CN111448306B - Anaerobic coryneform faecalis (Anaerofustis stercorihominis) and application thereof - Google Patents

Anaerobic coryneform faecalis (Anaerofustis stercorihominis) and application thereof Download PDF

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CN111448306B
CN111448306B CN201780094884.3A CN201780094884A CN111448306B CN 111448306 B CN111448306 B CN 111448306B CN 201780094884 A CN201780094884 A CN 201780094884A CN 111448306 B CN111448306 B CN 111448306B
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CN111448306A (en
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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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Abstract

Provided are a corynebacterium faecalis (Anaerofustis stercorihominis) and uses thereof, wherein the corynebacterium faecalis (Anaerofustis stercorihominis) has effects of preventing and/or treating inflammation-related diseases (such as inflammatory bowel disease (such as ulcerative enteritis, gastritis, common enteritis) and rheumatoid arthritis).

Description

Anaerobic coryneform faecalis (Anaerofustis stercorihominis) and application thereof
Technical Field
The invention relates to the field of microorganisms, in particular to anaerobic coryneform faecalis (Anaerofustis stercorihominis) and application thereof.
Background
Inflammatory bowel disease (inflammatory bowel disease, IBD) is a chronic inflammatory bowel disease of unknown etiology, which is easily repeated, severely affecting the quality of life of the patient. Modern medicine considers that factors responsible for Inflammatory Bowel Disease (IBD) are genetic, dietary, infectious, autoimmune, psychological factors, environmental, and the like. Inflammatory bowel diseases, including ulcerative enteritis (UC) and Crohn's Disease (CD), are all inflammation-related disorders.
Ulcerative enteritis (ulcerative colitis, UC) is an important disease type of inflammatory bowel disease (inflammatory bowel disease, IBD) whose pathogenesis is unknown, the main lesion being submucosa in the colonic mucosa, which is a chronic intestinal disease. Based on the current research, the cause of ulcerative enteritis is mainly considered to have host genetic susceptibility, intestinal flora and immune response of intestinal mucosa, clinical pathology is manifested by continuous abdominal pain, diarrhea and mucous bloody stool, and the disease is repeated, and the number of patients suffering from UC in China has a remarkable rising trend in recent years.
At present, the clinical medicine aiming at ulcerative enteritis (UC) mainly comprises salicylic acid, adrenoglucocorticoid and immune preparation. The salicylic acid medicine can well inhibit prostaglandin synthesis and remove oxygen free radicals, thereby achieving the purpose of relieving inflammatory reaction, but can only relieve in a short period of time, and cannot realize radical treatment. The common salicylic acid western medicine for clinically treating ulcerative enteritis (UC) is mainly sulfasalazine (SASP), and mainly aims at mild, moderate and chronic UC patients; adrenoglucocorticoid is the first drug of choice for patients with severe or explosive UC, such as betamethasone; immunosuppressants such as cyclosporine may inhibit ulcerative enteritis (UC) by inhibiting the production of T-cell IL-2 and affecting the progression of the immune response.
The existing three medicines aiming at ulcerative enteritis (UC) can relieve the UC to a certain extent, but have certain side effects, and the side effects of salicylic acid are rash, hepatotoxicity, leukopenia, anemia and the like caused by digestive tract reaction, headache, reticulocytosis, oligospermia and anaphylaxis, and the medicines have antibacterial effect and are easy to cause flora disorder and drug resistance enhancement. The adrenoglucocorticoid can cause side effects such as metabolic disturbance, water retention and the like of organisms, and can only be used as emergency medication and cannot be taken for a long time. Immunosuppressant treatment has great dependence on medicines, long treatment period, easy nephrotoxicity and secondary infection, and can be used as an auxiliary treatment means.
Therefore, there is an urgent need in the art to develop a new drug for treating and/or preventing inflammation-related diseases without toxic or side effects.
Disclosure of Invention
The invention aims to provide a novel medicine without toxic or side effect, which is used for treating and/or preventing inflammation-related diseases.
The first aspect of the invention provides a fecal anaerobic coryneform bacterium, characterized in that the fecal anaerobic coryneform bacterium is a fecal anaerobic coryneform bacterium (Anaerofustis stercorihominis).
In another preferred example, the 16s rDNA of the anaerobic coryneform fecal bacterium has the sequence set forth in SEQ ID No.: 1.
In another preferred embodiment, the anaerobic coryneform fecal bacteria is Anaerofustis stercorihominis AM-6 and accession number GDMCC 60087.
In another preferred embodiment, the anaerobic coryneform fecal bacteria are from the gut, animal manure, fermentation tanks, and/or anaerobic reactors.
In another preferred embodiment, the anaerobic coryneform fecal bacteria are derived from a human or non-human mammal.
In another preferred embodiment, the non-human mammal comprises a rodent (e.g., mouse, rat), primate (e.g., monkey).
In a second aspect, the present invention provides a composition comprising: (a) A safe and effective amount of the coryneform faecalis and/or a metabolite thereof according to the first aspect of the present invention; and (b) a food-acceptable carrier or a pharmaceutically acceptable carrier.
In another preferred embodiment, the composition further comprises a growth factor (preferably, a milk growth factor).
In another preferred embodiment, the composition is selected from the group consisting of: a food composition, a health composition, a pharmaceutical composition, a beverage composition, a feed composition, or a combination thereof.
In another preferred embodiment, the composition is an oral formulation.
In another preferred embodiment, the composition is a liquid formulation, a solid formulation, or a semi-solid formulation.
In another preferred embodiment, the composition is in a dosage form selected from the group consisting of: powders, tablets, dragees, capsules, granules, suspensions, solutions, syrups, drops, sublingual tablets, or combinations thereof.
In another preferred embodiment, the food composition comprises an emulsion product, a solution product, a powder product, or a suspension product.
In another preferred embodiment, the food composition comprises milk, milk powder, or emulsion.
In another preferred embodiment, the liquid formulation is selected from the group consisting of: a solution or suspension product.
In another preferred embodiment, the composition comprises 1X 10-1X 10 15 cfu/mL or cfu/g Anaerofustis stercorihominis AM25-6, preferably 1X 10 4 -1×10 10 cfu/mL or cfu/g Anaerofustis stercorihominis AM25-6, based on the total volume or weight of the composition.
In another preferred embodiment, the composition comprises 0.0001 to 99wt%, preferably 0.1 to 90wt% of the anaerobic coryneform faecalis and/or a metabolite thereof, based on the total weight of the composition.
In another preferred embodiment, the composition is in unit dosage form (tablet, capsule or vial) and the mass of the composition in each unit dosage form is from 0.05 to 5g, preferably from 0.1 to 1g.
In another preferred embodiment, the composition further comprises other probiotics and/or prebiotics.
In another preferred embodiment, the probiotic is selected from the group consisting of: lactic acid bacteria, bifidobacteria, lactobacillus acidophilus, or combinations thereof.
In another preferred embodiment, the prebiotic is selected from the group consisting of: fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), xylo-oligosaccharides (XOS), lactulose-oligosaccharides (LACT), soy Oligosaccharides (SOS), inulin (Inul in), oligosaccharides, or combinations thereof.
In another preferred embodiment, the composition further comprises a substance (e.g., a protectant) that helps to maintain the viability of the anaerobic coryneform fecal bacteria.
In another preferred embodiment, the substance (e.g., protectant) that helps to maintain the viability of the anaerobic bar bacteria is selected from the group consisting of: cysteine, glutathione, butyl hydroxy anisole, dibutyl methyl toluene, tocopherol, bamboo leaf antioxidants, D-isoascorbic acid, sodium salts thereof, sodium ascorbate, calcium ascorbate, phospholipids, vitamin C (ascorbic acid), vitamin E, or combinations thereof.
In another preferred embodiment, the weight ratio of the substance (e.g. protectant) that helps to maintain the viability of the anaerobic bar bacteria is from 0.1 to 2%, preferably from 0.5 to 1.5%, more preferably from 0.5 to 1.0%, based on the total weight of the composition.
In another preferred embodiment, the substance (e.g., protectant) that helps to maintain the viability of the anaerobic coryneform bacteria in the manure is present in an amount of 1mg to 20mg, preferably 5mg to 15mg, more preferably 5mg to 10mg, based on 1g of the composition.
In a third aspect the present invention provides the use of a coryneform faecalis according to the first aspect of the invention, or a composition according to the second aspect of the invention, for the preparation of a composition or formulation for the prevention and/or treatment of an inflammation-related disorder.
In another preferred embodiment, the inflammation-related disorder is selected from the group consisting of: inflammatory bowel disease, rheumatoid arthritis, or a combination thereof.
In another preferred embodiment, the inflammation-related disorder is selected from the group consisting of: ulcerative enteritis, gastritis, plain enteritis, or a combination thereof.
In another preferred embodiment, the formulation comprises a probiotic.
In a fourth aspect the present invention provides the use of a coryneform faecalis according to the first aspect of the invention, or a composition according to the second aspect of the invention, for the preparation of a composition or formulation for one or more uses selected from the group consisting of:
(i) Controlling a decrease in body weight of the mammal;
(ii) Lowering the Disease Activity Index (DAI) of the mammal;
(iii) Improving intestinal lesions in mammals.
In another preferred embodiment, the reduction in weight of the control mammal means a reduction in weight of the experimental group mammal of not more than 10%, preferably not more than 5%, more preferably not more than 2% as compared to the model group mammal.
In another preferred embodiment, the improving intestinal lesions in a mammal comprises slowing the shortening of the length of the colon, and/or reducing the inflammatory response of the colon.
In another preferred embodiment, the mammal comprises a human or a non-human mammal.
In another preferred embodiment, the non-human mammal comprises a rodent (e.g., mouse, rat), primate (e.g., monkey).
In a fifth aspect, the present invention provides a method of preparing a composition according to the second aspect of the invention, comprising the steps of:
the composition of the second aspect of the invention is formed by mixing the anaerobic coryneform faecalis and/or metabolite thereof of the first aspect of the invention with a food or pharmaceutical acceptable carrier.
In another preferred embodiment, the method further comprises the step of mixing with a growth factor.
In another preferred embodiment, the method further comprises the step of mixing with a substance (e.g., a protectant) that helps to maintain the viability of the anaerobic coryneform fecal bacteria.
In another preferred embodiment, the substance (e.g., protectant) that helps to maintain the viability of the anaerobic bar bacteria is selected from the group consisting of: cysteine, glutathione, butyl hydroxy anisole, dibutyl methyl toluene, tocopherol, bamboo leaf antioxidants, D-isoascorbic acid, sodium salts thereof, sodium ascorbate, calcium ascorbate, phospholipids, vitamin C (ascorbic acid), vitamin E, or combinations thereof.
In another preferred embodiment, the method further comprises the step of mixing with a probiotic, and/or a prebiotic.
In another preferred embodiment, the probiotic is selected from the group consisting of: lactic acid bacteria, bifidobacteria, lactobacillus acidophilus, or combinations thereof.
In another preferred embodiment, the prebiotic is selected from the group consisting of: fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), xylo-oligosaccharides (XOS), lactulose-oligosaccharides (LACT), soy Oligosaccharides (SOS), inulin (Inulin), oligosaccharides, or combinations thereof.
In another preferred embodiment, the growth factor is a milk growth factor.
In another preferred embodiment, the growth factor is selected from the group consisting of: vitamins, purines, pyrimidines, or combinations thereof.
In another preferred embodiment, the composition is an oral formulation.
The sixth aspect of the present invention provides a production method comprising the steps of:
(a) Culturing the anaerobic coryneform fecal bacteria according to the first aspect of the present invention under conditions suitable for culturing, thereby obtaining a culture product;
(b) Optionally, isolating the anaerobic coryneform fecal bacteria and/or metabolites thereof from the culture product; and/or
(c) Optionally, the culture product or the anaerobic coryneform fecal bacteria and/or the metabolites thereof obtained in the previous step are mixed with a food acceptable carrier or a pharmaceutically acceptable carrier, thereby producing the composition of the present invention.
In another preferred embodiment, before step (c), the method further comprises a step of mixing the culture product or the fecal anaerobic coryneform bacteria obtained in the previous step and/or a metabolite thereof with a growth factor.
In another preferred embodiment, the growth factor is a milk growth factor.
In another preferred embodiment, the growth factor is selected from the group consisting of: vitamins, purines, pyrimidines, or combinations thereof.
In another preferred embodiment, before step (c), the method further comprises the step of mixing the culture product or the fecal anaerobic coryneform bacteria obtained in the previous step and/or the metabolite thereof with a substance (such as a protective agent) that helps to maintain the activity of the fecal anaerobic coryneform bacteria.
In another preferred embodiment, before step (c), the method further comprises the step of mixing the culture product or the fecal anaerobic coryneform bacteria and/or its metabolites obtained in the previous step with probiotics and/or prebiotics.
In a seventh aspect, the invention provides a method of ameliorating intestinal lesions in a mammal by administering to said subject a composition according to the second aspect of the invention.
In another preferred embodiment, said administration comprises oral administration.
In another preferred embodiment, the dosage is 0.01-5g/50kg body weight/day, preferably 0.1-2g/50kg body weight/day.
In another preferred embodiment, the subject comprises a human or non-human mammal.
In another preferred embodiment, the non-human mammal comprises a rodent (e.g., mouse, rat), primate (e.g., monkey).
In another preferred embodiment, the method is non-diagnostic and non-therapeutic.
In an eighth aspect, the invention provides a method of controlling weight loss in a mammal, and/or reducing disease activity index in a mammal, characterized by administering to said subject a composition according to the second aspect of the invention.
In another preferred embodiment, said administration comprises oral administration.
In another preferred embodiment, the dosage is 0.01-5g/50kg body weight/day, preferably 0.1-2g/50kg body weight/day.
In another preferred embodiment, the subject comprises a human or non-human mammal.
In another preferred embodiment, the non-human mammal comprises a rodent (e.g., mouse, rat), primate (e.g., monkey).
In another preferred embodiment, the method is non-diagnostic and non-therapeutic.
In a ninth aspect, the present invention provides a method for preventing and/or treating an inflammation-related disorder, comprising the steps of:
administering to said subject a composition according to the second aspect of the invention, thereby preventing and/or treating an inflammation-related disorder.
In another preferred embodiment, said administration comprises oral administration.
In another preferred embodiment, the dosage is 0.01-5g/50kg body weight/day, preferably 0.1-2g/50kg body weight/day.
In another preferred embodiment, the subject comprises a human or non-human mammal.
In another preferred embodiment, the non-human mammal comprises a rodent (e.g., mouse, rat), primate (e.g., monkey).
In another preferred embodiment, the method is non-diagnostic and non-therapeutic.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
FIG. 1 shows a picture of a colony cultivated for 48h by human anaerobic coryneform bacteria Anaerofustis stercorihominis AM-6.
FIG. 2 shows a gram (1000-fold) of human manure anaerobioscles Anaerofustis stercorihominis AM-6 under a microscope.
FIG. 3 shows Control group, model group, VSL # Body weight changes in mice in the 3 and AM25-6 treatment groups. Figure 3 shows that the body weight of the control group mice remained essentially on a slow rise, and that the DSS-induced mice in the model group had a gradual decrease in body weight, day 7, with the body weight of the mice in the model group being most pronounced relative to the control group. While AM25-6 and VSL # 3 can slow the weight loss of UC mice, AM25-6 and VSL on day 7 # The control of weight loss in 3 mice was more pronounced relative to the model group. Indicating that these two groups of probiotics can control the weight loss caused by UC. By comparing the weight values of the groups on day 7, it was found that the weight of the mice in the AM25-6 group was slightly higher than that of VSL # 3, the AM25-6 is slightly better than VSL in controlling the weight loss of UC mice # 3。
FIG. 4 shows Control group, model group, VSL # Changes in DAI index in mice of the 3 and AM25-6 treatment groups. Drawing of the figureThe data of 4 indicate that the DAI of the Control group of mice maintained a positive constant low level, whereas the DSS-induced mice had progressively higher DAI due to a series of pathologies, the day 7 model group of mice became significantly more pronounced than the Control group, and the day 7 model group of mice reached the highest level of DAI. The probiotic intervention can control the elevation of DAI, with significant levels of AM25-6 at day 5 relative to the model group, and AM25-6 and VSL at day 7 # The DAI of group 3 is controlled to some extent relative to the model group. By day 7 DAI values, it was found that the DAI was slightly lower than the VSL in the AM25-6 mice # 3, it can be shown that AM25-6 has slightly better effect than VSL in controlling the rise of DAI in UC mice # 3。
Detailed Description
The inventor of the present invention has conducted extensive and intensive studies and experiments, and has unexpectedly found that the anaerobic coryneform fecal bacteria (Anaerofustis stercorihominis) has an effect of preventing and/or treating diseases associated with inflammation (such as inflammatory bowel disease (such as ulcerative enteritis, gastritis, general enteritis), and rheumatoid arthritis), and that feeding an active composition containing the anaerobic coryneform fecal bacteria of the present invention to a subject for feeding, the composition can control weight loss, reduce Disease Activity Index (DAI), improve intestinal lesions, and effectively alleviate diseases associated with inflammation (such as inflammatory bowel disease (such as ulcerative enteritis, gastritis, general enteritis), and rheumatoid arthritis). The present inventors have completed the present invention on the basis of this.
As used herein, the term "comprising" means that the various ingredients may be applied together in a mixture or composition of the invention. Thus, the terms "consisting essentially of and" consisting of are encompassed by the term "containing.
As used herein, the term "growth factor" includes milk growth factors, and specifically, includes nutrients of vitamins, purines, pyrimidines, or combinations thereof.
Wherein the vitamins include (but are not limited to): vitamin C, vitamin E, vitamin A precursor, vitamin B 6 Vitamin D 3 Vitamin K, folic acid, or a combination thereof;
the purine materials include (but are not limited to): a purine nucleoside, wherein the purine nucleoside comprises a 5' -phosphate of the purine nucleoside; the 5' -phosphate of purine nucleosides is selected from the group consisting of: inosinic acid (inosine-5 '-phosphate; IMP), guanylic acid (guanosine-5' -phosphate; GMP), xanthylic acid (xanthosine-5 '-phosphate; XMP), adenylate (adenosine-5' -phosphate; AMP), or a combination thereof;
the pyrimidine includes all substances containing pyrimidine structures.
As used herein, the terms "controlling a decrease in weight of a mammal", "slowing down a decrease in weight of a mammal", "controlling a decrease in weight of a mammal", and "slowing down a decrease in weight of a mammal" are used interchangeably to refer to a decrease in weight of a subject animal as a result of a progressive severity of inflammation during the course of a model of ulcerative colitis, as a percentage of the decrease in weight relative to the original weight. The higher the weight reduction degree is, the more serious the disease is, and the anaerobic coryneform fecal bacteria can control the weight reduction of experimental animals and alleviate the symptoms of the disease in the treatment process of ulcerative enteritis of mammals.
Disease Activity Index (DAI)
As used herein, the term "disease activity index" refers to a composite score that is made in conjunction with 3 conditions of percent weight loss, stool consistency, and stool bleeding in a patient (or diseased animal).
Anaerobic coryneform bacterium and application thereof
As used herein, the terms "anaerobic bar bacteria", "Anaerofustis stercorihominis", "anaerobic bar bacteria of the invention" are used interchangeably. In a preferred embodiment, the strain is Anaerofustis stercorihominis AM-6, accession number GDMCC 60087, isolated from human (preferably, healthy male) faeces. The physiological characteristics of the anaerobic coryneform faecalis are as follows: the anaerobic coryneform faecalis Anaerofustis stercorihominis AM-6 is separated by PYG culture medium, and the separation condition is anaerobic condition at 37 ℃. AM25-6 was light yellow in colony, smaller in colony, needle-tip-shaped and about 0.5mm in diameter, cultured in PYG medium for 2 days. The thallus has short rod shape, gram positive shape, and no spore or flagellum. The test results of catalase and oxidase are negative, and various carbohydrates including glucose, mannitol, lactose, sucrose, maltose, liu Chun, xylose, mannose, triose, raffinose, sorbitol, rhamnose and trehalose can be fermented, acetic acid, butyric acid, isovaleric acid, benzoic acid and lactic acid are mainly produced, and isobutyric acid, valeric acid, 3-methylbutyric acid, succinic acid, adipic acid and citric acid can be produced in small amounts. And the inventive anaerobic coryneform faecalis Anaerofustis stercorihominis AM-6 is relatively sensitive to 20 common antibiotics in table 2.
The invention provides application of corynebacterium faecalis in treating and/or preventing inflammation-related diseases (such as inflammatory bowel diseases (such as ulcerative enteritis, gastritis and common enteritis) and rheumatoid arthritis). Subjects were induced to model with DSS (sodium dextran sulfate), strain Anaerofustis stercorihominis AM25-6 had one or more uses selected from the group consisting of: (i) controlling weight loss in the subject; (ii) decreasing Disease Activity Index (DAI); (iii) improving the extent of intestinal lesions. According to a preferred embodiment of the present invention, C57bl/6 mice are used as test mice, and induced modeling is performed with DSS (dextran sodium sulfate) to obtain ulcerative enteritis (UC) model mice, and UC model mice treated with Anaerofustis stercorihominis AM-6 have a reduced weight loss compared with untreated control groups (model groups), and various indexes related to inflammation (such as inflammatory bowel disease (such as ulcerative enteritis, gastritis, plain enteritis) and rheumatoid arthritis) are improved, such as improving intestinal lesions (including reducing colon length shortening, reducing inflammatory reaction of colon, etc.), reducing Disease Activity Index (DAI), etc. Thus, the strain can be used for preventing and/or treating inflammation-related diseases (such as inflammatory bowel disease (such as ulcerative enteritis, gastritis, plain enteritis), rheumatoid arthritis).
Composition and application thereof
The present invention also provides a composition, preferably comprising a food composition, a health composition, a pharmaceutical composition, a beverage composition, or a feed composition, preferably a pharmaceutical composition. The composition comprises an effective amount of a fecal anaerobic coryneform bacterium, and in a preferred embodiment, the composition further comprises a growth factor (e.g., a milk growth factor). In a preferred embodiment, the composition further comprises a probiotic selected from the group consisting of: lactic acid bacteria, bifidobacteria, lactobacillus acidophilus, or combinations thereof; and/or a prebiotic selected from the group consisting of: fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), xylo-oligosaccharides (XOS), lactulose-oligosaccharides (LACT), soy Oligosaccharides (SOS), inulin (Inulin), oligosaccharides, or combinations thereof. In a preferred embodiment, the composition further comprises a substance (e.g., a protectant) selected from the group consisting of: cysteine, glutathione, butyl hydroxy anisole, dibutyl methyl toluene, tocopherol, bamboo leaf antioxidants, D-isoascorbic acid, sodium salts thereof, sodium ascorbate, calcium ascorbate, phospholipids, vitamin C (ascorbic acid), vitamin E, or combinations thereof. The weight ratio of the substance (such as a protective agent) which helps to maintain the activity of the anaerobic coryneform faecalis is 0.1-2%, preferably 0.5-1.5%, more preferably 0.5-1.0% based on the total weight of the composition.
In a preferred embodiment, the composition is a liquid formulation, a solid formulation, or a semi-solid formulation.
In a preferred embodiment, the liquid formulation is selected from the group consisting of: a solution or suspension product.
In a preferred embodiment, the composition is in a dosage form selected from the group consisting of: powders, tablets, dragees, capsules, granules, suspensions, solutions, syrups, drops, sublingual tablets, or combinations thereof.
The composition of the present invention may be administered in any form of an oral liquid, a tablet, an injection, an orally disintegrating tablet, a lyophilized powder preparation or a capsule, preferably an enteric-coated preparation (e.g., a capsule), and in the present invention, the excipient, the pharmaceutically acceptable vehicle and the carrier used in the present invention are selected mainly according to the characteristics of a suitable bacterium or its metabolite and the specific administration mode required, so that the bacterium or its metabolite can smoothly pass through the stomach to be absorbed by the person to be administered, unless otherwise specified. These substances may be selected according to the route of administration.
The compositions of the present invention may further comprise any additional excipients among those commonly used in pharmaceutical formulations, for example, in order to stabilize the composition itself, or to make it easy to disperse or to impart a suitable taste thereto.
Among the excipients, inulin, fructose, starch, xylo-oligosaccharides, silica, buffering agents and flavourings are suitable examples.
The pharmaceutical preparation of the present invention may further comprise auxiliary active ingredients.
Lactose, maltodextrin, dextrose, sucrose, sorbitol, mannose, starch, acacia, calcium phosphate, alginate, gelatin, calcium silicate, finely crystalline cellulose, polyvinylpyrrolidone (PVP), cellulose, water, syrup, methylcellulose, methylparaben, propylhydroxybenzoate, talc, magnesium stearate or mineral oil and the like may be used as carriers, excipients or diluents for the pharmaceutical compositions of the invention.
In addition, the pharmaceutical composition of the present invention may further comprise lubricants, wetting agents, emulsifiers, suspension stabilizers, preservatives, sweeteners, flavors and the like. The pharmaceutical composition of the present invention may be produced in enteric coated formulations by a variety of well known methods so that the active ingredient of the pharmaceutical composition, i.e., the microorganism, can pass smoothly through the stomach without being destroyed by gastric acid.
In addition, the microorganism of the present invention may be used in the form of a capsule prepared by a conventional method. For example, standard excipients and the cold dry microorganisms of the present invention are mixed to form pellets, which are then filled into gelatin capsules. In addition, the microorganisms and the medicaments of the present invention allow the use of excipients such as liquid gums, celluloses, silicates or mineral oils, etc. mixed to make suspensions or dispersions which can be filled into soft gelatin capsules.
The pharmaceutical composition of the present application can be prepared into enteric coated tablets for oral use. The term "enteric coating" in the present application includes all coatings which are allowed to be applied by conventional drugs, which are not degraded by gastric acid, but are sufficiently decomposed in the small intestine and rapidly released from the microorganisms of the present application. The casing of the present application is capable of being maintained in synthetic gastric acid, such as HCl solution at ph=1, for more than 2 hours at 36-38 ℃ and preferably disintegrated in synthetic intestinal fluid, such as buffer at ph=7.0, within 1.0 hour.
The enteric coating of the present application is coated at about 16-30mg, preferably 16-25mg, more preferably 16-20mg per tablet. The thickness of the enteric coating in the present application is 5-100. Mu.m, and the desirable thickness is 20-80. Mu.m. The enteric coating ingredients are selected from conventional polymers known per se.
Preferred casings of the application are prepared from copolymers of cellulose acetate phthalate or trimellitate polymers and methacrylic acid (e.g., copolymers containing greater than 40% methacrylic acid and methacrylic acid containing hydroxypropyl methylcellulose phthalate or ester derivatives thereof).
The viscosity of the cellulose acetate phthalate used in the casing of the present application is about 45-90cp, acetyl content 17-26%, phthalic acid content 30-40%. The cellulose acetate trimellitate used in the casing has a viscosity of about 5-21cp and an ethyl phthalide content of 17-26%. Cellulose trimellitate acetate is produced by Eastman koda corporation and can be used in the enteric coating material of the present application.
The hydroxypropyl methylcellulose phthalate used in the casings of the present invention generally has a molecular weight of 20,000 to 130,000 daltons, desirably a molecular weight of 80,000 to 100,000 daltons, a hydroxypropyl content of 5 to 10%, a methoxy content of 18 to 24%, and a phthaloyl content of 21 to 35%.
The hydroxypropyl methylcellulose phthalate used in the casing of the present invention is HP50, produced by Shin-Etsu Chemidnl co.ltd. HP50 contains 6-10% hydroxypropyl, 20-24% methoxy, 21-27% propyl, and has a molecular weight of 84,000 daltons. Another enteric coating material is HP55, HP55 containing 5-9% hydroxypropyl methylcellulose phthalate, 18-22% methoxy, 27-35% phthalic acid, and having a molecular weight of 78,000 daltons.
The casing of the present invention is prepared as follows: the enteric coating solution is sprayed 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., methylene chloride), or combinations thereof. Softeners such as di-n-butyl phthalate and glyceryl triacetate are added to the casing solution in a ratio of 1 part of casing to about 0.05 parts or about 0.3 parts of softener. The spraying process is preferably carried out continuously, the amount of sprayed material being controlled according to the conditions used for coating. The spray pressure can be adjusted at will and in general, the desired result can be obtained at an average pressure of 1 to 1.5 bar.
In the specification, "pharmaceutically effective amount" means an amount that is functionally or actively acceptable to and acceptable to humans and/or animals. For example, in the present invention, a composition containing 1X 10-1X 10 can be prepared 15 cfu/ml or cfu/g (may contain, in particular, 1X 10) 4 -1×10 10 cfu/ml or cfu/g; more particularly, may contain 1X 10 6 -1×10 10 cfu/ml or cfu/g) of anaerobiosis faecalis and/or a metabolite thereof.
When used in the preparation of a pharmaceutical composition, the effective dose of the anaerobic coryneform faecalis or metabolite thereof used may vary with the mode of administration and the severity of the disease to be treated. Dosage forms suitable for oral administration comprising about 1X 10-1X 10 intimately admixed with a solid or liquid pharmaceutically acceptable carrier 15 cfu/ml or cfu/g (preferably, may contain 1X 10) 4 -1×10 10 cfu/ml or cfu/g; more preferably, it may contain 1X 10 6 -1×10 10 cfu/ml or cfu/g) of active manure anaerobioscles or active ingredients produced by fermentation. This dosage regimen can be adjusted to provide the optimal therapeutic response. For example, separate doses may be administered several times per day, or the dose may be proportionally reduced, as dictated by the urgent need for the treatment of the condition.
The anaerobic coryneform faecalis or the metabolite thereof can be administered by oral administration and the like. The solid support comprises: starch, lactose, dicalcium phosphate, microcrystalline cellulose, sucrose and kaolin, while the liquid carrier includes: culture medium, polyethylene glycol, nonionic surfactant and edible oils (such as corn oil, peanut oil and sesame oil) as long as they are suitable for the characteristics of the anaerobic coryneform bacterium or its metabolites and the particular mode of administration desired. Adjuvants commonly used in the preparation of pharmaceutical compositions may also advantageously be included, for example flavouring agents, pigments, preservatives and antioxidants such as vitamin E, vitamin C, BHT and BHA.
Preferred pharmaceutical compositions are solid compositions, especially tablets and/or solid filled or liquid filled capsules, from the standpoint of ease of preparation and administration. Oral administration is preferred.
The composition of the invention is administered to the subject 1 or more times per day. The unit of administration dose means a dose which can be divided in form and which is suitable for use in humans or all other mammalian subjects. Each unit containing a pharmaceutically acceptable carrier and a therapeutically effective amount of a microorganism of the present invention. The amount administered will vary with the weight of the patient and the severity of the inflammation-associated disorder (e.g., inflammatory bowel disease (e.g., ulcerative enteritis, gastritis, plain enteritis), rheumatoid arthritis), the supplemental active ingredient included, and the microorganism employed. Further, if possible, the administration may be divided, and if necessary, the administration may be continued. Therefore, the amount administered does not limit the present invention. In addition, "composition" in the present invention means not only a pharmaceutical product but also a functional food and a health supplementary food. In a preferred embodiment, the composition comprises: beverages, foods, medicines, animal feeds, and the like.
In a preferred embodiment of the present invention, there is also provided a food composition comprising an effective amount of anaerobic coryneform faecalis and/or metabolites thereof, and the balance of a food acceptable carrier, said food composition being in a dosage form selected from the group consisting of solid, dairy, solution, powder, or suspension preparations. In a preferred embodiment, the food composition may further comprise a growth factor (e.g., milk growth factor). In a preferred embodiment, the composition further comprises a probiotic selected from the group consisting of: lactic acid bacteria, bifidobacteria, lactobacillus acidophilus, or combinations thereof; and/or a prebiotic selected from the group consisting of: fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), xylo-oligosaccharides (XOS), lactulose-oligosaccharides (LACT), soy Oligosaccharides (SOS), inulin (Inulin), oligosaccharides, or combinations thereof. In a preferred embodiment, the composition further comprises a substance (e.g., a protectant) selected from the group consisting of: cysteine, glutathione, butyl hydroxy anisole, dibutyl methyl toluene, tocopherol, bamboo leaf antioxidants, D-isoascorbic acid, sodium salts thereof, sodium ascorbate, calcium ascorbate, phospholipids, vitamin C (ascorbic acid), vitamin E, or combinations thereof.
In a preferred embodiment, the composition is formulated as follows:
1×10-1×10 15 cfu/mL of anaerobic coryneform faecalis and/or metabolites thereof; and a food or pharmaceutically acceptable carrier, and/or excipient.
In another preferred embodiment, the composition is formulated as follows:
1×10 4 -1×10 10 cfu/mL of anaerobic coryneform faecalis and/or metabolites thereof; and a food or pharmaceutically acceptable carrier, and/or excipient.
Microecological preparation
The microecological preparation is a biological preparation containing probiotics and metabolites or a dietary supplement capable of increasing the probiotics, and can achieve the aim of improving the health level of a human body by adjusting and maintaining microecological balance in intestinal tracts. Mainly comprises probiotics, prebiotics and synbiotics.
In the present invention, the microecological formulation comprises (a) a safe and effective amount of a fecal anaerobic coryneform bacterium and/or a metabolite thereof; and (b) a food-acceptable carrier or a pharmaceutically acceptable carrier. In a preferred embodiment, the formulation further comprises a growth factor (e.g., milk growth factor, preferably including vitamins, purines, and/or pyrimidines). In a preferred embodiment, the formulation further comprises a probiotic selected from the group consisting of: lactic acid bacteria, bifidobacteria, lactobacillus acidophilus, or combinations thereof; and/or a prebiotic selected from the group consisting of: fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), xylo-oligosaccharides (XOS), lactulose-oligosaccharides (LACT), soy Oligosaccharides (SOS), inulin (Inulin), oligosaccharides, or combinations thereof. In a preferred embodiment, the composition further comprises a substance (e.g., a protectant) selected from the group consisting of: cysteine, glutathione, butyl hydroxy anisole, dibutyl methyl toluene, tocopherol, bamboo leaf antioxidants, D-isoascorbic acid, sodium salts thereof, sodium ascorbate, calcium ascorbate, phospholipids, vitamin C (ascorbic acid), vitamin E, or combinations thereof.
Production method of anaerobic coryneform bacterium faecalis
In general, anaerobic coryneform bacteria can be produced by a conventional method.
In the present invention, there is provided a method capable of mass-producing anaerobic coryneform faecalis, comprising, in particular, the steps of:
(a) Culturing the anaerobic coryneform faecalis under the condition suitable for culturing, thereby obtaining a culture product;
(b) Optionally, isolating the anaerobic coryneform fecal bacteria and/or metabolites thereof from the culture product; and
(c) Optionally, the culture product or the anaerobic coryneform fecal bacteria and/or its metabolites obtained in the previous step are mixed with a food acceptable carrier or a pharmaceutically acceptable carrier, thereby producing a composition.
In the present invention, the conditions suitable for culturing refer to any conditions suitable for culturing the corynebacterium faecalis of the present invention, and in a preferred embodiment, the conditions suitable for culturing refer to anaerobic culture based on 37℃for 24 hours to 72 hours using PYG culture.
Method for improving intestinal lesions in mammals
In another preferred embodiment, the method comprises: the pharmaceutical composition, food composition, beverage composition, or combination thereof of the present invention is ingested. The subject includes a mammal, such as a human.
In another preferred embodiment, the method comprises: the pharmaceutical composition, food composition, or animal feed of the present invention, or a combination thereof, is ingested. The subject is an animal, preferably a murine, or a lagomorph.
Method for controlling weight loss in a mammal and/or for reducing Disease Activity Index (DAI) in a mammal
In another preferred embodiment, the method comprises: the pharmaceutical composition, food composition, beverage composition, or combination thereof of the present invention is ingested. The subject includes a mammal, such as a human.
In another preferred embodiment, the method comprises: the pharmaceutical composition, food composition, or animal feed of the present invention, or a combination thereof, is ingested. The subject is an animal, preferably a murine, or a lagomorph.
Method for preventing and/or treating inflammation-related diseases
In another preferred embodiment, the method comprises: the pharmaceutical composition, food composition, beverage composition, or combination thereof of the present invention is ingested. The subject includes a mammal, such as a human.
In another preferred embodiment, the method comprises: the pharmaceutical composition, food composition, or animal feed of the present invention, or a combination thereof, is ingested. The subject is an animal, preferably a murine, or a lagomorph.
Preservation of bacterial species
The strain manure anaerobic coryneform bacteria Anaerofustis stercorihominis AM-6 (the same as the preservation name) of the invention is preserved in the Guangdong province microorganism strain preservation center (GDMCC) 10-13 days in 2016, and the No. 59 building 5 of the Hirship 100 university in Guangzhou, address, and the preservation number is: GDMCC 60087.
The main advantages of the invention include:
(a) The anaerobic corynebacteria faecalis disclosed by the invention can be used for remarkably improving indexes (such as controlling weight loss, improving intestinal tract pathological degrees (including reducing colon length, reducing colon inflammatory response and the like), reducing Disease Activity Index (DAI) and the like) related to inflammatory diseases (such as inflammatory bowel diseases (such as ulcerative enteritis, gastritis and common enteritis).
(b) The treatment of the UC mice by the anaerobic corynebacteria in the invention is particularly characterized in that the weight reduction of the UC mice induced by DSS can be effectively controlled, the rise of the Disease Activity Index (DAI) of the mice is inhibited, and the colon change of the mice is improved.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions such as Sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or in accordance with the following "microorganisms: conditions described in the handbook (James Cappuccino and Natalie Sherman, pearson Education press) or as recommended by the manufacturer.
Unless otherwise indicated, the materials and reagents used in the examples were all commercially available products.
Example 1 isolation and identification of human feces anaerobic coryneform bacterium Anaerofustis stercorihominis AM-6
1.1 Isolation of AM25-6
The isolated sample was obtained from a healthy male feces, which was collected into sterile sample tubes and returned to the laboratory for sorting within 1 h. The collected fresh samples were immediately transferred to an anaerobic chamber, 0.2g of the samples were suspended in 1ml of sterile PBS (phosphate buffer), and thoroughly mixed by shaking. Strain isolation was performed using a gradient dilution plating method, and a PYG medium (available from the microorganism science and technology company of cycius) was used as the medium. The coated plate is placed in anaerobic culture at 37 ℃ and the anaerobic gas component is N 2 ∶CO 2 ∶H 2 =90:5:5. After 3 days of culture, single colonies are picked for streaking and purity division, and the pure culture of each single strain is obtained. Culturing the obtained pure culture strain to a concentration of about 10 9 cfu/ml, 400ul of fungus liquid is taken, 40% glycerol is added into 400ul of fungus liquid, the glycerol concentration reaches 20%, and then ultralow temperature preservation is carried out at-80 ℃.
1.2 16S rDNA identification
Extracting genome DNA of the isolated strain, performing PCR amplification by using a 16S rDNA universal primer, wherein the primer sequences are 27f (5'-AGAGTTTGATCATGGCTCAG-3' (SEQ ID NO: 2) and 1492r (5'-TAGGGTTACCTTGTTACGACTT-3' (SEQ ID NO: 3)), the amplification system is 10×PCR buffer,3uL, dNTP,2.5uL, 27F,0.5uL, 1492R,0.5uL, taq enzyme, 0.3uL, genome template, 1uL, ddH2O,18.2uL, and PCR amplification conditions are 95 ℃ pre-denatured for 4min, and then performing 30 cycles including denaturation at 95 ℃ for 30S, annealing at 57 ℃ for 40S, and extension at 72 ℃ for 1min for 30S, and performing electrophoresis detection and purification on the obtained 16S rDNA amplification product, 3730 sequencing to obtain 16S rDNA sequence (SEQ ID NO: 1) of 1425bp in length, and putting the 16S rNDA sequence of AM25-6 in EzBioCloud databasehttp：// www.ezbiocloud.net/identify)The bacterium having the highest homology (homology: 99.86%) to AM25-6 was Anaerofustis stercorihominis DSM 17244 (purchased from German collection of strains).
1.3 Physiological and biochemical characterization of AM25-6
The isolated AM25-6 colonies were cultured in PYG medium for 72h as follows: pale yellow, smaller colonies, needlepoint, translucent, about 0.2mm in diameter (fig. 1). The microscopic morphology of the AM26-6 cells was found to be short bar-like by gram staining and spore flagellum staining, gram positive, sporulation and flagellum free (FIG. 2). The results of the tests for catalase and oxidase were negative, and the utilization of the carbon source by AM25-6 was detected by API 20A (French Mei Liai), the results are shown in Table 1, wherein +represents a positive reaction, -represents a negative reaction, and w represents a weak positive reaction.
TABLE 1
1.4 Antibiotic susceptibility to AM25-6
The sensitivity of AM25-6 to 20 common antibiotics was tested by using a drug sensitive paper method, 100ul of the bacterial liquid of AM25-6 cultured to logarithmic phase was coated on a flat plate, antibiotic drug sensitive tablets (purchased from Hangzhou microbial agent Co., ltd.) were stuck on the surface of the flat plate, and the size of the inhibition zone was measured by culturing at 37℃for 48 hours, and the results are shown in Table 2.
TABLE 2
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The results show that AM25-6 is relatively sensitive to 20 common antibiotics and the strain safety is high.
EXAMPLE 2 bioactive substances of human Thermomyces lanuginosus Anaerofustis stercorihominis AM-6
This example mainly examined the production of metabolites of AM25-6 in PYG culture for 48h, mainly including the content of products of Short Chain Fatty Acids (SCFA), mainly including acetic acid, propionic acid, butyric acid, valeric acid, and organic acids, including 3-methylbutyric acid, quinic acid, lactic acid, oxalic acid, malonic acid, benzoic acid, maleic acid, succinic acid, fumaric acid, malic acid, adipic acid, tartaric acid, shikimic acid, citric acid, isocitric acid, and L-ascorbic acid, each standard of SCFA and organic acid was purchased from Sigma. The detection process is as follows:
2.1 Sample pretreatment
1ml of the AM25-6 bacterial liquid was centrifuged at 12000r/min for 5min, and the supernatant was prepared for detection of Short Chain Fatty Acids (SCFAs) and organic acids.
2.2 Determination of SCFA
SCFA was detected by Agilent weather chromatograph (GC-7890B, agilent), HP-INNOWax (Cross-Linked PEG), 30m×0.25mm×0.25um capillary column for analysis, hydrogen flame ion detector, GC parameters set as column temperature: 180-200 ℃; gasification chamber temperature: 240 ℃; detecting the temperature: 210 ℃; sample injection amount: 2. Mu.L; carrier gas flow rate: n (N) 2 50mL/min; hydrogen flow rate: 50mL/min; air flow rate: 600-700 ml/min.
2.3 Determination of organic acids
The organic acid was also detected using Agilent Meteorological chromatograph (GC-7890B, agilent), column temperature was 122-5532G DB-5ms (40 m 0.25mm 0.25 um): 270-290 ℃; sample inlet temperature: 250 ℃; gas flow rate: 0.86ml/min.
2.4 Experimental results
TABLE 3 Table 3
The results are shown in Table 3. The results show that AM25-6 has the ability to produce acetic acid, butyric acid, isovaleric acid, benzoic acid and lactic acid, and is capable of producing isobutyric acid, valeric acid, 3-methylbutyric acid, succinic acid, adipic acid and citric acid in small amounts.
EXAMPLE 3 treatment of UC mice with human Exophiala tenuifolia Anaerofustis stercorihominis AM-6
The mouse model selected in this example was: mice were fed with 0.15% DSS (Dextran sodium Sulfate, molecular weight 36000-50000, commercially available from MPBIO in the united states) for 7 days, a strain of C57bl/6 mice (commercially available from the center of Hubei medical laboratory animals), 8 weeks old, 20g±2g body weight, and raised in an SPF-rated murine room environment.
The test mice were divided into 4 groups at random, 12 per group, comprising:
Normal group (control group) -feeding with common feed;
model group-DSS modeling, each mouse perfused with 0.2ml PBS per day;
AM25-6 treatment group-DSS modeling, each mouse lavage 0.2ml of AM25-6 bacteria liquid every day;
VSL # 3 treatment group-DSS modeling, each mouse perfused with 0.2ml VSL per day # 3 (therapeutic probiotic drug purchased from alfassigma, USA, clinically used in UC);
the AM25-6 treatment process comprises the following steps: culturing for 24 hr AM25-6 bacterial liquid, centrifuging to collect bacterial cells, suspending with PBS (phosphate buffer), and adjusting bacterial concentration to 10 9 cfu/ml, 200ul of AM25-6 per day per mouse. VSL (vertical seismic profiling) # 3 also use PBS for suspending, and the concentration is adjusted to 10 9 cfu/ml, each mouse was perfused with 200ul per day.
Gastric lavage AM25-6 and VSL was initiated 3 days prior to DSS molding # 3, DSS was added to the drinking water of mice, modeling of UC was performed by free drinking for 7 days, and the weight and diet of mice were recorded dailyAnd drinking water, simultaneously observing the fecal character and fecal occult blood condition of the mice, and calculating Disease Activity Index (DAI) of the mice on the 1 st day, the 3 rd day, the 5 th day and the 7 th day respectively, wherein the DAI scoring standard is shown in Table 4. Mice were sacrificed after the end of the experiment, all mice were bled, necked off, colon was taken, photographed, weighed, and colon length was measured. Colon tissue was stored in-80 ℃ freezer and paraformaldehyde.
TABLE 4 DAI index scoring Table
Stool traits in the table: normal stool-shaped stool; loose stool-pasty, semi-formed stool that does not adhere to the anus; loose stool-loose water stool that can adhere to anus. Wherein the hematochezia condition: normal mice were positive for hematochezia; blood is red or brown to the naked eye; occult blood positivity is unobvious macroscopic bloody stool, and is detected by using tetramethyl benzidine. The DAI index is equal to the sum of three integrals of weight loss, stool character and stool occult blood.
The therapeutic effect of AM25-6 on DSS-induced UC model mice was examined by comparing body weight loss, DAI index and colon length, respectively, as follows.
3.1 Weight change
The body weight changes of the mice before and after treatment are shown in table 5 and fig. 3 below:
TABLE 5
The results in table 5 show that the body weight of the Control group (Control group) mice tended to increase slowly, the DSS-induced body weight of the 3 groups of mice all continued to decrease, and on day 3, the body weight decrease of the model group started significantly (P < 0.05) relative to the Control group, and on day 7, the degree of difference between the model group and the Control group was more pronounced (P < 0.01). While AM25-6 and VSL # 3 can slow the weight loss of UC mice, and on day 7, the control phase of weight loss of the mice in both groups Compared with the model group, the model group is obvious ▲ P < 0.05). Description of AM25-6 and VSL # 3 can control the weight loss caused by UC. The weight of the mice in the AM25-6 group was slightly higher than VSL on day 7 # 3, demonstrating the effect of AM25-6 on controlling weight loss in UC mice and VSL # 3 has equivalent effect and even slightly better effect than VSL # 3。
3.2 Variation of DAI index
DSS-induced ulcerative enteritis mice were shown to have changes in DAI index due to weight loss, stool traits and changes in hematocrit conditions, with changes in DAI index before and after treatment as shown in table 6 and fig. 4:
TABLE 6
The data in Table 6 and FIG. 4 show that the DAI of Control mice was substantially leveled, whereas with DSS induction, model group, AM25-6 and VSL # The DAI of the 3 mice gradually increased, the day three mice became significantly elevated relative to the control group (< P < 0.05), and the day 7 mice reached the highest level (relative to the control group (< P < 0.01). The bacterial intervention of the invention can control the rise of DAI, and the DAI of the mice in the AM25-6 group on the 5 th day is obviously controlled relative to the model group ▲ P < 0.1), AM25-6 and VSL on day 7 # The DAI of the mice in the 3 groups is obviously lower than that of the model group ▲ P < 0.05), and the DAI of the AM25-6 mice was slightly lower than that of VSL # 3, it can be demonstrated that AM25-6 has effect of controlling DAI elevation of UC mice and VSL # 3 is equivalent to or even slightly better than VSL # 3。
3.3 Variation of colon length
The colon tissue of the UC model mice was altered because the occurrence of ulcers and inflammation resulted in shortening of the colon tissue, and the colon length of the mice after the end of treatment was measured by dissection as shown in table 7.
TABLE 7
The results in table 7 show that the colon tissue shortening of the mice (model group) after 7 days of DSS induction was more severe, and very pronounced compared to the control group (P < 0.01). The bacteria AM25-6 and VSL of the present invention # 3 can significantly control the shortening of the colon of mice (very significant with respect to the model group, < P < 0.01). As can be seen from the data in the tables, the colon length ratio VSL of the AM25-6 mice group # The 3 groups of mice have slightly longer colon length, which can prove that the AM25-6 has slightly stronger capability of controlling the colon shortening of UC mice than VSL # 3. From the results, the strain AM25-6 of the invention can obviously slow down the lesions of the colon of mice, and the effect of the strain is similar to that of VSL # 3 is equivalent to or even slightly better than VSL # 3。
Example 4 food composition containing anaerobic coryneform faecalis Anaerofustis stercorihominis AM-6
Raw material ratios are shown in table 8.
TABLE 8
Raw materials Mass percent (%)
Anaerofustis stercorihominis AM25-6 0.5
Milk 90.0
White sugar 9.0
Vitamin C 0.5
Mixing milk and white sugar according to the above formula ratio, stirring to completely mix, preheating, homogenizing under 20Mpa, sterilizing at about 90deg.C for 5-10 min, cooling to 40-43deg.C, mixing with protective agent vitamin C, and inoculating 1-100×10 6 Anaerofustis stercorihominis AM25 bacteria/g and Anaerofustis stercorihominis AM-6 bacteria are prepared into the food composition.
EXAMPLE 5 pharmaceutical composition containing Excellent anaerobism faecalis Anaerofustis stercorihominis AM-6
The proportions of the raw materials are shown in Table 9.
TABLE 9
Mixing lactose, yeast powder and peptone with purified water at a certain proportion, preheating to 60-65deg.C, homogenizing under 20Mpa, sterilizing at about 90deg.C for 20-30 min, cooling to 36-38deg.C, mixing with vitamin C, and inoculating Anaerofustis stercorihominis AM-6 viable bacteria (1-50×10) 6 cfu/mL), fermenting at 36-38deg.C to pH 6.0, centrifuging, and lyophilizing to water content less than 3% to obtain Anaerofustis stercorihominis AM-6 strain lyophilized product. Weighing 0.5 g Anaerofustis stercorihominis AM-25-6 lyophilized product, mixing with maltodextrin and protectant (such as vitamin C and cysteine), and encapsulating to obtain pharmaceutical composition containing Anaerofustis stercorihominis AM-6 bacteria.
Example 6A method of preparing a medicament for treating inflammation-related disorders such as ulcerative enteritis (UC)
1. Preparing bacterial liquid: anaerofustis stercorihominis AM25-6 (1×10) 9 cfu/ml), anaerobic culture is carried out, and PYG culture medium is adopted as the anaerobic culture medium, and anaerobic fermentation is carried out for 2-3 days at 37 ℃.
2. Preparing a growth factor: mixing skimmed milk and casein, centrifuging, and ultrafiltering to obtain milk growth factor crude extract (containing vitamins, purines, and/or pyrimidine nutrients).
3. Drug or drug formulation preparation: adding 5 volumes (ml) of growth factors and 1 volume (ml) of protective agents (such as vitamin C and cysteine) into 100 volumes (ml) of Anaerofustis stercorihominis AM-6 fermented bacterial liquid, fully stirring and uniformly mixing, and then adding starch auxiliary materials (such as maltodextrin) to prepare the Anaerofustis stercorihominis AM-6-containing medicament or medicament dosage form.
All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.
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Claims (20)

1. The anaerobic coryneform fecal bacteria is characterized by being human anaerobic coryneform fecal bacteria (Anaerofustis stercorihominis), wherein the anaerobic coryneform fecal bacteria is Anaerofustis stercorihominis AM-6 and has a deposit number of GDMCC 60087.
2. A composition, wherein the composition comprises: (a) A safe and effective amount of the anaerobic coryneform fecal bacteria of claim 1; and (b) a pharmaceutically acceptable carrier.
3. The composition of claim 2, wherein the composition further comprises a growth factor.
4. A composition according to claim 3, wherein the growth factor is a milk growth factor obtained by mixing skim milk, casein, centrifuging, and ultrafiltration.
5. The composition of claim 2, wherein the composition comprises 1 x 10 4 -1×10 10 cfu/mL or cfu/g Anaerofustis stercorihominis AM25-6, based on the total volume or weight of the composition.
6. The composition of claim 2, wherein the composition further comprises other probiotics and/or prebiotics.
7. The composition of claim 2, further comprising a substance that helps maintain the viability of the anaerobic bar bacteria.
8. The composition of claim 7, wherein the substance that helps to maintain the viability of the anaerobic bar is selected from the group consisting of: cysteine, glutathione, butyl hydroxy anisole, dibutyl methyl toluene, tocopherol, bamboo leaf antioxidants, D-isoascorbic acid, sodium salts thereof, sodium ascorbate, calcium ascorbate, phospholipids, vitamin C, vitamin E, or combinations thereof.
9. The composition of claim 7, wherein the weight ratio of the substance that helps to maintain the viability of the anaerobic bar is from 0.1 to 2% based on the total weight of the composition.
10. The composition of claim 7, wherein the material that helps maintain the viability of the anaerobic bar is present in an amount of 0.5 to 1.5% by weight based on the total weight of the composition.
11. The composition of claim 7, wherein the material that helps maintain the viability of the anaerobic bar is present in an amount of 0.5 to 1.0% by weight based on the total weight of the composition.
12. The composition according to claim 7, wherein the substance which contributes to the maintenance of the activity of the anaerobic coryneform fecal bacteria is contained in an amount of 1mg to 20mg based on 1g of the composition.
13. The composition according to claim 7, wherein the substance which contributes to the maintenance of the activity of the anaerobic coryneform fecal bacteria is contained in an amount of 5mg to 15mg based on 1g of the composition.
14. The composition according to claim 7, wherein the substance which contributes to the maintenance of the activity of the anaerobic coryneform fecal bacteria is contained in an amount of 5mg to 10mg based on 1g of the composition.
15. Use of the anaerobic coryneform fecal bacteria of claim 1, or the composition of claim 2, in the manufacture of a composition or formulation for treating an inflammatory bowel disease selected from the group consisting of: ulcerative enteritis, plain enteritis, or a combination thereof.
16. Use of the anaerobic coryneform fecal bacteria of claim 1, or the composition of claim 2, in the preparation of a composition or formulation for one or more uses selected from the group consisting of:
(i) Controlling a decrease in body weight of the mammal;
(ii) Lowering the Disease Activity Index (DAI) of the mammal;
(iii) Improving intestinal lesions of a mammal,
wherein the mammal is a mouse with an inflammatory bowel disease selected from the group consisting of: ulcerative enteritis, plain enteritis, or a combination thereof.
17. A method of preparing the composition of claim 2, comprising the steps of:
Mixing the anaerobic coryneform fecal bacteria of claim 1 with a pharmaceutically acceptable carrier, thereby forming the composition of claim 2.
18. A method of manufacture comprising the steps of:
(a) Culturing the anaerobic coryneform fecal strain according to claim 1 under conditions suitable for the culture, thereby obtaining a culture product.
19. The method of manufacturing as claimed in claim 18, wherein said method further comprises the steps of:
(b) Separating the anaerobic coryneform fecal bacteria from the culture product.
20. The method of claim 19, wherein the method further comprises the steps of:
(c) The culture product or the manure anaerobic coryneform bacteria obtained in the last step is mixed with a pharmaceutically acceptable carrier, so that the composition of the invention is prepared.
CN201780094884.3A 2017-09-15 2017-09-15 Anaerobic coryneform faecalis (Anaerofustis stercorihominis) and application thereof Active CN111448306B (en)

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