CN111647039A - Method for extracting sheep embryo element active peptide - Google Patents
Method for extracting sheep embryo element active peptide Download PDFInfo
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- CN111647039A CN111647039A CN202010645994.8A CN202010645994A CN111647039A CN 111647039 A CN111647039 A CN 111647039A CN 202010645994 A CN202010645994 A CN 202010645994A CN 111647039 A CN111647039 A CN 111647039A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2/00—Peptides of undefined number of amino acids; Derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/02—Peptides of undefined number of amino acids; Derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
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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
- C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
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- C07K1/14—Extraction; Separation; Purification
- C07K1/30—Extraction; Separation; Purification by precipitation
- C07K1/303—Extraction; Separation; Purification by precipitation by salting out
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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
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- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
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Abstract
The invention relates to an extraction method of sheep embryo element active peptide, which comprises the following steps: taking out sheep embryo from mother body of Tibetan goat, cleaning and sterilizing, taking out viscera, umbilical cord and submaxillary gland of sheep embryo in sterile room below 4 deg.C, homogenizing for 4-6 min, adding into saline with pH of 2.5-3.5, stirring and extracting for 3.5-4.5h, centrifuging, and collecting supernatant; adding ammonium sulfate, standing overnight, collecting precipitate, dissolving the precipitate with 4-6 times of saline, nano-filtering, concentrating, and lyophilizing to obtain the final product. The extraction method has the advantages of simple process, high product yield, strong activity of the obtained active peptide and high purity.
Description
Technical Field
The invention belongs to the technical field of biology, relates to extraction of sheep embryo active substances, and particularly relates to an extraction method of sheep embryo bioactive peptide.
Background
Since 1912, the first discovery by professor karl, a substance (called sheep embryo element) capable of restoring cell activity in sheep embryo cells, was made by swiss scientists. Researchers all over the world are studying the components and functions of sheep embryo extract, and great achievements are achieved. The method is basically in the experimental stage in China, the process is complicated, the large-scale production is inconvenient, the yield is low, and the activity of the obtained active peptide is not high and is easy to activate.
The present invention has been made in view of the above circumstances.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides the method for extracting the sheep embryo bioactive peptide.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for extracting sheep embryo bioactive peptide comprises the following steps:
(1) taking out sheep embryos from the mother body of the Tibetan goat, washing and sterilizing, taking out the viscera, umbilical cord and submaxillary gland of the taken-out sheep embryos in a sterile room, homogenizing at low temperature for 4.5-5.5 minutes, then putting into saline with pH of 2.5-3.5, stirring and extracting for 3.5-4.5 hours, centrifuging, and collecting the supernatant;
(2) and (2) adding ammonium sulfate into the supernatant obtained in the step (1), saturating and standing overnight, collecting the precipitate, dissolving the precipitate with 4-6 times of saline water, performing nanofiltration, and freeze-drying to obtain the sheep embryo element active peptide.
The sheep embryo element active peptide freeze-dried powder obtained by the invention is preferably stored at a low temperature below 0 ℃ and a low temperature below-18 ℃, and can not be inactivated for 2 years.
Further, in the step (1), the zang goat is a zang goat living at an altitude of more than 3000 m and pregnant for more than 10 weeks.
Further, in the step (1), 75% alcohol is adopted for disinfection.
Further, in the step (1), glacial acetic acid is adopted to adjust the pH value of the brine.
Further, in the step (1) and the step (2), the saline water is NaCl solution, and the concentration of the saline water is 0.04-0.06 mol/L.
Further, in the step (1), 10 times of saline having a pH of 3.0 and a concentration of 0.05mol/L was added.
Further, in the step (1), the temperature of the homogenate is 0 to 4 ℃. The homogenization time is not too long, and the heat is slowly generated after more than 5 minutes; the time is less than 5 minutes, the cell disruption is incomplete, and the extraction rate is not high.
Further, in the step (1), the viscera, umbilical cord and submandibular gland of the removed sheep embryo are removed in a sterile room at 4 ℃ or lower, cooled to 0 ℃, and then homogenized at 0-4 ℃ for 5 minutes. During extraction, the temperature in the whole process is not more than 4 ℃, and the loss of active substances is reduced.
Further, in the step (1), the rotation speed of the homogenate is 11500 and 12500rpm, and the homogenization time is 5 min.
Further, the rotational speed of the homogenate was 12000 revolutions, and the time of the homogenate was 5 minutes.
Further, in the step (2), the nanofiltration is used for intercepting active substances with the molecular weight of 100-10000Da to form the sheep embryo extract stock solution. Microfiltration is carried out before nanofiltration.
The raw material of the sheep embryo extract extracted by the invention is from a pollution-free Tibetan goat in a plateau in a snow region, and the Tibetan goat has extremely strong life immunity due to severe climate, so the sheep embryo extract is an ideal raw material for extracting the sheep embryo extract.
Compared with the prior art, the invention has the beneficial effects that:
(1) the method for extracting the sheep embryo element active peptide adopts high-speed homogenate, and the acidic saline water is extracted at low temperature, so that the acidic protein precipitation is facilitated, and the extract (mainly immunoglobulin and anti-aging factors) has higher activity and higher yield;
(2) saturated precipitation extraction by adopting ammonium sulfate is simple to operate;
(3) nanofiltration concentration is adopted, interception and extraction are carried out according to the molecular weight section of the active substance, so that inorganic salt, water and macromolecular hybrid protein can be removed, the yield can be improved, and the working efficiency can be improved;
(4) the sheep embryo element active peptide extracted by the invention can play an important role in the medical treatment and beauty industry, can be used for treating stomachache and gastric ulcer, is used for beauty and skin care, and has the effects of removing wrinkles, tendering skin, ruddy skin and the like;
(5) the extraction method of the sheep embryo element active peptide has the advantages of high yield, strong activity of the obtained active peptide, high purity, low cost and small loss, and can be used for large-scale production.
(6) The sheep embryo element active peptide extracted by the invention is freeze-dried and then stored at low temperature, and the inactivation can be avoided for 2 years.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.
The saline solution in the invention is NaCl solution, and the solution in the invention is aqueous solution.
Example 1
The method for extracting the sheep embryo bioactive peptide comprises the following steps:
(1) taking out sheep embryos from the mother body of the Tibetan goat, washing and disinfecting the sheep embryos by using alcohol, taking out the viscera, umbilical cord and submandibular gland of the taken-out sheep embryos in a sterile room, homogenizing at the low temperature of below 10 ℃ for 4.5 minutes, then putting the sheep embryos in saline with the pH value of 3.0, stirring and extracting for 4 hours, centrifuging, and collecting supernatant;
(2) and (2) adding ammonium sulfate into the supernatant obtained in the step (1), saturating and standing overnight, collecting the precipitate, dissolving the precipitate with 5 times of saline, performing nanofiltration, and performing freeze-drying to obtain the sheep embryo element active peptide.
Example 2
The method for extracting the sheep embryo bioactive peptide comprises the following steps:
(1) taking out sheep embryos from the parents of Tibetan goats living at an altitude of more than 3000 meters and more than 10 weeks of pregnancy, washing and disinfecting the sheep embryos by using 75% alcohol, taking out the viscera, umbilical cords and submandibular glands of the taken-out sheep embryos in a sterile room at a temperature of less than 4 ℃, cooling the viscera, umbilical cords and submandibular glands to 0 ℃, homogenizing at a temperature of 0-4 ℃ for 4.5 minutes at a rotation speed of 12500rpm, wherein the temperature in the whole process is not more than 4 ℃ during extraction, and the loss of active substances is reduced; then putting the mixture into 10 times of saline water with the pH value of 2.5 and the concentration of 0.06mol/L, stirring and extracting for 4.5h, centrifuging and collecting supernate;
(2) adding ammonium sulfate into the supernatant obtained in the step (1), saturating and standing overnight, collecting precipitate, dissolving the precipitate with 4 times of saline water, performing microfiltration and impurity removal by using a filter membrane with the pore diameter of 0.22 micrometer to obtain filtrate, performing nanofiltration to intercept active substances with the molecular weight of 100-10000Da to form sheep embryo element stock solution, and performing freeze-drying to obtain freeze-dried powder, namely the sheep embryo element active peptide.
Example 3
The method for extracting the sheep embryo bioactive peptide comprises the following steps:
(1) taking out sheep embryos from a mother body of a Tibetan goat living at an altitude of more than 3000 m and more than 10 weeks of pregnancy, washing and disinfecting the sheep embryos by using 75% alcohol, taking out the liver, lung, heart, spleen, heart gland, kidney, umbilical cord and submandibular gland of the taken-out sheep embryos from a sterile room at 2 ℃, cooling the liver, lung, heart, spleen, heart gland, kidney, umbilical cord and submandibular gland to 0 ℃, then homogenizing at 0-4 ℃ for 5 minutes at the rotation speed of 12000rpm, wherein during extraction, the temperature (including the temperature of homogenizing) in the whole process is not more than 4 ℃, and the loss of active substances is reduced; then putting into 10 times of saline water with pH of 3.0 and concentration of 0.05mol/L, adjusting pH of the saline water with glacial acetic acid, stirring and extracting for 4h, centrifuging, and collecting supernatant;
(2) and (2) adding ammonium sulfate into the supernatant obtained in the step (1), saturating and standing overnight, collecting the precipitate, dissolving the precipitate with 5 times of saline water, performing microfiltration to remove impurities, performing nanofiltration to intercept active substances with the molecular weight of 100-10000Da to form a sheep embryo extract stock solution, concentrating, and performing freeze-drying to obtain the sheep embryo extract active peptide.
Comparative example 1
The extraction method of the sheep embryo bioactive peptide of the comparison example is only different from the extraction method of the example 3 in that the homogenization time is 3.5 minutes.
Comparative example 2
The extraction method of the sheep embryo bioactive peptide of the comparison example is only different from the extraction method of the example 3 in that the temperature of the homogenate is 4-8 ℃.
Comparative example 3
The extraction method of the sheep embryo bioactive peptide of the present comparative example is different from the extraction method of example 3 only in that the pH of the saline was not adjusted, but the saline was prepared with NaCl and water to have a concentration of 0.05 mol/L.
Test example 1
The sheep embryo bioactive peptides prepared in examples 1 to 3 and comparative examples 1 to 3 were analyzed for their properties, and the results are shown in Table 1.
TABLE 1
Performance of | Example 1 | Example 2 | Example 3 | Comparative example 1 | Comparative example 2 | Comparative example 3 |
Yield (‰) | 3.58 | 3.62 | 3.75 | 2.49 | 1.85 | 0.91 |
Purity (%) | 98.45 | 98.67 | 98.99 | 98.59 | 32.87 | 55.29 |
From table 1, it can be seen that the sheep embryo bioactive peptide obtained by the method of the present invention has high yield and high purity; the homogenization time is short, and the yield is low; the temperature of the homogenate is high or extraction is not carried out with acidic brine, the yield is low and the purity is low.
Test example 2
The lyophilized powder of ovine embryo extract active peptide obtained in example 3 was stored at 0-4 deg.C and-18 deg.C, and the antioxidant activity was measured and compared every month after one year.
The result shows that the sheep embryo element active peptide freeze-dried powder obtained by the invention can be stored at the temperature of 18 ℃ below zero and can not be inactivated for 2 years.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.
Claims (10)
1. The extraction method of the sheep embryo bioactive peptide is characterized by comprising the following steps:
(1) taking out sheep embryos from the mother body of the Tibetan goat, washing and sterilizing, taking out the viscera, umbilical cord and submaxillary gland of the taken-out sheep embryos in a sterile room, homogenizing at low temperature for 4.5-5.5 minutes, then putting into saline with pH of 2.5-3.5, stirring and extracting for 3.5-4.5 hours, centrifuging, and collecting the supernatant;
(2) and (2) adding ammonium sulfate into the supernatant obtained in the step (1), saturating and standing overnight, collecting the precipitate, dissolving the precipitate with 4-6 times of saline water, performing nanofiltration, and freeze-drying to obtain the sheep embryo element active peptide.
2. The method for extracting sheep embryo peptide as claimed in claim 1, wherein in step (1), the zang goat is a zang goat living at an altitude of more than 3000 m and more than 10 weeks gestation.
3. The method for extracting sheep embryo bioactive peptide as claimed in claim 1, wherein in step (1), 75% alcohol is used for sterilization.
4. The method for extracting sheep embryo bioactive peptide as claimed in claim 3, wherein in step (1), glacial acetic acid is used to adjust the pH of saline.
5. The method for extracting sheep embryo bioactive peptide as claimed in claim 1 or 4, wherein the saline solution in step (1) and step (2) is NaCl solution, and the concentration of the saline solution is 0.04-0.06 mol/L.
6. The method for extracting sheep embryo bioactive peptide as claimed in claim 5, wherein in step (1), the peptide is added into 10 times of saline with pH of 3.0 and concentration of 0.05 mol/L.
7. The method for extracting sheep embryo bioactive peptide as claimed in claim 1, wherein the temperature of the homogenate in step (1) is below 10 ℃.
8. The method for extracting ovine embryo bioactive peptide as claimed in claim 7, wherein the viscera, umbilical cord and submandibular gland of the extracted ovine embryo are taken out in a sterile room below 4 ℃, cooled to 0 ℃, and then homogenized at 0-4 ℃ for 5 minutes.
9. The method for extracting bioactive peptides derived from ovine embryo according to claim 1 or 7, wherein in step (1), the rotational speed of the homogenate is 11500-12500rpm, and the time of the homogenate is 5 min.
10. The method for extracting bioactive peptides of ovine embryo extract as claimed in claim 1, wherein in step (2), the nanofiltration is used to cut off active substances with molecular weight of 100-10000Da to form a stock solution of ovine embryo extract.
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Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1242196A (en) * | 1998-07-17 | 2000-01-26 | 西安阳光保健品开发有限责任公司 | Method for preparing goat fetal hormone injection |
CN1333294A (en) * | 2000-07-11 | 2002-01-30 | 张建军 | Novel process for extracting purified sheed fetal agent and method for testing biological activity thereof |
CN102329827A (en) * | 2011-07-12 | 2012-01-25 | 湖州康海斯生物科技有限公司 | Sheep placenta amino acid and low molecular weight polypeptide, and preparation method for sheep placenta amino acid and low molecular weight polypeptide |
CN103265612A (en) * | 2013-05-31 | 2013-08-28 | 中南大学 | Extraction method of sheep embryo active peptide |
KR20180116553A (en) * | 2017-04-17 | 2018-10-25 | 함양군 | Method for producing a Goat ginseng-Ovine placenta cream |
-
2020
- 2020-07-07 CN CN202010645994.8A patent/CN111647039A/en active Pending
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1242196A (en) * | 1998-07-17 | 2000-01-26 | 西安阳光保健品开发有限责任公司 | Method for preparing goat fetal hormone injection |
CN1333294A (en) * | 2000-07-11 | 2002-01-30 | 张建军 | Novel process for extracting purified sheed fetal agent and method for testing biological activity thereof |
CN102329827A (en) * | 2011-07-12 | 2012-01-25 | 湖州康海斯生物科技有限公司 | Sheep placenta amino acid and low molecular weight polypeptide, and preparation method for sheep placenta amino acid and low molecular weight polypeptide |
CN103265612A (en) * | 2013-05-31 | 2013-08-28 | 中南大学 | Extraction method of sheep embryo active peptide |
KR20180116553A (en) * | 2017-04-17 | 2018-10-25 | 함양군 | Method for producing a Goat ginseng-Ovine placenta cream |
Non-Patent Citations (2)
Title |
---|
蔡英姬等: "羊胚胎盘肤的制备及活性测定", 《延边大学医学学报》 * |
陆道培等: "胎盘球蛋白的制备及体内外免疫抑制作用", 《中国实验血液学杂志》 * |
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Application publication date: 20200911 |