CN102747054A - Process for extracting superoxide dismutase (SOD) - Google Patents
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Abstract
The invention discloses a process for extracting an enzyme and particularly relates to a process for extracting SOD. The process comprises the steps of crushing roxburgh rose fruits to obtaining roxburgh rose fruit blocks; leaching roxburgh rose fruit blocks to obtain a leaching liquid; conducting centrifugal separation on the leaching liquid to obtain an SOD crude enzyme preparation; conducting ultrafiltration on the SOD crude enzyme preparation to obtain an SOD solution; concentrating the SOD solution to obtain an SOD concentrated solution; and conducting centrifugal spray drying on the SOD concentrated solution to obtain the SOD. The process is a purely physical process for extracting the SOD, can be finished by usage of common simple devices and is simple in process and free of dangers; the temperature adopted in the extracting process is not high, conditions are mild, and the SOD can be stored at the normal temperature; a biotechnology keep-alive process and a purely physical treatment process are adopted, so that the process is free from environmental pollution and endogenous chemical pollution; and the development cost is reduced, and the process is applicable to large-scale extraction of the SOD of plants.
Description
Technical field
The present invention relates to a kind of process for extracting of enzyme, be specifically related to the process for extracting of a kind of superoxide-dismutase (SOD).
Background technology
SOD is the abbreviation of superoxide-dismutase (superoxide dismutase), and international EC is encoded to EC 1.15.1.1, belongs to the redox fermentoid.
SOD is a kind of active substance that can remove mistake polyradical in the body; This material is relevant closely with aging, tumour generation, autoimmune disorder and the radio-protective etc. of body, and is better to curative effect of disease such as treatment rheumatoid arthritis, cataract, urocystitis, tetter, lupus erythematosus.This enzyme no antigen, untoward reaction is little, and spinoff is little, is the medicinal enzyme that a kind of medical value is very high, have wide future.
SOD is divided into three kinds by the difference of metal prothetic group composition, and first kind is that (Cu.Zn-SOD), be green is present in the body cell slurry title that contains copper (Cu) zinc (Zn) metal prothetic group; Second kind is that (Mn-SOD), be purple is present in eukaryotic plastosome and the prokaryotic cell prokaryocyte title that contains manganese (Mn) metal prothetic group; The third is to contain the title of iron (Fe) metal prothetic group (Fe-SOD), be tawny is present in the prokaryotic cell prokaryocyte.
The distribution of SOD is very extensive, has separated obtaining up to now from various organisms such as bacterium, fungi, algae, fish, insect and mammals.But from finding SOD the earliest to the Application and Development of today to SOD, SOD mostly is a separation and Extraction from the blood of animal.Therefore, the mode of disclosed extraction SOD is most is that animal blood extracts and separates.
Because the resource-constrained of the animal blood of separation and Extraction SOD, cost are also very expensive, and then cause raw material nervous, cost increases.When separation and Extraction SOD, need repeatedly add animal blood this material of separating for several times again, make and cause chemical pollution easily by the separation-extraction technology very complicated.In addition, because what use is animal blood, also there be cross infection and other potentially dangerous of virus in the blood such as AIDS when extracting SOD.WHO was once appealed: the use of the SOD that stops at once from animal blood, extracting.
In addition, deriving from the SOD of animal blood, is the unicity enzyme that is called Cu.Zn-SOD) that contains copper (Cu) zinc (Zn) metal prothetic group.
Summary of the invention
The method that the purpose of this invention is to provide a kind of SOD of extraction, this method can obtain SOD from plant.
The process for extracting of SOD provided by the invention may further comprise the steps:
Single Roxburgh Rose Fruit is pulverized, obtained the Single Roxburgh Rose Fruit piece;
The Single Roxburgh Rose Fruit piece is carried out lixiviate, obtain vat liquor;
Vat liquor is carried out spinning, obtain the SOD crude enzyme liquid;
The SOD crude enzyme liquid is carried out ultrafiltration, obtain SOD solution;
With the SOD solution concentration, obtain the SOD liquid concentrator;
SOD liquid concentrator centrifugal spray drying is obtained SOD.
Optional, SOD liquid concentrator centrifugal spray drying is obtained pulverulent solids SOD.
Optional, wild thorn pear is really pulverized, obtain wild thorn pear fruit piece.
Optional, in 60 ~ 70 ℃ pure water, the Single Roxburgh Rose Fruit piece is carried out extracting at constant temperature 8 ~ 12h.
Optional, the SOD crude enzyme liquid is carried out ultrafiltration further comprises:
The SOD crude enzyme liquid through the one-level filter membrane, is collected the one-level trapped fluid;
With passing through the secondary filter membrane again behind the pure water dilution one-level trapped fluid, collect secondary through liquid and second entrapment liquid;
Through passing through three grades of filter membranes behind the liquid again, collect three grades of trapped fluids with pure water dilution secondary;
Second entrapment liquid and three grades of trapped fluids merging are obtained the solution into SOD.
Optional, one-level filter membrane molecular weight is 5000 ~ 10000, secondary filter membrane molecular weight is that 50000 ~ 80000, three grades of filter membrane molecular weight are 20000 ~ 40000.
Optional, SOD solution is carried out PH regulate, make its scope between 3.8 ~ 5.2.
Optional, before Single Roxburgh Rose Fruit is pulverized, also comprise the step of cleaning Single Roxburgh Rose Fruit.
Optional, the step of after cleaning, Single Roxburgh Rose Fruit being dried, and in drying course, add sulphur to Single Roxburgh Rose Fruit.
Optional, with SOD solution decompression vacuum concentration 18 ~ 30min, obtain the SOD liquid concentrator.
The process for extracting of SOD provided by the invention is that full physical method extracts SOD, only needs simple device commonly used to accomplish, and technology is simple, and is not dangerous; And employed temperature is not high in the leaching process, and mild condition can be preserved under the finished product SOD normal temperature; Owing to adopted biotechnology keep-alive technology, full physical treatment technology was not so both non-environmental-pollution had interior originality chemical pollution yet; And then the process for extracting of this SOD can reduce cost of development, is a kind of method that scale is extracted plant SOD that possesses.
Description of drawings
Fig. 1 is the method flow diagram that from wild thorn pear, extracts SOD that the embodiment of the invention 1 provides.
Fig. 2 is the method flow diagram that from wild thorn pear, extracts SOD that the embodiment of the invention 2 provides.
Embodiment
Below in conjunction with accompanying drawing technical scheme provided by the invention is described in detail, the description of this part only is exemplary and explanatory, should not be regarded as the restriction to public technology content of the present invention.
The applicant detects the content of SOD in plantation Rosa roxburghii and the wild thorn pear, and the plantation Rosa roxburghii contains SOD54000U/100g, and wild thorn pear contains SOD ≧ 810000U/100g.The content of SOD is very high in the wild thorn pear, and wild thorn pear generally is grown in the height of 800 meters to 1800 meters of height above sea level, has the extremely strong anti-opposite sex.It is generally acknowledged that the anti-opposite sex of organism is strong more, the content of SOD is just high more.
Embodiment 1:
Below be that the process for extracting that the embodiment of the invention provides is described.Please refer to Fig. 1, Fig. 1 is a kind of method flow diagram that from wild thorn pear, extracts SOD that the embodiment of the invention 1 provides, and this method may further comprise the steps:
Step 101: wild thorn pear is really pulverized, obtained wild thorn pear fruit piece.
Step 102: the wild thorn pear fruit piece lixiviate in water with obtaining obtains vat liquor.
Step 103: vat liquor is carried out spinning, get supernatant and obtain the SOD crude enzyme liquid.
Step 104: the SOD crude enzyme liquid is carried out ultrafiltration, filter out impurity and obtain SOD solution.
Step 105: SOD solution is concentrated, obtain the SOD liquid concentrator.
Step 106: SOD liquid concentrator centrifugal spray drying is obtained SOD.
The process for extracting of the SOD that this embodiment provides is that full physical method extracts, separates the SOD prozyme, only needs simple device commonly used to accomplish, and technology is simple, and is not dangerous; And employed temperature is not high in the leaching process, and mild condition can be preserved under the finished product SOD normal temperature; Owing to adopted biotechnology keep-alive technology, full physical treatment technology was not so both non-environmental-pollution had interior originality chemical pollution yet; And then the process for extracting of this SOD can reduce cost of development, is a kind of method that scale is extracted plant SOD that possesses.
The product that extracts with this method is the polymer of Cu.Zn-SOD, Mn-SOD and three kinds of enzymes of Fe-SOD.
The polymeric method of differentiating three kinds of enzymes is an electrophoretic method: separate through polyacrylamide gel electrophoresis, according to the relative molecular mass of Cu.Zn-SOD, Mn-SOD and Fe-SOD and electrically charged etc. nature difference, on gel strip, be distinguished into different bands.
Embodiment 2:
With reference to figure 2, what embodiment 2 provided really extracts the method for SOD with wild thorn pear, may further comprise the steps:
Step 101: wild thorn pear fruit water is cleaned.
Step 102: the wild thorn pear dried fruit is dry, and embodiment is that nature dries, and reaches water ratio and is 10% ~ 20% and get final product.
Step 103: with kibbler wild thorn pear is really pulverized,, obtained wild thorn pear fruit piece with 10 ~ 20 purpose sieves screening wild thorn pear fruit and impurity.
Step 104: take by weighing 2 kilograms of wild thorn pear fruit pieces and wrap with gauze is loose, put in 40 kilograms of pure water solvents, be warmed to 55 ℃ and keep these temperature-resistant lixiviates 24 hours, per 2 hours hand mixing once obtain vat liquor.
Step 105: with the vat liquor spinning, left standstill then 2 hours, get supernatant liquid and promptly get the SOD crude enzyme liquid.
Step 106: is 5000 one-level filter membrane with the SOD crude enzyme liquid through molecular weight, removes most of small-molecule substance, mainly is inorganic salt and organic acid and polysaccharide, collects one-level trapped fluid and one-level through liquid, dilutes 100 times of one-level trapped fluids with pure water.
Step 107: the one-level trapped fluid after will diluting is 50000 secondary filter membrane through molecular weight, collects second entrapment liquid and secondary through liquid, dilutes 100 times with pure water passing through liquid again.
Step 108: the secondary after will diluting is three grades of filter membranes of 20000 through liquid through molecular weight, collects three grades of trapped fluids.Second entrapment liquid, three grades of trapped fluids merging are SOD solution, and its staple is the polymer of Cu.Zn-SOD, Mn-SOD and three kinds of enzymes of Fe-SOD.
Step 109: with the one-level trapped fluid after the dilution SOD solution is carried out PH and regulate, make its pH value reach 3.8.
Step 110: will pass through the SOD solution decompression vacuum concentration that PH regulates, temperature remains on 80 ℃, and concentration time 18min obtains the SOD liquid concentrator.
Step 111: with SOD liquid concentrator centrifugal spray drying, the intake air temperature of drying machine with centrifugal spray is 180 ℃, and drop temperature can remain on about 50 ℃ like this.
Step 112: obtain SOD.
The polymeric method of differentiating three kinds of enzymes is an electrophoretic method: separate through polyacrylamide gel electrophoresis, according to the relative molecular mass of Cu.Zn-SOD, Mn-SOD and Fe-SOD and electrically charged etc. nature difference, on gel strip, be distinguished into different bands.
SOD is an antioxygen enzyme of single-minded removing ultra-oxygen anion free radical in the body, and its cleaning reaction is following:
O
2 -·+O
2 -·+2H
+→H
2O
2+O
2
SOD guarantees efficient removing (O through three approach
2 -).The variety of first utilization metal prothetic group form in case a kind of enzyme is obstructed, compensates with regard to available another kind; The 2nd, be utilized in the high conservative in the evolution, prevent variation; The 3rd, by the structure unity that shows height on the relevant position with catalysis, guarantee carrying out smoothly of catalysis.
Embodiment 3:
The method of really extracting SOD with wild thorn pear is provided, all identical except following steps with embodiment 2:
Step 104: 2 kilograms of wild thorn pear fruit pieces that take by weighing after the pulverizing are wrapped with gauze is loose, put in 100 kilograms of pure water solvents, are warmed to 90 ℃ and keep these temperature-resistant lixiviates 8 hours, and per 2 hours hand mixing once obtain vat liquor.
Step 106: is 10000 one-level filter membrane with the SOD crude enzyme liquid through molecular weight, removes most of small-molecule substance, mainly is inorganic salt and organic acid and polysaccharide, collects one-level trapped fluid and one-level through liquid, dilutes 100 times of one-level trapped fluids with pure water.
Step 107: the one-level trapped fluid after will diluting is 80000 secondary filter membrane through molecular weight, collects second entrapment liquid and secondary through liquid, dilutes 100 times with pure water passing through liquid again.
Step 108: the secondary after will diluting is three grades of filter membranes of 40000 through liquid through molecular weight, collects three grades of trapped fluids.Second entrapment liquid, three grades of trapped fluids merging are SOD solution, and its staple is the polymer of Cu.Zn-SOD, Mn-SOD and three kinds of enzymes of Fe-SOD.
Step 109: with the one-level trapped fluid after the dilution SOD solution is carried out PH and regulate, make its pH value reach 4.0.
Step 110: will pass through the SOD solution decompression vacuum concentration that PH regulates, temperature remains on 65 ℃, and concentration time 30min obtains the SOD liquid concentrator.
Step 111: with SOD liquid concentrator centrifugal spray drying, 160 ℃ of drying machine with centrifugal spray intake air temperatures, 20 ℃ of drop temperatures.
SOD is a kind of crucial metalloenzyme of defence oxidative damage in the organism, as the scavenging agent of single-minded ultra-oxygen anion free radical, has application widely at aspects such as treatment of diseases, food, makeup, agriculturals.
Embodiment 4:
The method of extracting SOD with the plantation Single Roxburgh Rose Fruit is provided, all identical except following steps with embodiment 2:
Step 104: 2 kilograms of plantation Single Roxburgh Rose Fruit pieces that take by weighing after the pulverizing are wrapped with gauze is loose, put in 140 kilograms of pure water solvents, are warmed to 70 ℃ and keep these temperature-resistant lixiviates 12 hours, and per 2 hours hand mixing once obtain vat liquor.
Step 109: with the one-level trapped fluid after the dilution SOD solution is carried out PH and regulate, make its pH value reach 5.2.
Step 110: will pass through the SOD solution decompression vacuum concentration that PH regulates, temperature remains on 75 ℃, and concentration time 20min obtains the SOD liquid concentrator.
Step 111: with SOD liquid concentrator centrifugal spray drying, 160 ℃ of drying machine with centrifugal spray intake air temperatures, 20 ℃ of drop temperatures.
The SOD that uses this method to extract is the synthesis of three kinds of enzymes, and first kind is 65.3% for Cu.Zn-SOD content; Second kind is 14.4% for Mn-SOD content; The third is that Fe-SOD content is 20.3%.
Embodiment 5:
The method of really extracting SOD with wild thorn pear is provided, all identical except following steps with embodiment 2:
Step 102: with wild thorn pear fruit heating, drying, wild thorn pear per ton really adds 6 gram sulphur and cooks protective material simultaneously, keeps the biotic component and the active substance of Rosa roxburghii dry fruit to greatest extent, reaches water ratio and is 10% ~ 20% and can stop oven dry.Wild thorn pear really adds the amount of sulphur and can confirm according to actual needs.
Step 104: 2 kilograms of wild thorn pear fruit pieces that take by weighing after the pulverizing are wrapped with gauze is loose, put in 100 kilograms of pure water solvents, are warmed to 60 ℃ and keep these temperature-resistant lixiviates 8 hours, and per 2 hours hand mixing once obtain vat liquor.
Step 109: with the one-level trapped fluid after the dilution SOD solution is carried out PH and regulate, make its pH value reach 4.5.
Step 110: will pass through the SOD solution decompression vacuum concentration that PH regulates, temperature remains on 70 ℃, and concentration time 20min obtains the SOD liquid concentrator.
Step 111: with SOD liquid concentrator centrifugal spray drying, 160 ℃ of drying machine with centrifugal spray intake air temperatures, 20 ℃ of drop temperatures.
Present method mainly is that full physical method extracts, separates SOD, only needs simple device commonly used to accomplish, and technology is simple, and is not dangerous; And employed temperature is not higher than 200 ℃ in the leaching process, and mild condition can be preserved under the finished product SOD normal temperature; Owing to adopted biotechnology keep-alive technology, full physical treatment technology was not so both non-environmental-pollution had interior originality chemical pollution yet; Reducing cost of development, is a kind of method that scale is extracted plant SOD that possesses.
Embodiment 6:
The method of really extracting SOD with wild thorn pear is provided, all identical except following steps with embodiment 2:
Step 102: with wild thorn pear fruit heating, drying, wild thorn pear per ton really adds 6 gram sulphur and cooks protective material simultaneously, keeps the biotic component and the active substance of Rosa roxburghii dry fruit to greatest extent, reaches water ratio and is 10% ~ 20% and can stop oven dry.
Step 104: 2 kilograms of wild thorn pear fruit pieces that take by weighing after the pulverizing are wrapped with gauze is loose, put in 100 kilograms of pure water solvents, are warmed to 65 ℃ and keep these temperature-resistant lixiviates 24 hours, and per 2 hours hand mixing once obtain vat liquor.
Step 109: the wild thorn pear juice with the one-level trapped fluid after the dilution carries out the PH adjusting to SOD solution, makes its pH value reach 5.0.
Step 110: will pass through the SOD solution decompression vacuum concentration that PH regulates, temperature remains on 80 ℃, and concentration time 18min obtains the SOD liquid concentrator.
Step 111: with SOD liquid concentrator centrifugal spray drying, 180 ℃ of intake air temperatures, 50 ℃ of drop temperatures.
This process for extracting has a extensive future in the world market, and domestic market demand is also very big, and third generation protective foods is only admitted by many advanced countries, can adapt to the demand and entering WTO and world market of development.For convenience has been opened in the production industrialization of the oxidation resistant product that directly utilizes plant resources.Be intended to strengthen the original innovation ability and the international competitiveness of China's functional food through research and industrialization demonstration to the functional food gordian technique.
Used concrete example among this paper technical scheme provided by the invention is set forth, the explanation of above embodiment just is used for helping to understand technical scheme provided by the invention.Should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention, can also carry out some improvement and modification the present invention; These improvement and modification also fall in the protection domain of claim of the present invention.
Claims (10)
1. the process for extracting of a superoxide-dismutase SOD is characterized in that, may further comprise the steps:
Single Roxburgh Rose Fruit is pulverized, obtained the Single Roxburgh Rose Fruit piece;
The Single Roxburgh Rose Fruit piece is carried out lixiviate, obtain vat liquor;
Vat liquor is carried out spinning, obtain the SOD crude enzyme liquid;
To contain the SOD crude enzyme liquid and carry out ultrafiltration, obtain SOD solution;
With the SOD solution concentration, obtain the SOD liquid concentrator;
SOD liquid concentrator centrifugal spray drying is obtained SOD.
2. the process for extracting of SOD as claimed in claim 1 is characterized in that, SOD liquid concentrator centrifugal spray drying is obtained pulverulent solids SOD.
3. the process for extracting of SOD as claimed in claim 1 is characterized in that, wild thorn pear is really pulverized, and obtains wild thorn pear fruit piece.
4. the process for extracting of SOD as claimed in claim 1 is characterized in that, in 60 ~ 70 ℃ pure water, the Single Roxburgh Rose Fruit piece is carried out extracting at constant temperature 8 ~ 12h.
5. the process for extracting of SOD as claimed in claim 1 is characterized in that, the SOD crude enzyme liquid is carried out ultrafiltration further comprise:
The SOD crude enzyme liquid through the one-level filter membrane, is collected the one-level trapped fluid;
With passing through the secondary filter membrane again behind the pure water dilution one-level trapped fluid, collect secondary through liquid and second entrapment liquid;
Through passing through three grades of filter membranes behind the liquid again, collect three grades of trapped fluids with pure water dilution secondary;
Second entrapment liquid and three grades of trapped fluids merging are obtained SOD solution.
6. the process for extracting of SOD as claimed in claim 5 is characterized in that, one-level filter membrane molecular weight is 5000 ~ 10000, and secondary filter membrane molecular weight is that 50000 ~ 80000, three grades of filter membrane molecular weight are 20000 ~ 40000.
7. like the process for extracting of each described SOD of claim 1 to 6, it is characterized in that, SOD solution is carried out PH regulate, make its scope between 3.8 ~ 5.2.
8. like the process for extracting of each described SOD of claim 1 to 6, it is characterized in that, before Single Roxburgh Rose Fruit is pulverized, also comprise the step of cleaning Single Roxburgh Rose Fruit.
9. the process for extracting of SOD as claimed in claim 8 is characterized in that, the step of after cleaning, Single Roxburgh Rose Fruit being dried, and in drying course, add sulphur to Single Roxburgh Rose Fruit.
10. like the process for extracting of each described SOD of claim 1 to 6, it is characterized in that,, obtain the SOD liquid concentrator SOD solution decompression vacuum concentration 18 ~ 30min.
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Cited By (13)
Publication number | Priority date | Publication date | Assignee | Title |
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CN106318919A (en) * | 2016-02-20 | 2017-01-11 | 武熙熙 | Method for preparing wild rosa SOD complex enzyme |
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