KR20140020685A - A novel lactococcus sp. strain and use thereof - Google Patents

Abstract

The present invention provides Lactococcus sp. KR-050L which is a novel strain and described in deposit number of KCTC 12170BP; a pharmaceutical composition for preventing or treating cancer or inflammatory diseases which includes Lactococcus sp. KR-050L culture medium, fungus body, or supernatant, extracts thereof, or fractions thereof as effective components; a composition for inhibiting activities for signal transducers and activators of transcription 3 (STAT3) which is induced by interleukin-6 (IL-6) or IL-11 which includes Lactococcus sp. KR-050L culture medium, Lactococcus sp. KR-050L fungus body, or supernatant, extracts thereof, or fractions thereof; foods, food additive, forage, or forage additive which includes the Lactococcus sp. KR-050L or the culture medium thereof; and personal hygienic goods which includes the Lactococcussp. KR-050L or culture thereof.

Description

Novel Lactococcus Species Strains and Their Uses {A novel Lactococcus sp. strain and use according}

The invention is a novel strain Lactococcus of Accession No. KCTC 12170BP sp .) KR-050L; Pharmaceutical composition for the prevention or treatment of inflammatory diseases or cancer comprising the lactococcus species KR-050L culture, cells or supernatants, extracts or fractions thereof as an active ingredient; Signal transducers STAT3 induced by IL-6 (interleukin-6) or IL-11 containing the Lactococcus species KR-050L culture solution, Lactococcus species KR-050L cells or supernatant, extracts or fractions thereof and inhibitory compositions for activators of transcription 3); Lactococcus species sp .) food, food additives, feed or feed additives comprising KR-050L or its culture; And the Lactococcus species sp .) to provide a personal hygiene product comprising KR-050L or its culture.

Interleukin-6 (IL-6) is a cytokine, also called B cell stimulatory factor 2 or interferon-β2 (INF-β2). IL-6 was found to be a differentiation factor involved in the activation of B lymphocytes. Subsequently, it was found to be a multifunctional cytokine that affects the function of various cells. IL-6 transfers its biological activity through two kinds of proteins present on the cell membrane, one is the IL-6 receptor, a protein to which IL-6 binds, and the other is gp130. IL-6 receptor is a membrane-bound protein having a molecular weight of about 80 kDa expressed through a cell membrane, and gp130 is a membrane protein having a molecular weight of about 130 kDa belonging to a non-ligand binding signal transmission. IL-6 first forms an IL-6 receptor and an IL-6 / IL-6 receptor complex and then binds to gp130. After binding of the ligand and the receptor, JAK2 (Janus kinase 2) is activated by transphosphorylation in the cell. Activated JAK2 phosphorylates several tyrosine residues in the receptor cytoplasmic domain, which causes STAT3 (signal transducers and activators of transcription with SH2 or other phosphotyrosine binding motifs). It acts as a docking site for proteins in the cytoplasm such as 3). STAT3 bound to the cytoplasmic domain of the receptor is phosphorylated by JAK2 and then isolated from the receptor. Activated STAT3 binds to each other in the cytoplasm to form a homo- or heterodimer, then enters the nucleus and binds to the recognition sequence of the target gene to increase transcription.

The IL-6-induced signaling system has been reported to be involved in inflammatory diseases and various cancer diseases. Therefore, inhibition of the IL-6-induced signaling system is useful for the prevention or treatment of these diseases. Currently, anti-IL-6 R antibodies are the most studied for the function of inhibiting the signaling system of IL-6. Inhibition of synovial cell growth for rheumatoid arthritis of the anti-IL-6 antibody has been reported, in addition to IL-6 products, such as plasmacytosis, hyperimmunoglobulinemia, anemia, nephritis, cachexia, woodpecker disease and angioplasty nephritis It has been disclosed as a therapeutic agent for diseases that contribute to. It has been described as a prophylactic or therapeutic agent for sensitive T cell related diseases such as multiple sclerosis, uveitis, chronic thyroiditis, hypersensitivity, contact dermatitis and atopic dermatitis and as a systemic lupus erythematosus treatment. In addition, the patent described as a treatment for Crohn's disease discloses an anti-IL-6 R antibody as an active ingredient. The anti-IL-6 R antibody has also been reported as an active ingredient in the treatment of pancreatitis, psoriasis. In addition, it has been disclosed as an active ingredient in the treatment of combustible idiopathic atrophy. However, these proteins can have epitopes that can be recognized as foreign proteins and can still be immunogenic when used as a therapeutic. However, small molecule compounds other than proteins are not recognized by this immune system, and much research is being conducted.

In addition, the IL-6 signaling system is associated with STAT3, an intermediate mediator, in relation to cancer diseases. It is reported to be involved in several forms of cancer, including myeloma, breast carcinoma, prostate cancer, brain tumor, head and neck carcinoma, melanoma, leukemia and lymphoma, especially chronic myeloid leukemia and multiple myeloma. Cells derived from both rat and human prostate cancer have been found to have structurally activated STAT3, which has been shown to be structurally activated in some acute leukemias and T cell lymphomas. Interestingly, STAT3 has been shown to structurally phosphorylate on serine residues in chronic lymphocytic leukemia. STAT3 has been found to be structurally active in myeloma tumor cells in both myeloid mononuclear cells and cultures from patients with multiple myeloma. These cells are resistant to Fas-mediated apoptosis and express high levels of Bcl-xL. STAT3 signaling has been shown to be essential for the survival of myeloma tumor cells by conferring resistance to apoptosis. On the other hand, recently, IL-6 is ideally secreted in a group of cancer patients induced by Ras, including pancreatic cancer, and by removing IL-6, it is possible to suppress tumor growth and angiogenesis by Ras as well as tumor size. It has been reported that can be reduced. In addition, the gp130 / JAK / STAT3 pathway caused by IL-6 has emerged as a new target in chemotherapy, as EGFR has been shown to activate STAT3 due to overexpression of IL-6 in lung adenocarcinoma. Doing.

On the other hand, interleukin-11 (IL-11) is an inflammatory cytokine belonging to the IL-6 family and has almost the same signaling system as IL-6, and its expression in hematopoietic, immune response, inflammation and various cancer cells It is known to play an important role in cancer progression. Recently, it has been reported that IL-11 binds to its receptors, IL-11Rα and gp130, to promote cell proliferation and cancer invasion in gastric and colorectal cancers, and smad7 is activated by IL-11 / STAT3 signaling and simultaneously TGFβ Blocking the smad activator that induces signals activates oncogenic programs (antiapoptotic genes, proangiogenic genes, and proliferative genes). Tumor induction has been reported. Thus, the gp130 / JAK / STAT3 pathway by IL-11 has emerged as a new target in chemotherapy.

The present inventors found a novel strain of Lactococcus sp . KR-050L ( Lactococcus sp . KR-050L) in natural resources, and the transcription and transcription of STAT3, an inflammation-related transcription factor activated by IL-6, using its extracts and fractions. It was confirmed to inhibit the activity and phosphorylation. In addition, the lactococcus sp. KR-050L extract and fractions thereof have excellent activity of inhibiting the signaling system induced by IL-6, and thus are used for the prevention or treatment of inflammatory diseases and cancers caused by IL-6. It was confirmed that the composition can be used to complete the present invention.

One object of the present invention is Lactococcus species of Accession No. KCTC 12170BP sp .) to provide KR-050L.

Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of inflammatory diseases or cancer comprising the lactococcus species KR-050L culture solution, cells or supernatant, extracts or fractions thereof as an active ingredient.

Still another object of the present invention is to provide a signal transducer of STAT3 induced by IL-6 (interleukin-6) or IL-11, which contains the L-tococcus sp. KR-050L culture, cells or supernatant, extracts or fractions thereof. and activators of transcription 3).

Another object of the present invention is the Lactococcus spp. sp .) To provide a food, food additive, feed or feed additive containing KR-050L or its culture.

Another object of the present invention is the Lactococcus spp. sp .) to provide a personal hygiene product comprising KR-050L or its culture.

As one embodiment for achieving the above object, the present invention is Lactococcus species of Accession No. KCTC 12170BP sp .) Provide KR-050L.

Lactococcus spp. Of the invention sp .) strain is the first isolate identified from fermented foods such as various kimchi and salted fish, excellent strains and flavors selected through various experiments. Single cells are of the cocci type (Fig. 1) having a size of 0.6 to 0.8 μm of short axis and 0.8 to 1.5 μm of long axis, and the flora is light cream color forming a circular colony in MRS agar medium. Analysis of the genes of the selected novel strain (SEQ ID NO: 1) to confirm that the strain has a high homology with the standard strain Lactococcus lactis ( Lactococcus species) sp .) It was named KR-050L, and it was deposited on March 21, 2012 to the Korean Collection for Type Culture (KCTC), and was assigned accession number KCTC 12170BP.

In the present invention, " Lactococcus species ( Lactococcus sp .) "is a genus Streptococcus group known as lactic acid bacteria (genus Streptococcus). Group N1 ) is a strain belonging to the lactic acid (lactic acid) bacteria. It is known as a homofermentor which produces a kind of product, for example lactic acid, as the main or sole product of glucose fermentation. These properties can be changed by adjusting the culture conditions such as pH, glucose concentration and nutritional restrictions. Gram-positive, catalase-negative, non-motile cocci, which are found alone or in pairs, are known to grow at low temperatures below 7 ° C. . To date, seven species have been identified: L. lactis , L. garvieae , L. plantarum , L. raffinolactis , L. piscium , L. chungangensis and L. fujiensis . These strains are widely used in the dairy industry for producing fermented dairy products such as cheese and yogurt. Especially widely used in industrial dairy fermentation is L. lactis . To be used for fermentation of dairy products, milk must be acidified quickly to reduce the growth of contaminating bacteria to lower the pH of the fermentation product, as well as to enhance the flavor, ie taste and aroma of the final product. In addition, in order to use it on an industrial scale, it must be one that can be easily grown and expanded at low cost.

As another aspect, the present invention provides a pharmaceutical composition for the prevention or treatment of inflammatory diseases or cancer comprising the lactococcus species KR-050L culture solution, cells or supernatants, extracts or fractions thereof as an active ingredient.

The term "culture liquid" of the present invention refers to the strain obtained by culturing the strain in a medium capable of supplying nutrients for the growth and survival of Lactococcus species KR-050L in vitro, the metabolites and extra nutrients thereof. Means the whole medium including, but also includes a culture solution from which the strain is removed after the strain culture. On the other hand, the liquid from which the cells are removed from the culture medium is also referred to as "supernatant", and the culture solution is left alone for a certain time to take only the liquid in the upper layer except the portion that has sunk in the lower layer, remove the cells through filtration, or centrifuge the culture solution It can be obtained by removing the precipitate of and taking only the liquid at the top. The "bacteria" refers to the strains of the present invention, and includes the strains themselves selected from the fermented foods or the strains isolated from the culture by culturing the strains. The cells can be obtained by centrifugation of the culture solution to take a portion that has sunk in the lower layer, or it can be obtained by allowing it to stand for a certain time and then removing the liquid at the top because it sinks to the lower layer of the culture by gravity.

Inflammatory diseases caused by IL-6 or IL-11 include osteoarthritis, rheumatoid arthritis, osteoporosis, plasmaacytosis, hyperimmunoglobulinemia, and anemia ), Nephritis, cachexia, livestock disease, vascular proliferative nephritis, multiple sclerosis, uveitis, chronic thyroiditis, delayed-type hypersensitivity, contact dermatitis, atopic dermatitis, systemic lupus erythematosus, Crohn's disease, pancreatitis, psoriasis, peptic idiopathic atrophy, diabetes mellitus and Alzheimer's disease. In addition, the cancer caused by IL-6 or IL-11 is pancreatic cancer, breast cancer, breast cancer, prostatic cancer, brain tumor, head and neck cancer, black Melanoma, myeloma, leukemia, lymphoma, hepatoma, gastric cancer, colon cancer, bone cancer, uterine cancer, ovary Cancer (ovarian cancer), rectal cancer, esophageal cancer, small intestine tumor, anal cancer, fallopian tube carcinoma, endometrial carcinoma, Cervical carcinoma, vaginal carcinoma, vulva carcinoma, Hodgkin's disease, bladder cancer, renal cancer, ureteral cancer, Renal cell carcinoma, renal pelvic carcinoma and central nervous system tumors.

The extract of the lactococcus species KR-050L culture solution, cells or supernatant of the present invention may be an extract extracted with ethyl acetate (EtOAc) or ethanol (ethanol, ethyl alcohol; EtOH). Furthermore, the fraction of the lactococcus species KR-050L culture solution, cells or supernatant of the present invention may be a fraction obtained by fractionating ethyl acetate extract with methanol.

Preferably, the culture medium obtained by incubating the lactococcus species KR-050L for several days to separate the cells and the supernatant by centrifugation, each volume of 2 to 20 times, preferably 3 to 5 times the volume of a nonpolar solvent ( For example, ethyl acetate may be used for extraction at 20 to 40 ° C., preferably at room temperature, for 12 hours to 4 days, and preferably for 3 days. The extraction method may use any known method such as cold extraction, reflux cooling extraction or ultrasonic extraction without limitation. Preferably it can be filtered under reduced pressure by extracting 1 to 5 successive times by cold extraction. Thereafter, the filtrate extract is concentrated under reduced pressure at 20 to 40 ° C., preferably at room temperature using a vacuum rotary concentrator, and fractionated with water, C1 to C6 lower alcohol, or a mixed solvent thereof to obtain an aqueous or lower alcohol soluble crude extract. .

According to a specific embodiment of the present invention, the lactococcus species KR-050L cell ethyl acetate extract or 100% methanol fraction thereof (FIG. 3), the lactococcus species KR-050L supernatant ethyl acetate extract and methanol fraction thereof, preferably It was confirmed that the ethyl acetate and ethanol extracts (FIG. 5) of 40-100% methanol fraction (FIG. 4) and lactococcus species KR-050L culture lyophilized (FIG. 5) were able to inhibit STAT3 activity in a concentration-dependent manner.

A pharmaceutical composition for preventing or treating an inflammatory disease or cancer of the present invention is an extract of Lactococcus sp. KR-050L culture, an extract of Lactococcus sp. KR-050L cells or supernatant, or a fraction thereof, preferably based on solids. 0.0001 to 10% by weight of the total weight may be included, and more preferably 0.001 to 1% by weight.

In addition, the pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" of the present invention means that it exhibits properties that are not toxic to the cells or humans exposed to the composition. The carrier can be used without limitation so long as it is known in the art such as buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, bases, excipients, lubricants. In addition, the pharmaceutical compositions of the present invention may be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, and the like, oral formulations, suppositories, and sterile injectable solutions, respectively, according to a conventional method. have. Furthermore, it can be used in the form of an external preparation for skin in the form of ointments, lotions, spray agents, patches, creams, powders, suspensions, gels or gels. Examples of carriers, excipients and diluents that can be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations are the lactic acid bacteria lactococcus sp. KR-050L extracts and fractions are prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium styrate and talc are also used. Liquid preparations for oral use may include various excipients, such as wetting agents, sweeteners, fragrances, preservatives, etc., in addition to water and liquid paraffin, which are commonly used to include suspensions, solutions, emulsions, and syrups. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Meanwhile, the pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term "administration" of the present invention means introducing a predetermined substance into an individual in an appropriate manner and the route of administration of the composition can be administered via any general route as long as it can reach the target tissue. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, nasal administration, pulmonary administration, rectal administration, but is not limited thereto.

The term "individual" refers to all animals, including rats, mice, livestock, etc., including humans, who may develop or may develop inflammatory diseases or cancers caused by IL-6 or IL-11. Preferably, it may be a mammal including a human.

The term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and not causing side effects, and the effective dose level is determined by the patient's sex, Including, but not limited to, medicaments and other medical fields that are used in combination, or in combination with, or in combination with, a pharmaceutically acceptable carrier, excipient, Can be readily determined by those skilled in the art according to known factors. Preferably, the extract of the Lactococcus sp. KR-050L culture solution of the present invention, the extract of Lactococcus sp. KR-050L cells or supernatant or fractions thereof is 0.0001 to 100 mg / kg body weight per day, preferably based on solids. May be administered at 0.001 to 100 mg / kg body weight. Dosing may include administering the recommended dose once daily, or in divided doses.

In another embodiment, the present invention provides a STAT3 (signal) induced by IL-6 (interleukin-6) or IL-11, which contains the Lactococcus sp. KR-050L culture, cell or supernatant, extracts or fractions thereof. It provides a composition for inhibiting the activation of transducers and activators of transcription 3).

Lactobacillus sp. KR-050L cultures, cells or supernatants of the present invention, extracts or fractions thereof are subjected to signal transducers and activators of transcription 3 (STAT3) induced by IL-6 (interleukin-6) or IL-11. Have inhibitory activity against. Therefore, it can be used as a composition for inhibiting the activity of STAT3 induced by IL-6 (interleukin-6) or IL-11.

In another aspect, the present invention is the Lactococcus species ( Lactococcus) sp .) Provides food, food additives, feed or feed additives comprising KR-050L or its culture.

The foods include not only health functional foods, but also general foods that are widely consumed by humans on a daily basis. When used as a health functional food, may be formulated into a conventional health functional food formulation known in the art together with a pharmaceutically acceptable carrier or additive. The health functional food may be prepared, for example, as a powder, a granule, a tablet, a capsule, a suspension, an emulsion, a syrup, a liquid, an extract, tea, jelly, The pharmaceutically acceptable carrier or additive may be any carrier or additive known to be usable in the art for the preparation of the formulation to be prepared.

Preferably the food is margarine, fat continuous or water continuous or bicontinuous spreads, fat reduced spreads, chocolate, or confectionary such as chocolate coating or chocolate fillings or bakery loaf. It can be used as an ice cream, ice cream coating, ice cream content, dressing, mayonnaise, yogurt, cheese, cream substitute, dry soup, drink, cereal bar, sauce, snack bar, dairy product, clinical nutrition or pediatric preparation. More preferably, it may be fermented food, and examples of fermented food include, but are not limited to, kimchi, vegetable fermented product, soybean paste, soy sauce, cheonggukjang, salted fish, fermented dairy products (fermented milk, yogurt, cheese) and the like.

Lactococcus species of the invention when used as feed additives sp .) KR-050L or its culture may be 20 to 90 wt% high concentrate or prepared in powder or granule form. The feed additives include organic acids such as citric acid, fumaric acid, adipic acid, lactic acid and malic acid, phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate and polyphosphate (polyphosphate), polyphenols, catechins, alpha-tocopherols and rosemary Extracts, vitamin C, green tea extracts, licorice extracts, chitosan, tannic acid, phytic acid, such as any one or more may be further included. When used as a feed, the composition may be formulated in a conventional feed form and may include common feed ingredients.

The feed additives and feeds may be selected from the group consisting of cereals, such as ground or crushed wheat, oats, barley, corn and rice; Feeds based on vegetable protein such as rapeseed, soybeans and sunflower; Animal protein feeds such as blood, meat, bone meal and fish meal; Sugar and dairy products, for example, may further include a dry ingredient consisting of various powdered milk and whey powder, and may further include a nutritional supplement, digestion and absorption enhancer, growth promoter and the like.

The feed additive may be administered to animals singly or in combination with other feed additives in edible carriers. The feed additives can also be administered to the animal either as a top dressing or they can be mixed directly with the animal feed or in a separate oral form separate from the feed. When the feed additive is administered separately from an animal feed, it can be prepared in an immediate release or sustained release formulation, in combination with a pharmaceutically acceptable edible carrier as is well known in the art. Such edible carriers may be solid or liquid, such as corn starch, lactose, sucrose, soy flakes, peanut oil, olive oil, sesame oil and propylene glycol. When a solid carrier is used, the feed additive can be a tablet, capsule, powder, troche or emulsion or top-dressing in finely divided form. When a liquid carrier is used, the feed additive can be a gelatin soft capsule, or a syrup or suspension, emulsion, or solution formulation.

The feed may comprise any protein-containing organic fructose commonly used to meet an animal's dietary needs. These protein-containing flours usually consist mainly of corn, soy flour, or corn / soy flour mix.

The feed additives and feeds may also contain adjuvants such as preservatives, stabilizers, wetting or emulsifying agents, solution promoters and the like. The feed additive may be used by being added to animal feed by infiltration, spraying or mixing.

The feed or feed additive of the present invention is applicable to many animal diets, including mammals, poultry and fish. The mammal can be used not only for pigs, cows, sheep, goats, experimental rodents, and experimental rodents, but also for pets (eg, dogs, cats). As poultry, chickens, turkeys, ducks, geese, pheasants, And can also be used for quail, etc., may be used in trout, etc. as the fish, but is not limited thereto.

In another aspect, the present invention is the Lactococcus species ( Lactococcus) sp .) Provide personal care products comprising KR-050L or its culture.

Preferably the personal hygiene products are soaps, cosmetics, wet wipes, tissue paper, shampoo, skin cream, face cream, toothpaste, lipstick, perfume, make-up, foundation, ball touch, mascara, eye shadow, sunscreen lotion, hair care products , Air freshener gel or cleaning gel.

The present invention is a novel lactic acid bacterium Lactococcus species KR-050L ( Lactococcus sp . KR-050L) strain, the lactococcus species KR-050L is excellent in taste and flavor, and exhibits inhibitory activity against STAT3 induced by IL-6 or IL-11, IL-6 or IL- It can be used as a composition for the prevention or treatment of inflammatory diseases or cancers caused by 11.

1 is an electron micrograph of Lactococcus sp. KR-050L (Lactococcus sp. KR-050L) according to an embodiment of the present invention.
2 is a diagram showing the gene sequence of Lactococcus species KR-050L according to an embodiment of the present invention.
Figure 3 is a diagram showing the inhibitory activity against the IL-6 induced luciferase expression of the ethyl acetate extract of Lactococcus sp. KR-050L cells according to an embodiment of the present invention and column chromatography fraction thereof.
4 is a diagram showing the inhibitory activity against IL-6 induced luciferase expression of ethyl acetate extract of the lactococcus spp. KR-050L supernatant and column chromatography fraction thereof according to an embodiment of the present invention.
5 is a diagram showing the inhibitory activity against IL-6 induced luciferase expression of ethyl acetate and ethanol crude extract of Lactobacillus sp. KR-050L culture lyophilisate according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  1: Isolation and selection of lactic acid bacteria

Samples were collected from fermented foods such as kimchi and salted fish to isolate the lactic acid bacteria strains from nature. The supernatant of the collected sample was diluted by mixing sterile water, shaken at 30 ° C. for 30 minutes, and diluted by 10 ⁴ times by agar plate dilution method to dilute 0.1 ml of MRS-Agar medium (Table 1). ) And spread evenly over the entire medium. After 3 to 5 days of incubation at 30 ° C in a thermostat, morphological analysis of the single colony showed that only the lactic acid bacteria strain was purely cultured, and then cultured on MRS-Agar slope medium, suspended in 20% glycerol, And stored in a 70 ° C freezer. Each colony was inoculated into a culture solution containing 8% skim milk powder and cultured after the final selection of strains having excellent taste and aroma (Table 2).

Example  2: Identification of selected strains

To identify the phylogenetic location of the selected strains, 571 bp including the internal transcribed spacer 1 of the 16S ribosomal RNA gene, the 5.8S ribosomal RNA gene and the internal transcription spacer 2 The base sequence of was analyzed. 16S rDNA fragment 5'-TCC GTA GGT GAA CCT GCG G-3 '(SEQ ID NO: 2) from the selected strains were amplified by performing PCR using a primer set, and then purified by amplifying products by an automatic sequence analysis device (Applied The nucleotide sequences were analyzed using a Biosystems model 373A automatic sequencer and an analysis kit (BigDye Terminator Cycle Sequencing kit, Perkin-Elmer Applied Biosystems). 16S rDNA of the finally selected strain was confirmed by homology analysis using the blast program of the GenBank showed a high homology with the standard strain Lactococcus lactis (SEQ ID NO: 1; Fig. 2) ). In addition, the analysis of the isolated lactic acid using a variety of biochemical methods, it was confirmed that it has a high similarity with the standard strain lactococcus lactis (Table 3). Therefore, the strain of the present invention was finally confirmed to have high homology with Lactococcus lactis, and the lactic acid bacteria strain of the present invention was lactococcus species KR-050L ( Lactococcus sp . KR-050L), and it was deposited on March 21, 2012 at the Korea Collection for Type Culture (KCTC) located at 125, Gwahak-ro, Yuseong-gu, Daejeon, Korea, with accession number KCTC 12170BP. Was given.

Example  3: Acid resistance  Experiment

Lactobacillus sp. KR-050L strain isolated and selected in Example 1 was inoculated into sterile MRS liquid medium. After culturing at 30 ° C for 24 hours, the strain was recovered by centrifugation at 9000 rpm for 25 minutes. Then, the isolated strain recovered in MRS liquid medium adjusted to pH 3.0 with HCl was inoculated at a concentration of 1.9 × 10 ⁹ CFU / ml and incubated for 90 minutes at 30 ℃. After inoculation with Lactococcus sp. KR-050L strain, each sample was collected every 30 minutes, incubated for 90 minutes, diluted in sterile saline solution, plated on MRS plate medium, incubated at 30 ° C for 72 hours, and then colonized on plate medium. The number of viable cells was determined by counting the numbers (Table 4).

Example  4: My bile  Experiment

In order to test the bile resistance of Lactococcus sp. KR-050L strain isolated and selected in Example 1, an artificial bile was prepared by preparing MRS medium containing 1, 2, 3, and 4% of oxgall, respectively. It was used as a liquid. Resistance to bile acids is one of the basic characteristics of probiotics microorganisms, as well as resistance to acidic conditions in the stomach.

The isolated lactococcus species KR-050L of the present invention were incubated at 30 ° C. for 24 hours and then centrifuged at 9000 rpm for 25 minutes to recover the cells. The recovered cells were washed twice with 0.01 M phosphate buffered saline (PBS) and incubated at 30 ° C. for 3 hours by adding MRS medium containing 1-4% ox bile in the same amount as the supernatant. Thereafter, by diluting with 0.85% salt buffer solution, the survival rate was calculated by measuring the colony number of the KR-050L strains lactococcus spp. Grown on MRS agar and grown. As a result, the number of viable cells tended to decrease compared with the initial number of inoculated bacilli after treatment of bacillus juice, but it was judged that the bacillus was possessed (Table 5).

Example  5: Lactococcus  Bell KR -050L extract and Fraction  Produce

<5.1> Lactococcus  Bell KR -050L culture and lyophilization

Lactic acid bacteria Lactococcus species KR-050L of the present invention was primarily cultured using MRS medium, and the second main culture was inoculated at a 1% concentration in a medium containing 6 to 8% skim milk (30%). Incubated for 48 hours at ℃. After lyophilization of a part of the culture, the lyophilized sample was aliquoted, and ethyl acetate (EtO Ac) or ethanol (EtOH) was added for 6 hours. The extract was filtered and concentrated under reduced pressure to give EtOAc and EtOH crude extract.

<5.2> Lactococcus  Bell KR Preparation of ethyl acetate extract of -050L cells

The culture broth of <5.1> was separated into cells and supernatant by centrifugation. EtOAc was added to the isolated cells and sonicated for 6 hours. The EtOAc extract obtained by filtration was concentrated under reduced pressure to give an EtOAc crude extract. Meanwhile, an equal amount of EtOAc was added to the supernatant, the mixture was mixed, and the above procedure was repeated three times to obtain a water-soluble fraction and an EtOAc-soluble fraction. Then, the EtOAc-soluble fraction was concentrated under reduced pressure to obtain an EtOAc-soluble extract.

<5.3> Methanol Fraction  Produce

The EtOAc-soluble fraction of the supernatant obtained in <5.2> was dissolved in 40% methanol (MeOH) and loaded into an ODS-SPE cartridge, followed by a solvent system (40% MeOH, 60% MeOH, 80) mixed with MeOH and water. % MeOH, 100% MeOH) was used as a mobile phase to perform column chromatography to finally obtain four methanol subfractions.

Example  6: Lactococcus  Bell KR Extract of -050L Cells and Cultures and Its Fraction of IL -6-induced STAT3 &Lt; / RTI &gt;

The following experiment was performed to determine the inhibitory effect of the expression of STAT3 induced by IL-6 of the extracts of lactococcus spp KR-050L and fractions thereof of the present invention.

<6.1> Transformant Preparation

PStat3-Luc and pcDNA3.1 (+) (Clontech laboratories, Palo Alto, Calif., USA) to Hep3B cells (ATCC HB-8064) as lipofectamin plus (Invitrogen, Carlsbad, CA, USA) Transfection. Two days later, hygromycin was treated at a concentration of 100 μg / ml to obtain clones stably expressing luciferase. It was confirmed through the luciferase assay that luciferase stably expressed in the obtained clone.

<6.2> IL -6 Reactivity STAT3  Reporter gene testing

The transfected cells were serum-free (serum starvation) with DMEM (GIBCO 119950965) and the samples were treated for 1 hour as follows, followed by the addition of 10 ng / ml IL-6 (R & D system, USA). Incubated for hours: 1. negative control (non-treated); 2. Positive Control (IL-6 10 ng / ml); And extracts and fractions (10, 30, 60 μg / ml).

The cultured cells were washed with PBS, 50 μl of lysis buffer (luciferase assay system, Promega, USA) was added and stirred for 20 minutes, and then 30 to 100 μl luciferase substrate (luciferase assay system, Promega, USA) was added. Within 5 minutes, the color development was measured with a luminometer (EG & G BERTHOLD, USA).

As a result of measuring the IL-6 induced STAT3 luciferase activity against the EtOAc extract and the fraction obtained from the first column chromatography of Lactococcus spp. KR-050L cells and cultures, the concentration dependent inhibitory activity of the extract and the fraction was determined. Shown (FIGS. 3 and 4). In addition, STAT3 luciferase activity induced by IL-6 was measured for extracts obtained by lyophilizing a culture of Lactobacillus lactococcus species KR-050L extracted with EtOAc or EtOH and concentrated with PBS. And EtOH extract showed a concentration-dependent inhibitory activity, but did not show inhibitory activity in the sample dissolved in PBS (Fig. 5).

Korea Research Institute of Bioscience and Biotechnology KCTC12170BP 20120321

<110> Korea Research Institute of Bioscience and Biotechnology <120> A novel Lactococcus sp. strain and use <130> PA120712 / KR <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 1409 <212> DNA Lactococcus sp. KR-050L <400> 1 tgcagttgag cgctgaagtt ggtacttgta ccgactggat gagcagcgaa cgggtgagta 60 acgcgtgggg aatctgcctt tgagcggggg acaacatttg gaaacgaatg ctaataccgc 120 ataaaaactt taaacacaag ttttaagttt gaaagatgca attgcatcac tcaaagatga 180 tcccgcgttg tattagctag ttggtgaggt aaaggctcac caaggcgatg atacatagcc 240 gacctgagag ggtgatcggc cacattggga ctgagacacg gcccaaactc ctacgggagg 300 cagcagtagg gaatcttcgg caatggacga aagtctgacc gagcaacgcc gcgtgagtga 360 agaaggtttt cggatcgtaa aactctgttg gtagagaaga acgttggtga gagtggaaag 420 ctcatcaagt gacggtaact acccagaaag ggacggctaa ctacgtgcca gcagccgcgg 480 taatacgtag gtcccgagcg ttgtccggat ttattgggcg taaagcgagc gcaggtggtt 540 tattaagtct ggtgtaaaag gcagtggctc aaccattgta tgcattggaa actggtagac 600 ttgagtgcag gagaggagag tggaattcca tgtgtagcgg tgaaatgcgt agatatatgg 660 aggaacaccg gtggcgaaag cggctctctg gcctgtaact gacactgagg ctcgaaagcg 720 tggggagcaa acaggattag ataccctggt agtccacgcc gtaaacgatg agtgctagat 780 gtagggagct ataagttctc tgtatcgcag ctaacgcaat aagcactccg cctggggagt 840 acgaccgcaa ggttgaaact caaaggaatt gacgggggcc cgcacaagcg gtggagcatg 900 tggtttaatt cgaagcaacg cgaagaacct taccaggtct tgacatactc gtgctattcc 960 tagagatagg aagttccttc gggacacggg atacaggtgg tgcatggttg tcgtcagctc 1020 gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa cccctattgt tagttgccat 1080 cattaagttg ggcactctaa cgagactgcc ggtgataaac cggaggaagg tggggatgac 1140 gtcaaatcat catgcccctt atgacctggg ctacacacgt gctacaatgg atggtacaac 1200 gagtcgcgag acagtgatgt ttagctaatc tcttaaaacc attctcagtt cggattgtag 1260 gctgcaactc gcctacatga agtcggaatc gctagtaatc gcggatcagc acgccgcggt 1320 gaatacgttc ccgggccttg tacacaccgc ccgtcacacc acgggagtgg gagtacccga 1380 agtaggtgcc taaccgcagg agggcgctc 1409 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 16S rDNA fragment <400> 2 tccgtaggtg aacctgcgg 19

Claims (13)

Lactococcus of Accession No. KCTC 12170BP sp .) KR-050L.
Claim 1 lactococcus species KR-050L culture, cells or supernatant, extracts or fractions thereof as an active ingredient pharmaceutical composition for the prevention or treatment of inflammatory diseases or cancer.
3. The method of claim 2,
Pharmaceutical composition characterized in that the inflammatory disease or cancer is caused by IL-6 or IL-11.
The method of claim 3,
The inflammatory diseases include osteoarthritis, rheumatoid arthritis, osteoporosis, plasmaacytosis, hyperimmunoglobulinemia, anemia, nephritis, cachexia Pastoral disease, vascular proliferative nephritis, multiple sclerosis, uveitis, chronic thyroiditis, delayed-type hypersensitivity, contact dermatitis, atopic dermatitis Atopic dermatitis, systemic lupus erythematosus, Crohn's disease, pancreatitis, psoriasis, arc idiopathic atrophy, diabetes mellitus, and Alzheimer's disease A pharmaceutical composition, which is a disease selected from the group consisting of Alzheimer's disease.
The method of claim 3,
The cancer includes pancreatic cancer, breast cancer, breast cancer, prostatic cancer, brain tumor, head and neck cancer, melanoma, myeloma, and leukemia. leukemia, lymphoma, hepatoma, gastric cancer, colon cancer, bone cancer, uterine cancer, ovarian cancer, rectal cancer, Esophageal cancer, small intestine tumor, anal cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma carcinoma, vulva carcinoma, Hodgkin's disease, bladder cancer, renal cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma And a cancer selected from the group consisting of central nervous system tumors. .
3. The method of claim 2,
The extract is a pharmaceutical composition that is extracted with ethyl acetate (Ethyl acetate) or ethanol (ethanol, ethyl alcohol; EtOH).
3. The method of claim 2,
The fraction is a pharmaceutical composition of ethyl acetate extract fractions with methanol.
3. The method of claim 2,
A pharmaceutical composition comprising 0.001 to 1% by weight of the total weight of the extract of the lactococcus species KR-050L culture, the extracts of the lactococcus species KR-050L cells or the supernatant or fractions thereof based on solids.
Signal transducers and activators of transcription 3 induced by IL-6 (interleukin-6) or IL-11 containing L-tococcus sp. KR-050L cultures, cells or supernatants, extracts or fractions thereof of claim 1 Composition for inhibiting activity).
Lactococcus species according to claim 1 sp .) Food, food additives, feed or feed additives comprising KR-050L or its culture.
11. The method of claim 10,
Food, food additives, feed or feed additives characterized in that the fermented food.
Lactococcus species according to claim 1 sp .) Personal care products comprising KR-050L or its culture.
The method of claim 12,
Soaps, Cosmetics, Wipes, Tissue Paper, Shampoos, Skin Creams, Face Creams, Toothpastes, Lipsticks, Fragrances, Make-Ups, Foundations, Ball-Touch, Mascara, Eyeshadow, Sunscreen Lotion, Hair Care Products, Air Freshener Gels & Cleansers A personal hygiene product selected from the group consisting of gels.

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