WO2023163318A1 - Fermentation product containing short-chain fatty acid and use thereof for alleviating, preventing, and treating obesity - Google Patents

Fermentation product containing short-chain fatty acid and use thereof for alleviating, preventing, and treating obesity Download PDF

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Publication number
WO2023163318A1
WO2023163318A1 PCT/KR2022/017492 KR2022017492W WO2023163318A1 WO 2023163318 A1 WO2023163318 A1 WO 2023163318A1 KR 2022017492 W KR2022017492 W KR 2022017492W WO 2023163318 A1 WO2023163318 A1 WO 2023163318A1
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weight
fermentation product
medium composition
composition
medium
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PCT/KR2022/017492
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French (fr)
Korean (ko)
Inventor
김유미
장기현
석창환
신지원
곽윤금상
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(주) 바이노텍
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Priority claimed from KR1020220148277A external-priority patent/KR102660139B1/en
Publication of WO2023163318A1 publication Critical patent/WO2023163318A1/en

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    • 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
    • A23L33/115—Fatty acids or derivatives thereof; Fats or oils
    • A23L33/12—Fatty acids or derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • 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
    • A61P3/04—Anorexiants; Antiobesity agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/52—Propionic acid; Butyric acids

Definitions

  • the present invention relates to the use of a fermentation product containing short-chain fatty acids for the improvement, prevention or treatment of overweight.
  • Garcinia cambogia In relation to weight loss, Garcinia cambogia, green tea extract, conjugated linolenic acid, and the like are used as body fat reduction supplements or functional food materials. Garcinia cambogia accounts for about 47% of the body fat reduction market by suppressing fat absorption, but side effects such as liver toxicity are appearing.
  • microorganisms live in symbiosis with the human body, performing various functions related to metabolic function control, immune system development, interaction with the nervous system, and maintenance of homeostasis of the human body, such as human digestive function or securing energy.
  • microorganisms living in the intestine can have a great impact on human health, including not only intestinal diseases, but also metabolic diseases such as obesity and diabetes, mental diseases such as depression and ADHD, and aging.
  • Short-chain fatty acids refer to organic acids having 6 carbon atoms or less, such as acetate, propionate, and butyrate. These short-chain fatty acids can have a lot of effects on our body. For example, cells constituting the mucous membrane of the large intestine maintain the wall of the large intestine using short-chain fatty acids as an energy source and can prevent bacteria from entering the body.
  • butyrate is absorbed into the colonic mucosa and plays an important role in proliferating mucosal epithelial cells of all digestive tracts and organs.
  • Butyrate activates the FFAR3 (Free Fatty Acid Receptor 3) cell membrane receptor in fat cells (PPAR- ⁇ correction or not), which prevents the accumulation of fat and uses energy for muscle and metabolism, which is very effective for weight loss and diet. Results have been disclosed.
  • Hua. V. In an animal model of obese mice by Lin et al, it was reported that when a high-fat diet containing butyrate was administered for 4 weeks, a significant reduction in body fat was reported (Hua V. Lin et al., PLoS One. 2012 ;7(4): e35240).
  • butyrate shows many clinical effects in reducing body fat, it has a very strong specific odor and is difficult to commercialize.
  • butyrate-producing microorganisms in the intestine produce butyrate in the process of decomposing resistant starch such as inulin, which is a water-soluble dietary fiber.
  • the present invention is intended to provide a fermentation product with a high content of short-chain fatty acids.
  • the present invention also aims to improve, prevent or treat overweight by using the fermentation product.
  • the present invention also seeks to provide a method for preparing the fermentation product.
  • the term "about” means within 10%, preferably within 5%, more preferably within 1% of a given value or range.
  • the present invention provides a fermentation product comprising a high concentration of short chain fatty acids.
  • the fermentation product is produced by inoculating a Clostridium strain into a medium composition and then fermenting it, in which case it contains a high concentration of short-chain fatty acids.
  • the present invention is a fermentation product comprising short-chain fatty acids
  • the fermentation product is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • yeast extract as a nitrogen source; and at least one of roasted soy flour or soy peptone;
  • the fermentation product comprises at least about 10 mM short-chain fatty acids.
  • a fermentation product comprising short chain fatty acids is provided.
  • the term "fermentation product” refers to a product of enzymatic or metabolic decomposition using microorganisms, and specifically, in the present invention, Clostridium (Clostridium ) may refer to a product cultured after inoculation of a strain.
  • “fermentation product” herein may be used interchangeably with “fermentation product” and the like.
  • the term “medium” or “medium composition” refers to a material in which nutrients necessary for culturing the microorganism or strain are mixed as main components, including water essential for survival and growth, as well as nutrients and growth supplies, etc.
  • the medium composition of the present application includes a composition and content optimal for producing short-chain fatty acids, particularly butyrate in high concentration, as a nitrogen source; and at least one of roasted soy flour or soy peptone; and at least one selected from the group consisting of inulin, chicory powder, maltodextrin and glucose as a carbon source as an essential component.
  • growth factors such as inorganic salts, amino acids and/or vitamins may be additionally included.
  • the medium composition of the present invention can be cultured while controlling temperature, pH, etc. under anaerobic conditions in a conventional medium.
  • Yeast extract, roasted soy flour, soy peptone, etc. included in the medium composition function as a nitrogen source, and the composition of the nitrogen source may be very important in the growth of microorganisms.
  • the medium composition includes the yeast extract and additionally includes at least one of roasted soybean flour or soy peptone, the amount of butyrate produced can be increased, and thus the improvement, prevention, or treatment effect of butyrate on overweight can be improved.
  • the nitrogen source may essentially include yeast extract and roasted soybean flour, or may essentially include yeast extract and soy peptone.
  • the yeast extract may be included in about 0.1 to 10% by weight, preferably about 0.5 to 5% by weight, more preferably about 0.5 to 1% by weight, such as about 0.5 or 1% by weight, based on the total weight of the medium composition. may be %.
  • Yeast extracts commercially available may be appropriately used, for example, those containing protein or amino acid components as a nitrogen source or may be purchased as Powdered Yeast Extract-II from Choheung Co., Ltd.
  • Roasted soybean flour may be included in about 0.1 to 10% by weight, preferably about 0.5 to 7% by weight, more preferably about 2 to 6% by weight, more preferably about 0.1 to 10% by weight based on the total weight of the medium composition. 1 to 5 weight percent, such as about 1, 3, or 5 weight percent.
  • Roasted soybean flour can be suitably used commercially available, for example, commercially available ones containing protein or amino acid components as a nitrogen source can be appropriately purchased and used (for example, available from Ilho Foods).
  • Soy peptone may be included in about 0.1 to 10% by weight, preferably about 0.5 to 7% by weight, more preferably about 2 to 6% by weight, more preferably about 0.1 to 10% by weight based on the total weight of the medium composition. 1 to 5% by weight, such as about 5% by weight. Soy peptone can be suitably used commercially available, and can be purchased from SOLABIA, for example.
  • the medium composition may further include skim milk powder as a nitrogen source.
  • Skim milk powder may be included in about 0.1 to 10% by weight, preferably about 0.5 to 7% by weight, more preferably about 2 to 6% by weight, more preferably about 0.1 to 10% by weight based on the total weight of the medium composition. 1 to 5% by weight, such as about 5% by weight. Skim milk powder commercially available can be suitably used, and can be purchased from Seoul Milk, for example.
  • the medium composition includes a carbon source as an essential component in addition to a nitrogen source, and the carbon source is at least one selected from the group consisting of inulin, chicory powder, maltodextrin, and glucose.
  • the carbon source may be included in about 1 to 40% by weight, preferably about 5 to 20% by weight, such as about 5, 10, and 13% by weight, based on the weight of the total medium composition.
  • the carbon source comprises alone one of inulin, chicory powder, maltodextrin and glucose, or consists essentially of inulin and chicory powder, or consists essentially of inulin, chicory powder and maltodextrin. can be included as
  • Each carbon source can affect the amount of butyrate produced.
  • inulin may be included in about 1 to 10% by weight, and within this range, the amount of butyrate produced may further increase. More preferably, it may be about 3 to 7% by weight, such as about 5% by weight.
  • Inulin commercially available commercially available products may be suitably used, and may be obtained from, for example, BIOGLAN.
  • Chicory powder may be included in about 1 to 10% by weight, preferably about 3 to 7% by weight, such as about 5% by weight, based on the weight of the total medium composition.
  • Chicory powder can be suitably used commercially available commercially, for example, it can be purchased from Joeun Herbs.
  • Maltodextrin may be included in about 1 to 10% by weight, preferably about 3 to 7% by weight, such as about 3 or 5% by weight, based on the weight of the total medium composition.
  • the maltodextrin may be, for example, indigestible maltodextrin.
  • Commercially available maltodextrins may be appropriately used, and may be purchased from Samyang Corporation, for example.
  • Glucose may be included in about 1 to 10% by weight, preferably about 3 to 7% by weight, such as about 5% by weight, based on the weight of the total medium composition. Glucose commercially available may be appropriately used, and may be purchased from Samyang Corporation, for example.
  • the medium composition consists of guar gum, sodium alginate, pectin and oligosaccharides (e.g., galactooligosaccharide, fructooligosaccharide, isomaltooligosaccharide, xylooligosaccharide, soybean oligosaccharide, lactulose, chitin oligosaccharide, isomaltooligosaccharide, etc.) as a carbon source. It may further include one or more selected from the group.
  • oligosaccharides e.g., galactooligosaccharide, fructooligosaccharide, isomaltooligosaccharide, xylooligosaccharide, soybean oligosaccharide, lactulose, chitin oligosaccharide, isomaltooligosaccharide, etc.
  • the guar gum may be included in about 0.1 to 5% by weight, preferably about 0.1 to 1% by weight, for example, about 0.3% by weight based on the weight of the total medium composition.
  • the sodium alginate may be included in about 0.1 to 5% by weight, preferably about 0.1 to 1% by weight, for example, about 0.3% by weight based on the weight of the total medium composition.
  • the pectin may be included in about 0.1 to 5% by weight, preferably about 0.1 to 1% by weight, for example, about 0.3% by weight based on the weight of the total medium composition.
  • the oligosaccharide eg, galactooligosaccharide, fructooligosaccharide, isomaltooligosaccharide, xylooligosaccharide, soybean oligosaccharide, lactulose, chitin oligosaccharide, isomaltooligosaccharide, etc.
  • the oligosaccharide is about 0.1 to 5 weight based on the weight of the total medium composition. %, preferably about 1 to 3% by weight, such as about 3% by weight.
  • the medium composition may further include one or more inorganic salts selected from the group consisting of sodium chloride, sodium dehydroacetate, dibasic potassium phosphate, magnesium sulfate, ammonium sulfate and calcium carbonate.
  • Each of the inorganic salts is about 0.01 to 5% by weight, preferably about 0.05 to 1% by weight, such as about 0.06% by weight, about 0.1% by weight, about 0.3% by weight, about 0.4% by weight, based on the weight of the total medium composition. %, about 0.5% by weight, or about 1% by weight.
  • the medium composition contains about 0.5% by weight of sodium chloride, about 0.3% by weight of sodium dehydroacetate, about 0.3% by weight of dibasic potassium phosphate, about 0.06% by weight of magnesium sulfate, about 0.4% by weight of ammonium sulfate and 1 calcium carbonate. Further includes % by weight.
  • the medium composition includes the above-mentioned nitrogen source and carbon source, and optionally the above-mentioned inorganic salts, the production of butyrate is increased, so that the effect of preventing overweight of the fermentation product may be further improved.
  • the medium composition based on the total weight of the medium composition, 0.1 to 10% by weight of yeast extract as a nitrogen source; and 0.1 to 10% by weight of roasted soybean flour or 0.1 to 10% by weight of soy peptone; and
  • At least one selected from the group consisting of 1 to 10% by weight of inulin, 1 to 10% by weight of chicory powder, 1 to 10% by weight of maltodextrin, and 1 to 10% by weight of glucose may be included.
  • the medium composition may further include 0.1 to 10% by weight of skim milk powder as a nitrogen source based on the total weight of the medium composition.
  • the medium composition may further include at least one selected from the group consisting of 0.1 to 5% by weight of guar gum, 0.1 to 5% by weight of sodium alginate, 0.1 to 5% by weight of pectin, and 0.1 to 5% by weight of oligosaccharide as a carbon source. there is.
  • the medium composition based on the total weight of the medium composition,
  • yeast extract as a nitrogen source
  • roasted soybean flour or 5% by weight of soy peptone
  • the medium composition may further include 5% by weight of skim milk powder as a nitrogen source based on the total weight of the medium composition.
  • the medium composition based on the total weight of the medium composition, at least one selected from the group consisting of 0.3% by weight of guar gum, 0.3% by weight of sodium alginate, 0.3% by weight of pectin, and 3% by weight of oligosaccharide as a carbon source Further can include
  • the medium composition also contains 0.5% by weight of sodium chloride, 0.3% by weight of sodium dehydroacetate, 0.3% by weight of potassium dihydrogen phosphate, 0.06% by weight of magnesium sulfate, 0.4% by weight of ammonium sulfate and carbonic acid, based on the total weight of the medium composition.
  • Calcium 1% by weight of inorganic salts may be further included.
  • the medium composition may have the composition of Examples 1 to 17 mentioned in Tables 1 and 2.
  • the improvement effect of the composition on overweight can be further improved.
  • the productivity of butyrate may be lowered, and the culture medium composition may not be dissolved well, precipitate may occur, or boiling may occur during sterilization.
  • the medium composition based on the total weight of the medium composition, yeast extract as a nitrogen source 0.5% by weight; And 5% by weight of soy peptone,
  • the medium composition When inorganic salts of 0.5% by weight of sodium chloride, 0.3% by weight of sodium dehydroacetate, 0.3% by weight of potassium dibasic phosphate, 0.06% by weight of magnesium sulfate, 0.4% by weight of ammonium sulfate, and 1% by weight of calcium carbonate are included, the medium composition
  • the solubility may be improved and the amount of butyrate produced may increase to about 90 mM or more, preferably about 100 mM or more, and more preferably about 200 mM or more.
  • the short chain fatty acid is butyrate, propionate, acetate, specifically butyrate.
  • the fermentation product of the present invention contains at least about 10 mM butyrate, such as at least about 20 mM, or at least about 30 mM, at least about 40 mM, at least about 50 mM, at least about 60 mM, at least about 70 mM, at least about 80 mM.
  • about 90 mM or more about 100 mM or more, about 110 mM or more, about 120 mM or more, about 130 mM or more, about 140 mM or more, about 150 mM or more, about 160 mM or more, about 170 mM or more, about 180 mM or more , It may include about 190 mM or more, about 200 mM or more, about 210 mM or more butyrate.
  • a composition containing short-chain fatty acids can be prepared by inoculating a Clostridium strain into the aforementioned medium composition and then fermenting (cultivating) the strain. This production method and culture conditions are described later.
  • the medium composition of the present invention does not contain RCM (Reinforced Clostridial Medium).
  • RCM is a medium used for culturing Clostridium and other anaerobic bacteria in the food and medical fields. This medium is used as an enrichment medium for microbiological testing of non-sterilized products, and is also a standard medium registered in USP, EP, and JP.
  • the medium includes Beef extract, Casein peptone, Dextrose, Sodium chloride, Sodium acetate, Yeast extract, Soluble Starch, L-Cysteine HCl, Agar, and the like, and is easily commercially available.
  • RCM is also used as a differential medium for sulfite reducing bacteria such as Clostridium perfringens in food and beverage fields. Since the use and purpose of RCM is difficult to use in food production as a reagent prepared for experiments, it is necessary not to use RCM in order to prepare commercial food compositions and the like.
  • the Clostridium strain is one of Clostridium butyricum, Clostridium tyrobutyricum, or Clostridium saccharoperbutylacetonicum. It includes the above, and specifically, it is Clostridium butyricum.
  • the present invention provides a yeast extract as a nitrogen source; and at least one of roasted soy flour or soy peptone; and at least one selected from the group consisting of inulin, chicory powder, maltodextrin, and glucose as a carbon source, and does not include an enriched Clostridia medium.
  • the medium composition can be used as a medium for producing short-chain fatty acids.
  • the medium composition may further include skim milk powder as a nitrogen source.
  • the medium composition may further include at least one selected from the group consisting of guar gum, sodium alginate, pectin, and oligosaccharides as a carbon source.
  • the medium composition may further include inorganic salts, for example, one or more inorganic salts selected from the group consisting of sodium chloride, sodium dehydroacetate, dibasic potassium phosphate, magnesium sulfate, ammonium sulfate and calcium carbonate.
  • inorganic salts for example, one or more inorganic salts selected from the group consisting of sodium chloride, sodium dehydroacetate, dibasic potassium phosphate, magnesium sulfate, ammonium sulfate and calcium carbonate.
  • the fermentation product of the present invention can be used for the amelioration, prevention or treatment of overweight.
  • the fermented composition of the present invention exhibits an effect of inhibiting differentiation of adipocytes, reducing fat accumulation, and further inhibiting triglyceride content.
  • the fermentation product of the present invention showed an effect of significantly reducing body weight in the high-fat diet group, and also reduced the percentage of body fat and the amount of body fat.
  • the fermented composition of the present invention can suppress the expression of mast cells and reduce body weight and fat mass, so that it can be used for improvement, prevention or treatment of overweight.
  • the fermentation product of the present invention also exhibits a concentration-dependent antioxidant effect, and furthermore, it has been confirmed that it reduces the level of TNF alpha, showing an effect on improving inflammatory diseases.
  • the present invention provides a composition comprising the fermentation product.
  • the composition may be used for improvement, prevention or treatment of overweight.
  • the present invention provides a pharmaceutical composition or veterinary composition for preventing or treating overweight, comprising the fermentation product.
  • the present invention provides a food composition or animal feed composition for improving or preventing overweight, comprising the fermentation product.
  • composition includes a product comprising a specified component in a specified amount and any product that results directly or indirectly from the combination of a specified component in a specified amount.
  • this term encompasses a product comprising an active ingredient and an inactive ingredient constituting a carrier, and includes any combination, complexation or aggregation of two or more ingredients or dissociation of one or more ingredients, or other types of It is intended to include any product resulting directly or indirectly by a reaction or interaction.
  • composition refers to a pharmaceutical composition for use as a medicine for humans, a veterinary composition for use as a medicine for animals, or a dietary product or food for humans or animals (e.g., functional food composition, health functional food, i.e., food, beverage, feed or pet food, or food, beverage, animal feed or pet food supplement); Accordingly, “composition” herein is used as a meaning including both “pharmaceutical composition", “veterinary composition", or “food composition”.
  • the composition may further include a pharmaceutically acceptable excipient, a veterinarily acceptable excipient, a food acceptable excipient, and the like, depending on each use.
  • phrases “pharmaceutically acceptable,” “veterinarily acceptable,” or “food acceptable” means, within the scope of sound medical or food judgment, to match a reasonable benefit/risk ratio, excessive toxicity, Refers to a compound, material, composition, carrier, and/or dosage form suitable for use in contact with human or animal tissue without irritation, allergic reaction, or other problem or complication.
  • phrases “pharmaceutically acceptable excipient”, “veterinarily acceptable excipient” or “food acceptable excipient” are generally safe, non-toxic, biologically and otherwise desirable pharmaceutical, veterinary or otherwise acceptable excipients. It means an excipient useful for preparing a food composition, and includes an excipient acceptable for human pharmaceutical use or animal veterinary use, and further for food use. As used in the specification and claims, “pharmaceutically acceptable excipient”, “veterinarily acceptable excipient”, or “food acceptable ingredient” includes both one or more of these excipients.
  • pharmaceutically, veterinarily, or food-acceptable excipients used in the formulation of the present invention may be diluents or inert carriers, lubricants, binders, or combinations thereof. Excipients used in the formulations of the present invention may further include fillers, anti-microbial agents, antioxidants, anti-caking agents, coating agents, or mixtures thereof. Any other pharmaceutical, veterinary, or food acceptable excipient may be used without limitation.
  • the composition according to the present invention may be a pharmaceutical or veterinary composition for improving, preventing or treating overweight, a food composition for improving overweight, and may be used for various other purposes.
  • the composition of the present invention may be used for anti-inflammatory or antioxidant purposes in addition to overweight improvement.
  • composition according to the present invention can be administered to the human body in a variety of ways, and can also be administered to other mammals.
  • other mammals may be domestic animals such as dogs, cats, rabbits, pigs, sheep, goats, dairy cows, horses, and cattle, and pets, but are not limited thereto.
  • the composition when the composition is a pharmaceutical or veterinary composition, the composition may be formulated into a variety of forms for parenteral or oral administration.
  • Representative formulations for parenteral administration include formulations for injection, and in this case, the form of the composition may be preferably an isotonic aqueous solution or suspension.
  • Formulations for injection may be prepared according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. For example, each component may be dissolved in saline or a buffer solution and formulated for injection.
  • Formulations for oral use may include powders, granules, tablets, pills, capsules, and the like, but are not limited thereto.
  • composition according to the present invention can be prepared in various formulations using a drug delivery system.
  • the drug delivery system may be liposomes, ethosomes, elastic ethosomes, targeting liposomes, microparticles, microcapsules, nanoparticles, nanocapsules, nanofibers, and the like, but is not limited thereto.
  • compositions for food include, for example, health functional foods, vitamin complexes, chewing gum, beverages, various foods, tea, various processed meat products, fish products, tofu, jelly, noodles, breads, health supplements, seasonings, sauces, confectionery, Candies, dairy products, other processed foods, fermented foods, natural seasonings, etc. can be used, but are not limited thereto.
  • the term "health functional food” refers to food manufactured and processed using raw materials or ingredients having useful functionality for the human body.
  • the above “functionality” means obtaining useful effects for health purposes such as regulating nutrients for the structure and function of the human body or physiological functions.
  • the health functional food of the present invention can be prepared by a method commonly used in the art, and can be prepared by adding raw materials and components commonly added in the art during manufacture.
  • the formulation of the health functional food may also be manufactured without limitation as long as the formulation is recognized as a health functional food.
  • yeast extract as a nitrogen source; and at least one of roasted soy flour or soy peptone;
  • Preparing a medium composition by dissolving and sterilizing at least one selected from the group consisting of inulin, chicory powder, maltodextrin and glucose as a carbon source in water;
  • Sterilization in step (a) may be performed by heating at a temperature of about 100 to 130° C. for about 10 to 20 minutes, and under these conditions, sterilization can be effectively achieved.
  • the heating temperature may be about 120° C.
  • the heating time may be about 15 minutes.
  • step (c) fermentation can be performed for about 1 to 15 days by putting an anaerobic pack in an anaerobic culture tank to maintain the culture state anaerobically. More preferably, the culture may be performed for about 1 to 5 days. At this time, the culture temperature may be about 25 ⁇ 37 °C. For example, the incubation period may be about 5 days and the incubation temperature may be about 37°C.
  • the fermented composition of the present invention can suppress the expression of mast cells and reduce body weight and fat mass, so that it can be effectively used for improvement, prevention or treatment of overweight.
  • 1 is a graph showing the results of measuring the cytotoxicity of compositions according to Examples in intestinal endothelial cells.
  • Figure 2 is a graph showing the results of measuring the antioxidant efficacy of the composition according to the embodiment.
  • Figure 3 is a graph showing the results of measuring the decrease in TNF alpha level in primary cultured splenocytes for the composition according to the Example.
  • Figure 4 is a graph showing the results of measuring the IgE level reduction in the plasma of animals with DSS-induced enteritis for the composition according to the Example.
  • Figure 5 is a graph showing the results of measuring the cytotoxicity evaluation on 3T3-L1 preadipocyte cells for the composition according to the example.
  • 6 is a graph showing the results of measuring the induction of differentiation from 3T3-L1 preadipocytes to adipocytes with respect to the composition according to the example.
  • Figure 9 is the result of measuring the weight change in obesity-induced mice on a high-fat diet according to the administration of the composition according to the embodiment.
  • a small amount of 1 platinum of Clostridium butyricum cultured on a solid medium is inoculated into a sterilized culture medium, and in order to maintain an anaerobic state in an incubator at 37 ° C, the medium is placed in an anaerobic culture tank 2.5 L, Incubated using a gas pack. Thereafter, the anaerobic culture status indicator paper was used to check the presence or absence of oxygen in the anaerobic culture tank, and the cells were cultured for a total of 5 days.
  • a composition was prepared under the same conditions as in Example 1, except that 3% by weight (based on the total culture medium) of roasted soy flour was added.
  • a composition was prepared under the same conditions as in Example 1, except that 5% by weight of roasted soybean flour (based on the total culture medium) was added.
  • a composition was prepared under the same conditions as in Example 3, except that 0.3% by weight of guar gum (based on the total culture medium) was additionally added.
  • a composition was prepared under the same conditions as in Example 3, except that 0.3% by weight of sodium alginate (based on the total culture medium) was additionally added.
  • a composition was prepared under the same conditions as in Example 3, except that 0.3% by weight (based on the total culture medium) of pectin was additionally added.
  • a composition was prepared under the same conditions as in Example 3, except that 3% by weight (based on the total culture medium) of fructooligosaccharide powder was additionally added.
  • a composition was prepared under the same conditions as in Example 3, except that 3% by weight (based on the total culture medium) of galacto-oligosaccharide was additionally added.
  • a composition was prepared under the same conditions as in Example 3, except that 3% by weight (based on the total culture medium) of indigestible maltodextrin was additionally added.
  • a small amount of 1 platinum of Clostridium butyricum cultured on a solid medium is inoculated into a sterilized culture medium, and in order to maintain an anaerobic state in a 37 ° C incubator, the medium is placed in an anaerobic culture tank 2.5 L, Incubated using a gas pack. Thereafter, the anaerobic culture status indicator paper was used to check the presence or absence of oxygen in the anaerobic culture tank, and the cells were cultured for a total of 5 days.
  • a composition was prepared under the same conditions as in Example 10, except that 5% by weight of chicory powder was added as a carbon source.
  • a composition was prepared under the same conditions as in Example 10, except that 5% by weight of indigestible maltodextrin was added as a carbon source.
  • a composition was prepared under the same conditions as in Example 10, except that 5% by weight of glucose was added as a carbon source.
  • Yeast extract 0.5% by weight as a nitrogen source 0.5% by weight as a nitrogen source; And a composition was prepared under the same conditions as in Example 10, except that 5% by weight of each of soy peptone and skim milk powder was added.
  • Yeast extract 0.5% by weight as a nitrogen source 0.5% by weight as a nitrogen source; And a composition was prepared under the same conditions as in Example 10, except that 5% by weight of each of soy peptone and skim milk powder and 5% by weight of chicory powder as a carbon source were added.
  • Yeast extract 0.5% by weight as a nitrogen source A composition was prepared under the same conditions as in Example 10, except that 5% by weight of each of soy peptone and skim milk powder and 5% by weight of indigestible maltodextrin as a carbon source were added.
  • Yeast extract 0.5% by weight as a nitrogen source A composition was prepared under the same conditions as in Example 10, except that 5% by weight of each of soy peptone and skim milk powder and 5% by weight of glucose as a carbon source were added.
  • RCM Reinforced Clostridial Medium
  • the prepared culture medium a small amount of 1 platinum is collected and inoculated with Clostridium butyricum cultured on RCM solid medium, and in order to maintain an anaerobic state in an incubator at 37 ° C, the medium is placed in a 2.5 L anaerobic culture tank , cultured using a gas pack. Thereafter, the anaerobic culture status indicator paper was used to check the presence or absence of oxygen inside the anaerobic culture tank, and the culture was carried out for a total of 5 days.
  • a composition was prepared under the same conditions as in Comparative Example 1, except that 1% by weight of inulin powder was added based on the total culture medium prior to sterilization.
  • a composition was prepared under the same conditions as in Comparative Example 2, except that 5% by weight of inulin powder was added based on the total culture medium.
  • a composition was prepared under the same conditions as in Comparative Example 2, except that 10% by weight of inulin powder was added based on the total culture medium.
  • a composition was prepared under the same conditions as in Comparative Example 2, except that 20% by weight of inulin powder was added based on the total culture medium.
  • a composition was prepared under the same conditions as in Comparative Example 2, except that 5% by weight of chicory powder was added based on the total culture medium instead of inulin powder.
  • a small amount of 1 platinum of Clostridium butyricum cultured on a solid medium is inoculated into a sterilized culture medium, and in order to maintain an anaerobic state in an incubator at 37 ° C, the medium is placed in an anaerobic culture tank 2.5 L, Incubated using a gas pack. Thereafter, the anaerobic culture status indicator paper was used to check the presence or absence of oxygen in the anaerobic culture tank, and the cells were cultured for a total of 5 days.
  • a composition was prepared under the same conditions as in Comparative Example 7, except that 1% by weight (based on the total culture medium) of whey protein isolate was added instead of soybean protein isolate.
  • a small amount of 1 platinum of Clostridium butyricum cultured on a solid medium is inoculated into a sterilized culture medium, and in order to maintain an anaerobic state in a 37 ° C. incubator, the medium is placed in an anaerobic culture tank 2.5 L, Incubated using a gas pack. Thereafter, the anaerobic culture status indicator paper was used to check the presence or absence of oxygen in the anaerobic culture tank, and the cells were cultured for a total of 5 days.
  • a composition was prepared under the same conditions as in Comparative Example 9, except that 5% by weight of chicory powder was added as a carbon source.
  • a composition was prepared under the same conditions as in Comparative Example 9, except that 5% by weight of indigestible maltodextrin was added as a carbon source.
  • a composition was prepared under the same conditions as in Comparative Example 9, except that 5% by weight of glucose was added as a carbon source.
  • compositions of Examples 1 to 17 and Comparative Examples 1 to 12 are summarized and shown in Tables 1 to 3 below.
  • Examples 1 to 3 include both inulin and chicory powder, and when Examples 1 to 3 are compared with Comparative Examples 7 and 8, there is a difference in including roasted soy flour as a composition of the medium composition.
  • roasted soy flour was produced, especially when the roasted soybean flour was 3% by weight (based on the total medium weight) (Example 2), and when the roasted soybean flour was 5% by weight (based on the total medium weight) (Example 3), It can be seen that the production of butyrate was markedly increased to 21.25 mM and 25.96 mM, respectively.
  • Example 4 to 9 compared to Example 3, which additionally include a carbon source in the medium composition, butyrate production was almost similar to Example 3 or slightly increased.
  • the medium compositions of Examples 10 to 17 include yeast extract and soy peptone as nitrogen sources, inulin, chicory powder, maltodextrin or glucose as carbon sources, sodium chloride, sodium dehydroacetate, dibasic potassium phosphate, magnesium sulfate, It further includes inorganic salts of ammonium sulfate and calcium carbonate.
  • yeast extract and soy peptone as nitrogen sources
  • inulin, chicory powder maltodextrin or glucose as carbon sources
  • sodium chloride sodium dehydroacetate
  • dibasic potassium phosphate sodium dehydroacetate
  • dibasic potassium phosphate dibasic potassium phosphate
  • magnesium sulfate magnesium sulfate
  • the solubility during the preparation of the medium composition was improved, and the amount of butyrate produced increased to 90 mM or more, and in particular, in Examples 10 to 12, it increased to 200 mM or more. did
  • the cell viability of the composition (fermentation product) of Example 3 was measured as a lyophilized sample through the MTT method.
  • IEC-18 cells Intestinal endothelial cells, IEC-18 cells, were cultured in DMEM (Dulbecco's Modified Eagle Medium, Hyclone Lab., USA) containing 10% fetal bovine serum (FBS), and then plated at 5 x 104 cells/cells in a 24 well plate. Divided into mL. After culturing for 48 hours, 100 ⁇ g/mL of LPS and the lyophilized sample were simultaneously treated. At this time, the lyophilized samples were reconstituted in an aqueous medium (water) at 10, 50, 100, 250, 500, and 1000 ug/ml, respectively. After the incubation was completed, the MTT reagent was treated and reacted to form formazan, and the supernatant was removed, treated with DMSO, and measured at 540 nm using a microplate reader ELISA device.
  • DMEM Dulbecco's Modified Eagle Medium, Hyclone Lab., USA
  • FBS feta
  • Example 3 The fermentation product of Example 3 was lyophilized, and antioxidant and anti-inflammatory activities were evaluated.
  • the antioxidant efficacy was verified through the DPPH experiment, and ascorbic acid (1 mg/mL) was used as a control and compared with the fermentation product.
  • the lyophilized samples were reconstituted in an aqueous medium (water) to 1, 2.5, 5, 7.5, and 10 ug/ml, respectively.
  • a 0.2 mM DPPH solution was used, and each sample, purified water, MeOH, and 0,2 mM DPPH were sequentially mixed in a 96 well plate and reacted in the dark for 30 minutes. After that, the optical density (OD) at a wavelength of 517 nm was calculated using a microplate reader ELISA device and compared with that of ascorbic acid (control).
  • a TNF alpha measurement experiment was conducted in spleen cells of a mouse animal model of Dextran Sulfate Sodium (DSS) enteritis.
  • the untreated mice were used as a normal control (CON)
  • the DSS mouse model was used as a negative control (DSS)
  • Butyric acid 5 mM, 10 mM, and Sulfasalazine 50 mg/kg were used as positive controls.
  • the freeze-dried sample was reconstituted at a low concentration of 100 ug/mL and a high concentration of 200 ug/mL in the same manner as in Experimental Example 3 and used.
  • TNF alpha measurement was performed using a TNF alpha Mouse ELISA kit.
  • the sample of the present invention showed a higher reducing ability than Butyric acid, and showed an effect similar to that of Sulfasalazine.
  • TNF alpha is a cytokine that plays an important role in various immune-mediated inflammatory diseases, and the reduced TNF alpha level indicates that the composition of the present invention is effective in improving inflammatory diseases.
  • a freeze-dried sample of the fermentation product of Example 3 was used to measure IgE levels in plasma of mouse animals with DSS-induced enteritis using a Mouse IgE ELISA kit.
  • the sample of Example 3 was reconstituted at a low concentration of 100 ug/mL and a high concentration of 200 ug/mL and used.
  • the butyrate fermentation product showed a concentration-dependent decrease in IgE level, and it was confirmed in FIG. 4 that the IgE level decreased the most at a high concentration (200 ug/mL) of the butyrate fermentation product. This indicates that the composition of the present invention is effective in improving inflammatory diseases.
  • the freeze-dried sample of the fermentation product of Example 3 was reconstituted in an aqueous medium (water) at concentrations of 25, 50, 100, and 200 ⁇ g/ml, respectively, and then treated with 3T3-L1 cells, respectively, followed by the MTT method. Cell viability was measured through The MTT method was performed in the same manner as in Experimental Example 2.
  • the freeze-dried sample of the butyrate fermentation product of Example 3 was reconstituted with an aqueous medium (water) and then treated at concentrations of 25, 50, and 100 ⁇ g/ml to induce differentiation of 3T3-L1 preadipocytes into mature adipocytes and treated with Oil Red-O staining.
  • 3T3-L1 cells, pre-adipocytes were cultured in DMEM (Dulbecco's Modified Eagle Medium, Hyclone Lab., USA) containing 10% Bovine calf serum, and then plated 1 x 10 in a 6-well plate. It was aliquoted at 5 cells/mL.
  • DMEM fetal bovine serum
  • FBS fetal bovine serum
  • Triglyceride Quantification Assay kit was used to measure the pure triglyceride (TG) content in mature adipocytes by treating the lyophilized product of the fermentation product of Example 3.
  • Differentiation-induced pre-adipocytes were homogenized using 5% NP-40 solution, incubated at 90 ° C for 5 minutes, and then reacted at room temperature for 5 minutes to completely dissolve TG. After that, it was reacted with Lipase at room temperature for 20 minutes and reacted with Triglyceride Probe and Triglyceride Enzyme Mix at room temperature for 60 minutes, and the light intensity at 570 nm wavelength was measured with a spectrophotometer.
  • the fermentation product of the present invention was reconstituted at concentrations of 25, 50, and 100 ⁇ g/ml using an aqueous medium (water) and used.
  • Freeze-dried samples of the fermentation product of Example 3 were reconstituted according to Table 7, and after oral administration according to Table 7 to 8-week-old C57BL6J mice with a high-fat diet, body weight changes were measured during the experimental period for 14 weeks.
  • Example 9 The effect of the fermentation product of Example 3 on reducing body fat in the obese animal model induced by the high-fat diet of Experimental Example 9 was measured by Dual energy X-ray Absorptiometry (DXA) to measure the volume of subcutaneous and visceral fat. The results are shown in FIG. 10 .
  • DXA Dual energy X-ray Absorptiometry
  • Example 9 To confirm the effect of the fermentation product of Example 3 on organ weight in the obese animal model induced by the high-fat diet of Experimental Example 9, the weight of liver, epididymis, groin or brown adipose tissue was measured.
  • FIG. 11 Compared to the normal diet group (NCD), the size of epididymal fat increased in the high-fat diet group (HFD) (FIG. 11-A), and compared to the simple high-fat diet group (HFD + vehicle), the liver in the butyrate fermentation product simultaneous administration group of the present invention , the weights of epididymal white fat, inguinal light fat, and brown adipose tissue were lower (FIG. 11-B).
  • the high-dose administration group (BF-1) showed a decrease in tissue weight that was almost similar to or lower than that of the positive control group (SB-0.5).
  • the fermentation product of the present invention according to the examples can inhibit the expression of mast cells, and in the experimental examples, it was confirmed that body weight and body fat mass were reduced. In particular, considering that as the concentration of butyrate increases, it is effective in reducing body weight and fat mass.
  • the butyrate content was about 26 mM
  • the other examples having similar or higher butyrate content were also similar. or will show a more effective weight loss effect.

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Abstract

The present invention relates to a fermentation product containing a short-chain fatty acid and a use thereof for alleviating, preventing, or treating obesity. The present invention relates to a fermentation product containing a short-chain fatty acid, wherein the fermentation product comprises: a yeast extract as a nitrogen source; at least one selected from the group consisting of fried soybean powder or soy peptone; and at least one selected from the group consisting of inulin, chicory powder, maltodextrin, and glucose as a carbon source, is produced by inoculating a Clostridium strain into a medium composition containing no reinforced clostridial media and fermenting same, and contains a short-chain fatty acid in an amount of 10 mM or higher.

Description

단쇄 지방산을 포함하는 발효 생성물 및 그의 과체중 개선, 예방 또는 치료 용도 Fermentation products containing short-chain fatty acids and uses thereof for the improvement, prevention or treatment of overweight
본 발명은 단쇄 지방산을 포함하는 발효 생성물 그의 과체중 개선, 예방 또는 치료 용도에 관한 것이다.The present invention relates to the use of a fermentation product containing short-chain fatty acids for the improvement, prevention or treatment of overweight.
전세계적으로 비만 치료에 대한 시장은 매년 약 20% 이상 증가하는 고성장 시장이며, 2025년에는 약 3억명 이상의 비만 환자가 생겨날 것으로 예상되고 있다. 국내 비만 인구도 갈수록 급증하는 추세이며 이에 따른 성인병의 증가도 매우 높아지고 있다. 또한 삶의 질과 미적 향상을 추구하는 현대 사회에서 체중 감소와 관련된 제품의 시장은 점점 증가하고 있는 추세이다. Worldwide, the market for obesity treatment is a high-growth market that increases by about 20% or more every year, and it is expected that more than 300 million obese patients will be created by 2025. The number of obese people in Korea is also rapidly increasing, and the increase in adult diseases is also very high. In addition, in modern society seeking quality of life and aesthetic improvement, the market for products related to weight loss is gradually increasing.
종래 치료제로서의 비만에 대한 약물은 대부분 식욕 억제에 관한 것이고, 이에 대한 부작용이 매우 커서 일반적인 사람들이 쉽게 복용하기에는 위험성이 매우 크다고 할 수 있다. Most of the drugs for obesity as a conventional therapeutic agent are related to appetite suppression, and the side effects thereof are very large, so it can be said that the risk is very high for ordinary people to take them easily.
체중 감소와 관련하여, 체지방 감소 보조제 또는 기능성 식품 소재로, 가르시니아 캄보지아, 녹차추출물, 공액 리놀렌산 등이 이용되고 있다. 가르시니아 캄보지아는 지방 흡수를 억제함으로써 체지방 감소 시장의 약 47 %를 차지하고 있으나, 간 독성 등 부작용이 나타나고 있다. In relation to weight loss, Garcinia cambogia, green tea extract, conjugated linolenic acid, and the like are used as body fat reduction supplements or functional food materials. Garcinia cambogia accounts for about 47% of the body fat reduction market by suppressing fat absorption, but side effects such as liver toxicity are appearing.
따라서, 부작용을 나타내지 않으면서 체지방 감소를 도모하는 신규 소재의 개발이 필요하다. Therefore, it is necessary to develop a novel material that promotes body fat reduction without showing side effects.
한편, 미생물은 우리 인체와 공생함으로써, 인간의 소화기능이나 에너지의 확보 등 대사 기능 조절과 면역계 발달, 신경계와의 상호작용, 인체의 항상성 유지와 관련된 다양한 기능을 수행한다. 특히 장내에 서식하는 미생물은 장 질환뿐만 아니라, 비만이나 당뇨병과 같은 대사 질환, 우울증이나 ADHD와 같은 정신질환, 그리고 노화 등 인간의 건강에 큰 영향을 미칠 수 있다. On the other hand, microorganisms live in symbiosis with the human body, performing various functions related to metabolic function control, immune system development, interaction with the nervous system, and maintenance of homeostasis of the human body, such as human digestive function or securing energy. In particular, microorganisms living in the intestine can have a great impact on human health, including not only intestinal diseases, but also metabolic diseases such as obesity and diabetes, mental diseases such as depression and ADHD, and aging.
위나 소장에서 소화되어 흡수되지 않고 대장까지 내려가는 저항성 전분(Resistant starch)을 대장 내에 서식하는 미생물이 먹이로 섭취하게 되면, 단쇄 지방산이 생성된다. 단쇄 지방산은 아세테이트(Acetate), 프로피오네이트(propionate), 부티레이트(butyrate)와 같이 탄소수 6개 이하의 유기산을 의미한다. 이러한 단쇄 지방산은 우리 인체에 굉장히 많은 영향을 끼칠 수 있다. 예를 들어, 대장 점막을 이루는 세포들은 단쇄 지방산을 에너지원으로 하여 대장벽을 유지하고, 세균이 몸속으로 침입하는 것을 막을 수 있다. When microorganisms living in the large intestine consume resistant starch, which is not digested and absorbed in the stomach or small intestine and goes down to the large intestine, short-chain fatty acids are produced. Short-chain fatty acids refer to organic acids having 6 carbon atoms or less, such as acetate, propionate, and butyrate. These short-chain fatty acids can have a lot of effects on our body. For example, cells constituting the mucous membrane of the large intestine maintain the wall of the large intestine using short-chain fatty acids as an energy source and can prevent bacteria from entering the body.
또한, 부티레이트는 대장 점막으로 흡수되어서 모든 소화관과 장기의 점막 상피 세포를 증식시키는데 중요한 역할을 한다. 부티레이트는 지방세포에 있는 FFAR3(Free fatty acid receptor3) 세포막 수용체를 활성화(PPAR-γ 수정여부)시키는데, 이는 지방의 축적을 방지하고 근육과 대사에 에너지를 이용하여 체중 감소 및 다이어트에 매우 효과적이라는 연구 결과가 개시되었다. Hua.V. Lin et al의 비만 쥐의 동물 모델에서, 부티레이트가 포함된 고지방 식이를 4주 동안 진행했을 때 굉장히 뚜렷하게 체지방의 감소를 나타낸다는 연구 결과가 보고되었다 (Hua V. Lin et al., PLoS One. 2012; 7(4): e35240). 또한, 부티레이트가 포함된 고지방 식이를 한 인체 적용시험에서도 체중감소, 장염증성 질환의 염증 감소 효과를 증명한 연구들이 나오고 있다. In addition, butyrate is absorbed into the colonic mucosa and plays an important role in proliferating mucosal epithelial cells of all digestive tracts and organs. Butyrate activates the FFAR3 (Free Fatty Acid Receptor 3) cell membrane receptor in fat cells (PPAR-γ correction or not), which prevents the accumulation of fat and uses energy for muscle and metabolism, which is very effective for weight loss and diet. Results have been disclosed. Hua. V. In an animal model of obese mice by Lin et al, it was reported that when a high-fat diet containing butyrate was administered for 4 weeks, a significant reduction in body fat was reported (Hua V. Lin et al., PLoS One. 2012 ;7(4): e35240). In addition, there are studies that have demonstrated the effect of reducing weight loss and reducing inflammation in intestinal inflammatory diseases in human body application tests with a high-fat diet containing butyrate.
다만, 전술한 것처럼, 부티레이트가 체지방 감소에 있어 많은 임상적 효과를 보이고 있으나, 특이취가 매우 강하여 상업적으로 제품화하기에 어려운 점이 있다. However, as described above, although butyrate shows many clinical effects in reducing body fat, it has a very strong specific odor and is difficult to commercialize.
버터와 같은 포화 지방산을 섭취할 때, 부티레이트를 섭취할 수 있으나, 이 경우 부티레이트가 매우 소량 포함되어 있으므로, 버터를 많이 섭취하여 생기는 체지방 증가가 더 문제가 될 수 있다. When saturated fatty acids such as butter are consumed, butyrate can be ingested, but in this case, butyrate is included in very small amounts, so the increase in body fat caused by consuming a lot of butter can be more of a problem.
이 외에, 장 내에서, 부티레이트를 생성하는 미생물이, 수용성 식이섬유인 이눌린과 같은 저항성 전분을 분해하는 과정에서 부티레이트를 생성하게 하는 방법이 있을 수 있다.In addition, there may be a method in which butyrate-producing microorganisms in the intestine produce butyrate in the process of decomposing resistant starch such as inulin, which is a water-soluble dietary fiber.
본 발명은 단쇄 지방산의 함량이 높은 발효 생성물을 제공하고자 한다.The present invention is intended to provide a fermentation product with a high content of short-chain fatty acids.
본 발명은 또한 상기 발효 생성물을 이용하여 과체중을 개선, 예방 또는 치료하고자 한다.The present invention also aims to improve, prevent or treat overweight by using the fermentation product.
본 발명은 또한 상기 발효 생성물의 제조 방법을 제공하고자 한다. The present invention also seeks to provide a method for preparing the fermentation product.
첨부한 도면을 참고로 하여 본 발명의 실시예에 대해 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있도록 상세히 설명한다. 본 발명은 여러 가지 상이한 형태로 구현될 수 있으며 여기에서 설명하는 실시예에 한정되지 않는다. 도면에서 본 발명을 명확하게 설명하기 위해서 설명과 관계없는 부분은 생략하였으며, 명세서 전체를 통하여 동일 또는 유사한 구성요소에 대해서는 동일한 도면부호가 사용되었다. 또한 널리 알려져 있는 공지기술의 경우 그 구체적인 설명은 생략한다.With reference to the accompanying drawings, embodiments of the present invention will be described in detail so that those skilled in the art can easily carry out the present invention. This invention may be embodied in many different forms and is not limited to the embodiments set forth herein. In order to clearly describe the present invention in the drawings, parts irrelevant to the description are omitted, and the same reference numerals are used for the same or similar components throughout the specification. In addition, in the case of widely known known technologies, detailed descriptions thereof will be omitted.
명세서 전체에서, 어떤 부분이 어떤 구성요소를 "포함"한다고 할 때, 이는 특별히 반대되는 기재가 없는 한 다른 구성요소를 제외하는 것이 아니라 다른 구성요소를 더 포함할 수 있는 것을 의미한다. Throughout the specification, when a certain component is said to "include", it means that it may further include other components without excluding other components unless otherwise stated.
본원에 사용된 용어 "약"은 주어진 수치 또는 범위의 10% 이내, 바람직하게는 5% 이내, 더욱 바람직하게는 1% 이내를 의미한다. As used herein, the term "about" means within 10%, preferably within 5%, more preferably within 1% of a given value or range.
단쇄 지방산 포함 발효 생성물Fermentation products containing short-chain fatty acids
일 실시태양에서, 본 발명은 단쇄 지방산을 고농도로 포함하는 발효 생성물을 제공한다. 상기 발효 생성물은 배지 조성물에 클로스트리디움(Clostridium) 균주를 접종한 후 발효시켜 생성되며, 이 경우 고농도의 단쇄 지방산을 포함한다. In one embodiment, the present invention provides a fermentation product comprising a high concentration of short chain fatty acids. The fermentation product is produced by inoculating a Clostridium strain into a medium composition and then fermenting it, in which case it contains a high concentration of short-chain fatty acids.
구체적 실시태양에서, 본 발명은 단쇄 지방산을 포함하는 발효 생성물로서, In a specific embodiment, the present invention is a fermentation product comprising short-chain fatty acids,
상기 발효 생성물은 The fermentation product is
질소원으로서 효모 추출물; 및 볶음 콩가루 또는 소이 펩톤 중 하나 이상; 및yeast extract as a nitrogen source; and at least one of roasted soy flour or soy peptone; and
탄소원으로서 이눌린(inulin), 치커리(chicory) 분말, 말토덱스트린 및 포도당에서 이루어진 군에서 선택된 하나 이상At least one selected from the group consisting of inulin, chicory powder, maltodextrin and glucose as a carbon source
을 포함하며, 강화 클로스트리디아 배지를 포함하지 않는 배지 조성물에 클로스트리디움(Clostridium) 균주를 접종한 후 발효시켜 생성되는 것이고, It is produced by inoculating a Clostridium strain into a medium composition that does not contain an enhanced Clostridia medium and then fermenting it,
상기 발효 생성물은 약 10 mM 이상의 단쇄 지방산을 포함하는 것인, Wherein the fermentation product comprises at least about 10 mM short-chain fatty acids.
단쇄 지방산을 포함하는 발효 생성물을 제공한다. A fermentation product comprising short chain fatty acids is provided.
본원에 사용된 용어 "발효 생성물"은 미생물을 이용한 효소적 또는 대사적 분해의 결과물을 의미하는 것으로, 구체적으로는 본 발명에서는 상기 또는 하기에서 구체적 실시태양으로 언급하는 배지 조성물에 클로스트리디움(Clostridium) 균주를 접종한 후 배양한 산물을 의미할 수 있다. 또한 본원에서 "발효 생성물"은 "발효물" 등과 혼용될 수 있다.As used herein, the term "fermentation product" refers to a product of enzymatic or metabolic decomposition using microorganisms, and specifically, in the present invention, Clostridium (Clostridium ) may refer to a product cultured after inoculation of a strain. In addition, "fermentation product" herein may be used interchangeably with "fermentation product" and the like.
본원에서 사용된 용어 "배지" 또는 "배지 조성물"은 상기 미생물 또는 균주를 배양하기 위해 필요로 하는 영양물질을 주성분으로 혼합한 물질을 의미하며, 생존 및 발육에 불가결한 물을 비롯하여 영양물질 및 발육인자 등을 공급한다. 나아가, 본원의 배지 조성물은 단쇄 지방산, 특히 부티레이트를 고농도로 생성하기에 최적의 조성과 함량을 포함하는 것으로서, 질소원으로서 ; 및 볶음 콩가루 또는 소이 펩톤 중 하나 이상; 및 탄소원으로서 이눌린(inulin), 치커리(chicory) 분말, 말토덱스트린 및 포도당에서 이루어진 군에서 선택된 하나 이상을 필수 성분으로 포함한다. 그 외 무기염류, 아미노산 및/또는 비타민과 같은 생육인자 등을 추가적으로 포함할 수 있다. 본 발명의 배지 조성물은 통상의 배지 내에서 혐기성 조건 하에서 온도, pH 등을 조절하면서 배양할 수 있다.As used herein, the term "medium" or "medium composition" refers to a material in which nutrients necessary for culturing the microorganism or strain are mixed as main components, including water essential for survival and growth, as well as nutrients and growth supplies, etc. Furthermore, the medium composition of the present application includes a composition and content optimal for producing short-chain fatty acids, particularly butyrate in high concentration, as a nitrogen source; and at least one of roasted soy flour or soy peptone; and at least one selected from the group consisting of inulin, chicory powder, maltodextrin and glucose as a carbon source as an essential component. In addition, growth factors such as inorganic salts, amino acids and/or vitamins may be additionally included. The medium composition of the present invention can be cultured while controlling temperature, pH, etc. under anaerobic conditions in a conventional medium.
상기 배지 조성물에 포함되는 효모 추출물, 볶음 콩가루, 소이 펩톤 등은 질소 공급원으로서 기능하고, 미생물의 생장에 있어 질소원의 구성은 매우 중요할 수 있다. 배지 조성물이 효모 추출물을 포함하면서, 추가로 볶음 콩가루 또는 소이 펩톤 중 하나 이상을 필수적으로 포함하는 경우, 부티레이트 생성량이 많아질 수 있고, 이로 인해 부티레이트의 과체중에 대한 개선, 예방 또는 치료 효과가 향상될 수 있다. Yeast extract, roasted soy flour, soy peptone, etc. included in the medium composition function as a nitrogen source, and the composition of the nitrogen source may be very important in the growth of microorganisms. When the medium composition includes the yeast extract and additionally includes at least one of roasted soybean flour or soy peptone, the amount of butyrate produced can be increased, and thus the improvement, prevention, or treatment effect of butyrate on overweight can be improved. can
일 구체예에서 상기 질소원은 효모 추출물 및 볶음 콩가루를 필수적으로 포함하거나, 효모 추출물 및 소이 펩톤를 필수적으로 포함할 수 있다.In one embodiment, the nitrogen source may essentially include yeast extract and roasted soybean flour, or may essentially include yeast extract and soy peptone.
효모 추출물은 배지 조성물의 전체 중량을 기준으로, 약 0.1 내지 10 중량%로 포함될 수 있고, 바람직하게는 약 0.5 내지 5 중량%, 보다 바람직하게는 약 0.5 내지 1 중량%, 예컨대 약 0.5 또는 1 중량%일 수 있다. 효모 추출물은 상업용으로 시판되는 것을 적절하게 사용할 수 있으며, 예컨대 질소원으로써 단백질 또는 아미노산 성분을 포함하는 것이거나 또는 ㈜ 조흥으로부터 분말이스트엑기스-II로써 구입된 것일 수 있다.The yeast extract may be included in about 0.1 to 10% by weight, preferably about 0.5 to 5% by weight, more preferably about 0.5 to 1% by weight, such as about 0.5 or 1% by weight, based on the total weight of the medium composition. may be %. Yeast extracts commercially available may be appropriately used, for example, those containing protein or amino acid components as a nitrogen source or may be purchased as Powdered Yeast Extract-II from Choheung Co., Ltd.
볶음 콩가루는 배지 조성물의 전체 중량을 기준으로, 약 0.1 내지 10 중량%로 포함될 수 있고, 바람직하게는 약 0.5 ~ 7 중량%, 보다 바람직하게는, 약 2 내지 6 중량%, 보다 바람직하게는 약 1 내지 5 중량%, 예컨대 약 1, 3, 또는 5 중량%일 수 있다. 볶음 콩가루는 상업용으로 시판되는 것을 적절하게 사용할 수 있으며, 예컨대 질소원으로써 단백질 또는 아미노산 성분을 포함하는 것으로서, 상업적으로 판매되는 것을 적절히 구입하여 사용할 수 있다(예컨대 일호식품으로부터 구입가능함).Roasted soybean flour may be included in about 0.1 to 10% by weight, preferably about 0.5 to 7% by weight, more preferably about 2 to 6% by weight, more preferably about 0.1 to 10% by weight based on the total weight of the medium composition. 1 to 5 weight percent, such as about 1, 3, or 5 weight percent. Roasted soybean flour can be suitably used commercially available, for example, commercially available ones containing protein or amino acid components as a nitrogen source can be appropriately purchased and used (for example, available from Ilho Foods).
소이 펩톤은 배지 조성물의 전체 중량을 기준으로, 약 0.1 내지 10 중량%로 포함될 수 있고, 바람직하게는 약 0.5 ~ 7 중량%, 보다 바람직하게는, 약 2 내지 6 중량%, 보다 바람직하게는 약 1 내지 5 중량%, 예컨대 약 5 중량%일 수 있다. 소이펩톤은 상업용으로 시판되는 것을 적절하게 사용할 수 있으며, 예컨대 SOLABIA로부터 구입할 수 있다.Soy peptone may be included in about 0.1 to 10% by weight, preferably about 0.5 to 7% by weight, more preferably about 2 to 6% by weight, more preferably about 0.1 to 10% by weight based on the total weight of the medium composition. 1 to 5% by weight, such as about 5% by weight. Soy peptone can be suitably used commercially available, and can be purchased from SOLABIA, for example.
상기 배지 조성물은 질소원으로서 탈지 분유를 추가로 포함할 수 있다. The medium composition may further include skim milk powder as a nitrogen source.
탈지 분유는 배지 조성물의 전체 중량을 기준으로, 약 0.1 내지 10 중량%로 포함될 수 있고, 바람직하게는 약 0.5 ~ 7 중량%, 보다 바람직하게는, 약 2 내지 6 중량%, 보다 바람직하게는 약 1 내지 5 중량%, 예컨대 약 5 중량%일 수 있다. 탈지분유는 상업용으로 시판되는 것을 적절하게 사용할 수 있으며, 예컨대 서울우유로부터 구입할 수 있다.Skim milk powder may be included in about 0.1 to 10% by weight, preferably about 0.5 to 7% by weight, more preferably about 2 to 6% by weight, more preferably about 0.1 to 10% by weight based on the total weight of the medium composition. 1 to 5% by weight, such as about 5% by weight. Skim milk powder commercially available can be suitably used, and can be purchased from Seoul Milk, for example.
배지 조성물은 질소원 이외에 탄소원을 필수 성분으로 포함하며, 탄소원은 이눌린(inulin), 치커리(chicory) 분말, 말토덱스트린 및 포도당에서 이루어진 군에서 선택된 하나 이상이다. 탄소원는 전체 배지 조성물의 중량을 기준으로, 약 1 ~ 40 중량%로 포함될 수 있고, 바람직하게는 약 5 ~ 20 중량%, 예컨대 약 5, 10, 13 중량%일 수 있다.The medium composition includes a carbon source as an essential component in addition to a nitrogen source, and the carbon source is at least one selected from the group consisting of inulin, chicory powder, maltodextrin, and glucose. The carbon source may be included in about 1 to 40% by weight, preferably about 5 to 20% by weight, such as about 5, 10, and 13% by weight, based on the weight of the total medium composition.
일 구체예에서, 상기 탄소원은 이눌린(inulin), 치커리(chicory) 분말, 말토덱스트린 및 포도당 중 하나를 단독으로 포함하거나, 이눌린 및 치커리 분말을 필수적으로 포함하거나, 이눌린, 치커리 분말 및 말토덱스트린을 필수적으로 포함할 수 있다.In one embodiment, the carbon source comprises alone one of inulin, chicory powder, maltodextrin and glucose, or consists essentially of inulin and chicory powder, or consists essentially of inulin, chicory powder and maltodextrin. can be included as
탄소원 각각은 부티레이트 생성량에 영향을 미칠 수 있다. 전체 배지 조성물의 중량을 기준으로, 이눌린은 약 1 ~ 10 중량%로 포함될 수 있고, 이러한 범위 내에서 부티레이트의 생성량이 더욱 증가할 수 있다. 보다 바람직하게는 약 3 ~ 7 중량%, 예컨대 약 5 중량%일 수 있다. 이눌린은 상업용으로 시판되는 것을 적절하게 사용할 수 있으며, 예컨대 BIOGLAN으로부터 구입할 수 있다.Each carbon source can affect the amount of butyrate produced. Based on the weight of the entire medium composition, inulin may be included in about 1 to 10% by weight, and within this range, the amount of butyrate produced may further increase. More preferably, it may be about 3 to 7% by weight, such as about 5% by weight. Inulin commercially available commercially available products may be suitably used, and may be obtained from, for example, BIOGLAN.
치커리 분말은 전체 배지 조성물의 중량을 기준으로, 약 1 ~ 10 중량%로 포함될 수 있고, 바람직하게는 약 3 ~ 7 중량%, 예컨대 약 5 중량%로 포함될 수 있다. 치커리 분말은 상업용으로 시판되는 것을 적절하게 사용할 수 있으며, 예컨대 조은약초로부터 구입할 수 있다.Chicory powder may be included in about 1 to 10% by weight, preferably about 3 to 7% by weight, such as about 5% by weight, based on the weight of the total medium composition. Chicory powder can be suitably used commercially available commercially, for example, it can be purchased from Joeun Herbs.
말토덱스트린은 전체 배지 조성물의 중량을 기준으로, 약 1 ~ 10 중량%로 포함될 수 있고, 바람직하게는 약 3 ~ 7 중량%, 예컨대 약 3 또는 5 중량%로 포함될 수 있다. 상기 말토덱스트린은 예컨대 난소화성 말토덱스트린일 수 있다. 말토덱스트린은 상업용으로 시판되는 것을 적절하게 사용할 수 있으며, 예컨대 삼양사로부터 구입할 수 있다.Maltodextrin may be included in about 1 to 10% by weight, preferably about 3 to 7% by weight, such as about 3 or 5% by weight, based on the weight of the total medium composition. The maltodextrin may be, for example, indigestible maltodextrin. Commercially available maltodextrins may be appropriately used, and may be purchased from Samyang Corporation, for example.
포도당은 전체 배지 조성물의 중량을 기준으로, 약 1 ~ 10 중량%로 포함될 수 있고, 바람직하게는 약 3 ~ 7 중량%, 예컨대 약 5 중량%로 포함될 수 있다. 포도당은 상업용으로 시판되는 것을 적절하게 사용할 수 있으며, 예컨대 삼양사로부터 구입할 수 있다.Glucose may be included in about 1 to 10% by weight, preferably about 3 to 7% by weight, such as about 5% by weight, based on the weight of the total medium composition. Glucose commercially available may be appropriately used, and may be purchased from Samyang Corporation, for example.
상기 배지 조성물은 탄소원으로서 구아검, 알긴산 나트륨, 펙틴 및 올리고당(예컨대, 갈락토올리고당, 프락토올리고당, 이소말토올리고당, 자일로올리고당, 대두올리고당, 락툴로스, 키틴올리고당, 이소말토올리고당 등)으로 이루어지는 군에서 선택되는 하나 이상을 추가로 포함할 수 있다. The medium composition consists of guar gum, sodium alginate, pectin and oligosaccharides (e.g., galactooligosaccharide, fructooligosaccharide, isomaltooligosaccharide, xylooligosaccharide, soybean oligosaccharide, lactulose, chitin oligosaccharide, isomaltooligosaccharide, etc.) as a carbon source. It may further include one or more selected from the group.
상기 구아검은 전체 배지 조성물의 중량을 기준으로, 약 0.1 내지 5 중량%, 바람직하게는 약 0.1 내지 1 중량%, 예컨대, 약 0.3 중량%로 포함될 수 있다.The guar gum may be included in about 0.1 to 5% by weight, preferably about 0.1 to 1% by weight, for example, about 0.3% by weight based on the weight of the total medium composition.
상기 알긴산나트륨은 전체 배지 조성물의 중량을 기준으로, 약 0.1 내지 5 중량%, 바람직하게는 약 0.1 내지 1 중량%, 예컨대, 약 0.3 중량%로 포함될 수 있다.The sodium alginate may be included in about 0.1 to 5% by weight, preferably about 0.1 to 1% by weight, for example, about 0.3% by weight based on the weight of the total medium composition.
상기 펙틴은 전체 배지 조성물의 중량을 기준으로, 약 0.1 내지 5 중량%, 바람직하게는 약 0.1 내지 1 중량%, 예컨대, 약 0.3 중량%로 포함될 수 있다.The pectin may be included in about 0.1 to 5% by weight, preferably about 0.1 to 1% by weight, for example, about 0.3% by weight based on the weight of the total medium composition.
상기 올리고당(예컨대, 갈락토올리고당, 프락토올리고당, 이소말토올리고당, 자일로올리고당, 대두올리고당, 락툴로스, 키틴올리고당, 이소말토올리고당 등)은 전체 배지 조성물의 중량을 기준으로, 약 0.1 내지 5 중량%, 바람직하게는 약 1 내지 3 중량%, 예컨대, 약 3 중량%로 포함될 수 있다.The oligosaccharide (eg, galactooligosaccharide, fructooligosaccharide, isomaltooligosaccharide, xylooligosaccharide, soybean oligosaccharide, lactulose, chitin oligosaccharide, isomaltooligosaccharide, etc.) is about 0.1 to 5 weight based on the weight of the total medium composition. %, preferably about 1 to 3% by weight, such as about 3% by weight.
상기 배지 조성물은 염화나트륨, 데히드로초산나트륨, 제2 인산칼륨, 황산마그네슘, 황산암모늄 및 탄산 칼슘으로 이루어지는 군에서 선택되는 하나 이상의 무기염류를 추가로 포함할 수 있다. 상기 무기염류 각각은 전체 배지 조성물의 중량을 기준으로, 약 0.01 내지 5 중량%, 바람직하게는 약 0.05 내지 1 중량%, 예컨대 약 0.06 중량%, 약 0.1 중량%, 약 0.3 중량%, 약 0.4 중량%, 약 0.5 중량%, 또는 약 1 중량%로 포함될 수 있다. 일 구체예에서, 상기 배지 조성물은 염화나트륨 약 0.5 중량%, 약 데히드로초산나트륨 0.3 중량%, 약 제2 인산칼륨 0.3 중량%, 약 황산마그네슘 0.06 중량%, 약 황산암모늄 0.4 중량% 및 탄산 칼슘 1 중량%를 추가로 포함한다. The medium composition may further include one or more inorganic salts selected from the group consisting of sodium chloride, sodium dehydroacetate, dibasic potassium phosphate, magnesium sulfate, ammonium sulfate and calcium carbonate. Each of the inorganic salts is about 0.01 to 5% by weight, preferably about 0.05 to 1% by weight, such as about 0.06% by weight, about 0.1% by weight, about 0.3% by weight, about 0.4% by weight, based on the weight of the total medium composition. %, about 0.5% by weight, or about 1% by weight. In one embodiment, the medium composition contains about 0.5% by weight of sodium chloride, about 0.3% by weight of sodium dehydroacetate, about 0.3% by weight of dibasic potassium phosphate, about 0.06% by weight of magnesium sulfate, about 0.4% by weight of ammonium sulfate and 1 calcium carbonate. Further includes % by weight.
배지 조성물이 상기 언급된 질소원, 탄소원을 포함하며, 선택적으로 상기 무기염류를 포함하는 경우, 부티레이트의 생성량이 증가되어, 이의 발효 생성물의 과체중 예방 효과가 더욱 우수해질 수 있다.When the medium composition includes the above-mentioned nitrogen source and carbon source, and optionally the above-mentioned inorganic salts, the production of butyrate is increased, so that the effect of preventing overweight of the fermentation product may be further improved.
일 구체예에서, 상기 배지 조성물은, 배지 조성물 전체 중량을 기준으로, 질소원으로서 효모 추출물 0.1 내지 10 중량%; 및 볶음 콩가루 0.1 내지 10 중량% 또는 소이 펩톤 0.1 내지 10 중량% 중 하나 이상; 및In one embodiment, the medium composition, based on the total weight of the medium composition, 0.1 to 10% by weight of yeast extract as a nitrogen source; and 0.1 to 10% by weight of roasted soybean flour or 0.1 to 10% by weight of soy peptone; and
탄소원으로서 이눌린(inulin) 1 내지 10 중량%, 치커리(chicory) 분말 1 내지 10 중량%, 말토덱스트린 1 내지 10 중량% 및 포도당 1 내지 10 중량%에서 이루어진 군에서 선택된 하나 이상을 포함할 수 있다.As a carbon source, at least one selected from the group consisting of 1 to 10% by weight of inulin, 1 to 10% by weight of chicory powder, 1 to 10% by weight of maltodextrin, and 1 to 10% by weight of glucose may be included.
이 때 상기 배지 조성물은, 상기 배지 조성물의 전체 중량을 기준으로, 질소원으로서 탈지 분유 0.1 내지 10 중량%를 추가로 포함할 수 있다.At this time, the medium composition may further include 0.1 to 10% by weight of skim milk powder as a nitrogen source based on the total weight of the medium composition.
또한 상기 배지 조성물은 탄소원으로서 구아검 0.1 내지 5 중량%, 알긴산 나트륨 0.1 내지 5 중량%, 펙틴 0.1 내지 5 중량% 및 올리고당 0.1 내지 5 중량%으로 이루어지는 군에서 선택되는 하나 이상을 추가로 포함할 수 있다.In addition, the medium composition may further include at least one selected from the group consisting of 0.1 to 5% by weight of guar gum, 0.1 to 5% by weight of sodium alginate, 0.1 to 5% by weight of pectin, and 0.1 to 5% by weight of oligosaccharide as a carbon source. there is.
또다른 구체예에서, 상기 배지 조성물은, 상기 배지 조성물의 전체 중량을 기준으로, In another embodiment, the medium composition, based on the total weight of the medium composition,
질소원으로서 효모 추출물 0.5 내지 1 중량%; 및 볶음 콩가루 1 내지 5 중량% 또는 소이 펩톤 5 중량% 중 하나 이상; 및0.5 to 1% by weight of yeast extract as a nitrogen source; and 1 to 5% by weight of roasted soybean flour or 5% by weight of soy peptone; and
탄소원으로서 이눌린(inulin) 5 중량%, 치커리(chicory) 분말 5 중량%, 말토덱스트린 3 내지 5 중량% 및 포도당 5 중량%에서 이루어진 군에서 선택된 하나 이상 At least one selected from the group consisting of 5% by weight of inulin, 5% by weight of chicory powder, 3 to 5% by weight of maltodextrin, and 5% by weight of glucose as a carbon source.
을 포함할 수 있다.can include
이 때 상기 배지 조성물은, 상기 배지 조성물의 전체 중량을 기준으로, 질소원으로서 탈지 분유 5 중량%를 추가로 포함할 수 있다.At this time, the medium composition may further include 5% by weight of skim milk powder as a nitrogen source based on the total weight of the medium composition.
또한 상기 배지 조성물은, 상기 배지 조성물의 전체 중량을 기준으로, 탄소원으로서 구아검 0.3 중량%, 알긴산 나트륨 0.3 중량%, 펙틴 0.3 중량% 및 올리고당 3 중량%으로 이루어지는 군에서 선택되는 하나 이상을 추가로 포함할 수 있다.In addition, the medium composition, based on the total weight of the medium composition, at least one selected from the group consisting of 0.3% by weight of guar gum, 0.3% by weight of sodium alginate, 0.3% by weight of pectin, and 3% by weight of oligosaccharide as a carbon source Further can include
상기 배지 조성물은, 또한 상기 배지 조성물의 전체 중량을 기준으로, 염화나트륨 0.5 중량%, 데히드로초산나트륨 0.3 중량%, 제2 인산칼륨 0.3 중량%, 황산마그네슘 0.06 중량%, 황산암모늄 0.4 중량% 및 탄산 칼슘 1 중량%의 무기염류를 추가로 포함할 수 있다. The medium composition also contains 0.5% by weight of sodium chloride, 0.3% by weight of sodium dehydroacetate, 0.3% by weight of potassium dihydrogen phosphate, 0.06% by weight of magnesium sulfate, 0.4% by weight of ammonium sulfate and carbonic acid, based on the total weight of the medium composition. Calcium 1% by weight of inorganic salts may be further included.
또다른 구체예에서, 상기 배지 조성물은 표 1 및 2에 언급된 실시예 1 내지 17의 조성을 가질 수 있다.In another embodiment, the medium composition may have the composition of Examples 1 to 17 mentioned in Tables 1 and 2.
이러한 수치범위 내에서, 조성물의 과체중에 대한 개선 효과가 더욱 향상될 수 있다. 또한, 이러한 수치범위를 벗어나게 되면, 부티레이트의 생산성이 낮아질 수 있고, 배양 배지 조성물을 제조할 때 용해가 잘 되지 않거나, 침전물이 발생하거나, 멸균 시 끓어 넘치는 현상이 발생할 수 있다. 특히 상기 배지 조성물은, 또한 상기 배지 조성물의 전체 중량을 기준으로, 질소원으로서 효모추출물 0.5 중량%; 및 소이펩톤 5 중량%를 포함하고, Within this numerical range, the improvement effect of the composition on overweight can be further improved. In addition, when the value is out of this range, the productivity of butyrate may be lowered, and the culture medium composition may not be dissolved well, precipitate may occur, or boiling may occur during sterilization. In particular, the medium composition, based on the total weight of the medium composition, yeast extract as a nitrogen source 0.5% by weight; And 5% by weight of soy peptone,
탄소원으로서 이눌린, 치커리 분말, 말토덱스트린 또는 포도당 각각 5 중량%를 포함하면서, As a carbon source, each containing 5% by weight of inulin, chicory powder, maltodextrin or glucose,
염화나트륨 0.5 중량%, 데히드로초산나트륨 0.3 중량%, 제2 인산칼륨 0.3 중량%, 황산마그네슘 0.06 중량%, 황산암모늄 0.4 중량% 및 탄산 칼슘 1 중량%의 무기염류를 추가로 포함하는 경우, 배지 조성물의 제조시 용해도가 향상되고 부티레이트의 생성량이 약 90 mM 이상, 바람직하게는 약 100 mM 이상, 보다 바람직하게는 약 200 mM 이상으로 증가할 수 있다. When inorganic salts of 0.5% by weight of sodium chloride, 0.3% by weight of sodium dehydroacetate, 0.3% by weight of potassium dibasic phosphate, 0.06% by weight of magnesium sulfate, 0.4% by weight of ammonium sulfate, and 1% by weight of calcium carbonate are included, the medium composition The solubility may be improved and the amount of butyrate produced may increase to about 90 mM or more, preferably about 100 mM or more, and more preferably about 200 mM or more.
또다른 구체예에서, 상기 단쇄 지방산은 부티레이트, 프로피오네이트, 아세테이트이며, 구체적으로, 부티레이트이다. 이에 따라 본 발명의 발효 생성물은 약 10 mM 이상의 부티레이트, 예컨대 약 20 mM 이상, 또는 약 30 mM 이상, 약 40 mM 이상, 약 50 mM 이상, 약 60 mM 이상, 약 70 mM 이상, 약 80 mM 이상, 약 90 mM 이상, 약 100 mM 이상, 약 110 mM 이상, 약 120 mM 이상, 약 130 mM 이상, 약 140 mM 이상, 약 150 mM 이상, 약 160 mM 이상, 약 170 mM 이상, 약 180 mM 이상, 약 190 mM 이상, 약 200 mM 이상, 약 210 mM 이상의 부티레이트를 포함하는 것일 수 있다.In another embodiment, the short chain fatty acid is butyrate, propionate, acetate, specifically butyrate. Accordingly, the fermentation product of the present invention contains at least about 10 mM butyrate, such as at least about 20 mM, or at least about 30 mM, at least about 40 mM, at least about 50 mM, at least about 60 mM, at least about 70 mM, at least about 80 mM. , about 90 mM or more, about 100 mM or more, about 110 mM or more, about 120 mM or more, about 130 mM or more, about 140 mM or more, about 150 mM or more, about 160 mM or more, about 170 mM or more, about 180 mM or more , It may include about 190 mM or more, about 200 mM or more, about 210 mM or more butyrate.
전술한 배지 조성물에 클로스트리디움(Clostridium) 균주를 접종한 후 발효(배양)시키면 단쇄 지방산을 포함하는 조성물이 제조될 수 있다. 이 제조 방법과 배양 조건에 대해서는 후술한다.A composition containing short-chain fatty acids can be prepared by inoculating a Clostridium strain into the aforementioned medium composition and then fermenting (cultivating) the strain. This production method and culture conditions are described later.
본 발명의 배지 조성물은 RCM(강화 클로스트리디아 배지; Reinforced Clostridial Medium)을 포함하지 않는다. RCM은 식품, 의료 분야 등에서 클로스트리디움과 기타 혐기성균의 배양에 사용되는 배지이다. 이 배지는 비멸균제품에 대한 미생물 검사 시 증균배지로 사용되며 USP, EP, JP 등에 등록된 표준 배지이기도 하다. 상기 배지는 Beef extract, Casein peptone, Dextrose, Sodium chloride, Sodium acetate, Yeast extract, Soluble Starch, L-Cysteine HCl, Agar 등을 포함하며, 상업적으로 쉽게 입수가능하다. RCM은 또한 식품, 음료 분야에서 Closteridium perfringens 같은 Sulfite reducing bacteria의 분별배지로도 이용되기도 한다. RCM의 용도 및 목적은 실험용으로 제조된 시약으로 식품 제조에 사용하기에는 어려움이 있으므로, 상업용 식품 조성물 등을 제조하기 위해서는 RCM을 사용하지 않는 것이 필요하다.The medium composition of the present invention does not contain RCM (Reinforced Clostridial Medium). RCM is a medium used for culturing Clostridium and other anaerobic bacteria in the food and medical fields. This medium is used as an enrichment medium for microbiological testing of non-sterilized products, and is also a standard medium registered in USP, EP, and JP. The medium includes Beef extract, Casein peptone, Dextrose, Sodium chloride, Sodium acetate, Yeast extract, Soluble Starch, L-Cysteine HCl, Agar, and the like, and is easily commercially available. RCM is also used as a differential medium for sulfite reducing bacteria such as Clostridium perfringens in food and beverage fields. Since the use and purpose of RCM is difficult to use in food production as a reagent prepared for experiments, it is necessary not to use RCM in order to prepare commercial food compositions and the like.
일 구체예에서, 상기 클로스트리디움 균주는, 클로스트리디움 부티리쿰(Clostridium butyricum), 클로스트리디움 타이로부티리쿰(Clostridium tyrobutyricum), 또는 클로스트리디움 사카로퍼부탈아세토니쿰(Clostridium saccharoperbutylacetonicum) 중 하나 이상을 포함하는 것이고, 구체적으로, 클로스트리디움 부티리쿰(Clostridium butyricum)이다.In one embodiment, the Clostridium strain is one of Clostridium butyricum, Clostridium tyrobutyricum, or Clostridium saccharoperbutylacetonicum. It includes the above, and specifically, it is Clostridium butyricum.
또다른 실시태양에서, 본 발명은 질소원으로서 효모 추출물; 및 볶음 콩가루 또는 소이 펩톤 중 하나 이상; 및 탄소원으로서 이눌린(inulin), 치커리(chicory) 분말, 말토덱스트린 및 포도당에서 이루어진 군에서 선택된 하나 이상 포함하며, 강화 클로스트리디아 배지를 포함하지 않는 배지 조성물을 제공한다. 상기 배지 조성물은 단쇄 지방산을 생성하기 위한 배지로서 사용될 수 있다.In another embodiment, the present invention provides a yeast extract as a nitrogen source; and at least one of roasted soy flour or soy peptone; and at least one selected from the group consisting of inulin, chicory powder, maltodextrin, and glucose as a carbon source, and does not include an enriched Clostridia medium. The medium composition can be used as a medium for producing short-chain fatty acids.
상기 배지 조성물은 질소원으로서 탈지 분유를 추가로 포함할 수 있다.The medium composition may further include skim milk powder as a nitrogen source.
상기 배지 조성물은 탄소원으로서 구아검, 알긴산 나트륨, 펙틴 및 올리고당으로 이루어지는 군에서 선택되는 하나 이상을 추가로 포함할 수 있다.The medium composition may further include at least one selected from the group consisting of guar gum, sodium alginate, pectin, and oligosaccharides as a carbon source.
상기 배지 조성물은 무기염류를 추가로 포함할 수 있으며, 예컨대 염화나트륨, 데히드로초산나트륨, 제2 인산칼륨, 황산마그네슘, 황산암모늄 및 탄산 칼슘으로 이루어지는 군에서 선택되는 하나 이상의 무기염류를 추가로 포함할 수 있다.The medium composition may further include inorganic salts, for example, one or more inorganic salts selected from the group consisting of sodium chloride, sodium dehydroacetate, dibasic potassium phosphate, magnesium sulfate, ammonium sulfate and calcium carbonate. can
상기 배지 조성물에 포함되는 각각의 성분의 구체적 종류 및 함량은 상기 언급된 바에 따른다. The specific types and contents of each component included in the medium composition are as described above.
발효 생성물의 체중 감량 용도Weight Loss Uses of Fermentation Products
또다른 실시태양에서, 본 발명의 발효 생성물은 과체중의 개선, 예방 또는 치료를 위해 사용될 수 있다. 구체적으로, 본 발명의 발효 조성물은 지방 세포 분화 억제 효과를 나타내며, 지방 축적 감소, 나아가 트리글리세라이드 함량 억제 효과를 나타낸다. 또한 본 발명의 발효 생성물은 고지방 식이군에서 체중이 유의적으로 감소시키는 효과를 나타내었으며, 체지방률 및 체지방량 역시 감소시켰다. 결국 본 발명의 발효 조성물은 비만 세포의 발현을 억제시킬 수 있고, 체중 및 체지방량 감소시켜, 과체중의 개선, 예방 또는 치료에 사용될 수 있다. In another embodiment, the fermentation product of the present invention can be used for the amelioration, prevention or treatment of overweight. Specifically, the fermented composition of the present invention exhibits an effect of inhibiting differentiation of adipocytes, reducing fat accumulation, and further inhibiting triglyceride content. In addition, the fermentation product of the present invention showed an effect of significantly reducing body weight in the high-fat diet group, and also reduced the percentage of body fat and the amount of body fat. As a result, the fermented composition of the present invention can suppress the expression of mast cells and reduce body weight and fat mass, so that it can be used for improvement, prevention or treatment of overweight.
본 발명의 발효 생성물은 또한 농도 의존적인 항산화 효과를 나타내며, 나아가 TNF alpha 수준을 감소시킴이 확인되어 염증성 질환 개선에도 효과를 나타낸다. The fermentation product of the present invention also exhibits a concentration-dependent antioxidant effect, and furthermore, it has been confirmed that it reduces the level of TNF alpha, showing an effect on improving inflammatory diseases.
이에 따라 본 발명은 상기 발효 생성물을 포함하는 조성물을 제공한다. 상기 조성물은 과체중의 개선, 예방 또는 치료 용도에 사용될 수 있다.Accordingly, the present invention provides a composition comprising the fermentation product. The composition may be used for improvement, prevention or treatment of overweight.
일 구체예에서, 본 발명은 상기 발효 생성물을 포함하는 과체중의 예방 또는 치료용 약학 조성물 또는 수의학 조성물을 제공한다. In one embodiment, the present invention provides a pharmaceutical composition or veterinary composition for preventing or treating overweight, comprising the fermentation product.
또다른 구체예에서, 본 발명은 상기 발효 생성물을 포함하는 과체중의 개선 또는 예방용 식품 조성물 또는 동물용 사료 조성물을 제공한다.In another embodiment, the present invention provides a food composition or animal feed composition for improving or preventing overweight, comprising the fermentation product.
본원에 사용된 용어 "조성물"은 특정 양으로의 특정 성분을 포함하는 생성물 및 특정 양으로의 특정 성분의 조합에 의해 직접적으로 또는 간접적으로 야기된 임의의 생성물을 포함한다. 그것이 약학 조성물에 관련될 때 이러한 용어는 활성 성분 및 담체를 구성하는 불활성 성분을 포함하는 생성물을 포함하고, 임의의 2종 이상의 성분의 조합, 복합화 또는 응집 또는 1종 이상의 성분의 해리, 다른 종류의 반응 또는 상호작용에 의해 직접적으로 또는 간접적으로 야기된 임의의 생성물을 포함하는 것으로 의도된다. As used herein, the term "composition" includes a product comprising a specified component in a specified amount and any product that results directly or indirectly from the combination of a specified component in a specified amount. As it relates to a pharmaceutical composition, this term encompasses a product comprising an active ingredient and an inactive ingredient constituting a carrier, and includes any combination, complexation or aggregation of two or more ingredients or dissociation of one or more ingredients, or other types of It is intended to include any product resulting directly or indirectly by a reaction or interaction.
본원에서 "조성물"은 인간에게 의약품으로 사용하기 위한 약학 조성물, 동물에게 의약품으로 사용하기 위한 수의학 조성물, 또는 사람 또는 동물용 식이 제품 또는 식품(예를 들어, 기능성 식품 조성물, 건강기능식품, 즉, 식품, 음료, 사료 또는 반려동물 식품, 또는 식품, 음료, 동물용 사료 또는 반려동물 식품 보충제) 등을 모두 포함한다. 이에 따라 본원에서의 "조성물"은 "약학 조성물", "수의학 조성물", 또는 "식품 조성물"을 모두 포함하는 의미로 사용된다. 상기 조성물은 각각 용도에 따라 약학성 허용되는 부형제, 수의학상 허용되는 부형제, 식품상 허용되는 부형제 등을 추가로 포함할 수 있다.As used herein, "composition" refers to a pharmaceutical composition for use as a medicine for humans, a veterinary composition for use as a medicine for animals, or a dietary product or food for humans or animals (e.g., functional food composition, health functional food, i.e., food, beverage, feed or pet food, or food, beverage, animal feed or pet food supplement); Accordingly, "composition" herein is used as a meaning including both "pharmaceutical composition", "veterinary composition", or "food composition". The composition may further include a pharmaceutically acceptable excipient, a veterinarily acceptable excipient, a food acceptable excipient, and the like, depending on each use.
본원에 사용된 어구 "약학상 허용되는", "수의학상 허용되는", 또는 "식품상 허용되는"은 합리적인 이익/위험 비와 어울리는, 건전한 의학적 또는 식품상 판단의 범주 내에서, 과독한 독성, 자극, 알레르기 반응, 또는 다른 문제 또는 합병증 없이 인간 또는 동물의 조직과 접촉하여 사용하기에 적합한 화합물, 물질, 조성물, 담체, 및/또는 투여 형태를 지칭한다.As used herein, the phrases "pharmaceutically acceptable," "veterinarily acceptable," or "food acceptable" means, within the scope of sound medical or food judgment, to match a reasonable benefit/risk ratio, excessive toxicity, Refers to a compound, material, composition, carrier, and/or dosage form suitable for use in contact with human or animal tissue without irritation, allergic reaction, or other problem or complication.
본원에 사용된 어구 "약학상 허용되는 부형제", "수의학상 허용되는 부형제" 또는 "식품상 허용되는 부형제"는 일반적으로 안전하고, 비독성이며, 생물학적으로도 다른 방식으로도 바람직한 약학, 수의학 또는 식품 조성물을 제조하는데 유용한 부형제를 의미하며, 인간 약학 용도 또는 동물 수의학 용도, 나아가 식품 용도를 위해 허용되는 부형제를 포함한다. 명세서 및 청구범위에 사용된 "약학상 허용되는 부형제", "수의학상 허용되는 부형제", 또는 "식품상 허용되는 성분"는 1종 이상의 이러한 부형제 둘 다를 포함한다. 예를 들어, 본 발명의 제제에 사용되는 약학상, 수의학상, 식품상 허용되는 부형제는 희석제 또는 불활성 담체, 윤활제, 결합제, 또는 이들의 조합일 수 있다. 본 발명의 제제에 사용되는 부형제는 충전제, 항-미생물제, 항산화제, 케이킹 방지제, 코팅제, 또는 이들의 혼합물을 추가로 포함할 수 있다. 그 외 약학상, 수의학상, 또는 식품성 허용되는 부형제라면 제한없이 사용될 수 있다.As used herein, the phrases "pharmaceutically acceptable excipient", "veterinarily acceptable excipient" or "food acceptable excipient" are generally safe, non-toxic, biologically and otherwise desirable pharmaceutical, veterinary or otherwise acceptable excipients. It means an excipient useful for preparing a food composition, and includes an excipient acceptable for human pharmaceutical use or animal veterinary use, and further for food use. As used in the specification and claims, "pharmaceutically acceptable excipient", "veterinarily acceptable excipient", or "food acceptable ingredient" includes both one or more of these excipients. For example, pharmaceutically, veterinarily, or food-acceptable excipients used in the formulation of the present invention may be diluents or inert carriers, lubricants, binders, or combinations thereof. Excipients used in the formulations of the present invention may further include fillers, anti-microbial agents, antioxidants, anti-caking agents, coating agents, or mixtures thereof. Any other pharmaceutical, veterinary, or food acceptable excipient may be used without limitation.
본 발명에 따른 조성물은 과체중의 개선, 예방 또는 치료용 약학 또는 수의학 조성물일 수도 있고, 과체중의 개선용 식품 조성물일 수도 있으며, 이외에도 다양한 용도로 사용될 수 있다. 예컨대, 본 발명의 조성물은 과체중 개선 이외에도, 항염증 또는 항산화 용도로 사용될 수 있다. The composition according to the present invention may be a pharmaceutical or veterinary composition for improving, preventing or treating overweight, a food composition for improving overweight, and may be used for various other purposes. For example, the composition of the present invention may be used for anti-inflammatory or antioxidant purposes in addition to overweight improvement.
본 발명에 따른 조성물은 다양한 방법으로 인체에 투여될 수 있고, 다른 포유류에도 투여될 수 있다. 여기서, 다른 포유류로는 개, 고양이, 토끼, 돼지, 양, 염소, 젖소, 말, 소 등의 가축, 애완 동물일 수 있으나, 이에 제한되지 않는다.The composition according to the present invention can be administered to the human body in a variety of ways, and can also be administered to other mammals. Here, other mammals may be domestic animals such as dogs, cats, rabbits, pigs, sheep, goats, dairy cows, horses, and cattle, and pets, but are not limited thereto.
조성물이 약학 또는 수의학 조성물인 경우, 상기 조성물은 다양한 비경구 또는 경구 투여용 형태로 제조될 수 있다. 비경구 투여용 제형의 대표적인 것으로 주사용 제형이 있고, 이때 조성물의 형태는 등장성 수용액 또는 현탁액인 것이 바람직할 수 있다. 주사용 제형은 적합한 분산제 또는 습윤제, 현탁화제를 사용하여 당업계에 공지된 기술에 따라 제조할 수 있다. 예를 들어, 각 성분을 식염수 또는 완충액에 용해시켜 주사용으로 제형화 할 수 있다. 경구용 제형으로는 분말, 과립, 정제, 환약, 캡슐 등이 있을 수 있고, 이에 제한되지 않는다. When the composition is a pharmaceutical or veterinary composition, the composition may be formulated into a variety of forms for parenteral or oral administration. Representative formulations for parenteral administration include formulations for injection, and in this case, the form of the composition may be preferably an isotonic aqueous solution or suspension. Formulations for injection may be prepared according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. For example, each component may be dissolved in saline or a buffer solution and formulated for injection. Formulations for oral use may include powders, granules, tablets, pills, capsules, and the like, but are not limited thereto.
본 발명에 따른 조성물은 약물 전달체를 이용하여 다양한 제제로 제조될 수 있다. 약물 전달 시스템은 리포좀, 에토좀, 탄성 에토좀, 타겟팅 리포좀, 마이크로입자, 마이크로캡슐, 나노입자, 나노캡슐, 나노 파이버 등일 수 있고, 이에 제한되지 않는다.The composition according to the present invention can be prepared in various formulations using a drug delivery system. The drug delivery system may be liposomes, ethosomes, elastic ethosomes, targeting liposomes, microparticles, microcapsules, nanoparticles, nanocapsules, nanofibers, and the like, but is not limited thereto.
조성물이 식품 조성물인 경우, 체지방 감소를 위한 식품, 예컨대 식품의 주원료, 부원료, 식품 첨가제, 기능성 식품, 음료 등으로 활용될 수 있다. 식품용 조성물은, 예를 들어, 건강기능식품, 비타민 복합제, 껌, 음료, 각종 식품류, 차, 각종 식육가공품, 어육제품 두부류, 묵류, 면류, 빵류, 건강보조식품, 조미식품, 소스류, 과자류, 캔디류, 유가공품, 기타 가공식품, 발효 식품, 천연조미료 등으로 활용될 수 있으나, 이에 제한되지 않는다.When the composition is a food composition, it may be used as a food for reducing body fat, such as a main ingredient, a supplementary ingredient, a food additive, a functional food, or a beverage. Compositions for food include, for example, health functional foods, vitamin complexes, chewing gum, beverages, various foods, tea, various processed meat products, fish products, tofu, jelly, noodles, breads, health supplements, seasonings, sauces, confectionery, Candies, dairy products, other processed foods, fermented foods, natural seasonings, etc. can be used, but are not limited thereto.
본 발명에서 용어 "건강기능식품"이란, 인체에 유용한 기능성을 가진 원료나 성분을 사용하여 제조 및 가공한 식품을 말한다. 상기 "기능성" 은 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건용도에 유용한 효과를 얻는 것을 의미한다. 본 발명의 건강기능식품은 당업계에서 통상적으로 사용되는 방법에 의하여 제조 가능하며, 제조시 당업계에서 통상적으로 첨가하는 원료 및 성분을 첨가하여 제조할 수 있다. 또한 상기 건강기능식품의 제형 또한 건강기능식품으로 인정되는 제형이면 제한없이 제조될 수 있다.In the present invention, the term "health functional food" refers to food manufactured and processed using raw materials or ingredients having useful functionality for the human body. The above "functionality" means obtaining useful effects for health purposes such as regulating nutrients for the structure and function of the human body or physiological functions. The health functional food of the present invention can be prepared by a method commonly used in the art, and can be prepared by adding raw materials and components commonly added in the art during manufacture. In addition, the formulation of the health functional food may also be manufactured without limitation as long as the formulation is recognized as a health functional food.
발효 생성물의 제조 방법Methods for producing fermentation products
또다른 실시태양에서 본 발명은In another embodiment, the present invention
(a) 질소원으로서 효모 추출물; 및 볶음 콩가루 또는 소이 펩톤 중 하나 이상; 및(a) yeast extract as a nitrogen source; and at least one of roasted soy flour or soy peptone; and
탄소원으로서 이눌린(inulin), 치커리(chicory) 분말, 말토덱스트린 및 포도당에서 이루어진 군에서 선택된 하나 이상을 물에 용해 및 멸균시켜 배지 조성물을 제조하는 단계;Preparing a medium composition by dissolving and sterilizing at least one selected from the group consisting of inulin, chicory powder, maltodextrin and glucose as a carbon source in water;
(b) 상기 배지 조성물에 클로스트리디움(Clostridium) 균주를 접종시키는 단계; 및(b) inoculating a Clostridium strain into the medium composition; and
(c) 상기 클로스트리디움 균주가 접종된 배지 조성물을 발효시켜 단쇄 지방산을 포함하는 발효 생성물을 제조하는 단계(c) preparing a fermentation product containing short-chain fatty acids by fermenting the medium composition inoculated with the Clostridium strain
를 포함하는, 상기 발효 생성물의 제조 방법을 제공한다.Including, it provides a method for producing the fermentation product.
단계 (a)에서 멸균은 약 100 ~ 130℃의 온도에서 약 10 ~ 20 분 동안 가열시킴으로써 수행될 수 있고, 이러한 조건에서, 멸균이 효과적으로 이루어질 수 있다. 예를 들어, 가열 온도는 약 120℃일 수 있고, 가열 시간은 약 15분 일 수 있다. Sterilization in step (a) may be performed by heating at a temperature of about 100 to 130° C. for about 10 to 20 minutes, and under these conditions, sterilization can be effectively achieved. For example, the heating temperature may be about 120° C., and the heating time may be about 15 minutes.
단계 (c)에서 발효는 혐기성 배양통에 혐기성 팩을 넣어 배양 상태를 혐기성으로 유지시켜 약 1 ~ 15일 동안 배양이 이루어질 수 있다. 보다 바람직하게는 약 1 ~ 5일 간 배양이 이루어질 수 있다. 이때, 배양 온도는 약 25 ~ 37℃일 수 있다. 예를 들어, 배양 기간은 약 5일이고, 배양 온도는 약 37℃일 수 있다.In step (c), fermentation can be performed for about 1 to 15 days by putting an anaerobic pack in an anaerobic culture tank to maintain the culture state anaerobically. More preferably, the culture may be performed for about 1 to 5 days. At this time, the culture temperature may be about 25 ~ 37 ℃. For example, the incubation period may be about 5 days and the incubation temperature may be about 37°C.
본 발명에 따르면, 단쇄 지방산, 예컨대 부티레이트를 고농도로 포함하는 발효 생성물을 제공할 수 있다. 이에 따라 본 발명의 발효 조성물은 비만 세포의 발현을 억제시킬 수 있고, 체중 및 체지방량 감소시켜, 과체중의 개선, 예방 또는 치료에 효과적으로 사용될 수 있다. According to the present invention, it is possible to provide a fermentation product containing a high concentration of short-chain fatty acids such as butyrate. Accordingly, the fermented composition of the present invention can suppress the expression of mast cells and reduce body weight and fat mass, so that it can be effectively used for improvement, prevention or treatment of overweight.
도 1은 실시예에 따른 조성물의 장관 내피 세포에서의 세포 독성을 측정한 결과를 나타내는 그래프이다.1 is a graph showing the results of measuring the cytotoxicity of compositions according to Examples in intestinal endothelial cells.
도 2는 실시예에 따른 조성물의 항산화 효능을 측정한 결과를 나타내는 그래프이다.Figure 2 is a graph showing the results of measuring the antioxidant efficacy of the composition according to the embodiment.
도 3은 실시예에 따른 조성물에 대한, 초대 배양한 비장세포에서의 티엔에프알파 레벨 감소를 측정한 결과를 나타내는 그래프이다.Figure 3 is a graph showing the results of measuring the decrease in TNF alpha level in primary cultured splenocytes for the composition according to the Example.
도 4는 실시예에 따른 조성물에 대한, DSS로 유도된 장염 동물의 혈장에서 IgE 레벨 감소를 측정한 결과를 나타내는 그래프이다.Figure 4 is a graph showing the results of measuring the IgE level reduction in the plasma of animals with DSS-induced enteritis for the composition according to the Example.
도 5는 실시예에 따른 조성물에 대한, 3T3-L1 프리아디포사이트 세포에 대한 세포 독성 평가를 측정한 결과를 나타내는 그래프이다.Figure 5 is a graph showing the results of measuring the cytotoxicity evaluation on 3T3-L1 preadipocyte cells for the composition according to the example.
도 6은 실시예에 따른 조성물에 대한, 3T3-L1 지방 전구 세포에서 지방 세포로 분화 유도 측정한 결과를 나타내는 그래프이다.6 is a graph showing the results of measuring the induction of differentiation from 3T3-L1 preadipocytes to adipocytes with respect to the composition according to the example.
도 7은 실시예에 따른 조성물을 오일레드오 염색으로 확인한 사진이다.7 is a photograph confirming the composition according to the Example by Oil Red O staining.
도 8은 실시예에 따른 조성물에 대해서, 순수한 트리글리세라이드(triglycerides, TG) 함량을 측정한 결과를 나타내는 그래프이다.8 is a graph showing the results of measuring the pure triglycerides (TG) content of the compositions according to Examples.
도 9는 실시예에 따른 조성물 투여에 따른 고지방 식이 비만 유도 mice에서 체중 변화를 측정한 결과이다.Figure 9 is the result of measuring the weight change in obesity-induced mice on a high-fat diet according to the administration of the composition according to the embodiment.
도 10은 실시예에 따른 조성물 투여에 따른 고지방 식이 비만 유도 mice에서 체지방률 및 체지방량을 측정한 결과이다.10 is a result of measuring the percentage of body fat and the amount of body fat in obesity-induced mice fed a high-fat diet according to administration of the composition according to the example.
도 11은 실시예에 따른 조성물 투여에 따른 고지방 식이 비만 유도 mice에서 장기 중량에 미치는 효과를 측정한 결과이다. 11 is a result of measuring the effect on organ weight in obesity-induced mice fed with a high-fat diet according to administration of the composition according to the Example.
이하에서는 실시예를 통해 본 발명을 보다 상세히 설명한다. 그러나, 본 발명은 하기 실시예에 한정되지 않는다.Hereinafter, the present invention will be described in more detail through examples. However, the present invention is not limited to the following examples.
실시예 1Example 1
전체 배양 배지의 중량을 기준으로, 효모 추출물 1 중량%, 볶음 콩가루 1 중량%를 첨가하고, 이눌린 5 중량% 및 치커리 분말 5 중량%를 정제수에 첨가한 후 용해시켰다(RCM(Reinforced Clostridial Medium)은 사용하지 않음). 오토클레이브를 사용하여 121℃에서 15 분 동안 멸균시킨 뒤 배양 배지로써 사용하였다.Based on the weight of the total culture medium, 1% by weight of yeast extract and 1% by weight of roasted soybean flour were added, and 5% by weight of inulin and 5% by weight of chicory powder were added to purified water and then dissolved (Reinforced Clostridial Medium (RCM) was not used). After sterilization at 121 ° C. for 15 minutes using an autoclave, it was used as a culture medium.
고체 배지에 배양된 클로스트리디움 부티리쿰을 1백금이 소량 채취하여 멸균이 된 배양 배지에 접종하고, 37℃ 인큐베이터(Incubator)에 혐기성 상태를 유지시키기 위해, 배지를 혐기 배양통 2.5 L에 넣고, 가스팩을 이용해 배양하였다. 이후 혐기 배양상태 표시종이를 이용해 혐기 배양통 내부의 산소 유무를 확인해 가며 총 5일 동안 정치배양하였다.A small amount of 1 platinum of Clostridium butyricum cultured on a solid medium is inoculated into a sterilized culture medium, and in order to maintain an anaerobic state in an incubator at 37 ° C, the medium is placed in an anaerobic culture tank 2.5 L, Incubated using a gas pack. Thereafter, the anaerobic culture status indicator paper was used to check the presence or absence of oxygen in the anaerobic culture tank, and the cells were cultured for a total of 5 days.
실시예 2Example 2
볶음 콩가루를 3 중량%(전체 배양 배지 기준)를 첨가한 것을 제외하고는 실시예 1과 동일한 조건으로 조성물을 제조하였다.A composition was prepared under the same conditions as in Example 1, except that 3% by weight (based on the total culture medium) of roasted soy flour was added.
실시예 3Example 3
볶음 콩가루를 5 중량%(전체 배양 배지 기준)를 첨가한 것을 제외하고는 실시예 1과 동일한 조건으로 조성물을 제조하였다.A composition was prepared under the same conditions as in Example 1, except that 5% by weight of roasted soybean flour (based on the total culture medium) was added.
실시예 4Example 4
구아검을 0.3 중량%(전체 배양 배지 기준)를 추가로 첨가한 것을 제외하고는 실시예 3과 동일한 조건으로 조성물을 제조하였다.A composition was prepared under the same conditions as in Example 3, except that 0.3% by weight of guar gum (based on the total culture medium) was additionally added.
실시예 5Example 5
알긴산나트륨을 0.3 중량%(전체 배양 배지 기준)를 추가로 첨가한 것을 제외하고는 실시예 3과 동일한 조건으로 조성물을 제조하였다.A composition was prepared under the same conditions as in Example 3, except that 0.3% by weight of sodium alginate (based on the total culture medium) was additionally added.
실시예 6Example 6
펙틴을 0.3 중량%(전체 배양 배지 기준)를 추가로 첨가한 것을 제외하고는 실시예 3과 동일한 조건으로 조성물을 제조하였다.A composition was prepared under the same conditions as in Example 3, except that 0.3% by weight (based on the total culture medium) of pectin was additionally added.
실시예 7Example 7
프락토올리고당 분말을 3 중량%(전체 배양 배지 기준)를 추가로 첨가한 것을 제외하고는 실시예 3과 동일한 조건으로 조성물을 제조하였다.A composition was prepared under the same conditions as in Example 3, except that 3% by weight (based on the total culture medium) of fructooligosaccharide powder was additionally added.
실시예 8Example 8
갈락토올리고당을 3 중량%(전체 배양 배지 기준)를 추가로 첨가한 것을 제외하고는 실시예 3과 동일한 조건으로 조성물을 제조하였다.A composition was prepared under the same conditions as in Example 3, except that 3% by weight (based on the total culture medium) of galacto-oligosaccharide was additionally added.
실시예 9Example 9
난소화성 말토덱스트린을 3 중량%(전체 배양 배지 기준)를 추가로 첨가한 것을 제외하고는 실시예 3과 동일한 조건으로 조성물을 제조하였다.A composition was prepared under the same conditions as in Example 3, except that 3% by weight (based on the total culture medium) of indigestible maltodextrin was additionally added.
실시예 10Example 10
전체 배양 배지의 중량을 기준으로, 질소원으로써 소이 펩톤 5 중량% 및 효모 추출물 0.5 중량%를 첨가하고, 탄소원으로써 이눌린 5 중량%, 무기염류로써 염화나트륨 0.5 중량%, 데히드로초산나트륨 0.3 중량%, 제2인산칼륨 0.3 중량%, 황산마그네슘 0.06 중량%, 황산암모늄 0.4 중량%, 탄산칼슘 1 중량%를 정제수에 첨가한 후 용해시킨 후 오토클레이브를 사용하여 121℃에서 15 분 동안 멸균시킨 뒤 배양 배지로써 사용하였다.Based on the weight of the total culture medium, 5% by weight of soy peptone and 0.5% by weight of yeast extract were added as a nitrogen source, 5% by weight of inulin as a carbon source, 0.5% by weight of sodium chloride as an inorganic salt, 0.3% by weight of sodium dehydroacetate, 0.3% by weight of potassium diphosphate, 0.06% by weight of magnesium sulfate, 0.4% by weight of ammonium sulfate, and 1% by weight of calcium carbonate were added to purified water and then dissolved. used
고체 배지에 배양된 클로스트리디움 부티리쿰을 1백금이 소량 채취하여 멸균이 된 배양 배지에 접종하고, 37℃ 인큐베이터(Incubator)에 혐기성 상태를 유지시키기 위해, 배지를 혐기 배양통 2.5 L에 넣고, 가스팩을 이용해 배양하였다. 이후 혐기 배양상태 표시종이를 이용해 혐기 배양통 내부의 산소 유무를 확인해 가며 총 5일 동안 정치배양하였다.A small amount of 1 platinum of Clostridium butyricum cultured on a solid medium is inoculated into a sterilized culture medium, and in order to maintain an anaerobic state in a 37 ° C incubator, the medium is placed in an anaerobic culture tank 2.5 L, Incubated using a gas pack. Thereafter, the anaerobic culture status indicator paper was used to check the presence or absence of oxygen in the anaerobic culture tank, and the cells were cultured for a total of 5 days.
실시예 11Example 11
탄소원으로써 치커리 분말 5 중량%를 첨가한 것을 제외하고는 실시예 10과 동일한 조건으로 조성물을 제조하였다.A composition was prepared under the same conditions as in Example 10, except that 5% by weight of chicory powder was added as a carbon source.
실시예 12Example 12
탄소원으로써 난소화성말토덱스트린 5 중량%를 첨가한 것을 제외하고는 실시예 10과 동일한 조건으로 조성물을 제조하였다.A composition was prepared under the same conditions as in Example 10, except that 5% by weight of indigestible maltodextrin was added as a carbon source.
실시예 13Example 13
탄소원으로써 포도당 5 중량%를 첨가한 것을 제외하고는 실시예 10과 동일한 조건으로 조성물을 제조하였다.A composition was prepared under the same conditions as in Example 10, except that 5% by weight of glucose was added as a carbon source.
실시예 14Example 14
질소원으로써 효모 추출물 0.5 중량%; 및 소이 펩톤과 탈지분유 각 5 중량%를 첨가한 것을 제외하고는 실시예 10과 동일한 조건으로 조성물을 제조하였다.Yeast extract 0.5% by weight as a nitrogen source; And a composition was prepared under the same conditions as in Example 10, except that 5% by weight of each of soy peptone and skim milk powder was added.
실시예 15Example 15
질소원으로써 효모 추출물 0.5 중량%; 및 소이 펩톤과 탈지분유 각 5 중량%, 탄소원으로써 치커리 분말 5 중량%를 첨가한 것을 제외하고는 실시예 10과 동일한 조건으로 조성물을 제조하였다.Yeast extract 0.5% by weight as a nitrogen source; And a composition was prepared under the same conditions as in Example 10, except that 5% by weight of each of soy peptone and skim milk powder and 5% by weight of chicory powder as a carbon source were added.
실시예 16Example 16
질소원으로써 효모 추출물 0.5 중량%; 및 소이 펩톤과 탈지분유 각 5 중량%, 탄소원으로써 난소화성말토덱스트린 5 중량%를 첨가한 것을 제외하고는 실시예 10과 동일한 조건으로 조성물을 제조하였다.Yeast extract 0.5% by weight as a nitrogen source; A composition was prepared under the same conditions as in Example 10, except that 5% by weight of each of soy peptone and skim milk powder and 5% by weight of indigestible maltodextrin as a carbon source were added.
실시예 17Example 17
질소원으로써 효모 추출물 0.5 중량%; 및 소이 펩톤과 탈지분유 각 5 중량%, 탄소원으로써 포도당 5 중량%를 첨가한 것을 제외하고는 실시예 10과 동일한 조건으로 조성물을 제조하였다.Yeast extract 0.5% by weight as a nitrogen source; A composition was prepared under the same conditions as in Example 10, except that 5% by weight of each of soy peptone and skim milk powder and 5% by weight of glucose as a carbon source were added.
비교예 1Comparative Example 1
RCM(Reinforced Clostridial Medium)을 38.4 g/L 농도로 정제수에 혼합 후 오토클레이브(Autoclave)를 사용하여 121℃에서 15 분 동안 멸균시킨 뒤 배양 배지로 사용하였다.RCM (Reinforced Clostridial Medium) was mixed with purified water at a concentration of 38.4 g/L, sterilized using an autoclave at 121 ° C. for 15 minutes, and then used as a culture medium.
제조한 배양 배지에 RCM 고체 배지에 배양된 클로스트리디움 부티리쿰을 1백금이 소량 채취하여 접종하고, 37℃ 인큐베이터(Incubator)에 혐기성 상태를 유지시키기 위해, 배지를 2.5 L의 혐기 배양통에 넣고, 가스팩을 이용해 배양하였다. 이 후 혐기 배양상태 표시종이를 이용해 혐기 배양통 내부의 산소 유무를 확인해 가며 총 5일 동안 정치배양하였다.In the prepared culture medium, a small amount of 1 platinum is collected and inoculated with Clostridium butyricum cultured on RCM solid medium, and in order to maintain an anaerobic state in an incubator at 37 ° C, the medium is placed in a 2.5 L anaerobic culture tank , cultured using a gas pack. Thereafter, the anaerobic culture status indicator paper was used to check the presence or absence of oxygen inside the anaerobic culture tank, and the culture was carried out for a total of 5 days.
비교예 2Comparative Example 2
멸균 이전에 전체 배양 배지 기준으로 이눌린 분말을 1 중량% 첨가한 것을 제외하고는, 비교예 1과 동일한 조건으로 조성물을 제조하였다.A composition was prepared under the same conditions as in Comparative Example 1, except that 1% by weight of inulin powder was added based on the total culture medium prior to sterilization.
비교예 3Comparative Example 3
전체 배양 배지 기준으로 이눌린 분말을 5 중량% 첨가한 것을 제외하고는, 비교예 2와 동일한 조건으로 조성물을 제조한다.A composition was prepared under the same conditions as in Comparative Example 2, except that 5% by weight of inulin powder was added based on the total culture medium.
비교예 4Comparative Example 4
전체 배양 배지 기준으로 이눌린 분말을 10 중량% 첨가한 것을 제외하고는, 비교예 2와 동일한 조건으로 조성물을 제조한다.A composition was prepared under the same conditions as in Comparative Example 2, except that 10% by weight of inulin powder was added based on the total culture medium.
비교예 5Comparative Example 5
전체 배양 배지 기준으로 이눌린 분말을 20 중량% 첨가한 것을 제외하고는, 비교예 2와 동일한 조건으로 조성물을 제조한다.A composition was prepared under the same conditions as in Comparative Example 2, except that 20% by weight of inulin powder was added based on the total culture medium.
비교예 6Comparative Example 6
이눌린 분말 대신, 치커리 분말을 전체 배양 배지 기준으로 5 중량% 첨가한 것을 제외하고는, 비교예 2와 동일한 조건으로 조성물을 제조한다.A composition was prepared under the same conditions as in Comparative Example 2, except that 5% by weight of chicory powder was added based on the total culture medium instead of inulin powder.
비교예 7Comparative Example 7
전체 배양 배지의 질량을 기준으로, RCM을 대체하여 효모 추출물 1 중량%, 분리대두단백 1 중량%를 첨가하고, 이눌린 5 중량% 및 치커리 분말 5 중량%를 정제수에 첨가한 후 용해시켰다. 오토클레이브를 사용하여 121℃에서 15 분 동안 멸균시킨 뒤 배양 배지로써 사용하였다.Based on the mass of the entire culture medium, 1% by weight of yeast extract and 1% by weight of isolate soybean protein were added to replace RCM, and 5% by weight of inulin and 5% by weight of chicory powder were added to purified water and dissolved. After sterilization at 121 ° C. for 15 minutes using an autoclave, it was used as a culture medium.
고체 배지에 배양된 클로스트리디움 부티리쿰을 1백금이 소량 채취하여 멸균이 된 배양 배지에 접종하고, 37℃인큐베이터(Incubator)에 혐기성 상태를 유지시키기 위해, 배지를 혐기 배양통 2.5 L에 넣고, 가스팩을 이용해 배양하였다. 이후 혐기 배양상태 표시종이를 이용해 혐기 배양통 내부의 산소 유무를 확인해 가며 총 5일 동안 정치배양하였다.A small amount of 1 platinum of Clostridium butyricum cultured on a solid medium is inoculated into a sterilized culture medium, and in order to maintain an anaerobic state in an incubator at 37 ° C, the medium is placed in an anaerobic culture tank 2.5 L, Incubated using a gas pack. Thereafter, the anaerobic culture status indicator paper was used to check the presence or absence of oxygen in the anaerobic culture tank, and the cells were cultured for a total of 5 days.
비교예 8Comparative Example 8
분리대두단백 대신 유청분리단백 1 중량%(전체 배양 배지 기준)를 첨가한 것을 제외하고는 비교예 7과 동일한 조건으로 조성물을 제조하였다.A composition was prepared under the same conditions as in Comparative Example 7, except that 1% by weight (based on the total culture medium) of whey protein isolate was added instead of soybean protein isolate.
비교예 9Comparative Example 9
전체 배양 배지의 중량을 기준으로, 질소원으로써 효모 추출물 0.5 중량% 및 탈지분유 5 중량%를 첨가하고, 탄소원으로써 이눌린 5 중량%, 무기염류로써 염화나트륨 0.5 중량%, 데히드로초산나트륨 0.3 중량%, 제2인산칼륨 0.3 중량%, 황산마그네슘 0.06 중량%, 황산암모늄 0.4 중량%, 탄산칼슘 1 중량%를 정제수에 첨가한 후 용해시킨 후 오토클레이브를 사용하여 121 ℃에서 15 분 동안 멸균시킨 뒤 배양 배지로써 사용하였다.Based on the weight of the entire culture medium, 0.5% by weight of yeast extract and 5% by weight of skim milk powder were added as a nitrogen source, 5% by weight of inulin as a carbon source, 0.5% by weight of sodium chloride and 0.3% by weight of sodium dehydroacetate as inorganic salts, 0.3% by weight of potassium diphosphate, 0.06% by weight of magnesium sulfate, 0.4% by weight of ammonium sulfate, and 1% by weight of calcium carbonate were added to purified water, dissolved, sterilized at 121 ° C. for 15 minutes using an autoclave, and then used as a culture medium. used
고체 배지에 배양된 클로스트리디움 부티리쿰을 1백금이 소량 채취하여 멸균이 된 배양 배지에 접종하고, 37 ℃ 인큐베이터(Incubator)에 혐기성 상태를 유지시키기 위해, 배지를 혐기 배양통 2.5 L에 넣고, 가스팩을 이용해 배양하였다. 이후 혐기 배양상태 표시종이를 이용해 혐기 배양통 내부의 산소 유무를 확인해 가며 총 5일 동안 정치배양하였다.A small amount of 1 platinum of Clostridium butyricum cultured on a solid medium is inoculated into a sterilized culture medium, and in order to maintain an anaerobic state in a 37 ° C. incubator, the medium is placed in an anaerobic culture tank 2.5 L, Incubated using a gas pack. Thereafter, the anaerobic culture status indicator paper was used to check the presence or absence of oxygen in the anaerobic culture tank, and the cells were cultured for a total of 5 days.
비교예 10Comparative Example 10
탄소원으로써 치커리 분말 5 중량%를 첨가한 것을 제외하고는 비교예 9와 동일한 조건으로 조성물을 제조하였다.A composition was prepared under the same conditions as in Comparative Example 9, except that 5% by weight of chicory powder was added as a carbon source.
비교예 11Comparative Example 11
탄소원으로써 난소화성말토덱스트린 5 중량%를 첨가한 것을 제외하고는 비교예 9와 동일한 조건으로 조성물을 제조하였다.A composition was prepared under the same conditions as in Comparative Example 9, except that 5% by weight of indigestible maltodextrin was added as a carbon source.
비교예 12Comparative Example 12
탄소원으로써 포도당 5 중량%를 첨가한 것을 제외하고는 비교예 9와 동일한 조건으로 조성물을 제조하였다.A composition was prepared under the same conditions as in Comparative Example 9, except that 5% by weight of glucose was added as a carbon source.
실시예 1 내지 17, 비교예 1 내지 12의 조성을 정리하여 아래 표 1 내지 3으로 나타내었다.The compositions of Examples 1 to 17 and Comparative Examples 1 to 12 are summarized and shown in Tables 1 to 3 below.
배양 조건
(중량%)culture conditions
(weight%) 		실시예Example
1One 22 33 44 55 66 77 88 99
RCMRCM -- -- --
이눌린 inulin 55 55 55 55 55 55 55 55 55
치커리 분말 chicory powder 55 55 55 55 55 55 55 55 55
구아검guar gum -- -- -- 0.30.3 -- -- -- -- --
알긴산나트륨sodium alginate -- -- -- -- 0.30.3 -- -- -- --
펙틴pectin -- -- -- -- -- 0.30.3 -- -- --
프락토올리고당분말Fructooligosaccharide Powder -- -- -- -- -- -- 33 -- --
갈락토올리고당Galactooligosaccharides -- -- -- -- -- -- -- 33 --
난소화성말토덱스트린indigestible maltodextrin -- -- -- -- -- -- -- -- 33
분리대두단백Soy Protein Isolate -- -- -- -- -- -- -- -- --
유청분리단백whey protein isolate -- -- -- -- -- -- -- -- --
볶음 콩가루Stir-fried Bean Flour 1One 33 55 55 55 55 55 55 55
효모 추출물 yeast extract 1One 1One 1One 1One 1One 1One 1One 1One 1One
배양 조건
(중량%)culture conditions
(weight%) 		실시예Example
1010 1111 1212 1313 1414 1515 1616 1717
소이 펩톤 Soy Peptone 55 55 55 55 55 55 55 55
탈지분유skim milk powder -- -- -- -- 55 55 55 55
효모 추출물yeast extract 0.50.5
이눌린 inulin 55 　 　 　 55 　 　 　
치커리 분말 chicory powder 　 55 　 　 　 55 　 　
난소화성말토덱스트린 indigestible maltodextrin 　 　 55 　 　 　 55 　
포도당 glucose 　 　 　 55 　 　 　 55
염화나트륨sodium chloride 0.50.5
데히드로초산나트륨sodium dehydroacetate 0.30.3
제2 인산칼륨dibasic potassium phosphate 0.30.3
황산마그네슘magnesium sulfate 0.060.06
황산암모늄Ammonium Sulphate 0.40.4
탄산 칼슘calcium carbonate 1One
배양 조건
(중량%)culture conditions
(weight%) 		비교예comparative example
1One 22 33 44 55 66 77 88 99 1010 1111 1212
RCMRCM 3.843.84 3.843.84 3.843.84 3.843.84 3.843.84 3.843.84 -- -- -- -- -- --
탈지분유skim milk powder -- -- -- -- -- -- -- -- 55 55 55 55
이눌린inulin -- 1 One 55 1010 2020 -- 55 55 55 -- -- --
치커리 분말chicory powder -- -- -- -- -- 55 55 55 -- 55 -- --
난소화성말토덱스트린indigestible maltodextrin -- -- -- -- -- -- -- -- -- -- 55 --
포도당glucose -- -- -- -- -- -- -- -- -- -- -- 55
분리대두단백Soy Protein Isolate -- -- -- -- -- -- 1One -- -- -- -- --
유청분리단백whey protein isolate -- -- -- -- -- -- -- 1One -- -- -- --
볶음 콩가루Stir-fried Bean Flour -- -- -- -- -- -- -- -- -- -- -- --
효모 추출물 yeast extract -- -- -- -- -- -- 1One 1One 0.50.5 0.50.5 0.50.5 0.50.5
염화나트륨sodium chloride -- -- -- -- -- -- -- -- 0.50.5 0.50.5 0.50.5 0.50.5
데히드로초산나트륨sodium dehydroacetate -- -- -- -- -- -- -- -- 0.30.3 0.30.3 0.30.3 0.30.3
제2 인산칼륨dibasic potassium phosphate -- -- -- -- -- -- -- -- 0.30.3 0.30.3 0.30.3 0.30.3
황산마그네슘magnesium sulfate -- -- -- -- -- -- -- -- 0.060.06 0.060.06 0.060.06 0.060.06
황산암모늄Ammonium Sulphate -- -- -- -- -- -- -- -- 0.40.4 0.40.4 0.40.4 0.40.4
탄산 칼슘calcium carbonate -- -- -- -- -- -- -- -- 1One 1One 1One 1One
실험예 1 : 클로스트리디움 부티리쿰(Experimental Example 1: Clostridium butyricum ( Clostridium butyricumClostridium butyricum ) 균주 발효 생성물의 부티레이트 함량 측정 실험) Butyrate content measurement experiment of strain fermentation product
상기 실시예 1 내지 17, 비교예 1 내지 12로 제조된 발효 생성물에 포함되어 있는 부티레이트를 분석하기 위해 HPLC(HPLC, Shimazu)로 부티레이트 함량을 측정하였고, 그 결과를 아래 표 4 및 5에 나타내었다.In order to analyze the butyrate contained in the fermentation products prepared in Examples 1 to 17 and Comparative Examples 1 to 12, the butyrate content was measured by HPLC (HPLC, Shimazu), and the results are shown in Tables 4 and 5 below. .
배양 조건
(중량%)culture conditions
(weight%) 		실시예Example
1One 22 33 44 55 66 77 88 99
RCMRCM -- -- --
이눌린 inulin 55 55 55 55 55 55 55 55 55
치커리 분말 chicory powder 55 55 55 55 55 55 55 55 55
구아검guar gum -- -- -- 0.30.3 -- -- -- -- --
알긴산나트륨sodium alginate -- -- -- -- 0.30.3 -- -- --
펙틴pectin -- -- -- -- -- 0.30.3 -- -- --
프락토올리고당분말Fructooligosaccharide Powder -- -- -- -- -- -- 33 -- --
갈락토올리고당Galactooligosaccharides -- -- -- -- -- -- -- 33 --
난소화성말토덱스트린indigestible maltodextrin -- -- -- -- -- -- -- -- 33
분리대두단백Soy Protein Isolate -- -- -- -- -- -- -- -- --
유청분리단백whey protein isolate -- -- -- -- -- -- -- -- --
볶음 콩가루Stir-fried Bean Flour 1One 33 55 55 55 55 55 55 55
효모 추출물 yeast extract 1One 1One 1One 1One 1One 1One 1One 1One 1One
부티레이트
(mM)butyrate
(mM) 		11.311.3 21.321.3 26.026.0 26.726.7 27.327.3 32.032.0 31.631.6 29.329.3 32.632.6
배양 조건
(중량%)culture conditions
(weight%) 		실시예Example
1010 1111 1212 1313 1414 1515 1616 1717
소이 펩톤 Soy Peptone 55 55 55 55 55 55 55 55
탈지분유skim milk powder -- -- -- -- 55 55 55 55
효모 추출물yeast extract 0.50.5
이눌린inulin 55 -　- -　- -　- 55 -　- -　- -　-
치커리 분말chicory powder -　- 55 -　- -　- -　- 55 -　- -　-
난소화성말토덱스트린indigestible maltodextrin -　- -　- 55 -　- -　- -　- 55 -　-
포도당glucose -　- -　- -　- 55 -　- -　- -　- 55
염화나트륨sodium chloride 0.50.5
데히드로초산나트륨sodium dehydroacetate 0.30.3
제2 인산칼륨dibasic potassium phosphate 0.30.3
황산마그네슘magnesium sulfate 0.060.06
황산암모늄Ammonium Sulphate 0.40.4
탄산 칼슘calcium carbonate 1One
부티레이트
(mM)butyrate
(mM) 		200200 215215 214214 171171 133133 133133 135135 9696
배양 조건
(중량%)culture conditions
(weight%) 		비교예comparative example
1One 22 33 44 55 66 77 88 99 1010 1111 1212
RCMRCM 3.843.84 3.843.84 3.843.84 3.843.84 3.843.84 3.843.84 -- -- -- -- -- --
탈지분유skim milk powder -- -- -- -- -- -- -- -- 55 55 55 55
이눌린inulin -- 1 One 55 1010 2020 -- 55 55 55 -- -- --
치커리 분말chicory powder -- -- -- -- -- 55 55 55 -- 55 -- --
난소화성말토덱스트린indigestible maltodextrin -- -- -- -- -- -- -- -- -- -- 55 --
포도당glucose -- -- -- -- -- -- -- -- -- -- -- 55
분리대두단백Soy Protein Isolate -- -- -- -- -- -- 1One -- -- -- -- --
유청분리단백whey protein isolate -- -- -- -- -- -- -- 1One -- -- -- --
볶음 콩가루Stir-fried Bean Flour -- -- -- -- -- -- -- -- -- -- -- --
효모 추출물 yeast extract -- -- -- -- -- -- 1One 1One 0.50.5 0.50.5 0.50.5 0.50.5
염화나트륨sodium chloride -- -- -- -- -- -- -- -- 0.50.5 0.50.5 0.50.5 0.50.5
데히드로초산나트륨sodium dehydroacetate -- -- -- -- -- -- -- -- 0.30.3 0.30.3 0.30.3 0.30.3
제2 인산칼륨dibasic potassium phosphate -- -- -- -- -- -- -- -- 0.30.3 0.30.3 0.30.3 0.30.3
황산마그네슘magnesium sulfate -- -- -- -- -- -- -- -- 0.060.06 0.060.06 0.060.06 0.060.06
황산암모늄Ammonium Sulphate -- -- -- -- -- -- -- -- 0.40.4 0.40.4 0.40.4 0.40.4
탄산 칼슘calcium carbonate -- -- -- -- -- -- -- -- 1One 1One 1One 1One
부티레이트
(mM)butyrate
(mM) 		2.42.4 10.710.7 16.716.7 14.814.8 13.913.9 3.93.9 -- -- -- -- -- --
표 4 및 5를 참조하면, RCM을 사용하는 비교예 1 내지 6을 먼저 비교하면, 저항성 전분인 이눌린이 포함되는 경우(비교예 2 내지 5)의 부티레이트 생성량이 이눌린이 포함되지 않은 경우(비교예 1 및 6)의 부티레이트 생성량에 비해 많은 것을 볼 수 있다. 비교예 6의 경우, 치커리 분말은 부티레이트 생성량에 크게 영향을 미치지 않았다.비교예 7 및 8의 경우, RCM이 사용되지 않았고, 이눌린 및 치커리 분말을 포함하며, 비교예 7은 효모 추출물과 분리대두단백을 포함하고, 비교예 8은 효모 추출물과 유청분리단백을 포함한다. 비교예 7 내지 12의 경우 부티레이트가 생성되지 않았다.Referring to Tables 4 and 5, comparing Comparative Examples 1 to 6 using RCM first, the amount of butyrate produced when inulin, which is a resistant starch, is included (Comparative Examples 2 to 5) when inulin is not included (Comparative Example Compared to the amount of butyrate produced in 1 and 6), a lot can be seen. In Comparative Example 6, chicory powder did not significantly affect the amount of butyrate production. In Comparative Examples 7 and 8, RCM was not used and inulin and chicory powder were included, and Comparative Example 7 was yeast extract and isolated soybean protein. Comparative Example 8 includes yeast extract and whey protein isolate. In Comparative Examples 7 to 12, butyrate was not produced.
실시예 1 내지 3은 이눌린과 치커리 분말을 모두 포함하는데, 실시예 1 내지 3을 비교예 7 및 8과 비교했을 때, 볶음 콩가루를 배지 조성물의 구성으로 포함하는 차이점이 있다. 이 경우, 부티레이트가 생성되었으며, 특히 볶음 콩가루가 3 중량%(전체 배지 중량 기준)인 경우(실시예 2), 그리고 볶음 콩가루가 5 중량%(전체 배지 중량 기준)인 경우(실시예 3), 부티레이트 생성량이 각각 21.25 mM, 25.96 mM로 현저하게 증가한 것을 확인할 수 있다. Examples 1 to 3 include both inulin and chicory powder, and when Examples 1 to 3 are compared with Comparative Examples 7 and 8, there is a difference in including roasted soy flour as a composition of the medium composition. In this case, butyrate was produced, especially when the roasted soybean flour was 3% by weight (based on the total medium weight) (Example 2), and when the roasted soybean flour was 5% by weight (based on the total medium weight) (Example 3), It can be seen that the production of butyrate was markedly increased to 21.25 mM and 25.96 mM, respectively.
실시예 4 내지 9는, 실시예 3과 비교하여, 배지 조성물에 탄소원을 추가로 포함하는 것인데, 부티레이트 생성량이 실시예 3과 거의 유사하거나 약간 증가하였다. Examples 4 to 9, compared to Example 3, which additionally include a carbon source in the medium composition, butyrate production was almost similar to Example 3 or slightly increased.
실시예 10 내지 17의 배지 조성물은, 질소원으로서 효모추출물 및 소이펩톤를 포함하고, 탄소원으로서 이눌린, 치커리 분말, 말토덱스트린 또는 포도당을 포함하면서, 염화나트륨, 데히드로초산나트륨, 제2 인산칼륨, 황산마그네슘, 황산암모늄 및 탄산 칼슘의 무기염류를 추가로 포함하는데, 이 경우, 배지 조성물의 제조시 용해도가 향상되었고, 부티레이트의 생성량이 90 mM 이상으로 증가하였으며, 특히 실시예 10 내지 12에서는 200 mM 이상으로 증가하였다.The medium compositions of Examples 10 to 17 include yeast extract and soy peptone as nitrogen sources, inulin, chicory powder, maltodextrin or glucose as carbon sources, sodium chloride, sodium dehydroacetate, dibasic potassium phosphate, magnesium sulfate, It further includes inorganic salts of ammonium sulfate and calcium carbonate. In this case, the solubility during the preparation of the medium composition was improved, and the amount of butyrate produced increased to 90 mM or more, and in particular, in Examples 10 to 12, it increased to 200 mM or more. did
하기 실험예들에서는, 실시예 3의 조성물을 활용하여 실험이 이루어졌다.In the following experimental examples, experiments were conducted using the composition of Example 3.
실험예 2 : 장관내피세포(IEC-18)에서의 부티레이트 발효 생성물의 세포독성평가 실험Experimental Example 2: Evaluation of cytotoxicity of butyrate fermentation products in intestinal endothelial cells (IEC-18)
실시예 3의 조성물(발효 생성물)을 동결건조 시료로 MTT법을 통한 세포 생존률을 측정하였다. The cell viability of the composition (fermentation product) of Example 3 was measured as a lyophilized sample through the MTT method.
장관내피세포인 IEC-18 세포를 10% 우태아혈청(fetal bovine serum, FBS)이 포함된 DMEM(Dulbecco's Modified Eagle Medium, Hyclone Lab., USA)에서 배양한 후 24 well plate에 5 x 104 cells/mL로 분주하였다. 48시간동안 배양한 후 LPS 100㎍/mL 과 상기 동결건조 시료를 동시에 처리하였다. 이때 동결건조 시료는 수성 매질(물)을 사용하여 각각 10, 50, 100, 250, 500, 1000 ug/ml로 재구성하였다. 배양이 끝난 후 MTT 시약을 처치하고 formazan을 형성할 수 있도록 반응시킨 뒤 상층액을 모두 제거하고 DMSO를 처치한 후 microplate reader ELISA 기기를 사용하여 540nm에서 측정하였다. Intestinal endothelial cells, IEC-18 cells, were cultured in DMEM (Dulbecco's Modified Eagle Medium, Hyclone Lab., USA) containing 10% fetal bovine serum (FBS), and then plated at 5 x 104 cells/cells in a 24 well plate. Divided into mL. After culturing for 48 hours, 100 μg/mL of LPS and the lyophilized sample were simultaneously treated. At this time, the lyophilized samples were reconstituted in an aqueous medium (water) at 10, 50, 100, 250, 500, and 1000 ug/ml, respectively. After the incubation was completed, the MTT reagent was treated and reacted to form formazan, and the supernatant was removed, treated with DMSO, and measured at 540 nm using a microplate reader ELISA device.
그 결과 농도 의존적으로 세포 사멸을 유도하지 않았으며, 최고 농도인 1000 μg/mL의 농도까지 세포 사멸을 실질적으로 유도하지 않았음을 도 1에서 확인하였다. 이로부터 본 발명의 부티레이트 발효 생성물은 세포독성을 나타내지 않음을 확인하였다.As a result, it was confirmed in FIG. 1 that cell death was not induced in a concentration-dependent manner, and cell death was not substantially induced up to the highest concentration of 1000 μg/mL. From this, it was confirmed that the butyrate fermentation product of the present invention did not exhibit cytotoxicity.
실험예 3 : 장관면역세포에서의 부티레이트 발효 생성물의 항산화 활성평가 실험Experimental Example 3: Evaluation of antioxidant activity of butyrate fermentation products in intestinal immune cells
실시예 3의 발효 생성물을 동결건조 된 시료로, 항산화 및 항염증 활성 평가를 실시하였다.The fermentation product of Example 3 was lyophilized, and antioxidant and anti-inflammatory activities were evaluated.
DPPH 실험을 통한 항산화 효능검증을 하였으며, 아스코르브산 (1 mg/mL)을 대조군으로 이용하여 발효 생성물과 비교하였다. 상기 동결건조 시료를 수성 매질(물)을 사용하여 각각 1, 2.5, 5, 7.5, 10 ug/ml로 재구성하였다. 0.2 mM DPPH 용액을 사용하였으며 96 well plate에 각각의 시료, 정제수, MeOH, 0,2 mM DPPH를 순차적으로 혼합한 뒤 암실에서 30분간 반응하였다. 그 후 Microplate reader ELISA 기기를 통해 517 nm 파장에서 나타나는 측정값(OD; optical density)을 계산하여 아스코르브산(대조군)과 비교하였다.The antioxidant efficacy was verified through the DPPH experiment, and ascorbic acid (1 mg/mL) was used as a control and compared with the fermentation product. The lyophilized samples were reconstituted in an aqueous medium (water) to 1, 2.5, 5, 7.5, and 10 ug/ml, respectively. A 0.2 mM DPPH solution was used, and each sample, purified water, MeOH, and 0,2 mM DPPH were sequentially mixed in a 96 well plate and reacted in the dark for 30 minutes. After that, the optical density (OD) at a wavelength of 517 nm was calculated using a microplate reader ELISA device and compared with that of ascorbic acid (control).
DPPH의 결과를 통하여 발효 생성물은 농도 의존적인 항산화 활동이 관찰되었음을 도 2에서 확인할 수 있었다.Through the results of DPPH, it was confirmed in FIG. 2 that the concentration-dependent antioxidant activity of the fermentation product was observed.
실험예 4 : 초대배양된 비장세포에서 발효를 통한 부티레이트 발효 생성물의 TNF alpha 측정평가 실험Experimental Example 4: TNF alpha measurement evaluation experiment of butyrate fermentation product through fermentation in primary cultured spleen cells
실시예 3의 발효 생성물의 동결건조 시료를 이용하여 Dextran Sulfate Sodium(DSS) 장염 마우스 동물 모델의 비장 세포에서 TNF alpha 측정실험을 진행하였다. 아무것도 처리하지 않은 것을 정상 대조군(CON)으로, DSS 마우스 모델을 음성 대조군(DSS)으로 하였으며, 그 외 Butyric acid 5 mM, 10 mM, Sulfasalazine 50 mg/kg을 양성 대조군으로 사용하였다. 상기 동결건조 시료는 실험예 3과 동일한 방법으로 100 ug/mL의 저농도 및 200 ug/mL의 고농도로 재구성하여 사용하였다. TNF alpha 측정은 TNF alpha Mouse ELISA kit를 사용하여 진행하였다.Using the freeze-dried sample of the fermentation product of Example 3, a TNF alpha measurement experiment was conducted in spleen cells of a mouse animal model of Dextran Sulfate Sodium (DSS) enteritis. The untreated mice were used as a normal control (CON), the DSS mouse model was used as a negative control (DSS), and Butyric acid 5 mM, 10 mM, and Sulfasalazine 50 mg/kg were used as positive controls. The freeze-dried sample was reconstituted at a low concentration of 100 ug/mL and a high concentration of 200 ug/mL in the same manner as in Experimental Example 3 and used. TNF alpha measurement was performed using a TNF alpha Mouse ELISA kit.
농도 의존적으로 TNF alpha 레벨이 감소되는 것을 도 3에서 확인할 수 있었다. 특히 본 발명의 시료는 Butyric acid보다 높은 감소능을 나타내었으며, Sulfasalazine과는 유사한 효과를 나타내었다. It was confirmed in FIG. 3 that the TNF alpha level was reduced in a concentration-dependent manner. In particular, the sample of the present invention showed a higher reducing ability than Butyric acid, and showed an effect similar to that of Sulfasalazine.
TNF alpha는 여러 면역 매개 염증성 질환에서 중요한 역할을 하는 사이토카인으로서, TNF alpha 레벨이 감소되었다는 것은 본 발명의 조성물이 염증성 질환 개선에 효과가 있음을 나타내는 것이다. TNF alpha is a cytokine that plays an important role in various immune-mediated inflammatory diseases, and the reduced TNF alpha level indicates that the composition of the present invention is effective in improving inflammatory diseases.
실험예 5 : DSS로 유도된 장염 동물의 혈장에서 염증인자인 IgE level 측정 평가 실험Experimental Example 5: IgE level measurement and evaluation test, an inflammatory factor, in plasma of animals with enteritis induced by DSS
실시예 3의 발효 생성물의 동결건조 시료로 DSS로 유도된 장염 마우스 동물의 혈장에서 IgE 레벨을 Mouse IgE ELISA kit를 사용하여 측정하였다. 사용된 시료 및 실험군 등에 대해서는 실험예 4와 동일한 방법을 이용하여, 실시예 3의 시료를 100 ug/mL의 저농도 및 200 ug/mL의 고농도로 재구성하여 사용하였다.A freeze-dried sample of the fermentation product of Example 3 was used to measure IgE levels in plasma of mouse animals with DSS-induced enteritis using a Mouse IgE ELISA kit. Using the same method as in Experimental Example 4 for the sample used and the experimental group, the sample of Example 3 was reconstituted at a low concentration of 100 ug/mL and a high concentration of 200 ug/mL and used.
부티레이트 발효 생성물이 농도 의존적으로 IgE 레벨이 감소하는 것을 나타내고 있으며, 부티레이트 발효 생성물 고농도 (200 ug/mL)에서 IgE 레벨이 가장 많이 감소된 것을 도 4에서 확인하였다. 이는 본 발명의 조성물이 염증성 질환 개선에 효과가 있음을 나타내는 것이다.The butyrate fermentation product showed a concentration-dependent decrease in IgE level, and it was confirmed in FIG. 4 that the IgE level decreased the most at a high concentration (200 ug/mL) of the butyrate fermentation product. This indicates that the composition of the present invention is effective in improving inflammatory diseases.
실험예 6 : 3T3-L1 preadipocyte에 대한 부티레이트 발효 생성물의 세포독성평가 실험Experimental Example 6: Evaluation of cytotoxicity of butyrate fermentation products on 3T3-L1 preadipocytes
실시예 3의 발효 생성물의 동결건조 시료를 수성 매질(물)을 사용하여 각각 25, 50, 100, 200 ㎍/㎖의 농도로 재구성한 후, 이를 3T3-L1 세포에 각각 처리한 후 MTT법을 통한 세포 생존률 측정을 진행하였다. MTT 법은 실험예 2와 동일한 방법으로 수행되었다. The freeze-dried sample of the fermentation product of Example 3 was reconstituted in an aqueous medium (water) at concentrations of 25, 50, 100, and 200 μg/ml, respectively, and then treated with 3T3-L1 cells, respectively, followed by the MTT method. Cell viability was measured through The MTT method was performed in the same manner as in Experimental Example 2.
그 결과를 도 5에 나타내었다. 부티레이트 발효 생성물을 100 ㎍/㎖ 농도로 처리시에는 세포 생존률이 93.5±4.2%였고, 200 ㎍/㎖ 농도로 처리시에는 84.1±1.8%를 나타내었다. 또한 25, 50, 100 ㎍/㎖ 농도에서는 세포 독성이 없음을 확인하였고, 이 농도를 하기 실험에서 사용하였다.The results are shown in FIG. 5 . When the butyrate fermentation product was treated at a concentration of 100 μg/ml, the cell viability was 93.5±4.2%, and when treated at a concentration of 200 μg/ml, it was 84.1±1.8%. In addition, it was confirmed that there was no cytotoxicity at the concentrations of 25, 50, and 100 μg/ml, and these concentrations were used in the following experiments.
실험예 7 : 3T3-L1 지방전구세포에 대한 부티레이트 발효 생성물의 지방세포분화 평가 및 Oil Red-O 염색 실험Experimental Example 7: Evaluation of adipocyte differentiation of butyrate fermentation products in 3T3-L1 preadipocytes and Oil Red-O staining experiment
실시예 3의 부티레이트 발효 생성물의 동결건조 시료를 수성매질(물)로 재구성 후 25, 50, 100 ㎍/㎖의 농도별로 처리하여, 3T3-L1 지방 전구세포를 성숙된 지방 세포로의 분화로 유도하고 이를 Oil Red-O 염색으로 처리하였다. 지방전구세포(pre-adipocytes)인 3T3-L1 세포를 10% 소혈청(Bovine calf serum)이 들어있는 DMEM(Dulbecco's Modified Eagle Medium, Hyclone Lab., USA)에서 배양한 후 6 well plate에 1 x 105 cells/mL로 분주하였다. 48시간동안 배양한 후 167 nM 인슐린(insulin), 500 mM 3-아이소부틸-1 메틸잔틴(isobutylmetylxanthine), 0.1 mM 덱사메타손(dexamethasone), 10% 우태아혈청(fetal bovine serum, FBS)이 포함된 DMEM 배지로 갈아주고 성숙한 지방세포로의 분화를 유도하였다. 이때 실시예 3의 부티레이트 발효 생성물의 동결건조 시료를 수성매질(물)로 재구성 후 25, 50, 100 ㎍/㎖의 농도별로 처리하여 7일간 배양하였다. 배양이 끝난 후 세포 배양액을 제거하고 세포를 PBS로 세척한 후 10% 포르말린(formalin)을 첨가하여 실온에서 1시간동안 세포를 고정시켰다. 1시간 후 포르말린을 모두 제거하고 증류수로 세척한 후 지질 성분만을 염색시키는 Oil-red O 시약을 넣고 10분간 염색시켰다. 염색이 끝난 후 증류수로 세척하고 이미지를 스캔 한 후 100% 이소프로판올(isopropanol)을 넣고 염색된 Oil-red O를 용출시켜 540nm 파장에서 흡광도를 측정하였다.The freeze-dried sample of the butyrate fermentation product of Example 3 was reconstituted with an aqueous medium (water) and then treated at concentrations of 25, 50, and 100 μg/ml to induce differentiation of 3T3-L1 preadipocytes into mature adipocytes and treated with Oil Red-O staining. 3T3-L1 cells, pre-adipocytes, were cultured in DMEM (Dulbecco's Modified Eagle Medium, Hyclone Lab., USA) containing 10% Bovine calf serum, and then plated 1 x 10 in a 6-well plate. It was aliquoted at 5 cells/mL. After incubation for 48 hours, DMEM containing 167 nM insulin, 500 mM 3-isobutyl-1 methylxanthine, 0.1 mM dexamethasone, and 10% fetal bovine serum (FBS) The culture medium was changed and differentiation into mature adipocytes was induced. At this time, the freeze-dried sample of the butyrate fermentation product of Example 3 was reconstituted with an aqueous medium (water), treated at concentrations of 25, 50, and 100 μg/ml and cultured for 7 days. After the incubation, the cell culture medium was removed, the cells were washed with PBS, and 10% formalin was added to fix the cells at room temperature for 1 hour. After 1 hour, all formalin was removed, washed with distilled water, and oil-red O reagent, which stains only lipid components, was added and stained for 10 minutes. After staining was finished, washed with distilled water, scanned the image, put 100% isopropanol, and eluted the stained Oil-red O to measure absorbance at a wavelength of 540 nm.
그 결과는 도 6에 나타내었다. 본 발명의 부티레이트 발효 생성물을 50 ㎍/㎖ 농도로 처리한 경우 34.1%, 100 ㎍/㎖에서 62.9% 만큼 지방 세포 분화가 억제되는 것이 확인되었다. 특히 100 ㎍/㎖에서는 양성 대조군인 부티레이트 1 mM 대비 더 우수한 지방 분화 억제를 나타내었다. The results are shown in FIG. 6 . It was confirmed that adipocyte differentiation was inhibited by 34.1% when the butyrate fermentation product of the present invention was treated at a concentration of 50 μg/ml and by 62.9% at 100 μg/ml. In particular, at 100 μg/ml, inhibition of adipogenesis was better than that of 1 mM butyrate, which is a positive control group.
또한 세포내 지방 축적 정도를 현미경으로 관찰하였고, 현미경 사진을 도 7에 나타내었다. 양성대조군인 부티레이트과 대비하여 본 발명의 발효물 100 ㎍/㎖에서 더 높은 지방 축적이 감소한 것을 확인하였다.In addition, the degree of intracellular fat accumulation was observed under a microscope, and a micrograph is shown in FIG. 7 . Compared to the positive control group, butyrate, it was confirmed that a higher fat accumulation was reduced in 100 μg/ml of the fermented product of the present invention.
실험예 8 : 부티레이트 발효 생성물의 처리를 통한 트리글리세라이드 함량 측정평가 실험Experimental Example 8: Triglyceride content measurement evaluation experiment through treatment of butyrate fermentation product
실시예 3의 발효 생성물의 동결건조물을 처리하여 성숙한 지방세포 내에 존재하는 순수한 트리글리세라이드(TG) 함량을 측정하기 위하여 Triglyceride Quantification Assay kit를 사용하였다. Triglyceride Quantification Assay kit was used to measure the pure triglyceride (TG) content in mature adipocytes by treating the lyophilized product of the fermentation product of Example 3.
분화 유도된 지방전구세포를 5% NP-40 solution을 이용해 균질화 시킨 후 90℃ 온도로 5분간 배양 후 상온에서 5분간 반응시키는 것을 반복하여 TG가 완전히 용해되도록 수행하였다. 그 후 Lipase와 함께 20분간 상온에서 반응시키고 Triglyceride Probe와 Triglyceride Enzyme Mix와 함께 60분간 상온에서 반응시켜 분광광도계로 570 nm 파장에서의 광도를 측정하였다. 본 발명의 발효 생성물은 수성매질(물)을 이용하여 25, 50, 100 ㎍/㎖ 농도로 재구성하여 사용되었다. Differentiation-induced pre-adipocytes were homogenized using 5% NP-40 solution, incubated at 90 ° C for 5 minutes, and then reacted at room temperature for 5 minutes to completely dissolve TG. After that, it was reacted with Lipase at room temperature for 20 minutes and reacted with Triglyceride Probe and Triglyceride Enzyme Mix at room temperature for 60 minutes, and the light intensity at 570 nm wavelength was measured with a spectrophotometer. The fermentation product of the present invention was reconstituted at concentrations of 25, 50, and 100 μg/ml using an aqueous medium (water) and used.
그 결과를 도 8에 나타내었다. 부티레이트 발효 생성물을 50 ㎍/㎖로 처리한 경우 23.7%, 100 ㎍/㎖로 처리한 경우 44.1%만큼 TG 함량이 억제되었음을 확인하였으며, 특히 100 ㎍/㎖에서는 양성 대조군인 부티레이트 1 mM 대비 더 우수한 TG 억제를 나타내었다.The results are shown in FIG. 8 . It was confirmed that the TG content was suppressed by 23.7% when the butyrate fermentation product was treated with 50 μg / ml and 44.1% when treated with 100 μg / ml. showed inhibition.
실험예 9 : 부티레이트 발효생성물의 고지방 식이로 비만 유도된 마우스에서 체중 변화 측정Experimental Example 9: Measurement of body weight change in mice induced with obesity by high-fat diet of butyrate fermentation product
군army 성별gender 동물 수number of animals 투여액량
(mL/kg)dose amount
(mL/kg) 		투여량
(g/kg)dose
(g/kg)
정상 식이군 (NCD + vehicle)Normal diet group (NCD + vehicle) Male Male 88 1010 --
고지방 식이군 (vehicle)High-fat diet group (vehicle) Male Male 88 1010 --
고지방 식이군 + 실시예3 발효생성물 저농도 0.5 g/kg (BF-0.5)High-fat diet group + low concentration of Example 3 fermentation product 0.5 g/kg (BF-0.5) Male Male 88 1010 0.50.5
고지방 식이군 + 실시예 3 발효생성물 고농도 1 g/kg (BF-1)High-fat diet group + Example 3 fermentation product high concentration 1 g / kg (BF-1) Male Male 88 1010 1One
고지방 식이군 + sodium butyrate 0.5 g/kg (SB-0.5)High-fat diet + sodium butyrate 0.5 g/kg (SB-0.5) Male Male 88 1010 0.50.5
실시예 3의 발효 생성물의 동결 건조 시료를 표 7에 따라 재구성하였고, 고지방 식이와 함께 8주령 C57BL6J mice에 상기 표 7에 따라 경구 투여한 후 14 주간 실험 기간 동안 체중변화를 측정하였다. Freeze-dried samples of the fermentation product of Example 3 were reconstituted according to Table 7, and after oral administration according to Table 7 to 8-week-old C57BL6J mice with a high-fat diet, body weight changes were measured during the experimental period for 14 weeks.
그 결과를 도 9에 나타내었다. 고지방 식이군(vehicle)에서 정상 식이군(NCD+vehicle)에 비해 체중이 유의적으로 증가하였다. 본 발명의 부티레이트 발효 생성물 및 고지방 식이 동시 투여군에서 체중이 유의적으로 감소하였으며, 특히 고농도 투여군(BF-1)은 양성 대조군(SB-0.5)과 거의 유사한 체중 감소를 나타내었다.The results are shown in FIG. 9 . Body weight increased significantly in the high-fat diet group (vehicle) compared to the normal diet group (NCD+vehicle). In the group administered with the butyrate fermentation product of the present invention and the high fat diet at the same time, the weight was significantly decreased, and in particular, the high concentration administration group (BF-1) showed a weight loss almost similar to that of the positive control group (SB-0.5).
실험예 10 : 부티레이트 발효생성물의 고지방 식이로 비만 유도된 마우스에서 체지방량 감소 측정Experimental Example 10: Measurement of body fat mass reduction in mice induced with obesity by high-fat diet of butyrate fermentation product
전술한 실험예 9의 고지방 식이로 유도된 비만 동물 모델에서 실시예 3의 발효 생성물의 체지방 감소에 미치는 영향을 Dual energy X-ray Absorptiometry (DXA)로 피하 및 내장 지방의 부피를 측정하였다. 그 결과를 도 10에 나타내었다.The effect of the fermentation product of Example 3 on reducing body fat in the obese animal model induced by the high-fat diet of Experimental Example 9 was measured by Dual energy X-ray Absorptiometry (DXA) to measure the volume of subcutaneous and visceral fat. The results are shown in FIG. 10 .
본 발명의 부티레이트 발효 생성물 및 고지방 식이(HFD) 동시 투여군에서 고지방 식이군(HFD + vehicle) 대비 유의적으로 체지방률 및 체지방량이 낮은 것을 확인하였다. It was confirmed that the butyrate fermentation product of the present invention and the high fat diet (HFD) co-administered group had significantly lower body fat percentage and body fat mass than the high fat diet group (HFD + vehicle).
실험예 11 : 부티레이트 발효생성물의 고지방 식이로 비만 유도된 마우스에서 장기 중량에 미치는 영향 분석Experimental Example 11: Analysis of the Effect of Butyrate Fermentation Product on Organ Weight in Obesity-Induced Mice with a High-Fat Diet
전술한 실험예 9의 고지방 식이로 유도된 비만 동물 모델에서 실시예 3의 발효생성물의 장기 중량에 미치는 영향을 확인하기 위해, 간, 부고환, 사타구니 또는 갈색 지방 조직의 중량을 측정하였다. To confirm the effect of the fermentation product of Example 3 on organ weight in the obese animal model induced by the high-fat diet of Experimental Example 9, the weight of liver, epididymis, groin or brown adipose tissue was measured.
그 결과를 도 11에 나타내었다. 정상 식이군(NCD)에 비해 고지방 식이군(HFD)에서 부고환 지방의 크기가 증가하였고(도 11-A), 단순 고지방 식이군(HFD + vehicle)에 비해 본 발명의 부티레이트 발효생성물 동시 투여군에서 간, 부고환 백색 지방, 사타구니 박색 지방, 갈색 지방 조직의 중량이 더 낮았다(도 11-B). 특히 고농도 투여군(BF-1)은 양성 대조군(SB-0.5)과 거의 유사하거나 더 낮은 조직 중량의 감소를 나타내었다. The results are shown in FIG. 11 . Compared to the normal diet group (NCD), the size of epididymal fat increased in the high-fat diet group (HFD) (FIG. 11-A), and compared to the simple high-fat diet group (HFD + vehicle), the liver in the butyrate fermentation product simultaneous administration group of the present invention , the weights of epididymal white fat, inguinal light fat, and brown adipose tissue were lower (FIG. 11-B). In particular, the high-dose administration group (BF-1) showed a decrease in tissue weight that was almost similar to or lower than that of the positive control group (SB-0.5).
실험예들의 결과를 참조하면, 실시예에 따른 본 발명의 발효 생성물은 비만 세포의 발현을 억제시킬 수 있고, 실험예들에서 체중 및 체지방량 감소를 확인하였다. 특히 부티레이트의 농도가 증가할 수록 체중 및 체지방량 감소에 효과적임을 고려할 때, 부티레이트 함량이 약 26 mM이었던 실시예 3에 따른 위 실험 결과를 통해, 부티레이트 함량이 이와 유사하거나 이보다 높은 나머지 실시예들 역시 유사하거나 또는 보다 효과적인 체중 감소 효과를 나타낼 것이다.Referring to the results of the experimental examples, the fermentation product of the present invention according to the examples can inhibit the expression of mast cells, and in the experimental examples, it was confirmed that body weight and body fat mass were reduced. In particular, considering that as the concentration of butyrate increases, it is effective in reducing body weight and fat mass. Through the above experimental results according to Example 3, in which the butyrate content was about 26 mM, the other examples having similar or higher butyrate content were also similar. or will show a more effective weight loss effect.
이상에서 본 발명의 바람직한 실시예에 대하여 상세하게 설명하였지만 본 발명의 권리범위는 이에 한정되는 것은 아니고 다음의 청구범위에서 정의하고 있는 본 발명의 기본 개념을 이용한 당업자의 여러 변형 및 개량 형태 또한 본 발명의 권리범위에 속하는 것이다.Although the preferred embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements of those skilled in the art using the basic concept of the present invention defined in the following claims are also made according to the present invention. falls within the scope of the rights of

Claims (19)

  1. 단쇄 지방산을 포함하는 발효 생성물로서, As a fermentation product containing short-chain fatty acids,
    상기 발효 생성물은, The fermentation product is
    질소원으로서 효모 추출물; 및 볶음 콩가루 또는 소이 펩톤에서 이루어진 군에서 선택된 하나 이상; 및yeast extract as a nitrogen source; And at least one selected from the group consisting of stir-fried soy flour or soy peptone; and
    탄소원으로서 이눌린(inulin), 치커리(chicory) 분말, 말토덱스트린 및 포도당에서 이루어진 군에서 선택된 하나 이상At least one selected from the group consisting of inulin, chicory powder, maltodextrin and glucose as a carbon source
    을 포함하며, 강화 클로스트리디아 배지를 포함하지 않는 배지 조성물에 클로스트리디움(Clostridium) 균주를 접종한 후 발효시켜 생성되는 것이고, It is produced by inoculating a Clostridium strain into a medium composition that does not contain an enhanced Clostridia medium and then fermenting it,
    상기 발효 생성물은 10 mM 이상의 단쇄 지방산을 포함하는 것인, Wherein the fermentation product contains 10 mM or more of short-chain fatty acids,
    단쇄 지방산을 포함하는 발효 생성물.A fermentation product containing short-chain fatty acids.
  2. 제1항에 있어서, 상기 단쇄 지방산은 부티레이트(butyrate)인, 발효 생성물.The fermentation product of claim 1 , wherein the short chain fatty acid is butyrate.
  3. 제1항에 있어서, 상기 발효 생성물은 20 mM 이상의 단쇄 지방산을 포함하는 것인, 발효 생성물.The fermentation product according to claim 1 , wherein the fermentation product comprises at least 20 mM short chain fatty acids.
  4. 제1항에 있어서, 상기 클로스트리디움 균주는 클로스트리디움 부티리쿰(Clostridium butyricum), 클로스트리디움 타이로부티리쿰(Clostridium tyrobutyricum), 또는 클로스트리디움 사카로퍼부탈아세토니쿰(Clostridium saccharoperbutylacetonicum) 중 하나 이상을 포함하는 것인, 발효 생성물.The method of claim 1, wherein the Clostridium strain is one of Clostridium butyricum, Clostridium tyrobutyricum, or Clostridium saccharoperbutylacetonicum A fermentation product comprising the above.
  5. 제1항에 있어서, 상기 클로스트리디움 균주는 클로스트리디움 부티리쿰(Clostridium butyricum)인, 발효 생성물.The fermentation product according to claim 1, wherein the Clostridium strain is Clostridium butyricum.
  6. 제1항에 있어서, 상기 배지 조성물은 질소원으로서 탈지 분유를 추가로 포함하는 것인, 발효 생성물.The fermentation product according to claim 1, wherein the medium composition further comprises skim milk powder as a nitrogen source.
  7. 제1항에 있어서, 상기 배지 조성물은 탄소원으로서 구아검, 알긴산 나트륨, 펙틴 및 올리고당으로 이루어지는 군에서 선택되는 하나 이상을 추가로 포함하는 것인, 발효 생성물.The fermentation product according to claim 1, wherein the medium composition further comprises at least one selected from the group consisting of guar gum, sodium alginate, pectin, and oligosaccharides as a carbon source.
  8. 제1항에 있어서, 상기 배지 조성물은 염화나트륨, 데히드로초산나트륨, 제2 인산칼륨, 황산마그네슘, 황산암모늄 및 탄산 칼슘으로 이루어지는 군에서 선택되는 하나 이상의 무기염류를 추가로 포함하는 것인, 발효 생성물.The fermentation product according to claim 1, wherein the medium composition further comprises one or more inorganic salts selected from the group consisting of sodium chloride, sodium dehydroacetate, dibasic potassium phosphate, magnesium sulfate, ammonium sulfate and calcium carbonate. .
  9. 제1항에 있어서, 상기 배지 조성물은, 상기 배지 조성물의 전체 중량을 기준으로, The method of claim 1, wherein the medium composition, based on the total weight of the medium composition,
    질소원으로서 효모 추출물 0.1 내지 10 중량%; 및 볶음 콩가루 0.1 내지 10 중량% 또는 소이 펩톤 0.1 내지 10 중량% 중 하나 이상; 및0.1 to 10% by weight of yeast extract as a nitrogen source; and 0.1 to 10% by weight of roasted soybean flour or 0.1 to 10% by weight of soy peptone; and
    탄소원으로서 이눌린(inulin) 1 내지 10 중량%, 치커리(chicory) 분말 1 내지 10 중량%, 말토덱스트린 1 내지 10 중량% 및 포도당 1 내지 10 중량%에서 이루어진 군에서 선택된 하나 이상 At least one selected from the group consisting of 1 to 10% by weight of inulin, 1 to 10% by weight of chicory powder, 1 to 10% by weight of maltodextrin, and 1 to 10% by weight of glucose as a carbon source.
    을 포함하는 것인, 발효 생성물.A fermentation product comprising a.
  10. 제9항에 있어서, 상기 배지 조성물은, 상기 배지 조성물의 전체 중량을 기준으로, 질소원으로서 탈지 분유 0.1 내지 10 중량%를 추가로 포함하는 것인, 발효 생성물.The fermentation product according to claim 9, wherein the medium composition further comprises 0.1 to 10% by weight of skim milk powder as a nitrogen source based on the total weight of the medium composition.
  11. 제9항에 있어서, 상기 배지 조성물은, 상기 배지 조성물의 전체 중량을 기준으로, 탄소원으로서 구아검 0.1 내지 5 중량%, 알긴산 나트륨 0.1 내지 5 중량%, 펙틴 0.1 내지 5 중량% 및 올리고당 0.1 내지 5 중량%으로 이루어지는 군에서 선택되는 하나 이상을 추가로 포함하는 것인, 발효 생성물.The method of claim 9, wherein the medium composition, based on the total weight of the medium composition, 0.1 to 5% by weight of guar gum, 0.1 to 5% by weight of sodium alginate, 0.1 to 5% by weight of pectin and 0.1 to 5% of oligosaccharides as carbon sources A fermentation product further comprising at least one selected from the group consisting of % by weight.
  12. 제1항에 있어서, 상기 배지 조성물은, 상기 배지 조성물의 전체 중량을 기준으로, The method of claim 1, wherein the medium composition, based on the total weight of the medium composition,
    질소원으로서 효모 추출물 0.5 내지 1 중량%; 및 볶음 콩가루 1 내지 5 중량% 또는 소이 펩톤 5 중량% 중 하나 이상; 및0.5 to 1% by weight of yeast extract as a nitrogen source; and 1 to 5% by weight of roasted soybean flour or 5% by weight of soy peptone; and
    탄소원으로서 이눌린(inulin) 5 중량%, 치커리(chicory) 분말 5 중량%, 말토덱스트린 3 내지 5 중량% 및 포도당 5 중량%에서 이루어진 군에서 선택된 하나 이상 At least one selected from the group consisting of 5% by weight of inulin, 5% by weight of chicory powder, 3 to 5% by weight of maltodextrin, and 5% by weight of glucose as a carbon source.
    을 포함하는 것인, 발효 생성물.A fermentation product comprising a.
  13. 제12항에 있어서, 상기 배지 조성물은, 상기 배지 조성물의 전체 중량을 기준으로, 질소원으로서 탈지 분유 5 중량%를 추가로 포함하는 것인, 발효 생성물.The fermentation product according to claim 12, wherein the medium composition further comprises 5% by weight of skim milk powder as a nitrogen source based on the total weight of the medium composition.
  14. 제12항에 있어서, 상기 배지 조성물은, 상기 배지 조성물의 전체 중량을 기준으로, 탄소원으로서 구아검 0.3 중량%, 알긴산 나트륨 0.3 중량%, 펙틴 0.3 중량% 및 올리고당 3 중량%으로 이루어지는 군에서 선택되는 하나 이상을 추가로 포함하는 것인, 발효 생성물.The method of claim 12, wherein the medium composition is selected from the group consisting of 0.3% by weight of guar gum, 0.3% by weight of sodium alginate, 0.3% by weight of pectin, and 3% by weight of oligosaccharide as a carbon source, based on the total weight of the medium composition. A fermentation product, further comprising one or more.
  15. 제12항에 있어서, 상기 배지 조성물은, 상기 배지 조성물의 전체 중량을 기준으로, 염화나트륨 0.5 중량%, 데히드로초산나트륨 0.3 중량%, 제2 인산칼륨 0.3 중량%, 황산마그네슘 0.06 중량%, 황산암모늄 0.4 중량% 및 탄산 칼슘 1 중량%를 추가로 포함하는 것인, 발효 생성물.The method of claim 12, wherein the medium composition, based on the total weight of the medium composition, sodium chloride 0.5% by weight, sodium dehydroacetate 0.3% by weight, potassium dibasic phosphate 0.3% by weight, magnesium sulfate 0.06% by weight, ammonium sulfate 0.4% by weight and 1% by weight of calcium carbonate.
  16. 제1항 내지 제15항 중 어느 한 항에 따른 발효 생성물을 포함하는 과체중의 예방 또는 치료용 약학 조성물.A pharmaceutical composition for preventing or treating overweight, comprising the fermentation product according to any one of claims 1 to 15.
  17. 제1항 내지 제15항 중 어느 한 항에 따른 발효 생성물을 포함하는 과체중의 개선 또는 예방용 식품 조성물.A food composition for improving or preventing overweight, comprising the fermentation product according to any one of claims 1 to 15.
  18. 제1항 내지 제15항 중 어느 한 항에 따른 발효 생성물을 포함하는 과체중의 개선 또는 예방용 동물용 사료 조성물.An animal feed composition for improving or preventing overweight, comprising the fermentation product according to any one of claims 1 to 15.
  19. (a) 질소원으로서 효모 추출물; 및 볶음 콩가루 또는 소이 펩톤 중 하나 이상; 및(a) yeast extract as a nitrogen source; and at least one of roasted soy flour or soy peptone; and
    탄소원으로서 이눌린(inulin), 치커리(chicory) 분말, 말토덱스트린 및 포도당에서 이루어진 군에서 선택된 하나 이상At least one selected from the group consisting of inulin, chicory powder, maltodextrin and glucose as a carbon source
    을 물에 용해 및 멸균시켜 배지 조성물을 제조하는 단계;Dissolving and sterilizing in water to prepare a medium composition;
    (b) 상기 배지 조성물에 클로스트리디움(Clostridium) 균주를 접종시키는 단계; 및(b) inoculating a Clostridium strain into the medium composition; and
    (c) 상기 클로스트리디움 균주가 접종된 배지 조성물을 발효시켜 단쇄 지방산을 포함하는 발효 생성물을 제조하는 단계(c) preparing a fermentation product containing short-chain fatty acids by fermenting the medium composition inoculated with the Clostridium strain
    를 포함하는, 제1항 내지 제15항 중 어느 한 항에 따른 발효 생성물의 제조 방법. A method for producing a fermentation product according to any one of claims 1 to 15, comprising a.
PCT/KR2022/017492 2022-02-23 2022-11-08 Fermentation product containing short-chain fatty acid and use thereof for alleviating, preventing, and treating obesity WO2023163318A1 (en)

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