KR20130049554A - Composition for preventing or treating atopic dermatitis - Google Patents

Abstract

PURPOSE: A composition for preventing or treating atopic dermatitis, containing four kinds of probiotics or cultures thereof is provided to treat atopic dermatitis. CONSTITUTION: Lactobacillus casei CBT(1) is deposited to a KCTC(Korean Collection for Type Culture) in the number of KCTC 11863BP. Lactobacillus platarum LP(1) is deposited to a KCTC in the number of KCTC 11867BP. Bifidobacterium animalis subsp. lactis CBT2501H is deposited to a KCTC in the number of KCTC 11903BP. A composition for preventing or treating atopic dermatitis contains the strains or the cultures thereof.

Description

Composition for preventing or treating atopy {COMPOSITION FOR PREVENTING OR TREATING ATOPIC DERMATITIS}

The present invention relates to novel lactic acid bacteria and compositions comprising the same, which are useful for the prevention or treatment of atopy, and more particularly, to food and pharmaceutical compositions useful for the prevention and treatment of atopy, including the novel lactic acid bacteria strains.

Atopic Dermatitis ("AD") is a chronic, recurrent inflammatory skin disease that usually begins in infancy or childhood, accompanied by pruritus, dry skin, and characteristic eczema. Atopic dermatitis is on the rise globally, and the prevalence is reported to be 20% of the population. Moreover, atopic dermatitis is not only a skin disease but also a signal of allergic march such as allergic asthma and rhinitis. However, the exact pathophysiology of atopic dermatitis has not yet been identified, and at present atopic dermatitis is a multifactorial disease associated with genetic susceptibility, a product of any lifestyle such as barrier function of damaged skin or internal organs, or the environment and eating habits. It is only known as an immune response to the increased antigen. Because treating atopic dermatitis is not easy, it is necessary to manage the alterable risk factors of atopic dermatitis and to adjust the immune response to atopic dermatitis.

In addition, immunological disorders have been reported in atopic patients. Skin lesions associated with atopic dermatitis patients show overproduction of IL-4, IL-5, IL-9, IL-10 and IL-13 by Th2-cell-mediated immune response . Moreover, atopic dermatitis is associated with increased serum immunoglobulin (IgE) concentrations and reduced production of interleukin-12 (IL-12) and interferon gamma (IFN-γ) produced by Th1 immune responses.

As a therapeutic agent for atopic dermatitis, antagonists of chemical transfer agents represented by antihistamines and steroids used as anti-inflammatory agents are mainly used. These drugs are all used symptomatically, and do not directly reduce the amount of serum immunoglobulin IgE, a major factor in onset. In addition, antihistamines cause side effects such as drowsiness and dry mouth, and steroid agents may cause side effects such as suppressing the entire immune system. TH ₂ Drugs that can alleviate the situation in which cells are significantly superior seem to allow for the fundamental treatment of allergies, but these drugs have not yet been developed.

Under this background, lactic acid bacteria having a function of inhibiting the production of IgE are attracting attention as useful supplements for atopic dermatitis. The treatment of atopic dermatitis by lactic acid bacteria is specific to the strain, and the atopic improvement effect of products currently used as food or drugs is not sufficient, so it is required to develop a composition useful for the treatment of atopic including probiotics having excellent effects. It is becoming.

The present invention is to meet the technical needs of the technical field to which the present invention belongs, one object of the present invention is to provide novel lactic acid bacteria strains that provide useful efficacy in the prevention and treatment of atopy.

Another object of the present invention is to provide a food and pharmaceutical composition effective for the prevention and treatment of atopy, including various strains of lactic acid bacteria.

One aspect of the present invention for achieving the above object is the Lactobacillus casei CBT (1) ( Lactobacillus internationally deposited in the Korean Collection for Type Culture (KCTC) Accession No. KCTC 11863BP casei CBT (1)) relates to lactic acid bacteria.

Another aspect of the present invention for achieving the above object is Lactobacillus plantarum LP (1) ( Lactobacillus internationally deposited as the accession number KCTC 11867BP to the Korea Collection for Type Culture (KCTC) platarum LP (1)) relates to lactic acid bacteria.

Another aspect of the present invention for achieving the above object is Bifidobacterium animalis lactis CBT2501H ( Bifidobacterium) deposited internationally as Accession No. KCTC 11903BP to the Korea Collection for Type Culture (KCTC). animalis subsp . lactis CBT2501H (11903BP)) relates to lactic acid bacteria.

Another aspect of the present invention for achieving the above object is Lactobacillus casei CBT (1) ( Lactobacillus casei CBT (1)) (KCTC 11863BP), Lactobacillus plantarum LP (1) ( Lactobacillus plantarum LP (1)) (KCTC11867BP)) , Lactobacillus rhamnosus LR3 ( Lactobacillus rhamnosus LR3) (KCTC 11868BP) , Bifitobacterium animalis lactis CBT2501H ( Bifidobacterium animalis subsp . lactis It relates to a composition for preventing or treating atopy comprising CBT2501H (KCTC 11903BP) or a culture thereof.

The composition for preventing or treating atopy, including the complex lactic acid bacteria of the present invention, may further include other lactic acid bacteria or a pharmaceutically acceptable carrier or excipient.

Since the composition of the present invention comprises four probiotic lactic acid bacteria or a culture thereof to alleviate the situation in which Th2 cells are dominant, the present invention can provide a composition useful for the treatment of atopy.

In addition, the composition for preventing or treating atopic dermatitis of the present invention directly reduces the amount of serum immunoglobulin IgE, which is a major factor in the onset of atopic dermatitis, and is useful as a composition for preventing or treating atopic dermatitis as a medicine, food, or dietary supplement. Can be.

Figure 1 compares the 16S rRNA sequences of Lactobacillus casei CBT (1) strain (KCTC 11863BP) and Lactobacillus casei ATCC 334.
Figure 2 shows the results of random amplified polymorphic DNA (RAPD) analysis of genome DNA of Lactobacillus casei CBT (1) strain and Lactobacillus casei ATCC 393.
FIG. 3 compares 16S rRNA sequences of Lactobacillus plantarum LP (1) strain (KCTC 11867BP) and Lactobacillus plantarum ^T (ATCC 14917) strains.
Figure 4 shows the results of RAPD analysis of genome DNA of Lactobacillus plantarum LP (1) strain and Lactobacillus plantarum ^T (ATCC 14917) strain.
5 is a graph showing the results of evaluating acid resistance and bile resistance of the Lactobacillus plantarum LP (1) strain.
Figure 6 compares the 16S rRNA sequences of Bifidobacterium animalis lactis CBT2501H strain and Bifidobacterium animalis lactis YIT 4121 strain (ATCC25527).
Figure 7 shows the results of RAPD analysis of genome DNA of Bifidobacterium animalis lactis CBT2501H strain and Bifidobacterium animalis ^T (ATCC 25527) strain.
8 is a graph showing the results of evaluation of acid resistance and heat resistance of Bifidobacterium animalis lactis CBT2501H strain.
9A is a graph showing the effect of the administration of the composition of the present invention on IgE concentration in vivo, and FIG. 9B is a graph showing the effect of the administration of the composition of the present invention on IL-4 concentration in vivo.
10 is a graph showing the effect of the administration of the lactic acid bacteria composition of the present invention on allergic symptoms of atopic dermatitis model animals (NC / Nga mouse).
Figure 11 is a photograph showing the change in the state of skin lesions after administration of the present invention for 6 weeks in the NC / Nga mouse model induced atopic dermatitis.
12 is a graph showing the effect of the administration of the lactic acid bacteria composition of the present invention on the IgE concentration and the production amount of Th2-associated cytokine IL-4 in the experimental groups.
FIG. 13 is a graph showing the administration effect of various lactic acid bacteria strains on the serum concentration of Th1-related cytokines in NC / Nga mice.
14 is a histopathological analysis showing the effect of repeated administration of various lactic acid bacteria preparations on atopic symptoms in atopic dermatitis model animals.

As used herein, the terms "treat" and "treatment" refer to the prevention, reduction, alleviation or elimination of symptoms of atopic dermatitis.

One aspect of the present invention relates to a novel lactic acid bacteria strain, Lactobacillus casei CBT (1) ( Lactobacillus internationally deposited with the Korean Collection for Type Culture (KCTC) Accession No. KCTC 11863BP casei CBT (1)) Lactobacillus, Lactobacillus Lactobacillus Lactobacillus Lactobacillus Internationally Deposited by Korea Biotechnology Research Institute (KCTC) 11867BP platarum LP (1)) Bifidobacterium animalis lactis CBT2501H ( Bifidobacterium) , internationally deposited as Lactobacillus and the Korea Institute of Biotechnology and Gene Access Bank No. KCTC 11903BP. animalis subsp . lactis CBT2501H)) lactic acid bacteria.

Another aspect of the present invention relates to a composition for preventing or treating atopy, the composition includes the following lactic acid bacteria species, these lactic acid bacteria strains have been deposited to the Korea Biotechnology Research Institute gene bank as follows.

1) Lactobacillus casei CBT (1) ( Lactobacillus casei CBT (1)) (KCTC 11863BP),

2) Lactobacillus plantarum LP (1) ( Lactobacillus plantarum LP (1)) (KCTC 11867BP) ,

3) Lactobacillus Lactobacillus rhamnosus LR3) (KCTC 11868BP) ,

4) Bifitobacterium animalis lactis CBT2501H ( Bifidobacterium animalis subsp . lactis CBT2501H (KCTC 11903BP)

The novel lactic acid bacteria of the present invention and compositions comprising them can inhibit the process of differentiation and maturation of Th2 cells by inhibiting the production of IL-4 and promoting the production of IL-12. Atopic dermatitis therapeutic agent by such a mechanism may be a therapeutic agent that can be used as a causative therapy for atopic diseases, and is more preferable because there is no risk of side effects such as antihistamines or steroids.

The composition may include four kinds of lactic acid bacteria in the same ratio, or may include lactic acid bacteria of the genus Lactobacillus and Bifidobacterium genus in a ratio of 2: 1 to 10: 1.

The composition of the present invention contains a mixture of lactic acid bacteria strains as an active ingredient in an amount of 10 ⁸ to 10 ¹² cfu / g, or a culture having an equal number of live bacteria, based on the total weight of the composition.

The composition of the present invention is Lactobacillus Lactobacillus in addition to the above-mentioned lactic acid bacteria salivarius ), Lactobacillus brevis ), Lactobacillus helveticus ), Lactobacillus fermentum ), Lactobacillus paracasei ), Lactobacillus delbrueckii ), Lactobacillus reuteri ), Lactobacillus buchneri ), Lactobacillus gasseri ), Lactobacillus johonsonii ), Lactobacillus kefir ), Lactococcus lactis ), Bifidobacterium ( Bifidobacterium) bifidum ), Bifidobacterium infantis ), Bifidobacterium pseudolongum ), Bifidobacterium thermofilum (B ifidobacterium themophilu m), Bifidobacterium adolescentis ( Bifidobacterium adolescentis ) may further comprise one or more lactic acid bacteria selected from the group consisting of.

The lactic acid bacteria strains may be grown in a general culture method for lactic acid bacteria, recovered in a separation process such as centrifugation, and may be prepared and used in a probiotic form by drying, for example, lyophilization.

The composition of the present invention may further comprise conventional pharmaceutically acceptable carriers and / or excipients in addition to the active ingredient, in addition to various pharmaceutically conventional additives such as binders, disintegrating agents, coatings, lubricants, etc. And can be formulated.

The composition of the present invention may be formulated in powder, granule, tablet, capsule or liquid form by mixing with the lactic acid bacteria and a suitable carrier, excipient, auxiliary active ingredient and the like. The composition of the present invention may also be enterically coated and commercialized using known methods to reach the small intestine after passing through the stomach and rapidly release the active microorganism into the intestine.

Excipients usable in the present invention include sugars such as sucrose, lactose, mannitol, glucose, and the like, starches such as corn starch, potato starch, rice starch, partially pregelatinized starch and the like. The binder is a naturally-occurring macromolecular substance such as dexatrin, sodium alginate, carrageenan, guar gum, acacia, agar, tragacanth, gelatin, gluten, hydroxypropylcellulose, methylcellulose, hydroxypropylmethyl Cellulose derivatives such as cellulose, ethyl cellulose, hydroxypropyl ethyl cellulose, carboxymethyl cellulose sodium, and polyvinylpyrrolidone, polyvinyl alcohol, polyvinylacetate, polyethylene glycol, polyacrylic acid, polymethacrylic acid and vinyl acetate resin It contains a polymer.

Degrading agents include cellulose derivatives such as carboxymethyl cellulose, carboxymethyl cellulose calcium, low-substituted hydroxypropyl cellulose, sodium carboxymethyl starch, hydroxypropyl starch, corn starch, potato starch, rice starch and partially pregelatinized starch. Starch can be used.

Examples of lubricants that can be used in the present invention include talc, stearic acid, calcium stearate, magnesium stearate, colloidal silica, hydrosilicic dioxide, various types of waxes and hydrogenated oils, and the like.

Coating agents include dimethylaminoethyl methacrylate-methacrylic acid copolymer, polyvinyl acetal diethylamino acetate, ethyl acrylate-methacrylic acid copolymer, ethyl acrylate-methyl methacrylate-chlorotrimethylammoniumethyl methacrylate. Water-insoluble polymers such as copolymers, ethyl cellulose, methacrylic acid-ethyl acrylate copolymers, hydroxypropyl methyl cellulose phthalates, hydroxypropyl methyl cellulose acetate succinate, and other polymers such as methyl cellulose, hydroxypropyl methyl cellulose Water-soluble polymers such as polyvinylpyrrolidone and polyethylene glycol, but are not necessarily limited thereto.

The dosage of the strain of lactic acid bacteria as an active ingredient in the composition for preventing or treating atopy of the present invention may be appropriately determined in consideration of the purpose of treatment or prevention, the type of patient to be treated or prevented, the symptoms, weight, age or sex of the patient. Can be. In general, for adult patients, at least 1 × 10 ⁶ live cells, preferably 1 × 10 ⁸ to 1 × 10 ¹² live cells, can be administered once or divided as needed.

The composition for preventing or treating lactic acid bacteria-containing atopy of the present invention may be administered or ingested as a medicine or food to humans or animals. The drug containing the composition for the prophylaxis or treatment of lactic acid bacteria-containing atopy of the present invention is one embodiment of the present invention, and the food containing the composition for the prophylaxis or treatment of lactic acid-containing atopy of the present invention is also an embodiment of the present invention. Examples of such foods include, but are not limited to, yogurt and fermented dairy products.

In general, changes in cytokine concentrations in atopic dermatitis are caused by the immune system based on the activation of Th2, where the differentiation of undifferentiated T-helper cells-1 (Th1) into T-helper cells-2 (Th2) is highly promoted. It is known. During the activation of these Th2 cells, Th2 cells produce interleukin-4 (IL-4), IL-5, IL-13, IL-9 and IL-10. Among them, IL-4 and IL-13 promote the synthesis of immunoglobulin E (IgE) and IgG1, and in the human body, they promote the synthesis of IgE and IgG4.

In the present invention, after the administration of the novel single lactic acid bacteria strains and mixed lactic acid bacteria containing them, the concentration pattern of cytokines related to the Th2 serum of the rat was examined, and showed a significant reduction of IgE and IL-4 than the non-lactic acid treated group. . In particular, IgE tended to decrease more significantly in the group treated with mixed lactic acid bacteria than in the group treated with the Lactobacillus strain alone, and the concentration of IgE in the positive control group was increased four times than that in the negative control group. The concentration of IgE in the rats was significantly decreased by three times the concentration of IgE in the positive control group, while the lactic acid bacteria treated group showed the largest decrease. Moreover, the concentration of IL-4 in all groups treated with lactic acid bacteria was significantly reduced than the positive control. Thus, administration of mixed lactic acid bacteria in animal models has been shown to be effective in alleviating the symptoms of atopic dermatitis.

In addition, the present invention is to reduce the concentration of IL-4 (Th2 group) and IL-12 and IFL-γ (Th1 group of cytokines) due to the administration of novel lactic acid bacteria or complex lactic acid bacteria containing four kinds of lactic acid bacteria strains It was confirmed that the symptoms of atopic dermatitis were alleviated. At the same time, the concentration of cytokine treated with mixed lactic acid bacteria was closer to the normal concentration than that of the cytokine of the group treated with the Lactobacillus strain alone. It was confirmed that more effective than lactic acid bacteria administration.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited by the following examples.

Example

One. Lactobacillus Kazei CBT (One) ( Lactobacillus casei CBT (1) Isolation and Identification of Strains

1-1. New microbial isolation

After serial dilution of 1 g of dairy products in sterile anaerobic water, 1 ml of each dilution was poured into BL (BL. BD. USA) solid medium and incubated under anaerobic conditions for 3 days.

The grown colonies were transferred to Lactobacillus selective medium (BL solid medium) to which BCP (Bromocresol purple, 0.17 g / L) was added, and then cultured under the same conditions for 3 days. The BCP indicator changes from purple to yellow when the lactic acid bacteria form lactic acid and the surrounding pH decreases. After selecting the lactic acid bacteria colonies whose color around the colonies changed to yellow color, biochemical and molecular biological identification were carried out. After that, one strain was tested that the antibiotic resistance of the strains met the criteria suggested by the European Food Safety Authority (EFSA). Selected.

1-2. Identification of new microorganisms

1) 16s rRNA  Sympathy

Genome DNA was extracted from the isolated strain and 16s rRNA sequences were analyzed.

The genome DNA was extracted from 1 ml of the pure culture of the strain isolated from dairy products using Accuprep genome extraction kit (Bioneer, Korea). Using the extracted DNA as a template, PCR (MyCycler, BIO-RAD, USA) was performed using a primer F (5'-AGAGTTTGATCCTGGCTCAG3 ') and primer R (5'-AAGGAGGTGATCCAGCC 3') as a template. PCR products were linked to the pGEM-Teasy vector (Promega, USA) to provide E. coli Strain DH5α was transformed and then plated on LB / x-gal / ampplate and incubated overnight at 37 ° C. After screening the recombinant plasmid containing the insert from the transformant, DNA sequencing was performed. DNA sequencing analysis using the Cluster V method of DNA star program L. casei ^T (ATCC 393) and L. casei ( ATCC 334). The gene sequence of the 16s rRNA of the isolated strain is L. casei ^T , as shown in FIG. (ATCC 393), 98.5%, and L. casei ( ATCC 334) showed 99.8% homology.

2) RAPD  ( Random Amplified Polymorphic DNA ) analysis

In the RAPD analysis, genome DNA was extracted from a strain isolated from dairy products, and PCR-RAPD (MyCycler, BIO-RAD, USA) was used as a template (GTG) ₅ (5'-GTGGTGGTGGTGGTG 3 ') primer. Was performed. The final product, the PCR product, was stained with EtBr and observed with G: BOX (SYNGENE, UK). The strain isolated from the RAPD results, L. casei ^T , as shown in Figure 2 (ATCC 393) and showed a different band pattern. Therefore, the strain isolated from dairy products from the above results was L. casei ^T It was confirmed that it is a new strain different from (ATCC 393).

3) Antibiotic Resistance Test

Antibiotic resistance experiments were performed using the micro-dilution method recommended by the European Food Safety Authority (EFSA), and each antibiotic was purchased from Sigma and Fluka.

Antibiotic concentrates were diluted to concentrations of 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5 μg / ml using MRS medium, and 100 μl of the diluted solution containing the antibiotics in 96-well plates. Inoculate aliquots and measure growth after 16 hours of incubation at 37 ° C after inoculating 1% of Lactobacillus casei CBT (1) pre-cultured in MRS liquid medium (Minimal Inhibition Concentration, “MIC ”).

In order to reduce the error of experiments, all experiments consisted of three sets, and after taking the average of these values, the final MIC value was determined. MIC for the strain was determined by the following method. At the same experimental time as the experimental group, 100 μl of the MRS culture medium without antibiotics was inoculated with 1% of the strain, and immediately stored at −4 ° C. Based on the OD value of the group that inhibited the growth of the strain at -4 ℃ after 16 hours, the concentration of antibiotics showing an OD value of less than that in the experimental group was defined as MIC. The calculated MIC values are shown in Table 1 below.

Lactobacillus casei CBT (1) isolated from dairy products was found to be lower than the antibiotic resistance criteria set out in EFSA, which does not seem to be resistant to the antibiotics used in the experiment.

Antibiotic (μg / ml) Spawn name AMP VAN GEN KAN ST Lactobacillus casei CBT (1) <2 > 256 <4 <64 <16 EFSA break point 2 nr 32 64 nr Spawn name ERM CLM QU + DA TET Lactobacillus casei CBT (1) <0.5 <0.03125 <0.25 <0.5 EFSA break point One One 4 4 ※ AMP; Ampicilin, VAN; Vancomycin, GEN; Gentamycin, KAN; Kanamycin, ST; Streptomycin, ERM; Erythomycin, CLM; Clindamycin, TET; Tetracycline, QU + DA; Quinupristin + Dalfopristin, nr; not required

Based on the above results, the strain isolated from dairy products was named Lactobacillus casei CBT (1), and on March 2, 2011, it was deposited with the microbial resource center (KCTC), a depository institution for Korean patent strains, and was assigned accession number KCTC 11863BP. .

2. Lactobacillus Flora Room  LP (1) ( Lactobacillus plantarum LP (One)  ( KCTC  11867 BP Separation and identification

2-1. Isolation of New Microorganisms

Kimchi, a traditional Korean fermented food, was serially diluted in sterile anaerobic water, and 1 ml of each diluted solution was poured into ManRogosaSharpe (MRS. BD. USA) solid medium and incubated under anaerobic conditions for 3 days. The grown colonies were picked up again on lactic acid bacteria selection medium to which BCP (Bromocresol purple, 0.17g / L) was added to MRS, and then cultured again under the same conditions for 3 days. After selecting the lactic acid bacteria colonies whose color around the colonies changed to yellow color, biochemical and molecular biological identification were performed, and one strain having the best physical properties (acid resistance and bile resistance) was finally selected.

2-2. Identification of new microorganisms

1) sugar usability analysis

The sugar availability was confirmed using the API CHL50 kit for the strain isolated from kimchi. Sugar usability check results were confirmed by typing in the API web (https://apiweb.biomerieux.com), and the identification results, as shown in Table 2, Lactobacillus plantarum ( Lactobacillus plantarum ) and 99.9% showed the same biochemical properties.

number temperament reaction number temperament reaction 0  Control - 25  Esculine + One  Glycerol - 26  Salicine + 2  Erythritol - 27  Cellobiose + 3  D-Arabinose - 28  Maltose + 4  L-Arabinose + 29  Lactose + 5  Ribose + 30  Melibiose + 6  D-Xylose - 31  Saccharose + 7  L-Xylose - 32  Trehalose + 8  Adonitol - 33  Inuline - 9  β-Methyl-xyloside - 34  Melezitose + 10  Galactose + 35  D-Raffinose + 11  D-Glucose + 36  Amidon - 12  D-Fructose + 37  Glycogene - 13  D-Mannose + 38  Xylitol - 14  L-Sorbose - 39  β-Gentiobiose + 15  Rhamnose - 40  D-Turanose + 16  Dulcitol - 41  D-Lyxose - 17  Inositol - 42  D-Tagatose - 18  Mannitol + 43  D-Fucose - 19  Sorbitol + 44  L-Fucose - 20 alpha -Methyl-D-mannoside + 45  D-Arabitol - 21 alpha -Methyl-D-glucoside - 46  L-Arabitol - 22  N-Acetyl glucosamine + 47  Gluconate + 23  Amygdaline + 48 2-Ceto-gluconate - 24  Arbutine + 49 5-Ceto-gluconate -

2) 16s rRNA  Sympathy

16s rRNA sequences were analyzed to identify new microorganisms using molecular biological methods. Genome DNA was extracted from 1 ml of pure culture broth isolated from Kimchi using Accuprep genome extraction kit (Bioneer, Korea). Using the extracted DNA as a template, PCR (MyCycler, BIO-RAD, USA) was performed using a primer F (5'-AGAGTTTGATCCTGGCTCAG3 ') and primer R (5'-AAGGAGGTGATCCAGCC 3') as a template. PCR products were linked to the pGEM-Teasy vector (Promega, USA) to provide E. coli Strain DH5α was transformed and then plated on LB / x-gal / ampplate and incubated overnight at 37 ° C. After screening, the recombinant plasmid containing the insert was isolated from the transformant, followed by 16s rRNA gene sequence analysis. 16s rRNA gene sequencing was performed using the Cluster V method of the DNA star program. Plastic taryum ^T jyunju were compared homology with (L. plantarum ^T) (14917 ATCC). 16s rRNA sequences of the isolated strains, as shown in FIG. Plastic taryum ^T (L. plantarum ^T) (14917 ATCC) and showed a homology of 99.5%.

3) RAPD  ( Random Amplified Polymorphic DNAD ) And Pulsed Field Come    Electrophoresis ( PFGE ) analysis

RAPD analysis was performed by extracting the genome DNA from Lactobacillus plantarum LP (1) isolated from kimchi, PCR-RAPD (MyCycler) using (GTG) ₅ (5'-GTGGTGGTGGTGGTG 3 ') primer as a template , BIO-RAD, USA). The final product, the PCR product, was stained with EtBr and observed with G: BOX (SYNGENE, UK). In addition, PFGE analysis was performed by producing a plug of pure cultured Lactobacillus plantarum LP (1) with a final OD of 4 and cutting the genome DNA using various enzymes, followed by electrophoresis to control L. plantarum ^T (ATCC 14917) for comparison. The strain isolated from the RAPD results, as shown in Figure 4, Lactobacillus plantarum LP (1) L. plantarum ^T The band pattern was similar to that of (ATCC 14917), but the PFGE result showed a different band pattern. Based on the above results, Lactobacillus plantarum LP (1), a microorganism isolated from kimchi, was L. plantarum ^T It was confirmed that it is a new strain different from (ATCC 14917).

4) Acid resistance  And My bile  evaluation

Acid resistance tests on Lactobacillus plantarum LP (1) isolated from kimchi were performed using artificial gastric fluid (NaCl 2g, pepsin 3.2g, concentrated hydrochloric acid 5.8ml / 1L, pH = 2.1). After inoculating 1% in ml, the number of viable cells was measured in MRS solid medium for 2 hours in units of 30 minutes. In addition, the biliary resistance test was inoculated with 1% of the pre-cultured isolated strain in MRS liquid medium with different Oxgall concentration, and the number of viable cells was measured using MRS solid medium 2 hours after the inoculation. The Oxgall concentration used at this time is 0, 0.25, 0.5, 0.75, 1%.

As a result of the acid resistance and bile resistance test, as shown in FIG. 5, the isolated strain Lactobacillus plantarum LP (1) is stably maintained in the acidic and bile conditions, so that the acid resistance and bile interference of the strain I could confirm it.

Based on the above results, the new microorganism isolated from Kimchi was named Lactobacillus plantarum LP (1), and on March 2, 2011, the microorganism resource center (KCTC), a depository institution for Korean patent strains, was deposited. ) Was given accession number KCTC 11867BP.

3. Bifidobacterium Animalis Lactis CBT2501H  ( Bifidobacterium animalis subsp . lactis CBT2501H  ( KCTC  11903 BP Separation and identification

3-1. New microbial isolation

After serial dilution of 1 g of human feces in sterile anaerobic water, 1 ml of each dilution was poured into BL (BL. BD. USA) solid medium and incubated under anaerobic conditions for 3 days.

The grown colonies were transferred to Lactobacillus selective medium (BL solid medium) to which BCP (Bromocresol purple, 0.17 g / L) was added, and then cultured under the same conditions for 3 days. After selecting the lactic acid bacteria colonies whose color around the colonies changed to yellow color, biochemical and molecular biological identifications were performed. After that, one strain was finally selected in which the antibiotic resistance of the strains satisfies the criteria suggested by EFSA.

3-2. Identification of new microorganisms

1) sugar usability analysis

The sugar availability was confirmed using AP150 CHB for the strain isolated from human feces. The sugar usability check result was confirmed by entering into the API web (https://apiweb.biomerieux.com).

number Carbohydrates reaction number Carbohydrates reaction 0  Control - 25  Esculine - One  Glycerol - 26  Salicine - 2  Erythritol - 27  Cellobiose - 3  D-Arabinose - 28  Maltose + 4  L-Arabinose - 29  Lactose + 5  Ribose + 30  Melibiose + 6  D-Xylose + 31  Saccharose + 7  L-Xylose - 32  Trehalose - 8  Adonitol - 33  Inuline - 9 β-Methyl-xyloside - 34  Melezitose - 10  Galactose + 35  D-Raffinose + 11  D-Glucose + 36  Amidon - 12  D-Fructose - 37  Glycogene - 13  D-Mannose - 38  Xylitol - 14  L-Sorbose - 39  β-Gentiobiose + 15  Rhamnose - 40  D-Turanose - 16  Dulcitol - 41  D-Lyxose - 17  Inositol - 42  D-Tagatose - 18  Mannitol - 43  D-Fucose - 19  Sorbitol - 44  L-Fucose - 20 alpha -Methyl-D-mannoside - 45  D-Arabitol - 21 alpha -Methyl-D-glucoside - 46  L-Arabitol - 22 N-Acetyl glucosamine - 47  Gluconate - 23 Amygdaline + 48 2-Ceto-gluconate - 24 Arbutine - 49 5-Ceto-gluconate -

2) 16s rRNA  Sympathy

Genome DNA was extracted from the isolated strain and 16s rRNA sequences were analyzed.

Genome DNA was extracted from 1 ml of pure culture medium from human feces using Accuprep genome extraction kit (Bioneer, Korea). Using the extracted DNA as a template, PCR (MyCycler, BIO-RAD, USA) was performed using a primer F (5'-AGAGTTTGATCCTGGCTCAG3 ') and primer R (5'-AAGGAGGTGATCCAGCC 3') as a template.

PCR products were linked to the pGEM-Teasy vector (Promega, USA) to provide E. coli Strain DH5α was transformed and then plated on LB / x-gal / ampplate and incubated overnight at 37 ° C. After screening the recombinant plasmid containing the insert from the transformant, DNA sequencing was performed. DNA nucleotide sequence analysis using the method of DNA star Cluster V program B. animalis subsp . lactis Homology was compared with strain YIT 4121. The 16s rDNA sequence of the isolated strain was B. animalis. subsp . lactis 100% homology with strain YIT 4121 (see FIG. 6).

3) Random Amplified Polymorphic DNA  ( RAPD ) analysis

In the RAPD analysis, genome DNA was extracted from a strain isolated from human feces, and PCR-RAPD (MyCycler, BIO-RAD, USA) was used as a template (GTG) ₅ (5'-GTGGTGGTGGTGGTG 3 ') primer. Was performed. The final product, the PCR product, was stained with EtBr and observed with G: BOX (SYNGENE, UK).

In the RAPD results, the isolated strain was B. animalis ^T , as shown in FIG. 7. (ATCC 25527) and showed a different band pattern.

Therefore, the strain isolated from human feces from the above results was B. animalis ^T (ATCC 25527) and other novel strains could be confirmed.

4) Acid resistance  And heat resistance experiment

Acid resistance experiments of Bifidobacterium animalis lactis CBT2501H isolated from human feces corrected gastric juice (NaCl 2g, pepsin 3.2g / 1L) to pre-cultured in BL liquid medium to pH 7.0, 3.0, 2.3 The viable cell count was measured in BL solid medium for 2 hours in 30 minutes after 1% inoculation in one 10ml.

On the other hand, in the heat resistance test, the bacterial strain was measured using BL solid medium for 90 minutes at 30 minute intervals while incubating at 52.5 ° C. after 1% inoculation of the isolated strain pre-cultivated in BL liquid medium. The isolated strain Bifidobacterium animalis lactis CBT2501H was very stable at pH = 7.0 and pH = 3.0 as a result of the acid resistance test, and it was confirmed that the viable cell number was stably maintained even at the heat resistance.

Based on the above results, the strain isolated from feces was identified as “ Bifidobacterium animalis lactis CBT2501H ( Bifidobacterium animalis subsp. It was named lactis CBT2501H strain and was deposited on March 2, 2011 to the microbial resource center (KCTC), a depository institution for Korean patent strains, and was given accession number KCTC 11903BP.

4. Preparation of atopy prevention / treatment composition

(1) Lactic acid bacteria fermentation medium

Fermentation medium for the cultivation of lactic acid bacteria strains contained in the composition of the present invention is made of 1% -10% (w / v) aqueous solution by mixing or mixing skim milk powder and soybean isolated protein, respectively 0.01% compared to the protein according to the lactic acid species Enzyme treatment is performed by adding a solution of proteolytic enzyme (Delvolase; DSM, Netherlands) in the range -1% (w / w). The enzyme treatment step was omitted for the Lactobacillus plantarum lactic acid bacterium.

After enzymatic treatment, glucose, lactose, fructose or sucrose in the range of 0.1% -5% (w / v), yeast extract in the range of 0.1% -5% (w / v), 0.1% Ionic components of soypeptone in the range -5% (w / v), ammonium citrate in the range of 0.01% -0.1% (w / v), sodium acetate, dipotassium poststate, magnesium sulfate, manganese sulfate, sodium chloride Lactic acid bacteria culture medium was prepared by adding and dissolving. The prepared lactic acid bacteria culture medium was sterilized using a heat exchanger.

(2) lactic acid bacteria culture and lactic acid bacteria production

Lactobacillus spawn pre-cultured in the lactic acid bacteria fermentation broth prepared in step (1) at a level of 0.1% -1% (v / v) and maintained at 37 ° C. and pH 5.0-6.0 to 8 hours − Fermentation was carried out for 18 hours. After completion of the lactic acid bacteria culture lactic acid bacteria were separated and concentrated using a continuous centrifuge (Separator). The isolated lactic acid bacteria were lyophilized by the addition of a cryoprotectant and then ground using a grinder to prepare lactic acid bacteria having a constant particle size.

(3) lactic acid bacteria sample preparation

Lactobacillus casei CBT (1) lactic acid bacteria (LC), Lactobacillus plantarum LP (1) lactic acid bacteria (LP), Lactobacillus rhamnosus LR3 lactic acid bacteria (LR) prepared in step (2), Bifidobacterium annie Malys lactis CBT2501H bifidus (BL) was orally administered to experimental animals once daily at a dose of 10 ⁸ CFU / head for 6 weeks.

Experimental Example

1. Animal and Experimental Design

In this experimental example, the efficacy of the combined administration of various lactic acid bacteria strains and four kinds of lactic acid bacteria of the present invention was positive for 8-week-old male NC / Nga mouse (Central LAB Seoul Inc. (Seoul, South Korea)) model in which atopic dermatitis was induced. Comparative evaluation was made with the control and negative controls. The negative control group is the normal group corresponding to the sham group, and the positive control group is the NC / Nga mouse group in which atopic dermatitis is induced by DNCB.

The experimental animals were acclimated to the environment for one week after arriving at the laboratory, and only animals that exhibited no special symptoms during this period were used in the experiment. During the acclimation period, animals were kept at 24 ± 1 ° C, 55 ± 10% relative humidity, light / dark cycles at 12/12 hours, and were freely available for commercial rodent feed and water.

All animal procedures and guidelines of the Animal Experiments (Guideline for Animal Experiments , CBTA-005). Mice were anesthetized with ether and shaved with scissors and razors. In this example, sensitization to DNCB and repeated inoculation resulted in skin lesions similar to atopic dermatitis. Specifically, to induce atopic dermatitis, mice were treated six times with 0.5% DNCB solution for two weeks and then four times with 1% DNCB solution for two weeks. DNCB solutions were prepared by adding acetone: olive oil in a 3: 1 ratio to DNCB.

2. Cytokine  In vitro analysis of concentration changes ( ex - vivo assay )

Whole blood cells from NC / Nga mice, an atopic dermatitis-induced animal model, were transferred to IEME medium (2 nM glutamex, 10% FES, 50 μmol betamercaptoethanol, 0.1% penicillin-streptomycin complex, pH 7.4, Gibco). Mix in 5 ratios. 50 μl of whole blood cells were placed in 150-μl IMEM medium (10% FBS, 0.1 penicillin-streptomycin complex (pH 7.4) contained in each well of a 96-well-plate and 5% CO ₂ Incubate for 24 hours in the incubator. Atopy environment made by adding 3 μg / ml of 1-chloro-2-2,4-dinitrobenzene (DNCB) to a solution of whole blood cells (50 μl per well) in acetone and ethanol at a ratio of 1: 4 After exposure, the cells were incubated for 12 hours. At this time, DNCB was purchased from Sigma (St. Louis, MO, USA) and used after recrystallization with acetone. In addition, whole blood cells treated with 1 μg / ml of lactic acid bacteria sample were incubated for 12 hours, and the supernatant was centrifuged at 4 ° C. and 1000 rpm for 10 minutes. The concentrations of IgE (Mouse IgE ELISA Kit, BD Bioscience) and IL-4 (Mouse IL-4 Immunoassay ELISA Kit, R & D Systems) of the supernatants obtained by whole blood cell culture test were measured and shown in FIG. 9.

In FIG. 9, (+) CTL is the result for a group of mice with DNCB-induced atopic dermatitis-like skin lesions, and (-) CTL is the result for a group of normal mice not showing DNCB-induced atopic dermatitis-like skin lesions. Indicates.

MIX, LC, LR, LP, and BP are the result of the administration of lactic acid bacteria to a group of mice with DNCB-induced atopic dermatitis-like skin lesions. “LC” is the result of administration of the Lactobacillus casei CBT (1) strain. , “LR” is the result of administration of Lactobacillus rhamnosus LR3 strain, “LP” is the result of administration of Lactobacillus plantarum LP1 strain, and “BL” is administration of Bifidobacterium animalis lactis BL2 strain. One result is shown, and "MIX" shows the result of mixing and administering four kinds of similar bacteria of the present invention.

9, the concentrations of IgE and IL-4 were all groups treated with lactic acid bacteria (LC, LR, LP and BL) or mixed lactic acid bacteria (MIX) when compared with the atopic dermatitis group ((+) CTL). Decreased significantly, especially in the group treated with lactic acid bacteria to a concentration close to the normal concentration (Fig. 9A). IL-4 concentrations that result in the synthesis of IgE were significantly lower in the lactic acid bacteria treated group than in the lactic acid bacteria-treated group, but there was no significant difference between the lactic acid bacteria treated group and the mixed lactic acid treated group (FIG. 9B). .

3. Through oral administration of lactic acid bacteria scratch  Score ( scratch score ) Improvement

Lactic acid bacteria or mixed lactic acid bacteria were orally administered to NC / Nga mice in which atopic dermatitis was induced with DNCB for 6 weeks. During the experiment, body weights were measured twice a week for six weeks. Scratch scores were measured at weekly intervals, and three rats from one group were randomly selected and moved to a new cage, followed by counting the number of times the rats scratched the face and back skin for 10 minutes and measured as an average value. The measured values were expressed as mean ± SD (n = 3), and * in FIG. 10 indicates P <0.05.

Scratch scores were measured by gross examination for dermatitis. When the lactic acid bacteria were administered during the experimental period, no weight loss due to stress due to oral administration of lactic acid bacteria or lactic acid bacteria was observed.

The scratch score, an indicator of atopic dermatitis, was consistently high in the positive control, while markedly decreased in all groups treated with lactic acid bacteria. Among the groups treated with lactic acid bacteria, the scratches were significantly reduced in the group treated with the mixed lactic acid bacteria (FIG. 10). Moreover, in the visual inspection of the inflammation of each group after the end of the experiment, it was confirmed that the degree of inflammation in all the groups treated with lactic acid bacteria improved close to the normal group (Fig. 11).

4. Serum from Oral Administration of Lactic Acid Bacteria Cytokine  Concentration change

Blood was collected from the heart of experimental animals under ether anesthesia, then plasma was separated by centrifugation and stored at -20 ° C until use. Plasma concentration of IgE, concentration of IL-4 (Mouse IL-5 Immunoassay ELISA Kit, R & D Systems), concentration of IL-12 (Mouse IL-12 p70 Immunoassay) using Luminex 100TM instrument set (Komabiotech, Seoul, South Korea) ELISA Kit), and the concentration of IFN-γ (Mouse IFN Immunoassay ELISA Kit) was measured 10 times, and the concentration of immunoglobulin G (IgG) was measured using a Luminex 100TM instrument set (Komabiotech, Seoul, South Korea) It was. The effect of repeated oral administration of lactic acid bacteria on serum total IgE and IL-4 in atopic dermatitis model animals is shown in FIG. 12, and repeated oral administration of lactic acid bacteria in serum of atopic dermatitis model animals And effects on the amount of INF-γ are shown in FIG. 13, respectively. In this example, the measurement data was treated with Graphpad Prism ^™ 5.0 to calculate the statistical parameters and mean and standard error of the group and to compare with other groups. Significance was determined using ANOVA ( P <0.05).

Scratch scores and visual inspection results of NC / Nga mice changed serum cytokine concentrations based on alleviation of atopic dermatitis. Referring to FIG. 12, the concentrations of IgE and IL-4 in the lactic acid bacteria-treated group were lower than those in the lactic acid bacteria-treated group, and in particular, the group treated with the mixed lactic acid bacteria (MIX) was lower than the groups administered with a single lactic acid bacteria strain.

Referring to Figure 13, a type of cytokine associated with T-helper cell 2 (Th-2), IL-12 and IFN- associated with Th-1 (T-helper cell 1) cytokine that inhibits the expression of IL-4 Measurement of the concentration of γ showed that the concentration of IL-12 was lower in the positive control than in the negative control. On the other hand, the concentration of IL-12 was significantly increased in all groups treated with lactic acid bacteria, and the concentration was similar to the group ((-) CTL) that did not cause atopic dermatitis.

The concentration of IFN-γ also increased similarly to IL-12. The concentration of IL-12 in all the groups treated with lactic acid bacteria increased markedly to the IL-12 concentration in the negative control group, but the concentration of IFN-γ was increased in all groups treated with lactic acid bacteria, especially in the group treated with mixed lactic acid bacteria. It increased even more when the lactic acid bacteria mixture of the four lactic acid bacteria of the present invention was administered.

4. Histopathological examination after oral administration of lactic acid bacteria

 To prepare skin samples for histopathological examination, skin tissues of mice with DNCB-induced atopic dermatitis were cut and fixed in 10% formalin solution for 24 hours and then washed with water. The skin tissue was dehydrated with alcohol (70%, 80%, 90%, 100% alcohol for 1 hour) and xylene (3 steps for 1 hour per step) and fixed to paraffin. Paraffin blocks were cut into 7 μm thick sections, stained with hematoxylin-eosin (H & E), and observed under a microscope. In addition, the skin tissue was stained with anti-IL-4 antibody (1:50 dilution; Santacruz Biotechnology, Inc., CA, USA) and diaminobenzidine DAB (2-min colorization) and photographed in the x200 microscope field. It was examined to observe the change in the number of immune cells. The results of observing changes in skin tissues taken from each group of NC / Nga mice are shown in FIG. 14. Referring to FIG. 14, as a result of H & E staining of skin tissue taken from each group of NC / Nga mice, epidermis (epidemises) and dermis (dermis) became thick, mast cells in a positive control group in which atopic dermatitis was induced. ) And eosinophils were increased over the negative control (FIG. 14A). Immunohistochemical (IHC) staining of IL-4 and IL-5 of skin tissues showed IL-5 in subcutaneous tissue of the positive control. The discovery of IL-5 indicates that AD-inducing agents, in particular Th2 cells, activate the immune system, thereby increasing IgE production in B cells and causing mast and basophils in this region. ), Resulting in an allergic reaction (FIGS. 14B and 14C).

In contrast, in atopic dermatitis-induced but lactobacillus-treated rat skin tissue (MIX, LC, LR, LP, BL), the epidermis and dermis thickened and the number of immune cells increased to normal levels.

The present invention can be practiced with various modifications and changes without departing from the spirit and scope thereof, as will be apparent to those skilled in the art. The specific embodiments described herein are merely for explaining preferred embodiments of the present invention and should not be construed as limiting the present invention. It is intended that the scope of protection of the present invention be defined by the appended claims, and that the various modifications and variations are intended to be included within the scope of the present invention.

Korea Biotechnology Research Institute KCTC11863BP 20110302 Korea Biotechnology Research Institute KCTC11867BP 20110302 Korea Biotechnology Research Institute KCTC11903BP 20110428 Korea Biotechnology Research Institute KCTC11868BP 20110302

Claims (12)

LBTobacillus casei CBT (1) ( Lactobacillus ), which was deposited internationally with the Korean Collection for Type Culture (KCTC) as accession number KCTC 11863BP. casei CBT (1)) lactic acid bacteria.
Lactobacillus Lactobacillus ( Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus ) platarum LP (1)) lactic acid bacteria.
Bifidobacterium animalis lactis CBT2501H ( Bifidobacterium ), internationally deposited as Korean Collection for Type Culture (KCTC), accession number KCTC 11903BP. animalis subsp. lactis CBT2501H) lactic acid bacteria.
Lactobacillus casei CBT (1) ( Lactobacillus casei CBT (1)) (KCTC 11863BP), 2) Lactobacillus plantarum LP (1) ( Lactobacillus plantarum LP (1) (KCTC11867BP)) , 3) Lactobacillus rhamnosus LR3 ( Lactobacillus rhamnosus) LR3 (KCTC 11868BP)) , 4) Bifitobacterium animalis lactis CBT2501H ( Bifidobacterium animalis subsp . lactis A composition for preventing or treating atopy comprising CBT2501H (KCTC 11903BP) or a culture thereof.
The method according to claim 4, wherein the composition comprises, based on the total weight of the composition, a lactic acid bacterium mixture as an active ingredient in an amount of 10 ⁸ to 10 ¹² CFU / g, or a culture having an equal number of live bacteria. Composition for preventing or treating atopy.
The method of claim 4, wherein the composition is Lactobacillus Salivarius ( Lactobacillus) salivarius ), Lactobacillus brevis ), Lactobacillus helveticus ), Lactobacillus fermentum ), Lactobacillus paracasei ), Lactobacillus delbrueckii ), Lactobacillus reuteri ), Lactobacillus buchneri ), Lactobacillus gasseri , Lactobacillus johonsonii ), Lactobacillus kefir , Lactococcus lactis ), Bifidobacterium bifidum , Bifidobacterium infantis infantis ), Bifidobacterium pseudolongum ), Bifidobacterium thermofilum (B ifidobacterium themophilu m), and Adolfo sentiseu Bifidobacterium (Bifidobacterium adolescentis ) a composition for preventing or treating atopy further comprising at least one lactic acid bacteria selected from the group consisting of.
The composition for preventing or treating atopy according to claim 4, wherein the composition further comprises a pharmaceutically acceptable carrier or excipient.
The composition for preventing or treating atopy according to claim 4, wherein the composition comprises Lactobacillus spp. And Bifidobacterium spp. In a ratio of 2: 1 to 10: 1.
The composition for preventing or treating atopy according to claim 4, wherein the composition comprises four kinds of lactic acid bacteria in the same ratio.
The composition for preventing or treating atopy according to claim 4, wherein the composition is prepared in a probiotic form by lyophilizing the lactic acid bacteria strains.
A drug comprising a composition for the prophylaxis or treatment of lactic acid bacteria-containing atopy according to any one of claims 4 to 10.
Food comprising a composition for the prophylaxis or treatment of lactic acid bacteria-containing atopy according to any one of claims 4 to 10.

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