Application of bifidobacterium animalis subsp. Lactis BX-245 in relieving inflammatory bowel disease

Abstract

The application of the animal bifidobacterium subsp. Lactis BX-245 in relieving the inflammatory bowel disease is characterized in that the animal bifidobacterium subsp. Lactis BX-245 is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.26973. The invention also discloses an application of the animal bifidobacterium subsp. Lactis BX-245 in relieving the inflammatory bowel disease. The research aims at discussing the relieving effect of the animal bifidobacterium subsp. Lactis BX-245 on IBD mice induced by dextran sodium sulfate (DSS), and 6-week male C57BL / 6J mice which normally eat and drink water are randomly divided into a normal group (NC group), a model group (IBD group), a common yoghourt (FM group), an animal bifidobacterium subsp. Lactis BX-245 bacterial powder group (BX-245 group) and BX-245 yoghourt (BFM group). The relieving effect of the animal bifidobacterium subsp. Lactis BX-245 and the fermented milk of the animal bifidobacterium subsp. Lactis BX-245 on the symptom of the mouse inflammatory bowel disease is evaluated through weight change, immune factors, targeted metabonomics and metagenomics, and data support is provided for application of the animal bifidobacterium subsp. Lactis BX-245 in products.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to application of animal Bifidobacterium lactis subspecies BX-245 in alleviating inflammatory bowel disease. Background Art

[0002] Inflammatory bowel disease (IBD), mainly including ulcerative colitis (UC) and Crohn's disease (CD), is a group of diseases characterized by long-term chronic inflammation of the intestine. Its cause is not yet fully understood, but it is generally believed to be caused by the combined effects of genetic susceptibility, immune system abnormalities and environmental factors. Symptoms of IBD patients include diarrhea, abdominal pain, weight loss, fever, etc. Severe cases can lead to complications such as malnutrition, intestinal perforation and cancer.

[0003] Existing treatments mainly include drug therapy and surgical treatment. Drug therapy includes immunosuppressants, anti-inflammatory drugs, antibiotics and biological agents. Although it can relieve symptoms, long-term use may bring side effects, such as immunosuppression, drug resistance, liver and kidney damage, etc. In addition, surgical treatment may be inevitable for some critically ill patients, but the risks brought by surgery and the postoperative recovery time are long, and some patients may experience complications or recurrence of the disease after surgery.

[0004] In addition, most existing probiotic preparations focus on promoting intestinal health. Different probiotic strains have quite different clinical performances, and no targeted application strategies are provided for problems such as abnormal immune response and damaged intestinal barrier that are unique to IBD patients. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide an application of animal Bifidobacterium lactis subspecies BX-245 for alleviating inflammatory bowel disease, which can effectively solve the problems raised in the above background technology.

[0006] To solve the above problems, the technical solution adopted by the present invention is: application of animal Bifidobacterium lactis subsp. lactis BX-245 to alleviate inflammatory bowel disease, characterized in that the animal Bifidobacterium lactis subsp. lactis BX-245 is preserved in the General Microbiological Culture Collection Center of China Microbiological Culture Collection Administration, and the preservation number is: CGMCC No.26973.

[0007] As a further preferred embodiment of the present invention, the invention relates to the use of animal Bifidobacterium lactis subspecies BX-245 in the preparation of a product / drug for alleviating inflammatory bowel disease.

[0008] As a further preferred embodiment of the present invention, animal Bifidobacterium lactis subspecies BX-245 is used to improve and reduce the levels of pro-inflammatory factors IL-6, TNF-α, and INF-γ in serum and to increase the level of anti-inflammatory factor IL-10.

[0009] The present study aims to explore the alleviating effect of animal Bifidobacterium lactis subspecies BX-245 on IBD mice induced by sodium dextran sulfate (DSS). Six-week-old male C57BL / 6J mice with normal food and water intake were randomly divided into a normal group (NC group), a model group (IBD group), a regular yogurt group (FM group), an animal Bifidobacterium lactis subspecies BX-245 bacterial powder group (BX-245 group) and a BX-245 yogurt group (BFM group). The alleviating effect of animal Bifidobacterium lactis subspecies BX-245 and its fermented milk on the symptoms of inflammatory bowel disease in mice was evaluated by weight changes, immune factors, targeted metabolomics and metagenomics, aiming to provide data support for the application of animal Bifidobacterium lactis subspecies BX-245 in products.

[0010] Compared with the prior art, the present invention provides an application of animal Bifidobacterium lactis subspecies BX-245 for alleviating inflammatory bowel disease, which has the following beneficial effects: 1. During the experimental period, animal Bifidobacterium lactis subspecies BX-245 can significantly increase the body weight of mice with inflammatory bowel disease, significantly improve the DAI score, reduce the levels of pro-inflammatory factors IL-6, TNF-α, and INF-γ in serum, and increase the level of anti-inflammatory factor IL-10, thereby alleviating the excessive immune response state of mice with inflammatory bowel disease.

[0011] 2. Compared with the NC group, DSS caused a decrease in the diversity of intestinal flora and intestinal flora imbalance in IBD mice. After BX-245 intervention, the diversity of intestinal flora was significantly restored and the metabolic pathway was improved. The main manifestations were that the relative abundance of Bifidobacterium pseudolongum and Bacteroides vulgatus was significantly reduced; the relative abundance of Adlercreutzia equolifaciens was significantly increased. The above three strains are related to inflammation and intestinal barrier. In summary, BX-245 can improve the related symptoms of inflammatory bowel disease mice by restoring intestinal flora imbalance.

[0012] 3. After BX-245 intervention, the levels of butyric acid, valeric acid, ursocholic acid, citric acid, and tryptophan in the feces of mice were significantly increased compared with those in the IBD group, indicating that BX-245 can improve the metabolic disorders of IBD mice caused by DSS modeling. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is the experimental design diagram of the present invention; Figure 2 This is a schematic diagram of the library construction process of the present invention; Figure 3 This is a graph showing the results of the mouse weight test; Figure 4 This is the disease activity index scoring result diagram; Figure 5 It is the colon length ratio of colon weight test result; Figure 6 HE staining and pathological scoring test results; Figure 7 This is a graph showing the results of immune factor testing; Figure 8 This is a diagram of the results of the short-chain fatty acid targeted metabolism test; Fig. 9 This is the result diagram of targeted metabolic testing of bile acid, amino acid and organic acid; Fig.10 This is a diagram of the results of tryptophan targeted metabolism detection; Fig.11 This is the result diagram of α diversity detection; Fig.12 This is the result diagram of β diversity detection; Fig.13 The results of the intestinal flora phylum level analysis are shown in Figure 2. Fig.14 This is a comparison chart of the relative abundance of different bacterial species; Fig.15 A figure showing comparative analysis of the difference in intestinal flora among different groups of mice based on bacterial species level; Fig.16 Figure 2 is a comparative analysis of the difference in intestinal flora of different groups of mice based on the bacterial species level; Fig.17 Three figures are provided for comparative analysis of the difference in intestinal flora of different groups of mice based on the bacterial species level; Fig.18 This is the result diagram of metabolic pathway analysis; Fig.19 This is the result of the correlation analysis between the different strains and the different metabolic pathways; Fig. 20 This is a table diagram of MRM conditions. DETAILED DESCRIPTION

[0014] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0015] The "Bifidobacterium animalis subsp. lactis BX-245" in the examples was purchased from Inner Mongolia Agricultural University.

[0016] As a specific embodiment of the present invention: 1. Purpose This study aimed to investigate the alleviating effect of animal Bifidobacterium lactis subsp. lactis BX-245 on dextran sulfate sodium (DSS)-induced IBD mice. Six-week-old male C57BL / 6J mice with normal food and water intake were randomly divided into a normal group (NC group), a model group (IBD group), a regular yogurt group (FM group), an animal Bifidobacterium lactis subsp. lactis BX-245 bacterial powder group (BX-245 group) and a BX-245 yogurt group (BFM group). The alleviating effect of animal Bifidobacterium lactis subsp. lactis BX-245 and its fermented milk on the symptoms of inflammatory bowel disease in mice was evaluated by weight changes, immune factors, targeted metabolomics and metagenomics, aiming to provide data support for the application of animal Bifidobacterium lactis subsp. lactis BX-245 in products.

[0017] 2. Experimental Materials and Methods 2.1 Main Materials Table 1 Main experimental material information Main experimental materials factory Pure milk (Pro: 3.6%, Fat: 4.4%) Inner Mongolia Mengniu Dairy (Group) Co., Ltd. sucrose Guangxi Fengtang Co., Ltd. Bifidobacterium animalis subsp. lactis BX-245 Key Laboratory of Dairy Biotechnology and Engineering, Ministry of Education, Inner Mongolia Agricultural University Commercial starter culture PYS-010 Beijing Ketuo Hengtong Biotechnology Co., Ltd. Dextran Sulfate Sodium MP Biomedicals, USA 2.2 Preparation of yogurt samples BX-245 yogurt: Using raw milk and 6.5% sucrose as raw materials, inoculate animal Bifidobacterium lactis subspecies BX-245 and commercial starter (PYS-010) for fermentation. 6.5% sucrose is completely dissolved in preheated milk, inoculated and fermented after homogenization, sterilization and cooling processes, and the fermentation end point is reached when the TA value is ≥70°T. After cooling to 20°C in a water bath, it is aseptically packaged, and after ripening at 2-6°C for 24 hours, it is aseptically packaged and stored at 4°C.

[0018] Ordinary yogurt: raw milk and 6.5% sucrose are used as raw materials, and commercial fermentation agent (PYS-010) is used for fermentation. 6.5% sucrose is completely dissolved in preheated milk, and inoculated and fermented after homogenization, sterilization, and cooling. When the TA value is ≥70°T, the fermentation end point is reached, and the product is cooled in a water bath to 20°C and then aseptically packaged. After being ripened at 2-6°C for 24 hours, it is aseptically packaged and stored at 4°C.

[0019] To ensure the number of live bacteria in yogurt during the experiment, BX-245 yogurt and ordinary yogurt were fermented in two batches respectively. The first batch of yogurt was gavaged on days 0-15 (the number of live bifidobacteria in BX-245 yogurt was 7.5×108CFU / mL), and the second batch of yogurt was gavaged on days 16-30 (the number of live bifidobacteria in BX-245 yogurt was 7.7×108CFU / mL). The daily gavage volume was 0.2mL / head.

[0020] 2.3 Experimental methods 2.3.1 IBD animal experiment design After adaptive feeding (-1 week to 0 week), 40 C57BL / 6J mice were randomly divided into 5 groups, with 8 mice in each group ( Figure 1 ). Control group (NC group), model group (IBD group), probiotic group (BX-245 group), BX-245 yogurt group (BFM group) and ordinary yogurt group (FM group).

[0021] During the adaptive feeding period and the experimental period, all mice had free access to food and water. On days 0 to 5, mice in the IBD group, BX-245 group, BFM group, and FM group were given 2.5% DSS drinking water, and mice in the NC group were given sterile water. On days 5 to 10, all mice were given sterile water. This process was one cycle, and a total of three cycles were performed to construct an inflammatory bowel disease mouse model.

[0022] Oral gavage: The NC group and the IBD group were given 0.2 mL / day of sterile saline by oral gavage starting from day 0, and the other groups were given 0.2 mL / day of bacterial solution (4×109 CFU / day, suspended in sterile saline every day) by oral gavage starting from day 0.

[0023] The BFM group was given 0.2 mL / day by gavage starting from day 0. The BX-245 yogurt group and the FM group were gavaged with 0.2 mL / day of ordinary yogurt starting from day 0.

[0024] Preparation of 2.5% DSS solution: Dissolve DSS powder thoroughly with sterile water and replace the DSS solution every two days.

[0025] Body weights of all C57BL / 6J mice were measured on days 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30 of the experiment, and feces were collected and DAI scores were performed on days 0, 5, 10, 15, 20, 25, and 30.

[0026] On the 30th day of the experiment, C57BL / 6J mice were killed and the blood supernatant, colon and its contents were collected to detect the levels of mouse immune factors (IL-6, IL-10, TNF-α, IFN-γ), as well as H&E staining and pathological scores of mouse colon.

[0027] 2.3.2 Disease activity index (DAI) score This study used the DAI scoring index to evaluate the degree of intestinal damage in colitis mice. The score mainly includes weight loss rate, fecal characteristics, and occult blood. During the experiment, the mental state, activity, and diet of the mice were observed, and the mice were weighed regularly. The DAI score was performed on the 0th, 5th, 10th, 15th, 20th, 25th, and 30th days of the experiment.

[0028] DAI scoring method: Take fresh feces (30 mg) and apply it evenly on the fecal occult blood test paper, and observe the fecal occult blood status and fecal properties. If blood can be directly seen or the feces are watery and the fecal occult blood test paper changes color to "++++" within 10 seconds, the score is 4 points. The feces are thin and paste-like and change color to "+++" within 30-60 seconds, the score is 3 points. The feces are thin and soft and change color to "++" within 60-120 seconds, the score is 2 points. The feces are soft and change color to "-" for more than 120 seconds, and the score is 0 points.

[0029] The weight change rate was calculated according to the formula. W1 represents the weight of mice one day before modeling (g), and W2 represents the weight of mice every day after modeling (g)

[0030] 2.3.3 Ratio of colon length to colon weight in mice Shortening of the colon is also one of the pathological manifestations of colitis in mice. After the mice were killed, the length of the mouse colon was collected and recorded. The colon mass of mice in different experimental groups was compared, and the state, color, expansion, ulceration, etc. of the colon were observed in a naturally stretched state. The weight was weighed, the length was measured, and the length to weight ratio of the colon was calculated. According to the ratio of the two, the edema of the colon tissue was analyzed. If the ratio of the two is smaller, the edema is more serious, and vice versa.

[0031] 2.3.4 Hematoxylin-eosin staining (HE) and pathological scoring of mouse colon After the mice were killed, 1 cm of the colon distal to the anus was taken for HE staining. Hematoxylin-eosin staining, referred to as HE staining, can accurately locate and characterize cells, extracellular matrix and other substances in the tissue, understand the structure and function of colon tissue, and through HE staining, the morphology of colon tissue, crypts and goblet cells can be observed, and pathological analysis and scoring can be performed to evaluate the protective effect of BX-245 on colon tissue damage in mice with inflammatory bowel disease.

[0032] Slice production process: Place the colon tissue in an embedding box → Soak the embedding box in 55%, 70%, 80% and 90%, 95%, 100% alcohol solutions for 40 min → Soak in two anhydrous ethanol solutions for 30 min each → Place the embedding box of the colon tissue in xylene I and xylene II in turn and soak for 0.5 h → Place the colon tissue in a container of xylene reagent and soak until it is transparent → Place it in paraffin → Wait for the paraffin to solidify → Trim the wax block → Install the microtome to slice → Flatten at constant temperature and moisture → Remove excess moisture → 60~65℃ constant temperature box → Remove the paraffin between tissues.

[0033] HE staining operation process: Take the slices out of the refrigerator → place at room temperature for 5 min → place in an oven at 60℃ for 40 min → take out the slices → place at room temperature for 10 min → place in xylene I for 15 min → xylene II for 15 min → 100% alcohol for 10 min → 100% alcohol for 10 min → 95% alcohol for 5 min → 85% alcohol for 5 min → wash with distilled water → stain with hematoxylin solution for 3 min → rinse with tap water → hematoxylin differentiation stain for 3 s → rinse with tap water → cell nuclei appear blue → rinse with tap water → drop eosin stain for 3 min → stop staining.

[0034] HE staining scoring criteria:

[0035] AB-PAS staining steps: 1. Dewaxing to water: xylene I for 5 minutes, xylene II for 5 minutes, xylene III for 5 minutes, anhydrous ethanol for 1 minute, 95% ethanol for 1 minute, 75% ethanol for 1 minute, and distilled water for 5 minutes; 2. Add Alcian blue staining solution for 10-20 minutes, and wash with distilled water 3 times, 1-2 minutes each time; 3. Place in oxidant for 5 minutes. Rinse with tap water and soak in distilled water twice; 4. Immerse in Schiff staining solution for 10-20 minutes; 5. Pour off the Schiff staining solution and rinse with running water for 10 minutes; 6. Stain the nucleus with hematoxylin staining solution for 1-2 minutes and wash with water; 7. Differentiate with acidic differentiation solution for 2-5 seconds, then wash with water; 8. Use Scott bluing solution to bluing for 3 minutes, then wash with water for 3 minutes; 9. Dehydration and transparency: 75% ethanol for 1 minute, 95% ethanol for 1 minute, anhydrous ethanol for 1 minute, xylene 3 times, 1-2 minutes each time, and neutral gum sealing.

[0036] 2.3.5 Mouse immune factor detection In this study, mouse interleukin 6 (IL-6), interleukin 10 (IL-10), tumor necrosis factor α (TNF-α), and mouse interferon gamma (INF-γ) ELISA kits were used to detect mouse serum IL-6, IL-10, TNF-α, IFN-γ and other cytokines to evaluate the effect of BX-245 on the intestinal immune balance of mice with inflammatory bowel disease.

[0037] Table 2 ELISA method project step Reagent and sample preparation 1. Before use, the sample and all components of the kit must be rewarmed for at least 120 minutes to ensure that they are fully rewarmed to room temperature; 2. Take out the concentrated washing solution from the refrigerator and heat it in a water bath to completely dissolve the crystals. Dilute the concentrated washing solution with distilled water at a ratio of 1:20; Substrate solution A and B should be fully mixed at a volume ratio of 1:1 before use and used within 15 minutes after mixing. Operating Procedure 1. Prepare the working solutions of various components of the kit according to the method described in the previous instructions; 2. Take out the required strips from the aluminum foil bag, seal the remaining strips with a ziplock bag and put them back in the refrigerator; 3. Set up standard wells, 0 value wells, blank wells and sample wells, add 50μL of standards of different concentrations to the standard wells, add 50μL of sample diluent to the 0 value wells, add nothing to the blank wells, and add 50μL of the sample to be tested to the sample wells; 4. Except for the blank wells, add 100μL of horseradish peroxidase (HRP)-labeled detection antibody to the standard wells, 0 value wells and sample wells; 5. Cover the reaction plate with a sealing film and incubate in a 37℃ water bath or thermostat in the dark for 60min; 6. Remove the sealing film, discard the liquid, pat dry on absorbent paper, fill each well with washing solution, let it stand for 20S, shake off the washing solution, pat dry on absorbent paper, and repeat this 5 times. If using an automatic plate washer, please wash the plate according to the plate washer operating procedures. Adding a soaking procedure for 30 seconds can improve the accuracy of the test. After washing, before adding the substrate, pat the reaction plate dry on a clean, non-chipping paper; 7. Mix substrates A and B in a 1:1 volume ratio and add 100μL of the substrate mixture to all wells. Cover the reaction plate with a sealing film and incubate in a 37℃ water bath or thermostat in the dark for 15 minutes; 8. Add 50μL of the stop solution to all wells and read the absorbance (OD value) of each well on the microplate reader. Result calculation 1. Use the concentration of the standard as the horizontal axis (6 standard wells, plus 1 0 value well, a total of 7 concentration points), and the corresponding absorbance (OD value) as the vertical axis. Use computer software and four-parameter logistic curve fitting (4-pl) to create a standard curve equation. Use the absorbance (OD value) of the sample to calculate the concentration value of the sample using the equation. 2. If the sample is diluted, the concentration value measured by the above method must be multiplied by the dilution factor to obtain the final concentration of the sample. 2.3.6 Extraction and sequencing of intestinal microbial metagenomic DNA from fecal samples Method for extracting intestinal microbial DNA from fecal samples - QIAamp® PowerFecal® Pro DNA Kit: Refer to the instructions for use of the kit: Briefly spin the PowerBead Pro tube to ensure the beads settle to the bottom. Add 250 mg of stool sample and 800 µL Solution CD1 and vortex briefly to mix; mount the PowerBead Pro tube horizontally on a 2 mL tube vortexer and vortex at maximum speed for 10 min; centrifuge at 15,000 × g for 1 min; transfer the supernatant to the provided clean 2 mL microcentrifuge tube; add 200 µL of Solution CD2 and vortex for 5 s; centrifuge at 15,000 × g for 1 min; transfer the supernatant to the provided clean 2 mL microcentrifuge tube; add 600 µL of Solution CD3 and vortex for 5 s; load 650 µL of lysate into the MB spin column and centrifuge at 15,000 × g for 1 min; discard the flow-through and repeat the previous step to ensure that all lysate passes through the MB spin column; place the MB spin column into the provided clean 2 mL collection tube Tube); add 500 µL of Solution EA to the MB spin column and centrifuge at 15,000 × g for 1 min; discard the flow-through and put the MB spin column back into the same 2 mL collection tube; add 500 µL of Solution C5 to the MB spin column and centrifuge at 15,000 × g for 1 min; discard the flow-through and put the MB spin column into a new clean 2 mL collection tube (Collection Tube); centrifuge at 15,000 × g for 2 min; put the MB spin column into a new clean 1.5 mL elution tube (Elution Tube); add 50-100 µL of Solution C6 to the center of the white filter membrane and centrifuge at 15,000 × g for 2 min; discard the MB spin column and collect the DNA. The purity of the DNA was checked using Nanodrop One ultra-micro-volume UV spectrophotometer and 0.6% agarose gel electrophoresis, and the DNA was placed in a -20 ℃ refrigerator for later use.

[0038] Method for purifying intestinal microbial DNA from fecal samples - magnetic bead method: Measure the volume of DNA and transfer it to a new 1.5 mL low DNA adsorption tube (DNA LoBind Tube), add 0.45× volume of DNA fragment sorting and purification magnetic beads, and flick to mix; place it on a rotating mixer and rotate for 10 min, remove it and let it stand at room temperature for 10 min; vortex for 5 seconds, place it on a magnetic rack, and wait for the magnetic beads to be adsorbed; transfer all the liquid to a clean 2 mL microcentrifuge tube (Microcentrifuge Tube); add 1.5 mL 75% ethanol, and immediately aspirate it after injection, and repeat once; dry the magnetic beads, but do not dry them to the extent of cracking, remove the low DNA adsorption tube from the magnetic rack, add 30 µL of nuclease-free water (Water Nuclease-Free) to dissolve it back, and flick to mix; place it on a rotating mixer and rotate for 10 min, remove it and let it stand at room temperature for 10 min; vortex for 5 seconds, place it on a magnetic rack, and wait for the magnetic beads to be adsorbed; transfer all the liquid to a new clean 1.5 mL low DNA adsorption tube to obtain purified DNA. The concentration of metagenomic DNA was detected using the Qubit analysis kit, and the purity of the metagenomic DNA was detected again using a NanoDrop spectrophotometer and 0.6% agarose gel electrophoresis to ensure that the purified DNA met the purity and quality for library construction and sequencing. DNA samples that met the requirements of subsequent experiments were temporarily stored in a -20°C refrigerator for subsequent experiments.

[0039] Quality requirements for intestinal microbial DNA in fecal samples: Total amount ≥2ug; Fragment length ≥20Kb Concentration ≥20ng / μL; Purity: OD 260 / 280 ≥1.8; OD 260 / 230 ≥1.5.

[0040] Reference for database construction process Figure 2 ① DNA fragmentation: DNA is fragmented into about 10Kb to fragment the genome.

[0041] ②Terminal modification: Use Taq polymerase to fill in the uneven ends and add protruding base A to both ends to produce sticky ends.

[0042] ③Add adapters. The ends of the PCR fragments after terminal modification have protruding A tails, while the adapters have protruding T tails. The adapters can be added to the two ends of the DNA fragments using ligase. The adapters of NEB are special ring structures connected by base U. After connecting the adapters, the base U is deleted to form a "Y"-shaped adapter, ensuring that each single sequence has different sequencing primers at both ends, so that different oligonucleotide sequences can be connected in subsequent PCR.

[0043] ④ Magnetic bead purification: Use special magnetic beads (AMPure XP Beads) to remove large fragments and various impurities, thereby obtaining library fragments with successful addition of adapters.

[0044] ⑤PCR amplification: Use primers complementary to the adapter to perform amplification.

[0045] ⑥ Second magnetic bead purification: Perform magnetic bead purification again to separate the product DNA fragments from impurities such as polymerase.

[0046] ⑦ Quality detection: Perform DNA concentration detection, agarose gel electrophoresis and fragment length detection to complete library construction.

[0047] Finally, the qualified DNA library was constructed and sequenced on the Illumina NovaSeq platform to generate paired-end sequences.

[0048] Data output statistics: Fecal samples from 40 mice were collected, including 8 in the NC group, 8 in the IBD group, 8 in the BX-245 group, 8 in the BFM group, and 8 in the FM group. Some samples were invalid due to too small sample size, DNA not meeting sequencing requirements, or sequencing failure, so DNA was repeatedly extracted, tested, and tested for purity, and library construction and PCR amplification were attempted.

[0049] 2.3.7 Determination of fecal short-chain fatty acids 2.3.7.1 Preparation of standards First, water was used to mix acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid and isovaleric acid standards to prepare a 100 mg / mL stock solution, and the stock solution was diluted with water to obtain a series of working standard solutions. Then, ether was used to mix hexanoic acid standards to prepare a 100 mg / mL stock solution, and the stock solution was diluted with ether to obtain a hexanoic acid working standard solution. Finally, ether was used to prepare an internal standard (4-methylvaleric acid) with a concentration of 375 μg / mL. Take 200 μL of the series of working standard solutions of the six acids, 100 μL of 15% phosphoric acid, 20 μL of hexanoic acid working standard solution, 20 μL of internal standard and 260 μL of ether and mix them to prepare ten standard curve points of 500 μg / mL, 250 μg / mL, 100 μg / mL, 50 μg / mL, 25 μg / mL, 10 μg / mL, 2 μg / mL, 0.5 μg / mL, 0.1 μg / mL, and 0.02 μg / mL. The stock solution is stored at -20°C and the working solution is prepared and used immediately.

[0050] 2.3.7.2 Metabolite extraction Accurately weigh 0.02g of feces into a 1.5mL sterile centrifuge tube, add 500μL of distilled water and 2 glass beads, and homogenize for 1min. Then centrifuge the sample (4℃, 12000rpm, 10min), and take 200μL of supernatant. Add 100μL of 15% phosphoric acid, 20μL of internal standard (4-methylvaleric acid) solution and 280μL of ether to the supernatant, homogenize for 1min, centrifuge the sample again (4℃, 12000rpm, 10min), and take the supernatant for testing.

[0051] 2.3.7.3 On-machine testing The parameters of the Thermo Trace 1300 gas phase system are as follows: the chromatographic column is an Agilent HP-INNOWAX capillary column (30m×0.25mm, 0.25μm); split injection, injection volume 1μL, split ratio 10:1; injection port temperature 250℃, ion source temperature 300℃, transfer line temperature 250℃; the program temperature is 90℃, then raised to 120℃ at 10℃ / min, then raised to 150℃ at 5℃ / min, and finally raised to 250℃ at 25℃ / min and maintained for 2min. The carrier gas is helium, and the carrier gas flow rate is 1.0mL / min. The mass spectrometry detection of metabolites was carried out on an ISQ 7000 mass spectrometer in single ion monitoring mode with an electron energy of 70eV.

[0052] 2.3.8 Fecal bile acid determination 2.3.8.1 Preparation of standards The bile acid standard was mixed with methanol to prepare a mixed standard stock solution with a concentration of 1000 μg / mL, and then diluted with 30% methanol to obtain ten standard curve points. Each stock solution and working standard solution were stored at -20°C.

[0053] 2.3.8.2 Metabolite extraction Accurately weigh 0.02g of feces in a 2mL centrifuge tube, accurately add 400μL of methanol (-20℃), and vortex for 60s. Add 2 steel beads, put it in a tissue grinder, grind it at 55Hz for 1min, and repeat the above operation at least twice. Then ultrasonicate at room temperature for 30min, centrifuge (12000rpm, 4℃, 10min), take 300μL of supernatant, add 600μL of distilled water to mix, and vortex for 30s. Take an appropriate amount of supernatant and add 30% methanol to dilute 10 times, filter the supernatant through a 0.22μm filter membrane, and add the filtrate to the test bottle.

[0054] 2.3.8.3 On-machine testing The parameters of EXion LC liquid chromatography are as follows: ACQUITY UPLC® BEH C18 column (2.1×100mm, 1.7μm), injection volume of 5μL, column temperature of 40℃, mobile phase of 0.01% formic acid water (A) and acetonitrile (B). Gradient elution conditions are 0-4min, 25%B; 4-9min, 25-30%B; 9-14min, 30-36%B; 14-18min, 36-38%B; 18-24min, 38-50%B; 24-32min, 50-75%B; 32-33min, 75-90%B; 33-35.5min, 90-25%B. The flow rate is 0.25mL / min.

[0055] The mass spectrometry detection of metabolites was performed by AB6500 Plus mass spectrometer in negative ionization mode. The ion source temperature was 500°C, the ion source voltage was -4500V, the collision gas was 6psi, the curtain gas was 30psi, the nebulizer gas and auxiliary gas were both 50psi, and multiple reaction monitoring was used for scanning.

[0056] 2.3.9 Fecal amino acid determination 2.3.9.1 Preparation of standards Accurately weigh 19 amino acid standards to prepare a mixed standard linear master solution with a concentration of 5 mg / mL. Dilute the linear master solution with methanol to obtain a series of concentrations. Prepare a certain concentration of L-Alanine-d4 and Phenylanine-d2 solutions and mix them to obtain the internal standard solution. The linear, internal standard and quality control master solutions and working solutions are all stored in a -20℃ refrigerator.

[0057] 2.3.9.2 Metabolite extraction Take a sample at a ratio of 1:1 and add it to mass spectrometry water, vortex mix to obtain a diluted sample; take 50 μL of the diluted sample, add 200 μL of a precipitant containing a mixed internal standard (acetonitrile: methanol = 1:1), vortex mix, let stand on ice for 30 min, and centrifuge at 12,000 r / min at 4°C for 10 min.

[0058] 2.3.9.3 On-machine testing The chromatographic column was ACQUITY UPLC BEH Amide (2.1×100 mm, 1.7 μm); the mobile phase was 5 mmol / L ammonium acetate aqueous solution containing 0.1% formic acid in phase A; acetonitrile containing 0.1% formic acid in phase B; the column temperature was 50°C; the injection volume was 1 μL; the flow rate was 0.3 mL / min; the chromatographic gradient was preset according to the standard coefficient; the mass spectrometry conditions were electrospray ionization source (ESI), positive ionization mode, ion source temperature of 550°C, ion source voltage of 5500V, curtain gas of 35 psi, nebulizer gas of 50 psi, and auxiliary gas of 60 psi. Multiple reaction monitoring was used for scanning.

[0059] 2.3.10 Determination of fecal vitamins 2.3.10.1 Preparation of standards Take the standard and dilute it with aqueous solution to obtain a series of standard working solutions with gradient contents. The contents of vitamin B1, vitamin B2, vitamin B6, and vitamin B9 standards are 0.10, 0.25, 0.50, 1.00, 2.50, 5.00, 10.00, 25.00, and 100.00 ng / mL, respectively; the contents of nicotinamide and nicotinic acid standards are 5.0, 12.5, 25.0, 50.0, 125.0, 250.0, 500.0, 1 250.0, and 5 000.0 ng / mL, respectively; the contents of vitamin B12 standards are 0.50, 1.25, 2.50, 5.00, 12.50, 25.00, 50.00, 125.00, and 500.00 ng / mL, respectively; and the content of nicotinamide-13C6 is 500 ng / mL. The contents of pyridoxal-D3 and folic acid-13C5 were both 20 ng / mL. Take 150 μL of standard working solution in a 1.5 mL centrifuge tube, 600 μL of methanol / acetonitrile (volume ratio 1:1) solution, 10 μL of internal standard solution, vortex mix to prepare the standard curve solution, and establish the standard curve using the isotope internal standard method.

[0060] 2.3.10.2 Metabolite extraction Weigh 0.2 g of fecal sample, 750 μL of methanol / acetonitrile / water (volume ratio 2:2:1) solution, 10 μL of internal standard solution, and vortex mix. The pre-treated sample solution was centrifuged at 14000 r / min and 4°C for 15 min in a low-temperature high-speed centrifuge; 500 μL of supernatant was passed through an Ostro plate and dried with nitrogen at 37°C; 100 μL of 0.3% formic acid aqueous solution was added for re-dissolution, and centrifuged at 14 000 r / min and 4°C for 10 min; the supernatant was taken into a sample injection vial and analyzed according to the above chromatographic conditions.

[0061] 2.3.10.3 On-machine testing Mass spectrometry analysis was performed using a 5500 QTRAP mass spectrometer (AB SCIEX) in positive ion mode. Ionization mode: ESI+, ion spray voltage: +4500 V, ion source temperature: 550°C, curtain gas (CUR) 40.00 psi, nebulizer gas (GS1) 55.00 psi, auxiliary gas (GS2) 55.00 psi, MRM mode was used to detect the ion pairs to be tested, and the MRM conditions are shown in Fig. 20 ; 2.3.11 Determination of fecal organic acids 2.3.11.1 Preparation of standards Accurately weigh appropriate amounts of oxalic acid, citric acid, succinic acid, tartaric acid, DL-malic acid, lactic acid, and acetic acid standards to prepare a mixed standard solution with a concentration of 1 to 200 µg / mL, of which the concentration of citric acid is 3 to 600 µg / mL, and store at 4°C for later use. The external standard method was used for quantification, and the standard curve was drawn with the mass concentration of the standard solution as the abscissa and the peak area as the ordinate to obtain the linear regression equation.

[0062] Malic acid has a right-handed isomer (D-malic acid) and a left-handed isomer (L-malic acid) due to its chiral isomer structure. DL-malic acid is a mixture of D-malic acid and L-malic acid. Therefore, two malic acid standard chromatographic peaks will appear during the detection, for a total of 8 chromatographic peaks.

[0063] 2.3.11.2 Metabolite extraction Weigh 0.05g of fecal sample into a 2mL homogenizer tube containing 1.0mm zirconium oxide beads, add 1mL of 50% methanol water, and use a homogenizer to homogenize the mixture at 3m / s for 30s, repeat twice. Centrifuge (12000×g, 10min) and take the supernatant; take 40μL of fecal supernatant, add 20μL of 200mM 3-NPH (3-nitrophenylhydrazine) and 20μL of 120mM EDC (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride)-6% pyridine, shake and centrifuge to mix, and incubate at 40℃ for 30min. Add 1920μL of 50% methanol water, shake and mix, and centrifuge. Take the supernatant, filter it with a 0.22um microporous membrane filter, and put it into a sample bottle for further analysis.

[0064] 2.3.11.3 On-machine testing The mobile phase contained 5% methanol water + 0.1% formic acid and 0.1% formic acid in methanol, with a flow rate of 0.4 mL / min and 1 μL injected each time. The column temperature was 40 °C. Mass spectrometry scanning was performed using an electrospray ionization (ESI) source and multiple reaction monitoring (MRM) mode. The spray gas and auxiliary heating gas were both 50 psi, and the air curtain gas was 35 psi. The desolvation gas temperature and ionization voltage were 550 °C and 4500 V, respectively. SCEIX OS software was used for quantitative analysis on a UPLC-MS / MS6500+ system (AB Sciex Pte. Ltd., Boston, USA).

[0065] 2.3.12 Data Analysis The non-parametric Kruskal-Wallis and Mann-Whitney rank sum tests were used to test the significance of differences between multiple groups and two groups of samples, respectively. The graphics were mainly drawn using R software (R version: 3.6.1, http: / / www.r-project.org / ) and Origin 2021. The data were expressed as mean ± standard error of the mean (SEM). For the structural analysis of fecal microbial communities, principal coordinates analysis (PCoA) based on Bray-Curtis distance was used to intuitively display the differences in intestinal flora of samples from different groups. Spearman correlation analysis was used to determine the correlation between differential strains of intestinal flora and metabolic pathways.

[0066] 3. Experimental results 3.1. Body weight measurement results Figure 3 In the figure are the weight changes and weight change rates of mice during the experiment, respectively. The significance level in the figure is set to 0.05, and significant differences are indicated by “*” (p< 0.05), “**” (p< 0.01), “***” (p< 0.001) and “****” (p< 0.0001).

[0067] During the experiment, the body weight of each mouse was measured. Figure 3The results show that after drinking 2.5% DSS, the weight of mice showed a downward trend, indicating that drinking 2.5% DSS will cause weight loss in mice. At the end of the experiment, the mean weights of the NC group, IBD group, BX-245 group, and BFM group were 25.16g, 22.40g, 23.61g, 23.81g, and 23.61g, respectively. Compared with the NC group, except for the FM group, the weights of the other groups were significantly reduced (BX-245 group p < 0.05, BFM group p < 0.05, IBD group p < 0.001). Compared with the IBD group, the weights of mice in the BX-245 group, BFM group, and FM group all showed an upward trend, but there was no significant difference. The weight change rate and the overall trend of weight change were the same. Compared with the NC group, the weight of the other groups was significantly reduced (IBD group p < 0.01, BX-245 group p < 0.05, BFM group p < 0.01, FM group p < 0.01).

[0068] 3.2 Disease Activity Index Score Results (Ref. Figure 4 ) The DAI scores of mice were measured every 5 days during the experiment. The DAI scores (calculated by weight loss, stool consistency, and fecal occult blood subscore) after 2.5% DSS administration were as follows: Figure 4 As shown. Compared with the NC group, the scores of the other groups increased significantly (p < 0.05). At the end of the experiment, the DAI scores of the IBD group, BX-245 group, BFM group, and FM group were 5.25 (SEM = 0.9682), 3.00 (SEM = 1.5), 3.38 (SEM = 0.8570), and 3.38 (SEM = 1.4948), respectively. Compared with the IBD group, the DAI scores of the BX-245 group, BFM group, and FM group were significantly reduced. This shows that BX-245 can effectively relieve IBD-related symptoms.

[0069] 3.3 Colon length to colon weight results (ref. Figure 5 ) After the experiment, the mice were killed and their colon tissues were collected to measure colon length and weight. Figure 5 Compared with the NC group, the colon length of mice in the other groups was significantly lower than the colon weight (p < 0.05), indicating that the colon of mice was severely edematous after drinking 2.5% DSS, and the inflammatory bowel disease mouse model was successfully established.

[0070] 3.4 HE staining and pathological scoring test results (ref. Figure 6 ) The H&E-stained colon tissue sections of the five groups of mice were observed and scored under a light microscope. The IBD group had the highest score among all the groups. HE staining of the IBD group showed multifocal ulcer formation, large-area epithelial cell degeneration and shedding, glandular structure atrophy and necrosis, and severe inflammatory cell infiltration. AB-PAS staining showed a decrease in the number of goblet cells. HE staining of colon tissue in the NC group showed that the intestinal mucosal epithelial structure was intact, the crypt structure was not atrophied, and no inflammatory cell infiltration was observed. AB-PAS staining showed blue staining of goblet cells. Compared with the NC group, the histological score of the IBD group increased significantly (p < 0.05). After oral administration of BX-245, HE staining of the BX-245 group showed focal ulcer formation, local epithelial cell degeneration and shedding, glandular structure atrophy and necrosis, and moderate inflammatory cell infiltration. AB-PAS staining showed a decrease in the number of goblet cells. Although HE staining was reduced under light microscopy, there was no significant difference in histological scores (p >0.05).

[0071] 3.5 Immune factor test results (reference Figure 7 ) The occurrence of IBD is related to factors such as imbalanced immune response, dysfunctional digestive tract flora, environmental and genetic susceptibility. Imbalanced immune response caused by over-activation is one of the main characteristics of IBD. INF-γ and TNF-α can stimulate the body to produce inflammatory responses and cause immune imbalance, and are currently considered to be most relevant to the inflammatory state of IBD. The imbalance of intestinal flora induced by IBD inhibits Treg cells, resulting in reduced IL-10 secretion, promoting the differentiation of Th1 cells and the release of Th17 cells, and disrupting the normal immune balance of the intestine. At the end of the experiment, the immune function of mice with inflammatory bowel disease was evaluated by detecting the levels of immune factors in the serum. The results of IL-6, IL-10, TNF-α, and INF-γ content are shown in Table 1. Figure 7 The average levels of IL-6, TNF-α, and INF-γ in the IBD group were 122.42 pg / mL, 618.57 pg / mL, and 668.75 pg / mL, respectively, which were significantly higher than those in the NC group. The average level of IL-10 was 228.10 pg / mL, which was significantly lower than that in the NC group. IL-10 is an anti-inflammatory factor, while IL-6, TNF-α, and INF-γ are pro-inflammatory factors. The results showed that the intestinal inflammation level of mice increased after drinking 2.5% DSS. After BX-245 intervention, the serum pro-inflammatory factor TNF-α level of mice in the BX-245 group, BFM group, and FM group was significantly reduced compared with the IBD group ( p <0.05), the levels of IL-6 and INF-γ in the BX-245 group and the BFM group were significantly lower than those in the IBD group ( p<0.05), and there was no significant difference in serum INF-γ level between BX-245 group and NC group (p >0.05). At the same time, the level of serum anti-inflammatory factor IL-10 in BX-245 group was increased, but no significance was detected (p >0.05). The above performance shows that oral administration of BX-245, BX-245 yogurt and ordinary yogurt have a certain improvement effect on the excessive immune response of the intestine, and oral administration of BX-245 is more excellent in improving the imbalance of immune response in mice.

[0072] 3.6 Targeted Metabolic Outcomes ( Figure 8-Figure 10 ) Intestinal flora metabolites, such as short-chain fatty acids, bile acids, vitamins, organic acids, and amino acids, play a key role in maintaining host health and normal physiological functions. Therefore, at the end of the experiment, the metabolic function of IBD mice was evaluated by detecting the content of metabolites in feces. The test results are shown in Figure 8-Figure 10 , Microbial fermentation of indigestible dietary fiber in the intestine produces short-chain fatty acids (SCFAs) as major products. Short-chain fatty acids can have an impact on the integrity of the human intestinal epithelial and mucosal barriers, immune responses, and the diversity of the microbiota. Therefore, alterations in SCFAs may affect inflammatory bowel disease. In inflammatory bowel disease, SCFAs are involved in the main pathogenic process and play an important role in the development of intestinal inflammation. Therefore, the contents of propionic acid (3.69μmol / g, 3.55μmol / g, 5.97μmol / g, 3.33μmol / g, 3.43μmol / g), butyric acid (6.24μmol / g, 1.89μmol / g, 3.59μmol / g, 2.06μmol / g, 2.02μmol / g), acetic acid (18.96μmol / g, 15.74μmol / g, 19.17μmol / g, 14.54μmol / g, 14.21μmol / g) and n-valeric acid (0.54μmol / g, 0.16μmol / g, 0.45μmol / g, 0.15μmol / g, 0.26μmol / g) in short-chain fatty acids in the NC group, IBD group, BX-245 group, BFM group and FM group were compared with each other. It was found that the butyric acid and valeric acid contents in the IBD group were significantly lower than those in the NC group, while the acetic acid content showed a decreasing trend compared with the NC group, but there was no significant difference. After BX-245 intervention, the butyric acid and valeric acid contents in the BX-245 group were significantly increased compared with the IBD group. The results showed that intragastric administration of BX-245 powder could significantly improve the butyric acid and valeric acid contents in the feces of IBD mice. In summary, BX-245 can improve the related symptoms of inflammatory bowel disease mice by reducing the content of SCFAs in the feces of mice.

[0073] Bile acids have become a key class of microbiota-related metabolites that are disturbed in patients with IBD. In recent years, metabolomics studies have shown that there are persistent defects in bile acid metabolism in patients with IBD, with increased primary bile acids and decreased secondary bile acids. Specific bile acid metabolites interact with intestinal epithelial cells and immune cells, leading to the inflammatory environment that occurs in IBD. Comparison of ursochial acid (0.58 μmol / g, 0.24 μmol / g, 0.43 μmol / g, 0.34 μmol / g, 0.36 μmol / g) in bile acids in the NC, IBD, BX-245, BFM, and FM groups revealed that the ursochial acid content in the IBD, BFM, and FM groups was significantly reduced compared with the NC group. After BX-245 intervention, the ursochial acid content in the BX-245 group was significantly increased compared with the IBD group. The results showed that intragastric administration of BX-245 powder could significantly improve the ursochial acid content in the feces of IBD mice. Ursocholic acid is a secondary bile acid, so BX-245 can reduce the content of secondary bile acids by reducing the content of ursocholic acid and thus improve the bile acid metabolism disorder in mice with inflammatory bowel disease.

[0074] Vitamins are a class of low-molecular organic compounds with biological activity. Although the amount required is very small, their role cannot be replaced by any nutrient. In the body, they can neither produce energy nor are they the raw materials for the formation of tissues, and the human body also needs very little of them. However, they have a huge effect on the overall metabolism of the body. If the human body lacks a certain vitamin for a long time or the supply is insufficient, it will cause metabolic disorders and pathological reactions. Therefore, the VB2 (0.04μmol / g, 0.01μmol / g, 0.02μmol / g, 0.02μmol / g, 0.02μmol / g) and VB5 (0.38μmol / g, 0.05μmol / g, 0.06μmol / g, 0.06μmol / g, 0.06μmol / g) in the NC group, IBD group, BX-245 group, BFM group and FM group were compared with each other. It was found that the VB2 and VB5 contents in the IBD group, BX-245 group, BFM group and FM group were significantly lower than those in the NC group, but there was no significant change after BX-245 intervention. The results showed that the contents of VB2 and VB5 in the feces of mice were significantly reduced after intragastric administration of 2.5% DSS.

[0075] Organic acids can enter the cell wall of bacteria, causing pH gradient changes inside and outside the bacteria, inhibiting bacterial growth. The suitable pH for the growth of several common pathogens is neutral to alkaline, such as 6.0-8.0 for Escherichia coli and 6.0-7.5 for Streptococcus, while probiotics such as lactic acid bacteria are suitable for reproduction in an acidic environment. Citric acid reduces the pH in the gastrointestinal tract, and probiotics such as lactic acid bacteria in the intestines have good growth conditions, thereby maintaining the normal balance of microbial flora in the digestive tract of livestock and poultry. Therefore, the citric acid in organic acids of the NC group, IBD group, BX-245 group, BFM group and FM group (1.47 μmol / g, 0.76 μmol / g, 1.20 μmol / g, 0.94 μmol / g, 0.78 μmol / g) were compared with each other. It was found that the citric acid content in the IBD group, BFM group and FM group was significantly reduced compared with the NC group. After BX-245 intervention, the citric acid content in the BX-245 group was significantly increased compared with the IBD group. The results showed that intragastric administration of BX-245 powder can significantly improve the citric acid content in the feces of IBD mice. In summary, BX-245 can promote the growth of probiotics in the intestine by reducing the citric acid content in the feces of mice.

[0076] Amino acids are an important class of substances, mainly composed of five elements: carbon, hydrogen, oxygen, nitrogen, and sulfur. It is known that 20 amino acids constitute proteins in the human body. Compared with the normal state, the levels of 20 amino acids in serum decrease to varying degrees during inflammatory diseases. Therefore, the tryptophan in the cholangiocarboxylic acid of the NC group, IBD group, BX-245 group, BFM group, and FM group (0.76μmol / g, 0.30μmol / g, 0.63μmol / g, 0.41μmol / g, 0.37μmol / g) was compared with each other. It was found that the tryptophan content in the IBD group and FM group was significantly reduced compared with the NC group. After BX-245 intervention, the tryptophan content in the BX-245 group was significantly increased compared with the IBD group. The results showed that intragastric administration of BX-245 bacterial powder can significantly improve the tryptophan content in the feces of IBD mice. Tryptophan also plays an important role in maintaining the integrity of the intestinal barrier and immune homeostasis. BX-245 can reduce the tryptophan content in the feces of mice with inflammatory bowel disease, thereby protecting the intestinal barrier and immune homeostasis.

[0077] 3.7 Metagenomic Results Intestinal flora imbalance is a key factor leading to the onset of IBD, and restoring normal intestinal flora is also an important way to alleviate IBD symptoms. Therefore, this experiment analyzed the characteristics and composition structure of the intestinal flora of each group of mice. In order to explore the effect of BX-245 on IBD intestinal flora, the α diversity of intestinal flora in the NC group, IBD group, BX-245 group, BFM group and FM group on the 30th day was calculated based on the Shannon index and Simpson index, and the changes in intestinal flora diversity in the NC group, IBD group, BX-245 group, BFM group and FM group were analyzed. At the same time, the principal coordinates analysis (PCoA) of the β diversity of the intestinal flora of mice was performed based on the Bray-Curtis distance.

[0078] The α diversity results are as follows Fig.11 As shown: the S index of the Shannon index had no significant difference among the five groups. The Simpson index and J index of the IBD group were significantly lower than those of the NC group. There were no significant differences among the other groups, indicating that DSS induction would reduce the diversity of the intestinal flora of mice. The non-weighted calculation method mainly considers the presence or absence of species, that is, if the species types of the two groups are the same, it means that the sample distance between the two groups is the smallest; the weighted method considers both the presence or absence of species and the abundance of species. This experiment used two calculation methods to analyze β diversity. The results are shown in the figure. Fig.12 There was a significant difference in β diversity between the NC group and the IBD group (P < 0.05), indicating that the intestinal flora structure of mice was damaged after oral administration of 2.5% DSS. After BX-245 intervention, although there was a significant difference between the BX-245 group and the NC group (P < 0.05), compared with the BFM group and the FM group, the BX-245 group had a more obvious trend of approaching the NC group, indicating that the ability of BX-245 to restore the intestinal flora structure of mice is higher than that of ordinary yogurt.

[0079] Table 3 β diversity significance

[0080] To evaluate the effect of Bifidobacterium animalis subsp. lactis BX-245 on the intestinal flora of IBD mice, the composition of the intestinal flora of each group of mice was analyzed at the phylum level. The results are shown in Fig.13As shown. There were 6 dominant bacterial phyla in the intestines of the three groups of mice, namely Firmicutes, Actinobacteria, Bacteroidetes, Verrucomicrobia, Deferribacteres and Proteobacteria. Among them, Actinobacteria (18.00%), Bacteroidetes (11.30%) and Firmicutes (52.88%) accounted for more than 90% of all groups. The intestinal flora of mice changed after DSS induction. The relative abundance of Actinobacteria in the intestinal flora of IBD group mice increased significantly compared with that of NC group, while the relative abundance of Bacteroidetes tended to increase, but there was no significant difference. There was no significant difference in the relative abundance of Firmicutes. After BX-245 intervention, the relative abundance of Actinobacteria in the intestinal flora of IBD mice could be increased and the relative abundance of Bacteroidetes could be reduced, but there was no significant difference.

[0081] The composition of the intestinal flora of each group of mice was further analyzed at the species level. Figure 14-17shown. The mice in each group were mainly composed of Lactobacillus murinus (28.23%), Lactobacillus reuteri (13.43%), Bifidobacterium pseudolongum (11.31%), Akkermansia muciniphila (9.78%), Mucispirillum schaedleri (4.82%), Lactobacillus johnsonii (3.51%), Bifidobacterium animalis (3.25%), Alistipes unclassified (2.80%), Enterorhabduscaecimuris (2.48%), Bacteroides thetaiotaomicron (2.38%), Bacteroides vulgatus (2.17%) Adlercreutzia equolifaciens (1.75%), Peptostreptococcaceae nonameunclassified (1.60%), Clostridium sp ASF502 (1.37%), Anaerotruncus sp G3 2012 (1.23%), Parabacteroides goldsteinii (1.20%). In order to compare the changes in intestinal bacterial species after oral administration of BX-245, a difference analysis was performed based on the bacterial species level, and a total of 9 different bacteria were identified between the two groups ( Fig.13), compared with the NC group, the relative abundance of Bifidobacterium pseudolongum, Bacteroides thetaiotaomicron, Bacteroides vulgatus, Lactobacillus reuteri, and Peptostreptococcaceae unclassified in the IBD group increased significantly; the relative abundance of Adlercreutzia equolifaciens, Alistipes unclassified, and Mucispirillum schaedleri decreased significantly. After BX-245 intervention, the relative abundance of Bifidobacterium pseudolongum and Bacteroides vulgatus in the BX-245 group decreased significantly compared with the IBD group; the relative abundance of Adlercreutzia equolifaciens increased significantly. Bifidobacterium animalis did not appear in both the NC group and the IBD group, but its relative abundance increased significantly after oral administration of BX-245.

[0082] Key metabolites of Bifidobacterium pseudolongum, HDCA and 12-ketolithocholic acid (12-KCAc), were demonstrated to inhibit the expression of colonic epithelial guanylate cyclase 1A (Gucy1A), thereby attenuating the onset of DSS-induced colitis. Adlercreutzia equolifaciens pellets and, to a lesser extent, their supernatants have anti-inflammatory properties in intestinal epithelial cells and hepatocytes by inhibiting the NF-κB pathway. Bacteroides vulgatus is one of the most abundant bacteria in the human gut and is widely considered to be a pathogen, a commensal capable of causing pathology in response to host and / or environmental triggers. In specific pathogen-free (SPF) mice lacking the intracellular bacterial sensor nod2, which is associated with CD, Bacteroides vulgatus was the sole cause of small intestinal damage.

[0083] refer to Fig.18A total of 269 metabolic pathways were annotated from mouse feces, and 3 metabolic pathways in the top 30 relative abundance showed significant differences among the three groups (P < 0.05), namely 3-dehydroquinoline biosynthesis (PWY-6163), uracil nucleotide biosynthesis (PWY-5686), and tRNA charging pathway (TRNA-CHARGING-PWY): The tRNA charging pathway is the process of aminoacylation of tRNA, during which amino acids are selected in protein synthesis, which is a two-step reaction involving the activation of the amino acid and then its transfer to tRNA. Compared with the NC group, the biosynthesis of 3-dehydroquinoline in the IBD group was significantly decreased, and after BX-245 intervention, the biosynthesis of 3-dehydroquinoline in the BX-245 group was significantly increased compared with the IBD group; compared with the NC group, there were no significant differences in uracil nucleotide biosynthesis and tRNA charging pathway between the IBD group and the BX-245 group, but after BX-245 intervention, the biosynthesis of uracil nucleotide and tRNA charging pathway in the BX-245 group were significantly increased compared with the IBD group.

[0084] In addition, to further explore the relationship between different bacterial species and different functions, correlation analysis was performed. Fig.19 The uracil nucleotide biosynthesis shown was significantly positively correlated with Adlercreutzia equolifaciens and Mucispirillumschaedleri, and significantly negatively correlated with Bacteroides vulgatus, Bacteroides thetaiotaomicron and Peptostreptococcaceae unclassified; the uracil nucleotide biosynthesis was significantly positively correlated with Alistipes unclassified, and significantly negatively correlated with Lactobacillus reuteri; 4. Conclusion (1) During the experimental period, animal Bifidobacterium lactis subspecies BX-245 can significantly increase the body weight of mice with inflammatory bowel disease, significantly improve the DAI score, reduce the levels of pro-inflammatory factors IL-6, TNF-α, and INF-γ in serum, and increase the level of anti-inflammatory factor IL-10, thereby alleviating the excessive immune response state of mice with inflammatory bowel disease.

[0085] (2) Compared with the NC group, DSS caused a decrease in the diversity of intestinal flora and intestinal flora imbalance in IBD mice. After intervention with BX-245, the diversity of intestinal flora was significantly restored and the metabolic pathway was improved. The main manifestations were that the relative abundance of Bifidobacterium pseudolongum and Bacteroides vulgatus was significantly reduced; the relative abundance of Adlercreutzia equolifaciens was significantly increased. The above three strains are related to inflammation and intestinal barrier. In summary, BX-245 can improve the related symptoms of inflammatory bowel disease mice by restoring intestinal flora imbalance.

[0086] (3) After BX-245 intervention, the levels of butyric acid, valeric acid, ursocholic acid, citric acid, and tryptophan in the feces of mice were significantly increased compared with those in the IBD group, indicating that BX-245 can improve the metabolic disorders of IBD mice induced by DSS modeling.

[0087] The above description is only a preferred embodiment of the present invention, and does not limit the patent scope of the present invention. All equivalent structural changes made by using the contents of the present invention specification and drawings under the inventive concept of the present invention, or directly / indirectly applied in other related technical fields are included in the patent protection scope of the present invention.

Claims

1. The use of animal Bifidobacterium lactis subspecies BX-245 for alleviating inflammatory bowel disease, characterized in that: The Bifidobacterium animalis subsp. lactis BX-245 is deposited in the General Microbiological Culture Collection Center of China Microbiological Culture Collection Administration, with the deposit number: CGMCC No.26973.

2. The use of animal Bifidobacterium lactis subspecies BX-245 for alleviating inflammatory bowel disease according to claim 1, characterized in that: The invention relates to the application of animal Bifidobacterium lactis subspecies BX-245 in the preparation of relief products / relief medicines for inflammatory bowel disease.

3. The use of animal Bifidobacterium lactis subspecies BX-245 for alleviating inflammatory bowel disease according to claim 1, characterized in that: Animal Bifidobacterium lactis subspecies BX-245 can improve and reduce the levels of pro-inflammatory factors IL-6, TNF-α, and INF-γ in serum and increase the level of anti-inflammatory factor IL-10.

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