CN110616207A - Method for extracting superoxide dismutase from plant - Google Patents
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- CN110616207A CN110616207A CN201911092718.7A CN201911092718A CN110616207A CN 110616207 A CN110616207 A CN 110616207A CN 201911092718 A CN201911092718 A CN 201911092718A CN 110616207 A CN110616207 A CN 110616207A
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0089—Oxidoreductases (1.) acting on superoxide as acceptor (1.15)
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- C12Y115/00—Oxidoreductases acting on superoxide as acceptor (1.15)
- C12Y115/01—Oxidoreductases acting on superoxide as acceptor (1.15) with NAD or NADP as acceptor (1.15.1)
- C12Y115/01001—Superoxide dismutase (1.15.1.1)
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Abstract
The invention discloses a method for extracting superoxide dismutase by using plants as raw materials, which utilizes the characteristic of combination of zinc and protease to activate and extract the superoxide dismutase in active liquid after wall breaking of the plants, thereby obtaining the ideal superoxide dismutase. The extraction method of the invention has low cost, simple process, convenient and practical operation, high activity of the extracted superoxide dismutase, good quality and long activity retention time, and can be directly used as filling raw materials of nutriments, health products, medicines or foods.
Description
Technical Field
The invention relates to a method for extracting superoxide dismutase, in particular to a method for extracting superoxide dismutase by taking plants as raw materials, and belongs to the technical field of biology.
Background
SOD is the abbreviation of superoxide dismutase (superoxide dismutase), the code of the international enzyme commission is EC 1.15.1.1, which belongs to the redox enzyme, the SOD is one of the biological important protective enzymes, can enhance the stress resistance of the organism, prevent aging, can remove the active substance of excessive free radicals in the organism, the substance is closely related to the aging, the tumorigenesis, the autoimmune disease, the radiation protection and the like of the organism, has better curative effect on the diseases of rheumatoid arthritis, cataract, cystitis, skin disease, lupus erythematosus and the like, the SOD has no antigenicity, the adverse reaction is small, the side effect is small, and the SOD is a medicinal enzyme with high medical value and wide prospect.
SOD has been widely distributed, and has been isolated from various organisms such as bacteria, fungi, algae, fish, insects, and mammals, and is classified into three types according to the difference of metal prosthetic group components, the first type is called (Cu.Zn-SOD) containing copper (Cu) and zinc (Zn) metal prosthetic groups, which is green and exists in the cytoplasm of the organism; the second one is called (Mn-SOD) containing manganese (Mn) metal prosthetic group, which is purple and exists in mitochondria and prokaryotic cells of eukaryotic cells; the third one is called (Fe-SOD) containing iron (Fe) metal auxiliary group, which is yellow brown and exists in prokaryotic cell.
However, from the earliest discovery of SOD to the development and application of SOD today, SOD is mostly separated and extracted from animal blood. Therefore, the methods for extracting SOD disclosed in the prior art are mostly animal blood extraction and separation.
Patent application No. 97108883 discloses a method for extracting SOD from earthworms, which comprises the following steps: washing earthworm, purifying, preparing SOD mother liquor from osmotic solution, rough filtering, centrifuging to prepare supernatant, ultrafiltering, reverse osmosis concentrating, freeze drying, etc. patent application No. 86100551 discloses a method for purifying superoxide dismutase (SOD), which comprises heating animal liver homogenate step by step under the condition of cupric salt, dialyzing, precipitating, removing impurities, and extracting SOD; salting out by adopting ammonium sulfate step by step to remove the impurity egg white, collecting the protein containing SOD, dialyzing, concentrating and drying to obtain the SOD with the specific activity of 2789 units/mg, but when the SOD is separated and extracted, the substance needs to be added for a plurality of times and separated again, so that the separation and extraction process is complicated and complicated, chemical pollution is easy to cause, and the scheme is difficult to implement due to the defects of raw materials or methods.
Disclosure of Invention
In view of the above, the present invention provides a method for extracting superoxide dismutase from plants, which utilizes the combination of zinc and protease to activate and extract superoxide dismutase in the active solution after wall breaking of the plants, thereby obtaining the ideal superoxide dismutase.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for extracting superoxide dismutase from plants comprises the following steps:
(1) crushing the plant raw materials to 80-100 meshes of diameter, adding an active protective agent for wall breaking treatment to obtain plant slurry for later use;
(2) keeping the temperature of the plant slurry at 50-55 ℃ for 25-35 min, and performing active proliferation fission;
(3) centrifuging the plant slurry subjected to active proliferation fission, wherein the rotation speed of the centrifugation is 4000-6000 rpm, the centrifugation time is 10-30 min, and removing precipitates to obtain an active solution;
(4) mixing equal amount of zinc gluconate and alkaline protease, dissolving with water, and adding ethanol to obtain mixed solution;
(5) and mixing the mixed solution and the active solution, precipitating at the constant temperature of 20-25 ℃ for 20-28 h, removing the precipitate, and drying at the temperature of-18 to-100 ℃ to obtain the superoxide dismutase.
The extraction method of the invention has low cost, simple process, convenient and practical operation, high activity of the extracted superoxide dismutase, good quality and long activity retention time, and can be directly used as filling raw materials of nutriments, health products, medicines or foods.
Further, the plant material is one or more of okra, sea buckthorn, pine needle and silphium perfoliatum.
The plant materials are common plants, have strong adaptability, high yield, easy purchase, low cost, and rich superoxide dismutase content.
Further, the adding amount of the active protective agent in the step (1) is 0.005-0.02 times of the mass of the plant raw materials, wherein the active protective agent is one or a mixture of more of trehalose, mannitol and lactose.
The activity protectant can effectively maintain the activity of superoxide dismutase in plant material.
Further, in the step (4), "zinc gluconate and alkaline protease are mixed and dissolved with water" is added with water with the mass 60 times of the sum of the mass of the zinc gluconate and the mass of the alkaline protease;
the ethanol is 95-97% in concentration, and the mass ratio of the ethanol to the mixture of zinc gluconate and alkaline protease with equal mass is 7: 3;
and (5) mixing the mixed solution and the active solution in a mass ratio of 6: 4.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all 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 invention.
In the following examples, the active protectant is trehalose, mannitol and lactose mixed in equal mass.
Example 1
The method for extracting superoxide dismutase by using plants as raw materials comprises the following steps:
(1) mixing and crushing okra, pine needles and silphium perfoliatum to 100 meshes of diameter, adding an active protective agent with the mass 0.02 times of that of the mixture of the okra, the pine needles and the silphium perfoliatum, and performing wall breaking treatment to obtain plant pulp for later use;
(2) keeping the plant slurry at 50 deg.C for 30min, and performing active proliferation fission;
(3) centrifuging the plant slurry subjected to active proliferation fission, wherein the rotation speed of the centrifugation is 4000rpm, the centrifugation time is 30min, and removing precipitates to obtain an active solution;
(4) mixing equal amounts of zinc gluconate and alkaline protease, dissolving with 60 times of water, and adding 97% ethanol until the mass ratio of ethanol to the mixture of zinc gluconate and alkaline protease is 7:3 to obtain a mixed solution;
(5) mixing the mixed solution and the active solution according to the mass ratio of 6:4, precipitating for 20-28 h at the constant temperature of 20 ℃, removing the precipitate, and drying at-80 ℃ to obtain the superoxide dismutase.
Example 2
The method for extracting superoxide dismutase by using plants as raw materials comprises the following steps:
(1) crushing the okra to 80 meshes, adding an active protective agent with the mass of 0.005 time of that of the okra, and breaking the wall to obtain plant pulp for later use;
(2) keeping the plant slurry at 50 deg.C for 25min, and performing active proliferation fission;
(3) centrifuging the plant slurry subjected to active proliferation fission, wherein the rotation speed of the centrifugation is 4000rpm, the centrifugation time is 10min, and removing precipitates to obtain an active solution;
(4) mixing equal amounts of zinc gluconate and alkaline protease, dissolving with 60 times of water, and adding 95% ethanol until the mass ratio of ethanol to the mixture of equal amounts of zinc gluconate and alkaline protease is 7:3 to obtain a mixed solution;
(5) mixing the mixed solution and the active solution at a mass ratio of 6:4, precipitating at 20 deg.C for 20 hr, removing precipitate, and drying at-100 deg.C to obtain superoxide dismutase.
Example 3
The plant materials include okra, fructus Hippophae, folium Pini, and Colophonium
The method for extracting superoxide dismutase by using plants as raw materials comprises the following steps:
(1) crushing okra and sea buckthorn to 100 meshes, adding an active protective agent with the mass 0.02 time of that of the mixture of the okra and the sea buckthorn, and breaking the wall to obtain plant pulp for later use;
(2) keeping the temperature of the plant slurry at 55 ℃ for 35min, and performing active proliferation fission;
(3) centrifuging the plant slurry subjected to active proliferation fission, wherein the rotation speed of the centrifugation is 6000rpm, the centrifugation time is 30min, and removing the precipitate to obtain an active solution;
(4) mixing equal amounts of zinc gluconate and alkaline protease, dissolving with 60 times of water, and adding 97% ethanol until the mass ratio of ethanol to the mixture of zinc gluconate and alkaline protease is 7:3 to obtain a mixed solution;
(5) mixing the mixed solution and the active solution at a mass ratio of 6:4, precipitating at a constant temperature of 25 ℃ for 28h, removing the precipitate, and drying at-18 ℃ to obtain the superoxide dismutase.
Example 4
The method for extracting superoxide dismutase by using plants as raw materials comprises the following steps:
(1) pulverizing fructus Hippophae to 90 mesh, adding active protectant 0.01 times of fructus Hippophae mass, breaking cell wall, and obtaining plant slurry;
(2) keeping the plant slurry at 52 deg.C for 30min, and performing active proliferation fission;
(3) centrifuging the plant slurry subjected to active proliferation fission, wherein the rotation speed of the centrifugation is 5000rpm, the centrifugation time is 20min, and removing the precipitate to obtain an active solution;
(4) mixing equal amounts of zinc gluconate and alkaline protease, dissolving with 60 times of water, and adding 95% ethanol until the mass ratio of ethanol to the mixture of equal amounts of zinc gluconate and alkaline protease is 7:3 to obtain a mixed solution;
(5) mixing the mixed solution and the active solution at a mass ratio of 6:4, precipitating at a constant temperature of 23 ℃ for 25h, removing the precipitate, and drying at-75 ℃ to obtain the superoxide dismutase.
Example 5
The method for extracting superoxide dismutase by using plants as raw materials comprises the following steps:
(1) crushing silphium perfoliatum to 100 meshes of diameter, adding an active protective agent with the mass of 0.02 times of that of the silphium perfoliatum, and performing wall breaking treatment to obtain plant slurry for later use;
(2) keeping the plant slurry at 50 deg.C for 30min, and performing active proliferation fission;
(3) centrifuging the plant slurry subjected to active proliferation fission, wherein the rotation speed of the centrifugation is 6000rpm, the centrifugation time is 10min, and removing the precipitate to obtain an active solution;
(4) mixing equal amounts of zinc gluconate and alkaline protease, dissolving with 60 times of water, and adding 95% ethanol until the mass ratio of ethanol to the mixture of equal amounts of zinc gluconate and alkaline protease is 7:3 to obtain a mixed solution;
(5) mixing the mixed solution and the active solution at a mass ratio of 6:4, precipitating at 20 deg.C for 25 hr, removing precipitate, and drying at-20 deg.C to obtain superoxide dismutase.
Claims (8)
1. A method for extracting superoxide dismutase by taking plants as raw materials is characterized by comprising the following steps:
(1) crushing plant raw materials, adding an active protective agent for wall breaking treatment to obtain plant slurry for later use;
(2) keeping the temperature of the plant slurry at 50-55 ℃ for 25-35 min, and performing active proliferation fission;
(3) centrifuging the plant slurry subjected to active proliferation fission, and removing precipitates to obtain an active liquid;
(4) mixing equal amount of zinc gluconate and alkaline protease, dissolving with water, and adding ethanol to obtain mixed solution;
(5) and mixing the mixed solution and the active solution, precipitating at the constant temperature of 20-25 ℃ for 20-28 h, removing the precipitate, and drying at the temperature of-18 to-100 ℃ to obtain the superoxide dismutase.
2. The method for extracting superoxide dismutase from plant as raw material according to claim 1, wherein the plant raw material is one or more of okra, sea buckthorn, pine needles and silphium perfoliatum.
3. The method for extracting superoxide dismutase from plant as raw material according to claim 1, wherein the pulverization in the step (1) is to pulverize to 80-100 mesh in diameter.
4. The method for extracting superoxide dismutase from plant as raw material according to claim 1, wherein the amount of the active protective agent added in step (1) is 0.005-0.02 times of the mass of the plant raw material.
5. The method for extracting superoxide dismutase from plant as raw material according to claim 1 or 4, characterized in that the active protective agent is one or more of trehalose, mannitol, lactose.
6. The method for extracting superoxide dismutase from plant as raw material according to claim 1, wherein the rotation speed of the centrifugation in the step (3) is 4000-6000 rpm, and the centrifugation time is 10-30 min.
7. The method for extracting superoxide dismutase from plant as the raw material according to claim 1, wherein the zinc gluconate is mixed with the alkaline protease in the step (4), and the dissolving with water is performed by adding water with a mass 60 times of the sum of the mass of the zinc gluconate and the mass of the alkaline protease;
the ethanol is ethanol with the concentration of 95-97;
the mass ratio of the ethanol to the mixture of zinc gluconate and alkaline protease with equal mass is 7: 3.
8. The method for extracting superoxide dismutase from plant as raw material as claimed in claim 1, wherein the mass ratio of the mixed solution and the active solution in the step (5) is 6: 4.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112646788A (en) * | 2020-12-28 | 2021-04-13 | 刘越伟 | Processing method for extracting superoxide dismutase from corn germ |
| CN113149745A (en) * | 2021-05-13 | 2021-07-23 | 王喜 | Preparation process for preparing biological nutrient solution by using SOD as raw material |
| CN113973983A (en) * | 2021-11-05 | 2022-01-28 | 广东真理农产品科技开发有限公司 | Insect extract-based nutrient solution preparation method and application |
| CN116764157A (en) * | 2023-01-12 | 2023-09-19 | 苏州盛仓生物环保科技有限公司 | Method for degrading toluene by enzyme oxidation |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112646788A (en) * | 2020-12-28 | 2021-04-13 | 刘越伟 | Processing method for extracting superoxide dismutase from corn germ |
| CN113149745A (en) * | 2021-05-13 | 2021-07-23 | 王喜 | Preparation process for preparing biological nutrient solution by using SOD as raw material |
| CN113973983A (en) * | 2021-11-05 | 2022-01-28 | 广东真理农产品科技开发有限公司 | Insect extract-based nutrient solution preparation method and application |
| CN116764157A (en) * | 2023-01-12 | 2023-09-19 | 苏州盛仓生物环保科技有限公司 | Method for degrading toluene by enzyme oxidation |
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