Bifidobacterium animalis subspecies lactis for relieving ulcerative colitis and constipation and its application

Abstract

The present invention discloses animal Bifidobacterium lactis subspecies for relieving ulcerative colitis and constipation and its application, belonging to the field of microorganisms. The present invention screens out a strain of animal Bifidobacterium lactis subspecies ( Bifidobacterium animalum subsp. milk ) LIHUO 01, which was deposited in Guangdong Microbiological Culture Collection Center on November 20, 2024, with the deposit number GDMCC No: 65512. This animal Bifidobacterium lactis subspecies LIHUO 01 has good in vitro probiotic properties, can relieve ulcerative colitis, improve constipation, and regulate intestinal flora, and has good application prospects in the preparation of foods or health products or medicines for relieving ulcerative colitis or improving constipation or regulating intestinal flora.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and particularly relates to animal Bifidobacterium lactis subspecies for relieving ulcerative colitis and constipation and application thereof. Background Art

[0002] Inflammatory bowel disease (IBD) is an inflammatory gastrointestinal disease with chronic and recurrent characteristics, including ulcerative colitis (UC) and Crohn's disease (CD). Ulcerative colitis is characterized by abdominal pain, blood in the stool, and weight loss. The etiology and pathogenesis of ulcerative colitis are believed to be related to multiple factors, generally believed to be related to genetic susceptibility, immune response disorders, intestinal flora disorders, environmental factors, diet, etc. At present, the mainstream drugs for relieving and treating ulcerative colitis are aminosalicylic acid, corticosteroids, TNF-α inhibitors, antibiotics, immunomodulators, lncRNA and miRNA, etc. Although they can improve clinical symptoms, the efficacy is still not ideal, and they can also cause potential adverse reactions such as abdominal pain, osteoporosis, infection, and immune-mediated toxicity. Some patients with difficult-to-treat inflammatory bowel disease still need surgical treatment. Although total rectal colectomy may cure the disease, postoperative complications are frequent, especially pouchitis and incontinence. Therefore, there is an urgent need to develop drugs or dietary supplements with low cost, high safety, and good effects in relieving or treating ulcerative colitis.

[0003] Constipation is a chronic gastrointestinal disease, which can be divided into functional constipation and organic constipation. Functional constipation is a chronic constipation caused by intestinal-related dysfunction. Functional constipation can be divided into outlet obstruction constipation, slow-transit constipation and mixed constipation. Slow-transit constipation is a motility disorder characterized by a significant increase in intestinal transit time, a decrease in the number of bowel movements and the amount of bowel movements, which affects the quality of life of individuals. The pathogenesis of slow-transit constipation is multifactorial and is generally believed to be related to colorectal motility disorders, mucosal immune imbalance, intestinal microecological imbalance, and abnormal brain-gut axis regulation (Liyun W, Payam G, Yi Y, et al. Regulation of enteric nervous system via sacral nerve stimulation inopioid-induced constipated rats. [J]. Frontiers in neuroscience, 2023, 171146883-1146883.). Most of the drugs used to treat chronic transit constipation are laxatives that cause diarrhea, such as chloride channel activators, emollients, hyperosmotic agents, lubricants, saline laxatives, serotonin antagonists and stimulants. However, these drugs are accompanied by side effects, such as diarrhea, cramps, and headaches. In order to overcome the side effects of laxatives, there is an urgent need to develop drugs or dietary supplements with lower costs, higher safety, and better effects in relieving or treating chronic transit constipation (JinJu J, Raja G, YooJeong J, et al. Multi-strain probiotics alleviate loperamide-induced constipation by adjusting the microbiome, serotonin, and short-chain fatty acids in rats. [J]. Frontiers in microbiology, 2023, 14 1174968-1174968.).

[0004] In recent years, probiotics have also been gradually used to relieve or treat ulcerative colitis, and probiotics have good safety and are beneficial to host health. Studies have reported that probiotics that have a relieving or therapeutic effect on ulcerative colitis include: Lactobacillus, Bifidobacterium, Streptococcus, Enterococcus, and Bacillus (Xu Kang, Wang Zhe, Xue Zhipeng, et al. Research progress on the role and mechanism of probiotics in relieving or treating inflammatory bowel disease [J]. Food Industry Science and Technology, 2022, 43 (20): 414-420. DOI:10.13386 / j.issn1002-0306.2021090166.). Studies have reported that probiotics that have an improvement or therapeutic effect on constipation include: Lactobacillus, Bifidobacterium, etc. Constipation can be relieved mainly by regulating the level of gastrointestinal regulatory peptides, regulating intestinal flora, and increasing the content of metabolites and organic acids (MAKIZAKI Y, MAEDA A, OIKAWA Y, et al. Alleviation of low-fiber diet-induced constipation by probiotic Bifidobacterium bifidum G9-1 is based on correction of gut microbiota dysbiosis [J]. Bioscience of Microbiota, Food and Health, 2019, 38 (2): 49-53.).

[0005] Breast milk has attracted much attention due to its human origin, long history of safe intake, adaptation to mucosal and substrate dairy products, and adaptation to the human intestine. Research reports that lactic acid bacteria were first isolated from breast milk in 2003 (Dong Xueyan, Jiang Tiemin, Liu Jichao, Chen Lijun. Research Progress on Lactic Acid Bacteria in Breast Milk [J]. Food Industry Science and Technology, 2016, 37(19): 391-395.). The sources of lactic acid bacteria in breast milk include exogenous and endogenous pathways. The types of lactic acid bacteria that can be isolated from breast milk reported in the study include Streptococcus ( Streptococcus ), Lactobacillus spp. Lactobacillus ), Lactococcus ( Lactococcus ), Enterococcus spp. Enterococcus ), Bifidobacterium spp. Bifidobacterium ), Leuconostoc spp. Leuconostoc ) and Pediococcus ( Pediococcus) etc. Lactic acid bacteria in breast milk are relatively safe, transferable, and have good antibacterial properties (Togo Amadou, Dufour Jean-Charles, Lagier Jean-Christophe, Dubourg Gregory, Raoult Didier, Million Matthieu. Repertoire of human breast and milk microbiota: a systematic review. [J]. Future microbiology, 2019, 14.).

[0006] For probiotic bifidobacteria to exert their probiotic effects in the host, they must have a certain tolerance to the low pH gastric acid environment and the high bile salt concentration in the intestinal environment. This requires that a certain number of live bacteria still exist after the strain passes through the gastrointestinal tract. However, most bifidobacteria have poor tolerance to gastrointestinal fluid and poor tolerance to the external environment.

[0007] In summary: Providing a strain of breast milk-derived Bifidobacterium lactis that has good in vitro probiotic properties and can relieve ulcerative colitis and improve constipation and its application have great practical significance. Summary of the invention

[0008] The purpose of the present invention is to disclose the application of a strain of breast milk-derived animal Bifidobacterium lactis subspecies LIHUO 01 in relieving ulcerative colitis and improving constipation, which can be used to prepare medicines capable of relieving ulcerative colitis, or foods or health products or medicines capable of improving constipation or regulating intestinal flora, and can be particularly used in medicines capable of relieving ulcerative colitis or probiotic products capable of improving constipation or regulating intestinal flora. In order to solve the problems existing in the above-mentioned prior art, the bacteria have good in vitro probiotic properties, can relieve ulcerative colitis, improve constipation, and can regulate intestinal flora.

[0009] To achieve the above object, the present invention provides the following solutions:

[0010] In the first aspect, the present invention discloses a strain of animal Bifidobacterium lactis subspecies LIHUO 01 from breast milk, which has good in vitro probiotic properties, relieves ulcerative colitis, improves constipation and regulates intestinal flora. The animal Bifidobacterium lactis subspecies LIHUO 01 has been deposited in the Guangdong Provincial Microbiological Culture Collection Center on November 20, 2024, with a preservation address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with a preservation number of GDMCC No: 65512 and a classification name of Bifidobacterium animalis subsp. lactis .

[0011] Furthermore, the breast milk-derived Bifidobacterium animalis subspecies lactis LIHUO 01 was isolated and screened from the breast milk of healthy volunteer pregnant women in Shangrao City, Jiangxi Province. The strain was sequenced and analyzed, and its 16S rDNA sequence fragment was compared with the nucleic acid sequence of NCBI's BLAST. The results showed that the similarity with the nucleic acid sequence of Bifidobacterium animalis subspecies lactis was 99.93%, indicating that the strain was Bifidobacterium animalis subspecies lactis, and it was named Bifidobacterium animalis subspecies lactis LIHUO 01.

[0012] Furthermore, the good in vitro probiotic properties refer to that the strain has good tolerance to artificial gastrointestinal fluid, the strain has strong hydrophobicity, the strain has a high scavenging rate for hydroxyl free radicals, and the strain has a strong inhibitory effect on Staphylococcus aureus CMCC26003 and Salmonella typhimurium CMCC 50115.

[0013] In the second aspect, the present invention discloses the use of a breast milk-derived animal Bifidobacterium lactis subspecies LIHUO 01 in the preparation of a product for relieving ulcerative colitis. Furthermore, the product for relieving ulcerative colitis includes but is not limited to products for relieving the symptoms of ulcerative colitis, relieving intestinal inflammation, maintaining the integrity of the intestinal barrier, reducing oxidative stress, regulating intestinal flora, or improving the level of short-chain fatty acids in metabolites. When the product is a product for relieving the symptoms of ulcerative colitis, the direction of action includes but is not limited to relieving weight loss, relieving shortening of the colon, and reducing the activity of myeloperoxidase (MPO) in colon tissue. When the product is used to relieve intestinal inflammation, the direction of action includes but is not limited to reducing the concentration of TNF-α, IL-1β, and IL-6 in colon tissue and downregulating the relative mRNA levels of TNF-α, IL-1β, and IL-6, increasing the concentration of IL-10 in colon tissue and upregulating the relative mRNA level of IL-10; downregulating the relative mRNA levels of NF-κB, COX-2, and iNOS in colon tissue, and upregulating the relative mRNA level of IκBα in colon tissue; reducing the concentration of IL-6 in serum and increasing the concentration of IL-10 in serum. When the product is used to maintain the integrity of the intestinal barrier, the direction of action includes but is not limited to 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. When the product is used to reduce oxidative stress, the direction of action includes but is not limited to increasing the activity of superoxide dismutase (SOD) in colon tissue, reducing the activity of catalase (CAT) in colon tissue and the concentration of malondialdehyde (MDA). When the product is a product for regulating intestinal flora, the direction of action is not limited to increasing the number of Firmicutes in cecal contents at the phylum level ( Firmicutes ) and decreased the relative abundance of Bacteroidetes ( Bacteroidota) relative abundance; at the genus level, the cecal contents of Ileobacterium ( Ileibacterium ), Lactobacillus spp. Lactobacillus ) 、 Enterobacter Enterorhabdus ) and decreased the relative abundance of Bacteroides ( Bacteroides When the product is a product for improving the level of short-chain fatty acids in metabolites, the direction of action includes but is not limited to increasing the concentration of acetic acid and propionic acid in colon contents.

[0014] The present invention also protects the preparation containing the Bifidobacterium animalis subspecies lactis LIHUO 01.

[0015] The animal Bifidobacterium lactis subspecies LIHUO 01 preparation is prepared by culturing the aforementioned animal Bifidobacterium lactis subspecies LIHUO 01, and the animal Bifidobacterium lactis subspecies LIHUO 01 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] Based on the above, the present invention also protects a microbial agent comprising the aforementioned Bifidobacterium animalis subspecies lactis LIHUO 01 or a preparation of Bifidobacterium animalis subspecies lactis LIHUO 01.

[0017] In the third aspect, the present invention discloses an application of a breast milk-derived animal Bifidobacterium lactis subspecies LIHUO 01 in the preparation of a product for improving constipation. Further, the product for improving constipation includes but is not limited to products that promote defecation, improve intestinal motility, regulate gastrointestinal regulatory peptide levels, promote the secretion of neurotransmitters, reduce the level of aquaporin, improve colon inflammation, regulate intestinal flora, or improve the level of short-chain fatty acids in metabolites. When the product is a product that promotes defecation, the direction of action includes but is not limited to increasing the number of fecal particles and increasing the water content of feces, and improving the gastrointestinal propulsion rate. When the product is a product that improves gastrointestinal motility, the direction of action includes but is not limited to increasing the content of mucus protein in colon tissue and upregulating the relative level of mRNA of c-Kit in colon tissue. When the product is a product that regulates the level of gastrointestinal regulatory peptides, the direction of action includes but is not limited to increasing the concentrations of motilin (MTL), gastrin (GAS), and substance P (SP) in serum, and reducing the concentrations of somatostatin (SS) and vasoactive intestinal peptide (VIP). When the product is a product that promotes the secretion of neurotransmitters, the direction of action includes but is not limited to increasing the concentration of acetylcholine (Ach) in serum and increasing the concentration of 5-hydroxytryptamine (5-HT) in colon tissue. When the product is a product that reduces the level of aquaporin, the direction of action includes but is not limited to reducing the concentration of aquaporin (AQP3) in colon tissue. When the product is a product that improves colon inflammation, the direction of action includes but is not limited to reducing the concentration of tumor necrosis factor-α (TNF-α) in colon tissue and increasing the concentration of interleukin 10 (IL-10) in colon tissue. When the product is a product that regulates intestinal flora, the direction of action includes but is not limited to increasing the Bifidobacterium genus ( Bifidobacterium ), Staphylococcus spp. Staphylococus ) 、 Corynebacterium Corynebacterium ), reducing the relative abundance of Lachnospiraceac _NK4A136_group, norank_f_ Oscillospiraceae 、norank_f_ Muribaculaceae When the product is a product for improving the level of short-chain fatty acids in metabolites, the direction of action includes but is not limited to increasing the concentration of isobutyric acid and isovaleric acid in the colon content.

[0018] In a fourth aspect, the present invention provides a product, comprising the aforementioned breast milk-derived Bifidobacterium animalis subspecies lactis LIHUO 01 or the aforementioned microbial agent or the aforementioned preparation of Bifidobacterium animalis subspecies lactis LIHUO 01.

[0019] Furthermore, the product is food, health care product or medicine.

[0020] The food or health product also includes additives, which are selected from any one or more of flavoring agents, colorants, fillers, disintegrants, sweeteners, lubricants, adhesives, and pH regulators.

[0021] Furthermore, the product includes at least one of a probiotic product, a nutritional supplement, an animal feed and a pet snack.

[0022] Furthermore, the probiotic product includes at least one of a fermented product, a milk beverage, a live bacteria preparation, milk powder and rice flour.

[0023] Furthermore, the product is a medicine.

[0024] Furthermore, the medicine is in the form of pills, powders, capsules, tablets, film-coated preparations, orally dissolving granules, liquid preparations, suppositories or enemas.

[0025] Furthermore, the medicine also includes a pharmaceutically acceptable carrier and / or excipient.

[0026] Furthermore, the medicine also includes pharmaceutical excipients, and the pharmaceutical excipients are selected from any one or more of excipients, buffers, emulsifiers, stabilizers, diluents, adhesives, preservatives, and lubricants.

[0027] In a fifth aspect, the present invention also discloses a method for culturing the aforementioned animal Bifidobacterium lactis subspecies LIHUO 01.

[0028] Specifically, the culture method comprises inoculating animal Bifidobacterium lactis subspecies LIHUO 01 into a culture medium and then performing conditional culture.

[0029] The culture medium includes but is not limited to MRS culture medium, modified MRS culture medium, RCM culture medium, Bifidobacterium BS culture medium, BBL agar culture medium. The conditional culture is preferably anaerobic culture. Further preferably, the conditional culture is anaerobic culture at 37°C.

[0030] The beneficial effects of the above technical solution of the present invention are as follows:

[0031] The present invention discloses the application of animal Bifidobacterium lactis subspecies LIHUO 01 derived from human milk in relieving ulcerative colitis, improving constipation, and regulating intestinal flora. Specifically, the animal Bifidobacterium lactis subspecies LIHUO 01 derived from human milk can be used to prepare products that can relieve ulcerative colitis, improve constipation, or regulate intestinal flora. The animal Bifidobacterium lactis subspecies LIHUO 01 derived from human milk of healthy volunteer parturients in Shangrao City, Jiangxi Province. In a mouse model of ulcerative colitis induced by dextran sulfate (DSS), animal Bifidobacterium lactis subspecies LIHUO 01 can alleviate the weight loss of mice with ulcerative colitis, 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 mRNA of NF-κB, COX-2, and iNOS in colon tissue, and up-regulate the relative level of mRNA of IκBα; reduce the concentration of IL-6 in serum and increase the concentration of IL-10 in serum. The strain can reduce the concentration of mannitol in serum, increase the relative level of Claudin-1 protein in colon tissue, and up-regulate the relative mRNA levels of Claudin-1, ZO-1, and MUC2 in colon tissue. The strain increases the activity of SOD in colon tissue and reduces the activity of CAT and the concentration of MDA in colon tissue. The strain increases the number of Firmicutes in cecal contents at the phylum level ( Firmicutes ) and decreased the relative abundance of Bacteroidetes ( Bacteroidota ) relative abundance; at the genus level, the cecal contents of Ileobacterium ( Ileibacterium ), Lactobacillus spp. Lactobacillus ) 、 Enterobacter Enterorhabdus ) and decreased the relative abundance of Bacteroides ( Bacteroides ). The strain can increase the concentration of acetic acid and propionic acid in the colon contents. In the rat model of constipation induced by loperamide hydrochloride (Lop), 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 concentration of MTL, GAS, and SP in serum, and reduce the concentration 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 Bifidobacterium genus ( Bifidobacterium ), Staphylococcus spp. Staphylococus ) 、 Corynebacterium Corynebacterium ), reducing the relative abundance of Lachnospiraceac _NK4A136_group, norank_f_ Oscillospiraceae 、norank_f_ Muribaculaceae The strain can increase the concentration of isobutyric acid and isovaleric acid in the colon contents.

[0032] Collection Instructions

[0033] Strain Name: Bifidobacterium animalis subsp. lactis LIHUO 01;

[0034] Chinese name of the strain: Bifidobacterium animalis subsp. lactis LIHUO 01;

[0035] Category naming: Bifidobacterium animalis subsp. lactis ;

[0036] Deposit number: GDMCC No:65512;

[0037] Depository: Guangdong Microbiological Culture Collection Center;

[0038] Deposit date: November 20, 2024;

[0039] Collection address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The colony morphology (left) and bacterial morphology (right) of Bifidobacterium animalis lactis subspecies LIHUO 01 in Example 1, wherein the scale bar in the bacterial morphology represents 5 μm.

[0041] Figure 2 1 is a phylogenetic tree based on the 16S r DNA sequence of Bifidobacterium animalis lactis subspecies LIHUO 01 in Example 1, wherein Stain LIHUO 01 represents Bifidobacterium animalis lactis subspecies LIHUO 01.

[0042] Figure 3 The inhibitory effects of animal Bifidobacterium lactis subsp. LIHUO 01 and animal Bifidobacterium lactis subsp. BB12 on two pathogenic bacteria in Example 5; the pathogenic bacteria targeted are A: Staphylococcus aureus CMCC 26003 and B: Salmonella typhimurium CMCC 50115; the MRS culture medium in the figure is a blank control.

[0043] Figure 4 is the weight change rate of mice in each group in Example 6, and the values ​​of the days on the horizontal axis are 15, 16, 17, 18, 19, 20, and 21 respectively.

[0044] Figure 5 DAI scores of mice in each group in Example 6, *** indicates P <0.001.

[0045] Figure 6 This is an example of colon measurement of each group of mice in Example 6.

[0046] Figure 7 The colon length statistics of each group of mice in Example 6, *** indicates P <0.001.

[0047] Figure 8 is the spleen index of each group of mice in Example 6, *** indicates P <0.001.

[0048] Fig. 9 is the colon MPO activity of each group of mice in Example 6, ** indicates P <0.01,*** indicates P <0.001.

[0049] Fig.10 This is HE staining of the colon of each group of mice in Example 7. All scale bars are 500 μm.

[0050] Fig.11 is the colon tissue pathological score of each group of mice in Example 7, ** indicates P <0.01,*** indicates P <0.001.

[0051] Fig.12 The concentrations of TNF-α, IL-1β, IL-6, and IL-10 in the colon of each group 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.

[0052] Fig.13 is the relative mRNA level of TNF-α, IL-1β, IL-6, and IL-10 in the colon of each group of mice in Example 8, ns represents P >0.05,* indicates P <0.05,** indicates P <0.01,*** indicates P <0.001.

[0053] Fig.14 The relative mRNA levels of NF-κB, IκBα, COX-2, and iNOS in the colon of each group of mice in Example 8, * indicatesP <0.05,** indicates P <0.01,*** indicates P <0.001.

[0054] Fig.15 is the serum IL6 and IL-10 concentration of each group of mice in Example 8, ns represents P >0.05,* indicates P <0.05,** indicates P <0.01,*** indicates P <0.001.

[0055] Fig.16 is the serum mannitol concentration of each group of mice in Example 9, *** indicates P <0.001.

[0056] Fig.17 is the relative level of Claudin-1 protein (tight junction protein) in the colon of each group of mice in Example 9, ns represents P >0.05,* indicates P <0.05、*** indicates P <0.001.

[0057] Fig.18 The relative mRNA levels of Claudin-1 (tight junction protein), ZO-1 (tight junction protein), and MUC2 (mucin) in the colon of each group of mice in Example 9, * indicates P <0.05,** indicates P <0.01,*** indicates P <0.001.

[0058] Fig.19 are the SOD activity, CAT activity and MDA concentration of the colon of each group of mice in Example 10, ns represents P >0.05,* indicates P <0.05,** indicates P <0.01,*** indicates P <0.001.

[0059] Fig. 20 is the Sobs index and Ace index of the cecal content microorganisms of each group of mice at the genus level in Example 11, ns represents P >0.05.

[0060] Fig.21 This is the principal component analysis (PCA) and principal coordinate analysis (PCoA) of the cecal content microbiome of each group of mice at the genus level in Example 11.

[0061] Fig. 22 The figure below shows the distribution of the microbiome of the cecal contents of each group of mice in Example 11 at the phylum and genus levels, wherein the upper figure shows the microbial distribution at the phylum level, and the lower figure shows the microbial distribution at the genus level.

[0062] Fig.23 This is the differential analysis of the microbiome of the cecal contents of each group of mice at the phylum level in Example 11, ns represents P >0.05,* indicates P <0.05.

[0063] Fig.24 This is a differential analysis of the microbiome of the cecal contents of each group of mice at the genus level in Example 11.

[0064] Fig.25 is the concentration of short-chain fatty acids in the colon contents of each group of mice in Example 12, ns represents P >0.05,* indicates P <0.05,** indicates P <0.01,*** indicates P <0.001.

[0065] Fig.26 is the feces water content and feces particle number of each group of rats in Example 13, ns represents P >0.05,* indicates P <0.05、*** indicates P <0.001.

[0066] Fig. 27 is the gastrointestinal propulsion rate of each group of rats in Example 13, ** indicates P <0.01,*** indicates P <0.001.

[0067] Fig.28 This is AB staining of the colon of each group of rats in Example 14, and the scale bar represents 500 μm.

[0068] Fig.29 is the relative level of c-Kit mRNA in the colon of each group of rats in Example 14, * indicates P <0.05、*** indicates P <0.001.

[0069] Fig.30 is the level of serum gastrointestinal regulatory peptides (MTL, GAS, SP, VIP, SS) in each group of rats in Example 15, ns represents P >0.05,* indicates P <0.05,** indicates P <0.01,*** indicates P<0.001.

[0070] Fig.31 is the serum Ach concentration of each group of rats in Example 16, *** indicates P <0.001.

[0071] Fig.32 is the colon 5-HT concentration of each group of rats in Example 16, * indicates P <0.05,** indicates P <0.01,*** indicates P <0.001.

[0072] Fig.33 is the colon AQP3 concentration of each group of rats in Example 17, *** indicates P <0.001.

[0073] Fig.34 is the concentration of colon inflammatory factors in each group of rats in Example 18, ns represents P >0.05、*** indicates P <0.001.

[0074] Fig.35 is the Sobs index and Ace index of the rat cecal content microorganisms based on the genus level in Example 19, and ns represents: P >0.05.

[0075] Fig.36 This is the principal component analysis (PCA) and principal coordinate analysis (PCoA) of the cecal content microbiome of each group of rats at the genus level in Example 19.

[0076] Fig.37 The figure below shows the distribution of the microbiome of the cecal contents of each group of rats in Example 19 at the phylum and genus levels, wherein the upper figure shows the microbial distribution at the phylum level, and the lower figure shows the microbial distribution at the genus level.

[0077] Fig.38 This is a differential analysis of the microbiome of the cecal contents of each group of rats at the genus level in Example 19.

[0078] Fig.39 is the concentration of short-chain fatty acids in the colon contents of each group of rats in Example 20, ns represents P >0.05,* indicates P <0.05,** indicates P <0.01. DETAILED DESCRIPTION

[0079] 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.

[0080] Example 1: Isolation, purification, identification and preservation of Bifidobacterium animalis subspecies lactis LIHUO 01

[0081] The specific steps are as follows:

[0082] (1) Collection of breast milk samples

[0083] The breast milk samples were obtained from the breast milk of healthy volunteers in Shangrao City, 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 manually collected the breast milk, discarded the first ten drops of breast milk, collected about 5 mL of breast milk into a sterile centrifuge tube, and quickly transported it back to the laboratory at low temperature to carry out strain isolation experiments.

[0084] (2) Isolation, purification, identification and preservation of strains

[0085] 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 by culture.

[0086] 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.

[0087] 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 (manufacturer: Solarbio, item number: Cat#G1060) to carry out Gram staining experiments on isolated strains. The isolated strains were stored in 25% glycerol at -80°C.

[0088] 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) was as follows: 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. PCR amplification program: 95°C pre-denaturation for 10 min, (95°C denaturation for 30 s, 55°C annealing for 30 s, 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 products of the strains were compared with NCBI to determine the genus and species information of the strains, and the phylogenetic tree was constructed using MEGA X.

[0089] (3) Detection of physiological and biochemical characteristics of strains

[0090] 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.

[0091] (4) Preservation of strains

[0092] The identified lactic acid bacteria were preserved in the Guangdong Microbiological Culture Collection Center (address: 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou).

[0093] The experimental results are as follows:

[0094] The colony morphology of the isolated strain LIHUO 01 is white, round, moist, opaque, and with neat edges (see Figure 1 The bacterial morphology is irregular rod-shaped, with "Y"-shaped branches, arranged singly or in pairs (see Figure 1 Strain LIHUO 01 is a catalase-negative, Gram-positive strain.

[0095] The sequencing results of the 16S rDNA PCR product of strain LIHUO 01 were compared with the NCBI BLAST. The results showed that the strain had a 99.93% similarity with the nucleic acid sequence of Bifidobacterium animalis subsp. lactis. The phylogenetic tree was constructed as shown in Figure 2, strain LIHUO 01 was identified as Bifidobacterium animalis subsp. lactis, hereinafter referred to as Bifidobacterium animalis subsp. lactis LIHUO 01. The physiological and biochemical characteristics of Bifidobacterium animalis subsp. lactis LIHUO 01 are shown in Table 1 (+ indicates positive, w indicates weak positive, and - indicates negative).

[0096] Table 1 Physiological and biochemical characteristics of Bifidobacterium animalis subsp. lactis LIHUO 01

[0097]

[0098] The animal Bifidobacterium lactis subspecies LIHUO 01 of the present invention was deposited in Guangdong Provincial Microbiological Culture Collection Center on November 20, 2024, with the deposit number being GDMCC No: 65512.

[0099] The 16S rDNA of strain LIHUO 01 is shown in SEQ ID NO.1:

[0100]

[0101] Example 2: Determination of the tolerance of Bifidobacterium animalis subsp. lactis LIHUO 01 to artificial gastrointestinal fluid

[0102] The specific steps are as follows:

[0103] (1) Preparation of test bacterial solution of Bifidobacterium animalis subsp. lactis LIHUO 01 and Bifidobacterium animalis subsp. lactis BB12 (commercial strain control)

[0104] The bacterial cultures of animal Bifidobacterium lactis subsp. LIHUO 01 and animal Bifidobacterium lactis subsp. 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 bacteria, and an equal volume of PBS solution was added to wash the bacteria twice, and an appropriate amount of PBS solution was added to resuspend the bacteria to prepare the test bacterial culture.

[0105] (2) Artificial gastrointestinal fluid tolerance test of Bifidobacterium animalis subspecies LIHUO 01 and Bifidobacterium animalis subspecies BB12

[0106] 1 mL of animal Bifidobacterium lactis subspecies LIHUO 01 and animal Bifidobacterium lactis subspecies BB12 were taken to determine the number of viable bacteria N0 at 0 h, 1 mL of animal Bifidobacterium lactis subspecies LIHUO 01 and animal Bifidobacterium lactis subspecies BB12 were 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, and 1 mL of animal Bifidobacterium lactis subspecies LIHUO 01 treated with artificial gastric juice for 3 h was taken to determine the number of viable bacteria N1. 01. 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.

[0107] The experimental results are as follows:

[0108] The survival rates of animal Bifidobacterium lactis subsp. LIHUO 01 treated in artificial gastric juice at pH 2.5 for 3 h, artificial intestinal juice at pH 8.0 for 8 h, and artificial gastrointestinal juice for 11 h were 98.06%, 79.70%, and 78.16%, respectively, and the survival rates were higher than those of the control strain animal Bifidobacterium lactis subsp. BB12 (88.48%, 77.40%, and 68.48%). The specific measured values ​​are shown in Table 2.

[0109] For Bifidobacterium to exert its probiotic effects in the host, it must have a certain tolerance to the low pH gastric acid environment and the high bile salt concentration in the intestinal environment. It is required that a certain number of live bacteria still exist after the strain passes through the gastrointestinal tract. The results of this experiment show that the animal Bifidobacterium lactis subspecies LIHUO 01 shows a high tolerance to artificial gastrointestinal fluid, which is conducive to its probiotic effects in the host.

[0110] Table 2 Tolerance of Bifidobacterium animalis subsp. lactis LIHUO 01 and Bifidobacterium animalis subsp. lactis BB12 in artificial gastrointestinal fluid

[0111]

[0112] Example 3: Determination of the surface hydrophobicity of Bifidobacterium animalis subspecies lactis LIHUO 01

[0113] The specific steps are as follows:

[0114] (1) Preparation of test bacterial solution of Bifidobacterium animalis subspecies LIHUO 01 and Bifidobacterium animalis subspecies BB12

[0115] Preparation of test bacterial solution: The bacterial cultures of animal Bifidobacterium lactis subsp. LIHUO 01 and animal Bifidobacterium lactis subsp. 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.

[0116] (2) Determination of surface hydrophobicity of Bifidobacterium animalis subspecies LIHUO 01 and Bifidobacterium animalis subspecies BB12

[0117] The surface hydrophobicity of the test strains was determined by hydrocarbon adsorption method. The test bacterial solution (bacterial concentration was 1.00×10 8 CFU / mL), 2 mL of the test bacterial solution was mixed with 2 mL of xylene, and allowed to stand at room temperature for 30 min. The aqueous phase was taken to measure the absorbance A1. The experiment was repeated 3 times, and the surface hydrophobicity of the strain was calculated according to the following formula: Surface hydrophobicity (%) = (A0-A1) / A0×100.

[0118] The experimental results are as follows:

[0119] The surface hydrophobicity of animal Bifidobacterium lactis subspecies LIHUO 01 was 90.87%, slightly higher than that of the control strain animal Bifidobacterium lactis subspecies BB12 (see Table 3). Strains with surface hydrophobicity rates higher than 70%, lower than 70%, higher than 30%, and lower than 30% were respectively strong hydrophobic, medium hydrophobic, and weak hydrophobic strains. The results showed that animal Bifidobacterium lactis subspecies LIHUO 01 was a strong hydrophobic strain.

[0120] Surface hydrophobicity is an important driving force for the nonspecific adhesion of strains to biological surfaces, and plays an important role in the process of strain adhesion and colonization to the host intestinal wall. The results of this experiment show that animal Bifidobacterium lactis subspecies LIHUO 01 has a strong surface hydrophobicity, which is conducive to its adhesion to the host intestinal epithelial cells and thus exerting a probiotic effect.

[0121] Table 3 Surface hydrophobicity of Bifidobacterium animalis subsp. lactis LIHUO 01 and Bifidobacterium animalis subsp. lactis BB12

[0122]

[0123] Example 4: Determination of the scavenging rate of hydroxyl free radicals by Bifidobacterium animalis subsp. lactis LIHUO 01

[0124] The specific steps are as follows:

[0125] (1) The method for preparing the test bacterial solution is the same as that in Example 3.

[0126] (2) Determination of the scavenging rate of hydroxyl free radicals by Bifidobacterium animalis subspecies LIHUO 01 and Bifidobacterium animalis subspecies BB12

[0127] The hydroxyl radical scavenging ability detection kit (manufacturer: Solarbio, product number: Cat# BC1325) was used to determine the hydroxyl radical scavenging rate of the test strain, and the experimental operation was carried out strictly in accordance with the instructions.

[0128] The experimental results are as follows:

[0129] The scavenging rate of animal Bifidobacterium lactis subspecies LIHUO 01 on hydroxyl free radicals was 50.23%, which was higher than that of the control strain animal Bifidobacterium lactis subspecies BB12 (see Table 4). The experimental results showed that animal Bifidobacterium lactis subspecies LIHUO 01 had a good scavenging rate on hydroxyl free radicals.

[0130] The strong oxidative property of hydroxyl radicals can degrade polysaccharides and DNA, kill red blood cells, decompose cell membranes, and induce cell necrosis. The results of this experiment showed that animal Bifidobacterium lactis subsp. LIHUO 01 had a certain ability to scavenge hydroxyl free radicals, indicating that the strain had in vitro antioxidant capacity.

[0131] Table 4 Scavenging rate of hydroxyl free radicals by Bifidobacterium animalis subspecies LIHUO 01 and Bifidobacterium animalis subspecies BB12

[0132]

[0133] Example 5: Determination of the antibacterial ability of Bifidobacterium animalis subspecies lactis LIHUO 01 against pathogenic bacteria

[0134] The specific steps are as follows:

[0135] (1) Preparation of fermentation broth of Bifidobacterium animalis subsp. LIHUO 01 and Bifidobacterium animalis subsp. BB12

[0136] After the test strains of Bifidobacterium animalis subsp. lactis LIHUO 01 and Bifidobacterium animalis subsp. lactis BB12 were activated twice, the concentration of 1×10 7 CFU / mL inoculation was inoculated into MRS liquid medium and cultured anaerobically at 37°C for 18 h. The bacterial solution was centrifuged at 8000 r / min for 10 min, and the fermentation supernatant was collected and filtered through a 0.22 μm microporous filter membrane. Two pathogenic bacteria, Staphylococcus aureus CMCC 26003 and Salmonella typhimurium CMCC 50115 (the strain was activated three times and the bacterial concentration was adjusted to 1×10 8 CFU / mL) was added to LB agar at about 45°C, a sterile 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.

[0137] The experimental results are as follows:

[0138] Animal Bifidobacterium lactis subspecies LIHUO 01 had different degrees of inhibitory effects on Staphylococcus aureus CMCC 26003 and Salmonella typhimurium CMCC 50115, and the inhibitory effects were higher than those of the control strain Animal Bifidobacterium lactis subspecies BB12. Figure 3 and Table 5.

[0139] Staphylococcus aureus and Salmonella typhimurium are prone to cause intestinal infections, diarrhea and other diseases. The results of this experiment showed that animal Bifidobacterium lactis subspecies LIHUO 01 had different degrees of inhibitory effects on Staphylococcus aureus CMCC 26003 and Salmonella typhimurium CMCC 50115, indicating that the strain can inhibit intestinal pathogens, thereby reducing the probability of intestinal infections, diarrhea and other diseases.

[0140] Table 5 Inhibitory effects of Bifidobacterium animalis subspecies LIHUO 01 and Bifidobacterium animalis subspecies BB12 on pathogenic bacteria

[0141]

[0142] Example 6: Effects of Bifidobacterium animalis subsp. lactis LIHUO 01 on the symptoms of DSS-induced ulcerative colitis in mice

[0143] This example verifies the effect of Bifidobacterium animalis subsp. lactis LIHUO 01 on the symptoms of DSS-induced ulcerative colitis in mice.

[0144] The specific steps are as follows:

[0145] (1) Preparation of Bifidobacterium animalis subsp. lactis LIHUO 01 bacterial suspension

[0146] The strains of animal Bifidobacterium lactis subsp. LIHUO 01 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.

[0147] (2) Construction of ulcerative colitis model and strain intervention

[0148] 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.

[0149] 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 01 group (n=10 / group).

[0150] 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 01 group were gavaged with animal Bifidobacterium lactis subsp. LIHUO 01 bacterial solution at a volume of 200 μL / mouse / day (the dose of animal Bifidobacterium lactis subsp. LIHUO 01 was 1.00×10 8 CFU / d); mice in all groups drank sterile water and had free access to food.

[0151] 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 01 group were gavaged with Bifidobacterium animalis subsp. lactis LIHUO 01 solution at a volume of 200 μL / mouse / day (the dose of Bifidobacterium animalis subsp. lactis LIHUO01 was 1.00×10 8 CFU / d); mice in the normal group drank sterile water; except for the normal group, mice in other groups drank sterile water containing 2.5% (w / v) DSS (DSS: dextran sulfate, manufacturer: MP, product number: YD08001); mice in all groups were free to eat.

[0152] (3) DAI score

[0153] On the 15th to 21st day, the body weight of the mice 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.

[0154] Table 6 DAI score table

[0155]

[0156] (4) Determination of mouse colon length and spleen index

[0157] 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%.

[0158] (5) Determination of myeloperoxidase (MPO) activity in colon tissue

[0159] 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.

[0160] 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 the BCA protein concentration assay kit (enhanced) (manufacturer: Biyuntian, item number: CatNo.P0010); the MPO activity of the colon tissues of mice in each treatment group was determined using the MPO test kit (manufacturer: Nanjing Jiancheng, item number: A044-1-1).

[0161] The experimental results are as follows:

[0162] 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 01 group decreased less than that in the model group (body weight decreased by 13.74% and 7.06% on the 21st day, respectively) (see Figure 4 ), indicating that the intervention of Bifidobacterium animalis subsp. lactis LIHUO 01 alleviated the body weight loss of DSS-induced ulcerative colitis mice to a certain extent.

[0163] 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 mice in the model group was significantly higher than that in the normal group, while the DAI score of the mice in the mesalazine group and the LIHUO 01 group was significantly lower than that in the model group (see Figure 5 ). This indicates that Bifidobacterium animalis subsp. lactis LIHUO 01 can reduce the susceptibility of DSS-induced ulcerative colitis in mice.

[0164] 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 01 group (6.69 cm and 6.88 cm, respectively) was significantly increased compared with the model group, indicating that the intervention of animal Bifidobacterium lactis subsp. LIHUO 01 alleviated the shortening of the colon of mice with ulcerative colitis caused by DSS to a certain extent (see Figure 6 , Figure 7 ).

[0165] 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 01 group was significantly lower than that in the model group (see Figure 8 Compared with the normal group, the MPO activity in the colon of the mice in the model group was significantly increased; compared with the model group, the MPO activity in the colon of the mice in the mesalazine group and the LIHUO 01 group was significantly decreased (see Fig. 9 ), indicating that Bifidobacterium animalis subsp. lactis LIHUO 01 could reduce the MPO activity in the colon of mice with DSS-induced ulcerative colitis.

[0166] The results of this experiment showed that the intervention of animal Bifidobacterium lactis subsp. LIHUO 01 can 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. This indicates that animal Bifidobacterium lactis subsp. LIHUO 01 can prevent and improve the symptoms of DSS-induced ulcerative colitis in mice.

[0167] Example 7: Effects of Bifidobacterium animalis subsp. lactis LIHUO 01 on colonic histological damage in mice with DSS-induced ulcerative colitis

[0168] The specific steps are as follows:

[0169] 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.

[0170] Table 7 Histopathological scoring table

[0171]

[0172] The experimental results are as follows:

[0173] The results of HE staining of the colon tissues of mice in each group and the results of the colon histopathological scoring of mice are shown in Fig.10 , Fig.11 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, with fewer crypts, severe inflammatory infiltration, and mucosal lesions; the colon of the mice in the mesalazine group and LIHUO 01 group had fewer inflammatory cells and less inflammatory infiltration than the model group. Compared with the normal group, the colon histopathological score of the mice in the model group increased significantly; while the colon histopathological score of the mice in the mesalazine group and LIHUO 01 group decreased significantly compared with the model group.

[0174] The results of this experiment showed that intervention with animal Bifidobacterium lactis subsp. LIHUO 01 could significantly reduce the pathological score of colon tissue in DSS-induced ulcerative colitis mice and reduce the destruction of colon tissue in DSS-induced ulcerative colitis mice.

[0175] Example 8: Effects of Bifidobacterium animalis subsp. lactis LIHUO 01 on inflammatory cytokines in colon tissue, genes related to NF-κB signaling pathway, and serum inflammatory cytokines in mice with DSS-induced ulcerative colitis

[0176] The specific steps are as follows:

[0177] The grouping, modeling, treatment of mice, and treatment of colon tissue samples were the same as in Example 6.

[0178] (1) Determination of inflammatory cytokine concentrations in colon tissue

[0179] 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).

[0180] (2) Real-time fluorescence quantitative PCR (RT-qPCR) detection of inflammatory factors and NF-κB signaling pathway-related genes in colon tissue

[0181] 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, IκBα, COX-2, and iNOS genes using a fluorescent 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, IκBα, COX-2, and iNOS in colon tissue were calculated by the method (GAPDH gene was used as the internal reference gene).

[0182] Table 8 Primer sequences

[0183]

[0184] 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.

[0185] 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).

[0186] (3) Detection of serum inflammatory cytokines

[0187] After the mouse blood samples were allowed to stand and stratified, they were centrifuged at 4°C and 3000 r / min for 15 min to obtain serum. The concentrations of IL-6 and IL-10 in the serum of each group of mice were determined using ELISA kits for IL-6 (manufacturer: Shanghai ELISA, catalog number: ml063159V) and IL-10 (manufacturer: Shanghai ELISA, catalog number: mlC50274-1).

[0188] The experimental results are as follows:

[0189] 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.12 and Fig.13 The relative mRNA levels of NF-κB, IκBα, COX-2, and iNOS in the colon tissue of each group of mice are shown in Fig.14 The concentrations of IL-6 and IL-10 in the serum of mice in each group are shown in Fig.15 .

[0190] Compared with the normal group, the concentrations and relative mRNA 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 mRNA levels of TNF-α, IL-1β, and IL-6 in the colon tissue of the mice in the mesalazine group and LIHUO 01 group were significantly lower than those in the model group. Compared with the normal group, the concentration and relative mRNA level of IL-10 in the colon tissue of the mice in the model group were significantly decreased, and the concentration and relative mRNA 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 mRNA level of IL-10 in the colon tissue of the mice in the LIHUO 01 group were significantly higher than those in the model group (see Fig.12 , Fig.13 ).

[0191] In order to investigate whether the NF-κB signaling pathway plays a role in regulating the anti-inflammatory mechanism of the LIHUO 01 group, the relative mRNA levels of related genes were further determined. Compared with the normal group, the relative mRNA levels of NF-κB, COX-2, and iNOS in the colon tissue of mice in the model group were significantly increased, while the relative mRNA levels of NF-κB, COX-2, and iNOS in the colon tissue of mice in the mesalazine group and LIHUO 01 group were 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 01 group was significantly increased compared with the model group (see Fig.14 ).

[0192] 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 01 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 was not significantly different from that in the model group; while the IL-10 concentration in the serum of the mice in the LIHUO 01 group was significantly higher than that in the model group (see Fig.15 ).

[0193] The results of this experiment showed that intervention with animal Bifidobacterium lactis subsp. LIHUO 01 could significantly reduce the concentration of proinflammatory factors TNF-α, IL-1β, and IL-6 in the colon tissue of mice with DSS-induced ulcerative colitis, and down-regulated the relative mRNA levels of TNF-α, IL-1β, and IL-6 genes; significantly increased the concentration of anti-inflammatory factor IL-10 in colon tissue and up-regulated the relative mRNA level of IL-10; significantly down-regulated the relative mRNA levels of NF-κB, COX-2, and iNOS in colon tissue, and significantly up-regulated the relative mRNA level of IκBα in colon tissue. Intervention with animal Bifidobacterium lactis subsp. LIHUO 01 could significantly reduce the concentration of proinflammatory factor IL-6 in the serum of mice with DSS-induced ulcerative colitis, and significantly increase the concentration of anti-inflammatory factor IL-10 in the serum. The results showed that intervention with animal Bifidobacterium lactis subspecies LIHUO01 can significantly reduce the concentration of pro-inflammatory factors in the colon and serum of mice with DSS-induced ulcerative colitis, significantly increase the concentration of anti-inflammatory factors in the colon and serum of mice with DSS-induced ulcerative colitis, effectively improve DSS-induced ulcerative colitis in mice, regulate disordered intestinal immunity, and alleviate colon damage in mice with DSS-induced ulcerative colitis by inhibiting the NF-κB signaling pathway.

[0194] Example 9: Effects of Bifidobacterium animalis subsp. lactis LIHUO 01 on the intestinal barrier of mice with DSS-induced ulcerative colitis

[0195] The specific steps are as follows:

[0196] 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.

[0197] (1) Determination of mannitol concentration in serum

[0198] Mannitol ELISA kit (manufacturer: Shanghai ELISA, catalog number: YJ710290) was used to measure the mannitol concentration in the serum of each group of mice.

[0199] (2) Determination of relative levels of tight junction proteins in colon tissue

[0200] The colon tissues of three mice were randomly selected from each group of mice 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, catalog number: CatNo.P0010). The protein concentration of all samples was adjusted to 3mg / mL, and 5× protein loading buffer (reducing) (manufacturer: Xinsaimei, catalog number: WB2001) 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 β-actin secondary antibody (manufacturer: Wuhan Tri-Tac, catalog number: RGAM001) and tight junction protein Claudin-1 secondary antibody (manufacturer: Wuhan Tri-Tac, catalog number: RGAR001) for 3 hours, 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 immunoreactive bands.

[0201] (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

[0202] 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 the method (GAPDH gene was used as the internal reference gene).

[0203] Table 9 Primer sequences

[0204]

[0205] 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.

[0206] The experimental results are as follows:

[0207] The concentration of mannitol in the serum of mice in each group was shown in Fig.16 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 01 group was significantly lower than that in the model group.

[0208] The relative levels of tight junction protein Claudin-1 in colon tissue of mice in each group are shown in Fig.17 The relative levels of claudin-1, ZO-1, and MUC2 related mRNA in the colon tissue of mice in each group are shown in Fig.18 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, and the relative level of tight junction protein Claudin-1 in the colon tissue of mice in the LIHUO 01 group was significantly increased compared with that in the model group. Compared with the normal group, the relative levels of tight junction protein Claudin-1, tight junction protein ZO-1 and mucin MUC2 mRNA in the colon tissue of mice in the model group were significantly decreased, while the relative levels of tight junction protein Claudin-1, tight junction protein ZO-1 and mucin MUC2 transcript mRNA in the colon tissue of mice in the mesalazine group and LIHUO 01 group were significantly increased compared with that in the model group.

[0209] The experimental results showed that the intervention of animal Bifidobacterium lactis subspecies LIHUO 01 could 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 in colon tissue, and significantly upregulate the relative mRNA levels of tight junction protein Claudin-1, tight junction protein ZO-1, and mucus protein MUC2. This indicates that the intervention of animal Bifidobacterium lactis subspecies LIHUO 01 can effectively improve the intestinal permeability of mice with DSS-induced ulcerative colitis and repair colon barrier damage.

[0210] Example 10: Effect of Bifidobacterium animalis subsp. lactis LIHUO 01 on the antioxidant capacity of colon tissue in mice with DSS-induced colitis

[0211] The specific steps are as follows:

[0212] 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.

[0213] The experimental results are as follows:

[0214] The activity of SOD, CAT and MDA in colon tissue of mice in each group are shown in Fig.19 Compared with the normal group, the SOD activity of the colon tissue of the mice in the model group was significantly decreased; the SOD activity of the colon tissue of the mice in the mesalazine group was not significantly different from that in the model group, while the SOD activity of the colon tissue of the mice in the LIHUO 01 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 the mice in the model group were significantly increased, while the CAT activity and MDA concentration of the colon tissue of the mice in the mesalazine group and the LIHUO 01 group were significantly decreased compared with those in the model group.

[0215] The oxidative stress of DSS-induced colitis mice was further evaluated by measuring the SOD activity, CAT activity, and MDA concentration in the colon tissue of mice. The results of this experiment showed that the intervention of animal Bifidobacterium lactis subspecies LIHUO 01 could reduce the CAT activity and MDA concentration in the colon of mice with ulcerative colitis, and significantly increase the SOD activity. This shows that animal Bifidobacterium lactis subspecies LIHUO 01 can reduce the level of oxidative stress and thus alleviate DSS-induced ulcerative colitis in mice.

[0216] Example 11: Effect of Bifidobacterium animalis subsp. lactis LIHUO 01 on intestinal flora of mice with DSS-induced ulcerative colitis

[0217] The specific steps are as follows:

[0218] 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.

[0219] 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.

[0220] The experimental results are as follows:

[0221] The Sobs index and Ace index of the cecal contents of each group of mice are shown in Fig. 20 Compared with the normal group, based on the genus level, the Sobs index and Ace index of the cecal contents of the mice in the model group increased, but the difference was not significant; the Sobs index and Ace index of the cecal contents of the mice in the mesalazine group increased compared with the model group, but the difference was not significant; while the Sobs index and Ace index of the cecal contents of the mice in the LIHUO01 group decreased compared with the model group, but the difference was not significant. This indicates that the intake of DSS leads to changes in the α-diversity of intestinal microorganisms in mice, and the intervention of animal Bifidobacterium lactis subsp. LIHUO 01 reverses these changes to a certain extent.

[0222] Principal component analysis (PCA) and principal coordinate analysis (PCoA) of the cecal content microorganisms of each group of mice based on genus level analysis (see Fig.21 ) showed that the normal group, LIHUO 01 group and model group showed different microbiome compositions, while the LIHUO01 group was closer to the normal group.

[0223] The distribution of microorganisms in the cecal contents of mice in each group at the phylum and genus levels is shown in Fig. 22Firmicutes ( Firmicutes ), Bacteroidetes ( Bacteroidota ) dominated the relative abundance of the microbiome in the cecal contents of mice in each group. Compared with the normal group, the Firmicutes ( Firmicutes ) was significantly reduced, while the relative abundance of Firmicutes ( Firmicutes Compared with the normal group, the relative abundance of Bacteroidetes in the cecal contents of mice in the model group was significantly higher than that in the model group. Bacteroidota ) was significantly increased, while the relative abundance of Bacteroidetes ( Bacteroidota ) was significantly lower than that in the model group (see Fig.23 ).

[0224] Ileobacter spp. Ileibacterium )、norank_f_ Muribaculaceae The relative abundance of Enterobacter genus in the cecal contents of mice in each group was dominant. Compared with the normal group, the cecal contents of the model group mice were Ileibacterium ), Lactobacillus spp. Lactobacillus ) decreased significantly, while norank_f_norank_o_ Clostridia _UCG-014, Bacteroides spp. Bacteroides ) were significantly increased. Compared with the model group, the relative abundance of Bacteroides ( Bacteroides ) decreased significantly, while the relative abundance of Ileobacter spp. Ileibacterium ), Lactobacillus spp. Lactobacillus ) 、 Enterobacter Enterorhabdus ) increased significantly (see Fig.24 ).

[0225] In this study, intervention with Bifidobacterium animalis subsp. lactis LIHUO 01 made the microbial composition of the cecal contents of DSS-induced ulcerative colitis mice closer to that of the normal group, and significantly increased the Firmicutes phylum in the cecal contents at the phylum level. Firmicutes ) and significantly reduced the relative abundance of Bacteroidetes ( Bacteroidota ) relative abundance; at the genus level, the cecal contents of Ileobacterium ( Ileibacterium ), Lactobacillus spp. Lactobacillus ) 、 Enterobacter Enterorhabdus ) and significantly reduced the relative abundance of Bacteroides ( Bacteroides ), indicating that the intervention of Bifidobacterium animalis subsp. lactis LIHUO 01 could regulate the changes in intestinal flora of mice with ulcerative colitis caused by DSS.

[0226] Example 12: Effects of Bifidobacterium animalis subsp. lactis LIHUO 01 on short-chain fatty acids in colonic contents of mice with DSS-induced ulcerative colitis

[0227] The specific steps are as follows:

[0228] The grouping, modeling and treatment of mice were the same as in Example 6, and the colon contents of mice in each group were collected under sterile conditions.

[0229] The colon contents of 5 mice in each group were randomly selected and sent to Shanghai Meiji Biopharmaceutical Technology Co., Ltd. to detect the content of short-chain fatty acids: acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, caproic acid, and isocaproic acid. The specific method is: weigh the standard and colon content samples 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 15min, 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 was high-purity helium (purity not less than 99.999%), flow rate was 1.0mL / min, and the temperature of the injection port was 180℃. Injection volume 1μL, split injection, split ratio 10:1, solvent extension 2.5min. Program temperature rise: the initial temperature of the column oven is 80℃, the temperature is raised to 120℃ at 20℃ / min, and then raised to 160℃ at 5℃ / min, and then maintained at 220℃ for 3min. Mass spectrometry conditions: electron impact ion source (EI), ion source temperature 230℃, quadrupole temperature 150℃, transmission line temperature 230℃, electron energy 70 eV. The scanning mode is selected ion scanning mode (SIM).

[0230] The experimental results are as follows:

[0231] The concentrations of short-chain fatty acids in the colon contents of mice in each group are shown in Fig.25 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 decreased, the concentrations of acetic acid and propionic acid in the colon contents of mice in the LIHUO 01 group were significantly increased compared with the model group, while there was no significant difference between the mesalazine group and the model group.

[0232] Short-chain fatty acids are absorbed by colonic epithelial cells and can also be used as energy storage to promote the proliferation of colonic epithelial cells and the growth of mucosa. The results of this experiment showed that animal Bifidobacterium lactis subspecies LIHUO 01 can increase the concentration of short-chain fatty acids acetic acid and propionic acid in the colon content of DSS-induced ulcerative colitis mice. This shows that animal Bifidobacterium lactis subspecies LIHUO 01 can improve the level of short-chain fatty acids in metabolites, thereby alleviating ulcerative colitis.

[0233] Example 13: Effects of Bifidobacterium animalis subsp. lactis LIHUO 01 on the symptoms of constipation in rats induced by loperamide hydrochloride (Lop)

[0234] The specific steps are as follows:

[0235] (1) Preparation of Bifidobacterium animalis subsp. lactis LIHUO 01 bacterial suspension

[0236] The strains of animal Bifidobacterium lactis subsp. LIHUO 01 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.

[0237] (2) Construction of constipation model, strain intervention, and determination of fecal particle number and fecal water content

[0238] 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.

[0239] 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 01 experimental group (n=8 / group).

[0240] 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 01 experimental group were gavaged with animal Bifidobacterium lactis subsp. LIHUO 01 bacterial solution at a volume of 1 mL / rat / day (the dosage of animal Bifidobacterium lactis subsp. LIHUO 01 was 1.00×10 8 CFU / d); rats in all groups drank sterile water and had free access to food.

[0241] 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 01 experimental group rats were gavaged with animal Bifidobacterium lactis subsp. LIHUO 01 bacterial solution at a volume of 1 mL / rat / day (the dose of animal Bifidobacterium lactis subsp. LIHUO 01 was 1.00×10 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℃ for 24 hours, cooled to room temperature in a dryer and weighed m2. The calculation formula for fecal water content is as follows: fecal water content (%) = (m1-m2) / m1×100%.

[0242] (3) Preparation of Evans blue solution

[0243] 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.

[0244] (4) Determination of gastrointestinal propulsion rate

[0245] 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 blood samples were collected, the abdomen of the rats was disinfected with 75% ethanol solution, and the abdomen was cut open along the midline of the abdomen using standard dissecting scissors. The complete gastrointestinal tissue of the rats was removed, and 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%.

[0246] 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.

[0247] The experimental results are as follows:

[0248] The fecal water content and fecal particle number 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 those in the constipation model group, and the fecal water content and fecal particle number of rats in the LIHUO 01 experimental group were significantly higher than those in the constipation model group (see Fig.26 ).

[0249] 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 blank group, while the gastrointestinal propulsion rate of rats in the polyethylene glycol 4000 powder group and the LIHUO 01 experimental group was significantly higher than that in the constipation model group (see Fig. 27 ).

[0250] The experimental results showed that the intervention of animal Bifidobacterium lactis subspecies LIHUO 01 can increase the fecal water content, fecal particle number, and gastrointestinal propulsion rate of rats with Lop-induced constipation, thereby improving the symptoms of constipation.

[0251] Example 14: Effects of Bifidobacterium animalis subsp. lactis LIHUO 01 on relative levels of mucin and c-Kit mRNA in colonic tissue of rats with Lop-induced constipation

[0252] The specific steps are as follows:

[0253] The grouping, modeling, treatment of rats, and processing of colon tissue samples were the same as in Example 13.

[0254] (1) AB staining of colon tissue

[0255] The colon tissues fixed in 4% paraformaldehyde solution were dehydrated, paraffin-embedded, sectioned at 5 μm, dewaxed, and stained with Alcian blue (AB).

[0256] (2) Real-time fluorescence quantitative PCR (RT-qPCR) detection of c-Kit mRNA gene in colon tissue

[0257] 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 the method (GAPDH gene was used as the internal reference gene).

[0258] Table 10 Primer sequences

[0259]

[0260] 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.

[0261] The experimental results are as follows:

[0262] 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.28 ), 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 01 experimental group was increased compared with that in the model group.

[0263] 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 01 experimental group was significantly increased compared with the constipation model group (see Fig.29 ).

[0264] 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 animal Bifidobacterium lactis subspecies LIHUO 01 can increase the mucus protein in the colon of rats with Lop-induced constipation, and significantly upregulate the expression of c-Kit gene in the colon tissue of rats with Lop-induced constipation, thereby improving intestinal motility and constipation.

[0265] Example 15: Effect of Bifidobacterium animalis subsp. lactis LIHUO 01 on serum gastrointestinal regulatory peptide levels in rats with Lop-induced constipation

[0266] The specific steps are as follows:

[0267] The grouping, modeling and treatment of rats were the same as in Example 13.

[0268] 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).

[0269] The experimental results are as follows:

[0270] 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 constipation model group; while the serum MTL, GAS, and SP concentrations of rats in the LIHUO 01 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 constipation model group increased significantly, while the serum VIP and SS concentrations of rats in the polyethylene glycol 4000 powder group and the LIHUO 01 experimental group decreased significantly compared with the constipation model group (see Fig.30 ).

[0271] MTL, GAS, and SP can promote gastrointestinal muscle contraction and pyloric sphincter relaxation, and promote intestinal peristalsis. SS and VIP are gastrointestinal regulatory peptides that inhibit intestinal peristalsis. The results of this experiment showed that the intervention of animal Bifidobacterium lactis subspecies LIHUO 01 can significantly increase the concentrations of MTL, GAS, and SP in the serum of rats with Lop-induced constipation, and significantly reduce the concentrations of VIP and SS; regulate the level of serum gastrointestinal regulatory peptides and improve constipation.

[0272] Example 16: Effects of Bifidobacterium animalis subsp. lactis LIHUO 01 on neurotransmitter levels in rats with Lop-induced constipation

[0273] The specific steps are as follows:

[0274] 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.

[0275] 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 treatment.

[0276] 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.

[0277] The experimental results are as follows:

[0278] Compared with the blank group, the serum Ach concentration of rats in the constipation model group decreased significantly, and the serum Ach concentration of rats in the polyethylene glycol 4000 powder group and the LIHUO 01 experimental group increased significantly compared with the constipation model group (see Fig.31 ).

[0279] 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 01 experimental group was significantly increased compared with the constipation model group (see Fig.32 ).

[0280] Acetylcholine (Ach) can excite gastrointestinal smooth muscle, increase contraction amplitude, tension and peristalsis, and promote gastric and intestinal fluid secretion. 5-hydroxytryptamine (5-HT) is an important neurotransmitter that can reduce the colon transit time of feces in rats. The results of this experiment showed that the intervention of animal Bifidobacterium lactis subsp. LIHUO 01 can significantly increase the serum Ach concentration and colon tissue 5-HT concentration in rats with Lop-induced constipation, thereby improving constipation.

[0281] Example 17: Effect of Bifidobacterium animalis subsp. lactis LIHUO 01 on colonic aquaporin 3 (AQP3) in rats with Lop-induced constipation

[0282] The specific steps are as follows:

[0283] The grouping, modeling and treatment of rats were the same as in Example 13.

[0284] The detection of protein content in colon tissue of each group of rats was the same as in Example 16; the concentration of AQP3 in colon tissue of each group of rats was determined using an AQP3 ELISA kit (manufacturer: Shanghai ELISA, catalog number: ml003095V).

[0285] The experimental results are as follows:

[0286] 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 rats in the polyethylene glycol 4000 powder group and the LIHUO 01 experimental group was significantly lower than that in the constipation model group (see Fig.33 ).

[0287] 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 animal Bifidobacterium lactis subspecies LIHUO 01 can significantly reduce the concentration of AQP3 in colon tissue of rats with Lop-induced constipation, thereby improving constipation.

[0288] Example 18: Effects of Bifidobacterium animalis subsp. lactis LIHUO 01 on inflammatory factors in colon tissue of rats with Lop-induced constipation

[0289] The specific steps are as follows:

[0290] The grouping, modeling and treatment of rats were the same as in Example 13.

[0291] 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).

[0292] The experimental results are as follows:

[0293] 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 01 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 the concentration of IL-10 in the colon tissue of rats in the polyethylene glycol 4000 powder group was not significantly different from that in the constipation model group, while the concentration of IL-10 in the colon tissue of rats in the LIHUO 01 experimental group was significantly higher than that in the constipation model group (see Fig.34 ).

[0294] The experimental results showed that the intervention of animal Bifidobacterium lactis subspecies LIHUO 01 can significantly reduce the concentration of pro-inflammatory factor TNF-α in the colon tissue of rats with Lop-induced constipation, and significantly increase the concentration of anti-inflammatory factor IL-10, thereby improving the inflammation caused by constipation.

[0295] Example 19: Effect of Bifidobacterium animalis subsp. lactis LIHUO 01 on intestinal flora of rats with Lop-induced constipation

[0296] The specific steps are as follows:

[0297] The grouping, modeling and treatment of rats were the same as in Example 13, and the cecal contents of rats in each group were collected under sterile conditions.

[0298] 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 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, 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.

[0299] The experimental results are as follows:

[0300] The Sobs index and Ace index of the α diversity of the cecal contents of rats in each group were shown in Fig.35 . Compared with the blank group, based on the genus level, the Sobs index of the microorganisms in the cecal contents of the rats in the constipation model group increased, but the difference was not significant; while the Sobs index of the microorganisms in the cecal contents of the rats in the polyethylene glycol 4000 powder group and the LIHUO 01 experimental group increased compared with the constipation model group, but the difference was not significant. Compared with the blank group, based on the genus level, the Ace index of the microorganisms in the cecal contents of the rats in the constipation model group decreased, but the difference was not significant; while the Ace index of the microorganisms in the cecal contents of the rats in the polyethylene glycol 4000 powder group and the LIHUO 01 experimental group increased compared with the constipation model group, but the difference was not significant.

[0301] 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.36 ) showed that the blank group, LIHUO 01 experimental group and constipation model group showed different microbiome compositions, while the LIHUO 01 experimental group was closer to the blank group.

[0302] The distribution of microorganisms in the cecal contents of rats in each group at the phylum and genus levels was shown in Fig.37 Firmicutes ( Firmicutes ), Actinobacteria ( Actinobacteriota ) were the dominant flora in the cecal contents of rats in the blank group and the LIHUO 01 experimental group; while Firmicutes ( Firmicutes ), Bacteroidetes ( Bacteroidota ) were the dominant flora in the cecal contents of rats in the constipation model group and the polyethylene glycol 4000 powder group.

[0303] Lactobacillus Lactobacillus )、norank_f_norank_o_ Clostridia _UCG-014, Rombutsella spp. Romboutsia ) dominated the microbiome of the cecal contents of rats in each group. Compared with the blank group, Staphylococcus aureus ( 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 01 experimental group. Lachnospiraceac _NK4A136_group, norank_f_ Oscillospiraceae 、norank_f_ Muribaculaceae The relative abundance of the genus Bifidobacterium ( Bifidobacterium ), Staphylococcus spp. Staphylococus ) 、 Corynebacterium Corynebacterium ) increased significantly (see Figure 38 ).

[0304] The results of this experiment showed that the intervention of animal Bifidobacterium lactis subspecies LIHUO 01 could significantly increase the Bifidobacterium genus in the cecal contents of rats with Lop-induced constipation at the genus level. Bifidobacterium ), Staphylococcus spp. Staphylococus ) 、 Corynebacterium Corynebacterium ) significantly decreased Lachnospiraceac _NK4A136_group, norank_f_ Oscillospiraceae 、norank_f_ Muribaculaceae , thereby regulating the relative abundance of intestinal flora.

[0305] Example 20: Effect of Bifidobacterium animalis subsp. lactis LIHUO 01 on short-chain fatty acids in colonic contents of rats with Lop-induced constipation

[0306] The specific steps are as follows:

[0307] 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.

[0308] 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. The specific experimental method was the same as Example 12.

[0309] The experimental results are as follows:

[0310] The concentrations of short-chain fatty acids in the colon contents of rats in each group were shown in Figure 39 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 01 experimental group were higher than those in the constipation model group, among which the concentrations of isobutyric acid and isovaleric acid were significantly higher than those in the constipation model group. Compared with the blank group, the concentration of isohexanoic acid in the colon contents of rats in the constipation model group was significantly reduced, while the concentration of isohexanoic acid in the colon contents of rats in the polyethylene glycol 4000 powder group was significantly higher than that in the constipation model group.

[0311] The results of this experiment showed that the intervention of Bifidobacterium lactis subsp. LIHUO 01 could increase the concentration of isobutyric acid and isovaleric acid in the colon contents of rats with Lop-induced constipation, improve the level of short-chain fatty acids in metabolites, and thus improve constipation.

Claims

1. A strain of Bifidobacterium animalis subsp. lactis ( Bifidobacterium animalis subsp. lactis LIHUO 01, characterized by: The preservation number of the animal Bifidobacterium lactis subspecies LIHUO 01 is GDMCC No: 65512, and it was preserved in the Guangdong Provincial Microbiological Culture Collection Center on November 20, 2024.

2. A preparation of Bifidobacterium animalis subsp. lactis LIHUO 01, characterized in that: The preparation is carried out by culturing the animal Bifidobacterium lactis subspecies LIHUO 01 as claimed in claim 1, wherein the animal Bifidobacterium lactis subspecies LIHUO 01 preparation comprises any one or more of live bacteria, fermentation broth, fermentation broth precipitate and freeze-dried powder.

3. A microbial agent comprising the Bifidobacterium animalis subspecies lactis LIHUO 01 of claim 1 or the preparation of Bifidobacterium animalis subspecies lactis LIHUO 01 of claim 2.

4. Use of the animal Bifidobacterium lactis subsp. LIHUO 01 according to claim 1 or the animal Bifidobacterium lactis subsp. LIHUO 01 preparation according to claim 2 or the microbial agent according to claim 3 in the preparation of a product for regulating intestinal flora.

5. Use of the animal Bifidobacterium lactis subspecies LIHUO 01 according to claim 1 or the animal Bifidobacterium lactis subspecies LIHUO 01 preparation according to claim 2 or the microbial agent according to claim 3 in the preparation of a medicine for relieving ulcerative colitis.

6. The use according to claim 5, characterized in that: The drug is used to relieve the symptoms of ulcerative colitis, relieve intestinal inflammation, maintain the integrity of the intestinal barrier, reduce oxidative stress, regulate intestinal flora or improve the level of short-chain fatty acids in metabolites.

7. Use of the animal Bifidobacterium lactis subsp. LIHUO 01 according to claim 1 or the animal Bifidobacterium lactis subsp. LIHUO 01 preparation according to claim 2 or the microbial agent according to claim 3 in the preparation of a product for improving constipation.

8. The use according to claim 7, characterized in that: The products are used to promote defecation, improve intestinal motility, regulate gastrointestinal regulatory peptide levels, promote neurotransmitter secretion, reduce water channel protein levels, improve colon inflammation, regulate intestinal flora or improve short-chain fatty acid levels in metabolites.

9. A product comprising the animal Bifidobacterium lactis subsp. LIHUO 01 according to claim 1 or the animal Bifidobacterium lactis subsp. LIHUO 01 preparation according to claim 2 or the microbial agent according to claim 3, characterized in that: The product is food, health product or medicine.

10. The product according to claim 9, characterized in that The product is a food or a health product, which also includes an additive, and the additive is selected from any one or more of a flavoring agent, a colorant, a filler, a disintegrant, a lubricant, a binder and a pH adjuster.

11. The product according to claim 9, characterized in that The product is a medicine, which also includes pharmaceutical excipients, which are selected from any one or more of buffers, emulsifiers, stabilizers, diluents, adhesives, preservatives and lubricants.

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