WO2018045492A1 - 长栖粪杆菌(Faecalibacterium longum)及其应用 - Google Patents

长栖粪杆菌(Faecalibacterium longum)及其应用 Download PDF

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WO2018045492A1
WO2018045492A1 PCT/CN2016/098246 CN2016098246W WO2018045492A1 WO 2018045492 A1 WO2018045492 A1 WO 2018045492A1 CN 2016098246 W CN2016098246 W CN 2016098246W WO 2018045492 A1 WO2018045492 A1 WO 2018045492A1
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composition
bacterium
acid
acceptable carrier
faecal
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PCT/CN2016/098246
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English (en)
French (fr)
Inventor
邹远强
薛文斌
肖亮
刘传
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深圳华大基因研究院
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Priority to US16/082,769 priority Critical patent/US10799540B2/en
Priority to JP2018546802A priority patent/JP6862464B2/ja
Priority to PCT/CN2016/098246 priority patent/WO2018045492A1/zh
Priority to EP16915428.3A priority patent/EP3511406A4/en
Priority to CN201680083550.1A priority patent/CN109219656A/zh
Publication of WO2018045492A1 publication Critical patent/WO2018045492A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • 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
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/20—Milk; Whey; Colostrum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • 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
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

  • the present invention is in the field of microbiology, and in particular, the present invention relates to the use of Faecalibacterium longum in the treatment and prevention of diabetes and related diseases, and also to a composition comprising the genus Diptera and its use.
  • a large number of symbiotic microorganisms are found in the human intestine. These symbiotic microbial communities constitute the "second organ" of the human body. They not only participate in the digestion and absorption of nutrients, but also play an important role in the maintenance of human health. In terms of the number of cells, the number of all cells of intestinal microbes is 10 times that of human cells. These large microbial populations mainly have two species: thick-walled bacteria and pseudobacteria. More and more studies have shown that the composition and diversity of intestinal microbes are closely related to the occurrence of multiple diseases, such as obesity, diabetes, irritable bowel syndrome, ulcerative enteritis, colon cancer, fatty liver and so on. Due to the influence of environment, diet, drugs, etc., the balance of intestinal microbes in the original state of health is broken, leading to intestinal dysfunction, which in turn induces the occurrence of the above diseases.
  • Diabetes is a disease caused by a variety of factors, such as hyperglycemia, mainly due to insufficient insulin secretion or metabolic disorders caused by insulin deficiency. Common risk factors for diabetes are genetic factors, environmental factors and malnutrition. Lifestyle habits.
  • Type 2 diabetes is a type of non-insulin-dependent diabetes mellitus, which accounts for 90% of all patients with diabetes. Numerous studies have shown that the incidence of type 2 diabetes is closely related to the disorder of the intestinal flora. The intestinal flora is closely related to the regulation of energy balance and inflammatory response between the host. The main cause of type 2 diabetes is insulin resistance and Caused by low levels of inflammatory response.
  • hypoglycemic drugs for the treatment of diabetes include sulfonylureas, ⁇ -glucosidase inhibitors and biguanides.
  • Sulfonamides mainly promote insulin secretion, but cause severe liver and kidney damage.
  • ⁇ -glucosidase inhibitors mainly inhibit the hydrolysis of carbohydrates by inhibiting the activity of ⁇ -amylase and intestinal ⁇ -glucosidase, thereby achieving the purpose of reducing postprandial blood glucose, but It is easy to cause adverse reactions such as bloating and diarrhea; biguanides can achieve hypoglycemic effects by regulating blood sugar transport, such as delaying sugar absorption, promoting glucose decomposition, inhibiting liver glucose production, and increasing glucose transfer protein. Biguanide drugs also cause some side effects, which can cause stomach upset, diarrhea, vomiting, and rash. It is prone to inactivation after long-term use.
  • Another object of the present invention is to provide a use of the bacterium of the genus Diquat for the treatment and prevention of diabetes and related diseases.
  • Another object of the present invention is to provide a pharmaceutical, beverage, food composition, or animal feed composition for the treatment and prevention of diabetes and related diseases thereof which is effective and has no toxic side effects.
  • Another object of the present invention is to provide a method of reducing body weight, fasting blood glucose and/or blood lipids and uses thereof.
  • Another object of the present invention is to provide a method for improving sugar tolerance and an application thereof.
  • a bacterium of the genus Herba which is Faecalibacterium longum.
  • sequence of the 16s rDNA of the faecal bacterium is shown as SEQ ID NO.: 1.
  • the faecal bacterium is Faecalibacterium longum CM04-06, and the accession number is CGMCC 1.5208.
  • a second aspect of the invention provides a composition
  • a composition comprising: (a) a safe and effective amount of the faecal bacterium of claim 1 and/or a metabolite thereof; and (b) food acceptable A carrier or a pharmaceutically acceptable carrier.
  • the composition further comprises a milk growth factor.
  • the composition is selected from the group consisting of a food composition, a health care composition, a pharmaceutical composition, a beverage composition, a feed composition, or a combination thereof.
  • the composition is an oral preparation.
  • the composition is a liquid formulation, a solid formulation, or a semi-solid formulation.
  • the dosage form of the composition is selected from the group consisting of powders, powders, tablets, dragees, capsules, granules, suspensions, solutions, syrups, drops, and tongues. Under the tablet.
  • the food composition comprises an emulsion preparation, a solution preparation, a powder preparation, or a suspension preparation.
  • the food composition comprises a dairy product, a milk powder, or an emulsion.
  • the liquid formulation is selected from the group consisting of a solution preparation or a suspension product.
  • the composition contains 1 x 10-1 x 10 10 cfu/mL or cfu/g Faecalibacterium longum CM04-06, preferably 1 x 10 4 - 1 x 10 10 cfu/mL or cfu /g Faecalibacterium longum CM04-06, based on the total volume or total weight of the composition.
  • the composition contains 0.0001 to 99% by weight, preferably 0.1 to 90% by weight (please review) the described faecal bacterium and/or its metabolite, to the composition Total weight.
  • the composition is a unit dosage form (one tablet, one capsule or one vial), and the mass of the composition in each unit dosage form is from 0.05 to 5 g, preferably from 0.1 to 1 g.
  • composition further contains other probiotics and/or prebiotics.
  • the probiotic is selected from the group consisting of lactic acid bacteria, bifidobacteria, Lactobacillus acidophilus, or a combination thereof.
  • the prebiotic is selected from the group consisting of oligofructose (FOS), galactooligosaccharide (GOS), xylooligosaccharide (XOS), oligofructose (LACT), and low soybean.
  • Glycan SOS
  • inulin or a combination thereof.
  • a third aspect of the invention provides the use of the composition of the invention according to the first aspect of the invention, or the composition of the second aspect of the invention, for the preparation of a medicament or preparation for selection
  • One or more uses of the group : (a) preventing and/or treating obesity; (b) lowering blood lipids; (c) preventing or treating cardiovascular disease; and/or (d) preventing and/or treating diabetes.
  • the formulation comprises a microecological formulation.
  • a fourth aspect of the invention provides a use of the composition according to the first aspect of the invention, or the composition of the second aspect of the invention, for the preparation of a medicament or a preparation for selection
  • HDL-C high density lipoprotein
  • LDL-C low density lipoprotein
  • the mammal comprises a human, a rodent (e.g., a rat, a mouse).
  • a rodent e.g., a rat, a mouse
  • the reducing blood lipid levels in the mammal comprises reducing total cholesterol (TC) levels and/or triglyceride levels.
  • the reducing blood glucose levels in the mammal comprises reducing fasting blood glucose levels.
  • composition of the second aspect of the invention is formed by mixing the faecal bacterium of the first aspect of the invention and/or its metabolite with a food acceptable carrier or a pharmaceutically acceptable carrier.
  • the method further comprises the step of mixing with a growth factor.
  • the growth factor is a milk growth factor.
  • the growth factor is selected from the group consisting of a vitamin, an anthraquinone, a pyrimidine, or a combination thereof.
  • the composition is an oral preparation.
  • a sixth aspect of the invention provides a method of production comprising the steps of:
  • composition of the bacterium of the genus of the genus Bacillus and/or its metabolite obtained in the previous step is mixed with a food-acceptable carrier or a pharmaceutically acceptable carrier to prepare a composition.
  • step (c) prior to step (c), the step of mixing the isolated long-neared Bacillus bacterium and/or its metabolite with the growth factor is further included.
  • the growth factor is a milk growth factor.
  • the growth factor is selected from the group consisting of a vitamin, an anthraquinone, a pyrimidine, or a combination thereof.
  • a seventh aspect of the present invention provides a method of reducing body weight, fasting blood glucose and/or blood lipid, which comprises administering to the subject the fecal bacterium of the first aspect of the invention and/or a metabolite thereof, or the second of the present invention The composition of the aspect.
  • the administration comprises oral administration.
  • the administration dose is 0.01-5 g / 50 kg body weight / day, preferably, 0.1-2 g / 50 kg body weight / day.
  • the subject comprises a mammal, such as a human.
  • An eighth aspect of the present invention provides a method for improving sugar tolerance, which comprises administering the subject of the first aspect of the present invention to the bacterium of the present invention and/or a metabolite thereof, or the second aspect of the present invention combination.
  • the administration comprises oral administration.
  • the administration dose is 0.01 to 5 g / 50 kg body weight / day, preferably 0.1 to 2 g / 50 kg body weight / day.
  • the subject comprises a mammal, such as a human.
  • Figure 1 shows a picture of colonies cultured for 3 days in Fascalibacterium longum.
  • Fig. 2 is a Gram-stained picture (1000 times) of a microorganism of the genus Faeccalibacterium longum of the present invention under a microscope.
  • Figure 3 shows the changes in body weight of the model group and the diabetic mice treated with E. faecalis CM04-06 and metformin.
  • Figure 4 shows the results of the model group and the fasting blood glucose (FBG) of D. faecalis CM04-06 and metformin in diabetic mice.
  • FBG fasting blood glucose
  • Figure 5 shows the glucose tolerance of the model group and the diabetic mice treated with E. faecalis CM04-06 and metformin.
  • Faecalibacterium longum has the function of preventing and treating diabetes and related diseases (such as cardiovascular diseases and obesity diseases), and will contain long feces.
  • diabetes and related diseases such as cardiovascular diseases and obesity diseases
  • the active composition of the bacillus was fed to the test subject, and the composition was found to inhibit weight gain, lower blood fat, lower fasting blood glucose, improve glucose tolerance, and effectively alleviate diabetes, cardiovascular and obesity.
  • the present invention has been completed on this basis.
  • the term “contains” means that the various ingredients can be used together in the mixture or composition of the invention. Therefore, the terms “consisting essentially of” and “consisting of” are encompassed by the term “contains.”
  • growth factor As used herein, the terms “growth factor”, “milk growth factor” are used interchangeably and all include nutrients of vitamins, terpenoids, pyrimidines, or combinations thereof.
  • the vitamins include, but are not limited to: vitamin C, vitamin E, vitamin A, vitamin A precursor, vitamin B 6 , vitamin D 3 , vitamin K, folic acid, or a combination thereof;
  • the terpenoid includes, but is not limited to, a purine nucleoside, wherein the purine nucleoside comprises a 5'-phosphate of a purine nucleoside, and the 5'-phosphate of the purine nucleoside is selected from the group consisting of: Inosinic acid (inosine-5'-phosphate; IMP), guanylic acid (guanosine-5'-phosphate; GMP), xanthosine (xanthine-5'-phosphate; XMP), Adenylate (adenosine-5'-phosphate; AMP), or a combination thereof;
  • the pyrimidine species include all substances containing a pyrimidine structure.
  • the terms "H. faecalis” and “Faecalibacterium longum” are used interchangeably.
  • the strain is Faecalibacterium longum CM04-06, deposited under the number CGMCC 1.5208, isolated from human feces.
  • the physiological characteristics of the faecal bacterium are as follows: The faecal bacterium is cultured in an anaerobic environment at 37 ° C for 2-3 days, the colony is yellowish white, the water content is high, a little sticky, nearly round, opaque, flat middle bulge, The diameter of the colony was about 2-3 mm; by Gram staining and microscopic observation, the CM04-06 cells were long rod-shaped, and the Gram staining reaction was negative. No spores and flagella were found. The diameter of the cells was about 1 um and the length.
  • the growth temperature ranges from 30 to 45 ° C
  • the pH ranges from 4.0 to 9.0
  • the optimum temperature and The pH is 37 ° C and pH 7.0.
  • the present invention provides the use of the bacterium of the genus Diquat for the treatment and prevention of diabetes and related diseases such as cardiovascular diseases and obesity diseases.
  • the subject ingests a high-fat diet, the strain Faecalibacterium longum CM04-06 has one or more uses selected from the group consisting of: (i) inhibiting the subject's weight gain; (ii) lowering blood lipids; (iii) lowering the fasting Blood glucose levels; (iv) increased glucose tolerance.
  • C57bL/6 mice are used as test mice, and fed with a high-fat diet and injected with streptavidin (STZ) to obtain a type 2 diabetes (T2D) model mouse, strain Faecalibacterium Longum CM04-06 treatment of T2D model mice, compared with the untreated control group (model group), their weight gain decreased and blood lipids decreased, and various diabetes-related indicators also decreased, such as fasting blood glucose levels
  • STZ streptavidin
  • the strain can be used for the prevention and treatment of diabetes and related diseases (such as cardiovascular diseases, obesity, etc.).
  • the invention also provides a composition, preferably a pharmaceutical composition.
  • the composition includes an effective amount of B. faecalis, and in a preferred embodiment, the composition further comprises a milk growth factor.
  • the composition further comprises a probiotic selected from the group consisting of lactic acid bacteria, bifidobacteria, Lactobacillus acidophilus, or a combination thereof; and/or a prebiotic selected from the group consisting of oligofructose ( FOS), galactooligosaccharide (GOS), xylooligosaccharide (XOS), oligofructose (LACT), soy oligosaccharide (SOS), inulin, or a combination thereof.
  • FOS oligofructose
  • GOS galactooligosaccharide
  • XOS xylooligosaccharide
  • LACT soy oligosaccharide
  • SOS soy oligosaccharide
  • the composition is a liquid formulation, a solid formulation, or a semi-solid formulation.
  • the liquid formulation is selected from the group consisting of a solution product or a suspension product.
  • the dosage form of the composition is selected from the group consisting of powders, powders, tablets, dragees, capsules, granules, suspensions, solutions, syrups, drops, and sublingual lozenge.
  • the pharmaceutical composition of the present invention can be administered in any form of a pharmaceutical tablet, an injection or a capsule, which comprises an excipient, a pharmaceutically acceptable vehicle and a carrier, which can be selected depending on the route of administration.
  • the pharmaceutical preparation of the present invention may further comprise an auxiliary active ingredient.
  • Lactose glucose, sucrose, sorbitol, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, fine crystalline cellulose, polyvinylpyrrolidone (PVP), cellulose, water, syrup, Methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate or mineral oil and the like can be used as a carrier, an excipient or a diluent of the pharmaceutical composition of the present invention.
  • PVP polyvinylpyrrolidone
  • the pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, an emulsifier, a suspension stabilizer, a preservative, a sweetener, a perfume, and the like.
  • the pharmaceutical composition of the present invention can be produced in a casing formulation by various known methods, so that the active ingredient of the pharmaceutical composition, that is, the microorganism, can smoothly pass through the stomach without being destroyed by gastric acid.
  • the microorganism of the present invention can be used in the form of a capsule prepared by a conventional method.
  • a standard excipient is mixed with the cold-dried microorganism of the present invention to prepare a pellet, and then the pellet is filled into a gelatin capsule.
  • the microorganisms and medicaments of the present invention allow the use of excipients such as liquid gums, celluloses, silicates or mineral oils to prepare suspensions or dispersions, which suspensions or dispersions can be filled into soft gelatin capsules. in.
  • the pharmaceutical composition of the present invention can be made into an enteric tablet for oral use.
  • casing in this application includes all coatings which are permitted for use with conventional drugs which are not degraded by gastric acid but which are sufficiently decomposed in the small intestine and rapidly release the microorganism of the present invention.
  • the casing of the present invention is coated at about 16-30 mg per tablet, preferably 16-25 mg, more preferably 16-20 mg.
  • the thickness of the casing is 5 to 100 ⁇ m, and the thickness is preferably 20 to 80 ⁇ m.
  • the casing component is selected from conventional polymers known to the public.
  • Preferred casings of the present invention are cellulose acetate phthalate polymers or trimellitate polymers and copolymers of methacrylic acid (for example, containing 40% or more of methacrylic acid and containing methylcellulose phthalic acid) Preparation of a copolymer of hydroxypropyl acrylate or its ester derivative of methacrylic acid).
  • the cellulose acetate phthalate used in the casing of the present invention has a viscosity of about 45 to 90 cp, an acetyl content of 17 to 26%, and a phthalic acid content of 30 to 40%.
  • the cellulose acetate terephthalate used in the casing has a viscosity of about 5-21 cs and an acetamidine content of 17-26%.
  • Cellulose acetate trimellitate is produced by Eastman Kodak Company and can be used in the casing material of the present invention.
  • the hydroxypropyl methylcellulose phthalate used in the casing of the present invention has a molecular weight of generally 20,000 to 130,000 Daltons, an ideal molecular weight of 80,000 to 100,000 Daltons, and a hydroxypropyl content of 5 to 10%.
  • the methoxy group content is 18-24%, and the phthaloyl group content is 21-35%.
  • HP50 Hydroxypropyl methylcellulose phthalic acid used in the casing of the present invention
  • HP50 contains 6-10% hydroxypropyl, 20-24% methoxy, 21-27% propyl and has a molecular weight of 84,000 Daltons.
  • Another casing material is HP55, HP55 contains 5-9% hydroxypropyl methylcellulose phthalate, 18-22% methoxy, 27-35% phthalic acid, and its molecular weight is 78,000 Daltons.
  • the casing of the present invention is prepared by spraying a casing solution onto the core using conventional methods. All solvents in the enteric coating process are alcohols (e.g., ethanol), ketones (e.g., acetone), halogenated hydrocarbon compounds (e.g., dichloromethane), or combinations thereof. Softeners such as di-n-butyl phthalate and triacetin are added to the casing solution in a ratio of about 1 part to about 0.05 part or about 0.3 parts of softener.
  • the spraying method is preferably carried out continuously, and the amount of the sprayed material can be controlled according to the conditions employed for the coating. Spray pressure can follow Intentional regulation, in general, achieves ideal results at an average pressure of 1-1.5 bar.
  • pharmaceutically effective amount is meant an amount that is functional or active to a human and/or animal and that is acceptable to humans and/or animals.
  • it may be prepared to contain 1 ⁇ 10 -1 ⁇ 10 10 cfu / ml or cfu / g (particularly, may contain 1 ⁇ 10 4 - 1 ⁇ 10 10 cfu / ml or cfu / g; more special
  • the preparation may contain 1 x 10 6 - 1 x 10 10 cfu/ml or cfu/g) of the bacterium of the genus Diquat and/or its metabolite.
  • the effective dosage of the faecal bacterium or its metabolites used may vary depending on the mode of administration and the severity of the disease to be treated.
  • a dosage form suitable for internal administration comprising about 1 x 10-1 x 10 10 cfu/ml or cfu/g in intimate admixture with a solid or liquid pharmaceutically acceptable carrier (particularly, may contain 1 x 10 4 -1 x 10 10 cfu/ml or cfu/g; more particularly, it may contain 1 ⁇ 10 6 -1 ⁇ 10 10 cfu/ml or cfu/g) of active active faecal bacteria or a fermentatively produced active ingredient.
  • This dosage regimen can be adjusted to provide an optimal therapeutic response. For example, several separate doses may be administered per day, or the dose may be proportionally reduced, as is critical to the condition of the treatment.
  • the A. faecalis or a metabolite thereof can be administered by oral administration or the like.
  • Solid carriers include: starch, lactose, dicalcium phosphate, microcrystalline cellulose, sucrose and kaolin, while liquid carriers include: medium, polyethylene glycol, nonionic surfactants and edible oils (such as corn oil, peanut oil). And sesame oil), as long as it is suitable for the characteristics of the faecal bacteria or its metabolites and the specific mode of administration required.
  • Adjuvants commonly used in the preparation of pharmaceutical compositions may also be advantageously included, such as flavoring agents, coloring agents, preservatives, and antioxidants such as vitamin E, vitamin C, BHT, and BHA.
  • compositions are solid compositions, especially tablets and solid filled or liquid filled capsules. Oral administration is preferred.
  • composition of the invention is administered to the individual for one or more administrations per day.
  • Dosage unit of administration means a dose that is formally separable and suitable for use in humans or all other mammalian individuals.
  • Each unit contains a pharmaceutically acceptable carrier and a therapeutically effective amount of the wood invention microorganism.
  • the amount administered varies depending on the weight of the patient and the severity of diabetes, the supplementary active ingredient included, and the microorganism used. Further, if possible, it can be administered separately and can be administered continuously if necessary. Therefore, the amount administered does not limit the invention.
  • the "composition" in the present invention means not only a medicine but also a functional food and a health supplement.
  • the composition comprises: a beverage, a food, a pharmaceutical, an animal feed, and the like.
  • a food composition comprising an effective amount of a faecal bacterium and/or a metabolite thereof, and a balance of a food acceptable carrier, said food combination
  • the dosage form is selected from the group consisting of a solid, a dairy, a solution, a powder, or a suspension.
  • the food composition may also contain a milk growth factor.
  • composition of the composition is as follows:
  • composition of the composition is as follows:
  • the microecological preparation is a biological preparation containing probiotics and metabolites or a dietary supplement which can increase probiotics, and can achieve the purpose of improving human health by regulating and maintaining the micro-ecological balance in the intestinal tract. It mainly includes probiotics, prebiotics and synbiotics.
  • the microecological preparation comprises (a) a safe and effective amount of the faecal bacterium and/or its metabolite; and (b) a food acceptable carrier or a pharmaceutically acceptable carrier.
  • the formulation further comprises a growth factor (such as milk growth factor, preferably including a vitamin, an anthraquinone, and/or a pyrimidine).
  • the bacterium of the genus Diquat can be obtained by a conventional method.
  • composition of the bacterium of the genus of the genus Bacillus and/or its metabolite obtained in the previous step is mixed with a food-acceptable carrier or a pharmaceutically acceptable carrier to prepare a composition.
  • the method comprises ingesting a pharmaceutical composition, a food composition, a beverage composition, or a combination thereof of the present invention.
  • the subject is a human.
  • the method comprises: ingesting the pharmaceutical composition of the present invention, a food composition, Or animal feed, or a combination thereof.
  • the subject is an animal, preferably a mouse, a rabbit.
  • the method comprises ingesting a pharmaceutical composition, a food composition, a beverage composition, or a combination thereof of the present invention.
  • the subject is a human.
  • the method comprises: ingesting a pharmaceutical composition, a food composition, or an animal feed of the invention, or a combination thereof.
  • the subject is an animal, preferably a mouse, a rabbit.
  • the strain of the genus Faecalibacterium longum CM04-06 (same as the preservation name) of the present invention has been deposited with the General Microbiology Center (CGMCC) of the China Microbial Culture Collection Management Committee on June 13, 2016, at the Chaoyang District, Beijing, China. No. 3, No. 1 Courtyard, Beichen West Road, deposit number: CGMCC 1.5208.
  • CGMCC General Microbiology Center
  • the bacterium of the present invention can significantly reduce body weight, lower blood lipids, and fasting blood sugar levels.
  • the long-lived bacterium of the present invention can significantly reduce indicators (such as cholesterol and triglycerides) associated with obesity and related diseases such as cardiovascular diseases.
  • the bacterium of the present invention can significantly reduce the levels of total cholesterol, triglyceride, and low density lipoprotein.
  • the microorganism of the present invention can significantly increase the level of high density lipoprotein.
  • the bacterium of the present invention can significantly improve the sugar tolerance.
  • ATCC 27768 Faecalibacterium prausnitzii ATCC 27768, hereinafter referred to as ATCC 27768, purchased From the American Type Culture Collection, the accession number is ATCC 27768.
  • CM04-06 The faecal bacterium CM04-06 (hereinafter referred to as CM04-06) of the present invention is isolated from a stool sample of a healthy child (male) in Shenzhen.
  • the environment for separation and culture is strictly anaerobic conditions.
  • the specific separation process is: taking about 0.2g of stool sample in the anaerobic operation box, using 1ml of sterile PBS for suspension and dispersion, fully shaking and mixing, and then performing gradient dilution coating.
  • the medium was anaerobic PYG medium (purchased from Huanqi Microbiology Technology Co., Ltd.), and the specific components are as follows:
  • the formulation of the inorganic salt solution is:
  • the coated plate was cultured under anaerobic conditions at 37 ° C for 3-4 days. After the growth of the surface of the plate was delayed, a single colony was picked and streaked until the pure culture strain was obtained, and the isolated strain was subjected to glycerol-80. °C frozen storage.
  • Genomic extraction The isolated strain was cultured, and when the concentration of the bacteria reached the order of 10 8 cfu/ml, 2 ml of the bacterial liquid was taken for genomic DNA extraction.
  • 16S rDNA sequence database alignment The 1372 bp 16S rDNA sequence was obtained by sequencing, and the sequence was aligned in the EzTaxon-e database to obtain the species classification information of the strain. According to the information of 16S rDNA, it can be preliminarily determined that CM04-06 is a new species belonging to the genus Faecalibacterium.
  • the 16S rDNA sequence of CM04-06 is SEQ ID NO.: 1, as follows:
  • CM04-06 used the 16S rDNA sequence to compare the EzTaxon-e database with the 16S rDNA sequence of CM04-06, and obtained the species closely related to CM04-06. These species and 16S rDNA of CM04-06 were obtained. The sequences were aligned to the sequence, and then the neighbor-joining phylogenetic tree was drawn using the Mega5 software.
  • Morphological characteristics CM04-06 was cultured for 2-3 days at 37 °C in an anaerobic environment. The colonies were yellowish white, with high water content, a little sticky, nearly round, opaque, flat middle bulge, and colony diameter. 2-3mm ( Figure 1).
  • CM04-06 is utilized in raffinose and trehalose, hydrolysis of escin and gelatin, and esterase (C4), proline arylamine, cystine Aromatic aminease, chymotrypsin, naphthol-AS-BI-phosphohydrolase, ⁇ -uronic acid chymase, ⁇ -glucose
  • esterase C4
  • proline arylamine cystine Aromatic aminease
  • chymotrypsin cystine Aromatic aminease
  • naphthol-AS-BI-phosphohydrolase ⁇ -uronic acid chymase
  • ⁇ -glucose There is a significant difference in the activity of chymase and N-acetyl-glucosamine, which proves that CM04-06 is a new species different from known bacteria.
  • ATCC 27768 The cells cultured to the stable phase of CM04-06 and Faecalibacterium prausnitzii ATCC 27768 (hereinafter referred to as ATCC 27768, purchased from the American Type Culture Collection (Accession No. ATCC 27768) were collected and then subjected to bacterial collection. Extraction and detection of cellular fatty acids. The content and difference of fatty acid components of the two strains of cells were analyzed by gas chromatography.
  • CM04-06 is a new species, which was named as Faecalibacterium longum.
  • SCFA short chain fatty acids
  • Sample preparation Take 1 ml of CM04-06 bacterial solution cultured for 48 hours, centrifuge at 12000 r/min for 5 min, and aspirate the supernatant for use.
  • the detector was a hydrogen flame ion detector, and the GC parameter was set to Column temperature: 180 ⁇ 200°C; gasification chamber temperature: 240°C; detection temperature: 210°C; injection volume: 2 ⁇ L; carrier gas flow rate: N 2 , 50mL/min; hydrogen flow rate: 50mL/min; air flow rate: 600 ⁇ 700mL/min.
  • the detection of extracellular polysaccharides is carried out by the phenolic phenol method.
  • the phenolic phenol can react with the free monosaccharides, oligosaccharides and hexoses in the polysaccharides, and the color produced is proportional to the absorbance, and the absorption wavelength is 490 nm.
  • the specific experimental process is as follows:
  • the experimental strain was cultured in PYG medium (formulation as in Example 1) for 2 days, treated with 10 ml of bacterial liquid in boiling water bath for 30 min, then centrifuged at 10000 r/min, and the supernatant was taken, and 80% trichloroacetic acid was added to a final concentration of 8%.
  • the protein was precipitated by treatment at 4 ° C overnight. Centrifuge at 10,000 r/min for 30 min, and adjust the pH of the supernatant to 6.0 with NaOH.
  • the content of extracellular polysaccharide in the CM04-06 fermentation broth cultured for 2 days was 233 mg/L.
  • T2D type 2 diabetes mellitus
  • STZ streptavidin
  • T2D mice The test mice were treated with C57bL/6 mice (purchased from Hubei Medical Experimental Animal Center) at 8 weeks of age. The experimental environment of the mice was SPF grade, and the feeding was performed for 1 week. The modeling method was induced by high fat plus STZ, and the fasting blood glucose level (FBG) reached 10 mM/L or more as a T2D model mouse.
  • FBG fasting blood glucose level
  • mice in the T2D model were administered with normal saline;
  • mice in T2D model were treated with CM04-06 bacterial solution
  • CM04-06 is cultured to a stable phase, and the concentration of bacteria is about 10 8 cfu/ml.
  • mice were sacrificed 2 months after the test. All the mice were taken from the eyeball for blood collection, neck removal, total serum cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL-C) and low density lipid. Protein cholesterol (LDL-C).
  • mice in the CM04-06 and metformin treatment groups gained less weight than the model group as the mice gradually gained weight. It indicated that CM04-06 can effectively slow the increase of body weight in mice (*P ⁇ 0.05/**P ⁇ 0.01). At the same time, by statistics, CM04-06 is more effective in controlling the weight gain of mice than metformin.
  • the blood lipids of the mice were examined after the end of the experiment, including total cholesterol (TC), triglyceride (TG), low density lipoprotein (LDLC) and high density lipoprotein (HDLC).
  • the test results are shown in Table 8.
  • the results showed that the low-density lipoprotein in the metformin group and the CM04-06 group was lower than the model group (*P value ⁇ 0.05), and the high-density lipoprotein was higher than the model group (*P value ⁇ 0.05), and the main function of high-density lipoprotein. It is to remove excess cholesterol and low-density lipoprotein in blood and cells, and has anti-atherosclerosis effect. It can be seen that CM04-06 can improve hyperlipidemia symptoms in diabetic mice. At the same time, compared with the metformin group, CM04-06 has a better effect on the improvement of blood lipids, including lower LDL content and higher HDL content.
  • Example 5 Food composition containing Faeccalibacterium longum CM04-06
  • Vitamin C 0.5
  • Example 6 Pharmaceutical composition containing Faeccalibacterium longum CM04-06
  • the ratio of raw materials is shown in Table 10.
  • lactose, yeast powder and peptone are mixed uniformly with purified water, preheated to 60-65 ° C, homogenized at 20 Mpa, sterilized at 90 ° C for 20-30 minutes, cooled to 36-38 ° C, mixed with vitamin C, and connected.
  • Faecalibacterium longum CM04-06 live bacteria (1-50 ⁇ 10 6 cfu/mL), fermented to pH 6.0 at 36-38 ° C, centrifuged, freeze-dried to a moisture content of less than 3%, ie, prepared Faecalibacterium longum CM04-06 bacteria frozen Dry matter.
  • 0.5 g of Faecalibacterium longum CM04-06 lyophilizate was weighed in an equal amount with maltodextrin and then filled into capsules to prepare a pharmaceutical composition containing Faecalibacterium longum CM04-06.
  • Faecalibacterium longum CM04-06 (1 ⁇ 10 9 cfu/ml) was subjected to anaerobic culture, and anaerobic medium was cultured in PYG medium for 2-3 days at 37 °C.
  • CM04-06 fermented bacterial solution Add 5 volumes of the above growth factor to 100 volumes of CM04-06 fermented bacterial solution, mix well and mix, then add starch adjuvant (such as maltodextrin) to prepare Faecalibacterium containing Longum CM04-06 drug.
  • starch adjuvant such as maltodextrin
  • the strain of the genus Faecalibacterium longum CM04-06 (same as the preservation name) of the present invention has been deposited with the General Microbiology Center (CGMCC) of the China Microbial Culture Collection Management Committee on June 13, 2016, at the Chaoyang District, Beijing, China. No. 3, No. 1 Courtyard, Beichen West Road, deposit number: CGMCC 1.5208.
  • CGMCC General Microbiology Center

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Abstract

提供了长栖粪杆菌(Faecalibacterium longum)及其应用，具体是长栖粪杆菌(Faecalibacterium longum)在治疗和预防糖尿病及其相关疾病中的用途，还提供了一种用于治疗和预防糖尿病及其相关疾病的组合物，包括药品、饮料、食品，或动物饲料组合物等，以及降低体重、空腹血糖和/或血脂、提高糖耐受能力的方法。

Description

长栖粪杆菌(Faecalibacterium longum)及其应用 技术领域
本发明属于微生物学领域，具体地，本发明涉及长栖粪杆菌(Faecalibacterium longum)在治疗和预防糖尿病及其相关疾病中的应用，也涉及包含长栖粪杆菌的组合物及其应用。
背景技术
人体肠道中寄居了大量的共生微生物，这些共生微生物群体组成了人体的“第二器官”，不仅协同宿主参与着营养物质的消化和吸收，而且对人体健康的维持起着重要的作用。从细胞数量来讲，肠道微生物所有细胞的数量是人体细胞数量的数10倍，这些庞大的微生物群体主要有物种组成是：厚壁菌门和拟杆菌门两大类。越来越多的研究表明，肠道微生物的组成和多样性同和多疾病的发生有着密切的关系，比如，肥胖、糖尿病、肠易激综合征、溃疡性肠炎、结肠癌、脂肪肝等。由于环境、饮食、药物等影响，打破了原有健康状态下的肠道微生物的平衡，导致肠道功能紊乱，进而诱发以上几种疾病的发生。
近几年，随着人们日常生活水平的提高，饮食结构逐渐失衡，肥胖、糖尿病等代谢性疾病的发病率迅速升高。据世界卫生组织统计，2014年全球约有4.22亿人患有糖尿病，截止2012年，因为糖尿病引起的死亡数量达到150万人。因此，糖尿病成为当下一个重大的公共健康问题。糖尿病是一种由多种因素引起的血糖异常过高的疾病，主要是由于胰岛素分泌不足或者胰岛素功能缺陷所引起的代谢性障碍，引起糖尿病发生的常见的高危因素有遗传因素，环境因素以及不良的生活习惯。糖尿病的发生会伴随着很多并发症的出现，比如，并发冠心病、高血压、心肌梗死、脑卒中、老年性痴呆、帕金森、肾病、视网膜病等，严重的降低患者额生活质量。其中二型糖尿病是一种非胰岛素依赖糖尿病，其患病人数占所有糖尿病的患者数量的90％。大量研究表明二型糖尿病的发病与肠道菌群的紊乱有着密切的联系，肠道菌群在与宿主之间调节能量平衡和炎症反应有着密切的联系，二型糖尿病的发病主要是胰岛素抵抗和低水平的炎症反应引起的。
目前用于治疗糖尿病的降糖药物有磺酰尿素类，α-葡萄糖甙酶抑制剂和双胍类，其中磺酰尿素类主要是促进胰岛素分泌，但是会引起严重的肝、肾损 伤，且过敏体质不宜服用；α-葡萄糖甙酶抑制剂主要是通过抑制α-淀粉酶和肠内α-葡萄糖甙酶的活性，抑制碳水化合物的水解，从而达到降低餐后血糖的目的，但是容易引发腹胀，腹泻等不良反应；双胍类药物可以通过调节血糖转运达到降糖目的，比如延缓糖吸收、促进葡萄糖的分解、抑制肝脏葡萄糖生成，以及增加葡萄糖转移蛋白量。双胍类药物也会带来一些副作用，容易引起肠胃不适、腹泻、呕吐、皮疹，长期服用容易出现失活。
因此本领域迫切需要开发一种的新的，无毒副作用的，用于治疗和预防糖尿病及其相关疾病的药物。
发明内容
本发明的另一目的是提供长栖粪杆菌在治疗和预防糖尿病及其相关疾病方面的用途。
本发明的另一目的是提供一种有效的无毒副作用的，用于治疗和预防糖尿病及其相关疾病的药品、饮料、食品组合物，或动物饲料组合物。
本发明的另一目的是提供一种降低体重、空腹血糖和/或血脂的方法及其应用。
本发明的另一目的是提供一种提高糖耐受能力的方法及其应用。
本发明第一方面提供了一种长栖粪杆菌，所述长栖粪杆菌为长栖粪杆菌(Faecalibacterium longum)。
在另一优选例中，所述的长栖粪杆菌的16s rDNA的序列如SEQ ID NO.:1所示。
在另一优选例中，所述长栖粪杆菌为Faecalibacterium longum CM04-06，保藏号为CGMCC 1.5208。
本发明第二方面提供了一种组合物，所述组合物包括：(a)安全有效量的权利要求1所述的长栖粪杆菌和/或其代谢产物；以及(b)食品上可接受的载体或药学上可接受的载体。
在另一优选例中，所述组合物还包括牛奶生长因子。
在另一优选例中，所述组合物选自下组：食品组合物、保健组合物、药物组合物、饮料组合物、饲料组合物、或其组合。
在另一优选例中，所述的组合物为口服制剂。
在另一优选例中，所述的组合物为液态制剂、固态制剂、半固态制剂。
在另一优选例中，所述的组合物的剂型选自下组：粉末剂、散剂、片剂、糖衣剂、胶囊剂、颗粒剂、悬浮剂、溶液剂、糖浆剂、滴剂、和舌下含片。
在另一优选例中，所述的食品组合物包括乳液制品、溶液制品、粉末制品、或悬浮液制品。
在另一优选例中，所述的食品组合物包括乳品、乳粉、或乳液。
在另一优选例中，所述的液态制剂选自下组：溶液制品或悬浮液制品。
在另一优选例中，所述组合物含有1×10-1×1010cfu/mL或cfu/g Faecalibacterium longum CM04-06，较佳地1×104-1×1010cfu/mL或cfu/g Faecalibacterium longum CM04-06，按所述组合物的总体积或总重量计。
在另一优选例中，所述的组合物中，含有0.0001－99wt％，较佳地0.1-90wt％(请复核)所述的长栖粪杆菌和/或其代谢产物，以所述组合物的总重量计。
在另一优选例中，所述的组合物为单元剂型(一片、一粒胶囊或一小瓶)，每个单元剂型中所述组合物的质量为0.05-5g，较佳地为0.1-1g。
在另一优选例中，所述的组合物还含有其它益生菌和/或益生元。
在另一优选例中，所述的益生菌选自下组：乳酸菌、双歧杆菌、嗜酸乳杆菌、或其组合。
在另一优选例中，所述的益生元选自下组：低聚果糖(FOS)、低聚半乳糖(GOS)、低聚木糖(XOS)、低聚乳果糖(LACT)、大豆低聚糖(SOS)、菊粉(Inulin)、或其组合。
本发明第三方面提供了一种本发明第一方面所述长栖粪杆菌、或本发明第二方面所述组合物的用途，用于制备药物或制剂，所述药物或制剂用于选自下组的一种或多种用途：(a)预防和/或治疗肥胖；(b)降低血脂；(c)预防或治疗心血管疾病；和/或(d)预防和/或治疗糖尿病。
在另一优选例中，所述制剂包括微生态制剂。
本发明第四方面提供了一种本发明第一方面所述长栖粪杆菌、或本发明第二方面所述组合物的用途，用于制备药物或制剂，所述药物或制剂用于选自下组的一种或多种用途：
(i)抑制哺乳动物的体重增长；
(ii)降低哺乳动物的血脂水平；
(iii)提高哺乳动物中的高密度脂蛋白(HDL-C)的水平；
(iv)降低哺乳动物中的低密度脂蛋白(LDL-C)水平；
(v)降低哺乳动物血糖水平；
(vi)提高哺乳动物糖耐受能力。
在另一优选例中，所述哺乳动物包括人、啮齿动物(如大鼠、小鼠)。
在另一优选例中，所述降低哺乳动物的血脂水平包括降低总胆固醇(TC)水平和/或甘油三酯水平。
在另一优选例中，所述降低哺乳动物血糖水平包括降低空腹血糖水平。
本发明第五方面提供了一种本发明第二方面所述组合物的制法，包括步骤：
将本发明第一方面所述的长栖粪杆菌和/或其代谢产物与食品上可接受的载体或药学上可接受的载体混合，从而形成本发明第二方面所述的组合物。
在另一优选例中，所述制法还包括与生长因子混合的步骤。
在另一优选例中，所述生长因子为牛奶生长因子。
在另一优选例中，所述生长因子选自下组：维生素类物质、嘌呤类物质、嘧啶类物质、或其组合。
在另一优选例中，所述组合物为口服制剂。
本发明第六方面提供了一种生产方法，包括步骤：
(a)在适合培养的条件下，对本发明第一方面所述的长栖粪杆菌进行培养，从而获得培养产物；
(b)任选地，从所述培养产物分离长栖粪杆菌菌体和/或其代谢产物；和
(c)任选地，将上一步骤分离获得的长栖粪杆菌菌体和/或其代谢产物与食品上可接受的载体或药学上可接受的载体混合，从而制得组合物。
在另一优选例中，在步骤(c)之前，还包括将分离获得的长栖粪杆菌菌体和/或其代谢产物与生长因子混合的步骤。
在另一优选例中，所述生长因子为牛奶生长因子。
在另一优选例中，所述生长因子选自下组：维生素类物质、嘌呤类物质、嘧啶类物质、或其组合。
本发明第七方面提供了一种降低体重、空腹血糖和/或血脂的方法，给所述对象施用本发明第一方面所述的长栖粪杆菌和/或其代谢产物、或本发明第二方面所述的组合物。
在另一优选例中，所述的施用包括口服。
在另一优选例中，所述的施用剂量为0.01-5g/50kg体重/天，较佳地， 0.1-2g/50kg体重/天。
在另一优选例中，所述的对象包括哺乳动物，如人。
本发明第八方面提供了一种提高糖耐受能力的方法，给所述对象施用本发明第一方面所述的长栖粪杆菌和/或其代谢产物、或本发明第二方面所述的组合物。
在另一优选例中，所述的施用包括口服。
在另一优选例中，所述的施用剂量为0.01-5g/50kg体重/天，较佳地，0.1-2g/50kg体重/天。
在另一优选例中，所述的对象包括哺乳动物，如人。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了长栖粪杆菌Faecalibacterium longum培养3天的菌落的图片。
图2为本发明的长栖粪杆菌Faecalibacterium longum在显微镜下的革兰氏染色图片(1000倍)。
图3显示了模型组以及长栖粪杆菌CM04-06和二甲双胍治疗的糖尿病小鼠体重的变化。
图4显示了模型组以及长栖粪杆菌CM04-06和二甲双胍治疗糖尿病小鼠空腹血糖(FBG)的结果。
图5显示了模型组以及长栖粪杆菌CM04-06和二甲双胍治疗的糖尿病小鼠葡萄糖耐受的情况。
具体实施方式
本发明人经过广泛而深入的研究和实验，意外地发现，长栖粪杆菌(Faecalibacterium longum)具有预防和治疗糖尿病及其相关疾病(如心血管疾病、肥胖疾病)的作用，将含有长栖粪杆菌的活性组合物饲喂食实验对象，发现该组合物能够抑制体重增加，降低血脂，降低空腹血糖，提高糖耐受能力，有效减轻糖尿病、心血管及肥胖等病症。在此基础上完成本发明。
如本文所用，术语“含有”表示各种成分可一起应用于本发明的混合物或组合物中。因此，术语“主要由...组成”和“由...组成”包含在术语“含有”中。
如本文所用，术语“生长因子”、“牛奶生长因子”可互换使用，均包括维生素类物质、嘌呤类物质、嘧啶类物质、或其组合的营养物质。
其中，所述维生素类物质包括(但并不限于)：维生素C、维生素E、维生素A、维生素A前体、维生素B6、维生素D3、维生素K、叶酸、或其组合；
所述嘌呤类物质包括(但并不限于)：嘌呤核苷，其中所述嘌呤核苷包括嘌呤核苷的5’-磷酸酯，所述嘌呤核苷的5’-磷酸酯选自下组：肌苷酸(肌苷-5’-磷酸酯；IMP)、鸟苷酸(鸟苷-5’-磷酸酯；GMP)、黄苷酸(黄嘌呤核苷-5’-磷酸酯；XMP)、腺苷酸(腺苷-5’-磷酸酯；AMP)、或其组合；
所述嘧啶类物质包括所有含嘧啶结构的物质。
长栖粪杆菌及其应用
如本文所用，术语“长栖粪杆菌”，“Faecalibacterium longum”可互换使用。在一个优选例中，所述菌株为Faecalibacterium longum CM04-06，保藏号为CGMCC 1.5208，分离自人的粪便中。长栖粪杆菌的生理特性如下：长栖粪杆菌在厌氧环境中37℃培养2-3天，菌落为黄白色，含水量较高，有点黏稠，近圆形，不透明，扁平中间凸起，菌落直径约2-3mm；通过革兰氏染色、显微观察，CM04-06菌体为长杆状，革兰氏染色反应为阴性，未发现芽孢和鞭毛，菌体的直径大约为1um，长度为4–10um；并且本发明所述的长栖粪杆菌的氧化酶和过氧化氢酶反应均为阴性，生长温度的范围是30-45℃，pH值范围是4.0-9.0，最适温度和pH值是37℃和pH 7.0。能耐受3％的NaCl。能发酵几种碳水化合物，包括甘露糖，棉籽糖，海藻糖，主要产生甲酸，乙酸，丁酸，乳酸，此外，还可发酵产生胞外多糖。
本发明提供了长栖粪杆菌在治疗和预防糖尿病及其相关疾病(如心血管疾病、肥胖疾病)方面的应用。受试者摄入高脂肪的食物，菌株Faecalibacterium longum CM04-06具有选自下组的一个或多个用途：(i)抑制该受试者体重增加；(ii)降低血脂；(iii)降低空腹血糖水平；(iv)提高糖耐受能力。 根据本发明的一个优选例，采用C57bL/6小鼠作为试验小鼠，用高脂饲料喂养和注射链霉尿素(STZ)进行诱导，从而获得2型糖尿病(T2D)模型小鼠，经菌株Faecalibacterium longum CM04-06来治疗的T2D模型小鼠，与未接受治疗的对照组(模型组)相比，其体重增加幅度减缓且血脂下降，并且各种与糖尿病相关的指标也下降，如空腹血糖水平，此外糖耐受能力也有显著改善。因此，所述菌株能够用以预防和治疗糖尿病及其相关疾病(如心血管疾病、肥胖等)。
组合物及其应用
本发明还提供了一种组合物，优选地，为药物组合物。所述组合物包括有效量的长栖粪杆菌，在一个优选例中，所述组合物还包括牛奶生长因子。在一个优选例中，所述组合物还包括选自下组的益生菌：乳酸菌、双歧杆菌、嗜酸乳杆菌、或其组合；和/或选自下组的益生元：低聚果糖(FOS)、低聚半乳糖(GOS)、低聚木糖(XOS)、低聚乳果糖(LACT)、大豆低聚糖(SOS)、菊粉(Inulin)、或其组合。
在一优选例中，所述的组合物为液态制剂、固态制剂、半固态制剂。
在一优选例中，所述的液态制剂选自下组：溶液制品或悬浮液制品。
在一优选例中，所述的组合物的剂型选自下组：粉末剂、散剂、片剂、糖衣剂、胶囊剂、颗粒剂、悬浮剂、溶液剂、糖浆剂、滴剂、和舌下含片。
本发明药物组合物可以以药物片剂，针剂或胶囊的任一种形式给药，所述药物制剂包括赋形剂、药物允许的媒介和载体，这些物质可根据给药途径进行选择。本发明中药物制剂可进一步包含辅助的活性组份。
乳糖、葡萄糖、蔗糖、山梨糖醇、甘露糖、淀粉、阿拉伯胶、磷酸钙、藻酸盐、明胶、硅酸钙、细结晶纤维素、聚乙烯吡咯烷酮(PVP)、纤维素、水、糖浆、甲基纤维素、羟基苯甲酸甲酯、羟基苯甲酸丙酯、滑石、硬脂酸镁或矿物油等都可用作本发明中药物组合物的载体、赋形剂或稀释剂等。
此外，本发明的药物组合物可进一步包括润滑剂、润湿剂、乳化剂、悬浮液稳定剂、防腐剂、甜味剂和香料等。本发明的药物组合物可通过多种公知的方法以肠衣制剂生产，以便于药物组合物的活性成分即微生物能顺利通过胃而不被胃酸所破坏。
另外，本发明的微生物可以常规方法制备的胶囊形式使用。例如，标准赋形剂和本发明的冷干微生物混合制成小球药丸，然后将药丸装填入明胶胶囊 中。此外，本发明的微生物和药物允许使用的赋形剂如液体胶、纤维素、硅酸盐或矿物油等混合制作悬浮液或分散液，这种悬浮液或分散液可装入软的明胶胶囊中。
本发明的药物组合物可制成肠衣片供口服使用。本申请中的术语—“肠衣”，包括所有常规药物允许使用的包衣，这些包衣不被胃酸降解，但在小肠中能充分分解并快速释放出本发明的微生物。木发明的肠衣能在合成胃酸如pH＝1的HCl溶液中在36-38℃维持2小时以上，并优选在合成肠液如pH＝7.0的缓冲液中在1.0小时内分解。
本发明的肠衣为以每片约16-30mg进行包衣，较佳地16-25mg，更佳地16-20mg进行包衣。本发明中肠衣厚度为5-100μm，理想的厚度为20-80μm。肠衣成分选自己公开知晓的常规聚合物。
本发明优选的肠衣由纤维素乙酸邻苯二甲酸酯聚合物或偏苯三酸酯聚合物以及异丁烯酸的共聚物(例如，含有40％以上异丁烯酸和含有甲基纤维素邻苯二甲酸羟丙酯或其酯类衍生物的异丁烯酸的共聚物)制备。
本发明中肠衣所使用的纤维素乙酸邻苯二甲酸酯的粘度为约45-90cp，乙酰含量17-26％，邻苯二甲酸含量30-40％。用于肠衣中的纤维素乙酸偏苯二酸酯粘度为约5-21cs，乙酞含量17-26％。纤维素乙酸偏苯三酸酯由Eastman科达公司生产，可用于本发明中的肠衣材料。
用于本发明肠衣中的羟丙基甲基纤维素邻苯二甲酸酷，分子量一般为20,000-130,000道尔顿，理想分子量为80,000-100,000道尔顿，羟丙基含量为5-10％，甲氧基含量为18-24％，邻苯二甲酰基含量为21-35％。
用于本发明肠衣中的羟丙基甲基纤维素邻苯二甲酸酷为HP50，由日本Shin-Etsu Chemidnl Co.Ltd.生产。HP50含有6-10％羟丙基，20-24％甲氧基，21-27％的丙基，其分子量为84,000道尔顿。另一种肠衣物质为HP55，HP55含有5-9％的羟丙基甲基纤维素邻苯二甲酸酯，18-22％甲氧基，27-35％的邻苯二甲酸，其分子量为78,000道尔顿。
本发明肠衣如下制备：使用常规方法将肠衣溶液喷雾到核心上。该肠包衣方法中所有溶剂为醇类(如乙醇)、酮类(如丙酮)、卤代烃化合物(如二氯甲烷)、或其组合物。将软化剂如二-正丁基邻苯二甲酸酯和三乙酸甘油酯加入到肠衣溶液中，其比例为1份包衣物对约0.05份或约0.3份软化剂。喷雾方法优选连续执行，所喷雾的料量可根据包衣所采用的条件进行控制。喷雾压力可随 意调节，一般而言，能在平均1-1.5巴压力下获得理想的结果。
说明书中“药物有效量”是指可对人和/或动物产生功能或活性的且可被人和/或动物所接受的量。比如，在本发明中，可制备含有1×10-1×1010cfu/ml或cfu/g(特别的，可含有1×104-1×1010cfu/ml或cfu/g；更特别地，可含有1×106-1×1010cfu/ml或cfu/g)的长栖粪杆菌和/或其代谢产物的制剂。
当用于制备药物组合物时，所用的长栖粪杆菌或其代谢产物的有效剂量可随施用的模式和待治疗的疾病的严重程度而变化。适用于内服的剂量形式，包含与固态或液态药学上可接受的载体密切混合的约1×10-1×1010cfu/ml或cfu/g(特别的，可含有1×104-1×1010cfu/ml或cfu/g；更特别地，可含有1×106-1×1010cfu/ml或cfu/g)的活性长栖粪杆菌或发酵产生的活性成分。可调节此剂量方案以提供最佳治疗应答。例如，由治疗状况的迫切要求，可每天给予若干次分开的剂量，或将剂量按比例地减少。
所述的长栖粪杆菌或其代谢产物可通过口服等途径给予。固态载体包括：淀粉、乳糖、磷酸二钙、微晶纤维素、蔗糖和白陶土，而液态载体包括：培养基、聚乙二醇、非离子型表面活性剂和食用油(如玉米油、花生油和芝麻油)，只要适合长栖粪杆菌或其代谢产物特性和所需的特定给药方式。在制备药物组合物中通常使用的佐剂也可有利地被包括，例如调味剂、色素、防腐剂和抗氧化剂如维生素E、维生素C、BHT和BHA。
从易于制备和给药的立场看，优选的药物组合物是固态组合物，尤其是片剂和固体填充或液体填充的胶囊。口服给药是优选的。
将本发明组合物施用给所述个体，每天给药1次或多次。给药剂量单位表示其形式上能分开且适用于人类或其他所有哺乳动物个体的剂量。每一单位含有药物允许的载体和有效治疗量的木发明微生物。给药量随病人的体重和糖尿病的严重程度、所包括的补充活性组份和所使用的微生物而变化。此外如可能，可分开给药，并且如需要可连续给药。因此，所述给药量不会对本发明造成限制。此外，本发明中的“组合物”不仅意味着药品而且表示可作为功能性食品和健康补充食品。在一个优选例中，所述组合物包括：饮料、食品、药品、动物饲料等。
在本发明的一个优选例中，还提供了一种食品组合物，它含有有效量的长栖粪杆菌和/或其代谢产物，以及余量的食品上可接受的载体，所述的食物组合物的剂型选自固体、乳品、溶液制品、粉末制品、或悬浮液制品。在一优选例中， 所述食品组合物还可含有牛奶生长因子。
在一优选例中，所述组合物的配方如下：
1×10-1×1010cfu/mL的长栖粪杆菌和/或其代谢产物；以及食品上或药学上可接受的载体，和/或赋形剂。
在另一优选例中，所述组合物的配方如下：
1×106-1×1010cfu/mL的长栖粪杆菌和/或其代谢产物；以及食品上或药学上可接受的载体，和/或赋形剂。
微生态制剂
微生态制剂是一种包含益生菌及代谢产物的生物制剂或者是可以增加益生菌的膳食补充剂，可以通过调节、维持肠道内微生态平衡,达到提高人体健康水平的目的。主要包括益生菌、益生元和合生元。
在本发明中，所述微生态制剂包括(a)安全有效量的长栖粪杆菌和/或其代谢产物；以及(b)食品上可接受的载体或药学上可接受的载体。在一优选例中，所述制剂还包括生长因子(如牛奶生长因子，较佳地，包括维生素类物质、嘌呤类物质、和/或嘧啶类物质)。
长栖粪杆菌的生产方法
通常，长栖粪杆菌可以用常规方法制得。
在本发明中，提供了一种能够大规模生产长栖粪杆菌的方法，具体地，包括如下步骤：
(a)在适合培养的条件下，对所述的长栖粪杆菌进行培养，从而获得培养产物；
(b)任选地，从所述培养产物分离长栖粪杆菌菌体和/或其代谢产物；和
(c)任选地，将上一步骤分离获得的长栖粪杆菌菌体和/或其代谢产物与食品上可接受的载体或药学上可接受的载体混合，从而制得组合物。
降低体重、空腹血糖和/或血脂的方法
在另一优选例中，所述方法包括：摄取本发明的药物组合物、食品组合物、饮料组合物、或其组合。所述实验对象为人。
在另一优选例中，所述方法包括：摄取本发明的药物组合物、食品组合物、 或动物饲料，或其组合。所述实验对象为动物，较佳地为鼠类，兔类。
提高糖耐受能力的方法
在另一优选例中，所述方法包括：摄取本发明的药物组合物、食品组合物、饮料组合物、或其组合。所述实验对象为人。
在另一优选例中，所述方法包括：摄取本发明的药物组合物、食品组合物、或动物饲料，或其组合。所述实验对象为动物，较佳地为鼠类，兔类。
菌种保藏
本发明的菌种长栖粪杆菌Faecalibacterium longum CM04-06(与保藏名称相同)已于2016年6月13日保藏在中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，地址中国北京市朝阳区北辰西路1号院3号，保藏号：CGMCC 1.5208。
本发明的主要优点包括：
(a)本发明的长栖粪杆菌能够显著降低体重、降低血脂、空腹血糖水平。
(b)本发明的长栖粪杆菌能够显著降低与肥胖及其相关疾病(如心血管疾病)相关的指标(如胆固醇和甘油三酯)
(c)本发明的长栖粪杆菌能够显著降低总胆固醇、甘油三酯、低密度脂蛋白的水平。
(d)本发明的长栖粪杆菌能够显著提高高密度脂蛋白的水平。
(e)本发明的长栖粪杆菌能够显著改善糖耐受能力。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York：Cold Spring Harbor Laboratory Press，1989)中所述的条件，或按照《微生物：实验手册》(James Cappuccino和Natalie Sherman编，Pearson Education 出版社)中所述的条件，或按照制造厂商所建议的条件。
如无特别说明，实施例所用的材料均为市售产品。
Faecalibacterium prausnitzii ATCC 27768，以下简称ATCC 27768，购 自美国模式培养物保藏所(American Type Culture Collection)，保藏号为ATCC 27768。
实施例1长栖粪杆菌CM04-06的筛选鉴定
1.1 CM04-06的分离培养
本发明所述长栖粪杆菌CM04-06(以下简称CM04-06)分离于深圳市一位健康的儿童(男性)的粪便样品。分离培养的环境是严格厌氧条件，具体的分离过程是：在厌氧操作箱中取0.2g左右的粪便样品，使用1ml无菌的PBS进行悬浮分散，充分震荡混匀后进行梯度稀释涂布，培养基采用厌氧的PYG培养基(购自环凯微生物科技公司)，具体组分见下表：
表1
组分 含量(1L)
蛋白胨 5g
胰化酪蛋白 5g
酵母粉 10g
牛肉膏 5g
葡萄糖 5g
K2HPO4 2g
醋酸钠 5g
Cysteine-HCl·H2O 0.5g
血红素 5mg
无机盐溶液 40ml
刃天青 1mg
蒸馏水 950ml
无机盐溶液的配方是：
无机盐组分 含量(1L)
CaCl2·2H2O 0.25g
MgSO4·7H2O 0.5g
K2HPO4 1g
KH2PO4 1g
NaHCO3 10g
NaCl 2g
涂布平板置于37℃厌氧条件下培养3-4天，待平板表面生长出菌落后，挑取单菌落进行划线分纯，直至获得纯培养菌株，分离到的菌株采用甘油进行-80℃冷冻保藏。
1.2长栖粪杆菌CM04-06的16S rDNA鉴定
(1)基因组提取：对分离到的菌株进行培养，待菌浓达到108cfu/ml数量级的时候取2ml的菌液进行基因组DNA的提取。
(2)16S rDNA的PCR扩增：以DNA作为模板进行16S rDNA扩增，扩增引物选用16S rDNA的通用扩增引物：8F-1492R(5’-AGAGTTTGATCATGGCTCAG-3’(SEQ ID NO.:2)和5’-TAGGGTTACCTTGTTACGACTT-3’(SEQ ID NO.:3))，PCR扩增程序是：
Figure PCTCN2016098246-appb-000001
Figure PCTCN2016098246-appb-000002
(3)纯化、测序：获得PCR产物进行磁珠纯化，然后进行电泳检测，16SrDNA的条带位置大约在1.5k左右，将纯化好的产物进行3730测序。
(4)16S rDNA序列数据库比对：通过测序获得1372bp的16S rDNA序列，将这段序列在EzTaxon-e数据库中进行比对，初步获得菌株的物种分类信息。根据16S rDNA的信息可以初步判定CM04-06是属于Faecalibacterium属的一个新种。
CM04-06的16S rDNA序列为SEQ ID NO.:1，如下所示：
Figure PCTCN2016098246-appb-000003
1.3 CM04-06的16S rDNA进化分析
CM04-06的进化分析使用16S rDNA序列，通过CM04-06的16S rDNA序列进行EzTaxon-e数据库的比对，获得同CM04-06亲缘关系较近的物种，将这些物种和CM04-06的16S rDNA序列进行对序列比对，然后使用Mega5软件绘制邻接法(neighbour-joining)进化树。
1.4 CM04-06的微生物学特征
(1)形态学特征：CM04-06在厌氧环境中37℃培养2-3天，菌落为黄白色，含水量较高，有点黏稠，近圆形，不透明，扁平中间凸起，菌落直径约2-3mm(图1)。
(2)显微学特征：在1000倍显微镜放大下，CM04-06的菌体为长杆状，革兰氏染色反应为阴性，未发现芽孢和鞭毛，菌体的直径大约为1um，长度为4–10um(图2)。
(3)生理生化特征：氧化酶和过氧化氢酶反应均为阴性，生长温度的范围是30-45℃，pH值范围是4.0-9.0，最适温度和pH值是37℃和pH 7.0。能耐受3％的NaCl。CM04-06同亲缘关系最近的参考菌ATCC 27768的生理生化反应(包括底物利用情况API 20A和酶反应API ZYM)的比较如下表：(+，表示阳性反应；-，表示阴性反应；w表示弱阳性反应)。
表2
Figure PCTCN2016098246-appb-000004
Figure PCTCN2016098246-appb-000005
通过以上CM04-06同参考菌的生理生化反应比较显示，CM04-06在棉籽糖和海藻糖的利用，七叶灵和明胶水解以及酯酶(C4)、缬氨酸芳胺酶、胱氨酸芳胺酶、胰凝乳蛋白酶、萘酚-AS-BI-磷酸水解酶、β-糖醛酸甙酶、α-葡萄糖 甙酶和N-乙酰-葡萄糖胺酶的活性有显著区别，可以证明CM04-06是一株不同于已知菌的新种。
1.5 细胞脂肪酸的分析
将培养至稳定期的CM04-06和Faecalibacterium prausnitzii ATCC 27768(以下简称ATCC 27768，购自美国模式培养物保藏所(American Type Culture Collection)，保藏号为ATCC 27768)的菌进行菌体收集，然后进行细胞脂肪酸的提取，检测。通过气相色谱分析两株菌细胞脂肪酸组分的含量及差异。
表3
Figure PCTCN2016098246-appb-000006
Figure PCTCN2016098246-appb-000007
综合以上CM04-06表型、16S rDNA序列以及生理生化反应显示，CM04-06是一株新种，将其命名为长栖粪杆菌Faecalibacterium longum。
实施例2长栖粪杆菌Faecalibacterium longum CM04-06的生物活性物质
2.1 短链脂肪酸(SCFA)的检测
(1)样品制备：取培养48h的CM04-06菌液1ml，12000r/min离心5min，吸取上清液，备用。
(2)SCFA的测定：短链脂肪酸的测定采用外标法，选用乙酸、丙酸、丁酸、戊酸进行标准曲线的制作。采用安捷伦气象色谱仪(GC-7890B,Agilent)，选用HP-INNOWax(Cross-Linked PEG)，30m×0.25mm×0.25um的毛细柱进行分析，检测器为氢火焰离子检测器，GC参数设置为柱温：180～200℃；气化室温度：240℃；检测温度：210℃；进样量：2μL；载气流量：N2，50mL/min；氢气流量：50mL/min；空气流量：600～700mL/min。
(3)结果：测定得出SCFA产量为：甲酸(7.62mmol/L)，乙酸(44.8mmol/L)，丁酸(40.03mmol/L)。
2.2 有机酸的检测
(1)样品制备：同2.1部分。
(2)有机酸的测定：有机酸的检测标准品选用：3-甲基丁酸，戊酸，奎宁酸，乳酸，草酸，丙二酸，苯甲酸，马来酸，丁二酸，反富马酸，苹果酸，己二酸，酒石酸，莽草酸，柠檬酸，异柠檬酸和L-抗坏血酸。仍然采用安捷伦气 象色谱仪(GC-7890B,Agilent)，色谱柱选用122-5532G DB-5ms(40m×0.25mm×0.25um)，柱温：270～290℃；进样口温度：250℃；气体流量:0.86mL/min。
(3)结果：测定得出有机酸产量详见下表(表4)。
表4
有机酸 3-甲基丁酸 戊酸 奎宁酸 乳酸 草酸 丙二酸
含量(mmol/L) 0.26 0.49 0 30.53 0 0
有机酸 苯甲酸 马来酸 丁二酸 反富马酸 苹果酸 己二酸
含量(mmol/L) 1.79 0 0.88 0 0 0.60
有机酸 酒石酸 莽草酸 柠檬酸 异柠檬酸 L-抗坏血酸  
含量(mmol/L) 0 0 0. 0 0.11  
实施例3、长栖粪杆菌Faecalibacterium longum CM04-06产胞外多糖的检测
胞外多糖的检测采用硫酸苯酚法，硫酸苯酚可以与游离的单糖、寡糖和多糖中的己糖等发生显色反应，产生的颜色同吸光度成正比，其吸收波长为490nm。具体实验过程如下：
(1)多糖的提取
实验菌株在PYG培养基(配方同实施例1)中培养2天，取10ml菌液沸水浴处理30min，然后10000r/min离心，取上清，加入80％三氯乙酸至终浓度为8％，4℃过夜处理，沉淀蛋白。取出10000r/min离心30min，用NaOH调节上清液的pH至6.0。加入2倍体积的无水乙醇进行多糖沉淀，4℃过夜处理，取出进行10000r/min离心30min，弃掉上清，沉淀用预热的蒸馏水进行溶解，然后转移至超滤管(3000Da滤径)中进行超滤，5000r/min离心30min，内管中截留的多糖转移至容量瓶中蒸馏水定容至10ml，备用。
(2)葡萄糖标准曲线的制作
精密称取标准葡萄糖20mg至100ml容量瓶中，加水至刻度线，然后分别配置20、40、60、80、100μg/ml的葡萄糖标准液。每组标准液取400ul，三个平行，依次加入400ul 5％的苯酚和1ml浓硫酸进行反应，沸水浴15min后冷却至室温，测量在490nm的吸光度。然后以吸光度作为纵坐标，葡萄糖标准液浓度为横坐 标绘制标准曲线。
(3)检测提取的多糖的浓度
领取多糖溶液400ul，依次加入400ul 5％的苯酚和1ml浓硫酸进行反应，沸水浴15min后冷却至室温，测量在490nm的吸光度。根据葡萄糖标准曲线计算多糖的浓度。
(4)结果
通过计算，培养2天的CM04-06发酵液中胞外多糖的含量为233mg/L。
实施例4 长栖粪杆菌Faecalibacterium longum CM04-06在糖尿病动物模型的体内试验
本实施例利用高脂饲料喂养和注射链霉尿素(STZ)诱导造成2型糖尿病(T2D)模型小鼠，通过灌胃长栖粪杆菌CM04-06治疗2个月，考察CM04-06对2型糖尿病(T2D)的治疗效果。
4.1 T2D小鼠的造模：试验小鼠采用C57bL/6小鼠(购自湖北医学实验动物中心)，8周龄，小鼠试验的环境为SPF级，适应性喂养1周进行分组造模。造模方法采用高脂加STZ诱导，空腹血糖值(FBG)达到10mM/L以上可作为T2D模型小鼠。
4.2 实验分组：
(1)模型(Model)组：T2D模型的小鼠采用生理盐水进行灌胃；
(2)CM04-06治疗组：T2D模型的小鼠采用CM04-06菌液进行灌胃治疗；
(3)阳性药物组：T2D模型的小鼠，采用阳性药物二甲双胍进行干预治疗。
4.3 菌液准备：CM04-06培养至稳定期，至菌浓约108cfu/ml数量级。
4.4 试验过程：按照分组情况进行实验，T2D成模后(FBG>10)开始进行治疗，治疗持续2个月。每日记录小鼠的进食活动情况，并称量体重，每周进行尾静脉采血，检测小鼠的空腹血糖值，在治疗第四周和实验结束时候测量小鼠的葡萄糖耐量(OGTT)。试验2个月后处死小鼠，所有小鼠摘眼球取血、脱颈、检测血清中总胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白胆固醇(HDL-C)和低密度脂蛋白胆固醇(LDL-C)。
4.5 实验结果及分析
(1)体重变化：如表5和图3所示，随着试验的进行小鼠的体重逐渐升高，CM04-06和二甲双胍治疗组的小鼠体重增加的幅度比模型组低，由此可以说明，CM04-06可以有效的减缓小鼠体重的增加(*P<0.05/**P<0.01)。同时通过统计，CM04-06在控制小鼠的体重增加要比二甲双胍更加有效。
表5
分组编号 1周 3周 5周 7周 9周
Model组(g) 22.48 24.99 28.42 31.81 33.45
CM04-06组(g) 21.56 22.76 24.59 25.41 25.65**
二甲双胍组(g) 21.54 23.42 25.23 26.6 27.49*
(2)CM04-06对小鼠空腹血糖的的实验结果
表6
分组编号 第一周 第三周 第五周 第七周 第九周
Model组(mmol/L) 15.5 16.8 17.2 16.9 15.2
CM04-06组(mmol/L) 16.2 15.2 13.4* 10.8** 8.2**
二甲双胍组(mmol/L) 16.7 15.8 14.6 12.1* 9.8**
根据表6和图4的结果显示随着治疗的进行，CM04-06和二甲双胍组的小鼠空腹血糖值逐渐下降，小鼠的血糖值趋向于正常，降糖的效果比较显著，说明CM04-06可以有效的降低血糖(同模型组比较*P值<0.05/**P<0.01)。同时CM04-06组小鼠的血糖值比二甲双胍组的血糖值更低，可见CM04-06降空腹血糖的能力优于二甲双胍。
(3)CM04-06对小鼠糖耐受的实验结果
通过对处死前小鼠的葡萄糖耐受(OGTT)情况测试(表7和图5)，结果显示在灌胃葡萄糖之后，30min小鼠的血糖值达到最高(18.2-22.5mmol/L)，然后二甲双胍组和CM04-06血糖匀速的下降，120min时将至9.8mmol/L和10.1mmol/L，而模型组小鼠的血糖为15.2，在这个时间点，CM04-06血糖值与模型组有显著的差异(*P值<0.05)，综合葡萄糖耐受的整个过程来看，相比之下，CM04-06可以有效的改善糖尿病小鼠的葡萄糖耐受不良的情况。在整个调节葡萄糖的过程中，CM04-06在各个时间点的血糖值比二甲双胍的低，由此可见 CM04-06对糖尿病小鼠葡萄糖耐受能力的改善效果更佳。
表7
分组编号 0min 30min 60min 90min 120min
Model组(mmol/L) 15.8 22.5 17.2 15.8 15.2
CM04-06组(mmol/L) 9 18.2 15 10.8* 9.8*
二甲双胍组(mmol/L) 9.2 19.5 16.1 10.9* 10.1*
(4)CM04-06对小鼠血脂的影响
通过实验结束后对小鼠的血脂进行检测，包括总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白(LDLC)和高密度脂蛋白(HDLC)，检测结果如表8所示。结果显示二甲双胍组和CM04-06组的低密度脂蛋白低于模型组(*P值<0.05)，同时，高密度脂蛋白高于模型组(*P值<0.05)，高密度脂蛋白主要功能是清除血液和细胞中过多的胆固醇和低密度脂蛋白，具有抗动脉粥样硬化的作用，由此可见CM04-06可以改善糖尿病小鼠的高血脂症状。同时相比于二甲双胍组，CM04-06对血脂的改善效果更好，包括低密度脂蛋白的含量更低，高密度脂蛋白含量更高。
表8
Figure PCTCN2016098246-appb-000008
实施例5 含长栖粪杆菌Faecalibacterium longum CM04-06的食品组合物
原料配比如表9。
表9
原料 质量百分比(％)
Faecalibacterium longum CM04-06 0.5
牛奶 90.0
白糖 9.0
维生素C 0.5
按照上述配方比例混合牛奶、白糖，搅拌至完全混合，预热，20Mpa压力均质，90℃左右杀菌5-10分钟，冷却至40-43℃，混入维生素C，接种1-100×106cfu/g的Faecalibacterium longum CM04-06菌，即制成含Faecalibacterium longum CM04-06菌的食品组合物。
实施例6 含长栖粪杆菌Faecalibacterium longum CM04-06的药物组合物
原料配比见表10。
表10
Figure PCTCN2016098246-appb-000009
按照比例将乳糖、酵母粉、蛋白胨以纯净水混合均匀，预热到60-65℃，20Mpa压力均质，90℃左右杀菌20-30分钟，冷却至36-38℃，混入维生素C，接入Faecalibacterium longum CM04-06活菌(1-50×106cfu/mL)，36-38℃发酵至pH值为6.0，离心，冷冻干燥至水份含量小于3％，即制备Faecalibacterium longumCM04-06菌冷冻干燥物。称取0.5克Faecalibacterium longum CM04-06冷冻干燥物与麦芽糊精等量混合后装入胶囊中，即制成含Faecalibacterium longumCM04-06菌的药物组合物。
实施例7 含长栖粪杆菌Faecalibacterium longum CM04-06的药物的制备
7.1 菌液准备：将Faecalibacterium longum CM04-06(1×109cfu/ml)进行厌 氧培养，厌氧培养基采用PYG培养基，经过37℃厌氧发酵2-3天。
7.2 生长因子制备：将脱脂牛奶、酪蛋白进行混合、离心、超滤获得牛奶生长因子粗提物(含有维生素类物质、嘌呤类物质、和/或嘧啶类物质的营养物质)。
7.3 药物剂型制作：将5体积上述生长因子加入到100体积的CM04-06发酵的菌液中，充分搅拌混匀，然后加入淀粉辅料(如麦芽糊精)，从而制备得到含有长栖粪杆菌Faecalibacterium longum CM04-06的药物。
菌种保藏
本发明的菌种长栖粪杆菌Faecalibacterium longum CM04-06(与保藏名称相同)已于2016年6月13日保藏在中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，地址中国北京市朝阳区北辰西路1号院3号，保藏号：CGMCC 1.5208。
本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种长栖粪杆菌，其特征在于，所述长栖粪杆菌为长栖粪杆菌(Faecalibacterium longum)。
  2. 如权利要求1所述的长栖粪杆菌，其特征在于，所述长栖粪杆菌为Faecalibacterium longum CM04-06，保藏号为CGMCC 1.5208。
  3. 一种组合物，其特征在于，所述组合物包括：(a)安全有效量的权利要求1所述的长栖粪杆菌和/或其代谢产物；以及(b)食品上可接受的载体或药学上可接受的载体。
  4. 如权利要求3所述的组合物，其特征在于，所述组合物还包括牛奶生长因子。
  5. 如权利要求3所述的组合物，其特征在于，所述组合物含有1×10-1×1010cfu/mL或cfu/g Faecalibacterium longum CM04-06，较佳地1×104-1×1010cfu/mL或cfu/g Faecalibacterium longum CM04-06，按所述组合物的总体积或总重量计。
  6. 一种权利要求1所述长栖粪杆菌、或权利要求3所述组合物的用途，其特征在于，用于制备药物或制剂，所述药物或制剂用于选自下组的一种或多种用途：(a)预防和/或治疗肥胖；(b)降低血脂；(c)预防或治疗心血管疾病；和/或(d)预防和/或治疗糖尿病。
  7. 一种权利要求1所述长栖粪杆菌、或权利要求3所述组合物的用途，其特征在于，用于制备药物或制剂，所述药物或制剂用于选自下组的一种或多种用途：
    (i)抑制哺乳动物的体重增长；
    (ii)降低哺乳动物的血脂水平；
    (iii)提高哺乳动物中的高密度脂蛋白(HDL-C)的水平；
    (iv)降低哺乳动物中的低密度脂蛋白(LDL-C)水平；
    (v)降低哺乳动物血糖水平；
    (vi)提高哺乳动物糖耐受能力。
  8. 如权利要求7所述的用途，其特征在于，所述降低哺乳动物的血脂水平包括降低总胆固醇(TC)水平和/或甘油三酯水平。
  9. 一种权利要3所述组合物的制法，其特征在于，包括步骤：
    将权利要求1所述的长栖粪杆菌和/或其代谢产物与食品上可接受的载体或药学上可接受的载体混合，从而形成权利要求3所述的组合物。
  10. 一种生产方法，其特征在于，包括步骤：
    (a)在适合培养的条件下，对权利要求1所述的长栖粪杆菌进行培养，从而获得培养产物；
    (b)任选地，从所述培养产物分离长栖粪杆菌菌体和/或其代谢产物；和
    (c)任选地，将上一步骤分离获得的长栖粪杆菌菌体和/或其代谢产物与食品上可接受的载体或药学上可接受的载体混合，从而制得组合物。
PCT/CN2016/098246 2016-09-06 2016-09-06 长栖粪杆菌(Faecalibacterium longum)及其应用 WO2018045492A1 (zh)

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JP2018546802A JP6862464B2 (ja) 2016-09-06 2016-09-06 フィーカリバクテリウム・ロンガム（Ｆａｅｃａｌｉｂａｃｔｅｒｉｕｍ ｌｏｎｇｕｍ）およびその使用
PCT/CN2016/098246 WO2018045492A1 (zh) 2016-09-06 2016-09-06 长栖粪杆菌(Faecalibacterium longum)及其应用
EP16915428.3A EP3511406A4 (en) 2016-09-06 2016-09-06 FAECALIBACTERIUM LONGUM AND ASSOCIATED APPLICATION
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