CN105734035A - High-quality white jade natto and plasmin and preparation method and application thereof - Google Patents
High-quality white jade natto and plasmin and preparation method and application thereof Download PDFInfo
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- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
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Abstract
The invention discloses a preparation method and application of high-quality white jade natto and high-activity plasmin.The method includes the steps of soaking and steaming white jade natto, inoculating bacillus subtilis for fermentation to prepare white jade natto, and freezing, drying and smashing white jade natto to prepare white jade natto freeze-dried powder.The method further includes the steps of conducting separation purification and freeze-drying on white jade plasmin to prepare white jade plasmin freeze-dried powder.Compared with natto prepared through a traditional method, the prepared white jade natto is low in ammonia odor, white and bright in color and luster and good in taste and has a function of reducing blood glucose.The white jade plasmin prepared through the method has higher activity of 45800 IU/g.The method is simple in process, short in cycle and beneficial to industrial production, the white jade natto has a function of treating diabetes, and the application field of natto is further widened.
Description
[technical field]
The present invention relates to biological technical field, particularly refer to a kind of high-quality natto and high activity fibrinolysin and its preparation method and application.
[background technology]
Technique for preparing natto with Semen sojae atricolor for raw material, by Semen sojae atricolor steaming and decocting and inoculate fermentation of bacillus subtilis prepare.The fibrinolysin contained in natto has the thrombolytic effect of targeting, belongs to the streptokinase albuminoid naturally occurred, and without hemorrhagic toxic and side effects.Therefore, natto is widely used as functional health-care food in prevention and treatment cardiovascular and cerebrovascular disease field.White jade natto is also known as three clear beans, and because its seed is white as a jade, cooked soup is clear like water, and tasty and refreshing clearing heat in QI system when eating, is the special product of Sanqingshan Mountain in addition, and therefore named " three clear beans ", is that ancient times submits imperial court's tribute bean.Sugared part of Semen Phaseoli Vulgaris, reducing sugar, protamine content are moderate, but calcium, phosphorus, copper, ferrum, zinc equal size are apparently higher than other bean products, and possibly together with the aminoacid of 10 kinds of needed by human, nutrition is very abundant.Three clear bean good palatabilities, old children all likes, and is suitable for frying, boils, the multiple cooking method such as stewes, and is used as the first-class raw material of fabricated snack, has the advantages that salty food, sweet food are all suitable, is deeply subject to the favor of consumer.And white jade natto medicinal function is extensive, has lung heat clearing, antiinflammatory, skin care, bone strengthening, stomach invigorating, the kidney invigorating, effects such as removing toxic substances of enriching blood, particularly that the action effect such as diabetes, cardiovascular and cerebrovascular disease is obvious.Although white jade natto is rich in multiple nutrients material, there is many health cares and disease treatment effect, although be special product of China, but the rare bean varieties that still few people know, and being mainly applied on dining table with food forms, its value is also far from obtaining effective exploitation.
The number of thrombosis type cardio-cerebral diseases is directly died from every year just up to 18,000,000 in the whole world, and indirect death toll is more than 30,000,000, close to the world the 1/3 of total death toll, has become as the number one killer of human health.China is populous, and pace of population aging accelerates, and the M & M of cardiovascular and cerebrovascular disease constantly rises, and every year because suffering from the dead number of cardiovascular and cerebrovascular disease up to 7,000,000 people, cardiovascular and cerebrovascular disease number of patients alreadys more than 300,000,000.Only the whole cardiovascular and cerebrovascular disease relevant market of China alreadys more than 1 trillion yuan.And cardiovascular and cerebrovascular disease primary pathogenic event behind is the formation of thrombosis.But, the treatment of current thrombotic disease mainly has surgical operation, conservative treatment and 3 kinds of methods of thrombolytic therapy all to there is certain defect.Surgery hands therapy is very risky, and operative site blood vessel is thinning, it is easy to cause that angiorrhexis is hemorrhage, and artificial blood vessel position is easier to again be formed thrombosis in addition；Conservative treatment refers mainly to long-term taking Western medicine, and Western medicine therapy allows patient's long-term taking antithrombotic reagent, including anticoagulant, medicament for resisting platelet aggregation and Altace Ramipril, reduces blood coagulation tendency.Long-term taking Western medicine accelerates vessel aging, blood vessel is made to become fragile thinning, cause that angiorrhexis causes serious hemorrhagic side effect, thrombolytic therapy is presently considered to be that treatment thrombotic disease is maximally efficient and reliable means, but, on market, thrombolytic drug is difficult to be popularized because there is the bottleneck problems such as half-life too short, low the causing bleeding property side effect of specificity, and is only used for injection rescue medication.Therefore, on market very lack can long-term taking and safely and effectively prevention and treatment thrombotic disease thrombolytic drug.
By traditional method, with the natto that Semen sojae atricolor is prepared for raw material, because it contains the natural targeting thrombolytic effect composition fibrinolysin that has, and internal effective acting time length, be considered as a new generation's thrombolytic drug without hemorrhagic side effect.But the natto that traditional method prepares has ammonia stink, color and luster dimness due to it, is difficult to be popularized at home.And preparing natto by the traditional method of fermented soybean, fibrinolysin content is low, active low, function singleness.Being because Semen sojae atricolor as raw material, simply provide the nutritional labeling of growth for bacillus subtilis, Semen sojae atricolor itself does not have effect of disease treatment and health care.
[summary of the invention]
It is an object of the invention to overcome tradition in current technical method to prepare, for raw material, the deficiency that natto exists with Semen sojae atricolor, reduce ammonia stink, improve appearance luster degree, improve fibrinolysin content and activity, and by changing the bean material with medical value, expand medicinal efficacy and the value of natto.Therefore, the soybean material in traditional method is replaced by the present invention nutritional labeling and kind is more rich, and has the white jade natto of multiple medical value.The natto prepared by the fermentation of white jade Bacillus subtilis natto is called white jade natto, and fibrinolysin contained therein is called white jade fibrinolysin.
For achieving the above object, the preparation method of high-quality white jade natto provided by the invention and high activity fibrinolysin, it comprises the steps:
1) white jade natto is soaked with culture fluid；Described culture fluid includes following composition: lactose 10g/L, trypsin 20g/L, Na2HPO4·12H2O5g/L、NaH2PO4·2H2O1.0g/L、MgSO40.5g/L、CaCL20.3g/L, pH7.0～7.5 of culture fluid；The mass volume ratio of described white jade natto and culture fluid is 1:3 (kg:L), and described soaking conditions is 25 DEG C and soaks 18h；
2) the white jade natto after immersion is carried out steaming and decocting；Described conditions of cooking is 0.1MPa, 121 DEG C of steaming and decocting 35min；
3) inoculation bacillus subtilis carries out solid fermentation；Described bacillus subtilis inoculation condition is after the white jade natto after steaming and decocting is naturally cooled to 40-50 DEG C, by the 10% of white jade natto weight inoculation bacillus subtilis seed liquor；Described seed liquor is to obtain through the 37 DEG C of cultivations of LB culture medium；Described white jade Bafillus natt inoculation condition is temperature 37 DEG C, humidity 75%～85%, material thickness 2～3cm, and fermentation time is 24h；
4) the white jade natto after solid fermentation prepares white jade natto lyophilized powder through lyophilization and pulverizing；
5) described white jade natto lyophilized powder through lixiviate, saltout, dialyse and four steps of ion exchange chromatography carry out the separation purification of white jade fibrinolysin, eventually pass lyophilization and prepare white jade fibrinolysin lyophilized powder.
Preferably, step 4) in white jade natto prepare white jade natto lyophilized powder through lyophilization and pulverizing, specifically comprise the following steps that
(4.1) lyophilization: freezed in～80 DEG C by white jade natto, is then placed in vacuum freeze drier, is 10Pa in vacuum, dry 24h under-50 DEG C of conditions of condenser temperature；
(4.2) by the cryodesiccated white jade natto pulverizer through screen cloth 80 order, white jade natto lyophilized powder is prepared.
Further, described step 5) in by white jade natto lyophilized powder separation and Extraction white jade fibrinolysin lyophilization, specifically comprise the following steps that
(5.1) lixiviate: mixed by mass volume ratio 0.5:1 with normal saline by white jade natto lyophilized powder, lixiviate 24h under 4 DEG C of conditions, then at 4 DEG C, 8000rpm is centrifuged 10min, and gained supernatant is the thick zyme extract of white jade fibrinolysin；
(5.2) saltout: thick zyme extract saturation be 25% and 65% ammonium sulfate and 0 DEG C of salt fractionation precipitation, in 4 DEG C, 8000rpm is centrifuged the PBS dissolving of 30min, gained precipitation 10mMpH7.5；
(5.3) dialysis: after saltouing, sample is placed in bag filter, is immersed in distilled water, every 6h 10%BaC12Whether have precipitation to produce in detection distilled water, until inspection does not measure precipitation, dialysis completes；
(5.4) gel permeation chromatography: above-mentioned dialysis solution is added SephdexG200 chromatographic column, pH7.5PBS buffer linear elution, collects the white jade fibrinolysin of eluting；
(5.5) lyophilization: freezed in～80 DEG C by the enzyme liquid collected, be then placed in vacuum freeze drier, is 10Pa in vacuum, and under-50 DEG C of conditions of condenser temperature, dry 24h, prepares white jade fibrinolysin lyophilized powder.
The application that the present invention also provides for a kind of high-quality white jade natto obtained by said method and high activity fibrinolysin has in effect of lowering blood sugar medicine in preparation.
Above-mentioned bacillus subtilis is BacillussubtilisQK, and by China typical culture collection center preservation, deposit number is CCTCCNO:M203078.
Above-mentioned white jade Bafillus natt inoculation condition is preferably, temperature 37 DEG C, humidity 75%～85%, material thickness 2～3cm, and fermentation time is 24h, and fermentation gained white jade natto ammonia stink is low, the white clean light of color and luster.
The present invention has the advantage that relative to existing Semen sojae atricolor fermentation of bacillus subtilis technology
(1) present invention prepares white jade natto and fibrinolysin ammonia stink are low, color and luster bright and clean, are not easily eaten person and accept.
(2) white jade natto nutrition content and abundant species, not only contribute to the healthy of the mankind, also bacillus subtilis bacteria growing and breeding it are beneficial to, it is remarkably improved fibrinolysin yield and activity, extracting the white jade natto plasmin activity after separating is 45800IU/g, is significantly higher than the plasmin activity that prior art prepares.
(3) white jade natto itself has antithrombotic and effect for the treatment of cardiovascular and cerebrovascular disease, with the white jade natto that white jade natto is prepared for raw material, there is antithrombotic efficacy too, the two is organically combined by fermentation of bacillus subtilis, and the therapeutic effect of the cardiovascular and cerebrovascular diseases such as thromboembolia type is more notable.
(4) white jade natto not only nutrient substance enriches, also have many disease treatments and health-care effect, there is lung heat clearing, antiinflammatory, skin care, bone strengthening, stomach invigorating, the kidney invigorating, effects such as removing toxic substances of enriching blood, particularly that the action effect such as diabetes, cardiovascular and cerebrovascular disease is obvious.Therefore, white jade natto has white above-mentioned many medicinal efficacies.
[detailed description of the invention]
Below in conjunction with embodiment, the present invention is described in further detail, but the present invention is not limited within scope of embodiments.
Embodiment one
(1) prepared by fermentation seed liquid
BacillussubtilisQK bacillus subtilis condition of culture is: with LB culture medium for seed culture medium, and pH is 7.2, and triangular flask liquid amount is 25ml/250ml, and cultivation temperature is 37 DEG C, and shaking speed is 180rpm, and cultivation cycle is 16h.
(2) white jade natto steaming and decocting
Take the white jade natto of 10kg full seed and carry out remove impurity cleaning, add the above-mentioned culture fluid of 30L and soak 18h in 25 DEG C, put into after the white jade natto soaked is drained in digesting apparatus with 0.1MPa, 121 DEG C of steaming and decocting 35min.
(3) inoculation fermentation
White jade natto after steaming and decocting naturally cools to 45 DEG C, 10% inoculation BacillussubtilisQK bacillus subtilis seed liquor of white jade natto weight is pressed under aseptic condition, then ferment with Zymolysis Equipment, fermentation condition is temperature 37 DEG C, humidity 80%, material thickness 3cm, and fermentation time is 24h.
(4) white jade natto lyophilized powder is prepared
White jade natto is freezed in～80 DEG C, is then placed in vacuum freeze drier, be 10Pa in vacuum, dry 24h under-50 DEG C of conditions of condenser temperature.By the cryodesiccated white jade natto pulverizer through 80 mesh sieves, prepare white jade natto lyophilized powder.
(5) prepared by white jade fibrinolysin lyophilized powder
Lixiviate: white jade natto lyophilized powder mixes by mass volume ratio 0.5:1 with normal saline, lixiviate 24h under 4 DEG C of conditions, then at 4 DEG C, 8000rpm is centrifuged 10min, and gained supernatant is the thick zyme extract of white jade fibrinolysin.
Saltout: thick zyme extract saturation be 25% and 65% ammonium sulfate and 0 DEG C of salt fractionation precipitation, in 4 DEG C, 8000rpm is centrifuged the PBS dissolving of 30min, gained precipitation 10mMpH7.5.
Dialysis: after saltouing, sample is placed in bag filter, is immersed in distilled water, every 6h 10%BaC12Whether have precipitation to produce in detection distilled water, until inspection does not measure precipitation, dialysis completes.
Gel permeation chromatography: above-mentioned dialysis solution is added SephdexG200 chromatographic column, pH7.5PBS buffer linear elution, collects the white jade fibrinolysin of eluting.
Lyophilization: freezed in～80 DEG C by the enzyme liquid collected, be then placed in vacuum freeze drier, is 10Pa in vacuum, and under-50 DEG C of conditions of condenser temperature, dry 24h, prepares white jade fibrinolysin lyophilized powder.
Embodiment two
Concrete grammar step is with embodiment one, it is distinctive in that: prepared by (1) fermentation seed liquid: cultivation temperature is 37.5 DEG C, (2) white jade natto steaming and decocting: add the above-mentioned culture fluid of 30L and soak 19h in 26 DEG C, put into after the white jade natto soaked is drained in digesting apparatus with 0.1MPa, 123 DEG C of steaming and decocting 36min.(3) inoculation fermentation: the white jade natto after steaming and decocting naturally cools to 45 DEG C；Fermentation condition is temperature 37.5 DEG C, humidity 75%, material thickness 2cm, and fermentation time is 24.5h.(4) white jade natto lyophilized powder is prepared: be 10Pa in vacuum, dry 24.5h under-50 DEG C of conditions of condenser temperature.By the cryodesiccated white jade natto pulverizer through 90 mesh sieves, prepare white jade natto lyophilized powder.(5) prepared by white jade fibrinolysin lyophilized powder: lixiviate 24.5h under 4 DEG C of conditions, and then at 5 DEG C, 8000rpm is centrifuged 11min；Thick zyme extract saturation is ammonium sulfate and 0 DEG C of salt fractionation precipitation of 25% and 65%, and in 5 DEG C, 8000rpm be centrifuged the PBS dissolving of 32min, gained precipitation 10mMpH7.5；Being 10Pa in vacuum, under-50 DEG C of conditions of condenser temperature, dry 24.5h, prepares white jade fibrinolysin lyophilized powder.
Embodiment three
Concrete grammar step is with embodiment one, it is distinctive in that: prepared by (1) fermentation seed liquid: cultivation temperature is 38 DEG C, (2) white jade natto steaming and decocting: add the above-mentioned culture fluid of 30L and soak 20h in 27 DEG C, put into after the white jade natto soaked is drained in digesting apparatus with 0.1MPa, 125 DEG C of steaming and decocting 37min.(3) inoculation fermentation: the white jade natto after steaming and decocting naturally cools to 50 DEG C；Fermentation condition is temperature 38 DEG C, humidity 85%, material thickness 3cm, and fermentation time is 25h.(4) white jade natto lyophilized powder is prepared: be 10Pa in vacuum, dry 25h under-50 DEG C of conditions of condenser temperature.By the cryodesiccated white jade natto pulverizer through 100 mesh sieves, prepare white jade natto lyophilized powder.(5) prepared by white jade fibrinolysin lyophilized powder: lixiviate 25h under 4 DEG C of conditions, and then at 6 DEG C, 8000rpm is centrifuged 12min；Thick zyme extract saturation is ammonium sulfate and 0 DEG C of salt fractionation precipitation of 25% and 65%, and in 6 DEG C, 8000rpm be centrifuged the PBS dissolving of 35min, gained precipitation 10mMpH7.5；Being 10Pa in vacuum, under-50 DEG C of conditions of condenser temperature, dry 25h, prepares white jade fibrinolysin lyophilized powder.
Embodiment four
The mensuration that fibrinolysin enzyme is lived uses agarose fibrin plate method, the urokinase of variable concentrations is configured to 20,40,60,80,100,120,140,160IU/mL series concentration, point sample, on flat board, is placed in 37 DEG C and cultivates 18h, measure fibrinolytic circle area, make enzyme work～area standard curve.Take the fibrinolysin 10 μ L of white jade natto lyophilized powder 0.1g or separation, be placed in 37 DEG C and cultivate 18h, measure fibrinolytic circle area, calculate corresponding enzyme according to urokinase activity standard curve and live.
According to the method for embodiment one, white jade natto raw material changing Semen sojae atricolor, preparation Semen sojae atricolor natto lyophilized powder and Semen sojae atricolor fibrinolysin into, enzyme activity determination result shows, it is 387IU/g that the enzyme of Semen sojae atricolor natto lyophilized powder is lived, and Semen sojae atricolor fibrinolysin lyophilized powder enzyme is lived as 21800IU/g.And white jade natto prepared in embodiment one and white jade plasmin activity respectively 675IU/g, 45800IU/g, far above Semen sojae atricolor natto lyophilized powder and plasmin activity.
Embodiment five
The hypoglycemic activity of white jade natto lyophilized powder
(1) diabetic mouse model is set up
Water 12h is can't help in mice fasting, lumbar injection streptozotocin (STZ) 30mg/kg body weight, every day 1 time, continuous 5 days.Become mould standard: this research continuous 2 days of mice fasting glucose of employing is more than 15.9mmol L~ 1For modeling Success criteria.
(2) test and packet
Laboratory animal is grouped: mice, male, body weight (18 ± 2) g.Taking mice 60, lottery is randomly divided into 6 groups: Normal group (10), model group (10), white jade natto lyophilized powder large, medium and small dosage group (each 10), insulin group (10).The large, medium and small dosage group of white jade natto lyophilized powder be administered respectively 10g/kg body weight, 5g/kg body weight, 2.5g/kg body weight.Adopt mice docking blood taking method with blood, measure blood sugar concentration.
(3) white jade natto lyophilized powder hypoglycemic activity
Table. white jade natto lyophilized powder impact (the mmol L on diabetic mice blood glucose～1)
Claims (4)
1. a preparation method for high-quality white jade natto and high activity fibrinolysin, it comprises the steps:
1) white jade natto is soaked with culture fluid；Described culture fluid includes following composition: lactose 10g/L, trypsin 20g/L, Na2HPO4·12H2O5g/L、NaH2PO4·2H2O1.0g/L、MgSO40.5g/L、CaCL20.3g/L, pH7.0～7.5 of culture fluid；The mass volume ratio of described white jade natto and culture fluid is 1:3 (kg:L), and described soaking conditions is 25～27 DEG C and soaks 18～20h；
2) the white jade natto after immersion is carried out steaming and decocting；Described conditions of cooking is 0.1MPa, 121～125 DEG C of steaming and decocting 35～37min；
3) inoculation bacillus subtilis carries out solid fermentation；Described bacillus subtilis inoculation condition is after the white jade natto after steaming and decocting is naturally cooled to 40-50 DEG C, by the 10% of white jade natto weight inoculation bacillus subtilis seed liquor；Described seed liquor is to obtain through 37～38 DEG C of cultivations of LB culture medium；Described white jade Bafillus natt inoculation condition is temperature 37～38 DEG C, humidity 75%～85%, material thickness 2～3cm, and fermentation time is 24～25h；
4) the white jade natto after solid fermentation prepares white jade natto lyophilized powder through lyophilization and pulverizing；
5) described white jade natto lyophilized powder through lixiviate, saltout, dialyse and four steps of ion exchange chromatography carry out the separation purification of white jade fibrinolysin, eventually pass lyophilization and prepare white jade fibrinolysin lyophilized powder.
2. the preparation method of high-quality white jade natto according to claim 1 and high activity fibrinolysin, it is characterised in that: step 4) in white jade natto prepare white jade natto lyophilized powder through lyophilization and pulverizing, specifically comprise the following steps that
(4.1) lyophilization: freezed in～80 DEG C by white jade natto, is then placed in vacuum freeze drier, is 10Pa in vacuum, dry 24～25h under-50 DEG C of conditions of condenser temperature；
(4.2) by the cryodesiccated white jade natto pulverizer through screen cloth 80～100 order, white jade natto lyophilized powder is prepared.
3. the preparation method of high-quality white jade natto according to claim 1 and 2 and high activity fibrinolysin, it is characterised in that: described step 5) in by white jade natto lyophilized powder separation and Extraction white jade fibrinolysin lyophilization, specifically comprise the following steps that
(5.1) lixiviate: mixed by mass volume ratio 0.5:1 with normal saline by white jade natto lyophilized powder, lixiviate 24～25h under 4 DEG C of conditions, then at 4～6 DEG C, 8000rpm is centrifuged 10～12min, and gained supernatant is the thick zyme extract of white jade fibrinolysin；
(5.2) saltout: thick zyme extract saturation be 25% and 65% ammonium sulfate and 0 DEG C of salt fractionation precipitation, in 4～6 DEG C, 8000rpm is centrifuged the PBS dissolving of 30～35min, gained precipitation 10mMpH7.5；
(5.3) dialysis: after saltouing, sample is placed in bag filter, is immersed in distilled water, every 6～6.5h 10%BaC12Whether have precipitation to produce in detection distilled water, until inspection does not measure precipitation, dialysis completes；
(5.4) gel permeation chromatography: above-mentioned dialysis solution is added SephdexG200 chromatographic column, pH7.5PBS buffer linear elution, collects the white jade fibrinolysin of eluting；
(5.5) lyophilization: freezed in～80 DEG C by the enzyme liquid collected, be then placed in vacuum freeze drier, is 10Pa in vacuum, and under-50 DEG C of conditions of condenser temperature, dry 24～25h, prepares white jade fibrinolysin lyophilized powder.
4. the application that the high-quality white jade natto obtained by claim 1 method and high activity fibrinolysin have in effect of lowering blood sugar medicine in preparation.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110368482A (en) * | 2019-07-18 | 2019-10-25 | 嫦娥创新（武汉）生物科技有限公司 | A kind of composition and its preparing the application in prevention and cure of cardiovascular disease drug |
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