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WO2023038419A1 - Souche de protection de la santé intestinale lactobacillus paracasei eps da-bacs possédant des effets favorisant la croissance de lactobacillus bifidus et inhibant les bactéries intestinales nuisibles clostridium difficile, et son polysaccharide - Google Patents

Souche de protection de la santé intestinale lactobacillus paracasei eps da-bacs possédant des effets favorisant la croissance de lactobacillus bifidus et inhibant les bactéries intestinales nuisibles clostridium difficile, et son polysaccharide Download PDF

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Publication number
WO2023038419A1
WO2023038419A1 PCT/KR2022/013422 KR2022013422W WO2023038419A1 WO 2023038419 A1 WO2023038419 A1 WO 2023038419A1 KR 2022013422 W KR2022013422 W KR 2022013422W WO 2023038419 A1 WO2023038419 A1 WO 2023038419A1
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WIPO (PCT)
Prior art keywords
eps
bacs
lactobacillus paracasei
exopolysaccharide
present
Prior art date
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Ceased
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PCT/KR2022/013422
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English (en)
Korean (ko)
Inventor
한상덕
한영선
박민주
조현일
이은석
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Dong A Pharmaceutical Co Ltd
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Dong A Pharmaceutical Co Ltd
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Publication of WO2023038419A1 publication Critical patent/WO2023038419A1/fr
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Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

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

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Abstract

La présente invention concerne la souche de protection de santé intestinale Lactobacillus paracasei EPS DA-BACS possédant des effets favorisant la croissance de Lactobacillus bifidus et inhibant les bactéries intestinales nuisibles Clostridium difficile, et son polysaccharide. La souche de la présente invention est caractérisée par la production d'exopolysaccharide (EPS) en tant que postbiotique, et présente l'avantage de posséder une stabilité gastro-intestinale et des effets d'inhibition des bactéries nuisibles et des eumycètes et d'atténuation de l'inflammation, d'inhiber Clostridium difficile, et d'offrir un effet prébiotique.
PCT/KR2022/013422 2021-09-13 2022-09-07 Souche de protection de la santé intestinale lactobacillus paracasei eps da-bacs possédant des effets favorisant la croissance de lactobacillus bifidus et inhibant les bactéries intestinales nuisibles clostridium difficile, et son polysaccharide Ceased WO2023038419A1 (fr)

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CN118879582A (zh) * 2024-09-19 2024-11-01 上海博诗康生物科技有限公司 一种细胞完整、分散均匀、稳定性强的后生元制备方法

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KR102374480B1 (ko) * 2021-09-13 2022-03-15 동아제약 주식회사 비피더스 유산균 생장촉진 및 장내 유해균 클로스트리듐 디피실리균 억제 효능을 갖는 장 건강 균주 락토바실러스 파라카제이 eps da-bacs 및 그 다당류
CN115161364B (zh) * 2022-09-06 2022-12-09 东北农业大学 提高副干酪乳杆菌jy062胞外多糖产量的制法
KR20240097985A (ko) * 2022-12-15 2024-06-27 주식회사 인트론바이오테크놀로지 클로스트리디오이데스 디피실리 (Clostridioides difficile) 균에 대하여 용균 활성을 갖는 항균단백질 CDL2200
KR102756859B1 (ko) * 2023-09-15 2025-01-22 (주) 옵트바이오 신규 유산균 및 이의 대사산물을 포함하는 조성물

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