WO2018107365A1 - 粪厌氧棒杆菌(Anaerostipes caccae)及其应用 - Google Patents

粪厌氧棒杆菌(Anaerostipes caccae)及其应用 Download PDF

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WO2018107365A1
WO2018107365A1 PCT/CN2016/109690 CN2016109690W WO2018107365A1 WO 2018107365 A1 WO2018107365 A1 WO 2018107365A1 CN 2016109690 W CN2016109690 W CN 2016109690W WO 2018107365 A1 WO2018107365 A1 WO 2018107365A1
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composition
acid
anaerobic
acceptable carrier
group
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PCT/CN2016/109690
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English (en)
French (fr)
Inventor
邹远强
薛文斌
肖亮
李晓平
余靖宏
刘传
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深圳华大基因研究院
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Priority to CN201680091523.9A priority Critical patent/CN110062806B/zh
Priority to PCT/CN2016/109690 priority patent/WO2018107365A1/zh
Publication of WO2018107365A1 publication Critical patent/WO2018107365A1/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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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

Definitions

  • the present invention is in the field of microbiology, and in particular, the present invention relates to the use of Anaerostipes caccae for the treatment and prevention of inflammation-related diseases, and to a composition comprising Corynebacterium faecalis and uses thereof.
  • Ulcerative enteritis is a type of inflammatory bowel disease (IBD). It is divided into colitis and proctitis according to the site of inflammation. The pathogenesis of this disease is not clear. It is mainly considered to be genetic susceptibility, intestinal flora, and intestine. Caused by the immune response of the mucosa. A large number of studies at home and abroad have shown that the incidence of UC is closely related to the imbalance of intestinal flora. The clinicopathological manifestations of UC are abdominal pain, diarrhea, and mucous bloody stools, and recurrent attacks.
  • UC disease mainly includes salicylic acid, glucocorticoids and immunological preparations.
  • Salicylic acid drugs can better inhibit prostaglandin synthesis and scavenge oxygen free radicals to achieve the purpose of relieving inflammatory reaction.
  • the main salicylic acid western medicine used in the treatment of UC is sulfasalazine (SASP), mainly for light Patients with moderate, moderate, and chronic UC; glucocorticoids are the first choice for patients with severe or explosive UC, such as betamethasone; immunosuppressive agents such as cyclosporine can inhibit immune response by inhibiting IL-2 production by T cells. Progress, thereby inhibiting UC.
  • All of the above three drugs can alleviate UC to a certain extent, but there are certain side effects.
  • the side effects of salicylic acid are gastrointestinal reactions, headache, reticulocyte increase, sperm reduction and rash caused by allergic reactions. Hepatotoxicity, leukopenia, anemia, etc.
  • Glucocorticoids can cause side effects such as metabolic disorders and water retention, and can only be used as an emergency medication and cannot be taken for a long time.
  • Immunosuppressive therapy is highly dependent on drugs, has a long treatment period, and is prone to cause nephrotoxicity and secondary infection. It can only be used as a means of adjuvant therapy.
  • Another object of the present invention is to provide a use of Corynebacterium faecalis in the treatment and/or prevention of inflammation-related diseases.
  • Another object of the present invention is to provide a pharmaceutical, beverage, food composition, or animal feed composition for the treatment and/or prevention of inflammation-related diseases which is effective and has no toxic side effects.
  • Another object of the present invention is to provide a method for improving intestinal lesions in mammals and uses thereof.
  • Another object of the present invention is to provide a method of slowing down the body weight of a mammal, and/or reducing a disease activity index (DAI) of a mammal, and uses thereof.
  • DAI disease activity index
  • a bacterium of Corynebacterium faecalis which is Anaerostipes caccae, and has a deposit number of GDMCC No.: 60088.
  • sequence of the 16s rDNA of the C. faecalis is shown in SEQ ID NO.: 1.
  • the Corynebacterium faecalis is Anaerostipes caccae AF04-45.
  • the anaerobic bacterium is from the intestines, animal feces, fermentation tanks, and/or anaerobic reactors.
  • a second aspect of the present invention provides a composition
  • a composition comprising: (a) a safe and effective amount of the Corynebacterium faecalis and/or a metabolite thereof according to the first aspect of the present invention; and (b) a food product An acceptable carrier or pharmaceutically acceptable carrier.
  • the composition further comprises a growth factor (preferably, a milk growth factor).
  • a growth factor preferably, 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, sublingual Tablets, or a combination thereof.
  • 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 15 cfu/mL or cfu/g Anaerostipes caccae AF04-45, preferably 1 x 10 4 - 1 x 10 10 cfu/mL or cfu /g Anaerostipes caccae AF04-45, 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 of the anaerobic bacteria of the genus E. faecalis and/or its metabolites, based on the total weight of the composition. meter.
  • 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.
  • the composition further contains a substance (e.g., a protective agent) that helps maintain the activity of the anaerobic bacterium.
  • a substance e.g., a protective agent
  • the substance (such as a protective agent) which contributes to maintaining the activity of Corynebacterium faecalis is selected from the group consisting of cysteine, glutathione, butylhydroxyanisole, dibutylmethyl Toluene, tocopherol, bamboo leaf antioxidant, D-isoascorbic acid and its sodium salt, sodium ascorbate, calcium ascorbate, phospholipid, vitamin C (ascorbic acid), vitamin E, or a combination thereof.
  • the weight ratio of the substance (e.g., protective agent) which contributes to maintaining the activity of the anaerobic bacteria of the genus Escherichia coli is 0.1 to 2%, preferably 0.5 to 1.5%, more preferably 0.5 to 1% based on the total weight of the composition.
  • the content of the substance (such as a protective agent) which contributes to maintaining the activity of the anaerobic bacteria of the genus Escherichia coli is 1 mg to 20 mg, preferably 5 mg to 15 mg, more preferably 1 g of the composition. , 5mg-10mg.
  • a third aspect of the invention provides a anaerobic bacterium of the first aspect of the invention, or the invention Use of the composition of the second aspect for the preparation of a medicament or formulation for the prevention and/or treatment of an inflammation-related disease.
  • the inflammation-related disease is selected from the group consisting of inflammatory bowel disease, rheumatoid arthritis, or a combination thereof.
  • the inflammation-related disease is selected from the group consisting of ulcerative enteritis, gastritis, common enteritis, or a combination thereof.
  • the formulation comprises a microecological formulation.
  • a fourth aspect of the invention provides the use of the composition of the second aspect of the invention, or the composition of the second aspect of the invention, for the preparation of a medicament or a preparation for the selection
  • the mammal comprises a human, a rodent (e.g., a rat, a mouse).
  • a rodent e.g., a rat, a mouse
  • the slowing down of the body weight of the mammal means that the body weight of the experimental group is reduced by no more than 15%, preferably no more than 10%, more preferably than the mammal of the model group. , no more than 5%, significantly lower than the reduction in body weight of the model group.
  • the slowing down the pathology of the mammalian gut includes slowing the shortening of the length of the colon, and/or reducing the inflammatory response of the colon.
  • the anaerobic bacterium of the first aspect of the invention and/or its metabolite is mixed with a food acceptable carrier or a pharmaceutically acceptable carrier to form the composition of the second aspect of the invention.
  • the method further comprises the step of mixing with a growth factor.
  • the method further comprises the step of mixing with a substance (e.g., a protective agent) that helps maintain the activity of the anaerobic bacterium.
  • a substance e.g., a protective agent
  • the substance (such as a protective agent) which contributes to maintaining the activity of Corynebacterium faecalis is selected from the group consisting of cysteine, glutathione, butylhydroxyanisole, dibutylmethyl Toluene, tocopherol, bamboo leaf antioxidant, D-isoascorbic acid and its sodium salt, sodium ascorbate, calcium ascorbate, phospholipid, vitamin C (ascorbic acid), vitamin E, or a combination thereof.
  • the method further comprises the step of mixing with a probiotic, and/or a prebiotic.
  • 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.
  • 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:
  • the culture product obtained in the previous step or the anaerobic bacillus bacillus and/or its metabolite is mixed with a food-acceptable carrier or a pharmaceutically acceptable carrier, thereby producing the present invention Said composition.
  • the step of mixing the culture product obtained in the previous step or the C. anaerobic bacillus cell and/or its metabolite with a 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.
  • the culture product obtained by the previous step or the anaerobic bacteria of the genus Corynebacterium and/or its metabolites are further included to help maintain fecal anaerobic conditions.
  • step (c) further comprising the culture product obtained in the previous step or the C. anaerobic bacillus cell and/or its metabolites and probiotics and/or prebiotics The step of mixing.
  • a seventh aspect of the invention provides a method of improving intestinal lesions in a mammal, the composition of the second aspect of the invention being administered to the subject.
  • 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.
  • An eighth aspect of the invention provides a method of slowing down the body weight of a mammal, and/or reducing a disease activity index (DAI) of a mammal, the composition of the second aspect of the invention being administered to the subject.
  • DAI disease activity index
  • 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 48 hours by Anaerostipes caccae AF04-45.
  • Figure 2 shows a Gram-stained picture (1000 times) of the microorganism E. anaerobicis caccae AF04-45 under a microscope.
  • Figure 3 shows the changes in body weight of Control group, model group, VSL # 3 and AF04-45 treatment groups.
  • Figure 4 shows the changes in the DAI index of the Control, Model, VSL # 3 and AF04-45 treated mice.
  • Figure 5 shows the length of the colon in the Control, Model, VSL # 3 and AF04-45 treatment groups.
  • Anaerostipes caccae has prevention and/or treatment of inflammation-related diseases (such as inflammatory bowel disease (such as ulcerative enteritis, gastritis, common enteritis). And rheumatoid arthritis), the active composition containing Corynebacterium faecalis was fed to the test subject, and the composition was found to slow down the weight loss, reduce the disease activity index (DAI), and improve intestinal lesions. Effectively reduce inflammation-related diseases (such as inflammatory bowel disease (such as ulcerative enteritis, gastritis, common enteritis), rheumatoid arthritis) and other diseases.
  • inflammation-related diseases such as inflammatory bowel disease (such as ulcerative enteritis, gastritis, common enteritis).
  • 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 includes milk growth factors, in particular, nutrients including 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; 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.
  • controlling the weight loss of a mammal and “reducing the weight loss of a mammal” are used interchangeably to mean that the mammal's body weight is also severely affected by the inflammation during the construction of the ulcerative enteritis model.
  • the decrease, the percentage of weight loss is the percentage of weight loss to the original body weight.
  • DAI Disease Activity Index
  • disease activity index refers to a combined score of the combined weight loss, stool viscosity, and stool bleeding in a patient (sick animal).
  • the terms "A. anaerobicus”, “Anaerostipes caccae”, “C. anaerobics of the invention” are used interchangeably.
  • the strain is Anaerostipes Caccae AF04-45, deposited under the symbol GDMCC No.: 60088, is isolated from the feces of a human (preferably, healthy female), in particular, from the intestine of an adult female.
  • the physiological characteristics of Corynebacterium faecalis are as follows: Anaerostipes caccae AF04-45 is anaerobic cultured at 37 °C for 2 days.
  • the colonies are pale yellow, convex, viscous, opaque, nearly round, and the edges are neat.
  • the diameter is about 2-3 mm; the microscopically amplified 1000 times of Anaerostipes caccae AF04-45 is rod-shaped, Gram-negative, and does not produce spores and flagella. Anaerostipes caccae AF04-45 is negative for catalase, negative for oxidase, and strictly anaerobic, with a survival rate of 100% in 0.3% of bile salts for 4 hours.
  • the present invention provides the use of Corynebacterium faecalis for treating and/or preventing inflammation-related diseases such as inflammatory bowel diseases such as ulcerative enteritis, gastritis, common enteritis, and rheumatoid arthritis.
  • inflammation-related diseases such as inflammatory bowel diseases such as ulcerative enteritis, gastritis, common enteritis, and rheumatoid arthritis.
  • the subject was induced to model with DSS (sodium dextran sulfate), and the strain Anaerostipes caccae AF04-45 had one or more uses selected from the group consisting of: (i) slowing down the subject's weight loss; (ii) Improve the degree of intestinal lesions; (iii) reduce the disease activity index (DAI).
  • DAI disease activity index
  • C57bl/6 mice are used as test mice, and induction modeling is performed using DSS (sodium dextran sulfate) to obtain a mouse model of ulcerative enteritis (UC) by Anaerostipes caccae AF04-
  • UC ulcerative enteritis
  • the weight loss was slowed compared with the untreated control group (model group), and various indicators related to inflammation-related diseases such as inflammatory bowel disease were also improved.
  • various indicators related to inflammation-related diseases such as inflammatory bowel disease were also improved.
  • the strain can be used for the prevention and/or treatment of inflammation-related diseases (such as inflammatory bowel disease (such as ulcerative enteritis, gastritis, common enteritis), rheumatoid arthritis).
  • the present invention also provides a composition, preferably the composition comprises a food composition, a health care composition, a pharmaceutical composition, a beverage composition, or a feed composition, preferably a pharmaceutical composition.
  • the composition comprises an effective amount of Corynebacterium faecalis, and in a preferred embodiment, the composition further comprises a growth factor (such as 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, oligosaccharides, or combinations thereof.
  • a probiotic selected from the group consisting of lactic acid bacteria, bifidobacteria, Lactobacillus acidophilus, or a combination thereof
  • a prebiotic selected from the group consisting of oligofructose ( FOS), galactooligosaccharide (GOS), xylooligosaccharide (XOS), oligofructose (LACT), soy oligosacc
  • the composition further comprises a substance (e.g., a protective agent) selected from the group consisting of cysteine, glutathione, butylated hydroxyanisole. Dibutylmethyltoluene, tocopherol, bamboo leaf antioxidant, D-isoascorbic acid and its sodium salt, sodium ascorbate, calcium ascorbate, phospholipid, vitamin C (ascorbic acid), vitamin E, or a combination thereof.
  • the weight ratio of the substance (e.g., protective agent) which contributes to maintaining the activity of the anaerobic bacteria of the genus Escherichia coli is 0.1 - 2%, preferably 0.5 - 1.5%, more preferably 0.5, based on the total weight of the composition. -1%.
  • 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, sublingual Slice, or group thereof Hehe.
  • composition of the present invention may be administered in the form of an oral liquid, a tablet, an injection, an orally disintegrating tablet, a lyophilized powder preparation or a capsule, preferably an enteric solvent type (e.g., a capsule), in the present invention, if there is no special
  • excipients, pharmaceutically acceptable vehicles and carriers used in the present invention are mainly selected according to the characteristics of the suitable bacteria or their metabolites and the specific administration mode required, and are advantageous for the bacteria or its metabolites to be smoothly passed through the stomach.
  • the drug is absorbed. These substances can be selected depending on the route of administration.
  • compositions of the present invention may further comprise any additional excipients in the excipients typically used in pharmaceutical formulations, for example, to stabilize the composition itself, or to readily disperse or impart a suitable taste.
  • inulin fructose, starch, xylooligosaccharide, silica, buffering agents, and flavoring agents are suitable examples.
  • the pharmaceutical preparation of the present invention may further comprise an auxiliary active ingredient.
  • 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 as used in this application includes coatings which are permitted for use with all 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 trimellitate 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.
  • Hydroxypropyl methylcellulose phthalate used in the casing of the present invention generally having a molecular weight of 20,000 to 130,000 Daltons, an ideal molecular weight of 80,000 to 100,000 Daltons, and a hydroxypropyl group content It is 5-10%, the methoxy group content is 18-24%, and the phthaloyl group content is 21-35%.
  • HP50 The hydroxypropylmethylcellulose phthalate used in the casing of the present invention is HP50, which is produced by Shin-Etsu Chemidnl Co. Ltd., Japan. 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.
  • the spray pressure can be adjusted at will, and in general, the desired result can be obtained 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 15 cfu / ml or cfu / g (particularly, may contain 1 ⁇ 10 4 - 1 ⁇ 10 10 cfu / ml or cfu / g; more special
  • the formulation may contain 1 x 10 6 - 1 x 10 10 cfu/ml or cfu/g) of Corynebacterium faecalis and/or its metabolites.
  • the effective dosage of the anaerobic bacillus or its metabolite used may vary depending on the mode of administration and the severity of the condition to be treated.
  • a dosage form suitable for internal administration comprising about 1 x 10-1 x 10 15 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 anaerobic bacteria or fermentation-produced active ingredients.
  • 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 anaerobic bacillus or its metabolite can be administered by oral 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 anaerobic bacillus 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 a microorganism of the invention.
  • the amount of administration varies with the patient's body weight and inflammation-related diseases (such as inflammatory bowel disease (such as ulcerative enteritis, gastritis, common enteritis), rheumatoid arthritis), cardiovascular disease (such as hypertension, hyperlipidemia, coronary heart disease)
  • inflammation-related diseases such as inflammatory bowel disease (such as ulcerative enteritis, gastritis, common enteritis), rheumatoid arthritis), cardiovascular disease (such as hypertension, hyperlipidemia, coronary heart disease)
  • inflammation-related diseases such as inflammatory bowel disease (such as ulcerative enteritis, gastritis, common enteritis), rheumatoid
  • 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 Corynebacterium faecalis and/or a metabolite thereof, and a balance of a food acceptable carrier, said food Group
  • the dosage form of the composition is selected from the group consisting of a solid, a dairy, a solution, a powder, or a suspension.
  • the food composition may further comprise a growth factor (such as 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, oligosaccharides, or combinations thereof.
  • a probiotic selected from the group consisting of lactic acid bacteria, bifidobacteria, Lactobacillus acidophilus, or a combination thereof
  • a prebiotic selected from the group consisting of oligofructose ( FOS), galactooligosaccharide (GOS), xylooligosaccharide (XOS), oligofructose (LACT), soy oligosacc
  • the composition further comprises a substance (e.g., a protective agent) selected from the group consisting of cysteine, glutathione, butylated hydroxyanisole. Dibutylmethyltoluene, tocopherol, bamboo leaf antioxidant, D-isoascorbic acid and its sodium salt, sodium ascorbate, calcium ascorbate, phospholipid, vitamin C (ascorbic acid), vitamin E, or a combination thereof.
  • a substance e.g., a protective agent
  • cysteine e.g., cysteine, glutathione, butylated hydroxyanisole.
  • Dibutylmethyltoluene, tocopherol, bamboo leaf antioxidant, D-isoascorbic acid and its sodium salt sodium ascorbate, calcium ascorbate, phospholipid, vitamin C (ascorbic acid), vitamin E, or a combination thereof.
  • a protective agent selected from the group consisting of cysteine, glutathione, butylated hydroxyanisole
  • 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 Corynebacterium faecalis and/or a metabolite thereof; 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 formulation 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, oligosaccharides, or combinations thereof.
  • a probiotic selected from the group consisting of lactic acid bacteria, bifidobacteria, Lactobacillus acidophilus, or a combination thereof
  • prebiotic selected from the group consisting of oligofructose (FOS) ), galactooligosaccharide (GOS), xylooligosaccharide (XOS), oligofructose (LACT), soy oligo
  • the composition further comprises a substance (e.g., a protective agent) selected from the group consisting of cysteine, glutathione, butylated hydroxyanisole. Dibutylmethyltoluene, tocopherol, bamboo leaf antioxidant, D-isoascorbic acid and its sodium salt, sodium ascorbate, calcium ascorbate, phospholipid, vitamin C (ascorbic acid), vitamin E, or a combination thereof.
  • a substance e.g., a protective agent
  • cysteine e.g., cysteine, glutathione, butylated hydroxyanisole.
  • Dibutylmethyltoluene, tocopherol, bamboo leaf antioxidant, D-isoascorbic acid and its sodium salt sodium ascorbate, calcium ascorbate, phospholipid, vitamin C (ascorbic acid), vitamin E, or a combination thereof.
  • a protective agent selected from the group consisting of cysteine, glutathione, butylated hydroxyanisole
  • anaerobic bacteria of the genus E. faecalis can be produced by a conventional method.
  • the culture product obtained in the previous step or the anaerobic bacillus bacillus and/or its metabolite 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 includes mammals such as humans.
  • 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 method comprises ingesting a pharmaceutical composition, a food composition, a beverage composition, or a combination thereof of the present invention.
  • the subject includes mammals such as humans.
  • 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.
  • Anaerostipes caccae AF04-45 (same as the preservation name) of the present invention has been deposited with the Guangdong Provincial Collection of Microbial Cultures on October 13, 2016. Address: No. 100, No. 100, Xianlie Middle Road, Guangzhou Building 5, Building No.: GDMCC No.: 60088.
  • the anaerobic bacterium of the present invention can significantly improve the indicators associated with inflammation-related diseases such as inflammatory bowel disease (such as slowing down weight loss, improving the degree of intestinal lesions (including slowing down the length of the colon, reducing inflammation of the colon) Reaction, etc.), reduce disease activity index (DAI), etc.).
  • inflammation-related diseases such as inflammatory bowel disease (such as slowing down weight loss, improving the degree of intestinal lesions (including slowing down the length of the colon, reducing inflammation of the colon) Reaction, etc.), reduce disease activity index (DAI), etc.).
  • VSL#3 purchased from Sigma-Tau, USA.
  • Anaerostipes caccae DSMZ 14662 purchased from the German Collection of Microorganisms, under the accession number DSMZ 14662.
  • Example 1 Isolation and Identification of Anaerostipes caccae AF04-45 (AF04-45)
  • the isolated samples were from fresh fecal samples from a healthy woman in Shenzhen.
  • the isolation medium was anaerobic PYG medium (purchased from Huanqi Microbiology Technology Co., Ltd.).
  • the composition of the medium is shown in Table 1-1 and Table 1-2:
  • Inorganic salt component Content (1L) CaCl 2 ⁇ 2H 2 O 0.25g MgSO 4 ⁇ 7H 2 O 0.5g K 2 HPO 4 1g KH 2 PO 4 1g NaHCO 3 10g NaCl 2g
  • the medium was prepared according to the above ingredients, and autoclaved at 115 ° C for 25 min.
  • the solid medium needs to be transferred to an anaerobic tank for pouring the plate, and dried for use.
  • Fresh feces were immediately transferred to an anaerobic tank, and about 0.2 g was suspended in sterilized PBS (phosphate buffer), thoroughly blown and dispersed, and then subjected to gradient dilution coating.
  • PBS phosphate buffer
  • the genomic DNA of the single bacteria was extracted, and the 16S rDNA amplification was carried out using DNA as a template, and the universal primers of 16S rDNA (8F-AGAGTTTGATCATGGCTCAG (SEQ ID NO.: 2) and 1492R-TAGGGTTACCTTGTTACGACTT (SEQ ID NO.: 3)) were purchased.
  • Self-generated Bioengineering (Shanghai) Co., Ltd. the amplification conditions are pre-denaturation at 95 °C for 4 min, then denaturation at 95 °C for 30 s, annealing at 57 °C for 40 s, extension at 72 °C for 1 min 30 s, 30 cycles.
  • the amplified PCR product was purified and sequenced at 3730 to obtain the full length 16S rDNA sequence of each strain.
  • the 16S rDNA sequence of AF04-45 is 1399 bp in length as shown in SEQ ID NO.: 1.
  • AF04-45 is similar to the 16S rDNA of the reference strain Anaerostipes caccae DSMZ 14662 (purchased from the German Collection of Microorganisms, deposited as DSMZ 14662). The maximum is 100%.
  • 16S rDNA identification it is basically confirmed that AF04-45 belongs to E. faecalis.
  • the 16S rDNA sequence of AF04-45 is SEQ ID NO.: 1, as follows:
  • the colonies of AF04-45 cultured on PYG plates at 37 ° C for 2 days were light yellow, convex, viscous, opaque, nearly round, with neat edges and a diameter of about 2-3 mm (Fig. 1).
  • AF04-45 is negative for catalase, negative for oxidase, and strictly anaerobic.
  • the survival rate of 3h in 0.3% bile salt is as high as 100%.
  • the substrate utilization of AF04-45 API 20A experimental results are shown in Table 2 below:
  • the bioactive substances of AF04-45 mainly investigate the production of short-chain fatty acids (SCFA) and organic acids.
  • AF04-45 was cultured for 48 hours, 1 ml of the bacterial solution was centrifuged at 12000 r/min for 5 min, and the supernatant was taken to prepare for detection of short-chain fatty acids (SCFA) and organic acids.
  • SCFA short-chain fatty acids
  • the short-chain fatty acid was determined by an external standard method, and acetic acid, propionic acid, butyric acid, and valeric acid were used to prepare a standard curve.
  • acetic acid, propionic acid, butyric acid, and valeric acid were used to prepare a standard curve.
  • HP-INNOWax Cross-Linked PEG
  • 30m ⁇ 0.25mm ⁇ 0.25um capillary column was used for analysis.
  • 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 SCFA yield was determined to be: 2.24 mmol/L acetic acid and 37.31 mmol/L butyric acid.
  • the selection criteria for organic acids are: 3-methylbutyric acid, valeric acid, quinic acid, lactic acid, oxalic acid, malonic acid, benzoic acid, maleic acid, succinic acid, anti-fumaric acid, malic acid, Diacid, tartaric acid, shikimic acid, citric acid, isocitric acid and L-ascorbic acid.
  • GC-7890B Agilent Meteorological Chromatograph
  • the column is selected from 122-5532G DB-5ms (40m ⁇ 0.25mm ⁇ 0.25um), column temperature: 270 ⁇ 290 ° C; inlet temperature: 250 ° C; gas Flow rate: 0.86 ml/min.
  • the content of organic acids in Anaerostipes caccae AF04-45 was determined as shown in Table 3 below:
  • AF04-45 was cultured to logarithmic growth phase. 100 ul of bacterial solution was applied to the plate (PYG medium). After application, the antibiotic susceptibility tablets were applied and 37°C was used. Incubate for 48 h under oxygen. The diameter of the zone of inhibition was measured, and the results are shown in Table 4.
  • AF04-45 was cultured to a stationary phase (about 24 h), centrifuged, and the cells were collected, suspended in PBS (PBS plus 0.05% cysteine hydrochloride), and the concentration was adjusted. Up to 10 9 cfu/ml, spare.
  • VSL # 3 Dissolve the powder of VSL # 3 (purchased from Sigma Tau, USA) into PBS (phosphate buffer), and adjust the concentration of the bacteria to 10 9 cfu/ml, and set aside.
  • mice The experimental mice were treated with C57bl/6 mice (purchased from Hubei Medical Experimental Animal Center), 8 weeks old, weighing 21g ⁇ 2g, and the breeding environment of mice was SPF grade. mold.
  • mice A total of 48 experimental mice, 12 mice in each group, including model group, AF04-45 treatment group, VSL # 3 positive drug group, Control group (blank control group), as follows:
  • AF04-45 treatment group 0.15% DSS induction for 7 days, and intragastric AF04-45 probiotics (live bacteria in PBS suspension, 10 9 cfu/ml)
  • VSL # 3 positive drug group 0.15% DSS was induced for 7 days, and VSL # 3 probiotics were administered (PBS suspension in PBS, 10 9 cfu/ml)
  • Control group normal feeding, no DSS induction
  • DSS induction and intragastric intervention were performed according to the grouping of mice.
  • the intervention method was performed by side-molding.
  • the body weight, diet and drinking water were recorded every day.
  • the fecal traits and fecal occult blood were observed on the first day.
  • the disease activity index (DAI) of the mice was calculated on the 3rd, 5th, and 7th days.
  • the DAI scores are shown in Table 5.
  • the experiment lasted for 7 days, and the daily gavage of probiotics and PBS was 200 ul.
  • the mice were sacrificed. All mice were bled, necked, colonic, photographed, weighed, and the length of the colon was measured. Knot The intestinal tissue was stored in a -80 ° C refrigerator and paraformaldehyde.
  • the DAI index is equal to the sum of the three points of weight, stool traits and fecal occult blood.
  • mice The body weights of the mice during the experimental procedure are shown in Table 6 and Figure 3.
  • the data in Table 6 showed that the body weight of the mice showed a downward trend with the induction of DSS.
  • the DSS-induced mice (model group) showed significant changes in body weight compared with the control group (*P ⁇ 0.001), the decrease in body weight is one of the indicators of the severity of UC, in which the weight loss of the model group is the most serious, and the weight of the AF04-45 group and the VSL#3 group on day 7 is significantly higher than that of the model group. ( ⁇ P ⁇ 0.05), indicating that AF04-45 can effectively reduce the weight loss of UC mice.
  • the DAI index is equal to the sum of the three points of body weight, stool traits, and fecal occult blood, and the DAI index of the mice on days 1, 3, 5, and 7 is calculated, and the results are shown in Table 7 and Figure 4.
  • AF04-45 can significantly improve the disease symptoms of UC, and the DAI index of AF04-45 mice on day 7 is significantly lower than that of VSL # 3 group. It can be seen that AF04-45 has more disease control effects on UC mice. it is good.
  • mice were sacrificed and dissected, and the colon tissue of the mice was taken out and the length was measured. Due to the DSS-induced UC model, a series of lesions in the colon tissue cause the colon to shorten, so the length of the colon is also one of the indicators for judging the severity of ulcerative enteritis in mice.
  • the results of measuring the colon length of each group of mice are shown in Table 8 and Figure 5.
  • Example 5 Food Composition Containing Anaerostipes caccae AF04-45
  • Example 6 Pharmaceutical composition containing Anaerostipes caccae AF04-45
  • 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 protective agent vitamin C, access Anaerostipes caccae AF04-45 viable (1-50 ⁇ 10 6 cfu / mL ), 36-38 °C fermentation to pH 6.0, centrifuged, freeze dried to a moisture content of less than 3%, i.e. prepared Anaerostipes caccae AF04- 45 bacteria freeze-dried.
  • Anaerostipes caccae AF04-45 lyophilizate 0.5 g was weighed in an equal amount with maltodextrin and then filled into capsules to prepare a pharmaceutical composition containing Anaerostipes caccae AF04-45.
  • Anaerostipes caccae AF04-45 (same as the preservation name) of the present invention has been deposited with the Guangdong Provincial Collection of Microbial Cultures on October 13, 2016. Address: No. 100, No. 100, Xianlie Middle Road, Guangzhou Building 5, Building No.: GDMCC No.: 60088.

Abstract

一种粪厌氧棒杆菌(Anaerostipes caccae)及其应用，具体地，提供了粪厌氧棒杆菌(Anaerostipes caccae)在治疗和/或预防炎症相关疾病的用途，还提供了一种用于治疗和/或预防炎症相关疾病的组合物，包括药品、饮料、食品，或动物饲料组合物等，以及改善哺乳动物肠道病变、减缓哺乳动物体重的下降、和/或降低哺乳动物的疾病活动指数(DAI)的方法。

Description

粪厌氧棒杆菌(Anaerostipes caccae)及其应用 技术领域
本发明属于微生物学领域，具体地，本发明涉及粪厌氧棒杆菌(Anaerostipes caccae)在治疗和预防炎症相关疾病中的应用，也涉及包含粪厌氧棒杆菌的组合物及其应用。
背景技术
溃疡性肠炎(UC)是属于炎症性肠病(IBD)的一种，根据炎症部位分为结肠炎和直肠炎，其发病机理尚未清楚，目前主要认为是遗传易感性、肠道菌群、肠粘膜的免疫反应引起的。国内外大量的研究表明，UC的发病与肠道菌群的失衡关系比较密切。UC的临床病理表现为腹痛、腹泻和黏液血便，且反复发作。
由于病理机制不明确，临床的治疗也缺乏特异性和针对性，目前，临床上针对UC病的用药主要有水杨酸类、糖皮质激素、免疫制剂。水杨酸类药物可以比较好的抑制前列腺素合成，清除氧自由基从而达到缓解炎症反应的目的，临床上治疗UC常见的水杨酸类西药主要是柳氮磺胺吡啶(SASP)，主要针对轻度、中度以及慢性UC患者；糖皮质激素是重症或者爆发性UC患者的首选用药，比如倍他米松；免疫抑制剂如环孢素可以通过抑制T细胞IL-2的产生，影响免疫反应的进展，从而对UC进行抑制。
上述三类药物均可以一定程度上对UC进行缓解，但是也都存在一定的副作用，水杨酸类的副作用是引发消化道反应、头痛、网织红细胞增多、精子减少及过敏反应引起的皮疹、肝毒性、白细胞减少、贫血等。糖皮质激素会导致机体代谢紊乱、水潴留等副作用，仅可作为应急用药，不能长期服用。免疫抑制剂治疗对药物依赖性较大，治疗周期长，容易引起肾毒性及二次感染，只能作为一种辅助治疗的手段。
因此本领域迫切需要开发一种的新的，无毒副作用的，用于治疗和预防炎症相关疾病的药物。
发明内容
本发明的另一目的是提供粪厌氧棒杆菌在治疗和/或预防炎症相关疾病的用途。
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/或预防炎症相关疾病的药品、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改善哺乳动物肠道病变的方法及其应用。
本发明的另一目的是提供一种减缓哺乳动物体重的下降、和/或降低哺乳动物的疾病活动指数(DAI)的方法及其应用。
本发明第一方面提供了一种粪厌氧棒杆菌，所述粪厌氧棒杆菌为粪厌氧棒杆菌(Anaerostipes caccae),保藏号为GDMCC No.:60088。
在另一优选例中，所述的粪厌氧棒杆菌的16s rDNA的序列如SEQ ID NO.:1所示。
在另一优选例中，所述粪厌氧棒杆菌为Anaerostipes caccae AF04-45。
在另有一优选例中，所述粪厌氧棒杆菌来自肠道、动物粪便、发酵池、和/或厌氧反应器。
本发明第二方面提供了一种组合物，所述组合物包括：(a)安全有效量的本发明第一方面所述的粪厌氧棒杆菌和/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，所述组合物还包括生长因子(较佳地，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、舌下含片、或其组合。
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1×10-1×1015cfu/mL或cfu/g Anaerostipes caccae AF04-45，较佳地1×104-1×1010cfu/mL或cfu/g Anaerostipes caccae AF04-45，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％所述的粪厌氧棒杆菌和/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)、寡聚糖、或其组合。
在另一优选例中，所述组合物还含有有助于保持粪厌氧棒杆菌活力的物质(如保护剂)。
在另一优选例中，所述有助于保持粪厌氧棒杆菌活力的物质(如保护剂)选自下组：半胱氨酸、谷胱甘肽、丁基羟基茴香醚、二丁基甲基甲苯、生育酚、竹叶抗氧化物、D-异抗坏血酸及其钠盐、抗坏血酸钠、抗坏血酸钙、磷脂、维生素C(抗坏血酸)、维生素E、或其组合。
在另一优选例中，所述有助于保持粪厌氧棒杆菌活力的物质(如保护剂)的重量比为0.1－2％，较佳地，0.5-1.5％，更佳地，0.5－1％，以所述组合物的总重计。
在另一优选例中，以组合物1g计，所述有助于保持粪厌氧棒杆菌活力的物质(如保护剂)的含量为1mg－20mg，较佳地，5mg－15mg，更佳地，5mg－10mg。
本发明第三方面提供了一种本发明第一方面所述粪厌氧棒杆菌、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预防和/或治疗炎症相关疾病。
在另一优选例中，所述炎症相关疾病选自下组：炎症性肠病、类风湿性关节炎、或其组合。
在另一优选例中，所述炎症相关疾病选自下组：溃疡性肠炎、胃炎、普通肠炎、或其组合。
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粪厌氧棒杆菌、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)减缓哺乳动物体重的下降；
(ii)降低哺乳动物的疾病活动指数(DAI)；
(iii)改善哺乳动物肠道病变。
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，所述减缓哺乳动物体重的下降指与模型组哺乳动物相比，实验组哺乳动物体重的降低幅度为不超过15％，较佳地，不超过10％，更佳地，不超过5％，显著低于模型组哺乳动物体重的降低幅度。
在另一优选例中，所述减缓哺乳动物肠道的病变包括减缓结肠长度的缩短、和/或减轻结肠炎症反应。
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粪厌氧棒杆菌和/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，所述制法还包括与有助于保持粪厌氧棒杆菌活力的物质(如保护剂)混合的步骤。
在另一优选例中，所述有助于保持粪厌氧棒杆菌活力的物质(如保护剂)选自下组：半胱氨酸、谷胱甘肽、丁基羟基茴香醚、二丁基甲基甲苯、生育酚、竹叶抗氧化物、D-异抗坏血酸及其钠盐、抗坏血酸钠、抗坏血酸钙、磷脂、维生素C(抗坏血酸)、维生素E、或其组合。
在另一优选例中，所述制法还包括与益生菌、和/或益生元混合的步骤。
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)、寡聚糖、或其组合。
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粪厌氧棒杆菌进行培 养，从而获得培养产物；
(b)任选地，从所述培养产物分离粪厌氧棒杆菌菌体和/或其代谢产物；和/或
(c)任选地，将上一步骤获得的培养产物或粪厌氧棒杆菌菌体和/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，从而制得本发明所述的组合物。
在另一优选例中，在步骤(c)之前，还包括将上一步骤获得的所述培养产物或所述粪厌氧棒杆菌菌体和/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。
在另一优选例中，在步骤(c)之前，还包括将上一步骤获得的所述培养产物或所述粪厌氧棒杆菌菌体和/或其代谢产物与有助于保持粪厌氧棒杆菌活力的物质(如保护剂)混合的步骤。
在另一优选例中，在步骤(c)之前，还包括将上一步骤获得的所述培养产物或所述粪厌氧棒杆菌菌体和/或其代谢产物与益生菌和/或益生元混合的步骤。
本发明第七方面提供了一种改善哺乳动物肠道病变的方法，给所述对象施用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减缓哺乳动物体重的下降、和/或降低哺乳动物的疾病活动指数(DAI)的方法，给所述对象施用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显示了粪厌氧棒杆菌Anaerostipes caccae AF04-45培养48h菌落的图片。
图2显示了粪厌氧棒杆菌Anaerostipes caccae AF04-45在显微镜下的革兰氏染色图片(1000倍)。
图3显示了Control组、模型组、VSL#3和AF04-45治疗组小鼠的体重的变化。
图4显示了Control组、模型组、VSL#3和AF04-45治疗组小鼠的DAI指数的变化。
图5显示了Control组、模型组、VSL#3和AF04-45治疗组小鼠结肠的长度。
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，粪厌氧棒杆菌(Anaerostipes caccae)具有预防和/或治疗炎症相关疾病(如炎症性肠病(如溃疡性肠炎、胃炎、普通肠炎)、类风湿性关节炎)的作用，将含有粪厌氧棒杆菌的活性组合物饲喂食实验对象，发现该组合物能够减缓体重降低，降低疾病活动指数(DAI)，改善肠道病变，可有效减轻炎症相关疾病(如炎症性肠病(如溃疡性肠炎、胃炎、普通肠炎)、类风湿性关节炎))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”包括牛奶生长因子，具体地，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。
如本文所用，术语“控制哺乳动物体重下降”、“减缓哺乳动物体重下降”可互换使用，是指哺乳动物在进行溃疡性肠炎模型构建过程中由于炎症的不断严重，实验动物的体重也随之下降，体重下降的百分比为下降体重占原始体重的百分比。体重下降的程度越高，疾病越严重，本发明的粪厌氧棒杆菌在对哺乳动物溃疡性肠炎治疗过程中可以控制实验动物的体重下降，减缓疾病的症状。
疾病活动指数(DAI)
如本文所用，术语“疾病活动指数”是指结合患者(患病动物)的体重下降百分率、大便粘稠度和大便出血情况3种情况进行综合评分。
粪厌氧棒杆菌及其应用
如本文所用，术语“粪厌氧棒杆菌”、“Anaerostipes caccae”、“本发明的粪厌氧棒杆菌”可互换使用。在一个优选例中，所述菌株为Anaerostipes  caccae AF04-45，保藏号为GDMCC No.:60088，分离自人(较佳地，健康女性)的粪便中，具体地，分离自成年女性的肠道中。粪厌氧棒杆菌的生理特性如下：粪厌氧棒杆菌(Anaerostipes caccae AF04-45)37℃厌氧培养2天的菌落为淡黄色，凸起，较粘稠，不透明，近圆形，边缘整齐，直径约2-3mm；显微镜下放大1000倍粪厌氧棒杆菌(Anaerostipes caccae AF04-45)的菌体为杆状，革兰氏阴性，不产芽孢和鞭毛。粪厌氧棒杆菌(Anaerostipes caccae AF04-45)为过氧化氢酶阴性，氧化酶阴性，严格厌氧，在0.3％的胆盐中4h的存活率高达100％。能发酵多种碳水化合物，包括葡萄糖、甘露糖、蔗糖、麦芽糖、木糖、甘露醇、山梨醇等，主要产生乳酸、丁二酸、戊酸、3-甲基丁酸、柠檬酸等，并且本发明的粪厌氧棒杆菌(Anaerostipes caccae AF04-45)对表4所示的20种抗生素均比较敏感。
本发明提供了粪厌氧棒杆菌在治疗和/或预防炎症相关疾病(如炎症性肠病(如溃疡性肠炎、胃炎、普通肠炎)、类风湿性关节炎)的应用。受试者用DSS(葡聚糖硫酸钠)进行诱导造模，菌株Anaerostipes caccae AF04-45具有选自下组的一个或多个用途：(i)减缓该受试者的体重降低；(ii)改善肠道病变程度；(iii)降低疾病活动指数(DAI)。根据本发明的一个优选例，采用C57bl/6小鼠作为试验小鼠，用DSS(葡聚糖硫酸钠)进行诱导造模，从而获得溃疡性肠炎(UC)模型小鼠，经Anaerostipes caccae AF04-45来治疗的UC模型小鼠，与未接受治疗的对照组(模型组)相比，其体重降低幅度减缓，并且各种与炎症相关疾病(如炎症性肠病)相关的指标也得到改善，如改善肠道病变程度(包括减缓结肠长度的缩短、和/或减轻结肠炎症反应)、降低疾病活动指数(DAI)等。因此，所述菌株能够用以预防和/或治疗炎症相关疾病(如炎症性肠病(如溃疡性肠炎、胃炎、普通肠炎)、类风湿性关节炎)。
组合物及其应用
本发明还提供了一种组合物，较佳地，所述组合物包括食品组合物、保健组合物、药物组合物、饮料组合物、或饲料组合物，优选地，为药物组合物。所述组合物包括有效量的粪厌氧棒杆菌，在一个优选例中，所述组合物还包括生长因子(如牛奶生长因子)。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)、寡聚糖、或其组合。在一个优选例中，所述组合物还包括选自下组的有助于保持粪厌氧棒杆菌活力的物质(如保护剂)：半胱氨酸、谷胱甘肽、丁基羟基茴香醚、二丁基甲基甲苯、生育酚、竹叶抗氧化物、D-异抗坏血酸及其钠盐、抗坏血酸钠、抗坏血酸钙、磷脂、维生素C(抗坏血酸)、维生素E、或其组合。以组合物的总重计，所述有助于保持粪厌氧棒杆菌活力的物质(如保护剂)的重量比为0.1—2％，较佳地，0.5-1.5％，更佳地，0.5－1％。
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、舌下含片、或其组 合。
本发明的组合物可以以口服液、片剂、针剂、口崩片、冻干粉制剂或胶囊的任一种形式给药，优选肠溶剂型(如胶囊)，在本发明中，如无特别说明，本发明所用的赋形剂、药物允许的媒介和载体主要是根据适合菌或其代谢产物特性和所需的特定给药方式进行选择，有利于菌或其代谢产物顺利通过胃而被给药者吸收。这些物质可根据给药途径进行选择。
本发明的组合物可进一步包含那些通常用于药物制剂的赋形剂中任何附加的赋形剂，为的是例如稳定组合物自身，或使其容易地分散或赋予其适宜的味道。
在所述赋形剂当中，菊粉、果糖、淀粉、低聚木糖、二氧化硅、缓冲试剂以及风味剂是合适的实例。
本发明中药物制剂可进一步包含辅助的活性组份。
乳糖、麦芽糊精、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。本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三酸酯粘度为约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酯，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酯为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1×10-1×1015cfu/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)的粪厌氧棒杆菌和/或其代谢产物的制剂。
当用于制备药物组合物时，所用的粪厌氧棒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1×10-1×1015cfu/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)的活性粪厌氧棒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。
所述的粪厌氧棒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)，只要适合粪厌氧棒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本发明微生物。给药量随病人的体重和炎症相关疾病(如炎症性肠病(如溃疡性肠炎、胃炎、普通肠炎)、类风湿性关节炎)、心血管疾病(如高血压、高血脂，冠心病)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，它含有有效量的粪厌氧棒杆菌和/或其代谢产物，以及余量的食品上可接受的载体，所述的食品组 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生长因子(如牛奶生长因子)。在一优选例中，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)、寡聚糖、或其组合。在一个优选例中，所述组合物还包括选自下组的有助于保持粪厌氧棒杆菌活力的物质(如保护剂)：半胱氨酸、谷胱甘肽、丁基羟基茴香醚、二丁基甲基甲苯、生育酚、竹叶抗氧化物、D-异抗坏血酸及其钠盐、抗坏血酸钠、抗坏血酸钙、磷脂、维生素C(抗坏血酸)、维生素E、或其组合。
在一优选例中，所述组合物的配方如下：
1×10-1×1015cfu/mL的粪厌氧棒杆菌和/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：
1×104-1×1010cfu/mL的粪厌氧棒杆菌和/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)安全有效量的粪厌氧棒杆菌和/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)。在一个优选例中，所述制剂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)、寡聚糖、或其组合。在一个优选例中，所述组合物还包括选自下组的有助于保持粪厌氧棒杆菌活力的物质(如保护剂)：半胱氨酸、谷胱甘肽、丁基羟基茴香醚、二丁基甲基甲苯、生育酚、竹叶抗氧化物、D-异抗坏血酸及其钠盐、抗坏血酸钠、抗坏血酸钙、磷脂、维生素C(抗坏血酸)、维生素E、或其组合。
粪厌氧棒杆菌的生产方法
通常，粪厌氧棒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粪厌氧棒杆菌的方法，具体地，包括如下步骤：
(a)在适合培养的条件下，对本发明所述的粪厌氧棒杆菌进行培养，从而获得培养产物；
(b)任选地，从所述培养产物分离粪厌氧棒杆菌菌体和/或其代谢产物；和
(c)任选地，将上一步骤获得的培养产物或粪厌氧棒杆菌菌体和/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。
改善哺乳动物肠道病变的方法
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。所述实验对象包括哺乳动物，如人。
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。
减缓哺乳动物体重的下降、和/或降低哺乳动物的疾病活动指数(DAI)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。所述实验对象包括哺乳动物，如人。
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
本发明的菌种粪厌氧棒杆菌Anaerostipes caccae AF04-45(与保藏名称相同)已于2016年10月13日保藏在广东省微生物菌种保藏中心，地址：广州市先烈中路100号大院59号楼5楼，保藏号：GDMCC No.:60088。
本发明的主要优点包括：
(a)本发明的粪厌氧棒杆菌能够显著改善与炎症相关疾病(如炎症性肠病)相关的指标(如减缓体重下降、改善肠道病变程度(包括减缓结肠长度的缩短、减轻结肠炎症反应等)、降低疾病活动指数(DAI)等)。
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，实施例所用的材料和试剂均为市售产品。
VSL#3，购自美国Sigma-Tau。
Anaerostipes caccae DSMZ 14662，购自德国微生物菌种保藏中心，保藏号为DSMZ 14662。
实施例1：粪厌氧棒杆菌Anaerostipes caccae AF04-45(简称AF04-45)的分离鉴定
1.1分离培养
分离样品来自于深圳一位健康女性的新鲜粪便样品，分离培养基为厌氧PYG培养基(购自环凯微生物科技公司)，培养基成分如表1-1、表1-2：
表1-1 PYG培养基成分
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
表1-2 无机盐溶液的配方
无机盐组分 含量(1L)
CaCl2·2H2O 0.25g
MgSO4·7H2O 0.5g
K2HPO4 1g
KH2PO4 1g
NaHCO3 10g
NaCl 2g
按照以上成分配制好培养基，115℃高压灭菌25min，固体培养基需要转入厌氧箱中进行平板的倾倒，晾干备用。
新鲜粪便立即转移至厌氧箱中，取约0.2g悬浮于灭菌的PBS(磷酸盐缓冲液)中，充分吹打分散，然后进行梯度稀释涂布，平板放置在厌氧箱中37℃培养3-5天，厌氧气体组成为氮气：氢气：二氧化碳＝90：5：5。待平板长出肉眼可见的菌落，挑取单个菌落进行划线分纯，并对分纯的单菌进行保藏和鉴定。
1.2 16S rDNA鉴定
提取单菌的基因组DNA，以DNA作为模板进行16S rDNA扩增，采用16S rDNA的通用引物(8F-AGAGTTTGATCATGGCTCAG(SEQ ID NO.:2)和1492R-TAGGGTTACCTTGTTACGACTT(SEQ ID NO.:3))(购自生工生物工程(上海)股份有限公司)，扩增条件是95℃预变性4min，然后95℃变性30s，57℃退火40s，72℃延伸1min30s，30个循环。扩增的PCR产物进行纯化，3730测序，获得每株菌的16S rDNA全长序列。其中AF04-45的16S rDNA序列长度为1399bp，如SEQ ID NO.:1所示。通过在NCBI的数据库比对，可以得出AF04-45同数据库中参考菌株Anaerostipes caccae(粪厌氧棒杆菌)DSMZ 14662(购自德国微生物菌种保藏中心，保藏号为DSMZ 14662)的16S rDNA相似度最高为100％。通过16S rDNA鉴定，基本可以确认AF04-45属于粪厌氧棒杆菌。
AF04-45的16S rDNA序列为SEQ ID NO.:1，如下所示：
Figure PCTCN2016109690-appb-000001
Figure PCTCN2016109690-appb-000002
1.3AF04-45的微生物特征和生理生化特征
(1)菌落形态
AF04-45在PYG平板上37℃培养2天的菌落为淡黄色，凸起，较粘稠，不透明，近圆形，边缘整齐，直径约2-3mm(图1)。
(2)菌体显微形态
显微镜下放大1000倍AF04-45的菌体为杆状，革兰氏阴性，不产芽孢和鞭毛(图2)。
(3)生理生化特征
AF04-45为过氧化氢酶阴性，氧化酶阴性，严格厌氧，在0.3％的胆盐中4h的存活率高达100％。AF04-45的底物利用情况API 20A实验结果如下表2：
表2
Figure PCTCN2016109690-appb-000003
实施例2粪厌氧棒杆菌Anaerostipes caccae AF04-45的生物活性物质
AF04-45的生物活性物质主要考察短链脂肪酸(SCFA)和有机酸产生情况。
(1)样品预处理
将AF04-45培养48h，取1ml菌液进行12000r/min离心5min，取上清，准备进行短链脂肪酸(SCFA)和有机酸的检测。
(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。
测定得出SCFA产量为：乙酸2.24mmol/L，丁酸37.31mmol/L。
(3)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。测得粪厌氧棒杆菌Anaerostipes caccae AF04-45有机酸的含量详见下表3：
表3
有机酸 3-甲基丁酸 戊酸 奎宁酸 乳酸 草酸 丙二酸
含量(mmol/L) 0.37 0.38 0 10.33 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) 0 0 0.45 0.02 0 0.05
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 0.33 0 0.11  
实施例3粪厌氧棒杆菌Anaerostipes caccae AF04-45的抗生素敏感情况
考察AF04-45对20种抗生素的敏感情况，将AF04-45培养至对数生长期，取100ul菌液进行平板(PYG培养基)涂布，涂抹均匀后贴上抗生素药敏片，37℃厌氧条件下培养48h。测量抑菌圈的直径，其结果详见表4。
表4
Figure PCTCN2016109690-appb-000004
Figure PCTCN2016109690-appb-000005
结果显示，AF04-45对这20种抗生素均比较敏感。
实施例4粪厌氧棒杆菌Anaerostipes caccae AF04-45对溃疡性肠炎的缓解情况
4.1灌胃菌株的准备
(1)AF04-45的培养：将AF04-45培养至稳定期(约24h)，离心，收集菌体，使用PBS(PBS添加0.05％的半胱氨酸盐酸盐)进行悬浮，调整菌浓至109cfu/ml，备用。
(2)VSL#3的准备：溶解VSL#3(购自美国Sigma Tau)的菌粉至PBS(磷酸盐缓冲液)中，同样调整菌浓至109cfu/ml，备用。
4.2试验小鼠的准备
(1)小鼠品系：实验小鼠采用C57bl/6小鼠(购自湖北医学实验动物中心)，8周龄，体重21g±2g，小鼠饲养环境为SPF级，适应性喂养1周进行造模。
(2)造模方法：UC模型小鼠采用DSS造模法，持续给小鼠饮用0.15％的DSS(葡聚糖硫酸钠，分子量36000-50000)(购自美国MPBiomedicals)7天。
(3)实验分组：试验小鼠共48只，每组12只小鼠，包括模型组、AF04-45治疗组、VSL#3阳性药组、Control组(空白对照组)，具体如下所示：
模型组：0.15％的DSS诱导7天，并灌胃PBS
AF04-45治疗组：0.15％的DSS诱导7天，并灌胃AF04-45益生菌(PBS悬浮的活菌，109cfu/ml)
VSL#3阳性药组：0.15％的DSS诱导7天，并灌胃VSL#3益生菌(PBS悬浮的活菌，109cfu/ml)
Control组：正常饲喂，不进行DSS诱导
4.3实验过程
按照小鼠分组情况进行DSS诱导和灌胃干预，干预方法采用边造模边治疗，每天记录小鼠体重、饮食和饮水情况，同时观察小鼠的粪便性状及粪便隐血情况，分别在第1天、第3天、第5天和第7天计算小鼠的疾病活动指数(DAI)，DAI评分详见表5。实验持续7天，益生菌和PBS的日灌胃量为200ul。实验结束后处死小鼠，所有小鼠取血、脱颈、取结肠、拍照、称重、量取结肠长度。结 肠组织保存于-80℃冰箱和多聚甲醛中。
表5
Figure PCTCN2016109690-appb-000006
DAI指数等于体重、大便性状以及大便隐血三个积分之和。
4.4实验结果
(1)小鼠的体重的变化
实验过程小鼠的体重记录见表6和图3。
表6
Figure PCTCN2016109690-appb-000007
表6的数据显示，随着DSS的诱导，小鼠的体重呈现下降趋势，到第5天后，DSS诱导的小鼠(模型组)相比Control组小鼠的体重出现显著的变化(*P<0.001)，体重的下降是UC严重程度的一个指标之一，其中模型组的体重下降的最严重，在第7天AF04-45组小鼠和VSL#3组小鼠的体重显著高于模型组(▲P<0.05)，说明AF04-45可以有效减缓UC小鼠体重的下降。
(2)小鼠的疾病活动指数(DAI)
DAI指数等于体重、大便性状以及大便隐血三个积分之和，分别计算小鼠在第1、3、5、7天的DAI指数，结果如表7和图4所示。
表7
Figure PCTCN2016109690-appb-000008
Figure PCTCN2016109690-appb-000009
随着DSS诱导，小鼠的DAI指数逐渐上升，到第7天小鼠的DAI值达到最高，Control组由于未进行DSS诱导，其DAI指数维持稳定，第5天和第7天的DAI同DSS诱导的小鼠(模型组)DAI存在显著性差异(**P<0.01)。在AF04-45和VSL#3的干预下，小鼠的疾病活动情况有一定的改善，第7天，AF04-45组和VSL#3组的DAI指数明显低于模型组(*P<0.5)。说明AF04-45可以明显改善UC的疾病症状，同时AF04-45组小鼠在第7天的DAI指数显著低于VSL#3组，由此可见，AF04-45对UC小鼠的疾病控制的更好。
(3)小鼠结肠长度的变化
实验结束后处死小鼠并进行解剖，取出小鼠的结肠组织，测量其长度。由于DSS诱导的UC模型，小鼠在结肠组织会发生一系列病变导致结肠缩短，因此结肠的长度变化也是评判小鼠溃疡性肠炎的严重程度的指标之一。通过测量各组小鼠的结肠长度结果如表8和图5所示。
表8
Figure PCTCN2016109690-appb-000010
结果显示，造模7天小鼠的结肠缩短比较严重，相对于模型组，正常小鼠的结肠长度要长于UC造模的模型小鼠(**P<0.01)，通过AF04-45和VSL#3的干预，可以显著减缓小鼠的结肠缩短(*P<0.05)。说明AF04-45在控制UC小鼠结肠病变有良好的效果。
实施例5含粪厌氧棒杆菌Anaerostipes caccae AF04-45的食品组合物
原料配比如表9。
表9
原料 质量百分比(％)
Anaerostipes caccae AF04-45 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℃，混入保护剂维生素C，接种1-100×106cfu/g的Anaerostipes caccae AF04-45菌，即制成含Anaerostipes caccae AF04-45菌的食品组合物。
实施例6含Anaerostipes caccae AF04-45的药物组合物
原料配比见表10。
表10
原料 质量百分比(％)
Anaerostipes caccae AF04-45 1.0％
乳糖 2.0％
酵母粉 2.0％
蛋白胨 1.0％
纯净水 93.5％
维生素C 0.5％
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℃，混入保护剂维生素C，接入Anaerostipes caccae AF04-45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Anaerostipes caccae AF04-45菌冷冻干燥物。称取0.5克Anaerostipes caccae AF04-45冷冻干燥物与麦芽糊精等量混合后装入胶囊中，即制成含Anaerostipes caccae AF04-45的药物组合物。
实施例7含粪厌氧棒杆菌Anaerostipes caccae AF04-45的药物的制备
7.1菌液准备：将Anaerostipes caccae AF04-45(1×109cfu/ml)进行厌氧培养，厌氧培养基采用实施例1的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体积生长因子和1体积的保护剂维生素C加入到100体积的AF04-45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)，从而制备得到含粪厌氧棒杆菌Anaerostipes caccae AF04-45的药物。
菌种保藏
本发明的菌种粪厌氧棒杆菌Anaerostipes caccae AF04-45(与保藏名称相同)已于2016年10月13日保藏在广东省微生物菌种保藏中心，地址：广州市先烈中路100号大院59号楼5楼，保藏号：GDMCC No.:60088。
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. 一种粪厌氧棒杆菌，其特征在于，所述粪厌氧棒杆菌为粪厌氧棒杆菌(Anaerostipes caccae),保藏号为GDMCC No.:60088。
  2. 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所述的粪厌氧棒杆菌和/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。
  3. 如权利要求2所述的组合物，其特征在于，所述的组合物还含有其它益生菌和/或益生元。
  4. 如权利要求2所述的组合物，其特征在于，所述组合物还含有有助于保持粪厌氧棒杆菌活力的物质(如保护剂)。
  5. 一种权利要求1所述粪厌氧棒杆菌、或权利要求2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预防和/或治疗炎症相关疾病。
  6. 一种权利要求1所述粪厌氧棒杆菌、或权利要求2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)减缓哺乳动物体重的下降；
    (ii)降低哺乳动物的疾病活动指数(DAI)；
    (iii)改善哺乳动物肠道病变。
  7. 一种权利要求2所述组合物的制法，其特征在于，包括步骤：
    将权利要求1所述的粪厌氧棒杆菌和/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2所述的组合物。
  8. 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所述的粪厌氧棒杆菌进行培养，从而获得培养产物；
    (b)任选地，从所述培养产物分离粪厌氧棒杆菌菌体和/或其代谢产物；和/或
    (c)任选地，将上一步骤获得的培养产物或粪厌氧棒杆菌菌体和/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。
  9. 一种改善哺乳动物肠道病变的方法，其特征在于，给所述对象施用权利要求2所述的组合物。
  10. 一种减缓哺乳动物体重的下降、和/或降低哺乳动物的疾病活动指数(DAI)的方法，其特征在于，给所述对象施用权利要求2所述的组合物。
PCT/CN2016/109690 2016-12-13 2016-12-13 粪厌氧棒杆菌(Anaerostipes caccae)及其应用 WO2018107365A1 (zh)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021143621A1 (zh) * 2020-01-13 2021-07-22 浙江大学 Anaerostipes sp B2131菌及其在炎症性肠病中的应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004085628A1 (en) * 2003-03-27 2004-10-07 Rowett Research Institute Lactic acid utilising bacteria and their therapeutic use
CN102325464A (zh) * 2009-02-23 2012-01-18 根特大学 用于缓解肠问题的方法和新菌株
CN103079582A (zh) * 2010-06-04 2013-05-01 国立大学法人东京大学 诱导调节性t细胞的增殖或积累的组合物
CN104160014A (zh) * 2011-12-01 2014-11-19 国立大学法人东京大学 诱导调节性t细胞的增殖或积累的人源细菌

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012142605A1 (en) * 2011-04-15 2012-10-18 Samaritan Health Services Rapid recolonization deployment agent
CN104546933A (zh) * 2014-09-30 2015-04-29 深圳华大基因科技有限公司 粪拟杆菌在治疗或预防类风湿性关节炎或其相关疾病中的应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004085628A1 (en) * 2003-03-27 2004-10-07 Rowett Research Institute Lactic acid utilising bacteria and their therapeutic use
CN102325464A (zh) * 2009-02-23 2012-01-18 根特大学 用于缓解肠问题的方法和新菌株
CN103079582A (zh) * 2010-06-04 2013-05-01 国立大学法人东京大学 诱导调节性t细胞的增殖或积累的组合物
CN104160014A (zh) * 2011-12-01 2014-11-19 国立大学法人东京大学 诱导调节性t细胞的增殖或积累的人源细菌

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ATARASHI, KOJI: "Induction of colonic regulatory T cells by indigenous Clostridium species", SCIENCE, vol. 331, no. 6015, 23 December 2010 (2010-12-23), pages 337 - 341, XP055598571, DOI: 10.1126/science.1198469 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021143621A1 (zh) * 2020-01-13 2021-07-22 浙江大学 Anaerostipes sp B2131菌及其在炎症性肠病中的应用

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