Pediococcus acidilactici and its application in improving stroke, fatty liver and uric acid metabolism

Abstract

The present invention discloses Pediococcus acidilactici and its application in improving stroke, fatty liver and uric acid metabolism, belonging to the technical field of marine microorganisms. Pediococcus acidilactici ) NHNK‑610, which was deposited in the China Center for Type Culture Collection on April 25, 2024, and its deposit number is CCTCCNO: M2024797. Experiments have shown that NHNK‑610 has the functions of dissolving blood clots, regulating endothelial cell coagulation factors, repairing aging blood vessels, promoting blood-brain barrier, inhibiting Klebsiella pneumoniae from producing alcohol, agglutinating Klebsiella pneumoniae, inhibiting virulence genes of Klebsiella pneumoniae, promoting alcohol metabolism, reducing alcohol damage, tolerating alcohol, degrading uric acid, inhibiting xanthine oxidase activity, promoting uric acid excretion, and increasing bacterial biofilm colonization on mucosal mucin and intestinal epithelial cells.

Description

Technical Field

[0001] The invention belongs to the technical field of marine microorganisms, and particularly relates to Pediococcus acidilactici and applications thereof in improving stroke, fatty liver and uric acid metabolism. Background Art

[0002] Mazzaella contains a variety of nutrients that are essential to the human body, such as calcium, iodine, iron, potassium, fiber, carotene and alginate. Its growth conditions are very harsh, and it can only grow in small quantities in unpolluted sea areas. Brown algae are now widely used in food, medicine and cosmetics: such as the application of fucoidan in adjuvant cancer treatment, and alginate has many effects such as soothing, anti-aging and moisturizing. There are a large number of bacteria colonizing the surface of large algae and having a complex and close relationship. The area where algae and bacteria interact closely is called the "algaesphere". The algaesphere microbiome is significantly different from the microorganisms in the surrounding seawater in terms of composition and function. The algaesphere microorganisms support the large algae host to achieve morphological development, adapt to environmental changes, release and sedimentation of algae spores, etc. by providing growth factors.

[0003] Stroke, also known as "stroke", is an acute cerebrovascular disease. It is a group of acute cerebrovascular diseases that cause brain tissue damage due to sudden rupture of brain blood vessels or blood vessel blockage that prevents blood from flowing into the brain. There are two types of stroke: ischemic stroke and hemorrhagic stroke. Ischemic stroke is caused by blood clots or arterial stenosis that prevents blood from reaching the brain, causing brain tissue damage; hemorrhagic stroke is caused by blood vessel rupture that causes blood to overflow into the surrounding brain tissue, thereby compressing or damaging brain cells. Current studies have found that an important area of ​​drug targets is the blood-brain barrier, which is a structure located around the brain that prevents unnecessary circulating cells and biomolecules from entering the brain. In the case of a stroke, the blood vessels in the brain are blocked or cut off, which damages the integrity of the blood-brain barrier, leading to neuronal death and the accumulation of toxic byproducts, and immune cells infiltrating the brain to aggravate inflammation.

[0004] Compared with the general population, patients with fatty liver have a significantly increased risk of stroke and myocardial infarction, and the more severe the fatty liver, the higher the risk. Fatty liver is closely related to obesity, metabolic abnormalities and insulin resistance syndrome, dyslipidemia and changes in intestinal flora. Studies have found that Klebsiella pneumoniae, a bacterium known to produce high concentrations of alcohol, is present in the feces of patients. After testing, the alcohol content produced by the flora isolated from the patient's intestine is about 4-6 times that of healthy people. Through metagenomic high-throughput sequencing of fecal samples from healthy controls, it was found that 61% of patients contained Klebsiella pneumoniae with high alcohol production capacity, while only 6.25% of the healthy group had such flora in their intestines. The mucus phenotype regulator rmpA, the enterobactin gene iroD and the galactosyltransferase-related gene wabG are common virulence genes in Klebsiella pneumoniae and are closely related to the pathogenicity of Klebsiella pneumoniae.

[0005] In addition, researchers isolated Klebsiella pneumoniae with high alcohol production capacity from the fecal samples of patients with the world's first bacterial autobrewery syndrome / non-alcoholic fatty liver disease. This bacterium continuously produces alcohol in the human body, causing persistent damage to the liver and intestines. Studies have found that endogenous alcohol production by intestinal flora Klebsiella pneumoniae can cause fatty liver in mice, and the fatty liver disease that occurs in mice fed with Klebsiella pneumoniae has similar molecular mechanisms to alcohol-mediated fatty liver disease. In addition, alcohol can affect liver function, significantly reducing the liver's ability to synthesize protein, which in turn causes a decrease in platelet production, prolongs bleeding time, and causes hemorrhagic stroke. Therefore, alcohol not only increases the risk of ischemic stroke, but also increases the occurrence of hemorrhagic stroke.

[0006] During metabolism in the body, alcohol inhibits the excretion of uric acid, causing uric acid to accumulate in the body. In addition, alcohol stimulates the liver to produce more uric acid. A blood uric acid level >420 umol / L is an independent risk factor for stroke, and increased blood uric acid in patients with acute stroke is also an independent risk factor for stroke recurrence. Epidemiological surveys have shown that high blood uric acid levels are positively correlated with the risk of early death after stroke, and increased blood uric acid levels can indicate a poor prognosis for patients 90 days after stroke.

[0007] Marine microorganisms have developed complex molecular adaptations to cope with these harsh conditions, affecting their primary and secondary metabolic pathways. These bioactive substances have important application values ​​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 object of the present invention is to provide a strain of Pediococcus acidilactici and its use in improving stroke, fatty liver and uric acid metabolism.

[0009] The first aspect of the present invention provides a strain of Pediococcus acidilactici ( Pediococcus acidilactici ), the lactic acid Pediococcus is lactic acid Pediococcus NHNK-610, which has been deposited in the China Center for Type Culture Collection on April 25, 2024, and its deposit number is CCTCC NO: M 2024797.

[0010] The second aspect of the present invention provides a method based on the Pediococcus acidilactici described in the first aspect. Pediococcus acidilactici ) products prepared.

[0011] The third aspect of the present invention provides the Pediococcus acidilactici as described in the first aspect above ( Pediococcus acidilactici ) or the use of the product as described in the second aspect above in the preparation of products for improving stroke, improving fatty liver and improving uric acid metabolism.

[0012] Furthermore, the improvement of stroke includes any one or more of dissolving blood clots, regulating endothelial cell coagulation factors, repairing aging blood vessels, and promoting the blood-brain barrier.

[0013] Furthermore, the improvement of fatty liver includes any one or more of inhibiting Klebsiella pneumoniae from producing alcohol, agglutinating Klebsiella pneumoniae, inhibiting virulence genes of Klebsiella pneumoniae, promoting alcohol metabolism, reducing alcohol damage, and tolerating alcohol.

[0014] Furthermore, the inhibition rate of alcohol production by Klebsiella pneumoniae is 12.50% to 15.79%.

[0015] Furthermore, the improvement of uric acid metabolism includes any one or more of degrading uric acid, inhibiting xanthine oxidase activity, and promoting uric acid excretion.

[0016] Furthermore, the degradation rate of uric acid is 59.02% to 63.07%, and the inhibition rate of xanthine oxidase activity is 38.01% to 56.00%.

[0017] Furthermore, the product also includes auxiliary materials and / or additives.

[0018] The fourth aspect of the present invention provides the Pediococcus acidilactici as described in the first aspect above ( Pediococcus acidilactici ) is used in colonizing mucosal mucins and / or bacterial biofilms to increase the colonization ability of mucosal mucins and intestinal epithelial cells.

[0019] The Pediococcus acidilactici NHNK-610 disclosed in the present invention has a deposit number of CCTCC NO: M 2024797. Experiments show that NHNK-610 has the functions of dissolving thrombus, regulating vascular endothelial cell coagulation factors, repairing aged blood vessels, promoting the increase of blood-brain barrier, inhibiting Klebsiella pneumoniae from producing alcohol, agglutinating Klebsiella pneumoniae, inhibiting virulence genes of Klebsiella pneumoniae, promoting alcohol metabolism, reducing alcohol damage, tolerating alcohol, degrading uric acid, inhibiting xanthine oxidase activity, promoting uric acid excretion, and increasing bacterial biofilm colonization on mucosal mucin and intestinal epithelial cells, and can be used to prepare products for improving stroke, improving fatty liver and improving uric acid metabolism.

[0020] Biological Deposit Description

[0021] Pediococcus acidilactici ( Pediococcus acidilactici ) NHNK-610, 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 April 25, 2024, and its deposit number is CCTCC NO: M 2024797. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a graph showing the detection results of Pediococcus acidilactici NHNK-610 in Gram staining microscopy in Example 1 of the present invention;

[0023] Figure 2 This is a plate image of artificial thrombus of the fermentation liquid of Pediococcus acidilactici NHNK-610 in Example 3 of the present invention;

[0024] Figure 3 This is a graph showing the agglutination test results of the inactivated bacteria of Pediococcus acidilactici NHNK-610 and Klebsiella pneumoniae in Example 8 of the present invention;

[0025] Figure 4 This is a graph showing the results of the experiment on the adsorption of mucin by live bacteria of Pediococcus acidilactici NHNK-610 and biofilm of Pediococcus acidilactici NHNK-610 in Example 19 of the present invention;

[0026] Figure 5 This is a graph showing the experimental results of adsorption of intestinal epithelial cells Caco-2 by live bacteria of Pediococcus acidilactici NHNK-610 and biofilm of Pediococcus acidilactici NHNK-610 in Example 20 of the present invention;

[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 a strain of Pediococcus acidilactici, a product 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 should be 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 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 implement and apply the technology of the present invention.

[0029] The lactic acid Pediococcus NHNK-610 described in the present invention is derived from dried sea antlers and is identified as lactic acid Pediococcus ( Pediococcus acidilactici ). This strain is Gram-positive and spherical under a microscope. The cells usually grow in pairs, not in chains. When growing on MRS plates, it forms round colonies with smooth, translucent surfaces, white in color, and neat edges. It grows evenly and turbidly in MRS liquid culture medium, and the bacteria form white precipitates after long-term storage. The optimal growth temperature is 37°C.

[0030] Pediococcus acidilactici ( Pediococcus acidilactici )NHNK-610, depository unit: China Center for Type Culture Collection, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Wuhan University, deposit date: April 25, 2024, deposit number: CCTCCNO: M2024797.

[0031] Furthermore, in the application described in the present invention, the lactic acid Pediococcus NHNK-610 provided by the present invention exists in the form of live bacteria or inactivated bacteria, or in the form of fermentation products (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: fermentation broth, fermentation products, live bacteria, and inactivated bacteria.

[0032] In the present invention, the improvement of stroke, fatty liver and uric acid metabolism includes dissolving blood clots, regulating endothelial cell coagulation factors, repairing aging blood vessels, promoting the increase of blood-brain barrier, inhibiting Klebsiella pneumoniae to produce alcohol, agglutinating Klebsiella pneumoniae, inhibiting virulence genes of Klebsiella pneumoniae, promoting alcohol metabolism, reducing alcohol damage, tolerating alcohol, degrading uric acid, inhibiting xanthine oxidase activity, promoting uric acid excretion, and increasing bacterial biofilm colonization of mucosal mucin and intestinal epithelial cells. Specifically:

[0033] In vitro experiments show that the lactic acid Pediococcus NHNK-610 of the present invention has the effect of dissolving thrombus. NHNK-610 has fibrinolytic activity and can form a transparent circle on an artificial thrombus plate, and the diameter of the transparent circle is 6.89 mm to 6.98 mm.

[0034] In vitro experiments show that the lactic acid Pediococcus acidilactici NHNK-610 of the present invention has the effect of down-regulating the tissue factor of human umbilical vein cell EA.hy926 fusion cell TF , relative expression multiples 0.08-0.19; Pediococcus acidilactici NHNK-610 has the ability to upregulate the thrombomodulin factor of human umbilical vein cell EA.hy926 fusion cells TM , the relative expression multiples were 1.15 to 1.27 times.

[0035] In vitro experiments show that the lactic acid Pediococcus acidilactici NHNK-610 of the present invention has the ability to down-regulate apoptosis-related pro-apoptotic protein genes of human umbilical vein cell fusion cells EA.hy926 BAX and caspase genes CASPASE-3 The relative expression multiple was 0.04 to 0.10.

[0036] In vitro experiments show that the lactic acid Pediococcus acidilactici NHNK-610 has the ability to upregulate the blood-brain barrier-related occludens gene bEed.3 in brain microvascular endothelial cells. CLND5 and CLND11 , tight junction protein gene ZO-1 and ZO-2 , occludin gene OCLN The relative expression multiples were 1.09 to 2.35 times.

[0037] In vitro experiments show that the fermentation product of Pediococcus acidilactici NHNK-610 of the present invention has the effect of inhibiting Klebsiella pneumoniae ( Klebsiellapneumoniae ) growth, with the inhibition rate reaching 29.73%~32.43%.

[0038] In vitro experiments show that the Pediococcus acidilactici NHNK-610 of the present invention has the function of agglutinating Klebsiella pneumoniae, and the agglutination rate after acting together with the pathogen for 30 minutes can reach 16.88% to 19.86%.

[0039] In vitro experiments show that the lactic acid Pediococcus NHNK-610 of the present invention has the ability to down-regulate the mucus phenotype regulatory gene of the virulence factor of Klebsiella pneumoniae rpmA , Enterobactin gene iroD and galactosyltransferase-related genes wabG The relative expression level was 0.03 to 0.84 times.

[0040] In vitro experiments show that the Pediococcus acidilactici NHNK-610 of the present invention has the effect of inhibiting the alcohol production of Klebsiella pneumoniae, and the inhibition rate reaches 12.50% to 16.95%.

[0041] In vitro experiments show that the lactic acid Pediococcus acidilactici NHNK-610 of the present invention has the effect of regulating the expression of genes related to alcohol damage in HepG2 liver cells, and upregulating the anti-inflammatory factor-related transforming growth factor gene. TGF-β and interleukin-10 gene IL-10 Expression of alcohol metabolism-related alcohol dehydrogenase-1B gene ADH1B and / or aldehyde dehydrogenase-type 2 gene ALDH2 The relative expression of α-tumor necrosis factor gene was downregulated. TNF-α The relative expression level was 0.28-0.34 times.

[0042] In vitro experiments show that the lactic acid Pediococcus NHNK-610 of the present invention has the effect of regulating the expression of genes related to alcohol damage in Caco-2 intestinal epithelial cells, upregulating the zonula occludens protein gene ZO-1 , tight junction protein gene OCLD and / or claudin-4 gene CLD4 Expression of alcohol metabolism-related alcohol dehydrogenase-1B gene ADH1B and / or aldehyde dehydrogenase-type 2 gene ALDH2 The relative expression levels were 1.14 to 12.33 times.

[0043] In vitro experiments show that the lactic acid Pediococcus NHNK-610 of the present invention has an alcohol tolerance effect, and the growth rate in a culture medium containing 10% alcohol is 263.16% to 293.75%.

[0044] In vitro experiments show that the Pediococcus acidilactici NHNK-610 of the present invention has the function of degrading uric acid, and the degradation rate is 59.94% to 53.62%.

[0045] In vitro experiments show that the Pediococcus acidilactici NHNK-610 of the present invention has the effect of inhibiting the activity of xanthine oxidase (XOD), and the inhibition rate is 38.10% to 40.00%.

[0046] In vitro experiments show that the lactic acid Pediococcus acidilactici NHNK-610 of the present invention has the effect of promoting uric acid excretion and upregulating the ATP binding cassette subfamily G member 2 gene of renal tubular epithelial cells uric acid transport related genes ABCG2 and glucose transporter 9 gene SLC2A9 The relative expression of ATP-binding cassette subfamily G member 2 gene in Caco-2 intestinal epithelial cells was upregulated. ABCG2 The relative expression levels were 1.19-6.46 times.

[0047] It should be noted that the reagents and consumables used in the present invention are all common commercial products. The present invention is further described in conjunction with the embodiments:

[0048] Example 1 Isolation of NHNK-610

[0049] See also Figure 1 Take an appropriate amount of dried sea antlers and soak them in sterile PBS for half an hour, then shake them several times. Centrifuge the sample at 6000r / min to remove the precipitate, streak the supernatant onto an MRS solid plate, and culture at 37℃ for 24h. Pick the colonies and repeatedly inoculate and screen until a uniform single colony is obtained, which is named NHNK-610.

[0050] Gram staining microscopy: strain NHNK-610 is a Gram-positive colony, spherical under the microscope, cells are generally grown in pairs, not arranged in chains; it grows on MRS plates, forming white, smooth and rounded translucent round colonies with neat edges; it grows evenly turbid in MRS culture medium, and the bacteria present white precipitate after long-term storage.

[0051] Example 2 Nucleic acid identification of NHNK-610

[0052] 1. 16S rDNA gene sequence analysis

[0053] Pick a single colony in MRS liquid medium, culture at 37℃ overnight, centrifuge at 8000 rpm for 1min to collect the bacteria, 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, and 72℃ extension for 10min.

[0054] 2. Results

[0055] The sequencing result of the PCR product is shown in SEQ ID NO.1. After homology comparison (BLASTN) with the standard sequence published in GenBank, it was concluded that the NHNK-610 strain was Pediococcus acidilactici ( Pediococcus acidilactici ).

[0056] Example 3 Fibrinolytic activity of NHNK-610

[0057] 1. Preparation of NHNK-610 fermentation solution

[0058] A single colony of NHNK-610 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.

[0059] 2. Artificial thrombus plate culture medium

[0060] Sample A: Dissolve 22 mg of fibrinogen in 10 mL of normal saline and place in a 37°C water bath for 5 to 10 minutes;

[0061] 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 min to 10 min;

[0062] Sample C: Weigh 0.1 g of agarose and dissolve it in 8 mL of normal saline. Heat to fully dissolve the agarose.

[0063] 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.

[0064] 3. NHNK-610 fibrinolytic activity assay

[0065] Take 2 μL of NHNK-610 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.

[0066] The measurement results of the fibrinolytic activity transparent zone diameter of NHNK-610 are shown in Table 1:

[0067] Table 1

[0068] ,

[0069] See also Figure 2 NHNK-610 has plasminogen activi ty, which makes the artificial thrombus plate present a transparent circle with a diameter of 6.89 mm to 6.98 mm, and can dissolve the fibrin artificial thrombus formed by thrombin.

[0070] Example 4 NHNK-610 regulates the expression of coagulation factor-related genes in endothelial cells EA.hy926

[0071] 1. Cell culture

[0072] Human umbilical vein endothelial cell fusion cells (EA.hy926) were cultured in DMEM medium containing 10% fetal bovine serum (FBS). The cells were cultured at 37°C and 5% CO2. When the cells were 80% to 90% confluent, they were subcultured.

[0073] 2. Preparation of NHNK-610 fermentation products

[0074] A single colony of NHNK-610 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%.

[0075] 3. NHNK-610 regulates the gene expression of coagulation-related factors in EA.hy926

[0076] 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-610 fermentation product was added to the experimental group, and an equal volume of DMEM culture medium containing MRS was added to the control group. After overnight culture, the cells were collected.

[0077] 4. Fluorescence quantitative PCR and data processing

[0078] 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 coagulation factor-related genes TF Anticoagulant factor-related genes TM expression.

[0079] The relative expression multiple of the gene in the control group is F=1, and 2 -ΔΔCT The F value of each sample is calculated by this method. Formula: F=2 -ΔΔCT ,in:

[0080] △CT 实验 =CT 实验 -CT 内参（实验） ;

[0081] △CT 对照 =CT 对照 -CT 内参（对照） ;

[0082] △△CT=△CT 实验 -△CT 对照 .

[0083] The results of NHNK-610 regulating the gene expression of coagulation-related factors in EA.hy926 are shown in Table 2:

[0084] Table 2

[0085] ,

[0086] The results showed that NHNK-610 fermentation products could down-regulate the genes of coagulation factors in EA.hy926 cells TF , up-regulating anticoagulant factor genes in EA.hy926 cells TM expression, reducing coagulation factors to reduce thrombosis.

[0087] Example 5 NHNK-610 promotes the repair of aged vascular endothelial cells EA.hy926

[0088] 1. Cell culture

[0089] Refer to Example 4;

[0090] 2. Preparation of NHNK-610 fermentation products

[0091] Refer to Example 4;

[0092] 3. NHNK-610 regulates the expression of apoptosis-related genes in aged endothelial cells EA.hy926

[0093] 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 the cells were 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-610 fermentation product was added to the experimental group, and DMEM medium containing 0.2% (v / v) MRS was added to the control group, and the culture was continued for 24 hours.

[0094] After culture, cells were collected, cell RNA was extracted using the Trzol method, and RNA was reverse transcribed into cDNA using a kit. The internal reference gene was glyceraldehyde-3-phosphate dehydrogenase GADPH , using qPCR fluorescence quantitative technology to detect BAX and CASPASE-3 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.

[0095] The results of NHNK-610 regulating the expression of apoptosis-related genes in aged EA.hy926 are shown in Table 3:

[0096] Table 3

[0097] ,

[0098] The results showed that NHNK-610 fermentation products could inhibit H2O2-induced cell apoptosis and down-regulate BAX andCASPASE-3 NHNK-610 fermentation products can reduce the degree of oxidative damage to vascular endothelial cells and repair aging blood vessels.

[0099] Example 6 NHNK-610 regulates the expression of blood-brain barrier-related genes in bEnd.3 cells

[0100] 1. Cell culture

[0101] Brain microvascular endothelial cells bEnd.3 were inoculated in DMEM medium containing 10% FBS serum and cultured at 37°C and 5% CO2. When the cells were 80% confluent, the cells were collected and inoculated in 6-well plates. After overnight culture, the supernatant was discarded. 100 μL and 1 mL of serum-free DMEM medium were added to each well of the experimental group, and 100 μL MR and 1 mL of serum-free DMEM medium were replaced in the control group. The cells were cultured at 37°C and 5% CO2 for 16 h.

[0102] 2. Preparation of NHNK-610 fermentation products

[0103] Refer to Example 4.

[0104] 3. NHNK-610 regulates the expression of blood-brain barrier-related genes in bEnd.3 cells

[0105] When the confluence of brain microvascular endothelial cells bEnd.3 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 for 24 h at 37 °C and 5% CO2. After the culture, 200 μL of NHNK-610 fermentation product and 2 mL of DMEM medium were added to the experimental group, and an equal volume of MRS and DMEM medium was added to the control group, and incubated for 18 h at 37 °C and 5% CO2.

[0106] After culture, cells were collected, cell RNA was extracted using Trizol method, and RNA was reverse transcribed into cDNA using a kit. qPCR fluorescence quantitative technology was used to detect the occludin factor CLND5 and CLND11 , tight junction protein factor ZO-1 and ZO-2 Occlusin factor OCLN The relative expression level of mRNA, the internal reference is β-actin ACTB The relative expression multiple of the gene in the control group is F=1, and 2 -ΔΔCT The F value of each sample was calculated by this method.

[0107] The results of NHNK-610 regulating the expression of blood-brain barrier-related genes in bEnd.3 cells are shown in Table 4:

[0108] Table 4

[0109] .

[0110] Example 7 Experiment on the inhibition of Klebsiella pneumoniae growth by NHNK-610 fermentation product

[0111] 1. Preparation of NHNK-610 fermentation products

[0112] A single colony of Pediococcus acidilactici NHNK-610 was selected and placed in MRS liquid medium. After culturing at 37°C for 48 h, the OD was adjusted to 0. 600 =0.3, 5000rpm to take the supernatant, and then filter with a 0.22μm filter membrane to obtain a sterile fermentation product.

[0113] 2. Preparation of Klebsiella pneumoniae suspension

[0114] Klebsiella pneumoniae CICC10870 was inoculated into BHI liquid medium at 1% (v / v) and cultured at 37°C for 24 h. After the culture, the cells were centrifuged at 5000 rpm and the OD was adjusted with BHI. 600 =0.3 for backup.

[0115] 3. Experiment on the inhibition of Klebsiella pneumoniae proliferation by NHNK-610 fermentation products

[0116] 3 mL of BHI medium and 0.3 mL of NHNK-610 fermentation product were added to a centrifuge tube. An equal volume of MRS medium was added to the control group. The suspension of Klebsiella pneumoniae was inoculated at 1% (v / v). After shaking culture at 37°C for 24 h, the absorbance at 600 nm was measured.

[0117] The results of NHNK-610 fermentation product inhibiting the growth of Klebsiella pneumoniae are shown in Table 5:

[0118] Table 5

[0119] ,

[0120] The results showed that NHNK-610 could reduce the proliferation of Klebsiella pneumoniae, with an inhibition rate of 29.73%~32.43%.

[0121] Example 8 NHNK-610 agglutination of Klebsiella pneumoniae

[0122] 1. Preparation of NHNK-610 inactivated bacteria

[0123] Pick a single colony of Pediococcus acidilactici NHNK-610 in MRS liquid medium, incubate at 37°C for 48 h, centrifuge at 5000 rpm for 10 min to obtain the precipitate, wash twice with PBS, then resuspend the bacteria in PBS and adjust the OD600 =0.3. Inactivated bacteria can be obtained by high pressure sterilization at 121℃ for 15min.

[0124] 2. Preparation of Klebsiella pneumoniae suspension

[0125] Klebsiella pneumoniae CICC 10870 was inoculated into BHI liquid medium at 1% (v / v) and cultured at 37°C for 24 h. After the culture, the cells were centrifuged at 5000 rpm and the OD was adjusted with PBS. 600 =0.3 for backup.

[0126] 3. Coagulation experiment

[0127] See also Figure 3 , the suspension of Klebsiella pneumoniae and NHNK-610 inactivated bacteria was mixed in a volume ratio of 1:1, and the mixed reaction liquid of NHNK-610, Klebsiella pneumoniae and NHNK-610 and Klebsiella pneumoniae was sampled at 30 minutes after the reaction, and the sampling range was the top 50 μL of the liquid surface, and then transferred to a 96-well plate to measure its absorbance at OD=600nm. At the same time, the agglutination precipitate was stained with Gram stain and its bacterial agglutination state was observed.

[0128] Calculation formula:

[0129] Agglutination rate (%) = [(Ax+Ay)-2Amix] / (Ax+Ay)×100%;

[0130] Ax represents the OD value of NHNK-610 alone at the reaction time. 600 Value, Ay represents the OD value of Klebsiella pneumoniae alone at the reaction time 600 Amix means the OD value measured at the reaction time after NHNK-610 and Klebsiella pneumoniae are mixed. 600 Numeric value.

[0131] The results of NHNK-610 inactivating bacteria and agglutinating Klebsiella pneumoniae are shown in Table 6:

[0132] Table 6

[0133] ,

[0134] The results showed that after 30 minutes of reaction, NHNK-610 inactivated bacteria could agglutinate Klebsiella pneumoniae.

[0135] Example 9: Experiment on the inhibition of virulence gene expression of Klebsiella pneumoniae by NHNK-610

[0136] 1. Preparation of NHNK-610 fermentation products and live bacterial suspension

[0137] A single colony of Pediococcus acidilactici NHNK-610 was selected and placed in MRS liquid medium, cultured at 37°C for 48 h, and then adjusted to OD 600 = 0.3, centrifuged at 5000 rpm for 10 min, and the supernatant was filtered with a 0.22 μm filter membrane to obtain a sterile fermentation product. The precipitated cells were washed twice with sterile PBS, and the cells were resuspended in PBS and the OD was adjusted. 600 =0.3, and NHNK-610 live bacterial suspension was obtained.

[0138] 2. Experiment on inhibiting the expression of virulence genes of Klebsiella pneumoniae

[0139] Klebsiella pneumoniae CICC 10870 was inoculated into fresh BHI medium at 1% (v / v), cultured at 37°C with shaking for 24 h, and the OD was adjusted to 0. 600 =0.3. Take 3mL of Klebsiella pneumoniae liquid, add 1mL of fresh BHI medium, 1mL of NHNK-610 fermentation product or live bacterial suspension, add an equal volume of PBS to the control group, and culture at 37℃ for 24h. After the culture, centrifuge at 8000rpm for 1min to obtain the cells, extract total RNA according to the kit instructions, detect RNA concentration and purity, and reverse transcribe it into cDNA. proc As the internal reference gene, qPCR detection was performed rpmA , iroD , wabG 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.

[0140] The results of NHNK-610 fermentation product inhibiting virulence genes of Klebsiella pneumoniae are shown in Table 7:

[0141] Table 7

[0142] ,

[0143] The results of NHNK-610 live bacteria inhibiting the virulence genes of Klebsiella pneumoniae are shown in Table 8:

[0144] Table 8

[0145] ,

[0146] The results showed that NHNK-610 could inhibit the virulence genes of Klebsiella pneumoniae rpmA , iroD , wabG expression, thereby reducing the pathogenicity of Klebsiella pneumoniae.

[0147] Example 10: Experiment on the inhibition of ethanol production by NHNK-610 in Klebsiella pneumoniae

[0148] 1. Preparation of NHNK-610 fermentation products and live bacterial suspension

[0149] A single colony of Pediococcus acidilactici NHNK-610 was selected and placed in MRS liquid medium, cultured at 37°C for 48 h, and then adjusted to OD 600 =1.0, centrifuge at 5000rpm for 10min, take the supernatant, and filter with a 0.22μm filter membrane to obtain the sterile fermentation product. Wash the precipitated bacteria twice with sterile PBS, resuspend the bacteria with PBS and adjust the OD 600 =1.0, and NHNK-610 live bacterial suspension was obtained.

[0150] 2. Preparation of Klebsiella pneumoniae suspension

[0151] Klebsiella pneumoniae CICC 10870 was inoculated into BHI liquid medium at 1% (v / v) and cultured at 37°C for 24 h. After the culture, the cells were centrifuged at 5000 rpm, resuspended in YPD liquid medium and adjusted to OD 600 =0.3 for backup.

[0152] 3. Experiment on NHNK-610 inhibiting ethanol production of Klebsiella pneumoniae

[0153] The fermentation product of NHNK-610 and live bacteria were added to the suspension of Klebsiella pneumoniae at 10% (v / v), and an equal volume of PBS was added to the control group. The cells were cultured at 37°C with shaking for 8 hours, and centrifuged at 5000 rpm for 10 minutes to obtain 1 ml of supernatant for later use.

[0154] Preparation of 5% potassium dichromate solution: weigh 5g potassium dichromate and dissolve it in 50ml water, add 10ml concentrated sulfuric acid, cool and make up to 100ml.

[0155] Potassium dichromate oxidation method to detect alcohol content: add 1 ml of Klebsiella pneumoniae supernatant and 2 ml of 5% potassium dichromate solution to a 10 mL centrifuge tube, heat in a 100°C water bath for 10 min, cool in running water for 5 min, take 100 μL and measure its absorbance at 600 nm. The results of NHNK-610 inhibiting Klebsiella pneumoniae from producing alcohol are shown in Table 9:

[0156] Table 9

[0157] ,

[0158] The results showed that NHNK-610 inhibited the production of alcohol by Klebsiella pneumoniae.

[0159] Example 11 NHNK-610 regulates the expression of genes related to alcohol-induced liver damage

[0160] 1. Preparation of NHNK-610 fermentation products and live bacterial suspension

[0161] Pick a single colony of NHNK-610 and place it in fresh MRS medium and culture it at 37°C for 24 h. Adjust the OD to 0. 600 =0.5, 5000rpm to take the supernatant, and then filter with a 0.22μm filter membrane to obtain a sterile fermentation product. Collect the centrifugal precipitated bacteria, wash twice with sterile PBS, resuspend the bacteria with DMEM medium and adjust the OD 600 =0.5, and a live bacterial suspension was obtained.

[0162] 2. Culture of human hepatocytes HepG2

[0163] HepG2 cells were activated with DMEM medium containing 10% FBS and 1% penicillin-streptomycin, cultured at 37°C and 5% CO2, and subcultured or plated after the cells were fused to 80%-90%.

[0164] 3. NHNK-610 regulates the expression of genes related to alcohol-induced liver cell damage

[0165] HepG2 cells were seeded in a 6-well cell culture plate at 1×10^6 cells / well and cultured for 12 hours until the cells adhered to the wall. The cell culture medium was removed, washed twice with sterile PBS, and 1.9 mL of DMEM culture medium and 100 μL of NHNK-610 fermentation product or live bacterial suspension were added respectively. An equal volume of DMEM culture medium was added to the control group. After 3 hours of treatment, 6% alcohol was added to induce damage and cultured for 24 hours at 37°C and 5% CO2. After the culture was completed, the supernatant was discarded, washed twice with sterile PBS, and then 1 mL of cell RNA extraction reagent was added to each well. Total RNA was extracted according to the reagent instructions and the concentration and purity were determined. After the extraction was completed, it was reverse transcribed into cDNA and determined by qPCR. TNF-α , TGF-β, IL-10 and ADH1B, ALDH2 The relative expression of the gene in the control group is F=1, and the expression of the gene in the control group is F=1. -ΔΔCT The F value of each sample was calculated by this method.

[0166] The results of NHNK-610 fermentation products regulating the gene expression of alcohol-induced damaged liver cells are shown in Table 10:

[0167] Table 10

[0168] ,

[0169] The results of NHNK-610 live bacteria regulating the gene expression of alcohol-induced damaged liver cells are shown in Table 11:

[0170] Table 11

[0171] ,

[0172] The results showed that NHNK-610 could upregulate anti-inflammatory factor genes TGF-β and alcohol metabolism genes ADH1B, ALDH2 downregulation of pro-inflammatory factor genes TNF-α NHNK-610 can alleviate the inflammation caused by alcohol-damaged liver cells and enhance the alcohol metabolism ability of liver cells.

[0173] Example 12 NHNK-610 regulates the expression of genes related to alcohol-induced damage to intestinal epithelial cells

[0174] 1. Preparation of NHNK-610 fermentation products, live bacteria and inactivated bacteria

[0175] Pick a single colony of NHNK-610 and place it in fresh MRS medium and culture it at 37°C for 24 h. Adjust the OD 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. Collect the centrifugal precipitated bacteria, wash twice with sterile PBS, resuspend the bacteria with DMEM medium and adjust the OD 600 =0.5, and a live bacterial suspension was obtained. Some precipitated live bacteria were washed twice with sterile PBS, sterilized by high pressure at 121°C for 15 min, and resuspended in DMEM medium to adjust the OD 600 =0.5, and inactivated bacteria were obtained.

[0176] 2. Cultivation of human intestinal epithelial cells Caco-2

[0177] Caco-2 cells were activated with DMEM medium containing 10% FBS and 1% penicillin-streptomycin, cultured at 37°C and 5% CO2, and subcultured or plated after the cells were fused to 80%-90%.

[0178] 3. NHNK-610 regulates the expression of genes related to alcohol-induced damage to intestinal epithelial cells

[0179] Caco-2 cells were seeded in a 6-well cell culture plate at 1×10^6 cells / well and cultured for 12 hours until the cells adhered to the wall. The cell culture medium was removed, washed twice with sterile PBS, and then 1.9 mL of DMEM culture medium and 100 μL of NHNK-610 live bacteria / fermentation product / inactivated bacterial suspension were added. An equal volume of DMEM culture medium was added to the control group. After 3 hours of treatment, 6% alcohol was added to induce damage and cultured for 24 hours at 37°C and 5% CO2. After the culture was completed, the supernatant was discarded, washed twice with sterile PBS, and then 1 mL of cell RNA extraction reagent was added to each well. Total RNA was extracted according to the reagent instructions and the concentration and purity were determined. After the extraction was completed, it was reverse transcribed into cDNA and determined by qPCR. ZO-1, OCLD, CLD-4 , ADH1B and ALDH2 The relative expression of the gene in the control group is F=1, and the expression of the gene in the control group is F=1. -ΔΔCT The F value of each sample was calculated by this method.

[0180] The results of NHNK-610 fermentation products regulating the gene expression of alcohol-induced damaged intestinal epithelial cells are shown in Table 12:

[0181] Table 12

[0182] ,

[0183] The results of NHNK-610 live bacteria regulating the gene expression of alcohol-induced damaged intestinal epithelial cells are shown in Table 13:

[0184] Table 13

[0185] ,

[0186] The results of NHNK-610 inactivated bacteria regulating the gene expression of alcohol-induced damaged intestinal epithelial cells are shown in Table 14:

[0187] Table 14

[0188] ,

[0189] Results showed that NHNK-610 upregulated intestinal barrier genes ZO-1 , OCLD , CLD-4 and alcohol metabolism genes ADH1B , ALDH2 NHNK-610 can alleviate alcohol-induced intestinal epithelial cell barrier damage and enhance the alcohol metabolism capacity of intestinal epithelial cells.

[0190] Example 13 NHNK-610 alcohol tolerance test

[0191] Inoculate NHNK-610 into fresh MRS medium at a 2% inoculation rate and culture at 37°C for 24 hours. After the culture is completed, inoculate into MRS medium containing 10% alcohol at a ratio of 2%. Take 100 μL of bacterial suspension, measure the absorbance at OD=600nm, and record the OD value before and after 48 hours of culture at 37°C.

[0192] Calculation formula: Growth rate (%) = (A1-A0) / A0*100%;

[0193] Among them, A1 represents the absorbance value after culture, and A0 represents the absorbance value before culture.

[0194] The results of the NHNK-610 alcohol tolerance test are shown in Table 15:

[0195] Table 15

[0196] ,

[0197] The results showed that NHNK-610 has the ability to tolerate alcohol growth and can grow in a culture medium containing 10% alcohol, with a growth rate of 263.16%~293.75%.

[0198] Example 14: Experiment on degradation of uric acid by NHNK-610

[0199] 1. Preparation of uric acid solution

[0200] First, prepare a 1 M NaOH solution, and then dissolve the uric acid powder in the hot NaOH solution to a final concentration of 25 mg / ml.

[0201] 2. NHNK-610 degradation of uric acid experiment

[0202] The NHNK-610 frozen tube bacterial solution was inoculated into fresh MRS medium at a ratio of 2% (v / v), cultured at 37°C for 24 h, centrifuged at 5000 rpm for 10 min to obtain bacterial pellets, washed with sterile PBS and resuspended, and the OD was adjusted. 600 =1.0. The prepared uric acid solution was added to the NHNK-610 bacterial suspension to make the final concentration of uric acid 500 mg / L. The bacterial suspension of the control group was replaced with PBS and cultured at 37°C for 48 hours. After the culture, the supernatant was taken by centrifugation at 5000 rpm for 10 minutes, and the absorbance at OD=510nm was measured with a uric acid detection kit. Degradation rate (%) = (1-absorbance of experimental group / absorbance of control group)*100.

[0203] The results of the uric acid degradation experiment of NHNK-610 are shown in Table 16:

[0204] Table 16

[0205] ,

[0206] The results showed that NHNK-610 could degrade uric acid, thereby reducing its accumulation.

[0207] Example 15 Experiment on the inhibition of xanthine oxidase activity by NHNK-610

[0208] 1. Preparation of NHNK-610 fermentation products

[0209] NHNK-610 glycerol tube bacterial solution was inoculated into fresh MRS medium at 2% (v / v), cultured at 37°C for 48 h, and then adjusted to OD with MRS. 600 =1.0, centrifuge at 5000rpm for 10min, take the supernatant, and filter through a 0.22μm filter membrane to obtain the fermentation product.

[0210] 2. Experiment on inhibition of xanthine oxidase (XOD) activity by NHNK-610 fermentation products

[0211] According to the instructions of Nanjing Jiancheng XOD kit, the experimental group added 25μL fermentation product and 25μL XOD enzyme solution, and the control group replaced the fermentation product with an equal volume of MRS. After the 20-minute color reaction, the absorbance of each group at OD=530nm was measured to calculate the inhibition rate of fermentation product on XOD. Inhibition rate (%) = (1-experimental group / control group)*100.

[0212] The results of NHNK-610 inhibiting xanthine oxidase (XOD) activity are shown in Table 17:

[0213] Table 17

[0214] ,

[0215] The results showed that the fermentation products of NHNK-610 could inhibit the activity of XOD enzyme and the formation of xanthine from hypoxanthine, thereby reducing the formation of uric acid.

[0216] Example 16 NHNK-610 regulates the expression of genes related to uric acid excretion in renal tubular epithelial cells

[0217] 1. Preparation of NHNK-610 fermentation products

[0218] Refer to Example 15.

[0219] 2. NHNK-610 regulates genes related to uric acid excretion in renal tubular epithelial cells

[0220] Renal tubular epithelial cell HKC cell culture and intervention:

[0221] Renal tubular epithelial cells HKC were inoculated in DMEM / F12 medium containing 10% FBS and cultured at 37°C and 5% CO2. When the cells were 80% confluent, the cells were collected and inoculated in 6-well plates and cultured overnight. 1% (V / V) fermentation product was added to each well of the experimental group; 1% (V / V) MRS was added to each well of the control group. Three parallels were set up in each group and cultured at 37°C and 5% CO2 for 16 hours.

[0222] RNA extraction and fluorescence quantitative PCR:

[0223] After the culture was completed, RIPA lysis buffer was added to lyse the cells, and total RNA was extracted. After testing the RNA concentration and purity, it was reverse transcribed into cDNA. GAPDH As the internal reference gene, qPCR fluorescence quantitative technology was used to detect the uric acid excretion-related gene ATP binding cassette subfamily G member 2 gene ABCG2 , solute carrier protein gene SLC2A9 expression.

[0224] Data processing:

[0225] The relative expression multiple of the gene in the control group is F=1, and 2 -ΔΔCT The F value of each sample was calculated by this method.

[0226] The results of NHNK-610 fermentation products regulating the expression of genes related to uric acid excretion in HKC cells are shown in Table 18:

[0227] Table 18

[0228] ,

[0229] The results showed that fermentation products upregulated HKC renal tubular epithelial cells ABCG2 and SLC2A9 The gene expression was upregulated by 5.87 to 38.89 times. NHNK-610 can promote the excretion of uric acid from the renal tubules.

[0230] Example 17 NHNK-610 regulates the expression of genes related to uric acid excretion in human intestinal epithelial cells Caco-2

[0231] 1. Preparation of NHNK-610 live bacteria and inactivated bacteria

[0232] The NHNK-610 frozen tube bacterial solution was inoculated into MRS medium at 2% and cultured at 37°C for 24 hours. The bacteria were collected by centrifugation at 5000 rpm for 10 minutes, washed with PBS and centrifuged to discard the supernatant. The bacteria were resuspended in DMEM and the OD was adjusted. 600 =0.5 to obtain a live bacterial suspension. The washed live bacteria were resuspended in PBS and the OD 600=0.5, sterilize at 121℃ for 15min, centrifuge and resuspend with an equal volume of DMEM culture medium to obtain an inactivated bacterial suspension.

[0233] 2. NHNK-610 regulates the expression of genes related to uric acid excretion in Caco-2 cells

[0234] Caco-2 cell culture and intervention treatment:

[0235] Caco-2 cells were inoculated in DMEM medium containing 10% FBS serum and cultured at 37°C and 5% CO2. When the cells were 80% confluent, the cells were collected and inoculated in 6-well plates. After overnight culture, the supernatant was discarded. 1 mL of live bacteria or inactivated bacteria and 1 mL of serum-free DMEM medium were added to each well of the experimental group, and the control group was replaced with an equal volume of DMEM, and cultured at 37°C and 5% CO2 for 16 h.

[0236] RNA extraction and fluorescence quantitative PCR:

[0237] After the culture was completed, RIPA lysis buffer was added to lyse the cells, and total RNA was extracted. After testing the RNA concentration and purity, it was reverse transcribed into cDNA. GAPDH As the internal reference gene, qPCR fluorescence quantitative technology was used to detect ABCG2 , ZO-1 , CLD4 and OCLD 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.

[0238] The results of NHNK-610 live bacteria regulating the expression of uric acid excretion related factors are shown in Table 19:

[0239] Table 19

[0240] ,

[0241] The results of NHNK-610 inactivated bacteria regulating the gene expression of alcohol-induced damaged intestinal epithelial cells are shown in Table 20:

[0242] Table 20

[0243] ,

[0244] The results showed that both live and inactivated NHNK-610 bacteria could upregulate genes related to uric acid excretion in human intestinal epithelial cells. ABCG2 The expression of uric acid in the intestine is enhanced. Upregulation of intestinal barrier-related genes ZO-1 , CLD4 , OCLD expression, strengthening the intestinal barrier and reducing the risk of uric acid metabolism caused by insufficient intestinal barrier.

[0245] Example 18 NHNK-610 forms biofilm

[0246] 1. Formation of NHNK-610 biofilm

[0247] A single colony of NHNK-610 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°C for 24 hours.

[0248] 2. Crystal violet staining

[0249] 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.

[0250] The amount of biofilm formation of NHNK-610 is shown in Table 21:

[0251] Table 21

[0252] ,

[0253] The results showed that NHNK-610 600 =0.2 in MRS medium at 37℃ for 24h to form biofilm.

[0254] Example 19 Determination of the adsorption capacity of NHNK-610 to mucin

[0255] 1. Preparation of NHNK-610 live bacterial suspension

[0256] Pick a single colony of NHNK-610 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 to obtain a live bacterial suspension.

[0257] 2. Preparation of NHNK-610 biofilm bacterial suspension

[0258] A single colony of NHNK-610 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. The bacteria were resuspended in DMEM culture medium and the OD was adjusted. 600 =0.5 to obtain biofilm bacterial suspension.

[0259] 3. NHNK-610 adhesion test to mucin

[0260] Reagent preparation

[0261] Mucin solution: weigh 10 mg of mucin and dissolve it in 10 mL of 50 mM Tris-HCL solution at 4°C overnight.

[0262] Blocking solution: Dissolve bovine serum albumin in PBS solution at a ratio of 2% (m / v).

[0263] Washing solution: Dissolve bovine serum albumin in PBS solution at a ratio of 0.1% (m / v).

[0264] Adsorption experiment of NHNK-610 bacterial liquid and biofilm on mucin

[0265] Immerse the coverslip in the mucin solution and coat at 4°C overnight. 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-610 live bacterial suspension or biofilm bacterial suspension, add it to the mucin-coated slide, 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.

[0266] See also Figure 4 NHNK-610 can absorb mucin, the main component of the mucosal layer. After NHNK-610 is coated with the biofilm formed by itself, it can increase the adsorption capacity of mucin.

[0267] Example 20 Determination of the Adhesion Ability of NHNK-610 to Human Intestinal Epithelial Cells Caco-2

[0268] 1. Preparation of NHNK-610 live bacteria and biofilm bacterial suspension

[0269] The preparation method is similar to that of Example 19.

[0270] 2. Determination of the adsorption capacity of NHNK-610 to Caco-2 cells

[0271] Caco-2 cells were inoculated in DMEM medium containing 10% FBS serum and cultured at 37°C and 5% CO2. When the confluence reached 80%, the cells were digested with trypsin and collected, and inoculated into a 6-well plate containing a slide for overnight culture. After the culture was completed, the supernatant was discarded, washed twice with sterile PBS, and 1 mL of DMEM medium without FBS serum and 1 mL of live bacterial suspension or biofilm bacterial suspension were added, and the incubation continued for 2 hours. After the incubation was completed, it was washed twice with PBS to remove non-adherent bacteria. The slide was fixed in 4% paraformaldehyde for 15 minutes, followed by Gram staining and photography.

[0272] See also Figure 5 , NHNK-610 biofilm can increase the adsorption of bacteria to intestinal epithelial cells.

[0273] 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 Pediococcus acidilactici ( Pediococcus acidilactici ), characterized in that, The lactic acid Pediococcus is lactic acid Pediococcus NHNK-610, which was deposited in the China Center for Type Culture Collection on April 25, 2024, and its deposit number is CCTCC NO: M 2024797.

2. Based on the Pediococcus acidilactici as claimed in claim 1 ( Pediococcus acidilactici ) products prepared.

3. The Pediococcus acidilactici according to claim 1 ( Pediococcus acidilactici ) or the product as claimed in claim 2 for use in the preparation of a product for improving stroke, improving fatty liver and improving uric acid metabolism, wherein the improvement of fatty liver is any one or more of inhibiting Klebsiella pneumoniae from producing alcohol, agglutinating Klebsiella pneumoniae, inhibiting virulence genes of Klebsiella pneumoniae, promoting alcohol metabolism, reducing alcohol damage, and tolerating alcohol.

4. The use according to claim 3, characterized in that: The improvement of stroke is any one or more of dissolving blood clots, regulating endothelial cell coagulation factors, repairing aging blood vessels, and promoting the blood-brain barrier.

5. The use according to claim 3, characterized in that: The improving uric acid metabolism is any one or more of degrading uric acid, inhibiting xanthine oxidase activity, and promoting uric acid excretion.

6. The use according to claim 3, characterized in that: The product also includes auxiliary materials and / or additives.

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