Remission application of Bifidobacterium animalis subsp. lactis BX-245 in inflammatory bowel disease

Abstract

Use of Bifidobacterium animalis subsp. lactis BX-245 in the preparation of a probiotic preparation for relieving inflammatory bowel disease, characterized in that the Bifidobacterium animalis subsp. lactis BX-245 is deposited in the China General Microbiological Culture Collection Center, with the deposit number: CGMCC No. 26973; the purpose of this invention is to explore the alleviating effect of Bifidobacterium animalis subsp. lactis BX-245 on dextran sulfate sodium (DSS)-induced IBD mice. Six-week-old male C57BL / 6J mice with normal food intake and water intake were randomly divided into a normal group (NC group), a model group (IBD group), a regular yogurt group (FM group), a Bifidobacterium animalis subsp. lactis BX-245 powder group (BX-245 group), and a BX-245 yogurt group (BFM group). The alleviating effect of Bifidobacterium animalis subsp. lactis BX-245 and its fermented milk on the symptoms of murine inflammatory bowel disease was evaluated by body weight changes, immune factors, targeted metabolomics, and metagenomics, aiming to provide data support for the application of Bifidobacterium animalis subsp. lactis BX-245 in products.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, specifically to the application of Bifidobacterium animalis subsp. lactis 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 etiology is not fully understood, but it is generally believed to be caused by the combined effects of genetic susceptibility, abnormal immune system, and environmental factors. Symptoms of IBD patients include diarrhea, abdominal pain, weight loss, fever, etc., and severe cases can lead to complications such as malnutrition, intestinal perforation, and canceration.

[0003] Existing treatment methods mainly include drug treatment and surgical treatment. Drug treatment includes immunosuppressants, anti-inflammatory drugs, antibiotics, and biological agents, etc. 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 severe patients, but the risks brought by surgery and the postoperative recovery time are long, and some patients may have complications or recurrence of the disease after surgery.

[0004] In addition, most of the existing probiotic preparations focus on the function of promoting intestinal health. Different probiotic strains have significant differences in clinical performance, and for the problems unique to IBD patients, such as abnormal immune response and damaged intestinal barrier, no targeted application strategies are provided. Summary of the Invention

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

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

[0007] As a further preferred solution of the present invention, it is based on the application of Bifidobacterium animalis subsp. lactis BX-245 in the preparation of products / drugs for alleviating inflammatory bowel disease.

[0008] As a further preferred embodiment of the present invention, the content of pro-inflammatory factors IL-6, TNF-α, and INF-γ in the serum is reduced and the content of anti-inflammatory factor IL-10 is increased by Bifidobacterium animalis subsp. lactis BX-245.

[0009] The purpose of this study is to investigate the alleviating effect of Bifidobacterium animalis subsp. lactis BX-245 on dextran sulfate sodium (DSS)-induced IBD mice. Six-week-old male C57BL / 6J mice with normal feeding and drinking were randomly divided into a normal group (NC group), a model group (IBD group), a regular yogurt group (FM group), a Bifidobacterium animalis subsp. lactis BX-245 powder group (BX-245 group), and a BX-245 yogurt group (BFM group). The alleviating effect of Bifidobacterium animalis subsp. lactis BX-245 and its fermented milk on the symptoms of murine inflammatory bowel disease was evaluated by body weight changes, immune factors, targeted metabolomics, and metagenomics, aiming to provide data support for the application of Bifidobacterium animalis subsp. lactis BX-245 in products.

[0010] Compared with the prior art, the present invention provides the alleviating application of Bifidobacterium animalis subsp. lactis BX-245 in inflammatory bowel disease, and has the following beneficial effects:

[0011] 1. During the experimental period, Bifidobacterium animalis subsp. lactis BX-245 can significantly increase the body weight of mice with inflammatory bowel disease, and at the same time can significantly increase the DAI score, and reduce the content of pro-inflammatory factors IL-6, TNF-α, and INF-γ in the serum and increase the content of anti-inflammatory factor IL-10, alleviating the over-immune response state of mice with inflammatory bowel disease.

[0012] 2. Compared with the NC group, DSS can cause a decrease in the diversity of the intestinal flora and intestinal flora dysregulation in IBD mice. After BX-245 intervention, the diversity of the intestinal flora can be significantly restored, and the metabolic pathway can be improved. The main manifestations are that the relative abundances of Bifidobacterium pseudolongum and Bacteroides vulgatus are significantly decreased; the relative abundance of Adlercreutzia equolifaciens is significantly increased. The above three strains are related to inflammation and the intestinal barrier. In summary, BX-245 can improve the related symptoms of mice with inflammatory bowel disease by restoring intestinal flora dysregulation.

[0013] 3. After BX-245 intervention, compared with the mice in the IBD group, the contents of butyric acid, n-valeric acid, ursodeoxycholic acid, citric acid, and tryptophan in the feces of mice are significantly increased. It shows that BX-245 can improve the metabolic disorder of mice with inflammatory bowel disease caused by DSS modeling. Description of the Drawings

[0014] Figure 1 It is the experimental design diagram of the present invention;

[0015] Figure 2 Schematic diagram of the library construction process of the present invention;

[0016] Figure 3 Graph of mouse body weight detection results;

[0017] Figure 4 Graph of disease activity index score results;

[0018] Figure 5 Results of colon length to colon weight detection;

[0019] Figure 6 Results of HE staining and pathological score detection;

[0020] Figure 7 Graph of immune factor detection results;

[0021] Figure 8 Graph of short-chain fatty acid targeted metabolism detection results;

[0022] Figure 9 Graph of bile acid, amino acid, and organic acid targeted metabolism detection results;

[0023] Figure 10 Graph of tryptophan targeted metabolism detection results;

[0024] Figure 11 Graph of α-diversity detection results;

[0025] Figure 12 Graph of β-diversity detection results;

[0026] Figure 13 Graph of intestinal flora phylum level analysis results

[0027] Figure 14 Graph of relative abundance comparison of differential strains;

[0028] Figure 15 Graph of comparative analysis of differential strains of intestinal flora in different groups of mice based on bacterial species level - Figure 1;

[0029] Figure 16 Graph of comparative analysis of differential strains of intestinal flora in different groups of mice based on bacterial species level - Figure 2;

[0030] Figure 17 Graph of comparative analysis of differential strains of intestinal flora in different groups of mice based on bacterial species level - Figure 3;

[0031] Figure 18 Graph of metabolic pathway analysis results;

[0032] Figure 19 Graph of correlation analysis results between differential strains and differential metabolic pathways;

[0033] Figure 20 It is a table diagram of MRM conditions. Specific implementation manners

[0034] The endpoints and any values within the ranges disclosed herein are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the values between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0035] "Bifidobacterium animalis subsp. lactis BX-245" in the examples was obtained by purchase and is derived from Inner Mongolia Agricultural University.

[0036] As a specific embodiment of the present invention:

[0037] 1. Experimental purpose

[0038] The purpose of this study was to investigate the alleviating effect of Bifidobacterium animalis subsp. lactis BX-245 on dextran sulfate sodium (DSS)-induced IBD mice. Six-week-old male C57BL / 6J mice with normal feeding and drinking were randomly divided into a normal group (NC group), a model group (IBD group), a common yogurt group (FM group), a Bifidobacterium animalis subsp. lactis BX-245 powder group (BX-245 group), and a BX-245 yogurt group (BFM group). The alleviating effect of Bifidobacterium animalis subsp. lactis BX-245 and its fermented milk on the symptoms of murine inflammatory bowel disease was evaluated by body weight changes, immune factors, targeted metabolomics, and metagenomics, aiming to provide data support for the application of Bifidobacterium animalis subsp. lactis BX-245 in products.

[0039] 2. Experimental materials and methods

[0040] 2.1 Main materials

[0041] Table 1 Information of main experimental materials

[0042]

[0043]

[0044] 2.2 Preparation of yogurt samples

[0045] BX-245 Yogurt: Using raw milk and 6.5% sucrose as raw materials, inoculate Bifidobacterium animalis subsp. lactis BX-245 and a commercial starter culture (PYS-010) for fermentation. Completely dissolve 6.5% sucrose in preheated milk, inoculate and ferment after homogenization, sterilization, and cooling processes. Wait until the TA value ≥ 70°T to reach the fermentation end point, cool in a water bath to 20°C and then aseptically package. After ripening at 2-6°C for 24 h, aseptically package and store at 4°C.

[0046] Ordinary Yogurt: Using raw milk and 6.5% sucrose as raw materials, ferment with a commercial starter culture (PYS-010). Completely dissolve 6.5% sucrose in preheated milk, inoculate and ferment after homogenization, sterilization, and cooling processes. Wait until the TA value ≥ 70°T to reach the fermentation end point, cool in a water bath to 20°C and then aseptically package. After ripening at 2-6°C for 24 h, aseptically package and store at 4°C.

[0047] To ensure the viable count of bacteria in yogurt during the experiment, BX-245 yogurt and ordinary yogurt were each fermented in two batches. The first batch of yogurt was gavaged from day 0 to day 15 (the viable count of Bifidobacterium in BX-245 yogurt was 7.5×108 CFU / mL), and the second batch of yogurt was gavaged from day 16 to day 30 (the viable count of Bifidobacterium in BX-245 yogurt was 7.7×108 CFU / mL). The daily gavage volume was 0.2 mL / animal.

[0048] 2.3 Experimental Methods

[0049] 2.3.1 IBD Animal Experiment Design

[0050] After 40 C57BL / 6J mice were adaptively fed (-1 week to 0 week), they were randomly divided into 5 groups, with 8 mice in each group ( Figure 1 ). Control group (NC group), model group (IBD group), probiotic group (B X-245 group), BX-245 yogurt group (BFM group), and ordinary yogurt group (FM group).

[0051] During the adaptive feeding period and the experimental period, all mice had free access to food and water. From day 0 to day 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. From day 5 to day 10, all mice were given sterile water. This process was one cycle, and a total of three cycles were carried out to construct a mouse model of inflammatory bowel disease.

[0052] Gavage: The NC group and the IBD group were given 0.2 mL / day of sterile normal saline by 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 normal saline daily) by gavage starting from day 0.

[0053] The BFM group was given 0.2 mL / day by gavage starting from day 0,

[0054] From day 0, the BX-245 yogurt group and the FM group were intragastrically administered with 0.2 mL / day of regular yogurt.

[0055] Preparation of 2.5% DSS solution: The DSS powder was fully stirred and dissolved in sterile water, and the DSS solution was changed every two days.

[0056] All C57BL / 6J mice were weighed on days 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30 during the experiment. Fecal samples were collected and DAI scores were determined on days 0, 5, 10, 15, 20, 25, and 30.

[0057] On day 30 of the experiment, after sacrificing the C57BL / 6J mice, blood supernatants, colons, and their contents were collected to detect the levels of mouse immune factors (IL-6, IL-10, TNF-α, IFN-γ), and H&E staining and pathological scoring of the mouse colon were performed.

[0058] 2.3.2 Disease activity index (DAI) scoring

[0059] In this study, the DAI scoring index was used to evaluate the degree of intestinal injury in colitis mice. This scoring mainly included the weight loss rate, fecal characteristics, and occult blood conditions. During the experiment, the mental state, activity, and diet of the mice were observed, and the mice were weighed regularly. DAI scores were determined on days 0, 5, 10, 15, 20, 25, and 30 of the experiment.

[0060] DAI scoring method: Take fresh feces (30 mg) and spread them evenly on the fecal occult blood test paper to observe the fecal occult blood condition and fecal characteristics. If blood can be directly seen or the feces are in a watery state and the fecal occult blood test paper changes color within 10 s, it is scored as "++++" with a score of 4 points; if the feces are in a thin paste state and change color within 30 - 60 s, it is scored as "+++" with a score of 3 points; if the feces are soft and change color within 60 - 120 s, it is scored as "++" with a score of 2 points; if the feces are soft and change color after more than 120 s, it is scored as "-" with a score of 0 points.

[0061] The weight change rate was calculated according to the formula. W1 represents the weight (g) of the mouse one day before model establishment, and W2 represents the weight (g) of the mouse every day after model establishment.

[0062]

[0063] 2.3.3 Mouse colon length to colon weight

[0064] Colonic shortening is also one of the pathological manifestations of murine colitis. After the mice were sacrificed, the colonic length of the mice was collected and recorded. The colonic mass of mice in different experimental groups was compared, and the colonic status, color, presence or absence of dilation, ulcers, etc. were observed in the natural stretched state. The weight was measured, its length was measured, and the colonic length-to-weight ratio was calculated. According to the ratio of the two, the edema condition of the colonic tissue was analyzed. If the ratio was smaller, the edema condition was more severe, and vice versa.

[0065] 2.3.4 HE (hematoxylin-eosin staining) staining and pathological scoring of murine colon

[0066] After the mice were sacrificed, 1 cm of the colon distal to the anus was taken for HE staining. Hematoxylin-eosin staining method, abbreviated as HE staining method, can accurately localize and qualitatively analyze cells, extracellular matrix and other substances in tissues, understand the organizational structure and function of the colon. Through HE staining, the morphological features of the colonic 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 the damage of colonic tissue in mice with inflammatory bowel disease.

[0067] Procedure for making sections:

[0068] The colonic tissue was placed in an embedding cassette → the embedding cassettes were respectively immersed in alcohol solutions with concentrations of 55%, 70%, 80%, 90%, 95% and 100% for 40 min → immersed in two absolute alcohols for 30 min each → the embedding cassettes with colonic tissue were successively placed in xylene I and xylene II for 0.5 h → the colonic tissue was placed in a container with xylene reagent and soaked until it became transparent → placed in paraffin → waited for the paraffin to solidify → trimmed the wax block → installed a microtome knife to cut sections → flattened the sections at a constant temperature with water → removed the excess water → placed in an incubator at 60 - 65 °C → removed the paraffin between tissues.

[0069] Procedure for HE staining operation:

[0070] The sections were taken out of the refrigerator → placed at room temperature for 5 min → put into an oven at 60 °C for 40 min → the sections were taken out → placed at room temperature for 10 min → put into 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 → rinsed with distilled water → stained with hematoxylin staining solution for 3 min → rinsed with tap water to remove the staining solution → differentiated and stained with hematoxylin for 3 s → rinsed with tap water → the cell nuclei showed blue → rinsed with tap water → dropped eosin staining solution and stained for 3 min → terminated the staining.

[0071] HE staining scoring criteria:

[0072]

[0073] AB-PAS staining steps:

[0074] 1. Dewax to water: 5 minutes in xylene I, 5 minutes in xylene II, 5 minutes in xylene III, 1 minute in absolute ethanol, 1 minute in 95% ethanol, 1 minute in 75% ethanol, and wash with distilled water for 5 minutes;

[0075] 2. Dropwise add Alcian blue staining solution for 10 - 20 minutes, wash with distilled water 3 times, 1 - 2 minutes each time;

[0076] 3. Oxidize in the oxidant for 5 minutes. Rinse with tap water and soak in distilled water 2 times;

[0077] 4. Incubate in Schiff staining solution for 10 - 20 minutes;

[0078] 5. Pour off the Schiff staining solution and rinse with running water for 10 minutes;

[0079] 6. Stain the nucleus with hematoxylin staining solution for 1 - 2 minutes and wash with water;

[0080] 7. Differentiate with acidic differentiating solution for 2 - 5 seconds and wash with water;

[0081] 8. Blue with Scott bluing solution for 3 minutes and wash with water for 3 minutes;

[0082] 9. Dehydrate and clear: 1 minute in 75% ethanol, 1 minute in 95% ethanol, 1 minute in absolute ethanol, 3 times in xylene, 1 - 2 minutes each time, and seal with neutral balsam.

[0083] 2.3.5 Detection of mouse immune factors

[0084] In this study, mouse interleukin 6 (IL-6), interleukin 10 (IL-10), tumor necrosis factor α (TNF-α), and mouse interferon γ (INF-γ) ELISA kits were used to detect cytokines such as IL-6, IL-10, TNF-α, and IFN-γ in mouse serum, and to evaluate the effect of BX-245 on the intestinal immune balance in mice with inflammatory bowel disease.

[0085] Table 2 Enzyme-linked immunosorbent assay method

[0086]

[0087]

[0088] 2.3.6 Extraction and sequencing of intestinal microbial metagenomic DNA from fecal samples

[0089] Method for extracting intestinal microbial DNA from fecal samples - Pro DNA Kit method: Refer to the kit instruction manual for the method:

[0090] Briefly rotate the PowerBead Pro tube to ensure that the beads settle to the bottom. Add 250 mg of fecal sample and 800 μL of Solution CD1, and vortex briefly to mix; fix the PowerBead Pro tube horizontally on a 2 mL tube vortex oscillator and vortex at maximum speed for 10 min; centrifuge at 15000×g for 1 min; transfer the supernatant to a clean 2 mL centrifuge tube (Microcentrifuge Tube) provided; add 200 μL of Solution CD2, and vortex for 5 s; centrifuge at 15000×g for 1 min; transfer the supernatant to a clean 2 mL centrifuge tube (Microcentrifuge Tube) provided; add 600 μL of Solution CD3, and vortex for 5 s; load 650 μL of lysis buffer into an MB spin column (MB Spin Column), and centrifuge at 15000×g for 1 min; discard the effluent, and repeat the previous step to ensure that all lysis buffer passes through the MB spin column; place the MB spin column into a clean 2 mL collection tube (Collection Tube) provided; add 500 μL of Solution EA to the MB spin column, and centrifuge at 15000×g for 1 min; discard the effluent, 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 15000×g for 1 min; discard the effluent, and place the MB spin column into a new clean 2 mL collection tube (Collection Tube) provided; centrifuge at 15000×g for 2 min; place the MB spin column into a new clean 1.5 mL elution tube (Elution Tube) provided; add 50 - 100 μL of Solution C6 to the center of the white filter membrane, and centrifuge at 15000×g for 2 min; discard the MB spin column and collect the DNA. Detect the purity of the DNA using a Nanodrop One ultra-micro ultraviolet spectrophotometer and 0.6% agarose gel electrophoresis, and store the DNA in a -20°C refrigerator for later use.

[0091] Method for purifying intestinal microbial DNA from fecal samples - magnetic bead method:

[0092] Measure the volume of DNA and transfer it to a new 1.5 mL low DNA-binding tube (DNA LoBind Tube). Add DNA fragment sorting and purification magnetic beads with a volume of 0.45×, and flick to mix well. Place it on a rotary mixer and rotate for 10 min. After removing, let it stand at room temperature for 10 min. Vortex for 5 s, place it on a magnetic stand, and wait for the magnetic beads to adsorb. Transfer all the liquid to a clean 2 mL centrifuge tube (Microcentrifuge Tube). Add 1.5 mL of 75% ethanol, immediately aspirate it after injection, and repeat once. Air-dry the magnetic beads, but do not let the magnetic beads dry to the extent of cracking. Remove the low DNA-binding tube from the magnetic stand, add 30 μL of nuclease-free water (Water Nuclease-Free) to resuspend, and flick to mix well. Place it on a rotary mixer and rotate for 10 min. After removing, let it stand at room temperature for 10 min. Vortex for 5 s, place it on a magnetic stand, and wait for the magnetic beads to adsorb. Transfer all the liquid to a new clean 1.5 mL low DNA-binding tube to obtain purified DNA. Use a Qubit analysis kit to detect the concentration of metagenomic DNA, and again use a NanoDrop spectrophotometer and 0.6% agarose gel electrophoresis to detect the purity of metagenomic DNA to ensure that the purified DNA meets the purity and quality requirements for library construction and sequencing. Temporarily store the DNA samples that meet the requirements of subsequent experiments in a -20°C refrigerator for subsequent experimental use. Quality requirements for intestinal microbial DNA in fecal samples:

[0093] Total amount ≥ 2 μg; fragment length ≥ 20 Kb, concentration ≥ 20 ng / μL; purity: OD 260 / 280 ≥ 1.8; OD 260 / 230 ≥ 1.5.

[0094] Library construction process reference Figure 2

[0095] ① DNA fragmentation. Fragment the DNA to about 10 Kb to fragment the genome.

[0096] ② End modification. Use Taq polymerase to fill in uneven ends and add protruding base A at both ends to generate sticky ends.

[0097] ③ Adapter addition. After end modification, the ends of the PCR fragments have protruding A tails, while the adapters have protruding T tails. Ligase can be used to add the adapters to both ends of the DNA fragments. The adapters from NEB are circular structures connected by special base U. After connecting the adapters, delete base U to form a "Y" - shaped adapter, ensuring that both ends of each single sequence are different sequencing primers, so that different oligonucleotide sequences can be ligated in subsequent PCR.

[0098] 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.

[0099] ⑤PCR amplification: Use primers complementary to the adapter to perform amplification.

[0100] ⑥ Second magnetic bead purification: Perform magnetic bead purification again to separate the product DNA fragments from impurities such as polymerase.

[0101] ⑦ Quality detection: Perform DNA concentration detection, agarose gel electrophoresis and fragment length detection to complete library construction.

[0102] Finally, the qualified DNA library was constructed and sequenced on the Illumina NovaSeq platform to generate double-end sequences.

[0103] Data output statistics:

[0104] 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.

[0105] 2.3.7 Determination of fecal short-chain fatty acids

[0106] 2.3.7.1 Preparation of Standards

[0107] 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 the hexanoic acid working standard solution, 20 μL of the 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.

[0108] 2.3.7.2 Metabolite extraction

[0109] Accurately weigh 0.02g of feces in 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.

[0110] 2.3.7.3 On-machine testing

[0111] 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 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.

[0112] 2.3.8 Fecal bile acid determination

[0113] 2.3.8.1 Preparation of Standards

[0114] 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.

[0115] 2.3.8.2 Metabolite extraction

[0116] 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 into a tissue grinder, grind 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.

[0117] 2.3.8.3 On-machine testing

[0118] The parameters of the EXion LC liquid chromatography are as follows: ACQUITY is used. BEH C18 chromatographic column (2.1×100 mm, 1.7 μm), injection volume is 5 μL, column temperature is 40 °C, mobile phase is 0.01% formic acid water (A) and acetonitrile (B). The gradient elution conditions are as follows: 0 - 4 min, 25% B; 4 - 9 min, 25 - 30% B; 9 - 14 min, 30 - 36% B; 14 - 18 min, 36 - 38% B; 18 - 24 min, 38 - 50% B; 24 - 32 min, 50 - 75% B; 32 - 33 min, 75 - 90% B; 33 - 35.5 min, 90 - 25% B. The flow rate is 0.25 mL / min.

[0119] The mass spectrometry detection of metabolites is completed by an AB6500 Plus mass spectrometer, using the negative ion ionization mode. The ion source temperature is 500 °C, the ion source voltage is -4500 V, the collision gas is 6 psi, the curtain gas is 30 psi, the nebulizing gas and the auxiliary gas are both 50 psi, and multiple reaction monitoring is used for scanning.

[0120] 2.3.9 Determination of fecal amino acids

[0121] 2.3.9.1 Preparation of standards

[0122] Accurately weigh 19 amino acid standards, prepare a mixed standard linear stock solution with a concentration of 5 mg / mL, and dilute the linear stock solution with methanol to obtain a series of concentrations. Prepare solutions of a certain concentration of L-Alanine-d4 and Phenylanine-d2, and mix well to obtain the internal standard solution. The stock solutions and working solutions of linear, internal standard, and quality control are all stored in a -20 °C refrigerator.

[0123] 2.3.9.2 Extraction of metabolites

[0124] Take the sample and add mass spectrometry water at a ratio of 1:1, vortex and mix well to obtain the diluted sample; take 50 μL of the diluted sample, add 200 μL of the precipitant containing the mixed internal standard (acetonitrile:methanol = 1:1), vortex and mix well, let stand on ice for 30 min, and centrifuge at 12000 r / min at 4 °C for 10 min.

[0125] 2.3.9.3 Detection on the machine

[0126] Chromatographic column: ACQUITY UPLC BEH Amide (2.1×100 mm, 1.7 μm); Mobile phase: Phase A is an aqueous solution of 5 mmol / L ammonium acetate containing 0.1% formic acid; Phase B is acetonitrile containing 0.1% formic acid; Column temperature is 50 °C; Injection volume is 1 μL; Flow rate is 0.3 mL / min; Chromatographic gradient is preset according to the standard coefficient; Mass spectrometry conditions are electrospray ionization source (ESI), positive ion ionization mode, ion source temperature is 550 °C, ion source voltage is 5500 V, curtain gas is 35 psi, nebulizing gas is 50 psi, auxiliary gas is 60 psi. Multiple reaction monitoring is used for scanning.

[0127] 2.3.10 Determination of fecal vitamins

[0128] 2.3.10.1 Preparation of standards

[0129] Take the standard products and dilute them with aqueous solution to prepare standard working solutions with a series of gradient contents. The prepared 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, 100.00 ng / mL in sequence; The prepared contents of nicotinamide and niacin standards are 5.0, 12.5, 25.0, 50.0, 125.0, 250.0, 500.0, 1250.0, 5000.0 ng / mL in sequence; The prepared contents of vitamin B12 standards are 0.50, 1.25, 2.50, 5.00, 12.50, 25.00, 50.00, 125.00, 500.00 ng / mL in sequence; The content of nicotinamide-13C6 is 500 ng / mL. The contents of pyridoxal-D3 and folic acid-13C5 are both 20 ng / mL. Take 150 μL of the standard working solution into

[0130] a 1.5 mL centrifuge tube, add 600 μL of methanol / acetonitrile (volume ratio 1:1) solution and 10 μL of internal standard solution, vortex and mix evenly to prepare a standard curve solution, and establish a standard curve using the isotope internal standard method.

[0131] 2.3.10.2 Extraction of metabolites

[0132] Weigh 0.2 g of fecal sample, add 750 μL of methanol / acetonitrile / water (volume ratio 2:2:1) solution and 10 μL of internal standard solution, and homogenize and vortex mix. Centrifuge the pretreated sample solution at 14000 r / min and 4 °C for 15 min using a low-temperature high-speed centrifuge; Take 500 μL of the supernatant, pass it through an Ostro plate, and dry it under nitrogen at 37 °C; Add 100 μL of 0.3% formic acid aqueous solution for reconstitution, centrifuge at 14000 r / min and 4 °C for 10 min; Take the supernatant into an injection vial and analyze it according to the above chromatographic conditions.

[0133] 2.3.10.3 On-machine testing

[0134] Mass spectrometry analysis was performed using a 5500QTRAP 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 Figure 20 ;

[0135] 2.3.11 Determination of fecal organic acids

[0136] 2.3.11.1 Preparation of Standards

[0137] 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. Use the external standard method for quantification, plot the standard curve with the mass concentration of the standard solution as the abscissa and the peak area as the ordinate, and obtain the linear regression equation.

[0138] Malic acid has a right-handed isomer (D-malic acid) and a left-handed isomer (L-malic acid) due to its chiral isomer structure, and 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.

[0139] 2.3.11.2 Metabolite extraction

[0140] Weigh 0.05g of fecal sample into a 2mL homogenizer tube containing 1.0mm zirconium oxide beads, add 1mL of 50% methanol water, 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, centrifuge, take the supernatant, filter it with a 0.22um microporous membrane filter, and put it into a sample bottle for further analysis.

[0141] 2.3.11.3 On-machine testing

[0142] The mobile phase contains 5% methanol water + 0.1% formic acid and 0.1% formic acid methanol, the flow rate is 0.4 mL / min, 1 μL is injected each time, and the column temperature is 40 °C. Mass spectrometry scanning is carried out using the electrospray ionization (ESI) source and the multiple reaction monitoring (MRM) mode. The spray gas and the auxiliary heating gas are both 50 psi, and the air curtain gas is 35 psi. The desolvation gas temperature and the ionization voltage are 550 °C and 4500 V respectively. The SCEIX OS software is used for quantitative analysis of the UPLC-MS / MS 6500+ system (AB Sciex Pte. Ltd., Boston, USA).

[0143] 2.3.12 Data analysis

[0144] The non-parametric Kruskal-Wallis and Mann-Whitney rank sum tests were used to perform significance tests on multiple groups and two groups of samples respectively. The plotting was mainly done using R software (R version: 3.6.1, http: / / www.r-project.org / ) and Origin 2021. The data was expressed as mean ± standard error of the mean (SEM). For the structural analysis of the fecal microbial community, principal coordinates analysis (PCoA) based on Bray-Curtis distance was used to visually display the differences in the intestinal flora of samples in different groups. The Spearman correlation coefficient was used to analyze and determine the correlation between the differential strains of the intestinal flora and the metabolic pathways.

[0145] 3. Experimental results

[0146] 3.1. Results of body weight measurement

[0147] Figure 3 Shown in the figure are the body weight changes and the body weight change rates of the mice during the experiment. The significance level in the figure was set at 0.05, and significant differences were indicated by "*" (p < 0.05), "**" (p < 0.01), "***" (p < 0.001), and "****" (p < 0.0001).

[0148] During the experiment, the body weight of each mouse was weighed. According to Figure 3The results showed that after drinking 2.5% DSS, the body weights of the mice showed a downward trend, indicating that drinking 2.5% DSS could cause weight loss in mice. At the end of the experiment, the average body weights of the NC group, IBD group, BX-245 group, BFM group, and FM group were 25.16 g, 22.40 g, 23.61 g, 23.81 g, and 23.61 g respectively. Compared with the NC group, the body weights of all other groups except the FM group were significantly reduced (p < 0.05 for the BX-245 group, p < 0.05 for the BFM group, p < 0.001 for the IBD group). Compared with the IBD group, the body weights of the mice in the BX-245 group, BFM group, and FM group showed an upward trend, but there was no significant difference. The weight change rate was the same as the overall trend of weight change. Compared with the NC group, the body weights of all other groups were significantly reduced (p < 0.01 for the IBD group, p < 0.05 for the BX-245 group, p < 0.01 for the BFM group, p < 0.01 for the FM group).

[0149] 3.2 Results of disease activity index scoring (refer to Figure 4 )

[0150] During the experiment, the mice were scored for DAI every 5 days. The results of the DAI scoring (calculated by weight loss, fecal consistency, and fecal occult blood subscores) after administration of 2.5% DSS are as Figure 4 shown. Compared with the NC group, the scores of all other groups were significantly increased (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 indicates that BX-245 can effectively relieve IBD-related symptoms.

[0151] 3.3 Results of colon length to colon weight (refer to Figure 5 )

[0152] After the experiment, the mice were sacrificed and the colon tissues were collected and measured for colon length and weight. The results are as Figure 5 shown. Compared with the NC group, the colon length to colon weight of the mice in all other groups was significantly reduced (p < 0.05), indicating that after drinking 2.5% DSS, the mice had severe colon edema, and the inflammatory bowel disease mouse model was successfully established.

[0153] 3.4 Results of HE staining and pathological scoring detection (refer to Figure 6 )

[0154] Observed the colon tissue sections of five groups of mice stained with H&E and scored them under a light microscope. The IBD group had the highest score among all groups. In the IBD group, multi-focal ulcer formation was visible in the HE staining, with extensive degeneration and exfoliation of epithelial cells, atrophy and necrosis of glandular structures, accompanied by severe inflammatory cell infiltration. The AB-PAS staining showed a decrease in the number of goblet cells. In the NC group, the HE staining of the colon tissue showed a complete intestinal mucosal epithelial structure, no atrophy of crypt structures, and no inflammatory cell infiltration. The AB-PAS staining showed blue-stained goblet cells. Compared with the NC group, the histological score of the IBD group increased significantly (p < 0.05). After intragastric administration of BX-245, focal ulcer formation was visible in the BX-245 group compared with the IBD group in the HE staining, with local epithelial cell degeneration and exfoliation, atrophy and necrosis of glandular structures, accompanied by moderate inflammatory cell infiltration. The AB-PAS staining showed a decrease in the number of goblet cells. Although a decrease in HE staining was found under the light microscope, there was no significant difference in the histological score (p > 0.05).

[0155] 3.5 Detection results of immune factors (refer to Figure 7 )

[0156] The occurrence of IBD is related to factors such as immune response imbalance, dysfunction of digestive tract flora, environment, and genetic susceptibility. The immune response imbalance caused by over-activation is one of the main characteristics of IBD. INF-γ and TNF-α can stimulate the body to produce an inflammatory response and cause immune imbalance in the body, and are currently considered to be most related to the inflammatory state of IBD. The imbalance of the intestinal flora induced by IBD inhibits Treg cells, resulting in a decrease in IL-10 secretion, promoting the differentiation of Th1 cells and the release of Th17 cells, and breaking the normal intestinal immune balance. 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 detection results of the contents of IL-6, IL-10, TNF-α, and INF-γ are shown in Figure 7, the average contents 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 content 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, compared with the IBD group, the levels of the pro-inflammatory factor TNF-α in the sera of mice in the BX-245 group, BFM group, and FM group were all significantly decreased (p < 0.05). The levels of IL-6 and INF-γ in the BX-245 group and BFM group were significantly lower than those in the IBD group (p < 0.05), and there was no significant difference in the level of serum INF-γ between the BX-245 group and the NC group (p > 0.05). At the same time, the level of the anti-inflammatory factor IL-10 in the sera of mice in the BX-245 group increased, but no significance was detected (p > 0.05). The above manifestations showed that intragastric administration of BX-245, BX-245 yogurt, and ordinary yogurt all had a certain improvement effect on the state of excessive immune response in the intestine, and intragastric administration of BX-245 was more excellent in improving the imbalance of immune response in mice.

[0157] 3.6 Targeted metabolic results ( Figures 8 - 10 )

[0158] Intestinal microbiota 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 contents of metabolites in feces, and the detection results are shown in Figures 8 - 10 ,

[0159] Microbial fermentation of indigestible dietary fiber in the intestine produces short-chain fatty acids (SCFAs) as the main product. Short-chain fatty acids can affect the integrity of the human intestinal epithelium and mucosal barrier, immune responses, and the diversity of the microbiota. Therefore, changes in SCFAs may affect inflammatory bowel disease. In inflammatory bowel disease, SCFAs are involved in the main pathogenic processes and play an important role in the development of intestinal inflammation. Therefore, propionic acid (3.69 μmol / g, 3.55 μmol / g, 5.97 μmol / g, 3.33 μmol / g, 3.43 μmol / g), butyric acid (6.24 μmol / g, 1.89 μmol / g, 3.59 μmol / g, 2.06 μmol / g, 2.02 μmol / g), acetic acid (18.96 μmol / g, 15.74 μmol / g, 19.17 μmol / g, 14.54 μmol / g, 14.21 μmol / g), and n-valeric acid (0.54 μmol / g, 0.16 μmol / g, 0.45 μmol / g, 0.15 μmol / g, 0.26 μmol / g) in SCFAs of the NC group, IBD group, BX-245 group, BFM group, and FM group were compared with each other. It was found that compared with the NC group, the contents of butyric acid and n-valeric acid in the IBD group were significantly decreased, and the content of acetic acid tended to decrease compared with the NC group, but there was no significant difference. After BX-245 intervention, compared with the IBD group, the contents of butyric acid and n-valeric acid in the BX-245 group were significantly increased. The results showed that intragastric administration of BX-245 bacterial powder could significantly improve the contents of butyric acid and n-valeric acid in the feces of IBD mice. In summary, BX-245 can improve the related symptoms of IBD mice by reducing the content of SCFAs in the feces of mice.

[0160] Bile acids have become a class of key microbiota-related metabolites disrupted in patients with IBD. In recent years, metabolomic studies have shown that there are persistent defects in bile acid metabolism in patients with IBD, with an increase in primary bile acids and a decrease in secondary bile acids. Specific bile acid metabolites interact with intestinal epithelial cells and immune cells, leading to the inflammatory environment that appears in IBD. Comparing chenodeoxycholic acid (0.58 μmol / g, 0.24 μmol / g, 0.43 μmol / g, 0.34 μmol / g, 0.36 μmol / g) in bile acids of the NC group, IBD group, BX-245 group, BFM group, and FM group with each other, it was found that compared with the NC group, the contents of chenodeoxycholic acid in the IBD group, BFM group, and FM group were significantly decreased. After BX-245 intervention, compared with the IBD group, the content of chenodeoxycholic acid in the BX-245 group was significantly increased. The results showed that intragastric administration of BX-245 bacterial powder could significantly improve the content of chenodeoxycholic acid in the feces of IBD mice. Chenodeoxycholic acid belongs to secondary bile acids. Therefore, BX-245 can improve the disorder of bile acid metabolism in IBD mice by reducing the content of chenodeoxycholic acid and thus reducing the content of secondary bile acids.

[0161] Vitamins are a class of low-molecular-weight organic compounds with biological activity. Although the requirement for them is very small, their functions cannot be replaced by any other nutrients. In the body, they can neither produce energy nor are they raw materials for forming tissues, and the human body's requirement for them is also very small. However, they have a huge effect on the overall metabolism in the body. If the human body lacks a certain vitamin or the supply is insufficient for a long time, it will cause metabolic disorders and pathological reactions. Therefore, VB2 (0.04 μmol / g, 0.01 μmol / g, 0.02 μmol / g, 0.02 μmol / g, 0.02 μmol / g) and VB5 (0.38 μmol / g, 0.05 μmol / g, 0.06 μmol / g, 0.06 μmol / g, 0.06 μmol / g) in vitamins of the NC group, IBD group, BX-245 group, BFM group and FM group were compared with each other. It was found that the contents of VB2 and VB5 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.

[0162] Organic acids can enter the cell wall of bacteria, causing a pH gradient change inside and outside the bacteria and inhibiting bacterial growth. The suitable pH for the growth of several common pathogenic bacteria is neutral to slightly alkaline. For example, the suitable pH for Escherichia coli is 6.0 - 8.0, and for Streptococcus is 6.0 - 7.5, while probiotics such as Lactobacillus are suitable for reproduction in an acidic environment. Citric acid can lower the pH in the gastrointestinal tract, providing good growth conditions for probiotics such as Lactobacillus in the intestine, thus maintaining the normal balance of the microbial flora in the digestive tract of livestock and poultry. Therefore, citric acid (1.47 μmol / g, 0.76 μmol / g, 1.20 μmol / g, 0.94 μmol / g, 0.78 μmol / g) in organic acids of the NC group, IBD group, BX-245 group, BFM group and FM group was compared with each other. It was found that the content of citric acid in the IBD group, BFM group and FM group was significantly lower than that in the NC group. After BX-245 intervention, the content of citric acid in the BX-245 group was significantly higher than that in the IBD group. The results showed that intragastric administration of BX-245 bacterial powder could significantly improve the content of citric acid in the feces of IBD mice. In summary, BX-245 can promote the growth of probiotics in the intestine by reducing the content of citric acid in the feces of mice.

[0163] Amino acids are an important class of substances, mainly composed of five elements: carbon, hydrogen, oxygen, nitrogen, and sulfur. There are 20 known amino acids that make up proteins in the human body. Compared with the normal state, during the process of inflammatory diseases, the levels of 20 amino acids in serum decrease to varying degrees. Therefore, the tryptophan in the bile amino acids of the NC group, IBD group, BX-245 group, BFM group, and FM group (0.76 μmol / g, 0.30 μmol / g, 0.63 μmol / g, 0.41 μmol / g, 0.37 μmol / g) was compared with each other. It was found that the content of tryptophan in the IBD group and FM group was significantly lower than that in the NC group. After BX-245 intervention, the content of tryptophan in the BX-245 group was significantly higher than that in the IBD group. The results showed that gavage with BX-245 bacterial powder could significantly improve the content of tryptophan 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 content of tryptophan in the feces of inflammatory bowel disease mice, thereby playing a role in protecting the intestinal barrier and immune homeostasis.

[0164] 3.7 Metagenomic results

[0165] Intestinal microbiota imbalance is a key factor leading to the onset of IBD, and restoring the normalization of the intestinal microbiota is also an important way to relieve IBD symptoms. Therefore, the characteristics and composition of the intestinal microbiota of mice in each group were analyzed in this experiment. To explore the effect of BX-245 on the IBD intestinal microbiota, the α-diversity of the intestinal microbiota of the samples on the 30th day of the NC group, IBD group, BX-245 group, BFM group, and FM group was calculated based on the Shannon index and Simpson index, and the changes in the intestinal microbiota diversity of the NC group, IBD group, BX-245 group, BFM group, and FM group were analyzed. At the same time, the principal co-ordinates analysis (PCoA) of the β-diversity of the mouse intestinal microbiota was performed based on the Bray-Curtis distance.

[0166] The results of α-diversity are as Figure 11 shown: There was no significant difference in the S index of the Shannon index among the five groups. Compared with the NC group, the Simpson index and J index in the IBD group were significantly decreased, and there was no significant difference among the other groups, indicating that DSS induction would reduce the diversity of the mouse intestinal microbiota. The unweighted calculation method mainly considers the presence or absence of species, that is, if the species types of two populations are the same, it means that the sample distance between the two populations is the smallest; the weighted method considers both the presence or absence of species and the species abundance. In this experiment, both calculation methods were used to analyze the β-diversity, and the results are as Figure 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 intragastric 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 showed a more obvious trend of approaching the NC group, indicating that BX-245 had a higher ability to restore the intestinal flora structure of mice than the ordinary yogurt of BX-245 yogurt.

[0167] Table 3 Significance of β-diversity

[0168]

[0169] 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 mice in each group was analyzed at the phylum level, and the results were as Figure 13 shown. There were 6 dominant 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. After DSS induction, the intestinal flora of mice changed. The relative abundance of Actinobacteria in the intestinal flora of IBD group mice increased significantly compared with the NC group, and 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 decreased, but there was no significant difference.

[0170] Further analysis of the composition of the intestinal flora of mice in each group at the species level showed the results as Figures 14 - 17As shown. The main bacteria in each group of mice were composed of sixteen species, namely 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%), Enterorhabdus caecimuris (2.48%), Bacteroides thetaiotaomicron (2.38%), Bacteroides vulgatus (2.17%), Adlercreutzia equolifaciens (1.75%), Peptostreptococcaceae noname unclassified (1.60%), Clostridium sp ASF502 (1.37%), Anaerotruncus sp G32012 (1.23%), Parabacteroides goldsteinii (1.20%). To compare the changes in the intestinal bacteria after intragastric administration of BX-245, differential analysis was performed at the bacterial species level, and a total of 9 differential bacteria were identified between the two groups ( Figure 13) Compared with the NC group, the relative abundances of Bifidobacterium pseudolongum, Bacteroides thetaiotaomicron, Bacteroides vulgatus, Lactobacillus reuteri, and Peptostreptococcaceae unclassified were significantly increased in the IBD group; the relative abundances of Adlercreutzia equolifaciens, Alistipes unclassified, and Mucispirillum schaedleri were significantly decreased. After BX-245 intervention, compared with the IBD group, the relative abundances of Bifidobacterium pseudolongum and Bacteroides vulgatus in the BX-245 group were significantly decreased; the relative abundance of Adlercreutzia equolifaciens was significantly increased. Bifidobacterium animalis was not present in either the NC group or the IBD group, but its relative abundance was significantly increased after gavage with BX-245.

[0171] Studies have shown that the key metabolites of Bifidobacterium pseudolongum, namely HDCA and 12-ketolithocholic acid (12-KCAc), can inhibit the expression of colonic epithelial guanylate cyclase 1A (Gucy1A), thereby reducing the onset of DSS-induced colitis. The precipitate and supernatant of Adlercreutzia equolifaciens have anti-inflammatory properties in intestinal epithelial cells and hepatocytes to a lesser extent by inhibiting the NF-κB pathway. Bacteroides vulgatus is one of the most abundant bacteria in the human intestine and is widely regarded as a pathogenic bacterium, a symbiont that can cause pathology in response to host and / or environmental triggers. In specific pathogen-free (SPF) mice lacking the intracellular bacterial sensor nod2 associated with CD, Bacteroides vulgatus is the sole cause of small intestine injury.

[0172] Reference Figure 18, a total of 269 metabolic pathways were annotated from mouse feces, and among the top 30 relative abundances, 3 metabolic pathways showed significant differences among the three groups (P<0.05), namely 3-dehydroquinic acid ester biosynthesis (PWY-6163), uridine nucleotide biosynthesis (PWY-5686), and tRNA charging pathway (TRNA-CHARGING-PWY). The tRNA charging pathway is the selection of amino acids during protein synthesis in the aminoacylation process of tRNA, which is a two-step reaction including activating the amino acid and then transferring it to tRNA. Among them, compared with the NC group, 3-dehydroquinic acid ester biosynthesis was significantly decreased in the IBD group, and after BX-245 intervention, compared with the IBD group, 3-dehydroquinic acid ester biosynthesis was significantly increased in the BX-245 group. There were no significant differences in uridine nucleotide biosynthesis and tRNA charging pathway between the IBD group and the BX-245 group compared with the NC group. However, after BX-245 intervention, compared with the IBD group, uridine nucleotide biosynthesis and tRNA charging pathway were significantly increased in the BX-245 group.

[0173] In addition, to further explore the relationship between differential strains and differential functions, a correlation analysis was performed. The results are as Figure 19 shown. Uridine nucleotide biosynthesis was significantly positively correlated with Adlercreutzia equolifaciens and Mucispirillum schaedleri, and significantly negatively correlated with Bacteroides vulgatus, Bacteroides thetaiotaomicron, and Peptostreptococcaceae unclassified. Uridine nucleotide biosynthesis was significantly positively correlated with Alistipes unclassified, and significantly negatively correlated with Lactobacillus reuteri.

[0174] 4. Conclusions

[0175] During the experimental period, Bifidobacterium animalis subsp. lactis BX-245 could significantly increase the body weight of mice with inflammatory bowel disease, significantly increase the DAI score, reduce the contents of pro-inflammatory factors IL-6, TNF-α, and INF-γ in the serum, and increase the content of anti-inflammatory factor IL-10, alleviating the excessive immune response state of mice with inflammatory bowel disease.

[0176] Compared with the NC group, DSS can cause a decrease in the diversity of the intestinal flora and intestinal flora dysregulation in IBD mice. After BX-245 intervention, the diversity of the intestinal flora can be significantly restored, and the metabolic pathway can be improved. The main manifestations are that the relative abundances of Bifidobacterium pseudolongum and Bacteroides vulgatus are significantly decreased; the relative abundance of Adlercreutzia equolifaciens is significantly increased. The above three strains are related to inflammation and the intestinal barrier. In summary, BX-245 can improve the related symptoms of mice with inflammatory bowel disease by restoring intestinal flora dysregulation.

[0177] (3) Compared with the IBD group mice after BX-245 intervention, the contents of butyric acid, n-valeric acid, ursodeoxycholic acid, citric acid, and tryptophan in the feces of mice are significantly increased. It shows that BX-245 can improve the metabolic disorder of mice with inflammatory bowel disease caused by DSS modeling.

[0178] The above are only the preferred embodiments of the present invention, and do not limit the patent scope of the present invention accordingly. Any equivalent structural transformation made by using the description and drawings of the present invention under the inventive concept of the present invention, or direct / indirect application in other related technical fields, is included in the patent protection scope of the present invention.

Claims

1. The use of animal Bifidobacterium lactis subspecies BX-245 in the preparation of a probiotic preparation for relieving ulcerative colitis, 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 in the preparation of a probiotic preparation for relieving ulcerative colitis according to claim 1, characterized in that: Animal Bifidobacterium lactis subspecies BX-245 can 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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