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

Abstract

The present invention discloses a Lactobacillus helveticus strain for improving thrombosis and vascular health and its products and applications, and relates to the field of microbial technology. The present invention discloses a Lactobacillus helveticus strain, which is Lactobacillus helveticus ( Lactobacillus helveticus ) NHNK‑614, which was deposited in the China Center for Type Culture Collection on July 1, 2024, and its deposit number is CCTCC NO:M 20241430. Experiments have shown that NHNK‑614 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 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 helveticus for improving thrombus and blood vessel health, and a product and application thereof. Background Art

[0002] In the fish intestinal microbiome, bacteria (aerobic, facultative anaerobic and obligate anaerobic) are the main colonizing microorganisms. Intestinal microorganisms play a very important role in the growth of fish, and their balance is related to the health of the fish body. The wide variety of microorganisms in the fish intestine play a very important role in inhibiting pathogenic microorganisms and can protect the host fish from pathogenic microorganisms.

[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. In addition, nitric oxide (NO) as a signal molecule can inhibit the activation and aggregation of platelets and reduce damage to vascular endothelial cells by counteracting free radicals. The anticoagulant system can remove unnecessary thrombi and make blood vessels smooth. Weak fibrinolysis leads 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 currently 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 apoptosis program.

[0007] Marine microorganisms 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 characteristics and metabolic processes, and marine microorganisms are more likely to synthesize structurally unique enzymes and secondary metabolites than terrestrial microorganisms. Exploring the application of marine fish gut microorganisms has important practical significance in expanding the value of the marine industry. Summary of the invention

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

[0009] The present invention provides Lactobacillus helveticus ( Lactobacillus helveticus ), which is Lactobacillus helveticus ( Lactobacillus helveticus ) NHNK-614, 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 July 1, 2024, and its deposit number is CCTCC NO: M 20241430 of Lactobacillus helveticus).

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

[0012] The preparation method of the fermentation / secretion product is as follows: the Lactobacillus helveticus ( Lactobacillus helveticus ) 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 helveticus ( Lactobacillus helveticus ) in the preparation of products for improving thrombosis.

[0014] The present invention also proposes the above-mentioned Lactobacillus helveticus ( Lactobacillus helveticus ) The use of the prepared product in the preparation of products for improving thrombosis.

[0015] 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.

[0016] The present invention also proposes the above-mentioned Lactobacillus helveticus ( Lactobacillus helveticus ) in the preparation of products for improving vascular health.

[0017] The present invention also proposes the above-mentioned Lactobacillus helveticus ( Lactobacillus helveticus ) Use of the prepared product in the preparation of products for improving vascular health.

[0018] 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.

[0019] The present invention also proposes the above-mentioned Lactobacillus helveticus ( Lactobacillus helveticus ) in any of the following:

[0020] (1) Colonization of mucosal mucin;

[0021] (2) Bacterial biofilm increases the ability to colonize mucosal mucins and intestinal epithelial cells.

[0022] The present invention also provides a product for improving thrombosis and vascular health, comprising the above-mentioned Lactobacillus helveticus ( Lactobacillus helveticus ), or as mentioned above, Lactobacillus helveticus ( Lactobacillus helveticus ) and acceptable excipients and / or adjuvants.

[0023] The Lactobacillus helveticus disclosed in the present invention ( Lactobacillus helveticus ), and its deposit number is CCTCCNO: M 20241430. Experiments have shown that NHNK-614 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 of mucin and intestinal epithelial cells, and can be used to prepare products that improve blood clots and vascular health.

[0024] Biological Deposit Description

[0025] Lactobacillus helveticus ( Lactobacillus helveticus ) NHNK-614, 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 July 1, 2024, and its deposit number is CCTCC NO: M 20241430. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Lactobacillus helveticus ( Lactobacillus helveticus ) MRS plate colony image and Gram staining image of NHNK-614;

[0027] Figure 2 Lactobacillus helveticus ( Lactobacillus helveticus ) Artificial thrombus plate image of NHNK-614;

[0028] Figure 3 Lactobacillus helveticus ( Lactobacillus helveticus ) Adsorption of NHNK-614 bacterial solution and biofilm to mucin;

[0029] Figure 4 Lactobacillus helveticus ( Lactobacillus helveticus ) Adsorption of NHNK-614 bacterial liquid and biofilm on intestinal epithelial cells.

[0030] The following specific implementation manner will further illustrate the present invention in conjunction with the above-mentioned drawings. DETAILED DESCRIPTION

[0031] The invention provides Lactobacillus helveticus and application thereof. Those skilled in the art can learn from the content of this article and appropriately improve the process parameters to realize. It is particularly noted 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 by preferred embodiments, and the relevant personnel can obviously change 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.

[0032] The fermented mucus lactobacillus NHNK-614 of the present invention is derived from the intestine of the flatfish and is identified as Lactobacillus helveticus ( Lactobacillus helveticus ). This strain is Gram-positive and short rod-shaped under a microscope. When grown on an MRS plate, it forms round colonies with smooth, opaque surfaces, milky white, and neat edges. It grows evenly in MRS liquid culture medium and can produce white precipitates when left to stand for a long time. The optimal growth temperature is 37°C. Lactobacillus helveticus ( Lactobacillus helveticus )NHNK-614, depository unit: China Center for Type Culture Collection, address: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, deposit date: July 1, 2024, deposit number: CCTCCNO: M 20241430.

[0033] Furthermore, in the application described in the present invention, the Lactobacillus helveticus NHNK-614 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 their components, extracellular polysaccharides, and compounds containing immunogenic components, preferably selected from: live bacteria, fermentation broth, fermentation / secretion products.

[0034] In vitro experiments show that the Lactobacillus helveticus NHNK-614 of the present invention has the effect of dissolving thrombus. NHNK-614 has fibrinolytic activity and can form a transparent circle on an artificial thrombus plate, and the diameter of the transparent circle is 7.19 mm to 7.45 mm.

[0035] In vitro experiments show that the Lactobacillus helveticus NHNK-614 of the present invention has the ability to upregulate the urokinase-type plasma plasminogen activator gene of human renal tubular epithelial cells HKC-1. UPA The relative expression multiple was 1.16 to 1.20 times.

[0036] In vitro experiments show that the Lactobacillus helveticus NHNK-614 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 plasminogen activator gene TPA The relative expression multiples were 2.12 to 4.44 times.

[0037] In vitro experiments show that the Lactobacillus helveticus NHNK-614 of the present invention has the effect of promoting the growth of human umbilical vein endothelial cell fusion cells EA.hy926.

[0038] In vitro experiments show that the Lactobacillus helveticus NHNK-614 of the present invention has the ability to down-regulate the tumor necrosis factor gene, an inflammatory factor, in human umbilical vein cell fusion cells EA.hy926. TNF-α , interleukin-6 gene IL-6 The relative expression was 0.65-0.89 times. Up-regulation of anti-apoptosis related factor B lymphocytoma-2 gene BCL-2 The relative expression multiples were 2.83 to 3.48 times.

[0039] In vitro experiments show that the Lactobacillus helveticus NHNK-614 of the present invention has the effect of promoting the repair of oxidative damage in human umbilical vein endothelial cell fusion cells EA.hy926, and promotes the oxidatively damaged EA.hy926 cells to produce superoxide dismutase SOD, glutathione GSH and nitric oxide NO, with a growth rate of 13.73% to 301.74%.

[0040] In vitro experiments show that the Lactobacillus helveticus NHNK-614 of the present invention has the functional ability of increasing the colonization of mucin and intestinal epithelial cells by bacterial biofilm.

[0041] The reagents and consumables used in the present invention are all common commercially available products. Now, in conjunction with the embodiments, the present invention is further described:

[0042] Example 1 Isolation of NHNK-614

[0043] The flathead mullet ( Sebastes schlegelii ), wipe the abdominal surface of the fish with 75% alcohol, cut off the stomach and anterior midgut of the fish with sterile tools, remove the contents, rinse twice with sterile saline, scrape the mucus on the inner wall of each organ, add it to sterile saline, vortex and shake well, take the supernatant and streak it on MRS solid plate, culture at 37℃ for 48h, pick white colonies and repeatedly streak and purify them until a single colony with regular and uniform shape is obtained, which is named NHNK-614.

[0044] Gram staining microscopy: strain NHNK-614 is a Gram-positive colony, which appears as a short rod under a microscope; it grows on an MRS plate and forms milky white, smooth and rounded opaque round colonies with neat edges; it grows evenly in MRS medium and becomes turbid, and the bacteria form white precipitates after being placed for a long time. Figure 1 shown.

[0045] Example 2 Nucleic acid identification of NHNK-614

[0046] 1. 16S rDNA gene sequence analysis:

[0047] Pick a single colony in MRS liquid medium, culture at 37℃ overnight, collect the bacteria by centrifugation at 8000 rpm for 1min, and operate according to the instructions of the Gram-positive bacteria DNA extraction kit. The primers used were bacterial 16S sequencing universal primers 27F and 1492R, and the PCR amplification system was 20μL. The PCR amplification program was 95℃ pre-denaturation for 5min, 94℃ for 15s, 57℃ for 15s, 72℃ for 1min, 35 cycles; 72℃ extension for 10min.

[0048] 2. Results

[0049] The PCR product was sequenced and compared with the standard sequences published in the GenBank database (BLASTN). It was concluded that the NHNK-614 strain was Lactobacillus helveticus ( Lactobacillus helveticus ).

[0050] Example 3 Fibrinolytic activity of NHNK-614

[0051] 1. Preparation of NHNK-614 fermentation solution

[0052] A single colony of NHNK-614 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, that is, fermentation liquid is obtained.

[0053] 2. Artificial thrombus plate culture medium

[0054] Sample A: Dissolve 22 mg of fibrinogen in 10 mL of normal saline and incubate in a 37°C water bath for 5 to 10 min.

[0055] Sample B: Take 5 mg of thrombin (concentration of 40 U / mg) and add it to 2 mL of normal saline and incubate in a 37°C water bath for 5-10 min;

[0056] Sample C: Weigh 0.1 g agarose and dissolve it in 8 mL saline. Heat to fully dissolve the agarose.

[0057] 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.

[0058] 3. NHNK-614 fibrinolytic activity assay

[0059] Take 2 μL of NHNK-614 fermented bacteria liquid and apply it on the artificial thrombus plate, set up three parallel plates, and incubate in a 37°C incubator for 24 hours. 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.

[0060] The results are shown in Table 1:

[0061] Table 1: NHNK-614 fibrinolytic activity transparent zone diameter measurement table

[0062]

[0063] like Figure 2 As shown, NHNK-614 has plasminogen activitv, which makes the artificial thrombus plate present a transparent circle with a diameter of 7.19 mm to 7.45 mm, and can dissolve the fibrin artificial thrombus formed by thrombin.

[0064] Example 4 NHNK-614 upregulates the expression of urokinase-related genes in human renal tubular epithelial cells

[0065] 1. Cell culture

[0066] 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.

[0067] 2. Preparation of NHNK-614 fermentation products

[0068] A single colony of NHNK-614 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%.

[0069] 3. NHNK-614 regulates urokinase gene expression in human renal tubular epithelial cells HKC-1

[0070] 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 suspension was cultured for 24 h at 37°C and 5% CO2. 2 ml of DMEM medium containing 0.2% (v / v) NHNK-614 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.

[0071] 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 the genes related to promoting urokinase expression 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:

[0072] △CT 实验 =CT 实验 -CT 内参（实验） ;

[0073] △CT 对照 =CT 对照 -CT 内参（对照） ;

[0074] △△CT=△CT 实验 -△CT 对照 .

[0075] The results are shown in Table 2:

[0076] Table 2: Results of NHNK-614 regulating the expression of urokinase-related genes in HKC-1 cells

[0077]

[0078] The results showed that NHNK-614 fermentation products could upregulate the urokinase-related gene in HKC-1 cells UPA The expression level of urokinase was upregulated to 1.16-1.20 times, promoting the production of urokinase to enhance fibrinolytic activity.

[0079] Example 5 NHNK-614 regulates the expression of anticoagulant factor-related genes in endothelial cells EA.hy926

[0080] 1. Cell culture

[0081] Human umbilical vein endothelial cell fusion cells (EA.hy926) were cultured in DMEM medium containing 10% (v / v) fetal bovine serum (FBS). The cells were cultured at 37°C and 5% CO2. When the cell fusion reached 80-90%, subculture was performed.

[0082] 2. Preparation of NHNK-614 fermentation products

[0083] The preparation method of NHNK-614 fermentation product refers to Example 4.

[0084] 3. NHNK-614 regulates the expression of anticoagulant factor-related genes in EA.hy926

[0085] 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-614 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.

[0086] 4. Fluorescence quantitative PCR and data processing

[0087] 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 was calculated by this method.

[0088] The results are shown in Table 3 below:

[0089] Table 3: NHNK-614 regulates the expression of anticoagulant factor-related genes in EA.hy926 cells

[0090]

[0091] The results showed that NHNK-614 fermentation products could upregulate anticoagulant factor-related genes in EA.hy926 cells TM , ENOS and TPA expression, increasing anticoagulant factors to reduce thrombosis.

[0092] Example 6 NHNK-614 promotes the growth of endothelial cells EA.hy926

[0093] 1. Cell culture

[0094] The cell culture method is as in Example 5.

[0095] 2. Preparation of NHNK-614 fermentation products

[0096] The preparation method of NHNK-614 fermentation product refers to Example 4.

[0097] 3. NHNK-614 promotes the growth of EA.hy926 cells

[0098] 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 DMEM culture medium containing 0.2% (v / v) NHNK-614 fermentation product was added. 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. The cell growth rate (%) = (experimental group-control group) / control group × 100%.

[0099] The results are shown in Table 4:

[0100] Table 4: Results of NHNK-614 promoting the growth of EA.hy926 cells

[0101]

[0102] The results showed that the fermentation products of NHNK-614 promoted the growth of EA.hy926 cells.

[0103] Example 7 NHNK-614 regulates the expression of genes related to oxidative damage in endothelial cells EA.hy926

[0104] 1. Cell culture

[0105] The cell culture method is as in Example 5.

[0106] 2. Preparation of NHNK-614 fermentation products

[0107] The preparation method of NHNK-614 fermentation product refers to Example 4.

[0108] 3. NHNK-614 regulates the expression of inflammation / apoptosis-related genes in EA.hy926 cells induced by H2O2

[0109] 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 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. DMEM medium containing 0.2% (v / v) NHNK-614 fermentation product was added to the experimental group, 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.

[0110] After culture, cells were collected, cellular RNA was extracted using the Trzol method, and RNA was reverse transcribed into cDNA using a kit. GAPDH The internal reference gene was detected by qPCR fluorescence quantitative technology. TNF-α , IL-6 and BCL-2 The relative expression of the gene in the control group was F=1, and the expression of -ΔΔCT The F value of each sample was calculated by this method.

[0111] The results are shown in Table 5:

[0112] Table 5: NHNK-614 regulates the expression of inflammation / apoptosis-related genes in EA.hy926 cells induced by H2O2 oxidative damage

[0113]

[0114] The results showed that NHNK-614 fermentation products could inhibit the inflammatory response and cell apoptosis induced by H2O2 and downregulate the genes of inflammatory factors. TNF-α and IL-6 Upregulation of anti-apoptotic factor genes BCL-2 expression.

[0115] Example 8 NHNK-614 promotes the repair of oxidatively damaged vascular endothelial cells EA.hy926

[0116] 1. Cell culture

[0117] The cell culture method is as in Example 5.

[0118] 2. Preparation of NHNK-614 fermentation products

[0119] The preparation method of NHNK-614 fermentation product refers to Example 4.

[0120] 3. NHNK-614 promotes the production of antioxidant factors in EA.hy926 cells induced by H2O2

[0121] 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-614 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.

[0122] 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, glutathione GSH and nitric oxide NO in the cell supernatant were detected using a kit.

[0123] Increase rate (%) = (experimental group - control group) / control group × 100%

[0124] The results are shown in Table 6:

[0125] Table 6: NHNK-614 promotes the production of antioxidant factors in H2O2-induced oxidative damage EA.hy926 cells

[0126]

[0127] The results showed that NHNK-614 fermentation products could increase the secretion of antioxidant factors such as superoxide dismutase SOD, glutathione GSH and nitric oxide NO by oxidatively damaged vascular endothelial cells, and promote the repair of oxidative damage to vascular endothelial cells.

[0128] Example 9 NHNK-614 forms biofilm

[0129] 1. NHNK-614 biofilm formation

[0130] A single colony of NHNK-614 was selected and placed in MRS liquid medium, cultured at 37°C for 24 h, and the OD was adjusted 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.

[0131] 2. Crystal violet staining

[0132] 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.

[0133] The results are shown in Table 7:

[0134] Table 7: NHNK-614 biofilm formation

[0135]

[0136] The results showed that NHNK-614 in OD 600 =0.2 in MRS medium at 37℃ for 24h to form biofilm.

[0137] Example 10 NHNK-614 adsorbs mucin

[0138] 1. Preparation of NHNK-614 live bacterial suspension

[0139] Pick a single colony of NHNK-614 in fresh MRS medium and culture at 37℃ with shaking for 24h. Collect the cells by centrifugation at 5000rpm for 10min, wash twice with sterile PBS, resuspend the cells in DMEM medium and adjust the OD 600 =0.5.

[0140] 2. Preparation of NHNK-614 biofilm

[0141] A single colony of NHNK-614 was selected and placed in MRS liquid medium, cultured at 37°C for 24 h, and the OD was adjusted 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.

[0142] 3. Determination of mucin adsorption capacity

[0143] (1) Reagent preparation

[0144] Mucin solution: Weigh 10 mg of mucin and dissolve it in 10 mL of 50 mM Tris-HCL solution at 4°C overnight.

[0145] Blocking solution: Dissolve bovine serum albumin in PBS solution at a ratio of 2% (m / v).

[0146] Washing solution: Dissolve bovine serum albumin in PBS solution at a ratio of 0.1% (m / v).

[0147] (2) Adsorption experiment of NHNK-614 bacterial liquid and biofilm on mucin

[0148] 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-614 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.

[0149] like Figure 3 The results showed that NHNK-614 could adsorb mucin, the main component of the mucosal layer, and that NHNK-614 could increase its adsorption capacity for mucin after coating the biofilm formed by itself.

[0150] Example 11 Adsorption of NHNK-614 to intestinal epithelial cells

[0151] 1. Preparation of NHNK-614 live bacterial suspension

[0152] The method for preparing the bacterial suspension is as described in Example 10.

[0153] 2. Preparation of NHNK-614 biofilm

[0154] The biofilm preparation method is described in Example 10.

[0155] 3. Cultivation of intestinal epithelial cells Caco-2

[0156] 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-614 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.

[0157] like Figure 4 The results showed that NHNK-614 could adsorb Caco-2 cells, and the adsorption capacity of NHNK-614 on Caco-2 cells could be increased after coating the biofilm formed by itself.

[0158] 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 helveticus ( Lactobacillus helveticus ), which is Lactobacillus helveticus ( Lactobacillus helveticus )NHNK-614 was deposited in the China Center for Type Culture Collection on July 1, 2024, and its deposit number is CCTCC NO: M 20241430.

2. Lactobacillus helveticus according to claim 1 ( Lactobacillus helveticus ) is characterized in that The product is Lactobacillus helveticus ( Lactobacillus helveticus ) of live bacteria, fermented bacterial liquid or fermented product, wherein the preparation method of the live bacteria is: the Lactobacillus helveticus ( Lactobacillus helveticus ) 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 helveticus ) 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: the Lactobacillus helveticus ( Lactobacillus helveticus ) 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. Lactobacillus helveticus as claimed in claim 1 ( Lactobacillus helveticus ) in the preparation of products for improving thrombosis.

4. Lactobacillus helveticus as claimed in claim 2 ( Lactobacillus helveticus ) The use of the prepared product in the preparation of products for improving thrombosis.

5. The use according to claim 3 or claim 4, 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.

6. Lactobacillus helveticus as claimed in claim 1 ( Lactobacillus helveticus ) 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 to vascular endothelial cells, and reducing inflammation and apoptosis of vascular endothelial cells.

7. Lactobacillus helveticus as claimed in claim 2 ( Lactobacillus helveticus ) The use of the prepared product in the preparation of products 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.

8. Lactobacillus helveticus as claimed in claim 1 ( Lactobacillus helveticus ) in any of the following: (1) Colonization of mucosal mucin; (2) Bacterial biofilm increases the ability to colonize mucosal mucins and intestinal epithelial cells.

9. A product for improving thrombosis and vascular health, characterized in that: comprising the Lactobacillus helveticus according to claim 1 ( Lactobacillus helveticus ), or the Lactobacillus helveticus according to claim 2 ( Lactobacillus helveticus ) and acceptable excipients and / or adjuvants.

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