CN109797115A - One plant of production Nattokinase and beta-glucosidase bacillus subtilis GZU05 and its application method - Google Patents
One plant of production Nattokinase and beta-glucosidase bacillus subtilis GZU05 and its application method Download PDFInfo
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- CN109797115A CN109797115A CN201910025309.9A CN201910025309A CN109797115A CN 109797115 A CN109797115 A CN 109797115A CN 201910025309 A CN201910025309 A CN 201910025309A CN 109797115 A CN109797115 A CN 109797115A
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- nattokinase
- gzu05
- bacillus subtilis
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- glucosidase
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- 239000000126 substance Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Abstract
One plant of production Nattokinase and beta-glucosidase bacillus subtilis GZU05, the microbiological classification of the bacillus subtilis are named as bacillus subtilis GZU05, Latin name:Bacillus subtilis.GZU05, on November 9th, 2018 in China typical culture collection center preservation, deposit number is CCTCC M 2018763.It is collected in Guizhou traditional zymotic bean product fermented soya bean.Application method is the bacterial strain GZU05 solid state fermentation natto culture medium culture that will be selected, and fermentation natto produces beta-glucosidase and Nattokinase, obtains the fermented soya bean product rich in Nattokinase.GZU05 of the invention can be used to high-valued conversion Gingko yellow ketoside and can produce the Nattokinase natto Nattokinase enzyme activity height produced of high level, and organoleptic properties are good, are suitable for being applied to production Nattokinase.
Description
Technical field
The present invention relates to microorganisms, further relate to enzyme, are furthermore related to bacillus amyloliquefaciens, are related to bacillus subtilis
The application method of bacterium.
Background technique
Beta-glucosidase (β-D-Glucosidase, EC3.2.1.21) is a kind of hydrolase from a wealth of sources.Its
Characteristic is β-D-Glucose glycosidic bond that hydrolyzable is incorporated into end irreducibility, while releasing β-glucose and matching accordingly
Base.Beta-glucosidase can be generated by yeast, aspergillus, trichoderma, bacterium etc..According to report both domestic and external, which be can be widely used for
The bioconversion of Flavonoid substances.
Microbial fermentation producing enzyme is an effective way of biological enzyme source.It is both domestic and external studies have shown that passing through microorganism
Enzymatic production can hydrolyze Gingko yellow ketoside into ginkgo flavone aglycone, and aglycon activity is made to be apparently higher than Gingko yellow ketoside.Gingko yellow
Ketone is the drug for the best prevention and treatment cardiovascular and cerebrovascular disease having now been found that, current GINKGO BILOBA EXTRACT is mainly extracted from ginkgo leaf
, but the GINKGO BILOBA EXTRACT from ginkgo leaf in advance mainly exists in the form of the Gingko yellow ketoside, and and Gingko yellow ketoside
Bioactivity is significantly smaller than ginkgo flavone aglycone, since the environmental-friendly selectivity of enzyme process is high, it is therefore necessary to utilize β-grape
Gingko yellow ketoside is converted into aglycon by glycosidase.
Though it is more for beta-glucosidase research achievement at present, it is mostly microbe-derived in nature, from biography
Being rarely reported in system fermented food.In addition, traditional enzyme cannot be such that Aglycones content maximizes, Gao Xuan substrate poor selectivity
It is also seldom that selecting property hydrolyzes the report that Gingko yellow ketoside is aglycon.Wu Yi etc. is using fixed business beta-glucosidase enzyme hydrolysis Gingko yellow
Ketoside achieves better result.But also seldom reported using non-commercial beta-glucosidase enzyme hydrolysis GINKGO BILOBA EXTRACT glycosides, due to
Business beta-glucosidase is expensive, and it is ginkgo that therefore, screening, which produces the bacterial strain of the beta-glucosidase of hydrolysis Gingko yellow ketoside,
The industrialized production of flavone aglycone lays the foundation, and reduces conversion cost with regard to particularly significant.
Thrombus disease affects the health of the mankind again, according to statistics, the whole world every year at least 12,000,000 patients because of thrombotic
Disease and it is dead, China there are about 2,600,000.Therefore the exploitation of thrombolysis medicine for treating thrombus object is always the hot spot concerning human health.At present
Clinically common thrombolysis medicine for treating thrombus object has streptokinase (Streptokinase, SK), urokinase (Urokinase, UK) and group
It knits type plasminogen activator (type plasminogenactivator, t-PA), but these drugs have administration pain, pair
The disadvantages of acting on big, expensive, half-life short, in contrast, Nattokinase (Nattokinase, NK) has been demonstrated have
Efficient thrombolytic effect not only can directly reduce fibrinogen, but also can promote to be catalyzed plasminogen turn
Plasmin is turned to, the synthesis of the internal thrombolysis factor is increased.And Nattokinase has orally available, safe, honest and clean
Valence and the fibrinolytic advantages such as strong, become the research hotspot of thrombolysis class product in recent years.
In Chinese patent database, there are many patent and the application part for being related to beta-glucosidase, such as
No. ZL2014106023384 " a kind of beta-glucosidase and β-glucosidase mutant and application ", ZL2015101756557
Number " height endurability beta-glucosidase and its application ", No. 2017102790450 " one plant of β-Portugal for producing hydrolysis Gingko yellow ketoside
Polyglycoside enzyme producing strains and its screening technique " etc.；But it has no and is related to the application part of bacillus subtilis GZU05.
Summary of the invention
The present invention is intended to provide one plant of production Nattokinase and beta-glucosidase bacillus subtilis GZU05, the bacterial strain pair
The beta-glucosidase and high-yield nattokinase of the fermentation produced selective hydrolysis Gingko yellow ketoside of fermented soya bean are to prevention and treatment cardiovascular and cerebrovascular
Disease all plays a significant role.
Another object of the present invention is to provide to be applied to production flavone aglycone and produces to be rich in receive by the bacillus subtilis
The method of beans kinases fermented soya bean.
The one plant of production Nattokinase and beta-glucosidase bacillus subtilis GZU05 that inventor provides, microbiology
Classification naming be bacillus subtilis GZU05, Latin name: Bacillus subtilis.GZU05, in 2018 11
In China typical culture collection center preservation, deposit number was CCTCC M 2018763 months 9 days, during depositary institution address is
State Wuhan, Wuhan University, postcode 430072, telephone number 027-68754952.
The bacillus subtilis GZU05 that inventor provides is acquired in the traditional zymotic bean product fermented soya bean of Guizhou,
It has the performance of the beta-glucosidase and high-yield nattokinase that produce selective hydrolysis Gingko yellow ketoside.
What inventor provided is rich in Nattokinase beans for be applied to production flavone aglycone and the production of the bacillus subtilis
The method of fermented soya beans, salted or other wise, comprising the following steps:
(1) fermented bean products sample suitably dilutes, and is coated on aobvious with the Gardenoside prepared in advance on Gardenoside colour developing plate
Color screening and culturing medium culture, it is big to filter out blue bacterium circle, and saturate bacterial strain, is crossed, separated, purified, through gram
Dyeing microscopic examination is after single culture, slant preservation and glycerol stocks is spare；
(2) GINKGO BILOBA EXTRACT is converted using bacterial strain fermentation liquor, measures β-glucosidase activity power；Nattokinase is measured simultaneously
Enzyme activity, carry out the sensory evaluation of natto product, it is high and comprehensive to pick out beta-glucosidase enzyme, Nattokinase enzyme activity
The optimal natto fermentation bacterial strain GZU05 of sensory evaluation；
(3) morphological analysis, physiological and biochemical test, 16S rRNA sequence are carried out to bacterial strain GZU05 to analyze, determines the bacterial strain
Belong to bacillus subtilis；
(4) bacillus subtilis GZU05 solid state fermentation natto culture medium culture, fermentation natto produce Nattokinase and β-Portugal
Polyglycoside enzyme, ferment 48~72h；Obtain the fermented soya bean product rich in Nattokinase.
In (1) step of the above method, the formula of the Gardenoside colour developing screening and culturing medium are as follows: ginkgo biloba p.e 5, sulphur
Sour magnesium 0.5, potassium dihydrogen phosphate 10, Gardenoside 0.1, sodium glutamate 10, ammonium sulfate 2, agar 20；The cultivation temperature is 37 DEG C,
Incubation time is 36~72h.
In (2) step of the above method, the unit of the enzyme activity is under determination condition, and every 1min hydrolyzes Gingko yellow
Enzyme amount needed for ketoside generates 1 μm of ol reduced sugar with glucose meter, is defined as an enzyme activity unit.
In (2) step of the above method, the measuring method of the beta-glucosidase are as follows: 15 ml plug test tube are taken,
Substrate 1.8mL is added, 3min is preheated at 50 DEG C, glucuroide enzyme solution 0.2mL, the water-bath 30min at 50 DEG C is added, is added
DNS reagent 3mL, boiling water bath 10min, cooling are settled to 15mL.Using the enzyme solution of inactivation as blank control, measures and inhale at 540nm
Light value measures the content of reducing sugar of generation；The Nattokinase enzyme activity determination method are as follows: take the thick enzyme of Nattokinase solid state fermentation
10 μ L point sample of liquid places 10min, moves into 37 DEG C of incubators, after keeping the temperature 16h in (four holes are one group parallel) on fibrin plate
It takes out, measures the diameter of dissolution circle with vernier caliper and calculate each dissolution and enclose area, according to urokinase standard curve regression equation
Calculate sample Nattokinase enzyme activity；The fermented soya bean product sensory evaluation method rich in Nattokinase are as follows: use after fermentation
Weighting method is given a mark, and carries out sensory evaluation in terms of wire drawing (viscosity), color, smell and posture 4 respectively.
In previous step method, the substrate be 1% Gingko yellow ketoside or amarogentin, Gardenoside, rutin, gentiobiose,
One of maltotriose, arbutin, laminarin, trehalose.
In (4) step of the above method, the culture medium is made from following method: soya bean being cleaned up, is rejected mould
It is rotten, damage by worms, deformity soya bean, remove impurity, rinsed well with deionized water, then precise, the dissolution of 4 times of volumes be added
1% salt and 1% deionized water of glucose sugar are totally submerged, soak at room temperature 18~for 24 hours to get.
In (4) step of the above method, the beta-glucosidase has highly selective, hydrolysis silver to Gingko yellow ketoside
Apricot yellow ketoside enzyme activity is 4.2U/g, and the enzyme can store 2~4d at room temperature, can store 7~10d at 4 DEG C, the enzymatic conversion ability
It is maintained at 45% or more, fermentation the produced Nattokinase enzyme activity of natto is more than 6000U/g.
Compared with prior art, the present invention having the advantage that 1. biological complex enzyme prepared by the present invention is to substrate ginkgo
The recovery of standard addition of flavones conversion is up to 88%, and beta-glucosidase is to the conversion ratio of substrate GINKGO BILOBA EXTRACT up to 45%；And it sends out
Fermenting process technique is simple with raw material, reduces the production cost of beta-glucosidase；And enzyme is to environment resistance document more before this
The beta-glucosidase of middle report significantly improves: tolerance pH2.8~5.5, and 40 DEG C~70 DEG C of tolerable temperature；And conversion reaction can connect
It is continuous to carry out, it is suitble to large-scale continuous production；2. the β-glucosidase activity of bacillus subtilis GZU05 of the invention is
4.022U/g, Nattokinase enzyme activity are 5786.1 ± 510.9, have the function of converting aglycon for Gingko yellow ketoside.And
Natto Nattokinase enzyme activity produced is high, and organoleptic properties are good, are easy to be accepted by the public；3. withered grass gemma of the invention
Bacillus GZU05 can be used to high-valued conversion Gingko yellow ketoside and can produce the Nattokinase of high level, while providing one kind
The cultural method of new production beta-glucosidase, Nattokinase, and it is highly-safe；It is moderate since soya bean is from a wealth of sources,
Reduce the production cost of beta-glucosidase, Nattokinase.Therefore the bacillus subtilis has a good application prospect.
Detailed description of the invention
Fig. 1 is concentration of glucose standard curve；
Fig. 2 is urokinase standard curve；
Fig. 3 is that bacterial strain GZU05 microscope (100x) observation is shone；
Fig. 4 is the systematic growth tree graph of bacterial strain GZU05.
Specific embodiment
Following embodiment is used to further illustrate the present invention:
1 seed liquid culture medium of embodiment, Nattokinase and the preparation of beta-glucosidase crude enzyme liquid
1) seed liquid culture medium (g/L) is prepared: glucose 10, yeast extract 5, beef extract 10, NaCl 5, from inclined-plane
Picking 2-3 ring lawn is inoculated into liquid seed culture medium, and in 37 DEG C, 18h is cultivated under conditions of 180r/min.
2) mass parts ratio, every liter of deionized water addition Nattokinase and beta-glucosidase crude enzyme liquid preparation method: are pressed
NaCl 10g, glucose 10g, heating are completely dissolved, dry beans after cooling: deionized water=1:4, which is totally submerged, impregnates commercially available non-turn
Transgenic soybean, soaking time are 18 hours.Draining is packed into the sterilizing of 50/250 triangular flask afterwards.Kind is inoculated with by the inoculum concentration of 2%-4%
On sub- liquid to the solid-state fermentation culture medium cooled down, 37 DEG C of culture 72h, every 12h rock once, form fermentation material.Fermentation material is taken,
It is extracted 24 hours at 4 DEG C with physiological saline extraction, with mortar grinder, then at 4 DEG C, centrifugal force is 8000-12000 × g's
Under the conditions of be centrifuged 12-18min, take supernatant, after clasmatosis beta-glucosidase, Nattokinase crude enzyme liquid；
Embodiment 2: the measurement of β-glucosidase activity power:
1) glucose standard curve is drawn: 1mg/ml anhydrous grape sugar juice 0,0.2mL, 0.4mL, 0.6mL are drawn,
0.8mL, 1.0mL are in plug test tube, moisturizing to 2mL, add DNS reagent 3mL, boil 10min after mixing in boiling water, cold
But add water to be settled to 15mL afterwards, survey absorbance under spectrophotometer 540nm wavelength；Using absorbance as ordinate, glucose amount
For abscissa, standard curve, equation of linear regression y=0.5841x+0.0044, R are drawn2=0.9991 (attached drawing 1)；
(2) measurement of β-glucosidase activity power: bacterial strain β-glucosidase activity power is detected by DNS method, is selected
The higher bacterial strain of enzyme activity；Take 15ml plug test tube that substrate (1% Gingko yellow ketoside or amarogentin, Gardenoside, reed is added
One of fourth, gentiobiose, maltotriose, arbutin, laminarin, trehalose) 1.8mL, 50 DEG C of preheating 3min, enzyme is added
Liquid 0.2mL, 50 DEG C of water-bath 30min, are added DNS reagent 3mL, boiling water bath 10min, and cooling is settled to 15mL.With the enzyme solution of inactivation
For blank control, light absorption value is measured at 540nm.According to β-grape in concentration of glucose standard curve regression equation calculation sample
Glycosidase enzyme activity.
The definition of β-glucosidase activity unit of force: under determination condition, every 1min hydrolysis Gingko yellow ketoside generates 1 μm of ol
Enzyme amount needed for reduced sugar (with glucose meter) is defined as 1 enzyme activity unit (U).
The enzyme activity of GZU05 fermentation the produced beta-glucosidase enzyme hydrolysis Gingko yellow ketoside of fermented soya bean is 3.696U/g, is higher than water
The enzyme activity for solving other substrates is shown to the highly selective of Gingko yellow ketoside.
Embodiment 3: Nattokinase enzyme activity determination
The definition of Nattokinase enzyme activity:, which measuring the diameter of dissolution circle with vernier caliper, and calculates each dissolution encloses area, according to
Urokinase standard curve regression equation calculation sample Nattokinase enzyme activity.
The production of urokinase standard curve: by urokinase standard items be formulated as respectively 248IU/m L, 496IU/m L,
744IU/m L, 992IU/m L and 1240IU/m L respectively take 10 μ L point samples (4 holes in the fibrinogen plate well newly prepared
It is parallel for 1 group), 10min is placed, is taken out after cultivating 16h at 37 DEG C, the diameter of measurement dissolution circle calculates each dissolution and encloses area；With
Dissolve area (x, the mm of circle2) it is abscissa, with urokinase enzyme activity (y, IU/mL) for ordinate, draw urokinase standard curve.
Equation of linear regression is y=4.2016x+10.874, R2=0.9994 (attached drawing 2).Then the natto for surveying GZU05 fermentation natto swashs
Enzyme activity is 6861U/g.
Embodiment 4: ultraviolet spectrophotometry micrometric measurement Nattokinase:
Ultraviolet spectrophotometer method is lived by the kind energy accurate response Nattokinase fibrinolytic that Japan Natto Kinase Association is established
One of measuring method and universally acknowledged natto kinase activity measuring method of property.The advantages of this method is objective to obtain
Fibrinolytic activity of nattokinase from natto content out, and various samples can be detected simultaneously.Due to agarose-fibrinogen plate assay measurement
Value easily with incubation time, slab-thickness variation and change, the poor unsuitable precise requirements of accuracy it is high sample measurement.Tool
Body method are as follows: 1.4mL Tris-HCl (50mM, pH 7.8) buffer and 0.4mL fibrinogen solution are added into test tube
0.1mL fibrin ferment (20U/mL) is added after 37 DEG C of incubation 5min in (7.2mg/mL), then incubates 10min at 37 DEG C and form artificial blood
The sample to be tested of 0.1mL is added in bolt, and 60min is incubated at 37 DEG C, and 2mL trichloroacetic acid (0.2moL/L) solution left standstill is added
20min terminates reaction, and 13000r/min is centrifuged 10min, supernatant is taken to measure absorbance at 275nm wavelength.Enzyme activity definition:
Every 1min enzyme amount required for absorbance increases by 0.01 at the 275nm is defined as the fibrin degradation enzyme activity of 1 unit.
Reach by the Nattokinase vigor of the Nattokinase after ultraviolet spectrophotometry micrometric measurement GZU05 solid state fermentation natto 36h
116FU/g。
The strains solid fermented natto sensory evaluation of embodiment 5:GZU05 and marking situation
(1) the strains solid fermented sensory evaluation of GZU05: the natto after fermentation after-ripening is given a mark using weighting method, respectively
Sensory evaluation is carried out in terms of wire drawing (viscosity), color, smell and character row state four, each index is divided into five grades, point
There are not 10 professional persons to judge, be averaged, standards of grading and score weight are shown in Table 1.
The sensory evaluation criteria of 1 fermented product of table
(2) the natto Analyses Methods for Sensory Evaluation Results of solid state fermentation
Good production bacterial strain is the basis for guaranteeing food fermentation, and the bacterial strain for selecting sense organ excellent is the prerequisite of subsequent fermentation
Condition.By GZU05 strain inoculated using soya bean as the solid fermentation culture medium of primary raw material, it is placed under identical fermentation condition and carries out
Culture, after fermentation, records length of string, color, smell and the mouthfeel score value of each strain fermentation product, and calculate comprehensive respectively
Closing scoring is 4.2.The natto color of fermentation is yellow to faint yellow, and stirring fermented soya bean has the phenomenon that long wire drawing, with light
Fragrance matter, smell coordination, posture are uniform, and the vigor of Nattokinase kinases is higher than 6000U/g (wet soya bean) in fermented soya bean.
Embodiment 6: colonial morphology identification, Physiology and biochemistry identification and 16SrRNA molecular biology identification
Bacterial strain observes and records colonial morphology, color in cultivating 48h on screening and culturing medium plate.
Gram's staining: bacterium colony on picking plate carry out smear, fixation, crystal violet just dye, mordant dyeing, decoloration, washing, kind
It is red redye, dry, microscopy.
The colony morphology characteristic of bacterial strain is observed, which has obvious blue bacterium circle, and protrusion has fold, Gram's staining
Afterwards, in optical microscopy oil microscopic observation cell morphological characteristic, as a result as shown in Fig. 3.
Physiological and biochemical property identification, the bacillus subtilis GZU05 physiology are carried out to the bacillus subtilis GZU05
Biochemical character-utilization of carbon source is as shown in table 2, the bacillus subtilis GZU05 physio-biochemical characteristics-enzyme activity, carbon assimilation
As shown in table 3.
The systematic growth tree graph of the bacillus subtilis GZU05 is as indicated at 4.
2 bacterial strain GZU05 physio-biochemical characteristics of table-produce acid using carbon source
Note :+: it is positive ,-: it is negative,
3 bacterial strain GZU05 physio-biochemical characteristics of table-enzyme activity, carbon assimilation
Note :+: positive reaction；: negative reaction；W: weakly positive reaction
It is attached: the 16S rRNA sequence of bacillus subtilis GZU05:
ATGGCGCGTGCTATACATGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGG CGGA
CGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGG GCTAATACCGGATGGT
TGTCTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCAC TTACAGATGGACCCGCGGCGCATTAGCT
AGTTGGTGAGGTAACGGCTCACCAAGGCGACGATG CGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTG
AGACACGGCCCAGACTCCTACGG GAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACG
CCGCGTGAGTG ATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGC
GGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATA CGTAGGTGGCAA
GCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGT CTGATGTGAAAGCCCCCGGCTCAA
CCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAG AAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAAT
GCGTAGAGATGTGGAGGAACACCAGTG GCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGT
GGGGAGCGAACAGGA TTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGC
CCC TTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTC AAAGGAAT
TGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAA GAACCTTACCAGGTCTTGAC
ATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCA GAGTGACAGGTGGTGCATGGTTGTCGTCAGCT
CGTGTCGTGAGATGTTGGGTTAAGTCCCGCA ACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCA
CTCTAAGGTGACTGCCGGT GACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGG
GCTACAC ACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATC TGTT
CTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGC GGATCAGCATGCCGCG
GTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAG AGTTTGTAACACCCGAAGTCGGTGAGGT
AACCTTTATGGAGCCAGCCGCCGAAAGGGGACCCC
Claims (9)
1. one plant of production Nattokinase and beta-glucosidase bacillus subtilis GZU05, it is characterised in that: the bacillus subtilis
Microbiological classification be named as bacillus subtilis GZU05, Latin name:Bacillus subtilisGZU05,
On November 9th, 2018 in China typical culture collection center preservation, deposit number is CCTCC M 2018763；Preservation list
Bit address is Wuhan, China, Wuhan University, postcode 430072, telephone number 027-68754952.
2. according to bacillus subtilis GZU05 described in claim 1, it is characterised in that: it is in Guizhou traditional zymotic bean product
It is acquired in fermented soya bean, it has the performance of the beta-glucosidase and high-yield nattokinase that produce selective hydrolysis Gingko yellow ketoside.
3. bacillus subtilis described in claim 1 is applied to production flavone aglycone and production rich in Nattokinase fermented soya bean
Method, comprising the following steps:
(1) fermented bean products sample suitably dilutes, and is coated on Gardenoside colour developing plate with the Gardenoside colour developing sieve prepared in advance
Culture medium culture is selected, it is big to filter out blue bacterium circle, and saturate bacterial strain, is crossed, separated, purified, through Gram's staining
Microscopy is after single culture, slant preservation and glycerol stocks is spare；
(2) GINKGO BILOBA EXTRACT is converted using bacterial strain fermentation liquor, measures β-glucosidase activity power；The enzyme of Nattokinase is measured simultaneously
Vigor carries out the sensory evaluation of natto product, picks out beta-glucosidase enzyme, Nattokinase enzyme activity height, and integrated sensory
Evaluate optimal natto fermentation bacterial strain GZU05；
(3) morphological analysis, physiological and biochemical test, 16S rRNA sequence are carried out to bacterial strain GZU05 to analyze, determines that the bacterial strain belongs to
Bacillus subtilis；
(4) bacillus subtilis GZU05 solid state fermentation natto culture medium culture, fermentation natto produce Nattokinase and β-glucose
Glycosides enzyme, ferment 48~72h；Obtain the fermented soya bean product rich in Nattokinase.
4. method as claimed in claim 3, it is characterised in that in (1) step of method, the Gardenoside colour developing screening and culturing
The formula of base are as follows: ginkgo biloba p.e 5, magnesium sulfate 0.5, potassium dihydrogen phosphate 10, Gardenoside 0.1, sodium glutamate 10, sulfuric acid
Ammonium 2, agar 20；The cultivation temperature is 37 DEG C, and incubation time is 36~72h.
5. method as claimed in claim 3, it is characterised in that in (2) step of method, the unit of the enzyme activity is to survey
Under fixed condition, enzyme amount needed for every 1min hydrolysis Gingko yellow ketoside generates 1 μm of ol reduced sugar with glucose meter is defined as an enzyme
Unit of activity.
6. method as claimed in claim 3, it is characterised in that in (2) step of method, the measurement of the beta-glucosidase
Method are as follows: take 15 ml plug test tube, substrate 1.8mL is added, 3min is preheated at 50 DEG C, glucuroide enzyme solution is added
0.2mL, the water-bath 30min at 50 DEG C, are added DNS reagent 3mL, boiling water bath 10min, and cooling is settled to 15mL.With the enzyme of inactivation
Liquid is blank control, and light absorption value is measured at 540nm, measures the content of reducing sugar of generation；The Nattokinase enzyme activity determination
Method are as follows: take 10 μ L point sample of Nattokinase solid state fermentation crude enzyme liquid in (four holes are one group parallel) on fibrin plate, place
10min moves into 37 DEG C of incubators, takes out after keeping the temperature 16h, measures the diameter of dissolution circle with vernier caliper and calculates each dissolution circle face
Product, according to urokinase standard curve regression equation calculation sample Nattokinase enzyme activity；The fermented soya bean product rich in Nattokinase
Sensory evaluation method are as follows: given a mark after fermentation using weighting method, respectively from wire drawing (viscosity), color, smell and posture 4
A aspect carries out sensory evaluation.
7. method as claimed in claim 6, it is characterised in that the substrate is 1% Gingko yellow ketoside or amarogentin, cape jasmine
One of glycosides, rutin, gentiobiose, maltotriose, arbutin, laminarin, trehalose.
8. method as claimed in claim 3, it is characterised in that in (4) step of method, the culture medium is following method system
: soya bean is cleaned up, reject go rotten, damage by worms, deformity soya bean, remove impurity, rinsed well with deionized water, then
Precise, the deionized water that the salt of the dissolution of 4 times of volumes 1% and 1% glucose sugar is added are totally submerged, room temperature
Impregnate 18~24 h to get.
9. method as claimed in claim 3, it is characterised in that in (4) step of method, the beta-glucosidase is to ginkgo
Flavonoid glycoside has highly selective, and hydrolysis Gingko yellow ketoside enzyme activity is 4.2U/g, and the enzyme can store 2~4d at room temperature, at 4 DEG C
7~10d can be stored, which is maintained at 45% or more, and fermentation the produced Nattokinase enzyme activity of natto is more than 6000 U/g.
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| CN110438027A (en) * | 2019-06-05 | 2019-11-12 | 贵州大学 | Produce the Bacillus amyloliquefaciens strain GUTU06 and its screening technique of a variety of enzymes |
| CN111154674A (en) * | 2020-01-09 | 2020-05-15 | 齐鲁工业大学 | Bacillus subtilis for fermenting pea natto and application thereof |
| CN112048524A (en) * | 2020-08-28 | 2020-12-08 | 哈尔滨工业大学 | Method for improving dark fermentation hydrogen production performance by using ginkgetin |
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| CN110438027A (en) * | 2019-06-05 | 2019-11-12 | 贵州大学 | Produce the Bacillus amyloliquefaciens strain GUTU06 and its screening technique of a variety of enzymes |
| CN110438027B (en) * | 2019-06-05 | 2022-08-26 | 贵州大学 | Bacillus amyloliquefaciens strain GUTU06 producing multiple enzymes and screening method thereof |
| CN111154674A (en) * | 2020-01-09 | 2020-05-15 | 齐鲁工业大学 | Bacillus subtilis for fermenting pea natto and application thereof |
| CN111154674B (en) * | 2020-01-09 | 2021-07-23 | 齐鲁工业大学 | Bacillus subtilis for fermenting pea natto and application thereof |
| CN112048524A (en) * | 2020-08-28 | 2020-12-08 | 哈尔滨工业大学 | Method for improving dark fermentation hydrogen production performance by using ginkgetin |
| CN113174335A (en) * | 2021-04-19 | 2021-07-27 | 广西大学 | Separation method of banana colletotrichum gloeosporioides |
| CN113174335B (en) * | 2021-04-19 | 2022-06-17 | 广西大学 | Separation method of banana colletotrichum gloeosporioides |
| 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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