WO2019194130A1 - Novel mucin-type glycoprotein and use thereof - Google Patents

Novel mucin-type glycoprotein and use thereof Download PDF

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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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mucin
type glycoprotein
bacteria
type
mol
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PCT/JP2019/014480
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French (fr)
Japanese (ja)
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宜之 宮本
森田　達也
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丸共バイオフーズ株式会社
国立大学法人静岡大学
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Priority to JP2020512235A priority Critical patent/JP7464931B2/en
Publication of WO2019194130A1 publication Critical patent/WO2019194130A1/en

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—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

[Problem] To provide a novel mucin-type glycoprotein having a large sulfate group content and having a unique composition or structure, and a use thereof. [Solution] Provided is a mucin-type glycoprotein comprising a sulfate group and a sialic acid, wherein the content of the sulfate group is at least 0.07 mol per 1 mol of saccharide bound to a core protein by an O-glycosidic bond, and the content of the sialic acid is at least 0.1 mol per 1 mol of saccharide bound to a core protein by an O-glycosidic bond. According to the present invention, it is possible to obtain a mucin-type glycoprotein having a novel composition or structure. In addition, according to the present invention, the mucin-type glycoprotein can effectively increase the number of Akkermansia bacteria and/or Bacteroides bacteria in a living human or animal body, and thus contribute to the prevention or treatment of diseases such as obesity, type 2 diabetes, hyperlipidemia, and so on.

Description

新規ムチン型糖タンパク質およびその用途Novel mucin-type glycoprotein and its use
　本発明は、硫酸基の含有量が大きいという新規な成分ないし構造を有するムチン型糖タンパク質およびその用途に関する。 The present invention relates to a mucin-type glycoprotein having a novel component or structure having a high sulfate group content and use thereof.
　ムチンは、動植物の粘液などに見られる粘性物質をいい、分子量１００万～１０００万の、糖含有量の高い糖タンパク質を主成分としている。この糖タンパク質の糖鎖の大部分は、コアタンパク質中のセリンまたはスレオニンの水酸基と糖鎖の還元末端に位置する糖（Ｎ－アセチルガラクトサミンである場合が多い）とがＯ－グリコシド結合を介して結合してなる比較的短い糖鎖であり、係る糖鎖は「ムチン型糖鎖」と呼ばれている（以下、ムチン型糖鎖を有する糖タンパク質をムチン型糖タンパク質という）。 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).
　ムチンは、粘膜上皮の保護や保湿、抗菌、潤滑等、種々の生理作用を有することが報告されており、動植物から抽出精製したムチンあるいはムチン型糖タンパク質が、健康食品や医薬品として利用されている。例えば、特許文献１には、特定のアミノ酸配列からなる繰り返し構造に糖鎖が結合していることを特徴とする新規ムチン型糖タンパク質と、これを健康増進、薬剤投与、疾患の治療もしくは予防等に用いることとが開示されている（請求項１、請求項１４）。また、特許文献２には、エイの皮や体表粘性物から採取されるムチンを有効成分とする、抗原特異的Ｔ細胞の増殖促進剤が開示されている（請求項１）。 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. . For example, 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). Further, 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).
特許第５０５７３８３号公報Japanese Patent No. 5057383 特許第５３５５６８２号公報Japanese Patent No. 5355682
　しかしながら、ムチン型糖タンパク質は、特にその糖鎖において、多種多様な成分ないし構造を採りうる。そのため、上記特許文献を鑑みても、有用な用途を提供しうる、新規な成分ないし構造を有するムチン型糖タンパク質の提供は未だ十分になされているとはいえない。本発明は係る課題を解決するために成されたものであって、新規な成分ないし構造を有するムチン型糖タンパク質およびその用途を提供することを目的とする。 However, 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.
　本発明者らは、鋭意研究の結果、エイから、硫酸基やシアル酸の含有量が大きく、特有の成分ないし構造を有する新規なムチン型糖タンパク質を単離・精製することに成功した。また、硫酸基の含有量が大きいムチン型糖タンパク質が、腸内のアッカーマンシア属細菌およびバクテロイデス属細菌の菌数を顕著に増加させることを見出した。さらに、ムチン型糖タンパク質において、スレオニンの含有割合が高いものが、よりアッカーマンシア属細菌の菌数増加作用を増強できることを見出した。 As a result of diligent research, 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. In addition, 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. Further, it was found that a mucin-type glycoprotein having a high content of threonine can enhance the effect of increasing the number of bacteria of the genus Ackermancia.
　ここで、下記非特許文献１～３では、アッカーマンシア属細菌であるアッカーマンシア　ムシニフィラは、当該細菌を投与することにより、マウスで、肥満の進行を抑制することや、脂肪塊の発達、インスリン抵抗性および脂質異常症を減少させること等が報告されている。
　非特許文献１；Hubert P. et al., NATURE MEDICINE, Vol. 23, No. 1, January 2017, pp. 107-116
　非特許文献２；Patrice D. Cani and Willem M. de Vos, Frontiers in Microbiology, Vol. 8, Art. 1765, 22 September 2017
　非特許文献３；Amandine Everard et al, PNAS, Vol. 110, No. 22, May 28, 2013, pp. 9066-9071 Here, in the following Non-Patent Documents 1 to 3, Ackermansia muciniphila, which is a bacterium of the genus Ackermancia, can suppress obesity progression, develop fat mass, insulin resistance in mice by administering the bacterium. It has been reported that sex and dyslipidemia are reduced.
Non-Patent Document 1; 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
　また、非特許文献４では、バクテロイデス属細菌であるバクテロイデス　テタイオタオミクロンもまた、体重減少後にその菌数が増加していることや、ラクチトールやポリデキストロースなどのプレバイオティクスと共投与することにより体重減少や血中の中性脂肪が減少することが報告されている。
　非特許文献４；Kaisa Olli et al, Frontiers in Nutrition, Vol. 3, Art. 15, 08 June 2016 In 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.
Non-Patent Document 4; Kaisa Olli et al, Frontiers in Nutrition, Vol. 3, Art. 15, 08 June 2016
　すなわち、本発明に係る新規ムチン型糖タンパク質をヒトや動物に摂取させて、その生体におけるアッカーマンシア属細菌および／またはバクテロイデス属細菌の菌数を増加させることにより、肥満や２型糖尿病、脂質異常症を予防ないし治療できると考えられる。そこで、これらの知見に基づいて、下記の各発明を完成した。 That is, by allowing humans and animals to ingest the novel mucin-type glycoprotein according to the present invention and increasing the number of bacteria of the genus Ackermannia and / or Bacteroides in the living body, obesity, type 2 diabetes, dyslipidemia It is thought that the disease can be prevented or treated. Accordingly, the following inventions have been completed based on these findings.
（１）硫酸基およびシアル酸を含有するムチン型糖タンパク質であって、前記硫酸基の含有量が、コアタンパク質にＯ－グリコシド結合している糖１モルに対し、０．０７モル以上であり、前記シアル酸の含有量が、コアタンパク質にＯ－グリコシド結合している糖１モルに対し、０．１モル以上である、前記ムチン型糖タンパク質。 (1) 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. The mucin-type glycoprotein, wherein the content of the sialic acid is 0.1 mol or more with respect to 1 mol of a sugar having an O-glycoside bond to the core protein.
（２）トリプトファン、メチオニンおよびシステインを除くアミノ酸の総量に占めるスレオニンの質量百分率が１５％以上である、（１）に記載のムチン型糖タンパク質。 (2) The mucin-type glycoprotein according to (1), wherein the mass percentage of threonine in the total amount of amino acids excluding tryptophan, methionine and cysteine is 15% or more.
（３）エイの皮および／または体表粘性物から採取される、（１）または（２）に記載のムチン型糖タンパク質。 (3) The mucin-type glycoprotein according to (1) or (2), which is collected from ray skin and / or body surface viscous material.
（４）（１）から（３）のいずれかに記載のムチン型糖タンパク質を有効成分とする、アッカーマンシア属細菌および／またはバクテロイデス属細菌の菌数増加剤。 (4) An agent for increasing the number of bacteria of the genus Ackermansia and / or the genus Bacteroides, comprising the mucin-type glycoprotein according to any one of (1) to (3) as an active ingredient.
（５）下記のムチン型糖タンパク質を有効成分とする、アッカーマンシア属細菌および／またはバクテロイデス属細菌の菌数増加剤；
　硫酸基を含有するムチン型糖タンパク質であって、前記硫酸基の含有量が、コアタンパク質にＯ－グリコシド結合している糖１モルに対し、０．０７モル以上である、前記ムチン型糖タンパク質。 (5) 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. .
（６）アッカーマンシア属細菌の菌数増加剤であって、前記ムチン型糖タンパク質において、トリプトファン、メチオニンおよびシステインを除くアミノ酸の総量に占めるスレオニンの質量百分率が１５％以上である、（５）に記載の菌数増加剤。 (6) An agent 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 agent for increasing the number of bacteria described.
（７）前記ムチン型糖タンパク質が、エイの皮および／または体表粘性物から採取されたものである、（５）または（６）に記載の菌数増加剤。 (7) The agent for increasing the number of bacteria according to (5) or (6), wherein the mucin-type glycoprotein is collected from ray skin and / or body surface viscous material.
（８）肥満、２型糖尿病および脂質異常症からなる群から選択される１以上の疾患の予防または治療に用いられることを特徴とする、（４）から（７）のいずれかに記載の菌数増加剤。 (8) The bacterium according to any one of (4) to (7), which is used for the prevention or treatment of one or more diseases selected from the group consisting of obesity, type 2 diabetes and dyslipidemia Number increasing agent.
（９）（１）から（３）のいずれかに記載のムチン型糖タンパク質を有効成分とする、アッカーマンシア属細菌および／またはバクテロイデス属細菌の菌数増加用食品組成物。 (9) 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.
（１０）下記のムチン型糖タンパク質を有効成分とする、アッカーマンシア属細菌および／またはバクテロイデス属細菌の菌数増加用食品組成物；
　硫酸基を含有するムチン型糖タンパク質であって、前記硫酸基の含有量が、コアタンパク質にＯ－グリコシド結合している糖１モルに対し、０．０７モル以上である、前記ムチン型糖タンパク質。 (10) 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. .
（１１）アッカーマンシア属細菌の菌数増加用食品組成物であって、前記ムチン型糖タンパク質において、トリプトファン、メチオニンおよびシステインを除いたアミノ酸の総量に占めるスレオニンの質量百分率が１５％以上である、（１０）に記載の菌数増加用食品組成物。 (11) 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).
（１２）前記ムチン型糖タンパク質が、エイの皮および／または体表粘性物から採取されたものである、（１０）または（１１）に記載の菌数増加用食品組成物。 (12) The food composition for increasing the number of bacteria according to (10) or (11), wherein the mucin-type glycoprotein is collected from ray skin and / or body surface viscous material.
（１３）肥満、２型糖尿病および脂質異常症からなる群から選択される１以上の疾患の予防または治療に用いられることを特徴とする、（９）から（１２）のいずれかに記載の菌数増加用食品組成物。 (13) The bacterium according to any one of (9) to (12), which is used for the prevention or treatment of one or more diseases selected from the group consisting of obesity, type 2 diabetes and dyslipidemia Number increasing food composition.
（１４）下記のムチン型糖タンパク質を有効成分とする、肥満、２型糖尿病および脂質異常症からなる群から選択される１以上の疾患の予防または治療剤；
　硫酸基を含有するムチン型糖タンパク質であって、前記硫酸基の含有量が、コアタンパク質にＯ－グリコシド結合している糖１モルに対し、０．０７モル以上である、前記ムチン型糖タンパク質。 (14) A prophylactic or therapeutic agent for one or more diseases selected from the group consisting of obesity, type 2 diabetes and dyslipidemia, 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. .
（１５）前記ムチン型糖タンパク質が、さらにシアル酸を含有するムチン型糖タンパク質であって、前記シアル酸の含有量が、コアタンパク質にＯ－グリコシド結合している糖１モルに対し、０．１モル以上である、（１４）に記載の予防または治療剤。 (15) 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 prophylactic or therapeutic agent according to (14), which is 1 mol or more.
（１６）前記ムチン型糖タンパク質が、トリプトファン、メチオニンおよびシステインを除くアミノ酸の総量に占めるスレオニンの質量百分率が１５％以上である、（１４）または（１５）に記載の予防または治療剤。 (16) The prophylactic or therapeutic agent according to (14) or (15), wherein the mucin-type glycoprotein has a mass percentage of threonine in the total amount of amino acids excluding tryptophan, methionine and cysteine of 15% or more.
（１７）前記ムチン型糖タンパク質が、エイの皮および／または体表粘性物から採取されたものである、（１４）～（１６）のいずれかに記載の予防または治療剤。 (17) The preventive or therapeutic agent according to any one of (14) to (16), wherein the mucin-type glycoprotein is collected from ray skin and / or body surface viscous material.
（１８）前記ムチン型糖タンパク質が、ヒトまたは動物の腸内におけるアッカーマンシア属細菌および／またはバクテロイデス属細菌の菌数を増加させるものである、（１４）～（１７）のいずれかに記載の予防または治療剤。 (18) 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.
（１９）下記のムチン型糖タンパク質を有効成分とする、肥満、２型糖尿病および脂質異常症からなる群から選択される１以上の疾患の予防または治療用食品組成物；
　硫酸基を含有するムチン型糖タンパク質であって、前記硫酸基の含有量が、コアタンパク質にＯ－グリコシド結合している糖１モルに対し、０．０７モル以上である、前記ムチン型糖タンパク質。 (19) A food composition for preventing or treating one or more diseases selected from the group consisting of obesity, type 2 diabetes and dyslipidemia, 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. .
（２０）前記ムチン型糖タンパク質が、さらにシアル酸を含有するムチン型糖タンパク質であって、前記シアル酸の含有量が、コアタンパク質にＯ－グリコシド結合している糖１モルに対し、０．１モル以上である、（１９）に記載の予防または治療用食品組成物。 (20) 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 food composition for prevention or treatment according to (19), which is 1 mol or more.
（２１）前記ムチン型糖タンパク質が、トリプトファン、メチオニンおよびシステインを除くアミノ酸の総量に占めるスレオニンの質量百分率が１５％以上である、（１９）または（２０）に記載の予防または治療用食品組成物。 (21) The food composition for prevention or treatment according to (19) or (20), wherein the mucin-type glycoprotein has a mass percentage of threonine in the total amount of amino acids excluding tryptophan, methionine and cysteine of 15% or more. .
（２２）前記ムチン型糖タンパク質が、エイの皮および／または体表粘性物から採取されたものである、（１９）～（２１）のいずれかに記載の予防または治療用食品組成物。 (22) The food composition for prevention or treatment according to any one of (19) to (21), wherein the mucin-type glycoprotein is collected from ray skin and / or body surface viscous material.
（２３）前記ムチン型糖タンパク質が、ヒトまたは動物の腸内におけるアッカーマンシア属細菌および／またはバクテロイデス属細菌の菌数を増加させるものである、（１９）～（２２）のいずれかに記載の予防または治療用食品組成物。 (23) 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.
（２４）　下記（ａ）および（ｂ）の工程を有する、肥満、２型糖尿病および脂質異常症からなる群から選択される１以上の疾患を予防または治療する方法；
　（ａ）前記疾患を罹患している、または、罹患する可能性があるヒトもしくは動物に、下記のムチン型糖タンパク質を摂取させる工程；
　硫酸基を含有するムチン型糖タンパク質であって、前記硫酸基の含有量が、コアタンパク質にＯ－グリコシド結合している糖１モルに対し、０．０７モル以上である、前記ムチン型糖タンパク質、
　（ｂ）前記ヒトまたは動物の腸内におけるアッカーマンシア属細菌および／またはバクテロイデス属細菌の菌数を増加させて、前記疾患を予防または治療する工程。 (24) 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.
（２５）前記ムチン型糖タンパク質が、さらにシアル酸を含有するムチン型糖タンパク質であって、前記シアル酸の含有量が、コアタンパク質にＯ－グリコシド結合している糖１モルに対し、０．１モル以上である、（２４）に記載の方法。 (25) 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.
（２６）前記ムチン型糖タンパク質が、トリプトファン、メチオニンおよびシステインを除くアミノ酸の総量に占めるスレオニンの質量百分率が１５％以上である、（２４）に記載の方法。 (26) The method according to (24), wherein the mucin-type glycoprotein has a mass percentage of threonine in the total amount of amino acids excluding tryptophan, methionine and cysteine of 15% or more.
（２７）前記ムチン型糖タンパク質が、エイの皮および／または体表粘性物から採取されたものである、（２４）に記載の方法。 (27) The method according to (24), wherein the mucin-type glycoprotein is collected from ray skin and / or body surface viscous material.
（２８）肥満、２型糖尿病および脂質異常症からなる群から選択される１以上の疾患の予防または治療用医薬品を製造するための、下記のムチン型糖タンパク質の使用；
　硫酸基を含有するムチン型糖タンパク質であって、前記硫酸基の含有量が、コアタンパク質にＯ－グリコシド結合している糖１モルに対し、０．０７モル以上である、前記ムチン型糖タンパク質。 (28) Use of the following 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;
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. .
（２９）前記ムチン型糖タンパク質が、さらにシアル酸を含有するムチン型糖タンパク質であって、前記シアル酸の含有量が、コアタンパク質にＯ－グリコシド結合している糖１モルに対し、０．１モル以上である、（２８）に記載の使用。 (29) 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.
（３０）前記ムチン型糖タンパク質が、トリプトファン、メチオニンおよびシステインを除くアミノ酸の総量に占めるスレオニンの質量百分率が１５％以上である、（２８）または（２９）に記載の使用。 (30) The use according to (28) or (29), wherein the mucin-type glycoprotein has a mass percentage of threonine in a total amount of amino acids excluding tryptophan, methionine and cysteine of 15% or more.
（３１）前記ムチン型糖タンパク質が、エイの皮および／または体表粘性物から採取されたものである、（２８）または（２９）に記載の使用。 (31) The use according to (28) or (29), wherein the mucin-type glycoprotein is collected from ray skin and / or body surface viscous material.
（３２）前記ムチン型糖タンパク質が、ヒトまたは動物の腸内におけるアッカーマンシア属細菌および／またはバクテロイデス属細菌の菌数を増加させるものである、（２８）または（２９）に記載の使用。 (32) The use according to (28) or (29), wherein the mucin-type glycoprotein increases the number of bacteria of the genus Ackermancia and / or Bacteroides in the intestines of humans or animals.
　本発明によれば、新規な成分ないし構造を有するムチン型糖タンパク質を得ることができる。また、本発明によれば、ヒトや動物の生体におけるアッカーマンシア属細菌および／またはバクテロイデス属細菌の菌数を効果的に増加させ、もって肥満や２型糖尿病、脂質異常症等の疾患の予防や治療に寄与することができる。 According to the present invention, a mucin-type glycoprotein having a novel component or structure can be obtained. In addition, according to the present invention, 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.
新規ムチン型糖タンパク質およびブタ胃粘膜ムチンを基質とするムシナーゼ反応により生じた還元糖の量を示すグラフである。It is a graph which shows the quantity of the reducing sugar produced | generated by the mucinase reaction which makes a novel mucin type | mold glycoprotein and porcine gastric mucosa mucin a substrate. ラット腸管粘膜ムチンを基質とするムシナーゼ反応により生じた還元糖の量を示す棒グラフである。It is a bar graph which shows the quantity of the reducing sugar produced | generated by the mucinase reaction which uses rat intestinal mucosa mucin as a substrate. ブタ胃粘膜ムチンを基質とするムシナーゼ反応により生じた還元糖の量を示す棒グラフである。It is a bar graph which shows the quantity of the reducing sugar produced | generated by the mucinase reaction which uses porcine gastric mucosa mucin as a substrate. 新規ムチン型糖タンパク質を基質とするムシナーゼ反応により生じた還元糖の量を示す棒グラフである。It is a bar graph which shows the quantity of the reducing sugar produced | generated by the mucinase reaction which uses a novel mucin type glycoprotein as a substrate. ムチン型糖タンパク質を摂取しないラット（対照群）、新規ムチン型糖タンパク質を摂取したラット（１．５％新規ムチン群）およびブタ胃粘膜ムチンを摂取したラット（１．５％ブタ胃粘膜ムチン群）の盲腸内容物に含まれるＯ－結合型糖の濃度を示す棒グラフである。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. 対照群、０．７５％新規ムチン群、１．５％新規ムチン群および１．５％ブタ胃粘膜ムチン群のラット盲腸内容物に存在する、各種腸内細菌由来の１６Ｓ　ｒＤＮＡコピー数を示す棒グラフである。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. ムチン型糖タンパク質を摂取しないラット（対照群）および新規ムチン型糖タンパク質を摂取したラット（ロットＩ～Ｖ群）の盲腸内容物に存在する、全真正細菌およびアッカーマンシア　ムシニフィラ由来の１６Ｓ　ｒＤＮＡコピー数を示す棒グラフである。Number of 16S rDNA copies derived from all eubacteria and Ackermancia muciniphila present in the cecal contents of rats not receiving mucin-type glycoprotein (control group) and rats receiving new mucin-type glycoprotein (groups I to V) It is a bar graph which shows.
　以下、本発明について詳細に説明する。本発明は下記〈１〉～〈７〉を提供する。
　〈１〉ムチン型糖タンパク質。
　〈２〉アッカーマンシア属細菌および／またはバクテロイデス属細菌（以下、まとめて「本細菌」という場合がある。）の菌数増加剤。
　〈３〉本細菌の菌数増加用食品組成物。
　〈４〉肥満、２型糖尿病および脂質異常症からなる群から選択される１以上の疾患（以下、「本疾患」という場合がある。）の予防または治療剤。
　〈５〉本疾患の予防または治療用食品組成物。
　〈６〉本疾患を予防または治療する方法。
　〈７〉本疾患の予防または治療用医薬品を製造するためのムチン型糖タンパク質の使用。 Hereinafter, the present invention will be described in detail. The present invention provides the following <1> to <7>.
<1> Mucin-type glycoprotein.
<2> An agent for increasing the number of bacteria of the genus Ackermancia and / or Bacteroides (hereinafter sometimes collectively referred to as “the present bacterium”).
<3> A food composition for increasing the number of bacteria of this bacterium.
<4> A preventive or therapeutic agent for one or more diseases selected from the group consisting of obesity, type 2 diabetes, and dyslipidemia (hereinafter sometimes referred to as “this disease”).
<5> A food composition for preventing or treating this disease.
<6> A method for preventing or treating this disease.
<7> Use of a mucin-type glycoprotein for producing a pharmaceutical product for preventing or treating this disease.
　本発明において「ムチン型糖タンパク質」とは、上述のとおり、ムチン型糖鎖を有する糖タンパク質をいう。また、「ムチン型糖鎖」とは、コアタンパク質中のセリンまたはスレオニンの水酸基にＯ－グリコシド結合を介して結合してなる糖鎖をいう。ムチン型糖鎖の還元末端に位置する糖、すなわち、コアタンパク質のセリン残基またはスレオニン残基とＯ－グリコシド結合している糖は、多くの場合、Ｎ－アセチルガラクトサミンである。 In the present invention, “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.
　本発明に係るムチン型糖タンパク質は、硫酸基を高い割合で含有することを特徴としている。具体的には、Ｏ－グリコシド結合している糖１モルに対し、硫酸基を０．０７モル以上という割合で含有している。硫酸基は、ムチン型糖鎖では非還元末端の糖残基に付加している場合が多いことから、本発明に係るムチン型糖タンパク質は、その糖鎖非還元末端の多くに硫酸基を有しているといえる。 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. In many cases, 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.
　本発明に係るムチン型糖タンパク質は、硫酸基に加えて、シアル酸を高い割合で含有するものであってもよい。具体的には、Ｏ－グリコシド結合している糖１モルに対し、シアル酸を０．１モル以上という割合で含有するものであってよい。シアル酸もまた、ムチン型糖鎖では非還元末端に位置している場合が多いことから、本発明に係るムチン型糖タンパク質は、その糖鎖非還元末端の多くにシアル酸を有するものであってよい。 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.
　本発明に係るムチン型糖タンパク質において、スレオニンの含有割合あるいは質量百分率が所定の値以上の場合は、アッカーマンシア属細菌の菌数増加効果をより高くすることができる。好ましいスレオニンの含有割合としては、例えば、２８ｍｇ／ｇ以上、２８．５ｍｇ／ｇ以上、２９ｍｇ／ｇ以上、２９．５ｍｇ／ｇ以上、３０ｍｇ／ｇ以上、３０．５ｍｇ／ｇ以上、３１ｍｇ／ｇ以上、３１．５ｍｇ／ｇ以上、３２ｍｇ／ｇ以上、３２．５ｍｇ／ｇ以上、３３ｍｇ／ｇ以上、３３．５ｍｇ／ｇ以上を挙げることができる。また、トリプトファン、メチオニンおよびシステインを除くアミノ酸の総量に占めるスレオニンの質量百分率として、好ましくは、１４％以上、１４．５％以上、１５％以上、１５．５％以上、１６％以上、１６．５％以上、１７％以上、１７．５％以上、１８％以上、１８．５％以上、１９％以上、１９．５％以上を例示することができる。 In the mucin-type glycoprotein according to the present invention, when the content ratio or mass percentage of threonine is a predetermined value or more, the effect of increasing the number of bacteria of the genus Ackermancia can be further increased. Preferable 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.
　なお、タンパク質におけるアミノ酸の測定値について、測定方法により相当の差異が生じるのは本発明の分野における技術常識である。この点、本発明に係るムチン型糖タンパク質におけるアミノ酸の含有割合や質量百分率は、後述する実施例６（２）に示すように、強酸によりタンパク質を加水分解した後、ポストカラムニンヒドリン法により誘導体化してクロマトグラフィーにより分離測定する方法により定量することが好ましい。 In addition, it is common technical knowledge in the field of the present invention that a considerable difference occurs in the measurement value of amino acids in proteins depending on the measurement method. In this regard, 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.
　本発明に係るムチン型糖タンパク質は、例えば、ガンギエイ等のエイ（板鰓亜綱エイ上目に属する軟骨魚類）の表皮や体表に付着している粘性物から採取することができる。後述する実施例においてはメガネカスベ属の表皮・体表粘性物とソコガンギエイ属の体表粘性物とを合わせて用いているが、このように複数種のエイに由来する原料を合わせて用いてもよく、単一種のエイに由来する原料を用いてもよい。 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. In the examples to be described later, 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.
　エイの皮や体表粘性物には本発明に係るムチン型糖タンパク質が豊富に含まれるため、これをそのまま用いてもよいが、精製・濃縮して用いてもよい。精製・濃縮する場合は、まず、原料にタンパク質分解酵素を作用させて夾雑タンパク質・ペプチドを低分子化する。タンパク質分解酵素は、アスパルティックプロテイナーゼ、金属プロテイナーゼ、セリンプロテイナーゼおよびチオールプロテイナーゼといったプロテイナーゼ（エンドペプチダーゼ）やペプチダーゼ（エキソペプチダーゼ）から、原料の由来等に応じて適宜選択して用いることができる。続いて、これを限外ろ過に供して低分子化した雑タンパク質・ペプチドを除去するとともに、高分子物質を濃縮すればよい。その他、原料をすり潰した後、適当な溶媒（水や生理食塩水、リン酸緩衝液等）を加えて撹拌、抽出し、遠心分離を行って上清を回収する方法を挙げることができる。また、ムチンクロット法、硫安分画による方法、バリウムイオンやカルシウムイオンの存在下でのアルコール分別による方法、セタブロン（セチルトリメチルアンモニウムブロマイド）や塩化セチルピリジニウム（ＣＰＣ）などの四級アミンの陽性界面活性剤を使用する沈澱法などによる精製方法を挙げることができる。 Since the skin and viscous body of the ray contain abundant mucin-type glycoproteins according to the present invention, they may be used as they are, or may be used after purification and concentration. When purifying and concentrating, first, a proteolytic enzyme is allowed to act on the raw material to reduce the molecular weight of the contaminating protein / peptide. 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. Subsequently, this is subjected to ultrafiltration to remove low molecular weight miscellaneous proteins / peptides and to concentrate the polymer substance. In addition, after crushing raw materials, an appropriate solvent (water, physiological saline, phosphate buffer, etc.) is added, stirred and extracted, and centrifuged to collect the supernatant. In addition, mucin clot method, ammonium sulfate fractionation method, alcohol fractionation method in the presence of barium ion and calcium ion, positive surface activity of quaternary amines such as cetablone (cetyltrimethylammonium bromide) and cetylpyridinium chloride (CPC) And a purification method such as a precipitation method using an agent.
　本発明に係るムチン型糖タンパク質は、ヒトまたは動物に経口摂取させることにより使用することができる。また、本発明に係るムチン型糖タンパク質は生体の腸内においてその機能を発揮することから、腸内に到達する方法で使用すればよく、例えば、有効成分を経腸栄養剤に添加して、これを、胃や小腸などの消化管に挿入したチューブを経由して経腸栄養法により投与する方法で使用してもよい。 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.
　本発明に係るムチン型糖タンパク質は、主として、消化管においてその作用（特定の腸内細菌に対する菌数増加作用）を発揮する。そのため、本発明に係るムチン型糖タンパク質をヒトや動物に対して用いる場合の摂取量（投与量）は、他の薬剤において一般的な体重当たりではなく、食物摂取量当たりで算出するのが妥当と考えられる。後述する実施例では、ムチン型糖タンパク質を１質量％程度含有する飼料を摂取したラットで効果が認められた（図６）。ここで、一般に、ラットはヒトと比較して基礎代謝が顕著に大きいため、ヒトでは、ラットで有効な投与量の１／４～１／２の投与量で有効な場合が多い。従って、当該実施例によれば、本発明に係るムチン型糖タンパク質をヒトに用いる場合の、１日当たりの摂取量は、「当該ヒト個体における１日の食物（固形物）摂取量の０．２５～０．５質量％程度の量」を例示することができる。 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). Here, in general, since 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.
　本発明に係るムチン型糖タンパク質は、硫酸基を高い割合で含有しているため、これを摂取したヒトや動物の腸内において、硫酸基分解酵素（スルファターゼ）を有する腸内細菌であるアッカーマンシア属細菌やバクテロイデス属細菌の菌数を特異的に増加させることができる。 Since 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. The number of genus bacteria and Bacteroides bacteria can be specifically increased.
　ここで、「アッカーマンシア属細菌」は、アッカーマンシア属に属する微生物をいう。係る微生物としては、例えば、アッカーマンシア　ムシニフィラ（Akkermansia muciniphila）を挙げることができる。アッカーマンシア　ムシニフィラは多くのヒトの腸内に生息する腸内細菌であり、上述のとおり、本細菌を投与することにより、マウスで、肥満の進行を抑制することや、脂肪塊の発達、インスリン抵抗性および脂質異常症を減少させること等が報告されている。 Here, “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.
　また、「バクテロイデス属細菌」は、バクテロイデス属に属する微生物をいう。係る微生物としては、例えば、バクテロイデス　テタイオタオミクロン（Bacteroides thetaiotaomicron）を挙げることができる。バクテロイデス属細菌もまた、腸内細菌叢を構成する細菌であり、上述のとおり、バクテロイデス　テタイオタオミクロンは、体重減少後にその菌数が増加していることや、ラクチトールやポリデキストロースなどのプレバイオティクスと共投与することにより体重減少や血中の中性脂肪が減少することが報告されている。 Also, “bacteroides 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.
　すなわち、本発明に係るムチン型糖タンパク質をヒトや動物に摂取させて、その生体における本細菌の菌数を増加させることにより、肥満や２型糖尿病、脂質異常症を予防ないし治療できると考えられる。このことから、本発明に係るムチン型糖タンパク質は、本疾患を予防または治療する用途に用いることができる。 That is, it is considered that obesity, type 2 diabetes, and dyslipidemia can be prevented or treated by ingesting the mucin-type glycoprotein according to the present invention into humans or animals and increasing the number of bacteria in the living body. . Therefore, the mucin-type glycoprotein according to the present invention can be used for the purpose of preventing or treating this disease.
　具体的には、例えば、本発明に係るムチン型糖タンパク質は、本細菌の菌数増加剤、本細菌の菌数増加用食品組成物、本疾患の予防もしくは治療剤、または、本疾患の予防もしくは治療用食品組成物の有効成分とすることができる。すなわち、本発明に係るムチン型糖タンパク質は、本疾患の予防または治療用医薬品を製造するために使用することができる。また、（ａ）本疾患を罹患している、または、罹患する可能性があるヒトもしくは動物に、本発明に係るムチン型糖タンパク質を摂取させる工程と、（ｂ）その腸内における本細菌の菌数を増加させて、前記疾患を予防または治療する工程とにより、本疾患を予防または治療することができる。 Specifically, for example, 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. Alternatively, 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.
　本発明の剤の形態としては、有効成分であるムチン型糖タンパク質のみからなるもののほか、適当な賦形剤や担体と合わせてなる、医薬品や食品添加剤、サプリメントなどの形態を挙げることができる。医薬品や食品添加剤、サプリメントの形態とする場合、その剤型としては、例えば、散剤、錠剤、糖衣剤、カプセル剤、顆粒剤、ドライシロップ剤、液剤、シロップ剤、ドロップ剤、ドリンク剤等の固形または液状の剤型を挙げることができる。係る各剤型の医薬品や食品添加剤、サプリメントは、当業者に公知の方法で製造することができる。 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. . In the case of pharmaceuticals, food additives, and supplements, 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.
　また、本発明の食品組成物の形態としては、有効成分であるムチン型糖タンパク質のみからなるもののほか、菓子や飲料、加工食品、健康食品、乳幼児食品などの通常の飲食物の形態を挙げることができる。飲食物の形態とする場合は、通常の製造過程で、有効成分を添加して製造することができる。 In addition, 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.
　本発明において、本細菌の「菌数を増加させる」とは、生体のいずれかの細胞ないし組織・器官における当該細菌の菌数を増加させることをいう。 In the present invention, “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.
　例えば、腸における本細菌の菌数は、腸の内容物または糞便中の当該細菌の菌数と相関していると考えられるため、腸内容物または糞便中の本細菌の菌数を計測することにより、腸において本細菌の菌数が増加したか否かを確認することができる。具体的には、例えば、本発明に係るムチン型糖タンパク質の摂取後の腸内容物または糞便を試料として、本細菌に特異的なプライマーを用いたリアルタイムＰＣＲ法を行って１６Ｓ ｒＤＮＡコピー数を計測する。本細菌に特異的なプライマーは、本細菌の公知の塩基配列に基づいて設計することができ、例えば配列番号１～４に示す配列からなるプライマーを用いることができる。また、簡便には、本細菌を定量するための市販のキットを用いて定量することもできる。 For example, since 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. Specifically, for example, 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. To do. 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. In addition, for convenience, it can be quantified using a commercially available kit for quantifying the bacterium.
　本細菌の１６Ｓ ｒＤＮＡコピー数と本細菌の菌数とは相関関係にあるため、１６Ｓ ｒＤＮＡコピー数は、菌数の指標とすることができる。よって、本細菌の１６Ｓ ｒＤＮＡコピー数を計測した結果、摂取後の糞便における１６Ｓ ｒＤＮＡコピー数が摂取前よりも大きければ、あるいは摂取後の盲腸内容物における１６Ｓ ｒＤＮＡコピー数が摂取していない被検体の盲腸内容物よりも大きければ、本発明に係るムチン型糖タンパク質により本細菌の菌数が増加したと判断することができる。 Since 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.
　以下、本発明について、各実施例に基づいて説明する。なお、本発明の技術的範囲は、これらの実施例によって示される特徴に限定されない。また、本実施例において、「％」は、特段の記載のない限り質量％（（ｗ／ｗ）％）を表す。 Hereinafter, the present invention will be described based on each example. Note that the technical scope of the present invention is not limited to the features shown by these examples. Further, in this example, “%” represents mass% ((w / w)%) unless otherwise specified.
＜実施例１＞新規ムチン型糖タンパク質の単離・精製
　真カスベ（エイ上目ガンギエイ目ガンギエイ科メガネカスべ属メガネカスべ、Raja pulchra Liu ）から、体表に付着している粘性物とともに表皮を剥ぎ取り、これを真カスベ由来原料とした。また、水カスべ（エイ上目ガンギエイ目ガンギエイ科ソコガンギエイ属ドブカスべ、Bathyraja smirnovi）から、体表に付着している粘性物を水洗により剥離して回収し、これを水カスベ由来原料とした。 <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. In addition, from 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 .
　真カスべ由来原料約２４０ｋｇおよび水カスべ由来原料約１２０ｋｇの合計約３６０ｋｇを斜軸ニーダー（ＧＮ１００／６０ＳＴ、サムソン）中へ投入し、チオールプロテイナーゼ製剤３００ｇを加えて５５℃で４時間攪拌した。続いて、金属プロテイナーゼおよびペプチダーゼの混合製剤３００ｇを加えて５５℃でさらに４時間インキュベートした。その後、９０℃で１０分間加熱処理することにより、酵素を失活させた。 About 360 kg of a total of about 240 kg of the raw material derived from the true casbet and about 120 kg of the water casbet material was put into an oblique axis kneader (GN100 / 60ST, Samsung), 300 g of the thiol proteinase preparation was added, and the mixture was stirred at 55 ° C. for 4 hours. Subsequently, 300 g of a mixed preparation of metalloproteinase and peptidase was added and further incubated at 55 ° C. for 4 hours. Thereafter, the enzyme was inactivated by heat treatment at 90 ° C. for 10 minutes.
　加熱処理後の酵素反応液に珪藻土（ラヂオライト＃１００、昭和化学工業）３５ｋｇを加え、フィルタープレス（ＦＰ－６６型、薮田機械）でろ過して、ろ液を回収した。このろ液を内部保持液として、限外ろ過膜（マイクローザＡＣＰ－３０１３Ｄ、分画分子量１３０００、旭化成）を用いて清水を加えながら限外ろ過を行うことにより、夾雑タンパク質やペプチド等の不純物を除去した。膜透過液の固形分濃度がブリックス計（ＭＡＳＴＥＲ－１０Ｐα、アタゴ）指針でおおよそゼロとなったことを確認したのち清水の注入を止め、そのまま運転を続けた。膜透過液がほとんど排出されなくなるまで濃縮を行って、約９０Ｌの内部保持液を得た。これを９０℃にて１０分間加熱殺菌した後、回転ディスク型噴霧乾燥機（ＤＡ２２０－１０Ｓ型、坂本技研）を用いて噴霧乾燥し、乾燥粉末１．４ｋｇを得た。以下、これを「新規ムチン型糖タンパク質」として用いた。 35 kg of diatomaceous earth (Radiolite # 100, Showa Chemical Co., Ltd.) was added to the enzyme reaction solution after the heat treatment, and the filtrate was collected by filtration with a filter press (FP-66 type, Iwata Machinery). By using this filtrate as an internal retentate and performing ultrafiltration while adding fresh water using an ultrafiltration membrane (Microza ACP-3013D, fractional molecular weight 13000, Asahi Kasei), impurities such as contaminating proteins and peptides can be removed. Removed. After confirming that the solid content concentration of the membrane permeate was approximately zero with a Brix meter (MASTER-10Pα, Atago), the injection of fresh water was stopped and the operation was continued. Concentration was performed until almost no membrane permeate was discharged to obtain about 90 L of an internal retentate. This was sterilized by heating at 90 ° C. for 10 minutes, and then spray-dried using a rotary disk type spray dryer (DA220-10S type, Sakamoto Giken) to obtain 1.4 kg of dry powder. Hereinafter, this was used as a “new mucin-type glycoprotein”.
＜比較例１＞ブタ胃粘膜ムチン
　比較例１として、ブタ胃粘膜ムチンの精製物を用いた。具体的には、市販のブタ胃粘膜ムチン（Mucin from porcine stomach TypeＩＩ、SIGMA-ALDRICH）５ｇをビーカーに量り取り、そこに１００倍量の０．１５ＭのＮａＣｌ水溶液を加えて懸濁した後、１ＭのＮａＯＨ水溶液でｐＨ７．５に調整した。これをホモジナイザーＰＴ－３１００（KINEMATICA）を用いて４℃にて１４５００回転／分（ｒｐｍ）で６０秒間均一化した後、吸引濾過することで濾液を得た。この濾液に２倍量の９０％エタノールを加えて転倒混和し、－３０℃で一晩放置した後、２３００×ｇ、４℃で１０分間遠心分離して上清を除去することによりエタノール沈殿を行った。沈殿物に０．１５ＭのＮａＣｌ水溶液を５００ｍＬ加え、ボルテックスミキサーを用いて懸濁した。これを再度エタノール沈殿させ、得られた沈殿物に蒸留水を１５０ｍＬ加え、ボルテックスミキサーを用いて懸濁した。この懸濁液を凍結乾燥したものを「ブタ胃粘膜ムチン」とし、重量を測定した。 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. Two times the amount of 90% ethanol was added to the filtrate and mixed by inversion. The mixture was allowed to stand at −30 ° C. overnight, and then centrifuged at 2300 × g for 10 minutes at 4 ° C. to remove the supernatant. went. To the precipitate, 500 mL of a 0.15 M NaCl aqueous solution was added and suspended using a vortex mixer. This was ethanol precipitated again, and 150 mL of distilled water was added to the resulting precipitate and suspended using a vortex mixer. The suspension obtained by freeze-drying was designated as “pig stomach mucosa mucin”, and the weight was measured.
＜比較例２＞ラット腸管粘膜ムチン
　比較例２として、ラットの腸管粘膜から分泌されるムチンを用いた。ムチンを摂取しないラットの盲腸内容物に含まれるムチンは、その腸管粘膜から分泌されるムチンであることから、下記（１）の条件下で飼育したラットを解剖して盲腸内容物を回収し、そこから下記（２）の方法により盲腸内容物ムチン画分を調製して、これを「ラット腸管粘膜ムチン」とした。 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”.
（１）ラットの飼育条件
　飼料：ムチンを含有しない標準精製飼料（ミルクカゼイン２５．０％、コーンスターチ６０．２５％、セルロースパウダー５．０％、コーンオイル５．０％、ビタミン混合物１．０％、ミネラル混合物３．５％、重酒石酸コリン０．２５％）。なお、ビタミンおよびミネラルは、ＡＩＮ－７６（米国国立栄養研究所（ＡＩＮ）から１９７７年に発表された、ラットの標準精製飼料のガイドライン）に準拠した混合物を用いた。
　飲料水：腸内細菌によるムチンの分解を抑制するため、飲料水中に下記の抗生物質を括弧内の終濃度となるよう添加した。ベンジルペニシリン（５０ｕｎｉｔ／ｍＬ）、ネオマイシン三硫酸塩（２ｍｇ／ｍＬ）、セフォペラゾンナトリウム（０．５ｍｇ／ｍＬ）。
　飼育期間：７日間 (1) 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
（２）盲腸内容物ムチン画分の調製
　盲腸内容物の凍結乾燥物２００ｍｇをガラス遠心管に量り取り、そこに３０倍量のリン酸緩衝生理食塩水（ｐＨ７．２）を加えて懸濁した後、１０分間煮沸殺菌した。これを３７℃の恒温振盪水槽に入れ、１２５ｒｐｍで９０分間振とうした。ホモジナイザーＰＴ－２１００（KINEMATICA) を用いて４℃にてレベル１１で２０秒間破砕して均一化した後、２００００×ｇ、４℃で３０分間の遠心分離を２回行って上清を回収した。これを５０ｍＬ容量のファルコンチューブに移し、３倍量の１００％エタノールを加えて転倒混和し、－３０℃で一晩放置した後、２３００×ｇ、４℃で１０分間遠心分離して上清を除去することによりエタノール沈殿を行った。沈殿物に０．１５ＭのＮａＣｌ水溶液を１５ｍＬ加え、ボルテックスミキサーを用いて懸濁した。これを再度エタノール沈殿させ、得られた沈殿物に蒸留水を５ｍＬ加え、ボルテックスミキサーを用いて懸濁した。この溶液を凍結乾燥したものを盲腸内容物ムチン画分とし、重量を測定した。 (2) Preparation of cecal content mucin fraction 200 mg of lyophilized cecal content was weighed into a glass centrifuge tube, and 30 times the amount of phosphate buffered saline (pH 7.2) was added and suspended. Thereafter, it was sterilized by boiling for 10 minutes. This was placed in a constant temperature shaking water bath at 37 ° C. and shaken at 125 rpm for 90 minutes. After homogenization by homogenizer PT-2100 (KINEMATICA) for 20 seconds at 4 ° C. and level 11 for 20 seconds, the mixture was centrifuged twice at 20000 × g and 4 ° C. for 30 minutes to recover the supernatant. Transfer this to a 50 mL Falcon tube, add 3 volumes of 100% ethanol, mix by inversion, leave at −30 ° C. overnight, centrifuge at 2300 × g, 4 ° C. for 10 minutes, and remove the supernatant. The ethanol precipitation was performed by removing. 15 mL of 0.15M NaCl aqueous solution was added to the precipitate, and suspended using a vortex mixer. This was ethanol precipitated again, and 5 mL of distilled water was added to the resulting precipitate and suspended using a vortex mixer. The lyophilized solution was used as the cecal content mucin fraction and weighed.
＜実施例２＞新規ムチン型糖タンパク質の評価：成分および構造
　実施例１の新規ムチン型糖タンパク質、比較例１のブタ胃粘膜ムチンおよび比較例２のラット腸管粘膜ムチンについて、下記（１）～（６）の方法により成分分析を行った。それぞれの粉末１０ｍｇを蒸留水４ｍＬに溶解してムチン溶液とし、分析に用いた。なお、新規ムチン型糖タンパク質は、異なる６つの原料からそれぞれ調製し（ロットＡ～Ｆとする）、さらに、各ロットから複数の試料（試料１～１３）を個別に採取（サンプリング）して解析した。また、ブタ胃粘膜ムチンおよびラット腸管粘膜ムチンも、試料を７つずつ（試料１～７）個別にサンプリングして解析した。 <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.
（１）タンパク質濃度の測定
　タンパク質濃度はＬｏｗｒｙ法に従って測定した。すなわち、標準試料にはウシ血清由来アルブミン (SIGMA-ALDRICH) を用い、蒸留水で２５－１００μｇ／ｍＬの希釈系列を作製した。測定試料は、ムチン溶液を蒸留水で２０倍に希釈した。１０％のＮａ２ＣＯ３を含有する０．５規定の水酸化ナトリウム水溶液と、０．５％のＣｕＳＯ４・５Ｈ２Ｏを含有する１％のクエン酸ナトリウム水溶液とを、体積比１０：１の割合で混合してＢｉｕｒｅｔ試薬を調製した。また、１．８Ｎのフェノール試薬 (ナカライテクス) を蒸留水で１２倍希釈して希釈フェノール試薬を調製した。 (1) Measurement of protein concentration 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 μg / 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.
　標準試料および測定試料各０．５ｍＬに、Ｂｉｕｒｅｔ試薬０．５ｍＬを加えて室温で１０分間放置した。希釈フェノール試薬１．５ｍＬを加えて撹拌し、３７℃で３０分間放置した。その後、室温で１５分間放置した後、７５０ｎｍにおける吸光度を測定した。標準試料の測定結果を基に、測定試料におけるタンパク質濃度を求め、新規ムチン型糖タンパク質またはブタ胃粘膜ムチン１ｍｇ当たりのタンパク質濃度（μｇ／ｍｇ）を算出した。 To each 0.5 mL of standard sample and measurement sample, 0.5 mL of Biuret reagent was added and left at room temperature for 10 minutes. The diluted phenol reagent 1.5mL was added and stirred, and it was left to stand at 37 degreeC for 30 minutes. Thereafter, after standing at room temperature for 15 minutes, the absorbance at 750 nm was measured. Based on the measurement results of the standard sample, the protein concentration in the measurement sample was determined, and the protein concentration (μg / mg) per mg of novel mucin-type glycoprotein or porcine gastric mucosa mucin was calculated.
（２）全糖濃度の測定
　全糖濃度はフェノール硫酸法に従って測定し、グルコース換算で算出した。すなわち、標準試料には１ｍｇ／ｍＬのＤ（+）－グルコース（Ｗａｋｏ）を用い、蒸留水で３１．２５～２５０μｇ／ｍＬの希釈系列を作製し、蒸留水のみのブランクを含め、それぞれ大試験管２本に１ｍＬずつ分注した。測定試料は、粉末ムチン５０ｍｇを蒸留水１０ｍＬに分散させたものを原液とし、それを蒸留水で１０、２０、４０および１００倍に希釈し、原液を含めそれぞれ大試験管２本に１ｍＬずつ分注した。 (2) Measurement of total sugar concentration The total sugar concentration was measured according to the phenol-sulfuric acid method and calculated in terms of glucose. In other words, 1 mg / mL D (+)-glucose (Wako) was used as a standard sample, a dilution series of 31.25 to 250 μg / 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.
　標準試料および測定試料に５％フェノール溶液を加え、ボルテックスミキサーで撹拌した。ホールピペットで濃硫酸５ｍＬを直接液面に加えるように添加し、ボルテックスミキサーで撹拌した。放置して試料を室温に戻した後、４８０ｎｍにおける吸光度を測定した。標準試料の測定結果を基に、測定試料における全糖濃度を求め、新規ムチン型糖タンパク質またはブタ胃粘膜ムチン１ｍｇ当たりの全糖濃度（μｇ／ｍｇ）を算出した。 A 5% phenol solution was added to the standard sample and the measurement sample, and the mixture was stirred with a vortex mixer. Concentrated sulfuric acid (5 mL) was added directly to the liquid level with a whole pipette, and the mixture was stirred with a vortex mixer. After allowing the sample to return to room temperature, the absorbance at 480 nm was measured. Based on the measurement result of the standard sample, the total sugar concentration in the measurement sample was determined, and the total sugar concentration (μg / mg) per 1 mg of novel mucin-type glycoprotein or porcine gastric mucosa mucin was calculated.
（３）Ｏ－結合型糖濃度の測定
　Ｏ－グリコシド結合している糖（Ｏ－結合型糖）の濃度は、Crowther RSらの方法に従って行った（Crowther RS, Wetmore RF、Fluorometric assay of O-linked glycoproteins by reaction with 2-cyanoacetamide、Anal. biochem.、第１６３巻、第１７０－１７４頁、１９８７年）。すなわち、アルカリ処理により糖タンパク質のＯ－グリコシド結合を切断し、生じる糖鎖還元末端に２－シアノアセトアミド（２－ＣＮＡ）を反応させて蛍光物質を生じさせ、係る蛍光強度を測定した。 (3) Measurement of 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.
　具体的には、標準試料にＮ－アセチルガラクトサミン（SIGMA-ALDRICH）を用い、蒸留水で０．１５６２５～１０μｇ／ｍＬの希釈系列を作製した。測定試料は、ムチン溶液を蒸留水で８０倍に希釈した。また、０．１５Ｍの水酸化ナトリウム水溶液と０．６Ｍの２－ＣＮＡ水溶液とを体積比５：１の割合で混合してアルカリ化２－ＣＮＡ溶液を調製した。標準試料および測定試料各１００μＬを１．５ｍＬ容量のマイクロチューブに分注し、アルカリ化２－ＣＮＡ溶液１２０μＬを加え、１００℃ で３０分加熱した。放置冷却して室温に戻した後、各マイクロチューブに０．６Ｍのホウ酸緩衝液（ｐＨ８．０）１ｍＬを加えて撹拌した。続いて、蛍光光度計（Ｆ－２０００、日立製作所)を用いて、３３６ｎｍの励起光にて、３８３ｎｍの蛍光を測定した。標準試料の測定結果を基に、測定試料におけるＯ－結合型糖の濃度を求め、新規ムチン型糖タンパク質またはブタ胃粘膜ムチン１ｍｇ当たりのＯ－結合型糖濃度（μｍｏｌ／ｍｇ）を算出した。 Specifically, N-acetylgalactosamine (SIGMA-ALDRICH) was used as a standard sample, and a dilution series of 0.15625 to 10 μg / mL was prepared with distilled water. As a measurement sample, a mucin solution was diluted 80 times with distilled water. Further, 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 μL of each of the standard sample and the measurement sample was dispensed into a 1.5 mL capacity microtube, 120 μL of the alkalized 2-CNA solution was added, and the mixture was heated at 100 ° C. for 30 minutes. After allowing to cool to room temperature, 1 mL of 0.6 M borate buffer (pH 8.0) was added to each microtube and stirred. Subsequently, fluorescence at 383 nm was measured with excitation light at 336 nm using a fluorometer (F-2000, Hitachi, Ltd.). Based on the measurement result of the standard sample, the concentration of O-linked saccharide in the measured sample was obtained, and the concentration of O-linked saccharide (μmol / mg) per 1 mg of novel mucin-type glycoprotein or porcine gastric mucosa mucin was calculated.
（４）硫酸基濃度の測定
　ムチン溶液を１０倍希釈した測定試料１００μＬを、１．５ｍＬ容量のマイクロチューブに分取し、１時間遠心濃縮を行って水分を完全に蒸発させた。その後、Ｍｉｌｌｉ－Ｑ水で調製した４Ｍ塩酸を２００μＬ加えて１００℃で４時間置くことにより酸加水分解を行った。続いて遠心濃縮を行い塩酸を蒸発させた。その後、Ｍｉｌｌｉ－Ｑ水３００μＬを加えて加水分解物を溶解させ、再度、遠心濃縮を行った。このＭｉｌｌｉ－Ｑ水による加水分解物の洗浄作業を計３回繰り返し、最終的に５００μＬのＭｉｌｌｉ－Ｑ水に溶解させた。これをフィルター (ＤＩＳＭＩＣ－１３ＨＰ、１３ＨＰ０２０ＡＮ、アドバンテック)でろ過したものを、下記条件のイオンクロマトグラフィーに供して硫酸イオン濃度を測定した。測定結果を基に、新規ムチン型糖タンパク質またはブタ胃粘膜ムチン１ｍｇ当たりの硫酸基濃度（μｍｏｌ／ｍｇ）を算出した。 (4) Measurement of sulfate group concentration 100 μL of a measurement sample obtained by diluting a mucin solution 10-fold was taken into a 1.5 mL capacity microtube and centrifuged for 1 hour to completely evaporate water. Thereafter, 200 μL of 4M hydrochloric acid prepared with Milli-Q water was added and placed at 100 ° C. for 4 hours for acid hydrolysis. Subsequently, centrifugal concentration was performed to evaporate hydrochloric acid. Thereafter, 300 μL of Milli-Q water was added to dissolve the hydrolyzate, and centrifugal concentration was performed again. This washing operation of the hydrolyzate with Milli-Q water was repeated 3 times in total, and finally dissolved in 500 μL of Milli-Q water. This was filtered through a filter (DISMIC-13HP, 13HP020AN, Advantech) and subjected to ion chromatography under the following conditions to measure the sulfate ion concentration. Based on the measurement results, the sulfate group concentration (μmol / mg) per mg of novel mucin-type glycoprotein or porcine gastric mucosa mucin was calculated.
　《イオンクロマトグラフィーの条件》
装置：イオンクロマトグラフ　ＩＣＳ－２０００（DIONEX）
検出器 ：電気伝導度検出器　ＤＳ６（DIONEX）
カラム ：ＩｏｎＰａｃＡＳ１７（４×２５０ｍｍ）（DIONEX）
ガードカラム：ＩｏｎＰａｃ ＡＧ１７Ｃ（DIONEX）
カラム温度：３０℃
溶離液：１８分間に５ｍＭから４０ｍＭまで直線的に濃度を高めた水酸化カリウム水溶液
流速：１ｍＬ／分 <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
（５）シアル酸濃度の測定
　標準試料にはＮ－アセチルノイラミン酸（ＮｅｕＡｃ）およびＮ－グリコリルノイラミン酸（ＮｅｕＧｃ）を用いた。測定試料は、ムチン溶液を蒸留水で２０倍に希釈した。測定試料５０μＬと６２．５ｍＭの塩酸２００μＬをガラスバイアル瓶に入れた。ボルテックスミキサーで撹拌後、８０℃に設定したヒートブロック中で１時間放置することにより酸加水分解を行ってシアル酸を遊離させた。その後、室温で２０分間放冷した。この溶液５０μＬをガラスバイアル瓶に分取し、ＤＭＢ溶液 (Coupling solution : 蒸留水 : DMB solution = ５：４：１（体積比）、シアル酸蛍光標識試薬キット、タカラバイオ) ２００μＬを加えた。ボルテックスミキサーで撹拌後、５０℃で２．５時間遮光下に放置することにより、遊離したシアル酸を１，２－ジアミノ－４，５－メチレンジオキシベンゼン（ＤＭＢ）により蛍光標識した。室温で放冷した後、下記条件の高速液体クロマトグラフィー（ＨＰＬＣ）に供した。標準試料の測定結果を元に検量線を作成し、新規ムチン型糖タンパク質またはブタ胃粘膜ムチン１ｍｇ当たりのシアル酸濃度（μｍｏｌ／ｍｇ）を算出した。 (5) Measurement of sialic acid concentration N-acetylneuraminic acid (NeuAc) and N-glycolylneuraminic acid (NeuGc) were used as standard samples. As a measurement sample, a mucin solution was diluted 20 times with distilled water. 50 μL of a measurement sample and 200 μL of 62.5 mM hydrochloric acid were placed in a glass vial. After stirring with a vortex mixer, 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. 50 μL of this solution was dispensed into a glass vial, and 200 μL of DMB solution (Coupling solution: distilled water: DMB solution = 5: 4: 1 (volume ratio), sialic acid fluorescent labeling reagent kit, Takara Bio) was added. After stirring with a vortex mixer, the free sialic acid was fluorescently labeled with 1,2-diamino-4,5-methylenedioxybenzene (DMB) by leaving it in the dark at 50 ° C. for 2.5 hours. After allowing to cool at room temperature, it was subjected to high performance liquid chromatography (HPLC) under the following conditions. A calibration curve was prepared based on the measurement results of the standard sample, and the sialic acid concentration (μmol / mg) per mg of novel mucin-type glycoprotein or porcine gastric mucosa mucin was calculated.
《ＨＰＬＣの条件》
装置 ：高速液体クロマトグラフ　ＬＣ－１０ＡＤ（島津製作所）
検出器：蛍光検出器　ＲＦ－１０ＡＸＬ（島津製作所）
励起波長：３７３ｎｍ、測定波長：４４８ｎｍ
カラム：ＴＳＫ　ｇｅｌ　ＯＤＳ－８０ＴＳ（TOSOH）
溶離液：Ａ液が水：メタノール＝９３：７（体積比）、Ｂ液がアセトニトリル：メタノール＝９３：７（体積比）として、Ａ：Ｂ＝９０：１０（体積比）からＡ：Ｂ＝４０：６０（体積比）まで直線的に濃度勾配をかけた溶液
流速：０．５ｍＬ／分
カラム温度：４０℃
注入量：１０μＬ << HPLC conditions >>
Apparatus: High-performance liquid chromatograph LC-10AD (Shimadzu Corporation)
Detector: Fluorescence detector RF-10AXL (Shimadzu Corporation)
Excitation wavelength: 373 nm, measurement wavelength: 448 nm
Column: TSK gel ODS-80TS (TOSOH)
Eluent: Liquid A is water: methanol = 93: 7 (volume ratio), liquid B is acetonitrile: methanol = 93: 7 (volume ratio), and A: B = 90: 10 (volume ratio) to A: B = Solution flow rate linearly gradient up to 40:60 (volume ratio): 0.5 mL / min Column temperature: 40 ° C.
Injection volume: 10 μL
（６）糖鎖を構成する単糖の測定
　標準試料にはMonosaccharide Mixture-11（Ｊ－オイルミルズ）を用いた。測定試料は、ムチン溶液を全糖量が１μｍｏｌ／ｍＬとなるように希釈した。試料５０μＬを１．５ｍＬ容量のエッペンチューブに分注し、８規定のトリフルオロ酢酸（ＴＦＡ）または塩酸 （ＨＣｌ）を加えた。これをヒートブロックを用いて１００℃に加熱し、ＴＦＡの場合は３時間、ＨＣｌの場合は４時間、放置することにより糖の酸加水分解を行った。その後、２０μＬを新しいエッペンチューブに移し、８０℃で１時間遠心濃縮した。続いて、２－プロパノール４０μＬを加え、再度、８０℃で遠心濃縮を行った。その後、ピリジン：メタノールを体積比１：９で混合した溶液を４０μＬ加え、ボルテックスミキサーで混和した。そこに、無水酢酸１０μＬを加え、再度ボルテックスミキサーで混和した後、室温で３０分放置した。続いて、８０℃で３０分間遠心濃縮し、蒸留水１０μＬおよびABEE Labeling solution（ABEE Labeling Kit、Ｊ－オイルミルズ）４０μＬを加え、ヒートブロックを用いて８０℃で１時間加熱した。その後、蒸留水２００μＬおよびクロロホルム２００μＬを加え、ボルテックスミキサーで混和した。１５０００×ｇ、室温にて５分間遠心分離した後、上層（水層）を回収してフィルター（DISMIC-13HP、13HP020AN（アドバンテック）濾過し、下記条件のＨＰＬＣに供した。標準試料の測定結果を元に、単糖の同定および定量を行った。その結果において検出されたグルコース、アラビノースおよびリボースはムチン型糖タンパク質の糖鎖構成糖ではないため、原料由来の夾雑物あるいは各試料の調製段階で混入したものと考えられる。したがって、これらの単糖は「その他の単糖」としてまとめて示した。 (6) Measurement of monosaccharides constituting sugar chains Monosaccharide Mixture-11 (J-Oil Mills) was used as a standard sample. As a measurement sample, the mucin solution was diluted so that the total sugar amount was 1 μmol / mL. 50 μL of the sample was dispensed into a 1.5 mL Eppendorf tube, and 8N trifluoroacetic acid (TFA) or hydrochloric acid (HCl) was added. This was heated to 100 ° C. using a heat block, and the sugar was hydrolyzed by allowing it to stand for 3 hours in the case of TFA and 4 hours in the case of HCl. Thereafter, 20 μL was transferred to a new Eppendorf tube and centrifuged at 80 ° C. for 1 hour. Subsequently, 40 μL of 2-propanol was added, and centrifugal concentration was performed again at 80 ° C. Thereafter, 40 μL of a solution in which pyridine: methanol was mixed at a volume ratio of 1: 9 was added and mixed with a vortex mixer. Thereto was added 10 μL of acetic anhydride, mixed again with a vortex mixer, and allowed to stand at room temperature for 30 minutes. Subsequently, the mixture was centrifuged at 80 ° C. for 30 minutes, 10 μL of distilled water and 40 μL of 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 μL of distilled water and 200 μL 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. Originally, monosaccharides were identified and quantified, and glucose, arabinose, and ribose detected in the results were not sugar chain constituent sugars of mucin-type glycoproteins. Therefore, these monosaccharides are collectively shown as “other monosaccharides”.
《ＨＰＬＣの条件》
装置 ：高速液体クロマトグラフ　ＬＣ－１０ＡＤ（島津製作所）
検出器：蛍光検出器　ＲＦ－１０ＡＸＬ（島津製作所）
励起波長：３０５ｎｍ、測定波長：３６０ｎｍ
カラム：Ｈｏｎｅｎｐａｋ　Ｃ１８（Ｊ－オイルミルズ）
溶離液：ホウ酸カリウム緩衝液（ｐＨ８．９）
流速：１ｍＬ／分
カラム温度：３０℃
注入量：１０μＬ << HPLC conditions >>
Apparatus: High-performance liquid chromatograph LC-10AD (Shimadzu Corporation)
Detector: Fluorescence detector RF-10AXL (Shimadzu Corporation)
Excitation wavelength: 305 nm, measurement wavelength: 360 nm
Column: Honenpak C18 (J-Oil Mills)
Eluent: potassium borate buffer (pH 8.9)
Flow rate: 1 mL / min Column temperature: 30 ° C
Injection volume: 10 μL
　以上の本実施例２（１）～（５）の結果を表１に、（６）の結果を表２に、それぞれ示す。
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000002
 The results of Example 2 (1) to (5) are shown in Table 1, and the result of (6) is shown in Table 2.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000002
　表１に示すように、ムチン型糖鎖量の指標となるＯ－結合型糖の濃度は、新規ムチン型糖タンパク質では０．４３４～０．５４８マイクロモル／ｍｇ（μｍｏｌ／ｍｇ）、ブタ胃粘膜ムチンでは０．７２８～０．７５２μｍｏｌ／ｍｇ、ラット腸管粘膜ムチンでは０．４３４～０．４９８μｍｏｌ／ｍｇであった。そして、硫酸基濃度は、新規ムチン型糖タンパク質では０．０３９～０．０９０μｍｏｌ／ｍｇであったのに対して、ブタ胃粘膜ムチンでは０．０３５～０．０４６μｍｏｌ／ｍｇ、ラット腸管粘膜ムチンでは０．０２１～０．０３７μｍｏｌ／ｍｇであった。すなわち、Ｏ－結合型糖１モルに対する硫酸基の量は、ブタ胃粘膜ムチンの０．０４７～０．０６３モル（０．０３５／０．７５２≒０．０４７～０．０４６／０．７２８≒０．０６３）、ラット腸管粘膜ムチンの０．０４２～０．０８５モル（０．０２１／０．４９８≒０．０４２～０．０３７／０．４３４≒０．０８５）に対して、新規ムチン型糖タンパク質では０．０７１～０．２０７モル（０．０３９／０．５４８≒０．０７１～０．０９０／０．４３４≒０．２０７）であり、新規ムチン型糖タンパク質において、顕著に大きいことが明らかになった。 As shown in Table 1, 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 (μmol / mg) for the novel mucin-type glycoprotein, It was 0.728 to 0.752 μmol / mg for mucosal mucin and 0.434 to 0.498 μmol / mg for rat intestinal mucosal mucin. The sulfate group concentration was 0.039 to 0.090 μmol / mg for the novel mucin-type glycoprotein, whereas 0.035 to 0.046 μmol / mg for the porcine gastric mucosa, and for the rat intestinal mucosa mucin. It was 0.021 to 0.037 μmol / mg. That is, 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.
　また、シアル酸濃度も、ブタ胃粘膜ムチンでは０．０４５～０．０５１μｍｏｌ／ｍｇであったのに対して、新規ムチン型糖タンパク質では０．０６８～０．０８５μｍｏｌ／ｍｇ、ラット腸管粘膜ムチンでは０．１８９～０．２０８μｍｏｌ／ｍｇであった。すなわち、Ｏ－結合型糖１モルに対するシアル酸の量は、ブタ胃粘膜ムチンの０．０６０～０．０７０モル（０．０４５／０．７５２≒０．０６０～０．０５１／０．７２８≒０．０７０）に対して、新規ムチン型糖タンパク質では０．１２４～０．１９６モル（０．０６８／０．５４８≒０．１２４～０．０８５／０．４３４≒０．１９６）、ラット腸管粘膜ムチンでは０．３８０～０．４７９モル（０．１８９／０．４９８≒０．３８０～０．２０８／０．４３４≒０．４７９）であり、新規ムチン型糖タンパク質およびラット腸管粘膜ムチンの方が顕著に大きいことが明らかになった。 In addition, the sialic acid concentration was 0.045 to 0.051 μmol / mg for porcine gastric mucosa mucin, whereas it was 0.068 to 0.085 μmol / mg for new mucin-type glycoprotein, and for rat intestinal mucosa mucin. It was 0.189 to 0.208 μmol / 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. 0.070), 0.124 to 0.196 mol (0.068 / 0.548≈0.124 to 0.085 / 0.434≈0.196) for the novel mucin-type glycoprotein, rat intestine In the mucosal mucin, it is 0.380 to 0.479 mol (0.189 / 0.498≈0.380 to 0.208 / 0.434≈0.479), which is a novel mucin-type glycoprotein and rat intestinal mucosal mucin. It became clear that the direction was significantly larger.
　一方、表２に示すように、新規ムチン型糖タンパク質の糖鎖は、ガラクトースをおよそ０．３～０．７μｍｏｌ／ｍｇ、フコースをおよそ０．３～０．５μｍｏｌ／ｍｇ、マンノースをおよそ０～０．０５μｍｏｌ／ｍｇ、Ｎ－アセチルグルコサミン（ＧｌｃＮＡｃ）をおよそ０．３～０．６μｍｏｌ／ｍｇ、Ｎ－アセチルガラクトサミン（ＧａｌＮＡｃ）をおよそ０．７～１．１μｍｏｌ／ｍｇ含有することが明らかになった。 On the other hand, as shown in Table 2, the sugar chain of the novel mucin-type glycoprotein has galactose of about 0.3 to 0.7 μmol / mg, fucose of about 0.3 to 0.5 μmol / mg, and mannose of about 0 to 0.05 μmol / mg, N-acetylglucosamine (GlcNAc) approximately 0.3 to 0.6 μmol / mg, N-acetylgalactosamine (GalNAc) approximately 0.7 to 1.1 μmol / mg It was.
　以上の結果から、新規ムチン型糖タンパク質は、ロット間の差異や測定誤差を鑑みても、Ｏ－グリコシド結合している糖１モルに対し、硫酸基を０．０７モル以上という高い割合で含有するという点で、新規な成分ないし構造を有することが明らかになった。また、新規ムチン型糖タンパク質は、硫酸基に加えてシアル酸の含有量も大きく、Ｏ－グリコシド結合している糖１モルに対し、シアル酸を０．１モル以上含有することが明らかになった。 Based on the above results, 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. In addition, 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.
　ここで、一般に、ムチン型糖鎖においては、硫酸基は糖鎖の非還元末端の糖残基に付加している場合が多い。従って、表１の測定結果において、暫定的に、Ｏ－結合型糖のモル数をムチン型糖鎖のモル数と見なし、全ての硫酸基がムチン型糖鎖の非還元末端に位置すると見なした場合、新規ムチン型糖タンパク質では、ムチン型糖鎖０．４８１モル（平均値）に対して、硫酸基は０．０６４モル（平均値）であるから、硫酸基による糖鎖非還元末端の封鎖率（末端キャッピング率）は、平均約１３．３％（（０．０６４／０．４８１）×１００≒１３．３１）となる。これに対して、ブタ胃粘膜ムチンでは、ムチン型糖鎖０．７４０モル（平均値）に対して、硫酸基は０．０４０モル（平均値）であるから、硫酸基による末端キャッピング率は、平均約５．４％（（０．０４０／０．７４０）×１００≒５．４１）に過ぎない。また、ラット腸管粘膜ムチンでも、ムチン型糖鎖０．４６６モル（平均値）に対して、硫酸基は０．０２６モル（平均値）であるから、硫酸基による末端キャッピング率は、平均約５．６％（（０．０２６／０．４６６）×１００≒５．５８）に過ぎない。すなわち、新規ムチン型糖タンパク質は多数の糖鎖非還元末端に硫酸基を含有し、係る末端キャッピング率は、ブタ胃粘膜ムチンやラット腸管粘膜ムチンなどの他のムチン型糖タンパク質と比較して２倍以上と顕著に高いことが明らかになった。このことから、新規ムチン型糖タンパク質は、多数の糖鎖非還元末端に硫酸基を含有するという特有の構造を有することが明らかになった。 Here, in general, in mucin-type sugar chains, 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. In this case, in the new mucin-type glycoprotein, since the sulfate group is 0.064 mol (average value) with respect to 0.481 mol (average value) of the mucin-type sugar chain, the sugar chain non-reducing end of the sulfate group is The blocking rate (terminal capping rate) is about 13.3% ((0.064 / 0.481) × 100≈13.31) on average. On the other hand, in 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). That is, 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.
＜実施例３＞新規ムチン型糖タンパク質の評価：ムチン非摂取ラットにおけるムシナーゼ活性
　実施例１の新規ムチン型糖タンパク質および比較例１のブタ胃粘膜ムチンについて、ムチンを摂取しないラットの糞便に含まれるムチン型糖鎖分解酵素（ムシナーゼ）によって分解される程度（ムシナーゼ活性）を分析した。なお、糞便中に存在するムシナーゼの大部分は腸内細菌に由来するものである。 <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.
（１）ムシナーゼ酵素液の調製
　比較例２（１）の標準精製飼料を摂取したラットから、糞便を採取した。採取から３時間以内の糞便に１００倍量（ｗ／ｖ） の０．０１Ｍ酢酸バッファー（ｐＨ５．５）を加えた後、糞便をハサミで細断し、氷中で２０分間放置することで軟化させた。ホモジナイザーＰＴ－２１００（KINEMATICA) を用いて、４℃にてレベル１１で３０秒間破砕して均一化したものをムシナーゼ酵素液とした。 (1) Preparation of mucinase enzyme solution Feces were collected from rats ingesting the standard purified feed of Comparative Example 2 (1). After adding 100 times (w / v) 0.01M acetic acid buffer (pH 5.5) to feces within 3 hours after collection, the feces are shredded with scissors and allowed to stand for 20 minutes in ice for softening I let you. A homogenizer PT-2100 (KINEMATICA) was used and crushed and homogenized at level 11 at 4 ° C. for 30 seconds to obtain a mucinase enzyme solution.
（２）ムシナーゼ反応
　新規ムチン型糖タンパク質またはブタ胃粘膜ムチン２００ｍｇを０．０１Ｍ酢酸バッファー（ｐＨ５．５）１０ｍＬに溶解し、一晩冷蔵保存して、これを基質溶液とした。ムシナーゼ酵素液を３０℃で２分間予備加温した後、ムシナーゼ酵素液０．９ｍＬに対して基質溶液０．１ｍＬを加え、３０℃で３０分置くことにより、ムシナーゼ反応を行った。その後、沸騰水中で３分間加熱して酵素を失活させることで反応を停止させ、これを反応後溶液とした。 (2) 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.
（３）還元糖濃度の測定
　反応後溶液中に遊離した還元糖の量を、Ｓｏｍｏｇｙｉ－Ｎｅｌｓｏｎ法により測定した。すなわち、まず、Ａ液（１５％硫酸銅溶液）とＢ液（蒸留水５００ｍＬにＮａ２ＣＯ３を１２．５ｇ、ロッシェル塩を１２．５ｇ、ＮａＨＣＯ３を１０ｇおよびＮａ２ＳＯ４を１００ｇ溶解した溶液）とを、体積比１：２５の割合で混合して、Ｓｏｍｏｇｙｉ試薬を調製した。また、蒸留水４５０ｍＬに（ＮＨ４）６Ｍｏ７Ｏ２４・４Ｈ２Ｏを２５ｇ溶解させた溶液と、Ｈ２ＳＯ４を２１ｍＬと、蒸留水２５ｍＬにＮａ２ＨＡｓＯ４・７Ｈ２Ｏを３ｇ溶解させた溶液とを混合し、３７℃で２４～４８時間加温して、Ｎｅｌｓｏｎ試薬を調製した。Ｎｅｌｓｏｎ試薬は常温で保存して、使用前には再度、３７℃で２４～４８時間加温した。 (3) Measurement of reducing sugar concentration The amount of reducing sugar released into the solution after the reaction was measured by the Somogyi-Nelson method. That is, first, solution A (15% copper sulfate solution) and solution B (12.5 g of Na 2 CO 3 , 12.5 g of Rochelle salt, 10 g of NaHCO 3 and 100 g of Na 2 SO 4 were dissolved in 500 mL of distilled water. Solution) was mixed at a volume ratio of 1:25 to prepare Somogyi reagent. Also, 25 g of (NH 4 ) 6 Mo 7 O 24 · 4H 2 O dissolved in 450 mL of distilled water, 21 mL of H 2 SO 4 , and 3 g of Na 2 HAsO 4 · 7H 2 O in 25 mL of distilled water The solution was mixed and heated at 37 ° C. for 24-48 hours to prepare a Nelson reagent. The Nelson reagent was stored at room temperature and warmed again at 37 ° C. for 24-48 hours before use.
　標準試料には１ｍｇ／ｍＬのＤ（＋）－グルコース（Ｗａｋｏ）を用い、蒸留水で２５～２００μｇ／ｍＬの希釈系列を作製した。標準試料または反応後溶液１ｍＬに対してＳｏｍｏｇｙｉ試薬１ｍＬを加え、ビー玉で試験管にふたをして沸騰水中で２０分間反応させた。その後直ちに氷水中で冷やすことで逆酸化を防ぐとともに反応を停止した。次いで、Ｎｅｌｓｏｎ試薬１ｍＬを加え、１５分間放置して発色させた後、これを４５０μＬ量り取り、３．３ｍＬの蒸留水に加えた。２３３０×ｇ、室温にて１０分間遠心分離して上清を回収し、６６０ｎｍで吸光度を測定した。標準試料の吸光度測定結果に基づき、検量線を作成した。この検量線を用いて、反応後溶液の吸光度測定結果から、反応後溶液中の還元糖濃度（ｎｍｏｌ／ｍＬ）を算出した。その結果を図１に示す。 1 mg / mL D (+)-glucose (Wako) was used as a standard sample, and a dilution series of 25 to 200 μg / mL was prepared with distilled water. 1 mL of Somogyi reagent was added to 1 mL of the standard sample or post-reaction solution, and the test tube was capped with a marble and allowed to react in boiling water for 20 minutes. Immediately after that, it was cooled in ice water to prevent reverse oxidation and to stop the reaction. Next, 1 mL of Nelson reagent was added and the color was allowed to stand for 15 minutes, and then 450 μL was weighed out and added to 3.3 mL of distilled water. 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.
　図１に示すように、新規ムチン型糖タンパク質およびブタ胃粘膜ムチンのいずれも、酵素反応時間が長いほど還元糖濃度が大きかった。すなわち、糞中に存在するムシナーゼにより、糖鎖が分解されて還元糖が生成したことが明らかになった。しかしながら、新規ムチン型糖タンパク質とブタ胃粘膜ムチンとで還元糖濃度を比較すると、酵素反応時間が１０分、２０分および３０分のいずれにおいても、新規ムチン型糖タンパク質の方が顕著に小さく、ブタ胃粘膜ムチンの約１／３～１／５の濃度であった。すなわち、新規ムチン型糖タンパク質に対するムシナーゼ活性は、ブタ胃粘膜ムチンと比較して顕著に小さかった。この結果から、新規ムチン型糖タンパク質は、ムチンを含有しない精製飼料を摂取したラットの腸内細菌によっては資化されにくい、特有の成分ないし構造を有することが明らかになった。 As shown in FIG. 1, 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. However, when comparing the reducing sugar concentration between the novel mucin-type glycoprotein and porcine gastric mucosa mucin, 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. That is, 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.
　一般に、ムチン型糖鎖の分解は、エキソ型のムシナーゼによって非還元末端から進行するが、糖鎖非還元末端に硫酸エステルやシアル酸が存在する場合は分解が阻害される。このことと、実施例２の結果とを鑑みると、新規ムチン型糖タンパク質に対するムシナーゼ活性が小さい理由は、新規ムチン型糖タンパク質の糖鎖非還元末端の多数に硫酸基やシアル酸が存在するためとも考えられる。 In general, 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. In view of this and the result of Example 2, 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.
＜実施例４＞新規ムチン型糖タンパク質の評価：ムチン摂取ラットにおけるムシナーゼ活性
　実施例１の新規ムチン型糖タンパク質、比較例１のブタ胃粘膜ムチンおよび比較例２のラット腸管粘膜ムチンについて、ムチンを摂取したラットの糞便に含まれるムシナーゼによって分解される程度（ムシナーゼ活性）を分析した。 <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.
（１）ラットの飼育
　７週齢（体重１５０～１７０ｇ）のＷｉｓｔａｒ系雄ラット（日本エスエルシー）１８匹を６匹ずつ３群に分け、対照群、１．５％新規ムチン群、１．５％ブタ胃粘膜ムチン群とした。各群につき、下記の飼料を水道水とともに自由摂取させながら、１５日間飼育した。飼育条件は、室温２４±１℃、相対湿度５５±５℃、１２時間の明暗周期（７：００－１９：００点灯）、ステンレス製ゲージ内での個別飼育とした。１２日目に糞便を採取した後、１５日目に解剖して盲腸を摘出し、切り開いて盲腸組織および盲腸内容物を回収した。
　　対照群：比較例２（１）の標準精製飼料。
　　１．５％新規ムチン群：比較例２（１）の標準精製飼料において、コーンスターチを置換することにより１．５％の新規ムチン型糖タンパク質を含有する飼料。
　　１．５％ブタ胃粘膜ムチン群：比較例２（１）の標準精製飼料において、コーンスターチを置換することにより１．５％のブタ胃粘膜ムチンを含有する飼料。 (1) Breeding of rats Seven 7-week-old (weight 150-170 g) Wistar male rats (Japan SLC) were divided into 3 groups of 6 rats, a control group, a 1.5% new mucin group, 1.5 % Porcine gastric mucosa mucin group. Each group was reared for 15 days while freely taking the following feed with tap water. Breeding conditions were a room temperature of 24 ± 1 ° C., a relative humidity of 55 ± 5 ° C., a 12 hour light / dark cycle (lighting from 7:00 to 19:00), and individual breeding in a stainless steel gauge. Feces were collected on the 12th day and then dissected on the 15th day to remove the cecum and cut open to collect cecal tissue and cecal contents.
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).
（２）ムシナーゼ酵素液の調製
　対照群、１．５％新規ムチン群および１．５％ブタ胃粘膜ムチン群の糞便から、実施例３（１）に記載の方法によりムシナーゼ酵素液を調製し、「対照群由来酵素」、「新規ムチン群由来酵素」および「ブタ胃粘膜ムチン群由来酵素」とした。また、これらのムシナーゼ酵素液中のタンパク質濃度を実施例２（１）に記載のＬｏｗｒｙ法に従って測定した。 (2) Preparation of mucinase enzyme solution From the stool of the control group, 1.5% new mucin group and 1.5% porcine gastric mucosa mucin group, 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).
（３）ムシナーゼ反応
　実施例１の新規ムチン型糖タンパク質、比較例１のブタ胃粘膜ムチンおよび比較例２のラット腸管粘膜ムチンの３種類を基質とし、本実施例４（２）のムシナーゼ酵素液を用いて、実施例３（２）に記載の方法によりムシナーゼ反応を行った。ただし、ラット腸管粘膜ムチンを基質とした反応では、ムシナーゼ酵素液を０．０１Ｍ酢酸バッファー（ｐＨ５．５）により４倍に希釈してから用いた。続いて、実施例３（３）に記載の方法により還元糖濃度を測定した。測定した還元糖濃度は、ムシナーゼ反応の時間およびムシナーゼ酵素液中のタンパク質量で除し、ムシナーゼ酵素液中のタンパク質１ｍｇによりムシナーゼ反応１分間当たりに生じた還元糖の量（ｎｍｏｌ／分／ｍｇタンパク質）として表した。ラット腸管粘膜ムチンを基質とした場合の結果を図２に、ブタ胃粘膜ムチンを基質とした場合の結果を図３に、新規ムチン型糖タンパク質を基質とした場合の結果を図４に、それぞれ示す。 (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). Was used to carry out a mucinase reaction by the method described in Example 3 (2). However, in the reaction using rat intestinal mucosa mucin as a substrate, 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). The measured reducing sugar concentration is divided by the time of the mucinase reaction and the amount of protein in the mucinase enzyme solution, and the amount of reducing sugar produced per minute of the mucinase reaction by 1 mg of protein in the mucinase enzyme solution (nmol / min / mg protein). ). 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, and FIG. 4 shows the results when new mucin-type glycoprotein is used as a substrate. Show.
　図２に示すように、ラット腸管粘膜ムチンを基質とした場合の還元糖の量は、対照群由来酵素では２９．６±２．１ｎｍｏｌ／分／ｍｇタンパク質であったのに対して、新規ムチン群由来酵素では９２．６±５．７ｎｍｏｌ／分／ｍｇタンパク質、ブタ胃粘膜ムチン群由来酵素では８７．６±１３．０ｎｍｏｌ／分／ｍｇタンパク質であり、いずれも対照群由来酵素と比較して有意に高かった。すなわち、ラット腸管粘膜ムチンに対するムシナーゼ活性は、新規ムチン型糖タンパク質またはブタ胃粘膜ムチンを摂取したラットの糞便に含まれるムシナーゼの方が、ムチンを摂取しないラットの糞便に含まれるムシナーゼよりも高かった。この結果から、新規ムチン型糖タンパク質またはブタ胃粘膜ムチンの摂取により、「腸管粘膜から分泌されるムチン」に対する分解活性が高い腸内細菌が誘導されることが明らかになった。 As shown in FIG. 2, 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. In other words, 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”.
　次に、図３に示すように、ブタ胃粘膜ムチンを基質とした場合の還元糖の量は、対照群由来酵素では７．６±１．８ｎｍｏｌ／分／ｍｇタンパク質であったのに対して、新規ムチン群由来酵素では２１．９±２．６ｎｍｏｌ／分／ｍｇタンパク質、ブタ胃粘膜ムチン群由来酵素では２３．６±５．３ｎｍｏｌ／分／ｍｇタンパク質であり、いずれも対照群由来酵素と比較して有意に高かった。すなわち、ブタ胃粘膜ムチンに対するムシナーゼ活性は、新規ムチン型糖タンパク質またはブタ胃粘膜ムチンを摂取したラットの糞便に含まれるムシナーゼの方が、ムチンを摂取しないラットの糞便に含まれるムシナーゼよりも高かった。この結果から、新規ムチン型糖タンパク質またはブタ胃粘膜ムチンの摂取により、「ブタ胃粘膜ムチン」に対する分解活性が高い腸内細菌が誘導されることが明らかになった。 Next, as shown in FIG. 3, 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. That is, 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".
　最後に、図４に示すように、新規ムチン型糖タンパク質を基質とした場合の還元糖の量は、対照群由来酵素では３．３±０．４ｎｍｏｌ／分／ｍｇタンパク質、ブタ胃粘膜ムチン群由来酵素では５．３±０．６ｎｍｏｌ／分／ｍｇタンパク質であったのに対して、新規ムチン群由来酵素では９．６±０．７ｎｍｏｌ／分／ｍｇタンパク質であり、対照群由来酵素およびブタ胃粘膜ムチン群由来酵素と比較して有意に高かった。すなわち、新規ムチン型糖タンパク質に対するムシナーゼ活性は、新規ムチン型糖タンパク質を摂取したラットの糞便に含まれるムシナーゼの方が、ムチンを摂取しないラットの糞便に含まれるムシナーゼはもとより、ブタ胃粘膜ムチンを摂取したラットの糞便に含まれるムシナーゼよりも高かった。この結果から、新規ムチン型糖タンパク質の摂取により、「新規ムチン型糖タンパク質」に対する分解活性が高い腸内細菌が誘導されることが明らかになった。 Finally, as shown in FIG. 4, when the novel mucin-type glycoprotein is used as a substrate, 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. In other words, 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".
　以上のとおり、新規ムチン型糖タンパク質またはブタ胃粘膜ムチンのいずれを摂取した場合も、ブタ胃粘膜ムチンに対する分解活性は同程度に高くなった（図３）のに対して、新規ムチン型糖タンパク質に対する分解活性は、新規ムチン型糖タンパク質を摂取した場合にのみ、高くなった（図４）。この結果から、新規ムチン型糖タンパク質は、ブタ胃粘膜ムチンの摂取により誘導される腸内細菌によっては分解されにくい、特有の成分ないし構造を有することが明らかになった。また、新規ムチン型糖タンパク質の摂取により、ブタ胃粘膜ムチンの摂取により誘導されるものとは異なる、特有の腸内細菌叢を誘導できることが明らかになった。 As described above, when either the novel mucin-type glycoprotein or the porcine gastric mucosa mucin was ingested, the degradation activity against the porcine gastric mucosa mucin increased to the same extent (FIG. 3), whereas the novel mucin-type glycoprotein The degrading activity against was increased only when a novel mucin-type glycoprotein was ingested (FIG. 4). From this result, it was revealed that the novel mucin-type glycoprotein has a unique component or structure that is difficult to be degraded by intestinal bacteria induced by ingestion of porcine gastric mucosa mucin. In addition, it has been clarified that the intake of a novel mucin-type glycoprotein can induce a unique intestinal bacterial flora different from that induced by the intake of porcine gastric mucosa.
（４）盲腸内容物中のＯ－結合型糖濃度の測定
　本実施例４（１）で回収した盲腸内容物から、比較例２（２）に記載の方法により盲腸内容物ムチン画分を調製した。ここで、ムチンを摂取しないラット（対照群）の盲腸内容物に含まれるムチンはラット腸管粘膜ムチンであるが、ムチンを摂取したラット（１．５％新規ムチン群、１．５％ブタ胃粘膜ムチン群）の盲腸内容物に含まれるムチンは、ラット腸管粘膜ムチンおよび摂取したムチンに由来する。 (4) Measurement of O-linked sugar concentration in cecal contents From the cecal contents recovered in Example 4 (1), a cecal content mucin fraction was prepared by the method described in Comparative Example 2 (2). did. Here, 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.
　調製した盲腸内容物ムチン画分に蒸留水を加えて５ｍＬに定容した後、さらに蒸留水で１０倍に希釈した。これを測定試料として、実施例２（３）に記載の方法により、Ｏ－グリコシド結合している糖（Ｏ－結合型糖）の濃度を測定した。標準試料の測定結果を基に、測定試料におけるＯ－結合型糖の濃度を求め、盲腸内容物ムチン画分１ｇ当たりのＯ－結合型糖濃度（μｍｏｌ／ｇ）を算出した。その結果を図５に示す。 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. Using this as a measurement sample, the concentration of O-glycosidically linked saccharide (O-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 (μmol / g) per 1 g of cecal content mucin fraction was calculated. The result is shown in FIG.
　図５に示すように、Ｏ－結合型糖濃度は、対照群では０．５０±０．０４μｍｏｌ／ｇであったのに対して、１．５％新規ムチン群では２．２６±０．４７μｍｏｌ／ｇであり、対照群と比較して有意に高かった。一方、１．５％ブタ胃粘膜ムチン群のＯ－結合型糖濃度は０．５４±０．１１μｍｏｌ／ｇであり、対照群と比較して有意差は無かった。ここで、Ｏ－結合型糖の濃度は、ムチン型糖鎖の量に比例すると考えられる。従って、新規ムチン型糖タンパク質を摂取したラットの腸内には、ブタ胃粘膜ムチンを摂取したラットの腸内と比較して、顕著に多くのムチン型糖鎖が残存していることが明らかになった。 As shown in FIG. 5, the O-linked sugar concentration was 0.50 ± 0.04 μmol / g in the control group, whereas 2.26 ± 0.47 μmol in the 1.5% new mucin group. / G, significantly higher than the control group. On the other hand, the O-linked sugar concentration in the 1.5% porcine gastric mucosa mucin group was 0.54 ± 0.11 μmol / g, which was not significantly different from the control group. Here, 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.
　この結果から、新規ムチン型糖タンパク質は、ブタ胃粘膜ムチンと比較してラット腸内で利用される速度が遅いことが明らかになった。その理由は、実施例３および本実施例４（３）で述べたように、新規ムチン型糖タンパク質が、これを摂取していない腸内細菌によっては資化されにくい、特有の成分ないし構造を有するためと考えられる。なお、盲腸組織の杯細胞数、クリプト長および盲腸内容物中のアンモニア濃度については、各群間に有意差はなかった。 From this result, it was revealed that the novel mucin-type glycoprotein is used at a slower rate in the rat intestine than the porcine gastric mucosa mucin. The reason is that, as described in Example 3 and Example 4 (3), 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. In addition, there was no significant difference between the groups regarding the number of goblet cells in the cecal tissue, the crypt length, and the ammonia concentration in the cecal contents.
＜実施例５＞新規ムチン型糖タンパク質の評価：腸内細菌への作用
　実施例１の新規ムチン型糖タンパク質および比較例１のブタ胃粘膜ムチンを摂取したラットにおける腸内細菌（全真正細菌、アッカーマンシア　ムシニフィラおよびバクテロイデス　テタイオタオミクロン）量の変化を、１６Ｓ　ｒＲＮＡ遺伝子を標的としたリアルタイムＰＣＲ法により分析した。 <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）ラットの飼育
　７週齢（体重１５０～１７０ｇ）のＷｉｓｔａｒ系雄ラット（日本エスエルシー）２４匹を６匹ずつ４群に分け、対照群、０．７５％新規ムチン群、１．５％新規ムチン群、１．５％ブタ胃粘膜ムチン群とした。各群につき、下記の飼料を水道水とともに自由摂取させながら、１４日間飼育した。飼育条件は、実施例４（１）に記載の条件と同様にした。その後、解剖して盲腸を摘出し、切り開いて盲腸内容物を回収した。
　　対照群：比較例２（１）の標準精製飼料。
　　０．７５％新規ムチン群：比較例２（１）の標準精製飼料において、コーンスターチを置換することにより０．７５％の新規ムチン型糖タンパク質を含有する飼料。
　　１．５％新規ムチン群：比較例２（１）の標準精製飼料において、コーンスターチを置換することにより１．５％の新規ムチン型糖タンパク質を含有する飼料。
　　１．５％ブタ胃粘膜ムチン群：比較例２（１）の標準精製飼料において、コーンスターチを置換することにより１．５％のブタ胃粘膜ムチンを含有する飼料。 (1) Rearing of rats 24 Wistar male rats (Japan SLC) 7 weeks old (body weight 150-170 g) were divided into 4 groups of 6 rats, control group, 0.75% new mucin group, 1.5 % New mucin group, 1.5% porcine gastric mucosa mucin group. Each group was bred for 14 days while freely feeding the following feed with tap water. Rearing conditions were the same as those described in Example 4 (1). Then, the cecum was excised and excised, and the cecum contents were collected by opening.
Control group: Standard purified feed of Comparative Example 2 (1).
0.75% new mucin group: A feed containing 0.75% of novel mucin-type glycoprotein by replacing corn starch in the 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).
（２）ＤＮＡの抽出
　盲腸内容物からのＤＮＡの抽出は、ＤＮＡ抽出キットISOFECAL for Beads Beating（ニッポンジーン）を用いて行った。具体的には、１５０ｍｇの盲腸内容物に１ｍＬのLysis Solution Fを加えて懸濁した後、全量を２ｍＬ容量のBeads Tubeへ移して混合した。続いて、ビーズ式破砕装置により、２７００ｒｐｍ、室温にて２分間ビーズ破砕した後、１２０００×ｇ、室温にて５分間遠心分離し、上清６００μＬを２ｍＬ容量のマイクロチューブに移した。Purification solutionを４００μＬ加えて混合した後、クロロホルムを６００μＬ加えて１５秒間ボルテックスミキサーにより混合した。１２０００×ｇ、室温にて５分間遠心分離し、上清８００μＬを１．５ｍＬ容量マイクロチューブに移してPrecipitation solutionを８００μＬ加えてボルテックスミキサーで混合した。２００００×ｇ、室温にて１５分間遠心分離して上清を除去した。沈殿物にWash solution 1 mLを加えて転倒混和した。２００００×ｇ、４℃にて１０分間遠心分離し、上清を除去して沈殿物に７０％エタノール１ｍＬとEtachinmate２μＬを加えてボルテックスミキサーで混合した。２００００×ｇ、４℃にて５分間遠心分離し、上清を除去して沈殿物を乾燥させた後、TE（ｐＨ８．０）１００μＬに溶解した。この溶液をＤＮＡ溶液とした。 (2) Extraction of DNA DNA was extracted from the cecum contents using a DNA extraction kit ISOFECAL for Beads Beating (Nippon Gene). Specifically, 1 mL of Lysis Solution F was added to and suspended in 150 mg of cecal contents, and then the entire amount was transferred to a 2 mL volume of Beads Tube and mixed. Subsequently, the beads were crushed by a bead type crusher at 2700 rpm and room temperature for 2 minutes, and then centrifuged at 12000 × g and room temperature for 5 minutes, and 600 μL of the supernatant was transferred to a 2 mL capacity microtube. 400 μL of the purification solution was added and mixed, then 600 μL of chloroform was added and mixed with a vortex mixer for 15 seconds. Centrifugation was performed at 12000 × g and room temperature for 5 minutes, 800 μL of the supernatant was transferred to a 1.5 mL capacity microtube, 800 μL of Precipitation solution was added, and the mixture was mixed with a vortex mixer. The supernatant was removed by centrifugation at 20000 × g for 15 minutes at room temperature. 1 mL of Wash solution was added to the precipitate and mixed by inversion. Centrifugation was performed at 20000 × g and 4 ° C. for 10 minutes, the supernatant was removed, 1 mL of 70% ethanol and 2 μL of Etachinmate were added to the precipitate, and mixed with a vortex mixer. After centrifugation at 20000 × g and 4 ° C. for 5 minutes, the supernatant was removed and the precipitate was dried, and then dissolved in 100 μL of TE (pH 8.0). This solution was used as a DNA solution.
（３）リアルタイムＰＣＲ
　リアルタイムＰＣＲはLightCycler（登録商標）Nano（Roche）を用いて、添付の使用書に従って行った。反応液の組成は、鋳型ＤＮＡ溶液が２μＬ、SYBR Premix EX Taq ＩＩ（タカラバイオ）が１０μＬ、１０μＭのセンスプライマー溶液が０．８μＬ、１０μＭのアンチセンスプライマー溶液が０．８μＬおよびＤＥＰＣ処理水が６．４μＬの計２０μＬとした。なお、鋳型ＤＮＡ溶液は、本実施例５（２）のＤＮＡ溶液を、全真正細菌を測定する場合は５００倍に、アッカーマンシア　ムシニフィラまたはバクテロイデス　テタイオタオミクロンを測定する場合は１００倍に、それぞれ希釈して用いた。既知濃度のＤＮＡを用いた測定により検量線を作成し、係る検量線から、測定結果に基づき盲腸内容物１ｇあたりの各細菌の１６Ｓ ｒＤＮＡコピー数を絶対定量した。その結果を図６に示す。また、各細菌に特異的なプライマーの配列を以下に示す。 (3) Real-time PCR
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 μL of template DNA solution, 10 μL of SYBR Premix EX Taq II (Takara Bio), 0.8 μL of 10 μM sense primer solution, 0.8 μL of 10 μM antisense primer solution, and 6 DEPC-treated water. 4 μL total 20 μL. 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.
　《アッカーマンシア　ムシニフィラ》
センスプライマー : ＣＡＧＣＡＣＧＴＧＡＡＧＧＴＧＧＧＧＡＣ（配列番号１）
アンチセンスプライマー：ＣＣＴＴＧＣＧＧＴＴＧＧＣＴＴＣＡＧＡＴ（配列番号２）
　《バクテロイデス　テタイオタオミクロン》
センスプライマー : ＧＣＡＡＡＣＴＧＧＡＧＡＴＧＧＣＧＡ（配列番号３）
アンチセンスプライマー：ＡＡＧＧＴＴＴＧＧＴＧＡＧＣＣＧＴＴＡ（配列番号４）
　《全真正細菌》
センスプライマー :ＣＧＧＣＡＡＣＧＡＧＣＧＣＡＡＣＣＣ（配列番号５）
アンチセンスプライマー：ＣＣＡＴＴＧＴＡＧＣＡＣＧＴＧＴＧＴＡＧＣＣ（配列番号６） 《Ackermancia Musinifira》
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)
《All Eubacteria》
Sense primer: CGGCAACGAGCCGCAACCC (SEQ ID NO: 5)
Antisense primer: CCATTGTAGCACGTGTGTAGCC (SEQ ID NO: 6)
　図６に示すように、全真正細菌の１６Ｓ ｒＤＮＡコピー数は、各群間に有意差はなかった。その一方で、アッカーマンシア　ムシニフィラの１６Ｓ ｒＤＮＡコピー数は、１．５％新規ムチン群で対照群に対して有意に大きく、０．７５％新規ムチン群でも、対照群と比較して、有意差はないものの大きかった。これに対して、ブタ胃粘膜ムチン群のアッカーマンシア　ムシニフィラの１６Ｓ ｒＤＮＡコピー数は、対照群と同等であった。また、バクテロイデス　テタイオタオミクロンの１６Ｓ ｒＤＮＡコピー数も、１．５％新規ムチン群で対照群に対して有意に大きく、０．７５％新規ムチン群でも、対照群と比較して、有意差はないものの大きかった。これに対して、ブタ胃粘膜ムチン群のバクテロイデス　テタイオタオミクロンの１６Ｓ ｒＤＮＡコピー数は、対照群と同等であった。 As shown in FIG. 6, the 16S rDNA copy number of all eubacteria was not significantly different between the groups. On the other hand, 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. In contrast, the 16S DNA copy number of Ackermansia muciniphila in the porcine gastric mucosa mucin group was equivalent to that in the control group. Also, 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. In contrast, the 16S DNA copy number of Bacteroides tetaiotaomicron in the porcine gastric mucosa mucin group was equivalent to that in the control group.
　すなわち、ラット盲腸内容物中のアッカーマンシア属細菌の１６Ｓ ｒＤＮＡコピー数およびバクテロイデス属細菌の１６Ｓ ｒＤＮＡコピー数は、新規ムチン型糖タンパク質の摂取により顕著に増大した。この結果から、新規ムチン型糖タンパク質は、アッカーマンシア属細菌およびバクテロイデス属細菌の菌数を増加させる作用を有することが明らかになった。 That is, 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.
　ここで、一般に、大多数の腸内細菌は硫酸基分解酵素（スルファターゼ）やシアル酸分解酵素（シアリダーゼ）を有していないために、硫酸基やシアル酸の含有量が高い糖鎖を資化できない。これに対して、アッカーマンシア属細菌およびバクテロイデス属細菌は、これらの酵素を有しているため、硫酸基やシアル酸の含有量が高い糖鎖を資化し、栄養源として利用できることが報告されている。 Here, in general, the majority of 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. On the other hand, Ackermansia bacteria and Bacteroides bacteria have these enzymes, and it has been reported that sugar chains with a high content of sulfate groups and sialic acids can be assimilated and used as nutrient sources. Yes.
　しかしながら、本実施例５では、いずれの群も、表１に示すようにシアル酸の含有量が相当に高いラット腸管粘膜ムチンを元来有するにもかかわらず、これらの細菌の菌数は大きく異なっていた。よって、摂取するムチン型糖タンパク質においてシアル酸の含有量が高いことは、アッカーマンシア属細菌およびバクテロイデス属細菌の増加要因とならないことが示唆される。そして、硫酸基の含有量が高い新規ムチン型糖タンパク質を摂取した群のみにおいてこれらの細菌が増加したことから、硫酸基の含有量が高いことが、これらの細菌の増加要因となることが示唆される。 However, in this Example 5, although all groups originally had rat intestinal mucosa mucin with a considerably high sialic acid content as shown in Table 1, the numbers of these bacteria differed greatly. It was. Therefore, it is suggested that the high content of sialic acid in the mucin-type glycoprotein to be ingested does not cause an increase in Ackermansia bacteria and Bacteroides bacteria. And since these bacteria increased only in the group that ingested a novel mucin-type glycoprotein with a high sulfate group content, it was suggested that a high sulfate group content would cause an increase in these bacteria. Is done.
　すなわち、硫酸基の含有量が高いムチン型糖タンパク質を摂取したラットの腸内では、アッカーマンシア属細菌やバクテロイデス属細菌が、当該ムチン型糖タンパク質を利用できない他の腸内細菌種と比較して優性となり、特異的に増加したものと考えられる。このことから、コアタンパク質にＯ－グリコシド結合している糖１モルに対し、硫酸基を０．０７モル以上という高い割合で含有するムチン型糖タンパク質は、アッカーマンシア属細菌およびバクテロイデス属細菌の菌数を増加させる作用を有することが明らかになった。 In other words, in the intestines of rats that have ingested mucin-type glycoproteins with a high sulfate group content, Ackermansia bacteria and Bacteroides bacteria are compared to other intestinal bacterial species that cannot use the mucin-type glycoproteins. It seems that it became dominant and increased specifically. Therefore, 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.
＜実施例６＞新規ムチン型糖タンパク質の評価：アッカーマンシア属細菌の菌数増加作用
（１）新規ムチン型糖タンパク質の調製および含有成分の解析
　実施例１に記載の方法により、異なる５つの原料から新規ムチン型糖タンパク質を調製し、ロットＩ～Ｖとした。ただし、ロットＩおよびロットＩＩＩは真カスベ由来原料：水カスベ由来原料＝２：１（重量比）の原料から、ロットＩＩは真カスベ由来原料のみから、ロットＩＶおよびロットＶは水カスベ由来原料のみから、それぞれ調製した。これらについて、実施例２（１）～（５）に記載の方法によりタンパク質濃度、全糖濃度、Ｏ－結合型糖濃度、硫酸基濃度およびシアル酸濃度を測定した。その結果を表３に示す。なお、表３において、±の左側は平均値、右側は標準誤差を示す。標準誤差を付した数値は、各ロットから３つの試料をサンプリングして、各試料につき三重反復測定した結果である。標準誤差を付していない数値は、各ロットから１つの試料をサンプリングして、各試料につき三重反復測定した平均値である。
Figure JPOXMLDOC01-appb-T000003
 <Example 6> Evaluation of novel mucin-type glycoprotein: Increase in the number of bacteria of genus Ackermancia (1) Preparation of new mucin-type glycoprotein and analysis of contained components Five different raw materials according to the method described in Example 1 A new mucin-type glycoprotein was prepared from lots I to V. However, lot I and lot III are raw material derived from true kasbe: raw material derived from water kasbe = 2: 1 (weight ratio), lot II is only from raw material derived from true kasbe, and lots IV and lot V are only raw material derived from water kasube. Respectively. For these, 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. In Table 3, the left side of ± shows the average value, and 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.
Figure JPOXMLDOC01-appb-T000003
　表３に示すように、新規ムチン型糖タンパク質は、いずれのロットにおいても、Ｏ－グリコシド結合している糖１モルに対し、硫酸基を０．０７モル以上、シアル酸を０．１モル以上という高い割合で含有することが確認された。 As shown in Table 3, 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.
（２）アミノ酸解析
　本実施例６（１）の新規ムチン型糖タンパク質について、下記の方法により各アミノ酸の含有割合（ｍｇ／ｇ）を測定した。また、測定結果に基づいて、総アミノ酸に占める各アミノ酸の質量百分率（％）を解析した。なお、下記の測定方法では、トリプトファン（Ｗ）、メチオニン（Ｍ）およびシステイン（Ｃ）は分解されるため、これらのアミノ酸は測定していない。また、「総アミノ酸」は「Ｗ、ＭおよびＣを除く総アミノ酸」を意味する。解析結果を表４に示す。表４において、含有割合は、ムチン型糖タンパク質１ｇ当たりの各アミノ酸の含有量（ｍｇ）で示す。また、質量百分率は、Ｗ、ＭおよびＣを除くアミノ酸の総量に占める各アミノ酸の質量の百分率で示す。数値は、各ロットから１つの試料をサンプリングして、各試料につき二重反復測定した平均値である。 (2) Amino acid analysis About the novel mucin-type glycoprotein of this Example 6 (1), the content rate (mg / g) of each amino acid was measured by the following method. Further, based on the measurement results, the mass percentage (%) of each amino acid in the total amino acids was analyzed. In the following measurement method, tryptophan (W), methionine (M) and cysteine (C) are decomposed, so these amino acids are not measured. In addition, “total amino acids” means “total amino acids excluding W, M and C”. The analysis results are shown in Table 4. In Table 4, the content ratio is indicated by the content (mg) of each amino acid per gram of mucin-type glycoprotein. The mass percentage is expressed as a percentage of the mass of each amino acid in the total amount of amino acids excluding W, M and C. The numerical value is an average value obtained by sampling one sample from each lot and performing duplicate measurement for each sample.
《アミノ酸の測定》
　ムチン型糖タンパク質５０ｍｇを２．０ｍＬの蒸留水に懸濁し、このうち０．５ｍＬを１ｍＬ容量のバキュームリアクションチューブ (ジーエルサイエンス) に量り入れた。そこへ０．２％の２－メルカプトエタノールを含む１２ｍｏｌ／Ｌの塩酸溶液を０．５ｍＬ加えてよく混合した後、真空ポンプ (ＤＴＣ－２２、ＵＬＶＡＣ) で脱気した。流動パラフィン (Ｗａｋｏ) で満たしたヒートブロック(ＨＦ６１、ヤマト科学)にリアクションチューブを入れて１１０℃で２４時間置くことにより、ムチン型糖タンパク質を酸加水分解した。リアクションチューブを室温に戻してチューブ内の内容物をメスフラスコに移した後、蒸留水で５回洗った。これに３ｍｏｌ／ＬのＮａＯＨ１．７５ｍＬを加えてｐＨ２．２付近に調整した後、０．０６７ｍｏｌ／Ｌのクエン酸ナトリウム緩衝液(ｐＨ２．２、Ｗａｋｏ) で２５ｍＬに定容した。これをメンブレンフィルター（ＤＩＳＭＩＣ－１３ＨＰ、１３ＨＰ０２０ＡＮ、アドバンテック）でろ過してろ液を回収し、アミノ酸分析に供した。アミノ酸分析は、分析用カラム (日立ハイテクパックドカラム＃２６２２ＰＨ、径４．６×６０ｍｍ、日立ハイテクノロジーズ) およびプレカラム (日立パックドカラム ＃２６５０Ｌ、径４．６×４０ｍｍ、日立ハイテクノロジーズ) を備えたＬ－８９００形高速アミノ酸分析計 (日立ハイテクノロジーズ) で行った。溶離液 (ＭＣＩ（商標）ＢＵＦＦＥＲ　Ｌ－８５００－ＰＨ－ＫＩＴ、三菱ケミカル) および反応液 (日立用ニンヒドリン発色溶液キット、Ｗａｋｏ) は市販のキットを用いた。アミノ酸の標品には、アミノ酸混合標準液　Ｈ型 (Ｗａｋｏ)１ｍＬを０．０６７ｍｏｌ／Ｌのクエン酸ナトリウム緩衝液 (ｐＨ２．２、Ｗａｋｏ) で２５ｍＬに定容したものを用いた。
Figure JPOXMLDOC01-appb-T000004
 <Measurement of amino acids>
50 mg of mucin-type glycoprotein was suspended in 2.0 mL of distilled water, and 0.5 mL of this was suspended in a 1 mL capacity vacuum reaction tube (GL Science). Thereto, 0.5 mL of a 12 mol / L hydrochloric acid solution containing 0.2% 2-mercaptoethanol was added and mixed well, followed by degassing with a vacuum pump (DTC-22, ULVAC). The mucin-type glycoprotein was acid hydrolyzed by placing a reaction tube in a heat block (HF61, Yamato Science) filled with liquid paraffin (Wako) and placing it at 110 ° C. for 24 hours. 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. To this, 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 ™ BUFFER L-8500-PH-KIT, Mitsubishi Chemical) and the reaction solution (Hitachi's ninhydrin coloring solution kit, Wako). As the amino acid preparation, 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).
Figure JPOXMLDOC01-appb-T000004
　表４に示すように、ロットＩＶは、ロットＩ～ＩＩＩおよびロットＶと比較して、セリンおよびスレオニンの含有割合および質量百分率が小さく、イソロイシン、チロシンおよびフェニルアラニンの含有割合および質量百分率が大きいことが明らかになった。 As shown in Table 4, 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.
（３）ラットの飼育
　７週齢（体重１４０～１６０ｇ）のＷｉｓｔａｒ系雄ラット（日本エスエルシー）３６匹を６匹ずつ６群に分け、対照群ならびにロットＩ～Ｖ群とした。各群につき、下記の飼料を水道水とともに自由摂取させながら、１４日間飼育した。飼育条件は、実施例４（１）に記載の条件と同様にした。その後、解剖して盲腸を摘出し、切り開いて盲腸内容物を回収した。
　　対照群：比較例２（１）の標準精製飼料において、コーンスターチを一部置換することにより５重量％のセルロースおよび１０重量％のスクロースを含有する飼料。
　　ロットＩ～Ｖ群：比較例２（１）の標準精製飼料において、コーンスターチを一部置換することにより１．２％の新規ムチン型糖タンパク質（本実施例６（１）のロットＩ～
Ｖ）を含有する飼料。 (3) Breeding of rats 36 7-week-old (140 to 160 g body weight) Wistar male rats (Japan SLC) were divided into 6 groups of 6 rats, which were used as a control group and lots I to V groups. Each group was bred for 14 days while freely feeding the following feed with tap water. Rearing conditions were the same as those described in Example 4 (1). Then, the cecum was excised and excised, and the cecum contents were collected by opening.
Control group: a feed containing 5% by weight cellulose and 10% by weight sucrose by partially replacing corn starch in the standard purified feed of Comparative Example 2 (1).
Lot I to V group: 1.2% novel mucin-type glycoprotein (lot I to lot of this Example 6 (1)) by partially replacing corn starch in the standard purified feed of Comparative Example 2 (1)
V) feed.
（４）ＤＮＡの抽出およびリアルタイムＰＣＲ
　回収した盲腸内容物から、実施例５（２）に記載の方法によりＤＮＡを抽出した。続いて、実施例５（３）に記載の方法によりリアルタイムＰＣＲを行い、全真正細菌およびアッカーマンシア　ムシニフィラの１６Ｓ　ｒＤＮＡコピー数を測定した。その結果を図７に示す。 (4) DNA extraction and real-time PCR
DNA was extracted from the collected cecal contents by the method described in Example 5 (2). Subsequently, real-time PCR was performed by the method described in Example 5 (3), and the number of 16S rDNA copies of all eubacteria and Ackermansia ciniphila was measured. The result is shown in FIG.
　図７に示すように、全真正細菌の１６Ｓ ｒＤＮＡコピー数は、各群間に有意差はなかった。その一方で、アッカーマンシア　ムシニフィラの１６Ｓ ｒＤＮＡコピー数は、ロットＩ群、ロットＩＩ群、ロットＩＩＩ群およびロットＶ群で対照群に対して有意に大きく、ロットＩＶ群でも、対照群と比較して、有意差はないものの大きかった。すなわち、ラット盲腸内容物中のアッカーマンシア属細菌の１６Ｓ ｒＤＮＡコピー数は、新規ムチン型糖タンパク質の摂取により増大した。この結果から、新規ムチン型糖タンパク質は、アッカーマンシア属細菌の菌数を増加させる作用を有することが明らかになった。 As shown in FIG. 7, the number of 16S rDNA copies of all eubacteria was not significantly different between the groups. On the other hand, 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.
　ここで、非特許文献５によれば、アッカーマンシア　ムシニフィラは、必須アミノ酸のうちのスレオニンを合成することができない（RESULTS AND DISCUSSION、第２段落第５－７行目）。また、非特許文献６によれば、アンモニアおよび他のアミノ酸を含み、かつＬ－スレオニンを含まない培地では、アッカーマンシア　ムシニフィラは増殖できない（Results and discussion 、第１段落第１４－１７行目、Fig. S1）。すなわち、アッカーマンシア　ムシニフィラは、生存ないし増殖に、外部からスレオニンを供給されることが必要といえる。
　非特許文献５；Ottman N. et al, Genome-scale model and omics analysis of metabolic capacities of Akkermansia muciniphila reveal a preferential mucin-degrading lifestyle, Appl Environ Microbiol, Volume 83, Issue 18, e01014-e01017, September 2017
　非特許文献６；Kees C. H. van der Ark et al., Model-driven design of a minimal medium for Akkermansia muciniphila confirms mucus adaptation, Microbial Biotechnology 11(1339-1353), January 2018, DOI: 10.1111/1751-7915.13033 Here, according to 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. et al, Genome-scale model and omics analysis of metabolic capacities of Akkermansia muciniphila reveal a preferential mucin-degrading lifestyle, Appl Environ Microbiol, Volume 83, Issue 18, e01014-e01017, September 2017
Non-Patent Document 6; Kees C. H. van der Ark et al., Model-driven design of a minimal medium for Akkermansia muciniphila confirms mucus adaptation, Microbial Biotechnology 11 (1339-1353), January 2018, DOI: 10.1111 / 1751-7915.13033
　この点、表４に示すように、ロットＩＶのムチン型糖タンパク質は、他のロットと比較して、スレオニンの含有割合がムチン型糖タンパク質１ｇあたり２８．５ｍｇ、総アミノ酸に占めるスレオニンの質量百分率が１４．２％と小さかった。このことから、ロットＩＶ群で、アッカーマンシア属細菌の菌数が増加しているものの、その増加の程度が比較的小さいのは、摂取させたムチン型糖タンパク質におけるスレオニンの含有割合ないし総アミノ酸に占める質量百分率が小さいためと考えられた。すなわち、この結果から、ムチン型糖タンパク質においてスレオニンの含有率が高いものが、より強いアッカーマンシア属細菌の菌数増加作用を有することが明らかになった。具体的には、高いアッカーマンシア属細菌の菌数増加効果を得るためには、ムチン型糖タンパク質において、スレオニンの含有割合が２９ｍｇ／ｇ以上、あるいは、トリプトファン、メチオニンおよびシステインを除くアミノ酸の総量に占めるスレオニンの質量百分率が１５％以上であることが好ましいことが明らかになった。 In this regard, as shown in Table 4, 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. Specifically, in order to obtain a high effect of increasing the number of bacteria of the genus Ackermancia, in the mucin-type glycoprotein, 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.

Claims (13)

  1. 　硫酸基およびシアル酸を含有するムチン型糖タンパク質であって、前記硫酸基の含有量が、コアタンパク質にＯ－グリコシド結合している糖１モルに対し、０．０７モル以上であり、前記シアル酸の含有量が、コアタンパク質にＯ－グリコシド結合している糖１モルに対し、０．１モル以上である、前記ムチン型糖タンパク質。 A mucin-type glycoprotein containing a sulfate group and sialic acid, wherein the sulfate group content is 0.07 mol or more with respect to 1 mol of a sugar having an O-glycoside bond to the core protein, The mucin-type glycoprotein, wherein the acid content is 0.1 mol or more with respect to 1 mol of a sugar having an O-glycoside bond to the core protein.
  2. 　トリプトファン、メチオニンおよびシステインを除くアミノ酸の総量に占めるスレオニンの質量百分率が１５％以上である、請求項１に記載のムチン型糖タンパク質。 The mucin-type glycoprotein according to claim 1, wherein the mass percentage of threonine in the total amount of amino acids excluding tryptophan, methionine and cysteine is 15% or more.
  3. 　エイの皮および／または体表粘性物から採取される、請求項１または請求項２に記載のムチン型糖タンパク質。 The mucin-type glycoprotein according to claim 1 or 2 collected from ray skin and / or body surface viscous material.
  4. 　請求項１から請求項３のいずれかに記載のムチン型糖タンパク質を有効成分とする、アッカーマンシア属細菌および／またはバクテロイデス属細菌の菌数増加剤。 An agent for increasing the number of bacteria of the genus Ackermancia and / or the genus Bacteroides, comprising the mucin-type glycoprotein according to any one of claims 1 to 3 as an active ingredient.
  5. 　下記のムチン型糖タンパク質を有効成分とする、アッカーマンシア属細菌および／またはバクテロイデス属細菌の菌数増加剤；
    　硫酸基を含有するムチン型糖タンパク質であって、前記硫酸基の含有量が、コアタンパク質にＯ－グリコシド結合している糖１モルに対し、０．０７モル以上である、前記ムチン型糖タンパク質。     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. .
  6. 　アッカーマンシア属細菌の菌数増加剤であって、前記ムチン型糖タンパク質において、トリプトファン、メチオニンおよびシステインを除くアミノ酸の総量に占めるスレオニンの質量百分率が１５％以上である、請求項５に記載の菌数増加剤。 The bacterium according to claim 5, which is an agent for increasing the number of bacteria of the genus Ackermansia, wherein the mass percentage of threonine in the total amount of amino acids excluding tryptophan, methionine and cysteine in the mucin-type glycoprotein is 15% or more. Number increasing agent.
  7. 　前記ムチン型糖タンパク質が、エイの皮および／または体表粘性物から採取されたものである、請求項５または請求項６に記載の菌数増加剤。 The agent for increasing the number of bacteria according to claim 5 or 6, wherein the mucin-type glycoprotein is collected from ray skin and / or body surface viscous material.
  8. 　肥満、２型糖尿病および脂質異常症からなる群から選択される１以上の疾患の予防または治療に用いられることを特徴とする、請求項４から請求項７のいずれかに記載の菌数増加剤。 The bacterial count increasing agent according to any one of claims 4 to 7, which is used for the prevention or treatment of one or more diseases selected from the group consisting of obesity, type 2 diabetes and dyslipidemia. .
  9. 　請求項１から請求項３のいずれかに記載のムチン型糖タンパク質を有効成分とする、アッカーマンシア属細菌および／またはバクテロイデス属細菌の菌数増加用食品組成物。 A food composition for increasing the number of bacteria of the genus Ackermancia and / or the genus Bacteroides, comprising the mucin-type glycoprotein according to any one of claims 1 to 3 as an active ingredient.
  10. 　下記のムチン型糖タンパク質を有効成分とする、アッカーマンシア属細菌および／またはバクテロイデス属細菌の菌数増加用食品組成物；
    　硫酸基を含有するムチン型糖タンパク質であって、前記硫酸基の含有量が、コアタンパク質にＯ－グリコシド結合している糖１モルに対し、０．０７モル以上である、前記ムチン型糖タンパク質。     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. .
  11. 　アッカーマンシア属細菌の菌数増加用食品組成物であって、前記ムチン型糖タンパク質において、トリプトファン、メチオニンおよびシステインを除いたアミノ酸の総量に占めるスレオニンの質量百分率が１５％以上である、請求項１０に記載の菌数増加用食品組成物。 The food composition for increasing the number of bacteria of the genus Ackermancia, wherein the mass percentage of threonine in the total amount of amino acids excluding tryptophan, methionine and cysteine in the mucin-type glycoprotein is 15% or more. The food composition for increasing the number of bacteria described in 1.
  12. 　前記ムチン型糖タンパク質が、エイの皮および／または体表粘性物から採取されたものである、請求項１０または請求項１１に記載の菌数増加用食品組成物。 The food composition for increasing the number of bacteria according to claim 10 or 11, wherein the mucin-type glycoprotein is collected from ray skin and / or body surface viscous material.
  13. 　肥満、２型糖尿病および脂質異常症からなる群から選択される１以上の疾患の予防または治療に用いられることを特徴とする、請求項９から請求項１２のいずれかに記載の菌数増加用食品組成物。 It is used for the prevention or treatment of one or more diseases selected from the group consisting of obesity, type 2 diabetes and dyslipidemia, for increasing the number of bacteria according to any one of claims 9 to 12 Food composition.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010107083A1 (en) * 2009-03-19 2010-09-23 丸共バイオフーズ株式会社 Proliferation promoter for antigen-specific t cell
WO2017178496A1 (en) * 2016-04-11 2017-10-19 Wageningen Universiteit Novel bacterial species

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010107083A1 (en) * 2009-03-19 2010-09-23 丸共バイオフーズ株式会社 Proliferation promoter for antigen-specific t cell
WO2017178496A1 (en) * 2016-04-11 2017-10-19 Wageningen Universiteit Novel bacterial species

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ROBERTON, A. M. ET AL.: "In vitro utilization of mucin by Bacteroides fragilis", APPL. ENVIRON. MICROBIOL., vol. 43, no. 2, February 1982 (1982-02-01), pages 325 - 330, XP055645000 *
SUMI, T. ET AL.: "Isolation and properties of a sialoglycoprotein from the skin mucus of the stingray Dasyatis akajei", FISHERIES SCIENCE, vol. 63, no. 3, 1997, pages 453 - 458, XP055645001 *

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