CN109706135A - A kind of Nattokinase liquid state fermentation culture medium and the fermentation process for producing Nattokinase - Google Patents
A kind of Nattokinase liquid state fermentation culture medium and the fermentation process for producing Nattokinase Download PDFInfo
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Abstract
The present invention discloses a kind of Nattokinase liquid state fermentation culture medium and produces the fermentation process of Nattokinase, wherein, it include the component of following quality: 1~3g of soybean powder, 1~3g of sucrose, 0.2~0.6g of glycerol, 0.01~0.03g of 0.1~0.3g of potassium dihydrogen phosphate, 1~2g of dipotassium hydrogen phosphate and calcium chloride in Nattokinase liquid state fermentation culture medium described in every 100mL, and the pH value of the Nattokinase liquid state fermentation culture medium is 7.2~7.6.Nattokinase liquid state fermentation culture medium provided by the invention significantly improves the yield of Nattokinase when obtaining Nattokinase for carrying out fermented and cultured to bacillus subtilis.
Description
Technical field
The present invention relates to technical field of bioengineering, in particular to a kind of Nattokinase liquid state fermentation culture medium and production are received
The fermentation process of beans kinases.
Background technique
The related disease as caused by thrombus seriously threatens human health, all far super other diseases of morbidity and mortality,
And with society development thrombotic diseases have the tendency that it is more and more fiery.According to statistics and report, thrombotic diseases still Cheng Gaofa
Situation, it is related to thrombotic diseases that the whole world about has more than 15,000,000 Died Patients every year, it is contemplated that arrive the year two thousand twenty, the disease cause or
Lethal patient's number will increase to 25,000,000 people, and China carries out the patient of thrombolytic drug therapy also up to 3,000,000 people or so every year.Cause
This, exploitation has significant thrombolytic effect and low-cost thrombolytic drug or functional food are concerned.Currently, clinical edible
Thrombolysis drug mainly organized type plasminogen activator (tissue-type plasminogen activator, t-
PA), urokinase (urokinase, UK) and streptokinase (streptokinase, SK), but the generally existing administration route of this kind of drug
Complexity, toxic side effect is larger, drug half-life is short and expensive.Therefore, with the Nattokinase of high thrombolysis activity
(nattokinase, NK) has received widespread attention.Research shows that NK can be by reducing fibrinogen, promoting catalysis fibre
Plasmin is converted into fibrin-ferment, increases internal three kinds of efficient thrombolysis of thrombolysis mechanism of thrombolysis factor synthesis.
In addition, NK also has platelet aggregation-against, reducing blood lipid and other effects, has in terms of preventing and treating cardiovascular and cerebrovascular disease and widely answer
With.Simultaneously because have can be by oral edible by NK, the highly-safe, advantages such as production cost is low, property is stables are sharp based on natto
Enzyme, which develops related thrombolysis class product, becomes research hotspot.
Nattokinase (NK) be the single-stranded peptide for being about 28ku by a relative molecular mass constitute have fibrinolytic
Alkaline serine protease.NK has good enzymatic activity within the scope of temperature is lower than 60 DEG C and pH 5-12.Currently, commercialization
The Nattokinase zymotechnique soybean of production is based on the solid-state fermentation process derived from Japan, but since solid-state fermentation process is generally deposited
The disadvantages of material sterilizing is not thorough, the production cycle is long, the on-line monitoring of zymotechnique index is difficult, lead to different batches of product matter
Measure unstable, production efficiency is low.Therefore, high-purity Nattokinase is prepared and turns to liquid-state fermentation technology by researcher, in the past 20
It is generally existing although domestic research and production in relation to Nattokinase liquid state fermentation is there are many report and example in many years
Purifying and aftertreatment technology are complicated, and product price is relatively high, is particularly present the relatively low problem of Nattokinase yield, cannot
Meet the increasingly increased market demand.
Summary of the invention
The main object of the present invention is the fermentation for proposing a kind of Nattokinase liquid state fermentation culture medium and producing Nattokinase
Method, it is intended to improve the yield of Nattokinase.
To achieve the above object, the present invention proposes that a kind of Nattokinase liquid state fermentation culture medium, natto described in every 100mL swash
Include the component of following quality in enzyme solution state fermentation medium:
1~3g of soybean powder, 1~3g of sucrose, 0.2~0.6g of glycerol, 0.1~0.3g of potassium dihydrogen phosphate, dipotassium hydrogen phosphate 1~
0.01~0.03g of 2g and calcium chloride, and the pH value of the Nattokinase liquid state fermentation culture medium is 7.2~7.6.
It preferably, further include the magnesium sulfate of 0.01~0.1g in Nattokinase liquid state fermentation culture medium described in every 100mL.
Preferably, in Nattokinase liquid state fermentation culture medium described in every 100mL: the soybean powder is 1.5g, the sucrose
For 3g, the glycerol be 0.4g, the potassium dihydrogen phosphate is 0.23g, the dipotassium hydrogen phosphate is 1.64g, the calcium chloride is
0.02g, the magnesium sulfate are 0.03g, and the pH value of the Nattokinase liquid state fermentation culture medium is 7.4.
To achieve the above object, the present invention also proposes a kind of fermentation process for producing Nattokinase, comprising the following steps:
Bacillus subtilis is inoculated into fermentation medium and carries out fermented and cultured, the fermentation liquid of acquisition is to contain natto
The product of kinases, wherein the fermentation medium is Nattokinase liquid state fermentation culture medium as described above.
Preferably, bacillus subtilis is inoculated into fermentation medium and carries out fermented and cultured, the fermentation liquid of acquisition is
In the step of product containing Nattokinase:
The fermentation temperature of the fermented and cultured is 26~37 DEG C, and fermentation time is 24~72h.
Preferably, bacillus subtilis is inoculated into fermentation medium and carries out fermented and cultured, the fermentation liquid of acquisition is
In the step of product containing Nattokinase:
The fermentation temperature of the fermented and cultured is 30 DEG C, fermentation time 48h.
Preferably, bacillus subtilis is inoculated into fermentation medium and carries out fermented and cultured, the fermentation liquid of acquisition is
Before the step of product containing Nattokinase, further includes:
Step S10, natto and Nattokinase product are added in physiological saline, in 25~40 DEG C, 150~220r/min item
4~6h of constant-temperature table culture under part, obtains the bacteria suspension containing bacillus subtilis, then carries out to the bacillus subtilis
It isolates and purifies, obtains the single colonie of bacillus subtilis；
Step S20, the single colonie of the bacillus subtilis is first added in LB liquid medium and is cultivated, inoculated to hair
Continue to cultivate in ferment culture medium, the bacterial strain of bacillus subtilis, the target after obtaining primary dcreening operation are then filtered out from cultured products
Bacterial strain；
Step S30, the aimed strain after the primary dcreening operation is subjected to plate streaking and obtains single colonie, then picking single bacterium is fallen within
After being cultivated in LB liquid medium, inoculates into fermentation medium and carry out fermented and cultured, then from fermented and cultured product again
The secondary bacterial strain for filtering out bacillus subtilis, the aimed strain after obtaining secondary screening, the as described bacillus subtilis；
Wherein, fermentation medium described in step S20 and S30 is Nattokinase liquid state fermentation culture as described above
Base.
Preferably, the bacillus subtilis is isolated and purified described in step S10, obtains bacillus subtilis
The step of single colonie, specifically includes:
After the bacteria suspension containing bacillus subtilis is diluted coating using dilution spread plate method, 25~40
Constant temperature incubation 18 at DEG C~for 24 hours, obtain the single colonie of bacillus subtilis.
Preferably, step S20 includes:
The single colonie of the bacillus subtilis is added in LB liquid medium, in 25~40 DEG C, 150~220r/min
Under the conditions of after 10~12h of constant-temperature table culture, inoculate into fermentation medium, in 25~40 DEG C, 150~220r/min condition
Lower constant-temperature table 18~30h of culture, obtains cultured products；
The thallus in the cultured products is removed, the culture solution obtained is on fibrin plate, 36.5~37.5
12~18h is cultivated under the conditions of DEG C, the aimed strain after obtaining primary dcreening operation.
Preferably, step S30 includes:
Plate streaking is carried out to the aimed strain after the primary dcreening operation and obtains single colonie；
The single colonie is added in LB liquid medium, constant-temperature table under the conditions of 25~40 DEG C, 150~220r/min
After cultivating 10~12h, inoculate into fermentation medium, constant-temperature table culture under the conditions of 25~40 DEG C, 150~220r/min
18~30h obtains fermented and cultured product；
Remove the thallus in the fermented and cultured product, the fermentation liquid obtained on fibrin plate, 36.5~
12~18h is cultivated under the conditions of 37.5 DEG C, the aimed strain after obtaining secondary screening, the as described bacillus subtilis.
In technical solution provided by the invention, with soybean powder, sucrose, glycerol, potassium dihydrogen phosphate, dipotassium hydrogen phosphate and chlorine
Change culture medium of the calcium as liquid state fermentation production Nattokinase, swashs for carrying out fermented and cultured acquisition natto to bacillus subtilis
When enzyme, the yield of Nattokinase is significantly improved.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention for those of ordinary skill in the art without creative efforts, can be with
Other relevant attached drawings are obtained according to these attached drawings.
Fig. 1 is the flow diagram of an embodiment of the fermentation process of production Nattokinase provided by the invention.
The embodiments will be further described with reference to the accompanying drawings for the realization, the function and the advantages of the object of the present invention.
Specific embodiment
It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention
Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, according to normal conditions or manufacturer builds
The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase
Product.
To solve to prepare the method for Nattokinase by liquid-state fermentation technology in the prior art, there are Nattokinase yield phases
To lower problem, the present invention proposes a kind of Nattokinase liquid state fermentation culture medium, Nattokinase liquid state fermentation described in every 100mL
It include the component of following quality: 1~3g of soybean powder, 1~3g of sucrose, 0.2~0.6g of glycerol, potassium dihydrogen phosphate 0.1 in culture medium
0.01~0.03g of~0.3g, 1~2g of dipotassium hydrogen phosphate and calcium chloride, and the pH of the Nattokinase liquid state fermentation culture medium
Value is 7.2~7.6, and remaining ingredient is distilled water.
In technical solution provided by the invention, with soybean powder, sucrose, glycerol, potassium dihydrogen phosphate, dipotassium hydrogen phosphate and chlorine
Change culture medium of the calcium as liquid state fermentation production Nattokinase, swashs for carrying out fermented and cultured acquisition natto to bacillus subtilis
When enzyme, the yield of Nattokinase is significantly improved；It is from a wealth of sources and low in cost simultaneously using soybean powder as main nitrogen,
Reduce the cost of material of culture medium.
It, can also be by a small amount of in the Nattokinase liquid state fermentation culture medium in another embodiment provided by the invention
The addition of magnesium sulfate, to further increase the yield of Nattokinase, the magnesium sulfate is in addition, in every 100mL culture medium
In additive amount no more than 0.1g, be more than this addition range, it is not significant to the raising of Nattokinase yield, it is preferable that at this
It further include the magnesium sulfate of 0.01~0.1g in embodiment, in Nattokinase liquid state fermentation culture medium described in every 100mL.
In a preferred embodiment provided by the invention, in Nattokinase liquid state fermentation culture medium described in every 100mL: described
Soybean powder is 1.5g, the sucrose is 3g, the glycerol is 0.4g, the potassium dihydrogen phosphate is 0.23g, the dipotassium hydrogen phosphate
It is 0.02g for 1.64g, the calcium chloride, the magnesium sulfate is 0.03g, and the pH of the Nattokinase liquid state fermentation culture medium
Value is 7.4, thus matches the Nattokinase liquid state fermentation culture medium of composition to the improvement effect of Nattokinase yield especially
Significantly.
Nattokinase liquid culture medium based on above-mentioned offer, the present invention is it is further proposed that a kind of Nattokinase that produces
Fermentation process, Fig. 1 show an embodiment of the fermentation process of production Nattokinase provided by the invention.Referring to Fig. 1, at this
In embodiment, it is described production Nattokinase fermentation process the following steps are included:
Step S40, bacillus subtilis is inoculated into fermentation medium and carries out fermented and cultured, the fermentation liquid of acquisition is
Product containing Nattokinase, wherein the fermentation medium is Nattokinase liquid state fermentation culture medium as described above.
Bacillus subtilis is inoculated into shown Nattokinase liquid state fermentation culture medium and carries out fermented and cultured, is fermented
After product, the processing such as is separated, is purified, is concentrated to the fermentation liquid in the tunning, can be obtained Nattokinase product,
The processing modes such as its separation, purification, concentration can be carried out using the common means of field of microbial fermentation, such as pass through centrifugation
Mode removes the thallus in the tunning, obtains fermentation liquid, is then purified, is dried to the fermentation liquid
Deng therefore not to repeat here.Wherein, inoculum concentration of the bacillus subtilis in the Nattokinase liquid state fermentation culture medium be not
Make specific restriction, such as can be any inoculum concentration in 0.5~10%, preferably 0.5~5%, more preferably 2%.
For Nattokinase liquid state fermentation culture medium provided by the invention, ferment to bacillus subtilis
During culture obtains Nattokinase, fermentation temperature should be suitable for fast-growth and the breeding of the bacillus subtilis,
In the present embodiment, the fermentation temperature of the fermented and cultured in step S40 is 26~37 DEG C, and fermentation time is 24~72h,
Under this fermentation condition, the bacillus subtilis energy fast-growth breeding, and a large amount of natto is generated during growth and breeding
Kinases.
It is highly preferred that the fermentation temperature of the fermented and cultured is 30 DEG C, fermentation time 48h receives under this fermentation condition
The yield of beans kinases is higher.It is received using Nattokinase liquid state fermentation culture medium provided by the invention by liquid-state fermentation technology preparation
When beans kinases, process regulation is simple, it is only necessary to the two parameters of the pH value and fermentation temperature of regulation culture base,
His parameter is not necessarily to Special controlling, and is not necessarily to Feeding medium among process, it is only necessary to which the preparation of Nattokinase can be completed in 24~72h, shorter
A large amount of Nattokinase product can be produced in production cycle, optimize the production work that liquid fermentation method prepares Nattokinase
Skill.
The bacillus subtilis can directly select existing bacterial strain, such as directly from market, purchase is obtained, can also be from containing
Have in the product of bacillus subtilis and obtained by screening, selects swash from the natto containing bacillus subtilis in the present embodiment
In enzyme preparation in such a way that screening obtains, that is, in the present embodiment, to bacillus subtilis carry out fermented and cultured it
Before, further include the steps that screening bacillus subtilis from Nattokinase product.Referring to Fig. 1, before step S40,
Further include:
Step S10, natto and Nattokinase product are added in physiological saline, in 25~40 DEG C, 150~220r/min item
4~6h of constant-temperature table culture under part, obtains the bacteria suspension containing bacillus subtilis, then carries out to the bacillus subtilis
It isolates and purifies, obtains the single colonie of bacillus subtilis；
Contain bacillus subtilis in the natto and Nattokinase product, by being isolated and purified after culture, can be obtained
The bacterium colony of bacillus subtilis, wherein the additive amount of the natto and Nattokinase product without limitation, if containing natto and
The ingredient of Nattokinase product, such as when specific operation, natto and natto can accordingly be added in every 30mL physiological saline
The mixture 2g of kinases product.
Further, in the present embodiment, the bacillus subtilis is isolated and purified described in step S10,
The step of obtaining the single colonie of bacillus subtilis, specifically includes: by the bacteria suspension containing bacillus subtilis using dilution
After coated panel method is diluted coating, the constant temperature incubation 18~for 24 hours at 25~40 DEG C obtains the single colonie of bacillus subtilis.
Step S20, the single colonie of the bacillus subtilis is first added in LB liquid medium and is cultivated, inoculated to hair
Continue to cultivate in ferment culture medium, the bacterial strain of bacillus subtilis, the target after obtaining primary dcreening operation are then filtered out from cultured products
Bacterial strain；
The LB culture medium is a kind of common culture medium in biochemical molecular experiment, generally with the culture medium come preculture bacterium
Kind, it expands strain at double, reaches requirement, liquid culture medium and solid medium can be divided into, selected in the present embodiment
LB liquid culture medium can be obtained directly from commercially available, can also be according to " Molecular Cloning:A Laboratory guide " (J. Pehanorm Brooker D.W.
Russell write) in formula and method prepare, this will not be repeated here.Further, in the present embodiment, step S20 packet
It includes:
Step S21, the single colonie of the bacillus subtilis is added in LB liquid medium, 25~40 DEG C, 150~
Under the conditions of 220r/min after 10~12h of constant-temperature table culture, inoculate to Nattokinase liquid state fermentation culture medium as described above
In, constant-temperature table 18~30h of culture, obtains cultured products under the conditions of 25~40 DEG C, 150~220r/min；
Step S22, remove the thallus in the cultured products, the culture solution obtained on fibrin plate,
12~18h is cultivated under the conditions of 36.5~37.5 DEG C, the aimed strain after obtaining primary dcreening operation.
Wherein, in the step s 21, the single colonie of the bacillus subtilis connects after LB liquid medium culture
Inoculum concentration when kind is into the fermentation medium is 0.5~5%, more preferably 1%, after obtaining the cultured products, is led to
Centrifugation removal thallus is crossed, then culture solution is applied to after being cultivated on fibrin plate, it is biggish several to pick out dissolution circle
Bacterial strain, as the aimed strain after the primary dcreening operation.
Step S30, the aimed strain after the primary dcreening operation is subjected to plate streaking and obtains single colonie, then picking single bacterium is fallen within
After being cultivated in LB liquid medium, inoculates to Nattokinase liquid state fermentation culture medium as described above and carry out fermented and cultured, so
Filter out the bacterial strain of bacillus subtilis again from fermented and cultured product afterwards, the aimed strain after obtaining secondary screening is as described
Bacillus subtilis；
In the present embodiment, step S30 includes:
Step S31, plate streaking is carried out to the aimed strain after the primary dcreening operation and obtains single colonie；
Step S32, the single colonie is added in LB liquid medium, under the conditions of 25~40 DEG C, 150~220r/min
It after 10~12h of constant-temperature table culture, inoculates into fermentation medium, constant temperature under the conditions of 25~40 DEG C, 150~220r/min
18~30h of shaking table culture obtains fermented and cultured product；
Step S33, remove the thallus in the fermented and cultured product, the fermentation liquid obtained on fibrin plate,
12~18h is cultivated under the conditions of 36.5~37.5 DEG C, the aimed strain after obtaining secondary screening, the as described bacillus subtilis.
Wherein, in step S31, the single colonie obtained by plate streaking is passing through LB liquid medium culture
Afterwards, inoculum concentration when being seeded in the fermentation medium is 0.5~5%, more preferably 1%, is obtaining the tunning
Afterwards, thallus is removed by centrifugation, then fermentation liquid is applied to after being cultivated on fibrin plate, it is maximum picks out dissolution circle
Bacterial strain obtains the withered grass gemma of Nattokinase as the aimed strain after the secondary screening for fermented and cultured in as step S40
Bacillus.
The aimed strain that middle step S10 is filtered out to step S30 through the embodiment of the present invention is sequenced by 16sr RNA,
Its sequencing result is as shown in SEQ ID NO:1, it was demonstrated that the aimed strain filtered out is bacillus subtilis Pseudomonas (Bacillus
Subtilis), meet screening expected purpose, can be used for the initial strains that liquid-state fermentation technology prepares Nattokinase.
Nattokinase is prepared using liquid state fermentation method provided in an embodiment of the present invention, the yield of Nattokinase reaches as high as
5000IU/mL or so, compared with the prior art in general liquid fermentation process (enzyme activity is about 2000IU/mL or so), produce
Amount improves 2~3 times, also has the advantages that simple process is easy to operate, with short production cycle.
Technical solution of the present invention is described in further detail below in conjunction with specific embodiments and the drawings, it should be understood that
Following embodiment is only used to explain the present invention, is not intended to limit the present invention.
Embodiment 1
(1) Nattokinase liquid state fermentation culture medium composition (being counted as unit of 100mL volume) are as follows: soybean powder 1.2g, sucrose
2.4g, glycerol 0.4g, potassium dihydrogen phosphate 0.23g, dipotassium hydrogen phosphate 1.64g and calcium chloride 0.02g, pH value 7.4.
(2) natto and the total 2g of Nattokinase product is taken to be added in the physiological saline of 30mL, in 28 DEG C, 180r/min condition
Lower constant-temperature table culture 5h, obtains the bacteria suspension containing bacillus subtilis；Above-mentioned bacteria suspension is diluted by decimal dilution method, it will
Concentration is 10-1、10-2、10-3、10-4、10-5、10-6Dilution respectively draw 100 μ L and be inoculated into LB culture medium flat plate surface respectively,
It is inverted in after smearing uniformly in the constant incubator that temperature setting is 28 DEG C and cultivates 20h, chosen the biggish bacterial strain of growth and divided
From purifying and numbering preservation, the single colonie of bacillus subtilis is obtained.
(3) bacillus subtilis that picking step (2) obtains single colonie be added LB liquid medium in, 28 DEG C,
Under the conditions of 180r/min after constant-temperature table culture 11h, the Nattokinase liquid in step (1) is seeded to according to 1% inoculum concentration
In fermentation medium, constant-temperature table culture for 24 hours, obtains cultured products under the conditions of 28 DEG C, 180r/min；Centrifugation removes the culture
Thallus in product takes 10 μ L culture solutions on fibrin plate, cultivates 15h under the conditions of 37 DEG C, and it is larger to select dissolution circle
Several bacterial strains, as the aimed strain after primary dcreening operation.
(4) plate streaking is carried out to the aimed strain after the primary dcreening operation obtained in step (3) and obtains single colonie；Picking single colonie
It is added in LB liquid medium, under the conditions of 28 DEG C, 180r/min after constant-temperature table culture 11h, is seeded to by 1% inoculum concentration
In Nattokinase liquid state fermentation culture medium in step (1), constant-temperature table culture for 24 hours, must be sent out under the conditions of 28 DEG C, 180r/min
Ferment cultured products；Centrifugation removes the thallus in the fermented and cultured product, takes 10 μ L fermentation liquids on fibrin plate, 37
15h is cultivated under the conditions of DEG C, is picked out dissolution and is enclosed maximum bacterial strain, as the aimed strain after secondary screening.The object bacteria filtered out
Strain turns out to be bacillus subtilis Pseudomonas (Bacillus through 16sr RNA sequencing (sequencing result is as shown in SEQ ID NO:1)
subtilis)。
(5) bacillus subtilis that step (4) obtain the natto in step (1) is inoculated into according to 2% inoculum concentration to swash
It in enzyme solution state fermentation medium, ferments for 24 hours at a temperature of 28 DEG C, is then centrifuged for removal thallus, the fermentation liquid of acquisition is to contain
The product of Nattokinase.
Fermentation liquid obtained in step (5) is taken, uses fibrin plate method measurement enzyme activity for 4820IU/mL.
Embodiment 2
It is same as Example 1, the difference is that, in step (1) Nattokinase liquid state fermentation culture medium composition (with
100mL volume is unit meter) are as follows: soybean powder 1.5g, sucrose 3.0g, glycerol 0.4g, potassium dihydrogen phosphate 0.23g, dipotassium hydrogen phosphate
1.64g, calcium chloride 0.02g and magnesium sulfate 0.03g, pH value 7.4；Fermentation temperature in step (5) is 30 DEG C, fermentation time
For 48h.
Use the enzyme activity of the final fermentation liquid obtained of fibrin plate method measurement for 5690IU/mL.
Embodiment 3
It is same as Example 1, the difference is that, in step (1) Nattokinase liquid state fermentation culture medium composition (with
100mL volume is unit meter) are as follows: soybean powder 2.0g, sucrose 2.0g, glycerol 0.5g, potassium dihydrogen phosphate 0.2g, dipotassium hydrogen phosphate
1.6g, calcium chloride 0.03g and magnesium sulfate 0.01g, pH value 7.4；Fermentation temperature in step (5) is 28 DEG C, fermentation time
For 36h.
Use the enzyme activity of the final fermentation liquid obtained of fibrin plate method measurement for 5330IU/mL.
Embodiment 4
It is same as Example 1, the difference is that, in step (1) Nattokinase liquid state fermentation culture medium composition (with
100mL volume is unit meter) are as follows: soybean powder 1.0g, sucrose 2.0g, glycerol 0.4g, potassium dihydrogen phosphate 0.3g, dipotassium hydrogen phosphate
2.0g, calcium chloride 0.02g and magnesium sulfate 0.04g, pH value 7.4；Fermentation temperature in step (5) is 28 DEG C, fermentation time
For 72h.
Use the enzyme activity of the final fermentation liquid obtained of fibrin plate method measurement for 4690IU/mL.
Embodiment 5
It is same as Example 1, the difference is that, in step (1) Nattokinase liquid state fermentation culture medium composition (with
100mL volume is unit meter) are as follows: soybean powder 1.0g, sucrose 2.0g, glycerol 0.4g, potassium dihydrogen phosphate 0.3g, dipotassium hydrogen phosphate
2.0g, calcium chloride 0.02g and magnesium sulfate 0.04g, pH value 7.4；Fermentation temperature in step (5) is 30 DEG C, fermentation time
For 36h.
Use the enzyme activity of the final fermentation liquid obtained of fibrin plate method measurement for 5120IU/mL.
Embodiment 6
It is same as Example 1, the difference is that, in step (1) Nattokinase liquid state fermentation culture medium composition (with
100mL volume is unit meter) are as follows: soybean powder 1.5g, sucrose 2.5g, glycerol 0.2g, potassium dihydrogen phosphate 0.2g, dipotassium hydrogen phosphate
1.5g, calcium chloride 0.02g and magnesium sulfate 0.03g, pH value 7.4；Fermentation temperature in step (5) is 33 DEG C, fermentation time
For 48h.
Use the enzyme activity of the final fermentation liquid obtained of fibrin plate method measurement for 5080IU/mL.
Embodiment 7
It is same as Example 1, the difference is that, in step (1) Nattokinase liquid state fermentation culture medium composition (with
100mL volume is unit meter) are as follows: soybean powder 1.5g, sucrose 1.5g, glycerol 0.4g, potassium dihydrogen phosphate 0.2g, dipotassium hydrogen phosphate
1.5g, calcium chloride 0.03g and magnesium sulfate 0.02g, pH value 7.4；Fermentation temperature in step (5) is 37 DEG C, fermentation time
For 36h.
Use the enzyme activity of the final fermentation liquid obtained of fibrin plate method measurement for 4750IU/mL.
Embodiment 8
It is same as Example 1, the difference is that, in step (1) Nattokinase liquid state fermentation culture medium composition (with
100mL volume is unit meter) are as follows: soybean powder 2.5g, sucrose 1.0g, glycerol 0.6g, potassium dihydrogen phosphate 0.1g, dipotassium hydrogen phosphate
1.7g, calcium chloride 0.01g and magnesium sulfate 0.1g, pH value 7.4；Fermentation temperature in step (5) is 26 DEG C, fermentation time is
54h。
Use the enzyme activity of the final fermentation liquid obtained of fibrin plate method measurement for 5530IU/mL.
Embodiment 9
It is same as Example 1, the difference is that, in step (1) Nattokinase liquid state fermentation culture medium composition (with
100mL volume is unit meter) are as follows: soybean powder 3.0g, sucrose 1.5g, glycerol 0.35g, potassium dihydrogen phosphate 0.25g, dipotassium hydrogen phosphate
1.0g, calcium chloride 0.03g and magnesium sulfate 0.08g, pH value 7.4；Fermentation temperature in step (5) is 35 DEG C, fermentation time
For 42h.
Use the enzyme activity of the final fermentation liquid obtained of fibrin plate method measurement for 5450IU/mL.
Embodiment 10
(1) Nattokinase liquid state fermentation culture medium composition (being counted as unit of 100mL volume): soybean powder 1.5g, sucrose
3.0g, glycerol 0.4g, potassium dihydrogen phosphate 0.23g, dipotassium hydrogen phosphate 1.64g, calcium chloride 0.02g and magnesium sulfate 0.03g, pH value
It is 7.2.
(2) natto and the total 2g of Nattokinase product is taken to be added in the physiological saline of 30mL, in 25 DEG C, 220r/min condition
Lower constant-temperature table culture 6h, obtains the bacteria suspension containing bacillus subtilis；Above-mentioned bacteria suspension is diluted by decimal dilution method, it will
Concentration is 10-1、10-2、10-3、10-4、10-5、10-6Dilution respectively draw 100 μ L and be inoculated into LB culture medium flat plate surface respectively,
It is inverted in the constant incubator that temperature setting is 25 DEG C and is cultivated for 24 hours after smearing uniformly, choose the biggish bacterial strain of growth and divided
From purifying and numbering preservation, the single colonie of bacillus subtilis is obtained.
(3) bacillus subtilis that picking step (2) obtains single colonie be added LB liquid medium in, 25 DEG C,
Under the conditions of 220r/min after constant-temperature table culture 12h, the Nattokinase liquid in step (1) is seeded to according to 1% inoculum concentration
In fermentation medium, constant-temperature table culture 26h, obtains cultured products under the conditions of 25 DEG C, 220r/min；Centrifugation removes the culture
Thallus in product takes 10 μ L culture solutions on fibrin plate, cultivates 18h under the conditions of 36.5 DEG C, select dissolution circle compared with
Big several bacterial strains, as the aimed strain after primary dcreening operation.
(4) plate streaking is carried out to the aimed strain after the primary dcreening operation obtained in step (3) and obtains single colonie；Picking single colonie
It is added in LB liquid medium, under the conditions of 25 DEG C, 220r/min after constant-temperature table culture 12h, is seeded to by 1% inoculum concentration
In Nattokinase liquid state fermentation culture medium in step (1), constant-temperature table culture 26h, must be sent out under the conditions of 25 DEG C, 220r/min
Ferment cultured products；Centrifugation removes the thallus in the fermented and cultured product, takes 10 μ L fermentation liquids on fibrin plate,
18h is cultivated under the conditions of 36.5 DEG C, dissolution is picked out and encloses maximum bacterial strain, as the aimed strain after secondary screening.The mesh filtered out
Mark bacterial strain turns out to be bacillus subtilis Pseudomonas (Bacillus through 16sr RNA sequencing (sequencing result is as shown in SEQ ID NO:1)
subtilis)。
(5) bacillus subtilis that step (4) obtain is inoculated into the Nattokinase liquid state fermentation culture medium in step (1)
In, ferment 72h at a temperature of 26 DEG C, is then centrifuged for removal thallus, and the fermentation liquid of acquisition is the product containing Nattokinase.
Fermentation liquid obtained in step (5) is taken, uses fibrin plate method measurement enzyme activity for 4960IU/mL.
Embodiment 11
(1) Nattokinase liquid state fermentation culture medium composition (being counted as unit of 100mL volume): soybean powder 1.5g, sucrose
3.0g, glycerol 0.4g, potassium dihydrogen phosphate 0.23g, dipotassium hydrogen phosphate 1.64g, calcium chloride 0.02g and magnesium sulfate 0.03g, pH value
It is 7.6.
(2) natto and the total 2g of Nattokinase product is taken to be added in the physiological saline of 30mL, in 40 DEG C, 150~220r/
Constant-temperature table culture 4h under the conditions of min, obtains the bacteria suspension containing bacillus subtilis；Above-mentioned bacteria suspension is pressed into decimal dilution method
Concentration is 10 by dilution-1、10-2、10-3、10-4、10-5、10-6Dilution respectively draw 100 μ L to be inoculated into LB culture medium respectively flat
Plate surface is inverted in the constant incubator that temperature setting is 40 DEG C after smearing uniformly and cultivates 18h, chooses and grow biggish bacterial strain
Preservation is isolated and purified and numbered, the single colonie of bacillus subtilis is obtained.
(3) bacillus subtilis that picking step (2) obtains single colonie be added LB liquid medium in, 40 DEG C,
Under the conditions of 150r/min after constant-temperature table culture 10h, the Nattokinase liquid in step (1) is seeded to according to 1% inoculum concentration
In fermentation medium, constant-temperature table culture 18h, obtains cultured products under the conditions of 40 DEG C, 150r/min；Centrifugation removes the culture
Thallus in product takes 10 μ L culture solutions on fibrin plate, cultivates 12h under the conditions of 37.5 DEG C, select dissolution circle compared with
Big several bacterial strains, as the aimed strain after primary dcreening operation.
(4) plate streaking is carried out to the aimed strain after the primary dcreening operation obtained in step (3) and obtains single colonie；Picking single colonie
It is added in LB liquid medium, under the conditions of 40 DEG C, 150r/min after constant-temperature table culture 10h, is seeded to by 1% inoculum concentration
In Nattokinase liquid state fermentation culture medium in step (1), constant-temperature table culture 18h, must be sent out under the conditions of 40 DEG C, 150r/min
Ferment cultured products；Centrifugation removes the thallus in the fermented and cultured product, takes 10 μ L fermentation liquids on fibrin plate,
12h is cultivated under the conditions of 37.5 DEG C, dissolution is picked out and encloses maximum bacterial strain, as the aimed strain after secondary screening.The mesh filtered out
Mark bacterial strain turns out to be bacillus subtilis Pseudomonas (Bacillus through 16sr RNA sequencing (sequencing result is as shown in SEQ ID NO:1)
subtilis)。
(5) bacillus subtilis that step (4) obtain is inoculated into the Nattokinase liquid state fermentation culture medium in step (1)
In, it ferments for 24 hours at a temperature of 37 DEG C, is then centrifuged for removal thallus, the fermentation liquid of acquisition is the product containing Nattokinase.
Fermentation liquid obtained in step (5) is taken, uses fibrin plate method measurement enzyme activity for 5240IU/mL.
Embodiment 12
(1) Nattokinase liquid state fermentation culture medium composition (being counted as unit of 100mL volume): soybean powder 1.5g, sucrose
3.0g, glycerol 0.4g, potassium dihydrogen phosphate 0.23g, dipotassium hydrogen phosphate 1.64g, calcium chloride 0.02g and magnesium sulfate 0.03g, pH value
It is 7.4.
(2) natto and the total 2g of Nattokinase product is taken to be added in the physiological saline of 30mL, in 30 DEG C, 200r/min condition
Lower constant-temperature table culture 5h, obtains the bacteria suspension containing bacillus subtilis；Above-mentioned bacteria suspension is diluted by decimal dilution method, it will
Concentration is 10-1、10-2、10-3、10-4、10-5、10-6Dilution respectively draw 100 μ L and be inoculated into LB culture medium flat plate surface respectively,
It is inverted in after smearing uniformly in the constant incubator that temperature setting is 30 DEG C and cultivates 22h, chosen the biggish bacterial strain of growth and divided
From purifying and numbering preservation, the single colonie of bacillus subtilis is obtained.
(3) bacillus subtilis that picking step (2) obtains single colonie be added LB liquid medium in, 30 DEG C,
Under the conditions of 200r/min after constant-temperature table culture 10h, the Nattokinase liquid in step (1) is seeded to according to 1% inoculum concentration
In fermentation medium, constant-temperature table culture 30h, obtains cultured products under the conditions of 30 DEG C, 200r/min；Centrifugation removes the culture
Thallus in product takes 10 μ L culture solutions on fibrin plate, cultivates 16h under the conditions of 37 DEG C, and it is larger to select dissolution circle
Several bacterial strains, as the aimed strain after primary dcreening operation.
(4) plate streaking is carried out to the aimed strain after the primary dcreening operation obtained in step (3) and obtains single colonie；Picking single colonie
It is added in LB liquid medium, under the conditions of 30 DEG C, 200r/min after constant-temperature table culture 10h, is seeded to by 1% inoculum concentration
In Nattokinase liquid state fermentation culture medium in step (1), constant-temperature table culture 30h, must be sent out under the conditions of 30 DEG C, 200r/min
Ferment cultured products；Centrifugation removes the thallus in the fermented and cultured product, takes 10 μ L fermentation liquids on fibrin plate, 37
16h is cultivated under the conditions of DEG C, is picked out dissolution and is enclosed maximum bacterial strain, as the aimed strain after secondary screening.The object bacteria filtered out
Strain turns out to be bacillus subtilis Pseudomonas (Bacillus through 16sr RNA sequencing (sequencing result is as shown in SEQ ID NO:1)
subtilis)。
(5) bacillus subtilis that step (4) obtain is inoculated into the Nattokinase liquid state fermentation culture medium in step (1)
In, ferment 48h at a temperature of 30 DEG C, is then centrifuged for removal thallus, and the fermentation liquid of acquisition is the product containing Nattokinase.
Fermentation liquid obtained in step (5) is taken, uses fibrin plate method measurement enzyme activity for 5380IU/mL.
The above is only a preferred embodiment of the present invention, is not intended to limit the scope of the invention, for this field
For technical staff, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any
Modification, equivalent replacement, improvement etc. should all be included within the scope of the present invention.
SEQUENCE LISTING
<110>Wuhan Polytechnic University
<120>fermentation process of a kind of Nattokinase liquid state fermentation culture medium and production Nattokinase
<130> 2018.12.05
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1461
<212> DNA
<213> Bacillus subtilis
<400> 1
gaggaactgt cggcttgcta tacatgcaag tcgagcggac agatgggagc ttgctccctg 60
atgttagcgg cggacgggtg agtaacacgt gggtaacctg cctgtaagac tgggataact 120
ccgggaaacc ggggctaata ccggatggtt gtttgaaccg catggttcaa acataaaagg 180
tggcttcggc taccacttac agatggaccc gcggcgcatt agctagttgg tgaggtaacg 240
gctcaccaag gcgacgatgc gtagccgacc tgagagggtg atcggccaca ctgggactga 300
gacacggccc agactcctac gggaggcagc agtagggaat cttccgcaat ggacgaaagt 360
ctgacggagc aacgccgcgt gagtgatgaa ggttttcgga tcgtaaagct ctgttgttag 420
ggaagaacaa gtaccgttcg aatagggcgg taccttgacg gtacctaacc agaaagccac 480
ggctaactac gtgccagcag ccgcggtaat acgtaggtgg caagcgttgt ccggaattat 540
tgggcgtaaa gggctcgcag gcggtttctt aagtctgatg tgaaagcccc cggctcaacc 600
ggggagggtc attggaaact ggggaacttg agtgcagaag aggagagtgg aattccacgt 660
gtagcggtga aatgcgtaga gatgtggagg aacaccagtg gcgaaggcga ctctctggtc 720
tgtaactgac gctgaggagc gaaagcgtgg ggagcgaaca ggattagata ccctggtagt 780
ccacgccgta aacgatgagt gctaagtgtt agggggtttc cgccccttag tgctgcagct 840
aacgcattaa gcactccgcc tggggagtac ggtcgcaaga ctgaaactca aaggaattga 900
cgggggcccg cacaagcggt ggagcatgtg gtttaattcg aagcaacgcg aagaacctta 960
ccaggtcttg acatcctctg acaatcctag agataggacg tccccttcgg gggcagagtg 1020
acaggtggtg catggttgtc gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac 1080
gagcgcaacc cttgatctta gttgccagca ttcagttggg cactctaagg tgactgccgg 1140
tgacaaaccg gaggaaggtg gggatgacgt caaatcatca tgccccttat gacctgggct 1200
acacacgtgc tacaatggac agaacaaagg gcagcgaaac cgcgaggtta agccaatccc 1260
acaaatctgt tctcagttcg gatcgcagtc tgcaactcga ctgcgtgaag ctggaatcgc 1320
tagtaatcgc ggatcagcat gccgcggtga atacgttccc gggccttgta cacaccgccc 1380
gtcacaccac gagagtttgt aacacccgaa gtcggtgagg taacctttta ggagccagcc 1440
gccgaagttg aacaaggtgg a 1461
Claims (10)
1. a kind of Nattokinase liquid state fermentation culture medium, which is characterized in that Nattokinase liquid state fermentation culture medium described in every 100mL
In include following quality component:
1~3g of soybean powder, 1~3g of sucrose, 0.2~0.6g of glycerol, 0.1~0.3g of potassium dihydrogen phosphate, 1~2g of dipotassium hydrogen phosphate with
And 0.01~0.03g of calcium chloride, and the pH value of the Nattokinase liquid state fermentation culture medium is 7.2~7.6.
2. Nattokinase liquid state fermentation culture medium as described in claim 1, which is characterized in that Nattokinase described in every 100mL
It further include the magnesium sulfate of 0.01~0.1g in liquid state fermentation culture medium.
3. Nattokinase liquid state fermentation culture medium as claimed in claim 2, which is characterized in that Nattokinase described in every 100mL
In liquid state fermentation culture medium: the soybean powder is 1.5g, the sucrose is 3g, the glycerol is 0.4g, the potassium dihydrogen phosphate
For 0.23g, the dipotassium hydrogen phosphate be 1.64g, the calcium chloride is 0.02g, the magnesium sulfate is 0.03g, and the natto
The pH value of kinases liquid state fermentation culture medium is 7.4.
4. a kind of fermentation process for producing Nattokinase, which comprises the following steps:
Bacillus subtilis is inoculated into fermentation medium and carries out fermented and cultured, the fermentation liquid of acquisition is to contain Nattokinase
Product, wherein the fermentation medium is Nattokinase liquid state fermentation culture as described in claims 1 to 3 any one
Base.
5. the fermentation process of production Nattokinase as claimed in claim 4, which is characterized in that bacillus subtilis to be inoculated into
Carry out fermented and cultured in fermentation medium, in the step of fermentation liquid of acquisition is the product containing Nattokinase:
The fermentation temperature of the fermented and cultured is 26~37 DEG C, and fermentation time is 24~72h.
6. the fermentation process of production Nattokinase as claimed in claim 5, which is characterized in that bacillus subtilis to be inoculated into
Carry out fermented and cultured in fermentation medium, in the step of fermentation liquid of acquisition is the product containing Nattokinase:
The fermentation temperature of the fermented and cultured is 30 DEG C, fermentation time 48h.
7. the fermentation process of production Nattokinase as claimed in claim 4, which is characterized in that bacillus subtilis to be inoculated into
Fermented and cultured is carried out in fermentation medium, before the step of fermentation liquid of acquisition is the product containing Nattokinase, further includes:
Step S10, natto and Nattokinase product are added in physiological saline, under the conditions of 25~40 DEG C, 150~220r/min
Constant-temperature table 4~6h of culture, obtains the bacteria suspension containing bacillus subtilis, then separates to the bacillus subtilis
Purifying, obtains the single colonie of bacillus subtilis；
Step S20, the single colonie of the bacillus subtilis is first added in LB liquid medium and is cultivated, inoculated to fermentation and train
It supports and continues to cultivate in base, the bacterial strain of bacillus subtilis, the aimed strain after obtaining primary dcreening operation are then filtered out from cultured products；
Step S30, the aimed strain after the primary dcreening operation is subjected to plate streaking and obtains single colonie, then picking single bacterium falls within LB liquid
After being cultivated in body culture medium, inoculates into fermentation medium and carry out fermented and cultured, then sieved again from fermented and cultured product
The bacterial strain for selecting bacillus subtilis, the aimed strain after obtaining secondary screening, the as described bacillus subtilis；
Wherein, fermentation medium described in step S20 and S30 is the Nattokinase as described in claims 1 to 3 any one
Liquid state fermentation culture medium.
8. the fermentation process of production Nattokinase as claimed in claim 7, which is characterized in that institute described in step S10
The step of stating bacillus subtilis to be isolated and purified, obtaining the single colonie of bacillus subtilis, specifically includes:
After the bacteria suspension containing bacillus subtilis is diluted coating using dilution spread plate method, at 25~40 DEG C
Constant temperature incubation 18~for 24 hours, obtain the single colonie of bacillus subtilis.
9. the fermentation process of production Nattokinase as claimed in claim 7, which is characterized in that step S20 includes:
The single colonie of the bacillus subtilis is added in LB liquid medium, in 25~40 DEG C, 150~220r/min condition
After 10~12h of lower constant-temperature table culture, inoculate into fermentation medium, it is permanent under the conditions of 25~40 DEG C, 150~220r/min
Warm 18~30h of shaking table culture, obtains cultured products；
The thallus in the cultured products is removed, the culture solution obtained is on fibrin plate, in 36.5~37.5 DEG C of items
12~18h is cultivated under part, the aimed strain after obtaining primary dcreening operation.
10. the fermentation process of production Nattokinase as claimed in claim 7, which is characterized in that step S30 includes:
Plate streaking is carried out to the aimed strain after the primary dcreening operation and obtains single colonie；
The single colonie is added in LB liquid medium, constant-temperature table culture under the conditions of 25~40 DEG C, 150~220r/min
After 10~12h, inoculate into fermentation medium, under the conditions of 25~40 DEG C, 150~220r/min constant-temperature table culture 18~
30h obtains fermented and cultured product；
Remove the thallus in the fermented and cultured product, the fermentation liquid obtained is on fibrin plate, 36.5~37.5
12~18h is cultivated under the conditions of DEG C, the aimed strain after obtaining secondary screening, the as described bacillus subtilis.
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