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WO2020091312A1 - Composition contenant des probiotiques en tant que principe actif pour prévenir ou traiter des lésions intestinales causées par la consommation d'alcool - Google Patents

Composition contenant des probiotiques en tant que principe actif pour prévenir ou traiter des lésions intestinales causées par la consommation d'alcool Download PDF

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WO2020091312A1
WO2020091312A1 PCT/KR2019/014095 KR2019014095W WO2020091312A1 WO 2020091312 A1 WO2020091312 A1 WO 2020091312A1 KR 2019014095 W KR2019014095 W KR 2019014095W WO 2020091312 A1 WO2020091312 A1 WO 2020091312A1
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Prior art keywords
lactobacillus
composition
lactic acid
ckdb001
acid bacteria
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Korean (ko)
Inventor
박나현
김우리
이인옥
김병국
최인석
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CKD Bio Corp
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CKD Bio Corp
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    • 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
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • 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]

Definitions

  • the present invention relates to a composition for preventing or treating alcoholic bowel damage comprising probiotics.
  • Alcohol is a favorite product that has been consumed for a long time to relieve stress or socialize. Small intake can help blood circulation and be beneficial to health, but chronic intake of excess can lead to liver disease such as hepatitis and cirrhosis. However, the liver is not the only organ affected by excessive alcohol intake. More than 80% of the alcohol consumed is absorbed into the body through the small intestine, so the effect of alcohol on the small intestine can never be overlooked.
  • the intestine is largely divided into a small intestine and a large intestine, and the small intestine can be further classified into a duodenum, a factory, and a ileum.
  • the effect of alcohol is different for each part of the intestine.
  • bleeding and inflammatory reactions due to damage to the intestinal mucosa are most prominent, nutrient absorption is impaired in the plant, and contraction of villi in the ileum increases, resulting in diarrhea.
  • the present inventors have made diligent research efforts to discover functional probiotic strains that can prevent or treat alcoholic bowel damage that can be applied as food and pharmaceutical products.
  • a total of 530 types of lactic acid bacteria were first selected from 17 strains that confirmed safety, stability, and industrial potential, and then 12 types of lactic acid bacteria with excellent intestinal adhesion were selected.
  • the cell survival rate due to alcohol was improved.
  • Seven strains that can be selected were selected second.
  • four strains that effectively lower the inflammatory cytokine expression increased due to alcohol were finally selected, and it was confirmed that the mixed strains of the four selected strains can effectively prevent inflammation of intestinal cells caused by alcohol than each single strain.
  • the four strains of the present invention are Lactobacillus bulgaricus CKDB001 (Accession No .: KCTC13669B), Lactobacillus helveticus CKDB001 (Accession No .: KCTC13670BP), Lactobacillus plantarum ) CKDB008 (Accession No .: KCTC13673BP), Bifidobacterium Bifidobacterium bifidum ) CKDB001 (accession number KCTC13114BP).
  • each of the selected four strains has excellent prevention or treatment effect against alcoholic intestinal damage, and in particular, these mixed strains can effectively prevent inflammation of intestinal cells caused by alcohol than each single strain.
  • the present invention has been completed by clarifying.
  • an object of the present invention is to provide a method for preventing or treating alcoholic bowel damage, enteritis, or inflammatory bowel disease, comprising the step of administering to a subject a lactic acid bacteria composition comprising each selected single strain or a mixed strain thereof. Is to do.
  • Probiotics is a microorganism that is beneficial to health when taken in an appropriate amount, and serves to protect the surface of the intestinal mucosa from various pathogens and foreign substances. Recently, various studies based on the hypothesis that probiotics will be involved in the protection of intestinal cells have been conducted as they show the effect of controlling intestinal mucosa defense and intestinal permeability in chronic inflammatory bowel disease.
  • the present inventors selected probiotics having an alcohol-protecting effect on epithelial cell lines for each organ of the small intestine, and developed a mixed strain of probiotics that can cover all damages of each small intestine.
  • the invention provides a composition comprising a lactic acid bacteria Lactobacillus bacteria (Lactobacillus) in lactic acid bacteria, Bifidobacterium (Bifidobacterium) in lactic acid bacteria, or a mixture thereof.
  • Lactobacillus bacteria Lactobacillus bacteria
  • Bifidobacterium Bifidobacterium
  • the Lactobacillus genus lactic acid bacteria are Lactobacillus bulgaricus , Lactobacillus helveticus , Lactobacillus helveticus , Lactobacillus plantarum , or mixtures thereof.
  • the Lactobacillus lactic acid bacteria are Lactobacillus bulgaricus CKDB001 (Accession No .: KCTC13669B), Lactobacillus helveticus CKDB001 (Accession No .: KCTC13670BP), Lactobacillus Lactobacillus plantarum CKDB008 (Accession No .: KCTC13673BP), or mixtures thereof.
  • the lactic acid bacteria in the Bifidobacterium genus Bifidobacterium bifidum ), Bifidobacterium lactis , or mixtures thereof are included in the Bifidobacterium genus Bifidobacterium bifidum ), Bifidobacterium lactis , or mixtures thereof.
  • the lactic acid bacteria of the genus Bifidobacterium is Bifidobacterium bifidum CKDB001 (Accession No. KCTC13114BP).
  • the lactic acid bacteria are Lactobacillus bulgaricus CKDB001 (Accession No .: KCTC13669BP), Lactobacillus helveticus CKDB001 (Accession No .: KCTC13670BP), Lactobacillus plantar ( Lactobacillus plantarum ) CKDB008 (Accession No .: KCTC13673BP), Bifidobacterium Bifidobacterium bifidum ) CKDB001 (Accession No. KCTC13114BP), or a mixed strain thereof.
  • Lactobacillus bulgaricus CKDB001 strain of the present invention was deposited on October 23, 2018 at the Korea Institute of Biotechnology and Microbiology Resource Center (Korean Collection for Type Culture, KCTC), and was assigned a deposit number KCTC 13669BP.
  • Lactobacillus helveticus CKDB001 strain of the present invention was deposited on October 23, 2018 at the Korea Institute of Biotechnology and Microbiology Resource Center (Korean Collection for Type Culture, KCTC), and was assigned a deposit number KCTC 13670BP.
  • Lactobacillus plantarum CKDB008 strain of the present invention was deposited on October 23, 2018 at the Korea Institute of Biotechnology and Microbiology Resource Center (Korean Collection for Type Culture, KCTC), and was assigned a deposit number KCTC 13673BP.
  • Bifidobacterium bifidum CKDB001 strain of the present invention was deposited on September 23, 2016 at the Korea Institute of Biotechnology and Microbiology Resource Center (Korean Collection for Type Culture, KCTC), and was assigned a deposit number KCTC13114BP.
  • the four strains were confirmed to be Gram positive, hemolytic, hemolytic, sporulation ability, and Catalase negative.
  • the sugar availability of the strain was analyzed using the API 50 CHL kit (Table 1), and gene identification was performed by analyzing the 16s rDNA sequence for strain identification (Table 2).
  • the lactic acid bacteria which are active ingredients of the composition of the present invention, are Lactobacillus rhamnosus ( Lactobacillus rhamnosus) , which are commercially available strains that have the ability to adhere to small intestine (colon) and colon epithelial cells. GG strain, LGG), so it has very good gut adhesion regardless of the intestine.
  • the lactic acid bacteria show a very high cell viability, similar to normal cells, as well as the commercial strain LGG strain despite ethanol treatment of small intestine (duodenum and ileum) epithelial cells.
  • the lactic acid bacteria significantly reduce the expression of inflammatory cytokines (IL-1 ⁇ and TNF ⁇ ) compared to the commercial strain LGG despite ethanol treatment of small intestine (duodenum and ileum) epithelial cells. Therefore, the lactic acid bacteria of the present invention shows a very good anti-inflammatory effect on the small intestine.
  • IL-1 ⁇ and TNF ⁇ inflammatory cytokines
  • the four types of lactic acid bacteria deposited in the final selection and depository of the present invention have excellent intestinal adhesion ability, cell viability, and anti-inflammatory effect of each strain, but when used as a mixed strain mixed with them, the anti-inflammatory effect of each single strain It has characteristics in that it has a better anti-inflammatory effect (synergy effect). Therefore, the lactic acid bacteria of the present invention is more useful when used as a mixed strain.
  • the strain, or a culture thereof, which is an active ingredient of the compositions of the present invention is a culture medium containing cells in addition to the cells isolated and / or purified from the strain, an extract of cells, a culture supernatant, a concentrate thereof, a concentrate, It is a dried product, and if necessary, a diluent, a diluent, and the like, and includes all of the state obtained by treating the culture medium and the culture.
  • the culture method, extraction method, separation method, concentration method, drying method, dilution method, etc. of the above-mentioned cell body are not particularly limited.
  • the medium for culturing the cells generally includes milk protein such as skim milk, whey, and casein, sugars, yeast extracts, etc., and various aerobic or anaerobic methods common to the culture method can be suitably used.
  • the culture temperature for example, 35 to 45 ° C is set, and during the culture, an alkali such as sodium hydroxide is used to use a neutralization culture method that maintains the pH of the medium from neutral to acidic, for example, about 5 to 6 pH. It might be.
  • a neutralization culture method such as a batch culture method can be used. After cultivation, the culture or the supernatant may be concentrated, dried, or diluted, if necessary.
  • the supernatant and the cells of the culture may be separated using a centrifugation method or a membrane separation method, and the cells may be recovered in a concentrated state.
  • the cells may be subjected to ultrasonic treatment or enzyme treatment to extract components in the cells, or the culture or supernatant, the cells or the extract may be dried. These can be used as an active ingredient of the composition of the present invention.
  • lactic acid bacteria which are active ingredients of the composition of the present invention, exhibit excellent cell viability and anti-inflammatory effects against the above-mentioned intestinal epithelial cell toxicity of alcohol. Therefore, the composition of the present invention has the purpose of preventing, improving or treating alcoholic bowel damage.
  • the lactic acid bacteria of the present invention exhibit the advantageous effects described above for various parts of the small intestine (duodenum, ileum) and large intestine.
  • the “intestine” of the intestinal injury refers to the small intestine and / or large intestine, and more specifically the “small intestine” refers to the duodenum, plant, and / or ileum. Most specifically, the small intestine refers to the duodenum and / or ileum.
  • the lactic acid bacteria of the present invention significantly reduce the expression of inflammatory cytokines in the intestine. Therefore, the composition of the present invention has the purpose of preventing, improving or treating enteritis or inflammatory bowel disease.
  • the enteritis includes bacterial enteritis, viral enteritis, and alcoholic enteritis.
  • the inflammatory bowel disease in the present invention is selected from the group consisting of Crohn's disease, Behcet's disease, and ulcerative colitis.
  • the lactic acid bacteria composition of the present invention may be prepared as a food composition having the above-described use, or a pharmaceutical composition.
  • the composition of the present invention when the composition of the present invention is prepared as a food composition, as an active ingredient, as well as the lactic acid bacteria, it may include a component that is conventionally added in food production.
  • the additive components include, for example, proteins, carbohydrates, fats, nutrients, seasonings and flavoring agents.
  • the carbohydrate include monosaccharides (eg, glucose, fructose, etc.), disaccharides (eg, maltose, sucrose, oligosaccharides, etc.) and polysaccharides (eg, dextrins, cyclodextrins, etc.).
  • Sugars and sugar alcohols such as xylitol, sorbitol, erythritol, etc.
  • natural flavoring agents taumatin, stevia extracts (e.g. rebaudioside A, glycyrrhizine, etc.)
  • synthetic flavoring agents sacharin, aspar Tom
  • citric acid liquid fructose
  • sugar glucose
  • acetic acid malic acid
  • fruit juice jujube extract or licorice extract
  • licorice extract may be additionally included in addition to the above-mentioned strain as an active ingredient of the present invention. have.
  • the food composition of the present invention includes processing forms of all natural materials such as food, functional food, nutritional supplement, health food and food additives.
  • Food compositions of this type can be prepared in various forms according to conventional methods known in the art.
  • the lactic acid bacteria themselves may be prepared in the form of tea, juice, and drink to be consumed or granulated, encapsulated, and powdered.
  • food products include beverages (including alcoholic beverages), fruits and processed foods thereof (eg, canned fruits, canned foods, jams, marmalades, etc.), fish, meat, and processed foods (eg ham, sausage corn beef, etc.) ), Breads and noodles (e.g. udon, buckwheat noodles, ramen, spaghetti, macaroni, etc.), juice, various drinks, cookies, syrup, dairy products (e.g.
  • yogurt, fermented milk, butter, cheese, etc. edible vegetable oil, margarine, Vegetable protein, retort food, frozen food, various seasonings (eg, miso, soy sauce, sauce, etc.) can be prepared by adding the lactic acid bacteria of the present invention.
  • lactic acid bacteria of the present invention in the form of food additives, it may be prepared and used in the form of a powder or a concentrate.
  • the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier included in the pharmaceutical composition of the present invention is commonly used in formulation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, but is not limited thereto It does not work.
  • the pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components.
  • a lubricant e.g., a talc, a kaolin, a kaolin, a kaolin, a kaolin, a kaolin, kaolin, kaolin, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, a talct, a talct, a talct, a stevia, glycerin, a stevia, glycerin, glycerin, g
  • the pharmaceutical composition of the present invention can be administered orally or parenterally, and is preferably applied by oral administration.
  • the pharmaceutical composition of the present invention may be formulated in various oral or parenteral dosage forms, but is not limited thereto.
  • Suitable dosages of the pharmaceutical compositions of the invention vary by factors such as formulation method, mode of administration, patient's age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion, and response sensitivity, Usually, a skilled doctor can easily determine and prescribe a dose (pharmaceutical effective amount) effective for the desired treatment or prevention.
  • the daily dosage of the pharmaceutical composition of the invention is 0.0001-100 mg / kg.
  • pharmaceutical effective amount as used herein means an amount sufficient to prevent or treat the aforementioned diseases.
  • treatment refers to reduction, suppression, sedation, or eradication of a disease state
  • prevention means to suppress the exacerbation of the disease state so that the disease state does not develop and includes the treatment.
  • Formulations for oral administration include, for example, tablets, pills, hard / soft capsules, liquids, suspensions, emulsifiers, and syrups. There are granules, elixirs, and the like, and these formulations may use one or more diluents or excipients, such as fillers, extenders, wetting agents, disintegrating agents, lubricants, binders, and surfactants, which are commonly used in addition to the active ingredients.
  • a disintegrating agent agar, starch, alginic acid or its sodium salt, calcium phosphate monohydrogen anhydride, etc.
  • magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidine, low-substituted hydroxypropylcellulose, and the like can be used.
  • lactose, dextrose, sucrose, mannitol, sorbitol, and cellulose. Glycine or the like can be used as a diluent, and in some cases, commonly known boiling mixtures, absorbents, colorants, flavoring agents, sweeteners and the like can be used together.
  • composition may be sterilized or contain preservatives, stabilizers, hydration or emulsification accelerators, salts for osmotic pressure control, adjuvants such as buffers, and other therapeutically useful substances, conventional methods of mixing, granulating or coating methods It can be formulated according to.
  • the pharmaceutical composition of the present invention is prepared in a unit dose form by formulating using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily carried out by those skilled in the art to which the present invention pertains. Or it can be manufactured by incorporating into a multi-dose container.
  • the formulation may be in the form of a solution, suspension, syrup or emulsion in an oil or aqueous medium, or may be in the form of ex-agent, powder, granule, tablet or capsule, and may further include a dispersant or stabilizer.
  • the present invention comprises the step of administering to the subject a composition comprising a lactic acid bacteria of the genus Lactobacillus (Lactobacillus), lactic acid bacteria of the genus Bifidobacterium, or mixtures thereof It provides a method of preventing or treating intestinal damage.
  • a composition comprising a lactic acid bacteria of the genus Lactobacillus (Lactobacillus), lactic acid bacteria of the genus Bifidobacterium, or mixtures thereof It provides a method of preventing or treating intestinal damage.
  • Alcoholic intestinal damage which is the target disease of the treatment or prevention method of the present invention, is as defined in relation to the disease to be treated of the pharmaceutical composition.
  • the subject is a mammal or human.
  • the present invention comprises the step of administering to the subject a composition comprising a lactobacillus (Lactobacillus) genus lactic acid bacteria, Bifidobacterium genus lactic acid bacteria, or mixtures thereof, Provides a method for preventing or treating enteritis or inflammatory bowel disease.
  • a lactobacillus Lactobacillus
  • Bifidobacterium genus lactic acid bacteria Bifidobacterium genus lactic acid bacteria
  • Enteritis or inflammatory bowel disease which is the target disease of the treatment or prevention method of the present invention, is as defined in relation to the target disease of the pharmaceutical composition.
  • the method for preventing or treating alcoholic intestinal damage, enteritis, or inflammatory bowel disease of the present invention includes a composition comprising the aforementioned Lactobacillus lactic acid bacteria, Bifidobacterium lactic acid bacteria, or mixtures thereof Since it is a method using the same active ingredient, the description thereof is omitted to avoid excessive complexity of the present specification for overlapping contents.
  • the present invention provides a composition for preventing or treating alcoholic bowel damage comprising probiotics as an active ingredient.
  • the compositions of the present invention relates to novel separation Lactobacillus own, Lactobacillus Bulgaria kusu (Lactobacillus bulgaricus) CKDB001 (accession number: KCTC13669B), Lactobacillus helveticus (Lactobacillus helveticus) CKDB001 (accession number: KCTC13670BP), Lactobacillus Planta column (Lactobacillus plantarum ) CKDB008 (Accession No .: KCTC13673BP), Bifidobacterium Bifidobacterium bifidum ) CKDB001 (Accession No. KCTC13114BP), or a mixed strain thereof.
  • the present invention Since the present invention has an excellent effect of reducing inflammation of intestinal cells due to alcohol stimulation, it can be usefully used as a food or therapeutic agent for preventing and improving alcoholic bowel damage.
  • FIGS. 1A and 1B are diagrams showing the intestinal epithelial cell adhesion ability of the probiotic strain of the present invention.
  • Figure 2a is a diagram showing the protective effect of alcoholic cell damage to the duodenal cell line (HUTU-80) of the probiotic strain of the present invention.
  • Figure 2b is a diagram showing the protective effect of alcoholic cell damage to the ileum cell line (IEC-18) of the probiotic strain of the present invention.
  • Figure 3a is a diagram showing the inhibitory effect of mRNA expression of the inflammatory cytokine IL-1 ⁇ in the duodenal cell line (HUTU-80) of the probiotic strain of the present invention.
  • Figure 3b is a diagram showing the inhibitory effect of mRNA expression of the inflammatory cytokine TNF ⁇ in the duodenal cell line (HUTU-80) of the probiotic strain of the present invention.
  • Figure 4a is a diagram showing the inhibitory effect of mRNA expression of the inflammatory cytokine IL-1 ⁇ in the ileal cell line (IEC-18) of the probiotic strain of the present invention.
  • Figure 4b is a diagram showing the inhibitory effect of mRNA expression of the inflammatory cytokine TNF ⁇ in the ileal cell line (IEC-18) of the probiotic strain of the present invention.
  • Figure 5a is a diagram showing the inhibitory effect of inflammatory cytokine expression in the ileal cell line (IEC-18) of the mixed strain of the present invention.
  • Figure 5b is a diagram showing the inhibitory effect of inflammatory cytokine expression in the duodenal cell line (HUTU-80) of the mixed strain of the present invention.
  • % used to indicate the concentration of a specific substance, unless otherwise specified, solids / solids (weight / weight)%, solids / liquids (weight / volume)%, and The liquid / liquid is (volume / volume)%.
  • Probiotics used in this experiment was a strain isolated from Chong Kun Dang Bio (CKDBiO, Korea) and was isolated from feces, crude milk and other fermented foods. The collected sample was suspended in sterile anaerobic water, and after dilution of the decimal, it was spread on MRS (ManRogosaSharpe) solid medium and BL (Blood-liver) solid medium. After incubation in an anaerobic chamber maintained at 5% CO 2 , 10% H 2 , and 85% N 2 atmospheric composition for 48 hours at 37 ° C., colonies having different forms were selected and purified. As a result of the experiment, a total of 530 lactic acid bacteria were isolated. Subsequently, the 530 strains of the isolated lactic acid bacteria were tested for hemolysis, whether gelatinase was produced, whether urease was produced, and whether bio-amine was produced. Was selected.
  • an MRS or BL liquid medium adjusted to pH 2.5 using hydrochloric acid was prepared. After inoculating each of the strains with 1% in the medium, after diluting with 10% of anaerobic dilution (pH 6.2) in order to measure the initial number of bacteria, 1 ml was dispensed and plated using the same solid medium, followed by incubation at 37 ° C for 48 hours. . MRS liquid medium and BL liquid medium having a pH of 2.5 inoculated with the strain were cultured in a 37 ° C incubator for 2 hours, and then cultured in the same solid medium in the same manner as above. After colonization, colonies were counted to measure resistance to pH.
  • the culture medium was cultured by setting the optimized growth medium and culture conditions for each strain, and then the cell recovery and freeze-drying process was performed in a conventional method (60 ° C freezer after centrifugation). After freezing, lyophilization was performed under operating conditions between 0 ° C and 45 ° C). After recovering the cells, 5-50% (volume / weight)% of maltodextrin or 5-50% (volume / weight) of trehalose or 5-50% (volume / weight) of cellulose is added to the concentrate compared to the concentrate, and then lyophilized and pulverized to prepare lactic acid bacteria raw material. Did. As a result, a total of 17 strains (Table 3) with the highest probability of industrialization due to the long-term storage stability and the highest original survival rate by species were selected. In the experiment, the powdered strain was suspended in an animal cell culture medium and used.
  • HUTU-80 duodenal epithelial cell line, Human
  • IEC-18 colon epithelial cell line, Rat
  • HT-29 colon epithelial cell line, Human
  • DMEM Dulbeco's Modified Eagle's Media
  • HT-29 used Rowell Park Memorial Institue (RPMI) 1640 medium
  • Penicillin / Streptomycin (P / S) for each medium.
  • Incubation was performed at 37 ° C and 5% CO 2 using a culture solution to which 1% and Fetal Bovine Serum (FBS) 10% were added.
  • FBS Fetal Bovine Serum
  • the present inventors measured the adhesion capacity to the intestinal epithelial cells (IEC-18) and the colon epithelial cells (HT-29) in order to confirm the intestinal adhesion ability of the probiotic strains of the present invention.
  • IEC-18 was dispensed into a 12-well cell culture dish at a concentration of 1 x 10 5 cells / mL and cultured in a cell incubator (37 ° C for 5 days, 5% CO 2 ) until a single layer.
  • the probiotic strain was diluted with 1 ⁇ 10 9 CFU / mL in DMEM without antibiotics.
  • IEC-18 achieves confluency, 1 mL of each fungus solution is dispensed into the cells and incubated in a cell incubator for 90 minutes. Thereafter, the cells were washed twice with PBS and treated with Trypsin-EDTA to remove the cells.
  • the number of viable cells is measured after incubation at 37 ° C. for 18 hours.
  • a commercial strain Lactobacillus rhamnosus GG was used, and the intestinal adhesion rate was calculated as shown in Equation 2.
  • Intestinal adhesion rate (viable cell count / initial viable cell count after 90 min incubation) x 100
  • B. bifidum is the strain that has superior intestinal adhesion ability than IEC L. GG (0.02%) in IEC-18.
  • CKDB001 0.18%
  • L. plantarum CKDB008 0.11%
  • S. thermophilus CKDB021 0.08%
  • L. salivarius CKDB001 0.05%)
  • L. bulgaricus CKDB001 0.04%
  • L. helveticus CKDB001 0.02%
  • L. fermentum Seven types of CKDB004 were identified (FIG. 1A).
  • HT-29 was dispensed in a 12 well cell culture dish at a concentration of 1 x 10 5 cells / mL and cultured in a cell culture medium (37 ° C., 5% CO 2 for about 5 days) until a monolayer.
  • Each probiotic strain was diluted with 1 ⁇ 10 9 CFU / mL in RPMI 1640 without antibiotics.
  • HT-29 formed confluency 1 mL of each fungus solution was dispensed into cells and cultured in a cell incubator for 90 minutes. Thereafter, the cells were washed twice with PBS and treated with Trypsin-EDTA to remove the cells.
  • the detached cells were serially diluted and inoculated onto an MRS agar plate, and the number of viable cells was measured after incubation at 37 ° C. for 18 hours.
  • a commercial strain Lactobacillus rhamnosus GG, was used, and the intestinal adhesion rate was calculated as shown in Equation 2 in the same manner as in 2.1 above.
  • Intestinal adhesion rate (viable cell count / initial viable cell count after 90 min incubation) x 100
  • the strain with superior intestinal adhesion ability than LGG was L. helveticus CKDB001 (8.17%), L. reuteri CKDB016 (4.88%), L. plantarum CKDB008 (4.41%), L. acidophilus CKDB007 (3.43%), L. bulgaricus CKDB001 (3.39%), L. casei 9 types of CKDB007 (3.32%), B. lactis CKD005 (3.22%), B. longum CKD004 (3.11%), and L. fermentum CKD004 (2.37%) were identified (FIG. 1B).
  • the present invention Probiotic Protective effect of alcoholic intestinal cell damage-Cell viability measurement ( MTT assay)
  • MTT assay was performed to evaluate the toxicity of alcohol to intestinal epithelial cells and the prophylactic effect of probiotics.
  • the MTT assay was conducted with 12 strains that were superior to LGG in the previous intestinal adhesion test.
  • HUTU-80 and IEC-18 were dispensed in a 96-well cell culture dish at a concentration of 1 ⁇ 10 5 cell / mL and cultured in a cell culture medium (37 ° C, 5% CO 2 ) until a single layer.
  • the probiotic strain was diluted with 1 ⁇ 10 6 CFU / mL in cell medium MEM without antibiotics.
  • 200 ⁇ L of each fungus was dispensed into the cells and cultured in a cell incubator for 90 minutes. Thereafter, the cells were washed twice with PBS, and cell culture medium containing ethanol was dispensed and reacted for 90 minutes.
  • DMSO Dimethyl sulfoxide
  • the cell viability was reduced to 33% compared to the normal group in the control group with only ethanol.
  • the comparative bacteria LGG was pretreated, the cell viability increased to 82%.
  • the cell viability was reduced to 59% compared to the normal group in the control group with only ethanol.
  • the comparative bacteria LGG was pretreated, the cell viability increased to 77%.
  • 5 cells showed higher cell viability than LGG.
  • B. bifidum CKDB001 , L. bulgaricus CKDB001 , L. fermentum CKDB004 , L. helveticus CKDB001 , and L. acidophilus CKDB007 showed a cell survival rate of 80% or more and showed a significant difference from the control group (FIG. 2B).
  • the present invention Probiotic Inhibitory effect of inflammatory cytokines in the intestine
  • RT-PCR was performed to confirm that the probiotic strain of the present invention inhibits mRNA expression of inflammatory cytokines induced by alcohol. Confirmation of the inflammatory cytokine mRNA shedding amount was conducted with seven strains that were superior to LGG in the previous intestinal adhesion test and MTT assay.
  • duodenal epithelial cell line HUTU-80
  • ileal epithelial cell line IEC-18
  • a 6-well cell culture dish at a concentration of 1 ⁇ 10 5 cells / mL, and then incubate until a single layer (37 °C, 5% CO 2).
  • Probiotics were diluted in 1MEM 10 CFU / mL in DMEM without antibiotics.
  • each fungus solution is dispensed into cells by 3 mL and cultured in a cell incubator for 90 minutes. Thereafter, the cells were washed twice with PBS, and cell culture medium containing ethanol was dispensed and reacted for 90 minutes.
  • the genomic base sequence used for PCR analysis is described in Table 4.
  • the expression level of IL-1 ⁇ mRNA was increased by 385% and the expression of TNF ⁇ by 357% compared to normal in the ethanol-only control group.
  • IL-1 ⁇ decreased to 235% and TNF ⁇ to 257%.
  • B. bifidum is the strain that has lower IL-1 ⁇ expression than LGG It was reduced to 155% with one CKDB001.
  • B. bifidum was the strain that lowered TNF ⁇ expression level than LGG.
  • Two types of CKDB008 were reduced to 130% and 142%, respectively. Therefore, two of the seven candidate strains were found to be able to reduce the inflammatory response of duodenal cells caused by alcohol more effectively than the comparative group LGG (FIGS. 3A and 3B).
  • IL-1 ⁇ mRNA expression was increased by 320% and TNF ⁇ expression was increased by 289% compared to normal.
  • IL-1 ⁇ decreased to 201% and TNF ⁇ to 223%.
  • L. bulgaricus is a strain that has lower IL-1 ⁇ expression than LGG. CKDB001 , L. helveticus CKDB001 , L. plantarum It was reduced to 145%, 166%, and 182%, respectively, with three types of CKDB008.
  • Strains with lower TNF ⁇ expression than LGG were L. helveticus It was reduced to 191% with one CKDB001. Therefore, three of the seven candidate strains were found to be able to reduce the inflammatory response of ileal epithelial cells due to alcohol more effectively than the comparative group LGG (FIGS. 4A and 4B).
  • mixed strain of the present invention (4 excellent strains for inhibiting the expression level of inflammatory cytokine mRNA: B. bifidum CKDB001, L. helveticus CKDB001, L. plantarum CKDB008, L. bulgaricus CKDB001) was tested to inhibit the expression of inflammatory cytokines in the intestine.
  • the experimental method was carried out in the same manner as in the above 4, except that the mixed strains of the above 4 were set as the experimental group.
  • the expression level of IL-1 ⁇ mRNA increased to 481% compared to normal, and decreased to 220% when pretreated with the comparative bacteria LGG.
  • L. helveticus CKDB001, L. plantarum CKDB008 and L. bulgaricus CKDB001 lowered IL-1 ⁇ mRNA expression levels to 110%, 202%, and 132%, respectively, and were found to be more effective than LGG.
  • IL-4 ⁇ mRNA expression was 103 when pretreated by mixing four bacteria. It was significantly lowered to%, showing a similar degree to Normal.
  • TNF ⁇ the expression level increased to 343% compared to normal when treated with 5% ethanol, and decreased to 150% when pretreated with the comparative bacteria LGG.
  • L. helveticus It was found that CKDB001 lowered the TNF ⁇ mRNA expression level to 104% and was more effective than LGG. When 4 bacteria were mixed and pretreated, it was lowered to 105%, and the effect of suppressing TNF ⁇ mRNA expression was found to be very excellent (FIG. 5A).
  • the expression level of IL-1 ⁇ mRNA increased to 427% compared to normal, and decreased to 217% when pretreated with the comparative group LGG.
  • B. bifidum CKDB001, L. plantarum CKDB008 lowered IL-1 ⁇ mRNA expression to 168% and 212%, respectively, and was found to be more effective than LGG.
  • IL-1 ⁇ mRNA expression was significantly lowered to 150%, synergistic. Appeared.
  • TNF ⁇ TNF ⁇
  • expression was increased to 411% compared to normal when treated with 2.5% ethanol, and decreased to 266% when pretreated with the comparative group LGG.
  • B. bifidum CKDB001, L. plantarum CKDB008 lowered the mRNA expression levels to 143% and 150%, respectively, and was found to be more effective than LGG, and when 4 bacteria were mixed and pretreated, the TNF ⁇ mRNA expression level was significantly lower to 138%, which was the most effective (FIG. 5B). .
  • the present inventors confirmed from the above results that the mixed strain of the present invention is more effective in suppressing inflammatory cytokine expression compared to a single strain in both ileum cell line and duodenal cell line.
  • each intestine is subdivided to verify the efficacy of the strain for each site.
  • 17 strains were selected from 530 types of lactic acid bacteria, which confirmed safety, stability, and industrial potential. After that, 12 species with excellent intestinal adhesion were selected, and among them, 7 strains capable of improving the cell viability due to alcohol were selected second.

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Abstract

La présente invention concerne une composition pour prévenir ou traiter des lésions intestinales causées par la consommation d'alcool, ladite composition contenant des probiotiques en tant que principe actif. La composition de la présente invention contient Lactobacillus bulgaricus CKDB001 (Numéro d'entrée: KCTC13669B), Lactobacillus helveticus CKDB001 (Numéro d'entrée: KCTC13670BP), Lactobacillus plantarum CKDB008 (Numéro d'entrée: KCTC13673BP), ou Bifidobacterium bifidum CKDB001 (Numéro d'entrée: KCTC13114BP), qui sont de nouvelles souches d'acide lactique isolées, ou une souche mélangée de celles-ci, ont un excellent effet de réduction de l'inflammation des cellules intestinales provoquée par une irritation causée par la consommation d'alcool, et peuvent ainsi avoir une utilisation utile en tant qu'aliment ou agent thérapeutique pour prévenir ou soulager une lésion intestinale causée par la consommation d'alcool.
PCT/KR2019/014095 2018-10-30 2019-10-24 Composition contenant des probiotiques en tant que principe actif pour prévenir ou traiter des lésions intestinales causées par la consommation d'alcool Ceased WO2020091312A1 (fr)

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KR102626672B1 (ko) * 2022-04-29 2024-01-18 한림대학교 산학협력단 락토바실러스 헬베티커스 ckdb001 균주를 포함하는 알코올성 간질환의 예방, 개선, 또는 치료용 조성물

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