CN107029217B - Superoxide dismutase composite protective agent and preparation method thereof - Google Patents
Superoxide dismutase composite protective agent and preparation method thereof Download PDFInfo
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- CN107029217B CN107029217B CN201710207537.9A CN201710207537A CN107029217B CN 107029217 B CN107029217 B CN 107029217B CN 201710207537 A CN201710207537 A CN 201710207537A CN 107029217 B CN107029217 B CN 107029217B
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- 102000019197 Superoxide Dismutase Human genes 0.000 title claims abstract description 55
- 108010012715 Superoxide dismutase Proteins 0.000 title claims abstract description 55
- 239000003223 protective agent Substances 0.000 title claims abstract description 52
- 239000002131 composite material Substances 0.000 title claims abstract description 36
- 238000002360 preparation method Methods 0.000 title claims abstract description 26
- 229910021642 ultra pure water Inorganic materials 0.000 claims abstract description 29
- 239000012498 ultrapure water Substances 0.000 claims abstract description 29
- 239000007795 chemical reaction product Substances 0.000 claims abstract description 28
- 239000000243 solution Substances 0.000 claims abstract description 28
- 239000011550 stock solution Substances 0.000 claims abstract description 28
- 230000001954 sterilising effect Effects 0.000 claims abstract description 27
- 150000001413 amino acids Chemical class 0.000 claims abstract description 16
- 238000010438 heat treatment Methods 0.000 claims abstract description 15
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 15
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 14
- 238000009835 boiling Methods 0.000 claims abstract description 14
- 238000001816 cooling Methods 0.000 claims abstract description 14
- 239000008103 glucose Substances 0.000 claims abstract description 14
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims abstract description 3
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 17
- 239000008101 lactose Substances 0.000 claims description 17
- 239000004475 Arginine Substances 0.000 claims description 15
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 15
- 239000002202 Polyethylene glycol Substances 0.000 claims description 13
- 229940006091 aloe polysaccharide Drugs 0.000 claims description 13
- 238000000034 method Methods 0.000 claims description 13
- 229920001223 polyethylene glycol Polymers 0.000 claims description 13
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 12
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 12
- 239000001110 calcium chloride Substances 0.000 claims description 12
- CHHHXKFHOYLYRE-UHFFFAOYSA-M 2,4-Hexadienoic acid, potassium salt (1:1), (2E,4E)- Chemical compound [K+].CC=CC=CC([O-])=O CHHHXKFHOYLYRE-UHFFFAOYSA-M 0.000 claims description 11
- 229940069338 potassium sorbate Drugs 0.000 claims description 11
- 235000010241 potassium sorbate Nutrition 0.000 claims description 11
- 239000004302 potassium sorbate Substances 0.000 claims description 11
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 3
- 239000004472 Lysine Substances 0.000 claims description 3
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims description 3
- 239000000126 substance Substances 0.000 claims description 3
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 claims description 2
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 claims description 2
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 15
- 239000000047 product Substances 0.000 abstract description 14
- 102000004190 Enzymes Human genes 0.000 abstract description 8
- 108090000790 Enzymes Proteins 0.000 abstract description 8
- 238000004108 freeze drying Methods 0.000 abstract description 3
- 230000001737 promoting effect Effects 0.000 abstract description 3
- 230000029663 wound healing Effects 0.000 abstract description 3
- 241000894006 Bacteria Species 0.000 abstract description 2
- 238000005265 energy consumption Methods 0.000 abstract description 2
- 230000002401 inhibitory effect Effects 0.000 abstract description 2
- 150000001875 compounds Chemical class 0.000 description 8
- 239000007787 solid Substances 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 239000003995 emulsifying agent Substances 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- WQGWDDDVZFFDIG-UHFFFAOYSA-N pyrogallol Chemical compound OC1=CC=CC(O)=C1O WQGWDDDVZFFDIG-UHFFFAOYSA-N 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 108091005508 Acid proteases Proteins 0.000 description 1
- 238000012356 Product development Methods 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003064 anti-oxidating effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 238000006701 autoxidation reaction Methods 0.000 description 1
- 230000003385 bacteriostatic effect Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000007323 disproportionation reaction Methods 0.000 description 1
- 230000009982 effect on human Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 229940079877 pyrogallol Drugs 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- -1 superoxide anion free radical Chemical class 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/44—Oxidoreductases (1)
- A61K38/446—Superoxide dismutase (1.15)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/02—Inorganic compounds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/12—Carboxylic acids; Salts or anhydrides thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
- A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
- A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- 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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- Proteomics, Peptides & Aminoacids (AREA)
- Inorganic Chemistry (AREA)
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- Immunology (AREA)
- Genetics & Genomics (AREA)
- General Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Gastroenterology & Hepatology (AREA)
- Cosmetics (AREA)
- Enzymes And Modification Thereof (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention relates to an enzyme composite protective agent and a preparation method thereof, wherein the preparation method comprises the following steps: (1) dissolving positively charged amino acid and glucose in ultrapure water in a molar ratio of 1:1 to prepare 0.6mol/L solution, heating the solution in a boiling water bath for 1 hour, and cooling to obtain a reaction product stock solution; (2) dissolving 34.2-57.5 g of synergist and 65mL of the reaction product stock solution obtained in the step (1) in ultrapure water, shaking up, and metering to 1L to obtain the composite protective agent before sterilization; (3) and (3) sterilizing the product obtained in the step (2) at 121 ℃ for 20min to obtain the superoxide dismutase composite protective agent. The protective agent not only improves the enzyme activity by 20 percent, but also greatly increases the stability, and increases the storage period at normal temperature by nearly one time; has the effects of promoting wound healing and inhibiting bacteria; does not need freeze-drying and other complex and high-energy-consumption steps, and has lower cost.
Description
Technical Field
The invention relates to an enzyme composite protective agent and a preparation method thereof, in particular to a superoxide dismutase composite protective agent and a preparation method thereof.
Background
Superoxide dismutase is known to be an antioxidant metalloenzyme existing in organisms, has the function of catalyzing superoxide anion free radical disproportionation to generate oxygen and hydrogen peroxide, and plays an important role in balance of organism oxidation and antioxidation.
Superoxide dismutase is a pure natural bioactive substance, has no toxic or side effect on human bodies, belongs to acid protease, and has activity and catalytic efficiency related to the content and physical and chemical factors of the environment. The activity of superoxide dismutase has poor stability at normal temperature and short biological half-life period, and can be slowly reduced along with the time under the natural environment, thereby limiting the clinical application of the superoxide dismutase.
CN104586766A discloses a superoxide dismutase preparation with stable enzymatic activity, which contains superoxide dismutase, an alcohol solvent, an emulsifier and oil, wherein the superoxide dismutase dispersed in the alcohol solvent is wrapped in the oil phase under the action of the emulsifier, so that a single-phase transparent alcohol-in-oil superoxide dismutase preparation is obtained, the degradation of the superoxide dismutase is avoided, the stability of the superoxide dismutase is enhanced, and the activity of the superoxide dismutase preparation is favorably exerted.
CN1245831 discloses a method for preparing superoxide dismutase with high temperature resistance and no inactivation, which comprises adding 2-8% solid protective agent into the extracted solid powdery superoxide dismutase, mixing them uniformly according to the above proportion, and freeze-drying, to ensure the stability of the enzyme activity of superoxide dismutase, and expand the product development and application range, relatively ensuring that the product containing superoxide dismutase is not easy to deteriorate in high temperature process and normal temperature storage.
However, there are the following problems: (1) the adoption of alcohol-in-oil preparations or solid protective agents can not improve the activity and stability of superoxide dismutase at the same time, thereby greatly limiting the application of the superoxide dismutase; (2) the preparation process is not simple enough and the cost is high.
Disclosure of Invention
In order to solve the above problems in the prior art, the present invention aims to provide a superoxide dismutase composite protective agent, which can simultaneously improve the activity and stability of superoxide dismutase.
The invention also aims to provide a method for preparing the superoxide dismutase composite protective agent. The method has simple process and low cost.
The technical scheme of the invention is as follows:
scheme one
The preparation method of the superoxide dismutase composite protective agent is characterized by comprising the following steps:
(1) dissolving positively charged amino acid and glucose in ultrapure water in a molar ratio of 1:1 to prepare 0.6mol/L solution, heating the solution in a boiling water bath for 1 hour, and cooling to obtain a reaction product stock solution;
(2) dissolving 34.2-57.5 g of synergist and 65mL of the reaction product stock solution obtained in the step (1) in ultrapure water, shaking up, and metering to 1L to obtain the composite protective agent before sterilization;
(3) and (3) sterilizing the product obtained in the step (2) at 121 ℃ for 20min to obtain the superoxide dismutase composite protective agent.
The further preferable scheme of the technical scheme of the invention is as follows: the positively charged amino acid is selected from one of lysine, arginine or histidine.
The further preferable scheme of the technical scheme of the invention is as follows: the synergist comprises 30-37 g of lactose, and the lactose is used for further improving the stability of the superoxide dismutase.
The further preferable scheme of the technical scheme of the invention is as follows: the synergist also comprises 10-13 g of calcium chloride, and the calcium chloride is used for further improving the stability of the superoxide dismutase.
The further preferable scheme of the technical scheme of the invention is as follows: the synergist also comprises 0.1-0.3 g of aloe polysaccharide and/or 0.05-0.2 g of polyethylene glycol, and the aloe polysaccharide and the polyethylene glycol have the functions of forming a film on the skin when the superoxide dismutase is used and promoting wound healing.
The further preferable scheme of the technical scheme of the invention is as follows: the synergist also comprises 5-7 g of potassium sorbate, and the potassium sorbate can play a bacteriostatic role.
The further preferable scheme of the technical scheme of the invention is as follows: the synergist comprises 30-37 g of lactose, 10-13 g of calcium chloride, 0.1-0.3 g of aloe polysaccharide, 0.05-0.2 g of polyethylene glycol and 5-7 g of potassium sorbate.
The further preferable scheme of the technical scheme of the invention is as follows: the synergist comprises 34.2g of lactose, 11.1g of calcium chloride, 0.2g of aloe polysaccharide, 0.1g of polyethylene glycol and 6g of potassium sorbate.
Scheme two
The superoxide dismutase composite protective agent is prepared by the preparation method of the superoxide dismutase composite protective agent according to any one of the schemes.
Compared with the prior art, the invention has the following advantages:
(1) the superoxide dismutase composite protective agent provided by the invention not only improves the enzyme activity by 20%, but also greatly increases the stability, and increases the storage period at normal temperature by nearly one time;
(2) the superoxide dismutase composite protective agent provided by the invention has the effects of promoting wound healing and inhibiting bacteria;
(3) the preparation method of the superoxide dismutase composite protective agent provided by the invention only comprises the conventional simple steps of water bath heating, physical mixing, sterilization and the like, does not need the complicated high-energy-consumption steps of freeze-drying and the like, and has lower cost.
Detailed Description
The invention is further described below with reference to specific embodiments.
1. The superoxide dismutase compound protective agent and the preparation method thereof provided by the invention have more optional factors, and various embodiments can be designed, so that the specific embodiments are only used as an exemplary illustration of the specific implementation mode of the invention, and do not limit the scope of the invention. The following examples are chosen to illustrate the invention in order to provide a thorough understanding of the invention and to enable those skilled in the art to practice the invention. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the spirit of the invention. All falling within the scope of the present invention.
Example 1
A preparation method of a superoxide dismutase composite protective agent is characterized by comprising the following steps:
(1) dissolving positively charged amino acid (arginine) and glucose in ultrapure water in a molar ratio of 1:1 to prepare 0.6mol/L solution, heating the solution in a boiling water bath for 1 hour, and cooling to obtain a reaction product stock solution;
(2) dissolving 65mL of a synergist (34.2 g of lactose) and the reaction product stock solution obtained in the step (1) in ultrapure water, shaking up, and metering to 1L to obtain a compound protective agent before sterilization;
(3) and (3) sterilizing the product obtained in the step (2) at 121 ℃ for 20min to obtain a sample 1.
Example 2
A preparation method of a superoxide dismutase composite protective agent is characterized by comprising the following steps:
(1) dissolving positively charged amino acid (arginine) and glucose in ultrapure water in a molar ratio of 1:1 to prepare 0.6mol/L solution, heating the solution in a boiling water bath for 1 hour, and cooling to obtain a reaction product stock solution;
(2) dissolving a synergist (34.2 g of lactose and 11.1g of calcium chloride) and 65mL of the reaction product stock solution obtained in the step (1) in ultrapure water, shaking up, and fixing the volume to 1L to obtain a composite protective agent before sterilization;
(3) and (3) sterilizing the product obtained in the step (2) at 121 ℃ for 20min to obtain a sample 2.
Example 3
A preparation method of a superoxide dismutase composite protective agent is characterized by comprising the following steps:
(1) dissolving positively charged amino acid (arginine) and glucose in ultrapure water in a molar ratio of 1:1 to prepare 0.6mol/L solution, heating the solution in a boiling water bath for 1 hour, and cooling to obtain a reaction product stock solution;
(2) dissolving 65mL of synergist (34.2 g of lactose and 0.2g of aloe polysaccharide) and the reaction product stock solution obtained in the step (1) in ultrapure water, shaking up, and metering to 1L to obtain the compound protective agent before sterilization;
(3) and (3) sterilizing the product obtained in the step (2) at 121 ℃ for 20min to obtain a sample 3.
Example 4
A preparation method of a superoxide dismutase composite protective agent is characterized by comprising the following steps:
(1) dissolving positively charged amino acid (arginine) and glucose in ultrapure water in a molar ratio of 1:1 to prepare 0.6mol/L solution, heating the solution in a boiling water bath for 1 hour, and cooling to obtain a reaction product stock solution;
(2) dissolving 65mL of synergist (34.2 g of lactose and 0.1g of polyethylene glycol) and the reaction product stock solution obtained in the step (1) in ultrapure water, shaking up, and fixing the volume to 1L to obtain the compound protective agent before sterilization;
(3) and (3) sterilizing the product obtained in the step (2) at 121 ℃ for 20min to obtain a sample 4.
Example 5
A preparation method of a superoxide dismutase composite protective agent is characterized by comprising the following steps:
(1) dissolving positively charged amino acid (arginine) and glucose in ultrapure water in a molar ratio of 1:1 to prepare 0.6mol/L solution, heating the solution in a boiling water bath for 1 hour, and cooling to obtain a reaction product stock solution;
(2) dissolving 65mL of a reaction product stock solution obtained in the step (1) with a synergist (34.2 g of lactose, 0.2g of aloe polysaccharide and 0.1g of polyethylene glycol) in ultrapure water, shaking up, and fixing the volume to 1L to obtain a compound protective agent before sterilization;
(3) and (3) sterilizing the product obtained in the step (2) at 121 ℃ for 20min to obtain a sample 5.
Example 6
A preparation method of a superoxide dismutase composite protective agent is characterized by comprising the following steps:
(1) dissolving positively charged amino acid (arginine) and glucose in ultrapure water in a molar ratio of 1:1 to prepare 0.6mol/L solution, heating the solution in a boiling water bath for 1 hour, and cooling to obtain a reaction product stock solution;
(2) dissolving 65mL of a synergist (34.2 g of lactose and 6g of potassium sorbate) and a reaction product stock solution obtained in the step (1) in ultrapure water, shaking up, and fixing the volume to 1L to obtain a compound protective agent before sterilization;
(3) and (3) sterilizing the product obtained in the step (2) at 121 ℃ for 20min to obtain a sample 6.
Example 7
A preparation method of a superoxide dismutase composite protective agent is characterized by comprising the following steps:
(1) dissolving positively charged amino acid (arginine) and glucose in ultrapure water in a molar ratio of 1:1 to prepare 0.6mol/L solution, heating the solution in a boiling water bath for 1 hour, and cooling to obtain a reaction product stock solution;
(2) dissolving 65mL of a reaction product stock solution obtained in the step (1) with a synergist (34.2 g of lactose, 11.1g of calcium chloride, 0.2g of aloe polysaccharide and 0.1g of polyethylene glycol) in ultrapure water, shaking up, and fixing the volume to 1L to obtain a compound protective agent before sterilization;
(3) the product obtained in the step (2) was sterilized at 121 ℃ for 20min to obtain sample 7.
Example 8
A preparation method of a superoxide dismutase composite protective agent is characterized by comprising the following steps:
(1) dissolving positively charged amino acid (arginine) and glucose in ultrapure water in a molar ratio of 1:1 to prepare 0.6mol/L solution, heating the solution in a boiling water bath for 1 hour, and cooling to obtain a reaction product stock solution;
(2) dissolving 65mL of a reaction product stock solution obtained in the step (1) with a synergist (30 g of lactose, 10g of calcium chloride, 0.1g of aloe polysaccharide, 0.05g of polyethylene glycol and 5g of potassium sorbate) in ultrapure water, shaking up, and fixing the volume to 1L to obtain a compound protective agent before sterilization;
(3) and (3) sterilizing the product obtained in the step (2) at 121 ℃ for 20min to obtain a sample 8.
Example 9
A preparation method of a superoxide dismutase composite protective agent is characterized by comprising the following steps:
(1) dissolving positively charged amino acid (arginine) and glucose in ultrapure water in a molar ratio of 1:1 to prepare 0.6mol/L solution, heating the solution in a boiling water bath for 1 hour, and cooling to obtain a reaction product stock solution;
(2) dissolving 65mL of a reaction product stock solution obtained in the step (1) with a synergist (34.2 g of lactose, 11.1g of calcium chloride, 0.2g of aloe polysaccharide, 0.1g of polyethylene glycol and 6g of potassium sorbate) in ultrapure water, shaking up, and metering to 1L to obtain a composite protective agent before sterilization;
(3) the product obtained in step (2) was sterilized at 121 ℃ for 20min to obtain sample 9.
Example 10
A preparation method of a superoxide dismutase composite protective agent is characterized by comprising the following steps:
(1) dissolving positively charged amino acid (arginine) and glucose in ultrapure water in a molar ratio of 1:1 to prepare 0.6mol/L solution, heating the solution in a boiling water bath for 1 hour, and cooling to obtain a reaction product stock solution;
(2) dissolving 65mL of a reaction product stock solution obtained in the step (1) with a synergist (lactose 37g, calcium chloride 13g, aloe polysaccharide 0.3g, polyethylene glycol 0.2g and potassium sorbate 7g) in ultrapure water, shaking up, and metering to 1L to obtain a composite protective agent before sterilization;
(3) and (3) sterilizing the product obtained in the step (2) at 121 ℃ for 20min to obtain a sample 10.
Example 11
This example differs from example 9 in that lysine was used instead of arginine to produce sample 11.
Example 12
This example differs from example 9 in that histidine was used instead of arginine to make sample 12.
Comparative example 1
Sterilized ultrapure water was used as control 1.
Comparative example 2
(1) Dissolving positively charged amino acid (arginine) and glucose in ultrapure water in a molar ratio of 1:1 to prepare 0.6mol/L solution, heating the solution in a boiling water bath for 1 hour, and cooling to obtain a reaction product stock solution;
(2) dissolving 65mL of the reaction product stock solution obtained in the step (1) in ultrapure water, shaking up, and metering the volume to 1L to obtain a composite protective agent before sterilization;
(3) sterilizing the product obtained in step (2) at 121 deg.C for 20min to obtain control 2.
Evaluation of the effects of the implementations
10mg of superoxide dismutase (enzyme activity is 4500U/mg) is dissolved in 20mL of sample or control.
1. Determination of enzyme Activity: pyrogallol autoxidation determination method is adopted.
2. The temperature acceleration method is used for measuring the normal-temperature storage period when the enzyme activity is lost by 10 percent.
TABLE 1 evaluation of the effects
Claims (7)
1. A method for preparing a superoxide dismutase composite protective agent is characterized by comprising the following steps: prepared by the following steps:
(1) dissolving positively charged amino acid and glucose in ultrapure water in a molar ratio of 1:1 to prepare 0.6mol/L solution, heating the solution in a boiling water bath for 1 hour, and cooling to obtain a reaction product stock solution;
(2) dissolving 34.2-57.5 g of synergist and 65mL of the reaction product stock solution obtained in the step (1) in ultrapure water, shaking up, and metering to 1L to obtain the composite protective agent before sterilization;
(3) sterilizing the substance obtained in the step (2) at 121 ℃ for 20min to obtain the superoxide dismutase composite protective agent; the positively charged amino acid is selected from one of lysine, arginine or histidine;
the synergist comprises 30-37 g of lactose.
2. The method for preparing the superoxide dismutase complex protective agent according to claim 1, characterized in that: the synergist also comprises 10-13 g of calcium chloride.
3. The method for preparing the superoxide dismutase complex protective agent according to claim 1, characterized in that: the synergist also comprises 0.1-0.3 g of aloe polysaccharide and/or 0.05-0.2 g of polyethylene glycol.
4. The method for preparing the superoxide dismutase complex protective agent according to claim 1, characterized in that: the synergist also comprises 5-7 g of potassium sorbate.
5. The method for preparing the superoxide dismutase complex protective agent according to claim 1, characterized in that: the synergist comprises 30-37 g of lactose, 10-13 g of calcium chloride, 0.1-0.3 g of aloe polysaccharide, 0.05-0.2 g of polyethylene glycol and 5-7 g of potassium sorbate.
6. The method for preparing the superoxide dismutase complex protective agent according to claim 5, wherein: the synergist comprises 34.2g of lactose, 11.1g of calcium chloride, 0.2g of aloe polysaccharide, 0.1g of polyethylene glycol and 6g of potassium sorbate.
7. The superoxide dismutase composite protective agent prepared by the preparation method of the superoxide dismutase composite protective agent according to any one of claims 1 to 6.
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