WO2023287202A1 - Composition for prevention, reduction or treatment of cancer, comprising lactiplantibacillus paraplantarum wikim0130 - Google Patents

Composition for prevention, reduction or treatment of cancer, comprising lactiplantibacillus paraplantarum wikim0130 Download PDF

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WO2023287202A1
WO2023287202A1 PCT/KR2022/010228 KR2022010228W WO2023287202A1 WO 2023287202 A1 WO2023287202 A1 WO 2023287202A1 KR 2022010228 W KR2022010228 W KR 2022010228W WO 2023287202 A1 WO2023287202 A1 WO 2023287202A1
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cancer
tumor
wikim0130
paraplantarum
lactiplantibacillus
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Korean (ko)
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최학종
윤미선
박효경
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한국식품연구원
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/38—Other non-alcoholic beverages
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

  • the present invention relates to a novel Lacti Plantibacillus paraplantarum strain isolated from kimchi and a composition for preventing, improving or treating cancer comprising the same.
  • probiotic bacteria is being considered in order to solve the toxicity of attenuated bacteria with tumor orientation under existing research, but in their case, low anticancer effects or low tumor selectivity have been observed.
  • Lactic acid bacteria-related technology with excellent anti-tumor effect based on kimchi lactobacillus which can overcome the safety and efficiency problems of bacteriotherapy while supplementing the limitations of existing anticancer drugs, has been rarely reported.
  • Development of useful compositions for prevention and treatment and securing of various resources are required.
  • An object of the present invention is to provide a novel lactic acid bacteria of the genus Lacti Plantibacillus paraplantarum having excellent efficacy in preventing or improving cancer.
  • the present inventors have tried to find a lactic acid bacterium strain showing the effect of preventing or improving cancer from traditional fermented foods, and as a result, a novel lactic acid bacterium strain of the genus Lactiplantibacillus, Lactiplantibacillus paraplantarum WiKim0130, was isolated. , which led to the identification and completion of the present invention.
  • the present invention provides Lactiplantibacillus paraplantarum WiKim0130 ( Lactiplantibacillus paraplantarum WiKim0130).
  • Lactiplantibacillus paraplantarum WiKim0130 of the present invention is a new strain of Lactiplantibacillus paraplantarum derived from kimchi. Although Lacti Plantibacillus paraplantarum WiKim0130 in the present invention was isolated and identified from kimchi, its acquisition route is not limited thereto.
  • the lactic acid bacteria strain isolated through the examples of the present invention was found to have a nucleic acid sequence of SEQ ID NO: 1 as a result of 16S rDNA sequencing analysis for identification and classification of microorganisms.
  • the microorganism of the present invention having the 16S rDNA nucleotide sequence of SEQ ID NO: 1 was Lactiplantibacillus paraplantarum WiKim0130 ( Lactiplantibacillus paraplantarum WiKim0130), and deposited with the Korea Microbial Conservation Center on April 22, 2021 (accession number KCCM12978P).
  • Lactiplantibacillus paraplantarum WiKim0130 of the present invention is a Gram-positive bacterium and is a facultative anaerobe capable of growing under both aerobic and anaerobic conditions, and does not form spores, has no motility, and cells take the form of bacilli. are taking
  • the growth temperature is 15 °C to 40 °C
  • the optimum growth temperature is 37 °C. It was possible to grow at 15°C, but it did not grow at 45°C.
  • the growth pH is 4.0 to 7.5
  • the optimum growth pH is 6.0 to 6.5.
  • the shape was circular, the elevation was convex, and the surface was smooth. Catalase, gas formation, and indole production were not observed, but lactic acid production was observed. Additional characteristics were observed through experiments using API 50 CH kit for identifying lactic acid bacteria from Biomerieux and described in the following examples.
  • the Lacti Plantibacillus paraplantarum WiKim0130 of the present invention is CT26 (mouse colon cancer), MC38 (mouse colon cancer), HT29 (human colon cancer), SW620 (human colon cancer) H1650 (human lung cancer) , A549 (human lung cancer), PANC1 (human pancreatic cancer), A375 (human skin cancer), and HepG2 (human liver cancer) cells show cytotoxicity, and the normal skin cell line CCD-986-sk shows cancer cell killing ability that does not show cytotoxicity Lactiplantibacillus paraplantarum WiKim0130 ( Lactiplantibacillus paraplantarum WiKim0130) strain was confirmed to have an antitumor effect.
  • CT26 (mouse colon cancer cells) treated with Lactiplantibacillus paraplantarum WiKim0130 strain was 142.6%, MC38 (mouse colon cancer cells) 303.9%, HT29 (human colon cancer cells) 165.4%, SW620 ( human colorectal cancer cells) was 193.4%, H1650 (human non-small cell lung cancer cells) was 156.9%, A549 (human non-small cell lung cancer cells) was 124.8%, PANC1 (human pancreatic cancer cells) was 134.5%, A375 (human skin cancer cells) was 128.2% %, HepG2 (human liver cancer cells) was 133.7%, confirming the ability to kill cancer cells according to the observation of LDH leakage. Therefore, the Lacti Plantibacillus paraplantarum WiKim0130 according to the present invention can be variously used for preventing, treating, or improving human or animal cancer.
  • the composition containing Lacti Plantibacillus paraplantarum WiKim0130 as an active ingredient is CT26 (mouse colon cancer cells), MC38 (mouse colon cancer cells), HT29 (human colon cancer cells), SW620 (human colorectal cancer cells), H1650 (human non-small cell lung cancer cells), A549 (human non-small cell lung cancer cells), PANC1 (human pancreatic cancer cells), A375 (human skin cancer cells), HepG2 (human liver cancer cells) are killed,
  • the composition may be used as an active ingredient of a composition for preventing, treating, or improving one or more cancers selected from colorectal cancer, lung cancer, and pancreatic cancer.
  • the present invention provides a composition for preventing, improving or treating cancer disease comprising Lacti Plantibacillus paraplantarum WiKim0130, a culture thereof, a lysate thereof, or an extract thereof as an active ingredient.
  • Lacti Plantibacillus paraplantarum WiKim0130 included in the composition according to the present invention may exist as live or dead cells, and may also exist in a dried or lyophilized form.
  • Types of lactic acid bacteria suitable for inclusion in various compositions and formulation methods are well known to those skilled in the art.
  • Lacti Plantibacillus paraplantarum WiKim0130 is a culture obtained by culturing in a known liquid medium or solid medium, a fermented product obtained by culturing the strain and additional components together, or the strain obtained by organic cultivation. It can be formulated in the form of a solvent-extracted extract, dissolution of the cell membrane of the strain, or a lysate (or lysate) obtained by crushing or homogenizing, but is not limited thereto.
  • the composition may be a composition containing a strain of Lacti Plantibacillus paraplantarum WiKim0130 present as live or dead bacteria.
  • the cancer disease is brain tumor, benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, cerebral lymphoma, oligodendroma, intracranial carcinoma, ependymoma, brainstem tumor, head and neck tumor, laryngeal cancer, oropharyngeal cancer, nasal cancer, Sinus cancer, nasopharyngeal cancer, salivary gland cancer, hypopharyngeal cancer, thyroid cancer, chest tumor, small cell lung cancer, non-small cell lung cancer, thymus cancer, mediastinal tumor, esophageal cancer, breast cancer, male breast cancer, abdominal tumor, stomach cancer, liver cancer, gallbladder cancer, bile duct cancer, Pancreatic cancer, small intestine cancer, colon cancer, anal cancer, bladder cancer, kidney cancer, male genital tumor, penile cancer, urethral cancer, prostate cancer, female genital tumor, cervical cancer, endometrial cancer
  • the composition may be a pharmaceutical composition for the prevention or treatment of cancer comprising a culture, lysate, fermented product or extract of the Lacti Plantibacillus paraplantarum WiKim0130 strain.
  • the pharmaceutical composition of the present invention can be prepared using a pharmaceutically suitable and physiologically acceptable adjuvant in addition to the Lacti Plantibacillus paraplantarum WiKim0130 strain.
  • a pharmaceutically suitable and physiologically acceptable adjuvant in addition to the Lacti Plantibacillus paraplantarum WiKim0130 strain.
  • an excipient a disintegrant, a sweetener, a binder, a coating agent, an expanding agent, a lubricant, a lubricant, or a flavoring agent may be used.
  • the pharmaceutical composition may be preferably formulated as a pharmaceutical composition by including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients for administration.
  • the active ingredient may be combined with an oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, or water.
  • suitable binders, lubricants, disintegrants and coloring agents may also be included in the mixture.
  • suitable binders include, but are not limited to, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tracacanth or sodium oleate, sodium stearate, magnesium stearate, sodium benzoate. ate, sodium acetate, sodium chloride, and the like.
  • Disintegrants include, but are not limited to, starch, methylcellulose, agar, bentonite, xanthan gum, and the like.
  • acceptable pharmaceutical carriers are sterile and biocompatible, and include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added if necessary.
  • diluents such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets.
  • the present invention provides a food composition for preventing or improving cancer comprising Lacti Plantibacillus paraplantarum WiKim0130, a culture thereof, a lysate thereof, a fermented product thereof, or an extract thereof as an active ingredient.
  • the food composition may include a form of health functional food or beverage, bar, or the like.
  • the food composition containing the strain as an active ingredient may include a beverage such as fermented milk.
  • the food composition according to the present invention can be formulated in the same way as the pharmaceutical composition and used as a functional food or added to various foods.
  • Foods to which the composition of the present invention can be added include, for example, beverages, vitamin complexes, health supplements, and the like.
  • the food composition of the present invention may include ingredients commonly added during food preparation, and include, for example, proteins, carbohydrates, fats, nutrients, seasonings, and flavoring agents.
  • examples of the aforementioned carbohydrates include monosaccharides such as glucose, fructose, and the like; disaccharides such as maltose, sucrose, oligosaccharides and the like; and polysaccharides such as conventional sugars such as dextrins and cyclodextrins and sugar alcohols such as xylitol, sorbitol and erythritol.
  • flavoring agents natural flavoring agents [thaumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.]) and synthetic flavoring agents (saccharin, aspartame, etc.) may be used.
  • thaumatin, stevia extract eg, rebaudioside A, glycyrrhizin, etc.
  • synthetic flavoring agents sacharin, aspartame, etc.
  • composition according to the present invention may be used as a feed additive or feed.
  • the composition When used as a feed additive, the composition may be at a high concentration of 20 to 90% or prepared in powder or granular form.
  • the feed additives are organic acids such as citric acid, fumaric acid, adipic acid, lactic acid, and malic acid, sodium phosphate, potassium phosphate, acid pyrophosphate, polyphosphate (polyphosphate), polyphenol, catechin, alpha-tocopherol, and rosemary. extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, phytic acid, and the like, or any one or more natural antioxidants may be further included.
  • the composition When used as a feed, the composition may be formulated in a conventional feed form, and may include a common feed component together.
  • the feed additives and feeds include grains such as pulverized or crushed wheat, oats, barley, corn and rice; vegetable protein feeds such as those based on rape, soybean, and sunflower; animal protein feed such as blood meal, meat meal, bone meal and fish meal; It may further include sugar and dairy products, for example, dry ingredients composed of various powdered milk and whey powder, etc., and may further include nutritional supplements, digestion and absorption enhancers, growth promoters, and the like.
  • the feed additive may be administered to animals alone or in combination with other feed additives in an edible carrier.
  • the feed additives can be easily administered to animals as a top dressing, directly mixed with animal feed, or in an oral formulation separate from feed.
  • a pharmaceutically acceptable edible carrier can be solid or liquid, for example corn starch, lactose, sucrose, soybean flakes, peanut oil, olive oil, sesame oil and propylene glycol.
  • the feed additive may be a tablet, capsule, powder, troche or sugar-containing tablet or top dressing in a microdispersible form.
  • the feed additive may be a gelatin soft capsule, or a syrup, suspension, emulsion, or solution formulation.
  • the feed additives and feeds may contain auxiliary agents, for example, preservatives, stabilizers, wetting agents or emulsifying agents, solution accelerators, and the like.
  • the feed additive may be used by adding it to the animal's feed by soaking, spraying or mixing.
  • the feed or feed additive of the present invention can be applied to a number of animal diets including mammals, poultry and fish.
  • animals include mammals, and mammals may be used for pigs, cows, sheep, goats, laboratory rodents, and laboratory rodents, as well as pets (eg, dogs and cats), but are not limited thereto. not.
  • the amount of the Lacti Plantibacillus paraplantarum WiKim0130 strain included in the composition according to the present invention may be about 10 6 to 10 12 cfu / g, for example, 10 7 to 10 11 cfu / g, 10 8 to 10 10 cfu/g.
  • administering a strain it is preferable to administer it in a viable state, and it can be killed before ingestion or administered in an attenuation state.
  • a sterilization process through a heat treatment process may be additionally performed.
  • the amount of strain and daily intake required to have the minimum efficacy may vary depending on the body or health condition of the consumer, but may generally be about 10 6 to 10 12 cfu/g, such as 10 7 to 10 11 cfu/g. , 10 8 to 10 10 cfu/g.
  • Lactiplantibacillus paraplantarum WiKim0130 is a lactic acid bacterium isolated from kimchi and exhibits excellent anticancer effects, so it can be used in various ways for the prevention, improvement, and treatment of cancer diseases.
  • 1 is a graph of inflammatory cytokine analysis for antitumor activity according to the treatment of the WiKim0130 strain according to the present invention ((a) IL-12p70, (b) TNF, (c) IL-6, (d) MCP-1) .
  • Figure 2 is a graph of in vitro anticancer activity efficacy analysis according to the treatment of the WiKim0130 strain according to the present invention ((a) CT26, MC38, HT29, and SW620, (b) H1650 and A549, (c) PANC1, (d) A375, (e) HepG2, (f) CCD986Sk).
  • FIG. 3 is a photograph (a) of tumor size change and a tumor volume change graph (b) of CT26 mouse colon cancer according to treatment with WiKim0130 strain according to the present invention.
  • Example One Lactiplantibacillus Paraplantarum by WiKim0130 Isolation, cultivation and characterization
  • the present inventors isolated kimchi lactic acid bacteria from kimchi with radish kimchi at the ripening stage. After crushing the kimchi sample and spreading the stock solution of the kimchi extract on MRS agar medium (Difco), it was incubated at 30 ° C for more than 24 hours. Single colonies formed after culturing were subcultured to select uniform colonies, and the selected uniform colonies were finally identified through 16S RNA sequencing.
  • a single colony of Lactiplantibacillus paraplantarum cultured on MRS agar medium was inoculated into 20 ml of MRS liquid medium and then cultured at 37° C. for 24 hours. After culturing, the cells were centrifuged at 8,000 rpm for 10 minutes to remove the culture medium, and washed three times with PBS (Phosphate Buffered Saline) solution to remove remaining media components.
  • PBS Phosphate Buffered Saline
  • the strain isolated through the examples of the present invention was found to have a nucleic acid sequence of SEQ ID NO: 1.
  • the microorganism of the present invention was Lactiplantibacillus paraplantarum WiKim0130 ( Lactiplantibacillus paraplantarum WiKim0130), and deposited with the Korea Microbial Conservation Center on April 22, 2021 (accession number KCCM12978P).
  • WiKim0130 Lactiplantibacillus paraplantarum The mycological characteristics of WiKim0130.
  • reaction substrate result 1 glycerol - 26 Salicin + 2 erythritol - 27 Cellobiose + 3 D Arabinose - 28 Maltose + 4 L arabinose - 29 Lactose - 5 Ribose D-Ribose - 30 Melibios - 6 D-Xylose D-Xylose - 31 sucrose + 7 L Xylose L-Xylose - 32 Trehalose + 8 Adonitol D-Adonitol - 33 Inulin - 9 methyl-BD-xylofuranocycle - 34 Melezitus + 10 D-galactose - 35 Raffinose - 11 D-glucose + 36 Amidon - 12 D-fructose + 37 Glycogen - 13 D-mannose + 38 xylitol - 14 L-Sorbose - 39 Gentiobios + 15FKASHTM - 40 D Tur
  • Example 2 Lactiplantibacillus Paraplantarum by WiKim0130 Inflammatory cytokine test results
  • Dendritic cells differentiated from bone marrow cells of mice transplanted with MC38 cancer cells were treated with Lacti Plantibacillus paraplantarum WiKim0130 to confirm the expression of anti-cancer active immune cytokines.
  • Bone marrow cells were obtained from mouse shinbones and femurs using RPMI medium and a syringe needle. The isolated bone marrow cells were cultured in RPMI 1640 medium containing granulocyte-macrophage colony-stimulating factor (GM-CSF), 10% heat-inactivated bovine serum, 50 ⁇ M ⁇ -mercaptoethanol, and 2 mM glutamine, and dendritic cells differentiated into cells.
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • Bone marrow-derived dendritic cells cultured in a 96-well plate at a density of 2 ⁇ 10 5 cells/ml were treated with Lactiplantibacillus paraplantarum WiKim0130 at a concentration of MOI 3 for 24 hours, and the supernatant was recovered to detect cytokines.
  • the analysis was performed, and LPS (100 ng/ml), which causes an inflammatory response and increases the expression of anticancer activity cytokines, was also compared, and shown in FIG. 1.
  • Example 3 Lactiplantibacillus Paraplantarum by WiKim0130 cancer cell ability to kill Verification result
  • the cells used in the anticancer activity efficacy test were CT26 (mouse colon cancer), MC38 (mouse colon cancer), HT29 (human colon cancer), SW620 (human colon cancer), H1650 (human lung cancer), A549 (human lung cancer), PANC1 (human pancreatic cancer), A375 (human skin cancer), HepG2 (human liver cancer) cells, and normal skin cell line CCD-986-sk were used.
  • CT26 and MC38 were subcultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in a 37°C, 5% CO 2 incubator, and cells in the growth phase were used for experiments.
  • the cultured CT26 and MC38 cells were treated with 0.05% trypsin-EDTA in a 37°C incubator for 3 minutes to detach the cells, and then washed twice with DPBS to prepare 1 X 10 4 cells/well.
  • HT29, SW620, H1650, A549, PANC1, A375, HepG2 were subcultured in RPMI medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37°C in a 5% CO 2 incubator. Experiments were performed using growing cells. The cultured HT29, SW620, H1650, A549, PANC1, A375, and HepG2 cells were treated with 0.05% trypsin-EDTA for 3 minutes in a 37°C incubator to detach the cells, wash twice with DPBS, and then 1 X 10 4 cells/well. prepared.
  • FBS fetal bovine serum
  • CCD-986sk Human-derived normal skin cell CCD-986sk was subcultured in RPMI medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in a 37°C, 5% CO 2 incubator, and the cells in the growing phase were used for experiments. .
  • the cultured CCD-986sk was treated with 0.05% trypsin-EDTA in a 37°C incubator for 3 minutes to detach the cells, and then washed twice with DPBS to prepare 1 X 10 4 cells/well.
  • human CCD-986-sk human skin cells
  • the cytotoxicity of human CCD-986-sk was 95.1%, similar to that of control cells, and it was observed that they survived about 5% better than cells not treated with Lacti Plantibacillus paraplantarum WiKim0130. Apoptosis of skin cells was not observed.
  • mice used in the experiment were supplied with male 5-week-old BALB/c mice (Orient Bio, Korea), and stabilized for 1 week in an animal breeding room in an SPF environment maintained at a room temperature of 20 ⁇ 2 ° C and a humidity of 55 ⁇ 15%. After a period, they were reared during the experimental period. Feed was supplied with common pellet feed without antibiotics, and water was allowed to be consumed at any time. All animals were reared, tested, and euthanized according to the protocol approved by the Animal Experiment Ethics Committee of the World Institute of Kimchi. Observation of tumor size change was performed by measuring the volume (mm 3 ) of the tumor using the formula of 3.14 x (length x height x width)/6.
  • CT26 mouse colon cancer cells ATCC, USA
  • CT26 mouse colon cancer cells were cultured at 5% CO 2 and 37°C using DMEM medium (Hyclone, USA) containing 10% fetal bovine serum and 1% penicillin-streptomycin.
  • a CT26 cancer cell-transplanted mouse tumor model was prepared and the anti-tumor efficacy was confirmed and shown in FIG. 3 .
  • Lacti Plantibacillus paraplantarum WiKim0130 was injected into the tumor-formed mouse through the tail vein.
  • Lactiplantibacillus paraplantarum WiKim0130 was prepared by quantifying the number of bacteria at 1 ⁇ 10 10 CFU/ml using PBS, and 0.1 ml (1 ⁇ 10 9 CFU) was injected into the tail vein of the experimental animals once, and PBS was administered to the negative control group. did Changes in the tumor size of CT26 mouse colon cancer over time were visually confirmed, and the volume of the tumor size was measured in the cancer model mouse. As a result, the change in the size of the tumor over time was visually confirmed for 10 to 13 days, and the volume of the tumor tissue in the cancer model mouse was also measured.

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Abstract

The present invention relates to a novel Lactiplantibacillus paraplantarum WiKim0130 isolated from kimchi and a composition comprising same. The Lactiplantibacillus paraplantarum WiKim0130, according to the present invention, exhibits an excellent anticancer effect and thus may be variously employed for the use of prevention, reduction and treatment of a cancer disease.

Description

락티플란티바실러스 파라플란타룸 WIKIM0130을 포함하는 암의 예방, 개선 또는 치료용 조성물 Composition for preventing, improving or treating cancer containing Lactiplantibacillus paraplantarum WIKIM0130
본 발명은 김치로부터 분리한 신규한 락티플란티바실러스 파라플란타룸 균주 및 이를 포함하는 암의 예방, 개선 또는 치료용 조성물에 관한 것이다.The present invention relates to a novel Lacti Plantibacillus paraplantarum strain isolated from kimchi and a composition for preventing, improving or treating cancer comprising the same.
의학기술의 발전으로 암환자의 생존율은 증가하고 있으나 아직도 높은 사망률을 보이고 있다. 실례로 2019년 우리나라 전체 사망자 중 암에 의한 사망자는 27.5%로 여전히 사망원인 1위로 기록되고 있다.Although the survival rate of cancer patients is increasing with the development of medical technology, the mortality rate is still high. For example, among all deaths in Korea in 2019, cancer deaths accounted for 27.5%, which is still the number one cause of death.
현재 임상에서 시행되는 암 치료 방법은 화학항암요법, 표적항암요법, 방사선 치료 등 많은 방법이 시도되고 있다. 하지만 여전히 암치료에 있어서 모든 암세포를 완전히 제거하지 못하고 정상세포에 대한 독성이 강하다. 그 이유로는 ① 불완전한 암 표적, ② 암 조직에 대한 불충분한 침투, ③ 종양이질성에 의한 제한적인 암 사멸효과 등을 들 수 있다. 실제로, 암 조직에 무질서하고 불완전하게 생성되는 혈관 때문에 ① 치료물질과 영양분이 잘 전달되지 않는 영역이 생기고, ② 여기에 생성되는 저산소/저포도당 영역의 세포들은 성장이 정지되어 대부분의 항암제나 방사선 치료에 내성을 갖게 된다. 또한, ③ 암세포의 빠른 성장은 혈관사이의 간극을 멀게하고, 혈류를 감소시키며 혈관으로부터 멀리 떨어진 암 조직으로의 약물전달은 더욱 감소하게 된다. Currently, many methods of cancer treatment, such as chemotherapy, targeted anticancer therapy, and radiation therapy, are being tried in clinical practice. However, it still fails to completely remove all cancer cells in cancer treatment and is highly toxic to normal cells. The reasons include ① incomplete cancer targeting, ② insufficient penetration into cancer tissues, and ③ limited cancer killing effect due to tumor heterogeneity. In fact, due to disorderly and incompletely formed blood vessels in cancer tissue, ① there is an area where treatment substances and nutrients are not delivered well, ② cells in the hypoxic/low glucose area generated here stop growing, and most of the anticancer drugs or radiation treatments become resistant to In addition, ③ rapid growth of cancer cells widens the gap between blood vessels, reduces blood flow, and further reduces drug delivery to cancer tissues far from blood vessels.
암 표적능을 지닌 박테리아 균주를 이용한 암 치료 연구는 세계적으로 관심이 증가하고 있으며 일부 유산균을 포함한 미생물은 종양으로 생성된 미세환경에서 특이적으로 성장하는 것이 보고되고 있다. 이러한 특징으로 박테리오테라피 기술, 즉 약독화 된 박테리아를 기반으로 하는 암 치료 연구가 꾸준하게 시도되고 있다. Research on cancer treatment using bacterial strains with cancer-targeting activity is increasing worldwide interest, and it has been reported that microorganisms, including some lactic acid bacteria, grow specifically in the microenvironment created by tumors. Due to these characteristics, research on cancer treatment based on bacteriotherapy technology, that is, attenuated bacteria, has been steadily attempted.
기존에 연구 중인 종양 지향성을 갖는 약독화 박테리아의 독성을 해결하고자 프로바이오틱 박테리아의 활용이 고려되고 있으나, 이들의 경우 항암 효과가 낮거나 또는 종양 선택성이 낮게 관찰되었다. 기존의 항암치료제의 한계를 보완하며 박테리오테라피의 안전성과 효율성 문제를 극복 할 수 있는 김치 유산균 기반 항종양 효과가 우수한 유산균 관련 기술은 드물게 보고 되어지고 있는 바, 효과가 우수한 유산균을 포함하는 고형암에 대한 예방 및 치료에 유용한 조성물의 개발 및 다양한 자원 확보가 요구되고 있다. The use of probiotic bacteria is being considered in order to solve the toxicity of attenuated bacteria with tumor orientation under existing research, but in their case, low anticancer effects or low tumor selectivity have been observed. Lactic acid bacteria-related technology with excellent anti-tumor effect based on kimchi lactobacillus, which can overcome the safety and efficiency problems of bacteriotherapy while supplementing the limitations of existing anticancer drugs, has been rarely reported. Development of useful compositions for prevention and treatment and securing of various resources are required.
본 발명은 암의 예방 또는 개선 효능이 우수한 신규한 락티플란티바실러스 파라플란타룸 속 유산균을 제공하고자 한다. An object of the present invention is to provide a novel lactic acid bacteria of the genus Lacti Plantibacillus paraplantarum having excellent efficacy in preventing or improving cancer.
이에, 본 발명자들은 전통발효식품으로부터 암의 예방 또는 개선 효과를 나타내는 유산균 균주를 찾고자 노력한 결과, 신규한 락티플란티바실러스 속 유산균 균주인 락티플란티바실러스 파라플란타룸 WiKim0130(Lactiplantibacillus paraplantarum WiKim0130)을 분리, 동정하여 본 발명을 완성하게 되었다.Accordingly, the present inventors have tried to find a lactic acid bacterium strain showing the effect of preventing or improving cancer from traditional fermented foods, and as a result, a novel lactic acid bacterium strain of the genus Lactiplantibacillus, Lactiplantibacillus paraplantarum WiKim0130, was isolated. , which led to the identification and completion of the present invention.
본 발명은 락티플란티바실러스 파라플란타룸 WiKim0130(Lactiplantibacillus paraplantarum WiKim0130)을 제공한다.The present invention provides Lactiplantibacillus paraplantarum WiKim0130 ( Lactiplantibacillus paraplantarum WiKim0130).
본 발명의 락티플란티바실러스 파라플란타룸 WiKim0130(Lactiplantibacillus paraplantarum WiKim0130)은 김치 유래의 락티플란티바실러스 파라플란타룸 신규 균주이다. 비록 본 발명에서의 락티플란티바실러스 파라플란타룸 WiKim0130을 김치에서 분리, 동정하기는 했으나, 이의 입수 경로가 이에 한정되는 것은 아니다. Lactiplantibacillus paraplantarum WiKim0130 of the present invention is a new strain of Lactiplantibacillus paraplantarum derived from kimchi. Although Lacti Plantibacillus paraplantarum WiKim0130 in the present invention was isolated and identified from kimchi, its acquisition route is not limited thereto.
본 발명에의 실시예를 통해 분리된 유산균 균주는 미생물의 동정 및 분류를 위한 16S rDNA 염기서열 분석 결과, SEQ ID NO: 1의 핵산서열을 갖는 것으로 나타났다. The lactic acid bacteria strain isolated through the examples of the present invention was found to have a nucleic acid sequence of SEQ ID NO: 1 as a result of 16S rDNA sequencing analysis for identification and classification of microorganisms.
따라서, SEQ ID NO: 1의 16S rDNA 염기서열을 갖는 본 발명의 미생물을 락티플란티바실러스 파라플란타룸 WiKim0130(Lactiplantibacillus paraplantarum WiKim0130)로 명명하였으며, 한국미생물보존센터에 2021년 04월 22일자로 기탁하였다(수탁번호 KCCM12978P).Therefore, the microorganism of the present invention having the 16S rDNA nucleotide sequence of SEQ ID NO: 1 was Lactiplantibacillus paraplantarum WiKim0130 ( Lactiplantibacillus paraplantarum WiKim0130), and deposited with the Korea Microbial Conservation Center on April 22, 2021 (accession number KCCM12978P).
본 발명의 락티플란티바실러스 파라플란타룸 WiKim0130은 그람양성균이고 호기적 조건과 혐기적 조건에서 모두 성장이 가능한 통성 혐기성(facultative anaerobe)이며, 포자를 형성하지 않고 운동성이 없으며 세포는 간균의 형태를 취하고 있다. 생육 온도는 15℃내지 40℃ 최적 생장온도는 37℃이다. 15℃에서 생육 가능하였으며, 45℃에서는 성장하지 않는다. 생육 pH는 4.0 내지 7.5이며, 최적 생장 pH는 6.0 내지 6.5이다. 형상은 원형이며 융기는 볼록하고 표면은 매끄러운 균락의 형태를 지녔으며, 카탈라제, 가스 형성 여부, 인돌 생산은 관찰되지 않았으며, 젖산 생산은 관찰되었다. Biomerieux 사의 유산균 동정용 API 50 CH kit를 이용한 실험으로 추가 특성을 관찰하여 하기 실시예에 기재하였다.Lactiplantibacillus paraplantarum WiKim0130 of the present invention is a Gram-positive bacterium and is a facultative anaerobe capable of growing under both aerobic and anaerobic conditions, and does not form spores, has no motility, and cells take the form of bacilli. are taking The growth temperature is 15 ℃ to 40 ℃, the optimum growth temperature is 37 ℃. It was possible to grow at 15℃, but it did not grow at 45℃. The growth pH is 4.0 to 7.5, and the optimum growth pH is 6.0 to 6.5. The shape was circular, the elevation was convex, and the surface was smooth. Catalase, gas formation, and indole production were not observed, but lactic acid production was observed. Additional characteristics were observed through experiments using API 50 CH kit for identifying lactic acid bacteria from Biomerieux and described in the following examples.
하기 실시예에서는, 본 발명의 락티플란티바실러스 파라플란타룸 WiKim0130이 CT26(생쥐 대장암)과 MC38(생쥐 대장암), HT29(사람 대장암), SW620(사람 대장암) H1650(사람 폐암), A549(사람 폐암), PANC1(사람 췌장암), A375(사람 피부암), HepG2(사람 간암) 세포에 세포 독성을 나타내며, 정상피부세포주인 CCD-986-sk에서는 세포독성이 나타나지 않는 암세포 사멸능과 BALB/c 생쥐를 이용한 CT26(생쥐 대장암)세포가 이식된 실험동물 모델을 이용하여 락티플란티바실러스 파라플란타룸 WiKim0130 (Lactiplantibacillus paraplantarum WiKim0130) 균주의 항종양 효과를 확인하였다. 락티플란티바실러스 파라플란타룸 WiKim0130 (Lactiplantibacillus paraplantarum WiKim0130) 균주가 처리된 CT26(생쥐 대장암세포)은 142.6%, MC38(생쥐 대장암세포)은 303.9%, HT29(사람 대장암세포)은 165.4%, SW620(사람 대장암세포)은 193.4%, H1650(사람 비소세포폐암세포)은 156.9%, A549(사람 비소세포폐암세포)은 124.8%, PANC1(사람 췌장암세포)은 134.5%, A375(사람 피부암세포)는 128.2%, HepG2(사람 간암세포)는 133.7%로 LDH leakage 관찰 따른 암세포 사멸능을 확인하였다. 따라서, 본 발명에 따른 락티플란티바실러스 파라플란타룸 WiKim0130은 사람 또는 동물의 암 예방, 치료 또는 개선 용도를 위해 다양하게 활용될 수 있다.In the following examples, the Lacti Plantibacillus paraplantarum WiKim0130 of the present invention is CT26 (mouse colon cancer), MC38 (mouse colon cancer), HT29 (human colon cancer), SW620 (human colon cancer) H1650 (human lung cancer) , A549 (human lung cancer), PANC1 (human pancreatic cancer), A375 (human skin cancer), and HepG2 (human liver cancer) cells show cytotoxicity, and the normal skin cell line CCD-986-sk shows cancer cell killing ability that does not show cytotoxicity Lactiplantibacillus paraplantarum WiKim0130 ( Lactiplantibacillus paraplantarum WiKim0130) strain was confirmed to have an antitumor effect. CT26 (mouse colon cancer cells) treated with Lactiplantibacillus paraplantarum WiKim0130 strain was 142.6%, MC38 (mouse colon cancer cells) 303.9%, HT29 (human colon cancer cells) 165.4%, SW620 ( human colorectal cancer cells) was 193.4%, H1650 (human non-small cell lung cancer cells) was 156.9%, A549 (human non-small cell lung cancer cells) was 124.8%, PANC1 (human pancreatic cancer cells) was 134.5%, A375 (human skin cancer cells) was 128.2% %, HepG2 (human liver cancer cells) was 133.7%, confirming the ability to kill cancer cells according to the observation of LDH leakage. Therefore, the Lacti Plantibacillus paraplantarum WiKim0130 according to the present invention can be variously used for preventing, treating, or improving human or animal cancer.
또한, 하기 실시예에서 확인할 수 있는 바와 같이, 락티플란티바실러스 파라플란타룸 WiKim0130을 유효성분으로 포함하는 조성물은 CT26(생쥐 대장암세포), MC38(생쥐 대장암세포), HT29(사람 대장암세포), SW620(사람 대장암세포), H1650(사람 비소세포폐암세포), A549(사람 비소세포폐암세포), PANC1(사람 췌장암세포), A375(사람 피부암세포), HepG2(사람 간암세포)를 사멸시켜, 상기 조성물은 대장암, 폐암 및 췌장암으로부터 선택되는 하나 이상의 암의 예방, 치료 또는 개선용 조성물의 유효 성분으로 사용될 수 있다.In addition, as can be seen in the following examples, the composition containing Lacti Plantibacillus paraplantarum WiKim0130 as an active ingredient is CT26 (mouse colon cancer cells), MC38 (mouse colon cancer cells), HT29 (human colon cancer cells), SW620 (human colorectal cancer cells), H1650 (human non-small cell lung cancer cells), A549 (human non-small cell lung cancer cells), PANC1 (human pancreatic cancer cells), A375 (human skin cancer cells), HepG2 (human liver cancer cells) are killed, The composition may be used as an active ingredient of a composition for preventing, treating, or improving one or more cancers selected from colorectal cancer, lung cancer, and pancreatic cancer.
본 발명은 락티플란티바실러스 파라플란타룸 WiKim0130, 이의 배양물, 이의 파쇄물, 또는 이의 추출물을 유효성분으로 포함하는 암 질환의 예방, 개선 또는 치료용 조성물을 제공한다.The present invention provides a composition for preventing, improving or treating cancer disease comprising Lacti Plantibacillus paraplantarum WiKim0130, a culture thereof, a lysate thereof, or an extract thereof as an active ingredient.
본 발명에 따른 조성물에 포함되는 락티플란티바실러스 파라플란타룸 WiKim0130은 생균체 또는 사균체로서 존재할 수 있으며, 또한 건조 또는 동결건조된 형태로 존재할 수도 있다. 다양한 조성물 내에 포함시키기 적합한 유산균의 형태 및 제제화 방법은 당업자에게 잘 알려져 있다. 예를 들어, 락티플란티바실러스 파라플란타룸 WiKim0130은 공지의 액체 배지 또는 고체 배지에서 배양시켜 수득한 배양물이거나, 상기 균주와 추가의 성분을 함께 배양하여 수득한 발효물이거나, 상기 균주를 유기용매로 추출한 추출물, 상기 균주의 세포막을 용해시키거나, 파쇄 또는 균질화 처리한 용해물(또는 파쇄물) 등의 형태로 제제화할 수 있으나 이에 제한되는 것은 아니다. Lacti Plantibacillus paraplantarum WiKim0130 included in the composition according to the present invention may exist as live or dead cells, and may also exist in a dried or lyophilized form. Types of lactic acid bacteria suitable for inclusion in various compositions and formulation methods are well known to those skilled in the art. For example, Lacti Plantibacillus paraplantarum WiKim0130 is a culture obtained by culturing in a known liquid medium or solid medium, a fermented product obtained by culturing the strain and additional components together, or the strain obtained by organic cultivation. It can be formulated in the form of a solvent-extracted extract, dissolution of the cell membrane of the strain, or a lysate (or lysate) obtained by crushing or homogenizing, but is not limited thereto.
한 구체예에서, 상기 조성물은 생균 또는 사균으로 존재하는 락티플란티바실러스 파라플란타룸 WiKim0130 균주를 포함하는 조성물일 수 있다.In one embodiment, the composition may be a composition containing a strain of Lacti Plantibacillus paraplantarum WiKim0130 present as live or dead bacteria.
본 발명에 있어서, 상기 암 질환은 뇌종양, 양성성상세포종, 악성성상세포종, 뇌하수체 선종, 뇌수막종, 뇌림프종, 핍지교종, 두개내인종, 상의세포종, 뇌간종양, 두경부 종양, 후두암, 구인두암, 비강암, 부비동암, 비인두암, 침샘암, 하인두암, 갑상선암, 흉부종양, 소세포성 폐암, 비소세포성 폐암, 흉선암, 종격동 종양, 식도암, 유방암, 남성유방암, 복부종양, 위암, 간암, 담낭암, 담도암, 췌장암, 소장암, 대장암, 항문암, 방광암, 신장암, 남성생식기종양, 음경암, 요도암, 전립선암, 여성생식기종양, 자궁경부암, 자궁내막암, 난소암, 자궁육종, 질암, 여성 외부생식기암, 여성요도암, 피부암, 골수종, 백혈병 및 악성림프종일 수 있으며, 바람직하게는 대장암, 폐암, 췌장암, 피부암 및 간암을 포함할 수 있다.In the present invention, the cancer disease is brain tumor, benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, cerebral lymphoma, oligodendroma, intracranial carcinoma, ependymoma, brainstem tumor, head and neck tumor, laryngeal cancer, oropharyngeal cancer, nasal cancer, Sinus cancer, nasopharyngeal cancer, salivary gland cancer, hypopharyngeal cancer, thyroid cancer, chest tumor, small cell lung cancer, non-small cell lung cancer, thymus cancer, mediastinal tumor, esophageal cancer, breast cancer, male breast cancer, abdominal tumor, stomach cancer, liver cancer, gallbladder cancer, bile duct cancer, Pancreatic cancer, small intestine cancer, colon cancer, anal cancer, bladder cancer, kidney cancer, male genital tumor, penile cancer, urethral cancer, prostate cancer, female genital tumor, cervical cancer, endometrial cancer, ovarian cancer, uterine sarcoma, vaginal cancer, external female It may be genital cancer, female urethral cancer, skin cancer, myeloma, leukemia, and malignant lymphoma, and preferably may include colorectal cancer, lung cancer, pancreatic cancer, skin cancer, and liver cancer.
한 구체예에서, 상기 조성물은 락티플란티바실러스 파라플란타룸 WiKim0130 균주의 배양물, 파쇄물, 발효물 또는 추출물을 포함하는 암 질환의 예방 또는 치료용 약학 조성물일 수 있다. In one embodiment, the composition may be a pharmaceutical composition for the prevention or treatment of cancer comprising a culture, lysate, fermented product or extract of the Lacti Plantibacillus paraplantarum WiKim0130 strain.
본 발명에 따른 조성물이 약제학적 조성물로 활용될 경우, 본 발명의 약제학적 조성물은 상기 락티플란티바실러스 파라플란타룸 WiKim0130 균주 이외에 약제학적으로 적합하고 생리학적으로 허용되는 보조제를 사용하여 제조될 수 있으며, 상기 보조제로는 부형제, 붕해제, 감미제, 결합제, 피복제, 팽창제, 윤활제, 활택제 또는 향미제 등을 사용할 수 있다.When the composition according to the present invention is used as a pharmaceutical composition, the pharmaceutical composition of the present invention can be prepared using a pharmaceutically suitable and physiologically acceptable adjuvant in addition to the Lacti Plantibacillus paraplantarum WiKim0130 strain. And, as the adjuvant, an excipient, a disintegrant, a sweetener, a binder, a coating agent, an expanding agent, a lubricant, a lubricant, or a flavoring agent may be used.
상기 약제학적 조성물은 투여를 위해서 상기 기재한 유효성분 이외에 추가로 약제학적으로 허용가능한 담체를 1종 이상 포함하여 약제학적 조성물로 바람직하게 제제화할 수 있다.The pharmaceutical composition may be preferably formulated as a pharmaceutical composition by including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients for administration.
예를 들어, 정제 또는 캡슐제의 형태로의 제제화를 위해, 유효성분은 에탄올, 글리세롤, 물 등과 같은 경구, 무독성의 약제학적으로 허용가능한 불활성 담체와 결합될 수 있다. 또한, 원하거나 필요한 경우, 적합한 결합제, 윤활제, 붕해제 및 발색제 또한 혼합물로 포함될 수 있다. 적합한 결합제는 이에 제한되는 것은 아니나, 녹말, 젤라틴, 글루코스 또는 베타-락토오스와 같은 천연당, 옥수수감미제, 아카시아, 트래커캔스 또는 소듐올레이트와 같은 천연 및 합성검, 소듐 스테아레이트, 마그네슘 스테아레이트, 소듐 벤조에이트, 소듐아세테이트, 소듐 클로라이드등을 포함한다. 붕해제는 이에 제한되는 것은 아니나, 녹말, 메틸셀룰로스, 아가, 벤토니트, 잔탄검 등을 포함한다. 액상 용액으로 제제화되는 조성물에 있어서 허용가능한 약제학적 담체로는, 멸균 및 생체에 적합한 것으로서, 식염수, 멸균수, 링거액, 완충식염수, 알부민 주사 용액, 덱스트로즈 용액, 말토덱스트린 용액, 글리세롤, 에탄올 및 이들 성분 중 1 성분이상을 혼합하여 사용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있다.For example, for formulation in the form of a tablet or capsule, the active ingredient may be combined with an oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, or water. In addition, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included in the mixture. Suitable binders include, but are not limited to, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tracacanth or sodium oleate, sodium stearate, magnesium stearate, sodium benzoate. ate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methylcellulose, agar, bentonite, xanthan gum, and the like. For compositions formulated as liquid solutions, acceptable pharmaceutical carriers are sterile and biocompatible, and include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added if necessary. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to prepare formulations for injections such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets.
나아가 해당 분야의 적절한 방법으로 Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA에 개시되어 있는 방법을 이용하여 각 질환에 따라 또는 성분에 따라 바람직하게 제제화할 수 있다.Furthermore, it can be preferably formulated according to each disease or component using a method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field.
한 구체예에서, 본 발명은 락티플란티바실러스 파라플란타룸 WiKim0130, 이의 배양물, 이의 파쇄물, 이의 발효물 또는 이의 추출물을 유효성분으로 포함하는 암의 예방 또는 개선용 식품 조성물을 제공한다. 상기 식품 조성물은 건강기능식품 또는 음료, 바 등의 형태를 포함할 수 있다. In one embodiment, the present invention provides a food composition for preventing or improving cancer comprising Lacti Plantibacillus paraplantarum WiKim0130, a culture thereof, a lysate thereof, a fermented product thereof, or an extract thereof as an active ingredient. The food composition may include a form of health functional food or beverage, bar, or the like.
본 발명에 있어서, 상기 균주를 유효성분으로 포함하는 식품 조성물은 발효유 등의 음료를 포함할 수 있다.In the present invention, the food composition containing the strain as an active ingredient may include a beverage such as fermented milk.
본 발명에 따른 식품 조성물은 상기 약제학적 조성물과 동일한 방식으로 제제화되어 기능성 식품으로 이용하거나, 각종 식품에 첨가할 수 있다. 본 발명의 조성물을 첨가할 수 있는 식품으로는 예를 들어, 음료류, 비타민복합제, 건강보조식품류 등이 있다.The food composition according to the present invention can be formulated in the same way as the pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, vitamin complexes, health supplements, and the like.
본 발명의 식품 조성물은 식품 제조시에 통상적으로 첨가되는 성분을 포함할 수 있으며, 예를 들어, 단백질, 탄수화물, 지방, 영양소, 조미제 및 향미제를 포함한다. 상술한 탄수화물의 예는 모노사카라이드, 예를 들어, 포도당, 과당 등; 디사카라이드, 예를 들어 말토스, 슈크로스, 올리고당 등; 및 폴리사카라이드, 예를 들어 덱스트린, 사이클로덱스트린 등과 같은 통상적인 당 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 향미제로서 천연 향미제 [타우마틴, 스테비아추출물 (예를 들어 레바우디오시드A, 글리시르히진 등]) 및 합성향미제(사카린, 아스파르탐 등)를 사용할 수 있다. 예컨대, 본 발명의 식품 조성물이 드링크제와 음료류로 제조되는 경우에는 구연산, 액상과당, 설탕, 포도당, 초산, 사과산, 과즙, 및 각 종 식물 추출액 등을 추가로 포함시킬 수 있다.The food composition of the present invention may include ingredients commonly added during food preparation, and include, for example, proteins, carbohydrates, fats, nutrients, seasonings, and flavoring agents. Examples of the aforementioned carbohydrates include monosaccharides such as glucose, fructose, and the like; disaccharides such as maltose, sucrose, oligosaccharides and the like; and polysaccharides such as conventional sugars such as dextrins and cyclodextrins and sugar alcohols such as xylitol, sorbitol and erythritol. As flavoring agents, natural flavoring agents [thaumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.]) and synthetic flavoring agents (saccharin, aspartame, etc.) may be used. For example, when the food composition of the present invention is made into drinks and beverages, citric acid, high fructose corn syrup, sugar, glucose, acetic acid, malic acid, fruit juice, and various plant extracts may be further included.
상기 본 발명에 따른 조성물은 사료첨가제 또는 사료로서 이용될 수 있다.The composition according to the present invention may be used as a feed additive or feed.
사료 첨가제로서 이용될 경우, 상기 조성물은 20 내지 90% 고농축액이거나 분말 또는 과립 형태로 제조될 수 있다. 상기 사료첨가제는 구연산, 후말산, 아디픽산, 젖산, 사과산등의 유기산이나 인산 나트륨, 인산 칼륨, 산성 피로인산염, 폴리인산염(중합인산염) 등의 인산염이나, 폴리페놀, 카테킨, 알파-토코페롤, 로즈마리 추출물, 비타민 C, 녹차 추출물, 감초 추출물, 키토산, 탄닌산, 피틴산 등의 천연 항산화제 중 어느 하나 또는 하나 이상을 추가로 포함할 수 있다. 사료로서 이용될 경우, 상기 조성물은 통상의 사료 형태로 제제화 될 수 있으며, 통상의 사료성분을 함께 포함할 수 있다.When used as a feed additive, the composition may be at a high concentration of 20 to 90% or prepared in powder or granular form. The feed additives are organic acids such as citric acid, fumaric acid, adipic acid, lactic acid, and malic acid, sodium phosphate, potassium phosphate, acid pyrophosphate, polyphosphate (polyphosphate), polyphenol, catechin, alpha-tocopherol, and rosemary. extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, phytic acid, and the like, or any one or more natural antioxidants may be further included. When used as a feed, the composition may be formulated in a conventional feed form, and may include a common feed component together.
상기 사료첨가제 및 사료는 곡물, 예를 들면 분쇄 또는 파쇄된 밀, 귀리, 보리, 옥수수 및 쌀; 식물성 단백질 사료, 예를 들면 평지, 콩, 및 해바라기를 주성분으로 하는 사료; 동물성 단백질 사료, 예를 들면 혈분, 육분, 골분 및 생선분; 당분 및 유제품, 예를 들면 각종 분유 및 유장 분말로 이루어지는 건조성분 등을 더 포함할 수 있으며, 이외에도 영양보충제, 소화 및 흡수향상제, 성장촉진제 등을 더 포함할 수 있다.The feed additives and feeds include grains such as pulverized or crushed wheat, oats, barley, corn and rice; vegetable protein feeds such as those based on rape, soybean, and sunflower; animal protein feed such as blood meal, meat meal, bone meal and fish meal; It may further include sugar and dairy products, for example, dry ingredients composed of various powdered milk and whey powder, etc., and may further include nutritional supplements, digestion and absorption enhancers, growth promoters, and the like.
상기 사료첨가제는 동물에게 단독으로 투여하거나 식용 담체 중에서 다른 사료첨가제와 조합하여 투여할 수도 있다. 또한, 상기 사료첨가제는 탑드레싱으로서 또는 이들을 동물사료에 직접 혼합하거나 또는 사료와 별도의 경구제형으로 용이하게 동물에게 투여할 수 있다. 상기 사료첨가제를 동물사료와 별도로 투여할 경우, 당해 기술분야에 잘 알려진 바와 같이 약제학적으로 허용가능한 식용 담체와 조합하여, 즉시 방출 또는 서방성 제형으로 제조할 수 있다. 이러한 식용 담체는 고체 또는 액체, 예를 들어 옥수수전분, 락토오스, 수크로오스, 콩플레이크, 땅콩유, 올리브유, 참깨유 및 프로필렌글리콜일 수 있다. 고체 담체가 사용될 경우, 사료첨가제는 정제, 캡슐제, 산제, 트로키제 또는 함당정제 또는 미분산성 형태의 탑 드레싱일 수 있다. 액체 담체가 사용될 경우, 사료첨가제는 젤라틴 연질 캡슐제, 또는 시럽제나 현탁액, 에멀젼제, 또는 용액제의 제형일 수 있다. The feed additive may be administered to animals alone or in combination with other feed additives in an edible carrier. In addition, the feed additives can be easily administered to animals as a top dressing, directly mixed with animal feed, or in an oral formulation separate from feed. When the feed additive is administered separately from animal feed, it can be prepared as an immediate release or sustained release formulation by combining it with a pharmaceutically acceptable edible carrier, as is well known in the art. Such edible carriers can be solid or liquid, for example corn starch, lactose, sucrose, soybean flakes, peanut oil, olive oil, sesame oil and propylene glycol. When a solid carrier is used, the feed additive may be a tablet, capsule, powder, troche or sugar-containing tablet or top dressing in a microdispersible form. When a liquid carrier is used, the feed additive may be a gelatin soft capsule, or a syrup, suspension, emulsion, or solution formulation.
또한, 상기 사료첨가제 및 사료는 보조제, 예를 들어 보존제, 안정화제, 습윤제 또는 유화제, 용액촉진제 등을 함유할 수 있다. 상기 사료첨가제는 침주, 분무 또는 혼합하여 동물의 사료에 첨가하여 이용될 수 있다.In addition, the feed additives and feeds may contain auxiliary agents, for example, preservatives, stabilizers, wetting agents or emulsifying agents, solution accelerators, and the like. The feed additive may be used by adding it to the animal's feed by soaking, spraying or mixing.
본 발명의 사료 또는 사료첨가제는 포유류, 가금 및 어류를 포함하는 다수의 동물식이에 적용할 수 있다.The feed or feed additive of the present invention can be applied to a number of animal diets including mammals, poultry and fish.
본 발명에 있어서 동물을 포유류를 포함하고 포유류로서 돼지, 소, 양, 염소, 실험용 설치동물, 및 실험용 설치동물뿐만 아니라, 애완동물(예: 개, 고양이) 등에게 사용할 수 있으나, 이에 한정되는 것은 아니다.In the present invention, animals include mammals, and mammals may be used for pigs, cows, sheep, goats, laboratory rodents, and laboratory rodents, as well as pets (eg, dogs and cats), but are not limited thereto. not.
본 발명에 따른 조성물에 포함되는 락티플란티바실러스 파라플란타룸 WiKim0130 균주의 양은 1회를 기준으로 약 106 내지 1012 cfu/g일 수 있으며, 예컨대 107 내지 1011 cfu/g, 108 내지 1010 cfu/g일 수 있다. 균주를 투여할 경우에는 생균 상태로 투여하는 것이 바람직하며, 섭취 전에 사멸시키거나 감쇄(attenuation) 상태로 투여할 수 있다. 또한, 배양 상등액 등을 사용하여 제조할 경우에는 열처리 과정을 통한 멸균화 과정을 추가적으로 거칠 수 있다. 최소의 효능을 가지는데 필요한 균주량 및 일일 섭취 정도는 섭취자의 신체 또는 건강상태에 따라 달라질 수 있으나, 일반적으로 약 106 내지 1012 cfu/g일 수 있으며, 예컨대 107 내지 1011 cfu/g, 108 내지 1010 cfu/g일 수 있다.The amount of the Lacti Plantibacillus paraplantarum WiKim0130 strain included in the composition according to the present invention may be about 10 6 to 10 12 cfu / g, for example, 10 7 to 10 11 cfu / g, 10 8 to 10 10 cfu/g. When administering a strain, it is preferable to administer it in a viable state, and it can be killed before ingestion or administered in an attenuation state. In addition, when manufacturing using a culture supernatant, etc., a sterilization process through a heat treatment process may be additionally performed. The amount of strain and daily intake required to have the minimum efficacy may vary depending on the body or health condition of the consumer, but may generally be about 10 6 to 10 12 cfu/g, such as 10 7 to 10 11 cfu/g. , 10 8 to 10 10 cfu/g.
본 발명의 이점 및 특징, 그리고 그것들을 달성하는 방법은 상세하게 후술되어있는 실시예들을 참조하면 명확해질 것이다. 그러나 본 발명은 이하에서 개시되는 실시예들에 한정되는 것이 아니라 서로 다른 다양한 형태로 구현될 것이며, 단지 본 실시예들은 본 발명의 개시가 완전하도록 하고, 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자에게 발명의 범주를 완전하게 알려주기 위해 제공되는 것이며, 본 발명은 청구항의 범주에 의해 정의될 뿐이다.The advantages and features of the present invention, and how to achieve them, will become clear with reference to the detailed description of the following embodiments. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in various different forms, and only the present embodiments will complete the disclosure of the present invention and allow common knowledge in the art to which the present invention belongs. It is provided to fully inform the holder of the scope of the invention, and the present invention is only defined by the scope of the claims.
본 발명에 따른 락티플란티바실러스 파라플란타룸 WiKim0130은 김치로부터 분리된 유산균으로서, 우수한 항암 효과를 나타내므로, 암 질환의 예방, 개선, 및 치료 용도를 위해 다양하게 활용될 수 있다.Lactiplantibacillus paraplantarum WiKim0130 according to the present invention is a lactic acid bacterium isolated from kimchi and exhibits excellent anticancer effects, so it can be used in various ways for the prevention, improvement, and treatment of cancer diseases.
도 1은 본 발명에 따른 WiKim0130 균주 처리에 따른 항종양 활성을 위한 염증성 사이토카인 분석 그래프이다((a) IL-12p70, (b) TNF, (c) IL-6, (d) MCP-1).1 is a graph of inflammatory cytokine analysis for antitumor activity according to the treatment of the WiKim0130 strain according to the present invention ((a) IL-12p70, (b) TNF, (c) IL-6, (d) MCP-1) .
도 2는 본 발명에 따른 WiKim0130 균주 처리에 따른 In vitro 항암 활성 효능 분석 그래프이다((a) CT26, MC38, HT29, 및 SW620, (b) H1650 및 A549, (c) PANC1, (d) A375, (e) HepG2, (f) CCD986Sk).Figure 2 is a graph of in vitro anticancer activity efficacy analysis according to the treatment of the WiKim0130 strain according to the present invention ((a) CT26, MC38, HT29, and SW620, (b) H1650 and A549, (c) PANC1, (d) A375, (e) HepG2, (f) CCD986Sk).
도 3은 본 발명에 따른 WiKim0130 균주 처리에 따른 CT26 생쥐 대장암의 종양 크기 변화 사진(a) 및 종양 부피 변화 그래프 (b)이다.3 is a photograph (a) of tumor size change and a tumor volume change graph (b) of CT26 mouse colon cancer according to treatment with WiKim0130 strain according to the present invention.
이하, 본 발명을 실시예를 통해 상세히 설명한다. 하기 실시예는 본 발명을 예시하는 것일 뿐 본 발명의 범위가 하기 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail through examples. The following examples are merely illustrative of the present invention, but the scope of the present invention is not limited to the following examples.
실시예Example 1: One: 락티플란티바실러스Lactiplantibacillus 파라플란타룸Paraplantarum WiKim0130의by WiKim0130 분리, 배양 및 특성 확인 Isolation, cultivation and characterization
본 발명자는 적숙기 깍두기 김치로부터 김치 유산균을 분리하였다. 김치 시료를 파쇄하고 김치 추출물의 원액을 MRS agar 배지(Difco)에 도말 후 30℃에서 24시간 이상 배양하였다. 배양 후 형성된 단일 콜로니를 계대배양하여 균단일 집락을 선별하였고, 선별된 균단일 집락은 16S RNA 염기서열 분석을 통하여 최종적으로 동정하였다.The present inventors isolated kimchi lactic acid bacteria from kimchi with radish kimchi at the ripening stage. After crushing the kimchi sample and spreading the stock solution of the kimchi extract on MRS agar medium (Difco), it was incubated at 30 ° C for more than 24 hours. Single colonies formed after culturing were subcultured to select uniform colonies, and the selected uniform colonies were finally identified through 16S RNA sequencing.
MRS agar 배지(Difco)에 배양된 락티플란티바실러스 파라플란타룸(Lactiplantibacillus paraplantarum)의 단일 콜로니를 MRS 액체배지 20 ml에 접종 후 37℃에서 24시간 동안 정치 배양하였다. 배양 후, 균체를 8,000 rpm에서 10분 동안 원심분리하여 배양액을 제거하고, PBS(Phosphate Buffered Saline)용액으로 3회 세척하여 남아 있는 배지 성분을 제거 하였다.A single colony of Lactiplantibacillus paraplantarum cultured on MRS agar medium (Difco) was inoculated into 20 ml of MRS liquid medium and then cultured at 37° C. for 24 hours. After culturing, the cells were centrifuged at 8,000 rpm for 10 minutes to remove the culture medium, and washed three times with PBS (Phosphate Buffered Saline) solution to remove remaining media components.
본 발명의 실시예를 통해 분리된 균주는 미생물의 동정을 위한 16S rDNA 염기서열 분석 결과, SEQ ID NO: 1의 핵산서열을 갖는 것으로 나타났다. As a result of 16S rDNA sequencing analysis for identification of microorganisms, the strain isolated through the examples of the present invention was found to have a nucleic acid sequence of SEQ ID NO: 1.
이에, 본 발명의 미생물을 락티플란티바실러스 파라플란타룸 WiKim0130 (Lactiplantibacillus paraplantarum WiKim0130)으로 명명하였으며, 한국미생물보존센터에 2021년 04월 22일자로 기탁하였다(수탁번호 KCCM12978P).Accordingly, the microorganism of the present invention was Lactiplantibacillus paraplantarum WiKim0130 ( Lactiplantibacillus paraplantarum WiKim0130), and deposited with the Korea Microbial Conservation Center on April 22, 2021 (accession number KCCM12978P).
다음으로, 락티플란티바실러스 파라플란타룸 WiKim0130 (Lactiplantibacillus paraplantarum WiKim0130)의 균학적 특성을 확인하였으며, 결과는 아래와 같다.Next, Lactiplantibacillus paraplantarum WiKim0130 ( Lactiplantibacillus paraplantarum The mycological characteristics of WiKim0130) were confirmed, and the results are as follows.
1) 균의 특성1) Characteristics of bacteria
① 세포의 형태: 간균① Cell type: Bacillus
② 그람(Gram) 염색: 양성② Gram staining: positive
③ 생육온도: 생장가능 생육온도 15~40℃③ Growth temperature: Growth possible growth temperature 15 ~ 40 ℃
최적 생장온도: 37℃Optimum growth temperature: 37℃
④ 생육 pH 생장가능 생육 pH 4.0~ 7.5④ Growth pH Growth possible Growth pH 4.0~ 7.5
최적 pH 6.0 ~ 6.5Optimal pH 6.0 ~ 6.5
⑤ 산소에 대한 영향: 통성혐기성⑤ Effect on oxygen: facultative anaerobic
2) 균락의 형태2) Type of colony
① 형상: 원형① Shape: Round
② 융기: 볼록② Uplift: Convex
③ 표면: 매끄러움(Smooth)③ Surface: Smooth
3) 카탈라제: -3) Catalase: -
4) 가스형성여부: -4) Gas formation: -
5) 15℃에서 생육: +5) Growth at 15°C: +
6) 45℃에서 생육: -6) Growth at 45°C: -
7) 인돌생산: -7) Indole production: -
8) 젖산생산: +8) Lactic acid production: +
9) Biomerieux 사의 유산균 동정용 API 50 CH kit를 이용한 실험9) Experiment using API 50 CH kit for identifying lactic acid bacteria from Biomerieux
반응 기질reactive substrate 결과result 반응기질 reaction substrate 결과result
1 글리세롤1 glycerol -- 26 살리신26 Salicin ++
2 에리스리톨2 erythritol -　- 27 셀로비오스27 Cellobiose ++
3 D 아라비노스3 D Arabinose -　- 28 말토스28 Maltose ++
4 L 아라비노스4 L arabinose -- 29 유당29 Lactose -　-
5 리보스 D-리보스5 Ribose D-Ribose -　- 30 멜리비오스30 Melibios -　-
6 D 크실로스 D-크실로스6 D-Xylose D-Xylose -　- 31 자당31 sucrose ++
7 L 크실로스 L-크실로스7 L Xylose L-Xylose -　- 32 트레할로스32 Trehalose ++
8 아도니톨 D-아도니톨8 Adonitol D-Adonitol -　- 33 이눌린33 Inulin -　-
9 메틸-BD-크실로푸라노시클9 methyl-BD-xylofuranocycle -　- 34 멜레지토스34 Melezitus ++
10 D-갈락토스10 D-galactose -　- 35 라피노스35 Raffinose -　-
11 D-포도당11 D-glucose ++ 36 아미돈36 Amidon -　-
12 D-과당12 D-fructose +　+ 37 글리코겐37 Glycogen -　-
13 D-만노스13 D-mannose ++ 38 크실리톨38 xylitol -　-
14 L-소르보스14 L-Sorbose -　- 39 겐티오비오스39 Gentiobios ++
15 FKASHTM15FKASHTM -　- 40 D 투라노스40 D Turanos --
16 둘시톨16 dulcitol -　- 41 D 라이소스41 D Lysos -　-
17 이노시톨17 Inositol -　- 42 D 타가토스42 D Tagatose -　-
18 만니톨18 mannitol ++ 43 D 푸코스43 D Fucos -　-
19 소르비톨19 Sorbitol -　- 44 L 푸코스44 L Fucose -　-
20 a-메틸-D-만노시드20 a-methyl-D-mannoside -　- 45 D 아라비톨45 D Arabitol -　-
21 a-메틸-D-클루코시드21 a-methyl-D-glucoside -　- 46 L 아라비톨46 L Arabitol -　-
22 N-아세틸-글루코사민22 N-acetyl-glucosamine ++ 47 글루코나테47 Gluconate --
23 아미그달린23 Amygdalin ++ 48 2-케토-글루코나테48 2-keto-gluconate --
24 아르부틴24 Arbutin ++ 49 5-케토-글루코나테49 5-keto-gluconate -　-
25 에스큘린25 Esculin ++ 　 　
실시예Example 2: 2: 락티플란티바실러스Lactiplantibacillus 파라플란타룸Paraplantarum WiKim0130의by WiKim0130 염증성 사이토카인 실험 결과 Inflammatory cytokine test results
MC38 암세포가 이식된 마우스의 골수 세포로부터 분화시킨 수지상 세포에 락티플란티바실러스 파라플란타룸 WiKim0130을 처리하여 항암 활성 면역 사이토카인 발현을 확인하였다. 마우스 정강이뼈 및 대퇴골을 RPMI 배지와 주사기 바늘을 이용하여 골수 세포를 수득하였다. 분리된 골수세포는 그래뉼로사이트-마크로파지 콜로니-자극인자(GM-CSF), 10%의 열-비활성화된 우혈청, 50 μM β머캅토에탄올, 2 mM 글루타민을 함유하는 RPMI 1640 배지에서 배양하며 수지상세포로 분화시켰다. 2 Х 105 cells/ml의 밀도로 96-웰 플레이트에 배양한 골수-유래 수지상세포에 MOI 3 의 농도로 락티플란티바실러스 파라플란타룸 WiKim0130을 24시간 동안 처리하고 상층액을 회수하여 사이토카인 분석을 실시하였으며, 염증 반응을 일으키며 항암 활성 사이토카인의 발현을 증가시키는 LPS (100 ng/ml)도 함께 비교하였고, 도 1에 나타내었다.Dendritic cells differentiated from bone marrow cells of mice transplanted with MC38 cancer cells were treated with Lacti Plantibacillus paraplantarum WiKim0130 to confirm the expression of anti-cancer active immune cytokines. Bone marrow cells were obtained from mouse shinbones and femurs using RPMI medium and a syringe needle. The isolated bone marrow cells were cultured in RPMI 1640 medium containing granulocyte-macrophage colony-stimulating factor (GM-CSF), 10% heat-inactivated bovine serum, 50 μM β-mercaptoethanol, and 2 mM glutamine, and dendritic cells differentiated into cells. Bone marrow-derived dendritic cells cultured in a 96-well plate at a density of 2 Х 10 5 cells/ml were treated with Lactiplantibacillus paraplantarum WiKim0130 at a concentration of MOI 3 for 24 hours, and the supernatant was recovered to detect cytokines. The analysis was performed, and LPS (100 ng/ml), which causes an inflammatory response and increases the expression of anticancer activity cytokines, was also compared, and shown in FIG. 1.
본 발명의 락티플란티바실러스 파라플란타룸WiKim0130을 골수유래 수지상세포에 처리하였을때 항암 면역반응을 활성화시키는 IL-12p70 발현이 8.3배 증가되고, 항암작용을 나타내는 TNF-a도 67.8배 증가, 항암 활성을 방해하는 TGF-β를 억제시키는 항암 면역반응에서 역할이 중요한 IL-6도 1695.1배 증가되었으며, 종양내로 면역세포의 침투를 유도하는 MCP-1도 10.2배 증가하였다.When bone marrow-derived dendritic cells were treated with Lacti Plantibacillus paraplantarum WiKim0130 of the present invention, the expression of IL-12p70, which activates the anti-cancer immune response, increased 8.3 times, and TNF-a, which shows anti-cancer activity, increased 67.8 times, anti-cancer IL-6, which plays an important role in the anti-cancer immune response that inhibits TGF-β, which interferes with its activity, was also increased by 1695.1 times, and MCP-1, which induces the infiltration of immune cells into tumors, was also increased by 10.2 times.
실시예Example 3: 3: 락티플란티바실러스Lactiplantibacillus 파라플란타룸Paraplantarum WiKim0130의by WiKim0130 암세포 cancer cell 사멸능ability to kill 확인 결과 Verification result
락티플란티바실러스 파라플란타룸 WiKim0130 (Lactiplantibacillus paraplantarum WiKim0130)의 항암 활성 효과를 관찰하기 위해 LDH assay kit를 이용하여 세포가 사멸될 때 방출되는 LDH leakage(%)를 측정하여 세포 사멸능(cell cytotoxicity)을 측정하였다. 항암 활성 효능실험에 사용된 세포는 CT26(생쥐 대장암)과 MC38(생쥐 대장암), HT29(사람 대장암), SW620(사람 대장암), H1650(사람 폐암), A549(사람 폐암), PANC1(사람 췌장암), A375(사람 피부암), HepG2(사람 간암) 세포 그리고 정상 피부세포주인 CCD-986-sk를 이용하였다.To observe the anti-cancer activity effect of Lactiplantibacillus paraplantarum WiKim0130 (cell cytotoxicity) by measuring LDH leakage (%) released when cells die using an LDH assay kit was measured. The cells used in the anticancer activity efficacy test were CT26 (mouse colon cancer), MC38 (mouse colon cancer), HT29 (human colon cancer), SW620 (human colon cancer), H1650 (human lung cancer), A549 (human lung cancer), PANC1 (human pancreatic cancer), A375 (human skin cancer), HepG2 (human liver cancer) cells, and normal skin cell line CCD-986-sk were used.
- 마우스 유래 암세포 CT26과 MC38은 10% fetal bovine serum (FBS)과 1% penicillin/streptomycin을 첨가한 DMEM 배지에 37℃, 5% CO2 배양기에서 계대배양하며 성장기의 세포를 사용하여 실험하였다. 배양된 CT26, MC38 세포는 0.05% trypsin-EDTA로 37℃ 배양기에서 3분간 처리하여 세포를 탈착한 다음 DPBS로 2회 세척 후 1 X 104 cells/well로 준비하였다.- Mouse-derived cancer cells CT26 and MC38 were subcultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in a 37°C, 5% CO 2 incubator, and cells in the growth phase were used for experiments. The cultured CT26 and MC38 cells were treated with 0.05% trypsin-EDTA in a 37°C incubator for 3 minutes to detach the cells, and then washed twice with DPBS to prepare 1 X 10 4 cells/well.
- 사람 유래 암세포 HT29, SW620, H1650, A549, PANC1, A375, HepG2는 10% fetal bovine serum (FBS)과 1% penicillin/streptomycin을 첨가한 RPMI 배지에 37℃, 5% CO2 배양기에서 계대배양하여 성장기의 세포를 사용하여 실험하였다. 배양된 HT29, SW620, H1650, A549, PANC1, A375, HepG2세포는 0.05% trypsin-EDTA로 37℃배양기에서 3분간 처리하여 세포를 탈착한 다음 DPBS로 2회 세척 후 1 X 104 cells/well로 준비하였다.- Human-derived cancer cells HT29, SW620, H1650, A549, PANC1, A375, HepG2 were subcultured in RPMI medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37°C in a 5% CO 2 incubator. Experiments were performed using growing cells. The cultured HT29, SW620, H1650, A549, PANC1, A375, and HepG2 cells were treated with 0.05% trypsin-EDTA for 3 minutes in a 37°C incubator to detach the cells, wash twice with DPBS, and then 1 X 10 4 cells/well. prepared.
- 사람 유래 정상피부세포 CCD-986sk는 10% fetal bovine serum (FBS)과 1% penicillin/streptomycin을 첨가한 RPMI 배지에 37℃, 5% CO2 배양기에서 계대배양하면서 성장기의 세포를 사용하여 실험하였다. 배양된 CCD-986sk는 0.05% trypsin-EDTA로 37℃ 배양기에서 3분간 처리하여 세포를 탈착한 다음 DPBS로 2회 세척 후 1 X 104 cells/well로 준비하였다.- Human-derived normal skin cell CCD-986sk was subcultured in RPMI medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in a 37°C, 5% CO 2 incubator, and the cells in the growing phase were used for experiments. . The cultured CCD-986sk was treated with 0.05% trypsin-EDTA in a 37°C incubator for 3 minutes to detach the cells, and then washed twice with DPBS to prepare 1 X 10 4 cells/well.
- 암세포 사멸능(cell viability assay)는 MOI3 농도로 처리한 다음 72시간 동안 배양하였고, 배양 후 LDH assay 시약을 처리한 후 흡광도(OD490 nm)를 측정하였다. 락티플란티바실러스 파라플란타룸 WiKim0130 를 처리하지 않은 세포의 흡광도를 100% 기준으로, 락티플란티바실러스 파라플란타룸 WiKim0130을 처리 한 세포의 LDH leakage (%)를 계산하였다.- Cancer cell viability assay was treated with MOI3 concentration and cultured for 72 hours. After culture, absorbance (OD490 nm) was measured after treatment with LDH assay reagent. LDH leakage (%) of cells treated with Lacti Planti Bacillus paraplantarum WiKim0130 was calculated based on 100% absorbance of cells not treated with Lacty Plantibacillus paraplantarum WiKim0130.
- 그 결과를 도 2의 그래프에 나타내었다. 대장암(3종), 폐암(2종), 췌장암(1종) 세포가 배양된 후 락티플란티바실러스 파라플란타룸 WiKim0130을 처리한 세포의 세포 사멸능을 관찰하였으며, 락티플란티바실러스 파라플란타룸 WiKim0130 (Lactiplantibacillus paraplantarum WiKim0130) 를 처리하지 않은 각각의 암세포를 100% 기준으로, 락티플란티바실러스 파라플란타룸 WiKim0130 (Lactiplantibacillus paraplantarum WiKim0130)를 처리한 CT26(생쥐 대장암세포)은 142.6%, MC38(생쥐 대장암세포)은 303.9%, HT29(사람 대장암세포)은 165.4%, SW620(사람 대장암세포)은 193.4%, H1650(사람 비소세포폐암세포)은 156.9%, A549(사람 비소세포폐암세포)은 124.8%, PANC1(사람 췌장암세포)은 134.5%, A375(사람 피부암세포)는 128.2%, HepG2(사람 간암세포)는 133.7%로 LDH leakage 관찰 따른 암세포 사멸능을 확인하였으며, 항암활성효과 판정은 정상세포주인 CCD-986-sk(사람 피부세포)의 세포독성은 95.1%로 대조군 세포와 비슷한 값으로 락티플란티바실러스 파라플란타룸 WiKim0130을 처리하지 않은 세포보다 약 5% 더 잘 생존하는 것으로 관찰되어 정상피부세포의 세포사멸은 관찰되지 않았다.- The results are shown in the graph of FIG. 2. After colon cancer (type 3), lung cancer (type 2), and pancreatic cancer (type 1) cells were cultured, the apoptotic ability of cells treated with Lactiplantibacillus paraplantarum WiKim0130 was observed. Based on 100% of each cancer cell not treated with Lantarum WiKim0130 ( Lactiplantibacillus paraplantarum WiKim0130), CT26 (mouse colon cancer cells) treated with Lactiplantibacillus paraplantarum WiKim0130 were 142.6%, MC38 ( mouse colon cancer cells) was 303.9%, HT29 (human colon cancer cells) was 165.4%, SW620 (human colon cancer cells) was 193.4%, H1650 (human non-small cell lung cancer cells) was 156.9%, A549 (human non-small cell lung cancer cells) was 124.8 %, PANC1 (human pancreatic cancer cells) was 134.5%, A375 (human skin cancer cells) was 128.2%, and HepG2 (human liver cancer cells) was 133.7%. The cytotoxicity of human CCD-986-sk (human skin cells) was 95.1%, similar to that of control cells, and it was observed that they survived about 5% better than cells not treated with Lacti Plantibacillus paraplantarum WiKim0130. Apoptosis of skin cells was not observed.
실시예Example 4: 4: 락티플란티바실러스Lactiplantibacillus 파라플란타룸Paraplantarum WiKim0130의by WiKim0130 항종양 효과 확인 Confirmation of antitumor effect
실험에 사용된 실험동물은 수컷 5주령의 BALB/c 마우스 (오리엔트 바이오, 한국)를 공급받았고, 실내 온도 20±2℃, 습도 55±15%가 유지되는 SPF 환경의 동물 사육실에서 1주일의 안정화 기간을 거쳐 실험기간 동안 사육하였다. 사료는 항생제가 첨가되지 않은 일반적인 팰렛 사료를 공급하였고, 물은 수시로 섭취할 수 있게 하였다. 실험 진행은 세계김치연구소 동물실험윤리위원회에서 승인된 프로토콜에 따라 모든 동물 사육, 실험 및 안락사를 실시하였다. 종양 크기 변화의 관찰은3.14 x (길이 x 높이 x 넓이)/6의 식을 사용하여 종양의 부피(mm3)를 측정하여 진행하였다.Experimental animals used in the experiment were supplied with male 5-week-old BALB/c mice (Orient Bio, Korea), and stabilized for 1 week in an animal breeding room in an SPF environment maintained at a room temperature of 20 ± 2 ° C and a humidity of 55 ± 15%. After a period, they were reared during the experimental period. Feed was supplied with common pellet feed without antibiotics, and water was allowed to be consumed at any time. All animals were reared, tested, and euthanized according to the protocol approved by the Animal Experiment Ethics Committee of the World Institute of Kimchi. Observation of tumor size change was performed by measuring the volume (mm 3 ) of the tumor using the formula of 3.14 x (length x height x width)/6.
1)One) CT26 항종양 효과 확인Confirmation of CT26 antitumor effect
- CT26 생쥐 대장암 세포(ATCC, 미국)는 구입하여 사용하였다. CT26 생쥐 대장암 세포는 10% 우태아혈청과 1% 페니실린-스트렙토마이신(penicillin-streptomycin)을 포함한 DMEM 배지(Hyclone, 미국)를 사용하여 5% CO2, 37℃ 조건에서 배양하였다.- CT26 mouse colon cancer cells (ATCC, USA) were purchased and used. CT26 mouse colon cancer cells were cultured at 5% CO 2 and 37°C using DMEM medium (Hyclone, USA) containing 10% fetal bovine serum and 1% penicillin-streptomycin.
- CT26세포이식 동물모델 제작을 위해서 6주령 BALB/c 마우스(18-21 g)를 실험에 이용하였다. 배양한 생쥐 대장암 세포 CT26는 1x105 를 수확 (harvest)한 후, 50 μl의 PBS에서 재부유한 후 마우스의 오른쪽 대퇴부(thigh) 피하에 주입하였다. 약 11~13일 후 종양이 형성되어 부피가 약 70~80 mm3 되면 효능 분석을 위한 실험을 수행하였다.- 6-week-old BALB/c mice (18-21 g) were used for the experiment to create an animal model for CT26 cell transplantation. The cultured mouse colon cancer cells CT26 were harvested at 1x10 5 , resuspended in 50 μl of PBS, and injected subcutaneously into the right thigh of the mouse. After about 11 to 13 days, when tumors were formed and the volume reached about 70 to 80 mm 3 , an experiment for efficacy analysis was performed.
- 락티플란티바실러스 파라플란타룸 WiKim0130 (Lactiplantibacillus paraplantarum WiKim0130) 균주의 항종양 효과를 확인하기 위하여 CT26 암세포 이식 생쥐 종양모델을 제작하여 항종양 효능을 확인하여 도 3에 나타내었다. 종양의 부피가 약 80~100 mm3 된 후, 종양이 형성된 마우스에 락티플란티바실러스 파라플란타룸 WiKim0130 를 꼬리정맥 주사하였다. 락티플란티바실러스 파라플란타룸 WiKim0130은 PBS를 이용하여 1Х1010 CFU/ml 균수로 정량하여 준비하였으며, 0.1 ml (1Х109 CFU)씩 실험동물에 1회 꼬리정맥주사하고, 음성대조군에는 PBS를 투여하였다. 시간 경과에 따라 CT26 생쥐 대장암의 종양 크기의 변화를 육안으로 확인하였고, 암모델 마우스에서 종양크기의 부피를 측정하였다. 그 결과, 시간 경과에 따른 종양의 크기의 변화를 10~13일 동안 육안으로 확인하였고, 암모델 마우스에서 종양조직의 부피도 측정하였다. 그 결과, 락티플란티바실러스 파라플란타룸 WiKim0130 유산균이 주사된 CT26(생쥐 대장암) 세포 이식 마우스 그룹에서는 종양이 대조군 대비 99.6% 감소되는 치료효과가 관찰되었다.- In order to confirm the anti-tumor effect of the Lactiplantibacillus paraplantarum WiKim0130 strain, a CT26 cancer cell-transplanted mouse tumor model was prepared and the anti-tumor efficacy was confirmed and shown in FIG. 3 . After the volume of the tumor reached about 80-100 mm 3 , Lacti Plantibacillus paraplantarum WiKim0130 was injected into the tumor-formed mouse through the tail vein. Lactiplantibacillus paraplantarum WiKim0130 was prepared by quantifying the number of bacteria at 1Х10 10 CFU/ml using PBS, and 0.1 ml (1Х10 9 CFU) was injected into the tail vein of the experimental animals once, and PBS was administered to the negative control group. did Changes in the tumor size of CT26 mouse colon cancer over time were visually confirmed, and the volume of the tumor size was measured in the cancer model mouse. As a result, the change in the size of the tumor over time was visually confirmed for 10 to 13 days, and the volume of the tumor tissue in the cancer model mouse was also measured. As a result, in the CT26 (mouse colon cancer) cell-transplanted mouse group injected with Lactiplantibacillus paraplantarum WiKim0130 lactic acid bacteria, a 99.6% reduction in tumors compared to the control group was observed.
본 발명의 유산균 락티플란티바실러스 파라플란타룸 WiKim0130 김치 유산균을 꼬리 정맥 주사 후 종양이 감소되는 효능이 관찰되었으며, 대조군(PBS)의 종양에 비해 평균 99.6% 감소됨으로서 그 종양 부피의 증가율이 유의적(P<0.001) 으로 현저하게 감소하는 것이 확인되었다.After tail vein injection of the Lactobacillus paraplantarum WiKim0130 Kimchi Lactobacillus of the present invention, the effect of reducing tumors was observed, and the tumor volume was reduced by an average of 99.6% compared to the tumor of the control group (PBS), and the increase rate of the tumor volume was significant. (P<0.001) It was confirmed that it decreased significantly.
[수탁번호][Accession number]
기탁기관명 : 한국미생물보존센터(국외)Name of depository organization: Korea Center for Microbial Conservation (overseas)
수탁번호 : KCCM12978PAccession number: KCCM12978P
수탁일자 : 20210422Entrusted date: 20210422
Figure PCTKR2022010228-appb-img-000001
Figure PCTKR2022010228-appb-img-000001

Claims (9)

  1. 수탁번호 KCCM12978P의 락티플란티바실러스 파라플란타룸 WiKim0130(Lactiplantibacillus paraplantarum WiKim0130).Accession number KCCM12978P's Lactiplantibacillus paraplantarum WiKim0130 ( Lactiplantibacillus paraplantarum WiKim0130).
  2. 락티플란티바실러스 파라플란타룸 WiKim0130(Lactiplantibacillus paraplantarum WiKim0130; 수탁번호 KCCM12978P), 이의 배양물, 이의 파쇄물 또는 이의 추출물을 유효성분으로 포함하는 암의 예방 또는 개선용 식품 조성물.Lactiplantibacillus paraplantarum WiKim0130 ( Lactiplantibacillus paraplantarum WiKim0130; Accession No. KCCM12978P), a culture thereof, a lysate thereof, or a food composition for preventing or improving cancer comprising a lysate thereof or an extract thereof as an active ingredient.
  3. 제2항에 있어서,According to claim 2,
    상기 암은 뇌종양, 양성성상세포종, 악성성상세포종, 뇌하수체 선종, 뇌수막종, 뇌림프종, 핍지교종, 두개내인종, 상의세포종, 뇌간종양, 두경부 종양, 후두암, 구인두암, 비강암, 부비동암, 비인두암, 침샘암, 하인두암, 갑상선암, 흉부종양, 소세포성 폐암, 비소세포성 폐암, 흉선암, 종격동 종양, 식도암, 유방암, 남성유방암, 복부종양, 위암, 간암, 담낭암, 담도암, 췌장암, 소장암, 대장암, 항문암, 방광암, 신장암, 남성생식기종양, 음경암, 요도암, 전립선암, 여성생식기종양, 자궁경부암, 자궁내막암, 난소암, 자궁육종, 질암, 여성 외부생식기암, 여성요도암, 피부암, 골수종, 백혈병 및 악성림프종을 포함하는 것인 식품 조성물.The cancer is brain tumor, benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, cerebral lymphoma, oligodendroma, intracranial race, ependymoma, brainstem tumor, head and neck tumor, laryngeal cancer, oropharyngeal cancer, nasal cancer, sinus cancer, nasopharyngeal cancer, Salivary gland cancer, hypopharyngeal cancer, thyroid cancer, chest tumor, small cell lung cancer, non-small cell lung cancer, thymus cancer, mediastinal tumor, esophageal cancer, breast cancer, male breast cancer, abdominal tumor, stomach cancer, liver cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, small intestine cancer, colon Cancer, anal cancer, bladder cancer, kidney cancer, male genital tumor, penile cancer, urethral cancer, prostate cancer, female genital tumor, cervical cancer, endometrial cancer, ovarian cancer, uterine sarcoma, vaginal cancer, female external genital cancer, female urethral cancer , A food composition comprising skin cancer, myeloma, leukemia and malignant lymphoma.
  4. 제2항에 있어서,According to claim 2,
    상기 식품은 건강기능식품인 식품 조성물.The food is a health functional food food composition.
  5. 제2항에 있어서,According to claim 2,
    상기 식품은 음료, 바 또는 발효유인 식품 조성물.The food composition is a beverage, bar or fermented milk.
  6. 락티플란티바실러스 파라플란타룸 WiKim0130(Lactiplantibacillus paraplantarum WiKim0130; 수탁번호 KCCM12978P) 또는 이의 배양물로 이루어지는 것인 발효용 유산균 스타터.A lactic acid bacteria starter for fermentation comprising Lactiplantibacillus paraplantarum WiKim0130 (Accession No. KCCM12978P) or a culture thereof.
  7. 락티플란티바실러스 파라플란타룸 WiKim0130(Lactiplantibacillus paraplantarum WiKim0130; 수탁번호 KCCM12978P), 이의 배양물, 이의 파쇄물 또는 이의 추출물을 유효성분으로 포함하는 사료 또는 사료 첨가제 조성물.A feed or feed additive composition comprising Lactiplantibacillus paraplantarum WiKim0130 ( Lactiplantibacillus paraplantarum WiKim0130; accession number KCCM12978P), a culture thereof, a lysate thereof, or an extract thereof as an active ingredient.
  8. 락티플란티바실러스 파라플란타룸 WiKim0130(Lactiplantibacillus paraplantarum WiKim0130; 수탁번호 KCCM12978P), 이의 배양물, 이의 파쇄물 또는 이의 추출물을 유효성분으로 포함하는 암 질환의 예방 또는 치료용 약학 조성물.Lactiplantibacillus paraplantarum WiKim0130 ( Lactiplantibacillus paraplantarum WiKim0130; accession number KCCM12978P), a pharmaceutical composition for preventing or treating cancer disease comprising a culture thereof, a lysate thereof, or an extract thereof as an active ingredient.
  9. 제8항에 있어서,According to claim 8,
    상기 암 질환은 뇌종양, 양성성상세포종, 악성성상세포종, 뇌하수체 선종, 뇌수막종, 뇌림프종, 핍지교종, 두개내인종, 상의세포종, 뇌간종양, 두경부 종양, 후두암, 구인두암, 비강암, 부비동암, 비인두암, 침샘암, 하인두암, 갑상선암, 흉부종양, 소세포성 폐암, 비소세포성 폐암, 흉선암, 종격동 종양, 식도암, 유방암, 남성유방암, 복부종양, 위암, 간암, 담낭암, 담도암, 췌장암, 소장암, 대장암, 항문암, 방광암, 신장암, 남성생식기종양, 음경암, 요도암, 전립선암, 여성생식기종양, 자궁경부암, 자궁내막암, 난소암, 자궁육종, 질암, 여성 외부생식기암, 여성요도암, 피부암, 골수종, 백혈병 및 악성림프종을 포함하는 것인 약학 조성물.The cancer diseases include brain tumor, benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, brain lymphoma, oligodendroglioma, intracranial race, ependymoma, brain stem tumor, head and neck tumor, laryngeal cancer, oropharyngeal cancer, nasal cavity cancer, sinus cancer, nasopharyngeal cancer , salivary gland cancer, hypopharyngeal cancer, thyroid cancer, chest tumor, small cell lung cancer, non-small cell lung cancer, thymus cancer, mediastinal tumor, esophageal cancer, breast cancer, male breast cancer, abdominal tumor, stomach cancer, liver cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, small intestine cancer, Colon cancer, anal cancer, bladder cancer, kidney cancer, male genital tumor, penile cancer, urethral cancer, prostate cancer, female genital tumor, cervical cancer, endometrial cancer, ovarian cancer, uterine sarcoma, vaginal cancer, female external genital cancer, female urethra A pharmaceutical composition comprising cancer, skin cancer, myeloma, leukemia and malignant lymphoma.
PCT/KR2022/010228 2021-07-13 2022-07-13 Composition for prevention, reduction or treatment of cancer, comprising lactiplantibacillus paraplantarum wikim0130 WO2023287202A1 (en)

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