Novel bifidobacterium longum and use thereof

Abstract

The present invention relates to novel Bifidobacterium longum SNUG50180, SNUG50432, and SNUG50527, and, more specifically, to: Bifidobacterium longum SNUG50180 having the effects of improving intestinal health and treating or preventing intestinal diseases, Bifidobacterium longum SNUG50432 having an effect of promoting immune anticancer activity and Bifidobacterium longum SNUG50527 having an effect of treating or preventing cancer, which are Bifidobacterium longum strains isolated from the feces of Korean individuals who have not taken antibiotics for at least 6 months; and uses thereof.

Description

Novel Bifidobacterium longum and its uses

[0001] The present invention relates to novel Bifidobacterium longum SNUG50180, SNUG50432 and SNUG50527 and their uses.

[0002]

[0003] A healthy human body is home to a symbiotic mix of microorganisms, numbering ten times the number of human cells. These microorganisms form a uniquely structured gut microbiota, tailored to each individual and specific disease. A healthy gut microbiota interacts intricately with the body, performing diverse functions such as maintaining intestinal homeostasis through the regulation of metabolism and immune responses. Microorganisms and their products, which possess antimicrobial and enzymatic activity and contribute to the balance of gut microbiota, are called probiotics. To be considered probiotics, they must be native to the human gut, non-pathogenic, non-toxic, and resistant to degradation during transit through the intestines. Furthermore, they must be sensitive to antibiotics used to prevent infection, but not harbor antibiotic-resistant plasmids. They must also be resistant to the acids, enzymes, and bile found in the gut environment. Probiotics can be supplied to humans or animals in the form of dried cells or fermented products to improve intestinal flora. In addition, efforts are being made to develop new anticancer treatments with fewer side effects and superior efficacy that can maintain balance by regulating the function of the immune system using prebiotics or overcome the shortcomings of currently used anticancer treatments.

[0004] Meanwhile, a correlation between an imbalance in the composition of the gut microbiota and an abnormal intestinal immune response has recently been discovered. Using next-generation sequencing, the gut microbiota of patients with inflammatory bowel disease and ulcerative colitis was analyzed. Compared to healthy controls, the patient group showed an imbalance in the gut microbiota, with decreased diversity and a predominance of Proteobacteria. Furthermore, a decrease in Ruminococcaceae and Lachnospiracaea, which produce short-chain fatty acids, and an increase in Enterobacteriaceae and Fusobacteriaceae, which are proinflammatory gut bacteria, were observed. In addition, inflammatory bowel diseases such as Crohn's disease show a Th1-like immune response, such as an increase in inflammatory cytokines such as interferon gamma (IFN-r) and tumor necrosis factor alpha (TNF-a), while ulcerative colitis shows a Th2 response, such as an increase in IL-13 along with an increase in the expression of Th17 cells.

[0005] Bifidobacterium species are among the earliest bacteria to colonize the intestinal microbiota of infants, and their stability and probiotic efficacy have been well-documented. In particular, Bifidobacterium longum is known to protect the intestines from infectious pathogens, strengthen barrier function, and enhance immunity.

[0006] The present inventors, noting that the health-promoting effects of probiotics are strain-specific rather than general characteristics of the genus and species, screened various Bifidobacterium strains to identify novel strains with excellent immune-enhancing effects, increased T cell and NK cell activity, changes in intestinal microflora, combined effects with immuno-oncology drugs, and tumor growth inhibition effects, and confirmed the excellent therapeutic effects of the strains, thereby completing the present invention.

[0007]

[0008] The present invention aims to provide a novel Bifidobacterium longum having an effect of improving intestinal health and treating or preventing intestinal diseases.

[0009] The purpose of the present invention is to provide a novel Bifidobacterium longum having tumor growth inhibition effect through combined use with an immuno-oncology agent.

[0010] The purpose of the present invention is to provide a novel Bifidobacterium longum having a therapeutic or preventive effect on cancer.

[0011] The present invention aims to provide a pharmaceutical composition for treating or preventing intestinal diseases and a food composition for improving intestinal health, which includes Bifidobacterium longum.

[0012] The present invention aims to provide a pharmaceutical composition for treating or preventing cancer, comprising Bifidobacterium longum.

[0013] 1. A Bifidobacterium longum strain isolated from the stool of a Korean who has not taken antibiotics for more than 6 months, wherein the Bifidobacterium longum is any one selected from the group consisting of Bifidobacterium longum SNUG50180 with the deposit number KCTC 15655BP, Bifidobacterium longum SNUG50432 with the deposit number KCTC 15656BP, and Bifidobacterium longum SNUG50527 with the deposit number KCTC 15657BP.

[0014] 2. In the above 1, Bifidobacterium longum SNUG50180 is a Bifidobacterium longum strain having the effect of improving intestinal health, treating or preventing intestinal diseases, or suppressing immune activity.

[0015] 3. In the above 1, Bifidobacterium longum SNUG50432 is a Bifidobacterium longum strain having a cancer treatment or prevention effect by promoting immune anticancer activity.

[0016] 4. In the above 1, Bifidobacterium longum SNUG50527 is a Bifidobacterium longum strain having a cancer treatment or prevention effect or an immune activity increasing effect.

[0017] 5. A pharmaceutical composition for treating or preventing inflammatory bowel disease, comprising at least one selected from the group consisting of a Bifidobacterium longum strain, a culture solution of the strain, a lysate of the strain, and an extract of the strain among any one of the above 1 to 4.

[0018] 6. A food composition for improving intestinal health, comprising at least one selected from the group consisting of a Bifidobacterium longum strain, a culture solution of the strain, a lysate of the strain, and an extract of the strain, of any one of the above 1 to 4.

[0019] 7. A pharmaceutical composition for treating or preventing cancer, comprising at least one selected from the group consisting of a Bifidobacterium longum strain, a culture solution of the strain, a lysate of the strain, and an extract of the strain among any one of the above 1 to 4.

[0020] 8. In the above 7, the cancer is any one selected from the group consisting of lung cancer, stomach cancer, liver cancer, pancreatic cancer, skin cancer, uterine cancer, ovarian cancer, colon cancer, small intestine cancer, thyroid cancer, parathyroid cancer, urethral cancer, bladder cancer, penile cancer, prostate cancer, multiple myeloma, leukemia, lymphoma, kidney cancer, renal cell carcinoma, and pelvic tumor. A pharmaceutical composition for treating or preventing cancer.

[0021] 9. A pharmaceutical composition for treating or preventing cancer, further comprising an immunotherapy agent, in the above 7.

[0022] 10. In the above 9, the immuno-oncology agent is a pharmaceutical composition for treating or preventing cancer, wherein the pharmaceutical composition is any one selected from the group consisting of an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti-CD28 antibody, and an anti-TIM-3 antibody.

[0023]

[0024] Bifidobacterium longum SNUG50180 of the present invention has the effect of improving intestinal health and treating or preventing intestinal diseases.

[0025] Bifidobacterium longum SNUG50180 of the present invention can reduce the secretion of inflammatory cytokines and suppress immune activity by inhibiting inflammation and T cell activity.

[0026] The Bifidobacterium longum SNUG50180 of the present invention can strengthen the tight junction of the intestinal wall of the large intestine, and can treat or improve colitis by suppressing weight loss and reduction in the length of the large intestine caused by colitis.

[0027] Bifidobacterium longum SNUG50432 of the present invention has a combined effect of promoting the activity of an immunotherapy agent when used in combination with an immunotherapy agent, and can effectively suppress tumor growth to treat cancer.

[0028] Bifidobacterium longum SNUG50527 of the present invention has an effect in treating or preventing cancer.

[0029] Bifidobacterium longum SNUG50527 of the present invention can promote the production of inflammatory cytokines and enhance immune activity by promoting T cell and NK cell activity.

[0030] Bifidobacterium longum SNUG50527 of the present invention can treat cancer by inhibiting tumor growth.

[0031]

[0032] Figure 1 shows the results of an in vitro experiment to confirm the immunomodulatory effect of Bifidobacterium longum SNUG50180, SNUG50432, and SNUG50527, in which the amount of cytokines was measured after treating mouse spleen cells with the strains.

[0033] Figures 2a and 2b show changes in the thymus organ indices of mice when administered Bifidobacterium longum SNUG50180 and SNUG50527.

[0034] Figure 2c shows the proliferation of spleen cells in mice administered with Bifidobacterium longum SNUG50180 and SNUG50527.

[0035] Figures 2d and 2e show the peritoneal macrophage phagocytic activity of mice administered with Bifidobacterium longum SNUG50180 and SNUG50527.

[0036] Figure 2f shows the metabolic activity of peritoneal macrophages in mice administered with Bifidobacterium longum SNUG50180 and SNUG50527.

[0037] Figures 3a to 3f show the analysis of T cell activity when Bifidobacterium longum SNUG50180 and SNUG50527 were administered.

[0038] Figures 3g to 3i show changes in the expression of CD274+ in CD11c+MHCII+ dendritic cells when Bifidobacterium longum SNUG50180 and SNUG50527 were administered.

[0039] Figures 4a to 4e show the analysis of the expression levels of cytokines and antimicrobial peptides in mouse ileum tissue when Bifidobacterium longum SNUG50180 and SNUG50527 were administered.

[0040] Figures 5a and 5b show changes in the intestinal flora when Bifidobacterium longum SNUG50180 and SNUG50527 were administered.

[0041] Figures 5c and 5d show changes in the intestinal flora of the Bifidobacterium longum SNUG50180 administration group.

[0042] Figures 5e and 5f show changes in the intestinal flora of the Bifidobacterium longum SNUG50527 administration group.

[0043] Figure 6a shows a schematic diagram of an experiment confirming the tumor growth inhibitory effect by administering Bifidobacterium longum.

[0044] Figures 6b and 6c show the tumor growth inhibitory effect in mice administered Bifidobacterium longum SNUG50180 and SNUG50527.

[0045] Figures 6d to 6f show the results of flow cytometry analysis of tumor tissues from mice administered Bifidobacterium longum SNUG50180 and SNUG50527.

[0046] Figures 6g to 6h show the results of transcriptome analysis of tumor tissues of mice administered Bifidobacterium longum SNUG50180 and SNUG50527.

[0047] Figures 7a and 7b show the results of body weight and disease activity index analysis according to enteritis in mice administered Bifidobacterium longum SNUG50180 and SNUG50527.

[0048] Figures 7c and 7d show histopathological changes in the colon of mice administered Bifidobacterium longum SNUG50180 and SNUG50527.

[0049] Figures 7e to 7i show the expression levels of inflammatory cytokines and chemokines and intestinal tight junction markers in mice administered Bifidobacterium longum SNUG50180 and SNUG50527.

[0050] Figure 8a shows the effect of promoting immunotherapy activity and the resulting tumor growth inhibition effect in mice administered Bifidobacterium longum SNUG50432.

[0051] Figures 8b to 8h show the results of confirming the infiltrating immune cells in tumor tissues of mice administered Bifidobacterium longum SNUG50432.

[0052]

[0053] The present invention provides novel Bifidobacterium longum SNUG50180, SNUG50432 and SNUG50527 and a composition comprising each strain.

[0054] Bifidobacterium longum SNUG50180, SNUG50432, and SNUG50527 were isolated from the intestinal flora of Koreans. The intestinal flora isolation samples were provided by stool from healthy individuals who had not taken antibiotics for 6 months.

[0055] Bifidobacterium longum SNUG50180, SNUG50432, and SNUG50527 were deposited at the Biological Resource Center (KCTC), Korea Research Institute of Bioscience and Biotechnology on October 23, 2023 (accession numbers of each strain: KCTC 15655BP, KCTC 15656BP, KCTC 15657BP).

[0056] Bifidobacterium longum SNUG50180 has been shown to be effective in treating or preventing intestinal diseases and improving intestinal health. Bifidobacterium longum SNUG50180 may treat or prevent inflammatory bowel disease.

[0057] Administration of Bifidobacterium longum SNUG50180 can restore weight loss and colon length reduction caused by colitis.

[0058] In one embodiment, a mouse model of colitis administered with Bifidobacterium longum SNUG50180 showed suppressed weight loss and colon length reduction, and significantly improved disease activity index (DAI) compared to a positive control group for colitis.

[0059] Administration of Bifidobacterium longum SNUG50180 can alleviate the symptoms of inflammatory bowel disease in vivo and alleviate the infiltration of inflammatory cells and destruction of the mucosal tissue layer in colonic tissue.

[0060] In one embodiment, colon tissue administered with Bifidobacterium longum SNUG50180 showed recovery of infiltration of inflammatory cells and destruction of mucosal tissue compared to the colitis positive control group.

[0061] Administration of Bifidobacterium longum SNUG50180 may help alleviate inflammatory bowel disease by reducing the expression of inflammation-related genes in intestinal tissue and strengthening tight junctions between intestinal epithelial cells. The decreased expression of inflammation-related genes may lead to a decrease in the expression of inflammatory cytokines and chemokines. Increased expression of Zo-1, which is associated with tight junctions between epithelial cells, may strengthen tight junctions between intestinal epithelial cells. Tight junctions between intestinal epithelial cells may prevent the leakage of harmful microorganisms and toxins, which can cause inflammation.

[0062] In one example, the expression of inflammatory cytokines TNFα, IFNγ, IL-17 and inflammatory chemokine CCL-2 was significantly reduced in colon tissue of mice administered Bifidobacterium longum SNUG50180.

[0063] In one example, expression of Zo-1 was significantly increased in colon tissue of mice administered Bifidobacterium longum SNUG50180.

[0064] Bifidobacterium longum SNUG50180 can regulate excessive immune activity induced by external factors.

[0065] Administration of Bifidobacterium longum SNUG50180 can modulate the activity of adaptive immunity, resulting in a decrease in the activity of CD25 or CD69+ T cells. Furthermore, administration of the strain can modulate excessive immune activity in the intestine by suppressing IL-4 expression.

[0066] In one example, in mice administered Bifidobacterium longum SNUG50180, the expression of CD25 and CD69 was reduced in CD4 T cells and CD8 T cells in the mesenteric lymph nodes.

[0067] In one example, the expression of IL-4 was significantly reduced in the ileum tissue of mice administered Bifidobacterium longum SNUG50180.

[0068] Administration of Bifidobacterium longum SNUG50180 can suppress immune activity and alleviate inflammation by reducing the expression of inflammatory cytokines.

[0069] In one example, mouse splenocytes treated with Bifidobacterium longum SNUG50180 showed a decrease in the expression of inflammatory cytokines IFNγ, TNFα, IL-4, IL-6, and IL-17A.

[0070] Administration of Bifidobacterium longum SNUG50180 may increase thymus organ markers and increase spleen cell proliferation by activating leukocytes.

[0071] Administration of Bifidobacterium longum SNUG50180 can alter the composition of the intestinal flora, specifically increasing beneficial bacteria and reducing harmful bacteria. In one example, the alpha diversity index using Fisher's index of the cecum of mice administered Bifidobacterium longum SNUG50180 significantly increased.

[0072] In one example, in the intestinal flora composition of mice administered Bifidobacterium longum SNUG50180, the beneficial bacteria Parabacteroides distasonis and Bacteroides caccae increased, and the harmful bacteria Prevotella, Bacteroides acidifaciens, Bacteroides fragilis, and Bacteroides ovatus decreased compared to the control group.

[0073] The present invention provides a pharmaceutical composition for treating or preventing intestinal diseases, comprising Bifidobacterium longum SNUG50180. The pharmaceutical composition for treating or preventing intestinal diseases of the present invention may comprise, as an active ingredient, at least one selected from the group consisting of the Bifidobacterium longum SNUG50180 strain, a culture solution of the strain, a lysate of the strain, and an extract of the strain.

[0074] The pharmaceutical composition for treating or preventing intestinal diseases of the present invention may be prepared in the form of capsules, tablets, granules, injections, ointments, or powders. The pharmaceutical composition may further comprise a combination of one or more of the strain of the present invention or a culture solution, lysate, or extract thereof, along with any other optional ingredients used in the art, such as carriers, excipients, diluents, preservatives, binders, solubilizers, pigments, or fragrances.

[0075] The present invention provides a food composition for improving intestinal health comprising Bifidobacterium longum SNUG50180. The food composition for improving intestinal health of the present invention may comprise, as an active ingredient, at least one selected from the group consisting of a Bifidobacterium longum SNUG50180 strain, a culture solution of the strain, a lysate of the strain, and an extract of the strain.

[0076] The food composition for improving intestinal health of the present invention can be manufactured in the form of various foods, such as beverages, tea, powders, granules, tablets, capsules, etc. The food composition may additionally include any other ingredients used in the relevant field, such as vitamins, electrolytes, flavoring agents, coloring agents, pH regulators, stabilizers, and preservatives, in addition to a combination of one or more of the strain of the present invention or its culture solution, lysate, and extract.

[0077] Bifidobacterium longum SNUG50432, when used in combination with immunotherapy, has a synergistic effect that enhances the activity of immunotherapy, thereby increasing the tumor growth inhibition efficacy. Co-administration of Bifidobacterium longum SNUG50432 with immunotherapy can further maximize the cancer treatment or prevention effect. Co-administration of this strain with immunotherapy can inhibit tumor growth and reduce the overall tumor volume.

[0078] In one embodiment, when Bifidobacterium longum SNUG50432 was co-administered with an immunotherapy anti-PD-1 antibody, tumor growth by cell line transplantation was significantly suppressed and tumor volume was reduced compared to when the immunotherapy or Bifidobacterium longum SNUG50432 was administered alone.

[0079] Co-administration of Bifidobacterium longum SNUG50432 and immunotherapy can suppress tumor growth by changing the proportion of immune cells within tumor tissue.

[0080] In one example, the proportion of CD4 T cells and NK cells expressing IFNγ, the expression levels of IFNγ and NK1.1 in NK cells, and the proportion of M1 macrophages increased in tumor tissues of mice co-administered with Bifidobacterium longum SNUG50432 and an immunotherapy anti-PD-1 antibody.

[0081] The present invention provides a pharmaceutical composition for treating or preventing cancer, comprising Bifidobacterium longum SNUG50432. The pharmaceutical composition for treating or preventing cancer may comprise, as an active ingredient, at least one selected from the group consisting of a Bifidobacterium longum SNUG50432 strain, a culture solution of the strain, a lysate of the strain, and an extract of the strain.

[0082] The pharmaceutical composition for treating or preventing cancer comprising Bifidobacterium longum SNUG50432 of the present invention may be administered together with an immunotherapy agent. Immunoassay agents that may be administered together with the pharmaceutical composition for treating or preventing cancer comprising Bifidobacterium longum SNUG50432 of the present invention include immune checkpoint inhibitors, immunocytotherapy agents, and antibody-drug conjugates. The immunotherapy agent may be, for example, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti-CD28 antibody, or an anti-TIM-3 antibody.

[0083] The pharmaceutical composition for treating or preventing cancer comprising Bifidobacterium longum SNUG50432 of the present invention may be prepared in the form of a capsule, tablet, granule, injection, ointment, or powder. The pharmaceutical composition may further comprise any other ingredients used in the art, such as a carrier, excipient, diluent, preservative, binder, solubilizer, colorant, or fragrance, in addition to a combination of one or more of the strain of the present invention or a culture solution, lysate, or extract thereof.

[0084] In the pharmaceutical composition for treating or preventing cancer of the present invention, the cancer may be, for example, lung cancer, stomach cancer, liver cancer, pancreatic cancer, skin cancer, uterine cancer, ovarian cancer, colon cancer, small intestine cancer, thyroid cancer, parathyroid cancer, urethral cancer, bladder cancer, penile cancer, prostate cancer, multiple myeloma, leukemia, lymphoma, kidney cancer, renal cell carcinoma, or vesicular tumor, but is not limited thereto.

[0085] Bifidobacterium longum SNUG50527 has cancer-preventive and therapeutic properties. Administration of Bifidobacterium longum SNUG50527 can inhibit tumor growth and reduce overall tumor volume.

[0086] In one example, when Bifidobacterium longum SNUG50527 was administered to mice, tumor growth caused by cell line transplantation was significantly inhibited and tumor volume was reduced.

[0087] Administration of Bifidobacterium longum SNUG50527 can inhibit tumor growth by changing the proportion of immune cells within tumor tissue.

[0088] In one example, the proportion of total CD8 T cells and CD8 T cells expressing IFNγ, the IFNγ expression levels of CD4 and CD8 T cells, and the NK1.1 expression levels of NK cells were increased in tumor tissues of mice administered with Bifidobacterium longum SNUG50527.

[0089] Bifidobacterium longum SNUG50527 can increase immune activity.

[0090] Administration of Bifidobacterium longum SNUG50527 can increase the activity of adaptive immunity and increase T cell activity. Administration of Bifidobacterium longum SNUG50527 can reduce the expression of CD274, which suppresses T cell activity, and thus increase the expression of CD25 and CD69, which indicate T cell activity.

[0091] In one example, in mice administered Bifidobacterium longum SNUG50527, CD274 expression on dendritic cells in mesenteric lymph nodes was decreased, and CD25 and CD69 expression on CD4 T cells and CD8 T cells was increased.

[0092] Administration of Bifidobacterium longum SNUG50527 can induce immune activation by increasing the expression of inflammatory cytokines.

[0093] In one example, the expression of inflammatory cytokines IFNγ, TNFα, IL-17A and antimicrobial peptide Reg3γ was significantly increased in the ileum tissue of mice administered Bifidobacterium longum SNUG50527.

[0094] In one example, the expression of inflammatory cytokines IFNγ, TNFα, IL-4, IL-6, IL-10, and IL-17A was significantly increased in splenocytes of mice administered Bifidobacterium longum SNUG50527.

[0095] Administration of Bifidobacterium longum SNUG50527 may increase the activity of innate immunity, increase thymus organ markers, and increase the proliferation of splenocytes.

[0096] Administration of Bifidobacterium longum SNUG50527 may increase phagocytic activity and increase the activity of macrophages.

[0097] In one example, the phagocytic activity of peritoneal macrophages in mice administered Bifidobacterium longum SNUG50527 was significantly increased.

[0098] In one embodiment, the metabolic activity of peritoneal macrophages in mice administered Bifidobacterium longum SNUG50527 was increased.

[0099] Administration of Bifidobacterium longum SNUG50527 can change the composition of the intestinal flora, specifically increasing beneficial bacteria and reducing harmful bacteria.

[0100] In one example, in the intestinal flora composition of mice administered Bifidobacterium longum SNUG50527, beneficial bacteria such as Parabacteroides distasonis and Bacteroides caccae increased, and harmful bacteria such as Akkermansia muciniphila, Ruminococcus gnavus, and Oscillospira decreased.

[0101] The present invention provides a pharmaceutical composition for treating or preventing cancer, comprising Bifidobacterium longum SNUG50527. The pharmaceutical composition for treating or preventing cancer may comprise, as an active ingredient, at least one selected from the group consisting of a Bifidobacterium longum SNUG50527 strain, a culture solution of the strain, a lysate of the strain, and an extract of the strain.

[0102] The pharmaceutical composition for treating or preventing cancer of the present invention may be prepared in the form of a capsule, tablet, granule, injection, ointment, or powder. The pharmaceutical composition may further comprise a combination of one or more of the strain of the present invention or a culture solution, lysate, or extract thereof, along with any other optional ingredients used in the art, such as a carrier, excipient, diluent, preservative, binder, solubilizer, colorant, or fragrance.

[0103] In the pharmaceutical composition for treating or preventing cancer of the present invention, the cancer may be, for example, lung cancer, stomach cancer, liver cancer, pancreatic cancer, skin cancer, uterine cancer, ovarian cancer, colon cancer, small intestine cancer, thyroid cancer, parathyroid cancer, urethral cancer, bladder cancer, penile cancer, prostate cancer, multiple myeloma, leukemia, lymphoma, kidney cancer, renal cell carcinoma, or vesicular tumor, but is not limited thereto.

[0104] Hereinafter, the present invention will be described in more detail with examples.

[0105]

[0106] Example

[0107] Example 1. Isolation and identification of a novel Bifidobacterium longum strain.

[0108] A fecal sample was provided from a Korean who had not taken antibiotics for 6 months, and a novel strain of Bifidobacterium longum was isolated from the sample's flora. The strain showed strong resistance to high concentrations of bile salts (2%) and low acidity (pH 4). Bifidobacterium longum strains SNUG50180, SNUG50432, and SNUG50527 were cultured on Lactobacilli MRS Agar (BD Difco) supplemented with 0.05% L-cysteine ​​hydrochloride at 37°C for 24 h under anaerobic conditions. The cells were collected by centrifugation at 3000 rpm and washed twice with 1X phosphate-buffered saline (PBS). After measuring the bacterial concentration, the bacterial colonies containing 1X PBS containing 20% ​​glycerol were stored at -80°C.

[0109] To identify the strain, genomic DNA of the strain was extracted using the Wizard genomic purification kit (Promega), and then PCR analysis was performed using the 27F / 1492R primers (SEQ ID NOs. 1 and 2) in Table 1, targeting the 16S rRNA gene. After purification using the QIAquick PCR purification kit (Qiagen), the base sequence was analyzed using an ABI3711 automatic sequencer (Thermo Fisher Scientific).

[0110] Sequence number: Forward (27F primer) 1 Reverse (1492R primer) 2

[0111] Using the analyzed sequence information, multiple comparisons were performed using CJ Bioscience's EzBioCloud program (http: / www.ezbiocloud.net / identify) to finally identify the strains. The new strains were named Bifidobacterium longum SNUG50180, SNUG50432, and SNUG50527, respectively, and deposited with the Biological Resource Center of the Korea Research Institute of Bioscience and Biotechnology on October 23, 2023.

[0112]

[0113] Example 2. Confirmation of the immunomodulatory effect of Bifidobacterium longum in vitro

[0114] An in vitro experiment was conducted to confirm the immunomodulatory effect of the Bifidobacterium longum strain of the present invention. Mouse splenocytes were cultured in RPMI 1640 (BD Difco) containing 10% FBS (BD Difco) and 1% penicillin / streptomycin. 2x10 cultured splenocytes 5 Place the dogs in a 96-well plate, and each strain has 4x10 6 colony-forming unit (CFU), PBS or 4x10 as control 6 Escherichia coli (E. coli) was added at 1 CFU. After treatment with 1 μg / ml of anti-CD3 antibody (OKT3, Thermo Fisher Scientific), which activates T cells, the cells were incubated at 37 °C for 72 hours.

[0115] The results of measuring the amount of each cytokine in the supernatant of cultured cells after strain treatment are shown in Figures 1a to 1f. Bifidobacterium longum SNUG50527 significantly increased the expression of inflammatory cytokines IFNγ, TNFα, IL-4, IL-6, IL-10, and IL-17A in the splenocyte co-culture group. Bifidobacterium longum SNUG50180 showed a decrease in the expression of inflammatory cytokines IFNγ, TNFα, IL-4, IL-6, and IL-17A compared to the SNUG50432 and SNUG50527 strains.

[0116]

[0117] Example 3. Confirmation of the immunomodulatory effect of Bifidobacterium longum in vivo

[0118] (1) Preparation for animal testing

[0119] An animal experiment was conducted to confirm in vivo immunomodulation by administration of Bifidobacterium longum (Seoul National University IACUC Approval Number: SNU-210614-4-2). Seven-week-old female SPF (specific pathogen-free) C57 / BL6 mice were divided into groups of 10 each and housed in cages equipped with a 12-h light / dark cycle and a constant temperature and humidity controller, with free access to food and water.

[0120] Bifidobacterium SNUG50527 or SNUG50180 strains were cultured in MRS liquid medium at 37°C under anaerobic conditions until reaching the exponential phase. The supernatant was removed and the strains were resuspended in PBS at a concentration of 1x10 9 After diluting to CFU / ml, it was administered to mice in the experimental group. After administering 200 μl daily via oral gavage for 21 days, the mice were necropsied.

[0121] (2) Confirmation of the effect of adaptive immune regulation

[0122] The thymus of the mouse was obtained, weighed, and the organ index, which is the ratio of the body weight of the mouse, was determined. As a result, it was confirmed that the thymus organ index of the Bifidobacterium-administered group significantly increased (Fig. 2a and Fig. 2b). In addition, the spleen of the mouse was obtained, washed twice with RPMI medium, and then 1x10 6 The cells were placed in a 96-well plate, treated with Concanavalin A (ConA), which activates T cells, and cultured at 37°C for 44 hours. Afterwards, 20 μl of WST-8 solution was added and cultured for an additional 4 hours. The absorbance at 470 nm was measured, and an increase in the proliferation of spleen cells in the Bifidobacterium-administered group was confirmed (Fig. 2c).

[0123] (3) Confirmation of innate immune regulation effect

[0124] After injecting 10 ml of PBS into the peritoneal cavity of the mouse obtained in Example 3 (1) above, the PBS was recovered and centrifuged at 400 x g for 5 minutes to obtain peritoneal macrophages. 2 x 10 5Peritoneal macrophages from dogs were placed in 96-well plates with DMEM medium and cultured at 37°C for 2 hours. The medium was removed, and DMEM containing 200 ng / ml LPS (Lipopolysaccharide) was treated and cultured for an additional 2 hours at 37°C. Phagocytic activity was compared using a Vibrant phagocytosis assay kit (Thermo Fisher Scientific) and a microplate reader (TECAN). As a result, it was confirmed that the phagocytic activity of peritoneal macrophages from mice administered Bifidobacterium longum SNUG50527 significantly increased (Fig. 2d and 2e). In addition, to confirm changes in the metabolic activity of peritoneal macrophages, LPS-treated peritoneal macrophages were cultured for 24 hours and then treated with 20 μl of WST-8 (cell counting kit 8, Abcam) solution. After an additional 4-hour incubation, the absorbance at 470 nm was measured using a microplate reader. As a result, an increase in macrophage metabolic activity was observed in mice administered SNUG50527 (Fig. 2f), indicating that oral administration of Bifidobacterium longum increases macrophage activity.

[0125] (4) Confirmation of changes in T cell activity

[0126] The mesenteric lymph nodes of the mice obtained in Example 3(1) were obtained and the cells were separated using a 100 μm cell strainer (SPL) and RPMI medium. Fc receptors were blocked with anti-mouse CD16 / 32 antibody (BD Bioscience), and T cells were stained using anti-CD3ε, anti-CD4, anti-CD8, anti-CD25, and anti-CD69 antibodies (BioLegend), and myeloid cells were stained using anti-F4 / 80, anti-CD11b, anti-CD11c, anti-CD80, anti-CD206, anti-CD274, and anti-mouse IA / IE, anti-Gr1 antibodies (BioLegend). Immune cell characteristics were analyzed using an LSR Fortessa X-20 flow cytometer, BD FACS Diva Software (BD Bioscience), and FlowJo software (BD Bioscience). The expression of CD25 and CD69, which indicate T cell activation, increased in CD4 T cells and CD8 T cells of the SNUG50527-treated group, and decreased in CD4 T cells and CD8 T cells of the SNUG50180-treated group (Figures 3a to 3f). In addition, the expression of CD274, which suppresses T cell activation, was significantly reduced in dendritic cells of the SNUG50527-treated group (Figures 3g to 3i). This indicates that administration of Bifidobacterium longum SNUG50527 increases the activity of CD4 and CD8 T cells by inhibiting the expression of CD274 on dendritic cells, thereby increasing the activity of adaptive immunity, whereas administration of Bifidobacterium longum SNUG50180 decreases the activity of adaptive immunity by inhibiting the activity of CD4 and CD8 T cells.

[0127] (5) Confirmation of changes in small intestinal tissue immune indicators due to administration of Bifidobacterium longum

[0128] The ileum tissue of the mouse of the above Example 3(1) was obtained, and some of the ileum samples were preserved in RNAlater (Thermo Fisher Scientific) solution and frozen at -80°C. An easy-spin total RNA extraction kit (Intron) was used to extract RNA, and the extracted RNA was immediately synthesized into cDNA using a high capacity RNA-to-cDNA kit (Thermo Fisher Scientific) in an equal amount. The gene expression level was analyzed using the roter-gene SYBR green PCR kit (Qiagen), and the primers in Table 2 below were used to target the inflammatory cytokines IFNγ, TNFα, IL-17A, the adaptive immune activation cytokine IL-4, and the antimicrobial peptide Reg3γ gene. The results of correcting the expression level with the GAPDH housekeeping gene are shown in Figures 4a to 4e.

[0129] Sequence number target gene forward 3 GAPDH reverse 4 GAPDH forward 5 IL-4 reverse 6 IL-4 forward 7 IL-17A reverse 8 IL-17A forward 9 IFNγ reverse 10 IFNγ forward 11 TNFα reverse 12 TNFα forward 13 Reg3γ reverse 14 Reg3γ

[0130] Compared to the control group, the expression of inflammatory cytokines IFNγ, TNFα, IL-17A and antimicrobial peptide Reg3γ significantly increased in the SNUG50527 administration group, while the expression of IL-4, which induces adaptive immune activity, significantly decreased in the SNUG50180 administration group. This suggests that administration of the strain SNUG50527 can induce intestinal immune activity by increasing the expression of inflammatory cytokines in the ileum tissue, and administration of SNUG50180 can suppress excessive intestinal immune activity by suppressing the adaptive immune activity of the ileum tissue through suppression of IL-4 expression.

[0131] (6) Analysis of changes in intestinal flora caused by Bifidobacterium longum

[0132] The cecal contents of the mice in Example 3 (1) above were obtained and frozen at -80°C. The frozen samples were subjected to total genomic DNA extraction using the QIAamp Fast DNA Stool Mini Kit (Qiagen), and large-scale base sequence data were generated targeting the V4-V5 region of the bacterial 16S rRNA gene. The Kraken2 pipeline (jhu.edu) was used to confirm the entire genetic information of the intestinal bacteria and to determine the structure of the mouse fecal flora. In addition, the alpha diversity index was calculated using Bracken (jhu.edu). The analysis results showed that the alpha diversity index using Fisher's index significantly increased in the SNUG50180-administered group, and that the administration of each Bifidobacterium longum changed the composition of the intestinal flora (Figs. 5a and 5b). Specifically, it suggests that beneficial bacteria were increased, harmful bacteria were reduced, and the structure of the intestinal flora was regulated.

[0133] In the Bifidobacterium longum administration group, the beneficial bacteria Parabacteroides distasonis and Bacteroides caccae commonly increased, and in the SNUG50180 administration group, the harmful bacteria Prevotella, Bacteroides acidifaciens, Bacteroides fragilis, and Bacteroides ovatus decreased compared to the control group (Fig. 5c and 5d). In the SNUG50527 administration group, the harmful bacteria Akkermansia muciniphila, Ruminococcus gnavus, and Oscillospira were reduced compared to the control group (Fig. 5e and Fig. 5f), indicating that administration of each Bifidobacterium longum can change the intestinal flora as described above and control the immune activity of the host.

[0134]

[0135] Example 4. Confirmation of the tumor growth inhibitory effect of Bifidobacterium longum.

[0136] (1) Confirmation of tumor growth inhibition effect in vivo

[0137] To confirm the tumor growth inhibitory effect of administration of Bifidobacterium longum, an animal experiment was conducted (Seoul National University IACUC Approval No.: SNU-220218-4-1). Specifically, MC38 cell line (Kerafast), a mouse colon cancer cell line, was cultured in DMEM medium, and 2x10 mice were injected with the same breeding environment as in Example 3 (1). 5Each mouse was injected subcutaneously. Mice in the experimental groups were administered each Bifidobacterium longum strain or PBS in the same manner as in Example 3. Tumor volumes were measured and recorded twice a week using calipers starting on the 7th day of administration, and on the 21st day of administration, the experiment was terminated after measuring the tumor volume and the mice were necropsied (Fig. 6a). It was confirmed that tumor growth was significantly inhibited in the SNUG50527 administration group (Figs. 6b and 6c), indicating that administration of Bifidobacterium longum SNUG50527 can inhibit tumor growth in vivo.

[0138] (2) Tumor tissue flow cytometry analysis

[0139] Tumor tissue (200-300 mg) was finely chopped and placed in PBS containing 400 μg / ml Collagenase (Sigma-Aldrich) and 4 μg / ml DNase I, and incubated with shaking at 37°C for 40 min. The tissue was filtered through a 100 μm strainer, centrifuged at 200 × g for 5 min, and treated with PBS containing 1% ethylenediaminetetraacetic acid (EDTA; Invitrogen). After washing with RPMI medium, leukocytes were separated by a concentration gradient using a 40% / 80% Percoll (Cytiva) solution. The isolated leukocytes were cultured for 4 hours in RPMI medium containing 50 ng / ml of Phorbol 12-myristate 13-acetate (PMA), 1 μg / ml of Ionomycin (Sigma-Aldrich), and 5 ng / ml of Brefeldin A (BioLegend). T cells were then stained with FVS510 (BD Bioscience), anti-CD16 / 32, anti-CD3, anti-CD45, anti-CD4, anti-CD8, anti-CD25, anti-Foxp3, anti-NK1.1, and anti-IFNγ antibodies (BioLegend), and bone marrow cells were stained with anti-CD11b, anti-CD11c, anti-CD80, anti-CD206, anti-CD274, and anti-mouse IA / IE antibodies (BioLegend), and flow cytometry analysis was performed. The analysis results confirmed that the proportion of CD8 T cells, the proportion of CD8 T cells expressing IFNγ, the IFNγ expression levels of CD4 and CD8 T cells, and the NK1.1 expression level of NK cells all increased in the tumor tissues of SNUG50527-administered mice (Figs. 6d to 6f). This indicates that oral administration of Bifidobacterium longum SNUG50527 can inhibit tumor growth by changing the proportion and characteristics of immune cells in tumor tissues.

[0140] (3) RNA sequence analysis of tumor tissue

[0141] RNA sequencing was performed on tumor tissues from four mice in each group. Specifically, a portion of mouse tumor tissues was treated with RNA later solution (Thermo Fisher Scientific), followed by RNA isolation, DNA and rRNA removal, RNA fragmentation, reverse transcription, and PCR amplification. mRNA libraries were constructed using the TruSeq Standard mRNA Library Prep Kit (Illumina) and NovaSeq6000 (Illumina), and sequence analysis was performed. Sequence information was preprocessed using the fastp pipeline, the HISAT2 pipeline (Kim et al., 2019), and SAMtools, and then analyzed using the Kraken2, featureCounts (Liao et al., 2013), and DESeq2 (Love et al., 2014) packages. The analysis results confirmed that in the tumor tissues of mice administered with Bifidobacterium longum SNUG50527, the expression of seven genes, including Tirap and IL-33, increased, and the expression of eight genes, including IL-27ra, Klrb1a, Ampd2, and Pdcd1, decreased (Figs. 6g to 6h). This indicates that oral administration of Bifidobacterium longum SNUG50527 inhibits tumor growth by altering the expression of immune-related genes in tumor tissues.

[0142]

[0143] Example 5. Confirmation of the in vivo inflammatory bowel disease improvement effect of Bifidobacterium longum.

[0144] (1) Confirmation of the effect of improving inflammatory bowel disease in vivo

[0145] To confirm the effect of Bifidobacterium longum administration on improving inflammatory bowel disease in vivo, an animal experiment was conducted (Seoul National University IACUC Approval Number: SNU-160602-9). Specifically, to establish a mouse model of colitis, 8-week-old female C57BL / 6 mice were divided into groups of 8 each and housed in cages equipped with a 12-h light / dark cycle and a constant temperature and humidity controller, with free access to food and water. To induce colitis, 2% dextran sulfate sodium (DSS, MP Biomedicals, USA) was dissolved in drinking water and administered for a total of 7 days. The normal control group was supplied with drinking water without added DSS, and the negative control group was administered 200 μl of PBS orally daily.

[0146] Mice in the experimental group were administered Bifidobacterium longum SNUG50527 and SNUG50180 strains. Each strain was cultured in MRS liquid medium at 37°C under anaerobic conditions until reaching the exponential phase. The supernatant was removed and the strain was resuspended in PBS at a concentration of 1x10 9 The solution was diluted to 10 CFU / ml. 200 μl was administered daily via oral gavage from the start of DSS administration until the end of the experiment. DSS administration was discontinued after 7 days, and body weight changes in the control and experimental mice were measured daily for 9 days while DSS-induced enteritis was induced. The experiment was terminated on the 9th day after the start of DSS administration, and the mice were necropsied.

[0147] Changes in mouse body weight and disease activity index (DAI) due to acute enteritis are shown in Fig. 7a. Compared to the normal control group, the DSS control group showed a significant decrease in body weight, confirming the establishment of a mouse model of colitis. Meanwhile, the SNUG50180 administration group showed significantly improved effects in body weight loss and disease activity index compared to the DSS positive control group. In addition, changes in intestinal length were measured, and it was confirmed that the SNUG50180 administration group showed a significant improvement in the extent of colon length reduction compared to the DSS control group (Fig. 7b).

[0148] (2) Observation of histopathological changes in the colon due to administration of Bifidobacterium longum

[0149] Hematoxylin & eosin staining was performed to observe histopathological changes in the colon caused by administration of a single strain of Bifidobacterium longum. After autopsy, the distal segment of the colon was fixed in 10% neutral formalin solution, and paraffin tissue specimens were sectioned into 5-μm-thick sections, stained with H&E reagent, and observed under a light microscope. The histological scores of the distal segments were then evaluated using Panoramic Viewer (3DHISTECH, Ltd). The histological scores were scored from 0 to 12 for four categories: epithelial loss, intestinal crypts, goblet cell depletion, and inflammatory cell infiltration. As a result, in the DSS positive control group, infiltration of inflammatory cells in the colon tissue and significant destruction of the mucosal tissue were observed compared to the normal control group, whereas histologically, it was confirmed that inflammation was alleviated in the SNUG50180-administered group compared to the DSS positive control group (Figs. 7c and 7d). This suggests that administration of Bifidobacterium longum SNUG50180 can alleviate symptoms of inflammatory bowel disease in vivo.

[0150] (3) Confirmation of the ability to control intestinal inflammation by administering Bifidobacterium longum

[0151] In order to confirm the ability of Bifidobacterium longum to control intestinal inflammation, cDNA was synthesized from the colon tissue of mice at the end of the experiment in Example 5 (1) as in Example 3 (5), and then the changes in gene expression levels were analyzed using the primers in Tables 2 and 3.

[0152] Sequence number target gene forward 15CCL-2 reverse 16CCL-2 forward 17ZO-1 reverse 18ZO-1

[0153] As a result, compared to the DSS positive control group, the expression of inflammatory cytokines TNFα, IFNγ, IL-17 and inflammatory chemokine CCL-2 was significantly reduced in the intestinal tissue of the SNUG50180 administration group, and the expression of Zo-1 associated with tight junctions between intestinal epithelial cells was significantly increased (Figs. 7e to 7i). This suggests that administration of Bifidobacterium longum SNUG50180 helped alleviate inflammatory bowel disease in vivo by reducing the expression of inflammation-related genes in intestinal tissue and strengthening tight junctions between intestinal epithelial cells.

[0154]

[0155] Example 6. Confirmation of the tumor growth inhibition effect of combined administration of Bifidobacterium longum and immunotherapy.

[0156] (1) Confirmation of tumor growth inhibition effect by combined use of Bifidobacterium longum and immunotherapy (in vivo)

[0157] To confirm the tumor growth inhibition effect of the combined use of Bifidobacterium longum and immunotherapy, an animal experiment was conducted (Seoul National University IACUC Approval No.: SNU-220218-4-1). Specifically, MC38 cell line (Kerafast), a mouse colon cancer cell line, was cultured in DMEM medium and administered to mice in the same breeding environment as in Example 3 (2x10 5Each mouse was injected subcutaneously. Afterwards, the mice in the experimental group were administered Bifidobacterium longum SNUG50432 or PBS orally daily as in Example 3, and simultaneously, 100 μg / 100 μl of an immunotherapy anti-PD-1 antibody or 100 μg / 100 μl of IgG2a were injected intraperitoneally on days 10, 13, and 17 after MC38 transplantation. Tumor volumes were measured twice a week using a caliper starting on day 7 after the start of administration, and on day 21 after the start of administration, the experiment was terminated and the mice were necropsied. As a result of the experiment, it was confirmed that tumor growth was significantly inhibited in the group administered with SNUG50432 and anti-PD-1 combination (Fig. 8a). This suggests that tumor growth can be inhibited in vivo through combination administration of an immunotherapy and Bifidobacterium longum SNUG50432.

[0158] (2) Tumor tissue flow cytometry analysis

[0159] Flow cytometry analysis was performed as in Example 4(2), and it was confirmed that the proportion of CD4 T cells and NK cells expressing IFNγ, the IFNγ and NK1.1 expression levels of NK cells, and the proportion of M1 macrophages increased in the tumor tissues of mice co-administered with anti-PD-1 and SNUG50432 (Figs. 8b to 8h). This indicates that co-administration of an immunotherapy and Bifidobacterium longum SNUG50432 inhibits tumor growth by changing the proportion and characteristics of immune cells in tumor tissues.

[0160]

[0161] Attached below are the deposit certificates and translations of the deposit certificates for Bifidobacterium longum SNUG50180, SNUG50432 and SNUG50527.

[0162]

[0163]

[0164]

[0165]

[0166]

[0167]

Claims

1. Bifidobacterium longum strain isolated from the stool of a Korean who had not taken antibiotics for more than 6 months. The above Bifidobacterium longum is any one selected from the group consisting of Bifidobacterium longum SNUG50180 with the deposit number KCTC 15655BP, Bifidobacterium longum SNUG50432 with the deposit number KCTC 15656BP, and Bifidobacterium longum SNUG50527 with the deposit number KCTC 15657BP. Bifidobacterium longum strain.

2. In claim 1, the Bifidobacterium longum SNUG50180 is a Bifidobacterium longum strain having the effect of improving intestinal health, treating or preventing intestinal diseases, or suppressing immune activity.

3. In claim 1, the Bifidobacterium longum SNUG50432 is a Bifidobacterium longum strain having a cancer treatment or prevention effect by promoting immune anticancer activity.

4. In claim 1, the Bifidobacterium longum SNUG50527 is a Bifidobacterium longum strain having a cancer treatment or prevention effect, or an immune activity increasing effect.

5. A pharmaceutical composition for treating or preventing inflammatory bowel disease, comprising at least one selected from the group consisting of a Bifidobacterium longum strain according to any one of claims 1 to 4, a culture solution of the strain, a lysate of the strain, and an extract of the strain.

6. A food composition for improving intestinal health, comprising at least one selected from the group consisting of a Bifidobacterium longum strain according to any one of claims 1 to 4, a culture solution of the strain, a lysate of the strain, and an extract of the strain.

7. A pharmaceutical composition for treating or preventing cancer, comprising at least one selected from the group consisting of a Bifidobacterium longum strain according to any one of claims 1 to 4, a culture solution of the strain, a lysate of the strain, and an extract of the strain.

8. A pharmaceutical composition for the treatment or prevention of cancer according to claim 7, wherein the cancer is any one selected from the group consisting of lung cancer, stomach cancer, liver cancer, pancreatic cancer, skin cancer, uterine cancer, ovarian cancer, colon cancer, small intestine cancer, thyroid cancer, parathyroid cancer, urethral cancer, bladder cancer, penile cancer, prostate cancer, multiple myeloma, leukemia, lymphoma, kidney cancer, renal cell carcinoma, and splenic tumor.

9. A pharmaceutical composition for treating or preventing cancer, further comprising an immunotherapy agent according to claim 7.

10. A pharmaceutical composition for treating or preventing cancer according to claim 9, wherein the immune anticancer agent is any one selected from the group consisting of an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti-CD28 antibody, and an anti-TIM-3 antibody.

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