CN102181411A - Method for extracting superoxide dismutase (SOD) from porcine blood red cells - Google Patents
Method for extracting superoxide dismutase (SOD) from porcine blood red cells Download PDFInfo
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- CN102181411A CN102181411A CN2011100816641A CN201110081664A CN102181411A CN 102181411 A CN102181411 A CN 102181411A CN 2011100816641 A CN2011100816641 A CN 2011100816641A CN 201110081664 A CN201110081664 A CN 201110081664A CN 102181411 A CN102181411 A CN 102181411A
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Abstract
The invention relates to a method for extracting superoxide dismutase (SOD) from porcine blood red cells, in the method, anticoagulation treatment is adopted for preventing blood coagulation and breaking of blood cells. Colloid mill breaking and low-temperature swelling are adopted for releasing matters dissolved in the blood cells to the maximum extent so as to get hemolysate. Thermal change is performed on the hemolysate twice for removing impurities, removing hemoglobin and a large number of impurity proteins so as to get SOD crude enzyme liquid, and ultrafiltration concentration, gradient elution, dialysis, salt removal and freeze-drying are further performed on the SOD crude enzyme liquid so as to get an SOD product. The method is simple to operate and easy to master; furthermore, by adopting the method, the product quality can be effectively improved, the production cost can be reduced, the production cycle can be shortened, the production efficiency can be improved, and the method is applicable to industrialized production.
Description
Technical field:
The present invention relates to a kind of extracting method of superoxide-dismutase, particularly a kind of method of from pig erythrocyte, extracting superoxide-dismutase SOD.
Background technology:
Superoxide-dismutase (Superoxide Dismutase is hereinafter to be referred as SOD) is a kind of metalloenzyme that can remove excessive free radicals in the body, has been found extensively to be present in the intravital multiple tissue of humans and animals.The effect that SOD has is anti-ageing, prevent pigment deposition, anti-inflammatory and protection body's immunity.Aspects such as food, healthcare products, makeup and agricultural have been widely used at present.
The existing method of extracting SOD from hemocyte is similar getting on the steps such as blood, anti-freezing, physiological saline washing and low temperature haemolysis of early stage.The foreign protein sex change in later stage adopts chloroform and Ethanol Method to remove oxyphorase mostly, clear liquid acetone precipitation then, chromatographic column on the throw out (Sephadex G-100 or DE-32 or metal chelate chromatography method), its shortcoming is: it is higher that (1) removes the oxyphorase cost with chloroform and ethanol, while chloroform or toxic reagent, the residual security of reagent does not guarantee; (2) removing in the process of oxyphorase with the chloroform alcohol method, because the coprecipitated effect of SOD and oxyphorase causes the SOD yield to reduce; (3) owing to the restriction of chromatographic column volume, the purification process amount is little, and equipment cost drops into high, is unfavorable for large-scale production.
Goal of the invention:
The object of the present invention is to provide the method for a kind of large-scale production SOD, this method technology is simple, the SOD purity of extraction and safe, simultaneously can reduce the cost input.
Summary of the invention:
As above design, technical scheme of the present invention is: a kind of method of extracting copper-zinc superoxide dismutase SOD from pig erythrocyte is characterized in that: finish according to the following step:
1. collect the pig blood of fresh and healthy;
2. pig blood being carried out anti-freezing handles;
3. the pig blood after the anti-freezing is carried out the high speed centrifugation processing and obtain blood cell liquid;
4. blood cell liquid is carried out the haemolysis fragmentation; Through colloidal mill fragmentation twice, the distilled water or the high purity water that add 2 times of volumes then spend the night 0 ℃ of-4 ℃ of swelling blood cell liquid, obtain hemolysate earlier;
5. above-mentioned hemolysate is obtained the SOD crude enzyme liquid by twice thermal change removal of impurities process; Concrete grammar is: adding earlier the food grade sodium salt in the hemolysate, to make saturation ratio be 4%-12%, add mantoquita and zinc salt then respectively, make its concentration reach 0.5mmol/L-3mmol/L, be incubated 10min-30min in 50 ℃ of-70 ℃ of thermostatic equipments, cooling separates 20min-30min with the 3-foot separating machine then, get supernatant liquor, separate with the tube type solid-liquid separating machine again, remove precipitation, collect supernatant liquor; Add mantoquita and zinc salt in the supernatant liquor, make its concentration reach 1mmol/L-6mmol/L, in 60 ℃ of-70 ℃ of following thermostatic equipments, be incubated 10min-30min, cooling separates 20-30min with the 3-foot separating machine then, gets supernatant liquor, separate with the tube type solid-liquid separating machine again, remove precipitation, collect supernatant liquor;
6. the supernatant liquor that 5. above-mentioned steps is obtained carries out ultrafiltration and concentration and obtains concentrated solution;
7. concentrated solution is carried out gradient elution; Concrete grammar is: concentrated solution is carried out multistage phosphate buffered saline buffer gradient elution, the elementary concentration of phosphate buffered saline buffer is 1mmol/L-3mmol/L, ultimate density is 50mmol/L-70mmol/L, the concentration gradient multiple of every two-stage is 2-5 times, be cooled to 1 ℃-5 ℃ earlier before every grade of concentration gradient of supernatant liquor is handled, add 1-2 cold acetone doubly then, rapidly frozen centrifugation 3-10min under 2000r/min-4000r/min, get precipitation, supernatant liquor reclaims as acetone; Precipitation is dissolved with 3-15 phosphate buffered saline buffer doubly, and frozen centrifugation 5-15min under 4000r/min-6000r/min gets supernatant rapidly, abandons precipitation;
8. the supernatant liquor that 7. above-mentioned steps is obtained is packed in the dialysis tubing at 0 ℃ of-4 ℃ of current downflow dialysis 24h-36h desalination of dialysing;
9. lyophilize: the good supernatant liquor lyophilize of will dialysing promptly makes the SOD product.
Above-mentioned antithrombotics is the food grade Trisodium Citrate, addition 4.0-6.0g/kg pig blood.
The sodium salt that adds in the above-mentioned thermal change removal of impurities process can be one or several the mixture in sodium-chlor, Sodium Bromide, SODIUMNITRATE, the sodium sulfate; The mantoquita that adds can be one or several mixtures in cupric chloride, copper sulfate or the cupric nitrate; The zinc salt that adds can be one or several mixtures in zinc chloride, zinc sulfate or the zinc nitrate.
The concrete grammar of above-mentioned supernatant liquor ultrafiltration and concentration is to be 6000 hollow-fibre membrane ultrafilter with supernatant liquor through molecular weight cut-off, constantly adds 4 ℃ of left and right sides high purity waters, obtains concentrated solution through concentrating again after removing small molecules foreign protein and metal ion.
Above-mentioned concentrated solution carries out the phosphate buffered saline buffer gradient elution of 2-7 level.
The used salt of above-mentioned phosphate buffered saline buffer is the two kinds or more of mixtures in Sodium phosphate dibasic, SODIUM PHOSPHATE, MONOBASIC, potassium primary phosphate, the dipotassium hydrogen phosphate, and its pH is between 6.5-8.0.
The used dialyzate of above-mentioned dialysis desalination is used distilled water in earlier stage, and the later stage can be with high purity water or PEG-6000/20000.
Invention has following advantage and positively effect:
1, the present invention has carried out the anti-freezing processing in order to prevent that blood coagulation and hemocyte from breaking.
2, the present invention has adopted the homogeneous crushing technology in the hemolytic process of cell, simultaneously in conjunction with cell low temperature swelling technology, not only broken to the full extent cell has discharged the cell Dissolve things inside, and whole process carries out at low temperatures, kept the SOD activity to the full extent.
3; the present invention does not adopt conventional chloroform alcohol method to remove oxyphorase; but adopt twice thermal change impurity removal method make the oxyphorase sex change method; mantoquita and zinc salt have been added in this method; not only SOD there is good provide protection; and improved the probability of foreign protein sex change; simultaneously in the temperature-dependent process, added sodium salt; sodium salt plays the effect that salt is separated in the thermal change process; suitable ionic strength can farthest reduce the coprecipitated effect in the thermal change process; simultaneously the SOD activity be can not reduce again, thereby SOD productive rate and activity improved because of too high ionic concn.And, in thermal change removal of impurities separating process, adopted the method for two kinds of separating machine couplings, not only remove small metaprotein, and improved production efficiency, shortened the production cycle.
4, the present invention has adopted the method for gradient elution in the purification step in later stage, and traditional technology often needs to adopt the chromatography column method, therefore the present invention can avoid too high equipment input of chromatography column method and the lower problem of chromatography column productive rate, so not only help suitability for industrialized production, and improved the SOD productive rate, can create higher profit.
Simultaneously, the acetone waste liquid that linear gradient elution method is produced in the process of later stage purifying can reclaim by simple distillation method, has therefore saved cost, is beneficial to large-scale production more.
Therefore, linear gradient elution method of the present invention is simple to operate, easier grasp in large-scale production.
Embodiment:
A kind of method of from pig erythrocyte, extracting copper-zinc superoxide dismutase SOD, finish according to the following step:
1, collects the pig blood of fresh and healthy; The pig blood of fresh and healthy is meant the animal blood of processing all from the large-scale slaughterhouse of fixed point, and all pigs only must be through the no eqpidemic disease of national Animal Quarantine department's check.
2, pig blood being carried out anti-freezing handles: the antithrombotics of employing is the food grade Trisodium Citrate, addition 4.0-6.0g/kg pig blood.
3, the high speed centrifugation of the pig blood after the anti-freezing by tubular-bowl centrifuge obtained the blood cell liquid that volume is 50L.The rotating speed of tubular-bowl centrifuge is 14000r/min-16000r/min.
4, blood cell is carried out the haemolysis fragmentation; Through colloidal mill fragmentation twice, the distilled water or the high purity water that add 2 times of volumes then spend the night 0 ℃ of-4 ℃ of swelling blood cell earlier;
5, in hemolysate, add sodium-chlor earlier, making its saturation ratio is 6%, add mantoquita and zinc salt then respectively, make its concentration reach lmmol/L, in 65 ℃ of thermostatic equipments, be incubated 15min, cooling, separate 30min with the 3-foot separating machine then, get supernatant, separate with the tube type solid-liquid separating machine again, remove precipitation, collect supernatant liquor; Add mantoquita and zinc salt in the supernatant liquor, make its concentration reach 2mmol/L, be incubated 15min in 70 ℃ of thermostatic equipments, cooling separates 30min with the 3-foot separating machine then, gets supernatant, separates with the tube type solid-liquid separating machine again, removes precipitation, collects supernatant liquor.
6, be 6000 hollow-fibre membrane ultrafilter with supernatant liquor through molecular weight cut-off, constantly add 4 ℃ of left and right sides high purity waters, remove small molecules foreign protein and metal ion after, concentrate and obtain concentrated solution.
7, concentrated solution is carried out 4 grades of phosphate buffered saline buffer gradient elutions, the elementary concentration of phosphate buffered saline buffer is 1.5mmol/L, and ultimate density is 40.5mmol/L, pH of buffer=7.0.The concentration gradient multiple of every two-stage is 3 times, is cooled to 1 ℃ earlier before every grade of concentration gradient of supernatant liquor is handled, and adds the cold acetone of 2 times of volumes then, and centrifugal 5min under 4000r/min gets precipitation rapidly, and supernatant liquor reclaims as acetone.Precipitation is dissolved with 6 times phosphate buffered saline buffer, and centrifugal 10min under 5000r/min gets supernatant rapidly, abandons precipitation.
8, supernatant liquor is packed in the dialysis tubing, at 0 ℃ of-4 ℃ of current downflow dialysis 24h, dialyzate is used distilled water in earlier stage, and the later stage concentrates with PEG-6000.
9, the supernatant liquor lyophilize that will dialyse and get well, the SOD product that promptly makes.
Claims (7)
1. method of extracting copper-zinc superoxide dismutase SOD from pig erythrocyte is characterized in that: finish according to the following step:
1. collect the pig blood of fresh and healthy;
2. pig blood being carried out anti-freezing handles;
3. the pig blood after the anti-freezing is carried out the high speed centrifugation processing and obtain blood cell liquid;
4. blood cell liquid is carried out the haemolysis fragmentation; Through colloidal mill fragmentation twice, the distilled water or the high purity water that add 2 times of volumes then spend the night 0 ℃ of-4 ℃ of swelling blood cell liquid, obtain hemolysate earlier;
5. above-mentioned hemolysate is obtained the SOD crude enzyme liquid by twice thermal change removal of impurities process; Concrete grammar is: adding earlier the food grade sodium salt in the hemolysate, to make saturation ratio be 4%-12%, add mantoquita and zinc salt then respectively, make its concentration reach 0.5mmol/L-3mmol/L, be incubated 10min-30min in 50 ℃ of-70 ℃ of thermostatic equipments, cooling separates 20min-30min with the 3-foot separating machine then, get supernatant liquor, separate with the tube type solid-liquid separating machine again, remove precipitation, collect supernatant liquor; Add mantoquita and zinc salt in the supernatant liquor, make its concentration reach lmmol/L-6mmol/L, in 60 ℃ of-70 ℃ of following thermostatic equipments, be incubated 10min-30min, cooling separates 20-30min with the 3-foot separating machine then, gets supernatant liquor, separate with the tube type solid-liquid separating machine again, remove precipitation, collect supernatant liquor;
6. the supernatant liquor that 5. above-mentioned steps is obtained carries out ultrafiltration and concentration and obtains concentrated solution;
7. concentrated solution is carried out gradient elution; Concrete grammar is: concentrated solution is carried out multistage phosphate buffered saline buffer gradient elution, the elementary concentration of phosphate buffered saline buffer is lmmol/L-3mmol/L, ultimate density is 50mmol/L-70mmol/L, the concentration gradient multiple of every two-stage is 2-5 times, be cooled to 1 ℃-5 ℃ earlier before every grade of concentration gradient of supernatant liquor is handled, add 1-2 cold acetone doubly then, rapidly frozen centrifugation 3-10min under 2000r/min-4000r/min, get precipitation, supernatant liquor reclaims as acetone; Precipitation is dissolved with 3-15 phosphate buffered saline buffer doubly, and frozen centrifugation 5-15min under 4000r/min-6000r/min gets supernatant rapidly, abandons precipitation;
8. the supernatant liquor that 7. above-mentioned steps is obtained is packed in the dialysis tubing at 0 ℃ of-4 ℃ of current downflow dialysis 24h-36h desalination of dialysing;
9. lyophilize: the good supernatant liquor lyophilize of will dialysing promptly makes the SOD product.
2. a kind of method of extracting copper-zinc superoxide dismutase SOD from pig erythrocyte according to claim 1, it is characterized in that: above-mentioned antithrombotics is the food grade Trisodium Citrate, addition 4.0-6.0g/kg pig blood.
3. a kind of method of extracting copper-zinc superoxide dismutase SOD from pig erythrocyte according to claim 1 is characterized in that: the sodium salt that adds in the above-mentioned thermal change removal of impurities process can be one or several the mixture in sodium-chlor, Sodium Bromide, SODIUMNITRATE, the sodium sulfate; The mantoquita that adds can be one or several mixtures in cupric chloride, copper sulfate or the cupric nitrate; The zinc salt that adds can be one or several mixtures in zinc chloride, zinc sulfate or the zinc nitrate.
4. a kind of method of from pig erythrocyte, extracting copper-zinc superoxide dismutase SOD according to claim 1, it is characterized in that: the concrete grammar of above-mentioned supernatant liquor ultrafiltration and concentration is to be 6000 hollow-fibre membrane ultrafilter through molecular weight cut-off with supernatant liquor, constantly add 4 ℃ of left and right sides high purity waters, obtain concentrated solution through concentrating again after removing small molecules foreign protein and metal ion.
5. a kind of method of extracting copper-zinc superoxide dismutase SOD from pig erythrocyte according to claim 1, it is characterized in that: above-mentioned concentrated solution carries out the phosphate buffered saline buffer gradient elution of 2-7 level.
6. a kind of method of from pig erythrocyte, extracting copper-zinc superoxide dismutase SOD according to claim 1, it is characterized in that: the used salt of above-mentioned phosphate buffered saline buffer is the two kinds or more of mixtures in Sodium phosphate dibasic, SODIUM PHOSPHATE, MONOBASIC, potassium primary phosphate, the dipotassium hydrogen phosphate, and its pH is between 6.5-8.0.
7. a kind of method of from pig erythrocyte, extracting copper-zinc superoxide dismutase SOD according to claim 1, it is characterized in that: the used dialyzate of above-mentioned dialysis desalination is used distilled water in earlier stage, and the later stage can be with high purity water or PEG-6000/20000.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103361320A (en) * | 2013-07-25 | 2013-10-23 | 天津核生科技生物工程有限公司 | Method of extracting SOD from milk |
| CN106119216A (en) * | 2016-07-08 | 2016-11-16 | 陈石良 | SOD and the joint production process of hemoglobin oligopeptide powder is extracted from poultry blood cell liquid |
| CN106399271A (en) * | 2016-11-01 | 2017-02-15 | 湖北宝迪农业科技有限公司 | Method for extracting superoxide dismutase from pig blood and superoxide dismutase |
| CN106754767A (en) * | 2016-09-08 | 2017-05-31 | 刘加明 | A kind of superoxide dismutase aliphatic acid inclusion compound and preparation method thereof |
| CN107164340A (en) * | 2017-07-13 | 2017-09-15 | 成都宏安生物科技有限公司 | The extracting method of superoxide dismutase in a kind of fresh porcine blood |
| CN109055325A (en) * | 2018-08-28 | 2018-12-21 | 佛山科学技术学院 | A kind of isolation and purification method of duck blood erythrocyte sod |
| CN112791103A (en) * | 2020-12-31 | 2021-05-14 | 四平华科生物技术有限责任公司 | Deer blood product and preparation method thereof |
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| CN1464050A (en) * | 2002-06-07 | 2003-12-31 | 王捷 | Process for extracting superoxide dismutase from fresh animal blood |
| CN1616655A (en) * | 2004-08-14 | 2005-05-18 | 李维标 | Method for preparing superoxide dismutase |
| CN101139576A (en) * | 2007-08-17 | 2008-03-12 | 浙江大学 | Method for extracting superoxide dismutase from erythrocyte |
| CN101353370A (en) * | 2008-09-09 | 2009-01-28 | 浙江大学 | Method for extracting hemoglobin and superoxide dismutase from livestock blood |
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Patent Citations (5)
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| CN1274754A (en) * | 2000-06-03 | 2000-11-29 | 吉林大学生命科学技术研究所 | Method of extracting high-purity copper-zinc superoxide dismutase from animal's blood |
| CN1464050A (en) * | 2002-06-07 | 2003-12-31 | 王捷 | Process for extracting superoxide dismutase from fresh animal blood |
| CN1616655A (en) * | 2004-08-14 | 2005-05-18 | 李维标 | Method for preparing superoxide dismutase |
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| CN101353370A (en) * | 2008-09-09 | 2009-01-28 | 浙江大学 | Method for extracting hemoglobin and superoxide dismutase from livestock blood |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103361320A (en) * | 2013-07-25 | 2013-10-23 | 天津核生科技生物工程有限公司 | Method of extracting SOD from milk |
| CN103361320B (en) * | 2013-07-25 | 2014-06-18 | 天津核生科技生物工程有限公司 | Method of extracting SOD from milk |
| CN106119216A (en) * | 2016-07-08 | 2016-11-16 | 陈石良 | SOD and the joint production process of hemoglobin oligopeptide powder is extracted from poultry blood cell liquid |
| CN106754767A (en) * | 2016-09-08 | 2017-05-31 | 刘加明 | A kind of superoxide dismutase aliphatic acid inclusion compound and preparation method thereof |
| CN106399271A (en) * | 2016-11-01 | 2017-02-15 | 湖北宝迪农业科技有限公司 | Method for extracting superoxide dismutase from pig blood and superoxide dismutase |
| CN106399271B (en) * | 2016-11-01 | 2020-02-18 | 湖北宝迪农业科技有限公司 | Method for extracting superoxide dismutase from pig blood and superoxide dismutase |
| CN107164340A (en) * | 2017-07-13 | 2017-09-15 | 成都宏安生物科技有限公司 | The extracting method of superoxide dismutase in a kind of fresh porcine blood |
| CN109055325A (en) * | 2018-08-28 | 2018-12-21 | 佛山科学技术学院 | A kind of isolation and purification method of duck blood erythrocyte sod |
| CN112791103A (en) * | 2020-12-31 | 2021-05-14 | 四平华科生物技术有限责任公司 | Deer blood product and preparation method thereof |
| CN112791103B (en) * | 2020-12-31 | 2024-04-19 | 四平华科生物技术有限责任公司 | Deer blood product and preparation method thereof |
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Application publication date: 20110914 |