WO2016093450A1 - 신규한 균주로 알러지, 아토피성 피부염, 비염, 가려움증 등의 개선치료 및 면역력조절 기능을 갖는 락토바실러스 플랜타럼 k-1 br 균주 - Google Patents

신규한 균주로 알러지, 아토피성 피부염, 비염, 가려움증 등의 개선치료 및 면역력조절 기능을 갖는 락토바실러스 플랜타럼 k-1 br 균주 Download PDF

Info

Publication number
WO2016093450A1
WO2016093450A1 PCT/KR2015/006103 KR2015006103W WO2016093450A1 WO 2016093450 A1 WO2016093450 A1 WO 2016093450A1 KR 2015006103 W KR2015006103 W KR 2015006103W WO 2016093450 A1 WO2016093450 A1 WO 2016093450A1
Authority
WO
WIPO (PCT)
Prior art keywords
lactobacillus plantarum
lactobacillus
lactic acid
strain
acid bacteria
Prior art date
Application number
PCT/KR2015/006103
Other languages
English (en)
French (fr)
Korean (ko)
Inventor
정용현
정진웅
Original Assignee
(주)바이오리듬
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by (주)바이오리듬 filed Critical (주)바이오리듬
Priority to CN201580067058.0A priority Critical patent/CN107109351A/zh
Publication of WO2016093450A1 publication Critical patent/WO2016093450A1/ko

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/746—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for lactic acid bacteria (Streptococcus; Lactococcus; Lactobacillus; Pediococcus; Enterococcus; Leuconostoc; Propionibacterium; Bifidobacterium; Sporolactobacillus)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus
    • C12R2001/25—Lactobacillus plantarum
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • 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.
  • 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).
  • 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 centuries, and is old enough to appear in Hippocrates' book.
  • Hippocrates (460-377 B.C.) report that fermented foods are effective for diarrhea patients.
  • 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.
  • 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.
  • inflammatory bowel disease Crohn's disease and colitis
  • 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.
  • 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.
  • 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.
  • lactic acid bacteria increase the immune mechanisms in our bodies to suppress the infection of pathogenic bacteria that cause disease.
  • 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,
  • 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.
  • pH gastric acid
  • intestinal motility a substance that influences the secretion of gastric acid and serous secretions.
  • mucus a substance that influences the secretion of gastric acid and serous secretions.
  • 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. ,
  • 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).
  • FIG. 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.
  • 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.
  • 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.
  • RBL basophilic leukemia
  • PMA phorbol 12'-myristate 13'-acetate
  • 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.
  • lactic acid bacteria are found in a variety of fermented foods, as well as anaerobic foods and plants, and are widely present in nature.
  • 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%.
  • 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.
  • 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).
  • 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.
  • 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).
  • 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.
  • 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.
  • 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 ⁇ g / 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.
  • Shiraganian buffer 119 mM NaCl, 5 mM KCl, 0.4 mM MgCl 2 , 25 mM PIPES, 40 mM NaOH, pH 7.2
  • Shiraganian buffer 5.6 mM glucose, 1 mM
  • 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 ⁇ g / 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 °C. After that,
  • hypotonic buffer (10mM Tris-HCl, 1.5mM MgCl 2 , 1mM DTT, 0.1% NP-40, pepstatin 5 ⁇ g / ml, aprotinin 5 ⁇ g / ml) was added and left on ice for 15 minutes after
  • the supernatant cytosol fraction was taken and again precipitated by extraction buffer (10mM Tris-HCl, 50mM KCl, 300mM NaCl, 1mM DTT, pepstatin 5 ⁇ g / ml, aprotinin 5 ⁇ g / 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.
  • extraction buffer 10mM Tris-HCl, 50mM KCl, 300mM NaCl, 1mM DTT, pepstatin 5 ⁇ g / ml, aprotinin 5 ⁇ g / ml.
  • 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.
  • 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). .
  • Passive dermal anaphylaxis model animal production was first performed by direct sensitization by injecting 10 ⁇ g of IgE serum against DNP-BSA (Dinitrophenol-bovine serum albumin) in physiological saline to the back of anesthetized mice with DNP-HSA.
  • DNP-BSA Dinitrophenol-bovine serum albumin
  • 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 °C, 0.6 N phosphoric acid .
  • 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.
  • 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 ⁇ g / ⁇ l 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.
  • Passive dermal anaphylaxis model animals were prepared by first injecting 10 ⁇ g of IgE serum to Dinitrophenol-bovine serum albumin (DNP-BSA) in physiological saline, and injecting them into the back of ether anesthetized mice.
  • DNP-BSA Dinitrophenol-bovine serum albumin
  • 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.
  • Example 7 Inhibitory Effects of Lactobacillus plantarum K-1 BR Lactic Acid Bacteria on IgE, TNF-a, IL-1beta, and IL-4 Expression
  • 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
  • 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.
  • 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 ⁇ g / ⁇ l 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.
  • 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:
  • 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.
  • 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.
  • 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.
  • Sample lysate (40 ⁇ g) 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).
  • PIERCE enhanced chemilluminescence system
  • 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).
  • Lactobacillus plantarum K-1 BR inhibited degranulation, but the effect was not strong.
  • 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.
  • K-1 BR increased NK and Tc cells decreased by cyclophosphamide (see FIG. 6).
  • 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.
  • PMA phorbol 12'-myristate 13'-acetate
  • 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-1 ⁇ and TNF-a by 92.7%, 94.5% and 99.2%, respectively.
  • LP also inhibited histamine-induced IgE by 93.6%.
  • 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-1 ⁇ and TNF-a. It can improve and treat diseases such as atopic dermatitis, rhinitis and pruritus.
  • 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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Mycology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Epidemiology (AREA)
  • Medical Informatics (AREA)
  • Alternative & Traditional Medicine (AREA)
  • Botany (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Plant Pathology (AREA)
  • Immunology (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Coloring Foods And Improving Nutritive Qualities (AREA)
PCT/KR2015/006103 2014-12-09 2015-06-17 신규한 균주로 알러지, 아토피성 피부염, 비염, 가려움증 등의 개선치료 및 면역력조절 기능을 갖는 락토바실러스 플랜타럼 k-1 br 균주 WO2016093450A1 (ko)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201580067058.0A CN107109351A (zh) 2014-12-09 2015-06-17 作为新型菌株的具有改善治疗过敏、遗传过敏性皮炎、鼻炎、搔痒症等及免疫力调节功能的植物乳杆菌k‑1 br菌株

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR10-2014-0175693 2014-12-09
KR1020140175693A KR20160069733A (ko) 2014-12-09 2014-12-09 신규한 균주로 알러지, 아토피성 피부염, 비염, 가려움증 등의 개선치료 및 면역력조절 기능을 갖는 Lactobacillus plantarum K-1 BR 균주

Publications (1)

Publication Number Publication Date
WO2016093450A1 true WO2016093450A1 (ko) 2016-06-16

Family

ID=56107606

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2015/006103 WO2016093450A1 (ko) 2014-12-09 2015-06-17 신규한 균주로 알러지, 아토피성 피부염, 비염, 가려움증 등의 개선치료 및 면역력조절 기능을 갖는 락토바실러스 플랜타럼 k-1 br 균주

Country Status (3)

Country Link
KR (1) KR20160069733A (zh)
CN (1) CN107109351A (zh)
WO (1) WO2016093450A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021162282A1 (ko) * 2020-02-13 2021-08-19 주식회사 케이티앤지 신규 락토바실러스 플란타룸 kc3 균주를 이용한 면역장애, 호흡기 염증 질환, 알레르기 및 천식의 예방 또는 치료용 조성물 및 이의 용도

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2018216532B2 (en) * 2017-01-31 2021-06-03 Navipharm Co, Ltd Novel lactic acid bacteria and use thereof
KR102185827B1 (ko) 2020-08-26 2020-12-03 주식회사 엔테로바이옴 아커만시아 뮤시니필라 균주를 포함하는 아토피성 질환 예방 또는 치료용 약학적 조성물
KR102185828B1 (ko) 2020-08-26 2020-12-03 주식회사 엔테로바이옴 피칼리박테리움 프로스니치 균주를 포함하는 아토피성 질환 예방 또는 치료용 약학적 조성물

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20060096851A (ko) * 2005-03-04 2006-09-13 백경수 알레르기 질환 예방 및 치료 기능을 가진 유산균 락토바실러스 프랜타룸
KR20130034764A (ko) * 2011-09-29 2013-04-08 정용현 신규한 균주로 항알러지, 아토피성 피부염 개선 및 면역력증진 기능을 갖는 Lactobacillus plantarum K-1과 이를 함유하는 미생물제제

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20050076374A (ko) 2004-01-20 2005-07-26 김수남 아토피와 항알러지 및 장내유해세균의 암모니아 생산을저해하는 활성을 갖는 두류 유산균 발효물을 함유한기능성 제품 및 그의 제조방법
KR101255050B1 (ko) * 2009-07-14 2013-04-16 씨제이제일제당 (주) 신규한 락토바실러스 플란타룸 및 이를 포함하는 조성물
KR101178217B1 (ko) * 2009-10-28 2012-09-07 씨제이제일제당 (주) 신규한 락토바실러스 플란타룸 및 이를 포함하는 조성물

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20060096851A (ko) * 2005-03-04 2006-09-13 백경수 알레르기 질환 예방 및 치료 기능을 가진 유산균 락토바실러스 프랜타룸
KR20130034764A (ko) * 2011-09-29 2013-04-08 정용현 신규한 균주로 항알러지, 아토피성 피부염 개선 및 면역력증진 기능을 갖는 Lactobacillus plantarum K-1과 이를 함유하는 미생물제제

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JANG, SE - EUN ET AL.: "Inhibitory Effect of Lactobacillus piantarum K-1 on Passive Cutaneous Anaphylaxis Reaction and Scratching Behavior in Mice.", ARCH. PHARM. RES., vol. 34, no. 12, December 2011 (2011-12-01), pages 2117 - 2123 *
JANG, SE EUN: "Anti-allergic Effects of Kimchi-derived Lactobacillus Plantarum K-1.", MASTER'S THESIS, August 2011 (2011-08-01) *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021162282A1 (ko) * 2020-02-13 2021-08-19 주식회사 케이티앤지 신규 락토바실러스 플란타룸 kc3 균주를 이용한 면역장애, 호흡기 염증 질환, 알레르기 및 천식의 예방 또는 치료용 조성물 및 이의 용도

Also Published As

Publication number Publication date
KR20160069733A (ko) 2016-06-17
CN107109351A (zh) 2017-08-29

Similar Documents

Publication Publication Date Title
Karami et al. Isolation and identification of probiotic Lactobacillus from local dairy and evaluating their antagonistic effect on pathogens
US10471111B2 (en) Lactobacillus and composition for preventing, improving, or treating degenerative brain diseases or cognitive function disorders
WO2019151843A1 (ko) 락토바실러스 플란타럼 kbl396 균주 및 그 용도
WO2011007922A1 (ko) 신규한 락토바실러스 플란타룸 및 이를 포함하는 조성물
WO2010064777A1 (ko) 신규한 락토바실러스 플란타룸 및 이를 포함하는 조성물
JP7088573B2 (ja) ラクトバチルス・ガッセリーｋｂｌ６９７株及びその使用
CN102597214B (zh) 新型植物乳杆菌及含该植物乳杆菌的组合物
WO2016093450A1 (ko) 신규한 균주로 알러지, 아토피성 피부염, 비염, 가려움증 등의 개선치료 및 면역력조절 기능을 갖는 락토바실러스 플랜타럼 k-1 br 균주
WO2011052996A2 (en) Novel lactobacillus plantarum and composition comprising the same
Tuo et al. Study of probiotic potential of four wild Lactobacillus rhamnosus strains
WO2017047962A1 (ko) 신규 유산균 및 퇴행성 뇌질환 또는 인지기능 장애의 예방, 개선 또는 치료용 조성물
KR20130034764A (ko) 신규한 균주로 항알러지, 아토피성 피부염 개선 및 면역력증진 기능을 갖는 Lactobacillus plantarum K-1과 이를 함유하는 미생물제제
KR20180044245A (ko) 변비 개선 효능을 갖는 신규한 유산균 및 이의 용도
WO2017131402A1 (ko) 면역조절 작용을 가는 사람 소화관 유래 신규 유산균 및 이의 용도
WO2019098810A2 (ko) 신규 유산균 및 이의 용도
WO2020091312A1 (ko) 프로바이오틱스를 유효성분으로 포함하는 알코올성 장손상 예방 또는 치료용 조성물
KR101838281B1 (ko) 알레르기성 질환의 개선 활성을 갖는 락토바실러스 커베터스 wikim53 및 이를 포함하는 조성물
KR101406168B1 (ko) 신규한 락토바실러스 플란타룸 및 이를 포함하는 조성물
CN1329505C (zh) 发酵乳酸杆菌GM－090及其在生产刺激INF－γ分泌及/或治疗过敏的药物中的用途
WO2010064778A1 (ko) 신규한 락토바실러스 플란타룸 및 이를 포함하는 조성물
US11058733B2 (en) Active substances of Bifidobacterium lactis GKK2, composition comprising the same and method of promoting longevity using the same
WO2018034492A1 (ko) 페디오코쿠스 펜토사세우스를 유효성분으로 포함하는 th1-매개 면역 질환, th17-매개 면역 질환 또는 th2-매개 면역 질환의 예방, 개선 또는 치료용 조성물
WO2022039561A1 (ko) 클로스트리디움 디피실리 감염의 치료 또는 예방용 조성물
WO2022039514A1 (ko) 락토바실러스 사케이 또는 이로부터 유래된 세포밖 소포체를 유효성분으로 포함하는 뇌질환의 치료용 조성물
WO2020111674A1 (ko) 녹차 유래 유산균을 포함하는 미세먼지에 의한 탈모 케어용 조성물

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15867274

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15867274

Country of ref document: EP

Kind code of ref document: A1