Lactobacillus kefiranofaciens and application thereof

Abstract

The invention discloses lactobacillus kefiranofaciens and application thereof, and belongs to the technical field of microorganisms. The strain number of the lactobacillus kefiranofaciens is 2503, the lactobacillus kefiranofaciens is preserved in the general microbiological center of the China Committee for Culture Collection of Microorganisms, and the preservation number of the lactobacillus kefiranofaciens is CGMCC NO.33828. The lactobacillus kefiranofaciens is named as Lactobacillus kefiranofaciens. The lactobacillus kefiranofaciens provided by the invention is strong in acid resistance and bile salt resistance, has the functions of resisting oxidation and scavenging free radicals, can be planted in intestinal tracts, improves the abundance of beneficial bacteria in the intestinal tracts and reduces the abundance of pathogenic bacteria, and has good prevention and treatment effects on inflammatory bowel diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to a Lactobacillus kefiranofaciens strain and its application. Background Art

[0002] Inflammatory bowel disease (IBD) is an immune-related disease characterized by chronic intestinal inflammation, mainly including Crohn's disease (CD) and ulcerative colitis (UC). The number of patients globally has exceeded 8 million, and the incidence rate is showing an increasing trend year by year, especially in developing countries. The pathogenesis of IBD is complex, involving multiple factors such as genetic susceptibility, intestinal flora dysbiosis, abnormal activation of the immune system, and damage to the intestinal barrier function.

[0003] Currently, the treatment of IBD mainly relies on drug therapy, including aminosalicylate drugs, corticosteroids, immunosuppressants, and biological agents, etc. Traditional drugs such as aminosalicylates (such as mesalazine) have limited effects on moderate to severe patients and are only suitable for mild cases; glucocorticoids (such as prednisone) can quickly control inflammation, but long-term use is likely to cause osteoporosis, infection, and metabolic disorders; immunosuppressants (such as azathioprine) may cause liver and kidney toxicity and bone marrow suppression. Biological agents such as anti-TNF-α monoclonal antibodies (such as adalimumab) and anti-integrin monoclonal antibodies (such as vedolizumab) can target and inhibit inflammation, but about 30% of patients have primary non-response, 50% of patients develop secondary failure over time, and they need to be administered by frequent injections, with high costs. In addition, long-term use may increase the risks of infection and tumors. Secondly, surgical treatment such as intestinal resection or ostomy still plays an important role in some patients with IBD. Although it can relieve acute symptoms, the postoperative recurrence rate is as high as 50%, and it is prone to pouchitis or flora dysbiosis, leading to new inflammation or even canceration. Therefore, it is necessary to provide new directions and more effective treatment measures for the treatment of IBD.

[0004] Probiotics are a class of active microorganisms beneficial to human health, such as Lactobacillus, Bifidobacterium, Lactococcus, Streptococcus, Bacillus, etc. Probiotics are widely used in the adjuvant treatment of diseases due to their good curative effects and high safety. Research shows that probiotics can inhibit the colonization of harmful bacteria and promote the colonization of beneficial bacteria, optimize the composition of the microbiota, thereby inducing an anti-inflammatory response and improving the intestinal barrier function.

[0005] Although there are precedents of using probiotics to relieve IBD-related clinical symptoms, the existing probiotics have the following barriers in the treatment of IBD: 1. Conventional probiotics (such as Lactobacillus and Bifidobacterium) have low survival rates in gastric acid (pH 2.0 - 3.0) and bile salt environments, resulting in insufficient viable bacteria reaching the intestine and poor treatment effects; 2. The intestinal barrier of IBD patients is damaged, leading to LPS translocation and bacterial invasion, exacerbating the inflammatory response. The existing probiotics are insufficient in repairing the intestinal barrier function of IBD patients, resulting in the continuous deterioration of inflammation; 3. The existing probiotic therapy lacks effective antioxidant intervention, making oxidative stress exacerbate the pathological process of IBD; 4. IBD patients are usually in immune imbalance (dysregulation of the pro-inflammatory / anti-inflammatory cytokine ratio), and the existing technology lacks precise regulation. Summary of the Invention

[0006] To improve the defects of the existing technology, the present invention provides a strain of Lactobacillus kefiranofaciens, and the strain has the following characteristics: 1. It has strong acid and bile salt tolerance, and can ensure that a sufficient amount of viable bacteria reach the intestine for colonization and play a therapeutic role; 2. It has strong antioxidant ability and can significantly relieve the damage of oxidative stress to intestinal epithelial cells; 3. It has the functions of repairing intestinal barrier and reconstructing immune balance; 4. It regulates the intestinal flora and restores the flora diversity; 5. It has a significant effect on alleviating weight loss, colon shortening and pathological damage in the DSS-induced IBD mouse model. In view of this, the present invention provides the application of the Lactobacillus kefiranofaciens in the preparation of drugs for treating IBD, the preparation of immunomodulators, and the preparation of intestinal microbiota regulators.

[0007] The present invention includes the following technical solutions: In the first aspect of the present invention, the present invention provides a strain of Lactobacillus kefiranofaciens ( Lactobacillus kefiranofaciens ), with the strain number 2503, deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, and the deposit number is CGMCC NO.33828. Hereinafter, it is simply referred to as Lactobacillus kefiranofaciens ( Lactobacillus kefiranofaciens ) 2503.

[0008] The 16S rRNA sequence of the Lactobacillus kefiranofaciens ( Lactobacillus kefiranofaciens ) 2503 is as shown in SEQ ID NO.1.

[0009] The Lactobacillus kefiranofaciens ( Lactobacillus kefiranofaciens ) 2503 of the present invention shows purple in Gram staining, the bacterial cells are long rod-shaped, about 5 - 30 μm in length and about 1 μm in width.

[0010] In the second aspect of the present invention, the present invention provides a culture of Lactobacillus kefiranofaciens, and the culture is obtained by culturing Lactobacillus kefiranofaciens ( Lactobacillus kefiranofaciensThe fermentation product obtained by culturing in a microbial culture medium.

[0011] The culture refers to the general term for the liquid or solid products (all substances in the culture vessel, i.e., the fermentation product) with a microbial population after artificial inoculation and culture, that is, the product 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 the subculture obtained by subculturing microorganisms, which can be a culture of a certain generation or a mixture of several generations.

[0012] In the present invention, the microbial culture medium used for culturing Lactobacillus kefiranofaciens Lactobacillus kefiranofaciens ) 2503 is a solid or liquid culture medium routinely prepared by those skilled in the art, and the present invention does not limit the components of the microbial culture medium.

[0013] The fermentation product includes Lactobacillus kefiranofaciens Lactobacillus kefiranofaciens )2503 and the metabolites of Lactobacillus kefiranofaciens Lactobacillus kefiranofaciens )2503.

[0014] In the third aspect of the present invention, the present invention provides a bacterial agent, which contains Lactobacillus kefiranofaciens Lactobacillus kefiranofaciens )2503 and / or the metabolites of Lactobacillus kefiranofaciens Lactobacillus kefiranofaciens )2503 and / or the culture described in the second aspect of the present invention.

[0015] The active ingredient in the bacterial agent is Lactobacillus kefiranofaciens Lactobacillus kefiranofaciens )2503 and / or its metabolites, or the culture of Lactobacillus kefiranofaciens Lactobacillus kefiranofaciens )2503. The content of the active ingredient in the bacterial agent is 1.0×10 8 -1.0×10 10 CFU / g. In a specific embodiment of the present invention, the content of the active ingredient in the bacterial agent is 1.0×10 9 CFU / g.

[0016] In a preferred embodiment of the present invention, the bacterial agent further includes an acceptable carrier, and the carrier includes 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 powder, which are commonly used in the art and have a protective effect on live bacteria.

[0017] In some embodiments of the present invention, the dosage form of the bacterial agent is selected from liquid preparation, emulsion, suspension, powder, granule, wettable powder, or water dispersible granule.

[0018] In the fourth aspect of the present invention, the present invention provides an application of Lactobacillus kefiranofaciens ( Lactobacillus kefiranofaciens ) 2503 described in the first aspect of the present invention, a culture of Lactobacillus kefiranofaciens ( Lactobacillus kefiranofaciens ) 2503 described in the second aspect of the present invention, 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 adjuvantly treating inflammatory bowel disease.

[0019] The inflammatory bowel disease includes Crohn's disease and ulcerative colitis.

[0020] The drug has at least one of the following functions: a1) Slowing down the weight loss of the patient; a2) Improving the fecal characteristics and bloody stools of the patient; a3) Improving the spleen index of the patient; a4) Slowing down the shortening of the patient's colon length; a5) Increasing the expression levels of MUC2, ZO-1 and Claudin-1 and repairing the intestinal barrier function of the patient; a6) Increasing the activities of antioxidant enzymes CAT, T-SOD, and GSH-Px and reducing the oxidative stress level of the patient; a7) Scavenging free radicals; a8) Reducing the levels of serum pro-inflammatory factors IL-1β, TNF-α and IL-6 in the patient and increasing the level of anti-inflammatory factor IL-10; a9) Increasing the abundance of intestinal beneficial bacteria and reducing the abundance of pathogenic bacteria, and restoring the intestinal flora diversity and metabolite levels of the patient.

[0021] In a specific example of the present invention, the increasing the abundance of intestinal beneficial bacteria and reducing the abundance of pathogenic bacteria includes: a9.1) At the phylum level, increasing the abundance of Bacteroidetes ( Bacteroidetes ) and reducing the abundance of Firmicutes ( Firmicutes ), and reversing the abnormal F / B ratio; a9.2) At the family level, increasing the abundances of Muribaculaceae ( Muribaculaceae ), Prevotellaceae ( Prevotellaceae ), and Lactobacillaceae ( Lactobacillaceae ), and reducing the abundance of Staphylococcaceae ( Staphylococcaceae ); a9.3) At the genus level, increasing the abundances of Muribaculum ( Muribaculum ) and unclassified_ Clostridia_ UCG_014 and reducing the abundances of Staphylococcus ( Staphylococcus ) 、 Odoribacter ( Odoribacter ) 、Abundance of Mammaliicoccus Mammaliicoccus ); a9.4) Enrichment of Lachnospiraceae Lachnospiraceae)、 Paraprevotella Paraprevotella)、 Clostridium Clostridium_sp_ASF356、 Lactobacillus kefiranofaciens Lactobacillus kefiranofacien) .

[0022] In a specific example of the present invention, the restoration of the metabolite levels of the patient's intestinal flora includes: a9.5) Restoring the levels of intestinal short-chain fatty acids (SCFAs), which include acetic acid, propionic acid, isobutyric acid, butyric acid, and isovaleric acid; a9.6) Restoring the levels of intestinal microbial indole metabolites, which include indole-3-carboxaldehyde, indole-3-acetic acid, indole-3-propionic acid, indole-3-acrylic acid, indole, and tryptamine.

[0023] In a fifth aspect of the present invention, the present invention provides an application of Lactobacillus kefiranofaciens Lactobacillus kefiranofaciens 2503 described in the first aspect of the present invention, the culture of Lactobacillus kefiranofaciens Lactobacillus kefiranofaciens 2503 described in the second aspect of the present invention, or the bacterial agent described in the third aspect of the present invention in the preparation of an antioxidant or free radical scavenger.

[0024] In a specific embodiment of the present invention, the free radicals include DPPH, ABTS, and hydroxyl radicals.

[0025] In a sixth aspect of the present invention, the present invention provides an application of Lactobacillus kefiranofaciens Lactobacillus kefiranofaciens 2503 described in the first aspect of the present invention, the culture of Lactobacillus kefiranofaciens Lactobacillus kefiranofaciens 2503 described in the second aspect of the present invention, or the bacterial agent described in the third aspect of the present invention in the preparation of an intestinal microflora regulator.

[0026] The intestinal microflora regulator has at least one of the following functions: b1) Increasing the abundance of beneficial intestinal bacteria; b2) Decreasing the abundance of pathogenic bacteria; b3) Regulating the diversity of the intestinal flora; b4) Regulating the levels of intestinal microbial metabolites.

[0027] In a seventh aspect of the present invention, the present invention provides an application of Lactobacillus kefiranofaciens Lactobacillus kefiranofaciens 2503 described in the first aspect of the present invention, the culture of Lactobacillus kefiranofaciens Lactobacillus kefiranofaciens 2503 described in the second aspect of the present invention, or the bacterial agent described in the third aspect of the present invention in the preparation of an immunomodulator.

[0028] The immunomodulator has at least one of the following functions: c1) Reducing the levels of pro-inflammatory factors IL-1β, TNF-α, and IL-6; c2) Increasing the level of anti-inflammatory factor IL-10.

[0029] The Lactobacillus kefiranofaciens 2503 provided by the present invention has the following obvious advantages: 1. The Lactobacillus kefiranofaciens 2503 provided by the present invention has excellent acid tolerance (pH 2.5 - 3.0) and bile salt tolerance (0.3%), ensuring sufficient viable bacteria to colonize the intestine and perform functions.

[0030] 2. The Lactobacillus kefiranofaciens 2503 provided by the present invention can significantly up-regulate the expression of ZO-1, Claudin-1, and MUC2, enhance the intestinal barrier function, reduce LPS translocation, and thus block the "intestinal leakage - inflammation" vicious cycle.

[0031] 3. The cell contents of the Lactobacillus kefiranofaciens 2503 provided by the present invention have strong free radical scavenging abilities (DPPH, ABTS, hydroxyl radicals), and can enhance the activities of T-SOD, CAT, and GSH-Px, and reduce the levels of MPO and MDA, effectively alleviating oxidative stress.

[0032] 4. The Lactobacillus kefiranofaciens 2503 provided by the present invention can significantly inhibit IL-1β, TNF-α, and IL-6, and promote the secretion of IL-10, achieving multi-target immune regulation.

[0033] 5. The Lactobacillus kefiranofaciens 2503 provided by the present invention can increase the abundances of beneficial bacteria such as Muribaculaceae , Lachnospiraceae etc., reduce the abundances of pathogenic bacteria such as Staphylococcus etc., promote the production of SCFA and tryptophan indole metabolites, activate the AhR / Cyp1a1 pathway, enhance the barrier function and regulate immunity.

[0034] 6. The Lactobacillus kefiranofaciens 2503 provided by the present invention is a natural strain without genetic modification, has high safety, can replace or be combined with existing drugs, and reduce the treatment cost and risk.

[0035] Deposit description Strain classification and naming: Lactobacillus kefiranofaciens Latin name of the strain: Lactobacillus kefiranofaciens Accession number registered in the preservation center: CGMCC NO.33828 Preservation institution: China General Microbiological Culture Collection Center Abbreviation of the preservation institution: CGMCC Address of the depositary institution: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing Date of deposit: March 17, 2025 Description of the drawings

[0036] Figure 1 It is the morphological diagram of Lactobacillus kefiranofaciens subsp. kefiranofaciens; a: colony; b&c: cell morphology; Figure 2 It is the test result diagram of the acid tolerance ability of Lactobacillus kefiranofaciens subsp. kefiranofaciens; Figure 3 It is the test result diagram of the bile salt tolerance ability of Lactobacillus kefiranofaciens subsp. kefiranofaciens; Figure 4 It is the test result diagram of the scavenging ability of Lactobacillus kefiranofaciens subsp. kefiranofaciens to free radicals; Figure 5 It is the progress diagram of the animal experiment of Lactobacillus kefiranofaciens subsp. kefiranofaciens; Figure 6 It is the effect of Lactobacillus kefiranofaciens subsp. kefiranofaciens on the body weight of mice; Figure 7 It is the effect of Lactobacillus kefiranofaciens subsp. kefiranofaciens on DAI; Figure 8 It is the effect of Lactobacillus kefiranofaciens subsp. kefiranofaciens on the colon length; Figure 9 It is the H&E staining of colon tissue (upper layer: 40×, lower layer: 100×); Figure 10 It is the pathological score of colon tissue; Figure 11 It is the effect of Lactobacillus kefiranofaciens subsp. kefiranofaciens on the expression of genes related to the intestinal barrier function of mice; Figure 12 It is the effect of Lactobacillus kefiranofaciens subsp. kefiranofaciens on the expression of proteins related to the intestinal barrier function of mice; Figure 13 It is the effect of Lactobacillus kefiranofaciens subsp. kefiranofaciens on the oxidative stress indexes in the homogenate of mouse colon tissue; Figure 14 It is the effect of Lactobacillus kefiranofaciens subsp. kefiranofaciens on the serum cytokines of mice; Figure 15 It is a Venn diagram; Figure 16 It is Alpha diversity; Figure 17 It is a PCoA analysis diagram; Figure 18 It is the species composition at the phylum level; Figure 19 It is the species composition at the family level; Figure 20 It is the species composition at the genus level; Figure 21 It is the phylogenetic cladogram of each group based on LEfSe analysis; Figure 22 It is a bar chart for LDA analysis; Figure 23 It is the influence on the content of SCFAs; Figure 24 It is tryptophan-targeted metabolomics; Figure 25 It is the content of indole metabolites derived from gut microbiota; Figure 26 It is the activation status of the AhR / Cyp1a1 signaling pathway. Specific implementation manners

[0037] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0038] The First Part: Experimental Materials and Methods 1.1 Experimental Materials 1.1.1 Test Strains Lactobacillus kefiranofaciens subsp. kefiranofaciens 2503 was isolated from Russian kefir grains, belonging to the family Lactobacillaceae and the genus Lactobacillus, and is currently preserved in the China General Microbiological Culture Collection Center (CGMCC NO. 33828).

[0039] 1.1.2 Culture Media Glucose 20.0 g, yeast extract powder 45.0 g, dipotassium hydrogen 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, made up to 1,000 mL with distilled water, adjusted to pH 6.2, autoclaved at 115 °C for 20 min.

[0040] 1.1.3 Experimental Mice In this experiment, 50 SPF-grade male Balb / C mice were used, purchased from Beijing Sibeifu Co., Ltd. The animals were housed in the barrier system animal experiment center of the College of Food Science and Engineering, Tianjin University of Science and Technology. The indoor conditions of the experiment center were: relative humidity: 55 ± 5%, 23 ± 1 °C, 12 h light-dark cycle, and the animals were allowed to freely feed and drink water. This experiment was approved by the Animal Protection and Use Committee of Tianjin University of Science and Technology (approval number: SCXK (Jing) 2024-0001), and the welfare ethics number was 2024030, and the animals were raised and managed in accordance with the "Guide for the Care and Use of Laboratory Animals".

[0041] 1.2 Experimental Methods 1.2.1 Isolation of Strains Under aseptic conditions, inoculate a sample of Russian kefir grains (about 5 g) into raw cow's milk, activate anaerobically at 37 °C for 72 h, and dilute serially to 10 -3 ~10 -6 concentration. Spread the plates until the modified MRS solid medium, and culture under anaerobic conditions (85% N2, 10% CO2, 5% H2) at 37 °C for 48 - 72 hours. Screen the target strains by colony morphology (raised colonies with a diameter of 1 - 2 mm, milky white, and neat edges). After purification by the three-zone streaking method, the typical characteristics of the finally obtained Lactobacillus kefiranofaciens include: Gram-positive rod-shaped, catalase-negative, and able to ferment lactose to produce acid.

[0042] 1.2.2 Strain morphological observation (1)Gram staining and observation Take a small amount of bacterial liquid and spread it evenly on a clean glass slide, and fix it by gently heating with a flame. Drop crystal violet for staining for 1 min, rinse with distilled water to remove excess stain; drop iodine solution for 1 min, rinse with distilled water; drop 95% ethanol for decolorization for 10 - 30 s; drop safranin for staining for 1 min, rinse with distilled water to remove excess stain; slightly heat and dry. Observe the staining results under an optical microscope (100×).

[0043] (2)Scanning electron microscopy observation Take 1 mL of bacterial liquid, centrifuge at 8000 r / min for 1 min, and collect the bacterial cell precipitate. Resuspend the bacterial cells with 0.1 M phosphate buffer and wash repeatedly 3 times to remove residual medium. Resuspend the washed bacterial cells in 2.5% glutaraldehyde solution and fix at 4 °C for 12 h. After fixation, wash 3 times with PBS to remove excess fixative. Immerse the fixed sample successively in ethanol solutions with gradients of 30%, 50%, 70%, 80%, and 90% for dehydration. After dehydration, wash the bacterial cells twice with absolute 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 bacterial cells under an accelerating voltage of 5 - 15 kV.

[0044] 1.2.3 Acid tolerance determination Use 5 mol / L hydrochloric acid to adjust the pH of MRS medium to 2, 3, and 4, sterilize and cool for later use. Inoculate the activated Lactobacillus kefiranofaciens 2503 at an inoculation amount of 4% into different media, sample and count at 0 h, 3 h, and 6 h respectively, and calculate the strain survival rate according to the formula.

[0045] Survival rate (%) = total viable bacteria after incubation / total viable bacteria before incubation × 100 Equation (1 - 1) 1.2.4 Determination of Bile Salt Tolerance Based on MRS medium, add bile salts with mass fractions of 0.1%, 0.2%, and 0.3% respectively. After sterilization and cooling, inoculate the activated Lactobacillus kefiranofaciens 2503 into different media at an inoculation amount of 4%, sample and count at 0 h, 3 h, and 6 h respectively, and calculate the strain survival rate according to formula (1-1).

[0046] 1.2.5 Determination of Free Radical Scavenging Ability (1)Sample Preparation Prepare two kinds of samples, namely the supernatant of the fermentation broth and the cell lysate extract in advance. Preparation of the supernatant of the fermentation broth: Centrifuge the cultured fermentation broth at 4°C and 8000 r / min for 10 min to obtain the supernatant of the fermentation broth for standby. Cell lysate extract: Centrifuge the cultured fermentation broth at 8000 r / min for 10 min, retain the cell precipitate, then wash it 3 times with PBS, resuspend the cells in PBS solution, and use an ultrasonic crusher to break the cells under ice bath conditions (power 400 W, total time 10 min, working for 5 s, intermittent for 5 s). Centrifuge the broken turbid liquid to collect the supernatant for standby.

[0047] (2)Determination of DPPH Free Radical Scavenging Ability Mix the samples with an equal volume of 0.2 mmol / L DPPH-absolute ethanol solution as the measurement tubes. Replace the DPPH solution in the interference tubes with absolute ethanol, and the blank tubes are prepared by mixing the DPPH solution with absolute ethanol. React in the dark for 30 min and measure the absorbance of the supernatant at 517 nm. The reaction system and calculation formula for the DPPH free radical scavenging rate are as follows: Table 1 Reaction System for Scavenging DPPH Free Radicals 。

[0048] Formula (1-2) Where: A0: OD value of the blank tube; A1: OD value of the sample tube; A2: OD value of the interference tube.

[0049] (3)Determination of ABTS Free Radical Scavenging Ability Mix the samples to be tested with an equal volume of ABTS + working solution as the measurement tubes. Replace the ABTS + working solution in the interference tubes with distilled water, and the blank tubes are prepared by mixing the ABTS + working solution with distilled water. React in the dark for 20 min and measure the absorbance of the supernatant at 734 nm. The reaction system and calculation formula for the ABTS + free radical scavenging rate are as follows: Table 2 Reaction system for scavenging ABTS free radicals 。

[0050] Formula (1-3) Where: A0: OD value of the blank tube; A1: OD value of the sample tube; A2: OD value of the interference tube.

[0051] (4) Determination of hydroxyl radical scavenging ability Respectively 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 the sample, and finally add 0.5 mL of H2O2. After standing and reacting at 37 °C for 60 min, measure the absorbance at 536 nm using an ultraviolet spectrophotometer. The reaction system and calculation formula for the hydroxyl radical scavenging rate are as follows: Table 3 Reaction system for scavenging hydroxyl radicals 。

[0052] Formula (1-4) Where: A0: OD value of the blank tube; A1: OD value of the control tube; A2: OD value of the sample tube.

[0053] 1.2.6 Preparation of bacterial powder After centrifuging the fermentation broth, wash it twice with physiological saline, then add 10% skim milk powder according to the ratio of protective agent: bacterial sludge (w / v) of 3:1. The freeze-drying thickness is 1 cm, pre-freeze at -80 °C for 2 h, and then freeze-dry the sample for 24 h. The activity of the obtained bacterial powder is 1.0×10 9 CFU / g.

[0054] 1.2.7 Animal experiment design After 50 male SPF-grade Balb / C mice were adaptively fed for one week, they 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 9CFU / kg). Preventive treatment interventions were carried out from day 0 to day 7 of the experiment: mice in the L-Lk and H-Lk groups were gavaged with 0.2 mL of bacterial suspension every day, mice in the 5-ASA group were gavaged with an equal volume of 5-aminosalicylic acid, and mice in the Control and IBD groups were gavaged with an equal volume of normal saline. From day 8 to day 14 of the experiment, except for the Control group, inflammatory bowel disease models were induced in the remaining groups by freely drinking a 3.0% dextran sulfate sodium (DSS) aqueous solution. The L-Lk, H-Lk, and 5-ASA groups maintained the original protocol interventions, and the Control and IBD groups continued to be gavaged with an equal volume of normal saline. The experimental procedure is as Figure 5 shown.

[0055] 1.2.8 Disease activity index scoring During the experiment, the body weight and fecal status of the mice were recorded, and the occult blood in the feces of the mice was measured using an occult blood detection kit for feces to perform disease activity index scoring. The scoring details are shown in the table. The DAI index is the average value of the total scores of the percentage of body weight loss, fecal status, and degree of blood in the stool.

[0056] Table 4 Disease activity index scoring criteria .

[0057] 1.2.9 Sample collection and preparation The experimental mice were fasted and water-deprived 24 h before sacrifice. Blood samples were obtained by orbital blood collection. The samples were allowed to stand at 4 °C for 30 min, centrifuged (3500 r / min for 15 min), and the supernatant was taken and stored at -80 °C in aliquots after subpackaging. After blood collection, the internal organs of the mice were removed, rinsed thoroughly with normal saline, and blotted dry with filter paper and weighed. The entire colon was removed, its length was measured and photographed. One part of the colon was fixed in 4% paraformaldehyde for histological analysis, and the other part was stored at -80 °C for later use.

[0058] 1.2.10 Organ index The tissues were weighed after pretreatment and calculated according to the following formula: Equation (1-5) Where: m1: organ weight, g; m2: mouse body weight, g.

[0059] 1.2.11 Colon H&E staining and pathological scoring The fixed colon tissues were rinsed with PBS solution and dehydrated successively with 80%, 90%, and 100% ethanol. After being cleared with xylene and embedded in paraffin, the tissues were cut into thin slices about 5 μm thick. The sections were placed in xylene to remove paraffin and hydrated step by step through gradient ethanol. They were stained with hematoxylin and eosin dyes (H&E) respectively, and the excess dyes were washed off. After mounting the slides, the staining effects were observed under a microscope, and the tissue morphology and pathological changes were evaluated according to the histological scoring criteria.

[0060] Table 5 Histopathological Scoring Criteria for Colon Tissues 。

[0061] 1.2.12 Immunofluorescence Staining of Colon The fixed colon tissues were sent to Wuhan Sevier Biotechnology Co., Ltd. for immunofluorescence staining. Images were observed and collected using a fluorescence microscope, and the expression levels of MUC2, ZO-1, Claudin-1, AhR, and Cyp1a1 were analyzed.

[0062] 1.2.13 Determination of Oxidative Stress Indexes in Colon The colon tissues were prepared into 10% homogenates, diluted according to the kit requirements, and the contents of SOD, CAT, GSH-Px, MDA, and MPO in the colon tissues were determined.

[0063] 1.2.14 Detection of Serum Factors An ELISA kit was used to detect the contents of LPS, IL-1β, TNF-α, IL-6, and IL-10 in the serum of mice.

[0064] 1.2.15 RT-qPCR Quantitative Analysis of Gene Expression 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.

[0065] Table 6 Reverse Transcription Reaction System 。

[0066] The primer sequences of the target genes required were queried in the NCBI database, as shown in Table 7, and were synthesized by Suzhou Genewiz Biotechnology Co., Ltd.

[0067] Table 7 Primer Sequences 。

[0068] The reverse transcription reaction system was prepared at low temperature. After mixing, ensure that all systems sank to the bottom of the tube. The reaction system is shown in Table 8.

[0069] Table 8 RT-qPCR Reaction System 。

[0070] Set the amplification program according to Table 9. After the reaction is completed, analyze the melting curve and record the Ct value.

[0071] Table 9 Real Time PCR reaction program 。

[0072] Using the GAPDH gene as an internal reference, calculate the relative expression level of the target gene mRNA by the 2 -△△Ct method.

[0073] 1.2.16 Changes in the intestinal flora of mice by high-throughput sequencing Collect the feces of mice in each group, store them at low temperature and send them to Beijing Biomarker Biotechnology Co., Ltd. for 16S rDNA sequencing, and use the online cloud platform to analyze the diversity of the intestinal flora and the differences in species composition of mice in each group.

[0074] 1.2.17 Determination of fecal short-chain fatty acids Accurately weigh 0.1 g of feces, resuspend it with 400 μL of saturated NaCl solution, add 20 μL of 10% H2SO4 for acidification, vortex for 2 min, and mix well until there are no particulate matters of mouse feces. Let it stand for 20 min. Add 500 μL of ether, shake well and let it stand for 10 min, then centrifuge (13,000 r / min, 15 min) and take the upper ether phase. Add 0.20 g of anhydrous Na2SO4 to the ether phase for dehydration, let it stand at 4℃ for 10 min, then centrifuge (13,000 r / min, 15 min), and take the upper ether phase through a 0.22 μm organic phase filter membrane. Analyze the content of short-chain fatty acids in the feces of mice in each group by gas chromatography, with the retention times of chromatographic-grade acetic acid, propionic acid, isobutyric acid, butyric acid, and isovaleric acid in the gas chromatography column as the reference.

[0075] 1.2.18 Determination of changes in fecal tryptophan metabolites Collect the feces of mice in each group, store them at low temperature and send them to Shanghai Majorbio Bio-pharm Technology Co., Ltd. for targeted metabolomics detection, and use the online cloud platform to analyze the changes in tryptophan metabolism of intestinal microorganisms in the blank group, model group, and high-dose probiotic group.

[0076] 1.2.19 Data statistics and analysis All data are expressed as mean ± standard deviation; one-way ANOVA and T-tests were performed on multiple groups of data to evaluate statistical significance at 95% and 99% confidence intervals; independent sample T-tests were used for comparisons between two groups. Graphpad Prism 8.0 software was used to analyze the data and plot the graphs. # indicates compared with the blank group, * indicates compared with the model group, * / # indicates (P<0.05), ** / ## indicates (P<0.01), *** / indicates (P<0.001).

[0077] Part II Experimental Results 2.1 Strain Morphological Observation As Figure 1 shown, Lactobacillus kefiranofaciens formed milky white colonies on the surface of the modified MRS solid medium with irregular edges; Gram staining was purple, and rod-shaped bacteria could be observed under an optical microscope; the individual morphology of the bacteria was observed using a scanning electron microscope, with lengths ranging from about 5 - 30 μm and widths of about 1 μm.

[0078] 2.2 Acid Tolerance Test The results are as Figure 2 shown. As the pH of the growth environment decreased and the incubation time extended for Lactobacillus kefiranofaciens, the strain survival rate gradually decreased. The highest survival rate of the strain was 63.84 ± 22.73% under the condition of incubating at pH 4 for 3 h; under the condition of incubating at pH = 2 for 6 h, the survival rate was only 7.87 ± 1.74% at the lowest.

[0079] 2.3 Bile Salt Tolerance Test The bile salt tolerance results are as Figure 3 shown. The highest survival rate of the strain was 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 strain survival rate was low, and the survival rate of the strain was almost 0 after incubating for 6 h.

[0080] 2.4 Free Radical Scavenging Ability Test The results are as Figure 4As shown, the scavenging abilities of the supernatant of Lactobacillus kefiranofaciens 2503 fermentation broth against DPPH, ABTS, and hydroxyl radicals reached 66.22 ± 5.16%, 29.32 ± 2.20%, and 29.12 ± 5.96%, respectively; the scavenging abilities of the cell contents against DPPH, ABTS, and hydroxyl radicals reached 70.30 ± 3.13%, 52.02 ± 5.59%, and 46.44 ± 10.14%, respectively. The free radical scavenging ability of the cell contents was better than that of the fermentation broth supernatant. The reason might be that the key antioxidant components synthesized by the bacteria (such as SOD, GSH-Px, CAT, etc.) were mainly stored inside the cells and needed to be released after cell disruption. Therefore, the ability to scavenge free radicals and antioxidant activity were stronger. In summary, Lactobacillus kefiranofaciens 2503 had good in vitro free radical scavenging activity and showed potential application value in alleviating oxidative stress and protecting the intestine from free radical damage.

[0081] 2.5 Effects of preventive and therapeutic treatment on IBD mice 2.5.1 Effects on mouse body weight By detecting the changes in mouse body weight, the effects of different intervention strategies on the IBD process can be reflected. As Figure 6 shown, during the entire experiment, the physiological status of the mice in the blank group (Control) was stable, and their body weight increased steadily, with a 6.09 ± 2.92% increase at the end of the experiment. The mice in the model group (IBD) showed a decrease in body weight from the second day of modeling, accompanied by symptoms of reduced activity. At the end of the experiment, the body weight decreased by 6.0 ± 2.32%, preliminarily indicating the successful establishment of the IBD model. The body weight of the mice in the positive drug group (5-ASA) fluctuated less, with a 0.85 ± 3.94% increase at the end of the experiment. The downward trend of body weight in the low-dose Lactobacillus kefiranofaciens group (L-Lk) was alleviated compared to the IBD group. The body weight tended to be stable from the fourth day of modeling, and the weight loss rate at the end of the experiment was approximately 2.19 ± 3.35%. After intervention with high-dose Lactobacillus kefiranofaciens (H-Lk), a better protective effect was shown. The body weight of the mice during modeling remained basically unchanged. The protective effect was equivalent to that of 5-ASA and was significantly better than that of the L-Lk group. Analyzing the comprehensive results of body weight changes, Lactobacillus kefiranofaciens 2503 could effectively improve the body weight loss induced by DSS, and the intervention effect showed a dose-dependent relationship.

[0082] 2.5.2 Effects on mouse DAI As Figure 7As shown, the DAI of the mice in the Control group remained stable throughout the experiment, almost zero. The DAI of the mice in the IBD group began to increase slowly from the first day of modeling; on the 4th day of DSS induction, symptoms such as unformed feces with blood streaks were visible; on the 5th day, the body weight began to drop sharply, the feces were watery stools, and there were residues of feces and blood around the anus of most mice. At the end of the experiment, the DAI reached a peak of 2.83 ± 0.39, showing a significant difference from the Control group, indicating that the IBD model was successfully established. The DAI of the mice in the 5-ASA group began to rise slowly on the 5th day of modeling and was 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 upward trend of DAI in the L-Lk group of mice was alleviated compared with that of the IBD group, and the final DAI score was 1.49 ± 0.42, significantly better than that of the IBD group, but the effect was weaker than that of the 5-ASA group. The DAI of the mice in the H-Lk group remained stable in the first 5 days of DSS treatment and began to rise slowly on the 6th day of DSS induction. The DAI at the end of the experiment was 1.10 ± 0.47, showing a preventive effect comparable to that of 5-ASA. Based on the comprehensive results of DAI changes, the intervention of Lactobacillus kefiranofaciens 2503 can significantly delay the progression of DSS-induced IBD.

[0083] Lactobacillus kefiranofaciens JK-24 disclosed in the prior art CN119752669 A still showed blood in the stool in mice under the intervention of a super-high dose of 10 10 CFU / kg (see the attached instructions of the prior art Figure 3 ), and the DAI score was about 2.5 (see the attached instructions of the prior art Figure 4 ), while Lactobacillus kefiranofaciens 2503 provided by the present invention can achieve a DAI of 1.10 ± 0.47 at a dose of 10 9 CFU / kg, with basically no blood in the stool. It shows that Lactobacillus kefiranofaciens provided by the present invention has significant advantages over the prior art in improving blood in the stool, fecal characteristics, and body weight.

[0084] 2.5.3 Effects on the organ indices of mice As shown in Table 10, the spleen index of the mice in the DSS-induced IBD model group increased significantly to 4.12 ± 0.58‰, indicating that a relatively serious inflammatory reaction occurred in the bodies of the mice in the IBD group. After intervention with Lactobacillus kefiranofaciens 2503, the spleen indices of the L-Lk and H-Lk groups decreased to 3.40 ± 0.61‰ and 3.74 ± 0.59‰ respectively, which were comparable to those of the 5-ASA group, indicating that Lactobacillus kefiranofaciens 2503 can effectively relieve the systemic immune abnormal activation caused by DSS induction. In addition, there were no significant differences in the heart, liver, lung, and kidney indices of the mice in each group, suggesting the safety of Lactobacillus kefiranofaciens 2503.

[0085] Table 10 Changes in the organ indices of mice in each group 。

[0086] 2.5.4 Effect on the length of mouse colon As Figure 8 shown, the colon of mice in the Control group was in good shape, without congestion or swelling. There were brown granular feces in the intestinal lumen, and the length was 8.29 ± 0.53 cm. After induction with DSS, the colon of mice in the IBD group was significantly shortened to 5.86 ± 0.74 cm (P < 0.001). At the same time, the intestinal lumen was swollen and the feces became loose, indicating that inflammation caused 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), and the feces were formed and the morphology was close to normal. The lengths of the colon tissue structures 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), and the edema and congestion conditions improved. The above results show that the preventive and therapeutic effects of Lactobacillus kefiranofaciens 2503 can improve the colon shortening and apparent morphology induced by DSS.

[0087] Lactobacillus kefiranofaciens JK-24 disclosed in the prior art CN119752669 A, under the intervention of an ultra-high dose of 10 10 CFU / kg, compared with the model group, the colon recovery rate was (5.2 - 4.5) / 4.5 = 16%. However, Lactobacillus kefiranofaciens 2503 provided by the present invention can achieve a colon recovery rate of (7.12 - 5.86) / 5.86 = 22% at a dose of 10 9 CFU / kg. This shows that Lactobacillus kefiranofaciens provided by the present invention is more beneficial to improving the colon shortening induced by DSS than the prior art.

[0088] 2.5.5 Effect on the pathological score of mouse colon The colon tissue damage of mice in each group was observed by HE staining ( Figure 9 ), and further, based on the histopathological scoring criteria, the degree of inflammation, crypt structure and lesion range were quantitatively evaluated ( Figure 10). The colon tissue structure of the mice in the Control group was intact, the crypt structure was clear, the epithelial surface layer was rich in a large number of goblet cells, and there was no infiltration of inflammatory cells in the submucosa, and the pathological score was 0.25 ± 0.46. Compared with the Control group, after DSS induction, the colon crypt structure of the mice in the IBD group was damaged by inflammation, the number of goblet cells decreased sharply, and dense inflammatory infiltration appeared in the submucosa, showing typical inflammatory pathological characteristics, and the pathological score increased significantly to 6.63 ± 1.06 (P < 0.001). Compared with the IBD group, the colon crypt structure of the mice in the 5-ASA and H-Lk groups was intact, the epithelial integrity was restored, the number of goblet cells increased, and the pathological scores decreased significantly to 2.25 ± 0.89 and 3.50 ± 0.93 respectively (P < 0.001); in the L-Lk group, the colon crypt structure was partially restored, and there was still infiltration of inflammatory cells in the submucosa, and the pathological score decreased significantly to 5.0 ± 1.19 (P < 0.05).

[0089] 2.5.6 Effects on the intestinal barrier of mice As Figure 11 shown, the transcriptional levels of the intestinal barrier function-related genes MUC2, ZO-1, and Claudin-1 in the mice in the IBD group decreased significantly to 28.64 ± 7.20%, 18.50 ± 6.16%, and 31.45 ± 6.98% of the Control group respectively (P < 0.001), indicating that DSS damaged the intestinal barrier function of the mice. The transcriptional levels of each gene in the 5-ASA group were significantly restored (P < 0.001). After intervention with high-dose Lactobacillus kefiranofaciens, the transcriptional levels of MUC2, ZO-1, and Claudin-1 were significantly increased and restored to 70.91 ± 24.14%, 84.42 ± 6.38%, and 65.82 ± 7.02% of the Control group respectively (P < 0.05); although the intervention with low-dose strains could up-regulate the expression of each gene, the effect was slightly lower than that of the high-dose group.

[0090] The immunofluorescence results further verified the data of RT-qPCR and more intuitively reflected the differences in the localization and expression of intestinal barrier proteins in each group of mice. The results were as 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.

[0091] 2.5.7 Effects on oxidative stress in mouse colon 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.

[0092] The results are as follows Figure 13As shown, compared with the Control group, due to the induction of DSS in IBD group mice, the activities of antioxidant enzymes such as CAT, T-SOD, and GSH-Px in colon homogenates decreased to 0.75±0.32, 49.78±2.19, and 192.03±16.42 U / mg prot respectively (P<0.001); the 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 triggered the disorder of oxidative stress homeostasis in the body. Compared with the IBD group, 5-ASA intervention could significantly increase the activities of antioxidant enzymes (P<0.05) and decrease the MPO activity and MDA content (P<0.001). The preventive and therapeutic effects of Lactobacillus kefiranofaciens 2503 showed a dose-dependence. Compared with the IBD group, in the colon tissue homogenates of H-Lk group mice, the activities of CAT and T-SOD increased to 1.12±0.22 and 61.37±4.80 U / mg prot respectively (P<0.05), and the 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). The above results indicate that Lactobacillus kefiranofaciens 2503 alleviates the intestinal inflammatory response caused by DSS by increasing the activities of antioxidant enzymes such as T-SOD and CAT, decreasing the MPO activity and MDA content, and reducing the damage of free radicals to the intestinal mucosa.

[0093] The present invention provides Lactobacillus kefiranofaciens 2503 at a dose of 10 9 CFU / kg. Compared with the model group, the increase rate of CAT activity was (1.12 - 0.75) / 0.75 = 49%; compared with the model group, the decrease rate of MPO was (0.39 - 0.15) / 0.39 = 62%, and the decrease rate of MDA was (0.57 - 0.37) / 0.57 = 35%. Compared with Lactobacillus kefiranofaciens JK-24 disclosed in the prior art CN119752669 A, the changes in CAT, MPO, and MDA were significantly more excellent, indicating that Lactobacillus kefiranofaciens 2503 is more beneficial to reducing the damage of free radicals to the intestinal mucosa.

[0094] 2.5.8 Effects on mouse serum cytokines As a metabolite of gut microbiota, the elevated level of LPS in serum reflects the disruption of intestinal barrier function and bacterial translocation. The level of LPS in serum is an important indicator for evaluating intestinal permeability. As key pro-inflammatory factors, the levels of IL-1β, TNF-α, and IL-6 are closely related to mucosal injury and neutrophil infiltration. As a key immunomodulatory factor, IL-10 can inhibit the production of pro-inflammatory cytokines and promote the function of regulatory T cells (Tregs). In this study, the regulatory effects of Lactobacillus kefiranofaciens 2503 on immune balance and intestinal barrier function were evaluated by detecting the concentrations of related indicators in mouse serum.

[0095] As Figure 14 shown, significant changes occurred in the serum factor levels of mice in the DSS-induced IBD model group. Compared with the Control group, the level of LPS, a marker of impaired intestinal barrier function, in the IBD group increased significantly to 239.71 ± 8.25 pg / mL (P < 0.001). Meanwhile, the levels of pro-inflammatory factors 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), and the level of anti-inflammatory factor IL-10 decreased to 533.59 ± 21.23 pg / mL (P < 0.001), indicating that DSS caused impaired intestinal barrier function in mice and systemic inflammatory responses occurred in the body. 5-ASA intervention showed significant efficacy. Compared with the IBD group, the levels of LPS, IL-1β, TNF-α, and IL-6 in mouse serum decreased 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 level of IL-10 recovered to 615.11 ± 29.64 pg / mL (P < 0.001). The intervention effect of Lactobacillus kefiranofaciens showed a dose-dependent characteristic. The levels of LPS, IL-1β, TNF-α, IL-6, and IL-10 in mouse serum 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).

[0096] These results indicate that Lactobacillus kefiranofaciens 2503 may improve the pathological damage of DSS-induced IBD through the synergistic mechanisms of repairing the intestinal barrier and regulating immunity. Its efficacy is dose-dependent, and it shows a regulatory efficacy comparable to that of 5-ASA in reducing LPS translocation and inhibiting pro-inflammatory factors and promoting anti-inflammatory factors.

[0097] In the present invention, the intervention of Lactobacillus kefiranofaciens 2503 has a promoting effect on the anti-inflammatory factor IL-10 in the serum. In contrast, Lactobacillus kefiranofaciens JK-24 disclosed in the prior art CN119752669 A further reduces the anti-inflammatory factor IL-10 on the basis of the model group. Those skilled in the art know that IL-10 is a key factor for inhibiting inflammation. Therefore, Lactobacillus kefiranofaciens 2503 provided by the present invention is more conducive to immune regulation.

[0098] 2.5.9 Effects on the intestinal flora of mice 2.5.9.1 OTU clustering analysis 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 the similarity of gene sequences, and is usually used to describe microbial populations with similarity higher than a certain threshold. Its main function is to group microorganisms according to similar characteristics for research and comparison.

[0099] As Figure 15 shown in the Venn diagram of the overlap between groups, the number of OTUs common to all five groups is 313, and the number of OTUs unique to the Control group is 1515; after induction with DSS, the number of OTUs unique to the IBD group is only 1073, indicating that DSS leads to a decrease in the diversity of the intestinal flora of mice; in contrast, after intervention with Lactobacillus kefiranofaciens 2503 and 5-ASA, the number of OTUs unique to each group increases to 1455, 1401, and 1176.

[0100] This result shows that DSS induction destroys the diversity of the intestinal flora of mice, and Lactobacillus kefiranofaciens 2503 can positively regulate the homeostasis of the intestinal flora.

[0101] 2.5.9.2 Alpha diversity analysis The α-diversity of the flora is a core index for evaluating the microbial community structure within a sample. High α-diversity of the intestinal flora usually indicates a healthy and functionally diverse intestinal flora, while low α-diversity may be related to some diseases or health problems. Commonly used indicators include the Chao1 index, ACE index, Shannon index, and Simpson index for comprehensive evaluation. The Chao1 index and ACE index can measure the species richness, while the Shannon index and Simpson index are used to measure the species diversity.

[0102] As Figure 16It can be seen that compared with the Control group, the richness index and diversity of the intestinal flora in the IBD group of mice were significantly reduced (P<0.01). Under the preventive and protective effects of Lactobacillus kefiranofaciens and 5-ASA, the above indicators all recovered to some extent. The intervention effect of Lactobacillus kefiranofaciens 2503 showed a dose-dependence, and the recovery effects of the richness index and diversity in the H-Lk group were better than those in the 5-ASA group. This result indicates that this strain can positively regulate the flora structure and restore the reduction of the flora α-diversity caused by DSS.

[0103] 2.5.9.3 Beta diversity analysis Beta diversity is a core index to measure the differences in the microbial community composition between different samples, and principal coordinates analysis (PCoA) is often used to analyze the Beta diversity of the flora in different samples. PCoA performs dimensionality reduction on multi-dimensional data based on the Bray-Curtis distance matrix, projects the differences between groups into a two-dimensional space, where the horizontal and vertical coordinates represent the principal coordinate axes with the largest explanatory power respectively. The closer the samples are, the higher the similarity of the flora structure.

[0104] As Figure 17 shown, there were significant differences in the microbial structures of the intestinal flora between the Control group and the IBD group, and the flora distributions of the two groups showed a completely separated state, indicating that DSS intervention led to changes in the intestinal flora structure. After preventive treatment with Lactobacillus kefiranofaciens 2503 and 5-ASA, the flora distribution approached the Control group, and the overlapping area between the H-Lk group and the Control group was the largest and the distance was the closest. The intervention effect of 5-ASA was equivalent to that of low-dose probiotics. This result indicates that Lactobacillus kefiranofaciens 2503 can restore the imbalance of the flora structure caused by DSS.

[0105] 2.5.9.4 Differences in intestinal flora composition To further understand the differences in the intestinal flora composition of mice in different groups, the richness and differences of the intestinal flora of mice were further analyzed at the phylum, family, and genus levels.

[0106] As Figure 18 shown, at the phylum level, the dominant phyla in each group were Bacteroidetes ( Bacteroidetes ), Firmicutes ( Firmicutes ), Desulfobacterota ( Desulfobacterota ), Actinobacteriota ( Actinobacteria ), and Proteobacteria ( Proteobacteria ). After DSS induction in the IBD group of mice, Firmicutes the relative abundance increased (Control group: 37.3%, IBD group: 48.5%), BacteroidotaThe abundance decreased (Control group: 55.5%, IBD group: 40.0%), resulting in Firmicutes / Bacteroidetes a significant increase in the ratio of (F / B) (P < 0.001). Studies have shown that the F / B in the healthy gut microbiota maintains a dynamic balance, and its abnormal changes are usually associated with microbiota disorders. 5-ASA has no significant regulatory effect on the F / B value. After the intervention of Lactobacillus kefiranofaciens 2503, by increasing the Bacteroidetes abundance and simultaneously inhibiting the Firmicutes overproliferation, the abnormal F / B ratio was reversed.

[0107] As Figure 19 shown, at the family level, compared with the Control group, the abundances of Muribaculaceae ( Muribaculaceae ), Prevotellaceae ( Prevotellaceae ), and Lactobacillaceae ( Lactobacillaceae ) with metabolic protection functions in the intestinal tracts of IBD group mice were significantly decreased (P < 0.05); the abundance of Staphylococcaceae ( Staphylococcaceae ) was significantly increased (P < 0.001). Lactobacillus kefiranofaciens 2503 exhibited multi-dimensional regulatory effects. The H-Lk group not only significantly inhibited the Staphylococcaceae overproliferation (P < 0.01), but also increased the Muribaculaceae , Prevotellaceae and Lactobacillaceae abundances (P < 0.05). Although the 5-ASA intervention could reduce the Staphylococcaceae level, its effect on the restoration of functional microbiota was weaker than that of the H-Lk group. Literature studies have shown that Staphylococcaceae could exacerbate the condition by disrupting the gut microbiota balance and inducing immune responses. For example, Staphylococcus aureus can produce toxins and pro-inflammatory factors, exacerbating intestinal inflammation, increasing the risk of bacterial translocation by disrupting the intestinal barrier function; Muribaculaceae , Prevotellaceae and Lactobacillaceae some members of could 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, certain Lactobacillaceae strains can metabolize tryptophan to generate indole compounds, and these metabolites play an anti-inflammatory role through the AhR signaling pathway and support the repair and regeneration of intestinal epithelial cells.

[0108] As Figure 20 shown, at the genus level, the top 8 genera in the intestinal tracts of Control group mice were the unnamed Labrys ( unclassified_Muribaculaceae ), Bacteroides ( Bacteroides ), Staphylococcus ( Staphylococcus ), unclassified_Clostridia_UCG_014、Lachnospiraceae_NK4A136_ group , Odoribacter,Odoribacter ), Mammaliicoccus Mammaliicoccus ), Muribaculum Muribaculum ). Compared with the Control group, the relative abundances of Bacteroides , Staphylococcus , Odoribacter and Mammaliicoccus in the intestines of IBD mice were significantly increased ( P < 0.05); while the abundances of Muribaculum and unclassified_Clostridia_UCG_014 decreased sharply. Under the intervention of Lactobacillus kefiranofaciens 2503 and 5-ASA, the above-mentioned dysbiosis was improved; compared with the IBD group, the abundances of Staphylococcus、Odoribacter、 Mammaliicoccus in the H-Lk group were significantly decreased ( P < 0.01), the abundance of Muribaculum was significantly increased ( P < 0.01), and the abundance of unclassified_Clostridia_UCG_014 was increased. Under the protective effect of 5-ASA, only the abundances of Bacteroides、 Staphylococcus , Mammaliicoccus and Odoribacter were decreased, and the abundance of Muribaculum could not be increased. The research shows that Bacteroides is a class of Gram-negative bacteria, and the LPS in its cell wall can activate the immune response of the host. Staphylococcus Some members such as Staphylococcus aureus can cause various types of infections, including skin infections, pneumonia, endocarditis, and osteomyelitis, etc. Mammaliicoccus is mainly related to mammals and may cause animal skin infections. Odoribacter is a Gram-negative bacterium, usually present in the intestines of humans and animals. The overgrowth of this bacterium may lead to the imbalance of the intestinal microbiota and is related to digestive system diseases such as irritable bowel syndrome. Muribaculum is mainly present in the intestines of mice and other mammals, helps maintain the balance of other beneficial bacteria in the intestine, prevents the overgrowth of harmful bacteria, and participates in the production of short-chain fatty acids. unclassified_Clostridia_UCG_014 belongs to Clostridia class. Although there is no clear evidence of its probiotic function, many Clostridium bacteria have been shown to participate in tryptophan metabolism.

[0109] 2.5.9.5 Differences in intestinal microbiota composition To further explore the effect of Lactobacillus kefiranofaciens 2503 on the microbiota of IBD mice, the LEfSe method was used to screen for differential species (LDA > 3.0) in the intestinal microbiota of mice in each group. The cladogram shows that ( Figure 21 ), the differential species in IBD mice are Bacteroides ( Bacteroides ), Ruminococcus flavefaciens ( Ruminococcus flavefaciens), Parabacteroides goldsteinii ( Parabacteroides ), Acinetobacter spp., ( Acinetobacter ); These genera have been shown to interfere with normal intestinal immune responses and increase the risk of intestinal inflammation. The differential species in the 5-ASA group were mainly Prevotellaceae ( Prevotellaceae ), and the Lactobacillus kefiranofaciens dose group was enriched in Lachnospiraceae ( Lachnospiraceae ), Prevotellaceae ( Paraprevotella ), Clostridium_sp_ASF356 and Lactobacillus kefiranofaciens ( Lactobacillus kefiranofaciens ). Linear discriminant analysis ( Figure 22 ) further verified that the IBD group was mainly enriched in Bacteroides ; the 5-ASA group was mainly enriched in Prevotellaceae ; after intervention with Lactobacillus kefiranofaciens 2503, it was mainly enriched in the abundance of Lachnospiraceae, Paraprevotella, uncultured_Muribaculaceae, Clostridium_sp_ ASF356, Alloprevotella, Lactobacillus kefiranofacien . The above results once again proved that intragastric administration of Lactobacillus kefiranofaciens 2503 could not only colonize in the mouse intestine but also counteract DSS-induced colitis by increasing the abundance of beneficial bacteria and decreasing the abundance of pathogenic bacteria in the intestine. Research shows that Lachnospiraceae members are the main producers of short-chain fatty acids, Paraprevotella is an effective trypsin-degrading symbiotic bacterium that can help maintain intestinal homeostasis and prevent pathogens, Clostridium_sp_ASF356 belongs to the genus Clostridium, and the microorganisms of this genus are often related to tryptophan metabolism.

[0110] 2.5.10 Effects on short-chain fatty acids in the mouse intestine SCFAs are key metabolites after the fermentation of dietary fiber by the gut microbiota, participating in the regulation of host immune responses and playing multiple roles in maintaining intestinal health, mainly composed of acetic acid, propionic acid, and butyric acid. Since the homeostasis of the gut microbiota is significantly disrupted during the pathogenesis of IBD, its ability to metabolize and produce SCFAs will also change accordingly. Therefore, the detection of SCFAs has become an effective means to evaluate the functional status of the gut microbiota.

[0111] The levels of SCFAs in the feces of mice in each group were as Figure 23As shown in the figure. The main SCFA levels in the feces of mice in the Control group were as follows: 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), isovaleric acid (0.09 ± 0.02 mg / g). Compared with the Control group, the contents of each SCFA in the IBD group were significantly reduced to 47.2%, 46.3%, 66.5%, 49.5%, and 32.9% of the Control group (P < 0.01). The improvement effect of Lactobacillus kefiranofaciens 2503 was dose-dependent. Compared with the IBD group, the H-Lk group significantly restored the levels of each SCFA: acetic acid (0.58 ± 0.08 mg / g), propionic acid (0.43 ± 0.05 mg / g), isobutyric acid (0.67 ± 0.07 mg / g), butyric acid (0.13 ± 0.02 mg / g), isovaleric acid (0.07 ± 0.01 mg / g) (P < 0.05). The 5-ASA group only had a weak improvement on isobutyric acid (P < 0.05) and had no significant restorative effect on the remaining SCFAs.

[0112] These results indicate that Lactobacillus kefiranofaciens 2503 drives the biosynthesis of SCFAs by enriching Muribaculaceae and Lachnospiraceae and other functional strains, while 5-ASA cannot restore the SCFA metabolic homeostasis due to the lack of the ability to regulate the flora.

[0113] 2.5.11 Effects on the intestinal tryptophan metabolism of mice 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. Among them, the metabolites produced by the indole pathway can enhance the intestinal barrier and regulate immunity by activating AhR. Intestinal flora analysis showed that after the intervention of Lactobacillus kefiranofaciens 2503, the flora related to tryptophan metabolism could be enriched, such as Lachnospiraceae , Clostridium_sp_ASF356 and unclassified_ Clostridia_UCG _014 The abundance. This suggests that 2503 may improve the symptoms of DSS-induced colitis by regulating tryptophan metabolism. Therefore, this study used targeted metabolomics to analyze the changes in tryptophan metabolites in the feces of mice.

[0114] As Figure 24 shown in (a) PCA, the distributions of tryptophan metabolites in the Control group and the IBD group showed an obvious separation trend, indicating that DSS treatment led to disorders 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 could partially restore the tryptophan metabolic homeostasis. Differential volcanoFigure 24 (b, c) showed that compared with the Control group, two metabolites in the feces of IBD group mice were significantly increased and one metabolite was significantly decreased; after the intervention of Lactobacillus kefiranofaciens, the changes in fecal metabolites were offset, resulting in a significant increase in one metabolite and a significant downregulation of four metabolites. Heat Figure 24 (d) Analysis showed that the tryptophan metabolism in the IBD group was biased towards the inflammatory pathway, specifically manifested as the accumulation of kynurenine and the decrease in the abundance of anti-inflammatory AhR ligands indoles. After the intervention of high-dose Lactobacillus kefiranofaciens, the tryptophan metabolism was reversed to the Control group, the AhR ligand substances increased, and the abundance of pro-inflammatory metabolites decreased. The bar chart data further quantified the changes in the levels of indole metabolites derived from gut microbiota.

[0115] As Figure 25 shown, the levels of core indole metabolites in the intestines of Control group mice were: 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), tryptamine (0.004 ± 0.003 ng / mg). The IBD group showed tryptophan metabolism disorder, and the 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; after the intervention of high-dose Lactobacillus kefiranofaciens, the levels of such indole metabolites increased and were close to those of the Control group. These results indicate that Lactobacillus kefiranofaciens 2503 can restore the tryptophan metabolism homeostasis in IBD mice to a certain extent.

[0116] 2.5.11 Effects on the expression of AhR / Cyp1a1 pathway AhR is a ligand-activated transcription factor that can regulate the intestinal immune balance by recognizing exogenous ligands such as dietary polyphenols and endogenous ligands (such as indole compounds metabolized by the microbiota). Under normal circumstances, AhR activation can promote the differentiation of regulatory T cells (Tregs) and inhibit the overactivation of Th17 cells, thereby maintaining immune tolerance; at the same time, it strengthens the intestinal epithelial barrier function by upregulating the secretion of tight junction proteins and mucins. As a key effector molecule of the AhR signal, Cyp1a1 not only participates in the metabolism of exogenous toxins but also reduces the damage of oxidative stress to the intestinal mucosa by scavenging reactive oxygen species. Previous studies have shown that indole substances in the mouse intestine can be increased after 2503 intervention, indicating that Lactobacillus kefiranofaciens may improve IBD by activating the AhR / Cyp1a1 signaling pathway. To further clarify the mechanism of action of Lactobacillus kefiranofaciens in preventing and treating IBD in mice, this study used immunofluorescence to detect the activation status of AhR / Cyp1a1.

[0117] The results are as Figure 26 shown. In the Control group, the colon tissue structure of mice was intact, the crypt structure was clear, and the fluorescence signals of AhR and Cyp1a1 were strong and evenly distributed in the nucleus. Compared with the Control group, in the IBD group, the localization of AhR and Cyp1a1 in the colon tissue of mice was mainly in the cell membrane, and the fluorescence signals in the nucleus were weak, showing a broken or discontinuous distribution. The fluorescence intensity was significantly reduced to only 49.45±6.81% and 59.53±9.84% of the Control group (P<0.05), indicating that the AhR / Cyp1a1 signaling pathway was inhibited in the colon of mice in the model group. In the 5-ASA group, the expression of AhR and Cyp1a1 was restored to 67.16±4.87% and 88.92±12.52% of the Control group. In the L-Lk group, the nuclear fluorescence intensity of AhR and Cyp1a1 was increased to 79.04±4.88% and 90.64±12.76% of the Control group (P<0.05); the intervention effect of the high dose was more significant. AhR and Cyp1a1 were mainly localized in the cell nucleus, and the fluorescence intensities reached 82.50±11.32% and 101.80±5.02% of the Control group (P<0.01), indicating that Lactobacillus kefiranofaciens 2503 can improve the symptoms of IBD in mice by activating the AhR / Cyp1a1 signaling pathway.

[0118] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A Lactobacillus kefiranofaciens, characterized in that, The Lactobacillus kefiranofaciens is Lactobacillus kefiranofaciens ( Lactobacillus kefiranofaciens Lactobacillus kefiranofaciens ) 2503, which is deposited in the China General Microbiological Culture Collection Center with the deposit number CGMCC NO. 33828.

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

3. The Lactobacillus kefiranofaciens culture according to claim 2, characterized in that, The fermentation product includes Lactobacillus kefiranofaciens ( Lactobacillus kefiranofaciens ), 2503 and the metabolites of Lactobacillus kefiranofaciens ( Lactobacillus kefiranofaciens ), 2503.

4. A bacterial agent, characterized in that: The bacterial agent contains Lactobacillus kefiranofaciens subsp. kefiranofaciens ( Lactobacillus kefiranofaciens ) 2503 and / or the metabolites of Lactobacillus kefiranofaciens subsp. kefiranofaciens ( Lactobacillus kefiranofaciens ) 2503 and / or the culture as claimed in claim 2 or 3.

5. Use of Lactobacillus kefiranofaciens ( Lactobacillus kefiranofaciens ) 2503 as claimed in claim 1, a culture of Lactobacillus kefiranofaciens ( Lactobacillus kefiranofaciens ) 2503 as claimed in claim 2 or 3, or the bacterial agent as claimed in claim 4 in the preparation of a medicament for preventing and / or treating and / or adjuvantly treating inflammatory bowel disease.

6. The application according to claim 5, characterized in that, The inflammatory bowel disease includes Crohn's disease and ulcerative colitis, and the drug has at least one of the following functions: a1) Slowing down the weight loss of the patient; a2) Improving the patient's fecal characteristics and blood in the stool; a3) Improving the patient's spleen index; a4) Slowing down the shortening of the patient's colon length; a5) Increasing the expression levels of MUC2, ZO-1 and Claudin-1 and repairing the intestinal barrier function of the patient; a6) Increasing the activities of antioxidant enzymes CAT, T-SOD, and GSH-Px and reducing the oxidative stress level of the patient; a7) Scavenging free radicals; a8) Reducing the levels of serum pro-inflammatory factors IL-1β, TNF-α and IL-6 in the patient and increasing the level of anti-inflammatory factor IL-10; a9) Increasing the abundance of beneficial intestinal bacteria and reducing the abundance of pathogenic bacteria, and restoring the intestinal flora diversity and metabolite levels of the patient.

7. The application according to claim 6, wherein The increasing the abundance of beneficial intestinal bacteria and reducing the abundance of pathogenic bacteria includes: a9.1) At the phylum level, increase the abundance of Bacteroidetes ( Bacteroidetes ), decrease the abundance of Firmicutes ( Firmicutes ), and reverse the abnormal F / B ratio; a9.2) At the family level, the abundances of Muribaculaceae ( Muribaculaceae ), Prevotellaceae ( Prevotellaceae ), and Lactobacillaceae ( Lactobacillaceae ) were increased, while the abundance of Staphylococcaceae ( Staphylococcaceae ) was decreased; a9.3) At the genus level, the abundances of Muribaculum and unclassified_Clostridia_ UCG_014 were increased, while the abundances of Staphylococcus and 、 Odoribacter and 、 Mammaliicoccus were decreased;​​ a9.4) Enrichment of Lachnospiraceae ( Lachnospiraceae), Paraprevotella Paraprevotella), Clostridium Clostridium_sp_ASF356, Lactobacillus kefiranofaciens ( Lactobacillus kefiranofacien) ; The restoring the intestinal flora metabolite levels of the patient includes: a9.5) Restoring the intestinal short-chain fatty acid levels, and the short-chain fatty acids include acetic acid, propionic acid, isobutyric acid, butyric acid and isovaleric acid; a9.6) Restoring the intestinal microbial indole metabolite levels, and the indole metabolites include indole-3-carboxaldehyde, indole-3-acetic acid, indole-3-propionic acid, indole-3-acrylic acid, indole and tryptamine.

8. Use of Lactobacillus kefiranofaciens ( Lactobacillus kefiranofaciens ) 2503 as claimed in claim 1, a culture of Lactobacillus kefiranofaciens ( Lactobacillus kefiranofaciens ) 2503 as claimed in claim 2 or 3, or the bacterial agent as claimed in claim 4 in the preparation of an antioxidant or a free radical scavenger.

9. Use of Lactobacillus kefiranofaciens ( Lactobacillus kefiranofaciens ) 2503 as claimed in claim 1, a culture of Lactobacillus kefiranofaciens ( Lactobacillus kefiranofaciens ) 2503 as claimed in claim 2 or 3, or the bacterial agent as claimed in claim 4 in the preparation of an intestinal microbial regulator.

10. Use of Lactobacillus kefiranofaciens ( Lactobacillus kefiranofaciens ) 2503, the culture of Lactobacillus kefiranofaciens ( Lactobacillus kefiranofaciens ) 2503 according to claim 2 or 3, or the bacterial agent according to claim 4 in the preparation of an immunomodulator.

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