Antithrombotic microbial preparation and preparation method thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to an antithrombotic microbial preparation and a preparation method thereof. The preparation method of the microbial preparation comprises the following steps: (1) activating plant lactobacillus to obtain a seed solution; inoculating the seed solution into a fermentation culture medium for fermentation to obtain a fermentation solution; (2) centrifuging the fermentation liquid, collecting thallus precipitate, and resuspending by using a freeze-drying protective agent to obtain resuspension liquid; and (3) performing vacuum freeze drying on the resuspension to obtain freeze-dried powder which is the microbial preparation. By taking the microbial preparation disclosed by the invention, the total volume of thrombus can be reduced, the content of solid components in the thrombus can be reduced, the occurrence rate of the thrombus is reduced, and the microbial preparation has a relatively good effect of preventing the formation of the thrombus. And the microbial preparation is safe and non-toxic, is convenient to take and has a wide market prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to an anti-thrombus microbial preparation and a preparation method thereof. BACKGROUND

[0002] Thrombosis is the pathological basis of common diseases such as ischemic heart disease, ischemic stroke and venous embolism. Therefore, anti-thrombus treatment is crucial for preventing and treating such diseases. The current main strategies include inhibiting the activation and aggregation of platelets, inhibiting the generation and activity of thrombin (i.e. anticoagulation), and activating the fibrinolytic system to promote thrombus dissolution.

[0003] The drugs currently used in clinical treatment of thrombotic diseases are mainly divided into three categories: anti-platelet drugs, anticoagulant drugs and thrombolytic drugs. Existing thrombolytic drugs generally have short half-lives, low fibrin specificity, easy bleeding side effects and high prices. Therefore, the development of new, efficient and safe thrombolytic drugs has broad application prospects. In recent years, active substances from microorganisms have shown great potential for anti-thrombus. Related studies have shown that the fermentation supernatant of Acetomicrobium and S. saprophyticus strain exhibits obvious thrombolytic effect in vitro and can effectively inhibit thrombosis in mice. In the prevention and treatment of thrombotic diseases (such as pulmonary embolism), microorganisms with anti-inflammatory and immunomodulatory functions are selected as auxiliary treatment candidates, and microbial preparations are prepared through fermentation, freeze-drying and other technologies, aiming to improve the microenvironment of thrombosis by regulating the immune state of the body.

[0004] Based on the above purpose, the present application provides an anti-thrombus microbial preparation and a preparation method thereof. SUMMARY

[0005] The first object of the present application is to provide a preparation method of an anti-thrombus microbial preparation.

[0006] The second object of the present application is to provide an anti-thrombus microbial preparation.

[0007] In order to achieve the above objects, the technical solution adopted by the present application is as follows: A preparation method of an anti-thrombus microbial preparation, the preparation method of the microbial preparation comprising the following steps: (1) Activating Lactobacillus plantarum to obtain a seed liquid; inoculating the seed liquid into a fermentation medium to obtain a fermentation liquid; the concentrations of the components in the fermentation medium are as follows: glucose 14-25 mg / mL, peptone 8-15 mg / mL, yeast extract 3.5-5.0 mg / mL, diammonium hydrogen citrate 2.0-3.2 mg / mL, ethyl-β-D-fructofuranoside 0.1-0.85 mg / mL, sodium acetate 3.4-6.0 mg / mL, magnesium sulfate 0.4-0.8 mg / mL, manganese sulfate 0.1-0.5 mg / mL, potassium dihydrogen phosphate 3.0-4.2 mg / mL, and Tween-80 0.5-2.0 mg / mL; the viable cell count of the seed liquid is ≥1×10 9 CFU / mL; (2) Centrifuging the fermentation liquid obtained in step (1) to collect a bacterial pellet, and resuspending the bacterial pellet with a freeze-drying protective agent to obtain a resuspension liquid; the freeze-drying protective agent comprises rutin ketose and L-glutamic acid sodium. (3) Vacuum freeze-drying the resuspension liquid obtained in step (2) to obtain a microbial preparation.

[0008] Preferably, the concentrations of the components in the fermentation medium of step (1) are as follows: glucose 19 mg / mL, peptone 11 mg / mL, yeast extract 4.2 mg / mL, diammonium hydrogen citrate 2.6 mg / mL, ethyl-β-D-fructofuranoside 0.45 mg / mL, sodium acetate 4.7 mg / mL, magnesium sulfate 0.6 mg / mL, manganese sulfate 0.3 mg / mL, potassium dihydrogen phosphate 3.6 mg / mL, and Tween-80 1.2 mg / mL.

[0009] Preferably, the specific steps of step (1) are as follows: inoculating Lactobacillus plantarum into MRS liquid medium at an inoculation amount of 2-4 v / v%, and culturing at 35-39°C for 10-18 h to obtain a seed liquid; inoculating the seed liquid into a fermentation medium at an inoculation amount of 3-5 v / v%, and culturing at 35-39°C for 35-40 h to obtain a fermentation liquid.

[0010] Preferably, the concentration of rutin ketose in the freeze-drying protective agent of step (2) is 10-15 g / L, and the concentration of L-glutamic acid sodium is 4.2-8.0 g / L.

[0011] Preferably, the mass ratio of the freeze-drying protective agent to the bacterial pellet in step (2) is (1-3):1; the centrifugation speed is 6000-8000 r / min, and the centrifugation time is 6-8 min.

[0012] An antithrombotic microbial preparation, which is prepared according to the preparation method of the antithrombotic microbial preparation described above.

[0013] Compared with the prior art, the present application has the following beneficial effects: The present application provides a preparation method of an anti-thrombus microbial preparation, by adding ethyl-beta-D-fructofuranoside in the fermentation medium and adding rutin ketose in the freeze-drying protective agent, the effective viable cell number of the Lactobacillus plantarum strain NCU0011190 in the microbial preparation is improved, and the death of the microbial body in the freeze-drying process is reduced. The test results show that by taking the microbial preparation of the present application, the total volume of thrombus can be reduced, the content of solid components in the thrombus can also be reduced, the incidence of thrombus can be reduced, and the effect of preventing thrombus formation is good. Moreover, the microbial preparation is safe, non-toxic, convenient to take, and has a broad market prospect. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 The figure is the detection result graph of the influence of the microbial preparation of different groups on the wet weight of thrombus in rats; Figure 2 The figure is the detection result graph of the influence of the microbial preparation of different groups on the dry weight of thrombus in rats; Figure 3 The figure is the detection result graph of the influence of the microbial preparation of different groups on the incidence of thrombus in rats. DETAILED DESCRIPTION

[0015] The technical solutions of the present application are further described below in combination with specific embodiments. However, those skilled in the art should understand that the following examples are only used to illustrate the present application, and should not be regarded as limiting the present application. The specific conditions not mentioned in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, such as those not specifically mentioned, are conventional products obtained through market channels.

[0016] The Lactobacillus plantarum strain NCU0011190 used in the embodiments of the present application is from the State Key Laboratory of Nanchang University, No. 235, Nanjing Road, Qingshanhu Campus, Nanchang, Jiangxi Province, China, and is preserved in the China General Microbiological Culture Collection Center, with the preservation number of CGMCC No. 33046, and the preservation date of December 12, 2024. Lactiplantibacillus plantarum

[0017] Example 1 A preparation method of an anti-thrombus microbial preparation includes the following steps: (1) inoculate the Lactobacillus plantarum NCU0011190 into the MRS liquid medium at an inoculation amount of 3 v / v %, and culture at 37℃ for 14 h to obtain a seed liquid; the viable cell number of the seed liquid is ≥1×10 9 CFU / mL.

[0018] ​(2) Take glucose, proteose peptone, yeast extract, diammonium hydrogen citrate, ethyl-β-D-fructofuranoside, sodium acetate, magnesium sulfate, manganese sulfate, potassium dihydrogen phosphate, Tween-80, and dissolve them in deionized water according to their respective concentrations, mix well, adjust pH to 6.0, sterilize at 115°C for 30 min to obtain a fermentation medium. Among them, the final concentrations of glucose, proteose peptone, yeast extract, diammonium hydrogen citrate, ethyl-β-D-fructofuranoside, sodium acetate, magnesium sulfate, manganese sulfate, potassium dihydrogen phosphate, and Tween-80 in the fermentation medium are 19 mg / mL, 11 mg / mL, 4.2 mg / mL, 2.6 mg / mL, 0.45 mg / mL, 4.7 mg / mL, 0.6 mg / mL, 0.3 mg / mL, 3.6 mg / mL, and 1.2 mg / mL, respectively. Seed liquid is inoculated into the fermentation medium at a inoculation amount of 4 v / v %, and fermented at 37°C for 37 h to obtain a fermentation broth.

[0019] (3) The fermentation broth obtained in step (2) is centrifuged at a speed of 7000 r / min for 7 min, and the bacterial precipitate is resuspended with a freeze-drying protective agent to obtain a resuspension. The mass ratio of the freeze-drying protective agent to the bacterial precipitate is 2:1; the concentration of rutin ketosugar in the freeze-drying protective agent is 12 g / L, and the concentration of L-glutamic acid sodium is 6.1 g / L, and the solvent is water.

[0020] (4) The resuspension obtained in step (3) is vacuum freeze-dried to obtain a microbial preparation.

[0021] Example 2 A method for preparing an anti-thrombotic microbial preparation includes the following steps: (1) Bacillus plantarum NCU0011190 is inoculated into MRS liquid medium at a inoculation amount of 2 v / v %, and cultured at 35°C for 10 h to obtain a seed liquid; the viable bacterial number of the seed liquid is ≥1×10 9 CFU / mL.

[0022] (2) Take glucose, proteose peptone, yeast extract, diammonium hydrogen citrate, ethyl-β-D-fructofuranoside, sodium acetate, magnesium sulfate, manganese sulfate, potassium dihydrogen phosphate, Tween-80, and dissolve them in deionized water according to their respective concentrations, mix well, adjust pH to 6.0, sterilize at 115°C for 30 min to obtain a fermentation medium. Among them, the final concentrations of glucose, proteose peptone, yeast extract, diammonium hydrogen citrate, ethyl-β-D-fructofuranoside, sodium acetate, magnesium sulfate, manganese sulfate, potassium dihydrogen phosphate, and Tween-80 in the fermentation medium are 14 mg / mL, 8 mg / mL, 3.5 mg / mL, 2.0 mg / mL, 0.1 mg / mL, 3.4 mg / mL, 0.4 mg / mL, 0.1 mg / mL, 3.0 mg / mL, and 0.5 mg / mL, respectively. Seed liquid is inoculated into the fermentation medium at a 3 v / v % inoculation amount, and fermented at 35°C for 35 h to obtain a fermentation broth.

[0023] (3) The fermentation broth obtained in step (2) is centrifuged at a speed of 6000 r / min for 6 min, and the bacterial precipitate is resuspended with a freeze-drying protective agent to obtain a resuspension. The mass ratio of the freeze-drying protective agent to the bacterial precipitate is 1:1; the concentration of rutin ketosugar in the freeze-drying protective agent is 10 g / L, and the concentration of L-glutamic acid sodium is 4.2 g / L, and the solvent is water.

[0024] (4) The resuspension obtained in step (3) is vacuum freeze-dried to obtain a microbial preparation.

[0025] Example 3 A method for preparing an antithrombotic microbial preparation includes the following steps: (1) Bacillus lactis NCU0011190 is inoculated into MRS liquid medium at a 4 v / v % inoculation amount, and cultured at 39°C for 18 h to obtain a seed liquid; the viable bacterial number of the seed liquid is ≥1×10 9 CFU / mL.

[0026] (2) Take glucose, proteose peptone, yeast extract, diammonium hydrogen citrate, ethyl-β-D-fructofuranoside, sodium acetate, magnesium sulfate, manganese sulfate, potassium dihydrogen phosphate, Tween-80, according to the respective concentration of use, fully dissolved in deionized water, mixed evenly, adjust pH to 6.0, sterilize at 115℃ for 30min to obtain the fermentation medium. Among them, the final concentration of glucose in the fermentation medium is 25 mg / mL, proteose peptone is 15 mg / mL, yeast extract is 5.0 mg / mL, diammonium hydrogen citrate is 3.2 mg / mL, ethyl-β-D-fructofuranoside is 0.85 mg / mL, sodium acetate is 6.0 mg / mL, magnesium sulfate is 0.8 mg / mL, manganese sulfate is 0.5 mg / mL, potassium dihydrogen phosphate is 4.2 mg / mL, and Tween-80 is 2.0 mg / mL. The seed liquid is inoculated into the fermentation medium at a inoculation amount of 5v / v%, and fermented at 39℃ for 40h to obtain the fermentation broth.

[0027] (3) The fermentation broth obtained in step (2) is centrifuged at a speed of 8000 r / min for 8 min, and the bacterial precipitate is resuspended with a freeze-drying protective agent to obtain a resuspension. The mass ratio of the freeze-drying protective agent to the bacterial precipitate is 3:1; the concentration of rutin ketosugar in the freeze-drying protective agent is 15g / L, and the concentration of L-glutamic acid sodium is 8.0g / L, and the solvent is water.

[0028] (4) The resuspension obtained in step (3) is vacuum freeze-dried to obtain a freeze-dried powder, which is the microbial preparation.

[0029] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that ethyl-β-D-fructofuranoside is omitted from the fermentation medium ingredients prepared in step (2) of Example 1, and the others are the same as Example 1.

[0030] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that rutin ketosugar is omitted from the freeze-drying protective agent ingredients, and the others are the same as Example 1.

[0031] Test Example 1 Effect of different medium ingredients on the viable cell number of Lactiplantibacillus plantarum NCU0011190 in the fermentation broth: In order to explore the viable cell number of Lactiplantibacillus plantarum NCU0011190 in the fermentation broth prepared in step (2) of Examples 1-3 and Comparative Example 1, we collected 1mL of the fermentation broth prepared in step (2) of Examples 1-3 and Comparative Example 1, and placed it in 9mL of sterile normal saline, and then diluted it by 10 times, and then diluted it to the required gradient. The effective viable cell number of Lactiplantibacillus plantarum NCU0011190 in the fermentation broth of different groups was determined by spread plate counting method, and the results are shown in Table 1.

[0032] Table 1 Determination of viable cell number of Bacillus velezensis NCU0011190 Group Total viable count (10 10 CFU / mL) Example 1 1.89 Example 2 1.87 Example 3 1.85 Comparative Example 1 1.46 The results are shown in Table 1, which are the effects of different medium components on the viable cell number of Bacillus velezensis NCU0011190 in the fermentation broth. As can be seen from Table 1, the total number of effective viable cells in the microbial preparation prepared by Examples 1-3 is higher than that of Comparative Example 1, and the total number of viable cells in the fermentation broth prepared by Example 1 is the highest, which is 1.89×10 10 CFU / mL. This shows that the ethyl-β-D-fructofuranoside component in the fermentation medium plays a very important role in fermentation, which can significantly improve the viable cell number of Bacillus velezensis NCU0011190 in the microbial preparation.

[0033] Test Example 2 Effect of freeze-drying protectant on strain survival rate: In order to explore the effect of the freeze-drying protectant prepared in step (3) of Examples 1-3 and Comparative Examples 1-2 on the survival rate of the strain, we took 1 mL of the resuspension prepared in step (3) of Examples 1-3 and Comparative Examples 1-2, and determined the viable cell number by plate colony counting method, which is recorded as the viable cell number before freeze-drying; the freeze-dried powder obtained after freeze-drying was dissolved with sterile distilled water, and the viable cell number was determined by plate colony counting method, which is recorded as the viable cell number after freeze-drying. The survival rate during freeze-drying was calculated according to the following formula: survival rate (%) = (viable cell number after freeze-drying / viable cell number before freeze-drying) × 100 , and the results are shown in Table 2.

[0034] Table 2 Effect of freeze-drying protectant on strain freeze-drying survival rate Group Freeze-dried survival rate (%) Example 1 91.3 Example 2 91.1 Example 3 89.7 Comparative Example 1 88.4 Comparative Example 2 75.2 The results are shown in Table 2, which are the effects of freeze-drying protectant on strain freeze-drying survival rate. As can be seen from the table, compared with Comparative Example 2, the freeze-drying protectant of Example 1-3 can significantly improve the strain freeze-drying survival rate, indicating that rutin ketose can improve the survival rate of the strain in the freeze-drying process.

[0035] Test Example 3 In vivo antithrombotic effect study of an antithrombotic microbial preparation The test selects 70 male SD rats with a body weight of 160-180 g. After adaptive feeding for 1 week, the rats are randomly divided into Example 1 group, Example 2 group, Example 3 group, Comparative Example 1 group, Comparative Example 2 group, model group, and positive control group, 10 rats in each group. The positive control group is given aspirin (100 mg / kg of body weight) by gavage. The model group is given normal saline (20 mL / kg of body weight) by gavage. 1 g of the microbial agent prepared in Examples 1-3 and Comparative Examples 1-2 is dissolved in 600 mL of normal saline to prepare a microbial agent solution; the Example 1-3 groups and Comparative Example 1-2 groups are given the microbial agent solution (20 mL / kg of body weight) of the corresponding group by gavage. After 2 weeks of administration, the rats are anesthetized with sodium pentobarbital, and fixed in a supine position. About 3 cm of skin is incised along the median line of the abdomen, the abdominal cavity is opened from the linea alba, the inferior vena cava is separated and ligated with a fine silk thread. Then the abdominal cavity is sutured. The rats are sacrificed after 4 hours of ligation, the abdominal cavity is opened, the blood vessel is clamped with an artery clamp 2 cm below the ligation, the blood in the lumen of the segment is sucked out, and the segment is cut longitudinally to observe whether thrombosis occurs. If thrombosis exists, the thrombus is taken out and weighed. Then the wet thrombus is placed in a 60℃ oven for 24 hours, and the dry weight is weighed again; and the incidence of thrombosis is calculated.

[0036] The results are shown in Figure 1 and Figure 2 , which are the detection results of the effects of the microbial agents of different groups on the wet weight and dry weight of thrombus in rats. The wet weight of thrombus reflects the total volume and water content of thrombus; the dry weight of thrombus reflects the content of solid components in thrombus. As can be seen from Figure 1 and Figure 2 , compared with Comparative Example 1, Comparative Example 2, and the model group, the anti-thrombotic microbial agent prepared in Example 1-3 of the application can significantly reduce the wet weight and dry weight of thrombus. This indicates that the microbial agent not only reduces the total volume of thrombus, but also reduces the content of solid components in thrombus, thereby significantly reducing the probability of thrombosis.

[0037] The results are shown in Figure 3 , which is the detection results of the effects of the microbial agents of different groups on the incidence of thrombus in rats. The incidence of thrombus can directly reflect the degree of tendency of blood coagulation to form thrombus in the blood vessels of rats. As can be seen from Figure 3 , compared with Comparative Example 1, Comparative Example 2, and the model group, the anti-thrombotic microbial agent prepared in Example 1-3 of the application can significantly reduce the incidence of thrombus, thereby reducing the probability of thrombosis. Therefore, the microbial agent has a good effect on preventing thrombosis.

[0038] Finally, it should be pointed out that the above embodiments are only used to illustrate the technical solutions of the present application, and are not intended to limit the present application. The basic principles and main features of the present application have been described above with specific embodiments, and some modifications or replacements can be made on the basis of the present application, but these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the present application.

Claims

1. A method for preparing an antithrombotic microbial preparation, characterized by, The preparation method of the microbial preparation comprises the following steps: (1) the Lactobacillus plantarum is activated to obtain a seed liquid; the seed liquid is inoculated into a fermentation medium to carry out fermentation to obtain a fermentation liquor; the concentration of each component in the fermentation medium is as follows: glucose 14-25 mg / mL, peptone 8-15 mg / mL, yeast extract 3.5-5.0 mg / mL, diammonium hydrogen citrate 2.0-3.2 mg / mL, ethyl-beta-D-fructofuranoside 0.1-0.85 mg / mL, sodium acetate 3.4-6.0 mg / mL, magnesium sulfate 0.4-0.8 mg / mL, manganese sulfate 0.1-0.5 mg / mL, potassium dihydrogen phosphate 3.0-4.2 mg / mL, Tween-80 0.5-2.0 mg / mL; the viable bacterial number of the seed liquid is ≥1×10 9 CFU / mL; (2) centrifuging the fermentation liquor obtained in step (1) to collect the bacterial precipitate, and resuspending the bacterial precipitate with a freeze-drying protective agent to obtain a resuspension; the freeze-drying protective agent comprises rutin ketose and L-glutamic acid sodium; (3) vacuum freeze-drying the resuspension obtained in step (2) to obtain the microbial preparation.

2. The method of claim 1, wherein the anti-thrombotic microbial preparation is prepared by the steps of: The concentration of each component in the fermentation medium in step (1) is as follows: glucose 19 mg / mL, protein peptone 11 mg / mL, yeast extract 4.2 mg / mL, diammonium hydrogen citrate 2.6 mg / mL, ethyl-beta-D-fructofuranoside 0.45 mg / mL, sodium acetate 4.7 mg / mL, magnesium sulfate 0.6 mg / mL, manganese sulfate 0.3 mg / mL, potassium dihydrogen phosphate 3.6 mg / mL, and Tween-80 1.2 mg / mL.

3. The method of claim 1, wherein the anti-thrombotic microbial preparation is prepared by the steps of: The specific steps of step (1) are as follows: inoculating Lactobacillus plantarum in a MRS liquid medium at an inoculation amount of 2-4 v / v%, and culturing at 35-39 DEG C for 10-18 h to obtain a seed liquid; inoculating the seed liquid in a fermentation medium at an inoculation amount of 3-5 v / v%, and fermenting at 35-39 DEG C for 35-40 h to obtain a fermentation liquor.

4. The method of claim 1, wherein the anti-thrombotic microbial preparation is prepared by the steps of: The concentration of rutin ketose in the freeze-drying protective agent in step (2) is 10-15 g / L, and the concentration of L-glutamic acid sodium is 4.2-8.0 g / L.

5. The method for preparing an antithrombotic microbial preparation according to claim 1, characterized in that, The mass ratio of the freeze-drying protective agent to the bacterial precipitate in step (2) is (1-3):1; the centrifugation speed is 6000-8000 r / min, and the centrifugation time is 6-8 min.

6. An antithrombotic microbial preparation, characterized by, The microbial preparation is prepared according to the preparation method of the antithrombotic microbial preparation in any one of claims 1-5.

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