CN111321090A - Bacillus subtilis and application thereof in producing nattokinase - Google Patents
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Abstract
The invention relates to a Bacillus subtilis YC4, which has the Latin literature name of Bacillus subtilis YC4 and is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M20191132. The invention also relates to application of the bacillus subtilis in producing nattokinase and a method for producing nattokinase by using the bacillus subtilis. The bacillus subtilis YC4 is obtained by using a strain screened from food as an initial strain through mutagenesis, is safe and nontoxic, and can be used for fermentation production of nattokinase-related products; meanwhile, the strain has the performance of high-yield nattokinase, the enzyme production performance is genetic and stable, the nattokinase yield is up to 12000U/mL, the industrial large-scale production of the nattokinase is favorably realized, and a foundation is provided for developing the nattokinase into a new generation thrombolytic medicine or functional food additive.
Description
Technical Field
The invention relates to the field of microbial fermentation, in particular to bacillus subtilis and application thereof in producing nattokinase.
Background
In modern society, thrombosis in human blood vessels causes serious cardiovascular diseases such as cerebral infarction and myocardial infarction, especially causes great harm to the health of middle-aged and elderly people, and millions of patients with thrombotic diseases worldwide have the defects that the current thrombolytic drugs such as urokinase, streptokinase, recombinant tissue plasminogen activator and the like have short half-life period, great side effect, high price, oral administration incapability and the like, and are difficult to become applicable mass drugs. The nattokinase has the advantages of good safety, rapid action, low cost, oral administration and the like, can be produced by fermenting the bacillus subtilis, is used for developing a new generation of thrombolytic agent or health food, and has wide prospect.
Nattokinase (NK), an alkaline serine protease having thrombolytic activity first found in natto (a traditional food in japan) by Sumi in japan in 1987, is produced by bacillus subtilis, has high affinity for fibrin, and exhibits strong thrombolytic activity compared to other proteases, and is capable of decreasing fibrinogen, promoting and catalyzing the conversion of plasminogen to plasmin, and increasing the synthesis of thrombolytics in vivo. Meanwhile, because the nattokinase has the advantages of oral taking, high safety, low price, strong fibrinolytic activity and the like, the development and research become the research focus of thrombolytic products in recent years. Although no nattokinase drug is available on the market at present, nattokinase has attracted more attention in various applications. The nattokinase is used as a functional food additive, has the advantages of food safety, low cost and lasting thrombolytic effect, can reduce blood viscosity and blood plasma coagulation factor II concentration by adding the nattokinase, and is beneficial to preventing and treating thrombotic diseases.
With the development of biotechnology, significant progress has been made in the field of microbial production of nattokinase. At present, the yield of nattokinase is improved by means of strain screening, genetic engineering, fermentation process control and the like. The high-yield and high-quality strains can greatly reduce the production cost and improve the production efficiency. Generally, the yield of a wild strain product obtained by screening and separating from the nature is very low, the product yield requirement of the fermentation industry on the strain is very high, and the wild strain is difficult to meet the large-scale industrial production condition of the nattokinase, so that the cultivation of a high-quality and high-yield microbial strain for improving the enzyme activity and the stability of the high-quality and high-yield microbial strain plays an important role in expanding the application of the nattokinase in the fields of medicine and commerce.
Disclosure of Invention
The invention provides the bacillus subtilis with excellent property and genetic stability for producing the nattokinase and the application thereof in producing the nattokinase for solving the technical problems.
The inventor screens bacillus subtilis which can produce nattokinase from natto food produced by Hokkaido, and uses the bacillus subtilis as an original strain to mutate to obtain a novel bacillus subtilis which can produce nattokinase and has excellent enzyme production performance.
The technical scheme for solving the technical problems is as follows: a Bacillus subtilis is named as Bacillus subtilis YC4, the Latin literature name is Bacillus subtilis YC4, the Bacillus subtilis is preserved in China Center for Type Culture Collection (CCTCC), the preservation number is CCTCC NO: M20191132, and the preservation date is 2019, 12 months and 30 days.
On the other hand, the invention also provides the application of the bacillus subtilis in producing nattokinase.
On the other hand, the invention also provides a method for producing nattokinase, which utilizes the bacillus subtilis to produce nattokinase.
Further, the seed solution of the bacillus subtilis is inoculated into a fermentation culture medium to be fermented to produce the nattokinase.
Further, the fermentation medium includes a carbon source, a nitrogen source, and inorganic salts.
Further, the fermentation medium comprises the following components: 10-30 g/L glucose, 10-30 g/L peptone, 1-3 g/L sodium dihydrogen phosphate, 1-3 g/L disodium hydrogen phosphate dodecahydrate, 0.3-0.5 g/L magnesium sulfate, 0.1-0.3 g/L calcium chloride, water as solvent, and pH 7.0-7.4.
Further, the fermentation medium comprises the following components: 10g/L glucose, 20g/L peptone, 1g/L sodium dihydrogen phosphate, 2g/L disodium hydrogen phosphate dodecahydrate, 0.5g/L magnesium sulfate, 0.2g/L calcium chloride, water as solvent, and pH 7.2.
Further, the fermentation conditions were: the temperature is 30-37 ℃, the culture time is 36-56 h, and shaking culture is carried out at 160-200 r/min.
Further, the fermentation conditions were: the temperature is 34-37 ℃, the culture time is 36-48 h, and shaking culture is carried out at 160-180 r/min.
The bacillus subtilis YC4 is obtained by using a strain screened from food as an initial strain through mutagenesis, is safe and nontoxic, and can be used for fermentation production of nattokinase-related products; meanwhile, the strain natto kinase has the high yield of 12000U/mL, has the performance of high yield of natto kinase, has stable genetic performance, is beneficial to realizing the industrial large-scale production of natto kinase, and provides a foundation for developing new generation thrombolytic drugs or functional food additives for natto kinase.
Detailed Description
The principles and features of this invention are described below in conjunction with specific embodiments, which are set forth merely to illustrate the invention and are not intended to limit the scope of the invention.
Example 1 isolation of Bacillus subtilis producing Nattokinase from commercial Natto food
The method for screening bacillus subtilis for producing nattokinase from natto food (purchased from Nippon mountain) comprises the following steps:
picking 4-5 natto grains with a sterilized spoon in a sterile super-clean workbench, adding the natto grains into a conical flask containing glass beads and sterile normal saline, uniformly mixing the natto grains with vibration, and carrying out water bath on the conical flask at 80 ℃ for 15min to kill redundant nutrients and mixed bacteria. Taking out, rapidly cooling to room temperature in ice bath, placing into a sterile ultra-clean workbench, shaking the conical flask to mix uniformly, and taking the bacterial liquid to dilute according to a gradient of 10 times. Each gradient was prepared by adding 100. mu.L of the bacterial suspension to a casein plate (5g/L casein, 1g/L glucose, 1g/L yeast powder, 1g/L K)2HPO4、0.5g/L K2HPO4、0.1g/L MgSO415g/L agar powder, pH is 7.0 ~ 7.4), with scribbling the glass stick with it coating evenly, when the suitable bacterial colony of size grows out on the flat board, observe the size of transparent circle, measure transparent circle diameter size and bacterial colony diameter size respectively, calculate the transparent circle diameter of every bacterial strain and the specific value H of bacterial colony diameter, pick the great bacterial colony of specific value and be in succession at the bacterial colony of specific valuePlate streaking is carried out on the solid medium until a pure single colony is obtained. Respectively selecting strains on the plate, inoculating the strains into a liquid culture medium for fermentation culture, centrifuging fermentation liquor, taking supernatant fluid as crude enzyme liquid, carrying out enzyme activity determination, screening out strains with the maximum enzyme activity, and storing. And (4) observing morphological characteristics, and determining the bacillus subtilis through molecular biological identification and gene library analysis and comparison.
Example 2 mutagenesis of Bacillus subtilis
(1) EMS mutagenesis of strains
Using the Bacillus subtilis producing nattokinase obtained in example 1 as a mutagenic starting strain, the original strain of Bacillus subtilis stored in a refrigerator is inoculated with an inoculating loop into a 250mL conical flask containing 50mL of seed culture medium (the components are 10g/L peptone, 5g/L yeast powder, 10g/L NaCl, 1000mL of sterile water, and the pH value is 7.0), and then the conical flask is moved into a constant-temperature shaking incubator to be cultured at constant temperature of 160r/min and 37 ℃. Culturing to logarithmic phase, placing the bacterial liquid in sterilized centrifuge tube, centrifuging at 8000rpm/min for 10min, collecting thallus precipitate, washing with sterile normal saline for 2-3 times, diluting, counting with blood counting plate, and diluting to 107～108Single cell suspension per mL.
Preparing 0.5mol/L mother liquor of Ethyl Methanesulfonate (EMS): 0.5mL of EMS stock was added to 10mL of phosphate buffer pH7.4, capped and the tube was gently swirled. Since EMS is easy to volatilize under the condition of high concentration, in order to be safe and prevent invalidation, a vessel required before preparation is placed in a refrigerator for precooling, and then the preparation is carried out in an ice bath. Adding 1mL EMS mother solution into 4mL diluted bacterial suspension, treating for 50min under ice bath condition, adding 2mL 5% Na2S2O3The reaction was terminated by the solution, and after centrifugation, the reaction mixture was washed with physiological saline and centrifuged several times. The pellet was suspended by adding 5mL of sterile water.
(2) Ultraviolet mutagenesis of strains
Adding 10mL of bacterial suspension treated by EMS into a sterile culture dish containing a magnetic rotor, placing the sterile culture dish on a magnetic stirrer, keeping the vertical irradiation distance of a 25W ultraviolet lamp and the culture dish containing the bacterial suspension and the magnetic rotor to be 30cm, closing the culture dish after covering the culture dish with a cover for ultraviolet irradiation for 30s under dark conditions, opening the magnetic stirrer, opening the cover, opening the ultraviolet lamp, stirring while irradiating, irradiating for 90s with ultraviolet, placing the mutagenized bacterial liquid in a refrigerator at 4 ℃ after irradiation, and storing the mutagenized bacterial liquid for 2h at low temperature.
(3) Assisted mutagenesis
Under the condition of red light, the bacterial liquid after ultraviolet mutagenesis is carried out 10-2、10-3、10-4And (3) performing gradient dilution, namely coating and culturing 0.1mL of diluted bacterial liquid on a screening culture medium containing 0.4% LiCl as an auxiliary mutagen, and performing 3 parallels on each group by using bacterial suspension without mutagenesis as a control.
Screening medium containing LiCl with mass fraction of 0.4% as auxiliary mutagen: 5g/L casein, 1g/L glucose, 1g/L yeast powder, 1g/L K2HPO4、0.5g/L K2HPO4、0.1g/L MgSO415g/L agar powder, the pH value is 7.0-7.4, LiCl solution is added when the solid culture medium is cooled to about 60 ℃, and the mixture is uniformly mixed and poured into a flat plate.
(4) Preliminary screening of mutagenized strains
The coated flat plate is placed in a constant temperature incubator upside down, constant temperature culture is carried out at 37 ℃ for 24h under the dark condition, when bacterial colonies with good growth state grow on the flat plate, 1-3 mL of saturated ammonium sulfate solution is added into the flat plate so as to observe the size of a transparent ring (non-decomposed casein reacts with the solution to generate white precipitate), the diameter D of the transparent ring and the diameter D of the bacterial colonies are respectively measured by a digital vernier caliper, the ratio of the diameter of the transparent ring to the diameter of the bacterial colonies is used for representing the enzyme production capability of the bacterial strains, therefore, screening is carried out according to the ratio of the diameter of the transparent ring to the diameter of the bacterial colonies, the larger ratio is that the enzyme production capability is stronger, 8 mutant bacterial strains with strong enzyme production capability are selected, and the well-grown mutant bacterial strains are cultured in a solid culture medium (comprising 10g/L of peptone, 5g/L of yeast powder, 10g/L of NaCl, 1000mL of sterile water and 20g/L, pH 7.0) until a pure single colony is obtained for storage.
TABLE 1 preliminary screening results for mutagenized strains
(5) Rescreening of mutagenized strains
The enzyme activity of the nattokinase is measured by an agarose-fibrin plate method.
The agarose-fibrin plate was prepared as follows: weighing 0.8-1.0 g of agarose powder, adding the agarose powder into 0.01mol/L phosphate buffer solution (pH 7.4-7.6) to prepare 1% agarose buffer solution, heating until the agarose buffer solution is completely dissolved, and then placing the agarose powder in a constant-temperature water bath kettle at 55-60 ℃ for later use. Weighing 100-110 mg of fibrinogen, adding the fibrinogen into 0.01mol/L phosphate buffer solution (pH 7.4-7.6) to obtain 2.0-2.2 mg/mL fibrinogen buffer solution, and carrying out water bath in a constant-temperature water bath kettle at 40-45 ℃ until the fibrinogen is completely dissolved. And after the agarose solution and the fibrinogen solution are uniformly mixed, adding thrombin, quickly pouring into a clean and dry culture dish, standing at room temperature, and fully solidifying into an opalescent fibrin plate for later use after cooling.
The unit of the national food and drug administration is U/mL when measuring the enzyme activity, so the unit of the enzyme activity of the nattokinase in the application is U/mL.
The method comprises the steps of precisely measuring urokinase samples, preparing the urokinase samples into concentration gradients of 50U/mL, 100U/mL, 200U/mL, 400U/mL, 600U/mL, 800U/mL and 1000U/mL by using normal saline, adding 10 mu L of the urokinase samples into a small hole of a fibrin flat plate perforated by a 3mL rubber head dropper, placing the urokinase flat plate in a 37 ℃ constant temperature incubator for 18h, taking out the urokinase flat plate, measuring the vertical diameter of a transparent ring, and expressing the product of the vertical diameter as the area A of the transparent ring, drawing a urokinase standard curve by taking the constant logarithm of the area of the transparent ring lgA as an abscissa and taking the constant logarithm of the enzyme activity lgc as an ordinate to obtain a regression equation, wherein the enzyme activity X of the nattokinase is c × D, wherein c is the nattokinase activity in a sample liquid calculated by the regression equation, U/mL, D is the dilution multiple of the sample liquid, and the fermentation supernatant needs to be diluted only when the diameter of the transparent ring is in a certain range and the linear relationship between lgc and the lA.
Inoculating the 8 strains screened after the preliminary screening to 50mL seedsCulturing in 250mL conical flask containing culture medium (10 g/L peptone, 5g/L yeast powder, 10g/L NaCl, 1000mL sterile water, pH 7.0) at 160r/min and 37 deg.C for 14h, inoculating seed solution at 5% (V/V) inoculum size to 50mL fermentation medium (10 g/L glucose, 20g/L peptone, 1g/L sodium dihydrogen phosphate, 2g/L disodium hydrogen phosphate dodecahydrate, 0.5g/L MgSO 0)4，0.2g/LCaCl2Adjusting pH to 7.2) in a 250mL conical flask, and performing shake culture at 37 ℃ for 48h at 200 r/min. 1mL of fermentation liquid is taken out and put in a centrifuge tube, and centrifuged for 5min at 10000r/min, and the supernatant is the crude enzyme liquid. Taking 10 mu L of supernatant fluid to be spotted on an agarose-fibrin plate orifice plate, standing for 10min, putting the plate in a constant temperature incubator to incubate for 18h at the constant temperature of 37 ℃, measuring the vertical diameter of a transparent ring by a digital display vernier caliper, and calculating the relative enzyme activity of the nattokinase through a urokinase standard curve (because the enzyme activity and the diameter of the transparent ring with the diameter of 15 mm-24 mm have a linear relation, the crude enzyme solution needs to be diluted when the enzyme activity and the diameter exceed the range). 3 strains with larger transparent rings are selected and compared with the original strain for the activity of the nattokinase, and the result is shown in the table 2:
TABLE 2 rescreening results of mutagenized strains
The Bacillus subtilis YC4(Bacillus subtilis YC4) is preserved in China Center for Type Culture Collection (CCTCC) in 2019, 12 months and 30 days, wherein the Bacillus subtilis YC4 has the highest nattokinase activity of 11242U/mL, and the preservation number is M20191132.
Example 3 genetic stability of Strain Bacillus subtilis YC4
The Bacillus subtilis YC4 obtained by re-screening in the example 2 is subjected to subculture, taken as the first generation, streaked every 24h to form a solid culture medium plate, continuously subcultured for 5 generations, inoculated into a 250mL conical flask filled with 50mL of seed culture medium (the components are 10g/L peptone, 5g/L yeast powder, 10g/L NaCl, 1000mL sterile water and pH 7.0) for each generation, inoculated into a 250mL conical flask filled with 50mL of fermentation culture medium by the inoculation amount of 5% (V/V) after shaking culture at constant temperature of 160r/min and 37 ℃ for 12h, inoculated into a 250mL conical flask filled with 50mL of fermentation culture medium by the shaking culture at constant temperature of 160r/min and 37 ℃ for 48h, centrifuged for 5min by 1mL of 10000r/min fermentation liquor, and the supernatant is the crude enzyme solution. Taking 10 mu L of supernatant to be spotted on an agarose-fibrin plate orifice plate, standing for 10min, putting the plate in a constant temperature incubator at 37 ℃ for incubation for 18h, measuring the vertical diameter of a transparent ring by a digital vernier caliper, and determining the passage stability, wherein the results are shown in Table 3:
TABLE 3 passage stability test results of Bacillus subtilis YC4 strain
6 passage tests show that the strain Bacillus subtilis YC4 obtained by mutagenesis has good enzyme production stability, the enzyme activity of the nattokinase is stabilized by about 12000U/mL, the enzyme activity is improved by about 147 percent compared with the nattokinase of the original strain, the mutagenesis effect is good, the production benefit is obviously increased, and the production cost is reduced.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (9)
1. The bacillus subtilis is characterized by being bacillus subtilis YC4, having a Latin literature name of Bacillus subtilis YC4 and being preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M20191132.
2. Use of the bacillus subtilis of claim 1 for the production of nattokinase.
3. A method for producing nattokinase, characterized in that the bacillus subtilis of claim 1 is used for producing nattokinase.
4. The method according to claim 3, wherein the seed solution of Bacillus subtilis is inoculated into a fermentation medium to produce nattokinase by fermentation.
5. The method of claim 4, wherein the fermentation medium comprises a carbon source, a nitrogen source, and inorganic salts.
6. The method of claim 5, wherein the fermentation medium comprises the following components: 10-30 g/L glucose, 10-30 g/L peptone, 1-3 g/L sodium dihydrogen phosphate, 1-3 g/L disodium hydrogen phosphate dodecahydrate, 0.3-0.5 g/L magnesium sulfate, 0.1-0.3 g/L calcium chloride, water as solvent, and pH 7.0-7.4.
7. The method of claim 6, wherein the fermentation medium comprises the following components: 10g/L glucose, 20g/L peptone, 1g/L sodium dihydrogen phosphate, 2g/L disodium hydrogen phosphate dodecahydrate, 0.5g/L magnesium sulfate, 0.2g/L calcium chloride, water as solvent, and pH 7.2.
8. The method according to any one of claims 3 to 7, wherein the fermentation conditions are: the temperature is 30-37 ℃, the culture time is 36-56 h, and shaking culture is carried out at 160-200 r/min.
9. The method of claim 8, wherein the fermentation conditions are: the temperature is 34-37 ℃, the culture time is 36-48 h, and shaking culture is carried out at 160-180 r/min.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113151074A (en) * | 2021-04-09 | 2021-07-23 | 江南大学 | Bacillus subtilis mutant strain for high yield of nattokinase and application thereof |
| CN113736684A (en) * | 2021-06-25 | 2021-12-03 | 郑州大学 | Method for preparing thrombolytic enzyme by fermentation of American ginseng endophyte |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113151074A (en) * | 2021-04-09 | 2021-07-23 | 江南大学 | Bacillus subtilis mutant strain for high yield of nattokinase and application thereof |
| CN113736684A (en) * | 2021-06-25 | 2021-12-03 | 郑州大学 | Method for preparing thrombolytic enzyme by fermentation of American ginseng endophyte |
| CN113736684B (en) * | 2021-06-25 | 2023-11-03 | 郑州大学 | Method for preparing thrombolytic enzyme by fermenting American ginseng endophyte |
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