WO2019205505A1 - 一种缓解内毒素感染的脆弱拟杆菌及其应用 - Google Patents

一种缓解内毒素感染的脆弱拟杆菌及其应用 Download PDF

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WO2019205505A1
WO2019205505A1 PCT/CN2018/110059 CN2018110059W WO2019205505A1 WO 2019205505 A1 WO2019205505 A1 WO 2019205505A1 CN 2018110059 W CN2018110059 W CN 2018110059W WO 2019205505 A1 WO2019205505 A1 WO 2019205505A1
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bacteroides fragilis
endotoxin
pharmaceutical composition
ccfm1020
tablet
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PCT/CN2018/110059
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English (en)
French (fr)
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翟齐啸
陈卫
谭惠子
陆文伟
田丰伟
赵建新
张灏
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江南大学
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    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
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    • A61K35/74—Bacteria
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    • A—HUMAN NECESSITIES
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    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K9/00—Medicinal preparations characterised by special physical form
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    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
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    • A61K9/2004—Excipients; Inactive ingredients
    • A61K9/2013—Organic compounds, e.g. phospholipids, fats
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    • A61K9/2004—Excipients; Inactive ingredients
    • A61K9/2022—Organic macromolecular compounds
    • A61K9/205—Polysaccharides, e.g. alginate, gums; Cyclodextrin
    • A61K9/2054—Cellulose; Cellulose derivatives, e.g. hydroxypropyl methylcellulose
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    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/2004—Excipients; Inactive ingredients
    • A61K9/2022—Organic macromolecular compounds
    • A61K9/205—Polysaccharides, e.g. alginate, gums; Cyclodextrin
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    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/04—Preserving or maintaining viable microorganisms
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

  • the invention relates to a fragile Bacteroides for relieving endotoxin infection and application thereof, and belongs to the technical field of microorganisms.
  • Endotoxin also known as lipopolysaccharide
  • the endotoxin structure is stable, mainly composed of polysaccharide O antigen, core polysaccharide and lipid A (lipid A).
  • Lipid A is a lipidated aglucosamine, which has a pyrogenic effect and is a toxic component of Gram-negative bacterial endotoxin.
  • endotoxin is released only when the bacteria die or dissolve the bacterial cells by artificial means, and the endotoxin structure is stable, only long time high temperature treatment or high temperature treatment with strong alkali, strong acid or strong oxidant can destroy it.
  • Biological activity is mainly composed of polysaccharide O antigen, core polysaccharide and lipid A (lipid A).
  • Normal intestinal lumen contains a large number of intestinal bacteria, which can selectively absorb nutrients and prevent the entry of microorganisms and their harmful metabolites.
  • the intestinal flora is disordered, releasing endotoxin, and excessive endotoxin can increase intestinal permeability, leading to a series of intestinal disorders caused by immune disorders.
  • the body's macrophages, neutrophils, endothelial cells, platelets, as well as the complement system and coagulation system causing microcirculatory disorders leading to endotoxin shock; more likely to aggravate the possibility of depression .
  • Studies have reported that at least 750,000 patients develop endotoxin-infected shock each year, of which more than 210,000 are killed.
  • Patent CN102406869A discloses an application method for reducing endotoxin levels by using traditional Chinese medicines such as rhubarb and lycium; it has been found that polymyxin can specifically bind to the active site lipid A in endotoxin, thereby neutralizing or inactivating its toxicity.
  • patent CN102470152A discloses the following uses of bacteria of six lactic acid bacteria, bifidobacteria or any mixture thereof: treatment of metabolism in mammals Endotoxemia, inhibition of bacterial displacement and regulation of lipid absorption. It has also been reported that bifidobacteria can treat neonatal necrotizing enterocolitis caused by endotoxin (Xiang Ling, et al.
  • Bacteroides is the highest content of Gram-negative bacteria in human intestinal symbiotic bacteria. It has a large number of carbohydrate degradation genes, strong environmental adaptability, and is closely related to host immune, metabolic and nervous systems. At present, it has been suggested that Bacteroides has potential physiological regulation function and may become a "new generation of probiotics".
  • the prior art CN105434476A discloses that the Bacteroides fragilis ZY-312 in the Bacteroides group can alleviate the ulcerative inflammation caused by the chemical damage caused by DSS to the intestine; the patents CN103120705A and CN103082294A respectively disclose the prevention of constipation and diarrhea.
  • Patent 103156888A discloses that a strain of Bacteroides fragilis can alleviate the inflammatory response caused by the hapten substance TNBS in intestinal tissues damaged by ethanol; it has also been found that Bacteroides fragilis ATCC25285 can alleviate the pathogenic bacteria Helicobacter pylori Colitis caused by colonization (Sarkis Mazmanian, et al. "A Microbial symbiosis factor ensur intestinal inflammatory disease”. "Nature”, 2008, 453).
  • the present invention summarizes the basic attempts of the prior art, and through a large number of experimental studies, screened a Bacteroides fragilis with endotoxin-relieving infection, and proved that it can regulate the intestinal immune pathway due to endotoxin and relieve inflammation in animal models. To relieve endotoxin infection. Its use in pharmaceutical compositions was further examined.
  • a first object of the present invention is to provide a Bacteroides fragilis, named Bacteroides fragilis CCFM1020, which is identified as Bacteroides fragilis CCFM1020 according to its gene sequence. It was deposited with the Guangdong Provincial Microbial Culture Collection on March 29, 2018. The deposit address is 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou, with the deposit number GDMCC No: 60342.
  • the Bacteroides fragilis CCFM1020 was submitted to the Guangdong Provincial Microbial Culture Collection and before the original number HCK-B3, so the numbers CCFM1020 and HCK-B3 were the same strain of Bacteroides.
  • the Bacteroides fragilis CCFM1020 has the following properties:
  • composition of the intestinal flora of mice infected with endotoxin can be stabilized.
  • Bacterial characteristics Gram-negative rod-shaped bacteria, the cells are arranged in a single or double arrangement, about 0.9-1.2 ⁇ m wide, 3-8 ⁇ m long, without spore formation.
  • Colony characteristics obvious colonies formed on the medium, the diameter is between 1-3mm, the front shape is round, the side shape is convex, the edges are neat, transparent or translucent, and the surface is moist and smooth.
  • the strain is a strict anaerobic bacterium, sensitive to oxygen, and grows optimally at a temperature of 30-37 ° C.
  • the highest and lowest initial growth pH is 8.0 and 5.0, and the optimum initial growth pH is 7.0.
  • a second object of the present invention is to provide the use of the Bacteroides fragilis CCFM1020 for the preparation of a pharmaceutical composition for alleviating endotoxin infection.
  • the pharmaceutical composition consists of a Bacteroides fragilis CCFM1020 agent and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier comprises one or more fillers, binders, wetting agents, disintegrants, lubricants, and flavors selected from the group consisting of pharmaceutically acceptable fillers, binders, wetting agents, disintegrating agents, lubricants, and flavoring agents. Carrier of the agent.
  • the pharmaceutical composition is in the form of a granule, capsule, tablet, pill or oral solution.
  • the Bacteroides fragilis CCFM 1020 weighs 15-35%, or 20-30% by weight of the pharmaceutical composition in the pharmaceutical composition.
  • the filler is understood to be an excipient diluent for increasing the weight and volume of the tablet to facilitate tableting, or an excipient absorbent for absorbing excess liquid components of the material.
  • the filler is selected from the group consisting of starch, sucrose, lactose, calcium sulfate or microcrystalline cellulose.
  • the binder is understood to mean that the bulk drug itself is not tacky or tacky and that a viscous material is added to facilitate granulation.
  • the binder is selected from the group consisting of cellulose derivatives, alginates, gelatin or polyvinylpyrrolidone.
  • the wetting agent is understood to mean a liquid in which the starting drug itself is non-tacky, but which can wet its pharmaceutically active excipients and induce its viscosity to form granules.
  • the wetting agent is selected from the group consisting of water, ethanol, starch or syrup.
  • the disintegrant is understood to be an excipient that can be added to a tablet to promote rapid disintegration of the tablet into fine particles in the gastrointestinal fluid.
  • the disintegrant is selected from the group consisting of sodium carboxymethyl starch, carboxypropyl cellulose, croscarmellose, agar, calcium carbonate or sodium hydrogencarbonate.
  • the lubricant is understood to be a chemical that facilitates the mobility of the tablet during granulation and facilitates demolding of the tablet.
  • the lubricant is selected from the group consisting of talc, calcium stearate, magnesium stearate, silica gel or polyethylene glycol.
  • the flavoring agent is to be understood as a pharmaceutical excipient used in a pharmaceutical product to improve or mask the undesirable odor and taste of the drug.
  • the flavoring agent is selected from sweeteners such as monosaccharide, sucrose, lecithin, orange syrup or cherry syrup; aromatics of lemon, fennel or peppermint; sodium alginate, gum arabic, gelatin, methylcellulose Or a sizing agent of sodium carboxymethyl cellulose; an effervescent agent of a mixture of citric acid, tartaric acid or sodium hydrogencarbonate.
  • the pharmaceutical composition is in the form of a granule, a capsule, a tablet, a pill or an oral solution, and the single dosage form contains a predetermined active substance to achieve a desired amount, for example, the present invention Bacteroides fragilis CCFM1020 bacterial agent.
  • a third object of the present invention is to provide the Bacteroides fragilis CCFM1020 microbial agent, which is a powder prepared by preparing a bacterial liquid containing the Bacteroides fragilis CCFM1020 by a conventional freeze-drying process or other processes, and contains the powder. 10 6 CFU/g or more active Bacteroides fragilis CCFM1020 cells.
  • the Bacteroides fragilis CCFM1020 microbial agent contains at least 10 6 CFU/g of active Bacteroides fragilis CCFM1020.
  • a fourth object of the present invention is to provide a method for culturing the Bacteroides fragilis CCFM1020 by inoculating the Bacteroides fragilis CCFM1020 into a medium and anaerobic culture at 35 to 37 °C.
  • the 12-18 h strain is cultured for a stationary phase.
  • the culture is carried out using BHI medium.
  • cysteine hydrochloride 1 g/L, hemin-11 g/L, and vitamin K 10.002 g/L are further added to the BHI medium.
  • a fifth object of the present invention is to provide a Bacteroides fragilis cryopreservation containing Bifidobacterium fragilis CCFM1020 of ⁇ 10 10 CFU/ml.
  • the cryopreservation agent is a bacterium solution in which the Bacteroides fragilis CCFM1020 is in a stable phase is washed with a phosphate buffer solution having a pH of 7.0 to 7.2 for 1-2 times, and then a protective agent is added thereto. Store at -80 ° C for use.
  • the protectant contains 1 g/L cysteine hydrochloride, 200 g/L glycerol.
  • a sixth object of the present invention is to provide a composition for non-pharmaceutical use containing the Bacteroides fragilis CCFM1020.
  • the invention also claims the use of the Bacteroides fragilis CCFM1020 as a fermenting microorganism in the production of products in the food and biological fields.
  • the Bacteroides fragilis CCFM1020 of the present invention has low immunogenicity, and can significantly restore the amount of inflammatory factors in the blood of mice and the number of Foxp3 + regulatory T cells in the spleen due to endotoxin infection to normal mice. Level and ability to stabilize the composition of the intestinal flora of endotoxin-infected mice.
  • the Bacteroides fragilis CCFM1020 can be used for preparing a pharmaceutical composition capable of alleviating endotoxin infection, and has broad application prospects.
  • Figure 1 shows the concentration of immune factors in blood and colon tissues of mice after oral administration of Bacteroides fragilis CCFM1020;
  • Figure 2 shows the regulatory effect of Bacteroides fragilis CCFM1020 on the levels of immune factors in blood of endotoxin-infected mice. * indicates that there is a significant difference between B. fragilis CCFM1020 and endotoxin-producing modules, * indicates p ⁇ 0.05, ** indicates p ⁇ 0.01;
  • Figure 3 shows the regulatory effect of Bacteroides fragilis CCFM1020 on the regulatory T cells in the spleen of endotoxin-infected mice. * indicates that there is a significant difference between the CCFM1020 group and the endotoxin-producing module, ** indicates p ⁇ 0.01;# Indicates that there is a significant difference between the endotoxin infection group and the control group, ## indicates p ⁇ 0.01;
  • Figure 4 shows the intestinal flora of the endotoxin-infected group; among them, D1LPSCTRL: the intestinal flora of the toxin-infected group in the first day of the experiment; D3LPSCTRL: the intestinal flora of the toxin-infected group in the third day of the experiment; D5LPSCTRL : Intestinal flora of mice in the toxin-infected group during the fifth day of the experiment;
  • Figure 5 shows the regulatory effect of Bacteroides fragilis CCFM1020 on the composition of intestinal flora in endotoxin-infected mice.
  • D1LPSBF the first day of the experiment
  • B. fragilis CCFM1020 interfered with the intestinal flora of endotoxin-infected mice
  • D3LPSBF the third day of the experiment
  • B. fragilis CCFM1020 interfered with the intestinal flora of endotoxin-infected mice
  • D5LPSBF experimental Five days of Bacteroides fragilis CCFM1020 interfered with the intestinal flora of endotoxin-infected mice.
  • Figure 6 is the relative abundance of Allobaculum in the intestinal flora of endotoxin-infected mice and the intestinal flora of endotoxin-infected mice.
  • Figure 7 is a relative abundance of Clostridium in the intestinal flora of endotoxin-infected mice and B. fragilis CCFM1020 in the intestinal flora of endotoxin-infected mice;
  • Figure 8 shows the relative abundance of Lactobacillus in the intestinal flora of endotoxin-infected mice and the intestinal flora of endotoxin-infected mice.
  • Bacteroides fragilis classified as Bacteroides fragilis, was deposited with the Guangdong Provincial Collection of Microorganisms and Cultures on March 29, 2018. The deposit address is 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou, China. For GDMCC No: 60342.
  • Example 1 Cultivation and preservation of Bacteroides fragilis CCFM1020
  • Preparation of medium Prepare brain heart infusion BHI medium (such as the product of Qingdao Haibo Biotechnology Co., Ltd.), dissolve in distilled water, and add cysteine hydrochloride 1g/L, hemin. 0.01 g / L, vitamin K1 0.002g / L, mixed evenly, and then adjusted its pH to 7.0, sterilized at 115-121 ° C for 15-20min, the medium was obtained.
  • BHI medium such as the product of Qingdao Haibo Biotechnology Co., Ltd.
  • Culture method The strain of Bacteroides fragilis CCFM1020 is inoculated according to the inoculation amount of 2-4% by weight of the above medium, and anaerobic culture is carried out at 37 ° C for 12-18 hours, which can reach a stable period.
  • protective agent 1 g/L of cysteine hydrochloride and 200 g/L of glycerin were weighed and uniformly dissolved in distilled water, and sterilized at 115-121 ° C for 15-20 min to obtain the protective agent.
  • Example 2 Tolerance dose experiment of Bacteroides fragilis CCFM1020 intragastrically administered mice
  • Example 3 Immunity tolerance test of Bacteroides fragilis CCFM1020 intragastrically administered mice
  • mice Twenty healthy female C57BL6 mice aged 6-8 weeks were randomly divided into two groups: negative control group and Bacteroides fragilis CCFM1020 intervention group, each group containing 10 mice.
  • the mice in the CCFM1020 intervention group were intragastrically administered with 0.1 ml of the above-mentioned B.
  • fragilis CCFM1020 cryopreservation agent (10 10 CFU/ml) every 24 hours, and the control mice were intragastrically administered once every 24 hours for 0.1 ml of the above cryopreservative.
  • the protective agent was administered to all mice after 5 days of continuous gavage, and the blood and colon tissues were separately lysed by RIPA lysate (product of Biyuntian Biotechnology Co., Ltd.), and then ELISA kit (Nanjing Senbega Biotechnology Co., Ltd.) was used. Company product) Determination of the concentration of immune factors.
  • Example 4 Modulation of immune factor levels in blood of endotoxin-infected mice by Bacteroides fragilis CCFM1020
  • mice Thirty healthy female C57BL6 mice aged 6-8 weeks were randomly divided into three groups: negative control group, endotoxin infection model group and Bacteroides fragilis CCFM1020 treatment group, each group containing 10 mice.
  • the endotoxin infection group and the Bacteroides fragilis CCFM1020 treatment group were intraperitoneally injected with 0.1 ml of endotoxin solution diluted with physiological saline (0.1 mg/kg) every 24 hours, and the control group and endotoxin-infected group were per hour.
  • mice of the Bacteroides fragilis CCFM1020 treatment group were intragastrically administered once every 24 hours, and 0.1 ml of the fragile Bacteroides fragilis CCFM1020 prepared in Example 1 (10 10 CFU) /ml), all mice were sacrificed after 5 consecutive days, and blood was taken for determination of the concentration of immune factors using an ELISA kit (Nanjing Senbega Biotechnology Co., Ltd.).
  • Example 5 Modulation of regulatory T cell content in spleen of endotoxin-infected mice by Bacteroides fragilis CCFM1020
  • mice Thirty healthy female C57BL6 mice aged 6-8 weeks were randomly divided into three groups: negative control group, endotoxin infection model group and Bacteroides fragilis CCFM1020 treatment group, each group containing 10 mice.
  • the endotoxin infection group and the Bacteroides fragilis CCFM1020 treatment group were intraperitoneally injected with 0.1 ml of endotoxin solution diluted with physiological saline (0.1 mg/kg) every 24 hours, and the control group and endotoxin-infected group were per hour.
  • mice of the Bacteroides fragilis CCFM1020 treatment group were intragastrically administered once every 24 hours to the above-mentioned fragile Bacteroides CCFM1020 cryopreservative (10 10 CFU/ml), and all the rats were sacrificed after 5 days.
  • the spleen tissue was lysed by erythrocyte lysate, and then labeled with CD4, CD25 and surface molecule Foxp3 using a mouse regulatory T cell staining kit (product of eBioscience), and then flow cytometry (FACSCalibur, BD) ) detecting changes in the content of regulatory T cells.
  • Example 6 Modulation of intestinal flora of endotoxin-infected mice by Bacteroides fragilis CCFM1020
  • mice Thirty healthy female C57BL6 mice aged 6-8 weeks were randomly divided into three groups: negative control group, endotoxin infection model group and Bacteroides fragilis CCFM1020 treatment group, each group containing 10 mice.
  • the endotoxin infection group and the Bacteroides fragilis CCFM1020 treatment group were intraperitoneally injected with 0.1 ml of endotoxin solution diluted with physiological saline (0.1 mg/kg) every 24 hours, and the control group and endotoxin-infected group were per hour.
  • mice of the Bacteroides fragilis CCFM1020 treatment group were intragastrically administered once every 24 hours to the above-mentioned fragile Bacteroides CCFM1020 cryopreservative (10 10 CFU/ml), and all the rats were sacrificed after 5 days.
  • Mouse. During the experiment, mouse feces were taken before, third, and fifth days of the first day of administration, and the fecal bacterial meteogenome was extracted using the Fast DNA Spin Kit for Feces kit (MP Biomedicals). After PCR amplification of the 16s V3-V4 region sequence, the composition of the intestinal flora in the fecal sample was passed through the second generation sequencer.
  • Fig. 4 to Fig. 8 The contents of Bacteroides and Absidia in the intestinal tract of endotoxin-infected mice were 50% and 40%, respectively, of which Bacteroides S24-7 accounted for 42.7 and Lactobacillus 12%. , Clostridium 8.2%, the content of Allobaculum associated with colitis increased significantly to 10%, resulting in a decrease in the diversity of the intestinal flora.
  • Bacteroides fragilis CCFM1020 After treatment with Bacteroides fragilis CCFM1020, the content of Bacteroides and thick-walled bacteria in the fecal flora of mice was adjusted to 60% and 30%, and the variation range of Bacteroides, Clostridium and Allobaculum was greatly reduced.
  • Example 6 indicate that the Bacteroides fragilis CCFM1020 of the present invention can attenuate the changes of the LPS to the intestinal flora, increase the diversity of the intestinal flora, stabilize the balance of the intestinal flora, and thereby prevent the endotoxin.
  • Bacteroides fragilis CCFM1020 of the present invention can help alleviate the intestinal flora composition and immune response disorder caused by endotoxin infection in mice.
  • Example 7 Preparation of a capsule product containing the Bacteroides fragilis CCFM1020 of the present invention
  • the Bacteroides fragilis CCFM1020 of the present invention was anaerobic cultured in BHI medium at 37 ° C for 24 hours, centrifuged at 5000 ° C for 15 min at 4 ° C, and washed 1-2 times with sterile phosphate buffer (pH 7.2). The cells were resuspended using the above protective agent to a final concentration of 10 10 CFU/ml. The bacterial suspension is added to the sodium alginate solution, and the mixture is thoroughly stirred to uniformly disperse the cells in the sodium alginate solution, and then the mixture is extruded into a calcium chloride solution to form a colloidal particle, which is statically solidified for 30 minutes, and the colloidal particles are collected by filtration. The collected micelles were freeze-dried for 48 hours to obtain a powder containing the Bacteroides fragilis CCFM1020 of the present invention, and the powder was placed in a commercially available pharmaceutical capsule to obtain the capsule product.
  • Example 8 Preparation of a tablet using the Bacteroides fragilis CCFM1020 of the present invention

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Abstract

提供了一种缓解内毒素感染的脆弱拟杆菌CCFM1020及其应用。所述脆弱拟杆菌具有低免疫原性，能够降低内毒素感染宿主血液中促炎因子的含量，增加抑炎因子的浓度，上调Foxp3+调节性T细胞的数量，及稳定肠道菌群组成。还提供了脆弱拟杆菌CCFM1020用于缓解内毒素感染的药物组合物。

Description

一种缓解内毒素感染的脆弱拟杆菌及其应用 技术领域
本发明涉及一种缓解内毒素感染的脆弱拟杆菌及其应用，属于微生物技术领域。
背景技术
内毒素，又称脂多糖，是革兰氏阴性菌细胞壁的重要组成组分。内毒素结构稳定，主要由多糖O抗原、核心多糖和类脂A(lipid A)三部分组成。类脂A是脂化的葡萄胺二糖，具有致热作用，是革兰氏阴性细菌内毒素的毒性成分。一般情况下，内毒素只有当细菌死亡溶解或用人工方法破坏菌细胞后才释放出来，而内毒素结构稳定，只有长时间的高温处理或以强碱、强酸或强氧化剂辅助高温处理才能破坏其的生物活性。
正常机体肠腔内含有大量的肠道细菌，肠粘膜能够选择性地吸收营养成分，并阻止微生物及其有害的代谢物进入。然而，当机体处在应激状态下或过量使用抗生素时，肠道菌群紊乱，释放出内毒素，过量的内毒素能够增加肠道通透性，导致一系列免疫失调引起的肠道疾病，通过屏障进入血液循环，作用于机体的巨噬细胞、中性粒细胞、内皮细胞、血小板，以及补体系统和凝血系统等，引起微循环紊乱而导致内毒素休克；更有加重抑郁症的可能性。已有研究报道，每年至少有75万例患者发生内毒素感染休克，其中超过21万人因此丧命。
目前，针对内毒素感染的治疗原则多主张减少内毒素的产生和吸收及改善内毒素引起的微循环障碍。专利CN102406869A公开了一种采用大黄、芍丹等中药降低内毒素水平的应用方法；有研究发现，多粘菌素能特异性结合内毒素中活性部位类脂A，从而中和或者灭活其毒性(吕根法、卫国等。“多粘菌素B拮抗内毒素的体外作用研究”，《第三军医大学学报》，2004，26，14:1252-1254)；CN106334540A、CN1864755等专利发明了用于血液灌流的内毒素吸附剂及其应用，用于清除机体内内毒素。然而，传统的中医药治疗对清除内毒素具有一定的作用，但疗效并不显著；抗生素的应用并不能有效地清除内毒素，反而具有一定的负面影响；血液净化法对内毒素有着良好的清除作用，但费用较高，且临床数据不足。
因此，通过调节肠道菌群抑制肠源性内毒素引起的炎症，对治疗或预防内毒素感染有广阔的应用前景。目前，已有一些专利或专利申请文献涉及通过肠道菌治疗内毒素感染，如专利CN102470152A公开了6种乳酸菌、双歧杆菌或它们的任何混合物的细菌的以下用途：在哺乳动物中治疗代谢性内毒素血症、抑制细菌位移和调节脂质吸收。也有研究报道，双歧杆菌能够治疗内毒素引起的新生儿坏死性小肠结肠炎(Xiang Ling,et al.“Protective Effects of Bifidobacterium on Intestinal Barrier Function in LPS-Induced Enterocyte Barrier Injury of Caco-2  Monolayers and in a Rat Nec Model”,《PLoS One》,2016,11(8):e0161635)。
拟杆菌是人体肠道共生菌中含量最高的一类革兰氏阴性菌，具有大量碳水化合物降解基因，环境适应力强，并且与宿主的免疫、代谢及神经系统均存在紧密联系。目前已有观点认为拟杆菌具备潜在的生理调节功能，可能成为“新一代益生菌”。例如，已有专利CN105434476A公开了拟杆菌群中的脆弱拟杆菌ZY-312能够缓解DSS对肠道造成的化学性损伤而引起的溃疡性炎症；专利CN103120705A及CN103082294A分别公开了具有的预防便秘及腹泻功能的脆弱拟杆菌；专利103156888A公开了一株脆弱拟杆菌能够缓解半抗原物质TNBS在被乙醇破坏的肠组织中引起的炎症反应；也有研究发现脆弱拟杆菌ATCC25285能够缓解由致病菌肝螺杆菌定植而引起的结肠炎(Sarkis Mazmanian,et al.“A Microbial symbiosis factor prevents intestinal inflammatory disease”.《Nature》,2008,453)。但目前并没有任何专利文献报道脆弱拟杆菌对内毒素感染的缓解作用，尤其未关注该类菌株改变肠道通透性等的作用途径。
本发明总结现有技术的基础尝试，通过大量实验研究，筛选出了一种具有缓解内毒素感染的脆弱拟杆菌，并证明其在动物模型中可调节因内毒素激活肠道免疫通路，缓解炎症，进而缓解内毒素感染。进一步考察了其在药物组合物中的应用。
发明内容
本发明的第一个目的是提供一种脆弱拟杆菌(Bacteroides fragilis)，命名为脆弱拟杆菌(Bacteroides fragilis)CCFM1020，依据其基因序列对该拟杆菌鉴定为脆弱拟杆菌(Bacteroides fragilis)CCFM1020，已于2018年3月29日保藏于广东省微生物菌种保藏中心，保藏地址为广州市先烈中路100号大院59号楼5楼，保藏编号为GDMCC No:60342。
所述脆弱拟杆菌CCFM1020在提交广东省微生物菌种保藏中心及之前使用原始编号HCK-B3，故编号CCFM1020及HCK-B3为同一株脆弱拟杆菌。
所述的脆弱拟杆菌CCFM1020具有下列性质：
1)具有低免疫原性，对小鼠血液及结肠组织中的免疫因子无显著性影响；
2)能够降低内毒素感染小鼠血液中促炎因子的含量，增加抑炎因子的浓度；
3)能够上调内毒素感染小鼠脾脏中Foxp3 +调节性T细胞的数量；
4)能够稳定内毒素感染小鼠的肠道菌群组成。
菌体特征：革兰氏染色阴性杆状细菌，菌体单个或成双排列，约0.9-1.2μm宽，3-8μm长，无芽孢形成。
菌落特征：在培养基上形成明显地菌落，直径在1-3mm之间，正面形态圆形，侧面形态呈凸起状，边缘整齐，透明或半透明，表面湿润光滑。
生长特性：该菌株为严格厌氧菌，对氧气敏感，在温度30-37℃生长最佳，最高和最低初始生长pH为8.0和5.0，最适初始生长pH为7.0。
本发明的第二个目的是提供所述脆弱拟杆菌CCFM1020在制备用于缓解内毒素感染的药物组合物方面的应用。
在本发明的一种实施方式中，所述的药物组合物是由脆弱拟杆菌CCFM1020菌剂与在药学上可接受的载体组成。
在本发明的一种实施方式中，在药学上可接受的载体包括一种或多种选自药学上通常使用的填充剂、粘合剂、润湿剂、崩解剂、润滑剂及矫味剂的载体。
在本发明的一种实施方式中，所述的药物组合物是颗粒剂、胶囊剂、片剂、丸剂或口服液剂型。
在本发明的一种实施方式中，所述脆弱拟杆菌CCFM1020在药物组合物中重量是所述药物组合物重量的15-35％，或20-30％。
在本发明的一种实施方式中，所述的填充剂应该理解为用于增加片剂重量和体积而便于压片的辅料稀释剂，或以吸收原料中多余液体成分的辅料吸收剂。所述填充剂选自淀粉、蔗糖、乳糖、硫酸钙或微晶纤维素。
在本发明的一种实施方式中，所述粘合剂应该理解为原料药物本身无粘性或粘性不足，需加入粘性物质以便于制粒。所述粘合剂选自纤维素衍生物、藻酸盐、明胶或聚乙烯吡咯烷酮。
在本发明的一种实施方式中，所述润湿剂应该理解为原料药物本身无粘性，但可润湿其药物原辅料并诱发其粘性而制成颗粒的液体。所述润湿剂选自水、乙醇、淀粉或糖浆。
在本发明的一种实施方式中，所述崩解剂应该理解为一种能够加入片剂中而促进其片剂在胃肠液中快速崩解成细小粒子的辅料。所述崩解剂选自羧甲基淀粉钠、羧丙纤维素、交联羧甲基纤维素、琼脂、碳酸钙或碳酸氢钠。
在本发明的一种实施方式中，所述润滑剂应该理解为一种有利于提高片剂在制粒过程中的流动性，有利于片剂脱模的化学物质。所述润滑剂选自滑石粉、硬脂酸钙、硬脂酸镁、微粉硅胶或聚乙二醇。
在本发明的一种实施方式中，所述矫味剂应该理解为在药品中用以改善或屏蔽药物不良气味和味道的药用辅料。所述的矫味剂选自如单糖浆、蔗糖、卵磷脂、橙皮糖浆或樱桃糖浆的甜味剂；柠檬、茴香或薄荷油的芳香剂；海藻酸钠、阿拉伯胶、明胶、甲基纤维素或羧甲基纤维素钠的胶浆剂；柠檬酸、酒石酸或碳酸氢钠混合物的泡腾剂。
在本发明的一种实施方式中，所述的药物组合物是颗粒剂、胶囊剂、片剂、丸剂或口服液剂型，单个剂型含有为达到所需药量的预定活性物质，例如本发明的脆弱拟杆菌CCFM1020菌剂。
本发明第三个目的是提供所述脆弱拟杆菌CCFM1020菌剂，所述菌剂是将含有所述脆弱拟杆菌CCFM1020的菌液通过常规冷冻干燥工艺制备或其他工艺制备所得到的粉剂，它含有10 6CFU/g以上的活性脆弱拟杆菌CCFM1020细胞。
在本发明的一种实施方式中，所述的脆弱拟杆菌CCFM1020菌剂含有至少10 6CFU/g的活性脆弱拟杆菌CCFM1020。
本发明的第四个目的是提供所述脆弱拟杆菌(Bacteroides fragilis)CCFM1020的培养方法，是将所述脆弱拟杆菌(Bacteroides fragilis)CCFM1020接种至培养基中，于35～37℃厌氧培养。
在本发明的一种实施方式中，培养12-18h菌株达稳定期。
在本发明的一种实施方式中，应用BHI培养基进行培养。
在本发明的一种实施方式中，所述BHI培养基中还加入半胱氨酸盐酸盐1g/L，氯化血红素0.01g/L，维生素K10.002g/L。
本发明的第五个目的是提供一种脆弱拟杆菌冻存剂，含有≥10 10CFU/ml的脆弱拟杆菌CCFM1020。
在本发明的一种实施方式中，所述冻存剂是将脆弱拟杆菌CCFM1020处于稳定期的菌液用pH7.0～7.2的磷酸盐缓冲液清洗1-2次后，加入保护剂，于-80℃保存备用。
在本发明的一种实施方式中，所述保护剂含有1g/L半胱氨酸盐酸盐，200g/L甘油。
本发明的第六个目的是提供含有所述脆弱拟杆菌CCFM1020的非药物用途的组合物。
本发明还要求保护所述脆弱拟杆菌CCFM1020在食品、生物领域作为发酵微生物在生产产品方面的应用。
有益效果：本发明的脆弱拟杆菌CCFM1020具有低免疫原性，可显著将因内毒素感染而造成的小鼠血液中炎症因子的含量与脾脏中Foxp3 +调节性T细胞的数量恢复至正常小鼠水平，并能够稳定内毒素感染小鼠的肠道菌群组成。所述的脆弱拟杆菌CCFM1020可用于制备能够缓解内毒素感染的药物组合物，具有广泛的应用前景。
附图说明
图1为小鼠灌胃脆弱拟杆菌CCFM1020后血液及结肠组织中免疫因子的浓度；
图2为脆弱拟杆菌CCFM1020对内毒素感染小鼠血液内免疫因子水平的调节作用，*表 示脆弱拟杆菌CCFM1020组与内毒素造模组存在显著性差异，*表示p<0.05，**表示p<0.01；
图3为脆弱拟杆菌CCFM1020对内毒素感染小鼠脾脏内调节性T细胞含量的调节作用，*表示脆弱拟杆菌CCFM1020组与内毒素造模组存在显著性差异，**表示p<0.01；#表示内毒素感染组与对照组存在显著性差异，##表示p<0.01；
图4为内毒素感染组小鼠肠道菌群；其中，D1LPSCTRL:实验第一天内毒素感染组小鼠肠道菌群；D3LPSCTRL:实验第三天内毒素感染组小鼠肠道菌群；D5LPSCTRL:实验第五天内毒素感染组小鼠肠道菌群；
图5为脆弱拟杆菌CCFM1020对内毒素感染小鼠肠道菌群组成的调节作用。其中，D1LPSBF:实验第一天脆弱拟杆菌CCFM1020干预内毒素感染组小鼠肠道菌群；D3LPSBF:实验第三天脆弱拟杆菌CCFM1020干预内毒素感染组小鼠肠道菌群；D5LPSBF:实验第五天脆弱拟杆菌CCFM1020干预内毒素感染组小鼠肠道菌群。
图6为Allobaculum在内毒素感染小鼠肠道菌群和脆弱拟杆菌CCFM1020干预内毒素感染组小鼠肠道菌群中的相对丰度；
图7为梭状杆菌在内毒素感染小鼠肠道菌群和脆弱拟杆菌CCFM1020干预内毒素感染组小鼠肠道菌群中的相对丰度；
图8为乳杆菌在内毒素感染小鼠肠道菌群和脆弱拟杆菌CCFM1020干预内毒素感染组小鼠肠道菌群中的相对丰度。
生物材料保藏
一种脆弱拟杆菌，分类学名称为Bacteroides fragilis，已于2018年3月29日保藏于广东省微生物菌种保藏中心，保藏地址为广州市先烈中路100号大院59号楼5楼，保藏编号为GDMCC No:60342。
具体实施方式
实施例1：脆弱拟杆菌CCFM1020的培养和保存
1、培养基的制备：配制脑心浸液BHI培养基(例如青岛海博生物技术有限公司的产品)，溶解于蒸馏水中，并加入半胱氨酸盐酸盐1g/L，氯化血红素0.01g/L，维生素K1 0.002g/L，混合均匀，然后调整其pH为7.0，115-121℃灭菌15-20min后，即得到所述培养基。
2、培养方法：脆弱拟杆菌CCFM1020菌种按照以上述培养基的重量计2-4％接种量接种，在37℃下厌氧培养12-18h，可达稳定期。
3、保护剂的制备：称取半胱氨酸盐酸盐1g/L，甘油200g/L，均匀溶解于蒸馏水中，115-121℃灭菌15-20min后，即得到所述保护剂。
4、保存方法：将脆弱拟杆菌CCFM1020稳定期菌液用无菌的磷酸盐缓冲液(pH7.2)清洗1-2次后，用所述的保护剂重悬达到浓度10 10CFU/ml，即得脆弱拟杆菌CCFM1020冻存剂，于-80℃保存备用。
实施例2：脆弱拟杆菌CCFM1020灌胃小鼠的耐受剂量实验
取6-8周龄健康雌性C57BL6小鼠10只，每24小时灌胃一次0.1ml的实施例1制备的脆弱拟杆菌CCFM1020冻存剂(10 10CFU/ml)，观察5日，记录小鼠体重及死亡状况。
结果如表1所示，喂食浓度为10 9CFU的脆弱拟杆菌CCFM1020未对小鼠造成任何负面影响，小鼠正常生长，无死亡现象产生，且无任何病理症状。
表1：喂食10 9CFU的脆弱拟杆菌CCFM1020对小鼠体重的影响
Figure PCTCN2018110059-appb-000001
实施例3：脆弱拟杆菌CCFM1020灌胃小鼠的免疫耐受实验
取6-8周龄健康雌性C57BL6小鼠20只，随机分为两组：阴性对照组及脆弱拟杆菌CCFM1020干预组，每组含小鼠10只。脆弱拟杆菌CCFM1020干预组小鼠每24小时灌胃一次0.1ml的上述脆弱拟杆菌CCFM1020冻存剂(10 10CFU/ml)，对照组小鼠则每24小时灌胃一次0.1ml上述冻存剂的保护剂，连续灌胃5天后处死所有小鼠，分别取血液及结肠组织经RIPA裂解液(碧云天生物技术有限公司产品)裂解匀浆后，采用ELISA试剂盒(南京森贝伽生物科技有限公司产品)测定免疫因子的浓度。
实验结果如附图1所示，脆弱拟杆菌CCFM1020干预组小鼠血液中的免疫因子TNF-α、IL-6、IL-10及CXCL-2及结肠组织中的免疫因子sIgA及IgE的含量与对照组小鼠无明显差别。因此，实施例3结果表明本发明脆弱拟杆菌CCFM1020不会刺激小鼠的免疫系统，具有低免疫原性。
实施例4：脆弱拟杆菌CCFM1020对内毒素感染小鼠血液中免疫因子水平的调节作用
取6-8周龄健康雌性C57BL6小鼠30只，随机分为三组：阴性对照组，内毒素感染模型组及脆弱拟杆菌CCFM1020治疗组，每组含小鼠10只。内毒素感染组及脆弱拟杆菌CCFM1020治疗组小鼠每24小时腹腔注射一次0.1ml由生理盐水稀释的内毒素溶液(0.1mg/kg)，对照组及内毒素感染组小鼠则每24小时灌胃一次0.1ml实施例1制备的冻存剂的保护剂，脆弱拟杆菌CCFM1020治疗组小鼠每24小时灌胃一次0.1ml的实施例1制备的含有 脆弱拟杆菌CCFM1020冻存剂(10 10CFU/ml)，连续5天后处死所有小鼠，取血液采用ELISA试剂盒(南京森贝伽生物科技有限公司产品)测定免疫因子的浓度。
实验结果如附图2所示，内毒素感染将小鼠血液中促炎因子TNF-α含量从522.93ng/L上升至566ng/L，而抑炎因子IL-10从686.83ng/L下降为567.68ng/L，而经过脆弱拟杆菌CCFM1020的治疗，显著性地降低了小鼠血液中TNF-α的含量，调整为450ng/L(p<0.05)，增加了IL-10的含量为692ng/L(p<0.01)，使得上述免疫因子达到正常小鼠水平。以上结果表明，本发明脆弱拟杆菌CCFM1020能够缓解内毒素感染造成的炎症反应。
实施例5：脆弱拟杆菌CCFM1020对内毒素感染小鼠脾脏中调节性T细胞含量的调节作用
取6-8周龄健康雌性C57BL6小鼠30只，随机分为三组：阴性对照组，内毒素感染模型组及脆弱拟杆菌CCFM1020治疗组，每组含小鼠10只。内毒素感染组及脆弱拟杆菌CCFM1020治疗组小鼠每24小时腹腔注射一次0.1ml由生理盐水稀释的内毒素溶液(0.1mg/kg)，对照组及内毒素感染组小鼠则每24小时灌胃一次0.1ml上述冻存剂的保护剂，脆弱拟杆菌CCFM1020治疗组小鼠每24小时灌胃一次0.1ml的上述脆弱拟杆菌CCFM1020冻存剂(10 10CFU/ml)，连续5天后处死所有小鼠，取脾脏组织经红细胞裂解液裂解匀浆后，采用小鼠调节性T细胞染色试剂盒(eBioscience公司产品)标记CD4、CD25及表面分子Foxp3后，通过流式细胞仪(FACSCalibur，BD公司)检测调节性T细胞的含量变化。
实验结果如附图3所示，内毒素感染显著减少了小鼠脾脏中Foxp3 +调节性T细胞，从7.69％降至5.69％(p<0.01)，而经脆弱拟杆菌CCFM1020治疗后，小鼠脾脏中调节性T细胞含量显著性地提高至7.73％(p<0.01)，接近对照组小鼠水平。实施例5结果表明，本发明脆弱拟杆菌CCFM1020缓解内毒素感染造成的炎症反应。
实施例6：脆弱拟杆菌CCFM1020对内毒素感染小鼠肠道菌群组成的调节作用
取6-8周龄健康雌性C57BL6小鼠30只，随机分为三组：阴性对照组，内毒素感染模型组及脆弱拟杆菌CCFM1020治疗组，每组含小鼠10只。内毒素感染组及脆弱拟杆菌CCFM1020治疗组小鼠每24小时腹腔注射一次0.1ml由生理盐水稀释的内毒素溶液(0.1mg/kg)，对照组及内毒素感染组小鼠则每24小时灌胃一次0.1ml上述冻存剂的保护剂，脆弱拟杆菌CCFM1020治疗组小鼠每24小时灌胃一次0.1ml的上述脆弱拟杆菌CCFM1020冻存剂(10 10CFU/ml)，连续5天后处死所有小鼠。实验过程中，分别于第一天灌胃前、第三天及第五天灌胃后，取小鼠粪便，采用Fast DNA Spin Kit for Feces试剂盒(MP Biomedicals公司产品)提取粪便细菌宏基因组后，对16s V3-V4区序列进行PCR扩增后，通过二代测序仪粪便样品中肠道菌群的组成差异。
实验结果如附图4至图8所示，内毒素感染小鼠肠道中拟杆菌门与厚壁菌门的含量分别为50％及40％，其中拟杆菌S24-7占42.7，乳杆菌12％，梭状杆菌8.2％，与结肠炎相关的Allobaculum的含量显著增加至10％，导致肠道菌群多样性的降低。而经过脆弱拟杆菌CCFM1020治疗后发现，小鼠粪便菌群中拟杆菌门与厚壁菌门的含量调整为60％及30％，其中拟杆菌、梭状杆菌与Allobaculum等变化幅度大大减小，分别占38.3％、8.7％及0.5％，并且乳杆菌含量有升高，约为15％。因此实施例6结果表明，本发明脆弱拟杆菌CCFM1020拟杆菌能够削弱了LPS对肠道菌群的改变，提高肠道菌群的多样性，稳定肠道菌群的平衡，从而预防因内毒素引起肠道菌群失调而可能导致的一系列疾病。
以上实例表明，本发明脆弱拟杆菌CCFM1020能够辅助缓解内毒素感染对小鼠造成的肠道菌群组成及免疫应答失调。
实施例7：制备含有本发明脆弱拟杆菌CCFM1020的胶囊制品
将本发明的脆弱拟杆菌CCFM1020在BHI培养基中于37℃厌氧培养24小时，在4℃于5000rpm条件下离心15min，用无菌的磷酸盐缓冲液(pH 7.2)冲洗1-2次，使用上述保护剂重悬菌体使菌体终浓度达到10 10CFU/ml。将菌悬液加入海藻酸钠溶液中，充分搅拌使得细胞均匀分散于海藻酸钠溶液中，然后将此混合液挤压到氯化钙溶液中形成胶粒，静止固化30min，过滤收集胶粒，将收集得到的胶粒进行冷冻干燥48小时，得到含本发明脆弱拟杆菌CCFM1020粉剂，将该粉剂装入市售的药用胶囊，即得所述的胶囊制品。
实施例8：利用本发明脆弱拟杆菌CCFM1020制备片剂
分别称取采用冷冻干燥方法制备的本发明脆弱拟杆菌CCFM1020菌粉制剂25.7重量份、淀粉55.0重量份、纤维素衍生物4.5重量份、羧甲基淀粉钠12.0重量份、滑石粉0.8重量份、蔗糖1.0重量份与水1.0重量份，混合，采用常规方法制成湿颗粒，然后使用中南制药机械厂生产的压片机进行压片，使用青州市益康中药机械有限公司生产的小型药物干燥机进行干燥，再包装得到本发明的片剂。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (15)

  1. 一种脆弱拟杆菌(Bacteroides fragilis)，已于2018年3月29日保藏于广东省微生物菌种保藏中心，保藏地址为广州市先烈中路100号大院59号楼5楼，保藏编号为GDMCC No:60342。
  2. 权利要求1所述的脆弱拟杆菌在制备用于缓解内毒素感染的药物组合物方面的应用。
  3. 一种药物组合物，其特征在于，含有权利要求1所述的脆弱拟杆菌和药学上可接受的载体。
  4. 根据权利要求3所述的药物组合物，其特征在于，所述在药学上可接受的载体包括一种或多种选自药学上通常使用的填充剂、粘合剂、润湿剂、崩解剂、润滑剂及矫味剂的载体。
  5. 根据权利要求4所述的药物组合物，其特征在于，所述的填充剂为用于增加片剂重量和体积而便于压片的辅料稀释剂，或以吸收原料中多余液体成分的辅料吸收剂。
  6. 根据权利要求4所述的药物组合物，其特征在于，所述粘合剂为原料药物本身无粘性或粘性不足，需加入粘性物质以便于制粒。
  7. 根据权利要求4所述的药物组合物，其特征在于，所述润湿剂为原料药物本身无粘性，但可润湿其药物原辅料并诱发其粘性而制成颗粒的液体。
  8. 根据权利要求4所述的药物组合物，其特征在于，所述崩解剂为一种能够加入片剂中而促进其片剂在胃肠液中快速崩解成细小粒子的辅料。
  9. 根据权利要求4所述的药物组合物，其特征在于，所述润滑剂为一种有利于提高片剂在制粒过程中的流动性，有利于片剂脱模的化学物质；所述矫味剂为在药品中用以改善或屏蔽药物不良气味和味道的药用辅料。
  10. 根据权利要求3或4所述的药物组合物，其特征在于，所述的药物组合物是颗粒剂、胶囊剂、片剂、丸剂或口服液剂型。
  11. 根据权利要求3～5任一所述的药物组合物，其特征在于，含有≥10 6CFU/ml或≥10 6CFU/g的权利要求1所述的脆弱拟杆菌。
  12. 含有权利要求1所述脆弱拟杆菌的菌剂，其特征在于，是将所述脆弱拟杆菌通过常规冷冻干燥工艺制备或其他工艺制备所得到的粉剂，含有10 6CFU/g以上的权利要求1所述的活性脆弱拟杆菌。
  13. 一种脆弱拟杆菌冻存剂，其特征在于，含有≥10 10CFU/ml的权利要求1所述的脆弱拟杆菌。
  14. 根据权利要求13所述的冻存剂，其特征在于，将权利要求1所述的脆弱拟杆菌处于稳定期的菌液用pH7.0～7.2的磷酸盐缓冲液清洗1-2次后，加入保护剂；所述保护剂含有1g/L半胱氨酸盐酸盐及200g/L甘油。
  15. 权利要求1所述的脆弱拟杆菌在非医药用途方面的应用。
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