WO2023038418A1 - Bifidobacterium bifidum eps da-laim strain for intestinal health, having effect of promoting growth of lactobacillus, and polysaccharides of same - Google Patents
Bifidobacterium bifidum eps da-laim strain for intestinal health, having effect of promoting growth of lactobacillus, and polysaccharides of same Download PDFInfo
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- WO2023038418A1 WO2023038418A1 PCT/KR2022/013421 KR2022013421W WO2023038418A1 WO 2023038418 A1 WO2023038418 A1 WO 2023038418A1 KR 2022013421 W KR2022013421 W KR 2022013421W WO 2023038418 A1 WO2023038418 A1 WO 2023038418A1
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- eps
- bifidobacterium bifidum
- laim
- kctc
- exopolysaccharide
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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- 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
Definitions
- the present invention relates to an intestinal health strain, Bifidobacterium bifidum EPS DA-LAIM, and a polysaccharide thereof having a Lactobacillus lactic acid bacterium growth promoting effect.
- Probiotics is a general term for microorganisms that are beneficial to the human body, and typically include lactic acid bacteria and bifidobacteria. After entering the human body, they adhere to intestinal epithelial cells to inhibit the growth of harmful microorganisms, improve diarrhea and constipation, and exhibit intestinal microbial balance, such as controlling intestinal microbial balance, as well as exhibiting physiological effects such as immune activity enhancement and anticancer activity. It maintains the balance of intestinal microorganisms and has a beneficial effect on human health.
- exopolysaccharide EPS
- the polysaccharide produced by Bifidobacterium has various and unique physical properties, so it can be used as a material for physical and functional functions, and it has excellent physiological functions such as anticancer effect, cholesterol suppression, immune regulation, and constipation improvement. this is gradually increasing.
- 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 bifidus bacteria that produce Ropy-EPS.
- the present invention is Bifidobacterium bifidum ( Bifidobacterium bifidum ) EPS DA-LAIM (KCTC 14638BP) is provided.
- 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 is Bifidobacterium bifidum ( Bifidobacterium bifidum ) EPS DA-LAIM (KCTC 14638BP), a pharmaceutical composition for preventing or treating diseases caused by excessive accumulation of active oxygen containing any one selected from dead cells and cultures thereof is provided.
- the present invention is Bifidobacterium bifidum ( Bifidobacterium bifidum ) EPS DA-LAIM (KCTC 14638BP), dead cells thereof, and a food composition for improving diseases caused by excessive accumulation of active oxygen containing any one selected from its culture are provided.
- the present invention is Bifidobacterium bifidum ( Bifidobacterium bifidum ) EPS DA-LAIM (KCTC 14638BP), a health functional food for improving diseases caused by excessive accumulation of active oxygen containing any one selected from dead cells and culture thereof is provided.
- the present invention is Bifidobacterium bifidum ( Bifidobacterium bifidum ) EPS DA-LAIM (KCTC 14638BP), provides a cosmetic composition for improving skin aging caused by excessive accumulation of active oxygen containing any one selected from dead cells and cultures thereof.
- the present invention is Bifidobacterium bifidum ( Bifidobacterium bifidum ) EPS (exopolysaccharide) produced by EPS DA-LAIM (KCTC 14638BP) is provided, and the EPS (exopolysaccharide) preferably includes exopolysaccharide (EPS) in the form of ropy secreted extracellularly do.
- EPS exopolysaccharide
- the EPS exopolysaccharide
- the EPS is preferably a) Bifidobacterium bifidum ( Bifidobacterium bifidum ) EPS DA-LAIM (KCTC 14638BP) 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);
- the present invention is Bifidobacterium bifidum ( Bifidobacterium bifidum )
- EPS exopolysaccharide
- the present invention is Bifidobacterium bifidum ( Bifidobacterium bifidum )
- EPS exopolysaccharide
- KCTC 14638BP KCTC 14638BP
- the present invention is Bifidobacterium bifidum ( Bifidobacterium bifidum ) Provides a health functional food for improving diseases caused by excessive accumulation of active oxygen containing EPS (exopolysaccharide) produced by EPS DA-LAIM (KCTC 14638BP).
- EPS exopolysaccharide
- the present invention is Bifidobacterium bifidum ( Bifidobacterium bifidum )
- EPS exopolysaccharide
- the present invention is Bifidobacterium bifidum ( Bifidobacterium bifidum ) EPS DA-LAIM (KCTC 14638BP), its dead cell, its culture and Bifidobacterium bifidum ( Bifidobacterium bifidum )
- EPS exopolysaccharide
- KCTC 14638BP EPS DA-LAIM
- the composition for promoting the growth of probiotics preferably Lactobacillus plantarum ( Lactobacillus plantarum ), Lactobacillus fermentum ( Lactobacillus fermentum ), Lactobacillus helveticus ( Lactobacillus helveticus ) Growth of any one or more probiotics selected It is good to promote
- the strain of the present invention is characterized by producing EPS (exopolysaccharide) as a postbiotics, and has the advantage of providing excellent antioxidant efficacy and prebiotics action ability to promote the growth of the genus Lactobacillus.
- Figure 1 shows the result of evaluating the antioxidant capacity of the strain of the present invention, scavenging activity (Scavening activity).
- Figure 2 is a result of evaluating the prebiotic effect of the strain of the present invention, Lactobacillus plantarum ( Lactobacillus plantarum ), Lactobacillus fermentum ( Lactobacillus fermentum ), Lactobacillus helveticus ( Lactobacillus helveticus ) for prebiotics It represents the prebiotic index.
- the present invention is Bifidobacterium bifidum ( Bifidobacterium bifidum ) EPS DA-LAIM (KCTC 14638BP) is provided.
- 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. It has been confirmed that the strain of the present invention has an excellent antioxidant effect and has a prebiotics action ability to promote the growth of the genus Lactobacillus.
- EPS exopolysaccharide
- the above strain is 'Bifidobacterium bifidum ( Bifidobacterium bifidum ) It was named 'EPS DA-LAIM', and was deposited with the Korea Research Institute of Bioscience and Biotechnology and assigned the accession number KCTC 14638BP on July 15, 2021.
- the strain preferably produces EPS (exopolysaccharide). Therefore, the present invention is a bifidobacterium bifidum ( Bifidobacterium bifidum ) Provides EPS (exopolysaccharide) produced by EPS DA-LAIM (KCTC 14638BP).
- the EPS exopolysaccharide
- the EPS is preferably a) Bifidobacterium bifidum ( Bifidobacterium bifidum ) EPS DA-LAIM (KCTC 14638BP) 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);
- 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 is Bifidobacterium bifidum ( Bifidobacterium bifidum ) EPS DA-LAIM (KCTC 14638BP), its dead cells, its culture, and EPS (exopolysaccharide) containing any one selected from the above-mentioned to provide a pharmaceutical composition for preventing or treating diseases caused by excessive accumulation of active oxygen .
- Antioxidant function is to suppress or slow down the oxidation reaction that occurs in the body.
- the oxidation reaction that occurs in the body induces aging and cell mutation to generate cancer cells. Therefore, the public is also interested in antioxidant action as a preventive measure to suppress such negative reactions in the body.
- diseases caused by excessive accumulation of active oxygen include, for example, cancer, inflammation, anemia, myocardial infarction, cerebral palsy, arteriosclerosis, diabetes, rheumatoid arthritis, Parkinson's disease, and autoimmune diseases It may be any one selected from, but is not limited thereto and may include all diseases accompanied by a reaction in which active oxygen is excessively accumulated without limitation.
- prevention refers to any action that suppresses or delays the onset of diseases affected by the administration of the pharmaceutical composition according to the present invention, that is, diseases caused by excessive accumulation of active oxygen.
- treatment refers to all activities that improve or beneficially change symptoms caused by diseases caused by excessive accumulation of active oxygen by the administration of the pharmaceutical composition according to the present invention.
- the Bifidobacterium bifidum of the present invention ( Bifidobacterium bifidum ) EPS DA-LAIM (KCTC 14638BP), a composition comprising any one selected from dead cells, cultures thereof, and EPS (exopolysaccharide) as an active ingredient, in addition to the above ingredients, an active ingredient exhibiting the same or similar function
- EPS exopolysaccharide
- the pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier in addition to Bifidobacterium bifidum EPS DA-LAIM.
- 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.
- Bifidobacterium bifidum EPS DA-LAIM may be included in an amount of 0.00001% to 99.99% by weight based on the total weight of the pharmaceutical composition, preferably 0.1% by weight. Weight% to 90% by weight, more preferably 0.1% to 70% by weight, more preferably 0.1% to 50% by weight, but not limited thereto, the condition of the subject to be administered, the type of specific disease, and the progress Depending on the degree, etc., it may be variously 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 diseases caused by excessive accumulation of active oxygen.
- 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.
- the present invention is Bifidobacterium bifidum ( Bifidobacterium bifidum ) EPS DA-LAIM (KCTC 14638BP), its dead cells, its culture, and EPS (exopolysaccharide) containing any one selected from the above provides a food composition for improving diseases caused by excessive accumulation of active oxygen.
- the term “improvement” refers to all activities that improve or beneficially change diseases caused by excessive accumulation of active oxygen by administration of the composition of the present invention.
- the Bifidobacterium bifidum EPS DA-LAIM is preferably included in an amount of 0.00001 to 50% by weight relative to the food composition . 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 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, alcohol, and vitamin complexes. And it may be any one selected from other health supplements, but is not necessarily limited thereto.
- the food composition of the present invention when used 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.
- the present invention is Bifidobacterium bifidum ( Bifidobacterium bifidum ) EPS DA-LAIM (KCTC 14638BP), its dead cells, its culture, and EPS (exopolysaccharide) containing any one selected from the above provides a health functional food for improving diseases caused by excessive accumulation of active oxygen.
- Bifidobacterium bifidum Bifidobacterium bifidum
- EPS DA-LAIM KCTC 14638BP
- 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 Health Functional Food Act 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 functions.
- the food composition and health functional food of the present invention may include additional ingredients that are commonly used and can improve odor, 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
- the content of Bifidobacterium bifidum EPS DA-LAIM is not particularly limited, and can be varied depending on the condition of the subject to be administered, the type of specific disease, the degree of progression, etc. there is. If necessary, it may also be included in the total content of food.
- the present invention is Bifidobacterium bifidum ( Bifidobacterium bifidum ) EPS DA-LAIM (KCTC 14638BP), its dead cell, its culture and Bifidobacterium bifidum ( Bifidobacterium bifidum )
- EPS exopolysaccharide
- the composition for promoting the growth of probiotics preferably Lactobacillus plantarum ( Lactobacillus plantarum ), Lactobacillus fermentum ( Lactobacillus fermentum ), Lactobacillus helveticus ( Lactobacillus helveticus ) Growth of any one or more probiotics selected It is good to promote
- Bifidobacterium bifidum Bifidobacterium bifidum
- EPS exopolysaccharide
- KCTC 14638BP EPS DA-LAIM
- KCTC 14638BP EPS DA-LAIM
- the composition for promoting the growth of probiotics claimed in the present invention is also called prebiotics in the art.
- the present invention is Bifidobacterium bifidum ( Bifidobacterium bifidum ) EPS DA-LAIM (KCTC 14638BP), its dead cells, its culture, and EPS (exopolysaccharide) containing any one selected from the above provides a cosmetic composition for improving skin aging due to excessive accumulation of active oxygen.
- Bifidobacterium bifidum Bifidobacterium bifidum
- EPS DA-LAIM KCTC 14638BP
- EPS exopolysaccharide
- the antioxidant effect of cosmetics means the ability to improve or prevent skin functions damaged by oxidation, through which skin aging can be prevented or improved.
- the Bifidobacterium bifidum EPS DA-LAIM 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.
- the content of Bifidobacterium bifidum EPS DA-LAIM is less than 0.0001% by weight, the antioxidant effect is insignificant, and when 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 other than the Bifidobacterium bifidum EPS DA-LAIM of the present invention as active ingredients, such as antioxidants , conventional adjuvants such as stabilizers, solubilizers, 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 100 ⁇ l were plated on TOS-MUP selective media ( Bifidobacterium selective media). 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 and spotted once more on a TOS-MUP selective medium. The plate was incubated anaerobically at 37° C. for 48 hours, and then it was confirmed once again that the colony grew well, and finally 129 bifidus candidate strains were selected.
- TOS-MUP selective media Bifidobacterium selective media
- the EPS production ability of 129 bifidobacteria isolated from feces of healthy Korean volunteers was quantitatively evaluated. Inoculate 1% of each strain in MRS broth (De Man, Rogosa and Sharpe, 0.05% L-cysteine) containing 10% sucrose as a sugar source, incubate anaerobically at 37 ° C for 48 hours, and centrifuge the culture medium at 4 ° C ( 10,000 ⁇ g, 25 min) to remove the cells, and to the supernatant, twice the volume of cooled 95% ethanol was slowly added to precipitate the EPS at 4 ° C for 15 to 24 hours. Thereafter, the precipitate was collected by centrifugation (10,000 ⁇ g, 25 min) at 4 ° C., and the remaining ethanol was dried and lyophilized to measure the weight of the solid content.
- MRS broth De Man, Rogosa and Sharpe, 0.05% L-cysteine
- Ropy-EPS Ropy-Exopolysaccharide
- EPS DA-LAIM As a result of measuring the EPS production ability of 129 bifidobacteria isolated from feces of healthy Korean volunteers and the ropy characteristics of the EPS, Bifidobacterium bifidum, one of the best EPS production species, Bifidobacterium bifidum , as shown in Table 1 EPS DA-LAIM was selected.
- the EPS DA-LAIM strain showed an excellent EPS production ability that was 3.6 times higher than that of other Bifidobacterium sp. A strand of 50 mm was formed, and it was confirmed that the EPS had ropy characteristics. Its 16S rRNA nucleotide sequence is shown in SEQ ID NO: 1.
- the DPPH radical scavenging activity was confirmed by colorimetric method. Specifically, the Bifidobacterium stock was inoculated to a concentration of 1% (v/v) in MRS broth (0.05% L-cysteine), and incubated at 32-37 ° C for 24-168 hours under anaerobic conditions. Cells were recovered by centrifuging 1 ml of the cultured bacterial solution at 13,000 rpm and 4° C. for 3 minutes, and at this time, the number of bacteria was approximately 5.0 ⁇ 10 8 CFU/ml. The cells were washed once with sterilized PBS buffer (pH 7.4) and dissolved in 1 ml of the same new buffer.
- PBS buffer pH 7.4
- DPPH scavenging activity (%) (1-(sample OD 517 -blank OD 517 )/control OD 517 )*100
- the DPPH scavenging activity of the EPS DA-LAIM strain was 59.6%, which was 1.9 times higher than that of vitamin C (0.1mg/ml), a representative antioxidant, and 3.2 times higher than that of the same strain of a famous domestic dry food product. (Fig. 1). 1 is a result of evaluating the antioxidant ability of the strain of the present invention.
- 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 4 It was added to 10% and reacted at 4°C for 2 hours.
- the reaction solution was centrifuged at 4 ° C (10,000 ⁇ g, 25 min) to remove precipitated proteins, and then 2 times of 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.
- MRS broth or TSB broth containing 2% EPS as a sugar source 100 ⁇ l of MRS broth or TSB broth containing 2% EPS as a sugar source was dispensed into a 96-well plate, and then the MRS broth contained intestinal beneficial bacteria activated the previous day ( lactic acid bacteria), the TSB broth was inoculated with 1% of the culture medium of intestinal harmful bacteria ( Escherichia coli ), respectively, and the OD 600 value after 24 hours was observed.
- 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.
- 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 a bifidobacterium bifidum EPS DA-LAIM strain (KCTC 14638BP) for intestinal health, having the effect of promoting growth of lactobacillus, polysaccharides produced from same, and a composition comprising same, for ameliorating diseases caused by the excessive accumulation of active oxygen.
Description
본 발명은 락토바실러스 유산균 생장촉진 효능을 갖는 장 건강 균주 비피도박테리움 비피덤 EPS DA-LAIM 및 그 다당류에 관한 것이다.The present invention relates to an intestinal health strain, Bifidobacterium bifidum EPS DA-LAIM, and a polysaccharide thereof having a Lactobacillus lactic acid bacterium growth promoting effect.
프로바이오틱스(probiotics)는 인체에 이로움을 주는 미생물을 총칭하는 말로, 대표적으로 유산균 및 비피더스균을 포함한다. 이들은 인체에 유입된 후 장내 상피세포에 부착하여 유해 미생물의 생육을 억제하고 설사와 변비를 개선하며 장내 미생물 균형을 조절하는 등의 정장작용을 나타낼 뿐 아니라 면역활성 증진, 항암 작용 등의 생리적 효능을 지녀 장내 미생물의 균형을 유지하고 인체건강에 유익한 영향을 미친다.Probiotics is a general term for microorganisms that are beneficial to the human body, and typically include lactic acid bacteria and bifidobacteria. After entering the human body, they adhere to intestinal epithelial cells to inhibit the growth of harmful microorganisms, improve diarrhea and constipation, and exhibit intestinal microbial balance, such as controlling intestinal microbial balance, as well as exhibiting physiological effects such as immune activity enhancement and anticancer activity. It maintains the balance of intestinal microorganisms and has a beneficial effect on human health.
한편, 일부 비피더스균은 EPS(exopolysaccharide)라고 불리우는 다당류를 생합성하는데, 이는 세포벽 주위에 협막을 생성하거나 세포벽 외부에 점질 형태로 축적되는 미생물의 대사산물 중 하나이다. 비피더스균이 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 비피더스균에 대한 관심이 점차 높아지고 있다.On the other hand, some bifidobacteria biosynthesize a polysaccharide called exopolysaccharide (EPS), 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 Bifidobacterium has various and unique physical properties, so it can be used as a material for physical and functional functions, and it has excellent physiological functions such as anticancer effect, cholesterol suppression, immune regulation, and constipation improvement. this is gradually increasing.
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 비피더스균을 발굴하여 제공하는 것을 목적으로 한다.An object of the present invention is to discover and provide bifidus bacteria that produce Ropy-EPS.
본 발명은 비피도박테리움 비피덤(Bifidobacterium bifidum) EPS DA-LAIM (KCTC 14638BP)을 제공한다.The present invention is Bifidobacterium bifidum ( Bifidobacterium bifidum ) EPS DA-LAIM (KCTC 14638BP) is provided.
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 비피도박테리움 비피덤(Bifidobacterium bifidum) EPS DA-LAIM (KCTC 14638BP), 그의 사균체 및 그의 배양물 중 선택되는 어느 하나를 함유하는 활성산소가 과량 축적되어 유발되는 질환의 예방 또는 치료용 약학 조성물을 제공한다.On the other hand, the present invention is Bifidobacterium bifidum ( Bifidobacterium bifidum ) EPS DA-LAIM (KCTC 14638BP), a pharmaceutical composition for preventing or treating diseases caused by excessive accumulation of active oxygen containing any one selected from dead cells and cultures thereof is provided.
한편, 본 발명은 비피도박테리움 비피덤(Bifidobacterium bifidum) EPS DA-LAIM (KCTC 14638BP), 그의 사균체 및 그의 배양물 중 선택되는 어느 하나를 함유하는 활성산소가 과량 축적되어 유발되는 질환의 개선용 식품 조성물을 제공한다.On the other hand, the present invention is Bifidobacterium bifidum ( Bifidobacterium bifidum ) EPS DA-LAIM (KCTC 14638BP), dead cells thereof, and a food composition for improving diseases caused by excessive accumulation of active oxygen containing any one selected from its culture are provided.
한편, 본 발명은 비피도박테리움 비피덤(Bifidobacterium bifidum) EPS DA-LAIM (KCTC 14638BP), 그의 사균체 및 그의 배양물 중 선택되는 어느 하나를 함유하는 활성산소가 과량 축적되어 유발되는 질환의 개선용 건강기능식품을 제공한다.On the other hand, the present invention is Bifidobacterium bifidum ( Bifidobacterium bifidum ) EPS DA-LAIM (KCTC 14638BP), a health functional food for improving diseases caused by excessive accumulation of active oxygen containing any one selected from dead cells and culture thereof is provided.
한편, 본 발명은 비피도박테리움 비피덤(Bifidobacterium bifidum) EPS DA-LAIM (KCTC 14638BP), 그의 사균체 및 그의 배양물 중 선택되는 어느 하나를 함유하는 활성산소의 과량 축적으로 말미암은 피부노화 개선용 화장료 조성물을 제공한다.On the other hand, the present invention is Bifidobacterium bifidum (Bifidobacterium bifidum) EPS DA-LAIM (KCTC 14638BP), provides a cosmetic composition for improving skin aging caused by excessive accumulation of active oxygen containing any one selected from dead cells and cultures thereof.
한편, 본 발명은 비피도박테리움 비피덤(Bifidobacterium bifidum) EPS DA-LAIM (KCTC 14638BP)이 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) 비피도박테리움 비피덤(Bifidobacterium bifidum) EPS DA-LAIM (KCTC 14638BP) 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 is Bifidobacterium bifidum ( Bifidobacterium bifidum ) EPS (exopolysaccharide) produced by EPS DA-LAIM (KCTC 14638BP) is provided, and the EPS (exopolysaccharide) preferably includes exopolysaccharide (EPS) in the form of ropy secreted extracellularly do. At this time, the EPS (exopolysaccharide) is preferably a) Bifidobacterium bifidum ( Bifidobacterium bifidum ) EPS DA-LAIM (KCTC 14638BP) 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 비피도박테리움 비피덤(Bifidobacterium bifidum) EPS DA-LAIM (KCTC 14638BP)이 생성하는 EPS(exopolysaccharide)를 함유하는 활성산소가 과량 축적되어 유발되는 질환의 예방 또는 치료용 약학 조성물을 제공한다.On the other hand, the present invention is Bifidobacterium bifidum ( Bifidobacterium bifidum ) Provided is a pharmaceutical composition for preventing or treating diseases caused by excessive accumulation of active oxygen containing EPS (exopolysaccharide) produced by EPS DA-LAIM (KCTC 14638BP).
한편, 본 발명은 비피도박테리움 비피덤(Bifidobacterium bifidum) EPS DA-LAIM (KCTC 14638BP)이 생성하는 EPS(exopolysaccharide)를 함유하는 활성산소가 과량 축적되어 유발되는 질환의 개선용 식품 조성물을 제공한다.On the other hand, the present invention is Bifidobacterium bifidum ( Bifidobacterium bifidum ) Provided is a food composition for improving diseases caused by excessive accumulation of active oxygen containing EPS (exopolysaccharide) produced by EPS DA-LAIM (KCTC 14638BP).
한편, 본 발명은 비피도박테리움 비피덤(Bifidobacterium bifidum) EPS DA-LAIM (KCTC 14638BP)이 생성하는 EPS(exopolysaccharide)를 함유하는 활성산소가 과량 축적되어 유발되는 질환의 개선용 건강기능식품을 제공한다.On the other hand, the present invention is Bifidobacterium bifidum ( Bifidobacterium bifidum ) Provides a health functional food for improving diseases caused by excessive accumulation of active oxygen containing EPS (exopolysaccharide) produced by EPS DA-LAIM (KCTC 14638BP).
한편, 본 발명은 비피도박테리움 비피덤(Bifidobacterium bifidum) EPS DA-LAIM (KCTC 14638BP)이 생성하는 EPS(exopolysaccharide)를 함유하는 활성산소의 과량 축적으로 말미암은 피부노화 개선용 화장료 조성물을 제공한다.On the other hand, the present invention is Bifidobacterium bifidum ( Bifidobacterium bifidum ) Provided is a cosmetic composition for improving skin aging caused by excessive accumulation of active oxygen containing EPS (exopolysaccharide) produced by EPS DA-LAIM (KCTC 14638BP).
한편, 본 발명은 비피도박테리움 비피덤(Bifidobacterium bifidum) EPS DA-LAIM (KCTC 14638BP), 그의 사균체, 그의 배양물 및 비피도박테리움 비피덤(Bifidobacterium bifidum) EPS DA-LAIM (KCTC 14638BP)이 생성하는 EPS(exopolysaccharide) 중 선택되는 어느 하나를 함유하는 프로바이오틱스 생육 촉진용 조성물을 제공한다. 이때, 상기 프로바이오틱스 생육 촉진용 조성물은, 바람직하게는 락토바실러스 플란타룸(Lactobacillus plantarum), 락토바실러스 퍼멘텀(Lactobacillus fermentum), 락토바실러스 헬베티쿠스(Lactobacillus helveticus) 중 선택되는 어느 하나 이상의 프로바이오틱스의 생육을 촉진시키는 것이 좋다.On the other hand, the present invention is Bifidobacterium bifidum ( Bifidobacterium bifidum ) EPS DA-LAIM (KCTC 14638BP), its dead cell, its culture and Bifidobacterium bifidum ( Bifidobacterium bifidum ) Provided is a composition for promoting the growth of probiotics containing any one selected from EPS (exopolysaccharide) produced by EPS DA-LAIM (KCTC 14638BP). At this time, the composition for promoting the growth of probiotics, preferably Lactobacillus plantarum ( Lactobacillus plantarum ), Lactobacillus fermentum ( Lactobacillus fermentum ), Lactobacillus helveticus ( Lactobacillus helveticus ) Growth of any one or more probiotics selected It is good to promote
본 발명의 균주는 포스트바이오틱스로서 EPS(exopolysaccharide)를 생성하는 것을 특징으로 하며, 우수한 항산화 효능 및 락토바실러스 속의 생장을 촉진하는 프리바이오틱스 작용 능력을 제공할 수 있는 장점이 있다.The strain of the present invention is characterized by producing EPS (exopolysaccharide) as a postbiotics, and has the advantage of providing excellent antioxidant efficacy and prebiotics action ability to promote the growth of the genus Lactobacillus.
도 1은 본 발명 균주의 항산화능을 평가한 결과로, 소거활성(Scavening activity)을 나타낸 것이다.Figure 1 shows the result of evaluating the antioxidant capacity of the strain of the present invention, scavenging activity (Scavening activity).
도 2는 본 발명 균주의 프리바이오틱 효과를 평가한 결과로, 락토바실러스 플란타룸(Lactobacillus plantarum), 락토바실러스 퍼멘텀(Lactobacillus fermentum), 락토바실러스 헬베티쿠스(Lactobacillus helveticus)에 대한 프리바이오틱 지수(prebiotic index)를 나타낸 것이다.Figure 2 is a result of evaluating the prebiotic effect of the strain of the present invention, Lactobacillus plantarum ( Lactobacillus plantarum ), Lactobacillus fermentum ( Lactobacillus fermentum ), Lactobacillus helveticus ( Lactobacillus helveticus ) for prebiotics It represents the prebiotic index.
본 발명은 비피도박테리움 비피덤(Bifidobacterium bifidum) EPS DA-LAIM (KCTC 14638BP)을 제공한다.The present invention is Bifidobacterium bifidum ( Bifidobacterium bifidum ) EPS DA-LAIM (KCTC 14638BP) is provided.
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 우수한 항산화 효능을 보유하며, 락토바실러스 속의 생장을 촉진하는 프리바이오틱스 작용 능력을 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. It has been confirmed that the strain of the present invention has an excellent antioxidant effect and has a prebiotics action ability to promote the growth of the genus Lactobacillus.
따라서, 본 발명에서는 상기한 균주를 '비피도박테리움 비피덤(Bifidobacterium bifidum) EPS DA-LAIM'이라 명명하였으며, 한국생명공학연구원에 기탁하여 2021년 07월 15일자로 수탁번호 KCTC 14638BP를 부여받았다.Therefore, in the present invention, the above strain is 'Bifidobacterium bifidum ( Bifidobacterium bifidum ) It was named 'EPS DA-LAIM', and was deposited with the Korea Research Institute of Bioscience and Biotechnology and assigned the accession number KCTC 14638BP on July 15, 2021.
또한, 본 발명에 있어, 상기 균주는, 바람직하게는 EPS(exopolysaccharide)를 생성하는 것으로 확인되었다. 따라서, 본 발명은 비피도박테리움 비피덤(Bifidobacterium bifidum) EPS DA-LAIM (KCTC 14638BP)이 생성하는 EPS(exopolysaccharide)를 제공한다.In addition, in the present invention, it was confirmed that the strain preferably produces EPS (exopolysaccharide). Therefore, the present invention is a bifidobacterium bifidum ( Bifidobacterium bifidum ) Provides EPS (exopolysaccharide) produced by EPS DA-LAIM (KCTC 14638BP).
이때, 상기 EPS(exopolysaccharide)는, 바람직하게는 a) 비피도박테리움 비피덤(Bifidobacterium bifidum) EPS DA-LAIM (KCTC 14638BP) 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) is preferably a) Bifidobacterium bifidum ( Bifidobacterium bifidum ) EPS DA-LAIM (KCTC 14638BP) 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 비피도박테리움 비피덤(Bifidobacterium bifidum) EPS DA-LAIM (KCTC 14638BP), 그의 사균체, 그의 배양물 및 상기한 EPS(exopolysaccharide) 중 선택되는 어느 하나를 함유하는 활성산소가 과량 축적되어 유발되는 질환의 예방 또는 치료용 약학 조성물을 제공한다.On the other hand, the present invention is Bifidobacterium bifidum ( Bifidobacterium bifidum ) EPS DA-LAIM (KCTC 14638BP), its dead cells, its culture, and EPS (exopolysaccharide) containing any one selected from the above-mentioned to provide a pharmaceutical composition for preventing or treating diseases caused by excessive accumulation of active oxygen .
항산화 기능은 체내에서 일어나는 산화반응을 억제시키거나 그 속도를 느리게 하는 것인데, 체내에서 일어나는 산화 반응은 노화의 유발과 세포의 변이를 유발하여 암세포를 생성한다. 따라서, 항산화 작용은 이런 체내의 부정적인 반응을 억제하기 위한 예방 방편으로 대중들도 관심이 많다.Antioxidant function is to suppress or slow down the oxidation reaction that occurs in the body. The oxidation reaction that occurs in the body induces aging and cell mutation to generate cancer cells. Therefore, the public is also interested in antioxidant action as a preventive measure to suppress such negative reactions in the body.
한편, 체내에서는 세포 호흡과 더불어 에너지에 해당하는 ATP생성이 주기능인 세포 내의 미토콘드리아가 호흡의 부산물인 자유 라디칼(free radical)을 생성하고, 이는 불안정하여, 지질, 핵산, 단백질 등의 다른 물질과 급격하고 무차별적으로 반응한다. 따라서 이런 자유 라디칼의 생성을 막을 수 있다면 체내의 산화를 예방한다고 볼 수 있으며, 간접적으로는 노화와 암을 예방할 수 있어서 관심이 크다.On the other hand, in the body, mitochondria within cells whose main function is to produce ATP, which corresponds to energy along with cellular respiration, generate free radicals, which are by-products of respiration, which are unstable and rapidly react with other substances such as lipids, nucleic acids, and proteins. and react indiscriminately. Therefore, if the generation of these free radicals can be prevented, it can be seen as preventing oxidation in the body, and indirectly, aging and cancer can be prevented, which is of great interest.
본 발명의 약학 조성물에 있어, 상기 활성산소가 과량 축적되어 유발되는 질환은, 일 예로 암, 염증, 빈혈, 심근경색, 뇌성마비, 동맥경화증, 당뇨, 류머티즘성 관절염, 파킨슨병, 및 자가면역질환 중 선택되는 어느 하나일 수 있으나, 이에 제한되지 않으며 활성산소가 과량 축적되는 반응을 수반하는 모든 질환을 제한 없이 포함할 수 있다.In the pharmaceutical composition of the present invention, diseases caused by excessive accumulation of active oxygen include, for example, cancer, inflammation, anemia, myocardial infarction, cerebral palsy, arteriosclerosis, diabetes, rheumatoid arthritis, Parkinson's disease, and autoimmune diseases It may be any one selected from, but is not limited thereto and may include all diseases accompanied by a reaction in which active oxygen is excessively accumulated without limitation.
본 발명에서 사용되는 용어, "예방"이란 본 발명에 따른 약학적 조성물의 투여에 의해 영향을 받는 질환, 즉 활성산소가 과량 축적되어 유발되는 질환을 억제하거나 발병을 지연시키는 모든 행위를 의미한다.As used herein, the term "prevention" refers to any action that suppresses or delays the onset of diseases affected by the administration of the pharmaceutical composition according to the present invention, that is, diseases caused by excessive accumulation of active oxygen.
본 발명에서 사용되는 용어, "치료"란 본 발명에 따른 약학적 조성물의 투여에 의해 활성산소가 과량 축적되어 유발되는 질환에 의한 증세가 호전되거나 이롭게 변경되는 모든 행위를 의미한다.As used herein, the term "treatment" refers to all activities that improve or beneficially change symptoms caused by diseases caused by excessive accumulation of active oxygen by the administration of the pharmaceutical composition according to the present invention.
본 발명의 상기 비피도박테리움 비피덤(Bifidobacterium bifidum) EPS DA-LAIM (KCTC 14638BP), 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 Bifidobacterium bifidum of the present invention ( Bifidobacterium bifidum ) EPS DA-LAIM (KCTC 14638BP), a composition comprising any one selected from dead cells, cultures thereof, and EPS (exopolysaccharide) as an active ingredient, in addition to the above ingredients, an active ingredient exhibiting the same or similar function One or more types may be included.
본 발명의 약학적 조성물은 비피도박테리움 비피덤(Bifidobacterium bifidum) EPS DA-LAIM 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 Bifidobacterium bifidum EPS DA-LAIM.
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, 비피도박테리움 비피덤(Bifidobacterium bifidum) EPS DA-LAIM은 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, Bifidobacterium bifidum EPS DA-LAIM may be included in an amount of 0.00001% to 99.99% by weight based on the total weight of the pharmaceutical composition, preferably 0.1% by weight. Weight% to 90% by weight, more preferably 0.1% to 70% by weight, more preferably 0.1% to 50% by weight, but not limited thereto, the condition of the subject to be administered, the type of specific disease, and the progress Depending on the degree, etc., it may be variously 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 활성산소가 과량 축적되어 유발되는 질환을 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 diseases caused by excessive accumulation of active oxygen.
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 비피도박테리움 비피덤(Bifidobacterium bifidum) EPS DA-LAIM (KCTC 14638BP), 그의 사균체, 그의 배양물 및 상기한 EPS(exopolysaccharide) 중 선택되는 어느 하나를 함유하는 활성산소가 과량 축적되어 유발되는 질환의 개선용 식품 조성물을 제공한다.On the other hand, the present invention is Bifidobacterium bifidum ( Bifidobacterium bifidum ) EPS DA-LAIM (KCTC 14638BP), its dead cells, its culture, and EPS (exopolysaccharide) containing any one selected from the above provides a food composition for improving diseases caused by excessive accumulation of active oxygen.
본 발명의 용어, "개선"이란, 본 발명의 조성물의 투여로 활성산소가 과량 축적되어 유발되는 질환이 호전 또는 이롭게 변경되는 모든 행위를 의미한다.As used herein, the term "improvement" refers to all activities that improve or beneficially change diseases caused by excessive accumulation of active oxygen by administration of the composition of the present invention.
본 발명의 활성산소가 과량 축적되어 유발되는 질환의 개선용 식품 조성물에 있어, 상기 비피도박테리움 비피덤(Bifidobacterium bifidum) EPS DA-LAIM은 바람직하게 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 diseases caused by excessive accumulation of active oxygen of the present invention, the Bifidobacterium bifidum EPS DA-LAIM is preferably included in an amount of 0.00001 to 50% by weight relative to the food composition . 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 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 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, alcohol, and vitamin complexes. And it may be any one selected from 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 the food composition of the present invention is used 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 비피도박테리움 비피덤(Bifidobacterium bifidum) EPS DA-LAIM (KCTC 14638BP), 그의 사균체, 그의 배양물 및 상기한 EPS(exopolysaccharide) 중 선택되는 어느 하나를 함유하는 활성산소가 과량 축적되어 유발되는 질환의 개선용 건강기능식품을 제공한다.On the other hand, the present invention is Bifidobacterium bifidum ( Bifidobacterium bifidum ) EPS DA-LAIM (KCTC 14638BP), its dead cells, its culture, and EPS (exopolysaccharide) containing any one selected from the above provides a health functional food for improving diseases caused by excessive accumulation of active oxygen.
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 Health Functional Food Act 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 functions.
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 odor, 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, 비피도박테리움 비피덤(Bifidobacterium bifidum) EPS DA-LAIM의 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 Bifidobacterium bifidum EPS DA-LAIM is not particularly limited, and can be varied depending on the condition of the subject to be administered, the type of specific disease, the degree of progression, etc. there is. If necessary, it may also be included in the total content of food.
본 발명은 비피도박테리움 비피덤(Bifidobacterium bifidum) EPS DA-LAIM (KCTC 14638BP), 그의 사균체, 그의 배양물 및 비피도박테리움 비피덤(Bifidobacterium bifidum) EPS DA-LAIM (KCTC 14638BP)이 생성하는 EPS(exopolysaccharide) 중 선택되는 어느 하나를 함유하는 프로바이오틱스 생육 촉진용 조성물을 제공한다.The present invention is Bifidobacterium bifidum ( Bifidobacterium bifidum ) EPS DA-LAIM (KCTC 14638BP), its dead cell, its culture and Bifidobacterium bifidum ( Bifidobacterium bifidum ) Provided is a composition for promoting the growth of probiotics containing any one selected from EPS (exopolysaccharide) produced by EPS DA-LAIM (KCTC 14638BP).
이때, 상기 프로바이오틱스 생육 촉진용 조성물은, 바람직하게는 락토바실러스 플란타룸(Lactobacillus plantarum), 락토바실러스 퍼멘텀(Lactobacillus fermentum), 락토바실러스 헬베티쿠스(Lactobacillus helveticus) 중 선택되는 어느 하나 이상의 프로바이오틱스의 생육을 촉진시키는 것이 좋다.At this time, the composition for promoting the growth of probiotics, preferably Lactobacillus plantarum ( Lactobacillus plantarum ), Lactobacillus fermentum ( Lactobacillus fermentum ), Lactobacillus helveticus ( Lactobacillus helveticus ) Growth of any one or more probiotics selected It is good to promote
본 발명에서는 비피도박테리움 비피덤(Bifidobacterium bifidum) EPS DA-LAIM (KCTC 14638BP)이 생성하는 EPS(exopolysaccharide)가 상기한 프로바이오틱스들의 생육을 이눌린 대비 효과적으로 증진시키는 것을 확인하였다. 따라서, 본 발명의 비피도박테리움 비피덤(Bifidobacterium bifidum) EPS DA-LAIM (KCTC 14638BP), 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, Bifidobacterium bifidum ( Bifidobacterium bifidum ) It was confirmed that EPS (exopolysaccharide) produced by EPS DA-LAIM (KCTC 14638BP) effectively promotes the growth of the probiotics compared to inulin. Therefore, Bifidobacterium bifidum of the present invention ( Bifidobacterium bifidum ) EPS DA-LAIM (KCTC 14638BP), its dead cells and its culture can also be said to 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 비피도박테리움 비피덤(Bifidobacterium bifidum) EPS DA-LAIM (KCTC 14638BP), 그의 사균체, 그의 배양물 및 상기한 EPS(exopolysaccharide) 중 선택되는 어느 하나를 함유하는 활성산소의 과량 축적으로 말미암은 피부노화 개선용 화장료 조성물을 제공한다.On the other hand, the present invention is Bifidobacterium bifidum ( Bifidobacterium bifidum ) EPS DA-LAIM (KCTC 14638BP), its dead cells, its culture, and EPS (exopolysaccharide) containing any one selected from the above provides a cosmetic composition for improving skin aging due to excessive accumulation of active oxygen.
화장품의 항산화 효능이라 함은 산화작용으로 손상된 피부 기능을 개선시키거나 예방하는 능력을 의미하는데, 이를 통해 피부노화를 방지 또는 개선할 수 있다. The antioxidant effect of cosmetics means the ability to improve or prevent skin functions damaged by oxidation, through which skin aging can be prevented or improved.
한편, 본 발명에 있어, 상기 비피도박테리움 비피덤(Bifidobacterium bifidum) EPS DA-LAIM은, 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. 비피도박테리움 비피덤(Bifidobacterium bifidum) EPS DA-LAIM의 함량이 0.0001중량% 미만인 경우에는 항산화 효과가 미미하고, 30.0중량% 초과할 경우 함유량 증가에 따른 뚜렷한 효과가 증가하지 않는다.Meanwhile, in the present invention, the Bifidobacterium bifidum EPS DA-LAIM 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. When the content of Bifidobacterium bifidum EPS DA-LAIM is less than 0.0001% by weight, the antioxidant effect is insignificant, and when 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 비피도박테리움 비피덤(Bifidobacterium bifidum) EPS DA-LAIM 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 other than the Bifidobacterium bifidum EPS DA-LAIM of the present invention as active ingredients, such as antioxidants , conventional adjuvants such as stabilizers, solubilizers, 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. 비피더스균 분리1. Isolation of Bifidobacteria
건강한 한국인 자원자의 대변을 수집한 후, 상기 대변을 멸균 생리식염수를 이용해 십진 희석법으로 단계별 희석한 다음, TOS-MUP 선택배지(Bifidobacterium selective media)에 100㎕씩 도말하였다. 도말한 플레이트를 37℃에서 48시간 동안 혐기배양한 뒤, 배양된 균주 중에서 단일 콜로니를 1차로 선별한 후 해당 콜로니를 TOS-MUP 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 비피더스 후보균주 129개를 선정하였다.After collecting the feces of healthy Korean volunteers, the feces were diluted step by step using sterile physiological saline by a decimal dilution method, and then 100 μl were plated on TOS-MUP selective media ( Bifidobacterium selective media). 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 and spotted once more on a TOS-MUP selective medium. The plate was incubated anaerobically at 37° C. for 48 hours, and then it was confirmed once again that the colony grew well, and finally 129 bifidus candidate strains were selected.
2. 비피더스균 동정2. Identification of Bifidobacteria
분리된 비피더스 후보균주를 대상으로 16S rRNA 염기서열 분석을 통한 동정을 수행하였다. 비피더스 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 비피더스 후보군들의 속(genus)과 종(species)을 확정지었다. 비피더스 후보균주 129개를 각각 동정한 결과, 모두 비피도박테리움 속(Bifidobacterium sp.)임이 밝혀져 대변으로부터 비피더스균이 잘 분리되었음을 확인하였다.Identification through 16S rRNA sequencing was performed on the isolated bifidus candidate strain. Genomic DNA was extracted and purified from the bifidus candidate strain, and the 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 Bifidus candidate group were determined. As a result of identifying 129 bifidus candidate strains, all of them were found to be of the genus Bifidobacterium sp., confirming that bifidobacteria were well separated from feces.
3. EPS 생성능 정량 평가3. Quantitative evaluation of EPS generating ability
건강한 한국인 자원자의 대변에서 자체분리한 129개 비피더스균의 EPS 생성능을 정량평가하였다. 10% 수크로오스를 당원으로 하는 MRS broth(De Man, Rogosa and Sharpe, 0.05% L-cysteine)에 각 균주를 1% 접종하여 37℃, 48시간 동안 혐기배양한 후 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. 이후, 침전물을 4℃에서 원심분리(10,000×g, 25min)해 회수하고, 남은 에탄올을 건조한 후 동결건조하여 고형분의 무게를 측정하였다.The EPS production ability of 129 bifidobacteria isolated from feces of healthy Korean volunteers was quantitatively evaluated. Inoculate 1% of each strain in MRS broth (De Man, Rogosa and Sharpe, 0.05% L-cysteine) containing 10% sucrose as a sugar source, incubate anaerobically at 37 ° C for 48 hours, and centrifuge the culture medium at 4 ° C ( 10,000 × g, 25 min) to remove the cells, and to the supernatant, twice the volume of cooled 95% ethanol was slowly added to precipitate the EPS at 4 ° C for 15 to 24 hours. Thereafter, the precipitate was collected by centrifugation (10,000 × g, 25 min) at 4 ° C., and the remaining ethanol was dried and lyophilized to measure the weight of the solid content.
4. EPS의 Ropy 특성 확인4. Checking EPS ropy characteristics
상기 129개 비피더스균의 Ropy-EPS(Ropy-Exopolysaccharide) 생성 능력을 측정하였다. 10% 수크로오스를 당원으로 하는 MRS 아가를 제조한 후 129개 비피더스균 각각을 스트레킹(streaking)하여 20~40℃에서 48~168시간 동안 혐기배양하였다. 배양이 완료된 후 멸균된 루프를 이용하여 단일 콜로니의 점성 물질을 확인하였고 늘어난 길이를 ㎜ 단위로 측정했다.The ability of the 129 bifidobacteria to produce Ropy-Exopolysaccharide (Ropy-EPS) was measured. After preparing MRS agar containing 10% sucrose as a sugar source, each of 129 bifidobacteria was streaked and anaerobically cultured at 20 to 40° C. for 48 to 168 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 129개 비피더스균의 EPS 생성능 및 그 EPS의 Ropy 특성을 측정한 결과, 표 1과 같이 EPS 생성이 가장 우수한 비피더스균 1종인 비피도박테리움 비피덤(Bifidobacterium bifidum) EPS DA-LAIM을 선정하였다. EPS DA-LAIM 균주는 함께 분리하였던 타 비피도박테리움 속(Bifidobacterium sp.) 균주 대비 3.6배, 국내 유명 건기식 제품의 동속동종 균주 대비 3.8배 높게 우수한 EPS 생성능을 나타내었고, 조건최적화 시 콜로니에서 최대 50㎜의 strand가 형성되어 상기 EPS가 Ropy 특성을 지님이 확인되었다. 이의 16S rRNA 염기서열은 서열번호 1에 나타내었다.As a result of measuring the EPS production ability of 129 bifidobacteria isolated from feces of healthy Korean volunteers and the ropy characteristics of the EPS, Bifidobacterium bifidum, one of the best EPS production species, Bifidobacterium bifidum , as shown in Table 1 EPS DA-LAIM was selected. The EPS DA-LAIM strain showed an excellent EPS production ability that was 3.6 times higher than that of other Bifidobacterium sp. A strand of 50 mm was formed, and it was confirmed that the EPS had ropy characteristics. Its 16S rRNA nucleotide sequence is shown in SEQ ID NO: 1.
| 균주명strain name | EPS 생성능EPS generating ability | EPS의 Ropy 특성EPS ropy characteristics |
| Bifidobacterium bifidum Bifidobacterium bifidum EPS DA-LAIMEPS DA-LAIM | 10.12 ㎎/㎖10.12 mg/mL | 조건최적화 시 50㎜ 생성When conditions are optimized, 50 mm is generated |
| 그 외 161개 균주161 other strains | 2.84 ㎎/㎖2.84 mg/mL | 미생성uncreated |
|
국내 건기식제품 Domestic dry food products
동속동종 균주homozygous strain |
2.64 ㎎/㎖2.64 mg/mL | 미생성uncreated |
[실험예 1: 분리균주의 DPPH 항산화능 평가][Experimental Example 1: DPPH antioxidant activity evaluation of isolated strains]
분리균주의 항산화능을 평가하기 위하여, DPPH 라디칼 소거능을 비색정량법으로 확인하였다. 구체적으로, 비피더스균 스톡을 MRS broth(0.05% L-cysteine)에 1%(v/v) 농도가 되도록 접종하고, 32~37oC에서 24~168시간 동안 혐기조건에서 정치배양하였다. 배양된 균액 1㎖를 13,000rpm, 4℃에서 3분간 원심분리하여 균체를 회수하였으며, 이때 균수는 대략 5.0×108 CFU/㎖가 되도록 하였다. 해당 균체를 멸균된 PBS 버퍼(pH 7.4)로 1번 세척하고 동일한 새 버퍼 1㎖에 용해하였다.In order to evaluate the antioxidant activity of the isolated strain, the DPPH radical scavenging activity was confirmed by colorimetric method. Specifically, the Bifidobacterium stock was inoculated to a concentration of 1% (v/v) in MRS broth (0.05% L-cysteine), and incubated at 32-37 ° C for 24-168 hours under anaerobic conditions. Cells were recovered by centrifuging 1 ml of the cultured bacterial solution at 13,000 rpm and 4° C. for 3 minutes, and at this time, the number of bacteria was approximately 5.0×10 8 CFU/ml. The cells were washed once with sterilized PBS buffer (pH 7.4) and dissolved in 1 ml of the same new buffer.
이후, 에탄올을 용매로 하여 제조한 0.2mM DPPH 용액 1㎖에 상기 비피더스 균체 1㎖를 첨가하여 섞어주었다. 해당 혼합액을 실온에서 30분간 빛을 차단한 채 반응시킨 후 반응이 끝나면 반응액을 13,000rpm, 4℃에서 3분간 원심분리하였다. 반응액의 상등액 200㎕를 96-웰 플레이트에 옮겨 517㎚에서 OD값을 측정하였다. 비교실험을 위해 대표적인 항산화물질인 아스코르브산(ascorbic acid; Vt. C, 0.1 ㎎/㎖) 1㎖에 DPPH 용액 1㎖를 첨가하여 반응시킨 양성대조군과 비피더스 균체 1㎖에 에탄올 1㎖를 첨가하여 반응시킨 블랭크(blank)군 및 PBS 버퍼 1㎖에 DPPH 용액 1㎖를 첨가하여 반응시킨 대조군(control)군도 함께 실험하였다. 또한, 동속동종 균주와의 비교를 위해 국내 유명 건기식 제품 내 동속동종 균주를 대상으로 동일 실험을 진행하였다. DPPH 항산화능을 정량화하기 위해, 하기와 같이 DPPH 소거활성(scavenging activity)을 계산해 군간 비교를 하였다 (식 1).Thereafter, 1 ml of the bifidus cells was added to 1 ml of a 0.2 mM DPPH solution prepared using ethanol as a solvent and mixed. The mixture was reacted at room temperature for 30 minutes while blocking light, and when the reaction was finished, the reaction mixture was centrifuged at 13,000 rpm and 4° C. for 3 minutes. 200 μl of the supernatant of the reaction solution was transferred to a 96-well plate and the OD value was measured at 517 nm. For comparative experiments, a positive control group reacted by adding 1 ml of DPPH solution to 1 ml of ascorbic acid (Vt. C, 0.1 mg/ml), a representative antioxidant, and 1 ml of ethanol was added to 1 ml of bifidus cells to react. A blank group and a control group reacted by adding 1 ml of DPPH solution to 1 ml of PBS buffer were also tested. In addition, for comparison with strains of the same type, the same experiment was conducted for strains of the same type in domestic famous dry season food products. In order to quantify the DPPH antioxidant capacity, the DPPH scavenging activity was calculated and compared between groups as follows (Equation 1).
[식 1][Equation 1]
DPPH scavenging activity (%) = (1-(sample OD517-blank OD517)/control OD517)*100DPPH scavenging activity (%) = (1-(sample OD 517 -blank OD 517 )/control OD 517 )*100
실험 결과, EPS DA-LAIM 균주의 DPPH 소거활성이 59.6%로, 대표적인 항산화물질인 비타민 C(0.1㎎/㎖)보다 1.9배, 국내 유명 건기식 제품의 동속동종 균주보다 3.2배 높은 항산화능을 나타내었다 (도 1). 도 1은 본 발명 균주의 항산화능을 평가한 결과이다.As a result of the experiment, the DPPH scavenging activity of the EPS DA-LAIM strain was 59.6%, which was 1.9 times higher than that of vitamin C (0.1mg/ml), a representative antioxidant, and 3.2 times higher than that of the same strain of a famous domestic dry food product. (Fig. 1). 1 is a result of evaluating the antioxidant ability of the strain of the present invention.
[실시예 2: EPS 분리 및 정제][Example 2: Separation and purification of EPS]
분리균주로부터 생성된 EPS를 분리정제하기 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, 25min)하여 침전된 단백질을 제거한 후 상등액에 2배의 냉각된 95% 에탄올을 서서히 가하여 4℃에서 15~24시간 동안 EPS를 침전시켰다.In order to separate and 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 4 It was added to 10% and reacted at 4°C for 2 hours. The reaction solution was centrifuged at 4 ° C (10,000 × g, 25 min) to remove precipitated proteins, and then 2 times of 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 2: EPS의 프리바이오틱 효능 평가][Experimental Example 2: Evaluation of prebiotic efficacy of EPS]
분리균주로부터 정제한 EPS가 프리바이오틱스 활성을 지니는지 확인하고자 2% EPS를 당원으로 하는 MRS broth 또는 TSB broth를 96-웰 플레이트에 100㎕씩 분주한 뒤 해당 MRS broth에는 전날 활성화한 장내 유익균(유산균)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% EPS as a sugar source was dispensed into a 96-well plate, and then the MRS broth contained intestinal beneficial bacteria activated the previous day ( lactic acid bacteria), the TSB broth was inoculated with 1% of the culture medium of intestinal harmful bacteria ( Escherichia coli ), respectively, and the OD 600 value after 24 hours was 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 활성을 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 2).To quantify prebiotic activity [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 2).
[식 2][Equation 2]
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 활성을 정량화할 수 있는 PI값을 측정해보았을 때, 락토바실러스 플란타룸(Lactobacillus plantarum), 락토바실러스 퍼멘텀(Lactobacillus fermentum), 락토바실러스 헬베티쿠스(Lactobacillus helveticus)에 대하여 대표적인 프리바이오틱스인 이눌린 대비 PI값이 각각 4.3배, 2.4배, 4.4배 높아, 본 발명 균주의 EPS가 이눌린보다 락토바실러스 속(Lactobacillus sp.)에 대한 프리바이오틱스 활성이 우수한 것이 관찰되었다 (도 2). 도 2는 본 발명 균주의 프리바이오틱 효과를 평가한 결과이다.As a result of the experiment, when the PI value that can quantify the prebiotic activity of EPS was measured, Lactobacillus plantarum , Lactobacillus fermentum , Lactobacillus helveticus ( Lactobacillus helveticus ) It was observed that the EPS of the strain of the present invention has better prebiotics activity against Lactobacillus sp. (Fig. 2). Figure 2 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 : KCTC14638BPAccession number: KCTC14638BP
수탁일자 : 20210715Entrusted date: 20210715
Claims (16)
- 비피도박테리움 비피덤(Bifidobacterium bifidum) EPS DA-LAIM (KCTC 14638BP).Bifidobacterium bifidum ( Bifidobacterium bifidum ) EPS DA-LAIM (KCTC 14638BP).
- 제1항에 있어서,According to claim 1,상기 균주는,The strain,포스트바이오틱스로서 EPS(exopolysaccharide)를 생성하는 것을 특징으로 하는 비피도박테리움 비피덤(Bifidobacterium bifidum) EPS DA-LAIM (KCTC 14638BP).Bifidobacterium bifidum characterized by producing EPS (exopolysaccharide) as postbiotics ( Bifidobacterium bifidum ) EPS DA-LAIM (KCTC 14638BP).
- 제2항에 있어서,According to claim 2,상기 EPS(exopolysaccharide)는,The EPS (exopolysaccharide),로우피(ropy) 형태의 EPS(exopolysaccharide)를 포함하는 것을 특징으로 하는 비피도박테리움 비피덤(Bifidobacterium bifidum) EPS DA-LAIM (KCTC 14638BP).Bifidobacterium bifidum characterized by containing EPS (exopolysaccharide) in the form of ropy ( Bifidobacterium bifidum ) EPS DA-LAIM (KCTC 14638BP).
- 비피도박테리움 비피덤(Bifidobacterium bifidum) EPS DA-LAIM (KCTC 14638BP), 그의 사균체 및 그의 배양물 중 선택되는 어느 하나를 함유하는 활성산소가 과량 축적되어 유발되는 질환의 예방 또는 치료용 약학 조성물.Bifidobacterium bifidum ( Bifidobacterium bifidum ) EPS DA-LAIM (KCTC 14638BP), a pharmaceutical composition for preventing or treating diseases caused by excessive accumulation of active oxygen containing any one selected from dead cells and cultures thereof.
- 비피도박테리움 비피덤(Bifidobacterium bifidum) EPS DA-LAIM (KCTC 14638BP), 그의 사균체 및 그의 배양물 중 선택되는 어느 하나를 함유하는 활성산소가 과량 축적되어 유발되는 질환의 개선용 식품 조성물.Bifidobacterium bifidum ( Bifidobacterium bifidum ) EPS DA-LAIM (KCTC 14638BP), a food composition for improving diseases caused by excessive accumulation of active oxygen containing any one selected from dead cells and cultures thereof.
- 비피도박테리움 비피덤(Bifidobacterium bifidum) EPS DA-LAIM (KCTC 14638BP), 그의 사균체 및 그의 배양물 중 선택되는 어느 하나를 함유하는 활성산소가 과량 축적되어 유발되는 질환의 개선용 건강기능식품.Bifidobacterium bifidum ( Bifidobacterium bifidum ) EPS DA-LAIM (KCTC 14638BP), a health functional food for improving diseases caused by excessive accumulation of active oxygen containing any one selected from dead cells and cultures thereof.
- 비피도박테리움 비피덤(Bifidobacterium bifidum) EPS DA-LAIM (KCTC 14638BP), 그의 사균체 및 그의 배양물 중 선택되는 어느 하나를 함유하는 활성산소의 과량 축적으로 말미암은 피부노화 개선용 화장료 조성물.Bifidobacterium bifidum ( Bifidobacterium bifidum ) EPS DA-LAIM (KCTC 14638BP), a cosmetic composition for improving skin aging caused by excessive accumulation of active oxygen containing any one selected from dead cells and culture thereof.
- 비피도박테리움 비피덤(Bifidobacterium bifidum) EPS DA-LAIM (KCTC 14638BP)이 생성하는 EPS(exopolysaccharide).Bifidobacterium bifidum ( Bifidobacterium bifidum ) EPS (exopolysaccharide) produced by DA-LAIM (KCTC 14638BP).
- 제8항에 있어서,According to claim 8,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).
- 제8항에 있어서,According to claim 8,상기 EPS(exopolysaccharide)는,The EPS (exopolysaccharide),a) 비피도박테리움 비피덤(Bifidobacterium bifidum) EPS DA-LAIM (KCTC 14638BP) 배양 상등액에 산성물질을 첨가하여 단백질을 침전 및 제거하는 단계; 및a) Bifidobacterium bifidum ( Bifidobacterium bifidum ) EPS DA-LAIM (KCTC 14638BP) Precipitating and removing proteins by adding an acidic material to the culture supernatant; andb) 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.
- 비피도박테리움 비피덤(Bifidobacterium bifidum) EPS DA-LAIM (KCTC 14638BP)이 생성하는 EPS(exopolysaccharide)를 함유하는 활성산소가 과량 축적되어 유발되는 질환의 예방 또는 치료용 약학 조성물.Bifidobacterium bifidum ( Bifidobacterium bifidum ) A pharmaceutical composition for preventing or treating diseases caused by excessive accumulation of active oxygen containing EPS (exopolysaccharide) produced by EPS DA-LAIM (KCTC 14638BP).
- 비피도박테리움 비피덤(Bifidobacterium bifidum) EPS DA-LAIM (KCTC 14638BP)이 생성하는 EPS(exopolysaccharide)를 함유하는 활성산소가 과량 축적되어 유발되는 질환의 개선용 식품 조성물.Bifidobacterium bifidum ( Bifidobacterium bifidum ) A food composition for improving diseases caused by excessive accumulation of active oxygen containing EPS (exopolysaccharide) produced by EPS DA-LAIM (KCTC 14638BP).
- 비피도박테리움 비피덤(Bifidobacterium bifidum) EPS DA-LAIM (KCTC 14638BP)이 생성하는 EPS(exopolysaccharide)를 함유하는 활성산소가 과량 축적되어 유발되는 질환의 개선용 건강기능식품. Bifidobacterium bifidum ( Bifidobacterium bifidum ) A health functional food for improving diseases caused by excessive accumulation of active oxygen containing EPS (exopolysaccharide) produced by EPS DA-LAIM (KCTC 14638BP).
- 비피도박테리움 비피덤(Bifidobacterium bifidum) EPS DA-LAIM (KCTC 14638BP)이 생성하는 EPS(exopolysaccharide)를 함유하는 활성산소의 과량 축적으로 말미암은 피부노화 개선용 화장료 조성물.Bifidobacterium bifidum ( Bifidobacterium bifidum ) A cosmetic composition for improving skin aging caused by excessive accumulation of active oxygen containing EPS (exopolysaccharide) produced by EPS DA-LAIM (KCTC 14638BP).
- 비피도박테리움 비피덤(Bifidobacterium bifidum) EPS DA-LAIM (KCTC 14638BP), 그의 사균체, 그의 배양물 및 비피도박테리움 비피덤(Bifidobacterium bifidum) EPS DA-LAIM (KCTC 14638BP)이 생성하는 EPS(exopolysaccharide) 중 선택되는 어느 하나를 함유하는 프로바이오틱스 생육 촉진용 조성물.Bifidobacterium bifidum ( Bifidobacterium bifidum ) EPS DA-LAIM (KCTC 14638BP), its dead cell, its culture and Bifidobacterium bifidum ( Bifidobacterium bifidum ) A composition for promoting the growth of probiotics containing any one selected from EPS (exopolysaccharide) produced by EPS DA-LAIM (KCTC 14638BP).
- 제15항에 있어서,According to claim 15,상기 프로바이오틱스 생육 촉진용 조성물은,The composition for promoting the growth of probiotics,락토바실러스 플란타룸(Lactobacillus plantarum), 락토바실러스 퍼멘텀(Lactobacillus fermentum), 락토바실러스 헬베티쿠스(Lactobacillus helveticus) 중 선택되는 어느 하나 이상의 프로바이오틱스의 생육을 촉진시키는 것을 특징으로 하는 프로바이오틱스 생육 촉진용 조성물.A composition for promoting the growth of probiotics, characterized in that it promotes the growth of any one or more probiotics selected from Lactobacillus plantarum , Lactobacillus fermentum , and Lactobacillus helveticus .
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| KR20050069440A (en) * | 2003-12-31 | 2005-07-05 | 재단법인서울대학교산학협력재단 | A new anti-cancer polysaccharide from the genus bifidobacterium |
| KR102421144B1 (en) * | 2021-09-10 | 2022-07-15 | 동아제약 주식회사 | Bifidobacterium bifidum EPS DA-LAIM promoting the growth of lactobacillus and polysaccharide therefrom |
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| KR102166596B1 (en) | 2018-09-21 | 2020-10-16 | 전북대학교 산학협력단 | Lactobacillus plantarum JDFM LP11 strain having antimicrobial activity, anti-aging activity and probiotics properties and uses thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR20050069440A (en) * | 2003-12-31 | 2005-07-05 | 재단법인서울대학교산학협력재단 | A new anti-cancer polysaccharide from the genus bifidobacterium |
| KR102421144B1 (en) * | 2021-09-10 | 2022-07-15 | 동아제약 주식회사 | Bifidobacterium bifidum EPS DA-LAIM promoting the growth of lactobacillus and polysaccharide therefrom |
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| HIDALGO-CANTABRANA CLAUDIO, ALGIERI FRANCESCA, RODRIGUEZ-NOGALES ALBA, VEZZA TERESA, MARTÍNEZ-CAMBLOR PABLO, MARGOLLES ABELARDO, R: "Effect of a Ropy Exopolysaccharide-Producing Bifidobacterium animalis subsp. lactis Strain Orally Administered on DSS-Induced Colitis Mice Model", FRONTIERS IN MICROBIOLOGY, vol. 7, 1 January 2016 (2016-01-01), pages 868, XP093043866, DOI: 10.3389/fmicb.2016.00868 * |
| LI SHENGJIE, HUANG RENHUI, SHAH NAGENDRA P., TAO XUEYING, XIONG YONGHUA, WEI HUA: "Antioxidant and antibacterial activities of exopolysaccharides from Bifidobacterium bifidum WBIN03 and Lactobacillus plantarum R315", JOURNAL OF DAIRY SCIENCE, AMERICAN DAIRY SCIENCE ASSOCIATION, US, vol. 97, no. 12, 1 December 2014 (2014-12-01), US , pages 7334 - 7343, XP093043864, ISSN: 0022-0302, DOI: 10.3168/jds.2014-7912 * |
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