WO2023038419A1 - Intestinal health strain lactobacillus paracasei eps da-bacs having lactobacillus bifidus growth promotion and harmful intestinal bacteria clostridium difficile inhibitory effects, and polysaccharide thereof - Google Patents

Intestinal health strain lactobacillus paracasei eps da-bacs having lactobacillus bifidus growth promotion and harmful intestinal bacteria clostridium difficile inhibitory effects, and polysaccharide thereof Download PDF

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WO2023038419A1
WO2023038419A1 PCT/KR2022/013422 KR2022013422W WO2023038419A1 WO 2023038419 A1 WO2023038419 A1 WO 2023038419A1 KR 2022013422 W KR2022013422 W KR 2022013422W WO 2023038419 A1 WO2023038419 A1 WO 2023038419A1
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eps
bacs
lactobacillus paracasei
exopolysaccharide
present
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PCT/KR2022/013422
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French (fr)
Korean (ko)
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한상덕
한영선
박민주
조현일
이은석
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동아제약 주식회사
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Publication of WO2023038419A1 publication Critical patent/WO2023038419A1/en

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • 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
    • 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
    • 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
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to an intestinal health strain, Lactobacillus paracasei EPS DA-BACS, and a polysaccharide thereof, which have an effect of promoting the growth of bifidus lactic acid bacteria and inhibiting the intestinal harmful bacteria Clostridium difficile .
  • Probiotics is a general term for microorganisms that are beneficial to the human body, and typically includes lactic acid bacteria. Characteristics that probiotics should have are that they have the human intestinal tract as their habitat, are sensitive to antibiotics, and have no pathogenicity or toxicity. In addition, while going to the intestine, it must have resistance to acid, bile, etc., and must express useful effects well.
  • lactic acid bacteria biosynthesize a polysaccharide called EPS (exopolysaccharide), which is one of the metabolites of microorganisms that form capsular membranes around cell walls or accumulate in the form of slime on the outside of cell walls.
  • EPS exopolysaccharide
  • the polysaccharide produced by lactic acid bacteria has various and unique physical properties, so it can be used as a material with physical properties and functions. It also has excellent physiological functions such as anti-cancer effects, cholesterol inhibition, immune regulation, and constipation improvement, and interest in lactic acid bacteria that produce EPS is gradually increasing. It is rising.
  • EPS is divided into a capsular form attached to the cell wall and a ropy form secreted extracellularly according to the structure and degree of polymerization.
  • Ropy-EPS is characterized by the formation of a viscous material in the form of solid lines in colonies, and provides relatively high viscosity, water retention and soft density.
  • literature has been reported that Ropy-EPS significantly increases the survival and attachment of lactic acid bacteria compared to Non-ropy-EPS in previous studies, lowers serum cholesterol, and suppresses hyperimmune reactions.
  • An object of the present invention is to discover and provide lactic acid bacteria that produce Ropy-EPS.
  • the present invention provides Lactobacillus paracasei EPS DA-BACS (KCTC14639BP).
  • the strain is preferably characterized by producing exopolysaccharide (EPS) as a postbiotics, and the EPS (exopolysaccharide) is preferably EPS in the form of ropy secreted extracellularly. It is characterized in that it contains (exopolysaccharide).
  • EPS exopolysaccharide
  • the present invention provides an antibacterial agent containing any one selected from Lactobacillus paracasei EPS DA-BACS (KCTC14639BP), dead cells thereof, and cultures thereof.
  • the antibacterial agent preferably Pseudomonas aeruginosa , Bacillus subtilis ( Bacillus subtilis ), Escherichia coli ( Escherichia coli ), Clostridium difficile bacteria ( Clostridium difficile ) and It is good to have antibacterial activity against Staphylococcus aureus .
  • the present invention provides an antifungal agent containing any one selected from Lactobacillus paracasei EPS DA-BACS (KCTC14639BP), dead cells thereof, and cultures thereof.
  • the antifungal agent preferably has antifungal activity against Aspergillus brasiliensis .
  • the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases containing any one selected from Lactobacillus paracasei EPS DA-BACS (KCTC14639BP), its dead cells and its culture.
  • the present invention provides a food composition for improving inflammatory diseases containing any one selected from Lactobacillus paracasei EPS DA-BACS (KCTC14639BP), its dead cells and its culture.
  • the present invention provides a health functional food for improving inflammatory diseases containing any one selected from Lactobacillus paracasei EPS DA-BACS (KCTC14639BP), its dead cells and its culture.
  • the present invention provides a cosmetic composition for preventing or improving skin inflammation containing any one selected from Lactobacillus paracasei EPS DA-BACS (KCTC14639BP), its dead cells and its culture.
  • the present invention provides EPS (exopolysaccharide) produced by Lactobacillus paracasei EPS DA-BACS (KCTC14639BP), and the EPS (exopolysaccharide) is preferably secreted extracellularly. ) characterized in that it comprises a form of EPS (exopolysaccharide).
  • the EPS exopolysaccharide
  • EPS exopolysaccharide
  • a) Lactobacillus paracasei Lactobacillus paracasei
  • EPS DA-BACS KCTC14639BP
  • the present invention provides an antibacterial agent containing EPS (exopolysaccharide) produced by Lactobacillus paracasei EPS DA-BACS (KCTC14639BP).
  • EPS exopolysaccharide
  • the antibacterial agent preferably Pseudomonas aeruginosa , Bacillus subtilis ( Bacillus subtilis ), Escherichia coli ( Escherichia coli ), Clostridium difficile bacteria ( Clostridium difficile ) and It is good to have antibacterial activity against Staphylococcus aureus .
  • the present invention provides an antifungal agent containing EPS (exopolysaccharide) produced by Lactobacillus paracasei EPS DA-BACS (KCTC14639BP).
  • EPS exopolysaccharide
  • the antifungal agent preferably has antifungal activity against Aspergillus brasiliensis .
  • the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases containing EPS (exopolysaccharide) produced by Lactobacillus paracasei EPS DA-BACS (KCTC14639BP).
  • EPS exopolysaccharide
  • Lactobacillus paracasei EPS DA-BACS KCTC14639BP
  • the present invention provides a food composition for improving inflammatory diseases containing EPS (exopolysaccharide) produced by Lactobacillus paracasei EPS DA-BACS (KCTC14639BP).
  • EPS exopolysaccharide
  • Lactobacillus paracasei EPS DA-BACS KCTC14639BP
  • the present invention provides a health functional food for improving inflammatory diseases containing EPS (exopolysaccharide) produced by Lactobacillus paracasei EPS DA-BACS (KCTC14639BP).
  • EPS exopolysaccharide
  • Lactobacillus paracasei EPS DA-BACS KCTC14639BP
  • the present invention provides a cosmetic composition for preventing or improving skin inflammation containing EPS (exopolysaccharide) produced by Lactobacillus paracasei EPS DA-BACS (KCTC14639BP).
  • EPS exopolysaccharide
  • Lactobacillus paracasei EPS DA-BACS KCTC14639BP
  • Lactobacillus paracasei Lactobacillus paracasei
  • EPS DA-BACS KCTC14639BP
  • its dead cells its culture
  • Lactobacillus paracasei Lactobacillus paracasei
  • EPS DA-BACS KCTC14639BP
  • the composition for promoting the growth of probiotics is preferably Lactobacillus gasseri, Bifidobacterium bifidum, Bifidobacterium animalis, Bifidobacterium animalis , Bifidobacterium pical ( Bifidobacterium faecale ) It is preferable to promote the growth of any one or more probiotics selected from among them.
  • the strain of the present invention is characterized by producing EPS (exopolysaccharide) as postbiotics, has gastrointestinal stability, harmful bacteria and fungi inhibition and inflammation relief, inhibits Clostridium difficile , and free It has the advantage of providing a biotic effect (prebiotic effect).
  • EPS exopolysaccharide
  • Figure 2 is a result of evaluating the anti-inflammatory activity of the strain of the present invention, it is shown for nitric oxide (NO) production.
  • NO nitric oxide
  • Figure 3 is the result of evaluating the growth inhibitory activity against Clostridium difficile of the strain of the present invention.
  • Figure 4 is the result of evaluating the prebiotic effect of the strains of the present invention, Lactobacillus gasseri, Bifidobacterium bifidum , Bifidobacterium animalis , Bifidobacterium It shows the prebiotic index for Bifidobacterium faecale .
  • the present invention provides Lactobacillus paracasei EPS DA-BACS (KCTC14639BP).
  • the strain discovered in the present invention is preferably characterized in that it produces EPS (exopolysaccharide) as a postbiotics, and the EPS (exopolysaccharide) is preferably secreted extracellularly. Characterized in that it contains exopolysaccharide (EPS) in the form of ropy.
  • the strain of the present invention has gastrointestinal stability, inhibits harmful bacteria and fungi, and alleviates inflammation induced by LPS, inhibits Clostridium difficile , and has a prebiotic effect, specifically the intestinal Proliferation of beneficial bacteria ( Bifidobacterium sp. ) and intestinal harmful bacteria ( Escherichia coli ) have been confirmed to have inhibitory effects.
  • the above strain was named ' Lactobacillus paracasei EPS DA-BACS', and was deposited with the Korea Research Institute of Bioscience and Biotechnology and assigned accession number KCTC14639BP on July 15, 2021.
  • the strain preferably produces EPS (exopolysaccharide). Accordingly, the present invention provides EPS (exopolysaccharide) produced by Lactobacillus paracasei EPS DA-BACS (KCTC14639BP).
  • the EPS exopolysaccharide
  • EPS exopolysaccharide
  • a) Lactobacillus paracasei Lactobacillus paracasei
  • EPS DA-BACS KCTC14639BP
  • trichloroacetic acid may be used as an acidic material in step a), and in this case, it is preferable to react at a low temperature for 1 to 6 hours.
  • ethanol may be used as an example of the hydrophilic organic solvent in step b. At this time, ethanol is preferably added slowly in a volume ratio of 1 to 3 times that of the supernatant using cooled 80 to 100% (v/v) ethanol and reacted at low temperature for 12 to 24 hours to precipitate the precipitate.
  • the trichloroacetic acid is added to a final concentration of 4 to 10% (w/v) and reacted at a low temperature of 0 to 6 ° C, and the ethanol is cooled to a temperature of -20 to -15 ° C. It is good to use what has been done.
  • EPS exopolysaccharide
  • the present invention provides an antibacterial agent containing any one selected from Lactobacillus paracasei EPS DA-BACS (KCTC14639BP), its dead cells, its culture, and the above EPS (exopolysaccharide).
  • the antibacterial agent preferably Pseudomonas aeruginosa , Bacillus subtilis ( Bacillus subtilis ), Escherichia coli ( Escherichia coli ), Clostridium difficile bacteria ( Clostridium difficile ) and It is good to have antibacterial activity against Staphylococcus aureus .
  • the present invention provides an antifungal agent containing any one selected from Lactobacillus paracasei EPS DA-BACS (KCTC14639BP), its dead cells, its culture, and the above EPS (exopolysaccharide).
  • the antifungal agent preferably has antifungal activity against Aspergillus brasiliensis .
  • the strain of the present invention has antibacterial and antifungal activity against the strain.
  • Lactobacillus paracasei Lactobacillus paracasei
  • EPS DA-BACS KCTC14639BP
  • EPS exopolysaccharide
  • the inflammatory disease for example, sepsis, disseminated intravascular coagulation (DIC), atherosclerosis, degenerative arthritis, periodontitis, Lymphadenopathy, lymphangitis, type 1 diabetes, rheumatoid arthritis, multiple sclerosis, cancer, metabolic disease, neurodegenerative Neurodegenerative disease, systemic sclerosis, polymyositis, polymyositis, inclusion body myositis, allergy, Alzheimer's disease, ankylosing spondylitis ), asthma, carpal tunnel syndrome, celiac disease, crohn's disease, intestinal diverticulum, eczema, fibrosis, systemic lupus erythematosus), pancreatitis, Parkinson's disease, psoriasis, Polymyalgia rheumatica, dermatosclerosis, vasculitis, gastritis, dermatitis, Myelitis, stomatitis, arthritis,
  • DIC disseminated
  • prevention refers to any action that suppresses or delays the onset of inflammatory diseases by administration of the pharmaceutical composition according to the present invention.
  • treatment refers to all activities in which symptoms caused by inflammatory diseases are improved or advantageously changed by administration of the pharmaceutical composition according to the present invention.
  • Lactobacillus paracasei EPS DA-BACS KCTC14639BP
  • a composition comprising any one selected from dead cells, cultures thereof, and EPS (exopolysaccharide) as an active ingredient
  • EPS exopolysaccharide
  • one or more active ingredients exhibiting the same or similar functions may be contained.
  • the pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier in addition to Lactobacillus paracasei EPS DA-BACS.
  • the type of carrier that can be used in the present invention is not particularly limited, and any carrier commonly used in the art may be used.
  • Non-limiting examples of the carrier include lactose, dextrose, sucrose, sorbitol, mannitol, saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, xylitol, erythritol, maltitol, maltodextrin, glycerol, ethanol, and the like. can These may be used alone or in combination of two or more.
  • the pharmaceutical composition of the present invention may be used by adding other pharmaceutically acceptable additives such as antioxidants, excipients, diluents, buffers or bacteriostats, surfactants, binders, fillers, bulking agents, wetting agents, disintegrants , a dispersant or a lubricant may be additionally added and used.
  • other pharmaceutically acceptable additives such as antioxidants, excipients, diluents, buffers or bacteriostats, surfactants, binders, fillers, bulking agents, wetting agents, disintegrants , a dispersant or a lubricant may be additionally added and used.
  • the Lactobacillus paracasei EPS DA-BACS may be included in an amount of 0.00001% to 99.99% by weight, preferably 0.1% to 90% by weight, based on the total weight of the pharmaceutical composition. %, more preferably from 0.1% to 70% by weight, more preferably from 0.1% to 50% by weight, but is not limited thereto, and may vary depending on the condition of the subject to be administered, the type of specific disease, the degree of progression, etc. can be changed. If necessary, it may be included in the entire content of the pharmaceutical composition.
  • the pharmaceutically effective amount and effective dosage of the pharmaceutical composition of the present invention may vary depending on the formulation method, administration method, administration time and/or route of administration of the pharmaceutical composition, and Type and degree of reaction, type of subject to be administered, age, weight, general health condition, symptom or severity of disease, sex, diet, excretion, drugs used simultaneously or at different times in the subject, or components of other compositions, etc. It can vary according to various factors including, and similar factors well known in the medical field, and those skilled in the art can easily determine and prescribe an effective dosage for the desired treatment.
  • the daily dosage of the pharmaceutical composition of the present invention is about 0.01 to 1,000 mg/kg, preferably 0.1 to 100 mg/kg, and may be administered once or several times a day.
  • Administration of the pharmaceutical composition of the present invention may be administered once a day, or may be divided and administered several times.
  • the pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. Considering all of the above factors, it can be administered in an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.
  • the pharmaceutical composition of the present invention may be additionally used in combination with various methods such as hormone therapy and drug therapy to prevent or treat inflammatory diseases.
  • the term "administration” means introducing the pharmaceutical composition of the present invention to a patient by any suitable method, and the route and method of administration of the pharmaceutical composition of the present invention may be independent, respectively, and the purpose Any administration route and administration method may be followed without particular limitation, as long as the pharmaceutical composition can reach the corresponding site.
  • the pharmaceutical composition may be administered by oral administration or parenteral administration, and may be formulated into various dosage forms suitable for oral administration or parenteral administration.
  • Non-limiting examples of preparations for oral administration using the pharmaceutical composition of the present invention include oily suspensions, troches, lozenges, tablets, aqueous suspensions, prepared powders, granules, emulsions, hard capsules, and soft capsules, syrups or elixirs; and the like.
  • a binder such as sorbitol, mannitol, starch, amylopectin, cellulose lactose, saccharose or gelatin; lubricating oils such as magnesium stearate, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax; excipients such as dicalcium phosphate and the like; A disintegrant such as corn starch or sweet potato starch may be used, and aromatics, syrups, sweeteners, and the like may also be used.
  • a liquid carrier such as fatty oil may be additionally used in addition to the above-mentioned materials.
  • intramuscular administration As a method for parenteral administration of the pharmaceutical composition of the present invention, intramuscular administration, transdermal administration, intravenous administration, intraperitoneal administration, or subcutaneous administration may be used, and a method of applying, spraying, or inhaling the composition to a diseased area It can also be used, but is not limited thereto.
  • Non-limiting examples of parenteral preparations using the pharmaceutical composition of the present invention include injection solutions, suppositories, ointments, powders for application, oils, powders for respiratory inhalation, aerosols for sprays, creams, and the like.
  • aqueous solutions In order to formulate the pharmaceutical composition of the present invention for parenteral administration, sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, external preparations, etc. may be used. Vegetable oils, propylene glycol, polyethylene glycol, injectable esters such as ethyl oleate, and the like can be used.
  • the pharmaceutical composition of the present invention is formulated as an injection solution
  • the pharmaceutical composition of the present invention is mixed in water together with a stabilizer or buffer to prepare a solution or suspension, which is used for unit administration in an ampoule or vial. can be formulated.
  • a propellant or the like may be mixed with additives so that the water-dispersed concentrate or wet powder is dispersed.
  • composition of the present invention When the pharmaceutical composition of the present invention is formulated into ointments, oils, creams, powders for application, skin external preparations, etc., animal oils, vegetable oils, waxes, paraffins, polyethylene glycols, silicones, bentonites, silicas, talc, starch, tras It can be formulated using Kant, cellulose derivatives, zinc oxide and the like as carriers.
  • Lactobacillus paracasei Lactobacillus paracasei
  • EPS DA-BACS KCTC14639BP
  • KCTC14639BP KCTC14639BP
  • EPS exopolysaccharide
  • improvement refers to all activities that improve or beneficially change inflammatory diseases by administration of the composition of the present invention.
  • the Lactobacillus paracasei EPS DA-BACS is preferably included in an amount of 0.00001 to 50% by weight relative to the food composition for improving inflammatory diseases. If it is less than 0.00001% by weight, the effect is insufficient, and if it exceeds 50% by weight, the increase in effect compared to the amount used is insignificant, which is uneconomical.
  • the food composition for improving inflammatory diseases of the present invention is, for example, noodles, gums, dairy products, ice creams, meats, grains, caffeinated beverages, general beverages, chocolates, breads, snacks, confectionery products, candies, pizzas, jellies, alcoholic beverages, It may be any one selected from alcohol, vitamin complexes, and other health supplements, but is not necessarily limited thereto.
  • the food composition of the present invention When using the food composition of the present invention as a food additive, it may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to conventional methods.
  • Lactobacillus paracasei Lactobacillus paracasei
  • EPS DA-BACS KCTC14639BP
  • its dead cells KCTC14639BP
  • EPS exopolysaccharide
  • health functional food refers to food manufactured and processed using raw materials or ingredients having functional properties useful for the human body in accordance with the Act on Health Functional Foods No. 6727, and "functional” refers to the structure of the human body. And it refers to intake for the purpose of obtaining useful effects for health purposes such as regulating nutrients for functions or physiological actions.
  • the food composition and health functional food of the present invention may include additional ingredients that are commonly used and can improve smell, taste, and visual properties.
  • it may include biotin, folate, panthotenic acid, vitamins A, C, D, E, B1, B2, B6, B12, niacin, and the like.
  • minerals such as chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), zinc (Zn), iron (Fe), and calcium (Ca) may be included.
  • amino acids such as cysteine, valine, lysine, and tryptophan may be included.
  • preservatives potassium sorbate, sodium benzoate, salicylic acid, sodium dihydroacetate, etc.
  • coloring agents tar colorant, etc.
  • coloring agents sodium nitrite, sodium nitrite, etc.
  • bleaching agents sodium sulfite
  • disinfectants bleaching powder and high bleaching powder, sodium hypochlorite, etc.
  • swelling agent alum, D-potassium hydrogen tartrate, etc.
  • strengthening agent emulsifier, thickener (thickener), coating agent, antioxidant (butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), etc.
  • Food additives such as seasonings (MSG monosodium glutamate, etc.), sweeteners (dulcin, cyclemate, saccharin, sodium, etc.), flavorings (vanillin, lactones, etc.), gum base, antifoaming agent, solvent, improver, etc. can be added.
  • the additive may be selected according to the type of food and
  • Lactobacillus paracasei EPS DA-BACS is not particularly limited, and may be variously changed depending on the condition of the subject to be administered, the type of specific disease, and the degree of progression. If necessary, it may also be included in the total content of food.
  • Lactobacillus paracasei Lactobacillus paracasei
  • EPS DA-BACS KCTC14639BP
  • Lactobacillus paracasei Lactobacillus paracasei
  • EPS DA-BACS KCTC14639BP
  • the composition for promoting the growth of probiotics is preferably Lactobacillus gasseri, Bifidobacterium bifidum, Bifidobacterium animalis, Bifidobacterium animalis , Bifidobacterium pical ( Bifidobacterium faecale ) It is preferable to promote the growth of any one or more probiotics selected from among them.
  • EPS exopolysaccharide
  • Lactobacillus paracasei EPS DA-BACS KCTC14639BP
  • the Lactobacillus paracasei EPS DA-BACS KCTC14639BP
  • its dead cells KCTC14639BP
  • the culture of the present invention also have the efficacy of promoting the growth of the above-mentioned probiotics.
  • the composition for promoting the growth of probiotics claimed in the present invention is also called prebiotics in the art.
  • the present invention is Lactobacillus paracasei ( Lactobacillus paracasei ) EPS DA-BACS (KCTC14639BP), its dead cells, its culture, and for preventing or improving skin inflammation containing any one selected from the above EPS (exopolysaccharide)
  • EPS exopolysaccharide
  • the skin inflammation may be, for example, any one selected from atopic dermatitis, contact dermatitis, seborrhea and acne, but is limited thereto It may include, without limitation, any skin inflammatory disease accompanied by an inflammatory reaction occurring in the skin.
  • the Lactobacillus paracasei EPS DA-BACS is preferably contained in an amount of 0.0001 to 30.0% by weight based on the total weight of the cosmetic composition. More preferably, it is good to contain 0.01 to 10% by weight based on the total weight of the cosmetic composition. If the content of Lactobacillus paracasei EPS DA-BACS is less than 0.0001% by weight, the effect of preventing or improving skin inflammation is insignificant, and if it exceeds 30.0% by weight, the obvious effect does not increase with the increase in the content.
  • the ingredients included in the cosmetic composition of the present invention may include ingredients commonly used in cosmetic compositions in addition to the Lactobacillus paracasei EPS DA-BACS of the present invention as active ingredients, such as antioxidants, stabilizers, solubilizers, conventional adjuvants such as agents, vitamins, pigments and flavors, and carriers.
  • the cosmetic composition of the present invention can be prepared in any formulation conventionally prepared in the art, for example, a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing , Oil, pack, massage cream and spray, etc., but may be formulated, but is not limited thereto. More specifically, it may be prepared in the form of softening lotion, nutrient lotion, nutrient cream, massage cream, essence, cleansing cream, cleansing foam, cleansing water, pack, spray or powder.
  • the formulation of the cosmetic composition of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide as a carrier component this can be used
  • a solvent, solubilizing agent or emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene fatty acid esters of glycol, 1,3-butyl glycol oil, glycerol aliphatic esters, polyethylene glycol or sorbitan.
  • the formulation of the cosmetic composition of the present invention is a suspension
  • a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester , microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tracanth, and the like
  • a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester
  • microcrystalline cellulose aluminum metahydroxide
  • bentonite agar or tracanth, and the like
  • lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, additional chlorofluorohydro propellants such as carbon, propane/butane or dimethyl ether.
  • the formulation of the cosmetic composition of the present invention is surfactant-containing cleansing
  • carrier components aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, Fatty acid amide ether sulfates, alkylamidobetaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives, or ethoxylated glycerol fatty acid esters may be used.
  • the cosmetic composition of the present invention is a soap, a surfactant-containing cleansing formulation, or a surfactant-free cleansing formulation
  • the soap is liquid soap, powder soap, solid soap, and oil soap
  • the surfactant-containing cleansing formulation is a cleansing foam, cleansing water, cleansing towel, and a cleansing pack
  • the surfactant-free cleansing formulation is a cleansing cream. , cleansing lotion, cleansing water and cleansing gel, but are not limited thereto.
  • the feces were diluted step by step using sterile physiological saline by a decimal dilution method, and then smeared on LBS selective media (Lactobacillus selective media) at 100 ⁇ l.
  • LBS selective media Lactobacillus selective media
  • Identification was performed through 16S rRNA sequencing for the isolated lactic acid bacteria candidate strains. Genomic DNA was extracted and purified from candidate Lactobacillus strains, and DNA was PCR amplified using primers for 16S rRNA gene sequencing. The homology analysis of the obtained 16S rRNA sequence was performed using BLAST software, and through this, the genus and species of each lactic acid bacteria candidate group were determined. As a result of identifying 261 candidate strains of lactic acid bacteria, respectively, it was found that they consisted of 225 Lactobacillus sp., 7 Weissella sp., and 29 Pediococcus .
  • EPS Extrapolysaccharide
  • EPS Extrapolysaccharide
  • Lactobacillus paracasei one of the lactic acid bacteria with the best EPS production, was tested as shown in Table 1.
  • EPS DA-BACS was selected. Its 16S rRNA nucleotide sequence is shown in SEQ ID NO: 1.
  • the isolated strain was inoculated into MRS broth (0.05% L-cysteine) and anaerobically cultured at 37° C. for 24 hours, followed by centrifugation and washing with PBS buffer to recover the cells.
  • the recovered cells were added to the artificial gastric fluid in an amount of 10% to a final concentration of 10 7 to 10 8 CFU/mL, and then cultured at 37°C. After 0 hour, 1.5 hour, and 3 hours, 1 ml each of the culture medium was collected, the number of viable cells was measured through dilution, and the survival rate was calculated relative to the number of initial inoculated bacteria (0 hour).
  • the survival rate was excellent at 143.5% after 3 hours compared to the number of initial inoculum of EPS DA-BACS, which was the highest among 12 strains of Lactobacillus paracasei homologous strains tested together It was a shame.
  • the average survival rate in artificial gastric juice of 12 isogeneous comparative strains tested together was 54.9%, which was about 2.6 times higher than that of EPS DA-BACS.
  • Artificial bile was prepared by adding oxgall filtered through a 0.25 ⁇ m membrane to MRS broth to a final concentration of 0.3%. Bacteria were inoculated into MRS broth (0.05% L-cysteine) and anaerobically cultured at 37° C. for 24 hours, followed by centrifugation and washing with PBS buffer to recover the cells. The recovered cells were added as much as 10% to the artificial bile solution to a final concentration of 10 7 to 10 8 CFU/mL, and then cultured at 37°C. After 0 hour, 1.5 hour, and 3 hours, 1 ml each of the culture medium was collected, and the number of viable cells was measured through dilution.
  • the EPS DA-BACS strain showed an excellent survival rate of 250% in artificial bile fluid.
  • Artificial pancreatic fluid was prepared by adding pancreatin filtered through a 0.25 ⁇ m membrane to MRS broth to a final concentration of 0.5%. Bacteria were inoculated into MRS broth (0.05% L-cysteine) and anaerobically cultured at 37° C. for 24 hours, followed by centrifugation and washing with PBS buffer to recover the cells. The recovered cells were added to the artificial pancreatic fluid in an amount of 10% to a final concentration of 10 7 to 10 8 CFU/mL, and then cultured at 37°C. After 0 hour, 1.5 hour, and 3 hours, 1 ml each of the culture medium was collected, and the number of viable cells was measured through dilution.
  • the EPS DA-BACS strain showed an excellent survival rate of 126.9% in artificial pancreatic fluid.
  • the lactic acid bacteria stock was inoculated to a concentration of 1% (v/v) in MRS broth (0.05% L-cysteine) and incubated at 32 to 37 o C for 24 to 168 hours. Political culture was carried out under anaerobic conditions. The lactic acid bacteria culture was centrifuged at 4,000 rpm for 10 minutes, and the supernatant was filtered to prepare a lactic acid bacteria culture.
  • the lactic acid bacteria culture was diluted to 10% using Mueller hinton II broth as a solvent, and then 200 ⁇ l was dispensed into each well of a 96-well plate.
  • the antibacterial activity evaluation strains Pseudomonas aeruginosa, Bacillus subtilis, Escherichia coli, Staphylococcus aureus ), which had been activated the previous day, were inoculated into the wells at 1% each.
  • the 96-well plate was incubated at 37° C. and the antibacterial activity was evaluated by measuring OD 600 at 0 hour and 24 hour, respectively.
  • Antibacterial activity evaluation classification Antibacterial activity evaluation strain Bacillus subtilis pseudomonas aeruginosa Staphylococcus aureus Escherichia coli Other homologous strains KCTC 3169 - - VG - KCTC 13090 - - VG - KCTC 3165 - - VG - KCTC 3189 - - VG - KCTC 5546 - - VG - KCTC 3510 - - VG VG KCTC 5058 VG VG VG KCTC 3074 - VG VG VG KCCM 40995 - - VG - KCCM 42830 - - VG - KCCM 32822 - - VG - KCCM 41276 - - VG VG strain of the present invention EPS DA-BACS - - - - Blank - - - - control group VG VG VG VG VG VG VG
  • Lactobacillus paracasei Lactobacillus paracasei
  • Pseudomonas aeruginosa Pseudomonas aeruginosa
  • Bacillus subtilis Bacillus subtilis
  • Escherichia coli Escherichia coli
  • Staphylococcus aureus Staphylococcus aureus
  • the antibacterial activity of the strain of the present invention is excellent because there is no strain controlling all four strains for evaluation of antibacterial activity, like EPS DA-BACS, among 12 strains of the same species.
  • the lactic acid bacteria stock was inoculated to a concentration of 1% (v / v) in MRS broth (0.05% L-cysteine) and cultured at 32-37 ° C for 18-24 hours under anaerobic conditions.
  • agar and 0.1MK 2 HPO 4 were added to the MRS broth and then autoclaved. After dispensing 300 ⁇ l of the corresponding MRS agar into a 24-well plate, the plate was air-dried for 30 minutes with the lid closed. 0.75 ⁇ l of the activated lactic acid bacteria culture medium was inoculated into the center of MRS agar hardened into wells, followed by anaerobic culture at 32-37° C. for 48 hours.
  • agar and 0.1MK 2 HPO 4 were added to YM (Yeast-Malt) broth and then autoclaved. After cooling the YM soft agar to 50 ° C. in a water bath, Aspergillus brasiliensis , an antifungal activity evaluation strain activated the previous day, was inoculated with 10 3 to 10 4 spores / ml. . 100 ⁇ l of YM soft agar inoculated with the antifungal activity evaluation strain was dispensed on MRS agar in which the lactic acid bacteria were cultured, and then air-dried for 30 minutes with the lid of the 24-well plate closed.
  • a blank group not inoculated with the antifungal activity evaluation strain on YM soft agar and a control group inoculated with 0.75 ⁇ l of sterilized peptone water instead of lactic acid bacteria were also tested.
  • the 24-well plate was incubated at 25° C. for up to 48 hours, and antifungal activity was visually confirmed.
  • MRS broth (0.05% L-cysteine) was inoculated with 1% of the strain of the present invention and anaerobically cultured at 37° C. for 24 hours, and then the number of viable cells was measured. Thereafter, cells were collected by centrifugation, washed with PBS buffer, and then heat-inactivated using an autoclave to prepare dead cells.
  • ESP DA-BACS cells (cells were dissolved in DMEM (Dulbecco's Modified Eagle's Medium) medium) were added to the RAW 264.7 cell line to a final concentration of 1.0 ⁇ 10 9 CFU / ml, reacted for 2 hours, and LPS 1 [mu]g/ml was added and incubated for 24 hours. Then, the amount of nitrogen monoxide produced in the culture medium was measured using an ELISA kit.
  • a positive control group treated with 1uM of dexamethasone, an anti-inflammatory drug, instead of cells, a group not treated with cells and LPS, and a group treated only with LPS were also tested.
  • the cell culture medium was centrifuged (10,000 ⁇ g, 25 min) at 4 ° C to remove the cells, and 2 times of cooled 95% ethanol was slowly added to the supernatant, and EPS was allowed to precipitate for 24 hours. The precipitate was collected by centrifugation (10,000 ⁇ g, 25min) at 4 ° C., and the remaining ethanol was dried and lyophilized to prepare crude EPS.
  • the cell culture medium was centrifuged (10,000 ⁇ g, 25 min) at 4 ° C to remove the cells, and trichloroacetic acid was added to the recovered supernatant to a final concentration of 4 to 10 % and reacted at 4°C for 2 hours.
  • the reaction solution was centrifuged (10,000 ⁇ g, 25 min) at 4 ° C to remove precipitated proteins, and then 2 times cold 95% ethanol was slowly added to the supernatant to precipitate EPS at 4 ° C for 15 to 24 hours.
  • the precipitate was collected by centrifugation (10,000 ⁇ g, 25 min) at 4 ° C, dissolved in tertiary distilled water, and dialysis-sack (M.W. cut off 10,000, Spectra / Por 6 membrane, Pre-wetted RC Tubing, Spectrum Laboratories, USA), dialyzed at 4° C. for 48 hours, and then lyophilized to prepare purified EPS.
  • Clostridium difficile Clostridium difficile
  • FIG. 3 is the result of evaluating the growth inhibitory activity against Clostridium difficile of the strain of the present invention.
  • EPS purified from the isolated strain has prebiotics activity
  • 100 ⁇ l of MRS broth or TSB broth containing 2% of the purified EPS as a glycogen was dispensed into a 96-well plate, and then the MRS broth was activated the previous day.
  • Cultures of beneficial bacteria (lactic acid bacteria and bifidobacteria) and 1% of cultures of intestinal harmful bacteria ( Escherichia coli ) were inoculated into the corresponding TSB broth, respectively, and OD 600 values after 24 hours were observed.
  • MRS broth or TSB broth with 2% inulin, a representative prebiotic, as a glycogen MRS broth or TSB broth to which no glycogen was added.
  • a blank group and a control group in which the same experiment was performed for MRS broth or TSB broth containing 2% glucose as a sugar source were also tested.
  • Prebiotic index Optical density of the growth of probiotic culture / Optical density of the growth of E. coli

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Abstract

The present invention relates to intestinal health strain Lactobacillus paracasei EPS DA-BACS having Lactobacillus bifidus growth promotion and harmful intestinal bacteria Clostridium difficile inhibitory effects, and polysaccharide thereof. The strain of the present invention is characterized by producing exopolysaccharide (EPS) as postbiotics, and has the advantage of having gastrointestinal stability and harmful bacteria and eumycetes inhibitory and inflammation relieving effects, inhibiting Clostridium difficile, and providing a prebiotic effect.

Description

비피더스 유산균 생장촉진 및 장내 유해균 클로스트리듐 디피실리균 억제 효능을 갖는 장 건강 균주 락토바실러스 파라카제이 EPS DA-BACS 및 그 다당류Lactobacillus paracasei EPS DA-BACS and its polysaccharides, which are intestinal health strains that promote the growth of bifidus lactic acid bacteria and inhibit the intestinal harmful bacteria Clostridium difficile
본 발명은 비피더스 유산균 생장촉진 및 장내 유해균 클로스트리듐 디피실리균(Clostridium difficile) 억제 효능을 갖는 장 건강 균주 락토바실러스 파라카제이 EPS DA-BACS 및 그 다당류에 관한 것이다.The present invention relates to an intestinal health strain, Lactobacillus paracasei EPS DA-BACS, and a polysaccharide thereof, which have an effect of promoting the growth of bifidus lactic acid bacteria and inhibiting the intestinal harmful bacteria Clostridium difficile .
프로바이오틱스(probiotics)는 인체에 이로움을 주는 미생물을 총칭하는 말로, 대표적으로 유산균을 포함한다. 프로바이오틱스가 갖추어야 할 특성은 사람의 장관을 서식지로 하고, 항생제에 민감하며 병원성 및 독성이 없어야 한다. 또한, 장으로 가는 동안 산, 담즙 등에 대한 내성을 갖추어야 하고, 유용한 효능을 잘 발현해야 한다.Probiotics is a general term for microorganisms that are beneficial to the human body, and typically includes lactic acid bacteria. Characteristics that probiotics should have are that they have the human intestinal tract as their habitat, are sensitive to antibiotics, and have no pathogenicity or toxicity. In addition, while going to the intestine, it must have resistance to acid, bile, etc., and must express useful effects well.
한편, 일부 유산균은 EPS(exopolysaccharide)라고 불리우는 다당류를 생합성하는데, 이는 세포벽 주위에 협막을 생성하거나 세포벽 외부에 점질 형태로 축적되는 미생물의 대사산물 중 하나이다. 유산균이 생성하는 다당류는 물성이 다양하고 독특하여 물성기능 소재로 활용될 수 있을 뿐 아니라 항암효과, 콜레스테롤 저해, 면역 조절, 변비 개선 등의 생리기능 활성이 뛰어나 EPS를 생성하는 유산균에 대한 관심이 점차 높아지고 있다.On the other hand, some lactic acid bacteria biosynthesize a polysaccharide called EPS (exopolysaccharide), which is one of the metabolites of microorganisms that form capsular membranes around cell walls or accumulate in the form of slime on the outside of cell walls. The polysaccharide produced by lactic acid bacteria has various and unique physical properties, so it can be used as a material with physical properties and functions. It also has excellent physiological functions such as anti-cancer effects, cholesterol inhibition, immune regulation, and constipation improvement, and interest in lactic acid bacteria that produce EPS is gradually increasing. It is rising.
EPS는 구조 및 중합도에 따라 세포벽에 부착되는 캡슐러(capsular) 형태와 세포외로 분비되는 로우피(ropy) 형태로 나뉘어진다. Ropy-EPS는 Non-ropy-EPS와 비교하였을 때 콜로니에서 실선모양의 점질물질이 형성되는 것이 특징이며, 상대적으로 높은 점도 및 보수력과 부드러운 밀도를 부여한다. 생리적 측면으로는 선행연구에서 Ropy-EPS가 Non-ropy-EPS 대비 유산균의 생존과 부착을 유의적으로 증가시키며, 혈청 콜레스테롤을 낮추고, 과잉면역반응을 억제한다는 문헌들이 보고되고 있다.EPS is divided into a capsular form attached to the cell wall and a ropy form secreted extracellularly according to the structure and degree of polymerization. Compared to Non-ropy-EPS, Ropy-EPS is characterized by the formation of a viscous material in the form of solid lines in colonies, and provides relatively high viscosity, water retention and soft density. In terms of physiological aspects, literature has been reported that Ropy-EPS significantly increases the survival and attachment of lactic acid bacteria compared to Non-ropy-EPS in previous studies, lowers serum cholesterol, and suppresses hyperimmune reactions.
본 발명은 Ropy-EPS를 생성하는 유산균을 발굴하여 제공하는 것을 목적으로 한다.An object of the present invention is to discover and provide lactic acid bacteria that produce Ropy-EPS.
본 발명은 락토바실러스 파라카제이(Lactobacillus paracasei) EPS DA-BACS (KCTC14639BP)을 제공한다.The present invention provides Lactobacillus paracasei EPS DA-BACS (KCTC14639BP).
본 발명에 있어, 상기 균주는, 바람직하게는 포스트바이오틱스로서 EPS(exopolysaccharide)를 생성하는 것을 특징으로 하며, 상기 EPS(exopolysaccharide)는, 바람직하게는 세포 외로 분비되는 로우피(ropy) 형태의 EPS(exopolysaccharide)를 포함하는 것을 특징으로 한다.In the present invention, the strain is preferably characterized by producing exopolysaccharide (EPS) as a postbiotics, and the EPS (exopolysaccharide) is preferably EPS in the form of ropy secreted extracellularly. It is characterized in that it contains (exopolysaccharide).
한편, 본 발명은 락토바실러스 파라카제이(Lactobacillus paracasei) EPS DA-BACS (KCTC14639BP), 그의 사균체 및 그의 배양물 중 선택되는 어느 하나를 함유하는 항균제를 제공한다. 이때, 상기 항균제는, 바람직하게는 녹농균(Pseudomonas aeruginosa), 고초균(Bacillus subtilis), 대장균(Escherichia coli), 클로스트리듐 디피실리균(Clostridium difficile) 및 황색포도구균(Staphylococcus aureus)에 대한 항균 활성을 가지는 것이 좋다.On the other hand, the present invention provides an antibacterial agent containing any one selected from Lactobacillus paracasei EPS DA-BACS (KCTC14639BP), dead cells thereof, and cultures thereof. At this time, the antibacterial agent, preferably Pseudomonas aeruginosa , Bacillus subtilis ( Bacillus subtilis ), Escherichia coli ( Escherichia coli ), Clostridium difficile bacteria ( Clostridium difficile ) and It is good to have antibacterial activity against Staphylococcus aureus .
한편, 본 발명은 락토바실러스 파라카제이(Lactobacillus paracasei) EPS DA-BACS (KCTC14639BP), 그의 사균체 및 그의 배양물 중 선택되는 어느 하나를 함유하는 항진균제를 제공한다. 이때, 상기 항진균제는, 바람직하게는 아스페길루스 브라실리언시스(Aspergillus brasiliensis)에 대한 항진균 활성을 가지는 것이 좋다.On the other hand, the present invention provides an antifungal agent containing any one selected from Lactobacillus paracasei EPS DA-BACS (KCTC14639BP), dead cells thereof, and cultures thereof. At this time, the antifungal agent preferably has antifungal activity against Aspergillus brasiliensis .
한편, 본 발명은 락토바실러스 파라카제이(Lactobacillus paracasei) EPS DA-BACS (KCTC14639BP), 그의 사균체 및 그의 배양물 중 선택되는 어느 하나를 함유하는 염증질환 예방 또는 치료용 약학 조성물을 제공한다.On the other hand, the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases containing any one selected from Lactobacillus paracasei EPS DA-BACS (KCTC14639BP), its dead cells and its culture.
한편, 본 발명은 락토바실러스 파라카제이(Lactobacillus paracasei) EPS DA-BACS (KCTC14639BP), 그의 사균체 및 그의 배양물 중 선택되는 어느 하나를 함유하는 염증질환 개선용 식품 조성물을 제공한다.On the other hand, the present invention provides a food composition for improving inflammatory diseases containing any one selected from Lactobacillus paracasei EPS DA-BACS (KCTC14639BP), its dead cells and its culture.
한편, 본 발명은 락토바실러스 파라카제이(Lactobacillus paracasei) EPS DA-BACS (KCTC14639BP), 그의 사균체 및 그의 배양물 중 선택되는 어느 하나를 함유하는 염증질환 개선용 건강기능식품을 제공한다.On the other hand, the present invention provides a health functional food for improving inflammatory diseases containing any one selected from Lactobacillus paracasei EPS DA-BACS (KCTC14639BP), its dead cells and its culture.
한편, 본 발명은 락토바실러스 파라카제이(Lactobacillus paracasei) EPS DA-BACS (KCTC14639BP), 그의 사균체 및 그의 배양물 중 선택되는 어느 하나를 함유하는 피부염증 예방 또는 개선용 화장료 조성물을 제공한다.On the other hand, the present invention provides a cosmetic composition for preventing or improving skin inflammation containing any one selected from Lactobacillus paracasei EPS DA-BACS (KCTC14639BP), its dead cells and its culture.
한편, 본 발명은 락토바실러스 파라카제이(Lactobacillus paracasei) EPS DA-BACS (KCTC14639BP)가 생성하는 EPS(exopolysaccharide)를 제공하며, 상기 EPS(exopolysaccharide)는, 바람직하게는 세포 외로 분비되는 로우피(ropy) 형태의 EPS(exopolysaccharide)를 포함하는 것을 특징으로 한다. 이때, 상기 EPS(exopolysaccharide)는, 바람직하게는 a) 락토바실러스 파라카제이(Lactobacillus paracasei) EPS DA-BACS (KCTC14639BP) 배양 상등액에 산성물질을 첨가하여 단백질을 침전 및 제거하는 단계; b) 상기 a) 단계의 단백질 제거액에 친수성 유기용매를 첨가하여 다당류를 추출하는 단계;를 포함하는 과정을 통해 수득되는 것이 좋다.On the other hand, the present invention provides EPS (exopolysaccharide) produced by Lactobacillus paracasei EPS DA-BACS (KCTC14639BP), and the EPS (exopolysaccharide) is preferably secreted extracellularly. ) characterized in that it comprises a form of EPS (exopolysaccharide). At this time, the EPS (exopolysaccharide), preferably a) Lactobacillus paracasei ( Lactobacillus paracasei ) EPS DA-BACS (KCTC14639BP) Precipitating and removing proteins by adding an acidic material to the culture supernatant; b) extracting the polysaccharide by adding a hydrophilic organic solvent to the protein removal liquid of step a);
한편, 본 발명은 락토바실러스 파라카제이(Lactobacillus paracasei) EPS DA-BACS (KCTC14639BP)가 생성하는 EPS(exopolysaccharide)를 함유하는 항균제를 제공한다. 이때, 상기 항균제는, 바람직하게는 녹농균(Pseudomonas aeruginosa), 고초균(Bacillus subtilis), 대장균(Escherichia coli), 클로스트리듐 디피실리균(Clostridium difficile) 및 황색포도구균(Staphylococcus aureus)에 대한 항균 활성을 가지는 것이 좋다.On the other hand, the present invention provides an antibacterial agent containing EPS (exopolysaccharide) produced by Lactobacillus paracasei EPS DA-BACS (KCTC14639BP). At this time, the antibacterial agent, preferably Pseudomonas aeruginosa , Bacillus subtilis ( Bacillus subtilis ), Escherichia coli ( Escherichia coli ), Clostridium difficile bacteria ( Clostridium difficile ) and It is good to have antibacterial activity against Staphylococcus aureus .
한편, 본 발명은 락토바실러스 파라카제이(Lactobacillus paracasei) EPS DA-BACS (KCTC14639BP)가 생성하는 EPS(exopolysaccharide)를 함유하는 항진균제를 제공한다. 이때, 상기 항진균제는, 바람직하게는 아스페길루스 브라실리언시스(Aspergillus brasiliensis)에 대한 항진균 활성을 가지는 것이 좋다.On the other hand, the present invention provides an antifungal agent containing EPS (exopolysaccharide) produced by Lactobacillus paracasei EPS DA-BACS (KCTC14639BP). At this time, the antifungal agent preferably has antifungal activity against Aspergillus brasiliensis .
한편, 본 발명은 락토바실러스 파라카제이(Lactobacillus paracasei) EPS DA-BACS (KCTC14639BP)가 생성하는 EPS(exopolysaccharide)를 함유하는 염증질환 예방 또는 치료용 약학 조성물을 제공한다.On the other hand, the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases containing EPS (exopolysaccharide) produced by Lactobacillus paracasei EPS DA-BACS (KCTC14639BP).
한편, 본 발명은 락토바실러스 파라카제이(Lactobacillus paracasei) EPS DA-BACS (KCTC14639BP)가 생성하는 EPS(exopolysaccharide)를 함유하는 염증질환 개선용 식품 조성물을 제공한다.On the other hand, the present invention provides a food composition for improving inflammatory diseases containing EPS (exopolysaccharide) produced by Lactobacillus paracasei EPS DA-BACS (KCTC14639BP).
한편, 본 발명은 락토바실러스 파라카제이(Lactobacillus paracasei) EPS DA-BACS (KCTC14639BP)가 생성하는 EPS(exopolysaccharide)를 함유하는 염증질환 개선용 건강기능식품을 제공한다.On the other hand, the present invention provides a health functional food for improving inflammatory diseases containing EPS (exopolysaccharide) produced by Lactobacillus paracasei EPS DA-BACS (KCTC14639BP).
한편, 본 발명은 락토바실러스 파라카제이(Lactobacillus paracasei) EPS DA-BACS (KCTC14639BP)가 생성하는 EPS(exopolysaccharide)를 함유하는 피부염증 예방 또는 개선용 화장료 조성물을 제공한다.On the other hand, the present invention provides a cosmetic composition for preventing or improving skin inflammation containing EPS (exopolysaccharide) produced by Lactobacillus paracasei EPS DA-BACS (KCTC14639BP).
한편, 본 발명은 락토바실러스 파라카제이(Lactobacillus paracasei) EPS DA-BACS (KCTC14639BP), 그의 사균체, 그의 배양물 및 락토바실러스 파라카제이(Lactobacillus paracasei) EPS DA-BACS (KCTC14639BP)가 생성하는 EPS(exopolysaccharide) 중 선택되는 어느 하나를 함유하는 프로바이오틱스 생육 촉진용 조성물을 제공한다. 이때, 상기 프로바이오틱스 생육 촉진용 조성물은, 바람직하게는 락토바실러스 가세리(Lactobacillus gasseri), 비피도박테리움 비피덤(Bifidobacterium bifidum), 비피도박테리움 애니말리스(Bifidobacterium animalis), 비피도박테리움 패이칼(Bifidobacterium faecale) 중 선택되는 어느 하나 이상의 프로바이오틱스의 생육을 촉진시키는 것이 좋다.On the other hand, the present invention Lactobacillus paracasei ( Lactobacillus paracasei ) EPS DA-BACS (KCTC14639BP), its dead cells, its culture and Lactobacillus paracasei ( Lactobacillus paracasei ) EPS DA-BACS (KCTC14639BP) produced It provides a composition for promoting the growth of probiotics containing any one selected from (exopolysaccharide). At this time, the composition for promoting the growth of probiotics is preferably Lactobacillus gasseri, Bifidobacterium bifidum, Bifidobacterium animalis, Bifidobacterium animalis , Bifidobacterium pical ( Bifidobacterium faecale ) It is preferable to promote the growth of any one or more probiotics selected from among them.
본 발명의 균주는 포스트바이오틱스로서 EPS(exopolysaccharide)를 생성하는 것을 특징으로 하며, 위장관 안정성, 유해균·진균 저해 및 염증완화 효과를 보유하며, 클로스트리듐 디피실리균(Clostridium difficile)을 저해하고 프리바이오틱 효과(prebiotic effect)를 제공할 수 있는 장점이 있다.The strain of the present invention is characterized by producing EPS (exopolysaccharide) as postbiotics, has gastrointestinal stability, harmful bacteria and fungi inhibition and inflammation relief, inhibits Clostridium difficile , and free It has the advantage of providing a biotic effect (prebiotic effect).
도 1은 본 발명 균주의 항진균 활성을 평가한 결과이다.1 is a result of evaluating the antifungal activity of the strain of the present invention.
도 2는 본 발명 균주의 항염증 활성을 평가한 결과로, 산화질소(NO) 생산량에 대해 나타낸 것이다.Figure 2 is a result of evaluating the anti-inflammatory activity of the strain of the present invention, it is shown for nitric oxide (NO) production.
도 3은 본 발명 균주의 클로스트리듐 디피실리균(Clostridium difficile)에 대한 생장저해 활성을 평가한 결과이다.Figure 3 is the result of evaluating the growth inhibitory activity against Clostridium difficile of the strain of the present invention.
도 4는 본 발명 균주의 프리바이오틱 효과를 평가한 결과로, 락토바실러스 가세리(Lactobacillus gasseri), 비피도박테리움 비피덤(Bifidobacterium bifidum), 비피도박테리움 애니말리스(Bifidobacterium animalis), 비피도박테리움 패이칼(Bifidobacterium faecale)에 대한 프리바이오틱 지수(prebiotic index)를 나타낸 것이다.Figure 4 is the result of evaluating the prebiotic effect of the strains of the present invention, Lactobacillus gasseri, Bifidobacterium bifidum , Bifidobacterium animalis , Bifidobacterium It shows the prebiotic index for Bifidobacterium faecale .
본 발명은 락토바실러스 파라카제이(Lactobacillus paracasei) EPS DA-BACS (KCTC14639BP)을 제공한다.The present invention provides Lactobacillus paracasei EPS DA-BACS (KCTC14639BP).
본 발명의 일 실시예에 의하면, 본 발명에서 발굴한 균주는 바람직하게는 포스트바이오틱스로서 EPS(exopolysaccharide)를 생성하는 것을 특징으로 하며, 상기 EPS(exopolysaccharide)는, 바람직하게는 세포 외로 분비되는 로우피(ropy) 형태의 EPS(exopolysaccharide)를 포함하는 것을 특징으로 한다. 본 발명의 균주는 위장관 안정성, 유해균·진균 저해 및 LPS로 유도된 염증의 완화 효과를 보유하며, 클로스트리듐 디피실리균(Clostridium difficile)을 저해하고 프리바이오틱 효과(prebiotic effect), 구체적으로 장내 유익균(Bifidobacterium sp.) 증식 및 장내 유해균(Escherichia coli)에 대한 저해 효과를 가짐을 확인한 바 있다.According to one embodiment of the present invention, the strain discovered in the present invention is preferably characterized in that it produces EPS (exopolysaccharide) as a postbiotics, and the EPS (exopolysaccharide) is preferably secreted extracellularly. Characterized in that it contains exopolysaccharide (EPS) in the form of ropy. The strain of the present invention has gastrointestinal stability, inhibits harmful bacteria and fungi, and alleviates inflammation induced by LPS, inhibits Clostridium difficile , and has a prebiotic effect, specifically the intestinal Proliferation of beneficial bacteria ( Bifidobacterium sp. ) and intestinal harmful bacteria ( Escherichia coli ) have been confirmed to have inhibitory effects.
따라서, 본 발명에서는 상기한 균주를 '락토바실러스 파라카제이(Lactobacillus paracasei) EPS DA-BACS'이라 명명하였으며, 한국생명공학연구원에 기탁하여 2021년 07월 15일자로 수탁번호 KCTC14639BP를 부여받았다.Therefore, in the present invention, the above strain was named ' Lactobacillus paracasei EPS DA-BACS', and was deposited with the Korea Research Institute of Bioscience and Biotechnology and assigned accession number KCTC14639BP on July 15, 2021.
또한, 본 발명에 있어, 상기 균주는, 바람직하게는 EPS(exopolysaccharide)를 생성하는 것으로 확인되었다. 따라서, 본 발명은 락토바실러스 파라카제이(Lactobacillus paracasei) EPS DA-BACS (KCTC14639BP)가 생성하는 EPS(exopolysaccharide)를 제공한다.In addition, in the present invention, it was confirmed that the strain preferably produces EPS (exopolysaccharide). Accordingly, the present invention provides EPS (exopolysaccharide) produced by Lactobacillus paracasei EPS DA-BACS (KCTC14639BP).
이때, 상기 EPS(exopolysaccharide)는, 바람직하게는 a) 락토바실러스 파라카제이(Lactobacillus paracasei) EPS DA-BACS (KCTC14639BP) 배양 상등액에 산성물질을 첨가하여 단백질을 침전 및 제거하는 단계; b) 상기 a) 단계의 단백질 제거액에 친수성 유기용매를 첨가하여 다당류를 추출하는 단계;를 포함하는 과정을 통해 수득되는 것이 좋다.At this time, the EPS (exopolysaccharide), preferably a) Lactobacillus paracasei ( Lactobacillus paracasei ) EPS DA-BACS (KCTC14639BP) Precipitating and removing proteins by adding an acidic material to the culture supernatant; b) extracting the polysaccharide by adding a hydrophilic organic solvent to the protein removal liquid of step a);
더욱 바람직하게는 상기 a) 단계의 산성물질로는 일 예로 트라이클로로아세트산을 사용할 수 있으며, 이 경우 저온에서 1~6시간 동안 반응시키는 것이 좋다. 또한, 상기 b) 단계의 친수성 유기용매로는 일 예로 에탄올을 사용할 수 있다. 이때, 에탄올은 냉각된 80~100%(v/v)의 에탄올을 이용하여 상등액의 1~3배의 부피비로 서서히 첨가하고 저온에서 12~24시간 동안 반응시켜 침전물을 침전시키는 것이 좋다.More preferably, trichloroacetic acid may be used as an acidic material in step a), and in this case, it is preferable to react at a low temperature for 1 to 6 hours. In addition, as an example of the hydrophilic organic solvent in step b), ethanol may be used. At this time, ethanol is preferably added slowly in a volume ratio of 1 to 3 times that of the supernatant using cooled 80 to 100% (v/v) ethanol and reacted at low temperature for 12 to 24 hours to precipitate the precipitate.
한편, 더욱 바람직하게 상기 트라이클로로아세트산은 최종농도가 4~10%(w/v)가 되도록 첨가하여 0~6℃ 저온에서 반응시키는 것이 좋고, 상기 에탄올은 -20~-15℃의 온도로 냉각된 것을 이용하는 것이 좋다.On the other hand, more preferably, the trichloroacetic acid is added to a final concentration of 4 to 10% (w/v) and reacted at a low temperature of 0 to 6 ° C, and the ethanol is cooled to a temperature of -20 to -15 ° C. It is good to use what has been done.
또한, 더욱 바람직하게 상기 단계 b) 이후에, 상기 b) 단계에서 수득한 침전물을 증류수에 용해하여 투석한 후, 건조하는 단계 c);를 더 포함하는 것이 좋다. 이를 통해 EPS(exopolysaccharide)를 고순도로 수득할 수 있다.In addition, more preferably, after step b), step c) of dissolving the precipitate obtained in step b) in distilled water, dialysis, and drying; it is good to further include. Through this, EPS (exopolysaccharide) can be obtained in high purity.
한편, 본 발명은 락토바실러스 파라카제이(Lactobacillus paracasei) EPS DA-BACS (KCTC14639BP), 그의 사균체, 그의 배양물 및 상기한 EPS(exopolysaccharide) 중 선택되는 어느 하나를 함유하는 항균제를 제공한다. 이때, 상기 항균제는, 바람직하게는 녹농균(Pseudomonas aeruginosa), 고초균(Bacillus subtilis), 대장균(Escherichia coli), 클로스트리듐 디피실리균(Clostridium difficile) 및 황색포도구균(Staphylococcus aureus)에 대한 항균 활성을 가지는 것이 좋다.On the other hand, the present invention provides an antibacterial agent containing any one selected from Lactobacillus paracasei EPS DA-BACS (KCTC14639BP), its dead cells, its culture, and the above EPS (exopolysaccharide). At this time, the antibacterial agent, preferably Pseudomonas aeruginosa , Bacillus subtilis ( Bacillus subtilis ), Escherichia coli ( Escherichia coli ), Clostridium difficile bacteria ( Clostridium difficile ) and It is good to have antibacterial activity against Staphylococcus aureus .
한편, 본 발명은 락토바실러스 파라카제이(Lactobacillus paracasei) EPS DA-BACS (KCTC14639BP), 그의 사균체, 그의 배양물 및 상기한 EPS(exopolysaccharide) 중 선택되는 어느 하나를 함유하는 항진균제를 제공한다. 이때, 상기 항진균제는, 바람직하게는 아스페길루스 브라실리언시스(Aspergillus brasiliensis)에 대한 항진균 활성을 가지는 것이 좋다. 본 발명의 일 실시예에서 본 발명의 균주가 상기한 균주에 대한 항균 및 항진균 활성을 가짐을 확인한 바 있다.On the other hand, the present invention provides an antifungal agent containing any one selected from Lactobacillus paracasei EPS DA-BACS (KCTC14639BP), its dead cells, its culture, and the above EPS (exopolysaccharide). At this time, the antifungal agent preferably has antifungal activity against Aspergillus brasiliensis . In one embodiment of the present invention, it was confirmed that the strain of the present invention has antibacterial and antifungal activity against the strain.
한편, 본 발명은 락토바실러스 파라카제이(Lactobacillus paracasei) EPS DA-BACS (KCTC14639BP), 그의 사균체, 그의 배양물 및 상기한 EPS(exopolysaccharide) 중 선택되는 어느 하나를 함유하는 염증질환 예방 또는 치료용 약학 조성물을 제공한다.On the other hand, the present invention Lactobacillus paracasei ( Lactobacillus paracasei ) EPS DA-BACS (KCTC14639BP), its dead cells, its culture and EPS (exopolysaccharide) for preventing or treating inflammatory diseases containing any one selected A pharmaceutical composition is provided.
본 발명의 약학 조성물에 있어, 상기 염증질환은, 일 예로 패혈증(sepsis), 파종혈관내응고(disseminated intravascular coagulation; DIC), 동맥경화(atherosclerosis), 퇴행성 관절염(degenerative arthritis), 치주염(periodontitis), 임파선염(lymphadenopathy), 림프관염(lymphangitis), 제1형 당뇨병(type 1 diabetes), 류마티스 관절염(rheumatoid arthritis), 다발경화증(multiple sclerosis), 암(cancer), 대사성 질환(metabolic disease), 신경퇴행성 질환(neurodegenerative disease), 전신경화증(systemic sclerosis), 다발성근염(polymyositis), 피부근염(polymyositis), 봉입체근염(inclusion body myositis), 알러지(allergy), 알츠하이머병(alzheimer's disease), 강직성 척추염(ankylosing spondylitis), 천식(asthma), 손목 터널 증후군(carpal tunnel syndrome), 셀리악병(celiac disease), 크론병(crohn's disease), 게실염(intestinal diverticulum), 습진(eczema), 섬유증(fibrosis), 루푸스(systemic lupus erythematosus), 췌장염(pancreatitis), 파킨슨병(Parkinson's disease), 건선(psoriasis), 류마티스성 다발근통(Polymyalgia rheumatica), 공피증(dermatosclerosis), 혈관염(vasculitis), 위염(gastritis), 피부염(dermatitis), 척수염(myelitis), 구내염(stomatitis), 관절염(arthritis), 비염(rhinitis), 편도염(tonisiltis), 결막염(conjunctivitis), 방광염(cystitis), 신장염(nephritis), 췌장염(pancreatitis), 간염(hepatitis), 충수염(appendicitis), 폐렴(pneumonia) 및 장염(gastroenteritis) 중 선택되는 어느 하나일 수 있으나, 이에 제한되지 않으며 염증성 반응을 수반하는 모든 질환을 제한 없이 포함할 수 있다.In the pharmaceutical composition of the present invention, the inflammatory disease, for example, sepsis, disseminated intravascular coagulation (DIC), atherosclerosis, degenerative arthritis, periodontitis, Lymphadenopathy, lymphangitis, type 1 diabetes, rheumatoid arthritis, multiple sclerosis, cancer, metabolic disease, neurodegenerative Neurodegenerative disease, systemic sclerosis, polymyositis, polymyositis, inclusion body myositis, allergy, Alzheimer's disease, ankylosing spondylitis ), asthma, carpal tunnel syndrome, celiac disease, crohn's disease, intestinal diverticulum, eczema, fibrosis, systemic lupus erythematosus), pancreatitis, Parkinson's disease, psoriasis, Polymyalgia rheumatica, dermatosclerosis, vasculitis, gastritis, dermatitis, Myelitis, stomatitis, arthritis, rhinitis, tonsillitis, conjunctivitis itis), cystitis, nephritis, pancreatitis, hepatitis, appendicitis, pneumonia, and gastroenteritis, but is not limited thereto. Any disease involving an inflammatory response may be included without limitation.
본 발명에서 사용되는 용어, "예방"이란 본 발명에 따른 약학적 조성물의 투여에 의해 염증질환을 억제하거나 발병을 지연시키는 모든 행위를 의미한다.As used herein, the term "prevention" refers to any action that suppresses or delays the onset of inflammatory diseases by administration of the pharmaceutical composition according to the present invention.
본 발명에서 사용되는 용어, "치료"란 본 발명에 따른 약학적 조성물의 투여에 의해 염증질환에 의한 증세가 호전되거나 이롭게 변경되는 모든 행위를 의미한다.As used herein, the term "treatment" refers to all activities in which symptoms caused by inflammatory diseases are improved or advantageously changed by administration of the pharmaceutical composition according to the present invention.
본 발명의 상기 락토바실러스 파라카제이(Lactobacillus paracasei) EPS DA-BACS (KCTC14639BP), 그의 사균체, 그의 배양물 및 상기한 EPS(exopolysaccharide) 중 선택되는 어느 하나를 유효성분으로 포함하는 조성물은 상기 성분에 추가로 동일 또는 유사한 기능을 나타내는 유효성분을 1종 이상 함유할 수 있다. The Lactobacillus paracasei EPS DA-BACS (KCTC14639BP) of the present invention, a composition comprising any one selected from dead cells, cultures thereof, and EPS (exopolysaccharide) as an active ingredient In addition, one or more active ingredients exhibiting the same or similar functions may be contained.
본 발명의 약학적 조성물은 락토바실러스 파라카제이 EPS DA-BACS 이외에 약학적으로 허용 가능한 담체를 추가로 포함할 수 있다.The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier in addition to Lactobacillus paracasei EPS DA-BACS.
본 발명에서 사용될 수 있는 담체의 종류는 특별히 제한되지 아니하며 당해 기술 분야에서 통상적으로 사용되는 담체라면 어느 것이든 사용할 수 있다. 상기 담체의 비제한적인 예로는, 락토오스, 덱스트로오스, 수크로오스, 솔비톨, 만니톨, 식염수, 멸균수, 링거액, 완충 식염수, 알부민 주사 용액, 자일리톨, 에리스리톨, 말티톨, 말토덱스트린, 글리세롤, 에탄올 등을 들 수 있다. 이들은 단독으로 사용되거나 2종 이상을 혼합하여 사용될 수 있다.The type of carrier that can be used in the present invention is not particularly limited, and any carrier commonly used in the art may be used. Non-limiting examples of the carrier include lactose, dextrose, sucrose, sorbitol, mannitol, saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, xylitol, erythritol, maltitol, maltodextrin, glycerol, ethanol, and the like. can These may be used alone or in combination of two or more.
또한, 본 발명의 약학적 조성물은 필요한 경우, 항산화제, 부형제, 희석제, 완충액 또는 정균제 등 기타 약학적으로 허용 가능한 첨가제들을 첨가하여 사용할 수 있으며, 계면 활성제, 결합제, 충진제, 증량제, 습윤제, 붕해제, 분산제 또는 윤활제 등을 부가적으로 첨가하여 사용할 수 있다.In addition, if necessary, the pharmaceutical composition of the present invention may be used by adding other pharmaceutically acceptable additives such as antioxidants, excipients, diluents, buffers or bacteriostats, surfactants, binders, fillers, bulking agents, wetting agents, disintegrants , a dispersant or a lubricant may be additionally added and used.
본 발명의 약학적 조성물에 있어서, 락토바실러스 파라카제이 EPS DA-BACS는 약학적 조성물의 전체의 중량을 기준으로 0.00001중량％ 내지 99.99중량％로 포함될 수 있으며, 바람직하게는 0.1중량% 내지 90중량%, 보다 바람직하게는 0.1중량% 내지 70중량%, 더욱 바람직하게는 0.1중량% 내지 50중량%로 포함될 수 있으나, 이에 한정되지 않으며 투여 대상의 상태, 구체적인 병증의 종류, 진행 정도 등에 따라 다양하게 변경될 수 있다. 필요한 경우, 약학적 조성물의 전체 함량으로도 포함될 수 있다.In the pharmaceutical composition of the present invention, the Lactobacillus paracasei EPS DA-BACS may be included in an amount of 0.00001% to 99.99% by weight, preferably 0.1% to 90% by weight, based on the total weight of the pharmaceutical composition. %, more preferably from 0.1% to 70% by weight, more preferably from 0.1% to 50% by weight, but is not limited thereto, and may vary depending on the condition of the subject to be administered, the type of specific disease, the degree of progression, etc. can be changed. If necessary, it may be included in the entire content of the pharmaceutical composition.
즉, 본 발명의 약학적 조성물의 약학적 유효량, 유효 투여량은 약학적 조성물의 제제화 방법, 투여 방식, 투여 시간 및/또는 투여 경로 등에 의해 다양해질 수 있으며, 약학적 조성물의 투여로 달성하고자 하는 반응의 종류와 정도, 투여 대상이 되는 개체의 종류, 연령, 체중, 일반적인 건강 상태, 질병의 증세나 정도, 성별, 식이, 배설, 해당 개체에 동시 또는 이시에 함께 사용되는 약물 기타 조성물의 성분 등을 비롯한 여러 인자 및 의약 분야에서 잘 알려진 유사 인자에 따라 다양해질 수 있으며, 당해 기술 분야에서 통상의 지식을 가진 자는 목적하는 치료에 효과적인 투여량을 용이하게 결정하고 처방할 수 있다. 예를 들어, 본 발명의 약학적 조성물의 일일 투여량은 약 0.01 내지 1,000mg/kg이고, 바람직하게는 0.1 내지 100mg/kg이며, 하루 일회 내지 수회에 나누어 투여할 수 있다.That is, the pharmaceutically effective amount and effective dosage of the pharmaceutical composition of the present invention may vary depending on the formulation method, administration method, administration time and/or route of administration of the pharmaceutical composition, and Type and degree of reaction, type of subject to be administered, age, weight, general health condition, symptom or severity of disease, sex, diet, excretion, drugs used simultaneously or at different times in the subject, or components of other compositions, etc. It can vary according to various factors including, and similar factors well known in the medical field, and those skilled in the art can easily determine and prescribe an effective dosage for the desired treatment. For example, the daily dosage of the pharmaceutical composition of the present invention is about 0.01 to 1,000 mg/kg, preferably 0.1 to 100 mg/kg, and may be administered once or several times a day.
본 발명의 약학적 조성물의 투여는 하루에 1회 투여될 수 있고, 수 회에 나누어 투여될 수도 있다. 본 발명의 약학적 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있다. 상기 요소를 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양으로 투여할 수 있으며, 이는 당업자에 의해 용이하게 결정될 수 있다.Administration of the pharmaceutical composition of the present invention may be administered once a day, or may be divided and administered several times. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. Considering all of the above factors, it can be administered in an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.
본 발명의 약학적 조성물은 염증질환을 예방 또는 치료하기 위하여 추가적으로 호르몬 치료, 약물치료 등의 다양한 방법들과 병용하여 사용될 수 있다.The pharmaceutical composition of the present invention may be additionally used in combination with various methods such as hormone therapy and drug therapy to prevent or treat inflammatory diseases.
본 발명에서 사용된 용어, "투여"는 어떠한 적절한 방법으로 환자에게 본 발명의 약학적 조성물을 도입하는 것을 의미하며, 본 발명의 약학적 조성물의 투여 경로 및 투여 방식은 각각 독립적일 수 있으며, 목적하는 해당 부위에 상기 약학적 조성물이 도달할 수 있는 한, 특별한 제한 없이 임의의 투여 경로 및 투여 방식에 따를 수 있다.As used herein, the term "administration" means introducing the pharmaceutical composition of the present invention to a patient by any suitable method, and the route and method of administration of the pharmaceutical composition of the present invention may be independent, respectively, and the purpose Any administration route and administration method may be followed without particular limitation, as long as the pharmaceutical composition can reach the corresponding site.
상기 약학적 조성물은 경구 투여 또는 비경구 투여 방식으로 투여할 수 있으며, 경구 투여 또는 비경구 투여를 위한 적합하고 다양한 제형으로 제제화되어 사용될 수 있다.The pharmaceutical composition may be administered by oral administration or parenteral administration, and may be formulated into various dosage forms suitable for oral administration or parenteral administration.
본 발명의 약학적 조성물을 이용한 경구 투여용 제제의 비제한적인 예로는, 유성 현탁액, 트로키제(troches), 로젠지(lozenge), 정제, 수용성 현탁액, 조제 분말, 과립, 에멀젼, 하드 캡슐, 소프트 캡슐, 시럽 또는 엘릭시르제 등을 들 수 있다.Non-limiting examples of preparations for oral administration using the pharmaceutical composition of the present invention include oily suspensions, troches, lozenges, tablets, aqueous suspensions, prepared powders, granules, emulsions, hard capsules, and soft capsules, syrups or elixirs; and the like.
본 발명의 약학적 조성물을 경구 투여용으로 제제화하기 위하여, 솔비톨, 만니톨, 전분, 아밀로펙틴, 셀룰로오스 락토오스, 사카로오스 또는 젤라틴 등과 같은 결합제; 스테아르산 마그네슘, 스테아르산 칼슘, 스테아릴 푸마르산 나트륨 또는 폴리에틸렌 글리콜 왁스 등과 같은 윤활유; 디칼슘 포스페이트 등과 같은 부형제; 옥수수 전분 또는 고구마 전분 등과 같은 붕해제 등을 사용할 수 있으며, 방향제, 시럽제, 감미제 등도 사용할 수 있다. 나아가 캡슐제의 경우에는 상기 언급한 물질 외에도 지방유와 같은 액체 담체 등을 추가로 사용할 수 있다.In order to formulate the pharmaceutical composition of the present invention for oral administration, a binder such as sorbitol, mannitol, starch, amylopectin, cellulose lactose, saccharose or gelatin; lubricating oils such as magnesium stearate, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax; excipients such as dicalcium phosphate and the like; A disintegrant such as corn starch or sweet potato starch may be used, and aromatics, syrups, sweeteners, and the like may also be used. Furthermore, in the case of capsules, a liquid carrier such as fatty oil may be additionally used in addition to the above-mentioned materials.
본 발명의 약학적 조성물의 비경구 투여 방법으로는, 근육 내 투여, 경피 투여, 정맥 내 투여, 복강 내 투여 또는 피하 투여 등을 이용할 수 있으며, 상기 조성물을 질환 부위에 도포하거나 분무, 흡입하는 방법 또한 이용할 수 있으나 이에 제한되지 않는다.As a method for parenteral administration of the pharmaceutical composition of the present invention, intramuscular administration, transdermal administration, intravenous administration, intraperitoneal administration, or subcutaneous administration may be used, and a method of applying, spraying, or inhaling the composition to a diseased area It can also be used, but is not limited thereto.
본 발명의 약학적 조성물을 이용한 비경구용 제제의 비제한적인 예로는, 주사액, 좌제, 연고, 도포용 파우더, 오일, 호흡기 흡입용 분말, 스프레이용 에어로졸제, 크림 등을 들 수 있다.Non-limiting examples of parenteral preparations using the pharmaceutical composition of the present invention include injection solutions, suppositories, ointments, powders for application, oils, powders for respiratory inhalation, aerosols for sprays, creams, and the like.
본 발명의 약학적 조성물을 비경구 투여용으로 제제화하기 위하여, 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결 건조 제제, 외용제 등을 사용할 수 있으며, 상기 비수성용제, 현탁제로는 올리브 오일과 같은 식물성 기름, 프로필렌글리콜, 폴리에틸렌글리콜, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다.In order to formulate the pharmaceutical composition of the present invention for parenteral administration, sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, external preparations, etc. may be used. Vegetable oils, propylene glycol, polyethylene glycol, injectable esters such as ethyl oleate, and the like can be used.
본 발명의 약학적 조성물을 주사액으로 제제화하는 경우, 본 발명의 약학적 조성물을 안정제 또는 완충제와 함께 물에서 혼합하여 용액 또는 현탁액으로 제조하고 이를 앰플(ampoule) 또는 바이알(vial)의 단위 투여용으로 제제화할 수 있다.When the pharmaceutical composition of the present invention is formulated as an injection solution, the pharmaceutical composition of the present invention is mixed in water together with a stabilizer or buffer to prepare a solution or suspension, which is used for unit administration in an ampoule or vial. can be formulated.
본 발명의 약학적 조성물을 에어로졸제로 제제화하는 경우, 수분산된 농축물 또는 습윤 분말이 분산되도록 추진제 등이 첨가제와 함께 배합할 수 있다.When the pharmaceutical composition of the present invention is formulated into an aerosol formulation, a propellant or the like may be mixed with additives so that the water-dispersed concentrate or wet powder is dispersed.
본 발명의 약학적 조성물을 연고, 오일, 크림, 도포용 파우더, 피부 외용제 등으로 제제화하는 경우에는, 동물성 유, 식물성 유, 왁스, 파라핀, 폴리에틸렌 글리콜, 실리콘, 벤토나이트, 실리카, 탈크, 전분, 트라칸트, 셀룰로오스 유도체, 산화 아연 등을 담체로 사용하여 제제화할 수 있다.When the pharmaceutical composition of the present invention is formulated into ointments, oils, creams, powders for application, skin external preparations, etc., animal oils, vegetable oils, waxes, paraffins, polyethylene glycols, silicones, bentonites, silicas, talc, starch, tras It can be formulated using Kant, cellulose derivatives, zinc oxide and the like as carriers.
한편, 본 발명은 락토바실러스 파라카제이(Lactobacillus paracasei) EPS DA-BACS (KCTC14639BP), 그의 사균체, 그의 배양물 및 상기한 EPS(exopolysaccharide) 중 선택되는 어느 하나를 함유하는 염증질환 개선용 식품 조성물을 제공한다.On the other hand, the present invention Lactobacillus paracasei ( Lactobacillus paracasei ) EPS DA-BACS (KCTC14639BP), its dead cells, its culture, and a food composition for improving inflammatory diseases containing any one selected from the above EPS (exopolysaccharide) provides
본 발명의 용어, "개선"이란, 본 발명의 조성물의 투여로 염증질환이 호전 또는 이롭게 변경되는 모든 행위를 의미한다.The term of the present invention, "improvement" refers to all activities that improve or beneficially change inflammatory diseases by administration of the composition of the present invention.
본 발명의 염증질환 개선용 식품 조성물에 있어, 상기 락토바실러스 파라카제이 EPS DA-BACS는 바람직하게 염증질환 개선용 식품 조성물 대비 0.00001~50 중량% 포함되는 것이 좋다. 0.00001 중량% 미만일 경우에는 그 효과가 미비하고, 50 중량%를 초과하는 경우에는 사용량 대비 효과 증가가 미미하여 비경제적이다.In the food composition for improving inflammatory diseases of the present invention, the Lactobacillus paracasei EPS DA-BACS is preferably included in an amount of 0.00001 to 50% by weight relative to the food composition for improving inflammatory diseases. If it is less than 0.00001% by weight, the effect is insufficient, and if it exceeds 50% by weight, the increase in effect compared to the amount used is insignificant, which is uneconomical.
본 발명의 염증질환 개선용 식품 조성물은 일 예로, 면류, 껌류, 유제품류, 아이스크림류, 육류, 곡류, 카페인 음료, 일반음료, 초콜릿, 빵류, 스낵류, 과자류, 사탕, 피자, 젤리, 알코올성 음료, 술, 비타민 복합제 및 그 밖의 건강보조식품류 중 선택되는 어느 하나일 수 있으나, 반드시 이에 한정되는 것은 아니다.The food composition for improving inflammatory diseases of the present invention is, for example, noodles, gums, dairy products, ice creams, meats, grains, caffeinated beverages, general beverages, chocolates, breads, snacks, confectionery products, candies, pizzas, jellies, alcoholic beverages, It may be any one selected from alcohol, vitamin complexes, and other health supplements, but is not necessarily limited thereto.
본 발명의 식품 조성물을 식품 첨가물로 사용할 경우, 이를 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다.When using the food composition of the present invention as a food additive, it may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to conventional methods.
한편, 본 발명은 락토바실러스 파라카제이(Lactobacillus paracasei) EPS DA-BACS (KCTC14639BP), 그의 사균체, 그의 배양물 및 상기한 EPS(exopolysaccharide) 중 선택되는 어느 하나를 함유하는 염증질환 개선용 건강기능식품을 제공한다.On the other hand, the present invention Lactobacillus paracasei ( Lactobacillus paracasei ) EPS DA-BACS (KCTC14639BP), its dead cells, its culture, and health function for improving inflammatory diseases containing any one selected from the above EPS (exopolysaccharide) provide food.
상기 "건강기능식품"이라 함은 건강기능식품에 관한 법률 제6727호에 따른 인체에 유용한 기능성을 가진 원료나 성분을 사용하여 제조 및 가공한 식품을 의미하며, "기능성"이라 함은 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건 용도에 유용한 효과를 얻을 목적으로 섭취하는 것을 의미한다.The term "health functional food" refers to food manufactured and processed using raw materials or ingredients having functional properties useful for the human body in accordance with the Act on Health Functional Foods No. 6727, and "functional" refers to the structure of the human body. And it refers to intake for the purpose of obtaining useful effects for health purposes such as regulating nutrients for functions or physiological actions.
본 발명의 식품 조성물 및 건강기능식품은 통상 사용되어 냄새, 맛, 시각 등을 향상시킬 수 있는 추가 성분을 포함할 수 있다. 예들 들어, 비오틴(biotin), 폴레이트(folate), 판토텐산(panthotenic acid), 비타민 A, C, D, E, B1, B2, B6, B12, 니아신(niacin) 등을 포함할 수 있다. 또한, 크롬(Cr), 마그네슘(Mg), 망간(Mn), 구리(Cu), 아연(Zn), 철(Fe), 칼슘(Ca) 등의 미네랄을 포함할 수 있다. 또한, 시스테인, 발린, 라이신, 트립토판 등의 아미노산을 포함할 수 있다. 또한, 방부제(소르빈산 칼륨, 벤조산나트륨, 살리실산, 디히드로초산나트륨 등), 착색제(타르색소 등), 발색제(아질산 나트륨, 아초산 나트륨 등), 표백제(아황산나트륨), 살균제(표백분과 고도 표백분, 차아염소산나트륨 등), 팽창제(명반, D-주석산수소칼륨 등), 강화제, 유화제, 증점제(호료), 피막제, 산화방지제(부틸히드록시아니졸(BHA), 부틸히드록시톨루엔(BHT) 등), 조미료(MSG 글루타민산나트륨 등), 감미료(둘신, 사이클레메이트, 사카린, 나트륨 등), 향료(바닐린, 락톤류 등), 검기초제, 거품억제제, 용제, 개량제 등의 식품 첨가물(food additives)을 첨가할 수 있다. 상기 첨가물은 식품의 종류에 따라 선별되고 적절한 양으로 사용될 수 있다.The food composition and health functional food of the present invention may include additional ingredients that are commonly used and can improve smell, taste, and visual properties. For example, it may include biotin, folate, panthotenic acid, vitamins A, C, D, E, B1, B2, B6, B12, niacin, and the like. In addition, minerals such as chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), zinc (Zn), iron (Fe), and calcium (Ca) may be included. In addition, amino acids such as cysteine, valine, lysine, and tryptophan may be included. In addition, preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dihydroacetate, etc.), coloring agents (tar colorant, etc.), coloring agents (sodium nitrite, sodium nitrite, etc.), bleaching agents (sodium sulfite), disinfectants (bleaching powder and high bleaching powder, sodium hypochlorite, etc.), swelling agent (alum, D-potassium hydrogen tartrate, etc.), strengthening agent, emulsifier, thickener (thickener), coating agent, antioxidant (butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), etc.) Food additives such as seasonings (MSG monosodium glutamate, etc.), sweeteners (dulcin, cyclemate, saccharin, sodium, etc.), flavorings (vanillin, lactones, etc.), gum base, antifoaming agent, solvent, improver, etc. can be added. The additive may be selected according to the type of food and used in an appropriate amount.
본 발명의 건강기능식품에 있어, 락토바실러스 파라카제이 EPS DA-BACS의 함량은 특별히 제한되지 않으며, 투여 대상의 상태, 구체적인 병증의 종류, 진행 정도 등에 따라 다양하게 변경될 수 있다. 필요한 경우, 식품의 전체 함량으로도 포함될 수 있다.In the health functional food of the present invention, the content of Lactobacillus paracasei EPS DA-BACS is not particularly limited, and may be variously changed depending on the condition of the subject to be administered, the type of specific disease, and the degree of progression. If necessary, it may also be included in the total content of food.
또한, 본 발명은 락토바실러스 파라카제이(Lactobacillus paracasei) EPS DA-BACS (KCTC14639BP), 그의 사균체, 그의 배양물 및 락토바실러스 파라카제이(Lactobacillus paracasei) EPS DA-BACS (KCTC14639BP)가 생성하는 EPS(exopolysaccharide) 중 선택되는 어느 하나를 함유하는 프로바이오틱스 생육 촉진용 조성물을 제공한다.In addition, the present invention Lactobacillus paracasei ( Lactobacillus paracasei ) EPS DA-BACS (KCTC14639BP), its dead cells, its culture and Lactobacillus paracasei ( Lactobacillus paracasei ) EPS produced by EPS DA-BACS (KCTC14639BP) It provides a composition for promoting the growth of probiotics containing any one selected from (exopolysaccharide).
이때, 상기 프로바이오틱스 생육 촉진용 조성물은, 바람직하게는 락토바실러스 가세리(Lactobacillus gasseri), 비피도박테리움 비피덤(Bifidobacterium bifidum), 비피도박테리움 애니말리스(Bifidobacterium animalis), 비피도박테리움 패이칼(Bifidobacterium faecale) 중 선택되는 어느 하나 이상의 프로바이오틱스의 생육을 촉진시키는 것이 좋다.At this time, the composition for promoting the growth of probiotics is preferably Lactobacillus gasseri, Bifidobacterium bifidum, Bifidobacterium animalis, Bifidobacterium animalis , Bifidobacterium pical ( Bifidobacterium faecale ) It is preferable to promote the growth of any one or more probiotics selected from among them.
본 발명에서는 락토바실러스 파라카제이(Lactobacillus paracasei) EPS DA-BACS (KCTC14639BP)가 생성하는 EPS(exopolysaccharide)가 상기한 프로바이오틱스들의 생육을 이눌린 대비 효과적으로 증진시키는 것을 확인하였다. 따라서, 본 발명의 락토바실러스 파라카제이(Lactobacillus paracasei) EPS DA-BACS (KCTC14639BP), 그의 사균체 및 그의 배양물 또한 상기한 프로바이오틱스들의 생육을 촉진시키는 효능을 보유한다 할 수 있다. 본 발명에서 청구하는 프로바이오틱스 생육 촉진용 조성물은 당업계에서 프리바이오틱스(prebiotics)로 불리기도 한다.In the present invention, it was confirmed that EPS (exopolysaccharide) produced by Lactobacillus paracasei EPS DA-BACS (KCTC14639BP) effectively promotes the growth of the probiotics compared to inulin. Therefore, it can be said that the Lactobacillus paracasei EPS DA-BACS (KCTC14639BP), its dead cells, and its culture of the present invention also have the efficacy of promoting the growth of the above-mentioned probiotics. The composition for promoting the growth of probiotics claimed in the present invention is also called prebiotics in the art.
한편, 본 발명은 락토바실러스 파라카제이(Lactobacillus paracasei) EPS DA-BACS (KCTC14639BP), 그의 사균체, 그의 배양물 및 상기한 EPS(exopolysaccharide) 중 선택되는 어느 하나를 함유하는 피부염증 예방 또는 개선용 화장료 조성물을 제공한다.On the other hand, the present invention is Lactobacillus paracasei ( Lactobacillus paracasei ) EPS DA-BACS (KCTC14639BP), its dead cells, its culture, and for preventing or improving skin inflammation containing any one selected from the above EPS (exopolysaccharide) A cosmetic composition is provided.
본 발명의 화장료 조성물에 있어, 상기 피부염증은, 일 예로 아토피성 피부염(atopic dermatitis), 접촉성 피부염(contact dermatitis), 지루성 피부염(seborrhea) 및 여드름 중 선택되는 어느 하나인 것일 수 있으나, 이에 제한되지 않으며 피부에서 일어나는 염증성 반응을 수반하는 모든 피부 염증성 질환을 제한 없이 포함할 수 있다.In the cosmetic composition of the present invention, the skin inflammation may be, for example, any one selected from atopic dermatitis, contact dermatitis, seborrhea and acne, but is limited thereto It may include, without limitation, any skin inflammatory disease accompanied by an inflammatory reaction occurring in the skin.
한편, 본 발명에 있어, 상기 락토바실러스 파라카제이 EPS DA-BACS는, 바람직하게 화장료 조성물 전체 중량에 대하여 0.0001~30.0중량%가 함유되는 것이 좋다. 더욱 바람직하게는 화장료 조성물 전체 중량에 대해서 0.01~10중량% 함유되는 것이 좋다. 락토바실러스 파라카제이 EPS DA-BACS의 함량이 0.0001중량% 미만인 경우에는 피부염증 예방 또는 개선 효과가 미미하고, 30.0중량% 초과할 경우 함유량 증가에 따른 뚜렷한 효과가 증가하지 않는다.Meanwhile, in the present invention, the Lactobacillus paracasei EPS DA-BACS is preferably contained in an amount of 0.0001 to 30.0% by weight based on the total weight of the cosmetic composition. More preferably, it is good to contain 0.01 to 10% by weight based on the total weight of the cosmetic composition. If the content of Lactobacillus paracasei EPS DA-BACS is less than 0.0001% by weight, the effect of preventing or improving skin inflammation is insignificant, and if it exceeds 30.0% by weight, the obvious effect does not increase with the increase in the content.
한편, 본 발명의 화장료 조성물에 포함되는 성분은 유효성분으로서 본 발명의 락토바실러스 파라카제이 EPS DA-BACS 이외에 화장료 조성물에 통상적으로 이용되는 성분들을 포함할 수 있으며, 예컨대 항산화제, 안정화제, 용해화제, 비타민, 안료 및 향료와 같은 통상적인 보조제, 그리고 담체를 포함한다.On the other hand, the ingredients included in the cosmetic composition of the present invention may include ingredients commonly used in cosmetic compositions in addition to the Lactobacillus paracasei EPS DA-BACS of the present invention as active ingredients, such as antioxidants, stabilizers, solubilizers, conventional adjuvants such as agents, vitamins, pigments and flavors, and carriers.
본 발명의 화장료 조성물은 당업계에서 통상적으로 제조되는 어떠한 제형으로도 제조될 수 있으며, 예를 들어, 용액, 현탁액, 유탁액, 페이스트, 겔, 크림, 로션, 파우더, 비누, 계면활성제-함유 클렌징, 오일, 팩, 마사지크림 및 스프레이 등으로 제형화될 수 있으나, 이에 한정되는 것은 아니다. 보다 상세하게는, 유연 화장수, 영양 화장수, 영양 크림, 마사지크림, 에센스, 클렌징 크림, 클렌징 폼, 클렌징 워터, 팩, 스프레이 또는 파우더의 제형으로 제조될 수 있다.The cosmetic composition of the present invention can be prepared in any formulation conventionally prepared in the art, for example, a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing , Oil, pack, massage cream and spray, etc., but may be formulated, but is not limited thereto. More specifically, it may be prepared in the form of softening lotion, nutrient lotion, nutrient cream, massage cream, essence, cleansing cream, cleansing foam, cleansing water, pack, spray or powder.
본 발명의 화장료 조성물의 제형이 페이스트, 크림 또는 겔인 경우에는 담체 성분으로서 동물성유, 식물성유, 왁스, 파라핀, 전분, 트라칸트, 셀룰로오스 유도체, 폴리에틸렌 글리콜, 실리콘, 벤토나이트, 실리카, 탈크 또는 산화아연 등이 이용될 수 있다.When the formulation of the cosmetic composition of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide as a carrier component this can be used
본 발명의 화장료 조성물의 제형이 용액 또는 유탁액인 경우에는 담체 성분으로서 용매, 용해화제 또는 유탁화제가 이용되고, 예컨대 물, 에탄올, 이소프로판올, 에틸 카보네이트, 에틸 아세테이트, 벤질 알코올, 벤질 벤조에이트, 프로필렌글리콜, 1,3-부틸글리콜 오일, 글리세롤 지방족 에스테르, 폴리에틸렌글리콜 또는 소르비탄의 지방산 에스테르가 있다.When the formulation of the cosmetic composition of the present invention is a solution or emulsion, a solvent, solubilizing agent or emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene fatty acid esters of glycol, 1,3-butyl glycol oil, glycerol aliphatic esters, polyethylene glycol or sorbitan.
본 발명의 화장료 조성물의 제형이 현탁액인 경우에는 담체 성분으로서 물, 에탄올 또는 프로필렌글리콜과 같은 액상의 희석제, 에톡실화 이소스테아릴 알코올, 폴리옥시에틸렌 소르비톨 에스테르 및 폴리옥시에틸렌 소르비탄 에스테르와 같은 현탁제, 미소 결정성 셀룰로오스, 알루미늄 메타히드록시드, 벤토나이트, 아가 또는 트라칸트 등이 이용될 수 있다.When the formulation of the cosmetic composition of the present invention is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester , microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tracanth, and the like can be used.
본 발명의 화장료 조성물의 제형이 파우더 또는 스프레이인 경우에는 담체 성분으로서 락토스, 탈크, 실리카, 알루미늄 히드록시드, 칼슘 실리케이트 또는 폴리아미드 파우더가 이용될 수 있고, 특히 스프레이인 경우에는 추가적으로 클로로플루오로히드로카본, 프로판/부탄 또는 디메틸 에테르와 같은 추진체를 포함할 수 있다.When the formulation of the cosmetic composition of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, additional chlorofluorohydro propellants such as carbon, propane/butane or dimethyl ether.
본 발명의 화장료 조성물의 제형이 계면활성제 함유 클렌징인 경우에는 담체 성분으로서 지방족 알코올 설페이트, 지방족 알코올 에테르 설페이트, 설포숙신산 모노에스테르, 이세티오네이트, 이미다졸리늄 유도체, 메틸타우레이트, 사르코시네이트, 지방산 아미드 에테르 설페이트, 알킬아미도베타인, 지방족 알코올, 지방산 글리세리드, 지방산 디에탄올아미드, 식물성 유, 라놀린 유도체 또는 에톡실화 글리세롤 지방산 에스테르 등이 이용될 수 있다.When the formulation of the cosmetic composition of the present invention is surfactant-containing cleansing, as carrier components, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, Fatty acid amide ether sulfates, alkylamidobetaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives, or ethoxylated glycerol fatty acid esters may be used.
본 발명의 화장료 조성물이 비누, 계면활성제 함유 클렌징 제형 또는 계면활성제 비함유 클렌징 제형일 경우, 피부에 도포한 후 닦아내거나 떼거나 물로 씻어낼 수도 있다. 구체적인 예로서, 상기 비누는 액상비누, 가루비누, 고형비누 및 오일비누이며, 상기 계면활성제 함유 클렌징 제형은 클렌징 폼, 클렌징 워터, 클렌징 수건 및 클렌징 팩이며, 상기 계면활성제 비 함유 클렌징 제형은 클렌징크림, 클렌징 로션, 클렌징 워터 및 클렌징 겔이며, 이에 한정되는 것은 아니다.When the cosmetic composition of the present invention is a soap, a surfactant-containing cleansing formulation, or a surfactant-free cleansing formulation, it may be applied to the skin and then wiped off, removed, or washed with water. As specific examples, the soap is liquid soap, powder soap, solid soap, and oil soap, the surfactant-containing cleansing formulation is a cleansing foam, cleansing water, cleansing towel, and a cleansing pack, and the surfactant-free cleansing formulation is a cleansing cream. , cleansing lotion, cleansing water and cleansing gel, but are not limited thereto.
이하, 본 발명의 내용을 하기 실시예 또는 실험예를 통해 더욱 상세히 설명하고자 한다. 다만, 본 발명의 권리범위가 하기 실시예 또는 실험예에만 한정되는 것은 아니고, 그와 등가의 기술적 사상의 변형까지를 포함한다.Hereinafter, the contents of the present invention will be described in more detail through the following examples or experimental examples. However, the scope of the present invention is not limited only to the following examples or experimental examples, and includes modifications of equivalent technical ideas.
[실시예 1: EPS 생성균주의 분리 및 동정][Example 1: Separation and identification of EPS producing strain]
1. 유산균 분리1. Isolation of lactic acid bacteria
건강한 한국인 자원자의 대변을 수집한 후, 상기 대변을 멸균 생리식염수를 이용해 십진 희석법으로 단계별 희석한 다음, LBS 선택배지(Lactobacillus selective media)에 100㎕씩 도말하였다. 도말한 플레이트를 37℃에서 48시간 동안 혐기배양한 뒤, 배양된 균주 중에서 단일 콜로니를 1차로 선별한 후 해당 콜로니를 LBS 선택배지에 한번 더 콜로니 픽킹(colony picking) 후 점적하였다. 점적한 플레이트를 37℃에서 48시간 동안 혐기배양한 뒤 다시 한번 해당 콜로니가 잘 자라는지 확인한 후 최종적으로 유산균 후보균주 261개를 선정하였다.After collecting feces from healthy Korean volunteers, the feces were diluted step by step using sterile physiological saline by a decimal dilution method, and then smeared on LBS selective media (Lactobacillus selective media) at 100 μl. After anaerobic culture of the plated plate at 37 ° C. for 48 hours, a single colony was first selected from among the cultured strains, and then the colony was picked once more on an LBS selective medium and then spotted. After anaerobically culturing the plated plate at 37 ° C. for 48 hours, it was confirmed once again that the colony grew well, and finally 261 candidate strains of lactic acid bacteria were selected.
2. 유산균 동정2. Identification of lactic acid bacteria
분리된 유산균 후보균주를 대상으로 16S rRNA 염기서열 분석을 통한 동정을 수행하였다. 유산균 후보균주로부터 지노믹(genomic) DNA를 추출 및 정제하고 16S rRNA 유전자 서열분석을 위해 프라이머를 사용해 DNA를 PCR 증폭하였다. 수득한 16S rRNA 염기서열의 상동성 분석은 BLAST 소프트웨어로부터 이루어졌으며, 이를 통해 각 유산균 후보군들의 속(genus)과 종(species)을 확정지었다. 유산균 후보균주 261개를 각각 동정한 결과, 락토바실러스 속(Lactobacillus sp.) 225개, 와이셀라 속(Weissella sp.) 7개, 페디오코커스(Pediococcus) 29개로 구성됨이 밝혀졌다.Identification was performed through 16S rRNA sequencing for the isolated lactic acid bacteria candidate strains. Genomic DNA was extracted and purified from candidate Lactobacillus strains, and DNA was PCR amplified using primers for 16S rRNA gene sequencing. The homology analysis of the obtained 16S rRNA sequence was performed using BLAST software, and through this, the genus and species of each lactic acid bacteria candidate group were determined. As a result of identifying 261 candidate strains of lactic acid bacteria, respectively, it was found that they consisted of 225 Lactobacillus sp., 7 Weissella sp., and 29 Pediococcus .
3. 균주의 EPS 생성여부 및 Ropy 특성3. Whether the strain produces EPS and characteristics of the ropy
건강한 한국인 자원자의 대변에서 자체 분리한 261개 유산균의 EPS(Exopolysaccharide) 생성여부 및 해당 EPS의 Ropy 특성을 측정하였다. 10% 수크로오스를 당원으로 하는 MRS 아가를 제조한 후 261개 유산균 각각을 스트레킹(streaking)하여 37℃에서 72시간 동안 혐기배양하였다. 배양이 완료된 후 멸균된 루프를 이용하여 단일 콜로니의 점성 물질을 확인하였고 늘어난 길이를 ㎜ 단위로 측정했다.The production of EPS (Exopolysaccharide) of 261 lactic acid bacteria isolated from feces of healthy Korean volunteers and the ropy characteristics of the EPS were measured. After preparing MRS agar containing 10% sucrose as a sugar source, each of 261 lactic acid bacteria was streaked and anaerobically cultured at 37° C. for 72 hours. After the incubation was complete, the viscous material of single colonies was identified using a sterile loop, and the length of the elongation was measured in mm.
건강한 한국인 자원자의 대변에서 자체분리한 261개 유산균의 EPS(Exopolysaccharide) 생성여부 및 해당 EPS의 Ropy 특성을 측정한 결과, 표 1과 같이 EPS 생성이 가장 우수한 유산균 1종인 락토바실러스 파라카제이(Lactobacillus paracasei) EPS DA-BACS을 선정하였다. 이의 16S rRNA 염기서열은 서열번호 1에 나타내었다.As a result of measuring the EPS (Exopolysaccharide) production of 261 lactic acid bacteria isolated from the feces of healthy Korean volunteers and the ropy characteristics of the EPS , Lactobacillus paracasei, one of the lactic acid bacteria with the best EPS production, was tested as shown in Table 1. ) EPS DA-BACS was selected. Its 16S rRNA nucleotide sequence is shown in SEQ ID NO: 1.
균주명strain name EPS의 Ropy 특성EPS ropy characteristics
Lactobacillus paracaseiLactobacillus paracasei EPS DA-BACS EPS DA-BACS 40mm 생성create 40mm
그 외 260개 균주260 other strains 97.3%는 미생성
2.7%는 최대 15mm 생성97.3% is non-generated
2.7% produces up to 15 mm
[실험예 1: 분리균주의 장내 생존성 평가][Experimental Example 1: Intestinal viability evaluation of isolated strains]
1. 인공위액에 대한 저항성1. Resistance to artificial gastric juice
인공위액은 1N HCl을 사용해 pH 3으로 조정한 MRS broth(De Man, Rogosa and Sharpe)에 0.25㎛ 막으로 필터링한 펩신을 최종농도가 1,000 unit/㎖가 되도록 첨가하여 제조하였다.Artificial gastric juice was prepared by adding pepsin filtered through a 0.25 μm membrane to MRS broth (De Man, Rogosa and Sharpe) adjusted to pH 3 with 1N HCl to a final concentration of 1,000 units/ml.
이후, MRS broth(0.05% L-cysteine)에 분리균주를 접종하여 37℃에서 24시간 동안 혐기배양한 뒤 원심분리 및 PBS버퍼로 세척하여 균체를 회수하였다. 회수된 균체를 상기의 인공위액에 10% 만큼 첨가해 최종농도가 107~108 CFU/㎖가 되도록 한 후 37℃에서 정치배양하였다. 0시간, 1.5시간, 3시간 후 배양액을 각각 1㎖씩 채취하여 희석을 통해 생균수를 측정하였으며, 초기접종균수(0시간) 대비 생존율을 계산하였다.Thereafter, the isolated strain was inoculated into MRS broth (0.05% L-cysteine) and anaerobically cultured at 37° C. for 24 hours, followed by centrifugation and washing with PBS buffer to recover the cells. The recovered cells were added to the artificial gastric fluid in an amount of 10% to a final concentration of 10 7 to 10 8 CFU/mL, and then cultured at 37°C. After 0 hour, 1.5 hour, and 3 hours, 1 ml each of the culture medium was collected, the number of viable cells was measured through dilution, and the survival rate was calculated relative to the number of initial inoculated bacteria (0 hour).
동속동종 균주와의 비교실험을 위해 타 락토바실러스 파라카제이(Lactobacillus paracasei) 균주 12개 스트레인(국내 미생물자원센터 KCTC·KCCM의 보유자원)도 동일한 방법으로 실험하여, 그 결과를 하기 표 2에 나타내었다.For comparative experiments with strains of the same species, 12 strains of other Lactobacillus paracasei strains (resources held by the Korea Microbial Resource Center KCTC KCCM) were also tested in the same way, and the results are shown in Table 2 below. was
인공위액에 대한 생존율Survival rate for artificial gastric juice
분류classification 균주 명strain name 0h 대비 3h에서의 생존율 (%)Survival rate at 3h versus 0h (%)
본 발명균주Inventive strain L. paracasei EPS DA-BACS L. paracasei EPS DA-BACS 143.5143.5
타 동속동종 균주Other homologous strains KCTC 3169KCTC 3169 63.263.2
KCTC 13090KCTC 13090 71.171.1
KCTC 3165KCTC 3165 59.559.5
KCTC 3189KCTC 3189 57.757.7
KCTC 5546KCTC 5546 40.640.6
KCTC 3510KCTC 3510 27.827.8
KCTC 5058KCTC 5058 105.5105.5
KCTC 3074KCTC 3074 8080
KCCM 40995KCCM 40995 55.255.2
KCCM 42830KCCM 42830 57.157.1
KCCM 32822KCCM 32822 0.0050.005
KCCM 41276KCCM 41276 4141
실험 결과, 표 2와 같이 EPS DA-BACS의 초기접종균수 대비 3시간 후 생존율이 143.5%로 우수하였으며, 이는 함께 실험한 타 락토바실러스 파라카제이(Lactobacillus paracasei) 동속동종 균주 12개 스트레인 중 가장 높은 수치였다. 함께 실험한 동속동종 비교균주 12개 스트레인의 인공위액 생존율 평균은 54.9%로, EPS DA-BACS의 생존율이 약 2.6배 높은 수치를 보였다.As a result of the experiment, as shown in Table 2, the survival rate was excellent at 143.5% after 3 hours compared to the number of initial inoculum of EPS DA-BACS, which was the highest among 12 strains of Lactobacillus paracasei homologous strains tested together It was a shame. The average survival rate in artificial gastric juice of 12 isogeneous comparative strains tested together was 54.9%, which was about 2.6 times higher than that of EPS DA-BACS.
2. 인공담즙액에 대한 저항성2. Resistance to artificial bile fluid
인공담즙액은 MRS broth에 0.25㎛ 막으로 필터링한 황소담즙(oxgall)을 최종농도가 0.3%가 되도록 첨가하여 제조하였다. MRS broth(0.05% L-cysteine)에 균을 접종하여 37℃에서 24시간 동안 혐기배양한 뒤 원심분리 및 PBS버퍼로 세척하여 균체를 회수하였다. 회수된 균체를 상기의 인공담즙액에 10% 만큼 첨가해 최종농도가 107~108 CFU/㎖가 되도록 한 후 37℃에서 정치배양하였다. 0시간, 1.5시간, 3시간 후 배양액을 각각 1㎖씩 채취하여 희석을 통해 생균수를 측정하였으며, 초기접종균수(0시간) 대비 생존율을 계산하여 하기 표 3에 나타내었다.Artificial bile was prepared by adding oxgall filtered through a 0.25 μm membrane to MRS broth to a final concentration of 0.3%. Bacteria were inoculated into MRS broth (0.05% L-cysteine) and anaerobically cultured at 37° C. for 24 hours, followed by centrifugation and washing with PBS buffer to recover the cells. The recovered cells were added as much as 10% to the artificial bile solution to a final concentration of 10 7 to 10 8 CFU/mL, and then cultured at 37°C. After 0 hour, 1.5 hour, and 3 hours, 1 ml each of the culture medium was collected, and the number of viable cells was measured through dilution.
인공담즙액에 대한 생존율Survival rate for artificial bile fluid
분류classification 균주 명strain name 0 h 대비 3 h에서의 생존율 (%)Survival rate at 3 h versus 0 h (%)
본 발명균주Inventive strain L. paracasei EPS DA-BACS L. paracasei EPS DA-BACS 250250
실험 결과, EPS DA-BACS 균주는 인공담즙액에서 250%라는 우수한 생존율을 나타냈다.As a result of the experiment, the EPS DA-BACS strain showed an excellent survival rate of 250% in artificial bile fluid.
3. 인공췌장액에 대한 저항성3. Resistance to artificial pancreatic fluid
인공췌장액은 MRS broth에 0.25㎛ 막으로 필터링한 판크레아틴을 최종농도가 0.5%가 되도록 첨가하여 제조하였다. MRS broth(0.05% L-cysteine)에 균을 접종하여 37℃에서 24시간 동안 혐기배양한 뒤 원심분리 및 PBS버퍼로 세척하여 균체를 회수하였다. 회수된 균체를 상기의 인공췌장액에 10% 만큼 첨가해 최종농도가 107~108 CFU/㎖가 되도록 한 후 37℃에서 정치배양하였다. 0시간, 1.5시간, 3시간 후 배양액을 각각 1㎖씩 채취하여 희석을 통해 생균수를 측정하였으며, 초기접종균수(0시간) 대비 생존율을 계산하여 하기 표 4에 나타내었다.Artificial pancreatic fluid was prepared by adding pancreatin filtered through a 0.25 μm membrane to MRS broth to a final concentration of 0.5%. Bacteria were inoculated into MRS broth (0.05% L-cysteine) and anaerobically cultured at 37° C. for 24 hours, followed by centrifugation and washing with PBS buffer to recover the cells. The recovered cells were added to the artificial pancreatic fluid in an amount of 10% to a final concentration of 10 7 to 10 8 CFU/mL, and then cultured at 37°C. After 0 hour, 1.5 hour, and 3 hours, 1 ml each of the culture medium was collected, and the number of viable cells was measured through dilution.
인공췌장액에 대한 생존율Survival rate for artificial pancreatic fluid
분류classification 균주 명strain name 0 h 대비 3 h에서의 생존율 (%)Survival rate at 3 h versus 0 h (%)
본 발명균주Inventive strain L. paracasei EPS DA-BACS L. paracasei EPS DA-BACS 126.9126.9
실험 결과, EPS DA-BACS 균주는 인공췌장액에서 126.9%라는 우수한 생존율을 나타냈다.As a result of the experiment, the EPS DA-BACS strain showed an excellent survival rate of 126.9% in artificial pancreatic fluid.
[실험예 2: 분리균주의 효능 규명][Experimental Example 2: Identification of the efficacy of the isolated strain]
1. 유해미생물에 대한 항균활성 평가1. Evaluation of antibacterial activity against harmful microorganisms
분리균주의 유해미생물에 대한 항균활성을 평가하기 위하여, 유산균 스톡을 MRS broth(0.05% L-cysteine)에 1%(v/v) 농도가 되도록 접종하고 32~37oC에서 24~168시간 동안 혐기조건에서 정치배양하였다. 상기 유산균 배양액을 4,000rpm에서 10분간 원심분리한 후 상층액을 필터여과하여 유산균 배양물로 제조하였다.In order to evaluate the antibacterial activity of the isolated strain against harmful microorganisms, the lactic acid bacteria stock was inoculated to a concentration of 1% (v/v) in MRS broth (0.05% L-cysteine) and incubated at 32 to 37 o C for 24 to 168 hours. Political culture was carried out under anaerobic conditions. The lactic acid bacteria culture was centrifuged at 4,000 rpm for 10 minutes, and the supernatant was filtered to prepare a lactic acid bacteria culture.
이후, Mueller hinton Ⅱ broth를 용매로 하여 유산균 배양물을 10%로 희석한 후 96-웰 플레이트의 각 웰에 200㎕씩 분주하였다. 해당 웰에 전날 활성화해두었던 항균활성 평가균주(Pseudomonas aeruginosa, Bacillus subtilis, Escherichia coli, Staphylococcus aureus)를 각각 1%씩 접종하였다. 상기 96-웰 플레이트를 37℃에서 배양하며 0시간, 24시간에 각각 OD600을 측정해 항균활성을 평가하였다.Thereafter, the lactic acid bacteria culture was diluted to 10% using Mueller hinton II broth as a solvent, and then 200 μl was dispensed into each well of a 96-well plate. The antibacterial activity evaluation strains ( Pseudomonas aeruginosa, Bacillus subtilis, Escherichia coli, Staphylococcus aureus ), which had been activated the previous day, were inoculated into the wells at 1% each. The 96-well plate was incubated at 37° C. and the antibacterial activity was evaluated by measuring OD 600 at 0 hour and 24 hour, respectively.
동속동종 균주와의 비교실험을 위해, 타 락토바실러스 파라카제이(Lactobacillus paracasei) 균주 12개 스트레인(국내 미생물자원센터 KCTC 및 KCCM의 보유자원)도 동일한 방법으로 실험하였으며, 10% 유산균 배양물에 항균활성 평가균주를 접종하지 않은 블랭크(blank)군과 10% 유산균 배양물 대신 Mueller hinton Ⅱ broth 200㎕를 분주해 항균활성 평가균주를 접종한 대조군도 함께 실험하였다. 그 결과를 하기 표 5에 나타내었다.For comparison experiments with strains of the same species, 12 strains of other Lactobacillus paracasei strains (resources held by KCTC and KCCM, Korea's Microbial Resource Center) were also tested in the same way, and 10% lactobacillus cultures were tested for antibacterial A blank group not inoculated with the activity evaluation strain and a control group inoculated with the antibacterial activity evaluation strain by dispensing 200 μl of Mueller hinton II broth instead of 10% lactic acid bacteria culture were also tested. The results are shown in Table 5 below.
항균활성 평가Antibacterial activity evaluation
분류classification 항균활성 평가균주Antibacterial activity evaluation strain
Bacillus subtilisBacillus subtilis pseudomonas aeruginosapseudomonas aeruginosa Staphylococcus aureusStaphylococcus aureus Escherichia coliEscherichia coli
타 동속동종균주Other homologous strains KCTC 3169KCTC 3169 -- -- VGVG --
KCTC 13090KCTC 13090 -- -- VGVG --
KCTC 3165KCTC 3165 -- -- VGVG --
KCTC 3189KCTC 3189 -- -- VGVG --
KCTC 5546KCTC 5546 -- -- VGVG --
KCTC 3510KCTC 3510 -- -- VGVG VGVG
KCTC 5058KCTC 5058 VGVG VGVG VGVG VGVG
KCTC 3074KCTC 3074 -- VGVG VGVG VGVG
KCCM 40995KCCM 40995 -- -- VGVG --
KCCM 42830KCCM 42830 -- -- VGVG --
KCCM 32822KCCM 32822 -- -- VGVG --
KCCM 41276KCCM 41276 -- -- VGVG VGVG
본 발명의 균주strain of the present invention EPS DA-BACSEPS DA-BACS -- -- -- --
BlankBlank -- -- -- --
대조군control group VGVG VGVG VGVG VGVG
- : No growth; VG : Visible growth- : No growth; VG: Visible growth
실험 결과, 락토바실러스 파라카제이(Lactobacillus paracasei) EPS DA-BACS 배양물이 첨가된 실험군에서는 녹농균(Pseudomonas aeruginosa), 고초균(Bacillus subtilis), 대장균 (Escherichia coli) 및 황색포도구균(Staphylococcus aureus) 4종 모두가 대조군과 비교하였을 때 생육이 완전히 저해됨을 확인하였다.As a result of the experiment, Lactobacillus paracasei (Lactobacillus paracasei) In the experimental group to which the EPS DA-BACS culture was added, Pseudomonas aeruginosa (Pseudomonas aeruginosa), Bacillus subtilis (Bacillus subtilis), Escherichia coli (Escherichia coli) and Staphylococcus aureus (Staphylococcus aureus) 4 types It was confirmed that growth was completely inhibited when all were compared to the control group.
또한, 동속동종 균주 12개 스트레인 중 EPS DA-BACS과 마찬가지로 항균활성 평가균주 4종 전부를 제어하는 균주가 없어 본 발명균주의 항균활성이 우수함을 확인하였다.In addition, it was confirmed that the antibacterial activity of the strain of the present invention is excellent because there is no strain controlling all four strains for evaluation of antibacterial activity, like EPS DA-BACS, among 12 strains of the same species.
2. 유해미생물에 대한 항진균 활성 평가2. Evaluation of antifungal activity against harmful microorganisms
항진균활성 평가에 [Raffael C. Inglin et al. High-throughput screening assays for antibacterial and antifungal activities of Lactobacillus species. Journal of Microbiological Methods. 114 (2015) 26-29]의 아가-스폿 어세이(agar-spot assay)를 적용하였다For evaluation of antifungal activity [Raffael C. Inglin et al. High-throughput screening assays for antibacterial and antifungal activities of Lactobacillus species. Journal of Microbiological Methods. 114 (2015) 26-29] was applied.
구체적으로, 유산균 스톡을 MRS broth(0.05% L-cysteine)에 1%(v/v) 농도가 되도록 접종하고 32~37oC에서 18~24시간 동안 혐기조건에서 정치배양하였다.Specifically, the lactic acid bacteria stock was inoculated to a concentration of 1% (v / v) in MRS broth (0.05% L-cysteine) and cultured at 32-37 ° C for 18-24 hours under anaerobic conditions.
이후, MRS broth에 1.5% 아가와 0.1M K2HPO4를 첨가한 후 오토클레이브하였다. 해당 MRS 아가를 24-웰 플레이트에 300㎕씩 분주한 뒤 플레이트의 뚜껑을 닫은 채 30분간 자연건조하였다. 상기 활성화한 유산균 배양액 0.75㎕를 웰에 굳힌 MRS 아가의 정가운데에 접종한 후 32~37℃에서 48시간 동안 혐기배양하였다. Thereafter, 1.5% agar and 0.1MK 2 HPO 4 were added to the MRS broth and then autoclaved. After dispensing 300 μl of the corresponding MRS agar into a 24-well plate, the plate was air-dried for 30 minutes with the lid closed. 0.75 μl of the activated lactic acid bacteria culture medium was inoculated into the center of MRS agar hardened into wells, followed by anaerobic culture at 32-37° C. for 48 hours.
그 후, YM(Yeast-Malt) broth에 0.5% 아가와 0.1M K2HPO4를 첨가한 후 오토클레이브하였다. 상기 YM 소프트 아가를 워터배스(water bath)에서 50℃로 식힌 후 전날 활성화해두었던 항진균활성 평가균주인 아스페길루스 브라실리언시스(Aspergillus brasiliensis)를 103~104 spores/㎖ 만큼 접종하였다. 항진균 활성 평가균주가 접종된 YM 소프트 아가를 상기 유산균이 배양된 MRS 아가 위에 100㎕씩 분주한 뒤 24-웰 플레이트의 뚜껑을 닫은 채 30분간 자연건조하였다. YM 소프트 아가에 항진균 활성 평가균주를 접종하지 않은 블랭크(blank)군과 유산균 대신 멸균 펩톤수 0.75㎕ 를 접종한 대조군도 함께 실험하였다. 상기 24-웰 플레이트를 25℃에서 48시간까지 배양하며 항진균활성을 육안으로 확인하였다.After that, 0.5% agar and 0.1MK 2 HPO 4 were added to YM (Yeast-Malt) broth and then autoclaved. After cooling the YM soft agar to 50 ° C. in a water bath, Aspergillus brasiliensis , an antifungal activity evaluation strain activated the previous day, was inoculated with 10 3 to 10 4 spores / ml. . 100 μl of YM soft agar inoculated with the antifungal activity evaluation strain was dispensed on MRS agar in which the lactic acid bacteria were cultured, and then air-dried for 30 minutes with the lid of the 24-well plate closed. A blank group not inoculated with the antifungal activity evaluation strain on YM soft agar and a control group inoculated with 0.75 μl of sterilized peptone water instead of lactic acid bacteria were also tested. The 24-well plate was incubated at 25° C. for up to 48 hours, and antifungal activity was visually confirmed.
실험 결과, EPS DA-BACS이 첨가된 실험군에서 아스페길루스 브라실리언시스의 생육이 해당 항진균 활성 평가균주가 접종되지 않은 블랭크(blank)와 동등 수준으로 저해됨을 확인하였다 (도 1). 도 1은 본 발명 균주의 항진균 활성을 평가한 결과이다.As a result of the experiment, it was confirmed that the growth of Aspergillus brasiliensis in the experimental group to which EPS DA-BACS was added was inhibited to the same level as that of the blank in which the antifungal activity evaluation strain was not inoculated (FIG. 1). 1 is a result of evaluating the antifungal activity of the strain of the present invention.
3. 염증완화 효능 평가3. Evaluation of anti-inflammatory efficacy
MRS broth(0.05% L-cysteine)에 본 발명균주를 1% 접종하여 37℃에서 24시간 동안 혐기배양한 후 생균수를 측정하였다. 그 후, 원심분리하고 PBS버퍼로 세척하여 균체를 회수한 뒤 오토클레이브를 통해 열-불활성화(heat-inactivation)하여 사균체로 제조하였다.MRS broth (0.05% L-cysteine) was inoculated with 1% of the strain of the present invention and anaerobically cultured at 37° C. for 24 hours, and then the number of viable cells was measured. Thereafter, cells were collected by centrifugation, washed with PBS buffer, and then heat-inactivated using an autoclave to prepare dead cells.
RAW 264.7 세포주에 ESP DA-BACS 균체(균체를 DMEM(Dulbecco's Modified Eagle's Medium) 배지에 용해하여 사용함)를 최종농도가 1.0×109 CFU/㎖가 되게끔 첨가하여 2시간 동안 반응시킨 후, LPS 1㎍/㎖를 첨가하여 24시간 동안 배양하였다. 그 후, ELISA 키트를 이용해 배양액에서 일산화질소 생성량을 측정하였다. 비교실험을 위해 균체 대신 염증완화약물인 덱사메타손 1uM을 처리한 양성대조군 및 균체 및 LPS를 처리하지 않은 군, LPS만 처리한 군도 함께 실험하였다.ESP DA-BACS cells (cells were dissolved in DMEM (Dulbecco's Modified Eagle's Medium) medium) were added to the RAW 264.7 cell line to a final concentration of 1.0 × 10 9 CFU / ml, reacted for 2 hours, and LPS 1 [mu]g/ml was added and incubated for 24 hours. Then, the amount of nitrogen monoxide produced in the culture medium was measured using an ELISA kit. For comparative experiments, a positive control group treated with 1uM of dexamethasone, an anti-inflammatory drug, instead of cells, a group not treated with cells and LPS, and a group treated only with LPS were also tested.
실험결과는 Graphpad Prism version 9.2.0을 이용하여 분석하였고, 일원배치 분산분석 및 Dunnett's test 사후검정으로 유의성을 검증하였다. The experimental results were analyzed using Graphpad Prism version 9.2.0, and significance was verified by one-way ANOVA and Dunnett's test post hoc test.
실험 결과, 1.0×109 CFU/㎖의 ESP DA-BACS 사균체가 대표적인 염증완화약물인 덱사메타손 1uM과 동등수준으로 RAW 264.7 세포주의 LPS 처리에 따른 일산화질소의 발생을 낮추는 것으로 확인하였다.As a result of the experiment, it was confirmed that 1.0×10 9 CFU/ml of ESP DA-BACS dead cells lowered the generation of nitrogen monoxide according to LPS treatment of the RAW 264.7 cell line to the same level as dexamethasone 1uM, a representative anti-inflammatory drug.
일원배치 분산분석을 통한 Dunnett's test 사후검정으로 통계분석 시, LPS만 처리한 군 대비 LPS와 덱사메타손 처리군 및 LPS와 균체 처리군 모두 유의수준 p<0.0001(****)로 유의하게 일산화질소의 발생이 낮아졌다 (도 2). 도 2는 본 발명 균주의 항염증 활성을 평가한 결과이다.Statistical analysis by Dunnett's test post hoc test through one-way analysis of variance showed that the LPS and dexamethasone treatment group and the LPS and cell culture treatment group showed a significant p<0.0001 (****) compared to the LPS only treatment group. incidence was lowered (Fig. 2). Figure 2 is the result of evaluating the anti-inflammatory activity of the strain of the present invention.
[실시예 2: EPS 분리 및 정제][Example 2: Separation and purification of EPS]
1. 분리1. Separation
분리균주로부터 생성된 EPS를 분리하기 위해 균체 배양액을 4℃에서 원심분리(10,000×g, 25 min)해 균체를 제거하고, 상등액에 2배의 냉각된 95% 에탄올을 서서히 가하여 4℃에서 15~24시간 동안 EPS를 침전시켰다. 침전물을 4℃에서 원심분리(10,000×g, 25min)해 회수하고, 남은 에탄올을 건조한 후 동결건조하여 crude EPS를 제조하였다.In order to isolate the EPS generated from the isolated strain, the cell culture medium was centrifuged (10,000 × g, 25 min) at 4 ° C to remove the cells, and 2 times of cooled 95% ethanol was slowly added to the supernatant, and EPS was allowed to precipitate for 24 hours. The precipitate was collected by centrifugation (10,000 × g, 25min) at 4 ° C., and the remaining ethanol was dried and lyophilized to prepare crude EPS.
2. 정제2. Tablets
분리균주로부터 생성된 EPS를 정제하기 위해 균체 배양액을 4℃에서 원심분리(10,000×g, 25 min)해 균체를 제거하고, 회수한 상등액에 트라이클로로아세트산(trichloroacetic acid)을 최종농도가 4~10%가 되도록 첨가해 4℃에서 2시간 동안 반응시켰다. 해당 반응액을 4℃에서 원심분리(10,000×g, 25 min)하여 침전된 단백질을 제거한 후 상등액에 2배의 냉각된 95% 에탄올을 서서히 가하여 4℃에서 15~24시간 동안 EPS를 침전시켰다.In order to purify the EPS generated from the isolated strain, the cell culture medium was centrifuged (10,000 × g, 25 min) at 4 ° C to remove the cells, and trichloroacetic acid was added to the recovered supernatant to a final concentration of 4 to 10 % and reacted at 4°C for 2 hours. The reaction solution was centrifuged (10,000 × g, 25 min) at 4 ° C to remove precipitated proteins, and then 2 times cold 95% ethanol was slowly added to the supernatant to precipitate EPS at 4 ° C for 15 to 24 hours.
이후, 침전물을 4℃에서 원심분리(10,000×g, 25min)해 회수한 후 3차 증류수에 용해하여 dialysis-sack(M.W. cut off 10,000, Spectra/Por 6 membrane, Pre-wetted RC Tubing, Spectrum Laboratories, USA)에 넣어 4℃, 48시간 동안 투석한 다음 동결건조하여 정제된 EPS를 제조하였다.Thereafter, the precipitate was collected by centrifugation (10,000 × g, 25 min) at 4 ° C, dissolved in tertiary distilled water, and dialysis-sack (M.W. cut off 10,000, Spectra / Por 6 membrane, Pre-wetted RC Tubing, Spectrum Laboratories, USA), dialyzed at 4° C. for 48 hours, and then lyophilized to prepare purified EPS.
[실험예 3: EPS의 항균 및 프리바이오틱 효능 평가][Experimental Example 3: Antibacterial and prebiotic efficacy evaluation of EPS]
1. 클로스트리듐 디피실리균(1. Clostridium difficile ( Clostridium difficileClostridium difficile )의 생장 저해) growth inhibition
분리균주로부터 분리한 EPS가 장내 유해균인 클로스트리듐 디피실리균(Clostridium difficile)의 생장을 저해하는지 평가하고자, EPS DA-BACS 균주의 crude EPS를 최종농도가 50㎎/㎖가 되도록 첨가한 TSB broth에 클로스트리듐 디피실리균을 1.0×105 CFU/㎖만큼 접종 후 37℃에서 혐기배양하며 배양시간별 OD600값을 측정하였다. 비교실험을 위해 crude EPS를 첨가하지 않은 TSB broth를 대상으로 동일실험을 진행한 군을 함께 실험하였다.In order to evaluate whether the EPS isolated from the isolated strain inhibits the growth of Clostridium difficile , an enteric harmful bacterium, crude EPS of the EPS DA-BACS strain is added to the final concentration of 50 mg / ㎖ TSB broth Clostridium difficile bacteria were inoculated by 1.0 × 10 5 CFU / ml, and then anaerobically cultured at 37 ° C., and the OD 600 value was measured for each culture time. For a comparative experiment, a group that performed the same experiment with TSB broth without adding crude EPS was tested together.
실험결과, EPS DA-BACS의 EPS가 첨가된 실험군에서 EPS가 첨가되지 않은 군 대비 클로스트리듐 디피실리균의 생장이 배양 시간에 따라 최대 67%까지 저해됨이 확인하였다 (도 3). 도 3은 본 발명 균주의 클로스트리듐 디피실리균(Clostridium difficile)에 대한 생장저해 활성을 평가한 결과이다.As a result of the experiment, it was confirmed that the growth of Clostridium difficile was inhibited by up to 67% according to the incubation time in the experimental group to which EPS of EPS DA-BACS was added compared to the group to which EPS was not added (FIG. 3). Figure 3 is the result of evaluating the growth inhibitory activity against Clostridium difficile of the strain of the present invention.
2. 프리바이오틱 효능 평가2. Evaluation of prebiotic efficacy
분리균주로부터 정제한 EPS가 프리바이오틱스 활성을 지니는지 확인하고자 정제된 EPS 2%를 당원으로 하는 MRS broth 또는 TSB broth를 96-웰 플레이트에 100㎕씩 분주한 뒤 해당 MRS broth에는 전날 활성화한 장내 유익균(유산균 및 비피더스균)의 배양액을, 해당 TSB broth에는 장내 유해균(Escherichia coli)의 배양액을 각각 1%씩 접종하여 24시간 후의 OD600값을 관찰하였다. 비교실험을 위해 대표적인 프리바이오틱스인 2% 이눌린(inulin)을 당원으로 하는 MRS broth 또는 TSB broth를 대상으로 동일실험을 진행한 군과 당원이 첨가되지 않은 MRS broth 또는 TSB broth를 대상으로 동일실험을 진행한 블랭크(blank) 군 및 2% 글루코오스를 당원으로 하는 MRS broth 또는 TSB broth를 대상으로 동일실험을 진행한 대조군도 함께 실험하였다.In order to confirm whether the EPS purified from the isolated strain has prebiotics activity, 100 μl of MRS broth or TSB broth containing 2% of the purified EPS as a glycogen was dispensed into a 96-well plate, and then the MRS broth was activated the previous day. Cultures of beneficial bacteria (lactic acid bacteria and bifidobacteria) and 1% of cultures of intestinal harmful bacteria ( Escherichia coli ) were inoculated into the corresponding TSB broth, respectively, and OD 600 values after 24 hours were observed. For comparative experiments, the same experiment was conducted on MRS broth or TSB broth with 2% inulin, a representative prebiotic, as a glycogen, and MRS broth or TSB broth to which no glycogen was added. A blank group and a control group in which the same experiment was performed for MRS broth or TSB broth containing 2% glucose as a sugar source were also tested.
프리바이오틱 효과를 정량화하기 위해 [Magdel-Din M. Hussein et al. Production and prebiotic activity of exopolysaccharides derived from some probiotics. Egyptian Pharmaceutical Journal. 14 (2015) 1-9]를 참고하여 하기와 같이 'prebiotic index'를 계산해 군간 비교를 하였다 (식 1).To quantify the prebiotic effect [Magdel-Din M. Hussein et al. Production and prebiotic activity of exopolysaccharides derived from some probiotics. Egyptian Pharmaceutical Journal. 14 (2015) 1-9], the 'prebiotic index' was calculated and compared between groups as follows (Equation 1).
[식 1][Equation 1]
Prebiotic index = Optical density of the growth of probiotic culture / Optical density of the growth of E. coli Prebiotic index = Optical density of the growth of probiotic culture / Optical density of the growth of E. coli
실험 결과, EPS의 프리바이오틱 효과를 정량화할 수 있는 PI값을 측정해보았을 때, 락토바실러스 가세리(Lactobacillus gasseri), 비피도박테리움 비피덤(Bifidobacterium bifidum), 비피도박테리움 애니말리스(Bifidobacterium animalis), 비피도박테리움 패이칼(Bifidobacterium faecale)에 대하여 대표적인 프리바이오틱스인 이눌린 대비 PI값이 각각 3.2배, 13배, 19배, 2배 높음을 확인할 수 있었다. 특히, 비피도박테리움 속(Bifidobacterium sp.)에 대하여 이눌린 대비 PI값이 우수한 것이 관찰되었다 (도 4). 도 4는 본 발명 균주의 프리바이오틱 효과를 평가한 결과이다.As a result of the experiment, when the PI value that can quantify the prebiotic effect of EPS was measured, Lactobacillus gasseri, Bifidobacterium bifidum , Bifidobacterium animalis ( Bifidobacterium animalis ), and Bifidobacterium faecale , it was confirmed that the PI value was 3.2 times, 13 times, 19 times, and 2 times higher than inulin, a representative prebiotics, respectively. In particular, it was observed that the PI value was excellent compared to inulin for Bifidobacterium sp. (FIG. 4). Figure 4 is the result of evaluating the prebiotic effect of the strain of the present invention.
[수탁번호][Accession number]
기탁기관명 : 한국생명공학연구원Name of Depositary Institution: Korea Research Institute of Bioscience and Biotechnology
수탁번호 : KCTC14639BPAccession number: KCTC14639BP
수탁일자 : 20210715Entrusted date: 20210715
Figure PCTKR2022013422-appb-img-000001
Figure PCTKR2022013422-appb-img-000001

Claims (24)

  1. 락토바실러스 파라카제이(Lactobacillus paracasei) EPS DA-BACS (KCTC14639BP). Lactobacillus paracasei EPS DA-BACS (KCTC14639BP).
  2. 제1항에 있어서,According to claim 1,
    상기 균주는,The strain,
    포스트바이오틱스로서 EPS(exopolysaccharide)를 생성하는 것을 특징으로 하는 락토바실러스 파라카제이(Lactobacillus paracasei) EPS DA-BACS (KCTC14639BP). Lactobacillus paracasei EPS DA-BACS (KCTC14639BP) characterized by producing EPS (exopolysaccharide) as postbiotics.
  3. 제2항에 있어서,According to claim 2,
    상기 EPS(exopolysaccharide)는,The EPS (exopolysaccharide),
    로우피(ropy) 형태의 EPS(exopolysaccharide)를 포함하는 것을 특징으로 하는 락토바실러스 파라카제이(Lactobacillus paracasei) EPS DA-BACS (KCTC14639BP).Lactobacillus paracasei EPS DA-BACS (KCTC14639BP), characterized in that it contains exopolysaccharide (EPS) in the form of ropy.
  4. 락토바실러스 파라카제이(Lactobacillus paracasei) EPS DA-BACS (KCTC14639BP), 그의 사균체 및 그의 배양물 중 선택되는 어느 하나를 함유하는 항균제.Lactobacillus paracasei ( Lactobacillus paracasei ) EPS DA-BACS (KCTC14639BP), an antibacterial agent containing any one selected from dead cells thereof and cultures thereof.
  5. 제4항에 있어서,According to claim 4,
    상기 항균제는,The antibacterial agent,
    녹농균(Pseudomonas aeruginosa), 고초균(Bacillus subtilis), 대장균(Escherichia coli), 클로스트리듐 디피실리균(Clostridium difficile) 및 황색포도구균(Staphylococcus aureus)에 대한 항균 활성을 가지는 것을 특징으로 하는 항균제. Pseudomonas aeruginosa ), Bacillus subtilis ( Bacillus subtilis ), Escherichia coli ( Escherichia coli ), Clostridium difficile bacteria ( Clostridium difficile ) and An antibacterial agent characterized by having antibacterial activity against Staphylococcus aureus .
  6. 락토바실러스 파라카제이(Lactobacillus paracasei) EPS DA-BACS (KCTC14639BP), 그의 사균체 및 그의 배양물 중 선택되는 어느 하나를 함유하는 항진균제.An antifungal agent containing any one selected from Lactobacillus paracasei EPS DA-BACS (KCTC14639BP), dead cells thereof, and cultures thereof.
  7. 제6항에 있어서,According to claim 6,
    상기 항진균제는,The antifungal agent,
    아스페길루스 브라실리언시스(Aspergillus brasiliensis)에 대한 항진균 활성을 가지는 것을 특징으로 하는 항진균제.An antifungal agent characterized by having antifungal activity against Aspergillus brasiliensis .
  8. 락토바실러스 파라카제이(Lactobacillus paracasei) EPS DA-BACS (KCTC14639BP), 그의 사균체 및 그의 배양물 중 선택되는 어느 하나를 함유하는 염증질환 예방 또는 치료용 약학 조성물.Lactobacillus paracasei ( Lactobacillus paracasei ) EPS DA-BACS (KCTC14639BP), a pharmaceutical composition for preventing or treating inflammatory diseases containing any one selected from dead cells and cultures thereof.
  9. 락토바실러스 파라카제이(Lactobacillus paracasei) EPS DA-BACS (KCTC14639BP), 그의 사균체 및 그의 배양물 중 선택되는 어느 하나를 함유하는 염증질환 개선용 식품 조성물.Lactobacillus paracasei ( Lactobacillus paracasei ) EPS DA-BACS (KCTC14639BP), a food composition for improving inflammatory diseases containing any one selected from its dead cells and its culture.
  10. 락토바실러스 파라카제이(Lactobacillus paracasei) EPS DA-BACS (KCTC14639BP), 그의 사균체 및 그의 배양물 중 선택되는 어느 하나를 함유하는 염증질환 개선용 건강기능식품.Lactobacillus paracasei ( Lactobacillus paracasei ) EPS DA-BACS (KCTC14639BP), a health functional food for improving inflammatory diseases containing any one selected from dead cells and culture thereof.
  11. 락토바실러스 파라카제이(Lactobacillus paracasei) EPS DA-BACS (KCTC14639BP), 그의 사균체 및 그의 배양물 중 선택되는 어느 하나를 함유하는 피부염증 예방 또는 개선용 화장료 조성물.Lactobacillus paracasei ( Lactobacillus paracasei ) EPS DA-BACS (KCTC14639BP), a cosmetic composition for preventing or improving skin inflammation containing any one selected from its dead cells and its culture.
  12. 락토바실러스 파라카제이(Lactobacillus paracasei) EPS DA-BACS (KCTC14639BP)가 생성하는 EPS(exopolysaccharide).EPS (exopolysaccharide) produced by Lactobacillus paracasei EPS DA-BACS (KCTC14639BP).
  13. 제12항에 있어서,According to claim 12,
    상기 EPS(exopolysaccharide)는,The EPS (exopolysaccharide),
    로우피(ropy) 형태의 EPS(exopolysaccharide)를 포함하는 것을 특징으로 하는 EPS(exopolysaccharide).An EPS (exopolysaccharide) comprising a ropy form of EPS (exopolysaccharide).
  14. 제12항에 있어서,According to claim 12,
    상기 EPS(exopolysaccharide)는,The EPS (exopolysaccharide),
    a) 락토바실러스 파라카제이(Lactobacillus paracasei) EPS DA-BACS (KCTC14639BP) 배양 상등액에 산성물질을 첨가하여 단백질을 침전 및 제거하는 단계; 및a) precipitating and removing proteins by adding an acidic material to the culture supernatant of Lactobacillus paracasei EPS DA-BACS (KCTC14639BP); and
    b) 상기 a) 단계의 단백질 제거액에 친수성 유기용매를 첨가하여 다당류를 추출하는 단계;를 포함하는 과정을 통해 수득되는 것을 특징으로 하는 EPS(exopolysaccharide).b) extracting polysaccharides by adding a hydrophilic organic solvent to the protein removal solution of step a); EPS (exopolysaccharide), characterized in that obtained through a process including.
  15. 락토바실러스 파라카제이(Lactobacillus paracasei) EPS DA-BACS (KCTC14639BP)가 생성하는 EPS(exopolysaccharide)를 함유하는 항균제.An antibacterial agent containing EPS (exopolysaccharide) produced by Lactobacillus paracasei EPS DA-BACS (KCTC14639BP).
  16. 제15항에 있어서,According to claim 15,
    상기 항균제는,The antibacterial agent,
    녹농균(Pseudomonas aeruginosa), 고초균(Bacillus subtilis), 대장균(Escherichia coli), 클로스트리듐 디피실리균(Clostridium difficile) 및 황색포도구균(Staphylococcus aureus)에 대한 항균 활성을 가지는 것을 특징으로 하는 항균제. Pseudomonas aeruginosa ), Bacillus subtilis ( Bacillus subtilis ), Escherichia coli ( Escherichia coli ), Clostridium difficile bacteria ( Clostridium difficile ) and An antibacterial agent characterized by having antibacterial activity against Staphylococcus aureus .
  17. 락토바실러스 파라카제이(Lactobacillus paracasei) EPS DA-BACS (KCTC14639BP)가 생성하는 EPS(exopolysaccharide)를 함유하는 항진균제.An antifungal agent containing EPS (exopolysaccharide) produced by Lactobacillus paracasei EPS DA-BACS (KCTC14639BP).
  18. 제17항에 있어서,According to claim 17,
    상기 항진균제는,The antifungal agent,
    아스페길루스 브라실리언시스(Aspergillus brasiliensis)에 대한 항진균 활성을 가지는 것을 특징으로 하는 항진균제.An antifungal agent characterized by having antifungal activity against Aspergillus brasiliensis .
  19. 락토바실러스 파라카제이(Lactobacillus paracasei) EPS DA-BACS (KCTC14639BP)가 생성하는 EPS(exopolysaccharide)를 함유하는 염증질환 예방 또는 치료용 약학 조성물.A pharmaceutical composition for preventing or treating inflammatory diseases containing EPS (exopolysaccharide) produced by Lactobacillus paracasei EPS DA-BACS (KCTC14639BP).
  20. 락토바실러스 파라카제이(Lactobacillus paracasei) EPS DA-BACS (KCTC14639BP)가 생성하는 EPS(exopolysaccharide)를 함유하는 염증질환 개선용 식품 조성물.A food composition for improving inflammatory diseases containing EPS (exopolysaccharide) produced by Lactobacillus paracasei EPS DA-BACS (KCTC14639BP).
  21. 락토바실러스 파라카제이(Lactobacillus paracasei) EPS DA-BACS (KCTC14639BP)가 생성하는 EPS(exopolysaccharide)를 함유하는 염증질환 개선용 건강기능식품.A health functional food for improving inflammatory diseases containing EPS (exopolysaccharide) produced by Lactobacillus paracasei EPS DA-BACS (KCTC14639BP).
  22. 락토바실러스 파라카제이(Lactobacillus paracasei) EPS DA-BACS (KCTC14639BP)가 생성하는 EPS(exopolysaccharide)를 함유하는 피부염증 예방 또는 개선용 화장료 조성물.A cosmetic composition for preventing or improving skin inflammation containing EPS (exopolysaccharide) produced by Lactobacillus paracasei EPS DA-BACS (KCTC14639BP).
  23. 락토바실러스 파라카제이(Lactobacillus paracasei) EPS DA-BACS (KCTC14639BP), 그의 사균체, 그의 배양물 및 락토바실러스 파라카제이(Lactobacillus paracasei) EPS DA-BACS (KCTC14639BP)가 생성하는 EPS(exopolysaccharide) 중 선택되는 어느 하나를 함유하는 프로바이오틱스 생육 촉진용 조성물.Selection of EPS (exopolysaccharide) produced by Lactobacillus paracasei EPS DA-BACS (KCTC14639BP), its dead cell, its culture and Lactobacillus paracasei EPS DA-BACS (KCTC14639BP) A composition for promoting the growth of probiotics containing any one of which is.
  24. 제23항에 있어서,According to claim 23,
    상기 프로바이오틱스 생육 촉진용 조성물은,The composition for promoting the growth of probiotics,
    락토바실러스 가세리(Lactobacillus gasseri), 비피도박테리움 비피덤(Bifidobacterium bifidum), 비피도박테리움 애니말리스(Bifidobacterium animalis), 비피도박테리움 패이칼(Bifidobacterium faecale) 중 선택되는 어느 하나 이상의 프로바이오틱스의 생육을 촉진시키는 것을 특징으로 하는 프로바이오틱스 생육 촉진용 조성물.Growth of one or more probiotics selected from Lactobacillus gasseri , Bifidobacterium bifidum, Bifidobacterium animalis , and Bifidobacterium faecale A composition for promoting the growth of probiotics, characterized in that promotes.
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