Probiotic composition for preventing and treating Anti-tumor treatment-related diarrhea and use thereof

Abstract

The present invention relates to the technical field of microorganisms. Provided are a probiotic composition and a use thereof in the preparation of a product for preventing and / or treating anti-tumor treatment-related diarrhea and other health conditions. Active ingredients of the probiotic composition comprise Bifidobacterium bifidum, Enterococcus avium, Ligilactobacillus salivarius, Limosilactobacillus fermentum, and Parabacteroides distasonis. The probiotic composition has good safety, auto-aggregation capacity, antioxidant capacity, short-chain fatty acid production capacity, in-vitro cell adhesion capacity, capacity to inhibit multiple pathogenic bacteria, and capacity to treat anti-tumor treatment-related diarrhea, etc.

Description

Probiotic composition for preventing and treating anti-tumor therapy-associated diarrhea and its application Technical Field

[0001] The present invention belongs to the field of microbial medicines and relates to a probiotic composition and an application thereof, and particularly relates to a composition containing five probiotics and an application thereof in preventing and / or treating anti-tumor therapy-associated diarrhea. Background Art

[0002] Cancer treatment-related diarrhea (CRD) is a common complication of various adjuvant anti-cancer therapies and is one of the symptoms that most significantly impacts health and quality of life. In severe cases, it can lead to treatment delays and decreased compliance, all of which can affect the long-term outcomes of cancer treatment and be potentially fatal. Data released by the International Agency for Research on Cancer, an agency of the World Health Organization, show that approximately 19.3 million new cancer cases were diagnosed worldwide in 2020, and the number is projected to reach 28.4 million by 2040. As many as 82% of cancer treatment patients experience side effects such as diarrhea.

[0003] Chemotherapy (particularly chemotherapy with traditional chemotherapies such as fluoropyrimidines and irinotecan (CPT-11), the first-line treatment for patients with metastatic colorectal cancer), radiotherapy, targeted therapies, and immunotherapy are the primary causes of anticancer therapy-related diarrhea, other than surgery. The overall incidence of diarrhea associated with fluoropyrimidines and irinotecan is 50% to 80%, with severe diarrhea (grade 3 to 5) exceeding 30%. Among patients receiving 5-fluorouracil (5-FU), 1% to 5% die from diarrhea. Approximately 70% of patients with abdominal or pelvic tumors require radiotherapy, of whom 60% to 70% experience acute symptoms such as abdominal pain, diarrhea, and bloody stools, while 30% to 40% develop chronic diarrhea. The incidence of diarrhea associated with targeted therapies (such as those using various tyrosine kinase inhibitors (TKIs) such as afatinib, ceritinib, erlotinib, and lapatinib) can be as high as over 90%. Immune checkpoint inhibitors often lead to immune enteritis. Diarrhea occurs in 1% to 45% of cancer patients treated with ipilimumab, nivolumab, and pembrolizumab.

[0004] The pathogenesis of anticancer therapy-related diarrhea, excluding surgical procedures, shares commonalities. Anticancer therapy-related diarrhea is multifactorial, with the following as the primary causative factors: 1) ionizing radiation from chemotherapy drugs and radiotherapy can cause oxidative stress and DNA damage; 2) intestinal mucosal damage leads to epithelial cell loss, decreased tight junction integrity, crypt epithelial cell death, and increased intestinal permeability; 3) hyperactivation of multiple signaling pathways, including nuclear factor κB, which stimulates cells to produce inflammatory factors, leading to mucosal inflammation; 4) damage to the small intestinal mucosa leads to bile acid malabsorption. Excessive bile acids (especially dihydroxy components) induce water and electrolyte secretion and increase intestinal motility, leading to diarrhea; 5) infection with common nosocomial pathogens (Salmonella, Shigella, Escherichia coli, and Clostridium difficile, among others); 6) bacterial translocation and intestinal flora imbalance, manifested as a decrease in probiotics and an increase in pathogenic bacteria; and 7) immune abnormalities, such as widespread T cell activation.

[0005] Currently, international clinical guidelines, such as "Guidance on the Management of Diarrhoea during Cancer Chemotherapy (2014)" and "Diarrhoea in Adult Cancer Patients: ESMO Clinical Practice Guidelines (2018)," recommend loperamide as the initial treatment for chemotherapy-induced diarrhea (CID), radiation enteritis, diarrhea associated with targeted therapies, and diarrhea associated with immune checkpoint inhibitors. The guidelines recommend octreotide for patients who fail to respond to loperamide. Loperamide should not be used for more than 48 hours. High-dose loperamide may also carry the risk of paralytic ileus and severe cardiotoxicity. Octreotide (the first-line treatment for patients with diarrhea scores of 3 or higher) can cause side effects such as gallstones, hyperglycemia, and impaired glucose tolerance. The guidelines also recommend budesonide as a second-line treatment for patients who fail to respond to loperamide. However, glucocorticoids can have systemic effects, increasing the risk of infection and further exacerbating viral and bacterial infections. Patients with grade 3-4 diarrhea and neutropenia can take oral antibiotics, but antibiotics can worsen diarrhea and increase the risk of Clostridium difficile infection. Cancer treatment usually involves a combination of multiple treatments. Combination therapy greatly increases the incidence of diarrhea. If diarrhea persists or if effective treatment is not taken after diarrhea, patients will experience symptoms such as electrolyte imbalance, dehydration, and anemia. Moderate or severe diarrhea can also cause serious complications manifested as neutropenia and systemic inflammatory response syndrome. Therefore, there is an urgent clinical need for a new generation of drugs that are safer and more effective for anti-tumor therapy-related diarrhea.

[0006] Intestinal probiotics are a class of active microorganisms that colonize the human intestine and alter the composition of the host's intestinal flora, benefiting the host. Intestinal probiotics have the advantages of high safety, minimal toxicity and side effects, and a wide range of indications and population coverage. Probiotic combinations can have a therapeutic effect on the complex pathogenesis of anti-tumor therapy-related diarrhea, primarily through: 1) Probiotics exert antioxidant functions by chelating metal ions, upregulating their own and host antioxidant enzymes and metabolites, and reducing the activity of ROS-producing enzymes; and 2) Probiotics can produce beneficial secondary metabolites, such as short-chain fatty acids, indole derivatives, and secondary bile acids. Beneficial secondary metabolites promote goblet cells to secrete mucin, promote epidermal cell repair, inhibit cell apoptosis, enhance tight junction proteins, and thus enhance intestinal barrier function; 3) Probiotics regulate immune responses by downregulating the NF-κβ pathway, inhibiting proinflammatory factors, and secreting anti-inflammatory factors, thereby playing an anti-inflammatory and immune-enhancing role; 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.

[0007] Chinese patent CN112694992B discloses a strain of Bifidobacterium bifidum that can alleviate diarrhea caused by enterotoxigenic Escherichia coli (ETEC). Chinese patent CN113234619B discloses a strain of Bifidobacterium bifidum that can alleviate acute intestinal damage. A small number of literature reports that Parabacteroides distasonis can alleviate intestinal inflammation. For example, M. Kverka et al. (Oral administration of Parabacteroides distasonis antigens attenuates experimental murine colitis through modulation of immunity and microbiota composition) reported that Parabacteroides distasonis can improve DSS-induced colitis in mice. Currently, only a small number of documents report the use of Lactobacillus salivarius in preventing and treating diarrhea. For example, patent CN110878267B discloses a strain of Lactobacillus salivarius ZLp4b that can significantly slow down and cure diarrhea in young animals. In addition to the above patents and literature, there are also some similar literature on the use of probiotics to prevent and treat diarrhea or intestinal inflammation. However, current probiotic preparations are mainly used to treat common diarrhea symptoms, and most of them are single strains, which makes it difficult to play a targeted therapeutic role against the complex pathogenesis of anti-tumor therapy-related diarrhea.

[0008] Summary of the Invention

[0009] The 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, a clonal strain of Enterococcus avium or a pure culture of Enterococcus avium; the third probiotic is selected from Lactobacillus salivarius, a progeny strain of Lactobacillus salivarius, a clonal strain of Lactobacillus salivarius or a pure culture of Lactobacillus salivarius; the fourth probiotic is selected from Lactobacillus fermentans, a progeny strain of Lactobacillus fermentans, a clonal strain of Lactobacillus fermentans or a pure culture of Lactobacillus fermentans; and the fifth probiotic is selected from Parabacteroides dieckii, a progeny strain of Parabacteroides dieckii, a clonal strain of Parabacteroides dieckii or a pure culture of Parabacteroides dieckii.

[0010] In some embodiments, the microbial deposition number of the Bifidobacterium bifidum is CCTCC NO: M2023349; the microbial deposition number of the Enterococcus avium is CCTCC NO: M2023350; the microbial deposition number of the Lactobacillus salivarius is CCTCC NO: M2023348; the microbial deposition number of the Lactobacillus mucimulus fermentans is CCTCC NO: M2023352; and the microbial deposition number of the Parabacteroides dienoides is CCTCC NO: M20222033.

[0011] In some embodiments, 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 number of CCTCC NO: M2023350, a cloned strain of Enterococcus avium with a microbial preservation number of CCTCC NO: M2023350, a cloned strain of Enterococcus avium with a microbial preservation number of CCTCC NO: M2023349 NO: M2023350 or the pure culture of the avian Enterococcus with a microbial preservation number of 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 clonal 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 mucilaginosus with a microbial preservation number of CCTCC NO: M2023352, a progeny strain of Lactobacillus mucilaginosus with a microbial preservation number of CCTCC NO: M2023352, or a pure culture of Lactobacillus mucilaginosus with a microbial preservation number of CCTCC NO: M2023348; NO: M2023352 or the pure culture of the fermented mucus Lactobacillus with a microbial preservation number of CCTCC NO: M2023352; and the fifth probiotic is selected from the group consisting of Parabacteroides dieffenbachia with a microbial preservation number of CCTCC NO: M20222033, a progeny strain of Parabacteroides dieffenbachia with a microbial preservation number of CCTCC NO: M20222033, a clonal strain of Parabacteroides dieffenbachia with a microbial preservation number of CCTCC NO: M20222033 or a pure culture of Parabacteroides dieffenbachia with a microbial preservation number of CCTCC NO: M20222033.

[0012] 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.

[0013] In some embodiments, the composition further contains excipients, and the excipients are selected from freeze-drying protectants, bacterial culture media, food additives, acceptable carriers or excipients in health products, and pharmaceutically acceptable carriers or excipients.

[0014] In some embodiments, in the microecological composition, 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).

[0015] The third aspect of the present invention provides a 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, or more 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). any four, any five, any six, any seven or eight of the proliferation; treatment, prevention and / or slow down Pseudomonas aeruginosa, Shigella dysenteriae, Staphylococcus aureus, Escherichia coli, Salmonella paratyphi B, Yersinia enterocolitica, Vibrio parahaemolyticus and Clostridium difficile any one, any two, any three, any four, any five, any six, any seven or eight of the tissue damage, disease or sub-health condition caused; improve the antioxidant capacity of the subject's intestine; treatment, prevention and / or slow down diarrhea caused by anti-tumor drugs; treatment, prevention and / or slow down intestinal tract caused by anti-tumor drugs Inflammation; treatment, prevention and / or slowing down of weight loss caused by anti-tumor drugs; treatment, prevention and / or slowing down of shortening of small intestine caused by anti-tumor drugs; treatment, prevention and / or slowing down of increase in small intestine thickness caused by anti-tumor drugs; treatment, prevention and / or slowing down of intestinal damage caused by anti-tumor drugs; treatment, prevention and / or slowing down of decrease in spleen weight to body weight ratio caused by anti-tumor drugs; treatment, prevention and / or slowing down of tissue damage, disease or sub-health condition caused by increased expression of any one, any two, any three or four of TNF-α, IL-1β, IL-22 and Bax conditions; treating, preventing and / or alleviating tissue damage, disease or sub-health condition caused by decreased expression of any one, any two or three of ZO-1, Occludin and AQP8; treating, preventing and / or alleviating diarrhea caused by radiotherapy; treating, preventing and / or alleviating weight loss caused by radiotherapy; treating, preventing and / or alleviating intestinal damage caused by radiotherapy; treating, preventing and / or alleviating intestinal inflammation caused by radiotherapy; and treating, preventing and / or alleviating decreased hematocrit, decreased white blood cell count, decreased lymphocyte count and / or decreased platelet count caused by anti-tumor drugs.

[0016] In some embodiments, the product is a food, a health product, or a medicine.

[0017] In some embodiments, the subject is selected from a human and a mouse.

[0018] In some embodiments, 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.

[0019] In some embodiments, the anti-tumor therapy-related toxic side effect is diarrhea.

[0020] In some embodiments, 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.

[0021] In some embodiments, the anti-tumor drug is selected from chemotherapy drugs, targeted drugs and immune checkpoint inhibitors.

[0022] In some embodiments, 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.

[0023] The fourth aspect of the present invention provides a method for preventing, treating or alleviating intestinal diseases, which comprises 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.

[0024] In some embodiments, the subject is selected from a human and a mouse.

[0025] In some embodiments, the administering to the subject is selected from oral administration, intraperitoneal injection, and oral gavage.

[0026] In some embodiments, the therapeutically effective amount is calculated based on the total bacterial content in the probiotic composition. 6-12 CFU (e.g., 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, 8×106 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, 6×10 9 CFU, 7×10 9 CFU, 8×10 9 CFU, 9×10 9 CFU, 1×10 10 CFU, 2×10 10 CFU, 3×10 10 CFU, 4×10 10 CFU, 5×10 10 CFU, 6×10 10 CFU, 7×10 10 CFU, 8×10 10 CFU, 9×10​​​​​​​​​​​​​​​​​​​​​CFU (any value or range between any two values) per day.

[0027] In some embodiments, 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.

[0028] The beneficial effects of the present invention are:

[0029] (1) 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; (2) 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 treatment-related diarrhea; (3) 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is a frontal photograph of the colony morphology of five strains.

[0031] Figure 2 is a photo of the co-culture characteristics of five single bacterial strains on BF839 agar medium.

[0032] FIG3 shows the probiotic composition and its single bacteria self-aggregation (co-aggregation) ability.

[0033] FIG4 shows the antibacterial activity test results of the probiotic composition and its single bacteria against pathogenic bacteria.

[0034] FIG5 shows the results of the adhesion test of the probiotic composition and its single bacteria to Caco2 cells. DETAILED DESCRIPTION

[0035] To make the objectives, technical solutions and advantages of the present invention more clear, the embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0036] Unless otherwise specified, the terms used herein have the common meanings in the biomedical field.

[0037] Unless otherwise specified, the specific temperature parameters in the present invention should be understood as constant temperature treatment, and variations within a certain temperature range are allowed (such as fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, ±1°C).

[0038] 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.

[0039] In some embodiments, the microbial deposit number of the Bifidobacterium bifidum is CCTCC NO: M2023349;

[0040] The microbial preservation number of the avian enterococcus is CCTCC NO: M2023350;

[0041] The microbial preservation number of the Lactobacillus salivarius is CCTCC NO: M2023348;

[0042] The microbial preservation number of the fermented Lactobacillus mucilaginosus is CCTCC NO: M2023352; and

[0043] The microbial preservation number of the Parabacteroides dienii is CCTCC NO: M20222033.

[0044] 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 deposited in the said deposit center; (2) a strain having the same genome as the strain described in (1); (3) a subculture strain without gene mutation 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), an inactivated product of the live bacterium, a lysate of the live bacterium or a fermentation product of the live bacterium, etc.

[0045] 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 passaged strains without genetic mutations. As known to those skilled in the art, minor mutations are usually inevitably introduced into strains through passage and application. When 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 connecting amino acid residues between two domains or the mutated amino acid residues are located within the higher-order structure of the protein and do not come into contact with immune cells, these mutations do not affect toxicity, immunogenicity, and biological activity). It is reasonable to expect that when these minor changes do not significantly affect the toxicity, immunogenicity, and biological activity of the offspring strains, the mutant strains can still achieve the purpose of the present invention, and the mutant 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. From the detection perspective, there is no substantial change in the toxicity, immunogenicity and biological activity of the strain, including but not limited to, the toxicity, immunogenicity and biological activity of the strain considered to be mutated within the limitations of the detection technology such as detection sensitivity and detection limit and the range of acceptable or unavoidable errors are the same as the strain contributed by the present invention. When 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 lines, animal species, age, sex, health status, culture conditions, etc., as well as foreseeable or unavoidable systematic errors. The active ingredient refers to a substance that plays a role in the production of biological effects. In the present invention, the active ingredient is a probiotic strain. By studying the co-cultivation characteristics of the five strains of the present invention, it was found that these strains do not inhibit each other, so they can be combined into compositions containing any 3, 4 or 5 types according to the efficacy characteristics of each strain, and it is reasonable to expect that the compositions of these strain combinations can simultaneously exert the efficacy of each strain in the group.

[0046] Tumor therapy-related diarrhea (TAD) or tumor-associated diarrhea (TAD) refers to diarrhea caused by various anti-tumor therapies that damage the intestinal mucosa, leading to an imbalance in intestinal absorption and secretion. Common TADs include chemotherapy-related diarrhea, radiotherapy-induced diarrhea, targeted therapy-induced diarrhea, and immune checkpoint inhibitor-induced diarrhea. Radiotherapy-induced diarrhea clinically manifests as symptoms of radiation enteritis or radiation-induced intestinal injury.

[0047] Chemotherapy refers to the use of non-selective chemical agents to kill tumor cells to achieve therapeutic goals. Chemotherapy is the mainstay of cancer treatment, alongside surgery and radiotherapy. Due to its lack of selectivity, chemotherapy can damage normal cells while killing tumor cells. Common chemotherapy drugs include, but are not limited to, antibiotics (such as doxorubicin, epirubicin, dactinomycin D, doxorubicin, daunorubicin, and their derivatives), taxanes (such as paclitaxel, docetaxel, nab-paclitaxel, and their derivatives), platinums (such as cisplatin, carboplatin, nedaplatin, oxaliplatin, lobaplatin, and their derivatives), alkylating agents (such as cyclophosphamide, nitrogen mustard, carmustine, and their derivatives), camptothecins (such as camptothecin, hydroxycamptothecin, topotecan, irinotecan, and their derivatives), and antimetabolites (such as capecitabine, gemcitabine, methotrexate, 5-fluorouracil, pemetrexed, cytarabine, and their derivatives).

[0048] Targeted therapy involves designing therapeutic drugs that target identified oncogenic sites at the cellular and molecular level. Once administered, the drug binds to and activates specific oncogenic sites, leading to the specific death of tumor cells. The emergence of new targeted drugs has transformed cancer treatment and ushered in an era of targeted therapy. Common targeted drugs include apatinib, axitinib, cabozantinib, sorafenib, and sunitinib.

[0049] Immune checkpoint inhibitors are monoclonal antibodies developed to target specific immune checkpoints. Their primary function is to block the interaction between tumor cells expressing immune checkpoints and immune cells, thereby preventing tumor cells from suppressing immune cells. Common immune checkpoint inhibitors include, but are not limited to, PD-1 / PD-L1 inhibitor antibodies (such as nivolumab and pembrolizumab) and CTLA-4 inhibitors (such as ipilimumab).

[0050] Radiotherapy refers to the use of radiation (such as alpha, beta, and gamma rays produced by radioisotopes, as well as x-rays, electron beams, proton beams, and other particle beams produced by various x-ray therapy devices or accelerators) to treat malignant tumors. Common radiotherapy methods include, but are not limited to, conventional radiotherapy, stereotactic radiotherapy, three-dimensional conformal radiotherapy, intensity-modulated radiotherapy, image-guided radiotherapy, volumetric arc-modulated radiotherapy, and proton radiotherapy.

[0051] The present invention provides a microecological composition, which contains any three, four or five of Bifidobacterium bifidum, Enterococcus avium, Ligilactobacillus salivarius, Limosilactobacillus fermentum and Parabacteroides distasonis, as well as auxiliary materials.

[0052] 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.

[0053] The excipients vary depending on the type of product being prepared, for example, food, health products or medicines can be prepared. Accordingly, the excipients can be selected from freeze-drying protectants, bacterial culture media, food additives, acceptable carriers or excipients in health products and pharmaceutically acceptable carriers or excipients.

[0054] The present invention provides use of a probiotic combination or a microecological composition in preparing food, health products or medicines.

[0055] 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 a subject.

[0056] 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.

[0057] The therapeutically effective amount or prophylactically effective amount is an amount that can achieve the desired therapeutic or prophylactic 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 the treating clinician 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 ~10 15 CFU, 10 3 ~10 14 CFU, 10 4 ~10 13 CFU, 10 5 ~10 12 CFU or 10 6 ~1012 CFU.

[0058] Pharmaceutically acceptable carrier refers to a pharmaceutical carrier that does not cause significant stimulation to the subject and does not eliminate the biological activity and characteristics of the probiotics administered. As known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, the 18th edition Mack Printing Company, 1990, pages 1289-1329), 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, binding agents, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, protective agents, etc. and combinations thereof. Unless conventional carriers are incompatible with active ingredients, it is considered that the carrier is used in therapeutic compositions or pharmaceutical compositions. The carrier can be selected to minimize the adverse side effects of the subject and / or minimize the inactivation of the active ingredient.

[0059] Excipients refer to substances added to pharmaceutical compositions to give the drug a certain shape or 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.

[0060] The microecological composition of the present invention can further contain a second beneficial active ingredient, for example, another probiotic, prebiotic, medicine, etc. with antidiarrheal function. Prebiotics help regulate the intestinal environment by promoting the growth of probiotics in the intestinal tract, thereby indirectly playing an antidiarrheal effect. Examples of the second beneficial active ingredient include but are not limited to Bacillus licheniformis, Bifidobacterium, Clostridium butyricum, oligofructose, galacto-oligosaccharide, isomaltooligosaccharide, oligoxylose, oligomannose, inulin, stachyose, soybean oligosaccharide, beta glucan, oligolactose, etc.

[0061] In some embodiments, the pharmaceutical composition is in the form of tablets, capsules, granules, solutions, suspensions, powders, etc.

[0062] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments derived by those skilled in the art based on the embodiments in the present application documents without creative work should fall within the scope of disclosure or protection of the present invention.

[0063] The culture medium used in the following examples was prepared as follows:

[0064] To prepare YCFA liquid medium: Add 10.0 g of peptone, 2.5 g of yeast extract, 0.45 mL of a 10 (w / w)% aqueous solution of MgSO₄·7H₂O, 0.45 mL of a 10 mg / mL aqueous solution of CaCl₂, 10 mL of TE141, 0.45 g of K₂HPO₄, 0.45 g of KH₂PO₄, 0.90 g of NaCl, and 3.2 mL of VFA-mix to 1 L of distilled water to prepare a solution. Deoxygenate the solution by nitrogen exchange and aliquot. Sterilize the aliquoted solution by moist heat at 121°C for 30 min and set aside.

[0065] 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 adjust the pH to 5.5 with 50% HCl to obtain an aqueous solution of nitrilotriacetic acid. 3.00 g of MgSO4·7H2O, 0.50 g of MnSO4·H2O, 1.00 g of NaCl, 0.10 g of FeSO4·7H2O, 0.18 g of CoSO4·7H2O, 0.10 g of CaCl2·2H2O, 0.18 g of ZnSO4·7H2O, 0.006 g of CuSO4·5H2O, 0.02 g of KAl(SO4)2·12H2O, 0.01 g of H3BO3, and 0.06 g of Na2MoO4·2H2O were added. 0.01 g, 0.03 g of NiCl2·6H2O, 0.03 mL of 10 mg / mL Na2SeO3·5H2O solution and 0.03 mL of 10 mg / mL Na2WO4·2H2O solution were added to the above nitrilotriacetic acid aqueous solution (the solution was stirred continuously during the addition to keep the solution clear) to obtain TE141.

[0066] Prepare 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. Adjust the pH of the solution to neutral with 5 M NaOH before use.

[0067] Preparation of triple-mix liquid medium (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 prepare a solution. Deoxygenate the solution by nitrogen exchange and aliquot. Sterilize the aliquoted solution by moist heat at 121°C for 30 minutes. Store the resulting triple-mix liquid medium in a cool, dry place.

[0068] Preparation of triple-mix solid culture medium: Add 5 g of agar powder to the aforementioned triple-mix liquid culture medium. Other steps are the same as those for the triple-mix liquid culture medium.

[0069] Preparation of BF839 liquid culture medium: 6.0 g of potato extract powder (Beijing Solebold Technology Co., Ltd., FA0270), 10.0 g of polyvalent peptone (Beijing Solebold Technology Co., Ltd., P8950-250), Dissolve 5.0 g of peptone (Haibo Biotechnology Co., Ltd., Qingdao High-Tech Industrial Park, HB8277), 0.3 g of sodium thioglycolate (Shanghai Aladdin Biochemical Technology Co., Ltd., S105664-25G), 5.0 g of yeast extract powder (Thermo Fisher Oxoid, LP0021B), 1.5 g of glucose (50-99-7, Chengdu Kelong Chemical Co., Ltd.), and 4.0 g of disodium hydrogen phosphate (7558-79-4, Chengdu Kelong Chemical Co., Ltd.) in 1 L of distilled water to prepare a solution. Deoxygenate the solution by nitrogen displacement and aliquot. Sterilize the solution by moist heat at 121°C for 15 min. Store the resulting culture medium in a cool, dry place.

[0070] Preparation of 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 prepare a mixture. Deoxygenate the mixture by nitrogen exchange and aliquot. Sterilize the mixture by moist heat at 121°C for 15 minutes. Store the resulting culture medium in a cool, dry place.

[0071] To prepare oxygen-free PBS: Dissolve 0.27 g of potassium dihydrogen phosphate, 1.42 g of sodium hydrogen phosphate, 8 g of sodium chloride, and 0.2 g of potassium chloride in 1 L of distilled water to prepare a mixture. Heat the mixture to a boil. Cool the mixture to room temperature and add 0.55 g of cysteine ​​hydrochloride. Stir the mixture until dissolved and adjust the pH to 6.5. Heat the mixture to a boil and maintain it at a slight boil for 30 minutes. After cooling, dispense the solution into 400 mL bottles using a quantitative dispenser under nitrogen flow. Stir the bottles with moist heat at 121°C for 30 minutes. Store the resulting PBS in a cool, dry place until needed.

[0072] GAM solid medium (Qingdao Haibo Biotechnology Co., Ltd., HB8462), TSB (Tryptone Soy Broth, Qingdao Haibo Biotechnology Co., Ltd., HB4114), and TSA (Tryptone Soy Agar, Qingdao Haibo Biotechnology Co., Ltd., HB4138) were prepared according to the instructions provided by the supplier. The ingredients were weighed and dissolved in water to form a mixture. The mixture was sterilized by moist heat at 121°C for 30 minutes to obtain the culture medium. The culture medium was stored in a cool, dry place.

[0073] Preparation of culture medium for Parabacteroides distichous Pdist-1: Dissolve 6 g of anhydrous glucose, 15 g of soy peptone, 10 g of yeast extract, 10 g of yeast peptone, 2 g of potassium dihydrogen phosphate, 2 g of disodium hydrogen phosphate, 0.2 g of magnesium sulfate, 0.01 g of manganese sulfate, 0.2 g of calcium chloride, 1 mL of Tween 80, and 0.5 g of cysteine ​​hydrochloride monohydrate in 1 L of distilled water. Deoxygenate the mixture by nitrogen displacement and aliquot. Sterilize the mixture at 121°C for 15 min.

[0074] To prepare the culture medium for Lactobacillus mucilaginosus Lferm-1, dissolve 30 g of anhydrous glucose, 15 g of soy peptone, 10 g of yeast extract powder, 5 g of sodium acetate, 2 g of potassium dihydrogen phosphate, 2 g of disodium hydrogen phosphate, 0.1 g of magnesium sulfate, 0.045 g of manganese sulfate, 1 mL of Tween 80, and 0.5 g of cysteine ​​hydrochloride monohydrate in 1 L of purified water. Deoxygenate the mixture by nitrogen displacement and aliquot. Sterilize the mixture by moist heat at 121°C for 15 min.

[0075] To prepare the culture medium for Lactobacillus salivarius Lsali-1, dissolve 24g of anhydrous glucose, 20g of soy peptone, 10g of yeast extract, 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 cysteine ​​hydrochloride monohydrate in 1L of purified water. Deoxygenate the mixture by nitrogen displacement and aliquot. Sterilize the mixture by moist heat at 121°C for 15 minutes.

[0076] To prepare the culture medium for Enterococcus avium Eaviu-1, dissolve 30 g of anhydrous glucose, 15 g of soy peptone, 10 g of yeast extract, 5 g of sodium acetate, 2 g of dipotassium hydrogen phosphate, 0.1 g of magnesium sulfate, 0.045 g of manganese sulfate, 1 mL of Tween 80, and 0.5 g of cysteine ​​hydrochloride monohydrate in 1 L of purified water. Deoxygenate the mixture by nitrogen displacement and aliquot. Sterilize the mixture by moist heat at 121°C for 15 min.

[0077] To prepare the culture medium for Bifidobacterium bifidum Bbifi-1 powder: Dissolve 20 g of anhydrous glucose, 40 g of soy peptone, 5 g of N-acetylglucosamine, 2 g of potassium dihydrogen phosphate, 2 g of disodium hydrogen phosphate, 0.1 g of magnesium sulfate, 0.045 g of manganese sulfate, 1 mL of Tween 80, and 0.5 g of cysteine ​​hydrochloride monohydrate in 1 L of purified water. Deoxygenate the mixture by nitrogen displacement and aliquot. Sterilize the mixture by moist heat at 121°C for 15 min.

[0078] The preparation method of freeze-drying protective agent is as follows:

[0079] Preparation of a Parabacteriodes dissimilars Pdist-1 lyoprotectant (also used as a lyoprotectant for animal dosing controls): Solution A: 6 g sucrose, 6 g trehalose, 2 g xylitol, 2 g sorbitol, 44 g purified water, sterilized at 115°C for 30 min. Solution B: 5 g monosodium glutamate, 15 g purified water, sterilized at 115°C for 20 min. Solution C: 4 g sodium ascorbate, 16 g purified water. Filter sterilize and set aside.

[0080] Preparation of lyoprotectant for Lactobacillus mucilaginosus Lferm-1, Lactobacillus salivarius Lsali-1, and Enterococcus avium Eaviu-1: Solution A: 8g sucrose, 8g trehalose, 44g purified water, sterilized at 115°C for 30 min. Solution B: 2g sodium glutamate, 2g arginine hydrochloride, 16g purified water, sterilized at 115°C for 30 min. Solution C: 4g sodium ascorbate, 16g purified water. Filter and sterilize until ready for use.

[0081] Preparation of the freeze-dried protective agent for Bifidobacterium bifidum Bbifi-1: Solution A: 6g sucrose, 6g trehalose, 2g xylitol, 2g sorbitol, 44g purified water, sterilized at 115°C for 30 min. Solution B: 2g arginine hydrochloride, 2g sodium glutamate, 16g purified water, sterilized at 115°C for 30 min. Solution C: 4g sodium ascorbyl palmitate, 16g purified water. Filter and sterilize until ready for use.

[0082] When used, the mixture is mixed at a volume ratio of 6:2:2 for solution A: solution B: solution C. For animal testing, the lyoprotectant is freeze-dried, crushed, and then prepared into a suspension with physiological saline.

[0083] Prepare a 0.1% Tween 80-PBS dilution: Add 3.58g of disodium hydrogen phosphate dodecahydrate, 0.27g of potassium dihydrogen phosphate, 8g of sodium chloride, and 1ml of Tween 80 to 1L of boiling water and dissolve using a glass glue rod. Add 0.5g of cysteine ​​hydrochloride monohydrate to the boiled solution. Open the Hungate apparatus and boil the solution again under nitrogen. After purging nitrogen for 20 minutes, dispense the solution into anaerobic bottles that have been deoxygenated with nitrogen. Cap and label the bottles. Autoclave the solution at 121°C for 15 minutes.

[0084] Example 1: Isolation and identification of strains

[0085] Fresh fecal samples were collected from several healthy human volunteers, and each fecal sample was processed independently. An appropriate amount of oxygen-free PBS was added to the fecal sample to obtain a mixture 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 10,000 rpm for 20 minutes, and then the supernatant was discarded and the precipitate was retained. An appropriate amount of oxygen-free PBS was added to the precipitate to resuspend the bacteria to obtain a suspension. An equal volume of 50 (v / v)% oxygen-free glycerol aqueous solution was added to the suspension and mixed thoroughly to obtain a bacterial mixture sample. The samples were divided into sample tubes, the sample tube bags were vacuumed, and then the sample tubes were stored in a -80°C refrigerator. The samples in each frozen sample tube were thawed separately. 0.5 mL of the thawed sample was resuspended in 4.5 mL of oxygen-free PBS and shaken to obtain a bacterial suspension. 0.5 mL of the bacterial suspension was diluted with 4.5 mL of anaerobic PBS under anaerobic conditions by shaking. The same method was used to dilute the sample tenfold to 10 -6 Dilution. Take the appropriate dilution of bacterial solution and mix it with YCFA liquid culture medium, then divide it into 384-well plates and culture it anaerobically at 37℃ for one week. Inoculate the bacterial solution in the well where bacteria have grown into YCFA culture medium and culture it for 48 hours before dividing the bacterial solution into two parts. Use MALDI-TOF-MS to detect one bacterial solution to preliminarily classify the isolated strains. After determining that the bacterial solution contains only bacteria with one genetic background (monoclonal strain), according to the mass spectrometry results, the other bacterial solution is inoculated into YCFA culture medium again for culture. One part is subjected to 16S rDNA gene amplification and sequencing, and the other part is added to 50 (v / v)% glycerol aqueous solution at a ratio of 1:1 (volume ratio) and mixed evenly before preservation.

[0086] The 16S rDNA gene sequence obtained by sequencing was compared with the NCBI Nucleotide database to further identify the species of the isolated strains. From the numerous strains whose species were further determined, five were selected for subsequent experiments of the present invention. Strain 1 had the highest sequence similarity (>99%) with a strain of Parabacteroides distasonis; therefore, strain 1 was named Parabacteroides distasonis Pdist-1 (abbreviated as Pdist-1). Strain 2 had the highest sequence similarity (100%) with a strain of Limosilactobacillus fermentum; therefore, strain 2 was named Lferm-1 (abbreviated as Lferm-1). Strain 3 had the highest sequence similarity (100.00%) with a strain of Lactobacillus salivarius, also known as Lactobacillus salivarius, with the Latin name Ligilactobacillus salivarius. Therefore, strain 3 was named Lactobacillus salivarius Lsali-1 (abbreviated as Lsali-1). Strain 4 had the highest sequence similarity (100.00%) with a strain of Enterococcus avium. Therefore, strain 4 was named Eaviu-1 (abbreviated as Eaviu-1). Strain 5 had the highest sequence similarity (99.86%) with a strain of Bifidobacterium bifidum. Therefore, strain 5 was named Bifidobacterium bifidum Bbifi-1 (abbreviated as Bbifi-1).

[0087] Parabacteroides dissimilaris (Pdist-1), Lactobacillus fermentans (Lferm-1), and Enterococcus avium (Eaviu-1) were inoculated into BF839 medium to observe their colony morphology. Lactobacillus salivarius (Lsali-1) and Bifidobacterium bifidum (Bbifi-1) were inoculated into a three-mix solid medium to observe their colony morphology. Frontal photographs of the colony morphology of the five strains are shown in Figure 1: A is a frontal photograph of Parabacteroides dissimilaris (Pdist-1); B is a frontal photograph of Lactobacillus fermentans (Lferm-1); C is a frontal photograph of Lactobacillus salivarius (Lsali-1); D is a frontal photograph of Enterococcus avium (Eaviu-1); and E is a frontal photograph of Bifidobacterium bifidum (Bbifi-1). All five strains exhibit white, opaque, round colonies with a raised center and a smooth, moist surface.

[0088] Example 2: Whole genome analysis of strains

[0089] The five strains obtained in Example 1 were inoculated into a three-mix liquid medium and cultured until late logarithmic growth. Whole-genome DNA was extracted from each strain and sequenced using the Illumina NovaSeq 6000 high-throughput sequencing platform. After genome sequence assembly and annotation, protein sequences were entered into the Virulence Factor Databases (VFDB) for virulence factor analysis. The results showed that none of the five strains possessed virulence factors in their genomes.

[0090] The novelty of the five strains was analyzed using the Average Nucleotide Identity (ANI) method. A whole-genome search was performed in Genbank, and the most similar strains were compared using fastANI (v1.33). The two strains most similar to the full genome of Parabacteroides distichous Pdist-1 were GCA_003462945.1 (ANI = 98.26%) and GCA_003459965.1 (ANI = 98.20%). The two strains most similar to the full genome of Lactobacillus fermentans Lferm-1 were GCA_003465085.1 (ANI = 99.32%) and GCA_024385625.1 (ANI = 99.29%). The two strains most similar to the complete genome of Lactobacillus salivarius Lsali-1 are GCA_009863605.1 (ANI = 99.94%) and GCA_009866185.1 (ANI = 99.87%). The two strains most similar to the complete genome of Enterococcus avium Eaviu-1 are GCA_018917545.1 (ANI = 98.75%) and GCA_018373135.1 (ANI = 98.62%). The two strains most similar to the complete genome of Bifidobacterium bifidum Bbifi-1 are GCA_003466395.1 (ANI = 99.01%) and GCA_003437945.1 (ANI = 99.00%). It can be seen that the species classification made in Example 1 is correct.

[0091] The strains Pdist-1, Eaviu-1, Lferm-1, Bbifi-1, and Lsali-1 isolated and cultured in the present invention were deposited with a patent application-approved depository institution, the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China. The culture name, taxonomic nomenclature, deposit date, identification survival date, and microbial deposit number are shown in Table 1.

[0092] Table 1. Statistics of strain preservation information

[0093] Example 3: Co-cultivation characteristics test between strains

[0094] 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 phase. A disposable sterile cotton swab was used to dip the bacterial solution of one of the strains and streaked three times in parallel on a BF839 solid culture medium. Then, the bacterial solutions of the remaining four strains were streaked once in a direction perpendicular to the first streak. After the streaked bacterial solution was dried, it was anaerobically cultured for 48 hours until the bacterial solution traces were obvious. The interaction relationship between the five bacterial strains is shown in Figure 2. As can be seen, there are no breakpoints at the intersection of the strains, indicating that there is no growth inhibition between the strains.

[0095] Example 4: Agglutination ability of single bacteria and bacterial combinations

[0096] Preparation of single bacterial suspension: strains Pdist-1, Eaviu-1, Lferm-1, Bbifi-1, Lsali-1 and the control strain Lactobacillus rhamnosus GG (LGG, CICC6141, purchased from the China Industrial Microbiology Culture Collection) were inoculated into three mixed liquid media at an initial inoculum of 2% and cultured anaerobically at 37°C until the late logarithmic growth stage. The cultured bacterial solution was centrifuged and washed three times with oxygen-free PBS. The bacterial cells were resuspended in an appropriate amount of PBS and the OD value of the bacterial solution was measured. 600 (Measure the absorbance of the bacterial solution at a wavelength of 600 nm) and use PBS as a diluent to dilute each bacterial solution to the same OD 600 value (0.5±0.1).

[0097] Preparation of bacterial composition suspension: dilute the above to the same OD 600 The Pdist-1, Eaviu-1, Lferm-1, Bbifi-1 and Lsali-1 bacterial solutions were mixed in equal volumes.

[0098] Take 15mL of diluted single bacterial suspension and mixed bacterial suspension respectively, and divide them into 5mL / tube, a total of 3 tubes, for parallel experiments. Measure the OD value of the bacterial suspension at the initial time 600 , recorded as A0; after standing at 37℃ for 24h, 1mL of the upper bacterial suspension was aspirated and the OD was measured again. 600 Value, denoted as A 24 . Self-aggregation rate / co-aggregation rate = (1-A 24 / A0)×100%, and the test results are expressed as Mean±SD (autoagglutination rate: agglutination rate between single strains; coagglutination rate: agglutination rate between different strains in a bacterial composition).

[0099] The results are shown in Figure 3. This indicates that both the bacterial composition and individual strains exhibited agglutination ability, with the order of agglutination ability (autoagglutination rate / coagglutination rate, agglutination rate) being: Eaviu-1 > LGG ≈ Pdist-1 > Lsali-1 ≈ Bbifi-1 ≈ bacterial composition > Lferm-1. Bacteria with greater agglutination ability are likely to have enhanced colonization ability and be more likely to form a protective barrier on the surface of host intestinal cells.

[0100] Example 5: Antioxidant capacity test of bacterial composition

[0101] Bacterial composition culture: After activation, the 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, with the inoculation amount of each strain being 1%, and cultured anaerobically at 37°C for 24 hours.

[0102] Sample Preparation: Take 0.5 mL of culture medium and centrifuge at 12,000 rpm for 20 minutes. Discard the supernatant and resuspend the cells in 0.5 mL of pre-chilled extract from a strain total antioxidant capacity assay kit (purchased from Beijing Solebao Technology Co., Ltd., BC1315). Transfer the cells to a sterilized screw-cap tube containing beads (Sigma-Aldrich, USA, G4649-1KG). Disrupt the cell walls once using a rapid sample preparation instrument (settings: 4.5 m / s for 30 seconds). Centrifuge at 12,000 rpm for 10 minutes at 4°C. Remove the supernatant and place on ice for analysis.

[0103] A BCA protein concentration assay kit (purchased from Beijing Solebaugh Technology Co., Ltd., PC0020) was used to draw a standard curve and detect BCA samples according to the kit's instructions.

[0104] The total antioxidant capacity of the strain was determined using a total antioxidant capacity assay kit according to the kit instructions and the standard curve. The total antioxidant capacity is expressed in μmol / mg prot.

[0105] The antioxidant capacity of the bacterial composition was 0.189 μmol / mg prot. The bacterial composition had a certain antioxidant capacity.

[0106] Example 6: Ability of single bacteria and bacterial combinations to produce short-chain fatty acids

[0107] Single bacteria: 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% inoculum size and cultured anaerobically at 37°C for 24 h.

[0108] 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% inoculum amount, and cultured anaerobically at 37°C for 24 hours.

[0109] Centrifuge the fermentation broth of a single bacterium and a bacterial combination separately, and remove 1 mL of the supernatant. Add 10 μL of formic acid to the supernatant, incubate at 4°C for 30 min, and centrifuge again. Remove the supernatant, filter it through a 0.22 μm filter, and set aside. Determine the content of short-chain fatty acids (SCFAs) in each filtered solution using a gas chromatograph. The relevant gas chromatography parameters are as follows: Instrument model: Agilent 7890A. Chromatographic column model: DB-WAX UI (30 m × 0.32 mm × 0.25 μm) capillary column. Column temperature: Initial temperature 80°C for 0.5 min; then increase to 180°C at 5°C / min and hold for 1 min; then increase to 220°C at 40°C / min and hold for 4 min. Inlet temperature: 220°C. Detector temperature: 250°C. Carrier gas: Hydrogen at 30 mL / min; Air at 300 mL / min. Tail flow rate: nitrogen 28.314 mL / min. Split ratio: 10:1. Injection volume: 1 μL. Four replicate injections of the same sample were performed, and the average value was calculated. Acid production was then calculated using the following formula: actual acid production (ppm) = measured acid production value of the strain minus the baseline value of the BF839 culture. Total SCFA production is the sum of acetic acid, propionic acid, butyric acid, isovaleric acid, and valeric acid.

[0110] Results: As shown in Table 2, the control strain LGG, the bacterial combination, and its individual strains all produced acetic acid. In addition to acetic acid, Pdist-1 also produced propionic acid and isovaleric acid. The total SCFA production of Bbifi-1, Pdist-1, and the bacterial combination was significantly higher than that of LGG, while the total SCFA production of Lsali-1, Eaviu-1, and Lferm-1 was comparable to that of LGG.

[0111] Table 2. Statistics of acid production of strains

[0112] Example 7: Ability of single bacteria and bacterial combinations to inhibit pathogenic bacteria

[0113] In this embodiment, 8 common pathogens that can cause diarrhea were selected for antibacterial ability testing. The source information of the pathogenic strains is as follows: 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. Salmonella paratyphi B (CMCC (B) 50094), purchased from the China Food and Drug Inspection Institute. Yersinia enterocolitica CMCC (B) 52204, purchased from the China Food and Drug Inspection Institute. Vibrio parahaemolyticus (ATCC 17802), purchased from the American Microbiological Collection. Clostridium difficile (CICC 22951) was purchased from the China Industrial Microbiology Culture Collection Center.

[0114] Single bacteria: Each single bacteria of the control strains LGG, Pdist-1, Eaviu-1, Lferm-1, Bbifi-1 and Lsali-1 was inoculated into the three mixed liquid culture media at an inoculum size of 5%, and cultured anaerobically for 48 hours to obtain the fermentation broth.

[0115] 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 a 1% inoculation rate, and anaerobically cultured for 48 hours to obtain a fermentation broth.

[0116] Pseudomonas aeruginosa, Shigella dysenteriae, Staphylococcus aureus, Escherichia coli, Salmonella paratyphi B, Yersinia enterocolitica and Vibrio parahaemolyticus were cultured aerobically in TSB medium until the logarithmic growth phase. The culture was diluted to 10 6 CFU / mL, take 200 μL and spread on TSA solid medium. C. difficile was cultured anaerobically in three-mixed liquid medium until the logarithmic growth phase. The culture was diluted to 10 6CFU / mL, 200 μL was spread on anaerobic GAM solid medium (supplemented with 5 v / v% horse serum, Beijing Solaibao Technology Co., Ltd., S9050). 3 Oxford cups were placed on each plate, and 200 μL of the fermentation broth to be tested (the aforementioned culture) was added to the Oxford cups. Clostridium difficile was cultured under anaerobic conditions, and the other 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. It can be seen that 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 a single bacterium in inhibiting certain pathogens. The bacterial composition is superior to the control strain LGG in terms of inhibiting Salmonella paratyphi B and Staphylococcus aureus, and its inhibitory ability against other pathogens is comparable to that of the control strain LGG.

[0117] Example 8: Adhesion test of single bacteria and bacterial combinations to Caco2 cells

[0118] Single bacteria: The control strains LGG, Pdist-1, Eaviu-1, Lferm-1, Bbifi-1, and Lsali-1 were inoculated into a three-mixed liquid medium at an initial inoculum volume of 2% and cultured anaerobically at 37°C until the late logarithmic growth phase. The cultured bacterial solution was washed twice by centrifugation with sterile PBS (Wuhan Boster Biotechnology Co., Ltd., PYG0021), and then the precipitated strain was diluted to 5×10 using DMEM (Thermo Fisher Scientific (China) Co., Ltd., C11995500BT) medium containing 10 v / v% FBS (Thermo Fisher Scientific (China) Co., Ltd., SH30084.03). 8 CFU / mL, set aside.

[0119] 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.

[0120] Caco-2 cells (Shangcheng Beina Chuanglian Biotechnology Co., Ltd., 350769) were adherent cells. Caco-2 cells were digested with trypsin cell digestion solution (Lanjieke Technology Co., Ltd., BL501A) preheated at 37°C. The digested Caco-2 cells were collected by centrifugation. The pellet was diluted with DMEM medium containing 10 v / v% FBS. 5×10 4 Caco-2 cells were seeded into 96-well plates at a density of CFU / well and cultured overnight in a 37°C carbon dioxide incubator for later use.

[0121] Each well of a 96-well plate was divided into two groups. 100 μL of the diluted single bacterial suspension or bacterial combination suspension was added to each group of 96-well cell culture plates containing Caco-2 cells. After addition, the 96-well cell culture plates were centrifuged at 1000 g for 1 minute. The wells corresponding to each bacterial suspension were divided into two groups: one group was incubated for 30 minutes, and the other group was incubated for 2 hours. After incubation, the cells were washed twice with sterile PBS to remove non-adherent bacteria. After washing, 50 μL of 0.25% trypsin cell digestion solution (Langjieke Technology Co., Ltd., BL501A) was added to each well, and the cells were incubated in a 37°C incubator. After the Caco-2 cells were digested and spherical, 150 μL of DMEM medium was added to each well and repeatedly pipetted for approximately 1 minute. After microscopic examination confirmed the digestion and separation of the cells and strains, 20 μL of the mixture was aspirated and diluted in 0.1% Tween 80-PBS in a 96-well plate in a 10-fold series. Pour the appropriate dilution gradient into the dissolved three-mix solid culture medium, culture at 37°C for 48 hours, and then count.

[0122] The results are shown in Figure 5. Figure A shows that after 30 minutes of adhesion of each strain to Caco2 cells, the adhesion ability of Enterococcus avium Eaviu-1, the bacterial composition, and LGG was comparable. Figure B shows the adhesion effect of each strain after 2 hours of adhesion. The adhesion ability of Bifidobacterium bifidum Bbifi-1 and Lactobacillus fermentum Lferm-1 was comparable to LGG, while the adhesion ability of Lactobacillus salivarius Lsali-1, Enterococcus avium Eaviu-1, and the bacterial composition was superior to LGG. The bacterial composition has better adhesion ability and is therefore more likely to colonize.

[0123] Example 9: Test of the therapeutic effect of the bacterial composition on mice with diarrhea caused by 5-fluorouracil (5-FU)

[0124] Preparation of bacterial composition:

[0125] Pdist-1, Lferm-1, Lsali-1, Eaviu-1, and Bbifi-1 were inoculated into their respective bacterial powder preparation media and incubated anaerobically at 37°C and 90 rpm for 16-24 hours to obtain a primary seed solution. Subsequently, each of the five primary seed solutions was transferred to its respective bacterial powder preparation media and incubated anaerobically at 37°C and 90 rpm for 10-15 hours to obtain a secondary seed solution. Each of the five secondary seed solutions was pumped into a fermenter containing the corresponding bacterial powder preparation media using a peristaltic pump for fermentation. After fermentation, the bacterial cells were collected by centrifugation. The corresponding lyoprotectant was added at a weight ratio of 1:1 to 1:2 to emulsify the sludge. Each sludge was freeze-dried and pulverized to obtain bacterial powder. An appropriate amount of the pulverized powder was taken to determine the viable cell count. Based on the viable cell count, the bacterial powders of each strain were mixed at an equal CFU ratio to obtain a bacterial composition powder. Finally, the bacterial composition powder was prepared 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.

[0126] Test method:

[0127] (1) Experimental design: 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. According to the initial weight of the mice, they were randomly divided into 8 groups (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, 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 bacterial composition group (1×10 9 CFU / mouse / day, the high-dose group of the bacterial composition (1×10 8 CFU / mouse / day, medium dose group of bacterial composition (1×10 7 CFU / mouse / day) and the low-dose group of bacterial composition (1×10 6CFU / mouse / day). The total experimental period was 9 days, designated as Days 1-9, with continuous gavage administration for 8 days from Days 1-8. Model establishment began on Day 3. Mice in the normal control group were injected intraperitoneally with 0.2 mL of normal saline per mouse. Mice in the remaining groups were intraperitoneally injected with 30 mg / kg body weight of 5-FU (5-fluorouracil, purchased from Tianjin Jinyao Pharmaceutical Co., Ltd., specification: 10 mL / vial, 0.25 g / 10 mL) for 4 consecutive days to induce a CID model in mice. Dissections were performed on Day 9. Specific experimental groupings and dosing schedules are shown in Table 3.

[0128] Table 3. Experimental groups and dosing regimens of bacterial compositions for the treatment of 5-FU-induced diarrhea in mice

[0129] Note: 5-FU: 5-fluorouracil; CFU: colony forming unit; d: day; ip: intraperitoneal injection; ig: oral administration; QD means once a day.

[0130] During the experiment, the animals were weighed daily and general observations were recorded (including but not limited to the animals' physical appearance, behavioral activity, respiration, glandular secretions, feces, etc.). The diarrhea of ​​each animal after modeling was observed and recorded, and scored. After euthanasia at the end of the experiment, mid-colon tissue samples were collected for qPCR testing of genes related to inflammatory factors (TNF-α, IL-1β) and tight junction proteins (ZO-1, Occludin).

[0131] (2) Diarrhea observation and scoring: The scoring standard for diarrhea was based on the diarrhea scoring method described in the study by Kurita A et al. (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). Mice were placed in a mouse cage lined with clean filter paper, with one mouse per cage. 0 points: hard stool, normal stool; 1 point: slightly wet stool, slightly wet stool, or soft stool; 2 points: moderately wet stool, unformed stool, and unclean perianal area; 3 points: severe diarrhea, loose stool, watery stool, and severe unclean perianal area. During the experimental period, the feces of mice were observed and scored every day. The total diarrhea score was the sum of the daily diarrhea scores.

[0132] (3) 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, and the cDNA was stored at -20℃ for future use. The relative transcription levels of the mRNA encoding genes of the pro-inflammatory factors IL-1β, TNF-α and tight junction proteins ZO-1 and Occludin-1 in the colon of each group of mice were detected by qRT-PCR method (primer sequences are shown in Table 7, and the internal reference gene is β-actin). Reaction procedure: 1) 95℃3min; 2) 95℃10s, 60℃30s, 95℃15s, 60℃1min, a total of 40 cycles; 3) 95℃10s. 2 -ΔΔCT Method for data analysis.

[0133] (4) Data statistics and analysis: The data such as body weight, diarrhea score and pathological examination results were expressed as mean ± standard deviation (Mean ± SEM), and one-way ANOVA statistical analysis was performed using SPSS statistical software 26.0.

[0134] Test results:

[0135] (1) The diarrhea score results are shown in Table 4.

[0136] Table 4. Diarrhea score results

[0137] Note: All data are expressed as mean ± standard deviation (SEM); for all groups compared with the model control group, "*" indicates P < 0.05, "**" indicates P < 0.01, "***" indicates P < 0.001, and "****" indicates P < 0.0001.

[0138] After 5-FU induction, diarrhea scores on Days 8 and 9 in the model group were significantly higher than those in the normal control group. All four doses of the bacterial composition significantly improved diarrhea in the animals, with significantly lower diarrhea scores on Days 8 and 9 and the total diarrhea score compared to the model control group. This suggests that the bacterial composition of the present invention can improve 5-FU-induced diarrhea.

[0139] (2) The qPCR test results are shown in Table 5.

[0140] Table 5. Effects of bacterial composition on colonic inflammation and intestinal barrier-related gene expression in mice with 5-FU-induced diarrhea

[0141] Note: All data are expressed as mean ± standard deviation (SEM); for all groups compared with the model control group, "*" indicates P < 0.05, "**" indicates P < 0.01, "***" indicates P < 0.001, and "****" indicates P < 0.0001.

[0142] Compared with the normal control group, the relative mRNA transcription levels of TNF-α and IL-1β genes in the model control group were significantly increased, while the relative mRNA transcription levels of ZO-1 and Occludin genes were significantly decreased. The high-dose bacterial composition and loperamide significantly reduced the relative mRNA transcription level of TNF-α. The high-dose, medium-high-dose, and medium-dose bacterial compositions significantly reduced the relative mRNA transcription level of IL-1β. The high-dose group, medium-dose group, and low-dose bacterial composition significantly increased the relative mRNA transcription level of ZO-1, which was comparable to the improvement effect of loperamide and Zhengchangsheng. The medium-high-dose and medium-dose bacterial compositions significantly increased the relative mRNA transcription level of Occludin, which was comparable to the improvement effect of Zhengchangsheng.

[0143] In summary, 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.

[0144] Example 10: Therapeutic effect of bacterial composition on irinotecan (CPT-11)-induced diarrhea in mice

[0145] The bacterial compositions used in Examples 10-12 were obtained by mixing five bacteria at equal CFU according to the method in Example 9. The CFU of the bacterial compositions mentioned in Examples 10-12 is the total CFU of the five bacteria.

[0146] Test method:

[0147] (1) Experimental design: 70 male Balb / c mice (purchased from Shanghai Jihui Experimental Animal Breeding Co., Ltd.) were housed in an SPF animal room. According to their body weight, they were randomly divided into a normal control group (no medication), a model control group (medication regimen: lyophilized protective agent 0.2 mL / mouse / day), a positive control (medication regimen: Bacillus licheniformis 2.5×10 8 CFU / mouse / day) group, bacterial composition high-dose group (dosage regimen: bacterial composition 1×10 9 CFU / mouse / day), medium and high dose groups of bacterial composition (dosage regimen: bacterial composition 1×10 8 CFU / mouse / day), medium-dose bacterial composition group (dosage regimen: bacterial composition 1×10 7 CFU / mouse / day), bacterial composition low-dose group (dosage regimen: bacterial composition 1×10 6 CFU / animal / day), 7 groups in total, 10 animals in each group. The entire experimental period was 10 days, recorded as D1 to D10.

[0148] Mice in the normal control group were injected abdominally with 0.2 mL of normal saline per mouse. Mice in the remaining groups were injected abdominally with 85 mg / kg body weight of CPT-11 to establish the model. Model establishment began on Day 3 and continued daily for 4 consecutive days from Day 3 to Day 6. Mice in the corresponding groups were administered intragastric administration once daily for 9 consecutive days, and autopsied on Day 10. Specific experimental groupings and dosing schedules are shown in Table 6.

[0149] Table 6. Experimental groups and dosing regimens of bacterial compositions for the treatment of CPT-11-induced diarrhea in mice

[0150] Note: CPT-11: irinotecan hydrochloride; CFU: colony forming unit; d: day; ip: intraperitoneal injection; ig: oral administration; QD means once a day.

[0151] During the experiment, animal weights were measured daily, and general observations were recorded (including but not limited to physical appearance, behavioral activity, respiration, glandular secretions, and fecal content). Post-modeling diarrhea was particularly observed and recorded, and scored. At the end of the experiment, whole blood samples were collected from each group via the peritoneal vein for routine blood tests. Following euthanasia, the intestines were harvested, and the length of the small intestine was measured. The spleens were weighed, and the spleen coefficient was calculated (spleen coefficient = spleen weight / body weight × 100). Samples were collected from five mice in each group near the cecum, snap-frozen in liquid nitrogen, and stored at -80°C. Samples were used for qPCR to measure mRNA expression levels of genes related to inflammatory factors (TNF-α, IL-1β, IL-22), tight junction proteins (ZO-1, Occludin), pro-apoptotic factors (Bax), and aquaporin (AQP8). The colorectal sections of the remaining five mice in each group were directly fixed in 10% formaldehyde and stained with hematoxylin and eosin for pathological examination.

[0152] (2) Diarrhea observation and scoring: The scoring criteria and operating method for diarrhea are the same as those in Example 9.

[0153] (3) Blood routine test: Whole blood samples were collected from each group of animals through the peritoneal vein. The samples were tested for blood routine using a fully automatic blood cell analyzer (Mindray).

[0154] (4) 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, and the cDNA was stored at -20℃ for future use. The relative transcription levels of mRNA of the pro-inflammatory factors TNF-α, IL-1β, 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 β-actin). Reaction procedure: 1) 95℃3min; 2) 95℃10s, 60℃30s, 95℃15s, 60℃1min, a total of 40 cycles; 3) 95℃10s. 2 -ΔΔCT Method for data analysis.

[0155] Table 7. qRT-PCR primer information

[0156] (5) Intestinal tissue pathology: The colorectal part was directly fixed in 10% formaldehyde solution and stained with HE for pathological examination.

[0157] (6) Data statistics and analysis: The data such as body weight, diarrhea score and pathological examination results were expressed as mean ± standard deviation (Mean ± SEM), and one-way ANOVA statistical analysis was performed using SPSS statistical software 26.0.

[0158] Test results:

[0159] (1) After intraperitoneal injection of irinotecan, 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), which indicated that the CID model was successfully constructed.

[0160] The diarrhea scores and body weight measurement results are shown in Tables 8 and 9, respectively.

[0161] Table 8. Effects of bacterial composition on diarrhea in CPT-11-induced CID model mice

[0162] Note: All data are expressed as mean ± standard deviation (SEM); for all groups compared with the model control group, "*" indicates P < 0.05, "**" indicates P < 0.01, "***" indicates P < 0.001, and "****" indicates P < 0.0001.

[0163] High-dose bacterial composition and Zhengchangsheng significantly reduced D10 diarrhea scores. Medium- and high-dose bacterial compositions showed a trend toward lowering D10 diarrhea scores. Compared to the model control group, the high-dose bacterial composition group showed a slightly greater decrease in D10 diarrhea scores than the positive control Zhengchangsheng group. Overall, the bacterial composition significantly improved diarrhea in mice with CPT-11-induced diarrhea, with a dose-dependent effect.

[0164] Table 9. Effects of bacterial composition on weight loss in mice with CPT-11-induced diarrhea

[0165] Note: All data are expressed as mean ± standard deviation (SEM); for all groups compared with the model control group, "*" indicates P < 0.05, "**" indicates P < 0.01, "***" indicates P < 0.001, and "****" indicates P < 0.0001.

[0166] The model mice continued to lose weight until the end of the study. High-dose, medium-high-dose, and medium-dose bacterial composition and Zhengchangsheng significantly alleviated weight loss in the model mice on Day 10, with the high-dose bacterial composition group achieving comparable relief to the Zhengchangsheng group.

[0167] (2) The results of intestine and spleen are shown in Table 10.

[0168] Table 10. Effects of bacterial composition on intestinal and spleen lesions in mice with CPT-11-induced diarrhea

[0169] Note: All data are expressed as mean ± standard deviation (SEM); for all groups compared with the model control group, "*" indicates P < 0.05, "**" indicates P < 0.01, "***" indicates P < 0.001, and "****" indicates P < 0.0001.

[0170] Small intestinal atrophy was significantly improved in the model mice treated with the high-dose, medium-high-dose, low-dose, and Zhengchangsheng groups of the bacterial composition. Small intestinal swelling was also significantly reduced in the model mice treated with the high-dose, medium-high-dose, medium-dose, and Zhengchangsheng groups of the bacterial composition. The medium-high, medium, and low-dose bacterial compositions and Zhengchangsheng significantly improved spleen atrophy in the model mice.

[0171] (3) The results of routine blood tests are shown in Table 11.

[0172] High-dose and medium-high-dose bacterial compositions significantly increased peripheral blood lymphocyte counts and decreased neutrophil counts in model mice. Zhengchangsheng (a Chinese herbal medicine) showed a trend toward increasing peripheral blood lymphocyte counts and significantly decreasing neutrophil counts in model mice. The percentage of lymphocytes in the peripheral blood of model mice in the high-dose, medium-high-dose, medium-dose, and low-dose bacterial composition groups, as well as the Zhengchangsheng (a Chinese herbal medicine) group, significantly increased, while the percentage of neutrophils significantly decreased.

[0173] Table 11. Blood routine test results of the bacterial composition on CPT-11-induced diarrhea mice

[0174] Note: All data are expressed as mean ± standard deviation (SEM); all groups are compared with the model control group, "*" indicates P < 0.05, "**" indicates P < 0.01, "***" indicates P < 0.001, and "****" indicates P < 0.0001

[0175] (4) The qPCR test results are shown in Table 12.

[0176] Table 12. Effects of bacterial composition on gene expression in colon tissue of mice with CPT-11-induced diarrhea

[0177] Note: All data are expressed as mean ± standard deviation (SEM); for all groups compared with the model control group, "*" indicates P < 0.05, and "**" indicates P < 0.01.

[0178] The mRNA expression of the inflammatory factor TNF-a in the colorectal region of the model mice was significantly downregulated in the high-dose, medium-high-dose, and Zhengchangsheng groups of the bacterial composition. The high-dose bacterial composition significantly downregulated the mRNA expression of the inflammatory factor IL-1β, with a trend of downregulation in the medium-high-dose and Zhengchangsheng groups compared to the model group. The mRNA expression levels of IL-22 in the high-dose and Zhengchangsheng groups also showed a decreasing trend. The expression of the tight junction protein-associated gene ZO-1 was significantly upregulated in the medium-dose and low-dose groups of the bacterial composition, as was the expression of the tight junction protein-associated gene Occludin. The mRNA expression of the apoptosis-related gene Bax was significantly reduced in the high-dose group of the bacterial composition, while the expression of the aquaporin AQP8 was significantly increased.

[0179] (5) The pathological examination results are shown in Table 13.

[0180] Table 13. Effects of bacterial composition on colorectal pathology scores in mice with CPT-11-induced diarrhea

[0181] Note: All data are expressed as mean ± standard deviation (SEM); for all groups compared with the model control group, "*" indicates P < 0.05, "**" indicates P < 0.01, and "****" indicates P < 0.0001.

[0182] The pathological scores of the high-dose, low-dose, and Zhengchangsheng groups of the bacterial composition were significantly lower than those of the model control group. The pathological scores of the high-dose and Zhengchangsheng groups showed a downward trend compared to the model group.

[0183] In summary, the bacterial composition can reduce CPT-11-induced diarrhea, reduce the mRNA expression of intestinal proinflammatory factors (TNF-a, IL-1β, IL-22) and pro-apoptotic factors (Bax), enhance the expression of tight junction proteins (ZO-1, Occludin) and water channel proteins (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.

[0184] Example 11: Therapeutic effect of bacterial composition on mice with radiation enteritis

[0185] (1) Experimental design: 80 male C57BL / 6 mice (purchased from Chengdu Yaokang Biotechnology Co., Ltd.) were raised in an SPF animal room. According to their body weight, the mice were randomly divided into a normal control group (dosage regimen: 0.2 mL of normal saline per day), a model control group (dosage regimen: 0.2 mL of lyophilized protective agent per day), a loperamide group (dosage regimen: 15 mg of loperamide per kg body weight), and a LGG (Lactobacillus rhamnosus GG, Shaanxi Zelang Biotechnology Co., Ltd.) group (dosage regimen: 1×10 9 CFU / mouse / day), bacterial composition high-dose group (dosage regimen: bacterial composition 1×10 9 CFU / mouse / day), medium and high dose groups of bacterial composition (dosage regimen: bacterial composition 1×10 8 CFU / mouse / day), medium-dose bacterial composition group (dosage regimen: bacterial composition 1×10 7 CFU / mouse / day), bacterial composition low-dose group (dosage regimen: bacterial composition 1×10 6CFU / mouse / day), with 8 groups of 10 mice each. Except for the normal control group, all other groups of mice underwent a single whole-abdomen X-ray irradiation at a dose of 11.5 Gy. The overall experimental period was 18 days, designated as Days 1 to 18. Each group was gavage-administered once daily for 17 consecutive days. Seven days after dosing, mice were irradiated on Day 8 and autopsied on Day 18. The experimental groupings and dosing schedule are shown in Table 14.

[0186] Table 14. Experimental groups and dosing regimens of bacterial compositions for the treatment of radiation enteritis in mice

[0187] Note: CFU: colony forming unit; d: day; ip: intraperitoneal injection; ig: oral administration; QD means once a day.

[0188] During the experiment, the animals were weighed daily and general observations were recorded (including but not limited to the animals' physical appearance, behavior, respiration, glandular secretions, and feces). Post-irradiation diarrhea was particularly observed and recorded, and scored. At the conclusion of the experiment, the animals were euthanized, and colorectal sections were fixed in 10% formaldehyde solution and stained with hematoxylin and eosin for pathological examination.

[0189] (2) Diarrhea observation and scoring: The scoring criteria and operating method for diarrhea are the same as those in Example 9.

[0190] (3) Intestinal tissue pathological examination: The colorectal part was directly fixed in 10% formaldehyde solution and subjected to pathological examination after HE staining.

[0191] (4) Data statistics and analysis: The data such as body weight, diarrhea score and pathological examination results were expressed as mean ± standard deviation (Mean ± SEM), and one-way ANOVA statistical analysis was performed using SPSS statistical software 26.0.

[0192] Experimental results: After intraperitoneal irradiation, the animals in the model control group showed obvious symptoms such as diarrhea, weight loss, intestinal mucosal damage and inflammatory infiltration, indicating that the mouse radiation enteritis model was successfully established.

[0193] (1) The diarrhea results are shown in Table 15.

[0194] Table 15. Effects of bacterial composition on diarrhea in mice with radiation enteritis model

[0195] Note: All data are expressed as mean ± standard deviation (SEM); all groups were compared with the model control group. "*" indicates P < 0.05, "**" indicates P < 0.01, "***" indicates P < 0.001, "****" indicates P < 0.0001, and " / " indicates not applicable.

[0196] Compared with the model control group, the total diarrhea scores of mice in the high-dose, medium-dose, and low-dose bacterial composition groups, the loperamide group, and the LGG group were significantly improved on D17. The total diarrhea score in the medium- and high-dose bacterial composition groups showed a downward trend on D17.

[0197] (2) The body weight results are shown in Table 16.

[0198] Table 16. Effects of bacterial composition on body weight changes in radiation enteritis model mice

[0199] Note: All data are expressed as mean ± standard deviation (SEM); for all groups compared with the model control group, "*" indicates P < 0.05, "**" indicates P < 0.01, "***" indicates P < 0.001, and "****" indicates P < 0.0001.

[0200] Compared with the model control group, the high-dose, low-dose, and LGG bacterial composition groups showed significant improvement in weight loss on Day 14. The low-dose bacterial composition significantly improved weight loss in model mice on Day 17. High- and medium-dose bacterial compositions, loperamide, and LGG all showed a trend toward restoring weight in model mice on Day 17.

[0201] (3) The colorectal pathological results are shown in Table 17.

[0202] Table 17. Effects of bacterial composition on colorectal pathological scores in radiation enteritis model mice

[0203] Note: All data are expressed as mean ± standard deviation (SEM); for all groups compared with the model control group, "*" indicates P < 0.05, "**" indicates P < 0.01, "***" indicates P < 0.001, and "****" indicates P < 0.0001.

[0204] Compared with the model control group, the pathological scores of the high-dose, low-dose, and LGG groups of the bacterial composition were significantly improved. The pathological scores of the colorectal area in the high-dose, medium-dose, and loperamide groups showed an improvement trend.

[0205] In summary, the bacterial composition has a significant therapeutic effect on mice with radiation enteritis induced by whole-abdomen irradiation with X-rays, improving the degree of diarrhea and weight loss, and alleviating the degree of intestinal lesions.

[0206] Example 12: Therapeutic effect of bacterial composition on 5-FU-induced tumor-bearing CID mice

[0207] Experimental design: 50 male BALB / c mice (purchased from Chengdu Yaokang Biotechnology Co., Ltd.) were housed in an SPF animal room. CT26 cells (purchased from Beijing Beina Biotechnology Co., Ltd.) were subcutaneously inoculated on the right flank of each mouse at a dose of 5×10 6 cells / mouse, the inoculation volume is 0.1mL. When the tumor volume grows to 100-150mm 3 At about 14 days, 42 mice were screened for subsequent experiments. The 42 mice were randomly divided into 7 groups according to tumor volume, with 6 mice in each group. The 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: loperamide 20 mg / kg body weight), and Zhengchangsheng group (dosage regimen: Bacillus licheniformis 2×10 8 CFU / mouse / day), bacterial composition high-dose group (dosage regimen: bacterial composition 1×10 9 CFU / mouse / day), medium-dose bacterial composition group (dosage regimen: bacterial composition 1×10 8 CFU / mouse / day) and the bacterial composition low-dose group (dosage regimen: bacterial composition 1×10 7 CFU / 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), with oral administration once a day (QD) for 9 consecutive days. On the 3rd, 4th, 5th and 6th days of administration, the normal control group was intraperitoneally injected with 0.2 ml of normal saline per mouse, and the mice in the other groups were intraperitoneally injected with 5-FU for modeling (50 mg / kg body weight, 10 ml / kg body weight). Compared with the normal control group, the tumor volume of mice in each group was reduced after 5-FU injection. The model control group developed severe diarrhea and weight loss, and caused significant abnormalities in routine blood indicators, indicating that the model was successfully constructed.

[0208] The specific dosing regimen is detailed in Table 18. After the experiment, whole blood samples were collected from the animals in each group by removing their eyeballs, and routine blood tests were performed using a five-differentiation blood cell analyzer for animals (Shenzhen Dima Biotechnology Co., Ltd.).

[0209] Table 18. Dosage regimen for 5-FU-induced tumor-bearing CID model

[0210] Note: ig means oral administration, QD means once a day.

[0211] Test results: The diarrhea results are shown in Table 19.

[0212] Table 19. Diarrhea scores of mice in each group

[0213] Note: All data are expressed as mean ± standard deviation (SEM); when the diarrhea scores of each group were compared with those of the model control group, "*" indicates P < 0.05, and "**" indicates P < 0.01.

[0214] Compared with the normal control group, the model control group began to experience diarrhea on Day 6, and the severity of diarrhea worsened over time. Compared with the model control group, the diarrhea score and total diarrhea score of the loperamide group were significantly reduced on Day 8; the low-dose bacterial composition significantly reduced the diarrhea scores on Day 8 and Day 9, as well as the total diarrhea score.

[0215] The results of routine blood tests are shown in Table 20.

[0216] Table 20. Blood routine results of 5-FU-induced tumor-bearing mouse model

[0217] Note: All data are expressed as mean ± standard deviation (SEM); for the comparison of blood routine data of each group with that of the model control group, “*” indicates P < 0.05, and “**” indicates P < 0.01.

[0218] Compared with the normal control group, the model control group had significantly decreased hematocrit, white blood cell count, lymphocyte count, and platelet count. Compared with the model control group, the red blood cell count and hematocrit were significantly increased in the medium-dose, low-dose, and whole intestine groups of the bacterial composition, and the lymphocyte count and platelet count were increased in the low-dose group.

[0219] In summary, all three doses of bacterial compositions showed a trend of improving diarrhea and routine blood tests; among them, the low-dose bacterial composition and loperamide could significantly improve diarrhea; the low-dose bacterial composition had the ability to significantly improve routine blood tests (lymphocyte count, red blood cell count, and platelet count).

[0220] It is understood from common technical knowledge that the present invention may be implemented by other embodiments that do not depart from its spirit or essential features. Therefore, the embodiments disclosed above are, in all respects, merely illustrative and not exclusive. All modifications within the scope of the present invention or equivalent to the scope of the present invention are intended to be encompassed by the present invention.

Claims

1. A probiotic combination, comprising a fifth probiotic and any two, three or four of the first probiotic, the second probiotic, the third probiotic and the fourth probiotic; The first probiotic is selected from Bifidobacterium bifidum, a progeny strain of the Bifidobacterium bifidum, a cloned strain of the Bifidobacterium bifidum, or a pure culture of the Bifidobacterium bifidum; The second probiotic is selected from Enterococcus avium, a progeny strain of Enterococcus avium, a cloned strain of Enterococcus avium, or a pure culture of Enterococcus avium; The third probiotic is selected from Lactobacillus salivarius, a progeny strain of the Lactobacillus salivarius, a cloned strain of the Lactobacillus salivarius, or a pure culture of the Lactobacillus salivarius; The fourth probiotic is selected from fermented Lactobacillus mucilaginosus, a progeny strain of the fermented Lactobacillus mucilaginosus, a clonal strain of the fermented Lactobacillus mucilaginosus, or a pure culture of the fermented Lactobacillus mucilaginosus; and The fifth probiotic is selected from Parabacteroides deaconii, a progeny strain of the Parabacteroides deaconii, a clonal strain of the Parabacteroides deaconii, or a pure culture of the Parabacteroides deaconii.

2. The probiotic composition according to claim 1, characterized in that 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; and The microbial preservation number of the Parabacteroides dienii is CCTCC NO: M20222033.

3. A microecological composition, wherein the probiotic combination according to claim 1 or 2 is used as an active material.

4. The microecological composition according to claim 3, characterized in that: The composition further contains auxiliary materials, which are 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.

5. The microecological composition according to claim 3, characterized in that: In the microecological composition, the content ratio of any two bacteria is 100 CFU: 1-10000 CFU, calculated based on the number of live bacterial cells.

6. Use of the probiotic combination according to claim 1 or 2 or the microecological composition according to any one of claims 3 to 5 in preparing a product for use alone or in combination with other microbial preparations and / or drugs to improve the health status of a subject; The improving the health condition of the subject is selected from: Inhibiting the proliferation of 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 in the body cavity of the subject; Treatment, prevention and / or mitigation of any one, any two, any three, any four, any five, any six, any seven or more of Pseudomonas aeruginosa, Shigella dysenteriae, Staphylococcus aureus, Escherichia coli, Salmonella paratyphi B, Yersinia enterocolitica, Vibrio parahaemolyticus and Clostridium difficile Eight kinds of tissue damage, disease or sub-health conditions caused by it; Improve the antioxidant capacity in the intestines of the subjects; Treat, prevent and / or alleviate diarrhea caused by antineoplastic drugs; Treat, prevent and / or reduce intestinal inflammation caused by anti-cancer drugs; Treat, prevent and / or slow down weight loss caused by antineoplastic drugs; Treat, prevent and / or slow down the shortening of small intestine caused by antineoplastic drugs; Treat, prevent and / or slow down the increase in small intestinal thickness caused by antineoplastic drugs; Treat, prevent and / or slow down intestinal damage caused by antineoplastic drugs; Treat, prevent and / or slow down the decrease in spleen weight to body weight ratio caused by anti-tumor drugs; Treating, preventing and / or alleviating tissue damage, disease or sub-health condition caused by increased expression of any one, any two, any three or four of TNF-α, IL-1β, IL-22 and Bax; Treating, preventing and / or alleviating tissue damage, disease or sub-health condition caused by decreased expression of any one, any two or three of ZO-1, Occludin and AQP8; Treat, prevent and / or relieve radiation-induced diarrhea; Treat, prevent and / or slow down radiation therapy-induced weight loss; Treat, prevent and / or slow down bowel damage caused by radiation therapy; Treat, prevent and / or reduce radiation-induced intestinal inflammation; and Treat, prevent and / or slow down the decrease in hematocrit, white blood cell count, lymphocyte count and / or platelet count caused by anti-cancer drugs.

7. The use according to claim 6, characterized in that The product is food, health product or medicine.

8. The use according to claim 6, characterized in that The subject is selected from humans and mice.

9. The use according to claim 6, characterized in that The anti-tumor drug is selected from chemotherapy drugs, targeted drugs and immune checkpoint inhibitors.

10. The use according to claim 9, characterized in that 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.

11. A method for preventing, treating or alleviating intestinal diseases, the method comprising administering a therapeutically effective amount of the microecological composition according to any one of claims 3 to 5 to a subject; The intestinal disease is selected from: Enteric 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 antineoplastic drugs; Intestinal inflammation caused by antineoplastic drugs; Intestinal damage caused by antineoplastic drugs; Radiation-induced diarrhea; Inflammation of the bowel caused by radiation therapy; and Intestinal damage caused by radiation therapy.

12. The method according to claim 11, characterized in that The subject is selected from humans and mice.

13. The method according to claim 11, characterized in that The administration to the subject is selected from oral administration, intraperitoneal injection, and oral gavage.

14. The method according to claim 11, characterized in that Calculated based on the total bacterial content in the probiotic composition, the therapeutically effective amount is 10 6-12 CFU per day.

15. The method according to claim 11, characterized in that 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.

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