KR102343239B1 - Novel strain of Lactobacillus plantarum having antimicrobial effect against pathogenic microorganism infection - Google Patents

Abstract

The present invention relates to microorganisms or fragments of microorganisms having an activity of inhibiting infection of various pathogenic microorganisms, including Staphylococcus aureus, specifically, containing microorganisms or fragments of microorganisms that enhance the expression of human beta-defensin-3 It relates to a composition that

Description

Lactobacillus having antimicrobial activity against infection of pathogenic microorganisms including Staphylococcus aureus {Novel strain of Lactobacillus plantarum having antimicrobial effect against pathogenic microorganism infection}

The present invention relates to a composition and a strain containing a lactic acid bacterium lysate, and specifically relates to a lactic acid bacterium having a higher activity of inhibiting infection of pathogenic microorganisms including Staphylococcus aureus than other strains.

Atopic dermatitis is a chronic inflammatory disease of the skin. Because it is not a simple skin disease, but a chronic systemic immune disease, treatment is not easy, and even if it is alleviated, the recurrence rate is high. Staphylococcus aureus is known to form colonies in more than 95% of the lesions of atopic dermatitis patients. In addition, it is said that there is a close relationship between the scale of colony formation and the severity of atopic dermatitis. Staphylococcus aureus secretes toxins along with inflammation to the skin, which can exacerbate atopic dermatitis. In addition, patients with atopic dermatitis have a reduced skin barrier function and are highly likely to develop complications due to secondary infections such as bacteria, viruses, and fungi. Therefore, if the infection of Staphylococcus aureus in the lesion area of atopic dermatitis patients is prevented, it is highly likely to be helpful in alleviating atopic dermatitis.

Therefore, antibiotic ointment is applied to the lesion area, or oral antibiotics are sometimes used in severe cases. However, frequent use of antibiotics results in antibiotic resistance. In other words, if you take antibiotics frequently, the existing antibiotics gradually stop working. Therefore, there is a need for research on new raw materials for atopic dermatitis treatment free from the problem of antibiotic resistance.

Accordingly, in the present application, human beta-defensin 3 (hBD-3), which is a kind of antibacterial peptide, from a lysate of a new lactic acid bacteria strain ' Lactobacillus plantarum KG (Lactobacillus plantarum KG)' was obtained from Lactobacillus plantarum sp. It was confirmed that it is expressed more than 500 times than that of various strains (KCTC 33133, 20124, 3105) in Thus, the present invention was completed.

It is an object of the present invention to provide a novel lactic acid bacterium having a high activity of inhibiting infection of pathogenic microorganisms including Staphylococcus aureus.

The present invention relates to a lactic acid bacterium belonging to the genus Lactobacillus, Lactobacillus plantarum KG (KCTC 14102BP) having a high activity of inhibiting infection of pathogenic microorganisms, including Staphylococcus aureus, and a lysate thereof.

The lactic acid bacteria according to the present invention has an infection inhibition activity of pathogenic microorganisms including Staphylococcus aureus by using the lysate obtained by crushing the lactic acid bacteria in a natural state without genetic manipulation of the lactic acid bacteria.

1 is a graph showing the results of measuring the mRNA expression level of the antibacterial peptide hBD-3 of Lactobacillus plantarum KG lysates and various Lactobacillus plantarum strain lysates.
Figure 2 is a graph showing the results of measuring the number of Staphylococcus aureus infection in skin epithelial cells when Lactobacillus plantarum KG lysates and various Lactobacillus plantarum strain lysates were pretreated.
3 is a graph showing the results of measuring the number of Staphylococcus aureus infection in skin epithelial cells when Lactobacillus plantarum KG lysates were pretreated by concentration.
Figure 4 is a graph showing the result that hBD-3 increased through Lactobacillus plantarum KG lysates directly reduces the number of Staphylococcus aureus.
Figure 5 is a graph showing the result that Lactobacillus plantarum KG lysates do not affect cytotoxicity.
6 is a graph showing the result that hBD-3 increased through Lactobacillus plantarum KG lysate directly reduces the number of other pathogenic microorganisms.

<Technical task>

The present invention relates to a microbial preparation having infection-suppressing activity of various pathogenic microorganisms, and more specifically, by increasing human beta-defensin-3 (hBD-3) expression in skin epithelial cells using microorganisms having infection-suppressing activity of pathogenic microorganisms The purpose is to provide a formulation.

<Technical solution>

The present invention provides Lactobacillus plantarum KG, which is a lactic acid bacterium isolated from kimchi.

<Production Example 1>

Manufacture of Lactobacillus plantarum KG lysates

The Lactobacillus plantarum KG strain of the present invention was inoculated in MRS broth and cultured at 37° C. for 20 hours. The culture solution was centrifuged at 8,000 rpm and 4° C. for 8 minutes to obtain cells, washed 3 times with sterile tertiary distilled water, and then crushed at 15,000 psi with a high pressure crusher, a total of 5 times. After freeze-drying, it was dissolved in DPBS (Welgene, Korea) and used for the test.

<Test Example 1>

Confirmation of hBD-3 expression level in Lactobacillus plantarum KG lysates

Skin epithelial cells (HaCaT) were prepared using Dulbecco's Modified Eagle's Medium (P/S, Welgene) containing 10% (v/v) fetal bovine serum (FBS, Welgene), 100 U/ml penicillin and 100 μg/ml streptomycin (P/S, Welgene). DMEM, Welgene, Korea). The incubator was maintained at a temperature of 37 °C, and _{an incubator in which 5% CO 2} was supplied was used. The cultured cells were used after culturing for 24 hours in a 6-well plate.

HaCaT cells, which are epithelial cells of the skin, were treated with ^{10 9} CFU of lysates of Lactobacillus plantarum K8 and lysates of Lactobacillus plantarum KG for 24, 48, 72 hours. RNA was extracted according to RNAiso Plus (TaKaRa) and product instructions. After synthesizing cDNA through PrimeScript ^TM RT Master Mix (TaKaRa), real-time PCR was performed using ^{CFX Connect TM} Real-Time PCR Detection System (Bio-Rad) and SYBR Premix Ex Taq ^{TM (TaKaRa).} . The primers used are as follows.

hBD-3: 5'-TCCATTATCTTCTTGTTTGCTTTGC-3' and 5'-TTCTGTAATGTGTTTATGATTCCTCCAT-3'

GAPDH: 5'-AAGGTCGGAGTCAACGGATT-3' and 5'-GCAGTGAGGGTCTCTCTCCT-3'

The results are shown in FIG. 1 .

As can be seen in FIG. 1 , it was confirmed that the hBD-3 mRNA expression level was increased 500-fold when ^{the Lactobacillus plantarum KG lysates of the present invention were treated with 10 9 CFU/ml for 72 hours.}

<Test Example 2>

Confirmation of Staphylococcus aureus infection in skin epithelial cells when Lactobacillus plantarum KG lysates were pretreated

Lactobacillus plantarum KG lysates obtained in Preparation Example of the present invention ^{were treated with 10 9} CFU of HaCaT cells, which are skin epithelial cells, for 72 hours. Then, Staphylococcus aureus was infected with 10 ⁷ CFU. After 6 hours, the culture medium was changed to a medium supplemented with Gentamicin antibiotic, and after 24 hours, HaCaT cells were lysed with hypertonic buffer. After spreading on BHI solid medium, which is a Staphylococcus aureus culture medium, CFU was counted the next day.

The results are shown in FIG. 2 .

As can be seen in FIG. 2 , when the Lactobacillus plantarum KG lysates of the present invention were ^{pretreated with 10 9} CFU/ml for 72 hours, Staphylococcus aureus infection in the skin epithelial cells was inhibited by 99% or more.

<Test Example 3>

Confirmation of Staphylococcus aureus infection in skin epithelial cells when Lactobacillus planta KG lysates were pretreated by concentration

All the test procedures were carried out in the same manner as in Test Example 2, but the Lactobacillus plantarum KG lysates in the initial stage of the test were conducted at various concentrations, not a single concentration.

The results are shown in FIG. 3 .

As can be seen in Figure 3, when the Lactobacillus plantarum KG lysates of the present invention were pretreated for 72 hours for each CFU, as the amount of CFU treatment of the Lactobacillus plantarum KG lysates increased, Staphylococcus aureus in the skin epithelial cells It can be seen that the infection is gradually suppressed.

<Test Example 4>

Confirmation of results that increased hBD-3 directly reduces the number of Staphylococcus aureus through Lactobacillus plantarum KG lysates

HaCaT cells were cultured for one day, washed 3 times with 1 ml DPBS (WelGene), and then the medium was replaced with DMEM (Welgene) containing no antibiotics. Thereafter, Lactobacillus plantarum KG lysates ^{were treated with 10 9} CFU/ml, for 72 hours. And only the culture medium was recovered, and this was named conditioned media. 10 μg of hBD-3 antibody (AF4435, R&D Systems) was added to 1 ml of conditioned media and stirred at 4° C. all day. After adding 20 μl SureBeads Protein G Magnetic Beads (BioRad), the mixture was stirred at 4° C. for 3 hours. Magnetic beads were removed by centrifugation at 2500 rpm for 1 minute, and since hBD-3 was removed from the obtained supernatant, it was named hBD-3 -. When beads were not used and hBD-3 was present, the supernatant was named hBD-3 +.

10, 20, and 50% of hBD-3 - and hBD-3 + were put in DMEM medium without antibiotics. Then, S. aureus was ^{treated with 10 4} CFU/ml for 1 hour, and then diluted and smeared on a Brain heart infusion (BHI) agar plate. The next day, the number of S. aureus was determined through colony counting.

The results are shown in FIG. 4 .

As can be seen in Figure 4, when hBD-3 is increased by treatment with Lactobacillus plantarum KG lysates of the present invention, it is removed through beads. Since hBD-3 is not present in the supernatant, the CFU of Staphylococcus aureus does not change even if the amount of the supernatant is increased. On the other hand, it was confirmed that the CFU of Staphylococcus aureus was significantly reduced when the amount of the supernatant containing hBD-3 was 50% because hBD-3 was not removed through beads. This shows that hBD-3 increased through Lactobacillus plantarum KG lysates can directly kill Staphylococcus aureus.

<Test Example 5>

Confirmation of results that Lactobacillus plantarum KG lysates do not affect cytotoxicity

HaCaT cells were cultured in 96 well plates. Thereafter, Lactobacillus plantarum KG lysates ^{were treated at 10 7} , 10 ⁸ , 10 ⁹ CFU/ml for 24, 48, 72 hours. The cell viability test was conducted using the EZ-Cytox Cell Viability Assay Kit (Daeil Lab Service, Korea) according to the instructions given. Absorbance was measured using an Eppendorf BioPhotometer, and absorbance was measured at 450 nm/590 nm.

The results are shown in FIG. 5 .

As can be seen in Figure 5, when the Lactobacillus plantarum KG lysates of the present invention were ^{treated with 10 9} CFU/ml for 72 hours, the absorbance showed a trend lower than that of the experimental group not treated for 72 hours, but there was no problem with significance. It was judged that there was no problem in cytotoxicity.

<Test Example 6>

Confirmation of results that increased hBD-3 directly reduces the number of harmful microorganisms through Lactobacillus plantarum KG lysates

HaCaT cells were cultured for one day, washed 3 times with 1 ml DPBS (WelGene), and then the medium was replaced with DMEM (Welgene) containing no antibiotics. Thereafter, Lactobacillus plantarum KG lysates ^{were treated with 10 9} CFU/ml, for 72 hours. And only the culture medium was recovered, and this was named conditioned media. 10 μg of hBD-3 antibody (AF4435, R&D Systems) was added to 1 ml of conditioned media and stirred at 4° C. all day. After adding 20 μl SureBeads Protein G Magnetic Beads (BioRad), the mixture was stirred at 4° C. for 3 hours. Magnetic beads were removed by centrifugation at 2500 rpm for 1 minute, and since hBD-3 was removed from the obtained supernatant, it was named hBD-3 -. When beads were not used and hBD-3 was present, the supernatant was named hBD-3 +.

50% of hBD-3 - and hBD-3 + were added to DMEM medium without antibiotics. And the pathogenic test bacteria described in [Table 2] below ^{were treated with 10 4} CFU/ml for 1 hour, and then diluted in a plate medium suitable for the growth of each pathogenic bacteria in [Table 2] and plated. The next day, the number of each bacteria was counted through colony counting.

The results are shown in FIG. 6 .

As can be seen in Figure 6, when hBD-3 is increased by treatment with Lactobacillus plantarum KG lysates of the present invention, it is removed through beads. Since hBD-3 is not present in the supernatant, the CFU of pathogenicity does not change even if the amount of the supernatant is increased. On the other hand, as in Test Example 4, when various pathogenic microorganisms were tested under the condition that the amount of the supernatant in which hBD-3 is present was 50%, it was confirmed that the CFU of the pathogenic microorganisms was significantly reduced. This shows that hBD-3 increased through Lactobacillus plantarum KG lysates can directly kill various pathogenic microorganisms.

Pathogenic microorganisms and suitable growth medium for antibacterial activity test pathogen Published strain code badge Acinetobacter baumannii NCCP 15998 Nutrient agar plate medium Acinetobacter baylyi ATCC 33305 Brain Heart Infusion agar plate medium Bordetella pertussis NCCP 13671 Bordet Gengou agar plate medium Pseudomonas aeruginosa NCCP 16100 Brain Heart Infusion agar plate medium Staphylococcus aureus NCCP 14560 Nutrient agar plate medium Streptococcus pneumoniae NCCP 14774 5% Sheep blood agar medium Streptococcus pyogenes NCCP 14783 Brain Heart Infusion agar plate medium Salmonella enteritidis KCDC 14546 Luria-Bertani (LB) agar plate medium Salmonella typhimurium KCDC 10328 Luria-Bertani (LB) agar plate medium Escherichia coli KCTC 2617 Luria-Bertani (LB) agar plate medium Haemophilus influenzae NCCP 14676 Gonococcal agr plate medium Klebsiella pneumoniae NCCP 16052 Nutrient agar plate medium Moraxella catarrhalis ATCC 43628 Brain Heart Infusion agar plate medium Yersinia enterocolitica KCDC 11129 Brain Heart Infusion agar plate medium Yersinia pseudotuberculosis KCDC 11125 Brain Heart Infusion agar plate medium

The pharmaceutical composition comprising the lactic acid bacteria or lactic acid bacteria disrupted body according to the present invention is in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, oral dosage forms, external preparations, suppositories, or sterile injection solutions according to a conventional method. It can be formulated and used, and may further include one or more additives selected from the group consisting of appropriate antibiotics, carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions.

Specifically, carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil can be used. For formulation, commonly used fillers, extenders, binders, wetting agents, and boron It can be prepared by using a diluent or excipient such as a release agent or a surfactant. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations include at least one excipient in the extract, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc. It can be prepared by mixing. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral use include suspensions, solutions, emulsions, syrups, etc., and various excipients, for example, wetting agents, sweeteners, fragrances, preservatives, etc. in addition to water and liquid paraffin, which are commonly used simple diluents, may be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base material for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.

In the pharmaceutical composition according to the present invention, the lactic acid bacteria or lactic acid bacteria crushed body may be included in an amount of 0.01 to 99.9 parts by weight, 0.1 to 90 parts by weight, 1 to 80 parts by weight, or 10 to 80 parts by weight based on 100 parts by weight of the pharmaceutical composition, but is limited thereto. However, it may vary depending on the patient's condition and the type and progression of the disease.

The lactic acid bacteria or lactic acid bacteria lysate according to the present invention has anti-inflammatory activity and can be used for prevention and treatment of inflammatory diseases or immune-related diseases. Accordingly, the present invention provides a pharmaceutical composition for the prevention and treatment of inflammatory diseases or immune-related diseases, including the lactic acid bacteria or lactic acid bacteria disrupted body, and the lactic acid bacteria or lactic acid bacteria disrupted body for the preparation of a medicament for the prevention and treatment of inflammatory diseases or immune-related diseases It provides a method for preventing and treating inflammatory diseases comprising administering to a subject a use and a therapeutically effective amount of lactic acid bacteria or lactic acid bacteria disrupted body.

Specifically, the inflammatory disease or immune-related disease is sepsis, arteriosclerosis, bacteremia, systemic inflammatory response syndrome, multiple organ dysfunction, cancer, osteoporosis, periodontitis, systemic lupus erythematosus, rheumatoid arthritis, osteoarthritis, juvenile chronic arthritis, spine Arthrosis, systemic sclerosis, idiopathic inflammatory myopathy, Sjoegren's syndrome, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia, autoimmune thrombocytopenia, thyroiditis, diabetes mellitus, immune-mediated kidney disease, central nervous system or Demyelination diseases of the peripheral nervous system, idiopathic demyelinating polyneuritis, Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuritis, hepatobiliary diseases, infectious or autoimmune chronic active hepatitis, primary biliary cirrhosis, granulation Hepatitis, sclerosing cholangitis, inflammatory bowel disease (IBD), ulcerative colitis, Crohn's disease, Irritable Bowel Syndrome, gluten-sensitive bowel disease, Hwi Whipple's disease, autoimmune or immune-mediated skin disease, vesicular skin disease erythema multiforme, contact dermatitis, psoriasis, allergic disease, asthma, allergic rhinitis, atopic dermatitis, food intolerance, urticaria, pulmonary immunity disease, eosinophilic pneumonia, idiopathic pulmonary fibrosis, hypersensitivity pneumonia, transplant-related disease, transplant rejection or graft-versus-host disease.

In one embodiment of the present invention, the lactic acid bacteria or lactic acid bacteria disrupted body may be administered to the subject by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dura mater or intracerebroventricular injection.

The preferred dosage of the lactic acid bacteria or lactic acid bacteria disrupted body according to the present invention may vary depending on the condition and weight of the patient, the type and extent of the disease, the drug form, the administration route and period, and may be appropriately selected by those skilled in the art. However, the daily dose is preferably 0.01 to 1,000 mg/kg, specifically, 0.1 to 1,000 mg/kg. Administration may be administered once a day or may be administered in several divided doses, thereby not limiting the scope of the present invention.

In the present invention, the 'subject' includes, but is not limited to, humans, orangutans, chimpanzees, mice, rats, dogs, cattle, chickens, pigs, goats, sheep, and the like.

The present invention also provides a food composition comprising the lactic acid bacteria or lactic acid bacteria disrupted body. The food composition is sepsis, arteriosclerosis, bacteremia, systemic inflammatory response syndrome, multiple organ dysfunction, cancer, osteoporosis, periodontitis, systemic lupus erythematosus, rheumatoid arthritis, osteoarthritis, juvenile chronic arthritis, spondyloarthropathy, systemic sclerosis, idiopathic Inflammatory myopathy, Sjoegren's syndrome, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia, autoimmune thrombocytopenia, thyroiditis, diabetes mellitus, immune-mediated kidney disease, demyelination of the central or peripheral nervous system Diseases, idiopathic demyelinating polyneuritis, Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuritis, hepatobiliary disease, infectious or autoimmune chronic active hepatitis, primary biliary cirrhosis, granulomatous hepatitis, sclerosing cholangitis , Inflammatory bowel IBD, Ulcerative colitis, Crohn's disease, Irritable Bowel Syndrome, gluten sensitive bowel disease, Whipple's disease, autoimmune or immune-mediated skin disease, vesicular skin disease erythema multiforme, contact dermatitis, psoriasis, allergic disease, asthma, allergic rhinitis, atopic dermatitis, food intolerance, urticaria, pulmonary immune disease, eosinophilic pneumonia, idiopathic lung Fibrosis, hypersensitivity pneumonia, transplant-related diseases, transplant rejection or graft-versus-host disease, such as inflammatory diseases or immune-related diseases, it can be used in the production of functional foods and general foods that can show the prevention and treatment effect.

In one embodiment of the present invention, the food composition is not limited thereto, but may further include one or more additives selected from the group consisting of organic acids, phosphates, antioxidants, lactose casein, dextrin, glucose, sugar and sorbitol. . The organic acid may be, but is not limited to, citric acid, humic acid, adipic acid, lactic acid or malic acid, and the phosphate salt may be, but is not limited to, sodium phosphate, potassium phosphate, acid pyrophosphate or polyphosphate (polyphosphate), and antioxidant The agent may be a natural antioxidant such as, but not limited to, polyphenols, catechins, alpha-tocopherol, rosemary extract, licorice extract, chitosan, tannic acid or phytic acid.

In the present invention, the lactic acid bacteria or lactic acid bacteria crushed body may be included in an amount of 0.01 to 99.9 parts by weight based on 100 parts by weight of the food composition, and specifically may be included in an amount of 1 to 90 parts by weight or 30 to 80 parts by weight.

In addition, the formulation of the food composition in one embodiment of the present invention is not limited thereto, but may be in solid, powder, granule, tablet, capsule or liquid form.

The food composition containing the lactic acid bacteria or lactic acid bacteria shreds according to the present invention is not limited thereto, but confectionery, sugars, ice cream products, dairy products, meat products, fish meat products, tofu or jelly products, edible oils and fats, noodles, teas, beverages, special nutrition Food, health supplements, seasonings, ice, ginseng products, pickled kimchi, raisins, fruits, vegetables, dried fruits or vegetables, cut products, fruit juices, vegetable juices, mixed juices thereof, chips, noodles, Processed livestock food, processed seafood, processed dairy food, fermented milk food, bean food, grain food, microbial fermented food, confectionery, confectionery, seasoning, processed meat, acidic beverage, licorice, herbs, etc. It can be used in the manufacture of foods. The form of the food includes, but is not limited to, solid, powder, granule, tablet, capsule, liquid or beverage form.

In another embodiment of the present invention, when the composition containing the lactic acid bacteria or lactic acid bacteria disrupted body is used in the production of health functional food, it may be used in an amount of 0.01 to 15% by weight of the total food weight, and when used in the preparation of beverages, the total It may be used in an amount of 0.02 to 10% by weight, or 0.3 to 1% by weight of the beverage.

In addition to the lactic acid bacteria or lactic acid bacteria crushed body, the food composition according to the present invention contains various nutrients, vitamins, minerals (electrolytes), synthetic flavoring agents and flavoring agents such as natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the food composition according to the present invention may contain pulp for the production of natural fruit juice, fruit juice beverage and vegetable beverage. The proportion of these additives is not limited thereto, but is generally selected from 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

The present invention also provides a cosmetic composition comprising the lactic acid bacteria or lactic acid bacteria disrupted body.

In the present invention, the lactic acid bacteria or crushed lactic acid bacteria may be included in an amount of 0.01 to 99.9 parts by weight based on 100 parts by weight of the cosmetic composition, and specifically may be included in an amount of 1 to 90 parts by weight or 30 to 80 parts by weight.

Cosmetics in which the cosmetic composition according to the present invention is used is not limited thereto in its formulation, but for example, softening lotion, nutrient lotion, massage cream, nutrient cream, pack, gel, essence, lipstick, makeup base, foundation, lotion, It may be included in ointments, gels, creams, cleansing agents, face washes, soaps, shampoos, rinses, treatments and cosmetics. Such cosmetics may include conventional ingredients such as water-based vitamins, oil-based vitamins, high-molecular peptides, high-molecular polysaccharides, and sphingolipids, and may be easily prepared according to techniques well known to those skilled in the art.

In addition to the above ingredients, additives commonly used in cosmetics, for example, vitamins, amino acids, proteins, surfactants, emulsifiers, fragrances, pigments, stabilizers, preservatives, antioxidants, sunscreens, pH adjusters and chelating agents are selected from the group consisting of One or more additives may be further included.

The present invention also provides a vaccine adjuvant comprising or consisting of the lactic acid bacteria or lactic acid bacteria disrupted body.

The lactic acid bacteria or lactic acid bacteria disrupted body according to the present invention can be used for various purposes. One preferred use is as an immunostimulatory agent or adjuvant for a pharmaceutical composition comprising an immunogenic polynucleotide, polypeptide, antibody, T-cell, or antigen-presenting cell (APC).

An adjuvant refers to a substrate that enhances the immune response to an antigen by inducing a faster antibody formation of the vaccine. The lactic acid bacteria or lactic acid bacteria lysate according to the present invention can increase the immune response to antigens by not only affecting the expression of inflammatory cytokines, but also affecting immune functions such as the expression of adherent molecules. It can also be used as a vaccine adjuvant because of its lack of immunogenicity.

One immune response that can be stimulated by the lactic acid bacteria or lactic acid bacteria lysate according to the present invention is of the Th1 or Th2 type, therefore, the adjuvant according to the present invention can be designed to preferentially induce an immune response of the Th1 or Th2 type. High concentrations of Th1-type cytokines (eg, TNF-α, IFN-γ, IL-2 and IL-12) tend to aid in the induction of cell-mediated immune responses to the administered antigen. In contrast, high concentrations of Th2-type cytokines (eg, IL-4, IL-5, IL-6, IL-10 and TNF-β) tend to aid in the induction of a humoral immune response. After application of the vaccine comprising lactic acid bacteria or lactic acid bacteria lysate according to the present invention, the patient will maintain an immune response comprising Th1- and Th2-type responses. If the response is predominantly Th1-type, the concentration of Th1-type cytokine will increase to a greater extent than that of Th2-type cytokine. The concentrations of these cytokines can be readily assessed using standard assays. For investigation of the cytokine family, see Mosmann and Coffman, Ann. Rev. Immunol. 7:145-173, 1989]. CpG-containing oligonucleotides (with unmethylated CpG dinucleotides) also induce predominantly Th1 responses, such oligonucleotides are well known and described, for example, in WO 96/02555.

Preferred adjuvants for use to induce a predominantly Th1 or Th2 type response may include the lactic acid bacteria or lactic acid bacteria disrupted body in combination with an aluminum salt.

<Preparation Example 2> Pharmaceutical containing lactic acid bacteria or lactic acid bacteria disrupted body

Preparation of powders

Lactobacillus or lactic acid bacteria lysate (analogues) 10 mg

Lactose 100 mg

talc 10 mg

The above ingredients are mixed and filled in an airtight bag to prepare a powder.

manufacture of tablets

5 mg of lactic acid bacteria or lactic acid bacteria lysate (analogues)

Corn Starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above ingredients, tablets are prepared by tableting according to a conventional manufacturing method of tablets.

Capsule preparation

Lactobacillus or lactic acid bacteria lysate (analogues) 10 mg

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium stearate 0.2 mg

According to a conventional capsule preparation method, the above ingredients are mixed and filled in a gelatin capsule to prepare a capsule.

manufacture of injections

Lactobacillus or lactic acid bacteria lysate (analogues) 50 mg

mannitol 180 mg

Sterile distilled water for injection 2974 mg

Na2HPO4·12H2O 26 mg

According to a conventional method for preparing injections, the content of the above ingredients per 1 ampoule (2 ml) is prepared.

Preparation of liquids

Lactobacillus or lactic acid bacteria lysate (analogues) 10 mg

10 g isomerized sugar

5 g of mannitol

Purified water appropriate amount

According to a conventional liquid preparation method, each component is added to purified water to dissolve, an appropriate amount of lemon flavor is added, the above components are mixed, purified water is added, and purified water is added to adjust the total to 100 ml, and then fill a brown bottle. Sterilize to prepare a solution.

<Production Example 3> Health food containing lactic acid bacteria or lactic acid bacteria crushed body

Lactobacillus or lactic acid bacteria lysate (analogues) 100 mg

appropriate amount of vitamin mixture

70 μg vitamin A acetate

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

0.2 μg of vitamin B12

Vitamin C 10 mg

Biotin 10 μg

Nicotinamide 1.7 mg

50 μg of folic acid

Calcium pantothenate 0.5 mg

Mineral mixture appropriate amount

Ferrous sulfate 1.75 mg

Zinc oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Dibasic calcium phosphate 55 mg

Potassium citrate 90 mg

Calcium carbonate 100 mg

Magnesium chloride 24.8 mg

The composition ratio of the vitamin and mineral mixture is relatively suitable for health food in a preferred embodiment, but the mixing ratio may be arbitrarily modified. , to prepare granules, and can be used for preparing health food compositions according to a conventional method.

<Preparation Example 4> Beverage containing lactic acid bacteria or lactic acid bacteria crushed body

Lactobacillus or lactic acid bacteria lysate (analogues) 100 mg

15 g vitamin C

100 g vitamin E (powder)

iron lactate 19.75 g

3.5 g zinc oxide

3.5 g of nicotinic acid amide

0.2 g vitamin A

0.25 g of vitamin B1

Vitamin B2 0.3g

water metering

After mixing the above ingredients according to the usual health drink manufacturing method, after stirring and heating at 85 ° C for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 liter container, sealed and sterilized, then refrigerated. It is used to prepare the health drink composition of the invention.

Although the composition ratio is prepared by mixing ingredients suitable for relatively favorite beverages in a preferred embodiment, the mixing ratio may be arbitrarily modified according to regional and national preferences such as demanding class, demanding country, and use.

<Preparation Example 5> Cosmetics containing lactic acid bacteria or lactic acid bacteria crushed body

Manufacture of flexible lotion (skin)

Lactobacillus or lactic acid bacteria lysate (analogues) 0.1% by weight

Glycerin 3.0 wt%

Butylene glycol 2.0 wt%

2.0% by weight of propylene glycol

Polyoxyethylene (60) hydrogenated castor oil 1.00 wt%

Ethanol 10.0 wt%

0.1% by weight of triethanolamine

preservative trace

pigment trace

spice trace

Purified water remaining

Manufacture of nutrient lotion (lotion)

Lactobacillus or lactic acid bacteria lysate (analogues) 0.1% by weight

1.70 wt% sitosterol

1.50 wt % polyglyceryl 2-oleate

Ceteareth-4 1.2 wt%

Cholesterol 1.5% by weight

dicetyl phosphate 0.4 wt%

5.0 wt% of concentrated glycerin

Sunflower oil 10.0 wt%

0.2% by weight of carboxyvinyl polymer

0.3% by weight of xanthan gum

preservative trace

spice trace

Purified water remaining

<Preparation Example 6> Adjuvant containing lactic acid bacteria or lactic acid bacteria disrupted body

Preparation of adjuvant containing lactic acid bacteria or lactic acid bacteria disrupted body

Lactobacillus or lactic acid bacteria lysate (analogues) 25㎍

Dioleoyl phosphatidylcholine (DOPC) 500㎍

Cholesterol 125㎍

Phosphate NaCl buffer and water balance

Total 0.5ml

An adjuvant consisting of the above ingredients is prepared as described in WO96/33739.

Preparation of influenza vaccine composition containing lactic acid bacteria or lactic acid bacteria lysate adjuvant ingredient amount per dose active ingredient Inactivated Split Virion 15 μg HA - A/New Calidonia/20/99 (H1N1) IVR-116 15 μg HA - A/New York/55/2004 (H3N2) NYMC x-157 15 μg HA - B/Jiangsu/10/2003 adjuvant -liposomes Dioleyl phosphatidylcholine (DOPC) 1,000 μg ·cholesterol 250 μg Lactobacillus or lactic acid bacteria shredder (Analogues) 50 μg

One dose of the influenza vaccine corresponds to 1 mL.

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Korea Institute of Biotechnology and Biotechnology KCTC14102BP 20200110

Claims (11)

As a lysate of Lactobacillus plantarum KG (Accession No.: KCTC 14102BP) having an activity of inhibiting infection of pathogenic microorganisms,
A lysate of Lactobacillus plantarum KG (Accession No.: KCTC 14102BP), characterized in that it has anti-infection activity of pathogenic microorganisms by enhancing the expression of human beta-defensin-3. delete delete According to claim 1, wherein the pathogenic microorganism is Staphylococcus aureus, Acinetobacter baumannii, Acinetobacter baylyi, Bordetella pertussis, Klebsiella Klebsiella pneumoniae, Moraxella catarrhalis, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pneumoniae, Streptococcus pyogenes, Escherichia coli, Salmonella Enteritidis, Salmonella Typhimurium, Haemophilus influenzae, E. enteritidis Colica (Yersinia enterocolitica) and Ersinia pseudotuberculosis (Yersinia pseudotuberculosis) is any one or more selected from, Lactobacillus plantarum KG (Accession No.: KCTC 14102BP) crushed body. delete According to claim 1, wherein the Lactobacillus plantarum KG (Accession No.: KCTC 14102BP) the crushed body, characterized in that the dried powder form, Lactobacillus plantarum KG (Accession No.: KCTC 14102BP) crushed body. The composition for food or health functional food for inhibiting infection of pathogenic microorganisms, comprising the lysate of Lactobacillus plantarum KG (Accession No.: KCTC 14102BP) according to claim 1. A cosmetic composition for inhibiting infection of pathogenic microorganisms comprising the lysate of Lactobacillus plantarum KG (Accession No.: KCTC 14102BP) according to claim 1. A composition for quasi-drugs for inhibiting infection of pathogenic microorganisms comprising the lysate of Lactobacillus plantarum KG (Accession No.: KCTC 14102BP) according to claim 1. A pharmaceutical composition for inhibiting infection of pathogenic microorganisms comprising a lysate of Lactobacillus plantarum KG (Accession No.: KCTC 14102BP) according to claim 1. A composition for inhibiting atopic dermatitis caused by infection of a pathogenic microorganism comprising a lysate of Lactobacillus plantarum KG (Accession No.: KCTC 14102BP) according to claim 1.

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