CN1060213C - Method for large scale production of corn superoxide dismutase (SOD) complex enzyme - Google Patents
Method for large scale production of corn superoxide dismutase (SOD) complex enzyme Download PDFInfo
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- CN1060213C CN1060213C CN99112607A CN99112607A CN1060213C CN 1060213 C CN1060213 C CN 1060213C CN 99112607 A CN99112607 A CN 99112607A CN 99112607 A CN99112607 A CN 99112607A CN 1060213 C CN1060213 C CN 1060213C
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- 102000019197 Superoxide Dismutase Human genes 0.000 title claims abstract description 41
- 108010012715 Superoxide dismutase Proteins 0.000 title claims abstract description 41
- 240000008042 Zea mays Species 0.000 title claims abstract description 29
- 235000002017 Zea mays subsp mays Nutrition 0.000 title claims abstract description 29
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 title claims abstract description 28
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- 102000004190 Enzymes Human genes 0.000 title claims abstract description 17
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- 238000011031 large-scale manufacturing process Methods 0.000 title abstract 2
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- GVJHHUAWPYXKBD-UHFFFAOYSA-N (±)-α-Tocopherol Chemical compound OC1=C(C)C(C)=C2OC(CCCC(C)CCCC(C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-UHFFFAOYSA-N 0.000 claims description 14
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- 229930003427 Vitamin E Natural products 0.000 claims description 7
- WIGCFUFOHFEKBI-UHFFFAOYSA-N gamma-tocopherol Natural products CC(C)CCCC(C)CCCC(C)CCCC1CCC2C(C)C(O)C(C)C(C)C2O1 WIGCFUFOHFEKBI-UHFFFAOYSA-N 0.000 claims description 7
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- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 6
- 108010059892 Cellulase Proteins 0.000 claims description 6
- 239000001110 calcium chloride Substances 0.000 claims description 6
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 6
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- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims description 4
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- HIVLDXAAFGCOFU-UHFFFAOYSA-N ammonium hydrosulfide Chemical compound [NH4+].[SH-] HIVLDXAAFGCOFU-UHFFFAOYSA-N 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
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- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 description 2
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- Coloring Foods And Improving Nutritive Qualities (AREA)
- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Cosmetics (AREA)
Abstract
A complex superoxide dismutase (SOD) enzyme widely exists in all aerobic organisms and has the function of removing free radicals which have extremely strong destructive capacity to cells. The free radicals are produced by organisms in poor surroundings and abnormal metabolism and are one of the prime reasons for the pathogenesis and the apoptosis of the cells. Thus, SOD has obvious effects of aging delaying, tumor inhibition, inflammation elimination and radioresistance. A preparation of the SOD can be applied to the fields of medicines, health-care products, food, beverages, cosmetics and domestic chemicals. After the SOD reacts with the free radicals, produced hydrogen peroxide is toxic to the cells, and the hydrogen peroxide can be removed by the action of catalase or peroxidase. The product of the present invention comprises the three enzymes, and thus, the product is called as complex SOD enzyme. The product has the advantages of thorough free radical clearance, strong antioxidative capacity and long time enzyme activity maintenance. Corn is used as a raw material, so the present invention has the advantages of low cost, easy obtainment, easy storage, safety and no poisonousness; the process is suitable for large-scale production and has favorable economic benefit and social benefit.
Description
The present invention relates to corn deep processing and Measurement for Biochemistry, particularly from corn kernel, produce the manufacture method of superoxide-dismutase (SOD) prozyme.
Superoxide-dismutase (SOD) extensively is present in the organism, and the disproportionation reaction of its catalyzed oxidation free radical can be removed the superoxide radical that organism is had strong toxic action, and organism is shielded.In recent years, scientific and technical personnel have studied SOD the effect of anti-inflammatory, anti-ageing, Antiradiation injury and inhibition tumor growth have been obtained positive effect, and the SOD that proves all kinds of biologies is all effective, many fields such as medicine, makeup, healthcare products and food SOD have been applied to, shown that it is a kind of economic and social benefit that has very much, the zymin that the utmost point has the exploitation future and has a extensive future.
At present, commercially available SOD preparation mostly extracted preparation from animal blood or tissue, and the portable pathogenic micro-organism of these raw materials is absolutely unsafe, and State Council has issued " blood products management rules " and limited in 1997.The SOD goods that have extract preparation from vegetables or fruit, fruits and vegetables are easy to rot when storing, and are the same with blood, are not the desirable feedstock of scale operation SOD.The main technique that above-mentioned raw materials prepares SOD is: fragmentation, sulphur ammonium precipitation, ultrafiltration, chromatography, concentrated, freeze-drying, and operation is long and complicated, the easy contaminate environment of sulphur ammonium, operation is long, loses enzyme activity easily, and chromatography and freeze-drying all are the technology that is difficult for scale operation.
Through international online retrieval world patent, United States Patent (USP), Chinese patent, biological abstracts, american agriculture digest, CAB Abstracts, U.S. chemical abstract and 4 kinds of domestic technical literatures, all less than finding and similar methods of the present invention.
The present invention provides a kind of corn that utilizes, through germinate, add phosphoric acid salt, pull an oar, add cellulase, pull an oar, add calcium chloride, filter, add saccharifying enzyme, diatomite filtration, centrifugal, ultrafiltration, vitaminize C, vitamin-E, potassium sorbate, freezing, melt centrifugal a series of process, produce the technology of superoxide-dismutase (SOD) prozyme, realize industrialized production.
Corn superoxide dismutase from corn (SOD) prozyme manufacture method, be with the raw material selection by winnowing, sterilization, germinate, add material quantity 1-2 pH7.8 doubly and contain the making beating of 60 mmole phosphate buffered saline buffers, beat the cellulase of thick slurry back adding 0.1-0.3% and beat screened stock again, the calcium chloride that adds 0.5-2%, filter, filtrate adds the saccharifying enzyme of 0.1-0.3%, filter with super-cell, 10000 rev/mins of filtrates are centrifugal, cross molecular weight and be 140,000 film, with the ultra-filtration membrane ultrafiltration of 4000-10000 molecular weight, concentrated solution adds the 0.3-0.5% vitamins C to filtrate by weight again, the potassium sorbate of 0.1-0.2% vitamin-E and 0.1-0.2%, freezing then, melt, centrifugal with 10000 rev/mins again, Co is used in bottling
60Irradiation-sterilize.
Liquid before freezing adds the Zulkovsky starch of 8-10% again, and spraying drying can get former powder, makes tablet or capsule, uses Co again
60Radiation sterilization gets final product.
The invention is novel, reasonable in design, made full use of the maize raw material that extensive resource is arranged, productive rate improves 30%, cost reduces 30-40%, and the production cycle reduces 1/3, can realize industrialized production, mainly show: (1) technology is simple, has saved easily contaminate environment and be difficult for the method for scale operation such as sulphur ammonium precipitation, chromatography and freeze-drying; (2) in the preparation except SOD, also have hydrogen peroxide and two kinds of enzymes of superoxide, these two kinds of enzymes can change into water with the hydrogen peroxide that free radical reaction produces with SOD, the also toxic effect of hydrogen peroxide pair cell is so prozyme removing free radical is completely.Another effect of prozyme is to keep the SOD activity more longways; (3) this technology can produce solid and two kinds of SOD preparations of liquid, and concentration can be regulated in production technique and control, so product can be used for using all fields of SOD; (4) the byproduct amount of starch of this technology is very big, reaches about 80% of corn starch contained therein, is an abundant starch resource.In a word, this invention has the keeping of raw material easily collecting, and technology is simple, and is with short production cycle, scale operation easily, product is renderd a service high, and enzyme retention period alive is long, safety non-toxic, product solid-liquid binary states and concentration can be regulated, by-product volume is big, and the economic worth advantages of higher is a low cost, high benefit industrialization technology.
Main contents of the present invention are as follows:
1. corn kernel is passed through selection by winnowing, the test percentage of germination will reach more than 70%, uses 50-100ppm then
Twice of flushing with clean water used in the chlorinated lime aqueous solution sterilization of concentration about 30-60 minute again.
2. will soak diel in the warm water of above-mentioned corn about 28 ℃, make the abundant imbibition of seed, emit surplus
Surplus moisture content.
3. corn kernel is germinateed, see that the about 1-2mm length of bud gets final product.
Will be in the process with 28 ℃ warm water washing several, in case the corn anaerobically fermenting.
4. with the corn kernel that germinates, constantly adding the phosphate buffered that pH7.8 contains 60 millimolar concentrations
Under the situation of liquid, reach the corase grind shelves with colloidal mill and break into thick slurry, the amount of buffer of adding is about corn
The 1-2 of weight doubly.
5. thick slurry is squeezed into receiver, adds corn weight about 0.2% every gram enzyme activity more than 2000 units
Cellulase stirs evenly.
6. the thinnest shelves that will slightly starch further with colloidal mill break into screened stock.
7. screened stock is squeezed into basin, adds corn weight 0.5-2% with the calcium chloride that anhydride calculates, stir evenly,
Left standstill 2-5 hour, and waited to occur to filter by flame filter press after the supernatant liquor.
8. filter cake is a starch, can make other products, adds the saccharifying enzyme of 0.1-0.3% (with jade in filtrate
Mi Chongliang is a radix, and saccharifying enzyme adds water makes it be every 100-150 unit/ml adding), stir evenly, put
Put half an hour.
9. filtrate is squeezed into the beverage pressure filter, is that flocculating aids filters with diatomite.
10. above-mentioned filtrate is centrifugal in 10000 rev/mins tubular-bowl centrifuge.
11. it is 140,000 ultra-filtration membrane that centrifugate is crossed molecular weight, collects the effluent liquid less than 140,000 molecular weight.Stream
Fluid concentrates by the neutral ultra-filtration membrane of 4000-10000 molecular weight again, makes it reach needed
SOD concentration.
12. the former powder of vitamin-E of the former powder of vitamins C of adding 0.3-0.5%, 0.1-0.2% reaches in concentrated solution
The potassium sorbate of 0.1-0.2% is inserted concentrated solution then it is frozen.
13. moving into indoor the making gradually of 20 ℃ of left and right sides temperature, the concentrated solution that will freeze melts, again at 10000 rev/mins
Tubular-bowl centrifuge in centrifugal.
14. centrifugate divided in the Plastic Bottle of packing into seal in gnotobasis, uses Co
60Dosage with 150rad
Sterilization is placed on and is lower than 10 ℃ of dry lucifuge places and deposits, and is liquid SOD product.
15. needn't be freezing with the liquid SOD (12) behind the ultrafiltration and concentration liquid adding auxiliary material, add SOD liquid weight
The Zulkovsky starch of 8-10% stirs evenly, and becomes the SOD powder by the spray-drier spray.This powder can
Film-making or capsule are packed in the Plastic Bottle, use Co
60Under the metering of 100rad, shine degerming, place
10 ℃ of dry lucifuge place preservations down, be solid SOD product.
16. product based on SOD, also contains hydrogen peroxide and peroxidase.
Most preferred embodiment of the present invention is as follows:
At a volume is 3m
3The germination groove in, add 1000 kilograms of the qualified corn kernels of the good percentage of germination of selection by winnowing, the warm water that adds 28 ℃ is to flooding corn, be incubated about 28 ℃ 24 hours, emit Yu Shui after 24 hours, the corn of imbibition is paved about 30 centimeters thickly impel germination in the germination groove, 28 ℃ of germination groove insulations, the end is the wire netting of aperture less than corn grain, draining and ventilation plant are arranged under the wire netting, corn grain heats up and will in time ventilate during germination, the surface exsiccation will in time be sprinkled water, after seeing germination the germination corn grain is broken thick slurry with colloidal mill, the water that will continue to flow in the time of the corn grain charging adds the phosphate buffered saline buffer of pH7.8, concentration is that 60 mmole weight are 2000Kg, the cellulase that in thick slurry, adds 2Kg, stir evenly, to slightly starch further and break into screened stock with colloidal mill, the calcium chloride that adds 10Kg in screened stock is treated to filter by plate-and-frame filter press after there is clear liquid on starch sinking upper strata, and filter cake is that starch has him to use, filtrate adds the commercially available saccharifying enzyme that is diluted to 120 units/ml concentration of 2Kg, filter by the beverage pressure filter after leaving standstill half an hour, will be coated with on the filter cloth of beverage pressure filter with diatomite, filtrate is centrifugal in 10000 rev/mins tubular-bowl centrifuge.Centrifugate is crossed the ultra-filtration membrane of 140,000 molecular weight earlier, collect effluent liquid, effluent liquid concentrates by the ultra-filtration membrane of 4000 molecular weight again, spissated degree as required, be advisable at 300-600 unit/ml as oral preparation, the vitamins C that concentrated solution is added concentrated solution weight 0.4%, 0.1% vitamin-E, 0.2% potassium sorbate stirs evenly the back at the freezing concentrated solution of refrigeration chamber, concentrated solution is moved to 20 ℃ of room temperatures after freezing to locate to place and wait to thaw, centrifugal with 10000 rev/mins in tubular-bowl centrifuge again after thawing, centrifugate is adorned bottle (according to dose, as 250-500ml) in sterilisable chamber, use Co
60Dose irradiation sterilization at 150rad.
Needn't be freezing with the ultrafiltration and concentration liquid that adds auxiliary material vitamin-E, vitamins C, potassium sorbate, adding is 8% Zulkovsky starch of radix with filtrate, stir evenly, spray at the spray-drier inner drying, promptly get solid SOD powder, the concentration unit of its every gram is about 10 times of ultrafiltration and concentration liquid, can make enteric coated tablet or capsule.
Enzyme activity detects index (u/ml)
| Project | Concentrated solution | Solid, powdery | Analytical procedure |
| Superoxide-dismutase | 608．8 | 2060 | The NBT photoreduction met hod |
| Catalase | 47．6 | 158．2 | Ultraviolet spectrophotometer method |
| Peroxidase | 54．3 | 183．5 | Guaiacol method |
Claims (2)
1. a corn superoxide dismutase from corn (SOD) prozyme manufacture method, be with the raw material selection by winnowing, sterilization, germinate, adding raw material weight 1-2 multiple pH7.8 contains the phosphate buffered saline buffer making beating of 60 mmoles, beat the cellulase of thick slurry back adding 0.1-0.3% and beat screened stock again, the calcium chloride that adds 0.5-2%, filter, filtrate adds the saccharifying enzyme of 0.1-0.3%, filter with super-cell, 10000 rev/mins of filtrates are centrifugal, cross molecular weight and be 140,000 film, filtrate is again with the ultrafiltration of 4000-10000 molecular weight ultra-filtration membrane, concentrated solution adds the 0.3-0.5% vitamins C by weight, the potassium sorbate of 0.1-0.2% vitamin-E and 0.1-0.2%, freezing then, melt, centrifugal with 10000 rev/mins again, Co is used in bottling again
60Irradiation-sterilize.
2. a corn superoxide dismutase from corn (SOD) prozyme manufacture method, be with the raw material selection by winnowing, sterilization, germinate, adding raw material weight 1-2 multiple pH7.8 contains the phosphate buffered saline buffer making beating of 60 mmoles, beat the cellulase of thick slurry back adding 0.1-0.3% and beat screened stock again, the calcium chloride that adds 0.5-2%, filter, filtrate adds the saccharifying enzyme of 0.1-0.3%, filter with super-cell, 10000 rev/mins of filtrates are centrifugal, cross molecular weight and be 140,000 film, filtrate is again with the ultrafiltration of 4000-10000 molecular weight ultra-filtration membrane, concentrated solution adds the 0.3-0.5% vitamins C by weight, the potassium sorbate of 0.1-0.2% vitamin-E and 0.1-0.2%, the Zulkovsky starch that adds 8-10% again, spraying drying can get former powder, make tablet or capsule, use Co again
60Radiation sterilization gets final product.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN99112607A CN1060213C (en) | 1999-01-07 | 1999-01-07 | Method for large scale production of corn superoxide dismutase (SOD) complex enzyme |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN99112607A CN1060213C (en) | 1999-01-07 | 1999-01-07 | Method for large scale production of corn superoxide dismutase (SOD) complex enzyme |
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| Publication Number | Publication Date |
|---|---|
| CN1224060A CN1224060A (en) | 1999-07-28 |
| CN1060213C true CN1060213C (en) | 2001-01-03 |
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| CN99112607A Expired - Fee Related CN1060213C (en) | 1999-01-07 | 1999-01-07 | Method for large scale production of corn superoxide dismutase (SOD) complex enzyme |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1303208C (en) * | 2003-07-23 | 2007-03-07 | 韩伯钧 | Extraction method for SOD complex enzyme coarse powder from corn |
| CN1313606C (en) * | 2003-07-23 | 2007-05-02 | 韩伯钧 | Extraction method for SOD complex enzyme fine powder from corn |
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| CN101892204B (en) * | 2010-06-18 | 2012-07-04 | 李作红 | Method for preparing corn superoxide dismutase from corn |
| CN101962636B (en) * | 2010-10-22 | 2012-12-05 | 江门市天成生物科技有限公司 | Preparation process of high-activity liquid papain |
| CN103421750B (en) * | 2012-05-23 | 2018-12-28 | 王兆非 | Industrialized preparing process and its rudiment room of superoxide dismutase complex enzyme are prepared using corn as raw material |
| CN103173423A (en) * | 2013-03-29 | 2013-06-26 | 湖南大学 | Method for extracting superoxide dismutase with high yield from corn |
| CN106501585B (en) * | 2016-12-09 | 2020-01-07 | 合肥中感微电子有限公司 | Overcharge detection circuit and battery protection system |
| CN112646789B (en) * | 2020-12-31 | 2022-08-19 | 王磊 | Industrial production method for extracting superoxide dismutase from Shenzhou grass |
| CN113373122A (en) * | 2021-07-29 | 2021-09-10 | 辽宁未来生物科技有限公司 | Preparation method of novel corn superoxide dismutase multienzyme |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1087678A (en) * | 1993-11-12 | 1994-06-08 | 沈阳农业大学 | A kind of method of from corn, extracting superoxide-dismutase (SOD) |
| CN1087947A (en) * | 1992-12-10 | 1994-06-15 | 赵慎志 | Produce the method for superoxide-dismutase from corn kernel |
-
1999
- 1999-01-07 CN CN99112607A patent/CN1060213C/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1087947A (en) * | 1992-12-10 | 1994-06-15 | 赵慎志 | Produce the method for superoxide-dismutase from corn kernel |
| CN1087678A (en) * | 1993-11-12 | 1994-06-08 | 沈阳农业大学 | A kind of method of from corn, extracting superoxide-dismutase (SOD) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1303208C (en) * | 2003-07-23 | 2007-03-07 | 韩伯钧 | Extraction method for SOD complex enzyme coarse powder from corn |
| CN1313606C (en) * | 2003-07-23 | 2007-05-02 | 韩伯钧 | Extraction method for SOD complex enzyme fine powder from corn |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1224060A (en) | 1999-07-28 |
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