WO2024063545A1 - Composition comprising lactobacillus plantarum strain for improving intestinal metabolite composition - Google Patents

Abstract

The present invention relates to a composition comprising a Lactobacillus plantarum strain for improving intestinal metabolite composition. The Lactobacillus plantarum strain according to one embodiment can reduce intestinal polyamines (for example, spermidine), and thus the present invention can be effectively used as a pharmaceutical composition, a food (a health functional food), a feed composition and an anti-cancer adjuvant, all of which are for preventing, alleviating, and treating cancer, or improving the prognosis thereof and aiding anticancer treatment.

Description

Composition for improving intestinal metabolite composition containing Lactobacillus plantarum strains

It relates to a composition for improving the composition of intestinal metabolites containing Lactobacillus plantarum strains.

Microbiome refers to microorganisms and their entire genetic information existing in a specific environment. In addition to the human body, microbiome information is being used in various fields such as animals, agriculture, the ocean, and the environment. In particular, with the development of human microbiome research based on the development of genetic information analysis and data analysis technology, based on this, microbiome information is being used. The growth of the diagnostic and healthcare industries is expected.

Human intestinal microorganisms not only decompose various substances that human enzymes cannot decompose and convert them into nutrients that human cells can absorb, but also play a role in preventing pathogen infection by inhibiting the growth of harmful bacteria originating from outside. These intestinal bacteria themselves or various metabolites secreted by the bacteria play a role in activating or regulating the body's immune response by stimulating numerous immune cells present in intestinal cells. In addition, due to the number and diversity of symbiotic microbial genes that are more than 100 times that of humans, the human microbiome is considered the second genome of humans, and its importance is recognized.

In particular, due to the advancement of human microbiome research, research results showing that intestinal microorganisms are directly or indirectly related to a number of human diseases are rapidly increasing, and research showing that intestinal microorganisms are also related to various cancers in the human body is also increasing.

Meanwhile, polyamines are known to play a role in the proliferation, differentiation, and development of eukaryotes. Polyamines include spermine, spermidine, and the diamine precursor putrescine and are low molecular weight organic polycations with two or more amino groups. The intracellular concentration of polyamines can be maintained within a certain physiological range through several regulatory mechanisms in normal cells. Additionally, polyamine metabolism is known to be dysregulated in many neoplastic conditions, including cancer. Polyamine levels are elevated in various types of cancer and a link between polyamine metabolism and oncogenic pathways such as mTOR and RAS pathways is known. Therefore, polyamines may have potential as therapeutic targets in the prevention and treatment of cancer.

Therefore, there is a need to develop substances for preventing or improving diseases by improving the composition of intestinal metabolites using the human microbiome.

One aspect is to provide a composition for improving the intestinal metabolite composition of an individual comprising a Lactobacillus plantarum strain, a culture of the strain, a lysate of the strain, or a mixture thereof as an active ingredient.

Another aspect is to provide a health functional food for improving the intestinal metabolite composition of an individual containing a Lactobacillus plantarum strain, a culture of the strain, a lysate of the strain, or a mixture thereof as an active ingredient. will be.

Another aspect is to provide a probiotic composition for improving the intestinal metabolite composition of an individual comprising a Lactobacillus plantarum strain, a culture of the strain, a lysate of the strain, or a mixture thereof.

Another aspect is to provide a food composition for improving the intestinal metabolite composition of an individual comprising a Lactobacillus plantarum strain, a culture of the strain, a lysate of the strain, or a mixture thereof.

Another aspect provides a feed composition for improving the intestinal metabolite composition of an individual comprising a Lactobacillus plantarum strain, a culture of the strain, lysate of the strain, or a mixture thereof as an active ingredient. will be.

Another aspect is a pharmaceutical composition for the prevention or treatment of proliferative diseases, specifically cancer, comprising a Lactobacillus plantarum strain, a culture of the strain, a lysate of the strain, or a mixture thereof as an active ingredient. is to provide.

Another aspect is to provide an anti-cancer adjuvant comprising a Lactobacillus plantarum strain, a culture of the strain, a lysate of the strain, or a mixture thereof as an active ingredient.

Another aspect is a method of improving the intestinal metabolite composition of a subject comprising administering to the subject an effective amount of a Lactobacillus plantarum strain, a culture of the strain, a lysate of the strain, or a mixture thereof. It is provided.

Another aspect is a method for preventing or treating cancer comprising administering to a subject an effective amount of a Lactobacillus plantarum strain, a culture of the strain, a lysate of the strain, or a mixture thereof. It is provided.

Another aspect provides the use of an effective amount of a Lactobacillus plantarum strain, a culture of the strain, a lysate of the strain, or a mixture thereof for the manufacture of a formulation for improving the intestinal metabolite composition of an individual. will be.

Another aspect is an effective amount of a Lactobacillus plantarum strain, a culture of the strain, a lysate of the strain, or a mixture thereof for the production of a pharmaceutical agent or health functional food for preventing or treating cancer. It provides a purpose.

One aspect is a composition for improving the intestinal metabolite composition of an individual comprising a Lactobacillus plantarum strain, a culture of the strain, a lysate of the strain, or a mixture thereof as an active ingredient (e.g., pharmaceutical composition) is provided.

Another aspect provides a method of improving the intestinal metabolite composition of a subject, comprising administering to the subject an effective amount of a Lactobacillus plantarum strain, a culture of the strain, a lysate of the strain, or a mixture thereof. do.

Another aspect provides the use of an effective amount of a Lactobacillus plantarum strain, a culture of the strain, a lysate of the strain, or a mixture thereof for the manufacture of a formulation for improving the intestinal metabolite composition of an individual. .

Lactobacillus is a genus of aerobic or facultative anaerobic Gram-positive bacilli that is widely distributed in nature. Microorganisms belonging to the Lactobacillus genus include Lactobacillus plantarum and Sakei. As a result of research to develop a new strain with excellent anticancer effect, the present inventors selected Lactobacillus plantarum GB104 as an anticancer candidate strain. The strain was deposited at the Korea Research Institute of Bioscience and Biotechnology Biological Resources Center under the deposit number KCTC14107BP on January 14, 2020. The strain corresponds to a probiotic strain, is harmless to the human body, and can be used without side effects.

The Lactobacillus ( Lactobacillus ) has been renamed to Limosilactobacillus or Lactiplantibacillus , and the changed strain names in this specification can be used interchangeably. For example, Lactobacillus plantrum was changed to Lactiplantibacillus plantrum .

In this specification, the term " Lactobacillus plantarum GB104" may be used together with L. Plantarum GB104 strain or Lactobacillus plantarum GB104 strain (Accession Number: KCTC14107BP).

In one embodiment, the strain may be a strain deposited under deposit number KCTC14107BP.

In one embodiment, the strain may be a strain containing a 16S rRNA gene consisting of the nucleotide sequence of SEQ ID NO: 1.

In one embodiment, the strain may be a strain having 16S rRNA comprising the nucleotide sequence of SEQ ID NO: 1 or a 16s rRNA comprising a nucleotide sequence having 97% or more nucleotide sequence identity thereto. Specifically, it has at least 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9% or 100% homology with the nucleotide sequence consisting of SEQ ID NO: 1 of the present specification.

In one embodiment, the strain may be live cells, dead cells, or a cytoplasmic fraction obtained by disrupting the strain, and preferably may be live cells.

As used herein, the term “culture” may be used interchangeably with “culture supernatant,” “culture supernatant,” “conditioned culture,” or “conditioned medium,” and can be used interchangeably with Lactobacillus strains to grow and survive in vitro. It may refer to the entire medium containing the strain, its metabolites, extra nutrients, etc. obtained by culturing the strain in a medium capable of supplying nutrients for a certain period of time. The culture refers to a product obtained by culturing a probiotic strain in a known medium, and the product may or may not include the strain itself. The medium may be selected from known liquid media or solid media, for example, MRS liquid medium, GAM liquid medium, MRS agar medium, GAM agar medium, and BL agar medium, but is not limited thereto.

As used herein, the term “lysate” may be used interchangeably with “lysate”, meaning a solution or suspension of cells of a microorganism such as Lactobacillus plantarum in an aqueous medium o broken down. Cell lysates include, for example, macromolecules such as DNA, RNA, proteins, peptides, carbohydrates, lipids, etc. and/or micromolecules such as amino acids, sugars, fatty acids, etc., or fractions thereof. The lysate also contains cell debris, which may be smooth or granular in structure.

The culture medium may include the culture medium itself, its concentrate, or freeze-dried product obtained by cultivating the strain, or the culture supernatant obtained by removing the strain from the culture medium, its concentrate, or freeze-dried product.

The culture medium may be obtained by culturing Lactobacillus plantarum in an appropriate medium (e.g., MRS plate medium) at a temperature of more than 10°C or less than 40°C for a certain period of time, for example, 4 to 50 hours. .

In one embodiment, the strain, the culture of the strain, or the lysate of the strain may contain acetylated spermidine.

In one embodiment, the strain, a culture of the strain, or lysate of the strain may contain a polyamine acetylation enzyme. More specifically, it may include an enzyme involved in acetylation of polyamines including spermidine.

In one embodiment, the acetylated spermidine may be N ¹ -acetylspermidine, N ⁸ -acetylspermidine, or N ¹ ,N ⁸ -diacetylspermidine.

In one embodiment, the strain, the culture of the strain, or the lysate of the strain may contain glutaric acid or glutaconic acid.

In one embodiment, the glutaric acid (glutarate or glutartic acid) may be 2-oxoglutarate or 2-hydroxyglutarate. Additionally, in one embodiment, the glutaconic acid may be trans-glutarconic acid.

In another embodiment, the improvement of the intestinal metabolite composition may include the following.

- Reduction of spermidine, a metabolite in the intestines or feces;

- Increase in acetylated spermidine, a metabolite in the intestines or feces;

- Reduction of polyamine metabolites in the intestines or feces;

- Decreased activity of polyamine synthetase in the intestine or feces; and

- Increased activity of polyamine degrading enzymes in the intestines or feces.

In one embodiment, the polyamine synthase may be ornithine decarboxylase (ODC), an enzyme that synthesizes spermidine from ornithine in cells, and the polyamine decomposition enzyme may be an enzyme that synthesizes spermidine from ornithine. It may be spermidine/spermine ^{N 1 -acetyltransferase} (SSAT), an enzyme that acetylates, but this is an example and is not limited thereto. Therefore, as the intestinal metabolite composition of the individual administered the GB104 strain of the present invention is improved, the activity of polyamine synthetase (e.g., ornithine decarboxylase) in the intestine or feces of the individual decreases, and polyamine decomposition occurs. The activity of enzymes (e.g. spermidine/spermine N ¹ -acetyltransferase) increases, the levels of ornithine and polyamines (e.g. spermidine) in the intestine or feces decrease, and acetylated Levels of polyamines (e.g., acetylated spermidine) may increase.

The intestinal microbial composition, also called the intestinal microbiota, may refer to the complex ecosystem within the gastrointestinal tract. Specifically, it consists of the entire microbial community in the gut, including bacteria, yeast, fungi, archaea, and viruses. The gut microbiota is responsible for colonic fermentation of dietary fiber, nutrient extraction, synthesis of certain vitamins, prevention of colonization by pathogens, maturation of the intestinal epithelium and immune system, release of metabolites into systemic tissues, and regulation of gastro-hormonal secretion and neurological functions. Maintains several functions. It is generally recognized that disturbance of the normal balance in the intestinal microbial composition can compromise intestinal barrier integrity, which is often observed in many different diseases. The Lactobacillus plantarum GB104 strain of the present invention can restore and/or maintain a health-beneficial intestinal microbial composition through regulation of the intestinal metabolite profile (specifically, polyamine metabolism) in an individual. Accordingly, the Lactobacillus plantarum strains herein may also further provide compositions for restoring and/or maintaining a health-beneficial intestinal microbial composition (or for preventing or treating disorders associated with impaired intestinal integrity). there is.

Polyamine metabolism may include polyamine biosynthesis, catabolism, and transport. Natural polyamines can be synthesized in the cytoplasm of all cells. This biosynthesis begins with L-methionine and L-ornithine, which are amino acids of the urea cycle, and putsrescine is formed through ornithine decarboxylation by ornithine decarboxylase (ODC). Putrescine is a polyamine precursor in mammalian cells that produces spermine and spermidine when the aminopropyl group is added by decarboxylated S-adenosylmethionine (dcSAM). This dcSAM is known to be produced by S-adenosylmethionine decarboxylase 1 (SAMDC or adenosylmethionine decarboxylase 1, AMD1).

The role of polyamine transport in metabolism has led to the concept that polyamines are transported into cells through specific polyamine transport systems (PTS). Polyamine uptake through PTS is upregulated in proliferating cells, including tumor cells, suggesting that PTS plays a role in regulating intracellular polyamine concentrations. Because polyamine levels are upregulated in various cancer cell types, polyamines may be common therapeutic targets for cancer treatment. Fast-growing cells, including tumor cells, exhibit higher activity of several enzymes involved in polyamine biosynthesis. Polyamine levels are elevated in cancer patients and may be correlated with cancer development. High polyamine levels are known to be associated with the progression of neuroblastoma, hepatocellular carcinoma (HCC), prostate cancer, lung cancer, breast cancer, stomach cancer, and colorectal cancer (CRC). It is known that reduced polyamine levels induce apoptosis in post-mitotic and senescent cells. Additionally, polyamines may play a role in establishing tumor immunity, promoting the growth of cancer cells through the excretion of sperm and their metabolites. Polyamines are also known to induce acquired chemical resistance to 5-fluorouracil and paclitaxel in colorectal and breast cancer. Reduction of polyamines may prevent tumor-induced immunosuppression by enhancing spontaneous IL-2 production, NK-cell activation, and recovery of T-lymphocyte populations without affecting tumor polyamines.

In one embodiment, the strain, the culture of the strain, or the lysate of the strain may promote the activity of intestinal immune cells or the expression of tight junction proteins between intestinal cells.

In one embodiment, the activity of the intestinal immune cells is an increase in the number of activated CD8 + T cells in immune cells, an increase in the ratio of activated CD8 + T cells in immune cells, or an increase in INF-γ secretion, more specifically activation. It may include increased secretion of cytokines or interferons from immune cells.

In another embodiment, the intestinal immune cells are within the small intestine or large intestine (e.g., small intestinal intraepithelial lymphocytes (IEL), small intestinal lamina propria (siLP), colonic lamina). may include immune cells within the propria (cLP)).

In one embodiment, the tight junction proteins include claudin-1, claudin-2, claudin-3, claudin-4, and claudin-5. It may include one or more selected from the group consisting of (claudin-5), Zonula Occludens (ZO)-1, ZO-2, ZO-3, and occludin.

Accordingly, the strain, the culture of the strain, or the lysate of the strain may be used to determine the activity of intestinal immune cells, specifically within the small intestine or large intestine (e.g., small intestine intraepithelial lymphocytes (IEL), small intestinal mucosa lamina propria). Antitumor activity by increasing the proportion of cytotoxic T cells (CD8+ T cells), which are directly involved in suppressing the growth of cancer cells, among immune cells within the intestinal lamina propria (siLP) and colonic lamina propria (cLP) This may lead to . In addition, the expression of intestinal cell tight junctions can be promoted or upregulated, thereby strengthening the intestinal barrier function.

Therefore, according to one embodiment, the Lactobacillus plantarum strain, a culture of the strain, lysate of the strain, or a mixture thereof prevents, improves, treats, or improves the prognosis of cancer by reducing intestinal polyamines. , it is provided as a pharmaceutical composition for anti-cancer support, food (health functional food), feed composition, or anti-cancer supplement.

In one embodiment, improving the intestinal metabolite composition of the subject may be for preventing or treating cancer.

The above cancers include stomach cancer, liver cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, gallbladder cancer, biliary tract cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, salivary gland cancer, and melanoma. It may be any one selected from the group consisting of cancer, bladder cancer, esophageal cancer, head and neck cancer, skin cancer, small intestine cancer, anal cancer, colon cancer, rectal cancer, kidney cancer, blood cancer, and lymphoma. In addition, the colon cancer may be a malignant tumor that occurs in any area selected from the group consisting of the ascending colon, transverse colon, descending colon, sigmoid colon, and rectal mucosa.

As used herein, the term “cancer” refers to a physiological condition in animals, typically characterized by abnormal or uncontrolled cell growth. Cancer and cancer pathology include, for example, metastasis, interference with normally functioning surrounding cells, release of cytokines or other secreted products at abnormal levels, inhibition or enhancement of inflammatory or immunological responses, neoplasia, and premalignancy. ), malignancy, or involvement of surrounding or distant tissues or organs, such as lymph node invasion.

The cancer may be gastrointestinal cancer or non-gastrointestinal cancer.

The gastrointestinal cancer is a malignant tumor that occurs in the gastrointestinal tract, such as the esophagus, stomach, small intestine, or large intestine. The gastrointestinal cancer includes, for example, esophageal cancer, gallbladder cancer, liver cancer, biliary tract cancer, pancreatic cancer, stomach cancer, small intestine cancer, colon cancer, colon cancer, and anal cancer. It may be one or more cancers selected from the group consisting of rectal cancer, but is not limited thereto, and in one example, it may be colon cancer.

The non-gastrointestinal cancer includes, without limitation, malignant tumors occurring in organs other than the gastrointestinal tract or digestive system, for example, hematological cancer, leukemia, acute myeloid leukemia, neuroblastoma, retinoblastoma, lung cancer, head and neck cancer, salivary gland cancer, melanoma, It may be, but is not limited to, laryngeal cancer, prostate cancer, breast cancer, bladder cancer, kidney cancer, multiple myeloma, cervical cancer, thyroid cancer, ovarian cancer, urethral cancer, skin cancer, osteosarcoma, glioblastoma, brain tumor, or lymphoma.

In one example of the present invention, the cancer may be colon cancer, and the colon cancer includes malignant tumors that occur in one or more regions selected from the group consisting of ascending colon, transverse colon, descending colon, sigmoid colon, and rectal mucosa. do. The colon cancer may be one or more types selected from the group consisting of adenocarcinoma, lymphoma, malignant carcinoid, leiomyosarcoma, Kaposi's sarcoma, and squamous cell carcinoma, but is not limited thereto.

The composition according to one embodiment may include 0.001% by weight to 80% by weight of the Lactobacillus plantarum strain based on the total weight of the composition. Additionally, the administered dose of the Lactobacillus plantarum strain may be 0.01 mg to 10,000 mg, 0.1 mg to 1000 mg, 1 mg to 100 mg, 0.01 mg to 1000 mg, 0.01 mg to 100 mg, 0.01 mg to 10 mg, or 0.01 mg to 1 mg. . The strain is included in the composition in a therapeutically effective amount or nutritionally effective concentration, for example, the strain is 10 ³ to 10 ¹⁶ CFU/g, 10 ³ to 10 ¹⁵ CFU/g, 10 ³ to 10 ¹⁴ CFU/g. g, 10 ³ to 10 ¹³ CFU/g, 10 ³ to 10 ¹² CFU/g, 10 ⁴ to 10 ¹⁶ CFU/g, 10 ⁴ to 10 ¹⁵ CFU/g, 10 ⁴ to 10 ¹⁴ CFU/g, 10 ⁴ to 10 ¹³ CFU/g, 10 ⁴ to 10 ¹² CFU/g, 10 ⁵ to 10 ¹⁶ CFU/g, 10 ⁵ to 10 ¹⁵ CFU/g, 10 ⁵ to 10 ¹⁴ CFU/g, 10 ⁵ to 10 ¹³ CFU/g , 10 ⁵ to 10 ¹² CFU/g, 10 ⁶ to 10 ¹³ CFU/g, 10 ⁶ to 10 ¹² CFU/g, 10 ⁷ to 10 ¹³ CFU/g, 10 ⁷ to 10 ¹² CFU/g, 10 ⁸ to 10 It may be included in an amount of ¹³ CFU/g or 10 ⁸ to 10 ¹² CFU/g, or may be included in the composition as a culture of live or dead cells in an equivalent number. Specifically, for adult patients, 1X10 ³ to 1X10 ¹⁶ CFU/g of live or dead cells may be administered once or in divided doses. However, the dosage may be prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, gender, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity, and those skilled in the art will Taking these factors into consideration, the dosage can be adjusted appropriately. The number of administrations can be one time or two or more times within the range of clinically acceptable side effects, and the administration site can be administered at one or two or more places. For animals other than humans, the dosage per kg (body weight) is the same as for humans, or, for example, the above-mentioned administration is based on the volume ratio (e.g., average value) of organs (e.g., heart, etc.) between the target animal and human. The converted dose can be administered. Possible routes of administration include oral, sublingual, parenteral (e.g., subcutaneous, intramuscular, intraarterial, intraperitoneal, intrathecal, or intravenous), rectal, topical (including transdermal), inhalation, and injection, or implantable device. Alternatively, it may include insertion of a substance. Examples of animals subject to treatment according to one embodiment include humans and other mammals, specifically humans, monkeys, mice, rats, rabbits, sheep, cows, dogs, horses, pigs, etc. Included. According to one embodiment, the composition includes killed dried strains, and can be administered in an amount of 1g to 10g, 0.5g to 1.5g, 2.5g to 3.5g, or 4.5g to 5.5g, once a day to 3 times. It may be administered once.

As used herein, the term "therapeutically effective amount" refers to an anticancer agent or method of the present invention for the method and use of the present invention that elicits a biological or medical response or desired therapeutic effect in the patient that researchers, doctors, or other clinicians wish to obtain. and the amount of a pharmaceutical composition containing an anticancer agent for use. The therapeutically effective amount of an anticancer agent may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the anticancer agent to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which the therapeutically beneficial effects outweigh any toxic or harmful effects.

The pharmaceutical composition according to one embodiment may include a pharmaceutically acceptable carrier and/or additive. For example, sterilized water, physiological saline, common buffers (phosphoric acid, citric acid, other organic acids, etc.), stabilizers, salts, antioxidants (ascorbic acid, etc.), surfactants, suspending agents, isotonic agents, or preservatives. can do. For topical administration, it may also include combinations with organic materials such as biopolymers and inorganic materials such as hydroxyapatite, specifically collagen matrices, polylactic acid polymers or copolymers, polyethylene glycol polymers or copolymers, and chemical derivatives thereof. You can.

In one embodiment, the pharmaceutical composition may be an oral formulation.

In one embodiment, the oral preparation comes in the form of tablets, pills, capsules, lozenges, granules, powders, suspensions, and sachets. , or it may be in the form of syrups.

When the pharmaceutical composition according to one embodiment is prepared in a formulation suitable for injection, the Lactobacillus bacteria may be dissolved or dispersed in a pharmaceutically acceptable carrier, or may be frozen in a dissolved or dispersed solution state. .

According to one embodiment, the pharmaceutical composition may be used as a suspending agent, solubilizing agent, stabilizer, isotonic agent, preservative, anti-adsorption agent, surfactant, diluent, excipient, pH adjuster, analgesic agent, etc., if necessary depending on the administration method or formulation. Buffers, reducing agents, antioxidants, etc. may be appropriately included. Pharmaceutically acceptable carriers and agents suitable for the present invention, including those exemplified above, are described in detail in Remington's Pharmaceutical Sciences, 19th ed., 1995. The pharmaceutical composition according to one embodiment is formulated in unit dosage form using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by a person skilled in the art to which the invention pertains. It can be manufactured by or by placing it in a multi-capacity container. The formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of powder, granules, tablets or capsules.

The pharmaceutical composition is administered in a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" means an amount sufficient to treat the disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, activity of the drug, and the type and severity of the patient's disease. It can be determined based on factors including sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, drugs used simultaneously, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.

Another aspect is to provide a health functional food for improving the intestinal metabolite composition of an individual containing a Lactobacillus plantarum strain, a culture of the strain, a lysate of the strain, or a mixture thereof as an active ingredient. will be.

In one embodiment, improving the intestinal metabolite composition of the subject may include proliferation of beneficial intestinal bacteria, suppression of harmful bacteria, improvement of intestinal health by regulating immunity, or improvement of bowel activity.

In one embodiment, the strain, the culture of the strain, or the lysate of the strain may contain acetylated spermidine or polyamine acetylation enzyme.

In one embodiment, the acetylated spermidine may be N ¹ -acetylspermidine, N ⁸ -acetylspermidine, or N ¹ ,N ⁸ -diacetylspermidine.

In one embodiment, the strain, the culture of the strain, or the lysate of the strain may contain glutaric acid or glutaconic acid.

In one embodiment, the glutaric acid (glutarate or glutartic acid) may be 2-oxoglutarate or 2-hydroxyglutarate. Additionally, in one embodiment, the glutaconic acid may be trans-glutarconic acid.

In one embodiment, improving intestinal metabolite composition may include:

- Reduction of spermidine, a metabolite in the intestines or feces;

- Increase in acetylated spermidine, a metabolite in the intestines or feces;

- Reduction of polyamine metabolites in the intestines or feces;

- Decreased activity of polyamine synthetase in the intestine or feces; and

- Increased activity of polyamine degrading enzymes in the intestines or feces.

In one embodiment, the strain, the culture of the strain, or the lysate of the strain may promote or up-regulate the activity of intestinal immune cells or the expression of tight junction proteins between intestinal cells.

In one embodiment, the activity of the intestinal immune cells is an increase in the number of activated CD8 + T cells in immune cells, an increase in the ratio of activated CD8 + T cells in immune cells, or an increase in INF-γ secretion, more specifically activation. It may include increased secretion of cytokines or interferons from immune cells.

In another embodiment, the intestinal immune cells are within the small intestine or large intestine (e.g., small intestinal intraepithelial lymphocytes (IEL), small intestinal lamina propria (siLP), colonic lamina). may include immune cells within the propria (cLP)).

In one embodiment, the tight junction proteins include claudin-1, claudin-2, claudin-3, claudin-4, and claudin-5. It may include one or more selected from the group consisting of (claudin-5), Zonula Occludens (ZO)-1, ZO-2, ZO-3, and occludin.

In one embodiment, the health functional food may be an oral preparation.

The strain, culture of the strain, lysate of the strain or a mixture thereof, route of administration, administration method, and administration dose are as described above.

In one embodiment, the health functional food may further include a foodologically acceptable carrier.

As used herein, the term “foodologically acceptable” means that the compound exhibits non-toxic properties to cells or humans exposed to the compound.

As used herein, the term “improvement” may refer to any action that at least reduces the severity of a parameter related to the condition being treated, for example, a symptom. At this time, the health functional food can be used simultaneously or separately with a drug for treatment before or after the onset of the disease in order to prevent or improve cancer.

In the health functional food, the active ingredient can be added directly to the food or used together with other foods or food ingredients, and can be used appropriately according to conventional methods. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use (prevention or improvement). In general, when manufacturing a food or beverage, the health functional food may be added in an amount of about 15% by weight or less, more specifically about 10% by weight or less, based on the raw materials. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be below the above range.

The health functional food may be formulated with one selected from the group consisting of tablets, pills, powders, granules, powders, capsules, and liquid formulations, further including one or more of carriers, diluents, excipients, and additives. Foods to which compounds according to one aspect can be added include various foods, powders, granules, tablets, capsules, syrups, beverages, gum, tea, vitamin complexes, health functional foods, etc.

Specific examples of the carriers, excipients, diluents and additives include lactose, dextrose, sucrose, sorbitol, mannitol, erythritol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium phosphate, calcium silicate, microcrystalline cellulose. , polyvinylpyrrolidone, cellulose, polyvinylpyrrolidone, methylcellulose, water, sugar syrup, methylcellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil. It may be at least one selected from.

In addition to containing the effective ingredients, the health functional food may contain other ingredients as essential ingredients without any particular restrictions. For example, like regular beverages, it may contain various flavoring agents or natural carbohydrates as additional ingredients. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose, etc.; disaccharides such as maltose, sucrose, etc.; and polysaccharides, such as common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. You can. The ratio of the natural carbohydrates can be appropriately determined by the selection of a person skilled in the art.

In addition to the above, health functional foods according to one aspect include various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavors, colorants and thickening agents (cheese, chocolate, etc.), pectic acid and salts thereof. , alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. These components can be used independently or in combination, and the proportions of these additives can also be appropriately selected by those skilled in the art.

The health functional food may be provided by mixing with conventionally known health functional food for preventing or improving cancer or other existing health functional food, and the health functional food for preventing or improving cancer is known to be a metabolic disease. It may be a health functional food for the prevention or improvement of, an existing health functional food, or a newly developed health functional food.

When the health functional food contains other health functional foods that have the effect of preventing or improving cancer, it is important to mix the amount to obtain the maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art. there is.

The food composition for preventing or improving cancer includes all forms such as functional food, nutritional supplement, health food, and food additives, and the above type of food composition It can be manufactured in various forms according to conventional methods known in the art.

Compositions herein may be considered food supplements. Food supplements, also known as dietary supplements or nutritional supplements, can be considered another pharmaceutical product. It is intended to supplement the diet and provide nutrients or beneficial ingredients that may not be available or consumed in sufficient amounts in the normal diet. Most food supplements are considered foods, but sometimes they are considered drugs, natural health products, or nutraceutical products. In the meaning of the present invention, food supplements include health functional foods. Food supplements are usually sold over the counter without a prescription. When food supplements take the form of pills or capsules, they contain the same excipients used in pharmaceuticals. However, food supplements may take the form of food fortified with some nutrients (e.g. infant formula). Accordingly, in certain embodiments, the compositions of the present invention are food supplements.

The composition according to the present invention can be administered as is or mixed with a suitable edible liquid or solid or in the form of tablets, pills, capsules, lozenges, granules, powders. ), suspensions, sachets, syrups, or may be freeze-dried in the form of unit doses. It may also be in the form of monodoses of a lyophilized composition that are mixed in a separate liquid container provided prior to administration.

The composition of the present invention may be included in various edible foods and foods such as milk products for infants. As used herein, the term “edible product” is broadly defined to include any product that can be ingested by an animal, in any form (e.g., a product that is absorbed by the sense organs). products that can be imported). The term "food product" is understood as an edible product that provides nutritional support to the body. Foods of particular interest are food supplements and infant formulas. Foods preferably include oatmeal porridge, lactic acid fermented foods, resistant starch, dietary fibers, carbohydrates, proteins and glycated proteins. It includes carrier materials such as glycosylated proteins. In certain embodiments, bacterial cells of the invention are homogenized with other ingredients, such as cereals or powdered milk, to form infant formula.

Another aspect provides a feed composition for improving the intestinal metabolite composition of an individual comprising a Lactobacillus plantarum strain, a culture of the strain, lysate of the strain, or a mixture thereof as an active ingredient. will be.

The strain, culture of the strain, lysate of the strain or a mixture thereof, route of administration, administration method, and administration dose are as described above.

The feed composition can be manufactured by adding the mixed strain composition in an appropriate effective concentration range according to various feed production methods known in the art, and can be used as a feed additive composition for the purpose of preventing or improving aging-related diseases.

The “feed” may mean any natural or artificial diet, meal, etc., or an ingredient of the meal, for or suitable for eating, ingestion, and digestion by an animal. The type of feed is not particularly limited, and feed commonly used in the technical field can be used. Non-limiting examples of the feed include plant feeds such as grains, root fruits, food processing by-products, algae, fiber, pharmaceutical by-products, oils, starches, cucurbits or grain by-products: proteins, non-lipids, Examples include animal feeds such as fats and oils, minerals, fats and oils, single-cell proteins, zooplanktons, or food.

Another aspect provides an anti-cancer adjuvant comprising a Lactobacillus plantarum strain, a culture of the strain, lysate of the strain, or a mixture thereof as an active ingredient.

The strain, culture of the strain, lysate of the strain or a mixture thereof, route of administration, administration method, and administration dose are as described above.

As used herein, “adjuvant” refers to an agent that assists the efficacy of the main drug, that is, an anticancer agent, to improve and/or enhance the therapeutic effect, or to prevent or alleviate the harmful effects of the main drug. . By improving the composition of intestinal metabolites, the Lactobacillus plantarum strain of the present invention can improve the anticancer effect of other anticancer agents without itself being burdensome to the human body.

Conventional treatments that can be used in combination with other anticancer agents may be selected from the group consisting of chemical anticancer agents for chemotherapy, targeted anticancer agents, antibody therapy, immunotherapy agents, and combinations thereof.

As used herein, the term “chemical anti-cancer agent” is also referred to as an anti-tumor drug (Antineoplastic agent) or a cytotoxic agent. It is a general term for drugs that exhibit anticancer activity mainly by acting directly on DNA to block DNA replication, transcription, and translation processes, or by interfering with the synthesis of nucleic acid precursors in metabolic pathways and inhibiting cell division. Specifically, the chemical anticancer agent may be any one selected from the group consisting of an alkylating agent, microtubule inhibitor, antimetabolite, and topoisomerase inhibitor. The anti-tumor drug acts not only on tumor cells but also on normal cells and exhibits cytotoxicity. Chemotherapy agents can be used for maintenance therapy. In addition, the term "maintenance therapy" in this specification refers to treating cancer with drugs after initial anti-cancer treatment, and refers to a treatment method performed to prevent or delay the recurrence of cancer.

As used herein, the term “targeted anticancer agent” refers to a treatment that kills cancer cells specifically by blocking signals involved in the growth and development of cancer by targeting specific proteins or specific genetic changes that occur frequently only in cancer cells. It is classified into monoclonal antibodies that react outside the cell and small molecule substances that act inside the cell. Monoclonal antibodies are anticancer drugs that block cancer cell-inducing signals transmitted outside the cell and act on initiation signals related to proliferation and death, while small molecule substances act on complex signaling that occurs inside cells.

Specifically, the targeted proteins are epidermal growth factor receptor (EGFR), vascular growth factor receptor (VEGFR), CD20, CD38, RNAK-L, BTK, Bcr-abl, PDGFR/FGFR family, MEK/RAF, and HER2/Neu. , Ubiquitin, JAK, MAP2K, ALK, PARP, tumor growth factor β receptor (TGFβR), Proteasome, Bcl-2, C-Met, VR1, VR2, VR3, c-kit, AXL, RET, Braf, DNA It may be methyltransferase (DNMT), CDK4/6, STING, etc.

As used herein, the term “antibody therapeutic agent” refers to a therapeutic agent that exhibits an anti-cancer effect using an antibody that recognizes a specific protein of cancer cells as an antigen. Antibody treatments may include Cetuximab, Trastuzumab, Emtansine, Emtansine, Rituximab, Ibritumomab, Tositumomab, Brentuximab, Ofatumumab, Obinutuzumab, Necitumumab, Bevacizumab, Ramucirumab, Nivolumab, Pembrolizumab, Atezolizumab, Durvalumab, Ipilimumab, etc.

As used herein, the term “immune anti-cancer agent” refers to a substance that inhibits the activity of immune checkpoint proteins that inhibit the differentiation, proliferation, and activity of immune cells, preventing cancer cells from exercising the function of evading the immune system. It is known to eliminate cancer cells. The immuno-anticancer agents include 2B4, 4-1BB (CD137), AaR, B7-H3, B7-H4, BAFFR, BTLA, CD2, CD7, CD27, CD28, CD30, CD40, CD80, CD83 ligand, CD86, CD160, CD200, CDS, CEACAM, CTLA-4, GITR, HVEM, ICAM-1, KIR, LAG-3, LAIR1, LFA-1 (CD 11 a/CD 18), LIGHT, NKG2C, NKp80, OX40, PD-1, PD- It may be an antibody against any one selected from the group consisting of L1, PD-L2, SLAMF7, TGFRp, TIGIT, Tim3, and VISTA. More specifically, anti-CTLA-4 antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-PD-L2 antibody, anti-B7-H4 antibody, anti-HVEM antibody, anti-TIM3 antibody, anti-GAL9 antibody, anti-LAG3 antibody, It may be any one selected from the group consisting of anti-VISTA antibody, anti-KIR antibody, anti-BTLA antibody, and anti-TIGIT antibody, but is not limited thereto.

The strain, its culture, or its lysate according to one embodiment may be administered in combination with the other anticancer drugs.

As used herein, the term “combination therapy” or “combination administration” or “in combination” refers to any form of simultaneous or concurrent treatment using at least two separate therapeutic agents. The components of the combination therapy may be administered simultaneously, sequentially, or in any order. The components may be administered in any suitable manner, in different doses or at different frequencies of administration or via different routes.

Specifically, the combined administration includes Lactobacillus Plantarum strains; And the anticancer agent or antibiotic may be administered simultaneously, or the anticancer agent or antibiotic may be administered after administering the Lactobacillus plantarum strain. The combination therapy according to the present invention is an efficacy that can be obtained by administering one or the rest of the components of the combination therapy at a conventional dose, for example, the efficacy measured through the degree of response, response rate, time to disease progression, or survival time. More therapeutically superior can be defined as being able to provide synergistic effects. For example, if the therapeutic efficacy is superior to the efficacy obtained by using each of the above alone, the efficacy of the combination treatment is synergistic. In particular, without harming one or more of the degree of response, rate of response, time to disease progression, and survival data, and in particular without harming the duration of response, than would occur when each component is used at its usual dose. , Lactobacillus plantarum strains with reduced or fewer problematic side effects; And if the usual dose of anticancer drugs or antibiotics can be reduced, a synergistic effect is considered to exist.

As used herein, the term “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 following 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 in batches with a specific time interval between administrations. The time interval may be the same or different between the respective administrations of the components of the combination therapy and may be selected, for example, in the range of 2 minutes to 96 hours, 1 day to 7 days or 1 week, 2 weeks or 3 weeks. . Generally, the time interval between administrations can range from minutes to hours, for example from 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.

According to the Lactobacillus plantarum strain according to one aspect, it is possible to reduce intestinal polyamines (e.g., spermidine), thereby preventing, improving, treating, or improving prognosis of cancer, and pharmaceutical compositions and foods for anti-cancer assistance. It has the effect of being useful as a (health functional food), feed composition, and anti-cancer supplement.

Figure 1 is a graph showing the relative difference in metabolite content in GB104 culture supernatant and MRS culture supernatant (control) as metabolic pathway through CE-TOF-MS.

Figure 2 is a table showing the change in trans-Glutaconic content among metabolites in GB104 culture supernatant, L. Plantarum F0077 strain, and MRS culture supernatant (control) as fold change based on the total strain.

Figure 3 is a table showing the change in content of spermidine and three types of acetylated spermidines among metabolites in GB104 culture supernatant and MRS culture supernatant (control) as fold change based on the overall average.

Figure 4 is a graph showing the relative difference in content of spermidine and three types of acetylated spermidine among metabolites in GB104 culture supernatant and MRS culture supernatant (control).

Figure 5 is a graph showing the relative changes in spermidine and acetylspermidine content after treatment of GB104 culture supernatant and MRS culture supernatant (control group) in colon cancer cell line HCT116.

Figure 6 is a graph showing changes in expression of polyamine synthase ODC and polyamine decomposition enzyme SSAT after treatment of colon cancer cell lines HCT116 and HT-29 with GB104 culture supernatant and MRS culture supernatant (control group); ODC: Ornithine decarboxylase, SSAT: Spermidine/spermine N ¹ -acetyltransferase.

Figure 7 is a graph showing the relative change in spermidine content in feces after treatment of GB104 and PBS (control group) in mice transplanted with colon cancer cells MC38, respectively.

Figure 8 is a graph showing changes in N ¹ -acetylspermidine/spermidine and ornithine content in tumors after treating mice transplanted with MC38 colon cancer cells with GB104 and PBS (control group), respectively.

Figure 9 is a graph showing the change in spermidine content in feces after antibiotic-treated mice were treated with GB104 and PBS (control group), respectively.

Figure 10 is a graph showing the change in the number of immune cells in the intestine after antibiotic-treated mice were treated with GB104 and PBS (control group), respectively.

Figure 11 is a graph showing changes in tight junction-related gene expression in intestinal tissue after antibiotic-treated mice were treated with GB104 and PBS (control group), respectively; ZO: Zonula Occludens.

Figure 12 is a graph showing the survival rate of cells after treating the colon cancer cell line HCT116 with culture supernatants of GB104 and other strains, respectively.

Figure 13 is a graph showing the cell cycle after treating the colon cancer cell line HCT116 with culture supernatants of GB104 and the comparison strain (WCFS1), respectively.

Figure 14 is a graph showing cell death after treating the colon cancer cell line HCT116 with culture supernatants of GB104 and the comparison strain (WCFS1), respectively.

Below, preferred embodiments are presented to aid understanding of the present invention. However, the following examples are provided only to make the present invention easier to understand, and the content of the present invention is not limited by the following examples. The embodiments may be subject to various changes, and the embodiments are not limited to the embodiments disclosed below and may be implemented in various forms.

Example 1. Lactobacillus Plantarum ( Lactobacillus plantarum ) Isolation and identification of strain GB104

Isolation and identification of Lactobacillus Plantarum GB104 strain was performed by the method described in Korean Patent Application No. 10-2020-0186738 and Korean Patent Application No. 10-2022-0080567. The above document is incorporated herein by reference in its entirety.

Briefly, Lactobacillus Plantarum GB104 was isolated from a vaginal sample of a healthy woman who visited the hospital for health checkup. First, vaginal samples were collected with a swab, inoculated into Rogosa SL (MRS) plate medium, and cultured in an anaerobic chamber at 37°C for 48 hours. When bacterial colonies grew, single colonies were subcultured onto new MRS plate medium for pure isolation. After pure isolation, the strain was cultured using MRS medium. Next, among the cultured strains, Lactobacillus plantarum has an inhibitory effect on fat cell accumulation and has low cytotoxicity. Strain GB104 was finally selected. To identify the final selected Lactobacillus Plantarum GB104 strain, the 16S rRNA gene sequence obtained through PCR using primers targeting the 16S rRNA gene was analyzed by Sanger sequencing method, and Lactobacillus Plantarum The 16S rRNA sequence of GB104 is shown as SEQ ID NO: 1. The present inventors named the GB104 strain as “ Lactobacillus plantarum GB104” (Accession number: KCTC 14107BP) and transferred it to the Korean collection for type cultures (KCTC) at the Korea Research Institute of Bioscience and Biotechnology in 2020. It was deposited on the 14th of February. In addition, Lactobacillus plantarum was changed to Lactiplantibacillus plantarum . In the following examples, the changed strain names of existing strains are described interchangeably.

Experimental Example 1. Analysis and content comparison results of polyamine and trans-Glutaconic acid in GB104 culture supernatant

Metabolome analysis of MRS culture supernatant (control) and GB104 culture supernatant was conducted by requesting Human Metabolome Technologies (HMT). Milli-Q water containing internal standards was added to the MRS culture supernatant and GB104 culture supernatant samples, and CE-TOF-MS analysis was performed. Cation and anion metabolite analysis was performed using the Agilent CE-TOF-MS system (Agilent Technologies Inc.), and the results are shown in Figure 1. Separation of metabolites was achieved through fused silica capillary. Putative metabolites analyzed in HMT were identified through the HMT internal library.

Figure 1 is a graph showing the relative difference in metabolite content in GB104 culture supernatant and MRS culture supernatant (control) as metabolic pathway through CE-TOF-MS.

Figure 2 is a table showing the change in trans-Glutaconic content among metabolites in GB104 culture supernatant, L. Plantarum F0077 strain, and MRS culture supernatant (control) as fold change based on the total strain.

Figure 3 is a table showing the change in content of spermidine and three types of acetylated spermidine among metabolites in GB104 culture supernatant and MRS culture supernatant (control) as fold change based on the overall average.

Figure 4 is a graph showing the relative difference in content of spermidine and three types of acetylated spermidine among metabolites in GB104 culture supernatant and MRS culture supernatant (control).

As shown in Figures 1 to 4, a total of 296 metabolites were analyzed, including both substances identified through the HMT internal library and unknown substances. Metabolites produced and destroyed by GB104 were further analyzed using the OPLS-DA model, which is used to confirm differences between the two groups, and 87 metabolites were selected out of a total of 296 metabolites. Among these metabolites, various metabolites such as amino acids, peptides, nucleosides, nucleotides, organic acids, and saccharides were included. What was particularly noteworthy was the change in polyamines, which are known to be involved in the proliferation of cancer cells. GB104 acetylated spermidine, a polyamine component, thereby reducing spermidine but increasing acetylated spermidine.

Experimental Example 2. Changes in metabolites and gene expression in cancer cells according to GB104 culture supernatant treatment

The effects of L. Plantarum GB104 culture supernatant on changes in metabolites and gene expression of human colon cancer cell lines were confirmed.

Human colon cancer cell line (HCT116) was distributed into each well of a 6-well plate at 2X10 ⁵ cells and cultured for 24 hours, then treated with the culture supernatant of L. Plantarum GB104 strain at a concentration of 10% and incubated at 37°C with 5% concentration. Cultured for 24 hours under CO ₂ conditions. The culture supernatant was obtained by culturing the L. Plantarum strain in MRS medium for 8, 16, and 24 hours, respectively, and then centrifuging the strain to precipitate the supernatant, which was then filtered through a 0.22 μm filter to obtain supernatant for each culture time.

Changes in the content of polyamines in cancer cells were conducted by performing metabolite extraction and HPLC-DAD analysis on HCT116 cell pellets treated with supernatant for each GB104 culture time, and the area values of each peak are shown in Figure 5. In addition, changes in the expression of genes that inhibit polyamine synthesis and promote degradation by the GB104 metabolite were analyzed using the QuantStudio 3 Real-Time PCR Instrument after RNA extraction and cDNA synthesis from HCT116 and HT-29 cell pellets treated with supernatants for each culture time. The mRNA expression level was confirmed, and the results are shown in Figure 6. Polyamines, which are involved in cell growth and proliferation, are especially synthesized in large quantities in cancer cells and play an important role in the proliferation of cancer cells. Polyamines are synthesized from ornithine in cells by ODC (Ornithine decarboxylase) and decomposed by acetylation by SSAT (Spermidine/spermine N ¹ -acetyltransferase). It has been reported that increased SSAT expression in cancer cells inhibits the proliferation of cancer cells, and high SSAT expression in cancer tissues leads to a good prognosis for anticancer treatment.

Figure 5 is a graph showing the relative changes in spermidine and acetylspermidine content after treatment of GB104 culture supernatant and MRS culture supernatant (control group) in colon cancer cell line HCT116.

Figure 6 is a graph showing changes in expression of polyamine synthase ODC and polyamine decomposition enzyme SSAT after treatment of colon cancer cell lines HCT116 and HT-29 with GB104 culture supernatant and MRS culture supernatant (control group); ODC: Ornithine decarboxylase, SSAT: Spermidine/spermine N ¹ -acetyltransferase.

As shown in Figure 5, spermidine decreased and acetylspermidine increased in HCT116 colon cancer cells treated with GB104 culture supernatant in a culture time-dependent manner. Through this, it was confirmed that GB104 acetylates polyamine.

As shown in Figure 6, in both colon cancer cell lines HCT116 and HT-29 treated with the culture supernatant of GB104, the expression of ODC, a polyamine synthase, was decreased, and the expression of SSAT, a polyamine degrading enzyme, was increased. Through this, it was confirmed that GB104 inhibits the proliferation of cancer cells caused by polyamines by inhibiting the synthesis and increasing the degradation of polyamines in cancer cells.

Experimental Example 3. Change in spermidine content in mouse model feces

3.1. Comparison of changes in spermidine content in feces of MC-38 mouse colon cancer model

Changes in spermidine content in feces and tumors due to administration of L. Plantarum GB104 strain were confirmed in the colon carcinoma MC-38 allograft model. A tumor model was established by subcutaneously injecting 100 ^μL of 2 On the 5th day of tumor cell injection, only mice with tumor sizes within the range of 20-40 mm ³ were selected and each group was randomly set, and then L. Plantarum GB104 strain was administered to the animal model at 1x10 ⁹ CFU per mouse. It was administered orally every day from the 6th day until just before the end of the test. On the 6th day of tumor cell injection (immediately before oral administration of GB104), feces were obtained from mice in all groups (0 day samples), and on day 12, feces were again obtained from mice in all groups (7 day samples). Tumor samples were obtained from mice at the end of the test, on day 20 of tumor cell injection. Extraction and derivatization of polyamines from mouse fecal and tumor samples were performed and HPLC-DAD analysis was performed. As a result, polyamines were detected in fecal and tumor samples, and it was confirmed that the change in polyamine content was caused by GB104. Changes in spermidine content in feces were expressed as a fold by dividing the area of the spermidine peak in the fecal sample after oral administration of GB104 for 7 days by the area of the spermidine peak in the fecal sample collected immediately before oral administration of GB104, and this value was expressed as a fold A comparison by star is shown in Figure 7. The change in acetylspermidine content in the tumor was expressed by dividing the area of the N ¹ -acetylspermidine peak by the area of the spermidine peak, and the change in ornithine content was shown in Figure 8 as the ornithine peak area value.

Figure 7 is a graph showing the relative change in spermidine content in feces after treatment of GB104 and PBS (control group) in mice transplanted with colon cancer cells MC38, respectively.

Figure 8 is a graph showing changes in N ¹ -acetylspermidine/spermidine and ornithine content in tumors after treating mice transplanted with MC38 colon cancer cells with GB104 and PBS (control group), respectively.

As shown in Figure 7, GB104 reduced spermidine content in a dose-dependent manner, and in particular, the change in spermidine content in the group administered GB104 (treatment dose 1 x ¹⁰⁹ CFU/200 μL/head) compared to the control group. decreased significantly.

As shown in Figure 8, the proportion of acetylspermidine was high in the tumors of mice treated with GB104, which means that GB104 not only weakens the proliferation ability of cancer cells by acetylating polyamines, but also facilitates the decomposition of polyamines. . In addition, ornithine, a substrate of polyamine synthetase ODC, increased in the tumors of mice treated with GB104, showing that GB104 inhibited the activity of polyamine synthetase.

3.2. Comparison of changes in spermidine content in feces of antibiotic-treated mouse colon cancer model

In an antibiotic-treated mouse model, changes in spermidine content in feces due to administration of L. Plantarum GB104 strain were confirmed. Antibiotics were mixed into sterilized drinking water and kept out of the light, and the mice were allowed to freely consume this drinking water for 7 days. Additionally, the antibiotic-treated drinking water was replaced every 2-3 days. From day 7 onwards, mice were provided with sterilized regular drinking water instead of drinking water containing antibiotics until the end of the test. Freeze-dried GB104 was suspended in D-PBS to adjust the dose to ¹ After the antibiotic treatment was terminated, feces were obtained from mice in all groups immediately before GB104 administration (0 day samples), and feces were again obtained from mice in all groups on the 7th day of oral administration of GB104 (7 day samples). Extraction and derivatization of polyamines from mouse fecal samples were performed and HPLC-DAD analysis was performed. Spermidine was detected in the fecal sample, and it was confirmed that the change in spermidine content was caused by GB104. The change in spermidine content was expressed as a fold by dividing the area of the spermidine peak of the fecal sample after oral administration of GB104 for 7 days by the area of the spermidine peak of the fecal sample collected immediately before oral administration of GB104, and this value was calculated for each group. Comparison was made and this is shown in Figure 9.

Figure 9 is a graph showing the change in spermidine content in feces after antibiotic-treated mice were treated with GB104 and PBS (control group), respectively.

As shown in Figure 9, the content of spermidine in feces decreased due to antibiotic treatment, but increased on the 7th day when the efficacy of the antibiotic decreased. However, this increase in spermidine was significantly reduced by GB104.

Experimental Example 4. Changes in the number of immune cells in the intestine according to GB104 administration

In an antibiotic-treated mouse model, changes in intestinal immune cells were confirmed by administration of L. Plantarum GB104 strain.

Antibiotics were mixed into sterilized drinking water and kept out of the light, and the mice were allowed to freely consume this drinking water for 7 days. Additionally, the antibiotic-treated drinking water was replaced every 2 to 3 days. From day 7 onwards, mice were provided with sterilized regular drinking water instead of drinking water containing antibiotics until the end of the test. Freeze-dried GB104 was suspended in D-PBS and orally administered at ¹ has been removed. The intestinal tissue was opened by making a longitudinal incision, washed with PBS, and cut into 1-2 cm long pieces. To remove epithelial cells, the cells were stirred for 20 minutes at 37°C with FACS buffer (PBS containing 3% FBS, 20 mM HEPES, 100 U/ml Penicillin, 100 μg/ml Streptomycin, 1 mM Sodium Pyruvate, and 10 mM EDTA). , Intraepithelial lymphocytes (IEL) were additionally isolated from the supernatant filtered from the small intestine. Pieces of intestinal tissue from which epithelial cells were removed were washed with PBS, finely chopped, and added to enzyme medium (small intestine: 400 U/ml Collagenase D, 10 μg/ml DNase I, large intestine: 800 U/ml Collagenase D, 10 μg/ml DNase I). RPMI 400 medium containing 3% FBS, 20 mM HEPES, 100 U/ml Penicillin, 100 μg/ml Streptomycin, 1 mM Sodium Pyruvate, and 1 mM NEAA) and stirred at 37°C for 45 minutes. The enzyme reaction was stopped by treatment with 10 mM EDTA, and the cells passed through the strainer were resuspended in 40% Percoll solution, and 75% Percoll solution was added thereto and centrifuged. After centrifugation, the middle layer was recovered and lamina propria (LP) cells were separated. The isolated immune cells were stained using a fluorescent antibody matching the marker of the cells to be identified and then analyzed using a FACSymphony device, and the results are shown in Figure 10.

Figure 10 is a graph showing the change in the number of immune cells in the intestine after antibiotic-treated mice were treated with GB104 and PBS (control group), respectively.

As shown in Figure 10, changes in immune cells in the small and large intestines were confirmed, and the group administered the L. Plantarum GB104 strain showed cytotoxic T cells (known as immune cells that directly inhibit cancer growth) compared to the control group. It was confirmed that the proportion of CD8 ⁺ T cells) was significantly increased, and the IFN-γ secreted by these cells was also significantly increased. Through this, it was confirmed that the L. Plantarum GB104 strain had an effect on the increase and activation of CD8 ⁺ anti-tumor immune T cells.

Experimental Example 5. Changes in intestinal tissue tight junction gene expression following GB104 administration

In an antibiotic-treated mouse model, changes in the expression of genes related to tight junctions in intestinal tissue due to administration of L. Plantarum GB104 strain were confirmed.

Antibiotics were mixed into sterilized drinking water and kept out of the light, and the mice were allowed to freely consume this drinking water for 7 days. Additionally, the antibiotic-treated drinking water was replaced every 2-3 days. From day 7 onwards, mice were provided with sterilized regular drinking water instead of drinking water containing antibiotics until the end of the test. Freeze-dried GB104 was suspended in D-PBS and adjusted to a dose of 1x10 ⁹ CFU/head, and then the test substance was orally administered at 200 μL per mouse daily for 14 days from the end of antibiotic treatment using an oral zonde. On the 14th day of oral administration of GB104, ileum tissue in the small intestine was extracted and disrupted, RNA was extracted and synthesized into cDNA, and tight junction-related genes ( ZO-1, Occludin, The mRNA expression level of Claudin-4 ) was confirmed, and the results are shown in Figure 11.

Figure 11 is a graph showing changes in tight junction-related gene expression in intestinal tissue after antibiotic-treated mice were treated with GB104 and PBS (control group), respectively; ZO: Zonula Occludens.

As shown in Figure 11, GB104 increased the expression level of tight junction genes ( ZO-1, Occludin, and Claudin-4 ) in small intestine tissue. Through this, it was confirmed that GB104 can enhance gut barrier function by increasing the expression of tight junction genes.

Experimental Example 6. Confirmation of cancer cell survival rate and cancer cell killing effect according to GB104 strain culture supernatant treatment

6.1. Confirmation of cancer cell survival rate according to GB104 strain culture supernatant treatment

Using a human colon cancer cell line, various L. Plantarum culture supernatants, including L. Plantarum GB104 strain, were each processed, and cell viability was screened through MTT analysis.

Human colon cancer cell line HCT116 cells were dispensed into each well of a 96-well plate at 2X10 ³ cells and cultured for 24 hours. Then, 10% culture supernatant of various L. Plantarum strains was added in a culture medium supplemented with DFMO and aminoguanidine. concentration and cultured for 72 hours under conditions of 37°C and 5% CO ₂ . The culture supernatant was obtained by culturing the L. Plantarum strain in MRS medium, precipitating the strain by centrifugation, collecting only the supernatant, and filtering it through a 0.22 μm filter. Using the Cell Proliferation Kit I (MTT) (Roche), treat the MTT solution to 0.5 mg/mL and then incubate for an additional 4 hours to dissolve the purple formazan crystals produced in living cells by metabolic activity with the solubilization solution. Absorbance was measured at 570 nm, and is shown in Figure 12.

Figure 12 is a graph showing the survival rate of cells after treating the colon cancer cell line HCT116 with culture supernatants of GB104 and other strains, respectively.

As shown in Figure 12, the various L. Plantarum culture supernatants did not show the same cancer cell growth inhibitory effect, and in particular, the GB104 culture supernatant showed an effect of reducing the cancer cell survival rate by about 95%, making it the most effective in inhibiting the growth of cancer cells. was confirmed.

6.2. Confirmation of cancer cell cycle according to treatment of GB104 strain culture supernatant

L. Plantarum GB104 culture supernatant was processed using the human colon cancer cell line, and changes in the cancer cell cycle were confirmed using flow cytometry.

Human colon cancer cell line HCT116 cells were distributed into each well of a 6-well plate at 5×10 ⁴ cells and cultured for 24 hours. Then, 10% of the culture supernatant of the L. Plantarum GB104 strain was added in a culture medium containing DFMO and aminoguanidine. concentration and cultured for 48 hours under conditions of 37°C and 5% CO ₂ . The culture supernatant was obtained by culturing the L. Plantarum strain in MRS medium, precipitating the strain by centrifugation, collecting only the supernatant, and filtering it through a 0.22 μm filter. After 48 hours, the cells were removed by treatment with trypsin-EDTA, harvested by centrifugation, fixed with 70% ethanol, and stored at 4°C for more than 1 hour. After treatment with RNase A and staining with PI (Propidium Iodine), the degree of death of cancer cells was confirmed through cell cycle analysis using a flow cytometer (BD FACSymphony A3 Cell Analyzer), which is shown in Figure 13.

Figure 13 is a graph showing the cell cycle after treating the colon cancer cell line HCT116 with culture supernatants of GB104 and the comparison strain (WCFS1), respectively.

As shown in Figure 13, it was confirmed that the sub-G1 region of cells treated with L. Plantarum GB104 culture supernatant increased about 9.8 times compared to control cells treated with MRS. Through this, it was confirmed that GB104 induces the death of cancer cells.

6.3. Confirmation of cancer cell killing effect following treatment of GB104 strain culture supernatant

L. Plantarum GB104 culture supernatant was treated using the human colon cancer cell line, and the cancer cell killing effect was confirmed using flow cytometry.

Human colon cancer cell line HCT116 cells were distributed into each well of a 6-well plate at 5×10 ⁴ cells and cultured for 24 hours. Then, 10% of the culture supernatant of the L. Plantarum GB104 strain was added in a culture medium containing DFMO and aminoguanidine. concentration and cultured for 48 hours under conditions of 37°C and 5% CO ₂ . The culture supernatant was obtained by culturing the L. Plantarum strain in MRS medium, precipitating the strain by centrifugation, collecting only the supernatant, and filtering it through a 0.22 μm filter. After 48 hours, the cells were removed by treatment with trypsin-EDTA and then harvested by centrifugation. After mixing the cells with 100 μL binding solution using the FITC Annexin V Apoptosis Detection Kit with 7-AAD (BioLegend), 5 μL FITC Annexin V and 5 μL 7-AAD were added, blocked from light at room temperature, and reacted for 15 minutes. Fluorescence measurement to confirm cancer cell death was confirmed using a flow cytometer (BD FACSymphony A3 Cell Analyzer), and is shown in Figure 14. Normally living cells are not labeled with Annexin V and 7-AAD, while cells in the early stage of apoptosis are labeled with Annexin V but not 7-AAD. Cells in the late stage of apoptosis can be identified by labeling both Annexin V and 7-AAD.

Figure 14 is a graph showing cell death after treating the colon cancer cell line HCT116 with culture supernatants of GB104 and the comparison strain (WCFS1), respectively.

As shown in Figure 14, it was confirmed that the early and late cell death of cancer cells treated with L. Plantarum GB104 culture supernatant increased about 4-fold compared to control cells treated with MRS. Through the above results, it was confirmed that GB104 is involved in inducing the initial death of cancer cells.

The description of the present invention described above is for illustrative purposes, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. will be. Therefore, the embodiments described above should be understood in all respects as illustrative and not restrictive.

[Accession number]

Name of depository institution: Korea Research Institute of Bioscience and Biotechnology

Accession number: KCTC14107BP

Trust date: 20200114

Claims (30)

1. A composition for improving the intestinal metabolite composition of an individual comprising a Lactobacillus plantarum strain, a culture of the strain, lysate of the strain, or a mixture thereof as an active ingredient.
2. The composition according to claim 1, wherein the strain is deposited under deposit number KCTC14107BP.
3. The composition according to claim 1, wherein the strain contains 16S rRNA of SEQ ID NO: 1.
4. The composition according to claim 1, wherein the strain, the culture of the strain, or the lysate of the strain contains acetylated spermidine.
5. The composition of claim 4, wherein the acetylated spermidine is N ¹ -acetylspermidine, N ⁸ -acetylspermidine, or N ¹ ,N ⁸ -diacetylspermidine.
6. The composition according to claim 1, wherein the strain, a culture of the strain, or a lysate of the strain contains a polyamine acetylation enzyme.
7. The composition according to claim 1, wherein the strain, the culture of the strain, or the lysate of the strain contains glutaric acid or glutaconic acid.
8. The composition according to claim 7, wherein the glutaric acid is 2-oxoglutaric acid or 2-hydroxyglutaric acid.
9. The composition of claim 1, wherein improving intestinal metabolite composition comprises any one or more of the following properties:

   - Reduction of spermidine, a metabolite in the intestines or feces;

   - Increase in acetylated spermidine, a metabolite in the intestines or feces;

   - Reduction of polyamine metabolites in the intestines or feces;

   - Decreased activity of polyamine synthetase in the intestine or feces; and

   - Increased activity of polyamine decomposition enzymes in the intestines or feces.
10. The composition according to claim 1, wherein the strain, the culture of the strain, or the lysate of the strain promotes the activity of intestinal immune cells or the expression of tight junction proteins between intestinal cells.
11. The composition according to claim 10, wherein the activity of intestinal immune cells includes an increase in the number of activated CD8+ T cells in immune cells, or an increase in INF-γ secretion.
12. The method of claim 10, wherein the tight junction protein is ZO (Zonula Occuludens)-1, ZO-2, ZO-3, Occludin, Claudin-1, Claudin-2, Claudin-3, and Claudin- A composition comprising at least one selected from the group consisting of 4.
13. A pharmaceutical composition for the prevention or treatment of cancer comprising a Lactobacillus plantarum strain, a culture of the strain, a lysate of the strain, or a mixture thereof as an active ingredient.
14. The pharmaceutical composition according to claim 13, wherein the strain is included in an amount of 10 ³ to 10 ¹⁶ cfu/g.
15. The pharmaceutical composition according to claim 13, which is an oral formulation.
16. The method of claim 13, wherein the cancer is stomach cancer, liver cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, pancreas cancer, gallbladder cancer, biliary tract cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, and retinoblastoma. , salivary gland cancer, melanoma, bladder cancer, esophagus cancer, head and neck cancer, skin cancer, small intestine cancer, anal cancer, colon cancer, rectal cancer, kidney cancer, blood cancer, and lymphoma.
17. The pharmaceutical composition according to claim 16, wherein the colon cancer occurs in any one site selected from the group consisting of ascending colon, transverse colon, descending colon, sigmoid colon, and rectal mucosa.
18. A health functional food for improving the intestinal metabolite composition of an individual comprising a Lactobacillus plantarum strain, a culture of the strain, lysate of the strain, or a mixture thereof as an active ingredient.
19. The health functional food according to claim 18, wherein the strain, the culture of the strain, or the lysate of the strain contains acetylated spermidine or polyamine acetylation enzyme.
20. The health functional food according to claim 18, wherein the strain, the culture of the strain, or the lysate of the strain contains glutaric acid or glutaconic acid.
21. The health functional food according to claim 18, wherein the improvement of intestinal metabolite composition includes the following:

    - Reduction of spermidine, a metabolite in the intestines or feces;

    - Increase in acetylated spermidine, a metabolite in the intestines or feces;

    - Reduction of polyamine metabolites in the intestines or feces;

    - Decreased activity of polyamine synthetase in the intestine or feces; and

    - Increased activity of polyamine degrading enzymes in the intestines or feces.
22. The health functional food according to claim 18, wherein the strain, the culture of the strain, or the lysate of the strain promotes the activity of intestinal immune cells or the expression of tight junction proteins between intestinal cells.
23. The health functional food according to claim 18, which is an oral preparation.
24. An anti-cancer adjuvant comprising a Lactobacillus plantarum strain, a culture of the strain, lysate of the strain, or a mixture thereof as an active ingredient.
25. A feed composition for improving the intestinal metabolite composition of an individual comprising a Lactobacillus plantarum strain, a culture of the strain, lysate of the strain, or a mixture thereof as an active ingredient.
26. A method for improving the intestinal metabolite composition of a subject comprising administering an effective amount of a Lactobacillus plantarum strain, a culture of the strain, a lysate of the strain, or a mixture thereof to a subject in need thereof. .
27. A method for preventing or treating cancer comprising administering an effective amount of a Lactobacillus plantarum strain, a culture of the strain, a lysate of the strain, or a mixture thereof to an individual in need thereof.
28. Use of a composition comprising a Lactobacillus plantarum strain, a culture of the strain, lysate of the strain, or a mixture thereof for the production of a formulation for improving the intestinal metabolite composition of an individual.
29. Use of a composition comprising a Lactobacillus plantarum strain, a culture of the strain, lysate of the strain, or a mixture thereof for the production of a pharmaceutical agent for preventing or treating cancer.
30. Use of a composition containing a Lactobacillus plantarum strain, a culture of the strain, lysate of the strain, or a mixture thereof for the production of a health functional food for preventing or treating cancer.

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