Probiotics and their uses

Abstract

The present invention discloses a probiotic containing Parabacteroides distasonis, which has a microorganism accession number CCTCC NO: M20222033, Limocilactobacillus fermentum, which has a microorganism accession number CCTCC NO: M2023352, Lactobacillus salivarius, which has a microorganism accession number CCTCC NO: M2023348, Enterococcus avium, which has a microorganism accession number CCTCC NO: M2023350, and Bifidobacterium bifidum, which has a microorganism accession number CCTCC NO: M2023349. The probiotic is safe, has antioxidant activity, and can inhibit pathogenic bacteria, improve colon damage, inhibit inflammation, and alleviate diarrhea caused by chemotherapy.

Description

[Technical Field]

[0001] The present invention is in the field of microbiology, and specifically relates to probiotics and their uses. [Background technology]

[0002] Intestinal microbiota are closely related to human health and are metaphorically referred to as the "microbial organ." The intestinal microbiota is an important component of the body. Under normal conditions, the intestinal microbiota maintains dynamic stability. They play important roles in many vital activities, including promoting nutrient digestion and absorption, maintaining normal intestinal physiological function, and regulating the immune system. However, the intestinal microbiota is easily affected by many factors, including environmental factors, dietary and lifestyle habits, psychological factors, disease status, tumor treatment, antibiotic use, and age. When affected by these factors, the human intestinal microbiota can develop intestinal disorders (dysbiosis). Intestinal disorders can manifest as a deficiency of beneficial intestinal bacteria, an overgrowth of pathogenic intestinal bacteria, impaired intestinal barrier function, and intestinal inflammation. Intestinal disorders can further lead to gastrointestinal disorders such as constipation, diarrhea, abdominal pain, and abdominal distension. Severe intestinal disorders can develop into diseases such as inflammatory bowel disease, ulcerative colitis, and irritable bowel syndrome, which have a significant impact on human health and quality of life.

[0003] Currently, increasing attention is being paid to the use of intestinal probiotics to improve intestinal health and prevent or treat intestinal diseases. Intestinal probiotics can maintain intestinal health by enhancing the function of the intestinal mucosal barrier, preventing the adhesion and colonization of pathogenic bacteria, and enhancing the immune response of the intestinal system. For example, Chinese invention patent application CN102711778A discloses animal Bifidobacterium lactis subsp. DN-173010, and mouse experiments and histological studies have verified that its fermented milk can alleviate ulcerative colitis. Patent application publication number CN107312726A discloses plant lactobacillus that can inhibit the growth of harmful bacteria such as Escherichia coli, Salmonella enterica, Streptococcus suis, and Staphylococcus aureus in the intestinal tract.

[0004] Probiotics are also used to prevent or improve the side effects of microbial flora dysbiosis caused by some drugs, such as antibiotics. Side effects associated with radiation therapy and chemotherapy are very common clinically. Chemotherapy-induced diarrhea (CID), also known as chemotherapy-induced diarrhea (CID), is one of the most common complications during chemotherapy in tumor patients. Although the development of CID is thought to be multifactorial, the pathogenesis of CID has not yet been fully elucidated. For example, in the case of 5-FU (5-fluorouracil), proliferating small intestinal cells are relatively sensitive to 5-FdUMP or 5-FUMP, which are produced by phosphorylation of 5-FU. 5-FdUMP or 5-FUMP damage the small intestinal mucosa and disrupt enterocyte division, causing necrosis of intestinal wall cells and widespread inflammation in the intestinal wall, leading to an imbalance in the number of small intestinal absorptive and secretory cells, which can result in diarrhea. Chemotherapy drugs also cause cellular DNA damage and mitochondrial dysfunction, resulting in the production of reactive oxygen species (ROS) and cell apoptosis. ROS can activate NF-κB and upregulate the expression of pro-inflammatory factors, resulting in damage to the intestinal epithelium, endothelium, and connective tissue. When the intestinal epithelium is damaged, harmful bacteria can easily colonize, destroying the intestinal microecosystem and inducing pathogenic bacterial infection, which promotes the occurrence and development of diarrhea.

[0005] Clinically, CID still lacks a unified and effective treatment. Generally speaking, the main goals of CID treatment are symptom control, patient relief, accelerated mucosal repair, and prevention of secondary infections. Common CID treatments include the use of antidiarrheal drugs, mucosal protective agents, and antibiotics, as well as high-dose loperamide and octreotide, a somatostatin analogue. However, these drugs have a single mechanism of action and significant side effects, making them unsuitable for long-term maintenance administration. For example, high-dose loperamide can pose a risk of paralytic ileus, while octreotide can cause side effects such as gallstones, hyperglycemia, and impaired glucose tolerance. Antibiotics can also kill beneficial intestinal bacteria, disrupt the structure of the bacterial flora, and lead to intestinal microecosystem damage.

[0006] The rich and diverse intestinal microbiota is a complex microbial ecosystem and an important natural barrier against enteric pathogens and other risk factors. M. Kverka et al. (Clinical and Experimental Immunology, 163:250-259, 2011) reported that Parabacteroides distasonis can ameliorate DSS-induced colitis in mice. Patent CN113215063B discloses Lactobacillus salivarius CPU-1, which can alleviate the toxicity and side effects of the chemotherapy drug temozolomide, focusing on the improvement of mucositis symptoms. Patents such as CN1511945A and CN86103736A investigate the use of multiple species of probiotics, including Lactobacillus fermentum, in the treatment or prevention of diarrhea. Patent CN113234619B discloses Bifidobacterium bifidum, which can alleviate acute intestinal injury. Inactivated Bacteroides fragilis ZY-312 is reported to be approved by the FDA for clinical trials in infectious diseases.

[0007] Although several studies have explored the potential of probiotics to prevent or treat multiple intestinal diseases, there remains a lack of clinically effective and safe probiotic intervention regimens (especially for diseases or conditions lacking effective treatments, such as CID). Developing novel probiotics to overcome the deficiencies of existing drugs, such as their high toxicity and side effects and single therapeutic mechanism, and providing effective treatment regimens for patients with tumor therapy-related diarrhea remains an urgent issue in the field of biopharmaceuticals. Summary of the Invention

[0008] In response to the drawbacks of the prior art, a first aspect of the present invention provides a probiotic composition, wherein the active ingredient in the probiotic composition comprises one or a combination of two or more of Bifidobacterium bifidum having a deposit number of CCTCC NO: M2023349 or a pure culture thereof, Enterococcus avium having a deposit number of CCTCC NO: M2023350 or a pure culture thereof, Lactobacillus salivarius having a deposit number of CCTCC NO: M2023348 or a pure culture thereof, Limosilactobacillus fermentum having a deposit number of CCTCC NO: M2023352 or a pure culture thereof, and Parabacteroides distasonis having a deposit number of CCTCC NO: M20222033 or a pure culture thereof.

[0009] In some embodiments, the probiotic composition further comprises a lyoprotectant, a food ingredient, a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable excipient.

[0010] A second aspect of the present invention provides an isolated Parabacteroides distasonis strain or a pure culture thereof, which has the microorganism deposit number CCTCC NO: M20222033.

[0011] A third aspect of the present invention provides an isolated strain of Limocilactobacillus fermentum or a pure culture thereof, having the microorganism deposit number CCTCC NO: M2023352.

[0012] A fourth aspect of the present invention provides an isolated Lactobacillus salivarius strain or a pure culture thereof, having the microorganism deposit number CCTCC NO: M2023348.

[0013] A fifth aspect of the present invention provides an isolated Enterococcus avium strain or a pure culture thereof, having the microorganism deposit number CCTCC NO: M2023350.

[0014] A sixth aspect of the present invention provides an isolated Bifidobacterium bifidum strain or a pure culture thereof, having the microorganism deposit number CCTCC NO: M2023349.

[0015] A seventh aspect of the present invention provides a method for preventing, treating or delaying intestinal disease, the method comprising administering to a subject a therapeutically effective amount of a probiotic composition according to the first aspect of the invention or a strain or a pure culture thereof according to any one of the second to sixth aspects of the invention.

[0016] In some embodiments, the subject is a mammal, and more preferably, the subject is a human.

[0017] In some embodiments, the probiotic composition according to the first aspect of the present invention or the strain or pure culture thereof according to any one of the second aspect of the present invention, the third aspect of the present invention, the fourth aspect of the present invention, the fifth aspect of the present invention and the sixth aspect of the present invention inhibits enteropathogenic bacteria in the subject, ameliorates colon damage in the subject, inhibits intestinal inflammation in the subject, repairs the intestinal barrier in the subject or ameliorates diarrhea symptoms in the subject.

[0018] In some embodiments, the intestinal disease is selected from enteropathogenic bacterial infection, diarrhea, and radiation enteritis, and preferably the enteropathogenic bacteria is one or more selected from Pseudomonas aeruginosa, Salmonella Paratyphi B, Yersinia enterocolitica, Staphylococcus aureus, Vibrio parahaemolyticus, Clostridium difficile, Shigella, and Escherichia coli, and preferably the diarrhea is diarrhea induced by an antitumor drug, and more preferably the diarrhea induced by an antitumor drug is diarrhea induced by a chemotherapy drug.

[0019] In some embodiments, the chemotherapy-induced diarrhea is diarrhea caused by one or more drugs selected from epirubicin, actinomycin D, doxorubicin, daunorubicin, paclitaxel, docetaxel, albumin-paclitaxel, cisplatin, carboplatin, nedaplatin, oxaliplatin, lobaplatin, cyclophosphamide, nitrogen mustard, carmustine, camptothecin, hydroxycamptothecin, topotecan, irinotecan, capecitabine, gemcitabine, methotrexate, 5-fluorouracil, pemetrexed, and cytarabine. [Brief explanation of the drawings]

[0020] [Figure 1] Frontal photographs of colony morphology of five strains. [Figure 2] FIG. 1 shows the experimental results of total antioxidant capacity of five strains. [Figure 3] This is a graph showing the results of bacteriostatic experiments on five strains. [Figure 4] These are the results of a test on Caco-2 cell barrier repair by Lactobacillus salivarius Lsali-1 and Enterococcus avium Eaviu-1. [Figure 5] 1 shows the results of an in vitro cell inflammation inhibition test of five strains of bacteria. [Figure 6] This shows the results of detecting the adhesive ability of five strains of bacteria to Caco2 cells. [Figure 7]This shows the diarrhea score and total diarrhea score of Parabacteroides distasonis Pdist-1 ameliorating 5-fluorouracil-induced diarrhea in a mouse model. [Figure 8] This figure shows the diarrhea score and total diarrhea score of the 5-fluorouracil-induced diarrhea mouse model ameliorated by Lactobacillus fermentum Lferm-1. [Figure 9] This figure shows the diarrhea score and total diarrhea score of Lactobacillus salivarius Lsali-1 improving a 5-fluorouracil-induced diarrhea mouse model. [Figure 10] Figure 1 shows the diarrhea score and total diarrhea score of Enterococcus avium Eaviu-1 ameliorating a 5-fluorouracil-induced diarrhea mouse model. [Figure 11] This shows the diarrhea score and total diarrhea score of Bifidobacterium bifidum Bbifi-1, which improves 5-fluorouracil-induced diarrhea in a mouse model. [Figure 12] This figure shows the diarrhea score and total diarrhea score of Bifidobacterium bifidum Bbifi-1 and DSM20456 ameliorating 5-fluorouracil-induced diarrhea in a mouse model. [Figure 13] Histopathological results of treating mice with 5-fluorouracil-induced diarrhea with Parabacteroides distasonis Pdist-1 and Limocylic Lactobacillus fermentum Lferm-1. The scale bar is 500 μm. [Figure 14] FIG. 10 shows the effects of four bacterial strains on the relative expression levels of colonic inflammatory factors and aquaporin genes in mice with 5-fluorouracil-induced diarrhea. [Figure 15] Therapeutic effect of Bifidobacterium bifidum Bbifi-1 strain on radiation enteritis-induced diarrhea in mice. [Figure 16] Photographs of the co-culture characteristics of five strains on BF839 agar medium. DETAILED DESCRIPTION OF THE INVENTION

[0021] In order to make the objectives, technical solutions and advantages of the present invention clearer, the following describes in more detail the embodiments of the present invention in conjunction with the drawings.

[0022] The present invention provides a probiotic composition for preventing, treating, or delaying intestinal diseases, the active ingredient of which comprises a therapeutically effective amount of any one or a combination of two or more of Bifidobacterium bifidum having deposit number CCTCC NO: M2023349 or a pure culture thereof, Enterococcus avium having deposit number CCTCC NO: M2023350 or a pure culture thereof, Lactobacillus salivarius having deposit number CCTCC NO: M2023348 or a pure culture thereof, Limosilactobacillus fermentum having deposit number CCTCC NO: M2023352 or a pure culture thereof, and Parabacteroides distasonis having deposit number CCTCC NO: M20222033 or a pure culture thereof.

[0023] The five strains with specific deposit numbers claimed in the present invention include, but are not limited to, (1) strains with specific deposit numbers stored in the depository center, (2) strains having the same genome as the strain described in (1), (3) passaged strains without genetic mutations based on (1) or (2) above, (4) passaged strains based on (1), (2), or (3) above that have accumulated minor mutations during passage but have not substantially changed in toxicity, immunogenicity, or biological activity, and (5) viable cells of any one of the strains described in (1) to (4) above, inactivated products of the viable cells, lysates of the viable cells, or fermentation products of the viable cells.

[0024] A strain having the same genome includes, but is not limited to, a strain having the same genetic background, i.e., a strain isolated from nature or from the body of an animal (including a human) with the same genome (same genetic background), that has been independently isolated and disclosed by another person after the priority date of the present invention. Conventional cultures are generally considered to be passaged strains without genetic mutations. As is known in the art, the introduction of minor mutations is usually unavoidable when a strain is passaged and used. If the mutation is a synonymous mutation occurring in a non-coding sequence region or a coding region, or a mutation that does not affect the toxicity, immunogenicity, or biological activity of the strain (e.g., a linking amino acid residue between two domains, or a minor mutation residue within the higher-order structure of a protein that does not contact immune cells and therefore does not affect toxicity, immunogenicity, or biological activity), it can be reasonably expected that the purpose of the present invention can still be achieved and that the progeny strain, derived from the contributing strain of the present invention, falls within the scope of the substantial technical contribution of the present invention. These minor variations still belong to the category of insubstantial variations and should be considered as mutant strains with no changes in toxicity, immunogenicity, and biological activity. From the perspective of detection, no substantial changes in toxicity, immunogenicity, and biological activity include, but are not limited to, considering the toxicity, immunogenicity, and biological activity to be the same within the limits of detection technology, such as detection sensitivity and detection limit, and within the range of acceptable or unavoidable errors. When measuring the toxicity, immunogenicity, and biological activity of the progeny of a strain using cells, animals, etc., differences that appear due to cell lines, animal breeds, age, sex, health status, culture conditions, etc., and predictable or unavoidable systematic errors belong to no substantial changes. An active ingredient refers to a substance that functions as a composition that generates a biological effect. In the present invention, the active ingredient is a probiotic strain.As a result of studying the co-culture characteristics of the five strains of the present invention, it was found that these strains do not inhibit each other two by two. Therefore, it is reasonably expected that they can be combined into compositions containing two, three, four or five strains depending on the efficacy characteristics of each strain, and that compositions combining these strains can simultaneously exert the efficacy of the strains within the group.

[0025] To better describe the preferred combinations of strains of the present invention, the following are defined: M1: Parabacteroides distasonis strain having the microorganism deposit number CCTCC NO: M20222033 or a pure culture thereof; M2: Enterococcus avium strain having the microorganism deposit number CCTCC NO: M2023350 or a pure culture thereof; M3: Limocilactobacillus fermentum strain having the microorganism deposit number CCTCC NO: M2023352 or a pure culture thereof; M4: Bifidobacterium bifidum strain having the microorganism deposit number CCTCC NO: M2023349 or a pure culture thereof; and M5: Lactobacillus salivarius strain having the microorganism deposit number CCTCC NO: M2023348 or a pure culture thereof.

[0026] In some embodiments, the active ingredients in the probiotic compositions described herein are any one, any two, any three, any four or five of M1, M2, M3, M4 and M5.

[0027] In some embodiments, the active ingredients in the probiotic composition are a combination of M1 and any one, any two, or any three selected from M2, M3, M4, and M5.

[0028] In some embodiments, the active ingredients in the probiotic composition are M2 in combination with any one, any two, or any three selected from M1, M3, M4, and M5.

[0029] In some embodiments, the active ingredients in the probiotic composition are M3 in combination with any one, any two, or any three selected from M1, M2, M4, and M5.

[0030] In some embodiments, the active ingredients in the probiotic composition are M4 in combination with any one, any two, or any three selected from M1, M2, M3, and M5.

[0031] In some embodiments, the active ingredients in the probiotic composition are a combination of M5 and any one, any two, or any three selected from M1, M2, M3, and M4.

[0032] In some embodiments, the active ingredient is a strain of a single species, and testing has demonstrated that even a single species of strain can clearly prevent, treat, or delay intestinal disease.

[0033] Thus, in some embodiments, the present invention provides use of Parabacteroides distasonis, having microorganism deposit number CCTCC NO: M20222033, or a pure culture thereof, in the manufacture of a formulation for preventing, delaying, or treating a disease or subcondition, wherein the prevention, delay, or treatment of the disease or subcondition includes preventing, treating, or delaying oxidative damage in the intestinal tract, inhibiting any one, any two, any three, any four, or any five of intestinal Pseudomonas aeruginosa, Salmonella Paratyphi B, Yersinia enterocolitica, Staphylococcus aureus, and Vibrio parahaemolyticus, increasing AQP8 expression, or tolerating, resisting, preventing, or alleviating diarrhea.

[0034] In some embodiments, the diarrhea is selected from chemotherapy-induced diarrhea.

[0035] In some embodiments, the chemotherapeutic agent is selected from 5-fluorouracil.

[0036] In some embodiments, the present invention provides use of a Limocilactobacillus fermentum having the microorganism deposit number CCTCC NO: M2023352 or a subculture thereof in the manufacture of a formulation for preventing, delaying, or treating a disease or subcondition, wherein the prevention, delaying, or treatment of a disease or subcondition includes preventing, treating, or delaying oxidative damage in the intestinal tract, inhibiting any one, any two, any three, or four of intestinal Salmonella Paratyphi B, Yersinia enterocolitica, Staphylococcus aureus, and Clostridium difficile, reducing TNF-α expression, reducing IL-6 expression, or tolerating, resisting, preventing, or alleviating diarrhea.

[0037] In some embodiments, the diarrhea is selected from chemotherapy-induced diarrhea.

[0038] In some embodiments, the chemotherapeutic agent is selected from 5-fluorouracil.

[0039] In some embodiments, the present invention provides use of Lactobacillus salivarius having Microorganism Deposit Number CCTCC NO: M2023348, or a subculture thereof, in the manufacture of a formulation for preventing, delaying, or treating a disease or subcondition, wherein the prevention, delaying, or treatment of the disease or subcondition includes preventing, treating, or delaying oxidative damage in the intestinal tract; inhibiting any one, any two, any three, any four, any five, any six, or seven of the following intestinal bacteria: Pseudomonas aeruginosa, Shigella flexneri, Salmonella Paratyphi B, Yersinia enterocolitica, Vibrio parahaemolyticus, Staphylococcus aureus, and Clostridium difficile; preventing, ameliorating, or repairing inflammation-induced intestinal barrier damage; reducing TNF-α expression; reducing IL-6 expression; reducing IL-1β expression; or tolerating, resisting, preventing, or alleviating diarrhea.

[0040] In some embodiments, the diarrhea is selected from chemotherapy-induced diarrhea.

[0041] In some embodiments, the present invention provides use of Enterococcus avium having Microorganism Deposit Number CCTCC NO: M2023350, or a subculture thereof, in the manufacture of a formulation for preventing, delaying, or treating a disease or subcondition, wherein the prevention, delaying, or treatment of the disease or subcondition includes preventing, treating, or delaying oxidative damage in the intestinal tract, inhibiting one or more of Pseudomonas aeruginosa and Clostridium difficile in the intestine, preventing, ameliorating, or repairing inflammation-induced intestinal barrier damage, reducing TNF-α expression, reducing IL-6 expression, or tolerating, resisting, preventing, or alleviating diarrhea.

[0042] In some embodiments, the diarrhea is selected from chemotherapy-induced diarrhea.

[0043] In some embodiments, the chemotherapeutic agent is selected from 5-fluorouracil.

[0044] In some embodiments, the present invention provides use of Bifidobacterium bifidum, having Microorganism Deposit Number CCTCC NO: M2023349, or a subculture thereof, in the manufacture of a formulation for preventing, delaying, or treating a disease or subcondition, wherein the prevention, delaying, or treatment of the disease or subcondition includes preventing, treating, or delaying oxidative damage in the intestinal tract, inhibiting any one, any two, any three, any four, any five, any six, or seven of the following intestinal bacteria: Pseudomonas aeruginosa, Shigella flexneri, Escherichia coli, Salmonella Paratyphi B, Yersinia enterocolitica, Vibrio parahaemolyticus, and Clostridium difficile, reducing TNF-α expression, or tolerating, resisting, preventing, or alleviating diarrhea.

[0045] In some embodiments, the diarrhea is selected from chemotherapy-induced diarrhea and radiation enteritis-induced diarrhea.

[0046] In some embodiments, the chemotherapeutic agent is selected from 5-fluorouracil.

[0047] In some embodiments, the radiation enteritis is radiation enteritis caused by abdominal X-ray irradiation.

[0048] A therapeutically effective amount refers to an amount of active ingredient effective to prevent, treat, alleviate, or ameliorate the symptoms or progression of a disease (e.g., intestinal disease: diarrhea, infection, inflammation, etc.), or to prolong the survival of the treated subject. Determining a therapeutically effective amount is well within the capabilities of those skilled in the art, particularly based on the detailed disclosure provided herein. A therapeutically effective amount or dose can be initially assessed in in vitro and cell culture assays, and then the dose can be adjusted in animal models to achieve a desired concentration or potency. Such information can be used to more accurately determine useful doses in humans.

[0049] In some embodiments, the probiotic composition administered to a subject in a single dose comprises 10 2 ~10 15 CFU, 10 3 ~10 14 CFU, 10 4 ~10 13 CFU, 10 5 ~10 12 CFU, 10 6 ~10 12 CFU, 10 7 ~10 11 CFU, or 10 8 ~10 10The probiotic dose may contain CFUs. The dosage may vary depending on the dosage form and route of administration used. The individual physician can select the exact dosage depending on the patient's condition. The dosage and interval can be individually adjusted to provide a sufficient amount of the active ingredient to induce the biological effect. Depending on the severity and responsiveness of the condition to be treated, administration may be single or multiple, with the course of treatment lasting from several days to several weeks, or until a cure is achieved or a diminution of the disease state is achieved. The amount of the composition administered will primarily be determined by the subject being treated, the severity of the condition, the method of administration, the judgment of the prescribing physician, etc.

[0050] The invention provides compositions in the form of normal foods, beverages, health foods, medical foods, or pharmaceuticals containing the probiotic compositions or strains described in the present invention. These normal foods, beverages, health foods, medical foods, or pharmaceuticals include various exemplary embodiments of the compositions of the present invention. These normal foods, beverages, health foods, medical foods, or pharmaceuticals may be manufactured or provided as probiotic powders, capsules, cereals, baby foods, health foods, or specific health foods, and may also be pharmaceutical capsules, tablets, powders, etc. The probiotic compositions of the present invention may further contain other beneficial active ingredients, such as another probiotic, prebiotic, or drug with anti-diarrheal function, where the prebiotic indirectly exerts its anti-diarrheal effect by promoting the growth of probiotics in the intestinal tract and helping to regulate the intestinal environment. Examples of the second class of beneficial active ingredients include, but are not limited to, Bacillus licheniformis, Bifidobacterium, Clostridium butyricum, fructooligosaccharides, galactooligosaccharides, isomaltooligosaccharides, xylooligosaccharides, mannanoligosaccharides, inulin, stachyose, soybean oligosaccharides, beta-dextran, lactosucrose, and the like.

[0051] "Any one" refers to selecting one option from the options provided. "Two or more" refers to selecting two, three, ... or all of the options available from the options provided.

[0052] Intestinal disease refers to infection, inflammation, diarrhea, or dysbiosis-like intestinal disease. In some embodiments, intestinal disease refers to enteropathogenic infection, colon damage, intestinal inflammation, intestinal barrier damage, and / or diarrhea.

[0053] In some embodiments, the intestinal disease is selected from enteropathogenic bacterial infection, diarrhea, and radiation enteritis, and preferably the enteropathogenic bacteria is one or more selected from Pseudomonas aeruginosa, Salmonella Paratyphi B, Yersinia enterocolitica, Staphylococcus aureus, Vibrio parahaemolyticus, Clostridium difficile, Shigella, and Escherichia coli, and preferably the diarrhea is diarrhea induced by an antitumor drug, and more preferably the diarrhea induced by an antitumor drug is diarrhea induced by a chemotherapy drug.

[0054] Enteric pathogens refer to bacteria that can cause intestinal infection or disease, and can invade the human body through contaminated food, water, direct contact, or insect vectors, resulting in various intestinal diseases. There are many types of enteric pathogens, and their survival and proliferation in the intestinal tract can disrupt the normal balance of intestinal flora, causing problems such as inflammation, diarrhea, and malabsorption.

[0055] In some embodiments, the enteric pathogen is one or more species selected from Pseudomonas aeruginosa, Salmonella paratyphi B, Yersinia enterocolitica, Staphylococcus aureus, Vibrio parahaemolyticus, Clostridium difficile, Shigella dysenteriae, and Escherichia coli. Radiation enteritis refers to intestinal complications caused by radiation therapy for pelvic, abdominal, or retroperitoneal malignant tumors. This inflammation can affect the small intestine, colon, and rectum, and is therefore also called radiation proctitis, colitis, or enteritis. The most common symptom of radiation enteritis is diarrhea, sometimes accompanied by mucus or blood in the stool. Other symptoms include abdominal pain, tenesmus, nausea and vomiting, abdominal distension, loss of appetite, and possibly weight loss. Diarrhea refers to a significant increase in bowel movements, usually more than three times per day, with thin, high-water stools (over 85%) and possibly mucus, pus, or undigested food. Causes of diarrhea include pathogenic bacterial infection, food poisoning, drug side effects, intestinal inflammation, and psychological factors.

[0056] In some embodiments, the diarrhea is antineoplastic drug-induced diarrhea. Antineoplastic drug-induced diarrhea: Antineoplastic drugs can be broadly classified into cytotoxic drugs, small molecule targeted drugs, monoclonal antibody drugs, immune checkpoint inhibitors, hormonal drugs, antibody-drug conjugates (ADCs), biological response modifiers, and other types of drugs based on their mechanism of action and drug source. Diarrhea caused by the use of these drugs is called antineoplastic drug-induced diarrhea, also known as antineoplastic treatment-related diarrhea / tumor-associated diarrhea.

[0057] In some embodiments, the diarrhea caused by antitumor drugs specifically refers to diarrhea caused by chemotherapy drugs.Cytotoxic drugs are generally also called chemotherapy drugs, and the mechanism of action of these drugs is mainly to destroy or interfere with the growth and division process of tumor cells, thereby inhibiting or killing cancer cells.Chemotherapeutic drugs are generally relatively toxic to rapidly dividing cells, so they not only affect cancer cells, but also may affect rapidly dividing cells in normal body tissues, such as hair follicle cells, gastrointestinal cells and bone marrow cells, which are common side effects of chemotherapy, such as hair loss, nausea, vomiting, diarrhea and bone marrow suppression.

[0058] In some embodiments, the chemotherapy-induced diarrhea is diarrhea caused by one or more drugs selected from epirubicin, actinomycin D, doxorubicin, daunorubicin, paclitaxel, docetaxel, albumin-paclitaxel, cisplatin, carboplatin, nedaplatin, oxaliplatin, lobaplatin, cyclophosphamide, nitrogen mustard, carmustine, camptothecin, hydroxycamptothecin, topotecan, irinotecan, capecitabine, gemcitabine, methotrexate, 5-fluorouracil, pemetrexed, and cytarabine.

[0059] The present invention further provides a method for preventing, treating or delaying intestinal disease, which method comprises administering to a subject a probiotic composition according to the first aspect of the invention or a strain or a pure culture thereof according to the second aspect of the invention. The subject may be an avian, mammalian or human subject, preferably a mammalian or human subject, more preferably a human subject.

[0060] The present invention further provides five isolated probiotic strains for preventing or treating intestinal diseases, which are Parabacteroides distasonis having the deposit number CCTCC NO: M20222033 or its progeny, clone strain, fermentate, lysate, extract and pure culture; Limosilactobacillus fermentum having the deposit number CCTCC NO: M2023352 or its progeny, clone strain, fermentate, lysate, extract and pure culture; Lactobacillus salivarius having the deposit number CCTCC NO: M2023348 or its progeny, clone strain, fermentate, lysate, extract and pure culture; and Lactobacillus salivarius having the deposit number CCTCC NO: M2023348 or its progeny, clone strain, fermentate, lysate, extract and pure culture; Enterococcus avium, having the deposit number CCTCC NO: M2023350, or its progeny strains, clones, fermentations, lysates, extracts and pure cultures, and Bifidobacterium bifidum, having the deposit number CCTCC NO: M2023349, or its progeny strains, clones, fermentations, lysates, extracts and pure cultures.

[0061] The technical solutions in the embodiments of the present invention are described below clearly and completely. Of course, the described embodiments are not all embodiments but only some of the embodiments of the present invention. All other embodiments that can be obtained by those skilled in the art without creative effort based on the embodiments of the present invention are within the scope of the present invention. Materials and equipment not described in the present invention are conventional materials and equipment in this field. Operational details not described in the present invention are conventional operations in this field. Software used in the present invention is operated in a conventional manner by referring to the software provider's instruction manual. Reagent kits used in the present invention are operated in a conventional manner by referring to the instruction manual of the reagent kit. It should be understood that specific parameters in the present invention are allowed to vary within a certain error range, for example, ±5%, unless otherwise specified. Temperature may vary within ±5°C, ±4°C, ±3°C, ±2°C, or ±1°C.

[0062] The methods for preparing media used in the following examples are as follows. Media not specifically described are prepared by methods commonly used in the art or are commercially available: Preparation of YCFA liquid medium: 10.0 g of peptone, 2.5 g of yeast extract, 0.45 mL of 10% (w / w) MgSO₄·7H₂O solution, 0.45 mL of 10 mg / mL CaCl₂ solution, 10 mL of TE141, 0.45 g of K₂HPO₄, 0.45 g of KH₂PO₄, 0.90 g of NaCl, and 3.2 mL of VFA-mix were added to 1 L of distilled water to obtain a solution. The solution was deoxygenated by N₂ replacement and then dispensed. The dispensed solution was sterilized at 121°C for 30 min using moist heat and then prepared for use.

[0063] Preparation of TE141: 1.50 g of nitrilotriacetic acid was added to 200 mL of pure water to obtain a solution, and an appropriate amount of NaOH was added to the solution until the solution became clear. 800 mL of water was then added to the solution, and the pH value was adjusted to 5.5 with 50% HCl to obtain an aqueous solution of nitrilotriacetic acid. 3.00 g of MgSO4·7H2O, 0.50 g of MnSO4·H2O, 1.00 g of NaCl, 0.10 g of FeSO4·7H2O, 0.18 g of CoSO4·7H2O, 0.10 g of CaCl2·2H2O, 0.18 g of ZnSO4·7H2O, 0.006 g of CuSO4·5H2O, 0.02 g of KAl(SO4)2·12H2O, 0.01 g of H3BO3, 0.01 g of Na2MoO4·2H2O, 0.03 g of NiCl2·6H2O, 0.03 mL of 10 mg / mL Na2SeO3·5H2O solution, and 0.03 mL of 10 mg / mL Na2WO4·2H2O solution were added to the above solution, and the solution was constantly stirred during the addition process to keep it clear.

[0064] Preparation of VFA-mix: 90 mL of acetic acid, 30 mL of propionic acid, 10 mL of n-valeric acid, 10 mL of isobutyric acid, and 10 mL of butyric acid were mixed uniformly to obtain a solution for use. The pH of the solution was adjusted to neutral with 5 M NaOH solution before use.

[0065] Preparation of triple-mix liquid medium (BHI + MRS + modified GAM): 19.25 g of BHI broth powder (Qingdao Haibo Biotechnology Co., Ltd., HB8297-5), 13.5 g of MRS broth powder (Guangdong Huankai Biotechnology Co., Ltd., 027312), and 15 g of modified GAM broth powder (Qingdao Haibo Biotechnology Co., Ltd., HB8518-3) were dissolved in 1 L of distilled water to obtain a solution. The solution was deoxygenated by nitrogen substitution and dispensed. The solution was then sterilized at 121 °C for 30 min using high-temperature moist heat.

[0066] Preparation of triple mixed solid medium (BHI + MRS + improved GAM): Increase 5 g of agar powder based on triple mixed liquid medium, and keep the other steps the same.

[0067] Preparation of double mixed medium (BHI + MRS): 19.25 g of BHI broth powder, 27.0 g of MRS broth powder, and 0.5 g of cysteine ​​hydrochloride monohydrate were dissolved in 1 L of distilled water to obtain a mixed solution. The mixed solution was deoxygenated, dispensed, and sterilized by high-temperature moist heat at 121°C for 15 min.

[0068] JPEG2026505892000002.jpg38170

[0069] Preparation of MRS broth: 54.0 g of MRS broth powder and 0.5 g of cysteine ​​hydrochloride monohydrate were weighed and dissolved in 1 L of distilled water, deoxygenated by N2 substitution, and sterilized at 121°C for 15 minutes.

[0070] Preparation of oxygen-free resazurin-free PBS: 0.27 g of potassium dihydrogen phosphate, 1.42 g of disodium hydrogen phosphate, 8 g of sodium chloride, and 0.2 g of potassium chloride were dissolved in 1 L of distilled water, heated to a boil, and cooled to room temperature. 0.55 g of cysteine ​​hydrochloride was added to the cooled solution, stirred to dissolve, and the pH was adjusted to 6.5. Using a volumetric dispenser, N2 was passed through, and the solution was heated to a boil and maintained at a gentle boil for 30 minutes. After cooling, the solution was dispensed into 400 mL bottles and sterilized at high temperature and moist heat for 30 minutes at 121°C.

[0071] MRS solid medium, GAM solid medium, TSB (tryptone soybean broth, Qingdao Haibo Biotechnology Co., Ltd., HB4114), TSA (tryptone soybean agar, Qingdao Haibo Biotechnology Co., Ltd., HB4138), and Brucella broth (Qingdao Haibo Biotechnology Co., Ltd., HB0241) media were prepared by weighing and dissolving according to the manufacturer's instructions, followed by high-temperature moist heat sterilization at 121°C for 30 minutes.

[0072] Preparation of Parabacteroides distasonis Pdist-1 powder production medium: 6 g of anhydrous glucose, 15 g of soybean peptone, 10 g of yeast extract powder, 10 g of yeast peptone, 2 g of potassium dihydrogen phosphate, 2 g of disodium hydrogen phosphate, 0.2 g of magnesium sulfate, 0.01 g of manganese sulfate, 0.2 g of calcium chloride, 1 mL of Tween 80, and 0.5 g of cysteine ​​hydrochloride monohydrate were dissolved in 1 L of distilled water. The mixture was deoxygenated by N2 substitution and dispensed. The mixture was sterilized at 121°C for 15 min.

[0073] Limosil Lactobacillus fermentum Lferm-1 powder production medium: 30 g of anhydrous glucose, 15 g of soybean peptone, 10 g of yeast extract powder, 5 g of sodium acetate, 2 g of potassium dihydrogen phosphate, 2 g of disodium hydrogen phosphate, 0.1 g of magnesium sulfate, 0.045 g of manganese sulfate, 1 mL of Tween 80, and 0.5 g of cysteine ​​hydrochloride monohydrate were dissolved in 1 L of purified water. The mixture was deoxygenated by N2 substitution and dispensed. The mixture was then sterilized at 121°C for 15 minutes using moist heat.

[0074] Preparation of Lactobacillus salivarius Lsali-1 powder production medium: 24 g of anhydrous glucose, 20 g of soybean peptone, 10 g of yeast extract powder, 10 g of peptone, 5 g of sodium acetate, 2 g of potassium dihydrogen phosphate, 2 g of disodium hydrogen phosphate, 0.1 g of magnesium sulfate, 0.045 g of manganese sulfate, 1 mL of Tween 80, and 0.5 g of cysteine ​​hydrochloride monohydrate were dissolved in 1 L of purified water. The mixture was deoxygenated by nitrogen substitution and dispensed. The mixture was then sterilized at 121°C for 15 minutes using moist heat.

[0075] Enterococcus avium Eaviu-1 powder production medium: 30 g of anhydrous glucose, 15 g of soybean peptone, 10 g of yeast powder, 5 g of sodium acetate, 2 g of dipotassium hydrogen phosphate, 0.1 g of magnesium sulfate, 0.045 g of manganese sulfate, 1 mL of Tween 80, and 0.5 g of cysteine ​​hydrochloride monohydrate were dissolved in 1 L of purified water. The mixture was deoxygenated by N2 substitution and dispensed. The mixture was sterilized at high temperature and humidity for 15 minutes at 121°C.

[0076] Bifidobacterium bifidum Bbifi-1 powder production medium: 20 g of anhydrous glucose, 40 g of soy peptone, 5 g of N-acetylglucosamine, 2 g of potassium dihydrogen phosphate, 2 g of disodium hydrogen phosphate, 0.1 g of magnesium sulfate, 0.045 g of manganese sulfate, 1 mL of Tween 80, and 0.5 g of cysteine ​​hydrochloride monohydrate were dissolved in 1 L of purified water. The mixture was deoxygenated by nitrogen substitution and dispensed. The mixture was then sterilized at 121°C for 15 minutes using moist heat.

[0077] Preparation of 0.1% Tween 80-PBS dilution: 3.58 g of disodium hydrogen phosphate dodecahydrate, 0.27 g of potassium dihydrogen phosphate, 8 g of sodium chloride, and 1 mL of Tween 80 were added to 1 L of boiling water and dissolved by stirring with a glass rod. 0.5 g of cysteine ​​hydrochloride monohydrate was added to the boiling solution. The Hungate apparatus was opened, and the solution was boiled again under N2 protection. After bubbling with N2 for 20 minutes, the solution was dispensed into anaerobic bottles that had been deoxygenated by passing N2 through. The bottles were stoppered, labeled, and sterilized at 121°C for 15 minutes.

[0078] Example 1: Isolation and identification of strains Fresh fecal samples were collected from several healthy human volunteers, and each fecal sample was processed independently. An appropriate amount of oxygen-free PBS was added to the fecal sample to obtain a mixture, which was then shaken to obtain a suspension. The suspension was filtered through gauze under N2 protection to obtain a filtrate. The filtrate was centrifuged at 10,000 rpm for 20 minutes, after which the supernatant was discarded and the precipitate was retained. An appropriate amount of oxygen-free PBS was added to the precipitate, and the bacterial cells were resuspended to obtain a suspension. An equal volume of 50% (v / v) oxygen-free glycerin aqueous solution was added to the suspension and thoroughly mixed to obtain a bacterial mixture sample. The sample was dispensed into sample tubes, which were then placed in a bag, vacuumed, and stored in a refrigerator at -80°C. Each frozen sample tube was thawed independently. 0.5 mL of the thawed sample was resuspended in 4.5 mL of oxygen-free PBS and shaken to obtain a bacterial suspension. Under anaerobic conditions, 0.5 mL of the bacterial suspension and 4.5 mL of anaerobic PBS were mixed uniformly by shaking and diluted. -6 The bacterial suspension was diluted tenfold to a dilution of 100%. The appropriate diluted bacterial suspension was homogenously mixed with YCFA liquid medium and then dispensed into a 384-well plate and incubated anaerobically at 37°C for one week. The bacterial suspension from the well where the bacteria had grown was inoculated into YCFA medium and incubated for 48 hours, after which the suspension was divided into two aliquots. One aliquot was detected by MALDI-TOF-MS, and the isolated strains were preliminarily classified into species. After confirming that the bacterial suspension contained only bacteria of a single genetic background (monoclonal strain), based on the mass spectrometry results, the other aliquot was inoculated into YCFA medium and incubated. One aliquot was subjected to 16S rDNA gene amplification and sequencing, and the other aliquot was added to 50% (v / v) glycerol solution at a 1:1 (volume ratio), mixed homogenously, and deposited.

[0079] The species of the isolated strains were further identified by aligning the 16S rDNA gene sequences obtained through sequencing with the NCBI Nucleotide database. Five strains with further confirmed species were selected for further experiments. Strain 1 had the highest sequence similarity (>99%) to one strain of Parabacteroides distasonis, and was therefore designated Parabacteroides distasonis Pdist-1 (abbreviated as Pdist-1). Strain 2 had the highest sequence similarity (100%) to one strain of Limosilactobacillus fermentum, and was therefore designated Limosilactobacillus fermentum Lferm-1 (abbreviated as Lferm-1). Strain 3 had the highest sequence similarity (100.00%) to one strain of Lactobacillus salivarius, and was therefore designated Lactobacillus salivarius Lsali-1 (abbreviated as Lsali-1). Strain 4 had the highest sequence similarity (100.00%) to one strain of Enterococcus avium, and was therefore designated Enterococcus avium Eaviu-1 (abbreviated as Eaviu-1). Strain 5 had the highest sequence similarity (99.86%) to one strain of Bifidobacterium bifidum, and was therefore designated Bifidobacterium bifidum Bbifi-1 (abbreviated as Bbifi-1).

[0080] Parabacteroides distasonis Pdist-1, Limocillous Lactobacillus fermentum Lferm-1, and Enterococcus avium Eaviu-1 were inoculated into BF839 medium and cultured to observe their colony morphology. Lactobacillus salivarius Lsali-1 and Bifidobacterium bifidum Bbifi-1 were inoculated into triple-mix solid medium and cultured to observe their colony morphology. Frontal photographs of the colony morphology of the five bacterial strains are shown in Figure 1. Of these, A is a frontal photograph of the colony morphology of Parabacteroides distasonis Pdist-1, B is a frontal photograph of the colony morphology of Limocillous Lactobacillus fermentum Lferm-1, C is a frontal photograph of the colony morphology of Lactobacillus salivarius Lsali-1, D is a frontal photograph of the colony morphology of Enterococcus avium Eaviu-1, and E is a frontal photograph of the colony morphology of Bifidobacterium bifidum Bbifi-1. As can be seen from this, all five bacterial strains exhibited white, opaque, circular colonies with a protruding center and a smooth, moist surface.

[0081] Example 2: Whole genome analysis of bacterial strains The five strains obtained in Example 1 were each inoculated into a triple-mix liquid medium and cultured until the bacteria reached the late logarithmic growth stage. Total genomic DNA was extracted from each strain and whole-genome sequencing was performed using the Illumina high-throughput sequencing platform NovaSeq 6000. After genome sequence assembly and annotation, the protein sequences were entered into the Virulence Factor Databases (VFDB) for virulence factor analysis. The results showed that none of the five strains contained virulence factors.

[0082] The novelty of the five strains was analyzed using the average nucleotide identity (ANI) method. A whole-genome search was performed in GenBank, and the closest strains were compared using fastANI (v1.33). The two closest strains to the whole genome of Parabacteroides distasonis Pdist-1 are GCA_003462945.1 (ANI = 98.26%) and GCA_003459965.1 (ANI = 98.20%), respectively. The two closest strains to the whole genome of Limocilactobacillus fermentum Lferm-1 are GCA_003465085.1 (ANI = 99.32%) and GCA_024385625.1 (ANI = 99.29%), respectively. The two closest matched strains for the complete genome of Lactobacillus salivarius Lsali-1 are GCA_009863605.1 (ANI = 99.94%) and GCA_009866185.1 (ANI = 99.87%), respectively. The two closest matched strains for the complete genome of Enterococcus avium Eaviu-1 are GCA_018917545.1 (ANI = 98.75%) and GCA_018373135.1 (ANI = 98.62%), respectively. The two closest matched strains for the complete genome of Bifidobacterium bifidum Bbifi-1 are GCA_003466395.1 (ANI = 99.01%) and GCA_003437945.1 (ANI = 99.00%), respectively. As can be seen from this, the species classification performed in Example 1 is correct.

[0083] The whole genome sequences of the five strains were annotated using emapper-2.1.9. The genome of Parabacteroides distasonis Pdist-1 contained genes encoding one isomeric bile acid production protein, two enzymes related to acetate production, three enzymes related to propionate production, one enzyme related to catalase (CAT) production, and one enzyme related to superoxide dismutase (SOD) production. The genome of Lactobacillus fermentum Lferm-1 contained one gene encoding an enzyme related to acetate production. The genome of Enterococcus salivarius Lsali-1 has genes encoding two acetate-related enzymes and one propionate-related enzyme, the genome of Enterococcus avium Eaviu-1 has two genes encoding acetate-related enzymes, one propionate-related enzyme, one gene encoding the SagA protein, and one gene encoding an SOD-related enzyme, and the genome of Bifidobacterium bifidum Bbifi-1 has genes encoding one acetate-related enzyme and one propionate-related enzyme.

[0084] The strains Pdist-1, Eaviu-1, Lferm-1, Bbifi-1, and Lsali-1 isolated and cultured in this invention have been submitted for deposit at the China Typical Culture Collection (CCTCC), located at Wuhan University, Wuhan, China. The names, taxonomic designations, dates of deposit, dates of viability confirmation, and microbial accession numbers of the cultures are listed in Table 1.

[0085] JPEG2026505892000003.jpg96170

[0086] Example 3: Hemolysis test Parabacteroides distasonis Pdist-1 was inoculated into a triple-mix liquid medium and cultured anaerobically at 37°C for 12 hours to obtain a bacterial solution containing the activated strain. Enterococcus faecalis (β-hemolytic, CICC23658, purchased from the China National Center for Industrial Microorganisms) was inoculated into a triple-mix liquid medium and cultured anaerobically at 37°C for 12 hours to obtain a bacterial solution containing the activated strain (positive control). The triple-mix liquid medium served as a negative control. 2.5 μL of each of the two bacterial solutions containing the activated strain and the negative control was inoculated onto Columbia blood plates (Shanghai Kemarjia Microbiology Technology Co., Ltd.), and three parallel experiments were performed for each sample. After 48 hours of anaerobically culture at 37°C, the Columbia blood plates were observed. Results: Enterococcus faecalis formed a clear, bordered, and completely transparent hemolytic ring around the colony, demonstrating beta-hemolysis. Parabacteroides distasonis Pdist-1 showed no change in the medium around the colony, demonstrating gamma-hemolysis, i.e., no hemolysis. The negative control showed no hemolytic ring. These results indicated that Parabacteroides distasonis Pdist-1 did not hemolyze.

[0087] Limocillinic Lactobacillus fermentum Lferm-1, Lactobacillus salivarius Lsali-1, Enterococcus avium Eaviu-1, and Bifidobacterium bifidum Bbifi-1 were inoculated into triple-mix liquid medium and cultured anaerobically at 37°C until late logarithmic growth. A bacterial suspension containing activated strains was obtained. Detection was performed using the liquid contact method with 2 mL of a 2% (v / v) fresh rabbit erythrocyte suspension in PBS (Beijing Borx Technology Co., Ltd.) and a bacterial suspension resuspended in 2 mL of sterile saline. An equal volume of sterile water served as the positive control, and an equal volume of sterile saline served as the negative control. Three parallel samples were set up for each group, and each sample was observed after 24 h of incubation. The results showed that the positive control caused hemolysis, but the negative controls, rimosyl Lactobacillus fermentum Lferm-1, Lactobacillus salivarius Lsali-1, Enterococcus avium Eaviu-1, and Bifidobacterium bifidum Bbifi-1, did not cause hemolysis.

[0088] Example 4: Antibiotic susceptibility testing According to the requirements for anaerobic antibiotic susceptibility testing in the "Technical Requirements for Antimicrobial Susceptibility Testing" of the "Health Industry Standards of the People's Republic of China" (standard number: WS / T 639-2018), the susceptibility of the strains to antibiotics was determined by the broth dilution method, and the MIC values ​​were recorded.

[0089] Final inoculum bacterial concentration is 5 x 10 5 The five bacterial suspensions were then aliquoted in 0.1 mL portions and placed in a 96-well plate containing an equal volume of the antibiotics, with the resulting CFU / mL and drug concentrations being 256 μg / mL, 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, and 0.125 μg / mL, respectively. Incubation: The 96-well plate was placed in a 37°C incubator and incubated anaerobically for 46 to 48 hours. The results are shown in Table 2 below.

[0090] JPEG2026505892000004.jpg104170Note: "-" indicates untested.

[0091] Example 5: Antioxidant experiments The five activated strains obtained in Example 1 were each inoculated into anoxic BF839 medium. LGG (Lactobacillus rhamnosus GG, CICC6141, purchased from the China Industrial Microorganisms Species Depositary) was used as a positive control. The following procedures were carried out in parallel for each of the six strains. Each strain was anaerobically cultured in anoxic BF839 medium at 37°C for 24 hours to obtain a culture solution.

[0092] JPEG2026505892000005.jpg94170

[0093] Example 6: Bacteriostatic ability test of strains against pathogenic bacteria Common enteropathogenic bacteria that can cause diarrhea, as shown in Table 3, were selected, and the bacteriostatic ability of the five strains obtained in Example 1 was detected.

[0094] JPEG2026505892000006.jpg80170

[0095] JPEG2026505892000007.jpg144170

[0096] Example 7: In vitro barrier repair test Caco-2 cells (purchased from Shangcheng Beina Chuanglian Biotechnology Co., Ltd., BNCC number: 350769) were seeded into a Transwell (permeable cell culture chamber). Caco-2 anchorage-dependent cells were digested using trypsin cell digestion solution preheated to 37°C. 1.1 × 10 cells were cultured in DMEM medium containing 10 (v / v)% FBS and 1 (w / v)% PS (DMEM medium purchased from Gibco, catalog number C11995500BT; FBS purchased from Gibco, catalog number 16000-044; PS is a penicillin and streptomycin mixture purchased from Gibco, catalog number 15140-122). 5 Caco-2 cells were seeded into a 24-well Transwell plate at a seeding density of 10 cells / well and cultured statically at 37°C in 5% CO2 for 21 days. Bacterial culture: 200 μL each of Lactobacillus salivarius Lsali-1 and Enterococcus avium Eaviu-1 cultures were taken from the bacterial deposit tubes and placed in 5 mL of double mixed medium (related reagents were deoxygenated beforehand) and cultured anaerobically for 24 hours in an electric thermostatic incubator at 37°C. Subculture was performed once and the culture was cultured anaerobically for 8 hours. 1 mL of the culture was taken and centrifuged at 12,000 rpm / min for 3 minutes. The strains were cultured at 10°C in DMEM medium containing 10% (v / v) FBS. 7The samples were diluted to CFU / mL and prepared for use. The same concentration of Lactobacillus rhamnosus GG (LGG, CICC 6141, China National Center for Industrial Microorganisms Depositary) served as a positive control. Effects of Lactobacillus salivarius Lsali-1 and Enterococcus avium Eaviu-1 on intestinal epithelial barrier function in a Caco-2 cell model: Four groups were established for each bacterium: a normal control group, a model group, a positive control group, and a bacterial group (Lactobacillus salivarius Lsali-1 group and Enterococcus avium Eaviu-1 group). A barrier damage model was established using the inflammatory factors IFN-γ (Pepro Tech, AF-300-02) and TNF-α (Pepro Tech, 300-01A) in the model group. After 21 days of culture, the Caco-2 cells differentiated to form a dense monolayer. The old medium in the lower chamber was then aspirated, and 800 μL of DMEM medium was added to the lower chamber of the normal control group. 800 μL of IFN-γ solution at a concentration of 10 ng / mL was added to the lower chambers of the model group, positive control group, and bacterial group. After 22 hours of static incubation in a 5% CO2 incubator at 37°C, the solution in the upper and lower chambers was removed. The normal control group received 200 μL of DMEM medium in the upper chamber and 800 μL of DMEM medium in the lower chamber. The model group received 200 μL of DMEM medium in the upper chamber. The positive control group received 200 μL of positive bacterial solution in the upper chamber. The bacterial group received 200 μL each of Lactobacillus salivarius Lsali-1 solution and Enterococcus avium Eaviu-1 solution in the upper chamber. The model, positive control, and bacterial groups received 800 μL of 50 ng / mL TNF-α solution in the lower chamber. After 5 hours of static incubation in a 5% CO2 incubator at 37°C, the transepithelial electrical resistance (TEER) values ​​of each group were measured.

[0097] The results are shown in Figure 4. Compared to the model group, the positive control group LGG significantly increased TEER values, demonstrating a clear repair effect on cell barrier damage. Similarly, compared to the model group, Lactobacillus salivarius Lsali-1 (A) and Enterococcus avium Eaviu-1 (B) also significantly increased TEER values, with effects similar to or equivalent to those of LGG. These results demonstrate that Lactobacillus salivarius Lsali-1 and Enterococcus avium Eaviu-1 can effectively alleviate barrier dysfunction caused by inflammatory factors (e.g., IFN-γ, TNF-α).

[0098] Example 8: In vitro cell inflammation inhibition test Polarization of THP-1 cells: RPMI-1640 medium (Thermo Fisher, C11875500BT) containing 10 (v / v)% FBS and PMA (phorbol 12-myristate 13-acetate, Sigma-Aldrich Company, P1585) at a final concentration of 100 ng / mL was used to culture 1 × 10 5 THP-1 cells were seeded into a 96-well plate at a density of 100 cells / well. The 96-well plate was placed in a 5% CO2 incubator and cultured at 37°C for 24 hours to allow for polarization into mature macrophages. Bacterial culture: 200 μL of Parabacteroides distasonis (Pdist-1), Lactobacillus fermentum (Lferm-1), Lactobacillus salivarius (Lsali-1), Enterococcus avium (Eaviu-1), and Bifidobacterium bifidum (Bbifi-1) cultures were inoculated into 5 mL of double-stranded mixed medium and cultured anaerobically at 37°C for 24 hours. After one subculture, the cultures were cultured anaerobically for 8 hours. One mL of the cultures was then taken and centrifuged at 5,000 rpm / min for 15 minutes. The above strains were cultured in RPMI-1640 medium containing 10 (v / v)% FBS at 5 × 10 7 CFU / mL, 2 × 10 6 CFU / mL, 5 × 10 7 CFU / mL, 5 × 10 7CFU / mL, 5 × 10 7 The cells were diluted to CFU / mL and prepared for use. Effect on TNF-α and IL-6 expression in THP-1 cells: After culturing mature THP-1 cells, the normal control group (untreated with bacteria or drugs) was replaced with RPMI-1640 medium containing 10% (v / v) FBS. The model group, positive control group (treated with dexamethasone), and test group (treated with each bacterial strain) were replaced with RPMI-1640 medium containing 10% (v / v) FBS, 100 ng / mL LPS (Sigma-Aldrich Company, L3024), and 20 ng / mL IFN-γ (PeproTech, AF-300-02), respectively, to model inflammatory macrophages. Each group was placed in a 5% CO2 incubator and cultured at 37°C for 24 hours. The medium was aspirated, and 100 μL of RPMI-1640 medium containing 10 (v / v)% FBS was added to the normal control group and model group, respectively. 100 μL of RPMI-1640 medium containing 10 (v / v)% FBS and dexamethasone (purchased from Sigma-Aldrich Company, D4902-25) with a final concentration of 25 μg / mL was added to the positive control group. 100 μL of each previously prepared bacterial solution was added to the test groups. The experimental results are shown in Figure 5. The model control group had significantly higher IL-6 and TNF-α expression than the normal control group (P<0.01). The positive control dexamethasone group significantly inhibited the expression of pro-inflammatory factors IL-6 and TNF-α in THP-1 cells (P<0.01). Compared with the model group, the expression of Lactobacillus fermentum Lferm-1, Lactobacillus salivarius Lsali-1, and Enterococcus avium Ea was significantly higher. viu-1 could significantly reduce the expression of the pro-inflammatory factor IL-6, and Lactobacillus fermentum Lferm-1, Lactobacillus salivarius Lsali-1, Enterococcus avium Eaviu-1, and Bifidobacterium bifidum Bbifi-1 could significantly reduce the expression of the pro-inflammatory factor TNF-α (P<0.05), indicating that these strains have anti-inflammatory effects in vitro.

[0099] Example 9: Test of adhesion ability to Caco-2 cells Culture of bacterial strains: 200 μL of Bifidobacterium bifidum Bbifi-1, Enterococcus avium Eaviu-1, Lactobacillus salivarius Lsali-1, Lactobacillus fermentum Lferm-1, and Parabacteroides distasonis Pdist-1 were inoculated into 5 mL of double-stranded mixed medium and cultured anaerobically at 37°C until the late logarithmic phase. The cultured bacterial solution was washed twice by centrifugation with sterile PBS (Wuhan Dr. De Bioengineering Co., Ltd., PYG0021). The strains were then cultured at 5 × 10 in DMEM (Thermo Fisher Scientific (China) Co., Ltd., C11995500BT) medium containing 10% (v / v) FBS (Thermo Fisher Scientific (China) Co., Ltd., SH30084.03). 8The bacteria were diluted to CFU / mL and prepared for use. The same concentration of Lactobacillus rhamnosus GG (LGG, CICC 6141, China National Center for Industrial Microorganisms Depositary) served as a positive control. 100 μL of the diluted bacterial suspension was added to each 96-well cell culture plate containing Caco-2 cells. After sample addition, the 96-well cell culture plate was placed in a horizontal centrifuge and centrifuged at 1000 g for 1 min. The wells corresponding to each bacterial suspension were divided into two subgroups. The two subgroups were incubated for 30 min and 2 h, respectively, and then washed twice with sterile PBS to remove any unadhered bacteria. After washing, 50 μL of trypsin cell digestion solution (Lanjieke Technology Co., Ltd., BL501A) was added to each well and placed in a 37°C incubator to digest the cells. After digestion and globularization of the Caco-2 cells, 150 μL of DMEM containing 10% (v / v) FBS was added to each well and pipetted repeatedly for approximately 1 minute. After confirming the isolation of the cells and bacterial strains by microscopic examination, 20 μL of the mixture was aspirated and diluted 10-fold in 0.1% Tween 80-PBS in a 96-well plate. The appropriate dilution gradient was selected, and the dissolved triple-mixed solid medium was poured into the plate. The plate was then incubated at 37°C for 48 hours and then counted. The results are shown in Figure 6. Figure 6A shows the adhesive effect of each bacterial strain when the incubation time was 30 minutes. Enterococcus avium Eaviu-1 had the same adhesive ability as the positive control group LGG. Figure 6B shows the adhesive effect of each bacterial strain when the incubation time was 2 hours. Bifidobacterium bifidum Bbifi-1 and limosiliac Lactobacillus fermentum Lferm-1 had the same adhesive ability as the positive control group LGG. Lactobacillus salivarius Lsali-1 and Enterococcus avium Eaviu-1 both had superior adhesive abilities than the positive control group LGG. Parabacteroides distasonis Pdist-1 had a certain adhesive ability, indicating that the five strains of bacteria have the potential to colonize the intestinal tract.

[0100] Example 10: Therapeutic effect on a mouse model of 5-fluorouracil (5-FU)-induced diarrhea Preparation of lyoprotectant for Parabacteroides distasonis Pdist-1: Solution A: 6g sucrose, 6g trehalose, 2g xylitol, 2g sorbitol, 44g purified water. Solution B: 5g sodium glutamate, 15g purified water, sterilized at 115°C for 20 minutes. Solution C: 4g sodium vitamin C, 16g purified water, filtered and sterilized for use. Preparation of lyoprotectant for Lactobacillus fermentum Lferm-1, Lactobacillus salivarius Lsali-1, and Enterococcus avium Eaviu-1: Solution A: 8g sucrose, 8g trehalose, 44g purified water, sterilized at 115°C for 30 minutes. Solution B: 2g sodium glutamate, 2g arginine hydrochloride, 16g purified water, sterilized at 115°C for 30 minutes. Solution C: 4g sodium vitamin C, 16g purified water. Preparation of lyophilized protective agent for Bifidobacterium bifidum Bbifi-1: Solution A: 6g sucrose, 6g trehalose, 2g xylitol, 2g sorbitol, 44g purified water, sterilized at 115°C for 30 minutes. Solution B: 2g arginine hydrochloride, 2g monosodium glutamate, 16g purified water, sterilized at 115°C for 30 minutes. Solution C: 4g sodium vitamin C, 16g purified water. Filter and sterilize for use.

[0101] The cryoprotectants for each strain were mixed at the volume ratio of solution A:solution B:solution C = 6:2:2 before use.

[0102] Production of bacterial powder: The five bacterial strains obtained in Example 1 were inoculated into the corresponding bacterial powder production medium and anaerobically cultured at 37°C and 90 rpm for 16 to 24 hours to obtain a primary seed solution. This was then transferred to a bacterial powder production medium and anaerobically cultured at 37°C and 90 rpm for 10 to 15 hours to obtain a secondary seed solution. The secondary seed solution was pumped using a peristaltic pump into a fermenter containing a bacterial powder production medium for fermentation and culture. After fermentation was stopped, the bacterial cells were collected by centrifugation. A lyoprotectant was added to the bacterial sludge at a weight ratio of 1:1 to 1:2, and the sludge was uniformly mixed to emulsify. The sludge was freeze-dried and crushed to obtain a bacterial powder. Before administration to animals, 1 x 10 9A bacterial suspension containing live CFU of bacteria was prepared in 0.2 mL of saline. Test animals were SPF-grade male Balb / c mice weighing 18-22 g, purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., and housed in an SPF-grade animal enclosure. Mice were randomly assigned to groups of five based on their initial body weight. Four groups were established for Parabacteroides distasonis Pdist-1, Limocillactobacillus fermentum Lferm-1, Enterococcus avium Eaviu-1, and Bifidobacterium bifidum Bbifi-1: a normal control group, a model control group, a positive control loperamide group, and a group of strains to be tested. Four groups were established for Lactobacillus salivarius Lsali-1: a normal control group, a model control group, a homologous control strain Lsali-3 group (Lsali-3 is another Lactobacillus salivarius strain isolated using the same method as in Example 1), and a strain Lsali-1 group to be tested. Experimental design: A mouse chemotherapy-associated diarrhea model was induced using 5-FU solution (5-fluorouracil, purchased from Tianjin Jinyao Pharmaceutical Co., Ltd., 10 mL per bottle, 0.25 g / 10 mL). The normal control group received an intraperitoneal injection of saline; all other groups received a single intraperitoneal injection of 5-FU at a modeling dose of 350 mg / kg body weight. The administration method for all groups was intragastric administration. The normal control group and model control group received intragastric administration of cryoprotectant. The positive control group received intragastric administration of loperamide (purchased from Xi'an Janssen Pharmaceutical Co., Ltd.) at 20 mg / kg body weight. The bacterial strain group awaiting measurement received 1 × 10 9 A suspension of the strains awaiting measurement was administered intragastrically at a dose of CFU / animal. The entire experiment lasted 9 days, designated D1-D9. Modeling treatment was performed on D3. The normal control group, model control group, and strain awaiting measurement group all received continuous intragastric administration of the test substance on D1-D5. The positive control group received continuous intragastric administration of loperamide on D1-D9. After administration on D5, the animals were observed for 4 consecutive days. The specific experimental groupings and administration regimens are shown in Table 4 below.

[0103] JPEG2026505892000009.jpg63170Note: 5-FU: 5-fluorouracil, CFU: colony forming unit, d: day

[0104] Diarrhea observation and scoring: Mice were placed in mouse cages lined with clean filter paper, one per cage. Hard feces were scored as 0 (normal); mild feces were scored as 1 (slightly wet or loose); moderate feces were scored as 2 (wet, unformed feces, and perianal soiling); and severe feces were scored as 3 (watery feces and severe perianal soiling). The feces of the mice were observed and scored daily during the experimental period. The total diarrhea score was the sum of the daily diarrhea scores.

[0105] Weight detection and weight change rate: During the experiment, the mice were weighed every day and the weight change rate was calculated as follows: weight change rate = (detected weight - initial weight) / initial weight x 100%.

[0106] After the experiment, all mice were dissected, and the entire cecum and colorectum were excised along with the anus. The length of the mouse colorectum was measured with a straightedge, with the end of the cecum as the zero point and the end of the rectum as the end point.

[0107] The results of the diarrhea experiment are shown in Figures 7 to 11. As can be seen, compared to the model control group, each of the five strains of bacteria had a clear improving effect on 5-FU-induced diarrhea, and the total diarrhea score was significantly reduced.

[0108] To compare the efficacy of Bifidobacterium bifidum Bbifi-1 and the standard strain DSM20456 (purchased from the German Collection of Microorganisms and Cell Cultures), a CID model was constructed as described above. As previously shown, the model may exhibit stable diarrhea after 4 days of CID modeling, i.e., on day 7. Therefore, the entire experimental period in this study was 7 days, designated days 1 to 7. On day 3, the normal control group received an intraperitoneal injection of saline. The other groups received a single intraperitoneal injection of 5-FU at a modeling dose of 350 mg / kg body weight. The Bbifi-1 and DSM20456 groups received daily injections of 1 × 10 9 CFU was administered intragastrically. During the experimental period, the feces of the mice were observed and scored daily, and the scoring criteria were the same as before. The specific regimen is shown in Table 5 below.

[0109] JPEG2026505892000010.jpg79170

[0110] The test results are shown in Figure 12. Compared to the model control group, Bbifi-1 has a more effective amelioration effect on 5-FU-induced diarrhea than the standard strain DSM20456.

[0111] Impact on colon injury After the animal experiments, the middle colon of each mouse was collected and fixed in 4% paraformaldehyde for 24 hours. The fixed colon tissue was dehydrated, cleared, infiltrated with paraffin, and embedded. The embedded colon tissue paraffin blocks were sectioned to a thickness of 5 μm, then spread and baked. The dried sections were then stained with conventional HE staining. Pathological changes were observed under a light microscope and scored according to Table 6 below. A total pathological score (the sum of the scores for each indicator) was calculated.

[0112] JPEG2026505892000011.jpg131170

[0113] As can be seen from Figures 1A and 2A in Figure 13, the normal control group had an intact colonic structure, with the mucosal layer, submucosa, muscularis layer, and serosal layer clearly visible. The model control group had necrosis and lysis of intestinal glands in the lamina propria, a decrease in goblet cells, lymphocyte infiltration, loosened connective tissue in the submucosa, and dilated blood vessels. The loperamide group had a relatively intact colonic structure, no obvious necrosis of mucosal epithelial cells, a significant increase in the number of goblet cells, and only a small amount of lymphocyte infiltration in the lamina propria. The Pdist-1 group had a relatively intact colonic mucosal structure, no obvious necrosis of mucosal epithelial cells, and a significant increase in the number of goblet cells compared to the model control group. The Lferm-1 group had a small amount of necrosis of mucosal epithelial cells with a small amount of lymphocyte infiltration in the colonic tissue, dilated intestinal glands, a slight decrease in the number of goblet cells, and mild edema with a small amount of lymphocyte infiltration in the submucosa. As can be seen from Figures 1B and 2B, obvious damage appeared in the mouse colon after 5-FU induction, and the total pathological score of the model control group was significantly higher than that of the normal control group (P<0.01). Parabacteroides distasonis Pdist-1 significantly reduced the total pathological score of the colon (P<0.05) after administration, improving colonic damage. Limocillous Lactobacillus fermentum Lferm-1 also significantly reduced the total pathological score of the colon (P<0.05), improving the degree of colonic damage.

[0114] Improvement of the relative mRNA transcription levels of IL-1β, TNF-α, and AQP8 in the mouse colon After the animal experiments, the middle colons of the mice were collected and stored in a refrigerator at -80°C. Total RNA was extracted from the colon tissues of each group of mice according to the instructions (ThermoFisher Scientific, catalog number 15596026) and reverse-transcribed into cDNA. The cDNA was then stored at -20°C for further use. qRT-PCR was used to detect the relative transcription levels of the pro-inflammatory factors IL-1β, TNF-α, and aquaporin 8 (AQP8) gene mRNA in the colons of each group of mice (primer sequences are listed in Table 7). The reaction program was 95°C for 3 min, 95°C for 20 s, 60°C for 45 s, and 72°C for 20 s, for a total of 39 cycles. -ΔΔCTThe data were analyzed using the statistical method, and significance analysis was performed on the data using the statistical software SPSS 24.0.

[0115] JPEG2026505892000012.jpg103170

[0116] The results are shown in Figure 14. As can be seen, compared with the normal control group, the model control group had significantly increased relative mRNA transcription levels of IL-1β and TNF-α, and significantly decreased relative mRNA transcription levels of AQP8. Compared with the model control group, administration of Pdist-1 significantly reduced the relative mRNA transcription levels of IL-1β and TNF-α (A, B) and significantly increased the relative mRNA transcription level of AQP8 (C), and administration of Lferm-1 significantly reduced the relative mRNA transcription level of TNF-α (D).

[0117] Example 11: Therapeutic effect of Bifidobacterium bifidum Bbifi-1 on radiation enteritis model mice The manufacturing methods for the freeze-dried protective agent and bacterial powder were the same as in Example 10. Test animals: 20 SPF-grade male C57BL / 6J mice weighing 20-25g were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. and housed in an SPF-grade animal breeding room. Test design: A mouse model of radiation enteritis was induced by abdominal X-ray irradiation. Mice were randomly divided into four groups, each with five mice, according to their initial body weight: normal control group, model control group, positive control group, and Bifidobacterium bifidum Bbifi-1 group. Except for the normal control group, which received no irradiation treatment, all other groups received a single abdominal X-ray irradiation modeling treatment with a modeling dose of 11.5Gy. All groups were administered intragastrically. The normal control group and model control group received intragastrically administered saline, and the positive control group received 1x10 9 CFU of Lactobacillus rhamnosus GG (LGG, purchased from Shaanxi Zelang Biotechnology Co., Ltd.) was administered intragastrically, and 1 × 10 Bifidobacterium bifidum Bbifi-1 group was administered intragastrically. 9CFU of Bifidobacterium bifidum Bbifi-1 was administered intragastrically. The entire experimental period was 13 days, designated D-2 to D-10. The mice were administered intragastrically for 12 consecutive days from D-2 to D-9, and then underwent a single abdominal X-ray irradiation on D0 (day 3).

[0118] Diarrhea observation and scoring: Mice were placed in mouse cages lined with clean filter paper, one per cage. Hard feces were scored as 0 (normal); mild feces were scored as 1 (slightly wet or loose); moderate feces were scored as 2 (wet, unformed feces, and perianal soiling); and severe feces were scored as 3 (watery feces and severe perianal soiling). The feces of the mice were observed and scored daily during the experimental period. The total diarrhea score was the sum of the daily diarrhea scores. On day 10 (day 13), the experimental animals were anesthetized with pentobarbital sodium and euthanized, followed by gross dissection and anatomical observation. The specific experimental groupings and dosing regimens are shown in Table 8 below.

[0119] JPEG2026505892000013.jpg90170Note: CFU: colony forming unit, d: day

[0120] The test results are shown in Figure 15, where A shows the diarrhea score curves for each group on days 1 to 12, and B shows the total diarrhea score for each group. Compared to the model control group, Bifidobacterium bifidum Bbifi-1 significantly improved diarrhea caused by X-ray irradiation, and the effect was comparable to that of the positive control group LGG (A). Both LGG and Bifidobacterium bifidum Bbifi-1 significantly reduced the total diarrhea score (P<0.05, B). These results clearly show that Bifidobacterium bifidum Bbifi-1 significantly improved X-ray irradiation-induced diarrhea in mice.

[0121] Example 12: Co-culture characteristics test between each strain The five bacterial strains obtained in Example 1 were each activated and cultured until the late logarithmic growth stage. A disposable sterile cotton swab was dipped in a bacterial suspension of one of the strains and drawn three parallel lines on BF839 solid medium. Then, a single line was drawn parallel to the first line with each of the remaining four bacterial strains, perpendicular to the first line. The drawn bacterial suspensions were then dried and cultured anaerobically for 48 hours until traces of the bacterial suspension were evident. The interaction relationships between the five bacterial strains are shown in Figure 16. As can be seen, the absence of breakpoints at the intersections of the strains indicates no growth inhibition between the strains.

[0122] As is clear from common general knowledge, the present invention can be realized in other embodiments without departing from the spirit or essential features thereof. Therefore, the above-disclosed embodiments are merely illustrative and not restrictive in all respects. All modifications within the scope of the present invention or within a range equivalent to the present invention are included in the present invention.

Claims

1. A probiotic composition, wherein the active ingredient in the probiotic composition comprises one or a combination of two or more of Bifidobacterium bifidum having a deposit number of CCTCC NO: M2023349 or a pure culture thereof, Enterococcus avium having a deposit number of CCTCC NO: M2023350 or a pure culture thereof, Lactobacillus salivarius having a deposit number of CCTCC NO: M2023348 or a pure culture thereof, Limosilactobacillus fermentum having a deposit number of CCTCC NO: M2023352 or a pure culture thereof, and Parabacteroides distasonis having a deposit number of CCTCC NO: M20222033 or a pure culture thereof.

2. 2. The probiotic composition of claim 1, further comprising a lyoprotectant, a food material, a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable excipient.

3. An isolated Parabacteroides distasonis strain or a pure culture thereof, having the microorganism deposit number CCTCC NO: M20222033.

4. An isolated Rimosilactobacillus fermentum strain or a pure culture thereof, having the microorganism deposit number CCTCC NO: M2023352.

5. An isolated Lactobacillus salivarius strain or a pure culture thereof, having the microorganism deposit number CCTCC NO: M2023348.

6. An isolated Enterococcus avium strain or a pure culture thereof, having the microorganism deposit number CCTCC NO: M2023350.

7. An isolated Bifidobacterium bifidum strain or a pure culture thereof, having the microorganism deposit number CCTCC NO: M2023349.

8. 10. A method for preventing, treating or delaying intestinal diseases, comprising administering to a subject a therapeutically effective amount of the probiotic composition of claim 1 or 2 or the strain or a pure culture thereof of any one of claims 3 to 7.

9. 9. The method of claim 8, wherein the subject is a mammal, and more preferably, the subject is a human.

10. 9. The method according to claim 8, wherein the probiotic composition according to claim 1 or 2 or the strain or pure culture thereof according to any one of claims 3 to 7 inhibits enteropathogenic bacteria in the subject, ameliorates colon damage in the subject, inhibits intestinal inflammation in the subject, repairs the intestinal barrier in the subject, or ameliorates diarrheal symptoms in the subject.

11. The method according to claim 8, wherein the intestinal disease is selected from enteropathogenic bacterial infection, diarrhea, and radiation enteritis, preferably the enteropathogenic bacteria is one or more selected from Pseudomonas aeruginosa, Salmonella Paratyphi B, Yersinia enterocolitica, Staphylococcus aureus, Vibrio parahaemolyticus, Clostridium difficile, Shigella, and Escherichia coli, and preferably the diarrhea is diarrhea induced by an antitumor drug, more preferably diarrhea induced by a chemotherapy drug.

12. 12. The method of claim 11, wherein the chemotherapy-induced diarrhea is diarrhea caused by one or more drugs selected from epirubicin, actinomycin D, doxorubicin, daunorubicin, paclitaxel, docetaxel, albumin-paclitaxel, cisplatin, carboplatin, nedaplatin, oxaliplatin, lobaplatin, cyclophosphamide, nitrogen mustard, carmustine, camptothecin, hydroxycamptothecin, topotecan, irinotecan, capecitabine, gemcitabine, methotrexate, 5-fluorouracil, pemetrexed, and cytarabine.

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