CN102604919B - Method for fermenting and producing plasmin by use of cordyceps militaris liquid - Google Patents

Method for fermenting and producing plasmin by use of cordyceps militaris liquid Download PDF

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CN102604919B
CN102604919B CN 201210111230 CN201210111230A CN102604919B CN 102604919 B CN102604919 B CN 102604919B CN 201210111230 CN201210111230 CN 201210111230 CN 201210111230 A CN201210111230 A CN 201210111230A CN 102604919 B CN102604919 B CN 102604919B
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plasmin
cordyceps militaris
liquid
expansin
fermentation
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CN102604919A (en
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姚强
宫志远
单洪涛
韩建东
王�琦
高能
孙涛
任鹏飞
万鲁长
任海霞
李瑾
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Institute of Agricultural Resources and Environment of Shandong Academy of Agricultural Sciences
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Institute of Agricultural Resources and Environment of Shandong Academy of Agricultural Sciences
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Priority to KR1020147008524A priority patent/KR101609603B1/en
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Priority to PCT/CN2013/000333 priority patent/WO2013155862A1/en
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6435—Plasmin (3.4.21.7), i.e. fibrinolysin

Abstract

The invention relates to a method for fermenting and producing plasmin by use of cordyceps militaris liquid. The method comprises the following steps of: (1) inoculating the cordyceps militaris strain into the PD liquid fermentation medium to perform activation culture, and transferring to a seed fermentation medium to perform seed culture to obtain seed liquid; (2) inoculating the seed liquid into a liquid fermentation medium according to a volume ratio of 5-15%, and performing liquid fermentation culture for 20-30 hours; adding an expansion protein solution, and continuing culture for 120-168 hours; and separating and removing cordyceps militaris mycelium to obtain plasmin fermentation supernate; and (3) extracting cordyceps militaris extracellular plasmin from the plasmin fermentation supernate to obtain the cordyceps militaris plasmin. According to the invention, the plant expansion protein and the optimized fermentation method are applied to the liquid fermentation production of cordyceps militaris plasmin, and the liquid fermentation output of the cordyceps militaris plasmin is remarkably increased to 93270U per liter of fermentation broth, which is over 2.69 times that of the traditional fermentation production method; and the method has perfect industrial application prospect.

Description

A kind of method of utilizing Cordyceps militaris fermentative production plasmin
Technical field
The present invention relates to a kind of method of utilizing the liquid fermenting production plasmin of Cordyceps militaris (L.) Link., belong to the bio-fermentation engineering field.
Background technology
Thrombotic disease is current harm humans health, causes one of the highest disease of mortality ratio, comprises acute myocardial infarction, ischemic heart disease, cardiovascular disorder, valvular heart disease, peripheral vascular disease, irregular pulse, hypertension, cerebral embolism, pulmonary infarction etc.Day by day become coercing human health.Estimate according to a research on " circulation: cardiovascular quality and the result " magazine that is published in American Heart Association, only owing to aging and population growth to the year two thousand thirty in 2010, the cardiovascular disease incidence rate will increase by 50% in worldwide year.The most effective and reliable means for the treatment of thromboembolic disorders are thrombolytic therapies at present, and therefore, the development of thrombolysis medicine for treating thrombus thing has become one of domestic and international study hotspot.The medicine that is used at present treatment of thrombotic disorders all has the shortcomings such as fibrin-specific is poor, Half-life in vivo is short, production cost is high, the heavy dose of continuous use of need in various degree.Some securities of searching are good, side effect is little, the thrombolytic drug of eutherapeutic natural origin so domestic and international experts and scholars turn one's attention to gradually.Cordyceps is China's tradition rare Chinese medicine, belonging to Ascomycota (Ascomycota), Ascomycetes (Ascomycetes), excrement shell bacterium subclass (Sordariomycetidae), Hypocreales (Hypocreales), Clavicipitaceae (Clavicipitaceae), Chinese caterpillar fungus Pseudomonas (Cordyceps Frey Link.), is a kind of entomogenous fungi.Among the people, usually be called as Cordyceps sinensis.Pharmaceutical research shows at strengthening immunity, antitumor, antibacterium and the aspect such as antimycotic to have good efficacy.Cordyceps militaris (L.) Link. (Cordyceps militaris) because of to the activeconstituents of Cordyceps sinensis on similar biochemical structure and medicinal function are arranged, enjoy in recent years all circles to pay close attention to.
Because rapid to the demand growth of Cordyceps militaris (L.) Link., the wild cordyceps militaris resource is plucked by madness and day by day rare, so its widespread use is subject to the serious restriction of medicine source resource.Research is found, identical by liquid fermenting production substantially on biochemical structure, physiological action with from Cordyccps-militaris-(L.)-link. Sporophore, mycelium, directly extracting the Chinese caterpillar fungus plasmin isoreactivity composition that obtains, and having that culture cycle is short, Production Flow Chart is easy to control, active substance is easy to the advantages such as extraction, the extraction content and the efficient that have been reported the plasmin isoreactivity material of Cordyceps militaris (L.) Link. all will be far above utilizing the artificial culture Chinese caterpillar fungus to produce and extract.But present Cordyceps militaris culture studies and application are still the starting stage, and many fermentation techniques and technique are still immature, and overall yield and fermentation level are lower, have limited the development of its suitability for industrialized production.
Expansin is the new albumen kind of a class of finding in plant cell wall.Studies show that, expansin has almost participated in the whole growth course of plant: increase the Magnocellular function except having, expansin forms at Growth of Cells, seed germination, the formation of root hair, root growth, leaf primordium, leaf grows, fruit maturation, organ break away from, and the pollen tube growth aspect plays an important role.In the various plants such as Arabidopis thaliana, tomato, strawberry, cotton, paddy rice and corn, all found expansin, be considered to it and be prevalent in the various dicotyledonous and monocotyledonous cell wallss.Test shows that expansin is a class cell walls zymoprotein, can induce cell walls extension and the pressure of acid dependence lax by the hydrogen bond that interrupts between the cell walls polymer, and then may or in growing process, play an important regulating and controlling factor in physiological regulating control and the cell walls extension process.Yet, about the mechanism of action of expansin clearly final conclusion not being arranged yet at present, the liquid fermenting that expansin is applied to the edible medicinal fungus Cordyceps militaris (L.) Link. carries out the production of the secondary meta-bolitess such as plasmin, is not reported at present both at home and abroad.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, a kind of method of utilizing expansin to improve Cordyceps militaris fermentative production plasmin output is provided.Technical solution of the present invention is specific as follows:
A kind of method of utilizing the liquid fermenting production plasmin of Cordyceps militaris (L.) Link. comprises the steps:
(1) Cordyceps militaris spawn is inoculated in the PD liquid fermentation medium, carries out activation culture, then by volume the inoculum size of percentage ratio 10～15% is transferred in the seed fermentation substratum, carry out seed culture, the seed culture condition is: 20～25 ℃ of temperature, and shaking table is cultivated 24～72h, gets seed liquor;
(2) step (1) being made seed liquor is inoculated in the liquid fermentation medium by 5～15% volume ratio, under the condition of 20～25 ℃ of temperature, liquid fermentation and culture 20～30h, then add expansin solution, the concentration that makes expansin is 1.0～3.5mg/mL, then continue to cultivate 120～168h, separate the removal cordyceps mycelium and get the plasmin fermented supernatant fluid;
(3) from the plasmin fermented supernatant fluid that step (2) makes, extract plasmin outside the Cordyceps militaris (L.) Link. born of the same parents, namely get the Cordyceps militaris (L.) Link. plasmin.
Preferred according to the present invention, the PD liquid fermentation medium in the described step (1), every liter of component is as follows:
Potato 200g, glucose 20g, distilled water is settled to 1000mL.
Preferred according to the present invention, the activation culture condition in the described step (1) is: shaking speed 100～160r/min, 20～25 ℃ of temperature, the dark activation 24～72h that cultivates.
Preferred according to the present invention, the seed fermentation substratum in the described step (1), every liter of component is as follows:
3g glucose, 2.5g yeast powder supernatant, 0.1g MgSO 4, 0.03g CaCl 2, 0.1g KH 2PO 4, 0.1gK 2HPO 4, the granulated glass sphere of 2～5 diameter 3～6mm.After the seed fermentation culture medium culturing, bacterium nodule number amount can improve more than 60%, the bacterium spherical diameter dwindles more than 40%, and mycelia is loose evenly.
Preferred according to the present invention, the liquid fermentation medium in the described step (2), every liter of component is as follows:
3g lactose, 2g molasses, 3g bean cake powder, 3g peptone, 0.2g MgSO 4, 0.03g CaCl 2, 0.3 g KH 2PO 4, 0.3 gK 2HPO 4
Preferred according to the present invention, in the described step (2), the concentration of expansin is 1.5～3.5mg/mL; Further preferred, the concentration of expansin is 1.5～3.0mg/mL; Most preferred, in the described step (2), the concentration of expansin is 2.0mg/mL.
Expansin solution in the described step (2) can prepare with reference to prior art, as adopt McQueen-Mason etc. at McQueen-Mason S J, Durachko D M, Cosgrove D J.Two endogenous proteins that induce cell wall extension in plants.Plant Cell, the method preparation of the record in 1992,4:1425～1433; Also can be prepared as follows expansin solution:
With broad bean or cucumber seeds through 0.05～0.15wt%HgCl 2Sterilization 4～6min, flowing water flushing 5～7h plants wet vermiculite, 25～28 ℃ of dark cultivations 4～6 days; Clip seedling epicotyl or root system 4～5cm put-20 ℃ of precooling 1～2h, add the homogenate buffer homogenate that is chilled in advance 0～4 ℃ after, with the nylon net filter of aperture 60～80 μ m, filter residue washs through homogenate buffer, then filter residue is added in the homogenate buffer, room temperature leaves standstill 1～3h, must leave standstill liquid; Add extracting solution in the liquid to leaving standstill, 0～4 ℃ of lower 24～30h that extracts filters, and the addition of pressing 0.3～0.5g/mL slowly adds (NH in filtrate 4) 2SO 4, add (NH 4) 2SO 4Constantly stir in the process, prevent (NH 4) 2SO 4Then partial over saturation leaves standstill 24～30h, the centrifugal 5～10min of 25000g under 4 ℃ of conditions, precipitation is redissolved with acidic buffer, the dialysis tubing dialysis of 4 ℃ of lower molecular weight 3000Da, dialyzate is got the expansin solution that supernatant liquor is preparation through the centrifugal 5～10min of 20000g.
In the above-mentioned expansin solution manufacturing method, described homogenate buffer component is: 25mmol/LHEPES (4-hydroxyethyl piperazine ethanesulfonic acid), 3mmol/L Na 2S 2O 5, 1mmol/L EDTA (ethylenediamine tetraacetic acid (EDTA)), 0.1wt%Triton X-100, pH 7.0; Described extracting solution component is: 25mmol/L 4-hydroxyethyl piperazine ethanesulfonic acid, 1.0mmol/L EDTA, 3mmol/L Na 2S 2O 5, 0.5mol/L NaCl, pH 6.8; Described acidic buffer preparation is: the 2.05g sodium-acetate is soluble in water, regulate pH to 4.0 with Glacial acetic acid, and water is settled to 1L.
Preferred according to the present invention, the separation described in the step (2) is centrifugation 10～15min under 4 ℃ of 12000r/min conditions.
Preferred according to the present invention, the method for the outer plasmin of extraction Cordyceps militaris (L.) Link. born of the same parents in the described step (3), step is as follows:
Under 0 ℃ of condition, add (NH after grinding by the addition of 0.14g/mL in the plasmin fermented supernatant fluid that makes to step (2) 4) 2SO 4, add (NH 4) 2SO 4Constantly stir in the process, prevent (NH 4) 2SO 4Partial over saturation, 0 ℃ staticly settles 4～8h and removes foreign protein, and 4 ℃ of centrifugal 10min of 12000rpm abandon precipitation, and the ammonium sulfate adding supernatant liquor after the addition of press 0.51g/mL will grind leaves standstill 4～8h; Under 4 ℃ of conditions, the centrifugal 10min of 12000rpm abandons supernatant liquor, is precipitated and dissolved in Sodium phosphate dibasic-citrate buffer solution; Being the dialysis tubing dialysis of 3000Da with molecular weight cut-off, is that the dialysis membrane of 3000Da carries out ultrafiltration and concentration with molecular weight cut-off then, removes micromolecular protein, namely gets Cordyceps militaris (L.) Link. born of the same parents plasmin outward.
Described Sodium phosphate dibasic-citrate buffer solution compound method is: 0.69g SODIUM PHOSPHATE, MONOBASIC, 0.012g citric acid are dissolved in the distilled water, are settled to 1L, pH 8.0.
The present invention compares with prior art and has the following advantages:
The present invention with the plant expansin and the fermentation process after optimizing be applied to the liquid fermenting production of Cordyceps militaris (L.) Link. plasmin, increased substantially the liquid fermenting output of Chinese caterpillar fungus plasmin, make output reach every liter of fermented liquid 93270U, be more than 2.69 times of traditional zymotic production method (Comparative Examples 1) output, have fabulous industrial applications prospect.
2, the present invention adopts the liquid aerobic fermentation method, be generally 6～8 days, with respect to prior art, output is high, cycle is short, need not to leave standstill and cultivate and induce the process such as synthetic product, production efficiency is higher, and the plasmin activity composition of the Cordyceps militaris (L.) Link. that produces can be directly used in the preparation of the thrombotic disease medicines such as Cardiovarscular, hypertension, cerebral embolism, pulmonary infarction;
3, expansin of the present invention can extract from most of dicotyledonous and monocotyledonss, wide material sources, cost is lower, preparation method of the present invention step after optimizing is also relatively simple, output is high, but scale extract to be produced, and the fermentative production of Cordyceps militaris (L.) Link. fibrinolytic enzyme active substance is had good promotion and promotes effect;
4, Cordyceps militaris zymotechnique of the present invention is simple, environment-protecting asepsis, and material cost is cheap, and full fermenting process is controlled, not limited by external environment condition, is fit to very much the industrial scale fermentor cultivation and produces.Present method also is applicable to other common cultivating champignon kind
5, the present invention is optimized the prescription of liquid fermentation medium and seed fermentation substratum, can increase substantially the output of the plasmin activity composition of Cordyceps militaris (L.) Link..
Description of drawings
Fig. 1 is that the expansin solution of different concns is to the influence curve of the plasmin output of Cordyceps militaris (L.) Link.;
Embodiment
Below in conjunction with embodiment the present invention is elaborated, but institute of the present invention protection domain is not limited to this.
Raw material and substratum
Cordyceps militaris (L.) Link. described in the embodiment (Cordyceps militaris) fermented bacterium, culture presevation number are CGMCC No.5.700 and CGMCC No.3.4655, available from Chinese common micro-organisms culture presevation administrative center.
Zymoplasm among the embodiment, Fibrinogen agar, urokinase standard substance are all contained big bio tech ltd available from Jinan;
The preparation process of the expansin solution described in the embodiment is as follows:
With broad bean (Vicia fabaL.; Available from Jinan Mao Feng seedling company limited) or cucumber (Cucumis sativus L. CV.JinnianNo.6; Available from the big beautiful kind industry in Jinan company limited) seed is through 0.15wt%HgCl 2Sterilization 6min, flowing water flushing 6h plants in the vermiculite that enters to wet, 27 ℃ of dark 4d that cultivate.Clip seedling epicotyl 4～5cm, namely the about 100g in vitellarium puts-20 ℃ of refrigerator precooling 1h, adds to be chilled in advance 4 ℃ homogenate buffer, behind the high-speed homogenization, with 70 μ m nylon net filters, filter residue washs through homogenate buffer, then filter residue is added in the homogenate buffer, room temperature leaves standstill 2h, must leave standstill liquid; Add extracting solution in the liquid to leaving standstill, 4 ℃ of lower 24h that extract filter, and the addition that filtrate is pressed 0.4g/mL slowly adds (NH in filtrate 4) 2SO 4, add (NH 4) 2SO 4Constantly stir in the process, prevent (NH 4) 2SO 4Partial over saturation, then leave standstill 24h, the centrifugal 5min of 25000g under 4 ℃ of conditions, precipitation is redissolved with acidic buffer, be polyvinylidene difluoride (PVDF) (PVDF) dialysis tubing (available from Beijing Bo Run Lai Te Science and Technology Ltd.) dialysis of 3000Da with molecular weight cut-off under 4 ℃ of conditions, dialyzate is got the expansin solution that supernatant liquor is preparation through the centrifugal 5min of 20000g, puts 4 ℃ of lower preservations.Other do not describe step can be with reference to McQueen-Mason etc. at McQueen-Mason S J, Durachko D M, Cosgrove D J.Two endogenous proteins that induce cell wall ext ension in plants.Plant Cell, 1992,4:1425～1433 and https: // description among the homes.bio.psu.edu/expansins/Protocols/Extraction.htm carries out.
Above-mentioned homogenate buffer component is: 25mmol/LHEPES (4-hydroxyethyl piperazine ethanesulfonic acid), 3mmol/LNa 2S 2O 5, 1mmol/L EDTA, 0.1wt%Triton X-100, pH 7.0;
The said extracted fluid component is: 25mmol/L HEPES, 1.0mmol/L EDTA, 3mmol/L Na 2S 2O 5, 0.5mol/LNaCl, pH 6.8;
Every liter of as follows preparation of described acidic buffer:
The 2.05g sodium-acetate is soluble in water, regulate pH to 4.0 with Glacial acetic acid, water is settled to 1L.
The measuring method of expansin strength of solution adopts the Xylene Brilliant Cyanine G method to detect, specifically can be with reference to (" fine works protein science experiment guide ", ISBN:703018086, date of publication 1900-1-1) the Xylene Brilliant Cyanine G method of record operates in, make typical curve with bovine serum albumin, expansin concentration is 0.31g/mL in the above-mentioned expansin solution after testing.
PD liquid fermentation medium described in the embodiment, every liter of component is as follows:
Potato 200g, glucose 20g, distilled water is settled to 1000mL.
Seed fermentation substratum described in the embodiment, every liter of component is as follows:
3g glucose, 2.5g yeast powder supernatant, 0.1g MgSO 4, 0.03g CaCl 2, 0.1g KH 2PO 4, 0.1gK 2HPO 4, the granulated glass sphere of 2～5 diameter 3～6mm.
Liquid fermentation medium described in the embodiment, every liter of component is as follows:
3g lactose, 2g molasses, 3g bean cake powder, 3g peptone, 0.2g MgSO 4, 0.03g CaCl 2, 0.3 g KH 2PO 4, 0.3g/L K 2HPO 4
Sodium phosphate dibasic described in the embodiment-citrate buffer solution compound method is: 0.69g SODIUM PHOSPHATE, MONOBASIC, 0.012g citric acid are dissolved in the distilled water, are settled to 1L, pH 8.0.
Embodiment 1:
A kind of liquid fermentation process that utilizes expansin to improve the plasmin composition output of Cordyceps militaris (L.) Link. comprises the steps:
(1) with Cordyceps militaris (L.) Link. (Cordyceps militaris) bacterial classification, bacterium numbering is CGMCC No.5.700, be inoculated in the PD liquid fermentation medium, carry out activation culture, the dark 48h that cultivates under the condition of 25 ℃ of temperature, shaking speed 150r/min, then by volume the inoculum size of percentage ratio 15% is transferred in the seed fermentation substratum, carry out seed culture, the seed culture condition is: 25 ℃ of temperature, and shaking table is cultivated 48h, gets seed liquor;
(2) step (1) being made seed liquor is inoculated in the liquid fermentation medium by 15% volume ratio, under the condition of 25 ℃ of temperature, liquid fermentation and culture 24h, then add expansin solution, the concentration that makes expansin is 1.5mg/mL, then continue to cultivate 144h, centrifugation 10min under the 12000r/min condition goes the mycelia precipitation to obtain fermented supernatant fluid;
(3) extract the method for plasmin outside the Cordyceps militaris (L.) Link. born of the same parents from the Cordyceps militaris (L.) Link. fermented supernatant fluid that step (2) makes, step is as follows:
Under 0 ℃ of condition, add (NH after grinding by the addition of 0.14g/mL in the plasmin fermented supernatant fluid that makes to step (2) 4) 2SO 4, add (NH 4) 2SO 4Constantly stir in the process, prevent (NH 4) 2SO 4Partial over saturation, 0 ℃ staticly settles 4h and removes foreign protein, and 4 ℃ of centrifugal 10min of 12000rpm abandon precipitation, and the ammonium sulfate adding supernatant liquor after the addition of press 0.51g/mL will grind leaves standstill 6h; Under 4 ℃ of conditions, the centrifugal 10min of 12000rpm abandons supernatant liquor, is precipitated and dissolved in Sodium phosphate dibasic-citrate buffer solution; Being the dialysis tubing dialysis of 3000Da with molecular weight cut-off, is that the dialysis membrane of 3000Da carries out ultrafiltration and concentration with molecular weight cut-off then, removes micromolecular protein, namely gets Cordyceps militaris (L.) Link. born of the same parents plasmin outward.
The preparation of Cordyceps militaris (L.) Link. plasmin testing sample
To be settled to 10mL with Sodium phosphate dibasic-citrate buffer solution by the plasmin that the 1L fermented liquid makes, and get 100 μ L and be diluted to 5mL and get Cordyceps militaris (L.) Link. plasmin testing sample.
The mensuration of Chinese caterpillar fungus plasmin activity composition
Adopt the fibrin plate method of this area routine, measuring method is referring to document (Astrup S, Mullertz.The fibrin Plate method for estimating of fibrinolytic activity[J] Archives of Biochemistry and Biophysics, 1952,40:346-351):
(1) making of fibrin plate:
The Fibrinogen agar-agar soln of zymoplasm with 37 ℃ mixed, namely generate scleroproein, be down flat immediately plate, after solidifying, be fibrin plate.The urokinase standard substance of point sample gradient concentration, 37 ℃ of constant-temperature incubation 18h survey its dissolving loop diameter, log (cm 2) be linear dependence with log (U/mL).Calculate the unit of activity of the suitable urokinase of Cordyceps militaris (L.) Link. plasmin testing sample according to the solusphere size of testing sample, represent the fibrinolytic of Cordyceps militaris (L.) Link. plasmin testing sample.
(2) mensuration of urokinase typical curve:
Compound concentration is the urokinase standardized solution of 100U/mL, 200U/mL, 300U/mL, 400U/mL, 500U/mL.Get the urokinase standardized solution 100 μ L point samples of above each concentration in fibrin plate, cultivate 6h for 37 ℃, two perpendicular diameter of vernier caliper measurement hydrolysis circle.As X-coordinate, the logarithm of urokinase concentration is ordinate zou, does the urokinase typical curve with the logarithm of the product of hydrolysis circle perpendicular diameter.
(3) mensuration of sample fibrinolytic:
Get 100 μ L Cordyceps militaris (L.) Link. plasmin testing sample point samples in the cup aperture of the Oxford of fibrin plate, cultivate 6h for 37 ℃, two perpendicular diameter of vernier caliper measurement hydrolysis circle.Fibrinolytic with reference to the Equation for Calculating testing sample of urokinase typical curve is 114.98U/mL.
Every liter of plasmin vigor corresponding to Cordyceps militaris (L.) Link. fermented liquid is: 114.98U/mL * (5mL/100 μ L) * 10mL=57490U; The result as shown in Figure 1.
Embodiment 2:
Liquid fermentation process as described in Example 1, difference are, under the condition of 25 ℃ of temperature, add expansin solution in the step (2), and the concentration that makes expansin is 2.0mg/mL, then continue to cultivate 144h.
Calculate after testing, the plasmin composition vigor of every milliliter of Cordyceps militaris (L.) Link. fermented liquid is 93.27U, and namely the plasmin composition vigor of every liter of Cordyceps militaris (L.) Link. fermented liquid is 93270U; The result as shown in Figure 1.
Embodiment 3:
Liquid fermentation process as described in Example 1, difference are, under the condition of 25 ℃ of temperature, add expansin solution in the step (2), and the concentration that makes expansin is 2.5mg/mL, then continue to cultivate 144h.
Calculate after testing, the plasmin composition vigor of every milliliter of Cordyceps militaris (L.) Link. fermented liquid is 81.32U, and namely the plasmin composition vigor of every liter of Cordyceps militaris (L.) Link. fermented liquid is 81320U; The result as shown in Figure 1.
Embodiment 4
Liquid fermentation process as described in Example 1, difference are, under the condition of 25 ℃ of temperature, add expansin solution in the step (2), and the concentration that makes expansin is 3.0mg/mL, then continue to cultivate 144h.
Calculate after testing, the plasmin composition vigor of every milliliter of Cordyceps militaris (L.) Link. fermented liquid is 82.02U, and namely the plasmin composition vigor of every liter of Cordyceps militaris (L.) Link. fermented liquid is 82020U; The result as shown in Figure 1.
Embodiment 5:
Liquid fermentation process as described in Example 1, difference are, under the condition of 25 ℃ of temperature, add expansin solution in the step (2), and the concentration that makes expansin is 3.5mg/mL, then continue to cultivate 144h.
Calculate after testing, the plasmin composition vigor of every milliliter of Cordyceps militaris (L.) Link. fermented liquid is 77.75U, and namely the plasmin composition vigor of every liter of Cordyceps militaris (L.) Link. fermented liquid is 77750U; The result as shown in Figure 1.
Embodiment 6:
Liquid fermentation process as described in Example 1, difference be, used Cordyceps militaris (L.) Link. (Cordyceps militaris) culture presevation of fermenting number is CGMCC No.3.4655, available from Chinese common micro-organisms culture presevation administrative center.Under the condition of 25 ℃ of temperature, add expansin solution in the step (2), the concentration that makes expansin is 2.0mg/mL, then continues to cultivate 144h.
Calculate after testing, the plasmin composition vigor of every milliliter of Cordyceps militaris (L.) Link. fermented liquid is 89.84U, and namely the plasmin composition vigor of every liter of Cordyceps militaris (L.) Link. fermented liquid is 89840U.
Comparative Examples 1
Liquid fermentation process as described in Example 1, difference are, the expansin solution that adds with the acidic buffer alternate embodiment that does not contain expansin in the described step (2), and under the condition that temperature is 25 ℃, liquid fermentation and culture 168h.
Calculate after testing, the plasmin composition vigor of every milliliter of Cordyceps militaris (L.) Link. fermented liquid is 34.66U, and namely the plasmin composition vigor of every liter of Cordyceps militaris (L.) Link. fermented liquid is 34660U; The result as shown in Figure 1.

Claims (2)

1. a method of utilizing the liquid fermenting production plasmin of Cordyceps militaris (L.) Link. is characterized in that, comprises the steps:
(1) Cordyceps militaris spawn is inoculated in the PD liquid fermentation medium, carry out activation culture, then by volume the inoculum size of percentage ratio 10～15 % is transferred in the seed fermentation substratum, carry out seed culture, the seed culture condition is: 20～25 ℃ of temperature, shaking table is cultivated 24～72 h, gets seed liquor;
(2) step (1) being made seed liquor is inoculated in the liquid fermentation medium by the volume ratio of 5～15 %, under the condition of 20～25 ℃ of temperature, liquid fermentation and culture 20～30 h, then add expansin solution, the concentration that makes expansin is 1.0～3.5 mg/mL, then continue to cultivate 120～168 h, separate the removal cordyceps mycelium and get the plasmin fermented supernatant fluid;
(3) from the plasmin fermented supernatant fluid that step (2) makes, extract plasmin outside the Cordyceps militaris (L.) Link. born of the same parents, namely get the Cordyceps militaris (L.) Link. plasmin;
Expansin solution in the described step (2) is prepared as follows:
With broad bean or cucumber seeds through 0.05～0.15 wt% HgCl 2Sterilization 4～6 min, flowing water flushing 5～7 h plant wet vermiculite, 25～28 ℃ of dark cultivations 4～6 days; Clip seedling epicotyl or root system 4～5 cm, put-20 ℃ of precooling 1～2 h, after adding the homogenate buffer homogenate that is chilled in advance 0～4 ℃, nylon net filter with aperture 60～80 μ m, filter residue washs through homogenate buffer, then filter residue is added in the homogenate buffer, room temperature leaves standstill 1～3 h, must leave standstill liquid; Add extracting solution in the liquid to leaving standstill, 0～4 ℃ of lower 24～30 h that extract filters, and the addition of pressing 0.3～0.5 g/ mL slowly adds (NH in filtrate 4) 2SO 4, add (NH 4) 2SO 4Constantly stir in the process, prevent (NH 4) 2SO 4Then partial over saturation leaves standstill 24～30 h, centrifugal 5～10 min of 25000 g under 4 ℃ of conditions, precipitation is redissolved with acidic buffer, the dialysis tubing dialysis of 4 ℃ of lower molecular weight 3000 Da, dialyzate is got the expansin solution that supernatant liquor is preparation through centrifugal 5～10 min of 20000 g;
Above-mentioned homogenate buffer component is: 25 mmol/L 4-hydroxyethyl piperazine ethanesulfonic acid, 3 mmol/L Na 2S 2O 5, 1 mmol/ L EDTA, 0.1 wt% Triton X-100, pH 7.0; Described extracting solution component is: 25 mmol/ L 4-hydroxyethyl piperazine ethanesulfonic acid, 1.0 mmol/ L ethylenediamine tetraacetic acid (EDTA)s, 3 mmol/ L Na 2S 2O 5, 0.5 mol/L NaCl, pH 6.8; Described acidic buffer preparation is: 2.05 g sodium-acetates are soluble in water, regulate pH to 4.0 with Glacial acetic acid, and water is settled to 1 L.
2. the method for claim 1 is characterized in that, the activation culture condition in the described step (1) is: shaking speed 100～160 r/min, 20～25 ℃ of temperature, dark activation 24～72 h that cultivate.
3 .The method of claim 1 is characterized in that, the seed fermentation substratum in the described step (1), and every liter of component is as follows:
3 g glucose, 2.5 g yeast powder supernatant, 0.1 g MgSO 4, 0.03 g CaCl 2, 0.1 g KH 2PO 4, 0.1 g K 2HPO 4, the granulated glass sphere of 2～5 diameter 3～6 mm.
4 .The method of claim 1 is characterized in that, the liquid fermentation medium in the described step (2), and every liter of component is as follows:
3 g lactose, 2 g molasses, 3 g bean cake powders, 3 g peptones, 0.2 g MgSO 4, 0.03 g CaCl 2, 0.3 g KH 2PO 4, 0.3 g K 2HPO 4
5 .The method of claim 1 is characterized in that, in the described step (2), the concentration of expansin is 1.5～3.5 mg/mL.
6 .Method as claimed in claim 5 is characterized in that, the concentration of expansin is 1.5～3.0 mg/mL.
7 .Method as claimed in claim 6 is characterized in that, the concentration of expansin is 2.0 mg/mL.
8 .The method of claim 1 is characterized in that, the separation described in the step (2) is centrifugation 10～15 min under 4 ℃ of 12000 r/min condition.
9 .The method of claim 1 is characterized in that, extracts the method for the outer plasmin of Cordyceps militaris (L.) Link. born of the same parents in the described step (3), and step is as follows:
Under 0 ℃ of condition, add (NH after grinding by the addition of 0.14 g/ mL in the plasmin fermented supernatant fluid that makes to step (2) 4) 2SO 4, add (NH 4) 2SO 4Constantly stir in the process, prevent (NH 4) 2SO 4Partial over saturation, 0 ℃ staticly settles 4～8 h and removes foreign protein, and 4 ℃ of 12000 centrifugal 10 min of rpm abandons precipitation, and the ammonium sulfate adding supernatant liquor after will grinding by the addition of 0.51 g/ mL leaves standstill 4～8 h; Under 4 ℃ of conditions, centrifugal 10 min of 12000 rpm abandon supernatant liquor, are precipitated and dissolved in Sodium phosphate dibasic-citrate buffer solution; Being the dialysis tubing dialysis of 3000 Da with molecular weight cut-off, is that the dialysis membrane of 3000 Da carries out ultrafiltration and concentration with molecular weight cut-off then, removes micromolecular protein, namely gets Cordyceps militaris (L.) Link. born of the same parents plasmin outward.
10 .Method as claimed in claim 9 is characterized in that, described Sodium phosphate dibasic-citrate buffer solution compound method is: 0.69 g SODIUM PHOSPHATE, MONOBASIC, 0.012 g citric acid are dissolved in the distilled water, are settled to 1 L, pH 8.0.
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