CN104212741A - Bacillus subtilis producing fermented chickpea having fibrinolysis and antioxidation functions, and its application - Google Patents
Bacillus subtilis producing fermented chickpea having fibrinolysis and antioxidation functions, and its application Download PDFInfo
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- CN104212741A CN104212741A CN201410401745.9A CN201410401745A CN104212741A CN 104212741 A CN104212741 A CN 104212741A CN 201410401745 A CN201410401745 A CN 201410401745A CN 104212741 A CN104212741 A CN 104212741A
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Abstract
The invention discloses a Bacillus subtilis producing fermented chickpea having fibrinolysis and antioxidation functions, and its application. The Bacillus subtilis is Bacillus subtilis DC-Tx, and is preserved in China General Microbiological Culture Collection Center at No.3 Beichen West Road, Chaoyang District, Beijing on July 15, 2014 with the preservation number of CGMCC NO.9462. The Bacillus subtilis DC-Tx highly producing natto kinase is screened in the invention, and can produce fermented chickpea having fibrinolysis and antioxidation functions. Results show that the fibrinolysis activity of the fermented chickpea produced by the above strain can reach 3000FU/g, the .OH clearance of the fermented chickpea can reach 88.5%, and the Fe<3+> reduction capability of the fermented chickpea is 3.33 times that of fresh chickpea. A fermented chickpea preparation method disclosed in the invention has the advantages of simple operation, low cost, short cycle, easy realization, abundant sources, easy obtaining and low cost of raw materials, no environmental pollution, low prices of used apparatuses and reagents, and easy realization.
Description
Technical field
The present invention relates to the subtilis that a kind of production has fibrinolytic and anti-oxidant function fermentation garbanzo, also relate to the application of this subtilis simultaneously, belong to microbial technology field.
Background technology
In recent years, due to reasons such as dietary unbalance, environmental degradation, inherited traits, blood vessel embolism class disease significantly increases, and health and the life security of the mankind in serious threat, die from every year cerebral infarction, myocardial infarction patient nearly millions of, occupy first of various disease.Thrombus is the prefered method for the treatment of this kind of disease, and therefore, the research and development with Hyperfibrinolysis thrombolytic drug receive the concern of people day by day, have vast potential for future development.One of focus of current thrombolytic drug research be produce orally active corroded rock mass food and protective foods---Nattokinase by fermentation using bacteria, but how not fully up to expectations the fibrinolytic activity of nattokinase from natto of the method gained is.
On the other hand, reactive oxygen species and free radicals is the secondary metabolite of human metabolism's process, always be in the running balance constantly producing and eliminate under normal circumstances, if but its too high levels, running balance is destroyed, will response to oxidative stress be caused, thus more than the 100 kind of common diseases such as cause cancer, coronary heart disease, atherosclerosis, diabetes, neural system are malfunctioning, immunizing power reduction, sacroiliitis.Its atherosclerosis can cause cardiovascular and cerebrovascular diseases again, and produce thrombus, therefore, reactive oxygen species and free radicals is also one of cause of disease of cardiovascular and cerebrovascular diseases.There are some researches show at present, supplement antioxidant by edible oxidation-resistance functional food, is that prevention comprises one of effective measure of the various diseases of cardiovascular and cerebrovascular diseases.
In sum, reactive oxygen species and free radicals can cause the various diseases comprising cardiovascular and cerebrovascular diseases, and there is the food of anti-oxidant function and protective foods can Scavenger of ROS and free radical, therefore also there is the effect of prevention cardiovascular and cerebrovascular diseases; And the thrombus that the protective foods solubilized of Hyperfibrinolysis has been formed, thus treatment cardiovascular and cerebrovascular diseases, if research and develop a kind of food with fibrinolytic and anti-oxidant function, then both there is the function that preventing cardiovascular disease occurs, result for the treatment of can be had again after thrombus produces, thus reach the object that prevention and therapy combines, but also there is no the report of correlative study at present.
Garbanzo (chickpea) formal name used at school Cicer arietinum L., be rich in the various saccharides of necessary for human, vegetable-protein and amino acid, Mierocrystalline cellulose, VITAMIN and inorganic salt, there is preventing hypertension and arteriosclerosis, reduction cholesterol and blood-fat and blood sugar, the effect such as diuresis, Cure for insomnia.There are some researches show, the enzymolysis product (Chickpea short-peptide) of Chickpea Protein, there is significant anti-oxidant function, also report is had to point out, use bacillus natto to ferment garbanzo, the Nattokinase of Hyperfibrinolysis can be obtained, but yet there are no the report of the fermentation garbanzo with fibrinolytic and anti-oxidant function.
Summary of the invention
The object of this invention is to provide subtilis and application thereof that a kind of production has fibrinolytic and anti-oxidant function fermentation garbanzo, subtilis of the present invention is adopted to produce fermentation garbanzo, the garbanzo tunning with Hyperfibrinolysis and remarkable antioxygenation can be obtained, and the method has simple to operate, cost is low, the cycle short advantage with being easy to realize, for the industrialization of Nattokinase provides technical support, and provide new thinking for its Application and Development research as functional foodstuff and protective foods.
In order to realize above object, the technical solution adopted in the present invention is to provide the subtilis that a kind of production has fibrinolytic and anti-oxidant function fermentation garbanzo, described subtilis is subtilis (Bacillus substilis) DC-Tx, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preserving number: CGMCC NO.9462, preservation date: on July 15th, 2014.
The present invention filters out the subtilis DC-Tx of high-yield nattokinase from fermented soya bean, adopts bacterial strain of the present invention, can produce the fermentation garbanzo with Hyperfibrinolysis and remarkable anti-oxidant function.Experimental result shows, the fermentation garbanzo that bacterial strain of the present invention is produced, and its fibrinolytic can reach 3000FU/g, and hydroxyl radical free radical (OH) clearance rate can reach 88.5%, Fe
3+reducing power is 3.33 times of fresh garbanzo.
The technical solution adopted in the present invention is also to provide a kind of subtilis producing the application had in fibrinolytic and anti-oxidant function fermentation garbanzo.
Subtilis of the present invention produces the method with fibrinolytic and anti-oxidant function fermentation garbanzo, comprises the following steps:
(1) picking subtilis is inoculated in LB liquid seed culture medium, and under 28-40 DEG C of condition, 120-180rpm shaking table constant temperature culture 12-18h, obtains subtilis seed liquor;
(2) by garbanzo soaked overnight, drain away the water, autoclaving, after being cooled to room temperature, according to the inoculum size inoculation subtilis seed liquor of 1-8%, cultivates 36-72h at 28-40 DEG C of condition bottom fermentation;
(3) by the garbanzo fermentation culture product cryogenic vacuum lyophilize of step (2), pulverize, cross 80-120 mesh sieve and get final product.
Described LB liquid seed culture medium comprises the raw material of following massfraction: peptone 1%, yeast powder 0.5%, NaCl 1%; PH6.8,121 DEG C of autoclaving 20min.
The cryodesiccated condition of described cryogenic vacuum is: precooling temperature ﹣ 50 DEG C, vacuum tightness 20-100Pa, freeze temperature≤﹣ 45 DEG C, freeze-drying time 24-48h.In the present invention, cryogenic vacuum lyophilize is selected in lyophilize, and does not adopt spraying dry, and object is fibrinolytic and anti-oxidant function in order to keep gained tunning better.
The present invention utilizes subtilis DC-Tx fermentative production to have the fermentation garbanzo of Hyperfibrinolysis and remarkable anti-oxidant function, and the method has simple to operate, and cost is low, the cycle short advantage with being easy to realize.Meanwhile, the present invention adopts garbanzo to be raw material, and abundance is easily got, and cost is low, non-environmental-pollution, and equipment used, reagent low price, be convenient to large-scale production.
Accompanying drawing explanation
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described in further detail.
Fig. 1 is that the present invention is fermented garbanzo fibrinolytic measurement result;
Fig. 2 is that the present invention is fermented garbanzo OH clearance rate measurement result; Wherein,
A is GSH, b is fermentation 36h garbanzo, and c is fermentation 48h garbanzo, and d is fermentation 72h garbanzo, and e is fresh garbanzo;
Fig. 3 is that the present invention is fermented garbanzo Fe
3+reducing power measurement result; Wherein,
A is GSH, b is fermentation 36h garbanzo, and c is fermentation 48h garbanzo, and d is fermentation 72h garbanzo, and e is fresh garbanzo.
Embodiment
Below in conjunction with embodiment, the specific embodiment of the present invention is described in further detail.
Embodiment 1
1, the screening of Nattokinase superior strain
The primary dcreening operation of 1.1 Nattokinase superior strains gets commercially available fermented soya bean, sample separation 10g, adds in 100mL sterilized water, abundant vibration, 100 DEG C of boiling water boil 5min, leave standstill a moment, get upper strata dirty solution, after gradient dilution, get the different dilution solution of 100 μ L respectively, be spread evenly across on LB flat board, 37 DEG C of incubated overnight, choosing single bacterium colony carries out streak culture, double.Single bacterium colony dibbling on picking LB separating plate, on fibrin plate, is cultivated 18h for 37 DEG C, is measured and dissolve loop diameter, and multiple sieve is carried out in the strain liquid fermentation of dissolving circle larger.
The multiple sieve of 1.2 Nattokinase superior strains is by bacterial strain larger for primary dcreening operation transparent circle diameter, with LB seed culture medium activation (37 DEG C, 150r/min, spend the night), inoculum size according to 5% accesses liquid fermentation medium respectively (in massfraction: 6% soyflour, 2% glucose, 0.06%CaCl
2, 0.07%MgSO
4, pH6.5) in, the phosphoric acid buffer dilution of 37 DEG C of fermentations 72h, its tunning pH7.4, get diluent 10 μ L, application of sample is 37 DEG C of constant temperature culture 18h on fibrin plate, measure transparent circle diameter, choose and dissolve the maximum bacterial strain of circle, called after DC-Tx.
The feature of bacterial strain DC-Tx is as follows:
Morphological specificity:
Dull and stereotyped upper 37 DEG C of LB cultivates 24h, and bacterial strain DC-Tx bacterium colony is rounded, and edge is micro-has lobate tooth, White-opalescent; Thalli morphology is little rod-short, long 3.0-4.0 μm, wide 0.8-1.0 μm, Gram-positive, raw in gemma, oval.
Physiological and biochemical property is in table 1.
The physiological and biochemical property of table 1 bacterial strain DC-Tx
16S rDNA checks order qualification:
Extract the genomic dna of bacterial strain DC-Tx, increase by 16S rDNA gene universal primer PCR; The PCR primer obtained, is connected with pGM-T carrier, transforms DH5 α competent cell; Screening positive clone, puies forward plasmid DNA and carries out enzyme and cut qualification; Connect correct positive colony and carry out 16S rDNA order-checking, its sequencing result is as follows:
Relatively find, the complete genome sequence of the 16S rDNA of bacterial strain DC-Tx all reaches 99% with the subtilis of announcing, bacillus amyloliquefaciens coincidence rate on the net.
The result of comprehensive above-mentioned morphological specificity, physiological and biochemical analysis and molecule order-checking qualification, shows that DC-Tx is a bacillus subtilis (Bacillus subtilis).
Embodiment 2
The present embodiment produces the method with fibrinolytic and anti-oxidant function fermentation garbanzo with subtilis DC-Tx, comprise the following steps:
(1) picking subtilis DC-Tx is inoculated in LB liquid seed culture medium, and under 28 DEG C of conditions, 180rpm shaking table constant temperature culture 15h, obtains subtilis DC-Tx seed liquor;
(2) by garbanzo soaked overnight, drain away the water, 121 DEG C of autoclaving 20min, after being cooled to room temperature, according to the inoculum size inoculation subtilis DC-Tx seed liquor of 8%, cultivate 36h at 28 DEG C of condition bottom fermentations;
(3) by the garbanzo fermentation culture product cryogenic vacuum lyophilize of step (2), pulverize, cross 100 mesh sieves and get final product; The cryodesiccated condition of cryogenic vacuum is: precooling temperature ﹣ 50 DEG C, vacuum tightness 20Pa, freeze temperature ﹣ 50 DEG C, freeze-drying time 24h.
Embodiment 3
The present embodiment produces the method with fibrinolytic and anti-oxidant function fermentation garbanzo with subtilis DC-Tx, comprise the following steps:
(1) picking subtilis DC-Tx is inoculated in LB liquid seed culture medium, and under 34 DEG C of conditions, 120rpm shaking table constant temperature culture 12h, obtains subtilis DC-Tx seed liquor;
(2) by garbanzo soaked overnight, drain away the water, 121 DEG C of autoclaving 20min, after being cooled to room temperature, according to the inoculum size inoculation subtilis DC-Tx seed liquor of 5%, cultivate 48h at 34 DEG C of condition bottom fermentations;
(3) by the garbanzo fermentation culture product cryogenic vacuum lyophilize of step (2), pulverize, cross 80 mesh sieves and get final product; The cryodesiccated condition of cryogenic vacuum is: precooling temperature ﹣ 50 DEG C, vacuum tightness 60Pa, freeze temperature ﹣ 55 DEG C, freeze-drying time 48h.
Embodiment 4
The present embodiment produces the method with fibrinolytic and anti-oxidant function fermentation garbanzo with subtilis DC-Tx, comprise the following steps:
(1) picking subtilis DC-Tx is inoculated in LB liquid seed culture medium, and under 40 DEG C of conditions, 150rpm shaking table constant temperature culture 18h, obtains subtilis DC-Tx seed liquor;
(2) by garbanzo soaked overnight, drain away the water, 121 DEG C of autoclaving 20min, after being cooled to room temperature, according to the inoculum size inoculation subtilis DC-Tx seed liquor of 1%, cultivate 72h at 40 DEG C of condition bottom fermentations;
(3) by the garbanzo fermentation culture product cryogenic vacuum lyophilize of step (2), pulverize, cross 120 mesh sieves and get final product; The cryodesiccated condition of cryogenic vacuum is: precooling temperature ﹣ 50 DEG C, vacuum tightness 100Pa, freeze temperature ﹣ 45 DEG C, freeze-drying time 36h.
Experimental example, the present invention are fermented the mensuration of garbanzo fibrinolytic and anti-oxidant function
The preparation of sample: the fermentation olecranon bean powder physiological saline suspension of preparation massfraction 2%, after the centrifugal 10min of 4 DEG C of lixiviate 4h, 5000 × g, after gained supernatant liquor 15000 × g repeated centrifugation 10min, supernatant liquor is used for the mensuration of fibrinolytic and anti-oxidant function.
1, the fibrinolytic that flat band method measures fermentation garbanzo is tieed up
The preparation of fibrin plate: 1g agarose is dissolved in the sodium phosphate buffer 100mL of 0.01M pH7.4, get this agarose solution 7.5mL, 50 DEG C of water bath heat preservation 5min, add the 100BP/mL zymoplasm 225 μ L being dissolved in physiological saline, mixing, 50 DEG C of water-bath 5min, get 7.5mL 3.6mg/mL bovine fibrinogen solution (being dissolved in the sodium phosphate buffer of 0.1M pH7.4), add in the zymoplasm-agarose solution of insulation, rapid mixing, pour the culture dish of diameter 9cm into, wait to coagulate.
The drafting of typical curve: the urokinase standard solution preparing 20,40,60,80 and 100IU/mL with sterile saline respectively, respectively get 10 μ L point samples on fibrin plate, 37 DEG C of insulation 18h, measure the diameter (see accompanying drawing 1) fibrin plate dissolving circle, get the mean diameter of three tests, calculate and dissolve circle area; With urokinase activity (IU/mL) for X-coordinate, dissolving circle area is ordinate zou, draws urokinase typical curve.
The mensuration of fibrinolytic: pipette 10 μ L fermentation olecranon bean powder vat liquors with micro sample adding appliance, point sample is on fibrin plate, and 7 DEG C of insulation 18h, measure diameter fibrin plate dissolving circle, calculates to dissolve and encloses area.According to typical curve, calculate fibrinolytic.
Measurement result finds, before 48h, with the prolongation of fermentation time, fibrinolytic significantly increases, and after 48h, fibrinolytic prolongation is in time substantially constant, and during fermentation 48h, tunning fibrinolytic can reach 3000FU/g.
2, garbanzo of fermenting removes the mensuration of OH ability
Get 50 μ L 3mM 1,10 phenanthrolenes (being dissolved in the sodium phosphate buffer of 0.1M pH7.4) to add in 96 orifice plates, add 50 μ L samples, mixing, adds 50 μ L FeSO
4(3mM) aqueous solution, mixing, adds 50 μ L massfraction 0.01% hydrogen peroxide (H
2o
2) the aqueous solution, cover lid, 37 DEG C of constant temperature 60min, measure the absorbancy at 536nm place, this is sample sets (A1), and Control distilled water replaces sample (A0), and Blank distilled water replaces sample and 0.01% hydrogen peroxide (A2).
With following formulae discovery sample to the clearance rate of OH: OH clearance rate (%)=[(A1-A0)/(A2-A0)] × 100%
Found that, the OH clearance rate of the garbanzo of fermentation 48h reaches 88.5%, it is the nearly twice that OH ability (47.7%) removed by gsh (GSH), and be significantly higher than the clearance rate (52.3%) (measurement result be shown in accompanying drawing 2) of fresh garbanzo to OH, illustrate that the present invention's fermentation garbanzo obtained of fermenting has very strong OH Scavenging activity.
3, garbanzo of fermenting reduction Fe
3+the mensuration of ability
Get 250 μ L samples to mix with the potassium ferricyanide aqueous solution of 250 μ L massfractions 1% (not containing sample in contrasting), 50 DEG C of water bath with thermostatic control 20min, after add 250 μ L massfraction 10% trichloroacetic acid solutions, the centrifugal 10min of 5000 × g after mixing, get 100 μ L supernatant liquors, add in 96 orifice plates, add 20 μ L massfraction 0.1%FeCl
3the aqueous solution, after add 80 μ L distilled water, mixing, mixed solution 25 DEG C of standing 10min, measure the absorbancy at supernatant liquor 700nm place.
Measurement result shows, fermentation garbanzo Fe
3+reducing power is the strongest when fermenting 72h, OD
700nmvalue is 0.40, though with GSH OD
700nmvalue (1.57) difference is comparatively large, but fresh garbanzo Fe
3+reducing power (OD
700nmvalue is 0.12) 3.33 times, show fermentation garbanzo there is stronger Fe
3+reducing power (see accompanying drawing 3).
Experimental result illustrates, the present invention's garbanzo of fermenting has very high fibrinolytic and stronger resistance of oxidation.The present invention is that the large-scale production of fermentation garbanzo provides a kind of new approach, and provides new thinking for its Application and Development research as functional foodstuff and protective foods.
Claims (5)
1. a production has the subtilis of fibrinolytic and anti-oxidant function fermentation garbanzo, it is characterized in that, described subtilis is subtilis (Bacillus subtilis) DC-Tx, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preserving number: CGMCC NO.9462, preservation date: on July 15th, 2014.
2. a subtilis as claimed in claim 1 is producing the application had in fibrinolytic and anti-oxidant function fermentation garbanzo.
3. subtilis as claimed in claim 1 produces a method with fibrinolytic and anti-oxidant function fermentation garbanzo, it is characterized in that, comprises the following steps:
(1) picking subtilis is inoculated in LB liquid seed culture medium, and under 28-40 DEG C of condition, 120-180rpm shaking table constant temperature culture 12-18h, obtains subtilis seed liquor;
(2) by garbanzo soaked overnight, drain away the water, autoclaving, after being cooled to room temperature, according to the inoculum size inoculation subtilis seed liquor of 1-8%, cultivates 36-72h at 28-40 DEG C of condition bottom fermentation;
(3) by the garbanzo fermentation culture product cryogenic vacuum lyophilize of step (2), pulverize, cross 80-120 mesh sieve and get final product.
4. subtilis according to claim 3 produces the method with fibrinolytic and anti-oxidant function fermentation garbanzo, it is characterized in that, described LB liquid seed culture medium comprises the raw material of following massfraction: peptone 1%, yeast powder 0.5%, NaCl 1%; PH6.8,121 DEG C of autoclaving 20min.
5. subtilis according to claim 3 produces the method with fibrinolytic and anti-oxidant function fermentation garbanzo, it is characterized in that, the cryodesiccated condition of described cryogenic vacuum is: precooling temperature ﹣ 50 DEG C, vacuum tightness 20-100Pa, freeze temperature≤﹣ 45 DEG C, freeze-drying time 24-48h.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105087447A (en) * | 2015-09-16 | 2015-11-25 | 河南省科学院生物研究所有限责任公司 | Microwave-resisting bacillus subtilis and application thereof in preparing nattokinase |
| CN107688019A (en) * | 2017-08-29 | 2018-02-13 | 宝鸡文理学院 | A kind of method of micromethod detection ascorbic acid to hydroxy radical inhibiting rate |
| CN111676156A (en) * | 2020-06-03 | 2020-09-18 | 青岛农业大学 | Bacillus belgii MRS for improving reduction activity and fermentation product and application thereof |
| CN115005433A (en) * | 2022-07-19 | 2022-09-06 | 湖北真福医药有限公司 | Bacillus subtilis fibrinolytic enzyme composition with effects of maintaining beauty and keeping young, preparation method and application |
| 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 |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103243040A (en) * | 2012-02-13 | 2013-08-14 | 中国科学院过程工程研究所 | Bacillus subtilis LSSE-22 and application thereof |
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2014
- 2014-08-15 CN CN201410401745.9A patent/CN104212741B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103243040A (en) * | 2012-02-13 | 2013-08-14 | 中国科学院过程工程研究所 | Bacillus subtilis LSSE-22 and application thereof |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105087447A (en) * | 2015-09-16 | 2015-11-25 | 河南省科学院生物研究所有限责任公司 | Microwave-resisting bacillus subtilis and application thereof in preparing nattokinase |
| CN105087447B (en) * | 2015-09-16 | 2018-08-21 | 河南省科学院生物研究所有限责任公司 | One plant of resistance to microwave bacillus subtilis and its application in Nattokinase preparation |
| CN107688019A (en) * | 2017-08-29 | 2018-02-13 | 宝鸡文理学院 | A kind of method of micromethod detection ascorbic acid to hydroxy radical inhibiting rate |
| CN111676156A (en) * | 2020-06-03 | 2020-09-18 | 青岛农业大学 | Bacillus belgii MRS for improving reduction activity and fermentation product and application thereof |
| 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 |
| CN115005433A (en) * | 2022-07-19 | 2022-09-06 | 湖北真福医药有限公司 | Bacillus subtilis fibrinolytic enzyme composition with effects of maintaining beauty and keeping young, preparation method and application |
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