Lactobacillus johnsonii, a strain that alleviates ulcerative colitis, and its application.

Abstract

This invention discloses a *Lactobacillus johnsonii* strain for alleviating ulcerative colitis and its applications, belonging to the field of microbial technology. The *Lactobacillus johnsonii* NSP009 strain of this invention can produce a large amount of indolelacic acid, which can improve weight loss and increased disease activity index caused by ulcerative colitis, while simultaneously inhibiting colonic atrophy, protecting the integrity of colonic tissue structure, and reducing intestinal inflammation levels, thus exhibiting a good alleviating effect on colitis. The *Lactobacillus johnsonii* NSP009 strain of this invention has very broad application prospects for preparing pharmaceutical compositions and fermented foods for alleviating ulcerative colitis.

Description

Technical Field

[0001] This invention relates to a Lactobacillus johnsonii strain that relieves ulcerative colitis and its application, belonging to the field of microbial technology. Background Technology

[0002] Inflammatory bowel disease (IBD) is a group of chronic inflammatory bowel diseases, including ulcerative colitis (UC) and Crohn's disease (CD). It is mainly characterized by symptoms such as diarrhea, abdominal pain, and rectal bleeding, severely impacting patients' health and quality of life. In my country, the number and incidence of UC patients are significantly higher than those of CD, and the trend is increasing year by year. Currently, clinical treatment for UC mainly uses aminosalicylic acids, corticosteroids, immunomodulators, and biologics, which primarily work by suppressing the immune response to achieve inflammatory remission. However, these drugs have certain limitations in treating colitis and may cause various side effects. Therefore, there is an urgent need to explore safe and effective intervention strategies.

[0003] Studies have shown that gut microbiota dysbiosis plays a significant role in the development and progression of IBD. Probiotics, as beneficial microorganisms, can regulate the gut microenvironment, thereby modulating host immunity and maintaining healthy intestinal barrier function, thus alleviating colitis-related symptoms. IBD patients often exhibit reduced abundance of *Lactobacillus* in their gut, accompanied by impaired aryl hydrocarbon receptor (AhR) activation. *Lactobacillus*, a common probiotic, has been extensively studied, and the beneficial effects of various *Lactobacillus* interventions on colitis have been reported. Furthermore, *Lactobacillus* has the ability to metabolize tryptophan to produce indole derivatives, some of which can act as AhR agonists. Activation of this receptor in the host mediates intestinal immune homeostasis, which is of great significance for improving colitis. Indole-3-lactic acid (ILA) is an effective AhR agonist and has been reported to activate CD4+ in vitro. + CD8αα + AhR in T cells regulates immune cell differentiation and mediates inflammatory remission; in addition, it can also activate human CD4 isolated under Th17 polarization conditions. + AhR in T cells promotes the production of the anti-inflammatory factor IL-22. Recent studies have also reported the ameliorative effects of ILA on colitis and colon cancer in mice.

[0004] Therefore, altering the host gut metabolic microenvironment through microbial agents is a promising intervention strategy with significant research value. Indolelacic acid is an important component of intestinal metabolites; however, no Lactobacillus johnsonii has yet been found to produce indolelacic acid. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, this invention provides *Lactobacillus johnsonii* and its application in the treatment of ulcerative colitis. This bacterium can produce large amounts of indolelacic acid, activating the AhR signaling pathway in colonic cells, and thus has a good effect on improving colitis.

[0006] This invention provides a strain of Lactobacillus johnsonii NSP009, which was deposited on March 8, 2023, at the Institute of Microbiology, Guangdong Academy of Sciences, at the address of Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No:63248.

[0007] The *Lactobacillus johnsonii* NSP009 strain was isolated from the colonic contents of DSS-induced colitis C57BL / 6 mice. Sequencing analysis revealed its 16S rDNA sequence to be shown in SEQ ID NO.1. Nucleic acid sequence alignment using NCBI showed a 99% similarity to *Lactobacillus johnsonii*, leading to its naming as *Lactobacillus johnsonii* NSP009.

[0008] The *Lactobacillus johnsonii* NSP009 described herein possesses the following properties: Cell characteristics: Gram-positive rod-shaped bacteria, non-spore-forming, non-flagellated, with a cell width and length of approximately 0.5-1.5 μm. Colony characteristics: Forms distinct colonies on culture media, with a diameter between 0.5-1 mm. The colonies are round on the upper surface, convex in the center, with neat edges, slightly whitish, opaque, and moist and smooth. Growth characteristics: This strain is anaerobic, with an optimal growth temperature of 36℃-38℃ and an optimal growth pH of 6.0-6.5. It grows well in glucose-containing media and enters the late logarithmic growth phase or early stationary phase within 16-24 hours.

[0009] The present invention also provides a microbial agent containing the aforementioned Lactobacillus johnsonii NSP009.

[0010] In one embodiment of the present invention, the viable count of Lactobacillus johnsonii NSP009 in the microbial agent is not less than 1 × 10⁻⁶. 6CFU / mL or 1×10 6 CFU / g.

[0011] The present invention also provides a product containing the aforementioned Lactobacillus johnsonii NSP009.

[0012] In one embodiment of the present invention, the viable count of Lactobacillus johnsonii NSP009 in the product is not less than 1 × 10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

[0013] In one embodiment of the present invention, the product is a pharmaceutical product.

[0014] In one embodiment of the present invention, the pharmaceutical product comprises Lactobacillus johnsonii NSP009, a drug carrier, and / or pharmaceutical excipients.

[0015] In one embodiment of the present invention, the dosage form of the medicine includes granules, capsules, tablets, pills, or oral liquids.

[0016] In one embodiment of the present invention, the pharmaceutical excipient is a pharmaceutically acceptable excipient.

[0017] In one embodiment of the present invention, the acceptable excipients include one or more commonly used thickeners, antioxidants, pH adjusters, emulsifiers, preservatives, fillers, binders, wetting agents, disintegrants, lubricants, and flavoring agents.

[0018] In one embodiment of the present invention, the filler is starch, sucrose, lactose, calcium sulfate and / or microcrystalline cellulose.

[0019] In one embodiment of the present invention, the adhesive is a cellulose derivative, alginate, gelatin, and / or polyvinylpyrrolidone.

[0020] In one embodiment of the present invention, the wetting agent is water, ethanol, starch and / or syrup.

[0021] In one embodiment of the present invention, the disintegrant is sodium carboxymethyl starch, carboxypropyl cellulose, croscarmellose, agar, calcium carbonate and / or sodium bicarbonate.

[0022] In one embodiment of the present invention, the lubricant is talc, calcium stearate, magnesium stearate, micronized silica gel, and / or polyethylene glycol.

[0023] In one embodiment of the present invention, the flavoring agent is a simple syrup, sucrose, lecithin, orange peel syrup, cherry syrup, lemon, fennel, peppermint oil, sodium alginate, gum arabic, gelatin, methylcellulose, sodium carboxymethyl cellulose, citric acid, tartaric acid and / or sodium bicarbonate.

[0024] The present invention also provides the use of the above-mentioned Lactobacillus johnsonii NSP009, or the above-mentioned microbial agent, in the preparation of products for the prevention and / or treatment of ulcerative colitis.

[0025] In one embodiment of the present invention, the product includes a pharmaceutical product.

[0026] In one embodiment of the present invention, the viable count of Lactobacillus johnsonii NSP009 in the product is not less than 1 × 10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

[0027] In one embodiment of the present invention, the pharmaceutical product comprises Lactobacillus johnsonii NSP009, a drug carrier, and / or pharmaceutical excipients.

[0028] In one embodiment of the present invention, the dosage form of the medicine includes granules, capsules, tablets, pills, or oral liquids.

[0029] In one embodiment of the present invention, the pharmaceutical excipient is a pharmaceutically acceptable excipient.

[0030] In one embodiment of the present invention, the acceptable excipients include one or more commonly used thickeners, antioxidants, pH adjusters, emulsifiers, preservatives, fillers, binders, wetting agents, disintegrants, lubricants, and flavoring agents.

[0031] In one embodiment of the present invention, the filler is starch, sucrose, lactose, calcium sulfate and / or microcrystalline cellulose.

[0032] In one embodiment of the present invention, the adhesive is a cellulose derivative, alginate, gelatin, and / or polyvinylpyrrolidone.

[0033] In one embodiment of the present invention, the wetting agent is water, ethanol, starch and / or syrup.

[0034] In one embodiment of the present invention, the disintegrant is sodium carboxymethyl starch, carboxypropyl cellulose, croscarmellose, agar, calcium carbonate and / or sodium bicarbonate.

[0035] In one embodiment of the present invention, the lubricant is talc, calcium stearate, magnesium stearate, micronized silica gel, and / or polyethylene glycol.

[0036] In one embodiment of the present invention, the flavoring agent is a simple syrup, sucrose, lecithin, orange peel syrup, cherry syrup, lemon, fennel, peppermint oil, sodium alginate, gum arabic, gelatin, methylcellulose, sodium carboxymethyl cellulose, citric acid, tartaric acid and / or sodium bicarbonate.

[0037] Beneficial effects

[0038] 1. The Lactobacillus johnsonii NSP009 strain of the present invention is a strain with colitis-improving effects screened from the intestines of colitis-affected mice. The Lactobacillus johnsonii NSP009 possesses the novel property of producing indolelacic acid; after 48 hours of fermentation, it can produce 20.02 mmol / L of indolelacic acid, which is 1.52 times that of the standard strain.

[0039] 2. The *Lactobacillus johnsonii* NSP009 described in this invention also possesses adjunctive therapeutic functions for ulcerative colitis. Mouse experiments showed that this strain resists weight loss, increased disease activity index, and colonic shortening caused by colitis, effectively improves colonic epithelial cell damage, and significantly reduces intestinal inflammation levels. Furthermore, this strain has the ability to produce large amounts of indolelacic acid, a metabolite that can act as an agonist of AhR, promoting the production of the anti-inflammatory factor IL-22. These results provide strong theoretical support for the adjunctive treatment of ulcerative colitis with probiotics.

[0040] Preservation of biological materials

[0041] A strain of Lactobacillus johnsonii, NSP009, was deposited at the Institute of Microbiology, Guangdong Academy of Sciences on March 8, 2023. Its taxonomic name is Lactobacillus johnsonii, and its accession number is GDMCCNo:63248. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences. Attached Figure Description

[0042] Figure 1 Changes in body weight and disease activity index in mice with colitis during intervention with Lactobacillus johnsonii NSP009; where A represents the changes in body weight of mice in each group during the intervention period, and B represents the changes in disease activity index of mice during the intervention period.

[0043] Figure 2Effects of Lactobacillus johnsonii NSP009 intervention on colon length in mice with colitis and representative colon images; where A is a representative colon image of each group of mice, and B is the colon length.

[0044] Figure 3 Effects of Lactobacillus johnsonii NSP009 intervention on colonic pathological damage in colitis mice; where A is a representative section of colonic tissue stained with HE, and B is the colonic pathological damage score.

[0045] Figure 4 Effects of Lactobacillus johnsonii NSP009 intervention on colonic inflammation levels in mice with colitis; where A represents myeloperoxidase (MPO) protein level, B represents TNF-α level, C represents IL-6 level, and D represents IL-22 level.

[0046] Figure 5 Effects of Lactobacillus johnsonii NSP009 intervention on the expression of AhR-related genes in the colon of colitis mice; where A represents the gene expression level of Ahr and B represents the gene expression level of Cyp1a1.

[0047] "*" indicates a significant difference compared to the model group (*: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001); data results in some tables are expressed as averages. Data analysis was performed using SPSS 24 with one-way ANOVA and Tukey's HSD post-hoc test. Different letters "a, b, c, etc." in the same column represent significant differences between groups (p<0.05). Detailed Implementation

[0048] The mice used in the following examples were purchased from Beijing Vital River Laboratory Animal Co., Ltd., and housed in a constant temperature (22±2℃) and constant humidity (55±10%) SPF environment with a 12-hour light / dark cycle, while being provided with free access to standard food and water. Experiments began after one week of acclimatization. The DSS (molecular weight 36,000-50,000 Da) used in the following examples were purchased from MP Company (USA). The MPO, TNF-α, IL-6, and IL-22EILSA kits used in the following examples were purchased from Nanjing Formex Biotechnology Co., Ltd.; the BCA protein concentration assay kit was purchased from Shanghai Beyotime Biotechnology Co., Ltd.; and the reverse transcription and qPCR kits were purchased from Takara Bio Inc. (Japan). All culture medium components used in the following examples were purchased from Shanghai Yuanye Co., Ltd.

[0049] The following examples involve culture media:

[0050] Preparation of enrichment medium (g / L): Potassium dihydrogen phosphate 1.0, sodium bicarbonate 3.0, potassium chloride 9.0, sodium chloride 9.0, anhydrous magnesium sulfate 1.2, potassium chloride dihydrate 0.2, ferrous sulfate heptahydrate 0.01, acid-hydrolyzed casein 1.4, tryptone 2.34, bacterial tryptone 2.34, yeast extract 2.1, cysteine ​​hydrochloride 1.6, bile salts 0.8, heme chloride 0.1, Tween 1.0, vitamin K1 0.01, β-glucan 5.0, dissolved in 1L distilled water, mixed thoroughly, then the pH was adjusted to 6.2-6.9, and sterilized at 115-121℃ for 15-20 min to obtain the enrichment medium.

[0051] MRS liquid culture medium (g / L): peptone 10.0, beef extract 8.0, yeast extract 4.0, glucose 20.0, dipotassium hydrogen phosphate 2.0, diammonium hydrogen citrate 2.0, sodium acetate 5.0, magnesium sulfate 0.2, manganese sulfate 0.04, Tween 1.0, dissolved in 1L of distilled water, and cysteine ​​hydrochloride 0.5-1g / L was added. The mixture was thoroughly mixed, and the pH was adjusted to 6.6-7.0. After sterilization at 115-121℃ for 15-20min, the MRS liquid culture medium was obtained.

[0052] Preparation of MRS solid medium: Add 1.5-2% agar to the MRS liquid medium. Mix well, then adjust the pH to 6.6-7.0, and sterilize at 115-121℃ for 15-20 min to obtain the MRS solid medium.

[0053] MRS selective medium (g / L): Peptone 10.0, beef extract 8.0, yeast extract 4.0, dipotassium hydrogen phosphate 2.0, diammonium hydrogen citrate 2.0, sodium acetate 5.0, magnesium sulfate 0.2, manganese sulfate 0.04, Tween 1.0, β-glucan 5, dissolved in 1L distilled water, and cysteine ​​hydrochloride 0.5-1g / L was added. The mixture was thoroughly mixed, and the pH was adjusted to 6.6-7.0. After sterilization at 115-121℃ for 15-20min, the liquid medium was obtained.

[0054] The detection methods involved in the following embodiments are as follows:

[0055] Disease Activity Index (DAI) Evaluation: Starting from day 0 of DSS treatment, the degree of diarrhea and bloody stool in mice were monitored daily. Combined with changes in body weight, the daily DAI (average of three indicators) was calculated according to the scoring criteria in Table 1, and the DAI change curve of mice during the modeling period was plotted.

[0056] Table 1 Scoring criteria for various indicators of the mouse disease activity index

[0057]

[0058] HE staining and pathological analysis of colon sections: Colon tissue was fixed by immersion in 4% paraformaldehyde solution for 24 hours, then dehydrated and embedded in paraffin, and cut into 3-4 μm thick paraffin sections for HE staining according to the following steps.

[0059] (1) Dewaxing paraffin sections to water: Dewax the sections sequentially with xylene I for 20 min, xylene II for 20 min, soak in anhydrous ethanol I for 5 min, soak in anhydrous ethanol II for 5 min, soak in 75% alcohol for 5 min, and wash with tap water.

[0060] (2) Hematoxylin staining: Immerse the sections in hematoxylin staining solution for 3-5 minutes, wash with tap water, use blue solution to reverse blue staining, and rinse with running water.

[0061] (3) Eosin staining: Dehydrate the sections in 85% ethanol and 95% ethanol for 5 min in sequence, and then soak them in eosin staining solution for 5 min.

[0062] (4) Dehydration and mounting: Soak the sections in anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, xylene I for 5 min, xylene II for 5 min, and finally mount with neutral resin.

[0063] (5) Microscopic examination and image acquisition and analysis.

[0064] The pathological score of tissue damage was determined according to the scoring criteria in Table 2. Histological score = epithelial damage score + inflammation severity score + lesion depth score.

[0065] Table 2. Histological Damage Scoring Criteria

[0066]

[0067] Determination of MPO and related cytokine levels in colon tissue: 30 mg of colon tissue was taken and mixed with sterile PBS and two 3 mm grinding beads at a ratio of 1:9 (w / v). The tissue was thoroughly homogenized using a homogenizer. The resulting homogenate was centrifuged at 10,000 rpm for 10 min at 4°C, and the supernatant was collected for subsequent assays. The levels of MPO, TNF-α, IL-6, and IL-22 in mouse colon tissue were detected using the corresponding ELISA kits. Specific operating procedures and precautions were performed according to the manufacturer's instructions. Additionally, the total protein concentration of the samples was determined using a BCA protein concentration assay kit, and the content of the target protein per mg of total protein was calculated.

[0068] RNA extraction and gene expression determination: Total RNA was extracted from colon tissue using Trizol reagent, and reverse transcription was performed on the total RNA using a reverse transcription kit. Using the cDNA obtained from reverse transcription as a template, a PCR reaction system (20 μL) was prepared using the TB Green Premix Ex Taq II kit (TAKARA), consisting of 10 μL TB Green Premix Ex Taq II (Tli RNaseH Plus), 6 μL dd H2O, 0.8 μL upstream primer, 0.8 μL downstream primer, 0.4 μL ROXReference Dye II, and 2 μL cDNA template. Primers are detailed in Table 3, with β-actin serving as the internal reference gene. Reaction conditions: Initial denaturation at 50℃ for 2 min, followed by 95℃ for 30 s; then 40 cycles of 95℃ for 20 s, 60℃ for 30 s, and 72℃ for 30 s; extension at 72℃ for 5 min, followed by termination of the reaction. Based on the Ct values ​​of the target gene and the internal reference gene, [the following steps were performed]. The relative expression level of the target gene is calculated using this method.

[0069] Table 3 Target gene and primer sequences

[0070]

[0071] Detection and quantification of indolelactic acid: Metabolites were extracted from bacterial culture broth and mouse colon contents using organic extraction. The metabolites were analyzed and detected using a QTRAP 4500 HPLC-MS / MS. Mass spectrometry data were imported into MultiQuant (v3.0.1) for quantitative analysis of the target substances based on the corresponding standard curves.

[0072] Example 1: Isolation and identification of Lactobacillus johnsonii NSP009

[0073] 1. Accumulation of fecal bacteria

[0074] Seven days after the establishment of the colitis model in mice, the colonic contents were collected and rapidly transferred to an anaerobic incubator. An appropriate amount of sterile PBS containing 0.1% L-cysteine ​​hydrochloride was added, and the mixture was vortexed and then passed through a 100 μm sterile cell sieve to obtain the bacterial culture. The bacterial culture was added to enrichment medium at 2% (v / v) and cultured under anaerobic conditions at 37°C with shaking for 24 h.

[0075] 2. Isolation and purification of Lactobacillus

[0076] Spread the diluted bacterial culture onto MRS solid medium and incubate for 2–3 days for selective culture of Lactobacillus. Select plates with appropriate colony counts, pick single colonies with neat edges, slightly white, opaque, moist, smooth surfaces, and uniform morphology from the solid medium, and inoculate them into 5 mL of liquid MRS selective medium. Incubate at 37°C under anaerobic conditions for 24 h to obtain purified culture.

[0077] 3. Preservation and Identification of Microbial Strains

[0078] The bacterial culture with the best viability from step 2 was used as an amplification template, and PCR amplification was performed using 16S universal primers (see Table 4). The PCR system is shown in Table 5, and amplification was carried out on a PCR instrument according to the following procedure: pre-denaturation at 95℃ for 5 min; followed by 29 cycles of 95℃ for 15 s, 60℃ for 15 s, and 72℃ for 45 s; extension at 72℃ for 5 min; and termination of the reaction by cooling to 4℃.

[0079] The amplified products were analyzed by 1% agarose gel electrophoresis and then subjected to first-generation sequencing. High-quality sequences were extracted from the sequencing data and submitted to NCBI for BLAST alignment to retrieve relevant sequence annotation information. The results showed that the sequence had over 99% homology with the 16S rDNA sequence of *Lactobacillus johnsonii*. This strain is now named *Lactobacillus johnsonii* NSP009 and deposited at the Institute of Microbiology, Guangdong Academy of Sciences.

[0080] Table 4 Primer Names and Sequences

[0081]

[0082] Table 5 PCR System

[0083]

[0084] 4. Determination of indolelactic acid production

[0085] Activated *Lactobacillus johnsonii* NSP009 and *Lactobacillus johnsonii* ATCC33200 were inoculated into MRS medium at a 2% (v / v) inoculum and cultured anaerobically at 37°C for 48 h. Indolelacic acid was detected by HPLC-MS / MS, and the signal intensity is shown in Table 6. The results show that the indolelacic acid yield of *Lactobacillus johnsonii* NSP009 in this invention is significantly higher than that of the standard strain *Lactobacillus johnsonii* ATCC33200, reaching 1.52 times that of the standard strain.

[0086] Table 6. Indolelactic acid production in bacterial culture broth

[0087]

[0088] Note: There are significant differences in the different representations of letters in the same column (P<0.05).

[0089] Example 2: Effects of Lactobacillus johnsonii NSP009 on body weight and disease activity index in mice with ulcerative colitis

[0090] The specific steps are as follows:

[0091] 1. Preparation of cryopreservative for Lactobacillus johnsonii NSP009:

[0092] (1) Culture method: In a sterile anaerobic environment, Lactobacillus johnsonii NSP009 was streaked on MRS solid medium and cultured under anaerobic conditions for 48 hours. After single colonies were formed, single colonies were picked and inoculated into MRS liquid medium. The culture was carried out anaerobically at 37°C for 16-24 hours to reach the stationary phase. The OD value at this time was 1.0-1.4, and the seed culture was prepared.

[0093] (2) Preparation of protective agent: Weigh 1 g / L of cysteine ​​hydrochloride and 200-300 g / L of glycerol according to the final concentration, dissolve them evenly in distilled water, and sterilize at 115-121℃ for 15-20 min.

[0094] (3) Preparation of cryoprotectant: After centrifuging the Lactobacillus johnsonii NSP009 seed culture cultured to the stable phase in step (1) (8000 rpm, 10 min, 4℃), wash it 1-2 times with sterile phosphate buffer (pH 7.2), and then resuspend the bacterial culture with the protectant prepared in step (2) to obtain Lactobacillus johnsonii NSP009 cryoprotectant, and store it at -80℃ for later use.

[0095] 2. Preparation of Lactobacillus johnsonii NSP009 inoculum:

[0096] (1) Activation of strains: The Lactobacillus johnsonii NSP009 cryopreservation agent prepared in step 1 was streaked on MRS solid medium and cultured under anaerobic conditions for 48 h. After single colonies were formed, they were inoculated into MRS liquid medium and cultured anaerobically at 37°C for 16-24 h to reach the stationary phase (OD value: 1.1-1.4).

[0097] (2) Preparation of bacterial agent: Take 100 μL of the culture medium obtained in step (1) at different dilution ratios and spread it on MRS solid medium. Count the number of colonies on the MRS solid plate and calculate the number of viable bacteria in the liquid medium of step (1). After washing 1-2 times with sterile phosphate buffer (pH 7.2), prepare the bacterial solution to a concentration of 1×10⁻⁶. 9 A formulation with a concentration of CFU / mL.

[0098] 3. Experimental methods:

[0099] This invention uses a 3% (w / v) DSS aqueous solution to induce ulcerative colitis in mice. Twenty-four healthy male C57BL / 6J mice aged 6 weeks were randomly divided into four groups (n=6 per group): normal group, DSS-induced colitis model group (hereinafter referred to as model group), Lactobacillus johnsonii NSP009+DSS (hereinafter referred to as NSP009 group), and mesalazine+DSS (hereinafter referred to as mesalazine group).

[0100] Preparation of mesalazine formulation: 30 mg mesalazine granules (Shanghai Aifa Pharmaceutical Co., Ltd.) were dissolved in 1 mL of 5% (w / v) sodium carboxymethyl cellulose aqueous solution to obtain a 30 mg / mL mesalazine formulation.

[0101] The experimental procedure is shown in Table 7. After a one-week adaptation period:

[0102] Normal group: Free access to purified water from day 0 to day 14, and 0.1 mL of sterile phosphate buffer was administered by gavage daily.

[0103] Model group: Free access to purified water from day 0 to day 7, free access to 3% DSS solution from day 8 to day 14, and 0.1 mL of sterile phosphate buffer by gavage daily from day 0 to day 14.

[0104] NSP009 group: Free access to purified water from day 0 to 7, free access to 3% DSS solution from day 8 to 14, and 0.1 mL of Lactobacillus johnsonii NSP009 preparation administered by gavage daily from day 0 to 14.

[0105] Mesalazine group: Free access to purified water from day 0 to day 7, free access to 3% DSS solution from day 8 to day 14, and 0.1 mL of mesalazine preparation administered by gavage daily from day 0 to day 14.

[0106] During DSS treatment, mouse weight, stool loosening, and stool bleeding were monitored at the same time every day.

[0107] Table 7 Experimental Procedure

[0108]

[0109] 4. Experimental Results:

[0110] The changes in body weight of mice in each group are as follows: Figure 1As shown in Figure A. The results showed that DSS treatment significantly reduced the body weight of mice (P<0.001), with the model group mice losing 77.74% of their initial body weight on the last day of DSS treatment. Intervention with Lactobacillus johnsonii NSP009 and mesalazine significantly reduced the weight loss in mice (on day 7 after DSS treatment, the body weight of mice in the NSP009 group and the mesalazine group recovered to 83.58% and 83.78% of their initial body weight, respectively, P<0.001), and there was no significant difference between the two groups (P>0.05).

[0111] Changes in disease activity index in each group of mice are as follows: Figure 1 As shown in Figure B. The results indicated that DSS treatment gradually increased the disease activity index, i.e., increased the degree of loose stools, bloody stools, and weight loss. Intervention with Lactobacillus johnsonii NSP009 and mesalazine significantly reduced the disease activity index in mice (on day 7 after DSS treatment, the disease activity index of mice in the NSP009 group and the mesalazine group recovered to 2.83 and 2.78, respectively, P<0.001), and there was no significant difference between the two groups (P>0.05).

[0112] The above results indicate that Lactobacillus johnsonii NSP009 of the present invention can significantly improve the body weight and disease activity index-related colitis phenotype in mice after DSS treatment, and its effect is no different from that of the positive control drug mesalazine.

[0113] Example 3: Effects of Lactobacillus johnsonii NSP009 on colon length and pathology in mice with ulcerative colitis

[0114] The specific steps are as follows:

[0115] The specific experimental method was the same as in Example 2. After the intervention ended on day 14, the mice were euthanized. The cecum and colon were quickly dissected and removed, the colon length was recorded, and the tissue was photographed. With the cecum as the proximal end and the anus as the distal end, a 1 cm section of colon tissue was taken and preserved in 4% paraformaldehyde solution.

[0116] DSS induces colitis and colonic atrophy in mice; therefore, colonic length is often used as one of the criteria for assessing the severity of colitis. Figure 2 As shown in A and B, the average colon length in the DSS group was 4.27 cm, which was 61.54% of the average colon length in the normal group. Intervention with Lactobacillus johnsonii NSP009 and mesalazine in mice significantly improved colon length shortening (P<0.0001), with average lengths of 5.03 cm and 5.00 cm, respectively.

[0117] HE staining and pathological analysis of colon tissue yielded representative section images and pathological scoring results for each group, as follows: Figure 3As shown in Figures A and B. Observations revealed that the colonic tissue structure of the normal group mice was intact, with neatly arranged epithelial cells and no obvious lesions. In the model group mice, the colonic tissue structure was incomplete, with disordered or even absent epithelial cells, destroyed crypt structures, congestion and edema of the mucosa and submucosa, and extensive inflammatory infiltration in the lamina propria and submucosa. Compared with the model group, both *Lactobacillus johnsonii* NSP009 and mesalazine significantly improved the pathological morphology of the colonic tissue, repaired mucosal damage, and reduced inflammatory cell invasion (P<0.0001). The colonic pathological score of the NSP009 group mice was 7.83, slightly lower than that of the mesalazine group mice (8.36).

[0118] The above results indicate that Lactobacillus johnsonii NSP009 can significantly improve colonic atrophy in colitis mice and protect the integrity of colonic tissue structure, with a slightly better effect than the mesalazine group.

[0119] Example 4: Effect of Lactobacillus johnsonii NSP009 on intestinal inflammation levels in mice with ulcerative colitis

[0120] The specific steps are as follows:

[0121] The specific experimental method was the same as in Example 2. After the intervention ended on day 14, the mice were euthanized. The colon tissue was preserved at -80°C for ELISA detection.

[0122] MPO is a protein produced by neutrophils and is an important marker of inflammatory response, widely used to reflect the inflammatory state of the intestinal mucosa. Figure 4 A. The level of colonic MPO in the model group mice was significantly higher than that in the normal group (P<0.0001). Lactobacillus johnsonii NSP009 and mesalazine significantly reduced the level of MPO in the intestine of colitis mice (P<0.0001). Compared with the model group, the MPO expression level in the NSP009 group was reduced by 0.73 times, which has a certain ameliorative effect on inflammation.

[0123] TNF-α and IL-6 are two important cytokines that can activate inflammatory cells and induce inflammatory responses; therefore, they are often considered pro-inflammatory factors. Figure 4B. The levels of TNF-α and IL-6 in the colon of model group mice were significantly higher than those in normal group (P<0.001), but intervention with Lactobacillus johnsonii NSP009 and mesalazine significantly reduced their levels in the colon (P<0.01) (TNF-α concentration: normal group: 9.21 pg / mg protein; model group: 62.67 pg / mg protein; NSP009 group: 38.27 pg / mg protein; IL-6 concentration: normal group: 40.27 pg / mg protein; model group: 116.18 pg / mg protein; NSP009 group: 61.94 pg / mg protein). IL-22 is a cytokine with anti-inflammatory effects. Compared with the model group, the intervention of Lactobacillus johnsonii NSP009 and mesalazine significantly increased the level of this cytokine (P<0.05) and restored it to near normal levels (normal group: 321.12 pg / mg protein; NSP009 group: 310.67 pg / mg protein).

[0124] The above results indicate that the Lactobacillus johnsonii NSP009 of the present invention can significantly downregulate the level of inflammation in colitis mice, reduce the level of pro-inflammatory cytokines, increase the level of anti-inflammatory cytokines, restore intestinal immune balance, and effectively control the occurrence and development of colitis.

[0125] Example 5: Effect of Lactobacillus johnsonii NSP009 on indolelactone levels in the intestines of mice with ulcerative colitis

[0126] The specific steps are as follows:

[0127] The specific experimental method was the same as in Example 2. After intervention on day 14, the mice were euthanized. The colon was quickly removed, and the colon contents were preserved at -80°C for indolelactone detection. Metabolites in the colon contents were extracted with 80% (v / v) methanol, and indolelactone was detected by HPLC-MS / MS. The concentrations are shown in Table 8. The results showed that *Lactobacillus johnsonii* NSP009 of this invention significantly increased the indolelactone level in colitis mice, restoring it to a level comparable to the normal group; compared with the model group, the indolelactone concentration increased by 1.65 times; compared with the mesalazine group, the indolelactone concentration increased by 1.46 times.

[0128] Table 8. Indolelactic acid concentration in mouse colon contents

[0129]

[0130] Note: There are significant differences in the different representations of letters in the same column (P<0.05).

[0131] Example 6: Effect of Lactobacillus johnsonii NSP009 on AhR pathway activation in the intestines of mice with ulcerative colitis

[0132] The specific experimental method was the same as in Example 2. After the intervention ended on day 14, the mice were euthanized. The colon tissue was rinsed with pre-cooled PBS, and 1 / 3 of it was soaked in RNA wait solution overnight and then frozen at -80°C for RNA extraction and qPCR experiments.

[0133] The expression of Ahr and its downstream key gene Cyp1a1 in the mouse intestine was detected by qPCR, and the results are as follows: Figure 5 As shown in A and B, the expression of Ahr and Cyp1a1 in the intestines of colitis mice was significantly decreased compared to the normal group (P<0.0001), reaching 48.21% and 34.34% of the normal group, respectively. Lactobacillus johnsonii NSP009 significantly upregulated the expression of these two genes (P<0.0001), reaching levels close to those of the normal group (Ahr and Cyp1a1 expression were 89.3% and 84.4% of the normal group, respectively). Furthermore, mesalazine had no significant effect on the expression of Ahr and Cyp1a1 (P>0.05); however, Lactobacillus johnsonii NSP009 showed significantly higher activation of these two genes than mesalazine (P<0.001).

[0134] The above results indicate that the Lactobacillus johnsonii NSP009 strain of the present invention can significantly upregulate the expression of Ahr and Cyp1a1 in colitis mice, effectively improving the impaired activation of the AhR signaling pathway in mice; the indolelacic acid produced by this strain may mediate the activation of this pathway.

[0135] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A microbial inoculant, characterized in that, Contains Lactobacillus johnsonii NSP009; said Lactobacillus johnsonii NSP009 was deposited on March 8, 2023 at the Institute of Microbiology, Guangdong Academy of Sciences, at the address of Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No:63248.

2. The microbial agent as described in claim 1, characterized in that, In the microbial agent, the viable count of Lactobacillus johnsonii NSP009 is not less than 1×10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

3. A medicine, characterized in that, The medicine contains the microbial agent as described in claim 1 or 2.

4. The medicine as described in claim 3, characterized in that, The viable count of *Lactobacillus johnsonii* in the drug is not less than 1 × 10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

5. The medicine as described in claim 4, characterized in that, The drug contains Lactobacillus johnsonii, and also contains a drug carrier and / or pharmaceutical excipients.

6. The use of the microbial agent according to claim 1 or 2 in the preparation of a medicine for the prevention and / or treatment of ulcerative colitis.

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