WO2016093450A1 - Lactobacillus plantarum k-1 br strain, as novel strain, having functions of immunity regulation and improved treatment of allergies, atopic dermatitis, rhinitis, itching, etc. - Google Patents

Abstract

The present invention relates to L. plantarum-K-1 BR (LP), which is lactobacillus that is separated from Kimchi and identified, the L. plantarum-K-1 BR (LP) exhibiting an anti-allergic effect; being capable of improved treatment of diseases such as allergic diseases, atopic dermatitis, rhinitis, pruritus, anaphylaxis, etc.; and having immunity regulation.

Description

[Revision according to Rule 26.10.07.2015] A new strain, Lactobacillus plantarum K-1 BR strain, which has improved treatment and immunity control functions such as allergy, atopic dermatitis, rhinitis and itching

The present invention causes a change in the intestinal flora (Microbiota) due to a combination of westernized diet, uniform living environment of civilization, irregular lifestyle and stress, resulting in various digestive inflammatory diseases, immune hypersensitivity diseases It relates to a Lactobacillus plantarum K-1 BR strain having improved treatment and immune system regulation functions such as atopic dermatitis, rhinitis, which is a kind of lactic acid bacteria against the occurrence of.

Allergy is a biochemical phenomenon that shows a specific and altered response to a foreign substance (antigen, Allergen). When the released substance causes symptoms, it is called allergen and the resulting disease is called allergic disease.

Lactic acid bacteria are bacteria that consume large amounts of carbohydrates and produce large amounts of organic acids such as lactic acid.

On the other hand, decaying bacteria are bacteria that produce odors by breaking down proteins in our bodies and producing ammonia, hydrogen sulfide, amines, indole, and phenol, like Bacteroides strains. Recently, lactic acid bacteria are called probiotics (for the life).

Currently, lactic acid bacteria are classified and the strains are classified into cocci and bacillus according to their shape, and cocci include Streptococcus, Pediococcus and Leuconostoc, and bacilli include Lactobacillus, Sporolactobacillus and Bifidobacterium.

Lactobacillus began with the history of mankind, and is old enough to appear in Hippocrates' book.

Hippocrates (460-377 B.C.) report that fermented foods are effective for diarrhea patients.

The first investigator to look for lactic acid bacteria was Antony van Leeuwenhoek, but was not a lactic acid bacterium detector, and the first lactic acid bacterium scientist was Louis Pasteur and thought it was a decayed bacterium, but in 1899, Tisser first identified lactic acid bacteria Bifidus from feces. In 1900, Moro, a pediatrician, studied the difference between the intestinal bacterial flora in feces of diarrhea and healthy children. Lactobacillus acidophilus, a lactic acid bacterium, was a beneficial bacterium.

Mechinnikov insisted that beneficial bacteria live in the digestive tract to survive. This argument is based on the premise that the intestinal bacteria live on beneficial lactic acid bacteria in our bodies. In our intestines, there are bacteria that work well and those that work bad. The balance of the long-term environment is broken down by various effects such as stress, fatigue, or aging. Lactobacillus has been shown to improve lactose intolerance, lactose intolerance, Lactobacillus constipation, Lactobacillus inhibiting harmful bacteria, Lactobacillus improving migraines, Lactobacillus liver protection, Lactobacillus antimutagenic effects, Lactobacillus skin beauty, It has the effect of improving the inflammatory bowel disease (Crohn's disease and colitis) of lactic acid bacteria, anti-cancer effect and immune enhancing effect, and anti-allergic effect.

When the balance of intestinal bacteria is broken, the intestinal condition worsens, constipation occurs, and the skin becomes rough. The adverse effects are not just there. The problem is that when the balance of the intestine is broken, it can cause various allergic diseases and various lifestyle diseases.

The intestine is the largest immune system in humans. That is, the intestine is closely related to the body's immunity and the state of the whole body. Phytolactic acid bacteria improve the health by increasing the good bacteria and balance the intestines.

Neutrophils are intermediates of leukocytes that manage immunity to eat and remove foreign substances such as bacteria that have penetrated the body. The increase in the number suggests that phyto-lactic acid bacteria revival immunity.

"The fermented food has gained in popularity recently after Korean scientists reported that 11 out of 13 chickens infected with AI started eating and recovering from Korean kimchi extract similar to sauerkraut."

Kimchi Lactobacillus, which overcomes and grows in the habitat environment of natural antibiotic materials such as garlic, ginger, red pepper powder, green onion, and salt, will have different cellular connection structure and different products. Lactobacillus produced by overcoming such an environment not only rejuvenates the skin or cells by repairing the intestinal environment, but also greatly enhances the body's immunity and greatly helps in preventing and improving allergic diseases such as pollen allergy and atopic dermatitis and lifestyle diseases. Becomes

Allergic disease is increasing every year, and it has risen more than 5% in the 2000s, and it is estimated that more than 10% are included in potential patients.

It is a pathological process of living organisms that is the result of antigen-antibody reactions that are the cause of allergy. The most common allergic reactions are bronchial asthma, allergic rhinitis, hay fever and atopic dermatitis. There is a fourth type such as

Allergic reactions such as anaphylaxis are caused by drugs such as antigens to Fc receptors, such as mast cells, binding of anti-IgE, lectin, stimulation of anaphylatoxin, synthetic cortical stimulating hormones such as calcium ionopore, compound K48 / 80, and codeine. Happens

In addition, lactic acid bacteria increase the immune mechanisms in our bodies to suppress the infection of pathogenic bacteria that cause disease. When the lactic acid bacteria activate the immune system, even if the virus invades our body, the defense ability increases. Kimchi lactic acid bacteria to fight the bird flu virus would be the same way. Therefore, identifying and identifying powerful lactic acid bacteria that benefit people will bring happiness through the maintenance of human health.

Korean Laid-Open Patent Publication No. 1020050076374 (2005.07.26) has an effect on anti-allergy and anti-atopy, and obtained by extracting and drying alcohol or polyethylreglycol in order to suppress the intestinal bacterial ammonia productivity. Beans or soybean extracts for the treatment of allergies and inflammation are included in the family Lactobacillus ashidophilus, Lactobacillus vulgaris, Lactobacillus confusus, Lactobacillus luteri, Lactobacillus cassia, Bifidobacterium braves, Bifidobacterium infantis, Soybeans that have the activity of inhibiting ammonia production of atopy and anti-allergic and enterotoxic bacteria obtained by inoculating at least one of Bifidobacterium Bifidum, Bifidobacterium thermofilum, Streptococcus Facecalis and Streptococcus thermophilus Functional products containing lactic acid bacteria fermentation products and methods for producing the same And the dog,

Effect of Kimchi Extracted Probiotics on Immunity Index in NC / Nga Mice in Atopic Animal Model, Korean Journal of Food Science and Technology, Vol. 40, No. 1, No. 197 (February 2008) pp.82-87 have.

The present invention has been made to solve the above problems, a number of microorganisms are living in the environment in which people live. It lives in various places from the human skin to the small intestine, and these bacteria are called normal bacterial flora. The gut flora, which is most closely related to human health, is the lactic acid bacteria.

Intestinal bacterial flora is affected by the host's environment, namely pH (gastric acid) in the digestive tract, intestinal motility, digestive enzymes, mucus, and bile. For example, when a person is stressed, the bowel movements stop, the secretion of gastric acid and serous secretions are high or low, and as a result, the intestinal bacterial flora may be out of balance and, in severe cases, myoclosis.

Therefore, in the situation of various digestive inflammatory diseases and immune hypersensitivity diseases such as atopic dermatitis known to be incurable of modern people or various diseases caused by lowered immunity, it is a health material that has been compared and evaluated through animal experiments. The purpose is to provide lactic acid bacteria strongly expressed from kimchi.

In order to solve the above problems, the present invention improves treatment and immunity of lactic acid bacteria L. plantarum-K-1 BR (LP) that inhibits AP-1 and NF-B activation from kimchi and improved atopic dermatitis, rhinitis, etc. It is a problem solving means of the present invention to provide a Lactobacillus plantarum K-1 BR strain (Accession Number KCCM11209P) having a control function.

The present invention causes a change in the intestinal flora (Microbiota) due to a complex cause of civilization, such as a uniform living environment, westernized diet, irregular lifestyle and stress, resulting in various digestive inflammatory diseases (diarrhea, ulcers) It can suppress the development of colitis, irritable bowel syndrome), immune hypersensitivity diseases (atopic dermatitis, asthma, various allergic diseases, diabetes, arthritis, etc.) and inflammatory diseases and various metabolic diseases, and can positively improve and treat the diseases. ,

It is expected that various diseases can be controlled by returning the damaged intestinal immune system to the intestinal flora of a healthy person. In addition, lactic acid bacteria can improve and treat immune diseases such as allergic diseases anaphylaxis, pruritus, etc. by regulating the intestinal immune system.

1 is a graph showing the inhibitory effect of IL-4, TNF-alpha expression (1A) and the activation of transcription factors NF-kappaB, AP-1 (1B) on PBL-stimulated RBL-2H3 cells of Lactobacillus plantarum K-1 BR

Figure 2 is a graph showing the inhibitory effect of Lactobacillus plantarum K-1 BR on IgE-induced passive dermal anaphylaxis reactions in mice.

Figure 3 is a graph showing the inhibitory effect of lactic acid bacteria Lactobacillus plantarum K-1 BR on histamine-induced pruritic action in mice. Antipruritic Effects of Lactic Acid Bacteria and Azelastine (3A) Anti-vascular Permeability of Lactic Acid Bacteria and Azelastine (3B).

4 is a graph showing the inhibitory effect of Lactobacillus plantarum K-1 BR on the expression of IL-4 (4A), TNF-alpha (4B), IL-1beta (4C) and IgE (4D) as histamine in mice.

5 is a graph showing the degranulation inhibitory effect of lactic acid bacteria K-1 BR

Figure 6 is a graph showing the effect of Lactobacillus K-1 BR on immunosuppressed mice with reduced T (6B) and NK (6A) cell numbers in the bone marrow and spleen induced by cyclocloamide.

Isolation and Identification of Novel Lactic Acid Bacteria

Kimchi crushed liquid was made by taking 10 g of kimchi. For kimchi crushing conditions, the mixture was thoroughly crushed at 2000 rpm for 2 minutes with a blender (RPM blender), diluted with 10 ^ 6 times of sterile purified water, plated in MRS (Man Rogosa Sharpe) agar medium, and cultured for 24 hours. Lactobacillus plantarum K-1 BR was prepared by separating and identifying 30 samples using API 50CHL kit.

The present invention searches for strains of NF-kB and AP-1 activation of rat basophilic leukemia (RBL) -2H3 cells induced by phorbol 12'-myristate 13'-acetate (PMA) from kimchi, and most strongly NF-kB. And the strain that inhibits the activation of AP-1 transcription factor is related to Lactobacillus plantarum K-1 BR, and on September 20, 2011 was deposited with the deposit number KCCM11209P to the Korea Microorganism Conservation Center.

Lactobacillus plantarum is a lactobacillus of the genus Lactobacillus, which is a rod-shaped bacilli and gram-positive.

In general, lactic acid bacteria are found in a variety of fermented foods, as well as anaerobic foods and plants, and are widely present in nature.

Its size is 0.7 ~ 1.0 × 3.0 ~ 8.0μ, and the optimum growth temperature is 29-33 ℃, and it is slightly aerobic. Lactobacillus plantarum is composed of arabinose, glucose, fructose, fructose, galactose, maltose, sucrose, dextran, and raffinose. And ferhalose (trehalose) and the like to produce lactic acid. Mainly separated from milk, cheese, butter, kefir, fermented sausage, fermented potatoes, grains, acid dough of bread, sesame like pickles or kimchi, tomatoes, etc. One of the lactic acid bacteria. In particular, lactobacillus plays an important role in the fermentation of Kimchi in Korea. Salt resistance is about 5.5%.

About 30 kinds of lactic acid bacteria are reported in Kimchi in Korea, and the kinds of bacteria grow according to fermentation, ripening and temperature of Kimchi. In the early stage of fermentation of Kimchi, a bacterium called Leuconostoc mesenteroides grows, and in the latter stage of fermentation, Lactobacillus plantarum, which is a stick-shaped lactobacillus with strong acid resistance, grows. Lactobacillus plantarum is a bacterium that grows mainly when kimchi is fermented and has a sour taste. It is resistant to acid and bile. In Europe, Lactobacillus plantarum is added to yogurt as a probiotic strain.

The Lactobacillus plantarum K-1 BR strain of the present invention analyzes Gram staining, general characteristics of the Lactobacillus plantarum K-1 BR strain, and API glycoavailability, and 16S after extracting DNA using QIAamp DNA Mini Kit for genetic analysis of the strain. Sequence comparison of 16S rRNA obtained by PCR and sequencing on the rRNA was performed (see Table 1 and Table 2).

It was Gram-positive bacillus, and the results of glucose analysis were shown in Table 1, and the results of sequencing were very similar to Lactobacillus plantarum, but not identical.

Table 1

No.	Our kind	Lactobacillus plantarum strain 1	Lactobacillus plantarum K-1 BR
One	Control	-	-
2	Glycerol	-	-
3	Erythritol	-	-
4	D-Arabinose	-	-
5	L-Arabinose	+	+
6	D-Ribose	+	+
7	D-Xylose	-	-
8	L-Xylose	-	-
9	Adonitol	-	-
10	Methyl-xyloside	-	-
11	D-Galactose	+	+
12	D-Glucose	+	+
13	D-Fructose	+	+
14	D-Mannose	+	+
15	L-Sorbose	-	-
16	Rhamnose	-	-
17	Dulcitol	-	-
18	Inositol	-	-
19	Mannitol	+	+
20	Sorbitol	+	+
21	Methyl-D-mannoside	+	+
22	Methyl-D-glucoside	-	-
23	N-Acetyl-glucosamine	+	+
24	Amydalin	+	+
25	Arbutine	+	+
26	Esulin	+	+
27	Salicin	+	+
28	Cellobiose	+	+
29	Maltose	+	+
30	Lactose	+	+
31	Melibiose	+	+
32	Sucrose	+	+
33	Trehalose	+	+
34	Inulin	-	-
35	Melesitose	+	+
36	Raffinose	+	+
37	Starch	-	-
38	Glycogen	-	-
39	Xylitol	-	-
40	Gentiobiose	+	+
41	D-Turanose	+	+
42	D-Lyxose	-	+
43	D-Tagatose	-	-
44	D-Fucose	-	-
45	L-Fucose	-	-
46	D-Arabitol	-	-
47	L-Arabitol	-	+
48	Gluconate	+	+
49	2-keto-gluconate	-	-
50	5-keto-glutonate	-	-

TABLE 2 16S rRNA Similarity of K-1 BR Strain with Lactobacillus plantarum

division	% Homology with K-1 BR strain
	Search	Align
Lactobacillus plantarum	99	99

Figure 1 shows the inhibitory effect of IL-4, TNF-alpha expression (1A) and transcription factors NF-kappaB, AP-1 (1B) inhibition of PBL-stimulated RBL-2H3 cells of Lactobacillus plantarum K-1 BR.

RBL-2H3 cells (2 × 10 ⁵ ) were stimulated with 20 nM PMA (PM), and L. plantarum K-1 BR (LPL, 0.2 × 10 ⁹ CFU / well; LPH, 110 ⁹ CFU / well) was treated. did. Normal group treated only vehicle instead of lactic acid bacteria. TNF- and IL-4 were measured by ELISA and NF-B and AP-1 by immunoblotting. Mean ± SD (n = 5). ^# p <0.05 vs. normal control group. ^* p <0.05 vs. PMA-treated group.

Figure 2 shows the inhibitory effect of Lactobacillus plantarum K-1 BR on IgE-induced passive dermal anaphylaxis reactions in mice. Lactobacillus and the control drug azelastine are administered once daily for 3 days. Normal group was vehicle only, CON was IgE-induced anaphylaxis, LPL was administered IgE and 1 × 10 ⁹ CFU L. plantarum K-1 BR, LPH was IgE and 1 × 10 ¹⁰ CFU L. plantarum K -1 BR lactic acid bacteria, AZ group was administered with IgE and 10 mg / kg azelastine. Mean ± SD (n = 5). ^# p <0.05 vs. normal control group. ^* p <0.05 vs. IgE-antigen complex-treated group.

Figure 3 shows the inhibitory effect of Lactobacillus plantarum K-1 BR on histamine-induced practicability in mice. Antipruritic Effects of Lactic Acid Bacteria and Azelastine (3A) Anti-vascular Permeability of Lactic Acid Bacteria and Azelastine (3B).

Lactobacillus and the control azelastine were administered once a day for 3 days. Normal group was vehicle only, CON was IgE to induce anaphylaxis, LPL was treated with histamine and 1 × 10 ⁹ CFU L. plantarum K-1 BR, LPH was treated with histamine and 1 × 10 ¹⁰ CFU L. plantarum K -1 BR lactic acid bacteria, AZ group was administered with histamine and 10 mg / kg azelastine. Mean ± SD (n = 5). ^# p <0.05 vs. normal control group. ^* p <0.05 vs. histamine-treated group.

Figure 4 shows the inhibitory effect of Lactobacillus plantarum K-1 BR on the expression of IL-4 (4A), TNF-alpha (4B), IL-1beta (4C) and IgE (4D) with histamine in mice. Lactobacillus and the control azelastine were administered once a day for 3 days. Normal group was vehicle only, CON was IgE-induced anaphylaxis, LPL was treated with histamine and 1 × 10 ⁹ CFU L. plantarum K-1 BR, LPH was histamine and 1 × 10 ¹⁰ CFU L. plantarum K -1 BR lactic acid bacteria, AZ group was administered with histamine and 10 mg / kg azelastine. Mean ± SD (n = 5). ^# p <0.05 vs. normal control group. ^* p <0.05 vs. IgE-antigen complex-treated group.

Figure 5 shows the degranulation inhibitory effect of lactic acid bacteria K-1 BR.

Figure 6 shows the effect of Lactobacillus K-1 BR on immunosuppressive mice with reduced T (6B) and NK (6A) cell numbers in cyclophosphamide-induced bone marrow and spleen.

Hereinafter, the specific configuration of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited only to the following Examples.

Example 1 Isolation and Identification of Novel Lactic Acid Bacteria

Kimchi crushed liquid was made by taking 10 g of kimchi. For kimchi crushing conditions, the mixture was thoroughly crushed at 2000 rpm for 2 minutes with a blender (RPM blender), diluted with 10 ^ 6 times of sterile purified water, plated in MRS (Man Rogosa Sharpe) agar medium, and cultured for 24 hours. Lactobacillus plantarum K-1 BR was prepared by separating and identifying 30 samples using API 50CHL kit.

16S rRNA sequence of Lactobacillus plantarum K-1 BR

<110> BIORHYTHM CO.LTD

<120> Lactobacillus plantarum, K-1 BR on antiallergic properties, anti

         level of immunity, microbial agent

<130> P-1412-1

<160> 1

<170> KopatentIn 2.0

<210> 1

<211> 504

<212> RNA

<213> Lactobacillus plantarum K-1 BR

<400> 1

agagtttgaa cctggctcag gacgaacgct ggcggcgtgc ctaatacatg caagtcgaac 60

gaactctggt attgattggt gcttgcatca tgatttacat ttgagtgagt ggcgaactgg 120

tgagtaacac gtgggaaacc tgcccagaag cgggggataa cacctggaaa cagatgctaa 180

taccgcataa caacttggac cgcatggtcc gagtttgaaa gatggcttcg gctatcactt 240

ttggatggtc ccgcggcgta ttagctagat ggtggggtaa cggctcacca tggcaatgat 300

acgtagccga cctgagaggg taatcggcca cattgggact gagacacggc ccaaactcct 360

acgggaggca gcagtaggga atcttccaca atggacgaaa gtctgatgga gcaacgccgc 420

gtgagtgaag aagggtttcg gctcgtaaaa ctctgttgtt aaagaagaac atatctgaga 480

gtaactgttc aggtatgacg gtat 504

Example 2 Measurement of TNF-a and IL-4

RBL-2H3 cells (3 × 10 ⁶ cells) were incubated in a 5% CO2 incubator at 37 ° C with moisture using DMEM, suspended cells were suspended by trypsin-EDTA solution, and then separated. Recovered and used for the experiment. RBL-2H3 cells were dispensed in 5 wells of 5 × 10 ⁵ cells / well in 24 wells, and then 0.5 μg / ml of IgE was added to the monoclonal cells of rats, and cultured in an incubator for 12 hours. The cells were washed with 0.5 ml of Shiraganian buffer (119 mM NaCl, 5 mM KCl, 0.4 mM MgCl ₂ , 25 mM PIPES, 40 mM NaOH, pH 7.2) and then 0.16 of Shiraganian buffer (5.6 mM glucose, 1 mM). CaCl ₂ , 0.1% BSA was added), and then incubated at 37 ° C. for 10 minutes.

Thereafter, 0.04 ml of lactic acid bacteria Lactobacillus plantarum K-1 BR (2 × 10 ⁸ or 1 × 10 ⁹ CFU / ml) treated for 30 minutes at 60 ° C. according to the present invention was added, followed by 0.02 ml of PMA 1 after 20 minutes. Pg / ml) was activated for 40 minutes at 37 ℃, centrifuged for 10 minutes at 2,000rpm, 0.025ml of the supernatant was transferred to 96 wells, IL-4 and TNF- with Endogen Elisa kit (USA) a was measured.

Example 3 Immunoblot analysis

RBL-2H3 cells (3 × 10 ⁶ cells) were incubated in a 5% CO2 incubator at 37 ° C with moisture using DMEM, suspended cells were suspended by trypsin-EDTA solution, and then separated. Recovered and used for the experiment. RBL-2H3 cells were dispensed in 5 wells of 5 × 10 ⁵ cells / well in 24 wells, and then 0.5 μg / ml of IgE was added to the monoclonal cells of rats, and cultured in an incubator for 12 hours. The cells were washed with 0.5 ml of siraganian buffer (119 mM NaCl, 5 mM KCl, 0.4 mM MgCl 2, 25 mM PIPES, 40 mM NaOH, pH 7.2), followed by 0.16 ml of shiraganian buffer (5.6 mM glucose (glucose). ), 1 mM CaCl ₂ , 0.1% BSA was added) and then incubated in the incubator for 10 minutes at 37 ℃. After that,

Lactobacillus plantarum K-1 BR (2 × 10 ⁸ or 1 × 10 ⁹ CFU / ml) treated with 30 minutes at 60 ° C. was added 0.04 ml, followed by 20 minutes, and then 0.02 ml of PMA 1 μg / ml. After activating the cells for 40 minutes at 37 ℃ and centrifuged for 10 minutes at 2,000rpm, hypotonic buffer (10mM Tris-HCl, 1.5mM MgCl ₂ , 1mM DTT, 0.1% NP-40, pepstatin 5㎍ / ㎖, aprotinin 5 Μg / ml) was added and left on ice for 15 minutes after suspension.

After centrifugation for 15 minutes at 4 ℃ and 12,000rpm, the supernatant cytosol fraction was taken and again precipitated by extraction buffer (10mM Tris-HCl, 50mM KCl, 300mM NaCl, 1mM DTT, pepstatin 5µg / ml, aprotinin 5µg / ml). 0.1 ml was added and the mixture was left on ice for 30 minutes after suspension, followed by centrifugation at 4 ° C and 12,000 rpm for 30 minutes, and the supernatant nuclear fraction was taken. After cell lysate (40㎍) was electrophoresed on 10% SDS polyacrylamide gel and transferred to PVDF (Millipore) membrane, the membrane was blocked with 2% skim milk solution dissolved in PBS for 1 hour at room temperature, and then again 1% skim milk. The primary antibody (NF-kB, AP-1, phosphor-AP-1, phosphor-NF-kB) diluted 1: 500 in the solution was slowly shaken for 2 hours. Discard the primary antibody dilution solution and wash twice with a washing solution (0.05% PBS-tween) for 10 minutes, and then slowly immersed the secondary antibody diluted 1: 5000 in 1% skim milk solution for 1 hour. The secondary antibody dilution solution was discarded as above, washed three times with a washing solution three times, and detected using an enhanced chemilluminescence system (PIERCE).

 As a result, Lactobacillus plantarum K-1 BR of Kimchi Lactobacillus inhibited the expression of IL-4 and TNF-a in the most strongly PMA-activated RBL-2H3 cells. [Fig. 1 (1A), (1B). Or Lactobacillus plantarum K-1 BR very favorably inhibited the activation of TNF-a transcription factor NF-kB (p-p65) and IL-4 transcription factor AP-1 (pc-jun). .

Example 4 Passive Skin Anaphylaxis (Antiallergic) Measurement Test

Passive dermal anaphylaxis model animal production was first performed by direct sensitization by injecting 10 μg of IgE serum against DNP-BSA (Dinitrophenol-bovine serum albumin) in physiological saline to the back of anesthetized mice with DNP-HSA. 0.2 ml of physiological saline containing 0.2 mg and 1.6 mg of evans blue was injected into the tail vein and killed by cervical dislocation 30 minutes later. Cut a portion of the back of the mouse, put it in a test tube, add 0.7 ml of 1N-KOH and incubate overnight at 37 ℃, 0.6 N phosphoric acid ^. 4 ml of acetone mixture (5:13) was added, followed by shaking and filtration to colorimetrically extract the extracted pigment at 620 nm. Samples were dissolved or suspended in physiological saline and administered orally or intraperitoneally one hour prior to challenge.

Lactobacillus plantarum K-1 BR (LPL, 1 × 10 ⁹ CFU / mouse; LPH, 1 × 10 ¹⁰ CFU / mouse) and azelastine (AZ, 10 mg / kg) were orally administered for 3 days prior to IgE administration.

The result is 1x10 ¹⁰ lactic acid bacteria Lactobacillus plantarum K-1 BR When administered, showed a more potent inhibitory effect of anaphylactic reaction than the control drug. [See Fig. 2]

Example 5 Antipruritic Effect

Lactobacillus in BALB / c mice at around 20 g body weight Lactobacillus plantarum K-1 BR Was administered orally for 3 days and placed in an acrylic observation box (22 × 22 × 24 cm) for 10 minutes after each last administration to purify the environment, and the back hairs were removed from the neck, and 300 μg / μl of histamine was added to a 29 gauge needle (gauge). After subcutaneous injection with neddle), immediately placed in an observation box, and the behavior of Soyang under unattended conditions was recorded by video camera for 1 hour. Soyang behavior is regarded as one act of scratching the back of the neck with the mouse's hind paw, and it is considered to be added once more than 1 second when performing continuous scratching, and actions such as scratching the ear, head, or licking the forefoot are It was not considered.

Lactobacillus (LPL, 1 × 10 ⁹ CFU / mouse; LPH, 1 × 10 ¹⁰ CFU / mouse) and azelastine (AZ, 10 mg / kg) were orally administered for 3 days prior to histamine administration.

As a result of 1x10 ¹⁰ lactic acid bacteria Lactobacillus plantarum K-1 BR Inhibition of AAC was significantly superior to the control drug when administered. [See Fig. 3 (3A)]

Example 6 Vascular Permeability Evaluation Experiment

Passive dermal anaphylaxis model animals were prepared by first injecting 10 μg of IgE serum to Dinitrophenol-bovine serum albumin (DNP-BSA) in physiological saline, and injecting them into the back of ether anesthetized mice. 0.2 ml of physiological saline containing 0.2 mg of HSA and 1.6 mg of evans blue was injected into the tail vein and killed by cervical dislocation 30 minutes later. Cut a portion of the back of the mouse, put it in a test tube, add 0.7 ml of 1N-KOH and incubate overnight at 37 degrees. To this test tube, 4 ml of 0.6 N phosphate and acetone mixture (5:13) were added, followed by shaking and filtration to colorimetrically determine the extracted pigment at 620 nm. Samples were dissolved or suspended in saline and administered orally or intraperitoneally one hour prior to challenge. Lactobacillus Lactobacillus plantarum K-1 BR (LPL, 1 × 10 ⁹ CFU / mouse; LPH, 1 × 10 ¹⁰ CFU / mouse) and azelastine (AZ, 10 mg / kg) were orally administered for 3 days prior to histamine administration.

As a result, when 1x10 ¹⁰ lactic acid bacteria were administered, the vascular permeability inhibitory effect was superior to that of the control drug. (See Fig. 3 (3B)]

Example 7 Inhibitory Effects of Lactobacillus plantarum K-1 BR Lactic Acid Bacteria on IgE, TNF-a, IL-1beta, and IL-4 Expression

Lactobacillus plantarum K-1 BR Lactobacillus plantarum in BALB / c mice before and after 20 g body weight Was administered orally for 3 days, and after 1 hour of the last administration, the back hair was removed, and 300 μg / μl of histamine was injected subcutaneously with a 29 gauge needle (gauge neddle), and 1 hour later, the injection site (1 × 1 cm) was cut out and It was eluted with cold lysis buffer (10 mM Tris, pH 7.5, 10 mM NaCl, 3 mM MgCl ₂ , 0.05% Nonidet P-40, 1 mM EGTA, 1: 100 protease inhibitor cocktail, and 1: 100 phosphatase inhibitor cocktail). The eluted solution was centrifuged at 2,700 g and 4 ^o C for 10 minutes. The supernatant was centrifuged at 20,800 × g, 4 ^° C. for 15 minutes (IgE, IL-4, TNF-a, IL-1beta analysis). Supernatants were transferred to 96 wells and measured by Enzyme-linked immunosorbent assay (ELISA, R & D systems (Minneapolis, MN, USA).

Lactobacillus plantarum K-1 BR (LPL, 1 × 10 ⁹ CFU / mouse; LPH, 1 × 10 ¹⁰ CFU / mouse) and azelastine (AZ, 10 mg / kg) were orally administered for 3 days prior to histamine administration.

As a result, 1x10 ⁹ , 1x10 ¹⁰ lactic acid bacteria Lactobacillus plantarum K-1 BR Inhibition of IgE, IL-4, TNF-a, IL-1beta expression was much better than the control drug when administered. (See Fig. 4 (4A), (4B), (4C), (4D)]

Example 8

Lactobacillus plantarum K-1 BR of Lactic Acid Bacteria

 Inhibitory effect of NF-kB and AP-1 activation

BALB / c mice weighing about 20 grams were orally administered with Lactobacillus plantarum K-1 BR for 3 days, and 1 hour after the last dose, the back of the neck was removed, and 300 μg / μl of histamine was injected subcutaneously with a 29 gauge needle (gauge neddle). 1 hour after injection, the injection site (1 × 1 cm) was cut out and ice-cold lysis buffer (10 mM Tris, pH 7.5, 10 mM NaCl, 3 mM MgCl ₂ , 0.05% Nonidet P-40,1mMEGTA, 1: 100 protease inhibitor eluted with cocktail, and 1: 100 phosphatase inhibitor cocktail. The eluted solution was centrifuged at 2,700 g and 4 ^o C for 10 minutes. This supernatant was centrifuged at 20,800 × g, 4 ^° C. for 15 minutes. This precipitate was washed by centrifugation three times with wash buffer (10 mM PIPES, pH 6.8, 300 mM sucrose, 3 mM MgCl ₂ , 1 mM MEGTA, 25 mM NaCl, 1: 100 protease inhibitor cocktail and 1: 100 phosphatase inhibitor cocktail). This precipitate was suspended and used for NF-kB, AP-1 immunoblot analysis.

Lactobacillus plantarum K-1 BR (LPL, 1 × 10 ⁹ CFU / mouse; LPH, 1 × 10 ¹⁰ CFU / mouse) and azelastine (AZ, 10 mg / kg) were orally administered for 3 days prior to histamine administration.

Immunoblot analysis

Sample lysate (40㎍) was electrophoresed on 10% SDS polyacrylamide gel and then transferred to PVDF (Millipore) membrane.The membrane was blocked with 2% skim milk solution dissolved in PBS for 1 hour at room temperature, and then again 1% skim milk. The primary antibody (NF-kB, AP-1, phosphor-AP-1, phosphor-NF-kB) diluted 1: 500 in the solution was slowly shaken for 2 hours. Discard the primary antibody dilution solution and wash twice with a washing solution (0.05% PBS-tween) for 10 minutes, and then slowly immersed the secondary antibody diluted 1: 5000 in 1% skim milk solution for 1 hour. The secondary antibody dilution solution was discarded as above, washed three times with a washing solution three times, and detected using an enhanced chemilluminescence system (PIERCE).

As a result of 1x10 ¹⁰ lactic acid bacteria Lactobacillus plantarum K-1 BR Administration of NF-kB, AP-1 was significantly inhibited compared to the control drug.

Example 9 Degranulation Test

RBL-2H3 cells were distributed in the Bank of Korea Cell Line and cultured in DMEM medium, 5% CO ₂ , 37 ° C. containing 10% FBS and L-glutamine. It was passaged 2-3 times a week.

Degranulation inhibitory activity of RBL-2H3 cells was measured by dispensing 0.8 mL of RBL-2H3 cells (5 × 10 ⁵ cells / ml) in a 24-well plate and incubating overnight with the addition of mouse monoclonal IgE (0.5ug / ml). box. The next day, the medium was removed and washed twice with 0.5 mL of siraganian buffer (119 mM NaCl, 5 mM KCl, 0.4 mM MgCl ₂ , 25 mM PIPES, 40 mM NaOH, pH 7.2). Again, 0.16 mL of Shiraganian buffer (5.6 mM glucose, 1 mM CaCl ₂ , 0.1% BSA added) was added and incubated at 37 ° C. for 10 minutes. Subsequently, 0.04 mL of the sample was added and incubated at 37 ° C. for 20 minutes. The reaction was stopped by adding 0.02 ml of antigen DNP-BSA (1 g / ml), incubating at 37 ° C. for 10 minutes, and standing on ice for 10 minutes. The degranulation inhibitory effect was evaluated by measuring the beta-hexosaminidase enzyme activity degranulated in the reaction solution. That is, after centrifugation of the reaction solution, 0.02 mL of supernatant and 96 mM p-NAG (p-nitrophenyl-N-acetyl-β-D-glucosaminide) dissolved in citrate buffer (0.1 M, pH 4.5) in a 96-well plate. 5 mL was made to react at 37 degreeC for 1 hour. Then, 0.2 mL of 0.1 M Na ₂ CO ₃ / NaHCO ₃ was added to stop the reaction, and the absorbance was measured at 405 nm of the ELISA reader.

As a result, Lactobacillus plantarum K-1 BR inhibited degranulation, but the effect was not strong.

Example 10 Immune Enhancer

Cytostaining for Identification of Phenotype of Cells

Immunosuppressed model animals were prepared with 300 mg / kg of cyclophosphamide in mice, and from the following day, Lactobacillus plantarum K-1 BR (1x10 ⁸ , 1x10 ⁹ CFU) was orally administered once daily for 3 days.

Splenocytes and bone marrow cells were collected and washed twice with FACS buffer, and the cells stained with a CD3 corresponding antibody having a wavelength of FITC or an NK1.1 antibody having a wavelength of PE in a dark room at 4 ° C. were stained twice. After washing, the cells were fixed with 1% paraformaldehyde dissolved in FACS buffer and analyzed by FACS.

As a result, K-1 BR increased NK and Tc cells decreased by cyclophosphamide (see FIG. 6).

In other words, LP is not only activated by the transcription factors NF-kB and c-jun but also by TNF-a and IL-4 in RBL-2H3 cells stimulated with phorbol 12'-myristate 13'-acetate (PMA). Expression was remarkably suppressed. In addition, LP showed a strong inhibitory effect on IgE-antigen-induced passive skin hypersensitivity reactions, and inhibited by 92.3% when ingested 1 × 10 ¹⁰ per mouse. Ingestion of 1 × 10 ¹⁰ CFU / LP of mice also inhibited histamine-induced practicability by 75.4%. LP significantly inhibited vascular osmoticity caused by histamine. LP inhibited histamine-induced increase in vascular osmotic pressure, which was directly proportional to the antipruritic effect of LP. LP (1 × 10 ¹⁰ CFU) inhibited the expression of histamine-induced IL-4, IL-1β and TNF-a by 92.7%, 94.5% and 99.2%, respectively. LP also inhibited histamine-induced IgE by 93.6%. As a result, LP suppresses the activation of transcription factors NF B and AP-1 in immune cells and regulates the expression of IgE-switching cytokine IL-4 and proinflammatory cytokines IL-1β and TNF-a. It can improve and treat diseases such as atopic dermatitis, rhinitis and pruritus.

The present invention has been made to solve the above problems, lactic acid bacteria Lactobacillus plantarum K-1 BR exhibits not only to increase immunity but also to anti-allergic effect, to improve and treat diseases such as allergic disease, atopic dermatitis, rhinitis, pruritus, etc. It can be used to improve disease treatment of various health foods, medicines, cosmetics, pets and livestock.

<110> BIORHYTHM CO.LTD

<120> Lactobacillus plantarum, K-1 BR on antiallergic properties, anti

         atopic dermatitis, anti rhinitis, anti pruritus. egulation of

         level of immunity, microbial agent

<130> P-1412-1

<160> 1

<170> KopatentIn 2.0

<210> 1

<211> 504

<212> RNA

<213> Lactobacillus plantarum K-1 BR

<400> 1

agagtttgaa cctggctcag gacgaacgct ggcggcgtgc ctaatacatg caagtcgaac 60

gaactctggt attgattggt gcttgcatca tgatttacat ttgagtgagt ggcgaactgg 120

tgagtaacac gtgggaaacc tgcccagaag cgggggataa cacctggaaa cagatgctaa 180

taccgcataa caacttggac cgcatggtcc gagtttgaaa gatggcttcg gctatcactt 240

ttggatggtc ccgcggcgta ttagctagat ggtggggtaa cggctcacca tggcaatgat 300

acgtagccga cctgagaggg taatcggcca cattgggact gagacacggc ccaaactcct 360

acgggaggca gcagtaggga atcttccaca atggacgaaa gtctgatgga gcaacgccgc 420

gtgagtgaag aagggtttcg gctcgtaaaa ctctgttgtt aaagaagaac atatctgaga 480

gtaactgttc aggtatgacg gtat 504

Claims (1)

1. Lactobacillus plantarum K-1 BR strain isolated from Kimchi to inhibit activation of NF-kB and AP-1 transcription factors (Accession No .; KCCM11209P)

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