Probiotic composition for preventing and treating antitumor treatment-related diarrhea and its use

Abstract

The present invention relates to the field of microbiology. The present invention relates to a probiotic composition and its use in the manufacture of a product for preventing and / or treating health conditions such as antitumor therapy-related diarrhea. The active ingredients of the probiotic composition include Bifidobacterium bifidum, Enterococcus avium, Lactobacillus salivarius, Rimosilactobacillus fermentum, and Parabacteroides distasonis. The probiotic composition has good safety profile, autoaggregation ability, antioxidant ability, short-chain fatty acid production ability, in vitro cell adhesion ability, the ability to inhibit multiple pathogenic bacteria, and the ability to treat antitumor therapy-related diarrhea.

Description

[Technical Field]

[0001] The present invention belongs to the field of microbial medicine and relates to a probiotic composition and its use, particularly to a composition containing five probiotics and its use in the prevention and / or treatment of anti-tumor therapy-related diarrhea. [Background technology]

[0002] Cancer treatment-related diarrhea (TCID) is a common complication of multiple antitumor adjuvant therapies and is one of the symptoms that has the greatest impact on health and quality of life. In severe cases, it can lead to treatment delays and reduced compliance, both of which can affect the long-term outcomes of oncology treatment and are potentially fatal. According to data released by the International Agency for Research on Cancer (IARC), a subsidiary of the World Health Organization, there were approximately 19.3 million new cancer cases worldwide in 2020, and this number is predicted to reach 28.4 million by 2040. As many as 82% of cancer treatment patients suffer from side effects such as diarrhea.

[0003] Chemotherapy (especially chemotherapy using conventional chemotherapy drugs such as fluoropyrimidines and irinotecan (CPT-11), which are first-line treatments for metastatic colorectal cancer patients), radiation therapy, targeted therapy, and immunotherapy are major causes of antitumor treatment-related diarrhea, other than surgery. The overall incidence of diarrhea associated with fluoropyrimidine and irinotecan treatment is 50% to 80%, with the incidence of severe (grade 3 to 5) diarrhea reaching over 30%. Of these, 1% to 5% of subjects treated with 5-fluorouracil (5-FU) died from diarrhea. Approximately 70% of patients with abdominal or pelvic tumors require radiation therapy, of which 60% to 70% experience acute symptoms such as abdominal pain, diarrhea, and bloody stool, and 30% to 40% experience chronic diarrhea. The incidence of diarrhea associated with targeted therapy (e.g., targeted therapy using multiple tyrosine kinase inhibitors (TKIs) such as afatinib, ceritinib, erlotinib, and lapatinib) can reach over 90%. Immune checkpoint inhibitors often cause immune-mediated enterocolitis. Diarrhea occurs in 1% to 45% of tumor patients treated with ipilimumab, nivolumab, and pembrolizumab.

[0004] There are common mechanisms underlying diarrhea associated with antitumor therapy other than surgery. Anti-tumor therapy-associated diarrhea is multifactorial, with the primary pathogenic factors being: 1) oxidative stress and DNA damage caused by chemotherapy and ionizing radiation during radiotherapy; 2) intestinal mucosal damage resulting in epithelial cell loss, reduced tight junction integrity, crypt epithelial cell death, and increased intestinal permeability; 3) overactivation of multiple signaling pathways, including nuclear factor-κB, stimulates cells to produce inflammatory factors, leading to mucosal inflammation; 4) small intestinal mucosal damage leads to bile acid malabsorption, and excess bile acids (especially dihydroxy components) induce water and electrolyte secretion, increasing intestinal peristalsis and resulting in diarrhea; 5) infection with common hospital-acquired pathogenic bacteria (e.g., Salmonella, Shigella, Escherichia coli, and Clostridium difficile); 6) bacterial translocation leading to intestinal dysbiosis, manifested as a decrease in probiotics and an increase in pathogenic bacteria; and 7) immune abnormalities, including general T cell activation.

[0005] Currently, international clinical guidelines, such as "Guidance on the management of diarrhea during cancer chemotherapy (2014)" and "Diarrhea in adult cancer patients: ESMO Clinical Practice Guidelines (2018)," recommend loperamide as the initial treatment for chemotherapy-induced diarrhea (CID), radiation enteritis, targeted therapy-associated diarrhea, and immune checkpoint inhibitor-associated diarrhea. The guidelines also recommend the use of octreotide for patients who do not respond to loperamide treatment. Loperamide administration should not exceed 48 hours. High-dose loperamide use may pose a risk of paralytic ileus and serious cardiotoxicity. Octreotide (the first-line drug for patients with diarrhea scores of grade 3 or higher) can induce side effects such as gallstones, hyperglycemia, and impaired glucose tolerance. Guidelines also recommend budesonide as a second-line treatment when loperamide therapy is ineffective. However, glucocorticoids exert systemic effects and can increase the risk of infection and further worsen viral and bacterial infections. Patients with grade 3–4 diarrhea accompanied by neutropenia can be given oral antibiotics, but antibiotics can worsen diarrhea and increase the risk of Clostridium difficile infection. Tumor treatments typically involve combination therapy with multiple treatment modalities. Combination therapy significantly increases the incidence of diarrhea. If diarrhea persists for a long time or if effective treatment is not administered after diarrhea, patients may develop symptoms such as electrolyte disturbances, dehydration, and anemia. Moderate or severe diarrhea can also lead to serious complications, manifesting as neutropenia and systemic inflammatory response syndrome. Therefore, there is a clinical need for safer and more effective next-generation drugs for the treatment of antitumor therapy-associated diarrhea.

[0006] Intestinal probiotics are a type of active microorganism that colonize and proliferate in the human intestinal tract, altering the composition of the host's intestinal microbiota and benefiting the host. Intestinal probiotics have the advantages of high safety, minimal toxicity and side effects, and a wide range of indications and target populations. Probiotic compositions can play a therapeutic role in the complex pathogenesis of antitumor therapy-associated diarrhea, primarily through the following: 1) probiotics exert antioxidant functions through metal ion chelating, upregulating self- and host-specific 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, and secondary bile acids. Beneficial secondary metabolites enhance intestinal barrier function by promoting the secretion of mucin by goblet cells, promoting the repair of epidermal cells, inhibiting cell apoptosis, and enhancing tight junction proteins. 3) Probiotics exert anti-inflammatory and immune-enhancing effects by modulating immune responses through methods such as downregulating the NF-κB pathway, inhibiting pro-inflammatory factors, and secreting anti-inflammatory factors. 4) Probiotics regulate the balance of intestinal microflora, reducing pathogenic bacteria and increasing probiotics, thereby maintaining a normal homeostatic environment in the intestinal tract. 5) Probiotics regulate bile acid metabolism through methods such as increasing bile salt hydrolase activity in the microflora and inhibiting the FXR-FGF15 signaling pathway, thereby alleviating bile acid-induced diarrhea. 6) Probiotics inhibit the growth of pathogenic bacteria through methods such as increasing the secretion of antibacterial proteins (defensins), producing antibacterial substances such as bacteriocins, and competing for adhesion sites with pathogens.

[0007] Chinese Patent CN112694992B discloses Bifidobacterium bifidum, which can alleviate diarrhea caused by enterotoxin-producing Escherichia coli (ETEC). Chinese Patent CN113234619B discloses Bifidobacterium bifidum, which can alleviate acute intestinal injury. A small amount of literature has reported that Parabacteroides distasonis has the ability to alleviate intestinal inflammation. For example, M. Kverka et al. (Oral administration of Parabacteroides distasonis antigens attenuates experimental murine colitis through modulation of immunity and microbiota composition) reported that Parabacteroides distasonis can ameliorate DSS-induced colitis in mice. Currently, there are only a few documents reporting the use of Lactobacillus salivarius in the prevention and treatment of diarrhea. For example, Patent CN110878267B discloses Lactobacillus salivarius ZLp4b, which can significantly delay and cure diarrhea in young animals. In addition to the above patents and publications, there are also several similar publications reporting the use of probiotics in the prevention and treatment of diarrhea or intestinal inflammation. However, current probiotic preparations are mainly used to treat general diarrheal symptoms, and are often single-strain preparations, making it very difficult to achieve targeted therapeutic effects on the complex pathogenetic mechanisms of anti-tumor therapy-related diarrhea. Summary of the Invention

[0008] A first aspect of the present invention provides a probiotic composition, which includes any three, any four, or any five of a first probiotic, a second probiotic, a third probiotic, a fourth probiotic, and a fifth probiotic (preferably, the probiotic composition includes the fifth probiotic and any two, or any five of the first probiotic, the second probiotic, the third probiotic, and the fourth probiotic). Preferably, the probiotic composition comprises three or four kinds of probiotics, more preferably, the probiotic composition comprises a fourth probiotic, a fifth probiotic, and any two or three kinds of the first probiotic, the second probiotic, and the third probiotic, and the first probiotic is Bifidobacterium bifidum, a descendant strain of the Bifidobacterium bifidum, a clone strain of the Bifidobacterium bifidum, or a pure strain of the Bifidobacterium bifidum. the second probiotic is selected from Enterococcus avium, a descendant strain of the Enterococcus avium, a clonal strain of the Enterococcus avium, or a pure culture of the Enterococcus avium; the third probiotic is selected from Lactobacillus salivarius, a descendant strain of the Lactobacillus salivarius, a clonal strain of the Lactobacillus salivarius, or a pure culture of the Lactobacillus salivarius; and the fourth probiotic is The fifth probiotic is selected from Rimosilactobacillus fermentum, a descendant strain of the Rimosilactobacillus fermentum, a clone strain of the Rimosilactobacillus fermentum, or a pure culture of the Rimosilactobacillus fermentum, and the fifth probiotic is selected from Parabacteroides disstasonis, a descendant strain of the Parabacteroides disstasonis, a clone strain of the Parabacteroides disstasonis, or a pure culture of the Parabacteroides disstasonis.

[0009] In some embodiments, the Bifidobacterium bifidum has a microbial deposit number of CCTCC NO: M2023349, the Enterococcus avium has a microbial deposit number of CCTCC NO: M2023350, the Lactobacillus salivarius has a microbial deposit number of CCTCC NO: M2023348, the Limosilactobacillus fermentum has a microbial deposit number of CCTCC NO: M2023352, and the Parabacteroides distasonis has a microbial deposit number of CCTCC NO: M20222033.

[0010] In some embodiments, the probiotic composition includes a first probiotic, a second probiotic, a third probiotic, a fourth probiotic, and a fifth probiotic, wherein the first probiotic is selected from Bifidobacterium bifidum having Microbial Deposit Number CCTCC NO:M2023349, a progeny strain of Bifidobacterium bifidum having Microbial Deposit Number CCTCC NO:M2023349, a clonal strain of Bifidobacterium bifidum having Microbial Deposit Number CCTCC NO:M2023349, or a pure culture of Bifidobacterium bifidum having Microbial Deposit Number CCTCC NO:M2023349; and the second probiotic is selected from Enterococcus avium having Microbial Deposit Number CCTCC NO:M2023350, a progeny strain of Bifidobacterium bifidum having Microbial Deposit Number CCTCC NO:M2023349, a clonal strain of Bifidobacterium bifidum having Microbial Deposit Number CCTCC NO:M2023349, or a pure culture of Bifidobacterium bifidum having Microbial Deposit Number CCTCC NO:M2023349. the third probiotic is selected from a progeny strain of the Enterococcus avium having the microorganism deposit number CCTCC NO:M2023350, a clonal strain of the Enterococcus avium having the microorganism deposit number CCTCC NO:M2023350, or a pure culture of the Enterococcus avium having the microorganism deposit number CCTCC NO:M2023350; and the third probiotic is selected from a Lactobacillus salivarius having the microorganism deposit number CCTCC NO:M2023348, a progeny strain of the Lactobacillus salivarius having the microorganism deposit number CCTCC NO:M2023348, a clonal strain of the Lactobacillus salivarius having the microorganism deposit number CCTCC NO:M2023348, or a pure culture of the Lactobacillus salivarius having the microorganism deposit number CCTCC NO:M2023348. and the fourth probiotic is selected from a pure culture of the Lactobacillus salivarius having the microorganism deposit number CCTCC NO: M2023348, a progeny strain of the Lactobacillus fermentum having the microorganism deposit number CCTCC NO: M2023352, a clone strain of the Lactobacillus fermentum having the microorganism deposit number CCTCC NO: M2023352, or a clone strain of the Lactobacillus fermentum having the microorganism deposit number CCTCC NO: M2023352.and the fifth probiotic is selected from a pure culture of the Limocilactobacillus fermentum having the microorganism deposit number CCTCC NO: M20222033, a descendant strain of the Parabacteroides disstasonis having the microorganism deposit number CCTCC NO: M20222033, a clonal strain of the Parabacteroides disstasonis having the microorganism deposit number CCTCC NO: M20222033, or a pure culture of the Parabacteroides disstasonis having the microorganism deposit number CCTCC NO: M20222033.

[0011] A second aspect of the present invention provides a microecological composition, said microecological composition comprising as an active ingredient the probiotic composition according to the first aspect of the present invention.

[0012] In some embodiments, the composition further comprises an auxiliary material selected from a lyoprotectant, a bacterial culture medium, a food additive, a carrier or auxiliary material acceptable for health foods, and a pharmaceutically acceptable carrier or auxiliary material.

[0013] In some embodiments, in the microecological composition, when calculated in terms of the number of viable bacteria, the content ratio of any two types of bacteria is 100 CFU:1 to 10,000 CFU (for example, 100 CFU:1 CFU, 2 CFU, 3 CFU, 4 CFU, 5 CFU, 6 CFU, 7 CFU, 8 CFU, 9 CFU, 10 CFU, 20 CFU, 30 CFU, 40 CFU, 50 CFU, 60 CFU, 70 CFU). , 80CFU, 90CFU, 100CFU, 200CFU, 300CFU, 400CFU, 500CFU, 600CFU, 700CFU, 800CFU, 900CFU, 1000CFU, 2000CFU, 3000CFU, 4000CFU, 5000CFU, 6000CFU, 7000CFU, 8000CFU, 9000CFU, 10000CFU).

[0014] A third aspect of the present invention provides the use of a probiotic composition according to the first aspect of the present invention or a microecology composition according to the second aspect of the present invention in the manufacture of a product for use alone or in combination with other microbial preparations and / or drugs to improve the health status of a subject, wherein improving the health status of said subject comprises reducing or eliminating any one of Pseudomonas aeruginosa, Shigella dysenteriae, Staphylococcus aureus, Escherichia coli, Salmonella Paratyphi B, Yersinia enterocolitica, Vibrio parahaemolyticus and Clostridium difficile in a body cavity (e.g. the intestinal cavity) of the subject. inhibiting the growth of any one, any two, any three, any four, any five, any six, any seven or eight of the following bacteria: Pseudomonas aeruginosa, Shigella dysenteriae, Staphylococcus aureus, Escherichia coli, Salmonella paratyphi B, Yersinia enterocolitica, Vibrio parahaemolyticus and Clostridium difficile; improving antioxidant capacity in the intestinal tract of a subject; treating or preventing diarrhea induced by antineoplastic agents. and / or delaying, treating, preventing and / or delaying intestinal inflammation caused by antineoplastic drugs, treating, preventing and / or delaying weight loss caused by antineoplastic drugs, treating, preventing and / or delaying shortening of small intestine length caused by antineoplastic drugs, treating, preventing and / or delaying increase in small intestine thickness caused by antineoplastic drugs, treating, preventing and / or delaying intestinal damage caused by antineoplastic drugs, treating, preventing and / or delaying decrease in spleen weight to body weight ratio caused by antineoplastic drugs, any one of TNF-α, IL-1β, IL-22 and Bax, any treating, preventing and / or delaying tissue damage, disease or sub-health conditions caused by increased expression of any two, three or four of ZO-1, Occludin and AQP8; treating, preventing and / or delaying tissue damage, disease or sub-health conditions caused by decreased expression of any one, two or three of ZO-1, Occludin and AQP8; treating, preventing and / or delaying diarrhea caused by radiation therapy; treating, preventing and / or delaying weight loss caused by radiation therapy; treating, preventing and / or delaying intestinal damage caused by radiation therapy; treating intestinal inflammation caused by radiation therapy;and treating, preventing, and / or delaying a decrease in hematocrit, a decrease in white blood cell count, a decrease in lymphocyte count, and / or a decrease in platelet count caused by an anti-tumor drug.

[0015] In some embodiments, the product is a food product, a health food 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 a probiotic composition according to the first aspect of the invention or a microecology composition according to the second aspect of the invention in the manufacture of a medicament for treating and / or preventing anti-tumor treatment-related toxicity and side effects.

[0018] In some embodiments, the anti-tumor treatment-related toxicity and side effect is diarrhea.

[0019] In some embodiments, the anti-tumor treatment-related toxicity and side effects are diarrhea due to an anti-tumor drug, or diarrhea due to radiation therapy, or intestinal damage due to radiation therapy.

[0020] In some embodiments, the anti-tumor agent is selected from a chemotherapeutic agent, a targeted agent, and an immune checkpoint inhibitor.

[0021] In some embodiments, the anti-tumor agent is selected from epirubicin, actinomycin D, doxorubicin, daunorubicin, paclitaxel, docetaxel, albumin paclitaxel, cisplatin, carboplatin, nedaplatin, oxaliplatin, lobaplatin, cyclophosphamide, nitrogen mustard, carmustine, camptothecin, hydroxycamptothecin, topotecan, irinotecan, capecitabine, gemcitabine, methotrexate, 5-fluorouracil, pemetrexed, cytarabine, apatinib, axitinib, cabozantinib, sorafenib, sunitinib, nivolumab, pembrolizumab, and ipilimumab.

[0022] A fourth aspect of the present invention provides a method of preventing, treating or delaying intestinal disease, the method comprising administering to a subject a therapeutically effective amount of a microecology composition according to the second aspect of the present invention, wherein the intestinal disease is selected from intestinal disease 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; antineoplastic drug-induced diarrhea; antineoplastic drug-induced intestinal inflammation; antineoplastic drug-induced intestinal damage; diarrhea due to radiation therapy; intestinal inflammation due to radiation therapy; and intestinal damage due to radiation therapy.

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

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

[0025] In some embodiments, the therapeutically effective amount is 10 or more times per day, calculated based on the total bacterial content of the probiotic composition. 6~12 CFU (e.g., 1 x 10 6 CFU, 2 × 10 6 CFU, 3 × 10 6 CFU, 4 × 106 CFU、5×10 6 CFU、6×10 6 CFU、7×10 6 CFU、8×10 6 CFU、9×10 6 CFU、1×10 7 CFU、2×10 7 CFU、3×10 7 CFU、4×10 7 CFU、5×10 7 CFU、6×10 7 CFU、7×10 7 CFU、8×10 7 CFU、9×10 7 CFU、1×10 8 CFU、2×10 8 CFU、3×10 8 CFU、4×10 8 CFU、5×10 8 CFU、6×10 8 CFU、7×10 8 CFU、8×10 8 CFU、9×10 8 CFU、1×10 9 CFU、2×10 9 CFU、3×10 9 CFU、4×10 9 CFU、5×10 9 CFU、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 11CFU, 7 × 10 11 CFU, 8 × 10 11 CFU, 9 × 10 11 CFU and 10 12 CFU (any one value or a range between any two values).

[0026] In some embodiments, the anti-tumor agent is selected from epirubicin, actinomycin D, doxorubicin, daunorubicin, paclitaxel, docetaxel, albumin paclitaxel, cisplatin, carboplatin, nedaplatin, oxaliplatin, lobaplatin, cyclophosphamide, nitrogen mustard, carmustine, camptothecin, hydroxycamptothecin, topotecan, irinotecan, capecitabine, gemcitabine, methotrexate, 5-fluorouracil, pemetrexed, cytarabine, apatinib, axitinib, cabozantinib, sorafenib, sunitinib, nivolumab, pembrolizumab, and ipilimumab.

[0027] The beneficial effects of the present invention are as follows: (1) The probiotic composition provided by the present invention contains probiotics of three, four, or five different species, with no mutual antagonism between the strains, no virulence factors, and good safety; (2) The probiotic composition provided by the present invention has the ability to self-aggregate / co-aggregate, antioxidant, produce short-chain fatty acids, inhibit multiple pathogenic bacteria, and has relatively strong in vitro adhesion ability, which can act synergistically against the complex pathogenic mechanisms of antitumor therapy-related diarrhea; and (3) The probiotic composition provided by the present invention can prevent and / or treat toxicity and side effects such as diarrhea, intestinal inflammation, and intestinal damage caused by chemotherapy or radiation therapy. [Brief explanation of the drawings]

[0028] [Figure 1] Frontal photographs of colony morphology of five strains. [Figure 2] Photographs of the characteristics of co-culture of five single strains of bacteria on BF839 agar medium. [Figure 3] 1 illustrates the probiotic composition and its ability to self-aggregate (co-aggregate) single bacteria. [Figure 4] The results of detecting the bacteriostatic activity of a probiotic composition and its single bacterium against pathogenic bacteria are shown. [Figure 5] 1 shows the results of detecting the adhesion ability of a probiotic composition and its single bacterium to Caco2 cells. DETAILED DESCRIPTION OF THE INVENTION

[0029] In order to make the objectives, technical solutions and advantages of the present invention clearer, the following describes in more detail the embodiments of the present invention in conjunction with the drawings.

[0030] Unless otherwise specified, terms used herein have their accepted meanings in the biopharmaceutical arts.

[0031] Unless otherwise specified, the specific temperature parameters in the present invention should be understood as isothermal treatments and are allowed to vary within certain temperature intervals (e.g., within ±5°C, ±4°C, ±3°C, ±2°C, ±1°C).

[0032] The present invention provides a probiotic composition, which comprises any three, four, or five of the following strains: Bifidobacterium bifidum, Enterococcus avium, Lactobacillus salivarius, Limosilactobacillus fermentum, and Parabacteroides distasonis.

[0033] In some embodiments, the Bifidobacterium bifidum microorganism deposit number is CCTCC NO: M2023349, The microorganism deposit number for the above Enterococcus avium is CCTCC NO: M2023350. The microorganism deposit number of the above Lactobacillus salivarius is CCTCC NO: M2023348. The microorganism deposit number of the above-mentioned Limocilactobacillus fermentum is CCTCC NO: M2023352, and The microorganism deposit number for the above Parabacteroides distasonis is CCTCC NO: M20222033.

[0034] The meaning of the strain with a specific deposit number according to the present invention includes, but is not limited to, (1) a strain with a specific deposit number stored in the depository center, (2) a strain having the same genome as the strain described in (1), (3) a passaged strain without genetic mutations based on (1) or (2) above, (4) a passaged strain based on (1), (2), or (3) above that has accumulated minor mutations during passage but has not substantially changed in toxicity, immunogenicity, or biological activity, and (5) a live cell of any one of the strains described in (1) to (4) above, an inactivated product of the live cell, a lysate of the live cell, or a fermentation product of the live cell.

[0035] Strains having the same genome include, but are not limited to, strains having the same genetic background, i.e., strains isolated from nature or from the body of an animal (including a human) with the same genome (same genetic background), which have been independently isolated and disclosed by others since the priority date of the present invention. Conventional cultures are generally considered to be passaged strains without genetic mutations. As known to those skilled in the art, when a strain is passaged and used, the introduction of minor mutations is usually unavoidable. If the mutations are synonymous mutations made in non-coding or coding regions, or mutations that do not affect the toxicity (biological safety), immunogenicity, or biological activity of the strain (for example, if possible, mutations are made in the connecting amino acid residues between two domains, or the mutated amino acid residues are located within the protein's higher-order structure and do not come into contact with immune cells, so that these mutations do not affect toxicity, immunogenicity, or biological activity), and if these minor changes do not have a significant effect on the toxicity, immunogenicity, or biological activity of the progeny strain, the mutated strain can still achieve the objectives of the present invention, and since the mutated strain is derived from the contributing strain of the present invention, it can be reasonably expected that the corresponding strain will still fall within the scope of the substantial technical contribution of the present invention. These minor mutations still belong to the category of non-substantial mutations and should be considered as mutant strains with no changes in toxicity, immunogenicity, or biological activity. From the perspective of detection, the absence of substantial changes in the toxicity, immunogenicity, and biological activity of a strain includes, but is not limited to, the assumption that the toxicity, immunogenicity, and biological activity of the mutated strain are the same as those of the strain contributing to the present invention, within the limitations of the detection technology, such as detection sensitivity and detection limit, and within the range of acceptable or unavoidable error. When measuring the toxicity, immunogenicity, and biological activity of the progeny of a strain using cells, animals, etc., differences that appear due to cell lines, animal breeds, age, sex, health status, culture conditions, etc., as well as predictable or unavoidable systematic errors, fall under the category of the absence of substantial changes. An active ingredient refers to a substance that functions as a composition that produces a biological effect. In the present invention, the active ingredient is a probiotic strain.As a result of studying the co-culture characteristics of the five strains of the present invention, it was found that these strains do not inhibit each other two by two, and therefore it is reasonably expected that they can be combined into compositions containing any three, four, or five strains depending on the efficacy characteristics of each strain, and that compositions combining these strains will be able to simultaneously exert the efficacy of each strain in the group.

[0036] Antitumor therapy-related diarrhea, or tumor-related diarrhea, refers to diarrhea caused by various antitumor therapies damaging the intestinal mucosa and resulting in an imbalance between intestinal absorption and secretion. Common antitumor therapy-related diarrhea include chemotherapy-related diarrhea, radiotherapy-induced diarrhea, targeted therapy-induced diarrhea, and immune checkpoint inhibitor-induced diarrhea. Radiotherapy-induced diarrhea clinically manifests as radiation enteritis or radiation intestinal injury.

[0037] Chemotherapy refers to the use of non-selective chemical drugs to kill tumor cells to achieve therapeutic goals. Chemotherapy is the main method of treating tumors, other than surgery and radiation therapy. Due to its lack of selectivity, chemotherapy can also damage normal cells while killing tumor cells. Common chemotherapeutic agents include, but are not limited to, antibiotic-based chemotherapeutic agents (e.g., epirubicin, actinomycin D, doxorubicin, daunorubicin and derivatives thereof, etc.), paclitaxel-based chemotherapeutic agents (e.g., paclitaxel, docetaxel, albumin paclitaxel and derivatives thereof, etc.), platinum-based chemotherapeutic agents (e.g., cisplatin, carboplatin, nedaplatin, oxaliplatin, lobaplatin and derivatives thereof, etc.), alkylating agent-based chemotherapeutic agents (e.g., cyclophosphamide, nitrogen mustard, carmustine and derivatives thereof, etc.), camptothecin-based chemotherapeutic agents (e.g., camptothecin, hydroxycamptothecin, topotecan, irinotecan and derivatives thereof, etc.), and antimetabolite-based chemotherapeutic agents (e.g., capecitabine, gemcitabine, methotrexate, 5-fluorouracil, pemetrexed, cytarabine and derivatives thereof, etc.).

[0038] Targeted therapy involves designing therapeutic drugs to target cancer sites that have already been identified at the cellular and molecular level. In targeted therapy, once a drug is introduced into the body, it specifically selects and binds to the cancer site, thereby killing tumor cells specifically. The emergence of new targeted drugs has changed the tumor treatment model and ushered in the era of targeted therapy. Common targeted drugs include apatinib, axitinib, cabozantinib, sorafenib, and sunitinib.

[0039] Immune checkpoint inhibitors are several monoclonal antibody-based drugs developed against corresponding immune checkpoints. The main effect of immune checkpoint inhibitors is to block the interaction between tumor cells that express immune checkpoints and immune cells, thereby blocking the inhibitory effect of immune cells by tumor cells. Common immune checkpoint inhibitors include, but are not limited to, PD-1 / PD-L1 inhibitor antibodies (e.g., nivolumab, pembrolizumab, etc.) and CTLA-4 inhibitors (e.g., ipilimumab, etc.).

[0040] Radiation therapy refers to a method of treating malignant tumors using radiation (e.g., alpha rays, beta rays, gamma rays generated by radioisotopes, and x-rays, electron beams, proton beams, and other particle beams generated by various x-ray therapy devices or accelerators). Commonly used radiation therapy methods include, but are not limited to, conventional radiation therapy, stereotactic body radiotherapy, three-dimensional conformal radiotherapy, intensity-modulated radiotherapy, image-guided radiotherapy, intensity-modulated arc therapy, proton beam therapy, etc.

[0041] The present invention provides a microecological composition, which comprises any three, four, or five of Bifidobacterium bifidum, Enterococcus avium, Lactobacillus salivarius, Limosilactobacillus fermentum, and Parabacteroides distasonis, and auxiliary materials.

[0042] A microecological composition refers to a bioactive preparation containing specific microbial species and / or their metabolites that promotes host health, increases disease resistance, or improves host physiology by restoring or optimizing the structure and function of the host's microbiota.

[0043] The above auxiliary materials vary depending on the type of product to be produced, for example, food, health food or pharmaceutical products can be produced, and correspondingly, the auxiliary materials may be selected from lyoprotectants, bacterial culture media, food additives, health food acceptable carriers or auxiliary materials and pharmaceutically acceptable carriers or auxiliary materials.

[0044] The present invention provides the use of a probiotic or microecological composition in the manufacture of a food, health food or pharmaceutical product.

[0045] When manufactured as a medicament, the probiotic or microecological composition is used in the manufacture of a medicament for use alone or in combination with other microbial preparations and / or drugs to improve the health status of a subject.

[0046] The present invention further provides a method of preventing, treating, or delaying intestinal disease, said method comprising administering to a subject a therapeutically effective amount of a microecological composition.

[0047] The therapeutically effective amount or prophylactically effective amount is a dose that can clinically achieve the desired therapeutic or prophylactic effect. 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 a clinician in light of the treatment of the patient's condition. In some embodiments, the total number of bacteria contained in a single use 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 ~10 12 It is a CFU.

[0048] A pharmaceutically acceptable carrier refers to a pharmaceutical carrier that does not cause significant irritation to a subject and does not eliminate the biological activity and properties of the administered probiotic. As known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th edition, Mack Printing Company, 1990, pp. 1289-1329), a pharmaceutically acceptable carrier can enhance or stabilize a composition or can be used to facilitate the manufacture of a composition. Pharmaceutically acceptable carriers may include solvents, dispersion media, coatings, surfactants, antioxidants, isotonicity agents, absorption delaying agents, salts, drug stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, protectants, and the like, and combinations thereof. Except where conventional carriers are incompatible with the active ingredient, their use in therapeutic or pharmaceutical compositions is contemplated. Carriers may be selected to minimize adverse side effects in a subject and / or minimize inactivation of the active ingredient.

[0049] An excipient refers to a substance added to a pharmaceutical composition so that the drug has a certain form or concentration, and examples thereof include sterilized water, saline, polyalkylene glycols (e.g., polyethylene glycol), vegetable oils, hydrogenated naphthalene, calcium bicarbonate, calcium phosphate, various sugars, various types of starch, cellulose derivatives, gelatin, etc.

[0050] The microecological compositions of the present invention may further comprise a second beneficial active ingredient, such as another probiotic, prebiotic, or drug with antidiarrheal properties. Prebiotics indirectly exert their antidiarrheal effects by promoting the growth of probiotics in the intestinal tract and helping to regulate the intestinal environment. Examples of second beneficial active ingredients include, but are not limited to, Bacillus licheniformis, Bifidobacterium, Clostridium butyricum, fructooligosaccharides, galactooligosaccharides, isomaltooligosaccharides, xylooligosaccharides, mannanoligosaccharides, inulin, stachyose, soybean oligosaccharides, beta-dextran, and lactosucrose.

[0051] In some embodiments, the dosage form of the pharmaceutical composition is a tablet, capsule, granule, solution, suspension, powder, or the like.

[0052] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Of course, the described embodiments are only some of the embodiments of the present invention, and are not all of the embodiments. Any other embodiments obtained based on the embodiments in the application documents of the present invention without requiring creative efforts by those skilled in the art should fall within the scope of the disclosure or claims of the present invention.

[0053] The method for preparing the media used in the following examples is as follows: Preparation of YCFA liquid medium: 10.0 g of peptone, 2.5 g of yeast extract, 0.45 mL of 10% (w / w) MgSO₄·7H₂O solution, 0.45 mL of 10 mg / mL CaCl₂ solution, 10 mL of TE141, 0.45 g of K₂HPO₄, 0.45 g of KH₂PO₄, 0.90 g of NaCl, and 3.2 mL of VFA-mix were added to 1 L of distilled water to obtain a solution. The solution was deoxygenated by N₂ replacement and then dispensed. The dispensed solution was sterilized at 121°C for 30 min using moist heat and then prepared for use.

[0054] Preparation of TE141: 1.50 g of nitrilotriacetic acid was added to 200 mL of pure water to obtain a solution. An appropriate amount of NaOH was added to the solution until the solution became clear, and then 800 mL of water was added to the solution, and the pH value was adjusted to 5.5 with 50% HCl to obtain an aqueous solution of nitrilotriacetic acid. MgSO4·7H2O 3.00g, MnSO4·H2O 0.50g, NaCl 1.00g, FeSO4·7H2O 0.10g, CoSO4·7H2O 0.18g, CaCl2·2H2O 0.10g, ZnSO4·7H2O 0.18g, CuSO4·5H2O 0.006g, KAl(SO4)2·12H2O 0.02g, H3BO30.01g, Na2MoO4·2H2O 0.01g, NiCl2·6H2O 0.03 g, 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 aqueous nitrilotriacetic acid solution (the solution was constantly stirred during the addition process to keep it clear), to obtain TE141.

[0055] Preparation of VFA-mix: 90 mL of acetic acid, 30 mL of propionic acid, 10 mL of n-valeric acid, 10 mL of isobutyric acid, and 10 mL of butyric acid were mixed uniformly to obtain a solution for use. Before use, the pH of the above solution was adjusted to neutral with 5 M NaOH.

[0056] Preparation of triple mixed liquid medium (BHI + MRS + modified GAM): 19.25 g of BHI broth powder (Qingdao Haibo Biotechnology Co., Ltd., HB8297-5), 13.5 g of MRS broth powder (Guangdong Huankai Biotechnology Co., Ltd., 027312), and 15 g of modified GAM broth powder (Qingdao Haibo Biotechnology Co., Ltd., HB8518-3) were dissolved in 1 L of distilled water to obtain a solution. The solution was deoxygenated by N2 replacement and then dispensed. The dispensed solution was sterilized at 121 °C for 30 min using high-temperature moist heat. The resulting triple mixed liquid medium was stored in a cool, dry place.

[0057] Preparation of triple mixed solid medium: Add 5 g of agar powder to the above triple mixed liquid medium as a base, and the other steps are the same as those for preparing triple mixed liquid medium.

[0058] JPEG2026508600000002.jpg55170

[0059] Preparation of BF839 solid medium: 50.4 g of BF839 solid agar (Qingdao Hi-Tech Industrial Park Haibo Biotechnology Co., Ltd., HB8805) was dissolved in 1 L of distilled water to obtain a mixture. The mixture was deoxygenated by nitrogen substitution and then dispensed. The mixture was sterilized at 121 °C for 15 min using high-temperature moist heat. The resulting medium was stored in a cool, dry place.

[0060] Preparation of oxygen-free PBS: 0.27 g of potassium dihydrogen phosphate, 1.42 g of disodium hydrogen phosphate, 8 g of sodium chloride, and 0.2 g of potassium chloride were dissolved in 1 L of distilled water. The mixture was heated to a boil, cooled to room temperature, and 0.55 g of cysteine ​​hydrochloride was added. The mixture was stirred to dissolve, and the pH was adjusted to 6.5. The mixture was heated to a boil and maintained at a gentle boil for 30 minutes. After cooling, the mixture was dispensed into 400 mL bottles using a volumetric dispenser under N2 flow. The bottles were sterilized at high temperature and humidity for 30 minutes at 121°C. The resulting PBS was stored in a cool, dry place for further use.

[0061] GAM solid medium (Qingdao Haibo Biotechnology Co., Ltd., HB8462), TSB (tryptone soy broth, Qingdao Haibo Biotechnology Co., Ltd., HB4114), and TSA (tryptone soy agar, Qingdao Haibo Biotechnology Co., Ltd., HB4138) were prepared by weighing the ingredients and dissolving them in water according to the steps specified in the manufacturer's instructions. The mixture was then sterilized at 121°C for 30 minutes using moist heat to obtain the medium. The medium was stored in a cool, dry place.

[0062] Preparation of Parabacteroides distasonis Pdist-1 powder production medium: 6 g of anhydrous glucose, 15 g of soybean peptone, 10 g of yeast extract powder, 10 g of yeast peptone, 2 g of potassium dihydrogen phosphate, 2 g of disodium hydrogen phosphate, 0.2 g of magnesium sulfate, 0.01 g of manganese sulfate, 0.2 g of calcium chloride, 1 mL of Tween 80, and 0.5 g of cysteine ​​hydrochloride monohydrate were dissolved in 1 L of distilled water. The mixture was deoxygenated by N2 substitution and dispensed. The mixture was sterilized at 121°C for 15 min.

[0063] Preparation of the medium for producing Limocillosilacocillus fermentum Lferm-1 powder: 30 g of anhydrous glucose, 15 g of soybean peptone, 10 g of yeast extract powder, 5 g of sodium acetate, 2 g of potassium dihydrogen phosphate, 2 g of disodium hydrogen phosphate, 0.1 g of magnesium sulfate, 0.045 g of manganese sulfate, 1 mL of Tween 80, and 0.5 g of cysteine ​​hydrochloride monohydrate were dissolved in 1 L of purified water. The mixture was deoxygenated by N2 substitution and dispensed. The mixture was then sterilized at 121°C for 15 minutes using moist heat.

[0064] Preparation of Lactobacillus salivarius Lsali-1 powder production medium: 24 g of anhydrous glucose, 20 g of soybean peptone, 10 g of yeast extract powder, 10 g of peptone, 5 g of sodium acetate, 2 g of potassium dihydrogen phosphate, 2 g of disodium hydrogen phosphate, 0.1 g of magnesium sulfate, 0.045 g of manganese sulfate, 1 mL of Tween 80, and 0.5 g of cysteine ​​hydrochloride monohydrate were dissolved in 1 L of purified water. The mixture was deoxygenated by nitrogen substitution and dispensed. The mixture was then sterilized at 121°C for 15 minutes using moist heat.

[0065] Preparation of Enterococcus avium Eaviu-1 powder production medium: 30 g of anhydrous glucose, 15 g of soy peptone, 10 g of yeast powder, 5 g of sodium acetate, 2 g of dipotassium hydrogen phosphate, 0.1 g of magnesium sulfate, 0.045 g of manganese sulfate, 1 mL of Tween 80, and 0.5 g of cysteine ​​hydrochloride monohydrate were dissolved in 1 L of purified water. The mixture was deoxygenated by N2 substitution and dispensed. The mixture was then sterilized at 121°C for 15 minutes using moist heat.

[0066] Preparation of Bifidobacterium bifidum Bbifi-1 powder production medium: 20 g of anhydrous glucose, 40 g of soy peptone, 5 g of N-acetylglucosamine, 2 g of potassium dihydrogen phosphate, 2 g of disodium hydrogen phosphate, 0.1 g of magnesium sulfate, 0.045 g of manganese sulfate, 1 mL of Tween 80, and 0.5 g of cysteine ​​hydrochloride monohydrate were dissolved in 1 L of purified water. The mixture was deoxygenated by nitrogen substitution and dispensed. The mixture was then sterilized at 121°C for 15 minutes using moist heat.

[0067] The method for preparing the lyoprotectant is as follows. Preparation of lyoprotectant for Parabacteroides distasonis Pdist-1 (also used as a lyoprotectant for animal administration control): Solution A: 6g sucrose, 6g trehalose, 2g xylitol, 2g sorbitol, 44g purified water, sterilized at 115°C for 30 minutes. Solution B: 5g monosodium glutamate, 15g purified water, sterilized at 115°C for 20 minutes. Solution C: 4g sodium vitamin C, 16g purified water. Filter and sterilize for use.

[0068] Preparation of lyophilized protective agents for Lactobacillus fermentum Lferm-1, Lactobacillus salivarius Lsali-1, and Enterococcus avium Eaviu-1: Solution A: 8g sucrose, 8g trehalose, and 44g purified water, sterilized at 115°C for 30 minutes. Solution B: 2g monosodium glutamate, 2g arginine hydrochloride, and 16g purified water, sterilized at 115°C for 30 minutes. Solution C: 4g sodium vitamin C, and 16g purified water. Filter and sterilize for use.

[0069] 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°C for 30 minutes. Solution B: 2g arginine hydrochloride, 2g monosodium glutamate, 16g purified water, sterilized at 115°C for 30 minutes. Solution C: 4g sodium vitamin C, 16g purified water. Filter and sterilize for use.

[0070] Before use, all the components were mixed at a volume ratio of solution A:solution B:solution C = 6:2:2. The lyoprotectant administered in the animal tests was prepared by freeze-drying the prepared lyoprotectant, pulverizing it, and preparing a suspension with physiological saline.

[0071] Preparation of 0.1% Tween 80-PBS dilution: 3.58 g of disodium hydrogen phosphate dodecahydrate, 0.27 g of potassium dihydrogen phosphate, 8 g of sodium chloride, and 1 mL of Tween 80 were added to 1 L of boiling water and dissolved by stirring with a glass rod. 0.5 g of cysteine ​​hydrochloride monohydrate was added to the boiling solution. The Hungate apparatus was opened, and the solution was boiled again under N2 protection. After bubbling with N2 for 20 minutes, the solution was dispensed into anaerobic bottles that had been deoxygenated by passing N2 through. The bottles were stoppered, labeled, and sterilized at 121°C for 15 minutes.

[0072] Example 1: Isolation and identification of strains Fresh fecal samples were collected from several healthy human volunteers, and each fecal sample was processed independently. An appropriate amount of oxygen-free PBS was added to the fecal sample to obtain a mixture, which was then shaken to obtain a suspension. The suspension was filtered through gauze under N2 protection to obtain a filtrate. The filtrate was centrifuged at 10,000 rpm for 20 minutes, after which the supernatant was discarded and the precipitate was retained. An appropriate amount of oxygen-free PBS was added to the precipitate, and the bacterial cells were resuspended to obtain a suspension. An equal volume of 50% (v / v) oxygen-free glycerin aqueous solution was added to the suspension and thoroughly mixed to obtain a bacterial mixture sample. The sample was dispensed into sample tubes, which were then placed in a bag, vacuumed, and stored in a refrigerator at -80°C. Each frozen sample tube was thawed independently. 0.5 mL of the thawed sample was resuspended in 4.5 mL of oxygen-free PBS and shaken to obtain a bacterial suspension. Under anaerobic conditions, 0.5 mL of the bacterial suspension and 4.5 mL of anaerobic PBS were mixed uniformly by shaking and diluted. -6 The bacterial suspension was diluted tenfold to a dilution of 100%. The appropriate diluted bacterial suspension was homogenously mixed with YCFA liquid medium and then dispensed into a 384-well plate and incubated anaerobically at 37°C for one week. The bacterial suspension from the well where the bacteria had grown was inoculated into YCFA medium and incubated for 48 hours, after which the suspension was divided into two aliquots. One aliquot was detected by MALDI-TOF-MS, and the isolated strains were preliminarily classified into species. After confirming that the bacterial suspension contained only bacteria of a single genetic background (monoclonal strain), based on the mass spectrometry results, the other aliquot was inoculated into YCFA medium and incubated. One aliquot was subjected to 16S rDNA gene amplification and sequencing, and the other aliquot was added to 50% (v / v) glycerol solution at a 1:1 (volume ratio), mixed homogenously, and deposited.

[0073] The species of the isolated strains were further identified by aligning the 16S rDNA gene sequences obtained through sequencing with the NCBI Nucleotide database. Five strains with further confirmed species were selected for further experiments. Strain 1 had the highest sequence similarity (>99%) to one strain of Parabacteroides distasonis, and was therefore designated Parabacteroides distasonis Pdist-1 (abbreviated as Pdist-1). Strain 2 had the highest sequence similarity (100%) to one strain of Limosilactobacillus fermentum, and was therefore designated Limosilactobacillus fermentum Lferm-1 (abbreviated as Lferm-1). Strain 3 had the highest sequence similarity (100.00%) to one strain of Lactobacillus salivarius, which is also known as Ligilactobacillus salivarius and has the Latin name Ligilactobacillus salivarius, so strain 3 was named Lactobacillus salivarius Lsali-1 (abbreviated as Lsali-1). Strain 4 had the highest sequence similarity (100.00%) to one strain of Enterococcus avium, so strain 4 was named Enterococcus avium Eaviu-1 (abbreviated as Eaviu-1). Because strain 5 had the highest sequence similarity (99.86%) with one strain of Bifidobacterium bifidum, strain 5 was designated Bifidobacterium bifidum Bbifi-1 (abbreviated as Bbifi-1).

[0074] Parabacteroides distasonis Pdist-1, Limocillous Lactobacillus fermentum Lferm-1, and Enterococcus avium Eaviu-1 were inoculated into BF839 medium and cultured to observe their colony morphology. Lactobacillus salivarius Lsali-1 and Bifidobacterium bifidum Bbifi-1 were inoculated into triple-mix solid medium and cultured to observe their colony morphology. Frontal photographs of the colony morphology of the five bacterial strains are shown in Figure 1. Of these, A is a frontal photograph of the colony morphology of Parabacteroides distasonis Pdist-1, B is a frontal photograph of the colony morphology of Limocillous Lactobacillus fermentum Lferm-1, C is a frontal photograph of the colony morphology of Lactobacillus salivarius Lsali-1, D is a frontal photograph of the colony morphology of Enterococcus avium Eaviu-1, and E is a frontal photograph of the colony morphology of Bifidobacterium bifidum Bbifi-1. As can be seen from this, all five bacterial strains exhibited white, opaque, circular colonies with a protruding center and a smooth, moist surface.

[0075] Example 2: Whole genome analysis of strains The five strains obtained in Example 1 were each inoculated into a triple-mix liquid medium and cultured until the bacteria reached the late logarithmic growth stage. Total genomic DNA was extracted from each strain and whole-genome sequencing was performed using the Illumina high-throughput sequencing platform NovaSeq 6000. After genome sequence assembly and annotation, the protein sequences were entered into the Virulence Factor Databases (VFDB) for virulence factor analysis. The results showed that none of the five strains contained virulence factors.

[0076] The novelty of the five strains was analyzed using the average nucleotide identity (ANI) method. A whole-genome search was performed in GenBank, and the closest strains were compared using fastANI (v1.33). The two closest strains to the whole genome of Parabacteroides distasonis Pdist-1 are GCA_003462945.1 (ANI = 98.26%) and GCA_003459965.1 (ANI = 98.20%), respectively. The two closest strains to the whole genome of Limocilactobacillus fermentum Lferm-1 are GCA_003465085.1 (ANI = 99.32%) and GCA_024385625.1 (ANI = 99.29%), respectively. The two closest matched strains for the complete genome of Lactobacillus salivarius Lsali-1 are GCA_009863605.1 (ANI = 99.94%) and GCA_009866185.1 (ANI = 99.87%), respectively. The two closest matched strains for the complete genome of Enterococcus avium Eaviu-1 are GCA_018917545.1 (ANI = 98.75%) and GCA_018373135.1 (ANI = 98.62%), respectively. The two closest matched strains for the complete genome of Bifidobacterium bifidum Bbifi-1 are GCA_003466395.1 (ANI = 99.01%) and GCA_003437945.1 (ANI = 99.00%), respectively. As can be seen from this, the species classification performed in Example 1 is correct.

[0077] The strains Pdist-1, Eaviu-1, Lferm-1, Bbifi-1, and Lsali-1 isolated and cultured in this invention have been submitted for deposit at the China Typical Culture Collection (CCTCC), located at Wuhan University, Wuhan, China. The names, taxonomic designations, dates of deposit, dates of viability confirmation, and microbial accession numbers of the cultures are listed in Table 1.

[0078] JPEG2026508600000003.jpg82170

[0079] Example 3: Co-culture characteristics test between each strain The bacterial strains Pdist-1, Eaviu-1, Lferm-1, Bbifi-1, and Lsali-1 obtained in Example 1 were each activated and cultured until late logarithmic growth. A disposable sterile cotton swab was dipped in a bacterial suspension of one of the strains and drawn three parallel lines on BF839 solid medium. Then, a single line was drawn parallel to the first line with each of the remaining four strains, perpendicular to the first line. The drawn bacterial suspensions were then dried and cultured anaerobically for 48 hours until traces of the bacterial suspension were evident. The interaction between each of the five bacterial strains is shown in Figure 2. As can be seen, the absence of breakpoints at the crossover points between the strains indicates no growth inhibition between the strains.

[0080] Example 4: Aggregation ability of single bacteria and bacterial compositions Preparation of single-cell suspension: The strains Pdist-1, Eaviu-1, Lferm-1, Bbifi-1, and Lsali-1, and the control strain Lactobacillus rhamnosus GG (LGG, CICC6141, purchased from the China Industrial Microorganisms Depository) were inoculated into triple-mix liquid media at an initial inoculum of 2% and cultured anaerobically at 37°C until the late logarithmic growth phase. The cultured cells were washed three times by centrifugation with oxygen-free PBS. After resuspending the cells in an appropriate amount of PBS, the OD of the culture was measured. 600 (Measure the absorbance value of the bacterial solution at a wavelength of 600 nm) and measure the same OD of each single bacterial solution using PBS as a diluent. 600 The solution was diluted to a value of (0.5±0.1).

[0081] Preparation of bacterial composition suspension: Same OD as above 600 Equal volumes of Pdist-1 bacterial solution, Eaviu-1 bacterial solution, Lferm-1 bacterial solution, Bbifi-1 bacterial solution, and Lsali-1 bacterial solution diluted to the specified values ​​were mixed uniformly.

[0082] 15 mL of each diluted single bacterial suspension and mixed bacterial suspension was taken and dispensed into three tubes at 5 mL each for parallel testing. 600After leaving the mixture at 37°C for 24 hours, 1 mL of the upper layer bacterial suspension was aspirated and the OD 600 The value was measured again and A 24 The self-agglutination rate / co-agglutination rate = (1-A 24 / A0) × 100%, and the detection results are expressed as Mean ± SD (autoagglutination rate: agglutination rate between single bacterial strains, coagglutination rate: agglutination rate between different bacterial strains in a bacterial composition).

[0083] JPEG2026508600000004.jpg27170

[0084] Example 5: Antioxidant capacity testing of bacterial compositions Cultivation of bacterial composition: After activating each single bacterium, Pdist-1, Eaviu-1, Lferm-1, Bbifi-1, and Lsali-1, they were mixed and inoculated into 5 mL of triple mixed medium. The inoculation amount of each strain was 1% and the mixture was cultured anaerobically at 37°C for 24 hours.

[0085] JPEG2026508600000005.jpg38170

[0086] JPEG2026508600000006.jpg16170

[0087] The antioxidant capacity of the samples was measured using a bacterial strain total antioxidant capacity detection reagent kit according to the kit's instructions, and based on the above calibration curve. The total antioxidant capacity unit is μmol / mg prot.

[0088] The antioxidant capacity of the bacterial composition is 0.189 μmol / mg prot. The bacterial composition has a certain antioxidant capacity.

[0089] Example 6: Short-chain fatty acid production capacity of single bacteria and bacterial compositions Single bacteria: The control strains LGG, Pdist-1, Eaviu-1, Lferm-1, Bbifi-1, and Lsali-1 were inoculated into BF839 liquid medium at a 5% inoculum size and cultured anaerobically at 37°C for 24 hours.

[0090] Bacterial composition: Pdist-1, Eaviu-1, Lferm-1, Bbifi-1 and Lsali-1 were mixed at an inoculum amount of 1% each and inoculated into BF839 liquid medium, followed by anaerobically culturing at 37°C for 24 hours.

[0091] The fermentation broths of the single bacterium and the bacterial composition were each centrifuged, and 1 mL of the supernatant was aspirated. 10 μL of formic acid was added to the supernatant, and the mixture was left to stand at 4°C for 30 minutes to oxidize. The supernatant was then aspirated and filtered through a 0.22 μm filter for use. The short-chain fatty acid (SCFA) content of each filtrate was measured by gas chromatography. The relevant gas chromatography parameters were as follows: Instrument model: Agilent 7890A. Chromatography column model: DB-WAX UI (30 m × 0.32 mm × 0.25 μm) capillary column. Column temperature: Initial temperature 80°C for 0.5 minutes, then increased to 180°C at 5°C / min for 1 minute, and then increased to 220°C at 40°C / min for 4 minutes. Sample inlet temperature: 220°C. Detector temperature: 250°C. Carrier gas: hydrogen gas 30 mL / min, air 300 mL / min. Makeup gas flow rate: nitrogen gas 28.314 mL / min. Split flow ratio: 10:1. Sample injection volume: 1 μL. Sample injection detection was repeated four times for the same sample, and the average value was calculated. Acid production was calculated according to the formula: Actual acid production of the strain (ppm) = measured acid production value of the strain - background value of BF839 medium. Total SCFA production is the sum of the production amounts of acetic acid, propionic acid, butyric acid, isovaleric acid, and valeric acid.

[0092] Results: As shown in Table 2, the control strain LGG, the bacterial composition, and the single bacteria all have the ability to produce acetic acid. In addition to producing acetic acid, Pdist-1 can also produce propionic acid and isovaleric acid. The total SCFA production of Bbifi-1, Pdist-1, and the bacterial composition was significantly superior to that of LGG, while the total SCFA production of Lsali-1, Eaviu-1, and Lferm-1 was comparable to that of LGG.

[0093] JPEG2026508600000007.jpg231170

[0094] Example 7: Pathogen-inhibiting ability of single bacteria and bacterial compositions In this example, eight common pathogenic bacteria that can cause diarrhea were selected to detect their bacteriostatic potential. The origins of the pathogenic bacterial strains are as follows: Pseudomonas aeruginosa (CMCC(B)10104) was purchased from the China Food and Drug Administration. Shigella dysenteriae (CMCC(B)51252) was purchased from the China Food and Drug Administration. Staphylococcus aureus (CMCC(B)26003) was purchased from the China Food and Drug Administration. Escherichia coli (CMCC(B)44102) was purchased from the China Food and Drug Administration. Salmonella paratyphi B (CMCC(B)50094) was purchased from the China Food and Drug Administration. Yersinia enterocolitica CMCC(B)52204 was purchased from the China Food and Drug Administration. Vibrio parahaemolyticus (ATCC 17802) was purchased from the American Type Culture Collection. Clostridium difficile (CICC 22951) was purchased from the China Industrial Microorganisms Species Depository.

[0095] Single bacteria: Each single bacteria of the control strains LGG, Pdist-1, Eaviu-1, Lferm-1, Bbifi-1 and Lsali-1 was inoculated into a triple mixed liquid medium at a 5% inoculum amount and cultured anaerobically for 48 hours to obtain a fermentation broth.

[0096] Bacterial composition: Pdist-1, Eaviu-1, Lferm-1, Bbifi-1 and Lsali-1 were mixed at an inoculum amount of 1% each and inoculated into a triple mixed liquid medium, which was then anaerobically cultured for 48 hours to obtain a fermentation broth.

[0097] JPEG2026508600000008.jpg94170

[0098] Example 8: Testing the ability of single bacteria and bacterial compositions to adhere to Caco2 cells Single bacteria: Each single bacteria of the control strains LGG, Pdist-1, Eaviu-1, Lferm-1, Bbifi-1, and Lsali-1 was inoculated into a triple-mix liquid medium at an initial inoculum of 2% and cultured anaerobically at 37°C until the late logarithmic growth phase. After the culture, the bacterial solution was washed twice by centrifugation with sterile PBS (Wuhan Dr. De Biotechnology Co., Ltd., PYG0021), and then the bacterial strains were precipitated at 5 × 10 in DMEM (Thermo Fisher Scientific (China) Co., Ltd., C11995500BT) medium containing 10 v / v% FBS (Thermo Fisher Scientific (China) Co., Ltd., SH30084.03). 8 It was diluted to CFU / mL and ready for use.

[0099] Bacterial composition: The bacterial composition was obtained by mixing equal volumes of the diluted bacterial solutions of each of the single bacteria Pdist-1, Eaviu-1, Lferm-1, Bbifi-1, and Lsali-1.

[0100] Caco-2 cells (Shangcheng Beina Chuanglian Biotechnology Co., Ltd., 350769) are anchorage-dependent cells. Caco-2 cells were digested with trypsin cell digestion solution (Lanjieke Technology Co., Ltd., BL501A) preheated to 37°C. The digested Caco-2 cells were collected by centrifugation. The precipitate was diluted with DMEM medium containing 10% FBS (v / v). 5 × 10 4 Caco-2 cells were seeded into a 96-well plate at a density of CFU / well and cultured overnight in a carbon dioxide incubator at 37°C before use.

[0101] Each well of a 96-well plate was divided into two groups, and 100 μL of each diluted single bacterial and bacterial composition suspension was added to a 96-well cell culture plate containing Caco-2 cells. After sample addition, the 96-well cell culture plate was placed in a horizontal centrifuge and centrifuged at 1000 g for 1 minute. The corresponding wells were divided into two subgroups, one of which was incubated for 30 minutes and the other for 2 hours. After incubation, the wells were washed twice with sterile PBS to remove any unadhered bacterial cells. After washing, 50 μL of 0.25% trypsin cell digestion solution (Lanjieke Technology Co., Ltd., BL501A) was added to each well, and the wells were placed in a 37°C incubator to digest the cells. After the Caco-2 cells had digested and formed a spherical shape, 150 μL of DMEM medium was added to each well, and the wells were pipetted repeatedly for approximately 1 minute. After confirming the isolation of cells and bacterial strains by microscopic examination, 20 μL of the mixture was aspirated and diluted 10-fold in 0.1% Tween 80-PBS in a 96-well plate. The appropriate dilution gradient was poured onto the melted triple-mix solid medium and counted after 48 h of incubation at 37°C.

[0102] The results are shown in Figure 5. Panel A shows that after 30 minutes of adhesion of each bacterial strain to Caco-2 cells, the adhesion ability of Enterococcus avium Eaviu-1 and the bacterial composition was equivalent to that of LGG. Panel B shows the adhesion effect of each bacterial strain after 2 hours of adhesion. Bifidobacterium bifidum Bbifi-1 and limocylic Lactobacillus fermentum Lferm-1 had similar adhesion ability to LGG, while Lactobacillus salivarius Lsali-1, Enterococcus avium Eaviu-1, and the bacterial composition all had superior adhesion ability to LGG. The bacterial compositions have relatively good adhesion ability, allowing them to settle and grow more easily.

[0103] Example 9: Testing the therapeutic effect of bacterial compositions on mice with 5-fluorouracil (5-FU)-induced diarrhea Preparation of bacterial composition: Pdist-1, Lferm-1, Lsali-1, Eaviu-1, and Bbifi-1 were inoculated into the corresponding bacterial powder production medium and anaerobically cultured at 37°C and 90 rpm for 16–24 h to obtain primary seed solutions. The five primary seed solutions were then transferred to the corresponding bacterial powder production medium and anaerobically cultured at 37°C and 90 rpm for 10–15 h to obtain secondary seed solutions. Each of the five secondary seed solutions was pumped using a peristaltic pump into a fermenter containing the corresponding bacterial powder production medium, where fermentation was continued. After fermentation was stopped, each bacterial cell was collected by centrifugation. The corresponding lyoprotectant was added to each sludge in a weight ratio of 1:1 to 1:2, and the sludge was uniformly mixed to form an emulsion. Each sludge was freeze-dried and crushed to obtain bacterial powder. An appropriate amount of the crushed bacterial powder was used to measure the viable cell count. The bacterial powders of each strain were mixed at equal CFU ratios according to the viable cell count to obtain bacterial composition powders. Finally, the bacterial composition powders were prepared into bacterial suspensions using saline, and animal tests were conducted. The bacterial suspensions were diluted with saline to obtain bacterial compositions with different bacterial doses.

[0104] Test Method: (1) Experimental Design: Ninety-six SPF-grade male Balb / c mice (purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) weighing 22-24 g were housed in an SPF-grade animal breeding room. Based on their initial body weight, the mice were randomly divided into eight groups (12 mice per group): normal control group, model control group, positive control loperamide group, positive control intestinal fertilizer group, high-dose bacterial composition group, medium-high-dose bacterial composition group, medium-dose bacterial composition group, and low-dose bacterial composition group. The eight groups were administered saline (0.2 mL / mouse / day), lyoprotectant (0.2 mL / mouse / day), loperamide (purchased from Henan Zhongjie Pharmaceutical Co., Ltd., 20 mg / kg body weight / mouse / day), intestinal fertilizer (containing live Bacillus licheniformis bacteria, Northeast Pharmaceutical Group Co., Ltd., 2 × 10 8 CFU / animal / day), high-dose bacterial composition (1 × 10 9 CFU / animal / day), medium- and high-dose bacterial composition (1 × 10 8 CFU / animal / day), medium dose bacterial composition (1 × 10 7 CFU / animal / day) and low-dose bacterial composition (1 × 10 6The mice were intragastrically administered 5-FU (CFU / mouse / day) for 8 consecutive days from D1 to D8. Modeling began on D3. Mice in the normal control group received an abdominal injection of 0.2 mL of saline per mouse. Mice in the remaining groups received intraperitoneal injections of 30 mg / kg body weight of 5-FU (5-fluorouracil, purchased from Tianjin Jinyao Pharmaceutical Co., Ltd., 10 mL per tube, 0.25 g / 10 mL) for 4 consecutive days to induce the mouse CID model. The mice were then sacrificed on D9. The specific experimental groupings and administration regimens are shown in Table 3.

[0105] JPEG2026508600000009.jpg108170Note: 5-FU: 5-fluorouracil, CFU: colony forming unit, d: day, ip: intraperitoneal injection, ig: intragastric administration, QD: once daily.

[0106] During the experimental period, animals were weighed daily and general observations were recorded (including, but not limited to, external signs, behavior, breathing, gland secretions, and fecal status). After modeling, each animal's diarrhea status was also carefully monitored and scored. After the experiment, the animals were euthanized, and tissue samples from the middle colon were collected for qPCR analysis of genes related to inflammatory factors (TNF-α, IL-1β) and tight junction proteins (ZO-1, Occludin).

[0107] (2) Observation and scoring of diarrhea: The diarrhea scoring criteria were based on the diarrhea scoring method used by Kurita A et al. (Modified irinotecan hydrochloride (CPT-11) administration schedule improves induction of delayed-onset diarrhea in rats. Kurita A et al., Cancer Chemother Pharmacol. 2000;46(3):211-20). Mice were placed in mouse cages lined with clean filter paper, one per cage. 0 points: hard, normal feces; 1 point: slightly moist, slightly moist, or loose feces; 2 points: moderately moist, unformed feces, and perianal uncleanliness; 3 points: severe diarrhea, muddy, watery, and severe perianal uncleanliness. The feces of the mice were observed and scored daily during the experimental period. The total diarrhea score was the sum of the diarrhea scores for each day.

[0108] (3) qPCR detection: Total RNA was extracted from the colon tissue of each mouse group using the Trizol method, reverse-transcribed into cDNA, and stored at -20°C for further use. qRT-PCR was used to detect the relative transcription levels of mRNA encoding the pro-inflammatory factors IL-1β and TNF-α, as well as the tight junction proteins ZO-1 and Occludin-1 in the colon of each mouse group (primer sequences are shown in Table 7, with β-actin as the internal reference gene). The reaction program was: 1) 95°C for 3 min, 2) 95°C for 10 s, 60°C for 30 s, 95°C for 15 s, 60°C for 1 min (40 cycles in total), and 3) 95°C for 10 s. -ΔΔCT The data were analyzed using the method.

[0109] (4) Data statistics and analysis: Data such as body weight, diarrhea score, and pathological examination results were expressed as mean ± standard deviation (Mean ± SEM), and statistical analysis was performed by One-way ANOVA using SPSS statistical software 26.0.

[0110] Test results: (1) The diarrhea score results are shown in Table 4.

[0111] JPEG2026508600000010.jpg83170Note: All data are expressed as mean ± standard deviation (SEM). All groups are compared with the model control group. "*" indicates P<0.05, "**" indicates P<0.01, "***" indicates P<0.001, and "****" indicates P<0.0001.

[0112] After 5-FU induction, the model group had significantly higher D8 and D9 diarrhea scores than the normal control group, and all four doses of the bacterial composition could significantly improve the animals' diarrhea condition, with the D8, D9 diarrhea scores and total diarrhea score significantly reduced compared to the model control group. This shows that the bacterial composition of the present invention can improve 5-FU-induced diarrhea.

[0113] (2) The qPCR detection results are shown in Table 5.

[0114] JPEG2026508600000011.jpg55170Note: All data are expressed as mean ± standard deviation (SEM). All groups are compared with the model control group. "*" indicates P<0.05, "**" indicates P<0.01, "***" indicates P<0.001, and "****" indicates P<0.0001.

[0115] Compared with the normal control group, the model control group showed significantly increased relative mRNA transcription levels of TNF-α and IL-1β genes, and significantly decreased relative mRNA transcription levels of ZO-1 and Occludin genes. High-dose bacterial composition and loperamide significantly reduced the relative mRNA transcription level of TNF-α. High-, medium-high-, and medium-dose bacterial composition significantly reduced the relative mRNA transcription level of IL-1β. The high-, medium-, and low-dose groups significantly increased the relative mRNA transcription level of ZO-1, achieving the same effect of improving intestinal health as loperamide. The medium-high-, and medium-dose bacterial composition significantly increased the relative mRNA transcription level of Occludin, achieving the same effect of improving intestinal health.

[0116] In summary, the bacterial composition of the present invention can significantly reduce the diarrhea score in a 5-FU-induced CID mouse model, and can play a significant therapeutic role in diarrhea by reducing inflammatory factors and increasing tight junction protein expression.

[0117] Example 10: Therapeutic effect of bacterial composition on irinotecan (CPT-11)-induced diarrhea in mice The bacterial compositions used in Examples 10 to 12 were obtained by mixing five types of bacteria at equal CFUs according to the method in Example 9. The CFU of the bacterial compositions described in Examples 10 to 12 is the total CFU of the five types of bacteria.

[0118] Test Method: (1) Experimental Design: Seventy male Balb / c mice (purchased from Shanghai Jihui Experimental Animal Breeding Co., Ltd.) were housed in an SPF-grade animal breeding room. Each group (10 mice) was randomly assigned based on body weight. The group consisted of a normal control (no treatment), a model control (treatment regimen: 0.2 mL of lyoprotectant / mouse / day), and a positive control intestinal stimulant (treatment regimen: 2.5 × 10 Bacillus licheniformis). 8 CFU / animal / day), and the high-dose bacterial composition group (administration regimen: bacterial composition 1 × 10 9 CFU / animal / day), medium- and high-dose bacterial composition group (administration regimen: bacterial composition 1 × 10 8CFU / animal / day), bacterial composition medium dose group (administration regimen: bacterial composition 1 × 10 7 CFU / animal / day) and the bacterial composition low dose group (administration regimen: bacterial composition 1 × 10 6 The test was conducted over a period of 10 days, designated D1 to D10.

[0119] Mice in the normal control group received an abdominal injection of 0.2 mL of saline per mouse. Mice in the remaining groups received an abdominal injection of 85 mg / kg body weight of CPT-11. Mice were modeled starting on day 3 and modeled once daily from days 3 to 6 for 4 consecutive days. Mice were intragastrically administered once daily for 9 consecutive days and then sacrificed on day 10. The specific experimental groupings and dosing regimens are shown in Table 6.

[0120] JPEG2026508600000012.jpg161170Note: CPT-11: irinotecan hydrochloride, CFU: colony forming unit, d: day, ip: intraperitoneal injection, ig: intragastric administration, QD: once daily.

[0121] During the experimental period, animals were weighed daily and general observations were recorded (including, but not limited to, the animal's external signs, behavior, breathing, gland secretions, and fecal status). Furthermore, the diarrheal status of each animal after modeling was also carefully monitored and scored. At the end of the experiment, whole blood samples were collected from the abdominal vein of each animal, and routine blood tests were performed. After euthanasia, the animals were euthanized, and the intestines were removed and the length of the small intestine was measured. The spleens were removed and weighed, and the spleen index (spleen weight / body weight × 100) was calculated. Samples were collected from the proximal end of the cecum of five mice per group. The samples were quickly frozen in liquid nitrogen and then stored frozen at -80°C. The samples were used for qPCR detection of the mRNA expression levels of related genes, such as inflammatory factors (TNF-α, IL-1β, IL-22), tight junction proteins (ZO-1, Occludin), proapoptotic factors (Bax), and aquaporin (AQP8). The colorectal sections of the remaining five mice in each group were directly fixed in 10% formaldehyde solution and stained with HE before pathological examination.

[0122] (2) Observation and scoring of diarrhea: The criteria for scoring diarrhea and the procedure were the same as in Example 9.

[0123] (3) Routine blood tests: Whole blood samples were collected from the abdominal vein of each animal group. Routine blood tests were performed on the samples using a fully automated blood cell analyzer (Mindray).

[0124] (4) qPCR detection: Total RNA was extracted from the colon tissue of each mouse group using the Trizol method, reverse-transcribed into cDNA, and stored at -20°C for further use. qPCR was used to detect the relative mRNA transcription levels of the pro-inflammatory factors TNF-α and IL-1β, the anti-inflammatory factor IL-22, the tight junction proteins ZO-1 and Occludin, the pro-apoptotic factor Bax, and the aquaporin AQP8 in the colon of each mouse group (primer sequences are listed in Table 7, with β-actin as the internal reference gene). The reaction program was: 1) 95°C for 3 min, 2) 95°C for 10 s, 60°C for 30 s, 95°C for 15 s, 60°C for 1 min (40 cycles in total), and 3) 95°C for 10 s. -ΔΔCT The data were analyzed using the method.

[0125] JPEG2026508600000013.jpg186170

[0126] (5) Pathological detection of intestinal tissue: The colorectal sections were directly fixed in 10% formaldehyde solution and stained with HE before pathological examination.

[0127] (6) Data statistics and analysis: Data such as body weight, diarrhea score, and pathological examination results were expressed as mean ± standard deviation (Mean ± SEM), and statistical analysis was performed by One-way ANOVA using SPSS statistical software 26.0.

[0128] Test results: (1) After induction by intraperitoneal injection of irinotecan, the mice in the model group developed obvious clinical disease-like symptoms (mainly including diarrhea, weight loss, intestinal atrophy, reduced spleen index, and abnormalities in peripheral blood lymphocyte and neutrophil counts), suggesting that the CID model was successfully established.

[0129] The diarrhea scores and body weight measurement results are shown in Tables 8 and 9, respectively.

[0130] JPEG2026508600000014.jpg67170Note: All data are expressed as mean ± standard deviation (SEM). All groups are compared with the model control group. "*" indicates P<0.05, "**" indicates P<0.01, "***" indicates P<0.001, and "****" indicates P<0.0001.

[0131] High doses of the bacterial composition and the intestinal stimulant can significantly reduce the D10 diarrhea total score. Medium and high doses of the bacterial composition tend to reduce the D10 diarrhea total score. Compared with the model control group, the high-dose bacterial composition group showed a slightly higher reduction in the D10 diarrhea total score than the positive control intestinal stimulant group. Overall, the bacterial composition significantly improved the diarrheal condition of mice with CPT-11-induced diarrhea, and tended to be dose-dependent.

[0132] JPEG2026508600000015.jpg78170Note: All data are expressed as mean ± standard deviation (SEM). All groups are compared with the model control group. "*" indicates P<0.05, "**" indicates P<0.01, "***" indicates P<0.001, and "****" indicates P<0.0001.

[0133] After modeling, the model mice continued to lose weight until the end of the study. High, medium-high, and medium doses of the bacterial composition and the intestinal stimulant significantly alleviated the weight loss on D10 in the model mice, and the degree of alleviation was similar in the high-dose bacterial composition treatment group and the intestinal stimulant treatment group.

[0134] (2) The results for the intestine and spleen are shown in Table 10.

[0135] JPEG2026508600000016.jpg63170Note: All data are expressed as mean ± standard deviation (SEM). All groups are compared with the model control group. "*" indicates P<0.05, "**" indicates P<0.01, "***" indicates P<0.001, and "****" indicates P<0.0001.

[0136] The high-dose, medium-high-dose, low-dose, and intestinal regulation groups of the bacterial composition significantly improved small intestinal atrophy in the model mice. The high-dose, medium-high-dose, medium-dose, and intestinal regulation groups of the bacterial composition also significantly improved small intestinal swelling in the model mice. The medium-high, medium, and low doses of the bacterial composition and intestinal regulation groups all significantly improved splenic atrophy in the model mice.

[0137] (3) The results of regular blood tests are shown in Table 11.

[0138] High-dose, medium-high-dose bacterial composition significantly increased the lymphocyte content and reduced the neutrophil content in the peripheral blood of model mice. The intestinal stimulant tended to increase the lymphocyte content and significantly reduced the neutrophil content in the peripheral blood of model mice. The high-dose, medium-high-dose, medium-dose, low-dose, and intestinal stimulant groups of the bacterial composition significantly increased the percentage of lymphocytes and significantly reduced the percentage of neutrophils in the peripheral blood of model mice.

[0139] JPEG2026508600000017.jpg77170 Note: All data are expressed as mean ± standard deviation (SEM). All groups are compared with the model control group. * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001, and **** indicates P<0.0001. (4) The qPCR detection results are shown in Table 12.

[0140] JPEG2026508600000018.jpg119170Note: All data are expressed as mean ± standard deviation (SEM). All groups are compared with the model control group. "*" indicates P<0.05, and "**" indicates P<0.01.

[0141] The high-dose, medium-high-dose, and intestinal regulation groups significantly downregulated the mRNA expression of the inflammatory factor TNF-α in the colorectal region of model mice. High-dose bacterial composition significantly downregulated the mRNA expression of the inflammatory factor IL-1β. The medium-high-dose bacterial composition and intestinal regulation groups showed a tendency for a consistent downregulation compared to the model group. The high-dose bacterial composition and intestinal regulation groups showed a tendency for reduced mRNA expression of IL-22. The medium-dose and low-dose bacterial composition groups significantly upregulated the expression of the tight junction protein-associated gene ZO-1, while the medium-high and low-dose bacterial composition groups significantly upregulated the expression of the tight junction protein-associated gene Occludin. The high-dose bacterial composition group significantly reduced the mRNA expression of the cell apoptosis-related gene Bax and significantly enhanced the expression of the aquaporin AQP8 in model mice.

[0142] (5) The pathological detection results are shown in Table 13.

[0143] JPEG2026508600000019.jpg82170Note: All data are expressed as mean ± standard deviation (SEM). All groups are compared with the model control group. "*" indicates P<0.05, "**" indicates P<0.01, and "****" indicates P<0.0001.

[0144] The high-medium-dose bacterial composition group, the low-dose bacterial composition group, and the regulated intestinal diet group had significantly lower pathological scores than the model control group. The high-dose bacterial composition group and the regulated intestinal diet group tended to have reduced pathological scores compared to the model group.

[0145] In summary, the bacterial composition reduced CPT-11-induced diarrhea, reduced the mRNA expression of intestinal pro-inflammatory factors (TNF-α, IL-1β, IL-22) and pro-apoptotic factor (Bax), and enhanced the mRNA expression of tight junction proteins (ZO-1, Occludin) and aquaporin (AQP8). This significantly improved symptoms such as CPT-11-induced diarrhea, weight loss, small intestinal swelling, splenic atrophy, abnormalities in peripheral blood immune cell (lymphocyte and neutrophil) counts, and intestinal inflammation in mice.

[0146] Example 11: Therapeutic effect of bacterial composition on radiation enteritis in mice (1) Study design: Eighty male C57BL / 6 mice (purchased from Chengdu Yaokang Biotechnology Co., Ltd.) were housed in an SPF-grade animal breeding room. Based on body weight, the mice were randomly divided into groups of 10 mice each: a normal control group (administration regimen: saline 0.2 mL / mouse / day), a model control group (administration regimen: lyoprotectant 0.2 mL / mouse / day), a loperamide group (administration regimen: loperamide 15 mg / kg body weight), and an LGG (Lactobacillus rhamnosus GG, Shaanxi Zelang Biotechnology Co., Ltd.) group (administration regimen: LGG 1 × 10 9 CFU / animal / day), bacterial composition high dose group (administration regimen: bacterial composition 1 × 10 9 CFU / animal / day), medium- and high-dose bacterial composition group (administration regimen: bacterial composition 1 × 10 8 CFU / animal / day), bacterial composition medium dose group (administration regimen: bacterial composition 1 × 10 7 CFU / animal / day), bacterial composition low dose group (administration regimen: bacterial composition 1 × 10 6 The mice were divided into eight groups (CFU / mouse / day). Except for the normal control group, all mice in the remaining groups received a single whole abdominal X-ray irradiation at a dose of 11.5 Gy. The entire test period was 18 days, designated D1 to D18. Each group received intragastric administration once daily for 17 consecutive days. After 7 days of administration, the mice were irradiated on D8 and dissected on D18. The test grouping and administration regimen are shown in Table 14.

[0147] JPEG2026508600000020.jpg109170Note: CFU: colony forming unit, d: day, ip: intraperitoneal injection, ig: intragastric administration, QD: once a day.

[0148] During the experiment, the animals were weighed daily and general observations were recorded (including, but not limited to, the animals' external signs, behavior, breathing, gland secretions, and fecal status). After irradiation modeling, the diarrhea status of each animal was also observed and recorded, and scored. At the end of the experiment, the animals were euthanized, and the colon and rectum were directly fixed in 10% formaldehyde solution and stained with HE for pathological examination.

[0149] (2) Observation and scoring of diarrhea: The criteria for scoring diarrhea and the procedure were the same as in Example 9.

[0150] (3) Pathological detection of intestinal tissue: The colorectal sections were directly fixed in 10% formaldehyde solution and stained with HE before pathological examination.

[0151] (4) Data statistics and analysis: Data such as body weight, diarrhea score, and pathological examination results were expressed as mean ± standard deviation (Mean ± SEM), and statistical analysis was performed by One-way ANOVA using SPSS statistical software 26.0.

[0152] Test results: After intraperitoneal irradiation, animals in the model control group developed obvious symptoms such as diarrhea, weight loss, intestinal mucosal damage, and inflammatory infiltration, suggesting that the mouse radiation enteritis model was successfully established.

[0153] (1) The diarrhea results are shown in Table 15.

[0154] JPEG2026508600000021.jpg64170Note: All data are expressed as mean ± standard deviation (SEM). All groups are compared with the model control group. "*" indicates P<0.05, "**" indicates P<0.01, "***" indicates P<0.001, "****" indicates P<0.0001, and " / " indicates not applicable.

[0155] Compared with the model control group, the high-dose, medium-dose, low-dose, loperamide, and LGG bacterial composition groups all showed significant improvements in the D17 diarrhea total scores of mice. The medium- and high-dose bacterial composition groups showed a tendency for the D17 diarrhea total score to be reduced.

[0156] (2) Body weight results are shown in Table 16.

[0157] JPEG2026508600000022.jpg68170Note: All data are expressed as mean ± standard deviation (SEM). All groups are compared with the model control group. "*" indicates P<0.05, "**" indicates P<0.01, "***" indicates P<0.001, and "****" indicates P<0.0001.

[0158] Compared with the model control group, the high-dose, low-dose, and LGG bacterial composition groups significantly improved the weight loss of mice on D14. The low-dose bacterial composition significantly improved the weight loss of model mice on D17. High-dose and medium-dose bacterial composition, loperamide, and LGG all tended to restore the weight of model mice on D17.

[0159] (3) The pathological results of the colorectum are shown in Table 17.

[0160] JPEG2026508600000023.jpg71170Note: All data are expressed as mean ± standard deviation (SEM). All groups are compared with the model control group. "*" indicates P<0.05, "**" indicates P<0.01, "***" indicates P<0.001, and "****" indicates P<0.0001.

[0161] Compared with the model control group, the high-dose bacterial composition group, the low-dose bacterial composition group, and the LGG group all showed significant improvements in pathological scores. The high-medium-dose bacterial composition group, the medium-dose bacterial composition group, and the loperamide group showed a tendency for improvements in pathological scores in the colorectal region.

[0162] In summary, the bacterial composition has a significant therapeutic effect on mice with radiation enteritis induced by whole abdominal X-ray irradiation, and can improve the severity of diarrhea and weight loss, and alleviate the severity of intestinal lesions.

[0163] Example 12: Therapeutic effect of bacterial composition on 5-FU-induced tumor-bearing CID mice Experimental design: Fifty male BALB / c mice (purchased from Chengdu Yaokang Biotechnology Co., Ltd.) were housed in an SPF-grade animal breeding room. Each mouse was subcutaneously inoculated with CT26 cells (purchased from Beijing Beina Biotechnology Co., Ltd.) in the right flank. The inoculation dose was 5 × 10 6 The inoculation volume is 0.1 mL. The tumor volume is 100-150 mm. 3 Once the tumors reached a certain size, 42 mice were screened for further experiments. The 42 mice were randomly divided into seven groups of six mice each according to tumor volume. The seven groups were the normal control group (administration regimen: 0.2 mL of lyoprotectant / mouse / day), the model control group (administration regimen: 0.2 mL of lyoprotectant / mouse / day), the positive control loperamide group (administration regimen: 20 mg of loperamide / kg body weight), and the intestinal stimulating group (administration regimen: 2 x 10 Bacillus licheniformis). 8 CFU / animal / day), bacterial composition high dose group (administration regimen: bacterial composition 1 × 10 9 CFU / animal / day), bacterial composition medium dose group (administration regimen: bacterial composition 1 × 10 8 CFU / animal / day) and the bacterial composition low dose group (administration regimen: bacterial composition 1 × 10 7The day of grouping was designated day 1 (D1), and administration began on day 1 (D1) and was administered intragastrically once daily (QD) for 9 consecutive days. On days 3, 4, 5, and 6 of administration, the normal control group received an intraperitoneal injection of 0.2 mL of saline per mouse, while the remaining groups received intraperitoneal injections of 5-FU (50 mg / kg body weight, 10 mL / kg body weight) for modeling. Compared to the normal control group, tumor volume in each group was reduced after 5-FU injection. The model control group experienced severe diarrhea and weight loss, as well as significant abnormalities in routine blood test indicators, suggesting successful model construction.

[0164] JPEG2026508600000024.jpg16170

[0165] JPEG2026508600000025.jpg108170Note: ig indicates oral intragastric administration and QD indicates once daily.

[0166] Test results: Diarrhea results are shown in Table 19.

[0167] JPEG2026508600000026.jpg70170Note: All data are expressed as mean ± standard deviation (SEM). When the diarrhea scores of each group were compared with the model control group, "*" indicates P<0.05 and "**" indicates P<0.01.

[0168] Compared with the normal control group, the model control group began to experience diarrhea on D6 and the severity of diarrhea worsened over time. Compared with the model control group, the loperamide group had significantly reduced diarrhea scores and total diarrhea scores on D8, D9, and the low-dose bacterial composition significantly reduced diarrhea scores and total diarrhea scores on D8, D9, and D9.

[0169] The results of routine blood tests are shown in Table 20.

[0170] JPEG2026508600000027.jpg70170Note: All data are expressed as mean ± standard deviation (SEM). Compared with the model control group, the regular blood test data of each group indicates P<0.05 and ** indicates P<0.01.

[0171] Compared with the normal control group, the model control group had significantly reduced hematocrit, white blood cell count, lymphocyte count, and platelet count. Compared with the model control group, the medium-dose bacterial composition group, low-dose group, and regulated intestinal diet group had significantly increased red blood cell count and hematocrit, and the low-dose bacterial composition group had increased lymphocyte count and platelet count.

[0172] In summary, all three doses of the bacterial composition tend to improve diarrhea and routine blood test indicators, among which the low-dose bacterial composition and loperamide can significantly improve diarrhea, and the low-dose bacterial composition has the ability to significantly improve routine blood test indicators (lymphocyte count, red blood cell count, and platelet count).

[0173] As is clear from common general knowledge, the present invention can be realized in other embodiments without departing from the spirit or essential features thereof. Therefore, the above-disclosed embodiments are merely illustrative and not restrictive in all respects. All modifications within the scope of the present invention or within a range equivalent to the present invention are included in the present invention.

Claims

1. A probiotic composition, wherein the probiotics include a fifth probiotic and any two, three, or four of a first probiotic, a second probiotic, a third probiotic, and a fourth probiotic; The first probiotic is selected from Bifidobacterium bifidum, a descendant strain of Bifidobacterium bifidum, a clonal strain of Bifidobacterium bifidum, or a pure culture of Bifidobacterium bifidum, and the microorganism deposit number of Bifidobacterium bifidum is CCTCC NO: M2023349; the second probiotic is selected from Enterococcus avium, a descendant strain of Enterococcus avium, a clonal strain of Enterococcus avium, or a pure culture of Enterococcus avium, and the Enterococcus avium microorganism deposit number is CCTCC NO: M2023350; the third probiotic is selected from Lactobacillus salivarius, a descendant strain of Lactobacillus salivarius, a clone strain of Lactobacillus salivarius, or a pure culture of Lactobacillus salivarius, and the microorganism deposit number of Lactobacillus salivarius is CCTCC NO: M2023348; The fourth probiotic is selected from Rimosilactobacillus fermentum, a descendant strain of Rimosilactobacillus fermentum, a clonal strain of Rimosilactobacillus fermentum, or a pure culture of Rimosilactobacillus fermentum, and the microorganism deposit number of Rimosilactobacillus fermentum is CCTCC NO: M2023352; and The fifth probiotic is selected from Parabacteroides distasonis, a descendant strain of Parabacteroides distasonis, a clonal strain of Parabacteroides distasonis, or a pure culture of Parabacteroides distasonis, and the microorganism deposit number of Parabacteroides distasonis is CCTCC NO: M20222033. Probiotic composition.

2. A microecological composition having the probiotic composition of claim 1 as an active ingredient.

3. The microecological composition of claim 2, characterized in that the composition further comprises an auxiliary material selected from the group consisting of a freeze-drying protection agent, a bacterial culture medium, a food additive, a carrier or auxiliary material acceptable for health foods, and a pharmaceutically acceptable carrier or auxiliary material.

4. The micro-eco-composition according to claim 2, wherein the ratio of the content of any two kinds of bacteria in the micro-eco-composition is 100 CFU: 1-10,000 CFU when calculated based on the number of viable bacteria.

5. Use of the probiotic composition of claim 1 or the microecological composition of any one of claims 2 to 4 in the manufacture of a product for use alone or in combination with other microbial preparations and / or drugs to improve the health status of a subject, comprising Improving the health status of the subject includes inhibiting the growth 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 a subject; treating, preventing and / or delaying tissue damage, disease or subconditions 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; improving antioxidant capacity in the intestinal tract of a subject; Treating, preventing and / or delaying diarrhea caused by antineoplastic drugs; Treating, preventing and / or delaying intestinal inflammation caused by anti-tumor drugs; Treating, preventing and / or delaying weight loss caused by antineoplastic drugs; Treating, preventing and / or delaying shortening of small intestine length caused by antineoplastic agents; Treating, preventing and / or delaying antineoplastic drug-induced increases in small intestinal thickness; Treating, preventing and / or delaying intestinal damage caused by anti-tumor drugs; Treating, preventing and / or delaying the decrease in spleen weight to body weight ratio caused by antineoplastic agents; Treating, preventing and / or delaying tissue damage, disease or sub-health conditions caused by elevated expression of any one, any two, any three or four of TNF-α, IL-1β, IL-22 and Bax; Treating, preventing and / or delaying tissue damage, disease or sub-health conditions caused by reducing the expression levels of any one, any two or all of ZO-1, Occludin and AQP8; Treating, preventing and / or delaying diarrhea caused by radiation therapy; Treating, preventing and / or delaying weight loss caused by radiation therapy; Treating, preventing, and / or delaying intestinal damage caused by radiation therapy; Treating, preventing and / or delaying intestinal inflammation caused by radiation therapy; and Treating, preventing and / or delaying the reduction in hematocrit, white blood cell count, lymphocyte count and / or platelet count caused by antineoplastic agents; Selected from use.

6. The use according to claim 5, characterized in that the product is a food, a health food or a medicine.

7. The use according to claim 5, characterized in that the subject is selected from humans and mice.

8. The use according to claim 5, characterized in that the antitumor drug is selected from chemotherapeutic drugs, targeted drugs and immune checkpoint inhibitors.

9. The use according to claim 8, characterized in that the antineoplastic agent is selected from the group consisting of epirubicin, actinomycin D, doxorubicin, daunorubicin, paclitaxel, docetaxel, albumin paclitaxel, cisplatin, carboplatin, nedaplatin, oxaliplatin, lobaplatin, cyclophosphamide, nitrogen mustard, carmustine, camptothecin, hydroxycamptothecin, topotecan, irinotecan, capecitabine, gemcitabine, methotrexate, 5-fluorouracil, pemetrexed, cytarabine, apatinib, axitinib, cabozantinib, sorafenib, sunitinib, nivolumab, pembrolizumab, and ipilimumab.

10. A method for preventing, treating or delaying intestinal disease, said method comprising administering to a subject a therapeutically effective amount of a microecological composition according to any one of claims 2 to 4, The intestinal disease is Intestinal diseases caused by any one, any two, any three, any four, any five, any six, any seven, or eight of the following bacteria: Pseudomonas aeruginosa, Shigella dysenteriae, Staphylococcus aureus, Escherichia coli, Salmonella paratyphi B, Yersinia enterocolitica, Vibrio parahaemolyticus, or Clostridium difficile; oxidative damage in the intestinal tract, diarrhea caused by antitumor drugs, Intestinal inflammation caused by antitumor drugs, Intestinal damage caused by antitumor drugs, Diarrhea caused by radiation therapy, intestinal inflammation caused by radiation therapy, and Intestinal damage caused by radiation therapy, Selected from method.

11. 11. The method of claim 10, wherein the subject is selected from a human and a mouse.

12. 11. The method of claim 10, wherein administering to the subject is selected from oral administration, intraperitoneal injection, and intragastric administration.

13. Calculated based on the total bacterial content in the probiotic composition, the therapeutically effective amount is 10 mg / day. ６～１２ The method of claim 10, characterized in that the cells are CFU.

14. 11. The method of claim 10, wherein the antitumor drug is selected from epirubicin, actinomycin D, doxorubicin, daunorubicin, paclitaxel, docetaxel, albumin paclitaxel, cisplatin, carboplatin, nedaplatin, oxaliplatin, lobaplatin, cyclophosphamide, nitrogen mustard, carmustine, camptothecin, hydroxycamptothecin, topotecan, irinotecan, capecitabine, gemcitabine, methotrexate, 5-fluorouracil, pemetrexed, cytarabine, apatinib, axitinib, cabozantinib, sorafenib, sunitinib, nivolumab, pembrolizumab, and ipilimumab.

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