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WO2019051790A1 - 单形巨单胞菌(Megamonas funiformis)及其应用 - Google Patents

单形巨单胞菌(Megamonas funiformis)及其应用 Download PDF

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Publication number
WO2019051790A1
WO2019051790A1 PCT/CN2017/101915 CN2017101915W WO2019051790A1 WO 2019051790 A1 WO2019051790 A1 WO 2019051790A1 CN 2017101915 W CN2017101915 W CN 2017101915W WO 2019051790 A1 WO2019051790 A1 WO 2019051790A1
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composition
group
monocytogenes
another preferred
mammal
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PCT/CN2017/101915
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English (en)
French (fr)
Inventor
邹远强
肖亮
李晓平
余靖宏
刘传
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BGI Shenzhen Co Ltd
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BGI Shenzhen Co Ltd
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Priority to US16/647,344 priority Critical patent/US11160837B2/en
Priority to PCT/CN2017/101915 priority patent/WO2019051790A1/zh
Priority to EP17925337.2A priority patent/EP3683303A4/en
Priority to JP2020515131A priority patent/JP6990303B2/ja
Priority to CN201811074629.5A priority patent/CN109504615A/zh
Publication of WO2019051790A1 publication Critical patent/WO2019051790A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • 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
    • A23L33/15—Vitamins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/175—Amino acids
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A61K9/0056—Mouth soluble or dispersible forms; Suckable, eatable, chewable coherent forms; Forms rapidly disintegrating in the mouth; Lozenges; Lollipops; Bite capsules; Baked products; Baits or other oral forms for animals
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    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
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    • A—HUMAN NECESSITIES
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    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A—HUMAN NECESSITIES
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    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
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    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K2035/11—Medicinal preparations comprising living procariotic cells
    • A61K2035/115—Probiotics
    • 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 relates to the field of microorganisms, and in particular to Megamonas funiformis and its use.
  • IBD Inflammatory bowel disease
  • UC ulcerative enteritis
  • CD Crohn's disease
  • Cardiovascular disease is the number one killer in developed countries and is becoming more common in developing countries. In recent years, China’s economy has developed rapidly, the people’s living standards have improved substantially, and the way of life is undergoing profound changes. The rhythm of work and life is accelerating, the calorie of diet increases, but physical activity is reduced. Cardiovascular risk factors such as obesity caused by unhealthy lifestyles have increased significantly, and the incidence of cardiovascular and cerebrovascular diseases has risen rapidly. Modern medical research believes that the basis of cardiovascular and cerebrovascular diseases is atherosclerosis, oxidized low-density lipoprotein (Ox-LDL) is the core of atherosclerosis.
  • Ox-LDL oxidized low-density lipoprotein
  • Ulcerative colitis is an important type of inflammatory bowel disease (IBD).
  • IBD inflammatory bowel disease
  • the cause of the disease is unknown.
  • the main lesion is in the submucosal layer of the colonic mucosa. It is a chronic disease. Intestinal disease.
  • the causes of ulcerative enteritis include host genetic susceptibility, intestinal flora and intestinal mucosal immune response.
  • the clinicopathological manifestations include persistent abdominal pain, diarrhea and mucous bloody stools, and the disease is repeated. The number of patients affected showed a clear upward trend.
  • the drugs for ulcerative enteritis mainly include salicylic acid, adrenal glucocorticoids, and immunological preparations.
  • Salicylic acid drugs can inhibit prostaglandin synthesis and scavenge oxygen Free radicals, in order to achieve the purpose of relieving inflammation, but can only be relieved in a short period of time, can not achieve radical cure.
  • the most common salicylate western medicine for the treatment of ulcerative enteritis (UC) is sulfasalazine (SASP), which is mainly for patients with mild, moderate and chronic UC.
  • Adrenal glucocorticoids are patients with severe or explosive UC.
  • Preferred drugs such as betamethasone; immunosuppressants such as cyclosporine can inhibit ulcerative enteritis (UC) by inhibiting the production of T-cell IL-2 and affecting the progression of the immune response.
  • the three drugs for ulcerative enteritis can alleviate UC to a certain extent, but there are also some side effects.
  • the side effects of salicylate are gastrointestinal reactions, headache, reticulocyte increase, Sperm reduction and allergic reactions caused by rash, hepatotoxicity, leukopenia, anemia, etc., these drugs also have antibacterial effects, easily lead to bacterial flora disorder and increased drug resistance.
  • Adrenal glucocorticoids can cause metabolic disorders, water retention and other side effects. They can only be used as emergency medications 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.
  • a first aspect of the invention provides a microorganism, which is Megamonas funiformis.
  • sequence of the 16s rDNA of the M. monocytogene is as shown in SEQ ID NO.: 1.
  • the M. monocytogenes is Megamonas funiformis AF24-28AC, and the accession number is GDMCC 60093.
  • the M. monocytogenes are from the gut, animal droppings, fermentation tanks, and/or anaerobic reactors.
  • the M. monocytogenes are derived from a human or a non-human mammal.
  • non-human mammals include rodents (e.g., mice, rats), primates (e.g., monkeys).
  • a second aspect of the invention provides a composition
  • a composition comprising: (a) a safe and effective amount The macromonomimella and/or metabolite thereof of the first aspect of the invention; and (b) a food acceptable carrier or a 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 Megamonas funiformis AF24-28AC, preferably 1 x 10 4 - 1 x 10 10 cfu/mL or cfu /g Megamonas funiformis AF24-28AC, based on the total volume or total weight of the composition.
  • the composition contains from 0.0001 to 99% by weight, preferably from 0.1 to 90% by weight, of the macromonas monocytogenes and/or metabolite thereof, to the total of the composition. Weight 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 contributes to the maintenance of the activity of M. monocytogenes.
  • a substance e.g., a protective agent
  • the substance (such as a protective agent) that contributes to maintaining the activity of M. monocytogenes is selected from the group consisting of cysteine, glutathione, butyl hydroxyanisole, dibutyl group 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 Macromonas monocytogenes is 0.1 to 2%, preferably 0.5 to 1.5%, more preferably 0.5. -1.0% 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 Macromonas monocytogenes is from 1 mg to 20 mg, preferably from 5 mg to 15 mg, more preferably in terms of 1 g of the composition. Ground, 5mg-10mg.
  • a third aspect of the invention provides the use of the macromonas of the first aspect of the invention, or the composition of the second aspect of the invention, for the preparation of a composition or formulation, the composition Or the formulation is for one or more uses selected from the group consisting of: (a) preventing and/or treating an inflammation-related disease; and/or (b) preventing and/or treating a cardiovascular 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 cardiovascular disease is selected from the group consisting of hypertension, hyperlipidemia, coronary heart disease, or a combination thereof.
  • the formulation comprises a microecological formulation.
  • a fourth aspect of the invention provides the use of the macromonas of the first aspect of the invention, or the composition of the second aspect of the invention, for the preparation of a composition or formulation, the composition Or the formulation is for one or more uses selected from the group consisting of:
  • said lowering blood lipid levels in a mammal comprises lowering cholesterol levels.
  • controlling the reduction in body weight of the mammal means that the body weight of the experimental group is reduced by no more than 10%, preferably no more than 5%, more preferably than the mammal of the model group. , no more than 2%.
  • the improving intestinal lesions in the mammal comprises slowing the shortening of the length of the colon, and/or reducing the inflammatory response of the colon.
  • the mammal comprises a human or a non-human mammal.
  • non-human mammals include rodents (e.g., mice, rats), primates (e.g., monkeys).
  • the macrophage 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 contributes to maintaining the activity of the microorganism.
  • a substance e.g., a protective agent
  • the substance (such as a protective agent) that contributes to maintaining the activity of M. monocytogenes is selected from the group consisting of cysteine, glutathione, butyl hydroxyanisole, dibutyl group 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 step of mixing the culture product obtained in the previous step or the microorganism of the microorganism Monmoralum 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.
  • step (c) further comprising the culture product obtained in the previous step or the microorganism of the macromonas and/or its metabolites and the help of maintaining the simplex A step of mixing substances that are active with macromonas (such as a protective agent).
  • step (c) further comprising the culture product obtained in the previous step or the macromonas cell and/or its metabolites and probiotics and/or probiotics
  • step (c) further comprising the culture product obtained in the previous step or the macromonas cell and/or its metabolites and probiotics and/or probiotics
  • the step of mixing the elements prior to step (c), further comprising the culture product obtained in the previous step or the macromonas cell and/or its metabolites and probiotics and/or probiotics.
  • 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 human or a non-human mammal.
  • non-human mammals include rodents (e.g., mice, rats), primates (e.g., monkeys).
  • the method is non-diagnostic and non-therapeutic.
  • An eighth aspect of the invention provides a method of reducing blood lipid levels in a mammal, controlling a decrease in body weight of the mammal, and/or reducing a disease activity index in the mammal, administering to the subject the composition of the second aspect of the invention .
  • 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 human or a non-human mammal.
  • non-human mammals include rodents (e.g., mice, rats), primates (e.g., monkeys).
  • the method is non-diagnostic and non-therapeutic.
  • a ninth aspect of the present invention provides a method of preventing and/or treating an inflammation-related disease, comprising the steps of:
  • composition of the second aspect of the invention is administered to the subject, thereby preventing and/or treating an inflammation-related disease.
  • 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 human or a non-human mammal.
  • non-human mammals include rodents (e.g., mice, rats), primates (e.g., monkeys).
  • the method is non-diagnostic and non-therapeutic.
  • a tenth aspect of the invention provides a method of preventing and/or treating cardiovascular diseases, comprising the steps of:
  • composition of the second aspect of the invention is administered to the subject to prevent and/or treat cardiovascular disease.
  • 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 human or a non-human mammal.
  • non-human mammals include rodents (e.g., mice, rats), primates (e.g., monkeys).
  • the method is non-diagnostic and non-therapeutic.
  • Figure 1 shows a picture of colonies cultured for 48 h in Megamonas funiformis AF24-28AC.
  • Figure 2 shows a Gram stained picture (1000 times) of Micromonas monocytogenes AF24-28AC under a microscope.
  • Figure 3 shows the standard curve for cholesterol detection.
  • Figure 4 shows changes in body weight of Control group, model group, VSL # 3 and AF24-28AC treatment groups.
  • Figure 5 shows the changes in the DAI index of the Control, Model, VSL # 3 and AF24-28AC treated mice.
  • the inventors have extensively and intensively studied and experimented and unexpectedly found that Megamonas funiformis has prevention and/or treatment of inflammation-related diseases (such as inflammatory bowel disease (such as ulcerative enteritis, gastritis, common The effects of enteritis, rheumatoid arthritis, and/or cardiovascular diseases (such as hypertension, hyperlipidemia, coronary heart disease), feeding the active composition containing the macromonas of the present invention to the test subject, and found
  • the composition can control body weight loss, lower blood fat, lower disease activity index (DAI), improve intestinal lesions, and can effectively reduce inflammation-related diseases (such as inflammatory bowel disease (such as ulcerative enteritis, gastritis, common enteritis), rheumatoid Sexual arthritis), alleviate cardiovascular diseases and other diseases.
  • inflammation-related diseases such as inflammatory bowel disease (such as ulcerative enteritis, gastritis, common enteritis), rheumatoid Sexual arthritis
  • DAI disease activity index
  • 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.
  • the term “controls the reduction in body weight of a mammal”, “slows down the weight of a mammal” “low”, “controlling the decline of mammalian body weight”, “reducing the decline of body weight in mammals” are used interchangeably, meaning that the mammals undergo constant inflammation during the construction of the ulcerative enteritis model, and the weight of the experimental animals also The decrease, the percentage of weight loss is the percentage of the weight loss to the original body weight. The higher the degree of weight loss, the more serious the disease, the macrobacterium of the present invention can control the experimental animals during the treatment of mammalian ulcerative enteritis Reduced body weight and slow down the symptoms of the disease.
  • DAI Disease Activity Index
  • disease activity index refers to a combination of three conditions, including weight loss percentage, stool viscosity, and stool bleeding, in a patient (or diseased animal).
  • the strain is Megamonas funiformis AF24-28AC, deposited under the number GDMCC 60093, isolated from the feces of a human (preferably, healthy female).
  • the physiological characteristics of M. monocytogenes were as follows: Megamonas funiformis AF24-28AC was isolated using PYG medium under the anaerobic conditions of 37 °C. The colonies of AF24-28AC cultured in PYG medium for 2 days were light yellow with irregular wavy edges, low water content and a diameter of about 2-3 mm.
  • the microscopic morphology of the cells is rod-shaped, Gram-negative, non-spore-forming and flagella.
  • Catalase and oxidase test results are negative, can ferment a variety of carbohydrates, including glucose, mannitol, lactose, sucrose, maltose, sulphate, xylose, arabinose, glycerol, cellobiose, mannitol , pine syrup, raffinose, sorbitol, rhamnose, trehalose, mainly produces acetic acid, propionic acid, isovaleric acid, benzoic acid and lactic acid, and can produce valeric acid, 3-methylbutyric acid, maleic acid in small amounts.
  • the Megamonas funiformis AF24-28AC of the present invention is resistant to penicillin, oxacillin and cefoperazone, and is sensitive to the other 17 antibiotics in Table 2.
  • the present invention provides the treatment and/or prevention of inflammation-related diseases such as inflammatory bowel diseases (such as ulcerative enteritis, gastritis, common enteritis), rheumatoid arthritis, and cardiovascular diseases (such as high).
  • inflammation-related diseases such as inflammatory bowel diseases (such as ulcerative enteritis, gastritis, common enteritis), rheumatoid arthritis, and cardiovascular diseases (such as high).
  • blood pressure hyperlipemia, coronary heart disease
  • the subject was induced to model with DSS (sodium dextran sulfate), and the strain Megamonas funiformis AF24-28AC has one or more uses selected from the group consisting of: (i) controlling the decrease in body weight of the subject; (ii) Reduce disease activity index (DAI); (iii) improve intestinal lesions; (iv) lower blood lipid levels.
  • DAI Reduce disease activity index
  • DAI Reduce disease activity index
  • improve intestinal lesions such as lower blood lipid levels.
  • C57bl/6 is small Rats were used as test mice, and induced by DSS (sodium dextran sulfate) to obtain ulcerative enteritis (UC) model mice, UC model mice treated with Megamonas funiformis AF24-28AC, and untreated Compared with the control group (model group), the weight loss was slowed down and blood lipids were decreased, and various inflammation-related diseases (such as inflammatory bowel disease (such as ulcerative enteritis, gastritis, common enteritis), rheumatoid arthritis) were associated.
  • DSS sodium dextran sulfate
  • the indicators have also been improved, such as improving the extent of intestinal lesions (including slowing the length of the colon, reducing the inflammatory response of the colon, etc.) and reducing the disease activity index (DAI). Therefore, the strain can be used for preventing and/or treating inflammation-related diseases (such as inflammatory bowel disease (such as ulcerative enteritis, gastritis, common enteritis), rheumatoid arthritis), cardiovascular diseases (such as hypertension, high Blood lipids, coronary heart disease).
  • inflammation-related diseases such as inflammatory bowel disease (such as ulcerative enteritis, gastritis, common enteritis), rheumatoid arthritis), cardiovascular diseases (such as hypertension, high Blood lipids, coronary heart disease).
  • 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 includes an effective amount of Macromonas monocytogenes, 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, and butyl hydroxy anise to help maintain the activity of M. monocytogenes.
  • a substance e.g., a protective agent
  • cysteine e.g., glutathione
  • butyl hydroxy anise to help maintain the activity of M. monocytogenes.
  • the weight ratio of the substance (e.g., protective agent) which contributes to maintaining the activity of Macromonas monocytogenes is 0.1 to 2%, preferably 0.5 to 1.5%, more preferably, based on the total weight of the composition. 0.5-1.0%.
  • 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 Tablet, or a combination thereof.
  • 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 mode of administration required, and the bacteria or its metabolites are smoothly passed through the stomach. It is absorbed by the administrator. 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 cp and an acetaminophen 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 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 preparation may contain 1 x 10 6 - 1 x 10 10 cfu/ml or cfu/g) of Macromonas monocytogenes and/or its metabolites.
  • the effective dosage of the microorganism, or its metabolite, 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 15 cfu/ml or cfu/g intimately mixed with a solid or liquid pharmaceutically acceptable carrier (preferably, may contain 1 x 10 4 -1) ⁇ 10 10 cfu/ml or cfu/g; more preferably, 1 ⁇ 10 6 - 1 ⁇ 10 10 cfu/ml or cfu/g) of active P. monocytogenes or a fermentatively produced active ingredient may be contained.
  • 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 M. monocytogene or a metabolite thereof can be administered by oral or the like.
  • the solid carrier includes: starch, lactose, dicalcium phosphate, microcrystalline cellulose, sucrose and kaolin, and the liquid carrier comprises: a medium, Polyethylene glycol, nonionic surfactants, and edible oils (such as corn oil, peanut oil, and sesame oil) are suitable for the characteristics of M. monocytogenes or their metabolites and the particular mode of administration desired.
  • 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/or 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), the severity of cardiovascular disease, and the supplementary active components included. It varies with the microorganisms 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 Macromonas monocytogenes and/or a metabolite thereof, and a balance of a food acceptable carrier,
  • the dosage form of the food 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, and butyl hydroxy anise to help maintain the activity of M. monocytogenes.
  • a substance e.g., a protective agent
  • cysteine e.g., glutathione
  • butyl hydroxy anise to help maintain the activity of M. monocytogenes.
  • 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 M. monocytogenes 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, and butyl hydroxy anise to help maintain the activity of M. monocytogenes.
  • a substance e.g., a protective agent
  • cysteine e.g., glutathione
  • butyl hydroxy anise to help maintain the activity of M. monocytogenes.
  • Macromonas monocytogenes can be produced by a conventional method.
  • the conditions suitable for culture refer to any conditions suitable for cultivating the fecal anaerobic baseball of the present invention.
  • the conditions suitable for culture refer to anaerobic culture at 37 ° C using PYG medium. 24h-72h.
  • 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.
  • 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.
  • 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 strain Mymagonas funiformis AF24-28AC (same name as the deposit) of the present invention has been deposited with the Guangdong Provincial Collection of Microorganisms and Culture Collections (GDMCC) on October 13, 2016, at the address of the 100th Xianlizhong Road, Guangzhou. Building 5, Building 5, 5th Floor, Deposit No.: GDMCC 60093.
  • the macromonas of the present invention can significantly improve indicators associated with inflammation-related diseases such as inflammatory bowel diseases (such as ulcerative enteritis, gastritis, common enteritis), rheumatoid arthritis (such as weight control) Decrease, improve the degree of intestinal lesions (including slowing the length of the colon, reducing the inflammation of the colon, etc.), reducing the disease activity index (DAI), etc.).
  • inflammation-related diseases such as inflammatory bowel diseases (such as ulcerative enteritis, gastritis, common enteritis), rheumatoid arthritis (such as weight control) Decrease, improve the degree of intestinal lesions (including slowing the length of the colon, reducing the inflammation of the colon, etc.), reducing the disease activity index (DAI), etc.).
  • the M. monocytogenes of the present invention are capable of significantly lowering blood lipids such as cholesterol levels.
  • the treatment of UC mice by the genus Megamonas funiformis AF24-28AC of the present invention can specifically control the decrease of body weight of UC mice induced by DSS and inhibit the disease activity index (DAI) of mice. Elevated, improves the changes in the colon of mice, and has a degrading effect on cholesterol.
  • DAI disease activity index
  • Example 1 Isolation and Identification of Megamonas funiformis AF24-28AC
  • the isolated sample was obtained from a healthy female feces, and the strain was isolated by gradient dilution plating.
  • the genomic DNA of the isolated strain was extracted and PCR amplified using 16S rDNA universal primers.
  • the primer sequences were 27f (5'-AGAGTTTGATCATGGCTCAG-3' (SEQ ID NO.: 2)) and 1492r (5'-TAGGGTTACCTTGTTACGACTT-3' (SEQ ID NO.: 3)), amplification conditions Don't do the following:
  • the obtained 16S rDNA amplification product was subjected to electrophoresis detection, purification, and 3730 sequencing to obtain a 16S rDNA sequence of length 1425 bp (SEQ ID NO.: 1), and the 16S rNDA sequence of AF24-28AC was used in the EzBioCloud database (http:// In the comparison between www.ezbiocloud.net/identify, the highest homology with AF24-28AC was Megamonas funiformis (Megamonas funiformis DSM 19343 (purchased from the German Collection of Cultures), with a similarity of 99.09%, through 16S rDNA identification, it can be confirmed that AF24-28AC belongs to the genus Macromonobacteria.
  • the isolated colonies of AF24-28AC cultured in PYG medium for 48 hours were characterized by light yellow color, irregular wavy edges, low water content and a diameter of about 2-3 mm (Fig. 1).
  • the microscopic morphology of the cells is rod-shaped, Gram-negative, non-spore-forming and flagella (Fig. 2).
  • the test results of catalase and oxidase were negative.
  • the utilization of carbon source by AF24-28AC was detected by API 20A (Mérieux, France). The results are shown in Table 1, where + indicates positive reaction and - indicates negative reaction. , w represents a weak positive reaction.
  • the drug sensitivity paper method was used to test the sensitivity of AF24-28AC to 20 common antibiotics. 100 ul of the bacterial solution of the AF24-28AC cultured in log phase was plated, and the antibiotic drug-sensitive tablets (purchased from Hangzhou microorganisms) were used. Reagent Co., Ltd.) was applied to the surface of the plate and cultured at 37 ° C for 48 hours to measure the size of the inhibition zone. The results are shown in Table 2.
  • AF24-28AC was resistant to penicillin, oxacillin and cefoperazone, and was sensitive to 17 other antibiotics.
  • SCFA short-chain fatty acids
  • organic acids include 3-methylbutyric acid, quinic acid, lactic acid, oxalic acid, malonic acid, benzoic acid, maleic acid, succinic acid, fufuic acid, malic acid, adipic acid, tartaric acid, shikimic acid, Citric acid, isocitric acid and L-ascorbic acid, standard for each SCFA and organic acid were purchased from Sigma.
  • Check The measurement process is as follows:
  • SCFA short-chain fatty acids
  • the SCFA was tested by Agilent Meteorological Chromatograph (GC-7890B, Agilent) using HP-INNOWax (Cross-Linked PEG), 30m ⁇ 0.25mm ⁇ 0.25um capillary column.
  • the detector is a hydrogen flame ion detector, GC.
  • the parameters are 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 organic acid was also detected by Agilent Meteorological Chromatograph (GC-7890B, Agilent).
  • the column was 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.
  • AF24-28AC has the ability to produce acetic acid, propionic acid, isovaleric acid, benzoic acid and lactic acid, and can produce valeric acid, 3-methylbutyric acid, maleic acid, succinic acid, malic acid in small amounts. , adipic acid and citric acid.
  • the method for determining the content of cholesterol is determined by the o-phthalaldehyde colorimetric method (OPA method), and the degradation ability of cholesterol is examined by the change of the cholesterol content of the strain in a certain concentration of cholesterol medium for a period of time.
  • OPA method o-phthalaldehyde colorimetric method
  • a certain amount of cholesterol was weighed and dissolved in ethanol at a concentration of 10 mg/mL, and sterilized by filtration. Add the prepared PYG medium to 10 mg/mL bile salt (autoclaving), 10% mass concentration of sodium thioglycolate (filter sterilization) and cholesterol, mix well, and then AF24 according to the inoculation amount of 3%.
  • -28AC was inoculated into the medium and cultured under anaerobic conditions at 37 ° C for 72 h.
  • the culture solution containing the cholesterol-containing PYG medium was centrifuged at 10,000 r/min, and the supernatant was collected for cholesterol detection, and the uninoculated cholesterol PYG medium was used as a blank control group.
  • the degradation of cholesterol is calculated according to the formula:
  • A the content of cholesterol in the cholesterol medium which is not inoculated
  • AF24-28AC has a certain ability to degrade cholesterol.
  • the mouse model selected in this example was: DSS (Dextran Sulfate, Na, purchased from MPBIO, USA) mouse model of ulcerative enteritis, continued to give mice 0.15% DSS (dextran) Sodium sulfate, molecular weight 36000-50000) 7 days, mouse strain was C57bl/6 mice (purchased from Hubei Medical Experimental Animal Center), 8 weeks old, weighing 20g ⁇ 2g, and reared in SPF-level rat room environment.
  • DSS Extran Sulfate, Na, purchased from MPBIO, USA
  • mouse strain was C57bl/6 mice (purchased from Hubei Medical Experimental Animal Center), 8 weeks old, weighing 20g ⁇ 2g, and reared in SPF-level rat room environment.
  • mice A total of 48 experimental mice were randomly divided into 4 groups, 12 in each group, including:
  • control group feeding with normal feed
  • VSL # 3 treatment group - DSS modeling each mouse perfused with 0.2 ml of probiotic VSL # 3 (purchased from Alfasigma, USA, clinically used for the treatment of probiotics for UC);
  • AF24-28AC treatment process culture 24h AF24-28AC bacterial solution, centrifuge to collect the cells, suspend with PBS (phosphate buffer), adjust the bacterial concentration to 10 9 cfu / ml, each mouse perfused 200ul per day AF24-28AC. VSL # 3 was also suspended in PBS, and the concentration was adjusted to 10 9 cfu/ml, and each mouse was given 200 ul per day.
  • PBS phosphate buffer
  • AF24-28AC and VSL # 3 were administered in the first 3 days of DSS modeling.
  • DSS was added to the drinking water of mice, and UC was modeled by free drinking for 7 days.
  • the mice were recorded daily for weight, diet and water.
  • the fecal traits and fecal occult blood of the mice were observed, and the disease activity index (DAI) of the mice was calculated on the first day, the third day, the fifth day, and the seventh day, respectively.
  • the DAI scoring standards are shown in Table 4.
  • the mice were sacrificed. All mice were bled, necked, colonic, photographed, weighed, and the length of the colon was measured. Colon tissue was stored in a -80 ° C refrigerator and paraformaldehyde.
  • Stool characteristics in the table normal stool - forming stool; loose stool - paste-like, semi-formed stool that does not adhere to the anus; loose stool - a thin watery stool that can adhere to the anus.
  • blood in the stool normal mice have positive blood in the stool; the blood in the naked eye is red or brown; the occult blood is positive in the blood of the naked eye, and the test is performed using tetramethylbenzidine.
  • the DAI index is equal to the sum of the three points of weight loss, stool traits, and fecal occult blood (?? Please explain the sum of the points, using formulas or words).
  • mice The body weight changes of mice before and after treatment are shown in Table 5 and Figure 4 below:
  • the data in Table 6 and Figure 5 show that the DAI of the Control group was almost the same, and with the DSS induction, the DAI of the model group, AF24-28AC and VSL # 3 mice gradually increased, and the third day model group mice DAI Compared with the control group, it began to increase significantly (*P ⁇ 0.05), and the DAI of the model group reached the highest level on the 7th day (P ⁇ 0.01 relative to the control group).
  • the intervention of the bacteria of the present invention can control the increase of DAI, and the DAI of the AF24-28AC group was significantly controlled on the 5th and 7th days compared with the control group ( ⁇ P ⁇ 0.05), and the AF24-28AC group was small.
  • the DAI of the mouse was lower than VSL # 3, and VSL # 3 was not significant in controlling DAI. Therefore, AF24-28AC was superior to VSL # 3 in controlling the increase of DAI in UC mice.
  • the colon tissue of the UC model mice changes, as the ulcer tissue and inflammation cause the colon tissue to shorten. After the end of the treatment, the colon length of the mouse measured by anatomy is shown in Table 7.
  • Example 5 Food composition containing Megamonas funiformis AF24-28AC
  • Example 6 Pharmaceutical composition containing Megamonas funiformis AF24-28AC
  • the raw material ratio is shown in Table 9.
  • 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.
  • Megamonas funiformis AF24-28AC 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, Megamonas funiformis AF24-28AC Freeze-dried.
  • Example 7 A method for preparing a medicament for treating an inflammation-related disease such as ulcerative enteritis (UC)
  • UC ulcerative enteritis
  • Anamonas funiformis AF24-28AC (1 ⁇ 10 9 cfu/ml) was subjected to anaerobic culture, and anaerobic medium was cultured in PYG for 2-3 days at 37 °C.
  • Preparation of drug or pharmaceutical dosage form 5 volumes (ml) of growth factor and 1 volume (ml) of a protective agent (such as vitamin C, cysteine) are added to 100 volumes (ml) of Megamonas funiformis AF24-28AC for fermentation. In the bacterial solution, mix well and then add a starch adjuvant (such as maltodextrin) to prepare a pharmaceutical or pharmaceutical dosage form containing Megamonas funiformis AF24-28AC.
  • a protective agent such as vitamin C, cysteine
  • Example 8 A method for preparing a medicament for treating cardiovascular diseases such as hyperlipemia
  • Anamonas funiformis AF24-28AC (1 ⁇ 10 9 cfu/ml) was subjected to anaerobic culture, and anaerobic medium was cultured in PYG for 2-3 days at 37 °C.
  • Preparation of drug or pharmaceutical dosage form 5 volumes (ml) of growth factor and 1 volume (ml) of a protective agent (such as vitamin C, cysteine) are added to 100 volumes (ml) of Megamonas funiformis AF24-28AC for fermentation. In the bacterial solution, mix well and then add a starch adjuvant (such as maltodextrin) to prepare a pharmaceutical or pharmaceutical dosage form containing Megamonas funiformis AF24-28AC.
  • a protective agent such as vitamin C, cysteine

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Abstract

提供了一种单形巨单胞菌(Megamonas funiformis)及其应用，具体地，单形巨单胞菌(Megamonas funiformis)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)、心血管疾病的作用。

Description

单形巨单胞菌(Megamonas funiformis)及其应用 技术领域
本发明涉及微生物领域，具体地，涉及单形巨单胞菌(Megamonas funiformis)及其应用。
背景技术
炎症性肠道疾病(inflammatory bowel disease，IBD)是一种病因不明的慢性炎症性肠道疾病，该疾病易反复，严重影响患者的生活质量。现代医学认为，引发炎症性肠道疾病(IBD)的因素有遗传、饮食、感染、自身免疫、心理因素以及环境等。炎症性肠道疾病包括溃疡性肠炎(UC)和克罗恩病(CD),均属于炎症相关疾病。
心血管疾病是发达国家的头号杀手，在发展中国家也日渐普遍。近年来，我国经济迅速发展，人民生活水平大幅度提高，生活方式正在发生深刻变化。工作和生活的节奏加快，饮食热量增加但体力活动减少。不良生活方式所引起的肥胖等心血管病危险因素显著增加，心脑血管病的发病率因而迅速上升。现代医学研究认为，心脑血管病的基础，是动脉粥样硬化、被氧化的低密度脂蛋白(Ox-LDL)是动脉粥样硬化的核心。目前国内的心脑血管病预防专利主要集中在活血化瘀降“血脂”或者“降胆固醇”降低低密度脂蛋白(LDL)方面。西方国家的心脑血管病预防主要集中在抗氧化降血脂或者降胆固醇方面，所以西方人用了大量抗氧化保健品预防心脑血管疾病没有收到显著效果，同样国内单纯使用活血化瘀中药或益气活血的中药保健品预防心脑血管疾病，虽然有效，但也是效果有限。
溃疡性肠炎(ulcerative colitis，UC)是炎症性肠道疾病(inflammatory bowel disease，IBD)的一种重要疾病类型，其发病原因不明，主要病变部位是在结肠粘膜的粘膜下层，属于一种慢性的肠道疾病。基于目前的研究，主要认为引发溃疡性肠炎的病因有宿主遗传易感性、肠道菌群以及肠粘膜的免疫反应，临床病理表现为持续腹痛、腹泻和黏液血便，且病情反复，近年来我国UC的患病人数呈明显上升趋势。
目前，临床上针对溃疡性肠炎(UC)的用药主要有水杨酸类、肾上腺糖皮质激素类、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，但也只能短期缓解，无法实现根治。临床上治疗溃疡性肠炎(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；肾上腺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，从而对溃疡性肠炎(UC)进行抑制。
现有针对溃疡性肠炎(UC)的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，这类药物同时也具有抗菌作用，容易引起菌群紊乱以及耐药性增强。肾上腺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新的，无毒副作用的，用于治疗和/或预防炎症相关疾病、心血管疾病的药物。
发明内容
本发明的目的在于提供一种新的，无毒副作用的，用于治疗和/或预防炎症相关疾病、心血管疾病的药物。
本发明第一方面提供了一种单形巨单胞菌，所述单形巨单胞菌为单形巨单胞菌(Megamonas funiformis)。
在另一优选例中，所述的单形巨单胞菌的16s rDNA的序列如SEQ ID NO.:1所示。
在另一优选例中，所述单形巨单胞菌为Megamonas funiformis AF24-28AC，保藏号为GDMCC 60093。
在另有一优选例中，所述单形巨单胞菌来自肠道、动物粪便、发酵池、和/或厌氧反应器。
在另一优选例中，所述单形巨单胞菌来源于人或非人哺乳动物。
在另一优选例中，所述非人哺乳动物包括啮齿动物(如小鼠、大鼠)、灵长类动物(如猴)。
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单形巨单胞菌和/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 Megamonas funiformis AF24-28AC，较佳地1×104-1×1010cfu/mL或cfu/g Megamonas funiformis AF24-28AC，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％所述的单形巨单胞菌和/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单形巨单胞菌活力的物质(如保护剂)。
在另一优选例中，所述有助于保持单形巨单胞菌活力的物质(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，所述有助于保持单形巨单胞菌活力的物质(如保护剂)的重量比为0.1-2％，较佳地，0.5-1.5％，更佳地，0.5-1.0％，以所述组合物的总重计。
在另一优选例中，以组合物1g计，所述有助于保持单形巨单胞菌活力的物质(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的单形巨单胞菌、或本发明第二方面所述的组合物的用途，用于制备组合物或制剂，所述组合物或制剂用于选自下组的一种或多种用途：(a)预防和/或治疗炎症相关疾病；和/或(b)预防和/或治疗心血管疾病。
在另一优选例中，所述炎症相关疾病选自下组：炎症性肠道疾病、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，所述心血管疾病选自下组：高血压、高血脂、冠心病、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的单形巨单胞菌、或本发明第二方面所述的组合物的用途，用于制备组合物或制剂，所述组合物或制剂用于选自下组的一种或多种用途：
(i)降低哺乳动物的血脂水平；
(ii)控制哺乳动物体重的降低；
(iii)降低哺乳动物的疾病活动指数(DAI)；
(iv)改善哺乳动物的肠道病变。
在另一优选例中，所述降低哺乳动物的血脂水平包括降低胆固醇水平。
在另一优选例中，所述控制哺乳动物体重的降低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10％，较佳地，不超过5％，更佳地，不超过2％。
在另一优选例中，所述改善哺乳动物的肠道病变包括减缓结肠长度的缩短、和/或减轻结肠炎症反应。
在另一优选例中，所述哺乳动物包括人或非人哺乳动物。
在另一优选例中，所述非人哺乳动物包括啮齿动物(如小鼠、大鼠)、灵长类动物(如猴)。
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单形巨单胞菌和/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单形巨单胞菌活力的物质(如保护剂)混合的步骤。
在另一优选例中，所述有助于保持单形巨单胞菌活力的物质(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单形巨单胞菌进行培养，从而获得培养产物；
(b)任选地，从所述培养产物分离单形巨单胞菌菌体和/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单形巨单胞菌菌体和/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单形巨单胞菌菌体和/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单形巨单胞菌菌体和/或其代谢产物与有助于保持单形巨单胞菌活力的物质(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单形巨单胞菌菌体和/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，所述的对象包括人或非人哺乳动物。
在另一优选例中，所述非人哺乳动物包括啮齿动物(如小鼠、大鼠)、灵长类动物(如猴)。
在另一优选例中，所述方法为非诊断性和非治疗性的。
本发明第八方面提供了一种降低哺乳动物的血脂水平、控制哺乳动物体重的降低和/或降低哺乳动物的疾病活动指数的方法，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，所述的对象包括人或非人哺乳动物。
在另一优选例中，所述非人哺乳动物包括啮齿动物(如小鼠、大鼠)、灵长类动物(如猴)。
在另一优选例中，所述方法为非诊断性和非治疗性的。
本发明第九方面提供了一种预防和/或治疗炎症相关疾病的方法，包括步骤：
给所述对象施用本发明第二方面所述的组合物，从而预防和/或治疗炎症相关疾病。
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，所述的对象包括人或非人哺乳动物。
在另一优选例中，所述非人哺乳动物包括啮齿动物(如小鼠、大鼠)、灵长类动物(如猴)。
在另一优选例中，所述方法为非诊断性和非治疗性的。
本发明第十方面提供了一种预防和/或治疗心血管疾病的方法，包括步骤：
给所述对象施用本发明第二方面所述的组合物，从而预防和/或治疗心血管疾病。
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，所述的对象包括人或非人哺乳动物。
在另一优选例中，所述非人哺乳动物包括啮齿动物(如小鼠、大鼠)、灵长类动物(如猴)。
在另一优选例中，所述方法为非诊断性和非治疗性的。
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显示了单形巨单胞菌Megamonas funiformis AF24-28AC培养48h菌落的图片。
图2显示了单形巨单胞菌Megamonas funiformis AF24-28AC在显微镜下的革兰氏染色图片(1000倍)。
图3显示了胆固醇检测的标准曲线。
图4显示了Control组、模型组、VSL#3和AF24-28AC治疗组小鼠的体重的变化。
图5显示了Control组、模型组、VSL#3和AF24-28AC治疗组小鼠的DAI指数的变化。
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，单形巨单胞菌(Megamonas funiformis)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)和/或心血管疾病(如高血压、高血脂，冠心病)的作用，将含有本发明的单形巨单胞菌的活性组合物饲喂食实验对象，发现该组合物能够控制体重降低，降低血脂，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)、减轻心血管疾病等病症。在此基础上，本发明人完成了本发明。
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“控制哺乳动物体重的降低”、“减缓哺乳动物体重的降 低”、“控制哺乳动物体重的下降”、“减缓哺乳动物体重的下降”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，本发明的单形巨单胞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体重的降低，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(或患病动物)的体重下降百分率、大便粘稠度和大便出血等3种情况进行综合评分。
单形巨单胞菌及其应用
如本文所用，术语“单形巨单胞菌”，“Megamonas funiformis”、“本发明的单形巨单胞菌”可互换使用。在一个优选例中，所述菌株为Megamonas funiformis AF24-28AC，保藏号为GDMCC 60093，分离自人(较佳地，健康女性)的粪便中。单形巨单胞菌的生理特性如下：单形巨单胞菌Megamonas funiformis AF24-28AC采用PYG培养基进行分离，分离条件为37℃厌氧条件。AF24-28AC在PYG培养基培养2天的菌落为浅黄色，边缘不规则波状，扁平，含水量低，直径约2-3mm。菌体的显微形态为杆状，革兰氏阴性、不产芽孢和鞭毛。过氧化氢酶和氧化酶的测试结果均为阴性，能发酵多种碳水化合物，包括葡萄糖、甘露醇、乳糖、蔗糖、麦芽糖、柳醇、木糖、阿拉伯糖、甘油、纤维二糖、甘露醇、松叁糖、棉籽糖、山梨醇、鼠李糖、海藻糖，主要产生乙酸、丙酸、异戊酸、苯甲酸和乳酸，可少量产生戊酸、3-甲基丁酸、马来酸、丁二酸、苹果酸、己二酸和柠檬酸。并且本发明的单形巨单胞菌Megamonas funiformis AF24-28AC对青霉素、苯唑西林、头孢哌酮有抗性，对表2中的其他17种抗生素均比较敏感。
本发明提供了单形巨单胞菌在治疗和/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)、心血管疾病(如高血压、高血脂，冠心病)的应用。受试者用DSS(葡聚糖硫酸钠)进行诱导造模，菌株Megamonas funiformis AF24-28AC具有选自下组的一个或多个用途：(i)控制该受试者体重的降低；(ii)降低疾病活动指数(DAI)；(iii)改善肠道病变程度；(iv)降低血脂水平。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Megamonas funiformis AF24-28AC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且血脂下降，并且各种与炎症相关疾病(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)相关的指标也得到改善，如改善肠道病变程度(包括减缓结肠长度缩短、减轻结肠炎症反应等)、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)、心血管疾病(如高血压、高血脂，冠心病)。
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单形巨单胞菌，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单形巨单胞菌活力的物质(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，所述有助于保持单形巨单胞菌活力的物质(如保护剂)的重量比为0.1-2％，较佳地，0.5-1.5％，更佳地，0.5-1.0％。
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5-21cp，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)的单形巨单胞菌和/或其代谢产物的制剂。
当用于制备药物组合物时，所用的单形巨单胞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(较佳地，可含有1×104-1×1010cfu/ml或cfu/g；更佳地，可含有1×106-1×1010cfu/ml或cfu/g)的活性单形巨单胞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。
所述的单形巨单胞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)，只要适合单形巨单胞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/或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，它含有有效量的单形巨单胞菌和/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单形巨单胞菌活力的物质(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的单形巨单胞菌和/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的单形巨单胞菌和/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)安全有效量的单形巨单胞菌和/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单形巨单胞菌活力的物质(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。
单形巨单胞菌的生产方法
通常，单形巨单胞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单形巨单胞菌的方法，具体地，包括如下步骤：
(a)在适合培养的条件下，对本发明所述的单形巨单胞菌进行培养，从而获得培养产物；
(b)任选地，从所述培养产物分离单形巨单胞菌菌体和/或其代谢产物；和
(c)任选地，将上一步骤获得的培养产物或单形巨单胞菌菌体和/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。
在本发明中，所述适合培养的条件指任何适于培养本发明的粪厌氧棒球菌的条件，在一优选实施方式中，所述适合培养的条件指采用PYG培养基于37℃厌氧培养24h-72h。
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。
控制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。
降低哺乳动物血脂的方法
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。
预防和/或治疗炎症相关疾病的方法
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。
预防和/或治疗心血管疾病的方法
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
本发明的菌种单形巨单胞菌Megamonas funiformis AF24-28AC(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保藏(GDMCC)，地址广州市先烈中路100号大院59号楼5楼，保藏号：GDMCC 60093。
本发明的主要优点包括：
(a)本发明的单形巨单胞菌能够显著改善与炎症相关疾病(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)相关的指标(如控制体重下降、改善肠道病变程度(包括减缓结肠长度缩短、减轻结肠炎症反应等)、降低疾病活动指数(DAI)等)。
(b)本发明的单形巨单胞菌能够显著降低血脂(如胆固醇水平)。
(c)本发明的单形巨单胞菌Megamonas funiformis AF24-28AC对UC小鼠的治疗具体表现为：可有效控制DSS诱导的UC小鼠体重的下降，抑制小鼠疾病活动指数(DAI)的升高，改善小鼠结肠的变化，并对胆固醇具有降解作用。
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。
实施例1：单形巨单胞菌Megamonas funiformis AF24-28AC的分离鉴定
分离样品来自于一位健康的女性粪便，采用梯度稀释涂平板方法进行菌株分离，培养基采用PYG培养基(购自环凯微生物科技公司)，进行厌氧培养，厌氧的气体组分为N2：CO2：H2＝90：5：5，培养48h，挑取单菌落进行划线分纯，获得每株单菌的纯培养。
对分离菌株的基因组DNA进行提取，采用16S rDNA通用引物进行PCR扩增，引物序列是27f(5’-AGAGTTTGATCATGGCTCAG-3’(SEQ ID NO.:2))和1492r(5’-TAGGGTTACCTTGTTACGACTT-3’(SEQ ID NO.:3))，扩增条件分 别如下：
Figure PCTCN2017101915-appb-000001
将获得的16S rDNA扩增产物进行电泳检测、纯化、3730测序，获得长度为1425bp的16S rDNA序列(SEQ ID NO.:1)，将AF24-28AC的16S rNDA序列在EzBioCloud数据库(http://www.ezbiocloud.net/identify)中进行比对，获得同AF24-28AC同源性最高的菌是Megamonas funiformis(Megamonas funiformis DSM 19343(购自德国菌种保藏中心)，相似度有99.09％，通过16S rDNA鉴定，基本可以确认AF24-28AC属于单形巨单胞菌。
Figure PCTCN2017101915-appb-000002
Figure PCTCN2017101915-appb-000003
(SEQ ID NO.:1)
1.1AF24-28AC的生理生化特征
分离到的AF24-28AC在PYG培养基中培养48h的菌落特征为：浅黄色，边缘不规则波状，扁平，含水量低，直径约2-3mm(图1)。菌体的显微形态为杆状，革兰氏阴性、不产芽孢和鞭毛(图2)。过氧化氢酶和氧化酶的测试结果均为阴性，AF24-28AC对碳源的利用情况通过API 20A(法国梅里埃)进行检测，其结果如表1，其中+表示阳性反应，-表示阴性反应，w表示弱阳性反应。
表1
Figure PCTCN2017101915-appb-000004
1.2 AF24-28AC的抗生素敏感情况
采用药敏纸片法对AF24-28AC对常见20种抗生素的敏感程度进行测试，取培养至对数期的AF24-28AC的菌液100ul进行平板涂布，将抗生素药敏片(购自杭州微生物试剂有限公司)贴在平板表面，37℃培养48h，测量抑菌圈大小，其结果如表2。
表2
Figure PCTCN2017101915-appb-000005
Figure PCTCN2017101915-appb-000006
结果显示，AF24-28AC对青霉素、苯唑西林、头孢哌酮有抗性，对其他17种抗生素均比较敏感。
实施例2单形巨单胞菌Megamonas funiformis AF24-28AC的生物活性物质
本实施例主要考察AF24-28AC在PYG培养48h的代谢产物的产生的情况，主要包括短链脂肪酸(SCFA)和有机酸的产物的含量，SCFA主要包括乙酸、丙酸、丁酸、戊酸，有机酸包括3-甲基丁酸，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，每种SCFA和有机酸的标准品均购自Sigma。检 测过程如下：
2.1样品预处理
取1ml AF24-28AC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。
2.2 SCFA的测定
SCFA的检测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。
2.3有机酸的测定
有机酸的检测也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。
4、实验结果
表3
Figure PCTCN2017101915-appb-000007
Figure PCTCN2017101915-appb-000008
表3的结果显示，AF24-28AC具有产生乙酸、丙酸、异戊酸、苯甲酸和乳酸的能力，能够少量产生戊酸、3-甲基丁酸、马来酸、丁二酸、苹果酸、己二酸和柠檬酸。
实施例3单形巨单胞菌Megamonas funiformis AF24-28AC的降胆固醇功能
胆固醇的含量测定方法采用邻苯二甲醛比色法(OPA法)，通过菌株在含一定浓度的胆固醇培养基中培养一段时间的胆固醇含量前后的变化来考察对胆固醇的降解能力。具体方如下：
(1)标准曲线的制作
精确量取0.5mg/mL的胆固醇标准溶液40uL，80uL，120uL，160uL，200uL于干净试管中，加入无水乙醇定容至1mL，每个试管中加入OPA 4mL(0.5mg邻苯二甲醛加入到1mL冰醋酸)，震荡混匀，室温静置10min，然后加入2mL的浓硫酸混匀，静置反应10min，于550nm处测定吸光度。以浓度作为横坐标，吸光度作为纵坐标绘制标准曲线(图3)，通过计算，线性回归的方程式为：y＝0.0085x+0.0072；相关系数R2为0.9992。
(2)胆固醇培养基的配制和实验菌株的培养
称取一定质量的胆固醇溶解于乙醇中，浓度为10mg/mL，过滤除菌。将配置好的PYG培养基分别加入10mg/mL的胆盐(高压灭菌)，10％质量浓度的巯基乙酸钠(过滤除菌)和胆固醇，充分混匀，然后按照3％的接种量将AF24-28AC接种至该培养基中，37℃厌氧条件下培养72h。
(3)胆固醇的测定
将含有胆固醇的PYG培养基培养好的菌液进行10000r/min的离心，收集上清，进行胆固醇检测，同时以未接种的胆固醇PYG培养基作为空白对照组。取500ul待测样品于干净的试管中，加入95％乙醇3ml和50％的KOH 2ml，震荡混匀，然后在60℃水浴中进行皂化反应10min，迅速进行冷却，加入5ml正己烷进行萃取，量取2.5ml有机相到另一洁净试管中，然后在60℃水浴中进行 氮气吹干，加入4ml 0.5g/L邻苯二甲醛乙酸溶液进行显色10min，添加2ml浓H2SO4反应10min，最后测量在550nm处的吸光值。
(4)胆固醇降解率的计算
胆固醇的降解按一下公式进行计算：
L＝(A-B)/A×100％
L:胆固醇降解率；
A：未接种菌的胆固醇培养基中胆固醇的含量；
B：培养菌之后胆固醇含量。
(5)胆固醇降解结果
通过计算，得到AF24-28AC的胆固醇降解率为68％。由此可见，AF24-28AC具有一定的胆固醇降解能力。
实施例4单形巨单胞菌Megamonas funiformis AF24-28AC对UC小鼠的治疗
本实施例所选取的小鼠模型为：DSS(硫酸葡聚糖钠Dextran Sulfate,Na，购自美国MPBIO)诱导的溃疡性肠炎小鼠模型，续给小鼠饮用0.15％的DSS(葡聚糖硫酸钠，分子量36000-50000)7天，小鼠品系为C57bl/6小鼠(购自湖北医学实验动物中心)，8周龄，体重20g±2g，在SPF级别的鼠房环境中饲养。
试验小鼠总计48只，随机分为4组，每组12只，包括：
正常组(control组)——采用普通饲料进行饲喂；
模型组——DSS造模，每只鼠每天灌胃0.2ml PBS；
AF24-28AC治疗组——DSS造模，每只鼠每天灌胃0.2ml AF24-28AC的菌液；
VSL#3治疗组——DSS造模，每只鼠每天灌胃0.2ml益生菌VSL#3(购自Alfasigma，USA，临床用于UC的治疗益生菌药物)；
AF24-28AC处理过程为：培养24h的AF24-28AC菌液，离心收集菌体，用PBS(磷酸缓冲液)进行悬浮，调整菌浓度至109cfu/ml，每只小鼠每天灌胃200ul的AF24-28AC。VSL#3也采用PBS进行悬浮，同样调节浓度至109cfu/ml，每只小鼠每天灌胃200ul。
在DSS造模的前3天开始灌胃AF24-28AC和VSL#3，DSS添加在小鼠饮水中，通过自由饮用进行UC的造模，持续7天，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评分标准详见表4。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4.DAI指数评分表
Figure PCTCN2017101915-appb-000009
表中的大便性状：正常大便-成形大便；松散大便-不粘附于肛门的糊状、半成型大便；稀便-可粘附于肛门的稀样水便。其中便血情况：正常小鼠便血为阳性；肉眼血便为红色或褐色；隐血阳性为不明显的肉眼血便，使用四甲基联苯胺进行检测。DAI指数等于体重下降、大便性状以及大便隐血三个积分之和(？？请对积分之和进行解释，用公式或文字都可以)。
以下，分别通过比较体重下降、DAI和结肠长度来考察AF24-28AC对DSS诱导的UC模型小鼠的治疗效果。
4.1体重变化
治疗前后小鼠的体重变化如下表5和图4所示：
表5
Figure PCTCN2017101915-appb-000010
表5的结果显示，Control组小鼠的体重呈缓慢升高的趋势，DSS诱导的3 组小鼠的体重均持续下降，在第3天，模型组相对于对照组体重下降开始显著(*P<0.05)，第7天，模型组和对照组之间的差异显著程度更加明显(**P<0.01)。而AF24-28AC和VSL#3的干预可以减缓UC小鼠体重的下降，在第7天，这两组小鼠的体重下降的控制相对于模型组比较显著(▲P<0.05)。说明AF24-28AC和VSL#3可以控制UC引起的体重下降情况。在第7天AF24-28AC组小鼠的体重略高于VSL#3，说明AF24-28AC在控制UC小鼠体重降低的效果可以达到VSL#3的效果。
4.2 DAI指数的变化
DSS诱导的溃疡性肠炎的小鼠由于体重下降、大便性状和便血情况的变化引起DAI指数的变化，小鼠DAI指数在治疗前后的变化如表6和图5：
表6
Figure PCTCN2017101915-appb-000011
表6和图5数据表明，Control组小鼠的DAI基本持平，而随着DSS诱导，模型组、AF24-28AC和VSL#3组小鼠的DAI逐渐升高，第三天模型组小鼠DAI相对于对照组变得开始显著升高(*P<0.05)，第7天模型组小鼠DAI达到最高水平(相对于control组**P<0.01)。本发明的菌的干预可以控制DAI的升高，AF24-28AC组小鼠在第5天和第7天的DAI相对于对照组得到了显著的控制(▲P<0.05)，AF24-28AC组小鼠的DAI低于VSL#3，且VSL#3在控制DAI方面不显著，因此，AF24-28AC在控制UC小鼠DAI升高的效果优于VSL#3。
4.3结肠长度的变化
UC模型小鼠的结肠组织会发生改变，因为溃疡和炎症的发生导致结肠组织缩短，治疗结束后，通过解剖测量的小鼠结肠长度见表7。
表7
Figure PCTCN2017101915-appb-000012
Figure PCTCN2017101915-appb-000013
表7的结果显示，在进行DSS诱导7天后的小鼠(模型组)的结肠组织缩短情况比较严重，与control组相比较非常显著(**P<0.01)。而而本发明的菌AF24-28AC和VSL#3的干预可以显著控制小鼠结肠的缩短(相对于模型组非常显著，*P<0.01)。通过表7中数据可以发现，AF24-28AC组小鼠的结肠长度比VSL#3组小鼠结肠长度长，因此，AF24-28AC在控制UC小鼠结肠缩短的能力略强于VSL#3。由此可见，本发明的菌AF24-28AC可以显著减缓小鼠结肠的病变，其效果与VSL#3相当，甚至略优于VSL#3。
实施例5含单形巨单胞菌Megamonas funiformis AF24-28AC的食品组合物
原料配比如表8。
表8
原料 质量百分比(％)
Megamonas funiformis AF24-28AC 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Megamonas funiformis AF24-28AC菌，即制成含Megamonas funiformis AF24-28AC菌的食品组合物。
实施例6含单形巨单胞菌Megamonas funiformis AF24-28AC的药物组合物
原料配比见表9。
表9
原料 质量百分比(％)
Megamonas funiformis AF24-28AC 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℃，混入维生素C，接入Megamonas funiformis AF24-28AC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Megamonas funiformis AF24-28AC菌冷冻干燥物。称取0.5克Megamonas funiformis AF24-28AC冷冻干燥物与麦芽糊精、保护剂(如维生素C、半胱氨酸)等量混合后装入胶囊中，即制成含Megamonas funiformis AF24-28AC菌的药物组合物。
实施例7一种用于治疗炎症相关疾病(如溃疡性肠炎(UC))的药物的制备方法
1、菌液准备：将Megamonas funiformis AF24-28AC(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。
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成分)。
3、药物或药物剂型制作：将5体积(ml)生长因子和1体积(ml)的保护剂(如维生素C、半胱氨酸)加入到100体积(ml)的Megamonas funiformis AF24-28AC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)，从而制备含有Megamonas funiformis AF24-28AC的药物或药物剂型。
实施例8一种用于治疗心血管疾病(如高血脂)的药物的制备方法
1、菌液准备：将Megamonas funiformis AF24-28AC(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。
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成分)。
3、药物或药物剂型制作：将5体积(ml)生长因子和1体积(ml)的保护剂(如维生素C、半胱氨酸)加入到100体积(ml)的Megamonas funiformis AF24-28AC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)，从而制备含有Megamonas funiformis AF24-28AC的药物或药物剂型。
在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 (16)

  1. 一种单形巨单胞菌，其特征在于，所述单形巨单胞菌为单形巨单胞菌(Megamonas funiformis)。
  2. 如权利要求1所述的单形巨单胞菌，其特征在于，所述的单形巨单胞菌的16s rDNA的序列如SEQ ID NO.:1所示。
  3. 如权利要求1所述的单形巨单胞菌，其特征在于，所述单形巨单胞菌为Megamonas funiformis AF24-28AC，保藏号为GDMCC 60093。
  4. 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所述的单形巨单胞菌和/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。
  5. 如权利要求4所述的组合物，其特征在于，所述组合物含有1×10-1×1015cfu/mL或cfu/g Megamonas funiformis AF24-28AC，较佳地1×104-1×1010cfu/mL或cfu/g Megamonas funiformis AF24-28AC，按所述组合物的总体积或总重量计。
  6. 如权利要求4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。
  7. 如权利要求4所述的组合物，其特征在于，所述组合物还含有有助于保持单形巨单胞菌活力的物质。
  8. 如权利要求7所述的组合物，其特征在于，所述有助于保持单形巨单胞菌活力的物质选自下组：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、维生素E、或其组合。
  9. 一种权利要求1所述的单形巨单胞菌、或权利要求4所述的组合物的用途，其特征在于，用于制备组合物或制剂，所述组合物或制剂用于选自下组的一种或多种用途：(a)预防和/或治疗炎症相关疾病；和/或(b)预防和/或治疗心血管疾病。
  10. 一种权利要求1所述的单形巨单胞菌、或权利要求4所述的组合物的用途，其特征在于，用于制备组合物或制剂，所述组合物或制剂用于选自下组的一种或多种用途：
    (i)降低哺乳动物的血脂水平；
    (ii)控制哺乳动物体重的降低；
    (iii)降低哺乳动物的疾病活动指数(DAI)；
    (iv)改善哺乳动物的肠道病变。
  11. 一种权利要求4所述组合物的制法，其特征在于，包括步骤：
    将权利要求1所述的单形巨单胞菌和/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4所述的组合物。
  12. 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所述的单形巨单胞菌进行培养，从而获得培养产物；
    (b)任选地，从所述培养产物分离单形巨单胞菌菌体和/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单形巨单胞菌菌体和/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。
  13. 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4所述的组合物。
  14. 一种降低哺乳动物的血脂水平、控制哺乳动物体重的降低和/或降低哺乳动物的疾病活动指数的方法，其特征在于，给所述对象施用权利要求4所述的组合物。
  15. 一种预防和/或治疗炎症相关疾病的方法，其特征在于，包括步骤：
    给所述对象施用权利要求4所述的组合物，从而预防和/或治疗炎症相关疾病。
  16. 一种预防和/或治疗心血管疾病的方法，其特征在于，包括步骤：
    给所述对象施用权利要求4所述的组合物，从而预防和/或治疗心血管疾病。
PCT/CN2017/101915 2017-09-15 2017-09-15 单形巨单胞菌(Megamonas funiformis)及其应用 Ceased WO2019051790A1 (zh)

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