Pediococcus pentosaceus strain ncu16v with prevention of thrombosis and cerebral ischemic injury, microbial inoculant comprising the same, and use thereof

Abstract

The application provides a Pediococcus pentosaceus strain NCU16V with thrombus prevention and cerebral ischemic injury prevention, a microbial inoculum containing the same and application, and relates to the technical field of microorganisms.The preservation number of the Pediococcus pentosaceus strain NCU16V is CGMCC No.35427.The Pediococcus pentosaceus strain NCU16V has good gastrointestinal digestion tolerance, and has a high survival rate after being digested in simulated artificial gastric juice and intestinal juice for 3 hours, as verified by the research of the application.Meanwhile, it is verified that the Pediococcus pentosaceus strain NCU16V and the microbial inoculum containing the same can be widely applied in drugs for preventing thrombosis, drugs for relieving brain tissue injury after cerebral ischemia and preparation of ginkgo fermented products with thrombosis prevention.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a Pediococcus pentosaceus strain NCU16V that has the ability to prevent thrombosis and cerebral ischemia-reperfusion injury, as well as microbial agents containing the same and their applications. Background Technology

[0002] Thrombotic diseases are the most prevalent manifestation of cardiovascular disease, and they have the highest morbidity and mortality rates. Among them, stroke and coronary heart disease are the most severe. The pathogenic factors of thrombotic diseases are blood vessel wall damage caused by various internal and external factors, leading to blood clotting. Blood vessel damage is accompanied by endothelial cell oxidation, which further exacerbates the damage and thrombus accumulation.

[0003] Existing drugs for preventing thrombosis include antiplatelet aggregation drugs such as aspirin, clopidogrel, favarin, and rivaroxaban. However, long-term use of these drugs often carries serious risks of bleeding and gastrointestinal damage. Meanwhile, existing drugs for improving brain tissue damage after cerebral ischemia include edaravone dexborneol, butylphenol, and piracetam. However, these drugs carry serious risks of liver and kidney damage, gastrointestinal mucosal damage, and bleeding. Some may also cause problems such as nerve excitation.

[0004] Therefore, it is both necessary and urgent to research and develop a microbial strain and its derived drugs that can both prevent thrombosis and alleviate brain tissue damage after cerebral ischemia without side effects.

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

[0006] The purpose of this invention is to provide a Pentosaccharide Pediococcus (Pediococcus pentosaceus) that can prevent thrombosis and cerebral ischemia-reperfusion injury. Pediococcus pentosaceus The strain NCU16V, when supplemented prophylactically, exhibits excellent preventative effects against thrombosis. Furthermore, it has been verified that the aforementioned Pediococcus pentosaceus strain NCU16V also has a technical effect in alleviating brain tissue damage following cerebral ischemia. In addition, the fermented product of ginkgo biloba obtained by fermentation with the Pediococcus pentosaceus strain NCU16V of this application shows a significantly enhanced effect in preventing thrombosis.

[0007] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted:

[0008] This invention provides a type of Pediococcus pentosaceus with antithrombotic and neuroprotective effects. Pediococcus pentosaceusThe *Pediococcus pentosaceus* strain NCU16V was deposited on July 28, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35427. The deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0009] Furthermore, the biological characteristics of the Pediococcus pentosaceus strain NCU16V include:

[0010] The survival rate of *Pediococcus pentosaceus* strain NCU16V after treatment in artificial gastric fluid for 3 hours was 90.2%–98.41%; the survival rate after treatment in artificial intestinal fluid for 3 hours was 92.5%–103.6%; the survival rate after treatment in MRS culture medium containing 0.3% bile salts for 3 hours was 86%–95.3%; and *Pediococcus pentosaceus* strain NCU16V could grow and reproduce normally in a culture environment with pH 3.0–7.0.

[0011] The present invention provides a microbial inoculant, which includes the above-mentioned Pediococcus pentosaceus strain NCU16V and excipients.

[0012] Furthermore, the microbial agent is the fermentation broth of Pediococcus pentosaceus strain NCU16V.

[0013] The above-mentioned Pediococcus pentosaceus strain NCU16V or the above-mentioned microbial agent provided by the present invention have the following applications:

[0014] (A) Application in the preparation of drugs for preventing thrombosis;

[0015] (B) Application in the preparation of drugs to alleviate brain tissue damage after cerebral ischemia;

[0016] (C) Application in the preparation of ginkgo fermentation products with antithrombotic properties.

[0017] Furthermore, the prevention of thrombosis includes:

[0018] Prophylactic supplementation with Pediococcus pentosaceus strain NCU16V has a preventive effect on thrombosis, specifically by reducing the content of thrombotic factors and alleviating oxidative stress damage in platelet activation and thrombosis.

[0019] Furthermore, the relief of thrombosis-induced oxidative stress includes: increasing the content of superoxide dismutase (SOD) in plasma, increasing the content of glutathione peroxidase (GSH-Px) in plasma, decreasing the content of reactive oxygen species (ROS) in plasma, and decreasing the content of malondialdehyde (MDA) in plasma.

[0020] Furthermore, the relief of brain tissue damage after cerebral ischemia includes: reducing brain cell apoptosis caused by cerebral ischemia.

[0021] Furthermore, the reduction of brain cell apoptosis caused by cerebral ischemia includes reducing the ischemic dead area of ​​brain tissue, reducing the expression of apoptosis factors, reducing calcium ion concentration, and reducing oxidative damage.

[0022] Furthermore, the drug for preventing thrombosis is the aforementioned microbial agent, which is suitable for oral administration to mammals at a unit dose, wherein the amount of Pediococcus pentosaceus strain NCU16V in the unit dose is 1.0 × 10⁻⁶. 7 ~1.0×10 11 CFU / mL;

[0023] And / or, the drug for relieving brain tissue damage after cerebral ischemia is the above-mentioned microbial agent, which is suitable for oral administration to mammals at a unit dose, wherein the amount of Pediococcus pentosaceus strain NCU16V in the unit dose is 1.0 × 10⁻⁶. 7 ~1.0×10 11 CFU / mL.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] This invention provides a Pentosacchariphyte strain that can prevent thrombosis and cerebral ischemia-reperfusion injury. Pediococcus pentosaceus The *Pediococcus pentosaceus* strain NCU16V was deposited on July 28, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35427, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. This application's research has verified that the aforementioned *Pediococcus pentosaceus* strain NCU16V exhibits good gastrointestinal digestive tolerance, showing a high survival rate after 3 hours of digestion in simulated gastric and intestinal fluids.

[0026] This application has verified that the prophylactic supplementation with the aforementioned Pediococcus pentosaceus strain NCU16V has a good preventive effect on thrombosis; at the same time, the aforementioned Pediococcus pentosaceus strain NCU16V also has the technical effect of alleviating brain tissue damage after cerebral ischemia; in addition, the ginkgo fermentation product fermented with the Pediococcus pentosaceus strain NCU16V of this application has a significantly improved effect on preventing thrombosis compared with ginkgo pulp or existing fermentation strains. Attached Figure Description

[0027] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0028] Figure 1 This is a comparative diagram showing the tolerance of 20 strains obtained from the strain screening provided in Example 1 of the present invention to gastrointestinal fluid.

[0029] Figure 2 This is a comparative diagram showing the bile salt tolerance of 20 strains obtained from the strain screening provided in Example 1 of the present invention.

[0030] Figure 3 This is a comparison chart of platelet aggregation rates of 20 strains obtained from strain screening provided in Example 1 of the present invention;

[0031] Figure 4 This is a hemolytic detection diagram of the Pediococcus pentosaceus strain NCU16V provided in Example 2 of the present invention;

[0032] Figure 5 This is a diagram of the biogenic amine synthesis verification experiment of the Pediococcus pentosaceus strain NCU16V provided in Example 2 of the present invention;

[0033] Figure 6 This is an experimental diagram of the antioxidant properties of the Pediococcus pentosaceus strain NCU16V provided in Example 2 of the present invention;

[0034] Figure 7 The diagram shows the changes in carotid artery blood flow in the blank group, model group, Pediococcus pentosaceus NCU16V group, and positive group provided in Example 3 of the present invention.

[0035] Figure 8a This is an image showing the HE staining results of the blank group provided in Example 3 of the present invention;

[0036] Figure 8b This is an image showing the HE staining results of the carotid artery thrombosis in the model group provided in Embodiment 3 of the present invention;

[0037] Figure 8c This is an image showing the HE staining results of carotid artery thrombosis in the Pediococcus pentosaceus NCU16V group provided in Example 3 of the present invention;

[0038] Figure 8d This is an image showing the HE staining results of the positive group of carotid artery thrombosis provided in Example 3 of the present invention;

[0039] Figure 9A bar chart comparing the thrombus lengths of the blank group, model group, Pediococcus pentosaccharis NCU16V group, and positive group provided in Example 3 of the present invention;

[0040] Figure 10 A comparison chart of thrombotic factor content in the blank group, model group, Pediococcus pentosaccharis NCU16V group, and positive group provided in Example 3 of the present invention;

[0041] Figure 11 TTC staining images of rat brain tissue from the sham-operated group, model group, Pediococcus pentosaceus NCU16V group, and positive group provided in Example 4 of this invention;

[0042] Figure 12 Fluo-4 AM staining images of calcium ions in rat brain tissue from the sham-operated group, model group, Pediococcus pentosaceus NCU16V group, and positive group provided in Example 4 of this invention;

[0043] Figure 13 The expression diagram of apoptosis factor in rat brain tissue cells of the sham-operated group, model group, Pediococcus pentosaceus NCU16V group and positive group provided in Example 4 of the present invention;

[0044] Figure 14 This is a comparative experimental diagram showing the effects of the blank group, model group, original pulp group, NCU16V ginkgo fermentation product group, NCU0011190 ginkgo fermentation product group, and positive control group on the black tail of mice provided in Example 5 of the present invention. Detailed Implementation

[0045] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0047] According to one aspect of the present invention, the present invention provides a Pentosacchariphyte strain that prevents thrombosis and cerebral ischemia-reperfusion injury. Pediococcus pentosaceus The Pediococcus pentosaceus strain NCU16V was deposited on July 28, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35427. The deposit date was July 28, 2025, and the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0048] The research and verification conducted in this application have shown that the aforementioned *Pediococcus pentosaceus* strain NCU16V exhibits good gastrointestinal digestive tolerance and a high survival rate after 3 hours of digestion in simulated gastric and intestinal fluids. Furthermore, this application has verified that prophylactic supplementation with the aforementioned *Pediococcus pentosaceus* strain NCU16V has a significant preventive effect against thrombosis. Additionally, the aforementioned *Pediococcus pentosaceus* strain NCU16V also has a technical effect in alleviating brain tissue damage following cerebral ischemia. Moreover, the fermented ginkgo product obtained through fermentation with the *Pediococcus pentosaceus* strain NCU16V of this application shows a significantly enhanced effect in preventing thrombosis.

[0049] In a preferred embodiment of the present invention, the biological characteristics of the Pediococcus pentosaceus strain NCU16V include:

[0050] The survival rate of *Pediococcus pentosaceus* strain NCU16V after treatment in artificial gastric fluid for 3 hours was 90.2%–98.41%; the survival rate after treatment in artificial intestinal fluid for 3 hours was 92.5%–103.6%; the survival rate after treatment in MRS culture medium containing 0.3% bile salts for 3 hours was 86%–95.3%; and *Pediococcus pentosaceus* strain NCU16V could grow and reproduce normally in a culture environment with pH 3.0–7.0.

[0051] According to one aspect of the present invention, a microbial inoculant comprises the above-mentioned Pediococcus pentosaceus strain NCU16V and excipients.

[0052] In a preferred embodiment of the present invention, the microbial agent is the fermentation broth of Pediococcus pentosaceus strain NCU16V.

[0053] According to one aspect of the present invention, the above-mentioned Pediococcus pentosaceus strain NCU16V or the above-mentioned microbial agent is used in the following aspects:

[0054] (A) Application in the preparation of drugs for preventing thrombosis;

[0055] (B) Application in the preparation of drugs to alleviate brain tissue damage after cerebral ischemia;

[0056] (C) Application in the preparation of ginkgo fermentation products with antithrombotic properties.

[0057] In a preferred embodiment of the present invention, the prevention of thrombosis includes:

[0058] Prophylactic supplementation with Pediococcus pentosaceus strain NCU16V has a preventive effect on thrombosis, specifically by alleviating thrombosis-induced oxidative stress and reducing the levels of thrombotic factors.

[0059] Specifically, the relief of thrombosis-induced oxidative stress includes: increasing the level of superoxide dismutase (SOD) in plasma, increasing the level of glutathione peroxidase (GSH-Px) in plasma, decreasing the level of reactive oxygen species (ROS) in plasma, and decreasing the level of malondialdehyde (MDA) in plasma.

[0060] The reduction of thrombotic factor levels includes: reducing PAF levels in plasma, reducing thromboxane B2 levels in plasma, and increasing 6-keto-PGF1α levels in plasma.

[0061] In a preferred embodiment of the present invention, the relief of brain tissue damage after cerebral ischemia includes: reducing brain cell apoptosis caused by cerebral ischemia.

[0062] Specifically, reducing brain cell apoptosis caused by cerebral ischemia includes reducing the ischemic dead area of ​​brain tissue, reducing the content of apoptosis factors, reducing calcium ion concentration, and reducing oxidative damage.

[0063] In a preferred embodiment of the present invention, the drug for preventing thrombosis is the aforementioned microbial agent, which is suitable for oral administration to mammals at a unit dose, wherein the amount of Pediococcus pentosaceus strain NCU16V in the unit dose is 1.0 × 10⁻⁶. 7 ~1.0×10 11 CFU / mL;

[0064] In a preferred embodiment of the present invention, the drug for alleviating brain tissue damage after cerebral ischemia is the aforementioned microbial agent, which is suitable for oral administration to mammals at a unit dose, wherein the amount of Pediococcus pentosaceus strain NCU16V in the unit dose is 1.0 × 10⁻⁶. 7 ~1.0×10 11 CFU / mL.

[0065] The technical solution of the present invention will be further described below with reference to the embodiments.

[0066] Example 1: Screening and acquisition of strain NCU16V

[0067] (a) Strain screening

[0068] (1) Spread the naturally fermented kimchi liquid from different places onto MRS solid plates containing 0.01% bromocresol purple or dilute it and spread it onto solid plates. Select strains with deep yellow colony color and different morphology, and purify them by streak culture to obtain 20 pure strains, numbered 1-20.

[0069] (2) The above 20 strains were subjected to simulated experiments of tolerance to gastrointestinal fluid digestion and tolerance to bile salts. After the strains were activated for 18 hours, 4 mL of activation solution was centrifuged and resuspended twice with physiological saline to obtain bacterial sludge.

[0070] The bacterial sludge was suspended in artificial gastric fluid (0.3% pepsin, 0.85% NaCl, pH adjusted to 2.5 with dilute hydrochloric acid), artificial intestinal fluid (0.1% trypsin, 0.68% potassium dihydrogen phosphate, pH=6.8), and MRS liquid medium containing 0.3% bile salts, respectively. Its tolerance to gastric fluid, intestinal fluid, and bile salts was analyzed. The culture was carried out at 37℃ for 3 h, and viable cell counts were performed at 0 h (A0) and 3 h (A1). Viable cell rate / % = [(A0-A1) / A0] 100.

[0071] Figure 1 This is a comparative diagram showing the tolerance of 20 strains obtained from the screening of strains provided in this embodiment to gastrointestinal fluid.

[0072] Figure 2 This is a comparative diagram showing the bile salt tolerance of 20 strains obtained from the screening of strains provided in this embodiment.

[0073] (II) Antiplatelet aggregation test:

[0074] (1) Preparation of washed mouse platelets:

[0075] 1. Eight male Kunming mice (18-22 g) were fasted the night before the experiment but given free access to water. The mice were anesthetized by intraperitoneal injection of 20% urethane (0.1 mL / 10 g), fixed on the operating table, and the linea alba was cut open. Careful care was taken to avoid major peritoneal vessels to fully expose the inferior vena cava. Blood was slowly drawn from the inferior vena cava using a syringe containing 3.8% sodium citrate (sodium citrate solution: whole blood = 1:9).

[0076] 2. After thoroughly mixing the blood with sodium citrate, 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 from the two mice into a 10mL centrifuge tube.

[0077] 3. Centrifuge at 200×g at room temperature for 16 min, aspirate the upper light white liquid, add PGE1 working solution to make the final concentration of PGE1 added this time 50 ng / mL, and mix gently.

[0078] 4. Centrifuge at 800×g for 10 min at room temperature. Discard the supernatant and add 2 mL of 1×Tyrode Buffer, 8 μL of EDTA solution, and 2 μL of PGE1 solution to bring the final concentrations of the latter two to 1 mM and 50 ng / mL, respectively. Resuspend the platelets by pipetting. Note that the pipetting motion should be as gentle as possible to avoid excessive shearing force that could lead to overactivation of platelets. Also, if the precipitate still contains a small number of red blood cells, discard them. Combine the platelet suspensions from all tubes to a total volume of approximately 6 mL.

[0079] 5. Centrifuge at 750×g for 10 min at room temperature, discard the supernatant, and retain the precipitate. Add an appropriate amount of 1×TyrodeBuffer, and gently mix by pipetting with a Pasteur pipette. The resuspended platelets are the washed platelets.

[0080] 6. Count the washed platelets using a hematology analyzer. Adjust the concentration of washed platelets to the required level using 1×Tyrode Buffer, depending on the experimental objective. Allow the platelets to stand at room temperature for about half an hour before starting the formal experiment.

[0081] (2) Platelet aggregation test:

[0082] Preheat the platelet aggregator and adjust the rotation speed to 1000 rpm; add the rotor to the platelet reaction vessel and aspirate 250 μL of mouse platelet-rich plasma (3 × 10⁻⁶). 8 Add the platelets (cFU / mL) to the reaction vessel, then add CaCl2 (final concentration 1 mM). Add an equal volume of culture supernatant of bacteria 1-20 (adjusted pH=7.0, 0.22μm membrane filtration) or the positive control drug acetylsalicylic acid according to the group. Incubate at 37℃ for 5 min. After incubation, place the reaction vessel in a platelet aggregator, adjust the baseline, add ADP to induce platelet aggregation, and record the platelet aggregation using software. Stop recording when the platelet aggregation reaches the highest aggregation rate, and express the highest aggregation rate.

[0083] Figure 3 This is a comparison chart of platelet aggregation rates of 20 strains obtained from the strain screening provided in this embodiment.

[0084] Based on the above, strain 16 has good acid and bile salt tolerance and gastrointestinal digestive ability, and its fermentation supernatant has a good ability to inhibit platelet aggregation. The strain 16 is named NCU16V.

[0085] (3) 16S identification of strain NCU16V:

[0086] 16S rRNA analysis was performed on the isolated strain 16 (named NCU16V) to preliminarily determine its taxonomic position.

[0087] Genomic DNA was extracted from the strain, and 16S rRNA was amplified using the extracted genomic DNA template. The amplification primers were:

[0088] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3', sequence as shown in SEQ ID NO:2;

[0089] 1492R: 5'-GGTTACCTTGTTACGACTT-3', sequence as shown in SEQ ID NO:3.

[0090] The amplified PCR product was purified and then sequenced using an ABI3730XL to obtain a 954bp 16S rRNA sequence. The 16S RNA sequence of the strain NCU16V is shown in SEQ ID NO:1.

[0091] The nucleotide sequence shown in SEQ ID No. 1 is as follows:

[0092]

[0093] The 16S RNA sequence of strain NCU16V was compared with the NCBI 16S ribosomal RNA sequences database, and the closest species was Pediococcus pentosaceus. Pediococcus pentosaceus With a similarity of 99.86%, the species classification information of this strain was preliminarily determined, and NCU16V was identified as a species of NCU16V. Pediococcus pentosaceus .

[0094] Meanwhile, as stated in this application Figure 1 , Figure 2 It is known that the survival rate of the *Pediococcus pentosaceus* strain NCU16V after treatment in artificial gastric fluid for 3 hours was 90.2%–98.41%; the survival rate of the *Pediococcus pentosaceus* strain NCU16V after treatment in artificial intestinal fluid for 3 hours was 92.5%–103.6%; the survival rate of the *Pediococcus pentosaceus* strain NCU16V after 3 hours in MRS culture medium containing 0.3% bile salts was 86%–95.3%; and the *Pediococcus pentosaceus* strain NCU16V could grow and reproduce normally in a culture environment with pH 3.0–7.0.

[0095] Example 2: Identification of Biological Characteristics

[0096] (a) Hemolytic test:

[0097] To verify the safety of the strain, a hemolytic test was performed on the *Pediococcus pentosaceus* strain NCU16V obtained in Example 1 above. *Staphylococcus aureus* ATCC6538 was used as a positive control strain.

[0098] The specific procedure is as follows: NCU16V is inoculated onto Columbia blood agar plates, and ATCC6538 is inoculated in the center of the plate at the same time. After inoculation, the plates are incubated at 37°C for 36 h. The hemolysis zone around the colonies is observed to determine whether hemolytic characteristics exist.

[0099] Figure 4 This is a hemolytic detection diagram of the Pediococcus pentosaceus strain NCU16V provided in this embodiment.

[0100] like Figure 4 As shown, NCU16V (the outer three colonies) did not form a clear hemolytic zone, while the positive control Staphylococcus aureus (the central colony) showed a clear hemolytic zone, indicating that NCU16V is non-hemolytic and proving the safety of the strain.

[0101] (II) Verification Experiment of Biogenic Amine Synthesis

[0102] Pediococcus pentosaceus strain NCU16V was inoculated into an amino acid decarboxylase test medium supplemented with 0.1% (w / v) tyrosine, with Salmonella ATCC14028 as a positive control. After inoculation, the samples were incubated at 37°C to observe color changes. After 60 h of incubation, the samples were taken out and the biogenic amine synthesis characteristics were determined based on the color. The test tubes with positive results (producing biogenic amines) remained purple, while the test tubes with negative results (not producing biogenic amines) turned yellow.

[0103] Figure 5 This is a diagram from the biogenic amine synthesis verification experiment of the Pediococcus pentosaceus strain NCU16V provided in this embodiment.

[0104] like Figure 5 It can be seen that the blank control group and the positive control Salmonella maintained their purple color, while NCU16V turned yellow, indicating that NCU16V cannot synthesize biogenic amines, further confirming its safety.

[0105] (III) Antioxidant properties

[0106] Pediococcus pentosaceus strain NCU16V was inoculated into MRS medium at a ratio of 0.1% and cultured at 37°C. Fermentation broth was collected at 12, 18, and 24 h to analyze the changes in antioxidant capacity. The fermentation broth was centrifuged and filtered through a 0.22 μm membrane. The scavenging rates of DPPH, ABTS, hydroxyl radicals, and superoxide anion radicals were then detected using a kit.

[0107] Figure 6 The figure shows the antioxidant performance of the Pediococcus pentosaceus strain NCU16V provided in this embodiment.

[0108] like Figure 6 It can be seen that as the culture time is extended, the antioxidant capacity of the fermentation broth gradually increases, but after 18 hours, it is basically at a high level of scavenging capacity.

[0109] Example 3: Prevention of ferric chloride-induced carotid artery thrombosis

[0110] (I) Animal acclimatization: Forty male SD rats, weighing 220-250g, were selected and randomly divided into four groups of 10 rats each after one week of acclimatization feeding: blank group, model group, Pediococcus pentosaceus NCU16V group, and positive group, as detailed below:

[0111] The Pediococcus pentosaceus NCU16V group was administered Pediococcus pentosaceus strain NCU16V bacterial suspension (1.0×10⁻⁶) by gavage daily. 9 The positive control group was given clopidogrel (20 mg / kg bw) by gavage daily, while the blank control group and the model group were given the same amount of normal saline by gavage daily for 14 consecutive days.

[0112] On day 14, after the last administration, the rats were kept on a fasting and water-free schedule for 12 hours. They were then anesthetized with an intraperitoneal injection of 10% chloral hydrate (7 mg / kg) and fixed in a supine position on the operating table. The skin of the rats' necks was prepared, and a longitudinal incision of about 3 cm was made along the midline of the neck using surgical scissors. The right common carotid artery was then bluntly dissected.

[0113] The model group, the Pediococcus pentosaceus NCU16V group, and the positive group used 1.5 cm x 1.0 cm filter paper strips soaked in 20% FeCl3 to wrap the isolated common carotid artery to induce carotid artery thrombosis. A 2.0 cm x 1.5 cm sterile surgical drape was placed under the filter paper strip to prevent FeCl3 from damaging other tissue structures. After 15 minutes, the two ends of the surgical drape were ligated with surgical sutures and the blood vessel was precisely cut to the same length as the width of the surgical drape for thrombosis analysis.

[0114] (II) Analysis and Testing:

[0115] (1) Use Doppler ultrasound to monitor blood flow in different groups and monitor the changes in carotid artery blood flow over time after modeling;

[0116] The blood flow in the carotid artery of different groups of rats and the vascular closure time caused by thrombosis were detected using Doppler ultrasound. The specific method was as follows: After applying a 20% FeCl3 filter paper strip to the rat's carotid artery, the blood flow signal in the carotid artery was immediately detected using a Doppler ultrasound probe and continuously monitored for 13 minutes. Changes in carotid artery blood flow in different groups of rats were recorded as follows: Figure 7 As shown.

[0117] Figure 7 The diagram shows the changes in carotid artery blood flow in the blank group, model group, Pediococcus pentosaceus NCU16V group, and positive group provided in this embodiment.

[0118] in: Figure 7 A represents the blank control group; Figure 7 B in the diagram represents the model group; Figure 7 C represents Pediococcus pentosaceus NCU16V group; Figure 7 Group D is the positive drug group.

[0119] Depend on Figure 7 It can be seen that the blood flow in the carotid artery of rats in the blank group was stable and normal, while the blood flow in the model group was significantly reduced due to severe thrombosis. The blood flow in the NCU16V group and the positive drug group was significantly higher than that in the model group, indicating that NCU16V and the positive drug significantly inhibited thrombosis and that NCU16V has a good effect in preventing thrombosis.

[0120] (2) By observing the carotid artery with HE staining, the pathological characteristics of vascular thrombosis were analyzed.

[0121] Figure 8aThis is an image showing the HE staining results of the carotid artery thrombosis in the blank group provided in this embodiment;

[0122] Figure 8b This is an image showing the HE staining results of the carotid artery thrombosis in the model group provided in this embodiment;

[0123] Figure 8c This is an image showing the HE staining results of carotid artery thrombosis in the Pediococcus pentosaceus NCU16V group provided in this embodiment;

[0124] Figure 8d This is an image showing the HE staining results of the positive group of carotid artery thrombosis provided in this embodiment;

[0125] like Figures 8a-8d As shown, there was no thrombus occlusion in the blank group, while the thrombus completely blocked the model group. Thromboembolism was significantly reduced in the NCU16V and positive group, confirming that NCU16V has a role in preventing thrombosis.

[0126] (3) Thrombus weight in each experimental group

[0127] After the model was established, the thrombus in the rat's carotid artery was cut off, weighed, and the weight of the thrombus was analyzed. Figure 9 As shown.

[0128] Figure 9 A bar chart comparing the thrombus lengths of the blank group, model group, Pediococcus pentosaccharis NCU16V group, and positive group provided in this embodiment;

[0129] Depend on Figure 9 It can be seen that the amount of thrombus formation in the model group was significantly higher than that in the control group, and prophylactic supplementation with the Pediococcus pentosus strain NCU16V of this application can reduce the amount of thrombus formation.

[0130] (4) Detection of oxidative stress indicators:

[0131] Immediately after the modeling was completed, rat serum was collected, and the changes in serum oxidative factors SOD, ROS, GSH-PX and MDA were analyzed using a kit. The results are shown in Table 1.

[0132] Table 1. Changes in serum oxidative factors SOD, ROS, GSH-PX, and MDA:

[0133]

[0134] As shown above, the levels of reactive oxygen species (ROS) and malondialdehyde (MDA) in the model group were significantly higher than those in the control group, while the levels of superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) were significantly lower. This indicates that the modeling process caused severe oxidative stress damage in rats. However, after prophylactic supplementation with NCU16V, ROS and MDA were significantly reduced, while SOD and GSH-PX were significantly increased, indicating that the oxidative stress damage was effectively alleviated. This confirms that prophylactic supplementation with NCU16V can alleviate oxidative stress damage caused by vascular injury and reduce thrombus formation.

[0135] (5) Detection of thrombosis factor indicators:

[0136] The specific detection method is as follows: Blood samples are centrifuged at 4℃ and 1000×g for 15 minutes within 30 minutes of collection. The supernatant is then used for ElisA detection. The specific steps are performed according to the kit's instruction manual. The detection results are as follows: Figure 10 As shown.

[0137] Figure 10 A comparison chart of thrombotic factor levels in the blank group, model group, Pediococcus pentosaceus NCU16V group, and positive group provided in this embodiment;

[0138] Depend on Figure 10 It was found that the levels of thrombosis inducers platelet-activating factor (PAF) and thromboxane 2 (TXB2) were significantly increased in the model group, while the levels of 6-Keto-PG-F-1 (prostacyclin metabolite), which inhibits thrombus formation, were significantly decreased. In contrast, in the Pediococcus pentosaceus NCU16V group and the positive group (clopidogrel) of the prophylactic supplement strain NCU16V used in this application, PAF and TXB2 were significantly decreased, while prostacyclin was significantly increased. This indicates that the use of NCU16V can significantly inhibit the production of thrombosis inducers and increase the levels of thrombosis inhibitors.

[0139] Example 4: Prevention of cerebral ischemia-reperfusion brain injury

[0140] (I) Forty male SD rats weighing 220-250g were selected and randomly divided into four groups of 10 rats each after one week of acclimatization: sham-operated group, model group, Pediococcus pentosaceus NCU16V group, and positive group.

[0141] The Pediococcus pentosaceus NCU16V group was administered Pediococcus pentosaceus strain NCU16V bacterial suspension (1.0×10⁻⁶) by gavage daily. 9 The positive group was given butylphenol (25 mg / kg bw) by gavage daily, while the sham-operated group and the model group were given the same amount of physiological saline. After 14 days of continuous culture, the model was established.

[0142] The modeling experiment was conducted as follows: Two hours after administration of the model group, positive group, and Pediococcus pentosaceus NCU16V group, rats were anesthetized, fixed in a supine position, and the right common carotid artery (CCA), internal carotid artery (ICA), and external carotid artery (ECA) were isolated. The ECA and CCA were ligated, and the distal end of the ICA was clamped with an arterial clamp. An incision was quickly made in the common carotid artery approximately 0.5 cm from the bifurcation of the ECA and ICA. A nylon suture (0.28 mm in diameter) coated with paraffin was inserted to a depth of 18.5 ± 0.5 mm to achieve cerebral ischemia due to middle cerebral artery occlusion. Approximately 1 cm of the nylon suture was left outside the ligation site, and the skin was sutured. Two hours later, the remaining suture end was gently pulled outwards until slight resistance was felt to achieve reperfusion of the middle cerebral artery.

[0143] The sham surgery group (blank group) had the common carotid artery, internal carotid artery and external carotid artery separated like other model mice, but only the CCA was ligated.

[0144] (II) Analysis and Testing

[0145] (1) Neurological function score:

[0146] The rats in each experimental group were assessed for neurological function using the Zea Longa scoring method. Normal function with no neurological deficits (0 points); inability to fully extend the forelimb contralateral to the injury (1 point); rotation of the body towards the paralyzed side while walking (2 points); tilting of the body towards the paralyzed side while walking (3 points); inability to walk spontaneously and loss of consciousness (4 points). See Table 2 for detailed results.

[0147] Table 2. Neurological function score results:

[0148]

[0149] As can be seen from the above, compared with the sham-operated group, the model group rats suffered significant damage to nerve cells after cerebral ischemia, resulting in paralysis symptoms. The prophylactic use of NCU16V and the positive control drug butylphenol can effectively alleviate this symptom and relieve the paralysis symptoms caused by cerebral ischemia in rats.

[0150] (2) TTC staining to observe brain tissue death

[0151] The brain tissue of rats in each experimental group after cerebral ischemia modeling was observed by TTC staining, specifically as follows: Figure 11 As shown.

[0152] Figure 11 TTC staining images of rat brain tissue from the sham-operated group, model group, Pediococcus pentosaceus NCU16V group, and positive group provided in Example 4 of this invention.

[0153] in: Figure 11 Group A represents the sham surgery group; Figure 11 B in the diagram represents the model group; Figure 11 C in the middle is the NCU16V group; Figure 11 Group D was the positive group (butylphenol group). The brain tissue death area of ​​rats after cerebral ischemia modeling in each experimental group is shown in Table 3.

[0154] Table 3. Area of ​​brain tissue death in rats after cerebral ischemia modeling in each experimental group:

[0155]

[0156] As shown above, compared to the sham-operated group, the model group rats experienced 50.47% brain cell death. Prophylactic use of NCU16V and positive control drugs significantly reduced brain cell death after cerebral ischemia, resulting in a smaller area of ​​brain cell damage. This indicates that prophylactic use of the *Pediococcus pentosaceus* strain NCU16V described in this application can significantly protect against neuronal damage caused by cerebral ischemia.

[0157] (3) Detection of calcium ions in ischemic brain tissue cells

[0158] The calcium ion concentration in the brain tissue of rats after cerebral ischemia modeling in each experimental group was analyzed by Fluo-4 AM staining. Specifically, ... Figure 12 As shown.

[0159] Figure 12 Fluo-4 AM staining images of calcium ions in rat brain tissue from the sham-operated group, model group, Pediococcus pentosaceus NCU16V group, and positive group provided in this embodiment.

[0160] As shown in the figure above, the intracellular calcium ion concentration in the nerve cells of rats in the sham-operated group was low. After cerebral ischemia, the intracellular calcium ion concentration in the rat nerve cells increased significantly, indicating that calcium ion influx occurred in nerve cells after ischemia, and the cytoplasmic calcium ion concentration increased. The high concentration of calcium ions can further induce inflammatory response. However, after supplementing with NCU16V and using positive control drugs, the fluorescence intensity of intracellular calcium ion concentration in nerve cells decreased significantly, indicating that NCU16V can alleviate nerve cell damage and protect nerve cells from damage after cerebral ischemia.

[0161] (4) Analysis of apoptosis factor expression

[0162] The specific detection method was as follows: Real-time quantitative PCR was used to analyze the gene expression levels of Bax, Bcl-2, and Caspase-3. Primers are shown in Table 4, and gene expression levels are as follows: Figure 13 As shown:

[0163] Table 4. Primer sequences for real-time PCR:

[0164]

[0165] Figure 13 The graph shows the expression of apoptosis factors in the brain tissue cells of rats in the sham-operated group, model group, Pediococcus pentosaceus NCU16V group, and positive group provided in this embodiment.

[0166] As shown in the attached figures, after cerebral ischemia, the expression of two apoptosis factors, Bax and Caspase-3, was significantly increased, while the expression of the apoptosis-inhibiting gene Bcl-2 was significantly decreased, indicating severe neuronal death. When NCU16V was used, the expression of apoptosis genes was significantly reduced, while the expression of apoptosis-inhibiting genes was increased, indicating that supplementing with NCU16V can significantly inhibit neuronal apoptosis after cerebral ischemia and protect neuronal cells from neuronal damage after ischemia.

[0167] Example 5

[0168] (I) Preparation of fermentation products of Pediococcus pentosaceus NCU16V ginkgo biloba:

[0169] (1) Preparation of raw pulp: Remove the shells from fresh ginkgo nuts, add water at a mass ratio of 1:5 (m / m) and then pulp to obtain ginkgo raw pulp.

[0170] (2) Fermentation: Pediococcus pentosaceus NCU16V was activated using MRS liquid medium. Ginkgo biloba pulp was sterilized at 85℃ for 30 min, cooled, and then inoculated for fermentation. The viable count was 1×10⁻⁶. 7 ~1×10 9 The inoculum of *Pediococcus pentosaceus* NCU16V was 1-2%, the fermentation temperature was 37℃, and the fermentation time was 48 h to obtain the fermented product of ginkgo.

[0171] (3) After fermentation, the fermented ginkgo product is sterilized at 500 MPa for 5 min and then frozen at -20℃. Thaw before use.

[0172] (II) Preparation of raw pulp: Remove the shells from fresh ginkgo nuts, add water at a mass ratio of 1:5 (W / W), and then pulp to obtain ginkgo raw pulp.

[0173] (III) At the same time, this experimental example provides "Lactobacillus plantarum NCU0011190" as a control to show that the Pediococcus pentosaceus strain NCU16V screened in this application has better antithrombotic effect than the existing Ginkgo biloba fermentation strain.

[0174] The plant lactobacillus ( Lactiplantibacillus plantarum The strain is NCU0011190, with accession number CGMCC No. 33046.

[0175] It should be noted that the *Lactobacillus plantarum* NCU0011190 of this application was deposited at the China General Microbiological Culture Collection Center (CGMCC) on December 12, 2024, with accession number CGMCC No. 33046 and deposit date of December 12, 2024. The deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0176] Preparation of fermentation products of ginkgo biloba by Lactobacillus plantarum NCU0011190:

[0177] (1) Preparation of raw pulp: Remove the shells from fresh ginkgo nuts, add water at a mass ratio of 1:5 (m / m) and then pulp to obtain ginkgo raw pulp.

[0178] (2) Fermentation: Lactobacillus plantarum NCU0011190 was activated using MRS liquid medium. Ginkgo biloba pulp was sterilized at 85℃ for 30 min, cooled, and then inoculated for fermentation. The viable count was 1×10⁻⁶. 7 ~1×10 9 The inoculum of *Lactobacillus plantarum* NCU0011190 was 1-2%, the fermentation temperature was 37℃, the fermentation time was 48 h, and after fermentation, it was sterilized by ultra-high pressure at 500 MPa for 5 min to obtain the fermented product of ginkgo.

[0179] (IV) Experimental Protocol: Sixty Kunming mice (18-20g) were randomly divided into four groups: blank group, model group, original pulp group, NCU16V ginkgo fermentation product group, NCU0011190 ginkgo fermentation product group, and positive control group, with 10 mice in each group; among them:

[0180] 1. The control group was given an equal volume of physiological saline by gavage daily;

[0181] 2. The model group was administered an equal volume of physiological saline by gavage daily;

[0182] 3. Ginkgo pulp group: Ginkgo pulp (15g / kg bw / d) from step (II) above was administered by gavage daily.

[0183] 4. NCU16V Ginkgo Fermentation Product Group: Daily gavage administration of the above steps (I) of Pediococcus pentosaceus NCU16V Ginkgo fermentation product (15g / kg bw / d).

[0184] 5. NCU0011190 Ginkgo Fermentation Product Group: Daily gavage administration of the above step (III) of Lactobacillus plantarum NCU0011190 Ginkgo fermentation product (15g / kg bw / d).

[0185] 6. Positive control group: Aspirin (30 mg / kg bw / d) was administered by gavage daily.

[0186] After administering the above dosage by gavage for 2 weeks (14 days), modeling was initiated. One hour after administration on day 14, mice in the model group, original pulp group, NCU16V ginkgo fermentation product group, NCU0011190 ginkgo fermentation product group, and positive control group were intraperitoneally injected with 0.06% carrageenan saline solution (100 mg / kg). The blank group was not injected.

[0187] On day 16, the formation of black tails in mice was observed. Blood was collected by enucleation and serum separation was performed to detect platelet p-selectin expression and serum inflammatory factors. At the same time, the total length of the mouse tail and the length of the black tail were measured with a ruler and photographed.

[0188] (iv) The experimental results are as follows:

[0189] Figure 14 This is a comparative experimental diagram showing the effects of the blank group, model group, original pulp group, NCU16V ginkgo fermentation product group, NCU0011190 ginkgo fermentation product group and positive control group on the black tail of mice provided in Example 5 of the present invention.

[0190] Depend on Figure 14 The results showed that no thrombus formation occurred in the tails of mice in the control group, while almost all tails of mice in the model group developed black tails, indicating severe thrombus formation. The NCU16V and positive control groups significantly reduced the length of the black tails, inhibiting thrombus formation. This indicates that NCU16V supplementation can effectively prevent carrageenan-induced thrombosis in mice.

[0191] Table 5. Plasma levels of p-selectin and inflammatory factors in mice:

[0192]

[0193] The levels of p-selectin, TNF-α, and IL-1β were detected using the ElisA kit, as shown in the table above. In the model group, p-selectin levels increased sharply, indicating severe platelet aggregation and increased thrombus formation, while the levels of inflammatory factors were significantly increased. In contrast, the NCU16V ginkgo fermentation product group significantly reduced the levels of platelet p-selectin and inflammatory factors.

[0194] Meanwhile, the NCU16V group of this application showed significantly better results than the Lactobacillus plantarum NCU0011190 group, which fully demonstrates that the Pediococcus pentosaceus strain NCU16V screened in this application has a better antithrombotic effect than the ginkgo fermentation product obtained by existing ginkgo fermentation strains.

Claims

1. A type of Pediococcus pentosaceus with preventive effects against thrombosis and cerebral ischemia-reperfusion injury ( Pediococcus pentosaceus strain NCU16V, characterized in that, The Pediococcus pentosaceus strain NCU16V was deposited at the China General Microbiological Culture Collection Center (CGMCC) on July 28, 2025, with accession number CGMCC No. 35427 and deposit date of July 28, 2025. The deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

2. A microbial inoculant, characterized in that, The microbial agent includes the Pediococcus pentosaceus strain NCU16V as described in claim 1 and excipients.

3. The use of the Pediococcus pentosaceus strain NCU16V as described in claim 1 or the microbial agent as described in claim 2 in the preparation of a drug for preventing thrombosis.

4. The use of the Pediococcus pentosaceus strain NCU16V as described in claim 1 or the microbial agent as described in claim 2 in the preparation of a drug to alleviate brain tissue damage after cerebral ischemia.

5. The application according to claim 4, characterized in that, The relief of brain tissue damage after cerebral ischemia includes: reducing brain cell apoptosis caused by cerebral ischemia.

6. The use of the Pediococcus pentosaceus strain NCU16V as described in claim 1 or the microbial agent as described in claim 2 in the preparation of a ginkgo fermentation product with antithrombotic effects.

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