Collinsella shenzhenensis and its applications

Abstract

Provided are the use of Collinsella shenzhenensis in treating and / or preventing inflammation-related diseases, cardiovascular diseases, and a composition for treating and / or preventing inflammation-related diseases, cardiovascular diseases, including a pharmaceutical product, a beverage, a food product, or an animal feed composition, etc., and a method for improving intestinal lesions of a mammal, reducing blood lipid levels of a mammal, controlling weight loss of a mammal, and / or reducing disease activity index (DAI) of a mammal.

Description

Technical Field

[0001] This invention belongs to the field of microbiology. Specifically, this invention relates to the application of Collinsella shenzhenensis in the treatment and prevention of inflammation-related diseases, and also to compositions containing Collinsella shenzhenensis and their applications. Background Technology

[0002] Ulcerative colitis (UC) is a type of inflammatory bowel disease (IBD). Based on the site of inflammation, it is classified as colitis or proctitis. Its pathogenesis is not fully understood, but it is currently believed to be caused by genetic susceptibility, gut microbiota, and immune responses of the intestinal mucosa. Numerous studies both domestically and internationally have shown a close relationship between the onset of UC and gut microbiota imbalance. The clinicopathological manifestations of UC include abdominal pain, diarrhea, and bloody mucus stools, with recurrent episodes.

[0003] Because the pathological mechanism is unclear, clinical treatment lacks specificity and targeting. Currently, the main medications used clinically for ulcerative colitis (UC) include salicylates, glucocorticoids, and immunosuppressants. Salicylates can effectively inhibit prostaglandin synthesis and scavenge oxygen free radicals, thereby alleviating the inflammatory response. The most common salicylate for UC treatment is sulfasalazine (SASP), primarily for mild, moderate, and chronic UC patients. Glucocorticoids are the first-line treatment for severe or fulminant UC patients, such as betamethasone. Immunosuppressants such as cyclosporine can inhibit the production of IL-2 on T cells, affecting the progression of the immune response and thus suppressing UC.

[0004] All three types of drugs mentioned above can alleviate UC to some extent, but they also have certain side effects. Salicylate derivatives can cause gastrointestinal reactions, headaches, increased reticulocyte count, decreased sperm count, and allergic reactions such as rashes, liver toxicity, leukopenia, and anemia. Glucocorticoids can cause metabolic disorders and water retention, and should only be used as emergency medication, not for long-term use. Immunosuppressant therapy has a high degree of drug dependence, a long treatment period, and is prone to causing nephrotoxicity and secondary infections; it can only be used as an adjunct therapy.

[0005] Therefore, there is an urgent need in this field to develop a new, non-toxic drug for the treatment and prevention of inflammation-related diseases. Summary of the Invention

[0006] Another object of the present invention is to provide the use of Collins bacteria in the treatment and / or prevention of inflammation-related diseases.

[0007] Another object of the present invention is to provide an effective, non-toxic, and non-toxic pharmaceutical, beverage, food composition, or animal feed composition for treating and / or preventing inflammation-related diseases.

[0008] Another objective of this invention is to provide a method for improving intestinal lesions in mammals and its application.

[0009] Another object of the present invention is to provide a method and its application for lowering blood lipids, controlling the loss of body weight in mammals, and / or reducing the disease activity index (DAI) in mammals.

[0010] Another object of the present invention is to provide the use of Collins bacteria in the treatment and / or prevention of cardiovascular-related diseases.

[0011] The first aspect of the present invention provides a Collins bacterium, wherein the Collins bacterium is Collins shenzhenensis.

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

[0013] In another preferred embodiment, the Collins bacterium is Collinsella shenzhenensis TF06-26, with accession number GDMCC NO.: 60090.

[0014] In another preferred embodiment, the Collins bacteria are derived from the intestines, animal feces, fermentation tanks, and / or anaerobic reactors.

[0015] A second aspect of the present invention provides a composition comprising: (a) a safe and effective amount of Collins bacteria and / or their metabolites as described in the first aspect of the present invention; and (b) a food-acceptable carrier or a pharmaceutically acceptable carrier.

[0016] In another preferred embodiment, the composition further includes a growth factor (preferably, milk growth factor).

[0017] In another preferred embodiment, the composition is selected from the group consisting of: food compositions, health care compositions, pharmaceutical compositions, beverage compositions, feed compositions, or combinations thereof.

[0018] In another preferred embodiment, the composition is an oral formulation.

[0019] In another preferred embodiment, the composition is a liquid formulation, a solid formulation, or a semi-solid formulation.

[0020] In another preferred embodiment, the dosage form of the composition is selected from the group consisting of powders, granules, tablets, sugar-coated tablets, capsules, granules, suspensions, solutions, syrups, drops, sublingual tablets, or combinations thereof.

[0021] In another preferred embodiment, the food composition includes an emulsion product, a solution product, a powder product, or a suspension product.

[0022] In another preferred embodiment, the food composition includes dairy products, milk powder, or emulsions.

[0023] In another preferred embodiment, the liquid formulation is selected from the group consisting of solution products or suspension products.

[0024] In another preferred embodiment, the composition contains 1×10⁻¹⁰ to 1×10⁻¹⁰ 15 cfu / mL or cfu / g Collinsella shenzhenensis TF06-26, preferably 1×10⁻⁶. 4 -1×10 10 cfu / mL or cfu / g Collinsella shenzhenensis TF06-26, based on the total volume or total weight of the composition.

[0025] In another preferred embodiment, the composition contains 0.0001-99 wt%, preferably 0.1-90 wt%, of the *Collinsella* and / or its metabolites, based on the total weight of the composition.

[0026] In another preferred embodiment, the composition is in a unit dosage form (a tablet, a capsule, or a vial), and the mass of the composition in each unit dosage form is 0.05-5g, preferably 0.1-1g.

[0027] In another preferred embodiment, the composition further contains other probiotics and / or prebiotics.

[0028] In another preferred embodiment, the probiotics are selected from the group consisting of lactic acid bacteria, bifidobacteria, lactobacillus acidophilus, or combinations thereof.

[0029] In another preferred embodiment, the prebiotic is selected from the group consisting of fructooligosaccharides (FOS), galactooligosaccharides (GOS), xylooligosaccharides (XOS), lactulose oligosaccharides (LACT), soybean oligosaccharides (SOS), inulin, oligosaccharides, or combinations thereof.

[0030] In another preferred embodiment, the composition also contains substances (such as protectants) that help maintain the viability of Collins bacteria.

[0031] In another preferred embodiment, the substance (such as a protectant) that helps maintain the viability of Collins bacteria is selected from the group consisting of: cysteine, glutathione, butylated hydroxyanisole, butylated methyltoluene, tocopherol, bamboo leaf antioxidant, D-isoascorbic acid and its sodium salt, sodium ascorbate, calcium ascorbate, phospholipids, vitamin C (ascorbic acid), vitamin E, or combinations thereof.

[0032] In another preferred embodiment, the substance (such as a protectant) that helps maintain the viability of Collins bacteria is present in a weight ratio of 0.1-2%, more preferably 0.5-1.5%, and even more preferably 0.5-1.0%, based on the total weight of the composition.

[0033] In another preferred embodiment, the content of the substance (such as a protectant) that helps maintain the viability of Collins bacteria is 1 mg to 20 mg per 1 g of the composition, more preferably 5 mg to 15 mg, and even more preferably 5 mg to 10 mg.

[0034] A third aspect of the present invention provides the use of the Collins bacterium described in the first aspect of the present invention, or the composition described in the second aspect of the present invention, for the preparation of a medicine or formulation for one or more uses selected from the group consisting of: (a) prevention and / or treatment of inflammation-related diseases; and / or (b) prevention and / or treatment of cardiovascular diseases.

[0035] In another preferred embodiment, the inflammation-related disease is selected from the group consisting of inflammatory bowel disease, rheumatoid arthritis, or a combination thereof.

[0036] In another preferred embodiment, the inflammation-related disease is selected from the group consisting of ulcerative colitis, gastritis, common enteritis, or a combination thereof.

[0037] In another preferred embodiment, the cardiovascular disease is selected from the group consisting of hypertension, hyperlipidemia, coronary heart disease, or a combination thereof.

[0038] In another preferred embodiment, the formulation includes a probiotic formulation.

[0039] A fourth aspect of the present invention provides the use of the *Collinus* strain described in the first aspect of the present invention, or the composition described in the second aspect of the present invention, for the preparation of a drug or formulation, said drug or formulation for one or more uses selected from the group consisting of:

[0040] (i) Reduce blood lipid levels in mammals;

[0041] (ii) Controlling the decrease in body weight in mammals;

[0042] (iii) Reduce the Disease Activity Index (DAI) in mammals;

[0043] (iv) Improves intestinal lesions in mammals.

[0044] In another preferred embodiment, the mammals include humans and rodents (such as rats and mice).

[0045] In another preferred embodiment, the reduction of blood lipid levels in mammals includes reducing cholesterol levels.

[0046] In another preferred embodiment, the controlled decrease in mammalian body weight refers to a decrease in body weight of the experimental group mammals compared to the model group mammals, which is no more than 10%, preferably no more than 5%, and more preferably no more than 2%.

[0047] In another preferred embodiment, the mitigation of mammalian intestinal pathology includes mitigating the shortening of colon length and / or reducing colonic inflammatory response.

[0048] The fifth aspect of the present invention provides a method for preparing the composition described in the second aspect of the present invention, comprising the steps of:

[0049] The Collins bacterium and / or its metabolites described in the first aspect of the present invention are mixed with a food-acceptable carrier or a pharmaceutically acceptable carrier to form the composition described in the second aspect of the present invention.

[0050] In another preferred embodiment, the preparation method further includes a step of mixing with growth factors.

[0051] In another preferred embodiment, the preparation method further includes the step of mixing with a substance that helps maintain the viability of Collins bacteria (such as a protectant).

[0052] In another preferred embodiment, the substance (such as a protectant) that helps maintain the viability of Collins bacteria is selected from the group consisting of: cysteine, glutathione, butylated hydroxyanisole, butylated methyltoluene, tocopherol, bamboo leaf antioxidant, D-isoascorbic acid and its sodium salt, sodium ascorbate, calcium ascorbate, phospholipids, vitamin C (ascorbic acid), vitamin E, or combinations thereof.

[0053] In another preferred embodiment, the preparation method further includes the step of mixing with probiotics and / or prebiotics.

[0054] In another preferred embodiment, the probiotics are selected from the group consisting of lactic acid bacteria, bifidobacteria, lactobacillus acidophilus, or combinations thereof.

[0055] In another preferred embodiment, the prebiotic is selected from the group consisting of fructooligosaccharides (FOS), galactooligosaccharides (GOS), xylooligosaccharides (XOS), lactulose oligosaccharides (LACT), soybean oligosaccharides (SOS), inulin, oligosaccharides, or combinations thereof.

[0056] In another preferred embodiment, the growth factor is milk growth factor.

[0057] In another preferred embodiment, the growth factor is selected from the group consisting of vitamins, purines, pyrimidines, or combinations thereof.

[0058] In another preferred embodiment, the composition is an oral formulation.

[0059] The sixth aspect of the present invention provides a production method, comprising the steps of:

[0060] (a) The Collins bacterium described in the first aspect of the present invention is cultured under suitable culture conditions to obtain a culture product;

[0061] (b) Optionally, isolate Collins bacteria cells and / or their metabolites from the culture product; and / or

[0062] (c) Optionally, the culture product or Collins bacteria cells and / or their metabolites obtained in the previous step are mixed with a food-acceptable carrier or a pharmaceutically acceptable carrier to prepare the composition of the present invention.

[0063] In another preferred embodiment, prior to step (c), the step further includes mixing the culture product obtained in the previous step or the Collins bacteria cells and / or their metabolites with growth factors.

[0064] In another preferred embodiment, the growth factor is milk growth factor.

[0065] In another preferred embodiment, the growth factor is selected from the group consisting of vitamins, purines, pyrimidines, or combinations thereof.

[0066] In another preferred embodiment, prior to step (c), the step further includes mixing the culture product obtained in the previous step or the Collins bacteria cells and / or their metabolites with a substance that helps maintain the viability of Collins bacteria (such as a protectant).

[0067] In another preferred embodiment, prior to step (c), the step further includes mixing the culture product obtained in the previous step or the Collins bacteria cells and / or their metabolites with probiotics and / or prebiotics.

[0068] The seventh aspect of the present invention provides a method for improving intestinal lesions in mammals by administering the composition described in the second aspect of the present invention to the subject.

[0069] In another preferred embodiment, the administration includes oral administration.

[0070] In another preferred embodiment, the dosage is 0.01-5g / 50kg body weight / day, more preferably 0.1-2g / 50kg body weight / day.

[0071] In another preferred embodiment, the object includes mammals, such as humans.

[0072] The eighth aspect of the present invention provides a method for lowering blood lipids, controlling weight loss in mammals, and / or reducing the disease activity index (DAI) in mammals, by administering the composition described in the second aspect of the present invention to the subject.

[0073] In another preferred embodiment, the administration includes oral administration.

[0074] In another preferred embodiment, the dosage is 0.01-5g / 50kg body weight / day, more preferably 0.1-2g / 50kg body weight / day.

[0075] In another preferred embodiment, the object includes mammals, such as humans.

[0076] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0077] Figure 1 The image shows a colony of *Collinsella shenzhenensis* cultured for 48 hours.

[0078] Figure 2 The image shows a Gram-stained image of Collinsella shenzhenensis under a microscope (1000x magnification).

[0079] Figure 3 The standard curve for cholesterol is shown.

[0080] Figure 4 The length of the colon in mice in the model group and the TF06-26 treatment group is shown. Detailed Implementation

[0081] Through extensive and in-depth research and experimentation, the inventors unexpectedly discovered that *Collinsella shenzhenensis* has preventive and / or therapeutic effects on inflammation-related diseases (such as inflammatory bowel disease (e.g., ulcerative colitis, gastritis, common enteritis), rheumatoid arthritis) and cardiovascular diseases (such as hypertension, hyperlipidemia, coronary heart disease). Feeding experimental subjects with an active composition containing *Collinsella shenzhenensis* revealed that this composition could control weight loss, lower blood lipids, reduce the disease activity index (DAI), improve intestinal lesions, and effectively alleviate symptoms of inflammation-related diseases (such as inflammatory bowel disease (e.g., ulcerative colitis, gastritis, common enteritis), rheumatoid arthritis), and cardiovascular diseases. This invention was completed based on these findings.

[0082] As used herein, the term "containing" means that various ingredients may be used together in the mixtures or compositions of the present invention. Therefore, the terms "consistent with..." and "composed of..." are included in the term "containing".

[0083] As used herein, the term "growth factor" includes milk growth factor, specifically including nutrients such as vitamins, purines, pyrimidines, or combinations thereof.

[0084] The vitamins mentioned include (but are not limited to): vitamin C, vitamin E, vitamin A, vitamin A precursor, vitamin B6, vitamin D3, vitamin K, folic acid, or combinations thereof;

[0085] The purine substances include (but are not limited to): purine nucleosides, wherein the purine nucleosides include 5'-phosphate esters of purine nucleosides; the 5'-phosphate esters of the purine nucleosides are selected from the group consisting of: inosine monophosphate (inosine-5'-phosphate; IMP), guanylic acid (guanosine-5'-phosphate; GMP), xanthocyanidin (xanthine nucleoside-5'-phosphate; XMP), adenosine monophosphate (adenosine-5'-phosphate; AMP), or combinations thereof;

[0086] The pyrimidine substances include all substances containing a pyrimidine structure.

[0087] As used herein, the terms "controlling weight loss in mammals" and "slowing down weight loss in mammals" are used interchangeably, referring to the decrease in body weight of experimental animals during the construction of an ulcerative colitis model due to the increasing severity of inflammation. The percentage of weight loss is the percentage of weight lost relative to the original body weight. The greater the degree of weight loss, the more severe the disease. The *Collinsella asiatica* strain of this invention can control weight loss in experimental animals and alleviate disease symptoms during the treatment of ulcerative colitis in mammals.

[0088] Disease Activity Index (DAI)

[0089] As used in this article, the term "disease activity index" refers to a comprehensive score that combines three factors: percentage weight loss in patients (or sick animals), stool consistency, and rectal bleeding.

[0090] Collins bacteria in Shenzhen and their applications

[0091] As used herein, the terms "Collinsella shenzhenensis", "Collinsella shenzhenensis", "Collinsella shenzhenensis of the present invention", and "Collinsella shenzhenensis of the present invention" are used interchangeably. In a preferred embodiment, the strain is Collinsella shenzhenensis TF06-26, with accession number GDMCC NO.: 60090, isolated from feces of humans (preferably, healthy women), specifically from the intestines of adolescent girls. The physiological characteristics of *Collinsella shenzhenensis* TF06-26 are as follows: After 48 hours of anaerobic culture, the colonies are white, raised, round, with neat edges, high water content, and opaque, with a colony diameter of 1.0 mm-2.0 mm. Gram staining, spore staining, and flagella staining were performed on *Collinsella shenzhenensis* TF06-26, and its morphology was observed under 1000x magnification. *Collinsella shenzhenensis* TF06-26 is Gram-negative, does not produce spores or flagella, and is a short rod-shaped bacterium. Collinsella shenzhenensis TF06-26 is catalase-negative, oxidase-negative, and non-motile. Its growth temperature range is 25-45℃, and its growth pH range is 5.0-8.0. It tolerates 2% NaCl and 0.3% bile salts. It can ferment various carbohydrates, including D-carbohydrate, N-acetylglucosamine, galactose, mannose, fructose, salicylate, maltose, melibiose, glucose, lactose, cellobiose, arbutin, and / or salicylate, primarily producing lactic acid, acetic acid, and butyric acid. Furthermore, this *Collinsella shenzhenensis* TF06-26 is resistant to kanamycin, amikacin, neomycin, and erythromycin, and sensitive to the remaining 16 antibiotics listed in Table 2. In addition, this *Collinsella shenzhenensis* can produce extremely high yields of extracellular polysaccharides (up to 284.49 mg / L, based on the total concentration of the fermentation broth).

[0092] This invention provides the application of *Collinsella shenzhenensis* in the treatment and / or prevention of inflammation-related diseases (such as inflammatory bowel disease (e.g., ulcerative colitis, gastritis, common enteritis), rheumatoid arthritis) and cardiovascular diseases (e.g., hypertension, hyperlipidemia, coronary heart disease). Subjects were induced to develop a model using DSS (sodium dextran sulfate). The strain *Collinsella shenzhenensis* TF06-26 has one or more uses selected from the group consisting of: (i) controlling weight loss in the subject; (ii) lowering blood lipids; (iii) improving the severity of intestinal lesions; and (iv) reducing the disease activity index (DAI). According to a preferred embodiment of the present invention, C57bl / 6 mice were used as experimental mice, and ulcerative colitis (UC) model mice were induced by DSS (sodium dextran sulfate) to obtain UC model mice. UC model mice treated with Collinsella shenzhenensis TF06-26 showed slower weight loss and lower blood lipids compared to the untreated control group (model group). Furthermore, various indicators related to inflammation-related diseases (such as inflammatory bowel disease (e.g., ulcerative colitis, gastritis, common enteritis), rheumatoid arthritis) were also improved, such as improved intestinal lesion severity (including slowing colon length shortening, reducing colonic inflammation, etc.) and reduced disease activity index (DAI). Therefore, the strain can be used to prevent and / or treat inflammation-related diseases (such as inflammatory bowel disease (e.g., ulcerative colitis, gastritis, common enteritis), rheumatoid arthritis) and cardiovascular diseases (such as hypertension, hyperlipidemia, coronary heart disease, etc.).

[0093] Compositions and their applications

[0094] The present invention also provides a composition, preferably comprising a food composition, a health care composition, a pharmaceutical composition, a beverage composition, or a feed composition, and more preferably, a pharmaceutical composition. The composition comprises an effective amount of *Collins spp.*, and in a preferred embodiment, the composition further comprises a growth factor (such as milk growth factor). In a preferred embodiment, the composition further comprises probiotics selected from the group consisting of lactic acid bacteria, bifidobacteria, *Lactobacillus acidophilus*, or combinations thereof; and / or prebiotics selected from the group consisting of fructooligosaccharides (FOS), galactooligosaccharides (GOS), xylooligosaccharides (XOS), lactulose oligosaccharides (LACT), soybean oligosaccharides (SOS), inulin, oligosaccharides, or combinations thereof. In a preferred embodiment, the composition further includes a substance (such as a protectant) selected from the group consisting of: cysteine, glutathione, butylated hydroxyanisole, butylated methyltoluene, tocopherol, bamboo leaf antioxidant, D-isoascorbic acid and its sodium salt, sodium ascorbate, calcium ascorbate, phospholipids, vitamin C (ascorbic acid), vitamin E, or combinations thereof. The weight percentage of the substance (such as a protectant) contributing to maintaining Collins viability is 0.1-2%, preferably 0.5-1.5%, and more preferably 0.5-1.0% based on the total weight of the composition.

[0095] In a preferred embodiment, the composition is a liquid formulation, a solid formulation, or a semi-solid formulation.

[0096] In a preferred embodiment, the liquid formulation is selected from the group consisting of solution products or suspension products.

[0097] In a preferred embodiment, the dosage form of the composition is selected from the group consisting of powders, granules, tablets, sugar-coated tablets, capsules, granules, suspensions, solutions, syrups, drops, sublingual tablets, or combinations thereof.

[0098] The compositions of the present invention can be administered in any form, such as oral liquid, tablets, injections, orally disintegrating tablets, lyophilized powder formulations, or capsules, preferably enteric-coated (e.g., capsules). In this invention, unless otherwise specified, the excipients, permissible media, and carriers used are primarily selected based on the characteristics of the suitable bacteria or their metabolites and the desired specific route of administration, facilitating the smooth passage of the bacteria or their metabolites through the stomach for absorption by the user. These substances can be selected according to the route of administration.

[0099] The compositions of the present invention may further include any additional excipients among those commonly used in pharmaceutical formulations, for example, to stabilize the composition itself, or to make it easily dispersed or to impart a suitable flavor.

[0100] Among the excipients, inulin, fructose, starch, xylooligosaccharides, silica, buffering agents, and flavoring agents are suitable examples.

[0101] The pharmaceutical formulation of the present invention may further include auxiliary active ingredients.

[0102] Lactose, maltodextrin, glucose, sucrose, sorbitol, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, fine crystalline cellulose, polyvinylpyrrolidone (PVP), cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, or mineral oil can all be used as carriers, excipients, or diluents of the pharmaceutical composition in this invention.

[0103] Furthermore, the pharmaceutical compositions of the present invention may further include lubricants, wetting agents, emulsifiers, suspension stabilizers, preservatives, sweeteners, and flavorings. The pharmaceutical compositions of the present invention can be produced as enteric-coated formulations using various known methods, so that the active ingredient of the pharmaceutical composition, i.e., the microorganism, can pass smoothly through the stomach without being destroyed by gastric acid.

[0104] Furthermore, the microorganisms of the present invention can be used in capsule form prepared by conventional methods. For example, standard excipients and the cryogenically dried microorganisms of the present invention are mixed to form small pellets, which are then filled into gelatin capsules. Additionally, the microorganisms of the present invention and permitted excipients for pharmaceuticals, such as liquid gums, cellulose, silicates, or mineral oils, can be mixed to form suspensions or dispersions that can be filled into soft gelatin capsules.

[0105] The pharmaceutical compositions of the present invention can be formulated into enteric-coated tablets for oral administration. The term "enteric-coated" as used in this application includes all coatings permitted for conventional pharmaceutical use that are not degraded by gastric acid but are readily decomposed in the small intestine, rapidly releasing the microorganisms of the present invention. The enteric-coated tablets of the present invention can be maintained at 36-38°C for more than 2 hours in synthetic gastric acid such as an HCl solution with pH=1, and preferably decompose within 1 hour in synthetic intestinal fluid such as a buffer solution with pH=7.0.

[0106] The casings of this invention are coated with approximately 16-30 mg per tablet, preferably 16-25 mg, and more preferably 16-20 mg. The casing thickness in this invention is 5-100 μm, ideally 20-80 μm. The casing components are selected from conventionally known polymers.

[0107] The preferred casings of the present invention are prepared from a copolymer of cellulose acetate phthalate polymer or trimellitate polymer and isobutylene acid (e.g., a copolymer of isobutylene acid containing more than 40% isobutylene acid and isobutylene acid containing methylcellulose phthalate hydroxypropyl ester or its ester derivative).

[0108] The cellulose phthalate acetate used in the casings of this invention has a viscosity of approximately 45-90 cp, an acetyl content of 17-26%, and a phthalic acid content of 30-40%. The cellulose trimellitate acetate used in the casings has a viscosity of approximately 5-21 cs and an acetyl content of 17-26%. The cellulose trimellitate acetate is produced by Eastman Kodak and can be used in the casing materials of this invention.

[0109] The hydroxypropyl methylcellulose phthalate used in the casings of this invention has a molecular weight of generally 20,000-130,000 Daltons, with an ideal molecular weight of 80,000-100,000 Daltons. The hydroxypropyl content is 5-10%, the methoxy content is 18-24%, and the phthaloyl content is 21-35%.

[0110] The hydroxypropyl methylcellulose phthalate used in the casings of this invention is HP50, manufactured by Shin-Etsu Chemidnl Co., Ltd., Japan. HP50 contains 6-10% hydroxypropyl, 20-24% methoxy, and 21-27% propyl, with a molecular weight of 84,000 Daltons. Another casing material is HP55, which contains 5-9% hydroxypropyl methylcellulose phthalate, 18-22% methoxy, and 27-35% phthalic acid, with a molecular weight of 78,000 Daltons.

[0111] The sausage casings of this invention are prepared as follows: a casing solution is sprayed onto the core using conventional methods. All solvents used in this casing coating method are alcohols (such as ethanol), ketones (such as acetone), halogenated hydrocarbon compounds (such as dichloromethane), or combinations thereof. A softener, such as di-n-butyl phthalate and triacetin, is added to the casing solution in a ratio of 1 part casing to approximately 0.05 parts or approximately 0.3 parts softener. The spraying method is preferably performed continuously, and the amount of material sprayed can be controlled according to the conditions used for coating. The spraying pressure can be adjusted arbitrarily; generally, ideal results can be obtained at an average pressure of 1-1.5 bar.

[0112] The term "effective amount of drug" in the instructions refers to an amount that can produce function or activity in humans and / or animals and is acceptable to humans and / or animals. For example, in this invention, a drug containing 1×10⁻¹⁰ to 1×10⁻¹⁰⁻¹ can be prepared. 15 CFU / ml or CFU / g (specifically, it may contain 1×10⁻⁶ CFU / ml). 4 -1×10 10 cfu / ml or cfu / g; more specifically, it may contain 1×10 6 -1×10 10 Preparations of *Colletotrichum sinensis* (cfu / ml or cfu / g) and / or its metabolites from Shenzhen.

[0113] When used to prepare pharmaceutical compositions, the effective dose of *Colletotrichum sinensis* or its metabolites may vary depending on the administration regimen and the severity of the disease being treated. Suitable dosage forms for oral administration comprise approximately 1 × 10⁻¹ to 1 × 10⁻¹ of the product, closely mixed with a pharmaceutically acceptable solid or liquid carrier. 15 cfu / ml or cfu / g (preferably, may contain 1×10⁻⁶ cfu / ml or cfu / g). 4 -1×10 10 cfu / ml or cfu / g; more preferably, it may contain 1×10 6 -1×10 10 The active ingredient is either *Cfu / ml* or *Cfu / g* of *Collinus* bacteria from Shenzhen or produced through fermentation. This dosage regimen can be adjusted to provide the optimal therapeutic response. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0114] The *Collinus* strain from Shenzhen or its metabolites can be administered orally. Solid carriers include starch, lactose, dicalcium phosphate, microcrystalline cellulose, sucrose, and kaolin, while liquid carriers include culture media, polyethylene glycol, nonionic surfactants, and edible oils (such as corn oil, peanut oil, and sesame oil), provided they are suitable for the characteristics of *Collinus* or its metabolites and the desired specific route of administration. Adjuvants commonly used in the preparation of pharmaceutical compositions may also be advantageously included, such as flavoring agents, colorings, preservatives, and antioxidants such as vitamin E, vitamin C, BHT, and BHA.

[0115] From the perspective of ease of preparation and administration, preferred pharmaceutical compositions are solid compositions, especially tablets and solid-filled or liquid-filled capsules. Oral administration is preferred.

[0116] The composition of the invention is administered to the individual once or more daily. Dosage units represent doses that are formally separable and applicable to human or all other mammalian individuals. Each unit contains a pharmaceutically permissible carrier and an effective therapeutic amount of the microorganisms of the invention. Dosage varies depending on the patient's weight and the severity of inflammation-related diseases (such as inflammatory bowel disease (e.g., ulcerative colitis, gastritis, common enteritis), rheumatoid arthritis), cardiovascular diseases (e.g., hypertension, hyperlipidemia, coronary heart disease), the included supplemental active ingredients, and the microorganisms used. Furthermore, if possible, it can be administered separately, and if necessary, it can be administered continuously. Therefore, the dosage does not limit the invention. Moreover, the term "composition" in this invention means not only a pharmaceutical product but also a product suitable as a functional food and health supplement. In a preferred embodiment, the composition includes: beverages, food, pharmaceuticals, animal feed, etc.

[0117] In a preferred embodiment of the present invention, a food composition is also provided, containing an effective amount of *Collinus spp.* and / or its metabolites, and the balance being a food-acceptable carrier. The dosage form of the food composition is selected from solid, dairy, solution, powder, or suspension products. In a preferred embodiment, the food composition may also contain growth factors (such as milk growth factor). In a preferred embodiment, the composition further includes probiotics selected from the group consisting of lactic acid bacteria, bifidobacteria, *Lactobacillus acidophilus*, or combinations thereof; and / or prebiotics selected from the group consisting of fructooligosaccharides (FOS), galactooligosaccharides (GOS), xylooligosaccharides (XOS), lactulose oligosaccharides (LACT), soybean oligosaccharides (SOS), inulin, oligosaccharides, or combinations thereof. In a preferred embodiment, the composition further includes a substance (such as a protectant) selected from the group consisting of: cysteine, glutathione, butylated hydroxyanisole, butylated methyltoluene, tocopherol, bamboo leaf antioxidant, D-isoascorbic acid and its sodium salt, sodium ascorbate, calcium ascorbate, phospholipids, vitamin C (ascorbic acid), vitamin E, or combinations thereof.

[0118] In a preferred embodiment, the composition is formulated as follows:

[0119] 1×10-1×10 15 Shenzhen Collins bacillus (cfu / mL) and / or its metabolites; and food- or pharmaceutically acceptable carriers and / or excipients.

[0120] In another preferred embodiment, the composition is formulated as follows:

[0121] 1×10 4 -1×10 10 Shenzhen Collins bacillus (cfu / mL) and / or its metabolites; and food- or pharmaceutically acceptable carriers and / or excipients.

[0122] Microecological preparations

[0123] Microecological preparations are biological agents containing probiotics and their metabolites, or dietary supplements that can increase probiotics. They can improve human health by regulating and maintaining the balance of the intestinal microecology. They mainly include probiotics, prebiotics, and synbiotics.

[0124] In this invention, the microecological preparation comprises (a) a safe and effective amount of *Collinus spp.* and / or its metabolites; and (b) a food-acceptable carrier or a pharmaceutically acceptable carrier. In a preferred embodiment, the preparation further comprises growth factors (such as milk growth factor, preferably including vitamins, purines, and / or pyrimidines). In another preferred embodiment, the preparation further comprises probiotics selected from the group consisting of lactic acid bacteria, bifidobacteria, *Lactobacillus acidophilus*, or combinations thereof; and / or prebiotics selected from the group consisting of fructooligosaccharides (FOS), galactooligosaccharides (GOS), xylooligosaccharides (XOS), lactulose oligosaccharides (LACT), soybean oligosaccharides (SOS), inulin, oligosaccharides, or combinations thereof. In a preferred embodiment, the composition further includes a substance (such as a protectant) selected from the group consisting of: cysteine, glutathione, butylated hydroxyanisole, butylated methyltoluene, tocopherol, bamboo leaf antioxidant, D-isoascorbic acid and its sodium salt, sodium ascorbate, calcium ascorbate, phospholipids, vitamin C (ascorbic acid), vitamin E, or combinations thereof.

[0125] Production method of Collins bacteria in Shenzhen

[0126] Typically, *Collins spp.* from Shenzhen can be prepared using conventional methods.

[0127] This invention provides a method for large-scale production of *Collins spp.*, specifically comprising the following steps:

[0128] (a) The Collins bacterium described in this invention is cultured under suitable culture conditions to obtain a culture product;

[0129] (b) Optionally, isolate Collins bacteria cells and / or their metabolites from the culture product; and

[0130] (c) Optionally, the culture product or Collins bacteria cells and / or their metabolites obtained in the previous step are mixed with a food-acceptable carrier or a pharmaceutically acceptable carrier to prepare a composition.

[0131] Methods to improve intestinal lesions in mammals

[0132] In another preferred embodiment, the method includes ingesting the pharmaceutical composition, food composition, beverage composition, or a combination thereof of the present invention. The experimental subjects include mammals, such as humans.

[0133] In another preferred embodiment, the method includes ingesting the pharmaceutical composition, food composition, or animal feed of the present invention, or a combination thereof. The experimental subject is an animal, preferably a rodent or rabbit.

[0134] Methods for controlling weight loss in mammals and / or reducing the disease activity index (DAI) in mammals.

[0135] In another preferred embodiment, the method includes ingesting the pharmaceutical composition, food composition, beverage composition, or a combination thereof of the present invention. The experimental subjects include mammals, such as humans.

[0136] In another preferred embodiment, the method includes ingesting the pharmaceutical composition, food composition, or animal feed of the present invention, or a combination thereof. The experimental subject is an animal, preferably a rodent or rabbit.

[0137] Methods to lower blood lipids

[0138] In another preferred embodiment, the method includes ingesting the pharmaceutical composition, food composition, beverage composition, or a combination thereof of the present invention. The experimental subjects include mammals, such as humans.

[0139] In another preferred embodiment, the method includes ingesting the pharmaceutical composition, food composition, or animal feed of the present invention, or a combination thereof. The experimental subject is an animal, preferably a rodent or rabbit.

[0140] strain preservation

[0141] The strain of this invention, Collinsella shenzhenensis TF06-26 (same as the deposit name), was deposited on October 13, 2016, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC NO.: 60090.

[0142] The main advantages of this invention include:

[0143] (a) The *Collinsella spp.* of the present invention can significantly reduce blood lipids (such as cholesterol levels).

[0144] (b) The Shenzhen Collins bacterium of the present invention can significantly improve indicators related to inflammation-related diseases (such as inflammatory bowel disease (such as ulcerative colitis, gastritis, common enteritis), rheumatoid arthritis) (such as controlling weight loss, improving the degree of intestinal lesions (including slowing down the shortening of colon length, reducing colonic inflammation response, etc.), and reducing the disease activity index (DAI).

[0145] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to conditions described in Microbiology: A Laboratory Manual (edited by James Cappuccino and Natalie Sherman, Pearson Education Press), or according to the manufacturer's recommendations.

[0146] Unless otherwise specified, all materials and reagents used in the examples are commercially available products.

[0147] VSL#3, purchased from Sigma-Tau, USA.

[0148] Collinsella aerofaciens JCM 10188, purchased from the Japan Microbial Culture Collection Center, accession number JCM 10188.

[0149] Example 1: Screening and identification of *Collins spp.* TF06-26 from Shenzhen

[0150] 1.1 Isolation of Collins bacterium TF06-26 (hereinafter referred to as TF06-26) from Shenzhen

[0151] (1) Sample collection

[0152] The *Collins spp.* TF06-26 strain of this invention was isolated from a fecal sample of a healthy teenage female volunteer in Shenzhen.

[0153] (2) Prepare the culture medium and PBS (phosphate buffer).

[0154] The culture medium used for strain isolation in this invention is anaerobic PYG medium (purchased from Huankai Microbial Technology Co., Ltd.), with the following specific components (1L): 5g peptone, 5g trypsin, 10g yeast extract, 5g beef extract, 5g glucose, 2g K2HPO4, 1mL Tween 80, 0.5g Cysteine-HCl·H2O, 5mg heme, 1uL vitamin K1, 40mL inorganic salt solution (each liter (L) contains 0.25g CaCl2·2H2O, 0.5g MgSO4·7H2O, 1g K2HPO4, 1g KH2PO4, 10g NaHCO3, 2g NaCl), 1mg resazurin, 950mL distilled water, and pH adjusted to 6.8–7.0. Sterilization conditions are 115℃ autoclaving for 25min. Solid culture media should be poured into plates in an anaerobic chamber.

[0155] Preparation of PBS: Weigh 8g NaCl, 0.2g KCl, 3.63g Na2HPO4·12H2O, 0.24g KH2PO4, and 0.5g cysteine ​​hydrochloride, dissolve them in 900ml double-distilled water, adjust the pH to 7.4 with hydrochloric acid and NaOH, add water to a final volume of 1L, purge with N2 to remove oxygen for 30s, seal the anaerobic bottle (tube), and then autoclave at 115℃ for 25min.

[0156] (3) Strain isolation

[0157] Freshly collected fecal samples were immediately transferred to an anaerobic chamber (the gas composition of the anaerobic chamber was: nitrogen: hydrogen: carbon dioxide = 90:5:5, v / v). Approximately 0.2g of fecal sample was suspended in PBS, thoroughly mixed, and serially diluted 10-fold. The samples were then plated and incubated at 37°C under anaerobic conditions for 2 days. Single colonies were selected for streak purification to obtain pure culture strains, which were then cryopreserved at -80°C with glycerol and freeze-dried under vacuum.

[0158] 1.2 16S rDNA Identification

[0159] The isolated pure cultures were subjected to 16S rDNA sequencing to obtain the taxonomic information of each strain. The strains were cultured in liquid PYG medium for 24 hours to reach a bacterial concentration of approximately 10⁻⁶. 8Genomic DNA was extracted from the bacterial culture at a concentration of cfu / ml. The extracted genomic DNA was used as a template for 16S rDNA PCR amplification. Universal 16S rDNA primers were used: 8F-1492R (5'-AGAGTTTGATCATGGCTCAG-3' (SEQ ID NO.: 2) and 5'-TAGGGTTACCTTGTTACGACTT-3' (SEQ ID NO.: 3)) (synthesized by Sangon Biotech (Shanghai) Co., Ltd.). The amplification program was: 95℃ pre-denaturation for 4 min, followed by 95℃ denaturation for 30 s, 57℃ annealing for 40 s, and 72℃ extension for 1 min 30 s, for 30 cycles. The amplified PCR products were purified and sequenced at 3730 nm to obtain the full-length 16S rDNA sequence for each strain. The 16S rDNA length of TF06-26 was 1380 bp, as shown in SEQ ID NO.: 1. By comparing with the EzTaxon-e database, the bacterium with the highest 16S rDNA sequence similarity between TF06-26 and the database is Collinsella aerofaciens JCM 10188.

[0160] The 16S rDNA sequence of TF06-26 is SEQ ID NO.: 1, as shown below:

[0161]

[0162] 1.3 Microbiological characteristics of TF06-26

[0163] (1) Morphological characteristics

[0164] The colonies of TF06-26 after 48 hours of anaerobic culture were white, raised, round, with neat edges, high water content, and opaque, with a colony diameter of 1.0mm-2.0mm. Figure 1 ).

[0165] (2) Microscopic features

[0166] Gram staining, spore staining, and flagella staining were performed on TF06-26, and the cell morphology of TF06-26 was observed under 1000x magnification. TF06-26 is Gram-negative, does not produce spores or flagella, and is short rod-shaped. Figure 2 ).

[0167] (3) Physiological and biochemical characteristics

[0168] TF06-26 is catalase-negative, oxidase-negative, and non-motile. Its growth temperature range is 25-45℃, its growth pH range is 5.0-8.0, its NaCl tolerance concentration is 2%, and its bile salt tolerance concentration is 0.3%. The substrate utilization (API 20A and API 50CHL) of TF06-26 and its closely related reference bacterium Collinsella aerofaciens JCM 10188 (purchased from the Japan Microbial Culture Collection, accession number JCM10188) is detailed in Table 1 (+ indicates a positive reaction; - indicates a negative reaction; w indicates a weak positive reaction).

[0169] Table 1

[0170]

[0171]

[0172] The comparison of carbon source utilization between TF06-26 and JCM10188 shows that TF06-26 and JCM10188 have significant differences in the utilization of lactose, sucrose, salicylate, galactose, fructose, mannose, arbutin, cellobiose, maltose, melibiose, trehalose, and 2-keto-gluconate. Therefore, it can be seen that TF06-26 and JCM10188 are not the same species.

[0173] 1.4 Antibiotic resistance

[0174] The sensitivity of TF06-26 to 20 antibiotics was investigated. TF06-26 was cultured to the logarithmic growth phase, and the bacterial culture was spread on PYG medium. After spreading evenly, antibiotic susceptibility testing strips were attached, and the culture was then placed in anaerobic conditions at 37℃ for 48 hours. The diameter of the inhibition zone was measured, and the results are shown in Table 2.

[0175] Table 2

[0176]

[0177] The results showed that TF06-26 was resistant to kanamycin, amikacin, neomycin and erythromycin, but sensitive to 16 other antibiotics.

[0178] 1.5 Genomic hybridization experiment of the novel species TF06-26 and its related strain JCM 10188

[0179] Based on the 16S rDNA alignment results of TF06-26, the closest related bacterium is Collinsella aerofaciens JCM 10188, with a 16S rDNA similarity of 99.9%. However, based on the 16S rDNA sequence, it is impossible to distinguish TF06-26 and JCM 10188 at the species level. Therefore, further DNA hybridization is required for confirmation.

[0180] DNA hybridization results showed that TF06-26 shared 51% homology with JCM 10188. According to Bergey's Manual of Bacteriological Identification, a DNA hybridization value higher than 70% indicates that the two strains belong to the same species. The DNA hybridization value between TF06-26 and JCM10188 was lower than 70%, therefore TF06-26 is a novel species distinct from known bacteria. Following the International Committee on Bacteriological Taxonomy (IBSP) naming conventions, this novel bacterium was named *Collinsella shenzhenensis* sp. nov., with TF06-26 designated as the type strain.

[0181] Example 2: Bioactive substances of *Collins tumefaciens* TF06-26 from Shenzhen

[0182] The bioactive substances in TF06-26 were mainly examined for the production of short-chain fatty acids (SCFA) and organic acids.

[0183] (1) Sample pretreatment

[0184] TF06-26 was cultured for 48 hours. 1 ml of the bacterial culture was centrifuged at 12000 r / min for 5 min. The supernatant was collected and prepared for the detection of short-chain fatty acids (SCFA) and organic acids.

[0185] (2) Determination of SCFA

[0186] Short-chain fatty acids were determined using the external standard method, with acetic acid, propionic acid, butyric acid, and valeric acid used to prepare standard curves. An Agilent gas chromatograph (GC-7890B) was used, employing an HP-INNOWax (Cross-Linked PEG) capillary column of 30m × 0.25mm × 0.25um. A flame ionization detector was used. GC parameters were set as follows: column temperature: 180–200℃; vaporization chamber temperature: 240℃; detection temperature: 210℃; injection volume: 2μL; carrier gas flow rate: N2, 50mL / min; hydrogen flow rate: 50mL / min; air flow rate: 600–700mL / min.

[0187] The measured SCFA yields were: acetic acid 19.88 mmol / L and butyric acid 4.37 mmol / L.

[0188] (3) Determination of organic acids

[0189] The following standards were selected for the detection of organic acids: 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. An Agilent gas chromatograph (GC-7890B) was used, with a 122-5532G DB-5ms column (40m × 0.25mm × 0.25um), column temperature: 270–290℃; injection port temperature: 250℃; gas flow rate: 0.86 ml / min. The organic acid content of *Collinsella shenzhenensis* TF06-26 is detailed in the table below:

[0190] Table 3

[0191]

[0192] Example 3: Extracellular polysaccharide production of *Collins spp.* TF06-26 from Shenzhen

[0193] Observation of TF06-26 colonies revealed high water content and a viscous culture, suggesting that TF06-26 possesses the ability to produce extracellular polysaccharides. The detection of extracellular polysaccharides employed the phenol sulfate method. Phenol sulfate reacts with free monosaccharides, oligosaccharides, and hexoses in polysaccharides, producing a color reaction. The color is directly proportional to the absorbance, with an absorption wavelength of 490 nm. The specific experimental procedure is as follows:

[0194] (1) Extraction of polysaccharides

[0195] The experimental strain was cultured in PY6 medium (formula same as in Example 1) for 3 days. 10 ml of the bacterial culture was boiled in a water bath for 30 min, then centrifuged at 10000 rpm. The supernatant was collected, and 80% trichloroacetic acid was added to a final concentration of 8%. The mixture was incubated overnight at 4°C to precipitate the protein. The supernatant was then centrifuged at 10000 rpm for 30 min, and the pH of the supernatant was adjusted to 6.0 with NaOH. Two volumes of anhydrous ethanol were added to precipitate the polysaccharides, and the mixture was incubated overnight at 4°C. The supernatant was then centrifuged at 10000 rpm for 30 min, and the supernatant was discarded. The precipitate was dissolved in preheated distilled water and then transferred to an ultrafiltration tube (3000 Da filter diameter) for ultrafiltration. The tube was centrifuged at 5000 rpm for 30 min. The polysaccharides retained in the inner tube were transferred to a volumetric flask and diluted to 10 ml with distilled water for later use.

[0196] (2) Preparation of glucose standard curve

[0197] Accurately weigh 20 mg of standard glucose into a 100 ml volumetric flask, add water to the mark, and then prepare glucose standard solutions of 20, 40, 60, 80, and 100 μg / ml. Take 400 μL of each standard solution, make three replicates, and sequentially add 400 μL of 5% phenol and 1 ml of concentrated sulfuric acid to each solution. After boiling in a water bath for 15 min, cool to room temperature and measure the absorbance at 490 nm. Then plot a standard curve with absorbance as the ordinate and glucose standard solution concentration as the abscissa.

[0198] (3) Detect the concentration of the extracted polysaccharides

[0199] Take 400 μL of polysaccharide solution, add 400 μL of 5% phenol and 1 mL of concentrated sulfuric acid sequentially, and react. After boiling in a water bath for 15 min, cool to room temperature and measure the absorbance at 490 nm. Calculate the concentration of polysaccharide based on the glucose standard curve.

[0200] (4) Results

[0201] Calculations showed that the extracellular polysaccharide content in the TF06-26 fermentation broth after 3 days of culture was 284.49 mg / L.

[0202] Example 4: Detection of the in vitro cholesterol-degrading function of *Collins tansii* TF06-26 from Shenzhen.

[0203] 4.1 Detection of bile salt tolerance in TF06-26

[0204] The human gut contains bile salts. If probiotics are to exert their beneficial effects in the gut, they need to have a certain tolerance to bile salts. Therefore, this invention investigated the tolerance of TF06-26 to bile salts in vitro.

[0205] A PYG liquid medium containing 0.2% bile salts was prepared. TF06-26 cells, cultured to the logarithmic growth phase, were inoculated into the 0.2% PYG medium at a rate of 10% and treated at 37°C for 2 hours. Plate counts were then performed, and the survival rate of TF06-26 cells in the 0.2% bile salt environment was calculated. The count results showed that TF06-26 cells could tolerate a 0.2% bile salt concentration, with a survival rate of 70%.

[0206] 4.2 Bile salt hydrolase activity of TF06-26

[0207] Bile saline hydrolase was detected using the TDCA method. TDAC plates were prepared by adding 4% TDAC (sodium taurodeoxycholate) and 0.37 g / L CaCl2 to PYG solid medium. TF06-26 was cultured to a concentration of approximately 10⁸ CFU / mL. 10 μL of bacterial suspension was dropped onto a 0.6 mm diameter filter paper disc, which was then placed on the TDAC plate surface and incubated at 37°C for 2 days. The white precipitate formed around the filter paper disc was observed; the diameter of the white precipitate represents the activity of bile saline hydrolase. Measurements showed that the white precipitate of TF06-26 was 9 mm, indicating that TF06-26 possessed bile saline hydrolase activity.

[0208] 4.3 In vitro cholesterol-lowering effect of TF06-26

[0209] The cholesterol content was determined using the o-phthalaldehyde colorimetric method (OPA method). The cholesterol degradation ability of the strain was examined by observing the changes in cholesterol content before and after cultivation in a medium containing a certain concentration of cholesterol for a period of time. The specific method is as follows:

[0210] (1) Preparation of cholesterol culture medium and culture of experimental strains

[0211] A certain mass 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 and mixed thoroughly. The test strain, TF06-26, was then inoculated into the medium at a 3% inoculum. Another commercial cholesterol-lowering probiotic, *Lactobacillus plantarum* Lp299v (purchased from Probi, Sweden), was also inoculated. These two strains were compared separately. Both strains were cultured anaerobically at 37°C for 72 hours.

[0212] (2) Construction of standard curve

[0213] 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 hm. 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.

[0214] (3) Determination of cholesterol in culture medium

[0215] 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. 1 ml of the sample was placed in a clean test tube, 6 ml of 95% ethanol and 4 ml of 50% KOH were added, and the mixture was shaken and mixed. The mixture was then subjected to saponification in a 60°C water bath for 10 min, followed by rapid cooling. 10 ml of n-hexane was added for extraction, and the mixture was thoroughly mixed. The mixture was allowed to stand at room temperature for 20 min. 8 ml of the organic phase (n-hexane layer) 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, and the mixture was allowed to stand at room temperature for 10 min. 2 ml of concentrated H₂SO₄ was added, and the reaction was allowed to continue for 10 min. Finally, the absorbance was measured at 550 hm.

[0216] (4) Calculation of cholesterol degradation rate

[0217] The cholesterol content in the culture medium before and after culture was calculated based on the standard curve. The cholesterol degradation rate was calculated using the following formula:

[0218] L = (AB) / A × 100%

[0219] L: Cholesterol degradation rate;

[0220] A: The cholesterol content in an uninoculated cholesterol culture medium;

[0221] B: Cholesterol content in the culture medium of the test strain after 48 hours of culture.

[0222] (5) Results of cholesterol degradation

[0223] Calculations showed that TF06-26 had a cholesterol degradation rate of 74.4%, while Lp299v had a degradation rate of 70%. This indicates that TF06-26 has a very significant cholesterol degradation capacity, and even a stronger cholesterol degradation capacity than Lp299v.

[0224] Cholesterol is the main component of blood lipids. Elevated cholesterol levels in plasma are closely related to the occurrence of atherosclerosis. Therefore, TF06-26 of the present invention can reduce blood lipids and reduce related indicators of atherosclerosis-related diseases (such as cardiovascular diseases).

[0225] Example 5: Intervention of *Collinsella shenzhenensis* TF06-26 in a mouse model of ulcerative colitis (UC).

[0226] The purpose of this embodiment is to investigate the therapeutic effect of Collinsella shenzhenensis TF06-26 on UC model mice, by comparing TF06-26, PBS, and positive control drug (VSL). # 3) (Purchased from Sigma Tau, USA) was used to intervene in UC model mice. The mice's weight, mortality rate, colon length, DAI index, and pathological changes in the intestinal mucosa were observed to evaluate the treatment effect of TF06-26 on UC.

[0227] 5.1 Experimental Materials

[0228] The experimental mice were C57bl / 6 mice (purchased from Hubei Medical Experimental Animal Center), 10 weeks old, weighing 28g±2g. The mice were housed in an SPF-grade environment and were acclimatized for 1 week before modeling.

[0229] 5.2 UC Modeling Method

[0230] The UC model was induced using 1.5% DSS (sodium dextran sulfate) (purchased from MPBiomedicals, USA) for 7 days.

[0231] 5.3 Experimental Grouping

[0232] A total of 56 experimental mice were randomly divided into four groups of 14 mice each, as follows:

[0233] Group 1: Control group (blank control group) – normal mice, without DSS induction.

[0234] Group 2: UC model group – DSS modeling was performed, and sterile PBS was administered via gavage.

[0235] Group 3: VSL # Treatment group 3 – DSS modeling and VSL # 3 Treatment (bacterial concentration 10) 9 cfu / ml)

[0236] Group 4: TF06-26 treatment group – DSS modeling was performed, and TF06-26 was used for treatment (bacterial concentration 10). 9 cfu / ml)

[0237] 5.4 Experimental Procedure

[0238] Mice were observed and recorded daily for their diet, water intake, activity, and other general conditions. They were also weighed, and the characteristics of their feces and the presence of fecal occult blood were observed. The DAI index was calculated (Table 4). The experimental intervention lasted for 7 days, with a daily gavage dose of 200 μL per mouse. After the experiment, mice were euthanized, and blood was collected from all mice. The mice were dislocated, their colons were harvested, photographed, weighed, and their length measured. The colon tissue was preserved at -80°C in paraformaldehyde.

[0239] Table 4 DAI Index Scoring Table

[0240]

[0241] The DAI index is equal to the sum of three scores: weight, stool characteristics, and fecal occult blood.

[0242] 5.5 Experimental Results

[0243] (1) Weight changes

[0244] The weight records of the mice during the experiment are shown in Table 5.

[0245] Table 5

[0246]

[0247] At the beginning of the modeling phase, the body weight levels of mice in each group were basically the same. However, with the induction of DSS, the body weight of the model group and the VSL group... # The body weight of mice in groups 3 and TF06-26 gradually decreased. On days 4 and 7, the body weight loss in the model group was significantly greater (compared to the control group *P<0.05). This indicates that DSS induction can lead to a significant decrease in mouse body weight; on day 7, the body weight of mice in the TF06-26 group was significantly higher than that in the model group. ▲ P < 0.05, indicating that TF06-26 can effectively control the weight loss in mice.

[0248] (2) Changes in the Disease Activity Index (DAI) of mice

[0249] The characteristics of the stool and the presence of fecal occult blood in mice were recorded on days 1, 4 and 7 after modeling. The DAI index was calculated, and the results are shown in Table 6.

[0250] Table 6

[0251]

[0252] As can be seen from the results in Table 6, after 7 days of treatment, VSL #Both treatment with TF06-26 and TF06-26 significantly reduced the disease activity index (DAI) in mice (compared to the model group *P<0.05). Furthermore, the DAI index in the TF06-26 group was lower than that of the positive control group (VSL) on day 7. # 3. This indicates that TF06-26 showed better disease improvement in UC mice than VSL. # 3.

[0253] (3) Changes in colon length in mice

[0254] In DSS-induced UC model mice, colonic ulceration typically occurs, leading to shortening of the colon. After the experiment, the colon length of each group of mice was measured by dissection. The results are shown in Table 7. Figure 4 As shown.

[0255] Table 7

[0256]

[0257] Table 7 and Figure 5 show that the colon length of mice in the TF06-26 group was significantly longer than that in the model group (*P < 0.05). Furthermore, the colon length of mice in the TF06-26 group was longer than that in the positive control group (VSL). # The longer duration of group 3 indicates that TF06-26 is more effective than VSL in improving the disease in UC mice. # 3. This shows that TF06-26 can significantly alleviate colonic lesions in mice.

[0258] Example 6: Food composition containing Collinsella shenzhenensis TF06-26

[0259] The raw material ratio is shown in Table 8.

[0260] Table 8

[0261]

[0262]

[0263] Mix milk and sugar according to the above formula ratio, stir until fully combined, preheat, homogenize under 20 MPa pressure, sterilize at about 90℃ for 5-10 minutes, cool to 40-43℃, mix in vitamin C as a protective agent, and inoculate with 1-100×10 6 Collinsella shenzhenensis TF06-26 cfu / g is used to prepare a food composition containing Collinsella shenzhenensis TF06-26.

[0264] Example 7: Pharmaceutical composition containing Collinsella shenzhenensis TF06-26

[0265] The raw material ratios are shown in Table 9.

[0266] Table 9

[0267] raw material Quality percentage (%) Collinsella shenzhenensis TF06-26 1.0％ lactose 2.0％ yeast powder 2.0％ peptone 1.0％ purified water 93.5％ Vitamin C 0.5％

[0268] Mix lactose, yeast powder, and peptone with purified water in the specified proportions until homogeneous. Preheat to 60-65℃, homogenize under 20 MPa pressure, sterilize at approximately 90℃ for 20-30 minutes, cool to 36-38℃, add vitamin C as a preservative, and inoculate with live Collinsella shenzhenensis TF06-26 bacteria (1-50 × 10⁻⁶). 6 The *Collinsella shenzhenensis* TF06-26 was prepared by fermentation at 36-38℃ until the pH reached 6.0, followed by centrifugation and freeze-drying until the moisture content was less than 3%. 0.5 g of the freeze-dried *Collinsella shenzhenensis* TF06-26 was weighed, mixed with an equal amount of maltodextrin, and then encapsulated to obtain a pharmaceutical composition containing *Collinsella shenzhenensis* TF06-26.

[0269] Example 8: Preparation of a drug containing Collinsella shenzhenensis TF06-26

[0270] 8.1 Preparation of bacterial culture: 1×10⁻⁶ Collinsella shenzhenensis TF06-26 9 The samples were anaerobic cultured at 37°C for 2-3 days using PYG medium (cfu / ml).

[0271] 8.2 Preparation of growth factors: Skim milk and casein are mixed, centrifuged, and ultrafiltered to obtain crude extract of milk growth factors (nutrients containing vitamins, purines, and / or pyrimidines).

[0272] 8.3 Drug formulation preparation: 5 volumes of growth factor and 1 volume of vitamin C protectant were added to 100 volumes of TF06-26 fermentation broth, stirred thoroughly, and then starch excipients (such as maltodextrin) were added to prepare a drug containing Collinsella shenzhenensis TF06-26.

[0273] strain preservation

[0274] The strain of this invention, Collinsella shenzhenensis TF06-26 (same as the deposit name), was deposited on October 13, 2016, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC NO.: 60090.

[0275] All references to this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims. sequence list <110> Shenzhen BGI Research Institute <120> Collinsella shenzhenensis and its applications <130> P2019-0499 <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1380 <212> DNA <213> Collinsella shenzhenensis TF06-26 <400> 1 cggcacccct ctccggaggg aagcgagtgg cgaacggctg agtaacacgt ggagaacctg 60 ccccctcccc cgggatagcc gcccgaaagg acgggtaata ccggataccc cggggtgccg 120 catggcaccc cggctaaagc cccgacggga ggggatggct ccgcggccca tcaggtagac 180 ggcggggtga cggcccaccg tgccgacaac gggtagccgg gttgagagac cgaccggcca 240 gattgggact gagacacggc ccagactcct acgggaggca gcagtgggga atcttgcgca 300 atggggggaa ccctgacgca gcgacgccgc gtgcgggacg gaggccttcg ggtcgtaaac 360 cgctttcagc agggaagagt caagactgta cctgcagaag aagccccggc taactacgtg 420 ccagcagccg cggtaatacg tagggggcga gcgttatccg gattcattgg gcgtaaagcg 480 cgcgtaggcg gcccggcagg ccgggggtcg aagcgggggg ctcaaccccc cgaagccccc 540 ggaacctccg cggcttgggt ccggtagggg agggtggaac acccggtgta gcggtggaat 600 gcgcagatat cgggtggaac accggtggcg aaggcggccc tctgggccga gaccgacgct 660 gaggcgcgaa agctggggga gcgaacagga ttagataccc tggtagtccc agccgtaaac 720 gatggacgct aggtgtgggg ggacgatccc cccgtgccgc agccaacgca ttaagcgtcc 780 cgcctgggga gtacggccgc aaggctaaaa ctcaaaggaa ttgacggggg cccgcacaag 840 cagcggagca tgtggcttaa ttcgaagcaa cgcgaagaac cttaccaggg cttgacatat 900 gggtgaagcg ggggagaccc cgtggccgag aggagcccat acaggtggtg catggctgtc 960 gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc cccgccgcgt 1020 gttgccatcg ggtgatgccg ggaacccacg cgggaccgcc gccgtcaagg cggaggaggg 1080 cggggacgac gtcaagtcat catgcccctt atgccctggg ctgcacacgt gctacaatgg 1140 ccggtacaga gggatgccac cccgcgaggg ggagcggatc ccggaaagcc ggccccagtt 1200 cggattgggg gctgcaaccc gcccccatga agtcggagtt gctagtaatc gcggatcagc 1260 atgccgcggt gaatgcgttc ccgggccttg tacacaccgc ccgtcacacc acccgagtcg 1320 tctgcacccg aagtcgccgg cccaaccgag aggggggagg cgccgaaggt gtggagggtg 1380

Claims

1. A type of Collins bacterium, characterized in that, The Collins bacteria are Collinsellashenzhenensis TF06-26, collection number GDMCC NO: 60090.

2. A composition, characterized in that, The composition comprises: (a) a safe and effective amount of Collins bacteria as described in claim 1; and (b) a pharmaceutically acceptable carrier.

3. The composition according to claim 2, characterized in that, The composition also includes growth factors.

4. The composition according to claim 2, characterized in that, The composition also contains other probiotics and / or prebiotics.

5. The composition according to claim 2, characterized in that, The composition also contains substances that help maintain the viability of Collins bacteria.

6. Use of the Collins bacterium of claim 1 or the composition of claim 2, characterized in that, Used to prepare a drug or formulation for one or more uses selected from the group consisting of: (a) prevention and / or treatment of inflammation-related diseases; and / or (b) prevention and / or treatment of cardiovascular diseases.

7. The use as described in claim 6, characterized in that, The inflammation-related diseases are selected from the following group: inflammatory bowel disease, rheumatoid arthritis, or a combination thereof.

8. The use as described in claim 6, characterized in that, The cardiovascular diseases mentioned are selected from the following group: hypertension, hyperlipidemia, coronary heart disease, or a combination thereof.

9. Use of the Collins bacterium of claim 1 or the composition of claim 2, characterized in that, Used for the preparation of a drug or formulation, said drug or formulation for one or more uses selected from the group consisting of: (i) Lowering blood lipid levels in mammals; (ii) Controlling the decrease in body weight in mammals; (iii) Reduce the Disease Activity Index (DAI) in mammals; (iv) Improves intestinal lesions in mammals.

10. A method for preparing the composition of claim 2, characterized in that, Including the following steps: The Collins bacterium of claim 1 is mixed with a pharmaceutically acceptable carrier to form the composition of claim 2.

11. The manufacturing method as described in claim 10, characterized in that, The preparation method also includes a step of mixing with growth factors.

12. The manufacturing method as described in claim 10, characterized in that, The preparation method also includes the step of mixing with a substance that helps maintain the viability of Collins bacteria.

13. The manufacturing method as described in claim 12, characterized in that, The substance that helps maintain the viability of Collins bacteria is a protective agent.

14. The manufacturing method as described in claim 10, characterized in that, The preparation method also includes the step of mixing with probiotics and / or prebiotics.

15. A production method, characterized in that, Including the following steps: (a) The Collins bacterium of claim 1 is cultured under suitable culture conditions to obtain a culture product.

16. The production method as described in claim 15, characterized in that, Further steps include: (b) Isolating Collins bacteria from the culture product; and / or (c) The culture product or Collins cells obtained in the previous step are mixed with a pharmaceutically acceptable carrier to prepare a composition.

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