KR101518866B1 - Lactobacillus plantarum and use thereof - Google Patents

Abstract

The present invention relates to a novel microbial Lactobacillus plantarum and uses thereof. Through the present invention, it is possible to obtain a probiotic effect, an immunity-enhancing effect, and an anticancer effect by utilizing the microorganism searched for, and cultivate immune cells such as the macrophage and the macrophage together so as to obtain a physiologically active substance, 1 alpha or TNF-alpha. ≪ / RTI >

Description

Lactobacillus plantarum and its use {Lactobacillus plantarum and use thereof}

The present invention relates to a novel microbial Lactobacillus plantarum and uses thereof.

Lactic acid bacteria such as Weissella spp., Leuconostoc spp., And Lactobacillus spp., Which are involved in the fermentation process of kimchi, are responsible for aging. According to recent studies, lactic acid probiotics have been shown to inhibit the growth of pathogenic microorganisms, in response to improved respiratory and allergic responses, improved immune function, and side effects from antibiotic administration. Lactic acid bacteria also have a function of lowering cholesterol (Huang Y. et al ., 2013), cancer prevention effect (Kumar. M, 2010) and lactose intolerance treatment (Saltzman JR, 1999). Recently, studies on the effect of lactic acid bacteria on immunity enhancement have been greatly activated.

Microbes such as Lactobacillus and Bifidobacterium control the expression of cytokines in immune cells such as macrophages and natural killer (NK) cells (Blum et al., 2002). ). Probiotics have been reported to affect the innate immune system and have specific effects on microorganisms (Perdiget al., 2001). Toll-like receptors (TLRs) are receptor proteins that play an important role in the innate immune system. The effects of probiotics may be accompanied by TLR-mediated immune responses (Suzuki et al., 2008). Cytokines directly or indirectly affect host defense systems. Soluble factors such as IL-1 and TNF-α secreted by monocytes appear to play an important role in the proliferation, activation, and differentiation of immune cells (Czarniecki, CW, 1993). In addition, IL-1 stimulates T-cell and B-cell proliferation, and TNF-α has a cytotoxic effect on tumor cells (Gill, HS, 1998). IL-1 is a major product of stimulated monocytes and is responsible for a variety of biological effects. The systemic effects of IL-1 include elevated circulating neutrophils, liver acute phase proteins, late sleeping waves, increased insulin levels and blood pressure (Dinarello, CA, 1987). TNF-α is also produced in a variety of cells, including activated macrophages and fibroblasts. Control of TNF-α production is important in maintaining the homeostasis of the immune system and in the prevention and treatment of immune diseases (Kang et al., 1996).

  The present invention has accomplished the present invention by isolating and identifying lactic acid bacteria from various kimchi, confirming probiotic function and immunological activity.

The present invention provides a newly isolated lactic acid bacterium, Lactobacillus plantarum (Accession No .: KCCM11426P), and the deposited Lactobacillus plantarum and immune cells are co-cultured to produce a physiological effect such as IL-1 alpha or TNF alpha The aim is to increase the active substance and to mass-produce the increased physiologically active substance.

Also provided is a pharmaceutical composition or composition for immunotherapy comprising the above-identified Lactobacillus plantarum, and a pharmaceutical composition for cancer therapy or a composition for cancer improvement comprising the above-identified Lactobacillus plantarum There is a purpose.

In the present invention, the cytokine is a leukocyte-mediated signaling language, the cytokine secreted from monocytes is referred to as monokine, and the cytokine secreted from lymphocytes is referred to as lymphokine. At this time, cytokines and interleukins are used as synonyms. Since the cytokine secreted from the white blood cells is a physiologically active substance that mediates the interactions between the white blood cells, INTER, which means between the interleukin, LEU, which means white blood, and KIN, which means the active substance, are called interleukins. After the amino acid sequence structure is determined, the interleukin is numbered. There are currently IL-1 to IL-19. It belongs to Interleukin, but uses the original name as it is.

Particularly, in the present invention, IL-1 activates T cells, differentiates and proliferates B cells, and exerts an NK cell action, thereby inducing an immunity-enhancing effect.

In the present invention, in the present invention, Tumor Necrosis Factor (TNF) is secreted in activated macrophages in the case of TNF alpha (Cachectin) and secreted in activated T cells in the case of TNF beta (Lymphotoxin). In addition to killing cancer cells, TNF also acts on normal cells and acts as a cofactor for the bacterial killing of eating cells, the activation of T cells, and the production of antibodies to B cells. In other words, the function of TNF stimulates the central heat of the brain, activates heat, activates immune cells including lymphocytes, and acts physiologically to control sleep, pain and appetite.

The term " immunopotentiating (pharmaceutical / food) composition "as used herein refers generally to a carrier which is a biocompatible material and an antigen or antigen-inducing substance carried on the carrier and, if desired, Quot; means a composition or preparation containing an immunomodulatory substance and / or one or more pharmaceutical additives. (Korean Patent No. 10-0574216). An "immune response" enriched by an immunoenhancing composition of the invention is an antigen-specific immune response induced by an antigen contained within the composition, or by an inducer contained therein. The activated immune response may be humoral immunity, mucosal immunity or cellular immunity, or a combination thereof. The term "antigen" is not limited to any particular species, as long as it is capable of eliciting an antigen-antibody response derived from the antigen.

Generally, antigens that produce antibodies that are effective in preventing and / or treating human or non-human mammal or avian diseases are selected. Thus, there may be mentioned, for example, "Vaccine Handbook" (edited by the National Institute of Health Alumni Association, published by Maruzen Co.), "Immunizing Agents" in Reminton's Pharmaceutical Sciences, 14th edition, 1990, Mack Publishing Co. , Section 75, pages 1426-1441 or the Physician's Desk Reference to Drugs Approved by the United States Food and Drug Administration, 46th edition, pages 208-209, 1992), but not limited to, . In addition, the following are also included, but are not limited thereto.

(1) for example, genetic recombination (transformation of toxic- or pathogenic-related genes), successive subculture (attenuated or non-pathogenic species as a result of autotransformation), formalin treatment, beta -propiolactone treatment, Viruses, mycoplasma, bacteria, parasites, toxins, cancer cells, etc. that have been attenuated by exposure to ultrasound, enzyme treatment, heating, etc., or non-toxic or non-pathogenic. (2) membrane surface proteins and nucleoproteins obtained from viruses, mycoplasma, bacteria, parasites, toxins, cancer cells and the like, proteoglycans, polypeptides, and the like obtained by, for example, chemical or enzymatic degradation, physical destruction, (3) a gene encoding an antigen capable of inducing a specific immunity against viruses, mycoplasma, bacteria, parasites, toxins, cancer cells and the like by using a corresponding virus, mycoplasma, bacteria , A parasite, a toxin, a cancer cell system, etc., identifying the gene, inserting it into a suitable vector, such as a plasmid, and expressing the gene in E. coli, yeast or animal cells, Synthetic peptides arranged such that the specific antigenicity is comparable or better. MAGE-1, MAGE-3, and BAGE, tissue-specific antigens such as tyrosinase, Mart-RAGE, and the like, although not limited thereto, 1, gp100 and gp75, and further, p15, Mucl, CEA, HPV E6, E7, HPR2 / neu.

"Antigen" includes, but is not limited to, antigens that are capable of eliciting an immune response that is associated with, or effective in treating, the onset of a disease, such as those mentioned below: cholera, whooping cough, fever, typhoid, meningitis, Inflammation, Japanese encephalitis, rubella, measles, yellow fever, mumps, hepatitis A, hepatitis B, infectious disease, gonorrhea, gonorrhea, poliomyelitis, colibacilllemia, rabies, diphtheria, botulism, Hepatitis C, chickenpox / herpes zoster, malaria, tuberculosis, candidiasis, tooth decay, acquired immunodeficiency syndrome, cancer (tumor), matitis, anthrax, brucellosis, mastoid lymphadenitis, Bordetella bronchitis, distemper, leukopenia, non-tracheitis, viral diarrhea, and pimelae (pneumoniae) addiction.

Antigens further include antigens that can induce an immune response that is effective in preventing infection of a virus, mycoplasma, bacterium, or parasite, as well as preventing the onset of a related disease and treating a patient suffering from such a disease, But are not limited to: Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Salmonellae, Meningococci B strain ( group B streptococci, group B streptococci, adenovirus, coronavirus, RS virus, human immunodeficiency virus I and II, herpes simplex I and II, CMV, EBV, Chlamydia trachomatis, Parvovirus, parainfluenza virus, calicivirus.

"Antigens" also include antigens used for animal production as well as for animal health. For example, this antigen is described in Vaccines in Agriculture, Immunological Applications to Animal Health and Production (edited by PRWood et al., CSIRO, 71-160, 1994). Such antigens include, but are not limited to, the antigens used in animal production such as those described below: 1) Repeating antigens; phosphorus-related peptides and releasing hormones such as ruthenicin-hormone releasing hormone, Antigens capable of inducing immune responses to dot-like follicle-releasing hormones, etc. 2) Antigens for animal growth and metabolism control; Growth hormone, growth hormone, steroid hormone, sex steroid hormone, plasma membrane antigen of adipocyte, lipid lipid, cortisol, adenocorticotropic hormone, adenocorticotropic hormone receptor,? -Adrenergic receptor , An antigen capable of inducing an immune response against an adenohippocylic hormone such as prolactin, ACTH, STH, TSH, LH, FSH, and the like; 3) environmental control antigen; Antigens capable of inducing an immune response to plant-related toxins, low molecular weight natural toxins.

In addition, the term "antigen" includes, but is not limited to any particular species capable of inducing a specific immune response to an antigen, and also includes an antigen capable of inducing a nonspecific immune response to the antigen. Antigens capable of inducing a nonspecific immune response to an antigen, such as those mentioned above, include staphylococcal enterotoxin, toxin shock syndrome toxin-1, exopolyadidermatitis toxin, CAP (membrane associated protein) and SPM Streptococcus pyogen-mitogen), for example, Miyagiken Ishikai Kaiho, Vol. 50 133-137, 1997), and so-called superantigens such as T-12 and NY-5.

The term "antigen-inducing agent" refers to a substance capable of inducing an antigen as described above in vivo, including but not limited to viruses, mycoplasma, bacteria, parasites, toxins , Plasmids and viruses containing nucleic acids encoding gene sequences for antigens capable of inducing specific immunity against tumor cells, and the like.

An antigenic substance that induces a specific immunity can be incorporated intact into the immunostimulatory composition without any modification, or further, to increase its antigenicity and / or its stability, for example, (1) (2) is supplemented with a suitable sugar (carbohydrate) chain, (3) is contained in a liposome, or (4) is a virus-liposome membrane fusion type Contained in liposomes or contained in virosomes obtained using (5) B30MDP [6-O- (2-tetradecylhexadecanoyl) -N-acetylmuramyl-L-alanyl-D-isoglutamine] .

In the present invention, the term "immunomodulating substance (substance having immunostimulatory, immunostimulating or immunomodulating activity)" is not limited to any particular species but includes, in particular, cytokines, chemokines, growth factors, ancillary peptides and DNA sequences, , Freund's complete adjuvant, Freund's incomplete adjuvant, Ischem, saponin, hexadecylamine, dimethyl dioctadecylammonium bromide, Abridin, cell wall skeleton component, cholera toxin, lipopolysaccharide endotoxin, cytokine Containing liposomes and Walter lead liposome-containing liposomes, 1,25-dihydroxyvitamin D3, and carboxyl vinyl polymers, arginine and sodium chloride. The term "cytokine" is not limited to any particular species as long as it has immunostimulatory activity, and includes, inter alia, IFN- ?, IFN- ?, IFN- ?, IL-1 ?, IL-1 ?, IL- 3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-12, TNF- ?, TNF- ?, and GM-CSF. For example, IL-1 [beta] and IL-2 are particularly preferred when accumulation of immunologically competent cells around the site of administration of the immunoconjugation composition and subsequent enhancement of antibody production is desired.

The amount of the immunomodulatory substance and the amount of the immuno-inducing antigenic substance contained in the immunopotentiating composition of the present invention may be arbitrarily adjusted depending on the blending ratio with respect to the biocompatible material and additives contained in the composition, and the shape or size of the composition. Using the composition of the present invention

The dose of the antigenic material to be administered may be approximately the same as that used for conventional administration methods (e.g., parenteral administration in the form of a solution or suspension). However, since the composition of the present invention has an excellent immunity-enhancing effect as described above, immunity can be sufficiently induced even in a smaller amount as compared with the usual administration method, and the dose is not limited to the antigenic substance, the composition And / or the immunomodulatory substance administered simultaneously with the antigenic substance, and the amount thereof.

The form or shape of the immunoenhancing composition of the present invention may be, for example, in the form of a solution, a suspension, a gel, a film, a sponge, a rod or a bar or a particle. The appropriate form can be selected to more effectively induce the immune response. A rod shape is preferred. More particularly preferred are coated or coated bar formulations such as those described in EP 659,406.

There is no particular limitation to the method of administering the immunoconjugation compositions of the present invention, including but not limited to parenteral administration, oral administration, nasal and / or pulmonary administration, injection with compressed air and embeding at the incision site or Conservation is included. The preferred method of administration can be selected depending on the form of the immunoenhancing composition in such a way that the immune response can be induced more effectively. In the case of a rod or a bar-shaped composition, parenteral administration or preservation at the incision site is preferable, but in the case of the particles, it can be directly applied to the incision site for preservation, Parenterally in the form of a suspension prepared by suspending the particles in a solvent for injection as described in Proc. (Helios Gene Gun System (Bio-Rad)) as described in Natl. Acad. Sci. USA, 93, 6291-6296 (1996).

Although the injectable solvent should be selected according to the characteristics of the biocompatible substance, antigenic substance and immunomodulating substance, the term "injectable solvent" means that 1) the particles can be dispersed in the solvent, 2) 3) the antigenic substance and the immunomodulatory substance can be retained in the particles when the particles are dispersed in the solvent, 4) the solvent is non-toxic, and 5) the solvent in which the particles are dispersed is non- It is not limited to adults, any particular species. For example, the injectable solvent referred to herein includes distilled water, physiological saline, phosphate buffer solution, soybean oil, sesame oil, peanut oil, cottonseed oil, MCT (medium chain fatty acid triglyceride), olive oil, corn oil, castor oil, Polyethylene glycol), PG (propylene glycol), and fatty acids (e.g., DOTMA, DOPE, DOGS, etc.) used to prepare liposomes.

In one embodiment of the present invention, there is provided Lactobacillus plantarum which is the deposit number KCCM 11426P. Wherein said Lactobacillus plantarum promotes IL-1 alpha production and provides a Lactobacillus plantarum characterized by increased cell viability, wherein the production of TNF-alpha is increased Lactobacillus plantarum is provided.

In one embodiment of the present invention, there is provided a pharmaceutical composition for enhancing immunity comprising Lactobacillus plantarum FBT 215 (KCCM11426P), wherein in such embodiments, the immunomodulator is enhanced by increased IL-1 alpha or TNF-alpha And a pharmaceutically acceptable carrier. In one embodiment of the invention, there is provided a food composition for improving immunity comprising Lactobacillus plantarum FBT 215 (KCCM11426P), wherein in such embodiments, an immunologic improvement due to increased IL-1 alpha or TNF-alpha Wherein said food composition is a food composition.

In one embodiment of the present invention, there is provided a pharmaceutical composition for the treatment of chemotherapy comprising Lactobacillus plantarum FBT 215 (KCCM11426P), wherein, in the above embodiments, the chemotherapeutic treatment with increased IL-1 alpha or TNF- And a pharmaceutically acceptable carrier. In one embodiment of the invention, there is provided a cancer food composition for improving cancer comprising Lactobacillus plantarum FBT 215 (KCCM11426P), wherein in this embodiment the cancer is improved due to increased IL-1 alpha or TNF-alpha ≪ / RTI >

In one embodiment of the present invention, there is provided a method for producing Lactobacillus plantarum FBT 215 (KCCM11426P) and culturing an macrophage cell line; And isolating the IL-1 alpha produced in said culturing step. In one embodiment of the invention, the step of culturing Lactobacillus plantarum FBT 215 (KCCM11426P) and the macrophage cell line; And separating the TNF alpha produced in said culturing step. The protein physiologically active substance such as IL-1 alpha or TNF-alpha can be easily separated and purified by conventional methods for separating and purifying proteins.

The composition of the present invention contains the above-mentioned effective ingredient of the present invention relative to the total weight of the composition. 0.1 to 50% by weight. The composition comprising the active ingredient of the present invention may further comprise suitable carriers, excipients or diluents according to conventional methods. 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. The composition according to the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols or the like, oral preparations, suppositories or sterilized injection solutions according to a conventional method have. More specifically, when formulating the composition, it can be prepared using a diluent or an excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, and the like. The solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like. These solid preparations may contain at least one or more excipients such as starch, calcium carbonate, Sucrose, lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, syrups and the like, and various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. in addition to commonly used diluents such as water and liquid paraffin . Formulations for parenteral administration include sterile 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. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The present invention may be varied depending on the age, sex and body weight of the patient, but it is generally administered in an amount of 0.01 to 500 mg / kg, preferably 0.1 to 100 mg / kg, once to several times per day . The dosage may also be increased or decreased depending on the route of administration, degree of disease, sex, weight, age, and the like. Thus, the dosage amounts are not intended to limit the scope of the invention in any manner.

The composition of the present invention can be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections. Since the active ingredient of the present invention has little toxicity and side effects, it can be safely used for prolonged use for preventive purposes.

The present invention provides a health functional food comprising the above-mentioned active ingredient of the present invention and a food-acceptable food supplementary additive, and various foods such as beverage, gum, tea, vitamin complex, , Pills, powders, granules, infusions, tablets, capsules or beverages. At this time, the amount of the active ingredient of the present invention in the food or beverage may be generally 0.01 to 15% by weight of the total food weight of the health food composition of the present invention, 0.02 To 10 g, preferably from 0.3 to 1 g.

Food supplementary additives as defined herein include food additives customary in the art, such as flavoring agents, flavoring agents, coloring agents, fillers, stabilizers, and the like. The health beverage composition according to the present invention is not particularly limited as to the components added in addition to the active ingredient of the present invention as an essential ingredient in the indicated ratios and may contain various flavors or natural carbohydrates as an additional ingredient . Examples of the natural carbohydrate include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; Polysaccharides such as dextrin, cyclodextrins; And sugar alcohols such as xylitol, sorbitol, and erythritol. (Taurine, stevia active ingredient (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavorings (saccharin, aspartame, etc.) are advantageously used as flavorings other than the above- . The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above-mentioned composition, the composition of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and intermediates (cheese, chocolate etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the compositions of the present invention may contain flesh for the production of natural fruit juices and fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not so critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

Materials and badges

In the present invention, MRS broth and MRS agar medium (MRS liquid medium + 1.5% agar) were prepared by using MRS medium (Difco, USA) for isolation and subculture of lactic acid bacteria. Sodium azide (Sigma, USA) was used to selectively isolate Gram-positive bacteria (Liasi, SA et al, 2009) and BCP (bromocresol purple, Sigma, USA) (Bettache, G. et al, 2012).

For the production of whey culture medium, whey powder supplied by Samky Oil Co., Ltd. was used. Yeast extract (Taekyung food, Korea) and ammonium citrate (Shinyo pure chemicals, Japan) were used as additive (Song, T. S., 2011) to hydrolyze whey. The preparation method and composition are shown in Table 1 (Composition of whey-based medium for the growth of lactic acid bacteria (Song Tae-seok, 2011)).

Whey powder (Samik dairy co., Korea) 25 g Alkalase (Novozyme, Denmark) 0.1 g Yeast extract (Taekyung food, Korea) 10 g Ammonium citrate (Shinyo pure chemicals, Japan) 2 g Distilled water (total) 1 L * pH adjusted to 7.0

Samples for microbial separation

Kimchi was purchased or collected to isolate lactic acid bacteria. Cabbage kimchi, mugimchi, pachimachi, dongchimi, and white kimchi were collected from restaurants and laboratory laboratories and stored at 4 ℃. Samples were suspended in physiological saline and the microorganisms attached to Kimchi were removed and diluted with Stomacher (Homogenius, MAYO) (Yoo Kwangwon et al., 2012).

Identification and Identification of Lactic Acid Bacteria

How to separate lactic acid bacteria

For the isolation of lactic acid bacteria, samples such as kimchi and fermented food were collected and separated. A 10 g sample was sequentially diluted in a 0.85% NaCl solution in a sequential decantation, and the sample was stained on MRS agar containing BCP (0.1 g / L, Yakuri pure chemicals co.) And sodium azide (0.1 g / L, Sigma) For 24 hours, and then colonies and Gram stain were used to isolate the lactic acid bacteria primarily. BCP is neutral to purple, but in acidity it changes to yellow and used for acid production confirmation, and sodium azide inhibits the growth of Gram-negative bacteria. Table 2 below shows the composition of the modified MRS medium.

Composition Content (g / L) Dextrose 20.0 Protease peptone 10.0 Beef extract 10.0 Yeast extract 5.0 Sodium acetate 5.0 Ammonium citrate 2.0 Polysorbate 80 1.0 Magnesium sulfate 0.1 Manganese sulfate 0.05 Dipotassium phosphate 2.0 Agar 15.0 Bromocresol purple 0.1 Sodium azide 0.1

* pH adjusted to 6.5

Lactic Acid Bacteria Isolate Immune Activity

Preparation of Lactic Acid Bacteria Samples

The lactic acid bacteria isolate was cultivated for 24 hours, and 1 ml of the lactic acid bacteria isolate was dispensed into an EP tube, and the supernatant liquid excluding the pellet was removed using a centrifuge (vision 15000 CFN II). After removing the supernatant, Hanks balanced salt solution (HBSS, Sigma), which is a solution to protect the pH and osmotic pressure of the cells after washing twice with physiological saline, was diluted 1: 9 with distilled water and mixed with the pellet. Lactic acid bacteria were killed by heat treatment in a water bath for 30 minutes (De Souza, BD, 2010). Each sample was appropriately diluted with HBSS to adjust the cell concentration to 0.1 (OD @ 610 nm).

Published macrophage cell line ( RAW  264.7)

RAW 264.7 cells are mouse-derived macrophages that secrete cytokines and are widely used for immunoassay and are readily detectable by microscopy (Raschke WC et al. , 1978). It was used in the molecular pharmacology laboratory of Yonsei University. RAW 264.7 cells were cultured in Dulbecco's modified Eagles minimal essential medium (DMEM, Sigma, USA) supplemented with 10% inactivated fetal bovine serum (FBS, Sigma, USA) and Penicillin-streptomycin (Sigma, USA). The cells were incubated on a Petri dish and observed under a microscope. The cells were subcultured at 3-4 days intervals.

Cell viability assay (Cell viability assay)

Cell viability was confirmed by MTT assay. The toxicity of the isolated lactic acid bacteria to cells was analyzed by measuring 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetraxolium bromide (MTT, Sigma) (Jin CY et al ., 2007). MTT assay is an assay that uses the ability of mitochondria to reduce MTT to formazan due to dehydrogenase action. 200,000 cells / ml of RAW 264.7 cells were dispensed in an amount of 180 쨉 l each, and 20 쨉 l of the microorganism samples were treated and cultured in a 5% CO ₂ incubator at 37 째 C for 3 days. After incubation, 100 μl of MTT solution (2 ~ 5 mg / ml, Sigma) was added, and treated for 4 hours at 37 ° C in a 5% CO ₂ incubator. The MTT solution was removed and 100 μl of DMSO (Dimethyl sulfoxide, Sigma) do. The reason for treating DMSO is that because formazan does not dissolve in water, it must be dissolved in DMSO to measure its absorbance (Twentyman, PR, 1987). After addition of DMSO, the OD value was measured at a wavelength of @ 540 nm after shaking on a rocker for 30 minutes and compared.

Enzyme-linked immunosorbent assay (ELISA)

Lactic acid bacteria samples were selected from MTT assay results. In the sandwich ELISA, the primary antibody is first coated on the plate and the sample to be measured is treated and attached to the antibody. The secondary antibody that reacts with the enzyme is treated and developed through an enzyme. Sulfuric acid is used to stop the enzyme reaction, and the amount of secretion is measured by observing the absorbance (John, RC, 2000). The ELISA kit was assayed for TNF-α and IL-1 α using Komabiotech's EzWay ^™ pink-ONE cytokine ELISA Kit. OD values were measured using a 450 nm wavelength with a microplate reader (Multiskan FC, Thermo) and compared.

Identification of lactic acid bacteria isolate

Biochemical Identification of Lactic Acid Bacteria

API 50 CH kit (Biomerieux, France) was used to determine the lactose-isolate isolates. Lactic acid bacteria isolates were plated on MRS agar and incubated at 37 ° C for 24 hours. The colonies formed were aseptically suspended in API CHL medium, dispensed into API 50 CH kit strips, and mineral oil added to anaerobic conditions. Lt; 0 > C for 48 hours.

Genetic identification of lactic acid bacteria isolate

Extraction of Lactic Acid Bacteria The extraction of genomic DNA was performed by using Silica gel membrane method (Dneasy Tissue Kit, Qiagen) from 5 ml of MRS liquid medium and incubated at 37 ° C for 1 day. 1 ml of the cultured cells was divided into EP tubes and centrifuged at 5,000 xg for 10 minutes to recover the cells. The cells were washed several times with physiological saline and suspended in 20 mg / ml lysozyme buffer. After allowing to stand for 30 minutes at 37 ° C in a constant temperature water bath, 25 μl of protein and partial enzyme K and 200 μl of AL buffer were added in order and suspended in a constant temperature water bath at 56 ° C for 30 minutes. After this time, DNA was extracted using DNA Tissue Kit method. The extracted genomic DNA was subjected to PCR using PTC 100 ™ (MJ research inc) using a PCR master mix and a 27 forward primer (AGAGTTTGATCMTGGCT CAG) and a 1492 reverse primer (TACGGYTACCTTGTTACGACTT). The PCR conditions were denaturation at 95 ° C for 5 minutes, denaturation at 94 ° C for 1 minute, primer attachment at 48 ° C for 1 minute, DNA extension at 72 ° C for 30 minutes, and finally 72 ° C for 5 minutes Final DNA extension was performed. PCR was performed using 16S rRNA sequence analysis by Cosmogenetech, Korea. Sequencing was performed by Applied Biosystems Automatic Sequencer ABI 3730xlAplied. The homology test of the nucleotide sequence was carried out by the BLAST of the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov) for the information of the GenBank database.

Caution for isolating lactic acid bacteria Probiotic  characteristic

Antibiotic susceptibility

Isolation of Lactic Acid Bacteria Confirming Immune Activity Antibiotic Sensitivity Antibiotic disc of BBL BD was used for the invention. Ampicillin (10 μg), Streptomycin (10 μg), Gentamycin (10 μg), Erythromycin (15 μg), Tetraoxycycline (30 μg), Neomycin (30 μg) and Colistin (10 μg) were used as antibiotics. After incubation for 24 hours at 37 ° C in an incubator, the medium was incubated with MRS light agar. The antibiotic disc was carefully placed on the MRS agar and cultured for 24 hours at 37 ° C in a clear zone (Kim, Hyunjoo, 2004).

Acid resistance and My bile

Artificial gastric juice was prepared to confirm the acid resistance of lactic acid bacteria isolate. Artificial gastric juice was prepared by adding 1% Pepsin (Sigma, USA) to the MRS liquid medium and pH 2.5, 3.0, 4.0 using HCl. FBT215 was cultured in an MRS liquid medium for 24 hours in a 37 ° C incubator. The supernatant was removed by centrifugation, washed twice with physiological saline, and the same amount of artificial gastric juice was added thereto. , And the number of surviving colonies of lactic acid bacteria was confirmed (Jin. LZ, 1998). The microorganism is suppressed or killed by the intestinal secreted bile. To have the function of probiotics, they must survive in oxgall-supplemented medium at concentrations higher than intestinal bile. Thus, the yeast must be resistant to proliferation in medium containing 0.3% (Gilliland. SE et al., 1984). The medium was prepared by adding 0.3% Oxoid to the MRS liquid medium for the biliary myogenous invention. Lactic acid bacteria treated for 2 hours in artificial gastric juice were centrifuged to remove artificial gastric juice and the same number of surviving colonies were observed at intervals of 3 hours with the same amount of MRS liquid medium supplemented with oxgal.

BSH  Detection method

The function of BSH catalyzes the separation of bound bile salts. Combined bile salts are more efficient than unbonded molecules in the emulsification and micelle formation of dietary fats. Because of this, BSH produced by lactic acid bacteria breaks down bile salts and makes them unable to absorb into the body. Therefore, the flat plate detection method was modified to confirm the activity of BSH (bile salt hydrolase). Lactic acid bacteria were streaked on MRS agar supplemented with 0.5% (w / v) taurodeoxycholic acid (TDCA, Sigma) and 0.035% (w / v) CaCl ₂ (Yakuri pure chemicals, Japan). An opaque ring around the colony of lactic acid bacteria was identified by incubating in an incubator for 5 days in an incubator (Unaerobic jar) (Dashkevicz, M. 1989).

Bacteriocin  Active measurement

It is an invention for confirming whether or not an effect of inhibiting the growth of a pathogenic microorganism in a substance produced by a lactic acid bacteria isolation strain. First, the isolated lactic acid bacteria cultured in the MRS liquid medium for 24 hours were centrifuged to obtain a supernatant. Since lactic acid produced during cultivation of lactic acid bacteria inhibits the growth of pathogenic microorganisms, it was adjusted to pH 7.0 with 1N NaOH and concentrated 10 times using a vacuum concentrator (Kilic. AO et al, 1996). E. coli O157 ATCC 35150, Staphylococcus aureus ATCC 25923 and Listeria monocytogenes KCCM 40307 were cultivated in a BHI liquid medium for 24 hours at 37 ° C and 200 rpm in a shaking incubator, and then cultured in BHI light agar medium and BHI agar medium And the effect of inhibiting the growth of cultured lactic acid bacteria was confirmed by paper disk method.

API ZYM kit Enzyme activity measurement

Twenty enzymatic activities were measured using API ZYM Kit (Biomerieux). The API ZYM kit is a kit that can identify enzymes produced by specific microorganisms by color change. The isolate of lactic acid bacteria was diluted to about 10 ⁵ CFU / ml with physiological saline, and 65 μl was inoculated into the strip. The inoculated cells were incubated in a 37 ° C incubator for 4 hours, and then ZYM A and ZYM B were added dropwise dropwise. After waiting at least 5 minutes, the color change was confirmed.

By using the microorganisms exploited through the present invention, useful physiologically active substances having immunity enhancement, anticancer effect and increased yield from the searched microorganisms, for example, substances such as Il-1 alpha or TNF-alpha Can be separated.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a schematic diagram for the production of a medium for searching for a desired microorganism according to one embodiment of the present invention. Fig.
Figure 2 is the cell viability for the invented microorganism of one embodiment of the present invention.
Figure 3 is the cell viability for the microorganism searched in one embodiment of the present invention.
Figure 4 is the concentration of TNF alpha produced by the invented microorganism in one embodiment of the present invention.
Figure 5 is the concentration of Il-1 alpha produced by the invented microorganism of one embodiment of the present invention.
Figure 6 is a blast result prepared for the identification of the microorganism searched in one embodiment of the present invention.
Figure 7 shows the results for bacteriocin production of the microorganism in accordance with one embodiment of the present invention. At this time, the left side is E. coli , the middle is L. monocytogenes , Staph . It is aureus .

The present invention will now be described in more detail with reference to the accompanying drawings and embodiments. It should be understood, however, that the following description is for the purpose of illustration only and is not intended to limit the scope of the invention described above.

Example 1. Search for lactic acid bacteria

Lactic acid bacteria isolation

In order to isolate lactic acid bacteria, Kimchi samples were taken and 10 g of the samples were sequentially decanted into physiological saline. The declining sample was plated on MRS agar containing BCP (0.1 g / L) and sodium azide (0.1 g / L) and cultured at 37 ° C for 24 hours to obtain 20 colonies that changed BCP to yellow . The colonies were cultured in MRS liquid medium, mixed with 50% glycerol and culture solution in a ratio of 1: 1, and stored in a freezer at 70 ° C in a low temperature freezer (Ilsin, Korea).

Example 2. Isolation of lactic acid bacteria immunity

Cell viability measurement

MTT detection method was repeatedly invented three times in 20 strains of isolated lactic acid bacteria. When the survival rate of the control group was considered to be 100%, the survival rate of FBT208 and FBT215 was more than 120% and the survival rate was higher than that of the FBT202 and FBT217 control groups, using 264.7 cells as a mouse derived macrophage. 3 weeks of FBT203, FBT211, and FBT220 showed no significant difference from the control group (see FIGS. 2 and 3).

Enzyme immune activity

RAW 264.7 cells, a mouse-derived macrophage, were cultured using seven strains identified after MTT detection. The ability of TNF-α and IL-1α to be confirmed by ELISA kit was compared. The highest level of TNF-α was 1105.3 ± 30.5 pg / ml for FBT 202 and 676.9 ± 22.2 pg / ml for FBT215. Overall, FBT215 was found to have the best cytokine production (see Figures 4 and 5)

.

Example 3 Identification of Lactic Acid Bacteria Isolate

Biochemical Identification of Lactic Acid Bacteria

An API 50 CH kit was used to identify FBT215 confirming immunological activity. FBT215 was streaked on MRS agar to obtain colonies, which were well diluted in CHL medium and incubated in a 37 ° C incubator for 48 hours. The results were as follows: L-arabinose, ribose, galactose, glucose, fructose, mannose, mannitol, sorbitol, N-acetyl-glucosamine, amygdalin, arbutin, esculin, salicin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, , gentiobiose, D-turanose, and gluconate (Table 3). Lactobacillus plantarum was identified as a 99.4% probability.

Carbohydrates FBT215 Carbohydrates FBT215 Control group - Esculin + Glycerol - Salicin + Erythritol - Cellobiose + D-Arabinose - Maltose + L-Arabinose + Lactose + Ribose + Melibiose + D-Xylose - Sucrose + L-Xylose - Trehalose + Adonitol - Inulin - beta -Methyl-D-xylose - Melezitose + Galactose + Raffinose - Glucose + Strach - Fructose + Glycogen - Mannose + Xylitol - Sorbose - Gentiobiose + Rhamnose - D-Turanose + Dulcitol - D-Lyxose - Inositol - D-Tagatose - Mannitol + D-Fucose - Sorbitol + L-Fucose - alpha -Methyl-D-Mannoside - D-arabitol - alpha -Methyl-D-glucoside - L-Arabitol - N-Acetyl-glucosamine + Gluconate + Amygdalin + 2-Keto-gluconate - Arbutin + 5-Ketogluconate -

+, positive; -, negative

FBT215 Genetic identification of

16S ribosomal RNA was analyzed for genetic identification of Lactobacillus plantarum FBT215. First, genomic DNA of FBT 215 was extracted and PCR was performed using PCR primer, 27 forward primer (AGAGTTTGATCMTGGCTCAG) and 1492 reverse primer (TACGGYTACCTTGTTACGACTT). Sequence analysis of 16s rRNA sequence was performed on the consignor (Cosmojin Tech). The homology test of the nucleotide sequence was carried out by the BLAST of the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov) for the information of the GenBank database (see FIG. 6). The probability of identification with Lactobacillus plantarum KCC-3 was 98% (1423/1450).

Example  4. FBT215 caution Probiotic  characteristic

Antibiotic susceptibility

To confirm antibiotic susceptibility, Lactobacillus plantarum FBT215 was inoculated on MRS agar agar, which was then stratified on MRS agar and antibiotic susceptibility was confirmed using an antibiotic paper disk (BBL ™ Sensi-Disc ™, BD, USA). Except of 7 antibiotics (Ampicillin, Streptomycin, Gentamycin, Erythromycin , Tetraoxycycline, Neomycin, Colistin of Colistin had a sensitivity to all antibiotics. There have resistance to Colistin, Colistin, so have a susceptibility to gram-negative bacteria L. plantarum FBT215 was found to be gram-positive.

Acid resistance and My bile

To confirm the acid resistance of FBT215, artificial gastric juice was prepared and its pH was adjusted to 2.5, 3.0, 4.0. FBT215 cultured in artificial gastric juice at each pH condition showed that the number of viable cells decreased with time, but it was found to be resistant to acidity in general. To confirm the biliary properties, the culture medium was treated with artificial gastric juice for 2 hours, and the medium was replaced with MRS broth supplemented with 0.3% of Oxgel. FBT215 was proliferated in medium containing bile for 3 hours, but after that, it was confirmed that the amount of viable cell decreased due to the influence of bile.

BSH  activation

FBT215 can be identified by streaking on an MRS agar supplemented with 0.5% (w / v) taurodeoxycholic acid (TDCA) and 0.035% (w / v) CaCl ₂ . Confirming that an opaque ring was formed around the colony of FBT215, it was confirmed that there was a bile decomposition enzyme.

Bacteriocin  activation

The pH of the isolated lactic acid bacterium was adjusted to neutral by separating the supernatant and concentrated 10 times at 40 ° C. E. coli O157 ATCC35150, Staphylococcus aureus ATCC25923, and Listeria bifidus were tested for the inhibition of growth of pathogenic bacteria using paper disk method. monocytogenes KCCM40307. < / RTI > It was confirmed that L. plantarum FBT215 does not produce a substance capable of inhibiting the growth of pathogenic bacteria (see FIG. 7).

API ZYM  Enzyme activity

Enzyme activity plays an important role in selecting probiotic strains. Lactobacillus plantarum FBT 215 showed seven enzymatic activities including luecin arylamidase, valine arylamidase, acid phosphate, naphtol-AS-BI-phosphohydrolase, β-Galactosidase, α-Glucodiase and β-Glucodiase.

Enzyme FBT215 LP1 Control group - - Alkaline phosphatase - - Esterase - - Esterase lipase - - Lipase - - Leucine arylamidase + + Valine arylamidase + + Crystine arylamidase - - Trypsin - - α-Chymotrypsin - - Acid phosphate + + Naphtol-AS-BI-phosphohydrolase + + α-Galactosidase - - β-Galactosidase + + β-Glucuronidase - - α-Glucodiase + + β-Glucodiase + + N-Acetyl-β-glucosaminidase - - α-Mannosidase - - α-Fucosidase - -

+, positive; -, negative

While the present invention has been described with reference to exemplary embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. You will know. In addition, many modifications may be made to adapt a particular situation and material to the teachings of the invention without departing from the essential scope thereof. Accordingly, it is intended that the invention not be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this invention, but that the invention be construed as including all embodiments falling within the scope of the appended claims.

Korea Microorganism Conservation Center (overseas) KCCM11426P 20130612

Claims (14)

Deposit No. KCCM11426P Lactobacillus plantarum FBT215. The method according to claim 1,
Lactobacillus plantarum FBT215 is a Lactobacillus plantarum FBT215 which promotes IL-1 alpha production. The method according to claim 1,
Lactobacillus plantarum characterized by increased macrophage survival rate. The method according to claim 1,
Lactobacillus plantarum FBT215 promoting the production of TNF-alpha. delete delete A food composition for improving immunity comprising the Lactobacillus plantarum FBT215 of claim 1. 8. The method of claim 7,
Lt; RTI ID = 0.0 > IL-1 < / RTI > alpha or TNF-alpha. delete delete A composition for improving cancer, comprising the Lactobacillus plantarum FBT215 of claim 1. 12. The method of claim 11,
Wherein cancer is ameliorated by increased IL-1 alpha or TNF-alpha. Culturing the Lactobacillus plantarum FBT215 of claim 1 and a macrophage cell line; And
And isolating the IL-1 alpha produced in said culturing step. Culturing the Lactobacillus plantarum FBT215 of claim 1 and a macrophage cell line; And
And isolating the TNF alpha produced in said culturing step.

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