WO2021040335A1 - Nouvelle souche symbiotique ayant une résistance dans une condition d'oxygène réactif et son utilisation - Google Patents

Nouvelle souche symbiotique ayant une résistance dans une condition d'oxygène réactif et son utilisation Download PDF

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WO2021040335A1
WO2021040335A1 PCT/KR2020/011192 KR2020011192W WO2021040335A1 WO 2021040335 A1 WO2021040335 A1 WO 2021040335A1 KR 2020011192 W KR2020011192 W KR 2020011192W WO 2021040335 A1 WO2021040335 A1 WO 2021040335A1
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strain
present
culture
resistance under
immune activity
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용동은
윤상선
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연세대학교 산학협력단
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/745—Bifidobacteria
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C12N1/205—Bacterial isolates
    • 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 a novel symbiotic microbial strain having resistance under reactive oxygen conditions and to a use thereof.
  • Reactive oxygen species or reactive oxygen species refer to chemically reactive molecules including oxygen atoms.
  • the active oxygen is a compound of oxygen generated in living organisms.Since it contains peroxygen ions and hydrogen peroxide and has unpaired electrons, it has very high reactivity and has a very strong oxidizing power that can attack living tissues and damage cells. Corresponds to oxygen.
  • Such reactive oxygen species can be generated even in normal metabolic processes, and it is reported that they play a role in regulating cellular signals and homeostasis.
  • concentration of free radicals increases rapidly in an individual as environmental stress, such as exposure to ultraviolet rays or high heat, and accordingly, damages the cell structure and causes various diseases.
  • oxidative stress when oxidative stress is present, the production of active oxygen increases or the antioxidant defense effect such as glutathione decreases to a significant level.
  • the effect of oxidative stress can vary depending on the magnitude of these changes, and if the level of disturbance is low, cells can overcome these conditions by themselves and return to their original state.
  • the level of disturbance by oxidative stress when the level of disturbance by oxidative stress is severe, cell death can occur, and even when the oxidative stress is at a moderate level, apoptosis can be triggered. Free radicals generated by the oxidative stress can directly damage the DNA present in the individual, and accordingly, various diseases such as aging, diabetes, hyperlipidemia, obesity, colon cancer, rheumatism, or asthma can be caused.
  • intestinal microorganisms are composed of strains and various microorganisms that coexist on the epithelial protective wall of the host, and these symbiotic strains are used to maintain and survive the individual's health. It has been reported to affect the physiological function of.
  • the symbiotic microbial colony (total of symbiotic microbes) is composed of various species and has various genes (microbiome), responding to external stimuli and minor environmental changes, and is very dynamic with the human body. Interact.
  • a healthy human body with a normal symbiotic microbial flora can overcome the invasion of pathogens through various antibacterial actions when pathogenic bacteria are infected in the intestines.
  • the immune system against pathogenic bacteria does not work normally when a broad spectrum antibiotic is continuously taken or exposed to oxidative stress conditions.
  • One object of the present invention is to provide a novel strain contained in the genus Bifidobacterium having resistance under reactive oxygen conditions, a culture solution thereof, a culture product, or an extract obtained therefrom.
  • Another object of the present invention is to provide a food composition for enhancing immune activity comprising the novel strain, a culture solution thereof, a culture product, or an extract obtained therefrom as an active ingredient.
  • Another object of the present invention is to provide a method for enhancing immune activity comprising administering to an individual the novel strain, a culture solution thereof, a culture product, or an extract obtained therefrom.
  • An embodiment of the present invention provides a strain contained in the genus Bifidobacterium.
  • the strain of the genus Bifidobacterium of the present invention may be a Bifidobacterium longum.
  • the Bifidobacterium longum of the present invention may be deposited with accession number KFCC11835P (Bifidobacterium longum YMC_19_03_38) or accession number KFCC11834P ( Bifidobacterium longum YMC_19_03_2).
  • the strains contained in the Bifidobacterium genus of the present invention are those strains that are resistant to anaerobic conditions and a culture environment containing excessive amounts of hydrogen peroxide among strains obtained from the feces of healthy humans. It was confirmed that the selected strain belongs to the genus Bifidobacterium through phylogenetic analysis using the genomic sequence and the nucleotide sequence of the 16s rRNA gene. Further, as a result of analyzing the genome sequence of each of the two deposited strains (YMC_19_03_38 and YMC_19_03_2), it was once again confirmed that it is a microorganism belonging to the genus Bifidobacterium containing a novel genome sequence.
  • the strain of the present invention may be one having resistance under reactive oxygen conditions.
  • the "resistance under active oxygen conditions" of the present invention is an environment in which active oxygen is inevitably generated during the electron transfer process for energy production in all organisms that breathe oxygen, or is generated by an inflammatory reaction caused by infection, In other words, it means the ability to survive without being killed in an oxidative stress environment.
  • the novel strain has resistance under anaerobic conditions and culture conditions containing an excessive amount of hydrogen peroxide, and thus resistance under reactive oxygen conditions, that is, a defense mechanism against oxidative stress (resistance to oxidative stress). Can have.
  • the novel strain of the present invention has resistance under reactive oxygen conditions, it is possible to maintain a normal microbial flora present in a target organ.
  • the strain of the present invention may have an ability to enhance immune activity.
  • the "enhancing immune activity" of the present invention refers to enhancing biodefensive ability by modulating an immune response.
  • Such enhancement of immune activity is one of important therapeutic and prophylactic strategies for reinforcing the body's defense mechanisms against various diseases, and it is possible to obtain an effect of enhancing immune activity by stimulating an immune response by increasing the activity of immune cells.
  • the strain may have an immune activity enhancing function, for example, infection by increasing the expression level of interleukin-1 (IL-1), a cytokine that mediates and regulates the innate immune response. And it is possible to mediate the inflammatory response of the host to other stimuli to enhance immune activity, but is not limited thereto.
  • IL-1 interleukin-1
  • a culture of the strain contained in the genus Bifidobacterium is provided.
  • the strain of the genus Bifidobacterium is the same as described in the strain, and thus is omitted to avoid excessive complexity of the specification.
  • the "culture product" of the present invention refers to a product obtained by culturing a microorganism in a known liquid or solid medium, and refers to a medium containing the microorganism.
  • the culture of the present invention is obtained after culturing the strain of the present invention in a medium, and the medium may be selected from a known liquid medium or solid medium used for culturing the strain of the genus Bifidobacterium.
  • the medium may be a GYSM medium, a humic acid agar medium, a Bennett's agar medium, or a malt extract agar medium, but is not limited thereto.
  • Another embodiment of the present invention provides a culture solution from which the strain has been removed from the culture of the strain contained in the genus Bifidobacterium.
  • the strain or culture of the genus Bifidobacterium is the same as described in the strain or culture, and thus is omitted to avoid excessive complexity of the specification.
  • the "culture solution” of the present invention refers to a product obtained by culturing microorganisms in a known liquid or solid medium, and is a concept in which microorganisms are not included.
  • the culture medium of the present invention refers to a liquid product from which the strain itself has been removed through a method such as filtration or centrifugation after culturing a predetermined strain in a liquid medium.
  • the filtration method of the present invention is not particularly limited, and the number of times the filtration is performed may be, for example, 1 to 10 times, 1 to 5 times, or 1 to 3 times.
  • the pore size of the filter paper may be performed using a filter paper having a pore size of 5 to 10 ⁇ m or a filter having a pore size of 0.1 to 1.0 ⁇ m, but is not limited thereto.
  • a culture of a strain contained in the genus Bifidobacterium or an extract of a culture solution there is provided a culture of a strain contained in the genus Bifidobacterium or an extract of a culture solution.
  • the strain, culture or culture of the genus Bifidobacterium is the same as described in the strain, culture or culture, and thus is omitted to avoid excessive complexity of the specification.
  • the extract of the present invention is a conventional extraction method known in the art, for example, solvent extraction, extraction by supercritical fluid extraction using carbon dioxide, extraction by extraction using ultrasonic waves, and constant molecular weight cut- It can be prepared using a method such as separation using an ultrafiltration membrane having an off value, separation by various chromatography (one prepared for separation according to size, charge, hydrophobicity, or affinity), or a combination thereof.
  • the extraction solvent used in the solvent extraction method of the present invention is water, a lower alcohol having 1 to 4 carbon atoms (e.g., methanol, ethanol, propanol, and butanol) or a mixture thereof, a hydrated lower alcohol, propylene glycol, 1,3 -Butylene glycol, glycerin, acetone, diethyl ether, ethyl acetate, butyl acetate, dichloromethane, chloroform, hexane, and may be selected from the group consisting of mixtures thereof, of which water, alcohol, hydrous alcohol, diethyl ether , Ethyl acetate, butyl acetate, chloroform, or may be selected from hexane, but is not limited thereto.
  • a lower alcohol having 1 to 4 carbon atoms e.g., methanol, ethanol, propanol, and butanol
  • a mixture thereof e.g., 1,3 -Buty
  • a food composition for enhancing immune activity comprising a strain contained in the genus Bifidobacterium , a culture thereof, a culture medium, or an extract obtained therefrom as an active ingredient.
  • the strains, cultures, cultures or extracts of the genus Bifidobacterium are the same as described in the strains, cultures, cultures or extracts, and thus are omitted to avoid excessive complexity of the specification.
  • the food of the present invention may be a health functional food.
  • the "health functional food” of the present invention refers to a food group or food composition that has added value to effect and express the function of the food by using physical, biochemical, biotechnological techniques, etc. to the food, It refers to a food processed by designing to sufficiently express the body's control functions related to disease prevention and recovery, etc. to the living body, which may further contain food additives acceptable for food, suitable carriers, excipients, and It may further include a diluent.
  • the function of the health functional food may be an immune activity enhancing function, for example, the expression level of interleukin-1 (IL-1), a cytokine that mediates and regulates the innate immune response. By increasing the inflammatory response of the host to infection and other stimuli can be mediated to enhance immune activity, but is not limited thereto.
  • IL-1 interleukin-1
  • the "enhancing immune activity" of the present invention refers to enhancing biodefensive ability by modulating an immune response.
  • Such enhancement of immune activity is one of the important therapeutic strategies for reinforcing body defense mechanisms against various diseases, and it is possible to obtain an effect of enhancing immune activity by stimulating an immune response by increasing the activity of immune cells.
  • the food composition of the present invention may be prepared in the form of various foods, for example, beverages, gums, teas, vitamin complexes, powders, granules, tablets, capsules, confectionery, rice cakes, bread, and the like.
  • the amount may be included in 0.001% by weight to 90% by weight, preferably 0.1% by weight to 40% by weight, and in the case of long-term intake use, the above Although it may be less than the range, since the active ingredient may be used in an amount greater than the above range when there is no problem in terms of safety, it is not limited thereto.
  • the food composition of the present invention is prepared in the form of a beverage
  • various flavoring agents or natural carbohydrates may be included as an additional component. That is, as natural carbohydrates, monosaccharides such as glucose, disaccharides such as fructose, and common sugars such as sucrose and polysaccharides, dextrins, cyclodextrins, and sugar alcohols such as xylitol, sorbitol, and erythritol can do.
  • flavoring agent examples include natural flavoring agents (taumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.).
  • other food compositions of the present invention include various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavors and natural flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof. , Organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonates used in carbonated beverages, and the like.
  • the components of the present invention may be used independently or in combination.
  • the proportion of these additives is not so critical, but is generally selected in the range of 0.1 to about 50 parts by weight per 100 parts by weight of the composition of the present invention.
  • Another embodiment of the present invention provides a probiotic composition comprising a strain contained in the genus Bifidobacterium as an active ingredient.
  • the strain of the genus Bifidobacterium is the same as described in the strain, and thus is omitted to avoid excessive complexity of the specification.
  • the "probiotic” of the present invention refers to an agent capable of improving the balance of the flora of a desired organ by using living microorganisms.
  • the strain of the present invention can be used as a probiotic composition for improving the health of an individual, and such a probiotic composition contains the strain itself, etc. contained in the Bifidobacterium of the present invention as an active ingredient, and an excipient or carrier is added Can be included as.
  • the probiotic composition of the present invention can be prepared and administered in a variety of formulations and methods. For example, by mixing a novel strain or a culture thereof according to the present invention with a carrier and flavor commonly used in the pharmaceutical field, such as tablets, troches, capsules, elixirs, syrups, powders, suspensions or granules, etc. It can be prepared and administered in a form.
  • a carrier a binder, a lubricant, a disintegrant, an excipient, a solubilizing agent, a dispersing agent, a stabilizer, a suspending agent, and the like can be used.
  • Another embodiment of the present invention provides a method for enhancing immune activity.
  • the method of the present invention includes the step of administering to a subject a strain contained in the genus Bifidobacterium , a culture solution thereof, a culture product, or an extract obtained therefrom.
  • the strain, culture, culture medium or extract of the genus Bifidobacterium is the same as described in the strain, culture medium, culture or extract, and thus is omitted to avoid excessive complexity of the specification.
  • the "individual" of the present invention refers to a mammal, including mice, livestock, etc., including humans with reduced immunity or in need of enhancing immune activity, but provided by the present invention Any individual with a possibility of enhancing immune activity by the above strain or the like may be included without limitation.
  • the method of the present invention may include administering to an individual an effective amount of the strain, a culture solution thereof, a culture product, or an extract obtained therefrom provided in the present invention.
  • the appropriate total daily use amount may be determined within the correct judgment range, and may be administered once or in several divided doses.
  • a specific effective amount for a specific individual is a specific strain, culture, culture medium or extract thereof, the age of the individual, including the type and degree of the reaction to be achieved, whether or not other agents are used in some cases, It is preferable to apply differently in consideration of body weight, general health status, sex and diet, administration time, administration route and administration period, and other active ingredients used simultaneously.
  • the novel strain of the present invention Since the novel strain of the present invention has resistance under anaerobic conditions and culture conditions containing an excess of hydrogen peroxide, it has resistance under reactive oxygen conditions, that is, a defense mechanism against oxidative stress. In addition, since the novel strain of the present invention has resistance under reactive oxygen conditions, it is possible to maintain a normal microbial flora present in a target organ. Therefore, the novel strain of the present invention can be very usefully used for maintaining a normal microbial flora or enhancing immune activity in an environment in which active oxygen is present.
  • FIG. 1 is a diagram showing an experimental design for inducing differentiation from human embryonic stem cells into vascular network tissues according to an embodiment of the present invention.
  • OCT4 which is a marker of undifferentiated stem cells
  • EOMES, MIXL1, and BRACHY which are markers of mesodermal cells, for mesodermal cells according to an embodiment of the present invention.
  • FIG 3 shows the result of measuring the OD 600 value in order to confirm the cultivation ability in the culture medium containing active oxygen of the selected strain according to an embodiment of the present invention.
  • An embodiment of the present invention relates to a strain contained in the genus Bifidobacterium.
  • One embodiment of the invention relates to a Bifidobacterium ronggum (Bifidobacterium longum) strain contained in the Bifidobacterium (Bifidobacterium).
  • Another embodiment of the present invention relates to a strain deposited with accession number KFCC11835P or accession number KFCC11834P, the strain contained in the genus Bifidobacterium.
  • Another embodiment of the present invention is a culture of the strain; Culture medium; It relates to an extract obtained therefrom.
  • Another embodiment of the present invention is a culture of the strain; Culture medium; It relates to a food composition for enhancing immune activity comprising the extract obtained therefrom as an active ingredient.
  • Another embodiment of the present invention relates to a probiotic composition
  • a probiotic composition comprising the strain as an active ingredient.
  • Another embodiment of the present invention is a culture of the strain; Culture medium; Or it relates to a method for enhancing immune activity comprising administering the extract obtained therefrom to a subject.
  • human embryonic stem cells were used, and the medium was replaced with Stemfit medium once every two days.
  • the Stemfit medium one containing b-FGF (20 ng/ml) was used.
  • Cells were cultured under a temperature condition of 5% CO 2 and 37°C, and the cells were passaged within 7 days.
  • embryonic stem cells were inoculated with 200,000 cells in RPMI1640 medium to which activin A (100 ng/ml) and CHIR99021 (3 ⁇ m) were added, followed by culture and endoderm Differentiation into sex cells was induced. Differentiation period was a total of 3 days, 0.2% by weight of FBS was added to the medium on the 2nd day, FBS was added to the medium in an amount of 2% by weight on the 3rd day, and B27 supplement was added as a supplement on the 1st day. I did.
  • FIG. 1 shows an experimental design diagram for inducing differentiation from human embryonic stem cells into vascular network tissues according to an embodiment of the present invention, and the following experiments were performed under the conditions shown in FIG. 1.
  • the human embryonic stem cells were inoculated in DMEM F/12 (KSR20%) medium to which 12 ⁇ M CHIR99021 (Tocris) was added, and under 5 vol% CO 2 conditions. Incubated for 2 days.
  • DMEM F/12 KSR20% medium to which 12 ⁇ M CHIR99021 (Tocris) was added, and under 5 vol% CO 2 conditions. Incubated for 2 days.
  • the mRNA expression levels of OCT4, markers of undifferentiated stem cells, and EOMES, MIXL1, and BRACHY, markers of mesodermal cells were measured using the primers shown in Table 1 below, as shown in FIG.
  • the level of expression of OCT4 mRNA which is a marker of undifferentiated stem cells, decreased, and the levels of mRNA expression of EOMES, MIXL1, and BRACHY, which are markers of mesodermal cells, increased, confirming that differentiation into mesodermal cells was induced.
  • OCT4 SEQ ID NO: 1 Forward direction 5'-GGGGTTCTATTTGGGAAGGTAT-3' SEQ ID NO: 2 Reverse 5'-TGTTGTCAGCTTCCTCCACC-3' EOMES SEQ ID NO: 3 Forward direction 5'-ATCATTACGAAACAGGGCAGGC-3' SEQ ID NO: 4 Reverse 5'-CGGGGTTGGTATTTGTGTAAGG-3' MIXL1 SEQ ID NO: 5 Forward direction 5'-ACGTCTTTCAGCGCCGAACAG-3' SEQ ID NO: 6 Reverse 5'-TTGGTTCGGGCAGGCAGTTCA-3' BRACHY SEQ ID NO: 7 Forward direction 5'-GTGCTGTCCCAGGTGGCTTACAGATG-3' SEQ ID NO: 8 Reverse 5'-CCTTAACAGCTCAACTCTAACTTG-3'
  • the vascular cell aggregate is inserted into a gel in which Matrigel and collagen I are mixed in a volume of 1:1, and then 15% FBS (Gibco), 100 ng/ml VEGFA and 100 ng DMEM:F12 medium containing /ml FGF-4 was covered and cultured for 6 days, and during culture, fresh medium was replaced every 2 to 3 days.
  • FBS Gibco
  • 100 ng/ml VEGFA and 100 ng DMEM:F12 medium containing /ml FGF-4 was covered and cultured for 6 days, and during culture, fresh medium was replaced every 2 to 3 days.
  • the posterior intestinal cells obtained in Preparation Example 2 and the vascular network tissue obtained in Preparation Example 3 were added with BMP4 30 ng/ml, 12 ⁇ M CHIR99021, EGF 100 ng/ml, VEGFA 30 ng/ml and FGF-4 30 ng/ml
  • BMP4 30 ng/ml, 12 ⁇ M CHIR99021, EGF 100 ng/ml, VEGFA 30 ng/ml and FGF-4 30 ng/ml Three-dimensional co-culture for 12 days in DMEM F/12 (KSR20%) was performed to induce differentiation of intestinal organoids including vascular tissues.
  • Intestinal organoids induced differentiation as described above were added with 30 ng/ml of BMP4, 12 ⁇ M CHIR99021, 100 ng/ml of EGF, 30 ng/ml of VEGFA and 30 ng/ml of FGF-4 using Matrigel. It was matured by subculture for 5 days in DMEM F/12 (KSR20%). Intestinal organoids were formed 20 days after initiation of co-culture of posterior intestinal cells and vascular network tissue.
  • the stool sample for obtaining the strain was diluted 10 -1 to 10 -10 times with saline.
  • the samples diluted 10 -1 , 10 -2 , 10 -10 times contain 2 mM hydrogen peroxide (H 2 O 2 ), containing a strain-specific culture medium of the genus Bifidobacterium.
  • H 2 O 2 hydrogen peroxide
  • the samples diluted 10 -1 , 10 -2 , 10 -10 times contain 2 mM hydrogen peroxide (H 2 O 2 ), containing a strain-specific culture medium of the genus Bifidobacterium.
  • Example 1 Each of the eight strains selected in Example 1 was dispensed into a liquid culture medium to contain the same components as the culture medium described in Example 1, and cultured in an anaerobic condition at 37° C. for 24 hours, and then an OD 600 value was determined. It was confirmed, and the results are shown in FIG. 3.
  • the control group the Bifidobacterium ronggum reference strains were used;; (tEc Typical E.Coli) ( Bifidobacterium longum Ref) and, typically E. coli.
  • S2 accession number: KFCC11834P ( Bifidobacterium longum YMC_19_03_2)
  • S10 and S38 accession number: KFCC11835P ( Bifidobacterium longum YMC_19_03_38)
  • KFCC11835P Bifidobacterium longum YMC_19_03_38
  • Example 2 S2 (Mac 0.5, 0.5 ⁇ l) or water (control) of Example 2 was injected into the intestinal organoid prepared in Preparation Example 4 of the present invention using a Micro Ingector, and cultured for 3 days. Then, RNA was extracted from the intestinal organoids according to a conventional method, RNA sequencing was performed, and an analysis program was used to identify genes with differences in RNA expression levels compared to the control group, and gene set enrichment analysis (Gene The gene set was analyzed using set enrichment analysis; GSEA) software (ver 4.0.3), and the results are shown in FIGS. 4 to 7.
  • GSEA set enrichment analysis
  • FIGS. 4 to 7 when the S2 strain according to the present invention was administered, the expression level of the gene encoding interferon-1 was remarkably increased (FIGS. 4 and 5 ), and the B cell receptor core gene ( The expression level of B cell receptor core genes) was remarkably increased (FIGS. 6 and 7 ).
  • the novel strain according to the present invention can impart resistance to free radicals by increasing the expression level of the gene encoding interferon-1 and the B cell receptor core gene, as well as activating the immune response to the individual It can be seen that the immunity of the body can be enhanced very effectively.
  • the novel strain of the present invention Since the novel strain of the present invention has resistance under anaerobic conditions and culture conditions containing an excess of hydrogen peroxide, it has resistance under reactive oxygen conditions, that is, a defense mechanism against oxidative stress. In addition, since the novel strain of the present invention has resistance under reactive oxygen conditions, it is possible to maintain a normal microbial flora present in a target organ. Therefore, the novel strain of the present invention can be very usefully used for maintaining a normal microbial flora or enhancing immune activity in an environment in which active oxygen is present.
  • SEQ ID NO: 1 OCT4 forward primer
  • SEQ ID NO: 2 OCT4 reverse primer
  • SEQ ID NO: 3 EOMES forward primer
  • SEQ ID NO: 4 EOMES reverse primer
  • SEQ ID NO: 5 MIXL1 forward primer
  • SEQ ID NO: 6 MTXL1 reverse primer
  • SEQ ID NO: 7 BRACHY forward primer
  • SEQ ID NO: 8 BRACHY reverse primer

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Abstract

La présente invention concerne une nouvelle souche de la présente invention ayant une résistance dans des conditions anaérobies et dans des conditions de culture contenant une quantité excessive de peroxyde d'hydrogène et présentant, par conséquent, une résistance dans des conditions d'oxygène réactif, c'est-à-dire un mécanisme de défense contre le stress oxydatif. De plus, étant donné que la nouvelle souche de la présente invention a une résistance dans des conditions d'oxygène réactif, il est possible de maintenir la flore microbienne normale présente dans un organe cible. Par conséquent, la nouvelle souche de la présente invention peut être utilisée de manière extrêmement utile pour maintenir une flore microbienne normale ou améliorer l'activité immunitaire dans un environnement dans lequel de l'oxygène actif est présent.
PCT/KR2020/011192 2019-08-23 2020-08-21 Nouvelle souche symbiotique ayant une résistance dans une condition d'oxygène réactif et son utilisation WO2021040335A1 (fr)

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KR20180089324A (ko) * 2017-01-31 2018-08-08 경희대학교 산학협력단 신규 유산균 및 이의 용도
US20180280453A1 (en) * 2015-08-27 2018-10-04 Alimentary Health Limited Use of bifidobacterium longum and an exopolysaccharide produced thereby

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KR101768678B1 (ko) * 2015-02-16 2017-08-18 (주) 피엘바이오 한국 장수마을에서 개발된 건강기능성이 우수한 비피도박테리움 롱검 아종 인판티스 bi9988
US20180280453A1 (en) * 2015-08-27 2018-10-04 Alimentary Health Limited Use of bifidobacterium longum and an exopolysaccharide produced thereby
KR20180089324A (ko) * 2017-01-31 2018-08-08 경희대학교 산학협력단 신규 유산균 및 이의 용도

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PARK HO-EUN, UM HYUN-BUM, LEE WAN-KYU: "The immune enhancing effects and characteristics of Bifidobacterium longum and Bifidobacterium breve for the probiotic use in humans and animals", JOURNAL OF BIOMEDICAL TRANSLATIONAL RESEARCH, vol. 19, no. 4, 1 December 2018 (2018-12-01), pages 65 - 72, XP055786822, ISSN: 2508-1357, DOI: 10.12729/jbtr.2018.19.4.065 *

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