Christensenella intestinihominis and uses thereof

Abstract

Provided are Christensenella intestinihominis and applications thereof, in particular, the use of Christensenella intestinihominis in the treatment and prevention of obesity and related diseases, and a composition for treating and preventing obesity and related diseases, including a pharmaceutical product, a beverage, a food product, or an animal feed composition, and the like, and a method for reducing body weight and / or blood lipids.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbiology, and in particular, the present application relates to the use of Christensenella intestinihominis in the treatment and prevention of obesity and its related diseases, and also relates to compositions comprising Christensenella intestinihominis and the use thereof. BACKGROUND

[0002] The human intestine is inhabited by a large number of symbiotic microorganisms, most of which are beneficial bacteria to the human body. These microorganisms not only cooperate with the host in the metabolism of substances and energy, but also play an important role in the maintenance of the health of the host. Many studies have shown that there is a close correlation between intestinal microorganisms and inflammatory and metabolic diseases such as irritable bowel syndrome (IBS), ulcerative colitis (UC), obesity, and type 2 diabetes. Through the research and development of intestinal probiotics, diseases caused by intestinal microbial imbalance can be effectively treated and prevented, especially intestinal inflammatory and metabolic diseases caused by bacterial flora disorder.

[0003] Short-chain fatty acids, including acetic acid, propionic acid, butyric acid, valeric acid, etc., are metabolic products of certain types of bacteria in the intestine, and the production of short-chain fatty acids plays an important role in the maintenance of intestinal health. Studies have shown that the content of short-chain fatty acid-producing bacteria such as Roseburia, Faecalibacterium, Eubacterium rectale, etc. in the intestine of healthy people is significantly different from that in the intestine of patients with type 2 diabetes.

[0004] Cholesterol, as a precursor of vitamin D synthesis, is of great significance to the human body. However, due to the unreasonable diet of most people, it is easy to cause the content of cholesterol in the body, especially in the blood, to be too high, and thus to cause obesity, diabetes, and a series of cardiovascular and cerebrovascular diseases such as coronary heart disease and atherosclerosis. Therefore, the reduction of cholesterol in the body is an effective way to prevent cardiovascular and cerebrovascular diseases.

[0005] Currently prescribed drugs for obesity treatment are sibutramine (Meridia by Abbott) and orlistat (Xenical by Roche). Sibutramine blocks the reabsorption of norepinephrine and serotonin, thereby regulating appetite and reducing the intake of food. However, this property of sibutramine as a substance acting on the central nervous system has side effects such as affecting heart rate and blood pressure. In contrast to sibutramine, orlistat acts locally. As an inhibitor of lipase, a fat-splitting enzyme of the stomach and small intestine, orlistat plays a role in hindering fat hydrolysis, thereby about 30% of fat taken in as food is not absorbed into the body and is excreted, thereby having a weight regulating effect. However, the undigested fat moves along the gastrointestinal track while causing side effects such as diarrhea, steatorrhea, which are not only uncomfortable, but also to the extent that normal social life is difficult.

[0006] Therefore, there is an urgent need in the art to develop a new, non-toxic side effect, drug for the treatment and prevention of obesity and its related diseases. SUMMARY

[0007] Another object of the present application is to provide the use of Christensenella in the treatment and prevention of obesity and its related diseases.

[0008] Another object of the present application is to provide an effective, non-toxic side effect, drug, beverage, food composition, or animal feed composition for the treatment and prevention of obesity and its related diseases.

[0009] Another object of the present application is to provide a method for reducing body weight and / or blood lipids and its use.

[0010] The first aspect of the present application provides a Christensenella, which is Christensenella intestinihominis.

[0011] In another preferred embodiment, the Christensenella is Christensenella intestinihominis.

[0012] In another preferred embodiment, the sequence of 16s rDNA of the Christensenella is shown in SEQ ID NO.: 1.

[0013] In another preferred embodiment, the Christensenella is Christensenella intestinihominis AF73-05CM02, with the accession number CGMCC 1.5207.

[0014] The second aspect of the present application provides a composition comprising: (a) a safe and effective amount of the Christensenella and / or metabolite thereof of the first aspect of the present application; and (b) a food acceptable carrier or a pharmaceutically acceptable carrier.

[0015] In another preferred embodiment, the composition is selected from the group consisting of a food composition, a health composition, a pharmaceutical composition, a beverage composition, a feed composition, or a combination thereof.

[0016] In another preferred embodiment, the composition is an oral preparation.

[0017] In another preferred embodiment, the composition is a liquid preparation, a solid preparation, or a semi-solid preparation.

[0018] In another preferred embodiment, the dosage form of the composition is selected from the group consisting of a powder, a granule, a tablet, a sugar-coated tablet, a capsule, a granule, a suspension, a solution, a syrup, a drop, and a sublingual tablet.

[0019] In another preferred embodiment, the food composition comprises a milk product, a solution product, a powder product, or a suspension product.

[0020] In another preferred embodiment, the food composition comprises a milk product, a milk powder, or a milk emulsion.

[0021] In another preferred embodiment, the liquid preparation is selected from the group consisting of a solution product or a suspension product.

[0022] In another preferred embodiment, the composition contains 1 x 10 10 cfu / mL or cfu / g Christensenella intestinihominis AF73-05CM02, preferably 1 x 10 4 -1 x 10 10 cfu / mL or cfu / g Christensenella intestinihominis AF73-05CM02, based on the total volume or the total weight of the composition.

[0023] In another preferred embodiment, the composition contains 0.0001-99 wt%, preferably 0.1-90 wt% (check) of the Christensenella and / or metabolite thereof, based on the total weight of the composition.

[0024] In another preferred embodiment, the composition is a unit dosage form (one tablet, one capsule, or one vial), and the mass of the composition in each unit dosage form is 0.05-5 g, preferably 0.1-1 g.

[0025] In another preferred embodiment, the composition further comprises other probiotics and / or prebiotics.

[0026] In another preferred embodiment, the probiotics are selected from the group consisting of lactic acid bacteria, bifidobacteria, Lactobacillus acidophilus, or combinations thereof.

[0027] In another preferred embodiment, the prebiotics are selected from the group consisting of fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), xylo-oligosaccharides (XOS), lactulose oligosaccharides (LACT), soybean oligosaccharides (SOS), inulin, or combinations thereof.

[0028] In another preferred embodiment, the use of the composition of the second aspect of the present application is for the preparation of a medicament or a formulation for one or more purposes selected from the group consisting of:

[0029] In another preferred embodiment, the formulation comprises a microecological formulation.

[0030] In another preferred embodiment, the use of the composition of the second aspect of the present application is for the preparation of a medicament or a formulation for one or more purposes selected from the group consisting of:

[0031] (i) lowering the cholesterol level in a mammal;

[0032] (ii) inhibiting weight gain in a mammal;

[0033] (iii) lowering the body fat ratio in a mammal;

[0034] (iv) lowering the blood lipid level in a mammal;

[0035] (v) increasing the level of high-density lipoprotein (HDL-C) in a mammal;

[0036] (vi) lowering the level of low-density lipoprotein (LDL-C) in a mammal.

[0037] In another preferred embodiment, the mammal comprises a human or a non-human mammal.

[0038] In another preferred embodiment, the non-human mammal comprises a rodent (e.g., a rat, a mouse).

[0039] In another preferred embodiment, the lowering of the blood lipid level in a mammal comprises lowering the total cholesterol (TC) level and / or the triglyceride level.

[0040] The fifth aspect of the present application provides a method for preparing the composition of the second aspect of the present application, comprising the steps of:

[0041] The Christensenella intestinihominis and / or its metabolite of the first aspect of the present application is mixed with a food-acceptable carrier or a pharmaceutically acceptable carrier, thereby forming the composition of the second aspect of the present application.

[0042] In another preferred embodiment, the composition is an oral preparation.

[0043] The sixth aspect of the present application provides a production method, comprising the steps of:

[0044] (a) culturing the Christensenella intestinihominis of the first aspect of the present application under suitable conditions for culturing, thereby obtaining a culture product;

[0045] (b) optionally, isolating the Christensenella intestinihominis and / or its metabolite from the culture product; and

[0046] (c) optionally, mixing the Christensenella intestinihominis and / or its metabolite isolated from the previous step with a food-acceptable carrier or a pharmaceutically acceptable carrier, thereby preparing the composition.

[0047] The seventh aspect of the present application provides a method for reducing body weight and / or blood lipid, comprising administering the Christensenella intestinihominis of the first aspect of the present application and / or its metabolite, or the composition of the second aspect of the present application to the subject.

[0048] In another preferred embodiment, the administration comprises oral administration.

[0049] In another preferred embodiment, the administration dose is 0.01-5 g / 50 kg body weight / day, preferably 0.1-2 g / 50 kg body weight / day.

[0050] In another preferred embodiment, the subject comprises a mammal, such as a human.

[0051] It should be understood that, within the scope of the present application, each of the technical features of the present application described above and each of the technical features specifically described in the following (such as the examples) can be combined with each other, thereby constituting a new or preferred technical solution. Due to the limited space, they are not listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0052] Figure 1 Gram staining picture (1000 times) of the intestinal Christensenella intestinihominis of the present application under a microscope.

[0053] Figure 2Pictures of colonies of Christensenella intestinihominis cultured for 4 days are shown.

[0054] Figure 3 A standard curve of cholesterol assay is shown. DETAILED DESCRIPTION

[0055] The present inventors have made extensive and in-depth research and experiments, and unexpectedly found that Christensenella intestinihominis has the effect of preventing and treating obesity and its related diseases (such as cardiovascular diseases, diabetes), and feeding experimental subjects with an active composition containing Christensenella intestinihominis, it is found that the composition can inhibit weight gain, reduce blood lipids, reduce cholesterol, reduce body fat ratio, reduce low-density lipoprotein levels, increase high-density lipoprotein levels, and effectively alleviate obesity, cardiovascular and diabetes and other diseases. On this basis, the present application is completed.

[0056] As used herein, the term "containing" means that various ingredients can be applied together in a mixture or composition of the present application. Therefore, the terms "consisting essentially of and "consisting of are included in the term "containing".

[0057] Christensenella and its application

[0058] As used herein, the terms "Christensenella", "Christensenella intestinihominis" can be used interchangeably. In a preferred example, the strain is Christensenella intestinihominis AF73-05CM02, with a preservation number of CGMCC 1.5207, isolated from human feces. The physiological characteristics of Christensenella are as follows: Christensenella is cultured in an anaerobic environment at 37℃ for 4-5 days, the colony is small, about 0.2mm in diameter, needle-like, grayish white and transparent; through gram staining and spore staining, observed under a microscope at 1000 magnification, the bacterial body is gram-positive, short rod-shaped, no spore, no flagellum, non-motile, the bacterial body is about 0.5um in diameter and about 1.0-2.0um in length, and appears as single bacterial body or short chain; and the oxidation enzyme and peroxidase reactions of the Christensenella of the present application are both negative, the temperature growth range is 30-42℃, the optimum growth temperature is 37-42℃, the pH tolerance range is 6.0-8.5, the optimum pH is 6.5-7.0, and it can tolerate 2% NaCl and 3% bile salts. It can ferment several carbohydrates, including glucose, mannitol, maltose, rhamnose, xylose, arabinose, mannose, raffinose, sorbitol, rhamnose, and in addition, it can also ferment to produce exopolysaccharide, mainly producing formic acid, acetic acid, butyric acid.

[0059] The present application provides the use of Christensenella in the treatment and prevention of obesity and its related diseases (e.g. cardiovascular disease, diabetes). A subject ingests a high-fat meal, and the strain Christensenella intestinihominis AF73-05CM02 has one or more uses selected from the group consisting of: (i) inhibiting weight gain in the subject; (ii) reducing blood lipids; (iii) reducing body fat ratio; (iv) reducing low-density lipoprotein levels; (v) increasing high-density lipoprotein levels. According to a preferred embodiment of the present application, C57BL / 6J male mice are used as test mice, which are fed with high-fat chow to obtain obese mice, and the obese mice are treated with the strain Christensenella intestinihominis AF73-05CM02. Compared with the control group (model group) without treatment, the obese mice treated with the strain Christensenella intestinihominis AF73-05CM02 have a slower weight gain and a lower blood lipid level, and various indicators related to obesity or cardiovascular disease are also reduced, such as body fat ratio, low-density lipoprotein level, and in addition, the high-density lipoprotein level is also significantly increased. Therefore, the strain can be used to prevent and treat obesity and its related diseases (e.g. cardiovascular disease, diabetes, etc.).

[0060] Compositions and uses thereof

[0061] The present application also provides a composition, preferably a pharmaceutical composition. The composition comprises an effective amount of Christensenella. In a preferred embodiment, the composition further comprises probiotics selected from the group consisting of lactic acid bacteria, Bifidobacterium, Lactobacillus acidophilus, or a combination thereof; and / or prebiotics selected from the group consisting of fructooligosaccharides (FOS), galactooligosaccharides (GOS), xylooligosaccharides (XOS), lactulose oligomers (LACT), soybean oligosaccharides (SOS), inulin, or a combination thereof.

[0062] In a preferred embodiment, the composition is a liquid preparation, a solid preparation, or a semi-solid preparation.

[0063] In a preferred embodiment, the liquid preparation is selected from the group consisting of a solution preparation or a suspension preparation.

[0064] In a preferred embodiment, the dosage form of the composition is selected from the group consisting of a powder, a granule, a tablet, a sugar-coated tablet, a capsule, a granule, a suspension, a solution, a syrup, a drop, and a sublingual tablet.

[0065] The pharmaceutical composition of the present application can be administered in any form of a pharmaceutical tablet, injection or capsule, and the pharmaceutical preparation includes excipients, pharmaceutically acceptable vehicles and carriers, which can be selected according to the administration route. The pharmaceutical preparation of the present application can further comprise an auxiliary active ingredient.

[0066] Lactose, glucose, sucrose, sorbitol, mannose, starch, acacia, calcium phosphate, alginate, gelatin, calcium silicate, fine crystalline cellulose, polyvinylpyrrolidone (PVP), cellulose, water, sugar syrup, methyl cellulose, methylparaben, propylparaben, talc, magnesium stearate or mineral oil, etc. can be used as a carrier, excipient or diluent, etc. for the pharmaceutical composition of the present application.

[0067] In addition, the pharmaceutical composition of the present application can further include lubricants, wetting agents, emulsifiers, suspension stabilizers, preservatives, sweetening agents and flavoring agents, etc. The pharmaceutical composition of the present application can be produced in an enteric coating formulation by various well-known methods so that the active ingredient of the pharmaceutical composition, i.e. the microorganism, can smoothly pass through the stomach without being destroyed by gastric acid.

[0068] In addition, the microorganism of the present application can be used in the form of a capsule prepared by a conventional method. For example, the standard excipient and the freeze-dried microorganism of the present application are mixed to prepare a small pellet, and then the pellet is filled into a gelatin capsule. In addition, the microorganism of the present application and the excipient allowed to be used for a medicine such as liquid gum, cellulose, silicate or mineral oil, etc. are mixed to prepare a suspension or dispersion, and the suspension or dispersion can be filled into a soft gelatin capsule.

[0069] The pharmaceutical composition of the present application can be prepared in an enteric coated tablet for oral administration. The term "enteric coating" in the present application includes all conventional pharmaceutical excipients allowed to be used for coating, which are not degraded by gastric acid but are sufficiently decomposed in the small intestine and rapidly release the microorganism of the present application. The enteric coating of the present application can be maintained for more than 2 hours at 36-38°C in a synthetic gastric acid such as HCl solution of pH=l, and is preferably decomposed within 1.0 hour in a synthetic intestinal fluid such as a buffer solution of pH=7.0.

[0070] The enteric coating of the present application is coated with about 16-30 mg per tablet, preferably 16-25 mg, and more preferably 16-20 mg. The enteric coating of the present application has a thickness of 5-100 μm, and desirably has a thickness of 20-80 μm. The enteric coating composition is selected from conventional polymers well known in the art.

[0071] The preferred enteric coating of the present application is prepared from a copolymer of cellulose acetate phthalate polymer or trimellitate polymer and methacrylic acid (e.g., a copolymer containing more than 40% of methacrylic acid and a copolymer of methacrylic acid containing methyl cellulose hydroxypropyl phthalate or ester derivatives thereof).

[0072] The cellulose acetate phthalate used in the present application has a viscosity of about 45-90 cps, an acetyl content of 17-26%, and a phthalic acid content of 30-40%. The cellulose acetate trimellitate used in the present application has a viscosity of about 5-21 cps, an acetyl content of 17-26%. The cellulose acetate trimellitate is produced by Eastman Kodak Co. and can be used in the present application.

[0073] The hydroxypropyl methylcellulose phthalate used in the present application has a molecular weight of about 20,000-130,000 daltons, preferably 80,000-100,000 daltons, a hydroxypropyl content of about 5-10%, a methoxyl content of about 18-24%, and a phthalyl content of about 21-35%.

[0074] The hydroxypropyl methylcellulose phthalate used in the present application is HP50, produced by Shin-Etsu Chemical Co. Ltd. of Japan. HP50 has a hydroxypropyl content of 6-10%, a methoxyl content of 20-24%, a phthalyl content of 21-27%, and a molecular weight of 84,000 daltons. Another coating material is HP55, which has a hydroxypropyl methylcellulose phthalate content of 5-9%, a methoxyl content of 18-22%, a phthalyl content of 27-35%, and a molecular weight of 78,000 daltons.

[0075] The coating of the present application is prepared by spraying the coating solution onto the core using conventional methods. The solvent used in the coating process is an alcohol (e.g., ethanol), a ketone (e.g., acetone), a halogenated hydrocarbon compound (e.g., methylene chloride), or a combination thereof. Softening agents such as di-n-butyl phthalate and triacetin can be added to the coating solution in a ratio of about 1 part of coating material to about 0.05 parts or about 0.3 parts of softening agent. The spraying process is preferably performed continuously, and the amount of material sprayed can be controlled according to the conditions used for the coating. The spraying pressure can be adjusted as desired, but generally, satisfactory results are obtained at an average pressure of about 1-1.5 bar.

[0076] The term "pharmaceutically effective amount" as used in the specification means an amount that is functional or active in and acceptable to humans and / or animals. For example, in the present application, a preparation of a Kristensen's bacterium and / or its metabolite can be prepared that contains 1 x 10 10 cfu / ml or cfu / g (in particular, it can contain 1 x 10 4 -1 x 10 10 cfu / ml or cfu / g; more particularly, it can contain 1 x 10 6 -1 x 10 10 cfu / ml or cfu / g).

[0077] When used to prepare a pharmaceutical composition, the effective dosage of the Kristensenia bacteria or metabolite thereof used can vary according to the mode of administration and the severity of the disease to be treated. Dosage forms suitable for internal administration include from about 1 x 10 10 cfu / ml or cfu / g (in particular, it can contain 1 x 10 4 -1 x 10 10 cfu / ml or cfu / g; more particularly, it can contain 1 x 10 6 -1 x 10 10 cfu / ml or cfu / g) of the active Kristensenia bacteria or metabolite thereof produced by fermentation. This dosage regimen can be adjusted to provide the optimum therapeutic response. For example, several divided doses can be administered daily or the dose can be proportionally reduced as indicated by the exigencies of the therapeutic situation.

[0078] The Kristensenia bacteria or metabolite thereof can be administered by oral or other routes. Solid carriers include starch, lactose, dicalcium phosphate, microcrystalline cellulose, sucrose and magnesium stearate, and liquid carriers include vegetable oils, such as corn, peanut and sesame oils, and water, and approved carriers for use in pharmaceutical compositions. Adjuvants commonly used in preparing pharmaceutical compositions can also be added, such as flavoring agents, coloring agents, preservatives and antioxidants, such as vitamin E, vitamin C, BHT and BHA.

[0079] From the standpoint of ease of preparation and administration, the preferred pharmaceutical composition is a solid composition, especially tablets and hard or liquid filled capsules. Oral administration is preferred.

[0080] The composition of the present application is administered to the individual one or more times per day. The dosage unit is that form in which the active ingredients are contained in a unitary amount appropriate to the subject to be treated. Each unit will contain a therapeutically effective amount of the microorganism of the present application in a pharmaceutically acceptable carrier. The amount administered will vary depending upon the weight and condition of the patient, the severity of the cardiovascular, diabetic or obesity condition, the supplementary active ingredients included, and the microorganism used. In addition, administration can be separated and, if necessary, continuous. Thus, the amount administered does not limit the present application. In addition, the "composition" in the present application means not only a pharmaceutical product but also a functional food and a health supplement food. In a preferred embodiment, the composition includes a beverage, a food, a pharmaceutical product, an animal feed, etc.

[0081] In one preferred embodiment of the present application, there is also provided a food composition comprising an effective amount of the Christensenella bacterium and / or its metabolite, and a remainder of a food acceptable carrier, said food composition being in a dosage form selected from a solid, a dairy, a solution preparation, a powder preparation, or a suspension preparation.

[0082] In one preferred embodiment, the composition is formulated as follows:

[0083] 1 x 10 10 Christensenella bacterium and / or its metabolite at a concentration of 1 x 10

[0084] In another preferred embodiment, the composition is formulated as follows:

[0085] 1 x 10 6 -1 x 10 10 Christensenella bacterium and / or its metabolite at a concentration of 1 x 10

[0086] Microecological preparation

[0087] A microecological preparation is a biological preparation containing probiotics and metabolites or a dietary supplement that can increase probiotics, which can achieve the purpose of improving the health level of human body by regulating and maintaining the microecological balance in the intestinal tract. It mainly includes probiotics, prebiotics and synbiotics.

[0088] In the present application, the microecological preparation comprises (a) a safe and effective amount of Christensenella bacterium and / or its metabolite; and (b) a food acceptable carrier or a pharmaceutically acceptable carrier.

[0089] Method for producing Christensenella bacterium

[0090] Generally, Christensenella bacterium can be prepared by conventional methods.

[0091] In the present application, a method for mass-producing Christensenella bacterium is provided, which specifically comprises the following steps:

[0092] (a) culturing the Christensenella bacterium under suitable conditions for cultivation, thereby obtaining a culture product;

[0093] (b) optionally, isolating Christensenella bacterium cells and / or its metabolites from the culture product; and

[0094] (c) optionally, mixing the Christensenella bacterium cells and / or its metabolites isolated in the previous step with a food acceptable carrier or a pharmaceutically acceptable carrier, thereby preparing a composition.

[0095] Methods of reducing body weight and / or blood lipids

[0096] In another preferred embodiment, the method comprises ingesting the pharmaceutical composition, the food composition, the beverage composition, or the combination thereof of the present application. The subject is a human.

[0097] In another preferred embodiment, the method comprises ingesting the pharmaceutical composition, the food composition, or the animal feed of the present application, or the combination thereof. The subject is an animal, preferably a murine, a lagomorph.

[0098] Bacterial strain preservation

[0099] The bacterial strain Christensenella intestinihominis AF73-05CM02 (same as the deposit name) of the present application has been deposited in the China General Microbiological Culture Collection Center (CGMCC) on June 13, 2016, at 1st North-Chen West Road, Yard 3, Chaoyang District, Beijing, China, with the accession number: CGMCC 1.5207.

[0100] The main advantages of the present application include:

[0101] (a) The Christensenella of the present application can significantly reduce body weight, reduce blood lipids, and reduce body fat ratio.

[0102] (b) The Christensenella of the present application can significantly reduce indicators (such as cholesterol and triglycerides) associated with obesity and its related diseases (such as cardiovascular diseases).

[0103] (c) The Christensenella of the present application can significantly reduce the levels of total cholesterol, triglycerides, and low-density lipoprotein.

[0104] (d) The Christensenella of the present application can significantly increase the level of high-density lipoprotein.

[0105] The present application is further described below in connection with specific examples. It should be understood that these examples are only used to illustrate the present application and are not used to limit the scope of the present application. The experimental methods in the following examples, if not specified, are generally carried out according to the conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions described in Microbiology: A Laboratory Manual (James Cappuccino and Natalie Sherman, Pearson Education Press), or the conditions recommended by the manufacturer.

[0106] The materials and reagents used in the examples are commercially available unless otherwise specified.

[0107] Christensenella minuta DSM 22607 (C. minuta DSM 22607), hereinafter referred to as DSM 22607, was purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), and the accession number is DSM 22607.

[0108] Example 1 Screening and identification of intestinal Christensenella AF73-05CM02 (hereinafter referred to as AF73-05CM02)

[0109] 1. Isolation of AF73-05CM02

[0110] The intestinal Christensenella AF73-05CM02 of the present application was isolated from a stool sample of a healthy male volunteer in Shenzhen. About 0.2 g of the stool sample was taken in an anaerobic operation box, suspended in sterile PBS, shaken and mixed, then gradient diluted and coated, and the isolation medium was PYG medium (purchased from HuanKai Microbial Science and Technology Co., Ltd.) 1L: peptone 5g, trypticase 5g, yeast powder 10g, beef extract 5g, glucose 5g, K2HPO4 2g, Tween 80 1mL, Cysteine-HCl·H2O 0.5g, hemin 5mg, vitamin K1 1uL, inorganic salt solution (containing CaCl2·2H2O 0.25g, MgSO4·7H2O 0.5g, K2HPO4 1g, KH2PO4 1g, NaHCO3 10g, NaCl 2g per L of inorganic salt solution) 40mL, resazurin 1mg, distilled water 950mL, pH 6.8-7.0, sterilized at 115℃ for 25min. After the plate was cultured at 37℃ for 5 days under anaerobic conditions, a single colony was picked and streaked for isolation, and the gas composition of the anaerobic environment was nitrogen:hydrogen:carbon dioxide = 90:5:5, v / v). The pure culture strain was obtained by streaking, and glycerol preservation and 16S rDNA identification were performed.

[0111] 2. Microbiological characteristics of AF73-05CM02

[0112] (1) Morphological characteristics

[0113] AF73-05CM02 had small colonies on PYG plates under anaerobic conditions at 37℃ for 4-5 days, with a diameter of about 0.2mm, needle-like, grayish white, transparent. Figure 2 ).

[0114] (2) Microscopic characteristics

[0115] The bacterial cell is gram-positive, short rod, no spore, no flagellum, non-motile, the diameter of the cell is about 0.5um, the length is about 1.0-2.0um, and it appears as single cell or short chain under microscope 1000 magnification by gram staining and spore staining. Figure 1

[0116] (3) Physiological and biochemical characteristics

[0117] AF73-05CM02 is catalase negative and oxidase negative, the temperature growth range is 30-42℃, the optimum growth temperature is 37-42℃, the pH tolerance range is 6.0-8.5, the optimum pH is 6.5-7.0, and it can tolerate 2% NaCl and 3% bile salt. The comparison of biochemical reactions of AF73-05CM02 and the closely related strain Christensenella minuta DSM 22607 (hereinafter referred to as DSM22607, purchased from DSMZ) in enzyme reaction (API ZYM) and substrate utilization (API20A) is as follows: (+, positive reaction; -, negative reaction; w, weak positive reaction)

[0118] Table 1

[0119]

[0120]

[0121]

[0122] The data in Table 1 shows that there are some differences in API reactions between AF73-05CM02 and the closely related strain, which are mainly reflected in the utilization of maltose, salicin, mannose, raffinose, and cottonseed sugar, the hydrolysis of escin, and the activity of β-glucosidase.

[0123] 3. 16S rDNA identification

[0124] AF73-05CM02 was cultured to the stationary phase, and 2mL of the cultured bacterial solution was taken for extraction of genomic DNA. The extracted genomic DNA was used as a template for amplification of 16S rDNA, and the amplification primers were 8F-1492R (5'-AGAGTTTGATCATGGCTCAG-3'(SEQ ID NO.:2) and 5'-TAGGGTTACCTTGTTACGACTT-3'(SEQ ID NO.:3).

[0125]

[0126] ​​

[0127]

[0128] The PCR product obtained by amplification was purified, and then sequenced by 3730 to obtain a 16S rDNA sequence of 1366 bp (SEQ ID NO. 1). The sequence was compared with the EzTaxon-e database to obtain the species classification information of the strain. According to the 16S rDNA information, it can be preliminarily determined that AF73-05CM02 belongs to the genus Christensenella.

[0129] SEQ ID No. 1

[0130]

[0131] 4. Evolution analysis of AF73-05CM02

[0132] The sequences of AF73-05CM02 and the model strain with a close sequence of 16S rDNA in the EzTaxon-e database were subjected to multiple sequence alignment, and then the phylogenetic tree was made by MEGA 5. The phylogenetic tree was constructed by using the maximum likelihood method.

[0133] 5. Analysis of cell fatty acids

[0134] AF73-05CM02 and C. minuta DSM 22607 were cultured, and the cells were collected after about 4-5 days of culture. The cell fatty acids were extracted, and then subjected to methylation. The content of fatty acid methyl ester of the two strains was analyzed by gas chromatography.

[0135] Table 2

[0136]

[0137]

[0138] According to the above morphological characteristics, microscopic characteristics, physiological and biochemical characteristics and 16S rDNA sequence characteristics of AF73-05CM02, it is shown that the strain is a new species from the perspective of taxonomy. The present application names it as Christensenella intestinihominis (intestinal Christensenella).

[0139] Example 2 Biological active substance of Christensenella intestinihominis AF73-05CM02

[0140] 1. Production and determination of active substances such as short-chain fatty acids (SCFA) and organic acids

[0141] (1) Sample pretreatment

[0142] Take 1 mL of the fermentation broth of AF73-05CM02 cultured for 72 h, centrifuge at 12000 r / min for 5 min, and take the supernatant for standby.

[0143] (2) Determination of SCFA

[0144] The determination of short-chain fatty acids uses the external standard method, and acetic acid, propionic acid, butyric acid and valeric acid are selected to make a standard curve. Agilent gas chromatograph (GC-7890B, Agilent) is used, HP-INNOWax (Cross-Linked PEG) capillary column with a length of 30 m, a diameter of 0.25 mm and a thickness of 0.25 um is selected for analysis, hydrogen flame ionization detector is used, and the GC parameter settings are as follows: column temperature: 180-200℃; gasification chamber temperature: 240℃; detection temperature: 210℃; sample size: 2 μL; carrier gas flow: N2, 50 mL / min; hydrogen flow: 50 mL / min; air flow: 600-700 ml / min.

[0145] The determination shows that the SCFA production is: formic acid 5.21 mmol / L, acetic acid 16.40 mmol / L, and butyric acid 1.63 mmol / L.

[0146] (3) Determination of organic acids

[0147] The detection standard of organic acids is selected as follows: 3-methylbutyric acid, valeric acid, quinic acid, lactic acid, oxalic acid, malonic acid, benzoic acid, maleic acid, succinic acid, trans-fumaric acid, malic acid, adipic acid, tartaric acid, shikimic acid, citric acid, isocitric acid and L-ascorbic acid. Agilent gas chromatograph (GC-7890B, Agilent) is still used, and 122-5532G DB-5ms (40 m x 0.25 mm x 0.25 um) is selected as the chromatographic column, with column temperature: 270-290℃; sample inlet temperature: 250℃; gas flow: 0.86 ml / min. The content of organic acids of Christensenella intestinihominis AF73-05CM02 is shown in the following table:

[0148] Table 3

[0149]

[0150] Example 3: Cholesterol degradation function of Christensenella intestinihominis AF73-05CM02 in vitro

[0151] The cholesterol content was determined using the o-phthalaldehyde colorimetric method (OPA method). The cholesterol degradation ability of the strain was assessed by observing changes in cholesterol content before and after cultivation in a medium containing a specific concentration of cholesterol for a period of time. The specific method is as follows:

[0152] (1) Construction of standard curve

[0153] Accurately measure 40 μL, 80 μL, 120 μL, 160 μL, and 200 μL of 0.5 mg / mL cholesterol standard solution into clean test tubes. Add anhydrous ethanol to each tube and bring the volume to 1 mL. Add 4 mL of OPA (0.5 mg o-phthalaldehyde added to 1 mL of glacial acetic acid) to each tube, vortex to mix, and let stand at room temperature for 10 min. Then add 2 mL of concentrated sulfuric acid, mix well, and let stand for 10 min. Measure the absorbance at 550 nm. Plot a standard curve with concentration on the x-axis and absorbance on the y-axis. Figure 3 The linear regression equation is calculated to be: y = 0.0085x + 0.0072; the correlation coefficient R0 is... 2 It is 0.9992.

[0154] (2) Preparation of cholesterol culture medium and culture of experimental strains

[0155] A certain amount of cholesterol was weighed and dissolved in ethanol to a concentration of 10 mg / mL, and then filtered to remove bacteria. 10 mg / mL of bile salts (autoclaved), 10% sodium thioglycolate (filtered to remove bacteria), and cholesterol were added to the prepared PYG medium (formulation same as in Example 1), and the mixture was thoroughly mixed. The test strain was then inoculated into the medium at a 3% inoculum and cultured anaerobically at 37°C for 72 h.

[0156] (3) Cholesterol determination

[0157] The bacterial culture in PYG medium containing cholesterol was centrifuged at 10,000 rpm, and the supernatant was collected for cholesterol detection. Uninoculated cholesterol-rich PYG medium was used as a blank control. 500 μL of the sample was placed in a clean test tube, and 3 mL of 95% ethanol and 2 mL of 50% KOH were added. The mixture was shaken and incubated in a 60°C water bath for 10 min for saponification. The mixture was then rapidly cooled, and 5 mL of n-hexane was added for extraction. 2.5 mL of the organic phase was transferred to another clean test tube and dried under nitrogen in a 60°C water bath. 4 mL of 0.5 g / L phthalaldehyde-acetic acid solution was added for color development for 10 min, followed by the addition of 2 mL of concentrated H₂SO₄ and a 10 min reaction. Finally, the absorbance was measured at 550 nm.

[0158] (4) Calculation of cholesterol degradation rate

[0159] The degradation of cholesterol was calculated according to the following formula:

[0160] L = (A - B) / A x 100%

[0161] L: the degradation rate of cholesterol;

[0162] A: the content of cholesterol in the cholesterol medium without inoculation of bacteria;

[0163] B: the content of cholesterol after cultivation of bacteria.

[0164] (5) Cholesterol degradation results

[0165] Through calculation, the cholesterol degradation rate of AF73-05CM02 was 46.6%. The results showed that AF73-05CM02 had certain cholesterol degradation ability, which could be further used in animal in vivo study.

[0166] Example 4, Detection of Exopolysaccharide Produced by Christensenella intestinihominis AF73-05CM02

[0167] The detection of exopolysaccharide used the sulfuric acid phenol method. Sulfuric acid phenol can have a color reaction with free monosaccharides, oligosaccharides and polysaccharides, and the color produced is proportional to the absorbance, with an absorption wavelength of 490 nm. The specific experimental process is as follows:

[0168] (1) Extraction of polysaccharide

[0169] The experimental strain was cultured in PYG medium (formula same as Example 1) for 3 days, 10 ml of bacterial solution was treated in boiling water bath for 30 min, then centrifuged at 10000 r / min, the supernatant was taken, 80% trichloroacetic acid was added to a final concentration of 8%, and it was treated overnight at 4°C to precipitate protein. The supernatant was taken and centrifuged at 10000 r / min for 30 min, and the pH of the supernatant was adjusted to 6.0 with NaOH. 2 times the volume of anhydrous ethanol was added for polysaccharide precipitation, and it was treated overnight at 4°C, then centrifuged at 10000 r / min for 30 min, the supernatant was discarded, and the precipitate was dissolved with preheated distilled water, then transferred to an ultrafiltration tube (3000 Da filter diameter) for ultrafiltration, centrifuged at 5000 r / min for 30 min, and the polysaccharide trapped in the inner tube was transferred to a volumetric flask and diluted to 10 ml with distilled water, ready for use.

[0170] (2) Preparation of glucose standard curve

[0171] Precise standard glucose 20mg to 100ml capacity bottle, add water to the scale line, then 20, 40, 60, 80, 100μg / ml glucose standard solution. Each group of standard liquid 400ul, three parallel, 400ul 5% phenol and 1ml concentrated sulfuric acid were added in turn for reaction, after boiling water bath for 15min, cooling to room temperature, measuring the absorbance at 490nm. Then with absorbance as the ordinate, glucose standard solution concentration as the abscissa draw standard curve.

[0172] (3) Detection of the concentration of the extracted polysaccharide

[0173] Take polysaccharide solution 400ul, 400ul 5% phenol and 1ml concentrated sulfuric acid were added in turn for reaction, after boiling water bath for 15min, cooling to room temperature, measuring the absorbance at 490nm. According to the glucose standard curve to calculate the concentration of polysaccharide.

[0174] (4) Results

[0175] By calculation, the content of the extracellular polysaccharide in AF73-05CM02 fermentation broth cultured for 3 days was 234mg / L.

[0176] Example 5 In vivo test of Christensenella intestinihominis AF73-05CM02 in an obese animal model

[0177] Preparation of experimental animals and test strains:

[0178] Experimental animals: C57BL / 6J male mice were used in the experiment, purchased from Guangdong Medical Laboratory Animal Center. Normal feeding, mouse age 6 weeks. A total of 30 mice, randomly divided into 3 groups, 10 in each group. The mouse room environment is SPF (Specific pathogen Free) level.

[0179] Test strains: AF73-05CM02 cultured normally, the culture medium is PYG liquid medium (formula same as example 1), 37℃ anaerobic conditions for 3d, to the bacteria concentration about 10 9 CFU / mL order of magnitude, can be used as experimental group gavage. In order to prevent the inactivation of the strain even death, every other day to replace the fresh bacteria liquid, bacteria liquid storage for 4℃ anaerobic storage.

[0180] Test process: The newly purchased mice were randomly divided into 3 groups: the first group was the control group (control group), the second group was the obesity model group (model group), and the third group was the bacterial agent treatment group. The first group was fed with ordinary feed, and the second and third groups were fed with high-fat feed (for obesity model modeling). After 5 weeks of feeding, the third group started to use AF73-05CM02 for gavage intervention, and the first and second groups were gavaged with an equal amount of culture medium, and the intervention lasted for 9 weeks. The amount of gavage bacteria solution was 0.15 mL / 10 g of body weight. The body weight, condition, food intake and other data of the mice were recorded every week before and after modeling, and before and after intervention. After the experiment, the mice were sacrificed, the fat content was recorded, the serum was collected, and the content of blood lipids in the serum was detected according to the kit instructions, including total cholesterol (TC), triglyceride (TG), high-density lipoprotein (HDLC) and low-density lipoprotein (LDLC).

[0181] Experimental results:

[0182] 1. The effect of AF73-05CM02 on the weight gain of obese model mice

[0183] Table 4-1

[0184]

[0185] Table 4-2

[0186]

[0187] By comparing the changes in the weight gain of mice in the test group after 9 weeks (Table 4-1 and Table 4-2), it can be found that AF73-05CM02 gavage intervention can effectively slow down the increase of the weight gain of mice (*P<0.05).

[0188] 2. The effect of AF73-05CM02 on the fat ratio of obese model mice

[0189] Table 5

[0190]

[0191] The results of Table 5 show that AF73-05CM02 can significantly reduce the fat ratio of obese model mice (*P<0.05).

[0192] 3. The effect of AF73-05CM02 on the blood lipids of experimental mice

[0193] Table 6-1

[0194]

[0195] Table 6-2

[0196]

[0197] The results of Tables 6-1 and 6-2 show that the intervention of AF73-05CM02 (Group 3) can effectively control the levels of TC, TG, and LDLC in blood and increase HDLC (*P<0.05). Among the main components of blood lipids are cholesterol and triglycerides, and the increase of the levels of cholesterol and triglycerides in blood plasma is closely related to the occurrence of atherosclerosis, while the main function of high-density lipoprotein is to remove excessive cholesterol and low-density lipoprotein in blood and cells, and it has the effect of anti-atherosclerosis. Therefore, AF73-05CM02 can reduce blood lipids, reduce the relevant indicators of atherosclerosis-related diseases (such as cardiovascular diseases), and at the same time significantly increase the level of high-density lipoprotein.

[0198] Example 6 Food composition containing Christensenella intestinihominis AF73-05CM02

[0199] The raw materials are mixed in the proportions shown in Table 7.

[0200] Table 7

[0201] Raw materials Mass percentage (%) Christensenella intestinihominis AF73-05CM02 0.5 Milk 90.0 White sugar 9.5

[0202] The milk and sugar are mixed in the above-mentioned proportions, stirred until completely mixed, preheated, homogenized at 20 Mpa, sterilized at about 90°C for 5-10 minutes, cooled to 40-43°C, inoculated with 1-100 x 10 6 cfu / g of Christensenella intestinihominis AF73-05CM02 bacteria, to produce a food composition containing Christensenella intestinihominis AF73-05CM02 bacteria.

[0203] Example 7 Pharmaceutical composition containing Christensenella intestinihominis

[0204] The raw materials are mixed in the proportions shown in Table 8.

[0205] Table 8

[0206]

[0207]

[0208] The lactose, yeast powder and peptone are mixed with pure water in proportion, preheated to 60-65℃, homogenized under 20Mpa pressure, sterilized at about 90℃ for 20-30 minutes, cooled to 36-38℃, inoculated with Christensenella intestinihominis AF73-05CM02 living bacteria (1-50×10 6 cfu / mL), fermented at 36-38℃ until the pH value is 6.0, centrifuged, freeze-dried to less than 3% moisture content, to prepare the Christensenella intestinihominis AF73-05CM02 freeze-dried material. 0.5g of the Christensenella intestinihominis AF73-05CM02 freeze-dried material is mixed with an equal amount of malt dextrin, and then loaded into a capsule to prepare a pharmaceutical composition containing Christensenella intestinihominis AF73-05CM02 bacteria.

[0209] Strain preservation

[0210] The strain Christensenella intestinihominis AF73-05CM02 (the same as the preservation name) of the application has been preserved in the China General Microbiological Culture Collection Center (CGMCC) on June 13, 2016, located at No. 1, Yiaonan West Road, Chaoyang District, Beijing, China, with the preservation number: CGMCC 1.5207.

[0211] All the documents mentioned in the application are cited as references in the application, just like each document is cited as a reference individually. In addition, it should be understood that the person skilled in the art can make various modifications or changes to the application after reading the above teaching of the application, and these equivalent forms also fall within the scope defined by the claims of the application. Sequence listing <110> Shenzhen Huada Gene Research Institute <120> Christensenella intestinihominis and application thereof <130> P2018-1413 <150> PCT / CN2016 / 098247 <151> 2016-09-06 <160> 3 <170> PatentIn version 3.5 <210> 1 <211> 1366 <212> DNA <213> Christensenella intestinihominis AF73-05CM02 <400> 1 ttgctctttg tgaagccctc gggtggaact gcgagtatac ttagtggcgg acgggtgagt 60 aacgcgtgag caatctgccc tgcaatgggg gacaacagtt ggaaacgact gctaataccg 120 catgagacca cgaaaccgca tggttttgag gtaaaaggat ttattcgatg caggatgagc 180 tcgcgtccca ttagatagtt ggtgaggtaa cggcccacca agtcaacgat gggtagccga 240 cctgagaggg tgatcggcca cactggaact gagacacggt ccagactcct acgggaggca 300 gcagtgggga atattgggca atgggggaaa ccctgaccca gcaacgccgc gtgagggaag 360 aaggtcttcg gattgtaaac ctttgtccta tgggacgaaa caaatgacgg taccatagga 420 ggaagctccg gctaactacg tgccagcagc cgcggtaata cgtagggagc aagcgttgtc 480 cggaattact gggcgtaaag ggtgcgtagg tggctatgta agtcagatgt gaaagaccgg 540 ggcttaaccc cggggttgca tttgaaactg tgtggcttga gtacaggaga gggaagtgga 600 attcctagtg tagcggtgaa atgcgtagat attaggagga acaccagtgg cgaaggcgac 660 tttctggact gtaactgaca ctgaagcacg aaagcgtggg gagcaaacag gattagatac 720 cctggtagtc cacgccgtaa acgatggata ctaggtgtgg ggcccgatag ggttccgtgc 780 cgaagctaac gcattaagta tcccgcctgg ggagtacgat cgcaaggttg aaactcaaag 840 gaattgacgg gggcccgcac aagcagcgga gcatgtggtt taattcgaag caacgcgaag 900 aaccttacca aggcttgaca tcctctgacg actgtagaga tacagtttcc cttcggggca 960 gagagacagg tggtgcatgg ttgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc 1020 gcaacgagcg caacccttat tgctagttgc cagcgcgtaa aggcgggaac tctagtgaga 1080 ctgccgggga caactcggag gaaggtgggg acgacgtcaa atcatcatgc cccttatgtc 1140 ttgggctaca cacgtgctac aatggccggt acaaagggca gcgaacccgt aaggggaagc 1200 gaatctcaaa aagccggtcc cagttcggat tgtgggctgc aacccgccca catgaagtcg 1260 gagttgctag taatcgcgaa tcagcatgtc gcggtgaatg cgttcccggg ccttgtacac 1320 accgcccgtc acaccacgga agttgggagc acccgaagcc agtgga 1366 <210> 2 <211> 20 <212> DNA <213> artificial sequence <400> 2 agagtttgat catggctcag 20 <210> 3 <211> 22 <212> DNA <213> artificial sequence <400> 3 tagggttacc ttgttacgac tt 22

Claims

1. A Christensenella bacterium, characterized in that, The Christensenella is Christensenella intestinihominis, and the Christensenella is Christensenella intestinihominis AF73-05CM02, with a preservation number of CGMCC 1.5207.

2. A composition characterized in that, The composition comprises: (a) a safe and effective amount of the Christensenella of claim 1; and (b) a food acceptable carrier or a pharmaceutically acceptable carrier.

3. The composition of claim 2, wherein The composition contains 1 x 10 -1 x 10 10 cfu / mL or cfu / g Christensenella intestinihominis AF73-05 CM02, per total volume or total weight of the composition.

4. The composition of claim 2, wherein The composition further comprises other probiotics and / or prebiotics.

5. Use of the Christensenella or the composition containing the Christensenella in the preparation of a preparation for one or more purposes selected from the group consisting of: (a) preventing and / or treating obesity; (b) reducing blood lipids; wherein The Christensenella is the Christensenella of claim 1.

6. Use of the Christensenella or the composition containing the Christensenella in the preparation of a preparation for one or more purposes selected from the group consisting of: (i) reducing the cholesterol level of a mammal; (ii) inhibiting the weight gain of a mammal; (iii) reducing the body fat ratio of a mammal; (iv) reducing the blood lipid level of a mammal; (v) increasing the level of high-density lipoprotein (HDL-C) in a mammal; (vi) reducing the low-density lipoprotein (LDL-C) level in a mammal; wherein The Christensenella is the Christensenella of claim 1.

7. The use of claim 6, wherein, The reduction of the blood lipid level of a mammal includes the reduction of the total cholesterol (TC) level and / or the triglyceride level.

8. A process for the preparation of the composition of claim 2, characterized in that, The steps include: mixing the Christensenella of claim 1 with a food acceptable carrier or a pharmaceutically acceptable carrier, thereby forming the composition of claim 2.

9. A production method characterized by comprising: The steps include: (a) culturing the Christensenella of claim 1 under conditions suitable for growth, thereby obtaining a culture product; (b) isolating the Christensenella cell mass from the culture product; and (c) mixing the Christensenella cell mass obtained by isolation in the previous step with a food acceptable carrier or a pharmaceutically acceptable carrier, thereby preparing the composition.

Images