Probiotic composition for preventing and treating anti-tumor treatment associated diarrhea and application thereof

Abstract

The invention belongs to the technical field of microorganisms. The invention relates to a probiotic composition and application thereof in preparation of products for preventing and / or treating health conditions such as anti-tumor treatment related diarrhea. The probiotic composition comprises the following active ingredients: bifidobacterium bifidum, enterococcus faecalis, lactobacillus salivarius, lactobacillus fermentum mucus and parabacteroides didysonii. The probiotic composition has good safety, self-agglutination capability, oxidation resistance, short-chain fatty acid production capability, in-vitro cell adhesion capability, capability of inhibiting a plurality of pathogenic bacteria, capability of treating anti-tumor treatment related diarrhea and the like.

Description

Technical Field

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

[0002] Cancer treatment–related diarrhea / tumor-associated diarrhea is a common complication of various adjuvant anti-tumor therapies and one of the symptoms that has the greatest impact on health and quality of life. In severe cases, it may also lead to delayed treatment and reduced compliance, all of which may affect the long-term outcomes of cancer treatment and are potentially fatal. According to data released by the International Agency for Research on Cancer under the World Health Organization, there will be approximately 19.3 million new cancer cases worldwide in 2020, and it is predicted that the number of new cancer cases worldwide will reach 28.4 million in 2040. As many as 82% of cancer treatment patients will suffer from side effects such as diarrhea.

[0003] Chemotherapy (especially chemotherapy using traditional chemotherapy drugs such as fluoropyrimidines and irinotecan (CPT-11), which are first-line drugs for patients with metastatic colorectal cancer), radiotherapy, targeted therapy, and immunotherapy are the main factors causing anti-tumor treatment-related diarrhea other than surgery. The overall incidence of diarrhea caused by fluoropyrimidines and irinotecan treatment is 50% to 80%, and the incidence of severe (grade 3 to 5) diarrhea is more than 30%, of which 1 to 5% of subjects using 5-fluorouracil (5-FU) died from diarrhea. About 70% of patients with abdominal or pelvic tumors require radiotherapy, of which 60% to 70% of patients will experience acute symptoms such as abdominal pain, diarrhea, and blood in the stool, and 30% to 40% of patients will experience chronic diarrhea. The incidence of diarrhea caused by targeted therapy (such as targeted therapy using various tyrosine kinase inhibitors (TKI) such as afatinib, ceritinib, erlotinib, and lapatinib) can be as high as 90% or more. Immune checkpoint inhibitors often lead to immune enteritis. When using ipilimumab, nivolumab, and pembrolizumab to treat cancer, 1% to 45% of cancer patients will experience diarrhea.

[0004] The pathogenesis of anti-tumor therapy-related diarrhea, excluding surgery, is common. Anti-tumor therapy-related diarrhea is caused by multiple factors, and its main pathogenic factors are as follows: 1) Chemotherapy drugs and ionizing radiation during radiotherapy can cause oxidative stress and DNA damage; 2) Intestinal mucosal damage leads to epithelial cell loss, reduced tight junction integrity, crypt epithelial cell death, and increased intestinal permeability; 3) Multiple signaling pathways such as nuclear factor NF-κB are highly activated, stimulating cells to produce inflammatory factors, leading to mucosal inflammation; 4) Damage to the small intestinal mucosa leads to poor bile acid absorption, and excessive bile acid (especially dihydroxy components) induces water and electrolyte secretion and increases intestinal peristalsis, leading to diarrhea; 5) Infection with common hospital-acquired pathogens (Salmonella, Shigella, Escherichia coli, Clostridium difficile, etc.); 6) Bacterial translocation and intestinal flora imbalance, manifested as a decrease in probiotics and an increase in pathogenic bacteria; 7) Immune abnormalities, such as the general activation of T cells.

[0005] At present, 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, targeted therapy-related diarrhea, and immune checkpoint inhibitor-related diarrhea. The guidelines recommend the use of octreotide for patients who are ineffective with loperamide. Loperamide should not be used for more than 48 hours. High-dose loperamide may also bring paralytic ileus and severe cardiac toxicity risks. Octreotide (first-line medication for patients with diarrhea scores above grade 3) can cause side effects such as gallstones, hyperglycemia, and impaired glucose tolerance. The guidelines also recommend budesonide as a second-line treatment when loperamide treatment is ineffective, but glucocorticoids can exert systemic effects, causing an increased risk of infection and further deterioration of viral, bacterial, and other infections. Patients with grade 3-4 diarrhea and neutropenia can take oral antibiotics, but antibiotics can cause worsening diarrhea and 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 lasts for a long time 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 need for a new generation of drugs that are safer and more effective for anti-tumor therapy-related diarrhea.

[0006] Intestinal probiotics are a type of active microorganisms that colonize in the human intestine and change the composition of the host's intestinal flora, which are beneficial to the host. Intestinal probiotics have the advantages of high safety, low toxicity and side effects, and a wide range of indications and populations. Probiotic combinations can play a therapeutic role in the complex pathogenesis of anti-tumor therapy-related diarrhea, mainly reflected in: 1) Probiotics play an antioxidant function by chelating metal ions, upregulating their own and host antioxidant enzymes and metabolites, and reducing the activity of ROS-producing enzymes; 2) Probiotics can produce beneficial secondary metabolites, such as short-chain fatty acids, indole derivatives, secondary bile acids, etc. Beneficial secondary metabolites promote goblet cells to secrete mucin, promote epidermal cell repair, inhibit cell apoptosis, enhance tight junction proteins, etc., thereby enhancing intestinal barrier function; 3) Probiotics regulate immune responses by downregulating the NF-κβ pathway, inhibiting proinflammatory factors, and secreting anti-inflammatory factors, thereby playing an anti-inflammatory and enhancing the body's immunity; 4) Probiotics regulate the balance of intestinal flora, reduce pathogenic bacteria, increase probiotics, and maintain a normal intestinal homeostatic environment; 5) Probiotics regulate bile acid metabolism by increasing the bile salt hydrolase activity of the flora, inhibiting the FXR-FGF15 signaling pathway, and relieving bile acid diarrhea; 6) Probiotics inhibit the growth of pathogenic bacteria by increasing the secretion of antimicrobial proteins (defensins), producing antimicrobial substances such as bacteriocins, and competing with pathogens for adhesion sites.

[0007] Chinese patent CN112694992B discloses a strain of Bifidobacterium bifidum that can relieve diarrhea caused by enterotoxigenic Escherichia coli (ETEC). Chinese patent CN113234619B discloses a strain of Bifidobacterium bifidum that can relieve acute intestinal damage. A small number of documents report that Parabacteroides distasonis has the effect of relieving intestinal inflammation, such as 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 Lactobacillus salivarius ZLp4b that can significantly slow down and cure diarrhea in young animals. In addition to the above patents and documents, there are also some similar documents on the use of probiotics for preventing and treating 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. Summary of the invention

[0008] A first aspect of the present invention provides a probiotic combination, wherein the probiotic combination contains any three, four or five of a first probiotic, a second probiotic, a third probiotic, a fourth probiotic and a fifth probiotic (preferably, the probiotic combination contains the fifth probiotic and any two, three or four of the first probiotic, the second probiotic, the third probiotic and the fourth probiotic; more preferably, the probiotic combination contains the fourth probiotic, the fifth probiotic and any two or three of the first probiotic, the second probiotic and the third probiotic); the first probiotic is selected from Bifidobacterium bifidum, a progeny strain of the Bifidobacterium bifidum, a cloned strain of the Bifidobacterium bifidum or the Bifidobacterium bifidum The second probiotic is selected from Enterococcus avium, a progeny strain of Enterococcus avium, 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 Lactobacillus salivarius, a cloned strain of Lactobacillus salivarius or a pure culture of Lactobacillus salivarius; the fourth probiotic is selected from Lactobacillus fermentative mucus, a progeny strain of Lactobacillus fermentative mucus, a cloned strain of Lactobacillus fermentative mucus or a pure culture of Lactobacillus fermentative mucus; and the fifth probiotic is selected from Parabacteroides deaconii, a progeny strain of Parabacteroides deaconii, a cloned strain of Parabacteroides deaconii or a pure culture of Parabacteroides deaconii.

[0009] In some embodiments, the microbial preservation number of the Bifidobacterium bifidum is CCTCC NO: M2023349; the microbial preservation number of the Enterococcus avium is CCTCC NO: M2023350; the microbial preservation number of the Lactobacillus salivarius is CCTCC NO: M2023348; the microbial preservation number of the Lactobacillus fermentans is CCTCC NO: M2023352; and the microbial preservation number of the Parabacteroides difficile is CCTCC NO: M20222033.

[0010] 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 Bifidobacterium bifidum with a microbial preservation number of CCTCC NO: M2023349. NO:M2023350, a progeny strain of the avian enterococcus with a microbial preservation number of CCTCC NO:M2023350, a cloned strain of the avian enterococcus with a microbial preservation number of CCTCC NO:M2023350, or a 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 cloned strain of Lactobacillus salivarius with a microbial preservation number of CCTCC NO:M2023348, or a pure culture of Lactobacillus salivarius with a microbial preservation number of CCTCC NO:M2023348; the fourth probiotic is selected from Lactobacillus salivarius with a microbial preservation number of CCTCC NO:M2023348. NO:M2023352, a progeny strain of the fermented mucus Lactobacillus with a microbial preservation number of CCTCCNO:M2023352, a clonal strain of the fermented mucus Lactobacillus with a microbial preservation number of CCTCC NO:M2023352, or a pure culture of the fermented mucus Lactobacillus with a microbial preservation number of CCTCC NO:M2023352; and the fifth probiotic is selected from the microbial preservation number of CCTCC NO:M20222033 of Parabacteroides difficile, a progeny strain of the Parabacteroides difficile with a microbial preservation number of CCTCC NO:M20222033, a clonal strain of the Parabacteroides difficile with a microbial preservation number of CCTCC NO:M20222033, or a pure culture of the Parabacteroides difficile with a microbial preservation number of CCTCC NO:M20222033.

[0011] 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.

[0012] In some embodiments, the composition further contains an excipient, and the excipient is selected from a freeze-drying protectant, a bacterial culture medium, a food additive, an acceptable carrier or excipient in a health product, and a pharmaceutically acceptable carrier or excipient.

[0013] 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).

[0014] The third aspect of the present invention provides the use of the probiotic combination described in the first aspect of the present invention or the microecological composition described in the second aspect of the present invention in the preparation of a product for use alone or in combination with other microbial preparations and / or drugs to improve the health of a subject; the improving the health of the subject is selected from: inhibiting any one, any two, any three, 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). The invention relates to treating, preventing and / or slowing down the proliferation of 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; improving the antioxidant capacity in the intestine of the subject; treating, preventing and / or slowing down diarrhea caused by anti-tumor drugs; treating, preventing and / or slowing down intestinal tract caused by anti-tumor drugs. inflammation; treating, preventing and / or slowing down weight loss caused by anti-tumor drugs; treating, preventing and / or slowing down shortening of small intestine length caused by anti-tumor drugs; treating, preventing and / or slowing down increase in small intestine thickness caused by anti-tumor drugs; treating, preventing and / or slowing down intestinal damage caused by anti-tumor drugs; treating, preventing and / or slowing down decrease in spleen weight to body weight ratio caused by anti-tumor drugs; treating, preventing and / or slowing down 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.

[0015] In some embodiments, the product is a food, a health product or a medicine.

[0016] In some embodiments, the subject is selected from a human and a mouse.

[0017] 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.

[0018] In some embodiments, the anti-tumor therapy-related toxic side effect is diarrhea.

[0019] 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.

[0020] In some embodiments, the anti-tumor drug is selected from chemotherapeutic drugs, targeted drugs and immune checkpoint inhibitors.

[0021] 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.

[0022] The fourth aspect of the present invention provides a method for preventing, treating or alleviating intestinal diseases, the method comprising administering a therapeutically effective amount of the microecological composition described in the second aspect of the present invention to a subject; the intestinal disease is selected from: intestinal diseases caused by any one, any two, any three, any four, any five, any six, any seven or eight of Pseudomonas aeruginosa, Shigella dysenteriae, Staphylococcus aureus, Escherichia coli, Salmonella paratyphi B, Yersinia enterocolitica, Vibrio parahaemolyticus, and Clostridium difficile; oxidative damage in the intestine; diarrhea caused by antitumor drugs; intestinal inflammation caused by antitumor drugs; intestinal damage caused by antitumor drugs; diarrhea caused by radiotherapy; intestinal inflammation caused by radiotherapy; and intestinal damage caused by radiotherapy.

[0023] In some embodiments, the subject is selected from a human and a mouse.

[0024] In some embodiments, the administration to the subject is selected from oral administration, intraperitoneal injection, and oral gavage.

[0025] In some embodiments, the therapeutically effective amount is 10 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 10 CFU, 1×10 11 CFU, 2×10 11 CFU, 3×10 11 CFU, 4×10 11 CFU, 5×10 11 CFU, 6×10 11 CFU, 7×10 11 CFU, 8×10 11 CFU, 9×10 11 CFU and 10 12CFU (any value or range between any two values) per day.

[0026] 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.

[0027] The beneficial effects of the present invention are:

[0028] (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 therapy-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

[0029] Figure 1 The following are front photos of the colony morphology of the five strains.

[0030] Figure 2 This is a characteristic photo of co-culture of five single bacterial strains on BF839 agar medium.

[0031] Figure 3 The probiotic composition and its single bacteria self-aggregation (co-aggregation) ability are shown.

[0032] Figure 4 The results of the antibacterial activity test of the probiotic composition and its single bacteria against pathogenic bacteria are shown.

[0033] Figure 5 The results of the adhesion test of the probiotic composition and its single bacteria to Caco2 cells are shown. DETAILED DESCRIPTION

[0034] In order to make the objectives, technical solutions and advantages of the present invention more clear, the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0035] Unless otherwise specified, the terms used herein have the common meanings in the biomedical field.

[0036] Unless otherwise specified, the specific temperature parameters in the present invention should be understood as constant temperature treatment, and variations are allowed within a certain temperature range (such as fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, ±1°C).

[0037] The invention provides a probiotic combination, which contains any three, four or five of Bifidobacterium bifidum, Enterococcus avium, Ligilactobacillus salivarius, Limosilactobacillus fermentum and Parabacteroides distasonis.

[0038] In some embodiments, the microbial deposit number of the Bifidobacterium bifidum is CCTCCNO: M2023349;

[0039] The microbial preservation number of the avian enterococcus is CCTCC NO: M2023350;

[0040] The microbial preservation number of the Lactobacillus salivarius is CCTCC NO: M2023348;

[0041] The microbial preservation number of the fermented mucus lactobacillus is CCTCC NO: M2023352; and

[0042] The microbial preservation number of the Parabacteroides dienii is CCTCC NO: M20222033.

[0043] The meaning of the strain with a specific deposit number of the present invention includes but is not limited to: (1) a strain with a specific deposit number stored in the said deposit center; (2) a strain having the same genome as the strain described in (1); (3) a subculture strain without gene mutations based on the aforementioned (1) or (2); (4) a subculture strain based on the aforementioned (1), (2) or (3) that accumulates minor mutations during subculture but has no substantial changes in toxicity, immunogenicity and biological activity; (5) a live bacterium based on any of the aforementioned strains (1)-(4), a killed product of the live bacterium, a lysate of the live bacterium or a fermentation product of the live bacterium, etc.

[0044] Strains with the same genome include, but are not limited to, strains with the same genetic background that have been independently isolated and disclosed by others after the priority date corresponding to the present invention, that is, strains isolated from nature or animals (including humans) with the same genome (same genetic background). Conventional cultures are generally considered to be subculture strains without gene mutations. As known to those skilled in the art, minor mutations are usually inevitably introduced into strains after subculture. 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 the connecting amino acid residues between two domains or the mutated amino acid residues are located inside the higher structure of the protein because they do not contact immune cells, these mutations do not affect toxicity, immunogenicity and biological activity). It can be reasonably expected that when these minor changes do not significantly affect the toxicity, immunogenicity and biological activity of the offspring strains, the mutated strains can still achieve the purpose of the present invention, and the mutated strains are derived from the strains contributed by the present invention, so the corresponding strains are still within the scope of the substantial technical contribution of the present invention. These minor mutations are still non-substantial mutations and should be regarded as mutant strains with no changes in toxicity, immunogenicity and biological activity. 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 as a mutation within the limits of the detection technology such as detection sensitivity and detection limit and acceptable or unavoidable errors are the same as the strain contributed by the present invention. 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 the differences in cell strains, animal breeds, age, gender, health conditions, culture conditions, etc. and the expected or unavoidable systematic errors. The active ingredient refers to a substance that plays a role in the components that produce 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 a composition containing any 3, 4 or 5 species according to the efficacy characteristics of each strain, and it is reasonable to expect that the composition of these strain combinations can simultaneously exert the efficacy of each strain in the group.

[0045] Anti-tumor therapy-related diarrhea or tumor-related diarrhea refers to diarrhea caused by various anti-tumor therapies that damage the intestinal mucosa, leading to imbalance in intestinal absorption and secretion. Common anti-tumor therapy-related diarrhea includes chemotherapy-related diarrhea, diarrhea caused by radiotherapy, diarrhea caused by targeted therapy, diarrhea caused by immune checkpoint inhibitors, etc. Radiotherapy-induced diarrhea is clinically manifested as symptoms of radiation enteritis or radiation intestinal injury.

[0046] Chemotherapy refers to the use of non-selective chemical drugs to kill tumor cells to achieve the purpose of treatment. Chemotherapy is the main means of treating tumors in addition to surgery and radiotherapy. Due to the lack of selectivity, chemotherapy will damage normal cells while killing tumor cells. Common chemotherapy drugs include but are not limited to antibiotic chemotherapy drugs (such as doxorubicin, epirubicin, actinomycin D, doxorubicin, daunorubicin and its derivatives, etc.), taxane chemotherapy drugs (such as paclitaxel, docetaxel, albumin paclitaxel and its derivatives, etc.), platinum chemotherapy drugs (such as cisplatin, carboplatin, nedaplatin, oxaliplatin, lobaplatin and its derivatives, etc.), alkylating agent chemotherapy drugs (such as cyclophosphamide, nitrogen mustard, carmustine and its derivatives, etc.), camptothecin chemotherapy drugs (such as camptothecin, hydroxycamptothecin, topotecan, irinotecan and its derivatives, etc.) and antimetabolite chemotherapy drugs (such as capecitabine, gemcitabine, methotrexate, 5-fluorouracil, pemetrexed, cytarabine and its derivatives, etc.).

[0047] Targeted therapy is to design corresponding therapeutic drugs for the identified carcinogenic sites at the cellular and molecular level. In targeted therapy, the drug will specifically select the carcinogenic sites to combine and act to specifically kill tumor cells. The emergence of new targeted drugs has changed the tumor treatment model and opened up the era of targeted therapy. Common targeted drugs include apatinib, axitinib, cabozantinib, sorafenib, sunitinib, etc.

[0048] Immune checkpoint inhibitors are monoclonal antibody drugs developed for corresponding immune checkpoints. The main function of immune checkpoint inhibitors is to block the interaction between tumor cells expressing immune checkpoints and immune cells, thereby blocking the inhibitory effect of tumor cells on immune cells. Common immune checkpoint inhibitors include but are not limited to PD-1 / PD-L1 inhibitor antibodies (such as nivolumab, pembrolizumab, etc.) and CTLA-4 inhibitors (such as ipilimumab, etc.).

[0049] Radiotherapy refers to the use of radiation (such as alpha rays, beta rays, gamma rays produced by radioisotopes, and x-rays, electron beams, proton beams and other particle beams produced by various x-ray therapy devices or accelerators) to treat malignant tumors. Commonly used radiotherapy methods include but are not limited to conventional radiotherapy, stereotactic radiotherapy, three-dimensional conformal radiotherapy, intensity modulated radiotherapy, image-guided radiotherapy, volumetric arc intensity modulated radiotherapy, proton radiotherapy, etc.

[0050] The invention provides a microecological composition, which contains any three, four or five of Bifidobacterium bifidum, Enterococcus avium, Ligilactobacillus salivarius, Limosilactobacillus fermentum and Parabacteroides distasonis and auxiliary materials.

[0051] 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.

[0052] The auxiliary materials vary depending on the type of product being prepared, for example, food, health products or medicines can be prepared. Accordingly, the auxiliary materials can be selected from freeze-drying protective agents, bacterial culture media, food additives, acceptable carriers or auxiliary materials in health products and pharmaceutically acceptable carriers or auxiliary materials.

[0053] The invention provides use of a probiotic combination or a microecological composition in preparing food, health products or medicines.

[0054] When prepared into medicines, the probiotic combination or microecological composition is used in the preparation of medicines for use alone or in combination with other microbial preparations and / or drugs to improve the health status of subjects.

[0055] 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.

[0056] The therapeutically effective amount or the 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 clinician treating 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.

[0057] A pharmaceutically acceptable carrier refers to a pharmaceutical carrier that does not cause significant stimulation to a 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. A pharmaceutically acceptable carrier may include a solvent, a dispersion medium, a coating, a surfactant, an antioxidant, an isotonic agent, an absorption delay agent, a salt, a drug stabilizer, a binding agent, an excipient, a disintegrant, a lubricant, a sweetener, a flavoring agent, a dye, a protective agent, etc. and a combination thereof. Unless a conventional carrier is incompatible with an active ingredient, it is considered that the carrier is used in a therapeutic composition or a pharmaceutical composition. The carrier may be selected to minimize the adverse side effects of the subject and / or minimize the inactivation of the active ingredient.

[0058] Excipients refer to substances added to pharmaceutical compositions to give the drug a certain shape or a certain concentration, for example, sterile water, physiological saline, polyalkylene glycols (such as polyethylene glycol), vegetable oils, hydrogenated naphthalene, calcium bicarbonate, calcium phosphate, various sugars, various types of starch, cellulose derivatives, gelatin, etc.

[0059] The microecological composition of the present invention can further contain a second beneficial active ingredient, for example, another probiotic, prebiotic, medicine, etc. with antidiarrhea function. Prebiotics help regulate the intestinal environment by promoting the growth of probiotics in the intestine, thereby indirectly playing an antidiarrhea 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.

[0060] In some embodiments, the dosage form of the pharmaceutical composition is tablets, capsules, granules, solutions, suspensions, powders, etc.

[0061] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those skilled in the art based on the embodiments in the application documents of the present invention without creative work should belong to the scope of disclosure or protection of the present invention.

[0062] The culture medium preparation method used in the following examples is as follows:

[0063] Preparation of YCFA liquid medium: Add 10.0 g of peptone, 2.5 g of yeast extract, 0.45 mL of 10 (w / w)% MgSO4·7H2O aqueous solution, 0.45 mL of 10 mg / mL CaCl2 aqueous solution, 110 mL of TE14, 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 obtain a solution. Perform N2 replacement and deoxygenation on the solution and package it separately. Sterilize the packaged solution at 121°C for 30 minutes and set aside.

[0064] Preparation of TE141: Add 1.50 g of nitrilotriacetic acid to 200 mL of pure water to obtain a solution. Add an appropriate amount of NaOH to the solution until the solution becomes clear, then add 800 mL of water to the solution, and then adjust the pH value to 5.5 with 50% HCl to obtain a nitrilotriacetic acid aqueous solution. Add 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 0.03 g of Na2MoO4·2H2O. 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.

[0065] Preparation of VFA-mix: Mix 90 mL of acetic acid, 30 mL of propionic acid, 10 mL of n-valeric acid, 10 mL of isobutyric acid and 10 mL of butyric acid to obtain a solution for use. Adjust the pH of the solution to neutral with 5M NaOH before use.

[0066] Preparation of triple-mixed liquid culture 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 obtain a solution. Perform N2 replacement and deoxygenation on the solution and package it. Sterilize the packaged solution at 121°C for 30 minutes. Store the obtained triple-mixed liquid culture medium in a cool and dry place.

[0067] Preparation of three-mix solid culture medium: add 5 g of agar powder to the above three-mix liquid culture medium, and the other steps are the same as the preparation of three-mix liquid culture medium.

[0068] Preparation of BF839 liquid culture medium: 6.0 g potato powder (Beijing Solebow Technology Co., Ltd., FA0270), 10.0 g polyvalent peptone (Beijing Solebow Technology Co., Ltd., P8950-250), 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 (Chengdu Kelong Chemical Co., Ltd., 50-99-7) and 4.0 g of disodium hydrogen phosphate (Chengdu Kelong Chemical Co., Ltd., 7558-79-4) were dissolved in 1 L of distilled water to obtain a solution. The solution was deoxygenated by N2 replacement and aliquoted. The solution was sterilized by high temperature and wet heat at 121°C for 15 min. The obtained culture medium was stored in a cool and dry place.

[0069] Preparation of BF839 solid culture medium: Dissolve 50.4 g of BF839 solid agar (HB8805, Qingdao Hi-Tech Industrial Park Haibo Biotechnology Co., Ltd.) in 1 L of distilled water to obtain a mixture. Perform N2 replacement and deoxygenation on the mixture and package it separately. Sterilize the mixture at 121°C for 15 minutes. Store the obtained culture medium in a cool and dry place.

[0070] Preparation of oxygen-free PBS: Dissolve 0.27 g of potassium dihydrogen phosphate, 1.42 g of disodium hydrogen phosphate, 8 g of sodium chloride and 0.2 g of potassium chloride in 1 L of distilled water to obtain a mixture. Heat and boil the mixture, cool the mixture to room temperature and add 0.55 g of cysteine ​​hydrochloride thereto, stir the mixture to dissolve it and adjust the pH to 6.5. Heat the mixture to boiling and keep it at a slight boiling state for 30 minutes. After cooling, use a quantitative dispenser to dispense into 400 mL / bottle under N2 conditions. Sterilize the bottle at 121°C for 30 minutes. Store the obtained PBS in a cool and dry place for later use.

[0071] GAM solid medium (Qingdao Haibo Biotechnology Co., Ltd., HB8462), TSB (Tryptone Soy Broth, Qingdao Haibo Biotechnology Co., Ltd., HB4114), TSA (Tryptone Soy Agar, Qingdao Haibo Biotechnology Co., Ltd., HB4138) medium were prepared according to the steps specified in the instructions provided by the supplier. The materials were weighed and dissolved in water to obtain a mixture. The mixture was sterilized at 121°C for 30 minutes to obtain the culture medium. The culture medium was stored in a cool and dry place.

[0072] Preparation of culture medium for the powder of Parabacteroides distiliensis Pdist-1: Dissolve 6g of anhydrous glucose, 15g of soy peptone, 10g of yeast extract powder, 10g of yeast peptone, 2g of potassium dihydrogen phosphate, 2g of disodium hydrogen phosphate, 0.2g of magnesium sulfate, 0.01g of manganese sulfate, 0.2g of calcium chloride, 1mL of Tween 80 and 0.5g of monohydrated cysteine ​​hydrochloride in 1L of distilled water. Deoxygenate the mixture with N2 and dispense it. Sterilize the mixture at 121℃ for 15min.

[0073] Preparation of culture medium for fermentation of Lactobacillus mucilaginosus Lferm-1 powder: Dissolve 30g of anhydrous glucose, 15g of soy peptone, 10g of yeast extract powder, 5g of sodium acetate, 2g of potassium dihydrogen phosphate, 2g of disodium hydrogen phosphate, 0.1g of magnesium sulfate, 0.045g of manganese sulfate, 1mL of Tween 80 and 0.5g of cysteine ​​hydrochloride monohydrate in 1L of purified water. Deoxygenate the mixture with N2 replacement and aliquot. Sterilize the mixture at 121℃ for 15min.

[0074] Preparation of culture medium for Lactobacillus salivarius Lsali-1 powder: Dissolve 24g of anhydrous glucose, 20g of soy peptone, 10g of yeast extract powder, 10g of peptone, 5g of sodium acetate, 2g of potassium dihydrogen phosphate, 2g of disodium hydrogen phosphate, 0.1g of magnesium sulfate, 0.045g of manganese sulfate, 1ml of Tween 80 and 0.5g of monohydrated cysteine ​​hydrochloride in 1L of purified water. Deoxygenate the mixture with N2 replacement and aliquot. Sterilize the mixture at 121℃ for 15min.

[0075] Preparation of culture medium for Enterococcus avium Eaviu-1 powder: Dissolve 30g of anhydrous glucose, 15g of soy peptone, 10g of yeast powder, 5g of sodium acetate, 2g of dipotassium hydrogen phosphate, 0.1g of magnesium sulfate, 0.045g of manganese sulfate, 1mL of Tween 80 and 0.5g of monohydrated cysteine ​​hydrochloride in 1L of purified water. Deoxygenate the mixture with N2 replacement and aliquot. Sterilize the mixture at 121℃ for 15min.

[0076] Preparation of culture medium for preparation of Bifidobacterium bifidum Bbifi-1 powder: Dissolve 20g of anhydrous glucose, 40g of soy peptone, 5g of N-acetylglucosamine, 2g of potassium dihydrogen phosphate, 2g of disodium hydrogen phosphate, 0.1g of magnesium sulfate, 0.045g of manganese sulfate, 1mL of Tween 80 and 0.5g of monohydrated cysteine ​​hydrochloride in 1L of purified water. Deoxygenate the mixture by N2 replacement and aliquot. Sterilize the mixture by high temperature and wet heat at 121℃ for 15min.

[0077] The preparation method of freeze-dried protective agent is as follows:

[0078] Preparation of Parabacteroides distristii Pdist-1 lyophilized protective agent (also used as a lyophilized protective agent for animal administration control): Solution A: 6g sucrose, 6g trehalose, 2g xylitol, 2g sorbitol, 44g purified water, sterilized at 115℃ for 30min. Solution B: 5g sodium glutamate, 15g purified water; sterilized at 115℃ for 20min. Solution C: 4g sodium vitamin C, 16g purified water. Filter and sterilize for later use.

[0079] Preparation of freeze-dried protective agent for fermented Lactobacillus mucus Lferm-1, Lactobacillus salivarius Lsali-1, and Enterococcus avium Eaviu-1: Liquid A: 8g sucrose, 8g trehalose, 44g purified water, sterilized at 115℃ for 30min. Liquid B: 2g sodium glutamate, 2g arginine hydrochloride, 16g purified water, sterilized at 115℃ for 30min. Liquid C: 4g sodium vitamin C, 16g purified water. Filter and sterilize for later use.

[0080] Preparation of 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℃ for 30min. Solution B: 2g arginine hydrochloride, 2g sodium glutamate, 16g purified water, sterilized at 115℃ for 30min. Solution C: 4g sodium vitamin C, 16g purified water. Filter and sterilize for later use.

[0081] When used, they are mixed in a volume ratio of A liquid: B liquid: C liquid = 6:2:2. The lyophilized protective agent for animal testing is prepared by freeze-drying the prepared lyophilized protective agent, crushing it, and preparing it into a suspension with physiological saline for use.

[0082] Preparation of 0.1% Tween 80-PBS dilution: Add 3.58g disodium hydrogen phosphate dodecahydrate, 0.27g potassium dihydrogen phosphate, 8g sodium chloride and 1ml Tween 80 to 1L boiling water and dissolve with a glass rod and glue stick. Add 0.5g cysteine ​​hydrochloride monohydrate to the boiled solution. Open the Hungate apparatus and boil the solution again under N2 protection. After blowing N2 for 20 minutes, dispense the solution into anaerobic bottles that have been deoxygenated with N2. Cover the bottle with a stopper and label it, and sterilize the solution at 121℃ for 15 minutes.

[0083] Example 1: Isolation and identification of strains

[0084] Fresh stool samples were collected from several healthy human volunteers, and each stool sample was processed independently. An appropriate amount of anaerobic PBS was added to the stool sample to obtain a mixture and the mixture was shaken to obtain a suspension. Under N2 protection, the suspension was filtered through 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 anaerobic PBS was added to the precipitate to resuspend the bacteria to obtain a suspension. An equal volume of 50 (v / v)% anaerobic glycerol aqueous solution was added to the suspension and mixed thoroughly to obtain a bacterial mixed solution sample. The samples were divided into sample tubes, the sample 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 independently. 0.5 mL of the thawed sample was resuspended in 4.5 mL of anaerobic 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 and mixing. 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 part of the 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, another part of the 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.

[0085] The 16S rDNA gene sequence obtained by sequencing was compared with the NCBI Nucleotide database to further identify the species of the isolated strain. Five strains were selected from the many strains whose species were further determined for subsequent experiments of the present invention. Strain 1 had the highest sequence similarity (>99%) with a strain of Parabacteroides distasonis, also known as Parabacteroides distasonis, so strain 1 was named Parabacteroides distasonis Pdist-1 (referred to as Pdist-1). Strain 2 had the highest sequence similarity (100%) with a strain of Limosilactobacillus fermentum, formerly known as fermented Lactobacillus, so strain 2 was named fermented Lactobacillus Lferm-1 (referred to as Lferm-1). Strain 3 has the highest sequence similarity (100.00%) with a strain of Lactobacillus salivarius, which is also called Lactobacillus salivarius, and its Latin name is Ligilactobacillus salivarius. Therefore, strain 3 is named Lactobacillus salivarius Lsali-1 (abbreviated as Lsali-1). Strain 4 has the highest sequence similarity (100.00%) with a strain of Enterococcus avium, so strain 4 is named Enterococcus avium Eaviu-1 (abbreviated as Eaviu-1). Strain 5 has the highest sequence similarity (99.86%) with a strain of Bifidobacterium bifidum, so strain 5 is named Bifidobacterium bifidum Bbifi-1 (abbreviated as Bbifi-1).

[0086] Parabacteroides distist-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 three mixed solid medium to observe their colony morphology. The front photos of the colony morphology of the above five strains are shown in Figure 1 , among which A is a frontal photo of the colony morphology of Parabacteroides distist-1; B is a frontal photo of the colony morphology of Lactobacillus fermentans Lferm-1; C is a frontal photo of the colony morphology of Lactobacillus sali-1; D is a frontal photo of the colony morphology of Enterococcus avium Eaviu-1; E is a frontal photo of the colony morphology of Bifidobacterium bifidum Bbifi-1. It can be seen that the five strains are all white opaque round colonies with a convex middle and a smooth and moist surface.

[0087] Example 2: Whole genome analysis of strains

[0088] The five strains obtained in Example 1 were inoculated into three mixed liquid mediums, and the bacteria were cultured to the late logarithmic growth stage. The whole genome DNA of each strain was extracted and the whole genome was sequenced using the Illumina high-throughput sequencing platform NovaSeq 6000. After the genome sequence was assembled and annotated, the protein sequence was entered into the virulence gene library Virulence Factor Databases (VFDB) for virulence factor analysis. The results showed that none of the five strains had virulence factors in their genomes.

[0089] The average nucleotide similarity (ANI) method was used to analyze the novelty of the five strains. A whole genome search was performed in Genbank, and the most similar strains were compared using fastANI (v1.33). The two strains closest to the whole genome of Parabacteroides distichous Pdist-1 were GCA_003462945.1 (ANI = 98.26%) and GCA_003459965.1 (ANI = 98.20%). The two strains closest to the whole genome of Lactobacillus fermentans Lferm-1 were GCA_003465085.1 (ANI = 99.32%) and GCA_024385625.1 (ANI = 99.29%). The two strains closest to the whole genome of Lactobacillus salivarius Lsali-1 are GCA_009863605.1 (ANI = 99.94%) and GCA_009866185.1 (ANI = 99.87%). The two strains closest to the whole genome of Enterococcus avium Eaviu-1 are GCA_018917545.1 (ANI = 98.75%) and GCA_018373135.1 (ANI = 98.62%). The two strains closest to the whole 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.

[0090] The strains Pdist-1, Eaviu-1, Lferm-1, Bbifi-1, and Lsali-1 isolated and cultured by the present invention were submitted to the depository recognized by the patent procedure for deposit. The depository is the China Center for Type Culture Collection (CCTCC); the address is Wuhan University, Wuhan, China; the culture name, classification nomenclature, deposit date, identification survival date, and microbial deposit number are shown in Table 1.

[0091] Table 1. Statistical table of strain preservation information

[0092]

[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 the 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, anaerobically cultured for 48 hours until the bacterial solution traces were obvious. The interaction relationship between the five bacterial strains is shown in the figure. Figure 2 As shown. It can be seen that there is no breakpoint at the intersection of the strains, indicating that there is no growth inhibition between the strains.

[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 control strain Lactobacillus rhamnosus GG (LGG, CICC6141, purchased from China Industrial Microbiological Culture Collection Center) were inoculated into three mixed liquid culture media at an initial inoculation rate of 2%, and cultured anaerobically at 37°C until the late logarithmic growth period. The cultured bacterial solution was centrifuged and washed three times with anaerobic PBS. The bacterial solution was resuspended with an appropriate amount of PBS and the OD 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 single 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 15 mL of diluted single bacterial suspension and mixed bacterial suspension respectively, and pack them into 5 mL / tube, a total of 3 tubes, for parallel experiments. Measure the OD 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-agglutination rate / co-agglutination 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 as follows Figure 3 As shown. It can be seen that the bacterial composition and its single bacteria have agglutination ability, and the agglutination ability (self-agglutination rate / co-agglutination rate, agglutination rate) is: Eaviu-1>LGG≈Pdist-1>Lsali-1≈Bbifi-1≈bacterial composition>Lferm-1. Bacteria with better agglutination ability may have better colonization ability and are 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, each single bacteria Pdist-1, Eaviu-1, Lferm-1, Bbifi-1 and Lsali-1 were mixed and inoculated into 5 mL of three-mixed culture medium. The inoculation amount of each strain was 1%, and the culture was anaerobically carried out at 37°C for 24 hours.

[0102] Sample processing: Take 0.5mL of culture solution, centrifuge at 12000rpm for 20min, discard the supernatant, and resuspend the bacteria with 0.5ml of the extract in the pre-cooled strain total antioxidant capacity detection kit (the kit was purchased from Beijing Solebow Technology Co., Ltd., BC1315). Transfer the bacteria to a sterilized screw cap tube containing beads (Sigma-Aldrich, USA, G4649-1KG). Use a fast sample preparation instrument to shake the bacterial cell wall once (parameter setting: 4.5m / s, 30s), centrifuge at 12000rpm and 4℃ for 10min, and take the supernatant and place it on ice for testing.

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

[0104] The total antioxidant capacity detection kit of the strain was used to determine the antioxidant capacity of the sample according to the instructions of the kit and the standard curve mentioned above. The total antioxidant capacity unit is 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% inoculation rate 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% inoculation rate, and cultured anaerobically at 37°C for 24 hours.

[0109] Centrifuge the fermentation broth of single bacteria and bacterial combination respectively, and take 1mL of supernatant. Add 10μL of formic acid to the supernatant, let it stand at 4℃ for 30min to acidify, and centrifuge again. Take the supernatant, filter it with a 0.22μm filter, and set aside. Use gas chromatograph to determine the content of short chain fatty acids (SCFAs) in each filtered solution. The relevant parameters of gas chromatography are as follows. Instrument model: Agilent 7890A. Chromatographic column model: DB-WAX UI (30m×0.32mm×0.25μm) capillary column. Column temperature: initial temperature 80℃, last for 0.5min; then increase to 180℃ at 5℃ / min, last for 1min; then increase to 220℃ at 40℃ / min, last for 4min. Inlet temperature: 220℃. Detector temperature: 250℃. Carrier gas: hydrogen 30mL / min; air 300mL / min. Tail blow flow rate: nitrogen 28.314mL / min. Split ratio: 10:1. Injection volume: 1μL. Repeat the injection test of the same sample 4 times, calculate the average value, and calculate the acid production according to the following formula: actual acid production of the strain (ppm) = acid production value of the strain measured - basic value of BF839 culture. The total SCFAs production is the sum of the production 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 composition and its single bacteria all have the ability to produce acetic acid. In addition to acetic acid, Pdist-1 can also produce propionic acid and isovaleric acid. The total SCFAs production of Bbifi-1, Pdist-1 and the bacterial composition is significantly better than that of LGG, and the total SCFAs production of Lsali-1, Eaviu-1 and Lferm-1 is equivalent to that of LGG.

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

[0112]

[0113]

[0114] Example 7: Ability of single bacteria and bacterial combinations to inhibit pathogenic bacteria

[0115] In this embodiment, 8 common pathogenic bacteria that can cause diarrhea are selected for antibacterial ability detection. The source information of the pathogenic strains is as follows: Pseudomonas aeruginosa (CMCC (B) 10104), purchased from China Food and Drug Inspection Institute. Shigella dysenteriae (CMCC (B) 51252), purchased from China Food and Drug Inspection Institute. Staphylococcus aureus (CMCC (B) 26003), purchased from China Food and Drug Inspection Institute. Escherichia coli (CMCC (B) 44102), purchased from China Food and Drug Inspection Institute. Salmonella paratyphi (CMCC (B) 50094), purchased from China Food and Drug Inspection Institute. Yersinia enterocolitica CMCC (B) 52204, purchased from China Food and Drug Inspection Institute. Vibrio parahaemolyticus (ATCC 17802), purchased from the American Microbiological Strain Collection Center. Clostridium difficile (CICC 22951) was purchased from China Industrial Microbiological Culture Collection Center.

[0116] Single bacteria: Each single bacteria of the control strains LGG, Pdist-1, Eaviu-1, Lferm-1, Bbifi-1 and Lsali-1 was inoculated into three mixed liquid culture media at an inoculation rate of 5%, and the fermentation liquid was obtained by anaerobically culturing for 48 hours.

[0117] Bacterial composition: Pdist-1, Eaviu-1, Lferm-1, Bbifi-1 and Lsali-1 were mixed and inoculated into a three-mixed liquid culture medium at an inoculation rate of 1% and anaerobically cultured for 48 hours to obtain a fermentation broth.

[0118] 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 apply it 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 6 CFU / mL, take 200μL and spread it on anaerobic GAM solid medium (add 5v / v% horse serum, Beijing Solebow Technology Co., Ltd., S9050). Place 3 Oxford cups on each plate, add 200μL of the fermentation liquid to be tested (the aforementioned culture) to the Oxford cups, C. difficile is cultured under anaerobic conditions, and the other pathogens are cultured under aerobic conditions. After culturing at 37℃ for 24h, measure the diameter of the inhibition zone and calculate the average value. The results are as follows Figure 4As shown. 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 single bacteria in inhibiting certain pathogens. The bacterial composition is superior to the control strain LGG in inhibiting the activity of Salmonella paratyphi B and Staphylococcus aureus, and its inhibitory ability on other pathogens is equivalent to that of the control strain LGG.

[0119] Example 8: Testing of the adhesion ability of single bacteria and bacterial combinations to Caco2 cells

[0120] Single bacteria: The control strains LGG, Pdist-1, Eaviu-1, Lferm-1, Bbifi-1 and Lsali-1 were inoculated into three mixed liquid medium at an initial inoculum of 2% and cultured anaerobically at 37°C until the late logarithmic growth period. The cultured bacterial solution was centrifuged and washed twice with sterile PBS (Wuhan Boster Biotechnology Co., Ltd., PYG0021), and then the strains in the precipitate were diluted to 5×10 using DMEM (Thermo Fisher Scientific (China) Co., Ltd., C11995500BT) medium containing 10v / v% FBS (Thermo Fisher Scientific (China) Co., Ltd., SH30084.03). 8 CFU / mL, set aside.

[0121] 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.

[0122] Caco-2 cells (Shangcheng Beina Chuanglian Biotechnology Co., Ltd., 350769) were adherent cells. Caco-2 cells were digested with 37°C preheated trypsin cell digestion solution (Lanjieke Technology Co., Ltd., BL501A). The digested Caco-2 cells were collected by centrifugation. The precipitate was diluted with DMEM medium containing 10 v / v% FBS. 5×10 4 The Caco-2 cells were seeded into a 96-well plate at a density of CFU / well and cultured overnight in a 37°C carbon dioxide incubator for later use.

[0123] Divide each well of the 96-well plate into two groups, and take 100 μL of diluted single bacteria and bacterial composition suspensions in each group and add them to the 96-well cell culture plate containing Caco-2 cells. After the addition, place the 96-well cell culture plate in a horizontal centrifuge and centrifuge at 1000g for 1 min. The wells corresponding to each bacterial solution are divided into two groups, one group is incubated for 30 min, and the other group is incubated for 2 h. After incubation, wash twice with sterile PBS to wash away non-adherent bacteria. After washing, add 50 μL of 0.25% trypsin cell digestion solution (Lanjieke Technology Co., Ltd., BL501A) to each well and place it in a 37°C incubator to digest the cells. After the Caco-2 cells are digested and present spherical shapes, add 150 μL of DMEM culture medium to each well and blow repeatedly for about 1 min. After the microscopic examination confirms that the cells and strains are digested and separated, take 20 μL of the above mixture and dilute it 10 times in a 96-well plate with 0.1% Tween 80-PBS. Pour the appropriate dilution gradient into the dissolved three-mix solid culture medium, culture at 37°C for 48 hours, and then count.

[0124] The results are as follows Figure 5 As shown. A shows that after each strain adhered to Caco2 cells for 30 minutes, the adhesion ability of avian enterococcus Eaviu-1, bacterial composition and LGG was equivalent. Figure B shows the adhesion effect of each strain when the adhesion time was 2 hours. The adhesion ability of Bifidobacterium bifidum Bbifi-1 and fermented mucus Lactobacillus Lferm-1 was equivalent to LGG, while the adhesion ability of Lactobacillus salivarius Lsali-1, avian enterococcus Eaviu-1 and bacterial composition was better than LGG. The bacterial composition has better adhesion ability and is therefore easier to colonize.

[0125] Example 9: Test on the therapeutic effect of bacterial composition on mice with diarrhea caused by 5-fluorouracil (5-FU)

[0126] Preparation of bacterial composition:

[0127] Pdist-1, Lferm-1, Lsali-1, Eaviu-1, and Bbifi-1 were inoculated into the corresponding culture medium for preparing bacterial powder, and cultured anaerobically at 37°C and 90rpm for 16 to 24 hours to obtain the first-level seed solution. Subsequently, the five first-level seed solutions were transferred to the corresponding culture medium for preparing bacterial powder, and cultured anaerobically at 37°C and 90rpm for 10 to 15 hours to obtain the second-level seed solution. The five second-level seed solutions were pumped into the fermentation tank containing the corresponding culture medium for preparing bacterial powder by peristaltic pumps for fermentation and culture. After stopping the fermentation, the bacteria were collected by centrifugation. The corresponding freeze-dried protective agent was added according to the ratio of 1:1 to 1:2 (weight ratio) between the bacterial mud and the freeze-dried protective agent, and the emulsified bacterial mud was mixed. The bacterial mud was freeze-dried and crushed to obtain the bacterial powder. Take an appropriate amount of each crushed bacterial powder for viable bacterial count determination. According to the viable bacterial count, the bacterial powder of each strain was mixed in an equal CFU ratio to obtain the bacterial composition bacterial powder. Finally, the bacterial composition bacterial powder was configured into a bacterial suspension using physiological saline for animal testing. The bacterial suspension was diluted with physiological saline to obtain bacterial compositions with different bacterial doses.

[0128] Test method:

[0129] (1) Experimental design: 96 SPF male Balb / c mice weighing 22-24 g (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were raised in an SPF animal room. The mice were randomly divided into 8 groups according to their initial weight (normal control group, model control group, positive control loperamide group, positive control Zhengchangsheng group, bacterial composition high-dose group, bacterial composition medium-high-dose group, bacterial composition medium-dose group, and bacterial composition low-dose group), with 12 mice in each group. The above 8 groups were gavaged with normal saline (0.2 mL / mouse / day), lyophilized protective agent (0.2 mL / mouse / day), loperamide (purchased from Henan Zhongjie Pharmaceutical Co., Ltd., 20 mg / kg body weight / mouse / day), Zhengchangsheng (containing live Bacillus licheniformis, Northeast Pharmaceutical Group Co., Ltd., 2×10 8 CFU / mouse / day), high-dose group of bacterial composition (1×10 9 CFU / mouse / day, medium and high dose groups of bacterial composition (1×10 8 CFU / each / 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 overall experimental period was 9 days, recorded as D1-D9, and D1-D8 was administered by gavage for 8 days. Modeling began on D3, and the normal control group mice were injected with 0.2 mL of normal saline / mouse in the abdomen. The other groups of mice were injected with 30 mg / kg body weight of 5-FU (5-fluorouracil, purchased from Tianjin Jinyao Pharmaceutical Co., Ltd., with a specification of 10 mL / vial, 0.25 g / 10 mL) for 4 consecutive days to induce the mouse CID model, and the mice were dissected on D9. The specific experimental groups and dosing regimens are shown in Table 3.

[0130] Table 3. Experimental groups and dosing regimen of bacterial composition in treating 5-FU-induced diarrhea mice

[0131]

[0132] Note: 5-FU: 5-fluorouracil; CFU: colony forming unit; d: day; ip: intraperitoneal injection; ig: intragastric administration; QD means once a day.

[0133] During the experiment, the weight of the animals was measured every day, and general observations were recorded (observation items included but were not limited to the appearance of the animals, behavioral activities, respiration, glandular secretions, feces, etc.), and the diarrhea of ​​each animal after modeling was observed and recorded and scored. After euthanasia at the end of the experiment, mid-colon tissue samples were collected for qPCR detection of genes related to inflammatory factors (TNF-α, IL-1β) and tight junction proteins (ZO-1, Occludin).

[0134] (2) Diarrhea observation and scoring: The scoring standard for diarrhea refers to the diarrhea scoring method in the study of 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). The mice were placed in mouse cages padded with clean filter paper, with one mouse in each cage. 0 points: hard stool, normal stool; 1 point: mildly 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 the mice were observed and scored every day. The total diarrhea score was the sum of the daily diarrhea scores.

[0135] (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 colon proinflammatory factors IL-1β, TNF-α, and tight junction proteins ZO-1 and Occludin-1 in 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.

[0136] (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.

[0137] Test results:

[0138] (1) The diarrhea score results are shown in Table 4.

[0139] Table 4. Diarrhea score results

[0140]

[0141] Note: All data are 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] The diarrhea scores of the model group at D8 and D9 after 5-FU induction were significantly higher than those of the normal control group. The four doses of the bacterial composition can significantly improve the diarrhea of ​​the animals, and the diarrhea scores at D8 and D9 and the total diarrhea score are significantly lower than those of the model control group. It can be seen that the bacterial composition of the present invention can improve 5-FU-induced diarrhea.

[0143] (2) The qPCR test results are shown in Table 5.

[0144] Table 5. Effects of bacterial composition on colonic inflammation and intestinal barrier-related gene expression in mice with 5-FU-induced diarrhea

[0145]

[0146] Note: All data are 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.

[0147] 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, and the relative mRNA transcription levels of ZO-1 and Occludin genes were significantly decreased. High-dose bacterial composition and loperamide can significantly reduce the relative mRNA transcription level of TNF-α. High-dose, medium-high-dose and medium-dose bacterial composition can significantly reduce the relative mRNA transcription level of IL-1β. The high-dose group, medium-dose group and low-dose bacterial composition can significantly increase the relative mRNA transcription level of ZO-1, which is comparable to the improvement effect of loperamide and Zhengchangsheng. Medium-high-dose and medium-dose bacterial compositions can significantly increase the relative mRNA transcription level of Occludin, which is comparable to the improvement effect of Zhengchangsheng.

[0148] 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.

[0149] Example 10: Therapeutic effect of bacterial composition on irinotecan (CPT-11)-induced diarrhea in mice

[0150] The bacterial composition used in Examples 10-12 was obtained by mixing five kinds of bacteria at equal CFU according to the method in Example 9. The CFU of the bacterial composition mentioned in Examples 10-12 is the total CFU of the five kinds of bacteria.

[0151] Test method:

[0152] (1) Experimental design: 70 male Balb / c mice (purchased from Shanghai Jihui Experimental Animal Breeding Co., Ltd.) were raised in an SPF animal room and randomly divided into a normal control group (no medication), a model control group (dosage regimen: lyophilized protective agent 0.2 mL / mouse / day), a positive control (dosage regimen: Bacillus licheniformis 2.5×10 8 CFU / each / day) group, bacterial composition high-dose group (dosage regimen: bacterial composition 1×10 9 CFU / each / day), medium-high dose group of bacterial composition (dosage regimen: bacterial composition 1×10 8 CFU / each / day), medium-dose group of bacterial composition (dosage regimen: bacterial composition 1×10 7 CFU / each / day), low-dose bacterial composition group (dosage regimen: bacterial composition 1×106 CFU / animal / day), 7 groups in total, 10 animals in each group. The overall test period was 10 days, recorded as D1 to D10.

[0153] The normal control group mice were injected with 0.2 mL of saline per mouse in the abdomen, and the other groups of mice were injected with 85 mg / kg body weight of CPT-11 in the abdomen to establish the model. The model was established from D3 and continued for 4 days from D3 to D6, once a day. The corresponding groups were gavaged and administered once a day for 9 consecutive days, and the animals were dissected on D10. The specific experimental groups and dosing schedules are shown in Table 6.

[0154] Table 6. Experimental groups and dosing regimen of bacterial composition for treating CPT-11-induced diarrhea in mice

[0155]

[0156] Note: CPT-11: irinotecan hydrochloride; CFU: colony forming unit; d: day; ip: intraperitoneal injection; ig: intragastric administration; QD means once a day.

[0157] During the experiment, the weight of the animals was measured every day, and general observations were made (observation items included but were not limited to the appearance of the animals, behavioral activities, respiration, glandular secretions, feces, etc.), and the diarrhea of ​​each animal after modeling was observed and recorded and scored. At the end of the experiment, whole blood samples were collected from the animals in each group through the abdominal vena cava for routine blood tests. After the animals were euthanized, the intestines were taken and the length of the small intestine was measured respectively; the spleen was weighed and the spleen coefficient was calculated (spleen coefficient = spleen weight / body weight × 100). Samples were collected from 5 mice in each group near the cecum, and the samples were frozen in liquid nitrogen and stored at -80 ° C. The samples were used for qPCR to detect the mRNA expression levels of related genes such as inflammatory factors (TNF-a, IL-1β, IL-22), tight junction proteins (ZO-1, Occludin), pro-apoptotic factors (Bax), and water channel proteins (AQP8). The colorectal parts of the remaining 5 mice in each group were directly fixed in 10% formaldehyde solution and stained with HE for pathological examination.

[0158] (2) Diarrhea observation and scoring: The scoring criteria and operating method for diarrhea are the same as those in Example 9.

[0159] (3) Routine blood test: Whole blood samples were collected from animals in each group through the peritoneal vein. The samples were tested for routine blood tests using a fully automatic blood cell analyzer (Mindray).

[0160] (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.

[0161] Table 7. qRT-PCR primer information

[0162]

[0163]

[0164] (5) Intestinal tissue pathological examination: The colorectal parts were directly fixed in 10% formaldehyde solution and subjected to pathological examination after HE staining.

[0165] (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.

[0166] Test results:

[0167] (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), indicating that the CID model was successfully constructed.

[0168] The diarrhea scores and body weight measurement results are shown in Tables 8 and 9, respectively.

[0169] Table 8. Effects of bacterial composition on diarrhea in CPT-11-induced CID model mice

[0170]

[0171] Note: All data are 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.

[0172] High-dose bacterial composition and Zhengchangsheng can significantly reduce the total score of D10 diarrhea. The medium- and high-dose bacterial composition has a tendency to reduce the total score of D10 diarrhea. Compared with the model control group, the total score of D10 diarrhea in the high-dose bacterial composition group decreased slightly more than that in the positive control Zhengchangsheng group. In general, the bacterial composition significantly improved the diarrhea of ​​mice with CPT-11-induced diarrhea, and had a dose-dependent trend.

[0173] Table 9. Effect of bacterial composition on weight loss in mice with CPT-11-induced diarrhea

[0174]

[0175] Note: All data are 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.

[0176] The body weight of the model mice continued to decrease after modeling until the end of the experiment. High-dose, medium-high-dose, medium-dose bacterial composition and Zhengchangsheng can significantly alleviate the weight loss of model mice on D10, and the degree of relief in the high-dose bacterial composition treatment group is comparable to that in the Zhengchangsheng group.

[0177] (2) The results of intestine and spleen are shown in Table 10.

[0178] Table 10. Effects of bacterial composition on intestinal and spleen lesions in mice with CPT-11-induced diarrhea

[0179]

[0180] Note: All data are 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.

[0181] The small intestinal atrophy of the model mice in the high-dose, medium-high-dose, low-dose and Zhengchangsheng groups of the bacterial composition was significantly improved. The small intestinal swelling of the model mice in the high-dose, medium-high-dose, medium-dose and Zhengchangsheng groups of the bacterial composition was also significantly improved. The medium-high-dose, medium-dose, low-dose bacterial composition and Zhengchangsheng can significantly improve the spleen atrophy of the model mice.

[0182] (3) The results of routine blood tests are shown in Table 11.

[0183] High-dose and medium-high-dose bacterial compositions can significantly increase the lymphocyte content in the peripheral blood of model mice and reduce the neutrophil content. Zhengchangsheng has a tendency to increase the lymphocyte content in the peripheral blood of model mice and can significantly reduce the neutrophil content. The percentage of lymphocytes in the peripheral blood of model mice in the high-dose group, medium-high-dose group, medium-dose group, low-dose group and Zhengchangsheng group of bacterial compositions was significantly increased, and the percentage of neutrophils was significantly reduced.

[0184] Table 11. Blood routine test results of bacterial composition on CPT-11-induced diarrhea mice

[0185]

[0186]

[0187] Note: All data are 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

[0188] (4) The qPCR test results are shown in Table 12.

[0189] Table 12. Effects of bacterial composition on gene expression in colon tissue of mice with CPT-11-induced diarrhea

[0190]

[0191] Note: All data are mean ± standard deviation (SEM); all groups were compared with the model control group, "*" indicates P < 0.05, and "**" indicates P < 0.01.

[0192] The mRNA expression of inflammatory factor TNF-a in the colorectal area of ​​model mice in the high-dose, medium-high-dose and Zhengchangsheng groups of the bacterial composition was significantly downregulated; the high-dose bacterial composition can significantly downregulate the mRNA expression of inflammatory factor IL-1β, and the medium-high-dose and Zhengchangsheng groups of the bacterial composition have a certain downward trend compared with the model group. The mRNA expression level of IL-22 in the high-dose and Zhengchangsheng groups of the bacterial composition has a downward trend. The expression of tight junction protein-related gene ZO-1 in the medium-dose and low-dose groups of the bacterial composition is significantly upregulated, and the expression of tight junction protein-related gene Occludin in the medium-dose and low-dose groups of the bacterial composition is significantly upregulated. The mRNA expression of apoptosis-related gene Bax in the model mice in the high-dose group of the bacterial composition is significantly reduced, and the expression of water channel protein AQP8 is significantly enhanced.

[0193] (5) The pathological test results are shown in Table 13.

[0194] Table 13. Effect of bacterial composition on colorectal pathology scores in mice with CPT-11-induced diarrhea

[0195]

[0196] Note: All data are 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.

[0197] The pathological scores of the high-dose group, low-dose group and Zhengchangsheng group of the bacterial composition were significantly lower than those of the model control group. The pathological scores of the high-dose group and Zhengchangsheng group of the bacterial composition showed a decreasing trend compared with the model group.

[0198] 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 aquaporin (AQP8) mRNA, and significantly improve the symptoms of CPT-11-induced diarrhea, weight loss, small intestinal swelling, spleen atrophy, abnormal number of peripheral blood immune cells (lymphocytes and neutrophils), and intestinal inflammation in mice.

[0199] Example 11: Therapeutic effect of bacterial composition on mice with radiation enteritis

[0200] (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 / mouse / day), a model control group (dosage regimen: 0.2 mL of lyophilized protective agent / mouse / day), a loperamide group (dosage regimen: loperamide 15 mg / kg body weight), and a LGG (Lactobacillus rhamnosus GG, Shaanxi Zelang Biotechnology Co., Ltd.) group (dosage regimen: LGG 1×10 9 CFU / each / day), bacterial composition high-dose group (dosage regimen: bacterial composition 1×10 9 CFU / each / day), medium-high dose group of bacterial composition (dosage regimen: bacterial composition 1×10 8 CFU / each / day), medium-dose group of bacterial composition (dosage regimen: bacterial composition 1×10 7 CFU / each / day), low-dose bacterial composition group (dosage regimen: bacterial composition 1×10 6CFU / mouse / day), 8 groups in total, 10 mice in each group. Except for the normal control group, all other groups of mice were subjected to a single full abdominal X-ray irradiation, with an irradiation dose of 11.5Gy. The overall experimental period was 18 days, recorded as D1 to D18. Each group was gavaged and administered once a day for 17 consecutive days. After 7 days of administration, irradiation was performed on D8 and dissection was performed on D18. The experimental grouping and dosing regimen are shown in Table 14.

[0201] Table 14. Experimental groups and dosing regimens of bacterial compositions for treating radiation enteritis in mice

[0202]

[0203]

[0204] Note: CFU: colony forming unit; d: day; ip: intraperitoneal injection; ig: oral administration; QD means once a day.

[0205] During the experiment, the weight of the animals was measured every day, and general observations were recorded (observation items included but were not limited to the appearance of the animals, behavioral activities, respiration, glandular secretions, feces, etc.), and the diarrhea of ​​each animal after irradiation modeling was observed and recorded and scored. At the end of the experiment, the animals were euthanized, and the colorectal part was directly fixed in 10% formaldehyde solution and stained with HE for pathological examination.

[0206] (2) Diarrhea observation and scoring: The scoring criteria and operating method for diarrhea are the same as those in Example 9.

[0207] (3) Intestinal tissue pathological examination: The colorectal parts were directly fixed in 10% formaldehyde solution and subjected to pathological examination after HE staining.

[0208] (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.

[0209] Experimental results: The animals in the model control group showed obvious symptoms such as diarrhea, weight loss, intestinal mucosal damage and inflammatory infiltration after intraperitoneal irradiation, indicating that the mouse radiation enteritis model was successfully established.

[0210] (1) The diarrhea results are shown in Table 15.

[0211] Table 15. Effect of bacterial composition on diarrhea in mice with radiation enteritis model

[0212]

[0213]

[0214] Note: All data are 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.

[0215] Compared with the model control group, the total diarrhea scores of mice in the high-dose, medium-dose, low-dose, loperamide and LGG groups of the bacterial composition were significantly improved on D17. The total diarrhea scores of the high-dose bacterial composition group on D17 tended to decrease.

[0216] (2) Body weight results are shown in Table 16.

[0217] Table 16. Effect of bacterial composition on body weight changes in radiation enteritis model mice

[0218]

[0219] Note: All data are 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.

[0220] Compared with the model control group, the weight loss of mice in the high-dose, low-dose and LGG groups of the bacterial composition was significantly improved at D14. The low-dose bacterial composition can significantly improve the weight loss of model mice at D17. High-dose and medium-dose bacterial compositions, loperamide and LGG all tend to restore the weight of model mice at D17.

[0221] (3) The colorectal pathological results are shown in Table 17.

[0222] Table 17. Effect of bacterial composition on colorectal pathological scores in radiation enteritis model mice

[0223]

[0224]

[0225] Note: All data are 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.

[0226] Compared with the model control group, the pathological scores of the high-dose group, low-dose group and LGG group of the bacterial composition were significantly improved. The pathological scores of the colorectal area of ​​the high-dose group, medium-dose group and loperamide group of the bacterial composition showed an improvement trend.

[0227] In summary, the bacterial composition has a significant therapeutic effect on mice with radiation enteritis induced by whole-abdomen irradiation with X-rays, and can improve the degree of diarrhea and weight loss, and alleviate the degree of intestinal lesions.

[0228] Example 12: Therapeutic effect of bacterial composition on 5-FU-induced tumor-bearing CID mice

[0229] Experimental design: 50 male BALB / c mice (purchased from Chengdu Yaokang Biotechnology Co., Ltd.) were raised in an SPF animal room. CT26 cells (purchased from Beijing Beina Biotechnology Co., Ltd.) were subcutaneously inoculated on the right rib 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 14:00, 42 mice were screened for subsequent experiments. The 42 mice were randomly divided into 7 groups according to the 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), whole intestine group (dosage regimen: Bacillus licheniformis 2×10 8 CFU / each / day), bacterial composition high-dose group (dosage regimen: bacterial composition 1×10 9 CFU / each / day), medium-dose group of bacterial composition (dosage regimen: bacterial composition 1×10 8 CFU / each / day) and the low-dose group of the bacterial composition (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.

[0230] The specific dosing regimen is shown in Table 18. After the experiment, the eyeballs of the animals in each group were removed to collect whole blood samples, and blood routine tests were performed using a five-category blood cell analyzer for animals (Shenzhen Dima Biotechnology Co., Ltd.).

[0231] Table 18. Dosage regimen for 5-FU-induced tumor-bearing CID model

[0232]

[0233]

[0234] Note: ig means oral administration, QD means once a day.

[0235] Test results: The diarrhea results are shown in Table 19.

[0236] Table 19. Diarrhea scores of mice in each group

[0237]

[0238] Note: All data are expressed as mean ± standard deviation (SEM); compared with the model control group, the diarrhea scores of each group were compared, "*" indicates P < 0.05, and "**" indicates P < 0.01.

[0239] Compared with the normal control group, the model control group began to experience diarrhea on D6, and the degree 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 D8; the low-dose bacterial composition significantly reduced the diarrhea scores and total diarrhea scores on D8 and D9.

[0240] The results of blood routine examination are shown in Table 20.

[0241] Table 20. Blood routine test results of 5-FU-induced tumor-bearing mouse model

[0242]

[0243] Note: All data are mean ± standard deviation (SEM); when the blood routine data of each group were compared with those of the model control group, "*" indicates P < 0.05, and "**" indicates P < 0.01.

[0244] Compared with the normal control group, the hematocrit, white blood cell count, lymphocyte count and platelet count of the model control group were significantly decreased. Compared with the model control group, the red blood cell count and hematocrit of the medium-dose group, low-dose group and whole intestine group of the bacterial composition were significantly increased, and the lymphocyte count and platelet count of the low-dose group of the bacterial composition were increased.

[0245] In summary, the three doses of bacterial composition all have a tendency to improve diarrhea and routine blood indicators; among them, the low-dose bacterial composition and loperamide can significantly improve diarrhea; the low-dose bacterial composition has the ability to significantly improve routine blood indicators (lymphocyte count, red blood cell count and platelet count).

[0246] It is known from common technical knowledge that the present invention can be implemented by other embodiments that do not deviate from its spirit or essential features. Therefore, the above disclosed embodiments are only illustrative in all respects and are not exclusive. All changes within the scope of the present invention or within the scope equivalent to the present invention are included in the present invention.

Claims

1. A probiotic combination, comprising any three, any four or five of a first probiotic, a second probiotic, a third probiotic, a fourth probiotic and a fifth 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; Treat, prevent and / or slow down tissue damage, disease or sub-health condition 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; 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.

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