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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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 invention relates to bacillus subtilis for producing fermented chickpeas with fibrinolysis and antioxidant functions, and also relates to application of the bacillus subtilis, belonging to the technical field of microorganisms.
Background
In recent years, due to unbalanced diet, deterioration of environment, genetic traits and the like, vascular embolism diseases are greatly increased, which seriously threatens human health and life safety, and patients who die of cerebral infarction and myocardial infarction every year are as high as millions and live first in various diseases. Therefore, the research and development of thrombolytic drugs with high fibrinolytic activity are receiving more and more attention, and the thrombolytic drugs have a wide development prospect. At present, one focus of thrombolytic drug research is to produce orally effective thrombolytic functional food and health food-nattokinase by bacterial fermentation, but the fibrinolytic activity of the nattokinase obtained by the method is much unsatisfactory.
On the other hand, active oxygen and free radicals are secondary metabolites in the metabolic process of the human body, and are always in dynamic equilibrium of continuous generation and elimination under normal conditions, but if the content of the active oxygen and the free radicals is too high, the dynamic equilibrium is destroyed, and oxidative stress reaction is caused, so that more than 100 common diseases such as cancer, coronary heart disease, atherosclerosis, diabetes, nervous system failure, immunity reduction, arthritis and the like are caused. In addition, atherosclerosis can cause cardiovascular and cerebrovascular diseases and generate thrombus, so that active oxygen and free radicals are also one of the causes of the cardiovascular and cerebrovascular diseases. At present, researches show that the supplement of antioxidant substances by eating antioxidant functional food is one of effective measures for preventing various diseases including cardiovascular and cerebrovascular diseases.
In conclusion, active oxygen and free radicals can cause various diseases including cardiovascular and cerebrovascular diseases, and foods and health-care foods with antioxidant functions can eliminate the active oxygen and the free radicals, so that the food and the health-care foods also have the effect of preventing the cardiovascular and cerebrovascular diseases; the health food with high fibrinolytic activity can dissolve thrombus formed so as to treat cardiovascular and cerebrovascular diseases, and if a food with fibrinolytic activity and antioxidant function can be developed, the health food not only has the function of preventing cardiovascular diseases, but also has the treatment effect after thrombus is generated so as to achieve the purpose of combining prevention and treatment, but no related research report exists at present.
Chickpea (chickpea) with the scientific name of Cicer arietinum L is rich in various saccharides, vegetable proteins, amino acids, cellulose, vitamins and inorganic salts required by human beings, and has the effects of preventing hypertension and arteriosclerosis, reducing cholesterol, blood fat and blood sugar, promoting urination, treating insomnia and the like. Researches show that enzymolysis products (chick pea oligopeptide) of chick pea protein have obvious antioxidant function, and reports indicate that natto kinase with high fibrinolytic activity can be obtained by fermenting chick pea with bacillus natto, but reports of fermented chick pea with fibrinolytic activity and antioxidant function are not available at present.
Disclosure of Invention
The invention aims to provide the bacillus subtilis for producing the fermented chickpea with fibrinolytic and antioxidant functions and the application thereof.
In order to achieve the above object, the technical scheme adopted by the present invention is to provide a Bacillus subtilis for producing fermented chickpeas with fibrinolysis and antioxidant functions, wherein the Bacillus subtilis is Bacillus subtilis DC-Tx (Bacillus subtilis), and the storage unit: china general microbiological culture Collection center, preservation Address: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, on Beijing, with a registration number: CGMCC NO.9462, preservation date: 7, month and 15 days 2014.
The bacillus subtilis DC-Tx with high nattokinase yield is screened from fermented soybeans, and the fermented chickpeas with high fibrinolytic activity and remarkable antioxidant function can be produced by adopting the strain. The experimental result shows that the fibrinolytic activity of the fermented chickpea produced by the bacterial strain can reach 3000FU/g, the hydroxyl free radical (OH) clearance rate can reach 88.5 percent, and Fe3+The reducing power is 3.33 times of that of fresh chickpeas.
The technical scheme adopted by the invention also provides the application of the bacillus subtilis in the production of fermented chickpeas with fibrinolysis and antioxidant functions.
The method for producing fermented chickpeas with fibrinolysis and antioxidant functions by using the bacillus subtilis comprises the following steps:
(1) selecting bacillus subtilis, inoculating the bacillus subtilis to an LB liquid seed culture medium, and culturing for 12-18h at the temperature of 28-40 ℃ by using a 120-plus 180rpm shaking table at constant temperature to obtain a bacillus subtilis seed liquid;
(2) soaking chickpeas overnight, draining water, autoclaving, cooling to room temperature, inoculating Bacillus subtilis seed solution according to the inoculation amount of 1-8%, and fermenting and culturing at 28-40 deg.C for 36-72 h;
(3) and (3) freezing and drying the fermentation culture product of the chickpeas in the step (2) at a low temperature in vacuum, crushing, and sieving with a sieve of 80-120 meshes to obtain the chickpeas.
The LB liquid seed culture medium comprises the following raw materials in percentage by mass: peptone 1%, yeast powder 0.5%, NaCl 1%; sterilizing at 121 deg.C for 20min at pH 6.8.
The low-temperature vacuum freeze drying conditions are as follows: pre-cooling at-50 deg.C under vacuum degree of 20-100Pa, lyophilizing at a temperature of no more than-45 deg.C, and lyophilizing for 24-48 h. In the invention, low-temperature vacuum freeze drying is selected for freeze drying, and spray drying is not adopted, so that the fibrinolysis and antioxidant functions of the obtained fermentation product are better maintained.
The fermentation chickpea with high fibrinolytic activity and remarkable anti-oxidation function is produced by utilizing the fermentation of the bacillus subtilis DC-Tx, and the method has the advantages of simple operation, low cost, short period and easy realization. Meanwhile, chickpeas are adopted as raw materials, so that the chickpeas are rich in sources, easy to obtain, low in cost, free of environmental pollution, low in price of used equipment and reagents and convenient for large-scale production.
Drawings
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 shows the results of measurement of fibrinolytic activity of fermented chick pea according to the present invention;
FIG. 2 shows the result of measurement of the clearance of fermented chick pea OH in the present invention; wherein,
a is GSH, b is fermented chickpeas of 36h, c is fermented chickpeas of 48h, d is fermented chickpeas of 72h, and e is fresh chickpeas;
FIG. 3 shows fermented chick pea Fe of the present invention3+The result of the reduction ability measurement; wherein,
a is GSH, b is fermented 36h chickpeas, c is fermented 48h chickpeas, d is fermented 72h chickpeas, and e is fresh chickpeas.
Detailed Description
The following examples further illustrate the embodiments of the present invention in detail.
Example 1
1. Screening of natto kinase high-producing strain
1.1 Primary screening of Nattokinase high-yield strain, taking commercial fermented soya beans, separating a sample by 10g, adding into 100mL of sterile water, fully oscillating, boiling in boiling water at 100 ℃ for 5min, standing for a moment, taking upper layer turbid liquid, after gradient dilution, respectively taking 100 mu L of solutions with different dilutions, uniformly coating the solutions on an LB plate, overnight culturing at 37 ℃, selecting a single colony for streak culture, and continuously carrying out twice. Picking single colony on LB separating plate to dibble onto fibrin plate, culturing at 37 deg.C for 18h, measuring diameter of lysis ring, fermenting with large lysis ring strain liquid, and rescreening.
1.2 rescreening Nattokinase high-yield strain, activating the strain with larger diameter of the primary screening transparent ring with LB seed culture medium (37 ℃, 150r/min, overnight), inoculating the strain into liquid fermentation culture medium (by mass fraction, 6% of soybean flour, 2% of glucose, 0.06% of CaCl) according to 5% of inoculum size2，0.07％MgSO4pH6.5), fermenting at 37 ℃ for 72h, diluting the fermentation product with phosphate buffer solution with pH7.4, taking 10 mu L of the dilution, adding the dilution on a fibrin plate, culturing at constant temperature of 37 ℃ for 18h, measuring the diameter of a transparent ring, and selecting the strain with the largest dissolving ring, wherein the strain is named as DC-Tx.
The characteristics of the strain DC-Tx are as follows:
morphological characteristics:
culturing on LB plate at 37 deg.C for 24h to obtain circular strain DC-Tx colony with slightly leaf-shaped teeth at edge, and white and opaque; the shape of the thallus is small and short rod, the length is 3.0-4.0 μm, the width is 0.8-1.0 μm, gram positive, spore is middle, and the thallus is oval.
The physiological and biochemical characteristics are shown in Table 1.
TABLE 1 physiological and biochemical characteristics of the Strain DC-Tx
16S rDNA sequencing identification:
extracting genome DNA of the strain DC-Tx, and performing PCR amplification by using a 16S rDNA gene universal primer; connecting the obtained PCR product with a pGM-T vector, and transforming DH5 alpha competent cells; screening positive clones, and carrying out enzyme digestion identification on the DNA of the quality-improved grains; the correct positive clones were ligated for 16S rDNA sequencing, with the following sequencing results:
the comparison shows that the coincidence rate of the full gene sequence of the 16S rDNA of the strain DC-Tx and bacillus subtilis and bacillus amyloliquefaciens published on the net reaches 99 percent.
The results of the morphological characteristics, the physiological and biochemical analysis and the molecular sequencing identification are integrated, and the result shows that the DC-Tx is a strain of Bacillus subtilis.
Example 2
The method for producing fermented chickpeas with fibrinolytic and antioxidant functions by using bacillus subtilis DC-Tx comprises the following steps:
(1) selecting bacillus subtilis DC-Tx, inoculating the bacillus subtilis DC-Tx in an LB liquid seed culture medium, and culturing for 15h at the constant temperature of 28 ℃ and in a shaking table at 180rpm to obtain bacillus subtilis DC-Tx seed liquid;
(2) soaking chickpeas overnight, draining water, autoclaving at 121 ℃ for 20min, cooling to room temperature, inoculating Bacillus subtilis DC-Tx seed solution according to the inoculation amount of 8%, and fermenting and culturing at 28 ℃ for 36 h;
(3) freezing and drying the chickpea fermentation culture product obtained in the step (2) at low temperature in vacuum, crushing, and sieving with a 100-mesh sieve to obtain the chickpea fermentation culture product; the low-temperature vacuum freeze drying conditions are as follows: pre-cooling at-50 deg.C under vacuum degree of 20Pa, lyophilizing at-50 deg.C for 24 h.
Example 3
The method for producing fermented chickpeas with fibrinolytic and antioxidant functions by using bacillus subtilis DC-Tx comprises the following steps:
(1) selecting bacillus subtilis DC-Tx, inoculating the bacillus subtilis DC-Tx in an LB liquid seed culture medium, and culturing for 12 hours at the constant temperature of 34 ℃ in a shaking table at 120rpm to obtain bacillus subtilis DC-Tx seed liquid;
(2) soaking chickpeas overnight, draining water, autoclaving at 121 ℃ for 20min, cooling to room temperature, inoculating Bacillus subtilis DC-Tx seed solution according to the inoculation amount of 5%, and fermenting and culturing at 34 ℃ for 48 h;
(3) freezing and drying the chickpea fermentation culture product obtained in the step (2) at low temperature in vacuum, crushing, and sieving with a 80-mesh sieve to obtain the chickpea fermentation culture product; the low-temperature vacuum freeze drying conditions are as follows: pre-cooling at-50 deg.C under vacuum degree of 60Pa, lyophilizing at-55 deg.C for 48 h.
Example 4
The method for producing fermented chickpeas with fibrinolytic and antioxidant functions by using bacillus subtilis DC-Tx comprises the following steps:
(1) selecting bacillus subtilis DC-Tx, inoculating the bacillus subtilis DC-Tx in an LB liquid seed culture medium, and culturing for 18h at 40 ℃ in a shaking table at 150rpm to obtain bacillus subtilis DC-Tx seed liquid;
(2) soaking chickpeas overnight, draining water, autoclaving at 121 ℃ for 20min, cooling to room temperature, inoculating bacillus subtilis DC-Tx seed solution according to the inoculation amount of 1%, and fermenting and culturing at 40 ℃ for 72 h;
(3) freezing and drying the chickpea fermentation culture product obtained in the step (2) at low temperature in vacuum, crushing, and sieving with a 120-mesh sieve to obtain the chickpea fermentation culture product; the low-temperature vacuum freeze drying conditions are as follows: pre-cooling at-50 deg.C under vacuum degree of 100Pa, lyophilizing at-45 deg.C for 36 h.
Experimental example, measurement of fibrinolysis and antioxidant function of fermented chickpea of the invention
Preparation of a sample: preparing 2% mass fraction of fermented chickpea powder normal saline suspension, leaching at 4 ℃ for 4h, centrifuging at 5000 Xg for 10min, repeatedly centrifuging the obtained supernatant at 15000 Xg for 10min, and measuring fibrinolysis and antioxidant functions of the supernatant.
1. Method for measuring fibrinolytic activity of fermented chickpeas by using vascular placard method
Preparation of fibrin plate: dissolving 1g agarose in 100mL sodium phosphate buffer solution with 0.01M pH7.4, taking 7.5mL agarose solution, preserving heat in 50 ℃ water bath for 5min, adding 225 μ L thrombin with 100BP/mL thrombin dissolved in normal saline, mixing, preserving heat in 50 ℃ water bath for 5min, taking 7.5mL bovine fibrinogen solution with 3.6mg/mL (dissolved in 0.1M pH7.4 sodium phosphate buffer solution), adding into the heat-preserved thrombin-agarose solution, quickly mixing, pouring into a culture dish with the diameter of 9cm, and waiting for coagulation.
Drawing a standard curve: respectively preparing 20, 40, 60, 80 and 100IU/mL urokinase standard solutions by using sterilized normal saline, spotting 10 microliter of the urokinase standard solutions on a fibrin plate, preserving the temperature at 37 ℃ for 18 hours, measuring the diameter of a lysis ring on the fibrin plate (see figure 1), and calculating the area of the lysis ring by taking the average diameter of three tests; urokinase activity (IU/mL) is taken as an abscissa, and the area of a lysis ring is taken as an ordinate, and a urokinase standard curve is drawn.
Determination of fibrinolytic Activity: transferring 10 μ L of fermented semen Ciceris Arietini powder leaching solution with a microsyringe, spotting on a fibrin plate, keeping the temperature at 7 deg.C for 18h, measuring the diameter of dissolving ring on the fibrin plate, and calculating the area of the dissolving ring. Fibrinolytic activity was calculated according to the standard curve.
The measurement result shows that before 48 hours, the fibrinolytic activity is obviously increased along with the prolonging of the fermentation time, after 48 hours, the fibrinolytic activity is basically unchanged along with the prolonging of the fermentation time, and the fibrinolytic activity of the fermentation product can reach 3000FU/g when the fermentation time is 48 hours.
2. Determination of OH scavenging ability of fermented chick pea
Adding 50 μ L of 3mM 1,10 phenanthroline (dissolved in 0.1M sodium phosphate buffer solution of pH 7.4) into 96-well plate, adding 50 μ L of sample, mixing, adding 50 μ L of FeSO4(3mM) water solution, mixing well, adding 50 μ L hydrogen peroxide (H) with mass fraction of 0.01%2O2) The sample group (A1) was obtained by measuring the absorbance at 536nm of the aqueous solution (A) at 37 ℃ for 60 minutes with a lid closed, Control replaced the sample with distilled water (A0), Blank replaced the sample with distilled water and 0.01% hydrogen peroxide (A2).
The sample clearance for OH was calculated using the following formula: OH clearance (%) [ (a1-a0)/(a2-a0) ] x 100%
As a result, the OH clearance of the chickpeas fermented for 48h reaches 88.5%, which is nearly twice of the Glutathione (GSH) clearance OH capacity (47.7%), and is significantly higher than the OH clearance (52.3%) of the fresh chickpeas (the measurement result is shown in figure 2), which indicates that the fermented chickpeas obtained by the fermentation method have strong OH clearance capacity.
3. Reduction of Fe by fermented chickpeas3+Determination of Capacity
Mixing 250 μ L of sample with 250 μ L of potassium ferricyanide aqueous solution with mass fraction of 1% (without sample in control), heating in 50 deg.C constant temperature water bath for 20min, adding 250 μ L of trichloroacetic acid aqueous solution with mass fraction of 10%, mixing, centrifuging at 5000 × g for 10min, collecting 100 μ L of supernatant, adding into 96-well plate, adding 20 μ L of FeCl with mass fraction of 0.1%3Adding 80 μ L distilled water into the water solution, mixing, standing the mixture at 25 deg.C for 10min, and measuring absorbance of the supernatant at 700 nm.
The measurement result shows that the fermented chickpea Fe3+The reduction capacity is strongest when the fermentation is carried out for 72 hours, and OD700nmA value of 0.40, although it is OD relative to GSH700nmThe value (1.57) is greatly different, but is fresh chickpea Fe3+Reducing ability (OD)700nmHas a value of0.12) of the fermented chickpeas, indicating that the fermented chickpeas have stronger Fe3+Reducing power (see FIG. 3).
The experimental result shows that the fermented chickpea has very high fibrinolytic activity and strong antioxidant capacity. The invention provides a new way for the large-scale production of the fermented chickpeas and provides a new idea for the development, application and research of the chickpeas as functional foods and health-care foods.
Claims (5)
1. The Bacillus subtilis for producing fermented chickpeas with fibrinolysis and antioxidant functions is Bacillus subtilis DC-Tx (Bacillus subtilis), and the preservation unit is as follows: china general microbiological culture Collection center, preservation Address: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, on Beijing, with a registration number: CGMCC NO.9462, preservation date: 7, month and 15 days 2014.
2. Use of the bacillus subtilis of claim 1 for the production of fermented chickpeas having fibrinolytic and antioxidant properties.
3. A method for producing fermented chickpeas with fibrinolytic and antioxidant functions using bacillus subtilis according to claim 1, comprising the steps of:
(1) selecting bacillus subtilis, inoculating the bacillus subtilis to an LB liquid seed culture medium, and culturing for 12-18h at the temperature of 28-40 ℃ by using a 120-plus 180rpm shaking table at constant temperature to obtain a bacillus subtilis seed liquid;
(2) soaking chickpeas overnight, draining water, autoclaving, cooling to room temperature, inoculating Bacillus subtilis seed solution according to the inoculation amount of 1-8%, and fermenting and culturing at 28-40 deg.C for 36-72 h;
(3) and (3) freezing and drying the fermentation culture product of the chickpeas in the step (2) at a low temperature in vacuum, crushing, and sieving with a sieve of 80-120 meshes to obtain the chickpeas.
4. The method for producing fermented chickpeas with fibrinolytic and antioxidant functions by using the bacillus subtilis as claimed in claim 3, wherein the LB liquid seed culture medium comprises the following raw materials in percentage by mass: peptone 1%, yeast powder 0.5%, NaCl 1%; sterilizing at 121 deg.C for 20min at pH 6.8.
5. The method for producing fermented chickpeas with fibrinolytic and antioxidant functions by using bacillus subtilis as claimed in claim 3, wherein the conditions of low-temperature vacuum freeze-drying are as follows: pre-cooling at-50 deg.C under vacuum degree of 20-100Pa, lyophilizing at a temperature of no more than-45 deg.C, and lyophilizing for 24-48 h.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 | A strain of Bacillus velesi MRS with improved reducing activity and its fermented product and application |
| 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 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| 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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| 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 |
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| 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 | A strain of Bacillus velesi MRS with improved reducing activity and its fermented product 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 |
| 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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