WO2024181369A1 - 脂肪前駆細胞分化促進剤、及びインスリン抵抗性改善剤 - Google Patents

脂肪前駆細胞分化促進剤、及びインスリン抵抗性改善剤 Download PDF

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WO2024181369A1
WO2024181369A1 PCT/JP2024/006844 JP2024006844W WO2024181369A1 WO 2024181369 A1 WO2024181369 A1 WO 2024181369A1 JP 2024006844 W JP2024006844 W JP 2024006844W WO 2024181369 A1 WO2024181369 A1 WO 2024181369A1
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lactic acid
fructobacillus
acid bacteria
strain
insulin resistance
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French (fr)
Japanese (ja)
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悠司 時本
秀聡 井戸垣
孝治 西川
優菜 枡田
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Osaka Soda Co Ltd
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Osaka Soda Co Ltd
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Priority to KR1020257030298A priority Critical patent/KR20250156727A/ko
Priority to JP2025503883A priority patent/JPWO2024181369A1/ja
Priority to CN202480014939.5A priority patent/CN120981172A/zh
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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 OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Preparation or treatment thereof
    • A23L2/52—Adding ingredients
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/13—Nucleic 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7084—Compounds having two nucleosides or nucleotides, e.g. nicotinamide-adenine dinucleotide, flavine-adenine dinucleotide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/326—Foods, ingredients or supplements having a functional effect on health having effect on cardiovascular health
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/3262—Foods, ingredients or supplements having a functional effect on health having an effect on blood cholesterol
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/328—Foods, ingredients or supplements having a functional effect on health having effect on glycaemic control and diabetes

Definitions

  • the present invention relates to an agent for promoting differentiation of adipose precursor cells and an agent for improving insulin resistance.
  • fat cells as endocrine organs, produce and secrete various cytokines and hormone-like substances, such as adiponectin, leptin, resistin, TNF ⁇ , and free fatty acids.
  • cytokines and hormone-like substances such as adiponectin, leptin, resistin, TNF ⁇ , and free fatty acids.
  • the enlarged fat cells seen in obesity are not only involved in excess fat accumulation, but are also said to oversecrete resistin, TNF ⁇ , free fatty acids, and other substances that impair insulin signaling in skeletal muscles and the liver and induce insulin resistance. This is thought to be the cause of insulin resistance, leading to lifestyle-related diseases such as hyperlipidemia, arteriosclerosis, high blood pressure, and diabetes.
  • adipocytes are known to secrete adiponectin, a cytokine that promotes glucose and lipid metabolism and has anti-inflammatory effects. Therefore, in order to prevent and improve lifestyle-related diseases, it is considered important to prevent the production of enlarged adipocytes.
  • Thiazolidine derivatives are known as a component of therapeutic drugs that improve insulin resistance.
  • the mechanism of action of these therapeutic drugs is as follows: the derivatives bind to the receptor PPAR ⁇ , increase the activity of PPAR ⁇ , and induce differentiation of preadipocytes into normal adipocytes that are not enlarged, while inducing apoptosis of enlarged adipocytes.
  • Differentiated adipocytes actively secrete adiponectin, which activates the metabolism of sugar and lipids, promotes the uptake of sugar from the blood, and improves insulin resistance.
  • Such functions of adipocytes lead to the prevention and treatment of not only diabetes but also hyperlipidemia. Based on the above, if we can promote the differentiation of preadipocytes and increase the number of normal adipocytes, we can expect to be able to prevent or improve insulin resistance.
  • Patent Document 1 an adipose precursor cell differentiation promoter containing an extract of Rubus suavissimus S. Lee as an active ingredient
  • Patent Document 2 a preadipocyte differentiation promoter containing acreatine, an acreatine analog, or an extract of Sarcachemikan as an active ingredient
  • Patent Document 3 discloses that a barley fermentation product of lactic acid bacteria, such as Leuconostoc mesenteroides or Lactobacillus plantarum, has an adipocyte differentiation promoting effect.
  • lactic acid bacteria, cultures of said lactic acid bacteria, culture supernatants of said lactic acid bacteria, and/or extracts of said lactic acid bacteria have an adipocyte differentiation promoting effect.
  • Patent Document 4 discloses an insulin resistance improving agent containing two or more aglycones selected from the group consisting of trans-p-coumaric acid, caffeic acid, and ferulic acid as active ingredients. However, it needs to be extracted from a natural material called plum vinegar polyphenol.
  • the present invention aims to provide an agent for promoting differentiation of adipose precursor cells and an agent for improving insulin resistance. It also aims to provide a lactic acid bacterium, Fructobacillus fructosus OS-1010 strain (accession number: NITE BP-03818), which has the effect of promoting differentiation of adipose precursor cells and the effect of improving insulin resistance.
  • the present inventors have newly discovered that certain lactic acid bacteria and/or components derived from certain lactic acid bacteria have the effect of promoting the differentiation of adipose precursor cells, and have completed the present invention.
  • Item 1 An agent for promoting differentiation of adipose precursor cells, comprising a lactic acid bacterium belonging to the genus Fructobacillus, a culture of the lactic acid bacterium, a culture supernatant of the lactic acid bacterium, and/or an extract of the lactic acid bacterium.
  • Item 2. The agent for promoting adipose precursor cell differentiation according to Item 1, wherein the lactic acid bacterium is Fructobacillus fructosus, Fructobacillus tropaeoi, or Fructobacillus durionis.
  • the lactic acid bacteria is selected from the group consisting of Fructobacillus fructosus NBRC3516 strain, Fructobacillus fructosus OS-1010 strain (accession number: NITE BP-03818), Fructobacillus tropaeoilu RD012353 strain (accession number: NITE BP-02765), Fructobacillus tropaeoilu RD012354 strain (accession number: NITE BP-02766), Fructobacillus tropaeoilu RD012355 strain (accession number: NITE BP-02767), Fructobacillus tropaeoilu RD012356 strain (accession number: NITE BP-02768), Fructobacillus tropaeoilu RD012357 strain (accession number: NITE BP-02769), Fructobacillus tropaeoilu RD012358 strain (accession number: NITE BP-02
  • Item 3 The fat precursor cell differentiation promoter according to Item 1 or 2, wherein the fat precursor cell differentiation promoter is Fructobacillus subtilis RD011727 strain (Accession Number: NITE BP-02766), or Fructobacillus durionis RD011727 strain (Accession Number: NITE BP-02764).
  • the agent for promoting adipose precursor cell differentiation according to any one of Items 1 to 3, comprising nicotinamide adenine dinucleotide and nicotinamide mononucleotide, and a content of nicotinamide mononucleotide per 1 part by weight of nicotinamide adenine dinucleotide is 0.001 parts by weight or more and 10 parts by weight or less.
  • Item 5. A food, drink, cosmetic, or pharmaceutical product comprising the preadipocyte differentiation promoter according to any one of Items 1 to 4.
  • Item 6. A lactic acid bacterium which is Fructobacillus fructosus OS-1010 strain (accession number: NITE BP-03818).
  • An insulin resistance improving agent comprising a lactic acid bacterium belonging to the genus Fructobacillus, a culture of the lactic acid bacterium, a culture supernatant of the lactic acid bacterium, and/or an extract of the lactic acid bacterium.
  • Item 8 The insulin resistance improving agent according to Item 7, wherein the lactic acid bacterium is Fructobacillus fructosus, Fructobacillus tropaeoi, or Fructobacillus durionis.
  • the lactic acid bacterium is Fructobacillus fructosus NBRC3516 strain, Fructobacillus fructosus OS-1010 strain (Accession Number: NITE BP-03818), Fructobacillus tropaeoilu RD012353 strain (Accession Number: NITE BP-02765), Fructobacillus tropaeoilu RD012354 strain (Accession Number: NITE BP-02766), Fructobacillus tropaeoilu RD012355 strain (Accession Number: NITE BP-02767), Fructobacillus tropaeoilu RD012356 strain (Accession Number: NITE BP-02768), Fructobacillus tropaeoilu RD012357 strain (Accession Number: NITE BP-02769), Fructobacillus tropaeoilu RD012358 strain (Accession Number: NITE BP-02769), Fruc
  • Item 9 The insulin sensitizer according to Item 7 or 8, wherein the Fructobacillus subtilis strain is Fructobacillus erythropoeis RD011727 strain (Accession Number: NITE BP-02764).
  • Item 10 The insulin resistance improving agent according to any one of Items 7 to 9, comprising nicotinamide adenine dinucleotide and nicotinamide mononucleotide, wherein the content of nicotinamide mononucleotide per part by weight of nicotinamide adenine dinucleotide is 0.001 parts by weight or more and 10 parts by weight or less.
  • Item 11 A food, drink, cosmetic, or pharmaceutical product comprising the insulin resistance improving agent according to any one of Items 7 to 10.
  • the present invention provides a novel material that is useful as an active ingredient for an agent for promoting differentiation of adipose precursor cells and an agent for improving insulin resistance.
  • the present invention provides lactic acid bacteria belonging to the genus Fructobacillus, a culture of said lactic acid bacteria, a culture supernatant of said lactic acid bacteria, and/or an extract of said lactic acid bacteria as an active ingredient for an agent for promoting differentiation of adipose precursor cells and an agent for improving insulin resistance.
  • lactic acid bacteria belonging to Fructobacillus fructosus e.g., Fructobacillus fructosus NBRC3516 strain ... us
  • OS-1010 strain accesion number: NITE BP-038178
  • lactic acid bacteria belonging to Fructobacillus tropaeoilus e.g., Fructobacillus tropaeoilus
  • RD012353 strain accesion number: NITE BP-02 765
  • Fructobacillus tropaeoiI RD012354 strain accesion Number: NITE BP-02766
  • Fructobacillus durionis genus lactic acid bacteria e.g., Fructobacillus durionis RD011727 strain (Accession Number: NITE BP-02764)
  • a culture of the lactic acid bacteria, a culture supernatant of the lactic acid bacteria, and/or an extract of the lactic acid bacteria have the effect of promoting differentiation in adi
  • FIG. 1 is a graph showing the amount of triglyceride accumulated in adipocytes in an evaluation of the effect of promoting differentiation of adipocyte precursor cells using lactic acid bacteria extract A1 prepared from Fructobacillus fructosus NBRC3516 strain and lactic acid bacteria extract B1 prepared from Fructobacillus fructosus OS-1010 strain (accession number: NITE BP-03818).
  • FIG. 1 is a graph showing the amount of triglyceride accumulation in adipocytes in an evaluation of the induction of insulin resistance in adipocyte precursor cells using TFN- ⁇ .
  • 1 is a graph showing the amount of triglyceride accumulated in adipocytes in an evaluation of the effect of improving insulin resistance in adipose precursor cells using lactic acid bacteria extract C1 prepared from Fructobacillus tropaeoilus RD012353 strain (accession number: NITE BP-02765).
  • 1 is a graph showing the amount of triglyceride accumulated in adipocytes in an evaluation of the effect of improving insulin resistance in adipose precursor cells using lactic acid bacteria extract D1 prepared from Fructobacillus tropaeoiyl RD012354 strain (accession number: NITE BP-02766).
  • the preadipocyte differentiation promoter and insulin resistance improver of the present invention are characterized in that they each contain, as active ingredients, lactic acid bacteria belonging to the genus Fructobacillus, a culture of the lactic acid bacteria, a culture supernatant of the lactic acid bacteria, and/or an extract of the lactic acid bacteria.
  • the preadipocyte differentiation promoter and insulin resistance improver are described in detail below.
  • the active ingredients of the preadipocyte differentiation promoter and insulin resistance improving agent of the present invention are lactic acid bacteria belonging to the genus Fructobacillus, a culture of the lactic acid bacteria, a culture supernatant of the lactic acid bacteria, and/or an extract of the lactic acid bacteria.
  • Lactic acid bacteria belonging to the genus Fructobacillus are lactic acid bacteria that produce nicotinamide mononucleotide (NMN) and/or nicotinamide adenine dinucleotide (NAD). Furthermore, the lactic acid bacteria belonging to the genus Fructobacillus are preferably lactic acid bacteria that produce at least NAD, and more preferably lactic acid bacteria that produce both NMN and NAD.
  • Examples of lactic acid bacteria belonging to the genus Fructobacillus include Fructobacillus durionis, Fructobacillus tropaeoilu, and Fructobacillus fructosus.
  • a more specific example of a lactic acid bacterium belonging to the genus Fructobacillus is preferably Fructobacillus fructosus, and among these, Fructobacillus fructosus NBRC3516 strain and Fructobacillus fructosus OS-1010 strain can be mentioned.
  • the Fructobacillus fructosus OS-1010 strain has been entrusted domestically to the National Institute of Technology and Evaluation's Patent Microorganisms Depository Center (NPMD) (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) on February 3, 2023 under the accession number NITE BP-03818.
  • NPMD National Institute of Technology and Evaluation's Patent Microorganisms Depository Center
  • the Fructobacillus durionis RD011727 strain has been internationally entrusted to the National Institute of Technology and Evaluation, Patent Microorganisms Depository Center (NPMD) (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) on October 28, 2020 under the accession number NITE BP-02764.
  • NPMD Patent Microorganisms Depository Center
  • the Fructobacillus tropaeoill RD012353 strain has been internationally entrusted to the National Institute of Technology and Evaluation, Patent Microorganisms Depository Center (NPMD) (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) on October 28, 2020 under the accession number NITE BP-02765.
  • NPMD Patent Microorganisms Depository Center
  • the Fructobacillus tropaeoill RD012354 strain has been internationally entrusted to the National Institute of Technology and Evaluation, Patent Microorganisms Depository Center (NPMD) (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) on October 28, 2020 under the accession number NITE BP-02766.
  • NPMD Patent Microorganisms Depository Center
  • Fructobacillus fructosus NBRC3516 strain Fructobacillus fructosus OS-1010 strain (Accession Number: NITE BP-03818), Fructobacillus tropaeoilu RD012353 strain (Accession Number: NITE BP-02765), Fructobacillus tropaeoilu Fructobacillus tropaeoilu strain RD012354 (accession number: NITE BP-02766) and Fructobacillus durionis strain RD011727 (accession number: NITE BP-02764) are lactic acid bacteria that produce nicotinamide mononucleotide (NMN) and/or nicotinamide adenine dinucleotide (NAD) when subjected to the lactic acid bacteria culture method described below.
  • NPN nicotinamide mononucleotide
  • NAD nicotinamide adenine dinucleotide
  • Fructobacillus fructosus NBRC3516 strain Fructobacillus fructosus OS-1010 strain, Fructobacillus tropaeoilu RD012353 strain, Fructobacillus Lactic acid bacteria, such as Fructobacillus tropaeoiI RD012354 strain and Fructobacillus durionis RD011727 strain, cultures of the lactic acid bacteria, culture supernatants of the lactic acid bacteria, and/or extracts of the lactic acid bacteria, can efficiently promote differentiation of adipose precursor cells.
  • Fructobacillus lactic acid bacteria may be used alone or in combination with multiple species.
  • Fructobacillus fructosus is preferred from the viewpoint of more effectively improving the effect of promoting differentiation of adipose precursor cells.
  • Fructobacillus lactic acid bacteria from the viewpoint of more effectively improving the effect of promoting differentiation of fat precursor cells, Fructobacillus fructosus NBRC3516 strain and Fructobacillus fructosus OS-1010 strain are preferred, with Fructobacillus fructosus OS-1010 strain being more preferred.
  • Fructobacillus fructosus NBRC3516 strain Fructobacillus fructosus OS-1010 strain, Fructobacillus tropaeoilu RD012353 strain, Fructobacillus Lactic acid bacteria, such as Fructobacillus tropaeoiI RD012354 strain and Fructobacillus durionis RD011727 strain, cultures of the lactic acid bacteria, culture supernatants of the lactic acid bacteria, and/or extracts of the lactic acid bacteria, can improve insulin resistance.
  • Fructobacillus lactic acid bacteria may be used alone or in combination with multiple species.
  • Fructobacillus lactic acid bacteria Fructobacillus fructosus, Fructobacillus tropaeoi, and Fructobacillus durionis are preferred from the viewpoint of more effectively improving the insulin resistance improving effect.
  • Fructobacillus fructosus NBRC3516 strain Fructobacillus fructosus OS-1010 strain, Fructobacillus tropaeoilu (Fructobacillus tropaeoilu) RD0 12353 strain, Fructobacillus tropaeoilu RD012354 strain, and Fructobacillus durionis RD011727 strain are preferred, and Fructobacillus fructosus OS-1010 strain is more preferred.
  • the form of the active ingredient is at least any one of the above-mentioned specific lactic acid bacteria, a culture of the lactic acid bacteria, a culture supernatant of the lactic acid bacteria, and an extract of the lactic acid bacteria.
  • the active ingredient may be used in any one of these forms alone or in combination of two or more forms.
  • the active ingredient when the active ingredient is in the form of lactic acid bacteria, the active ingredient includes the bacterial components of the lactic acid bacteria and products that are or were present in the lactic acid bacteria.
  • the lactic acid bacteria may be either live or killed. In the case of killed bacteria, the bacterial cells may be crushed. In the case of crushed bacteria, some of the bacterial components may be removed.
  • the lactic acid bacteria may be in the form of bacterial powder dried by means of freeze-drying, shelf drying, spray drying, or the like.
  • Lactic acid bacteria can be obtained by a production method including a step of culturing one or more of the above-mentioned specific lactic acid bacteria, and a step of separating and recovering the bacterial cells.
  • the method carried out in the separation and recovery step includes solid-liquid separation and washing of the bacterial cells.
  • the production method can include other steps in addition to the above steps.
  • the other steps include a resting cell reaction step (which can be carried out after the culturing step, and preferably after the separation and recovery step), a drying step (which can be carried out after the separation and recovery step or the resting cell reaction), a bacterial cell crushing step (which can be carried out after the separation and recovery step, the resting cell reaction step or the drying step), etc.
  • Culture media that can be used in the culturing process include those used for expansion culture (pre-culture medium) and those used for production culture (main culture medium).
  • the main culture medium can be prepared based on the medium used as the pre-culture medium, with the addition of additives.
  • the medium is preferably a liquid medium, but may be an agar medium.
  • the medium In addition to a carbon source, the medium generally contains a nitrogen source, minerals, etc.
  • Carbon sources include carbohydrates and carbohydrate materials.
  • Carbohydrates include sugars (monosaccharides, disaccharides, oligosaccharides), polysaccharides, and sugar alcohols.
  • Carbohydrates include lactose, sucrose, glucose, starch, xylitol, dextrose, and the like.
  • the carbohydrate material may be any organic composition containing carbohydrates, and examples of such carbohydrate materials include milk and its processed products (skim milk powder, whey, milk powder, condensed milk, etc.), soy milk and its processed products (soy milk hydrolysate, etc.), grains, fruits, vegetables, and other foods.
  • Milk may be derived from any mammal, such as cows, goats, sheep, buffalo, camels, llamas, donkeys, yaks, horses, and reindeer.
  • the carbohydrates may be isolated or may be contained in the carbohydrate material.
  • fructose (carbohydrate) may be used in the form contained in fruits (carbohydrate material).
  • These carbon sources may be used alone or in combination of multiple types. Among these carbon sources, glucose is preferred.
  • the concentration of the carbon source in the medium is not particularly limited and may be set appropriately depending on the type of medium and the culture method, but examples of this include 0.5 to 4 w/w%, preferably 1 to 3 w/w%, and more preferably 1.5 to 2.5 w/w%.
  • any inorganic or organic nitrogen source can be used.
  • proteins such as yeast extract (e.g., brewer's yeast), meat extract, and casein; protein hydrolysates such as peptone (e.g., protease peptone), peptides such as peptides; nitrogen-containing salts such as ammonium salts (e.g., ammonium citrate), and nitrates.
  • peptone e.g., protease peptone
  • peptides such as peptides
  • nitrogen-containing salts such as ammonium salts (e.g., ammonium citrate), and nitrates.
  • nitrogen sources may be used alone or in combination.
  • the concentration of the nitrogen source in the medium is not particularly limited and may be set appropriately depending on the type of medium, culture method, etc., but in the case of proteins, for example, 0.3 to 4 w/w%, preferably 0.5 to 3 w/w%, more preferably 1 to 2 w/w%; in the case of peptides, for example, 0.1 to 2 w/w%, preferably 0.3 to 1.8 w/w%, more preferably 0.5 to 1.5 w/w%; in the case of nitrogen-containing salts, for example, 0.03 to 1.5 w/w%, preferably 0.05 to 1 w/w%, more preferably 0.1 to 0.5 w/w%.
  • minerals include manganese (e.g., manganese salts such as manganese sulfate), zinc, iron, sodium (e.g., sodium salts such as sodium acetate), potassium (e.g., potassium salts such as dipotassium hydrogen sulfate and dipotassium hydrogen phosphate), magnesium (e.g., magnesium salts such as magnesium sulfate), calcium, phosphorus (e.g., phosphates such as dipotassium hydrogen phosphate), sulfur (e.g., sulfates such as manganese sulfate, potassium hydrogen sulfate, and magnesium sulfate), trace elements, etc.
  • manganese e.g., manganese salts such as manganese sulfate
  • zinc iron
  • sodium e.g., sodium salts such as sodium acetate
  • potassium e.g., potassium salts such as dipotassium hydrogen sulfate and dipotassium hydrogen phosphate
  • magnesium
  • concentration of minerals in the medium there are no particular limitations on the concentration of minerals in the medium, and it may be set appropriately depending on the type of medium, the culture method, etc., but in the case of manganese salts, for example, 0.001 to 0.01 w/w%, preferably 0.003 to 0.008 w/w%, can be mentioned; in the case of sodium salts, for example, 0.05 to 1.5 w/w%, preferably 0.1 to 1 w/w%, can be mentioned; in the case of magnesium salts, for example, 0.001 to 0.02 w/w%, preferably 0.005 to 0.015 w/w%, can be mentioned; in the case of potassium salts, for example, 0.05 to 1 w/w%, preferably 0.1 to 0.5 w/w%, can be mentioned; in the case of phosphates, for example, 0.05 to 1 w/w%, preferably 0.1 to 0.5 w/w%, can be mentioned; and in the case of sulfates, 0.00
  • the medium may contain other components such as vitamins (such as B vitamins), surfactants (non-ionic surfactants (such as Tween), anionic surfactants (such as SDS), antibacterial agents (such as triclosan), and antibiotics (such as monesin).
  • vitamins such as B vitamins
  • surfactants non-ionic surfactants (such as Tween)
  • anionic surfactants such as SDS
  • antibacterial agents such as triclosan
  • antibiotics such as monesin
  • the concentration of other components in the medium there are no particular limitations on the concentration of other components in the medium, and it may be set appropriately depending on the type of other components, the type of medium, the culture method, etc., but when a surfactant is included, the concentration of the surfactant is, for example, 0.01 to 0.5 w/w%, preferably 0.05 to 0.3 w/w%.
  • Culture conditions are not particularly limited as long as they allow the growth of Fructobacillus lactic acid bacteria.
  • the culture temperature may be any temperature that is optimal for the Fructobacillus lactic acid bacteria to be cultured, for example, 26 to 40°C, preferably 27 to 38°C, more preferably 28 to 36°C, and even more preferably 29 to 34°C.
  • the culture time may be set appropriately according to the type of Fructobacillus lactic acid bacteria to be actually cultured, for example, 4 to 48 hours, preferably 8 to 36 hours, and more preferably 12 to 24 hours.
  • the culture liquid may or may not need to be stirred during the culture.
  • the above-mentioned culture may be carried out as a production culture (main culture) for a certain period of time, and before that, an expansion culture (preculture) may be carried out in a small amount of medium (for example, 1/6 to 1/4 of the main culture medium in terms of volume ratio).
  • the culture conditions for the preculture may be set appropriately according to the type of Fructobacillus lactic acid bacteria, and the above-mentioned conditions may be adopted.
  • the culture obtained in the preculture may be inoculated into the main culture medium so that the OD660 is, for example, 0.01 to 0.04, preferably 0.01 to 0.03.
  • solid-liquid separation of the culture liquid can be performed by filtration using filter paper, centrifugation, decantation, screw press, roller press, rotary drum screen, belt screen, vibrating screen, multi-plate vibrating filter, vacuum dehydration, pressurized dehydration, belt press, centrifugal concentration dehydration, multi-plate dehydration, etc., and the resulting bacterial cells can be washed.
  • the resting cell process can increase the nicotinamide mononucleotide content in lactic acid bacteria.
  • the liquid used in the resting cell reaction step can be any liquid that can cause the resting cell reaction of lactic acid bacteria belonging to the genus Fructobacillus without any particular restrictions.
  • water, a buffer solution, an organic solvent, etc. can be used.
  • the pH of the liquid used in the resting cell reaction is, for example, 4.0 to 10.0, preferably 5.0 to 9.0, and more preferably 5.5 to 7.5.
  • the buffer solution examples include acetate buffer, phosphate buffer, borate buffer, carbonate buffer, citrate buffer, Tris buffer, HEPES buffer, etc.
  • buffer solution examples include KHC8H4O4 -NaOH (pH 4.0), CH3COOH - CH3COONa (pH 4.0), MES-NaOH (pH 5.0 ) , CH3COOH - CH3COONa (pH 5.0), KH2PO4 - K2HPO4 (pH 6.0 ) , MES-NaOH (pH 6.0 ) , KH2PO4-K2HPO4 (pH 7.0 ) , PIPES-NaOH (pH 7.0 ), HEPES-NaOH (pH 8.0 ) , H3BO4 -NaOH (pH 8.0), CHES-NaOH ( pH 9.0), H3BO4 - NaOH (pH 9.0), H2CO3 - NaHCO3 (pH 10.0), CHES-NaOH (pH 10.0), etc. are preferred, with KH 2 PO 4 —K 2 HPO 4 , H 3 BO 4
  • organic solvent examples include aromatic compounds such as benzene and benzonitrile, ketones such as acetone, acetylacetone, and methyl ethyl ketone, fatty acid esters such as ethyl acetate, butyl acetate, ethyl butyrate, and ethyl formate, ethers such as diethyl ether, dipropyl ether, dibutyl ether, tetrahydrofuran, and 1,4-dioxane, halogenated hydrocarbons such as dichloromethane, chloroform, and dichloroethane, 1,2-propanediol, 1,2- Examples include diols such as butanediol, 1,3-butanediol, 1,4-butanediol, 2,3-butanediol, 1,2-hexanediol, 1,6-hexan
  • liquids water and buffer solutions are preferred as the liquid used in the resting cell reaction.
  • the reaction temperature in the resting cell reaction is, for example, 21 to 37°C, preferably 24 to 28°C.
  • the reaction time is, for example, 0.1 to 24 hours, preferably 6 to 12 hours.
  • the resting cell reaction can be carried out by suspending lactic acid bacteria belonging to the genus Fructobacillus in the above liquid and leaving it to stand, stirring or shaking.
  • the resting cell reaction may be carried out without using the above liquid by adjusting the pH of the culture solution containing lactic acid bacteria belonging to the genus Fructobacillus to a range of 4.0 to 10.0, preferably 5.0 to 9.0, and more preferably 5.5 to 7.5.
  • the bacteria can be dried using drying methods such as freeze drying, shelf drying, and spray drying.
  • the dried cells can be disrupted by any method.
  • some of the cell components may be removed from the disrupted cells.
  • Lactic acid bacteria culture When the active ingredient is in the form of a culture of lactic acid bacteria, the active ingredient contains, together with the lactic acid bacteria, a medium component containing a product of the lactic acid bacteria.
  • the culture of lactic acid bacteria may be a culture produced using one of the above-mentioned specific lactic acid bacteria alone, a culture produced using a combination of multiple types, or a mixture obtained by mixing cultures produced using multiple types each alone.
  • the medium components contained in the culture may be those from which some of the medium components used in culturing the lactic acid bacteria have been removed.
  • the lactic acid bacteria contained in the culture may be either live or dead. In the case of dead bacteria, the cells may be crushed. In the case of crushed bacteria, some of the cell components may be removed.
  • the lactic acid bacteria culture can be obtained by a production method including a step of culturing one or more of the above-mentioned specific lactic acid bacteria.
  • the production method can include other steps in addition to the above-mentioned steps.
  • the other steps may include a step of removing a portion of the medium components, a step of subjecting the lactic acid bacteria to a resting cell reaction, and/or a drying step. Details of each step are as described above in "1-2-1. Preparation of lactic acid bacteria.”
  • culture supernatant of lactic acid bacteria When the active ingredient is in the form of a culture supernatant of lactic acid bacteria, the active ingredient includes a medium component containing a product of the lactic acid bacteria.
  • the culture supernatant of the lactic acid bacteria may be a culture supernatant produced using one of the above-mentioned specific lactic acid bacteria alone, a culture supernatant produced using a combination of multiple types, or a mixture obtained by mixing culture supernatants produced using multiple types each alone.
  • the medium components contained in the culture supernatant may have some of the medium components used in culturing the lactic acid bacteria removed.
  • the culture supernatant of lactic acid bacteria can be obtained by a production method including a step of culturing one or more of the above-mentioned specific lactic acid bacteria, and a step of separating and recovering the culture supernatant.
  • the production method may include other steps in addition to the above-mentioned steps.
  • the other steps may include a step of removing a portion of the medium components, a step of subjecting the lactic acid bacteria to a resting cell reaction, and/or a drying step. Details of each step are as described above in "1-2-1. Preparation of lactic acid bacteria".
  • the active ingredient is in the form of an extract of lactic acid bacteria
  • the active ingredient is a multi-component composition obtained by subjecting the above-mentioned lactic acid bacteria or a culture of lactic acid bacteria to an extraction treatment.
  • the extraction solvent used in the preparation of the extract include polar solvents such as water; monohydric lower alcohols having 1 to 4 carbon atoms, such as ethanol and isopropanol; polyhydric alcohols, such as 1,3-butylene glycol, propylene glycol, and glycerin; and mixtures thereof.
  • the temperature condition during the extraction is preferably room temperature (e.g., 5 to 35°C, preferably 20 to 30°C).
  • Specific methods for preparing the extract include, for example, a method of preparing a suspension containing the above-mentioned lactic acid bacteria or a culture of lactic acid bacteria or a pulverized product thereof in an extraction solvent, and subjecting the suspension to solid-liquid separation to obtain an extract liquid (extract liquid), or a method of drying the extract liquid as necessary to obtain a powder (extract powder).
  • the lactic acid bacteria extract may be an extract produced using one of the above-mentioned specified lactic acid bacteria alone, or may be an extract produced using a combination of multiple types of lactic acid bacteria, or may be a mixture obtained by mixing extracts produced using multiple types of lactic acid bacteria each alone.
  • the lactic acid bacteria belonging to the genus Fructobacilus which are the active ingredients used in the present invention
  • the culture of the lactic acid bacteria, the culture supernatant of the lactic acid bacteria, and the extract of the lactic acid bacteria all contain not only NMN and/or NAD produced by the lactic acid bacteria, but also other metabolites (other than NMN and NAD) produced by the lactic acid bacteria and/or bacterial components, etc. It is believed that the excellent effect of promoting differentiation of preadipocytes and the excellent effect of improving insulin resistance according to the present invention are achieved by the combination of these components.
  • the content of NMN is not particularly limited, but the content (content ratio) of NMN per 1 part by weight of NAD is, for example, 0.001 to 10 parts by weight.
  • the content of NMN per 1 part by weight of NAD is preferably 0.005 to 5.0 parts by weight, more preferably 0.005 to 3.0 parts by weight, even more preferably 0.008 to 2.0 parts by weight, particularly preferably 0.01 to 1.5 parts by weight, and even more preferably 0.02 to 1.0 parts by weight.
  • the content (content ratio) of NMN per mole of NAD is not particularly limited, but may be, for example, 0.001 moles or more and 10 moles or less, and preferably 0.005 moles or more and 5 moles or less. In a more preferred embodiment, the content of NMN per mole of NAD is preferably 0.01 moles or more and 3 moles or less, more preferably 0.02 moles or more and 2 moles or less, and even more preferably 0.02 moles or more and 1.5 moles or less.
  • the amount of active ingredient contained in each of the adipose precursor cell differentiation promoter and insulin resistance improving agent of the present invention is not particularly limited, and can be appropriately determined depending on the effect of promoting adipose precursor cell differentiation and the effect of improving insulin resistance to be imparted.
  • the content of the active ingredient contained in each of the preadipocyte differentiation promoter and insulin resistance improving agent of the present invention is, for example, 0.00001 to 10% by weight, preferably 0.0001 to 1% by weight, in terms of the dry weight of Fructobacillus lactic acid bacteria.
  • the above dry weight equivalent refers to the dry weight of the active ingredient when the active ingredient is a lactic acid bacterium or a culture of lactic acid bacteria, refers to the dry weight of the lactic acid bacteria used to obtain the culture supernatant when the active ingredient is a culture supernatant of lactic acid bacteria, and refers to the dry weight of the lactic acid bacteria used to obtain the extract when the active ingredient is an extract.
  • the preadipocyte differentiation promoter and insulin resistance improving agent of the present invention may or may not contain other pharmacological ingredients, as necessary, in addition to the above-mentioned active ingredients.
  • pharmacological ingredients include anti-inflammatory agents, antioxidants, bactericides, cooling agents, vitamins, mucopolysaccharides, etc.
  • the preadipocyte differentiation promoter and insulin resistance improver of the present invention may or may not contain a base and/or additives as necessary to obtain a desired formulation.
  • bases and/or additives are not particularly limited as long as they are pharma- ceutically acceptable, and examples of such bases and/or additives include aqueous bases such as water and monohydric lower alcohols having 1 to 4 carbon atoms (ethanol, isopropanol, etc.); oil bases such as naturally derived oils (vegetable oils, animal oils, and processed oils thereof), mineral oils, ester oils, fatty acid alkyl esters, fatty acids, fatty acid esters, and higher alcohols; surfactants; polyhydric alcohols (glycerin, propylene glycol, dipropylene glycol, 1,3-butylene glycol, etc.); and additives such as cooling agents, preservatives, flavoring agents, colorants, viscosity agents, pH adjusters, wetting agents, stabilizers, antioxidants, ultraviolet absorbers, chelating agents,
  • the formulation form of the preadipocyte differentiation promoter and insulin resistance improver of the present invention is not particularly limited, and may be any of liquid, semi-solid (cream, gel, ointment, paste), solid (granules, fine granules, powder, tablet, capsule), etc.
  • the preadipocyte differentiation promoter and insulin resistance improver of the present invention may be a non-emulsified formulation such as an aqueous formulation or an oil-based formulation, or an emulsified formulation such as an oil-in-water emulsion formulation or a water-in-oil emulsion formulation.
  • product categories for the preadipocyte differentiation promoter and insulin resistance improving agent of the present invention include foods and beverages, cosmetics, and pharmaceuticals.
  • Food and drink products are not particularly limited, but examples include fermented milk (drinkable yogurt, etc.), lactic acid bacteria drinks, milk drinks (coffee milk, fruit milk, etc.), tea drinks (green tea, black tea, oolong tea, etc.), fruit and vegetable drinks (drinks containing fruit juices such as orange, apple, grape, etc., and vegetable juices such as tomato, carrot, etc.), alcoholic drinks (beer, sparkling wine, wine, etc.), carbonated drinks, soft drinks, water-based drinks, and other beverages; and processed foods such as fermented milk (set yogurt, soft yogurt, etc.), sweets, instant foods, and seasonings.
  • Functional foods are also included as food and drink.
  • Functional foods refer to foods that have a certain functionality for the living body, and examples include health functional foods such as foods for specified health uses (including conditionally designated foods for specified health uses) and foods with nutritional functions, functional food, foods with functional claims, special purpose foods, nutritional supplements, health supplements, supplements (for example, tablets, coated tablets, sugar-coated tablets, capsules, liquids, and other dosage forms), and beauty foods (for example, diet foods).
  • functional foods may be special purpose foods such as foods for the sick, powdered milk for pregnant and lactating women, infant formula, foods for the elderly, and foods for caregivers.
  • Cosmetics include topical preparations for the skin and topical preparations for the mucous membranes, and more specifically include basic cosmetics such as lotions, milky lotions, creams, essences, gels, packs, sheet masks, and lip balms; skin cleansers such as face washes, makeup removers (including cleansing agents), exfoliants, and body shampoos; body care cosmetics such as sunscreens, body gels, body massage agents, antiperspirants, deodorants, hair removers, and bath additives; makeup cosmetics such as foundations, face powders, lipsticks, blushers, eye shadows, eyeliners, mascaras, and eyebrow inks; nail cosmetics such as manicures and nail removers; hair cosmetics such as hair styling agents, shampoos, conditioners, rinses, and hair growth agents; and oral cosmetics such as liquid toothpaste, toothpaste, mouthwash, and mouth sprays.
  • basic cosmetics such as lotions, milky lotions, creams, essences, gels, packs, sheet masks, and lip balms
  • Medicines include oral preparations and topical preparations (including quasi-drugs) including topical preparations for skin and topical preparations for mucous membranes.
  • Oral preparations may be in the form of tablets, coated tablets, sugar-coated tablets, capsules, and liquids, while topical preparations may be in the form of liquids (including lotions, sprays, aerosols, and emulsions), foams, ointments, creams, gels, patches, etc.
  • food and beverage products are preferred from the viewpoint of further enhancing the effect of promoting differentiation of fat precursor cells and the effect of improving insulin resistance.
  • the adipose precursor cell differentiation promoter and insulin resistance improving agent of the present invention are used for preventing or improving hyperinsulinemia, non-insulin-dependent diabetes mellitus, dyslipidemia, hypertension, obesity, and arteriosclerotic diseases caused by insulin resistance.
  • the amount of the active ingredient can be, for example, 0.01 to 200 mg/kg/day, preferably 0.1 to 20 mg/kg/day, and can be administered internally 1 to 5 times a day.
  • the amount of active ingredient can be, for example, 0.0001 to 0.2 mg/ cm2 , preferably 0.001 to 0.02 mg/ cm2 , applied 1 to 5 times a day.
  • Sample A (Lactic Acid Bacteria Extract A1)
  • Fructobacillus fructosus NBRC3516 strain was inoculated into 30 ml of MRS medium (preculture medium) manufactured by Difco and cultured at 30°C for 24 hours under static expansion.
  • the obtained culture solution was inoculated into 1 L of MRS medium (main culture medium) so that the OD660 was 0.02, and cultured with stirring at 30°C for 12 hours at pH 6.0 to 7.0.
  • the obtained culture solution was centrifuged to recover the bacterial cells.
  • the recovered bacterial cells were washed with 1 L of 0.85 w/w% KCl aqueous solution.
  • the washed bacterial cells were centrifuged again to recover the bacterial cells.
  • the recovered bacterial cells were sterilized and then freeze-dried to obtain lactic acid bacteria powder A.
  • lactic acid bacteria powder A 20 g was finely ground in a mortar and suspended in 100 mL of sterilized water to prepare a suspension. The suspension was stirred at room temperature (approximately 25°C) for 1 hour and centrifuged (12,000 rpm x 10 min), after which the supernatant was filtered through a 0.2 ⁇ m filter to obtain lactic acid bacteria extract A1.
  • Sample B (Lactic Acid Bacteria Extract B1)
  • a lactic acid bacteria extract B1 was obtained in the same manner as in the preparation of sample A in 1., except that the lactic acid bacteria was changed to Fructobacillus fructosus OS-1010 strain.
  • NMN nicotinamide mononucleotide
  • NAD nicotinamide adenine dinucleotide
  • mouse fetal fibroblast cell line 3T3-L1 National Research and Development Agency, National Institutes of Biomedical Innovation, Health and Nutrition, JCRB Cell Bank No.: JCRB9014
  • the cells were seeded in a 12-well plate at 3 ⁇ 10 4 cells per well, and cultured in DMEM medium containing 10% FBS at 37° C. in a 5% CO 2 environment until the cells became confluent.
  • Example 2 lactic acid bacteria extract A1 to a final concentration of 1.0% by volume (v/v%) in Example 1, and lactic acid bacteria extract B1 to a final concentration of 1.0% by volume (v/v%) in Example 2, which was added to a medium prepared by adding dexamethasone and isobutylmethylxanthine (IBMX) to final concentrations of 1 ⁇ M and 0.5 mM, respectively, to DMEM medium containing 10% FBS, and cultured at 37° C. in a 5% CO environment for two days.
  • IBMX isobutylmethylxanthine
  • Example 2 After removing the culture medium from each well, sterilized water was added to each well in Comparative Example 1, lactic acid bacteria extract A1 to a final concentration of 1.0% by volume (v/v%) in Example 1, and lactic acid bacteria extract B1 to a final concentration of 1.0% by volume (v/v%) in Example 2, and a medium prepared by adding insulin to a final concentration of 10 ⁇ g/ml in DMEM medium containing 10% FBS was added thereto, and the medium was added to each well and cultured at 37° C. in a 5% CO2 environment for 2 days. After removing the culture medium from each well, DMEM medium containing 10% FBS was added to each well, and the cells were cultured at 37° C. in a 5% CO 2 environment for 3 days.
  • NMN nicotinamide mononucleotide
  • NAD nicotinamide adenine dinucleotide
  • the preadipocytes used were the mouse fetal fibroblast cell line 3T3-L1 (JCRB Cell Bank No. JCRB9014, National Institutes of Biomedical Innovation, Health and Nutrition), a model of preadipocytes.
  • the cells were seeded in a 6-well plate so that the number of cells was 1 ⁇ 10 5 per well, and cultured in DMEM medium containing 10% FBS at 37° C. in a 5% CO 2 environment until the cells became confluent.
  • Test Example 1 was carried out under the following conditions. TNF- ⁇ was added to a DMEM medium containing 10% FBS so that the final concentration was 10 ng/ml, and dexamethasone, isobutylmethylxanthine (IBMX), and insulin were added to final concentrations of 1 ⁇ M, 0.5 mM, and 10 ⁇ g/ml, respectively. The resulting medium was added to each well, and cultured at 37°C in a 5% CO2 environment for 2 days.
  • IBMX isobutylmethylxanthine
  • insulin was added to a final concentration of 10 ⁇ g/ml in DMEM medium containing 10% FBS, and the resulting medium was added to each well.
  • the cells were then cultured at 37°C in a 5% CO2 environment for 4 days.
  • the control group received TNF- ⁇ at a final concentration of 10 ng/ml
  • the groups received lactic acid bacteria extract A1 to lactic acid bacteria extract E1.
  • lactic acid bacteria extract A1 was used at a final concentration of 1.0% by volume and TNF- ⁇ was used at a final concentration of 10 ng/ml
  • TNF- ⁇ was used at a final concentration of 10 ng/ml
  • lactic acid bacteria extract B1 was used at a final concentration of 1.0% by volume and TNF- ⁇ was used at a final concentration of 10 ng/ml
  • TNF- ⁇ was used at a final concentration of 10 ng/ml
  • lactic acid bacteria extract C1 was used at a final concentration of 1.0% by volume and TNF- ⁇ was used at a final concentration of 10 ng/ml.
  • Example 6 lactic acid bacteria extract D1 was used at a final concentration of 1.0% by volume and TNF- ⁇ was used at a final concentration of 10 ng/ml; in Comparative Example 5, TNF- ⁇ was used at a final concentration of 10 ng/ml; in Example 7, lactic acid bacteria extract E1 was used at a final concentration of 1.0% by volume and TNF- ⁇ was used at a final concentration of 10 ng/ml; and in Comparative Example 6, TNF- ⁇ was used at a final concentration of 10 ng/ml.
  • the results of Test Example 1 are shown in Figure 2.
  • the results of Example 3 are shown in Figure 3, the results of Example 4 in Figure 4, the results of Example 5 in Figure 5, the results of Example 6 in Figure 6, and the results of Example 7 in Figure 7.
  • Lactic Acid Bacteria Extract A1 Lactic Acid Bacteria Extract B1, Lactic Acid Bacteria Extract C1, Lactic Acid Bacteria Extract D1 and Lactic Acid Bacteria Extract E1 significantly improved the amount of triglyceride accumulation in adipocytes in which insulin resistance was induced by TNF- ⁇ . This confirmed that Lactic Acid Bacteria Extract A1, Lactic Acid Bacteria Extract B1, Lactic Acid Bacteria Extract C1, Lactic Acid Bacteria Extract D1 and Lactic Acid Bacteria Extract E1 improved insulin resistance in preadipocytes.
  • the present invention provides a novel material that is useful as an active ingredient for an agent for promoting differentiation of adipose precursor cells and an agent for improving insulin resistance.
  • the present invention provides lactic acid bacteria belonging to the genus Fructobacillus, a culture of said lactic acid bacteria, a culture supernatant of said lactic acid bacteria, and/or an extract of said lactic acid bacteria as an active ingredient for an agent for promoting differentiation of adipose precursor cells and an agent for improving insulin resistance.

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WO2019182044A1 (ja) * 2018-03-22 2019-09-26 オリエンタル酵母工業株式会社 多分化能性幹細胞分化促進剤
WO2022138656A1 (ja) * 2020-12-25 2022-06-30 株式会社大阪ソーダ ニコチンアミドモノヌクレオチドの製造方法

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