Lactobacillus casei and its application in improving alcohol, uric acid and thrombus metabolism products

Abstract

The invention discloses a Lactobacillus casei and its application in improving alcohol, uric acid and thrombus metabolism products, and relates to the field of microbial technology. The Lactobacillus casei (Lacticaseibacillus casei) NHNK‑609 disclosed in the invention has been deposited in the China Center for Type Culture Collection on March 7, 2024, and its deposit number is CCTCC NO: M 2024422. NHNK‑609 has the functions of inhibiting the growth and alcohol production of Klebsiella pneumoniae, agglutinating Klebsiella pneumoniae, inhibiting the virulence genes of Klebsiella pneumoniae, promoting alcohol metabolism, reducing alcohol damage, tolerating alcohol, degrading uric acid, inhibiting xanthine oxidase activity, promoting uric acid excretion, dissolving thrombus, and increasing bacterial biofilm colonization of mucosal mucin and intestinal epithelial cells. It can be used to prepare products that improve alcohol, uric acid and thrombus metabolism.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to Lactobacillus casei and its application in improving alcohol, uric acid and thrombus metabolism products. Background Art

[0002] The ocean is the largest biosphere on Earth and the largest repository of species diversity in the world. Marine microorganisms are one of the earliest sources of marine bioactive substances known to people, including algae, bacteria, fungi, protozoa and other microorganisms. These bioactive substances have important application value in various fields such as food, medicine, and cosmetics.

[0003] More than 60% of people with NAFLD have a bacterium in their intestines that produces large amounts of alcohol, Klebsiella pneumoniae ( Klebsiella pneumoniae ), while only 6.25% of healthy people have Klebsiella pneumoniae in their intestines. Studies have shown that endogenous alcohol produced by Klebsiella pneumoniae can cause non-alcoholic fatty liver disease in mice. 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. Therefore, people who drink a lot of alcohol for a long time are more likely to have increased uric acid.

[0004] Uric acid is the final product of purine metabolism in the human body. Disorders in purine metabolism and uric acid excretion are the main factors causing gout. One-third of the uric acid in the human body is excreted through the intestines, so increasing the decomposition and excretion of uric acid in the intestines is a feasible way to alleviate uric acid accumulation.

[0005] The liver plays an important role in the metabolism of uric acid, regulating the synthesis and decomposition of uric acid, and converting excess uric acid into other substances for excretion. If the liver function is abnormal, such as cirrhosis, hepatitis and other diseases, it will affect the liver's ability to metabolize uric acid, resulting in reduced uric acid excretion and increased uric acid concentration in the blood.

[0006] The human body excretes uric acid mainly through the kidneys and intestines, and about 70% of uric acid is excreted through the kidneys. Recent studies have shown that intestinal flora can affect purine metabolism, xanthine oxidase (XOD), xanthine dehydrogenase (XHD) and urease activity or the composition of short-chain fatty acids, thereby changing the production and excretion of uric acid.

[0007] Venous thromboembolism is the third most common cardiovascular disease after heart disease and stroke. Studies have shown that there is a positive correlation between serum uric acid levels and the risk of venous thromboembolism. The serum uric acid level of patients with venous thromboembolism is significantly higher than that of the control group, and serum uric acid ≥325 mmol / L is associated with an increased risk of recurrence of venous thromboembolism. Thrombosis refers to the formation of clots in blood vessels or the heart by blood components in the human body. The blood in the human cardiovascular system remains in a fluid state, and the two mutually antagonistic systems of coagulation and anticoagulation maintain a balance, so that the body's normal blood circulation is guaranteed. After the coagulation factors interact with fibrinogen, the formed reticular fibrin can entangle blood cells and platelet clots, thereby forming thrombi. Thrombotic diseases include myocardial infarction, cerebral thrombosis, pulmonary embolism, limb artery embolism, limb vein thrombosis, disseminated intravascular coagulation, etc., which seriously endanger the physical and mental health of patients.

[0008] 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. Therefore, it is of great practical significance to provide a marine industry-related product developed using microbial technology. Summary of the invention

[0009] In view of this, the present invention provides a Lactobacillus casei and its application in improving alcohol, uric acid and thrombus metabolism products.

[0010] The present invention provides Lactobacillus casei ( Lacticaseibacillus casei ), which is Lactobacillus casei ( Lacticaseibacillus casei ) NHNK-609, which was deposited in the China Center for Type Culture Collection (CCTCC for short, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Wuhan University, Postal Code 430072) on March 7, 2024, and its preservation number is CCTCC NO: M 2024422).

[0011] Further, according to the above-mentioned Lactobacillus casei ( Lacticaseibacillus casei ) prepared by Lactobacillus casei ( Lacticaseibacillus casei ) of live bacteria, inactivated bacteria, fermentation broth or fermentation products.

[0012] The present invention also proposes the above-mentioned Lactobacillus casei ( Lacticaseibacillus casei ) or the above-mentioned Lactobacillus casei ( Lacticaseibacillus casei ) Use of the prepared product in the preparation of products for improving alcohol metabolism.

[0013] Furthermore, the improvement of alcohol metabolism includes at least one of the following a)-h):

[0014] a) Inhibit Klebsiella pneumoniae ( Klebsiella pneumoniae ) growing and / or producing alcohol;

[0015] b) Agglutination of Klebsiella pneumoniae ( Klebsiella pneumoniae );

[0016] c) downregulation of Klebsiella pneumoniae ( Klebsiella pneumoniae ) Virulence genes iroD and wabG at least one of the expressions;

[0017] d) Upregulation of genes related to alcohol metabolism ADH1B and / or ALDH2 Expression;

[0018] e) Upregulation of genes related to alcohol-induced damage and anti-inflammatory factors TGF-β and / or IL-10 Expression;

[0019] f) Upregulation of genes related to alcohol-induced intestinal barrier damage ZO-1 , OCLD and CLD4 at least one of the expressions;

[0020] g) Down-regulation of the gene related to alcohol-induced injury pro-inflammatory factors and tumor necrosis factor α gene TNF-α and interleukin-6 gene IL-6 Expression;

[0021] h) Alcohol tolerance.

[0022] The present invention also proposes the above-mentioned Lactobacillus casei ( Lacticaseibacillus casei ) or the above-mentioned Lactobacillus casei ( Lacticaseibacillus casei ) Use of the prepared product in the preparation of products for improving uric acid metabolism.

[0023] Furthermore, the improvement of uric acid metabolism includes at least one of the following a)-c):

[0024] a) Degradation of uric acid;

[0025] b) Inhibit xanthine oxidase activity;

[0026] c) Promote uric acid excretion including upregulating uric acid transport-related genes ABCG2 and SLC2A9 at least one of the expressions.

[0027] The present invention also proposes the above-mentioned Lactobacillus casei ( Lacticaseibacillus casei ) or the above-mentioned Lactobacillus casei ( Lacticaseibacillus casei ) Use of the prepared product in the preparation of products for improving thrombus metabolism.

[0028] Furthermore, the improvement of thrombus metabolism is to dissolve the fibrin thrombus formed by thrombin.

[0029] The present invention also proposes the above-mentioned Lactobacillus casei ( Lacticaseibacillus casei ) in any of the following:

[0030] (1) Colonization of mucosal mucin;

[0031] (2) Bacterial biofilm increases the ability to colonize mucosal mucins and intestinal epithelial cells.

[0032] The present invention also provides a product for improving the metabolism of alcohol, uric acid and thrombus, comprising the above-mentioned Lactobacillus casei ( Lacticaseibacillus casei ), or Lactobacillus casei as described above ( Lacticaseibacillus casei ) and acceptable excipients and / or adjuvants.

[0033] Lactobacillus casei disclosed by the present invention ( Lacticaseibacillus casei ), and its deposit number is CCTCCNO: M 2024422. Experiments have shown that NHNK-609 has the functions of inhibiting the growth and alcohol production of Klebsiella pneumoniae, agglutinating Klebsiella pneumoniae, inhibiting the virulence genes of Klebsiella pneumoniae, promoting alcohol metabolism, reducing alcohol damage, tolerance to alcohol, degrading uric acid, inhibiting xanthine oxidase activity, promoting uric acid excretion, dissolving thrombus, and increasing bacterial biofilm colonization on mucosal mucin and intestinal epithelial cells.

[0034] Biological Deposit Description

[0035] Lactobacillus casei Lacticaseibacillus casei ) NHNK-609, was deposited in the China Center for Type Culture Collection (CCTCC for short, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Wuhan University, Postal Code 430072) on March 7, 2024, and its deposit number is CCTCC NO: M 2024422. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The MRS plate colony image and Gram staining image of Lactobacillus casei NHNK-609 in the present invention;

[0037] Figure 2 The figure is a result of the agglutination experiment between the inactivated bacteria of Lactobacillus casei NHNK-609 and Klebsiella pneumoniae in the present invention;

[0038] Figure 3 It is a plate image of artificial thrombus of Lactobacillus casei NHNK-609 fermentation liquid in the present invention;

[0039] Figure 4 This is a graph showing the results of the experiment on the adsorption of mucin by live Lactobacillus casei NHNK-609 bacteria and NHNK-609 biofilm in the present invention;

[0040] Figure 5 This is a graph showing the experimental results of the adsorption of live Lactobacillus casei NHNK-609 bacteria and NHNK-609 biofilm on intestinal epithelial cells Caco-2 in the present invention.

[0041] The following specific implementation manner will further illustrate the present invention in conjunction with the above-mentioned drawings. DETAILED DESCRIPTION

[0042] The invention provides Lactobacillus casei and application thereof. Those skilled in the art can learn from this article content, and suitably improve the process parameter and realize. It is particularly important to point out that all similar replacements and changes are obvious to those skilled in the art, and they are all considered to be included in the present invention. Method and application of the present invention have been described by preferred embodiments, and relevant personnel can obviously change or suitably change and combine the method and application of this article without departing from the content of the present invention, spirit and scope, to realize and apply the technology of the present invention.

[0043] The Lactobacillus casei strain NHNK-609 of the present invention is derived from the intestine of Abalone diversicolor and is identified as Lactobacillus casei ( Lacticaseibacillus casei ). This strain is Gram-positive and has a rod-shaped curved arrangement under a microscope. When grown on an MRS plate, it forms round colonies with a smooth, translucent surface, white in color, and neat edges. It grows evenly in an MRS liquid medium and has a white precipitate after long-term storage. The optimal growth temperature is 37°C.

[0044] Lactobacillus casei Lacticaseibacillus casei )NHNK-609, depository unit: China Center for Type Culture Collection, address: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, deposit date: March 7, 2024, deposit number: CCTCC NO: M 2024422.

[0045] Furthermore, in the application described in the present invention, the Lactobacillus casei NHNK-609 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 products, live bacteria, inactivated bacteria, and fermentation bacterial broth.

[0046] In vitro experiments show that the fermentation product of Lactobacillus casei NHNK-609 of the present invention has the effect of inhibiting Klebsiella pneumoniae ( Klebsiella pneumoniae) growth, with the inhibition rate reaching 62.16%~63.89%.

[0047] In vitro experiments show that the Lactobacillus casei NHNK-609 of the present invention has the effect of agglutinating Klebsiella pneumoniae, and the agglutination rate is 14.67-21.05%.

[0048] In vitro experiments show that the Lactobacillus casei NHNK-609 of the present invention has the ability to inhibit the gene expression of the virulence factor enterobactin of Klebsiella pneumoniae. iroD and galactosyltransferase-related genes wabG The relative gene expression was downregulated to 0.04~0.83 times.

[0049] In vitro experiments show that the Lactobacillus casei NHNK-609 of the present invention has the effect of inhibiting the alcohol production of Klebsiella pneumoniae, and the inhibition rate reaches 20.25% to 23.85%.

[0050] In vitro experiments show that the Lactobacillus casei NHNK-609 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 aldehyde dehydrogenase type 2 gene ALDH2 The relative expression of α-TNF was 1.11-6.75 times. TNF-α and interleukin-6 gene IL- 6 The relative expression level was 0.21-0.81 times.

[0051] In vitro experiments show that the Lactobacillus casei NHNK-609 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 expression of 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 1.11 to 4.55 times.

[0052] In vitro experiments show that the Lactobacillus casei NHNK-609 of the present invention has an alcohol tolerance effect, and the growth rate in a culture medium containing 10% alcohol is 33.33% to 47.37%.

[0053] In vitro experiments show that the Lactobacillus casei NHNK-609 of the present invention has the function of degrading uric acid, and the degradation rate is 59.02% to 63.07%.

[0054] In vitro experiments show that the Lactobacillus casei NHNK-609 of the present invention has the effect of inhibiting the activity of xanthine oxidase (XOD), and the inhibition rate is 38.01% to 56.00%.

[0055] In vitro experiments show that the Lactobacillus casei NHNK-609 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 level was 2.06 to 2.26 times.

[0056] In vitro experiments show that the Lactobacillus casei NHNK-609 of the present invention has thrombus dissolving activity, i.e., has fibrinolytic activity, dissolves thrombi formed by fibrinogen and thrombin, and forms a transparent circle on the thrombus plate with a diameter of 6.41 cm to 7.42 cm.

[0057] The reagents and consumables used in the present invention are all common commercially available products. The present invention is further described below in conjunction with the embodiments:

[0058] Example 1 Isolation of NHNK-609

[0059] The sample was obtained from an adult individual of Abalone variegata obtained from a farm, with a shell length of 5 cm and fasting for 3 days. The digestive tract was obtained by dissection with sterile tools, rinsed with sterile saline for 3 times, and the intestinal tissue was broken by homogenization and grinding. The homogenate was collected in 1 ml of sterile saline, and the supernatant was streaked on an MRS solid plate after gradient dilution. After constant temperature culture at 37°C for 24 to 48 hours, white colonies were picked and repeatedly inoculated and screened until a uniform single colony was obtained, named NHNK-609.

[0060] Gram staining microscopy: strain NHNK-609 is a Gram-positive colony, which is arranged in a rod-shaped curve under a microscope; it grows on an MRS plate and forms white, smooth, round, semi-transparent circular colonies with neat edges; it grows evenly in MRS liquid culture medium and becomes turbid and white precipitates after long-term storage. Figure 1 shown.

[0061] Example 2 Nucleic acid identification of NHNK-609

[0062] 1. 16S rDNA gene sequence analysis:

[0063] 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.

[0064] 2. Results

[0065] The PCR product was sequenced and compared with the standard sequences published in the GenBank database (BLASTN), and the NHNK-609 strain was identified as Lactobacillus casei ( Lacticaseibacillus casei ).

[0066] Example 3 Experiment on the inhibition of Klebsiella pneumoniae growth by NHNK-609 fermentation product

[0067] 1. Preparation of NHNK-609 fermentation products

[0068] A single colony of Lactobacillus casei NHNK-609 was selected and placed in MRS liquid medium, incubated at 37°C for 48 h, and then adjusted to OD 600 =0.3, 5000rpm to take the supernatant, and then filter with a 0.22μm filter membrane to obtain a sterile fermentation product.

[0069] 2. Preparation of Klebsiella pneumoniae bacterial suspension

[0070] Klebsiella pneumoniae CICC 10870 was inoculated into BHI liquid medium at 1% (v / v) and cultured at 37°C with shaking 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.

[0071] 3. Experiment on the inhibition of Klebsiella pneumoniae proliferation by NHNK-609 fermentation products

[0072] 3 mL of BHI medium and 0.3 mL of NHNK-609 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.

[0073] The results are shown in Table 1 below:

[0074] Table 1: NHNK-609 fermentation products inhibit the growth of Klebsiella pneumoniae

[0075]

[0076] The results showed that NHNK-609 could reduce the proliferation of Klebsiella pneumoniae, with an inhibition rate of 62.16%~63.89%.

[0077] Example 4 NHNK-609 agglutination test of Klebsiella pneumoniae

[0078] 1. Preparation of NHNK-609 inactivated bacteria

[0079] A single colony of Lactobacillus casei NHNK-609 was selected and placed in MRS liquid medium, incubated at 37°C for 48 h, centrifuged at 5000 rpm for 10 min, and the precipitate was washed twice with PBS. The cells were then resuspended in PBS and the OD was adjusted. 600 =0.3. Inactivated bacteria can be obtained by high pressure sterilization at 121℃ for 15 min.

[0080] 2. Preparation of Klebsiella pneumoniae suspension

[0081] 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.

[0082] 3. Coagulation experiment

[0083] The suspension of Klebsiella pneumoniae and NHNK-609 inactivated bacteria was mixed in a volume ratio of 1:1. After 30 minutes of reaction, samples of NHNK-609, Klebsiella pneumoniae, and the mixed reaction liquid of NHNK-609 and Klebsiella pneumoniae were taken. The sampling range was the top 50 μL of the liquid surface. After aspiration, it was transferred to a 96-well plate and its absorbance at OD = 600 nm was measured. At the same time, the agglutination precipitate was stained with Gram stain and its bacterial agglutination state was observed. Figure 2 shown.

[0084] Calculation formula:

[0085] Agglutination rate (%) = [(Ax+Ay)-2Amix] / (Ax+Ay)×100%;

[0086] Note: Ax: OD measured by NHNK-609 alone at the reaction time 600 Value; Ay: OD of Klebsiella pneumoniae alone measured at the reaction time 600Value; Amix: OD measured at the reaction time after NHNK-609 and Klebsiella pneumoniae were mixed 600 Numeric value.

[0087] The results are shown in Table 2 below:

[0088] Table 2

[0089]

[0090] The results showed that after 30 minutes of reaction, NHNK-609 inactivated bacteria could agglutinate Klebsiella pneumoniae.

[0091] Example 5 Experiment on the inhibition of virulence gene expression of Klebsiella pneumoniae by NHNK-609

[0092] 1. Preparation of NHNK-609 fermentation products and live bacterial suspension

[0093] A single colony of Lactobacillus casei NHNK-609 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-609 live bacterial suspension was obtained.

[0094] 2. Experiment on inhibiting the expression of virulence genes of Klebsiella pneumoniae

[0095] Klebsiella pneumoniae glycerol culture 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-609 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 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.

[0096] Formula: F=2 -ΔΔCT ,in:

[0097] △CT 实验 =CT实验 -CT 内参（实验）;

[0098] △CT 对照 =CT 对照 -CT 内参（对照）;

[0099] △△CT=△CT 实验 -△CT 对照。

[0100] The results are shown in Table 3-4 below:

[0101] Table 3

[0102]

[0103] Table 4

[0104]

[0105] The results showed that NHNK-609 could inhibit the virulence genes of Klebsiella pneumoniae iroD , wabG expression, thereby reducing the pathogenicity of Klebsiella pneumoniae.

[0106] Example 6 Experiment on the inhibition of ethanol production by Klebsiella pneumoniae by NHNK-609

[0107] 1. Preparation of NHNK-609 fermentation products and live bacterial suspension

[0108] A single colony of Lactobacillus casei NHNK-609 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-609 live bacterial suspension was obtained.

[0109] 2. Preparation of Klebsiella pneumoniae suspension

[0110] 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.

[0111] 3. Experiment on NHNK-609 inhibiting ethanol production of Klebsiella pneumoniae

[0112] The fermentation product of NHNK-609 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 min to obtain 1 ml of supernatant for later use.

[0113] 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.

[0114] Potassium dichromate oxidation method for detecting 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.

[0115] The calculation formula and results are shown in Table 5:

[0116] Table 5: NHNK-609 inhibits alcohol production by Klebsiella pneumoniae

[0117]

[0118] The results showed that NHNK-609 inhibited the production of alcohol by Klebsiella pneumoniae.

[0119] Example 7 NHNK-609 regulates the expression of genes related to alcohol-induced liver cell damage

[0120] 1. Preparation of NHNK-609 fermentation products and live bacterial suspension

[0121] Pick a single colony of NHNK-609 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.

[0122] 2. Culture of human hepatocytes HepG2

[0123] HepG2 cells were activated with DMEM medium containing 10% FBS and 1% penicillin-streptomycin, and cultured at 37°C and 5% CO2. When the cells were 80-90% confluent, they were passaged or plated.

[0124] 4. NHNK-609 regulates the expression of genes related to alcohol-induced liver cell damage

[0125] 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-609 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, IL-6 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.

[0126] The results are shown in Table 6-7 below:

[0127] Table 6

[0128]

[0129] Table 7

[0130]

[0131] The results showed that NHNK-609 could upregulate anti-inflammatory factor genes TGF-β, IL-10 and alcohol metabolism genes ADH1B, ALDH2 downregulation of pro-inflammatory factor genes TNF-α, IL-6 NHNK-609 can alleviate the inflammation caused by alcohol-damaged liver cells and enhance the alcohol metabolism ability of liver cells.

[0132] Example 8 NHNK-609 regulates the expression of genes related to alcohol-induced damage to intestinal epithelial cells

[0133] 1. Preparation of NHNK-609

[0134] Pick a single colony of NHNK-609 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 OD600 =0.5, and inactivated bacteria were obtained.

[0135] 2. Cultivation of human intestinal epithelial cells Caco-2

[0136] Caco-2 cells were activated with DMEM medium containing 10% FBS and 1% penicillin-streptomycin, and cultured at 37°C and 5% CO2. When the cells were 80-90% confluent, they were passaged or plated.

[0137] 4. NHNK-609 regulates the expression of genes related to alcohol-induced damage to intestinal epithelial cells

[0138] 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-609 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.

[0139] The results are shown in Tables 8-10 below:

[0140] Table 8

[0141]

[0142] Table 9

[0143]

[0144] Table 10

[0145]

[0146] Results showed that NHNK-609 upregulated intestinal barrier genes ZO-1, OCLD, CLD-4 and alcohol metabolism genes ADH1B, ALDH2 NHNK-609 can alleviate alcohol-induced intestinal epithelial cell barrier damage and enhance the alcohol metabolism ability of intestinal epithelial cells.

[0147] Example 9 NHNK-609 alcohol tolerance test

[0148] Inoculate NHNK-609 into fresh MRS medium at a 2% inoculation rate and culture at 37°C for 24h. After the culture is completed, inoculate into MRS medium containing 10% alcohol at a ratio of 2%. Take 100μL of bacterial suspension, measure the absorbance at OD=600nm, and record the OD value before and after 48 hours of culture at 37°C. Calculation formula: Growth rate (%) = (A1-A0) / A0*100% (A1: absorbance value after culture; A0: absorbance value before culture)

[0149] The results are shown in Table 11 below.

[0150] Table 11

[0151]

[0152] The results showed that NHNK-609 has the ability to tolerate alcohol growth and can grow in a culture medium containing 10% alcohol, with a growth rate of 33.33%~47.37%.

[0153] Example 10 Experiment on degradation of uric acid by NHNK-609

[0154] 1. Preparation of uric acid solution

[0155] 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.

[0156] 2. NHNK-609 degradation of uric acid experiment

[0157] The NHNK-609 frozen tube bacterial solution was inoculated into fresh MRS medium at a ratio of 2% (v / v), incubated 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-609 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 was completed, the supernatant was taken by centrifugation at 5000rpm for 10 minutes, and the absorbance at OD=510nm was measured using a uric acid detection kit. Degradation rate (%) = (1-absorbance of the experimental group / absorbance of the control group) * 100. The results are shown in Table 12 below:

[0158] Table 12

[0159]

[0160] The results showed that NHNK-609 degraded uric acid, thereby promoting uric acid metabolism.

[0161] Example 11 Experiment on the inhibition of xanthine oxidase (XOD) activity by NHNK-609

[0162] 1. Preparation of NHNK-609 fermentation products

[0163] NHNK-609 glycerol tube bacterial solution was inoculated into fresh MRS medium at 2% (v / v) and cultured at 37°C for 48 h. After that, the OD was adjusted to 0. 600 =1.0, centrifuge at 5000rpm for 10min, take the supernatant, and filter through a 0.22μm filter membrane to obtain the fermentation product.

[0164] 2. Experiment on inhibition of xanthine oxidase (XOD) activity by NHNK-609 fermentation products

[0165] 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 development reaction, the absorbance of each group at OD=530nm was measured to calculate the inhibition rate of the fermentation product on XOD. Inhibition rate (%) = (1-experimental group / control group) * 100, the results are shown in Table 13 below:

[0166] Table 13

[0167]

[0168] The results showed that the fermentation product of NHNK-609 could significantly inhibit the activity of XOD enzyme and inhibit the formation of xanthine from hypoxanthine, thereby reducing the formation of uric acid.

[0169] Example 12 NHNK-609 regulates the expression of genes related to uric acid excretion in renal tubular epithelial cells

[0170] 1. Preparation of NHNK-609 fermentation products

[0171] The method for preparing the fermentation product is as described in Example 7.

[0172] 2. NHNK-609 regulates genes related to uric acid excretion in renal tubular epithelial cells

[0173] (1) HKC cell culture and intervention

[0174] 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.

[0175] (2) RNA extraction and fluorescence quantitative PCR

[0176] 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 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.

[0177] The results are shown in Table 14 below.

[0178] Table 14

[0179]

[0180] The results showed that NHNK-609 fermentation products upregulated uric acid excretion-related genes in HKC renal tubular epithelial cells ABCG2 and SLC2A9 NHNK-609 can promote the excretion of uric acid from the renal tubules.

[0181] Example 13 NHNK-609 regulates the expression of uric acid excretion genes and intestinal barrier-related genes in human intestinal epithelial cells Caco-2

[0182] 1. Preparation of NHNK-609 live and inactivated bacteria

[0183] The NHNK-609 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.

[0184] 2. NHNK-609 regulates the expression of uric acid excretion genes and intestinal barrier-related genes in Caco-2 cells

[0185] (1) Caco-2 cell culture and intervention treatment

[0186] 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.

[0187] (2) RNA extraction and fluorescence quantitative PCR

[0188] 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 Expression of intestinal barrier-related gene zonula occludens gene ZO-1 , tight junction protein-4 gene CLD4 and tight junction protein genes OCLD expression.

[0189] (3) Data processing

[0190] 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.

[0191] The results are shown in Tables 15-16 below.

[0192] Table 15

[0193]

[0194] Table 16

[0195]

[0196] The results showed that both live and inactivated NHNK-609 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 hyperuricemia caused by insufficient intestinal barrier.

[0197] Example 14 Fibrinolytic activity of NHNK-609

[0198] 1. Preparation of NHNK-609 fermentation solution

[0199] A single colony of NHNK-609 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.

[0200] 2. Fibrin plate culture medium

[0201] Sample A: Dissolve 22 mg of fibrinogen in 10 mL of normal saline and incubate in a 37°C water bath for 5 to 10 min.

[0202] Sample B: Take 5 mg of thrombin (concentration of 40 U / mg) and add it to 2 mL of normal saline and incubate in a 37°C water bath for 5-10 min;

[0203] Sample C: Weigh 0.1 g agarose and dissolve it in 8 mL saline. Heat to fully dissolve the agarose.

[0204] Quickly add sample B into sample C, and finally add sample A. Pour the mixture into a plate and wait for the fibrin plate to solidify before use.

[0205] 3. NHNK-609 fibrinolytic activity assay

[0206] Take 2 μL of NHNK-609 fermentation liquid and apply it on the fibrin 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.

[0207] The results are shown in Table 17:

[0208] Table 17

[0209]

[0210] like Figure 3 As shown, NHNK-614 has plasminogen activitv, which makes the fibrin plate present transparent circles with a diameter of 6.41 cm to 7.42 cm, and can dissolve the fibrin artificial thrombus formed by thrombin.

[0211] Example 15 NHNK-609 forms biofilm

[0212] 1. Formation of NHNK-609 biofilm

[0213] A single colony of NHNK-609 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 degrees for 24 hours.

[0214] 2. Crystal violet staining

[0215] After the incubation, discard the supernatant, add 100 μL sterile PBS to each well and wash twice, then add 100 μL 4% paraformaldehyde fixative to each well and fix at room temperature for 30 minutes. Discard the fixative, add 100 μL crystal violet to each well and stain at room temperature for 30 minutes. After staining, wash twice with sterile PBS and dry, add 100 μL anhydrous ethanol to each well, let stand for 1 minute, and measure the absorbance at 600 nm.

[0216] The results are shown in Table 18:

[0217] Table 18: NHNK-609 biofilm formation

[0218]

[0219] The results showed that NHNK-609 in OD 600 =0.2 in MRS medium at 37℃ for 24h to form biofilm.

[0220] Example 16 Determination of the adsorption capacity of NHNK-609 to mucin

[0221] 1. Preparation of NHNK-609 live bacterial suspension

[0222] Pick a single colony of NHNK-609 in fresh MRS medium and culture at 37°C for 24 hours. Centrifuge at 5000 rpm for 10 minutes to collect the cells, wash twice with sterile PBS, resuspend the cells in DMEM medium and adjust the OD 600 =0.5 to obtain a live bacterial suspension.

[0223] 2. Preparation of NHNK-609 biofilm bacterial suspension

[0224] A single colony of NHNK-609 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 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.

[0225] 3. NHNK-609 adhesion test to mucin

[0226] (1) Reagent preparation

[0227] Mucin solution: Weigh 10 mg of mucin and dissolve it in 10 mL of 50 mM Tris-HCL solution at 4°C overnight.

[0228] Blocking solution: Dissolve bovine serum albumin in PBS solution at a ratio of 2% (m / v).

[0229] Washing solution: Dissolve bovine serum albumin in PBS solution at a ratio of 0.1% (m / v).

[0230] (2) Adsorption experiment of NHNK-609 bacterial liquid and biofilm on mucin

[0231] 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-609 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.

[0232] like Figure 4 The results showed that NHNK-609 could adsorb mucin, the main component of the mucosal layer, and that NHNK-609 could increase its adsorption capacity for mucin after coating the biofilm formed by itself.

[0233] Example 17 Determination of the Adhesion Ability of NHNK-609 to Human Intestinal Epithelial Cells Caco-2

[0234] 1. Preparation of NHNK-609 live bacteria and biofilm bacterial suspension

[0235] The preparation method is similar to that of Example 16.

[0236] 2. Determination of the adsorption capacity of NHNK-609 to Caco-2 cells

[0237] 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.

[0238] The results showed that NHNK-609 biofilm can increase the adsorption of bacteria to intestinal epithelial cells, such as Figure 5 shown.

[0239] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A Lactobacillus casei ( Lacticaseibacillus casei ), which is Lactobacillus casei ( Lacticaseibacillus casei ) NHNK-609 was deposited in the China Center for Type Culture Collection on March 7, 2024, and its deposit number is CCTCC NO: M 2024422.

2. Lactobacillus casei as claimed in claim 1 ( Lacticaseibacillus casei ) in the preparation of a product for improving alcohol metabolism, wherein the product for improving alcohol metabolism is an improvement in Klebsiella pneumoniae ( Klebsiella pneumoniae ) caused by non-alcoholic fatty liver disease products.

3. Lactobacillus casei as claimed in claim 1 ( Lacticaseibacillus casei ) in the preparation of a product for improving uric acid metabolism, wherein the improvement of uric acid metabolism is the degradation of uric acid.

4. Lactobacillus casei as claimed in claim 1 ( Lacticaseibacillus casei ) in the preparation of a product for improving thrombus metabolism, wherein the improved thrombus metabolism is the dissolution of fibrin thrombus formed by thrombin.

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