Bifidobacterium breve for relieving ulcerative colitis and improving constipation and application thereof

Abstract

The invention discloses bifidobacterium breve for relieving ulcerative colitis and improving constipation and application of the bifidobacterium breve, and belongs to the field of microorganisms. The bifidobacterium breve LIHUO 02 provided by the invention has the preservation number of GDMCC No: 65513, and is preserved in the Guangdong Microbial Culture Collection Center on November 20, 2024, and the preservation number of the bifidobacterium breve LIHUO 02 is CGMCC No: 65513. The probiotic performance of the bifidobacterium breve LIHUO 02 is verified through a plurality of experiments, and the strain has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and specifically relates to a strain of Bifidobacterium breve for relieving ulcerative colitis and improving constipation and an application thereof. Background Art

[0002] Microecological therapy treats diseases by supplementing bacterial preparations that are beneficial to the human body, restoring the ecological balance of normal intestinal flora, and resisting the colonization and invasion of pathogens.

[0003] Bifidobacterium is a probiotic that can be used in microecological therapy. Existing technologies have revealed that it can be used for the prevention and treatment of acute and chronic diarrhea, various enteritis and intestinal dysbacteriosis; as an auxiliary medication 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: Bifidobacterium breve CBT BR3 improves goblet cell function by inducing aromatic hydrocarbon receptors in vitro, reducing epithelial cell permeability, and promoting goblet cell regeneration, thereby alleviating DSS-induced ulcerative colitis (Seul IP, Hyung JK, Jongwook Y, et al. Bifidobacterium breve CBT BR3 is effective at relievingintestinal inflammation by augmenting goblet cell regeneration. [J]. Journal of gastroenterology and hepatology, 2023, 38 (8): 1346-1354.).

[0005] 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 bowel movements or incomplete defecation. Its etiology is still unclear, however, genetic, lifestyle, mental and behavioral factors are associated with it. Functional constipation can be divided into: outlet obstruction constipation, slow transit constipation and mixed constipation. Treatment of slow transit constipation includes lifestyle adjustments, traditional drugs (such as laxatives, prokinetic drugs, secretagogues, microecological regulators and traditional Chinese medicine), psychotherapy, enema, acupuncture, biofeedback therapy and surgery. These treatments (used alone or in combination) increase bowel movement frequency, improve stool consistency, and relieve constipation-related symptoms. However, the efficacy of these methods will eventually weaken, leading to dependence or relapse.

[0006] Therefore, providing a strain of Bifidobacterium breve that has good in vitro probiotic properties, strong gastrointestinal tolerance, and can relieve ulcerative colitis and improve constipation, and its application have important value and application prospects. Summary of the invention

[0007] The purpose of the present invention is to disclose a strain of Bifidobacterium breve using breast milk as a screening source, verify its probiotic properties through multiple experiments, explore its potential value, and develop its application value.

[0008] At present, the probiotics that can both relieve colitis and improve constipation in patents include 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.

[0009] The breve Bifidobacterium of the present invention is LIHUO 02 Bifidobacterium breve, and its related applications include at least: relieving ulcerative colitis and improving constipation, and can be used to prepare food or health products or medicines that can relieve ulcerative colitis or improve constipation or regulate intestinal flora, and can be particularly used in probiotic products that can relieve ulcerative colitis or improve constipation or regulate intestinal flora. The problems existing in the prior art are solved, and the bacteria have good in vitro probiotic properties, relieve ulcerative colitis, improve constipation, and regulate intestinal flora.

[0010] To achieve the above object, the present invention provides the following solutions: In a first aspect, the present invention discloses a novel strain of Bifidobacterium breve.

[0011] The Bifidobacterium breve is Bifidobacterium breve LIHUO 02, which was deposited in Guangdong Microbiological Culture Collection Center on November 20, 2024, with the deposit address being 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou, with the deposit number being GDMCC No: 65513, and the classification name being Bifidobacterium breve .

[0012] Furthermore, the breast milk-derived Bifidobacterium breve LIHUO 02 was isolated and screened from the breast milk of healthy volunteer parturients in Nanchang, Jiangxi Province. The colony is white, round, moist, opaque, and has neat edges; the bacterial morphology is an irregular rod-shaped, single or paired Gram-positive strain. The strain was sequenced and analyzed, and its 16S rDNA sequence was compared with the NCBI BLAST nucleic acid sequence. The result showed that the strain was Bifidobacterium breve, and it was named Bifidobacterium breve LIHUO02, and the fragment of its 16S rDNA can be found in SEQ ID NO.1.

[0013] SEQ ID NO.1:

[0014] Furthermore, the strain has good in vitro probiotic properties, including but not limited to: good tolerance to artificial gastrointestinal fluid, high adhesion rate to Caco-2 cells, high clearance rate of DPPH, and strong inhibitory effect on Escherichia coli O157:H7 and Salmonella Enteritidis CMCC5004.

[0015] The present invention also discloses a preparation of the Bifidobacterium breve LIHUO 02, which is prepared by culturing the Bifidobacterium breve LIHUO 02. The preparation includes but is not limited to any one or more of live bacteria, dead bacteria, fermentation broth, fermentation broth supernatant, fermentation broth precipitate, and freeze-dried powder.

[0016] The present invention also discloses a microbial agent comprising the aforementioned Bifidobacterium breve LIHUO 02 or a preparation thereof.

[0017] In a second aspect, the present invention discloses the use of the aforementioned Bifidobacterium breve LIHUO 02 in the preparation of a product for relieving ulcerative colitis.

[0018] Furthermore, the product for relieving ulcerative colitis is a product that relieves the symptoms of ulcerative colitis, relieves intestinal inflammation, maintains the integrity of the intestinal barrier, reduces oxidative stress, regulates intestinal flora, and improves the level of short-chain fatty acids in metabolites.

[0019] The product is a product for relieving the symptoms of ulcerative colitis mainly by relieving weight loss, relieving colon shortening, and reducing the MPO activity of colon tissue.

[0020] The product relieves intestinal inflammation mainly by reducing the concentrations of TNF-α, IL-1β, IL-6 in colon tissue and down-regulating the relative levels of mRNA, increasing the concentration of IL-10 in colon tissue and up-regulating the relative level of mRNA; down-regulating the relative level of NF-κB mRNA in colon tissue and up-regulating the relative level of IκBα mRNA; and reducing the IL-6 concentration in serum.

[0021] The product maintains the integrity of the intestinal barrier mainly by reducing the concentration of mannitol in serum, increasing the relative level of tight junction protein Claudin-1, and upregulating the relative mRNA levels of tight junction protein Claudin-1, tight junction protein ZO-1, and mucus protein MUC2.

[0022] The product reduces oxidative stress mainly by increasing the SOD activity of colon tissue and reducing the CAT activity and MDA concentration of colon tissue.

[0023] The product regulates the intestinal flora mainly by increasing the genus level of Lactobacillus ( Lactobacillus ) 、Lachnospiraceae_UCG-006 significantly reduced the relative abundance of Desulfovibrio spp. Desulfovibrio )、 Clostridium_sensu_stricto _1 and Escherichia coli-Shigella spp. ( Escherichia-Shigella ), the product improved the level of short-chain fatty acids in metabolites mainly by increasing the concentration of acetate in colonic contents.

[0024] In a third aspect, the present invention provides use of the aforementioned Bifidobacterium breve LIHUO 02 in preparing a product for improving constipation.

[0025] 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 colon inflammation, regulates intestinal flora, and improves the level of short-chain fatty acids in metabolites.

[0026] The product is a product for promoting defecation, mainly by increasing the number of fecal particles and the water content of feces, and improving the gastrointestinal propulsion rate.

[0027] The product is a product for improving gastrointestinal motility, which mainly increases the content of mucus protein in colon tissue, upregulates the relative level of c-Kit mRNA in colon tissue, regulates the level of serum gastrointestinal regulatory peptide, increases the concentrations of MTL, GAS, and SP in serum, and reduces the concentrations of SS and VIP.

[0028] The product is a product that promotes the secretion of neurotransmitters mainly by increasing the Ach concentration in serum and the 5-HT concentration in colon tissue.

[0029] The product is a product for reducing the level of aquaporin and mainly reduces the concentration of aquaporin AQP3 in colon tissue.

[0030] The product is a product for improving colon inflammation mainly by reducing the concentration of TNF-α in colon tissue and increasing the concentration of IL-10 in colon tissue.

[0031] The product modulates the intestinal flora mainly by significantly increasing the genus level of Staphylococcus in the cecal content ( Staphylococus ) 、 Corynebacterium Corynebacterium ), Bifidobacterium spp. ( Bifidobacterium ) significantly decreased Lachnospiraceac _NK4A136_group, norank_f_ Oscillospiraceae 、norank_f_ Eubacterium_coprostanoligencs _group, norank_f_ Muribaculaceae The relative abundance of genus.

[0032] The product improves the level of short-chain fatty acids in metabolites primarily by increasing the concentration of isocaproic acid in colonic contents.

[0033] In a fourth aspect, the present invention provides a product, wherein the product contains the aforementioned Bifidobacterium breve LIHUO 02 or a preparation or a microbial agent.

[0034] Furthermore, the product is a food or a health product.

[0035] Furthermore, the product includes at least one of a probiotic product, a nutritional supplement, an animal feed and a pet snack.

[0036] Furthermore, the probiotic product includes at least one of a fermented product, a milk beverage, a live bacteria preparation, milk powder and rice flour.

[0037] Furthermore, the product is a medicine.

[0038] Furthermore, the medicine is in the form of pills, powders, capsules, tablets, film-coated preparations, orally dissolving granules, liquid preparations, suppositories or enemas.

[0039] Furthermore, the medicine also includes a pharmaceutically acceptable carrier and / or excipient.

[0040] Preferably, the product is an oral product.

[0041] The present invention also protects a method for culturing Bifidobacterium breve LIHUO 02. The culturing method may be a technology that has been disclosed or undisclosed in the prior art. The culturing method is used to cultivate Bifidobacterium breve LIHUO 02 and is within the protection scope of the present invention.

[0042] Generally speaking, the culture method may include inoculating Bifidobacterium breve LIHUO 02 into MRS medium, modified MRS medium, RCM medium, Bifidobacterium BS medium or BBL agar medium.

[0043] The beneficial effects of the present invention are as follows: The present invention discloses the use of breast milk-derived Bifidobacterium breve LIHUO 02 in relieving ulcerative colitis, improving constipation, and regulating intestinal flora. The breast milk-derived Bifidobacterium breve LIHUO 02 can be used to prepare products that can relieve ulcerative colitis or improve constipation or regulate intestinal flora. In a DSS (dextran sulfate)-induced ulcerative colitis model, Bifidobacterium breve LIHUO 02 can alleviate the weight loss of ulcerative colitis mice, alleviate the shortening of the colon, and reduce the MPO activity of colon tissue. The strain can reduce the concentration of TNF-α, IL-1β, and IL-6 in colon tissue and down-regulate the relative level of mRNA, increase the concentration of IL-10 in colon tissue and up-regulate the relative level of mRNA; down-regulate the relative level of NF-κB mRNA in colon tissue, and up-regulate the relative level of IκBα mRNA; and reduce the concentration of IL-6 in serum. The strain can reduce the concentration of mannitol in serum, increase the relative level of tight junction protein Claudin-1 in colon tissue, and upregulate the relative mRNA levels of tight junction protein Claudin-1, tight junction protein ZO-1, and mucus protein MUC2. The strain increases the activity of SOD in colon tissue, reduces the activity of CAT and MDA in colon tissue. At the genus level, the Lactobacillus genus ( Lactobacillus ) 、Lachnospiraceae _UCG-006, reducing the relative abundance of Desulfovibrio spp. Desulfovibrio )、 Clostridium_sensu_stricto _1. Escherichia coli-Shigella spp. ( Escherichia-Shigella ) and the strain can increase the relative abundance of acetic acid concentration in colon contents. In the constipation rat model induced by Lop (loperamide hydrochloride), the strain can increase the number of fecal particles and the water content of feces, and increase the gastrointestinal propulsion rate. The strain can increase the content of mucus protein in colon tissue, upregulate the relative level of c-Kit mRNA in colon tissue, and regulate the level of serum gastrointestinal regulatory peptides: increase the concentrations of MTL, GAS, and SP in serum, and reduce the concentrations of SS and VIP. The strain can increase the concentration of Ach in serum and increase the concentration of 5-HT in colon tissue. The strain can reduce the concentration of water channel protein AQP3 in colon tissue. The strain can reduce the concentration of TNF-α in colon tissue and increase the concentration of IL-10 in colon tissue. The strain increases the cecal contents of Staphylococcus aureus ( Staphylococus ) 、 Corynebacterium Corynebacterium ), Bifidobacterium spp. ( Bifidobacterium ) significantly decreased Lachnospiraceac _NK4A136_group, norank_f_ Oscillospiraceae 、norank_f_ Eubacterium_coprostanoligencs _group, norank_f_ Muribaculaceae The relative abundance of the genus. This strain can increase the concentration of isocaproic acid in the colonic contents.

[0044] Collection Instructions Strain Name: Bifidobacterium breve LIHUO 02; Chinese name of strain: Bifidobacterium breve LIHUO 02; Category naming: Bifidobacterium breve ; Deposit number: GDMCC No:65513; Depository: Guangdong Microbiological Culture Collection Center; Deposit date: November 20, 2024; Collection address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The colony morphology and bacterial morphology of strain LIHUO 02. The left picture shows the colony morphology on the culture medium, and the right picture shows the bacterial morphology under a microscope (scale bar marked 5μm).

[0046] Figure 2 This is the inhibitory effect of Bifidobacterium breve LIHUO 02 and Bifidobacterium animalis BB12 on two pathogenic bacteria in Example 5, wherein A is against Escherichia coli O157:H7, and B is against Salmonella Enteritidis CMCC 50041.

[0047] Figure 3 This is the weight change rate of mice in Example 6, and the day values ​​on the horizontal axis are 15, 16, 17, 18, 19, 20, and 21, respectively.

[0048] Figure 4 This is the DAI score of mice in Example 6, *** indicates P <0.001.

[0049] Figure 5 This is a photograph of the mouse colon in Example 6.

[0050] Figure 6 The statistical results of the mouse colon length in Example 6, *** indicates P <0.001.

[0051] Figure 7 is the spleen index of mice in Example 6, *** indicates P <0.001.

[0052] Figure 8 This is the colonic MPO activity of mice in Example 6, ** indicates P <0.01,*** indicates P <0.001.

[0053] Fig. 9 This is HE staining of mouse colon in Example 7 (2×), and the scale bar represents 500 μm.

[0054] Fig.10 This is the colon histopathological score of mice in Example 7, ** indicates P <0.01,*** indicates P <0.001.

[0055] Fig.11 The concentrations of TNF-α, IL-1β, IL-6, and IL-10 in the colon tissue of mice in Example 8 can be distinguished by the ordinate axis, where ns represents P >0.05,* indicates P <0.05,** indicates P <0.01,*** indicates P <0.001.

[0056] Fig.12 The relative mRNA levels of TNF-α, IL-1β, IL-6, and IL-10 in the colon tissue of mice in Example 8, ns represents P >0.05,* indicates P <0.05,** indicates P <0.01,*** indicates P <0.001.

[0057] Fig.13 The relative mRNA levels of NF-κB and IκBα in the colon tissue of mice in Example 8, * indicates P <0.05,** indicates P <0.01,*** indicates P <0.001.

[0058] Fig.14 is the concentration of IL6 and IL-10 in the mouse serum of Example 8, ns represents P >0.05,* indicates P <0.05,** indicates P <0.01,*** indicates P <0.001.

[0059] Fig.15 is the mannitol concentration in the mouse serum of Example 9, *** indicates P <0.001.

[0060] Fig.16 is the relative level of the tight junction protein Claudin-1 in the mouse colon tissue in Example 9, ns represents P >0.05,* indicates P <0.05,** indicatesP <0.01.

[0061] Fig.17 The relative mRNA levels of tight junction protein Claudin-1, tight junction protein ZO-1, and mucin MUC2 in mouse colon tissue in Example 9, ns represents P >0.05,* indicates P <0.05,*** indicates P <0.001.

[0062] Fig.18 The SOD activity, CAT activity and MDA concentration of the mouse colon tissue in Example 10 are shown in ns. P >0.05,* indicates P <0.05,** indicates P <0.01,*** indicates P <0.001.

[0063] Fig.19 The Sobs index and Ace index of the mouse cecal content microorganisms based on the genus level in Example 11, ns represents P >0.05,* indicates P <0.05.

[0064] Fig. 20 This is the principal component analysis (PCA) and principal coordinate analysis (PCoA) of the mouse cecal content microbiome at the genus level in Example 11.

[0065] Fig.21 The distribution of the microbiome of the cecal contents of mice in Example 11 at the phylum and genus levels, wherein the upper figure is the microbial distribution at the phylum level, and the lower figure is the microbial distribution at the genus level.

[0066] Fig. 22 This is the differential analysis of the microbiome of the mouse cecal contents at the genus level in Example 11.

[0067] Fig.23 is the concentration of short-chain fatty acids in the colon content of mice in Example 12, ns represents P >0.05,* indicates P <0.05,*** indicates P <0.001.

[0068] Fig.24 The feces water content and feces particle number of rats in Example 13, ns represents P >0.05,* indicates P <0.05,** indicates P <0.01,*** indicates P <0.001.

[0069] Fig.25 is the gastrointestinal propulsion rate of rats in Example 13, ** indicates P <0.01,*** indicates P <0.001.

[0070] Fig.26 This is the AB staining of rat colon in Example 14, and the scale bars all represent 500 μm.

[0071] Fig. 27 The relative level of c-Kit mRNA in rat colon tissue in Example 14, * indicates P <0.05,*** indicates P <0.001.

[0072] Fig.28 is the level of gastrointestinal regulatory peptide in rat serum in Example 15, ns represents P >0.05,* indicates P <0.05,** indicates P <0.01,*** indicates P <0.001.

[0073] Fig.29 is the serum Ach concentration of rats in Example 16, ** indicates P <0.01,*** indicates P <0.001.

[0074] Fig.30 5-HT concentration in rat colon tissue of Example 16, * indicates P <0.05,*** indicates P <0.001.

[0075] Fig.31 is the AQP3 concentration in rat colon tissue in Example 17, *** indicates P <0.001.

[0076] Fig.32 is the concentration of inflammatory factors in rat colon tissue in Example 18, ns represents P >0.05,** indicates P <0.01,*** indicates P <0.001.

[0077] Fig.33 The Sobs index and Simpson index of rat cecal content microorganisms based on the genus level in Example 19, ns represents P >0.05.

[0078] Fig.34This is the principal component analysis (PCA) and principal coordinate analysis (PCoA) of the rat cecal content microbiome at the genus level in Example 19.

[0079] Fig.35 The distribution of the microbiome of the cecal contents of rats in Example 19 at the phylum and genus levels, wherein the upper figure is the microbial distribution at the phylum level, and the lower figure is the microbial distribution at the genus level.

[0080] Fig.36 This is the differential analysis of the microbiome of rat cecal contents at the genus level in Example 19.

[0081] Fig.37 is the concentration of short-chain fatty acids in the colon content of rats in Example 20, ns represents P >0.05,* indicates P <0.05. DETAILED DESCRIPTION

[0082] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0083] Example 1: Isolation, purification, identification and preservation of Bifidobacterium breve LIHUO 02 The specific steps are as follows: (1) Collection of breast milk samples The breast milk samples were obtained from breast milk of healthy volunteers in Nanchang, Jiangxi Province, and the volunteers had signed informed consent. The breast milk sample collectors wore sterile rubber gloves, washed the breasts and surrounding areas of the mothers with sterile water before collecting the samples, and collected the breast milk manually, discarding the first ten drops of breast milk, collecting about 5 mL of breast milk into a sterile centrifuge tube, and transporting it back to the laboratory at low temperature for strain isolation experiments.

[0084] (2) Isolation, purification, identification and preservation of strains Isolation of strains: In an anaerobic workstation, the collected breast milk samples were diluted in a sterile PBS solution and 10 -1 , 10 -2 The dilution was spread on a modified MRS plate containing 0.05% cysteine ​​hydrochloride and cultured anaerobically at 37°C for 72 hours. The isolated strains with different colony morphologies in the breast milk samples were obtained through culture.

[0085] Purification of isolated strains: observe the colony morphology on the modified MRS plate, pick out bacteria with inconsistent colony morphology and streak them onto new modified MRS plates, culture anaerobically at 37°C for 48h, and perform purification five times in sequence to obtain pure isolated strains.

[0086] Identification and preservation of isolated strains: Pick a single colony of the purified isolated strain and put it into a 3% hydrogen peroxide solution to disperse the bacteria. 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. Use a Gram staining kit to carry out the Gram staining experiment of the isolated strain. The isolated strain is stored in 25% glycerol at -80°C.

[0087] The genome of the isolated strain was extracted using a bacterial genomic DNA extraction kit (manufacturer: Beijing Tiangen Biochemical, catalog number: Cat.#DP302-02). The 16S rDNA sequence of the strain was amplified by PCR. The PCR reaction system (50 μL) included 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 enzyme-free ultrapure water. The PCR amplification program was as follows: 95°C pre-denaturation for 10 min, (95°C denaturation for 30 s, 55°C annealing for 30 s, and 72°C extension for 1 min) for a total of 30 cycles, and 72°C end extension for 10 min. The PCR product was detected by 1.0% agarose gel electrophoresis, and the successfully amplified product was sent to Shanghai Saiheng Biotechnology Co., Ltd. for sequencing. The sequencing results of the 16S rDNA PCR product of the strain were compared with NCBI to determine the genus and species information of the strain.

[0088] (3) Detection of physiological and biochemical characteristics of strains The physiological and biochemical characteristics of the isolated strains were sent to CICC for testing, and the detection method was referred to FMIC-QO01-001-2015 Microbiological Detection of Bacterial Multiphase Identification Detection Method.

[0089] (4) Collection of strains The identified lactic acid bacteria were deposited in the Guangdong Microbiological Culture Collection Center (address: 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou) and obtained the preservation number of the strain.

[0090] The experimental results are as follows: The colony morphology of the isolated strain LIHUO 02 is white, round, moist, opaque, and has neat edges; the bacterial body morphology is irregular rod-shaped, arranged singly or in pairs (see Figure 1 ). Strain LIHUO 02 was a Gram-positive strain with negative catalase test.

[0091] The sequencing results of the PCR product of 16S rDNA of strain LIHUO 02 were compared with NCBI BLAST, 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).

[0092] Bifidobacterium breve LIHUO 02 was deposited in Guangdong Provincial Microbiological Culture Collection Center on November 20, 2024, with the deposit number GDMCC No: 65513.

[0093] Table 1 Physiological and biochemical characteristics of Bifidobacterium breve LIHUO 02

[0094] Example 2: Determination of the tolerance of Bifidobacterium breve LIHUO 02 to artificial gastrointestinal fluid The specific steps are as follows: (1) Preparation of test bacterial liquid of Bifidobacterium breve LIHUO 02 and Bifidobacterium animalis subsp. lactis BB12 Preparation of test bacterial solution: The bacterial cultures of Bifidobacterium breve LIHUO 02 and Bifidobacterium animalis subsp. lactis BB12 were inoculated into MRS liquid culture medium respectively and cultured anaerobically at 37°C for 18 h. After the strains were activated three times, they were centrifuged and the supernatant was discarded to collect the cells, and an equal volume of PBS solution was added to wash the cells twice, and an appropriate amount of PBS solution was added to resuspend the cells to prepare the test bacterial solution.

[0095] (2) Artificial gastrointestinal fluid tolerance test of Bifidobacterium breve LIHUO 02 and Bifidobacterium animalis subsp. lactis BB12 1 mL of the test bacterial solution of Bifidobacterium breve LIHUO 02 and Bifidobacterium animalis lactis subsp. BB12 was taken to determine the number of viable bacteria N0 at 0 h. 1 mL of the test bacterial solution of Bifidobacterium breve LIHUO 02 and Bifidobacterium animalis lactis subsp. BB12 was taken to 9 mL of artificial gastric juice (containing 0.3% pepsin) at pH 2.5, and the number of viable bacteria N1 was determined after 3 h at 37 ° C. 1 mL of Bifidobacterium breve LIHUO 02 treated with artificial gastric juice for 3 h was taken to determine the number of viable bacteria N1. 02. The bacterial liquid of Bifidobacterium animalis subsp. lactis BB12 was transferred to 9 mL of artificial intestinal fluid (containing 0.1% trypsin and 1.8% bile salts) at pH 8.0. The number of viable bacteria N2 was determined after 7 h at 37°C. The experiment was repeated 3 times, and the survival rate of the strain was calculated according to the following formula: artificial gastric fluid survival rate (%) = (N1 / N0) × 100, artificial intestinal fluid survival rate (%) = (N2 / N1) × 100, artificial gastrointestinal fluid survival rate (%) = (N2 / N0) × 100.

[0096] The experimental results are as follows: The survival rates of Bifidobacterium breve LIHUO 02 strains treated in artificial gastric juice at pH 2.5 for 3 hours, artificial intestinal juice at pH 8.0 for 8 hours, and artificial gastrointestinal juice for 11 hours were 87.38%, 88.56%, and 77.38%, respectively, and the survival rate in artificial gastrointestinal juice was higher than that of the control strain Bifidobacterium animalis subsp. lactis BB12 (see Table 2). The results showed that Bifidobacterium breve LIHUO 02 showed high tolerance to artificial gastrointestinal juice.

[0097] Table 2 Survival rate of Bifidobacterium breve LIHUO 02 and Bifidobacterium animalis subsp. lactis BB12 in artificial gastrointestinal fluid

[0098] Example 3: Determination of the Adhesion Ability of Bifidobacterium breve LIHUO 02 to Caco-2 Cells The specific steps are as follows: (1) Preparation of test bacterial liquid of Bifidobacterium breve LIHUO 02 and Bifidobacterium animalis subsp. lactis BB12 Preparation of test bacterial solution: The bacterial cultures of Bifidobacterium breve LIHUO 02 and Bifidobacterium animalis subsp. lactis BB12 were inoculated into MRS liquid medium and cultured anaerobically at 37°C for 18 h. After the strains were activated three times, they were centrifuged and the supernatant was discarded to collect the cells. An equal volume of PBS solution was added to wash the cells twice, 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.

[0099] (2) Determination of the adhesion ability of Bifidobacterium breve LIHUO 02 and Bifidobacterium animalis subsp. lactis BB12 to Caco-2 cells Caco-2 cells were inoculated into a culture flask containing fresh DMEM culture medium, and the cell adhesion was observed. The culture medium in the culture flask was aspirated, and the PBS solution was added to rinse twice to remove the residual culture medium; after the PBS solution was completely aspirated, trypsin was added, and the bottle was placed in an incubator at 37°C for digestion; during digestion, the cells were taken out and observed every 3 minutes until the edges of some cells began to fall off, and the digesting trypsin was aspirated and the cells were collected into a centrifuge tube containing complete culture medium, and the cell suspension in the centrifuge tube was gently blown to disperse the cells; the cells were collected by centrifugation. 0.4% table blue solution was added at a ratio of 10:1, mixed, and 20 μL was taken to the cell counting plate for counting. The cells were gently resuspended with fresh DMEM culture medium and the Caco-2 cell concentration was adjusted to 1.00×10 5 The cells were inoculated into a 24-well cell culture plate, 1 mL per well, and incubated at 37°C in a 5% CO2 incubator until the cells grew into a monolayer. After washing twice with sterile PBS solution, 1 mL of 1.00×10 8CFU (denoted as N3) of Bifidobacterium breve LIHUO 02 and Bifidobacterium animalis lactis subsp. BB12 in fresh DMEM culture medium and incubate at 37°C in a 5% CO2 incubator for 2 h.

[0100] Wash the cells in each well of the 24-well plate 5 times with PBS solution to elute the non-adherent lactic acid bacteria and metabolic secretions; add 500 μL of 0.25% trypsin-EDTA to each well for digestion for 3 minutes, and then add 500 μL of complete medium to terminate the digestion; collect the solution in each well, perform 10-fold gradient dilution, and detect the number of viable bacteria after culture (recorded as N4) by plate colony counting method. Adhesion rate (%) = N4 / N3×100.

[0101] The experimental results are as follows: 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 results of this experiment showed that Bifidobacterium breve LIHUO 02 had good adhesion to Caco-2 cells, which was conducive to its probiotic properties in the host.

[0102] Table 3 Adhesion rate of Bifidobacterium breve LIHUO 02 and Bifidobacterium animalis subsp. lactis BB12 to Caco-2 cells

[0103] Example 4: Determination of the scavenging rate of DPPH by Bifidobacterium breve LIHUO 02 The specific steps are as follows: (1) Preparation of test bacterial liquid of Bifidobacterium breve LIHUO 02 and Bifidobacterium animalis subsp. lactis BB12 The method for preparing the test bacterial solution is the same as that in Example 3.

[0104] (2) Determination of the scavenging rate of DPPH by Bifidobacterium breve LIHUO 02 and Bifidobacterium animalis subsp. lactis BB12 Sample group: 1 mL of test bacterial solution (bacterial concentration is 1×10 8 CFU / mL) and 1mL 0.2mM DPPH solution, mix well and react in the dark for 30min, centrifuge at 6000r / min for 10min, and measure the absorbance of the supernatant solution at 517nm A2. Blank group: equal volume of anhydrous ethanol instead of DPPH solution (A3), control group: equal volume of PBS solution instead of test bacterial solution (A4). The experiment was repeated 3 times, and the scavenging rate of the strain for DPPH was calculated according to the following formula. DPPH scavenging rate (%) = [1-(A2-A3) / A4]×100.

[0105] The experimental results are as follows: The scavenging rate of Bifidobacterium breve LIHUO 02 on DPPH was 52.04%, which was higher than that of the control strain Bifidobacterium animalis subsp. lactis BB12 (see Table 4). The experimental results showed that Bifidobacterium breve LIHUO 02 had a certain scavenging rate on DPPH.

[0106] The strong oxidative property of DPPH free radicals can degrade polysaccharides and DNA, kill red blood cells, decompose cell membranes, and induce cell necrosis. The results of this experiment showed that Bifidobacterium breve LIHUO 02 had a certain ability to scavenge DPPH, indicating that the strain had in vitro antioxidant capacity.

[0107] Table 4 The scavenging rate of DPPH by Bifidobacterium breve LIHUO 02 and Bifidobacterium animalis subsp. lactis BB12

[0108] Example 5: Determination of the antibacterial ability of Bifidobacterium breve LIHUO 02 against pathogenic bacteria The specific steps are as follows: (1) Preparation of fermentation broth of Bifidobacterium breve LIHUO 02 and Bifidobacterium animalis subsp. lactis BB12 After the test strains of Bifidobacterium breve LIHUO 02 and Bifidobacterium animalis lactis subsp. BB12 were activated twice, the concentration of 1.00 × 10 7 CFU / mL inoculation was inoculated into MRS liquid medium, and cultured anaerobically at 37℃ for 18h. The bacterial solution was centrifuged at 8000r / min for 10min, and the fermentation supernatant was collected and filtered through a 0.22μm microporous filter membrane. Two pathogenic bacteria, Escherichia coli O157:H7 and Salmonella enteritidis CMCC 50041 (the strain was activated three times and the bacterial concentration was adjusted to 1.00×10 8 CFU / mL) was added to LB agar at about 45°C, a sterilized Oxford cup was placed on the LB agar plate, 20 mL of LB agar containing indicator bacteria was injected into the plate, and after the plate solidified, 150 μL of the fermentation supernatant of the test strain was added to the wells and cultured at 37°C for 24 h. Whether there was an inhibition zone around the wells was observed, and a vernier caliper was used to measure the diameter (mm) of various inhibition zones. The experiment was repeated 3 times.

[0109] The experimental results are as follows: Bifidobacterium breve LIHUO 02 had different degrees of inhibitory effect on Escherichia coli O157:H7 and Salmonella Enteritidis CMCC 50041, and the inhibitory effect was higher than that of the control strain Bifidobacterium animalis subsp. lactis BB12 (see Figure 2 , Table 5).

[0110] Escherichia coli and Salmonella can easily cause intestinal infections, diarrhea and other diseases. In this experiment, Bifidobacterium breve LIHUO 02 had different degrees of inhibition on Escherichia coli O157:H7 and Salmonella enteritidis CMCC 50041, indicating that the strain can inhibit intestinal pathogens, thereby reducing the probability of intestinal infections, diarrhea and other diseases.

[0111] Table 5 Inhibitory effects of Bifidobacterium breve LIHUO 02 and Bifidobacterium animalis subsp. lactis BB12 on pathogenic bacteria

[0112] Example 6: Effects of Bifidobacterium breve LIHUO 02 on the symptoms of DSS-induced ulcerative colitis in mice The specific steps are as follows: (1) Preparation of Bifidobacterium breve LIHUO 02 bacterial suspension The strains of Bifidobacterium breve LIHUO 02 were inoculated into MRS liquid medium and cultured anaerobically at 37°C for 18 h. After the strains were activated three times, they were centrifuged and the supernatant was discarded to collect the cells. An equal volume of PBS solution was added to wash the cells twice. An appropriate amount of PBS solution was added to resuspend the cells and adjust the cell concentration to 5.00×10 8 CFU / mL.

[0113] (2) Construction of ulcerative colitis model and strain intervention 7-8 weeks old specific pathogen-free (SPF) C57 / BL6 male mice were purchased from Changsha Tianqin Biotechnology Co., Ltd. The mice were raised at a temperature of 23±2°C, a humidity of 50-60%, and a 12-h light-dark cycle. The entire experimental period was 21 days.

[0114] On day 1-7, mice in all groups drank sterile water and had free access to food. On day 8, mice were randomly divided into normal group, model group, mesalazine group and LIHUO 02 group (n=10 / group).

[0115] On day 8-14, the mice in the normal group and the model group were gavaged with PBS solution at a volume of 200 μL / mouse / day, the mice in the mesalazine group were gavaged with mesalazine solution at a volume of 200 μL / mouse / day (the dose of mesalazine was 0.2 g / kg・BW / day, the manufacturer of mesalazine enteric-coated tablets was Sunflower Pharmaceutical, item number: 221227), and the mice in the LIHUO 02 group were gavaged with Bifidobacterium breve LIHUO02 at a volume of 200 μL / mouse / day (the dose of Bifidobacterium breve LIHUO 02 was 1.00×10 8 CFU / d); mice in all groups drank sterile water and had free access to food.

[0116] On day 15-21, the mice in the normal group and the model group were gavaged with PBS solution at a volume of 200 μL / mouse / day, the mice in the mesalazine group were gavaged with mesalazine solution at a volume of 200 μL / mouse / day (the dose of mesalazine was 0.2 g / kg・BW / day), and the mice in the LIHUO 02 group were gavaged with Bifidobacterium breve LIHUO 02 at a volume of 200 μL / mouse / day (the dose of Bifidobacterium breve LIHUO 02 was 1.00×10 8 CFU / d); the normal group drank sterile water; except the normal group, the other groups drank sterile water containing 2.5% (w / v) DSS (DSS: dextran sulfate, manufacturer: MP, product number: YD08001).

[0117] (3) DAI score On the 15th to 21st day, the body weight of mice in each group was measured every day, the hardness of the feces and the blood in the feces were observed, and the disease activity index (DAI) of the mice was scored according to the scoring rules in Table 6.

[0118] Table 6 DAI score table

[0119] (4) Mouse anatomy, colon length, and spleen index determination After the gavage on the 21st day, the mice were fasted but not watered for 12 hours. Each mouse was gavaged with mannitol at a dose of 0.6 g / kg・BW and fasted for 6 hours before being dissected. The mice were anesthetized with isoflurane and euthanized. After the eyeballs of all mice were removed and blood samples were collected, the abdomen of the mice was disinfected with 75% ethanol solution. The abdomen was cut open along the midline of the abdomen using standard dissecting scissors, the entire colon was removed, and the mesentery around the colon was removed; the cecum was found, the small intestine at the upper end of the cecum was cut off, and the cecum and colon were retained; the length of the colon was measured and photographed starting from the junction of the cecum and colon. 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%.

[0120] (5) Determination of myeloperoxidase (MPO) activity in colon tissue The colon tissue was washed with ice-cold saline, and about 1 cm of the distal colon tissue was cut and fixed in 4% paraformaldehyde solution; the remaining colon tissues were collected into sterile enzyme-free cryopreservation tubes, first quick-frozen in liquid nitrogen, and then stored at -80°C for testing.

[0121] The colon tissues of mice in each group were weighed and homogenized in a pre-cooled buffer provided by the manufacturer at a ratio of 1:19 (W / V) using a homogenizer. The protein content of the colon tissues of mice in each group was determined using a BCA protein concentration assay kit (enhanced) (manufacturer: Biyuntian, item number: CatNo.P0010); the MPO activity of the colon tissues of mice in each group was determined using an MPO test kit (manufacturer: Nanjing Jiancheng, item number: A044-1-1).

[0122] The experimental results are as follows: Due to the intervention of DSS, the body weight of mice in the model group decreased significantly compared with that in the normal group (body weight decreased by 19.89% on the 21st day), and the body weight of mice in the mesalazine group and LIHUO 02 group decreased less than that in the model group (body weight decreased by 13.74% and 8.92% on the 21st day, respectively) (see Figure 3 ), indicating that the intervention of Bifidobacterium breve LIHUO 02 alleviated the weight loss of DSS-induced ulcerative colitis mice to a certain extent.

[0123] The severity of DSS-induced ulcerative colitis in mice was investigated 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 ). The results showed that Bifidobacterium breve LIHUO 02 could reduce the susceptibility of DSS-induced ulcerative colitis in mice.

[0124] DSS-induced ulcerative colitis in mice is associated with colon pathology. Compared with the normal group (7.75 cm), the colon length of mice in the model group (5.13 cm) was significantly shortened; the colon length of mice in the mesalazine group and LIHUO 02 group (6.69 cm and 6.77 cm, respectively) was significantly increased compared with the model group, indicating that the intervention of Bifidobacterium breve LIHUO 02 alleviated the shortening of the mouse colon caused by DSS to a certain extent (see Figure 5 , Figure 6 ).

[0125] The spleen index of mice in the model group was significantly higher than that in the normal group, while the spleen index of mice in the mesalazine group and LIHUO 02 group was significantly lower than that in the model group (see Figure 7 Compared with the normal group, the MPO activity of the model group mice colon was significantly increased; compared with the model group, the MPO activity of the mesalazine group and LIHUO 02 group mice was significantly decreased (see Figure 8 ), indicating that Bifidobacterium breve LIHUO02 could reduce the colonic MPO activity in DSS-induced ulcerative colitis mice.

[0126] The results of this experiment showed that Bifidobacterium breve LIHUO 02 intervention could reduce the weight loss, DAI score, increase colon length, reduce spleen index, and reduce MPO activity in colon tissue of mice with DSS-induced ulcerative colitis, indicating that Bifidobacterium breve LIHUO 02 can prevent and improve the symptoms of DSS-induced ulcerative colitis in mice.

[0127] Example 7: Effects of Bifidobacterium breve LIHUO 02 on colonic histological damage in mice with DSS-induced ulcerative colitis The specific steps are as follows: The grouping, modeling, treatment of mice, and treatment of colon tissue samples were the same as in Example 6. The colon tissues fixed in 4% paraformaldehyde solution were dehydrated, paraffin-embedded, 5 μm sliced, dewaxed, and stained with hematoxylin-eosin (H&E staining) in sequence. The inflammatory cell infiltration and mucosal tissue damage of the colon tissue were observed under a microscope, and the histopathological scores were performed with reference to Table 7.

[0128] Table 7 Histopathological scoring table

[0129] The experimental results are as follows: The results of HE staining of colon tissues of mice in each group and the results of mouse pathological scores are shown in Fig. 9 , Fig.10 The goblet cells in the colon of the mice in the normal group were abundant, with no crypt destruction and no inflammatory infiltration, while the goblet cells in the colon of the mice in the model group were damaged, the crypts were reduced, the inflammatory infiltration was severe, and the mucosal layer was lesions; the inflammatory infiltrating cells and inflammatory infiltration in the mesalazine group and LIHUO 02 group were reduced compared with the model group. Compared with the normal group, the colon tissue pathology score of the mice in the model group was significantly increased; while the colon tissue pathology score of the mice in the mesalazine group and LIHUO 02 group was significantly decreased compared with the model group.

[0130] The results of this experiment showed that intervention with Bifidobacterium breve LIHUO 02 could significantly reduce the pathological score of colon tissue in mice with DSS-induced ulcerative colitis and reduce the damage of colon tissue in mice caused by DSS.

[0131] Example 8: Effects of Bifidobacterium breve LIHUO 02 on inflammatory cytokines in colon tissue of mice with DSS-induced ulcerative colitis, real-time fluorescence quantitative PCR (RT-qPCR) of genes related to the NF-κB signaling pathway, and serum inflammatory cytokines The specific steps are as follows: The grouping, modeling, treatment of mice, and treatment of colon tissue samples were the same as in Example 6.

[0132] (1) Determination of inflammatory cytokine content in colon tissue The method for determining the protein content of the colon tissue of each group of mice was the same as in Example 6; the concentrations of TNF-α, IL-6, IL-1β, and IL-10 in the colon tissue of each group of mice were determined using enzyme-linked immunosorbent assay kits (manufacturer: Shanghai ELISA, catalog number: ml002595V), IL-6 (manufacturer: Shanghai ELISA, catalog number: ml063159V), IL-1β (manufacturer: Shanghai ELISA, catalog number: ml002095V), and IL-10 (manufacturer: Shanghai ELISA, catalog number: mlC50274-1).

[0133] (2) Real-time fluorescence quantitative PCR (RT-qPCR) detection of inflammatory factors and NF-κB signaling pathway-related genes in colon tissue The total RNA of the colon tissue of each group of mice was extracted using an RNA extraction kit (manufacturer: Takara, catalog number: 9767), and the quality and concentration of the total RNA were tested. 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, catalog 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, catalog number: 820A). The primers are shown in Table 8. -ΔΔCt The relative levels of mRNA of inflammatory factors TNF-α, IL-6, IL-1β, IL-10, NF-κB, and IκBα in colon tissue were calculated by this method (GAPDH gene was used as the internal reference gene).

[0134] Table 8 Primer sequences

[0135] 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. 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). (3) Detection of serum inflammatory cytokines After the blood samples of each group of mice were allowed to stand and stratified, they were centrifuged at 4°C and 3000r / min for 15min to obtain serum. The concentrations of serum, IL-6, and IL-10 of mice in each treatment group were determined using ELISA kits for IL-6 (manufacturer: Shanghai ELISA, catalog number: ml063159V) and IL-10 (manufacturer: Shanghai ELISA, catalog number: mlC50274-1).

[0136] The experimental results are as follows: The concentrations and relative mRNA levels of TNF-α, IL-1β, IL-6, and IL-10 in the colon tissues of mice in each group are shown in Fig.11 , Fig.12 The relative mRNA levels of NF-κB and IκBα in the colon tissue of each group of mice are shown in Fig.13 The concentrations of IL-6 and IL-10 in the serum of mice in each group are shown in Fig.14 .

[0137] Compared with the normal group, the concentrations and relative levels of TNF-α, IL-1β, and IL-6 in the colon tissue of the mice in the model group were significantly increased, while the concentrations and relative levels of TNF-α, IL-1β, and IL-6 in the colon tissue of the mice in the mesalazine group and LIHUO 02 group were significantly lower than those in the model group. Compared with the normal group, the concentration and relative level of IL-10 in the colon tissue of the mice in the model group were significantly decreased, and the concentration and relative level of IL-10 in the colon tissue of the mice in the mesalazine group were not significantly different from those in the model group; while the concentration and relative level of IL-10 in the colon tissue of the mice in the LIHUO 02 group were significantly higher than those in the model group (see Fig.11 , Fig.12 ).

[0138] In order to investigate whether the NF-κB signaling pathway plays a role in regulating the anti-inflammatory mechanism of Bifidobacterium breve LIHUO 02, the relative mRNA levels of related genes were further determined. Compared with the normal group, the relative mRNA level of NF-κB in the colon tissue of mice in the model group was significantly increased, while the relative mRNA level of NF-κB in the colon tissue of mice in the mesalazine group and LIHUO 02 group was significantly decreased compared with the model group. Compared with the normal group, the relative mRNA level of IκBα in the colon tissue of mice in the model group was significantly decreased, while the relative mRNA level of IκBα in the colon tissue of mice in the mesalazine group and LIHUO 02 group was significantly increased compared with the model group (see Fig.13 ).

[0139] Compared with the normal group, the IL-6 concentration in the serum of the mice in the model group was significantly increased; while the IL-6 concentration in the serum of the mice in the mesalazine group and LIHUO 02 group was significantly lower than that in the model group. Compared with the normal group, the IL-10 concentration in the serum of the mice in the model group was significantly decreased, and the IL-10 concentration in the serum of the mice in the mesalazine group and LIHUO 02 group was not significantly different from that in the model group (see Fig.14 ).

[0140] The results of this experiment showed that intervention with Bifidobacterium breve LIHUO 02 could significantly reduce the concentrations of TNF-α, IL-1β, and IL-6 in the colon tissue of mice with DSS-induced ulcerative colitis, 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 tissue and up-regulate the relative mRNA level of IL-10; significantly down-regulate the relative mRNA level of NF-κB in the colon tissue, and significantly up-regulate the relative mRNA level of IκBα in the colon tissue. Intervention with Bifidobacterium breve LIHUO 02 could significantly reduce the concentration of IL-6 in the serum of mice with DSS-induced ulcerative colitis. The results showed that intervention with Bifidobacterium breve LIHUO 02 could significantly reduce the concentrations of pro-inflammatory factors in the colon and serum of mice with DSS-induced ulcerative colitis, significantly increase the concentrations of anti-inflammatory factors in the colon of mice with DSS-induced ulcerative colitis, effectively improve DSS-induced ulcerative colitis in mice, regulate disordered intestinal immunity, and alleviate DSS-induced colon damage in mice by inhibiting the NF-κB signaling pathway.

[0141] Example 9: Effects of Bifidobacterium breve LIHUO 02 on the intestinal barrier of mice with DSS-induced ulcerative colitis The specific steps are as follows: 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.

[0142] (1) Determination of mannitol concentration in serum Mannitol ELISA kit (manufacturer: Shanghai ELISA, catalog number: YJ710290) was used to measure the concentration of mannitol in the serum of each group of mice.

[0143] (2) Determination of relative levels of tight junction proteins in colon tissue The colon tissues of three mice were randomly selected from each group to determine the content of tight junction proteins. The colon tissues of mice in each group were weighed and homogenized in pre-cooled RIPA lysis buffer using a homogenizer, and centrifuged at 4°C and 12000r / min for 20min. The protein content of the colon tissues of mice in each group was determined using the BCA protein concentration assay kit (enhanced) (manufacturer: Biyuntian, item number: Cat No.P0010). The protein concentration of all samples was adjusted to 3mg / mL, and 6× loading buffer was added and treated at 100°C for 5min. Samples were added to each lane and electrophoresed on SDS-PAGE gel. The proteins on the gel were transferred to a PVDF membrane, placed in an incubation box, blocked for 15min, and then the PVDF membrane was washed 3 times with 1×TBST washing solution. Incubate with β-actin primary antibody (manufacturer: Wuhan Tri-Tac, catalog number: 66009-1-Ig) and tight junction protein Claudin-1 primary antibody (manufacturer: Wuhan Tri-Tac, catalog number: 13050-1-AP) at 4°C overnight. Recover the primary antibody, wash the PVDF membrane three times with 1×TBST cleaning solution, then add the secondary antibody for 3h, and wash the PVDF membrane three times with 1×TBST cleaning solution. Use chemiluminescence imaging system and enhanced chemiluminescence (ECL) preparation to develop and photograph the immune reaction bands.

[0144] (3) Real-time fluorescence quantitative PCR (RT-qPCR) detection of tight junction protein ZO-1, tight junction protein Claudin-1 and mucin MUC-2 genes in colon tissue The total RNA of the colon tissue of each group of mice was extracted using an RNA extraction kit (manufacturer: Takara, catalog number: 9767), and the quality and concentration of the total RNA were tested. 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, catalog number: RR092A). Real-time fluorescence quantitative PCR was performed on the tight junction protein ZO-1, tight junction protein Claudin-1 and mucin MUC-2 genes using a fluorescent quantitative PCR kit (manufacturer: Takara, catalog number: 820A). The primers are shown in Table 9. -ΔΔCt The relative mRNA levels of tight junction protein ZO-1, tight junction protein Claudin-1 and mucin MUC-2 genes in colon tissue were calculated by this method (GAPDH gene was used as the internal reference gene).

[0145] Table 9 Primer sequences

[0146] 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. The experimental results are as follows: The concentration of mannitol in the serum of mice in each group was shown in Fig.15 Compared with the normal group, the mannitol concentration in the serum of the mice in the model group was significantly increased, while the mannitol concentration in the serum of the mice in the mesalazine group and LIHUO 02 group was significantly lower than that in the model group.

[0147] The relative levels of tight junction protein Claudin-1 in the colon tissue of mice in each group are shown in Fig.16 The relative mRNA levels of tight junction protein Claudin-1, tight junction protein ZO-1, and mucin MUC2 in the colon tissue of mice in each group are shown in Fig.17 . Compared with the normal group, the relative level of tight junction protein Claudin-1 in the colon tissue of mice in the model group was significantly decreased, the relative level of tight junction protein Claudin-1 in the colon tissue of mice in the mesalazine group was not significantly different from that in the model group, while the relative level of tight junction protein Claudin-1 in the colon tissue of mice in the LIHUO02 group was significantly increased compared with that in the model group. Compared with the normal group, the relative mRNA levels of tight junction protein Claudin-1, tight junction protein ZO-1, and mucin MUC2 in the colon tissue of mice in the model group were significantly decreased, the relative mRNA levels of tight junction protein ZO-1 and mucin MUC2 in the colon tissue of mice in the mesalazine group were significantly increased compared with those in the model group; while the relative mRNA levels of tight junction protein Claudin-1, tight junction protein ZO-1, and mucin MUC2 in the colon tissue of mice in the LIHUO 02 group were significantly increased compared with those in the model group. The results of this experiment showed that the intervention of Bifidobacterium breve LIHUO 02 can significantly reduce the concentration of mannitol in the serum of mice with DSS-induced ulcerative colitis, significantly increase the relative level of tight junction protein Claudin-1, and significantly upregulate the relative mRNA levels of tight junction protein Claudin-1, tight junction protein ZO-1, and mucus protein MUC2. This shows that the intervention of Bifidobacterium breve LIHUO02 can effectively improve the intestinal permeability of mice with DSS-induced ulcerative colitis and repair intestinal barrier damage.

[0148] Example 10: Effect of Bifidobacterium breve LIHUO 02 on the antioxidant capacity of colon tissue in mice with DSS-induced ulcerative colitis The specific steps are as follows: The method for determining the protein content of the colon tissue of each group of mice was the same as in Example 6. The total superoxide dismutase (SOD) assay kit (manufacturer: Nanjing Jiancheng, item number: A001-3-2), catalase (CAT) (manufacturer: Nanjing Jiancheng, item number: A007-1-1), and malondialdehyde (MDA) (manufacturer: Nanjing Jiancheng, item number: A003-1-2) assay kits were used to determine the SOD activity, CAT activity, and MDA concentration of the colon tissue of each group of mice.

[0149] The experimental results are as follows: The activity of SOD, CAT and MDA in colon tissue of mice in each group are shown in Fig.18 .

[0150] The SOD activity, CAT activity, and MDA concentration of the colon tissue 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 the colon tissue of mice in the model group was significantly reduced; the SOD activity of the colon tissue of mice in the mesalazine group was not significantly different from that in the model group, while the SOD activity of the colon tissue of mice in the LIHUO 02 group was significantly increased compared with that in the model group. Compared with the normal group, the CAT activity and MDA concentration of the colon tissue of mice in the model group were significantly increased, while the CAT activity and MDA concentration of the colon tissue of mice in the mesalazine and LIHUO 02 groups were significantly reduced compared with those in the model group.

[0151] The results of this experiment showed that the intervention of Bifidobacterium breve LIHUO 02 could reduce the colonic CAT activity and MDA concentration of mice with ulcerative colitis and increase the SOD activity. This showed that Bifidobacterium breve LIHUO 02 could alleviate DSS-induced colitis in mice by reducing the level of oxidative stress.

[0152] Example 11: Effect of Bifidobacterium breve LIHUO 02 on intestinal flora of mice with DSS-induced ulcerative colitis The specific steps are as follows: The grouping, modeling and treatment of mice were the same as in Example 6, and the cecal content samples of mice were collected under sterile conditions.

[0153] The cecal contents of 6 mice in each group were randomly selected and sent to Shanghai Meiji Biopharmaceutical Technology Co., Ltd. for detection of microbial diversity. The total DNA of the cecal contents was extracted. After the concentration and purity of the total DNA were detected, the V3-V4 region of 16SrDNA was amplified using 338F and 806R. Uparse software (version 7.0.1090) was used to cluster the sequences into the same taxonomic unit (OTU). All sequences were divided into OTUs according to different similarity levels, and bioinformatics statistical analysis was performed on OTUs at a similarity level of 97%. The Bayesian algorithm in the RDP classifier was used to perform taxonomic analysis on the representative sequences of OTUs at a similarity level of 97%. The Qiime platform was used to analyze the alpha diversity and beta diversity at the genus level and the proportion and difference analysis of different species in samples of different groups. Principal component analysis (PCA) and principal coordinate analysis (PCoA) were performed based on the weighted UniFrac distance; R language was used for drawing.

[0154] The experimental results are as follows: The Sobs index and Ace index of the cecal contents of each group of mice are shown in Fig.19 . Compared with the normal group, the Sobs index of the cecal content microorganisms in the model group was significantly increased based on the genus level; the Sobs index of the cecal content microorganisms in the mesalazine group was higher than that in the model group, but the difference was not significant; the Sobs index of the cecal content microorganisms in the LIHUO 02 group was lower than that in the model group, but the difference was not significant. Compared with the normal group, the Ace index of the cecal content microorganisms in the model group was increased based on the genus level, but the difference was not significant; the Ace index of the cecal content microorganisms in the mesalazine group was higher than that in the model group, but the difference was not significant; the Ace index of the cecal content microorganisms in the LIHUO 02 group was lower than that in the model group, but the difference was not significant. It shows that the intake of DSS leads to changes in the microbial diversity of mice, and the intervention of Bifidobacterium breve LIHUO 02 reverses these changes to a certain extent.

[0155] Principal component analysis (PCA) and principal coordinate analysis (PCoA) of the cecal contents of each group of mice based on genus level analysis (see Fig. 20 ) showed that the normal group, LIHUO 02 group and model group showed different microbiome compositions.

[0156] The distribution of microorganisms in the cecal contents of mice in each group at the phylum and genus levels is shown in Fig.21 Firmicutes ( Firmicutes ), Bacteroidetes ( Bacteroidota) dominated the relative abundance of the microbiome in the cecal contents of each group of mice. Ileibacterium )、norank_f_ Muribaculaceae 、norank_f_norank_o_ Clostridia The relative abundance of the genus _UCG-014 was dominant in the microbiome of the cecal contents of each group of mice.

[0157] Compared with the normal group, the cecal contents of the model group mice were higher in Ileobacter spp. Ileibacterium ), Lactobacillus spp. Lactobacillus ) decreased significantly, norank_f_norank_o_ Clostridia _UCG-014, Bacteroides spp. Bacteroides ) were significantly increased. Compared with the model group, the relative abundance of Desulfovibrio spp. in the cecal contents of mice in the LIHUO 02 group was Desulfovibrio )、 Clostridium _ sensu_stricto _1. Escherichia coli-Shigella spp. ( Escherichia-Shigella ) decreased significantly, while the relative abundance of Lactobacillus ( Lactobacillus ) 、 Lachnospiraceae The relative abundance of _UCG-006 increased significantly (see Fig. 22 ).

[0158] The experimental results showed that the intervention of Bifidobacterium breve LIHUO 02 could make the microbial composition of cecal contents closer to that of the normal group, and could significantly increase the Lactobacillus genus in the cecal contents of DSS-induced colitis mice ( Lactobacillus ) 、 Lachnospiraceae _UCG-006 significantly reduced the relative abundance of Desulfovibrio spp. Desulfovibrio )、 Clostridium_sensu_stricto _1. Escherichia coli-Shigella spp. ( Escherichia-Shigella ), indicating that the intervention of Bifidobacterium breve LIHUO 02 could reverse the changes in intestinal flora of mice with ulcerative colitis caused by DSS.

[0159] Example 12: Effects of Bifidobacterium breve LIHUO 02 on short-chain fatty acids in colonic contents of mice with DSS-induced ulcerative colitis The specific steps are as follows: The grouping, modeling and treatment of mice were the same as in Example 6, and the colon contents of mice were collected under sterile conditions.

[0160] The colon contents of 5 mice randomly selected from each group were sent to Shanghai Meiji Biopharmaceutical Technology Co., Ltd. to detect the content of short-chain fatty acids. The specific method is: weigh the standard and colon content samples respectively in the grinding tube, add 0.5% phosphoric acid solution (containing the internal standard 2-ethylbutyric acid 10μg / mL). The sample was frozen and ground and centrifuged at 4 ℃ and 13000g for 15 min, the supernatant was taken and extracted with n-butanol solvent, vortexed and mixed, and then low-temperature ultrasonicated for 10min, centrifuged at 4 ℃ and 13000g for 5min, and the supernatant solution was taken for GC-MC detection. Chromatographic conditions: HP FFAP capillary column (30m×0.25mm×0.25μm), carrier gas is high-purity helium (purity not less than 99.999%), flow rate is 1.0mL / min, and the temperature of the injection port is 180℃. The injection volume is 1μL, split injection, split ratio is 10:1, and the solvent is extended for 2.5min. Program temperature rise: The initial temperature of the column oven is 80°C, which is then raised to 120°C at 20°C / min, then raised to 160°C at 5°C / min, and then maintained at 220°C for 3 min. Mass spectrometry conditions: electron impact ion source (EI), ion source temperature 230°C, quadrupole temperature 150°C, transmission line temperature 230°C, electron energy 70 eV. The scanning mode is selected ion scanning mode (SIM).

[0161] The experimental results are as follows: The concentrations of short-chain fatty acids in the colon contents of mice in each group are shown in Fig.23 Compared with the normal group, the concentrations of acetic acid, propionic acid, butyric acid, isobutyric acid, and isovaleric acid in the colon contents of mice in the model group were significantly reduced, while the concentration of acetic acid in the colon contents of mice in the Bifidobacterium breve LIHUO 02 intervention group was significantly higher than that in the model group, while there was no significant difference between the mesalazine group and the model group. This indicates that Bifidobacterium breve LIHUO 02 can improve the level of short-chain fatty acids in the colon contents of mice with DSS-induced ulcerative colitis.

[0162] Example 13: Effect of Bifidobacterium breve LIHUO 02 on the symptoms of loperamide hydrochloride (Lop)-induced constipation in rats The specific steps are as follows: (1) Preparation of Bifidobacterium breve LIHUO 02 bacterial suspension The strains of Bifidobacterium breve LIHUO 02 were inoculated into MRS liquid medium and cultured anaerobically at 37°C for 18 h. After the strains were activated three times, they were centrifuged and the supernatant was discarded to collect the cells. An equal volume of PBS solution was added to wash the cells twice. An appropriate amount of PBS solution was added to resuspend the cells and adjust the cell concentration to 1.00×10 8 CFU / mL.

[0163] (2) Construction of constipation model, strain intervention, and determination of fecal particle number and fecal water content 6-8 weeks old specific pathogen-free (SPF) SD male rats were purchased from Changsha Tianqin Biotechnology Co., Ltd. Rats were raised at a temperature of 23±2℃, a humidity of 50-60%, and a 12-h light-dark cycle. The entire experimental period was 21 days.

[0164] On the 1st day to the 7th day, rats in all groups drank sterile water and had free access to food. On the 8th day, the rats were divided into the blank group, the constipation model group, the polyethylene glycol 4000 powder group, and the LIHUO 02 experimental group (n=8 / group).

[0165] From d 8 to 14, the rats in the blank group and constipation model group were gavaged with PBS solution at a volume of 1 mL / rat / day, the rats in the polyethylene glycol 4000 powder group were gavaged with polyethylene glycol 4000 powder solution at a volume of 1 mL / rat / day (the dosage of polyethylene glycol 4000 powder was 0.3 g / kg・BW / day; the manufacturer of polyethylene glycol 4000 powder was Forlax, the product number was W11636), and the rats in the LIHUO 02 experimental group were gavaged with Bifidobacterium breve LIHUO 02 solution at a volume of 1 mL / rat / day (the dosage of Bifidobacterium breve LIHUO 02 was 1.00×10 8 CFU / d); rats in all groups drank sterile water and had free access to food.

[0166] From 15th to 21st day, the blank group rats were intraperitoneally injected with saline twice at a volume of 1 mL / rat / day; the other groups rats were intraperitoneally injected with loperamide hydrochloride twice at a volume of 1 mL / rat / day (loperamide hydrochloride dose: 16 mg / kg・BW / d; loperamide hydrochloride manufacturer: Aladdin, item number: L129465). The blank group and constipation model group rats were gavaged with PBS solution at a volume of 1 mL / rat / day, the polyethylene glycol 4000 powder group rats were gavaged with polyethylene glycol 4000 powder solution at a volume of 1 mL / rat / day (polyethylene glycol 4000 powder dose: 0.3 g / kg・BW / d), and the LIHUO 02 experimental group was gavaged with 1.00×10 Bifidobacterium breve LIHUO 02 bacterial solution at a volume of 1 mL / rat / day. 8 CFU / d. Rats in all groups drank sterile water and had free access to food. On the 21st day, the feces of each rat were collected within 1 hour, the number of fecal particles was recorded, and the weight was m1; the rat feces were dried at 60°C for 24 hours, cooled to room temperature in a dryer, and weighed m2. The calculation formula for fecal moisture content is as follows: fecal moisture content (%) = (m1-m2) / m1×100%.

[0167] (3) Preparation of Evans blue solution Dissolve 2.5 g carboxymethyl cellulose, 8 g milk powder, 4 g sucrose, 4 g starch and 3.5 mL 1% Evans blue solution in distilled water, mix well with a vortex mixer, and prepare 150 mL of Evans blue semi-solid paste. Prepare before use.

[0168] (4) Rat dissection After the gavage on the 21st day, the rats were fasted and deprived of water for 12 hours. Each rat was gavaged with 2 mL of Evans blue paste. Blood samples were collected after eye sockets were punctured at 15 minutes and the rats were killed. After the blood samples were collected, the abdomen of the rats was disinfected with 75% ethanol solution. The abdomen was cut open along the midline of the abdomen using standard dissecting scissors to remove the complete gastrointestinal tissue of the rats. The distance from the position of the pyloric sphincter of the rat to the end of the carbon powder propulsion and the total intestinal length of the small intestine were measured and recorded with a ruler to measure the travel distance of the Evans blue paste from the pylorus. The gastrointestinal propulsion rate (GIP) was calculated based on the following formula: GIP = Evans blue paste travel distance (cm) / total length of small intestine (cm) × 100%.

[0169] The colon tissue was washed with ice-cold saline, and about 1 cm of the distal colon tissue was cut and fixed in 4% paraformaldehyde solution; the remaining colon tissue was collected into sterile enzyme-free cryopreservation tubes, first quick-frozen in liquid nitrogen, and then stored at -80°C for testing.

[0170] Experimental results: The fecal water content and the number of fecal particles of rats in the constipation model group were significantly lower than those in the blank group, the fecal water content of rats in the polyethylene glycol 4000 powder group was significantly higher than that in the model group, and the fecal water content and the number of fecal particles of rats in the LIHUO 02 experimental group were significantly higher than those in the model group (see Fig.24 ).

[0171] Due to the intervention of loperamide hydrochloride (Lop), the gastrointestinal propulsion rate of rats in the constipation model group was significantly lower than that in the normal group, while the gastrointestinal propulsion rate of rats in the polyethylene glycol 4000 powder group and the LIHUO 02 experimental group was significantly higher than that in the model group (see Fig.25 ).

[0172] 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 rats with Lop-induced constipation, thereby improving the symptoms of constipation.

[0173] Example 14: Effects of Bifidobacterium breve LIHUO 02 on relative levels of mucin and c-Kit mRNA in colonic tissue of rats with Lop-induced constipation The specific steps are as follows: The grouping, modeling, treatment of rats, and processing of colon tissue samples were the same as in Example 13.

[0174] (1) AB staining of colon tissue The colon tissues of rats in each group fixed in 4% paraformaldehyde solution were dehydrated, paraffin-embedded, 5 μm sliced, dewaxed, and stained with Alcian blue (AB) in sequence.

[0175] (2) Real-time fluorescence quantitative PCR (RT-qPCR) detection of c-Kit mRNA in colon tissue The total RNA of the colon tissue of each group of rats was extracted using an RNA extraction kit (manufacturer: Takara, catalog number: 9767), and the quality and concentration of the total RNA were tested. 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, catalog number: RR092A). Real-time fluorescence quantitative PCR was performed on the c-Kit gene using a fluorescent quantitative PCR kit (manufacturer: Takara, catalog number: 820A). The primers are shown in Table 10. -ΔΔCt The relative level of c-Kit mRNA in colon tissue was calculated by this method (GAPDH gene was used as the internal reference gene).

[0176] Table 10 Primer sequences

[0177] Primer sequence reference: 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. The experimental results are as follows: Alcian blue (AB) staining was used to detect the distribution of mucus-secreting goblet cells in the colon tissue of rats in each group (see Fig.26 ), the results showed that compared with the blank group, the content of mucin in the colon tissue of rats in the constipation model group was decreased, while the content of mucin in the colon tissue of rats in the polyethylene glycol 4000 powder group and the LIHUO 02 experimental group was increased compared with that in the model group.

[0178] Compared with the blank group, the relative level of c-Kit mRNA in the colon tissue of rats in the constipation model group was significantly decreased, while the relative level of c-Kit mRNA in the colon tissue of rats in the polyethylene glycol 4000 powder group and the LIHUO 02 experimental group was significantly increased compared with the model group (see Fig. 27 ).

[0179] Interstitial cells of Cajal (ICC) are cells that regulate colonic motility, and their distribution is closely related to intestinal motility. 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 can increase the mucus protein in the colon of rats with Lop-induced constipation, and significantly upregulate the relative mRNA level of the c-Kit gene in the colon tissue of rats with Lop-induced constipation, thereby improving intestinal motility and constipation.

[0180] Example 15: Effect of Bifidobacterium breve LIHUO 02 on serum gastrointestinal regulatory peptide levels in rats with Lop-induced constipation The specific steps are as follows: The grouping, modeling and treatment of rats were the same as in Example 13.

[0181] After the blood samples of rats in each group were allowed to stand and stratified, they were centrifuged at 4°C and 3000r / min for 15min to obtain serum. The concentrations of Gas, MTL, SS, SP, and VIP in the serum of rats in each group were determined using enzyme-linked immunosorbent assay kits for gastrin (Gas) (manufacturer: Nanjing Jiancheng, item number: H239-1-2), motilin (MTL) (manufacturer: Nanjing Jiancheng, item number: H182-1-2), somatostatin (SS) (manufacturer: Nanjing Jiancheng, item number: H092), substance P (SP) (manufacturer: Nanjing Jiancheng, item number: H218-1-2), and vasoactive peptide (VIP) (manufacturer: Nanjing Jiancheng, item number: H219-1-2).

[0182] The experimental results are as follows: Compared with the blank group, the serum MTL, GAS, and SP concentrations of rats in the constipation model group decreased significantly, while the serum MTL and GAS concentrations of rats in the polyethylene glycol 4000 powder group increased significantly compared with the model group; while the serum MTL, GAS, and SP concentrations of rats in the LIHUO 02 experimental group increased significantly compared with the constipation model group. Compared with the blank group, the serum VIP and SS concentrations of rats in the model group increased significantly, while the serum VIP and SS concentrations of rats in the polyethylene glycol 4000 powder group and the LIHUO 02 experimental group decreased significantly compared with the constipation model group (see Fig.28 ).

[0183] MTL, GAS, and SP can promote gastrointestinal muscle contraction and relaxation of the pyloric sphincter, and promote intestinal motility. SS and VIP are gastrointestinal regulatory peptides that inhibit intestinal motility. The results of this experiment showed that the intervention of Bifidobacterium breve LIHUO 02 significantly increased the concentrations of MTL, GAS, and SP in the serum of rats with Lop-induced constipation, and significantly reduced the concentrations of VIP and SS; indicating that Bifidobacterium breve LIHUO 02 can improve constipation by regulating the level of serum gastrointestinal regulatory peptides.

[0184] Example 16: Effects of Bifidobacterium breve LIHUO 02 on neurotransmitter levels in rats with Lop-induced constipation The specific embodiments are as follows: 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.

[0185] The acetylcholine (Ach) enzyme-linked immunosorbent assay kit (manufacturer: Shanghai ELISA, catalog number: ml003048V) was used to measure the serum Ach concentration of rats in each group.

[0186] The colon tissues of rats in each group were weighed and homogenized in a 1:9 (W / V) pre-cooled PBS solution using a tissue homogenizer. The protein content of the colon tissues of rats in each group was determined using the BCA protein concentration assay kit (enhanced) (manufacturer: Biyuntian, item number: Cat No. P0010); the 5-hydroxytryptamine (5-HT) enzyme-linked immunosorbent assay kit (manufacturer: Shanghai ELISA, item number: ml059511V) was used to determine the concentration of 5-HT in the colon tissues of rats in each treatment group.

[0187] The experimental results are as follows: Compared with the blank group, the Ach concentration in the serum of rats in the constipation model group was significantly decreased, and the Ach concentration in the serum of rats in the polyethylene glycol 4000 powder group and the LIHUO 02 experimental group was significantly higher than that in the constipation model group (see Fig.29 ).

[0188] Compared with the blank group, the 5-HT concentration in the colon tissue of rats in the constipation model group was significantly decreased, while the 5-HT concentration in the colon tissue of the polyethylene glycol 4000 powder group and the LIHUO 02 experimental group was significantly increased compared with the constipation model group (see Fig.30 ).

[0189] Acetylcholine (Ach) can excite gastrointestinal smooth muscle, increase contraction amplitude, tension and peristalsis, and promote gastric and intestinal fluid secretion. 5-HT is an important neurotransmitter that can reduce the colon transit time of rat feces. The results of this experiment showed that the intervention of Bifidobacterium breve LIHUO 02 can significantly increase the serum Ach concentration and colon tissue 5-HT concentration of rats with Lop-induced constipation, thereby improving constipation.

[0190] Example 17: Effect of Bifidobacterium breve LIHUO 02 on AQP3 concentration in colon tissue of rats with Lop-induced constipation The specific steps are as follows: The grouping, modeling and treatment of rats were the same as in Example 13.

[0191] The detection of protein content in colon tissue of rats in each group was the same as in Example 16; the AQP3 concentration in colon tissue of rats in each treatment group was determined using an AQP3 ELISA kit (manufacturer: Shanghai ELISA, catalog number: ml003095V).

[0192] The experimental results are as follows: Compared with the blank group, the AQP3 concentration in the colon tissue of rats in the constipation model group was significantly increased, while the AQP3 concentration 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 Fig.31 ).

[0193] AQP3 is mainly expressed in colonic mucosal epithelial cells and is believed to play a key role in controlling water transport in the colon. The results of this experiment showed that the intervention of Bifidobacterium breve LIHUO 02 could significantly reduce the concentration of AQP3 in colonic tissue of rats with Lop-induced constipation, thereby improving constipation.

[0194] Example 18: Effects of Bifidobacterium breve LIHUO 02 on inflammatory factors in colon tissue of rats with Lop-induced constipation The experimental steps are as follows: The grouping, modeling and treatment of rats were the same as in Example 13.

[0195] The method for detecting the protein content in the colon tissue of each group of rats was the same as in Example 16; the concentrations of TNF-α and IL-10 in the colon tissue of each group of rats were determined using ELISA kits for TNF-α (manufacturer: Shanghai ELISA, item number: ml002595V) and IL-10 (manufacturer: Shanghai ELISA, item number: mlC50274-1).

[0196] The experimental results are as follows: Compared with the blank group, the concentration of TNF-α in the colon tissue of rats in the constipation model group was significantly increased, and the concentration of TNF-α in the colon tissue of rats in the polyethylene glycol 4000 powder group and the LIHUO 02 experimental group was significantly lower than that in the constipation model group. Compared with the blank group, the concentration of IL-10 in the colon tissue of rats in the constipation model group was significantly decreased, and there was no significant difference between the polyethylene glycol 4000 powder group and the constipation model group, while the concentration of IL-10 in the LIHUO 02 experimental group was significantly higher than that in the constipation model group (see Fig.32 ).

[0197] The results of this experiment showed that the intervention of Bifidobacterium breve LIHUO 02 could significantly reduce the concentration of pro-inflammatory factor TNF-α and significantly increase the concentration of anti-inflammatory factor IL-10 in the colon tissue of rats with Lop-induced constipation, thereby improving colon inflammation caused by constipation.

[0198] Example 19: Effect of Bifidobacterium breve LIHUO 02 on intestinal flora of rats with Lop-induced constipation The specific steps are as follows: The grouping, modeling and treatment of rats were the same as in Example 12, and the cecal contents of the rats were collected under sterile conditions.

[0199] The cecal contents of 6 rats were randomly selected from each group and sent to Shanghai Meiji Biopharmaceutical Technology Co., Ltd. for detection of microbial diversity. Total DNA from rat cecal contents was extracted. After the concentration and purity of total DNA were detected, the V3-V4 region of 16S rDNA was amplified using 338F and 806R. Uparse (version 7.0.1090) was used to cluster the sequences into the same taxonomic unit (OTU). All sequences were divided into OTUs according to different similarity levels, and bioinformatics statistical analysis was performed on OTUs at a similarity level of 97%. The Bayesian algorithm in the RDP classifier was used to perform taxonomic analysis on the representative sequences of OTUs at a similarity level of 97%. The Qiime platform was used to analyze the alpha diversity and beta diversity at the genus level, the proportion of different species in samples from different groups, and the species difference analysis. Principal component analysis (PCA) and principal coordinate analysis (PCoA) were performed based on weighted UniFrac distance; R language was used for drawing.

[0200] The experimental results are as follows: The Sobs index and Simpson index of the α diversity of the cecal contents of rats in each group are shown in Fig.33 . Compared with the blank group, based on the genus level, the Sobs index of the cecal content microorganisms in the constipation model group rats increased, but the difference was not significant; the Sobs index of the cecal content microorganisms 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 cecal content microorganisms in the LIHUO 02 experimental group decreased compared with the constipation model group, but the difference was not significant. Compared with the blank group, based on the genus level, the Simpson index of the cecal content microorganisms in the constipation model group rats decreased, but the difference was not significant; while the Simpson index of the cecal content microorganisms 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.

[0201] Principal component analysis (PCA) and principal coordinate analysis (PCoA) of the cecal content microorganisms of each group of rats based on genus level analysis (see Fig.34 ) showed that the blank group, LIHUO 02 experimental group and constipation model group showed different microbiome compositions, while the LIHUO 02 experimental group was closer to the blank group.

[0202] The distribution of microorganisms in the cecal contents of rats in each group at the phylum and genus levels was shown in Fig.35 Firmicutes ( Firmicutes ), Actinobacteria ( Actinobacteriota ) were the dominant flora in the cecum contents of rats in the blank group and the LIHUO 02 experimental group; while Firmicutes ( Firmicutes ), Bacteroidetes ( Bacteroidota ), Actinobacteria ( Actinobacteriota ) were the dominant flora in the cecal contents of rats in the constipation model group and the polyethylene glycol 4000 powder group. Clostridia _UCG-014, Lactobacillus spp. Lactobacillus ), Rombutsiella ( Romboutsia ) dominated the microbiome of the cecal contents of rats in each group.

[0203] Compared with the blank group, the number of Staphylococcus spp. in the cecal contents of rats in the constipation model group was Staphylococus ), Coprococcus ( Coprococcus ), Corynebacterium ( Corynebacterium ) decreased significantly, while Lachnospiraceac _NK4A136_group, norank_f_ Oscillospiraceae 、norank_f_ Muribaculaceae Compared with the constipation model group, the relative abundance of the genus was significantly increased 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 abundance of the genus Staphylococcus ( Staphylococus ) 、 Corynebacterium Corynebacterium ), Bifidobacterium spp. ( Bifidobacterium ) increased significantly (see Figure 36 ).

[0204] The results of this experiment showed that the intervention of Bifidobacterium breve LIHUO 02 could significantly increase the number of Staphylococcus spp. in the cecal contents of rats with Lop-induced constipation. Staphylococus ) 、 Corynebacterium Corynebacterium ), Bifidobacterium spp. ( Bifidobacterium ) significantly decreased Lachnospiraceac _NK4A136_group, norank_f_ Oscillospiraceae 、norank_f_ Eubacterium_coprostanoligencs _group, norank_f_ Muribaculaceae The relative abundance of the genera regulates the intestinal flora.

[0205] Example 20: Effect of Bifidobacterium breve LIHUO 02 on short-chain fatty acids in colonic contents of rats with Lop-induced constipation The specific steps are as follows: The grouping, modeling and treatment of rats were the same as in Example 13, and the colon contents of the rats were collected under sterile conditions.

[0206] The colon contents of 5 rats randomly selected from each group were sent to Shanghai Meiji Biopharmaceutical Technology Co., Ltd. to detect the concentration of short-chain fatty acids. For specific experimental methods, refer to Example 12.

[0207] The experimental results are as follows: The concentrations of short-chain fatty acids in the colon contents of rats in each group were shown in Figure 37 Compared with the blank group, the concentrations of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, and caproic acid in the colon contents of the constipation model group were not significantly different; while the concentrations of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, and caproic acid in the colon contents of rats in the LIHUO 02 experimental group were higher than those in the constipation model group. Compared with the blank group, the concentration of isocaproic acid in the colon contents of rats in the constipation model group was significantly reduced, while the concentration of isocaproic acid in the colon contents of rats in the polyethylene glycol 4000 powder group and the LIHUO 02 experimental group was significantly higher than that in the constipation model group.

[0208] The results of this experiment showed that the intervention of Bifidobacterium breve LIHUO 02 could increase the concentration of isocaproic acid in the colon contents of rats with Lop-induced constipation, indicating that this strain can improve constipation by improving the level of short-chain fatty acids.

Claims

1. A strain of Bifidobacterium breve ( Bifidobacterium breve LIHUO 02, characterized by: The deposit number is GDMCC No: 65513, and it was deposited in Guangdong Provincial Microbiological Culture Collection Center on November 20, 2024.

2. The preparation of Bifidobacterium breve LIHUO 02 according to claim 1, characterized in that The preparation is carried out by culturing the Bifidobacterium breve LIHUO 02 according to claim 1, wherein the preparation comprises any one or more of live bacteria, dead bacteria, fermentation broth, fermentation broth supernatant, fermentation broth precipitate and freeze-dried powder.

3. A microbial agent comprising the Bifidobacterium breve LIHUO 02 of claim 1 or the preparation of claim 2.

4. Use of the Bifidobacterium breve LIHUO 02 according to claim 1, the preparation according to claim 2, or the microbial agent according to claim 3 in the preparation of a product for relieving ulcerative colitis.

5. The use according to claim 4, characterized in that: The relief of ulcerative colitis includes any one or more of relief of ulcerative colitis symptoms, relief of intestinal inflammation, maintenance of intestinal barrier integrity, reduction of oxidative stress, regulation of intestinal flora, or improvement of short-chain fatty acid levels in metabolites.

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

7. The use according to claim 5, characterized in that: The method for relieving intestinal inflammation includes reducing the concentration of TNF-α, IL-1β, and IL-6 in colon tissue and down-regulating the relative level of mRNA, increasing the concentration of IL-10 in colon tissue and up-regulating the relative level of mRNA; down-regulating the relative level of NF-κB mRNA in colon tissue and up-regulating the relative level of IκBα mRNA; and reducing the concentration of IL-6 in serum.

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

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

10. The use according to claim 5, characterized in that: The intestinal flora is regulated mainly by increasing the number of Lactobacillus genus in the cecal contents at the genus level. 、Lachnospiraceae _UCG-006, reducing the relative abundance of Desulfovibrio, Clostridium_sensu_stricto _1 and the relative abundance of Escherichia coli-Shigella; the product improves the level of short-chain fatty acids in metabolites mainly by increasing the concentration of acetic acid in the colon contents.

11. Use of the Bifidobacterium breve LIHUO 02 according to claim 1, the preparation according to claim 2, or the microbial agent according to claim 3 in preparing a product for improving constipation.

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

13. The use according to claim 12, characterized in that: The defecation promotion includes increasing the number of feces particles, increasing the water content of feces or increasing the gastrointestinal propulsion rate.

14. The use according to claim 12, characterized in that: The improvement of gastrointestinal motility includes increasing the content of mucus protein in colon tissue, upregulating the relative level of c-Kit mRNA in colon tissue, regulating the level of serum gastrointestinal regulatory peptide, increasing the concentration of MTL, GAS, SP in serum or reducing the concentration of SS and VIP.

15. The use according to claim 12, characterized in that: The promotion of neurotransmitter secretion includes increasing the Ach concentration in serum or increasing the 5-HT concentration in colon tissue.

16. The use according to claim 12, characterized in that: The reducing of the water channel protein level includes reducing the concentration of the water channel protein AQP3 in colon tissue.

17. The use according to claim 12, characterized in that: The improvement of colon inflammation includes reducing the concentration of TNF-α in colon tissue or increasing the concentration of IL-10 in colon tissue.

18. The use according to claim 12, characterized in that: The described modulation of the intestinal flora included a significant increase in cecal contents at the genus level of Staphylococcus 、 The relative abundance of Corynebacterium and Bifidobacterium decreased significantly. Lachnospiraceac _NK4A136_group, norank_f_ Oscillospiraceae 、norank_f_ Eubacterium_ coprostanoligencs _group, norank_f_ Muribaculaceae The relative abundance of the genus; the improved levels of short-chain fatty acids in metabolites include increasing the concentration of isocaproic acid in colonic contents.

19. A product comprising the Bifidobacterium breve LIHUO 02 of claim 1 or the preparation of claim 2 or the microbial agent of claim 3.

20. The product according to claim 19, characterized in that The product is an oral product, and the product includes food, health care product or medicine.

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