WO2025198344A1 - Novel lactic acid bacteria and use thereof - Google Patents

Abstract

The present invention relates to a novel Bifidobacterium adolescentis strain or an extracellular vesicle thereof, a novel Bifidobacterium longum<i /> strain or an extracellular vesicle thereof, and anticancer uses thereof. Bifidobacterium adolescentis DS0019<i /> KCTC15776BP KCTC or an extracellular vesicle thereof, Bifidobacterium longum DS4022 KCTC15778BP or an extracellular vesicle thereof (EV), or Bifidobacterium longum DS4148 KCTC15777BP or an extracellular vesicle thereof according to the present invention has anticancer activity due to inducing apoptosis of cancer cells, and thus can be effectively used as a composition for preventing, treating, or alleviating cancer in humans or animals.

Description

Novel lactic acid bacteria and their uses

The present invention relates to a novel lactic acid bacterium and its use, and more specifically, to a novel Bifidobacterium adolecentis. It relates to a strain or its extracellular vesicles and its anticancer use, and also to a novel Bifidobacterium longum It relates to a strain or extracellular vesicles thereof and anticancer use thereof.

Probiotics are live bacteria that thrive in the human intestines, where a variety of microorganisms thrive. They promote the growth of beneficial bacteria. They live symbiotically in the human digestive system, breaking down fiber and complex proteins into essential nutrients. They also inhibit the growth of E. coli and other harmful bacteria, alleviate diarrhea and constipation, and aid in vitamin synthesis and lower blood cholesterol. Among these probiotics, lactic acid bacteria ferment sugars to obtain energy and produce large amounts of lactic acid. They are widely distributed in nature, including agricultural products, foods, and the bodies of humans and animals. They are widely used in the fermentation of cheese, fermented milk, kimchi, and bread. Generally, consuming lactic acid bacteria suppresses harmful bacteria in the gut and increases beneficial bacteria that aid in the digestion, absorption, and breakdown of food. Therefore, consuming lactic acid bacteria has been reported to have various beneficial effects, including lowering blood cholesterol, enhancing immunity, suppressing endogenous infections, improving liver cirrhosis, and having anticancer effects. In addition, research is currently underway to enhance the efficacy of natural products fermented with lactic acid bacteria. This homeostasis of intestinal microbes is maintained through continuous interaction with the body's immune system. However, if the balance is disrupted (dysbiosis) due to Western eating habits or indiscriminate antibiotic use, it can lead to chronic diseases, including cancer. Therefore, research in the field of pharmabiotics on the influence of intestinal microbes on the effectiveness of anticancer treatment has recently attracted significant attention.

Meanwhile, cancer has a high mortality rate worldwide, and in Western societies, it is the second most common cause of death after cardiovascular disease. In particular, Westernized diets, the widespread consumption of high-fat diets, rapid increases in environmental pollutants, and increased alcohol consumption have led to a steady increase in the incidence of cancers such as colon, breast, and prostate. Furthermore, lung cancer is on the rise due to an aging population, increased smoking, and air pollution. Given this reality, there is a pressing need to develop anticancer agents that can facilitate early cancer prevention and treatment, thereby contributing to improved human health, a better quality of life, and a better global health.

In addition, it has been reported that prokaryotic cells such as bacteria and eukaryotic host cells such as humans secrete vesicles outside the cells, and that the secreted vesicles perform various functions. Extracellular vesicles secreted from bacteria contain endotoxins (lipopolysaccharide; LPS) and bacterial proteins and genes, and are commonly called nanovesicles because they are 20-1000 nanometers (nm) in size. It has been reported that extracellular vesicles are found in various secretions, excretions, or tissue washings of humans or animals, and it has been reported that extracellular vesicles present in tissues reflect the state of the tissue that secretes the vesicles, and that they can be used to diagnose and treat diseases.

Accordingly, the present inventors confirmed that Bifidobacterium adolecentis strain or its extracellular vesicles and Bifidobacterium longum (from a newborn) and Bifidobacterium longum (from an elderly person) strain or its extracellular vesicles exhibit excellent therapeutic effects on cancer, and completed the present invention.

The purpose of the present invention is to provide a method for treating Bifidobacterium adolecentis DS0019. ( Bifidobacterium adolescentis DS0019) KCTC15776BP is provided.

Another object of the present invention is to provide a method for treating a disease caused by Bifidobacterium adolecentis DS0019. ( Bifidobacterium adolescentis DS0019) KCTC15776BP or extracellular vesicles (EV) thereof are provided as a pharmaceutical composition for preventing or treating cancer.

Another object of the present invention is to provide a method for treating a disease caused by Bifidobacterium adolecentis DS0019. ( Bifidobacterium adolescentis DS0019) KCTC15776BP or an extracellular vesicle thereof is provided as a food composition for preventing or improving cancer.

Another object of the present invention is to provide Bifidobacterium longum DS4148 KCTC15777BP.

Another object of the present invention is to provide Bifidobacterium longum DS4022 KCTC15778BP.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, comprising Bifidobacterium longum DS4148 KCTC15777BP or its extracellular vesicles (EV); or Bifidobacterium longum DS4022 KCTC15778BP or its extracellular vesicles (EV).

Another object of the present invention is to provide a food composition for preventing or improving cancer, comprising Bifidobacterium longum DS4148 KCTC15777BP or its extracellular vesicles (EV); or Bifidobacterium longum DS4022 KCTC15778BP or its extracellular vesicles (EV).

To avoid confusion due to overlapping content, the description of redundant content will be omitted below. In other words, the content of the invention is not limited to the content described below, and the content of the invention should be interpreted based on the overall content of the invention.

Hereinafter, the present invention will be described in detail.

The present invention provides Bifidobacterium adolescentis DS0019 (Deposit institution: Korea Research Institute of Bioscience and Biotechnology, Deposit date: January 17, 2024, Accession number: KCTC15776BP).

The Bifidobacterium adolescentis DS0019 strain of the present invention is characterized as a novel probiotic isolated and identified from the oral cavity of an adult.

The 16S rDNA base sequence for identification and classification of the Bifidobacterium adolescentis DS0019 strain of the present invention includes SEQ ID NO: 1 attached to this specification. Accordingly, the Bifidobacterium adolescentis DS0019 strain of the present invention may include the 16S rDNA of SEQ ID NO: 1.

The present invention provides Bifidobacterium longum DS4022 (Deposit institution: Korea Research Institute of Bioscience and Biotechnology, Deposit date: January 17, 2024, Accession number: KCTC15778BP).

The Bifidobacterium longum DS4022 strain of the present invention is characterized as a novel probiotic isolated and identified from feces derived from the elderly.

The 16S rDNA base sequence for identification and classification of the Bifidobacterium longum DS4022 strain of the present invention is as shown in SEQ ID NO: 4 attached to this specification. Therefore, the Bifidobacterium longum DS4022 strain of the present invention may include the 16S rDNA of SEQ ID NO: 4.

The present invention provides Bifidobacterium longum DS4148 (Deposit institution: Korea Research Institute of Bioscience and Biotechnology, Deposit date: January 17, 2024, Accession number: KCTC15777BP).

The Bifidobacterium longum DS4148 strain of the present invention is characterized as a novel probiotic isolated and identified from feces of a newborn.

The 16S rDNA base sequence for identification and classification of the Bifidobacterium longum DS4148 strain of the present invention is as shown in SEQ ID NO: 5 attached to this specification. Therefore, the Bifidobacterium longum DS4148 strain of the present invention may include the 16S rDNA of SEQ ID NO: 5.

The strain according to the present invention can be utilized in various forms, such as live cells, dead cells, cultures, lysates, or extracts. The strain according to the present invention, regardless of its form, exhibits results equivalent to or higher than the above-mentioned effects (especially considering live cells) under conditions involving the strain.

Probiotics refer to living bacteria, while dead bacteria refer to live bacteria cultured under certain conditions and then the effective ingredients are separated and extracted through methods such as heat drying, pressurization, and drug treatment.

The above culture refers to a product obtained by culturing lactic acid bacteria in a known liquid medium or solid medium, and is a concept including a strain according to the present invention. The product may include lactic acid bacteria. The medium may be selected from known liquid media or solid media, and may be, for example, MRS liquid medium, GAM liquid medium, MRS agar medium, GAM agar medium, or BL agar medium, but is not limited thereto.

The above-mentioned lysate refers to a form in which live cells, dead cells, or cultures thereof are separated and processed through mechanical or chemical methods. For example, the lysate form can be produced through bead mills, presses, sonicators, microfluidizers, enzyme treatment, etc.

The above extract means one obtained by extracting live cells, dead cells and/or fragments using a commonly known extraction method (a known extraction solvent (e.g., water, C1 to C4 alcohol (methanol, ethanol, etc.))).

In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising Bifidobacterium adolescentis DS0019 KCTC15776BP or extracellular vesicles (EV) thereof.

The above Bifidobacterium adolescentis DS0019 ( Bifidobacterium adolescentis DS0019) KCTC15776BP may include 16s rRNA of sequence number 1.

The present invention provides a pharmaceutical composition for preventing or treating cancer, comprising Bifidobacterium longum DS4022 KCTC15778BP or its extracellular vesicles (EV); or Bifidobacterium longum DS4148 KCTC15777BP or its extracellular vesicles (EV).

In addition, the present invention Bifidobacterium longum DS4022 A pharmaceutical composition for preventing or treating cancer comprising ( Bifidobacterium longum DS4022) KCTC15778BP or extracellular vesicles (EV) thereof is provided.

The above Bifidobacterium longum DS4022 strain may include 16s rRNA of sequence number 4.

In addition, the present invention relates to Bifidobacterium longum DS4148 A pharmaceutical composition for preventing or treating cancer comprising ( Bifidobacterium longum DS4148) KCTC15777BP or extracellular vesicles (EV) thereof is provided.

The above Bifidobacterium longum DS4148 strain may include 16s rRNA of sequence number 5.

The form of the strain according to the present invention can be used in various forms such as live cells, dead cells, cultures, lysates or extracts, but is not limited thereto.

In the present invention, “extracellular vesicles (EV)” refers to particles secreted from cells and released into the extracellular space, and are also called exosomes, ectosomes, microvesicles, microparticles, exosome-like vesicles, etc. Extracellular vesicles were observed in electron microscopic studies to originate from specific intracellular compartments called multivesicular bodies (MVBs) and to be released and secreted outside of the cell, rather than being directly separated from the plasma membrane. That is, when fusion of multivesicular bodies and the plasma membrane occurs, the vesicles are released into the extracellular environment, which are called extracellular vesicles.

Extracellular vesicles (EVs) facilitate the exchange of materials (proteins, lipids, and genetic material) between cells and function as mediators for physiological and pathological signaling. Extracellular vesicles are broadly categorized into exosomes and microvesicles. Exosomes vary in size depending on their biological origin. They are intraluminal vesicles created when the endosomal membrane of multi-vesicular endosomes matures, and are secreted when multi-vesicular endosomes fuse with the cell surface. Microvesicles, measuring 50-1000 nm in size, are vesicles secreted outside the cell by protrusion of the plasma membrane. Each cell produces different EVs depending on its physiological state and secretes EVs with specific lipid/protein/nucleic acid compositions. The above “extracellular vesicles” is used to mean exosomes and microvesicles.

The above extracellular vesicles may have an average diameter of 20 nm to 400 nm, preferably 50 nm to 300 nm, more preferably 100 nm to 250 nm.

The exosomes or extracellular vesicles included in the composition of the present invention are contained in large quantities in a culture solution (e.g., culture supernatant) of Bifidobacterium adolescentis DS0019, Bifidobacterium longum DS4022, or Bifidobacterium longum DS4148.

The above extracellular vesicles can be obtained using an extracellular vesicle (EV) extraction method known in the art, and are not limited thereto, but can be obtained by an extraction method including, for example, the following steps:

1) A step of culturing Bifidobacterium adolescentis or Bifidobacterium longum ;

2) The above Bifidobacterium adolecentis or Bifidobacterium longum Step of recovering the culture supernatant;

3) A step of centrifuging the recovered cell culture supernatant to remove cell residue; and

4) A step of obtaining exosomes separated and purified using one selected from the group consisting of tangential flow filtration (TFF), ultracentrifugation, size exclusion chromatography, and an exosome isolation kit from the cell culture supernatant from which the cell debris has been removed.

In culturing the above Bifidobacterium adolecentis or Bifidobacterium longum, a culture medium generally used for strain culture may be used. For example, it may be a strain cultured under a culture medium such as MRS broth.

The recovery of the culture supernatant in step 2) above may be the recovery of the culture medium, i.e., the conditioned medium. The recovered cell culture supernatant contains cell debris and extracellular vesicles derived from Bifidobacterium longum, and the cell debris contained in the cell culture supernatant can be removed by centrifugation in step 3).

After removing these cell debris, the cell culture supernatant can be separated and purified to obtain extracellular vesicles using one or more selected from the group consisting of tangential flow filtration (TFF), ultracentrifugation, size exclusion chromatography, and an exosome isolation kit.

In one embodiment, the present invention can purify extracellular vesicles of Bifidobacterium adolescentis DS0019, Bifidobacterium longum DS4022, or Bifidobacterium longum DS4148 with high purity through filtration and ultracentrifugation.

The term “isolation” in this specification includes both a process of selectively obtaining a target substance (e.g., exosome) from a biological sample (e.g., Bifidobacterium longum culture) (positive isolation) and a process of selectively removing impurities other than the target substance (negative isolation). Therefore, the term “isolation” can be used with the same meaning as “obtain,” “extract,” and “purify.” In the present invention, the process for isolating exosomes or extracellular vesicles may be performed using any method commonly used in the art without limitation, for example, utilizing the commercially available exosome isolation kit (e.g., EXO-BB, ExoQuick ® -ULTRA, ExoQuick ® -TC, Capturem TM Exosome Isolation Kit, Total Exosome Isolation Kit, ExoTrap TM Exosome Isolation Spin Column Kit, Exo2DTM, etc.), or including separation based on the difference in specific gravity between components in a solution (e.g., centrifugation), separation based on size (e.g., ultrafiltration or vacuum filter), separation based on affinity for a specific substrate (e.g., affinity chromatography), but is not limited thereto, and any method commonly used in the art as a separation method based on the unique physical properties of a target substance in a heterogeneous sample may be used without limitation.

In the present invention, "cancer" is a disease related to the regulation of cell death, and refers to a disease caused by excessive cell proliferation when the normal apoptosis balance is disrupted. These abnormally proliferating cells sometimes invade surrounding tissues and organs, forming a mass and destroying or deforming the normal structure of the body. This condition is called cancer. In general, a tumor refers to a mass that has grown abnormally due to the autonomous excessive growth of body tissues, and can be classified as benign tumors and malignant tumors. Malignant tumors grow much faster than benign tumors and can metastasize by infiltrating surrounding tissues, posing a threat to life. Such malignant tumors are commonly referred to as "cancer."

The above cancers include breast cancer, brain cancer, nerve cancer, colon cancer, lung cancer, small cell lung cancer, bronchial cancer, stomach cancer, liver cancer, biliary tract cancer, gallbladder cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, nasopharyngeal cancer, oropharyngeal cancer, laryngeal cancer, oral cancer, salivary gland cancer, melanoma (melanoma of the skin, eye, or organ), uterine cancer, ovarian cancer, rectal cancer, anal cancer, colon cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, vaginal cancer, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, peritoneal cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal cord tumor, It may be any one selected from the group consisting of brainstem glioma, pituitary adenoma and metastatic cancer of other organs thereof, and preferably breast cancer.

The composition according to the present invention can have anticancer activity by inducing death of cancer cells for the above cancer diseases.

The above pharmaceutical composition may be administered in combination with an anticancer agent.

When administered in combination with anticancer drugs, it can exhibit synergistic effects. These synergistic effects encompass all aspects of cancer treatment, including enhanced anticancer effects and improved anticancer effects against resistant cancers.

In the present invention, the "anticancer agent" may be selected from the group consisting of chemotherapeutic agents, targeted anticancer agents, anticancer viruses, antibody therapeutics, immunotherapy agents, and combinations thereof for chemotherapy. Specifically, the anticancer agent may comprise an antibody, an antigen-binding fragment, or a fusion protein comprising one or more single-domain antibodies, or antigen-binding fragments thereof, and one or more additional polypeptides. For example, the fusion protein may comprise one or more single-domain antibodies and a constant region or an Fc region, and the one or more single-domain antibodies, or antigen-binding fragments thereof, may be noncovalently or covalently conjugated to an antigen.

In the present invention, "chemotherapeutic agent" is also referred to as an antitumor drug or cytotoxic agent. It is a general term for drugs that exhibit anticancer activity by acting directly on DNA to block DNA replication, transcription, and translation processes, interfering with the synthesis of nucleic acid precursors in metabolic pathways, and inhibiting cell division. These antitumor drugs exhibit cytotoxicity not only on tumor cells but also on normal cells. Chemotherapeutic agents can be used for maintenance therapy. Furthermore, maintenance therapy refers to a treatment method implemented after initial chemotherapy to prevent or delay cancer recurrence.

Specifically, the chemotherapeutic agent may be any one selected from the group consisting of an alkylating agent, a microtubule inhibitor, an anti-metabolite, and a topoisomerase inhibitor. The alkylating agent may be any one selected from the group consisting of mechlorethamine, chlorambucil, busulfan, cyclophosphamide, ifosfamide, melphalan, thiotepa, altretamine, procarbazine, streptozotocin, carmustine (BCNU), lomustine (CCNU), dacarbazine, cisplatin, carboplatin, and oxaliplatin. The microtubule inhibitor may be any one selected from the group consisting of docetaxel, vinblastine (Velban), Oncovin, and vinorelbine (Navelbine). The antimetabolite may be any one selected from the group consisting of fluorouracil, doxifluridine, gemcitabine, capecitabine, cytarabine, fludarabine, methotrexate, pemetrexed, and mercaptopurine. The topoisomerase inhibitor may be any one selected from the group consisting of Hycamtin (Topocan), Irinotecan (Camptosar), Etoposide (Vepesid), Paclitaxel, Bleomycin (Blenoxane), Doxorubicin, and Daunorubicin (Cerubidine).

In the present invention, a "targeted anticancer agent" is a therapeutic agent that specifically kills cancer cells by targeting specific proteins or genetic changes that are abundant only in cancer cells and blocking signals involved in cancer growth and development. They are classified into monoclonal antibodies that react outside the cell and small molecules that act inside the cell. Monoclonal antibodies are anticancer agents that block cancer cell-inducing signals transmitted outside the cell, acting on initiating signals related to proliferation and apoptosis, while small molecules act on complex signaling that occurs inside the cell.

Specifically, the targeted proteins may be EGFR, VEGFR, CD20, CD38, RNAK-L, BTK, Bcr-abl, PDGFR/FGFR family, MEK/RAF, HER2/Neu, Ubiquitin, JAK, MAP2K, ALK, PARP, TGFβRI, Proteasome, Bcl-2, C-Met, VR1, VR2, VR3, c-kit, AXL, RET, Braf, DNMT, CDK4/6, STING, Trop-2, PD-L1 (programmed death-ligand 1), PARP (Poly ADP-ribose polymerase), PIK3CAα, AKT1/2/3, etc.

Specifically, the targeted anticancer drugs include abciximab, adalimumab, basiliximab, bezlotoxumab, canakinumab, daclizumab, denosumab, efalizumab, golimumab, inflectra, natalizumab, and olaratumab. Omalizumab, palipizumab, panitumumab, trastuzumab, T-DM1 (Trastuzumab emtansine), trastuzumab deruxtecan, sacituzumab govitecan, pertuzumab, cetuximab, rituximab, tocilizumab, secukinumab, ustekinumab, bevacizumab, atezolizumab, avelumab, durvalumab, palbociclib, ribociclib, abemaciclib, It may be any one selected from the group consisting of talazoparib, alpelisib, capivasertib, CDNs, SB11285, and DMXAA.

Additionally, the anticancer agent may include an antihormonal agent for antihormonal therapy. Specifically, the antihormonal agent has an anticancer effect in patient groups that have estrogen receptors (ER) and progesterone receptors (PR) and can kill cancer cells or prevent cancer. Examples of the antihormonal agent include tamoxifen (Tamoxifen, Nolvadex, etc.), aromatase inhibitors (Letrozole, Lenara, etc.), fulvestrant, goserelin (Zoladex, etc.), and leuprolide (Luprin, Lupron).

In one specific example, the anticancer agent administered in combination with Bifidobacterium adolescentis DS0019 KCTC15776BP or its extracellular vesicles may include at least one selected from the group consisting of doxorubicin, paclitaxel, and docetaxel.

In one specific example, the anticancer agent administered in combination with Bifidobacterium longum DS4022 KCTC15778BP or its extracellular vesicles may include at least one selected from the group consisting of doxorubicin, paclitaxel, and docetaxel.

In one specific example, Bifidobacterium longum DS4148 ( Bifidobacterium longum DS4148) KCTC15777BP or an anticancer agent administered in combination with its extracellular vesicles may include at least one selected from the group consisting of doxorubicin, paclitaxel, and docetaxel.

As used herein, "combination therapy," "combination administration," or "in combination" refers to any form of simultaneous or concurrent treatment using at least two separate therapeutic agents. The components of combination therapy may be administered simultaneously, sequentially, or in any order. The components may be administered in different dosages, at different administration frequencies, or via different routes, as appropriate.

The above anticancer agent may be administered simultaneously, sequentially, or separately from the composition according to the present invention.

Specifically, the combined administration is administered simultaneously with Bifidobacterium adolescentis DS0019 KCTC15776BP or its extracellular vesicles; Bifidobacterium longum DS4022 KCTC15778BP or its extracellular vesicles (EV); or Bifidobacterium longum DS4148 KCTC15777BP or its extracellular vesicles (EV), and an anticancer agent, or Bifidobacterium adolescentis DS0019 KCTC15776BP or its extracellular vesicles; Bifidobacterium longum DS4022 ( Bifidobacterium longum DS4022) KCTC15778BP or its extracellular vesicles (EV); or Bifidobacterium longum DS4148 ( Bifidobacterium longum DS4148) KCTC15777BP or its extracellular vesicles (EV) may be administered followed by administration of an anticancer agent. The combination therapy according to the present invention may be defined as providing a synergistic effect if the efficacy, as measured by, for example, the degree of response, the rate of response, the time until disease progression, or the duration of survival, is therapeutically superior to the efficacy that can be obtained by administering one or the other of the components of the combination therapy at a conventional dose. For example, the efficacy of the combination therapy is synergistic if the efficacy is therapeutically superior to the efficacy obtained by using each of the above alone. In particular, a synergistic effect is considered to exist if the usual dose of Bifidobacterium adolescentis DS0019 KCTC15776BP or its extracellular vesicles; Bifidobacterium longum DS4148 KCTC15777BP or its extracellular vesicles; or Bifidobacterium longum DS4022 KCTC15778BP or its extracellular vesicles, and the anticancer agent can be reduced without harming one or more of the degree of response, the rate of response, the time to disease progression, and the survival data, and in particular without harming the duration of response, and with a reduction and/or fewer problematic side effects than when each component is used at its usual dose.

In one specific example, the synergistic effect of the combined administration may be to reduce the dosage or frequency of administration of the existing anticancer agent. Specifically, Bifidobacterium adolescentis DS0019 KCTC15776BP or extracellular vesicles thereof; Bifidobacterium longum DS4148 KCTC15777BP or extracellular vesicles thereof; Or, when Bifidobacterium longum DS4022 ( Bifidobacterium longum DS4022) KCTC15778BP or its extracellular vesicles are administered in combination with an anticancer agent (e.g., Adriamycin, Paclitaxel, Docetaxel, etc.), it may exhibit the same or similar anticancer activity at a lower dose or a smaller number of administrations than the dose of the anticancer agent (e.g., Adriamycin, Paclitaxel, Docetaxel, etc.) administered alone.

Therefore, the combined administration of Bifidobacterium adolescentis DS0019 KCTC15776BP or its extracellular vesicles; Bifidobacterium longum DS4148 KCTC15777BP or its extracellular vesicles; or Bifidobacterium longum DS4022 KCTC15778BP or its extracellular vesicles has a synergistic effect that can reduce the usual dose and frequency of anticancer agents.

In the present invention, “administered simultaneously” is not particularly limited and means that the components of the combination therapy are administered substantially simultaneously, for example, as a mixture or in an immediately subsequent sequence.

As used herein, the term "sequentially administered" is not particularly limited and means that the components of the combination therapy are not administered simultaneously, but are administered one by one or simultaneously with a specific time interval between administrations. The time intervals may be the same or different between the administrations of each of the components of the combination therapy, and may be selected from the range of, for example, 2 minutes to 96 hours, 1 day to 7 days, or 1 week, 2 weeks, or 3 weeks. Typically, the time interval between administrations may range from several minutes to several hours, for example, 2 minutes to 72 hours, 30 minutes to 24 hours, or 1 to 12 hours. Additional examples include time intervals ranging from 24 to 96 hours, 12 to 36 hours, 8 to 24 hours, and 6 to 12 hours.

In the present invention, “prevention” means any act of inhibiting or delaying the onset of a cancer-related disease by administering Bifidobacterium adolescentis DS0019 KCTC15776BP or extracellular vesicles thereof; Bifidobacterium longum DS4148 KCTC15777BP or extracellular vesicles thereof; or Bifidobacterium longum DS4022 KCTC15778BP or extracellular vesicles thereof according to the present invention.

In the present invention, “treatment” means any act of improving or beneficially changing the symptoms of a cancer-related disease by administering Bifidobacterium adolescentis DS0019 KCTC15776BP or its extracellular vesicles; Bifidobacterium longum DS4148 KCTC15777BP or its extracellular vesicles; or Bifidobacterium longum DS4022 KCTC15778BP or its extracellular vesicles according to the present invention.

The pharmaceutical composition of the present invention can be used as a pharmaceutical composition comprising a pharmaceutically effective amount of Bifidobacterium adolescentis DS0019 KCTC15776BP or extracellular vesicles (EV) thereof; and/or a pharmaceutically acceptable carrier.

The pharmaceutical composition of the present invention can be used as a pharmaceutical composition comprising a pharmaceutically effective amount of Bifidobacterium longum DS4022 KCTC15778BP or extracellular vesicles (EV) thereof; and/or a pharmaceutically acceptable carrier.

The pharmaceutical composition of the present invention can be used as a pharmaceutical composition comprising a pharmaceutically effective amount of Bifidobacterium longum DS4148 KCTC15777BP or extracellular vesicles (EV) thereof; and/or a pharmaceutically acceptable carrier.

The term "pharmaceutically effective amount" as used herein means an amount sufficient to achieve the efficacy or activity of the above-mentioned Bifidobacterium adolescentis DS0019 KCTC15776BP or extracellular vesicles thereof; Bifidobacterium longum DS4148 KCTC15777BP or extracellular vesicles thereof; or Bifidobacterium longum DS4022 KCTC15778BP or extracellular vesicles thereof.

Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are those commonly used in formulation, and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.

The pharmaceutical composition according to the present invention can be administered to mammals, including humans, via various routes. Any commonly used route of administration may be used, including oral, transdermal, intravenous, intramuscular, and subcutaneous administration. Preferably, the composition is administered orally or applied to the skin.

The suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, administration method, patient's age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity, and an ordinary skilled physician can easily determine and prescribe a dosage effective for the desired treatment or prevention. In addition, the dosage may vary depending on the degree of concentration, but may be administered in one or several divided doses of 10 μl/kg to 1 ml/kg body weight. For example, it is preferable that the extracellular vesicles are included in the composition of the present invention in an amount of about 1 to 150 μg/ml, or 1 to 100 μg/ml. In addition, the extracellular vesicles may be included in a particle number of, for example, 1 X 10 ⁷ to 1 X 10 ¹² particles/ml, and the dosage thereof may be appropriately changed and administered depending on the subject to which it is applied. It should be understood that the actual dosage of the active ingredient should be determined in light of various related factors such as the disease to be treated, the severity of the disease, the route of administration, the patient's weight, age, and sex, and therefore, the dosage does not limit the scope of the present invention in any way.

That is, in this specification, "effective amount" or "therapeutically effective amount" means the amount required to delay or completely stop the onset or progression of a specific disease to be treated, and the effective amount included in the food composition or pharmaceutical composition of the present invention or the treatment method or treatment use of the present invention means the amount required to achieve the effect of preventing, improving, or treating cancer. Therefore, the effective amount can be adjusted according to various factors including the type of disease, the severity of the disease, the type and content of other ingredients contained in the composition, and the patient's age, weight, general health condition, sex, and diet, administration time, administration route, treatment period, and concurrently used drugs. It is obvious to those skilled in the art that an appropriate total daily use amount can be determined by a treating physician within the scope of sound medical judgment.

The pharmaceutical composition of the present invention can be manufactured in the form of a unit dose or can be manufactured by inserting it into a multi-dose container by formulating it using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains, and the like. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granules, tablets, capsules or gel (e.g., hydrogel), and may additionally include a dispersant or stabilizer.

The present invention provides Bifidobacterium adolescentis DS0019 KCTC15776BP or its extracellular vesicles for use in the prevention or treatment of cancer.

The present invention provides Bifidobacterium longum DS4022 KCTC15778BP or its extracellular vesicles for use in the prevention or treatment of cancer.

The present invention provides Bifidobacterium longum DS4148 KCTC15777BP or its extracellular vesicles for use in the prevention or treatment of cancer.

The present invention provides the use of Bifidobacterium adolescentis DS0019 KCTC15776BP or its extracellular vesicles in the manufacture of a medicament for treating cancer.

The present invention provides the use of Bifidobacterium longum DS4022 KCTC15778BP or its extracellular vesicles in the manufacture of a medicament for treating cancer.

The present invention provides the use of Bifidobacterium longum DS4148 KCTC15777BP or its extracellular vesicles in the manufacture of a medicament for treating cancer.

The present invention provides a method for preventing or treating cancer, comprising administering Bifidobacterium adolescentis DS0019 KCTC15776BP or its extracellular vesicles to a subject in need thereof.

The present invention provides a method for preventing or treating cancer, comprising administering Bifidobacterium longum DS4022 KCTC15778BP or its extracellular vesicles to a subject in need thereof.

The present invention provides a method for preventing or treating cancer, comprising administering Bifidobacterium longum DS4148 KCTC15777BP or its extracellular vesicles to a subject in need thereof.

The present invention provides a composition comprising Bifidobacterium adolescentis DS0019 KCTC15776BP or extracellular vesicles thereof.

The present invention provides a composition comprising Bifidobacterium longum DS4022 KCTC15778BP or extracellular vesicles thereof.

The present invention provides a composition comprising Bifidobacterium longum DS4148 KCTC15777BP or extracellular vesicles thereof.

The term "subject" or "individual" in this specification refers to a mammal that is the object of treatment, observation or experiment, and may preferably be a human or animal in need of prevention and/or treatment of cancer.

The present invention also provides an anticancer adjuvant comprising Bifidobacterium adolescentis DS0019 KCTC15776BP or extracellular vesicles thereof.

The present invention also provides an anticancer adjuvant comprising Bifidobacterium longum DS4022 KCTC15778BP or extracellular vesicles thereof.

The present invention also provides an anticancer adjuvant comprising Bifidobacterium longum DS4148 KCTC15777BP or extracellular vesicles thereof.

In the present invention, “anticancer adjuvant” means a preparation that can improve, enhance or increase the anticancer effect of an anticancer agent.

For example, when used in combination with an anticancer agent, a preparation that can improve, enhance, or augment the anticancer effect of the anticancer agent can be used as an anticancer adjuvant. As another example, when used in combination with an anticancer agent, a preparation that exhibits concentration-dependent anticancer activity at a level that does not exhibit anticancer activity on its own, can be used as an anticancer adjuvant that can improve, enhance, or augment the anticancer effect of the anticancer agent. In this case, the anticancer adjuvant can be used as either an anticancer agent or an anticancer adjuvant depending on the treatment concentration, and can be used as an anticancer adjuvant within a treatment concentration range where it does not exhibit anticancer activity on its own.

Anticancer drugs whose anticancer activity can be enhanced by the above anticancer adjuvant include, but are not particularly limited to, breast cancer, brain cancer, nerve cancer, colon cancer, lung cancer, small cell lung cancer, bronchial cancer, stomach cancer, liver cancer, biliary tract cancer, gallbladder cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, nasopharyngeal cancer, oropharyngeal cancer, laryngeal cancer, oral cancer, salivary gland cancer, melanoma (melanoma in the skin, eye, or organ), uterine cancer, ovarian cancer, rectal cancer, anal cancer, colon cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, vaginal cancer, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, peritoneal cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic It may be an anticancer agent used for any one selected from the group consisting of carcinoma, CNS tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, pituitary adenoma, and metastatic cancer of other organs thereof.

Preferably, it is an anticancer agent useful for the prevention or treatment of breast cancer, and more preferably, it is an anticancer agent useful for the prevention or treatment of hormone receptor-positive breast cancer. Examples thereof include doxorubicin, paclitaxel, or docetaxel.

Accordingly, the anticancer adjuvant according to the present invention may be an anticancer adjuvant that improves, enhances or increases the anticancer activity of adriamycin (doxorubicin), paclitaxel or docetaxel against breast cancer.

The present invention provides a food composition for preventing or improving cancer, comprising Bifidobacterium adolescentis DS0019 KCTC15776BP or its extracellular vesicles.

The present invention provides a food composition for preventing or improving cancer, comprising Bifidobacterium longum DS4022 KCTC15778BP or its extracellular vesicles.

The present invention provides a food composition for preventing or improving cancer, comprising Bifidobacterium longum DS4148 KCTC15777BP or its extracellular vesicles.

In the present invention, “improvement” means any act of improving the bad condition of a cancer-related disease by administering or ingesting the composition of the present invention to a subject.

When the food composition of the present invention is used as a food additive, the food composition may be added as is or used together with other foods or food ingredients, and may be used appropriately according to conventional methods. Generally, when manufacturing a food or beverage, the food composition of the present invention may be added in an amount of 15% by weight or less, preferably 10% by weight or less, relative to the raw materials.

There are no specific restrictions on the types of the above foods. Examples of foods to which the above substances can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and all foods in the conventional sense are included.

The above beverage may contain various flavoring agents or natural carbohydrates as additional ingredients. The above-mentioned natural carbohydrates may include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, natural sweeteners such as dextrin and cyclodextrin, or synthetic sweeteners such as saccharin and aspartame. The proportion of the natural carbohydrates may be appropriately determined by those skilled in the art.

In addition to the above, the food composition of the present invention may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the food composition of the present invention may contain fruit pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks. These ingredients may be used independently or in combination. The proportions of these additives may also be appropriately selected by those skilled in the art.

The food composition of the present invention can also be used as a health functional food. A health functional food is a food that emphasizes the bioregulatory function of food, and is a food that has added value by using physical, biochemical, and biotechnological methods to act and express for a specific purpose. The food composition of the present invention can be used as a health functional food. The ingredients of such a health functional food are designed and processed to sufficiently exert the body's body regulatory functions related to biodefense, regulation of body rhythm, and prevention and recovery from disease. The food composition may contain food-acceptable food additives, sweeteners, or functional raw materials. When the strain of the present invention is used as a health functional food (or health functional beverage additive), the novel strain may be added as is or used in combination with other foods or food ingredients, and may be used appropriately according to conventional methods. The mixing amount of the strains may be appropriately determined depending on the intended use (prevention, health or improvement, therapeutic treatment).

The Bifidobacterium adolescentis DS0019 ( Bifidobacterium adolescentis DS0019) KCTC15776BP or its extracellular vesicles (EV); Bifidobacterium longum DS4148 ( Bifidobacterium longum DS4148) KCTC15777BP or its extracellular vesicles (EV); or Bifidobacterium longum DS4022 ( Bifidobacterium longum DS4022) KCTC15778BP or its extracellular vesicles (EV); of the present invention induces the death of cancer cells and has anticancer activity, and thus can be usefully used as a composition for the prevention, treatment, or improvement of cancer in humans or animals. Through this, it has excellent effects as a pharmaceutical composition or a food composition.

Figure 1a is an image showing the results of confirming the size of extracellular vesicles (EV) of Bifidobacterium adolescentis DS0019 through transmission electron microscopy (TEM), Figure 1b is an image showing the results of confirming the size of extracellular vesicles (EV) of Bifidobacterium longum DS4022 KCTC15778BP (derived from an elderly person) through transmission electron microscopy (TEM), and Figure 1c is an image showing the results of confirming the size of extracellular vesicles (EV) of Bifidobacterium longum DS4148 KCTC15777BP (derived from a newborn) through transmission electron microscopy (TEM).

Figure 2a is a graph showing the results of confirming the anticancer activity of extracellular vesicles (EVs) derived from Bifidobacterium adolecentis DS0019 through cell viability after treating experimental cells with EVs for 24 hours, and Figure 2b is a graph showing the results of confirming the anticancer activity of EVs derived from Bifidobacterium longum strain through cell viability after treating experimental cells with EVs for 24 hours (Bifidobacterium adolecentis DS0019 KCTC15776BP strain: B.adolescentis, Bifidobacterium longum DS4022 KCTC15778BP strain: Elderly, Bifidobacterium longum DS4148 KCTC15777BP strain: Infant).

Figure 3a is a bar graph showing the results of confirming anticancer activity when Bifidobacterium adolescentis DS0019 extracellular vesicles (EV) and doxorubicin were combined (* p < 0.05), Figure 3b is a bar graph showing the results of confirming anticancer activity when Bifidobacterium longum DS4022 extracellular vesicles (EV) and doxorubicin were combined (*** p < 0.001), and Figure 3c is a bar graph showing the results of confirming anticancer activity when Bifidobacterium longum DS4148 extracellular vesicles (EV) and doxorubicin were combined (*** p < 0.001).

FIG. 4a is a diagram showing the results of a proteomics analysis to determine the effect of extracellular vesicles (EVs) of Bifidobacterium adolescentis DS0019 on breast cancer cells, FIG. 4b is a diagram showing the results of a proteomics analysis to determine the effect of extracellular vesicles (EVs) of Bifidobacterium longum DS4022 on breast cancer cells, and FIG. 4c is a diagram showing the results of a proteomics analysis to determine the effect of extracellular vesicles (EVs) of Bifidobacterium longum DS4148 on breast cancer cells.

Figure 5a is a schematic diagram showing the process of measuring CFU/ml using the flat plate smear method, and Figure 5b is an actual image showing the results using the flat plate smear method.

Figure 6a is a schematic diagram showing the process of calculating the dry weight of a strain, and Figure 6b is a diagram showing actual images of a bacterial pellet before drying, a bacterial pellet that is not completely dried, and a bacterial pellet that is completely dried.

Figure 7 is a schematic diagram showing the process of producing a heat-treated strain (dead cells).

Figure 8a is a graph showing the absorbance and dry weight of Bifidobacterium adolecentis DS0019, Figure 8b is a graph showing the absorbance and dry weight of Bifidobacterium longum DS4022, and Figure 8c is a graph showing the absorbance and dry weight of Bifidobacterium longum DS4148.

Figure 9a is a graph showing the results of measuring the effect of heat-treated Bifidobacterium adolecentis DS0019 on MCF-7 breast cancer cells for 48 hours, and Figure 9b is a graph showing the results of measuring the effect of heat-treated Bifidobacterium adolecentis DS0019 on MCF-7 breast cancer cells for 72 hours (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).

Figure 10a is a graph showing the results of measuring the effect of heat-treated Bifidobacterium adolecentis DS0019 on SK-BR-3 breast cancer cells for 48 hours, and Figure 10b is a graph showing the results of measuring the effect of heat-treated Bifidobacterium adolecentis DS0019 on SK-BR-3 breast cancer cells for 72 hours (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).

Figure 11a is a graph showing the results of measuring the effect of heat-treated Bifidobacterium longum DS4022 on MCF-7 breast cancer cells for 48 hours, and Figure 11b is a graph showing the results of measuring the effect of heat-treated Bifidobacterium longum DS4022 on MCF-7 breast cancer cells for 72 hours (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).

Figure 12a is a graph showing the results of measuring the effect of heat-treated Bifidobacterium longum DS4022 on SK-BR-3 breast cancer cells for 48 hours, and Figure 12b is a graph showing the results of measuring the effect of heat-treated Bifidobacterium longum DS4022 on SK-BR-3 breast cancer cells for 72 hours (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).

Figure 13a is a graph showing the results of measuring the effect of heat-treated Bifidobacterium longum DS4148 on MCF-7 breast cancer cells for 48 hours, and Figure 13b is a graph showing the results of measuring the effect of heat-treated Bifidobacterium longum DS4148 on MCF-7 breast cancer cells for 72 hours (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).

Figure 14a is a graph showing the results of measuring the effect of heat-treated Bifidobacterium longum DS4148 on SK-BR-3 breast cancer cells for 48 hours, and Figure 14b is a graph showing the results of measuring the effect of heat-treated Bifidobacterium longum DS4148 on SK-BR-3 breast cancer cells for 72 hours (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).

Figure 15a is a graph showing the results of measuring the cytotoxicity of MCF-7 breast cancer cell lines when treated for 48 hours according to the concentration of heat-treated Bifidobacterium adolecentis DS0019, and Figure 15b is a graph showing the results of measuring the cytotoxicity of MCF-7 breast cancer cell lines when treated for 72 hours according to the concentration of heat-treated Bifidobacterium adolecentis DS0019 (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).

Figure 16a is a graph showing the results of measuring the cytotoxicity of the SK-BR-3 breast cancer cell line when treated for 48 hours according to the concentration of heat-treated Bifidobacterium adolecentis DS0019, and Figure 16b is a graph showing the results of measuring the cytotoxicity of the SK-BR-3 breast cancer cell line when treated for 72 hours according to the concentration of heat-treated Bifidobacterium adolecentis DS0019 (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).

Figure 17a is a graph showing the results of measuring the cytotoxicity of MCF-7 breast cancer cell lines when treated for 48 hours according to the concentration of heat-treated Bifidobacterium longum DS4022, and Figure 17b is a graph showing the results of measuring the cytotoxicity of MCF-7 breast cancer cell lines when treated for 72 hours according to the concentration of heat-treated Bifidobacterium longum DS4022 (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).

Figure 18a is a graph showing the results of measuring the cytotoxicity of the SK-BR-3 breast cancer cell line when treated for 48 hours according to the concentration of heat-treated Bifidobacterium longum DS4022, and Figure 18b is a graph showing the results of measuring the cytotoxicity of the SK-BR-3 breast cancer cell line when treated for 72 hours according to the concentration of heat-treated Bifidobacterium longum DS4022 (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).

Figure 19a is a graph showing the results of measuring the cytotoxicity of MCF-7 breast cancer cell lines when treated for 48 hours according to the concentration of heat-treated Bifidobacterium longum DS4148, and Figure 19b is a graph showing the results of measuring the cytotoxicity of MCF-7 breast cancer cell lines when treated for 72 hours according to the concentration of heat-treated Bifidobacterium longum DS4148 (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).

Figure 20a is a graph showing the results of measuring the cytotoxicity of the SK-BR-3 breast cancer cell line when treated for 48 hours according to the concentration of heat-treated Bifidobacterium longum DS4248, and Figure 20b is a graph showing the results of measuring the cytotoxicity of the SK-BR-3 breast cancer cell line when treated for 72 hours according to the concentration of heat-treated Bifidobacterium longum DS4148 (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).

Hereinafter, preferred examples are presented to aid understanding of the present invention. However, the following examples are provided solely to facilitate understanding of the present invention and are not intended to limit the scope of the present invention.

<Example 1> Extraction of extracellular vesicles (EVs)

1. Isolation and identification of strains

(1) Originated from Bifidobacterium adolecentis

The Korea Research Institute of Bioscience and Biotechnology isolated Bifidobacterium adolescentis DS0019, a human commensal microbial strain, from the oral cavity of an adult. The strain was isolated by scraping the oral mucosa with a sterile cotton swab, suspending the swab in 1 ml of physiological saline, serially diluting it, and spreading 100 μl of the diluted solution on MRS plate medium, culturing it anaerobically at 37°C for 2-3 days, and then isolating the strain from the medium.

Phylogenetic identification of Bifidobacterium adolescentis DS0019 was performed through base sequence analysis of the 16S rRNA gene, and compared with the 16S rRNA genes of Bifidobacterium strains based on the similarity of the primary and secondary structures of the 16S rRNA gene using the PHYDIT program. Specifically, to identify the strain, the base sequence of the 16S rRNA of the DS0019 strain was analyzed using universal primers 27F (5'-AGA GTT TGA TCM TGG CTC A-3', SEQ ID NO: 2) and 1492R (5'-TAC GGY TAC CTT GTT ACG ACT T-3', SEQ ID NO: 3).

As a result, the base sequence of 16S rRNA showed 99.68% homology with the standard strain Bifidobacterium adolecentis KCTC 3216(T), which is a conventional strain, confirming that the strain of the invention is a novel strain, and its 16S rRNA sequence is shown in sequence number 1.

Then, it was deposited at the Microbial Resource Center of the Korea Research Institute of Bioscience and Biotechnology on January 17, 2024, and was assigned the accession number KCTC15776BP.

(2) Origin of Bifidobacterium longum

Bifidobacterium longum DS4022 and DS4148 ( Bifidobacterium longum DS4022, Bifidobacterium longum DS4148), human commensal microbial strains isolated from the feces of the elderly and newborns at the Korea Research Institute of Bioscience and Biotechnology, were used in the experiment. Phylogenetic identification of Bifidobacterium longum DS4022 and DS4148 ( Bifidobacterium longum DS4022, Bifidobacterium longum DS4148) was performed through base sequence analysis of the 16S rRNA gene, and the 16S rRNA genes of Bifidobacterium strains were compared with the 16S rRNA genes of other Bifidobacterium strains based on the similarity of the primary and secondary structures of the 16S rRNA gene using the PHYDIT program.

As a result of sequence comparison analysis, both strains were confirmed as new strains, and the 16S rRNA sequence of Bifidobacterium longum DS4022 is shown in sequence number 4, and the 16S rRNA sequence of Bifidobacterium longum DS4148 is shown in sequence number 5.

Then, it was deposited at the Microbial Resource Center of the Korea Research Institute of Bioscience and Biotechnology on January 17, 2024, and Bifidobacterium longum DS4022 was assigned the accession number KCTC15778BP.

In addition, Bifidobacterium longum DS4148 was deposited at the Microbial Resource Center of the Korea Research Institute of Bioscience and Biotechnology on January 17, 2024, and was assigned the accession number KCTC15777BP.

2. Extraction of extracellular vesicles

Bifidobacterium adolescentis DS0019, Bifidobacterium longum DS4022, and DS4148 were each inoculated into MRS broth (MB cell, Korea ) supplemented with 0.5 g/L L-cysteine HCl (MB cell, Korea), and cultured in an anaerobic incubator (MGC Anaerobic jar, Mitsubishi gas chemical company inc, Japan) containing an anaerobic gas pack (AnaeroPouch-Anaero, Mitsubishi gas chemical company inc, Japan) at 37°C. Afterwards, the culture medium was centrifuged at 7,000 g, 4 ° C, for 30 minutes in a high-speed centrifuge (LABOGENE, 2236R, GRF-L1000-6 rotor, Denmark) to separate only the supernatant. Then, the supernatant was filtered of cell debris using a syringe filter with a pore size of 0.45 μm (25 mm FLL/MLS Acrylic Transparent membrane.PES 0.45 μm blst Sterile, GVS North America, USA). The obtained supernatant was concentrated using a tangential flow filtration (TFF) cassette system (Masterflex, Satorious AG, Germany) equipped with a 100 kDa filter (Vivaflow 200 PES 100,000 MWCO, Satorious AG, Germany). Afterwards, the extract was further filtered using a 0.22 μm syringe filter (25 mm FLL/MLS Acrylic Transparent membrane. PES 0.22 μm blst Sterile, GVS North America, USA). The filtered extract was ultracentrifuged at 160,000 g for 2 h at 4°C using a Type 70 Ti Rotor (Beckman Instruments, Brea, CA, USA). The extracellular vesicle pellet was then obtained and resuspended in PBS. Each extracted extracellular vesicle (EV) was stored at -80°C.

<Example 2> Verification of extracellular vesicles (EVs) derived from Bifidobacterium adolecentis or Bifidobacterium longum

The size and morphology of the extracellular vesicles (EVs) derived from Bifidobacterium adolecentis or Bifidobacterium longum extracted in Example 1 were confirmed using a transmission electron microscope (TEM). Each EV solution was diluted with PBS, and 10 μl of the resulting suspension and uranyl acetate (2%) were dropped onto a 300-mesh copper grid (EMS, Hatfield, PA, USA) to perform negative staining. An H-7650 transmission electron microscope (Hithachi, Japan) was used for TEM, and the results are shown in Figures 1a to 1c.

As can be seen from the transmission electron microscope images of FIGS. 1A to 1C, it was confirmed that extracellular vesicles derived from Bifidobacterium adolecentis DS0019, Bifidobacterium longum DS4022, or Bifidobacterium longum DS4148 according to the present invention were produced.

Additionally, the dynamic light scattering distribution of the EV solution was measured using Nasonight (Malvern NanoSight LM10, Malvern Instruments, Worcestershire, UK) and ISO 19430 (Particle tracking analysis-PTA method) method and NanoSight NTA 3.4 Analytical software, and the results are shown in Table 1 below.

division medium
Mean
(nm) Mode
Mode
(nm) standard deviation
SD
(nm) Water concentration
Number Conc.
(particles/mL) Number of verified tracer particles
Number of Valid Tracks Analysis Quality Inspection*
Quality Check DS0019 strain 170.0 121.6 67.5 1.57 X 10 ¹² 4331.3 Pass DS4022 strain 195.3 144.5 82.2 1.59 X 10 ¹¹ 4246.0 Pass DS4148 strain 208.6 173.7 80.2 5.85 X 10 ¹¹ 2963.3 Pass

* Quality Check Criteria: Pass(Number of Valid Track >500), Fail (Number of Valid Track ≤500)

As can be confirmed from the results measured by the nanoparticle tracking analyzer (NTA) in Table 1 above, the mode (Mode) of the extracellular vesicles of Bifidobacterium adolescentis DS0019 ( Bifidobacterium adolescentis DS0019) was 121.6 nm, the mean (Mean) was 170.0 nm, and the width of the size distribution (standard deviation, SD) was 67.5 nm. In addition, the measured particle number concentration of the sample was measured as 1.57 x 10 ¹² particles/mL (dilution factor X 1000). Due to its small size, it can enter the blood vessels and mediate autocrine, paracrine, and endocrine effects through blood circulation, and can control the progression of diseases such as cancer.

In addition, the mean (Mean) of the extracellular vesicles of Bifidobacterium longum DS4022 derived from the elderly was observed to be 195.3 nm, the mode was 144.5 nm, and the width of the size distribution (standard deviation, SD) was 82.2 nm. In addition, the measured particle number concentration of the sample was measured to be 1.59 x10 ¹¹ particles/mL (dilution factor X 1000). Due to its small size, it can enter the blood vessels and mediate autocrine, paracrine, and endocrine effects through blood circulation, and can regulate the progression of diseases such as cancer.

In addition, the mean (Mean) of the extracellular vesicles of the Bifidobacterium longum DS4148 strain derived from newborns was observed to be 208.6 nm, the mode was 173.7 nm, and the width of the size distribution (standard deviation, SD) was 80.2 nm. In addition, the measured particle number concentration of the sample was determined to be 5.85 x 10 ¹¹ particles/mL (dilution factor X 1000). Due to its small size, it can enter the blood vessels and mediate autocrine, paracrine, and endocrine effects through blood circulation, and can regulate the progression of diseases such as cancer.

<Example 3> Viability analysis of breast cancer cell lines

The effects of extracellular vesicles (EVs) from Bifidobacterium adolecentis (DS0019, Korean Collection for Type Cultures, Korea), Bifidobacterium longum (DS4022, Korean Collection for Type Cultures, Korea), and Bifidobacterium longum (DS4148, Korean Collection for Type Cultures, Korea) on breast cancer cells were investigated using the MCF7 cell line (Korea Cell Line Bank, Seoul, Korea). A total of 5 × 10 ³ cells were cultured in RPMI-1640 (Welgene, Korea) medium supplemented with 10% FBS (fetal bovine serum, Gibco, USA) and 1% antibiotic (antibiotic-antimycotic, Gibco, USA). EVs were treated after 24 h of culture. Control cells were treated with PBS, and experimental cells were treated with EVs at two-fold intervals at a concentration of 1 μg/ml for 24 h. Afterwards, the number of viable cells was confirmed by performing an MTT assay, and the results are shown in Figures 2a and 2b.

As can be seen in Fig. 2a, the extracellular vesicles of Bifidobacterium adolescentis DS0019 according to the present invention induced apoptosis in the breast cancer cell line MCF-7, indicating that it has anticancer activity.

In addition, as can be confirmed in FIG. 2b, the extracellular vesicles of Bifidobacterium longum DS4022 derived from the elderly or Bifidobacterium longum DS4148 derived from the newborn according to the present invention induced apoptosis in the breast cancer cell line MCF-7, indicating that they have anticancer activity.

<Example 4> Confirmation of the efficacy of combined treatment with doxorubicin and extracellular vesicles (EVs) derived from Bifidobacterium adolecentis.

The effects of combined treatment with doxorubicin (DOX, Adriamycin) and extracellular vesicles (EVs) from Bifidobacterium adolecentis DS0019 (Korean Collection for Type Cultures, Korea) on breast cancer cells were investigated using MCF7 cell lines (Korea Cell Line Bank, Seoul, Korea). A total of 5 × 10 ³ cells were cultured in RPMI-1640 (Welgene, Korea) medium supplemented with 10% FBS (Fetal bovine serum, Gibco, USA) and 1% antibiotic (antibiotic-antimycotic, Gibco, USA). EVs were treated after 24 h of incubation. Specifically, MCF7 cells were simultaneously treated with 1 μM doxorubicin (DOX) and 10 μg/mL EVs from Bifidobacterium adolecentis DS0019 for 48 h, and the results were compared with the control group treated with doxorubicin alone or with no treatment. Afterwards, the number of viable cells was confirmed by performing an MTT assay, and the results are shown in Figure 3a.

As shown in Fig. 3a, the group treated with 1 μM doxorubicin and EVs of Bifidobacterium adolecentis DS0019 showed a 17.7% cell reduction compared to the group that was not treated. This confirmed the synergistic effect of the combined use of the anticancer drug (doxorubicin) and EVs of Bifidobacterium adolecentis DS0019.

<Example 5> In breast cancer cell lines B. longum Confirmation of enhanced anticancer efficacy after treatment with EVs derived from the DS4022 strain

Using MCF cell line (Korea Cell Line Bank, Seoul, Korea), doxorubicin (DOX, Adriamycin) and Bifidobacterium longum The effect of combined treatment with extracellular vesicles (EVs) from DS4022 (Korean Collection for Type Cultures, Korea) on breast cancer cells was investigated. A total of 5 × 10 ³ cells were cultured in RPMI-1640 (Welgene, Korea) medium supplemented with 10% FBS (Fetal bovine serum, Gibco, USA) and 1% antibiotics (antibiotic-antimycotic, Gibco, USA). EVs were treated after 24 h of culture. Specifically, MCF7 cells were simultaneously treated with 0.1 and 1 μM doxorubicin (DOX) and 100 μg/mL EVs from Bifidobacterium longum DS4022 for 48 h, and the treatment was compared with the control group treated with doxorubicin alone or with no treatment. Afterwards, the number of viable cells was confirmed by MTT assay, and the results are shown in Fig. 3b.

As shown in Fig. 3b, the groups treated with 0.1 and 1 μM doxorubicin and EVs of Bifidobacterium longum DS4022 showed a cell reduction of 29.3% and 23.3%, respectively, compared to the group that was not treated. This confirmed the synergistic effect of the combined use of anticancer drug (doxorubicin) and EVs of Bifidobacterium longum DS4022.

<Example 6> In breast cancer cell lines B. longum Confirmation of enhanced anticancer efficacy after treatment with EVs derived from the DS4148 strain

Using MCF cell line (Korea Cell Line Bank, Seoul, Korea), doxorubicin (DOX, Adriamycin) and Bifidobacterium longum The effect of combined treatment with extracellular vesicles (EVs) from DS4148 (Korean Collection for Type Cultures, Korea) on breast cancer cells was investigated. A total of 5 × 10 ³ cells were cultured in RPMI-1640 (Welgene, Korea) medium supplemented with 10% FBS (Fetal bovine serum, Gibco, USA) and 1% antibiotics (antibiotic-antimycotic, Gibco, USA). EVs were treated after 24 h of culture. Specifically, MCF7 cells were simultaneously treated with 0.1 μM doxorubicin (DOX) and 100 μg/mL EVs from Bifidobacterium longum DS4148 for 48 h, and compared with the control group treated with doxorubicin alone or with no treatment. Afterwards, the number of viable cells was determined by MTT assay, and the results are shown in Fig. 3c.

As shown in Fig. 3c, the group treated with 0.1 μM doxorubicin and EVs of Bifidobacterium longum DS4148 showed a 56.9% cell reduction compared to the group that was not treated. This confirmed the synergistic effect of the combined use of anticancer drug (doxorubicin) and EVs of Bifidobacterium longum DS4148.

<Example 7> Proteomics analysis of cellular responses after EV treatment in breast cancer cell lines

The effects of extracellular vesicles (EVs) from Bifidobacterium adolescentis DS0019, Bifidobacterium longum DS4022, and Bifidobacterium longum DS4148 on breast cancer cells were investigated using proteomics analysis using MCF7 cell lines (Korea Cell Line Bank, Seoul, Korea). A total of 2 × 10 ⁶ cells were cultured in RPMI-1640 (Welgene, Korea) medium supplemented with 10% FBS (Fetal bovine serum, Gibco, USA) and 1% antibiotic-antimycotic, Gibco, USA. EVs were treated after 24 h of culture. Among the control cells, the negative control was treated with PBS, and the positive control was treated with Adriamycin (doxorubicin). The cells of the experimental groups were treated with EV at a concentration of 320 μg/mL ( Bifidobacterium adolescentis DS0019), 320 μg/mL ( B. longum DS4022 strain - derived from elderly people), or 2.56 g/mL ( B. longum DS4148 strain - derived from newborns) for 24 hours. The treated cells were then harvested and subjected to proteomics. The above concentrations were treated as the amount of protein that resulted in 80% viable cell numbers.

The experiment was repeated three times, focusing on the DS0019 experimental groups (A1, A2, A3) and the PBS-treated control group (PBS 1, 2, 3). Proteins with a difference of 1.5 times or more compared to the control group and a p-value of less than 0.05 are displayed as a heat map in Fig. 4a, with increased proteins displayed in red and decreased proteins displayed in blue. Specifically, proteins that increased in breast cancer cells treated with DS0019 include HMOX1, ENDOD1, SDF2, IGF2, CTSL, and MAVS, while decreased proteins include MCM4, MPG, RTF2, MYADM, and CYB561. The increased proteins are proteins related to oxidative stress regulation, induction of immune response in stress response, and quality of extracellular vesicle proteins under stress conditions. In other words, proteins related to protein degradation and immune response regulation were increased. The common mechanisms of the decreased proteins are proteins involved in DNA repair and maintaining genome stability, regulating cell growth and proliferation, and maintaining redox reactions and cell membrane functions. Among these proteins, MCM proteins, in particular, are known to be overexpressed not only in breast cancer but also in cervical cancer and Hodgkin's lymphoma, and are involved in promoting cancer progression by increasing cell proliferation. It was found that the decrease in MCM4 protein inhibited the proliferation of breast cancer cells. Furthermore, MPG is associated with breast, lung, and colon cancer, and suppressing MPG protein can reduce the DNA repair capacity of cancer cells, potentially enhancing the efficacy of anticancer treatments.

In addition, the experiment was repeated three times focusing on the DS4022 experimental groups (E1, E2, E3) and the PBS-treated control group (PBS 1, 2, 3), and proteins with a difference of more than 1.5 times compared to the control group and a p-value of less than 0.05 were displayed as a heat map in Fig. 4b, where increased proteins are displayed in red and decreased proteins are displayed in blue. Specifically, proteins that increased in breast cancer cells treated with DS4022 include COL1A2, KRT86, FAU, PFN1, ATP6V1F, etc., and proteins that decreased include UBE2C, RHBDD2, WDR54, etc. Here, the increased proteins play an important role in intracellular protein quality control, oxidative stress regulation, and immune response regulation, and the decreased proteins are involved in tumor growth and cell growth regulation.

In addition, the experiment was repeated three times focusing on the DS4148 experimental group (I1, I2, I3) and the PBS-treated control group (PBS 1, 2, 3), and the proteins with a difference of more than 1.5 times compared to the control group and a p-value of less than 0.05 were displayed as a heat map in Fig. 4c, where increased proteins are displayed in red and decreased proteins are displayed in blue. Specifically, proteins that increased in breast cancer cells treated with DS4148 include FDPS, SELENOH, S100A8, SNAPIN, etc., and proteins that decreased include TRIP12, MACF1, ANKRD31, FMOD, etc. Here, the increased proteins are involved in cell signaling, maintenance of cell homeostasis, regulation of oxidative stress, regulation of inflammatory response, and regulation of immune response, and the decreased proteins are proteins related to the development of cancer in relation to DNA repair regulation, signaling, and immunity.

Therefore, through Figures 4a to 4c, it was found that DS0019, DS4022, and DS4148 were directly involved in the anticancer effect of breast cancer cells through changes in protein expression.

<Example 8> Preparation of killed cells of Bifidobacterium adolecentis or Bifidobacterium longum strains

1. Dry weight measurement of Bifidobacterium adolecentis or Bifidobacterium longum strains based on absorbance

When measuring CFU/ml using the flat plate method, it was confirmed that accurate CFU measurement was difficult in Bifidobacterium longum strains except for Bifidobacterium adolecentis strains due to differences in oxygen tolerance of Bifidobacterium strains, which are anaerobic strains. When CFU was titrated based on the flat plate method and the wet weight of the strains was measured using a tube, the difference in the mass of the bacteria was more than 10 times, reconfirming that the flat plate method is not a suitable method for measuring the weight of the strains. Figure 5a shows a schematic diagram of the flat plate method, and Figure 5b shows an actual image showing the results using the flat plate method.

Therefore, the dry weight was measured based on absorbance to measure the weight of the strain as follows.

Bifidobacterium adolescentis DS0019, Bifidobacterium longum DS4022, and Bifidobacterium longum DS4148 were cultured in MRS broth (MB cell, Korea) supplemented with 0.02% L-cysteine HCl (MB cell, Korea) for 4 days, respectively. The cultured strain colonies were inoculated into MRS liquid medium containing 0.02% L- cysteine HCl (MB cell, Korea) and cultured for 24, 48, and 72 h. The absorbance of the bacterial cultures was then measured at 600 nm using a spectrophotometer. After measuring the absorbance, 5 ml of the bacterial culture was centrifuged at 3,000 g and 4°C for 20 minutes, the supernatant was removed, and 1 ml of DPBS was added to wash the bacterial pellet.

Afterwards, the washed bacterial pellet and 4 ml of DPBS were added to an empty tube whose weight had been measured, and the process of centrifuging at 3,000 g and 4 °C for 20 minutes to remove the supernatant was repeated twice. The bacterial pellet from which the supernatant had been removed was dried in a dry oven at 40 °C for more than 24 hours until the color of the bacterial pellet became transparent. This process is shown in Fig. 6a, and Fig. 6b shows actual images of the bacterial pellet before drying, the bacterial pellet that was not completely dried, and the bacterial pellet that was completely dried.

The dry weight of the bacterial pellets cultured for 24, 48, and 72 hours was measured to measure the values at various absorbances, and the method for obtaining the dry weight is shown in Equation 1 below. In addition, the absorbance and the dry weight according to Equation 1 below are shown graphically in Figures 8a to 8c.

[Formula 1]

The linear regression equation of the graph (Fig. 8a) according to Bifidobacterium adolescentis DS0019 related to the above formula 1 is shown in formula 2 below, the linear regression equation of the graph (Fig. 8b) according to Bifidobacterium longum DS4022 related to the above formula 1 is shown in formula 3 below, and the linear regression equation of the graph (Fig. 8c) according to Bifidobacterium longum DS4148 is shown in formula 4 below.

[Formula 2]

Y=1.895X + 0.005

(R ² =0.964, where R ² stands for coefficient of determination)

[Formula 3]

Y=1.630X - 0.031

(R ² =0.845, where R ² stands for coefficient of determination)

[Formula 4]

Y=1.618X - 0.024

(R ² =0.828, where R ² stands for coefficient of determination)

2. Preparation of heat-treated strains (dead cells)

2-1. Preparation of Bifidobacterium adolecentis DS0019 killed cells

The strain was divided into 1.5 ml tubes so that the dry weight of the strain obtained by substituting the absorbance value into the above equation 2 was 10 mg/ml, and the strain was inactivated in a heating block at 80°C for 30 minutes to produce dead cells of the Bifidobacterium adolecentis strain. Afterwards, a portion of the sample was inoculated onto an MRS plate medium containing 0.02% L-cysteine HCl to confirm inactivation. The sample was stored in an ice box until the experiment to minimize other unnecessary reactions. This process is schematically shown in Fig. 7.

2-2. Preparation of Bifidobacterium longum DS4022 killed cells

The strain was divided into 1.5 ml tubes so that the dry weight of Bifidobacterium longum DS4022 obtained by substituting the absorbance value into the above equation 3 was 10 mg/ml, and the strain was inactivated in a heating block at 80°C for 30 minutes to prepare dead cells of the Bifidobacterium longum DS4022 strain.

Subsequently, a portion of the sample was inoculated onto MRS plate medium containing 0.02% L-cysteine HCl to confirm inactivation. The sample was stored in an ice box until the experiment to minimize unnecessary reactions. This process is schematically illustrated in Figure 7.

2-3. Preparation of Bifidobacterium longum DS4148 killed cells

The strain was divided into 1.5 ml tubes so that the dry weight of Bifidobacterium longum DS4148 obtained by substituting the absorbance value into the above equation 4 was 10 mg/ml, and the strain was inactivated in a heating block at 80°C for 30 minutes to prepare dead cells of the Bifidobacterium longum DS4148 strain.

Subsequently, a portion of the sample was inoculated onto MRS plate medium containing 0.02% L-cysteine HCl to confirm inactivation. The sample was stored in an ice box until the experiment to minimize unnecessary reactions. This process is schematically illustrated in Figure 7.

<Example 9> Analysis of breast cancer cell viability according to the concentration of Bifidobacterium adolecentis DS0019 strain (dead cells)

1. Breast cancer cell survival rate measurement process

Breast cancer cell lines MCF-7 and SK-BR-3 were seeded in 96-well plates at a density of 10,000 cells per 100 μl of RPMI1640 medium containing 10% fetal bovine serum and 1% antibiotic-antimycotic, and sufficient cell attachment was confirmed after 24 hours.

Afterwards, the killed Bifidobacterium adolescentis DS0019 sample prepared at a concentration of 10 mg/ml was diluted in RPMI1640 medium to 1 mg/ml, and diluted 10-fold to prepare samples of 1000, 100, 10, 1, and 0.1 μg/ml. The existing medium was removed, and 100 μl of each diluted sample was dispensed. After 24, 48, and 72 hours of dispensing, the medium containing the sample (Bifidobacterium adolescentis DS0019) was carefully removed using a multi-pipette. Afterwards, 100 μl of DPBS was dispensed, and the medium containing the killed Bifidobacterium adolecentis DS0019 was washed to remove unnecessary components, and the washed residual medium was carefully removed using a multi-pipette. After that, 100 μl of RPMI1640 was dispensed, and 10 μl of 5 mg/ml MTT assay (thiazolyl blue tetrazolium bromide) was additionally dispensed, and incubated at 37°C for 4 hours. The medium was removed using a multi-pipette, and 100 μl of DMSO was dispensed. Incubated at 37°C for 30 minutes in a dark place to completely dissolve the formazan crystals, and the breast cancer cell viability was measured by measuring the absorbance at 540 nm using a plate reader, which is shown in the graphs in Figs. 9a, 9b, 10a, and 10b.

2. MCF-7 breast cancer cell viability according to strain concentration and treatment time

2-1. The effect of heat-treated Bifidobacterium adolecentis DS0019 (killed cells) on MCF-7 breast cancer cells was measured for 48 hours.

Looking at Figure 9a, compared to the control group, no significant change was observed at low concentrations (0.1 to 100 μg/ml), but at high concentrations (1000 μg/ml), the viability of MCF-7 breast cancer cells was significantly reduced, confirming that there was an anticancer effect on MCF-7 breast cancer cells at a concentration of 1000 μg/ml, and through this, a concentration-dependent cell number reduction trend was observed.

2-2. The effect of heat-treated Bifidobacterium adolecentis DS0019 (killed cells) on MCF-7 breast cancer cells was measured for 72 hours.

Looking at Figure 9b, it was confirmed that the cell viability was lower overall than after 48 hours, and that the effect of the strain increased over time. It was confirmed that the cell viability decreased as the concentration increased from 0.1 μg/ml, 1 μg/ml, and 10 μg/ml, and when treated for 72 hours, a statistically significant anticancer effect was confirmed even at low concentrations, and in particular, the cell viability was confirmed to be the lowest at 1000 μg/ml.

Therefore, it was found that the longer the treatment time, the more the cell viability decreased, and the breast cancer cell inhibitory effect became stronger over time. In addition, it was confirmed that there was a statistically significant anticancer effect even at low concentrations when treated for 72 hours, confirming the anticancer effect dependent on the treatment time.

3. SK-BR-3 breast cancer cell viability according to strain concentration and treatment time

3-1. The effect of heat-treated Bifidobacterium adolecentis DS0019 (killed cells) on SK-BR-3 breast cancer cells was measured for 48 hours.

Looking at Figure 10a, there is no significant difference in cell viability compared to the control group at most concentrations (0.1, 1, 10, 100 μg/ml), but at a high concentration of 1000 μg/ml, the cell viability decreases to about 50% or less, confirming that the anticancer effect on SK-BR-3 breast cancer cells is high at high concentrations.

3-2. The effect of heat-treated Bifidobacterium adolecentis DS0019 (killed cells) on SK-BR-3 breast cancer cells was measured for 72 hours.

Looking at Figure 10b, it was confirmed that cell viability was lower overall after 48 hours, and the effect of the strain increased over time. At low concentrations (0.1, 1, 10, 100 μg/ml), cell viability was similar to the control group, and at high concentrations (1000 μg/ml), cell viability decreased significantly, confirming that high concentrations exhibited a strong anticancer effect against breast cancer.

<Example 10> Analysis of breast cancer cell viability according to the concentration of Bifidobacterium longum DS4022 strain (dead cells)

1. Breast cancer cell survival rate measurement process

Breast cancer cell lines MCF-7 and SK-BR-3 were seeded in 96-well plates at a density of 10,000 cells per 100 μl of RPMI1640 medium containing 10% fetal bovine serum and 1% antibiotic-antimycotic, and sufficient cell attachment was confirmed after 24 hours.

Afterwards, the killed Bifidobacterium longum DS4022 sample prepared at a concentration of 10 mg/ml was diluted in RPMI1640 medium to 1 mg/ml, and diluted 10-fold to prepare samples of 1000, 100, 10, 1, and 0.1 μg/ml. The existing medium was removed, and 100 μl of each diluted sample was dispensed. After 24, 48, and 72 hours of dispensing, the medium containing the sample (Bifidobacterium longum DS4022) was carefully removed using a multi-pipette. Afterwards, 100 μl of DPBS was dispensed to wash the medium containing the killed Bifidobacterium longum DS4022 to remove unnecessary components, and the washed residual medium was carefully removed using a multi-pipette. After that, 100 μl of RPMI1640 was dispensed, and 10 μl of 5 mg/ml MTT assay (thiazolyl blue tetrazolium bromide) was additionally dispensed and incubated at 37°C for 4 hours. The medium was removed using a multi-pipette, and 100 μl of DMSO was dispensed. The medium was incubated at 37°C for 30 minutes in a dark place to completely dissolve the formazan crystals, and the absorbance was measured at 540 nm using a plate reader to determine the breast cancer cell viability, which is shown in the graphs in Figs. 11a, 11b, 12a, and 12b.

2. MCF-7 breast cancer cell viability according to strain concentration and treatment time

2-1. The effect of heat-treated Bifidobacterium longum DS4022 (killed cells) on MCF-7 breast cancer cells was measured for 48 hours.

Looking at Figure 11a, compared to the control group, at a concentration of 0.1 μg/ml, the cell viability was lower than that of the control group, but there was no significant difference. At concentrations of 1, 10, and 100 μg/ml, the cell viability was lower than that of 0.1 μg/ml, but there was almost no difference in cell viability depending on the concentration. At the highest concentration of 1000 μg/ml, the viability of MCF-7 breast cancer cells was reduced the most, confirming that there was a cell growth inhibition or cell death effect above a certain concentration.

2-2. The effect of heat-treated Bifidobacterium longum DS4022 (killed cells) on MCF-7 breast cancer cells was measured for 72 hours.

Looking at Figure 11b, at a concentration of 0.1 μg/ml, the viability of MCF-7 breast cancer cells decreased slightly compared to the control group, at a concentration of 1 μg/ml, the viability of MCF-7 breast cancer cells decreased more than at a concentration of 0.1 μg/ml, and at a concentration of 10 μg/ml, the viability of MCF-7 breast cancer cells decreased to a level similar to that of the 1 μg/ml concentration. In addition, the viability of MCF-7 breast cancer cells decreased further as the concentration increased from 100 μg/ml to 1000 μg/ml, indicating that cytotoxicity was strong at concentrations above 100 μg/ml, and the cell viability decreased the most at a concentration of 1000 μg/ml, confirming that the anticancer effect against breast cancer was high at high concentrations.

3. SK-BR-3 breast cancer cell viability according to strain concentration and treatment time

3-1. The effect of heat-treated Bifidobacterium longum DS4022 (killed cells) on SK-BR-3 breast cancer cells was measured for 48 hours.

Looking at Figure 12a, the viability of SK-BR-3 breast cancer cells was similar to or slightly decreased compared to the control group at 0.1, 10, and 100 μg/ml. In addition, the viability of SK-BR-3 breast cancer cells was confirmed to be the lowest at 1 and 1000 μg/ml. This shows that although cell viability is not proportional to the concentration, it has a cell growth inhibition or cell death effect at a certain concentration, and it was confirmed that the anticancer effect against SK-BR-3 breast cancer was the best at 1000 μg/ml.

3-2. The effect of heat-treated Bifidobacterium longum DS4022 (killed cells) on SK-BR-3 breast cancer cells was measured for 72 hours.

Looking at Fig. 12b, it was confirmed that the cell viability was lower overall than after 48 hours, and the effect of the strain increased over time. At concentrations of 0.1 and 1 μg/ml, it was confirmed that the viability of SK-BR-3 breast cancer cells was significantly reduced compared to the control group, and at a concentration of 10 μg/ml, the viability of SK-BR-3 breast cancer cells was similar to the control group, and the viability of SK-BR-3 breast cancer cells was the lowest at the highest concentration of 1000 μg/ml. This showed that there was an effect of inhibiting the growth of SK-BR-3 breast cancer cells or inducing SK-BR-3 breast cancer cell death at a specific concentration, and it was confirmed that the anticancer effect on SK-BR-3 breast cancer was the best at 1000 μg/ml.

<Example 11> Analysis of breast cancer cell viability according to the concentration of Bifidobacterium longum DS4148 strain (dead cells)

1. Breast cancer cell survival rate measurement process

Breast cancer cell lines MCF-7 and SK-BR-3 were seeded in 96-well plates at a density of 10,000 cells per 100 μl of RPMI1640 medium containing 10% fetal bovine serum and 1% antibiotic-antimycotic, and sufficient cell attachment was confirmed after 24 hours.

Afterwards, the killed Bifidobacterium longum DS4148 sample prepared at a concentration of 10 mg/ml was diluted in RPMI1640 medium to 1 mg/ml, and diluted 10-fold to prepare samples of 1000, 100, 10, 1, and 0.1 μg/ml. The existing medium was removed, and 100 μl of each diluted sample was dispensed. After 24, 48, and 72 hours of dispensing, the medium containing the sample (Bifidobacterium longum DS4148) was carefully removed using a multi-pipette. Afterwards, 100 μl of DPBS was dispensed to wash the medium containing the killed Bifidobacterium longum DS4148 to remove unnecessary components, and the washed residual medium was carefully removed using a multi-pipette. After that, 100 μl of RPMI1640 was dispensed, and 10 μl of 5 mg/ml MTT assay (thiazolyl blue tetrazolium bromide) was additionally dispensed and incubated at 37°C for 4 hours. The medium was removed using a multi-pipette, and 100 μl of DMSO was dispensed. The medium was incubated at 37°C for 30 minutes in a dark place to completely dissolve the formazan crystals, and the breast cancer cell viability was measured by measuring the absorbance at 540 nm using a plate reader, which is shown in the graphs in Figs. 13a, 13b, 14a, and 14b.

2. MCF-7 breast cancer cell viability according to strain concentration and treatment time

2-1. The effect of heat-treated Bifidobacterium longum DS4148 (killed cells) on MCF-7 breast cancer cells was measured for 48 hours.

Looking at Fig. 13a, as the concentration increased from 0.1 to 1000 μg/ml, the viability of MCF-7 breast cancer cells tended to decrease, and in particular, the viability of MCF-7 breast cancer cells was the lowest at the highest concentration of 1000 μg/ml. This indicates that the viability of breast cancer cells tends to be dependent on the concentration of heat-treated Bifidobacterium longum DS4148, and that the anticancer effect on breast cancer cells is high at high concentrations.

2-2. The effect of heat-treated Bifidobacterium longum DS4148 (killed cells) on MCF-7 breast cancer cells was measured for 72 hours.

Looking at Figure 13b, it was confirmed that the viability of MCF-7 breast cancer cells was clearly reduced overall compared to the control group, and at concentrations of 1, 10, and 100 μg/ml, the viability of MCF-7 breast cancer cells tended to gradually decrease as the concentration increased, but it was maintained almost constant. Meanwhile, the viability of MCF-7 breast cancer cells was lowest at the lowest concentration of 0.1 μg/ml and the highest concentration of 1000 μg/ml. This shows that although it is not clear that the viability of MCF-7 breast cancer cells decreases in a concentration-dependent manner of heat-treated Bifidobacterium longum DS4148, it was found that overall, it had an anticancer effect on MCF-7 breast cancer cells at all concentrations.

3. SK-BR-3 breast cancer cell viability according to strain concentration and treatment time

3-1. The effect of heat-treated Bifidobacterium longum DS4148 (killed cells) on SK-BR-3 breast cancer cells was measured for 48 hours.

Looking at Figure 14a, at 0.1 μg/ml, the viability of SK-BR-3 breast cancer cells was higher than that of the control group, and at concentrations of 1, 10, and 100 μg/ml, the viability of SK-BR-3 breast cancer cells tended to increase as the concentration increased. Meanwhile, it was confirmed that the viability of SK-BR-3 breast cancer cells was the lowest at the highest concentration of 1000 μg/ml, which indicates that although cell viability is not proportional to the concentration, there is a cell growth inhibition or cell death effect at a specific concentration, and it was confirmed that the anticancer effect on the SK-BR-3 breast cancer cell line was the best at 1000 μg/ml.

3-2. The effect of heat-treated Bifidobacterium longum DS4148 (killed cells) on SK-BR-3 breast cancer cells was measured for 72 hours.

Looking at Figure 14b, at low concentrations of 0.1 and 1 μg/ml, the survival rate of SK-BR-3 breast cancer cells was higher than that of the control group, and at 10 and 100 μg/ml, the survival rate of SK-BR-3 breast cancer cells was lower than that of the control group, but showed a tendency to increase as the concentration increased, and the survival rate of SK-BR-3 breast cancer cells was lowest at 1000 μg/ml.

Through this, it was confirmed that the anticancer effect against the SK-BR-3 breast cancer cell line was the best at the highest concentration of 1000 μg/ml, although there was no tendency for cell viability to decrease as the concentration increased.

<Example 12> Analysis of cytotoxicity of breast cancer cell lines according to the concentration of Bifidobacterium adolecentis DS0019 strain (killed cells)

1. Cytotoxicity of MCF-7 breast cancer cell line according to strain concentration and treatment time.

1-1. 48-hour processing

Figure 15a is a graph showing the results of measuring the cytotoxicity of the MCF-7 breast cancer cell line when treated for 48 hours with different concentrations of heat-treated Bifidobacterium adolecentis DS0019 (killed cells). According to Figure 15a, there was no significant change at low concentrations (0.1, 1, 10, 100 μg/ml), and at the lowest concentration of 0.1 μg/ml, cytotoxicity actually decreased, and at the highest concentration of 1000 μg/ml, cytotoxicity was the highest, confirming that only high concentrations induced strong cytotoxicity.

1-2. 72-hour processing

Figure 15b is a graph showing the results of measuring the cytotoxicity of the MCF-7 breast cancer cell line according to the concentration of heat-treated Bifidobacterium adolecentis DS0019 (killed cells) when treated for 72 hours. According to Figure 15b, cytotoxicity increased overall, and strong cytotoxicity was confirmed at concentrations of 0.1, 1, 10, 100, and 1000 μg/ml. Therefore, it was found that killed Bifidobacterium adolecentis DS0019 cells can induce cytotoxicity in MCF-7 cells over time even at low concentrations, and that significant toxicity occurred even at low concentrations during long-term treatment (72 hours), indicating that the sensitivity tends to increase as the treatment time increases.

2. SK-BR-3 according to strain concentration and treatment time Cytotoxicity of breast cancer cell lines

2-1. 48-hour processing

Figure 16a is a graph showing the results of measuring the cytotoxicity of the SK-BR-3 breast cancer cell line when treated for 48 hours with different concentrations of heat-treated Bifidobacterium adolecentis DS0019 (killed cells). According to Figure 16a, there was no significant change at low concentrations (0.1, 1, 10, 100 μg/ml), and at the lowest concentration of 0.1 μg/ml, cytotoxicity actually decreased, and at the highest concentration of 1000 μg/ml, cytotoxicity was the highest, confirming that strong cytotoxicity was induced at high concentrations.

2-2. 72-hour processing

Figure 16b is a graph showing the results of measuring the cytotoxicity of the SK-BR-3 breast cancer cell line when treated for 72 hours with different concentrations of heat-treated Bifidobacterium adolecentis DS0019 (killed cells). According to Figure 16b, the cytotoxicity increased only slightly at low concentrations (0.1, 1, 10, 100 μg/ml), and the highest cytotoxicity was observed at the highest concentration of 1000 μg/ml, confirming that the cytotoxicity was strongest at high concentrations. In addition, it was confirmed that cytotoxicity occurred at a lower concentration in SK-BR-3 cells at 72 hours than at 48 hours, indicating that cytotoxicity was more evident with long-term exposure.

Therefore, it was confirmed that there was a statistically significant anticancer effect even at low concentrations when treated for 72 hours, and it was found that 72 hours of treatment was more effective in cell death than 48 hours of treatment at the same dose. In addition, heat-treated Bifidobacterium adolecentis DS0019 (killed cells) showed a cell death effect in MCF7 and SK-BR-3 breast cancer cell lines, indicating that it is effective in luminal and HER2 subtype breast cancer among breast cancers. Among them, it was confirmed that a more effective anticancer effect was shown in the luminal subtype, which accounts for more than 70% of breast cancer, when treated for 72 hours.

<Example 13> Analysis of cytotoxicity of breast cancer cell lines according to the concentration of Bifidobacterium longum DS4022 strain (killed cells)

1. Cytotoxicity of MCF-7 breast cancer cell line according to strain concentration and treatment time.

1-1. 48-hour processing

Figure 17a is a graph showing the results of measuring the cytotoxicity of the MCF-7 breast cancer cell line when treated for 48 hours with different concentrations of heat-treated Bifidobacterium longum DS4022 (killed cells). According to Figure 17a, at the lowest concentration of 0.1 μg/ml, cytotoxicity was minimal, and at 1, 10, and 100 μg/ml, there was no clear trend of increasing or decreasing cytotoxicity, but it remained almost constant, and it was higher than at the lowest concentration of 0.1 μg/ml. The cytotoxicity was highest at the highest concentration of 1000 μg/ml, confirming that strong cytotoxicity was induced at high concentrations.

1-2. 72-hour processing

Figure 17b is a graph showing the results of measuring the cytotoxicity of the MCF-7 breast cancer cell line when treated for 72 hours with different concentrations of heat-treated Bifidobacterium longum DS4022 (killed cells). According to Figure 17b, cytotoxicity tends to increase as the concentration increases, and cytotoxicity was highest at the highest concentration of 1000 μg/ml.

Through this, it was confirmed that the higher the concentration, the more cytotoxicity increases, and also, it was found that significant toxicity occurs even at low concentrations during long-term treatment (72 hours), indicating that sensitivity tends to increase as the treatment time increases.

2. SK-BR-3 according to strain concentration and treatment time Cytotoxicity of breast cancer cell lines

2-1. 48-hour processing

Figure 18a is a graph showing the results of measuring the cytotoxicity of the SK-BR-3 breast cancer cell line when treated for 48 hours with various concentrations of heat-treated Bifidobacterium longum DS4022 (killed cells). According to Figure 18a, cytotoxicity generally increased with increasing concentration, with 1 and 1000 μg/ml exhibiting the highest cytotoxicity. This confirms that cytotoxicity is high at specific concentrations.

2-2. 72-hour processing

Figure 18b is a graph showing the results of measuring the cytotoxicity of the SK-BR-3 breast cancer cell line when treated for 72 hours with different concentrations of heat-treated Bifidobacterium longum DS4022 (killed cells). According to Figure 18b, the cytotoxicity was significantly higher at low concentrations (0.1 and 1 μg/ml) and high concentrations (1000 μg/ml) than at intermediate concentrations (10 and 100 μg/ml). This confirmed that cytotoxicity was high at a specific concentration. In addition, in SK-BR-3 cells, cytotoxicity was generally higher at 72 hours than at 48 hours, indicating that cytotoxicity becomes more pronounced with long-term exposure.

<Example 14> Analysis of cytotoxicity of breast cancer cell lines according to the concentration of Bifidobacterium longum DS4148 strain (killed cells)

1. Cytotoxicity of MCF-7 breast cancer cell line according to strain concentration and treatment time.

1-1. 48-hour processing

Figure 19a is a graph showing the results of measuring the cytotoxicity of the MCF-7 breast cancer cell line when treated for 48 hours according to the concentration of heat-treated Bifidobacterium longum DS4148 (killed cells). According to Figure 19a, at low concentrations (0.1 and 1 μg/ml), cytotoxicity was almost non-existent and no significant change was observed. At intermediate concentrations (10, 100 μg/ml), the cytotoxicity did not show a clear trend of increasing or decreasing with concentration but remained almost constant. At high concentration (1000 μg/ml), cytotoxicity was the highest. This indicates that strong cytotoxicity was induced at high concentrations.

1-2. 72-hour processing

Figure 19b is a graph showing the results of measuring the cytotoxicity of the MCF-7 breast cancer cell line when treated for 72 hours according to the concentration of heat-treated Bifidobacterium longum DS4148 (killed cells). According to Figure 19b, in the concentration range of 1 to 1000 μg/ml, cytotoxicity tended to increase as the concentration increased, but exceptionally, cytotoxicity was highest at the lowest concentration (0.1 μg/ml) and the highest concentration (1000 μg/ml). This confirmed that cytotoxicity was high at a specific concentration, and compared to the 48-hour treatment, cytotoxicity was higher overall in the case of long-term treatment (72 hours), showing a tendency for sensitivity to increase as the treatment time increased.

2. SK-BR-3 according to strain concentration and treatment time Cytotoxicity of breast cancer cell lines

2-1. 48-hour processing

Figure 20a is a graph showing the results of measuring the cytotoxicity of the SK-BR-3 breast cancer cell line when treated for 48 hours according to the concentration of heat-treated Bifidobacterium longum DS4148 (killed cells). According to Figure 20a, the cytotoxicity levels were negative (minus) at concentrations of 0.1, 10, and 100 μg/ml, indicating no cytotoxicity. At a concentration of 1000 μg/ml, cytotoxicity was relatively the highest compared to other low concentrations, but the degree was not significant, indicating that the overall cytotoxicity ratio was not high.

2-2. 72-hour processing

Figure 20b is a graph showing the results of measuring the cytotoxicity of the SK-BR-3 breast cancer cell line when treated for 72 hours according to the concentration of heat-treated Bifidobacterium longum DS4148 (dead cells). According to Figure 20b, at low concentrations (0.1 and 1 μg/ml), the degree of cytotoxicity was negative (minus), indicating no cytotoxicity. Overall, there was no clear tendency for cytotoxicity to decrease or increase as the concentration increased, but cytotoxicity was high at the highest concentration (1000 μg/ml). This confirmed that cytotoxicity was high at high concentrations. In addition, in SK-BR-3 cells, cytotoxicity was generally higher at 72 hours than at 48 hours, indicating that cytotoxicity was more evident with long-term exposure.

Therefore, even with the same dose, it was confirmed that there was a statistically significant anticancer effect even at low concentrations when treated for 72 hours, and it was found that 72 hours of treatment was more effective in cell death than 48 hours of treatment. In addition, heat-treated Bifidobacterium adolecentis DS0019 (killed cells), Bifidobacterium longum DS4022 (killed cells), and heat-treated Bifidobacterium longum DS4148 (killed cells) showed a cell death effect in MCF7 and SK-BR-3 breast cancer cell lines, indicating that they are effective in liminal and HER2 subtype breast cancer among breast cancers. Among them, it was confirmed that a more effective anticancer effect was shown in the luminal subtype, which accounts for more than 70% of breast cancer, when treated for 72 hours.

[Accession number]

Name of depositor: Korea Research Institute of Bioscience and Biotechnology, Biological Resource Center (KCTC)

Accession number: KCTC15776BP

Date of acceptance: 20240117

Name of depositor: Korea Research Institute of Bioscience and Biotechnology, Biological Resource Center (KCTC)

Accession number: KCTC15778BP

Date of acceptance: 20240117

Name of depositor: Korea Research Institute of Bioscience and Biotechnology, Biological Resource Center (KCTC)

Accession number: KCTC15777BP

Date of acceptance: 20240117

Claims (17)

Bifidobacterium adolecentis DS0019 ( Bifidobacterium adolescentis DS0019) KCTC15776BP. In the first paragraph, the Bifidobacterium adolecentis DS0019 ( Bifidobacterium adolescentis DS0019) KCTC15776BP is Bifidobacterium adolescentis DS0019 containing 16s rRNA of sequence number 1. ( Bifidobacterium adolescentis DS0019) KCTC15776BP. Bifidobacterium longum DS4022 ( Bifidobacterium longum DS4022) KCTC15778BP. In the third paragraph, the Bifidobacterium longum DS4022 (Bifidobacterium longumDS4022) KCTC15778BP is Bifidobacterium longum DS4022 containing 16s rRNA of sequence number 4. (Bifidobacterium longumDS4022) KCTC15778BP. Bifidobacterium longum DS4148 ( Bifidobacterium longum DS4148) KCTC15777BP. In the fifth paragraph, the Bifidobacterium longum DS4148 ( Bifidobacterium longum DS4148) KCTC15777BP is Bifidobacterium longum DS4148 containing 16s rRNA of sequence number 5. ( Bifidobacterium longum DS4148) KCTC15777BP. Bifidobacterium adolecentis DS0019 A pharmaceutical composition for preventing or treating cancer, comprising ( Bifidobacterium adolescentis DS0019) KCTC15776BP or its extracellular vesicles (EV); Bifidobacterium longum DS4022 ( Bifidobacterium longum DS4022) KCTC15778BP or its extracellular vesicles (EV); or Bifidobacterium longum DS4148 ( Bifidobacterium longum DS4148) KCTC15777BP or its extracellular vesicles (EV). In claim 7, the Bifidobacterium adolecentis DS0019 ( Bifidobacterium adolescentis DS0019) KCTC15776BP is a pharmaceutical composition for preventing or treating cancer, comprising 16s rRNA of sequence number 1. In the seventh paragraph, the Bifidobacterium longum DS4022 ( Bifidobacterium longum DS4022) KCTC15778BP is a pharmaceutical composition for preventing or treating cancer, comprising 16s rRNA of sequence number 4. In the seventh paragraph, the Bifidobacterium longum DS4148 ( Bifidobacterium longum DS4148) KCTC15777BP is a pharmaceutical composition for preventing or treating cancer, comprising 16s rRNA of sequence number 5. A pharmaceutical composition for preventing or treating cancer, wherein the extracellular vesicles in claim 7 have an average diameter of 20 nm to 400 nm. In claim 7, the cancer is breast cancer, brain cancer, nerve cancer, colon cancer, lung cancer, small cell lung cancer, bronchial cancer, stomach cancer, liver cancer, biliary tract cancer, gallbladder cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, nasopharyngeal cancer, oropharyngeal cancer, laryngeal cancer, oral cancer, salivary gland cancer, skin, eye or organ melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, colon cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, vaginal cancer, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, peritoneal cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal cord tumor, A pharmaceutical composition for the prevention or treatment of cancer, wherein the composition is any one selected from the group consisting of brainstem glioma, pituitary adenoma, and metastatic cancer of other organs thereof. A pharmaceutical composition for preventing or treating cancer, wherein the pharmaceutical composition is administered in combination with an anticancer agent in claim 7. A pharmaceutical composition for preventing or treating cancer, wherein the anticancer agent is administered simultaneously, sequentially, or separately in claim 13. A pharmaceutical composition for preventing or treating cancer, wherein the anticancer agent comprises at least one selected from the group consisting of doxorubicin, paclitaxel, and docetaxel. Bifidobacterium adolecentis DS0019 A food composition for preventing or improving cancer, comprising ( Bifidobacterium adolescentis DS0019) KCTC15776BP or extracellular vesicles thereof; ( Bifidobacterium longum DS4022) KCTC15778BP or extracellular vesicles (EV) thereof; or ( Bifidobacterium longum DS4148) KCTC15777BP or extracellular vesicles (EV) thereof. In claim 16, the cancer is breast cancer, brain cancer, nerve cancer, colon cancer, lung cancer, small cell lung cancer, bronchial cancer, stomach cancer, liver cancer, biliary tract cancer, gallbladder cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, nasopharyngeal cancer, oropharyngeal cancer, laryngeal cancer, oral cancer, salivary gland cancer, skin, eye or organ melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, colon cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, vaginal cancer, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, peritoneal cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal cord tumor, A food composition for preventing or improving cancer, wherein the food composition is any one selected from the group consisting of brainstem glioma, pituitary adenoma, and metastatic cancer of other organs thereof.