CN1224060A

CN1224060A - Method for large scale production of corn superoxide dismutase (SOD) complex enzyme

Info

Publication number: CN1224060A
Application number: CN 99112607
Authority: CN
Inventors: 陈欣; 王成余; 王元志
Original assignee: 王元志
Current assignee: Liaoning Tiancheng Biological Engineering Co., Ltd.
Priority date: 1999-01-07
Filing date: 1999-01-07
Publication date: 1999-07-28
Anticipated expiration: 2019-01-07
Also published as: CN1060213C

Abstract

A process for large-scale preparing complex SOD enzyme using corn as raw material contains SOD for removing free radicals to delay senility, suppress cancers, relieve inflammation and resist against radiation, catalase and peroxidase for removing hydrogen peroxide generated after the action of SOD on free radicals. Its advantages are removing free radicals thoroughly, high antioxidizing power, durable enzyme activity and low cost.

Description

Corn superoxide dismutase from corn (SOD) prozyme method for large scale production

The present invention relates to corn deep processing and Measurement for Biochemistry, particularly from corn kernel, produce the method for large scale production 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 method for large scale production, be with the raw material selection by winnowing, sterilization, germinate, adding material quantity 1-2 PH7.8 doubly contains the phosphate buffered saline buffer making beating of 60mMol, 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%, sterilizes about 30-60 minute with the chlorinated lime aqueous solution of 50-100ppm concentration then, uses twice of flushing with clean water again.

2. will soak diel in the warm water of above-mentioned corn about 28 ℃, make the abundant imbibition of seed, emit residue 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, contain under the situation of phosphate buffered saline buffer of 60mMol concentration constantly adding PH7.8, reach the corase grind shelves with colloidal mill and break into thick slurry, the 1-2 that the amount of buffer of adding is about corn weight is doubly.

5. thick slurry is squeezed into receiver, adds the cellulase of corn weight about 0.2% every gram enzyme activity more than 2000 units, 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, and stirs evenly, and leaves standstill 2-5 hour, waits 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 (with corn weight is radix, and saccharifying enzyme adds water makes it be every 100-150 unit/ml adding) of 0.1-0.3% in filtrate, stirs evenly, and places 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.Effluent liquid concentrates by the neutral ultra-filtration membrane of 4000-10000 molecular weight again, makes it reach needed SOD concentration.

12. in concentrated solution, add the former powder of vitamins C, the former powder of vitamin-E of 0.1-0.2% and the potassium sorbate of 0.1-0.2% of 0.3-0.5%, then concentrated solution inserted 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, centrifugal in 10000 rev/mins tubular-bowl centrifuge again.

14. centrifugate divided in the Plastic Bottle of packing into seal in gnotobasis, uses Co ⁶⁰With the dosage sterilization of 150rad, be placed on and be lower than 10 ℃ of dry lucifuge places and deposit, be liquid SOD product.

15. needn't be freezing with the liquid SOD (12) behind the ultrafiltration and concentration liquid adding auxiliary material, add the Zulkovsky starch of SOD liquid weight 8-10%, stir evenly, become the SOD powder by the spray-drier spray.But packing into, this powder film-making or capsule in the Plastic Bottle, use Co ⁶⁰Under the metering of 100rad, shine degerming, be placed on 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 60mMol, weight is 2000Kg, the cellulase that in thick slurry, adds 2Kg, stir evenly, will slightly starch further and break into screened stock, in screened stock, add the calcium chloride of 10Kg with colloidal mill, treat to filter by plate-and-frame filter press after there is clear liquid on starch sinking upper strata, filter cake is that starch has his usefulness, and filtrate adds the commercially available saccharifying enzyme that is diluted to 120 units/ml concentration of 2Kg, filters 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
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	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

1. a corn superoxide dismutase from corn (SOD) prozyme method for large scale production, be with the raw material selection by winnowing, sterilization, germinate, adding material quantity 1-2 multiple PH7.8 contains the phosphate buffered saline buffer making beating of 60mMol, 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. manufacture method according to claim 1, freezing preceding liquid 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.