A strain of Propionibacterium freudenreichii subspecies shermanii and its application in preparing products for improving fatty liver

Abstract

The present invention relates to the field of marine microbial technology, and in particular provides a strain of Propionibacterium freudenreichii subspecies schreberi and its application in a product for improving fatty liver. The provided Propionibacterium freudenreichii subspecies schreberi NHNK‑616 is deposited in the China Center for Type Culture Collection, and the deposit number is CCTCC NO: M 20241429. Experiments show that NHNK‑616 has the functions of inhibiting the growth of Klebsiella pneumoniae, inhibiting the production of alcohol, agglutinating Klebsiella pneumoniae, inhibiting the virulence gene of Klebsiella pneumoniae, promoting alcohol metabolism, reducing alcohol damage, and tolerating alcohol, and can be used to prepare a product for improving fatty liver, wherein the product for improving fatty liver includes a product for improving non-alcoholic fatty liver disease and a product for improving alcohol metabolism.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a strain of Propionibacterium freudenreichii subspecies shermanii and application thereof in preparing a product for improving fatty liver. Background Art

[0002] Xu's flathead scorpionfish Sebastods schlegelii ) is a cold-water, near-shore, bottom-feeding carnivorous fish. In the fish intestinal microbiome, bacteria (aerobic, facultative anaerobic, and obligate anaerobic) are the main colonizing microorganisms. Intestinal microorganisms play a very important role in the growth of fish, and their balance is related to the health of the fish body. The wide variety of microorganisms in the fish intestine play a very important role in inhibiting pathogenic microorganisms and can protect the host fish from pathogenic microorganisms.

[0003] Nonalcoholic fatty liver disease (NAFLD) Nonalcoholic fatty liver disease NAFLD refers to a disease in which the fat content in the liver increases abnormally without alcohol intake. More and more evidence shows that NAFLD is closely related to obesity, metabolic abnormalities and insulin resistance syndrome, dyslipidemia and changes in intestinal flora. Studies have found that Klebsiella pneumoniae ( Klebsiella pneumoniae ), which is a bacterium known to produce high concentrations of alcohol. After testing, the flora isolated from the patient's intestines produced about 4-6 times the alcohol content of healthy people. Metagenomic high-throughput sequencing of fecal samples from NAFLD patients and healthy controls found that 61% of patients contained Klebsiella pneumoniae with high alcohol production ability, while only 6.25% of the healthy group had such flora in their intestines.

[0004] Mucus phenotype regulator rmpA , Enterobactin gene iroD and galactosyltransferase-related genes wabG It is a common virulence gene in Klebsiella pneumoniae and is closely related to the pathogenicity of Klebsiella pneumoniae. In addition, researchers isolated Klebsiella pneumoniae with high alcohol production ability from the stool samples of patients with bacterial autobrewery syndrome / non-alcoholic fatty liver disease, the first case in the world. This bacterium will continuously produce alcohol in the human body, causing persistent damage to the liver and intestines.

[0005] The study found that endogenous alcohol production by intestinal bacteria Klebsiella pneumoniae can cause NAFLD 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. These findings not only explain the similar pathological characteristics between NAFLD and alcoholic fatty liver, but also provide new ideas for the clinical diagnosis and treatment of fatty liver caused by this type of bacteria.

[0006] Marine microorganisms have developed complex molecular adaptations to cope with these harsh conditions, affecting their primary and secondary metabolic pathways. This has led to the evolution of unique physiological characteristics and metabolic processes, and marine microorganisms are more likely to synthesize structurally unique enzymes and secondary metabolites than terrestrial microorganisms. Exploring the application of marine fish gut microorganisms has important practical significance in expanding the value of the marine industry. Summary of the invention

[0007] In view of this, the present invention provides a strain of Propionibacterium freudenreichii subsp. schreiberi and its application. The provided Propionibacterium freudenreichii subsp. schreiberi NHNK-616 is deposited in the China Center for Type Culture Collection, and the deposit number is CCTCC NO: M20241429. Experiments show that NHNK-616 has the functions of inhibiting the growth of Klebsiella pneumoniae, inhibiting the production of alcohol, agglutinating Klebsiella pneumoniae, inhibiting the virulence gene of Klebsiella pneumoniae, promoting alcohol metabolism, reducing alcohol damage, and tolerating alcohol, and can be used to prepare a product for improving fatty liver, wherein the product for improving fatty liver includes a product for improving non-alcoholic fatty liver disease and a product for improving alcohol metabolism.

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

[0009] The inventors first provided a strain of Propionibacterium freudenreichii subspecies NHNK-616 ( Propionibacterium freudenreichii subsp. shermanii NHNK-616), with the deposit number of CCTCC NO: M 20241429, which was deposited in China Center for Type Culture Collection on July 1, 2024.

[0010] The above-mentioned Propionibacterium freudenreichii subsp. schreiberi NHNK-616 was derived from the intestine of the flatfish Xu's and was identified as Propionibacterium freudenreichii subsp. schreiberi by 16S rDNA ( Propionibacterium freudenreichii subsp. shermanii ). This strain is Gram-positive, and appears as a short rod or rod-shaped bacillus under a microscope, arranged in single or multiple arrangements, often in a short chain form; it grows on MRS plates and can form round colonies with moist white surfaces, neat edges, smooth protrusions; it grows evenly in MRS liquid culture medium and is turbid, and white precipitates may appear after long-term static culture. The optimal growth temperature is 30°C, and it is anaerobic.

[0011] The present invention also provides the use of the above-mentioned Propionibacterium freudenreichii subsp. shenkelii NHNK-616 in products for improving fatty liver, wherein the products for improving fatty liver include products for improving non-alcoholic fatty liver disease and products for improving alcohol metabolism.

[0012] When preparing a product for improving non-alcoholic fatty liver disease, its application mechanism includes but is not limited to any one or more of the following: inhibiting the growth of Klebsiella pneumoniae, inhibiting the production of alcohol, agglutinating Klebsiella pneumoniae, and inhibiting the virulence gene of Klebsiella pneumoniae.

[0013] In vitro experiments show that the fermentation product of Propionibacterium freudenreichii subsp. shenkelii NHNK-616 of the present invention has the effect of inhibiting the growth of Klebsiella pneumoniae, and the inhibition rate reaches 42.86%-48.89%.

[0014] In vitro experiments show that the Propionibacterium freudenreichii subspecies shermanii NHNK-616 of the present invention has the effect of inhibiting the production of alcohol by Klebsiella pneumoniae, and the inhibition rate reaches 20.39%-24.81%.

[0015] In vitro experiments show that the Propionibacterium freudenreichii subspecies shermanii NHNK-616 of the present invention has the function of agglutinating Klebsiella pneumoniae, and the agglutination rate is 12.20-18.64%.

[0016] In vitro experiments show that the Propionibacterium freudenreichii subspecies NHNK-616 of the present invention has the ability to inhibit the gene encoding the virulence factor enterobactin of Klebsiella pneumoniae. iroD and galactosyltransferase-related genes wabG The relative gene expression was downregulated to 0.44-0.84 times.

[0017] When preparing a product for improving alcohol metabolism, the improved alcohol metabolism is at least one of promoting alcohol metabolism, reducing alcohol damage, and tolerating alcohol, and its application mechanism includes but is not limited to any one or more of the following:

[0018] a) Upregulation of genes related to alcohol metabolism ADH1B and / or ALDH2 Expression;

[0019] b) Upregulation of anti-inflammatory factor genes induced by alcohol TGF-β and / or IL-10 Expression;

[0020] c) Down-regulation of genes that promote inflammation in alcohol-induced damage TNF-α , IL-6 At least one of;

[0021] d) Upregulation of genes related to alcohol-induced intestinal barrier damage ZO-1 , OCLD and CLD4 at least one of the expressions;

[0022] e) Tolerance to alcohol.

[0023] In vitro experiments show that the Propionibacterium freudenreichii subspecies NHNK-616 of the present invention has the effect of regulating the expression of genes related to alcohol damage in HepG2 liver cells: upregulating the anti-inflammatory factor-related transforming growth factor gene TGF-β and interleukin-10 gene IL-10 The relative expression of alcohol metabolism-related alcohol dehydrogenase-1B gene was 1.59-3.55 times. ADH1B and aldehyde dehydrogenase type 2 gene ALDH2 The relative expression of α-tumor necrosis factor gene was downregulated. TNF-α and interleukin-6 gene IL-6 The relative expression level was 0.16-0.74 times.

[0024] In vitro experiments show that the Propionibacterium freudenreichii subspecies NHNK-616 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 claudin-4 gene CLD4 The relative expression of alcohol metabolism-related alcohol dehydrogenase-1B gene was 1.25-2.26 times. ADH1B and / or aldehyde dehydrogenase type 2 gene ALDH2 The relative expression levels were 1.14-8.07 times.

[0025] In vitro experiments show that the Propionibacterium freudenreichii subspecies shermanii NHNK-616 of the present invention has an alcohol tolerance effect, and the growth rate in a culture medium containing 10% alcohol is 327.34%-350.00%.

[0026] In summary, the beneficial effects of the present invention are as follows:

[0027] The provided Propionibacterium freudenreichii subspecies shermanii NHNK-616 has the functions of inhibiting the growth of Klebsiella pneumoniae and the production of alcohol, agglutinating Klebsiella pneumoniae, inhibiting the virulence gene of Klebsiella pneumoniae, promoting alcohol metabolism, reducing alcohol damage and tolerating alcohol.

[0028] The inventor has made a biological deposit of Propionibacterium freudenreichii subsp. NHNK-616, and the deposit information is as follows:

[0029] Storage time: July 1, 2024

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

[0031] Deposit number: CCTCC NO: M 20241429

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

[0033] Classification name: Propionibacterium freudenreichii subsp. NHNK-616 ( Propionibacterium freudenreichii subsp. shermanii NHNK-616). BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The MRS plate colony image of Propionibacterium freudenreichii subspecies NHNK-616 in the present invention;

[0035] Figure 2 The Gram staining diagram of Propionibacterium freudenreichii subspecies NHNK-616 in the present invention;

[0036] Figure 3 The photos are of the inactivated Propionibacterium freudenreichii subsp. shermanii NHNK-616 combined with Klebsiella pneumoniae in the present invention; (a) is NHNK-616, (b) is Klebsiella pneumoniae, and (c) is a mixture of NHNK-616 and Klebsiella pneumoniae. DETAILED DESCRIPTION

[0037] The present invention provides Propionibacterium freudenreichii subsp. shermanii and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar substitutions 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 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.

[0038] The Propionibacterium freudenreichii subspecies NHNK-616 of the present invention is derived from the intestine of the flatfish Xu's and is identified as Propionibacterium freudenreichii subspecies NHNK-616 by 16S rDNA. Propionibacterium freudenreichii subsp. shermanii ). This strain is Gram-positive, and appears as a short rod or rod-shaped bacillus under a microscope, arranged in single or multiple arrangements, often in a short chain form; it grows on MRS plates and can form round colonies with moist white surfaces, neat edges, smooth protrusions; it grows evenly in MRS liquid culture medium and is turbid, and white precipitates may appear after long-term static culture. The optimal growth temperature is 30°C, and it is anaerobic.

[0039] Furthermore, in the application described in the present invention, the Propionibacterium freudenreichii subsp. schermerii NHNK-616 provided by the present invention exists in the form of being sterilized or not, or in the form of a fermentation product (i.e., supernatant), and the derivative form is preferably selected from: metabolites, metabolic biological products, prebiotics, cell walls and their components, extracellular polysaccharides, and compounds containing immunogenic components, preferably selected from: live bacteria, fermentation products, and inactivated bacteria.

[0040] The reagents and consumables used in the present invention are all common commercially available products. The present invention is now further described in conjunction with the embodiments.

[0041] Example 1 Isolation of NHNK-616.

[0042] The flathead mullet obtained from a marine cage culture base was wiped with 75% alcohol, and the stomach, pyloric caeca and anterior midgut were cut off with sterile instruments, and the contents were removed. The fish was rinsed twice with sterile saline, and the mucus on the inner wall of each organ was scraped and added to sterile saline. After vortexing and shaking, it was spread on MRS solid culture medium and placed in an anaerobic bag. After constant temperature culture at 30℃ for 48h, white colonies were picked and repeatedly streaked and screened until a uniform single colony was obtained, which was named NHNK-616.

[0043] Gram staining microscopy: strain NHNK-616 is a Gram-positive colony, which appears as short rod-shaped or rod-shaped bacilli under a microscope, arranged in single or multiple arrangements, often in a short chain form; it grows on MRS plates and can form round colonies with moist white surfaces, neat edges, and smooth protrusions; it can grow evenly in MRS culture medium in a turbid state, and the bacteria will form white precipitates after being placed for a long time. Figure 1 and Figure 2 shown.

[0044] Example 2 Nucleic acid identification of NHNK-616.

[0045] 1. 16S rDNA gene sequence analysis:

[0046] Pick a single colony in MRS liquid medium, culture it anaerobically at 30℃ 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.

[0047] 2. Results:

[0048] The PCR product was sequenced and compared with the standard sequence published in the GenBank database for homology (BLASTN), and it was found that the NHNK-616 strain obtained was Propionibacterium freudenreichii subsp. Propionibacterium freudenreichii subsp. shermanii ).

[0049] Example 3 Experiment on the inhibition of Klebsiella pneumoniae growth by NHNK-616 fermentation product.

[0050] 1. Preparation of NHNK-616 fermentation product:

[0051] A single colony of Propionibacterium freudenreichii subsp. NHNK-616 was selected and placed in MRS liquid medium, incubated anaerobically at 30°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 the fermentation product.

[0052] 2. Preparation of Klebsiella pneumoniae suspension:

[0053] 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, OD was adjusted with BHI. 600 =0.3, and obtain Klebsiella pneumoniae suspension.

[0054] 3. Experiment on inhibition of Klebsiella pneumoniae proliferation by NHNK-616 fermentation products:

[0055] 3 mL of BHI medium and 0.3 mL of NHNK-616 fermentation product were added to a centrifuge tube. An equal volume of MRS medium was added to the control group. The Klebsiella pneumoniae suspension obtained in the previous step was inoculated at 1% (v / v). After shaking and culturing at 37°C for 24 hours, the absorbance at 600 nm was measured. The results are shown in Table 1 below:

[0056] Table 1 Inhibition of Klebsiella pneumoniae growth by NHNK-616 fermentation products

[0057] .

[0058] The results showed that NHNK-616 could reduce the growth and proliferation of Klebsiella pneumoniae, with an inhibition rate of 42.86-48.89%.

[0059] Example 4 NHNK-616 agglutination experiment on Klebsiella pneumoniae

[0060] 1. Preparation of NHNK-616 inactivated bacteria:

[0061] A single colony of Propionibacterium freudenreichii subsp. NHNK-616 was selected and placed in MRS liquid medium, incubated anaerobically at 30°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. High pressure sterilization at 121℃ for 15 min can obtain a bacterial suspension inactivated with the inactivated bacteria.

[0062] 2. Preparation of Klebsiella pneumoniae suspension:

[0063] The preparation method of Klebsiella pneumoniae suspension is as described in Example 3. The bacteria are resuspended in PBS and the OD is adjusted. 600 =0.3.

[0064] 3. Coagulation experiment:

[0065] The suspension of Klebsiella pneumoniae and the suspension of inactivated bacteria of NHNK-616 were mixed in a volume ratio of 1:1. After standing for 30 minutes, the reaction liquids of NHNK-616 alone, Klebsiella pneumoniae, and the mixed reaction liquid of NHNK-616 and Klebsiella pneumoniae were sampled. The sampling range was the top 50 μL of the liquid surface. After aspiration, the liquid 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. The results are as follows: Figure 3 shown.

[0066] The calculation formula of agglutination rate is as follows:

[0067] Agglutination rate (%) = [(Ax+Ay)-2Amix] / (Ax+Ay)×100%;

[0068] Note: Ax: OD of NHNK-616 alone measured at the reaction time 600 Value; Ay: OD of Klebsiella pneumoniae alone measured at the reaction time 600 Value; Amix: OD measured at the reaction time after NHNK-616 and Klebsiella pneumoniae were mixed 600 The results are shown in Table 2 below:

[0069] Table 2

[0070] .

[0071] The results showed that after 30 minutes of reaction, NHNK-616 inactivated bacteria could agglutinate Klebsiella pneumoniae, with an agglutination rate of 12.20%-18.64%.

[0072] Example 5 Experiment on the inhibition of virulence gene expression of Klebsiella pneumoniae by NHNK-616.

[0073] 1. Preparation of NHNK-616 fermentation product and live bacterial suspension:

[0074] A single colony of Propionibacterium freudenreichii subsp. NHNK-616 was selected and placed in MRS liquid medium, incubated anaerobically at 30°C for 48 h, and then adjusted to OD 600 = 0.3, centrifuged at 5000 rpm for 10 min, and filtered the supernatant with a 0.22 μm filter membrane to obtain the 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-616 live bacterial suspension was obtained.

[0075] 2. Experiment on inhibiting the expression of virulence genes of Klebsiella pneumoniae:

[0076] 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.

[0077] Take 3 mL of Klebsiella pneumoniae liquid, add 1 mL of fresh BHI medium, 1 mL of NHNK-616 fermentation product or live bacterial suspension, and add an equal volume of PBS to the control group. Incubate at 37°C for 24 h. After the culture, centrifuge at 8000 rpm for 1 min 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.

[0078] Formula: F=2 -ΔΔCT ,in:

[0079] △CT 实验 =CT 实验 -CT 内参（实验） ;

[0080] △CT 对照 =CT 对照 -CT 内参（对照） ;

[0081] △△CT=△CT 实验 -△CT 对照 .

[0082] The results are shown in Tables 3 and 4 below:

[0083] Table 3

[0084]

[0085] Table 4

[0086] .

[0087] The results showed that NHNK-616 could inhibit the virulence genes of Klebsiella pneumoniae iroD , wabG The relative expression level was 0.44-0.84 times, thereby reducing the pathogenicity of Klebsiella pneumoniae.

[0088] Example 6 Experiment on the inhibition of ethanol production by Klebsiella pneumoniae by NHNK-616.

[0089] 1. Preparation of NHNK-616 fermentation product and live bacterial suspension:

[0090] A single colony of Propionibacterium freudenreichii subsp. NHNK-616 was selected and placed in MRS liquid medium and cultured anaerobically at 30°C for 48 h. The OD was adjusted to 0. 600 =1.0, centrifuge at 5000rpm for 10min, take the supernatant, and filter with a 0.22μm filter membrane to obtain the fermentation product. Wash the precipitated bacteria twice with sterile PBS, resuspend the bacteria with MRS and adjust the OD 600 =1.0, and NHNK-616 live bacterial suspension was obtained.

[0091] 2. Preparation of Klebsiella pneumoniae suspension:

[0092] 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.

[0093] 3. Experiment on NHNK-616 inhibiting alcohol production of Klebsiella pneumoniae:

[0094] The fermentation product of NHNK-616 and live bacteria were added to the suspension of Klebsiella pneumoniae at 10% (v / v), and an equal volume of MRS 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 collect 1 mL of supernatant for later use.

[0095] 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.

[0096] Potassium dichromate oxidation method for detecting alcohol content: add 1 mL of Klebsiella pneumoniae supernatant and 2 mL of 5% potassium dichromate solution into 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.

[0097] The calculation formula and results are shown in Table 5:

[0098] Table 5 NHNK-616 inhibits the production of alcohol by Klebsiella pneumoniae

[0099] .

[0100] The results showed that NHNK-616 inhibited the production of alcohol by Klebsiella pneumoniae with an inhibition rate of 20.39-24.81%.

[0101] Example 7 NHNK-616 regulates the expression of genes related to alcohol-induced liver cell damage.

[0102] 1. Preparation of NHNK-616 fermentation product and live bacterial suspension:

[0103] Pick a single colony of NHNK-616 and place it in fresh MRS medium for anaerobically culture at 30°C for 24 h. Adjust the OD to 0. 600 =0.5, 5000rpm, take the supernatant, and then filter with a 0.22μm filter membrane to obtain the 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.

[0104] 2. Culture of human hepatocytes HepG2:

[0105] HepG2 cells were activated with DMEM medium containing 10% (v / v) FBS and 1% (v / v) penicillin-streptomycin, and cultured at 37°C and 5% CO2. After the cells were fused to 80-90%, they were passaged or plated.

[0106] 3. NHNK-616 regulates the expression of genes related to alcohol-induced liver cell damage:

[0107] HepG2 cells were cultured at 1×10 6Each well was inoculated into a 6-well cell culture plate and cultured for 12 hours until the cells adhered to the wall. The cell culture medium was removed, and the cells were washed twice with sterile PBS. 1.9 mL of DMEM culture medium and 100 μL of NHNK-616 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 the cells were cultured for 24 hours at 37°C and 5% CO2. After the culture was completed, the supernatant was discarded, and the cells were washed twice with sterile PBS. Subsequently, 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 the method described in Example 5.

[0108] The results are shown in Tables 6 and 7 below:

[0109] Table 6

[0110]

[0111] Table 7

[0112] .

[0113] The results showed that NHNK-616 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-616 can alleviate the inflammation caused by alcohol-damaged liver cells and enhance the alcohol metabolism ability of liver cells.

[0114] Example 8 NHNK-616 regulates the expression of genes related to alcohol-induced damage to intestinal epithelial cells.

[0115] 1. Preparation of NHNK-616 fermentation product, live bacterial suspension and inactivated bacterial cells:

[0116] Pick a single colony of NHNK-616 and place it in fresh MRS medium for anaerobically culture at 30°C for 24 h. Adjust the OD to 0. 600=0.5, 5000rpm, take the supernatant, and then filter with a 0.22μm filter membrane to obtain the 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.

[0117] 2. Culture of human intestinal epithelial cells Caco-2:

[0118] Caco-2 cells were activated with DMEM medium containing 10% (v / v) FBS and 1% (v / v) penicillin-streptomycin, cultured at 37°C and 5% CO2, and subcultured or plated after the cells were 80-90% confluent.

[0119] 3. NHNK-616 regulates the expression of genes related to alcohol-induced damage to intestinal epithelial cells:

[0120] Caco-2 cells were cultured at a rate of 1×10 6 Each well was inoculated into a 6-well cell culture plate 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-616 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 the method described in Example 5.

[0121] The results are shown in Tables 8, 9 and 10 below:

[0122] Table 8

[0123]

[0124] Table 9

[0125]

[0126] Table 10

[0127] .

[0128] Results showed that NHNK-616 upregulated intestinal barrier genes ZO-1 , OCLD , CLD-4 and alcohol metabolism genes ADH1B , ALDH2 NHNK-616 can alleviate alcohol-induced intestinal epithelial cell barrier damage and enhance the alcohol metabolism ability of intestinal epithelial cells.

[0129] Example 9 Alcohol tolerance test of NHNK-616.

[0130] A single colony of Propionibacterium freudenreichii subsp. NHNK-616 was selected and placed in MRS liquid medium. After culturing at 30°C to the stationary phase, it was inoculated into MRS medium containing 10% alcohol at a ratio of 2% (v / v). The culture was treated at 30°C for 48 hours, and 100 μL of the bacterial solution was taken to detect the OD using an ELISA reader. 600 Absorbance value, record the OD value before and after 48 hours of cultivation.

[0131] Calculation formula: Growth rate (%) = (A1-A0) / A0×100%.

[0132] Note: A1: OD was measured after 48h of culture under 10% alcohol conditions. 600 Value; A0: initial inoculation OD 600 Numeric value.

[0133] The results are shown in Table 11 below:

[0134] Table 11

[0135] .

[0136] The results showed that NHNK-616 was tolerant to alcohol, with a growth rate of 327.34%-350.00% in a culture medium containing 10% alcohol.

[0137] 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 Propionibacterium freudenreichii subsp. Propionibacterium freudenreichii subsp. shermanii ) NHNK-616, characterized in that The deposit number is CCTCC NO: M 20241429, and it was deposited in the China Center for Type Culture Collection on July 1, 2024.

2. Use of the Propionibacterium freudenreichii subsp. shenkelii NHNK-616 described in claim 1 in the preparation of a product for improving non-alcoholic fatty liver disease caused by alcohol-producing Klebsiella pneumoniae.

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