Lactobacillus saké and its product application in improving aged blood vessels and venous thrombosis

Abstract

The present invention relates to the technical field of marine microorganisms, and provides a sake lactobacillus NHNK-615 and a product application thereof for improving aged blood vessels and venous thrombosis. The sake lactobacillus NHNK-615 is deposited in the China Center for Type Culture Collection, and the deposit number is CCTCC NO: M 20241428. Experiments show that NHNK-615 has the functions of dissolving thrombus, promoting the production of urokinase, increasing the anticoagulant factor of venous endothelial cells, promoting the growth of venous endothelial cells, promoting the repair of oxidative damage of venous endothelial cells, reducing inflammation and apoptosis of venous endothelial cells, and increasing the colonization of mucin and intestinal epithelial cells by bacterial biofilm, and can be used to prepare products for improving aged blood vessels and venous thrombosis.

Description

Technical Field

[0001] The invention relates to the technical field of marine microorganisms, and in particular to a strain of Lactobacillus sakei and its application in improving aged blood vessels and venous thrombosis. Background Art

[0002] Xu's flathead scorpionfish Sebastesschlegelii ) is widely distributed in the offshore reef areas of Bohai Sea, Yellow Sea and East China Sea in China, and is also distributed along the coasts of Japan and North Korea. It is a cold-water near-shore bottom carnivorous fish. 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 the clot formed by blood components in the human body in the blood vessels or heart. Thrombotic diseases include myocardial infarction, cerebral thrombosis, pulmonary embolism, limb arterial embolism, limb venous thrombosis, disseminated intravascular coagulation, etc., which seriously endanger the physical and mental health of patients. After the coagulation factors react with fibrinogen, the formed reticular fibrin can entangle blood cells and platelets to form clots, and then form thrombi. Thrombin modulating protein (TM) in the anticoagulant system and tissue plasminogen activator (tPA) in the fibrinolytic system are important proteins that play the role of inhibiting and clearing 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 fighting 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.

[0004] When blood vessels are damaged, inflammatory cells secrete inflammatory cytokines (such as IL-6 , TNF-α ), which will further lead to accelerated apoptosis of vascular stromal cells and smooth muscle cells, which also shows that inflammatory cytokines can cause secondary damage to the vascular wall.

[0005] B-lymphocytic neoplasm-2 ( B-cell lymphoma-2 , BCL-2 ) gene family and its related proteins are the earliest genes studied in relation to apoptosis, and are currently the most important gene family regulating cell apoptosis. BCL-2 The gene inhibits cell apoptosis caused by various factors by regulating mechanisms such as mitochondrial permeability, thereby prolonging cell viability. BCL-2It 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. BCL-2 Can block apoptosis.

[0006] 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

[0007] In view of this, the present invention provides a strain of Lactobacillus sakei and its product application for improving aged blood vessels and venous thrombosis. The Lactobacillus sakei NHNK-615 is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20241428. Experiments show that NHNK-615 has the functions of dissolving thrombus, promoting the production of urokinase, increasing the anticoagulant factor of venous endothelial cells, promoting the growth of venous endothelial cells, promoting the repair of oxidative damage of venous endothelial cells, reducing inflammation and apoptosis of venous endothelial cells, and increasing the colonization of mucin and intestinal epithelial cells by bacterial biofilm, and can be used to prepare products for improving aged blood vessels and venous thrombosis.

[0008] In order to achieve the purpose of the present invention, the inventor provides the following technical solutions:

[0009] The inventors first provided a strain of Lactobacillus NHNK-615 ( Latilactobacillus sakei NHNK-615), with the deposit number of CCTCC NO: M 20241428, which was deposited in China Center for Type Culture Collection on July 1, 2024.

[0010] The above-mentioned Lactobacillus NHNK-615 was derived from the intestine of Scorpionfish and was identified as Lactobacillus NHNK-615 by 16S rDNA ( Latilactobacillus sakei ). This strain is Gram-positive and short rod-shaped under a microscope; it grows on MRS plates to form round colonies with smooth, opaque surfaces, milky white, and neat edges; it grows evenly in MRS liquid culture medium and is turbid, and white precipitates may appear when the bacteria are left to stand for a long time. The optimal growth temperature is 37°C.

[0011] The present invention also provides the application of the above-mentioned Lactobacillus sakei NHNK-615 in products for improving aged blood vessels and venous thrombosis, specifically, the products can be used for preparing products for improving aged blood vessels and venous thrombosis.

[0012] The improvement of aging blood vessels and venous thrombosis includes at least one of dissolving thrombus, promoting the production of urokinase, increasing anticoagulant factors of venous endothelial cells, promoting the growth of venous endothelial cells, promoting the repair of oxidative damage of venous endothelial cells, reducing inflammation and apoptosis of venous endothelial cells, and increasing the colonization of mucin and intestinal epithelial cells by bacterial biofilm, and its application mechanism is any one or more of the following:

[0013] (A1) Dissolve blood clots;

[0014] (A2) Promote urokinase production: Upregulate urokinase-related genes in human renal tubular epithelial cells HKC-1 UPA Expression;

[0015] (A3) Increase the anticoagulant factor of venous endothelial cells: Upregulation of the anticoagulant factor gene in human umbilical vein cell fusion cells EA.hy926 ENOS , TM and TPA at least one of the expressions;

[0016] (B1) Promote the growth of venous endothelial cells;

[0017] (B2) promoting the repair of oxidative damage in venous endothelial cells and promoting the production of at least one of superoxide dismutase (SOD) and glutathione (GSH) in oxidatively damaged human umbilical vein cell fusion cells EA.hy926;

[0018] (B3) Reducing inflammation and apoptosis of venous endothelial cells: Downregulating inflammatory factor genes in oxidatively damaged human umbilical vein cell EA.hy926 fusion cells TNF-α and IL-6 Upregulation of at least one of the anti-apoptotic factor genes BCL-2 Expression;

[0019] (C1) Bacterial biofilm increases the ability to colonize mucosal mucins and intestinal epithelial cells.

[0020] In vitro experiments show that the thrombus dissolving agent has plasmin activity, dissolves thrombi formed by fibrinogen and thrombin, and can form a transparent circle on an artificial thrombus plate, with a diameter of 7.00 mm to 7.21 mm.

[0021] In vitro experiments showed that Lactobacillus sakei NHNK-615 up-regulated the gene of urokinase-type plasma plasminogen activator in human renal tubular epithelial cells HKC-1. UPA The relative expression multiple was 1.19-1.32 times.

[0022] In vitro experiments showed that NHNK-615 could upregulate the endothelial nitric oxide synthase gene in human umbilical vein cell EA.hy926 fusion cells. ENOS , thrombomodulin gene TM , tissue plasminogen activator gene TPA The relative expression multiples were 1.26-2.31 times.

[0023] In vitro experiments showed that Lactobacillus sakei NHNK-615 has the effect of promoting the repair of oxidatively damaged human umbilical vein cell fusion cells EA.hy926, and promoting the production of superoxide dismutase SOD and glutathione GSH in oxidatively damaged EA.hy926 cells, with a growth rate of 13.86%-22.00%.

[0024] In vitro experiments showed that NHNK-615 could down-regulate the inflammatory factor tumor necrosis factor gene in EA.hy926 fusion cells damaged by oxidative damage TNF-α , interleukin-6 gene IL-6 The relative expression was 0.80-0.89 times. Up-regulation of anti-apoptotic factor B lymphocytoma-2 gene BCL-2 The relative expression multiples were 1.22-2.57 times.

[0025] In vitro experiments showed that the adsorption capacity of Lactobacillus sakei NHNK-615 on Caco-2 cells increased after the bacteria produced biofilm, indicating that it can increase the colonization ability on mucosal mucin and intestinal epithelial cells.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] Sake Lactobacillus NHNK-615 has the functions of dissolving blood clots, promoting the production of urokinase, increasing the anticoagulant factor of venous endothelial cells, promoting the growth of venous endothelial cells, promoting the repair of oxidative damage of venous endothelial cells, reducing inflammation and apoptosis of venous endothelial cells, and the bacterial biofilm increases the colonization of mucin and intestinal epithelial cells. It can be used to prepare products to improve aging blood vessels and venous thrombosis.

[0028] The inventor has made a biological deposit of Lactobacillus sakei NHNK-615, and the deposit information is as follows:

[0029] Storage time: July 1, 2024

[0030] Name of depository: China Center for Type Culture Collection

[0031] Deposit number: CCTCC NO: M 20241428

[0032] Address of depository: Wuhan University, Wuhan, China

[0033] Classification and naming: Lactobacillus NHNK-615 ( Latilactobacillus sakei NHNK-615). BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the MRS plate colony image of the sake-spreading Lactobacillus NHNK-615 of the present invention;

[0035] Figure 2 This is a Gram staining image of Lactobacillus sakei NHNK-615 of the present invention;

[0036] Figure 3 This is a diagram showing the diameter of the transparent circle of fibrinolytic activity of the fermented bacterial liquid of Lactobacillus NHNK-615 in sake brewing according to the present invention.

[0037] In the figure, (a) is control MRS, (b) is NHNK-615 parallel 1, (c) is NHNK-615 parallel 2, and (d) is NHNK-615 parallel 3;

[0038] Figure 4 This is a graph showing the adsorption of mucin by the bacterial solution and biofilm of Lactobacillus sakei NHNK-615 in the present invention.

[0039] In the figure, (a) shows NHNK-615 adsorbing mucin, and (b) shows NHNK-615 biofilm adsorbing mucin;

[0040] Figure 5 This is a graph showing the adsorption of live Lactobacillus NHNK-615 and its biofilm to intestinal epithelial cells Caco-2.

[0041] In the figure, (a) is Caco-2 cells, (b) is NHNK-615 adsorbed Caco-2 cells, and (c) is NHNK-615 biofilm adsorbed Caco-2 cells. DETAILED DESCRIPTION

[0042] The present invention provides sake widely distributed lactobacillus NHNK-615 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 by way of example, 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.

[0043] The present invention relates to the Lactobacillus NHNK-615, which is derived from the intestine of Scorpionfish xu, and is identified as Lactobacillus NHNK-615 by 16S rDNA. Latilactobacillus sakei ). This strain is Gram-positive and short rod-shaped under a microscope; it grows on MRS plates to form round colonies with smooth, opaque surfaces, milky white, and neat edges; it grows evenly in MRS liquid culture medium and is turbid, and white precipitates may appear when the bacteria are left to stand for a long time. The optimal growth temperature is 37°C.

[0044] Furthermore, in the application described in the present invention, the sake lactobacillus NHNK-615 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); or in the form of a derivative, 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, fermentation / secretion products.

[0045] The reagents, consumables and biological products used in the present invention are all common commercially available products. The present invention is now further described in conjunction with the embodiments.

[0046] Example 1 Isolation of NHNK-615.

[0047] The flathead mullet ( Sebastesschlegelii ), 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-615.

[0048] Gram staining microscopy: strain NHNK-615 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 left for a long time. Figure 1 and Figure 2 shown.

[0049] Example 2 Nucleic acid identification of NHNK-615.

[0050] 1. 16S rDNA gene sequence analysis:

[0051] 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.

[0052] 2. Results:

[0053] The PCR product was sequenced and its nucleotide sequence was shown in SEQ ID NO.1. After homology comparison (BLASTN) with the standard sequence published in the GenBank database, it was found that the NHNK-615 strain was Lactobacillus sakei ( Latilactobacillus sakei ).

[0054] Example 3 Fibrinolytic activity of NHNK-615.

[0055] 1. Preparation of NHNK-615 fermentation solution:

[0056] A single colony of NHNK-615 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.

[0057] 2. Artificial thrombus plate culture medium:

[0058] Sample A: Dissolve 22 mg of fibrinogen in 10 mL of normal saline and incubate in a 37°C water bath for 5-10 min.

[0059] Sample B: Take 5 mg of thrombin (concentration 40 U / mg) and add it to 2 mL of normal saline and place in a 37°C water bath for 5-10 min;

[0060] Sample C: Weigh 0.1 g agarose and dissolve it in 8 mL normal saline. Heat to fully dissolve the agarose.

[0061] Quickly add sample B into sample C, and finally add sample A. Pour into a plate after mixing evenly, and wait for the artificial thrombus plate to solidify before use.

[0062] 3. NHNK-615 fibrinolytic activity assay:

[0063] Take 2 μL of NHNK-615 fermentation liquid and apply it on the artificial thrombus plate, set up three parallel and control MRS, 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.

[0064] The results are shown in Table 1 below:

[0065] Table 1 Diameter of transparent zone of fibrinolytic activity of NHNK-615 fermented bacterial liquid

[0066]

[0067] like Figure 3 As shown, NHNK-615 has plasminogen activitv, which makes the artificial thrombus plate present a transparent circle with a diameter of 7.00 mm to 7.21 mm, and can dissolve the fibrin artificial thrombus formed by thrombin.

[0068] Example 4 NHNK-615 up-regulates the expression of urokinase-related genes in human renal tubular epithelial cells.

[0069] 1. Cell culture:

[0070] Human renal tubular epithelial cells HKC-1 were cultured in DMEM medium containing 10% (v / v) FBS at 37°C and 5% CO 2 When the cell fusion reaches 80-90%, the cells are passaged.

[0071] 2. Preparation of NHNK-615 fermentation products:

[0072] A single colony of NHNK-615 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%.

[0073] 3. NHNK-615 fermentation products regulate the expression of urokinase gene in human renal tubular epithelial cells HKC-1:

[0074] When the confluence of HKC-1 cells reached about 90%, the adherent cells were digested with trypsin into a cell suspension and added to a 6-well plate at 37°C and 5% CO. 2 The cells were cultured for 24 h under the same conditions. 2 mL of DMEM medium containing 0.2% (v / v) NHNK-615 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.

[0075] 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:

[0076] △CT 实验 =CT 实验 -CT 内参（实验） ;

[0077] △CT 对照 =CT 对照 -CT 内参（对照） ;

[0078] △△CT=△CT 实验 -△CT 对照 .

[0079] The results are shown in Table 2 below:

[0080] Table 2 NHNK-615 fermentation products promote the expression of urokinase-related genes in HKC-1 cells

[0081]

[0082] The results showed that NHNK-615 fermentation products could upregulate the urokinase-related gene in HKC-1 cells UPA The expression level of urokinase was upregulated to 1.19-1.32 times, promoting the production of urokinase to enhance fibrinolytic activity.

[0083] Example 5 NHNK-615 regulates the expression of anticoagulant factor-related genes in venous endothelial cells EA.hy926.

[0084] 1. Cell culture:

[0085] Human umbilical vein cell fusion cells (EA.hy926) were cultured in DMEM medium containing 10% (v / v) fetal bovine serum (FBS) at 37°C and 5% CO 2 When the cell fusion reaches 80-90%, subculture is performed.

[0086] 2. Preparation of NHNK-615 fermentation products:

[0087] The preparation method of NHNK-615 fermentation product is referred to Example 4.

[0088] 3. NHNK-615 fermentation products regulate the expression of anticoagulant factor-related genes in EA.hy926:

[0089] When the degree of fusion of human umbilical vein cell EA.hy926 reached about 90%, the cells were collected with trypsin and the cell suspension was added to a 6-well plate and incubated at 37°C and 5% CO 2 After culturing for 24 h, the culture medium was removed and the cells were washed once with sterile PBS. 2 mL of DMEM medium containing 0.2% (v / v) NHNK-615 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. After overnight culture, the cells were collected.

[0090] 4. Fluorescence quantitative PCR and data processing:

[0091] 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.

[0092] The results are shown in Table 3 below:

[0093] Table 3 NHNK-615 fermentation products up-regulate the expression of anticoagulant factor genes in EA.hy926

[0094]

[0095] The results showed that NHNK-615 fermentation products could upregulate anticoagulant factor-related genes in EA.hy926 cells TM , ENOS and TPA expression, increasing anticoagulant factors to reduce thrombosis.

[0096] Example 6 NHNK-615 promotes the growth of venous endothelial cells EA.hy926.

[0097] 1. Cell culture:

[0098] The cell culture method is as in Example 5.

[0099] 2. Preparation of NHNK-615 fermentation products:

[0100] The preparation method of NHNK-615 fermentation product is referred to Example 4.

[0101] 3. Experiment on promoting the growth of EA.hy926 by fermentation products of NHNK-615:

[0102] When the degree of fusion of human umbilical vein cell EA.hy926 reached about 90%, trypsin was used to digest the 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% CO 2 24h under the conditions. After 24h, the culture medium in the 96-well plate was discarded, and DMEM medium containing 0.2% (v / v) NHNK-615 fermentation product was added. The control group was an equal volume of DMEM medium containing 0.2% (v / v) MRS, and the culture was continued for 24h. After the culture was completed, the number of cells was detected according to the steps in the CCK-8 kit manual. Cell growth rate (%) = (experimental group-control group) / control group × 100%.

[0103] The results are shown in Table 4 below:

[0104] Table 4 Effects of NHNK-615 fermentation products on the growth of EA.hy926 cells

[0105]

[0106] The results showed that the fermentation products of NHNK-615 promoted the growth of EA.hy926 cells.

[0107] Example 7 NHNK-615 regulates the expression of genes related to oxidative damage in venous endothelial cells EA.hy926.

[0108] 1. Cell culture:

[0109] The cell culture method is as in Example 5.

[0110] 2. Preparation of NHNK-615 fermentation products:

[0111] The preparation method of NHNK-615 fermentation product is referred to Example 4.

[0112] 3. NHNK-615 fermentation products regulate the expression of genes related to oxidative damage in endothelial cells EA.hy926:

[0113] When the degree of fusion of human umbilical vein cell EA.hy926 reached about 90%, the adherent cells were digested with trypsin into a cell suspension and added to a 6-well plate at 37°C and 5% CO 2 After the culture was completed, 2 mL of 1.5 mM H 2 O 2 DMEM medium, 37°C, 5% CO 2The cells were incubated for 3 h under the same conditions, and the culture medium was discarded after 3 h. The experimental group was added with DMEM medium containing 0.2% (v / v) NHNK-615 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 h.

[0114] After culture, cells were collected, cell RNA was extracted using the Trizol 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 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.

[0115] The results are shown in Table 5 below:

[0116] Table 5 NHNK-615 fermentation products regulate the expression of genes related to oxidative damage in EA.hy926

[0117]

[0118] The results showed that the fermentation product of NHNK-615 could inhibit H 2 O 2 Induced inflammatory response and cell apoptosis, down-regulation of inflammatory factor genes TNF-α and IL-6 Upregulation of anti-apoptotic factor genes BCL-2 expression.

[0119] Example 8 NHNK-615 promotes the repair of oxidatively damaged venous endothelial cells EA.hy926.

[0120] 1. Cell culture:

[0121] The cell culture method is as in Example 5.

[0122] 2. Preparation of NHNK-615 fermentation products:

[0123] The preparation method of NHNK-615 fermentation product is referred to Example 4.

[0124] 3. Repair of oxidative damage of EA.hy926 by NHNK-615 fermentation products:

[0125] 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 at 37°C and 5% CO. 2 After the culture was completed, 2 mL of 1.5 mM H 2 O2 DMEM medium, 37°C, 5% CO 2 Incubate for 3 h under the same conditions, and discard H 2 O 2 The experimental group was added with DMEM medium containing 0.2% (v / v) NHNK-615 fermentation product, and the control group was added with DMEM medium containing 0.2% (v / v) MRS, and the culture was continued for 24 h.

[0126] 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.

[0127] Increase rate (%) = (experimental group - control group) / control group × 100%

[0128] The results are shown in Table 6 below:

[0129] Table 6 NHNK-615 fermentation products promote the repair of oxidatively damaged EA.hy926

[0130]

[0131] The results showed that NHNK-615 fermentation products could increase the secretion of antioxidant factors such as superoxide dismutase SOD and glutathione GSH by oxidatively damaged venous endothelial cells, and promote the repair of oxidatively damaged vascular endothelial cells.

[0132] Example 9 NHNK-615 forms biofilm.

[0133] 1. Formation of NHNK-615 biofilm:

[0134] A single colony of NHNK-615 was picked up 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°C for 24 hours.

[0135] 2. Crystal violet staining:

[0136] After the incubation, the supernatant was discarded, 100 μL of sterile PBS was added to each well for washing twice, and then 100 μL of 4% paraformaldehyde fixative was added to each well for fixing at room temperature for 30 min. The fixative was discarded, 100 μL of crystal violet was added to each well, and staining was performed at room temperature for 30 min. After staining, the cells were washed twice with sterile PBS and dried, and 100 μL of anhydrous ethanol was added to each well. After standing for 1 min, the absorbance at 600 nm was measured.

[0137] The results are shown in Table 7 below:

[0138] Table 7 NHNK-615 biofilm formation

[0139]

[0140] The results showed that NHNK-615 had a 600 =0.2 in MRS medium at 37℃ for 24h to form biofilm.

[0141] Example 10 NHNK-615 adsorbs mucin.

[0142] 1. Preparation of NHNK-615 live bacterial suspension:

[0143] Pick a single colony of NHNK-615 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.

[0144] 2. Preparation of NHNK-615 biofilm:

[0145] A single colony of NHNK-615 was picked up 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°C 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.

[0146] 3. Determination of mucin adsorption capacity

[0147] (1) Reagent preparation

[0148] Mucin solution: weigh 10 mg of mucin and dissolve it in 10 mL of 50 mM Tris-HCl solution at 4°C overnight.

[0149] Blocking solution: Dissolve bovine serum albumin in PBS solution at a ratio of 2% (m / v).

[0150] Washing solution: Dissolve bovine serum albumin in PBS solution at a ratio of 0.1% (m / v).

[0151] (2) Adsorption experiment of NHNK-615 bacterial liquid and biofilm on mucin

[0152] 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-615 live bacterial suspension or biofilm bacterial suspension, drop it on 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 paraformaldehyde solution for 30 minutes, then perform Gram staining and observe under a microscope.

[0153] like Figure 4 The results showed that NHNK-615 could adsorb mucin, the main component of the mucosal layer, and that NHNK-615 could increase its adsorption capacity for mucin after coating the biofilm formed by itself.

[0154] Example 11 NHNK-615 adsorbs to intestinal epithelial cells.

[0155] 1. The preparation of NHNK-615 live bacterial suspension and biofilm was carried out according to Example 10.

[0156] 2. Culture of human colorectal adenocarcinoma cells Caco-2:

[0157] Human colorectal adenocarcinoma cells Caco-2 were inoculated in DMEM complete medium containing 10% FBS and 1% penicillin-streptomycin at 37°C and 5% CO. 2 Culture under the same conditions for 1-2 days, and collect the cells when the cell fusion reaches 80%-90%. First, place a cell slide at the bottom of a 6-well plate, and then 6 The cells were seeded into 6-well plates at 100 μl / well and cultured for 24 h. After the cells adhered to the wall, the culture medium was removed and washed twice with PBS. Then, 1.5 mL of DMEM culture medium and 0.5 mL of NHNK-615 bacterial suspension were added to each well and incubated at 37 °C for 2 h. After the incubation, the slides were removed and fixed with 4% paraformaldehyde solution for 30 min, followed by Gram staining and observation under a microscope.

[0158] The results are as follows Figure 5 As shown, NHNK-615 can adsorb Caco-2 cells, and the adsorption capacity of NHNK-615 to Caco-2 cells can be increased after the biofilm formed by NHNK-615 is coated.

[0159] 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 strain of Lactobacillus sakei ( Latilactobacillus sakei )NHNK-615, whose deposit number is CCTCCNO: M 20241428, was deposited in the China Center for Type Culture Collection on July 1, 2024.

2. Use of the Lactobacillus sakei NHNK-615 according to claim 1 in the preparation of a product for improving venous thrombosis.

3. The use of Lactobacillus sakei NHNK-615 according to claim 2 in the preparation of a product for improving venous thrombosis, characterized in that: Its application mechanism includes but is not limited to any one or more of the following: (A1) Dissolve blood clots; (A2) Promotes urokinase production and upregulates urokinase-related genes in human renal tubular epithelial cells HKC-1 UPA Expression; (A3) Increases anticoagulant factor in venous endothelial cells and upregulates anticoagulant factor gene in human umbilical vein cell fusion cell EA.hy926 ENOS , TM and TPA at least one of the expressions; (B1) Promote the growth of venous endothelial cells; (B2) promoting the repair of oxidative damage in venous endothelial cells and promoting the production of at least one of superoxide dismutase (SOD) and glutathione (GSH) in oxidatively damaged human umbilical vein cell fusion cells EA.hy926; (B3) Reduces inflammation and apoptosis of venous endothelial cells and downregulates inflammatory factor genes in oxidatively damaged human umbilical vein cell EA.hy926 fusion cells TNF-α and IL-6 Upregulation of at least one of the anti-apoptotic factor genes BCL-2 Expression; (C1) Bacterial biofilm increases the ability to colonize mucosal mucins and intestinal epithelial cells.

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