WO2024193633A1 - 一种益生菌及其应用 - Google Patents

一种益生菌及其应用 Download PDF

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WO2024193633A1
WO2024193633A1 PCT/CN2024/082932 CN2024082932W WO2024193633A1 WO 2024193633 A1 WO2024193633 A1 WO 2024193633A1 CN 2024082932 W CN2024082932 W CN 2024082932W WO 2024193633 A1 WO2024193633 A1 WO 2024193633A1
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strain
cctcc
diarrhea
intestinal
pure culture
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PCT/CN2024/082932
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English (en)
French (fr)
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李璟欣
张旭朏
傅芳
崔雅倩
金美玉
项晨
肖源灵
陈欣
税君瑞
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四川厌氧生物科技有限责任公司
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Publication of WO2024193633A1 publication Critical patent/WO2024193633A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the technical field of microorganisms, and in particular relates to a probiotic and an application thereof.
  • Intestinal microorganisms are closely related to human health and are vividly called "microbial organs". Intestinal flora is an important part of the body. Under normal conditions, intestinal flora maintains dynamic stability. Intestinal microorganisms play an important role in many life activities such as promoting digestion and absorption of nutrients, maintaining normal physiological functions of the intestine, and regulating the body's immunity. However, intestinal flora is easily affected by many factors such as environmental factors, diet and lifestyle, mental factors, disease status, tumor treatment, antibiotic use, and age. After being affected by the above factors, the human intestinal flora may suffer from intestinal disorders (intestinal flora imbalance).
  • Intestinal disorders can be manifested as the lack of beneficial intestinal bacteria, the overgrowth of intestinal pathogens, impaired intestinal barrier function, and intestinal inflammation. Intestinal disorders can further cause gastrointestinal diseases such as constipation, diarrhea, abdominal pain, and bloating in the host. Severe intestinal disorders can develop into diseases such as inflammatory bowel disease, ulcerative colitis, and irritable bowel syndrome. The aforementioned diseases greatly affect human health and quality of life.
  • Intestinal probiotics can enhance the barrier function of the intestinal mucosa, prevent the adhesion and colonization of pathogens, enhance the immune response of the intestinal system, etc., thereby maintaining intestinal health.
  • Chinese invention patent application CN 102711778A discloses a strain of animal Bifidobacterium lactis subspecies DN-173010, and verified through mouse experiments and histological studies that its fermented milk can alleviate ulcerative colitis.
  • the patent application text with publication number CN107312726A discloses a plant lactobacillus, which can inhibit the growth of harmful bacteria such as Escherichia coli, Salmonella, Streptococcus suis and Staphylococcus aureus in the intestine.
  • Probiotics are also used to prevent or improve the side effects of bacterial flora disorders caused by some drugs such as antibiotics.
  • side effects related to chemotherapy and radiotherapy are very common.
  • Diarrhea caused by chemotherapy drugs also known as chemotherapy-related diarrhea (CID)
  • CID chemotherapy-related diarrhea
  • the occurrence of CID is considered to be multifactorial, but CID The pathogenesis of dysentery is still not very clear.
  • 5-FU 5-fluorouracil
  • proliferating intestinal cells are more sensitive to 5-FdUMP or 5-FUMP produced by the phosphorylation of 5-FU.
  • 5-FdUMP or 5-FUMP can cause damage to the small intestinal mucosa and interfere with the division of intestinal cells, causing necrosis of intestinal wall cells and extensive inflammation of the intestinal wall, resulting in an imbalance in the number of absorptive and secretory cells in the small intestine, leading to diarrhea.
  • chemotherapy drugs can also cause cell DNA damage and mitochondrial dysfunction, leading to ROS production and cell apoptosis.
  • ROS can activate NF- ⁇ B, further upregulate the expression of proinflammatory factors, and then lead to damage to the intestinal epithelium, endothelium and connective tissue.
  • harmful bacteria are very easy to colonize, the intestinal microecology is destroyed, and then pathogenic bacteria infection is caused, which promotes the occurrence and development of diarrhea.
  • CID still lacks a unified and effective treatment in clinical practice.
  • the main purpose of CID treatment is to control symptoms, relieve patients' pain, accelerate mucosal repair and prevent secondary infection.
  • General CID treatment includes the use of antidiarrheal drugs, mucosal protection drugs and antibacterial drugs, as well as high-dose use of loperamide and even somatostatin-like drugs such as octreotide.
  • the above-mentioned drugs have a single mechanism of action and large side effects, and are not suitable for long-term maintenance medication.
  • high-dose use of loperamide may cause the risk of paralytic intestinal obstruction; the use of octreotide may cause side effects such as gallstones, hyperglycemia, and impaired glucose tolerance; antibacterial drugs may further kill beneficial intestinal bacteria, destroy the flora structure, and lead to intestinal microecological disorders.
  • Patent CN113215063B discloses that a Lactobacillus salivarius CPU-1 can alleviate the toxic side effects caused by the chemotherapy drug temozolomide, which focuses on the improvement of mucosal inflammation symptoms by Lactobacillus salivarius CPU-1.
  • Patents such as CN1511945A and CN86103736A study the use of various probiotics including Lactobacillus fermentum in the treatment or prevention of diarrhea.
  • Patent CN113234619B discloses a strain of Bifidobacterium bifidum that can alleviate acute intestinal damage.
  • An inactivated Bacteroides fragilis ZY-312 was reported to be approved by the FDA for CID clinical trials.
  • the active ingredients in the probiotic composition include any one or a combination of two or more of the following: Bifidobacterium bifidum or its pure culture with a preservation number of CCTCC NO: M 2023349, Enterococcus avium or its pure culture with a preservation number of CCTCC NO: M 2023350, Lactobacillus salivarius or its pure culture with a preservation number of CCTCC NO: M 2023348, Lactobacillus mucosa or its pure culture with a preservation number of CCTCC NO: M 2023352, and Parabacteroides difficile or its pure culture with a preservation number of CCTCC NO: M 20222033.
  • the probiotic composition further contains a lyophilization protectant, a food material, a pharmaceutically acceptable carrier and/or a pharmaceutically acceptable excipient.
  • the second aspect of the present invention provides an isolated Parabacteroides dieffenbachia strain or its pure culture, and the microbial preservation number of the Parabacteroides dieffenbachia strain is CCTCC NO: M20222033.
  • the third aspect of the present invention provides an isolated fermentation Lactobacillus mucosa strain or its pure culture, and the microbial preservation number of the fermentation Lactobacillus mucosa strain is CCTCC NO: M2023352.
  • the fourth aspect of the present invention provides an isolated Lactobacillus salivarius strain or its pure culture, and the microbial preservation number of the Lactobacillus salivarius strain is CCTCC NO: M2023348.
  • the fifth aspect of the present invention provides an isolated avian Enterococcus strain or its pure culture, and the microbial preservation number of the avian Enterococcus strain is CCTCC NO: M2023350.
  • the sixth aspect of the present invention provides an isolated Bifidobacterium bifidum strain or its pure culture, and the microbial preservation number of the Bifidobacterium bifidum strain is CCTCC NO: M2023349.
  • the seventh aspect of the present invention provides a method for preventing, treating or alleviating intestinal diseases, the method comprising administering a therapeutically effective amount of the probiotic composition described in the first aspect of the present invention or the strain or pure culture thereof described in any one of the second to sixth aspects of the present invention to a subject.
  • the subject is a mammal, and more preferably the subject is a human.
  • the probiotic composition of the first aspect of the present invention or the strain or pure culture thereof of any one of the second aspect, the third aspect, the fourth aspect, the fifth aspect and the sixth aspect of the present invention inhibits the intestinal pathogens of the subject, improves the colon damage of the subject, inhibits the intestinal inflammation of the subject, repairs the intestinal barrier or improve diarrhea symptoms in the subject.
  • the intestinal disease is selected from intestinal pathogen infection, diarrhea and radiation enteritis, and preferably the intestinal pathogen is selected from one or more of Pseudomonas aeruginosa, Salmonella paratyphi B, Yersinia enterocolitica, Staphylococcus aureus, Vibrio parahaemolyticus, Clostridium difficile, Shigella and Escherichia coli; preferably, the diarrhea is diarrhea caused by anti-tumor drugs, and further preferably, the diarrhea caused by anti-tumor drugs is diarrhea caused by chemotherapy drugs.
  • the diarrhea caused by the chemotherapy drug is diarrhea caused by one or more drugs selected from the group consisting of doxorubicin, epirubicin, dactinomycin 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.
  • drugs selected from the group consisting of doxorubicin, epirubicin, dactinomycin D, doxorubicin, daunorubicin, paclitaxel, docetaxel, albumin-paclitaxel, cisplatin,
  • Figure 1 is a frontal photograph of the colony morphology of the five strains.
  • FIG. 2 is a graph showing the total antioxidant capacity experimental results of five strains.
  • Figure 3 is a graph showing the results of the antibacterial experiment on five strains.
  • FIG. 4 shows the experimental results of Lactobacillus salivarius Lsali-1 and Enterococcus avium Eaviu-1 on Caco-2 cell barrier repair.
  • Figure 5 shows the results of the in vitro cell inflammation inhibition test of 5 bacterial strains.
  • FIG. 6 shows the results of the adhesion test of 5 bacterial strains to Caco2 cells.
  • Figure 7 shows the diarrhea score and total diarrhea score of the 5-fluorouracil-induced diarrhea mouse model improved by Parabacteroides distachyon Pdist-1.
  • Figure 8 shows the diarrhea score and total diarrhea score of the 5-fluorouracil-induced diarrhea mouse model improved by fermented Lactobacillus mucinus Lferm-1.
  • Figure 9 shows the diarrhea score and total diarrhea score of Lactobacillus salivarius Lsali-1 in improving the 5-fluorouracil-induced diarrhea mouse model.
  • Figure 10 shows the diarrhea score and total diarrhea score of Enterococcus avium Eaviu-1 in improving the 5-fluorouracil-induced diarrhea mouse model.
  • FIG11 is a graph showing the diarrhea score and total diarrhea score of the 5-fluorouracil-induced diarrhea mouse model improved by Bifidobacterium bifidum Bbifi-1.
  • FIG. 12 is a graph showing the improvement of the diarrhea score and total diarrhea score in the 5-fluorouracil-induced diarrhea mouse model by Bifidobacterium bifidum Bbifi-1 and DSM20456.
  • FIG. 13 shows the histopathological results of mice with 5-fluorouracil-induced diarrhea treated with Parabacteroides distist-1 and Lactobacillus fermentans Lferm-1.
  • the scale bar is 500 ⁇ m.
  • FIG. 14 is a graph showing the effects of four strains on the relative expression levels of colon inflammatory factors and aquaporin genes in mice with 5-fluorouracil-induced diarrhea.
  • FIG. 15 shows the therapeutic effect of Bifidobacterium bifidum Bbifi-1 strain on mice with diarrhea caused by radiation enteritis.
  • FIG. 16 is a photograph showing the co-cultivation characteristics of five strains on BF839 agar medium.
  • the present invention provides a probiotic composition for preventing, treating or alleviating intestinal diseases, the active ingredients of which include a therapeutically effective amount of any one or a combination of two or more selected from the following: Bifidobacterium bifidum or its pure culture with a preservation number of CCTCC NO: M 2023349, Enterococcus avium or its pure culture with a preservation number of CCTCC NO: M 2023350, Lactobacillus salivarius or its pure culture with a preservation number of CCTCC NO: M 2023348, Lactobacillus mucosa or its pure culture with a preservation number of CCTCC NO: M 2023352, and Parabacteroides difficile or its pure culture with a preservation number of CCTCC NO: M 20222033.
  • the five strains with specific deposit numbers for which protection is sought in the present invention include, but are not limited to: (1) strains with specific deposit numbers stored in the said deposit center; (2) strains having the same genome as the strains described in (1); (3) subculture strains without gene mutations based on the aforementioned (1) or (2); (4) subculture strains based on the aforementioned (1), (2) or (3) that have accumulated minor mutations during subculture but have no substantial changes in toxicity, immunogenicity and biological activity; (5) live bacteria based on any of the aforementioned strains (1)-(4), inactivated products of the said live bacteria, lysates of the said live bacteria or fermentation products of the said live bacteria, etc.
  • Strains with the same genome include, but are not limited to, strains with the same genetic background that have been independently isolated and disclosed by others after the priority date of the present invention, that is, strains isolated from nature or animals (including humans) with the same genome (same genetic background).
  • Conventional cultures are generally considered to be passage strains without gene mutations. As is known in the art, passage and application of strains usually inevitably lead to Minor mutations.
  • mutations occur in non-coding sequence regions or synonymous mutations in coding regions, or mutations that do not affect the toxicity, immunogenicity, and biological activity of the strain (for example, it may be a connecting amino acid residue between two domains, or a residue located inside the higher-level structure of a protein that does not contact immune cells and does not affect toxicity, immunogenicity, and biological activity), it can be reasonably expected that when these minor changes do not significantly affect the toxicity, immunogenicity, and biological activity of the offspring strains, the purpose of the present invention can still be achieved, and they are derived from the strains contributed by the present invention, and therefore are still within the scope of the substantial technical contribution of the present invention.
  • minor mutations are still non-substantial mutations and should be regarded as mutant strains with no changes in toxicity, immunogenicity, and biological activity.
  • detection there is no substantial change in toxicity, immunogenicity, and biological activity, including but not limited to, within the limitations of detection technology such as detection sensitivity and detection limit and within the range of acceptable or unavoidable errors, the toxicity, immunogenicity, and biological activity are considered to be the same.
  • the toxicity, immunogenicity and biological activity of the offspring of the strains are measured using cells, animals, etc., and there is no substantial change due to differences in cell strains, animal species, age, gender, health status, culture conditions, etc., as well as predictable or unavoidable systematic errors.
  • Active ingredients refer to substances that play a role in the components that produce biological effects.
  • the active ingredient is a probiotic strain.
  • M1 Parabacteroides dieldrin strain or its pure culture with microbial preservation number CCTCC NO: M20222033
  • M2 Enterococcus avium strain or its pure culture with microbial preservation number CCTCC NO: M20233503
  • M3 Lactobacillus fermentatus strain or its pure culture with microbial preservation number CCTCC NO: M2023352
  • M4 Bifidobacterium bifidum strain or its pure culture with microbial preservation number CCTCC NO: M2023349
  • M5 Lactobacillus salivarius strain or its pure culture with microbial preservation number CCTCC NO: M2023348.
  • the active ingredient in the probiotic composition of the present invention is any one, any two, any three, any four or five of M1, M2, M3, M4 and M5.
  • the active ingredient in the probiotic composition is a combination of M1 and any one, any two or any three selected from M2, M3, M4 and M5.
  • the active ingredient in the probiotic composition is a combination of M2 and any one, any two or any three selected from M1, M3, M4 and M5.
  • the active ingredient in the probiotic composition is a combination of M3 and any one, any two or any three selected from M1, M2, M4 and M5.
  • the active ingredient in the probiotic composition is a combination of M4 and any one, any two or any three selected from M1, M2, M3 and M5.
  • the active ingredient in the probiotic composition is a combination of M5 and any one, any two, or any three selected from M1, M2, M3, and M4.
  • the active ingredient is a strain of a single genus, and experiments have shown that even strains of a single genus can play a significant role in preventing, treating or alleviating intestinal diseases.
  • the present invention provides a use of Parabacteroides dieldrinii with a microbial preservation number of CCTCC NO: M20222033 or a pure culture thereof in the preparation of a preparation for preventing, alleviating or treating a disease or a sub-healthy state;
  • the prevention, alleviating or treating a disease or a sub-healthy state includes: preventing, treating or alleviating oxidative damage in the intestine; inhibiting any one, any two, any three, any four or five of Pseudomonas aeruginosa, Salmonella paratyphi B, Yersinia enterocolitica, Staphylococcus aureus and Vibrio parahaemolyticus in the intestine; increasing the expression of AQP8; tolerating, resisting, preventing or alleviating diarrhea.
  • the diarrhea is selected from diarrhea caused by chemotherapy drugs.
  • the chemotherapeutic drug is selected from 5-fluorouracil.
  • the present invention provides a use of a fermented mucus lactobacillus or its subcultured bacteria with a microbial preservation number of CCTCC NO: M2023352 in the preparation of a preparation for preventing a disease or sub-health state, alleviating a disease or sub-health state, or treating a disease or sub-health state; the prevention of a disease or sub-health state, alleviating a disease or sub-health state, or treating a disease or sub-health state includes: preventing, treating or alleviating oxidative damage in the intestine; inhibiting any one, any two, any three or four of Salmonella Paratyphi B, Yersinia enterocolitica, Staphylococcus aureus and Clostridium difficile in the intestine; reducing the expression of TNF- ⁇ ; reducing the expression of IL-6; tolerating, resisting, preventing or alleviating diarrhea.
  • the diarrhea is selected from diarrhea caused by chemotherapeutic drugs.
  • the chemotherapeutic agent is selected from 5-fluorouracil.
  • the present invention provides a use of Lactobacillus salivarius or its subcultured bacteria with a microbial deposit number of CCTCC NO: M2023348 in the preparation of a preparation for preventing a disease or a subhealthy state, alleviating a disease or a subhealthy state, or treating a disease or a subhealthy state;
  • the Preventing disease or sub-health state, alleviating disease or sub-health state or treating disease or sub-health state includes: preventing, treating or alleviating oxidative damage in the intestine; inhibiting any one, any two, any three, any four, any five, any six or seven of Pseudomonas aeruginosa, Shigella, Salmonella Paratyphi B, Yersinia enterocolitica, Vibrio parahaemolyticus, Staphylococcus aureus and Clostridium difficile in the intestine; preventing, improving or repairing intestinal barrier damage caused by inflammation; reducing TNF- ⁇ expression; reducing IL
  • the diarrhea is selected from diarrhea caused by chemotherapeutic drugs.
  • the present invention provides the use of avian Enterococcus or its subcultured bacteria with a microbial preservation number of CCTCC NO: M2023350 in preparing a preparation for preventing disease or sub-health state, alleviating disease or sub-health state, or treating disease or sub-health state;
  • the prevention of disease or sub-health state, alleviating disease or sub-health state, or treating disease or sub-health state includes: preventing, treating or alleviating oxidative damage in the intestine; inhibiting either or both of Pseudomonas aeruginosa and Clostridium difficile in the intestine; preventing, improving or repairing intestinal barrier damage caused by inflammation; reducing TNF- ⁇ expression; reducing IL-6 expression; tolerating, resisting, preventing or alleviating diarrhea.
  • the diarrhea is selected from diarrhea caused by chemotherapeutic drugs.
  • the chemotherapeutic agent is selected from 5-fluorouracil.
  • the present invention provides a use of Bifidobacterium bifidum or its subcultured bacteria with a microbial preservation number of CCTCC NO: M2023349 in preparing a preparation for preventing a disease or a sub-healthy state, alleviating a disease or a sub-healthy state, or treating a disease or a sub-healthy state;
  • the prevention of a disease or a sub-healthy state, alleviating a disease or a sub-healthy state, or treating a disease or a sub-healthy state includes: preventing, treating or alleviating oxidative damage in the intestine; inhibiting any one, any two, any three, any four, any five, any six or seven of Pseudomonas aeruginosa, Shigella, Escherichia coli, Salmonella paratyphi B, Yersinia enterocolitica, Vibrio parahaemolyticus and Clostridium difficile in the intestine; reducing the expression of
  • the diarrhea is selected from diarrhea caused by chemotherapeutic drugs and diarrhea caused by radiation enteritis.
  • the chemotherapeutic agent is selected from 5-fluorouracil.
  • the radiation enteritis is radiation enteritis caused by X-ray abdominal irradiation.
  • the therapeutically effective amount refers to an amount that effectively prevents, treats, alleviates or improves symptoms (e.g., intestinal diseases: diarrhea, The amount of active ingredient that reduces the symptoms or progression of an infection, inflammation, etc., or prolongs the survival of a subject treated.
  • the determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.
  • a therapeutically effective amount or dose can be initially estimated from in vitro and cell culture assays, and doses are formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.
  • the probiotic composition administered to a subject at a single time contains 10 2 to 10 15 CFU, 10 3 to 10 14 CFU, 10 4 to 10 13 CFU, 10 5 to 10 12 CFU, 10 6 to 10 12 CFU, 10 7 to 10 11 CFU, 10 8 to 10 10 CFU, or 10 8 to 10 10 CFU of probiotics.
  • the dosage can vary depending on the dosage form used and the route of administration used. The individual physician can select the exact dosage according to the patient's condition. The dosage and interval can be adjusted individually to provide a sufficient amount of active ingredients to induce a biological effect.
  • administration can be single or multiple administrations, and the course of treatment lasts from several days to several weeks or until a cure is achieved or a reduction in the disease state is achieved.
  • amount of the composition to be administered will depend on the subject to be treated, the severity of the disease, the mode of administration, the judgment of the prescribing physician, etc.
  • the invention provides a composition in the form of common food, beverage, health product, medical food or medicine, which comprises the probiotic composition or strain of the present invention.
  • These common foods, beverages, health products, medical foods or medicines include various exemplary embodiments of the composition of the present invention.
  • These common foods, beverages, health products, medical foods or medicines can be made or provided as probiotic powder, capsules, cereals, baby food, health food or food for specific health purposes, or can be pharmaceutical capsules, tablets, powders, etc.
  • the probiotic composition of the present invention can also further contain other beneficial active ingredients, such as another probiotic, prebiotic or medicine with antidiarrhea function, and prebiotics help regulate the intestinal environment by promoting the growth of probiotics in the intestine, thereby indirectly playing an antidiarrhea effect.
  • beneficial active ingredients such as another probiotic, prebiotic or medicine with antidiarrhea function
  • Examples of the second beneficial active ingredient include, but are not limited to, Bacillus licheniformis, Bifidobacterium, Clostridium butyricum, oligofructose, galacto-oligosaccharide, isomaltooligosaccharide, oligoxylose, oligomannose, inulin, stachyose, soybean oligosaccharide, beta glucan, oligolactose, etc.
  • Intestinal diseases refer to intestinal diseases such as infection, inflammation, diarrhea or dysbacteriosis.
  • intestinal diseases refer to intestinal pathogen infection, colon damage, intestinal inflammation, intestinal barrier damage and/or diarrhea.
  • the intestinal disease is selected from the group consisting of intestinal pathogen infection, diarrhea and radiation Enteritis
  • the intestinal pathogens are selected from one or more of Pseudomonas aeruginosa, Salmonella paratyphi B, Yersinia enterocolitica, Staphylococcus aureus, Vibrio parahaemolyticus, Clostridium difficile, Shigella and Escherichia coli; preferably the diarrhea is diarrhea caused by anti-tumor drugs, and further preferably the diarrhea caused by anti-tumor drugs is diarrhea caused by chemotherapy drugs.
  • Enteric pathogens refer to bacteria that can cause intestinal infections or diseases. They can enter the human body through contaminated food, water, direct contact or insect vectors, leading to various intestinal diseases. There are many types of intestinal pathogens, and their survival and reproduction in the intestines may disrupt the normal balance of intestinal flora, causing inflammation, diarrhea, malabsorption and other problems.
  • the intestinal pathogen is selected from one or more of Pseudomonas aeruginosa, Salmonella paratyphi B, Yersinia enterocolitica, Staphylococcus aureus, Vibrio parahaemolyticus, Clostridium difficile, Shigella and Escherichia coli.
  • Radiation enteritis refers to intestinal complications caused by pelvic, abdominal and retroperitoneal malignancies after radiotherapy. This inflammation can involve the small intestine, colon and rectum, so it is also called radiation proctitis, colitis, and enteritis. The most common symptom of radiation enteritis is diarrhea, sometimes accompanied by mucus or bloody stools.
  • Diarrhea refers to a significant increase in the number of bowel movements, usually more than 3 times a day, thin stools with high water content (more than 85%), and may be accompanied by symptoms of mucus, pus and blood or undigested food.
  • the causes of diarrhea include pathogen infection, food poisoning, drug side effects, intestinal inflammation, psychological factors, etc.
  • the diarrhea is diarrhea caused by anti-tumor drugs.
  • Diarrhea caused by anti-tumor drugs Anti-tumor drugs can be roughly divided into cytotoxic drugs, small molecule targeted drugs, monoclonal antibody drugs, immune checkpoint inhibitors, hormone drugs, antibody-drug conjugates (ADCs), biological response modifiers and other drugs according to their mechanism of action and drug source. Diarrhea caused by the use of these drugs is called diarrhea caused by anti-tumor drugs, also known as cancer treatment-related diarrhea/tumor-associated diarrhea.
  • the diarrhea caused by the anti-tumor drug refers specifically to diarrhea caused by chemotherapy drugs.
  • Cytotoxic drugs are also commonly referred to as chemotherapy drugs.
  • the mechanism of action of these drugs is mainly to inhibit or kill cancer cells by destroying or interfering with the growth and division process of tumor cells.
  • chemotherapy drugs are generally highly toxic to rapidly dividing cells, they not only act on cancer cells, but may also affect rapidly dividing cells in normal body tissues, such as hair follicle cells, gastrointestinal cells, and bone marrow cells. This is a common side effect of chemotherapy, such as hair loss, nausea, vomiting, diarrhea, and bone marrow suppression.
  • the diarrhea caused by the chemotherapy drug is diarrhea caused by one or more drugs selected from the group consisting of doxorubicin, epirubicin, dactinomycin 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.
  • drugs selected from the group consisting of doxorubicin, epirubicin, dactinomycin D, doxorubicin, daunorubicin, paclitaxel, docetaxel, albumin-paclitaxel, cisplatin,
  • the present invention also provides a method for preventing, treating or alleviating intestinal diseases, the method comprising administering the probiotic composition of the first aspect of the present invention or the strain or pure culture thereof of the second aspect of the present invention to a subject.
  • the subject may be avian, mammal or human, preferably mammal or human, more preferably human.
  • the present invention also provides five isolated probiotic strains for preventing or treating intestinal diseases, which are: a Parabacteroides distasonis or its progeny strains, cloned strains, fermentations, lysates, extracts and pure cultures, the Parabacteroides distasonis preservation number is CCTCC NO: M20222033; a Limosilactobacillus fermentum or its progeny strains, cloned strains, fermentations, lysates, extracts and pure cultures, the fermentation Lactobacillus is preserved with CCTCC NO: M2023352; a Lactobacillus salivarius (Lactobacillus salivarius) or its progeny strains, cloned strains, fermentations, lysates, extracts and pure cultures, the preservation number of Lactobacillus salivarius is CCTCC NO: M 2023348; an avian Enterococcus (Enterococcus avium) or its progeny strains, cloned strains
  • the specific parameters in the present invention should be understood as allowing variations within a certain error range, such as within the ⁇ 5% range.
  • the temperature can fluctuate within the range of ⁇ 5°C, ⁇ 4°C, ⁇ 3°C, ⁇ 2°C, ⁇ 1°C.
  • the culture medium used in the following examples is prepared as follows. If not otherwise specified, it can be prepared by common methods in the art or purchased commercially:
  • YCFA liquid medium 10.0 g of peptone, 2.5 g of yeast extract, 0.45 mL of 10 (w/w)% MgSO 4 ⁇ 7H 2 O aqueous solution, 0.45 mL of 10 mg/mL CaCl 2 aqueous solution, 10 mL of TE141, 0.45 g of K 2 HPO 4 , 0.45 g of KH 2 PO 4 , 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 N 2 replacement and aliquoted. The aliquoted solution was sterilized by high temperature and wet heat at 121°C for 30 min and set aside.
  • TE141 Preparation of TE141: Add 1.50 g of nitrilotriacetic acid to 200 mL of pure water to obtain a solution, add an appropriate amount of NaOH to the solution until the solution becomes clear, then add 800 mL of water to the solution, and then adjust the pH to 5.5 with 50% HCl to obtain an aqueous solution of nitrilotriacetic acid.
  • VFA-mix 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 to obtain a solution for use. Before use, adjust the pH to neutral with 5 M NaOH solution.
  • BHI+MRS+modified GAM three mixed liquid medium
  • 19.25g of BHI broth powder Qingdao Haibo Biotechnology Co., Ltd., HB8297-5
  • 13.5g of MRS broth powder (Guangdong Huankai Biotechnology Co., Ltd., 027312)
  • 15g of modified GAM broth powder Qingdao Haibo Biotechnology Co., Ltd., HB8518-3) were dissolved in 1L of distilled water to obtain a solution.
  • the solution was deoxygenated by N2 replacement and aliquoted, and the solution was sterilized at 121°C for 30min.
  • BHI+MRS second mixed culture medium
  • BF839 culture medium 6.0 g potato powder (Beijing Solebow Technology Co., Ltd., FA0270), 10.0 g polyvalent peptone (Beijing Solebow Technology Co., Ltd., P8950-250), 5.0 g of peptone (Haibo Biotechnology Co., Ltd., Qingdao High-Tech Industrial Park, HB8277), 0.3 g of sodium thioglycolate, 5.0 g of yeast extract powder (Thermo Fisher Oxoid, LP0021B), 1.5 g of glucose, and 4.0 g of disodium hydrogen phosphate were dissolved in 1 L of distilled water to obtain a mixed solution. The mixed solution was deoxygenated by N2 replacement and aliquoted, and the mixed solution was sterilized at 121°C for 30 min.
  • MRS broth Preparation of MRS broth: Weigh 54.0 g of MRS broth powder and 0.5 g of cysteine hydrochloride monohydrate, dissolve in 1 L of distilled water, deoxygenate with N2 , and sterilize at 121°C for 15 min.
  • Preparation of anaerobic and resazurin-free PBS Dissolve 0.27g potassium dihydrogen phosphate, 1.42g disodium hydrogen phosphate, 8g sodium chloride and 0.2g potassium chloride in 1L of distilled water, heat to boil, and cool to room temperature. Add 0.55g cysteine hydrochloride to the cooled solution, stir to dissolve, and adjust the pH to 6.5. Install a quantitative dispenser and pass N2 , heat the solution to boiling, keep it at a slight boiling state for 30 minutes, and after the solution is cooled, divide it into 400mL/bottle and sterilize it at 121°C for 30 minutes.
  • MRS solid medium GAM solid medium
  • TSB Teryptone Soy Broth, Qingdao Haibo Biotechnology Co., Ltd., HB4114
  • TSA Teryptone Soy Agar, Qingdao Haibo Biotechnology Co., Ltd., HB4138
  • Brucella broth Qingdao Haibo Biotechnology Co., Ltd., HB0241
  • Preparation of culture medium for the powder of Parabacteroides distiliensis Pdist-1 Dissolve 6g of anhydrous glucose, 15g of soy peptone, 10g of yeast extract powder, 10g of yeast peptone, 2g of potassium dihydrogen phosphate, 2g of disodium hydrogen phosphate, 0.2g of magnesium sulfate, 0.01g of manganese sulfate, 0.2g of calcium chloride, 1mL of Tween 80 and 0.5g of monohydrated cysteine hydrochloride in 1L of distilled water. Deoxygenate the mixture with N2 and dispense it. Sterilize the mixture at 121°C for 15min.
  • Preparation of culture medium for fermentation of Lactobacillus mucilaginosus Lferm-1 powder Dissolve 30g of anhydrous glucose, 15g of soy peptone, 10g of yeast extract powder, 5g of sodium acetate, 2g of potassium dihydrogen phosphate, 2g of disodium hydrogen phosphate, 0.1g of magnesium sulfate, 0.045g of manganese sulfate, 1mL of Tween 80 and 0.5g of monohydrated cysteine hydrochloride in 1L of purified water. Deoxygenate the mixture with N2 replacement and aliquot. Sterilize the mixture at 121°C for 15min.
  • Preparation of culture medium for Lactobacillus salivarius Lsali-1 powder 24 g of anhydrous glucose, 20 g of soy 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 cysteine monohydrate Dissolve 0.5 g of the acid salt in 1 L of purified water. Deoxygenate the mixture with N2 and dispense. Sterilize the mixture at 121°C for 15 min.
  • Preparation of culture medium for Enterococcus avium Eaviu-1 powder Dissolve 30g of anhydrous glucose, 15g of soy peptone, 10g of yeast powder, 5g of sodium acetate, 2g of dipotassium hydrogen phosphate, 0.1g of magnesium sulfate, 0.045g of manganese sulfate, 1mL of Tween 80 and 0.5g of monohydrated cysteine hydrochloride in 1L of purified water. Deoxygenate the mixture with N2 and dispense. Sterilize the mixture at 121°C for 15min.
  • Preparation of culture medium for Bifidobacterium bifidum Bbifi-1 powder Dissolve 20g of anhydrous glucose, 40g of soy peptone, 5g of N-acetylglucosamine, 2g of potassium dihydrogen phosphate, 2g of disodium hydrogen phosphate, 0.1g of magnesium sulfate, 0.045g of manganese sulfate, 1mL of Tween 80 and 0.5g of monohydrated cysteine hydrochloride in 1L of purified water. Deoxygenate the mixture with N2 replacement and aliquot. Sterilize the mixture at 121°C for 15min.
  • Tween 80-PBS dilution Add 3.58g disodium hydrogen phosphate dodecahydrate, 0.27g potassium dihydrogen phosphate, 8g sodium chloride and 1ml Tween 80 to 1L boiling water and dissolve with a glass rod and glue stick. Add 0.5g cysteine hydrochloride monohydrate to the boiled solution. Open the Hungate apparatus and boil the solution again under N2 protection. After blowing N2 for 20 minutes, dispense the solution into anaerobic bottles that have been deoxygenated with N2 . Cover the bottles with stoppers and label them well, and sterilize the solution at 121°C for 15 minutes.
  • Fresh stool samples were collected from several healthy human volunteers, and each stool sample was operated independently. An appropriate amount of anaerobic PBS was added to the stool sample to obtain a mixture and the mixture was shaken to obtain a suspension. Under N2 protection, the suspension was filtered with gauze to obtain a filtrate. The filtrate was centrifuged at 10000rpm for 20min, and then the supernatant was discarded and the precipitate was retained. An appropriate amount of anaerobic PBS was added to the precipitate to resuspend the bacteria to obtain a suspension. An equal volume of 50 (v/v)% anaerobic glycerol aqueous solution was added to the suspension and mixed thoroughly to obtain a bacterial mixed solution sample.
  • the samples were divided into sample tubes, the sample tubes were vacuumed, and then the sample tubes were stored in a -80°C refrigerator.
  • the samples in each frozen sample tube were thawed independently.
  • 0.5mL of the thawed sample was resuspended in 4.5mL of anaerobic PBS and shaken to obtain a bacterial suspension.
  • 0.5mL of the bacterial suspension was diluted with 4.5mL of anaerobic PBS under anaerobic conditions. The same method was used to dilute the sample tenfold to a dilution of 10-6 .
  • one portion was subjected to 16S rDNA gene amplification and sequencing, and the other portion was added to a 50 (v/v)% glycerol aqueous solution at a 1:1 (volume ratio) and mixed evenly before preservation.
  • the 16S rDNA gene sequence obtained by sequencing was compared with the NCBI Nucleotide database to further identify the species of the isolated strains.
  • Five strains were selected from the many strains whose species were further determined for subsequent experiments of the present invention.
  • Strain 1 had the highest sequence similarity with a strain of Parabacteroides distasonis (>99%), so strain 1 was named Parabacteroides distasonis Pdist-1 (referred to as Pdist-1).
  • Strain 2 had the highest sequence similarity with a strain of Limosilactobacillus fermentum (100%), so strain 2 was named Lactobacillus fermentum Lferm-1 (referred to as Lferm-1).
  • Strain 3 had the highest sequence similarity with a strain of Lactobacillus salivarius (100.00%), so strain 3 was named Lactobacillus salivarius Lsali-1 (referred to as Lsali-1).
  • Strain 4 had the highest sequence similarity with a strain of Enterococcus avium (100.00%), so strain 4 was named Eaviu-1 (Eaviu-1 for short).
  • Strain 5 had the highest sequence similarity with a strain of Bifidobacterium bifidum (99.86%), so strain 5 was named Bifidobacterium bifidum Bbifi-1 (Bbifi-1 for short).
  • Parabacteroides dissimilar Pdist-1, fermented mucus Lactobacillus Lferm-1, and avian enterococcus Eaviu-1 were inoculated into BF839 culture medium to observe their colony morphology. Lactobacillus sali-1 and Bifidobacterium bifidum Bbifi-1 were inoculated into three mixed solid culture medium to observe their colony morphology.
  • the front photos of the colony morphology of the above-mentioned 5 strains are shown in Figure 1, among which A is a front photo of the colony morphology of Parabacteroides dissimilar Pdist-1; B is a front photo of the colony morphology of fermented mucus Lactobacillus Lferm-1; C is a front photo of the colony morphology of Lactobacillus sali-1; D is a front photo of the colony morphology of avian enterococcus Eaviu-1; E is a front photo of the colony morphology of Bifidobacterium bifidum Bbifi-1. It can be seen that the 5 strains are white opaque round colonies with a convex middle and a smooth and moist surface.
  • Example 1 The five strains obtained in Example 1 were inoculated into three mixed liquid culture media, and the bacteria were cultured to the late logarithmic growth stage. The whole genome DNA of each strain was extracted, and the whole genome was sequenced using the Illumina high-throughput sequencing platform NovaSeq 6000. After the genome sequence was assembled and annotated, the protein sequence was entered into the virulence gene library Virulence Factor Databases (VFDB) for virulence factor analysis. The results showed that none of the five strains had virulence factors in their genomes.
  • VFDB Virulence Factor Databases
  • the novelty of the five strains was analyzed using the Average Nucleotide Identity (ANI) method.
  • ANI Average Nucleotide Identity
  • Genbank Genbank
  • fastANI v1.33
  • the whole genome sequences of the five strains were annotated using emapper-2.1.9, and it was found that the genome of Parabacteroides distist-1 had genes encoding 1 isomeric bile acid protein, 2 acetate-related enzymes, 3 propionate-related enzymes, 1 CAT (catalase)-related enzyme, and 1 SOD (superoxide dismutase)-related enzyme; the genome of Lactobacillus fermentative mucus Lferm-1 had 1 gene encoding acetate-related enzyme; the genome of Lactobacillus salivarius Lsali-1 had genes encoding 2 acetate-related enzymes and 1 propionate-related enzyme; the genome of Lactobacillus salivarius Eaviu-1 had 2 genes encoding acetate-related enzymes, 1 propionate-related enzyme, 1 SagA protein, and 1 SOD-related enzyme; the genome of Bifidobacterium bifidum Bbifi-1 had genes encoding 1 acetate-related enzyme and 1 prop
  • the strains Pdist-1, Eaviu-1, Lferm-1, Bbifi-1, and Lsali-1 isolated and cultured in the present invention were submitted to the depository recognized by the patent procedure for deposit.
  • the depository is the China Center for Type Culture Collection (CCTCC); the address is Wuhan University, Wuhan, China; the culture name, classification nomenclature, deposit date, identification survival date, and microbial deposit number are shown in Table 1.
  • the Parabacteroides dissinensis Pdist-1 was inoculated into a three-mixed liquid medium and cultured anaerobically at 37°C for 12 hours to obtain a bacterial solution containing an activated strain.
  • Enterococcus faecalis ( ⁇ -hemolytic, CICC23658, purchased from the China Industrial Microbiological Culture Collection Management Center) was inoculated into a three-mixed liquid medium and cultured anaerobically at 37°C for 12 hours to obtain a bacterial solution containing an activated strain (as a positive control).
  • the three-mixed liquid medium was used as a negative control.
  • Fermented mucus lactobacillus Lferm-1, salivarius Lsali-1, avian enterococcus Eaviu-1, and bifidobacterium bifidum Bbifi-1 were inoculated into three mixed liquid culture media, and cultured anaerobically at 37°C until the late logarithmic growth period to obtain a bacterial solution containing activated strains.
  • 2mL2 (v/v)% fresh rabbit red blood cell PBS suspension (Beijing Borsi Technology Co., Ltd.) and the bacterial suspension resuspended with 2mL sterile saline for liquid contact detection.
  • Example 1 The five strains obtained in Example 1 were activated and inoculated into anaerobic BF839 medium.
  • LGG Lactobacillus rhamnosus GG, CICC6141, purchased from China Industrial Microbiological Culture Collection Center
  • the following operations were performed in parallel on the six strains. All strains were anaerobically cultured at 37°C for 24 hours in anaerobic BF839 medium to obtain culture solution.
  • the BCA protein concentration determination kit (the kit was purchased from Beijing Solaibao Technology Co., Ltd., PC0020) was used to draw a standard curve and detect BCA samples according to the instructions of the kit.
  • the strain total antioxidant capacity detection kit (the kit was purchased from Beijing Solaibao Technology Co., Ltd., BC1315) was used to determine the antioxidant capacity of the sample in combination with the standard curve according to the instructions of the kit.
  • the unit of total antioxidant capacity is ⁇ mol/mg prot.
  • the experimental results are shown in Figure 2.
  • the total antioxidant capacity of Lactobacillus salivarius Lsali-1 and Enterococcus avium Eaviu-1 is significantly better than that of the positive control strain LGG; the total antioxidant capacity of Parabacteroides dissimilaris Pdist-1, Lactobacillus fermentatus Lferm-1, and Bifidobacterium bifidum Bbifi-1 is equivalent to that of the positive control strain LGG. This shows that these strains have strong antioxidant capacity.
  • Preparation of fermentation broth Inoculate the activated 5 strains into a three-mixed liquid medium and culture them anaerobically at 37°C for 48 hours to obtain the fermentation broth.
  • Preparation and coating of pathogenic bacteria Salmonella paratyphi B, Yersinia enterocolitica, Staphylococcus aureus, Pseudomonas aeruginosa, Shigella, Escherichia coli and Vibrio parahaemolyticus were activated by TSB broth medium (tryptone soy broth, Qingdao Haibo Biotechnology Co., Ltd., HB4114), diluted in TSB broth medium to reach the appropriate concentration, and 0.2 mL of the diluted bacterial solution was spread on TSA solid medium (tryptone soy agar, Qingdao Haibo Biotechnology Co., Ltd., HB4138).
  • Clostridium difficile was anaerobic activated and transferred, and then diluted to the appropriate concentration in a three-mixed liquid medium, and 0.2 mL of the diluted bacterial solution was spread on anaerobic GAM solid medium (supplemented with 5 (v/v)% horse serum, Beijing Solebold Technology Co., Ltd., S9050).
  • Co-culture with pathogenic bacteria Place 3 sterilized Oxford cups on the coated pathogenic bacteria plate, and add 0.2 mL of the strain fermentation liquid to the Oxford cup. Place in an anaerobic culture box, culture the plate upright for 24 hours, and measure the size of the inhibition zone with a vernier caliper. The experimental results are shown in Figure 3.
  • Pdist-1 has inhibitory ability against Pseudomonas aeruginosa, Salmonella paratyphi B, Yersinia enterocolitica, Staphylococcus aureus and Vibrio parahaemolyticus (A); Lactobacillus fermentum Lferm-1 has inhibitory ability against Salmonella paratyphi B, Yersinia enterocolitica, Staphylococcus aureus and Clostridium difficile (B); Lactobacillus salivarius Lsali-1 has inhibitory ability against Pseudomonas aeruginosa, Shigella, Salmonella paratyphi B The strain had an antibacterial effect on Pseudomonas aeruginosa, Shigella, Escherichia coli, Salmonella paratyphi B, Yersinia enterocolitica, Vibrio parahaemolyticus, Staphylococcus aureus and Clostridium difficile (C); Enterococcus
  • Caco-2 cells (purchased from Beina Chuanglian Biotechnology Co., Ltd., BNCC No.: 350769) were seeded into Transwell (permeable cell culture chamber): Caco-2 adherent cells were digested with trypsin cell digestion solution preheated at 37°C.
  • Caco-2 cells were seeded into 24-well Transwell at a seeding density of 1.1 ⁇ 10 5 cells/well using 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, penicillin-streptomycin mixture, purchased from Gibco, catalog number 15140-122), and cultured at 5% CO 2 and 37°C for 21 days.
  • DMEM medium purchased from Gibco, catalog number C11995500BT
  • FBS purchased from Gibco, catalog number 16000-044
  • PS penicillin-streptomycin mixture, purchased from Gibco, catalog number 15140-122
  • Strain culture Take 200 ⁇ L of Lactobacillus salivarius Lsali-1 and Enterococcus avium Eaviu-1 from the bacterial storage and add them to 5 mL of the second mixed culture medium (the relevant reagents are deoxygenated in advance), and culture anaerobically in a 37°C electric constant temperature incubator for 24 hours. Subculture once and culture anaerobically for 8 hours. Take 1 mL of the bacterial solution and centrifuge at 12000 rpm/min for 3 minutes. Use DMEM containing 10 (v/v)% FBS The strain was diluted to 10 7 CFU/mL in the culture medium and set aside.
  • Lactobacillus rhamnosus GG LGG, CICC 6141, China Industrial Microbiological Culture Collection Center
  • GG Lactobacillus rhamnosus GG
  • bacterial group Lactobacillus salivarius Lsali-1 group and Enterococcus avium Eaviu-1 group.
  • the old culture medium in the lower chamber was removed, 800 ⁇ L DMEM culture medium was added to the lower chamber of the normal control group, and 800 ⁇ L IFN- ⁇ solution with a concentration of 10 ng/mL was added to the lower chamber of the model group, positive control group and bacterial group.
  • the cells were placed in a 5% carbon dioxide incubator and cultured at 37°C for 22 hours.
  • the solution in the upper and lower chambers was removed, 200 ⁇ L DMEM culture medium was added to the upper chamber of the normal control group, and 800 ⁇ L DMEM culture medium was added to the lower chamber; 200 ⁇ L DMEM culture medium was added to the upper chamber of the model group; 200 ⁇ L positive bacterial solution was added to the upper chamber of the positive control group; 200 ⁇ L Lactobacillus salivarius Lsali-1 bacterial solution and Enterococcus avium Eaviu-1 bacterial solution were added to the upper chamber of the bacterial group; 800 ⁇ L TNF- ⁇ solution with a concentration of 50 ng/mL was added to the lower chamber of the model group, positive control group and bacterial group.
  • the cells were placed in a 5% carbon dioxide incubator at 37°C for 5 hours, and then the transmembrane electrical resistance (TEER) of the cell monolayer of each group was measured.
  • TEER transmembrane electrical resistance
  • THP-1 cell polarization THP-1 cells were inoculated in 96-well plates at a seeding density of 1 ⁇ 10 5 cells/well using RPMI-1640 medium (Thermo Fisher, C11875500BT) containing 10 (v/v)% FBS and a final concentration of 100 ng/mL PMA (phorbol 12-myristate 13-acetate, Sigma-Aldrich Company, P1585).
  • the 96-well plate was placed in a 5% CO 2 incubator and cultured at 37°C for 24 hours to polarize it into mature macrophages.
  • Strain culture 200 ⁇ L of the bacterial suspensions of the tested Parabacteroides distist-1, Lactobacillus fermentans Lferm-1, Lactobacillus sali-1, Enterococcus avium Eaviu-1, and Bifidobacterium bifidum Bbifi-1 were inoculated from the bacterial storage to 5 mL of the second mixed culture medium and cultured anaerobically at 37°C for 24 hours. After the transfer, anaerobic culture was performed for 8 h. Take 1 mL of the test bacterial solution and 5000 Centrifuge at rpm/min for 15 min.
  • TNF- ⁇ and IL-6 Effect on the expression of TNF- ⁇ and IL-6 in THP-1 cells: After the mature THP-1 cells were cultured, the normal control group (no bacteria or drug treatment) was replaced with RPMI-1640 medium containing 10 (v/v)% FBS; the model group, positive control group (dexamethasone treatment) and experimental group (each strain treatment) were replaced with RPMI-1640 medium containing 10 (v/v)% FBS, a final concentration of 100 ng/mL LPS (Sigma-Aldrich Company, L3024) and 20 ng/mL IFN- ⁇ (PeproTech, AF-300-02) to model inflammatory macrophages. Each group was placed in a 5% CO 2 incubator and cultured at 37°C for 24 hours.
  • the culture medium was aspirated, and 100 ⁇ L of RPMI-1640 culture medium containing 10 (v/v)% FBS was added to the normal control group and the model group, respectively; 100 ⁇ L of RPMI-1640 culture medium containing 10 (v/v)% FBS and dexamethasone (purchased from Sigma-Aldrich Company, D4902-25) with a final concentration of 25 ⁇ g/mL was added to the positive control group; 100 ⁇ L of each bacterial solution prepared in advance was added to the experimental group.
  • TNF- ⁇ content was detected using a Human TNF- ⁇ (Tumor Necrosis Factor Alpha) ELISA kit (purchased from Wuhan Elaruite Biotechnology Co., Ltd., E-EL-H0109c), and the IL-6 content was detected using a Human IL-6 (Interleukin 6) ELISA kit (purchased from Wuhan Elaruite Biotechnology Co., Ltd., E-EL-H6156).
  • TNF- ⁇ Human TNF- ⁇ (Tumor Necrosis Factor Alpha) ELISA kit
  • E-EL-H0109c Human IL-6
  • Human IL-6 (Interleukin 6) ELISA kit purchasedd from Wuhan Elaruite Biotechnology Co., Ltd., E-EL-H6156.
  • the experimental results are shown in Figure 5.
  • Strain culture 200 ⁇ L of Bifidobacterium bifidum Bbifi-1, Enterococcus avium Eaviu-1, Lactobacillus salivarius Lsali-1, Lactobacillus fermentum Lferm-1, and Parabacteroides distist-1 were inoculated into 5 mL of mixed medium and cultured anaerobically at 37°C until the late logarithmic growth phase.
  • the cultured bacterial solution was washed twice by centrifugation with sterile PBS (Wuhan Boster Biotechnology Co., Ltd., PYG0021), and then washed with 10 (v/v)% FBS (Thermo Fisher Scientific (China) Co., Ltd., SH30084.03)
  • the strain was diluted to 5 ⁇ 10 8 CFU/mL in DMEM (Thermo Fisher Scientific (China) Co., Ltd., C11995500BT) medium for later use.
  • the same concentration of Lactobacillus rhamnosus GG LGG, CICC 6141, China Industrial Microbiological Culture Collection Center was used as the positive control group.
  • 100 ⁇ L of the diluted bacterial suspension was added to the 96-well cell culture plate containing Caco-2 cells. After the addition, the 96-well cell culture plate was placed in a horizontal centrifuge and centrifuged at 1000g for 1 min. The wells corresponding to each bacterial solution were divided into two groups. After incubation for 30 min and 2 h, the two groups were washed twice with sterile PBS to wash away the non-adherent strains. After washing, 50 ⁇ L of trypsin cell digestion solution (Langjieke Technology Co., Ltd., BL501A) was added to each well and placed in a 37°C incubator to digest the cells.
  • trypsin cell digestion solution Liangjieke Technology Co., Ltd., BL501A
  • the adhesion ability of avian Enterococcus Eaviu-1 is equivalent to that of the positive control group LGG;
  • Figure 6B shows the adhesion effect of each strain when the incubation time is 2 hours.
  • the adhesion ability of Bifidobacterium bifidum Bbifi-1 and Lactobacillus fermentum Lferm-1 is equivalent to that of the positive control group LGG
  • the adhesion ability of Lactobacillus salivarius Lsali-1 and avian Enterococcus Eaviu-1 are better than that of the positive control group LGG
  • Parabacteroides dissinensis Pdist-1 has a certain adhesion ability, indicating that the five strains have the potential to colonize in the intestine.
  • Liquid A 6g sucrose, 6g trehalose, 2g xylitol, 2g sorbitol, 44g purified water.
  • Liquid B 5g sodium glutamate, 15g purified water; sterilize at 115°C for 20min.
  • Liquid C 4g sodium vitamin C, 16g purified water. Filter and sterilize for later use.
  • Liquid A 8g sucrose, 8g trehalose, 44g purified water, sterilize at 115°C for 30min.
  • Liquid B 2g sodium glutamate, 2g arginine hydrochloride, 16g purified water, sterilize at 115°C for 30min.
  • Liquid C 4g sodium vitamin C, 16g purified water.
  • Solution A 6g sucrose, 6g trehalose, 2g xylitol, 2g sorbitol, 44g purified water, sterilized at 115°C for 30min.
  • Solution B 2g arginine hydrochloride, 2g sodium glutamate, 16g purified water, sterilized at 115°C for 30min.
  • Solution C 4g sodium vitamin C, 16g purified water. Filter and sterilize for later use.
  • Preparation of bacterial powder The five strains obtained in Example 1 were inoculated into the corresponding bacterial powder preparation medium respectively. Anaerobic culture was performed at 37°C and 90rpm for 16 to 24 hours to obtain the first-level seed liquid. Subsequently, the culture medium was transferred to the bacterial powder and anaerobically cultured at 37°C and 90rpm for 10 to 15 hours to obtain the second-level seed liquid. The second-level seed liquid was pumped into the fermenter containing the bacterial powder culture medium with a peristaltic pump for fermentation and culture. After the fermentation was stopped, the bacteria were collected by centrifugation.
  • the lyophilization protective agent was added at a weight ratio of 1:1 to 1:2 between the bacterial mud and the lyophilization protective agent, and the emulsified bacterial mud was mixed.
  • the bacterial powder was obtained after freeze-drying and crushing. Before animal administration, the bacterial powder containing 1 ⁇ 10 9 CFU of live bacteria was prepared into a bacterial suspension using 0.2mL of physiological saline.
  • Experimental animals SPF-grade male Balb/c mice, weighing 18 to 22g, were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. and were raised in an SPF-grade animal room. According to the initial weight of mice, the mice were randomly divided into groups, with 5 mice in each group.
  • Parabacteroides distist-1, fermented mucus lactobacillus Lferm-1, avian enterococcus Eaviu-1, and bifidobacterium bifidum Bbifi-1 were each set up in 4 groups, namely, normal control group, model control group, positive control loperamide group, and strain group to be tested.
  • Lactobacillus salivarius Lsali-1 was set up in 4 groups, normal control group, model control group, same control strain Lsali-3 group (Lsali-3 was another strain of Lactobacillus salivarius strain isolated by the method consistent with Example 1) and strain Lsali-1 group to be tested.
  • 5-FU solution (5-fluorouracil, purchased from Tianjin Jinyao Pharmaceutical Co., Ltd., specification 10mL/branch, 0.25g/10mL) induced mice chemotherapy-related diarrhea model. Except for the normal control group intraperitoneal injection of normal saline, other groups were subjected to 5-FU single intraperitoneal injection modeling treatment, and the modeling dose was 350mg/kg body weight. All groups were given drugs by gavage.
  • the normal control group and the model control group were gavaged with lyophilized protective agent; the positive control group was gavaged with 20 mg/kg body weight loperamide (purchased from Xi'an Janssen Pharmaceutical Co., Ltd.); the test strain group was gavaged with the test strain suspension at a dose of 1 ⁇ 10 9 CFU/individual.
  • the overall experimental period was 9 days, recorded as D1-D9, and the modeling treatment was performed on D3.
  • the normal control group, the model control group, and the test strain group were continuously gavaged with the test substance on D1-D5, and the positive control group was continuously gavaged with loperamide on D1-D9. After the end of the administration on D5, they were observed for 4 consecutive days.
  • the specific experimental groups and dosing regimens are shown in Table 4 below.
  • 5-FU 5-fluorouracil
  • CFU colony forming unit
  • d day
  • mice Place mice in a mouse cage lined with clean filter paper, one per cage. Hard stools and normal stools are scored as 0 points; mild, slightly wet stools or soft stools are scored as 1 point; moderate, wet stools, unformed stools and unclean perianal area are scored as 2 points; severe, loose stools and severe unclean perianal area are scored as 3 points. During the experimental period, the feces of mice were observed and scored every day. The total diarrhea score was the sum of the daily diarrhea scores.
  • mice were dissected and the entire cecum and colorectum were removed together with the anus.
  • the length of the colorectum of the mice was measured with a ruler, with the end of the cecum as the zero point and the end of the rectum as the end point.
  • the modeling dose was 350 mg/kg body weight, and the Bbifi-1 group and the DSM20456 group were gavaged with 1 ⁇ 10 9 CFU every day. During the experimental period, the feces of mice were observed and scored every day, and the scoring criteria remained the same as before.
  • the specific plan is shown in Table 5 below.
  • the mid-segment colon of the mice was collected and fixed in 4% paraformaldehyde for 24 h.
  • the fixed colon tissue was dehydrated, transparent, waxed and embedded in sequence.
  • the embedded colon tissue wax block was sliced to a thickness of 5 ⁇ m, followed by spreading and baking, and the dried slices were subjected to conventional HE staining).
  • the pathological changes were observed under an optical microscope, and the pathological scores were performed according to the following Table 6, and the total pathological score (the sum of the scores of each index) was calculated.
  • the colon structure of the normal control group was intact, and the mucosal layer, submucosa, muscular layer and serosa were clearly visible; in the model control group, the colon basement showed necrosis and dissolution of intestinal glands, decreased goblet cells, lymphocyte infiltration, loose connective tissue in the submucosa, and dilated blood vessels; in the loperamide group, the colon structure was relatively intact, no obvious necrosis of mucosal epithelial cells was observed, the number of goblet cells increased significantly, and only a small amount of lymphocyte infiltration was observed in the basement; in the Pdist-1 group, the colon mucosal layer structure was relatively intact, no obvious necrosis of mucosal epithelial cells was observed, and the number of goblet cells increased significantly compared with the model control group; in the Lferm-1 group, a small amount of mucosal epithelial cell necrosis was observed in the colon tissue, accompanied by a small amount
  • the mouse colon showed obvious damage 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).
  • the total pathological score of the colon was significantly reduced (P ⁇ 0.05) and the colon damage was improved.
  • Lactobacillus fermentum Lferm-1 can also significantly reduce the total pathological score of the colon (P ⁇ 0.05) and improve the degree of colon damage.
  • the mid-colon of the mice was collected and stored in a -80°C refrigerator.
  • the reagent ThermoFisher Scientific, Cat. No. 15596026
  • the total RNA of the colon tissue of each group of mice was extracted and reverse transcribed into cDNA, which was stored at -20°C for standby use.
  • qRT-PCR was used to detect the relative transcription levels of the mRNA of the colon pro-inflammatory factors IL-1 ⁇ , TNF- ⁇ and aquaporin 8 (AQP8) in each group of mice (primer sequences are shown in Table 7). Reaction procedure: 95°C 3min, 95°C 20s, 60°C 45s, 72°C 20s, for a total of 39 cycles.
  • the 2 - ⁇ CT method was used for analysis, and the data were analyzed for significance using SPSS24.0 statistical software.
  • Example 11 Therapeutic effect of Bifidobacterium bifidum Bbifi-1 on radiation enteritis model mice
  • mice 20 SPF male C57BL/6J mice, weighing 20-25g, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., and raised in an SPF animal room.
  • Experimental design The radiation enteritis mouse model was induced by X-ray abdominal irradiation. According to the initial weight of the mice, they were randomly divided into 4 groups, namely normal control group, model control group, positive control group and Bifidobacterium bifidum Bbifi-1 group, with 5 mice in each group.
  • mice were subjected to a single X-ray abdominal irradiation modeling treatment with a modeling dose of 11.5Gy. All groups were given drugs by gavage.
  • the normal control group and the model control group were gavaged with normal saline; the positive control group was gavaged with 1 ⁇ 10 9 CFU of Lactobacillus rhamnosus GG (LGG, purchased from Shaanxi Zelang Biotechnology Co., Ltd.); the Bifidobacterium bifidum Bbifi-1 group was gavaged with 1 ⁇ 10 9 CFU of Bifidobacterium bifidum Bbifi-1.
  • the overall experimental period was 13 days, recorded as D-2 to D10. D-2 to D9 were continuous.
  • the drug was administered by gavage for 12 days, and the abdomen of the mice was irradiated with a single X-ray on D0 (day 3).
  • mice Place mice in mouse cages lined with clean filter paper, 1 per cage. Hard stools and normal stools are considered 0 points; mild, slightly wet stools or soft stools are considered 1 point; moderate, wet stools, unformed stools and unclean perianal areas are considered 2 points; severe, loose stools and severe unclean perianal areas are considered 3 points.
  • the total diarrhea score is the sum of the daily diarrhea scores.
  • D10 Day 13
  • the experimental animals were euthanized by sodium pentobarbital anesthesia and gross dissection and anatomical observation were performed. See Table 8 below for specific experimental groups and dosing regimens.
  • test results are shown in Figure 15, where A is the diarrhea score curve of each group on days 1-12, and B is the total diarrhea score of each group. It can be seen that compared with the model control group, Bifidobacterium bifidum Bbifi-1 has a significant improvement effect on diarrhea caused by X-ray irradiation, and the effect is equivalent to that of the positive control group LGG (A); LGG and Bifidobacterium bifidum Bbifi-1 can significantly reduce the total diarrhea score (P ⁇ 0.05, B). The above results show that Bifidobacterium bifidum Bbifi-1 has a significant improvement effect on diarrhea in mice induced by X-ray irradiation.
  • the five strains obtained in Example 1 were activated and cultured to the late logarithmic period. Use a disposable sterile cotton swab to dip the bacterial solution of one of the strains and streak three times in parallel on the BF839 solid culture medium, and then the bacterial solutions of the remaining four strains were streaked once in parallel in a direction perpendicular to the first streak. After the streaked bacterial solution was dried, anaerobically cultured for 48 hours until the bacterial solution traces were obvious. The interaction relationship between the five bacterial strains is shown in Figure 16. It can be seen that there is no breakpoint at the intersection of the strains, indicating that there is no growth inhibition between the strains.

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Abstract

本发明公开了一种益生菌，该益生菌包括微生物保藏编号为CCTCC NO：M20222033的迪氏副拟杆菌、微生物保藏编号为CCTCC NO：M2023352的发酵黏液乳杆菌、微生物保藏编号为CCTCC NO：M2023348的唾液乳杆菌、微生物保藏编号为CCTCC NO：M2023350的鸟肠球菌和微生物保藏编号为CCTCC NO：M2023349的两歧双歧杆菌。前述益生菌安全性好，具备抗氧化活性，能够抑制致病菌，能够改善结肠损伤，能够抑制炎症，能够缓解化疗药引起的腹泻。

Description

一种益生菌及其应用 技术领域
本发明属于微生物技术领域，具体涉及一种益生菌及其应用。
背景技术
肠道微生物与人体健康息息相关，且被形象的称为“微生物器官”。肠道菌群为机体重要的组成部分。正常状况下肠道菌群保持动态稳定。肠道微生物在促进营养物质消化吸收、维持肠道正常生理功能、调节机体免疫等众多生命活动中发挥重要的作用。然而肠道菌群容易受到环境因素、饮食和生活习惯、精神因素、疾病状态、肿瘤治疗、抗生素使用以及年龄等众多因素的影响。人体肠道菌群在受到上述因素影响后可能发生肠道紊乱(肠道菌群失调)。肠道紊乱可表现为肠道有益菌的缺失、肠道病原菌的过度繁殖、肠道屏障功能受损以及肠道炎症等疾病。肠道紊乱可进一步引起宿主的便秘、腹泻、腹痛、腹胀等胃肠道疾病。严重的肠道紊乱可发展成炎症性肠病、溃疡性结肠炎、肠易激综合征等疾病。前述疾病极大地影响人体健康和生活质量。
目前，利用肠道益生菌来改善肠道健康以及预防或治疗肠道疾病越来越受到关注。肠道益生菌可以增强肠道黏膜屏障功能、阻止病原菌的黏附和定殖、增强肠道系统的免疫反应等，从而达到维护肠道健康的作用。例如，中国发明专利申请CN 102711778A公开了一株动物双歧杆菌乳亚种DN-173010，并通过小鼠实验和组织学研究验证了其发酵乳能减轻溃疡性结肠炎。公开号为CN107312726A的专利申请文本公开了一种植物乳杆菌，此植物乳杆菌可抑制肠道内大肠杆菌、沙门氏菌、猪链球菌以及金黄色葡萄球菌等有害菌的生长。
益生菌也被用来预防或改善一些药物如抗生素等引起的菌群紊乱的副作用。在临床上，与放化疗相关的副作用是很常见。化疗药物引起的腹泻，也被称为化疗相关性腹泻(Chemotherapy induced diarrhea，CID)，是肿瘤患者化疗过程中最常见的并发症之一。CID的发生被认为是多因素的，但是CID 的发病机制尚不十分明确。以5-FU(5-氟尿嘧啶)为例，增殖的小肠细胞对5-FU被磷酸化产生的5-FdUMP或者5-FUMP较敏感。5-FdUMP或者5-FUMP能导致小肠黏膜损伤并干扰肠细胞的分裂，引起肠壁细胞坏死及肠壁的广泛炎症，造成小肠吸收和分泌细胞数量失衡，进而导致腹泻。另外，化疗药物还会造成细胞DNA损伤和线粒体功能障碍，导致ROS产生和细胞凋亡。ROS可以激活NF-κB，进一步上调促炎因子的表达，进而导致肠道上皮、内皮和结缔组织的损伤。在肠上皮发生损伤的情况下，有害细菌极易定殖，肠道微生态遭到破坏，进而造成致病菌感染，促进腹泻发生与发展。
CID在临床上仍然缺乏统一有效的治疗手段。总的来说，CID治疗的主要目的是控制症状，减轻患者痛苦，加速黏膜修复并预防继发性感染。一般CID治疗包括应用止泻药物、黏膜保护药物和抗菌药物，以及大剂量使用洛哌丁胺甚至生长抑素类药物奥曲肽。但上述药物的作用机制单一、副作用大，不适合长期维持性用药。例如，大剂量使用洛哌丁胺可能引起麻痹性肠梗阻的风险；使用奥曲肽可能引发胆结石、高血糖、糖耐量异常等副作用；抗菌药物可能进一步杀死肠道有益菌，破坏菌群结构，导致肠道微生态紊乱。
高丰富度、高多样性的肠道菌群是一个复杂的微生物生态系统，是对抗肠道病原体以及其他危险因素的重要天然屏障。M.Kverka等人(Clinical and Experimental Immunology,163:250–259，2011)报道了迪氏副拟杆菌能够改善DSS诱导的小鼠结肠炎。专利CN113215063B公开了一种唾液乳杆菌CPU-1可缓解化疗药物替莫唑胺所引起的毒副作用，其侧重的是唾液乳杆菌CPU-1对黏膜炎症状的改善。CN1511945A和CN86103736A等专利研究了包括发酵乳杆菌在内的多种益生菌在治疗或预防腹泻中的用途。专利CN113234619B公开了一株能够缓解急性肠道损伤的两歧双歧杆菌。一种灭活型的脆弱拟杆菌(Bacteroides fragilis)ZY-312被报道经FDA批准开展CID临床试验。
尽管一些研究已经探索了益生菌预防或治疗多种肠道疾病的可能性，但临床上仍然缺乏有效、安全的益生菌干预方案(尤其是干预如CID这样缺少有效治疗手段的疾病或症状)。开发新型益生菌，克服已有药物毒副作用大、治疗机制单一的缺陷，为肿瘤治疗相关性腹泻患者提供有效治疗方案仍是医药生物领域亟需解决的问题。
发明内容
针对现有技术存在的不足，本发明第一方面提供了一种益生菌组合物，所述益生菌组合物中的活性成分包括以下任一种或两种及以上的组合：保藏编号为CCTCC NO：M 2023349的两歧双歧杆菌或其纯培养物、保藏编号为CCTCC NO：M 2023350的鸟肠球菌或其纯培养物、保藏编号为CCTCC NO：M 2023348的唾液乳杆菌或其纯培养物、保藏编号为CCTCC NO：M 2023352的发酵黏液乳杆菌或其纯培养物以及保藏编号为CCTCC NO：M 20222033的迪氏副拟杆菌或其纯培养物。
在一些实施方式中，所述益生菌组合物中还含有冻干保护剂、食品材料、药学上可接受的载体和/或药学上可接受的赋形剂。
本发明第二方面提供了一种分离的迪氏副拟杆菌菌株或其纯培养物，所述迪氏副拟杆菌菌株的微生物保藏编号为CCTCC NO:M20222033。
本发明第三方面提供了一种分离的发酵黏液乳杆菌菌株或其纯培养物，所述发酵黏液乳杆菌菌株的微生物保藏编号为CCTCC NO：M2023352。
本发明第四方面提供了一种分离的唾液乳杆菌菌株或其纯培养物，所述唾液乳杆菌菌株的微生物保藏编号为CCTCC NO:M2023348。
本发明第五方面提供了一种分离的鸟肠球菌菌株或其纯培养物，所述鸟肠球菌菌株的微生物保藏编号为CCTCC NO：M2023350。
本发明第六方面提供了一种分离的两歧双歧杆菌菌株或其纯培养物，所述两歧双歧杆菌菌株的微生物保藏编号为CCTCC NO：M2023349。
本发明第七方面提供了一种预防、治疗或减缓肠道疾病的方法，所述方法包括，将治疗有效量的本发明第一方面所述的益生菌组合物或本发明第二至第六方面中任一方面所述的菌株或其纯培养物施用给受试者。
在一些实施方式中，所述受试者为哺乳动物，进一步优选所述受试者为人。
在一些实施方式中，本发明第一方面所述的益生菌组合物或本发明第二方面、本发明第三方面、本发明第四方面、本发明第五方面和本发明第六方面中的任一方面所述的菌株或其纯培养物抑制所述受试者的肠道病原菌、改善所述受试者的结肠损伤、抑制所述受试者的肠道炎症、修复所述受试者的 肠屏障或改善所述受试者的腹泻症状。
在一些实施方式中，所述肠道疾病选自肠道病原菌感染、腹泻和放射性肠炎，优选所述肠道病原菌选自铜绿假单胞菌、乙型副伤寒沙门氏菌、小肠结肠炎耶尔森菌、金黄色葡萄球菌、副溶血性弧菌、艰难梭菌、志贺氏菌和大肠杆菌中的一种或多种；优选所述腹泻为抗肿瘤药物引起的腹泻，进一步优选抗肿瘤药物引起的腹泻为化疗药物引起的腹泻。
在一些实施方式中，所述化疗药物引起的腹泻是选自以下一种或多种药物引起的腹泻：阿霉素、表阿霉素、放线菌素D、多柔比星、柔红霉素、紫杉醇、多西他赛、白蛋白紫杉醇、顺铂、卡铂、奈达铂、草酸铂、洛铂、环磷酰胺、氮芥、卡莫司汀、喜树碱、羟基喜树碱、拓扑替康、伊立替康、卡培他滨、吉西他滨、甲氨蝶呤、5-氟尿嘧啶、培美曲塞和阿糖胞苷。
附图说明
图1为5个菌株的菌落形态正面照片。
图2为5个菌株总抗氧化能力实验结果图。
图3为5个菌株抑菌实验结果图。
图4为唾液乳杆菌Lsali-1与鸟肠球菌Eaviu-1对Caco-2细胞屏障修复的试验结果。
图5为5株菌体外细胞炎症抑制试验结果。
图6为5株菌对Caco2细胞的黏附能力检测结果。
图7为迪氏副拟杆菌Pdist-1改善5-氟尿嘧啶致腹泻小鼠模型的腹泻评分和腹泻总分图。
图8为发酵黏液乳杆菌Lferm-1改善5-氟尿嘧啶致腹泻小鼠模型的腹泻评分和腹泻总分图。
图9为唾液乳杆菌Lsali-1改善5-氟尿嘧啶致腹泻小鼠模型的腹泻评分和腹泻总分图。
图10为鸟肠球菌Eaviu-1改善5-氟尿嘧啶致腹泻小鼠模型的腹泻评分和腹泻总分图。
图11为两歧双歧杆菌Bbifi-1改善5-氟尿嘧啶致腹泻小鼠模型的腹泻评分和腹泻总分图。
图12为两歧双歧杆菌Bbifi-1与DSM20456改善5-氟尿嘧啶致腹泻小鼠模型的腹泻评分和腹泻总分图。
图13为迪氏副拟杆菌Pdist-1和发酵黏液乳杆菌Lferm-1治疗5-氟尿嘧啶致腹泻小鼠的组织病理学结果，比例尺为500μm。
图14为4个菌株对5-氟尿嘧啶致腹泻小鼠的结肠炎性因子和水通道蛋白基因相对表达量影响结果图。
图15为两歧双歧杆菌Bbifi-1菌株对放射性肠炎致腹泻小鼠的治疗效果。
图16为五个菌株在BF839琼脂培养基上的共培养特征照片。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚，下面将结合附图对本发明实施方式作进一步地详细描述。
本发明提供了预防、治疗或减缓肠道疾病的益生菌组合物，其活性成分包括治疗有效量的选自以下任一种或两种及以上的组合：保藏编号为CCTCC NO：M 2023349的两歧双歧杆菌或其纯培养物、保藏编号为CCTCC NO：M 2023350的鸟肠球菌或其纯培养物、保藏编号为CCTCC NO：M 2023348的唾液乳杆菌或其纯培养物、保藏编号为CCTCC NO：M 2023352的发酵黏液乳杆菌或其纯培养物、保藏编号为CCTCC NO：M 20222033的迪氏副拟杆菌或其纯培养物。
本发明所请求保护的特定保藏编号的5个菌株，其涵义包括但不限于：(1)存放于所述保藏中心的特定保藏号的菌株；(2)与(1)所述菌株具有相同基因组的菌株；(3)基于前述(1)或(2)的没有基因突变的传代菌株；(4)基于前述(1)、(2)或(3)的在传代中积累微小突变的，但毒性、免疫原性与生物活性没有实质变化的传代菌株；(5)基于前述(1)-(4)任一所述菌株的活菌、所述活菌的灭活物、所述活菌的裂解物或所述活菌的发酵产物等。
具有相同基因组的菌株包括但不限于本发明对应优先权日以后被他人独立分离并公开的具备相同遗传背景的菌株，即，基因组相同(遗传背景相同)的从自然界或动物(包括人)体内分离的菌株。常规培养物通常被认为是没有基因突变的传代菌株。如本领域所知，菌株经传代应用通常不可避免会引 入微小突变。当突变发生在非编码序列区或者编码区的同义突变或者不影响菌株毒性、免疫原性与生物活性的突变(比如，可能是两个结构域之间的连接氨基酸残基，或者位于蛋白质高级结构内部因不与免疫细胞接触而不影响毒性、免疫原性与生物活性的微小突变的残基)，可以合理预期，当这些微小变化没有明显影响后代毒株的毒性、免疫原性与生物活性的情况下，仍然能实现本发明的目的，且其源于本发明贡献的菌株，因此仍在本发明的实质技术贡献范围内。这些微小的突变仍属于非实质性突变，应当视为毒性、免疫原性与生物活性没有变化的突变菌株。在检测角度，毒性、免疫原性与生物活性没有实质变化，包括担不限于，在检测灵敏度、检测限等检测技术的局限性和可接受或不可避免误差的范围内视为毒性、免疫原性与生物活性是相同的。用细胞、动物等测定菌株后代的毒性、免疫原性与生物活性，由于细胞品系、动物品种、年龄、性别、健康状况、培养条件等体现的差别以及可预期或不可避免的系统误差属于没有实质性变化。活性成分是指起到发生生物效应的组分的物质。在本发明中，活性成分是益生菌菌株。通过研究本发明的五种菌株的共培养特性发现，这些菌株两两之间互不抑制，因而可以根据每株菌的功效特点组配成含有2种、3种、4种或5种的组合物，并合理预期这些菌株组配的组合物能同时发挥组内菌株的功效。
为更好地说明本发明菌株的适宜的组合，拟定：M1：微生物保藏编号为CCTCC NO：M20222033的迪氏副拟杆菌菌株或其纯培养物；M2：微生物保藏编号为CCTCC NO：M20233503的鸟肠球菌菌株或其纯培养物；M3：微生物保藏编号为CCTCC NO：M2023352的发酵黏液乳杆菌菌株或其纯培养物；M4：微生物保藏编号为CCTCC NO：M2023349的两歧双歧杆菌菌株或其纯培养物；M5：微生物保藏编号为CCTCC NO：M2023348的唾液乳杆菌菌株或其纯培养物。
在一些实施方式中，本发明所述益生菌组合物中的活性成分为M1、M2、M3、M4和M5中的任一种、任两种、任三种、任四种或五种。
在一些实施方式中，所述益生菌组合物中的活性成分为M1与选自M2、M3、M4和M5中的任一种、任两种或任三种的组合。
在一些实施方式中，所述益生菌组合物中的活性成分为M2与选自M1、M3、M4和M5中的任一种、任两种或任三种的组合。
在一些实施方式中，所述益生菌组合物中的活性成分为M3与选自M1、M2、M4和M5中的任一种、任两种或任三种的组合。
在一些实施方式中，所述益生菌组合物中的活性成分为M4与选自M1、M2、M3和M5中的任一种、任两种或任三种的组合。
在一些实施方式中，所述益生菌组合物中的活性成分为M5与选自M1、M2、M3和M4中的任一种、任两种或任三种的组合。
在一些实施方式中，活性成分是单一种属的菌株，试验证明即便是仅含单一种属的菌株也能发挥明显的预防、治疗或减缓肠道疾病的作用。
因此，在一些实施方式中，本发明提供了微生物保藏编号为CCTCC NO：M20222033的迪氏副拟杆菌或其纯培养物在制备用于预防、减缓或者治疗疾病或亚健康状态的制剂中的用途；所述预防、减缓或者治疗疾病或亚健康状态包括：预防、治疗或减缓肠道内的氧化损伤；抑制肠道内铜绿假单胞菌、乙型副伤寒沙门氏菌、小肠结肠炎耶尔森菌、金黄色葡萄球菌和副溶血性弧菌中的任一种、任两种、任三种、任四种或五种；增加AQP8表达量；耐受、抵抗、预防或缓解腹泻。
在一些实施方式中，所述腹泻选自化疗药物引起的腹泻。
在一些实施方式中，所述化疗药物选自5-氟尿嘧啶。
在一些实施方式中，本发明提供了微生物保藏编号为CCTCC NO：M2023352的发酵黏液乳杆菌或其传代细菌在制备用于预防疾病或亚健康状态，减缓疾病或亚健康状态或者治疗疾病或亚健康状态的制剂中的用途；所述预防疾病或亚健康状态，减缓疾病或亚健康状态或者治疗疾病或亚健康状态包括：预防、治疗或减缓肠道内的氧化损伤；抑制肠道内乙型副伤寒沙门氏菌、小肠结肠炎耶尔森菌、金黄色葡萄球菌和艰难梭菌中的任一种、任两种、任三种或四种；降低TNF-α表达量；降低IL-6表达量；耐受、抵抗、预防或缓解腹泻。
在一些实施方式中，所述腹泻选自化疗药引起的腹泻。
在一些实施方式中，所述化疗药选自5-氟尿嘧啶。
在一些实施方式中，本发明提供了微生物保藏编号为CCTCC NO：M2023348的唾液乳杆菌或其传代细菌在制备用于预防疾病或亚健康状态，减缓疾病或亚健康状态或者治疗疾病或亚健康状态的制剂中的用途；所述预 防疾病或亚健康状态，减缓疾病或亚健康状态或者治疗疾病或亚健康状态包括：预防、治疗或减缓肠道内的氧化损伤；抑制肠道内铜绿假单胞菌、志贺氏菌、乙型副伤寒沙门氏菌、小肠结肠炎耶尔森菌、副溶血性弧菌、金黄色葡萄球菌和艰难梭菌中的任一种、任两种、任三种、任四种、任五种、任六种或七种；预防、改善或修复炎症引起的肠道屏障损伤；降低TNF-α表达量；降低IL-6表达量；降低IL-1β表达量；耐受、抵抗、预防或缓解腹泻。
在一些实施方式中，所述腹泻选自化疗药引起的腹泻。
在一些实施方式中，本发明提供了微生物保藏编号为CCTCC NO：M2023350的鸟肠球菌或其传代细菌在制备用于预防疾病或亚健康状态，减缓疾病或亚健康状态或者治疗疾病或亚健康状态的制剂中的用途；所述预防疾病或亚健康状态，减缓疾病或亚健康状态或者治疗疾病或亚健康状态包括：预防、治疗或减缓肠道内的氧化损伤；抑制肠道内铜绿假单胞菌与艰难梭菌中的任一种或两种；预防、改善或修复炎症引起的肠道屏障损伤；降低TNF-α表达量；降低IL-6表达量；耐受、抵抗、预防或缓解腹泻。
在一些实施方式中，所述腹泻选自化疗药引起的腹泻。
在一些实施方式中，所述化疗药选自5-氟尿嘧啶。
在一些实施方式中，本发明提供了微生物保藏编号为CCTCC NO：M2023349的两歧双歧杆菌或其传代细菌在制备用于预防疾病或亚健康状态，减缓疾病或亚健康状态或者治疗疾病或亚健康状态的制剂中的用途；所述预防疾病或亚健康状态，减缓疾病或亚健康状态或者治疗疾病或亚健康状态包括：预防、治疗或减缓肠道内的氧化损伤；抑制肠道内铜绿假单胞菌、志贺氏菌、大肠杆菌、乙型副伤寒沙门氏菌、小肠结肠炎耶尔森菌、副溶血性弧菌和艰难梭菌中的任一种、任两种、任三种、任四种、任五种、任六种或七种；降低TNF-α表达量；耐受、抵抗、预防或缓解腹泻。
在一些实施方式中，所述腹泻选自化疗药引起的腹泻与放射性肠炎引起的腹泻。
在一些实施方式中，所述化疗药选自5-氟尿嘧啶。
在一些实施方式中，所述放射性肠炎是X射线腹部辐照引起的放射性肠炎。
治疗有效量是指有效预防、治疗、减轻或改善病症(例如肠道疾病：腹泻、 感染、炎症等)的症状或进展或延长接受治疗的受试者存活时间的活性成分的量。治疗有效量的确定完全在本领域技术人员的能力范围内，尤其是根据本发明提供的详细公开内容。治疗有效量或剂量可以最初从体外和细胞培养测定中评估，在动物模型中配制剂量以达到所需的浓度或效价。此类信息可用于更准确地确定人体的有用剂量。
在一些实施方式中，单次施用给受试者的益生菌组合物中含有102～1015CFU、103～1014CFU、104～1013CFU、105～1012CFU、106～1012CFU、107～1011CFU、108～1010CFU、或108～1010CFU的益生菌量。剂量可以根据使用的剂型和使用的给药途径而变化。个体医生可以根据患者的状况选择确切的剂量。可以单独调整剂量和间隔以提供足够量的活性成分以诱导生物效应。根据待治疗病症的严重性和反应性，给药可以是单次或多次给药，治疗过程持续数天至数周或直至达到治愈或实现疾病状态的减轻。主要的，要施用的组合物的量将取决于被治疗的对象、疾病的严重程度、施用的方式、处方医师的判断等。
发明提供普通食物、饮料、保健品、医疗食品或药品形式的组合物，其包含本发明所述的益生菌组合物或菌株。这些普通食物、饮料、保健品、医疗食品或药品包含本发明组合物的各种示例性实施方式。这些普通食物、饮料、保健品、医疗食品或药品可以制成或提供为益生菌粉、胶囊、谷物、婴儿食品、健康食品或特定健康用途的食品，也可以为药物胶囊、药片、粉剂等。本发明的益生菌组合物还可以进一步含有其他有益的活性成分，例如另一种具有止泻功能的益生菌、益生元或药物等，益生元通过对肠道内益生菌的促生长，帮助调节肠道内环境，从而间接发挥止泻作用。第二种有益的活性成分的例子包括但不限于地衣芽孢杆菌、双歧杆菌、丁酸梭菌、低聚果糖、低聚半乳糖、低聚异麦芽糖、低聚木糖、低聚甘露糖、菊粉、水苏糖、大豆低聚糖、β葡聚糖、低聚乳果糖等。
任一种是指从所提供的选项中任选其中一种。两种及以上是指从所提供的选项中任选其中两种、三种……直至选择所有可选项作为选择方案。
肠道疾病是指感染、炎症、腹泻或菌群失调类肠道疾病。在一些实施方式中，肠道疾病是指肠道病原菌感染、结肠损伤、肠道炎症、肠屏障损伤和/或腹泻。
在一些实施方式中，所述肠道疾病选自肠道病原菌感染、腹泻和放射性 肠炎，优选所述肠道病原菌选自铜绿假单胞菌、乙型副伤寒沙门氏菌、小肠结肠炎耶尔森菌、金黄色葡萄球菌、副溶血性弧菌、艰难梭菌、志贺氏菌和大肠杆菌中的一种或多种；优选所述腹泻为抗肿瘤药物引起的腹泻，进一步优选抗肿瘤药物引起的腹泻为化疗药物引起的腹泻。
肠道病原菌是指那些能够引起肠道感染或疾病的细菌，其可以通过污染的食物、水、直接接触或昆虫媒介等途径进入人体，导致各种肠道疾病。肠道病原菌的种类繁多，它们在肠道中的生存和繁殖可能会破坏肠道的正常菌群平衡，引发炎症、腹泻、吸收障碍等问题。
在一些实施方式中，所述肠道病原菌选自铜绿假单胞菌、乙型副伤寒沙门氏菌、小肠结肠炎耶尔森菌、金黄色葡萄球菌、副溶血性弧菌、艰难梭菌、志贺氏菌和大肠杆菌中的一种或多种。放射性肠炎是指盆腔、腹腔、腹膜后恶性肿瘤在接受放射治疗后引起的肠道并发症。这种炎症可以累及小肠、结肠和直肠，因此也被称为放射性直肠炎、结肠炎、小肠炎。放射性肠炎最常见症状是腹泻，有时伴有粘液或血便，其他症状还包括腹痛、里急后重、恶心和呕吐、腹胀、食欲减退并可能伴随体重减轻。腹泻是指排便次数明显增多，通常超过每天3次，粪便稀薄且含水量高(超过85％)，可能伴有黏液、脓血或未消化食物的症状。腹泻的原因包括病原菌感染、食物中毒、药物副作用、肠道炎症、心理因素等。
在一些实施方式中，所述腹泻是抗肿瘤药物引起的腹泻。抗肿瘤药物引起的腹泻：抗肿瘤药物根据作用机制和药物来源，可以大致分为细胞毒药物、小分子靶向药物、单克隆抗体药物、免疫检查点抑制剂、激素类药物、抗体偶联药物(ADCs)、生物反应调节剂和其他类药物。使用这些药物引起的腹泻称为抗肿瘤药物引起的腹泻，也被称为抗肿瘤治疗相关性腹泻/肿瘤相关性腹泻(cancer treatment–related diarrhea/tumor-associated diarrhea)。
在一些实施方式中，所述抗肿瘤药物引起的腹泻特指化疗药物引起的腹泻。细胞毒药物通常也称为化疗药物，这些药物的作用机制主要是通过破坏或干扰肿瘤细胞的生长和分裂过程，从而抑制或杀死癌细胞。由于化疗药物通常对快速分裂的细胞有较强的毒性，它们不仅作用于癌细胞，也可能影响正常身体组织中快速分裂的细胞，如毛囊细胞、胃肠道细胞和骨髓细胞，这就是化疗常见的副作用，如脱发、恶心、呕吐、腹泻和骨髓抑制等。
在一些实施方式中，所述化疗药物引起的腹泻是选自以下一种或多种药物引起的腹泻：阿霉素、表阿霉素、放线菌素D、多柔比星、柔红霉素、紫杉醇、多西他赛、白蛋白紫杉醇、顺铂、卡铂、奈达铂、草酸铂、洛铂、环磷酰胺、氮芥、卡莫司汀、喜树碱、羟基喜树碱、拓扑替康、伊立替康、卡培他滨、吉西他滨、甲氨蝶呤、5-氟尿嘧啶、培美曲塞和阿糖胞苷。
本发明还提供了预防、治疗或减缓肠道疾病的方法，所述方法包括，将本发明第一方面所述的益生菌组合物或本发明第二方面所述的菌株或其纯培养物施用给受试者。受试者可以是禽类、哺乳动物和人，优选为哺乳动物和人，更优选为人。
本发明还提供了分离的预防或治疗肠道疾病的5株益生菌菌株，分别是：一种迪氏副拟杆菌(Parabacteroides distasonis)或其子代菌株、克隆菌株、发酵物、裂解物、提取物和纯培养物，所述迪氏副拟杆菌保藏号为CCTCC NO:M20222033；一种发酵黏液乳杆菌(Limosilactobacillus fermentum)或其子代菌株、克隆菌株、发酵物、裂解物、提取物和纯培养物，所述发酵黏液乳杆菌的保藏号为CCTCC NO：M2023352；一种唾液乳杆菌(Lactobacillus salivarius)或其子代菌株、克隆菌株、发酵物、裂解物、提取物和纯培养物，所述唾液乳杆菌的保藏号为CCTCC NO:M 2023348；一种鸟肠球菌(Enterococcus avium)或其子代菌株、克隆菌株、发酵物、裂解物、提取物和纯培养物，所述鸟肠球菌的保藏号为CCTCC NO：M2023350；和一种两歧双歧杆菌(Bifidobacterium bifidum)或其子代菌株、克隆菌株、发酵物、裂解物、提取物和纯培养物，所述两歧双歧杆菌的保藏号为CCTCC NO：M2023349。
下面将对本发明实施例中的技术方案进行清楚、完整地描述。显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例，基于本发明中的实施例，本领域技术人员在没有做出创造性劳动的前提下所获得的所有其它实施例，均应属于本发明保护的范围。本发明没有说明的材料与仪器为本领域常规材料与仪器。本发明没有说明的操作细节为本领域常规操作。本发明所用软件参照软件提供者的使用说明通过常规方法进行操作。本发明所用试剂盒参照试剂盒使用说明书通过常规方法进行操作。本发明中的特定参数，如无特殊说明，应理解为允许在一定误差范围，如±5％区间内存在变 动。温度可在±5℃、±4℃、±3℃、±2℃、±1℃的范围内波动。
以下实施例中所用到的培养基配制方法如下，未特殊说明的可通过本领域常用方法制备或商购获得：
YCFA液体培养基的配制：将蛋白胨10.0g，酵母提取物2.5g，10(w/w)％的MgSO4·7H2O水溶液0.45mL，10mg/mL的CaCl2水溶液0.45mL，TE141 10mL，K2HPO4 0.45g，KH2PO4 0.45g，NaCl 0.90g和VFA-mix 3.2mL加入1L的蒸馏水中得到溶液。对溶液进行N2置换除氧并分装。121℃高温湿热灭菌分装的溶液30min，备用。
TE141的配制：将次氮基三乙酸1.50g加入到200mL纯水中得到溶液，向溶液中加入适量NaOH至溶液变澄清，再向溶液中加水800mL，再用50％HCl调节pH值至5.5，得到次氮基三乙酸水溶液。将MgSO4·7H2O 3.00g，MnSO4·H2O 0.50g，NaCl 1.00g，FeSO4·7H2O 0.10g，CoSO4·7H2O 0.18g，CaCl2·2H2O 0.10g，ZnSO4·7H2O 0.18g，CuSO4·5H2O 0.006g，KAl(SO4)2·12H2O 0.02g，H3BO3 0.01g，Na2MoO4·2H2O 0.01g，NiCl2·6H2O 0.03g，10mg/mL的Na2SeO3·5H2O溶液0.03mL和10mg/mL的Na2WO4·2H2O溶液0.03mL加入上述溶液中，加入过程中不断搅拌溶液，保持溶液澄清。
VFA-mix的配制：将乙酸90mL，丙酸30mL，正戊酸10mL，异丁酸10mL和丁酸10mL混匀得到溶液备用。使用前用5M浓度NaOH溶液溶液至pH调至中性。
三混液体培养基(BHI+MRS+改良GAM)的配制：将BHI肉汤粉末(青岛海博生物技术有限公司，HB8297-5)19.25g，MRS肉汤粉末(广东环凯生物科技有限公司，027312)13.5g，改良GAM肉汤粉末(青岛海博生物技术有限公司，HB8518-3)15g，溶解于1L的蒸馏水中得到溶液。对溶液进行N2置换除氧并分装，121℃高温湿热灭菌溶液30min。
三混固体培养基(BHI+MRS+改良GAM)的配制：三混液体培养基的基础上增加5g琼脂粉，其他步骤相同。
二混培养基(BHI+MRS)的配制：将BHI肉汤粉末19.25g，MRS肉汤粉末27.0g和一水合半胱氨酸盐酸盐0.5g溶解于1L的蒸馏水中得到混合液。对混合液进行除氧分装和121℃高温湿热灭菌15min。
BF839培养基的配制：将土豆浸粉(北京索莱宝科技有限公司，FA0270)6.0g，多价蛋白胨(北京索莱宝科技有限公司，P8950-250)10.0g，胨(青岛高科技工业园海博生物技术有限公司，HB8277)5.0g，硫代乙醇酸钠0.3g，酵母浸粉(Thermo Fisher Oxoid，LP0021B)5.0g，葡萄糖1.5g，磷酸氢二钠4.0g溶解于1L的蒸馏水中得到混合液。对混合液用N2置换除氧并分装，121℃高温湿热灭菌混合液30min。
MRS肉汤的配制：称取MRS肉汤粉末54.0g，一水合半胱氨酸盐酸盐0.5g，溶解于1L的蒸馏水中，N2置换除氧，121℃灭菌15min。
无氧无刃天青PBS的配制：将磷酸二氢钾0.27g，磷酸氢二钠1.42g，氯化钠8g和氯化钾0.2g溶解于1L的蒸馏水中，加热煮沸，冷却至室温。在冷却的溶液中加入0.55g半胱氨酸盐酸盐，搅拌溶解后调节pH至6.5。装上定量分液器并通N2，将溶液加热至沸腾，在微沸状态下保持30min，溶液冷却后分装为400mL/瓶，121℃高温湿热灭菌30min。
MRS固体培养基、GAM固体培养基、TSB(胰蛋白胨大豆肉汤，青岛海博生物技术有限公司，HB4114)、TSA(胰蛋白胨大豆琼脂，青岛海博生物技术有限公司，HB4138)、布氏肉汤(青岛海博生物技术有限公司，HB0241)培养基的配制均按照说明书进行称量溶解，121℃高温湿热灭菌30min。
迪氏副拟杆菌Pdist-1菌粉制备培养基配制：将无水葡萄糖6g，大豆蛋白胨15g，酵母浸粉10g，酵母蛋白胨10g，磷酸二氢钾2g，磷酸氢二钠2g，硫酸镁0.2g，硫酸锰0.01g，氯化钙0.2g，吐温80 1mL和一水合半胱氨酸盐酸盐0.5g溶解于1L的蒸馏水中。对混合液用N2置换除氧，分装。121℃灭菌混合液15min。
发酵黏液乳杆菌Lferm-1菌粉制备培养基：将无水葡萄糖30g，大豆蛋白胨15g，酵母浸粉10g，乙酸钠5g，磷酸二氢钾2g，磷酸氢二钠2g，硫酸镁0.1g，硫酸锰0.045g，吐温80 1mL和一水合半胱氨酸盐酸盐0.5g溶解于1L的纯化水中。对混合液用N2置换除氧，分装。121℃高温湿热灭菌混合液15min。
唾液乳杆菌Lsali-1菌粉制备培养基配制：将无水葡萄糖24g，大豆蛋白胨20g，酵母浸粉10g，蛋白胨10g，乙酸钠5g，磷酸二氢钾2g，磷酸氢二钠2g，硫酸镁0.1g，硫酸锰0.045g，吐温80 1ml和一水合半胱氨酸盐 酸盐0.5g溶解于1L的纯化水中。对混合液用N2置换除氧，分装。121℃高温湿热灭菌混合液15min。
鸟肠球菌Eaviu-1菌粉制备培养基：将无水葡萄糖30g，大豆蛋白胨15g，酵母粉10g，乙酸钠5g，磷酸氢二钾2g，硫酸镁0.1g，硫酸锰0.045g，吐温80 1mL和一水合半胱氨酸盐酸盐0.5g溶解于1L的纯化水中。对混合液用N2置换除氧，分装。121℃高温湿热灭菌混合液15min。
两歧双歧杆菌Bbifi-1菌粉制备培养基：将无水葡萄糖20g，大豆蛋白胨40g，N-乙酰氨基葡萄糖5g，磷酸二氢钾2g，磷酸氢二钠2g，硫酸镁0.1g，硫酸锰0.045g，吐温80 1mL和一水合半胱氨酸盐酸盐0.5g溶解于1L的纯化水中。对混合液用N2置换除氧，分装。121℃高温湿热灭菌混合液15min。
0.1％吐温80-PBS稀释液制备：将十二水磷酸氢二钠3.58g，磷酸二氢钾0.27g，氯化钠8g和吐温80 1ml加入沸水1L，用玻璃棒胶棒溶解。将一水合半胱氨酸盐酸盐0.5g加入前述煮沸后的溶液。打开亨盖特装置，使溶液在N2保护下再次煮沸，吹N220 min后，把溶液分装至已通N2除氧的厌氧瓶中。加盖塞子并贴好标签纸，121℃高温灭菌溶液15min。
实施例1：菌株分离鉴定
采集若干名健康人类志愿者的新鲜粪便样本，分别独立地操作每份粪便样本。在粪便样本中加入适量无氧PBS得到混合物并震荡混合物以便得到悬液。在N2保护下，用纱布过滤悬液得到滤液。在10000rpm条件下离心滤液20min，然后弃去上清液并保留沉淀。在沉淀中加入适量无氧PBS重悬菌体得到悬液。在悬液中加入等体积的50(v/v)％的无氧甘油水溶液充分混匀得到细菌混合液样本。用样本管分装样本，对样本管套袋抽真空，然后于-80℃冰箱保存样本管。分别独立地解冻每支冻存的样本管中的样本。将0.5mL解冻后的样本重悬于4.5mL无氧PBS中振荡混匀，得到菌悬液。厌氧条件下将0.5mL菌悬液与4.5mL厌氧PBS振荡混匀进行稀释。用相同的方法依次十倍梯度稀释至10-6稀释度。取适当稀释度菌液与YCFA液体培养基混匀后分装到384孔板中，37℃厌氧培养一周。将已生长细菌的孔中的菌液接种到YCFA培养基当中培养48h后把菌液分成两份。利用MALDI-TOF-MS检测一份菌液以对分离菌株进行种属初步分类。确定菌液中只含有一种遗传背景的细菌(单克隆菌株)后，根据质谱结果，将另一份菌液再次接种到YCFA 培养基培养后，一份进行16S rDNA基因扩增并测序，并将另一份按1：1(体积比)加入50(v/v)％的甘油水溶液混合均匀后保藏。
将测序得到的16S rDNA基因序列与NCBI Nucleotide数据库做比对以进一步鉴定所分离菌株的种属。从进一步确定种属的众多菌株中选择5个进行本发明的后续实验。菌株1与一株迪氏副拟杆菌(Parabacteroides distasonis)的序列相似度最高(>99％)，因此，将菌株1命名为迪氏副拟杆菌Pdist-1(简称Pdist-1)。菌株2与一株发酵黏液乳杆菌(Limosilactobacillus fermentum)的序列相似度最高(100％)，因此，将菌株2命名为发酵黏液乳杆菌Lferm-1(简称Lferm-1)。菌株3与一株唾液乳杆菌(Lactobacillus salivarius)的序列相似度最高(100.00％)，因此，将菌株3命名为唾液乳杆菌Lsali-1(简称Lsali-1)。菌株4与一株鸟肠球菌Enterococcus avium的序列相似度最高(100.00％)，因此，将菌株4命名为鸟肠球菌Eaviu-1(简称Eaviu-1)。菌株5与一株两歧双歧杆菌Bifidobacterium bifidum的序列相似度最高(99.86％)，因此，将菌株5命名为两歧双歧杆菌Bbifi-1(简称Bbifi-1)。
分别将迪氏副拟杆菌Pdist-1、发酵黏液乳杆菌Lferm-1、鸟肠球菌Eaviu-1接种至BF839培养基培养以观察其菌落形态。分别将唾液乳杆菌Lsali-1、两歧双歧杆菌Bbifi-1接种至三混固体培养基培养以观察其菌落形态。前述5个菌株培养的菌落形态正面照片见图1，其中，A为迪氏副拟杆菌Pdist-1菌落形态正面照片；B为发酵黏液乳杆菌Lferm-1菌落形态正面照片；C为唾液乳杆菌Lsali-1菌落形态正面照片；D为鸟肠球菌Eaviu-1菌落形态正面照片；E为两歧双歧杆菌Bbifi-1菌落形态正面照片。由此可见，5株菌均呈白色不透明状圆形菌落，中间凸起、表面光滑湿润。
实施例2：菌株的全基因组分析
将实施例1所得5个菌株分别接种至三混液体培养基中，培养细菌至对数生长后期。提取各个菌株全基因组DNA，利用Illumina高通量测序平台NovaSeq 6000进行全基因组测序。基因组序列组装及注释后，将蛋白序列输入毒力基因库Virulence Factor Databases(VFDB)进行毒力因子分析。结果显示，5个菌株的基因组中都不具有毒力因子。
利用平均核苷酸相似度(Average Nucleotide Identity，ANI)法进行5个菌株的新颖性分析。在Genbank中进行全基因组搜索，通过fastANI(v1.33) 比较最近似的菌株。与迪氏副拟杆菌Pdist-1全基因组最相近的两个菌株分别为GCA_003462945.1(ANI＝98.26％)和GCA_003459965.1(ANI＝98.20％)。与发酵黏液乳杆菌Lferm-1全基因组最相近的两个菌株分别为GCA_003465085.1(ANI＝99.32％)和GCA_024385625.1(ANI＝99.29％)。与唾液乳杆菌Lsali-1全基因组最相近的两个菌株分别为GCA_009863605.1(ANI＝99.94％)和GCA_009866185.1(ANI＝99.87％)。与鸟肠球菌Eaviu-1全基因组最相近的两个菌株分别为GCA_018917545.1(ANI＝98.75％)和GCA_018373135.1(ANI＝98.62％)。与两歧双歧杆菌Bbifi-1全基因组最相近的两个菌株分别为GCA_003466395.1(ANI＝99.01％)和GCA_003437945.1(ANI＝99.00％)。由此可见，实施例1所做出的种属分类是正确的。
通过emapper-2.1.9对5个菌株的全基因组序列进行注释，发现迪氏副拟杆菌Pdist-1的基因组具有编码1个产异构胆汁酸蛋白、2个产乙酸相关酶、3个产丙酸相关酶、1个产CAT(过氧化氢酶)相关酶、1个产SOD(超氧化物歧化酶)相关酶的基因；发酵黏液乳杆菌Lferm-1的基因组具有1个编码产乙酸相关酶的基因；唾液乳杆菌Lsali-1的基因组具有编码2个产乙酸相关酶和1个产丙酸相关酶的基因；唾液乳杆菌Eaviu-1的基因组具有2个编码产乙酸相关酶、1个产丙酸相关酶、1个编码SagA蛋白、1个编码SOD相关酶的基因；两歧双歧杆菌Bbifi-1的基因组具有编码1个产乙酸相关酶和1个产丙酸相关酶的基因。
将本发明所分离培养得到的菌株Pdist-1、Eaviu-1、Lferm-1、Bbifi-1、Lsali-1分别提交至专利程序认可的保藏机构保藏。保藏单位为中国典型培养物保藏中心(CCTCC)；地址为中国，武汉，武汉大学；培养物名称、分类命名、保藏日期、鉴定存活日期、微生物保藏编号分别见表1。
表1.菌株保藏信息统计表

实施例3：溶血试验
将迪氏副拟杆菌Pdist-1接种至三混液体培养基中，37℃厌氧培养12h，得到含有活化菌株的菌液。将粪肠球菌(β溶血，CICC23658，购自中国工业微生物菌种保藏管理中心)接种至三混液体培养基中，37℃厌氧培养12h，得到含有活化菌株的菌液(作为阳性对照)。以三混液体培养基作为阴性对照。分别取2.5μL含有活化菌株的两种菌液和阴性对照接种至哥伦比亚血平板(上海科玛嘉微生物技术有限公司)，每个样本设置3个平行试验。于37℃厌氧培养48h后观察哥伦比亚血平板。结果：粪肠球菌菌落周围形成界限明显、完全透明的溶血环，为β溶血；迪氏副拟杆菌Pdist-1菌落周围的培养基没有变化，为γ溶血，即不溶血；阴性对照没有溶血环。结果显示，迪氏副拟杆菌Pdist-1不溶血。
分别将发酵黏液乳杆菌Lferm-1、唾液乳杆菌Lsali-1、鸟肠球菌Eaviu-1、两歧双歧杆菌Bbifi-1接种至三混液体培养基中，37℃厌氧培养至对数生长后期，得到含有活化菌株的菌液。取2mL2(v/v)％新鲜兔红细胞PBS悬液(北京博尔西科技有限公司)与用2mL无菌生理盐水重悬后的菌悬液进行液体接触法检测。以等体积无菌水作为阳性对照，以等体积无菌生理盐水作为阴性对照。每组设置3个平行，静置24h后对各样本进行观察。结果显示，阳性对照发生溶血；阴性对照、发酵黏液乳杆菌Lferm-1、唾液乳杆菌Lsali-1菌、鸟肠球菌Eaviu-1、两歧双歧杆菌Bbifi-1均不溶血。
实施例4：抗生素敏感试验
根据《中华人民共和国卫生行业标准》之“抗菌药物敏感性试验的技术要求”(标准号：WS/T 639-2018)中厌氧菌抗生素敏感性试验的要求，采用肉汤稀释法测定菌株对抗生素的敏感性，记录MIC值。
将制备好的5种菌悬液按顺序依次吸取0.1mL至等体积的已制备好的含 抗菌药物的96孔板中，使最终接种细菌浓度为5×105CFU/mL，药物浓度分别为256μg/mL、128μg/mL、64μg/mL、32μg/mL、16μg/mL、8μg/mL、4μg/mL、2μg/mL、1μg/mL、0.5μg/mL、0.25μg/mL、0.125μg/mL。孵育：将上述96孔板置于37℃培养箱中厌氧孵育46h～48h。结果如下表2所示。
表2.5种菌对14种抗生素的MIC值(μg/mL)
注：其中“-”代表未测试。
实施例5：抗氧化实验
分别将活化的实施例1所得5个菌株接种至无氧BF839培养基中。以LGG(鼠李糖乳杆菌Lactobacillus rhamnosus GG，CICC6141，购自中国工业微生物菌种保藏管理中心)作为阳性对照。六个菌株分别平行地进行如下操作。所有菌株均在无氧BF839培养基中37℃厌氧培养24h，得到培养菌液。
在12000rpm离心0.5mL培养菌液20min，弃上清，用0.5mL菌株总抗氧化能力检测试剂盒(试剂盒购自北京索莱宝科技有限公司，BC1315)中的预冷提取液重悬，将重悬液转移至灭菌的装有beads(购买自美国Sigma 公司，G4649-1KG)的螺帽管中；用快速样品制备仪振荡破壁一次(参数设置：4.5m/s，30s)；12000rpm 4℃离心10min，取上清置冰上备用。采用BCA蛋白浓度测定试剂盒(试剂盒购自北京索莱宝科技有限公司，PC0020)，按照试剂盒的说明书操作绘制标准曲线并检测BCA样品。采用菌株总抗氧化能力检测试剂盒(试剂盒购自北京索莱宝科技有限公司，BC1315)，按照试剂盒的说明书操作结合标准曲线测定样本的抗氧化能力。总抗氧化能力单位μmol/mg prot。实验结果参见图2。唾液乳杆菌Lsali-1、鸟肠球菌Eaviu-1的总抗氧化能力显著优于阳性对照菌株LGG；迪氏副拟杆菌Pdist-1、发酵黏液乳杆菌Lferm-1、两歧双歧杆菌Bbifi-1的总抗氧化能力与阳性对照菌株LGG相当。由此可见，这些菌株都具有较强的抗氧化能力。
实施例6：菌株对致病菌的抑菌能力测试
选取表3所示常见的可导致腹泻的肠道致病菌检测实施例1所得5个菌株的抑菌能力。
表3.致病菌株来源信息
菌株发酵液制备：将经活化5个菌株接种于三混液体培养基中，于37℃厌氧培养48h，获得发酵液。致病菌制备与涂布：乙型副伤寒沙门氏菌、小 肠结肠炎耶尔森菌、金黄色葡萄球菌、铜绿假单胞菌、志贺氏菌、大肠杆菌和副溶血性弧菌经TSB肉汤培养基(胰蛋白胨大豆肉汤，青岛海博生物技术有限公司，HB4114)活化后，于TSB肉汤培养基中稀释达到合适浓度，取0.2mL稀释好的菌液于TSA固体培养基(胰蛋白胨大豆琼脂，青岛海博生物技术有限公司，HB4138)上涂布。艰难梭菌经厌氧活化转接后，于三混液体培养基中稀释到合适浓度，取0.2mL稀释好的菌液涂布于无氧GAM固体培养基(添加5(v/v)％马血清，北京索莱宝科技有限公司，S9050)上。与致病菌共培养：放置3支灭菌牛津杯于涂布好的致病菌平板上，在牛津杯中加入0.2mL菌株发酵液。放入厌氧培养盒，平皿正置培养24h，用游标卡尺测量抑菌圈大小。实验结果参见图3。由此可见，迪氏副拟杆菌Pdist-1对铜绿假单胞菌、乙型副伤寒沙门氏菌、小肠结肠炎耶尔森菌、金黄色葡萄球菌和副溶血性弧菌均有抑制能力(A)；发酵黏液乳杆菌Lferm-1对乙型副伤寒沙门氏菌、小肠结肠炎耶尔森菌、金黄色葡萄球菌和艰难梭菌有抑菌能力(B)；唾液乳杆菌Lsali-1对铜绿假单胞菌、志贺氏菌、乙型副伤寒沙门氏菌、小肠结肠炎耶尔森菌、副溶血性弧菌、金黄色葡萄球菌和艰难梭菌均有抑菌能力(C)；鸟肠球菌Eaviu-1对铜绿假单胞菌及艰难梭菌均有抑菌能力，且对艰难梭菌抑菌能力较强(D)；两歧双歧杆菌Bbifi-1铜绿假单胞菌、志贺氏菌、大肠杆菌、乙型副伤寒沙门氏菌、小肠结肠炎耶尔森菌、副溶血性弧菌和艰难梭菌均有抑菌能力(E)。
实施例7：体外屏障修复试验
Caco-2细胞(购买自商城北纳创联生物科技有限公司，BNCC编号：350769)种入Transwell(可穿透性细胞培养小室)：使用37℃预热的胰酶细胞消化液消化Caco-2贴壁细胞。用含10(v/v)％FBS和1(w/v)％PS的DMEM培养基(DMEM培养基，购于Gibco，货号C11995500BT；FBS，购于Gibco，货号16000-044；PS，青链霉素混合液，购于Gibco，货号15140-122)以1.1×105个细胞/孔的接种密度将Caco-2细胞接种于24孔的Transwell中，5％CO2、37℃静置培养21d。菌株培养：分别从菌保管中取200μL唾液乳杆菌Lsali-1菌液和鸟肠球菌Eaviu-1菌液至5mL二混培养基中(相关试剂提前除氧)，37℃电热恒温培养箱厌氧培养24h。传代接种一次，厌氧培养8h。取1mL菌液，12000rpm/min离心3min。用含10(v/v)％FBS的DMEM 培养基稀释菌株至107CFU/mL，待用。以相同浓度的鼠李糖乳杆菌GG(LGG，CICC 6141，中国工业微生物菌种保藏管理中心)作为阳性对照。唾液乳杆菌Lsali-1和鸟肠球菌Eaviu-1对Caco-2细胞模型中肠上皮屏障功能的影响：针对每种细菌，分别设置4组，分别为正常对照组、模型组、阳性对照组、细菌组(唾液乳杆菌Lsali-1组和鸟肠球菌Eaviu-1组)。使用炎症因子IFN-γ(Pepro Tech，AF-300-02)和TNF-α(Pepro Tech，300-01A)构建屏障损伤模型作为模型组。Caco-2细胞培养21d后，待其分化形成致密单层细胞，吸去下室旧培养基，正常对照组下室加入800μL DMEM培养基，模型组、阳性对照组以及细菌组下室分别加入800μL浓度为10ng/mL的IFN-γ溶液。置于5％二氧化碳培养箱中，37℃静置培养22h后，吸去上室及下室溶液，正常对照组上室加入200μL DMEM培养基，下室加入800μL DMEM培养基；模型组上室加入200μL DMEM培养基；阳性对照组，上室加入200μL阳性菌液；细菌组上室分别加入200μL唾液乳杆菌Lsali-1菌液和和鸟肠球菌Eaviu-1菌液；模型组、阳性对照组和细菌组下室加入800μL浓度为50ng/mL的TNF-α溶液。置于5％二氧化碳培养箱中，37℃静置培养5h后，检测各组别细胞单层跨膜电阻(TEER)值。
结果如图4所示。由此可见，与模型组相比，阳性对照组LGG可显著增加TEER值，表明对细胞屏障损伤具有明显的修复作用。同样，与模型组相比，唾液乳杆菌Lsali-1(A)和鸟肠球菌Eaviu-1(B)也可以显著增加TEER值，且与LGG效果相似或相当。结果表明唾液乳杆菌Lsali-1和鸟肠球菌Eaviu-1可有效缓解炎症因子(如IFN-γ、TNF-α)造成的屏障功能障碍。
实施例8：体外细胞炎症抑制试验
THP-1细胞极化：使用含10(v/v)％FBS和终浓度为100ng/mL PMA(佛波醇12-十四酸酯13-乙酸酯，Sigma-Aldrich Company,P1585)的RPMI-1640培养基(Thermo Fisher，C11875500BT)，以1×105个细胞/孔的接种密度将THP-1细胞接种于96孔板中。将96孔板置于5％ CO2培养箱，37℃培养24h使其极化成为成熟巨噬细胞。菌株培养：分别从菌保管中接种待测迪氏副拟杆菌Pdist-1、发酵黏液乳杆菌Lferm-1、唾液乳杆菌Lsali-1、鸟肠球菌Eaviu-1和两歧双歧杆菌Bbifi-1菌液200μL至5mL二混培养基中，37℃厌氧培养24h。转接一次后，厌氧培养8h。取1mL待测菌液，5000 rpm/min离心15min。用含10(v/v)％FBS的RPMI-1640培养基分别依次把前述菌株稀释至5×107CFU/mL、2×106CFU/mL、5×107CFU/mL、5×107CFU/mL、5×107CFU/mL备用。对THP-1细胞表达TNF-α和IL-6的影响：THP-1成熟细胞培养好后，正常对照组(无细菌或药物处理)更换含10(v/v)％FBS的RPMI-1640培养基；模型组、阳性对照组(地塞米松处理)及试验组(各菌株处理)分别更换含10(v/v)％FBS、终浓度为100ng/mL LPS(Sigma-Aldrich Company,L3024)和20ng/mL IFN-γ(PeproTech，AF-300-02)的RPMI-1640培养基，进行炎症型巨噬细胞的造模。各组均置于5％ CO2培养箱中，37℃培养24h。吸去培养基，正常对照组和模型组分别添加100μL含10(v/v)％FBS的RPMI-1640培养基；阳性对照组添加100μL含10(v/v)％FBS和终浓度为25μg/mL的地塞米松(购于Sigma-Aldrich Company，D4902-25)的RPMI-1640培养基；试验组添加100μL前期制备好的各待测菌液。置于5％ CO2培养箱，37℃培养24h后，各组分别吸取80μL细胞培养液，4℃，5000rpm/min，离心15min，收集上清液，使用Human TNF-α(Tumor Necrosis Factor Alpha)ELISA试剂盒检测TNF-α含量(购于武汉伊莱瑞特生物科技股份有限公司，E-EL-H0109c)，使用HumanIL-6(Interleukin 6)ELISA试剂盒(购于武汉伊莱瑞特生物科技股份有限公司，E-EL-H6156)检测IL-6含量。实验结果如图5所示。由此可见，模型对照组细胞IL-6、TNF-α的表达显著高于正常对照组(P<0.01)；阳性对照地塞米松组能够显著抑制THP-1细胞中促炎因子IL-6、TNF-α的表达(P<0.01)；与模型组相比，发酵黏液乳杆菌Lferm-1、唾液乳杆菌Lsali-1、鸟肠球菌Eaviu-1能够显著降低促炎因子IL-6的表达，发酵黏液乳杆菌Lferm-1、唾液乳杆菌Lsali-1、鸟肠球菌Eaviu-1、两歧双歧杆菌Bbifi-1能够显著降低促炎因子TNF-α的表达(P<0.05)，表明这些菌株在体外具有抗炎作用。
实施例9：对Caco-2细胞的黏附能力测试
菌株培养：分别接种两歧双歧杆菌Bbifi-1菌液、鸟肠球菌Eaviu-1菌液、唾液乳杆菌Lsali-1菌液、发酵黏液乳杆菌Lferm-1菌液、迪氏副拟杆菌Pdist-1菌液200μL至5mL二混培养基中，37℃厌氧培养至对数生长后期。将培养后菌液以无菌PBS(武汉博士德生物工程有限公司，PYG0021)离心洗涤2次，使用含10(v/v)％FBS(赛默飞世尔科技(中国)有限公司，SH30084.03) 的DMEM(赛默飞世尔科技(中国)有限公司，C11995500BT)培养基将菌株稀释为5×108CFU/mL，待用。以相同浓度的鼠李糖乳杆菌(Lactobacillus rhamnosus)GG(LGG，CICC 6141，中国工业微生物菌种保藏管理中心)作为阳性对照组。分别将100μL稀释后菌悬液加入含Caco-2细胞的96孔细胞培养板。加样完毕后，96孔细胞培养板置于水平离心机中，1000g离心1min。每种菌液对应的孔分两小组，两小组分别孵育30min和2h后，用无菌PBS洗涤2次，洗去未黏附菌株。洗涤后每孔加入50μL胰酶细胞消化液(兰杰柯科技有限公司，BL501A)，置于37℃培养箱消化细胞。待Caco-2细胞消化变成球状后，每孔加入150μL含10(v/v)％FBS的DMEM培养基，反复吹打约1min。待显微镜检确定细胞和菌株消化分离后，吸取20μL上述混合液用0.1％吐温80-PBS在96孔板依次进行10倍梯度稀释，选择合适稀释梯度倾注已溶解的三混固体培养基，37℃培养48h后计数。结果如图6所示，图6A为孵育时间为30min时各菌株黏附效果，鸟肠球菌Eaviu-1与阳性对照组LGG黏附能力相当；图6B为孵育时间为2h时各菌株黏附效果，两歧双歧杆菌Bbifi-1、发酵乳杆菌Lferm-1黏附能力与阳性对照组LGG相当，唾液乳杆菌Lsali-1、鸟肠球菌Eaviu-1黏附能力均优于阳性对照组LGG，迪氏副拟杆菌Pdist-1具有一定黏附力，说明5株菌具有在肠道定殖的潜力。
实施例10：对5-氟尿嘧啶(5-FU)诱导腹泻小鼠模型的治疗效果
迪氏副拟杆菌Pdist-1冻干保护剂配制：A液：蔗糖6g，海藻糖6g，木糖醇2g，山梨醇2g，纯化水44g。B液：谷氨酸钠5g，纯化水15g；115℃灭菌20min。C液：维生素C钠4g，纯化水16g。过滤除菌备用。发酵黏液乳杆菌Lferm-1、唾液乳杆菌Lsali-1、鸟肠球菌Eaviu-1冻干保护剂配制：A液：蔗糖8g，海藻糖8g，纯化水44g，115℃灭菌30min。B液：谷氨酸钠2g，精氨酸盐酸盐2g，纯化水16g，115℃灭菌30min。C液：维生素C钠4g，纯化水16g。两歧双歧杆菌Bbifi-1冻干保护剂配制：A液：蔗糖6g，海藻糖6g，木糖醇2g，山梨醇2g，纯化水44g，115℃灭菌30min。B液：精氨酸盐酸盐2g，谷氨酸钠2g，纯化水16g，115℃灭菌30min。C液：维生素C钠4g，纯化水16g。过滤除菌备用。
各菌株的冻干保护剂在使用时均按体积比A液:B液:C液＝6:2:2混合。
菌粉制备：将实施例1所得5个菌株分别接种至相应的菌粉制备培养基， 37℃、90rpm厌氧培养16～24h，得到一级种子液。随后，转接至菌粉制备培养基，37℃、90rpm厌氧培养10～15h，得到二级种子液。将二级种子液用蠕动泵泵入含菌粉制备培养基的发酵罐中，发酵培养。停止发酵后离心收集菌体。按菌泥和冻干保护剂重量比1:1～1:2添加冻干保护剂，混匀乳化菌泥。冻干、粉碎后即得菌粉。动物给药前使用0.2mL生理盐水将含1×109CFU活菌的菌粉配制成菌悬液。试验动物：SPF级雄性Balb/c小鼠，体重18～22g，购自北京维通利华实验动物技术有限公司，饲养于SPF级动物房。根据小鼠初始体重随机分组，每组5只，迪氏副拟杆菌Pdist-1、发酵黏液乳杆菌Lferm-1、鸟肠球菌Eaviu-1、两歧双歧杆菌Bbifi-1各自设4组，分别为正常对照组、模型对照组、阳性对照洛哌丁胺组和待测菌株组。唾液乳杆菌Lsali-1设4组，正常对照组、模型对照组、同种对照菌株Lsali-3组(Lsali-3为用实施例1相通的方法分离得到的另一株唾液乳杆菌菌株)和待测菌株Lsali-1组。试验设计：5-FU溶液(5-氟尿嘧啶，购自天津金耀药业有限公司，规格10mL/支，0.25g/10mL)诱导小鼠化疗相关性腹泻模型。除正常对照组腹腔注射生理盐水外，其他组别均进行5-FU单次腹腔注射造模处理，造模剂量为350mg/kg体重。所有组别给药方式为灌胃，正常对照组、模型对照组灌胃冻干保护剂；阳性对照组灌胃20mg/kg体重洛哌丁胺(购自西安杨森制药有限公司)；待测菌株组按1×109CFU/只的剂量灌胃待测菌株菌悬液。总体实验周期为9天，记为D1-D9，D3进行造模处理，正常对照组、模型对照组、待测菌株组均D1-D5连续灌胃受试物，阳性对照组D1-D9连续灌胃洛哌丁胺，在D5给药结束后，连续观察4天。具体实验分组和给药方案见下表4。
表4.治疗5-FU致腹泻小鼠的实验分组和给药方案

注：5-FU:5-氟尿嘧啶；CFU:colony forming unit菌落形成单位；d:天
腹泻观察与评分：将小鼠置于垫有洁净滤纸的小鼠笼内，每笼1只。粪便硬，正常便视为0分；轻度、轻微湿便或软便视为1分；中度，湿便、粪便不成形且肛周不洁视为2分；重度，稀便且严重肛周不洁视为3分。实验周期内，每天对小鼠粪便进行观察、评分。腹泻总分为每天腹泻评分的总和。
体重检测及体重变化率：实验周期内，每天称量小鼠体重，计算体重变化率，体重变化率＝(检测体重-初始体重)/初始体重×100％。
实验结束后，解剖所有小鼠，连同肛门将整个盲肠与结直肠一起剖离。以盲肠末端为零点、直肠末端为终点用直尺测量小鼠结直肠长度。
腹泻实验结果如图7到图11所示。由此可见，与模型对照组相比，五株菌分别对5-FU导致的腹泻均具有明显改善作用，腹泻总分显著降低。
为对比两歧双歧杆菌Bbifi-1与两歧双歧杆菌标准菌株DSM20456(购自德国微生物与细胞培养物保藏中心)的药效差异，按前述方式构建CID模型。前期数据表明CID造模4天后即D7模型即可出现稳定的腹泻，故本次试验总体实验周期共7天，记为D1-D7，D3时除正常对照组腹腔注射生理盐水外，其他组别进行5-FU单次腹腔注射造模处理，造模剂量为350mg/kg体重，Bbifi-1组和DSM20456组每天灌胃1×109CFU。实验周期内，每天对小鼠粪便进行观察、评分，评分标准与之前保持一致。具体方案如下表5。
表5.治疗5-FU致腹泻小鼠的实验分组和给药方案

试验结果如图12所示，与模型对照组相比，Bbifi-1对5-FU导致的腹泻改善效果优于标准菌株DSM20456。
对结肠损伤的影响
动物实验结束后，采集小鼠中段结肠，于4％多聚甲醛中固定24h。将固定好的结肠组织依次进行脱水、透明、浸蜡和包埋。将包埋好的结肠组织蜡块进行切片，厚度为5μm，随后进行展片和烤片，将干燥的切片进行常规HE染色)。在光学显微镜下观察其病理变化，按照下表6对其进行病理评分，并计算病理评分总得分(各项指标得分总和)。
表6.结肠组织病理学评分指标及说明

由图13中1A和2A可知，正常对照组结肠结构完整，黏膜层、黏膜下层、肌层和浆膜层清晰可见；模型对照组结肠固有层可见肠腺坏死溶解，杯状细胞减少，淋巴细胞浸润，黏膜下层结缔组织疏松，血管扩张；洛哌丁胺组结肠结构较为完整，黏膜上皮细胞未见明显坏死，杯状细胞数量明显增多，固有层仅见少量淋巴细胞浸润；Pdist-1组结肠黏膜层结构较完整，黏膜上皮细胞未见明显坏死，杯状细胞数量较模型对照组明显增多；Lferm-1组结肠组织可见少量黏膜上皮细胞坏死，伴少量淋巴细胞浸润；少量肠腺可见扩张；杯状细胞数量轻度减少；黏膜下层可见轻度水肿，伴少量淋巴细胞浸润。由图13中1B和2B可知，5-FU诱导后小鼠结肠出现明显损伤，模型对照组病理总分显著高于正常对照组(P<0.01)。迪氏副拟杆菌Pdist-1给药后可显著降低结肠的病理总得分(P<0.05)，改善结肠损伤。发酵黏液乳杆菌Lferm-1也可以显著降低结肠的病理总得分(P<0.05)，改善结肠损伤程度。
对小鼠结肠IL-1β、TNF-α和AQP8的mRNA相对转录水平的改善
动物实验结束后，采集小鼠中段结肠，保存于-80℃冰箱。按照试剂(ThermoFisher Scientific,货号15596026)说明书提取各组小鼠结肠组织总RNA，并反转录成cDNA，保存于-20℃备用。用qRT-PCR检测各组小鼠结肠促炎因子IL-1β、TNF-α以及水通道蛋白8(AQP8)基因mRNA的相对转录水平(引物序列见表7)。反应程序：95℃3min，95℃20s，60℃45s，72℃20s，共39个循环。采用2-ΔΔCT法进行分析，通过SPSS24.0统计软件对数据进行显著性分析。
表7.qRT-PCR引物信息
结果如图14，由此可见，与正常对照组相比，模型对照组IL-1β、TNF-α的mRNA相对转录水平显著升高，AQP8的mRNA相对转录水平显著降低。与模型对照组相比，Pdist-1给药后可显著降低IL-1β和TNF-α的mRNA相对转录水平(A、B)，同时显著增加AQP8的mRNA相对转录水平(C)，Lferm-1给药后可显著降低TNF-α的mRNA相对转录水平(D)。
实施例11：两歧双歧杆菌Bbifi-1对放射性肠炎模型小鼠的治疗效果
冻干保护剂和菌粉制备方法同实施例10。试验动物：20只SPF级雄性C57BL/6J小鼠，体重20-25g，购自北京维通利华实验动物技术有限公司，饲养于SPF级动物房。试验设计：采用X射线腹部辐照诱导放射性肠炎小鼠模型。根据小鼠初始体重随机分为4组，分别为正常对照组、模型对照组、阳性对照组和两歧双歧杆菌Bbifi-1组，每组5只。除正常对照组不做辐照处理，其他组别均进行单次X射线腹部辐照造模处理，造模剂量为11.5Gy。所有组别给药方式均为灌胃，正常对照组、模型对照组灌胃生理盐水；阳性对照组灌胃1×109CFU鼠李糖乳杆菌GG(Lactobacillus rhamnosus GG，LGG，购自陕西泽朗生物科技有限公司)；两歧双歧杆菌Bbifi-1组灌胃1×109CFU两歧双歧杆菌Bbifi-1。总体实验周期为13天，记为D-2～D10。D-2～D9连续 灌胃给药12天，D0(第3天)进行小鼠X射线腹部单次辐照处理。
腹泻观察与评分：将小鼠置于垫有洁净滤纸的小鼠笼内，每笼1只。粪便硬，正常便视为0分；轻度、轻微湿便或软便视为1分；中度，湿便、粪便不成形且肛周不洁视为2分；重度，稀便且严重肛周不洁视为3分。实验周期内，每天对小鼠粪便进行观察、评分。腹泻总分为每天腹泻评分的总和。在D10(第13天)实验动物经戊巴比妥钠麻醉安乐死后行大体解剖及解剖观察。具体实验分组和给药方案见下表8。
表8.两歧双歧杆菌Bbifi-1治疗X射线致腹泻小鼠试验分组和给药方案
注：CFU:菌落形成单位；d:天
试验结果如图15所示，A是第1-12天各组腹泻评分曲线，B是各组腹泻总分图。由此可见，与模型对照组相比，两歧双歧杆菌Bbifi-1对X射线辐照引起的腹泻有显著的改善作用，且与阳性对照组LGG的效果相当(A)；LGG和两歧双歧杆菌Bbifi-1均可显著降低腹泻总分(P<0.05，B)。以上结果表明两歧双歧杆菌Bbifi-1对X射线辐照诱导的小鼠腹泻有显著改善作用。
实施例12：各菌株之间共培养特性测试
将实施例1所得5个菌株分别活化培养至对数后期。用一次性无菌棉签蘸取其中一个菌株的菌液在BF839固体培养基上平行划线三次，然后其余四个菌株的菌液分别与第一次划线垂直的方向上平行划线1次。待划线菌液晾干后厌氧培养48h至菌液痕迹明显。五株菌各菌株之间的互作关系如图16所示。由此可见，各菌株交叉处无断点，说明各菌株之间无生长抑制现象。
由技术常识可知，本发明可以通过其它的不脱离其精神实质或必要特征的实施方案来实现。因此，上述公开的实施方案，就各方面而言，都只是举例说明，并不是仅有的。所有在本发明范围内或在等同于本发明的范围内的改变均被本发明包含。

Claims (12)

  1. 一种益生菌组合物，所述益生菌组合物中的活性成分包括以下任一种或两种及以上的组合：保藏编号为CCTCC NO：M 2023349的两歧双歧杆菌或其纯培养物、保藏编号为CCTCC NO：M 2023350的鸟肠球菌或其纯培养物、保藏编号为CCTCC NO：M 2023348的唾液乳杆菌或其纯培养物、保藏编号为CCTCC NO：M 2023352的发酵黏液乳杆菌或其纯培养物以及保藏编号为CCTCC NO：M 20222033的迪氏副拟杆菌或其纯培养物。
  2. 根据权利要求1所述的益生菌组合物，其特征在于，所述益生菌组合物中还含有冻干保护剂、食品材料、药学上可接受的载体和/或药学上可接受的赋形剂。
  3. 一种分离的迪氏副拟杆菌菌株或其纯培养物，所述迪氏副拟杆菌菌株的微生物保藏编号为CCTCC NO:M20222033。
  4. 一种分离的发酵黏液乳杆菌菌株或其纯培养物，所述发酵黏液乳杆菌菌株的微生物保藏编号为CCTCC NO：M2023352。
  5. 一种分离的唾液乳杆菌菌株或其纯培养物，所述唾液乳杆菌菌株的微生物保藏编号为CCTCC NO:M2023348。
  6. 一种分离的鸟肠球菌菌株或其纯培养物，所述鸟肠球菌菌株的微生物保藏编号为CCTCC NO：M2023350。
  7. 一种分离的两歧双歧杆菌菌株或其纯培养物，所述两歧双歧杆菌菌株的微生物保藏编号为CCTCC NO：M2023349。
  8. 一种预防、治疗或减缓肠道疾病的方法，所述方法包括，将治疗有效量的权利要求1或2所述的益生菌组合物或权利要求3-7任意一项所述的菌株或其纯培养物施用给受试者。
  9. 根据权利要求8所述的方法，其特征在于，所述受试者为哺乳动物，进一步优选所述受试者为人。
  10. 根据权利要求8所述的方法，其特征在于，权利要求1或2所述的益生菌组合物或权利要求3-7任意一项所述的菌株或其纯培养物抑制所述受试者的肠道病原菌、改善所述受试者的结肠损伤、抑制所述受试者的肠道炎症、修复所述受试者的肠屏障或改善所述受试者的腹泻症状。
  11. 根据权利要求8所述的方法，其特征在于，所述肠道疾病选自肠道病原菌感染、腹泻和放射性肠炎，优选所述肠道病原菌选自铜绿假单胞菌、乙型副伤寒沙门氏菌、小肠结肠炎耶尔森菌、金黄色葡萄球菌、副溶血性弧菌、艰难梭菌、志贺氏菌和大肠杆菌中的一种或多种；优选所述腹泻为抗肿瘤药物引起的腹泻，进一步优选抗肿瘤药物引起的腹泻为化疗药物引起的腹泻。
  12. 根据权利要求11所述的益生菌组合物，其中，所述化疗药物引起的腹泻是选自以下一种或多种药物引起的腹泻：阿霉素、表阿霉素、放线菌素D、多柔比星、柔红霉素、紫杉醇、多西他赛、白蛋白紫杉醇、顺铂、卡铂、奈达铂、草酸铂、洛铂、环磷酰胺、氮芥、卡莫司汀、喜树碱、羟基喜树碱、拓扑替康、伊立替康、卡培他滨、吉西他滨、甲氨蝶呤、5-氟尿嘧啶、培美曲塞和阿糖胞苷。
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