CN110049772A

CN110049772A - Shenzhen Collins bacterium (Collinsella shenzhenensis) and its application

Info

Publication number: CN110049772A
Application number: CN201680091522.4A
Authority: CN
Inventors: 邹远强; 薛文斌; 肖亮; 李晓平; 余靖宏; 刘传
Original assignee: BGI Shenzhen Co Ltd
Current assignee: BGI Shenzhen Co Ltd
Priority date: 2016-12-13
Filing date: 2016-12-13
Publication date: 2019-07-23
Also published as: WO2018107364A1

Abstract

Provide purposes of Shenzhen Collins bacterium (Collinsella shenzhenensis) in treatment and/or prevention of inflammation related disease, cardiovascular disease, it additionally provides a kind of for treating and/or the composition of prevention of inflammation related disease, cardiovascular disease, including drug, beverage, food, or animal feed composition etc., and improve the method for mammalian gut lesion, the blood lipid level for reducing mammal, the decline of control weight of mammal, and/or the disease activity index (DAI) of reduction mammal.

Description

Shenzhen Collins bacterium (Collinsella shenzhenensis) and its application

Technical field

The invention belongs to microbiological arts, in particular it relates to which Shenzhen Collins bacterium (Collinsella shenzhenensis) is treating and preventing the application in inflammation related disease, are also related to composition and its application comprising Shenzhen Collins bacterium.

Background technique

Ulcerative enteritis (UC) is the one kind for belonging to inflammatory bowel disease (IBD), it is divided into colitis and rectitis according to inflammation part, its pathogenesis is not yet clear, caused by the immune response for being mainly considered genetic predisposition, intestinal flora, intestinal mucosa at present.It is a large amount of studies have shown that the morbidity of UC and the unbalance Relationship Comparison of intestinal flora are close both at home and abroad.The Clinical pathology of UC is abdominal pain, diarrhea and mucus bloody stool, and recurrent exerbation.

Since pathomechanism is indefinite, clinical treatment also lacks specificity and specific aim, currently, clinically mainly having salicylic acid, glucocorticoid, immune formulation for the medication of UC disease.Salicylates can be relatively good inhibition prostaglandin synthesis, scavenging activated oxygen is to achieve the purpose that alleviate inflammatory reaction, clinical treatment UC common salicylic acid Western medicine is mainly salicylazosulfapyridine (SASP), mainly for slight, moderate and chronic UC patient；Glucocorticoid is the preferred medication of severe or explosive UC patient, such as betamethasone；Immunosuppressor such as cyclosporine can influence the progress of immune response, to inhibit to UC by the generation of inhibition T cell IL-2.

Above-mentioned three classes drug can to a certain extent alleviate UC, but also all there is certain side effect, the side effect of salicylic acid is fash, hepatotoxicity wind agitation, oligoleukocythemia, anaemia caused by causing digestive tract reaction, headache, ceticulocytosis, oligozoospermia and allergic reaction etc..Glucocorticoid will lead to the side effects such as organism metabolic disorder, water retention, only can be used as emergency medication, cannot take for a long time.Immunosuppressant treatment is larger to drug dependence, and treatment cycle is long, easily causes renal toxicity and superinfection, can only be as a kind of means of adjuvant treatment.

Therefore a kind of new there is an urgent need in the art to develop, it has no toxic side effect, for treating and preventing the drug of inflammation related disease.

Summary of the invention

It is a further object of the present invention to provide Collins bacterium in treatment and/or the purposes of prevention of inflammation related disease.

It is a further object of the present invention to provide one kind effectively to have no toxic side effect, for treating and/or the drug of prevention of inflammation related disease, beverage, food compositions or animal feed composition.

It is a further object of the present invention to provide a kind of method and its application for improving mammalian gut lesion.

It is a further object of the present invention to provide a kind of reduction blood lipid, control the decline of weight of mammal, and/or the method and its application for the disease activity index (DAI) for reducing mammal.

It is a further object of the present invention to provide Collins bacterium in the application for treating and/or preventing cardiovascular related diseases.

First aspect present invention provides a kind of Collins bacterium, and the Collins bacterium is Shenzhen Collins bacterium (Collinsella shenzhenensis).

In another preferred example, the sequence of the 16s rDNA of the Collins bacterium is as shown in SEQ ID NO.:1.

In another preferred example, the Collins bacterium is Collinsella shenzhenensis TF06-26, and deposit number is GDMCC NO.:60090.

Have in a preference separately, the Collins bacterium comes from enteron aisle, animal wastes, fermentation vat, and/or anaerobic reactor.

Second aspect of the present invention provides a kind of composition, and the composition includes: Collins bacterium and/or its metabolite described in the first aspect present invention of (a) safe and effective amount；And (b) acceptable carrier or pharmaceutically acceptable carrier on food.

In another preferred example, the composition further includes growth factor (preferably, milk growth factor).

In another preferred example, the composition is selected from the group: food compositions, health composition, pharmaceutical composition, beverage composition for treating dental erosion, fodder compound, or combinations thereof.

In another preferred example, the composition is oral preparation.

In another preferred example, the composition is liquid formulation, solid formulation, semisolid preparations.

In another preferred example, the dosage form of the composition is selected from the group: powder agent, powder, tablet, sugar-coat agent, capsule, granule, suspending agent, solution, syrup, drops, sublingual lozenge, or combinations thereof.

In another preferred example, the food compositions include latex product, solution product, pulverulent product or suspension product.

In another preferred example, the food compositions include dairy products, milk powder or lotion.

In another preferred example, the liquid formulation is selected from the group: solution product or suspension product.

In another preferred example, the composition contains 1 × 10-1 × 10¹⁵Cfu/mL or cfu/g Collinsella shenzhenensis TF06-26, preferably 1 × 10⁴-1×10¹⁰Cfu/mL or cfu/g Collinsella shenzhenensis TF06-26, by the total volume or total weight of the composition.

In another preferred example, in the composition, containing 0.0001-99wt%, preferably Collins bacterium and/or its metabolite described in 0.1-90wt%, with the total weight of the composition.

In another preferred example, the composition is unit dosage form (an a piece of, capsule or a bottle), and the quality of composition described in each unit dosage form is 0.05-5g, preferably 0.1-1g.

In another preferred example, the composition also contains other probiotics and/or prebiotics.

In another preferred example, the probiotics is selected from the group: lactic acid bacteria, Bifidobacterium, lactobacillus acidophilus, or combinations thereof.

In another preferred example, the prebiotics are selected from the group: oligofructose (FOS), galactooligosaccharide (GOS), xylo-oligosaccharide (XOS), lactosucrose (LACT), soyabean oligosaccharides (SOS), inulin (Inulin), oligosaccharide, or combinations thereof.

In another preferred example, the composition also contains the substance (such as protective agent) for helping to maintain Collins's bacterium vigor.

In another preferred example, the substance (such as protective agent) for helping to maintain Collins's bacterium vigor is selected from the group: cysteine, glutathione, butylated hydroxy anisole, dibutylmethyl toluene, tocopherol, bamboo leaf antioxidant, D-araboascorbic acid and its sodium salt, sodium ascorbate, Calcium Ascorbate, phosphatide, vitamin C (ascorbic acid), vitamin E, or combinations thereof.

In another preferred example, the weight ratio of the substance (such as protective agent) for helping to maintain Collins's bacterium vigor is 0.1-2%, preferably, 0.5-1.5%, more preferably, 0.5-1.0%, with total restatement of the composition.

In another preferred example, in terms of composition 1g, the content of the substance (such as protective agent) for helping to maintain Collins's bacterium vigor is 1mg-20mg, preferably, 5mg-15mg, more preferably, 5mg-10mg.

Third aspect present invention provides Collins bacterium described in a kind of first aspect present invention or the present invention second The purposes of the aspect composition, is used to prepare drug or preparation, and the drug or preparation are used for one or more purposes selected from the group below: (a) prevention and or treatment inflammation related disease；And/or (b) prevent and/or treat cardiovascular disease.

In another preferred example, the inflammation related disease is selected from the group: inflammatory bowel disease, rheumatoid arthritis, or combinations thereof.

In another preferred example, the inflammation related disease is selected from the group: ulcerative enteritis, gastritis, common enteritis, or combinations thereof.

In another preferred example, the cardiovascular disease is selected from the group: hypertension, hyperlipidemia, coronary heart disease, or combinations thereof.

In another preferred example, the preparation includes probiotics.

Fourth aspect present invention provides the purposes of composition described in Collins bacterium described in a kind of first aspect present invention or second aspect of the present invention, is used to prepare drug or preparation, and the drug or preparation are used for one or more purposes selected from the group below:

(i) blood lipid level of mammal is reduced；

(ii) decline of weight of mammal is controlled；

(iii) disease activity index (DAI) of mammal is reduced；

(iv) improve mammalian gut lesion.

In another preferred example, the mammal includes people, rodent (such as rat, mouse).

In another preferred example, the blood lipid level for reducing mammal includes reducing cholesterol levels.

In another preferred example, the decline of the control weight of mammal refers to compared with model group mammal, and the reduction amplitude of experimental group weight of mammal is, preferably, being no more than 5%, more preferably, to be no more than 2% no more than 10%.

In another preferred example, the lesion for slowing down mammalian gut includes slowing down the shortening of colon lengths, and/or mitigating colitis to react.

Fifth aspect present invention provides a kind of preparation method of composition described in second aspect of the present invention, comprising steps of

Collins bacterium described in first aspect present invention and/or its metabolite are mixed with carrier acceptable on food or pharmaceutically acceptable carrier, to form composition described in second aspect of the present invention.

In another preferred example, the preparation method further includes the steps that mixing with growth factor.

In another preferred example, the preparation method further includes the steps that mixing with the substance (such as protective agent) for helping to maintain Collins's bacterium vigor.

In another preferred example, the preparation method further includes the steps that mixing with probiotics, and/or prebiotics.

In another preferred example, the growth factor is milk growth factor.

In another preferred example, the growth factor is selected from the group: vitamin substances, purine substance, pyrimidine substance, or combinations thereof.

In another preferred example, the composition is oral preparation.

Sixth aspect present invention provides a kind of production method, comprising steps of

(a) under conditions of being suitble to culture, the Collins bacterium is cultivated to the first aspect of the present invention, to obtain cultured products；

(b) optionally, Collins bacterium thallus and/or its metabolite are separated from the cultured products；And/or

(c) optionally, the cultured products of previous step acquisition or Collins bacterium thallus and/or its metabolite are mixed with carrier acceptable on food or pharmaceutically acceptable carrier, so that composition of the present invention be made.

In another preferred example, before step (c), further include the steps that mixing the cultured products of previous step acquisition or the Collins bacterium thallus and/or its metabolite with growth factor.

In another preferred example, the growth factor is milk growth factor.

In another preferred example; before step (c), further include the steps that mixing the cultured products of previous step acquisition or the Collins bacterium thallus and/or its metabolite with the substance (such as protective agent) for helping to maintain Collins's bacterium vigor.

In another preferred example, before step (c), further include the steps that mixing the cultured products of previous step acquisition or the Collins bacterium thallus and/or its metabolite with probiotics and/or prebiotics.

Seventh aspect present invention provides a kind of method for improving mammalian gut lesion, to composition described in object application second aspect of the present invention.

In another preferred example, the application includes oral.

In another preferred example, the administration dosage is 0.01-5g/50kg body weight/day, preferably, 0.1-2g/50kg body weight/day.

In another preferred example, the object includes mammal, such as people.

Eighth aspect present invention provides a kind of method of decline for reducing blood lipid, controlling weight of mammal, and/or the disease activity index (DAI) for reducing mammal, to composition described in object application second aspect of the present invention.

In another preferred example, the application includes oral.

In another preferred example, the object includes mammal, such as people.

It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and it can be combined with each other between each technical characteristic specifically described in below (e.g. embodiment), to form a new or preferred technical solution.Due to space limitations, I will not repeat them here.

Detailed description of the invention

Fig. 1 shows the picture of Shenzhen Collins bacterium Collinsella shenzhenensis culture 48h bacterium colony.

Fig. 2 shows that the leather of Shenzhen Collins bacterium Collinsella shenzhenensis under the microscope is blue Albert'stain Albert picture (1000 times).

Fig. 3 shows the standard curve of cholesterol.

Fig. 4 shows the length of model group and TF06-26 treatment group mouse Colon.

Specific embodiment

The present inventor after extensive and in-depth study and experiment, it is surprised to find that, Shenzhen Collins bacterium (Collinsella shenzhenensis) has prevention and or treatment inflammation related disease (such as inflammatory bowel disease (such as ulcerative enteritis, gastritis, common enteritis), rheumatoid arthritis), cardiovascular disease (such as hypertension, hyperlipidemia, coronary heart disease) effect, food experimental subjects will be fed containing the active compound of Shenzhen Collins bacterium, it was found that the composition can control weight loss, reduce blood lipid, it reduces disease activity index (DAI), improve enteron aisle lesion, inflammation related disease (such as inflammatory bowel disease (such as ulcerative enteritis can effectively be mitigated, gastritis, common enteritis), rheumatoid arthritis), mitigate the illnesss such as cardiovascular disease.The present invention is completed on this basis.

As used herein, term " containing " indicates that various composition can be applied in mixture or composition of the invention together.Therefore, term " mainly by ... form " and " consist of " were included in term " containing ".

As used herein, term " growth factor " includes milk growth factor, specifically, including vitamin substances, purine substance, pyrimidine substance, or combinations thereof nutriment.

Wherein, the vitamin substances include (but being not limited to): vitamin C, vitamin E, vitamin A, vitamin A precursor, vitamin B₆, vitamin D₃, vitamin K, folic acid, or combinations thereof；

The purine substance includes (but being not limited to): purine nucleosides, wherein the purine nucleosides includes 5 '-phosphates of purine nucleosides；5 '-phosphates of the purine nucleosides are selected from the group: inosinicacid (inosine -5 '-phosphate；IMP), guanylic acid (guanosine -5 '-phosphate；GMP), xanthosine monophosphate (xanthosine -5 '-phosphate；XMP), adenylate (5'-AMP ester；AMP), or combinations thereof；

The pyrimidine substance includes all substances containing pyrimidine structure.

As used herein, term " decline of control weight of mammal ", " slowing down weight of mammal decline " are used interchangeably, refer to that mammal is continuous serious due to inflammation in carrying out ulcerative enteritis model construction process, the weight of experimental animal also declines therewith, and the percentage of weight loss is the percentage for declining weight and accounting for original weight.The degree of weight loss is higher, and disease is more serious, and Shenzhen Collins bacterium of the invention can control the weight loss of experimental animal in mammal ulcerative enteritis therapeutic process, slows down the symptom of disease.

Disease activity index (DAI)

As used herein, term " disease activity index " refers to that the weight loss percentage in conjunction with patient's (or infected animal), stool 3 kinds of situations such as viscosity and fecal blood carry out comprehensive score.

Shenzhen Collins bacterium and its application

As used herein, term " Collins bacterium ", " Shenzhen Collins bacterium ", " Collinsella shenzhenensis ", " Shenzhen Collins bacterium of the invention ", " Collins bacterium of the invention " can be mutual Change use.In a preferred embodiment, the bacterial strain is Collinsella shenzhenensis TF06-26, and deposit number is GDMCC NO.:60090, is isolated from the excrement of people's (preferably, healthy women), specifically, is isolated from the enteron aisle of Young Female.The physiological property of Shenzhen Collins bacterium is as follows: the bacterium colony of Shenzhen Collins bacterium (Collinsella shenzhenensis TF06-26) Anaerobic culturel 48h is white, and protrusion is round, neat in edge, water content is more, opaque, colony diameter 1.0mm-2.0mm；By carrying out Gram's staining, spore staining, flagella staining respectively to Shenzhen Collins bacterium (Collinsella shenzhenensis TF06-26), the thalli morphology of Shenzhen Collins bacterium (Collinsella shenzhenensis TF06-26) is observed under 1000 times of amplifications.Shenzhen Collins bacterium (Collinsella shenzhenensis TF06-26) is Gram-negative, does not produce gemma and flagellum, rod-short.Shenzhen Collins bacterium (Collinsella shenzhenensis TF06-26) is negative catalase, and oxidase negative does not have motility, growth temperature range is 25-45 DEG C, growth pH value range is 5.0-8.0, and NaCl tolerable concentration is 2%, and cholate tolerable concentration is 0.3%.Can ferment multiple kinds of carbohydrate, including D- rock sugar, N- acetyl-aminoglucose, galactolipin, mannose, fructose, salicin, maltose, melibiose, glucose, lactose, cellobiose, Arbutin, and/or salicin, it is main to generate lactic acid, acetic acid, butyric acid, and Shenzhen Collins bacterium (Collinsella shenzhenensis TF06-26) of the invention is to kanamycins, amikacin, neomycin and erythromycin are resistant, to remaining 16 kinds of antibiotic sensitive in table 2, furthermore, Shenzhen Collins bacterium of the invention also can produce the exocellular polysaccharide (up to 284.49mg/L) of very high throughput, in terms of the total concentration of fermentation liquid.

The present invention provides Shenzhen Collins bacterium in treatment and/or the application of prevention of inflammation related disease (such as inflammatory bowel disease (such as ulcerative enteritis, gastritis, common enteritis), rheumatoid arthritis), cardiovascular disease (such as hypertension, hyperlipidemia, coronary heart disease).Subject carries out induction modeling with DSS (dextran sulfate sodium), and bacterial strain Collinsella shenzhenensis TF06-26 has one or more purposes selected from the group below: (i) controls the weight loss of the subject；(ii) blood lipid is reduced；(iii) improve enteron aisle lesion degree；(iv) disease activity index (DAI) is reduced.A preference according to the present invention, using C57bl/6 mouse as test mice, induction modeling is carried out with DSS (dextran sulfate sodium), to obtain ulcerative enteritis (UC) model mice, the UC model mice treated through Collinsella shenzhenensis TF06-26, compared with the control group (model group) for not receiving treatment, its weight loss amplitude slows down and rate and blood-lipid decreased, and various and inflammation related disease (such as inflammatory bowel disease (such as ulcerative enteritis, gastritis, common enteritis), rheumatoid arthritis) relevant index also improved, such as improve enteron aisle lesion degree (including slow down colon lengths shortening, mitigate colitis reaction etc.), reduce disease activity index (DAI) etc..Therefore, the bacterial strain can be to prevention and or treatment inflammation related disease (such as inflammatory bowel disease (such as ulcerative enteritis, gastritis, common enteritis), rheumatoid arthritis), cardiovascular disease (such as hypertension, hyperlipidemia, coronary heart disease disease).

Composition and its application

The present invention also provides a kind of compositions, preferably, the composition includes food compositions, health composition, pharmaceutical composition, beverage composition for treating dental erosion or fodder compound, it is preferable that are pharmaceutical composition.The composition includes a effective amount of Shenzhen Collins bacterium, and in a preferred embodiment, the composition further includes growth factor (such as milk growth factor).In a preferred embodiment, the composition further includes probiotics selected from the group below: lactic acid bacteria, Bifidobacterium, lactobacillus acidophilus, or combinations thereof；And/or prebiotics selected from the group below: oligofructose (FOS), galactooligosaccharide (GOS), xylo-oligosaccharide (XOS), lactosucrose (LACT), soyabean oligosaccharides (SOS), inulin (Inulin), oligosaccharide, or combinations thereof.In a preferred embodiment, the composition further includes the substance (such as protective agent) selected from the group below for helping to maintain Collins's bacterium vigor: cysteine, paddy Guang Sweet peptide, butylated hydroxy anisole, dibutylmethyl toluene, tocopherol, bamboo leaf antioxidant, D-araboascorbic acid and its sodium salt, sodium ascorbate, Calcium Ascorbate, phosphatide, vitamin C (ascorbic acid), vitamin E, or combinations thereof.With total restatement of composition, the weight ratio of the substance (such as protective agent) for helping to maintain Collins's bacterium vigor is 0.1-2%, preferably, 0.5-1.5%, more preferably, 0.5-1.0%.

In a preferred example, the composition is liquid formulation, solid formulation, semisolid preparations.

In a preferred example, the liquid formulation is selected from the group: solution product or suspension product.

In a preferred example, the dosage form of the composition is selected from the group: powder agent, powder, tablet, sugar-coat agent, capsule, granule, suspending agent, solution, syrup, drops, sublingual lozenge, or combinations thereof.

Composition of the invention can by oral solution, tablet, injection, oral disintegrating tablet, freeze-dried powder preparation or capsule it is any in the form of be administered, it is preferred that enteric dosage form (such as capsule), in the present invention, unless otherwise instructed, the medium and carrier that excipient used in the present invention, drug allow be mainly according to being suitble to bacterium or its metabolite characteristic and required specific administration mode to select, be conducive to bacterium or its metabolite passes through stomach and the person of being administered absorbs.These substances can be selected according to administration route.

Composition of the invention can further include any additional excipient in the excipient that those are commonly used in pharmaceutical preparation, in order that for example stable composition itself, or keep its readily dispersed or assign its suitable taste.

In the excipient, inulin, fructose, starch, xylo-oligosaccharide, silica, buffer reagent and flavouring agent are suitable examples.

Pharmaceutical formulations of the present invention can further include the active constituent of auxiliary.

Lactose, maltodextrin, glucose, sucrose, D-sorbite, mannose, starch, Arabic gum, calcium phosphate, alginates, gelatin, calcium silicates, fine crystallization cellulose, polyvinylpyrrolidone (PVP), cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, nipasol, talcum, magnesium stearate or mineral oil etc. are used as the carrier, excipient or diluent etc. of pharmaceutical composition in the present invention.

In addition, pharmaceutical composition of the invention can further comprise lubricant, wetting agent, emulsifier, suspension stabiliser, preservative, sweetener and fragrance etc..Pharmaceutical composition of the invention can be produced by a variety of well known methods with enteric-coated preparations, in order to which active constituent, that is, microorganism of pharmaceutical composition can pass through stomach without being destroyed by gastric acid.

In addition, the capsule form that microorganism of the invention can prepare in conventional manner uses.For example, standard excipients and cold dry microorganism of the invention are mixed and made into bead pill, then pill is packed into gelatine capsule.In addition, microorganism and drug of the invention allow using excipient such as liquid glue, cellulose, silicate or mineral oil etc. suspension or dispersion liquid is made by mixing, this suspension or dispersion liquid can be fitted into soft gelatine capsule.

Pharmaceutical composition of the invention can be made into casing piece for being administered orally.Term-" casing " in the application, including all conventional medicines allow using coating, these coatings can decompose sufficiently and quick release goes out microorganism of the invention not by gastric acid degradation in small intestine.Casing of the invention can maintain 2 hours or more in the HCl solution of synthesis gastric acid such as pH=1 at 36-38 DEG C, and preferably decompose in 1.0 hours in the buffer of synthesis intestinal juice such as pH=7.0.

Casing of the invention is to be coated with every agreement that contracts a film or TV play to an actor or actress 16-30mg, and preferably 16-25mg, more preferably 16-20mg are coated.Casing thickness is 5-100 μm in the present invention, preferably with a thickness of 20-80 μm.Casing ingredient selects oneself open conventional polymer known.

Currently preferred casing is by cellulosic phthalic acetate polymer or trimellitic acid polyisocyanate polyaddition The preparation of the copolymer (for example, copolymer of the methacrylate containing 40% or more methacrylate and containing methyl cellulose phthalate hydroxypropyl acrylate or its ester derivative) of object and methacrylate.

The viscosity of cellulosic phthalic acetate used in casing is about 45-90cp, acetyl content 17-26%, phthalate content 30-40% in the present invention.It is about 5-21cs, second phthalein content 17-26% for the cellulose acetate trimellitate viscosity in casing.Cellulose acetate trimellitate is produced by Eastman Kodak company, the enteric materials that can be used in the present invention.

For the hydroxypropyl methylcellulose phthalate in casing of the present invention, molecular weight is generally 20,000-130,000 dalton; desired molecular weight is 80,000-100,000 dalton; hydroxypropyl content is 5-10%, and methoxyl content 18-24%, phthalyl content is 21-35%.

It is HP50 for the hydroxypropyl methylcellulose phthalate in casing of the present invention, is produced by Japanese Shin-Etsu Chemidnl Co.Ltd..HP50 contains 6-10% hydroxypropyl, 20-24% methoxyl group, the propyl of 21-27%, 84,000 dalton of molecular weight.Another Enteric materials are HP55, and HP55 contains the hydroxypropyl methylcellulose phthalate of 5-9%, 18-22% methoxyl group, the phthalic acid of 27-35%, 78,000 dalton of molecular weight.

Casing of the present invention is prepared as follows: casing solution being sprayed in core using conventional method.In the enteric coating method all solvents be alcohols (such as ethyl alcohol), ketone (such as acetone), halogenated hydrocarbon compound (such as methylene chloride), or combinations thereof object.Softening agent such as di-n-butyl phthalic acid ester and glyceryl triacetate are added in casing solution, its ratio be 1 part of coatingss to about 0.05 part or about 0.3 part of softening agent.Spray method preferably continuously performs, and spraying doses can be controlled according to used condition is coated.Atomisation pressure can be adjusted arbitrarily, it is however generally that, ideal result can be obtained under average 1-1.5 bar pressure.

" medicine effective quantity ", which refers to, in specification to generate function or amount that is active and being received by people and/or animal to people and/or animal.For example, in the present invention, can prepare containing 1 × 10-1 × 10¹⁵Cfu/ml or cfu/g (particularly, can contain 1 × 10⁴-1×10¹⁰Cfu/ml or cfu/g；More particularly, 1 × 10 can be contained⁶-1×10¹⁰Cfu/ml or cfu/g) Shenzhen Collins bacterium and/or its metabolite preparation.

When being used to prepare pharmaceutical composition, the effective dose of Shenzhen Collins bacterium used or its metabolite can change with the mode and the severity of disease to be treated of application.Suitable for dosage form for oral administration, include and solid-state or intimately mixed about 1 × 10-1 × 10 of liquid pharmaceutically acceptable carrier¹⁵Cfu/ml or cfu/g are (preferably, can contain 1 × 10⁴-1×10¹⁰Cfu/ml or cfu/g；More preferably, 1 × 10 can be contained⁶-1×10¹⁰Cfu/ml or cfu/g) active Shenzhen Collins bacterium or fermentation generate active constituent.This dosage is adjusted to provide optimal treatment response.For example, dosage separated several times can be given once daily, or dosage is reduced pari passu by an urgent demand for the treatment of situation.

Shenzhen Collins bacterium or its metabolite can be given by the approach such as oral.Solid-state carrier includes: starch, lactose, Dicalcium Phosphate, microcrystalline cellulose, sucrose and white bole, and liquid carrier includes: culture medium, polyethylene glycol, nonionic surface active agent and edible oil (such as corn oil, peanut oil and sesame oil), as long as being suitble to Collins bacterium or its metabolite characteristic and required specific administration mode.Usually used adjuvant can also advantageously be included in preparation pharmaceutical composition, such as flavoring agent, pigment, preservative and antioxidant such as vitamin E, vitamin C, BHT and BHA.

In terms of easily prepared and administration position, preferred pharmaceutical composition is the capsule of solid-state composition, especially tablet and solid-filling or liquid filling.Oral administration is preferred.

The present composition is administered to the individual, is administered once daily or repeatedly.Dosage unit indicates that it can separate in form and be suitable for the mankind or the dosage of other all mammalian subjects.Per unit contains the carrier of drug permission and the microorganism of the present invention of effective therapeutic dose.Dosage with patient weight and inflammation Related disease (such as inflammatory bowel disease (such as ulcerative enteritis, gastritis, common enteritis), rheumatoid arthritis), the severity of cardiovascular disease (such as hypertension, hyperlipidemia, coronary heart disease), included supplement active constituent and used microorganism and change.Furthermore as possible, it can separate and be administered, and as needs can successive administration.Therefore, the dosage will not cause to limit to the present invention.In addition, " composition " in the present invention does not mean only that drug and expression can be used as functional food and healthy supplement.In a preferred embodiment, the composition includes: beverage, food, drug, animal feed etc..

In a preference of the invention, additionally provide a kind of food compositions, it contains a effective amount of Shenzhen Collins bacterium and/or its metabolite, and acceptable carrier on the food of surplus, the dosage form of the food compositions are selected from solid, dairy products, solution product, pulverulent product or suspension product.In a preferred example, the food compositions can also contain growth factor (such as milk growth factor).In a preferred example, the composition further includes probiotics selected from the group below: lactic acid bacteria, Bifidobacterium, lactobacillus acidophilus, or combinations thereof；And/or prebiotics selected from the group below: oligofructose (FOS), galactooligosaccharide (GOS), xylo-oligosaccharide (XOS), lactosucrose (LACT), soyabean oligosaccharides (SOS), inulin (Inulin), oligosaccharide, or combinations thereof.In a preferred embodiment, the composition further includes the substance (such as protective agent) selected from the group below for helping to maintain Collins's bacterium vigor: cysteine, glutathione, butylated hydroxy anisole, dibutylmethyl toluene, tocopherol, bamboo leaf antioxidant, D-araboascorbic acid and its sodium salt, sodium ascorbate, Calcium Ascorbate, phosphatide, vitamin C (ascorbic acid), vitamin E, or combinations thereof.

In a preferred example, the formula of the composition is as follows:

1×10-1×10¹⁵Shenzhen Collins bacterium of cfu/mL and/or its metabolite；And on food or pharmaceutically acceptable carrier and/or excipient.

In another preferred example, the formula of the composition is as follows:

1×10⁴-1×10¹⁰Shenzhen Collins bacterium of cfu/mL and/or its metabolite；And on food or pharmaceutically acceptable carrier and/or excipient.

Probiotics

Probiotics are the dietary supplements that a kind of biological agent comprising probiotics and metabolite can either increase probiotics, can achieve the purpose that improve human health level by adjusting, maintaining microecological balance in enteron aisle.It mainly include probiotics, prebiotics and synbiotic.

In the present invention, the probiotics include (a) Shenzhen Collins bacterium and/or its metabolite of safe and effective amount；And (b) acceptable carrier or pharmaceutically acceptable carrier on food.In a preferred example, the preparation further includes growth factor (such as milk growth factor, preferably, including vitamin substances, purine substance, and/or pyrimidine substance).In a preferred embodiment, the preparation further includes probiotics selected from the group below: lactic acid bacteria, Bifidobacterium, lactobacillus acidophilus, or combinations thereof；And/or prebiotics selected from the group below: oligofructose (FOS), galactooligosaccharide (GOS), xylo-oligosaccharide (XOS), lactosucrose (LACT), soyabean oligosaccharides (SOS), inulin (Inulin), oligosaccharide, or combinations thereof.In a preferred embodiment, the composition further includes the substance (such as protective agent) selected from the group below for helping to maintain Collins's bacterium vigor: cysteine, glutathione, butylated hydroxy anisole, dibutylmethyl toluene, tocopherol, bamboo leaf antioxidant, D-araboascorbic acid and its sodium salt, sodium ascorbate, Calcium Ascorbate, phosphatide, vitamin C (ascorbic acid), vitamin E, or combinations thereof.

The production method of Shenzhen Collins bacterium

In general, Shenzhen Collins bacterium can be made with conventional method.

In the present invention, the method that Shenzhen Collins bacterium can be mass produced by providing one kind specifically includes the following steps:

(a) under conditions of being suitble to culture, Collins bacterium of the present invention is cultivated, to obtain cultured products；

(b) optionally, Collins bacterium thallus and/or its metabolite are separated from the cultured products；With

(c) optionally, the cultured products of previous step acquisition or Collins bacterium thallus and/or its metabolite are mixed with carrier acceptable on food or pharmaceutically acceptable carrier, so that composition be made.

Improve the method for mammalian gut lesion

In another preferred example, which comprises absorb pharmaceutical composition of the invention, food compositions, beverage composition for treating dental erosion, or combinations thereof.The experimental subjects includes mammal, such as people.

In another preferred example, which comprises pharmaceutical composition of the invention, food compositions or animal feed are absorbed, or combinations thereof.The experimental subjects is animal, preferably muroid, Lagomorpha.

The method for controlling the decline of weight of mammal, and/or reducing the disease activity index (DAI) of mammal

The method for reducing blood lipid

Culture presevation

Strain Shenzhen Collins bacterium Collinsella shenzhenensis TF06-26 (identical as preservation title) of the invention is deposited in Guangdong Province's Culture Collection on October 13rd, 2016,5 building, the building of compound the 59th of address Xianlie Middle Road, Guangzhou City 100, deposit number: GDMCC NO.:60090.

Main advantages of the present invention include:

(a) Shenzhen Collins bacterium of the invention can significantly reduce blood lipid (such as cholesterol levels).

(b) Shenzhen Collins bacterium of the invention can significantly improve index relevant to inflammation related disease (such as inflammatory bowel disease (such as ulcerative enteritis, gastritis, common enteritis), rheumatoid arthritis) (such as control weight loss improves enteron aisle lesion degree (including slow down colon lengths and shorten, mitigate colitis reaction), reduces disease activity index (DAI)).

Present invention will be further explained below with reference to specific examples.It should be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition such as Sambrook et al., molecular cloning: laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989) condition described in, or (James Cappuccino and Natalie Sherman are compiled according to " microorganism: laboratory manual ", Pearson Education publishing house) described in condition, or according to the normal condition proposed by manufacturer.

Unless otherwise instructed, material and reagent used in embodiment are commercial product.

VSL#3 is purchased from U.S. Sigma-Tau.

Collinsella aerofaciens JCM 10188, purchased from Japanese Culture Collection, deposit number is JCM 10188.

The screening and identification of 1 Shenzhen Collins bacterium TF06-26 of embodiment

The separation of 1.1 Shenzhen Collins bacterium TF06-26 (abbreviation TF06-26)

(1) sample acquires

Shenzhen Collins bacterium TF06-26 of the invention is isolated from the fecal specimens of a healthy adolescent female volunteers of Shenzhen.

(2) prepare culture medium and PBS (phosphate buffer)

The culture medium of the used strain isolation of the present invention is the PYG culture medium (being purchased from Huan Kai microorganism scientific & technical corporation) of anaerobism, and specific ingredient is (1L): peptone 5g, pancreas casein 5g, yeast powder 10g, beef extract 5g, glucose 5g, K₂HPO₄2g, Tween 80 1mL, Cysteine-HClH₂O 0.5g, ferroheme 5mg, vitamin K₁1uL, (every liter of (L) inorganic salt solution contains CaCl to inorganic salt solution₂·2H₂O0.25g, MgSO₄·7H₂O 0.5g, K₂HPO₄1g, KH₂PO₄1g, NaHCO₃10g, NaCl 2g) 40mL, resazurin 1mg, distilled water 950mL, adjust pH to 6.8~7.0.Sterilising conditions are 115 DEG C of high pressure sterilization 25min.Solid medium need in anaerobic operation case pour plate.

The preparation of PBS: NaCl 8g, KCl 0.2g, Na are weighed₂HPO₄·12H₂O 3.63g, KH₂PO₄0.24g, cysteine hydrochloride 0.5g are dissolved in 900ml distilled water, with hydrochloric acid and NaOH tune pH value to 7.4, are added water to be settled to 1L, are passed through N₂Deoxygenation 30s, anaerobism bottle (pipe) sealing, then 115 DEG C of high pressure sterilization 25min, spare.

(3) strain isolation

The fresh excreta sample of collection is immediately transferred into the anaerobic box (gas composition of anaerobic box are as follows: nitrogen: hydrogen: carbon dioxide=90:5:5, v/v), about 0.2g excrement is taken to suspend in PBS, it mixes well, carries out the gradient dilution that 10 times are unit, then apply plate, it is cultivated 2 days under 37 DEG C of anaerobic conditions, selection single colonie carries out scribing line purifying, obtains pure culture bacterial strain, and carry out -80 DEG C of Freezing Glycerine preservations and lyophil preservation.

1.2 16S rDNA identification

16S rDNA sequencing is carried out to isolated pure culture bacterial strain, obtains the classification information of every plant of bacterium.Bacterial strain is cultivated 24 hours in the PYG culture medium of liquid, reaches bacterium dense about 10⁸Cfu/ml, genome extraction is carried out to bacterium solution, 16S rDNA PCR amplification is carried out using the genomic DNA of extraction as template, amplimer uses 16S rDNA universal primer: 8F-1492R (5 '-AGAGTTTGATCATGGCTCAG-3 ' (SEQ ID NO.:2) and 5 '-TAGGGTTACCTTGTTACGACTT-3 ' (SEQ ID NO.:3)) (synthesis is in Sangon Biotech (Shanghai) Co., Ltd.), amplification program is: 95 DEG C of initial denaturation 4min, then 95 DEG C of denaturation 30s, 57 DEG C of annealing 40s, 72 DEG C of extension 1min30s, 30 circulations.The PCR product of amplification is purified, 3730 sequencings, obtains the 16S rDNA full length sequence of every plant of bacterium, the 16S rDNA length of TF06-26 is 1380bp, as shown in SEQ ID NO.:1.By comparing in EzTaxon-e database, TF06-26 is Collinsella aerofaciens JCM with the highest bacterium of database 16S rDNA sequence similarity 10188。

The 16S rDNA sequence of TF06-26 is SEQ ID NO.:1, as follows:

The microbial characteristic of 1.3 TF06-26

(1) morphological feature

The bacterium colony of TF06-26 Anaerobic culturel 48h is white, and protrusion, round, neat in edge, water content is more, opaque, and colony diameter is 1.0mm-2.0mm (Fig. 1).

(2) microscopic features

By carrying out Gram's staining, spore staining, flagella staining respectively to TF06-26, the thalli morphology of TF06-26 is observed under 1000 times of amplifications.TF06-26 is Gram-negative, does not produce gemma and flagellum, rod-short (Fig. 2).

(3) physiological and biochemical property

TF06-26 is negative catalase, and oxidase negative does not have motility, and growth temperature range is 25-45 DEG C, and growth pH value range is 5.0-8.0, and NaCl tolerable concentration is 2%, and cholate tolerable concentration is 0.3%.TF06-26 and nearly edge refer to bacterium Collinsella aerofaciens JCM 10188 (purchased from Japanese Culture Collection, deposit number be JCM 10188) substrate utilization power (API 20A and API50CHL) see Table 1 for details (+, indicate positive reaction；, indicate negative reaction；W indicates weakly positive reaction).

Table 1

It is shown compared with the utilization of carbon source situation of JCM10188 by the above TF06-26, TF06-26 is same JCM10188 has significantly different in the utilization of lactose, sucrose, salicin, galactolipin, fructose, mannose, Arbutin, cellobiose, maltose, melibiose, trehalose and 2- keto-D-gluconate salt, it can be seen that TF06-26 is not the same species with JCM10188.

1.4 antibiotic resistance

TF06-26 is investigated to the sensitive situations of 20 kinds of antibiotic, TF06-26 is cultivated to logarithmic growth phase, it takes the bacterium solution of TF06-26 to carry out plate (PYG culture medium) coating, sticks antibiotic drug sensitive piece after smearing uniformly, be then placed under 37 DEG C of anaerobic conditions and cultivate 48h.The diameter of inhibition zone is measured, the results are shown in Table 2.

Table 2

TF06-26 is resistant to kanamycins, amikacin, neomycin and erythromycin as the result is shown, to other antibiotic sensitives in 16.

1.5 new species TF06-26 are tested with the genomic hybridization of relationship bacterial strain JCM 10188

With reference to the 16S rDNA comparison result of TF06-26, the nearest bacterium of affiliation is Collinsella aerofaciens JCM 10188,16S rDNA similarity is 99.9%, from the point of view of 16S rDNA sequence, TF06-26 and JCM 10188 can not be subjected to kind of a horizontal differentiation, it is therefore desirable to carry out DNA hybridization and further confirm.

DNA hybridization the results show that TF06-26 with JCM 10188 homology be 51%.According to " primary Jie Shi Bacteria Identification handbook ", the DNA hybridization value of two plants of bacterium is higher than 70%, it is possible to determine that this two plants of Pseudomonas are in the same species, and the DNA hybridization value of TF06-26 and JCM 10188 are lower than 70%, so TF06-26, which is one plant, is different from synxenic novel bacterial.According to the international division bacteria committee (IBSP) bacterium naming rule, the new bacterium of this strain is named as the type strain of Collinsella shenzhenensis sp.nov, TF06-26 as this kind.

The bioactive substance of 2 Shenzhen Collins bacterium TF06-26 of embodiment

The bioactive substance of TF06-26 mainly investigates short chain fatty acids (SCFA) and organic acid production.

(1) sample pretreatment

TF06-26 is cultivated into 48h, takes 1ml bacterium solution to carry out 12000r/min centrifugation 5min, takes supernatant, be ready for the detection of short chain fatty acids (SCFA) and organic acid.

(2) measurement of SCFA

The measurement of short chain fatty acids uses external standard method, and acetic acid, propionic acid, butyric acid, valeric acid is selected to carry out the production of standard curve.Using Agilent gas chromatograph (GC-7890B, Agilent), HP-INNOWax (Cross-Linked PEG) is selected, 30m × 0.25mm × 0.25um capillary column is analyzed, detector is hydrogen flame ionization detector, and GC parameter is set as column temperature: 180~200 DEG C；Gasify room temperature: 240 DEG C；Detection temperature: 210 DEG C；Sample volume: 2 μ L；Carrier gas flux: N₂, 50mL/min；Hydrogen flowing quantity: 50mL/min；Air mass flow: 600~700ml/min.

Measurement obtains SCFA yield are as follows: acetic acid 19.88mmol/L, butyric acid 4.37mmol/L.

(3) measurement of organic acid

The examination criteria product of organic acid are selected: 3 Methylbutanoic acid, valeric acid, quininic acid, lactic acid, oxalic acid, malonic acid, benzoic acid, maleic acid, succinic acid, anti-fumaric acid, malic acid, adipic acid, tartaric acid, shikimic acid, citric acid, isocitric acid and L-AA.Still Agilent gas chromatograph (GC-7890B, Agilent) is used, chromatographic column selects 122-5532G DB-5ms (40m × 0.25mm × 0.25um), column temperature: 270~290 DEG C；Injector temperature: 250 DEG C；Gas flow: 0.86ml/min.The content for measuring Shenzhen Collins's bacterium Collinsella shenzhenensis TF06-26 organic acid is detailed in following table:

Table 3

The exocellular polysaccharide production of 3 Shenzhen Collins bacterium TF06-26 of embodiment

Bacterium colony by observing TF06-26 can be found that bacterium colony water content is higher, and bacterium solution culture discovery is more sticky, can speculate the ability that TF06-26 has extracellular polysaccharide.The detection of exocellular polysaccharide uses Phenol-sulphate acid method, and with the hexose etc. in free monosaccharide, oligosaccharides and polysaccharide chromogenic reaction can occur for phenol sulfuric acid, and the color of generation is directly proportional with absorbance, absorbing wavelength 490nm.Specific experiment process is as follows:

(1) extraction of polysaccharide

Experimental strain takes 10ml bacterium solution boiling water bath to handle 30min culture 3 days in PYG culture medium (formula is with example 1), and then 10000r/min is centrifuged, and takes supernatant, and 80% trichloroacetic acid is added to final concentration of 8%, 4 DEG C of processing overnight, protein precipitation.It takes out 10000r/min and is centrifuged 30min, the pH to 6.0 of supernatant is adjusted with NaOH.The dehydrated alcohol that 2 times of volumes are added carries out polysaccharide precipitation, and 4 DEG C of processing overnight take out progress 10000r/min and are centrifuged 30min, discard supernatant, and the distilled water of precipitating preheating is dissolved, then shifted Ultrafiltration is carried out into super filter tube (3000Da filters diameter), 5000r/min is centrifuged 30min, and the polysaccharide retained in inner tube is transferred to distilled water in volumetric flask and is settled to 10ml, spare.

(2) production of glucose standard curve

Precision weighs standard glucose 20mg into 100ml volumetric flask, adds water to graduation mark, and the glucose standard of 20,40,60,80,100 μ g/ml is then respectively configured.Every group of titer takes 400ul, and three parallel, and the phenol and the 1ml concentrated sulfuric acid for sequentially adding 400ul 5% are reacted, and is cooled to room temperature after boiling water bath 15min, absorbance of the measurement in 490nm.Then using absorbance as ordinate, glucose standard concentration is that abscissa draws standard curve.

(3) concentration of the polysaccharide of Detection and Extraction

Polysaccharide solution 400ul is taken, the phenol and the 1ml concentrated sulfuric acid for sequentially adding 400ul 5% are reacted, and are cooled to room temperature after boiling water bath 15min, absorbance of the measurement in 490nm.The concentration of polysaccharide is calculated according to glucose standard curve.

(4) result

By calculating, the content of exocellular polysaccharide is 284.49mg/L in the TF06-26 fermentation liquid of culture 3 days.

The detection of 4 Shenzhen Collins's bacterium TF06-26 external degradation cholesterol function of embodiment

4.1TF06-26 the detection of cholate tolerance

Contain cholate, the tolerance that probiotics if necessary to play prebiotic effect in enteron aisle then needs that it is certain to have cholate in human body intestinal canal, therefore the present invention has investigated TF06-26 in vitro to the tolerance situation of cholate.

Prepare the PYG fluid nutrient medium for being 0.2% containing gallbladder salinity, it will be in PYG culture medium of the TF06-26 of culture to logarithmic growth phase according to 10% inoculum concentration to 0.2%, 37 DEG C of processing 2h, then it is diluted plate count, plate count is diluted to the TF06-26 before inoculation simultaneously, calculates survival rate of the TF06-26 in 0.2% cholate environment.Show that TF06-26 is resistant to the cholate of 0.2% concentration by count results, survival rate reaches 70%.

The bile salt hydrolase activity of 4.2TF06-26

Bile salt hydrolase is detected using TDCA method, prepares TDAC plate, the CaCl of TDAC (Taurodeoxycholate sodium) and 0.37g/L of addition 4% in PYG solid medium₂, TF06-26 is cultivated to concentration about 10⁸Cfu/ml takes 10ul bacterium solution drop on the filter paper that diameter is 0.6mm, and filter paper is placed in TDAC planar surface, and 37 DEG C are cultivated 2 days, and the white precipitate situation that observation filter paper periphery generates, the diameter of white precipitate represents the activity of bile salt hydrolase.By measurement, the white precipitate of TF06-26 is 9mm, shows that TF06-26 has the activity of bile salt hydrolase.

4.3TF06-26 external norcholesterol situation

The content assaying method of cholesterol uses o-phthalaldehyde colorimetric method (OPA method), is investigating the degradation capability to cholesterol containing the variation before and after the cholesterol level for cultivating a period of time in certain density cholesterol culture medium by bacterial strain.Specific side is as follows:

(1) culture of the preparation of cholesterol culture medium and experimental strain

The cholesterol for weighing certain mass is dissolved in ethyl alcohol, concentration 10mg/mL, filtration sterilization.Configured PYG culture medium is separately added into the cholate (high pressure sterilization) of 10mg/mL, the sodium thioglycolate (filtration sterilization) and cholesterol of 10% mass concentration, it mixes well, then strain to be tested is seeded in the culture medium according to 3% inoculum concentration, wherein strain to be tested is TF06-26, and other one plant of business norcholesterol probiotics lactobacillus plantarum Lp299v (being purchased from Sweden Probi company), both bacterium are made comparisons respectively, both bacterium all cultivate 72h under 37 DEG C of anaerobic conditions.

(2) production of standard curve

The cholesterol standard solution 40uL of accurate measuring 0.5mg/mL, 80uL, 120uL, 160uL, 200uL is in clean tube, dehydrated alcohol is added and is settled to 1mL, OPA 4mL (0.5mg o-phthalaldehyde is added to 1mL glacial acetic acid) is added in each test tube, concussion mixes, it is stored at room temperature 10min, then the concentrated sulfuric acid that 2mL is added mixes, and stands reaction 10min, absorbance is measured at 550nm.Using concentration as abscissa, absorbance draws standard curve (Fig. 3) as ordinate, by calculating, the equation of linear regression are as follows: y=0.0085x+0.0072；Coefficient R²It is 0.9992.

(3) in culture medium cholesterol measurement

The centrifugation that the cultured bacterium solution of PYG culture medium containing cholesterol is carried out to 10000r/min, collects supernatant, carries out cholesterol detection, while using nonvaccinated cholesterol PYG culture medium as blank control group.It takes 1ml sample to be tested in clean test tube, the KOH 4ml of 95% ethyl alcohol 6ml and 50% is added, concussion mixes, then saponification 10min is carried out in 60 DEG C of water-baths, it is cooled down rapidly, 10ml n-hexane is added and is extracted, mixes well, it is stored at room temperature 20min, 8ml organic phase (n-hexane layer) is measured into another clean tube, is then dried with nitrogen in 60 DEG C of water-baths, 4ml 0.5g/L o-phthalaldehyde acetic acid solution is added, it is stored at room temperature 10min, adds the dense H of 2ml₂SO₄10min is reacted, the light absorption value at 550nm is finally measured.

(4) calculating of degrading rate of cholesterol

The content of cholesterol in the culture medium of culture front and back is calculated according to standard curve, the degradation rate of cholesterol is calculated as follows:

L=(A-B)/A × 100%

L: degrading rate of cholesterol；

A: it is not inoculated with the content of cholesterol in the cholesterol culture medium of bacterium；

B: the content of cholesterol in strain to be tested culture 48h culture solution.

(5) cholesterol degradation result

By calculating, the degrading rate of cholesterol for obtaining TF06-26 is 74.4%, and Lp299v degradation rate has 70%, thus illustrates that TF06-26 has the cholesterol degradation ability of highly significant, or even has stronger cholesterol degradation ability than Lp299v.

Cholesterol is the main ingredient in blood lipid, the raising of cholesterol levels and atherosclerosis have close relationship in blood plasma, therefore TF06-26 of the invention can reduce blood lipid, reduce the index of correlation of atherosclerosis related disease (such as cardiovascular disease).

Intervention situation of the 5 Shenzhen Collins bacterium Collinsella shenzhenensis TF06-26 of embodiment to ulcerative enteritis (UC) mouse model

The purpose of the present embodiment is to investigate Shenzhen Collins bacterium Collinsella shenzhenensis TF06-26 to the therapeutic effect of UC model mice, by comparing TF06-26, PBS and positive drug (VSL^#3) it (is purchased from U.S. Sigma Tau) and therapeutic intervention, the treatment condition for observing the pathological hallmarks such as weight, the death rate, colon lengths, DAI index and the intestinal mucosa pathological change of mouse to evaluate TF06-26 to UC is carried out to UC model mice.

5.1 experimental material

Experiment mice is SPF grades using C57bl/6 mouse (being purchased from Hubei medical experiment animal center), 10 week old, weight 28g ± 2g, mouse feeding environment, adaptable fed progress modeling in 1 week.

5.2UC modeling method

The modeling method of UC is induced using 1.5%DSS (dextran sulfate sodium) (being purchased from U.S. MPBiomedicals), and the duration is 7 days.

5.3 experimental group

Experiment mice totally 56, four groups are randomly divided into, it is as follows to be grouped situation for every group of 14 mouse:

First group: Control group (blank control group) --- normal mouse does not carry out DSS induction

Second group: UC model group --- DSS modeling, and stomach-filling is carried out using sterile PBS

Third group: VSL^#3 treatment groups --- DSS modeling, and use VSL^#3 treatment (bacterium dense 10⁹cfu/ml)

4th group: TF06-26 treatment group --- DSS modeling, and (bacterium dense 10 is treated using TF06-26⁹cfu/ml)

5.4 experimentation

The ordinary circumstances such as the diet, drinking-water, activity of mouse are observed and recorded daily, and it weighs, observes the fecal character and fecal occult blood situation of mouse, calculate DAI index (table 4), Experiment intervention is treated 7 days by a definite date, and the day stomach-filling amount of probiotics is 200ul/.Mouse is put to death after experiment, all mouse take blood, de- neck, take colon, take pictures, weighing, measuring colon lengths.Colonic tissue is stored in -80 DEG C of refrigerators and paraformaldehyde.

4 DAI index score table of table

DAI index is equal to the sum of weight, stool and stool blood three integrals.

5.5 experimental result

(1) changes of weight

The weight record of experimentation mouse is shown in Table 5.

Table 5

Starting the modeling stage, the weight levels of each group mouse are almost the same, with the induction of DSS, model group, VSL^#The weight of 3 and TF06-26 group mouse is gradually reduced, and to the 4th day and the 7th day, the weight loss of model group mouse was than more significant (relative to P < 0.05 Control group *).It can be found that the weight of mouse can be caused to be remarkably decreased by the induction of DSS, at the 7th day, the weight of TF06-26 group mouse be significantly higher than model group (^▲P < 0.05), illustrate that TF06-26 can effectively control mouse weight decline.

(2) variation of the disease activity index (DAI) of mouse

Record mouse calculates DAI index, the results are shown in Table 6 in the 1st day, the 4th day and the 7th day stool of modeling and stool blood situation respectively.

Table 6

From the results shown in Table 6, after treatment 7 days, VSL^#3 and TF06-26 treats the disease activity index (P < 0.05 DAI index * relative to model group) that mouse can be significantly reduced.TF06-26 group is lower than positive drug VSL in the DAI index of the 7th day mouse simultaneously^#3, illustrate that TF06-26 improves situation to the disease of UC mouse and is better than VSL^#3。

(3) variation of mouse Colon length

The colon of DSS induction UC model mice generally will appear ulcer, and length is caused to shorten, and by dissecting mouse after experiment, the colon lengths of each group mouse be measured, as a result as shown in table 7 and Fig. 4.

Table 7

Table 7 and Fig. 5 show that the length of the colon of TF06-26 group mouse will be considerably longer than model group mouse (value < 0.05 * P), while the length of the colon of TF06-26 group mouse is than positive drug VSL^#3 groups of length illustrates that TF06-26 improves situation to the disease of UC mouse and is better than VSL^#3.It can be seen that TF06-26 can significantly slow the lesion of mouse Colon.

The food compositions of the 6 bacterium Collinsella shenzhenensis of Collins containing Shenzhen TF06-26 of embodiment

Raw material proportioning such as table 8.

Table 8

Raw material

Mass percent (%)

Collinsella shenzhenensis TF06-26	0.5
Milk	90.0
White sugar	9.0
Vitamin C	0.5

According to above-mentioned formula rate mixing milk, white sugar, stirring preheats, 20Mpa pressure homogeneous to being thoroughly mixed, 90 DEG C or so sterilization 5-10 minutes, be cooled to 40-43 DEG C, be mixed into protective agent vitamin C, inoculation 1-100 × 10⁶The Collinsella shenzhenensis TF06-26 bacterium of cfu/g, that is, be made the food compositions of the TF06-26 bacterium of shenzhenensis containing Collinsella.

The pharmaceutical composition of the 7 bacterium Collinsella shenzhenensis of Collins containing Shenzhen TF06-26 of embodiment

Raw material proportioning is shown in Table 9.

Table 9

Raw material	Mass percent (%)
Collinsella shenzhenensis TF06-26	1.0%
Lactose	2.0%
Yeast powder	2.0%
Peptone	1.0%
Pure water	93.5%
Vitamin C	0.5%

Proportionally lactose, yeast powder, peptone are uniformly mixed with pure water; it is preheating to 60-65 DEG C; 20Mpa pressure homogeneous; 90 DEG C or so sterilization 20-30 minutes; it is cooled to 36-38 DEG C; it is mixed into protective agent vitamin C, accesses Collinsella shenzhenensis TF06-26 viable bacteria (1-50 × 10⁶Cfu/mL), 36-38 DEG C of fermentation to pH value is 6.0, centrifugation, and freeze-drying less than 3%, that is, prepares Collinsella shenzhenensis TF06-26 bacterium freeze-drying object to water content.It is fitted into capsule after weighing 0.5 gram of Collinsella shenzhenensis TF06-26 freeze-drying object and maltodextrin mixed in equal amounts, that is, the pharmaceutical composition of the TF06-26 bacterium of shenzhenensis containing Collinsella is made.

The preparation of the drug of the 8 bacterium Collinsella shenzhenensis of Collins containing Shenzhen TF06-26 of embodiment

8.1 bacterium solution prepares: by Collinsella shenzhenensis TF06-26 (1 × 10⁹Cfu/ml) carry out Anaerobic culturel, anaerobic culture medium use PYG culture medium, by 37 DEG C anaerobic fermentation 2-3 days.

The preparation of 8.2 growth factors: skim milk, casein are mixed, are centrifuged, ultrafiltration obtains milk growth factor crude extract (nutriment containing vitamin substances, purine substance, and/or pyrimidine substance).

The production of 8.3 pharmaceutical dosage forms: the protective agent vitamin C of the 5 volume growth factors and 1 volume is added in the bacterium solution of TF06-26 fermentation of 100 volumes; mixing is sufficiently stirred; then starch supplementary material (such as maltodextrin) is added, so that the drug of the bacterium Collinsella shenzhenensis of Collins containing Shenzhen TF06-26 be prepared.

Culture presevation

All documents that the present invention refers to are incorporated herein by reference, as if each reference was individually incorporated by reference.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can make various modifications or changes to the present invention, these equivalent forms also fall within the scope of the appended claims of the present application.

Claims

A kind of Collins bacterium, which is characterized in that the Collins bacterium is Shenzhen Collins bacterium (Collinsella shenzhenensis).
A kind of composition, which is characterized in that the composition includes: Collins bacterium described in claim 1 and/or its metabolite of (a) safe and effective amount；And (b) acceptable carrier or pharmaceutically acceptable carrier on food.
Composition as claimed in claim 2, which is characterized in that the composition also contains other probiotics and/or prebiotics.
Composition as described in claim 2, which is characterized in that the composition also contains the substance (such as protective agent) for helping to maintain Collins's bacterium vigor.
A kind of purposes of composition described in Collins bacterium described in claim 1 or claim 2, it is characterized in that, it is used to prepare drug or preparation, the drug or preparation are used for one or more purposes selected from the group below: (a) prevention and or treatment inflammation related disease；And/or (b) prevent and/or treat cardiovascular disease.
The purposes of composition described in Collins bacterium described in a kind of claim 1 or claim 2, which is characterized in that be used to prepare drug or preparation, the drug or preparation are used for one or more purposes selected from the group below:

(i) blood lipid level of mammal is reduced；

(ii) decline of weight of mammal is controlled；

(iii) disease activity index (DAI) of mammal is reduced；

(iv) improve mammalian gut lesion.
A kind of preparation method of composition described in claim 2, which is characterized in that comprising steps of

Collins bacterium described in claim 1 and/or its metabolite are mixed with carrier acceptable on food or pharmaceutically acceptable carrier, to form composition as claimed in claim 2.
A kind of production method, which is characterized in that comprising steps of

(a) under conditions of being suitble to culture, Collins bacterium described in claim 1 is cultivated, to obtain cultured products；

(b) optionally, Collins bacterium thallus and/or its metabolite are separated from the cultured products；And/or

(c) optionally, the cultured products of previous step acquisition or Collins bacterium thallus and/or its metabolite are mixed with carrier acceptable on food or pharmaceutically acceptable carrier, so that composition be made.
A method of improving mammalian gut lesion, which is characterized in that apply composition as claimed in claim 2 to the object.
A method of blood lipid is reduced, the decline of weight of mammal is controlled, and/or reduces the disease activity index (DAI) of mammal, which is characterized in that applies composition as claimed in claim 2 to the object.