CN116396380A - Method for extracting cow leather collagen for preparing antibody - Google Patents
Method for extracting cow leather collagen for preparing antibody Download PDFInfo
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- CN116396380A CN116396380A CN202310528403.2A CN202310528403A CN116396380A CN 116396380 A CN116396380 A CN 116396380A CN 202310528403 A CN202310528403 A CN 202310528403A CN 116396380 A CN116396380 A CN 116396380A
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- 102000008186 Collagen Human genes 0.000 title claims abstract description 78
- 108010035532 Collagen Proteins 0.000 title claims abstract description 78
- 229920001436 collagen Polymers 0.000 title claims abstract description 78
- 239000010985 leather Substances 0.000 title claims abstract description 27
- 238000000034 method Methods 0.000 title claims description 16
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 58
- 239000000243 solution Substances 0.000 claims abstract description 55
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 42
- 239000002244 precipitate Substances 0.000 claims abstract description 32
- 102000057297 Pepsin A Human genes 0.000 claims abstract description 31
- 108090000284 Pepsin A Proteins 0.000 claims abstract description 31
- 229940111202 pepsin Drugs 0.000 claims abstract description 31
- 238000000605 extraction Methods 0.000 claims abstract description 30
- 239000011780 sodium chloride Substances 0.000 claims abstract description 29
- 238000005185 salting out Methods 0.000 claims abstract description 27
- 239000012535 impurity Substances 0.000 claims abstract description 25
- 239000012153 distilled water Substances 0.000 claims abstract description 23
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 23
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims abstract description 14
- 239000008055 phosphate buffer solution Substances 0.000 claims abstract description 14
- 239000006228 supernatant Substances 0.000 claims abstract description 14
- 239000000047 product Substances 0.000 claims abstract description 12
- 239000002994 raw material Substances 0.000 claims abstract description 5
- 241000283690 Bos taurus Species 0.000 claims description 29
- 239000007788 liquid Substances 0.000 claims description 27
- 238000004140 cleaning Methods 0.000 claims description 22
- 239000007787 solid Substances 0.000 claims description 21
- 238000009777 vacuum freeze-drying Methods 0.000 claims description 14
- 229910019142 PO4 Inorganic materials 0.000 claims description 13
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 13
- 239000010452 phosphate Substances 0.000 claims description 13
- 210000004003 subcutaneous fat Anatomy 0.000 claims description 11
- 238000009966 trimming Methods 0.000 claims description 11
- 238000002791 soaking Methods 0.000 claims description 8
- 238000000502 dialysis Methods 0.000 claims description 5
- 230000008569 process Effects 0.000 claims description 5
- 238000005406 washing Methods 0.000 claims description 2
- 230000016784 immunoglobulin production Effects 0.000 claims 3
- 102000004190 Enzymes Human genes 0.000 abstract description 26
- 108090000790 Enzymes Proteins 0.000 abstract description 26
- 229940088598 enzyme Drugs 0.000 abstract description 26
- 102000004169 proteins and genes Human genes 0.000 abstract description 6
- 108090000623 proteins and genes Proteins 0.000 abstract description 6
- 230000002378 acidificating effect Effects 0.000 abstract description 3
- 230000004071 biological effect Effects 0.000 abstract description 3
- 238000005119 centrifugation Methods 0.000 abstract description 3
- 241000894006 Bacteria Species 0.000 abstract description 2
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- 230000000052 comparative effect Effects 0.000 description 18
- 238000004108 freeze drying Methods 0.000 description 9
- 238000004321 preservation Methods 0.000 description 8
- 239000004365 Protease Substances 0.000 description 4
- 108090000526 Papain Proteins 0.000 description 3
- 102000004142 Trypsin Human genes 0.000 description 3
- 108090000631 Trypsin Proteins 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 230000007071 enzymatic hydrolysis Effects 0.000 description 3
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 3
- 229940055729 papain Drugs 0.000 description 3
- 235000019834 papain Nutrition 0.000 description 3
- 239000004753 textile Substances 0.000 description 3
- 239000012588 trypsin Substances 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 2
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- 241001465754 Metazoa Species 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
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Classifications
-
- 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, cold insoluble globulin [CIG]
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/10—Process efficiency
Abstract
The invention relates to the technical field of collagen extraction, and discloses an extraction method of cow leather collagen for preparing antibodies, which comprises the following steps: step 1, carrying out enzymolysis on a pretreated cow leather raw material by using a phosphate buffer solution containing pepsin, and carrying out enzymolysis for 8-10 hours at the pH=2.0-2.5 and the temperature of 35-39 ℃; step 2, centrifuging the obtained product after enzymolysis, taking supernatant, and adding NaCl for salting out; salting out and centrifuging, wherein the precipitate is crude collagen; dissolving crude collagen in acetic acid solution, dialyzing in disodium hydrogen phosphate solution, and dialyzing in distilled water to obtain cow leather collagen. The invention only uses a single enzyme to carry out enzymolysis under an acidic condition, so that the operation flow is more convenient while the efficiency is not reduced, and impurities such as bacteria, viruses, endotoxin, impurity proteins and the like are removed by means of repeated centrifugation, chromatography and the like, and the collagen with reserved biological activity is extracted.
Description
Technical Field
The invention belongs to the technical field of collagen extraction, and particularly relates to an extraction method of cow leather collagen for preparing antibodies.
Background
Collagen is a structural protein widely existing in extracellular matrixes of human and animals, is also a functional protein, is closely related to cell proliferation, differentiation, exercise, immunity, joint lubrication, wound healing and the like, and is widely applied to the fields of biomedical research, bioengineering, biological medicine, cosmetics, chemical industry, food and the like. China is one of countries using textiles earlier, but leather textiles have the problem of difficult detection. The immunodetection method in the biological field can effectively solve the problem, and the method for extracting the collagen of the cowhide to prepare the antibody is important in the process of detecting leather textile cultural relics by using specific immunity.
Disclosure of Invention
In view of the above, the present invention provides an extraction method of bovine collagen for preparing antibodies, comprising the steps of:
According to the invention, after the step 2 is performed with vacuum freeze drying, the cowhide is crushed, so that the enzymolysis is more complete, and the enzymolysis time can be reduced. In the invention, pepsin is used for enzymolysis in the step 3, because the pepsin has the best enzymolysis effect on the cow leather. Too low an enzymolysis temperature can reduce the enzymolysis efficiency, while too high an enzymolysis temperature can inactivate collagen and lose the extraction value; too short a time for enzymolysis can lead to pepsin not yet reaching the purpose of destroying the cell structure; too low or too high a pH can cause inactivation of the enzyme, and can also cause denaturation and inactivation of collagen; the enzyme adding amount and the feed liquid ratio can influence the enzymolysis speed, the speed is reduced due to too low, and waste is caused due to too high. Salting out uses neutral salt to destroy two factors of stable existence of protein in solution, so that protein is precipitated; dialysis can remove contaminants, particularly heavy metals and proteases, which have deleterious effects on proteins.
Further preferably, in step 1, the enzyme addition amount of the pepsin-containing phosphate buffer solution is 2.0-4.0wt%.
Further preferably, in step 1, the mass-to-volume ratio of the feed liquid in the enzymolysis process is 1:20-40.
Further preferably, in step 1, the pretreatment is: cleaning cowhide, cleaning subcutaneous fat and surface hair of the cowhide, trimming into small blocks, soaking in phosphate solution, washing, and draining; and then carrying out vacuum freeze drying, crushing, soaking in NaCl solution, and removing salt-soluble impurities to obtain solid precipitate, namely the pretreated cow leather raw material.
Further preferably, the salt-soluble impurities are removed and centrifuged at 5000-8000r/min for 4-15min to obtain a solid precipitate.
Further preferably, the pH of the phosphate solution is 5-8; the vacuum freeze drying time is 12-48h; the mass-volume ratio of the feed liquid in the process of soaking in the NaCl solution is 0.5-3:45-60, the mass concentration of the NaCl solution is 6-18%, and the soaking time is 12-14h.
Further preferably, in the step 2, the centrifugation is 8000-12000r/min for 7-20min.
Further preferably, in step 2, salting out is performed when NaCl is added to the supernatant to 2 to 5 mol/L.
Further preferably, in the step 2, the concentration of the acetic acid solution is 0.3-0.9mol/L.
Further preferably, in the step 2, the concentration of the disodium hydrogen phosphate is 0.01-0.1mol/L, and the dialysis time is 1-4d; the dialysis time in the distilled water is 1-4d.
Compared with the prior art, the invention has the following advantages: only a single enzyme is used for enzymolysis under an acidic condition, the operation flow is more convenient while the efficiency is not reduced, and impurities such as bacteria, viruses, endotoxin, impurity proteins and the like are removed by means of repeated centrifugation, chromatography and the like, so that the collagen with reserved biological activity is extracted.
Drawings
FIG. 1 shows the collagen extraction rates of the different enzymes of comparative example 1 under respective enzymatic hydrolysis conditions;
FIG. 2 shows the collagen extraction yield of example 2 and example 3
FIG. 3 shows the collagen extraction rate of pigskin under different pH conditions of enzymolysis in comparative example 2;
FIG. 4 shows the collagen extraction rate of pigskin under different enzymolysis temperature conditions in comparative example 3;
FIG. 5 shows the collagen extraction rate of pigskin under different enzymolysis time conditions in comparative example 4;
FIG. 6 shows the collagen extraction rate of pigskin under different enzyme dosage conditions in comparative example 5;
FIG. 7 shows the collagen extraction rate of pigskin under different feed ratios in comparative example 6.
Detailed Description
The technical contents and effects of the present invention will be further described in detail with reference to examples, but the present invention is not limited thereto.
Example 1
An extraction method of cow leather collagen for preparing antibody, comprising the following steps:
step 1: after cleaning fresh cowhide, cleaning subcutaneous fat and surface hair of cowhide with a surgical knife, and trimming pigskin into square blocks of 1cm×1cm with scissors. After the cowhide is soaked in phosphate solution with pH=7.5, the cowhide is washed by distilled water for 4 times, and is subjected to vacuum freeze drying for 48 hours after draining, and is soaked in 10wt% NaCl solution for 12 hours according to a feed liquid ratio of 1:50 (w/v) after being crushed, so that salt-soluble impurities are removed. After removing impurities, centrifuging for 10min at 8000r/min to obtain a solid precipitate.
Step 2: the solid precipitate is subjected to enzymolysis by pepsin, and the ratio of the feed to the liquid is 1:20 (w/v) in a phosphate buffer solution containing pepsin with an enzyme content of 2.0wt%, at a pH=2.0 and a temperature of 37℃for 8 hours.
Step 3: centrifuging the obtained product for 10min at 8000r/min after enzymolysis, collecting supernatant, adding NaCl to 4mol/L, and salting out; centrifuging for 10min at 8000r/min after salting out, and reserving the precipitate to obtain crude collagen. Dissolving crude collagen in 0.5mol/L acetic acid solution, dialyzing in 0.02mol/L disodium hydrogen phosphate solution for 2d, dialyzing in distilled water for 1d to obtain cow leather collagen, and freeze drying for preservation.
Example 2
Step 1: after cleaning fresh cowhide, cleaning subcutaneous fat and surface hair of cowhide with a surgical knife, and trimming pigskin into square blocks of 1cm×1cm with scissors. After the cowhide is soaked in phosphate solution with pH=7.5, the cowhide is washed by distilled water for 4 times, and is subjected to vacuum freeze drying for 48 hours after draining, and is soaked in 10wt% NaCl solution for 12 hours according to a feed liquid ratio of 1:50 (w/v) after being crushed, so that salt-soluble impurities are removed. After removing impurities, the mixture is centrifuged for 5min at 4000r/min to obtain a solid precipitate.
Step 2: the solid precipitate is subjected to enzymolysis by pepsin, and the ratio of the feed to the liquid is 1:20 (w/v) in a phosphate buffer solution containing pepsin with an enzyme content of 2.0wt%, at a pH=2.0 and a temperature of 37℃for 8 hours.
Step 3: centrifuging the obtained product for 10min at 8000r/min after enzymolysis, collecting supernatant, adding NaCl to 4mol/L, and salting out; centrifuging at 4000r/min for 5min after salting out, and reserving sediment to obtain crude collagen. Dissolving crude collagen in 0.5mol/L acetic acid solution, dialyzing in 0.02mol/L disodium hydrogen phosphate solution for 2d, dialyzing in distilled water for 1d to obtain cow leather collagen, and freeze drying for preservation.
Example 3
Step 1: after cleaning fresh cowhide, cleaning subcutaneous fat and surface hair of cowhide with a surgical knife, and trimming pigskin into square blocks of 1cm×1cm with scissors. After the cowhide is soaked in phosphate solution with pH=7.5, the cowhide is washed by distilled water for 4 times, and is subjected to vacuum freeze drying for 48 hours after draining, and is soaked in 5wt% NaCl solution for 12 hours according to a feed liquid ratio of 1:50 (w/v) after being crushed, so that salt-soluble impurities are removed. After removing impurities, centrifuging for 10min at 8000r/min to obtain a solid precipitate.
Step 2: the solid precipitate is subjected to enzymolysis by pepsin, and the ratio of the feed to the liquid is 1:20 (w/v) in a phosphate buffer solution containing pepsin with an enzyme content of 2.0wt%, at a pH=2.0 and a temperature of 37℃for 8 hours.
Step 3: centrifuging the obtained product for 10min at 8000r/min after enzymolysis, collecting supernatant, adding NaCl to 2mol/L, and salting out; centrifuging for 10min at 8000r/min after salting out, and reserving the precipitate to obtain crude collagen. Dissolving crude collagen in 0.3mol/L acetic acid solution, dialyzing in 0.01mol/L disodium hydrogen phosphate solution for 2d, dialyzing in distilled water for 1d, and freeze drying and preserving to obtain cow leather collagen.
Comparative example 1 (differing from example 1 in the use of trypsin or papain)
Step 1: after cleaning fresh cowhide, cleaning subcutaneous fat and surface hair of cowhide with a surgical knife, and trimming pigskin into square blocks of 1cm×1cm with scissors. After the cowhide is soaked in phosphate solution with pH=7.5, the cowhide is washed by distilled water for 4 times, and is subjected to vacuum freeze drying for 48 hours after draining, and is soaked in 10wt% NaCl solution for 12 hours according to a feed liquid ratio of 1:50 (w/v) after being crushed, so that salt-soluble impurities are removed. After removing impurities, centrifuging for 10min at 8000r/min to obtain a solid precipitate.
Step 2: carrying out enzymolysis on the solid precipitate by using trypsin or papain, wherein the ratio of the feed to the liquid is 1:20 (w/v) in a phosphate buffer solution containing pepsin with an enzyme content of 2.0wt%, wherein the reaction condition of trypsin is pH=8.0, the temperature is 42 ℃, and the enzymolysis is carried out for 8 hours (the reaction condition of papain is pH=6.0, the temperature is 55 ℃, and the enzymolysis is carried out for 8 hours);
step 3: centrifuging the obtained product for 10min at 8000r/min after enzymolysis, collecting supernatant, adding NaCl to 4mol/L, and salting out; centrifuging for 10min at 8000r/min after salting out, and reserving the precipitate to obtain crude collagen. Dissolving crude collagen in 0.5mol/L acetic acid solution, dialyzing in 0.02mol/L disodium hydrogen phosphate solution for 2d, dialyzing in distilled water for 1d to obtain cow leather collagen, and freeze drying for preservation.
Comparative example 2 (the difference from example 1 is the pH at the time of enzymatic hydrolysis)
Step 1: after cleaning fresh cowhide, cleaning subcutaneous fat and surface hair of cowhide with a surgical knife, and trimming pigskin into square blocks of 1cm×1cm with scissors. After the cowhide is soaked in phosphate solution with pH=7.5, the cowhide is washed by distilled water for 4 times, and is subjected to vacuum freeze drying for 48 hours after draining, and is soaked in 10wt% NaCl solution for 12 hours according to a feed liquid ratio of 1:50 (w/v) after being crushed, so that salt-soluble impurities are removed. After removing impurities, centrifuging for 10min at 8000r/min to obtain a solid precipitate.
Step 2: the solid precipitate is subjected to enzymolysis by pepsin, and the ratio of the feed to the liquid is 1:20 (w/v) in a phosphate buffer solution containing pepsin with an enzyme content of 2.0wt%, respectively, carrying out enzymolysis for 8 hours at 37 ℃ under the conditions of pH values of 1.5, 2.0, 2.5, 3.0 and 3.5.
Step 3: centrifuging the obtained product for 10min at 8000r/min after enzymolysis, collecting supernatant, adding NaCl to 4mol/L, and salting out; centrifuging for 10min at 8000r/min after salting out, and reserving the precipitate to obtain crude collagen. Dissolving crude collagen in 0.5mol/L acetic acid solution, dialyzing in 0.02mol/L disodium hydrogen phosphate solution for 2d, dialyzing in distilled water for 1d to obtain cow leather collagen, and freeze drying for preservation.
Comparative example 3 (the difference from example 1 is the temperature at which the enzyme is digested)
Step 1: after cleaning fresh cowhide, cleaning subcutaneous fat and surface hair of cowhide with a surgical knife, and trimming pigskin into square blocks of 1cm×1cm with scissors. After the cowhide is soaked in phosphate solution with pH=7.5, the cowhide is washed by distilled water for 4 times, and is subjected to vacuum freeze drying for 48 hours after draining, and is soaked in 10wt% NaCl solution for 12 hours according to a feed liquid ratio of 1:50 (w/v) after being crushed, so that salt-soluble impurities are removed. After removing impurities, centrifuging for 10min at 8000r/min to obtain a solid precipitate.
Step 2: the solid precipitate is subjected to enzymolysis by pepsin, and the ratio of the feed to the liquid is 1:20 (w/v) in a pepsin-containing phosphate buffer solution having an enzyme content of 2.0wt%, at ph=2.0, the enzyme was digested for 8 hours at a temperature of 27 ℃, 32 ℃, 37 ℃, 42 ℃, 47 ℃, respectively.
Step 3: centrifuging the obtained product for 10min at 8000r/min after enzymolysis, collecting supernatant, adding NaCl to 4mol/L, and salting out; centrifuging for 10min at 8000r/min after salting out, and reserving the precipitate to obtain crude collagen. Dissolving crude collagen in 0.5mol/L acetic acid solution, dialyzing in 0.02mol/L disodium hydrogen phosphate solution for 2d, dialyzing in distilled water for 1d to obtain cow leather collagen, and freeze drying for preservation.
Comparative example 4 (the difference from example 1 is the time at which the enzyme is digested)
Step 1: after cleaning fresh cowhide, cleaning subcutaneous fat and surface hair of cowhide with a surgical knife, and trimming pigskin into square blocks of 1cm×1cm with scissors. After the cowhide is soaked in phosphate solution with pH=7.5, the cowhide is washed by distilled water for 4 times, and is subjected to vacuum freeze drying for 48 hours after draining, and is soaked in 10wt% NaCl solution for 12 hours according to a feed liquid ratio of 1:50 (w/v) after being crushed, so that salt-soluble impurities are removed. After removing impurities, centrifuging for 10min at 8000r/min to obtain a solid precipitate.
Step 2: the solid precipitate is subjected to enzymolysis by pepsin, and the ratio of the feed to the liquid is 1:20 (w/v) in a phosphate buffer solution containing pepsin with an enzyme content of 2.0wt%, respectively performing enzymolysis for 2h, 4h, 6h, 8h and 10h at a pH=2.0 and a temperature of 37 ℃.
Step 3: centrifuging the obtained product for 10min at 8000r/min after enzymolysis, collecting supernatant, adding NaCl to 4mol/L, and salting out; centrifuging for 10min at 8000r/min after salting out, and reserving the precipitate to obtain crude collagen. Dissolving crude collagen in 0.5mol/L acetic acid solution, dialyzing in 0.02mol/L disodium hydrogen phosphate solution for 2d, dialyzing in distilled water for 1d to obtain cow leather collagen, and freeze drying for preservation.
Comparative example 5 (the difference from example 1 is the amount of enzyme added during the enzymatic hydrolysis)
Step 1: after cleaning fresh cowhide, cleaning subcutaneous fat and surface hair of cowhide with a surgical knife, and trimming pigskin into square blocks of 1cm×1cm with scissors. After the cowhide is soaked in phosphate solution with pH=7.5, the cowhide is washed by distilled water for 4 times, and is subjected to vacuum freeze drying for 48 hours after draining, and is soaked in 10wt% NaCl solution for 12 hours according to a feed liquid ratio of 1:50 (w/v) after being crushed, so that salt-soluble impurities are removed. After removing impurities, centrifuging for 10min at 8000r/min to obtain a solid precipitate.
Step 2: the solid precipitate is subjected to enzymolysis by pepsin, and the ratio of the feed to the liquid is 1:20 The enzyme addition amounts (w/v) were 1wt%, 2wt%, 3wt%, 4wt% and 5wt% respectively in pepsin-containing phosphate buffer solution, and the enzyme was hydrolyzed at 37℃for 8 hours at pH=2.0.
Step 3: centrifuging the obtained product for 10min at 8000r/min after enzymolysis, collecting supernatant, adding NaCl to 4mol/L, and salting out; centrifuging for 10min at 8000r/min after salting out, and reserving the precipitate to obtain crude collagen. Dissolving crude collagen in 0.5mol/L acetic acid solution, dialyzing in 0.02mol/L disodium hydrogen phosphate solution for 2d, dialyzing in distilled water for 1d to obtain cow leather collagen, and freeze drying for preservation.
Comparative example 6 (the difference from example 1 is the ratio of feed to liquid during enzymolysis)
Step 1: after cleaning fresh cowhide, cleaning subcutaneous fat and surface hair of cowhide with a surgical knife, and trimming pigskin into square blocks of 1cm×1cm with scissors. After the cowhide is soaked in phosphate solution with pH=7.5, the cowhide is washed by distilled water for 4 times, and is subjected to vacuum freeze drying for 48 hours after draining, and is soaked in 10wt% NaCl solution for 12 hours according to a feed liquid ratio of 1:50 (w/v) after being crushed, so that salt-soluble impurities are removed. After removing impurities, centrifuging for 10min at 8000r/min to obtain a solid precipitate.
Step 2: the solid precipitate was subjected to enzymolysis with pepsin in a phosphate buffer solution containing pepsin at a feed liquid ratio of 1:10, 1:20, 1:30, 1:40, 1:50 (w/v) and an enzyme addition amount of 2.0wt%, at a temperature of 37 ℃ for 8 hours at ph=2.0.
Step 3: centrifuging the obtained product for 10min at 8000r/min after enzymolysis, collecting supernatant, adding NaCl to 4mol/L, and salting out; centrifuging for 10min at 8000r/min after salting out, and reserving the precipitate to obtain crude collagen. Dissolving crude collagen in 0.5mol/L acetic acid solution, dialyzing in 0.02mol/L disodium hydrogen phosphate solution for 2d, dialyzing in distilled water for 1d to obtain cow leather collagen, and freeze drying for preservation.
As shown in FIG. 1, comparative example 1 shows that pepsin reacts with cow leather better than the other two enzymes, and the hydrolysis degree is more thorough, so that the collagen extraction rate is highest among the three.
The collagen extraction yield obtained in examples 2-3 is shown in FIG. 2.
As shown in FIG. 3, comparative example 2 shows that the collagen extraction rate shows a tendency of a gradual decrease with increasing pH between 1.5 and 2.5, and the pepsin extraction rate is highest at pH between 2.0 and 2.5.
As shown in FIG. 4, comparative example 3 shows that the collagen extraction rate shows a tendency of a gradual decrease with increasing temperature in the range of 27-47 ℃, and the extraction rate of pepsin to collagen is highest at about 37 ℃.
As shown in FIG. 5, comparative example 4 shows that the extraction rate of collagen from pigskin increases slowly with the prolongation of the enzymolysis time between 2 and 6 hours, and increases rapidly between 6 and 8 hours, and the extraction rate after 8 hours becomes gradually gentle, so that the extraction rate of collagen from pepsin is higher with the enzymolysis time between 8 and 12 hours.
As shown in FIG. 6, comparative example 5 shows that with the change of the enzyme addition amount, the collagen extraction rate shows the change rule of the rising and falling between 1% and 5% of the enzyme addition amount, and the collagen extraction rate is higher and can reach more than 40% when the enzyme addition amount is 2-4%.
As shown in fig. 7, comparative example 6 shows that the collagen extraction rate shows a change rule of a rising and falling with an increase of the feed-liquid ratio. In the range of 1:20-1:40, the extraction rate can still be improved along with the increase of the feed-liquid ratio, but the more preferable feed-liquid ratio is 1:20, and then the cost can be increased along with the lower increase amplitude.
In conclusion, the invention only uses a single enzyme to carry out enzymolysis under an acidic condition, the efficiency is not reduced, meanwhile, the collagen with the reserved biological activity is extracted, the extraction rate of the collagen by pepsin during enzymolysis under different conditions is greatly different, and under the cooperation of the limiting conditions of the invention, the extraction rate of the pepsin to the collagen can reach a higher level.
The above examples of the present invention are merely illustrative of the present invention and are not intended to limit the embodiments of the present invention. Other variations and modifications of the present invention will be apparent to those of ordinary skill in the art in light of the foregoing description. Not all embodiments are exhaustive. Obvious changes and modifications which are extended by the technical proposal of the invention are still within the protection scope of the invention.
Claims (8)
1. An extraction method of cow leather collagen for preparing antibody is characterized by comprising the following steps:
step 1, carrying out enzymolysis on a pretreated cow leather raw material by using a phosphate buffer solution containing pepsin, and carrying out enzymolysis for 8-10 hours at the pH=2.0-2.5 and the temperature of 35-39 ℃;
step 2, centrifuging the obtained product after enzymolysis, taking supernatant, and adding NaCl for salting out; salting out and centrifuging, wherein the precipitate is crude collagen; dissolving crude collagen in acetic acid solution, dialyzing in disodium hydrogen phosphate solution, and dialyzing in distilled water to obtain cow leather collagen.
2. The method for extracting bovine collagen from antibody according to claim 1, wherein the pepsin-containing phosphate buffer solution is added in an amount of 2.0 to 4.0wt% in step 1.
3. The method for extracting bovine collagen from antibody production according to claim 1, wherein in step 1, the mass-to-volume ratio of the feed liquid in the enzymolysis process is 1:20-40.
4. The method for extracting bovine collagen for antibody production according to any one of claims 1 to 3, wherein in step 1, the pretreatment is: cleaning cowhide, cleaning subcutaneous fat and surface hair of the cowhide, trimming into small blocks, soaking in phosphate solution, washing, and draining; and then carrying out vacuum freeze drying, crushing, soaking in NaCl solution, and removing salt-soluble impurities to obtain solid precipitate, namely the pretreated cow leather raw material.
5. The method for extracting bovine collagen from antibody according to claim 4, wherein the pH of the phosphate solution is 5-8; the vacuum freeze drying time is 12-48h; the mass-volume ratio of the feed liquid in the process of soaking in the NaCl solution is 0.5-3:45-60, the mass concentration of the NaCl solution is 6-18%, and the soaking time is 12-14h.
6. The method for extracting bovine collagen for antibody production according to claim 1, wherein in step 2, salting out is performed while adding NaCl to 2-5mol/L in the supernatant.
7. The method for extracting bovine collagen from antibody according to claim 1, wherein the concentration of the acetic acid solution in the step 2 is 0.3-0.9mol/L.
8. The method for extracting bovine collagen from antibody according to claim 1, wherein the concentration of disodium hydrogen phosphate is 0.01-0.1mol/L and the dialysis time is 1-4d in the step 2; the dialysis time in the distilled water is 1-4d.
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