Lactobacillus crispatus for improving thrombosis and vascular health and its products and applications

Abstract

The present invention discloses a Lactobacillus crispatus strain for improving thrombosis and vascular health and its products and applications, and relates to the technical field of microorganisms. The present invention discloses a Lactobacillus crispatus strain, which is Lactobacillus crispatus ( Lactobacillus crispatus ) NHNK‑608, which was deposited in the China Center for Type Culture Collection on March 7, 2024, and its deposit number is CCTCC NO: M 2024421. Experiments have shown that NHNK‑608 has the functions of dissolving blood clots, promoting the production of urokinase, increasing the anticoagulant factor of vascular endothelial cells, promoting the growth of vascular endothelial cells, promoting the repair of oxidative damage of vascular endothelial cells, and reducing inflammation and apoptosis of vascular endothelial cells. The bacterial biofilm increases the colonization ability of mucin and intestinal epithelial cells, and can be used to prepare products that improve blood clots and vascular health.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a Lactobacillus crispatus that improves thrombus and blood vessel health, and a product and application thereof. Background Art

[0002] Sea cucumber is an aquatic species with extremely high nutritional and economic value. The rapid development of sea cucumber farming has become a very important part of marine aquaculture. Edible species usually contain rich symbiotic bacteria, which are involved in the host's nutrient absorption, energy metabolism, growth and health. The study found that the imitation sea cucumber ( Japanese Apostichopus ) had the highest richness in the gut bacterial community. Marine microbes have developed complex molecular adaptations to cope with these harsh conditions, affecting their primary and secondary metabolic pathways. This has led to the evolution of unique physiological traits and metabolic processes, and marine microbes are more likely to synthesize structurally unique enzymes and secondary metabolites than terrestrial microbes.

[0003] Thrombosis refers to a clot formed by blood components in the human body in blood vessels or the heart. Thrombotic diseases include myocardial infarction, cerebral thrombosis, pulmonary embolism, limb artery embolism, limb vein thrombosis, disseminated intravascular coagulation, etc., which seriously endanger the patient's physical and mental health.

[0004] After the coagulation factors react with fibrinogen, the formed reticular fibrin can entangle blood cells and platelets to form clots, and then form thrombi. There is an anticoagulant system in the blood that dissolves fibrin and resists its dissolution. Thrombin modulator (TM) in the anticoagulant system and tissue plasminogen activator (tPA) in the fibrinolytic system are important proteins that inhibit and clear thrombi, and can reduce damage to vascular endothelial cells by fighting free radicals. The anticoagulant system can remove unnecessary thrombi and make blood vessels smooth. Weak fibrinolysis can lead to thrombus formation or excessive fibrin precipitation; excessive fibrinolysis can cause excessive consumption of blood coagulation factors. The fibrinolytic system is of great significance in limiting the expansion of blood coagulation range and maintaining blood flow.

[0005] When blood vessels are damaged, inflammatory cells will secrete inflammatory cytokines (such as IL-6, TNF-α), which will further accelerate the apoptosis of vascular stromal cells and smooth muscle cells. This also shows that inflammatory cytokines will cause secondary damage to the blood vessel wall.

[0006] The B-cell lymphoma-2 (Bcl2) gene family and its related proteins are the earliest genes related to apoptosis, and are also the most important gene family regulating cell apoptosis. The BCL-2 gene inhibits apoptosis caused by various factors by regulating mitochondrial permeability and other mechanisms, thereby prolonging cell viability. BCL-2 is an important member of the anti-apoptotic protein subfamily, which has the function of inhibiting cell apoptosis / promoting cell survival. It is generally located in mitochondria or endoplasmic reticulum, and BCL-2 can block the apoptotic program. BCL-2 and the caspase (Caspase) family play an important role in regulating cell apoptosis. Modern studies have shown that the CASPASE cascade system is directly involved in mediating cell apoptosis, and plays a vital role in the signal induction, transmission and amplification of different pathways of cell apoptosis. CASPASE-3 is involved in regulating a variety of programmed death modes including apoptosis, pyroptosis, autophagy and necrosis. CASPASE-3 is a key executor of the cell apoptosis pathway and mediates cell apoptosis.

[0007] Marine microorganisms are one of the earliest sources of marine bioactive substances known to people, including algae, bacteria, fungi, protozoa and other microorganisms. These bioactive substances have important application value in various fields such as medicine, food, and cosmetics. Therefore, it is of great practical significance to provide a marine industry-related product developed using microbial technology. Summary of the invention

[0008] In view of this, the present invention provides a Lactobacillus crispatus and a product and application thereof for improving thrombosis and vascular health.

[0009] The present invention provides Lactobacillus crispatus ( Lactobacillus crispatus ), which is Lactobacillus crispatus ( Lactobacillus crispatus ) NHNK-608, which was deposited in the China Center for Type Culture Collection (CCTCC for short, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Wuhan University, Postal Code 430072) on March 7, 2024, and its preservation number is CCTCC NO: M 2024421 of Lactobacillus crispatus).

[0010] Further, according to the above-mentioned Lactobacillus crispatus ( Lactobacillus crispatus ) prepared by Lactobacillus crispatus ( Lactobacillus crispatus ) of live bacteria, fermented bacterial liquid or fermentation / secretion product, wherein the preparation method of the live bacteria is: the Lactobacillus crispatus ( Lactobacillus crispatus ) inoculated into a culture medium, cultured at 37° C. for 46 to 50 h, obtained a fermentation broth, centrifuged to obtain a precipitate, and washed the precipitate to obtain the viable bacteria;

[0011] The preparation method of the fermented bacterial liquid is as follows: Lactobacillus crispatus ) was inoculated into the culture medium and cultured at 37°C for 46-50h to obtain the fermentation liquid;

[0012] The method for preparing the fermentation / secretion product comprises: Lactobacillus curly ) was inoculated into the culture medium and cultured at 37°C for 46-50 h to obtain the fermentation broth. The supernatant was collected by centrifugation and filtered at a precision of 0.22 μm. The filtrate obtained was the fermentation / secretion product.

[0013] The present invention also proposes the above-mentioned Lactobacillus crispatus ( Lactobacillus crispatus ) or the above-mentioned Lactobacillus crispatus ( Lactobacillus crispatus ) The use of the prepared product in the preparation of products for improving thrombosis.

[0014] Furthermore, the improvement of thrombosis includes any one or more of dissolving thrombus, promoting the production of urokinase and increasing anticoagulant factor of vascular endothelial cells.

[0015] The present invention also proposes the above-mentioned Lactobacillus crispatus ( Lactobacillus crispatus ) or the above-mentioned Lactobacillus crispatus ( Lactobacillus crispatus ) Use of the prepared product in the preparation of products for improving vascular health.

[0016] Furthermore, the improvement of vascular health includes any one or more of promoting the growth of vascular endothelial cells, promoting the repair of oxidative damage of vascular endothelial cells, and reducing inflammation and apoptosis of vascular endothelial cells.

[0017] The present invention also proposes the above-mentioned Lactobacillus crispatus ( Lactobacillus crispatus ) in any of the following:

[0018] (1) Colonization of mucosal mucin;

[0019] (2) Bacterial biofilm increases the ability to colonize mucosal mucins and intestinal epithelial cells.

[0020] The present invention also provides a product for improving thrombosis and vascular health, comprising the above-mentioned Lactobacillus crispatus ( Lactobacillus crispatus ) or the above-mentioned preparations, and acceptable excipients and / or adjuvants.

[0021] The Lactobacillus crispatus disclosed in the present invention ( Lactobacillus crispatus), and its deposit number is CCTCC NO:M 2024421. Experiments have shown that NHNK-608 has the functions of dissolving blood clots, promoting the production of urokinase, increasing anticoagulant factors in vascular endothelial cells, promoting the growth of vascular endothelial cells, promoting the repair of oxidative damage in vascular endothelial cells, and reducing inflammation and apoptosis of vascular endothelial cells. The bacterial biofilm increases the colonization of mucin and intestinal epithelial cells, and can be used to prepare products that improve blood clots and vascular health.

[0022] Biological Deposit Description

[0023] Lactobacillus crispatus ( Lactobacillus crispatus ) NHNK-608, was deposited in the China Center for Type Culture Collection (CCTCC for short, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Wuhan University, Postal Code 430072) on March 7, 2024, and its deposit number is CCTCC NO: M 2024421. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 For the Lactobacillus crispatus ( Lactobacillus crispatus ) Artificial thrombus plate image of NHNK-608;

[0025] Figure 2 For the Lactobacillus crispatus ( Lactobacillus crispatus ) NHNK-608 bacterial liquid and biofilm adsorption diagram of mucin, where (a) is Lactobacillus crispatus ( Lactobacillus crispatus ) The adsorption of NHNK-608 bacterial solution to mucin, (b) Lactobacillus crispatus ( Lactobacillus crispatus ) Adsorption of NHNK-608 biofilm to mucin;

[0026] Figure 3 For the Lactobacillus crispatus ( Lactobacillus crispatus ) Adsorption diagram of NHNK-608 bacterial liquid and biofilm on intestinal epithelial cells, among which (a) is Lactobacillus crispatus ( Lactobacillus crispatus ) NHNK-608 bacterial solution adsorption to intestinal epithelial cells, (b) Lactobacillus crispatus ( Lactobacillus crispatus ) Adsorption of NHNK-608 biofilm to intestinal epithelial cells.

[0027] The following specific implementation manner will further illustrate the present invention in conjunction with the above-mentioned drawings. DETAILED DESCRIPTION

[0028] The present invention provides Lactobacillus crispatus and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

[0029] The fermented mucus lactobacillus NHNK-608 of the present invention is derived from the intestinal tract of sea cucumber and is identified as Lactobacillus crispatus ( Lactobacillus crispatus ). This strain is Gram-positive and slightly curved in rod shape under a microscope. It is suitable for static growth in MRS liquid medium at 37°C, and the bacteria present white precipitation. When grown on MRS plates, it can form smooth white semi-transparent circular colonies with neat surface edges.

[0030] Lactobacillus crispatus ( Lactobacillus crispatus )NHNK-608, depository unit: China Center for Type Culture Collection, address: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, deposit date: March 7, 2024, deposit number: CCTCC NO: M 2024421.

[0031] Furthermore, in the application described in the present invention, the Lactobacillus crispatus NHNK-608 provided by the present invention exists in the form of being sterilized or not, or in the form of a fermentation product (i.e., supernatant), and the derivative form is preferably selected from: metabolites, metabolic biological products, prebiotics, cell walls and components thereof, extracellular polysaccharides, and compounds containing immunogenic components, preferably selected from: live bacteria, fermentation broth, and fermentation / secretion products.

[0032] In vitro experiments show that the Lactobacillus crispatus NHNK-608 of the present invention has the effect of dissolving thrombus. NHNK-608 has fibrinolytic activity and can form a transparent circle on an artificial thrombus plate, and the diameter of the transparent circle is 7.22 mm to 7.37 mm.

[0033] In vitro experiments show that the Lactobacillus crispatus NHNK-608 of the present invention has the ability to upregulate the gene of urokinase-type plasma plasminogen activator in human renal tubular epithelial cells HKC-1. UPA The relative expression multiple was 1.17 to 1.26 times.

[0034] In vitro experiments show that the Lactobacillus crispatus NHNK-608 of the present invention has the ability to upregulate the endothelial nitric oxide synthase gene of human umbilical vein cell EA.hy926 fusion cells. ENOS , thrombomodulin gene TM, Tissue-type plasminogen activator gene TPA The expression has a relative expression multiple of 3.64 to 8.35 times.

[0035] In vitro experiments showed that Lactobacillus crispatus NHNK-608 of the present invention has the effect of promoting the growth of EA.hy926, a human umbilical vein endothelial cell fusion cell, with a cell growth rate of 10.36% - 14.83%.

[0036] In vitro experiments showed that Lactobacillus crispatus NHNK-608 of the present invention has the effect of down-regulating the expression of tumor necrosis factor gene, an inflammation-related factor of EA.hy926, a human umbilical vein cell fusion cell TNF-α , interleukin-6 gene IL-6 and caspase-3 gene, an apoptosis factor CASPASE-3 The expression has a relative expression multiple of 0.49 to 0.82 times. It up-regulates the expression of B-cell lymphoma-2 gene, an anti-apoptosis related factor BCL-2 The expression has a relative expression multiple of 1.47 to 1.97 times.

[0037] In vitro experiments showed that Lactobacillus crispatus NHNK-608 of the present invention has the effect of promoting the repair of oxidative damage of EA.hy926, a human umbilical vein endothelial cell fusion cell, and promoting the production of superoxide dismutase SOD and glutathione GSH by EA.hy926 cells with oxidative damage, with a growth rate of 13.73% - 29.17%.

[0038] In vitro experiments showed that Lactobacillus crispatus NHNK-608 of the present invention has the ability to increase the colonization ability of the bacterial biofilm on mucin and intestinal epithelial cells.

[0039] The reagents and consumables used in the present invention are all ordinary commercially available products. Now, in combination with the examples, the present invention will be further elaborated:

[0040] Example 1 Isolation of NHNK-608

[0041] Samples of live Apostichopus japonicus were taken from Qingdao market. The body surface of the sea cucumber was wiped with a cotton ball dipped in sterile normal saline. The intestine of the sea cucumber was taken out and cut into pieces with sterile surgical scissors and scalpel, and rinsed 2 - 3 times with 0.9% sterile normal saline. The rinsed and cut sea cucumber intestine was put into a triangular flask containing MRS liquid medium with small glass beads for enrichment culture at 37°C and 160 r / min on a shaker for 24 h. An appropriate amount of the cultured bacterial liquid was spread on MRS solid medium and placed in an anaerobic bag. After incubation at 37°C for 48 h, white colonies were picked and repeatedly streaked and screened until uniform single colonies were obtained, which were named NHNK-608.

[0042] Gram staining microscopy: The strain NHNK-608 is a Gram-positive colony, showing slender rods under the microscope, slightly bent into chains, without flagella and spores; it grows on MRS plates, forming white colonies with irregular edges, in flakes, and convex in the center; it can grow evenly and turbidly in MRS medium, and the bacteria form white precipitates after long-term placement.

[0043] Example 2 Nucleic acid identification of NHNK-608

[0044] 1. 16S rDNA gene sequence analysis:

[0045] Pick a single colony and inoculate it into MRS liquid medium, culture overnight at 37 °C, centrifuge at 8000 rpm for 1 min to collect the bacteria, and operate according to the instructions of the Gram-positive bacteria DNA extraction kit. The primers used are the universal primers 27F and 1492R for bacterial 16S sequencing, and the PCR amplification system is 20 μL. The PCR amplification program is pre-denaturation at 95 °C for 5 min, 94 °C for 15 s, 57 °C for 15 s, 72 °C for 1 min, for 35 cycles; extension at 72 °C for 10 min.

[0046] 2. Results

[0047] After sequencing the PCR product and comparing its homology with the published standard sequences in the GenBank database (BLASTN), it was found that the strain NHNK-608 is Lactobacillus crispatus ( Lactobacillus crispatus ).

[0048] Example 3 Fibrinolytic activity of NHNK-608

[0049] 1. Preparation of NHNK-608 fermented broth

[0050] Pick a single colony of NHNK-608 and inoculate it into MRS liquid medium, statically culture at 37 °C for 48 h, and adjust it to OD 600 = 1.0 with MRS to obtain the fermented broth.

[0051] 2. Artificial thrombus plate medium

[0052] Sample A: Dissolve 22 mg of fibrinogen in 10 mL of physiological saline and water bath at 37 °C for 5 - 10 min;

[0053] Sample B: Add 5 mg of thrombin (concentration 40 U / mg) to 2 mL of physiological saline and water bath at 37 °C for 5 - 10 min;

[0054] Sample C: Weigh 0.1 g of agarose and dissolve it in 8 mL of physiological saline, and heat to fully dissolve the agarose.

[0055] Quickly add sample B into sample C, and finally add sample A. Pour the mixture into a plate after mixing evenly. Wait for the artificial thrombus plate to solidify before use.

[0056] 3. NHNK-608 fibrinolytic activity assay

[0057] Take 2μL of NHNK-608 fermented bacteria liquid and apply it on the artificial thrombus plate, set up three parallels, and apply an equal volume of MRS to the control group, and incubate in a 37℃ incubator for 24h. Fibrinogen will form fibrin artificial thrombus through the action of thrombin. After the incubation, observe whether there is a transparent circle formed by dissolving the artificial thrombus, and measure the diameter of the transparent circle.

[0058] The results are as follows:

[0059]

[0060] like Figure 1 As shown, NHNK-608 has plasminogen activitv, which makes the artificial thrombus plate present a transparent circle with a diameter of 7.22 mm to 7.37 mm, and can dissolve the fibrin artificial thrombus formed by thrombin.

[0061] Example 4 NHNK-608 upregulates the expression of urokinase-related genes in human renal tubular epithelial cells

[0062] 1. Cell culture

[0063] Human renal tubular epithelial cells HKC-1 were cultured in DMEM medium containing 10% (v / v) FBS at 37°C and 5% CO2. When the cell confluence reached 80-90%, the cells were passaged.

[0064] 2. Preparation of NHNK-608 fermentation products

[0065] A single colony of NHNK-608 was selected and placed in MRS liquid medium. After culturing at 37°C for 48 h, the OD was adjusted to 0. 600 =1.0, 5000rpm to take the supernatant, and then filter with a 0.22μm filter membrane to obtain a sterile fermentation product. The prepared fermentation product was added to the DMEM culture medium at a volume ratio of 0.2%.

[0066] 3. NHNK-608 fermentation products regulate the expression of urokinase gene in human renal tubular epithelial cells HKC-1

[0067] When the confluence of HKC-1 cells reached about 90%, the adherent cells were digested into a cell suspension using 0.25% trypsin and added to a 6-well plate. The cells were cultured for 24 h at 37°C and 5% CO2. 2 ml of DMEM medium containing 0.2% (v / v) NHNK-608 fermentation product was added to the experimental group, and an equal volume of DMEM medium containing 0.2% (v / v) MRS was added to the control group. The cells were collected after 24 h of culture.

[0068] The collected HKC-1 cells were used to extract RNA using the Trizol method, and the RNA was reverse transcribed into cDNA using a kit. GAPDH As the internal reference gene, fluorescence quantitative PCR was used to determine urokinase-related genes UPA The relative expression of the control group gene is F=1, using 2 -ΔΔCT The F value of each sample is calculated by this method. Formula: F=2 -ΔΔCT ,in:

[0069] △CT 实验 =CT 实验 -CT 内参（实验） ;

[0070] △CT 对照 =CT 对照 -CT 内参（对照） ;

[0071] △△CT=△CT 实验 -△CT 对照 .

[0072] The results are as follows:

[0073]

[0074] The results showed that NHNK-608 fermentation products could upregulate the urokinase-related gene in HKC-1 cells UPA The expression level of urokinase was upregulated to 1.17-1.26 times, promoting the production of urokinase to enhance fibrinolytic activity.

[0075] Example 5 NHNK-608 regulates the expression of anticoagulant factor-related genes in endothelial cells EA.hy926

[0076] 1. Endothelial cell culture

[0077] Human umbilical vein endothelial cell fusion cells (EA.hy926) were cultured in DMEM medium containing 10% (v / v) fetal bovine serum (FBS) at 37°C and 5% CO2. When the cell fusion reached 80-90%, subculture was performed.

[0078] 2. Preparation of NHNK-608 fermentation products

[0079] The preparation method of NHNK-608 fermentation product is referred to Example 4.

[0080] 3. NHNK-608 fermentation products regulate the expression of anticoagulant factor-related genes in EA.hy926

[0081] When the fusion degree of human umbilical vein endothelial cell EA.hy926 reached about 90%, the cells were collected with trypsin, and the cell suspension was added to a 6-well plate and cultured for 24 hours at 37°C and 5% CO2. After the culture, the culture medium was removed and washed once with sterile PBS. 2 ml of DMEM culture medium containing 0.2% (v / v) NHNK-608 fermentation product was added to the experimental group, and an equal volume of DMEM culture medium containing 0.2% (v / v) MRS was added to the control group. The cells were collected after overnight culture.

[0082] 4. Fluorescence quantitative PCR and data processing

[0083] RNA was extracted from cells using the Trizol method and reverse transcribed into cDNA. GAPDH As the internal reference gene, qPCR fluorescence quantitative technology was used to detect anticoagulant factor-related genes TM , ENOS and TPA The relative expression of the control group gene is F=1, using 2 -ΔΔCT The F value of each sample is calculated by this method. Formula: F=2 -ΔΔCT .

[0084] The results are shown in the table below:

[0085]

[0086] The results showed that NHNK-608 fermentation products could upregulate anticoagulant factor-related genes in EA.hy926 cells TM , ENOS and TPA expression, increasing anticoagulant factors to reduce thrombosis.

[0087] Example 6 NHNK-608 promotes the growth of endothelial cells EA.hy926

[0088] 1. Endothelial cell culture

[0089] The cell culture method is as in Example 5.

[0090] 2. Preparation of NHNK-608 fermentation products

[0091] The preparation method of NHNK-608 fermentation product is referred to Example 4.

[0092] 3. Experiment on promoting the growth of EA.hy926 by fermentation products of NHNK-608

[0093] When the fusion degree of human umbilical vein endothelial cell EA.hy926 reached about 90%, it was digested with trypsin to form a cell suspension, and the cell suspension was adjusted to 20,000 cells / mL with complete culture medium. 100 μl of cell suspension was added to a 96-well plate, and the 96-well plate was placed at 37°C and 5% CO2 for 24 hours. After 24 hours, the culture medium in the 96-well plate was discarded, and the control group was an equal volume of DMEM culture medium containing 0.2% (v / v) MRS, and culture was continued for 24 hours. After the culture was completed, the cell number was detected according to the steps in the CCK-8 kit manual, and the cell growth rate (%) = (experimental group-control group) / control group × 100%.

[0094] The results are as follows:

[0095]

[0096] The results showed that the fermentation products of NHNK-608 promoted the growth of EA.hy926 cells.

[0097] Example 7 NHNK-608 regulates the expression of genes related to oxidative damage in endothelial cells EA.hy926

[0098] 1. Endothelial cell culture

[0099] The cell culture method is as in Example 5.

[0100] 2. Preparation of NHNK-608 fermentation products

[0101] The preparation method of NHNK-608 fermentation product is referred to Example 4.

[0102] 3. NHNK-608 fermentation products regulate the expression of genes related to oxidative damage in endothelial cells EA.hy926

[0103] When the fusion degree of human umbilical vein endothelial cell EA.hy926 reached about 90%, the adherent cells were digested into a cell suspension using 0.25% trypsin and added to a 6-well plate, and cultured at 37°C and 5% CO2 for 24 hours. After the culture, 2 ml of DMEM medium containing 1.5 mM H2O2 was added, and incubated at 37°C and 5% CO2 for 3 hours. The culture medium was discarded after 3 hours. The experimental group was added with DMEM medium containing 0.2% (v / v) NHNK-608 fermentation product, and the control group was added with an equal volume of DMEM medium containing 0.2% (v / v) MRS, and culture was continued for 24 hours.

[0104] After culture, cells were collected, cellular RNA was extracted using the Trzol method, and RNA was reverse transcribed into cDNA using a kit. GAPDH As the internal reference gene, qPCR fluorescence quantitative technology was used to detect the pro-inflammatory factor genes TNF-α , IL-6 , CASPASE-3 and anti-apoptotic factor genes BCL-2 The relative expression of the control group gene is F=1, using 2 -ΔΔCT The F value of each sample was calculated by this method.

[0105] The results are as follows:

[0106]

[0107] The results showed that NHNK-608 fermentation products could inhibit the inflammatory response and cell apoptosis induced by H2O2 and downregulate the genes of pro-inflammatory factors. TNF-α , IL-6 and apoptosis genes CASPASE-3 Upregulation of anti-apoptotic factor genes BCL-2 expression.

[0108] Example 8 NHNK-608 promotes the repair of oxidatively damaged vascular endothelial cells EA.hy926

[0109] 1. Endothelial cell culture

[0110] The cell culture method is as in Example 5.

[0111] 2. Preparation of NHNK-608 fermentation products

[0112] The preparation method of NHNK-608 fermentation product is referred to Example 4.

[0113] 3. Repair of oxidative damage of EA.hy926 by NHNK-608 fermentation products

[0114] When the confluence of EA.hy926 reached about 90%, the adherent cells were digested with trypsin into a cell suspension and added to a 6-well plate, and cultured at 37°C and 5% CO2 for 24 hours. After the culture, 2 ml of DMEM medium containing 1.5 mM H2O2 was added, and the cells were incubated at 37°C and 5% CO2 for 3 hours. The H2O2 culture medium was discarded after 3 hours. The experimental group was added with DMEM medium containing 0.2% (v / v) NHNK-608 fermentation product, and the control group was added with an equal volume of DMEM medium containing 0.2% (v / v) MRS, and the culture was continued for 24 hours.

[0115] The cell culture medium was centrifuged at 4°C and 1200 rpm for 10 min to obtain the supernatant, and the contents of antioxidant factors superoxide dismutase SOD and glutathione GSH in the cell supernatant were detected using a kit.

[0116] Increase rate (%) = (experimental group - control group) / control group × 100%

[0117] The results are as follows:

[0118]

[0119] The results showed that NHNK-608 fermentation products could increase the secretion of antioxidant factors such as superoxide dismutase SOD and glutathione GSH by vascular endothelial cells, and promote the repair of oxidative damage to vascular endothelial cells.

[0120] Example 9 NHNK-608 forms biofilm

[0121] 1. Formation of NHNK-608 biofilm

[0122] Pick a single colony of NHNK-608 and place it in MRS liquid medium, incubate at 37°C for 24 h, and adjust the OD to 0. 600 =0.2, 100 μl of bacterial solution was added to each well of a 96-well plate, with 3 parallels per group, and then cultured at 37 degrees for 24 hours.

[0123] 2. Crystal violet staining

[0124] After the incubation, discard the supernatant, add 100 μL sterile PBS to each well and wash twice, then add 100 μL 4% paraformaldehyde fixative to each well and fix at room temperature for 30 minutes. Discard the fixative, add 100 μL crystal violet to each well and stain at room temperature for 30 minutes. After staining, wash twice with sterile PBS and dry, add 100 μL anhydrous ethanol to each well, let stand for 1 minute, and measure the absorbance at 600 nm.

[0125] The results are as follows:

[0126] NHNK-608 biofilm formation

[0127]

[0128] The results showed that NHNK-608 in OD 600 =0.2 in MRS medium at 37℃ for 24h to form biofilm.

[0129] Example 10 NHNK-608 adsorbs mucin

[0130] 1. Preparation of NHNK-608 live bacterial suspension

[0131] Pick a single colony of NHNK-608 in fresh MRS medium and culture at 37℃ for 24h. Centrifuge at 5000rpm for 10min to collect the cells, wash twice with sterile PBS, resuspend the cells in DMEM medium and adjust the OD 600 =0.5.

[0132] 2. Preparation of NHNK-608 biofilm

[0133] Pick a single colony of NHNK-608 and place it in MRS liquid medium, incubate at 37°C for 24 h, and adjust the OD to 0. 600 =0.2 was added to the bacterial culture dish, and then cultured at 37 degrees for 24 hours. After the culture was completed, the upper culture medium was discarded, the bottom biofilm was resuspended after washing twice with PBS, and the bacteria were collected by centrifugation at 5000rpm for 10min, and the bacteria were resuspended with DMEM culture medium and the OD was adjusted. 600 =0.5.

[0134] 3. Determination of mucin adsorption capacity

[0135] (1) Reagent preparation

[0136] Mucin solution: Weigh 10 mg of mucin and dissolve it in 10 mL of 50 mM Tris-HCL solution at 4°C overnight.

[0137] Blocking solution: Dissolve bovine serum albumin in PBS solution at a ratio of 2% (m / v).

[0138] Washing solution: Dissolve bovine serum albumin in PBS solution at a ratio of 0.1% (m / v).

[0139] (2) Adsorption experiment of NHNK-608 bacterial liquid and biofilm on mucin

[0140] Soak the coverslip in the mucin solution and coat overnight at 4°C. After coating, discard the supernatant. Wash twice with cleaning solution, soak in blocking solution, and incubate for 2 hours at room temperature. After incubation, rinse twice with cleaning solution. Take 100μL of NHNK-608 live bacterial suspension or biofilm bacterial suspension, add it to the mucin-coated coverslip, and incubate at 37°C for 2 hours. After incubation, rinse twice with cleaning solution to remove non-adherent bacteria. After drying, fix with 4% paraformaldehyde solution for 30 minutes, then perform Gram staining and observe under a microscope.

[0141] like Figure 2The results showed that NHNK-608 could adsorb mucin, the main component of the mucosal layer, and that NHNK-608 could increase its adsorption capacity for mucin after coating the biofilm formed by itself.

[0142] Example 11 Adsorption of NHNK-608 to intestinal epithelial cells

[0143] 1. Preparation of NHNK-608 live bacterial suspension

[0144] The method for preparing the bacterial suspension is as described in Example 10.

[0145] 2. Preparation of NHNK-608 biofilm

[0146] The biofilm preparation method is described in Example 10.

[0147] 3. Cultivation of intestinal epithelial cells Caco-2

[0148] Human colorectal adenocarcinoma cells Caco-2 were inoculated in DMEM complete medium containing 10% FBS and 1% penicillin-streptomycin double antibody, cultured at 37°C and 5% CO2 for 1-2 days, and the cells were collected when the cell fusion reached 80%~90%. First, a cell slide was placed at the bottom of a 6-well plate, and then the cells were inoculated into the 6-well plate at 1×10^6 / well and cultured for 24 hours. After the cells adhered to the wall, the culture medium was removed, washed twice with PBS, and then 1.5mL DMEM culture medium and 0.5mL NHNK-608 bacterial suspension were added to each well and incubated at 37°C for 2 hours. After the incubation, the slide was removed and fixed with 4% paraformaldehyde solution for 30 minutes, followed by Gram staining and observation under a microscope.

[0149] like Figure 3 The results showed that NHNK-608 could adsorb Caco-2 cells, and the adsorption capacity of NHNK-608 to Caco-2 cells could be increased after the biofilm formed by NHNK-608 itself was coated.

[0150] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A Lactobacillus crispatus ( Lactobacillus crispatus ), which is Lactobacillus crispatus ( Lactobacillus crispatus ) NHNK-608 was deposited in the China Center for Type Culture Collection on March 7, 2024, and its deposit number is CCTCC NO: M 2024421.

2. The Lactobacillus crispatus according to claim 1 ( Lactobacillus crispatus ) is characterized in that The product is Lactobacillus crispatus ( Lactobacillus crispatus ) of live bacteria, fermented bacterial liquid or fermented product, wherein the preparation method of the live bacteria is: the Lactobacillus crispatus ( Lactobacillus crispatus ) inoculated into a culture medium, cultured at 37° C. for 46 to 50 h, obtained a fermentation broth, centrifuged to obtain a precipitate, and washed the precipitate to obtain the viable bacteria; The preparation method of the fermented bacterial liquid is as follows: Lactobacillus crispatus ) was inoculated into the culture medium and cultured at 37°C for 46-50h to obtain the fermentation liquid; The preparation method of the fermentation product is: Lactobacillus crispatus ) was inoculated into the culture medium and cultured at 37°C for 46-50 h to obtain the fermentation broth. The supernatant was collected by centrifugation and filtered at a precision of 0.22 μm. The filtrate obtained was the fermentation product.

3. The Lactobacillus crispatus according to claim 1 ( Lactobacillus crispatus ) or the product according to claim 2 in the preparation of a product for improving thrombosis.

4. The use according to claim 3, characterized in that: The improvement of thrombosis includes any one or more of dissolving thrombus, promoting the production of urokinase and increasing the anticoagulant factor of vascular endothelial cells.

5. The Lactobacillus crispatus according to claim 1 ( Lactobacillus crispatus ) or the product of claim 2 in the preparation of a product for improving vascular health, wherein the improvement of vascular health includes any one or more of promoting the growth of vascular endothelial cells, promoting the repair of oxidative damage of vascular endothelial cells, and reducing inflammation and apoptosis of vascular endothelial cells.

6. The Lactobacillus crispatus according to claim 1 ( Lactobacillus crispatus ) in any of the following: (1) Colonization of mucosal mucin; (2) Bacterial biofilm increases the ability to colonize mucosal mucins and intestinal epithelial cells.

7. A product for improving thrombosis and vascular health, characterized in that: comprising the Lactobacillus crispatus according to claim 1 ( Lactobacillus crispatus ) or the product as claimed in claim 2, and acceptable excipients and / or auxiliary agents.

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