A strain of Leuconostoc mesenteroides subspecies mesenteroides for improving gastritis, its products and its application

Abstract

The present invention discloses a strain of Leuconostoc mesenteroides subsp. mesenteroides for improving gastritis, a product and an application thereof. Leuconostoc mesenteroides subsp.mesenteroides ) NHNK‑613, which was deposited in the China Center for Type Culture Collection on July 1, 2024, and its deposit number is CCTCCNO: M20241431. Experiments have shown that NHNK‑613 has the functions of inhibiting the growth of gastritis pathogens, inhibiting the formation of biofilms of gastritis pathogens, agglutinating gastritis pathogens, regulating the virulence genes of gastritis pathogens, inhibiting the production of urease of gastritis pathogens, and reducing inflammation induced by gastritis pathogens. The bacterial biofilm increases the adhesion to gastric mucosal mucin and gastric epithelial cells, and can be used to prepare products to improve gastritis.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a strain of Leuconostoc mesenteroides subspecies mesenteroides for improving gastritis, a product and an application thereof. Background Art

[0002] Sea hyacinth is a wild and natural deep-sea plant in the brown algae family. It 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. The area where algae and bacteria interact closely is called the "algal sphere". The algal sphere microbiome is significantly different from the microorganisms in the surrounding seawater in terms of composition and function. The algal sphere microorganisms support the large algae host to achieve morphological development, adapt to environmental changes, release and settle algal spores, etc. by providing growth factors.

[0003] Gastric cancer (GC) is the fifth most common cancer worldwide. Helicobacter pylori (H.pylori) is a major risk factor for GC and has been classified as a type I carcinogen. H. pylori infection promotes gastritis, atrophy, and intestinal metaplasia (IM). Among H. pylori-infected individuals, only 1%-3% eventually develop GC, so it is inferred that other factors are involved, and emerging evidence suggests that there is a large number of non-H. pylori microbiota in the gastric mucosa, and their dysbiosis may play a role in gastric carcinogenesis.

[0004] The researchers characterized the gastric microbiota of Helicobacter pylori-negative patients and found Streptococcus anginosus at different stages of gastric tumorigenesis, from superficial gastritis, atrophic gastritis, intestinal metaplasia to gastric cancer. Streptococcus anginosus is a Gram-positive, non-spore-forming, non-motile bacterium that is mainly found in the oral cavity, nasopharynx, gastrointestinal tract and vagina, and can cause invasive suppurative infections such as abscesses. Streptococcus anginosus has a significant adaptability to low pH conditions (pH 3-5), which may contribute to its survival in the gastric mucosa. In the study, Streptococcus anginosus was identified as a non-Helicobacter pylori pathogen that can rapidly cause acute gastritis after long-term infection of conventional mice and germ-free mice, leading to chronic gastritis, gastric mucus metaplasia and dysplasia. Streptococcus anginosus accelerates the occurrence of gastric cancer. Studies have found that the surface protein TMPC of Streptococcus anginosus directly interacts with the gastric epithelial cell receptor annexin A2 (ANXA2), causing it to colonize in the gastric mucosa and mediate the activation of the mitogen-activated protein kinase (MAPK) signaling pathway. In addition, infection with Streptococcus anginosus can also lead to the destruction of tight junctions between cells, thereby affecting the barrier function of the gastric mucosa. Therefore, Streptococcus anginosus accelerates the occurrence of tumors by promoting proliferation and destroying the integrity of the gastric mucosal barrier.

[0005] Urease is a high-affinity, high-activity, high-molecular-weight protein. Urease catalyzes the hydrolysis of urea into ammonia and carbon dioxide to neutralize gastric acid, thereby increasing the pH of the stomach and providing a suitable environment for the survival and proliferation of Helicobacter pylori and Streptococcus anginosus. At the same time, due to long-term stimulation by ammonia, gastric epithelial cells undergo proliferation, apoptosis, shedding, and atrophy, which can also become a predisposing factor for cancer.

[0006] Given that macroalgae-attached microbial communities 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. Marine microorganisms are more likely to synthesize structurally unique enzymes and secondary metabolites than terrestrial microorganisms. Exploring the application of marine microorganisms has important practical significance in expanding the value of the marine industry. Summary of the invention

[0007] In view of this, the object of the present invention is to provide a strain of Leuconostoc mesenteroides subspecies mesenteroides for improving gastritis, a product and an application thereof.

[0008] In a first aspect, the present invention provides a strain of Leuconostoc mesenteroides subsp. mesenteroides ( Leuconostoc mesenteroides subsp. mesenteroides ), the Leuconostoc mesenteroides subspecies mesenteroides is Leuconostoc mesenteroides subspecies mesenteroides NHNK-613, which was deposited in the China Center for Type Culture Collection on July 1, 2024, and its preservation number is CCTCC NO: M 20241431.

[0009] The second aspect of the present invention provides a method for improving gastritis based on the method of the first aspect. Leuconostoc mesenteroides subsp. mesenteroides ) prepared, wherein the product is the Leuconostoc mesenteroides subspecies mesenteroides ( Leuconostoc mesenteroides subsp. mesenteroides ) of live bacteria, fermentation / secretory products or inactivated bacteria.

[0010] Furthermore, the preparation method of the live bacteria is:

[0011] The Leuconostoc mesenteroides subsp. mesenteroides ( Leuconostoc mesenteroides subsp. mesenteroides ) is inoculated into a culture medium, cultured at 30° C. for 46 h to 50 h, a fermentation liquid is obtained, a precipitate is obtained by centrifugation, and the precipitate is washed to obtain the live bacteria;

[0012] The preparation method of the fermentation / secretion product is:

[0013] The Leuconostoc mesenteroides subsp. mesenteroides ( Leuconostoc mesenteroides subsp. mesenteroides) is inoculated into a culture medium, cultured at 30° C. for 46 h to 50 h, a fermentation broth is obtained, the supernatant is obtained by centrifugation, and the supernatant is filtered with a precision of 0.22 μm. The obtained filtrate is the fermentation / secretion product;

[0014] The preparation method of the inactivated bacteria is as follows:

[0015] The Leuconostoc mesenteroides subsp. mesenteroides ( Leuconostoc mesenteroides subsp. mesenteroides ) is inoculated into a culture medium, cultured at 30°C for 46h~50h to obtain a fermentation broth, centrifuged to obtain a precipitate, washed, and inactivated at 100°C~130°C for 10 min~30 min to obtain the inactivated bacteria.

[0016] The third aspect of the present invention provides a method for improving gastritis according to the first aspect. Leuconostoc mesenteroides subsp. mesenteroides ) or the use of the product as described in the second aspect above in the preparation of a product for improving gastritis.

[0017] Furthermore, the improvement of gastritis includes inhibiting the growth of Streptococcus anginosus and its biofilm formation, inhibiting the formation of composite biofilm of Streptococcus anginosus and Helicobacter pylori, down-regulating the expression of virulence genome of Streptococcus anginosus and inhibiting the production of urease of Streptococcus anginosus.

[0018] Furthermore, the pharyngitis streptococcus virulence genome comprises tmpc , fbp62 , cpsE At least one of .

[0019] Furthermore, the improvement of gastritis also includes down-regulating the expression of a first inflammatory factor genome caused by infection with Streptococcus anginosus and down-regulating the expression of a second inflammatory factor genome caused by co-infection with Streptococcus anginosus and Helicobacter pylori.

[0020] Furthermore, the first inflammatory factor genome and the second inflammatory factor genome both include INOS COX-2, TNF-α and IL-8 .

[0021] Furthermore, the improvement of gastritis also includes down-regulating the expression of the Helicobacter pylori virulence genome and agglutinating Helicobacter pylori, wherein the Helicobacter pylori virulence genome includes cagA , vacA , babA At least one of .

[0022] Furthermore, the improvement of gastritis also includes colonizing gastric mucosal mucin and bacterial biofilm to increase the colonization ability of gastric mucosal mucin and gastric epithelial cells.

[0023] The present invention discloses Leuconostoc mesenteroides subspecies mesenteroides NHNK-613, whose deposit number is CCTCC NO: M20241431. Experiments show that NHNK-613 has the functions of inhibiting the growth of gastritis pathogens, inhibiting the formation of biofilm of gastritis pathogens, agglutinating gastritis pathogens, regulating the virulence genes of gastritis pathogens, inhibiting the production of urease of gastritis pathogens, and reducing inflammation induced by gastritis pathogens infection. The bacterial biofilm increases the adhesion to gastric mucosal mucin and gastric epithelial cells, and can be used to prepare products for improving gastritis.

[0024] Biological Deposit Description

[0025] Leuconostoc mesenteroides subsp. mesenteroides ( Leuconostoc mesenteroides subsp. mesenteroides ) NHNK-613, deposited on July 1, 2024 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), and its deposit number is CCTCC NO: M 20241431. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a graph showing the detection results of Leuconostoc mesenteroides subspecies mesenteroides NHNK-613 in Gram staining microscopy for improving gastritis in Example 1 of the present invention;

[0027] Figure 2 This is a result diagram of an experiment on inhibiting the formation of biofilm of gastritis pathogens by using Leuconostoc mesenteroides subspecies NHNK-613 for improving gastritis in Example 4 of the present invention;

[0028] Figure 3 This is a graph showing the results of an experiment on agglutination of gastritis pathogens by Leuconostoc mesenteroides subspecies NHNK-613 for improving gastritis in Example 5 of the present invention;

[0029] Figure 4 This is a result diagram of a gastric mucin adsorption experiment conducted on Leuconostoc mesenteroides subspecies NHNK-613 for improving gastritis in Example 11 of the present invention;

[0030] Figure 5 This is a graph showing the results of an adsorption experiment on gastric epithelial cells of Leuconostoc mesenteroides subspecies NHNK-613 for improving gastritis in Example 12 of the present invention;

[0031] The following specific implementation manner will further illustrate the present invention in conjunction with the above-mentioned drawings. DETAILED DESCRIPTION

[0032] The present invention provides a strain of Leuconostoc mesenteroides subspecies mesenteroides for improving gastritis, a product and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0033] The Leuconostoc mesenteroides subspecies mesenteroides NHNK-613 described in the present invention is derived from dried sea antlers and is identified as Leuconostoc mesenteroides subspecies mesenteroides ( Leuconostoc mesenteroides subsp. mesenteroides ). This strain is Gram-positive and appears spherical, bean-shaped or short-stalked under a microscope, some of which are arranged in pairs or short chains; it grows on MRS plates to form round colonies with smooth, opaque surfaces, milky white, and neat edges; it grows evenly in MRS liquid culture medium and is turbid, and white precipitates may appear when the bacteria are left to stand for a long time. The optimal growth temperature is 30°C, it is microaerobic, and it grows well in anaerobic culture.

[0034] Leuconostoc mesenteroides subsp. mesenteroides ( Leuconostoc mesenteroides subsp. mesenteroides )NHNK-613, depository unit: China Center for Type Culture Collection, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Wuhan University, deposit date: July 1, 2024, deposit number: CCTCCNO: M20241431.

[0035] Furthermore, the Leuconostoc mesenteroides subspecies mesenteroides NHNK-613 provided by the present invention exists in the form of live bacteria, sterilized inactivated bacteria, or fermentation / secretion products (i.e., supernatant), or derivatives in the application or product described in the present invention. The derivatives are 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 / secretion products, and inactivated bacteria.

[0036] In the present invention, the improvement of gastritis includes at least one of inhibiting the growth of gastritis pathogens, inhibiting the formation of gastritis pathogen biofilms, agglutinating gastritis pathogens, regulating gastritis pathogen virulence genes, inhibiting gastritis pathogen urease production, reducing the function of gastritis pathogen infection-induced inflammation, and increasing the adhesion of bacterial biofilms to gastric mucosal mucin and gastric epithelial cells, specifically:

[0037] In vitro experiments show that the fermentation product of Leuconostoc mesenteroides subspecies NHNK-613 of the present invention has the effect of inhibiting the growth of gastritis pathogens Streptococcus anginosus and Helicobacter pylori, and the inhibition rate reaches 31.33% to 47.64%.

[0038] In vitro experiments show that the Leuconostoc mesenteroides subspecies mesenteroides NHNK-613 of the present invention has the effect of inhibiting the biofilm of gastritis pathogenic bacteria Streptococcus anginosus, Helicobacter pylori biofilm and Streptococcus anginosus-Helicobacter pylori, and the biofilm inhibition rate is 23.99% to 62.98%.

[0039] In vitro experiments show that the Leuconostoc mesenteroides subspecies mesenteroides NHNK-613 of the present invention has the function of agglutinating Helicobacter pylori, a gastritis pathogen, and the agglutination rate can reach 34.95% to 35.35% after acting on Helicobacter pylori for 30 minutes.

[0040] In vitro experiments show that the intestinal Leuconostoc mesenteroides subspecies NHNK-613 of the present invention has the ability to inhibit the outer membrane lipoprotein gene of Streptococcus anginosus tmpc , cell wall anchor protein gene fbp62 Capsular polysaccharide protein cpsE , the relative gene expression was downregulated to 0.55-0.89 times.

[0041] In vitro experiments show that the intestinal Leuconostoc mesenteroides subspecies NHNK-613 of the present invention has the ability to inhibit the cytotoxin-associated protein A gene of Helicobacter pylori cytotoxin and colonization-related genes cagA , vacuolating toxin gene vacA , blood group antigen binding adhesin gene babA , the relative gene expression was downregulated to 0.27-0.82 times.

[0042] In vitro experiments show that the fermentation product of Leuconostoc mesenteroides subspecies NHNK-613 of the present invention has the effect of inhibiting the production of urease of gastritis pathogen Streptococcus anginosus, and the inhibition rate is 12.20% to 20.78%.

[0043] In vitro experiments show that the Leuconostoc mesenteroides subspecies mesenteroides NHNK-613 of the present invention has the effect of reducing inflammation induced by gastritis pathogenic bacteria infection, down-regulating the expression of inflammatory factor genes cyclooxygenase-2 gene COX-2, nitric oxide synthase gene INOS, tumor necrosis factor-α gene TNF-α and / or interleukin-8 gene IL-8 caused by pharyngitis Streptococcus infection of GES-1 gastric epithelial cells, and the relative expression amount is down-regulated to 0.45 times to 0.88 times; down-regulating the expression of inflammatory factor genes cyclooxygenase-2 gene COX-2, nitric oxide synthase gene INOS, tumor necrosis factor-α gene TNF-α and / or interleukin-8 gene IL-8 caused by pharyngitis Streptococcus and Helicobacter pylori co-infection of GES-1 gastric epithelial cells, and the relative expression amount is down-regulated to 0.44 times to 0.68 times.

[0044] In vitro experiments show that the Leuconostoc mesenteroides subspecies NHNK-613 of the present invention has the ability of bacterial biofilm to increase the adhesion to gastric mucosal mucin and gastric epithelial cells.

[0045] The preparation method of the live bacteria is as follows:

[0046] The Leuconostoc mesenteroides subsp. mesenteroides ( Leuconostoc mesenteroides subsp. mesenteroides ) is inoculated into a culture medium, cultured at 30° C. for 46 h to 50 h, a fermentation liquid is obtained, a precipitate is obtained by centrifugation, and the precipitate is washed to obtain the live bacteria;

[0047] The preparation method of the fermentation / secretion product is:

[0048] The Leuconostoc mesenteroides subsp. mesenteroides ( Leuconostoc mesenteroides subsp. mesenteroides ) is inoculated into a culture medium, cultured at 30° C. for 46 h to 50 h, a fermentation broth is obtained, the supernatant is obtained by centrifugation, and the supernatant is filtered with a precision of 0.22 μm. The obtained filtrate is the fermentation / secretion product;

[0049] The preparation method of the inactivated bacteria is as follows:

[0050] The Leuconostoc mesenteroides subsp. mesenteroides ( Leuconostoc mesenteroides subsp. mesenteroides ) is inoculated into a culture medium, cultured at 30°C for 46h~50h to obtain a fermentation broth, centrifuged to obtain a precipitate, washed, and inactivated at 100°C~130°C for 10 min~30 min to obtain the inactivated bacteria.

[0051] It should be noted that the reagents and consumables used in the present invention are all common commercial products. The present invention is further described in conjunction with the embodiments:

[0052] Example 1 Isolation of NHNK-613

[0053] See also Figure 1 Take an appropriate amount of dried antlers and soak them in sterile PBS for half an hour, then shake them several times. Centrifuge the sample at 6000r / min to remove the precipitate, streak the supernatant onto MRS medium, and culture at 30℃ for 48h. Pick the white colonies and repeatedly streak them for purification until a single colony with regular and uniform shape is obtained, which is named NHNK-613.

[0054] Gram staining microscopy: strain NHNK-613 is a Gram-positive colony, spherical under the microscope, some arranged in pairs or short chains; it grows on MRS plates and forms milky white, smooth and rounded opaque round colonies with neat edges; it grows evenly turbid in MRS culture medium, and the bacteria present white precipitation after long-term storage.

[0055] Example 2 Nucleic acid identification of NHNK-613

[0056] 1. 16S rDNA gene sequence analysis

[0057] Pick a single colony in MRS liquid medium, culture at 37℃ overnight, centrifuge at 8000 rpm for 1min to collect the bacteria, and operate according to the instructions of the Gram-positive bacteria DNA extraction kit. The primers used were bacterial 16S sequencing universal primers 27F and 1492R, and the PCR amplification system was 20μL. The PCR amplification program was 95℃ pre-denaturation for 5min, 94℃ for 15s, 57℃ for 15s, 72℃ for 1min, 35 cycles, and 72℃ extension for 10min.

[0058] 2. Results

[0059] The sequencing result of the PCR product is shown in SEQ ID NO.1. After homology comparison (BLASTN) with the standard sequence published in GenBank, it was concluded that the NHNK-613 strain was Leuconostoc mesenteroides subsp. mesenteroides ( Leuconostoc mesenteroides subsp. mesenteroides ).

[0060] Example 3 Experiment on the inhibition of gastritis pathogen growth by NHNK-613 fermentation product

[0061] 1. Preparation of NHNK-613 fermentation products

[0062] A single colony of Leuconostoc mesenteroides subsp. mesenteroides NHNK-613 was selected and placed in MRS liquid medium. After culturing at 30°C for 48 h, the OD was adjusted to 0. 600 =0.3, 5000rpm to take the supernatant, and then filter with a 0.22μm filter membrane to obtain a sterile fermentation product.

[0063] 2. Cultivation of gastritis pathogens and preparation of bacterial suspension

[0064] An appropriate amount of Helicobacter pylori BNCC364207 was inoculated on Columbia blood agar plates and cultured at 37°C and 10% CO2 for 5 days. After the culture was completed, the bacterial moss was scraped with an inoculation loop and dissolved in BHI containing 10% fetal bovine serum (FBS) to adjust the OD 600 =0.3 for backup.

[0065] Streptococcus anginosus BNCC354394 was inoculated into BHI liquid medium at a ratio of 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.

[0066] 3. Experiment on the inhibition of gastritis pathogen proliferation by NHNK-613 fermentation products

[0067] Helicobacter pylori: Take 3 mL of BHI medium containing 10% FBS and 0.3 mL of NHNK-613 fermentation product and add them to a centrifuge tube, inoculate Helicobacter pylori culture at a ratio of 1% (v / v), add 0.3 mL of MRS medium to the control group, culture at 37°C, 10% CO2 for 48 hours, and measure the absorbance at 600 nm.

[0068] Streptococcus anginosus: Take 3 mL of BHI medium and 0.3 mL of NHNK-613 fermentation product and add them to a centrifuge tube. Inoculate Streptococcus anginosus liquid at a ratio of 1% (v / v). Add 0.3 mL of MRS medium to the control group. Culture with shaking at 37°C for 24 hours and measure the absorbance at 600 nm.

[0069] The results of NHNK-613 fermentation products inhibiting the growth of gastritis pathogens are shown in Table 1:

[0070] Table 1

[0071] ,

[0072] The results showed that the fermentation products of NHNK-613 had the effect of reducing the proliferation of gastritis pathogens.

[0073] Example 4: Experiment on the inhibition of biofilm formation of gastritis pathogens by NHNK-613

[0074] 1. Preparation of NHNK-613 fermentation products

[0075] The preparation method is as in Example 3;

[0076] 2. Cultivation of gastritis pathogens and preparation of bacterial suspension

[0077] The methods for culturing Helicobacter pylori and Streptococcus anginosus and preparing bacterial suspensions are as described in Example 3.

[0078] 3. Experiment on the inhibition of biofilm formation of gastritis pathogens by NHNK-613 fermentation products

[0079] Single biofilm: 100 μL Helicobacter pylori or Streptococcus anginosus was added to each well of the experimental group, and 100 μL NHNK-613 fermentation product was added to each well. The control group was added with an equal volume of MRS medium. Each group was set up in triplicate. Cultured at 37°C for 24 h.

[0080] Composite biofilm: 50 μL Helicobacter pylori and 50 μL Streptococcus anginosus were added to each well of the experimental group, and 100 μL NHNK-613 fermentation product was added to each well, and the same volume of MRS medium was added to the control group. Each group was set up in triplicate. Cultured at 37°C for 24 h.

[0081] After the culture, the supernatant was discarded, 100 μL of sterile PBS was added to each well for washing twice, and then 100 μL of 4% paraformaldehyde fixative was added to each well for fixing at room temperature for 30 min. The fixative was discarded, 100 μL of crystal violet was added to each well, and staining was performed at room temperature for 30 min. After staining, the cells were washed twice with sterile PBS and dried, 100 μL of anhydrous ethanol was added to each well, and the absorbance at 600 nm was measured after standing for 1 min, and the biofilm formation rate was calculated according to the formula.

[0082] The calculation formula and results of NHNK-613 fermentation products inhibiting the biofilm formation of gastritis pathogens are shown in Table 2:

[0083] Table 2

[0084] ,

[0085] See also Figure 2 The results showed that the fermentation product of NHNK-613 could inhibit the biofilms of Helicobacter pylori and Streptococcus anginosus, and could also inhibit the composite biofilms formed by Helicobacter pylori and Streptococcus anginosus.

[0086] Example 5: NHNK-613 Agglutination of Gastritis Pathogens

[0087] 1. Preparation of NHNK-613 inactivated bacteria

[0088] A single colony of Leuconostoc mesenteroides subsp. mesenteroides NHNK-613 was selected and placed in MRS liquid culture medium, incubated at 30°C for 48 h, and centrifuged at 5000 rpm for 10 min to obtain a precipitate. The precipitate was washed with sterile PBS, and the bacteria were resuspended in PBS and the OD was adjusted. 600 =0.3. Autoclave at 121℃ for 15min to obtain inactivated bacteria;

[0089] 2. Preparation of Helicobacter pylori suspension

[0090] The preparation method of Helicobacter pylori bacterial solution was as described in Example 3, and the bacterial cells were resuspended in PBS and the OD was adjusted. 600 =0.3.

[0091] 3. Coagulation

[0092] The bacterial suspension of Helicobacter pylori and inactivated NHNK-613 bacteria were mixed in a volume ratio of 1:1, and the agglutination was observed after standing for 30 minutes.

[0093] 4. Index determination:

[0094] After 30 minutes of reaction, samples of NHNK-613 alone, Helicobacter pylori, and the mixed reaction liquid of NHNK-613 and Helicobacter pylori were taken, and the sampling range was the top 50 μL of the liquid surface. After aspiration, the samples were transferred to a 96-well plate and the absorbance at OD=600nm was measured. At the same time, the agglutination precipitate was stained with Gram stain to observe the agglutination state of the bacteria.

[0095] Calculation formula:

[0096] Agglutination rate (%) = [(Ax+Ay)-2Amix] / (Ax+Ay)×100%;

[0097] Ax represents the OD value of NHNK-613 alone at the reaction time. 600 Value; Ay represents the OD value of Helicobacter pylori alone measured at the reaction time 600 Value; Amix means OD measured at the reaction time after NHNK-613 and Helicobacter pylori are mixed. 600 Numeric value.

[0098] The agglutination effect of NHNK-613 inactivated bacteria on pathogens is shown in Table 3:

[0099] Table 3

[0100] ,

[0101] See also Figure 3 ,The results showed that after 30 min of reaction, NHNK-613 inactivated bacteria could ,effectively agglutinate Helicobacter pylori.

[0102] Example 6 Experiment on the inhibition of virulence gene expression of Streptococcus anginosus by NHNK-613

[0103] 1. Preparation of NHNK-613 fermentation products, live bacteria and inactivated bacteria

[0104] A single colony of Leuconostoc mesenteroides subsp. mesenteroides NHNK-613 was selected and placed in MRS liquid medium and cultured at 30°C for 48 h. The OD was adjusted to 0. 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 the NHNK-613 live bacterial suspension was obtained. Another part of the live bacteria was sterilized at 121℃ for 15min to obtain inactivated bacteria.

[0105] 2. Experiment on inhibiting the expression of virulence genes of Streptococcus anginosus

[0106] The frozen tube of Streptococcus anginosus was inoculated into fresh BHI medium at a ratio of 1% (v / v), and cultured at 37℃ with shaking for 24h. The OD was adjusted to 600 =0.3. Take 3mL of cultured Streptococcus anginosus, add 1mL of NHNK-613 fermentation product (the control group added an equal amount of MRS medium), live bacteria or inactivated bacteria (the control group added an equal amount of PBS), and 1mL of fresh BHI medium, 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. gyrB As the internal reference gene, qPCR detection was performed tmpc , fbp62 , cpsE 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.

[0107] Formula: F=2 -ΔΔCT ,in:

[0108] △CT 实验 =CT 实验 -CT 内参（实验） ;

[0109] △CT 对照 =CT 对照 -CT 内参（对照） ;

[0110] △△CT=△CT 实验 -△CT 对照 .

[0111] The results are shown in Table 4, Table 5 and Table 6:

[0112] Table 4

[0113] ,

[0114] Table 5

[0115] ,

[0116] Table 6

[0117] ,

[0118] The results showed that NHNK-613 fermentation products, live bacteria and inactivated bacteria could inhibit the virulence genes of Streptococcus anginosus. tmpc , fbp62 and cpsE expression, thereby reducing the pathogenicity of Streptococcus anginosus.

[0119] Example 7 Experiment on the inhibition of Helicobacter pylori virulence gene expression by NHNK-613

[0120] 1. Preparation of NHNK-613 fermentation products, live bacteria and inactivated bacteria

[0121] The preparation method is similar to that of Example 6.

[0122] 2. Experiment on inhibiting the expression of Helicobacter pylori virulence genes

[0123] The culture method of Helicobacter pylori is as described in Example 3. The cultured Helicobacter pylori was scraped off the Columbia blood agar plate, washed twice with PBS, and then resuspended in BHI liquid containing 10% fetal bovine serum (FBS). The OD 600 =0.3. Take 3mL of Helicobacter pylori culture, add 1mL of NHNK-613 fermentation product (the control group added an equal amount of MRS culture medium), live bacteria or inactivated bacteria (the control group added an equal volume of PBS), and 2mL of BHI culture medium containing 10% FBS, and culture at 37℃ and 10% CO2 for 24h. After the culture, centrifuge at 8000rpm for 1min to obtain the bacteria, extract total RNA according to the kit instructions, detect RNA concentration and purity, and reverse transcribe it into cDNA. 16S rRNA As the internal reference gene, qPCR fluorescence quantitative technology was used to detect cagA , vacA and babA 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 the method described in Example 6.

[0124] The results are shown in Table 7, Table 8 and Table 9:

[0125] Table 7

[0126] ,

[0127] Table 8

[0128] ,

[0129] Table 9

[0130] ,

[0131] The results showed that live and inactivated bacteria of NHNK-613 could inhibit the virulence genes of Helicobacter pylori cagA , vacA and babA expression, thereby reducing the pathogenicity of Helicobacter pylori.

[0132] Example 8 Experiment on the inhibition of urease production by NHNK-613 in Streptococcus pharyngitis

[0133] 1. Preparation of NHNK-613 fermentation products and inactivated bacteria

[0134] A single colony of Leuconostoc mesenteroides subsp. mesenteroides NHNK-613 was selected and placed in MRS liquid medium, cultured at 30°C for 48 hours, adjusted to OD600=0.3 with MRS, centrifuged at 5000rpm for 10 minutes, and the supernatant was filtered with a 0.22μm filter membrane to obtain a sterile fermentation product. The precipitated bacteria were washed twice with MRS, resuspended with MRS and adjusted to OD600=0.3, and sterilized at 121°C for 15 minutes to obtain inactivated bacteria.

[0135] 2. Preparation of Streptococcus anginosus suspension

[0136] The preparation method is similar to that of Example 3.

[0137] 3. Experiment on the inhibition of urease production by Streptococcus anginosus by NHNK-613 fermentation products

[0138] Add 2 ml of Streptococcus anginosus liquid, 2 ml of BHI, 1 ml of NHNK-613 fermentation product or inactivated bacteria to the centrifuge tube, add an equal amount of MRS medium to the control group, shake culture at 37°C, centrifuge for 6 hours and take out 50 μL of supernatant for use. Urease indicator formula: 0.9% NaCl, 20 mmol / L urea, 14 μg / mL phenol red, adjust pH to 6.8 (color change point of phenol red) with HCl. Take 150 μL of urease indicator and mix it with 50 μL of culture supernatant at different time points, react at room temperature for 15 minutes, and measure its absorbance at 550 nm.

[0139] The calculation formula and results of the inhibition of urease production of Streptococcus anginosus by NHNK-613 fermentation products are shown in Table 10:

[0140] Table 10

[0141] ,

[0142] The results showed that NHNK-613 can effectively inhibit the production of urease of Streptococcus anginosus, with the inhibition rate reaching 12.20%~20.78%.

[0143] Example 9 NHNK-613 reduces gastric epithelial cell inflammation induced by pathogen infection

[0144] 1. Preparation of NHNK-613 fermentation products and inactivated bacteria

[0145] A single colony of Leuconostoc mesenteroides subsp. mesenteroides NHNK-613 was selected and placed in MRS liquid culture medium, cultured at 30°C for 48 h, and then adjusted to OD 0. 600 = 0.5, 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 bacteria were washed twice with sterile PBS, sterilized at 121 ° C for 15 min, and resuspended in DMEM medium without FBS and penicillin-streptomycin to an OD of 600 =0.5, and inactivated bacteria were obtained.

[0146] 2. Cultivation of gastritis pathogens and preparation of bacterial suspension

[0147] The culture method of Streptococcus anginosus and Helicobacter pylori is referred to Example 3. After the culture is completed, the bacteria are resuspended in DMEM medium without FBS and penicillin-streptomycin, and the OD is adjusted. 600 =0.5 for backup.

[0148] 3. Cultivation of human gastric epithelial cells GES-1

[0149] GES-1 cells were activated with DMEM medium containing 10% FBS and 1% penicillin-streptomycin, cultured at 37°C and 5% CO2, and subcultured or plated after the cells were fused to 80%-90%.

[0150] 4. Experiment on reducing gastric epithelial cell inflammation induced by pathogen infection by NHNK-613

[0151] GES-1 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, and the cells were washed twice with sterile PBS. Then, 100 μL of NHNK-613 inactivated bacteria was added (the control group was replaced with an equal volume of DMEM culture medium). After 2 hours of treatment, 100 μL of Streptococcus anginosus or a mixed bacterial solution of 50 μL Streptococcus anginosus and 50 μL Helicobacter pylori was added, and 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. 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. GADPH As the internal reference gene, the qPCR method was used to determine COX-2 , IL-8 , INOS , TNF-α 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 6.

[0152] The results are shown in Table 11, Table 12 and Table 13:

[0153] Table 11

[0154] ,

[0155] Table 12

[0156] ,

[0157] Table 13

[0158] ,

[0159] The results showed that NHNK-613 inactivated bacteria can reduce gastric epithelial cell inflammation caused by Streptococcus anginosus infection and Helicobacter pylori-Streptococcus anginosus co-infection, and inhibit the expression of genes related to inflammatory factors.

[0160] Example 10 NHNK-613 forms biofilm

[0161] 1. Formation of NHNK-613 biofilm

[0162] A single colony of NHNK-613 was selected and placed in MRS liquid medium, cultured at 30°C for 24 h, and the OD was adjusted to 0. 600 =0.2, 100 μl of bacterial solution was added to each well of a 96-well plate, with 3 parallels per group, and then cultured at 37°C for 24 hours.

[0163] 2. Crystal violet staining

[0164] After the culture was completed, the supernatant was discarded, 100 μL of sterile PBS was added to each well for washing twice, and then 100 μL of 4% paraformaldehyde fixative was added to each well for fixing at room temperature for 30 minutes. The fixative was discarded, 100 μL of crystal violet was added to each well, and staining was performed at room temperature for 30 minutes. After staining, the cells were washed twice with sterile PBS and dried, 100 μL of anhydrous ethanol was added to each well, and the absorbance at 600 nm was measured after standing for 1 minute. The results of the biofilm formation of NHNK-613 are shown in Table 14:

[0165] Table 14

[0166] ,

[0167] The results showed that NHNK-613 in OD 600 =0.2 in MRS medium at 30℃ for 24h to form biofilm.

[0168] Example 11: Adsorption experiment of NHNK-613 on gastric mucin

[0169] 1. Preparation of NHNK-613 live bacterial suspension

[0170] Pick a single colony of NHNK-613 in fresh MRS medium and culture at 30℃ for 24h. Centrifuge at 5000rpm for 10min to collect the cells, wash twice with sterile PBS, resuspend the cells in DMEM medium and adjust the OD 600 =0.5.

[0171] 2. Preparation of NHNK-613 biofilm

[0172] A single colony of NHNK-613 was selected and placed in MRS liquid medium, cultured at 30°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 30°C for 24 hours. After the culture was completed, the upper culture medium was discarded, the bottom biofilm was resuspended after washing twice with PBS, and the bacteria were collected by centrifugation at 5000rpm for 10min, and the bacteria were resuspended with DMEM culture medium and the OD was adjusted. 600 =0.5.

[0173] 3. Determination of gastric mucin adsorption capacity

[0174] (1) Reagent preparation

[0175] Gastric mucin solution: weigh 10 mg of gastric mucin and dissolve it in 10 mL of 50 mM Tris-HCL solution at 4°C overnight.

[0176] Blocking solution: Dissolve bovine serum albumin in PBS solution at a ratio of 2% (m / v).

[0177] Washing solution: Dissolve bovine serum albumin in PBS solution at a ratio of 0.1% (m / v).

[0178] (2) Adsorption experiment of NHNK-613 bacterial liquid and biofilm on gastric mucin

[0179] Immerse the coverslip in the gastric mucin solution and coat it 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-613 live bacterial suspension or biofilm bacterial suspension, drop it on the gastric 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.

[0180] See also Figure 4NHNK-613 can absorb gastric mucin, the main component of the gastric mucosa layer. After NHNK-613 is coated with the biofilm formed by itself, it can increase the adsorption capacity of gastric mucin.

[0181] Example 12 Adsorption experiment of NHNK-613 on gastric epithelial cells

[0182] 1. Preparation of NHNK-613 live bacteria and biofilm bacterial suspension

[0183] The preparation method is similar to that of Example 11.

[0184] 2. Culture of gastric epithelial cells GES-1

[0185] GES-1 cells were inoculated in DMEM complete medium containing 10% FBS and 1% penicillin-streptomycin double antibody, cultured at 37°C and 5% CO2 for 1d-2d, and the cells were collected when the cell fusion reached 80%~90%. First, a cell slide was placed at the bottom of a 6-well plate, and then the cells were inoculated into the 6-well plate at 1×10^6 / well and cultured for 24h. After the cells adhered to the wall, the culture medium was removed, washed twice with PBS, and then 1.5mL DMEM culture medium and 0.5mL NHNK-613 bacterial suspension were added to each well and incubated at 37°C for 2h. After the incubation, the slide was removed and fixed with 4% paraformaldehyde solution for 30min, followed by Gram staining and observation under a microscope.

[0186] See also Figure 5 NHNK-613 can adsorb gastric epithelial cells. After NHNK-613 is coated with its own biofilm, it can increase its adsorption capacity to gastric epithelial cells.

[0187] 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 Leuconostoc mesenteroides subsp. mesenteroides that improves gastritis ( Leuconostoc mesenteroides subsp. mesenteroides ), characterized in that, The Enterobacter mesenteroides subspecies is Enterobacter mesenteroides subspecies NHNK-613, which was deposited in the China Center for Type Culture Collection on July 1, 2024, and its preservation number is CCTCC NO: M20241431.

2. Based on the Leuconostoc mesenteroides subspecies mesenteroides for improving gastritis as claimed in claim 1 ( Leuconostoc mesenteroides subsp. mesenteroides ) is characterized in that The product is the Leuconostoc mesenteroides subspecies mesenteroides ( Leuconostoc mesenteroides subsp. mesenteroides ) of live bacteria, fermentation supernatant or inactivated bacteria.

3. The product according to claim 2, characterized in that The preparation method of the live bacteria is as follows: The Leuconostoc mesenteroides subsp. mesenteroides ( Leuconostoc mesenteroides subsp. mesenteroides ) is inoculated into a culture medium, cultured at 30° C. for 46 h to 50 h, a fermentation liquid is obtained, the precipitate is obtained by centrifugation, and the precipitate is washed to obtain the live bacteria; The preparation method of the fermentation supernatant is: The Leuconostoc mesenteroides subsp. mesenteroides ( Leuconostoc mesenteroides subsp. mesenteroides ) is inoculated into a culture medium, cultured at 30° C. for 46 h to 50 h, a fermentation liquid is obtained, the supernatant is obtained by centrifugation, and the supernatant is filtered with a precision of 0.22 μm. The obtained filtrate is the fermentation supernatant; The preparation method of the inactivated bacteria is as follows: The Leuconostoc mesenteroides subsp. mesenteroides ( Leuconostoc mesenteroides subsp. mesenteroides ) is inoculated into a culture medium, cultured at 30°C for 46h~50h to obtain a fermentation broth, centrifuged to obtain a precipitate, washed, and inactivated at 100°C~130°C for 10 min~30 min to obtain the inactivated bacteria.

4. The Leuconostoc mesenteroides subspecies mesenteroides for improving gastritis according to claim 1 Leuconostoc mesenteroides subsp. mesenteroides ) or the use of the preparation as claimed in claim 2 in the preparation of a product for improving gastritis caused by Helicobacter pylori and Streptococcus anginosus.

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