KR20190048732A - Lactobacillus johnsonii IDCC9203 having effect of preventing and treating inflammation, and uses thereof - Google Patents

Abstract

The present invention relates to a lactobacillus johnsonii IDCC9203 strain which highly expresses acid phosphatase, and has an effect of preventing and treating inflammation, and to a culture thereof, dead bacteria thereof, and a use thereof. The new strain Lb. johnsonii IDCC9203 of the present invention shows high bioavailability due to high activity of acid phosphatase, has multiple excellent functionalities, such as a high activity level of β-galactosidase and excellent anti-inflammatory efficacy, and is highly useful in industrial use since the new strain is a safe strain without cytotoxicity which does not generate biogenic amine, glucuronidase, etc.

Description

Lactobacillus Johnson IDECCIA 9203 < / RTI > and its use (Lactobacillus johnsonii IDCC9203 having an effect of preventing and treating inflammation, and uses thereof)

The present invention relates to Lactobacillus Johnson Iridis strain 9203, a culture thereof, a bacterium thereof and a use thereof, which are characterized by high expression of an acid phosphatase and an effect of preventing and treating inflammation.

Lactic acid bacterial preparations are being actively developed for the purpose of providing a beneficial effect on the health function of animals including humans. The probiotic bacteria must meet all of the several requirements related to toxicity deficiency, viability, adhesion and beneficial effects. These probiotic features are strain-dependent, even in the same species of bacteria. Therefore, it is important to find such strains with excellent performance for all probiotic requirements.

Inflammation is mediated by intracellular signaling proteins (cytokines) produced by macrophages and dendritic cells in the epithelium in response to antigenic stimulants. Immediately after contact with antigens such as endotoxins, such as LPS, antigen-presenting cells (including dendritic cells) in epithelium transmit these signals to their own macrophages, which in turn induce Th-1 type responses , Where a pro-inflammatory cytokine, including TNF- [alpha], IL-1 [beta], IL-6 and IL-12, is produced by macrophages. These cytokines then stimulate native killer cells, T-cells and other cells to produce interferon gamma (IFN gamma), a key mediator of inflammation. IFN gamma enhances the above-described responses that cause inflammatory reactions and toxicity. In particular, inflammation in the intestine is affected by lactose intolerance. Lactose intolerance may cause enteritis due to chronic diarrhea as well as acute diarrhea, and chronic enteritis may cause colon cancer (Chung, C.-W. 1999. Potential correlation between lactose intolerance and cancer occurrence, J. Korean Ass. Cancer Prevention, 4: 52-60).

Although various lactic acid bacteria have been discovered due to the needs of lactic acid bacterial preparations for treating and ameliorating such inflammatory phenomena, they have provided virtually only one usefulness, and they have not only provided toxicity deficiency, viability, And limitations in which beneficial effects can not be combined.

Phytate, on the other hand, is a substance found in food seeds such as rice, wheat, corn and legumes, and is a typical substance inhibiting the absorption of calcium in food. Phytate accounts for approximately 1 to 6.34% of the food normally consumed and is known to consume large quantities of phytate in countries with grain stocks. It is mainly hydrolyzed to phytic acid by an enzyme called phytase, but the enzyme is not present in animals such as human, chicken, pig, and mouse, and phytate can not be digested / absorbed. Phytate strongly binds to metal ions such as calcium, zinc, magnesium and iron present in foods to inhibit the absorption of minerals in the body (Patent Document 1; Korean Patent No. 10-1475833). According to Korean Patent No. 10-1475833, ingestion of phytate is known to cause autoimmune disease, and it has been reported that decomposition of phytate can reduce such toxicity.

In contrast, US 5834286 (Patent Document 2) discloses that phytate-degrading activity of Acid phosphatase (EC 3.1.3.2.) Has phytate-degrading activity, and discloses the synergy effect of phytate decomposition by the combination of Phytase and Acid phosphatase . It has also been reported that phytate-degrading activity of lactic acid bacteria is caused by non-specific acid phosphatase rather than phytase activity (Non-Patent Document 1). In addition, Acid phosphatase degrades the casein phosphopeptide in milk and promotes calcium absorption. Although there is a need to utilize the acid phosphatase activity of lactic acid bacteria due to the availability of the enzyme to humans and animals, it has been known that the expression level and the activity variation of the enzyme are large depending on the specific lactic acid bacteria strain.

Biological amine (BA) is a low molecular substance produced by the decarboxylation of free amino acid by microorganisms present during the storage and fermentation of food. It is known as putrescine (PUT), cadaverine (CAD), tryptamine (PHE), spermidine (SPD), spermine (SPM), histamine (HIS), tyramine (TYR), agmatine (AGM) and serotonin (SER). Ingestion of food containing excessive amounts of bio-amines or the failure to metabolize normal bio-amines due to physical anomalies can lead to fatal toxicity. Representative symptoms that occur after oral ingestion of the above-mentioned excessive amounts of bio-amines through food include nausea, respiratory distress, hot flush, sweating heart palpitation, headache ), Rash and bright red rash oral burning, hypertension and hypotension. Biological amines are also known as precursors of carcinogenic nitroso compounds, of which tyramine (tyramine) is known as a precursor of major mutants. According to KLAPACOVA et al., (2015) . The johnsonii KB2-1 strain is known to produce cadaverine.

Thus, there is still a need for the development of new lactic acid bacteria because of the need for better multi-functionality and limitations of existing strains.

Korean Patent No. 10-1475833 US 5834286

 M. Zamudio et al., Lactobacillus plantarum phytase activity is due to non-specific acid phosphatase, Letters in Applied Microbiology 2001, 32, 181-184.  Miroslav Kolosta et al., CHARACTERISATION OF LACTOBACILLI FROM EWE'S AND GOAT'S MILK FOR THEIR FURTHER PROCESSING RE-UTILIZATION, Potravinarstvo, vol. 8, 2014, no. 1, p. 130-134.  Dorota Zielinska et al., In Vitro Screening of Selected Probiotic Properties of Lactobacillus Strains Isolated from Traditional Fermented Cabbage and Cucumber, Curr Microbiol. 2015 Feb; 70 (2): 183-94.  KLAPACOVA et al. Antimicrobially active lactobacilli from goats' milk does not produce biogenic amines, Journal of Food & Nutrition Research. 2015, Vol. 54 Issue 3, p270-274. 5p.

Therefore, the inventors of the present invention have been studying microbial materials exhibiting multifunctional properties that are safe and useful when applied to a living body, and have found that Lb. acidophosphatase exhibiting high activity of acid phosphatase. The strain was firstly identified and obtained the novel strain of johnsonii IDCC9203. The strain has high activity of beta-galactosidase activity as well as human usefulness due to high activity of acid phosphatase, exhibits excellent anti-inflammatory activity, Which is a non-cytotoxic, safe strain, and completed the present invention.

Accordingly, an object of the present invention is to provide a strain or a bacterium of Lactobacillus johnsonii IDCC9203 (strain accession number: KCTC 10923BP) which is high in acid phosphatase activity.

Another object of the present invention is to provide a pharmaceutical composition for antiinflammation comprising the strain Lactobacillus johnsonii IDCC9203 (strain accession number: KCTC 10923BP), a culture thereof or a bacterium thereof as an active ingredient .

In order to achieve the above object, the present invention provides a strain of Lactobacillus johnsonii IDCC9203 (strain accession number: KCTC 10923BP) or a bacterium thereof having high acidity phosphatase activity.

In order to accomplish another object of the present invention, the present invention provides an anti-inflammatory pharmacological agent comprising Lactobacillus johnsonii IDCC9203 (strain accession number: KCTC 10923BP), a culture thereof or a bacterium thereof as an active ingredient Gt;

Hereinafter, the present invention will be described in detail.

Lactobacillus johnsonii strains highly expressing acid phosphatase (EC 3.1.3.2), as opposed to known strains known to correspond to Lactobacillus johnsonii species, have been disclosed for the first time in the present invention .

The present inventors firstly isolated the acid phosphatase high expression Lactobacillus johnsonii IDCC9203 (strain accession number: KCTC 10923BP) and confirmed its usefulness. The Lactobacillus Johnson IDCC9203 of the present invention is wild type, which is distinguished from a transformant produced by a recombinant gene technology, and since it has a considerable difficulty in identifying and isolating a useful bacterium in the natural world, . The Lactobacillus Johnson IDCC9203 strain of the present invention is characterized by high expression of an acid phosphatase to show a high level of bioavailability due to acid phosphatase. For example, It not only degrades the harmful phytate but also promotes the absorption of calcium.

In addition, the novel strain Lactobacillus Johnson IDCC9203 of the present invention has a high level of beta-galactosidase activity and thus has a high lactose degrading ability and thus has an excellent effect of improving lactose intolerance and exhibits excellent anti-inflammatory activity, Feature. In addition, the Lactobacillus Johnson IDCC9203 strain of the present invention is excellent in intestinal adhesiveness, excellent in acid resistance and bile resistance, and exhibits remarkable performance as a probiotic.

Accordingly, the present invention provides a strain expressing Lactobacillus johnsonii IDCC9203 (strain accession number: KCTC 10923BP) which expresses acid phosphatase (EC 3.1.3.2).

The bacterium of the above-mentioned 'Lactobacillus Johnson IDCC9203' includes not only the live bacteria itself obtained from the culture medium but also any form of processing (in particular, processed forms of live bacteria) to the lactic acid bacteria known to those skilled in the art. And the like.

The present invention also provides a bacterium of Lactobacillus Johnson IDCC9203 strain or a culture thereof.

The term " cultivation " in the present invention means all the activities performed in order to grow the microorganism under an appropriately artificially controlled environmental condition. In the present invention, the term " cultured product " , And includes a workpiece derived from the culture medium itself such as the culture medium itself cultured in the medium (i.e., the culture medium itself containing the bacteria), or a workpiece subjected to heat treatment in the culture medium. Preferably, the culture of the present invention may be a culture solution containing Lactobacillus johnsonii IDCC9203, or a workpiece subjected to heat treatment in the culture solution.

 The term " dead cells " in the present invention refers to cells which have been sterilized by heating, pressurization, drug treatment or the like. The production method of the dead cells is not particularly limited as long as it is by the method of lactic acid bacterial strain known in the art. For example, the dead cells of the present invention may be produced by the quenching method (by heat treatment) including heat treatment. The heat treatment may be performed only on the live cells isolated from the culture broth, or may be performed on the broth containing the live cells. The heat treatment temperature is not particularly limited as long as it maintains the shape of the cells and sterilizes other common bacteria. However, the heat treatment temperature may be 80 to 150 ° C, preferably 80 to 110 ° C .

The time for the heat treatment is not particularly limited as long as it is a condition that a person skilled in the art can obtain a desired production quality in consideration of the above temperature condition, but it can preferably proceed for 5 to 15 minutes. When heated for less than 5 minutes, the bacteria may not die completely, and when heated for a long time in excess of 15 minutes, the denaturation of the cell component may occur and the desired effect may not be exhibited.

During the heat treatment process for producing the dead cells, a person skilled in the art can add any pretreatment or additional conditions for the production efficiency at a line which does not affect the intrinsic functionality of the dead bacteria of the present invention. For example, distilled water can be added to and mixed with the pre-heat-treated cells. When the amount of distilled water is less than or less than the weight of the cells, the color of the raw material becomes darker due to the heat affected by heating, whereas when the amount of distilled water is 1 to 2 times the weight of the cell mass, It becomes sensually excellent.

The sanitized bacteria may be subjected to any additional process for the production of the mycological preparation (formulation), for example, concentration, drying, and the like. The drying method is not particularly limited as long as it is used in the art to dry the lactic acid bacteria. For example, the drying method may be a wind drying method or a freeze drying method.

The live bacteria and dead bacteria of the novel strain Lactobacillus zone Sony IDCC9203 of the present invention all have a remarkable anti-inflammatory effect. This is well illustrated in the specification of the present invention. The live bacteria or dead cells of the novel strain of the present invention are remarkably effective in inhibiting the inflammatory cytokines TNF- ?, IL-1? And IL-6. In addition, the Lactobacillus johnsonii strain IDCC9203 (strain accession number: KCTC 10923BP) of the present invention is free from cytotoxicity, including not producing biomolecules, glucuronidase, etc., (Cosmetics), or pharmaceuticals. Accordingly, the present invention provides a pharmaceutical composition for antiinflammation comprising as an active ingredient a strain of Lactobacillus johnsonii IDCC9203 (strain accession number: KCTC 10923BP), a culture thereof or a bacterium thereof.

The pharmaceutical composition of the present invention is not particularly limited as long as it is an inflammatory disease and can be applied for prevention and / or treatment thereof, but is preferably applicable to inflammatory bowel disease or inflammatory skin diseases. The inflammatory bowel disease may be selected from the group consisting of Crohn's disease, ulcerative colitis, intestinal Behcet's disease, and ischemic enteritis, but the present invention is not limited thereto. The inflammatory skin disease may be selected from the group consisting of acne, hypersensitive dermatitis and the like, but is not limited thereto.

The pharmaceutical composition of the present invention may further contain Lactobacillus Johnson IDCC9203, a culture thereof or a bacterium thereof alone or in combination with one or more pharmaceutically acceptable carriers, excipients or diluents. The term " pharmaceutically acceptable " as used herein means a non-toxic composition which is physiologically acceptable and which, when administered to humans, does not inhibit the action of the active ingredient and does not normally cause an allergic reaction such as gastrointestinal disorder, dizziness, .

The pharmaceutically acceptable carrier may further include, for example, a carrier for oral administration or a carrier for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. In addition, the carrier for parenteral administration may contain water, a suitable oil, a saline solution, an aqueous glucose and a glycol, and may further contain a stabilizer and a preservative. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. Other pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, Pa., 1995).

The pharmaceutical composition of the present invention can be administered to mammals including humans by any method. For example, it can be administered orally or parenterally (for example, a coating method). The pharmaceutical composition of the present invention may be formulated into oral or parenteral dosage forms according to the route of administration as described above. In the case of pharmaceutical preparations for oral administration, the composition of the present invention may be formulated into granules, tablets, pills, tablets, capsules, solutions, gels, syrups, slurries, suspensions, wafers and the like using methods known in the art . For example, an oral preparation can be obtained by combining the active ingredient with a solid excipient, then milling it, adding suitable auxiliaries, and then processing the mixture into a granular mixture. Examples of suitable excipients include, but are not limited to, sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol, and starches including corn starch, wheat starch, rice starch and potato starch, Cellulose such as methylcellulose, sodium carboxymethylcellulose and hydroxypropylmethyl-cellulose and the like, fillers such as gelatin, polyvinylpyrrolidone and the like. In addition, crosslinked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may optionally be added as a disintegrant. Further, the pharmaceutical composition of the present invention may further comprise an anti-coagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and an antiseptic agent.

In the case of a preparation for parenteral administration, it may be formulated by a method known in the art in the form of injection, cream, lotion, external ointment, oil, gel, and nasal aspirate. These formulations are described in Remington's Pharmaceutical Science, 15th edition, 1975. Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour, a commonly known formulary for all pharmaceutical chemistries.

The total effective amount of the Lactobacillus Johnson IDCC 9203 or its bacterium of the present invention may be administered to a patient in a single dose and may be administered in a fractionated treatment protocol administered over a prolonged period of time in multiple doses, ≪ / RTI > In the pharmaceutical composition of the present invention, the content of the active ingredient may be varied depending on the degree of the disease. Preferably, the total total volume of Lactobacillus Johnson IDCC9203 or bacterium thereof may be from about 0.01 mg to about 1,000 mg, and most preferably from 0.1 mg to 100 mg, per kg body weight of the patient per day on a solids content basis. More preferably, the daily intake amount may be from 1 x 10 ⁵ to 1 x 10 ¹⁰ CFU / kg, preferably from 1 x 10 ⁶ to 1 x 10 ⁹ CFU / kg, based on the number of cells, It may be ingested or divided into several doses, but is not limited thereto. However, the dose of the lactobacillus Johnson IDCC9203 or its bacterium of the present invention may be varied depending on various factors such as the route of administration and the number of treatments of the pharmaceutical composition as well as the age, weight, health, sex, severity of disease, The present inventors have found that the strain of the present invention and the bacterium cells of the present invention can be effectively used as a preventive, ameliorating or therapeutic agent for inflammation according to a specific use A suitable effective dose will be determined. The pharmaceutical composition according to the present invention is not particularly limited to its formulation, administration route and administration method as long as the effect of the present invention is exhibited.

The new strain Lb. johnsonii IDCC9203 exhibits a high level of bioavailability due to high activity of acid phosphatase as well as a high level of beta-galactosidase activity and excellent anti-inflammatory activity. . In addition, the novel strain of the present invention is a safe, non-cytotoxic strain that does not produce bioamines, glucuronidase, and the like.

Fig. 1 shows the ability of 24 strains isolated from infant feces to inhibit NO production (% strain: Lb. johnsonii IDCC9203).
2 is Lb. johnsonii represents the hydrophobic properties of intestinal adhesion related IDCC9203.
Fig. 3 Lb. (Fig. 3a: TNF-α, Fig. 3b: IL-6, Fig. 3c: IL-1β) by inhibition of inflammatory cytokine production by treatment concentration of johnsonii IDCC9203.

Hereinafter, the present invention will be described in detail.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

≪ Example 1 >

New strain Lactobacillus johnsonii  Isolation and identification of IDCC9203

<1-1> Selection of strain

Twenty - four kinds of lactic acid bacteria were isolated from infant feces and their NO production inhibitory activities were examined. Each lactic acid bacterium was centrifuged after completion of the culture to obtain cells, which were treated by heat-killed and quenched at 100 ° C for 15 minutes. RAW 264.7 cells (1 × 10 ⁵ / mL) were first cultured in DMEM and stimulated with LPS (2 μl / ml) and the heat-killed (1 × 10 ⁵ / ml) subject lactic acid bacteria for 24 hours. NO concentration was determined by measuring the amount of nitrite in the cell culture supernatant using the Griess reagent (Sigma, USA) according to the manufacturer's instructions. The cultured RAW264.7 cells were mixed with 150 μL of the cell culture supernatant and 150 μL Griess reagent, centrifuged at 1,000 × g for 10 minutes, and then incubated at room temperature for 10 minutes. Absorbance was measured at 540 nm using a microplate reader and compared based on the calibration curve formed with sodium nitrite. The NO inhibitory activity of the strain was expressed as a relative ratio (%) to the NO concentration of the positive control. Positive controls in RAW264.7 only treated LPS.

As a result of the measurement, it was confirmed that the three isolates showed the highest NO production inhibiting ability. The strain No. 3 was finally selected and identified.

<1-2> Identification of strain

The taxonomic species were identified by analyzing the 16S rRNA gene sequence of the final selection strain (Solgent, Korea). As a result, Lactobacillus johnsonii (99.1%) was obtained, and Lb. It was named johnsonii IDCC9203. The novel microorganism of the present invention identified above was deposited as " Lactobacillus johnsonii IDCC9203" on Mar. 15, 2006 in the Korean Collection for Type Cultures at 52, Eun-dong, Yuseong-gu, Daejeon, Republic of Korea KCTC 10923BP).

&Lt; Example 2 >

New strain Lactobacillus johnsonii  Comparative evaluation of enzyme activity of IDCC9203

The enzyme activity of the isolated strain was assayed using API ZYM kit (BioMérieux), and the known Lb. johnsonii KCTC3801 ^T, Lb. johnsonii KB2-1, Lb. and johnsonii K4 were compared with each other. Lb. The enzymatic activity of the johnsonii KB2-1 strain is described by Miroslav Kolosta et al. (2014) ', and Lb. The enzyme activity of the johnsonii K4 strain was referred to 'Dorota Zielinska et al., (2015)'.

As shown in Table 1, the new strain Lb. johnsonii IDCC9203 exhibited significantly higher activity of Acid phosphatase, an enzyme essential for the hydrolysis of phosphopeptides, than known same strain strains. Acid phosphatase decomposes casein phosphopeptide in milk to promote calcium absorption. On the other hand, the novel strain of the present invention can be safely used as a probiotics because it lacks the β-glucuronidase activity, which is a harmful enzyme that liberates benzopyrene, which is a carcinogen, by decomposing the benzopyrene and glucuronic acid complexes. In addition, the new strain of the present invention is highly active in the activity of β-galactosidase, which is a lactose-degrading enzyme, and may be useful for improving lactose intolerance

&Lt; Example 3 >

New strain Lactobacillus johnsonii  Evaluation of intestinal adhesiveness of IDCC9203

If the cell surface hydrophobicity of the lactic acid bacteria is strong, the adhesion to the intestines can be done well because it can strengthen hydrophobic bonds with the membranes. The Lb. To determine the cell surface hydrophobicity of johnsonii IDCC9203, the degree of attachment to hydrocarbon was measured. N-hexadecane and xylen were used as hydrocarbons for the determination of hydrophobicity. The absorbance (A ₀ ) at 660 nm was measured using a phosphoric acid buffer solution and the absorbance (A ₀ ) at 660 nm. The suspension was mixed with n-hexadecane and xylen ) Were added in the same amount, and the mixture was thoroughly mixed with a vortex mixer. After allowing the two layers to separate, the absorbance (A1) at 660 nm of the suspension excluding the organic solvent was measured and the degree of attachment of the lactic acid bacteria to the solvent was calculated by the following formula:

Surface hydrophobicity (%) = 100 x (A0 - A1) / A0.

As a result of the measurement, the new strain Lb. The johnsonii IDCC9203 showed more than 80% hydrophobicity, which was higher than that of the same strain of the same species ( Lb. johnsonii KCTC3801 ^T ) (see FIG. 2). As a result, it was confirmed that the novel strain of the present invention was excellent in the ability to bind to leaves.

<Example 4>

New strain Lactobacillus johnsonii  Evaluation of antibiotic resistance of IDCC9203

For the antibiotics of Table 2, MRS solid medium was prepared by adding each antibiotic at a concentration of 0.25 ~ 512 / / ml in a 2-fold dilution method. The microorganism cultured for 18 hours was inoculated into a solid medium containing 10 ⁵ / spot of antibiotics and cultured at 37 ° C for 24 to 48 hours to measure the minimum inhibitory concentration (MIC). Based on the EFSA breakpoint, the isolates were tested for resistance to each antibiotic and the results are shown in Table 2 below.

Antibiotic Concentration Unit: μg / mL AMP VAN GEN KAN ST ERM CM TET Lb. johnsonii IDCC9203 of MIC 0.5 > 128 8 32 32 0.5 0.25 One EPSA breakpoint 2 - 32 64 - One One 4 AMP: ampicillin; VAN; Vancomycin; GEN: Gentamicin; KAN: kanamycin; ST: streptomycin; ERM: erythromycin; CM: chloramphenicol; TET: tetracycline; (-): Not required

As shown in Table 2 above, the new strain of the present invention was found to be below the antibiotic resistance standard proposed by EFSA for all the antibiotics used in the experiment. Therefore, Lb. johnsonii IDCC9203 has been identified as a safe strain with no concern for antibiotic resistance.

&Lt; Example 5 >

New strain Lactobacillus johnsonii  Safety evaluation of IDCC9203 (evaluation of non-bioamine formation)

If you eat foods containing excess biogenic amine (BA) or if your body does not metabolize normal bio-amines, it can cause fatal toxicity. Biological amines are produced through the decarboxylation of free amino acids by microorganisms present during the storage and fermentation of food. According to KLAPACOVA et al., (2015) ', the previously known Lb. The johnsonii KB2-1 strain is known to produce cadaverine.

Thus, it was tested whether the new strain of the present invention produced a biogenic amine, specifically, the following method. The amino acid precursors (L-tyrosine disodium salt, L-histidine monohydrochloride monohydrate, L-ornithine monohydrochloride, L-lysine monohydrochloride, monohydrochloride)) was added a 0.1% MRS Lb. create a separate liquid culture medium johnsonii IDCC9203 was inoculated 1% in the MRS liquid medium containing the amino acid precursor, and then passaged 5 to 10 times to activate the decarboxylase. (0.5% of tryptone, 0.5% of yeast extract, 0.5% of cocoon extract, 0.5% of sodium chloride, 0.25% of glucose, 0.05% of tween-80, 0.02% of magnesium sulfate, 0.005% of manganese sulfate, 0.004% 0.2% salt, 0.001% thiamine, KH ₂ PO ₄ 0.2%, calcium carbonate 0.01%, pyridoxal-5-phosphate 0.005%, amino acid 1%, bromocresol purple 0.006% and agar 2% 5.3). The cells were cultured at 37 ° C for 24 to 48 hours to determine the color change to purple, and the ability to produce biogenic amines was determined. The bromocresol purple in the decarbonated enzyme medium is yellow at pH 5.2 but turns purple as the pH rises to 6.8. Therefore, the production of bio-amines can be confirmed by using a substance that turns purple when the pH rises due to the formation of bio-amines.

Tyramine Histamine Futuresyn Cadaverine Lb. johnsonii IDCC9203 - - - -

As shown in Table 3, the new strain Lb. Analysis of the biosurfactant-producing ability of johnsonii IDCC9203 revealed that all strains of tyramine, histamine, putrescine, and cadaverine were negative. Thus, the strain of the present invention was found to be safe because it lacks the ability to produce biogenic amines capable of inducing an irritable immune response .

&Lt; Example 6 >

New strain Lactobacillus johnsonii  Evaluation of anti-inflammatory efficacy of IDCC9203

Lb. johnsonii IDCC9203 was cultured in MRS broth for 2 hours at 37 ° C and then collected by 10,000 × g centrifugation for 15 minutes. After washing the cells twice with 1X PBS buffer, the samples were heated at 100 ° C for 10 minutes and then used for anti-inflammatory effect analysis.

Mouse macrophage RAW 264.7 cells were treated with phenol red-free Dulbecco's modified 10% fetal bovine serum (FBS, Atlas, Fort Collins, CO, USA) and 1% penicillin (Sigma-Aldrich, St. Louis, The cells were cultured in a medium of Eagle medium (DMEM, Gibco-BRL, Gaithersburg, MD, USA) at 37 ° C and 5% CO ₂ .

Lb. Raw 264.7 cells cultured in order to determine the effect of the johnsonii IDCC9203 strain on inflammatory cytokine production were collected using a scrapper or trypsinize, and then placed in a 15 ml falcon tube and centrifuged at 500 rpm for 5 minutes. After removal of the supernatant, 1 ml of complete media was added and resuspended to count the number of cells per ml. 2 ml of diluted cells in each well of a 6-well plate was added and incubated overnight. The medium of each well of the 6-well plate was washed and then washed with PBS. After 2 ml of fresh medium was dispensed, each well was treated with 4 ul of LPS (final LPS concentration 2 μl / ml), and 1 mM dexamethasone was added as a positive control (final concentration: 20 μl 10 uM / ml). The negative control was treated with PBS, the remaining wells were treated with LPS, and Lb. johnsonii IDCC9203 1X10 ⁵ ~ 1X10 ⁷ cells / mL were treated at each concentration and then cultured overnight for 24 hours. The culture broth of each well was transferred to a 15 ml falcon tube and centrifuged at 9,500 rpm for 5 minutes. The supernatant of each tube was collected separately and used for cytokine experiments. The inhibition of the inflammatory cytokines TNF-α, IL-6 and IL-1β was confirmed by ELISA kit (R & D system).

As shown in Fig. 3, Lb. The effect of johnsonii IDCC9203 on TNF-α, IL-6 and IL-1β, which are inflammatory cytokines, was remarkable. Thus, it can be confirmed that the strain of the present invention has a remarkable effect on relieving inflammation.

As described above, the present invention relates to a Lactobacillus Johnson Iridis strain 9203, a culture thereof, a bacterium thereof and a method for the same, which are characterized by high expression of an acid phosphatase and an effect of preventing and treating inflammation Lt; / RTI &gt;

The new strain Lb. johnsonii IDCC9203 exhibits a high level of bioavailability due to high activity of acid phosphatase as well as a high level of beta-galactosidase activity and excellent anti-inflammatory activity. . In addition, since the novel strain of the present invention is a safe strain without cytotoxicity that does not produce bioamines, glucuronidase and the like, it is highly industrially applicable.

Korea Biotechnology Research Institute KCTC10923BP 20060315

Claims (2)

A pharmaceutical composition for antiinflammatory activity comprising a strain of Lactobacillus johnsonii IDCC9203 (strain accession number: KCTC 10923BP), a culture thereof or a bacterium thereof as an active ingredient.
A pharmaceutical composition for preventing or treating an inflammatory disease comprising Lactobacillus johnsonii IDCC 9203 (strain accession number: KCTC 10923BP) strain, a culture thereof or a bacterium thereof as an active ingredient.

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