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 21
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 18
- 238000005238 degreasing Methods 0.000 claims description 18
- 239000011734 sodium Substances 0.000 claims description 18
- 229910052708 sodium Inorganic materials 0.000 claims description 18
- 150000003839 salts Chemical class 0.000 claims description 17
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 16
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 14
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- 125000003275 alpha amino acid group Chemical group 0.000 description 2
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- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 1
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- 210000003205 muscle Anatomy 0.000 description 1
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- 229940055695 pancreatin Drugs 0.000 description 1
- 229940055729 papain Drugs 0.000 description 1
- 235000019834 papain Nutrition 0.000 description 1
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- 210000002435 tendon Anatomy 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6478—Aspartic endopeptidases (3.4.23)
- C12N9/6481—Pepsins (3.4.23.1; 3.4.23.2; 3.4.23.3)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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Abstract
The invention discloses a collagen extraction process for removing pepsin residues, which comprises the following steps: s100: preparing calcium alginate pepsin microspheres; s200: calcium alginate pepsin was used to extract collagen. The immobilized pepsin microsphere has the advantages of simple preparation method, simple and convenient operation and mild reaction conditions. In the collagen extraction process, the sensitized telopeptide of the collagen can be effectively sheared and removed, and can be separated and removed by a simple centrifugal method, so that the aim of no pepsin residue in the extracted collagen is fulfilled, and the collagen material more suitable for clinical application is prepared.
Description
Technical Field
The invention relates to a collagen extraction process for removing pepsin residues, and belongs to the technical field of collagen purification.
Background
Collagen is the most major structural protein of extracellular matrix, is the protein with the largest content in human body, exists in tissues such as muscle, tendon and skin of human body, and plays an important role in protecting tissue organs. Collagen has found wide application in a variety of fields. In the skin aging process, the collagen is supplemented to increase skin elasticity, keep skin smooth and soft, and play a role in beautifying and resisting aging; in terms of food and health care, collagen is often used in the production of sausage casings, food additives, nutritional supplements, and the like; biological materials such as surgical sutures, collagen sponges and carriers for bioactive substances are commonly used in medicine, and collagen is also used as a raw material for preparation.
Accordingly, there has been a great deal of research on extraction of collagen, and at present, the main sources of collagen are skin and bones of mammals such as pigs, cows and sheep, and the extraction means mainly comprise alkali, acid, enzyme, salt and the like. The enzyme commonly used in the enzymatic method comprises pepsin, pancreatin, papain and the like, and the method has the advantages of mild action, high reaction speed, high substrate hydrolysis speed, no environmental pollution and the like. Because of the characteristic that the collagen can only be dissolved under the acidic condition, and the pepsin has activity under the acidic condition, the extraction mode of acid and pepsin becomes an effective collagen extraction method, in particular to a low-cost and high-efficiency extraction mode in the production process. In the extraction, the pH value of the solution is generally increased, pepsin is inactivated to stop the enzyme digestion reaction, and simultaneously, collagen precipitation is separated out and then solid-liquid separation is carried out. Although the conventional operation can control the degree of collagen digestion, there is no effective means to remove pepsin, which is easy to cause pepsin residue in the extracted collagen, and pepsin on the market is basically extracted from pig stomach, so that there is a new risk of virus hidden danger in the production of the extracted collagen, and clinical application risk is caused.
The enzyme immobilization method is to seal the enzyme in a certain immobilization space, and can play the role of the enzyme and recycle the enzyme. The prior researches show that the immobilized enzyme has longer storage time, higher activity maintenance and easy separation and recovery compared with the common enzyme, and achieves the effects of high repeated use rate and reduced cost.
The method has the advantages that the sensitivity characteristic of sodium alginate to calcium ions is utilized to prepare the calcium alginate immobilized pepsin microsphere, and the calcium alginate immobilized pepsin microsphere is applied to a collagen production and extraction process, on one hand, immobilized pepsin can still cut off collagen end peptide at fixed points, so that collagen with uniform molecular mass and a triple helix structure is obtained; on the other hand, the immobilized pepsin microspheres can be separated from the collagen by a quick and simple centrifugation or filtration mode. Therefore, the invention provides a simple and effective method for removing pepsin residues in the collagen extraction process, so as to improve the collagen production process, reduce the heat source and the virus risk and improve the clinical use safety.
Disclosure of Invention
The invention aims to provide a collagen extraction process for removing pepsin residues, which is characterized in that pepsin is immobilized and then subjected to enzyme digestion reaction, and separation is performed after enzymolysis, so that the purpose of removing the pepsin residues is achieved.
In order to solve the technical problems, the invention adopts the following technical scheme:
a collagen extraction process for removing pepsin residues, which comprises the following steps:
s100: preparing calcium alginate pepsin microspheres;
s200: calcium alginate pepsin was used to extract collagen.
In the foregoing collagen extraction process for removing pepsin residues, the step S200 further includes the following steps: degreasing pig skin blocks with degreasing liquid, washing with salt, soaking the pig skin blocks in acetic acid solution, adding calcium alginate pepsin into the acetic acid solution, and removing calcium alginate pepsin microspheres and other solid impurities in the solution after enzymolysis to obtain clean enzymolysis liquid; and adjusting the pH value of the enzymolysis liquid to 7-8, and separating out collagen.
In the aforementioned collagen extraction process for removing pepsin residues, the step S200 specifically includes the following steps:
s210: cutting the pigskin into small pieces after shaving and fat-removing, degreasing with degreasing liquid, and washing with salt, wherein the pigskin small pieces are washed with water after washing with salt;
s220: soaking the pigskin small pieces treated in the step S210 in 0.5M acetic acid solution for 12-36h, homogenizing and homogenizing the soaked pigskin small pieces, wherein the solid-to-liquid ratio is 1:50-100; the soaking is to break and homogenize the pig skin fully, which is difficult to break. The more complete the homogenization, the higher the pigskin collagen extraction rate.
S230: adding calcium alginate pepsin microspheres, wherein the mass ratio of the calcium alginate pepsin microspheres to pigskin small pieces is 1:100-200, the enzymolysis temperature is 10-20 ℃, and the enzymolysis time is 12-24 hours; enzymolysis, enzyme cutting collagen with pepsin, and cutting sensitized telopeptide.
S240: after the enzymolysis is finished, centrifuging or filtering the reaction solution by a screen, and removing calcium alginate pepsin microspheres and other solid impurities to obtain clean enzymolysis solution;
s250: regulating the pH value of the enzymolysis liquid obtained in the step S240 to 7-8 by using sodium hydroxide, precipitating collagen sediment, then adding 10% sodium chloride solution, and salting out for 12-24 hours;
s260: collecting the salted-out collagen precipitate after centrifugation, and washing with water for 2 times to obtain the atelopeptide collagen without pepsin residues.
In the aforementioned collagen extraction process for removing pepsin residues, the step S200 specifically includes the following steps:
s210: cutting pig skin into 5mm small pieces after shaving and fat-removing, degreasing with degreasing liquid, adding salt for washing, and washing the pig skin small pieces with water after salt washing;
s220: soaking the pigskin small pieces treated in the step S210 in 0.5M acetic acid solution for 18h, homogenizing and homogenizing the soaked pigskin small pieces, wherein the solid-to-liquid ratio is 1:67;
s230: adding calcium alginate pepsin microspheres, wherein the mass ratio of the calcium alginate pepsin microspheres to pigskin small pieces is 1:100, the enzymolysis temperature is 12 ℃, and the enzymolysis time is 15 hours;
s240: after the enzymolysis is finished, centrifuging or filtering the reaction solution by a screen, and removing calcium alginate pepsin microspheres and other solid impurities to obtain clean enzymolysis solution;
s250: adjusting the pH value of the enzymolysis liquid obtained in the step S240 to 7 by using sodium hydroxide, precipitating collagen sediment, then adding 10% sodium chloride solution, and salting out for 19h;
s260: collecting the salted-out collagen precipitate after centrifugation, and washing with water for 2 times to obtain the atelopeptide collagen without pepsin residues.
In the aforementioned collagen extraction process for removing pepsin residues, the step S100 includes the following steps: adding pure water into sodium alginate for dissolution to prepare sodium alginate solution; adding pepsin with different mass into the sodium alginate solution to obtain sodium alginate-pepsin solution; dripping sodium alginate-pepsin solution into calcium chloride solution to solidify calcium alginate into microspheres; removing impurities in the fusion liquid to obtain the pepsin immobilized microsphere.
In the aforementioned collagen extraction process for removing pepsin residues, the step S100 specifically includes the following steps:
s110: adding pure water into sodium alginate for dissolution to prepare sodium alginate solution with concentration of 1% -4%;
s120: adding pepsin with different mass into the sodium alginate solution, fully and uniformly stirring, standing to remove bubbles, and obtaining sodium alginate-pepsin solution with the concentration of 1 mg/ml-3 mg/ml;
s130: dripping sodium alginate-pepsin solution into 0.5-2.5% calcium chloride solution, standing to solidify calcium alginate into microspheres, and solidifying at 4deg.C overnight;
s140: and (3) removing the excessive calcium chloride solution in the step (S130), adding pure water, standing and washing for 3 times, and centrifuging to remove the aqueous solution to obtain the pepsin immobilized microsphere.
In the aforementioned collagen extraction process for removing pepsin residues, the step S100 specifically includes the following steps:
s110: adding pure water into sodium alginate to dissolve, and preparing sodium alginate solution with the concentration of 2%;
s120: adding pepsin with different mass into the sodium alginate solution, fully and uniformly stirring, standing to remove bubbles, and obtaining sodium alginate-pepsin solution with the concentration of 1 mg/ml;
s130: dripping sodium alginate-pepsin solution into 1.5% calcium chloride solution, standing to solidify calcium alginate into microsphere, and solidifying at 4deg.C overnight;
s140: and (3) removing the excessive calcium chloride solution in the step (S130), adding pure water, standing and washing for 3 times, and centrifuging to remove the aqueous solution to obtain the pepsin immobilized microsphere.
Compared with the prior art, the immobilized pepsin microsphere has the advantages of simple preparation method, simple and convenient operation and mild reaction conditions. In the collagen extraction process, the sensitized telopeptide of the collagen can be effectively sheared and removed, and can be separated and removed by a simple centrifugal method, so that the aim of no pepsin residue in the extracted collagen is fulfilled, and the collagen material more suitable for clinical application is prepared.
Drawings
FIG. 1 is an appearance diagram of immobilized sodium alginate pepsin microspheres;
FIG. 2 is a mass spectrum of collagen prepared by cleavage of the immobilized pepsin microspheres in example 1;
FIG. 3 is a mass spectrum of collagen prepared by cleavage of example 2 using immobilized pepsin microspheres;
FIG. 4 is a mass spectrum of collagen prepared by enzyme digestion of immobilized pepsin microspheres in example 3.
Detailed Description
Example 1 of the present invention: a collagen extraction process for removing pepsin residues, which comprises the following steps:
first, S100: preparing the calcium alginate pepsin microsphere.
The preparation method of the calcium alginate pepsin microsphere specifically comprises the following steps:
s110: adding pure water into sodium alginate to dissolve the sodium alginate to prepare a sodium alginate solution with the concentration of 1%;
s120: adding pepsin with different mass into the sodium alginate solution, fully and uniformly stirring, standing to remove bubbles, and obtaining sodium alginate-pepsin solution with the concentration of 1 mg/ml;
s130: dripping sodium alginate-pepsin solution into 0.5% calcium chloride solution, standing to solidify calcium alginate into microspheres, and solidifying at 4deg.C overnight;
s140: and (3) removing the excessive calcium chloride solution in the step (S130), adding pure water, standing and washing for 3 times, and centrifuging to remove the aqueous solution to obtain the pepsin immobilized microsphere.
Then, S200: calcium alginate pepsin was used to extract collagen.
The method for extracting collagen by using the calcium alginate pepsin microspheres specifically comprises the following steps:
s210: cutting pig skin into 5mm small pieces after shaving and fat-removing, degreasing with degreasing liquid, adding salt for washing, and washing the pig skin small pieces with water after salt washing;
s220: soaking the pigskin small pieces treated in the step S210 in 0.5M acetic acid solution for 18h, homogenizing and homogenizing the soaked pigskin small pieces, wherein the solid-to-liquid ratio is 1:100;
s230: adding calcium alginate pepsin microspheres, wherein the mass ratio of the calcium alginate pepsin microspheres to pigskin small pieces is 1:100, the enzymolysis temperature is 12 ℃, and the enzymolysis time is 15 hours;
s240: after the enzymolysis is finished, centrifuging or filtering the reaction solution by a screen, and removing calcium alginate pepsin microspheres and other solid impurities to obtain clean enzymolysis solution;
s250: adjusting the pH value of the enzymolysis liquid obtained in the step S240 to 7 by using sodium hydroxide, precipitating collagen sediment, then adding 10% sodium chloride solution, and salting out for 19h;
s260: collecting the salted-out collagen precipitate after centrifugation, and washing with water for 2 times to obtain the atelopeptide collagen without pepsin residues.
Example 2: a collagen extraction process for removing pepsin residues, which comprises the following steps:
first, S100: preparing the calcium alginate pepsin microsphere.
The preparation method of the calcium alginate pepsin microsphere specifically comprises the following steps:
s110: adding pure water into sodium alginate to dissolve the sodium alginate to prepare a sodium alginate solution with the concentration of 4%;
s120: adding pepsin with different mass into the sodium alginate solution, fully and uniformly stirring, standing to remove bubbles, and obtaining sodium alginate-pepsin solution with the concentration of 3 mg/ml;
s130: dripping sodium alginate-pepsin solution into 2.5% calcium chloride solution, standing to solidify calcium alginate into microsphere, and solidifying at 4deg.C overnight;
s140: and (3) removing the excessive calcium chloride solution in the step (S130), adding pure water, standing and washing for 3 times, and centrifuging to remove the aqueous solution to obtain the pepsin immobilized microsphere.
Then, S200: calcium alginate pepsin was used to extract collagen.
The method for extracting collagen by using the calcium alginate pepsin microspheres specifically comprises the following steps:
s210: cutting pig skin into 5mm small pieces after shaving and fat-removing, degreasing with degreasing liquid, adding salt for washing, and washing the pig skin small pieces with water after salt washing;
s220: soaking the pigskin small pieces treated in the step S210 in 0.5M acetic acid solution for 18h, homogenizing and homogenizing the soaked pigskin small pieces, wherein the solid-to-liquid ratio is 1:100;
s230: adding calcium alginate pepsin microspheres, wherein the mass ratio of the calcium alginate pepsin microspheres to pigskin small pieces is 1:100, the enzymolysis temperature is 12 ℃, and the enzymolysis time is 15 hours;
s240: after the enzymolysis is finished, centrifuging or filtering the reaction solution by a screen, and removing calcium alginate pepsin microspheres and other solid impurities to obtain clean enzymolysis solution;
s250: adjusting the pH value of the enzymolysis liquid obtained in the step S240 to 7 by using sodium hydroxide, precipitating collagen sediment, then adding 10% sodium chloride solution, and salting out for 19h;
s260: collecting the salted-out collagen precipitate after centrifugation, and washing with water for 2 times to obtain the atelopeptide collagen without pepsin residues.
Example 3: a collagen extraction process for removing pepsin residues, which comprises the following steps:
first, S100: preparing the calcium alginate pepsin microsphere.
The preparation method of the calcium alginate pepsin microsphere specifically comprises the following steps:
s110: adding pure water into sodium alginate to dissolve, and preparing sodium alginate solution with the concentration of 2%;
s120: adding pepsin with different mass into the sodium alginate solution, fully and uniformly stirring, standing to remove bubbles, and obtaining sodium alginate-pepsin solution with the concentration of 1 mg/ml;
s130: dripping sodium alginate-pepsin solution into 1.5% calcium chloride solution, standing to solidify calcium alginate into microsphere, and solidifying at 4deg.C overnight;
s140: and (3) removing the excessive calcium chloride solution in the step (S130), adding pure water, standing and washing for 3 times, and centrifuging to remove the aqueous solution to obtain the pepsin immobilized microsphere.
Then, S200: calcium alginate pepsin was used to extract collagen.
The method for extracting collagen by using the calcium alginate pepsin microspheres specifically comprises the following steps:
s210: cutting pig skin into 5mm small pieces after shaving and fat-removing, degreasing with degreasing liquid, adding salt for washing, and washing the pig skin small pieces with water after salt washing;
s220: soaking the pigskin small pieces treated in the step S210 in 0.5M acetic acid solution for 18h, homogenizing and homogenizing the soaked pigskin small pieces, wherein the solid-to-liquid ratio is 1:100;
s230: adding calcium alginate pepsin microspheres, wherein the mass ratio of the calcium alginate pepsin microspheres to pigskin small pieces is 1:100, the enzymolysis temperature is 12 ℃, and the enzymolysis time is 15 hours;
s240: after the enzymolysis is finished, centrifuging or filtering the reaction solution by a screen, and removing calcium alginate pepsin microspheres and other solid impurities to obtain clean enzymolysis solution;
s250: adjusting the pH value of the enzymolysis liquid obtained in the step S240 to 7 by using sodium hydroxide, precipitating collagen sediment, then adding 10% sodium chloride solution, and salting out for 19h;
s260: collecting the salted-out collagen precipitate after centrifugation, and washing with water for 2 times to obtain the atelopeptide collagen without pepsin residues.
Correspondingly, in order to meet the requirements of the sterile preparation in production, the clean enzymolysis liquid obtained in the step S240 can be subjected to sterile filtration through a 0.22 mu m membrane, and then the sterile collagen product can be obtained through sterile operation. Or freeze-drying the collagen obtained in the step S260 and then sterilizing by irradiation. Meeting different process requirements. The invention mainly provides a preparation method of collagen for removing pepsin residues in production, so as to optimize the production process and improve the production quality of the collagen.
And (3) effect measurement:
enzyme digestion effect: the collagen obtained according to the example is subjected to mass spectrometry detection, and compared with the porcine type I collagen alpha 1 chain amino acid sequence, as shown in figure 2, the labeled sequence is the collagen amino acid sequence obtained according to the example, and the result shows that the N-terminal peptide sequence and the C-terminal peptide sequence are already excised, so that the immobilized pepsin microsphere has the same enzyme digestion effect.
Residual measurement: the pepsin residue in the prepared collagen solid sample is measured by using a pig Pepsin (PG) enzyme-linked immunosorbent assay (ELISA) kit of Emblica officinalis of Shanghai, the detection limit is ng/ml, and a collagen sample prepared by adding no pepsin is used as a control. The obtained results show (Table I), compared with the non-immobilized pepsin, the pepsin residue can not be detected in the collagen prepared by pepsin enzyme cutting with calcium alginate immobilized, so that the collagen preparation without pepsin residue can be realized, and the product quality and the clinical application safety are improved.
Table-pepsin residue measurement
Remarks: n.d. =undetected.
Claims (7)
1. The extraction process for removing the pepsin residue is characterized by comprising the following steps of:
s100: preparing calcium alginate pepsin microspheres;
s200: the calcium alginate pepsin microsphere is used for extracting collagen.
2. The process for extracting collagen from pepsin according to claim 1, wherein the step S200 further comprises the following steps: degreasing pig skin blocks with degreasing liquid, washing with salt, soaking the pig skin blocks in acetic acid solution, adding calcium alginate pepsin into the acetic acid solution, and removing calcium alginate pepsin microspheres and other solid impurities in the solution after enzymolysis to obtain clean enzymolysis liquid; and adjusting the pH value of the enzymolysis liquid to 7-8, and separating out collagen.
3. The process for extracting collagen from pepsin residue according to claim 1, wherein the step S200 comprises the steps of:
s210: cutting the pigskin into small pieces after shaving and fat-removing, degreasing with degreasing liquid, and washing with salt, wherein the pigskin small pieces are washed with water after washing with salt;
s220: soaking the pigskin small pieces treated in the step S210 in 0.5M acetic acid solution for 12-36h, homogenizing and homogenizing the soaked pigskin small pieces, wherein the solid-to-liquid ratio is 1:50-100;
s230: adding calcium alginate pepsin microspheres, wherein the mass ratio of the calcium alginate pepsin microspheres to pigskin small pieces is 1:100-200, the enzymolysis temperature is 10-20 ℃, and the enzymolysis time is 12-24 hours;
s240: after the enzymolysis is finished, centrifuging or filtering the reaction solution by a screen, and removing calcium alginate pepsin microspheres and other solid impurities to obtain clean enzymolysis solution;
s250: regulating the pH value of the enzymolysis liquid obtained in the step S240 to 7-8 by using sodium hydroxide, precipitating collagen sediment, then adding 10% sodium chloride solution, and salting out for 12-24 hours;
s260: collecting the salted-out collagen precipitate after centrifugation, and washing with water for 2 times to obtain the atelopeptide collagen without pepsin residues.
4. The process for extracting collagen from pepsin residue according to claim 1, wherein the step S200 comprises the steps of:
s210: cutting pig skin into 5mm small pieces after shaving and fat-removing, degreasing with degreasing liquid, adding salt for washing, and washing the pig skin small pieces with water after salt washing;
s220: soaking the pigskin small pieces treated in the step S210 in 0.5M acetic acid solution for 18h, homogenizing and homogenizing the soaked pigskin small pieces, wherein the solid-to-liquid ratio is 1:100;
s230: adding calcium alginate pepsin microspheres, wherein the mass ratio of the calcium alginate pepsin microspheres to pigskin small pieces is 1:100, the enzymolysis temperature is 12 ℃, and the enzymolysis time is 15 hours;
s240: after the enzymolysis is finished, centrifuging or filtering the reaction solution by a screen, and removing calcium alginate pepsin microspheres and other solid impurities to obtain clean enzymolysis solution;
s250: adjusting the pH value of the enzymolysis liquid obtained in the step S240 to 7 by using sodium hydroxide, precipitating collagen sediment, then adding 10% sodium chloride solution, and salting out for 19h;
s260: collecting the salted-out collagen precipitate after centrifugation, and washing with water for 2 times to obtain the atelopeptide collagen without pepsin residues.
5. The process for extracting collagen from pepsin residues according to claim 1, wherein step S100 comprises the following steps: adding pure water into sodium alginate for dissolution to prepare sodium alginate solution; adding pepsin with different mass into the sodium alginate solution to obtain sodium alginate-pepsin solution; dripping sodium alginate-pepsin solution into calcium chloride solution to solidify calcium alginate into microspheres; removing impurities in the fusion liquid to obtain the pepsin immobilized microsphere.
6. The process for extracting collagen from pepsin residue according to claim 1, wherein the step S100 comprises the steps of:
s110: adding pure water into sodium alginate for dissolution to prepare sodium alginate solution with concentration of 1% -4%;
s120: adding pepsin with different mass into the sodium alginate solution, fully and uniformly stirring, standing to remove bubbles, and obtaining sodium alginate-pepsin solution with the concentration of 1 mg/ml-3 mg/ml;
s130: dripping sodium alginate-pepsin solution into 0.5-2.5% calcium chloride solution, standing to solidify calcium alginate into microspheres, and solidifying at 4deg.C overnight;
s140: and (3) removing the excessive calcium chloride solution in the step (S130), adding pure water, standing and washing for 3 times, and centrifuging to remove the aqueous solution to obtain the pepsin immobilized microsphere.
7. The process for extracting collagen from pepsin residue according to claim 1, wherein the step S100 comprises the steps of:
s110: adding pure water into sodium alginate to dissolve, and preparing sodium alginate solution with the concentration of 2%;
s120: adding pepsin with different mass into the sodium alginate solution, fully and uniformly stirring, standing to remove bubbles, and obtaining sodium alginate-pepsin solution with the concentration of 1 mg/ml;
s130: dripping sodium alginate-pepsin solution into 1.5% calcium chloride solution, standing to solidify calcium alginate into microsphere, and solidifying at 4deg.C overnight;
s140: and (3) removing the excessive calcium chloride solution in the step (S130), adding pure water, standing and washing for 3 times, and centrifuging to remove the aqueous solution to obtain the pepsin immobilized microsphere.
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