CN101104848A - Method for preparing plant superoxide dismutase - Google Patents
Method for preparing plant superoxide dismutase Download PDFInfo
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- CN101104848A CN101104848A CNA200610052491XA CN200610052491A CN101104848A CN 101104848 A CN101104848 A CN 101104848A CN A200610052491X A CNA200610052491X A CN A200610052491XA CN 200610052491 A CN200610052491 A CN 200610052491A CN 101104848 A CN101104848 A CN 101104848A
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Abstract
The method which is to prepare a plant superoxide dismatase has a plurality of procedures, including pulverizing and slurrying each raw materials separately; mixing the slurrying products; adding alkaline ion liquid to make the products undergo anaerobic fermentation; adding saturated buffer solution; putting oxidant together with extraction agent into a fermentation chamber, dehydrating and making the products sedimentate; filtrating the products, cooling the filtrate in anaerobic vacuum; adding dehydrated oxidant and making the filtrate sedimentate to get raw enzyme; pump filtering the raw enzyme and separating the filtrate by centrifugation, freezing and drying the sediment in vacuum; adding reducing sugar substrate to the products and adding masking agent and then bathing the products in warm water and pump filtering the products; adding dehydration oxidant to the filtrate in step (7) and placing the filtrate stationary in an anaerobic environment, then pump filtering and separating the filtrate by centrifugation and freezing and drying the filtrate with a vacuum machine. In this way the superoxide dismatase is obtained. Compared with prior art, the invention has the advantages that: the SOD of the invention, based on cheap Chinese herbal medicine and soybean meals and combined with the merits of the Chinese herbal medicine and soybean meals which coordinate mutually and undergo anaerobic fermentation, has high activity in restraining germ enzymes, and is hygienic and pollution-free. With good antiphlogosis effect, the SOD can be taken orally or applied externally.
Description
Technical field
The present invention relates to a kind of preparation method of enzyme, relate in particular to the preparation method of a kind of plant superoxide dismutase, belong to the biochemical engineering technical field.
Background technology
Superoxide dismutase (superoxide, dismutase, be called for short: SOD) be a kind of metalloenzyme, can catalysis ultra-oxygen anion free radical generation disproportionation, eliminate cell injury such as the caused biomacromolecule peroxidation of ultra-oxygen anion free radical, have anti-inflammatory and get rid of illness, the effect of health and beauty.SOD extensively is applied in many industries such as medicine, healthcare products, food and daily use chemicals at present.
Existing SOD prepares mainly based on three kinds of raw materials, and the first, be the source with animal body tissue or blood, the second, be raw material sources with the microorganism, the 3rd, be raw material sources with the plant.It is more expensive generally to be with animal body tissue or blood that the SOD of feedstock production exists price, short shortcoming of transformation period in the body, moreover, the appearance on a large scale of mad cow disease and bird flu now, the quality and the security of raw material are doubted, microorganism mainly utilizes it to ferment biosynthesizing, because microorganism is subject to Effect of Environmental, its rate of recovery of gained SOD and activity can not get guaranteeing, because the domestic search time of this respect is shorter, do not set up it and correspondingly improve unified quality standard, be difficult to the monitoring product quality.Interim in March in 2005 the 1st of " Chengdu aviation Vocationl Technical College journal " by the continue article of black having delivered " extraction of plant superoxide dismutase, modify its applied research of preparation technology's agent " of gold, this article discloses the thermally denature precipitator method and has saltoutd and metal-chelate closes that affinity chromatography prepares and the purification superoxide dismutase, above-mentioned these methods do not have effective measures to be suppressed to virus in the raw material and unwanted bacteria, it is not high to make the SOD activity, and the equipment input cost is big in the technology, is not suitable for large-scale industrial production.
Summary of the invention
Technical problem to be solved by this invention provides a kind of health, is fit to the preparation method of the plant superoxide dismutase of technical scale production.
The present invention solves the problems of the technologies described above the technical scheme that is adopted: the preparation method of a kind of plant superoxide dismutase is characterized in that comprising the steps:
(1) with dregs of beans, black nightshade, bittersweet, Herba ajugae ciliatae (Herba Ajugae Ajuga ciliata Bge.), mock-strawberry separated pulverizing, the making beating cleaned;
(2) product after the making beating in the combining step (1), adding 3～4 times of weight is 0.5～2% basic ion liquid to merging after product, mass percent, anaerobically fermenting 1～12 month;
(3) fermented after, add saturated damping fluid, be 7～8 until the pH value, anaerobism left standstill 4～6 hours;
(4) add oxygenant and extraction agent in fermenting container, the dehydration precipitation, filter, get filtrate, add the dehydration oxygenant, under-10～0 ℃, anaerobism vacuum freezing 1～4 hour, the amount of above-mentioned oxygenant interpolation is 1～100% of the middle liquor capacity of step (3), and the amount that above-mentioned extraction agent adds is 15～80% of the middle liquor capacity of step (3), and the amount that above-mentioned dehydration oxygenant adds is 15～80% of the middle liquor capacity of step (3);
(5) precipitation obtains the crude product enzyme, adopts 80～400 order filter cloth suction filtrations, then centrifugation, and taking precipitate was in-30～0 ℃ of vacuum lyophilization 8～72 hours;
(6) be that 10～15% reducing sugar substratum adds above-mentioned product to 40～60 times of weight product, mass percent in step (5), add 0.2～0.3 times of weight again and be 1.5～3% sequestering agent to reducing sugar substratum, mass percent, temperature is bathed under 78～90 ℃ of conditions, temperature bath product temperature to 65～75 ℃ is taken out;
(7) in 10～20 minutes, be cooled to 16～20 ℃, adopt 400～600 order filter cloth suction filtrations;
(8) get filtrate, add the dehydration oxygenant, anaerobism left standstill 2～4 hours, suction filtration, centrifugation obtained product, in-30～0 ℃ of vacuum machine lyophilize 24～72 hours, again through normal temperature vacuum drying 24～72 hours, promptly get superoxide dismutase, the amount that above-mentioned dehydration oxygenant adds is in the step (7) behind the suction filtration 15～80% of liquor capacity
Wherein the mass ratio between each raw material is 1～100: 1～100 in the step (1): 1～100: 1～100: 1～100.
Further improve the quality and the activity of gained enzyme, the raw material in the described step (1) also comprises Spreading Hedyotis Herb and Herba Scutellariae Barbatae, and the mass ratio between each material is 1～100: 1～100: 1～100: 1～100: 1～100: 1～100: 1～100.
Further improve the quality and the activity of gained enzyme, raw material in the described step (1) also comprises Herba Commelinae, humulus grass and Herba Houttuyniae, and the mass ratio between each material is 1～100: 1～100: 1～100: 1～100: 1～100: 1～100: 1～100: 1～100: 1～100: 1～100.
Basic ion liquid in the described step (2) adopts sodium hydroxide solution, Klorvess Liquid or sodium chloride solution, damping fluid in the step (3) adopts citric acid, Trisodium Citrate, acetic acid, sodium-acetate, potassium primary phosphate or sodium chloride solution, oxygenant in the step (4) adopts 100% ethanol, mass percent is 1～20% sodium hydroxide solution, mass percent is 10～40% hydrogen peroxide, mass percent is that 5～38% hydrochloric acid or mass percent are 10～98% sulfuric acid, extraction agent adopts acetone, methylene dichloride or trichloromethane, dehydration oxygenant in the step (4) adopts ethanol, trichloromethane, acetone, methylene dichloride or metabisulfite solution, reducing sugar substratum in the step (6) adopts maltose substratum or dextran substratum, sequestering agent in the step (6) adopts EDTA solution, polyoxyethylene glycol or above-mentioned two kinds mixture, the dehydration oxygenant in the step (8) adopts 100% ethanol, mass percent is 1～20% sodium hydroxide solution, mass percent is 10～40% hydrogen peroxide, mass percent is that 5～38% hydrochloric acid or mass percent are 10～98% sulfuric acid.
Be further to reduce the anaerobically fermenting time and to improve the anaerobically fermenting effect, add mass percent in the described step (2) before the anaerobically fermenting and be the throw out after filtering in 20～100% the step (4).
Induce activator to use, before bathing, temperature adds the upper strata material behind the suction filtration in the step (6) in the described step (6), and through-20～-10 ℃ of lyophilizes, the amount that this upper strata material adds is that the middle product of step (1) merges 10～50% of back mixture quality before adding behind this upper strata material suction filtration.
Compared with prior art, the invention has the advantages that: the cheap herbal medicine such as black nightshade, bittersweet, Herba ajugae ciliatae (Herba Ajugae Ajuga ciliata Bge.), mock-strawberry that will have the detoxify and promote the subsdence of swelling anticancer function cooperates with the dregs of beans that contains a large amount of metal ions, respectively get the chief, collaborative mutually, through anaerobically fermenting, make health pollution-free have suppress the very high SOD of germ enzymic activity, directly can take or be coated with outward, and has good anti-inflammatory effect, and can be according to concrete illness, suitably add wherein one or more raw materials, can improve curative effect pointedly; Recycling, fermentation is induced activation to improve the quality of products greatly simultaneously and is shortened preparation time repeatedly; SOD according to the present invention makes can preserve at normal temperatures, is fit to long-distance transport, has reduced the storage and transport expense, and used each raw materials cost of the present invention is cheap, the equipment less investment, and simple and safe operation process, the staff easily learns easily meeting; Owing to produce the gas of absorption and inhibition germ in the technological process, so if this product of scale operation can improve local air ambient.
Description of drawings
Fig. 1 is an embodiment of the invention process flow sheet
Embodiment
Embodiment describes in further detail the present invention below in conjunction with accompanying drawing.
Embodiment, with reference to figure 1, the preparation method of plant superoxide dismutase comprises the steps: in the present embodiment
(1) 100 gram dregs of beans, the 50 cron certain herbaceous plants with big flowers that will clean, 50 gram bittersweets, 50 gram Herba ajugae ciliatae (Herba Ajugae Ajuga ciliata Bge.)s, 50 gram mock-strawberrys are white, 50 gram flower Herba Hedyotidis Diffusaes, 50 gram Herba Scutellariae Barbataes, 50 gram Herba Commelinaes, 50 gram humulus grass and 50 gram Herba Houttuyniae separated pulverizing, making beating;
(2) product after the making beating in the combining step (1), adding weight 3.5 times (generally being controlled at 3～4 times) is the basic ion liquid of 1.5% (generally being controlled at 0.5～2%) in merging after product, mass percent, present embodiment adopts sodium chloride solution as basic ion liquid, anaerobically fermenting 2 months, if raw material is done, can the proper extension fermentation time, general anaerobically fermenting time range is controlled at for 1～December;
(3) fermented after, add saturated damping fluid, present embodiment adopts citric acid solution, is about 7 until pH, anaerobism leaves standstill 5 hours (generally being controlled at 4～6 hours);
(4) add oxygenant and extraction agent in fermenting container, the dehydration precipitation, the siphon upper strata stillness of night, filter, get filtrate, merge clear liquid and filtrate, add the dehydration oxygenant, in-5 ℃ of (generally being controlled at-10～0 ℃) anaerobism vacuum freezings 2 hours (generally being controlled at 1～4 hour), the amount that above-mentioned oxygenant adds is 50% (generally being controlled at 1～100%) of liquor capacity in the step (3), it is the sodium hydroxide solution of 15% (generally being controlled at 1～20% times) that present embodiment adopts mass percent, the amount that extraction agent adds is 50% (generally being controlled at 15～80%) of liquor capacity in the step (3), present embodiment adopts acetone, and the amount that above-mentioned dehydration oxygenant adds is 50% (generally being controlled at 15～80%) of liquor capacity in the step (3), and present embodiment adopts ethanol;
(5) precipitation obtains the crude product enzyme, adopt 300 orders (generally being controlled at 80～400 orders) filter cloth suction filtration, then with 3000 rev/mins, hydro-extracting cage work 4 minutes, taking precipitate is in-10 ℃ of (generally being controlled at-30～0 ℃) vacuum freezedryings 32 hours (generally being controlled at 8～72 hours);
(6) 50 times of (generally being controlled at 40～60 times) product, mass percents in step (5) of weight are 12% the reducing sugar substratum of (as be controlled at 10～15%) add above-mentioned product to, adding 0.25 times of weight (generally being controlled at 0.2～0.3 times) again is the sequestering agent (adding when temperature reaches 45 ℃) of 2% (generally being controlled at 1.5～3%) in reducing sugar substratum, mass percent, present embodiment adopts EDTA solution, temperature is bathed under 80 ℃ of (generally being controlled at 78～90 ℃) conditions, temperature is bathed product to 70 ℃ of temperature (generally being controlled at 65～75 ℃), takes out;
(7) in 18 minutes, (generally be controlled at 16～20 minutes), be cooled to 18 ℃ (generally being controlled at 16～20 ℃), adopt 500 orders (generally being controlled at 400～600 orders) filter cloth suction filtration;
(8) get filtrate, add in the filtrate that the dehydration oxygenant obtains to step (7), leave standstill 3 hours (generally being controlled at 2～4 hours), 500 orders (generally being controlled at 400～600 orders) filter cloth suction filtration, with 3000 rev/mins, hydro-extracting cage work 4 minutes, taking precipitate, behind-20 ℃ of freezing vacuum machines 24 hours (generally being controlled at 24～72 hours), again through dry 48 hours (generally being controlled at 24～72 hours) of normal temperature vacuum, promptly get high-quality superoxide dismutase 50 grams, the amount that above-mentioned dehydration oxygenant adds is 50% (generally being controlled at 15～80%) of liquor capacity behind the middle suction filtration of step (7), and present embodiment adopts ethanol.
Wherein, originally can suitably add wherein one or more raw materials according to concrete illness, the run-of-the-mill ratio is controlled at 1～100.Cheap dregs of beans also can substitute with soybean or soya bean, and its effect is the same.
Basic ion liquid in the step (2) can also adopt Repone K or sodium-chlor; Damping fluid in the step (3) can also adopt Trisodium Citrate, acetic acid, sodium-acetate, potassium primary phosphate, SODIUM PHOSPHATE, MONOBASIC or sodium chloride solution; It is that 10～40% hydrogen peroxide, mass percent are that 5～38% hydrochloric acid or mass percent are 10～98% sulfuric acid etc. that oxygenant in the step (4) can also adopt mass percent, and extraction agent can also adopt methylene dichloride or trichloromethane; Dehydration oxygenant in the step (4) can also adopt acetone, trichloromethane, methylene dichloride or metabisulfite solution etc.; Reducing sugar substratum in the step (6) can adopt maltose substratum or dextran substratum, adopts the maltose substratum in the present embodiment; Sequestering agent in the step (6) can also adopt the mixture of polyoxyethylene glycol or EDTA and ethylene glycol.
Can add mass percent in the step (2) before the anaerobically fermenting and be the precipitation after filtering in 20～100% the step (4), add the throw out (for the product after fermentation last time) after filtering in 50% the step (4) in the present embodiment, recycling, ferment repeatedly.
Induce activator to use, before temperature is bathed, add the upper strata material behind the suction filtration in step (7) in the step (6), the amount of interpolation be in the step (1) product merge add behind 30% (generally being controlled at 10～50%) of back mixture quality and this upper strata material suction filtration before-20 ℃ of lyophilizes of process.
Be further to save auxiliary material and to improve ferment effect, the solution separating that suction filtration reclaims in step (5) and the step (8) is extracted the mother liquor that is left behind the reagent and can be added in the basic ion liquid in the step 2 and recycle,
The SOD that present embodiment makes was on July 22nd, 2002, check through academy of agricultural sciences, Shanghai City animal and veterinary institute, recording enzymic activity is 3456U/mg, be saved on March 10th, 2006 under the normal temperature, survey same sample once more, recording enzymic activity is 2589 U/mg, viability is 75%, the used major equipment of this technology, as follows: the visible spectrophotometric machine of 721 electronics, refrigerator, 50 liters of plastic tanks (as anaerobic fermentation tank), the vacuum filtration device, distiller, LG-5 or LG-2 vacuum freeze drier (producing) by the ShangHai City centrifugal Machine Institute, whizzer, hydro-extracting cage and cooling refrigeration equipment.
The SOD that technology makes according to the present invention has good antiinflammation, oral dosage is each 500/mg, per 12 hours once oral, can make pulvis, aqua and capsule, all can use with various ringworms, constipation, polyarthritis, rheumatism cardiac muscle pain, suppurative ulcer, nerve swells and ache and acute and chronic gastroenteritis, the SOD that has had the present invention of a plurality of case proof to make has good antiinflammation.
Raw material in the present embodiment also can adopt following five kinds to prepare: dregs of beans, black nightshade, bittersweet, Herba ajugae ciliatae (Herba Ajugae Ajuga ciliata Bge.) and mock-strawberry, and its SOD that makes also has good antiinflammation, and other are identical with embodiment 1.
Raw material in the present embodiment also can adopt following eight kinds to prepare: dregs of beans, black nightshade, bittersweet, Herba ajugae ciliatae (Herba Ajugae Ajuga ciliata Bge.), mock-strawberry, Herba Commelinae, humulus grass and Herba Houttuyniae, and its SOD that makes also has good antiinflammation, and other are identical with embodiment 1.
Claims (6)
1. the preparation method of a kind of plant superoxide dismutase is characterized in that comprising the steps:
(1) with dregs of beans, black nightshade, bittersweet, Herba ajugae ciliatae (Herba Ajugae Ajuga ciliata Bge.), mock-strawberry separated pulverizing, the making beating cleaned;
(2) product after the making beating in the combining step (1), adding 3～4 times of weight is 0.5～2% basic ion liquid to merging after product, mass percent, anaerobically fermenting 1～12 month;
(3) fermented after, add saturated damping fluid, be 7～8 until the pH value, anaerobism left standstill 4～6 hours;
(4) add oxygenant and extraction agent in fermenting container, the dehydration precipitation, filter, get filtrate, add the dehydration oxygenant, under-10～0 ℃, anaerobism vacuum freezing 1～4 hour, the amount of above-mentioned oxygenant interpolation is 1～100% of the middle liquor capacity of step (3), and the amount that above-mentioned extraction agent adds is 15～80% of the middle liquor capacity of step (3), and the amount that above-mentioned dehydration oxygenant adds is 15～80% of the middle liquor capacity of step (3);
(5) precipitation obtains the crude product enzyme, adopts 80～400 order filter cloth suction filtrations, then centrifugation, and taking precipitate was in-30～0 ℃ of vacuum lyophilization 8～72 hours;
(6) be that 10～15% reducing sugar substratum adds above-mentioned product to 40～60 times of weight product, mass percent in step (5), add 0.2～0.3 times of weight again and be 1.5～3% sequestering agent to reducing sugar substratum, mass percent, temperature is bathed under 78～90 ℃ of conditions, temperature bath product temperature to 65～75 ℃ is taken out;
(7) in 10～20 minutes, be cooled to 16～20 ℃, adopt 400～600 order filter cloth suction filtrations;
(8) get filtrate, add the dehydration oxygenant, anaerobism left standstill 2～4 hours, suction filtration, centrifugation obtained product, in-30～0 ℃ of vacuum machine lyophilize 24～72 hours, again through normal temperature vacuum drying 24～72 hours, promptly get superoxide dismutase, the amount that above-mentioned dehydration oxygenant adds is in the step (7) behind the suction filtration 15～80% of liquor capacity
Wherein the mass ratio between each raw material is 1～100: 1～100 in the step (1): 1～100: 1～100: 1～100.
2. the preparation method of plant superoxide dismutase according to claim 1, it is characterized in that the raw material in the described step (1) also comprises Spreading Hedyotis Herb and Herba Scutellariae Barbatae, and the mass ratio between each material is 1～100: 1～100: 1～100: 1～100: 1～100: 1～100: 1～100.
3. the preparation method of plant superoxide dismutase according to claim 2, it is characterized in that the raw material in the described step (1) also comprises Herba Commelinae, humulus grass and Herba Houttuyniae, and the mass ratio between each material is 1～100: 1～100: 1～100: 1～100: 1～100: 1～100: 1～100: 1～100: 1～100: 1～100.
4. according to the preparation method of the arbitrary described plant superoxide dismutase of claim 1 to 3, it is characterized in that the basic ion liquid in the described step (2) adopts sodium hydroxide solution, Klorvess Liquid or sodium chloride solution, damping fluid in the step (3) adopts citric acid, Trisodium Citrate, acetic acid, sodium-acetate, potassium primary phosphate or sodium chloride solution, oxygenant in the step (4) adopts 100% ethanol, mass percent is 1～20% sodium hydroxide solution, mass percent is 10～40% hydrogen peroxide, mass percent is that 5～38% hydrochloric acid or mass percent are 10～98% sulfuric acid, extraction agent adopts acetone, methylene dichloride or trichloromethane, dehydration oxygenant in the step (4) adopts ethanol, trichloromethane, acetone, methylene dichloride or metabisulfite solution, reducing sugar substratum in the step (6) adopts maltose substratum or dextran substratum, sequestering agent in the step (6) adopts EDTA solution, polyoxyethylene glycol or above-mentioned two kinds mixture, the dehydration oxygenant in the step (8) adopts 100% ethanol, mass percent is 1～20% sodium hydroxide solution, mass percent is 10～40% hydrogen peroxide, mass percent is that 5～38% hydrochloric acid or mass percent are 10～98% sulfuric acid.
5. according to the preparation method of the arbitrary described plant superoxide dismutase of claim 1 to 3, it is characterized in that adding before the anaerobically fermenting in the described step (2) mass percent and be the throw out after filtering in 20～100% the step (4).
6. according to the preparation method of the arbitrary described plant superoxide dismutase of claim 1 to 3, it is characterized in that before temperature is bathed, adding the upper strata material behind the suction filtration in the step (6) in the described step (6), and through-20～-10 ℃ of lyophilizes, the amount that this upper strata material adds is that the middle product of step (1) merges 10～50% of back mixture quality before adding behind this upper strata material suction filtration.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103805575A (en) * | 2014-01-13 | 2014-05-21 | 李健 | Oxygen-enriched and anaerobic alternating catalytic production method of plant superoxide dismutase |
| CN109845594A (en) * | 2016-05-30 | 2019-06-07 | 路平 | A kind of implantation methods of multienzyme crops containing SOD and its application |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1618461A (en) * | 2004-07-19 | 2005-05-25 | 王成余 | Composition of nano-SOD and vegetative medicine or its extractives, and its prepn. method |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103805575A (en) * | 2014-01-13 | 2014-05-21 | 李健 | Oxygen-enriched and anaerobic alternating catalytic production method of plant superoxide dismutase |
| CN109845594A (en) * | 2016-05-30 | 2019-06-07 | 路平 | A kind of implantation methods of multienzyme crops containing SOD and its application |
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Effective date of registration: 20180509 Address after: 315010 room 10-4, 8 cool street, Haishu District, Ningbo, Zhejiang. Patentee after: Ningbo Royal ginseng Trade Co.,Ltd. Address before: 315806 room 404, building C, Huasheng commercial and residential building, 12 Lane East gangue Town, Beilun District, Ningbo, Zhejiang. Patentee before: Wang Haibao |
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Denomination of invention: A preparation method for plant superoxide dismutase Effective date of registration: 20230724 Granted publication date: 20110420 Pledgee: Zhejiang Tailong Commercial Bank Co.,Ltd. Ningbo Haishu sub branch Pledgor: Ningbo Royal ginseng Trade Co.,Ltd. Registration number: Y2023980049439 |