A strain of Bifidobacterium breve for relieving ulcerative colitis and improving constipation and its application

Abstract

The present invention discloses a Bifidobacterium breve strain for alleviating ulcerative colitis and improving constipation and its application, belonging to the field of microorganisms. The Bifidobacterium breve strain involved in the present invention ( Bifidobacterium breve ) LIHUO 02 has a deposit number of GDMCC No: 65513 and was deposited at the Guangdong Microbial Culture Collection Center on November 20, 2024. The present invention has verified the probiotic properties of Bifidobacterium breve LIHUO 02 through various experiments, and this strain has good application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of microorganisms, and particularly relates to a Bifidobacterium breve strain for alleviating ulcerative colitis and improving constipation and its application. Background Art

[0002] Microecological therapy treats diseases by supplementing beneficial flora preparations for the human body, restoring the ecological balance of the normal intestinal flora, and resisting the colonization and invasion of pathogenic bacteria.

[0003] Bifidobacterium is a probiotic that can be used in microecological therapy. The prior art has disclosed that it can be used for the prevention and treatment of acute and chronic diarrhea, various enteritis and intestinal flora disorders; as an adjuvant drug for inflammatory bowel disease; for the prevention and treatment of constipation and intestinal dysfunction, etc.

[0004] Some Bifidobacterium breve and their applications in microecological regulation have been disclosed in the prior art:

[0005] Bifidobacterium breve CBT BR3 alleviates DSS-induced ulcerative colitis by in vitro inducing the aryl hydrocarbon receptor, reducing epithelial cell permeability, and promoting goblet cell regeneration to improve goblet cell function (Seul I P, Hyung J K, Jongwook Y, et al. Bifidobacterium breve CBT BR3 is effective at relieving intestinal inflammation by augmenting goblet cell regeneration. [J]. Journal of gastroenterology and hepatology, 2023, 38 (8): 1346-1354.).

[0006] Constipation is a chronic gastrointestinal disease, which can be divided into functional constipation and organic constipation. Functional constipation is a functional intestinal disease characterized by difficult defecation, hard stools, reduced defecation frequency or incomplete defecation. Its etiology is still unclear. However, genetic, lifestyle, mental and behavioral factors are related to it. Functional constipation can be divided into: outlet obstructive constipation, slow transit constipation and mixed constipation. The treatment of slow transit constipation includes lifestyle adjustment, traditional drugs (such as laxatives, prokinetic drugs, secretagogues, microecological regulators and traditional Chinese medicine), psychotherapy, enema, acupuncture, biofeedback therapy and surgery. These treatment methods (used alone or in combination) increase the defecation frequency, improve the fecal consistency, and relieve constipation-related symptoms. However, the efficacy of these methods will ultimately weaken, leading to dependence or recurrence.

[0007] Therefore, it is of great value and application prospect to provide a strain of Bifidobacterium breve with good in vitro probiotic properties, strong gastrointestinal tolerance, and the ability to relieve ulcerative colitis and improve constipation, as well as its applications. Summary of the Invention

[0008] The object of the present invention is to disclose a strain of Bifidobacterium breve screened from breast milk, verify its probiotic properties through various experiments, explore its potential value, and develop its application value.

[0009] Among the currently reviewed patents, the probiotic that can both relieve colitis and improve constipation is Lactobacillus rhamnosus. The present invention fills the gap that breast milk-derived Bifidobacterium breve has the dual functions of relieving ulcerative colitis and improving constipation.

[0010] The Bifidobacterium breve of the present invention is Bifidobacterium breve LIHUO 02, and its related applications at least include: applications for relieving ulcerative colitis and improving constipation, which can be used to prepare drugs capable of relieving ulcerative colitis, or foods, health products or drugs for improving constipation or regulating the intestinal flora. In particular, it can be used in probiotic drugs for relieving ulcerative colitis, or probiotic products for improving constipation or regulating the intestinal flora. It solves the problems existing in the prior art, and this strain has good in vitro probiotic properties, the ability to relieve ulcerative colitis, improve constipation, and regulate the intestinal flora.

[0011] To achieve the above object, the present invention provides the following solutions:

[0012] In the first aspect, the present invention discloses a new strain of Bifidobacterium breve.

[0013] The Bifidobacterium breve is Bifidobacterium breve LIHUO 02, which was deposited at the Guangdong Provincial Microbial Culture Collection Center on November 20, 2024. The deposit address is the 5th floor of Building 59, No. 100, Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No: 65513. The taxonomic name is Bifidobacterium breve .

[0014] Furthermore, the breast milk-derived Bifidobacterium breve LIHUO 02 was isolated and screened from the breast milk of healthy volunteer parturients in the Nanchang area of Jiangxi Province. The colonies are white, round, with a moist surface, opaque, and neat edges; the cell morphology is irregular rod-shaped, and the Gram-positive strains are arranged singly or in pairs. After sequencing analysis, the 16S rDNA sequence of this strain was compared with the nucleic acid sequence by BLAST in NCBI. The results showed that this strain is Bifidobacterium breve, which was named Bifidobacterium breve LIHUO02. The fragment of its 16S rDNA can be seen in SEQ ID NO.1.

[0015] SEQ ID NO.1:

[0016]

[0017] Furthermore, the strain has good probiotic characteristics in vitro, including but not limited to: good tolerance to artificial gastrointestinal fluids, high adhesion rate to Caco-2 cells, high clearance rate of DPPH, and strong inhibitory effects on Escherichia coli O157:H7 and Salmonella enteritidis CMCC5004.

[0018] The present invention also discloses a preparation of the Bifidobacterium breve LIHUO 02, which is prepared by culturing the aforementioned Bifidobacterium breve LIHUO 02. The preparation includes but not limited to any one or more of live bacteria, dead bacteria, fermentation broth, fermentation broth supernatant, fermentation broth precipitate, and freeze-dried powder.

[0019] The present invention also discloses a microbial agent comprising the aforementioned Bifidobacterium breve LIHUO 02 or the preparation.

[0020] In a second aspect, the present invention discloses the use of the aforementioned Bifidobacterium breve LIHUO 02 in the preparation of a product for alleviating ulcerative colitis.

[0021] Furthermore, the product for alleviating ulcerative colitis is a product for alleviating ulcerative colitis symptoms, alleviating intestinal inflammation, maintaining the integrity of the intestinal barrier, reducing oxidative stress, regulating the intestinal flora, and improving the level of short-chain fatty acids in metabolites.

[0022] The product for alleviating ulcerative colitis symptoms mainly alleviates weight loss, alleviates colon shortening, and reduces the MPO activity in colon tissue.

[0023] The product for alleviating intestinal inflammation mainly reduces the concentrations of TNF-α, IL-1β, and IL-6 in colon tissue and downregulates the relative mRNA levels, increases the concentration of IL-10 in colon tissue and upregulates the relative mRNA levels; downregulates the relative mRNA level of NF-κB in colon tissue and upregulates the relative mRNA level of IκBα; reduces the concentration of IL-6 in serum.

[0024] The product for maintaining the integrity of the intestinal barrier mainly reduces the concentration of mannitol in serum, increases the relative level of the tight junction protein Claudin-1, and upregulates the relative mRNA levels of the tight junction proteins Claudin-1, ZO-1, and the mucin MUC2.

[0025] The product for reducing oxidative stress mainly increases the SOD activity in colon tissue and reduces the CAT activity and MDA concentration in colon tissue.

[0026] The product for regulating the intestinal flora mainly increases the Lactobacillus genus in cecal contents at the genus level ( Lactobacillus ) 、LachnospiraceaeThe relative abundance of _UCG-006 was significantly reduced in Desulfovibrio ( Desulfovibrio ), Clostridium_sensu_stricto _1 and Escherichia-Shigella ( Escherichia-Shigella ). The product improves the level of short-chain fatty acids in metabolites mainly by increasing the concentration of acetic acid in the colonic contents.

[0027] Thirdly, the present invention provides the use of the aforementioned Bifidobacterium breve LIHUO 02 in the preparation of a product for improving constipation.

[0028] Furthermore, the product for improving constipation can be a product that promotes defecation, improves intestinal motility, promotes the secretion of neurotransmitters, reduces the level of aquaporin, improves colonic inflammation, regulates the intestinal flora, and improves the level of short-chain fatty acids in metabolites.

[0029] The product is a product for promoting defecation, mainly by increasing the number of fecal particles and the water content of feces, and increasing the gastrointestinal propulsion rate.

[0030] The product for improving gastrointestinal motility mainly increases the content of mucin in the colonic tissue, up-regulates the relative level of mRNA of c-Kit in the colonic tissue, regulates the level of serum gastrointestinal regulatory peptides, increases the concentrations of MTL, GAS, and SP in the serum, and reduces the concentrations of SS and VIP.

[0031] The product for promoting the secretion of neurotransmitters mainly increases the concentration of Ach in the serum and the concentration of 5-HT in the colonic tissue.

[0032] The product for reducing the level of aquaporin mainly reduces the concentration of aquaporin AQP3 in the colonic tissue.

[0033] The product for improving colonic inflammation mainly reduces the concentration of TNF-α in the colonic tissue and increases the concentration of IL-10 in the colonic tissue.

[0034] The product regulates the intestinal flora mainly by significantly increasing the relative abundances of Staphylococcus ( Staphylococus ), 、 Corynebacterium ( Corynebacterium ), and Bifidobacterium ( Bifidobacterium ) in the cecal contents at the genus level, and significantly reducing the relative abundances of Lachnospiraceac _NK4A136_group, norank_f_ Oscillospiraceae _, norank_f_ Eubacterium_coprostanoligencs _group, and norank_f_ Muribaculaceae genus.

[0035] The product described above improves the levels of short-chain fatty acids in metabolites mainly by increasing the concentration of isocaproic acid in colonic contents.

[0036] In a fourth aspect, the present invention provides a product containing the aforementioned Bifidobacterium breve LIHUO 02 or a preparation or a microbial agent.

[0037] Furthermore, the product is a food or a health product.

[0038] Furthermore, the product includes at least one of probiotic products, nutritional health products, animal feeds, and pet snacks.

[0039] Furthermore, the probiotic product includes at least one of fermented products, milk beverages, live bacterial preparations, milk powder, and rice flour.

[0040] Furthermore, the product is a medicine.

[0041] Furthermore, the medicine is in the form of pills, powders, capsules, tablets, film-coated agents, orally soluble granules, liquids, suppositories, or enemas.

[0042] Furthermore, the medicine also includes a pharmaceutically acceptable carrier and / or excipient.

[0043] Preferably, the product is an oral product.

[0044] The present invention also protects the cultivation method of Bifidobacterium breve LIHUO 02. The cultivation method can be a technology that has been publicly disclosed or not publicly disclosed in the prior art. Any cultivation method for culturing Bifidobacterium breve LIHUO 02 is within the scope of protection of the present invention.

[0045] Generally, the cultivation method may include inoculating Bifidobacterium breve LIHUO 02 into MRS medium, modified MRS medium, RCM medium, Bifidobacterium BS medium, or BBL agar medium.

[0046] The beneficial effects of the present invention are as follows:

[0047] The present invention discloses the application of breast milk-derived Bifidobacterium breve LIHUO 02 in alleviating ulcerative colitis, improving constipation, and regulating the intestinal flora. The breast milk-derived Bifidobacterium breve LIHUO 02 can be used to prepare a drug capable of alleviating ulcerative colitis or a product for improving constipation or regulating the intestinal flora. In a DSS (dextran sulfate sodium)-induced ulcerative colitis model, Bifidobacterium breve LIHUO 02 can alleviate the weight loss of ulcerative colitis mice, relieve the shortening of the colon, and reduce the MPO activity in the colon tissue. This strain can reduce the concentrations of TNF-α, IL-1β, and IL-6 in the colon tissue and down-regulate the relative mRNA levels, increase the concentration of IL-10 in the colon tissue and up-regulate the relative mRNA levels; down-regulate the relative mRNA level of NF-κB in the colon tissue and up-regulate the relative mRNA level of IκBα; reduce the concentration of IL-6 in the serum. This strain can reduce the concentration of mannitol in the serum, increase the relative level of the tight junction protein Claudin-1 in the colon tissue, and up-regulate the relative mRNA levels of the tight junction proteins Claudin-1, ZO-1, and the mucin MUC2. This strain increases the SOD activity in the colon tissue and reduces the CAT activity and MDA concentration in the colon tissue. At the genus level, it increases the relative abundance of Lactobacillus ( Lactobacillus ) 、Lachnospiraceae _UCG-006 in the cecal contents, and reduces the relative abundances of Desulfovibrio ( Desulfovibrio ), Clostridium_sensu_stricto _1, Escherichia-Shigella ( Escherichia-Shigella ). This strain can increase the concentration of acetic acid in the colon contents. In a Lop (loperamide hydrochloride)-induced constipation rat model, this strain can increase the number of fecal pellets, increase the water content of feces, and increase the gastrointestinal propulsion rate. This strain can increase the mucin content in the colon tissue, up-regulate the relative mRNA level of c-Kit in the colon tissue, and regulate the serum gastrointestinal regulatory peptide levels: increase the concentrations of MTL, GAS, and SP in the serum, and reduce the concentrations of SS and VIP. This strain can increase the concentration of Ach in the serum and increase the concentration of 5-HT in the colon tissue. This strain can reduce the concentration of the aquaporin AQP3 in the colon tissue. This strain can reduce the concentration of TNF-α in the colon tissue and increase the concentration of IL-10 in the colon tissue. At the genus level, this strain increases the relative abundances of Staphylococcus ( Staphylococus ), 、 Corynebacterium ( Corynebacterium ), and Bifidobacterium ( Bifidobacterium ) in the cecal contents, and significantly reduces Lachnospiraceac _NK4A136_group, norank_f_ Oscillospiraceae , norank_f_ Eubacterium_coprostanoligencs _group, norank_f_ Muribaculaceae genus relative abundances. This strain can increase the concentration of isocaproic acid in the colon contents.

[0048] Depositing Description

[0049] Name of Strain: Bifidobacterium breve LIHUO 02;

[0050] Chinese Name of Strain: Bifidobacterium breve LIHUO 02;

[0051] Taxonomic Nomenclature: Bifidobacterium breve ;

[0052] Deposit Number: GDMCC No:65513;

[0053] Depositary Institution: Guangdong Provincial Culture Collection of Microorganisms;

[0054] Deposit Date: November 20, 2024;

[0055] Deposit Address: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou. Description of Drawings

[0056] Figure 1 Colony morphology and cell morphology of strain LIHUO 02, where the left figure shows the colony morphology on the culture medium and the right figure shows the cell morphology under the microscope (scale bar marked 5μm).

[0057] Figure 2 Inhibitory effects of Bifidobacterium breve LIHUO 02 and Bifidobacterium animalis BB12 on 2 pathogenic bacteria in Example 5, where A is against Escherichia coli O157:H7 and B is against Salmonella enteritidis CMCC 50041.

[0058] Figure 3 For the change rate of mouse body weight in Example 6, the numerical values of the number of days on the horizontal axis are 15, 16, 17, 18, 19, 20, 21 in sequence.

[0059] Figure 4 For the DAI score of mice in Example 6, *** indicates P <0.001.

[0060] Figure 5 Schematic diagram of mouse colon photos in Example 6.

[0061] Figure 6 For the statistical results of mouse colon length in Example 6, *** indicates P <0.001.

[0062] Figure 7 For the spleen index of mice in Example 6, *** indicates P <0.001.

[0063] Figure 8 For the MPO activity in the colon of the mice in Example 6, ** indicates P < 0.01, *** indicates P < 0.001.

[0064] Figure 9 For the HE staining (2×) of the colon of the mice in Example 7, the scale bar indicates 500 μm.

[0065] Figure 10 For the histopathological score of the colon tissue of the mice in Example 7, ** indicates P < 0.01, *** indicates P < 0.001.

[0066] Figure 11 For the concentrations of TNF-α, IL-1β, IL-6, and IL-10 in the colon tissue of the mice in Example 8, they can be distinguished by the vertical axis. Among them, ns indicates P > 0.05, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001.

[0067] Figure 12 For the relative mRNA levels of TNF-α, IL-1β, IL-6, and IL-10 in the colon tissue of the mice in Example 8, ns indicates P > 0.05, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001.

[0068] Figure 13 For the relative mRNA levels of NF-κB and IκBα in the colon tissue of the mice in Example 8, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001.

[0069] Figure 14 For the concentrations of IL6 and IL-10 in the serum of the mice in Example 8, ns indicates P > 0.05, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001.

[0070] Figure 15 For the concentration of mannitol in the serum of the mice in Example 9, *** indicates P < 0.001.

[0071] Figure 16 The relative levels of the tight junction protein Claudin-1 in the mouse colon tissue of Example 9, ns indicates P > 0.05, * indicates P < 0.05, ** indicates P < 0.01.

[0072] Figure 17 The relative mRNA levels of the tight junction protein Claudin-1, tight junction protein ZO-1, and mucin MUC2 in the mouse colon tissue of Example 9, ns indicates P > 0.05, * indicates P < 0.05, *** indicates P < 0.001.

[0073] Figure 18 The SOD activity, CAT activity, and MDA concentration in the mouse colon tissue of Example 10, ns indicates P > 0.05, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001.

[0074] Figure 19 The Sobs index and Ace index of the microbiota in the mouse cecal content at the genus level of Example 11, ns indicates P > 0.05, * indicates P < 0.05.

[0075] Figure 20 Principal component analysis (PCA) and principal coordinates analysis (PCoA) of the microbiota in the mouse cecal content at the genus level of Example 11.

[0076] Figure 21 The distribution of the microbiota in the mouse cecal content at the phylum and genus levels of Example 11. Among them, the upper figure is the microbial distribution at the phylum level, and the lower figure is the microbial distribution at the genus level.

[0077] Figure 22 Differential analysis of the microbiota in the mouse cecal content at the genus level of Example 11.

[0078] Figure 23 The short-chain fatty acid concentration in the mouse colon content of Example 12, ns indicates P > 0.05, * indicates P < 0.05, *** indicates P < 0.001.

[0079] Figure 24For the water content and number of fecal particles of the rats in Example 13, ns indicates P > 0.05, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001.

[0080] Figure 25 For the gastrointestinal propulsion rate of the rats in Example 13, ** indicates P < 0.01, *** indicates P < 0.001.

[0081] Figure 26 For the AB staining of the colon of the rats in Example 14, the scale bars all indicate 500 μm.

[0082] Figure 27 For the relative level of c-Kit mRNA in the colon tissue of the rats in Example 14, * indicates P < 0.05, *** indicates P < 0.001.

[0083] Figure 28 For the levels of gastrointestinal regulatory peptides in the serum of the rats in Example 15, ns indicates P > 0.05, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001.

[0084] Figure 29 For the concentration of Ach in the serum of the rats in Example 16, ** indicates P < 0.01, *** indicates P < 0.001.

[0085] Figure 30 For the concentration of 5-HT in the colon tissue of the rats in Example 16, * indicates P < 0.05, *** indicates P < 0.001.

[0086] Figure 31 For the concentration of AQP3 in the colon tissue of the rats in Example 17, *** indicates P < 0.001.

[0087] Figure 32 For the concentration of inflammatory factors in the colon tissue of the rats in Example 18, ns indicates P > 0.05, ** indicates P < 0.01, *** indicates P < 0.001.

[0088] Figure 33 For Example 19, the Sobs index and Simpson index of the rat cecal content microorganisms at the genus level. ns indicates P > 0.05.

[0089] Figure 34 For Example 19, the principal component analysis (PCA) and principal coordinates analysis (PCoA) of the rat cecal content microbiome at the genus level.

[0090] Figure 35 For Example 19, the distribution of the rat cecal content microbiome at the phylum and genus levels. Among them, the upper figure shows the microbial distribution at the phylum level, and the lower figure shows the microbial distribution at the genus level.

[0091] Figure 36 For Example 19, the differential analysis of the rat cecal content microbiome at the genus level.

[0092] Figure 37 For Example 20, the short-chain fatty acid concentration in the rat colon content. ns indicates P > 0.05, * indicates P < 0.05. Detailed implementation manners

[0093] The present invention will be further described in detail below in conjunction with specific embodiments. The following embodiments are not used to limit the present invention, but only to illustrate the present invention. The experimental methods used in the following embodiments are generally carried out under conventional conditions if not otherwise specified. The materials, reagents, etc. used in the following embodiments can be obtained from commercial sources if not otherwise specified.

[0094] Example 1: Isolation, purification, identification, and preservation of Bifidobacterium breve LIHUO 02

[0095] The specific steps are as follows:

[0096] (1) Collection of breast milk samples

[0097] The breast milk samples were from the milk of healthy volunteer parturients in Nanchang City, Jiangxi Province. The volunteers had signed informed consent forms. The breast milk sample collectors wore sterile rubber gloves. Before collecting the samples, the breasts and surrounding areas of the parturients were washed with sterile water. The breast milk was collected manually, and the first ten drops of breast milk were discarded. About 5 mL of breast milk was collected into a sterile centrifuge tube and quickly transported back to the laboratory at low temperature for the isolation experiment of strains.

[0098] (2) Isolation, purification, identification, and preservation of strains

[0099] Isolation of strains: In an anaerobic workstation, the collected breast milk samples were gradient-diluted in a sterile PBS solution, and 10-1 , 10 -2 The diluent was spread on a modified MRS plate containing 0.05% cysteine hydrochloride and anaerobically cultured at 37°C for 72 h. Isolated strains with different colony morphologies in the breast milk samples were obtained through cultivation.

[0100] Purification of the isolated strains: Observe the colony morphologies on the modified MRS plate, pick the bacteria with inconsistent colony morphologies and streak them onto a new modified MRS plate, and anaerobically culture at 37°C for 48 h. Purification was carried out 5 times in sequence to obtain pure isolated strains.

[0101] Identification and preservation of the isolated strains: Pick a single colony of the purified isolated strain into 3% hydrogen peroxide solution to disperse the bacteria, and observe whether bubbles are generated within half a minute. If bubbles are generated, it indicates that the strain is a catalase-positive strain; if no bubbles are generated, it indicates that the strain is a catalase-negative strain. The Gram staining experiment of the isolated strains was carried out using a Gram staining kit. And the isolated strains were preserved in 25% glycerol at -80°C.

[0102] The genomic DNA of the isolated strains was extracted using a bacterial genomic DNA extraction kit (manufacturer: Tiangen Biochemical Technology (Beijing) Co., Ltd., product number: Cat.#DP302-02). The 16S rDNA sequence of the strain was amplified by PCR. The PCR reaction system (50 μL): 2 μL of genomic DNA, 2 μL of primer 27F (10 μmol / L), 2 μL of primer 1492R (10 μmol / L), 25 μL of 2×Flash PCR MasterMix (Dye), and 19 μL of sterile and enzyme-free ultrapure water. The PCR amplification program: pre-denaturation at 95°C for 10 min, (denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min) for a total of 30 cycles, and terminal extension at 72°C for 10 min. The PCR products were detected by 1.0% agarose gel electrophoresis, and the successfully amplified products were sent to Shanghai Saiheng Biotechnology Co., Ltd. for sequencing. The sequencing results of the PCR products of the 16S rDNA of the strain were compared with those in NCBI to determine the genus and species information of the strain.

[0103] (3)Detection of the physiological and biochemical characteristics of the strain

[0104] The detection of the physiological and biochemical characteristics of the isolated strains was sent to CICC for detection, and the detection method referred to the microbiological detection method for polyphasic identification of bacteria in FMIC-QO01-001-2015.

[0105] (4)Depositing of the strain

[0106] The identified lactic acid bacteria were deposited in the Guangdong Microbial Culture Collection Center (Address: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou) to obtain the deposit number of the strain.

[0107] The experimental results are as follows:

[0108] The colony morphology of the isolated strain LIHUO 02 is that the colony is white, round, with a moist surface, opaque, and has a neat edge; the cell morphology is irregular rod-shaped, arranged singly or in pairs (see Figure 1 ). Strain LIHUO 02 is negative for the catalase test and is a Gram-positive strain.

[0109] The sequencing result of the PCR product of the 16S rDNA of strain LIHUO 02 was compared with the nucleic acid sequence by BLAST of NCBI, and strain LIHUO 02 was identified as Bifidobacterium breve. The physiological and biochemical characteristics of Bifidobacterium breve LIHUO 02 are shown in Table 1 (Note: + indicates positive, - indicates negative).

[0110] Bifidobacterium breve LIHUO 02 was deposited at the Guangdong Provincial Microbial Culture Collection Center on November 20, 2024, with the deposit number GDMCC No: 65513.

[0111] Table 1 Physiological and biochemical characteristics of Bifidobacterium breve LIHUO 02

[0112]

[0113] Example 2: Determination of the tolerance of Bifidobacterium breve LIHUO 02 to artificial gastrointestinal fluid

[0114] The specific steps are as follows:

[0115] (1) Preparation of the test bacterial solutions of Bifidobacterium breve LIHUO 02 and Bifidobacterium animalis subsp. lactis BB12

[0116] Preparation of the test bacterial solutions: The strain solutions of Bifidobacterium breve LIHUO 02 and Bifidobacterium animalis subsp. lactis BB12 were respectively inoculated into MRS liquid medium and anaerobically cultured at 37°C for 18 h; after the strains were activated 3 times, the supernatant was discarded by centrifugation, the cells were collected, washed 2 times with an equal volume of PBS solution, and an appropriate amount of PBS solution was added to resuspend the cells to prepare the test bacterial solutions.

[0117] (2) Determination of the tolerance of Bifidobacterium breve LIHUO 02 and Bifidobacterium animalis subsp. lactis BB12 to artificial gastrointestinal fluid

[0118] 1 mL of the test bacterial suspensions of Bifidobacterium breve LIHUO 02 and Bifidobacterium animalis subsp. lactis BB12 were respectively taken to determine the viable count N0 of the test bacterial suspensions at 0 h. 1 mL of the test bacterial suspensions of Bifidobacterium breve LIHUO 02 and Bifidobacterium animalis subsp. lactis BB12 were respectively transferred to 9 mL of artificial gastric juice at pH 2.5 (containing 0.3% pepsin), and the viable count N1 was determined after incubation at 37 °C for 3 h. 1 mL of the bacterial suspensions of Bifidobacterium breve LIHUO 02 and Bifidobacterium animalis subsp. lactis BB12 that had been treated with artificial gastric juice for 3 h were respectively transferred to 9 mL of artificial intestinal juice at pH 8.0 (containing 0.1% trypsin and 1.8% bile salts), and the viable count N2 was determined after incubation at 37 °C for 7 h. The experiment was repeated 3 times, and the survival rate of the strains was calculated according to the following formula: survival rate in artificial gastric juice (%) = (N1 / N0) × 100, survival rate in artificial intestinal juice (%) = (N2 / N1) × 100, survival rate in artificial gastric and intestinal juices (%) = (N2 / N0) × 100.

[0119] The experimental results are as follows:

[0120] The survival rates of Bifidobacterium breve LIHUO 02 after treatment in artificial gastric juice at pH 2.5 for 3 h, in artificial intestinal juice at pH 8.0 for 8 h, and in artificial gastric and intestinal juices for 11 h were 87.38%, 88.56%, and 77.38% respectively, and the survival rate in artificial gastric and intestinal juices was higher than that of the control strain Bifidobacterium animalis subsp. lactis BB12 (see Table 2). The results showed that Bifidobacterium breve LIHUO 02 exhibited high tolerance to artificial gastric and intestinal juices.

[0121] Table 2 Survival rates of Bifidobacterium breve LIHUO 02 and Bifidobacterium animalis subsp. lactis BB12 in artificial gastric and intestinal juices

[0122]

[0123] Example 3: Determination of the adhesion ability of Bifidobacterium breve LIHUO 02 to Caco-2 cells

[0124] The specific steps are as follows:

[0125] (1) Preparation of the test bacterial suspensions of Bifidobacterium breve LIHUO 02 and Bifidobacterium animalis subsp. lactis BB12

[0126] Preparation of the test bacterial suspensions: The seed suspensions of Bifidobacterium breve LIHUO 02 and Bifidobacterium animalis subsp. lactis BB12 were respectively inoculated into MRS liquid medium and anaerobically cultured at 37 °C for 18 h; after the strains were activated 3 times, the supernatant was discarded by centrifugation, the cells were collected, washed twice with PBS solution of equal volume, and resuspended in an appropriate amount of PBS solution to adjust the cell concentration to 1.00×10 8 CFU / mL.

[0127] (2)Determination of the adhesion ability of Bifidobacterium breve LIHUO 02 and Bifidobacterium animalis subsp. lactis BB12 to Caco-2 cells

[0128] Caco-2 cells were inoculated into a culture flask containing fresh DMEM medium. The cell adhesion situation was observed. The medium in the culture flask was aspirated, and the flask was rinsed twice with PBS solution to remove the residual medium. After aspirating all the rinsed PBS solution, trypsin was added, and the flask was shaken to evenly cover the bottom of the flask with trypsin, and then placed in an incubator at 37 °C for digestion. During digestion, it was taken out every 3 minutes to observe. When the edges of some cells began to fall off, the trypsin being digested was aspirated and the cells were pipetted into a centrifuge tube containing complete medium. The cell suspension in the centrifuge tube was gently pipetted to disperse the cells. The cells were collected by centrifugation. According to a ratio of 10:1, 0.4% trypan blue solution was added, mixed evenly, and 20 μL was taken to a cell counting chamber for counting. The cells were gently resuspended with fresh DMEM medium and the concentration of Caco-2 cells was adjusted to 1.00×10 5 cells, and inoculated into a 24-well cell culture plate, 1 mL per well, and incubated in a 5% CO2 incubator at 37 °C until the cells grew to a monolayer. After washing twice with sterile PBS solution, 1 mL of fresh DMEM medium containing 1.00×10 8 CFU (denoted as N3) of Bifidobacterium breve LIHUO 02 and Bifidobacterium animalis subsp. lactis BB12 was added respectively, and incubated in a 5% CO2 incubator at 37 °C for 2 h.

[0129] The cells in each well of the 24-well plate were washed 5 times with PBS solution to elute the non-adherent lactic acid bacteria and metabolic secretions; 500 μL of 0.25% trypsin-EDTA was added to each well for digestion for 3 min, and then 500 μL of complete medium was added to terminate the digestion; the solution in each well was collected, serially diluted 10-fold, and the viable cell count after culture (denoted as N4) was detected by the plate colony counting method. Adhesion rate (%) = N4 / N3×100.

[0130] The experimental results are as follows:

[0131] The adhesion rate of Bifidobacterium breve LIHUO 02 to Caco-2 cells was 24.2%, which was higher than that of the control strain Bifidobacterium animalis subsp. lactis BB12. The adhesion of strains to intestinal cells plays an important role in the efficacy of strains in the host. The experimental results show that Bifidobacterium breve LIHUO 02 has good adhesion to Caco-2 cells, which is beneficial to its exerting probiotic characteristics in the host.

[0132] Table 3 Adhesion rates of Bifidobacterium breve LIHUO 02 and Bifidobacterium animalis subsp. lactis BB12 to Caco-2 cells

[0133]

[0134] Example 4: Determination of the scavenging rate of Bifidobacterium breve LIHUO 02 on DPPH

[0135] The specific steps are as follows:

[0136] (1) Preparation of the test bacterial solutions of Bifidobacterium breve LIHUO 02 and Bifidobacterium animalis subsp. lactis BB12

[0137] The method for preparing the test bacterial solutions is the same as that in Example 3.

[0138] (2) Determination of the antibacterial ability of Bifidobacterium breve LIHUO 02 and Bifidobacterium animalis subsp. lactis BB12 against pathogenic bacteria

[0139] Sample group: 1 mL of the test bacterial solution (bacterial cell concentration is 1×10 8 CFU / mL) and 1 mL of 0.2 mM DPPH solution are mixed well and reacted in the dark for 30 min, centrifuged at 6000 r / min for 10 min, and the absorbance A2 of the supernatant solution is measured at 517 nm. Blank group: Equal volume of absolute ethanol is used to replace the DPPH solution (A3). Control group: Equal volume of PBS solution is used to replace the test bacterial solution (A4). The experiment is repeated 3 times, and the scavenging rate of the strain on DPPH is calculated according to the following formula. Scavenging rate of DPPH (%) = [1 - (A2 - A3) / A4]×100.

[0140] The experimental results are as follows:

[0141] The scavenging rate of Bifidobacterium breve LIHUO 02 on DPPH is 52.04%, which is higher than that of the control strain Bifidobacterium animalis subsp. lactis BB12 (see Table 4). The experimental results show that Bifidobacterium breve LIHUO 02 has a certain scavenging rate on DPPH.

[0142] The strong oxidation property of DPPH free radicals can degrade polysaccharides and DNA, kill red blood cells, decompose cell membranes, and induce cell necrosis. The experimental results of this experiment show that Bifidobacterium breve LIHUO 02 has a certain scavenging ability on DPPH, indicating that this strain has in vitro antioxidant ability.

[0143] Table 4 Scavenging rates of Bifidobacterium breve LIHUO 02 and Bifidobacterium animalis subsp. lactis BB12 on DPPH

[0144]

[0145] Example 5: Determination of the antibacterial ability of Bifidobacterium breve LIHUO 02 against pathogenic bacteria

[0146] The specific steps are as follows:

[0147] (1) Preparation of fermentation broths of test strains Bifidobacterium breve LIHUO 02 and Bifidobacterium animalis subsp. lactis BB12

[0148] After being activated twice, the test strains Bifidobacterium breve LIHUO 02 and Bifidobacterium animalis subsp. lactis BB12 were inoculated into MRS liquid medium at an inoculum size of 1.00×10 7 CFU / mL, and anaerobically cultured at 37°C for 18 h. The bacterial liquid was centrifuged at 8000 r / min for 10 min, and the fermentation supernatant was collected and filtered through a 0.22 μm microporous membrane. Two pathogenic bacteria, Escherichia coli O157:H7 and Salmonella enteritidis CMCC 50041 (after being activated three times, the cell concentration was adjusted to 1.00×10 8 CFU / mL), were added to LB agar at about 45°C. The sterilized Oxford cups were placed on the LB agar plates, and 20 mL of LB agar containing the indicator bacteria was poured into the plates. After the plates solidified, 150 μL of the fermentation supernatant of the test strains was added to the wells, and cultured at 37°C for 24 h. Whether an inhibition zone appeared around the wells was observed, and the diameter (mm) of various inhibition zones was measured using a vernier caliper. The experiment was repeated 3 times.

[0149] The experimental results are as follows:

[0150] Bifidobacterium breve LIHUO 02 had varying degrees of inhibitory effects on Escherichia coli O157:H7 and Salmonella enteritidis CMCC 50041, and was higher than that of the control strain Bifidobacterium animalis subsp. lactis BB12 (see Figure 2 , Table 5).

[0151] Escherichia coli and Salmonella can easily cause intestinal infections, diarrhea and other diseases. In this experiment, Bifidobacterium breve LIHUO 02 had varying degrees of inhibitory effects on Escherichia coli O157:H7 and Salmonella enteritidis CMCC 50041, indicating that this strain can inhibit intestinal pathogenic bacteria, thereby reducing the probability of occurrence of intestinal infections, diarrhea and other diseases.

[0152] Table 5 Inhibitory effects of Bifidobacterium breve LIHUO 02 and Bifidobacterium animalis subsp. lactis BB12 on pathogenic bacteria

[0153]

[0154] Example 6: Effect of Bifidobacterium breve LIHUO 02 on symptoms of DSS-induced ulcerative colitis mice

[0155] The specific steps are as follows:

[0156] (1)Preparation of Bifidobacterium breve LIHUO 02 bacterial suspension

[0157] The bacterial strain solution of Bifidobacterium breve LIHUO 02 was inoculated into MRS liquid medium respectively and anaerobically cultured at 37°C for 18 h. After the strain was activated three times, the supernatant was discarded by centrifugation, and the thalli were collected. The thalli were washed twice with PBS solution of equal volume, and an appropriate amount of PBS solution was added to resuspend the thalli and adjust the thalli concentration to 5.00×10 8 CFU / mL.

[0158] (2)Construction of ulcerative colitis model and intervention of strains

[0159] 7-8-week-old specific pathogen-free (SPF) male C57 / BL6 mice were purchased from Changsha Tianqin Biotechnology Co., Ltd. The mice were raised under the conditions of temperature 23±2°C, humidity 50-60%, and 12 h light-dark cycle. The whole experimental period was 21 d.

[0160] From day 1 to day 7, all groups of mice drank sterile water and ate freely. On day 8, the mice were randomly grouped (n = 10 mice / group), which were the normal group, the model group, the mesalazine group, and the LIHUO 02 group.

[0161] From day 8 to day 14, the normal group and the model group were gavaged with PBS solution at a dose of 200 μL / mouse / d, the mesalazine group was gavaged with mesalazine solution at a dose of 200 μL / mouse / d (the dose of mesalazine was 0.2 g / kg·BW / d, manufacturer of mesalazine enteric-coated tablets: Sunflower Pharmaceutical Industry, product number: 221227), and the LIHUO 02 group was gavaged with Bifidobacterium breve LIHUO 02 at a dose of 200 μL / mouse / d (the dose of Bifidobacterium breve LIHUO 02 was 1.00×10 8 CFU / d); all groups of mice drank sterile water and ate freely.

[0162] From day 15 to day 21, the normal group and the model group were gavaged with PBS solution at a dose of 200 μL / mouse / d, the mesalazine group was gavaged with mesalazine solution at a dose of 200 μL / mouse / d (the dose of mesalazine was 0.2 g / kg·BW / d), and the LIHUO 02 group was gavaged with Bifidobacterium breve LIHUO 02 at a dose of 200 μL / mouse / d (the dose of Bifidobacterium breve LIHUO 02 was 1.00×10 8 CFU / d); the normal group drank sterile water; except for the normal group, the other groups drank sterile water containing 2.5% (w / v) DSS (DSS: dextran sulfate sodium, manufacturer: MP, product number: YD08001).

[0163] (3)DAI score

[0164] The body weights of mice in each group were measured daily from the 15th to the 21st day, the hardness of mouse feces and the presence of bloody stools in mice were observed, and the disease activity index (DAI) of mice was scored according to the scoring rules in Table 6.

[0165] Table 6 DAI Scoring Table

[0166]

[0167] (4)Mouse dissection, determination of colon length and spleen index

[0168] After the end of gavage on the 21st day, the mice were fasted but allowed to drink water for 12 h. Each mouse was gavaged with mannitol at a dose of 0.6 g / kg·BW and then fasted and water-deprived for 6 h before dissection. The mice were anesthetized with isoflurane and euthanized. After collecting blood samples by eye enucleation from all mice, the abdominal cavity of the mice was disinfected with 75% ethanol solution. The abdomen was incised along the midline of the abdomen using a standard dissecting scissors, and the entire colon was removed, and the mesentery around the colon was removed; the cecum was found, and the small intestine at the upper end of the cecum was removed, leaving the cecum and colon parts; starting from the junction of the cecum and colon, the colon length was measured and photographed. The spleen was removed and weighed, and the spleen index was calculated according to the following formula: Spleen index = spleen weight (mg) / body weight (g) × 100%.

[0169] (5)Determination of the myeloperoxidase (MPO) activity of colon tissues

[0170] The colon tissues were washed with ice-cold physiological saline, about 1 cm of distal colon tissue was cut and fixed in 4% paraformaldehyde solution; the remaining colon tissues were collected into sterile enzyme-free cryopreservation tubes, first frozen in liquid nitrogen, and then stored at -80 °C for later measurement.

[0171] The colon tissues of mice in each group were weighed and homogenized using a homogenizer in a pre-cooled buffer provided by the manufacturer at a ratio of 1:19 (W / V). The protein content of colon tissues of mice in each group was determined using a BCA protein concentration assay kit (enhanced) (manufacturer: Beyotime, catalog number: CatNo.P0010); the MPO activity of colon tissues of mice in each group was determined using an MPO test kit (manufacturer: Nanjing Jiancheng, catalog number: A044-1-1).

[0172] The experimental results are as follows:

[0173] Due to the intervention of DSS, the body weight of mice in the model group decreased significantly compared with the normal group (the body weight decreased by 19.89% on the 21st day), and the body weight of mice in the mesalazine group and the LIHUO 02 group decreased less than that in the model group (the body weights decreased by 13.74% and 8.92% respectively on the 21st day) (see Figure 3 ), indicating that the intervention of Bifidobacterium breve LIHUO 02 alleviated the decrease in body weight of DSS-induced ulcerative colitis mice to a certain extent.

[0174] The severity of DSS-induced ulcerative colitis in mice was explored by examining the DAI score. On the 21st day, the DAI score of the model group was significantly higher than that of the normal group, while the DAI scores of the mesalazine group and the LIHUO 02 group were significantly lower than those of the model group (see Figure 4 ). It is shown that: Bifidobacterium breve LIHUO 02 can reduce the susceptibility of DSS-induced ulcerative colitis in mice.

[0175] DSS-induced ulcerative colitis in mice is related to colonic pathogenesis. Compared with the normal group (7.75 cm), the colonic length of the model group mice (5.13 cm) was significantly shortened; the colonic lengths of the mesalazine group and the LIHUO 02 group mice (6.69 cm and 6.77 cm respectively) were significantly increased compared with the model group, indicating that: the intervention of Bifidobacterium breve LIHUO 02 alleviated the shortening of the colon in mice caused by DSS to a certain extent (see Figure 5 、 Figure 6 ).

[0176] The spleen index of the model group mice was significantly higher than that of the normal group, while the spleen indices of the mesalazine group and the LIHUO 02 group mice were significantly lower than those of the model group (see Figure 7 ). Compared with the normal group, the MPO activity in the colon of the model group mice was significantly increased; compared with the model group, the MPO activities of the mesalazine group and the LIHUO 02 group mice were significantly decreased (see Figure 8 ), indicating that Bifidobacterium breve LIHUO02 can reduce the MPO activity in the colon of DSS-induced ulcerative colitis mice.

[0177] The results of this experiment showed that the intervention of Bifidobacterium breve LIHUO 02 could reduce the weight loss of DSS-induced ulcerative colitis mice, reduce the DAI score, increase the colonic length, reduce the spleen index, and reduce the MPO activity in the colonic tissue. It is shown that Bifidobacterium breve LIHUO 02 can prevent and improve the symptoms of DSS-induced ulcerative colitis in mice.

[0178] Example 7: Effect of Bifidobacterium breve LIHUO 02 on histological damage of the colon in DSS-induced ulcerative colitis mice

[0179] The specific steps are as follows:

[0180] The grouping, modeling, treatment of mice, and treatment of colon tissue samples were the same as in Example 6. The colon tissue fixed in 4% paraformaldehyde solution was dehydrated, paraffin-embedded, sectioned at 5 μm, dewaxed, and stained with hematoxylin-eosin (H&E staining) in sequence. The inflammatory cell infiltration and mucosal tissue damage in the colon tissue were observed under a microscope, and the histopathological score was referred to Table 7.

[0181] Table 7 Histopathological Scoring Table

[0182]

[0183] The experimental results are as follows:

[0184] The HE staining results of the colon tissues of mice in each group and the pathological scoring results of mice are shown in Figure 9 、 Figure 10 In the normal group, the goblet cells in the colon of mice were abundant, there was no crypt destruction, and no inflammatory infiltration. In the model group, the goblet cells in the colon of mice were damaged, the crypts were reduced, the inflammatory infiltration was severe, and the mucosal layer was diseased; in the mesalazine group and the LIHUO 02 group, the number of inflammatory infiltrating cells and the inflammatory infiltration were reduced compared with the model group. Compared with the normal group, the pathological score of the colon tissues of the model group mice increased significantly; while the pathological scores of the colon tissues of the mesalazine group and the LIHUO 02 group mice decreased significantly compared with the model group.

[0185] The results of this experiment showed that the intervention of Bifidobacterium breve LIHUO 02 could significantly reduce the pathological score of the colon tissues of mice with DSS-induced ulcerative colitis and reduce the damage of the colon tissues of mice caused by DSS.

[0186] Example 8: Effects of Bifidobacterium breve LIHUO 02 on Inflammatory Cytokines, Real-time Fluorescent Quantitative PCR (RT-qPCR) of Genes Related to NF-κB Signaling Pathway, and Serum Inflammatory Cytokines in Colon Tissues of Mice with DSS-induced Ulcerative Colitis

[0187] The specific steps are as follows:

[0188] The grouping, modeling, treatment of mice, and treatment of colon tissue samples were the same as in Example 6.

[0189] (1)Determination of the content of inflammatory cytokines in colon tissues

[0190] The method for determining the protein content of the colon tissues of mice in each group was the same as in Example 6; ELISA kits for TNF-α (manufacturer: Shanghai Enzyme-linked, product number: ml002595V), IL-6 (manufacturer: Shanghai Enzyme-linked, product number: ml063159V), IL-1β (manufacturer: Shanghai Enzyme-linked, product number: ml002095V), and IL-10 (manufacturer: Shanghai Enzyme-linked, product number: mlC50274-1) were used to determine the concentrations of TNF-α, IL-6, IL-1β, and IL-10 in the colon tissues of mice in each group.

[0191] (2)Detection of real-time fluorescent quantitative PCR (RT-qPCR) of inflammatory factors and genes related to NF-κB signaling pathway in colon tissues

[0192] Total RNA of colon tissues of mice in each group was extracted using an RNA extraction kit (manufacturer: Takara, product number: 9767), and the quality and concentration of the total RNA were detected. The RNA purity A260 / 280 was not less than 1.8. 1 μg of total RNA was reverse transcribed into cDNA using an RNA reverse transcription kit (manufacturer: Takara, product number: RR092A). Real-time fluorescence quantitative PCR was performed on TNF-α, IL-6, IL-1β, IL-10, NF-κB, and IκBα genes using a fluorescence quantitative PCR kit (manufacturer: Takara, product number: 820A). The primers are shown in Table 8. Using the 2 -ΔΔCt -ΔΔCt method to calculate the relative levels of mRNA of inflammatory factors TNF-α, IL-6, IL-1β, IL-10, NF-κB, and IκBα in colon tissues (using the GAPDH gene as the internal reference gene).

[0193] Table 8 Primer sequences

[0194]

[0195] References for primer sequences: MengMeng N, HuanXin G, JunWu C, et al. Bifidobacterium breve Alleviates DSS-Induced Colitis in Mice by Maintaining the Mucosal and Epithelial Barriers and Modulating Gut Microbes [J]. Nutrients, 2022, 14 (18): 3671-3671.

[0196] Jialu S, Qinggang X, Yingxue Y, et al. Gut microbiota modulation and anti-inflammatory properties of mixed lactobacilli in dextran sodium sulfate-induced colitis in mice. [J]. Food&function, 2021, 12 (11).

[0197] (3)Detection of serum inflammatory cytokines

[0198] After the blood samples of each group of mice were allowed to stand and separate, serum was obtained by centrifugation at 3000 r / min for 15 min at 4°C. ELISA kits for IL-6 (manufacturer: Shanghai Enzyme-linked, product number: ml063159V) and IL-10 (manufacturer: Shanghai Enzyme-linked, product number: mlC50274-1) were used to measure the concentrations of IL-6 and IL-10 in the sera of mice in each treatment group.

[0199] The experimental results are as follows:

[0200] The concentrations of TNF-α, IL-1β, IL-6, IL-10 and the relative levels of mRNA in the colon tissues of each group of mice are shown in Figure 11 and Figure 12 . The relative levels of mRNA of NF-κB and IκBα in the colon tissues of each group of mice are shown in Figure 13 . The concentrations of IL-6 and IL-10 in the sera of each group of mice are shown in Figure 14 .

[0201] Compared with the normal group, the relative levels of TNF-α, IL-1β, IL-6 concentrations and mRNA in the colon tissues of mice in the model group were significantly increased, while the relative levels of TNF-α, IL-1β, IL-6 concentrations and mRNA in the colon tissues of mice in the mesalazine group and LIHUO 02 group were significantly decreased compared with the model group. Compared with the normal group, the relative levels of IL-10 concentration and mRNA in the colon tissues of mice in the model group were significantly decreased, and there was no significant difference in the relative levels of IL-10 concentration and mRNA in the colon tissues of mice in the mesalazine group compared with the model group; while the relative levels of IL-10 concentration and mRNA in the colon tissues of mice in the LIHUO 02 group were significantly increased compared with the model group (see Figure 11 and Figure 12 ).

[0202] To investigate whether the NF-κB signaling pathway plays a role in the regulation of the anti-inflammatory mechanism of Bifidobacterium breve LIHUO 02, the relative levels of mRNA of related genes were further measured. Compared with the normal group, the relative level of NF-κB mRNA in the colon tissues of mice in the model group was significantly increased, while the relative levels of NF-κB mRNA in the colon tissues of mice in the mesalazine group and LIHUO 02 group were significantly decreased compared with the model group. Compared with the normal group, the relative level of IκBα mRNA in the colon tissues of mice in the model group was significantly decreased, while the relative levels of IκBα mRNA in the colon tissues of mice in the mesalazine group and LIHUO 02 group were significantly increased compared with the model group (see Figure 13 ).

[0203] Compared with the normal group, the concentration of IL-6 in the serum of mice in the model group was significantly increased; while the concentration of IL-6 in the serum of mice in the mesalazine group and the LIHUO 02 group was significantly lower than that in the model group. Compared with the normal group, the concentration of IL-10 in the serum of mice in the model group was significantly decreased, and there was no significant difference in the concentration of IL-10 in the serum of mice in the mesalazine group and the LIHUO 02 group compared with the model group (see Figure 14 )

[0204] The results of this experiment showed that the intervention of Bifidobacterium breve LIHUO 02 could significantly reduce the concentrations of TNF-α, IL-1β, and IL-6 in the colon tissues of DSS-induced ulcerative colitis mice and down-regulate the relative mRNA levels of TNF-α, IL-1β, and IL-6; significantly increase the concentration of IL-10 in the colon tissues and up-regulate the relative mRNA level of IL-10; significantly down-regulate the relative mRNA level of NF-κB in the colon tissues and significantly up-regulate the relative mRNA level of IκBα in the colon tissues. The intervention of Bifidobacterium breve LIHUO 02 could significantly reduce the concentration of IL-6 in the serum of DSS-induced ulcerative colitis mice. It is shown that the intervention of Bifidobacterium breve LIHUO 02 can significantly reduce the concentrations of pro-inflammatory factors in the colon and serum of DSS-induced ulcerative colitis mice, significantly increase the concentration of anti-inflammatory factors in the colon of DSS-induced ulcerative colitis mice, effectively improve DSS-induced ulcerative colitis in mice, regulate the disordered intestinal immunity, and reduce the colon injury caused by DSS in mice by inhibiting the NF-κB signaling pathway.

[0205] Example 9: Effect of Bifidobacterium breve LIHUO 02 on the intestinal barrier of DSS-induced ulcerative colitis mice

[0206] The specific steps are as follows:

[0207] The grouping, modeling, and treatment of mice were the same as in Example 6. The treatment of mouse serum samples was the same as in Example 8.

[0208] (1) Determination of the concentration of mannitol in serum

[0209] The mannitol enzyme-linked immunosorbent assay kit (manufacturer: Shanghai Enzyme-linked, product number: YJ710290) was used to determine the concentration of mannitol in the serum of each group of mice.

[0210] (2) Determination of the relative level of tight junction proteins in colon tissues

[0211] For each group, the colon tissues of 3 randomly selected mice were used to measure the content of tight junction proteins. The colon tissues of each group of mice were weighed and homogenized in pre-cooled RIPA lysis buffer using a homogenizer for homogenization of colon tissues, and then centrifuged at 4°C and 12,000 r / min for 20 min. The protein content of the colon tissues of each group of mice was measured using a BCA Protein Concentration Assay Kit (Enhanced) (manufacturer: Beyotime, product number: Cat No.P0010). The protein concentrations of all samples were adjusted to 3 mg / mL, and 6×loading buffer was added and treated at 100°C for 5 min. Samples were added to each lane and electrophoresed on an SDS-PAGE gel. The proteins on the gel were transferred to a PVDF membrane, placed in an incubation box, blocked for 15 min, and then the PVDF membrane was washed 3 times with 1×TBST washing solution. They were incubated overnight at 4°C with anti-β-actin primary antibody (manufacturer: Wuhan Sanying, product number: 66009-1-Ig) and anti-tight junction protein Claudin-1 primary antibody (manufacturer: Wuhan Sanying, product number: 13050-1-AP) respectively. The primary antibodies were recovered, and the PVDF membrane was washed 3 times with 1×TBST washing solution, then secondary antibody was added and incubated for 3 h, and the PVDF membrane was washed 3 times with 1×TBST washing solution. An enhanced chemiluminescence (ECL) reagent and a chemiluminescence imaging system were used to develop and photograph the immunoreactive bands.

[0212] (3)Real-time fluorescence quantitative PCR (RT-qPCR) detection of tight junction proteins ZO-1, Claudin-1 and mucin MUC-2 genes in colon tissues

[0213] Total RNA of the colon tissues of each group of mice was extracted using an RNA extraction kit (manufacturer: Takara, product number: 9767), and the quality and concentration of the total RNA were detected. The RNA purity A260 / 280 was not less than 1.8. 1 μg of total RNA was reverse transcribed into cDNA using an RNA reverse transcription kit (manufacturer: Takara, product number: RR092A). The tight junction protein ZO-1, Claudin-1 and mucin MUC-2 genes were subjected to real-time fluorescence quantitative PCR using a fluorescence quantitative PCR kit (manufacturer: Takara, product number: 820A), and the primers are shown in Table 9. The 2 -ΔΔCt -ΔΔCt method was used to calculate the relative mRNA levels of tight junction proteins ZO-1, Claudin-1 and mucin MUC-2 genes in colon tissues (using the GAPDH gene as the internal reference gene).

[0214] Table 9 Primer sequences

[0215]

[0216] Primer sequence reference: MengMeng N, HuanXin G, JunWu C, et al. Bifidobacterium breve Alleviates DSS-Induced Colitis in Mice by Maintaining the Mucosal and Epithelial Barriers and Modulating Gut Microbes [J]. Nutrients, 2022, 14 (18): 3671-3671.

[0217] The experimental results are as follows:

[0218] The mannitol concentration in the serum of mice in each group is shown in Figure 15 . Compared with the normal group, the mannitol concentration in the serum of mice in the model group was significantly increased, while the mannitol concentration in the serum of mice in the mesalazine group and the LIHUO 02 group was significantly decreased compared with the model group.

[0219] The relative levels of the tight junction protein Claudin-1 in the colon tissues of mice in each group are shown in Figure 16 . The relative mRNA levels of the tight junction proteins Claudin-1, ZO-1, and the mucin MUC2 in the colon tissues of mice in each group are shown in Figure 17 . Compared with the normal group, the relative level of the tight junction protein Claudin-1 in the colon tissues of mice in the model group was significantly decreased. There was no significant difference in the relative level of the tight junction protein Claudin-1 in the colon tissues of mice in the mesalazine group compared with the model group, while the relative level of the tight junction protein Claudin-1 in the colon tissues of mice in the LIHUO02 group was significantly increased compared with the model group. Compared with the normal group, the relative mRNA levels of the tight junction proteins Claudin-1, ZO-1, and the mucin MUC2 in the colon tissues of mice in the model group were significantly decreased. The relative mRNA levels of the tight junction protein ZO-1 and the mucin MUC2 in the colon tissues of mice in the mesalazine group were significantly increased compared with the model group; while the relative mRNA levels of the tight junction proteins Claudin-1, ZO-1, and the mucin MUC2 in the colon tissues of mice in the LIHUO 02 group were significantly increased compared with the model group

[0220] The results of this experiment showed that the intervention of Bifidobacterium breve LIHUO 02 could significantly reduce the concentration of mannitol in the serum of DSS-induced ulcerative colitis mice, significantly increase the relative level of tight junction protein Claudin-1, and significantly up-regulate the relative mRNA levels of tight junction proteins Claudin-1, ZO-1, and mucin MUC2. It was indicated that the intervention of Bifidobacterium breve LIHUO 02 could effectively improve the intestinal permeability of DSS-induced ulcerative colitis mice and repair the intestinal barrier damage.

[0221] Example 10: Effect of Bifidobacterium breve LIHUO 02 on the antioxidant capacity of colon tissues of DSS-induced ulcerative colitis mice

[0222] The specific steps were as follows:

[0223] The method for measuring the protein content of colon tissues of mice in each group was the same as that in Example 6. Kits for measuring total superoxide dismutase (SOD) (manufacturer: Nanjing Jiancheng, product number: A001-3-2), catalase (CAT) (manufacturer: Nanjing Jiancheng, product number: A007-1-1), and malondialdehyde (MDA) (manufacturer: Nanjing Jiancheng, product number: A003-1-2) were used to measure the SOD activity, CAT activity, and MDA concentration of colon tissues of mice in each group.

[0224] The experimental results were as follows:

[0225] The SOD activity, CAT activity, and MDA concentration of colon tissues of mice in each group are shown in Figure 18 .

[0226] The SOD activity, CAT activity, and MDA concentration of colon tissues of mice in each group were measured to further evaluate the oxidative stress of DSS-induced colitis mice. Compared with the normal group, the SOD activity of colon tissues of model group mice was significantly decreased; there was no significant difference in the SOD activity of colon tissues of mesalazine group mice compared with the model group, while the SOD activity of colon tissues of LIHUO 02 group mice was significantly increased compared with the model group. Compared with the normal group, the CAT activity and MDA concentration of colon tissues of model group mice were significantly increased, while the CAT activity and MDA concentration of colon tissues of mesalazine group and LIHUO 02 group mice were significantly decreased compared with the model group.

[0227] The results of this experiment showed that the intervention of Bifidobacterium breve LIHUO 02 could reduce the CAT activity and MDA concentration of colon in ulcerative colitis mice and increase the SOD activity. It was indicated that Bifidobacterium breve LIHUO 02 could relieve DSS-induced colitis in mice by reducing the level of oxidative stress.

[0228] Example 11: Effects of Bifidobacterium breve LIHUO 02 on the Intestinal Flora of Mice with DSS-Induced Ulcerative Colitis

[0229] The specific steps are as follows:

[0230] The grouping, modeling, and treatment of the mice were the same as in Example 6. The cecal content samples of the mice were collected under sterile conditions.

[0231] The cecal contents of 6 randomly selected mice from each group were sent to Shanghai Majorbio Bio-Pharm Technology Co., Ltd. for the detection of microbial diversity. The total DNA of the cecal contents was extracted. After detecting the concentration and purity of the total DNA, the V3-V4 region of the 16S rDNA was amplified using 338F and 806R. The Uparse software (version 7.0.1090) was used to cluster into the same taxonomic units (OTUs). According to different similarity levels, all sequences were divided into OTUs, and the bioinformatics statistical analysis of the OTUs at the 97% similarity level was performed. The RDP classifier (classifier) was used to perform taxonomic analysis on the representative sequences of the OTUs at the 97% similarity level. The Qiime platform was used to analyze the alpha diversity and beta diversity at the genus level, the proportions and differences of different species in the samples of different groups, and the principal component analysis (PCA) and principal coordinate analysis (PCoA) were performed based on the weighted UniFrac distance; R language was used for plotting.

[0232] The experimental results are as follows:

[0233] The Sobs index and Ace index of the microorganisms in the cecal contents of the mice in each group are shown in Figure 19 . Compared with the normal group, based on the genus level, the Sobs index of the microorganisms in the cecal contents of the model group increased significantly; the Sobs index of the microorganisms in the cecal contents of the mesalazine group increased compared with the model group, but the difference was not significant; the Sobs index of the microorganisms in the cecal contents of the LIHUO 02 group decreased compared with the model group, but the difference was not significant. Compared with the normal group, based on the genus level, the Ace index of the microorganisms in the cecal contents of the model group increased, but the difference was not significant; the Ace index of the microorganisms in the cecal contents of the mesalazine group increased compared with the model group, but the difference was not significant; the Ace index of the microorganisms in the cecal contents of the LIHUO 02 group decreased compared with the model group, but the difference was not significant. It shows that the intake of DSS led to changes in the microbial diversity of the mice, and the intervention of Bifidobacterium breve LIHUO 02 reversed these changes to a certain extent.

[0234] The principal component analysis (PCA) and principal coordinate analysis (PCoA) of the microorganisms in the cecal contents of the mice in each group based on the genus level analysis (see Figure 20 ) showed that the normal group, the LIHUO 02 group, and the model group showed different microbiome compositions.

[0235] The distribution of the microbiota in the cecal contents of each group of mice at the phylum and genus levels is shown in Figure 21 . Firmicutes ( Firmicutes ), Bacteroidetes ( Bacteroidota ) were dominant in the relative abundances of the microbiota in the cecal contents of each group of mice. The relative abundances of the genera Eubacterium rectale ( Ileibacterium ), norank_f_ Muribaculaceae , norank_f_norank_o_ Clostridia _UCG-014 were dominant in the relative abundances of the microbiota in the cecal contents of each group of mice.

[0236] Compared with the normal group, the relative abundances of the genera Eubacterium rectale ( Ileibacterium ) and Lactobacillus ( Lactobacillus ) in the cecal contents of the model group mice were significantly decreased, while the relative abundances of norank_f_norank_o_ Clostridia _UCG-014 and Bacteroides ( Bacteroides ) were significantly increased. Compared with the model group, the relative abundances of the genera Desulfovibrio ( Desulfovibrio ), Clostridium _ sensu_stricto _1, Escherichia-Shigella ( Escherichia-Shigella ) in the cecal contents of the LIHUO 02 group mice were significantly decreased, while the relative abundance of Lactobacillus ( Lactobacillus ) 、 Lachnospiraceae _UCG-006 was significantly increased (see Figure 22 ).

[0237] The experimental results showed that the intervention of Bifidobacterium breve LIHUO 02 could make the microbiota composition of the cecal contents closer to that of the normal group, significantly increase the relative abundance of Lactobacillus ( Lactobacillus ) 、 Lachnospiraceae _UCG-006 in the cecal contents of DSS-induced colitis mice, and significantly decrease the relative abundances of Desulfovibrio ( Desulfovibrio ), Clostridium_sensu_stricto _1, Escherichia-Shigella ( Escherichia-Shigella ), indicating that the intervention of Bifidobacterium breve LIHUO 02 could reverse the changes in the intestinal microbiota of DSS-induced ulcerative colitis mice.

[0238] Example 12: Effect of Bifidobacterium breve LIHUO 02 on short-chain fatty acids in the colonic contents of DSS-induced ulcerative colitis mice

[0239] The specific steps are as follows:

[0240] The grouping, model establishment, and treatment of the mice were the same as in Example 6. The colonic contents of the mice were collected under sterile conditions.

[0241] The colonic contents of 5 randomly selected mice in each group were sent to Shanghai Majorbio Bio-Pharm Technology Co., Ltd. to detect the content of short-chain fatty acids. The specific method was as follows: Weighed the standard product and the colonic content sample into a grinding tube respectively, and added 0.5% phosphoric acid solution (containing 10 μg / mL of internal standard 2-ethylbutyric acid). The samples were frozen and ground, centrifuged at 4 °C and 13,000 g for 15 min, the supernatant was taken and extracted with n-butanol solvent, vortexed and mixed evenly, and then ultrasonically treated at low temperature for 10 min, centrifuged at 4 °C and 13,000 g for 5 min, and the supernatant solution was taken for GC-MC detection. Chromatographic conditions: HP FFAP capillary column (30 m × 0.25 mm × 0.25 μm), the carrier gas was high-purity helium (purity not less than 99.999%), the flow rate was 1.0 mL / min, and the temperature of the injection port was 180 °C. The injection volume was 1 μL, split injection, split ratio 10:1, solvent delay 2.5 min. Program temperature rise: The initial temperature of the column oven was 80 °C, programmed to rise to 120 °C at a rate of 20 °C / min, then to 160 °C at a rate of 5 °C / min, and finally held at 220 °C for 3 min. Mass spectrometry conditions: Electron impact ionization source (EI), ion source temperature 230 °C, quadrupole temperature 150 °C, transfer line temperature 230 °C, electron energy 70 eV. The scanning mode was selected ion scanning mode (SIM).

[0242] The experimental results are as follows:

[0243] The concentrations of short-chain fatty acids in the colonic contents of mice in each group are shown in Figure 23 . Compared with the normal group, the concentrations of acetic acid, propionic acid, butyric acid, isobutyric acid, and isovaleric acid in the colonic contents of the model group mice were significantly decreased. However, the concentration of acetic acid in the colonic contents of the mice in the Bifidobacterium breve LIHUO 02 intervention group was significantly increased compared with the model group, while there was no significant difference in the mesalazine group compared with the model group. It shows that Bifidobacterium breve LIHUO 02 can improve the level of short-chain fatty acids in the colonic contents of mice with ulcerative colitis induced by DSS.

[0244] Example 13: Effect of Bifidobacterium breve LIHUO 02 on the symptoms of constipation rats induced by loperamide hydrochloride (Lop)

[0245] The specific steps are as follows:

[0246] (1) Preparation of Bifidobacterium breve LIHUO 02 bacterial suspension

[0247] The strain solution of Bifidobacterium breve LIHUO 02 was inoculated into MRS liquid medium respectively and anaerobically cultured at 37°C for 18 h. After the strain was activated three times, the supernatant was discarded by centrifugation, and the cells were collected. The cells were washed twice with PBS solution of equal volume, and an appropriate amount of PBS solution was added to resuspend the cells and adjust the cell concentration to 1.00×10 8 CFU / mL.

[0248] (2) Construction of constipation model, intervention of strains, determination of fecal particle number and fecal water content

[0249] 6-8-week-old specific pathogen-free (SPF) male SD rats were purchased from Changsha Tianqin Biotechnology Co., Ltd. The rats were raised under the conditions of temperature 23±2°C, humidity 50-60%, and 12-h light-dark cycle. The whole experimental period was 21 d.

[0250] From the 1st day to the 7th day, rats in all groups drank sterile water and ate freely. On the 8th day, they were grouped in a snake-like pattern (n = 8 rats / group), namely the blank group, the constipation model group, the polyethylene glycol 4000 powder group, and the LIHUO 02 experimental group.

[0251] From the 8th day to the 14th day, rats in the blank group and the constipation model group were gavaged with PBS solution at a dose of 1 mL / rat / d, rats in the polyethylene glycol 4000 powder group were gavaged with polyethylene glycol 4000 powder solution at a dose of 1 mL / rat / d (dose of polyethylene glycol 4000 powder: 0.3 g / kg·BW / d; manufacturer of polyethylene glycol 4000 powder: Forlax, product number: W11636), and rats in the LIHUO 02 experimental group were gavaged with Bifidobacterium breve LIHUO 02 strain solution at a dose of 1 mL / rat / d (dose of Bifidobacterium breve LIHUO 02: 1.00×10 8 CFU / d); rats in all groups drank sterile water and ate freely.

[0252] From the 15th day to the 21st day, rats in the blank group were intraperitoneally injected with normal saline twice at a dose of 1 mL / rat / d; rats in the other groups were intraperitoneally injected with loperamide hydrochloride twice at a dose of 1 mL / rat / d (dose of loperamide hydrochloride: 16 mg / kg·BW / d; manufacturer of loperamide hydrochloride: Aladdin, product number: L129465). Rats in the blank group and the constipation model group were gavaged with PBS solution at a dose of 1 mL / rat / d, rats in the polyethylene glycol 4000 powder group were gavaged with polyethylene glycol 4000 powder solution at a dose of 1 mL / rat / d (dose of polyethylene glycol 4000 powder: 0.3 g / kg·BW / d), and rats in the LIHUO 02 experimental group were gavaged with Bifidobacterium breve LIHUO 02 strain solution at a dose of 1.00×10 8CFU / d. All groups of rats were given sterile water to drink and had free access to food. On the 21st day, the feces of each rat were collected within 1 hour, the number of fecal pellets was recorded, and the weight was measured as m1; the rat feces were dried in an oven at 60 °C for 24 hours, cooled to room temperature in a desiccator, and weighed as m2. The formula for calculating fecal water content is as follows: Fecal water content (%) = (m1 - m2) / m1 × 100%.

[0253] (3)Preparation of Evans blue solution

[0254] Take 2.5 g of carboxymethyl cellulose, 8 g of milk powder, 4 g of sucrose, 4 g of starch, and 3.5 mL of 1% Evans blue solution, dissolve them in distilled water, and mix well with a vortex mixer to prepare 150 mL of Evans blue semi-solid paste. Prepare it fresh for use.

[0255] (4)Rat dissection

[0256] After the intragastric gavage on the 21st day, the rats were fasted and water-deprived for 12 hours. Each rat was intragastrically administered 2 mL of Evans blue paste. After 15 minutes, blood samples were collected by puncturing the orbital cavity, and the rats were sacrificed; after collecting the blood samples, the abdomen of the rats was disinfected with 75% ethanol solution, and the abdominal cavity was incised along the midline of the abdomen with a standard anatomical scissors to take out the complete gastrointestinal tissues of the rats. The distance from the position of the pyloric sphincter to the end of carbon powder propulsion and the total intestinal length of the small intestine were measured with a ruler, and the advancing distance of the Evans blue paste from the pylorus was measured. The gastrointestinal propulsion rate (GIP) was calculated based on the following formula: GIP = advancing distance of Evans blue paste (cm) / total length of small intestine (cm) × 100%.

[0257] The colon tissues were washed with ice-cold normal saline, about 1 cm of distal colon tissues were cut and fixed in 4% paraformaldehyde solution; the remaining colon tissues were collected into sterile and enzyme-free cryopreservation tubes, first frozen in liquid nitrogen, and then stored at -80 °C for further testing.

[0258] Experimental results:

[0259] The fecal water content and the number of fecal pellets in the rats of the constipation model group were significantly lower than those in the blank group. The fecal water content in the rats of the polyethylene glycol 4000 powder group was significantly increased compared with that in the model group, while the fecal water content and the number of fecal pellets in the rats of the LIHUO 02 experimental group were significantly increased compared with those in the model group (see Figure 24 ).

[0260] Due to the intervention of loperamide hydrochloride (Lop), the gastrointestinal propulsion rate in the rats of the constipation model group was significantly lower than that in the normal group, while the gastrointestinal propulsion rates in the rats of the polyethylene glycol 4000 powder group and the LIHUO 02 experimental group were significantly increased compared with those in the model group (see Figure 25 ).

[0261] The results of this experiment showed that the intervention of Bifidobacterium breve LIHUO 02 could increase the fecal water content, fecal particle number, and gastrointestinal propulsion rate of Lop-induced constipated rats, thereby improving the symptoms of constipation.

[0262] Example 14: Effects of Bifidobacterium breve LIHUO 02 on the relative levels of mucin and c-Kit mRNA in the colon tissues of Lop-induced constipated rats

[0263] The specific steps are as follows:

[0264] The grouping, modeling, treatment of rats, and treatment of colon tissue samples were the same as those in Example 13.

[0265] (1) AB staining of colon tissues

[0266] The colon tissues of rats in each group fixed in 4% paraformaldehyde solution were dehydrated, paraffin-embedded, sectioned at 5 μm, dewaxed, and stained with Alcian blue (AB) in turn.

[0267] (2) Real-time fluorescence quantitative PCR (RT-qPCR) detection of c-Kit mRNA in colon tissues

[0268] The total RNA of colon tissues of rats in each group was extracted using an RNA extraction kit (manufacturer: Takara, product number: 9767), and the quality and concentration of the total RNA were detected. The RNA purity A260 / 280 was not less than 1.8. 1 μg of total RNA was reverse transcribed into cDNA using an RNA reverse transcription kit (manufacturer: Takara, product number: RR092A). The c-Kit gene was subjected to real-time fluorescence quantitative PCR using a fluorescence quantitative PCR kit (manufacturer: Takara, product number: 820A), and the primers are shown in Table 10. The relative level of c-Kit mRNA in colon tissues (using the GAPDH gene as the internal reference gene) was calculated by the 2 -ΔΔCt -ΔΔCt method.

[0269] Table 10 Primer sequences

[0270]

[0271] References for primer sequences: Wenhui L, Aimin Z. The potential of Quercetin to protect against loperamide-induced constipation in rats [J]. Food Science & Nutrition, 2021, 9 (6): 3297-3307.

[0272] The experimental results are as follows:

[0273] Alcian blue (AB) staining was used to detect the distribution of mucus-secreting goblet cells in the colonic tissues of rats in each group (see Figure 26 ). The results showed that compared with the blank group, the content of mucin in the colonic tissues of rats in the constipation model group decreased, while the content of mucin in the colonic tissues of rats in the polyethylene glycol 4000 powder group and the LIHUO 02 experimental group increased compared with the model group.

[0274] Compared with the blank group, the relative mRNA level of c-Kit in the colonic tissues of rats in the constipation model group decreased significantly, while the relative mRNA level of c-Kit in the colonic tissues of rats in the polyethylene glycol 4000 powder group and the LIHUO 02 experimental group increased significantly compared with the model group (see Figure 27 ).

[0275] Interstitial cells of Cajal (ICC) are cells that regulate colonic peristalsis, and their distribution is closely related to intestinal peristalsis. The natural ligand of ICC is c-Kit, and the level of c-Kit can directly reflect the level of interstitial cells. The results of this experiment showed that the intervention of Bifidobacterium breve LIHUO 02 could increase the mucin in the colon of Lop-induced constipated rats and significantly up-regulate the relative mRNA level of the c-Kit gene in the colonic tissues of Lop-induced constipated rats, thereby improving intestinal peristalsis and relieving constipation.

[0276] Example 15: Effect of Bifidobacterium breve LIHUO 02 on the levels of gastrointestinal regulatory peptides in the serum of Lop-induced constipated rats

[0277] The specific steps are as follows:

[0278] The grouping, modeling, and treatment of rats were the same as in Example 13.

[0279] After the blood samples of rats in each group were allowed to stand and separate, serum was obtained by centrifugation at 4°C and 3000 r / min for 15 min. Enzyme-linked immunosorbent assay kits for gastrin (Gas) (manufacturer: Nanjing Jiancheng, product number: H239-1-2), motilin (MTL) (manufacturer: Nanjing Jiancheng, product number: H182-1-2), somatostatin (SS) (manufacturer: Nanjing Jiancheng, product number: H092), substance P (SP) (manufacturer: Nanjing Jiancheng, product number: H218-1-2), and vasoactive intestinal peptide (VIP) (manufacturer: Nanjing Jiancheng, product number: H219-1-2) were used to measure the concentrations of Gas, MTL, SS, SP, and VIP in the serum of rats in each group.

[0280] The experimental results are as follows:

[0281] Compared with the blank group, the concentrations of MTL, GAS, and SP in the serum of rats in the constipation model group were significantly decreased. The concentrations of MTL and GAS in the serum of rats in the polyethylene glycol 4000 powder group were significantly increased compared with the model group; while the concentrations of MTL, GAS, and SP in the serum of rats in the LIHUO 02 experimental group were significantly increased compared with the constipation model group. Compared with the blank group, the concentrations of VIP and SS in the serum of rats in the model group were significantly increased, while the concentrations of VIP and SS in the serum of rats in the polyethylene glycol 4000 powder group and the LIHUO 02 experimental group were significantly decreased compared with the constipation model group (see Figure 28 )

[0282] MTL, GAS, and SP can promote gastrointestinal muscle contraction and relaxation of the pyloric sphincter, and promote intestinal peristalsis. SS and VIP are inhibitory gastrointestinal regulatory peptides that can inhibit intestinal peristalsis. The results of this experiment showed that the intervention of Bifidobacterium breve LIHUO 02 could significantly increase the concentrations of MTL, GAS, and SP in the serum of Lop-induced constipated rats, and significantly decrease the concentrations of VIP and SS; indicating that Bifidobacterium breve LIHUO 02 can improve constipation by regulating the levels of serum gastrointestinal regulatory peptides.

[0283] Example 16: Effect of Bifidobacterium breve LIHUO 02 on the levels of neurotransmitters in Lop-induced constipated rats

[0284] The specific implementation examples are as follows:

[0285] The grouping, modeling, and treatment of rats were the same as in Example 13. The treatment of rat serum samples was the same as in Example 15.

[0286] An acetylcholine (Ach) enzyme-linked immunosorbent assay kit (manufacturer: Shanghai Enzyme-linked, product number: ml003048V) was used to measure the concentration of Ach in the serum of rats in each group.

[0287] The colon tissues of rats in each group were weighed and homogenized in a pre-cooled PBS solution at a ratio of 1:9 (W / V) using a tissue homogenizer. A BCA protein concentration assay kit (enhanced type) (manufacturer: Beyotime, product number: Cat No.P0010) was used to measure the protein content of the colon tissues of rats in each group; a 5-hydroxytryptamine (5-HT) enzyme-linked immunosorbent assay kit (manufacturer: Shanghai Enzyme-linked, product number: ml059511V) was used to measure the concentration of 5-HT in the colon tissues of rats in each treatment group.

[0288] The experimental results are as follows:

[0289] Compared with the blank group, the concentration of Ach in the serum of rats in the constipation model group was significantly decreased. The concentrations of Ach in the serum of rats in the polyethylene glycol 4000 powder group and the LIHUO 02 experimental group were significantly increased compared with the constipation model group (see Figure 29 )

[0290] Compared with the blank group, the concentration of 5-HT in the colon tissue of rats in the constipation model group decreased significantly, while the concentration of 5-HT in the colon tissue of the polyethylene glycol 4000 powder group and the LIHUO 02 experimental group was significantly higher than that in the constipation model group (see Figure 30 ).

[0291] Acetylcholine (Ach) can excite gastrointestinal smooth muscle, increase the contraction amplitude, tension and peristalsis, and promote the secretion of gastric juice and intestinal juice. 5-HT is an important neurotransmitter that can reduce the colonic transit time of rat feces. The results of this experiment show that the intervention of Bifidobacterium breve LIHUO 02 can significantly increase the serum Ach concentration and the colon tissue 5-HT concentration in Lop-induced constipated rats, thereby improving constipation.

[0292] Example 17: Effect of Bifidobacterium breve LIHUO 02 on the concentration of AQP3 in the colon tissue of Lop-induced constipated rats

[0293] The specific steps are as follows:

[0294] The grouping, modeling and treatment of rats were the same as in Example 13.

[0295] The detection of the protein content in the colon tissue of rats in each group was the same as in Example 16; the AQP3 concentration in the colon tissue of rats in each treatment group was measured using an AQP3 enzyme-linked immunosorbent assay kit (manufacturer: Shanghai Enzyme-linked, product number: ml003095V).

[0296] The experimental results are as follows:

[0297] Compared with the blank group, the concentration of AQP3 in the colon tissue of rats in the constipation model group increased significantly, while the concentration of AQP3 in the colon tissue of the polyethylene glycol 4000 powder group and the LIHUO 02 experimental group was significantly lower than that in the constipation model group (see Figure 31 ).

[0298] AQP3 is mainly expressed in colonic mucosal epithelial cells and is considered to play a key role in controlling colonic water transport. The results of this experiment show that the intervention of Bifidobacterium breve LIHUO 02 can significantly reduce the AQP3 concentration in the colon tissue of Lop-induced constipated rats, thereby improving constipation.

[0299] Example 18: Effect of Bifidobacterium breve LIHUO 02 on inflammatory factors in the colon tissue of Lop-induced constipated rats

[0300] The experimental steps are as follows:

[0301] The grouping, modeling and treatment of rats were the same as in Example 13.

[0302] The method for detecting the protein content in the colon tissues of rats in each group was the same as that in Example 16; ELISA kits for TNF-α (manufacturer: Shanghai Enzyme-linked, product number: ml002595V) and IL-10 (manufacturer: Shanghai Enzyme-linked, product number: mlC50274-1) were used to measure the concentrations of TNF-α and IL-10 in the colon tissues of rats in each group.

[0303] The experimental results are as follows:

[0304] Compared with the blank group, the concentration of TNF-α in the colon tissues of rats in the constipation model group was significantly increased, and the concentrations of TNF-α in the colon tissues of rats in the polyethylene glycol 4000 powder group and the LIHUO 02 experimental group were significantly lower than those in the constipation model group. Compared with the blank group, the concentration of IL-10 in the colon tissues of rats in the constipation model group was significantly decreased. There was no significant difference in the polyethylene glycol 4000 powder group compared with the constipation model group, while the LIHUO 02 experimental group was significantly increased compared with the constipation model group (see Figure 32 )

[0305] The results of this experiment showed that the intervention of Bifidobacterium breve LIHUO 02 could significantly reduce the concentration of the pro-inflammatory factor TNF-α in the colon tissues of Lop-induced constipated rats and significantly increase the concentration of the anti-inflammatory factor IL-10, thereby improving the colon inflammation caused by constipation.

[0306] Example 19: Effect of Bifidobacterium breve LIHUO 02 on the intestinal flora of Lop-induced constipated rats

[0307] The specific steps are as follows:

[0308] The grouping, modeling, and treatment of rats were the same as those in Example 12. Under sterile conditions, the cecal contents of rats were collected.

[0309] The cecal contents of 6 randomly selected rats in each group were sent to Shanghai Majorbio Bio-Pharm Technology Co., Ltd. for detecting microbial diversity. The total DNA of the cecal contents of rats was extracted. After detecting the concentration and purity of the total DNA, the V3-V4 region of 16S rDNA was amplified using 338F and 806R. Uparse (version 7.0.1090) was used to cluster into the same taxonomic units (OTUs). According to different similarity levels, all sequences were divided into OTUs, and bioinformatics statistical analysis was performed on the OTUs at the 97% similarity level. The Bayesian algorithm in the RDP classifier was used for taxonomic analysis of the representative sequences of OTUs at the 97% similarity level. The Qiime platform was used to analyze the α-diversity and β-diversity at the genus level, the proportions of different species in different group samples, and the species difference analysis. Principal component analysis (PCA) and principal coordinate analysis (PCoA) were performed based on the weighted UniFrac distance; R language was used for plotting.

[0310] The experimental results are as follows:

[0311] The Sobs index and Simpson index (Simpson index) of the α-diversity of the microorganisms in the cecal contents of rats in each group are shown in Figure 33 . Compared with the blank group, at the genus level, the Sobs index of the microorganisms in the cecal contents of rats in the constipation model group increased, but the difference was not significant; the Sobs index of the microorganisms in the cecal contents of rats in the polyethylene glycol 4000 powder group increased compared with the constipation model group, but the difference was not significant; while the Sobs index of the microorganisms in the cecal contents of rats in the LIHUO 02 experimental group decreased compared with the constipation model group, but the difference was not significant. Compared with the blank group, at the genus level, the Simpson index of the microorganisms in the cecal contents of rats in the constipation model group decreased, but the difference was not significant; while the Simpson index of the microorganisms in the cecal contents of rats in the polyethylene glycol 4000 powder group and the LIHUO 02 experimental group increased compared with the constipation model group, but the difference was not significant.

[0312] Principal component analysis (PCA) and principal coordinates analysis (PCoA) of the microorganisms in the cecal contents of rats in each group based on the genus level (see Figure 34 ) showed that different microbiome compositions were shown in the blank group, the LIHUO 02 experimental group and the constipation model group, and the LIHUO 02 experimental group was closer to the blank group.

[0313] The distribution of the microorganisms in the cecal contents of rats in each group at the phylum and genus levels is shown in Figure 35 . Firmicutes ( Firmicutes ), Actinobacteria ( Actinobacteriota ) were the dominant flora in the cecal contents of rats in the blank group and the LIHUO 02 experimental group; while Firmicutes ( Firmicutes ), Bacteroidetes ( Bacteroidota ), Actinobacteria ( Actinobacteriota ) were the dominant flora of the microorganisms in the cecal contents of rats in the constipation model group and the polyethylene glycol 4000 powder group. norank_f_norank_o_ Clostridia _UCG-014, Lactobacillus ( Lactobacillus ), Romboutsia ( Romboutsia ) were dominant in the microbiome of the cecal contents of rats in each group.

[0314] Compared with the blank group, the relative abundances of Staphylococcus ( Staphylococus ), Coprococcus ( Coprococcus ), Corynebacterium ( Corynebacterium ) in the cecal contents of rats in the constipation model group were significantly reduced, while Lachnospiraceac _NK4A136_group, norank_f_ Oscillospiraceae , norank_f_ MuribaculaceaeThe relative abundance of the genus was significantly increased. Compared with the constipation model group, in the cecal contents of rats in the LIHUO 02 experimental group Lachnospiraceac _NK4A136_group, norank_f_ Oscillospiraceae , norank_f_ Eubacterium_coprostanoligencs _group, norank_f_ Muribaculaceae The relative abundances of the genera were significantly decreased, while the relative abundances of Staphylococcus ( Staphylococus ), 、 Corynebacterium ( Corynebacterium ), and Bifidobacterium ( Bifidobacterium ) were significantly increased (see Figure 36 ).

[0315] The results of this experiment showed that the intervention of Bifidobacterium breve LIHUO 02 could significantly increase the relative abundances of Staphylococcus ( Staphylococus ), 、 Corynebacterium ( Corynebacterium ), and Bifidobacterium ( Bifidobacterium ) in the cecal contents of Lop-induced constipated rats, and significantly decrease the relative abundances of Lachnospiraceac _NK4A136_group, norank_f_ Oscillospiraceae , norank_f_ Eubacterium_coprostanoligencs _group, norank_f_ Muribaculaceae genera, thereby regulating the intestinal flora.

[0316] Example 20: Effect of Bifidobacterium breve LIHUO 02 on short-chain fatty acids in the colonic contents of Lop-induced constipated rats

[0317] The specific steps are as follows:

[0318] The rats were grouped, modeled, and treated as in Example 13, and the colonic contents of the rats were collected under sterile conditions.

[0319] The colonic contents of 5 randomly selected rats in each group were sent to Shanghai Majorbio Bio-Pharm Technology Co., Ltd. to detect the concentration of short-chain fatty acids. The specific experimental method refers to Example 12.

[0320] The experimental results are as follows:

[0321] The concentrations of short-chain fatty acids in the colonic contents of rats in each group are shown in Figure 37Compared with the blank group, there were no significant differences in the concentrations of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, and caproic acid in the large colon contents of the constipation model group; while the concentrations of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, and caproic acid in the colon contents of the rats in the LIHUO 02 experimental group were increased compared with those in the constipation model group. Compared with the blank group, the concentration of isohexanoic acid in the colon contents of the rats in the constipation model group was significantly decreased, while the concentration of isohexanoic acid in the colon contents of the rats in the polyethylene glycol 4000 powder group and the LIHUO 02 experimental group was significantly increased compared with that in the constipation model group.

[0322] The results of this experiment showed that the intervention of Bifidobacterium breve LIHUO 02 could increase the concentration of isohexanoic acid in the colon contents of Lop-induced constipated rats, indicating that this strain could improve constipation by improving the short-chain fatty acid level.

Claims

1. A strain of Bifidobacterium breve ( Bifidobacterium breve ), LIHUO 02, characterized in that The preservation number is GDMCC No: 65513, and it was preserved in the Guangdong Provincial Microbial Culture Collection Center on November 20, 2024.

2. The preparation of Bifidobacterium breve LIHUO 02 according to claim 1, characterized in that, It is prepared by culturing the Bifidobacterium breve LIHUO 02 described in claim 1, and the preparation includes any one or more of live bacteria, fermentation broth, fermentation broth precipitate, and freeze-dried powder.

3. A microbial inoculum comprising the Bifidobacterium breve LIHUO 02 described in claim 1 or the preparation described in claim 2.

4. Use of the Bifidobacterium breve LIHUO 02 described in claim 1, the preparation described in claim 2, or the microbial inoculum described in claim 3 in the preparation of a drug for relieving ulcerative colitis.

5. The application according to claim 4, characterized in that, The relieving of ulcerative colitis includes any one or more of relieving ulcerative colitis symptoms, relieving intestinal inflammation, maintaining the integrity of the intestinal barrier, reducing oxidative stress, regulating the intestinal flora, or improving the level of short-chain fatty acids in metabolites.

6. The application according to claim 5, characterized in that, The relieving of ulcerative colitis symptoms includes any one or more of relieving weight loss, relieving colon shortening, or reducing the MPO activity in colon tissue.

7. The application according to claim 5, wherein The relieving of intestinal inflammation includes reducing the concentrations of TNF-α, IL-1β, and IL-6 in colon tissue and downregulating the relative mRNA levels, increasing the concentration of IL-10 in colon tissue and upregulating the relative mRNA levels; downregulating the relative mRNA level of NF-κB in colon tissue and upregulating the relative mRNA level of IκBα; reducing the concentration of IL-6 in serum.

8. The application according to claim 5, characterized in that, The maintaining of the integrity of the intestinal barrier includes reducing the concentration of mannitol in serum, increasing the relative level of the tight junction protein Claudin-1 in colon tissue, and upregulating the relative mRNA levels of the tight junction proteins Claudin-1, ZO-1, and the mucin MUC2.

9. The application according to claim 5, wherein The reducing of oxidative stress includes increasing the SOD activity in colon tissue and reducing the MDA activity and CAT concentration in colon tissue.

10. The application according to claim 5, wherein The regulation of the intestinal flora mainly increases the relative abundance of Lactobacillus in the cecal contents at the genus level. 、Lachnospiraceae _UCG-006, and reduces the relative abundances of Desulfovibrio, Clostridium_sensu_stricto _1, and Escherichia-Shigella; the improvement of the short-chain fatty acid level in the metabolites mainly increases the concentration of acetic acid in the colonic contents.

12. Use of the Bifidobacterium breve LIHUO 02 described in claim 1, the preparation described in claim 2, or the microbial inoculum described in claim 3 in the preparation of a product for improving constipation.

12. The application according to claim 11, wherein The improving of constipation includes promoting defecation, enhancing intestinal motility, promoting the secretion of neurotransmitters, reducing the level of aquaporin, improving colon inflammation, regulating the intestinal flora, or improving the level of short-chain fatty acids in metabolites.

13. The application according to claim 12, wherein The promoting of defecation includes increasing the number of fecal particles, increasing the water content of feces, or increasing the gastrointestinal propulsion rate.

14. The application according to claim 12, wherein The enhancing of intestinal motility includes increasing the content of mucin in colon tissue, upregulating the relative mRNA level of c-Kit in colon tissue, regulating the level of serum gastrointestinal regulatory peptides, increasing the concentrations of MTL, GAS, and SP in serum, or reducing the concentrations of SS and VIP.

15. The application according to claim 12, wherein The promoting of the secretion of neurotransmitters includes increasing the concentration of Ach in serum or increasing the concentration of 5-HT in colon tissue.

16. The application according to claim 12, wherein The reduction of aquaporin levels includes reducing the concentration of aquaporin AQP3 in colonic tissue.

17. The application according to claim 12, wherein The improvement of colonic inflammation includes reducing the concentration of TNF-α in colonic tissue or increasing the concentration of IL-10 in colonic tissue.

18. The application according to claim 12, wherein The described modulation of the gut microbiota includes a significant increase in the relative abundances of Staphylococcus, Corynebacterium, and Bifidobacterium in the cecal contents at the genus level 、 and a significant decrease in the relative abundances of Lachnospiraceac _NK4A136_group, norank_f_ Oscillospiraceae , norank_f_ Eubacterium_ coprostanoligencs _group, norank_f_ Muribaculaceae genus; The described improvement in the levels of short-chain fatty acids in metabolites includes an increase in the concentration of isocaproic acid in the colonic contents.

19. A product comprising the Bifidobacterium breve LIHUO 02 according to claim 1, or the preparation according to claim 2, or the microbial agent according to claim 3, characterized in that, The product is a product for improving constipation, or the product is a medicine for relieving ulcerative colitis.

20. The product according to claim 19, wherein, The product is an oral product, and the product for improving constipation includes food or health products or medicines.

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