WO2024251031A1 - 一种防治抗肿瘤治疗相关性腹泻的益生菌组合物及其应用 - Google Patents

一种防治抗肿瘤治疗相关性腹泻的益生菌组合物及其应用 Download PDF

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WO2024251031A1
WO2024251031A1 PCT/CN2024/096562 CN2024096562W WO2024251031A1 WO 2024251031 A1 WO2024251031 A1 WO 2024251031A1 CN 2024096562 W CN2024096562 W CN 2024096562W WO 2024251031 A1 WO2024251031 A1 WO 2024251031A1
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Prior art keywords
probiotic
caused
diarrhea
strain
treat
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English (en)
French (fr)
Inventor
承磊
李璟欣
张旭朏
兰燕
高翔
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Sichuan Anaerobic Biotechnology Co Ltd
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Sichuan Anaerobic Biotechnology Co Ltd
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Priority to KR1020257029233A priority Critical patent/KR20250141794A/ko
Priority to JP2025553531A priority patent/JP2026508600A/ja
Priority to US19/163,427 priority patent/US20260115237A1/en
Priority to EP24818563.9A priority patent/EP4644531A1/en
Publication of WO2024251031A1 publication Critical patent/WO2024251031A1/zh
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • 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
    • 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
    • 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
    • A61K35/745—Bifidobacteria
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/12—Antidiarrhoeals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • 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; 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
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/32—Foods, ingredients or supplements having a functional effect on health having an effect on the health of the digestive tract
    • A23V2200/3204—Probiotics, living bacteria to be ingested for action in the digestive tract
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/46—Streptococcus ; Enterococcus; Lactococcus
    • 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 present invention belongs to the field of microbial medicines and relates to a probiotic composition and an application thereof, and specifically relates to a composition containing five probiotics and an application thereof in preventing and/or treating anti-tumor therapy-associated diarrhea.
  • Cancer treatment-related diarrhea/tumor-associated diarrhea is a common complication of various adjuvant anti-tumor therapies. It is one of the symptoms that has the greatest impact on health and quality of life. In severe cases, it may also lead to delayed treatment and reduced compliance, which may affect the long-term outcome of tumor treatment and is potentially fatal. According to data released by the International Agency for Research on Cancer under the World Health Organization, there will be approximately 19.3 million new cancer cases worldwide in 2020, and it is predicted that the number of new cancer cases worldwide will reach 28.4 million in 2040. As many as 82% of cancer treatment patients will suffer from side effects such as diarrhea.
  • Chemotherapy especially chemotherapy using traditional chemotherapy drugs such as fluoropyrimidines and irinotecan (CPT-11), which are first-line drugs for patients with metastatic colorectal cancer
  • radiotherapy targeted therapy
  • immunotherapy are the main factors causing anti-tumor treatment-related diarrhea other than surgery.
  • the overall incidence of diarrhea caused by fluoropyrimidines and irinotecan treatment is 50% to 80%, and the incidence of severe (grade 3 to 5) diarrhea is more than 30%, of which 1 to 5% of subjects using 5-fluorouracil (5-FU) died from diarrhea.
  • TKI tyrosine kinase inhibitors
  • afatinib, ceritinib, erlotinib, and lapatinib can be as high as 90% or more.
  • Immune checkpoint inhibitors often lead to immune enteritis.
  • ipilimumab, nivolumab, and pembrolizumab to treat cancer, 1% to 45% of cancer patients will experience diarrhea.
  • Anti-tumor therapy-related diarrhea is caused by multiple factors, and its main pathogenic factors are the following: 1) Chemotherapy drugs and ionizing radiation during radiotherapy can cause oxidative stress and DNA damage; 2) Intestinal mucosal damage leads to epithelial cell loss, reduced tight junction integrity, crypt epithelial cell death, and increased intestinal permeability; 3) Multiple signaling pathways such as nuclear factor NF- ⁇ B are highly activated, stimulating cells to produce inflammatory factors, leading to mucosal inflammation; 4) Damage to the small intestinal mucosa leads to poor bile acid absorption, and excessive bile acid (especially dihydroxy components) induces water and electrolyte secretion and increases intestinal peristalsis, leading to diarrhea; 5) Common hospital-acquired pathogens (Salmonella, Shigella, Escherichia coli, Clostridium difficile, etc.) infection; 6) Bacteria Dislocation, intestinal f
  • Octreotide (the first-line drug for patients with diarrhea scores above grade 3) can cause side effects such as gallstones, hyperglycemia, and impaired glucose tolerance.
  • the guidelines also recommend budesonide as a second-line treatment when loperamide treatment is ineffective, but glucocorticoids can exert systemic effects, causing an increased risk of infection and further deterioration of viral, bacterial and other infections.
  • Patients with grade 3-4 diarrhea and neutropenia can take oral antibiotics, but antibiotics can cause worsening diarrhea and an increased risk of Clostridium difficile infection. Cancer treatment usually involves a combination of multiple treatments. Combination therapy greatly increases the incidence of diarrhea.
  • Intestinal probiotics are a type of active microorganisms that colonize in the human intestine and change the composition of the host's intestinal flora, which are beneficial to the host. Intestinal probiotics have the advantages of high safety, low toxicity and side effects, and a wide range of indications and populations. Probiotic combinations can play a therapeutic role in the complex pathogenesis of anti-tumor therapy-related diarrhea, mainly reflected in: 1) Probiotics play an antioxidant function by chelating metal ions, upregulating their own and host antioxidant enzymes and metabolites, and reducing the activity of ROS-producing enzymes; 2) Probiotics can produce beneficial secondary metabolites, such as short-chain fatty acids, indole derivatives, secondary bile acids, etc.
  • Beneficial secondary metabolites promote goblet cells to secrete mucin, promote epidermal cell repair, inhibit cell apoptosis, enhance tight junction proteins, etc., thereby enhancing intestinal barrier function; 3) Probiotics regulate immune responses by downregulating the NF- ⁇ pathway, inhibiting proinflammatory factors, and secreting anti-inflammatory factors, thereby playing an anti-inflammatory and enhancing the body's immunity; 4) Probiotics regulate the balance of intestinal flora, reduce pathogenic bacteria, increase probiotics, and maintain a normal intestinal homeostatic environment; 5) Probiotics regulate bile acid metabolism by increasing the bile salt hydrolase activity of the flora, inhibiting the FXR-FGF15 signaling pathway, and relieving bile acid diarrhea; 6) Probiotics inhibit the growth of pathogenic bacteria by increasing the secretion of antimicrobial proteins (defensins), producing antimicrobial substances such as bacteriocins, and competing with pathogens for adhesion sites.
  • antimicrobial proteins defensins
  • Chinese patent CN112694992B discloses a strain of Bifidobacterium bifidum that can relieve diarrhea caused by enterotoxigenic Escherichia coli (ETEC).
  • Chinese patent CN113234619B discloses a strain of Bifidobacterium bifidum that can relieve acute intestinal damage.
  • patent CN110878267B discloses a Lactobacillus salivarius ZLp4b that can significantly slow down and cure diarrhea in young animals.
  • probiotics to prevent and treat diarrhea or intestinal inflammation.
  • current probiotic preparations are mainly used to treat common diarrhea symptoms, and most of them are single strains, which are difficult to play a targeted therapeutic role against the complex pathogenesis of anti-tumor therapy-related diarrhea.
  • a first aspect of the present invention provides a probiotic combination, wherein the probiotic combination contains any three, four or five of a first probiotic, a second probiotic, a third probiotic, a fourth probiotic and a fifth probiotic (preferably, the probiotic combination contains the fifth probiotic and any two, three or four of the first probiotic, the second probiotic, the third probiotic and the fourth probiotic; more preferably, the probiotic combination contains the fourth probiotic, the fifth probiotic and any two or three of the first probiotic, the second probiotic and the third probiotic);
  • the first probiotic is selected from Bifidobacterium bifidum, a progeny strain of the Bifidobacterium bifidum, a cloned strain of the Bifidobacterium bifidum or the Bifidobacterium bifidum
  • the second probiotic is selected from Enterococcus avium, a progeny strain of Enterococcus avium,
  • the microbial preservation number of the Bifidobacterium bifidum is CCTCC NO: M2023349; the microbial preservation number of the Enterococcus avium is CCTCC NO: M2023350; the microbial preservation number of the Lactobacillus salivarius is CCTCC NO: M2023348; the microbial preservation number of the Lactobacillus mucis fermentans is CCTCC NO: M2023352; and the microbial preservation number of the Parabacteroides difficile is CCTCC NO: M20222033.
  • the probiotic combination contains a first probiotic, a second probiotic, a third probiotic, a fourth probiotic and a fifth probiotic;
  • the first probiotic is selected from Bifidobacterium bifidum with a microbial preservation number of CCTCC NO: M2023349, a progeny strain of the Bifidobacterium bifidum with a microbial preservation number of CCTCC NO: M2023349, a cloned strain of the Bifidobacterium bifidum with a microbial preservation number of CCTCC NO: M2023349 or a pure culture of the Bifidobacterium bifidum with a microbial preservation number of CCTCC NO: M2023349;
  • the second probiotic is selected from Enterococcus avium with a microbial preservation number of CCTCC NO: M2023350, a progeny strain of Enterococcus avium with a microbial preservation
  • the cloned strain of the avian enterococcus of NO:M2023350 or the microbial deposit number is CCTCC NO:M2023350;
  • the third probiotic is selected from Lactobacillus salivarius with a microbial preservation number of CCTCC NO:M2023348, a progeny strain of Lactobacillus salivarius with a microbial preservation number of CCTCC NO:M2023348, a cloned strain of Lactobacillus salivarius with a microbial preservation number of CCTCC NO:M2023348, or a pure culture of Lactobacillus salivarius with a microbial preservation number of CCTCC NO:M2023348;
  • the fourth probiotic is selected from Lactobacillus mucosa fermented with a microbial preservation number of CCTCC NO:M2023352, a progeny strain of Lactobacillus mucosa fermented with a microbial preservation number of CCTCC NO:M2023352
  • the fifth probiotic is selected from the group consisting of Parabacteroides deaconii with a microbial preservation number of CCTCC NO:M20222033, a progeny strain of Parabacteroides deaconii with a microbial preservation number of CCTCC NO:M20222033, a clonal strain of Parabacteroides deaconii with a microbial preservation number of CCTCC NO:M20222033, or a pure culture of Parabacteroides deaconii with a microbial preservation number of CCTCC NO:M20222033.
  • the second aspect of the present invention provides a microecological composition, wherein the microecological composition uses the probiotic combination described in the first aspect of the present invention as an active material.
  • the composition further contains an excipient, and the excipient is selected from a freeze-drying protectant, a bacterial culture medium, a food additive, an acceptable carrier or excipient in a health product, and a pharmaceutically acceptable carrier or excipient.
  • the excipient is selected from a freeze-drying protectant, a bacterial culture medium, a food additive, an acceptable carrier or excipient in a health product, and a pharmaceutically acceptable carrier or excipient.
  • the content ratio of any two bacteria is 100CFU: 1-10000CFU (for example, 100CFU: 1CFU, 2CFU, 3CFU, 4CFU, 5CFU, 6CFU, 7CFU, 8CFU, 9CFU, 10CFU, 20CFU, 30CFU, 40CFU, 50CFU, 60CFU, 70CFU, 80 CFU, 90CFU, 100CFU, 200CFU, 300CFU, 400CFU, 500CFU, 600CFU, 700CFU, 800CFU, 900CFU, 1000CF U, any value among 2000CFU, 3000CFU, 4000CFU, 5000CFU, 6000CFU, 7000CFU, 8000CFU, 9000CFU, 10000CFU).
  • the third aspect of the present invention provides the use of the probiotic combination described in the first aspect of the present invention or the microecological composition described in the second aspect of the present invention in the preparation of a product for use alone or in combination with other microbial preparations and/or drugs to improve the health of a subject; the improving the health of the subject is selected from: inhibiting any one, any two, any three, any four, any five of Pseudomonas aeruginosa, Shigella dysenteriae, Staphylococcus aureus, Escherichia coli, Salmonella paratyphi B, Yersinia enterocolitica, Vibrio parahaemolyticus and Clostridium difficile in the subject's body cavity (e.g., intestinal cavity).
  • the improving the health of the subject is selected from: inhibiting any one, any two, any three, any four, any five of Pseudomonas aeruginosa, Shigella dysenteriae, Staphylococcus aure
  • the product is a food, a health product or a medicine.
  • the subject is selected from a human and a mouse.
  • the present invention provides the use of the probiotic combination described in the first aspect of the present invention or the microecological composition described in the second aspect of the present invention in the preparation of a drug for treating and/or preventing toxic side effects associated with anti-tumor therapy.
  • the anti-tumor therapy-related toxic side effect is diarrhea.
  • the anti-tumor therapy-related toxic side effects are diarrhea caused by anti-tumor drugs, diarrhea caused by radiotherapy, or intestinal damage caused by radiotherapy.
  • the anti-tumor drug is selected from chemotherapeutic drugs, targeted drugs and immune checkpoint inhibitors.
  • the anti-tumor drug is selected from 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, cytarabine, apatinib, axitinib, cabozantinib, sorafenib, sunitinib, nivolumab, pembrolizumab, and ipilimumab.
  • the fourth 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 microecological composition described in the second aspect of the present invention to a subject;
  • the intestinal disease is selected from: intestinal diseases caused by any one, any two, any three, any four, any five, any six, any seven or eight of Pseudomonas aeruginosa, Shigella dysenteriae, Staphylococcus aureus, Escherichia coli, Salmonella paratyphi B, Yersinia enterocolitica, Vibrio parahaemolyticus, and Clostridium difficile; oxidative damage in the intestine; diarrhea caused by antitumor drugs; intestinal inflammation caused by antitumor drugs; intestinal damage caused by antitumor drugs; diarrhea caused by radiotherapy; intestinal inflammation caused by radiotherapy; and intestinal damage caused by radiotherapy.
  • the subject is selected from a human and a mouse.
  • the administration to the subject is selected from oral administration, intraperitoneal injection, and oral gavage.
  • the therapeutically effective dose is 10 6-12 CFU (for example, 1 ⁇ 10 6 CFU, 2 ⁇ 10 6 CFU, 3 ⁇ 10 6 CFU, 4 ⁇ 10 6 CFU, 5 ⁇ 10 6 CFU, 6 ⁇ 10 6 CFU, 7 ⁇ 10 6 CFU, 8 ⁇ 10 6 CFU, 9 ⁇ 10 6 CFU, 1 ⁇ 10 7 CFU, 2 ⁇ 10 7 CFU, 3 ⁇ 10 7 CFU, 4 ⁇ 10 7 CFU, 5 ⁇ 10 7 CFU, 6 ⁇ 10 7 CFU, 7 ⁇ 10 7 CFU, 8 ⁇ 10 7 CFU, 9 ⁇ 10 7 CFU, 1 ⁇ 10 8 CFU, 2 ⁇ 10 8 CFU, 3 ⁇ 10 8 CFU, 4 ⁇ 10 8 CFU, 5 ⁇ 10 8 CFU, 6 ⁇ 10 8 CFU, 7 ⁇ 10 8 CFU, 8 ⁇ 10 8 CFU, 9 ⁇ 10 8 CFU, 1 ⁇ 10 9 CFU, 2 ⁇ 10 9 CFU, 3 ⁇ 10 9 CFU, 4 ⁇ 10 9 CFU, 5 ⁇ 10 9 CFU,
  • the anti-tumor drug is selected from 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, cytarabine, apatinib, axitinib, cabozantinib, sorafenib, sunitinib, nivolumab, pembrolizumab and ipilimumab.
  • the probiotic combination provided by the present invention contains 3, 4 or 5 probiotics of different species, and there is no mutual antagonism between the strains, and there are no virulence factors and good safety;
  • the probiotic combination provided by the present invention has the ability of self-aggregation/co-aggregation, anti-oxidation, production of short-chain fatty acids, inhibition of multiple pathogens, and strong in vitro adhesion ability, and can synergistically play a role in the complex pathogenesis of anti-tumor therapy-related diarrhea;
  • the probiotic combination provided by the present invention can prevent and/or treat toxic side effects such as diarrhea, intestinal inflammation and intestinal damage caused by chemotherapy drugs or radiotherapy.
  • Figure 1 is a frontal photograph of the colony morphology of the five strains.
  • Figure 2 is a photo of the co-culture characteristics of five strains of bacteria on BF839 agar medium.
  • FIG. 3 shows the probiotic composition and its single bacteria self-aggregation (co-aggregation) ability.
  • FIG. 4 shows the results of the antibacterial activity test of the probiotic composition and its single bacteria against pathogenic bacteria.
  • FIG. 5 shows the results of the test of the adhesion ability of the probiotic composition and its single bacteria to Caco2 cells.
  • the specific temperature parameters in the present invention should be understood as constant temperature treatment, and allow There are fluctuations within a certain temperature range (such as fluctuations within the range of ⁇ 5°C, ⁇ 4°C, ⁇ 3°C, ⁇ 2°C, ⁇ 1°C).
  • the present invention provides a probiotic combination, which contains any three, four or five of Bifidobacterium bifidum, Enterococcus avium, Ligilactobacillus salivarius, Limosilactobacillus fermentum and Parabacteroides distasonis.
  • the microbial preservation number of the Bifidobacterium bifidum is CCTCC NO: M2023349;
  • the microbial preservation number of the avian enterococcus is CCTCC NO: M2023350;
  • the microbial preservation number of the Lactobacillus salivarius is CCTCC NO: M2023348;
  • the microbial preservation number of the fermented mucus lactobacillus is CCTCC NO: M2023352;
  • the microbial preservation number of the Parabacteroides dienii is CCTCC NO:M20222033.
  • the meaning of the strain with a specific deposit number of the present invention includes but is not limited to: (1) a strain with a specific deposit number stored in the said deposit center; (2) a strain having the same genome as the strain described in (1); (3) a subculture strain without gene mutations based on the aforementioned (1) or (2); (4) a subculture strain based on the aforementioned (1), (2) or (3) that accumulates minor mutations during subculture but has no substantial changes in toxicity, immunogenicity and biological activity; (5) a live bacterium based on any of the aforementioned strains (1)-(4), a killed product of the live bacterium, a lysate of the live bacterium or a fermentation product of the live bacterium, 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 corresponding to 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 subculture strains without gene mutations. As known to those skilled in the art, minor mutations are usually inevitably introduced into strains after subculture.
  • mutations occur in non-coding sequence regions or synonymous mutations in coding regions or mutations that do not affect the toxicity (biosafety), immunogenicity and biological activity of the strain (for example, it is possible that mutations occur in the connecting amino acid residues between two domains or the mutated amino acid residues are located inside the higher structure of the protein because they do not contact immune cells, these mutations do not affect toxicity, immunogenicity and biological activity).
  • the mutated strains can still achieve the purpose of the present invention, and the mutated strains are derived from the strains contributed by the present invention, so the corresponding strains are still within the scope of the substantial technical contribution of the present invention.
  • These minor mutations are still non-substantial mutations and should be regarded as mutant strains with no changes in toxicity, immunogenicity and biological activity.
  • the toxicity, immunogenicity and biological activity of the strain including but not limited to, the toxicity, immunogenicity and biological activity of the strain considered as a mutation within the limits of detection technology such as detection sensitivity and detection limit and acceptable or unavoidable errors are the same as the strain contributed by the present invention.
  • the toxicity, immunogenicity and biological activity of the offspring of the strain are measured using cells, animals, etc., there is no substantial change due to differences in cell strains, animal species, age, sex, health status, culture conditions, etc., as well as predictable or unavoidable systematic errors.
  • Active ingredients refer to substances that act as components that produce biological effects. In the present invention, the active ingredient is a probiotic strain.
  • Anti-tumor therapy-related diarrhea or tumor-related diarrhea refers to diarrhea caused by various anti-tumor therapies that damage the intestinal mucosa, leading to imbalance in intestinal absorption and secretion.
  • Common anti-tumor therapy-related diarrhea includes chemotherapy-related diarrhea, diarrhea caused by radiotherapy, diarrhea caused by targeted therapy, diarrhea caused by immune checkpoint inhibitors, etc.
  • Radiotherapy-induced diarrhea is clinically manifested as symptoms of radiation enteritis or radiation intestinal injury.
  • Chemotherapy refers to the use of non-selective chemical drugs to kill tumor cells to achieve the purpose of treatment. Chemotherapy is the main means of treating tumors in addition to surgery and radiotherapy. Due to the lack of selectivity, chemotherapy will damage normal cells while killing tumor cells.
  • Common chemotherapy drugs include but are not limited to antibiotic chemotherapy drugs (such as doxorubicin, epirubicin, actinomycin D, doxorubicin, daunorubicin and its derivatives, etc.), taxane chemotherapy drugs (such as paclitaxel, docetaxel, albumin paclitaxel and its derivatives, etc.), platinum chemotherapy drugs (such as cisplatin, carboplatin, nedaplatin, oxaliplatin, lobaplatin and its derivatives, etc.), alkylating agent chemotherapy drugs (such as cyclophosphamide, nitrogen mustard, carmustine and its derivatives, etc.), camptothecin chemotherapy drugs (such as camptothecin, hydroxycamptothecin, topotecan, irinotecan and its derivatives, etc.) and antimetabolite chemotherapy drugs (such as capecitabine, gemcitabine, methotrexate, 5-fluorouracil, pemetrexed, cytarabine and
  • Targeted therapy is to design corresponding therapeutic drugs for the identified carcinogenic sites at the cellular and molecular level.
  • the drug will specifically select the carcinogenic sites to combine and act to specifically kill tumor cells.
  • the emergence of new targeted drugs has changed the tumor treatment model and opened up the era of targeted therapy.
  • Common targeted drugs include apatinib, axitinib, cabozantinib, sorafenib, sunitinib, etc.
  • Immune checkpoint inhibitors are monoclonal antibody drugs developed for corresponding immune checkpoints.
  • the main function of immune checkpoint inhibitors is to block the interaction between tumor cells expressing immune checkpoints and immune cells, thereby blocking the inhibitory effect of tumor cells on immune cells.
  • Common immune checkpoint inhibitors include but are not limited to PD-1/PD-L1 inhibitor antibodies (such as nivolumab, pembrolizumab, etc.) and CTLA-4 inhibitors (such as ipilimumab, etc.).
  • Radiotherapy refers to the use of radiation (such as alpha rays, beta rays, gamma rays produced by radioisotopes, and x-rays, electron beams, proton beams and other particle beams produced by various x-ray therapy devices or accelerators) to treat malignant tumors.
  • radiation such as alpha rays, beta rays, gamma rays produced by radioisotopes, and x-rays, electron beams, proton beams and other particle beams produced by various x-ray therapy devices or accelerators
  • Commonly used radiotherapy methods include but are not limited to conventional radiotherapy, stereotactic radiotherapy, three-dimensional conformal radiotherapy, intensity modulated radiotherapy, image-guided radiotherapy, volumetric arc intensity modulated radiotherapy, proton radiotherapy, etc.
  • the present invention provides a microecological composition, which contains any three or four of Bifidobacterium bifidum, Enterococcus avium, Ligilactobacillus salivarius, Limosilactobacillus fermentum, and Parabacteroides distasonis. 1 or 5 kinds and auxiliary materials.
  • a microecological composition refers to a biologically active preparation that contains specific microbial species and/or their metabolites.
  • the composition promotes the host's health, enhances resistance to disease, or improves the host's physiological function by restoring or optimizing the structure and function of the microbial community in the host.
  • the auxiliary materials vary depending on the type of product being prepared, for example, food, health products or medicines can be prepared. Accordingly, the auxiliary materials can be selected from freeze-drying protective agents, bacterial culture media, food additives, acceptable carriers or auxiliary materials in health products and pharmaceutically acceptable carriers or auxiliary materials.
  • the invention provides use of a probiotic combination or a microecological composition in preparing food, health products or medicines.
  • the probiotic combination or microecological composition When prepared into medicines, the probiotic combination or microecological composition is used in the preparation of medicines for use alone or in combination with other microbial preparations and/or drugs to improve the health status of subjects.
  • the present invention also provides a method for preventing, treating or alleviating intestinal diseases, which comprises administering a therapeutically effective amount of the microecological composition to a subject.
  • the therapeutically effective amount or preventive effective amount is an amount that can achieve the desired therapeutic or preventive effect clinically. In some embodiments, the therapeutically effective amount does not induce or cause undesirable side effects. In some embodiments, the therapeutically effective amount induces or causes side effects, but only causes side effects that are acceptable to clinicians in view of the patient's condition. In some embodiments, the total number of bacteria contained in a single dose or a single effective amount is 10 2 to 10 15 CFU, 10 3 to 10 14 CFU, 10 4 to 10 13 CFU, 10 5 to 10 12 CFU or 10 6 to 10 12 CFU.
  • a pharmaceutically acceptable carrier refers to a pharmaceutical carrier that does not cause significant irritation to the subject and does not eliminate the biological activity and properties of the administered probiotics.
  • a pharmaceutically acceptable carrier can enhance or stabilize the composition, or can be used to promote the preparation of the composition.
  • Pharmaceutically acceptable carriers may include solvents, dispersion media, coatings, surfactants, antioxidants, isotonic agents, absorption delay agents, salts, drug stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, protective agents, etc. and combinations thereof.
  • the carrier is considered to be used in a therapeutic composition or a pharmaceutical composition.
  • the carrier may be selected to minimize adverse side effects to the subject and/or minimize the inactivation of the active ingredient.
  • Excipients refer to substances added to pharmaceutical compositions to give the drug a certain shape or a certain concentration, for example, sterile water, physiological saline, polyalkylene glycols (such as polyethylene glycol), vegetable oils, hydrogenated naphthalene, calcium bicarbonate, calcium phosphate, various sugars, various types of starch, cellulose derivatives, gelatin, etc.
  • the microecological composition of the present invention may further contain a second beneficial active ingredient, for example, another probiotic, prebiotic, or drug with antidiarrhea function.
  • a second beneficial active ingredient for example, another probiotic, prebiotic, or drug with antidiarrhea function.
  • Prebiotics help regulate the intestinal environment by promoting the growth of probiotics in the intestine, thereby indirectly exerting an antidiarrhea effect.
  • Examples of the second beneficial active ingredient include, but are not limited to, Bacillus licheniformis, Bifidobacterium, Clostridium butyricum, oligofructose, Galacto-oligosaccharides, isomalto-oligosaccharides, xylo-oligosaccharides, manno-oligosaccharides, inulin, stachyose, soybean oligosaccharides, beta-glucan, lactofructo-oligosaccharides, etc.
  • the dosage form of the pharmaceutical composition is tablets, capsules, granules, solutions, suspensions, powders, etc.
  • the culture medium preparation method used in the following examples is as follows:
  • 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 value to 5.5 with 50% HCl to obtain a nitrilotriacetic acid aqueous solution.
  • 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. Adjust the pH of the above solution to neutral with 5M NaOH before use.
  • BHI+MRS+modified GAM Dissolve 19.25 g of BHI broth powder (Qingdao Haibo Biotechnology Co., Ltd., HB8297-5), 13.5 g of MRS broth powder (Guangdong Huankai Biotechnology Co., Ltd., 027312), and 15 g of modified GAM broth powder (Qingdao Haibo Biotechnology Co., Ltd., HB8518-3) in 1 L of distilled water to obtain a solution. Perform N2 replacement and deoxygenation on the solution and package it. Sterilize the packaged solution at 121°C for 30 minutes. Store the obtained triple-mixed liquid culture medium in a cool and dry place.
  • Preparation of three-mix solid culture medium add 5 g of agar powder to the above three-mix liquid culture medium, and the other steps are the same as the preparation of three-mix liquid culture medium.
  • BF839 liquid 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), Peptone (Haibo Biotechnology Co., Ltd., Qingdao High-Tech Industrial Park, HB8277) 5.0 g, sodium thioglycolate (Shanghai Aladdin Biochemical Technology Co., Ltd., S105664-25G) 0.3 g, yeast extract powder 5.0 g of (Thermo Fisher Oxoid, LP0021B), 1.5 g of glucose (Chengdu Kelong Chemical Co., Ltd., 50-99-7) and 4.0 g of disodium hydrogen phosphate (Chengdu Kelong Chemical Co., Ltd., 7558-79-4) were dissolved in 1 L of distilled water to obtain a solution. The solution was deoxygenated by N2 replacement and aliquoted.
  • BF839 solid culture medium Dissolve 50.4 g of BF839 solid agar (HB8805, Haibo Biotechnology Co., Ltd., Qingdao High-Tech Industrial Park) in 1 L of distilled water to obtain a mixture. Perform N2 replacement and deoxygenation on the mixture and package it. Sterilize the mixture at 121°C for 15 minutes. Store the obtained culture medium in a cool and dry place.
  • Preparation of oxygen-free PBS Dissolve 0.27 g of potassium dihydrogen phosphate, 1.42 g of disodium hydrogen phosphate, 8 g of sodium chloride and 0.2 g of potassium chloride in 1 L of distilled water to obtain a mixture. Heat and boil the mixture, cool the mixture to room temperature and add 0.55 g of cysteine hydrochloride thereto, stir the mixture to dissolve it and adjust the pH to 6.5. Heat the mixture to boiling and keep it at a slight boiling state for 30 minutes. After cooling, use a quantitative dispenser to dispense into 400 mL/bottle under N2 conditions. Sterilize the bottle at 121°C for 30 minutes. Store the obtained PBS in a cool and dry place for later use.
  • GAM solid medium (Qingdao Haibo Biotechnology Co., Ltd., HB8462), TSB (Tryptone Soy Broth, Qingdao Haibo Biotechnology Co., Ltd., HB4114), TSA (Tryptone Soy Agar, Qingdao Haibo Biotechnology Co., Ltd., HB4138) medium were prepared according to the steps specified in the instructions provided by the supplier. The materials were weighed and dissolved in water to obtain a mixture. The mixture was sterilized at 121°C for 30 minutes to obtain the culture medium. The culture medium was stored in a cool and dry place.
  • 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 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 cysteine hydrochloride monohydrate were dissolved in 1L of purified water.
  • the mixed solution was deoxygenated by N2 replacement and aliquoted.
  • the mixed solution was sterilized by high temperature and wet heat at 121°C for 15min.
  • Preparation of culture medium for Lactobacillus salivarius Lsali-1 powder Dissolve 24g of anhydrous glucose, 20g of soy peptone, 10g of yeast extract powder, 10g of peptone, 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 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 preparation of 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 by N2 replacement and aliquot. Sterilize the mixture by high temperature and wet heat at 121°C for 15min.
  • the preparation method of freeze-dried protective agent is as follows:
  • Solution A 6g sucrose, 6g trehalose, 2g xylitol, 2g sorbitol, 44g purified water, sterilized at 115°C for 30min.
  • Solution B 5g sodium glutamate, 15g purified water; sterilized at 115°C for 20min.
  • Solution C 4g sodium vitamin C, 16g purified water. Filter and sterilize for later use.
  • Liquid A 8g sucrose, 8g trehalose, 44g purified water, sterilized at 115°C for 30min.
  • Liquid B 2g sodium glutamate, 2g arginine hydrochloride, 16g purified water, sterilized at 115°C for 30min.
  • Liquid C 4g sodium vitamin C, 16g purified water. Filter and sterilize for later use.
  • 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.
  • the lyophilized protective agent for animal testing is prepared by freeze-drying the prepared lyophilized protective agent, crushing it, and preparing it into a suspension with physiological saline for use.
  • 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 processed 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 .
  • the 16S rDNA gene sequence obtained by sequencing was compared with the NCBI Nucleotide database to further identify the species of the isolated strain.
  • 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 (>99%) with a strain of Parabacteroides distasonis, so strain 1 was named Parabacteroides distasonis Pdist-1 (referred to as Pdist-1).
  • Strain 2 had the highest sequence similarity (100%) with a strain of Limosilactobacillus fermentum, so strain 2 was named Lferm-1 (referred to as Lferm-1).
  • Strain 3 has the highest sequence similarity with a strain of Lactobacillus salivarius (100.00%), which is also called Ligilactobacillus salivarius. Therefore, strain 3 was named Lactobacillus salivarius Lsali-1 (abbreviated as Lsali-1).
  • Strain 4 has the highest sequence similarity with a strain of Enterococcus avium (100.00%), so strain 4 was named Eaviu-1 (abbreviated as Eaviu-1).
  • Strain 5 has the highest sequence similarity with a strain of Bifidobacterium bifidum (99.86%), so strain 5 was named Bifidobacterium bifidum Bbifi-1 (abbreviated as Bbifi-1).
  • 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 average nucleotide similarity (ANI) method was used to analyze the novelty of the five strains.
  • a whole genome search was performed in Genbank, and the most similar strains were compared using fastANI (v1.33).
  • the strains Pdist-1, Eaviu-1, Lferm-1, Bbifi-1, and Lsali-1 isolated and cultured by 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 strains Pdist-1, Eaviu-1, Lferm-1, Bbifi-1 and Lsali-1 obtained in Example 1 were activated and cultured to the late logarithmic period.
  • the interaction relationship between the five bacterial strains is shown in Figure 2. 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.
  • Example 4 Agglutination ability of single bacteria and bacterial combinations
  • strains Pdist-1, Eaviu-1, Lferm-1, Bbifi-1, Lsali-1 and control strain Lactobacillus rhamnosus GG (LGG, CICC6141, purchased from China Industrial Microbiological Culture Collection Center) were inoculated with 2% initial inoculum. Plant in a three-mixed liquid medium and culture anaerobically at 37°C until the late logarithmic growth stage. Wash the cultured bacterial solution three times by centrifugation with anaerobic PBS.
  • Preparation of bacterial composition suspension Mix the Pdist-1 bacterial solution, Eaviu-1 bacterial solution, Lferm-1 bacterial solution, Bbifi-1 bacterial solution and Lsali-1 bacterial solution diluted to the same OD600 value in equal volumes.
  • Example 5 Antioxidant capacity test of bacterial composition
  • Bacterial composition culture After activation, each single bacteria Pdist-1, Eaviu-1, Lferm-1, Bbifi-1 and Lsali-1 were mixed and inoculated into 5 mL of three-mixed culture medium. The inoculation amount of each strain was 1%, and the culture was anaerobically carried out at 37°C for 24 hours.
  • Sample processing Take 0.5mL of culture solution, centrifuge at 12000rpm for 20min, discard the supernatant, and resuspend the bacteria with 0.5ml of the extract in the pre-cooled strain total antioxidant capacity detection kit (the kit was purchased from Beijing Solebow Technology Co., Ltd., BC1315). Transfer the bacteria to a sterilized screw-cap tube containing beads (Sigma-Aldrich, USA, G4649-1KG). Use a rapid sample preparation instrument to oscillate and break the bacterial cell wall once (parameter setting: 4.5m/s, 30s), centrifuge at 12000rpm 4°C for 10min, and take the supernatant and place it on ice for testing.
  • a BCA protein concentration determination kit (the kit was purchased from Beijing Solebow Technology Co., Ltd., PC0020) was used to draw a standard curve and detect BCA samples according to the instructions of the kit.
  • the total antioxidant capacity detection kit of strains was used to determine the antioxidant capacity of the samples according to the instructions of the kit and the standard curve mentioned above.
  • the total antioxidant capacity unit is ⁇ mol/mg prot.
  • the antioxidant capacity of the bacterial composition is 0.189 ⁇ mol/mg prot.
  • the bacterial composition has a certain antioxidant capacity.
  • Example 6 Ability of single bacteria and bacterial combinations to produce short-chain fatty acids
  • the control strains LGG, Pdist-1, Eaviu-1, Lferm-1, Bbifi-1 and Lsali-1 were inoculated into BF839 liquid culture medium at a 5% inoculation rate and cultured anaerobically at 37°C for 24 h.
  • Bacterial composition Pdist-1, Eaviu-1, Lferm-1, Bbifi-1 and Lsali-1 were mixed and inoculated into BF839 liquid culture medium at a 1% inoculation rate, and cultured anaerobically at 37°C for 24 hours.
  • results As shown in Table 2, the control strain LGG, the bacterial composition and its single bacteria all have the ability to produce acetic acid. In addition to acetic acid, Pdist-1 can also produce propionic acid and isovaleric acid. The total SCFAs production of Bbifi-1, Pdist-1 and the bacterial composition is significantly better than that of LGG, and the total SCFAs production of Lsali-1, Eaviu-1 and Lferm-1 is equivalent to that of LGG.
  • Example 7 Ability of single bacteria and bacterial combinations to inhibit pathogenic bacteria
  • CMCC (B) 10104 Pseudomonas aeruginosa (CMCC (B) 10104), purchased from the China Food and Drug Inspection Institute. Shigella dysenteriae (CMCC (B) 51252), purchased from the China Food and Drug Inspection Institute. Staphylococcus aureus (CMCC (B) 26003), purchased from the China Food and Drug Inspection Institute. Escherichia coli (CMCC (B) 44102), purchased from the China Food and Drug Inspection Institute. Institute. Salmonella paratyphi B (CMCC(B)50094) was purchased from China Food and Drug Inspection Institute.
  • CMCC (B) 10104 Pseudomonas aeruginosa (CMCC (B) 10104), purchased from the China Food and Drug Inspection Institute. Shigella dysenteriae (CMCC (B) 51252), purchased from the China Food and Drug Inspection Institute. Staphylococcus aureus (CMCC (B) 26003), purchased from the China Food and Drug Inspection Institute. Escherichi
  • Yersinia enterocolitica CMCC(B)52204 was purchased from China Food and Drug Inspection Institute. Vibrio parahaemolyticus (ATCC 17802) was purchased from American Microbiological Culture Collection. Clostridium difficile (CICC 22951) was purchased from China Industrial Microbiological Culture Collection Administration Center.
  • Bacterial composition Pdist-1, Eaviu-1, Lferm-1, Bbifi-1 and Lsali-1 were mixed and inoculated into a three-mixed liquid culture medium at an inoculation rate of 1% and anaerobically cultured for 48 hours to obtain a fermentation broth.
  • Pseudomonas aeruginosa Shigella dysenteriae, Staphylococcus aureus, Escherichia coli, Salmonella paratyphi B, Yersinia enterocolitica and Vibrio parahaemolyticus were cultured aerobically with TSB medium to the logarithmic growth phase.
  • the culture was diluted with TSB medium to 10 6 CFU/mL, and 200 ⁇ L was applied to TSA solid medium.
  • Clostridium difficile was cultured anaerobically with three mixed liquid medium to the logarithmic growth phase.
  • the culture was diluted with three mixed liquid medium to 10 6 CFU/mL, and 200 ⁇ L was applied to anaerobic GAM solid medium (added with 5v/v% horse serum, Beijing Solebow Technology Co., Ltd., S9050).
  • Three Oxford cups were placed in each plate, and 200 ⁇ L of the fermentation liquid to be tested (the aforementioned culture) was added to the Oxford cup.
  • Clostridium difficile was cultured under anaerobic conditions, and the remaining pathogens were cultured under aerobic conditions. After culturing at 37°C for 24 hours, the diameter of the inhibition zone was measured and the average value was calculated. The results are shown in Figure 4.
  • the bacterial composition has an inhibitory effect on the growth of Pseudomonas aeruginosa, Shigella dysenteriae, Salmonella paratyphi B, Yersinia enterocolitica, Vibrio parahaemolyticus, Staphylococcus aureus, and Clostridium difficile.
  • the bacterial composition makes up for the weakness of single bacteria in inhibiting certain pathogens.
  • the bacterial composition is superior to the control strain LGG in inhibiting the activity of Salmonella paratyphi B and Staphylococcus aureus, and its inhibitory ability on other pathogens is equivalent to that of the control strain LGG.
  • Example 8 Testing of the adhesion ability of single bacteria and bacterial combinations to Caco2 cells
  • the control strains LGG, Pdist-1, Eaviu-1, Lferm-1, Bbifi-1 and Lsali-1 were inoculated into three mixed liquid medium at an initial inoculum volume of 2%, and cultured anaerobically at 37°C until the late logarithmic growth period.
  • the cultured bacterial solution was washed twice by centrifugation with sterile PBS (Wuhan Boster Biotechnology Co., Ltd., PYG0021), and then the strains in the precipitate were diluted to 5 ⁇ 10 8 CFU/mL using DMEM (Thermo Fisher Scientific (China) Co., Ltd., C11995500BT) medium containing 10 v /v% FBS (Thermo Fisher Scientific (China) Co., Ltd., SH30084.03) for later use.
  • DMEM Thermo Fisher Scientific (China) Co., Ltd., C11995500BT
  • 10 v /v% FBS Thermo Fisher Scientific (China) Co., Ltd., SH30084.03
  • Bacterial composition The diluted bacterial solutions of Pdist-1, Eaviu-1, Lferm-1, Bbifi-1 and Lsali-1 were mixed in equal volumes to obtain a bacterial composition.
  • Caco-2 cells (Shangcheng Beina Chuanglian Biotechnology Co., Ltd., 350769) are adherent cells. Digest Caco-2 cells with 37°C preheated trypsin cell digestion solution (Lanjieke Technology Co., Ltd., BL501A). Collect digested Caco-2 cells by centrifugation. Dilute the precipitate with DMEM medium containing 10v/v% FBS. Inoculate Caco-2 cells in a 96-well plate at a density of 5 ⁇ 10 4 CFU/well, place in a 37°C carbon dioxide incubator for overnight culture, and set aside.
  • Figure 5 shows that after each strain adhered to Caco2 cells for 30 minutes, the adhesion ability of avian enterococcus Eaviu-1, bacterial composition and LGG was equivalent.
  • Figure B shows the adhesion effect of each strain when the adhesion time was 2 hours.
  • the adhesion ability of Bifidobacterium bifidum Bbifi-1 and fermented mucus Lactobacillus Lferm-1 was equivalent to LGG, and the adhesion ability of Lactobacillus salivarius Lsali-1, avian enterococcus Eaviu-1 and bacterial composition was better than LGG.
  • the bacterial composition has better adhesion ability and is therefore easier to colonize.
  • Example 9 Test on the therapeutic effect of bacterial composition on mice with diarrhea caused by 5-fluorouracil (5-FU)
  • Pdist-1, Lferm-1, Lsali-1, Eaviu-1, and Bbifi-1 were inoculated into the corresponding culture medium for preparing bacterial powder, and cultured anaerobically at 37°C and 90rpm for 16 to 24 hours to obtain the first-level seed solution.
  • the five first-level seed solutions were transferred to the corresponding culture medium for preparing bacterial powder, and cultured anaerobically at 37°C and 90rpm for 10 to 15 hours to obtain the second-level seed solution.
  • the five second-level seed solutions were pumped into the fermentation tank containing the corresponding culture medium for preparing bacterial powder by peristaltic pumps for fermentation and culture. After stopping the fermentation, the bacteria were collected by centrifugation.
  • the corresponding freeze-dried protective agent was added according to the ratio of 1:1 to 1:2 (weight ratio) between the bacterial mud and the freeze-dried protective agent, and the emulsified bacterial mud was mixed.
  • the bacterial mud was freeze-dried and crushed to obtain the bacterial powder. Take an appropriate amount of each crushed bacterial powder for viable bacterial count determination. According to the viable bacterial count, the bacterial powder of each strain was mixed in an equal CFU ratio to obtain the bacterial composition bacterial powder.
  • the bacterial composition bacterial powder was configured into a bacterial suspension using physiological saline for animal testing. The bacterial suspension was diluted with physiological saline to obtain bacterial compositions with different bacterial doses.
  • mice 96 SPF male Balb/c mice weighing 22-24 g (purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.) were raised in an SPF animal room. The mice were randomly divided into 8 groups according to their initial weight (normal control group, model control group, positive control loperamide group, positive control Zhengchangsheng group, bacterial composition high-dose group, bacterial composition medium-high-dose group, bacterial composition medium-dose group, and bacterial composition low-dose group), with 12 mice in each group.
  • the above 8 groups were gavaged with normal saline (0.2 mL/mouse/day), lyophilized protective agent (0.2 mL/mouse/day), loperamide (purchased from Henan Zhongjie Pharmaceutical Co., Ltd., 20 mg/kg body weight/mouse/day), Zhengchangsheng (containing live Bacillus licheniformis, Northeast Pharmaceutical Group Co., Ltd., 2 ⁇ 10 8 CFU/mouse/day), high-dose group of bacterial composition (1 ⁇ 10 9 CFU/mouse/day), medium-high-dose group of bacterial composition (1 ⁇ 10 8 CFU/mouse/day), medium-dose group of bacterial composition (1 ⁇ 10 7 CFU/mouse/day) and low-dose group of bacterial composition (1 ⁇ 10 6 CFU/mouse/day).
  • mice were injected with 0.2 mL of normal saline per mouse in the abdomen.
  • the other groups of mice were injected with 30 mg/kg body weight of 5-FU (5-fluorouracil, purchased from Tianjin Jinyao Pharmaceutical Co., Ltd., with a specification of 10 mL/vial, 0.25 g/10 mL) for 4 consecutive days to induce the mouse CID model, and the mice were dissected on D9.
  • 5-FU 5-FU
  • 5-FU 5-fluorouracil
  • CFU colony forming unit
  • d day
  • i.p intraperitoneal injection
  • i.g oral administration
  • QD means once a day.
  • the weight of the animals was measured every day, and general observations were recorded (observation items included but were not limited to the appearance of the animals, behavioral activities, respiration, glandular secretions, feces, etc.), and the diarrhea of each animal after modeling was observed and recorded and scored.
  • mid-colon tissue samples were collected for qPCR detection of genes related to inflammatory factors (TNF- ⁇ , IL-1 ⁇ ) and tight junction proteins (ZO-1, Occludin).
  • qPCR detection The total RNA of the colon tissue of each group of mice was extracted by Trizol method, and the total RNA was reverse transcribed into cDNA, which was stored at -20°C for future use.
  • the 2 - ⁇ CT method was used for data analysis.
  • the diarrhea scores of the model group at D8 and D9 after 5-FU induction were significantly higher than those of the normal control group.
  • the four doses of the bacterial composition can significantly improve the diarrhea of the animals, and the diarrhea scores at D8 and D9 and the total diarrhea score are significantly lower than those of the model control group. It can be seen that the bacterial composition of the present invention can improve 5-FU-induced diarrhea.
  • High-dose bacterial composition and loperamide can significantly reduce the relative mRNA transcription level of TNF- ⁇ .
  • High-dose, medium-high-dose and medium-dose bacterial composition can significantly reduce the relative mRNA transcription level of IL-1 ⁇ .
  • the high-dose group, medium-dose group and low-dose bacterial composition can significantly increase the relative mRNA transcription level of ZO-1, which is comparable to the improvement effect of loperamide and Zhengchangsheng.
  • Medium-high-dose and medium-dose bacterial compositions can significantly increase the relative mRNA transcription level of Occludin, which is comparable to the improvement effect of Zhengchangsheng.
  • the bacterial composition of the present invention can significantly reduce the diarrhea score of the CID mouse model induced by 5-FU, and can play a significant therapeutic effect on diarrhea by reducing inflammatory factors and increasing the expression of tight junction proteins.
  • Example 10 Therapeutic effect of bacterial composition on irinotecan (CPT-11)-induced diarrhea in mice
  • the bacterial composition used in Examples 10-12 was obtained by mixing five kinds of bacteria at equal CFU according to the method in Example 9.
  • the CFU of the bacterial composition mentioned in Examples 10-12 is the CFU of five kinds of bacteria.
  • mice The normal control group mice were injected with 0.2 mL of saline per mouse in the abdomen, and the other groups of mice were injected with 85 mg/kg body weight of CPT-11 in the abdomen to establish the model.
  • the model was established from D3 and continued for 4 days from D3 to D6, once a day.
  • the corresponding groups were gavaged and administered once a day for 9 consecutive days, and the animals were dissected on D10.
  • the specific experimental groups and dosing schedules are shown in Table 6.
  • CPT-11 irinotecan hydrochloride
  • CFU colony forming unit
  • d day
  • i.p intraperitoneal injection
  • i.g oral administration
  • QD means once a day.
  • the weight of the animals was measured every day, and general observations were made (observations included but were not limited to the animals' appearance, behavior, breathing, glandular secretions, feces, etc.), with a focus on The diarrhea of each animal after modeling was observed and recorded and scored.
  • Samples were collected from 5 mice in each group near the cecum, and the samples were frozen in liquid nitrogen and stored at -80 ° C. The samples were used for qPCR to detect the mRNA expression levels of related genes such as inflammatory factors (TNF-a, IL-1 ⁇ , IL-22), tight junction proteins (ZO-1, Occludin), pro-apoptotic factors (Bax), and aquaporins (AQP8).
  • the colorectal parts of the remaining 5 mice in each group were directly fixed in 10% formaldehyde solution and stained with HE for pathological examination.
  • Routine blood test Whole blood samples were collected from animals in each group through the peritoneal vein. The samples were tested for routine blood tests using a fully automatic blood cell analyzer (Mindray).
  • RNA of the colon tissue of each group of mice was extracted by Trizol method, and the total RNA was reverse transcribed into cDNA, which was stored at -20°C for future use.
  • the relative transcription levels of mRNA of the pro-inflammatory factors TNF- ⁇ , IL-1 ⁇ , anti-inflammatory factor IL-22, tight junction proteins ZO-1 and Occludin, pro-apoptotic factor Bax, and water channel protein AQP8 genes in the colon of each group of mice were detected by qPCR method (primer sequences are shown in Table 7, and the internal reference gene is ⁇ -actin).
  • Intestinal tissue pathological examination The colorectal parts were directly fixed in 10% formaldehyde solution and subjected to pathological examination after HE staining.
  • mice in the model group showed obvious clinical disease-like manifestations (mainly including diarrhea, weight loss, intestinal atrophy, decreased spleen coefficient, and abnormal numbers of peripheral blood lymphocytes and neutrophils), indicating that the CID model was successfully constructed.
  • the weight of the model mice continued to decrease after modeling until the end of the experiment.
  • High-dose, medium-high-dose, medium-dose bacterial composition and whole intestine can significantly alleviate the weight loss of model mice on D10, and the bacterial composition
  • the degree of relief in the high-dose compound treatment group was comparable to that in the Zhengchangsheng group.
  • the small intestinal atrophy of the model mice in the high-dose, medium-high-dose, low-dose and Zhengchangsheng groups of the bacterial composition was significantly improved.
  • the small intestinal swelling of the model mice in the high-dose, medium-high-dose, medium-dose and Zhengchangsheng groups of the bacterial composition was also significantly improved.
  • the medium-high-dose, medium-dose, low-dose bacterial composition and Zhengchangsheng can significantly improve the spleen atrophy of the model mice.
  • the expression of tight junction protein-related gene ZO-1 in the medium-dose and low-dose groups of the bacterial composition is significantly upregulated, and the expression of tight junction protein-related gene Occludin in the medium-dose and low-dose groups of the bacterial composition is significantly upregulated.
  • the mRNA expression of apoptosis-related gene Bax in the model mice in the high-dose group of the bacterial composition is significantly reduced, and the expression of water channel protein AQP8 is significantly enhanced.
  • the pathological scores of the high-dose group, low-dose group and Zhengchangsheng group of the bacterial composition were significantly lower than those of the model control group.
  • the pathological scores of the high-dose group and Zhengchangsheng group of the bacterial composition showed a decreasing trend compared with the model group.
  • the bacterial composition can reduce CPT-11-induced diarrhea, reduce the mRNA expression of intestinal proinflammatory factors (TNF-a, IL-1 ⁇ , IL-22) and pro-apoptotic factors (Bax), enhance the expression of tight junction proteins (ZO-1, Occludin) and aquaporin (AQP8) mRNA, and significantly improve the symptoms of CPT-11-induced diarrhea, weight loss, small intestinal swelling, spleen atrophy, abnormal number of peripheral blood immune cells (lymphocytes and neutrophils), and intestinal inflammation in mice.
  • TNF-a, IL-1 ⁇ , IL-22 intestinal proinflammatory factors
  • Bax pro-apoptotic factors
  • ZO-1, Occludin tight junction proteins
  • AQP8 aquaporin
  • Example 11 Therapeutic effect of bacterial composition on mice with radiation enteritis
  • mice 80 male C57BL/6 mice (purchased from Chengdu Yaokang Biotechnology Co., Ltd.) were housed in an SPF animal room. The mice were randomly divided into a normal control group (dosage regimen: 0.2 mL of normal saline/mouse/day), a model control group (dosage regimen: 0.2 mL of lyophilized protective agent/mouse/day), a loperamide group (dosage regimen: 15 mg/kg body weight of loperamide), a LGG (Lactobacillus rhamnosus GG, Shaanxi Zelang Biotechnology Co., Ltd.) group (dosage regimen: 1 ⁇ 10 9 CFU of LGG/mouse/day), a high-dose group of the bacterial composition (dosage regimen: 1 ⁇ 10 9 CFU of the bacterial composition/mouse/day), a medium-high-dose group of the bacterial composition (dosage regimen: 1 ⁇ 10 8 CFU of the bacterial
  • mice in other groups were subjected to a single full abdominal X-ray irradiation with an irradiation dose of 11.5 Gy.
  • the overall experimental period was 18 days, recorded as D1 to D18. Each group was gavaged and administered once a day for 17 consecutive days. After 7 days of administration, irradiation was performed on D8 and autopsy was performed on D18.
  • the experimental grouping and dosing regimen are shown in Table 14.
  • CFU colony forming unit
  • d day
  • i.p intraperitoneal injection
  • i.g oral administration
  • QD means once a day.
  • the weight of the animals was measured every day, and general observations were recorded (observation items included but were not limited to the appearance of the animals, behavioral activities, respiration, glandular secretions, feces, etc.), and the diarrhea of each animal after irradiation modeling was observed and recorded and scored.
  • the animals were euthanized, and the colorectal part was directly fixed in 10% formaldehyde solution and stained with HE for pathological examination.
  • mice in the model control group showed obvious symptoms such as diarrhea, weight loss, intestinal mucosal damage and inflammatory infiltration after intraperitoneal irradiation, indicating that the mouse radiation enteritis model was successfully established.
  • mice in the high-dose, medium-dose, low-dose, loperamide and LGG groups of the bacterial composition were significantly improved on D17.
  • the total diarrhea scores of the high-dose bacterial composition group on D17 tended to decrease.
  • the weight loss of mice in the high-dose, low-dose and LGG groups of the bacterial composition was significantly improved at D14.
  • the low-dose bacterial composition can significantly improve the weight loss of model mice at D17.
  • High-dose and medium-dose bacterial compositions, loperamide and LGG all tend to restore the weight of model mice at D17.
  • the pathological scores of the high-dose group, low-dose group and LGG group of the bacterial composition were significantly improved.
  • the pathological scores of the colorectal area of the high-dose group, medium-dose group and loperamide group of the bacterial composition showed an improvement trend.
  • the bacterial composition has a significant therapeutic effect on mice with radiation enteritis induced by whole-abdomen irradiation with X-rays, and can improve the degree of diarrhea and weight loss, and alleviate the degree of intestinal lesions.
  • Example 12 Therapeutic effect of bacterial composition on 5-FU-induced tumor-bearing CID mice
  • mice 50 male BALB/c mice (purchased from Chengdu Yaokang Biotechnology Co., Ltd.) were raised in an SPF animal room.
  • CT26 cells purchased from Beijing Beina Biotechnology Co., Ltd.
  • an inoculation dose 5 ⁇ 10 6 cells/mouse and an inoculation volume of 0.1 mL.
  • 42 mice were selected for subsequent experiments.
  • the aforementioned 42 mice were randomly divided into 7 groups according to the tumor volume, with 6 mice in each group.
  • the above 7 groups were normal control group (dosage regimen: 0.2 mL of lyophilized protective agent/mouse/day), model control group (dosage regimen: 0.2 mL of lyophilized protective agent/mouse/day), positive control loperamide group (dosage regimen: 20 mg/kg body weight of loperamide), whole intestine group (dosage regimen: 2 ⁇ 10 8 CFU of Bacillus licheniformis/mouse/day), high-dose group of bacterial composition (dosage regimen: 1 ⁇ 10 9 CFU of bacterial composition/mouse/day), medium-dose group of bacterial composition (dosage regimen: 1 ⁇ 10 8 CFU of bacterial composition/mouse/day) and low-dose group of bacterial composition (dosage regimen: 1 ⁇ 10 7 CFU of bacterial composition/mouse/day).
  • the day of grouping was set as the first day of the experiment (D1), and the drug administration started on the first day (D1), and the drug was administered by gavage once a day (QD) for 9 consecutive days.
  • the normal control group was intraperitoneally injected with 0.2 ml of normal saline per mouse, and the other groups of mice were intraperitoneally injected with 5-FU for modeling (50 mg/kg body weight, 10 ml/kg body weight).
  • 5-FU for modeling (50 mg/kg body weight, 10 ml/kg body weight).
  • the model control group had severe diarrhea and weight loss, and caused significant abnormalities in blood routine indicators, indicating that the model was successfully constructed.
  • the specific dosing regimen is shown in Table 18. After the experiment, the eyeballs of the animals in each group were removed to collect whole blood samples, and blood routine tests were performed using a five-category blood cell analyzer for animals (Shenzhen Dima Biotechnology Co., Ltd.).
  • the model control group began to experience diarrhea on D6, and the degree of diarrhea worsened over time.
  • the diarrhea score and total diarrhea score of the loperamide group were significantly reduced on D8; the low-dose bacterial composition significantly reduced the diarrhea scores and total diarrhea scores on D8 and D9.
  • the hematocrit, white blood cell count, lymphocyte count and platelet count of the model control group were significantly decreased.
  • the red blood cell count and hematocrit of the medium-dose group, low-dose group and whole intestine group of the bacterial composition were significantly increased, and the lymphocyte count and platelet count of the low-dose group of the bacterial composition were increased.
  • the three doses of bacterial composition all have a tendency to improve diarrhea and routine blood indicators; among them, the low-dose bacterial composition and loperamide can significantly improve diarrhea; the low-dose bacterial composition has the ability to significantly improve routine blood indicators (lymphocyte count, red blood cell count and platelet count).

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Abstract

属于微生物技术领域。提供了一种益生菌组合物及其在制备用于预防和/或治疗抗肿瘤治疗相关性腹泻等健康状况的产品中的应用。该益生菌组合物的活性成分包括两歧双歧杆菌、鸟肠球菌、唾液乳杆菌、发酵黏液乳杆菌和迪氏副拟杆菌。该益生菌组合物具有良好的安全性，自凝集能力，抗氧化能力、产短链脂肪酸能力、体外细胞黏附能力、抑制多种病原菌的能力以及对抗肿瘤治疗相关性腹泻治疗能力等。

Description

一种防治抗肿瘤治疗相关性腹泻的益生菌组合物及其应用 技术领域
本发明属于微生物药物领域，涉及一种益生菌组合物及其应用，具体涉及一种含有五种益生菌的组合物及其在预防和/或治疗抗肿瘤治疗相关性腹泻中的应用。
背景技术
抗肿瘤治疗相关性腹泻/肿瘤相关性腹泻(cancer treatment–related diarrhea/tumor-associated diarrhea)是多种抗肿瘤辅助治疗的常见并发症，是对健康与生活质量影响最大的症状之一，严重时还可能导致治疗延迟和依从性降低，这些均可能影响肿瘤治疗的长期结局，具有潜在致命性。世界卫生组织下属的国际癌症研究机构发布的数据显示，2020年全球新增癌症病例约1930万，预测2040年全球新增癌症病例将达到2840万。多达82％的癌症治疗患者会受到腹泻等副作用的困扰。
化疗(特别是使用治疗转移性结直肠癌患者的一线药物氟嘧啶类药物和伊立替康(CPT-11)等传统的化疗药物的化疗)、放疗、靶向疗法以及免疫疗法是手术以外引起抗肿瘤治疗相关性腹泻的主要因素。氟嘧啶类药物和伊立替康治疗引起的腹泻总体发生率为50％～80％，重度(3级～5级)腹泻的发生率达30％以上，其中1～5％使用5-氟尿嘧啶(5-FU)的受试者因腹泻死亡。腹部或盆腔肿瘤患者中，约70％需放射治疗，其中60％～70％的患者会出现腹痛、腹泻、便血等急性症状，30％～40％的患者会出现慢性腹泻。靶向疗法(如采用阿法替尼、塞瑞替尼、厄洛替尼、拉帕替尼等多种酪氨酸激酶抑制剂(TKI)的靶向疗法)引起的腹泻发生率可高达90％以上。免疫检查点抑制剂常导致免疫性肠炎。使用伊匹木单抗、纳武利尤单抗和帕博利珠单抗治疗肿瘤时，1％～45％肿瘤患者会发生腹泻。
手术除外的抗肿瘤治疗相关性腹泻的发病机理存在共性。抗肿瘤治疗相关性腹泻是由多因素共同导致的，其主要致病因素为以下几种：1)化疗药物及放疗过程中的电离辐射会造成氧化压力及DNA损伤；2)肠黏膜损伤导致上皮细胞丢失、紧密连接完整性降低、隐窝上皮细胞死亡，肠通透性增加；3)细胞核因子NF-κB等多种信号通路被高度激活，刺激细胞产生炎症因子，导致黏膜炎症；4)小肠粘膜受损导致胆汁酸吸收不良，过量的胆汁酸(尤其是二羟基成分)会诱导水和电解质分泌并增加肠蠕动，导致腹泻；5)常见医院内致病菌(沙门氏菌、志贺氏菌、大肠埃希菌和艰难梭菌等)感染；6)细菌 移位，肠道菌群失调，表现为益生菌减少和致病菌增多；7)免疫异常，如T细胞的普遍激活等。
目前，国际临床指南如“Guidance on the management of diarrhoea during cancer chemotherapy(2014)”和“Diarrhoea in adult cancer patients:ESMO Clinical Practice Guidelines(2018)”等推荐化疗相关性腹泻(Chemotherapy-induced Diarrhea，CID)、放射性肠炎、靶向疗法相关性腹泻以及免疫检查点抑制剂相关性腹泻的初始治疗药物均为洛哌丁胺。指南推荐洛哌丁胺治疗无效的患者使用奥曲肽。洛哌丁胺用药不得超过48h。大剂量使用洛哌丁胺还可能带来麻痹性肠梗阻和严重的心脏毒性风险。奥曲肽(腹泻评分3级以上患者的一线用药)可引发胆结石、高血糖、糖耐量异常等副作用。指南也推荐布地奈德作为洛哌丁胺治疗无效时的二线治疗药物，但糖皮质激素可发挥全身作用，引起感染风险增加和病毒、细菌等感染进一步恶化。3～4级腹泻伴中性粒细胞减少的患者可口服抗生素，但抗生素可引起腹泻加重以及艰难梭菌感染风险增加。肿瘤治疗通常包含多种治疗方式联合治疗。联合治疗使得腹泻发生率大大增加。如果长期腹泻或在腹泻之后未能采取有效治疗，患者会出现电解质紊乱、脱水和贫血等症状。中度或重度腹泻还会出现以中性粒细胞减少和全身炎症反应综合征为表现的严重并发症。因此，临床上迫切需要针对抗肿瘤治疗相关性腹泻更加安全有效的新一代药物。
肠道益生菌是定殖在人体肠道内，改变宿主肠道菌群组成的一类对宿主有益的活性微生物。肠道益生菌具有安全性高、毒副作用小、适应症及覆盖人群广泛等优势。益生菌组合能够针对抗肿瘤治疗相关性腹泻的复杂发病机理起到治疗作用，主要体现在：1)益生菌通过螯合金属离子、上调自身及宿主抗氧化酶及代谢产物以及降低产ROS的酶活性等方式起到抗氧化功能；2)益生菌能够产生有益次级代谢产物，如短链脂肪酸、吲哚衍生物、次级胆汁酸等。有益次级代谢产物促进杯状细胞分泌粘蛋白、促进表皮细胞修复、抑制细胞凋亡、增强紧密连接蛋白等进而增强肠屏障功能；3)益生菌通过下调NF-κβ通路、抑制促炎因子、分泌抗炎因子等方式调节免疫反应，从而起到抗炎和增强机体免疫力的作用；4)益生菌调节肠道菌群平衡，减少致病菌，增加益生菌，维持肠道正常内稳态环境；5)益生菌通过增加菌群的胆盐水解酶活性、抑制FXR-FGF15信号通路等方式调节胆汁酸代谢，缓解胆汁酸型腹泻；6)益生菌通过增加抗菌蛋白(防御素)分泌、产生细菌素等抗菌物质并与病原体竞争黏附位点等方式抑制致病菌生长。
中国专利CN112694992B公开了一株两歧双歧杆菌可缓解产肠毒素大肠杆菌(ETEC)引起的腹泻。中国专利CN113234619B公开了一株能够缓解急性肠道损伤的两歧双歧杆菌。有少量文献报道了迪氏副拟杆菌具有缓解肠道炎症的作用，如M.Kverka等人(Oral administration of Parabacteroides distasonis antigens attenuates experimental murine colitis through modulation of immunity and microbiota composition)报道了迪氏副拟杆菌能够改善DSS诱 导的小鼠结肠炎。目前仅有少量文件报道了唾液乳杆菌在防治腹泻方面的应用，例如专利CN110878267B公开了一种唾液乳杆菌ZLp4b可明显减缓并治愈幼畜腹泻。除上述专利和文献外，还有一些类似的益生菌用于防治腹泻或肠道炎症的文献。但目前的益生菌制剂主要用于治疗普通腹泻症状，且多为单菌株，很难针对抗肿瘤治疗相关性腹泻的复杂发病机制起到针对性治疗作用。
发明内容
本发明第一方面提供了一种益生菌组合，所述益生菌组合中含有第一益生菌、第二益生菌、第三益生菌、第四益生菌和第五益生菌中的任三种、任四种或五种(优选地，所述益生菌组合中含有第五益生菌以及第一益生菌、第二益生菌、第三益生菌和第四益生菌中的任两种、任三种或四种；更优选地，所述益生菌组合中含有第四益生菌、第五益生菌以及第一益生菌、第二益生菌和第三益生菌中的任两种或任三种)；所述第一益生菌选自两歧双歧杆菌、所述两歧双歧杆菌的子代菌株、所述两歧双歧杆菌的克隆菌株或所述两歧双歧杆菌的纯培养物；所述第二益生菌选自鸟肠球菌、所述鸟肠球菌的子代菌株、所述鸟肠球菌的克隆菌株或所述鸟肠球菌的纯培养物；所述第三益生菌选自唾液乳杆菌、所述唾液乳杆菌的子代菌株、所述唾液乳杆菌的克隆菌株或所述唾液乳杆菌的纯培养物；所述第四益生菌选自发酵黏液乳杆菌、所述发酵黏液乳杆菌的子代菌株、所述发酵黏液乳杆菌的克隆菌株或所述发酵黏液乳杆菌的纯培养物；以及所述第五益生菌选自迪氏副拟杆菌、所述迪氏副拟杆菌的子代菌株、所述迪氏副拟杆菌的克隆菌株或所述迪氏副拟杆菌的纯培养物。
在一些实施方式中，所述两歧双歧杆菌的微生物保藏编号为CCTCC NO:M2023349；所述鸟肠球菌的微生物保藏编号为CCTCC NO:M2023350；所述唾液乳杆菌的微生物保藏编号为CCTCC NO:M2023348；所述发酵黏液乳杆菌的微生物保藏编号为CCTCC NO:M2023352；以及所述迪氏副拟杆菌的微生物保藏编号为CCTCC NO:M20222033。
在一些实施方式中，所述益生菌组合中含有第一益生菌、第二益生菌、第三益生菌、第四益生菌和第五益生菌；所述第一益生菌选自微生物保藏编号为CCTCC NO:M2023349的两歧双歧杆菌、微生物保藏编号为CCTCC NO:M2023349的所述两歧双歧杆菌的子代菌株、微生物保藏编号为CCTCC NO:M2023349的所述两歧双歧杆菌的克隆菌株或微生物保藏编号为CCTCC NO:M2023349的所述两歧双歧杆菌的纯培养物；所述第二益生菌选自微生物保藏编号为CCTCC NO:M2023350的鸟肠球菌、微生物保藏编号为CCTCC NO:M2023350的所述鸟肠球菌的子代菌株、微生物保藏编号为CCTCC NO:M2023350的所述鸟肠球菌的克隆菌株或微生物保藏编号为CCTCC  NO:M2023350的所述鸟肠球菌的纯培养物；所述第三益生菌选自微生物保藏编号为CCTCC NO:M2023348的唾液乳杆菌、微生物保藏编号为CCTCC NO:M2023348的所述唾液乳杆菌的子代菌株、微生物保藏编号为CCTCC NO:M2023348的所述唾液乳杆菌的克隆菌株或微生物保藏编号为CCTCC NO:M2023348的所述唾液乳杆菌的纯培养物；所述第四益生菌选自微生物保藏编号为CCTCC NO:M2023352的发酵黏液乳杆菌、微生物保藏编号为CCTCC NO:M2023352的所述发酵黏液乳杆菌的子代菌株、微生物保藏编号为CCTCC NO:M2023352的所述发酵黏液乳杆菌的克隆菌株或微生物保藏编号为CCTCC NO:M2023352的所述发酵黏液乳杆菌的纯培养物；以及所述第五益生菌选自微生物保藏编号为CCTCC NO:M20222033的迪氏副拟杆菌、微生物保藏编号为CCTCC NO:M20222033的所述迪氏副拟杆菌的子代菌株、微生物保藏编号为CCTCC NO:M20222033的所述迪氏副拟杆菌的克隆菌株或微生物保藏编号为CCTCC NO:M20222033的所述迪氏副拟杆菌的纯培养物。
本发明第二方面提供了一种微生态组合物，所述微生态组合物以本发明第一方面所述的益生菌组合为活性材料。
在一些实施方式中，所述组合物中还含有辅料，所述辅料选自冻干保护剂、细菌培养基、食品添加剂、保健品中可接受的载体或辅料、和药学上可接受的载体或辅料。
在一些实施方式中，在所述微生态组合物中，按照活细菌菌体数量计，任意两种细菌的含量比为100CFU：1-10000CFU(例如，100CFU：1CFU、2CFU、3CFU、4CFU、5CFU、6CFU、7CFU、8CFU、9CFU、10CFU、20CFU、30CFU、40CFU、50CFU、60CFU、70CFU、80CFU、90CFU、100CFU、200CFU、300CFU、400CFU、500CFU、600CFU、700CFU、800CFU、900CFU、1000CFU、2000CFU、3000CFU、4000CFU、5000CFU、6000CFU、7000CFU、8000CFU、9000CFU、10000CFU中任一值)。
本发明第三方面提供了本发明第一方面所述的益生菌组合或本发明第二方面所述的微生态组合物在制备用于单独使用或者用于与其他微生物制剂和/或药物联合使用以改善受试者的健康状况的产品中的用途；所述改善受试者的健康状况选自：抑制受试者体腔(比如，肠腔)内的铜绿假单胞菌、痢疾志贺氏菌、金黄色葡萄球菌、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、任七种或八种的增殖；治疗、预防和/或减缓铜绿假单胞菌、痢疾志贺氏菌、金黄色葡萄球菌、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、任七种或八种引起的组织损伤、疾病或亚健康状况；改善受试者肠道内抗氧化的能力；治疗、预防和/或减缓抗肿瘤药物引起的腹泻；治疗、预防和/或减缓抗肿瘤药物引起的肠道炎症；治疗、预防和/或减 缓抗肿瘤药物引起的体重下降；治疗、预防和/或减缓抗肿瘤药物引起的小肠长度缩短；治疗、预防和/或减缓抗肿瘤药物引起的小肠厚度增加；治疗、预防和/或减缓抗肿瘤药物引起的肠损伤；治疗、预防和/或减缓抗肿瘤药物引起的脾脏重量与体重比值减小；治疗、预防和/或减缓TNF-α、IL-1β、IL-22和Bax中的任一种、任两种、任三种或四种表达量升高引起的组织损伤、疾病或亚健康状况；治疗、预防和/或减缓ZO-1、Occludin和AQP8的任一种、任两种或三种表达量下降引起的组织损伤、疾病或亚健康状况；治疗、预防和/或减缓放疗引起的腹泻；治疗、预防和/或减缓放疗引起的体重下降；治疗、预防和/或减缓放疗引起的肠损伤；治疗、预防和/或减缓放疗引起的肠道炎症；以及治疗、预防和/或减缓抗肿瘤药物引起的红细胞压积降低、白细胞数量降低、淋巴细胞数量降低和/或血小板数量降低。
在一些实施方式中，所述产品为食品、保健品或药品。
在一些实施方式中，所述受试者选自人和小鼠。
在一些实施方式中，本发明提供了本发明第一方面所述的益生菌组合或第二方面所述的微生态组合物在制备治疗和/或预防抗肿瘤治疗相关性毒副作用的药物中的应用。
在一些实施方式中，所述抗肿瘤治疗相关性毒副作用为腹泻。
在一些实施方式中，所述抗肿瘤治疗相关性毒副作用为抗肿瘤药物引起的腹泻、或放疗引起的腹泻，或放疗引起的肠损伤。
在一些实施方式中，所述抗肿瘤药物选自化疗药物、靶向药物和免疫检查点抑制剂。
在一些实施方式中，所述抗肿瘤药物选自阿霉素、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、阿糖胞苷、阿帕替尼、阿西替尼、卡博替尼、索拉非尼、舒尼替尼、纳武利尤单抗、帕博利珠单抗、伊匹木单抗。
本发明第四方面提供了一种预防、治疗或减缓肠道疾病的方法，所述方法为将治疗有效量的本发明第二方面所述的微生态组合物施用给受试者；所述肠道疾病选自：铜绿假单胞菌、痢疾志贺氏菌、金黄色葡萄球菌、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、艰难梭菌中的任一种、任两种、任三种、任四种、任五种、任六种、任七种或八种引起的肠道疾病；肠道内的氧化损伤；抗肿瘤药物引起的腹泻；抗肿瘤药物引起的肠道炎症；抗肿瘤药物引起的肠道损伤；放疗引起的腹泻；放疗引起的肠道炎症；以及放疗引起的肠道损伤。
在一些实施方式中，所述受试者选自人和小鼠。
在一些实施方式中，所述施用给受试者选自口服、腹腔注射、灌胃。
在一些实施方式中，按照所述益生菌组合物中的全部细菌含量计算，所 述治疗有效量为106-12CFU(比如，1×106CFU、2×106CFU、3×106CFU、4×106CFU、5×106CFU、6×106CFU、7×106CFU、8×106CFU、9×106CFU、1×107CFU、2×107CFU、3×107CFU、4×107CFU、5×107CFU、6×107CFU、7×107CFU、8×107CFU、9×107CFU、1×108CFU、2×108CFU、3×108CFU、4×108CFU、5×108CFU、6×108CFU、7×108CFU、8×108CFU、9×108CFU、1×109CFU、2×109CFU、3×109CFU、4×109CFU、5×109CFU、6×109CFU、7×109CFU、8×109CFU、9×109CFU、1×1010CFU、2×1010CFU、3×1010CFU、4×1010CFU、5×1010CFU、6×1010CFU、7×1010CFU、8×1010CFU、9×1010CFU、1×1011CFU、2×1011CFU、3×1011CFU、4×1011CFU、5×1011CFU、6×1011CFU、7×1011CFU、8×1011CFU、9×1011CFU与1012CFU中任一值或任两值之间的范围)每天。
在一些实施方式中，所述抗肿瘤药物选自阿霉素、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、阿糖胞苷、阿帕替尼、阿西替尼、卡博替尼、索拉非尼、舒尼替尼、纳武利尤单抗、帕博利珠单抗和伊匹木单抗。
本发明的有益效果在于：
(1)本发明提供的益生菌组合含有3种、4种或5种不同种属的益生菌，各菌株之间没有相互拮抗，且无毒力因子、安全性良好；(2)本发明提供的益生菌组合具有自凝集/共凝集、抗氧化、产短链脂肪酸、抑制多种病原菌的能力，且具有较强的体外黏附能力，可针对抗肿瘤治疗相关性腹泻的复杂发病机制协同发挥作用；(3)本发明提供的益生菌组合可以预防和/或治疗化疗药物或放射治疗引起的腹泻、肠道炎症和肠道损伤等毒副作用。
附图说明
图1为5个菌株的菌落形态正面照片。
图2为五株单菌在BF839琼脂培养基上的共培养特征照片。
图3示出了益生菌组合物及其单菌自凝集(共凝集)能力。
图4示出了益生菌组合物及其单菌对致病菌的抑菌活力检测结果。
图5示出了益生菌组合物及其单菌对Caco2细胞的黏附能力检测结果。
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。
除非有特别说明，本文中使用的术语具有生物医药领域通用的含义。
如无特殊说明，本发明中的特定温度参数应被理解为恒温处理，并允许 在一定温度区间内存在变动(如在±5℃、±4℃、±3℃、±2℃、±1℃的范围内波动)。
本发明提供了一种益生菌组合，所述益生菌组合含有两歧双歧杆菌(Bifidobacterium bifidum)，鸟肠球菌(Enterococcus avium)，唾液乳杆菌(Ligilactobacillus salivarius)，发酵黏液乳杆菌(Limosilactobacillus fermentum)，迪氏副拟杆菌(Parabacteroides distasonis)中的的任三种、任四种或五种。
在一些实施方式中，所述两歧双歧杆菌的微生物保藏编号为CCTCC NO:M2023349；
所述鸟肠球菌的微生物保藏编号为CCTCC NO:M2023350；
所述唾液乳杆菌的微生物保藏编号为CCTCC NO:M2023348；
所述发酵黏液乳杆菌的微生物保藏编号为CCTCC NO:M2023352；以及
所述迪氏副拟杆菌的微生物保藏编号为CCTCC NO:M20222033。
本发明的特定保藏编号的菌株的涵义包括但不限于：(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。如本领域技术人员所知，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(生物安全性)、免疫原性与生物活性的突变(比如，可能的情况是突变发生在两个结构域之间的连接氨基酸残基或者突变的氨基酸残基位于蛋白质高级结构内部因不与免疫细胞接触，因而这些突变不影响毒性、免疫原性与生物活性)。可以合理预期，当这些微小变化没有明显影响后代菌株的毒性、免疫原性与生物活性的情况下，突变的菌株仍然能实现本发明的目的，且突变的菌株源于本发明贡献的菌株，因此相应的菌株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，菌株的毒性、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突变的菌株毒性、免疫原性与生物活性与本发明贡献的菌株是相同的。用细胞、动物等测定菌株后代的毒性、免疫原性与生物活性时，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任3种、任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各菌株的功效。
抗肿瘤治疗相关性腹泻或肿瘤相关性腹泻是指各种抗肿瘤治疗对肠道粘膜造成损伤导致肠道吸收和分泌失衡引起的腹泻。常见的抗肿瘤治疗相关性腹泻包括化疗相关性腹泻、放疗引起的腹泻、靶向治疗引起的腹泻、免疫检查点抑制剂引起的腹泻等。放疗引起的腹泻在临床中表现为放射性肠炎或放射性肠损伤的症状。
化疗是指使用非选择性化学药物手段杀灭肿瘤细胞达到治疗目的。化疗是除了手术、放疗外对肿瘤治疗的主要手段。因为缺少选择性，在杀伤肿瘤细胞的同时化疗也会损伤正常细胞。常见化疗药物包括但不限于抗生素类化疗药(如阿霉素、表阿霉素、放线菌素D、多柔比星、柔红霉素及其衍生物等)、紫杉醇类化疗药(如紫杉醇、多西他赛、白蛋白紫杉醇及其衍生物等)、铂类化疗药(如顺铂、卡铂、奈达铂、草酸铂、洛铂及其衍生物等)、烷化剂类化疗药(如环磷酰胺、氮芥、卡莫司汀及其衍生物等)、喜树碱类化疗药(如喜树碱、羟基喜树碱、拓扑替康、伊立替康及其衍生物等)以及抗代谢化疗药(如卡培他滨、吉西他滨、甲氨蝶呤、5-氟尿嘧啶、培美曲塞、阿糖胞苷及其衍生物等)。
靶向治疗是在细胞分子水平上针对已经明确的致癌位点来设计相应的治疗药物。靶向治疗中，药物进入体内会特异地选择致癌位点来相结合发生作用以使肿瘤细胞特异性死亡。新型靶向药物的出现改变了肿瘤治疗模式并开辟了靶向治疗时代。常见靶向药物包括阿帕替尼、阿西替尼、卡博替尼、索拉非尼、舒尼替尼等。
免疫检查点抑制剂是针对相应的免疫检查点开发的一些单抗类药物。免疫检查点抑制剂的主要作用为阻断表达免疫检查点的肿瘤细胞与免疫细胞之间的作用，从而阻断肿瘤细胞对免疫细胞的抑制作用。常见的免疫检查点抑制剂包括但不限于PD-1/PD-L1抑制剂抗体(如纳武利尤单抗、帕博利珠单抗等)和CTLA-4抑制剂(如伊匹木单抗等)。
放疗是指利用放射线(如放射性同位素产生的α射线、β射线、γ射线，以及各类x射线治疗装置或加速器产生的x射线、电子线、质子束及其它粒子束等)治疗恶性肿瘤的方法。常用放射治疗方法包括但不限于常规放射治疗、立体定位放射治疗、三维适形放射治疗、调强放射治疗、图像引导放射治疗、容积弧形调强放射治疗、质子放射疗法等。
本发明提供了一种微生态组合物，所述微生态组合物含有两歧双歧杆菌(Bifidobacterium bifidum)，鸟肠球菌(Enterococcus avium)，唾液乳杆菌(Ligilactobacillus salivarius)，发酵黏液乳杆菌(Limosilactobacillus fermentum)，迪氏副拟杆菌(Parabacteroides distasonis)中的任三种、任四 种或五种以及辅料。
微生态组合物是指一种生物活性制剂，其包含特定微生物种类和/或其代谢产物，该组合物通过恢复或优化宿主体内的微生物群落结构和功能，促进宿主的健康状态，增强对疾病的抵抗力，或改善宿主的生理功能。
所述辅料根据制备的产品类型而不同，例如可制备食品、保健品或药品，相应地，辅料可选自冻干保护剂、细菌培养基、食品添加剂、保健品中可接受的载体或辅料和药学上可接受的载体或辅料。
本发明提供了益生菌组合或微生态组合物在制备食品、保健品或药品中的用途。
当制备成药品时，所述的益生菌组合或微生态组合物在制备用于单独使用或者用于与其他微生物制剂和/或药物联合使用以改善受试者的健康状况的药品中的用途。
本发明还提供了预防、治疗或减缓肠道疾病的方法，所述方法为将治疗有效量的微生态组合物施用给受试者。
所述治疗有效量或预防有效量是在临床上能够实现所需治疗性或预防性效果的用量。在一些实施方式中，治疗有效量不诱导或不引起不希望的副作用。在一些实施方式中，治疗有效量诱发或引起副作用，但仅引起鉴于患者的病症治疗临床医师可接受的副作用。在一些实施方式中，单次使用剂量或单次有效量中含有的总菌数为102～1015CFU、103～1014CFU、104～1013CFU、105～1012CFU或106～1012CFU。
药学上可接受的载体是指不对受试者引起显著刺激且不消除所施用的益生菌的生物活性及特性的药学载体。正如本领域技术人员已知的(参见例如Remington's Pharmaceutical Sciences,第18版MackPrinting Company,1990,第1289-1329页)，药学上可接受的载体可增强或稳定组合物，或可用于促进组合物的制备。药学上可接受的载体可包括溶剂、分散介质、涂层、表面活性剂、抗氧化剂、等渗剂、吸收延迟剂、盐、药物稳定剂、结合剂、赋形剂、崩解剂、润滑剂、甜味剂、调味剂、染料、保护剂等及其组合。除非常规载体与活性成分不相容，否则考虑将载体用于治疗性组合物或药物组合物中。载体可经选择以使受试者的不利副作用降至最低和/或使活性成分的失活降至最低。
赋形剂是指添加至药物组合物中以使药物具有一定形状或一定浓度的物质，例如，无菌水、生理盐水、聚亚烷基二醇(诸如聚乙二醇)、植物油、氢化萘、碳酸氢钙、磷酸钙、多种糖、各种类型淀粉、纤维素衍生物、明胶等。
本发明的微生态组合物还可以进一步含有第二种有益的活性成分，例如，另一种具有止泻功能的益生菌、益生元、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。
在一些实施方式中，药物组合物的剂型为片剂、胶囊剂、颗粒剂、溶液剂、混悬剂、粉剂等。
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。本领域技术人员在没有做出创造性劳动的前提下基于本发明申请文件中的实施例所获得的所有其它实施例均应属于本发明公开或保护的范围。
以下实施例中所用到的培养基配制方法如下：
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，NiCl2·6H2O0.03g，10mg/mL的Na2SeO3·5H2O溶液0.03mL和10mg/mL的Na2WO4·2H2O溶液0.03mL加入上述次氮基三乙酸水溶液中(加入过程中不断搅拌溶液，保持溶液澄清)得到TE141。
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 NaOH将前述溶液至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℃高温湿热灭菌分装的溶液30min。阴凉、干燥处存放所得三混液体培养基。
三混固体培养基的配制：在前述三混液体培养基的基础上再加入琼脂粉5g，其他步骤与三混液体培养基的配制相同。
BF839液体培养基的配制：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(上海阿拉丁生化科技股份有限公司，S105664-25G)0.3g，酵母浸粉 (Thermo Fisher Oxoid，LP0021B)5.0g，葡萄糖(成都市科隆化学品有限公司，50-99-7)1.5g和磷酸氢二钠(成都市科隆化学品有限公司，7558-79-4)4.0g溶解于1L的蒸馏水中得到溶液。N2置换溶液除氧并分装。121℃高温湿热灭菌溶液15min。阴凉、干燥处存放所得培养基。
BF839固体培养基的配制：将BF839固体琼脂(青岛高科技工业园海博生物技术有限公司，HB8805)50.4g溶解于1L的蒸馏水中得到混合物。对混合物进行N2置换除氧并分装。121℃高温湿热灭菌混合物15min。阴凉、干燥处存放得到的培养基。
无氧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的蒸馏水中得到混合物。加热煮沸混合物，冷却混合物至室温并在其中加入0.55g半胱氨酸盐酸盐，搅拌混合物使其溶解后调节pH至6.5。加热混合物至沸腾，在微沸状态下保持30min。冷却后在通N2条件下用定量分液器分装为400mL/瓶。121℃高温湿热灭菌瓶子30min。阴凉、干燥处存放得到的PBS备用。
GAM固体培养基(青岛海博生物技术有限公司，HB8462)、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)培养基的配制均按照供货商提供的说明书中规定的步骤进行称量材料溶解于水得到混合物。121℃高温湿热灭菌混合物30min得到培养基。阴凉、干燥处存放培养基。
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菌粉制备培养基的配制：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菌粉制备培养基的配制：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菌粉制备培养基的配制：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。
冻干保护剂配制方法如下：
迪氏副拟杆菌Pdist-1冻干保护剂(也作为动物给药对照用冻干保护剂)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：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。过滤除菌备用。
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。
使用时均按A液:B液:C液＝6:2:2的体积比混合。动物试验给药的冻干保护剂，是将配置好的冻干保护剂冻干后粉碎，并用生理盐水配置成悬液来使用。
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％)，唾液乳杆菌也称作联合唾液乳杆菌，拉丁名为Ligilactobacillus salivarius，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。
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：各菌株之间共培养特性测试
将实施例1所得菌株Pdist-1、Eaviu-1、Lferm-1、Bbifi-1和Lsali-1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，然后用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2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。
实施例4：单菌和细菌组合物的凝集能力
单菌悬液的配制：以2％的初始接种量将菌株Pdist-1、Eaviu-1、Lferm-1、Bbifi-1、Lsali-1和对照菌株鼠李糖乳杆菌杆菌GG(Lactobacillus rhamnosus GG，LGG，CICC6141，购买自中国工业微生物菌种保藏管理中心)分别接 种于三混液体培养基中，37℃厌氧培养至对数生长后期。以无氧PBS对培养后菌液进行3次离心洗涤。用适量PBS重悬菌体后测定菌液OD600(测定菌液在600nm波长处的吸光值)并用PBS作为稀释液将各单菌菌液稀释至同一OD600值(0.5±0.1)。
细菌组合物悬液的配制：将前述稀释为同一OD600值的Pdist-1菌液、Eaviu-1菌液、Lferm-1菌液、Bbifi-1菌液和Lsali-1菌液以等体积混匀。
分别取稀释后单菌悬液、混合菌悬液15mL，分装为5mL/支，共3支，用于平行试验。测量初始时间菌液OD600，记作A0；37℃静置24h后，吸取上层菌悬液1mL再次测量OD600值，记作A24。自凝集率/共凝集率＝(1-A24/A0)×100％，检测结果以Mean±S.D表示(自凝集率：单菌株之间的凝集率；共凝集率：细菌组合物中不同菌株之间的凝集率)。
结果如图3所示。由此可见，细菌组合物及其单菌都具有凝集能力，凝集能力(自凝集率/共凝集率，凝集率)依次为：Eaviu-1＞LGG≈Pdist-1＞Lsali-1≈Bbifi-1≈细菌组合物＞Lferm-1。凝集能力越好的细菌定殖能力可能越好并且容易在宿主肠细胞表面形成保护性屏障。
实施例5：细菌组合物的抗氧化能力测试
细菌组合物培养：将各单菌Pdist-1、Eaviu-1、Lferm-1、Bbifi-1和Lsali-1活化后混合接种至5mL三混培养基中，各菌株接种量均为1％，37℃厌氧培养24h。
样本处理：取0.5mL培养菌液，12000rpm离心20min，弃上清，用0.5ml预冷的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)中的提取液重悬菌体。将菌体转移至灭好菌的装有beads(美国Sigma-Aldrich公司，G4649-1KG)的螺帽管中。用快速样品制备仪振荡破菌体壁一次(参数设置：4.5m/s，30s)后，12000rpm 4℃离心10min，取上清置冰上待测。
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，按照试剂盒的说明书操作结合前述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。
细菌组合物的抗氧化能力为0.189μmol/mg prot。细菌组合物具有一定的抗氧化能力。
实施例6：单菌和细菌组合物产短链脂肪酸的能力
单菌：以5％接种量将对照菌株LGG、Pdist-1、Eaviu-1、Lferm-1、Bbifi-1和Lsali-1分别接种于BF839液体培养基中，37℃厌氧培养24h。
细菌组合物：分别以1％接种量将Pdist-1、Eaviu-1、Lferm-1、Bbifi-1和Lsali-1混合接种于BF839液体培养基中，37℃厌氧培养24h。
分别离心单菌及细菌组合物发酵液，吸取1mL上清液。在上清中加入10μL甲酸，4℃静置酸化30min，再次离心。吸取上清液，使用0.22μm滤 器过滤，备用。使用气相色谱仪测定各滤出溶液中的短链脂肪酸(short chain fatty acid，SCFAs)含量。气相色谱相关参数如下。仪器型号：Agilent 7890A。色谱柱型号：DB-WAX UI(30m×0.32mm×0.25μm)毛细管柱。柱温：初始温度80℃，持续0.5min；再以5℃/min上升至180℃，持续1min；再以40℃/min上升至220℃，持续4min。进样口温度：220℃。检测器温度：250℃。载气：氢气30mL/min；空气300mL/min。尾吹流量：氮气28.314mL/min。分流比：10:1。进样量：1μL。同一样品重复进样检测4次，计算平均值，并按以下公式计算产酸量，菌株实际产酸量(ppm)＝菌株测定产酸值-BF839培养基本底值，总SCFAs产量为乙酸、丙酸、丁酸、异戊酸和戊酸的产量之和。
结果：如表2，对照菌株LGG、细菌组合物及其单菌均有产乙酸能力。除产乙酸以外，Pdist-1还能够产丙酸、异戊酸。Bbifi-1、Pdist-1、细菌组合物总产SCFAs量明显优于LGG，Lsali-1、Eaviu-1、Lferm-1总产SCFAs量与LGG相当。
表2.菌株产酸量统计表

实施例7：单菌和细菌组合物的抑制病原菌能力
本实施例选取8种常见的可导致腹泻的致病菌进行抑菌能力检测。致病菌株来源信息如下：铜绿假单胞菌(CMCC(B)10104)，购买自中国食品药品检定研究院。痢疾志贺氏菌(CMCC(B)51252)，购买自中国食品药品检定研究院。金黄色葡萄球菌(CMCC(B)26003)，购买自中国食品药品检定研究院。大肠杆菌(CMCC(B)44102)，购买自中国食品药品检定 研究院。乙型副伤寒沙门氏菌(CMCC(B)50094)，购买自中国食品药品检定研究院。小肠结肠炎耶尔森菌CMCC(B)52204，购买自中国食品药品检定研究院。副溶血性弧菌(ATCC 17802)，购买自美国微生物菌株保藏中心。艰难梭菌(CICC 22951)，购买自中国工业微生物菌种保藏管理中心。
单菌：将对照菌株LGG、Pdist-1、Eaviu-1、Lferm-1、Bbifi-1和Lsali-1中各单菌以5％的接种量分别接种至三混液体培养基中，厌氧培养48h获得发酵液。
细菌组合物：分别以1％接种量将Pdist-1、Eaviu-1、Lferm-1、Bbifi-1和Lsali-1混合接种于三混液体培养基厌氧培养48h获得发酵液。
将铜绿假单胞菌、痢疾志贺氏菌、金黄色葡萄球菌、大肠杆菌、乙型副伤寒沙门氏菌、小肠结肠炎耶尔森菌和副溶血性弧菌分别用TSB培养基好氧培养至对数生长期。用TSB培养基稀释培养物至106CFU/mL，取200μL涂布于TSA固体培养基。将艰难梭菌用三混液体培养基厌氧培养至对数生长期。用三混液体培养基稀释培养物至106CFU/mL，取200μL涂布于无氧GAM固体培养基(添加5v/v％马血清，北京索莱宝科技有限公司，S9050)。每个平板中放置3个牛津杯，牛津杯中加入200μL待测发酵液(前述培养物)，艰难梭菌在厌氧条件下培养，其余致病菌均在有氧条件下培养。37℃培养24h后，测量抑菌圈直径并计算平均值。结果如图4所示。由此可见，细菌组合物对铜绿假单胞菌、痢疾志贺氏菌、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、艰难梭菌的生长均有抑制作用。细菌组合物弥补了单菌在抑制某些致病菌上的弱势。细菌组合物在抑制乙型副伤寒沙门氏菌及金黄色葡萄球菌活性方面优于对照菌株LGG，对其余致病菌的抑制能力与对照菌株LGG相当。
实施例8：单菌和细菌组合物对Caco2细胞的黏附能力测试
单菌：分别以初始接种量2％将对照菌株LGG、Pdist-1、Eaviu-1、Lferm-1、Bbifi-1和Lsali-1中各单菌接种于三混液体培养基中，37℃厌氧培养至对数生长后期。将培养后菌液用无菌PBS(武汉博士德生物工程有限公司，PYG0021)离心洗涤2次，然后使用含10v/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)培养基将沉淀中的菌株稀释为5×108CFU/mL，待用。
细菌组合物：将前述稀释后Pdist-1、Eaviu-1、Lferm-1、Bbifi-1、Lsali-1中各单菌菌液按照等体积进行混合以得到细菌组合物。
Caco-2细胞(商城北纳创联生物科技有限公司，350769)为贴壁细胞。用37℃预热的胰酶细胞消化液(兰杰柯科技有限公司，BL501A)消化Caco-2细胞。离心收集消化的Caco-2细胞。用含10v/v％FBS的DMEM培养基稀释沉淀。以5×104CFU/孔的密度将Caco-2细胞接种于96孔板中，置于37℃二氧化碳培养箱中过夜培养，待用。
把96孔板各孔分两组，每一组分别取100μL稀释后单菌及细菌组合物 悬液加入含Caco-2细胞的96孔细胞培养板。加样完毕后，将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，一组孵育30min，另一组孵育2h。孵育后，用无菌PBS洗涤2次以便洗去未黏附菌体。洗涤后每孔加入50μL0.25％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细胞被消化呈现球状后，每孔加入150μL DMEM培养基，反复吹打约1min。待显微镜检查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。将合适稀释梯度倾注到已溶解的三混固体培养基，37℃培养48h后计数。
结果如图5所示。A显示各菌株与Caco2细胞黏附30min后，鸟肠球菌Eaviu-1、细菌组合物与LGG黏附能力相当。图B示出了黏附时间为2h时各菌株黏附效果，两歧双歧杆菌Bbifi-1、发酵黏液乳杆菌Lferm-1黏附能力与LGG相当，唾液乳杆菌Lsali-1、鸟肠球菌Eaviu-1、细菌组合物黏附能力均优于LGG，细菌组合物具有较好的黏附能力因此更容易定殖。
实施例9：细菌组合物对5-氟尿嘧啶(5-FU)致腹泻小鼠的治疗效果测试
细菌组合物制备：
将Pdist-1、Lferm-1、Lsali-1、Eaviu-1、Bbifi-1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，将五种一级种子液分别转接至相应的菌粉制备培养基，37℃、90rpm厌氧培养10～15h，得到二级种子液。将五种二级种子液分别用蠕动泵泵入含相应的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各菌体。分别按菌泥和冻干保护剂1:1～1:2(重量比)添加相应的冻干保护剂，混匀乳化菌泥。把各菌泥冻干、粉碎后即得菌粉。取适量粉碎后的各菌粉进行活菌数测定。根据活菌数将各菌株的菌粉按照等CFU比例进行混合获得细菌组合物菌粉。最后利用生理盐水将细菌组合物菌粉配置成菌悬液进行动物试验。使用生理盐水稀释菌悬液得到不同细菌剂量的细菌组合物。
试验方法：
(1)试验设计：将体重22～24g的96只SPF级雄性Balb/c小鼠(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为8组(分别为正常对照组、模型对照组、阳性对照洛哌丁胺组、阳性对照整肠生组、细菌组合物高剂量组、细菌组合物中高剂量组、细菌组合物中剂量组、细菌组合物低剂量组)，每组12只。前述8个组分别依次灌胃给予生理盐水(0.2mL/只/天)、冻干保护剂(0.2mL/只/天)、洛哌丁胺(购自河南中杰药业有限公司，20mg/kg体重/只/天)、整肠生(含地衣芽胞杆菌活菌，东北制药集团有限公司，2×108CFU/只/天)，细菌组合物高剂量组(1×109CFU/只/天)，细菌组合物中高剂量组(1×108CFU/只/天)，细菌组合物中剂量组(1×107CFU/只/天)以及细菌组合物低剂量组(1×106CFU/ 只/天)。总体实验周期为9天，记为D1-D9，D1-D8连续灌胃给药8天。D3开始造模，正常对照组小鼠腹部注射0.2mL生理盐水/只，其余各组小鼠均使用30mg/kg体重的5-FU(5-氟尿嘧啶，购自天津金耀药业有限公司，规格为10mL/支，0.25g/10mL)连续腹腔注射4天诱导小鼠CID模型，D9解剖。具体实验分组和给药方案见表3。
表3.细菌组合物治疗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；i.p:腹腔注射；i.g：灌胃给药；QD表示每天一次。
实验期间每天检测动物体重，进行一般观察记录(观察项目包括但不限于动物的外观体征、行为活动、呼吸、腺体分泌、粪便情况等)，并重点观察并记录造模后每只动物的腹泻情况并进行评分。试验结束进行安乐死后，采集中段结肠组织样本进行炎症因子(TNF-α、IL-1β)和紧密链接蛋白(ZO-1、Occludin)相关基因的qPCR检测。
(2)腹泻观察与评分：腹泻的评分标准参照Kurita A等研究(Modified  irinotecan hydrochloride(CPT-11)administration schedule improves induction of delayed-onset diarrhea in rats.Kurita A et al.,Cancer Chemother Pharmacol.2000；46(3):211-20)中的腹泻评分方法。将小鼠置于垫有洁净滤纸的小鼠笼内，每笼1只。0分：粪便硬、正常便；1分：轻度湿便、轻微湿便或软便；2分：中度湿便、粪便不成形且肛周不洁；3分：重度腹泻，稀便、水样便且严重肛周不洁。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。
(3)qPCR检测：采用Trizol法提取各组小鼠结肠组织总RNA，将总RNA反转录成cDNA，保存cDNA于-20℃备用。用qRT-PCR方法检测各组小鼠结肠促炎因子IL-1β、TNF-α以及紧密连接蛋白ZO-1和Occludin-1编码基因mRNA的相对转录水平(引物序列见表7，内参基因为β-actin)。反应程序：1)95℃3min；2)95℃10s，60℃30s，95℃15s，60℃1min，共40个循环；3)95℃10s。采用2-ΔΔCT法进行数据分析。
(4)数据统计与分析：对体重、腹泻评分和病理学检查结果等数据以均数±标准差(Mean±SEM)表示，使用SPSS统计软件26.0进行One-way ANOVA统计学分析。
试验结果：
(1)腹泻评分结果如表4所示。
表4.腹泻评分结果
注：所有数据均采用平均值(Mean)±标准差(SEM)；所有组与模型对照组相比，“*”表示P<0.05，“**”表示P<0.01，“***”表示P<0.001，“****”表示P<0.0001。
5-FU诱导后模型组D8、D9腹泻评分显著高于正常对照组，细菌组合物的4个剂量均能显著改善动物腹泻情况，D8、D9腹泻评分和腹泻总分与模型对照组相比显著降低。由此可见，本发明的细菌组合物能够改善5-FU诱导的腹泻。
(2)qPCR检测结果如表5所示。
表5.细菌组合物对5-FU致腹泻小鼠结肠组织炎症和肠屏障相关基因表达的影响
注：所有数据均采用平均值(Mean)±标准差(SEM)；所有组与模型对照组相比，“*”表示P<0.05，“**”表示P<0.01，“***”表示P<0.001，“****”表示P<0.0001。
与正常对照组相比，模型对照组TNF-α、IL-1β基因的mRNA相对转录水平显著升高，ZO-1、Occludin基因的mRNA相对转录水平显著降低。高剂量细菌组合物和洛哌丁胺可显著降低TNF-α的mRNA相对转录水平。高剂量、中高剂量和中剂量细菌组合物可显著降低IL-1β的mRNA相对转录水平。高剂量组、中剂量组和低剂量细菌组合物可显著升高ZO-1的mRNA相对转录水平，与洛哌丁胺和整肠生改善效果相当。中高剂量和中剂量细菌组合物可显著升高Occludin的mRNA相对转录水平，与整肠生改善效果相当。
综上所述，本发明的细菌组合物能显著降低5-FU诱导的CID小鼠模型腹泻评分，能通过降低炎症因子和增加紧密连接蛋白表达量，对腹泻起到显著治疗作用。
实施例10：细菌组合物对伊立替康(CPT-11)致腹泻小鼠的治疗效果
实施例10-12中所用细菌组合物是按照实施例9中的方法将五种细菌按照等CFU混合得到的。实施例10-12所提及的细菌组合物的CFU是五种细 菌的总CFU。
试验方法：
(1)试验设计：将雄性Balb/c小鼠(购自上海吉辉试验动物饲养有限公司)70只饲养于SPF级动物房。根据体重随机分为正常对照组(不给药)、模型对照组(给药方案：冻干保护剂0.2mL/只/天)、阳性对照整肠生(给药方案：地衣芽胞杆菌2.5×108CFU/只/天)组、细菌组合物高剂量组(给药方案：细菌组合物1×109CFU/只/天)、细菌组合物中高剂量组(给药方案：细菌组合物1×108CFU/只/天)、细菌组合物中剂量组(给药方案：细菌组合物1×107CFU/只/天)、细菌组合物低剂量组(给药方案：细菌组合物1×106CFU/只/天)，共7组，每组10只。整体试验周期为10天，记为D1～D10。
正常对照组小鼠腹部注射0.2mL生理盐水/只，其余各组小鼠均用85mg/kg体重的CPT-11进行腹部注射造模，D3开始造模，D3～D6连续造模4天，每天1次。对应组别分别进行灌胃给药，每天给药一次，连续9天，D10解剖。具体实验分组和给药方案见表6。
表6.细菌组合物治疗CPT-11致腹泻小鼠的实验分组和给药方案
注：CPT-11:盐酸伊利替康；CFU:colony forming unit菌落形成单位；d:天；i.p:腹腔注射；i.g：灌胃给药；QD表示每天一次。
实验期间每天检测动物体重，进行一般观察记录(观察项目包括但不限于动物的外观体征、行为活动、呼吸、腺体分泌、粪便情况等)，并重点观 察并记录造模后每只动物的腹泻情况并进行评分。试验结束时，各组动物经腹腔静脉采集全血样本，进行血常规检测。动物安乐死后，取肠道，分别测量小肠的长度；取脾脏称重，计算脾脏系数(脾脏系数＝脾脏重量/体重×100)。每组5只小鼠于近盲肠端采集样品，液氮急冻样品后，于-80℃冷冻保存样品。样品用于qPCR检测炎性因子(TNF-a、IL-1β、IL-22)、紧密连接蛋白(ZO-1、Occludin)、促凋亡因子(Bax)、水通道蛋白(AQP8)等相关基因mRNA表达水平。每组剩余5只小鼠的结直肠部位在10％甲醛溶液中直接固定，用HE染色后进行病理学检查。
(2)腹泻观察与评分：腹泻的评分标准与操作方法与实施例9相同。
(3)血常规检测：各组动物经腹腔静脉采集全血样本。使用全自动血液细胞分析仪(Mindray)对样本进行血常规检测。
(4)qPCR检测：采用Trizol法提取各组小鼠结肠组织总RNA，将总RNA反转录成cDNA，保存cDNA于-20℃备用。用qPCR方法检测各组小鼠结肠促炎因子TNF-α、IL-1β、抗炎因子IL-22，紧密连接蛋白ZO-1和Occludin，促凋亡因子Bax，水通道蛋白AQP8基因的mRNA相对转录水平(引物序列见表7，内参基因为β-actin)。反应程序：1)95℃3min；2)95℃10s，60℃30s，95℃15s，60℃1min，共40个循环；3)95℃10s。采用2-ΔΔCT法进行数据分析。
表7.qRT-PCR引物信息

(5)肠道组织病理检测：结直肠部位在10％甲醛溶液中直接固定，HE染色后进行病理学检查。
(6)数据统计与分析：对体重、腹泻评分和病理学检查结果等数据以均数±标准差(Mean±SEM)表示，使用SPSS统计软件26.0进行One-way ANOVA统计学分析。
试验结果：
(1)模型组小鼠经伊立替康腹腔注射诱导后出现明显临床疾病类似表现(主要包括腹泻、体重下降、肠道萎缩、脾脏系数下降和外周血淋巴细胞数量和中性粒细胞数量异常)，这提示CID模型构建成功。
腹泻评分和体重测量结果分别如表8、表9所示。
表8.细菌组合物对CPT-11诱导的CID模型小鼠腹泻的影响
注：所有数据均采用平均值(Mean)±标准差(SEM)；所有组与模型对照组相比，“*”表示P<0.05，“**”表示P<0.01，“***”表示P<0.001，“****”表示P<0.0001。
高剂量细菌组合物和整肠生能显著降低D10腹泻总分。中高剂量细菌组合物有降低D10腹泻总分的趋势。相比模型对照组，细菌组合物高剂量组D10腹泻总分下降幅度略高于阳性对照整肠生组。总的来说，细菌组合物对CPT-11致腹泻小鼠的腹泻情况有显著改善，且具有剂量依赖趋势。
表9.细菌组合物对CPT-11致腹泻小鼠体重下降的影响
注：所有数据均采用平均值(Mean)±标准差(SEM)；所有组与模型对照组相比，“*”表示P<0.05，“**”表示P<0.01，“***”表示P<0.001，“****”表示P<0.0001。
模型小鼠造模后体重持续下降，直到试验终点。高剂量、中高剂量、中剂量细菌组合物和整肠生均能显著缓解模型小鼠D10的体重下降，且细菌组 合物高剂量治疗组与整肠生组的缓解程度相当。
(2)肠道与脾脏结果如表10所示。
表10.细菌组合物对CPT-11致腹泻小鼠的肠道和脾脏病变的影响
注：所有数据均采用平均值(Mean)±标准差(SEM)；所有组与模型对照组相比，“*”表示P<0.05，“**”表示P<0.01，“***”表示P<0.001，“****”表示P<0.0001。
细菌组合物高剂量组、中高剂量组、低剂量组和整肠生组模型小鼠的小肠萎缩均得到了显著改善。细菌组合物高剂量组、中高剂量组、中剂量组和整肠生组模型小鼠的小肠肿胀也有明显改善。中高剂量、中剂量、低剂量细菌组合物和整肠生均能显著改善模型小鼠脾脏萎缩情况。
(3)血常规结果如表11所示。
高剂量、中高剂量细菌组合物能显著升高模型小鼠外周血中淋巴细胞含量和降低中性粒细胞含量。整肠生具有升高模型小鼠外周血淋巴细胞含量的趋势，能显著降低中性粒细胞含量。细菌组合物高剂量组、中高剂量组、中剂量组、低剂量组和整肠生组模型小鼠外周血中淋巴细胞百分占比显著升高，中性粒细胞百分占比显著降低。
表11.细菌组合物对CPT-11致腹泻小鼠的血常规结果

注：所有数据均采用平均值(Mean)±标准差(SEM)；所有组与模型对照组相比，“*”表示P<0.05，“**”表示P<0.01，“***”表示P<0.001，“****”表示P<0.0001
(4)qPCR检测结果如表12所示。
表12.细菌组合物对CPT-11致腹泻小鼠结肠组织基因表达的影响
注：所有数据均采用平均值(Mean)±标准差(SEM)；所有组与模型对照组相比，“*”表示P<0.05，“**”表示P<0.01。
细菌组合物高剂量组、中高剂量组和整肠生组模型小鼠结直肠部位的炎症因子TNF-a的mRNA表达显著下调；高剂量细菌组合物能显著下调炎症因子IL-1βmRNA表达，细菌组合物中高剂量组和整肠生组相比模型组有一定下调趋势。细菌组合物高剂量组和整肠生组IL-22的mRNA表达水平有降低趋势。细菌组合物中剂量组、低剂量组紧密连接蛋白相关基因ZO-1的表达显著上调，细菌组合物中高剂量组、低剂量组紧密连接蛋白相关基因Occludin的表达显著上调。细菌组合物高剂量组模型小鼠细胞凋亡相关基因Bax的mRNA的表达显著降低，水通道蛋白AQP8的表达显著增强。
(5)病理检测结果如表13所示。
表13.细菌组合物对CPT-11致腹泻小鼠结直肠病理评分的影响
注：所有数据均采用平均值(Mean)±标准差(SEM)；所有组与模型对照组相比，“*”表示P<0.05，“**”表示P<0.01，“****”表示P<0.0001。
细菌组合物中高剂量组、低剂量组和整肠生组的病理评分显著低于模型对照组。细菌组合物高剂量组和整肠生组病理评分相比模型组有降低趋势。
综上所述，细菌组合物可降低CPT-11诱导的腹泻，降低肠道促炎因子(TNF-a、IL-1β、IL-22)和促凋亡因子(Bax)的mRNA表达，增强紧密连接蛋白(ZO-1、Occludin)和水通道蛋白(AQP8)mRNA的表达，对CPT-11诱导的小鼠腹泻、体重下降、小肠肿胀、脾脏萎缩、外周血免疫细胞(淋巴细胞数量和中性粒细胞)数量异常以及肠道炎症等症状有明显改善。
实施例11：细菌组合物对放射性肠炎小鼠的治疗效果
(1)试验设计：将雄性C57BL/6小鼠80只(购自成都药康生物科技有限公司)饲养于SPF级动物房。根据体重把前述小鼠随机分为正常对照组(给药方案：生理盐水0.2mL/只/天)、模型对照组(给药方案：冻干保护剂0.2mL/只/天)、洛哌丁胺组(给药方案：洛哌丁胺15mg/kg体重)、LGG(Lactobacillus rhamnosus GG，鼠李糖乳杆菌GG，陕西泽朗生物科技有限公司)组(给药方案：LGG 1×109CFU/只/天)、细菌组合物高剂量组(给药方案：细菌组合物1×109CFU/只/天)、细菌组合物中高剂量组(给药方案：细菌组合物1×108CFU/只/天)、细菌组合物中剂量组(给药方案：细菌组合物1×107CFU/只/天)、细菌组合物低剂量组(给药方案：细菌组合物1×106CFU/只/天)，共8组，每组10只。除正常对照组以外，其余各组小鼠均进行单次全腹部X射线辐照，辐照剂量为11.5Gy。整体试验周期为18天，记为D1～D18。各组别分别灌胃给药，每天给药一次，连续17天。给药7天后，于D8进行辐照，D18解剖。试验分组和给药方案如表14所示。
表14.细菌组合物治疗放射性肠炎小鼠的实验分组和给药方案

注：CFU:colony forming unit菌落形成单位；d:天；i.p:腹腔注射；i.g:灌胃给药；QD表示每天一次。
实验期间每天检测动物体重，并进行一般观察记录(观察项目包括但不限于动物的外观体征、行为活动、呼吸、腺体分泌、粪便情况等)，并重点观察并记录辐照造模后每只动物的腹泻情况并进行评分。试验结束时动物安乐死，取结直肠部分10％甲醛溶液中直接固定，HE染色后进行病理学检查。
(2)腹泻观察与评分：腹泻的评分标准与操作方法与实施例9相同。
(3)肠道组织病理检测：结直肠部位在10％甲醛溶液中直接固定，HE染色后进行病理学检查。
(4)数据统计与分析：对体重、腹泻评分和病理学检查结果等数据以均数±标准差(Mean±SEM)表示，使用SPSS统计软件26.0进行One-way ANOVA统计学分析。
试验结果：模型对照组动物经腹腔辐照后出现明显的腹泻、体重下降、肠黏膜损伤以及炎症浸润等症状，提示小鼠放射性肠炎模型构建成功。
(1)腹泻结果如表15所示。
表15.细菌组合物对放射性肠炎模型小鼠腹泻情况的影响

注：所有数据均采用平均值(Mean)±标准差(SEM)；所有组与模型对照组相比，“*”表示P<0.05，“**”表示P<0.01，“***”表示P<0.001，“****”表示P<0.0001，“/”表示不适用。
与模型对照组相比，细菌组合物高剂量组、中剂量组、低剂量组，洛哌丁胺组和LGG组小鼠的D17腹泻总分均有显著改善。细菌组合物中高剂量组D17腹泻总分有降低的趋势。
(2)体重结果如表16所示。
表16.细菌组合物对放射性肠炎模型小鼠体重变化的影响
注：所有数据均采用平均值(Mean)±标准差(SEM)；所有组与模型对照组相比，“*”表示P<0.05，“**”表示P<0.01，“***”表示P<0.001，“****”表示P<0.0001。
与模型对照组相比，细菌组合物高剂量组、低剂量组和LGG组小鼠D14的体重下降情况有显著改善。低剂量细菌组合物能显著改善模型小鼠D17的体重下降情况。高剂量、中剂量细菌组合物，洛哌丁胺和LGG均有恢复模型小鼠D17体重的趋势。
(3)结直肠病理结果如表17所示。
表17.细菌组合物对放射性肠炎模型小鼠结直肠部位病理评分的影响

注：所有数据均采用平均值(Mean)±标准差(SEM)；所有组与模型对照组相比，“*”表示P<0.05，“**”表示P<0.01，“***”表示P<0.001，“****”表示P<0.0001。
与模型对照组相比，细菌组合物高剂量组、低剂量组和LGG组的病理评分均有显著改善。细菌组合物中高剂量组、中剂量组和洛哌丁胺组结直肠部位的病理评分有改善趋势。
综上所述，细菌组合物对X射线全腹部辐照诱导的放射性肠炎小鼠有显著的治疗效果，能改善腹泻程度和体重下降，缓解肠道病变程度。
实施例12：细菌组合物对5-FU诱导荷瘤CID小鼠的治疗效果
试验设计：将雄性BALB/c小鼠(购自成都药康生物科技有限公司)50只饲养于SPF级动物房。于每只小鼠右侧肋部皮下接种CT26细胞(购买自北京北纳生物科技有限公司)，接种剂量为5×106个细胞/只，接种体积为0.1mL。待肿瘤体积生长至100-150mm3左右时，筛选42只小鼠进行后续实验。根据肿瘤体积随机把前述42只小鼠分为7组，每组6只。前述7组分别为正常对照组(给药方案：冻干保护剂0.2mL/只/天)、模型对照组(给药方案：冻干保护剂0.2mL/只/天)、阳性对照洛哌丁胺组(给药方案：洛哌丁胺20mg/kg体重)、整肠生组(给药方案：地衣芽胞杆菌2×108CFU/只/天)、细菌组合物高剂量组(给药方案：细菌组合物1×109CFU/只/天)、细菌组合物中剂量组(给药方案：细菌组合物1×108CFU/只/天)和细菌组合物低剂量组(给药方案：细菌组合物1×107CFU/只/天)。分组当天设为试验第1天(D1)，给药开始于第1天(D1)，每天灌胃给药1次(QD)，连续给药9天。在给药的第3天、第4天、第5天、第6天时，正常对照组腹腔注射生理盐水0.2ml/只，其余组小鼠均腹腔注射5-FU进行造模(50mg/kg体重，10ml/kg体重)。相比正常对照组，5-FU注射后各组小鼠肿瘤体积均降低。模型对照组出现严重腹泻和体重减轻，并造成显著血常规指标异常，提示模型构建成功。
具体给药方案详见表18。试验结束后，各组动物经摘取眼球采集全血样本，采用动物用五分类血液细胞分析仪(深圳市帝迈生物技术有限公司)进行血常规检测。
表18.5-FU诱导荷瘤CID模型给药方案

注：i.g.表示经口灌胃，QD表示每天一次。
试验结果：腹泻结果如表19所示。
表19.各组小鼠腹泻评分
注：所有数据均采用平均值(Mean)±标准差(SEM)；各组腹泻评分与模型对照组相比，“*”表示P<0.05，“**”表示P<0.01。
与正常对照组相比，模型对照组在D6开始出现腹泻，且随着时间的进展腹泻程度加剧。与模型对照组相比，洛哌丁胺组D8腹泻评分和腹泻总分显著降低；低剂量细菌组合物显著降低了D8、D9腹泻评分和腹泻总分。
血常规结果如表20所示。
表20.5-FU诱导的荷瘤小鼠模型血常规结果
注：所有数据均采用平均值(Mean)±标准差(SEM)；各组血常规数据与模型对照组相比，“*”表示P<0.05，“**”表示P<0.01。
与正常对照组相比，模型对照组的红细胞压积、白细胞数量、淋巴细胞数量和血小板数量显著降低。与模型对照组相比，细菌组合物中剂量组、低剂量组和整肠生组红细胞数量和红细胞压积显著升高，细菌组合物低剂量组淋巴细胞数量和血小板数量升高。
综上所述，三个剂量的细菌组合物均有改善腹泻和血常规指标的趋势；其中低剂量细菌组合物和洛哌丁胺可显著改善腹泻；低剂量细菌组合物具有显著改善血常规指标(淋巴细胞数量、红细胞数量和血小板数量)的能力。
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 (15)

  1. 一种益生菌组合，所述益生菌组合中含有第五益生菌以及第一益生菌、第二益生菌、第三益生菌和第四益生菌中的任两种、任三种或四种；
    所述第一益生菌选自两歧双歧杆菌、所述两歧双歧杆菌的子代菌株、所述两歧双歧杆菌的克隆菌株或所述两歧双歧杆菌的纯培养物；
    所述第二益生菌选自鸟肠球菌、所述鸟肠球菌的子代菌株、所述鸟肠球菌的克隆菌株或所述鸟肠球菌的纯培养物；
    所述第三益生菌选自唾液乳杆菌、所述唾液乳杆菌的子代菌株、所述唾液乳杆菌的克隆菌株或所述唾液乳杆菌的纯培养物；
    所述第四益生菌选自发酵黏液乳杆菌、所述发酵黏液乳杆菌的子代菌株、所述发酵黏液乳杆菌的克隆菌株或所述发酵黏液乳杆菌的纯培养物；以及
    所述第五益生菌选自迪氏副拟杆菌、所述迪氏副拟杆菌的子代菌株、所述迪氏副拟杆菌的克隆菌株或所述迪氏副拟杆菌的纯培养物。
  2. 如权利要求1所述的益生菌组合物，其特征在于，所述两歧双歧杆菌的微生物保藏编号为CCTCC NO:M2023349；
    所述鸟肠球菌的微生物保藏编号为CCTCC NO:M2023350；
    所述唾液乳杆菌的微生物保藏编号为CCTCC NO:M2023348；
    所述发酵黏液乳杆菌的微生物保藏编号为CCTCC NO:M2023352；以及
    所述迪氏副拟杆菌的微生物保藏编号为CCTCC NO:M20222033。
  3. 一种微生态组合物，所述微生态组合物以权利要求1或2所述的益生菌组合为活性材料。
  4. 如权利要求3所述的微生态组合物，其特征在于，所述组合物中还含有辅料，所述辅料选自冻干保护剂、细菌培养基、食品添加剂、保健品中可接受的载体或辅料和药学上可接受的载体或辅料。
  5. 如权利要求3所述的微生态组合物，其特征在于，在所述微生态组合物中，按照活细菌菌体数量计，任意两种细菌的含量比为100CFU：1-10000CFU。
  6. 权利要求1或2所述的益生菌组合或3-5中任一项所述的微生态组合物在制备用于单独使用或者用于与其他微生物制剂和/或药物联合使用以改善受试者的健康状况的产品中的用途；
    所述改善受试者的健康状况选自：
    抑制受试者体腔内的铜绿假单胞菌、痢疾志贺氏菌、金黄色葡萄球菌、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、任七种或八种的增殖；
    治疗、预防和/或减缓铜绿假单胞菌、痢疾志贺氏菌、金黄色葡萄球菌、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、任七种或 八种引起的组织损伤、疾病或亚健康状况；
    改善受试者肠道内抗氧化的能力；
    治疗、预防和/或减缓抗肿瘤药物引起的腹泻；
    治疗、预防和/或减缓抗肿瘤药物引起的肠道炎症；
    治疗、预防和/或减缓抗肿瘤药物引起的体重下降；
    治疗、预防和/或减缓抗肿瘤药物引起的小肠长度缩短；
    治疗、预防和/或减缓抗肿瘤药物引起的小肠厚度增加；
    治疗、预防和/或减缓抗肿瘤药物引起的肠损伤；
    治疗、预防和/或减缓抗肿瘤药物引起的脾脏重量与体重比值减小；
    治疗、预防和/或减缓TNF-α、IL-1β、IL-22和Bax中的任一种、任两种、任三种或四种表达量升高引起的组织损伤、疾病或亚健康状况；
    治疗、预防和/或减缓ZO-1、Occludin和AQP8的任一种、任两种或三种表达量下降引起的组织损伤、疾病或亚健康状况；
    治疗、预防和/或减缓放疗引起的腹泻；
    治疗、预防和/或减缓放疗引起的体重下降；
    治疗、预防和/或减缓放疗引起的肠损伤；
    治疗、预防和/或减缓放疗引起的肠道炎症；以及
    治疗、预防和/或减缓抗肿瘤药物引起的红细胞压积降低、白细胞数量降低、淋巴细胞数量降低和/或血小板数量降低。
  7. 如权利要求6所述的用途，其特征在于，所述产品为食品、保健品或药品。
  8. 如权利要求6所述的用途，其特征在于，所述受试者选自人和小鼠。
  9. 如权利要求6所述的用途，其特征在于，所述抗肿瘤药物选自化疗药物、靶向药物和免疫检查点抑制剂。
  10. 如权利要求9所述的用途，其特征在于，所述抗肿瘤药物选自阿霉素、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、阿糖胞苷、阿帕替尼、阿西替尼、卡博替尼、索拉非尼、舒尼替尼、纳武利尤单抗、帕博利珠单抗、伊匹木单抗。
  11. 一种预防、治疗或减缓肠道疾病的方法，所述方法为将治疗有效量的权利要求3-5中任一项所述的微生态组合物施用给受试者；
    所述肠道疾病选自：
    铜绿假单胞菌、痢疾志贺氏菌、金黄色葡萄球菌、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、艰难梭菌中的任一种、任两种、任三种、任四种、任五种、任六种、任七种或八种引起的肠道疾病；
    肠道内的氧化损伤；
    抗肿瘤药物引起的腹泻；
    抗肿瘤药物引起的肠道炎症；
    抗肿瘤药物引起的肠道损伤；
    放疗引起的腹泻；
    放疗引起的肠道炎症；以及
    放疗引起的肠道损伤。
  12. 如权利要求11所述的方法，其特征在于，所述受试者选自人和小鼠。
  13. 如权利要求11所述的方法，其特征在于，所述施用给受试者选自口服、腹腔注射、灌胃。
  14. 如权利要求11所述的方法，其特征在于，按照所述益生菌组合物中的全部细菌含量计算，所述治疗有效量为106-12CFU每天。
  15. 如权利要求11所述的方法，其特征在于，所述抗肿瘤药物选自阿霉素、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、阿糖胞苷、阿帕替尼、阿西替尼、卡博替尼、索拉非尼、舒尼替尼、纳武利尤单抗、帕博利珠单抗和伊匹木单抗。
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