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WO2019194130A1 - Nouvelle glycoprotéine de type mucine et utilisation associée - Google Patents

Nouvelle glycoprotéine de type mucine et utilisation associée Download PDF

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Publication number
WO2019194130A1
WO2019194130A1 PCT/JP2019/014480 JP2019014480W WO2019194130A1 WO 2019194130 A1 WO2019194130 A1 WO 2019194130A1 JP 2019014480 W JP2019014480 W JP 2019014480W WO 2019194130 A1 WO2019194130 A1 WO 2019194130A1
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Prior art keywords
mucin
type glycoprotein
bacteria
type
mol
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Japanese (ja)
Inventor
宜之 宮本
森田　達也
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MARUKYOU BIO FOODS CO Ltd
Shizuoka University NUC
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MARUKYOU BIO FOODS CO Ltd
Shizuoka University NUC
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Priority to JP2020512235A priority Critical patent/JP7464931B2/ja
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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
    • A23L33/17—Amino acids, peptides or proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology

Definitions

  • the present invention relates to a mucin-type glycoprotein having a novel component or structure having a high sulfate group content and use thereof.
  • Mucin is a viscous substance found in the mucus of animals and plants, and is mainly composed of glycoprotein with a molecular weight of 1 million to 10 million and high sugar content. Most of the sugar chains of this glycoprotein are composed of the hydroxyl group of serine or threonine in the core protein and the sugar located at the reducing end of the sugar chain (often N-acetylgalactosamine) via an O-glycoside bond. It is a relatively short sugar chain formed by binding, and this sugar chain is called a “mucin-type sugar chain” (hereinafter, a glycoprotein having a mucin-type sugar chain is referred to as a mucin-type glycoprotein).
  • Mucin has been reported to have various physiological effects such as protection of mucosal epithelium, moisturizing, antibacterial, lubrication, etc.
  • Mucin or mucin-type glycoprotein extracted and purified from animals and plants are used as health foods and pharmaceuticals.
  • Patent Document 1 discloses a novel mucin-type glycoprotein characterized in that a sugar chain is bound to a repeating structure composed of a specific amino acid sequence, health promotion, drug administration, treatment or prevention of a disease, etc. (Claim 1, Claim 14).
  • Patent Document 2 discloses an antigen-specific T cell proliferation promoter containing mucin collected from ray skin or body surface viscous material as an active ingredient (Claim 1).
  • mucin-type glycoproteins can take a wide variety of components or structures, particularly in their sugar chains. Therefore, even in view of the above-mentioned patent documents, it cannot be said that mucin-type glycoproteins having novel components or structures that can provide useful applications have been sufficiently provided.
  • the present invention has been made to solve such problems, and an object of the present invention is to provide a mucin-type glycoprotein having a novel component or structure and use thereof.
  • the present inventors have succeeded in isolating and purifying a novel mucin-type glycoprotein having a high content of sulfate groups and sialic acid and having a specific component or structure from A.
  • the present inventors have found that mucin-type glycoproteins with a high sulfate group content significantly increase the number of bacteria of the genus Ackermancia and Bacteroides in the intestine.
  • a mucin-type glycoprotein having a high content of threonine can enhance the effect of increasing the number of bacteria of the genus Ackermancia.
  • Non-Patent Document 1 Hubert P. et al., NATURE MEDICINE, Vol. 23, No. 1, January 2017, pp. 107-116
  • Non-Patent Literature 2 Hubert P. et al., NATURE MEDICINE, Vol. 23, No. 1, January 2017, pp. 107-116
  • Non-Patent Literature 2 Hubert P. et al., NATURE MEDICINE, Vol. 23, No. 1, January 2017, pp. 107-116
  • Non-Patent Literature 2 Patrice D. Cani and Willem M. de Vos, Frontiers in Microbiology, Vol. 8, Art. 1765, 22 September 2017
  • Non-Patent Document 3 Amandine Everard et al, PNAS, Vol. 110, No. 22, May 28, 2013, pp. 9066-9071
  • Non-Patent Document 4 Bacteroides teiootaomicron, a Bacteroides genus bacterium, also increases in the number of bacteria after weight loss, and is administered in combination with prebiotics such as lactitol and polydextrose. It has been reported that there is a decrease and a reduction in neutral fat in the blood.
  • a mucin-type glycoprotein containing a sulfate group and sialic acid wherein the content of the sulfate group is 0.07 mol or more with respect to 1 mol of a sugar having an O-glycoside bond to the core protein.
  • An agent for increasing the number of bacteria of the genus Ackermancia and / or Bacteroides comprising the following mucin-type glycoprotein as an active ingredient; A mucin-type glycoprotein containing a sulfate group, wherein the content of the sulfate group is 0.07 mol or more with respect to 1 mol of a sugar having an O-glycoside bond to the core protein. .
  • a food composition for increasing the number of bacteria of the genus Ackermancia and / or Bacteroides comprising the mucin-type glycoprotein according to any one of (1) to (3) as an active ingredient.
  • a food composition for increasing the number of bacteria of the genus Ackermancia and / or Bacteroides comprising the following mucin-type glycoprotein as an active ingredient; A mucin-type glycoprotein containing a sulfate group, wherein the content of the sulfate group is 0.07 mol or more with respect to 1 mol of a sugar having an O-glycoside bond to the core protein. .
  • a food composition for increasing the number of bacteria of the genus Ackermancia wherein, in the mucin-type glycoprotein, the mass percentage of threonine in the total amount of amino acids excluding tryptophan, methionine and cysteine is 15% or more.
  • the food composition for increasing the number of bacteria according to (10).
  • the mucin-type glycoprotein is a mucin-type glycoprotein further containing sialic acid, and the content of the sialic acid is 0. 1 mol per saccharide that is O-glycosidically bonded to the core protein.
  • the mucin-type glycoprotein increases the number of bacteria of the genus Ackermancia and / or Bacteroides in the intestines of humans or animals, according to any one of (14) to (17) Prophylactic or therapeutic agent.
  • the mucin-type glycoprotein is a mucin-type glycoprotein further containing sialic acid, and the content of the sialic acid is 0. 1 mol relative to 1 mol of an O-glycoside bond to the core protein.
  • the mucin-type glycoprotein increases the number of bacteria of the genus Ackermancia and / or Bacteroides in the intestines of humans or animals, according to any one of (19) to (22) A food composition for prevention or treatment.
  • a method for preventing or treating one or more diseases selected from the group consisting of obesity, type 2 diabetes and dyslipidemia comprising the following steps (a) and (b): (A) A step of causing a human or animal suffering from or possibly suffering from the above disease to take the following mucin-type glycoprotein; A mucin-type glycoprotein containing a sulfate group, wherein the content of the sulfate group is 0.07 mol or more with respect to 1 mol of a sugar having an O-glycoside bond to the core protein. , (B) A step of preventing or treating the disease by increasing the number of bacteria of the genus Ackermancia and / or Bacteroides in the intestine of the human or animal.
  • the mucin-type glycoprotein is a mucin-type glycoprotein further containing sialic acid, and the content of the sialic acid is 0. 1 mol per sugar of O-glycosidic bond to the core protein.
  • the method as described in (24) which is 1 mol or more.
  • mucin-type glycoprotein for producing a medicament for the prevention or treatment of one or more diseases selected from the group consisting of obesity, type 2 diabetes and dyslipidemia;
  • the mucin-type glycoprotein is a mucin-type glycoprotein further containing sialic acid, and the content of the sialic acid is 0. 1 mol per 1 mol of an O-glycoside bond to the core protein. Use as described in (28) which is 1 mol or more.
  • a mucin-type glycoprotein having a novel component or structure can be obtained.
  • the number of bacteria of the genus Ackermancia and / or Bacteroides in the living body of humans and animals is effectively increased, thereby preventing diseases such as obesity, type 2 diabetes and dyslipidemia. Can contribute to treatment.
  • Rats that did not take mucin-type glycoprotein (control group), rats that took new mucin-type glycoprotein (1.5% new mucin group), and rats that took porcine gastric mucosa mucin (1.5% porcine gastric mucosa mucin group) ) Is a bar graph showing the concentration of O-linked sugar contained in the cecal content. Bar graph showing the number of 16S rDNA copies derived from various intestinal bacteria present in rat cecal contents of the control group, 0.75% new mucin group, 1.5% new mucin group and 1.5% porcine gastric mucosa mucin group It is.
  • the present invention provides the following ⁇ 1> to ⁇ 7>.
  • “mucin-type glycoprotein” refers to a glycoprotein having a mucin-type sugar chain as described above.
  • the “mucin-type sugar chain” refers to a sugar chain formed by binding to the hydroxyl group of serine or threonine in the core protein via an O-glycoside bond.
  • the sugar located at the reducing end of the mucin-type sugar chain, that is, the sugar that is O-glycosidically bonded to the serine residue or threonine residue of the core protein is often N-acetylgalactosamine.
  • the mucin-type glycoprotein according to the present invention is characterized by containing a high proportion of sulfate groups. Specifically, it contains a sulfate group in a ratio of 0.07 mol or more per mol of O-glycosidic sugar.
  • the mucin-type glycoprotein according to the present invention has a sulfate group at the non-reducing end of the sugar chain because the sulfate group is often added to the sugar residue at the non-reducing end of the mucin-type sugar chain. It can be said that.
  • the mucin-type glycoprotein according to the present invention may contain a high proportion of sialic acid in addition to sulfate groups. Specifically, sialic acid may be contained in a ratio of 0.1 mol or more per mol of O-glycosidic sugar. Since sialic acid is also often located at the non-reducing end of mucin-type sugar chains, the mucin-type glycoprotein according to the present invention has sialic acid at many of the sugar chain non-reducing ends. It's okay.
  • threonine content is, for example, 28 mg / g or more, 28.5 mg / g or more, 29 mg / g or more, 29.5 mg / g or more, 30 mg / g or more, 30.5 mg / g or more, 31 mg / g or more. 31.5 mg / g or more, 32 mg / g or more, 32.5 mg / g or more, 33 mg / g or more, 33.5 mg / g or more.
  • the mass percentage of threonine in the total amount of amino acids excluding tryptophan, methionine and cysteine is preferably 14% or more, 14.5% or more, 15% or more, 15.5% or more, 16% or more, 16.5 %, 17%, 17.5%, 18%, 18.5%, 19%, 19.5% or more.
  • the amino acid content and mass percentage in the mucin-type glycoprotein according to the present invention are derivatized by the post-column hydrin method after hydrolyzing the protein with a strong acid, as shown in Example 6 (2) described later. It is preferable to quantify by a method of separating and measuring by chromatography.
  • the mucin-type glycoprotein according to the present invention can be collected from, for example, a sticky substance adhering to the epidermis or body surface of rays (cartilage fish belonging to the order of the Panaxiae ray) such as Gangi ray.
  • a sticky substance adhering to the epidermis or body surface of rays such as Gangi ray.
  • the skin / body surface viscous material of the genus Kasugabebe and the body surface viscous material of the genus Sokogangei are used in combination, but it is also possible to use raw materials derived from multiple types of rays in this way. It is also possible to use a raw material derived from a single type of ray.
  • proteolytic enzymes can be appropriately selected from proteinases (endopeptidases) such as aspartic proteinases, metalloproteinases, serine proteinases and thiol proteinases, and peptidases (exopeptidases) according to the origin of the raw materials.
  • the mucin-type glycoprotein according to the present invention can be used by ingestion by humans or animals. Further, since the mucin-type glycoprotein according to the present invention exerts its function in the intestine of a living body, it may be used in a method that reaches the intestine, for example, by adding an active ingredient to an enteral nutrient, You may use this by the method of administering by enteral nutrition through the tube inserted in digestive tracts, such as the stomach and the small intestine.
  • the mucin-type glycoprotein according to the present invention mainly exerts its action in the gastrointestinal tract (an action to increase the number of bacteria against specific intestinal bacteria). Therefore, when using the mucin-type glycoprotein according to the present invention for humans and animals, it is appropriate to calculate the intake (dose) per food intake, not per general body weight for other drugs. it is conceivable that. In the examples described later, the effect was observed in rats fed a feed containing about 1% by mass of mucin-type glycoprotein (FIG. 6).
  • the basal metabolism of rats is remarkably larger than that of humans, in humans, effective doses of 1/4 to 1/2 of those effective in rats are often effective. Therefore, according to this example, when the mucin-type glycoprotein according to the present invention is used in humans, the daily intake is “0.25 of the daily food (solid matter) intake in the human individual. An amount of about 0.5% by mass can be exemplified.
  • the mucin-type glycoprotein according to the present invention contains a high proportion of sulfate groups, Ackermancia, which is an enteric bacterium having a sulfate group-degrading enzyme (sulfatase), in the intestines of humans and animals who ingest it.
  • Ackermancia which is an enteric bacterium having a sulfate group-degrading enzyme (sulfatase)
  • the number of genus bacteria and Bacteroides bacteria can be specifically increased.
  • Ackermansia bacteria refers to microorganisms belonging to the genus Ackermancia.
  • Examples of such microorganisms include Akkermansia muciniphila.
  • Ackermansia muciniphila is an intestinal bacterium that lives in the intestines of many humans. As described above, administration of this bacterium suppresses obesity progression, fat mass development, and insulin resistance in mice. It has been reported that sex and dyslipidemia are reduced.
  • bacteria bacteria refers to microorganisms belonging to the genus Bacteroides. Examples of such microorganisms include Bacteroides-thetaiotaomicron. Bacteroides spp. Are also bacteria that make up the intestinal flora. As mentioned above, Bacteroides tetaiotaomicron has increased in number after weight loss and prebiotics such as lactitol and polydextrose. It has been reported that co-administration with the drug reduces body weight loss and triglycerides in the blood.
  • the mucin-type glycoprotein according to the present invention can be used for the purpose of preventing or treating this disease.
  • the mucin-type glycoprotein according to the present invention is an agent for increasing the number of bacteria of this bacterium, a food composition for increasing the number of bacteria of this bacterium, a preventive or therapeutic agent for this disease, or prevention of this disease.
  • it can be used as an active ingredient of a therapeutic food composition. That is, the mucin-type glycoprotein according to the present invention can be used for producing a pharmaceutical product for preventing or treating this disease. And (a) ingesting the mucin-type glycoprotein according to the present invention to a human or animal suffering from or possibly suffering from the disease, and (b) the bacteria in the intestine. This disease can be prevented or treated by increasing the number of bacteria and preventing or treating the disease.
  • Examples of the form of the agent of the present invention include forms consisting only of mucin-type glycoprotein which is an active ingredient, and forms such as pharmaceuticals, food additives and supplements combined with appropriate excipients and carriers.
  • the dosage forms include, for example, powders, tablets, dragees, capsules, granules, dry syrups, liquids, syrups, drops, drinks, etc. Or a liquid dosage form can be mentioned.
  • Such pharmaceutical products, food additives and supplements can be produced by methods known to those skilled in the art.
  • the form of the food composition of the present invention includes forms of ordinary foods and drinks such as confectionery and beverages, processed foods, health foods, infant foods, in addition to those composed only of mucin-type glycoprotein which is an active ingredient. Can do. When making it into the form of food and drink, it can be produced by adding an active ingredient in a normal production process.
  • “increasing the number of bacteria” of the present bacterium means increasing the number of the bacterium in any cell or tissue / organ of the living body.
  • the number of bacteria in the intestine is thought to correlate with the number of bacteria in the intestinal contents or feces, measure the number of bacteria in the intestinal contents or feces. Thus, it can be confirmed whether or not the number of the bacteria in the intestine has increased.
  • the intestinal contents or stool after ingestion of the mucin-type glycoprotein according to the present invention is used as a sample, and a real-time PCR method using a primer specific to this bacterium is performed to measure the 16S rDNA copy number.
  • Primers specific to this bacterium can be designed based on the known base sequences of this bacterium, and for example, primers comprising the sequences shown in SEQ ID NOs: 1 to 4 can be used.
  • it can be quantified using a commercially available kit for quantifying the bacterium.
  • the 16S rDNA copy number of this bacterium and the number of bacteria of this bacterium are correlated, the 16S rDNA copy number can be used as an index of the number of bacteria. Therefore, as a result of measuring the 16S16rDNA copy number of this bacterium, if the 16S rDNA copy number in the stool after ingestion is greater than that before ingestion, or the 16S rDNA copy number in the cecal contents after ingestion has not been ingested If it is larger than the cecal content, it can be determined that the number of bacteria of this bacterium has increased due to the mucin-type glycoprotein according to the present invention.
  • Example 1 Isolation and purification of a novel mucin-type glycoprotein Peel off the epidermis along with the sticky substance adhering to the body surface from a true kasbe (Raja pulchra Liu). This was used as a raw material derived from true Kasube.
  • the water casbe the upper eye moth moth, the genus Scorpio genus genus Dobkasbe, Bathyraja smirnovi
  • the sticky substance adhering to the body surface is removed by washing with water, and this is used as the raw material derived from water casbe .
  • Comparative Example 1 Porcine Gastric Mucosal Mucin As Comparative Example 1, a purified product of porcine gastric mucosal mucin was used. Specifically, 5 g of commercially available porcine stomach mucosa mucin (Mucin from porcine stomach Type II, SIGMA-ALDRICH) was weighed into a beaker, and 100 times the amount of 0.15 M NaCl aqueous solution was added and suspended therein. PH was adjusted to 7.5 with an aqueous NaOH solution. This was homogenized with a homogenizer PT-3100 (KINEMATICA) at 4500C for 14 seconds at 14500 revolutions / minute (rpm), and then subjected to suction filtration to obtain a filtrate.
  • a homogenizer PT-3100 KINEMATICA
  • Comparative Example 2 Rat Intestinal Mucosal Mucin As Comparative Example 2, mucin secreted from the rat intestinal mucosa was used. Since the mucin contained in the cecal contents of rats that do not take mucin is mucin secreted from the intestinal mucosa, the rat bred under the conditions of (1) below is dissected and the cecal contents are recovered, From there, a cecal content mucin fraction was prepared by the method of (2) below, and this was designated as “rat intestinal mucosa mucin”.
  • Rat breeding conditions Feed Standard purified feed containing no mucin (milk casein 25.0%, corn starch 60.25%, cellulose powder 5.0%, corn oil 5.0%, vitamin mixture 1.0% , 3.5% mineral mixture, 0.25% choline bitartrate). For vitamins and minerals, a mixture compliant with AIN-76 (Guidelines for standard purified feed for rats published in 1977 by the National Institute of Nutrition (AIN)) was used. Drinking water: In order to suppress the degradation of mucin by enteric bacteria, the following antibiotics were added to the drinking water to the final concentration in parentheses. Benzylpenicillin (50 units / mL), neomycin trisulfate (2 mg / mL), cefoperazone sodium (0.5 mg / mL). Breeding period: 7 days
  • Example 2 Evaluation of novel mucin-type glycoprotein: components and structure Regarding the novel mucin-type glycoprotein of Example 1, the porcine gastric mucosa mucin of Comparative Example 1 and the rat intestinal mucosa mucin of Comparative Example 2, the following (1) to Component analysis was performed by the method (6). 10 mg of each powder was dissolved in 4 mL of distilled water to make a mucin solution and used for analysis. In addition, new mucin-type glycoproteins are prepared from six different raw materials (referred to as lots A to F), and a plurality of samples (samples 1 to 13) from each lot are individually collected (sampled) and analyzed. did. In addition, porcine gastric mucosal mucin and rat intestinal mucosal mucin were also analyzed by sampling 7 samples (samples 1 to 7) individually.
  • the protein concentration was measured according to the Lowry method. Specifically, bovine serum-derived albumin (SIGMA-ALDRICH) was used as the standard sample, and a dilution series of 25-100 ⁇ g / mL was prepared with distilled water. As a measurement sample, a mucin solution was diluted 20 times with distilled water. A 0.5N sodium hydroxide aqueous solution containing 10% Na 2 CO 3 and a 1% sodium citrate aqueous solution containing 0.5% CuSO 4 .5H 2 O were mixed at a volume ratio of 10: 1. Biuret reagent was prepared by mixing at a ratio of Further, a diluted phenol reagent was prepared by diluting 1.8N phenol reagent (Nacalai Tex) 12 times with distilled water.
  • SIGMA-ALDRICH bovine serum-derived albumin
  • the total sugar concentration was measured according to the phenol-sulfuric acid method and calculated in terms of glucose.
  • 1 mg / mL D (+)-glucose (Wako) was used as a standard sample, a dilution series of 31.25 to 250 ⁇ g / mL was prepared with distilled water, and a large test was performed including a blank of distilled water only. 1 mL each was dispensed into two tubes.
  • the measurement sample is a stock solution of 50 mg of powdered mucin dispersed in 10 mL of distilled water, which is diluted 10, 20, 40, and 100 times with distilled water and divided into 1 mL each for two large test tubes including the stock solution. Noted.
  • O-linked sugar concentration The concentration of O-glycoside-linked sugar (O-linked sugar) was determined according to the method of Crowther RS et al. (Crowther RS, Wetmore RF, Fluorometric assay of O- linked glycoproteins by reaction with 2-cyanoacetamide, Anal. biochem., 163, 170-174, 1987). That is, the O-glycoside bond of glycoprotein was cleaved by alkali treatment, and the resulting sugar chain reducing end was reacted with 2-cyanoacetamide (2-CNA) to produce a fluorescent substance, and the fluorescence intensity was measured.
  • 2-cyanoacetamide 2-cyanoacetamide
  • N-acetylgalactosamine SIGMA-ALDRICH
  • a dilution series of 0.15625 to 10 ⁇ g / mL was prepared with distilled water.
  • a mucin solution was diluted 80 times with distilled water.
  • an alkalinized 2-CNA solution was prepared by mixing a 0.15 M aqueous sodium hydroxide solution and a 0.6 M 2-CNA aqueous solution in a volume ratio of 5: 1. 100 ⁇ L of each of the standard sample and the measurement sample was dispensed into a 1.5 mL capacity microtube, 120 ⁇ L of the alkalized 2-CNA solution was added, and the mixture was heated at 100 ° C.
  • Ion chromatography conditions Equipment: Ion chromatograph ICS-2000 (DIONEX) Detector: Electrical conductivity detector DS6 (DIONEX) Column: IonPacAS17 (4 ⁇ 250 mm) (DIONEX) Guard column: IonPac AG17C (DIONEX) Column temperature: 30 ° C Eluent: potassium hydroxide aqueous solution whose concentration is linearly increased from 5 mM to 40 mM in 18 minutes Flow rate: 1 mL / min
  • N-acetylneuraminic acid (NeuAc) and N-glycolylneuraminic acid (NeuGc) were used as standard samples.
  • a mucin solution was diluted 20 times with distilled water.
  • 50 ⁇ L of a measurement sample and 200 ⁇ L of 62.5 mM hydrochloric acid were placed in a glass vial.
  • the sialic acid was liberated by acid hydrolysis by leaving it in a heat block set at 80 ° C. for 1 hour. Then, it was left to cool at room temperature for 20 minutes.
  • ABEE Labeling solution (ABEE Labeling Kit, J-Oil Mills) were added, and the mixture was heated at 80 ° C. for 1 hour using a heat block. Thereafter, 200 ⁇ L of distilled water and 200 ⁇ L of chloroform were added and mixed with a vortex mixer. After centrifugation at 15000 ⁇ g for 5 minutes at room temperature, the upper layer (aqueous layer) was recovered, filtered (DISMIC-13HP, 13HP020AN (Advantech)), and subjected to HPLC under the following conditions.
  • Example 2 (1) to (5) are shown in Table 1, and the result of (6) is shown in Table 2.
  • the concentration of O-linked saccharide serving as an index of the amount of mucin-type sugar chains is 0.434 to 0.548 micromol / mg ( ⁇ mol / mg) for the novel mucin-type glycoprotein, It was 0.728 to 0.752 ⁇ mol / mg for mucosal mucin and 0.434 to 0.498 ⁇ mol / mg for rat intestinal mucosal mucin.
  • the sulfate group concentration was 0.039 to 0.090 ⁇ mol / mg for the novel mucin-type glycoprotein, whereas 0.035 to 0.046 ⁇ mol / mg for the porcine gastric mucosa, and for the rat intestinal mucosa mucin.
  • the amount of sulfate group per mole of O-linked sugar is 0.047 to 0.063 mole (0.035 / 0.752 ⁇ 0.047 to 0.046 / 0.728 ⁇ ) of porcine gastric mucosa mucin.
  • 0.063 0.042-0.085 mol of rat intestinal mucosa mucin (0.021 / 0.498 ⁇ 0.042-0.037 / 0.434 ⁇ 0.085)
  • a novel mucin type In glycoprotein it is 0.071 to 0.207 mol (0.039 / 0.548 ⁇ 0.071 to 0.090 / 0.434 ⁇ 0.207), and it is remarkably large in the novel mucin type glycoprotein. Became clear.
  • the sialic acid concentration was 0.045 to 0.051 ⁇ mol / mg for porcine gastric mucosa mucin, whereas it was 0.068 to 0.085 ⁇ mol / mg for new mucin-type glycoprotein, and for rat intestinal mucosa mucin. It was 0.189 to 0.208 ⁇ mol / mg. That is, the amount of sialic acid per mole of O-linked sugar is 0.060 to 0.070 mole (0.045 / 0.752 ⁇ 0.060 to 0.051 / 0.728 ⁇ ) of porcine gastric mucosa mucin.
  • the sugar chain of the novel mucin-type glycoprotein has galactose of about 0.3 to 0.7 ⁇ mol / mg, fucose of about 0.3 to 0.5 ⁇ mol / mg, and mannose of about 0 to 0.05 ⁇ mol / mg, N-acetylglucosamine (GlcNAc) approximately 0.3 to 0.6 ⁇ mol / mg, N-acetylgalactosamine (GalNAc) approximately 0.7 to 1.1 ⁇ mol / mg It was.
  • the new mucin-type glycoprotein contains a high proportion of 0.07 mol or more of sulfate groups with respect to 1 mol of O-glycoside-bonded sugar even in consideration of lot-to-lot differences and measurement errors. It has become clear that it has a novel component or structure.
  • the novel mucin-type glycoprotein has a high sialic acid content in addition to the sulfate group, and it is clear that 0.1 mol or more of sialic acid is contained per 1 mol of O-glycoside-bonded sugar. It was.
  • the sulfate group is often added to the sugar residue at the non-reducing end of the sugar chain. Therefore, in the measurement results of Table 1, the number of moles of O-linked sugar is assumed to be the number of moles of mucin-type sugar chains, and all sulfate groups are considered to be located at the non-reducing end of the mucin-type sugar chains.
  • the sugar chain non-reducing end of the sulfate group is about 13.3% ((0.064 / 0.481) ⁇ 100 ⁇ 13.31) on average.
  • porcine gastric mucosa mucin since the sulfate group is 0.040 mol (average value) with respect to 0.740 mol (average value) of mucin-type sugar chains, the end capping rate due to sulfate groups is The average is only about 5.4% ((0.040 / 0.740) ⁇ 100 ⁇ 5.41). Further, in rat intestinal mucosa mucin, since the sulfate group is 0.026 mol (average value) with respect to 0.466 mol (average value) of mucin-type sugar chains, the end capping rate due to sulfate groups is about 5 on average. .6% ((0.026 / 0.466) ⁇ 100 ⁇ 5.58).
  • the novel mucin-type glycoprotein contains sulfate groups at many non-reducing sugar chains, and the end capping rate is 2 compared with other mucin-type glycoproteins such as porcine gastric mucosa mucin and rat intestinal mucosa mucin. It became clear that it was significantly higher than twice. From this, it was revealed that the novel mucin-type glycoprotein has a unique structure in which a sulfate group is contained at a number of sugar chain non-reducing ends.
  • Example 3 Evaluation of novel mucin-type glycoprotein: mucinase activity in rats not ingesting mucin
  • the novel mucin-type glycoprotein of Example 1 and the porcine gastric mucosa mucin of Comparative Example 1 are contained in the stool of rats not ingesting mucin
  • the degree of degradation (mucinase activity) by mucin-type sugar chain degrading enzyme (mucinase) was analyzed. Note that most of the mucinase present in stool is derived from intestinal bacteria.
  • Mucinase reaction 200 mg of a novel mucin-type glycoprotein or porcine gastric mucosa mucin was dissolved in 10 mL of 0.01 M acetate buffer (pH 5.5) and stored refrigerated overnight to obtain a substrate solution. After preheating the mucinase enzyme solution at 30 ° C. for 2 minutes, 0.1 mL of the substrate solution was added to 0.9 mL of the mucinase enzyme solution and placed at 30 ° C. for 30 minutes to carry out the mucinase reaction. Thereafter, the reaction was stopped by heating in boiling water for 3 minutes to deactivate the enzyme, and this was used as a post-reaction solution.
  • the supernatant was collected by centrifugation at 2330 ⁇ g for 10 minutes at room temperature, and the absorbance was measured at 660 nm.
  • a calibration curve was prepared based on the absorbance measurement result of the standard sample. Using this calibration curve, the reducing sugar concentration (nmol / mL) in the post-reaction solution was calculated from the absorbance measurement result of the post-reaction solution. The result is shown in FIG.
  • both the new mucin-type glycoprotein and the porcine gastric mucosa mucin had a higher reducing sugar concentration as the enzyme reaction time was longer. That is, it became clear that sugar chains were decomposed by mucinase present in feces to produce reducing sugar.
  • the novel mucin-type glycoprotein is significantly smaller at any of the enzyme reaction times of 10 minutes, 20 minutes and 30 minutes, The concentration of porcine gastric mucosa mucin was about 1/3 to 1/5.
  • the mucinase activity for the novel mucin-type glycoprotein was significantly smaller than that of porcine gastric mucosa mucin. From this result, it was revealed that the novel mucin-type glycoprotein has a unique component or structure that is difficult to be assimilated by intestinal bacteria of rats that have ingested a purified feed containing no mucin.
  • the degradation of mucin-type sugar chains proceeds from the non-reducing end by exo-type mucinase, but the degradation is inhibited when a sulfate ester or sialic acid is present at the non-reducing end of the sugar chain.
  • a sulfate ester or sialic acid is present at the non-reducing end of the sugar chain.
  • the reason why the mucinase activity for the novel mucin-type glycoprotein is small is that sulfate groups and sialic acids are present in many of the sugar chain non-reducing ends of the novel mucin-type glycoprotein. You might also say that.
  • Example 4 Evaluation of novel mucin-type glycoprotein: mucinase activity in mucin-ingested rats The mucin of mucin-containing mucin in Example 1 and porcine gastric mucosal mucin in Comparative Example 1 and rat intestinal mucosal mucin in Comparative Example 2 The degree of degradation by mucinase contained in the ingested rat feces (mucinase activity) was analyzed.
  • Control group Standard purified feed of Comparative Example 2 (1).
  • 1.5% new mucin group A feed containing 1.5% of new mucin-type glycoprotein by replacing corn starch in the standard purified feed of Comparative Example 2 (1).
  • 1.5% porcine gastric mucosa mucin group A feed containing 1.5% porcine gastric mucosa mucin by replacing corn starch in the standard purified feed of Comparative Example 2 (1).
  • a mucinase enzyme solution was prepared by the method described in Example 3 (1). These were designated as “control group-derived enzyme”, “new mucin group-derived enzyme” and “porcine gastric mucosa mucin group-derived enzyme”. Moreover, the protein concentration in these mucinase enzyme solutions was measured according to the Lowry method described in Example 2 (1).
  • Example 3 (3) Mucinase reaction
  • the three mucin-type glycoproteins of Example 1, porcine gastric mucosal mucin of Comparative Example 1 and rat intestinal mucosal mucin of Comparative Example 2 were used as substrates, and the mucinase enzyme solution of Example 4 (2).
  • the mucinase enzyme solution of Example 4 (2) was used to carry out a mucinase reaction by the method described in Example 3 (2).
  • the mucinase enzyme solution was used after being diluted 4-fold with 0.01 M acetate buffer (pH 5.5). Subsequently, the reducing sugar concentration was measured by the method described in Example 3 (3).
  • FIG. 2 shows the results when rat intestinal mucosa mucin is used as a substrate
  • FIG. 3 shows the results when porcine gastric mucosa mucin is used as a substrate
  • FIG. 4 shows the results when new mucin-type glycoprotein is used as a substrate. Show.
  • the amount of reducing sugar when rat intestinal mucosa mucin was used as a substrate was 29.6 ⁇ 2.1 nmol / min / mg protein for the enzyme derived from the control group, whereas the novel mucin was used.
  • the group-derived enzyme is 92.6 ⁇ 5.7 nmol / min / mg protein
  • the porcine gastric mucosa mucin-group enzyme is 87.6 ⁇ 13.0 nmol / min / mg protein, both compared with the control group-derived enzyme Significantly higher.
  • mucinase activity in rat intestinal mucosal mucin was higher in the mucinase contained in the stool of rats that received the novel mucin-type glycoprotein or porcine gastric mucosa mucin than in the stool of rats that did not take mucin . From these results, it was clarified that ingestion of a novel mucin-type glycoprotein or porcine gastric mucosa induces intestinal bacteria having high degrading activity against “mucin secreted from the intestinal mucosa”.
  • the amount of reducing sugar when porcine gastric mucosa mucin was used as a substrate was 7.6 ⁇ 1.8 nmol / min / mg protein for the control group-derived enzyme.
  • the enzyme derived from the new mucin group is 21.9 ⁇ 2.6 nmol / min / mg protein, and the enzyme derived from the porcine gastric mucosa mucin group is 23.6 ⁇ 5.3 nmol / min / mg protein. It was significantly higher compared.
  • mucinase activity in porcine gastric mucosal mucin was higher in mucinase contained in the stool of rats ingesting the novel mucin-type glycoprotein or porcine gastric mucosal mucin than in the stool of rats not ingesting mucin . From these results, it was clarified that ingestion of a novel mucin-type glycoprotein or porcine gastric mucous mucin induces intestinal bacteria having a high degrading activity against "pig gastric mucosal mucin".
  • the amount of reducing sugar is 3.3 ⁇ 0.4 nmol / min / mg protein for the control group-derived enzyme
  • the porcine gastric mucosa mucin group The enzyme derived from the enzyme was 5.3 ⁇ 0.6 nmol / min / mg protein, whereas the enzyme derived from the new mucin group was 9.6 ⁇ 0.7 nmol / min / mg protein. It was significantly higher than the enzyme derived from the gastric mucosa group.
  • the mucinase activity for the novel mucin-type glycoprotein was determined not only by the mucinase contained in the stool of the rat that ingested the novel mucin-type glycoprotein, but also by the mucous mucin contained in the feces of the rat that did not take the mucin. It was higher than the mucinase contained in the stool of the ingested rat. From this result, it was clarified that ingestion of a novel mucin-type glycoprotein induces intestinal bacteria having a high degrading activity against "new mucin-type glycoprotein".
  • a cecal content mucin fraction was prepared by the method described in Comparative Example 2 (2). did.
  • the mucin contained in the cecal contents of rats not ingesting mucin (control group) is rat intestinal mucosa mucin, but rats ingested mucin (1.5% new mucin group, 1.5% porcine gastric mucosa)
  • the mucin contained in the cecal contents of the mucin group is derived from rat intestinal mucosa mucin and ingested mucin.
  • Distilled water was added to the prepared cecal content mucin fraction and the volume was adjusted to 5 mL, and then diluted 10 times with distilled water.
  • concentration of O-glycosidically linked saccharide was measured by the method described in Example 2 (3). Based on the measurement result of the standard sample, the concentration of O-linked sugar in the measured sample was determined, and the O-linked sugar concentration ( ⁇ mol / g) per 1 g of cecal content mucin fraction was calculated. The result is shown in FIG.
  • the O-linked sugar concentration was 0.50 ⁇ 0.04 ⁇ mol / g in the control group, whereas 2.26 ⁇ 0.47 ⁇ mol in the 1.5% new mucin group. / G, significantly higher than the control group.
  • the O-linked sugar concentration in the 1.5% porcine gastric mucosa mucin group was 0.54 ⁇ 0.11 ⁇ mol / g, which was not significantly different from the control group.
  • the concentration of O-linked sugar is considered to be proportional to the amount of mucin-type sugar chain. Therefore, it is clear that significantly more mucin-type glycans remain in the intestine of rats fed with a novel mucin-type glycoprotein than in the intestines of rats fed porcine gastric mucosa. became.
  • the novel mucin-type glycoprotein is used at a slower rate in the rat intestine than the porcine gastric mucosa mucin.
  • the novel mucin-type glycoprotein has a unique component or structure that is not easily assimilated by intestinal bacteria that have not ingested it. It is thought to have.
  • Example 5 Evaluation of novel mucin-type glycoprotein: action on intestinal bacteria Enterobacteria (all eubacteria, rats) ingesting the novel mucin-type glycoprotein of Example 1 and porcine gastric mucosa mucin of Comparative Example 1 Changes in the amount of Ackermansia muciniphila and Bacteroides teiotaomicron were analyzed by real-time PCR targeting the 16S rRNA gene.
  • 1.5% new mucin group A feed containing 1.5% of new mucin-type glycoprotein by replacing corn starch in the standard purified feed of Comparative Example 2 (1).
  • 1.5% porcine gastric mucosa mucin group A feed containing 1.5% porcine gastric mucosa mucin by replacing corn starch in the standard purified feed of Comparative Example 2 (1).
  • Real-time PCR was performed using LightCycler (registered trademark) Nano (Roche) according to the attached instruction manual.
  • the composition of the reaction solution was 2 ⁇ L of template DNA solution, 10 ⁇ L of SYBR Premix EX Taq II (Takara Bio), 0.8 ⁇ L of 10 ⁇ M sense primer solution, 0.8 ⁇ L of 10 ⁇ M antisense primer solution, and 6 DEPC-treated water. 4 ⁇ L total 20 ⁇ L.
  • the template DNA solution was diluted 500-fold when measuring all eubacteria, and 100-fold when measuring Ackermansia cinophila or Bacteroidestetaiotamicron. Used.
  • a calibration curve was prepared by measurement using a known concentration of DNA, and the 16S rDNA copy number of each bacterium per cecal content was absolutely determined from the calibration curve based on the measurement result. The result is shown in FIG.
  • the primer sequences specific to each bacterium are shown below.
  • Sense primer CAGCACGTGAAGGTGGGGAC (SEQ ID NO: 1)
  • Antisense primer CCTTGCCGGTTGCTTCAGAT (SEQ ID NO: 2)
  • Bacteroides Tetaiotaomicron Sense primer: GCAAACTGGGAGATGGCGA (SEQ ID NO: 3)
  • Antisense primer AAGGTTTGGTGCCGTTA (SEQ ID NO: 4)
  • Antisense primer CCATTGTAGCACGTGTGTAGCC (SEQ ID NO: 6)
  • the 16S rDNA copy number of all eubacteria was not significantly different between the groups.
  • the 16S rDNA copy number of Ackermancia mucinifira was significantly higher in the 1.5% new mucin group than in the control group, and the 0.75% new mucin group was also significantly different from the control group. There was nothing big.
  • the 16S DNA copy number of Ackermansia muciniphila in the porcine gastric mucosa mucin group was equivalent to that in the control group.
  • the 16S rDNA copy number of Bacteroides tetaiotaomicron was significantly higher in the 1.5% new mucin group than in the control group, and there was no significant difference in the 0.75% new mucin group as compared with the control group. It was big.
  • the 16S DNA copy number of Bacteroides tetaiotaomicron in the porcine gastric mucosa mucin group was equivalent to that in the control group.
  • the 16S rDNA copy number of the Ackermancia genus bacteria and the 16S rDNA copy number of the Bacteroides bacteria in the rat cecal contents were significantly increased by ingestion of the novel mucin-type glycoprotein. From these results, it was revealed that the novel mucin-type glycoprotein has an action of increasing the number of bacteria of the genus Ackermancia and Bacteroides.
  • enterobacteria have no sulfate group-degrading enzyme (sulfatase) or sialic acid-degrading enzyme (sialidase), so they assimilate sugar chains with high sulfate group and sialic acid content. Can not.
  • sulfate group-degrading enzyme sulfatase
  • sialic acid-degrading enzyme sialidase
  • mucin-type glycoproteins containing sulfate groups at a high ratio of 0.07 mol or more with respect to 1 mol of O-glycosidic linkage to the core protein are bacteria of Ackermancia bacteria and Bacteroides bacteria. It has become clear that it has the effect of increasing the number.
  • the protein concentration, total sugar concentration, O-linked sugar concentration, sulfate group concentration and sialic acid concentration were measured by the methods described in Examples 2 (1) to (5).
  • the results are shown in Table 3.
  • the left side of ⁇ shows the average value
  • the right side shows the standard error.
  • the numerical value with the standard error is the result of sampling three samples from each lot and performing triplicate measurements for each sample.
  • the numerical value without the standard error is an average value obtained by sampling one sample from each lot and measuring triplicate for each sample.
  • the novel mucin-type glycoprotein has a sulfate group of 0.07 mol or more and sialic acid of 0.1 mol or more per mol of O-glycoside-bonded sugar in any lot. It was confirmed that it was contained at a high ratio.
  • HF61 Yamato Science
  • the reaction tube was returned to room temperature and the contents in the tube were transferred to a volumetric flask and then washed 5 times with distilled water.
  • 1.75 mL of 3 mol / L NaOH was added to adjust the pH to around 2.2, and the volume was adjusted to 25 mL with 0.067 mol / L sodium citrate buffer (pH 2.2, Wako).
  • This was filtered through a membrane filter (DISMIC-13HP, 13HP020AN, Advantech), and the filtrate was recovered and subjected to amino acid analysis.
  • the amino acid analysis was performed using an analytical column (Hitachi High-Tech Packed Column # 2622PH, diameter 4.6 ⁇ 60 mm, Hitachi High-Technologies) and a precolumn (Hitachi Packed Column # 2650L, diameter 4.6 ⁇ 40 mm, Hitachi High-Technologies). -8900 high-speed amino acid analyzer (Hitachi High-Technologies).
  • Commercially available kits were used for the eluent (MCI TM BUFFER L-8500-PH-KIT, Mitsubishi Chemical) and the reaction solution (Hitachi's ninhydrin coloring solution kit, Wako).
  • an amino acid mixed standard solution type H (Wako) 1 mL was used which was fixed to 25 mL with 0.067 mol / L sodium citrate buffer (pH 2.2, Wako).
  • lot IV has a smaller content and mass percentage of serine and threonine and a larger content and mass percentage of isoleucine, tyrosine and phenylalanine than lots I to III and lot V. It was revealed.
  • the number of 16S rDNA copies of all eubacteria was not significantly different between the groups.
  • the 16S rDNA copy number of Ackermancia mucinifira was significantly larger than the control group in the lot I group, the lot II group, the lot III group and the lot V group, and also compared with the control group in the lot IV group. Although there was no significant difference, it was large. That is, the 16S rDNA copy number of Ackermancia bacteria in rat cecal contents was increased by ingestion of a novel mucin-type glycoprotein. From this result, it was revealed that the novel mucin-type glycoprotein has an action of increasing the number of bacteria of the genus Ackermancia.
  • Non-Patent Document 5 Ackermancia muciniphila cannot synthesize threonine among essential amino acids (RESULTS AND DISCUSSION, 2nd paragraph, lines 5-7). Further, according to Non-Patent Document 6, Ackermancia cinophila cannot grow on a medium containing ammonia and other amino acids and not containing L-threonine (Results and discussion, first paragraph, lines 14-17, Fig. 17). S1). In other words, it can be said that Ackermancia sinifira needs to be supplied with threonine from the outside for survival or proliferation.
  • Non-Patent Document 5 Ottman N.
  • the mucin-type glycoprotein of lot IV has a threonine content ratio of 28.5 mg / g mucin-type glycoprotein and the mass percentage of threonine in the total amino acids as compared to other lots. was as small as 14.2%. From this, although the number of bacteria of the genus Ackermancia is increasing in the lot IV group, the degree of increase is relatively small because the threonine content ratio or total amino acids in the ingested mucin-type glycoprotein This was thought to be due to the small mass percentage occupied. That is, this result revealed that mucin-type glycoproteins with a high threonine content have a stronger effect of increasing the number of bacteria of the genus Ackermancia.
  • the content ratio of threonine is 29 mg / g or more, or the total amount of amino acids excluding tryptophan, methionine and cysteine. It was found that the threonine occupying mass percentage is preferably 15% or more.

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Abstract

Le problème décrit par la présente invention concerne la fourniture d'une nouvelle glycoprotéine de type mucine ayant une teneur élevée en groupe sulfate et ayant une composition ou une structure unique, ainsi qu'une utilisation associée. La solution selon l'invention porte sur une glycoprotéine de type mucine comprenant un groupe sulfate et un acide sialique, la teneur en groupe sulfate étant d'au moins 0,07 mole pour 1 mole de saccharide lié à une protéine noyau par une liaison O-glycosidique, et la teneur en acide sialique étant d'au moins 0,1 mole pour 1 mole de saccharide lié à une protéine noyau par une liaison O-glycosidique. Selon la présente invention, il est possible d'obtenir une glycoprotéine de type mucine ayant une nouvelle composition ou structure. De plus, selon la présente invention, la glycoprotéine de type mucine peut augmenter de manière efficace le nombre de bactéries Akkermansia et/ou de bactéries Bacteroides dans le corps d'un être humain ou d'un animal vivant, et peut ainsi contribuer à la prévention ou au traitement de maladies telles que l'obésité, le diabète de type 2, l'hyperlipidémie, etc.
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WO2023176950A1 (fr) 2022-03-18 2023-09-21 株式会社明治 Composition pour lutter contre la prolifération d'une bactérie dans l'intestin, et son utilisation

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WO2017178496A1 (fr) * 2016-04-11 2017-10-19 Wageningen Universiteit Nouvelles espèces bactériennes

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WO2010107083A1 (fr) * 2009-03-19 2010-09-23 丸共バイオフーズ株式会社 Promoteur de la prolifération des lymphocytes t spécifiques de l'antigène
WO2017178496A1 (fr) * 2016-04-11 2017-10-19 Wageningen Universiteit Nouvelles espèces bactériennes

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* Cited by examiner, † Cited by third party
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WO2023176950A1 (fr) 2022-03-18 2023-09-21 株式会社明治 Composition pour lutter contre la prolifération d'une bactérie dans l'intestin, et son utilisation

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