CN106399271B - Method for extracting superoxide dismutase from pig blood and superoxide dismutase - Google Patents
Method for extracting superoxide dismutase from pig blood and superoxide dismutase Download PDFInfo
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- CN106399271B CN106399271B CN201610936334.9A CN201610936334A CN106399271B CN 106399271 B CN106399271 B CN 106399271B CN 201610936334 A CN201610936334 A CN 201610936334A CN 106399271 B CN106399271 B CN 106399271B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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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- 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)
Abstract
The present invention provides a method for extracting superoxide dismutase from pig blood and said superoxide dismutase, and is characterized by that in the course of extracting and separating superoxide dismutase by using pig blood as raw material, it does not use any organic solvent, so that it can prevent environmental pollution and toxic action to enzyme activity, and can simplify production process. In the method, lauroyl chloride is used as a protective agent in the purification process of the superoxide dismutase to protect the superoxide dismutase so as to avoid the damage of heat, acid and alkali on the superoxide dismutase, and the crude superoxide dismutase is further purified and refined by combining thermal denaturation and isoelectric point precipitation technologies, so that the method has the characteristics of short production period, low cost and suitability for industrial mass production. The prepared superoxide dismutase has high activity, and the unit enzyme activity reaches above 4500U/mg dry powder.
Description
Technical Field
The invention relates to the field of organic matter separation and purification, in particular to a method for extracting superoxide dismutase from pig blood and the superoxide dismutase.
Background
Superoxide Dismutase (SOD for short) is a vital antioxidant enzyme existing in organisms and is an internationally accepted Superoxide anion radical (O)2-) The specific scavenger plays a vital role in balancing the oxidation and antioxidation systems of organisms and avoiding free radical damage. The efficacy of SOD on human body can be classified as 'one-clearing four-resisting': "Yiqing" can effectively remove superoxide anion radical in organism, and protect human body cell from poisoning by oxygen radical; "four-resistant" means anti-aging, anti-disease, anti-radiation and anti-fatigue.
In recent years, the technology for preparing superoxide dismutase (SOD) by taking animal blood as a raw material is greatly improved, some of the technology realize large-scale production, and a few of the technology have already applied for invention patents, for example, CN03106275.X discloses a novel method for quickly extracting superoxide dismutase from animal blood, wherein alcohol and copper chloride are added into fresh animal blood to remove impure proteins such as hemoglobin, and then acetone fractional precipitation and freeze drying are carried out to prepare SOD finished products; CN200410041723.2 discloses a method for preparing superoxide dismutase, which adopts fresh pig blood clots as raw materials, and comprises the complex process flows of adding copper chloride for thermal denaturation reaction, ethanol-chloroform extraction, acetone fractional precipitation, column chromatography purification, dialysis, freeze-drying and the like, and finally light blue Cu, Zn-SOD is obtained. CN200410071087.8 discloses a new method for extracting superoxide dismutase from animal blood, which comprises adding sodium citrate into animal blood for anticoagulation, directly adding glucose, copper chloride and sodium chloride as protective agents into whole blood without centrifugation for removing foreign protein by thermal denaturation, then carrying out acetone solvent fractionation, and freeze-drying to obtain SOD lyophilized powder. CN200810148899.6 discloses a method for preparing superoxide dismutase, which comprises adding protein precipitator into animal blood clot to remove foreign protein, filtering, centrifuging, ultrafiltering, and lyophilizing to obtain lyophilized powder. CN201110228061.X discloses a new process for extracting superoxide dismutase SOD from animal blood cell liquid, which mainly comprises hemolysis, thermal denaturation, ultrafiltration concentration, acetone fractional precipitation, ion exchange column chromatography, etc. to obtain SOD refined product.
The prior technical scheme for extracting SOD from animal blood cell liquid basically uses harmful chemical reagents and organic solvents, which bring secondary pollution to products and environment, have high production cost and great operation difficulty, and lead to resource waste because high-quality hemoglobin after SOD extraction is seriously polluted and cannot be comprehensively utilized.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a method for extracting superoxide dismutase from pig blood, which aims to solve the problem that harmful chemical reagents and organic solvents are adopted in the existing preparation method for preparing superoxide dismutase from animal fresh blood, so that secondary pollution is caused.
The second purpose of the invention is to provide the superoxide dismutase prepared by the method for extracting the superoxide dismutase from the pig blood, and the superoxide dismutase freeze-dried powder has the advantages of high additional value, high biological activity and the like.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
a method for extracting superoxide dismutase from pig blood comprises the following steps:
(1) adding sodium citrate into fresh pig blood for anticoagulation treatment, centrifuging, collecting red blood cells to obtain blood cell liquid, adding water, and stirring to obtain hemolytic liquid;
(2) adjusting the pH of the hemolytic liquid obtained in the step (1) to 5.2-5.5 by using acid, heating to 60-65 ℃, preserving heat, then cooling and filtering to obtain filtrate;
(3) adjusting the pH value of the filtrate obtained in the step (2) to 6.2-6.5 by using alkali, heating to 70-75 ℃, preserving heat, then cooling and filtering to obtain a filtrate;
(4) carrying out ultrafiltration concentration on the filtrate obtained in the step (3) to obtain a crude SOD solution, heating the crude SOD solution to raise the temperature, adjusting the pH value to 8.5-9.0 by using alkali, dropwise adding a protective agent A, keeping the temperature, cooling after keeping the temperature, centrifuging and collecting supernatant to obtain an SOD enzyme solution;
(5) and (3) adding a protective agent B into the SOD enzyme solution obtained in the step (5), uniformly stirring, and freeze-drying to obtain the SOD freeze-dried powder.
The method for extracting superoxide dismutase from pig blood provided by the application does not use any organic solvent, avoids environmental pollution and toxic hazard to enzyme activity, and simplifies the production process. Compared with purification methods such as ammonium sulfate salting-out method, acetone fractional precipitation method, ion exchange resin or molecular sieve column chromatography and the like reported in many patents at present, the method has the characteristics of short production period, low cost and suitability for industrial mass production.
According to the method for extracting superoxide dismutase from pig blood, in the step (1), qualified fresh pig blood is selected, sodium citrate is added for anticoagulation treatment, the mass of the sodium citrate is 0.5-1.0% of that of the pig blood, the sodium citrate has good metal ion complexing ability and can be complexed with Ca2+、Mg2+And Fe2+The binding of the metal ions into soluble complexes deprives these ions of the opportunity to form insoluble complexes, thereby slowing solidification. Centrifuging to obtain blood cell liquid and plasma liquid, and spray drying to obtain plasma protein powder. In the step (2) and the step (3), the hemolytic liquid is first adjusted to 5.2EAnd 5.5, adjusting the temperature to 6.2-6.5, and then performing heating, heat preservation and cooling operations, wherein in the operation process, the heating and cooling are rapidly performed, and the heating and cooling are completed in as short a time as possible so as to prevent the pig blood quality from changing and influencing the obtained superoxide dismutase freeze-dried powder. And (4) carrying out ultrafiltration concentration to obtain a crude SOD solution, heating the crude SOD solution to raise the temperature, adjusting the pH value to 8.5-9.0 by using alkali, and adding a protective agent, wherein the protective agent has the function of avoiding the damage of heat, acid and alkali on SOD. And combines thermal denaturation and isoelectric point precipitation technologies to further purify and refine the SOD crude product, and compared with purification methods such as ammonium sulfate salting-out method, acetone fractional precipitation method, ion exchange resin or molecular sieve column chromatography and the like reported in many patents at present, the method has the characteristics of short production period, low cost and suitability for industrial scale production.
Preferably, in step (2), the acid is one of hydrochloric acid, sulfuric acid or phosphoric acid, and more preferably, the acid is hydrochloric acid.
The acid is used for adjusting the pH value of the solution, and the inorganic acid is adopted in the application, and the organic acid is not added.
Preferably, in the step (2), in the process of heating to 60-65 ℃, preserving heat, then cooling and filtering to obtain filtrate, the method specifically comprises the following steps: and (3) heating to 60-65 ℃, preserving heat for 30-60 minutes, cooling to below 45 ℃, and then performing filter pressing by using a plate-and-frame filter press to obtain filter pressing filtrate.
The temperature is quickly reduced to below 45 ℃, and the plate-and-frame filter press is adopted for filter pressing, so that the filtering efficiency is improved.
Preferably, in the step (3) and the step (4), the alkali is one of sodium hydroxide, potassium hydroxide or calcium hydroxide, and more preferably sodium hydroxide.
The alkali is used for adjusting the pH value of the solution, and the inorganic alkali is adopted in the application.
Preferably, in the step (3), in the process of heating to 70-75 ℃ and preserving heat, then cooling and filtering to obtain the filtrate, the method specifically comprises the following steps:
and (3) heating to 70-75 ℃, preserving the heat for 20-30 minutes, cooling to 35-45 ℃, and then performing filter pressing by using a plate-and-frame filter press to obtain filter pressing filtrate.
Preferably, in the step (4), ultrafiltration concentration is performed by adopting an ultrafiltration membrane device with the molecular weight of 10000Da, and the concentration is performed until the volume of the filtrate in the step (3) is 1/5-1/10.
Preferably, in the step (4), the temperature is increased to 45-50 ℃.
Preferably, in the step (4), in the process of dropping the protective agent a, preserving heat, cooling after preserving heat, centrifuging and collecting supernatant, the method specifically comprises the following steps:
dripping lauroyl chloride accounting for 0.01-0.03 percent of the mass of the SOD crude enzyme solution, preserving the heat for 1-2 hours, cooling to below 20 ℃ after preserving the heat, centrifuging and collecting supernate;
more preferably, the rotating speed of the centrifugation is 5000-10000 r/min, and the time is 5-10 minutes.
Preferably, in the step (5), the protective agent B is one or more of trehalose, lactose and mannitol;
more preferably, the addition amount of the protecting agent B is 2.5-3% of the mass of the SOD enzyme solution in the step (4).
Trehalose is a non-reducing sugar consisting of two glucose molecules with 1, 1-glycosidic bonds, has 3 isomers, namely trehalose (α), iso-trehalose (β) and new trehalose (α), and has a non-specific protective effect on various bioactive substances.
Lactose is white crystalline granules or powder; no bad smell, slightly sweet taste. As an adsorption dispersant for powdery food coloring matter, reducing coloring matter concentration, facilitating use and reducing discoloration during storage.
Mannitol is an isomer of sorbitol, and two alcohols have different hydroxy orientations on carbon atom II and have a molecular formula of C6H14O6And the molecular weight is 182.17. Is easily dissolved in water, is white transparent solid, and has sweet taste similar to sucrose. Has the function of preventing bonding.
The superoxide dismutase freeze-dried powder prepared by the method for extracting the superoxide dismutase from the pig blood.
Superoxide dismutase (SOD) is a biological enzyme widely existing in the nature, and can be divided into copper-zinc SOD, manganese SOD and iron SOD according to the different types of metals.
SOD can catalyze the reaction of eliminating superoxide anion free radical. A radical is an atom or group of atoms, molecules or ions having an unpaired electron. Under normal physiological conditions, free radicals are continuously generated in the organism, and the generation and elimination of the free radicals are in equilibrium. However, in some pathological conditions, when the amount of free radicals produced is large, the free radicals will damage biological macromolecules such as DNA, protein and lipid, and cause the generation of body diseases. Because the free radicals have high chemical activity and are intermediate metabolites of various biochemical reactions in human life activities, the attack of biomacromolecules by the free radicals to cause tissue damage is the root of the occurrence and development of many diseases. Therefore, SOD plays an important role in protecting organisms from being damaged by superoxide anion free radicals, resisting radiation, resisting tumors, delaying organism aging and the like.
SOD has the function of eliminating excessive superoxide anion free radicals generated in the metabolic process of an organism, and can delay the aging phenomenon caused by the invasion of the free radicals, namely delaying skin aging and lipofuscin precipitation. The aging free radical theory considers that aging is a random and destructive action result from free radicals generated in the normal metabolic process of the body, and the main mechanisms of the aging of the body caused by the free radicals can be summarized as the following three aspects: (1) reducing the cross-linking polymerization of biological macromolecules and the accumulation of lipofuscin; (2) slowing down the damage and reduction of organ tissue cells; (3) preventing the reduction of immunity.
SOD can improve the resistance of human body to free radical damage induced diseases, and the resistance of the human body to free radical damage induced diseases mainly comprises tumor, inflammation, emphysema, cataract, autoimmune diseases and the like.
SOD can increase free radical in human bodyThe resistance of the boundary induction factors, such as smoke, radiation, toxic chemicals and toxic drugs, enhances the adaptability of the body to the external environment. Ionizing radiation causes the production of O in vivo2-And OH, etc. OH has a great damaging effect on the body and the site where OH is produced is often the site of action, so there is little chance of damaging the biological macromolecules far away from the OH producing site. O is2-The damage action of (2) is far less than that of OH, but it can diffuse from the site of production to other sites, and reacts with H in the presence of an iron ion chelating agent to form OH, which causes damage to DNA, biological membranes, etc. Fumes, toxic chemicals and drugs also produce excessive amounts of O2-Damage to the body, SOD can also reduce these damages.
SOD can also eliminate body fatigue and enhance the adaptability to overload large amount of exercise. During the overload exercise, some tissue cells in the body will alternately produce temporary ischemia and re-perfusion to cause re-perfusion damage after ischemia and muscle fatigue and damage due to the increased lactic acid amount. If SOD is supplied before exercise, the muscle can be protected from the above phenomenon. The free radical level in vivo can be obviously increased by single strong movement, and the excessive free radicals are used as initiators to attack unsaturated fatty acid in cell membranes to cause lipid peroxidation, thereby causing the membrane fluidity to be reduced and the brittleness to be enhanced. One characteristic of peroxidized lipids is the formation of malondialdehyde, which forms crosslinks between phospholipids and may also form brown products with hemoglobin. After the body is strenuously exercised, the ATP content is reduced, the creatine kinase activity is temporarily reduced, the AMP content is increased, IMP is promoted to be increased under the action of adenine transaminase, hypoxanthine is generated under the action of nucleotidase and nucleosidase, and excessive hypoxanthine forms excessive O under the action of hypoxanthine oxidase after the exercise2-. Therefore, the supplement of exogenous SOD can effectively inhibit cell damage caused by movement.
The SOD functions on the principle that superoxide anion (O)2-) Is a main free radical in the living body, and O is often the main radical2-Is harmful to the body and is also one of the causes of aging. SOD is an important antioxidant enzyme for eliminating oxygen free radicals, and can catalyze O2-Subjecting it to disproportionation reaction to produce O2And H2O2；H2O2And generating nontoxic H under the action of Catalase (CAT)2O and O2Thereby playing a role of anti-aging.
The superoxide dismutase prepared by the method has high activity, and the unit enzyme activity reaches above 4500U/mg dry powder.
Compared with the prior art, the invention has the beneficial effects that:
1) the method for extracting superoxide dismutase from pig blood provided by the application does not use any organic solvent in the process of extracting and separating SOD by taking pig blood as a raw material, avoids environmental pollution and toxic hazard to enzyme activity, and simplifies the production process.
2) According to the method for extracting superoxide dismutase from pig blood, lauroyl chloride is used as a protective agent in the SOD purification process to protect SOD so as to avoid the damage of heat, acid and alkali to the SOD, and the crude SOD product is further purified and refined by combining thermal denaturation and isoelectric point precipitation technologies.
3) The superoxide dismutase prepared by the method for extracting the superoxide dismutase from the pig blood has high activity, and the unit enzyme activity reaches above 4500U/mg dry powder.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Example 1
(1) Collecting 1000L of qualified fresh pig blood, adding food-grade sodium citrate with the mass of 0.5% of the fresh pig blood for anticoagulation treatment, continuously separating and centrifuging by adopting a tubular centrifuge 16300r/mim, collecting red blood cells to obtain blood cell liquid 420L and blood plasma liquid 580L, adding water with the same volume as the blood cell liquid, and stirring for 60 minutes to obtain 840L of hemolytic liquid;
(2) adjusting the pH of the hemolytic solution obtained in the step (1) to 5.2 by using HCl, rapidly heating to 60 ℃, preserving heat for 50 minutes, rapidly cooling to below 45 ℃, and performing pressure filtration by using a plate-and-frame filter press to obtain 560L of filtrate;
(3) adjusting the pH value of the filtrate obtained in the step (2) to 6.2 by using NaOH, rapidly heating to 70 ℃, preserving heat for 20 minutes, then rapidly cooling to below 40 ℃, and performing filter pressing by using a plate-and-frame filter press to obtain 465L of filtrate;
(4) carrying out ultrafiltration concentration on the filtrate obtained in the step (3) by an ultrafiltration membrane device with the molecular weight cutoff of 10000Da, concentrating to 1/10 of the original volume to obtain 46L of SOD crude enzyme solution, heating the SOD crude enzyme solution to 45 ℃, adjusting the pH value to 9.0 by using alkali, dropwise adding lauroyl chloride with the mass of 0.02% of that of the SOD crude enzyme solution, keeping the temperature for 1 hour, rapidly cooling to below 20 ℃, centrifuging at 5000r/min for 10min, and collecting supernatant to obtain the SOD enzyme solution with blue-green color;
(5) and (3) adding trehalose accounting for 2.5% of the mass of the SOD enzyme solution in the step (5), uniformly stirring, and freeze-drying to obtain 55g of light blue green SOD freeze-dried powder.
Example 2
(1) Collecting 1000L of qualified fresh pig blood, adding food-grade sodium citrate with the mass of 0.6% of the fresh pig blood for anticoagulation treatment, adopting a tubular centrifuge 16300r/mim, continuously separating and centrifuging, collecting red blood cells to obtain 450L of blood cell liquid and 550L of plasma liquid, adding water with the same volume as the blood cell liquid, and stirring for 40 minutes to obtain 900L of hemolytic liquid;
(2) adjusting the pH of the hemolytic solution obtained in the step (1) to 5.5 by using HCl, rapidly heating to 60 ℃, preserving heat for 40 minutes, rapidly cooling to below 45 ℃, and performing pressure filtration by using a plate-and-frame filter press to obtain 610L of filtrate;
(3) adjusting the pH value of the filtrate obtained in the step (2) to 6.3 by using NaOH, rapidly heating to 70 ℃, preserving heat for 25 minutes, then rapidly cooling to below 45 ℃, and carrying out filter pressing by using a plate-and-frame filter press to obtain 520L of filtrate;
(4) carrying out ultrafiltration concentration on the filtrate obtained in the step (3) by an ultrafiltration membrane device with the molecular weight cutoff of 10000Da, concentrating to 1/10 of the original volume to obtain 52L of SOD crude enzyme solution, heating the SOD crude enzyme solution to 45 ℃, adjusting the pH value to 9.0 by using alkali, dropwise adding lauroyl chloride with the mass of 0.03 percent of that of the SOD crude enzyme solution, keeping the temperature for 1 hour, rapidly cooling to below 20 ℃, centrifuging at 5000r/min for 10min, and collecting supernatant to obtain the SOD enzyme solution with blue-green color;
(5) and (3) adding lactose accounting for 2.5 percent of the mass of the SOD enzyme solution in the step (5), uniformly stirring, and freeze-drying to obtain 51g of light blue green SOD freeze-dried powder.
Example 3
(1) 2000L of qualified fresh pig blood is collected, food-grade sodium citrate with the mass of 1.0% of that of the fresh pig blood is added into the fresh pig blood for anticoagulation treatment, a tubular centrifuge 16300r/mim is adopted for continuous separation and centrifugation, red blood cells are collected to obtain 930L of blood cell liquid and 1070L of blood plasma liquid, water with the same volume as the blood cell liquid is added into the blood cell liquid, and stirring is carried out for 60 minutes to obtain 1860L of hemolytic liquid;
(2) adjusting the pH of the hemolytic solution obtained in the step (1) to 5.3 by using phosphoric acid, rapidly heating to 65 ℃, preserving heat for 50 minutes, rapidly cooling to below 45 ℃, and performing pressure filtration by using a plate-and-frame filter press to obtain a filtrate 1230L;
(3) adjusting the pH value of the filtrate obtained in the step (2) to 6.5 by using KOH, quickly heating to 75 ℃, preserving the temperature for 30 minutes, then quickly cooling to below 45 ℃, and carrying out filter pressing by using a plate-and-frame filter press to obtain 1050L of filtrate;
(4) carrying out ultrafiltration concentration on the filtrate obtained in the step (3) by an ultrafiltration membrane device with the molecular weight cutoff of 10000Da, concentrating to 1/10 of the original volume to obtain 100L of SOD crude enzyme solution, heating the SOD crude enzyme solution to 45 ℃, adjusting the pH value to 9.0 by using alkali, dropwise adding lauroyl chloride with the mass of 0.02% of that of the SOD crude enzyme solution, keeping the temperature for 1 hour, rapidly cooling to below 20 ℃, centrifuging at 5000r/min for 10min, and collecting supernatant to obtain the SOD enzyme solution with blue-green color;
(5) and (3) adding lactose accounting for 2.5 percent of the mass of the SOD enzyme solution in the step (5), uniformly stirring, and freeze-drying to obtain 107g of light blue green SOD freeze-dried powder.
Example 4
(1) 2000L of qualified fresh pig blood is collected, food-grade sodium citrate with the mass of 0.7 percent of that of the fresh pig blood is added into the fresh pig blood for anticoagulation treatment, a tubular centrifuge 16300r/mim is adopted for continuous separation and centrifugation, red blood cells are collected to obtain 930L of blood cell liquid and 1070L of blood plasma liquid, water with the same volume as the blood cell liquid is added into the blood cell liquid, and stirring is carried out for 60 minutes to obtain 1860L of hemolytic liquid;
(2) adjusting the pH of the hemolytic liquid obtained in the step (1) to 5.5 by using phosphoric acid, rapidly heating to 65 ℃, preserving heat for 30 minutes, rapidly cooling to below 45 ℃, and performing pressure filtration by using a plate-and-frame filter press to obtain a filtrate 1130L;
(3) adjusting the pH value of the filtrate obtained in the step (2) to 6.5 by using KOH, quickly heating to 70 ℃, preserving the temperature for 20 minutes, then quickly cooling to below 45 ℃, and carrying out filter pressing by using a plate-and-frame filter press to obtain 1050L of filtrate;
(4) carrying out ultrafiltration concentration on the filtrate obtained in the step (3) by an ultrafiltration membrane device with the molecular weight cutoff of 10000Da, concentrating to 1/5 of the original volume to obtain 210L of SOD crude enzyme solution, heating the SOD crude enzyme solution to 50 ℃, adjusting the pH value to 8.5 by using alkali, dropwise adding lauroyl chloride with the mass of 0.03 percent of that of the SOD crude enzyme solution, keeping the temperature for 1 hour, rapidly cooling to below 20 ℃, centrifuging for 5min at 10000r/min, and collecting supernatant to obtain the SOD enzyme solution with blue-green color;
(5) and (3) adding lactose accounting for 3% of the mass of the SOD enzyme solution in the step (5), uniformly stirring, and freeze-drying to obtain 107g of light blue green SOD freeze-dried powder.
Experimental example 1 Activity test of superoxide dismutase lyophilized powder
The superoxide dismutase freeze-dried powder provided by the embodiments 1 to 4 of the application is subjected to an enzyme activity test, and is compared with a patent with the application number of CN201110228061. X. The results of the experiment are shown in table 1.
TABLE 1 enzymatic Activity test results of superoxide dismutase lyophilized powders
Experiments prove that the superoxide dismutase prepared by the method for extracting the superoxide dismutase from the pig blood has high activity, and the unit enzyme activity reaches above 4500U/mg dry powder.
In conclusion, the method for extracting superoxide dismutase from pig blood provided by the application does not use any organic solvent in the process of extracting and separating SOD by taking pig blood as a raw material, avoids environmental pollution and toxic hazard to enzyme activity, and simplifies the production process. In the process of SOD purification, lauroyl chloride is used as a protective agent to protect SOD so as to avoid the damage of heat, acid and alkali to the SOD, and the crude SOD product is further purified and refined by combining thermal denaturation and isoelectric point precipitation technologies, so that the method has the characteristics of short production period, low cost and suitability for industrial mass production. The prepared superoxide dismutase has high activity, and the unit enzyme activity reaches above 4500U/mg dry powder.
While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.
Claims (10)
1. A method for extracting superoxide dismutase from pig blood is characterized by comprising the following steps:
(1) adding sodium citrate into fresh pig blood for anticoagulation treatment, centrifuging, collecting red blood cells to obtain blood cell liquid, adding water, and stirring to obtain hemolytic liquid;
(2) adjusting the pH of the hemolytic liquid obtained in the step (1) to 5.2-5.5 by using acid, heating to 60-65 ℃, preserving heat, then cooling and filtering to obtain filtrate;
(3) adjusting the pH value of the filtrate obtained in the step (2) to 6.2-6.5 by using alkali, heating to 70-75 ℃, preserving heat, then cooling and filtering to obtain a filtrate;
(4) carrying out ultrafiltration concentration on the filtrate obtained in the step (3) to obtain a crude SOD enzyme solution, heating the crude SOD enzyme solution to raise the temperature, adjusting the pH value to 8.5-9.0 by using alkali, dropwise adding lauroyl chloride accounting for 0.01-0.03% of the mass of the crude SOD enzyme solution, keeping the temperature for 1-2 hours, cooling to below 20 ℃ after keeping the temperature, centrifuging and collecting supernatant to obtain the SOD enzyme solution;
(5) adding a protective agent B into the SOD enzyme solution obtained in the step (5), uniformly stirring, and freeze-drying to obtain SOD freeze-dried powder;
the protective agent B is one or the combination of more of trehalose, lactose and mannitol;
the addition amount of the protective agent B is 2.5-3% of the mass of the SOD enzyme solution in the step (4).
2. The method for extracting superoxide dismutase from pig blood as claimed in claim 1, wherein in the step (2), the acid is one of hydrochloric acid, sulfuric acid or phosphoric acid.
3. The method for extracting superoxide dismutase from pig blood as claimed in claim 1, wherein in the step (2), the acid is hydrochloric acid.
4. The method for extracting superoxide dismutase from pig blood as claimed in claim 1, wherein in the step (2), the method comprises the following steps in the process of heating to 60-65 ℃, preserving heat, cooling and filtering to obtain filtrate: and (3) heating to 60-65 ℃, preserving heat for 30-60 minutes, cooling to below 45 ℃, and then performing filter pressing by using a plate-and-frame filter press to obtain filter pressing filtrate.
5. The method for extracting superoxide dismutase from pig blood as claimed in claim 1, wherein in the step (3) and the step (4), the alkali is one of sodium hydroxide, potassium hydroxide or calcium hydroxide.
6. The method for extracting superoxide dismutase from pig blood as claimed in claim 1, wherein in the step (3) and the step (4), the alkali is sodium hydroxide.
7. The method for extracting superoxide dismutase from pig blood as claimed in claim 1, wherein in the step (3), the method comprises the following steps in the process of heating to 70-75 ℃, preserving heat, cooling and filtering to obtain filtrate:
and (3) heating to 70-75 ℃, preserving the heat for 20-30 minutes, cooling to 35-45 ℃, and then performing filter pressing by using a plate-and-frame filter press to obtain filter pressing filtrate.
8. The method for extracting superoxide dismutase from pig blood as claimed in claim 1, wherein in the step (4), the ultrafiltration concentration is performed by using an ultrafiltration membrane device with a molecular weight of 10000Da, and the ultrafiltration concentration is performed until the volume of the filtrate obtained in the step (3) is 1/5-1/10.
9. The method for extracting superoxide dismutase from pig blood as claimed in claim 1, wherein in the step (4), the temperature is raised to 45-50 ℃.
10. The method for extracting superoxide dismutase from pig blood as claimed in claim 1, wherein in the step (4), the rotation speed of the centrifugation is 5000 to 10000r/min, and the time is 5 to 10 minutes.
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101353370A (en) * | 2008-09-09 | 2009-01-28 | 浙江大学 | Method for extracting hemoglobin and superoxide dismutase from livestock blood |
| CN101629167A (en) * | 2009-08-26 | 2010-01-20 | 四川大学 | Method for extracting superoxide dismutase |
| CN102181411A (en) * | 2011-04-01 | 2011-09-14 | 黑龙江宝迪肉类食品有限公司 | Method for extracting superoxide dismutase (SOD) from porcine blood red cells |
| CN102250851A (en) * | 2011-08-10 | 2011-11-23 | 天津宝迪农业科技股份有限公司 | Novel process for extracting superoxide dismutase SOD from animal blood cell liquid |
| CN102628036A (en) * | 2012-03-19 | 2012-08-08 | 湖北奥彤生物制品科技有限公司 | Novel impurity-removal purification method for superoxide dismutase SOD production |
| CN103397003A (en) * | 2013-07-22 | 2013-11-20 | 南京宝迪农业科技有限公司 | New process of technology for extracting superoxide dismutase from animal blood |
| CN103436500A (en) * | 2013-08-22 | 2013-12-11 | 慈溪市迈康生物科技有限公司 | Method for extracting superoxide dismutase from animal blood |
| CN104611310A (en) * | 2015-03-10 | 2015-05-13 | 陈石良 | Technology for extracting proteins and superoxide dismutase (SOD) from horseradish tree leaves |
-
2016
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Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101353370A (en) * | 2008-09-09 | 2009-01-28 | 浙江大学 | Method for extracting hemoglobin and superoxide dismutase from livestock blood |
| CN101629167A (en) * | 2009-08-26 | 2010-01-20 | 四川大学 | Method for extracting superoxide dismutase |
| CN102181411A (en) * | 2011-04-01 | 2011-09-14 | 黑龙江宝迪肉类食品有限公司 | Method for extracting superoxide dismutase (SOD) from porcine blood red cells |
| CN102250851A (en) * | 2011-08-10 | 2011-11-23 | 天津宝迪农业科技股份有限公司 | Novel process for extracting superoxide dismutase SOD from animal blood cell liquid |
| CN102628036A (en) * | 2012-03-19 | 2012-08-08 | 湖北奥彤生物制品科技有限公司 | Novel impurity-removal purification method for superoxide dismutase SOD production |
| CN103397003A (en) * | 2013-07-22 | 2013-11-20 | 南京宝迪农业科技有限公司 | New process of technology for extracting superoxide dismutase from animal blood |
| CN103436500A (en) * | 2013-08-22 | 2013-12-11 | 慈溪市迈康生物科技有限公司 | Method for extracting superoxide dismutase from animal blood |
| CN104611310A (en) * | 2015-03-10 | 2015-05-13 | 陈石良 | Technology for extracting proteins and superoxide dismutase (SOD) from horseradish tree leaves |
Non-Patent Citations (1)
| Title |
|---|
| 月桂酰氯修饰羊血超氧化物歧化酶工艺优化及酶学性质;杨学山等;《食品科学》;20141231;第35卷(第7期);128-131 * |
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