CN106974940B

CN106974940B - Application of probiotics of scleritis in treating and preventing obesity and related diseases

Info

Publication number: CN106974940B
Application number: CN201610032106.9A
Authority: CN
Inventors: 冯强; 张东亚
Original assignee: BGI Shenzhen Co Ltd
Current assignee: BGI Shenzhen Co Ltd
Priority date: 2016-01-15
Filing date: 2016-01-15
Publication date: 2020-08-25
Anticipated expiration: 2036-01-15
Also published as: CN106974940A

Abstract

The invention discloses an application of a scleritis probiotic in treating and preventing obesity-related diseases, in particular to a composition or a preparation prepared from the scleritis probiotic, wherein the composition or the preparation is used for one or more of the following purposes: (a) prevention and/or treatment of obesity; (b) reducing blood fat; (c) preventing or treating cardiovascular diseases; and/or (d) the prevention and/or treatment of diabetes. The probiotics for the thick-wall fungi can obviously reduce the weight, the blood fat and the body fat ratio.

Description

Application of probiotics of scleritis in treating and preventing obesity and related diseases

Technical Field

The invention belongs to the field of microbiology, and particularly relates to application of Probiotics of firmicutes in treating and preventing obesity and related diseases, and also relates to a composition containing the Probiotics of the firmicutes and application thereof.

Background

There are a large number of commensal microorganisms in humans, most of which reside in the human intestinal tract, in amounts exceeding 1000 trillion (10 trillion)¹⁴Order of magnitude) of more than 10 times the total number of human cells. In a long evolutionary process, intestinal microorganisms and human beings achieve good cooperation and play a crucial role in nutrition, metabolism and immunity of human bodies, and many researchers regard the intestinal microflora of the human bodies as an organ of the human bodies or a second genome of the human bodies, wherein the abundant genetic information contained in the intestinal microflora is closely related to the health of the human bodies. Through the research on nearly ten thousand samples of hundreds of diseases such as diabetes, coronary heart disease, obesity, colon cancer and the like,some specific species show significant association with disease, and these results provide a completely new direction in clinical assessment and diagnosis of disease and later intervention.

Obesity is a chronic disease, and various factors can cause obesity, and the origin of the obesity is not clearly researched. Obesity is also an inducing factor of a series of diseases such as hypertension, diabetes, coronary heart disease, cystic disease, osteoarthritis, sleep apnea, respiratory disorders, uterine tumors, prostate cancer, breast cancer, colon cancer and the like. According to NIH reports, about 9700 million americans are currently overweight and obese, with type II diabetes associated with obesity reaching about 1510 million people, and about 20 million people die of obesity-related diseases each year.

Obesity is usually an excess of body fat due to a change in physiological or biochemical functions. Fats generally include neutral lipids, phospholipids, and cholesterol. The increase in fat is due to energy intake being greater than energy expenditure. Obesity is, in principle, of two types: (a) simple obesity (simple obesity) and (b) secondary obesity (secondary obesity). Simple obesity can be classified into congenital obesity (obesity) and acquired obesity (obesity), and the number of simple obese patients can account for more than 95% of the total obesity. Congenital obesity is caused by a large number of adipocytes and is common in childhood obesity. Acquired obesity is caused by larger size adipocytes and is common in adult obesity. Secondary obesity, also known as symptomatic obesity, is usually caused by endocrine or metabolic disorders.

There are currently five strategies for treating obesity: diet, exercise, behavior therapy, medication and rehabilitation (theripteic operation). Which strategy is taken depends primarily on the patient's health risk factors and the rate and effect of weight loss, and these strategies may be selected or combined to treat obese patients. The rate and effect of weight loss is influenced by a number of factors such as age, height, family history and risk factors. Diet-exercise therapy, i.e., eating low-calorie, low-fat foods and performing aerobic exercise, but this method is generally considered unsuccessful for the general public and requires constant adherence for a long period of time; the operation for removing the fat in the body can achieve the effect of immediate effect, but has a plurality of limitations, such as operation risk, difficulty in lasting fat removal effect, high cost and the like.

Drug therapy is currently the primary clinical treatment for obesity and its obesity-related diseases (e.g., diabetes). The mechanisms of drug therapy include appetite suppression, increased energy expenditure, stimulation of fat movement, reduction of triglyceride synthesis, and inhibition of fat absorption. The main drugs at present are: phenylpropanolamine (PPA), freshman (orlistat, xenolith iii) and nominatine (sibutramine, reductil tm). Hyperglycemia in some diabetic patients is still not adequately controlled by dietary and/or exercise therapy or the use of the above therapeutic compounds. For these patients, exogenous insulin should be used. The use of exogenous insulin is an expensive and painful procedure for the patient and can cause a number of complications for the patient. For example, an incorrect calculation of the insulin dose may result in an insulin response (hypoglycemia) due to no meals or abnormal exercise. In addition, local or systemic allergies or immune resistance to drugs may also occur with drugs.

At present, no effective method and medicament for treating and preventing obesity and related diseases with small side effect exist in the field.

There is therefore a great need in the art to develop new, non-toxic side effects for the treatment and prevention of obesity and its related diseases.

Disclosure of Invention

It is an object of the present invention to provide the use of Probiotics of firmicutes for the treatment and prevention of obesity and related diseases.

It is another object of the present invention to provide a pharmaceutical, beverage, food composition, or animal feed composition for the treatment and prevention of obesity and related diseases, which is effective without toxic and side effects.

It is another object of the present invention to provide a method for reducing body weight and/or blood glucose and uses thereof.

In a first aspect, the present invention provides the use of a firmicutes probiotic for the preparation of a composition or formulation for one or more uses selected from the group consisting of: (a) prevention and/or treatment of obesity; (b) reducing blood fat; (c) preventing or treating cardiovascular diseases; and/or (d) preventing and/or treating diabetes, wherein the firmicutes probiotic is selected from the group consisting of: associated enterococcus faecalis (Coprococcus eutactus), alisteria turbidicola (Dialister invisus), or a combination thereof.

In another preferred example, the firmicutes probiotic comprises associated coprococcus faecalis (coprococcus faecalis).

In another preferred embodiment, the associated enterococcus faecalis (Coprococcus eutactus) is selected from the group consisting of: coprococcus eutactus ATCC27759, Coprococcus eutactus ATCC 51897, Coprococcus eutactus L2-50, or a combination thereof.

In another preferred example, the firmicutes probiotic comprises the species alisterinaviruses turbidivorans (dialistrinvisions).

In another preferred example, the bacterium alisterium turbidivorans (Dialister invisus) is selected from the group consisting of: dialister services DSM 15470, Dialister services E2.20, Dialister services E9.48, or a combination thereof.

In another preferred example, the firmicutes probiotics comprise one or more selected from table 3.

In another preferred embodiment, the firmicutes are selected from table 3 and are from the same or different genera.

In another preferred embodiment, the composition is selected from the group consisting of: a food composition, a nutraceutical composition, a pharmaceutical composition, a beverage composition, a feed composition, or a combination thereof.

In another preferred embodiment, the composition is an oral preparation.

In another preferred embodiment, the composition is a liquid preparation, a solid preparation or a semi-solid preparation.

In another preferred embodiment, the dosage form of the composition is selected from the group consisting of: powders, tablets, dragees, capsules, granules, suspensions, solutions, syrups, drops, and sublingual tablets.

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 an emulsion.

In another preferred embodiment, the liquid formulation is selected from the group consisting of: solution preparations or suspension preparations.

In a second aspect, the present invention provides the use of a firmicutes probiotic for the preparation of a composition or formulation for one or more uses selected from the group consisting of: (i) reducing the level of monocyte chemotactic protein-1 (MCP-1) in a mammal; and/or (ii) improving Leptin resistance and increasing in vivo sensitivity to Leptin, wherein the firmicutes probiotic is selected from the group consisting of: associated enterococcus faecalis (Coprococcus eutactus), alistericum turbidivorans (dialistrinvisions), or a combination thereof.

In another preferred embodiment, the composition or formulation is also used independently or additionally for one or more uses selected from the group consisting of:

(iii) inhibiting weight gain in a mammal;

(iv) reducing the body-to-fat ratio (fat weight/body weight ratio) of a mammal;

(v) lowering blood lipid levels in a mammal;

(vi) increasing the level of High Density Lipoprotein (HDLC) in a mammal;

(vii) reducing Low Density Lipoprotein (LDLC) levels in a mammal.

In another preferred embodiment, the mammal includes a human, a rodent (e.g., rat, mouse).

In another preferred embodiment, said lowering of blood lipid levels in a mammal comprises lowering Total Cholesterol (TC) levels and/or triglyceride levels.

In a third aspect the present invention provides a composition for use in the treatment and/or prevention of obesity, the composition comprising: (i) a safe and effective amount of a firmicutes probiotic; and (ii) a food-or pharmaceutically acceptable carrier; wherein the Probiotics of the firmicutes are selected from the group consisting of: associated enterococcus faecalis (Coprococcus eutactus), alisteria turbidicola (Dialister invisus), or a combination thereof.

In another preferred embodiment, the composition comprises 1 × 10-1 × 10²⁰cfu/mL or cfu/g of Probiotics of firmicutes, preferably 1 × 10⁴-1×10¹⁵cfu/mL or cfu/g of firmicutes probiotic, based on the total volume or total weight of the composition.

In another preferred embodiment, the composition comprises 0.0001-99 wt%, preferably 0.1-90 wt% of the pachycephalus probiotics, 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 0.05-5g, preferably 0.1-1 g.

In another preferred embodiment, the composition further comprises other probiotics and/or prebiotics.

In another preferred embodiment, the other 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: fructooligosaccharides (FOS), Galactooligosaccharides (GOS), Xylooligosaccharides (XOS), Lactosucrose (LACT), Soy Oligosaccharides (SOS), Inulin (Inulin), or combinations thereof.

In a fourth aspect, the present invention provides a method for preparing a composition according to the third aspect of the present invention, comprising the steps of:

mixing (i) a firmicutes probiotic with (ii) a food-acceptable carrier or a pharmaceutically acceptable carrier to form the composition according to the third aspect of the invention.

In another preferred embodiment, the composition is an oral preparation.

In a fifth aspect, the present invention provides a method of reducing body weight and/or blood lipid by administering to the subject (i) a firmicutes probiotic or a composition according to the third aspect of the invention.

In another preferred embodiment, said administering 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 mammal, such as a human.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows the weight gain of groups of mice after gavage associated with fecal cocci compared to before gavage.

FIG. 2 shows the weight gain of groups of mice after gastric lavage with turbid Alicase bacteria compared to those before gastric lavage.

FIG. 3 shows the weight gain of the groups of mice after the gavage combination compared to before gavage.

FIG. 4 shows the body-fat ratio of the groups of mice 9 weeks after gastric lavage of associated enterococcus faecalis.

FIG. 5 shows the body-to-fat ratio of groups of mice 9 weeks after gastric lavage with turbid Alicase bacteria.

FIG. 6 shows the body-to-fat ratio of the groups of mice after 9 weeks of the gastric lavage of the combination bacteria.

FIG. 7 shows the effect of intragastric fecal associated bacteria on blood lipids.

FIG. 8 shows the effect of Salmonella enterica on blood lipids.

FIG. 9 shows the effect of the Zygosaccharomyces on blood lipid.

FIG. 10 shows the effect of stomach-lavage associated enterococcus faecalis on monocyte chemotactic protein-1 (MCP-1).

FIG. 11 shows the effect of Salmonella enterica on monocyte chemotactic protein-1 (MCP-1) by Salmonella enterica.

FIG. 12 shows the effect of Comptobacterium gavage on monocyte chemoattractant protein-1 (MCP-1).

FIG. 13 shows the effect of intragastric associated enterococcus faecalis on Leptin (LEP).

FIG. 14 shows the effect of Salmonella enterica on Leptin (LEP) by Salmonella enterica.

FIG. 15 shows the effect of COMBINATION OF GASTRIC on Leptin (LEP).

Detailed Description

The present inventors have made extensive and intensive studies and experiments, and have unexpectedly found that associated with coprococcus and/or diligenous bacteria (Dialister invisus) have/has an effect of preventing and treating obesity and related diseases (such as cardiovascular diseases), and when a subject is fed with an active composition comprising the foregoing firmicutes, the composition is found to be capable of inhibiting weight gain, reducing body fat ratio, reducing blood lipid, and effectively reducing cardiovascular diseases and obesity. The present invention has been completed based on this finding.

As used herein, the term "comprising" means that the various ingredients can 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 "comprising.

As used herein, the "body-to-fat ratio" refers to the ratio of fat weight/body weight.

The invention relates to a pachycete probiotic and application thereof

As used herein, the "firmicutes probiotic of the present invention" refers to a group of firmicutes bacteria that are commensal in the gut, gram positive, rod-shaped, such as clostridia XIVa and IV clusters (Clostridium cluster XIVa and IV), including some species of the genera Clostridium (Clostridium), Eubacterium, Ruminococcus (Ruminococcus) and sulphate reducing bacteria (Anaerofilum), among others. By interacting with other intestinal microorganisms, such probiotics play an important role in the balance of the intestinal flora and, at the same time, also perform other specific or necessary functions.

In the present invention, the "firmicutes probiotic of the present invention" refers to a mixture of one or more of 2 genera of associated coprococcus (coprococcus eutactus), alisteria turbidicola (Dialister invisus) or a mixture of one or more of a plurality of bacteria in each genus.

Among them, Coprococcus eutactus is an obligate anaerobe, immotile, gram-positive when in a paired or catenulated paired state, but discolors rapidly. In a medium containing a fermentable carbohydrate, it has a slightly elongated shape. However, the bacteria are generally circular and have a diameter of 0.7 to 1.3. mu.m.

The Salmonella enterica (Diarister invisus) is an obligate anaerobe, immotile coccobacillus (0.3-0.4X 0.3-0.6 μm) and gram-negative. The form of the polymer can be single dispersion, or pair, short chain, small cluster, etc.

The invention provides application of probiotics of scleritis in treating and preventing obesity and related diseases (such as cardiovascular diseases). The subject ingests a high fat diet and the firmicutes probiotic has (i) an effect of inhibiting weight gain in the subject; (ii) reducing blood fat; and (iii) the ability to reduce body-to-fat ratio. According to a preferred embodiment of the present invention, C57BL/6J male mice fed with a high fat diet that leads to obesity, treated with a firmicutes probiotic of the present invention (e.g., associated coprococcus, alisteria turbinata or a combination thereof) show a reduced weight gain and reduced blood fat, and a reduced index of various obesity or cardiovascular diseases, such as Leptin (LEP) and monocyte chemotactic protein-1 (MCP-1), compared to untreated controls. Therefore, the chlamydophila probiotics (such as associated coprococcus faecalis, aschert turbinatus or combination thereof) can be used for preventing and treating obesity and diseases caused by obesity, such as cardiovascular diseases and the like.

Composition and application thereof

The invention also provides a composition, preferably a pharmaceutical composition. The composition comprises an effective amount of a firmicutes probiotic of the present invention (such as associated coprococcus, poriferous or a combination thereof), 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: fructooligosaccharides (FOS), Galactooligosaccharides (GOS), Xylooligosaccharides (XOS), Lactosucrose (LACT), Soy Oligosaccharides (SOS), Inulin (Inulin), or combinations thereof.

In a preferred embodiment, the composition is a liquid preparation, a solid preparation or a semisolid preparation.

In a preferred embodiment, the liquid formulation is selected from the group consisting of: solution preparations or suspension preparations.

In a preferred embodiment, the dosage form of the composition is selected from the group consisting of: powders, tablets, dragees, capsules, granules, suspensions, solutions, syrups, drops, and sublingual tablets.

The pharmaceutical composition of the present invention may be administered in any form of pharmaceutical tablets, injections or capsules, which includes excipients, pharmaceutically acceptable vehicles and carriers, which may be selected according to the administration route. The pharmaceutical preparation of the present invention may further comprise auxiliary active ingredients.

Lactose, glucose, sucrose, sorbitol, mannose, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, fine crystalline cellulose, polyvinylpyrrolidone (PVP), cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, or the like can be used as the carrier, excipient, diluent, or the like of the pharmaceutical composition of the present invention.

In addition, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, emulsifiers, suspension stabilizers, preservatives, sweeteners, flavors, and the like. The pharmaceutical compositions of the present invention may be manufactured in enteric-coated formulations by a variety of well-known methods so that the active ingredient of the pharmaceutical composition, i.e., the microorganism, passes through the stomach without being destroyed by stomach acid.

In addition, the microorganism of the present invention can be used in the form of a capsule prepared by a conventional method. For example, standard excipients are mixed with the lyophilized microorganisms of the present invention to form pellets, which are then filled into gelatin capsules. In addition, the microorganisms of the present invention and the pharmaceutically acceptable excipients such as liquid gums, celluloses, silicates or mineral oils are mixed to make a suspension or dispersion, which can be filled into soft gelatin capsules.

The pharmaceutical composition of the present invention can be made into enteric coated tablets for oral administration. The term "enteric coating" in the present application includes all coatings which are approved for use with conventional drugs, which are not degraded by gastric acid, but which are sufficiently decomposed in the small intestine to rapidly release the microorganisms of the present invention. The casings of the invention can be maintained at 36-38 ℃ for more than 2 hours in synthetic gastric acid, e.g. HCl solution at pH 1, and preferably disintegrate within 1.0 hour in synthetic intestinal fluid, e.g. buffer at pH 7.0.

The enteric coating of the invention is coated at about 16-30mg, preferably 16-25mg, more preferably 16-20mg per tablet. The thickness of the casing is 5-100 μm, and the ideal thickness is 20-80 μm. The enteric coating composition is selected from conventional polymers known per se.

Preferred casings for use in the present invention are prepared from cellulose acetate phthalate polymers or trimellitate polymers and copolymers of methacrylic acid (e.g., copolymers containing greater than 40% methacrylic acid and methacrylic acid containing hydroxypropyl methylcellulose phthalate or its ester derivatives).

The cellulose acetate phthalate used in the enteric coating of the present invention has a viscosity of about 45 to 90cp, an acetyl content of 17 to 26%, and a phthalic acid content of 30 to 40%. The cellulose acetate trimellitate used in the enteric coating has a viscosity of about 5-21cp and an phthalide content of 17-26%. Cellulose acetate trimellitate is manufactured by Eastman Kodak company and can be used for the casing material in the present invention.

The hydroxypropyl methyl cellulose phthalate used in the enteric coating of the invention has a molecular weight of generally 20,000-.

Hydroxypropyl methylcellulose phthalate, which is used in the casing of the present invention, was HP50, produced by Shin-etsu chemidl co.ltd. HP50 contains 6-10% hydroxypropyl, 20-24% methoxy, and 21-27% propyl, and has a molecular weight of 84,000 daltons. Another enteric material is HP55, HP55 contains 5-9% hydroxypropyl methylcellulose phthalate, 18-22% methoxyl, 27-35% phthalic acid, and has a molecular weight of 78,000 daltons.

The sausage casing of the 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., dichloromethane), or combinations thereof. Softeners, such as di-n-butyl phthalate and glyceryl triacetate, are added to the enteric coating solution in a ratio of 1 part of the garment to about 0.05 parts or about 0.3 parts softener. The spraying process is preferably carried out continuously, the amount of material sprayed being controlled according to the conditions employed for coating. The spray pressure can be adjusted at will, and in general, the desired results are obtained at an average pressure of 1-1.5 Pa.

For example, in the present invention, a composition comprising 1 × 10-1 × 10 is prepared to provide a composition comprising a pharmaceutically effective amount of the compound of the present invention²⁰cfu/ml or cfu/g (in particular, 1 × 10 can be contained⁴-1×10¹⁵cfu/ml or cfu/g, more particularly, 1 × 10⁶-1×10¹¹cfu/ml or cfu/g) of a probiotic of the invention (e.g. associated coprococcus, alisteria turbinata or combinations thereof)。

When used in the preparation of pharmaceutical compositions, the effective dosage of the firmicutes (e.g., coprococcus, alisteria turbinata, or combinations thereof) of the present invention employed may vary with the mode of administration and the severity of the condition to be treated, dosage forms suitable for oral administration comprising from about 1 × 10 to 1 × 10 intimately admixed with a solid or liquid pharmaceutically acceptable carrier²⁰cfu/ml or cfu/g (in particular, 1 × 10 can be contained⁴-1×10¹⁵cfu/ml or cfu/g, more particularly, 1 × 10⁶-1×10¹¹cfu/ml or cfu/g) of active firmicutes probiotic bacteria (such as associated coprococcus, alisteria turbidivora or combinations thereof). This dosage regimen may be adjusted to provide the best therapeutic response. For example, divided doses may be administered several times per day, or the dose may be proportionally reduced, as may be required by the urgency of the condition being treated.

The probiotic bacteria of the genus firmicutes (such as associated coprococcus faecalis, asbestosis turbinatum or combinations thereof) can be administered orally or the like. The solid support comprises: starch, lactose, dicalcium phosphate, microcrystalline cellulose, sucrose and kaolin, and liquid carriers include: culture medium, polyethylene glycol, nonionic surfactant, and edible oil (such as corn oil, peanut oil, and sesame oil) as appropriate for the characteristics of probiotic bacteria of the firmicutes (such as coprococcus faecalis, alisteria turbinata, or a combination thereof) 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, colouring agents, preservatives and antioxidants such as vitamin E, vitamin C, BHT and BHA.

Preferred pharmaceutical compositions are solid compositions, especially tablets and solid-filled or liquid-filled capsules, from the standpoint of ease of preparation and administration. Oral administration is preferred.

The composition of the present invention is administered to the subject 1 or more times per day. Dosage units for administration represent dosages which can be divided formally and which are suitable for human beings or all other mammalian subjects. Each unit containing a pharmaceutically acceptable carrier and a therapeutically effective amount of a microorganism of the invention. The amount administered will vary with the weight and severity of the obesity of the patient, the supplemental active ingredients included and the microorganism used. Furthermore, the administration can be divided, if possible, and can be continued, if desired. Therefore, the amount to be administered is not a limitation of the present invention. Further, the "composition" in the present invention means not only a pharmaceutical but also a functional food and a health supplement food. In a preferred embodiment, the composition comprises: beverages, foods, pharmaceuticals, 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 a firmicutes probiotic (e.g. associated coprococcus, poriferous or a combination thereof) and the balance a food acceptable carrier, said food composition being in a form selected from the group consisting of solid, dairy, solution, powder, and suspension.

In a preferred embodiment, the formulation of the composition is as follows:

1×10-1×10²⁰cfu/mL of Probiotics of firmicutes (such as associated enterococcus faecalis, Allium turbidivorum or combinations thereof); and a food-acceptable or pharmaceutically acceptable carrier, and/or an excipient.

In another preferred embodiment, the formulation of the composition is as follows:

1×10⁶-1×10¹¹cfu/mL of Probiotics of firmicutes (such as associated enterococcus faecalis, Allium turbidivorum or combinations thereof); and a food-acceptable or pharmaceutically acceptable carrier, and/or an excipient.

Method for reducing body weight and/or blood lipids

In another preferred example, the method comprises: ingesting a pharmaceutical composition, food composition, beverage composition, or a combination thereof, of the present invention. The subject is a human.

In another preferred example, the method comprises: ingesting a pharmaceutical composition, food composition, or animal feed, or a combination thereof, of the present invention. The experimental object is an animal, preferably a mouse or a rabbit.

The main advantages of the invention include:

(a) the chlamydophila probiotics (such as associated coprococcus faecalis, turbid alisteria or a combination thereof) can obviously reduce weight, blood fat and body fat ratio.

(b) The chlamydophila probiotics (such as associated coprococcus faecalis, asbester opathiae or a combination thereof) can obviously reduce indexes (such as cholesterol and triglyceride) related to obesity and related diseases (such as cardiovascular diseases).

(c) The chlamydophila probiotics (such as associated coprococcus faecalis, ascharis brevicompactum or combination thereof) can obviously reduce the levels of total cholesterol, triglyceride and low-density lipoprotein.

(d) The chlamydophila probiotics (such as associated coprococcus faecalis, ascharis opathia or a combination thereof) can obviously improve the level of high-density lipoprotein.

(e) The chlamydophila probiotics (such as associated coprococcus faecalis, ascharis brevipedunculatus or a combination thereof) can improve insulin resistance and reduce the risk of atherosclerosis and cardiovascular diseases.

(f) The chlamydophila probiotics (such as associated coprococcus faecalis, asbester turbinatus or a combination thereof) can obviously reduce the level of monocyte chemotactic protein-1 (MCP-1).

(g) The chlamydophila probiotics (such as associated coprococcus faecalis, turbid alisteria or a combination thereof) can effectively improve Leptin resistance accompanied by obesity and improve sensitivity to Leptin in vivo.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: the conditions described in the laboratory Manual (New York: Cold Spring Harbor laboratory Press, 1989), or according to the microorganism: the conditions described in the handbook of experiments (James Cappuccino and Natalise Sherman eds., Pearson Edurion Press) or as recommended by the manufacturer.

Example 1 food compositions containing bacterial components (e.g. associated enterococcus faecalis, Aphanothece turbidivorans, or combinations thereof)

The raw material formulation is shown in table 1.

TABLE 1 food composition formula

Raw materials	Mass percent (%)
		Bacterial composition	0.5
Milk	90.0
		White sugar	9.5

The bacterial components in the formulas 1-6 are single bacterial components and respectively comprise Coprococcus euticus ATCC27759, Coprococcus euticus ATCC 51897, Coprococcus euticus L2-50, Dialisticiivisus DSM 15470, Dialisticis invisus E2.20 and Dialisticis invisus E9.48.

The bacteria component in the formula 7 is a mixture of any two or more (preferably 2 or 3) of the above 6 bacteria (in a weight ratio of 1:1 or 1:1: 1).

Mixing milk and white sugar according to the above formula ratio, stirring to completely mix, preheating, homogenizing under 20Mpa, sterilizing at 90 deg.C for 5-10 min, cooling to 40-43 deg.C, inoculating 1-100 × 10⁶cfu/g of bacteria component, and making into food composition containing bacteria component (such as associated enterococcus faecalis, or Allium fischeri or their combination).

Example 2

Pharmaceutical compositions comprising bacterial components (e.g. associated coprococcus, poriferous or combinations thereof)

The raw material ratio is shown in table 2.

TABLE 2 pharmaceutical composition formulations

Raw materials	Mass percent (%)
		Bacterial composition	1.0％
Lactose	2.0％
		Yeast powder	2.0％
Peptone	1.0％
		Purified water	94.0％

The bacteria component in formula 7 is a mixture of any two or more (preferably 2 or 3) of the above 6 bacteria (weight ratio is 1:1 or 1:1: 1). mixing lactose, yeast powder and peptone with purified water at a certain proportion, preheating to 60-65 deg.C, homogenizing under 20MPa, sterilizing at 90 deg.C for 20-30 min, cooling to 36-38 deg.C, inoculating bacteria component (1-50 × 10)⁶cfu/mL), fermentation at 36-38 deg.CUntil the pH value is 6.0, centrifuging, and freeze-drying until the water content is less than 3% to obtain freeze-dried product containing bacteria components. Weighing 0.5 g of lyophilized product containing bacteria component, mixing with maltodextrin in equal amount, and encapsulating to obtain medicinal composition containing bacteria component (such as associated enterococcus faecalis, turbid Alister bacteria or their combination).

Example 3 therapeutic Effect on obese model mice

Experimental materials:

mice: c57BL/6J male mice (purchased from Guangdong provincial animal center for medical laboratory) were purchased as normal breeding mice, 6 weeks old. The mice were grown in the same environment and fed the same diet.

6 sclerite probiotics are obtained from a preservation organization and are stored in Shenzhen Hua Dagenen institute. Meanwhile, Lactobacillus plantarum (Lactobacillus plantarum) is selected from China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.8198 and used as a control group (LP group) to be cultured in MRS culture solution for 24-48h at 37 ℃.

The source information of the 6 types of probiotic bacteria of firmicutes is shown in table 3. All strains were verified by 16S rDNA sequencing and the experiment was started.

The strain information is shown in Table 3.

TABLE 3 information on the strains

Wherein, the Coprococcus euticus L2-50 (strain 3) is derived from Rowell research Institute (Barcenella A, Pryde SE, Martin JC et al. Photogenic relationships of butyl-reducing Bacteria from the Human Gut. applied and environmental Microbiology 2000; 66: 1654) -1661.), Rowettresearch Institute (Barcelia A, Pryde SE, Martin JC et al. Photogenic relationships). Dialiser insights E2.20 (fungus 5) and Dialiser insights E9.48 (fungus 6) are both from the Dental Institute of Dental research Institute of Hospital, King's College of King, London, England (Department of Microbiology, Dental Institute, Guy's Tower, Guy's Hospital, King's College London, UK; J.Downs, M.Munson and W.G.Wade, Dialisioninavissu. nov, isolated from the human organ, International Journal of systematic and evolution Microbiology (2003),53, 1937. 1940).

High fat diet (HF): containing 78.8% basal diet, 1% cholesterol, 10% egg yolk powder, 10% lard and 0.2% bile salts, purchased from Nantong Temilion feed science and technology, Inc.

Ordinary maintenance feed: purchased from the centre of medical laboratory animals in Guangdong province.

The experimental method comprises the following steps:

the method comprises the steps of selecting normally fed C57BL/6J adult male mice, randomly grouping the mice into a control group (CK), a microbial inoculum group (a bacterium 1 group, a bacterium 2 group, a bacterium 3 group, a bacterium 4 group, a bacterium 5 group, a bacterium 6 group, a bacterium 1+ bacterium 5 group, a bacterium 2+ bacterium 6 group, a bacterium 3+ bacterium 4 group), a control microbial inoculum group (LP, Lactobacillus plantarum CGMCC No.8198) and an obesity model group (HF), wherein 10 mice in each group freely feed and drink water in an SPF (Specific pathogen Free) environment. The LP group, HF group and microbial agent group were fed with high-fat diet, and CK group was fed with ordinary maintenance diet. After feeding for 4 weeks, the bacterium agent group and the LP group begin to perfuse corresponding strain liquid; the HF group and the CK group were gavaged with the same amount of medium for 9 weeks.

The bacteria feeding amount is 0.15mL/10g body weight, and the bacteria concentration is 1 × 10⁷cfu/mL, concentration after concentration 1 × 10⁸cfu/mL, every other day, the bacterial solution should be cultured in advance, activated every week to ensure freshness, the concentration is measured and adjusted to 1 × 10⁸cfu/mL. For a single microbial inoculum group, taking corresponding bacterial liquid and irrigating the stomach according to the dosage; for the mixed bacteria group, the single bacteria liquid is mixed in equal proportion and then is irrigated into the stomach according to the dosage.

In the experimental period, the weight, state, food intake and other data of the mice are recorded every week. The last week of the experiment each group of mice was subjected to a glucose tolerance (OGTT) test. After the experiment, the mice were sacrificed, the fat weight was recorded, and serum was taken and the blood lipid and protein factor content were measured using an Elisa kit.

The experimental results are as follows:

(1) effect of associated enterococcus faecalis, Salmonella turbinata or combinations thereof on body weight in mice.

TABLE 4 weight gain of mice in each group after gavage associated with fecal cocci compared to before gavage (FIG. 1)

Note: the data in the tables are mean ± standard deviation, and no identical letter after the number indicates significant difference (p <0.05) for any two sets of data in each column, as is the case for tables 5-15.

TABLE 5 weight gain after gastric lavage of turbid mice wearing Alister bacteria compared to before gastric lavage (FIG. 2)

TABLE 6 weight gain of mice in each group after intragastric administration of the combination bacteria compared to before intragastric administration (FIG. 3)

The results are shown in tables 4 to 6 and FIGS. 1 to 3. The results show that associated enterococcus faecalis, alisteria turbinata, or a combination thereof is effective in reducing weight gain in obese model mice (P < 0.05).

(2) Effect of associated enterococcus faecalis, alisteria turbinata or a combination thereof on body-to-lipid ratio.

TABLE 7 body-to-fat ratio of mice of each group 9 weeks after gavage associated with fecal cocci (FIG. 4)

Grouping	Fat weight/body weight × 100%
		CK	2.83±0.13e
Bacterium
	1	4.11±0.10c
Bacterium
	2	4.00±0.26cd
Bacterium
	3	3.85±0.19d
LP			5.39±0.34b
	HF	7.48±0.46a

TABLE 8 body fat ratio of mice in each group 9 weeks after gastric lavage with turbid Alicase (FIG. 5)

Grouping	Fat weight/body weight × 100%
		CK	2.91±0.14e
Bacterium
	4	3.97±0.22cd
Bacterium
	5	4.16±0.23c
Bacterium
	6	3.76±0.20d
LP			5.21±0.24b
	HF	746±064a

TABLE 9 body-fat ratio of mice in each group 9 weeks after gavage of Comptobacterium (FIG. 6)

Grouping	Fat weight/body weight × 100%
		CK	2.90±0.13d
Bacterium
	1+5	3.73±0.19c
Bacterium
	2+6	3.87±0.23c
Bacterium
	3+4	3.93±0.22c
LP			5.42±0.17b
	HF	756±055a

The results are shown in tables 7 to 9 and FIGS. 4 to 6. The results show that associated coprococcus, aschersonia turbinata or the combination thereof can obviously reduce the body-fat ratio of the obese model mouse (P < 0.05).

(3) The effect of associated coprococcus, turbid brewsteria or their combination on blood lipids.

TABLE 10 blood lipid content of mice of each group 9 weeks after gavage associated with fecal cocci (FIG. 7)

Grouping	TC(mmol/L)	TG(mmol/L)	LDLC(mmol/L)	HDLC(mmol/L)
					CK	3.811±0.144d	0.914±0.048d	1.260±0.057e	3.354±0.166a
Bacterium 1	4.819±0.198c	1.041±0.080c	1.516±0.084c	3.529±0.123a
					Bacterium 2	4.790±0.226c	1.052±0.059c	1.479±0.083cd	3.510±0.160a
Bacterium 3	4.823±0.251c	1.073±0.057c	1.457±0.102d	3.479±0.158a
					LP	5.385±0.253b	1.234±0.094b	1.872±0.124b	2.743±0.139b
HF	6299±0257a	1303±0076a	2381±0157a	2193±0104c

TABLE 11 lipid levels in mice of groups 9 weeks after gastric lavage with turbid Alister bacteria (FIG. 8)

Grouping	TC(mmol/L)	TG(mmol/L)	LDLC(mmol/L)	HDLC(mmol/L)
					CK	3.892±0.154d	0.983±0.047c	1.250±0.036d	3.376±0.202a
Bacterium 4	4.896±0.230c	1.012±0.072c	1.395±0.082c	3.497±0.093a
					Bacterium 5	4.906±0.247c	1.014±0.054c	1.353±0.076c	3.439±0.160a
Bacterium 6	4.917±0.201c	1.069±0.070c	1.364±0.071c	3.421±0.195a
					LP	5.258±0.302b	1.216±0.088b	1.831±0.131b	2.741±0.100b
HF	6.390±0.290a	1.295±0.064a	2.357±0.126a	2.153±0.105c

TABLE 12 blood lipid content of mice in each group 9 weeks after gavage of the combination bacteria (FIG. 9)

Grouping	TC(mmol/L)	TG(mmol/L)	LDLC(mmol/L)	HDLC(mmol/L)
					CK	3.902±0.111d	0.964±0.046d	1.213±0.060d	3.287±0.239a
Bacterium 1+5	4.822±0.139c	1.019±0.067c	1.369±0.077c	3.324±0.177a
					Bacterium 2+6	4.904±0.182c	1.017±0.061c	1.367±0.077c	3.249±0.215a
Bacterium 3+4	4.834±0.192c	1.009±0.060c	1.381±0.082c	3.252±0.140a
					LP	5.498±0.280b	1.213±0.061b	1.742±0.105b	2.621±0.106b
HF	6.511±0.252a	1.304±0.094a	2.357±0.166a	2.196±0.096c

The results are shown in FIGS. 7 to 9 and tables 10 to 12. The main components of blood lipids are cholesterol and triglycerides, and elevated plasma cholesterol and triglyceride levels are associated with the development of atherosclerosis. The results show that associated coprococcus, aschersonia turbinata or the combination bacteria thereof can reduce blood fat and relevant indexes of atherosclerosis related diseases (such as cardiovascular diseases). Moreover, the effect of associated coprococcus, alliella turbinata or a combination thereof on reducing Total Cholesterol (TC), total Triglycerides (TG) and Low Density Lipoprotein (LDLC), and increasing High Density Lipoprotein (HDLC) is particularly significant (P < 0.05).

(4) Effect of associated enterococcus faecalis, Allium turbidicola or a combination thereof on Leptin (LEP), monocyte chemotactic protein-1 (MCP-1).

TABLE 13 content of Leptin (LEP) and monocyte chemotactic protein-1 (MCP-1) in each group of mice 9 weeks after gavage associated with enterococcus faecalis (FIG. 10, FIG. 13)

TABLE 14 content of Leptin (Leptin, LEP), monocyte chemotactic protein-1 (MCP-1) in groups of mice 9 weeks after gastric lavage with Aliskiers (FIG. 11, FIG. 14)

Grouping	MCP-1(pg/ml)	LEP(pg/ml)
			CK	343.07±44.17c	1162.93±96.41c
Bacterium
	4	339.99±39.03c	1158.48±130.63c
Bacterium
	5	338.79±31.89c	1166.22±136.26c
Bacterium
	6	336.81±38.48c	1158.43±111.51c
LP				365.95±48.92b	1264.09±158.29b
	HF	383.04±45.92a	1401.25±147.52a

TABLE 15 Leptin (LEP) and monocyte chemotactic protein-1 (MCP-1) content of each group of mice 9 weeks after gavage combined bacteria (FIG. 12, FIG. 15)

Grouping	MCP-1(pg/ml)	LEP(pg/ml)
			CK	336.55±38.88c	1164.35±106.96c
Bacterium
	1+5	335.85±31.27c	1173.25±114.83c
Bacterium
	2+6	333.48±31.21c	1169.25±132.61c
Bacterium
	3+4	330.36±35.21c	1176.85±109.40c
LP				366.47±24.75b	1259.06±144.03b
	HF	384.12±44.29a	1401.18±179.66a

The results are shown in FIGS. 10 to 15 and tables 13 to 15. The results show that associated coprococcus, the Ayuri brevicompactum or the combined bacteria thereof can obviously reduce the content of Leptin (LEP) and monocyte chemotactic protein-1 (MCP-1) in the serum of an obesity model mouse (P < 0.05).

The results show that associated coprococcus, the turbid alisteria or the combination thereof can improve leptin resistance and improve the sensitivity to Leptin (LEP) in vivo; in addition, the level of MCP-1 in serum is reduced after the treatment of associated coprococcus, turbid brewster or the combined bacteria thereof, which is beneficial to improving insulin resistance and reducing the risk of atherosclerosis and cardiovascular diseases.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. Use of a probiotic of the firmicutes species for the preparation of a composition or formulation for one or more uses selected from the group consisting of: (a) prevention and/or treatment of obesity; and/or (b) preventing or treating a cardiovascular disease, wherein the firmicutes probiotic is a turbid Brewster bacterium (Diarister invisus), the cardiovascular disease being selected from the group consisting of: atherosclerosis and hyperlipidemia.

2. The use of claim 1, wherein the composition or formulation further comprises Coprococcus eutactus.

3. The use according to claim 2, wherein the associated enterococcus faecalis (Coprococcus eutactus) is selected from the group consisting of: coprococcus eutactus ATCC27759, Coprococcus eutactus ATCC 51897, Coprococcus eutactus L2-50, or a combination thereof.

4. The use according to claim 1, wherein the composition or formulation is also used independently or additionally for one or more uses selected from the group consisting of:

(i) inhibiting weight gain in a mammal;

(ii) Reducing the body-to-fat ratio of the mammal;

(iii) Reducing blood lipid levels in a mammal.

5. The use of claim 4, wherein said lowering the blood lipid level of a mammal comprises:

(i) increasing the level of High Density Lipoprotein (HDLC) in a mammal;

(ii) reducing Low Density Lipoprotein (LDLC) levels in a mammal.

6. A composition for the treatment and/or prevention of obesity, the composition comprising: (i) a safe and effective amount of a firmicutes probiotic; and (ii) a pharmaceutically acceptable carrier; wherein the Probiotics of the firmicutes is a combination of associated Coprococcus eutacter (Coprococcus eutactus) and Ahrister turbidicola (Diarister invisus).

7. The composition of claim 6, wherein said composition comprises 1 × 10-1 × 10²⁰cfu/mL or cfu/g of firmicutes probiotic, based on the total volume or total weight of the composition.

8. The composition of claim 7, wherein said composition comprises 1 × 10⁴-1×10¹⁵cfu/mL or cfu/g of firmicutes probiotic, based on the total volume or total weight of the composition.

9. The composition of claim 6, wherein said composition comprises from 0.0001 to 99 wt% of said firmicutes probiotic, based on the total weight of said composition.

10. The composition of claim 9, wherein said firmicutes probiotic is present in an amount of 0.1 to 90 wt.%, based on the total weight of the composition.

11. The composition of claim 6, wherein the composition further comprises other probiotics and/or prebiotics.

12. A method of making the composition of claim 6, comprising the steps of:

mixing (i) a firmicutes probiotic, with (ii) a pharmaceutically acceptable carrier, thereby forming the composition of claim 6; wherein the Probiotics of the firmicutes is a combination of associated Coprococcus eutacter (Coprococcus eutactus) and Ahrister turbidicola (Diarister invisus).