Phytobacterium plantarum NCU0011190 with thrombus prevention effect and application of phytobacterium plantarum NCU0011190

Abstract

The invention provides a plant lactobacillus NCU0011190 with a thrombus prevention effect and application of the plant lactobacillus NCU0011190, and relates to the technical field of microorganisms. The preservation number of the bacterial strain NCU0011190 of the plant lactobacillus is CGMCC (China General Microbiological Culture Collection Center) No.33046. According to the present invention, the research results show that the plant lactobacillus strain NCU0011190 has characteristics of good digestion tolerance, high survival rate in the artificial gastrointestinal fluid, and platelet aggregation resistance, and the plant lactobacillus strain NCU0011190 has characteristics of platelet aggregation resistance. Besides, the plant lactobacillus strain NCU0011190 disclosed by the invention can effectively reduce sensitization and toxicity components in ginkgo puree through a fermentation degradation method, a fermented ginkgo fermentation product has a good antithrombotic effect, and the fermentation of the NCU0011190 not only maintains the original functional activity of ginkgo, but also has a certain synergistic effect.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to Lactobacillus plantarum NCU0011190 with thrombosis prevention function and application thereof. Background Art

[0002] In modern society, thrombosis in human blood vessels will lead to serious cardiovascular diseases such as cerebral infarction and myocardial infarction, which will cause great harm to the health of middle-aged and elderly people. Therefore, it is of great significance to study safe and effective anticoagulant and antithrombotic drugs suitable for long-term oral administration.

[0003] At the same time, ginkgo nuts are rich in nutrients and functional ingredients. In addition to starch, protein, fat, and sugar, they also contain a certain amount of vitamins, riboflavin, carotene, flavonoids, terpene lactones, alkaloids, polysaccharides, amino acids, ginkgolic acid, ginkgo biloba phenol, sterols, and trace elements such as calcium, phosphorus, iron, potassium, and magnesium. The nutrients and functional ingredients contained in ginkgo nuts have many medical and health effects on the human body. However, ginkgo nuts contain certain allergenic and toxic ingredients, which greatly restricts the development of the ginkgo food processing industry.

[0004] Therefore, it is necessary and urgent to research and develop a safe and effective microbial agent, which not only has anti-thrombotic effect, but also can reduce the allergenic and toxic components of ginkgo by fermentation degradation and improve the anti-thrombotic effect of fermented ginkgo products.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The object of the present invention is to provide a plant lactobacillus ( Lactiplantibacillus plantarum ) strain NCU0011190, which not only has anti-thrombotic effects, but also can reduce the allergenic and toxic components of ginkgo through fermentation degradation and improve the anti-thrombotic effect of ginkgo fermentation products after fermentation.

[0007] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted: The present invention provides a plant lactobacillus ( Lactiplantibacillus plantarum ) strain NCU0011190, the plant lactobacillus strain was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on December 12, 2024, and was classified and named plant lactobacillus Lactiplantibacillus plantaru m, the deposit number is CGMCC No.33046.

[0008] The present invention provides a microbial agent, which comprises the above-mentioned Lactobacillus plantarum strain NCU0011190 and its metabolites.

[0009] The present invention provides an application of the plant lactobacillus strain NCU0011190 or the microbial agent in fermentation and degradation of ginkgo allergenic and toxic components.

[0010] The present invention provides a method for fermenting and degrading allergenic and toxic components of ginkgo by using the Lactobacillus plantarum strain NCU0011190, the method comprising: (A) Preparation of puree: fresh ginkgo nuts are shelled, and water is added in a mass ratio of 1:5 (m / m) and the mixture is pulped to obtain ginkgo nut puree.

[0011] (B) Fermentation: Lactobacillus plantarum NCU0011190 was activated using MRS liquid culture medium. The ginkgo puree was sterilized at 85°C for 30 min and inoculated for fermentation after cooling. The inoculation amount of NCU0011190 activation liquid was 1-2%, the fermentation temperature was 37°C, and the fermentation time was 48 h.

[0012] The invention provides a ginkgo fermentation product prepared by the method.

[0013] Furthermore, the content of total ginkgolic acid in the ginkgo fermentation product is 0.7-2.0 μg / g.

[0014] The invention provides an application of the Lactobacillus plantarum strain NCU0011190, the microbial agent or the ginkgo fermentation product in the preparation of a drug for preventing and treating thrombosis.

[0015] The present invention provides a drug for preventing and treating thrombosis, the drug comprising Lactobacillus plantarum strain NCU0011190; and / or, the above-mentioned microbial agents; And / or, the above-mentioned ginkgo fermentation product.

[0016] Furthermore, the drug is a microbial agent, which is suitable for oral administration to mammals in a unit dose, and the amount of Lactobacillus plantarum strain NCU0011190 in the unit dose is 1.0×10 7 ~1.0×10 10 CFU / mL.

[0017] Furthermore, the medicine comprises a ginkgo fermentation product, which is suitable for oral administration to mammals in a unit dose, and the amount of the ginkgo fermentation product in the unit dose is 5 to 100 g.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a plant lactobacillus strain, wherein the plant lactobacillus strain NCU0011190 has been deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration Committee on December 12, 2024, with a deposit number of CGMCC No. 33046. The present application study verified that the plant lactobacillus strain NCU0011190 has good gastrointestinal digestion tolerance, a high survival rate after 3 h of digestion in simulated artificial gastric juice and intestinal juice, and the plant lactobacillus strain NCU0011190 also has an anti-thrombotic effect.

[0019] In addition, the plant lactobacillus strain NCU0011190 of the present application can effectively reduce the allergenic and toxic components in ginkgo puree through the fermentation degradation method, and the allergenic and toxicity of the ginkgo fermentation product is significantly reduced. At the same time, the ginkgo fermentation product after fermentation can not only maintain the original functional components in the anti-thrombotic effect, but also has a certain synergistic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1a A comparative diagram of the gastric juice tolerance and intestinal juice tolerance of the screening strains CH001 to CH021 provided in Example 1 of the present invention; Figure 1b A comparative diagram of the gastric juice tolerance and intestinal juice tolerance of the screening strains CH022 to CH042 provided in Example 1 of the present invention; Figure 1c A comparative diagram of the gastric juice tolerance and intestinal juice tolerance of the screening strains CH043 to CH063 provided in Example 1 of the present invention; Figure 2a A comparative diagram of the anti-platelet aggregation performance of the screening strains CH001-CH022 provided in Example 1 of the present invention; Figure 2b A comparative diagram of the anti-platelet aggregation performance of the screening strains CH023-CH044 provided in Example 1 of the present invention; Figure 2c A comparative diagram of the anti-platelet aggregation performance of the screening strains CH045-CH063 provided in Example 1 of the present invention; Figure 3a A comparative diagram of three coagulation tests of the screening strains CH001-CH021 provided in Example 1 of the present invention; Figure 3b A comparative diagram of three coagulation tests of the screening strains CH022 to CH042 provided in Example 1 of the present invention; Figure 3c A comparative diagram of three coagulation tests of the screening strains CH043-CH063 provided in Example 1 of the present invention; Figure 4 This is a cell adhesion test diagram of the strain NCU0011190 provided in Example 4 of the present invention; Figure 5 This is a bar graph comparing the effects of the strain NCU0011190, the model group and the positive control group provided in Example 5 of the present invention on the black tail rate of mice in the carrageenan-induced thrombosis model; Figure 6 This is an experimental comparison diagram of the effects of strain NCU0011190, model group and positive control group provided in Example 5 of the present invention on the black tail rate of mice in the carrageenan-induced thrombosis model; Figure 7 A comparison of histopathological HE staining of carrageenan thrombosis model mice by strain NCU0011190, model group and positive control group provided in Example 5 of the present invention; Figure 8a A bar graph comparing the serum inflammatory factor TNF-α content of carrageenan thrombosis model mice with the strain NCU0011190, the model group and the positive control group provided in Example 5 of the present invention; Figure 8b A bar graph comparing the serum inflammatory factor NF-κB content of carrageenan thrombosis model mice with the strain NCU0011190, the model group and the positive control group provided in Example 5 of the present invention; Figure 8c A bar graph comparing the serum inflammatory factor IL-6 content of carrageenan thrombosis model mice with the strain NCU0011190, the model group and the positive control group provided in Example 5 of the present invention; Figure 8d A bar graph comparing the serum inflammatory factor IL-1β content of carrageenan thrombosis model mice with the strain NCU0011190, the model group and the positive control group provided in Example 5 of the present invention; Fig. 9 This is a comparison chart of the degradation effect of ginkgolic acid of the strain NCU0011190 provided in Example 6 of the present invention; Fig.10 A bar graph comparing the thrombus lengths of the ginkgo fermentation product obtained by fermentation and degradation of the strain NCU0011190 provided in Example 7 of the present invention and the model group and the positive control group; Fig.11A bar chart comparing the thrombus weight of the ginkgo fermentation product obtained by fermentation and degradation of the strain NCU0011190 provided in Example 7 of the present invention and the model group and the positive control group; Fig.12 This is a comparison diagram of the ginkgo fermentation product obtained by fermentation and degradation of the strain NCU0011190 provided in Example 7 of the present invention and the actual thrombus in the model group and the positive control group. DETAILED DESCRIPTION

[0022] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] According to one aspect of the present invention, a plant lactobacillus ( Lactiplantibacillus plantarum ) strain, the plant lactobacillus strain NCU0011190 has been deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on December 12, 2024, and is classified and named plant lactobacillus Lactiplantibacillus plantaru m, the deposit number is CGMCC No.33046.

[0024] The present application is to obtain a microbial strain that has both anti-thrombotic effects and can reduce the allergenic and toxic components of ginkgo nuts by fermentation degradation and improve the anti-thrombotic effect of fermented ginkgo nuts. In the study, it was found that: (I) Lactobacillus plantarum strain NCU0011190, which has good tolerance, high survival rate in artificial gastric juice, sustainable growth and reproduction in artificial intestinal juice, good anti-platelet aggregation effect in vitro, and can prolong the APTT time. The anti-thrombotic effect of the above-mentioned Lactobacillus plantarum strain NCU0011190 has been demonstrated through in vivo experiments.

[0025] (ii) The plant lactobacillus strain NCU0011190 can effectively reduce the allergenic and toxic components in ginkgo puree through fermentation and degradation. The allergenic and toxic effects of ginkgo fermentation products are significantly reduced. At the same time, the anti-thrombotic effect of ginkgo fermentation products after fermentation can not only maintain the original functional components, but also have a certain synergistic effect.

[0026] According to one aspect of the present invention, a microbial agent is provided, comprising Lactobacillus plantarum strain NCU0011190 and metabolites thereof.

[0027] In a preferred embodiment of the present invention, the microbial agent is a fermentation broth of Lactobacillus plantarum strain NCU0011190, and the live bacteria concentration of Lactobacillus plantarum strain NCU0011190 in the fermentation broth is 1.0×10 7 ~1.0×10 9 CFU / mL.

[0028] The Lactobacillus plantarum strain NCU0011190 or the microbial agent containing the same provided by the present invention can be widely used in the fermentation degradation of ginkgo allergenic and toxic components.

[0029] According to one aspect of the present invention, a method for fermenting and degrading allergenic and toxic components of ginkgo using Lactobacillus plantarum strain NCU0011190 comprises: (A) Preparation of puree: fresh ginkgo nuts are shelled, and water is added in a mass ratio of 1:5 (m / m) and the mixture is pulped to obtain ginkgo nut puree.

[0030] (B) Fermentation: Lactobacillus plantarum NCU0011190 was activated using MRS liquid culture medium. The ginkgo puree was sterilized at 85°C for 30 min and then inoculated for fermentation after cooling. The inoculation amount of NCU0011190 activation solution was 1-2%, the fermentation temperature was 37°C, and the fermentation time was 48 h.

[0031] According to one aspect of the present invention, the present invention provides a ginkgo fermentation product prepared by the method of fermenting and degrading the allergenic and toxic components of ginkgo using the above-mentioned Lactobacillus plantarum strain NCU0011190, wherein the content of total ginkgolic acid in the ginkgo fermentation product is 0.7-1.5 μg / g.

[0032] According to one aspect of the present invention, the Lactobacillus plantarum strain NCU0011190, the above-mentioned microbial agent or ginkgo fermentation product provided by the present invention can be widely used in the preparation process of drugs for preventing and treating thrombosis.

[0033] According to one aspect of the present invention, a drug for preventing and treating thrombosis comprises Lactobacillus plantarum strain NCU0011190; and / or, the above-mentioned microbial agents; And / or, the above-mentioned ginkgo fermentation product.

[0034] In a preferred embodiment of the present invention, the drug is a microbial agent, which is suitable for oral administration to mammals in a unit dose, and the amount of Lactobacillus plantarum strain NCU0011190 in the unit dose is 1.0×10 7 ~1.0×10 10 CFU / mL.

[0035] In a preferred embodiment of the present invention, the drug is a ginkgo fermentation product, which is suitable for oral administration to mammals in a unit dose, and the amount of the ginkgo fermentation product in the unit dose is 5 to 100 g.

[0036] The technical solution of the present invention will be further described below in conjunction with embodiments.

[0037] Example 1 Screening and Acquisition of Strain NCU0011190 1. Screening of strains Take naturally fermented kimchi samples and perform preliminary screening on plates, which are MRS bromocresol purple solid culture medium. The morphology and color of the colonies are observed by plating on the plates to obtain possible strains of lactic acid bacteria.

[0038] (II) In this example, the gastric juice tolerance and intestinal juice tolerance of the 63 lactic acid bacteria strains (CH001-CH063) obtained in the above preliminary screening were investigated.

[0039] The specific inspection methods are as follows: (1) Gastric juice tolerance test: 100 mL of culture medium was centrifuged at 3000 r / min for 10 min. After removing the supernatant, the culture medium was diluted and resuspended with 10 mL of glucose solution. The resuspended medium was inoculated into commercial artificial gastric juice at a volume fraction of 1% and mixed evenly. The culture was shaken at 37°C and 100 r / min. Samples were taken at 0 and 3 h for live lactic acid bacteria count.

[0040] (2) Intestinal fluid tolerance test: 100 mL of culture medium was centrifuged at 3000 r / min for 10 min. After removing the supernatant, the culture medium was diluted and resuspended with 10 mL of glucose solution. The resuspended medium was inoculated into commercial artificial intestinal fluid at a volume fraction of 1% and mixed evenly. The culture was shaken at 37°C and 100 r / min. Samples were taken at 0 and 3 h for live lactic acid bacteria count.

[0041] For detailed investigation results, see Figure 1a~Figure 1c .

[0042] Figure 1a This is a comparative diagram of the gastric juice tolerance and intestinal juice tolerance of the screening strains CH001~CH021 provided in this example.

[0043] Figure 1b This is a comparative diagram of the gastric juice tolerance and intestinal juice tolerance of the screening strains CH022~CH042 provided in this example.

[0044] Figure 1c This is a comparative diagram of the gastric juice tolerance and intestinal juice tolerance of the screening strains CH043~CH063 provided in this example.

[0045] Figure 1a~Figure 1c The dot-dash line in the figure is the standard line indicating 80% survival rate of the screened strains in resistance to intestinal juice digestion and gastric juice digestion. A value higher than 80% indicates that the strain has better tolerance.

[0046] Depend on Figure 1a~Figure 1c It can be seen that the gastric juice tolerance and intestinal juice digestion tolerance of strains 1, 3, 5, 10, 19, 23, 25, 31, 33, 39, 49, 50, 53, 54, 55, 62, and 63 are higher than 80% of the strains, indicating that these 17 strains have good tolerance to gastrointestinal digestion.

[0047] (III) In this example, three tests on anti-platelet aggregation and coagulation were conducted on the 63 lactic acid strains obtained from the preliminary screening.

[0048] The specific inspection methods are as follows: (1) Antiplatelet aggregation: Preparation of washed platelets in mice: 1) Eight male Kunming mice (18-22 g) were fasted the night before the experiment and allowed to drink water freely. They were anesthetized by intraperitoneal injection of 20% urethane (0.1 mL / 10 g), fixed on the operating table, cut open along the linea alba, carefully bypassed the peritoneal large blood vessels, fully exposed the inferior vena cava, and blood was slowly drawn from the inferior vena cava with a syringe containing 3.8% sodium citrate (sodium citrate solution: whole blood = 1:9).

[0049] 2) After the blood and sodium citrate are thoroughly mixed, add an equal volume of 1×Tyrodes Buffer and mix well. Add PGE1 working solution to make the final concentration of PGE1 50 ng / mL, mix gently, and combine the blood of the two mice into a 10 mL centrifuge tube.

[0050] 3) Centrifuge at 200×g for 16 min at room temperature, aspirate the pale white liquid on the upper layer, add PGE1 working solution to make the final concentration of PGE1 added this time to 50 ng / mL, and mix gently.

[0051] 4) Centrifuge at 800×g for 10 min at room temperature, discard the supernatant, add 2 mL 1×Tyrode Buffer, 8 μL ETA solution and 2 μL PGE1 solution, so that the final concentrations of the latter two are 1 mM and 50 ng / mL, respectively. Resuspend with a Pasteur pipette. Note that during this process, the blowing action should be as gentle as possible to avoid excessive activation of platelets due to excessive shear force; in addition, if there are still some red blood cells in the precipitate, discard the remaining red blood cells. Combine the platelet suspensions in each tube to a total volume of about 6 mL.

[0052] 5) Centrifuge at 750×g for 10 min at room temperature, discard the supernatant, and keep the precipitate. Add an appropriate amount of 1×TyrodeBuffer, and gently pipette with a Pasteur pipette to mix. The resuspended platelets are the washed platelets.

[0053] 6) Count the washed platelets using a blood cell analyzer. Adjust the washed platelets to the required concentration using 1×Tyrode Buffer according to the purpose of the experiment. Allow the platelets to stand at room temperature for about half an hour before starting the formal experiment. Platelet aggregation experiment: preheat the platelet aggregometer and adjust the speed to 1000 rpm; add the rotor to the platelet reaction cup, draw 250 μL of mouse platelet-rich plasma (3×108 cells / mL) into the reaction cup, then add CaCl2 (final concentration of 1 mM), add equal volumes of lactic acid bacteria culture centrifugal supernatant (adjust pH=7.0) or positive drug acetylsalicylic acid according to the group, incubate at 37°C for 5 min, after incubation, place the reaction cup into the platelet aggregometer, adjust the baseline, add ADP to induce platelet aggregation, and use software to record platelet aggregation. Stop recording when platelet aggregation reaches the highest aggregation rate, expressed as the highest aggregation rate.

[0054] For detailed investigation results, see Figure 2a~Figure 2c .

[0055] Figure 2a This is a comparative diagram of the anti-platelet aggregation performance of the screened strains CH001~CH022 provided in this example.

[0056] Figure 2b This is a comparative diagram of the anti-platelet aggregation performance of the screened strains CH023~CH044 provided in this example.

[0057] Figure 2c This is a comparative diagram of the anti-platelet aggregation performance of the screened strains CH045~CH063 provided in this example.

[0058] Figure 2a~Figure 2c The dotted line in the figure is the standard line using 5uM acetylsalicylic acid (positive control) as the judgment standard. The platelet aggregation rate data of 5uM acetylsalicylic acid is 31.7%.

[0059] This application Figure 2a~Figure 2c In the test, the platelet aggregation rate was 31.7% as the standard to judge the anti-platelet aggregation performance of the screened strains. Strains with a platelet aggregation rate lower than 31.7% showed better anti-platelet aggregation effect. Figure 2c The MRS in the sample was used as blank control.

[0060] Depend on Figure 2a~Figure 2cIt can be seen that the anti-platelet aggregation effect of strains 8, 22, 30, 37, 42, 48, 52, 55, 57, 59, 60, and 63 is better than that of the positive control 5 micromolar acetylsalicylic acid, indicating that these 12 strains have good anti-platelet aggregation potential.

[0061] (2) Three coagulation tests Prothrombin time (PT): Take 0.1 mL of plasma to be tested, add 10 uL of the supernatant of the centrifuged NCU0011190 bacterial solution, and incubate at 37°C for 30 min; add 0.2 mL of 37°C pre-warmed PT reagent to the above solution, and record the coagulation time with a coagulation analyzer.

[0062] Activated partial thromboplastin time (APTT): Take 0.1 mL of plasma to be tested, add 10 uL of NCU0011190 bacterial solution centrifuged supernatant, and incubate at 37°C for 30 min; add 0.1 mL of 37°C pre-warmed APTT reagent to the above solution and incubate at 37°C for 5 min; add 0.1 mL of 37°C pre-warmed 0.025 mmol / L CaCl2 solution and record the coagulation time with a coagulation analyzer.

[0063] Thrombin time (TT): Take 0.1 mL of plasma to be tested, add 10 uL of the supernatant of the centrifuged NCU0011190 bacterial solution, and incubate at 37°C for 30 min; add 0.1 mL of TT reagent to the above solution, and record the coagulation time with a coagulation analyzer.

[0064] Prolonged APTT indicates that the coagulation factors of the intrinsic coagulation pathway and the common coagulation pathway are inhibited, prolonged PT indicates that the coagulation factors of the extrinsic coagulation pathway and the common coagulation pathway are inhibited, and prolonged TT indicates that the common coagulation pathway is inhibited; For detailed investigation results, see Figure 3a~Figure 3c .

[0065] Figure 3a This is a comparative diagram of the three coagulation tests of the screening strains CH001~CH021 provided in this embodiment.

[0066] Figure 3b This is a comparative diagram of the three coagulation tests of the screening strains CH022~CH042 provided in this embodiment.

[0067] Figure 3c This is a comparative diagram of the three coagulation tests of the screening strains CH043~CH063 provided in this embodiment.

[0068] Depend on Figure 3a~Figure 3cIt can be seen that the fermentation supernatants of strains 61, 59, 29, 24, 55, 57, 34, 30, 23, and 48 can significantly prolong the APTT time compared with the blank control MRS; the fermentation supernatants of strains 15, 14, 16, 8, 41, 43, 42, and 63 can prolong the PT time compared with the blank control MRS; the fermentation supernatants of strains 24, 21, 4, 3, 18, 27, 33, 32, 28, and 43 can prolong the TT time compared with the blank control MRS. Since lactic acid bacteria fermentation broth mainly improves thrombotic diseases by inhibiting inflammatory response, and inflammatory factors mainly participate in thrombosis through the exogenous coagulation pathway, APTT is mainly used as a reference.

[0069] In summary, the screening results of gastrointestinal fluid tolerance, antiplatelet aggregation effect and prolonged APTT time were obtained.

[0070] Through screening research, the present application obtained that strain No. 55 (self-numbered: NCU0011190) has good tolerance to gastrointestinal fluid, with a survival rate of 106.25% after 3 hours in simulated artificial gastric fluid and a survival rate of 92.77% after 3 hours in artificial intestinal fluid; at the same time, the supernatant of strain No. 55 can significantly prolong the APTT time, so strain No. 55 (self-numbered: NCU0011190) was selected as an excellent anti-thrombotic lactic acid bacterium.

[0071] Example 2 16S Identification of Strain NCU0011190: The isolated strain (strain named NCU0011190) was subjected to 16S rRNA analysis to preliminarily determine its taxonomic status.

[0072] The genomic DNA of the strain was extracted, and the 16S rRNA was amplified using the extracted genomic DNA template. The amplification primers were: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3', the sequence of which is shown in SEQ ID NO: 2; 1492R: 5'-GGTTACCTTGTTACGACTT-3', the sequence is shown in SEQ ID NO: 3.

[0073] The nucleotide sequence shown in SEQ ID NO:2 is as follows: AGAGTTTGATCCTGGCTCAG.

[0074] The nucleotide sequence shown in SEQ ID NO:3 is as follows: GGTTACCTTGTTACGACTT. The amplified PCR product was purified and then sequenced by ABI3730XL to obtain a 954 bp 16S rRNA sequence. The 16S RNA sequence of the strain NCU0011190 is shown in SEQ ID NO:1.

[0075]

[0076] The 16S RNA sequence of strain NCU0011190 was compared with the NCBI 16S ribosomal RNA sequences database, and the closest species was Lactobacillus plantarum ( Lactiplantibacillus plantarum ), the similarity is 99.93%, and the species classification information of this strain is preliminarily determined, and the species NCU0011190 is determined to be Lactiplantibacillus plantarum .

[0077] The applicant has studied the above-mentioned Lactobacillus plantarum ( Lactiplantibacillus plantarum ) strain NCU0011190 (bacterium No. 55 in Example 1) was deposited, and the plant lactobacillus strain was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on December 12, 2024, and was classified and named plant lactobacillus Lactiplantibacillus plantaru m, the deposit number is CGMCC No.33046.

[0078] Example 3 Antibacterial performance test of strain NCU0011190: The antibacterial performance of strain NCU0011190 was tested using the disc diffusion method. The test results are shown in the table below.

[0079]

[0080] From the above, it can be seen that the strain NCU0011190 of the present application has a good inhibitory effect on both Gram-positive pathogens and Gram-negative pathogens.

[0081] Example 4 Strain NCU0011190 Cell Adhesion Test: The specific detection methods are as follows: Caco-2 cells were inoculated into 6-well plates containing coverslips and cultured in an incubator at 37°C and 5% CO2. The medium was changed every other day. When the cells grew to a monolayer, they were washed once with sterile PBS (pH 7.2). 1 mL of bacterial suspension (1x10 8 CFU / mL) for 1 h. Wash 5 times with sterile PBS to remove non-adherent bacteria. Fix with methanol for 30 min, dry naturally and then perform Gram staining, and observe the adhesion under a microscope. Bacterial adhesion count to Caco-2 cells: Cell culture and treatment are the same as before, except that the bacterial suspension is directly added to the cell culture well without placing a coverslip, washed with PBS and lysed with 1% TritonX-100 for 10 min, and counted on the plate after gradient dilution. In addition, select the culture wells that have grown to a single layer for cell counting. Make 3 parallels for each treatment, and calculate the average number of bacteria adhering to each cell.

[0082] For detailed results, see Figure 4 .

[0083] Figure 4 This is a graph showing the adhesion test of strain NCU0011190 provided in this example.

[0084] Example 5 In vivo experiment on mice with strain NCU0011190.

[0085] 1. Improvement of carrageenan thrombosis (model, bacteria, positive control) Thirty Kunming mice (18-20 g) were randomly divided into a model group, an NCU0011190 group (1×10 9 CFU / kg bw / d), positive control group (aspirin: 150 mg / kg bw / d), 10 mice in each group, and the drug was administered for 14 consecutive days. One hour after administration on the 12th and 14th days, the mice were intraperitoneally injected with 0.06% carrageenan saline solution (100 mg / kg). The formation of black tails on the tails of mice was observed after 15 and 16 days. One hour after administration on the 16th day, blood was collected by eyeball extraction for serum separation. The total length of the tail and the length of the black tail of the mouse were measured with a ruler and photographed. Then the mice were euthanized and samples of the tail, liver, kidney, etc. were collected and fixed in 10% neutral formalin. The sections were stained with hematoxylin and eosin (HE) and histopathological observation was performed under a microscope.

[0086] Figure 5 This is a bar graph comparing the effects of the strain NCU0011190, model group and positive control group provided in this example on the black tail rate of mice in the carrageenan-induced thrombosis model.

[0087] Figure 6 This is an experimental comparison chart of the effects of strain NCU0011190, model group and positive control group provided in this example on the black tail rate of mice in the carrageenan-induced thrombosis model.

[0088] (II) Histopathological HE staining HE staining sections of mouse tail, kidney and liver tissues: (1) Fix the tissue with 4% paraformaldehyde for 24 hours, then remove, trim, dehydrate and dip in wax; (2) Embed the tissue in wax, then place in a -20℃ refrigerator to cool and solidify, and prepare for sectioning after solidification; (3) Use a paraffin slicer to slice the tissue, and then seal it in a glass slide for later use. (4) Dewax the cut sections, stain the nuclei with hematoxylin, and then stain the cytoplasm with eosin; (5) After staining, dehydrate the slides, observe and photograph under a microscope, and analyze the results. Figure 7This is a comparison of the histopathological HE staining of carrageenan thrombosis model mice using the strain NCU0011190, model group and positive control group provided in this example.

[0089] (III) Inflammatory factors Elisa was used to detect the levels of inflammatory factors TNF-α, IL-6, IL-1β and NF-κB in mouse plasma. After the administration, the eyeballs of each group of mice were removed and blood was collected. After standing at room temperature for 30 min, the blood was centrifuged at 3000 r / min for 10 min, and the upper serum was taken and detected by Elisa. For specific detection steps, please refer to the instructions of each kit.

[0090] Figure 8a This is a bar graph comparing the serum inflammatory factor TNF-α content of carrageenan thrombosis model mice with strain NCU0011190, model group and positive control group provided in this example.

[0091] Figure 8b This is a bar graph comparing the serum inflammatory factor NF-κB content of carrageenan thrombosis model mice with strain NCU0011190, model group and positive control group provided in this example.

[0092] Figure 8c This is a bar graph comparing the serum inflammatory factor IL-6 content of carrageenan thrombosis model mice with strain NCU0011190, model group and positive control group provided in this example.

[0093] Figure 8d This is a bar graph comparing the serum inflammatory factor IL-1β content of carrageenan thrombosis model mice with strain NCU0011190, model group and positive control group provided in this example.

[0094] Example 6: Fermentation and degradation of ginkgo toxic components by strain NCU0011190 1. NCU0011190 Group: The method for fermenting and degrading the allergenic and toxic components of ginkgo by using the plant lactobacillus strain NCU0011190 comprises: (1) Preparation of puree: peel fresh ginkgo nuts, add water in a mass ratio of 1:5 (m / m), and beat the mixture to obtain ginkgo nut puree.

[0095] (2) Fermentation: Lactobacillus plantarum NCU0011190 was activated using MRS liquid medium. The ginkgo pulp was sterilized at 85°C for 30 min and inoculated for fermentation after cooling. The number of viable bacteria was 1×10 7 ~1×10 9 The inoculation amount of NCU0011190 activation solution was 1~2%, the fermentation temperature was 37℃, and the fermentation time was 48 h.

[0096] (3) Take 2 g of fermented ginkgo sample, add 10 times the volume of 70% ethanol solution, place in an ultrasonic cleaner, ultrasonically treat for 30 min, extract twice, filter, combine the extracts, and measure the volume. Concentrate the filtrate appropriately to an extract, and extract with 50 mL of n-hexane three times. Collect and combine the n-hexane layer solution, rotary evaporate at 60 °C, evaporate to near dryness, cool to room temperature, dissolve with methanol and dilute to a certain volume to obtain the test solution for HPLC analysis.

[0097] The HPLC analysis is to calculate the sum of the areas of five chromatographic peaks of ginkgo acid, namely, ginkgolic acid (C15:1), ginkgolic acid (C13:0), heptadecadiene ginkgolic acid (C17:2), heptadecadiene ginkgolic acid (C17:1) and hydrogenated ginkgolic acid (C15:0) and quantitatively calculate the total content of ginkgolic acid compounds in ginkgo fruit powder using a standard curve.

[0098] The chromatographic conditions were as follows: chromatographic column: ShimNex CS C18, 5 μm (4.6 mm * 250 mm); mobile phase: methanol-10% acetic acid water (92:8); flow rate: 1.0 mL / min; column temperature: 35 °C; UV detection wavelength: 310 nm.

[0099] (ii) CH042 group: except replacing Lactobacillus plantarum NCU0011190 with strain CH042, the rest is the same as NCU0011190 group.

[0100] (III) CH048 group: except replacing Lactobacillus plantarum NCU0011190 with strain CH048, the rest is the same as NCU0011190 group.

[0101] (IV) CH059 group: except replacing Lactobacillus plantarum NCU0011190 with strain CH059, the rest is the same as NCU0011190 group.

[0102] (V) CH010 group: except replacing Lactobacillus plantarum NCU0011190 with strain CH010, the rest is the same as NCU0011190 group.

[0103] (VI) CH057 group: except replacing Lactobacillus plantarum NCU0011190 with strain CH057, the rest is the same as NCU0011190 group.

[0104] (VII) Puree group: Peel the fresh ginkgo nuts, add water at a mass ratio of 1:5 (m / m) and beat to obtain ginkgo puree. Then take 2g of ginkgo puree, add 10 times the volume of 70% ethanol solution, place it in an ultrasonic cleaner, ultrasonically treat it for 30 min, extract it twice, filter it, combine the extracts, and measure the volume. The filtrate was appropriately concentrated to an extract, and extracted with 50 mL of n-hexane for 3 times. Collect and combine the n-hexane layer solution, rotary evaporate at 60 °C, evaporate to near dryness, cool to room temperature, dissolve it with methanol and make up to a certain volume to obtain the test solution for HPLC analysis.

[0105] The above test groups of this example were compared and verified. After fermentation with NCU0011190, the total ginkgolic acid content decreased from 3.18 μg / g to 0.76 μg / g, a decrease of 76%, which was better than the degradation effects of the other five strains.

[0106] Note: After testing and identification, CH042, CH048, and CH059 are Lactobacillus plantarum; CH010 is Lactobacillus brevis; and CH057 is Lactobacillus casei.

[0107] Fig. 9 This is a comparison chart of the degradation effect of total ginkgolic acid of the strain NCU0011190 provided in this example.

[0108] Specifically, the test results of the five types of ginkgolic acid are as follows:

[0109] Example 7 The ginkgo fermentation product obtained by fermentation and degradation of strain NCU0011190 in Example 6 was subjected to an anti-thrombotic effect test and analysis.

[0110] The specific method is as follows: SD rats (180-200 g), half male and half female, were weighed and randomly divided into a model group, a fermented ginkgo group (10 mg / kg bw / d), and a positive control group (clopidogrel: 30 mg / kg bw / d), with 10 rats in each group.

[0111] After one week of adaptation, rats were gavaged according to the dosage for 2 weeks before modeling. Rats were anesthetized by intraperitoneal injection of sodium pentobarbital (100 µg / g), and DHM or DMSO was injected into the tail vein. 5 min after administration, the anesthetized rats were fixed in a supine position on the operating table, the neck skin was cut open, and the subcutaneous tissue was separated with surgical curved forceps. After the common carotid artery was exposed, the fascia wrapped around the blood vessel was carefully peeled off with forceps. The separated carotid artery was embedded in the groove of the ultrasonic blood flowmeter probe, and the ultrasonic blood flowmeter was used to automatically detect the blood flow before the carotid artery was injured by FeCl3. 1mm×3mm filter paper was fully soaked with 12% FeCl3 and covered on the carotid artery. After continuous stimulation for 1 min, the filter paper was washed away from the blood vessel with saline. After the filter paper was removed, the carotid artery was immediately embedded in the groove of the ultrasonic blood flowmeter probe, and the ultrasonic blood flowmeter was used to automatically detect the changes in blood flow over time after the carotid artery was injured by FeCl3. If the blood flow completely stopped and did not resume within 20 min, the time point when the blood flow completely stopped was recorded as the vascular occlusion time. If the blood flow did not completely stop, the maximum recording time was 50 min.

[0112] Specific results: After experimental measurement, the thrombus lengths of the model group, fermented ginkgo group and positive control group were 7.13, 5.15 and 5.02 mm, respectively; the thrombus weights were 3.46, 2.62 and 2.13 mg, respectively; compared with the model group, the weight and length of the thrombus in the fermented ginkgo group were significantly reduced, and there was no significant difference between the fermented ginkgo group and the positive control group, indicating that fermented ginkgo can significantly reduce the formation of thrombus.

[0113] Fig.10 A bar graph comparing the thrombus lengths of the ginkgo fermentation product obtained by fermentation and degradation of the strain NCU0011190 provided in Example 7 of the present invention and the model group and the positive control group; Fig.11 A bar chart comparing the thrombus weight of the ginkgo fermentation product obtained by fermentation and degradation of the strain NCU0011190 provided in Example 7 of the present invention and the model group and the positive control group; Fig.12 This is a comparison diagram of the ginkgo fermentation product obtained by fermentation and degradation of the strain NCU0011190 provided in Example 7 of the present invention and the actual thrombus in the model group and the positive control group.

[0114] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A plant lactobacillus with thrombosis prevention effect ( Lactiplantibacillus plantarum ) strain NCU0011190, characterized in that The plant lactobacillus strain was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on December 12, 2024, with the deposit number CGMCC No.33046.

2. A microbial agent, characterized in that: The microbial agent comprises the plant lactobacillus strain NCU0011190 and its metabolites according to claim 1.

3. Use of the plant lactobacillus strain NCU0011190 described in claim 1 or the microbial agent described in claim 2 in fermentation and degradation of allergenic and toxic components of ginkgo.

4. A method for fermenting and degrading the allergenic and toxic components of ginkgo using the Lactobacillus plantarum strain NCU0011190 of claim 1, characterized in that: The method comprises: (A) Preparation of puree: fresh ginkgo nuts are shelled, and water is added in a mass ratio of 1:5 (m / m) and then pulped to obtain ginkgo nut puree; (B) Fermentation: Lactobacillus plantarum NCU0011190 was activated using MRS liquid culture medium. The ginkgo puree was sterilized at 85°C for 30 min and then inoculated for fermentation after cooling. The inoculation amount of NCU0011190 activation solution was 1-2%, the fermentation temperature was 37°C, and the fermentation time was 48 h.

5. The ginkgo fermentation product prepared by the method of claim 4.

6. The ginkgo fermentation product according to claim 5, characterized in that: The content of total ginkgolic acid in the ginkgo fermentation product is 0.7-2.0 μg / g, wherein: The content of ginkgolic acid C15:1 is 0.5~1.5μg / g, the content of ginkgolic acid C13:0 is 0.05~0.15μg / g, the content of heptadecanedienoic acid C17:2 is 0.05~0.2μg / g, the content of heptadecanedienoic acid C17:1 is 0.1~0.3μg / g, and the content of hydrogenated ginkgolic acid C15:0 is 0.005~0.03μg / g.

7. Use of the Lactobacillus plantarum strain NCU0011190 according to claim 1, the microbial agent according to claim 2, or the ginkgo fermentation product according to claim 5 or 6 in the preparation of drugs for preventing and treating thrombosis.

8. A drug for preventing and treating thrombosis, characterized in that: The drug comprises the Lactobacillus plantarum strain NCU0011190 according to claim 1; And / or, the microbial agent according to claim 2; And / or, the ginkgo fermentation product according to claim 5 or 6.

9. The drug for preventing and treating thrombosis according to claim 8, characterized in that: The drug is the microbial agent according to claim 2; The microbial agent is suitable for oral administration to mammals in a unit dose, and the amount of Lactobacillus plantarum strain NCU0011190 in the unit dose is 1.0×10 7 ~1.0×10 10 CFU / mL.

10. The drug for preventing and treating thrombosis according to claim 8, characterized in that: The medicine comprises the ginkgo fermentation product according to claim 5 or 6; The ginkgo fermentation product is suitable for oral use in mammals in a unit dose, and the amount of the ginkgo fermentation product in the unit dose is 5-100g.

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