WO2023142877A1 - 脆弱拟杆菌在改善和治疗腹泻中的应用 - Google Patents

脆弱拟杆菌在改善和治疗腹泻中的应用 Download PDF

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WO2023142877A1
WO2023142877A1 PCT/CN2022/143593 CN2022143593W WO2023142877A1 WO 2023142877 A1 WO2023142877 A1 WO 2023142877A1 CN 2022143593 W CN2022143593 W CN 2022143593W WO 2023142877 A1 WO2023142877 A1 WO 2023142877A1
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diarrhea
bacteroides fragilis
solution
group
inactivated
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French (fr)
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刘洋洋
王晔
智发朝
郑丽君
王薇
常秀娟
李平
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广州知易生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/065—Microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • 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
    • A61P1/12—Antidiarrhoeals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention requires that the patent application number submitted to the State Intellectual Property Office of China on January 25, 2022 is 202210089886.6, and the title of the invention is "Application of Bacteroides fragilis in Improving and Treating Diarrhea” and submitted to the State Intellectual Property Office of China on October 28, 2022.
  • the submitted patent application number is 202211339890.X, and the invention title is "Application of Bacteroides fragilis in improving and treating diarrhea".
  • the entire contents of these two prior applications are hereby incorporated by reference.
  • the present invention relates to the application of Bacteroides fragilis, in particular to the application of Bacteroides fragilis in improving and/or treating diarrhea.
  • Diarrhea is a gastrointestinal disorder characterized by larger than usual and more frequent (more than 3 bowel movements per day) watery or soft stools. In extreme cases, more than 20 liters of fluid can be lost per day. At the same time, it is also one of the important diseases that endanger the health of the body. It seems to be a minor disease, but its fatality rate is second only to tumors, diabetes, etc., and ranks in the forefront of the mortality rate of many diseases. According to the data released by the World Health Organization in 2018, diarrheal diseases are the second leading cause of death among children under the age of 5 in the world, and about 525,000 children under the age of five die from diarrheal diseases every year. The prevalence of diarrhea in adults should not be underestimated. Most adults have diarrhea at least once a year. When intestinal infectious diseases are prevalent, they spread widely, and the resulting harm is even greater.
  • Diarrhea is not an independent disease, but a common manifestation of many diseases.
  • the cause of diarrhea can be divided into infectious diarrhea and non-infectious diarrhea.
  • Infectious diarrhea is mostly caused by viral, bacterial, fungal and parasitic infections, and may be accompanied by symptoms such as abdominal pain, fever, and leukocytosis; non-infectious diarrhea includes food, chemical poisoning, drug-induced diarrhea, and bait-induced diarrhea , symptomatic diarrhea, allergic diarrhea, and other congenital metabolic disease diarrhea.
  • Symptomatic treatment such as montmorillonite powder, loperamide, protein tannin, bismuth subcarbonate, aluminum hydroxide gel, etc.
  • drug symptomatic treatment such as montmorillonite powder, loperamide, protein tannin, bismuth subcarbonate, aluminum hydroxide gel, etc.
  • Antibiotics such as norfloxacin, levofloxacin, moxifloxacin, metronidazole, etc.
  • Symptomatic treatment should be supplemented with antidiarrheal drugs.
  • the treatment of chronic diarrhea is mostly long-term intermittent treatment, through dietary adjustment, supplemented by non-central reaction drugs such as montmorillonite powder and bismuth subcarbonate, and microecological regulators such as bifidobacterium live bacteria and bacillus licheniformis live bacteria .
  • non-central reaction drugs such as montmorillonite powder and bismuth subcarbonate
  • microecological regulators such as bifidobacterium live bacteria and bacillus licheniformis live bacteria .
  • Probiotics are a type of active microorganisms that are beneficial to the host by colonizing the human body and changing the composition of the flora in a certain part of the host.
  • Paraprobiotics are defined as "non-viable microbial cells which, if administered orally or topically in sufficient quantities, confer a benefit to the user", which includes both morphologically incomplete microbial cells and morphologically intact microbial cells (see Taverniti V , Guglielmetti S.
  • the present invention provides the application of Bacteroides fragilis in improving and/or treating diarrhea.
  • the present invention adopts the following technical solutions:
  • the present invention provides the use of Bacteroides fragilis in the preparation of a composition for improving and/or treating diarrhea, and the Bacteroides fragilis is live or inactivated.
  • Bacteroides fragilis is selected from Bacteroides fragilis ZY-312 with the deposit number CGMCC No.10685.
  • the diarrhea is infectious diarrhea or non-infectious diarrhea.
  • the infectious diarrhea is one or more of viral infectious diarrhea, bacterial/fungal infectious diarrhea and parasitic infectious diarrhea; and/or, the non-infectious diarrhea is non-infectious inflammatory diarrhea One or more of , tumor-induced diarrhea, malabsorption diarrhea, exercise-induced diarrhea, intestinal flora-related diarrhea and drug-related diarrhea.
  • the viral infectious diarrhea is one or more of Norovirus, Rotavirus, Sarovirus, Astrovirus, Adenovirus, Enterovirus, Coronavirus, etc.
  • the bacterial infection is Salmonella , Shigella, Campylobacter, Yersinia enterocolitica, Vibrio cholerae, toxigenic Escherichia coli, Staphylococcus aureus, diarrhea-causing Escherichia coli, etc.
  • fungi Infections are Candida, Mucor and Aspergillus, etc.
  • parasitic infections are Giardia blue (Giardia), Entamoeba histolytica, Cryptosporidium, Cyclosporidium, Blastocystis hominis, Trichinella and Schistosoma etc.
  • said malabsorptive diarrhea is secondary small intestinal malabsorptive diarrhea, preferably, said secondary small intestinal malabsorptive diarrhea is dyspepsia diarrhea, further preferably, said dyspepsia
  • the sexual diarrhea is lactose malabsorption diarrhea; and/or, the intestinal flora-related diarrhea is Clostridium difficile disorder-related diarrhea; and/or, the drug in the drug-related diarrhea is a laxative (phenolphthalein, tomato Lavender, castor oil, etc.), hypertonic drugs (magnesium sulfate, sodium sulfate, etc.), antibiotics (penicillins, cephalosporins, lincomycin, clindamycin, etc.), chemotherapy drugs (erubicin , docetaxel, fluorouracil, hydroxycamptothecin, etc.), antihypertensive drugs (propranolol, reserpine, methyldopa, etc.), antiarrhythmic drugs (cardiac glycosides
  • the drugs are laxatives, high One or more of osmotic drugs and antibiotics.
  • the Bacteroides fragilis is an inactivated bacterium, further preferably, the Bacteroides fragilis is an inactivated bacterium with a complete form and/or an inactivated bacterium with an incomplete form.
  • the above-mentioned Bacteroides fragilis is killed by any one or more methods of dry heat, moist heat, filtration, organic solvents, chemical reagents, ultraviolet or infrared rays, fermentation, freeze-drying, genetic recombination, genetic modification or transformation. Live.
  • the Bacteroides fragilis is live Bacteroides fragilis powder or inactivated bacteria powder, and the inactivated Bacteroides fragilis powder is fermented, washed and centrifuged with aqueous sodium chloride solution, resuspended by adding excipients, and sterilized. Live and dry steps are prepared.
  • the inactivated bacteria powder of Bacteroides fragilis is prepared by the following method, including the following steps:
  • the fermentation liquid is centrifuged, and the thallus is collected, and the weight-to-volume ratio of the thallus to the sodium chloride aqueous solution is 1g: (10-30) mL, and the sodium chloride aqueous solution is added for washing and centrifugation to obtain the washed After the bacteria;
  • step (4) drying the stock solution of inactivated bacteria obtained in step (4) until the residual moisture is lower than 5 wt%, to obtain the inactivated bacteria powder of Bacteroides fragilis.
  • step (2) the number of bacteria in the fermentation broth reaches above 10 8 CFU/mL.
  • the mass concentration of the aqueous sodium chloride solution is 0.6-1.5wt%, preferably 0.65-1.2wt%, more preferably 0.8-1.0wt%, most preferably 0.85- 0.95 wt%, eg 0.9 wt% sodium chloride aqueous solution.
  • the excipients include mannitol, sorbitol, maltodextrin, lactose, sodium chloride, maltose, sucrose, glucose, trehalose, dextran, proline, lysine, alanine at least one of acid, casein, and skim milk.
  • the weight-to-volume ratio of the bacteria to the first excipient solution is 1 g:(5-40) mL.
  • step (3) in the first excipient solution, the mass fraction of the excipient is 4-30 wt%, and the excipient has the above-mentioned meanings.
  • the solvent of the first excipient solution is selected from aqueous sodium chloride solution, wherein the aqueous sodium chloride solution has the above-mentioned meanings. Further preferably, the solvent of the first excipient solution is selected from physiological saline, such as 0.9 wt% sodium chloride aqueous solution.
  • the inactivation treatment method is selected from at least one of heat inactivation, freeze inactivation or chemical inactivation, preferably heat inactivation.
  • the temperature of the heat inactivation is 60-100° C.
  • the time of heat inactivation is 10-60 min.
  • a second excipient solution is added to make the total weight of the inactivated bacteria stock solution consistent with the weight of the bacterial cell solution before inactivation in step (3).
  • said second excipient solution is the same or different from said first excipient solution.
  • the mass fraction of the excipient is 4-30 wt%, and the excipient has the above-mentioned meanings.
  • the solvent of the second excipient solution is selected from aqueous sodium chloride solution, wherein the aqueous sodium chloride solution has the above-mentioned meanings.
  • the solvent of the first excipient solution is selected from physiological saline, such as 0.9 wt% sodium chloride aqueous solution.
  • the second excipient solution is the same as the first excipient solution.
  • the drying method is selected from vacuum freeze drying and/or spray drying, preferably vacuum freeze drying.
  • the vacuum freeze-drying conditions include: a freezing temperature of -20-40° C., a freezing time of 1-3 hours, and a vacuum degree of 0.20-0.25 mbar.
  • the vacuum freeze-drying process includes: prefreezing at -40 ⁇ 2°C for 1 to 3 hours, then prefreezing at -20 ⁇ 2°C for 0.5 to 1 hour, and finally prefreezing at -40 ⁇ 2°C for 0.5 to 2 hours, Under 0.25mbar vacuum degree, the inactivated bacteria powder is prepared by drying once and analyzing and drying.
  • the conditions of the centrifugation are not specifically limited, as long as the desired centrifugation effect can be achieved, for example, the centrifugation speed is 10000-20000 rpm.
  • the composition may be any one of pharmaceutical composition, food, health product or food additive.
  • the pharmaceutical composition contains a pharmaceutically effective dose of inactivated Bacteroides fragilis ZY-312 bacterial powder with a preservation number of CGMCC No. 10685.
  • the dosage form of the pharmaceutical composition is pill, tablet, granule, capsule, powder, suspension, oral liquid or enema, etc.
  • the pharmaceutical composition is administered orally or enemaly.
  • the administration period of the pharmaceutical composition is intermittent administration, periodic administration, continuous administration or long-term administration.
  • the present invention proves through different animal diarrhea model tests that Bacteroides fragilis, especially Bacteroides fragilis ZY-312 live bacteria, lysate and inactivated bacteria with the preservation number CGMCC No. 10685, has the ability to improve and treat infectious diarrhea and non The role of infectious diarrhea.
  • the inactivated Bacteroides fragilis powder with complete form provided by the present invention has a better effect on improving infectious diarrhea and non-infectious diarrhea under different concentration formulations, and has no side effects on the body, and has a good application prospect.
  • Fig. 1 is Bacteroides fragilis ZY-312 anaerobic culture of the present invention Bacteroides colony morphology figure;
  • Fig. 2 is Bacteroides fragilis ZY-312 Gram staining microscopic examination picture of the present invention
  • Fig. 3 is the transmission electron micrograph of Bacteroides fragilis ZY-312 inactivated bacterium powder of the present invention
  • ISGs interferon-stimulated genes
  • lfit1, lfit2, lfit3, Oasl2 and Rsad2 are interferon-stimulated genes (ISGs).
  • ISGs interferon-stimulated genes
  • Fig. 5 is a bar graph of the total oocyst number of a single New Zealand rabbit in each group during the treatment in Example 12 of the present invention.
  • Bacteroides fragilis ZY-312 Bacteroides fragilis ZY-312
  • deposit number CGMCC No.10685 Bacteroides fragilis ZY-312 was isolated and obtained by the applicant unit of the present invention, and has been authorized for patent protection (patent number 201510459408.X). According to the provisions of the patent examination guidelines, the public can buy it from commercial channels or has been authorized without preservation, that is, No deposit certificate is required.
  • Bacteroides fragilis ZY-312 was cultured on a blood plate for 48 hours, and it was slightly convex, translucent, white, smooth, non-hemolytic, and the diameter of the colony was between 1 and 3 mm, see Figure 1.
  • Bacteroides fragilis ZY-312 was examined by Gram staining. It is a Gram-negative bacterium with a typical rod shape, blunt rounded ends and dense staining. The uncolored part in the middle of the bacteria is like a vacuole. figure 2.
  • Enrichment Select a single colony from the anaerobic cultured colonies in Example 1 and inoculate it in TSB (Tryptone Soy Broth, containing 5% fetal bovine serum) to carry out enrichment fermentation culture, and the obtained bacterial solution is preserved for future use.
  • TSB Teryptone Soy Broth, containing 5% fetal bovine serum
  • Live Bacteroides fragilis solution use the bacteria solution prepared in step (1) to measure the number of bacteria with a McFarland tube, and dilute to 10 6 CFU/mL with normal saline, 10 7 CFU/mL, 10 8 CFU/mL, 10 9 CFU/mL and 10 10 CFU/mL, save for future use.
  • Bacteroides fragilis lysate The Bacteroides fragilis lysate was prepared by ultrasonic disruption. Specific steps: use 5 mL of live Bacteroides fragilis solution prepared in step (2), and lyse it for 30 minutes with an ultrasonic instrument, On for 10 seconds, and Off for 10 seconds. Centrifuge at °C, filter and set aside.
  • Bacteroides fragilis inactivated bacterial liquid the inactivated Bacteroides fragilis liquid is prepared by high temperature or ultraviolet irradiation. Specific steps: Take 5 mL of 10 7 Cells/mL and 10 9 Cells/mL live Bacteroides fragilis solution prepared in step (2) respectively, put them in a beaker, and place the beaker containing the bacteria solution in a constant temperature water bath at 100°C Put it in the pot for 20-30 minutes or put it in the ultraviolet environment for 30-60 minutes to prepare the inactivated bacteria liquid of Bacteroides fragilis.
  • step (3) Add excipients to the inactivated bacteria slime collected in step (2) to make the total weight consistent with the weight of the bacteria liquid before inactivation, stir to dissolve completely, and obtain the inactivated bacteria powder stock solution.
  • step (3) Vacuum freeze-dry the stock solution of inactivated bacterial powder obtained in step (3), pre-freeze at -40 ⁇ 2°C for 1 to 3 hours, then pre-freeze at -20 ⁇ 2°C for 0.5 to 1 hour, and finally freeze at -40 ⁇ 2°C Pre-freeze for 0.5-2 hours, and prepare inactivated bacterial powder by drying once (-5 ⁇ 2°C and 0 ⁇ 2°C) and analyzing and drying (35 ⁇ 2°C) under 0.25mbar vacuum degree, and the bacterial count of the bacterial powder reaches 1 ⁇ More than 10 11 Cells/g. It can be seen from the microscopic examination picture in Fig. 3 that the morphology of the bacteria in the prepared inactivated bacteria powder is complete.
  • This example prepares samples for use in the following examples.
  • mice 60 SPF-grade Kunming mice were selected for the experiment, half male and half male, weighing 18-22 g, and each experimental mouse was assigned a unique number. Prior to grouping animals, the item number, species/strain, sex, cage number, and animal number should be marked on the cage label.
  • Use BioBook software to randomly group mice according to their initial body weight and sex, and divide them into 6 groups, namely blank group (group 1), model group (group 2), positive control group (group 3, atropine sulfate solution 0.0030g/mL) , low (group 4), medium (group 5), high (group 6) doses of Bacteroides fragilis ZY-312 inactivated bacteria powder groups obtained in Example 2, 10 mice in each group.
  • Preparation of senna leaf solution weigh senna leaf, add water and boil for about 10 minutes, filter, and concentrate the filtrate to a liquid medicine of 1.33 g/mL under reduced pressure.
  • Modeling of senna diarrhea model Fasting was started 4 hours before senna administration, free drinking water, 10 mice (blank group) were intragastrically administered the same amount of normal saline, and the remaining 50 mice were intragastrically administered at 15 ⁇ L/g body weight Give 1.33g/mL senna medicinal liquid, 2 times a day, for 4 consecutive days. After modeling, 50 model mice were randomly divided into corresponding groups.
  • Dosing regimen Except for the blank group (group 1) and the model group (group 2) who were given the same amount of normal saline by intragastric administration, the other groups were given the corresponding drugs (about 0.2 mL) by intragastric administration at a rate of 10 ⁇ l/g body weight. Continuous administration for 7 days. After the treatment, the mice were placed in mouse cages lined with filter paper, one mouse per cage, and the filter paper was changed every 1 hour. Continuous observation was performed for 5 hours, and the number of loose stools and total stools of each mouse every 1 hour were recorded. Calculate loose stool rate, loose stool grade and diarrhea index.
  • Mouse stools can be divided into five types: normal stools, normal-looking but watery stools, soft stools with abnormal shapes, watery stools, and mucus stools.
  • the first two are regarded as normal stools, and the latter three are diarrhea stools.
  • Dry stools and loose stools are distinguished by whether there is stain on the filter paper.
  • the number of feces is 1 time per grain or per pile (those who cannot distinguish the number of grains).
  • Diarrhea index loose stool rate ⁇ loose stool grade.
  • the loose stool ratio is the ratio of the number of loose stools discharged by each mouse to the total number of stools.
  • Loose stool grade refers to the grading of the size of the stained area formed by the filter paper polluted by loose stool, which is divided into 4 grades: grade 1 for less than 1 cm, grade 2 for 1-1.9 cm, grade 3 for 2-3 cm, and grade 4 for more than 3 cm.
  • the results are shown in Table 2.
  • the diarrhea index of the positive control group and the inactivated Bacteroides fragilis powder in each dose group was significantly lower than that of the model group (P ⁇ 0.01). It shows that the inactivated bacteria powder of Bacteroides fragilis provided by the present invention can effectively improve the symptoms of diarrhea in mice, and there is a dose effect.
  • mice were randomly divided into 7 groups, namely blank group (group 1), model group (group 2), positive control group (group 3, Imodium 4mg/kg), Bacteroides fragilis Live bacterial liquid (group 4), lysate (group 5), inactivated bacterial liquid (group 6) and inactivated bacterial powder (group 7) groups, 10 mice in each group. After 4 days of continuous oral gavage of senna leaves, Bacteroides fragilis was treated once a day in the morning and in the afternoon for 4 consecutive days. The behavioral signs and clinical symptoms of the mice were observed every day, and the diarrhea was mainly observed and recorded. After the treatment, the mice were dissected for pathological observation.
  • the positive control group (Imodium) showed a significant curative effect (P ⁇ 0.05) on the second day of treatment, and was completely cured on the third day;
  • the bacterial liquid and inactivated bacterial powder groups showed significant curative effect on the third day (P ⁇ 0.05), and were basically completely cured on the fifth day. It shows that the live Bacteroides fragilis liquid, the lysate, the inactivated bacteria liquid and the inactivated bacteria powder provided by the present invention can obviously improve the symptoms of diarrhea in mice.
  • Modeling of magnesium sulfate diarrhea model fasting was started 4 hours before magnesium sulfate administration, free drinking water, 10 mice (blank group) were intragastrically administered the same amount of normal saline, and the remaining 60 mice were intragastrically administered 0.8g at 10 ⁇ l/g body weight /mL magnesium sulfate solution, 2 times a day, for 4 consecutive days. After modeling, 60 model mice were randomly divided into corresponding groups.
  • Dosing regimen Except for the blank group (group 1) and the model group (group 2) who were given the same amount of normal saline by intragastric administration, the other groups were given the corresponding drugs (about 0.2 mL) by intragastric administration at a rate of 10 ⁇ l/g body weight. Continuous administration for 7 days. After the treatment, the mice were placed in mouse cages lined with filter paper, one mouse per cage, and the filter paper was changed every 1 hour. Continuous observation was performed for 5 hours, and the number of loose stools and total stools of each mouse every 1 hour were recorded. Calculate loose stool rate, loose stool grade and diarrhea index.
  • Mouse stools can be divided into five types: normal stools, normal-looking but watery stools, soft stools with abnormal shapes, watery stools, and mucus stools.
  • the first two are regarded as normal stools, and the latter three are diarrhea stools.
  • Dry stools and loose stools are distinguished by whether there is stain on the filter paper.
  • the number of feces is 1 time per grain or per pile (those who cannot distinguish the number of grains).
  • Diarrhea index loose stool rate ⁇ loose stool grade.
  • the loose stool ratio is the ratio of the number of loose stools discharged by each mouse to the total number of stools.
  • Loose stool grade refers to the grading of the size of the stained area formed by the filter paper polluted by loose stool, which is divided into 4 grades: grade 1 for less than 1 cm, grade 2 for 1-1.9 cm, grade 3 for 2-3 cm, and grade 4 for more than 3 cm.
  • the experimental results showed that the diarrhea index of the positive control group, each dose group of inactivated Bacteroides fragilis powder and the live bacteria group was significantly lower than that of the model group (P ⁇ 0.01); but the diarrhea index of the live Bacteroides fragilis group was significantly higher than that of the model group.
  • the same dose of Bacteroides fragilis inactivated bacteria group show that the inactivated Bacteroides fragilis bacteria powder provided by the present invention can effectively improve the diarrhea symptoms of mice, and there is a dose effect, and the drug effect is better than that of the live Bacteroides fragilis bacteria.
  • Escherichia coli preserved on the slant was inoculated on a nutrient agar plate, cultured at 37°C for 24 hours, then picked several colonies and inoculated in MH broth, cultured at 37°C for 24 hours, diluted with sterilized physiological saline, and placed in a 0.5 McFarland turbidity tube Turbidity was used to adjust the bacterial content to 3 ⁇ 10 8 CFU/mL.
  • mice with a body weight of about 20 g were selected, and 0.2 mL of Escherichia coli bacterial liquid with a bacterial content of 3 ⁇ 10 8 CFU/mL was injected intraperitoneally to induce diarrhea in the mice.
  • the positive control group was intragastric administration of 0.01 g/mL berberine hydrochloride aqueous solution, the test grouping and method were the same as in Example 3, continuous observation was performed for 5 hours, and the diarrhea index of the mice was recorded. See Table 7 for specific experimental groupings and dosing regimens.
  • antibiotics (clindamycin, ampicillin, streptomycin) were combined at a mass ratio of 1:1:1 to construct an antibiotic-associated diarrhea model, followed by 1 ⁇ 10 9 Cells/mL NCTC 9343 inactivated bacterial powder Suspension, 1 ⁇ 10 9 Cells/mL Bacteroides fragilis inactivated bacterial powder suspension and 1 ⁇ 10 9 CFU/mL Bacteroides fragilis live bacteria suspension (prepared in Example 2) were treated, and the fragile Efficacy of Bacteroides inactivated bacteria powder.
  • the animals in each group were given 0.2 mL of 180 mg/mL triple antibiotic solution by intraperitoneal injection, and the blank group was injected with the same amount of normal saline, once a day, for 7 consecutive days; Imodium suspension, 1 ⁇ 10 9 Cells/mL NCTC 9343 inactivated bacterial powder, 1 ⁇ 10 9 Cells/mL Bacteroides fragilis inactivated bacterial powder suspension and 1 ⁇ 10 9 CFU/mL Bacteroides fragilis Live bacteria suspension, 2 times a day, once in the morning and afternoon, for 7 consecutive days. The behavioral signs and clinical symptoms of the rats were observed every day. After the treatment, the rats were dissected for pathological observation.
  • Clostridium difficile diarrhea model was established by clindamycin intraperitoneal injection combined with Clostridium difficile gavage, followed by 1 ⁇ 10 9 Cells/ml NCTC 9343 inactivated bacterial powder suspension and 1 ⁇ 10 9 Cells/ml Bacteroides fragilis inactivated bacterial powder suspension and 1 ⁇ 10 9 CFU/ml live Bacteroides fragilis powder suspension (prepared in Example 2) were used for treatment, and the curative effect of the inactivated Bacteroides fragilis bacterial powder was observed.
  • Clostridium difficile preserved on the slant was inoculated on CCFA agar plate, cultivated in an anaerobic gas mixture (80% N 2 -10% CO 2 -10% H 2 ) incubator at 37°C for 72 hours, and then picked several colonies to inoculate Incubate in CCFA broth for 72 hours at 37°C, dilute with sterilized normal saline, use 0.5 McFarland turbidimetric tube to measure the turbidity, and adjust the bacterial content to 3 ⁇ 10 8 CFU/mL.
  • anaerobic gas mixture 80% N 2 -10% CO 2 -10% H 2
  • Sixty female C57BL/6 mice aged 6-8 weeks and weighing 18-22 g were randomly divided into 6 groups, namely blank group (group 1), model group (group 2), positive control group (group 3, easy Montin 4mg/kg), NCTC 9343 inactivated bacteria powder group (group 4, 1 ⁇ 10 9 Cells/mL), Bacteroides fragilis inactivated bacteria powder group (group 5, 1 ⁇ 10 9 Cells/mL) and Bacteroides fragilis Bacillus live bacteria group (group 6, 1 ⁇ 10 9 CFU/mL), 10 rats in each group.
  • 6 groups namely blank group (group 1), model group (group 2), positive control group (group 3, easy Montin 4mg/kg), NCTC 9343 inactivated bacteria powder group (group 4, 1 ⁇ 10 9 Cells/mL), Bacteroides fragilis inactivated bacteria powder group (group 5, 1 ⁇ 10 9 Cells/mL) and Bacteroides fragilis Bacillus live bacteria group (group 6, 1 ⁇ 10 9 CFU/mL), 10 rats in each group.
  • mice in each group were given 100 mg/kg clindamycin solution by intraperitoneal injection, and the blank group was injected with the same amount (0.1 mL/10 g body weight) of normal saline, once a day for 3 consecutive days; 3 ⁇ 10 8 CFU were given by intragastric administration on the fourth day /mL Clostridium difficile solution, once a day, for 7 consecutive days; on the 11th day, the animals in the corresponding group were given intragastrically (0.1mL/10g body weight) normal saline, imodium suspension, 1 ⁇ 10 9 Cells/mL NCTC 9343 inactivated bacterial powder suspension and Bacteroides fragilis inactivated bacterial powder suspension, 2 times a day, once in the morning and once in the afternoon, for 7 consecutive days.
  • mice were euthanized on the morning of the 18th day and the mice were dissected for pathological observation.
  • the pathological scoring criteria are as follows (refer to Talamisu TSUKAHARA, Yoshie IWASAKI, Keizo NAKAYAMA et al.Microscopic structure of the large intestinal mucosa in piglets during an antibiotic-associated diarrhea[J].vet.Med.Sci,2003,65(3): 301 -306.):
  • 3 points a large number (>3X) of severe edema with multiple submucosal expansion.
  • Table 11 and Table 12 show that the diarrhea score and pathological score of the positive control group and the Bacteroides fragilis inactivated bacterial powder group were significantly lower than those of the model group (P ⁇ 0.01), while the diarrhea score and pathological score of the NCTC 9343 inactivated bacterial powder group Both were higher, but also significantly lower than the model group (P ⁇ 0.05); the diarrhea score and pathological score of the live Bacteroides fragilis powder group were significantly lower than those of the model group (P ⁇ 0.01), but the pathological score and the inactivated Bacteroides fragilis The bacteria powder group had statistical significance (P ⁇ 0.05).
  • the feed was formulated according to the AOAC recommended formula (reference: The Official Methods of Analysis of the AOAC.14th ed.US, 1984,877), as shown in Table 13.
  • a indicates that 1kg of basic feed contains: vitamin A (dry, stable) 20000IU, vitamin D (dry, stable) 2000IU, vitamin E (dry, stable) 100IU, vitamin K 5mg, choline 2000mg, inositol 100mg, p-amino Benzoic acid 100mg, niacin 40mg, calcium pantothenate 40mg, vitamin B 1 5mg, vitamin B 2 8mg, vitamin B 6 5mg, vitamin B 12 0.03mg, folic acid 2mg, biotin 0.4mg.
  • b means 1kg of basic feed contains: KH 2 PO 4 194.5g, CaCO 3 190.7g, NaCl 69.6g, MgSO 4 28.65g, FeSO 4 ⁇ 7H 2 O 13.5g, MnSO 4 ⁇ H 2 O 2.05g, KI 0.395g, ZnSO 4 ⁇ 7H 2 O 0.274g, CuSO 4 ⁇ 5H 2 O 0.239g, CoCl 2 ⁇ 6H 2 O 0.012g.
  • a total of 80 SPF-grade Wistar juvenile rats aged 3-4 weeks were selected, half male and half male. After one week of adaptive feeding, the rats were reared in single cages with filter paper and iron frames on the bottom of the cages. They were randomly divided into two groups, namely, the blank group (group 1, 10 rats) and the model group (70 rats). The young mice in the control group were fed with basal feed, and the rats in the model group were fed with high-lactose feed.
  • mice were randomly divided into a model group (group 2), a free recovery group (group 3), a positive control group (group 4, whole intestines 300 mg/kg) and Bacteroides fragilis live bacteria liquid (group 5), lysate (group 6), inactivated bacterial liquid (group 7) and inactivated bacterial powder (group 8) groups, 10 mice in each group.
  • group 2 a model group
  • group 3 a free recovery group
  • group 4 whole intestines 300 mg/kg
  • group 6 lysate
  • inactivated bacterial liquid group 7
  • inactivated bacterial powder group 8 groups
  • Example 3 As in Example 3, observe the behavioral signs and clinical symptoms of the mice every day, and record the diarrhea situation and score of each Wistar rat, and detect the serum IL-6 level after the experiment.
  • Rotavirus is the main cause of severe dehydrating gastroenteritis in 5-year-old children, and its incidence is high in autumn and winter.
  • the host cell receptor binding sites are VP4 and VP7 proteins.
  • the model of viral diarrhea was established by intragastric administration of rotavirus Wa strain to mice, and then respectively treated with NCTC9343 live/inactivated bacterial liquid, Bacteroides fragilis ZY-312 live/inactivated bacterial liquid, Bacteroides fragilis ZY-312 Inactivated bacterial powder and lysate were used for treatment, and the curative effect of live Bacteroides fragilis liquid, inactivated bacterial liquid, inactivated bacterial powder and lysate on rotavirus-induced diarrhea in mice was observed.
  • Three-day-old SPF grade Kunming suckling mice were selected, half male and half male, and the suckling mice were randomly divided into 10 groups, 8 rats in each group: blank group (group 1), model group (group 2), NCTC 9343 live bacterial solution group (group 3, 1 ⁇ 10 9 CFU/mL), NCTC 9343 inactivated bacteria solution group (group 4, 1 ⁇ 10 9 Cells/mL), low-dose live Bacteroides fragilis solution group (group 5, 1 ⁇ 10 5 CFU/mL) , Bacteroides fragilis live bacteria liquid medium dose group (group 6, 1 ⁇ 10 7 CFU/mL), Bacteroides fragilis live bacteria liquid high dose group (group 7, 1 ⁇ 10 9 CFU/mL), Bacteroides fragilis inactivated Low-dose bacterial solution group (group 8, 1 ⁇ 10 5 Cells/mL), middle-dose Bacteroides fragilis inactivated bacterial solution group (group 9, 1 ⁇ 10 7 Cells/mL), high-dose Bacteroides fragilis inactivated bacterial solution
  • the suckling mice in group2-group12 were orally inoculated with 50 ⁇ L of Wa strain rotavirus solution filtered through a 0.22 ⁇ m filter membrane with a titer of 1 ⁇ 10 7 PFU, and the suckling mice in group1 were orally administered 50 ⁇ L of PBS as a control.
  • the treated group of suckling mice had yellow watery stools, and the feces were collected to prepare a feces suspension, and 100 ⁇ L of the supernatant was sucked with a pipette gun, and added vertically to the rotavirus colloidal gold detection kit (Hangzhou AI In the sample hole of Biomedicine Co., Ltd., if red bands appear in the quality control area and detection area within 10min-20min, it indicates that the rotavirus is positive, the modeling is successful, and treatment can be carried out twice a day for 7 consecutive days .
  • mice Observe the behavioral signs and clinical symptoms of the mice every day, focus on observing and recording the diarrhea situation; collect fecal samples at the end of modeling and treatment, centrifuge and take the supernatant for Elisa (Millipore company) detection to detect the amount of rotavirus antigen in the feces; At the end of the experiment, the mice were sacrificed by neck dislocation, the small intestine tissue was collected to extract RNA, and the expression of interferon-stimulated genes (ISGs) was detected by qPCR. See Table 17 for specific experimental groupings and dosing regimens.
  • ISGs interferon-stimulated genes
  • Table 18 shows the diarrhea score of suckling mice at the beginning of treatment and 7 days after treatment, and table 19 shows the amount of rotavirus antigen in feces.
  • the rat diarrhea scores of the NCTC 9343 live bacteria and inactivated bacteria groups (P ⁇ 0.05) and the Bacteroides fragilis ZY-312 groups (P ⁇ 0.0001) were all significantly lower In the model group, it shows that the Bacteroides fragilis ZY-312 provided by the present invention can effectively improve the symptoms of diarrhea caused by the virus; at the same time, it was found that in the feces of the Bacteroides fragilis live bacteria liquid, inactivated bacteria liquid, inactivated bacteria powder, and lysate group Compared with the model group, the amount of rotavirus antigen was significantly reduced (P ⁇ 0.001), while there was no significant difference between the NCTC 9343 live and inactivated bacteria groups and the model group, indicating that B.
  • fragilis ZY-312 may improve diarrhea and reduce the body's virus It is related to the antigen load, and reduces the severity of diarrhea by reducing the host's viral antigen load.
  • the results in Figure 4 show that, compared with the model group, the expression of interferon-stimulated genes (ISGs) in each group of Bacteroides fragilis ZY-312 was significantly increased (P ⁇ 0.05), and the expression of ISGs drives infection and non-infection.
  • ISGs interferon-stimulated genes
  • Bacteroides fragilis can upregulate the expression of ISGs, initiate dendritic cells to connect innate immunity and adaptive immunity through interferon-related pathways, and induce Restricts the antiviral state, induces apoptosis in infected cells and regulates immune cell subsets critical for antiviral responses, coordinates host immune responses to viruses, produces potent antiviral activity, and improves diarrhea.
  • the effect of Bacteroides fragilis ZY-312 in each group was better than that of live and inactivated bacteria of NCTC 9343 (P ⁇ 0.05).
  • Example 11 The resistance of Bacteroides fragilis to norovirus infection
  • Human norovirus causes more than 90% of non-bacterial gastroenteritis worldwide, with a high incidence in spring and autumn, causing severe morbidity and mortality worldwide.
  • the two genotypes that infect humans are mainly GI and GII.
  • the human body is selected to transmit through the fecal-oral route after ingesting the encapsulated virus particles. After an incubation period of 24-48 hours, the virus will cause symptomatic diarrhea and vomiting within the next 12-60 hours.
  • Norovirus mainly infects people aged 5-17 and adults. It causes diarrhea and also produces upper respiratory symptoms.
  • HBGAs tissue blood group antigen
  • Escherichia coli BL21 competent cells were used to express GII.17 type norovirus (NoV) P protein (GenBank number: KU557839), and the protein size and concentration were determined by SDS-PAGE electrophoresis. Dilute boiled human type A/type B saliva 1:1000 with PBS and coat on a 96-well enzyme-linked plate overnight (100 ⁇ L/well), and block with 5% skimmed milk; protein P (GII.17) (1 ⁇ g /mL) was pre-incubated with Bacteroides fragilis serially diluted with 1% skim milk at 37°C for 1 hour, then added to a 96-well plate, and 100 ⁇ L mouse anti-NoV polyantiserum (1:3000, self-made) was added to each well, at 37°C Incubate for 1 h; add HRP-goat anti-mouse IgG antibody (1:6000, Abcam) 37% and incubate for 1 h; wash 5 times with 0.05% PBS-
  • each group of Bacteroides fragilis could block the binding of NoV-HBGAs (P ⁇ 0.05), and the OD 450 value was significantly reduced (P ⁇ 0.05).
  • 1 ⁇ 10 9 Cells/ mL of Bacteroides fragilis had a blocking rate of nearly 50% (P ⁇ 0.0001).
  • the OD 450 value decreases, and the ability to block the binding of NoV-HBGAs is stronger, which shows that Bacteroides fragilis can weaken the infectivity of the virus in a dose-dependent manner, and is effective against Norovirus. With strong antiviral activity, it can improve and resist diseases caused by norovirus, such as diarrhea.
  • New Zealand white rabbits were administered intragastrically with Cryptosporidium cuniculus oocysts (extracted from a single positive fecal sample in a rabbit farm in Jilin) to establish a model of parasite-associated diarrhea, followed by live Bacteroides fragilis and inactivated bacteria Powder and lysate (prepared in embodiment 2) are treated, observe the curative effect of Bacteroides fragilis live bacteria liquid, inactivated bacteria powder and lysate.
  • 35-day-old New Zealand white rabbits were selected and randomly divided into 6 groups, 8 rabbits in each group: blank group (group 1), model group (group 2), positive control group (nitazoxan) (group 3), fragile model group Bacillus inactivated bacteria powder group (group 4), Bacteroides fragilis lysate group (group 5), Bacteroides fragilis viable bacteria solution group (group 6).
  • New Zealand white rabbits in group2-group6 were intragastrically inoculated with 1.5 ⁇ 104 Cryptosporidium oocysts, and the blank group were intragastrically inoculated with normal saline.
  • the New Zealand rabbits After 3 days of gavage, the New Zealand rabbits had yellow watery stools, and the stools were collected to prepare a stool suspension, and the structure with oocysts was observed under a microscope, which indicated that the model was successfully established and could be treated.
  • the blank group was given normal saline, and the treatment group was given twice a day for 7 consecutive days. Diarrhea in New Zealand white rabbits was observed every day. Fecal samples were collected on the first day of treatment and at the end of treatment, and the number of oocysts was observed under a microscope. See Table 23 for specific experimental groupings and dosing regimens.
  • the number of oocysts in the daily feces of New Zealand rabbits during the administration period is shown in Table 24, and the total number of oocysts per New Zealand rabbit during the treatment period is shown in Figure 5.
  • the oocyst quantity in the New Zealand rabbit feces of the positive control group (nitazoxanide) and each group of Bacteroides fragilis is significantly lower than that of the model group (P ⁇ 0.01), illustrating that the present invention
  • the provided Bacteroides fragilis can effectively improve the symptoms of diarrhea caused by Cryptosporidium.
  • the amount of oocysts in the feces of New Zealand rabbits in the live Bacteroides fragilis liquid group was zero at the end point, indicating that the live Bacteroides fragilis liquid can significantly Improve parasite-associated diarrhea (P ⁇ 0.0001).

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Abstract

提供了脆弱拟杆菌在改善和/或治疗腹泻中的应用。通过不同的小鼠腹泻模型试验证明，保藏编号为CGMCC No.10685的脆弱拟杆菌ZY-312或其灭活菌，具有改善和治疗腹泻的作用。脆弱拟杆菌灭活菌粉在不同浓度配方下，对改善感染性腹泻或非感染性腹泻效果好，且对机体没有副作用。

Description

脆弱拟杆菌在改善和治疗腹泻中的应用
相关申请的交叉引用
本发明要求2022年01月25日向中国国家知识产权局提交的专利申请号为202210089886.6，发明名称为“脆弱拟杆菌在改善和治疗腹泻中的应用”和2022年10月28日向中国国家知识产权局提交的专利申请号为202211339890.X，发明名称为“脆弱拟杆菌在改善和治疗腹泻中的应用”的在先申请的优先权。该两件在先申请的全文通过引用的方式结合于本发明中。
技术领域
本发明涉及一种脆弱拟杆菌的应用，具体而言，涉及脆弱拟杆菌在改善和/或治疗腹泻中的应用。
背景技术
腹泻是一种胃肠道疾病，其特征为较平常大量且频繁(每天排便多于3次)的水状或软性大便。在极端情况下，每天可损失超过20升的液体。同时也是危害机体健康的重要疾病之一，它看起来是一种小病，但其致死率却仅次于肿瘤、糖尿病等，在诸多疾病死亡率中均位居前列。世界卫生组织2018年发布的资料显示，腹泻病是造成全球5岁以下儿童死亡的第二大原因，每年约有52.5万名五岁以下儿童死于腹泻病。成人腹泻的流行情况也不容小觑，大多数成年人每年至少有过一次腹泻，肠道传染病流行时波及面广，由此造成的危害性更大。
腹泻不是一种独立的疾病，而是很多疾病的一个共同表现。腹泻的病因可分为感染性腹泻和非感染性腹泻。感染性腹泻多是由病毒、细菌、真菌和寄生虫感染所致，可伴有腹痛、发热及白细胞增多等症状；非感染性腹泻，包括食品、化学品中毒、药源性腹泻、食饵性腹泻、症状性腹泻、过敏性腹泻、及其他先天代谢性疾病腹泻等。
当前，临床对于腹泻的治疗主要在于药物对症治疗，如蒙脱石散、咯派丁胺、鞣酸蛋白、次碳酸铋、氢氧化铝凝胶等。根据症状的持续时间可区分急性腹泻和慢性腹泻，其症状分别持续不到两周及至少两周。治疗感染性急性腹泻首选抗生素(如氟哌酸、左氧氟沙星、莫西沙星、甲硝唑等)进行病因治疗，但仅仅使用抗生素进行治疗，会增加患者肠胃的副作用，严重时甚至会加重病情，因此需辅以止泻药进行对症治疗。而慢性腹泻的治疗多为长期间歇性治疗，通过饮食调整，辅以如蒙脱石散、次碳酸铋等无中枢反应药物，以及双歧杆菌活菌、地衣芽孢杆菌活菌等微生态调节剂。
共生细菌在肠道稳态中起着重要作用，并对肠道完整性具有一定的保护作用。因此，采用益生 菌治疗腹泻成为近年来研究的热点。益生菌是通过定植在人体内，改变宿主某一部位菌群组成的一类对宿主有益的活性微生物。副益生菌被定义为“无活力的微生物细胞，如果口服或局部使用足够数量，即可为使用者带来好处”，该定义包括形态不完整的微生物细胞和形态完整的微生物细胞(参考Taverniti V,Guglielmetti S.The immunomodulatory properties of probiotic microorganisms beyond their viability(ghost probiotics:proposal of paraprobiotic concept)[J].Genes&Nutrition,2011,6(3):261-274.))。主流研究观点认为，破碎的微生物细胞能够更好地释放功能分子，因此具有更好的效果；但对于完整的灭活微生物细胞，没有统一的观点能够说明其在某些应用中优于活菌的原因。目前针对副益生菌的研究集中在乳杆菌、双歧杆菌等一代益生菌上，研究方向相对固定。因此，有必要开发新型副益生菌，扩展副益生菌的适应症范围。
发明内容
针对上述问题，本发明提供了脆弱拟杆菌在改善和/或治疗腹泻中的应用。
为了更好地解决上述问题，本发明采用如下技术方案：
第一方面，本发明提供了脆弱拟杆菌在制备改善和/或治疗腹泻的组合物中的应用，所述脆弱拟杆菌为活菌或灭活菌。
在本发明的实施方案中，脆弱拟杆菌选自保藏编号为CGMCC No.10685的脆弱拟杆菌ZY-312。
根据本发明的实施方案，所述腹泻为感染性腹泻或非感染性腹泻。
优选的，所述感染性腹泻为病毒感染性腹泻、细菌/真菌感染性腹泻和寄生虫感染性腹泻中的一种或多种；和/或，所述非感染性腹泻为非感染性炎症腹泻、肿瘤性腹泻、吸收不良性腹泻、运动性腹泻、肠道菌群失调相关性腹泻和药物相关性腹泻中的一种或多种。
优选的，所述病毒感染性腹泻为诺如病毒、轮状病毒、札如病毒、星状病毒及腺病毒、肠道病毒、冠状病毒等中的一种或多种；细菌感染为沙门菌属、志贺菌属、弯曲杆菌属、小肠结肠炎耶尔森氏菌、霍乱弧菌、产毒性大肠杆菌、金黄色葡萄球菌、致泻性大肠埃希菌等中的一种或多种；真菌感染为念珠菌、毛霉菌和曲霉菌等；寄生虫感染为蓝贾第鞭毛虫(贾第虫)、溶组织内阿米巴、隐孢子虫、环孢子虫、人芽囊原虫、旋毛虫和血吸虫等(补充完整)；所述吸收不良性腹泻为继发性小肠吸收不良性腹泻，优选的，所述继发性小肠吸收不良性腹泻为消化不良性腹泻，进一步优选的，所述消化不良性腹泻为乳糖吸收不良性腹泻；和/或，所述肠道菌群失调相关腹泻为艰难梭菌失调相关性腹泻；和/或，所述药物相关性腹泻中的药物为泻药(酚酞、番泻叶、蓖麻油等)、高渗性药(硫酸镁、硫酸钠等)、抗生素(青霉素类、头孢菌素类、林可霉素、克林霉素等)、化疗药(表柔比星、多西他赛、氟尿嘧啶、羟基喜树碱等)、抗高血压药(普萘洛尔、利血平、甲基多巴等)、抗心律失常药(强心苷类、奎尼丁等)、利尿药(呋塞米、依他尼酸等)、降脂药(氯贝丁酯、考来烯胺等)中的一种或多种，进一步优选的，所述药物为泻药、高渗性药和抗生素中的一 种或多种。
优选的，所述脆弱拟杆菌为灭活菌，进一步优选的，所述脆弱拟杆菌为形态完整的灭活菌和/或形态不完整的灭活菌。
优选的，上述脆弱拟杆菌是通过干热、湿热、过滤、有机溶剂、化学试剂、紫外或红外射线、发酵、冻干、基因重组、基因修饰或改造的方法中的任意一种或多种灭活的。
优选的，所述脆弱拟杆菌为脆弱拟杆菌活菌粉或灭活菌粉，所述脆弱拟杆菌灭活菌粉经过发酵培养、氯化钠水溶液洗涤与离心、加入赋形剂重悬、灭活和干燥步骤制备得到。
进一步优选的，所述脆弱拟杆菌灭活菌粉通过以下方法制备获得，包括以下步骤：
(1)取脆弱拟杆菌发酵培养；
(2)发酵培养结束后，对发酵液进行离心，收集菌体，按菌体与氯化钠水溶液的重量体积比为1g:(10～30)mL加入氯化钠水溶液洗涤、离心，得到洗涤后菌体；
(3)向洗涤后菌体中加入第一赋形剂溶液混合重悬得到菌体溶液，再进行灭活处理，离心，收集灭活菌泥；
(4)向步骤(3)获得的灭活菌泥中加入第二赋形剂溶液，得灭活菌原液；
(5)将步骤(4)获得的灭活菌原液干燥至残留水分低于5wt％，即得脆弱拟杆菌灭活菌粉。
根据本发明的实施方案，步骤(2)中，发酵液的菌数达到10 8CFU/mL以上。
根据本发明的实施方案，步骤(2)中，所述氯化钠水溶液的质量浓度为0.6-1.5wt％，优选为0.65-1.2wt％，更优选为0.8-1.0wt％，最优选0.85-0.95wt％，例如为0.9wt％的氯化钠水溶液。
根据本发明的实施方案，所述赋形剂包括甘露醇、山梨醇、麦芽糊精、乳糖、氯化钠、麦芽糖、蔗糖、葡萄糖、海藻糖、右旋糖酐、脯氨酸、赖氨酸、丙氨酸、酪蛋白、脱脂乳中的至少一种。
根据本发明的实施方案，步骤(3)中，所述菌体与第一赋形剂溶液的重量体积比为1g:(5～40)mL。
根据本发明的实施方案，步骤(3)中，所述第一赋形剂溶液中，赋形剂的质量分数为4～30wt％，所述赋形剂具有如上所述含义。
根据本发明的实施方案，步骤(3)中，所述第一赋形剂溶液的溶剂选自氯化钠水溶液，其中，所述氯化钠水溶液具有如上所述含义。进一步优选地，所述第一赋形剂溶液的溶剂选自生理盐水，例如0.9wt％氯化钠水溶液。
根据本发明的实施方案，步骤(3)中，所述灭活处理的方法选自热灭活、冷冻灭活或者化学灭活中的至少一种，优选为热灭活。
示例性地，所述热灭活的温度为60～100℃，热灭活的时间为10～60min。
根据本发明的实施方案，步骤(4)中，加入第二赋形剂溶液使灭活菌原液的总重量与步骤(3) 灭活前的菌体溶液重量一致。优选地，所述第二赋形剂溶液与所述第一赋形剂溶液相同或不相同。
根据本发明的实施方案，所述第二赋形剂溶液中，赋形剂的质量分数为4～30wt％，所述赋形剂具有如上所述含义。
优选地，所述第二赋形剂溶液的溶剂选自氯化钠水溶液，其中，所述氯化钠水溶液具有如上所述含义。进一步优选地，所述第一赋形剂溶液的溶剂选自生理盐水，例如0.9wt％氯化钠水溶液。
示例性地，所述第二赋形剂溶液与所述第一赋形剂溶液相同。
根据本发明的实施方案，步骤(5)中，所述干燥的方式选自真空冷冻干燥和/或喷雾干燥，优选为真空冷冻干燥。
示例性地，所述真空冷冻干燥的条件包括：冷冻温度为-20～-40℃，冷冻时间为1～3小时，真空度为0.20～0.25mbar。
示例性地，所述真空冷冻干燥的工艺包括：-40±2℃预冻1～3小时后，-20±2℃预冻0.5～1h，最后-40±2℃再预冻0.5～2h，0.25mbar真空度下经一次干燥、解析干燥制备成灭活菌粉。
根据本发明的实施方案，所述制备方法中，对所述离心的条件不做具体限定，只要能实现所需的离心效果即可，例如所述离心的转速为10000～20000rpm。
根据本发明的实施方案，上述的应用和/或用途中，组合物可以是药物组合物、食品、保健品或食品添加剂中的任意一种。
优选的，上述的应用和/或用途中，所述的药物组合物含有药学有效剂量的保藏编号为CGMCC No.10685的灭活的脆弱拟杆菌ZY-312菌粉。
上述的药物组合物中，其中，药物组合物的剂型为丸剂、片剂、颗粒剂、胶囊、散剂、混悬剂、口服液或灌肠剂等。
优选地，所述药物组合物通过口服或灌肠的形式给药。
上述的药物组合物，其中，所述药物组合物的给药周期为间歇给药、周期性给药、持续给药或长期给药。
与现有技术相比，本发明的有益效果是：
本发明通过不同的动物腹泻模型试验证明，脆弱拟杆菌，特别是保藏编号为CGMCC No.10685的脆弱拟杆菌ZY-312活菌、裂解液、灭活菌，具有改善和治疗感染性腹泻和非感染性腹泻的作用。本发明提供的形态完整的脆弱拟杆菌灭活菌粉在不同浓度配方下，对改善感染性腹泻和非感染性腹泻效果更好且对机体没有副作用，具有很好的应用前景。
附图说明
图1为本发明的脆弱拟杆菌ZY-312厌氧培养后菌落形态图；
图2为本发明的脆弱拟杆菌ZY-312革兰氏染色镜检图；
图3为本发明的脆弱拟杆菌ZY-312灭活菌粉的透射电子显微镜图；
图4A-E为本发明实施例10各组乳鼠小肠组织中干扰素刺激基因(ISGs)的表达量；
注:lfit1、lfit2、lfit3、Oasl2和Rsad2为干扰素刺激基因(ISGs)。与模型组比较，*P<0.05，**P<0.01，***P<0.001，****P<0.0001；与NCTC 9343活菌液组比较， #P<0.05， ##P<0.01， ###P<0.001；与NCTC 9343灭活菌液组比较， +P<0.05， +P<0.01， +++P<0.001，T tests；
图5为本发明实施例12治疗期间各组单只新西兰兔总卵囊数的柱形图。
本发明在实施过程中所使用的微生物菌种已于2015年4月2日在中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)(北京市朝阳区北辰西路1号院3号)保藏。分类命名：脆弱拟杆菌ZY-312(Bacteroides fragilis ZY-312)，保藏编号CGMCC No.10685。脆弱拟杆菌ZY-312由本发明申请单位自行分离获得，并且已经在授权专利保护(专利号201510459408.X)，按照专利审查指南的规定，公众能够从商业渠道买到或已经授权，不用保藏，即不用提供保藏证明。
具体实施方式
下文将结合具体实施例对本发明的技术方案做更进一步的详细说明。应当理解，下列实施例仅为示例性地说明和解释本发明，而不应被解释为对本发明保护范围的限制。凡基于本发明上述内容所实现的技术均涵盖在本发明旨在保护的范围内。
除非另有说明，以下实施例中使用的原料和试剂均为市售商品，或者可以通过已知方法制备。
以下实施例和对比例中，如无特殊说明，均采用本领域已知的方法发酵培养脆弱拟杆菌，其中，脆弱拟杆菌的保藏号为CGMCC NO.10685的脆弱拟杆菌ZY-312。
实施例1脆弱拟杆菌的发酵培养
将脆弱拟杆菌ZY-312菌种划线接种于血平皿，厌氧培养48h。观察菌落形态特征、染色特性、大小、球杆状和分布情况等。
菌落特征：脆弱拟杆菌ZY-312在血平皿上培养48h后，呈现圆形微凸、半透明、白色、表面光滑、不溶血，菌落直径在1～3mm之间，参见图1。
显微镜下形态：脆弱拟杆菌ZY-312进行革兰氏染色镜检，为革兰阴性细菌，呈现典型的杆状，两端钝圆而浓染，菌体中间不着色部分形如空泡，参见图2。
选取单个菌落接种于培养基中进行发酵培养8小时(温度为37℃)，所得菌液离心沉淀，转速3000r/min，离心15min，去上清，收集沉淀物，即得脆弱拟杆菌ZY-312菌泥。
实施例2样品制备
一、脆弱拟杆菌活菌液、裂解液及灭活菌液的制备
(1)增菌：从实施例1厌氧培养的菌落中选取单个菌落接种于TSB(胰蛋白胨大豆肉汤，含5％胎牛血清)中进行增菌发酵培养，所得菌液保存备用。
(2)脆弱拟杆菌活菌液：将步骤(1)制备的菌液，用麦氏比浊管做菌数测定，用生理盐水稀释至10 6CFU/mL，10 7CFU/mL，10 8CFU/mL，10 9CFU/mL和10 10CFU/mL，保存备用。
(3)脆弱拟杆菌裂解液：通过超声破碎的方法制备脆弱拟杆菌裂解液。具体步骤：采用步骤(2)制备的脆弱拟杆菌活菌液5mL，经超声仪裂解30min，On 10秒，Off 10秒，该体系裂解菌体效率达到99％，裂解后经6000rpm、10min、4℃离心，过滤后备用。
(4)脆弱拟杆菌灭活菌液：采用高温或紫外照射的方法制备脆弱拟杆菌灭活菌液。具体步骤：分别取步骤(2)制备的10 7Cells/mL和10 9Cells/mL的脆弱拟杆菌活菌液5mL置于烧杯中，并将盛有菌液的烧杯置于100℃的恒温水浴锅中20～30min或置于紫外线环境30～60min，便可制备脆弱拟杆菌灭活菌液。
二、脆弱拟杆菌灭活菌粉的制备
(1)取实施例1制得的脆弱拟杆菌发酵液，对发酵液进行离心处理，收集湿菌体，按菌体：生理盐水＝1:(10～30)(m:v)比例加入生理盐水对菌泥进行重悬洗涤，再次离心收集洗涤后的菌体。m代表质量，v代表体积。
(2)对步骤(1)所得菌体加入5％麦芽糊精+0.9％氯化钠混合成的赋形剂，按菌体：赋形剂＝1:(5～15)(m:m)比例进行添加，搅拌分散后在(70～100)±5℃热灭活(20～40)±5分钟后离心，收集菌泥。
(3)在步骤(2)收集的灭活菌泥中加入赋形剂使总重量与灭活前菌液重量一致，搅拌完全溶解，得灭活菌粉原液。
(4)将步骤(3)所得的灭活菌粉原液进行真空冷冻干燥，-40±2℃预冻1～3h后，-20±2℃预冻0.5～1h，最后-40±2℃再预冻0.5～2h，0.25mbar真空度下经一次干燥(-5±2℃和0±2℃)、解析干燥(35±2℃)制备成灭活菌粉，菌粉的菌数达到1×10 11Cells/g以上。由图3镜检图可以看出，制备的灭活菌粉中的菌体形态完整。
用以上同样的方法制得NCTC 9343灭活菌粉。
本实施例制备样品用于以下实施例。
实施例3脆弱拟杆菌灭活菌粉对番泻叶致腹泻小鼠的影响
一、实验方法
本实施例选用60只SPF级昆明小鼠进行实验，雌雄各半，体重18～22g，每只实验用小鼠均分配有一个唯一编号。在对动物进行分组之前，应在鼠笼的标签上标注项目编号、种属/品系、性别、笼号及动物号。使用BioBook软件根据小鼠的初始体重和性别进行随机分组，分为6组，即空白组 (group 1)，模型组(group 2)，阳性对照组(group 3，硫酸阿托品溶液0.0030g/mL)，低(group 4)、中(group 5)、高(group 6)剂量实施例2获得的脆弱拟杆菌ZY-312灭活菌粉组，每组小鼠10只。
番泻叶溶液的制备：称取番泻叶加水煮沸约10min，过滤，滤液减压浓缩1.33g/mL的药液。
番泻叶腹泻模型造模：番泻叶给药前4h开始禁食，自由饮水，10只小鼠(空白组)灌胃等量生理盐水，其余50只小鼠均按15μL/g体重灌胃给予1.33g/mL的番泻叶药液，每天2次，连续4天。造模结束后将50只造模小鼠再随机分为相应组别。
给药方案：除空白组(group 1)和模型组(group 2)灌胃给予等量生理盐水外，其余各组按10μl/g体重分别进行单次灌胃给予相应药物(约0.2mL)，连续给药治疗7天。治疗结束后，将小鼠置于垫有滤纸的小鼠笼内，每笼1只，每隔1h换一次滤纸，连续观察5h，记录每只小鼠每1h内的稀便数、总便数，并计算稀便率、稀便级和腹泻指数。
小鼠大便可分为5种：正常便、外形正常但含水分多、外形不正常的软便、水样便及黏液便。将前2种视为正常便，后3种为腹泻便。干便与稀便以滤纸上有无污迹为区别标准。粪便次数以每粒或每堆(不能分清粒数者)为1次。
腹泻指数＝稀便率×稀便级。其中，稀便率为每只小鼠所排稀便数与总便数之比。稀便级为以稀便污染滤纸形成污迹面积的大小定级，分为4级：小于1cm为l级，1～1.9cm为2级，2～3cm为3级，大于3cm为4级。
统计时先逐个统计每一堆湿便的级数，然后将该鼠所有稀便级数相加除以稀便次数，得到稀便的平均级数即稀便级。级数直径的测量：粪便性状为圆形则直接测量直径，若粪便性状为椭圆形则测量最长的和近似圆形的直径，二者相加再除以2。
具体实验分组和给药方案见表1。
表1脆弱拟杆菌灭活菌粉对番泻叶致泻小鼠的影响实验分组和给药方案
Figure PCTCN2022143593-appb-000001
Figure PCTCN2022143593-appb-000002
二、实验结果
表2脆弱拟杆菌灭活菌粉对番泻叶致泻小鼠的影响
Figure PCTCN2022143593-appb-000003
注:与模型组比较，**P<0.01,*P<0.05。
结果如表2所示，阳性对照组、脆弱拟杆菌灭活菌粉各剂量组腹泻指数均极显著低于模型组(P＜0.01)。说明本发明提供的脆弱拟杆菌灭活菌粉可有效改善小鼠腹泻症状，且存在剂量效应。
实施例4脆弱拟杆菌对番泻叶致腹泻小鼠的影响
一、实验方法
同实施例3。不同的是70只SPF级昆明小鼠，随机分为7组，即空白组(group 1)，模型组(group 2)，阳性对照组(group 3，易蒙停4mg/kg)，脆弱拟杆菌活菌液(group 4)、裂解液(group 5)、灭活菌液(group 6)和灭活菌粉(group 7)组，每组小鼠10只。番泻叶连续灌胃造模4天后，进行脆弱拟杆菌治疗，每天上、下午各一次，连续治疗4天。每天观察小鼠行为表征、临床症状等，重点观察腹泻情况并记录。治疗结束后解剖小鼠，进行病理观察。
具体实验分组和给药方案见表3。
表3脆弱拟杆菌对番泻叶致泻小鼠的影响实验分组和给药方案
Figure PCTCN2022143593-appb-000004
Figure PCTCN2022143593-appb-000005
二、实验结果
表4脆弱拟杆菌治疗后各组的软便动物数/动物总数的变化结果
Figure PCTCN2022143593-appb-000006
注:与模型组比较，*P<0.05。
如表4所示，阳性对照组(易蒙停)在治疗第2天就表现出显著疗效(P＜0.05)，在第3天完全治愈；而脆弱拟杆菌活菌液、裂解液、灭活菌液、灭活菌粉组在第3天表现出显著疗效(P＜0.05)，在第5天基本可完全治愈。说明本发明提供的脆弱拟杆菌活菌液、裂解液、灭活菌液、灭活菌粉均具有明显改善小鼠腹泻症状的作用。
实施例5脆弱拟杆菌对硫酸镁致腹泻小鼠的影响
一、实验方法
本实施例选用70只SPF级昆明小鼠进行实验，雌雄各半，体重18～22g，每只实验用小鼠均分配有一个唯一编号。在对动物进行分组之前，应在鼠笼的标签上标注项目编号、种属/品系、性别、笼号及动物号。使用BioBook软件根据小鼠的初始体重和性别进行随机分组，分为6组，即空白组(group 1)，模型组(group 2)，阳性对照组(group 3，0.8g/mL硫酸镁溶液)，灭活菌低(group 4)、中(group 5)、高(group 6)剂量实施例2获得的脆弱拟杆菌ZY-312灭活菌粉组及实施例2获得的活菌组(group 7)，每组小鼠10只。
硫酸镁腹泻模型造模：硫酸镁给药前4h开始禁食，自由饮水，10只小鼠(空白组)灌胃等量生理盐水，其余60只小鼠按10μl/g体重灌胃给予0.8g/mL硫酸镁溶液，每天2次，连续4天。造模结束后将60只造模小鼠再随机分为相应组别。
给药方案：除空白组(group 1)和模型组(group 2)灌胃给予等量生理盐水外，其余各组按10μl/g体重分别进行单次灌胃给予相应药物(约0.2mL)，连续给药治疗7天。治疗结束后，将小鼠置于垫有滤纸的小鼠笼内，每笼1只，每隔1h换一次滤纸，连续观察5h，记录每只小鼠每1h内的稀便数、总便数，并计算稀便率、稀便级和腹泻指数。
小鼠大便可分为5种：正常便、外形正常但含水分多、外形不正常的软便、水样便及黏液便。将前2种视为正常便，后3种为腹泻便。干便与稀便以滤纸上有无污迹为区别标准。粪便次数以每粒或每堆(不能分清粒数者)为1次。
腹泻指数＝稀便率×稀便级。其中，稀便率为每只小鼠所排稀便数与总便数之比。稀便级为以稀便污染滤纸形成污迹面积的大小定级，分为4级：小于1cm为l级，1～1.9cm为2级，2～3cm为3级，大于3cm为4级。
统计时先逐个统计每一堆湿便的级数，然后将该鼠所有稀便级数相加除以稀便次数，得到稀便的平均级数即稀便级。级数直径的测量：粪便性状为圆形则直接测量直径，若粪便性状为椭圆形则测量最长的和近似圆形的直径，二者相加再除以2。具体实验分组和给药方案见表5。
表5脆弱拟杆菌对硫酸镁致泻小鼠的影响实验分组和给药方案
Figure PCTCN2022143593-appb-000007
二、实验结果
表6脆弱拟杆菌对硫酸镁致泻小鼠的影响
Figure PCTCN2022143593-appb-000008
注:与模型组比较，**P<0.01,*P<0.05；与活菌组比较， ##P<0.01。
实验结果表明，阳性对照组、脆弱拟杆菌灭活菌粉各剂量组及活菌组腹泻指数均极显著低于模型组(P＜0.01)；但脆弱拟杆菌活菌组的腹泻指数明显高于同等剂量的脆弱拟杆菌灭活菌组。上述结果说明本发明提供的脆弱拟杆菌灭活菌粉可有效改善小鼠腹泻症状，并存在剂量效应，且药效优于脆弱拟杆菌活菌。
实施例6脆弱拟杆菌对大肠埃希菌致腹泻小鼠的影响
一、实验方法
(1)菌液的制备
保存于斜面的大肠埃希菌接种于营养琼脂平板，37℃培养24h，再挑取数个菌落接种于MH肉汤，37℃培养24h，用灭菌生理盐水稀释，以0.5麦氏比浊管比浊，调其含菌量为3×10 8CFU/mL。
(2)对大肠埃希菌腹泻模型小鼠的治疗试验
选择体重在20g左右的小鼠，通过腹腔注射0.2mL含菌量为3×10 8CFU/mL的大肠埃希菌菌液致小鼠腹泻。阳性对照组为灌胃给药0.01g/mL盐酸小檗碱水溶液，试验分组及方法同实施例3，连续观察5h，记录小鼠腹泻指数。具体实验分组和给药方案见表7。
表7脆弱拟杆菌对大肠埃希菌致泻小鼠的影响实验分组和给药方案
Figure PCTCN2022143593-appb-000009
Figure PCTCN2022143593-appb-000010
二、实验结果
表8脆弱拟杆菌对大肠埃希菌致泻小鼠的影响
Figure PCTCN2022143593-appb-000011
注:与模型组比较，**P<0.01,*P<0.05；与活菌组比较， ##P<0.01。
表8结果显示，阳性对照组、脆弱拟杆菌灭活菌粉各剂量组及活菌组腹泻指数均明显低于模型组(P＜0.01)，但脆弱拟杆菌活菌组的腹泻指数明显高于同等剂量的脆弱拟杆菌灭活菌组。上述 结果说明本发明提供的脆弱拟杆菌灭活菌粉可有效改善腹泻症状，且存在剂量效应。
实施例7脆弱拟杆菌对抗生素致腹泻大鼠的影响
用三种抗生素(克林霉素、氨苄西林、链霉素)质量比1∶1∶1联用构建抗生素相关性腹泻模型，随后分别用1×10 9Cells/mL的NCTC 9343灭活菌粉混悬液、1×10 9Cells/mL的脆弱拟杆菌灭活菌粉混悬液和1×10 9CFU/mL的脆弱拟杆菌活菌混悬液(实施例2制备)进行治疗，观察脆弱拟杆菌灭活菌粉的疗效。
一、实验方法
选取7-8周龄，体重200～250g的雌性SD大鼠60只，随机分为6组，即空白组(group 1)，模型组(group 2)，阳性对照组(group 3，易蒙停1.5mg/kg)、NCTC 9343灭活菌粉组(group 4，1×10 9Cells/mL)、脆弱拟杆菌灭活菌粉组(group 5，1×10 9Cells/mL)和脆弱拟杆菌活菌组(group 6，1×10 9CFU/mL)，每组10只。
各组动物腹腔注射给予180mg/mL的三联抗生素溶液0.2mL，空白组注射等量生理盐水，每天1次，连续7天；第8天相应组动物分别灌胃(1mL/100g体重)生理盐水、易蒙停混悬液、1×10 9Cells/mL的NCTC 9343灭活菌粉、1×10 9Cells/mL脆弱拟杆菌灭活菌粉混悬液和1×10 9CFU/mL脆弱拟杆菌活菌混悬液，每天2次，上下午各一次，连续7天。每天观察大鼠行为表征、临床症状等。治疗结束后解剖大鼠，进行病理观察。
分别在第7天下午(造模结束)、第14天下午(试验结束)采用单笼收集4h内每只大鼠的粪便，并称湿重，然后放置80℃烘箱3h将粪便烘干后称干重并记录。按公式计算：粪便含水量(％)＝(粪便湿重－粪便干重)/粪便湿重×100。
具体实验分组和给药方案见表9。
表9脆弱拟杆菌对抗生素致腹泻大鼠的影响实验分组和给药方案
Figure PCTCN2022143593-appb-000012
Figure PCTCN2022143593-appb-000013
二、实验结果
表10脆弱拟杆菌对抗生素致腹泻大鼠的影响
Figure PCTCN2022143593-appb-000014
注:与模型组比较，***P<0.001，**P<0.01，*P<0.05；与NCTC9343灭活菌粉组比较， ##P<0.01；与脆弱拟杆菌活菌粉组比较， @P<0.05。
表10结果显示，脆弱拟杆菌灭活菌粉组粪便含水量与阳性对照组相当，均极显著低于模型组(P<0.001)，而NCTC 9343灭活菌粉组粪便含水量较高，但也显著低于模型组(P<0.05)，脆弱拟杆菌活菌粉组粪便含水量显著低于模型组(P<0.001)，但与脆弱拟杆菌灭活菌粉组有统计学意义(P<0.05)。上述结果说明本发明提供的脆弱拟杆菌灭活菌粉可有效改善抗生素相关性腹泻症状，且疗效强度与易蒙停相当，优于NCTC 9343灭活菌粉(P＜0.01)与脆弱拟杆菌活菌组(P<0.05)。实施例8脆弱拟杆菌对艰难梭菌致小鼠腹泻的影响
用克林霉素腹腔注射联合艰难梭菌灌胃构建艰难梭菌性腹泻模型，随后分别用1×10 9Cells/ml的NCTC 9343灭活菌粉混悬液、1×10 9Cells/ml的脆弱拟杆菌灭活菌粉混悬液和1×10 9CFU/ml的脆弱拟杆菌活菌粉混悬液(实施例2制备)进行治疗，观察脆弱拟杆菌灭活菌粉的疗效。
一、实验方法
1.菌液的制备
保存于斜面的艰难梭菌接种于CCFA琼脂平板上，37℃，厌氧气体混合物(80％N 2-10％CO 2-10％H 2)培养箱中培养72h，再挑取数个菌落接种于CCFA肉汤中，37℃培养72h，用灭菌生理盐水稀释，以0.5麦氏比浊管比浊，调其含菌量为3×10 8CFU/mL。
2.对艰难梭菌腹泻模型小鼠的治疗试验
选取6-8周龄，体重18～22g的雌性C57BL/6小鼠60只，随机分为6组，即空白组(group 1)，模型组(group 2)，阳性对照组(group 3，易蒙停4mg/kg)、NCTC 9343灭活菌粉组(group 4，1×10 9Cells/mL)、脆弱拟杆菌灭活菌粉组(group 5，1×10 9Cells/mL)和脆弱拟杆菌活菌组(group 6，1×10 9CFU/mL)，每组10只。
各组动物腹腔注射给予100mg/kg的克林霉素溶液，空白组注射等量(0.1mL/10g体重)生理盐水，每天1次，连续3天；第4天灌胃给予3×10 8CFU/mL艰难梭菌菌液，每天1次，连续7天；第11天相应组动物分别灌胃(0.1mL/10g体重)生理盐水、易蒙停混悬液、1×10 9Cells/mL的NCTC 9343灭活菌粉混悬液和脆弱拟杆菌灭活菌粉混悬液，每天2次，上下午各一次，连续7天。
每天观察小鼠行为表征、临床症状等。每天记录各只小鼠的腹泻情况，腹泻的评分标准参照Kurita A等研究中的腹泻评分方法。0分：大便正常或没有；1分：轻度腹泻，大便可见轻微湿软；2分：中度腹泻，大便较湿且不成形，并且有轻度的肛周着色；3分：重度腹泻，水样便并伴有重度肛周着色(参考Kurita A,Kado S,Kaneda N,et al.Modified irinotecan hydrochloride(CPT-11)administration schedule improves induetion of delayed-onset diarrhea in rats[J].Cancer Chem other Pharmacol,2000,46(3):211-220))。
治疗结束，第18天上午安乐死后解剖小鼠，进行病理观察。病理评分标准如下(参考Talamisu TSUKAHARA,Yoshie IWASAKI,Keizo NAKAYAMA et al.Microscopic structure of the large intestinal mucosa in piglets during an antibiotic-associated diarrhea[J].vet.Med.Sci,2003,65(3):301-306.)：
(1)水肿
0分：无水肿。
1分：只有少量(＜2X)多发性黏膜下扩张的轻微性水肿。
2分：中量(2～3X)多发性黏膜下扩张的中度水肿。
3分：大量(＞3X)多发性黏膜下扩张的重度水肿。
4分：弥漫性黏膜下扩张的重度水肿。
(2)炎性细胞浸润
0分：无炎症。
1分：少量多灶性中性粒细胞浸润。
2分：中度多灶性中性粒细胞浸润(累及更多的黏膜下层)。
3分：大量多灶性甚至聚集性中性粒细胞浸润(累及更多的黏膜下层及肌层)。
4分：病灶累及同3分，但出现了脓肿或更广泛的肌层受累。
(3)肠上皮损伤
0分：无肠上皮损伤改变。
1分：少量多灶性浅表上皮损伤(空泡出现，个别细胞凋亡，绒毛顶端衰减/坏死)。
2分：中量多灶性浅表上皮损伤(空泡出现，个别细胞凋亡，绒毛顶端衰减/坏死)。
3分：大量多灶性上皮损伤空泡出现，个别细胞凋亡，绒毛顶端衰减/坏死)±伪膜形成(管腔内有含中性粒细胞，脱落上皮的纤维性渗出)。
4分：在3分基础上出现更明显的伪膜或者上皮溃疡(病灶处上皮完全性脱落)。
二、实验结果
表11脆弱拟杆菌对艰难梭菌致小鼠腹泻评分的影响
Figure PCTCN2022143593-appb-000015
注:与模型组比较，**P<0.01,*P<0.05。
表12脆弱拟杆菌对艰难梭菌致腹泻小鼠病理评分变化的影响
Figure PCTCN2022143593-appb-000016
Figure PCTCN2022143593-appb-000017
注:与模型组比较，**P<0.01,*P<0.05；与NCTC 9343灭活菌粉组比较， ##P<0.01；与脆弱拟杆菌活菌组比较， @P<0.05。
表11和表12结果显示，阳性对照组和脆弱拟杆菌灭活菌粉组腹泻评分和病理评分均显著低于模型组(P<0.01)，而NCTC 9343灭活菌粉组腹泻评分和病理评分均较高，但也显著低于模型组(P<0.05)；脆弱拟杆菌活菌粉组腹泻评分和病理评分均显著低于模型组(P<0.01)，但病理评分与脆弱拟杆菌灭活菌粉组比较有统计学意义(P<0.05)。上述结果说明本发明提供的脆弱拟杆菌灭活菌粉可有效改善艰难梭菌感染性腹泻症状，且疗效较强，优于NCTC 9343灭活菌粉(P<0.01)和脆弱拟杆菌活粉(P<0.05)。
实施例9脆弱拟杆菌对高乳糖致小鼠慢性腹泻的影响
用高乳糖饲料喂养Wistar大鼠21天，构建慢性腹泻模型，随后分别用实施例2制备的脆弱拟杆菌活菌液、裂解液、灭活菌液及灭活菌粉进行治疗，观察脆弱拟杆菌对慢性腹泻的疗效。
一、实验方法
1.饲料的制备
饲料按AOAC推荐配方进行配制(参考：The Official Mehods of Analysis of the AOAC.14th ed.US,1984,877)，具体如表13所示。
表13饲料各成分含量(％)
Figure PCTCN2022143593-appb-000018
注：a表示1kg基础饲料含：维生素A(干、稳定)20000IU，维生素D(干、稳定)2000IU，维生素E(干、稳定)100IU，维生素K 5mg，胆碱2000mg，肌醇100mg，对氨基苯甲酸100mg，尼克酸40mg.泛酸钙40mg，维生素B 1 5mg，维生素B 2 8mg，维生素B 6 5mg，维生素B 12 0.03mg，叶酸2mg，生物素0.4mg。
b表示1kg基础饲料含：KH 2PO 4 194.5g，CaCO 3 190.7g，NaCl 69.6g，MgSO 4 28.65g，FeSO 4·7H 2O 13.5g，MnSO 4·H 2O 2.05g，KI 0.395g，ZnSO 4·7H 2O 0.274g，CuSO 4·5H 2O 0.239g，CoCl 2·6H 2O 0.012g。
2.对高乳糖致慢性腹泻小鼠的治疗试验
选取3～4周龄的SPF级Wistar幼龄大鼠80只，雌雄各半，适应性喂养1周后，大鼠予单笼饲养，笼底垫滤纸和铁架。随机分为2组，即空白组(group 1，10只)和造模组(70只)，对照组 幼鼠予基础饲料喂养，造模组予高乳糖饲料喂养。21天后造得慢性腹泻模型，将60只慢性腹泻幼龄大鼠随机分为模型组(group 2)，自由恢复组(group 3)，阳性对照组(group 4，整肠生300mg/kg)和脆弱拟杆菌活菌液(group 5)、裂解液(group 6)、灭活菌液(group 7)和灭活菌粉(group 8)组，每组小鼠10只。第22天按组别进行灌胃给药(0.1mL/10g体重)，每天2次，上下午各一次，连续14天。每天观察小鼠行为表征、临床症状等，重点观察腹泻情况并记录。第35天麻醉后进行腹主动脉取血并解剖大鼠，进行病理观察。
具体实验分组和给药方案见表14。
表14脆弱拟杆菌对高乳糖致慢性腹泻大鼠的影响实验分组和给药方案
Figure PCTCN2022143593-appb-000019
同实施例3，每天观察小鼠行为表征、临床症状等，并记录各只Wistar大鼠的腹泻情况与评分，实验结束后检测血清IL-6水平。
二、实验结果
表15脆弱拟杆菌对高乳糖致慢性腹泻Wistar大鼠体重与腹泻评分的影响
Figure PCTCN2022143593-appb-000020
Figure PCTCN2022143593-appb-000021
注:与模型组比较，*P<0.05,**P<0.01,***P<0.001。
表16脆弱拟杆菌对高乳糖致慢性腹泻Wistar大鼠血清IL-6的影响
Figure PCTCN2022143593-appb-000022
注:与模型组比较，*P<0.05，**P<0.01。
表15和表16结果显示，试验结束后，阳性对照组和脆弱拟杆菌各组的大鼠体重均显著高于模型组、腹泻评分和血清IL-6含量均显著低于模型组(P＜0.01)，说明本发明提供的脆弱拟杆菌可有效改善慢性腹泻症状，机制可能与降低促炎因子IL-6的表达有关；同时发现，脆弱拟杆菌各组体重、腹泻评分及IL-6含量与阳性组比较均无显著性差异，说明本发明提供的脆弱拟杆菌治疗慢性腹泻的疗效与整肠生(地衣芽孢杆菌活菌胶囊)相当。
实施例10脆弱拟杆菌对轮状病毒致小鼠腹泻的影响
轮状病毒是导致5岁儿童严重脱水性肠胃炎的主要原因，在秋冬季节高发，宿主细胞受体结合位点为VP4蛋白和VP7蛋白。采用轮状病毒Wa株灌胃小鼠构建病毒性腹泻模型，随后分别用NCTC9343活菌液/灭活菌液、脆弱拟杆菌ZY-312活菌液/灭活菌液、脆弱拟杆菌ZY-312灭活菌粉和裂解液进行治疗，观察脆弱拟杆菌活菌液、灭活菌液、灭活菌粉和裂解液治疗轮状病毒致小鼠腹泻的疗效。
一.实验方法
选用3日龄SPF级昆明乳鼠，雌雄各半，乳鼠随机分成10组，每组8只：空白组(group 1)、模型组(group 2)、NCTC 9343活菌液组(group 3，1×10 9CFU/mL)、NCTC 9343灭活菌液组(group 4，1×10 9Cells/mL)、脆弱拟杆菌活菌液低剂量组(group 5，1×10 5CFU/mL)、脆弱拟杆菌活菌 液中剂量组(group 6，1×10 7CFU/mL)、脆弱拟杆菌活菌液高剂量组(group 7，1×10 9CFU/mL)、脆弱拟杆菌灭活菌液低剂量组(group 8，1×10 5Cells/mL)、脆弱拟杆菌灭活菌液中剂量组(group 9，1×10 7Cells/mL)、脆弱拟杆菌灭活菌液高剂量组(group 10，1×10 9Cells/mL)、脆弱拟杆菌灭活菌粉组(group 11，1×10 9Cells/mL)和脆弱拟杆菌裂解液组(group 12，1×10 9Cells/mL)。乳鼠恒温饥饿处理1h后，group2-group12组口服接种经0.22μm滤膜过滤滴度为1×10 7PFU的Wa株轮状病毒液50μL，group1组乳鼠灌胃50μL PBS作为对照。灌胃攻毒24h后，处理组乳鼠出现黄色稀水样便，收集粪便制备粪便悬液，用移液枪吸取100μL上清液，垂直滴加在轮状病毒胶体金检测试剂盒(杭州艾博医药有限公司)的加样孔中，10min-20min内观察质控区及检测区出现红色条带，表明轮状病毒阳性，造模成功，可以进行治疗处理，每天两次，连续治疗7天。每天观察小鼠行为表征、临床症状等，重点观察腹泻情况并记录；造模结束及治疗结束收集粪便样品，离心取上清进行Elisa(Millipore公司)检测，检测粪便中的轮状病毒抗原量；实验终点采用断颈法处死小鼠，取小肠组织提取RNA，qPCR法检测干扰素刺激基因(ISGs)的表达。具体实验分组和给药方案见表17。
表17实验分组和给药方案
Figure PCTCN2022143593-appb-000023
Figure PCTCN2022143593-appb-000024
二.实验结果
治疗开始及治疗7天后乳鼠的腹泻腹泻评分如表18所示，粪便中的轮状病毒抗原量如表19所示。
表18脆弱拟杆菌对轮状病毒致小鼠腹泻评分的影响
Figure PCTCN2022143593-appb-000025
注:与模型组比较，***P<0.001，****P<0.0001；与NCTC 9343活菌液组比较， #P<0.05， ###P<0.001， ####P<0.0001；与NCTC 9343灭活菌液组比较， +P<0.05， +++P<0.001，T tests。
表19粪便抗原量
Figure PCTCN2022143593-appb-000026
注:与模型组比较，***P<0.001；与NCTC 9343活菌液组比较， #P<0.05， ##P<0.01；与NCTC 9343灭活菌液组比较， +++P<0.001，T tests。
根据表18和表19的数据可知，试验结束后，NCTC 9343活菌与灭活菌组(P<0.05)以及脆弱拟杆菌ZY-312各组(P<0.0001)的大鼠腹泻评分均显著低于模型组，说明本发明提供的脆弱拟杆菌ZY-312可有效改善病毒所致腹泻症状；同时发现，脆弱拟杆菌活菌液、灭活菌液、灭活菌粉、裂解液组的粪便中的轮状病毒抗原量与模型组比较显著减少(P<0.001)，而NCTC 9343活菌和灭活菌组与模型组无显著性差异，说明脆弱拟杆菌ZY-312改善腹泻可能与减少机体病毒抗原载量有关，通过减少宿主的病毒抗原载量，减轻腹泻的严重程度。与此同时，图4的结果显示，相比模型组，脆弱拟杆菌ZY-312各组的干扰素刺激基因(ISGs)的表达量显著升高(P<0.05)，ISGs的表达驱动感染和未被感染的邻近细胞的抗病毒状态，直接干扰病毒的复制和传播，说明脆弱拟杆菌能够上调ISGs的表达，通过干扰素相关途径启动树突状细胞连接先天免疫和适应性免疫，在细胞中诱导限制性抗病毒状态、诱导受感染细胞凋亡和调节对抗病毒反应至关重要的免疫细胞亚群，协调宿主对病毒的免疫反应，产生强大的抗病毒活性，改善腹泻。脆弱拟杆菌ZY-312各组效果优于NCTC 9343活菌与灭活菌(P<0.05)。脆弱拟杆菌ZY-312菌液低中高剂量对病毒性腹泻的改善作用：高剂量>中剂量≈低剂量，活菌的效果与灭活菌相当。
实施例11脆弱拟杆菌对诺如病毒感染的抵抗作用
人诺如病毒导致全球超过90％的非细菌性胃肠炎，在春季和秋季高发，在全球范围内引起严 重的发病率和死亡率，感染人的两个基因型主要是GI和GII。选取人体在摄入包裹的病毒粒子后通过粪口途径传播，经过24-48小时的潜伏期后，病毒会在接下来的12-60小时内引起症状性腹泻和呕吐。诺如病毒主要感染5-17岁以及成年人群，在引发腹泻的同时还会产生上呼吸道症状，其结合位点是VP1蛋白的P结构，负责识别宿主的组织血型抗原(Histoblood group antigen，HBGAs)受体，阻断诺如病毒与HBGAs的结合，可以减少病毒对宿主细胞的附着，减弱病毒对宿主的传染性，改善其引起的腹泻。
一.实验方法
采用大肠杆菌BL21感受态细胞表达GII.17型诺如病毒(NoV)P蛋白(GenBank号：KU557839)，通过SDS-PAGE电泳确定蛋白大小和浓度。将煮沸的人A型/B型唾液1：1000采用PBS稀释包被在96孔酶联板板上过夜(100μL/孔)，采用5％脱脂奶封闭；将P蛋白(GII.17)(1μg/mL)与采用1％脱脂奶连续稀释的脆弱拟杆菌37℃预孵育1小时后，加入96孔板中，每孔加入100μL小鼠抗NoV多抗血清(1：3000，自制)，37℃孵育1h；加入HRP-羊抗鼠IgG抗体(1：6000，Abcam)37％孵育1h；以上步骤均以0.05％PBS—Tween20洗涤5次；加入含TMB的底物缓冲液(现配，100μL/孔)，避光室温放置10min显色；加入50μL 2mol/L H 2SO 4终止反应，于酶标仪上450nm波长处测定OD值。实验分组和给药方案如表20所示，脆弱拟杆菌ZY-312菌液给药浓度如表21所示。
表20脆弱拟杆菌抗诺如病毒实验分组
Figure PCTCN2022143593-appb-000027
Figure PCTCN2022143593-appb-000028
表21脆弱拟杆菌活菌液与灭活菌液给药浓度
Figure PCTCN2022143593-appb-000029
二.实验结果
通过阻断率来评价脆弱拟杆菌对NoV-HBGAs结合的阻断能力，阻断率＝(1-OD 450实验组/OD 450阳性对照组平均值)×100％。结果如表22所示。
表22脆弱拟杆菌对NoV-HBGAs结合的阻断率
Figure PCTCN2022143593-appb-000030
Figure PCTCN2022143593-appb-000031
注：与阳性对照组比较，*P<0.05，**P<0.01，***P<0.001，****P<0.0001，T tests。
根据表22的结果显示，与阳性对照组相比，脆弱拟杆菌各组均能够阻断NoV-HBGAs结合(P<0.05)，OD 450值显著降低(P<0.05)，1×10 9Cells/mL的脆弱拟杆菌的阻断率为近50％(P<0.0001)。且随着脆弱拟杆菌的给药浓度增加，OD 450值下降，阻断NoV-HBGAs结合的能力越强，以此说明脆弱拟杆菌可剂量依赖性地减弱病毒的传染性，对诺如病毒有强大的抗病毒活性，对诺如病毒所致疾病，如腹泻，具有改善和抵抗作用。脆弱拟杆菌菌液低中高剂量对病毒性腹泻的改善作用：高剂量>中剂量>低剂量，活菌的效果与灭活菌相当。
实施例12脆弱拟杆菌对隐孢子虫感染的免疫活性兔腹泻的影响
采用隐孢子虫(Cryptosporidium cuniculus)卵囊(从吉林养兔场的单个阳性粪便样本中提取)灌胃新西兰白兔构建寄生虫相关性腹泻模型，随后分别用脆弱拟杆菌活菌液、灭活菌粉和裂解液(实施例2制备)进行治疗，观察脆弱拟杆菌活菌液、灭活菌粉和裂解液的疗效。
一.实验方法
选用35日龄的新西兰白兔，随机分成6组，每组8只：空白组(group 1)、模型组(group 2)、阳性对照组(硝噻醋柳胺)(group 3)、脆弱拟杆菌灭活菌粉组(group 4)、脆弱拟杆菌裂解液组(group 5)、脆弱拟杆菌活菌液组(group 6)。group2-group6组新西兰白兔灌胃接种1.5×10 4个隐孢子虫卵囊，空白组灌胃生理盐水。灌胃3天后，新西兰兔出现黄色稀水样便，收集粪便制备粪便悬液，于显微镜下观察有卵囊的结构，表明造模成功，可以进行治疗处理。空白组给予生理盐水，治疗组每天给药两次，连续治疗7天。每天观察新西兰白兔腹泻情况，治疗第1天和治疗结束收集粪便样品，显微镜下观察卵囊数量。具体实验分组和给药方案见表23。
表23脆弱拟杆菌活菌、裂解液和灭活菌粉对隐孢子虫致新西兰兔腹泻的实验分组和给药方案
Figure PCTCN2022143593-appb-000032
Figure PCTCN2022143593-appb-000033
二.实验结果
给药期间新西兰兔每日粪便中的卵囊数量如表24所示，治疗期间每只新西兰兔总卵囊数量如图5所示。
表24给药期间新西兰兔每日粪便中的卵囊数量
Figure PCTCN2022143593-appb-000034
注:与模型组比较(T tests)，*P<0.05，**P<0.01，***P<0.001，****P<0.0001。
根据表24的数据可知，试验结束后，阳性对照组(硝噻醋柳胺)和脆弱拟杆菌各组新西兰兔粪便中的卵囊数量均显著低于模型组(P<0.01)，说明本发明提供的脆弱拟杆菌可有效改善隐孢子虫所致腹泻症状，其中，脆弱拟杆菌活菌液组在终点时，新西兰兔粪便中的卵囊量为零，说明脆弱拟杆菌活菌液可以显著的改善寄生虫相关性腹泻(P<0.0001)。根据图5可知，治疗期间，阳性对照组(硝噻醋柳胺)和脆弱拟杆菌各组每只新西兰兔粪便中的总卵囊数显著低于模型组(P<0.0001)，且脆弱拟杆菌裂解液组和脆弱拟杆菌活菌液、灭活菌粉组每只新西兰兔粪便中的总卵囊数显著低于阳性对照组(硝噻醋柳胺)(P<0.0001)，说明脆弱拟杆菌对隐孢子虫所致腹泻的治疗效果优于现 有药物硝噻醋柳胺。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (9)

  1. 脆弱拟杆菌在制备改善和/或治疗腹泻的组合物中的应用，其特征在于，所述脆弱拟杆菌为活菌或灭活菌或裂解液。
  2. 根据权利要求1所述的应用，其特征在于，所述脆弱拟杆菌选自保藏编号为CGMCC No.10685的脆弱拟杆菌ZY-312。
  3. 根据权利要求1或2所述的应用，其特征在于，所述腹泻为感染性腹泻或非感染性腹泻；
    优选的，所述感染性腹泻包括病毒感染性腹泻、细菌感染性腹泻、真菌感染性腹泻和寄生虫感染性腹泻中的一种或多种；和/或，所述非感染性腹泻包括非感染性炎症腹泻、肿瘤性腹泻、吸收不良性腹泻、运动性腹泻、肠道菌群失调相关性腹泻和药物相关性腹泻中的一种或多种；
    进一步优选的，所述病毒感染性腹泻包括诺如病毒、轮状病毒、札如病毒、星状病毒及腺病毒、肠道病毒、冠状病毒中的一种或多种；
    优选的，所述病毒性腹泻为诺如病毒或轮状病毒感染导致的腹泻；
    所述细菌感染包括沙门菌属、志贺菌属、弯曲杆菌属、小肠结肠炎耶尔森氏菌、霍乱弧菌、产毒性大肠杆菌、金黄色葡萄球菌、致泻性大肠埃希菌中的一种或多种；
    所述真菌感染包括念珠菌、毛霉菌和曲霉菌中的一种或多种；
    所述寄生虫感染包括蓝贾第鞭毛虫(贾第虫)、溶组织内阿米巴、隐孢子虫、环孢子虫、人芽囊原虫、旋毛虫和血吸虫中的一种或多种；
    所述吸收不良性腹泻为继发性小肠吸收不良性腹泻；更进一步优选的，所述继发性小肠吸收不良性腹泻为消化不良性腹泻，更进一步优选的，所述消化不良性腹泻为乳糖吸收不良性腹泻；
    所述肠道菌群失调相关腹泻为艰难梭菌失调相关性腹泻；
    所述药物相关性腹泻中的药物为泻药(酚酞、番泻叶、蓖麻油等)、高渗性药(硫酸镁、硫酸钠等)、抗生素(青霉素类、头孢菌素类、林可霉素、克林霉素等)、化疗药(表柔比星、多西他赛、氟尿嘧啶、羟基喜树碱等)、抗高血压药(普萘洛尔、利血平、甲基多巴等)、抗心律失常药(强心苷类、奎尼丁等)、利尿药(呋塞米、依他尼酸等)、降脂药(氯贝丁酯、考来烯胺等)中的一种或多种；更进一步优选的，所述药物为泻药、高渗性药和抗生素中的一种或多种。
  4. 根据权利要求1-3任一项所述的应用，其特征在于，所述脆弱拟杆菌为活菌或者形态完 整的灭活菌和/或形态不完整的灭活菌或裂解液。
  5. 根据权利要求4所述的应用，其特征在于，所述脆弱拟杆菌是通过干热、湿热、过滤、有机溶剂、化学试剂、紫外或红外射线、发酵、冻干、基因重组、基因修饰或改造的方法中的任意一种或多种灭活的。
  6. 根据权利要求4所述的应用，其特征在于，所述脆弱拟杆菌为脆弱拟杆菌活菌液或灭活菌粉，所述脆弱拟杆菌灭活菌粉经过发酵培养、氯化钠水溶液洗涤与离心、加入赋形剂重悬、灭活和干燥步骤制备得到；
    优选的，所述脆弱拟杆菌灭活菌粉通过以下方法制备获得，包括以下步骤：
    (1)取脆弱拟杆菌发酵培养；
    (2)发酵培养结束后，对发酵液进行离心，收集菌体，按菌体与氯化钠水溶液的重量体积比为1g:(10～30)mL加入氯化钠水溶液洗涤、离心，得到洗涤后菌体；
    (3)向洗涤后菌体中加入第一赋形剂溶液混合重悬得到菌体溶液，再进行灭活处理，离心，收集灭活菌泥；
    (4)向步骤(3)获得的灭活菌泥中加入第二赋形剂溶液，得灭活菌原液；
    (5)将步骤(4)获得的灭活菌原液干燥至残留水分低于5wt％，即得脆弱拟杆菌灭活菌粉；
    根据本发明的实施方案，步骤(2)中，发酵液的菌数达到10 8CFU/mL以上；
    根据本发明的实施方案，步骤(2)中，所述氯化钠水溶液的质量浓度为0.6-1.5wt％，优选为0.65-1.2wt％，更优选为0.8-1.0wt％，最优选0.85-0.95wt％，例如为0.9wt％的氯化钠水溶液；
    根据本发明的实施方案，所述赋形剂包括甘露醇、山梨醇、麦芽糊精、乳糖、氯化钠、麦芽糖、蔗糖、葡萄糖、海藻糖、右旋糖酐、脯氨酸、赖氨酸、丙氨酸、酪蛋白、脱脂乳中的至少一种；
    根据本发明的实施方案，步骤(3)中，所述菌体与第一赋形剂溶液的重量体积比为1g:(5～40)mL；
    根据本发明的实施方案，步骤(3)中，所述第一赋形剂溶液中，赋形剂的质量分数为4～30wt％，所述赋形剂具有如上所述含义；
    根据本发明的实施方案，步骤(3)中，所述第一赋形剂溶液的溶剂选自氯化钠水溶液，其中，所述氯化钠水溶液具有如上所述含义。进一步优选地，所述第一赋形剂溶液的溶剂选自生理盐水，例如0.9wt％氯化钠水溶液；
    根据本发明的实施方案，步骤(3)中，所述灭活处理的方法选自热灭活、冷冻灭活或者 化学灭活中的至少一种，优选为热灭活；
    示例性地，所述热灭活的温度为60～100℃，热灭活的时间为10～60min；
    根据本发明的实施方案，步骤(4)中，加入第二赋形剂溶液使灭活菌原液的总重量与步骤(3)灭活前的菌体溶液重量一致；优选地，所述第二赋形剂溶液与所述第一赋形剂溶液相同或不相同；
    根据本发明的实施方案，所述第二赋形剂溶液中，赋形剂的质量分数为4～30wt％，所述赋形剂具有如上所述含义；
    优选地，所述第二赋形剂溶液的溶剂选自氯化钠水溶液，其中，所述氯化钠水溶液具有如上所述含义；进一步优选地，所述第一赋形剂溶液的溶剂选自生理盐水，例如0.9wt％氯化钠水溶液；
    示例性地，所述第二赋形剂溶液与所述第一赋形剂溶液相同；
    根据本发明的实施方案，步骤(5)中，所述干燥的方式选自真空冷冻干燥和/或喷雾干燥，优选为真空冷冻干燥；
    示例性地，所述真空冷冻干燥的条件包括：冷冻温度为-20～-40℃，冷冻时间为1～3小时，真空度为0.20～0.25mbar；
    示例性地，所述真空冷冻干燥的工艺包括：-40±2℃预冻1～3小时后，-20±2℃预冻0.5～1h，最后-40±2℃再预冻0.5～2h，0.25mbar真空度下经一次干燥、解析干燥制备成灭活菌粉；
    根据本发明的实施方案，所述制备方法中，对所述离心的条件不做具体限定，只要能实现所需的离心效果即可，例如所述离心的转速为10000～20000rpm。
  7. 根据权利要求1-6任一项所述的应用，其特征在于，所述组合物可以是药物组合物、食品、保健品或食品添加剂中的任意一种。
  8. 根据权利要求7所述的应用，其特征在于，所述的药物组合物含有药学有效剂量的权利要求6所述的脆弱拟杆菌灭活菌粉。
  9. 根据权利要求8所述的应用，其特征在于，所述药物组合物的剂型为丸剂、片剂、颗粒剂、胶囊、散剂、混悬剂、口服液或灌肠剂；
    优选的，所述药物组合物通过口服或灌肠的形式给药；
    优选的，所述药物组合物的给药周期为间歇给药、周期性给药、持续给药或长期给药。
PCT/CN2022/143593 2022-01-25 2022-12-29 脆弱拟杆菌在改善和治疗腹泻中的应用 WO2023142877A1 (zh)

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