CN102220258B - Bacillus subtilis generating nattokinase and method for producing nattokinase by fermenting same - Google Patents
Bacillus subtilis generating nattokinase and method for producing nattokinase by fermenting same Download PDFInfo
- Publication number
- CN102220258B CN102220258B CN 201110099838 CN201110099838A CN102220258B CN 102220258 B CN102220258 B CN 102220258B CN 201110099838 CN201110099838 CN 201110099838 CN 201110099838 A CN201110099838 A CN 201110099838A CN 102220258 B CN102220258 B CN 102220258B
- Authority
- CN
- China
- Prior art keywords
- nattokinase
- beans
- dregs
- water
- fermentation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 108010073682 nattokinase Proteins 0.000 title claims abstract description 41
- 229940086319 nattokinase Drugs 0.000 title claims abstract description 40
- 244000063299 Bacillus subtilis Species 0.000 title claims abstract description 11
- 235000014469 Bacillus subtilis Nutrition 0.000 title claims abstract description 11
- 238000004519 manufacturing process Methods 0.000 title abstract description 4
- 244000046052 Phaseolus vulgaris Species 0.000 claims abstract description 39
- 235000010627 Phaseolus vulgaris Nutrition 0.000 claims abstract description 39
- 238000000855 fermentation Methods 0.000 claims abstract description 18
- 230000004151 fermentation Effects 0.000 claims abstract description 18
- 238000000034 method Methods 0.000 claims abstract description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 51
- 238000011081 inoculation Methods 0.000 claims description 13
- 239000002054 inoculum Substances 0.000 claims description 11
- 238000003756 stirring Methods 0.000 claims description 10
- 230000001954 sterilising effect Effects 0.000 claims description 9
- 238000004659 sterilization and disinfection Methods 0.000 claims description 8
- 238000004321 preservation Methods 0.000 claims description 7
- 238000012262 fermentative production Methods 0.000 claims description 5
- 244000005700 microbiome Species 0.000 claims description 4
- 238000010563 solid-state fermentation Methods 0.000 abstract description 4
- 239000002994 raw material Substances 0.000 abstract description 3
- 241001052560 Thallis Species 0.000 abstract 1
- 230000002255 enzymatic effect Effects 0.000 abstract 1
- 230000002349 favourable effect Effects 0.000 abstract 1
- 238000009629 microbiological culture Methods 0.000 abstract 1
- 230000008520 organization Effects 0.000 abstract 1
- 239000002609 medium Substances 0.000 description 25
- 108090000790 Enzymes Proteins 0.000 description 17
- 102000004190 Enzymes Human genes 0.000 description 17
- 229940088598 enzyme Drugs 0.000 description 17
- 239000000243 solution Substances 0.000 description 16
- 239000012153 distilled water Substances 0.000 description 13
- 238000002156 mixing Methods 0.000 description 9
- 239000007787 solid Substances 0.000 description 9
- 108010049003 Fibrinogen Proteins 0.000 description 7
- 102000008946 Fibrinogen Human genes 0.000 description 7
- 229940012952 fibrinogen Drugs 0.000 description 7
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 6
- 239000007853 buffer solution Substances 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 238000002835 absorbance Methods 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 108090000190 Thrombin Proteins 0.000 description 3
- 208000007536 Thrombosis Diseases 0.000 description 3
- 229920004890 Triton X-100 Polymers 0.000 description 3
- 239000013504 Triton X-100 Substances 0.000 description 3
- 102000003990 Urokinase-type plasminogen activator Human genes 0.000 description 3
- 108090000435 Urokinase-type plasminogen activator Proteins 0.000 description 3
- 239000011521 glass Substances 0.000 description 3
- 238000007654 immersion Methods 0.000 description 3
- 239000011259 mixed solution Substances 0.000 description 3
- 239000012488 sample solution Substances 0.000 description 3
- 229960004072 thrombin Drugs 0.000 description 3
- 229960005356 urokinase Drugs 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 239000008351 acetate buffer Substances 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 239000012470 diluted sample Substances 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000001723 fibrinogenic effect Effects 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 235000013557 nattō Nutrition 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 238000011218 seed culture Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 230000002537 thrombolytic effect Effects 0.000 description 2
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 101710184263 Alkaline serine protease Proteins 0.000 description 1
- FBPFZTCFMRRESA-ZXXMMSQZSA-N D-iditol Chemical compound OC[C@@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-ZXXMMSQZSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- DPDMMXDBJGCCQC-UHFFFAOYSA-N [Na].[Cl] Chemical compound [Na].[Cl] DPDMMXDBJGCCQC-UHFFFAOYSA-N 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004939 coking Methods 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 235000012054 meals Nutrition 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 235000011837 pasties Nutrition 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 230000005477 standard model Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 238000003805 vibration mixing Methods 0.000 description 1
Images
Landscapes
- Enzymes And Modification Thereof (AREA)
Abstract
The invention relates to bacillus subtilis generating nattokinase and a method for producing nattokinase by fermenting the same. The bacillus subtilis generating nattokinase is named as TK-1, the class name is bacillus subtilis, the collection number is CGMCC No.4731, the collection date is 4/2/2011, the collection address is: 3# yard, No.1 West Beichen Road, Chaoyang District, Beijing, and the collection organization is China General Microbiological Culture Collection Center. A fermentation medium contains bean pulp, and the fermentation method is mainly characterized in that the fermentation medium contains the bean pulp. According to the invention, the bacillus subtilis generates nattokinase through solid state fermentation of bean pulp serving as a raw material; the bean pulp is relatively cheap and has high protein content, thereby being favorable for the growth of thalli; the maximum enzymatic activity of the obtained nattokinase reaches 5,670 FU per gram of dry basis; and the maximum viable count can reach 7-109 cfu per gram of dry basis.
Description
Technical field
The invention belongs to microorganism field, especially a kind of method of producing Nattokinase subtilis and fermentative production Nattokinase thereof.
Background technology
Nattokinase is a kind of a kind of alkaline serine protease that is produced by bacillus natto, has stronger thrombolysis characteristic, utilizes the solid state fermentation mode to produce Nattokinase, has the characteristics such as simple, with low cost.Nattokinase can directly decompose thrombus, and the wet natto thrombolytic effect of every gram is equivalent to urokinase 1600IU.Nattokinase is to reaching 8-12 hour the action time of thrombus, and urokinase only has 30 minutes action time.Urokinase can only be injected, and Nattokinase is that injectable also can be oral, and can play a role in capillary vessel.Natto eats 1000 in Japan, is a kind of safe edible thrombus prevention agent.At present, utilize the kinds of culture medium of fermentation of bacillus subtilis product Nattokinase a lot, mostly cost compare is high.
Summary of the invention
The purpose of this invention is to provide a kind of method of producing Nattokinase subtilis and this strain fermentation production Nattokinase, present method is take dregs of beans as prepared using subtilis Produced by Solid-state Fermentation Nattokinase.
The present invention realizes that the technical scheme of purpose is as follows:
A kind of product Nattokinase subtilis, name is called TK-1, specific name Bacillius subtilis, deposit number is: CGMCC No.4731, preservation date: on April 2nd, 2011, the preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center.
A kind of sharp method that the Nattokinase fermentation of bacillus subtilis is produced Nattokinase of producing, fermention medium comprises dregs of beans.
And described fermention medium is: dregs of beans: the weight ratio of water is 1: 1-2, pH6.5-7.5, the dregs of beans 3-4h that is soaked in water.
And fermention medium is defined as: dregs of beans: the weight ratio of water is 1: 1.2, pH7.0, the dregs of beans 3-4h that is soaked in water.
And described fermention medium sterilization is: through 110 ℃ of autoclaving 20min.
And the inoculation of fermentation and culture condition are: with the seed liquor inoculum size of 5-15%, will produce in Nattokinase subtilis TK-1 (CGMCC No.4731) the seed liquor access fermention medium, and stir, culture temperature 30-45 ℃, incubator humidity 60-70%RH cultivates 24h.
And the inoculation of fermentation and culture condition are: the seed liquor inoculum size with 10%, seed liquor is accessed in the fermention medium, and stir, 37 ℃ of culture temperature, incubator humidity 65%RH cultivates 24h.
Advantage of the present invention and positively effect are:
1, the subtilis among the present invention produces Nattokinase take dregs of beans as raw material by solid fermentation, value of the meal is just lower, and protein content is very high in the dregs of beans, is beneficial to the growth of thalline, the Nattokinase enzyme work that obtains is up to the 5670FU/g butt, and viable count is the highest can to reach 7*10
9Cfu/g.
2, the present invention is fermented take the food grade dregs of beans as main raw material and is produced Nattokinase, dregs of beans is a kind of byproduct that obtains behind the soybean extracting bean oil, contain protein 40-50%, its main component also has amino acid, abundant trace element and the fat of less content, water content is generally at 10%-20%, and abundant nutritive ingredient can satisfy the bacillus natto growth and produce the needs of enzyme, the present domestic record of only producing Nattokinase take dregs of beans as the prepared using bacillus natto that also do not have.
Description of drawings:
The liquid seed liquor growth curve of Fig. 1 subtilis of the present invention;
Fig. 2 subtilis solid medium of the present invention growth curve;
Fig. 3 subtilis solid state fermentation of the present invention dregs of beans produces the Nattokinase curve determination.
Embodiment
Below in conjunction with embodiment, the present invention is further described, and following embodiment is illustrative, is not determinate, can not limit protection scope of the present invention with following embodiment.
A kind of product Nattokinase subtilis, name is called TK-1, specific name Bacillius subtilis, deposit number is: CGMCC No.4731, preservation date: on April 2nd, 2011, the preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center.
1, slant medium: extractum carnis 0.5%, peptone 1%, sodium-chlor 0.5%, agar 2%, pH7.0～7.2,121 ℃ sterilization 20 minutes, culture condition: cultivate 48h for 37 ℃.
Bacterial strain subtilis TK-1 was cultivating 48 hours under 37 ℃ of temperature on the slant medium, in slant tube, add the 20ml sterilized water, scrape the lawn that takes a morsel in sterilized water with transfering loop, the vibration mixing, make bacteria suspension, get the 1ml bacterial suspension inoculation in the 500ml Erlenmeyer flask that fills the liquid seed culture medium of 99ml, in 37 ℃, cultivated 8 hours on the 150rpm shaking table, obtain seed liquor.
2, seed culture
Liquid seed liquor substratum: peptone 1%, glucose 2%, dipotassium hydrogen phosphate 0.1%, sal epsom 0.05%, pH7.0～7.2.Culture condition: get the subtilis that preserves on the inclined-plane and be inoculated in liquid seed liquor substratum, 37 ℃, 150rpm cultivates, and surveys the OD value under the 600nm condition of every two hours taking a sample.Logarithmic phase is at 6-16h.The time of determining the inoculation fermentation substratum is 8 hours.And the mensuration growth curve, see Fig. 1.
3, solid-state cultivation (fermentation culture)
Fermention medium: fresh dregs of beans 20g, water addition ratio 1: 1.2, the pH7.0 of water.3-4h is soaked in water.
Inoculation and culture condition: the seed liquor inoculum size with 10%, seed liquor is accessed in the solid medium, 37 ℃ of culture temperature, incubator humidity 65%RH cultivated 24 hours, and measured growth curve, and sampling in per 2 hours detects viable count, sees Fig. 2.Sampling in per 2 hours, every 10g substratum 50ml physiological saline, after 4 hours, 4500rpm 20min centrifuging and taking supernatant liquor is pressed the enzyme activity determination method and is surveyed the Nattokinase vigor, sees Fig. 34 ℃ of lower lixiviates.As seen from the figure, best fermentation time is 24 hours.
4, contain different concns bean pulp fermentation medium sterilization situation
Example 1
Dregs of beans not adding distil water soaks, through 121 ℃ of autoclaving 20min.
Result: dregs of beans generation brown stain, small part dregs of beans coking pasty state.
Example 2
Dregs of beans adds distilled water with 1: 1.2 water addition ratio, mixes thoroughly, behind the immersion 3-4h, through 121 ℃ of autoclaving 20min.
The result: the dregs of beans browning degree is lighter.
Example 3
Dregs of beans adds distilled water with 1: 1.2 water addition ratio, mixes thoroughly, behind the immersion 3-4h, through 115 ℃ of autoclaving 30min.
The result: the dregs of beans browning degree is lighter.
Example 4
Dregs of beans adds distilled water with 1: 1.2 water addition ratio, mixes thoroughly, behind the immersion 3-4h, through 110 ℃ of autoclaving 20min.
The result: brown stain does not occur in dregs of beans, and sterilization is rear without miscellaneous bacteria.
With example 4 sterilization method sterilization fermentation substratum, can reach best sterilising effect, also solved the problem that brown stain occurs behind the medium sterilization.
5, produce the method for Nattokinase subtilis TK-1 fermentative production Nattokinase, embodiment is as follows
The seed liquor that following examples are used is the seed liquor that obtains in the step 1.
Embodiment 1
Fermention medium: fresh dregs of beans 20g, water addition ratio 1: 1.2, the pH7.0 of water.3-4h is soaked in water.
Inoculation and culture condition: the seed liquor inoculum size with 10%, seed liquor is accessed in the solid medium, be caught broken substratum with aseptic nipper, and stir, make seed liquor and substratum mixing, 37 ℃ of culture temperature, incubator humidity 65%RH.Cultivate 24h.
Recording enzyme alive is the 5670FU/g butt
Embodiment 2
Fermention medium: fresh dregs of beans 20g, water addition ratio 1: 2, the pH7.5 of water.3-4h is soaked in water.
Inoculation and culture condition: the seed liquor inoculum size with 10%, seed liquor is accessed in the solid medium, be caught broken substratum with aseptic nipper, and stir, make seed liquor and substratum mixing, 37 ℃ of culture temperature, incubator humidity 65%RH.Cultivate 24h.
Recording enzyme alive is the 2500FU/g butt
Embodiment 3
Fermention medium: fresh dregs of beans 20g, water addition ratio 1: 1.2, the pH7.0 of water.3-4h is soaked in water.
Inoculation and culture condition: the seed liquor inoculum size with 10%, seed liquor is accessed in the solid medium, be caught broken substratum with aseptic nipper, and stir, make seed liquor and substratum mixing, 40 ℃ of culture temperature, incubator humidity 65%RH.Cultivate 24h.
Recording enzyme alive is the 2250FU/g butt
Fermention medium: fresh dregs of beans 20g, water addition ratio 1: 1.2, the pH7.0 of water.3-4h is soaked in water.
Inoculation and culture condition: the seed liquor inoculum size with 20%, seed liquor is accessed in the solid medium, be caught broken substratum with aseptic nipper, and stir, make seed liquor and substratum mixing, 37 ℃ of culture temperature, incubator humidity 65%RH.Cultivate 24h.
Recording enzyme alive is the 4250FU/g butt
Embodiment 5
Fermention medium: fresh dregs of beans 20g, water addition ratio 1: 1, the pH7.0 of water.3-4h is soaked in water.
Inoculation and culture condition: the seed liquor inoculum size with 10%, seed liquor is accessed in the solid medium, be caught broken substratum with aseptic nipper, and stir, make seed liquor and substratum mixing, 37 ℃ of culture temperature, incubator humidity 65%RH.Cultivate 24h.
Recording enzyme alive is the 3550FU/g butt
The result
Live height as foundation take enzyme, and fermention medium is defined as: fresh dregs of beans 20g, water addition ratio 1: 1.2 (weight ratio), the pH7.0 of water.3-4h is soaked in water; Inoculation with culture condition is: the seed liquor inoculum size with 10%, seed liquor is accessed in the solid medium, and be caught broken substratum with aseptic nipper, and stir, make seed liquor and substratum mixing, 37 ℃ of culture temperature, incubator humidity 65%RH cultivates 24h.
It is as follows to measure enzyme used method and reagent alive among the present invention:
Enzyme activity determination: crude enzyme liquid preparation: 1 gram fermented product uses the 5ml stroke-physiological saline solution under 4 ℃, lixiviate 4 hours, and vat liquor centrifugal 20 minutes through 4500rpm, supernatant liquor is crude enzyme liquid.
The definition of enzyme unit alive: a unit of enzyme activity (FU) is defined as: under the condition of specifying experiment flow, 1 minute, the absorbance of filtrate at the 275nm place increased by 0.01 required enzyme amount.
Agents useful for same and collocation method are as follows:
(1) 0.05mol/L borate buffer solution (pH8.5 contains NaCl): 19.07gNa
2B
4O
710H
2O and 9.0gNaCl are dissolved in the 900ml distilled water.Use the high salt concentration acid for adjusting pH, be settled to 1000ml with distilled water.
(2) 0.2mol/L Tricholroacetic Acid (TCA) solution: with dissolving 32.68gTCA in the distilled water, be dissolved to 1000ml.
(3) 0.72% fibrinogen solutions: draw the 10ml0.05mol/L borate buffer solution in erlenmeyer flask with pipette, add 96mg Fibrinogen (SIGMA, FIBRINOGEN (Fraction I) Type I-S:From Bovine Plasma, PRODUCT NUMBER F8630), bottle remains stands.Pulverize insolubles until fully dissolving with glass stick.With filter paper filtering mixture (ADVANTEC, No.6,7cm), remove insolubles.Prepare to use.(note I).
(4) thrombin solution: with 0.05mol/L borate buffer solution dissolving zymoplasm (SIGMA, From Bovine Plasma, PRODUCT NUMBER T6634), make 1000U/mL solution.Installed in the bottle freezing preservation in piecemeal minute.Before the use, zymoplasm is mixing in the 0.05mol/L borate buffer solution.
(5) 1mol/L acetum: with dissolved in distilled water 60.1g acetic acid, be dissolved to 1000ml.
(6) 1mol/L acetate buffer (pH6.0): with dissolved in distilled water 129.6g CH
3COONa3H
2O.Add 47.6ml 1mol/L acetum, and be settled to 1000ml with distilled water.
(7) 10%Triton X-100 solution: heating for dissolving 10g Triton X-100 in distilled water, and be settled to 100ml with distilled water.(note II)
(8) diluent: with dissolved in distilled water 0.344g (ultimate density 2mmol/L) CaSO
42H
2O and 0.585g (ultimate density 10mmol/L) NaCl.Add 2.0ml 1mol/L acetate buffer solution (pH6.0), 0.5ml (ultimate density 0.005%) 10%Triton X-100 solution is settled to 1000ml with distilled water.
Prepare sample solution
Make the correction absorbance of filtrate between 0.04 and 0.08 with the diluted sample.Concentrated solution concentration is usually between 0.67-1.33FU/g.
Experimental procedure
1. 1.4ml 0.05mol/L borate buffer solution and 0.4ml 0.72% fibrinogen solution are divided to install in the same test tube, fibrinogen solution was hatched 5 minutes in 37 ± 0.3 ℃ of water baths in advance.
2. add 0.01ml thrombin solution, mixing.(note III)
3.10 after minute, add the 0.1ml sample solution, mixed 5 seconds, hatch for 37 ± 0.3 ℃.
4. the reaction beginning mixed respectively 5 seconds in the time of rear 20 and 40 minutes.
5.60 after minute, add 2ml 0.2mol/L TCA solution termination reaction, hatched 20 minutes for 37 ± 0.3 ℃.
6. mixed solution is moved in the Eppendorf tube centrifugal 5 minutes of 15000rpm.
7. carefully the 1ml supernatant liquor is moved in the Glass tubing with pasteur pipet, 275nm reads at the place absorbance (AT).
(blank test)
8. 1.4ml 0.05mol/L borate buffer solution and 0.4ml 0.72% fibrinogen solution are divided to install in the same test tube, fibrinogen solution was hatched 5 minutes in 37 ± 0.3 ℃ of water baths in advance.
9. add 0.01ml thrombin solution, mixing.
10.10 after minute, add 2ml 0.2mol/L TCA solution, mixed 5 minutes.
11. in mixed solution, add the 0.1ml sample solution, mixed 5 seconds, hatched 20 minutes for 37 ± 0.3 ℃.
12. mixed solution is moved in the Eppendorf tube centrifugal 5 minutes of 15000rpm.
13. carefully the 1ml supernatant liquor is moved in the Glass tubing with pasteur pipet, 275nm reads at the place absorbance (AB).(note IV)
Calculate: Nattokinase vigor (FU/g)=(AT-AB)/0.01*1/60*1/0.1*D, wherein D: diluted sample rate
Note
The I enzyme activity is different because of fibrinogenic share.Therefore, when fibrinogenic share changes, be necessary multiply by correction coefficient and proofread and correct enzyme activity.
Under the II room temperature, diluent can be preserved 15 days.
III mixes with agitator at every turn
IV is for the accuracy of detected result, and standard model should detect batch simultaneously chemical examination at each.
Below with reference to specific embodiment method of the present invention is described more specifically, but the present invention is not limited to specific embodiment.
Claims (5)
1. one kind is produced the Nattokinase subtilis, it is characterized in that: name is called TK-1, specific name Bacillius subtilis, deposit number is: CGMCC No.4731, preservation date: on April 2nd, 2011, the preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center.
2. the method that the Nattokinase fermentation of bacillus subtilis is produced Nattokinase is produced in a utilization as claimed in claim 1, and it is characterized in that: fermention medium comprises dregs of beans;
Described fermention medium is: dregs of beans: the weight ratio of water is 1:1-2, pH6.5-7.5, the dregs of beans 3-4h that is soaked in water;
Inoculation and the culture condition of fermentation are: with the seed liquor inoculum size of 5-15%, to produce in the Nattokinase subtilis TK-1CGMCC No.4731 seed liquor access fermention medium, and stir culture temperature 30-45 ℃, incubator humidity 60-70%RH cultivates 24h.
3. the method for fermentative production Nattokinase according to claim 2, it is characterized in that: described fermention medium is: dregs of beans: the weight ratio of water is 1:1.2, pH7.0, the dregs of beans 3-4h that is soaked in water.
4. the method for fermentative production Nattokinase according to claim 2 is characterized in that: described fermention medium sterilization is: through 110 ℃ of autoclaving 20min.
5. the method for fermentative production Nattokinase according to claim 2, it is characterized in that: the inoculation of fermentation and culture condition are: the seed liquor inoculum size with 10%, access seed liquor in the fermention medium, stir, 37 ℃ of culture temperature, incubator humidity 65%RH cultivates 24h.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110099838 CN102220258B (en) | 2011-04-20 | 2011-04-20 | Bacillus subtilis generating nattokinase and method for producing nattokinase by fermenting same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110099838 CN102220258B (en) | 2011-04-20 | 2011-04-20 | Bacillus subtilis generating nattokinase and method for producing nattokinase by fermenting same |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102220258A CN102220258A (en) | 2011-10-19 |
| CN102220258B true CN102220258B (en) | 2013-03-13 |
Family
ID=44776967
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201110099838 Active CN102220258B (en) | 2011-04-20 | 2011-04-20 | Bacillus subtilis generating nattokinase and method for producing nattokinase by fermenting same |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102220258B (en) |
Families Citing this family (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102433287B (en) * | 2011-12-28 | 2013-03-13 | 天津科技大学 | Direct-vat bacillus subtilis starter and preparation method thereof |
| CN102943060B (en) * | 2012-11-22 | 2014-08-20 | 中国农业科学院农产品加工研究所 | Strain for nattokinase with high activity and thermal stability and fermented product thereof |
| CN103320347B (en) * | 2013-05-06 | 2015-05-20 | 重庆师范大学 | Bacillus subtilis with thrombolysis activity, and fermentation method thereof |
| CN103255156B (en) * | 2013-05-30 | 2014-12-17 | 中国农业科学院农产品加工研究所 | Site mutant nattokinase gene high in activity and thermal stability, and application thereof |
| CN104531833A (en) * | 2014-12-18 | 2015-04-22 | 天津市百奥生物技术有限公司 | Nattokinase activity measurement method |
| CN105087447B (en) * | 2015-09-16 | 2018-08-21 | 河南省科学院生物研究所有限责任公司 | One plant of resistance to microwave bacillus subtilis and its application in Nattokinase preparation |
| CN105349451A (en) * | 2015-10-22 | 2016-02-24 | 张淑莲 | Bacillus subtilis natto and breeding method and application thereof |
| CN107047778A (en) * | 2016-11-29 | 2017-08-18 | 天津科技大学 | Bacillus coagulans mixed fungus fermentation bean food and preparation method thereof |
| CN107099487B (en) * | 2017-06-27 | 2020-07-24 | 南京工业大学 | Bacillus subtilis with high nattokinase secretion and application thereof |
| CN110432324B (en) * | 2019-05-10 | 2020-11-10 | 江南大学 | Bacillus subtilis with antithrombotic function and application thereof |
| CN113151074A (en) * | 2021-04-09 | 2021-07-23 | 江南大学 | Bacillus subtilis mutant strain for high yield of nattokinase and application thereof |
| CN115404227A (en) * | 2022-09-22 | 2022-11-29 | 南京工业大学 | Method for producing nattokinase by utilizing solid state fermentation of fructus cannabis seed meal |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1100142C (en) * | 2000-05-22 | 2003-01-29 | 北京孚曼生物技术有限公司 | Process for preparing natto kinase and its application |
| CN1451745A (en) * | 2002-04-15 | 2003-10-29 | 黑龙江省科学院应用微生物研究所 | Natto kinase produced using Bacillus subtilis, and producing method and use thereof |
-
2011
- 2011-04-20 CN CN 201110099838 patent/CN102220258B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1100142C (en) * | 2000-05-22 | 2003-01-29 | 北京孚曼生物技术有限公司 | Process for preparing natto kinase and its application |
| CN1451745A (en) * | 2002-04-15 | 2003-10-29 | 黑龙江省科学院应用微生物研究所 | Natto kinase produced using Bacillus subtilis, and producing method and use thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102220258A (en) | 2011-10-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102220258B (en) | Bacillus subtilis generating nattokinase and method for producing nattokinase by fermenting same | |
| CN102660461B (en) | Microbial preparation for shortening tobacco fermentation period and application of microbial preparation | |
| CN102199050B (en) | Composite microbial fertilizer and preparation method thereof | |
| CN105695367B (en) | A kind of compounding microbial inoculum FX of degrading straw and its application | |
| CN101100660B (en) | Method for producing cellulose by microorganism mixing fermentation | |
| CN104692844A (en) | Preparation method of bioorganic fertilizer | |
| CN102174423B (en) | Bacillus licheniformis CH15 for degrading straws and bacterial agent thereof | |
| CN105907649A (en) | Fungus for degrading pectin and cellulose in tobacco stems and application of fungus | |
| CN102168055A (en) | Bacillus subtilis fermenting method with high rate of maturing gemma | |
| CN107502563A (en) | One plant of kluyveromyces marxianus bacterium and its method for producing β glucuroides | |
| CN106811438A (en) | A kind of straw degradative acidifying microbial inoculum and preparation method thereof | |
| CN105602853B (en) | One plant of angstrom of Mo Senluosashi bacterial strain and its application in Pu'er tea production | |
| CN102634460B (en) | Rhizopus oryzae RH1-5 and separating and culturing method thereof | |
| CN106755179B (en) | A kind of culture medium suitable for bacteria cellulose fermentation | |
| CN118318964A (en) | Natto fermentation product with high yield and capable of maintaining normal platelet aggregation function and preparation method thereof | |
| CN103103135B (en) | Aspergillus oryzae strain and method for producing fungal alpha-amylase by liquid-state fermentation of aspergillus oryzae strain | |
| CN110204376A (en) | A kind of charcoal composite biological organic fertilizer and preparation method thereof | |
| CN101705217B (en) | Method for co-producing xylanase and cellulase | |
| CN103392920B (en) | Fermentation method of soybean hulls | |
| CN105779328A (en) | Method for culturing clostridium thermocellum | |
| CN101805760A (en) | Method for producing microbial oil comprising arachidonic acid | |
| CN105175275B (en) | A kind of isolation and purification method of L ornithine | |
| CN110616153A (en) | Application of trichoderma koningii LCCC30119 in production of cellulase and tobacco fermentation | |
| CN108611293B (en) | Formula and preparation method of bio-enzyme strain in bio-enzyme pulping process | |
| CN102851220B (en) | Yeast strain capable of high-yield production of beta-galactosidase, and its application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C41 | Transfer of patent application or patent right or utility model | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20151019 Address after: 211300, No. 1, No. 15 Tan Avenue, Gaochun Economic Development Zone, Jiangsu, Nanjing Patentee after: Nanjing Royal plaque Biotechnology Co., Ltd. Address before: 300457 No. thirteen, 29 Avenue, Tanggu economic and Technological Development Zone, Tianjin Patentee before: Tianjin University of Science & Technology |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: Bacillus subtilis generating nattokinase and method for producing nattokinase by fermenting same Effective date of registration: 20160112 Granted publication date: 20130313 Pledgee: Gaochun Jiangsu rural commercial bank Limited by Share Ltd Pledgor: Nanjing Royal plaque Biotechnology Co., Ltd. Registration number: 2016320000001 |
|
| PLDC | Enforcement, change and cancellation of contracts on pledge of patent right or utility model | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20170510 Granted publication date: 20130313 Pledgee: Gaochun Jiangsu rural commercial bank Limited by Share Ltd Pledgor: Nanjing Royal plaque Biotechnology Co., Ltd. Registration number: 2016320000001 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: Bacillus subtilis generating nattokinase and method for producing nattokinase by fermenting same Effective date of registration: 20170531 Granted publication date: 20130313 Pledgee: Gaochun Jiangsu rural commercial bank Limited by Share Ltd Pledgor: Nanjing Royal plaque Biotechnology Co., Ltd. Registration number: 2017320000017 |