CN105087447B - One plant of resistance to microwave bacillus subtilis and its application in Nattokinase preparation - Google Patents
One plant of resistance to microwave bacillus subtilis and its application in Nattokinase preparation Download PDFInfo
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Abstract
Application the invention discloses one plant of resistance to microwave bacillus subtilis and its in Nattokinase preparation, the bacillus subtilis are bacillus subtilis (Bacillus subtilis) LB 18, depositary institution：China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, preserving number：CGMCC NO.10835, preservation date：On May 22nd, 2015.The present invention filters out the bacillus subtilis strain of resistance to microwave LB 18 from fermented soya bean, bacterial strain using the present invention, the Nattokinase product (bean-containing capsule) with Hyperfibrinolysis and rich in bacillus subtilis viable bacteria can be obtained, the Nattokinase product not only has decompression, thrombolysis and other effects, also improve function of intestinal canal, improve the effects that immunity of organisms, research and development for Nattokinase as functional food and health food provide new thinking, make it have more wide development prospect.
Description
Technical field
Application the present invention relates to one plant of resistance to microwave bacillus subtilis and its in Nattokinase preparation, belongs to microorganism
Technical field.
Background technology
With the adjustment of people's the accelerating rhythm of life and eating habit, the incidence of thrombotic diseases greatly improves, sternly
The health and life security that threaten the mankind again, it is up to millions of to die of cerebral infarction, the patient of myocardial infarction every year, occupies various diseases
First of disease.Currently, the prefered method for the treatment of cardiovascular and cerebrovascular disease is thrombus, therefore, there is Hyperfibrinolysis thrombolytic drug
The research and development of (such as Nattokinase) become one of research hotspot.
Nattokinase is that natto (natto) is produced by bacillus subtilis (Bacillus subtilis) during the fermentation
The raw serine protease with Hyperfibrinolysis, the enzyme not only have significant thrombolysis activity, can also swash intravital
Plasminogen increases the content of endogenous fibrinolytic enzyme, has a variety of medical work(such as blood pressure lowering, anti-oxidant, antitumor and thrombus dissolving
Effect, and compared with traditional Thrombolytic agent, Nattokinase is not easy to cause bleeding, no antigen, long half time, safe nothing
The advantages that malicious, of low cost and oral effective, thus with the potentiality that exploitation is prevention cardiovascular and cerebrovascular disease health food.From
1987 by Dr.Sumi for the first time after being separated in natto, Nattokinase is just by the extensive pass of world related researcher
Note.
At the same time, due to the influence of eating habit, operating pressure and environment etc., the incidence of intestines problem also have by
The gesture that year increases, significant impact is caused to people’s lives and work.Enteric microorganism is also known as second set of genome of the mankind,
Generation with a variety of intestines problems such as metabolic disease, immunity disease even tumour has direct relation.Therefore, improve enteron aisle
Microenvironment, adjusting intestinal flora are the key that prevention and treatment intestines problems, and beneficial bacteria of intestinal tract is also 21 century newtype drug
The direction of research and development.
Nattokinase fermentation strain --- bacillus subtilis is human body probiotics, and live bacteria agent has a variety of lifes to human body
Object function.First, bacillus subtilis can inhibit pathogenic bacteria to grow, and promote growth of probiotics, so as to improve intestinal microecology
Environment prevents the intestines problems such as dysentery, enteritis, constipation；Secondly, bacillus subtilis, can by secreting organic acid, bacteriocin etc.
To enhance body's immunity；Furthermore bacillus subtilis is proliferated in enteron aisle, body gut metabolism can be adjusted；Finally, withered
Careless bacillus can promote digesting and assimilating for enteral nutrition substance by secreting various enzymes and vitamin substances.
And during existing Nattokinase product industrialization production, since the drying of tunning needs to use microwave
Therefore vacuum dryer, the bacillus subtilis viable bacteria which can kill most of fermenting and producing Nattokinase receive
Bacillus subtilis viable bacteria content is not high in beans kinases product.If the Nattokinase Producing Strain of one plant of resistance to microwave can be filtered out
Strain, so that it may which, to obtain the Nattokinase product rich in bacillus subtilis viable bacteria, which not only has Nattokinase product
Decompression, thrombolysis and other effects also contain abundant bacillus subtilis viable bacteria, improve function of intestinal canal, prevention and treatment to play
The effect of intestines problem, following development prospect is self-evident, but the report of related fields not yet domestic at present.
Invention content
Application the object of the present invention is to provide one plant of resistance to microwave bacillus subtilis and its in Nattokinase preparation, with
It solves in Nattokinase production process since bacillus subtilis is intolerant to microwave radiation, and is difficult to obtain rich in bacillus subtilis
The technical issues of Nattokinase product (bean-containing capsule) of viable bacteria.
To achieve the goals above, the technical solution adopted in the present invention is to provide one plant of resistance to microwave bacillus subtilis,
The bacillus subtilis is bacillus subtilis (Bacillus subtilis) LB-18, depositary institution：China Microbiological bacterium
Kind preservation administration committee common micro-organisms center, preservation address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, preserving number：
CGMCC NO.10835, preservation date：On May 22nd, 2015.
The present invention filters out the bacillus subtilis strain of resistance to microwave LB-18 from fermented soya bean, bacterial strain using the present invention, can be with
Obtain the Nattokinase product (bean-containing capsule) with Hyperfibrinolysis and rich in bacillus subtilis viable bacteria.Experimental result table
Bright, the Nattokinase product fibrinolytic of bacterial strain production of the present invention is up to 2800FU/g；Bacillus subtilis viable bacteria content is
1.26×108A/g is several times of existing Nattokinase tunning.
The technical solution adopted in the present invention, which also resides in, provides a bacillus subtilis in production rich in bacillus subtilis
Application in terms of the Hyperfibrinolysis Nattokinase product of bacterium viable bacteria.
The technical solution adopted in the present invention, which also resides in, provides bacillus subtilis production rich in bacillus subtilis
The method of the Hyperfibrinolysis Nattokinase of viable bacteria, includes the following steps：
(1) from inclined-plane, picking bacillus subtilis LB-18 is inoculated in LB liquid seed culture mediums, under the conditions of 25-40 DEG C,
120-180r/min shaking table constant temperature incubation 12-18h, obtain bacillus subtilis seed liquor；
(2) soybean soaking is stayed overnight, is drained away the water, high pressure sterilization after being cooled to room temperature, connects according to the inoculum concentration of 1-8%
Kind of bacillus subtilis seed liquor, fermented and cultured 36-72h under the conditions of 25-40 DEG C；
(3) it by the soya bean fermented and cultured product micro-wave vacuum of step (2), crushes, crosses 80-120 mesh and sieve to obtain the final product.
The LB liquid seed culture mediums include the raw material of following mass fraction：Peptone 1%, yeast powder 0.5%, NaCl
1%；PH6.8,121 DEG C of high pressure sterilization 20min.
The drying condition of the micro-wave vacuum equipment is：Drying temperature≤40 DEG C, vacuum degree -0.06MPa, microwave work(
Rate 8kW, 2450 ± 50MHz of microwave frequency, drying time 3-8h.
Bacillus subtilis using the present invention produces Nattokinase product, can not only obtain having Hyperfibrinolysis
Nattokinase, but also be a kind of active bacteria formulation rich in bacillus subtilis, to existing decompression, thrombolysis and other effects, also
The effect for improving function of intestinal canal, improving immunity of organisms carries for Nattokinase as the research and development of functional food and health food
New thinking has been supplied, more wide development prospect is made it have.Meanwhile the present invention uses soya bean for raw material, abundance is easy
It takes, at low cost, non-environmental-pollution, device therefor, reagent price are cheap, are convenient for large-scale production.
Description of the drawings
The specific implementation mode of the present invention is described in further detail below in conjunction with attached drawing.
Fig. 1 is bacterial strain LB-18 of the present invention and other Nattokinases produce the survival after the Co60 irradiation of bacterial strain various dose
Rate；
Fig. 2 is bacterial strain LB-18 of the present invention and other Nattokinases produce the survival after bacterial strain different time microwave irradiation
Rate.
Specific implementation mode
The specific implementation mode of the present invention is described in further detail with reference to embodiments.
The screening of 1 Nattokinase superior strain of embodiment
The primary dcreening operation of 1.1 Nattokinase superior strains takes commercially available fermented soya bean, weighs sample 10g, is added in 100mL sterile waters, fills
Point oscillation is broken up, and 100 DEG C of boiling water boil 5min, stand a moment, takes upper layer dirty solution, after gradient dilution, takes 100 μ L different respectively
The solution of dilution is spread evenly across on LB tablets, and 37 DEG C are incubated overnight, and is chosen single bacterium colony and is carried out scribing line culture, twice in succession.It chooses
It takes in the single bacterium colony dibbling to fibrin plate on LB separation tablets, 37 DEG C of culture 18h measure dissolving loop diameter, dissolving circle
Larger strain liquid fermentation carries out secondary screening.
The secondary screening of 1.2 Nattokinase superior strains lives the larger bacterial strain of primary dcreening operation dissolving loop diameter with LB seed culture mediums
Change (37 DEG C, 150r/min, overnight), liquid fermentation medium is respectively connected to (based on mass fraction according to 5% inoculum concentration：6%
Soy meal, 2% glucose, 0.06%CaCl2, 0.07%MgSO4, pH6.5) in, 37 DEG C of fermentations 72h, tunning 0.05M,
The phosphate buffer of pH7.4 dilutes, and takes 10 μ L of dilution, is loaded onto 37 DEG C of constant temperature incubation 18h on fibrin plate, measures molten
Loop diameter is solved, dissolving is chosen and encloses larger bacterial strain, carry out the screening of radiation hardness bacterial strain.
The screening of 2 radiation hardness Nattokinase superior strain of embodiment
2.1 nitrosoguanidine mutagenesis produce the relatively high bacterial strain of natto kinase activity after taking secondary screening, are inoculated in liquid LB cultures
In base, 37 DEG C, 150r/min, be incubated overnight after, transfer in LB liquid medium again (inoculum concentration 1%), 37 DEG C, 150r/
Min shaking table cultures are to logarithmic growth later stage (about 5-8h), and after microscopy observes thalli morphology, 8000r/min centrifuges 5min and collects bacterium
Body is resuspended in 0.1M PBS buffer solution after sodium phosphate buffer (PBS) washing thalline of 0.1M, pH7.0 of sterilizing
In (make bacterial concentration be 106-108A/mL), 2mg/mL nitrosoguanidines (NTG) solution is added and (is matched with the PBS of 0.1M, pH7.0
System), make its final concentration of 0.2mg/mL, 35 DEG C, after 150r/min shaking table mutagenesis 1h, wash 3 times with 0.1M, pH7.0 PBS and remove
NTG, thalline is gone to be resuspended in PBS.
2.2 resistance to Co60The thalline Co that the screening of irradiation Nattokinase superior strain will be resuspended in PBS after NTG mutagenesis60
Irradiation, first time irradiation dose are 100Gy；Gradient dilution applies LB tablets after irradiation, calculates lethality；Bacterium then is collected by centrifugation
Body, is added sterilizing LB liquid medium, 37 DEG C, 150r/min shaking tables cultivated again to late log phase (about 5h), microscopy observes bacterium
After volume morphing, thalline is collected, and be resuspended in PBS buffer solution, carries out second of Co60It irradiates (dosage gradually increases), such as
Co is repeated in this60Irradiation, dosage used is followed successively by 100,200,400,600,800,1000Gy.Through Co60After irradiating repeatedly,
Obtain one plant of resistance to Co60The bacterial strain LB-18 of irradiation (result is shown in attached drawing 1).
The resistance to microwave properties of 2.3 bacterial strain LB-18, which measure, is inoculated into bacterial strain LB-18 sterilized LB liquid medium, 37 DEG C,
150r/min shaking tables were cultivated again to the logarithmic growth later stage, after washing 2 times with mass fraction 0.9%NaCl, used mass fraction
0.9%NaCl is resuspended, and is diluted to 108A cell/mL, takes 1mL bacterium solutions that 9mL mass fraction 9%NaCl are added, and mixing is placed on ice
On block, be adjusted to medium power, carry out microwave irradiation, irradiation time is respectively 3,5,7min, each sample sets 3 repetitions, irradiates
Gradient dilution applies LB tablets afterwards, calculates Survival probability of bacteria, compares that (result is shown in attached drawing with Nattokinase production bacterial strain DC-Tx and R
2)。
The feature of bacterial strain LB-18 is as follows：
Morphological feature：
For 24 hours, bacterial strain LB-18 bacterium colonies are in subcircular, and edge is micro- to have lobate tooth, surface to have fold for 37 DEG C of cultures on LB tablets,
Milky is opaque, toughness；Microscope hypothallus be in elongated rod shape, 3.0-8.0 μm long, 0.6-0.8 μm wide, Gram-positive,
It is raw in gemma, ellipse.
Physiological and biochemical property is shown in Table 1.
The physiological and biochemical property of 1 bacterial strain LB-18 of table
Note：+ indicate positive；Indicate negative
16S rDNA sequencing identifications：
The genomic DNA for extracting bacterial strain LB-18 is expanded with 16S rDNA gene universal primer PCRs；Obtained PCR product,
It is connect with pGM-T carriers, converts DH5 α competent cells；Screening positive clone puies forward Plasmid DNA and carries out digestion identification；Connection is just
True positive colony carries out 16SrDNA sequencings, as a result with the existing sequence in ncbi database it was found that, bacterial strain LB-18's
The complete genome sequence of 16SrDNA reaches 100% with the bacillus subtilis coincidence rate announced on the net.Sequencing result is as follows：
GCATTACTGTTCATCTATCGCGGCTGGCTCCATAAAGGTTACCTCACCGACTTCGGGTGTTACAAACTC
TCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGAT
TCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGCTTAACCTCGCGGT
TTCGCTGCCCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCC
CCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGC
GCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCCCCCG
AAGGGGACGTCCTATCTCTAGGATTGTCAGAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAAC
CACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAG
TGCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAACCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTA
CCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTTACAGACCAGAGAGTCGCCTTCGCC
ACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTCC
CCAGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGAGCCCTTTAC
GCCCAATAATTCCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTCTGG
TTAGGTACCGTCAAGGTGCCGCCCTATTTGAACGGCACTTGTCTTCCCTAACACAGAGCTTTACGATCCGAAAACCT
TCATCACTCACGCGGCGTTGCTCGTCAGACTTTCGTCATGCGAGATCCTACTGCTGCCTCCGTAGGAGTCTGGGCGT
GTCTCAGTCCCATGTGCGATCACCTTCTCAGTCGCTACCCATCGTCGCCTGGTGGCGTACCTCACCACTAGCTATGG
CCCCGGTCAATGTATTGTTGCAGAGCCACTTTATGTCTGAACCTTGCGTTCACAACCATCCGGGATAGCCGGTTCCG
GATAATACACGAGTCTTTAC
In summary morphological feature, physiological and biochemical analysis and molecule sequencing identification as a result, showing that LB-18 is one plant of withered grass
Bacillus (Bacillus subtilis).
Embodiment 3
The method of Hyperfibrinolysis Nattokinase of the present embodiment production rich in bacillus subtilis viable bacteria, including following step
Suddenly：
(1) from inclined-plane, picking bacillus subtilis LB-18 is inoculated in LB liquid seed culture mediums, under the conditions of 25 DEG C,
180r/min shaking table constant temperature incubation 18h, obtain bacillus subtilis LB-18 seed liquors；
(2) soybean soaking is stayed overnight, is drained away the water, 121 DEG C of high pressure sterilization 20min, after being cooled to room temperature, according to 8%
Inoculum concentration is inoculated with bacillus subtilis LB-18 seed liquors, fermented and cultured 72h under the conditions of 25 DEG C；
(3) the soya bean fermented and cultured product of step (2) dry, pulverize with micro-wave vacuum equipment, sieves with 100 mesh sieve i.e.
；The drying condition of the micro-wave vacuum equipment is：20 DEG C, vacuum degree -0.06MPa, microwave power 8kW of drying temperature,
Microwave frequency 2400MHz, drying time 8h.
Embodiment 4
The method of Hyperfibrinolysis Nattokinase of the present embodiment production rich in bacillus subtilis viable bacteria, including following step
Suddenly：
(1) from inclined-plane, picking bacillus subtilis LB-18 is inoculated in LB liquid seed culture mediums, under the conditions of 28 DEG C,
120r/min shaking table constant temperature incubation 14h, obtain bacillus subtilis LB-18 seed liquors；
(2) soybean soaking is stayed overnight, is drained away the water, 121 DEG C of high pressure sterilization 20min, after being cooled to room temperature, according to 5%
Inoculum concentration is inoculated with bacillus subtilis LB-18 seed liquors, fermented and cultured 48h under the conditions of 28 DEG C；
(3) the soya bean fermented and cultured production of step (2) dry, pulverize with object micro-wave vacuum equipment, crossing 80 mesh sieve is
；The drying condition of the micro-wave vacuum equipment is：30 DEG C, vacuum degree -0.06MPa, microwave power 8kW of drying temperature,
Microwave frequency 2500MHz, drying time 5h.
Embodiment 5
The method of Hyperfibrinolysis Nattokinase of the present embodiment production rich in bacillus subtilis viable bacteria, including following step
Suddenly：
(1) from inclined-plane, picking bacillus subtilis LB-18 is inoculated in LB liquid seed culture mediums, under the conditions of 40 DEG C,
150r/min shaking table constant temperature incubation 12h, obtain bacillus subtilis LB-18 seed liquors；
(2) soybean soaking is stayed overnight, is drained away the water, 121 DEG C of high pressure sterilization 20min, after being cooled to room temperature, according to 1%
Inoculum concentration is inoculated with bacillus subtilis LB-18 seed liquors, fermented and cultured 36h under the conditions of 40 DEG C；
(3) the soya bean fermented and cultured product of step (2) dry, pulverize with micro-wave vacuum equipment, crossing 120 mesh sieve is
；The drying condition of the micro-wave vacuum equipment is：40 DEG C, vacuum degree -0.06MPa, microwave power 8kW of drying temperature,
Microwave frequency 2450MHz, drying time 3h.
Experimental example
The measurement of fibrinolytic activity of nattokinase from natto and viable count of the present invention
1, fiber flat band method measures the fibrinolytic of produced Nattokinase dry powder
The preparation of sample：Prepare the Nattokinase dry powder physiological saline of the bacterial strain LB-18 of the present invention productions of mass fraction 2%
Suspension, 4 DEG C of extraction 4h, after 5000r/min centrifuges 10min, after gained supernatant 15000r/min repeated centrifugations 10min, supernatant
Liquid is used for the measurement of fibrinolytic.
The preparation of fibrin plate：1g agaroses are dissolved in the sodium phosphate buffer 100mL of 0.01M, pH7.4, are taken
Agarose solution 7.5mL, 50 DEG C of water-baths keep the temperature 5min, the 225 μ L of 100BP/mL fibrin ferments for being dissolved in physiological saline are added, mix
Even, 50 DEG C of water-bath 5min take 3.6mg/mL bovine fibrinogen solution (sodium phosphate buffer for being dissolved in 0.1M, pH7.4)
7.5mL is added in fibrin ferment-agarose solution of heat preservation, and rapid mixing pours into the culture dish of diameter 9cm, waits coagulating.
The drafting of standard curve：Prepare the urokinase mark of 20,40,60,80 and 100IU/mL respectively with sterile saline
Quasi- product solution respectively takes 10 μ L point samples on fibrin plate, and 37 DEG C of heat preservation 18h measure dissolving circle on fibrin plate
Diameter takes the average diameter tested three times, calculates dissolving circle area；With urokinase activity (IU/mL) for abscissa, dissolving circle face
Product is ordinate, draws urokinase standard curve.
The measurement of fibrinolytic：Pipette 10 μ L natto powder leaching liquors with micro sample adding appliance, point sample on fibrin plate,
7 DEG C of heat preservation 18h, measure the diameter that circle is dissolved on fibrin plate, calculate dissolving circle area.According to standard curve, calculate fine
Molten activity.
Measurement result finds that before 48h, with the extension of fermentation time, fibrinolytic dramatically increases, after 48h, fibrinolytic
Extension at any time is basically unchanged, ferment 48h when tunning fibrinolytic can reach 2800FU/g.
2, the Nattokinase dry powder viable count of bacterial strain LB-18 fermenting and producings, which measures, takes 1g bacterial strain LB-18 productions of the present invention
Nattokinase dry powder is suspended in the 100mL sterile waters containing bead, concussion 30min, after gradient dilution, takes 100 μ L differences dilute
The dilution for releasing multiple is coated on LB tablets, and each gradient does 3 repetitions, and 37 DEG C are incubated overnight, according to what is grown on tablet
Clump count calculates the viable count in every gram of Nattokinase dry powder, and other Nattokinase production strain fermentation products is used in combination to make comparisons
(the results are shown in Table 2).
Fibrinolytic and viable count compare in 2 different strains of table fermentation Nattokinase dry powder
| Sample | Natto kinase activity (FU/g) | Viable count (a/g) in sample |
| DC-Tx | 3058 | 3.02×107 |
| R | 2956 | 6.78×106 |
| LB-18 | 2847 | 1.26×108 |
| Z | 2690 | 2.95×107 |
| Sh2 | 2435 | 3.71×107 |
| S2 | 2783 | 4.28×106 |
| d5 | 2433 | 7.82×106 |
| d6 | 3125 | 4.01×107 |
| J | 2347 | 5.33×106 |
| L | 2379 | 9.75×106 |
The experiment results show that the Nattokinase product (bean-containing capsule) of bacterial strain LB-18 productions of the present invention is with very high
Fibrinolytic, and it is rich in bacillus subtilis viable bacteria.The present invention is Nattokinase as functional food and health food
Development and application research provides new thinking, has more wide development prospect.
Claims (3)
1. one plant of resistance to microwave bacillus subtilis LB-18, Classification And Nomenclature is bacillus subtilis（Bacillus subtilis）
LB-18, on May 22nd, 2015 are preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number：
CGMCC NO.10835, preservation address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
2. a kind of resistance to microwave bacillus subtilis LB-18 as described in claim 1 is rich in bacillus subtilis viable bacteria in production
Hyperfibrinolysis Nattokinase in application.
3. a kind of resistance to microwave bacillus subtilis LB-18 as claimed in claim 2 is rich in bacillus subtilis viable bacteria in production
Hyperfibrinolysis Nattokinase in application process, which is characterized in that include the following steps：
（1）From inclined-plane, picking bacillus subtilis LB-18 is inoculated in LB liquid seed culture mediums, under the conditions of 25-40 DEG C, 120-
180r/min shaking table constant temperature incubation 12-18h, obtain bacillus subtilis seed liquor；
The LB liquid seed culture mediums include the raw material of following mass fraction：Peptone 1%, yeast powder 0.5%, NaCl 1%；
PH6.8,121 DEG C of high pressure sterilization 20min；
（2）Soybean soaking is stayed overnight, is drained away the water, after being cooled to room temperature, withered grass is inoculated with according to the inoculum concentration of 1-8% for high pressure sterilization
Bacillus seed liquor, fermented and cultured 36-72h under the conditions of 25-40 DEG C；
（3）By step（2）Soya bean fermented and cultured product micro-wave vacuum, crush, cross 80-120 mesh sieve to obtain the final product；
The drying condition of the micro-wave vacuum equipment is：Drying temperature≤40 DEG C, vacuum degree -0.06MPa, microwave power
8kW, 2450 ± 50MHz of microwave frequency, drying time 3-8h.
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| CN107950900A (en) * | 2017-12-19 | 2018-04-24 | 黑龙江省中医药科学院 | A kind of preparation method of the compound fermented soya bean of Hyperfibrinolysis |
| CN113234627B (en) * | 2021-05-13 | 2023-10-27 | 江南大学 | Bacillus subtilis for high yield of nattokinase and application thereof |
| CN113907272A (en) * | 2021-08-20 | 2022-01-11 | 上海交通大学 | Production method of low-ammonia natto and application of tea polyphenol in low-ammonia natto |
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| CN115317399A (en) * | 2022-07-07 | 2022-11-11 | 湖北真福医药有限公司 | Anti-aging composition and preparation method and application thereof |
| CN116790565A (en) * | 2023-08-14 | 2023-09-22 | 广东牧豆人农业科技有限公司 | Method for extracting nattokinase by utilizing bacillus natto fermented bean dregs |
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