A kühwein-like Lactobacillus strain and its application

Abstract

The present invention discloses a kühwein-like Lactobacillus and its application, belonging to the field of microbial technology. Lactobacillus kefiranofaciens ) strain number 2503, deposited with the General Microbiology Center of the China National Center for Microbiological Culture Collection under the accession number CGMCC No. 33828. The kumiss-like Lactobacillus provided by the present invention has strong acid and bile salt tolerance, antioxidant and free radical scavenging properties, and can colonize the intestines, increasing the abundance of beneficial bacteria and reducing the abundance of pathogenic bacteria. It has excellent preventive and therapeutic effects on inflammatory bowel disease.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a küsbeck-like lactobacillus strain and application thereof. Background Art

[0002] Inflammatory bowel disease (IBD) is an immune-related disorder characterized by chronic intestinal inflammation, primarily including Crohn's disease (CD) and ulcerative colitis (UC). The global incidence of IBD exceeds 8 million, and its incidence is increasing annually, particularly in developing countries. The pathogenesis of IBD is complex, involving the interplay of multiple factors, including genetic susceptibility, intestinal dysbiosis, abnormal immune system activation, and impaired intestinal barrier function.

[0003] Current treatment for IBD relies primarily on medications, including aminosalicylates, corticosteroids, immunosuppressants, and biologics. Traditional medications, such as aminosalicylates (e.g., mesalamine), have limited efficacy in patients with moderate to severe disease and are only suitable for mild cases. While glucocorticoids (e.g., prednisone) can rapidly control inflammation, long-term use can lead to osteoporosis, infection, and metabolic disorders. Immunosuppressants (e.g., azathioprine) can cause hepatotoxicity, renal toxicity, and bone marrow suppression. Biologics, such as anti-TNF-α monoclonal antibodies (e.g., adalimumab) and anti-integrin monoclonal antibodies (e.g., vedolizumab), can target inflammation, but approximately 30% of patients experience primary nonresponse, and 50% experience secondary failure over time. Frequent injections are required, leading to high costs. Furthermore, long-term use may increase the risk of infection and cancer. Secondly, IBD still plays a significant role in some patients. While surgical treatments such as intestinal resection or ostomy can alleviate acute symptoms, the recurrence rate is as high as 50%. It can also easily trigger pouch inflammation or dysbiosis, leading to new inflammation and even cancer. Therefore, new directions and more effective treatments are needed for IBD.

[0004] Probiotics are a class of living microorganisms that benefit human health, including species such as Lactobacillus, Bifidobacterium, Lactococcus, Streptococcus, and Bacillus. Due to their proven efficacy and safety, probiotics are widely used as an adjunctive treatment for diseases. Studies have shown that probiotics can inhibit the colonization of harmful bacteria and promote the colonization of beneficial bacteria, optimizing the composition of the microbiome, thereby inducing anti-inflammatory responses and improving intestinal barrier function.

[0005] Although there are precedents for using probiotics to alleviate IBD-related clinical symptoms, the following barriers exist in the current use of probiotics in the treatment of IBD: 1. Conventional probiotics (such as lactic acid bacteria and bifidobacteria) have a low survival rate in gastric acid (pH 2.0-3.0) and bile salt environments, resulting in insufficient numbers of live bacteria reaching the intestine and poor therapeutic effects; 2. The intestinal barrier of IBD patients is damaged, leading to LPS translocation, bacterial invasion, and exacerbated inflammatory responses. Current probiotics are insufficient to repair the intestinal barrier function of IBD patients, leading to continued worsening of inflammation; 3. Current probiotic therapies lack effective antioxidant intervention, causing oxidative stress to exacerbate the pathological process of IBD; 4. IBD patients are usually in an immune imbalance (imbalanced ratio of pro-inflammatory / anti-inflammatory factors), and existing technologies lack precise regulation. Summary of the Invention

[0006] In order to improve the defects of the prior art, the present invention provides a strain of Kühm-like Lactobacillus, which has the following characteristics: 1. Strong acid and bile salt resistance, which can ensure that sufficient live bacteria reach the intestine to colonize and exert a therapeutic effect; 2. Strong antioxidant capacity, which can significantly alleviate the damage of oxidative stress to intestinal epithelial cells; 3. It has the function of repairing the intestinal barrier and rebuilding the immune balance; 4. It regulates the intestinal flora and restores the diversity of the flora; 5. It has a significant effect of alleviating weight loss, colon shortening and pathological damage in the DSS-induced IBD mouse model. In view of this, the present invention provides a use of the Kühm-like Lactobacillus in the preparation of drugs for treating IBD, the preparation of immunomodulators, and the preparation of intestinal microbial regulators.

[0007] The present invention includes the following technical solutions:

[0008] In a first aspect of the present invention, the present invention provides a kumiss-like Lactobacillus ( Lactobacillus kefiranofaciens ), strain number 2503, deposited in the General Microbiology Center of China Culture Collection Administration Committee, deposit number CGMCC NO.33828. Lactobacillus kefiranofaciens )2503.

[0009] The kumiss-like Lactobacillus ( Lactobacillus kefiranofaciens ) The 16S rRNA sequence of 2503 is shown as SEQ ID NO.1.

[0010] The kumiss-like Lactobacillus of the present invention ( Lactobacillus kefiranofaciens )2503 Gram staining is purple, and the bacteria are long rods, about 5-30 μm in length and about 1 μm in width.

[0011] In a second aspect of the present invention, the present invention provides a culture of Kühm-like Lactobacillus, wherein the culture is obtained by Lactobacillus kefiranofaciens)2503 is a fermentation product obtained by culturing in a microbial culture medium.

[0012] The culture refers to a general term for liquid or solid products (all substances in the culture container, i.e., fermentation products) that grow a microbial community after artificial inoculation and cultivation, that is, products obtained by growing and / or amplifying microorganisms. It can be a biologically pure culture of microorganisms, or it can contain a certain amount of culture medium, metabolites and / or other components produced during the culture process. It also includes subculture cultures obtained by subculturing microorganisms, which can be cultures of a certain generation or a mixture of several generations.

[0013] The present invention cultivates kumiss-like Lactobacillus ( Lactobacillus kefiranofaciens ) The microbial culture medium used in 2503 is a solid culture medium or liquid culture medium conventionally prepared by those skilled in the art, and the present invention does not limit the components of the microbial culture medium.

[0014] The fermentation product includes kumiss-like Lactobacillus ( Lactobacillus kefiranofaciens ) 2503 and Kühm-like Lactobacillus ( Lactobacillus kefiranofaciens ) metabolites of 2503.

[0015] In the third aspect of the present invention, the present invention provides a bacterial agent, wherein the bacterial agent contains the kumiss-like Lactobacillus ( Lactobacillus kefiranofaciens ) 2503 and / or Küsbeckia lactis ( Lactobacillus kefiranofaciens ) metabolites of 2503 and / or the culture described in the second aspect of the present invention.

[0016] The active ingredient in the bacterial agent is Kühm-like Lactobacillus ( Lactobacillus kefiranofaciens ) 2503 and / or its metabolites, or Küsbeckia lactis ( Lactobacillus kefiranofaciens ) 2503 cultures, the content of active ingredients in the bacterial agent is 1.0×10 8 -1.0×10 10 CFU / g. In one embodiment of the present invention, the content of active ingredient in the bacterial agent is 1.0×10 9 CFU / g.

[0017] In a preferred embodiment of the present invention, the bacterial agent further comprises an acceptable carrier, which may include a solid carrier or a liquid carrier. The carrier is selected from one or a combination of two or more of montmorillonite, silica, diatomaceous earth, straw powder, corn flour, starch, and soybean flour, which are commonly used in the art and have a protective effect on live bacteria.

[0018] In some embodiments of the present invention, the formulation of the bacterial agent is selected from liquid preparations, emulsions, suspensions, powders, granules, wettable powders or water dispersible granules.

[0019] In the fourth aspect of the present invention, the present invention provides a kumiss-like Lactobacillus ( Lactobacillus kefiranofaciens ) 2503. The kumiss-like Lactobacillus described in the second aspect of the present invention ( Lactobacillus kefiranofaciens ) Use of the 2503 culture or the bacterial agent described in the third aspect of the present invention in the preparation of a drug for preventing and / or treating and / or assisting in the treatment of inflammatory bowel disease.

[0020] The inflammatory bowel diseases include Crohn's disease and ulcerative colitis.

[0021] The drug has at least one of the following functions:

[0022] a1) slow down the patient’s weight loss;

[0023] a2) Improve the patient’s stool characteristics and blood in stool;

[0024] a3) Improve the patient's spleen index;

[0025] a4) Slow down the shortening of the patient’s colon length;

[0026] a5) Increase the expression levels of MUC2, ZO-1, and Claudin-1, and repair the intestinal barrier function of patients;

[0027] a6) Increase the activity of antioxidant enzymes CAT, T-SOD, and GSH-Px, and reduce the patient's oxidative stress level;

[0028] a7) scavenging free radicals;

[0029] a8) Reduce the levels of pro-inflammatory factors IL-1β, TNF-α, and IL-6 in the patient's serum, and increase the level of the anti-inflammatory factor IL-10;

[0030] a9) Increase the abundance of beneficial intestinal bacteria and reduce the abundance of pathogenic bacteria, and restore the diversity of the patient's intestinal flora and metabolite levels.

[0031] In a specific embodiment of the present invention, the increasing the abundance of beneficial intestinal bacteria and reducing the abundance of pathogenic bacteria include:

[0032] a9.1) At the phylum level, add Bacteroidetes ( Bacteroidetes ) abundance, and reduced the abundance of Firmicutes ( Firmicutes ) abundance, reversing the abnormal F / B ratio;

[0033] a9.2) At the family level, add Rhabditiaceae ( Muribaculaceae ), Prevotellaceae ( Prevotellaceae )、Lactobacillus family( Lactobacillaceae ) abundance, and reduced Staphylococcus family ( Staphylococcaceae ) abundance;

[0034] a9.3) At the genus level, add the genus Rhabditis Muribaculum )and unclassified_ Clostridia_ UCG_014 abundance, and reduced Staphylococcus spp. ( Staphylococcus ) 、 Odorobacter spp. Odoribacter ) 、 Mammalian cocci ( Mammaliicoccus ) abundance;

[0035] a9.4) Enrichment of Lachnospiraceae ( Lachnospiraceae)、 Paraprevotella spp. Paraprevotella)、 Clostridium Clostridium_sp_ASF356、 Kühm-like Lactobacillus ( Lactobacillus kefiranofacien) .

[0036] In a specific embodiment of the present invention, restoring the patient's intestinal flora metabolite level includes:

[0037] a9.5) Restoring intestinal short-chain fatty acids (SCFAs) levels, including acetate, propionate, isobutyrate, butyrate, and isovaleric acid;

[0038] a9.6) Restoring the levels of indole metabolites from the gut microbiome, including indole-3-carboxaldehyde, indole-3-acetic acid, indole-3-propionic acid, indole-3-acrylic acid, indole, and tryptamine.

[0039] In a fifth aspect of the present invention, the present invention provides a kumiss-like Lactobacillus ( Lactobacillus kefiranofaciens ) 2503. The kumiss-like Lactobacillus described in the second aspect of the present invention ( Lactobacillus kefiranofaciens ) Use of the 2503 culture or the bacterial agent described in the third aspect of the present invention in the preparation of antioxidants or free radical scavengers.

[0040] In a specific embodiment of the present invention, the free radicals include DPPH, ABTS and hydroxyl radicals.

[0041] In the sixth aspect of the present invention, the present invention provides a kumiss-like Lactobacillus ( Lactobacillus kefiranofaciens ) 2503. The kumiss-like Lactobacillus described in the second aspect of the present invention ( Lactobacillus kefiranofaciens ) Use of the 2503 culture or the bacterial agent described in the third aspect of the present invention in the preparation of an intestinal microbial regulator.

[0042] The intestinal microbial regulator has at least one of the following functions:

[0043] b1) Increase the abundance of beneficial intestinal bacteria;

[0044] b2) reduce the abundance of pathogenic bacteria;

[0045] b3) regulating the diversity of intestinal flora;

[0046] b4) Regulate the levels of intestinal microbial metabolites.

[0047] In the seventh aspect of the present invention, the present invention provides a kumiss-like Lactobacillus ( Lactobacillus kefiranofaciens ) 2503. The kumiss-like Lactobacillus described in the second aspect of the present invention ( Lactobacillus kefiranofaciens ) Use of the 2503 culture or the bacterial agent described in the third aspect of the present invention in the preparation of an immunomodulator.

[0048] The immunomodulator has at least one of the following functions:

[0049] c1) Reduce the levels of pro-inflammatory cytokines IL-1β, TNF-α, and IL-6;

[0050] c2) Increase the level of anti-inflammatory factor IL-10.

[0051] The kühwein-like Lactobacillus 2503 provided by the present invention has the following obvious advantages:

[0052] 1. The kumiss-like Lactobacillus 2503 provided by the present invention has excellent acid resistance (pH 2.5-3.0) and bile salt resistance (0.3%), ensuring that sufficient live bacteria can colonize the intestine and function.

[0053] 2. The kühm-like Lactobacillus 2503 provided by the present invention can significantly upregulate the expression of ZO-1, Claudin-1 and MUC2, enhance intestinal barrier function, reduce LPS translocation, and thus block the vicious cycle of "leaky gut-inflammation".

[0054] 3. The kumiss-like Lactobacillus 2503 cell contents provided by the present invention have strong free radical scavenging ability (DPPH, ABTS, hydroxyl radicals), and enhance the activities of T-SOD, CAT, and GSH-Px, reduce the levels of MPO and MDA, and effectively alleviate oxidative stress.

[0055] 4. The kühwein-like Lactobacillus 2503 provided by the present invention can significantly inhibit IL-1β, TNF-α, and IL-6, promote IL-10 secretion, and achieve multi-target immune regulation.

[0056] 5. The kumiss-like Lactobacillus 2503 provided by the present invention can improve Muribaculaceae 、 Lachnospiraceae The abundance of beneficial bacteria such as Staphylococcus It can inhibit pathogenic bacteria such as leukemia and melanoma, promote the production of SCFA and tryptophan indole metabolites, activate the AhR / Cyp1a1 pathway, enhance barrier function and regulate immunity.

[0057] 6. The kumiss-like Lactobacillus 2503 provided by the present invention is a natural strain, has no genetic modification, is highly safe, and can replace or combine with existing drugs to reduce treatment costs and risks.

[0058] Deposit Description

[0059] Strain classification and nomenclature: Kühm-like Lactobacillus

[0060] Latin name of strain: Lactobacillus kefiranofaciens

[0061] CGMCC registration number: CGMCC NO.33828

[0062] Depository: General Microbiology Center of China Culture Collection Administration

[0063] Abbreviation of depository institution: CGMCC

[0064] Address of the depository: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0065] Deposit date: March 17, 2025 BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 The morphology of Kühm-like Lactobacillus; a: colony; b&c: bacterial morphology;

[0067] Figure 2 This is the result of the acid resistance test of Kühm-like Lactobacillus;

[0068] Figure 3 This is the result of the bile salt tolerance test of Kühm-like Lactobacillus;

[0069] Figure 4 This is a graph showing the results of testing the free radical scavenging ability of Kühm-like Lactobacillus;

[0070] Figure 5 This is a progress chart of animal experiments with Kühm-like Lactobacillus;

[0071] Figure 6 The effect of Lactobacillus kumiss on the body weight of mice;

[0072] Figure 7 The effect of Kühm-like Lactobacillus on DAI;

[0073] Figure 8 The effect of Lactobacillus kumiss on colon length;

[0074] Figure 9 H&E staining of colon tissue (upper layer: 40×, lower layer: 100×);

[0075] Figure 10 colon histopathology was scored;

[0076] Figure 11 The effect of Lactobacillus kumiss on the expression of genes related to intestinal barrier function in mice;

[0077] Figure 12 The effect of Lactobacillus kumiss on the expression of proteins related to intestinal barrier function in mice;

[0078] Figure 13 Effects of Lactobacillus kumiss on oxidative stress indices in mouse colon tissue homogenate;

[0079] Figure 14 The effect of Lactobacillus kumiss on serum cytokines in mice;

[0080] Figure 15 It is a Venn diagram;

[0081] Figure 16 for Alpha diversity;

[0082] Figure 17 This is the PCoA analysis diagram;

[0083] Figure 18 Species composition at the phylum level;

[0084] Figure 19 The species composition is at the family level;

[0085] Figure 20 Species composition at the genus level;

[0086] Figure 21 is the evolutionary branch diagram of each group based on LEfSe analysis;

[0087] Figure 22 Analyze histograms for LDA;

[0088] Figure 23 for the effect on SCFAs content;

[0089] Figure 24 for tryptophan-targeted metabolomics;

[0090] Figure 25 is the content of indole metabolites derived from intestinal microorganisms;

[0091] Figure 26 Activation of the AhR / Cyp1a1 signaling pathway. DETAILED DESCRIPTION

[0092] The technical solutions in the embodiments of the present invention will be described clearly and completely below. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative work shall fall within the scope of protection of the present invention.

[0093] Part I Experimental Materials and Methods

[0094] 1.1 Experimental Materials

[0095] 1.1.1 Test strains

[0096] Kfusin-like Lactobacillus 2503 was isolated from Russian kefir grains and belongs to the Lactobacillaceae family and the genus Lactobacillus. It is currently preserved in the China General Microbiological Culture Collection Center (CGMCC NO. 33828).

[0097] 1.1.2 Culture medium

[0098] Glucose 20.0 g, yeast extract 45.0 g, potassium dihydrogen phosphate 0.5 g, manganese sulfate 0.2 g, sodium chloride 0.15 g, cysteine ​​hydrochloride 1.4 g, sodium acetate 15 g, Tween-80 1.0 g, dilute to 1,000 mL with distilled water, adjust pH to 6.2, and autoclave at 115°C for 20 min.

[0099] 1.1.3 Experimental mice

[0100] This experiment used 50 SPF-grade male Balb / C mice purchased from Beijing Sibeifu Co., Ltd. The animals were housed at the Barrier System Animal Laboratory Center, School of Food Science and Engineering, Tianjin University of Science and Technology. The experimental room conditions were: relative humidity: 55 ± 5%, 23 ± 1°C, 12-h light-dark cycle, and free access to food and drinking water. This experiment was approved by the Tianjin University of Science and Technology Animal Care and Use Committee (approval number: SCXK (Beijing) 2024-0001), with welfare and ethics number 2024030, and was maintained in accordance with the Guide for the Care and Use of Laboratory Animals.

[0101] 1.2 Experimental Methods

[0102] 1.2.1 Isolation of strains

[0103] Under sterile conditions, a sample of Russian kefir grains (about 5 g) was inoculated into raw milk, anaerobically activated at 37°C for 72 h, and then graded diluted to 10 -3 ~10 -6concentration. Plate onto modified MRS solid medium and incubate at 37°C under anaerobic conditions (85% N₂, 10% CO₂, 5% H₂) for 48–72 hours. Initially isolate the target strain by colony morphology (milky white, raised colonies with neat margins, 1–2 mm in diameter). Purify using the three-zone streak method. Typical characteristics of the resulting kumiss-like Lactobacillus include: Gram-positive rods, catalase-negative growth, and the ability to ferment lactose to produce acid.

[0104] 1.2.2 Morphological observation of strains

[0105] (1) Gram staining and observation

[0106] Apply a small amount of bacterial suspension evenly to a clean glass slide and fix with a gentle flame. Stain with crystal violet for 1 minute, rinse with distilled water, and remove excess stain. Add iodine solution for 1 minute, then rinse with distilled water. Decolorize with 95% ethanol for 10-30 seconds. Stain with safranin for 1 minute, then rinse with distilled water. Dry under low heat. Observe the staining results under an optical microscope (100×).

[0107] (2) Scanning electron microscopy observation

[0108] Take 1 mL of bacterial solution, centrifuge at 8000 r / min for 1 min, and collect the bacterial precipitate. Resuspend the bacteria in 0.1 M phosphate buffer and wash repeatedly 3 times to remove the residual culture medium. Resuspend the washed bacteria in 2.5% glutaraldehyde solution and fix at 4 °C for 12 h. After fixation, wash 3 times with PBS to remove excess fixative. Dehydrate the fixed sample in a gradient of 30%, 50%, 70%, 80%, and 90% ethanol solution. After dehydration, wash the bacteria twice with anhydrous ethanol, fix the dried sample on the sample stage, and use an ion sputtering instrument to spray a 10-15 nm thick gold film on the surface of the sample to enhance conductivity. Observe and photograph the surface morphology of the bacteria under an accelerating voltage of 5-15 kV.

[0109] 1.2.3 Acid resistance test

[0110] Adjust the pH of MRS culture medium to 2, 3, or 4 using 5 mol / L hydrochloric acid, sterilize, and cool before use. Inoculate the activated Lactobacillus kumiss-like 2503 into different culture media at a 4% inoculum. Samples were collected and counted at 0, 3, and 6 hours, and the survival rate was calculated according to the formula.

[0111] Survival rate (%) = total viable bacteria count after incubation / total viable bacteria count before incubation × 100 Formula (1-1)

[0112] 1.2.4 Bile salt tolerance test

[0113] To the MRS culture medium, 0.1%, 0.2%, and 0.3% of ox bile salts were added, respectively. After sterilization and cooling, the activated kumiss-like Lactobacillus 2503 was inoculated into different culture media at a 4% inoculum size. Samples were taken and counted at 0 h, 3 h, and 6 h, and the survival rate of the strain was calculated according to formula (1-1).

[0114] 1.2.5 Free radical scavenging ability determination

[0115] (1) Sample preparation

[0116] Prepare two samples in advance: fermentation broth supernatant and cell disruption extract. To prepare the fermentation broth supernatant, centrifuge the fermentation broth at 8000 rpm for 10 minutes at 4°C to obtain the fermentation supernatant. To prepare the cell disruption extract, centrifuge the fermentation broth at 8000 rpm for 10 minutes, retaining the bacterial pellet. Wash the pellet three times with PBS and resuspend it in PBS. Ultrasonicate the cells on ice (400 W, 10 minutes, 5 seconds on, 5 seconds off). Centrifuge the turbid liquid and collect the supernatant.

[0117] (2) Determination of DPPH free radical scavenging ability

[0118] The samples were mixed with equal volumes of a 0.2 mmol / L DPPH-anhydrous ethanol solution. The DPPH solution in the interference tube was replaced with anhydrous ethanol, and the blank tube consisted of a mixture of DPPH solution and anhydrous ethanol. The reaction was allowed to proceed in the dark for 30 minutes, and the absorbance of the supernatant was measured at 517 nm. The reaction system and calculation formula for DPPH free radical scavenging efficiency are as follows:

[0119] Table 1 Reaction system for scavenging DPPH free radicals

[0120] .

[0121] Formula (1-2)

[0122] Where: A0: OD value of the blank tube; A1: OD value of the sample tube; A2: OD value of the interference tube.

[0123] (3) Determination of ABTS free radical scavenging ability

[0124] The samples to be tested were mixed with ABTS + Mix equal volumes of working solution as the measuring tube and interfere with the ABTS tube. + The working solution was replaced with distilled water, and the blank tube was replaced with ABTS +Mix the working solution with distilled water. Incubate in the dark for 20 minutes and measure the absorbance of the supernatant at 734 nm. + The free radical scavenging rate reaction system and calculation formula are as follows:

[0125] Table 2 Reaction system for scavenging ABTS free radicals

[0126] .

[0127] Formula (1-3)

[0128] Where: A0: OD value of the blank tube; A1: OD value of the sample tube; A2: OD value of the interference tube.

[0129] (4) Determination of hydroxyl radical scavenging ability

[0130] Take 1 mL of distilled water, 0.5 mL of o-phenanthroline solution, 0.5 mL of ferrous sulfate solution, and 0.5 mL of sample, mix them, and finally add 0.5 mL of H2O2. After standing for 60 minutes at 37°C, the absorbance is measured at 536 nm using a UV spectrophotometer. The reaction system and calculation formula of hydroxyl radical scavenging rate are as follows:

[0131] Table 3 Reaction system for scavenging hydroxyl radicals

[0132] .

[0133] Formula (1-4)

[0134] Where: A0: OD value of the blank tube; A1: OD value of the control tube; A2: OD value of the sample tube.

[0135] 1.2.6 Preparation of bacterial powder

[0136] After centrifugation, the fermentation broth was washed twice with physiological saline, and then 10% skim milk powder was added at a ratio of 3:1 between the protective agent and the bacterial slurry (w / v). The freeze-dried sample was frozen at -80 °C for 2 h, and then the sample was freeze-dried for 24 h before testing. The activity of the obtained bacterial powder was 1.0 × 10 9 CFU / g.

[0137] 1.2.7 Animal Experiment Design

[0138] After acclimation for one week, 50 male SPF Balb / C mice were randomly divided into five groups (n=10) according to body weight: blank control group (Control), inflammatory bowel disease model group (IBD), positive drug group (5-ASA, 5-aminosalicylic acid, 0.01 g / mL), low-dose Lactobacillus kefiranofaciens group (L-Lk, Lactobacillus kefiranofaciens 10 8 CFU / kg) and high-dose group (H-Lk, 10 9 CFU / kg). Preventive treatment interventions were performed on days 0-7 of the experiment: the L-Lk and H-Lk groups were gavaged with 0.2 mL of bacterial suspension every day, the 5-ASA group was gavaged with an equal volume of 5-aminosalicylic acid, and the Control and IBD groups were gavaged with an equal volume of normal saline. On days 8-14 of the experiment, except for the Control group, the inflammatory bowel disease model was induced by free drinking of 3.0% dextran sodium sulfate (DSS) aqueous solution in the other groups. The L-Lk, H-Lk and 5-ASA groups maintained the original intervention plan, and the Control and IBD groups continued to be gavaged with an equal volume of normal saline. The experimental process is as follows Figure 5 shown.

[0139] 1.2.8 Disease Activity Index Score

[0140] During the experiment, the weight and fecal status of the mice were recorded, and the occult blood content of the feces of the mice was measured using a fecal occult blood test kit. The disease activity index score was calculated. The scoring details are shown in the table. The DAI index is the average of the total score of the percentage of weight loss, fecal status, and the degree of blood in the stool.

[0141] Table 4 Disease Activity Index Scoring Criteria

[0142] .

[0143] 1.2.9 Sample collection and preparation

[0144] Mice were fasted for 24 hours before sacrifice. Blood samples were obtained by eye extraction, incubated at 4°C for 30 minutes, and centrifuged (3500 rpm for 15 minutes) to obtain the supernatant, which was aliquoted and stored at -80°C. After blood collection, the mice's internal organs were removed, rinsed with saline, blotted dry with filter paper, and weighed. The entire colon was removed, its length measured, and photographed. A portion was fixed in 4% paraformaldehyde for histological analysis, while the remaining portion was cryopreserved at -80°C until further use.

[0145] 1.2.10 Organ Index

[0146] The tissue was weighed after pretreatment and calculated according to the following formula:

[0147] Formula (1-5)

[0148] Where: m1: organ weight, g; m2: mouse body weight, g.

[0149] 1.2.11 Colon H&E staining and pathological scoring

[0150] The fixed colon tissue was rinsed with PBS and dehydrated with 80%, 90%, and 100% ethanol, respectively. The tissue was cleared with xylene, embedded in paraffin, and cut into approximately 5 μm sections. The sections were placed in xylene to remove the paraffin and then rehydrated in a series of ethanol gradients. The sections were stained with hematoxylin and eosin (H&E) stains, and the excess dye was washed away. After mounting, the staining was observed microscopically, and tissue morphology and pathological changes were assessed according to histological scoring criteria.

[0151] Table 5 Colon histopathology scoring criteria

[0152] .

[0153] 1.2.12 Colon immunofluorescence staining

[0154] The fixed colon tissues were sent to Wuhan Sevier Biotechnology Co., Ltd. for immunofluorescence staining. Fluorescence microscopy was used to observe and collect images, and the expression levels of MUC2, ZO-1, Claudin-1, AhR, and Cyp1a1 were analyzed.

[0155] 1.2.13 Determination of colonic oxidative stress indicators

[0156] The colon tissue was prepared into a 10% homogenate and diluted according to the requirements of the kit to determine the contents of SOD, CAT, GSH-Px, MDA, and MPO in the colon tissue.

[0157] 1.2.14 Serum factor detection

[0158] ELISA kits were used to detect the levels of LPS, IL-1β, TNF-α, IL-6, and IL-10 in mouse serum.

[0159] 1.2.15 Quantitative analysis of gene expression by RT-qPCR

[0160] RNA was extracted from the colon tissues of mice in each group using Trizol reagent, and cDNA was synthesized using a reverse transcription kit. The reaction system is shown in Table 6.

[0161] Table 6 Reverse transcription reaction system

[0162] .

[0163] The primer sequences of the desired target genes were queried in the NCBI database, as shown in Table 7, and synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.

[0164] Table 7 Primer sequences

[0165] .

[0166] Prepare the reverse transcription reaction system at low temperature. Mix thoroughly to ensure that all the system settles to the bottom of the tube. The reaction system is shown in Table 8.

[0167] Table 8 RT-qPCR reaction system

[0168] .

[0169] Set the amplification program according to Table 9. After the reaction is completed, analyze the melting curve and record the Ct value.

[0170] Table 9 Real-time PCR reaction program

[0171] .

[0172] GAPDH gene was used as internal reference and 2 -△△Ct The relative expression level of target gene mRNA was calculated by this method.

[0173] 1.2.16 High-throughput sequencing of changes in mouse intestinal flora

[0174] Feces of mice in each group were collected, cryopreserved and sent to Beijing Biomarker Biotechnology Co., Ltd. for 16S rDNA sequencing. The intestinal flora diversity and species composition differences of mice in each group were analyzed using an online cloud platform.

[0175] 1.2.17 Determination of fecal short-chain fatty acids

[0176] Accurately weigh 0.1 g of feces and resuspend in 400 μL of saturated NaCl solution. Add 20 μL of 10% H₂SO₄ to acidify the mixture, vortex for 2 minutes, and thoroughly mix until all fecal particles are removed. Allow to stand for 20 minutes. Add 500 μL of ether, vortex to mix thoroughly, and allow to stand for 10 minutes. Centrifuge (13,000 rpm for 15 minutes) and collect the upper ether phase. Dehydrate the ether phase by adding 0.20 g of anhydrous Na₂SO₄. Allow to stand at 4°C for 10 minutes, then centrifuge (13,000 rpm for 15 minutes). The upper ether phase was filtered through a 0.22 μm organic filter. Gas chromatography was used to analyze the content of short-chain fatty acids in the feces of each group of mice, using the retention times of chromatography-grade acetic acid, propionic acid, isobutyric acid, butyric acid, and isovaleric acid on the gas chromatography column as a reference.

[0177] 1.2.18 Determination of changes in fecal tryptophan metabolites

[0178] The feces of mice in each group were collected, stored at low temperature and sent to Shanghai Meiji Biotechnology Co., Ltd. for targeted metabolomics testing. The changes in intestinal microbial tryptophan metabolism in the blank group, model group and high-dose probiotic group were analyzed using an online cloud platform.

[0179] 1.2.19 Data Statistics and Analysis

[0180] All data are presented as mean ± standard deviation. Multiple groups were analyzed using one-way analysis of variance and T-tests to assess statistical significance at 95% and 99% confidence intervals. Comparisons between two groups were performed using independent-sample T-tests. Data were analyzed and graphed using Graphpad Prism 8.0 software. # indicates comparison with the blank group, * indicates comparison with the model group, * / # ​​indicates (P < 0.05), ** / ## indicates (P < 0.01), and *** / ### indicates (P < 0.001).

[0181] Part II Experimental Results

[0182] 2.1 Morphological observation of strains

[0183] like Figure 1 As shown, Kühm-like Lactobacillus forms milky white colonies with irregular edges on the surface of modified MRS solid culture medium; Gram staining is purple, and the bacteria can be observed as long rods under an optical microscope; the individual morphology of bacteria observed using a scanning electron microscope shows that the length varies from about 5-30 μm and the width is about 1 μm.

[0184] 2.2 Acid resistance test

[0185] The results are as follows Figure 2 As shown in the data, the survival rate of Lactobacillus kumiss-like strains gradually decreased with the decrease of pH in the growth environment and the extension of incubation time. The highest survival rate was 63.84±22.73% when incubated at pH 4 for 3 h; the lowest survival rate was only 7.87±1.74% when incubated at pH = 2 for 6 h.

[0186] 2.3 Bile salt tolerance test

[0187] Bile salt tolerance results Figure 3 As shown in the figure, the survival rate of the strain was the highest at 83.4±11.55% when incubated in a 0.1% bile salt environment for 3 h. In a high concentration 0.3% bile salt environment, the survival rate of the strain was lower, and after incubation for 6 h, the survival rate of the strain was almost 0.

[0188] 2.4 Free radical scavenging ability test

[0189] The results are as followsFigure 4 As shown, the fermentation supernatant of Lactobacillus kumissiformis 2503 exhibited scavenging abilities for DPPH, ABTS, and hydroxyl radicals of 66.22±5.16%, 29.32±2.20%, and 29.12±5.96%, respectively; the cell contents exhibited scavenging abilities for DPPH, ABTS, and hydroxyl radicals of 70.30±3.13%, 52.02±5.59%, and 46.44±10.14%, respectively. The cell contents exhibited superior free radical scavenging abilities compared to the fermentation supernatant, likely due to the fact that key antioxidant components synthesized by the bacteria (such as SOD, GSH-Px, and CAT) are primarily stored intracellularly and require cell disruption for release, resulting in enhanced free radical scavenging and antioxidant activities. In summary, Lactobacillus kumissiformis 2503 exhibits excellent free radical scavenging activity in vitro and demonstrates potential application in alleviating oxidative stress and protecting the intestine from free radical damage.

[0190] 2.5 Preventive and therapeutic effects on IBD mice

[0191] 2.5.1 Effects on Mouse Body Weight

[0192] By detecting the changes in mouse body weight, we can reflect the effects of different intervention strategies on the progression of IBD. Figure 6 As shown, throughout the experiment, mice in the blank group (Control) maintained a stable physiological state and steadily gained weight, reaching a 6.09±2.92% increase by the end of the experiment. Mice in the model group (IBD) experienced weight loss and decreased activity from day 2 of modeling, with a 6.0±2.32% decrease by the end of the experiment, preliminarily indicating a successful IBD model. Mice in the positive drug group (5-ASA) experienced minimal weight fluctuations, with a 0.85±3.94% increase by the end of the experiment. The low-dose Lactobacillus kleiner-like (L-Lk) group exhibited a more moderate weight loss trend than the IBD group, with weight stabilization from day 4 of modeling, and a weight loss rate of approximately 2.19±3.35% by the end of the experiment. High-dose Lactobacillus kleiner-like (H-Lk) intervention demonstrated a superior protective effect, maintaining virtually unchanged weight during the modeling period. This protective effect was comparable to that of the 5-ASA group and significantly superior to that of the L-Lk group. Comprehensive analysis of weight change results showed that Kühm-like Lactobacillus 2503 could effectively improve DSS-induced weight loss, and the intervention effect was dose-dependent.

[0193] 2.5.2 Effects on DAI in mice

[0194] like Figure 7As shown, the DAI of mice in the control group remained stable throughout the experiment, almost at 0. The DAI of mice in the IBD group slowly increased from the first day of modeling. On the fourth day of DSS induction, unformed and bloody feces were observed. Starting on the fifth day, weight loss began to drastically decrease, and feces became watery. Most mice had feces and blood residue around the anus. At the end of the experiment, the DAI peaked at 2.83±0.39, significantly different from the control group, indicating that the IBD model was successfully established. The DAI of mice in the 5-ASA group began to slowly increase on the fifth day of modeling, reaching 1.13±0.55 at the end of the experiment, significantly lower than that of the IBD group, suggesting that 5-ASA has a significant therapeutic effect. The increasing DAI trend in mice in the L-Lk group was alleviated compared to the IBD group, with a final DAI score of 1.49±0.42, significantly better than the IBD group, but weaker than the 5-ASA group. The DAI of H-Lk mice remained stable during the first five days of DSS treatment and began to slowly increase on the sixth day of DSS induction, reaching 1.10±0.47 at the end of the experiment, demonstrating a preventive effect comparable to that of 5-ASA. Based on the DAI changes, Lactobacillus kumiss-like 2503 treatment significantly delayed the progression of DSS-induced IBD.

[0195] Prior art CN119752669 A discloses a kumiss-like lactobacillus JK-24 in 10 10 Even with the ultra-high dose of CFU / kg, mice still had bloody stools (see the attached document of the prior art manual). Figure 3 ), the DAI score is around 2.5 (see the appendix of the prior art specification Figure 4 ), while the kumiss-like Lactobacillus 2503 provided by the present invention was 9 A DAI of 1.10±0.47 was achieved at a CFU / kg dose, with virtually no blood in the stool, indicating that the kühm-like Lactobacillus provided by the present invention has significant advantages over existing technologies in improving blood in the stool, stool characteristics, and body weight.

[0196] 2.5.3 Effects on Mouse Organ Indexes

[0197] As shown in Table 10, the spleen index of mice in the DSS-induced IBD model group significantly increased to 4.12±0.58‰, indicating a more severe inflammatory response in the IBD mice. After intervention with L. kumiss 2503, the spleen index in the L-Lk and H-Lk groups decreased to 3.40±0.61‰ and 3.74±0.59‰, respectively, comparable to that in the 5-ASA group. This indicates that L. kumiss 2503 can effectively alleviate the abnormal systemic immune activation induced by DSS. Furthermore, there were no significant differences in the heart, liver, lung, and kidney indexes among the mice, demonstrating the safety of L. kumiss 2503.

[0198] Table 10 Changes in organ indexes of mice in each group

[0199] .

[0200] 2.5.4 Effects on the length of mouse colon

[0201] like Figure 8 As shown, the colons of mice in the control group were intact, with no signs of congestion or swelling. Brown, granular feces measuring 8.29±0.53 cm were present in the intestinal lumen. Following DSS induction, the colons of mice in the IBD group significantly shortened to 5.86±0.74 cm (P<0.001), with concomitant intestinal swelling and loose feces, indicating inflammation-induced colon damage. Compared with the IBD group, the colon length of mice in the 5-ASA group recovered to 7.34±0.63 cm (P<0.01), with fecal formation and morphology approaching normal. The intact colon lengths of mice in the L-Lk and H-Lk groups were 6.67±0.42 cm and 7.12±0.55 cm, respectively (P<0.05), with improved edema and congestion. These results demonstrate that preventive treatment with Lactobacillus kumiss 2503 can ameliorate DSS-induced colon shortening and morphology.

[0202] Prior art CN119752669 A discloses a kumiss-like lactobacillus JK-24 in 10 10 Under the ultra-high dose intervention of CFU / kg, the colon recovery rate was (5.2-4.5) / 4.5=16% compared with the model group, while the kumquat-like Lactobacillus 2503 provided by the present invention was 9 The colon recovery rate at a CFU / kg dose is (7.12-5.86) / 5.86=22%, indicating that the kühm-like Lactobacillus provided by the present invention is more effective than the prior art in improving the colon shortening induced by DSS.

[0203] 2.5.5 Effects on colon pathology scores in mice

[0204] HE staining was used to observe the damage of colon tissue in each group of mice ( Figure 9 ), and further quantitatively evaluated the degree of inflammation, crypt structure, and lesion extent based on histopathological scoring criteria ( Figure 10The colonic histology of mice in the control group was intact, with clear crypt structures, abundant goblet cells in the epithelial surface, and no inflammatory cell infiltration in the submucosal layer. The pathological score was 0.25±0.46. Compared with the control group, mice in the IBD group showed inflammatory destruction of the colonic crypt structure, a sharp decrease in the number of goblet cells, and dense inflammatory infiltration in the submucosal layer after DSS induction, showing typical inflammatory pathological features. The pathological score was significantly increased to 6.63±1.06 (P<0.001). Compared with the IBD group, the colonic crypt structure of mice in the 5-ASA and H-Lk groups was intact, epithelial integrity was restored, and the number of goblet cells increased. The pathological scores were significantly reduced to 2.25±0.89 and 3.50±0.93, respectively (P<0.001). The colonic crypt structure of mice in the L-Lk group was partially restored, but inflammatory cell infiltration was still present in the submucosal layer. The pathological score was significantly reduced to 5.0±1.19 (P<0.05).

[0205] 2.5.6 Effects on the intestinal barrier of mice

[0206] like Figure 11 As shown, the transcript levels of intestinal barrier function-related genes MUC2, ZO-1, and Claudin-1 in IBD mice were significantly reduced to 28.64±7.20%, 18.50±6.16%, and 31.45±6.98%, respectively, compared to those in the control group (P<0.001), indicating that DSS disrupted the intestinal barrier function of mice. Transcription levels of these genes were significantly restored in the 5-ASA group (P<0.001). High-dose Lactobacillus kleissleroides treatment significantly increased the transcript levels of MUC2, ZO-1, and Claudin-1 to 70.91±24.14%, 84.42±6.38%, and 65.82±7.02%, respectively, compared to the control group (P<0.05). Although low-dose treatment with the strain upregulated the expression of these genes, the effect was slightly less pronounced than in the high-dose group.

[0207] Immunofluorescence results further verified the RT-qPCR data and more intuitively reflected the differences in the localization and expression of intestinal barrier proteins in each group of mice. Figure 12As shown, MUC2 was densely punctately distributed in the colon of mice in the control group, while ZO-1 and Claudin-1 were continuously arranged along the epithelial cell membrane, forming a complete tight junction network. In the IBD group, the fluorescence signals of MUC2, ZO-1, and Claudin-1 were weakened and sparsely distributed, and Merge reflected the destruction of the mucus layer and the disintegration of tight junctions. In the 5-ASA group, the fluorescence intensity and protein localization of MUC2, ZO-1, and Claudin-1 were well restored. In the L-Lk group, the fluorescence intensity of MUC2 was restored to some extent and evenly distributed in goblet cells, but the fluorescence intensity localization of ZO-1 and Claudin-1 showed discontinuity. In the H-Lk group, the fluorescence intensity of intestinal barrier proteins in mice was well restored and evenly distributed in the colon tissue, forming a complete connection network.

[0208] 2.5.7 Effects on oxidative stress in mouse colon

[0209] Oxidative stress is a key pathological mechanism of IBD. Its essence is that reactive oxygen species (ROS) and free radicals exceed the capacity of the antioxidant defense system, leading to cellular damage and dysfunction. As the largest digestive organ, the intestine is inevitably exposed to foreign matter and reactive oxygen species. Due to damage to intestinal epithelial cells, IBD mice exhibit deficient antioxidant defense mechanisms, resulting in an inability to effectively remove excess reactive oxygen species. These factors work together to significantly increase oxidative stress levels in the colon. In this study, we evaluated the effects of intervention with Lactobacillus kumiss on colonic oxidative stress in mice by measuring the activity of the antioxidant enzymes CAT, T-SOD, and GSH-Px, and the activity or content of the oxidative damage markers MPO and MDA.

[0210] The results are as follows Figure 13As shown, compared with the control group, DSS-induced reduction in the activities of antioxidant enzymes such as CAT, T-SOD, and GSH-Px in the colon homogenate of mice in the IBD group decreased to 0.75±0.32, 49.78±2.19, and 192.03±16.42 U / mg prot, respectively (P<0.001). MPO activity and MDA content increased to 0.39±0.07 U / g and 0.57±0.08 nmol / mg prot, respectively (P<0.001), indicating that DSS induces oxidative stress homeostasis. Compared with the IBD group, 5-ASA treatment significantly increased antioxidant enzyme activities (P<0.05) and decreased MPO activity and MDA content (P<0.001). The preventive and therapeutic effects of Lactobacillus kumiss 2503 were dose-dependent. Compared with the IBD group, in the colonic tissue homogenate of mice in the H-Lk group, CAT and T-SOD activities increased to 1.12±0.22 and 61.37±4.80 U / mg prot, respectively (P<0.05), while MPO activity and MDA content decreased to 0.15±0.04 U / g and 0.37±0.07 nmol / mg prot, respectively (P<0.001). These results suggest that Lactobacillus kumiss 2503 alleviates DSS-induced intestinal inflammation by increasing the activities of antioxidant enzymes such as T-SOD and CAT and reducing MPO activity and MDA content, thereby reducing free radical damage to the intestinal mucosa.

[0211] The present invention provides a method for preparing a lactobacillus kumiss-like lactobacillus 2503 in 10 9 At the CFU / kg dose, compared to the model group, the CAT activity increased by (1.12-0.75) / 0.75 = 49%; compared to the model group, the MPO decreased by (0.39-0.15) / 0.39 = 62%, and the MDA decreased by (0.57-0.37) / 0.57 = 35%. Compared to the prior art CN119752669 A disclosed in Lactobacillus kumiss JK-24, the changes in CAT, MPO, and MDA were significantly superior, indicating that Lactobacillus kumiss 2503 is more beneficial in reducing free radical damage to the intestinal mucosa.

[0212] 2.5.8 Effects on Mouse Serum Cytokines

[0213] As a metabolite of intestinal flora, elevated levels of LPS in serum reflect disruption of intestinal barrier function and bacterial translocation. Serum LPS levels are an important indicator for assessing intestinal permeability. Increased levels of IL-1β, TNF-α, and IL-6, key proinflammatory cytokines, are closely associated with mucosal damage and neutrophil infiltration. IL-10, a key immunomodulator, can inhibit the production of proinflammatory cytokines and promote regulatory T cell (Treg) function. This study evaluated the regulatory effects of Lactobacillus kumissiformis 2503 on immune balance and intestinal barrier function by measuring the concentrations of these indicators in mouse serum.

[0214] like Figure 14 As shown, serum factor levels in mice in the DSS-induced IBD model group showed significant changes. Compared with the control group, the level of LPS, a marker of intestinal barrier function impairment, in the IBD group was significantly increased to 239.71±8.25 pg / mL (P<0.001). Meanwhile, the levels of pro-inflammatory cytokines IL-1β, TNF-α, and IL-6 increased to 86.08±4.25, 687.81±55.89, and 120.85±6.99 pg / mL, respectively (P<0.001), while the level of anti-inflammatory cytokine IL-10 decreased to 533.59±21.23 pg / mL (P<0.001). This indicates that DSS-induced intestinal barrier function impairment in mice resulted in a systemic inflammatory response. 5-ASA intervention showed significant therapeutic effects. Compared with the IBD group, the levels of LPS, IL-1β, TNF-α and IL-6 in the mouse serum were reduced to 204.15±7.75, 59.29±2.58, 569.58±67.38 and 87.17±8.63 pg / mL, respectively (P<0.01), and the IL-10 level recovered to 615.11±29.64 pg / mL (P<0.001). The intervention effect of Lactobacillus kleissinus was dose-dependent. The levels of LPS, IL-1β, TNF-α, IL-6, and IL-10 in the serum of mice in the H-Lk group recovered to 200.53±10.11, 76.04±1.43, 591.63±39.73, 81.25±9.72, and 651.79±45.73 pg / mL, respectively (P<0.05).

[0215] These results suggest that Lactobacillus kumissiformis 2503 may improve DSS-induced IBD pathology by repairing the intestinal barrier and modulating immune synergy. Its efficacy is dose-dependent and demonstrates comparable efficacy to 5-ASA in reducing LPS translocation, inhibiting pro-inflammatory factors, and enhancing anti-inflammatory factors.

[0216] In the present invention, the intervention of Kühm-like Lactobacillus 2503 increased the anti-inflammatory factor IL-10 in serum, while the Kühm-like Lactobacillus JK-24 disclosed in the prior art CN119752669 A further reduced the anti-inflammatory factor IL-10 compared with the model group. Those skilled in the art are aware that IL-10 is a key factor in inhibiting inflammation, so the Kühm-like Lactobacillus 2503 provided by the present invention is more conducive to immune regulation.

[0217] 2.5.9 Effects on the intestinal flora of mice

[0218] 2.5.9.1 OTU cluster analysis

[0219] The Operational Taxonomic Unit (OTU) is a concept used in ecology and microbiology to classify and describe microbial communities. Specifically, an OTU is a taxonomic unit defined based on genetic sequence similarity and is typically used to describe microbial populations with similarity above a certain threshold. Its primary purpose is to group microorganisms based on similar characteristics, allowing for their study and comparison.

[0220] like Figure 15 The Venn diagram of the overlap between groups showed that the number of OTUs common to the five groups was 313, and the number of OTUs unique to the Control group was 1515; after DSS induction, the number of OTUs unique to the IBD group was only 1073, indicating that DSS caused a decrease in the diversity of the mouse intestinal flora; in contrast, after intervention with Kruskal Lactobacillus 2503 and 5-ASA, the number of OTUs unique to each group increased to 1455, 1401, and 1176, respectively.

[0221] This result indicates that DSS induces the destruction of intestinal flora diversity in mice, and Kühm-like Lactobacillus 2503 can positively regulate intestinal flora homeostasis.

[0222] 2.5.9.2 Alpha Diversity Analysis

[0223] Alpha diversity of the microbiome is a core indicator for assessing the structure of the microbial community within a sample. High alpha diversity generally indicates a healthy and functionally diverse gut microbiome, while low alpha diversity may be associated with certain diseases or health issues. Commonly used indicators include the Chao1 Index, ACE Index, Shannon Index, and Simpson Index for comprehensive assessment. The Chao1 Index and ACE Index measure species richness, while the Shannon Index and Simpson Index measure species diversity.

[0224] Depend on Figure 16Compared with the control group, the richness index and diversity of the intestinal microbiota in the IBD group mice were significantly reduced (P<0.01). Under the preventive and protective effects of Lactobacillus kumiss and 5-ASA, these indices recovered. The intervention effect of Lactobacillus kumiss 2503 was dose-dependent, with the H-Lk group showing greater restoration of richness index and diversity than the 5-ASA group. This result suggests that this strain can positively regulate microbial structure and restore the reduced α-diversity of the microbiota caused by DSS.

[0225] 2.5.9.3 Beta Diversity Analysis

[0226] Beta diversity is a core metric for measuring differences in microbial community composition between samples. Principal coordinates analysis (PCoA) is often used to analyze beta diversity in different sample microbiotas. PCoA reduces the dimensionality of multidimensional data based on the Bray-Curtis distance matrix, projecting inter-group differences into a two-dimensional space. The horizontal and vertical axes represent the principal coordinate axes with the highest explanatory power, respectively. Closer distances between samples indicate greater similarity in microbial community structure.

[0227] like Figure 17 As shown, significant differences in the intestinal microbial composition between the control and IBD groups were observed, with the two groups exhibiting completely separate microbial distributions, suggesting that DSS intervention led to changes in the intestinal microbial composition. After prophylactic treatment with Lactobacillus kumiss 2503 and 5-ASA, the microbial composition shifted closer to that of the control group, with the H-Lk group exhibiting the greatest overlap and closest distance to the control group. The intervention effect of 5-ASA was comparable to that of a low-dose probiotic. This result suggests that Lactobacillus kumiss 2503 can restore the imbalanced microbial composition caused by DSS.

[0228] 2.5.9.4 Differences in intestinal flora composition

[0229] In order to gain a deeper understanding of the differences in the composition of intestinal flora in different groups of mice, the richness and differences of the mouse intestinal flora were further analyzed at the phylum, family, and genus levels.

[0230] like Figure 18 As shown, at the phylum level, the dominant phylum in each group was Bacteroidetes ( Bacteroidetes ), Firmicutes ( Firmicutes ), Desulfobacteria ( Desulfobacterota ), Actinobacteria ( Actinobacteria ) and Proteobacteria ( Proteobacteria ). After DSS induction, IBD mice FirmicutesThe relative abundance increased (Control group: 37.3%, IBD group: 48.5%), Bacteroidota The abundance of β-catenin decreased (Control group: 55.5%, IBD group: 40.0%), leading to Firmicutes / Bacteroidetes The ratio (F / B) of 5-ASA increased significantly (P<0.001). Studies have shown that the F / B ratio in healthy intestinal flora maintains a dynamic balance, and its abnormal changes are usually related to flora disorders. 5-ASA has no significant regulatory effect on the F / B ratio. After intervention with Lactobacillus kumiss 2503, it can be improved by Bacteroidetes abundance, while suppressing Firmicutes Excessive proliferation, reversing abnormal F / B ratio.

[0231] like Figure 19 As shown, at the family level, compared with the control group, the IBD group mice had more Bacillus family ( Muribaculaceae ), Prevotellaceae ( Prevotellaceae )、Lactobacillus family( Lactobacillaceae ) abundance decreased significantly (P<0.05); Staphylococcus family ( Staphylococcaceae ) abundance was significantly increased (P<0.001). Kumquat-like Lactobacillus 2503 showed a multi-dimensional regulatory effect. The H-Lk group not only significantly inhibited Staphylococcaceae Overproliferation (P<0.01) and increased Muribaculaceae 、 Prevotellaceae and Lactobacillaceae Although 5-ASA intervention can reduce Staphylococcaceae level, but its effect on the recovery of functional flora is weaker than that of the H-Lk group. Staphylococcaceae It can aggravate the condition by disrupting the balance of intestinal flora and inducing immune responses. For example, Staphylococcus aureus can produce toxins and pro-inflammatory factors, exacerbating intestinal inflammation and increasing the risk of bacterial translocation by destroying the intestinal barrier function. Muribaculaceae 、 Prevotellaceae and Lactobacillaceae Some members of the gut microbiome can participate in the decomposition of complex carbohydrates to produce short-chain fatty acids, playing an important role in maintaining the diversity and stability of the gut microbiome; in addition, some Lactobacillaceae The strain can metabolize tryptophan to produce indole compounds, which exert anti-inflammatory effects through the AhR signaling pathway and support the repair and regeneration of intestinal epithelial cells.

[0232] like Figure 20 As shown, at the genus level, the top 8 bacterial genera in the intestine of the Control group mice include the unnamed Labiatae genus ( unclassified_Muribaculaceae ), Bacteroides ( Bacteroides ), Staphylococcus spp. (Staphylococcus )、 unclassified_Clostridia_UCG_014、Lachnospiraceae_NK4A136_ group 、Odorobacter spp. Odoribacter ), Mammalian cocci ( Mammaliicoccus ), Rhabditis spp. ( Muribaculum Compared with the Control group, the IBD group mice had Bacteroides 、 Staphylococcus 、 Odoribacter and Mammaliicoccus The relative abundance increased significantly ( P <0.05); and Muribaculum and unclassified_Clostridia_UCG_014 Under the intervention of Kühm-like Lactobacillus 2503 and 5-ASA, the above-mentioned bacterial imbalance was improved; compared with the IBD group, the H-Lk group Staphylococcus、Odoribacter、 Mammaliicoccus The abundance was significantly reduced ( P <0.01), Muribaculum The abundance increased significantly ( P <0.01) and improved unclassified_Clostridia_UCG_014 Abundance. Under the protective effect of 5-ASA, only Bacteroides、 Staphylococcus 、 Mammaliicoccus and Odoribacter The abundance of Muribaculum The abundance of Bacteroides It is a type of Gram-negative bacteria whose LPS in the cell wall can activate the host's immune response. Staphylococcus Some members of the bacterium, such as Staphylococcus aureus, can cause various types of infections, including skin infections, pneumonia, endocarditis, and osteomyelitis. Mammaliicoccus Primarily associated with mammals, it may cause skin infections in animals. Odoribacter It is a Gram-negative bacterium that is commonly found in the intestines of humans and animals. Overgrowth of this bacterium may lead to an imbalance in the intestinal microbiota and is associated with digestive system diseases such as irritable bowel syndrome. Muribaculum It mainly exists in the intestines of mice and other mammals, helps maintain the balance of other beneficial bacteria in the intestines, prevents the overgrowth of harmful bacteria, and participates in the production of short-chain fatty acids. unclassified_Clostridia_UCG_014 It belongs to the class Clostridium. Although there is no clear evidence that this bacterium has a probiotic function, many Clostridium bacteria have been shown to be involved in tryptophan metabolism.

[0233] 2.5.9.5 Differences in intestinal flora composition

[0234] To further explore the effect of Kühm-like Lactobacillus 2503 on the microbiota of IBD mice, the LEfSe method was used to screen the differential species in the intestinal microbiota of each group of mice (LDA>3.0). The phylogenetic tree showed that (Figure 21 ), the differential species in the IBD group mice included Bacteroides ( Bacteroides ), Ruminococcus xanthophylla ( Ruminococcus flavefaciens ), Parabacteroides archaeopteris ( Parabacteroides ), Acinetobacter spp., ( Acinetobacter These bacteria have been shown to interfere with normal intestinal immune responses and increase the risk of intestinal inflammation. The main species with differences in the 5-ASA group were Paraprevotella ( Prevotellaceae ), the Kumiss-like Lactobacillus dose group was enriched with Lachnospiraceae ( Lachnospiraceae ), Paraprevotella spp. ( Paraprevotella )、 Clostridium_sp_ASF356 and Kühm-like Lactobacillus ( Lactobacillus kefiranofaciens Linear Discriminant Analysis ( Figure 22 ) further verified that the IBD group was mainly enriched Bacteroides ; 5-ASA group was mainly enriched Prevotellaceae ; After intervention with Kühm-like Lactobacillus 2503, the main enrichment Lachnospiraceae, Paraprevotella, uncultured_Muribaculaceae, Clostridium_sp_ ASF356, Alloprevotella, Lactobacillus kefiranofacien The above results once again prove that oral administration of Küsbeck-like Lactobacillus 2503 can not only colonize the mouse intestine but also fight against DSS-induced colitis by increasing the abundance of beneficial bacteria and reducing the abundance of pathogenic bacteria in the intestine. Lachnospiraceae The members of the Paraprevotella is a potent trypsin-degrading commensal bacterium that helps maintain intestinal homeostasis and protect against pathogens, Clostridium_sp_ASF356 It belongs to the genus Clostridium, which is often related to tryptophan metabolism.

[0235] 2.5.10 Effects on Short-Chain Fatty Acids in Mouse Intestines

[0236] SCFAs are key metabolites produced by the fermentation of dietary fiber by the gut microbiome. They participate in regulating the host's immune response and play a multifaceted role in maintaining intestinal health. They are primarily composed of acetic acid, propionic acid, and butyric acid. Because the homeostasis of the gut microbiome is significantly disrupted during the pathogenesis of IBD, its ability to metabolize and produce SCFAs also changes accordingly. Therefore, the detection of SCFAs has become an effective means of assessing the functional status of the gut microbiome.

[0237] The levels of SCFAs in the feces of mice in each group were as follows Figure 23The levels of the major SCFAs in the feces of mice in the control group were: acetic acid (0.69±0.06 mg / g), propionic acid (0.44±0.04 mg / g), isobutyric acid (0.72±0.03 mg / g), butyric acid (0.13±0.01 mg / g), and isovaleric acid (0.09±0.02 mg / g). Compared with the control group, the levels of these SCFAs in the IBD group were significantly reduced to 47.2%, 46.3%, 66.5%, 49.5%, and 32.9%, respectively (P<0.01). The improvement effect of Lactobacillus kumissiformis 2503 was dose-dependent. Compared with the IBD group, the H-Lk group significantly restored the levels of various SCFAs: acetate (0.58±0.08 mg / g), propionate (0.43±0.05 mg / g), isobutyrate (0.67±0.07 mg / g), butyrate (0.13±0.02 mg / g), and isovaleric acid (0.07±0.01 mg / g) (P<0.05). The 5-ASA group only slightly improved isobutyrate (P<0.05), with no significant effect on the other SCFAs.

[0238] These results indicate that Kühm-like Lactobacillus 2503 was enriched Muribaculaceae and Lachnospiraceae Functional strains such as 5-ASA drive the biosynthesis of SCFAs, while 5-ASA cannot restore the metabolic homeostasis of SCFAs due to its lack of microbial regulatory ability.

[0239] 2.5.11 Effects on tryptophan metabolism in the intestine of mice

[0240] Tryptophan metabolism is one of the important pathways for the interaction between intestinal microorganisms and the host. Its metabolic pathways mainly include the indole pathway, the 5-hydroxytryptamine pathway, and the kynurenine pathway. The metabolites produced by the indole pathway can enhance the intestinal barrier and regulate immunity by activating AhR. Intestinal flora analysis showed that after intervention with Küsbeck-like Lactobacillus 2503, the flora involved in tryptophan metabolism, such as Lachnospiraceae 、 Clostridium_sp_ASF356 and unclassified_ Clostridia_UCG _014 This suggests that 2503 may improve the symptoms of DSS-induced colitis by regulating tryptophan metabolism. Therefore, this study used targeted metabolomics to analyze changes in tryptophan metabolites in mouse feces.

[0241] like Figure 24(a) PCA shows that the distribution of tryptophan metabolites in the control group and the IBD group showed a clear separation trend, indicating that DSS treatment caused disorder in tryptophan metabolism in mice; the sample points of the high-dose 2503 treatment group were clustered in the overlapping area of ​​the blank group and the model group, indicating that 2503 intervention can partially restore tryptophan metabolic homeostasis. Figure 24 (b, c) show that compared with the control group, two metabolites in the feces of mice in the IBD group increased significantly, and one metabolite decreased significantly; intervention with Lactobacillus kumiss-like offset the changes in metabolites in the feces, resulting in a significant increase in one metabolite and a significant downregulation of four metabolites. Figure 24 (d) Analysis showed that tryptophan metabolism in the IBD group was biased toward inflammatory pathways, as evidenced by the accumulation of kynurenine and a decrease in the abundance of anti-inflammatory AhR ligands, indoles. High-dose Lactobacillus kumiss-like treatment reversed tryptophan metabolism to that of the control group, with an increase in AhR ligands and a decrease in the abundance of pro-inflammatory metabolites. The bar graph further quantified the changes in the levels of gut microbial-derived indole metabolites.

[0242] like Figure 25 As shown, the levels of core indole metabolites in the intestines of control mice were as follows: indole-3-carboxaldehyde (1.53±0.80 ng / mg), indole-3-acetic acid (0.80±0.31 ng / mg), indole-3-propionic acid (0.04±0.02 ng / mg), indole-3-acrylic acid (0.002±0.001 ng / mg), indole (0.42±0.20 ng / mg), and tryptamine (0.004±0.003 ng / mg). The IBD group exhibited disrupted tryptophan metabolism, with levels of indole-3-carboxaldehyde, indole-3-propionic acid, indole-3-acrylic acid, indole, and tryptamine decreased by 17.6%, 30.5%, 17.3%, 19.1%, and 42.5%, respectively. Following high-dose Lactobacillus kumiss-like treatment, levels of these indole metabolites increased, approaching those of the control group. These results indicate that Kfushinone 2503 can restore tryptophan metabolic homeostasis in IBD mice to a certain extent.

[0243] 2.5.11 Effects on AhR / Cyp1a1 Pathway Expression

[0244] AhR is a ligand-activated transcription factor that regulates intestinal immune homeostasis by recognizing exogenous ligands such as dietary polyphenols and endogenous ligands such as indoles metabolized by the microbiome. Under normal circumstances, AhR activation promotes the differentiation of regulatory T cells (Tregs) and inhibits the overactivation of Th17 cells, thereby maintaining immune tolerance. It also strengthens intestinal epithelial barrier function by upregulating tight junction proteins and mucin secretion. Cyp1a1, a key effector molecule of AhR signaling, not only metabolizes exogenous toxins but also mitigates oxidative stress-induced damage to the intestinal mucosa by scavenging reactive oxygen species. Previous studies have shown that 2503 treatment can increase indole production in the intestines of mice, suggesting that Lactobacillus kumiss may improve IBD by activating the AhR / Cyp1a1 signaling pathway. To further clarify the mechanism of action of Lactobacillus kumiss in preventing and treating IBD in mice, this study used immunofluorescence to assess AhR / Cyp1a1 activation.

[0245] The results are as follows Figure 26 As shown, the colonic tissue of mice in the control group was intact, with clear crypt structures, and strong AhR and Cyp1a1 fluorescence signals with uniform nuclear distribution. Compared with the control group, AhR and Cyp1a1 in the colonic tissue of mice in the IBD group were primarily localized in the cell membrane, with weak nuclear fluorescence signals showing a fragmented or discontinuous distribution. The fluorescence intensity was significantly reduced to only 49.45±6.81% and 59.53±9.84% of that in the control group (P<0.05), indicating that the AhR / Cyp1a1 signaling pathway was inhibited in the colon of model mice. In the 5-ASA group, AhR and Cyp1a1 expression recovered to 67.16±4.87% and 88.92±12.52% of that in the control group, respectively. The nuclear fluorescence intensity of AhR and Cyp1a1 in the L-Lk group increased to 79.04±4.88% and 90.64±12.76% of that in the Control group (P<0.05); the high-dose intervention effect was more significant, with AhR and Cyp1a1 mainly localized in the cell nucleus, with fluorescence intensities reaching 82.50±11.32% and 101.80±5.02% of that in the Control group, respectively (P<0.01), indicating that Kühm-like Lactobacillus 2503 can improve IBD symptoms in mice by activating the AhR / Cyp1a1 signaling pathway.

[0246] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A kumiss-like Lactobacillus, characterized in that The kumiss-like lactobacillus is kumiss-like lactobacillus ( Lactobacillus kefiranofaciens )2503, deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit number CGMCC NO.33828.

2. A kumiss-like Lactobacillus culture, characterized in that The culture is the kumiss-like Lactobacillus ( Lactobacillus kefiranofaciens )2503 is a fermentation product obtained by culturing in a microbial culture medium.

3. The kühwein-like Lactobacillus culture according to claim 2, characterized in that The fermentation product includes kumiss-like Lactobacillus ( Lactobacillus kefiranofaciens ) 2503 and Kühm-like Lactobacillus ( Lactobacillus kefiranofaciens ) metabolites of 2503.

4. A bacterial agent, characterized in that The bacterial agent contains the kumiss-like Lactobacillus ( Lactobacillus kefiranofaciens ) 2503 and / or the culture described in claim 2 or 3.

5. The kumiss-like Lactobacillus according to claim 1 ( Lactobacillus kefiranofaciens ) 2503. The kumiss-like Lactobacillus of claim 2 or 3 ( Lactobacillus kefiranofaciens ) Use of the culture 2503 or the bacterial agent according to claim 4 in the preparation of a medicament for preventing, treating or assisting in the treatment of inflammatory bowel disease, wherein the inflammatory bowel disease is ulcerative colitis.

6. The use according to claim 5, characterized in that The drug has at least one of the following functions: a1) slow down the patient’s weight loss; a2) Improve the patient’s stool characteristics and blood in stool; a3) Improve the patient's spleen index; a4) Slow down the shortening of the patient’s colon length; a5) Increase the expression levels of MUC2, ZO-1, and Claudin-1, and repair the intestinal barrier function of patients; a6) Increase the activity of antioxidant enzymes CAT, T-SOD, and GSH-Px, and reduce the patient's oxidative stress level; a7) scavenging free radicals; a8) Reduce the levels of pro-inflammatory factors IL-1β, TNF-α, and IL-6 in the patient's serum, and increase the level of the anti-inflammatory factor IL-10; a9) Increase the abundance of beneficial intestinal bacteria and reduce the abundance of pathogenic bacteria, and restore the diversity of the patient's intestinal flora and metabolite levels.

7. The use according to claim 6, characterized in that The method of increasing the abundance of beneficial intestinal bacteria and reducing the abundance of pathogenic bacteria includes: a9.1) At the phylum level, add Bacteroidetes ( Bacteroidetes ) abundance, and reduced the abundance of Firmicutes ( Firmicutes ) abundance, reversing the abnormal F / B ratio; a9.2) At the family level, add Rhabditiaceae ( Muribaculaceae ), Prevotellaceae ( Prevotellaceae )、Lactobacillus family( Lactobacillaceae ) abundance, and reduced Staphylococcus family ( Staphylococcaceae ) abundance; a9.3) At the genus level, add Rhabditis spp. Muribaculum )and unclassified_Clostridia_ UCG_014 abundance, and reduced Staphylococcus spp. ( Staphylococcus ) 、 Odorobacter spp. Odoribacter ) 、 Mammalian cocci ( Mammaliicoccus ) abundance; a9.4) Enrichment of Lachnospiraceae ( Lachnospiraceae), Paraprevotella spp. Paraprevotella), Clostridium Clostridium_sp_ASF356, Kühm-like Lactobacillus ( Lactobacillus kefiranofacien) ; Restoring the patient's intestinal flora metabolite level includes: a9.5) Restoring intestinal short-chain fatty acid levels, including acetate, propionate, isobutyrate, butyrate, and isovaleric acid; a9.6) Restoring gut microbial indole metabolite levels, including indole-3-carboxaldehyde, indole-3-acetic acid, indole-3-propionic acid, indole-3-acrylic acid, indole, and tryptamine.

8. The kumiss-like Lactobacillus according to claim 1 ( Lactobacillus kefiranofaciens ) 2503. The kumiss-like Lactobacillus of claim 2 or 3 ( Lactobacillus kefiranofaciens ) Use of the culture 2503 or the bacterial agent described in claim 4 in the preparation of an antioxidant or free radical scavenger, wherein the antioxidant or free radical scavenger is a non-pharmaceutical product, not suitable for human or animal treatment, and is suitable for other non-medical purposes.

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