A strain of Pediococcus pentosaceus and its application in improving urticaria and alcohol metabolism products

Abstract

The present invention relates to the technical field of marine microorganisms, and in particular to a strain of Pediococcus pentosaceus and its application in products for improving urticaria and alcohol metabolism. The provided Pediococcus pentosaceus NHNK-617 is deposited in the China Center for Type Culture Collection, and the deposit number is CCTCC NO: M 20241432. Experiments show that NHNK-617 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 products for improving chronic urticaria and alcohol metabolism.

Description

Technical Field

[0001] The invention relates to the technical field of marine microorganisms, in particular to a strain of Pediococcus pentosaceus and application thereof in improving urticaria and alcohol metabolism products. Background Art

[0002] Sea antlers ( Mazzaella ) is rich in calcium, iodine, iron, potassium, fiber, carotene, alginate and other nutrients that are indispensable to the human body. Its growth conditions are very harsh, and it can only grow in small quantities in unpolluted sea areas. There are a large number of bacteria colonizing the surface of large algae and have a complex and close relationship. The area where algae and bacteria interact closely is called the "algal sphere". The algal sphere microorganisms are significantly different from those in the surrounding seawater in terms of composition and function. Algal sphere microorganisms support the large algae host to achieve morphological development, adapt to environmental changes, release and sedimentation of algal spores, etc. by providing growth factors.

[0003] Chronic urticaria presents as spontaneous wheals or angioedema with a duration of more than 6 weeks, often accompanied by severe pruritus. The gut microbiota of patients with chronic urticaria and healthy controls was investigated by metagenomic sequencing and targeted metabolomics, and the differences in functional relevance were evaluated. The results showed that the gut microbiota of patients with chronic urticaria was significantly different from that of healthy controls, and chronic urticaria was associated with reduced levels of short-chain fatty acid (SCFA) producers and Klebsiella pneumoniae ( Klebsiella pneumoniae ) significantly correlated with an increase in pathogens. After Klebsiella pneumoniae was transplanted into the mouse intestine, recipient mice showed increased allergic responses and increased blood lipopolysaccharide levels. Lipopolysaccharide promotes IgE-induced activation in vitro and increases allergic inflammatory responses in vivo.

[0004] Urticaria presents with localized edema due to dilation of small blood vessels and increased permeability in the skin and mucous membranes. Klebsiella pneumoniae has a high alcohol production capacity and continuously produces alcohol in the human body, causing vasodilation and increased permeability, while causing persistent damage to the liver and intestines. The endogenous alcohol produced by intestinal Klebsiella pneumoniae reduces the tight junction proteins ZO-1, claudin-1 and claudin-4, adhesion junction proteins β-catenin and E-cadherin, and desmosome plaque proteins in intestinal epithelial cells, thereby destroying the integrity of the intestinal barrier and increasing the severity of chronic urticaria. 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.

[0005] 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

[0006] In view of the gaps in the prior art, the present invention provides a Pediococcus pentosaceus and its application. The Pediococcus pentosaceus NHNK-617 provided is deposited in the China Center for Type Culture Collection, and the deposit number is CCTCC NO: M 20241432. Experiments show that NHNK-617 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 products for improving chronic urticaria and alcohol metabolism.

[0007] In order to achieve the purpose of the present invention, the inventor provides the following technical solutions:

[0008] The inventors first provided a strain of Pediococcus pentosaceus NHNK-617 ( Pediococcus pentosaceus NHNK-617), with the deposit number of CCTCC NO: M 20241432, which was deposited in China Center for Type Culture Collection on July 1, 2024.

[0009] The above-mentioned Pediococcus pentosaceus NHNK-617 was derived from dried sea antlers and was identified as Pediococcus pentosaceus by 16S rDNA ( Pediococcus pentosaceus ). This strain is Gram-positive, and appears as a spherical cell under a microscope, most of which grow in teams or quadruplets. When growing on an MRS plate, it can form round colonies with a moist surface, white color, neat edges, smooth and raised edges. In MRS liquid culture medium, it grows evenly and turbidly, and white precipitation may appear after long-term static culture. The optimal growth temperature is 37°C, and it is anaerobic.

[0010] The present invention also provides the use of the Pediococcus pentosaceus NHNK-617 in products for improving chronic urticaria.

[0011] The application mechanism of improving chronic urticaria 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.

[0012] In vitro experiments show that the fermentation product of Pediococcus pentosaceus NHNK-617 of the present invention has the effect of inhibiting the growth of Klebsiella pneumoniae, and the inhibition rate reaches 59.46%-61.11%.

[0013] In vitro experiments show that the Pediococcus pentosaceus NHNK-617 of the present invention has the effect of inhibiting the production of alcohol by Klebsiella pneumoniae, and the inhibition rate reaches 12.50%-18.47%.

[0014] In vitro experiments show that the Pediococcus pentosaceus NHNK-617 of the present invention has the function of agglutinating Klebsiella pneumoniae, and the agglutination rate is 15.38-17.95%.

[0015] In vitro experiments show that the NHNK-617 of the present invention has the ability to down-regulate the mucus phenotype regulatory gene of the virulence factor of Klebsiella pneumoniae. A rpmA , Enterobactin gene iroD and galactosyltransferase-related genes wabG The relative expression level was 0.01-0.57 times.

[0016] The present invention also provides the use of the Pediococcus pentosaceus NHNK-617 in improving alcohol metabolism products.

[0017] 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) Upregulation of genes related to alcohol-induced intestinal barrier damage ZO-1 , OCLD and CLD4 at least one of the expressions;

[0021] d) Alcohol tolerance.

[0022] In vitro experiments show that the Pediococcus pentosaceus NHNK-617 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 The relative expression of alcohol metabolism-related alcohol dehydrogenase-1B gene was 2.10-5.87 times. ADH1B and aldehyde dehydrogenase type 2 gene ALDH2 The relative expression levels were 1.16-7.93 times.

[0023] In vitro experiments show that the Pediococcus pentosaceus NHNK-617 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 The relative expression of alcohol metabolism-related alcohol dehydrogenase-1B gene was 1.09-3.80 times. ADH1 B and / or aldehyde dehydrogenase type 2 gene ALDH2 The relative expression level was 1.11-4.86 times.

[0024] In vitro experiments show that the Pediococcus pentosaceus NHNK-617 of the present invention has an alcohol tolerance effect, and the growth rate in a culture medium containing 10% alcohol is 62.50%-66.67%.

[0025] In summary, the beneficial effects of the present invention are as follows:

[0026] The provided Pediococcus pentosaceus NHNK-617 has the functions of inhibiting the growth and alcohol production of Klebsiella pneumoniae, agglutinating Klebsiella pneumoniae, inhibiting the virulence gene of Klebsiella pneumoniae, promoting alcohol metabolism, reducing alcohol damage and tolerating alcohol.

[0027] The inventors have made a biological deposit of Pediococcus pentosaceus NHNK-617, and the deposit information is as follows:

[0028] Storage time: July 1, 2024

[0029] Name of depository: China Center for Type Culture Collection

[0030] Deposit number: CCTCC NO: M 20241432

[0031] Address of depository: Wuhan University, Wuhan, China

[0032] Classification name: Pediococcus pentosaceus NHNK-617 ( Pediococcus pentosaceus NHNK-617). BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the MRS plate colony image of Pediococcus pentosaceus NHNK-617 in the present invention;

[0034] Figure 2 This is a Gram staining image of Pediococcus pentosaceus NHNK-617 in the present invention;

[0035] Figure 3 This is a photo of the inactivated bacteria of Pediococcus pentosaceus NHNK-617 combined with Klebsiella pneumoniae in the present invention;

[0036] In the figure, (a) is NHNK-617, (b) is Klebsiella pneumoniae, and (c) is a mixture of NHNK-617 and Klebsiella pneumoniae. DETAILED DESCRIPTION

[0037] The present invention provides Pediococcus pentosaceus 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 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 by way of example, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0038] The Pediococcus pentosaceus NHNK-617 of the present invention is derived from dried sea antlers and is identified as Pediococcus pentosaceus ( Pediococcus pentosaceus ). This strain is Gram-positive, and appears as a spherical cell under a microscope, most of which grow in teams or quadruplets. When growing on an MRS plate, it can form round colonies with a moist surface, white color, neat edges, smooth and raised edges. In MRS liquid culture medium, it grows evenly and turbidly, and white precipitation may appear after long-term static culture. The optimal growth temperature is 37°C, and it is anaerobic.

[0039] Furthermore, in the application described in the present invention, the Pediococcus pentosaceus NHNK-617 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 components thereof, 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-617.

[0042] Take an appropriate amount of dried sea antlers and soak them in sterile PBS. After shaking several times, centrifuge at 6000r / min for 10min to remove the precipitate. Take the supernatant and streak it on MRS solid culture medium. After constant temperature culture at 37℃ for 48h, pick white colonies and repeatedly streak and screen until a single colony with regular and uniform shape is obtained, which is named NHNK-617.

[0043] Gram staining microscopy: strain NHNK-617 is a Gram-positive colony, with spherical cells and non-spore shape. Most of them are in groups or tetrads, and a few are single cells. It grows on MRS plates, with round colonies, milky white surface, smooth protrusions, neat edges, and opaque; it can grow evenly in MRS medium, 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-617.

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

[0046] 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.

[0047] 2. Results:

[0048] The PCR product was sequenced and compared with the standard sequences published in the GenBank database for homology (BLASTN), and it was found that the NHNK-617 strain was Pediococcus pentosaceus ( Pediococcus pentosaceus ).

[0049] Example 3 Experiment on the inhibition of Klebsiella pneumoniae growth by NHNK-617 fermentation product.

[0050] 1. Preparation of NHNK-617 fermentation products:

[0051] A single colony of Pediococcus pentosaceus NHNK-617 was selected and placed in MRS liquid medium, incubated anaerobically 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 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 the Klebsiella pneumoniae suspension for later use.

[0054] 3. Experiment on inhibition of Klebsiella pneumoniae proliferation by NHNK-617 fermentation products:

[0055] 3 mL of BHI medium and 0.3 mL of NHNK-617 fermentation product were added to a centrifuge tube, and an equal volume of MRS medium was added to the control group. The above-mentioned Klebsiella pneumoniae suspension was inoculated at 1% (v / v), and the absorbance at 600 nm was measured after shaking culture at 37°C for 24 hours.

[0056] The results are shown in Table 1 below:

[0057] Table 1 Inhibition of Klebsiella pneumoniae growth by NHNK-617 fermentation products

[0058]

[0059] The results showed that NHNK-617 could reduce the growth and proliferation of Klebsiella pneumoniae, with an inhibition rate of 59.46%-61.11%.

[0060] Example 4 NHNK-617 agglutination experiment on Klebsiella pneumoniae

[0061] 1. Preparation of NHNK-617 inactivated bacteria:

[0062] A single colony of Pediococcus pentosaceus NHNK-617 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. High pressure sterilization at 121℃ for 15 min can obtain a bacterial suspension inactivated with the inactivated bacteria.

[0063] 2. Preparation of Klebsiella pneumoniae suspension:

[0064] 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.

[0065] 3. Coagulation experiment:

[0066] The suspension of Klebsiella pneumoniae and NHNK-617 inactivated bacteria was mixed in a volume ratio of 1:1. After standing for 30 minutes, samples of NHNK-617, Klebsiella pneumoniae, and the mixed reaction liquid of NHNK-617 and Klebsiella pneumoniae were collected. 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 3 shown.

[0067] The calculation formula of agglutination rate is as follows:

[0068] Agglutination rate (%) = [(Ax+Ay)-2Amix] / (Ax+Ay)×100%;

[0069] Note: Ax: OD of NHNK-617 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-617 and Klebsiella pneumoniae were mixed 600 Numeric value.

[0070] The results are shown in Table 2 below:

[0071] Table 2 Agglutination of pathogens by inactivated NHNK-617

[0072]

[0073] The results showed that after 30 minutes of reaction, NHNK-617 inactivated bacteria could agglutinate Klebsiella pneumoniae, with an agglutination rate of 15.38%-17.95%.

[0074] Example 5 Experiment on the inhibition of virulence gene expression of Klebsiella pneumoniae by NHNK-617.

[0075] 1. Preparation of NHNK-617 fermentation product and live bacterial suspension:

[0076] A single colony of Pediococcus pentosaceus NHNK-617 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 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 the NHNK-617 live bacterial suspension was obtained.

[0077] 2. Experiment on inhibiting the expression of virulence genes of Klebsiella pneumoniae:

[0078] 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.

[0079] Take 3 mL of Klebsiella pneumoniae culture, add 1 mL of fresh BHI medium, 1 mL of NHNK-617 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 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.

[0080] Formula: F=2 -ΔΔCT ,in:

[0081] △CT 实验 =CT 实验 -CT 内参（实验） ;

[0082] △CT 对照 =CT 对照 -CT 内参（对照） ;

[0083] △△CT=△CT 实验 -△CT 对照 .

[0084] The results are shown in Tables 3 and 4 below:

[0085] Table 3 NHNK-617 fermentation products inhibit virulence genes of Klebsiella pneumoniae

[0086]

[0087] Table 4 NHNK-617 live bacteria inhibit virulence genes of Klebsiella pneumoniae

[0088]

[0089] The results showed that NHNK-617 could downregulate virulence genes of Klebsiella pneumoniae rpmA , iroD , wabG The relative expression level was 0.01-0.57 times, thereby reducing the pathogenicity of Klebsiella pneumoniae.

[0090] Example 6 Experiment on the inhibition of ethanol production by Klebsiella pneumoniae by NHNK-617.

[0091] 1. Preparation of NHNK-617 fermentation product and live bacterial suspension:

[0092] A single colony of Pediococcus pentosaceus NHNK-617 was selected and placed in MRS liquid medium, cultured at 37°C for 48 h, and 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-617 live bacterial suspension was obtained.

[0093] 2. Preparation of Klebsiella pneumoniae suspension:

[0094] Klebsiella pneumoniae 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, resuspended in YPD liquid medium and adjusted to OD 600 =0.3 for backup.

[0095] 3. Experiment on NHNK-617 inhibiting alcohol production of Klebsiella pneumoniae:

[0096] The fermentation product of NHNK-617 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.

[0097] 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.

[0098] 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.

[0099] The calculation formula and results are shown in Table 5 below:

[0100] Table 5 NHNK-617 inhibits the production of alcohol by Klebsiella pneumoniae

[0101]

[0102] The results showed that NHNK-617 inhibited the production of alcohol by Klebsiella pneumoniae with an inhibition rate of 12.50%-18.47%.

[0103] Example 7 NHNK-617 regulates the expression of genes related to alcohol-induced liver cell damage.

[0104] 1. Preparation of NHNK-617 fermentation product and live bacterial suspension:

[0105] Pick a single colony of NHNK-617 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, 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.

[0106] 2. Culture of human hepatocytes HepG2:

[0107] 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.

[0108] 3. NHNK-617 regulates the expression of genes related to alcohol-induced liver cell damage:

[0109] HepG2 cells were cultured at 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, and the plates were washed twice with sterile PBS. 1.9 mL of DMEM culture medium and 100 μL of NHNK-617 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 plates were cultured for 24 hours at 37°C and 5% CO2. After the culture was completed, the supernatant was discarded, and the plates 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. 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 the method described in Example 5.

[0110] The results are shown in Tables 6 and 7 below:

[0111] Table 6 NHNK-617 fermentation products regulate gene expression in alcohol-induced damaged hepatocytes

[0112]

[0113] Table 7 NHNK-617 live bacteria regulates gene expression in alcohol-induced damaged hepatocytes

[0114]

[0115] The results showed that NHNK-617 could upregulate anti-inflammatory factor genes TGF-β , IL-10 and alcohol metabolism genes ADH1B , ALDH2 It can alleviate the inflammation caused by alcohol-damaged liver cells and improve the alcohol metabolism ability of liver cells.

[0116] Example 8 NHNK-617 regulates the expression of genes related to alcohol-induced damage to intestinal epithelial cells.

[0117] 1. Preparation of NHNK-617 fermentation products, live bacterial suspension and inactivated bacterial cells:

[0118] Pick a single colony of NHNK-617 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, 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.

[0119] 2. Culture of human intestinal epithelial cells Caco-2:

[0120] Caco-2 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.

[0121] 3. NHNK-617 regulates the expression of genes related to alcohol-induced damage to intestinal epithelial cells

[0122] Caco-2 cells were cultured at a rate of 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, washed twice with sterile PBS, and then 1.9 mL of DMEM culture medium and 100 μL of NHNK-617 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, 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.

[0123] The results are shown in Tables 8, 9 and 10 below:

[0124] Table 8 NHNK-617 fermentation products regulate gene expression in alcohol-induced intestinal epithelial cells

[0125]

[0126] Table 9 NHNK-617 live bacteria regulates gene expression in alcohol-induced intestinal epithelial cells

[0127]

[0128] Table 10 NHNK-617 inactivated bacteria regulates gene expression in alcohol-induced intestinal epithelial cells

[0129]

[0130] Results showed that NHNK-617 upregulated intestinal barrier genes ZO-1 , OCLD , CLD-4 and alcohol metabolism genes ADH1B , ALDH2 NHNK-617 can alleviate alcohol-induced intestinal epithelial cell barrier damage and enhance the alcohol metabolism ability of intestinal epithelial cells.

[0131] Example 9 Alcohol tolerance test of NHNK-617

[0132] A single colony of Pediococcus pentosaceus NHNK-617 was selected and placed in MRS liquid medium. After culturing at 37°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 37°C for 48 hours. 100 μL of the bacterial solution was taken and the OD was detected using an ELISA reader. 600 Absorbance value, record the OD value before and after 48 hours of cultivation. Calculation formula: Growth rate (%) = (A1-A0) / A0 × 100%

[0133] Note: A1: OD was measured after 48h of culture under 10% alcohol conditions. 600 Value; A0: initial inoculation OD 600 Numeric value.

[0134] The calculation results are shown in Table 11:

[0135] Table 11 Growth rate of NHNK-617 in 10% alcohol MRS medium

[0136]

[0137] The results showed that NHNK-617 was tolerant to alcohol, and the growth rate in a medium containing 10% alcohol was 62.50%-66.67%.

[0138] 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 pentosaceus NHNK-617 ( Pediococcus pentosaceus NHNK-617, characterized in that The deposit number is CCTCC NO: M 20241432, and it was deposited in the China Center for Type Culture Collection on July 1, 2024.

2. Use of the Pediococcus pentosaceus NHNK-617 according to claim 1 in preparing a product for inhibiting the growth of Klebsiella pneumoniae and inhibiting the production of alcohol in vitro.

3. Use of the Pediococcus pentosaceus NHNK-617 according to claim 1 in the preparation of a product for agglutinating Klebsiella pneumoniae in vitro.

4. The Pediococcus pentosaceus NHNK-617 of claim 1 is used to prepare a method for down-regulating virulence genes of Klebsiella pneumoniae in vitro rpmA , iD and wbG Application of at least one expressed product.

5. Use of the Pediococcus pentosaceus NHNK-617 according to claim 1 in the preparation of products for improving alcohol metabolism.

Images