Use of bacteroides fragilis in improving and treating diarrhea

Abstract

The application provides application of Bacteroides fragilis in improving and / or treating diarrhea. It is proved by different mouse diarrhea model tests that the Bacteroides fragilis ZY-312 with the preservation number of CGMCC No. 10685 or inactivated bacteria thereof have the effects of improving and treating diarrhea. The inactivated bacteria powder of the Bacteroides fragilis has better effects on improving infectious diarrhea or non-infectious diarrhea under different concentration formulations, has no side effects on the body, has a good edible and application prospect, and provides a good product suitable for human consumption for health care and improvement of diarrhea.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority and benefit to Chinese Patent Application No. 202210089886.6, filed on January 25, 2022, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the application of Bacteroides fragilis, and more specifically, to the application of Bacteroides fragilis in improving and / or treating diarrhea. Background Technology

[0004] Diarrhea is a gastrointestinal disorder characterized by large, frequent, and unusually large amounts of watery or soft stools (more than three times a day). In extreme cases, more than 20 liters of fluid can be lost daily. It is also a significant health hazard; although it may seem like a minor illness, its mortality rate is second only to cancer and diabetes, ranking among the highest of all diseases. Data released by the World Health Organization in 2018 shows that diarrheal diseases are the second leading cause of death in children under five worldwide, with approximately 525,000 children dying from diarrheal diseases each year. The prevalence of diarrhea in adults is also considerable; most adults experience diarrhea at least once a year, and the widespread impact of infectious intestinal diseases during epidemics can cause even greater harm.

[0005] Diarrhea is not a disease in itself, but rather a common symptom of many diseases. The causes 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 poisoning, chemical poisoning, drug-induced diarrhea, dietary diarrhea, symptomatic diarrhea, allergic diarrhea, and diarrhea caused by other congenital metabolic diseases.

[0006] Currently, clinical treatment for diarrhea mainly focuses on symptomatic drug therapy, such as montmorillonite powder, fluphenazine, tannic acid protein, bismuth subcarbonate, and aluminum hydroxide gel. Diarrhea can be differentiated into acute and chronic diarrhea based on the duration of symptoms, which last less than two weeks and at least two weeks, respectively. For infectious acute diarrhea, antibiotics (such as norfloxacin, levofloxacin, moxifloxacin, and metronidazole) are the first-line treatment for etiological treatment. However, using antibiotics alone can increase gastrointestinal side effects and may even worsen the condition in severe cases; therefore, antidiarrheal medications are often used as adjunctive therapy. Chronic diarrhea is often treated with long-term intermittent methods, including dietary adjustments, supplemented with non-centrally acting drugs such as montmorillonite powder and bismuth subcarbonate, as well as probiotics such as live Bifidobacterium and live Bacillus licheniformis.

[0007] Symbiotic bacteria play an important role in intestinal homeostasis and have a certain protective effect on intestinal integrity. Therefore, the use of probiotics to treat diarrhea has become a research hotspot in recent years. Probiotics are a class of live microorganisms that are beneficial to the host by colonizing the human body and changing the composition of the microbial community in a certain part of the host. Paraprobiotics are defined as "inactive microbial cells that can bring benefits to the user if taken orally or applied topically in sufficient quantities." This definition includes both morphologically incomplete and morphologically complete microbial cells (see 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.). Mainstream research suggests that broken microbial cells can release functional molecules better and therefore have better effects; however, there is no unified view on why intact inactivated microbial cells are superior to live bacteria in some applications. Current research on probiotics focuses on first-generation probiotics such as Lactobacillus and Bifidobacterium, and the research direction is relatively fixed. Therefore, it is necessary to develop novel probiotics to expand the range of indications for probiotics. Summary of the Invention

[0008] To address the above-mentioned problems, the present invention provides the application of Bacteroides fragilis in improving and / or treating diarrhea.

[0009] To better solve the above problems, the present invention adopts the following technical solution:

[0010] In a first aspect, the present invention provides the use of Bacteroides fragilis in the preparation of compositions for improving and / or treating diarrhea, wherein the Bacteroides fragilis is a live or inactivated bacterium.

[0011] In an embodiment of the present invention, Bacteroides fragilis is selected from Bacteroides fragilis ZY-312 with accession number CGMCC No.10685.

[0012] According to an embodiment of the present invention, the diarrhea is infectious diarrhea or non-infectious diarrhea.

[0013] Preferably, 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 one or more of non-infectious inflammatory diarrhea, tumor-related diarrhea, malabsorption diarrhea, exercise-induced diarrhea, gut microbiota dysbiosis-related diarrhea, and drug-induced diarrhea.

[0014] Preferably, the viral infectious diarrhea is one or more of norovirus, rotavirus, zarjor virus, astrovirus, adenovirus, enterovirus, coronavirus, etc.; the bacterial infection is one or more of Salmonella spp., Shigella spp., Campylobacter spp., Yersinia enterocolitica, Vibrio cholerae, enterotoxigenic Escherichia coli, Staphylococcus aureus, diarrhea-causing Escherichia coli, etc.; the fungal infection is Candida, Mucor, Aspergillus, etc.; the parasitic infection is Giardia lamblia, Entamoeba histolytica, Cryptosporidium, Cyclospora, Blastocystis hominis, Trichinella, and Schistosoma, etc. (to be completed); the malabsorption diarrhea is secondary small intestinal malabsorption diarrhea, preferably, the secondary small intestinal malabsorption diarrhea is indigestion diarrhea, and more preferably, the... The dyspeptic diarrhea is lactose malabsorption diarrhea; and / or, the intestinal flora imbalance-related diarrhea is Clostridium difficile dysbiosis-related diarrhea; and / or, the drugs in the drug-related diarrhea are one or more of the following: laxatives (phenolphthalein, senna, castor oil, etc.), hypertonic drugs (magnesium sulfate, sodium sulfate, etc.), antibiotics (penicillins, cephalosporins, lincomycin, clindamycin, etc.), chemotherapy drugs (epirarubicin, docetaxel, fluorouracil, hydroxycamptothecin, etc.), antihypertensive drugs (propranolol, reserpine, methyldopa, etc.), antiarrhythmic drugs (cardiac glycosides, quinidine, etc.), diuretics (furosemide, ethacrynic acid, etc.), and lipid-lowering drugs (clofibrate, cholestyramine, etc.). More preferably, the drugs are one or more of the following: laxatives, hypertonic drugs, and antibiotics.

[0015] Preferably, the Bacteroides fragilis is an inactivated bacterium; more preferably, the Bacteroides fragilis is an inactivated bacterium with intact morphology and / or an inactivated bacterium with incomplete morphology.

[0016] Preferably, the aforementioned Bacteroides fragilis is inactivated by any one or more of the following methods: dry heat, moist heat, filtration, organic solvents, chemical reagents, ultraviolet or infrared radiation, fermentation, freeze drying, gene recombination, gene modification or alteration.

[0017] Preferably, the Bacteroides fragilis is a live or inactivated Bacteroides fragilis powder, which is prepared by fermentation culture, washing and centrifugation with sodium chloride aqueous solution, resuspension with excipients, inactivation and drying.

[0018] More preferably, the Bacteroides fragilis inactivated powder is prepared by the following method, including the following steps:

[0019] (1) Take Bacteroides fragilis for fermentation culture;

[0020] (2) After the fermentation culture is completed, the fermentation broth is centrifuged and the cells are collected. The cells are washed and centrifuged with sodium chloride aqueous solution at a weight-volume ratio of 1g:(10-30)mL to obtain the washed cells.

[0021] (3) Add the first excipient solution to the washed bacterial cells, mix and resuspend to obtain a bacterial cell solution, then perform inactivation treatment, centrifuge, and collect the inactivated bacterial sludge;

[0022] (4) Add the second excipient solution to the inactivated sludge obtained in step (3) to obtain the inactivated sludge stock solution;

[0023] (5) Dry the inactivated bacterial stock solution obtained in step (4) until the residual moisture content is less than 5 wt% to obtain Bacteroides fragilis inactivated bacterial powder.

[0024] According to an embodiment of the present invention, in step (2), the bacterial count of the fermentation broth reaches 10. 8 CFU / mL or higher.

[0025] According to an embodiment of the present invention, in step (2), the mass concentration of the sodium chloride aqueous solution is 0.6-1.5 wt%, preferably 0.65-1.2 wt%, more preferably 0.8-1.0 wt%, and most preferably 0.85-0.95 wt%, for example, a 0.9 wt% sodium chloride aqueous solution.

[0026] According to an embodiment of the present invention, the excipient includes at least one selected from mannitol, sorbitol, maltodextrin, lactose, sodium chloride, maltose, sucrose, glucose, trehalose, dextran, proline, lysine, alanine, casein, and skim milk.

[0027] According to an embodiment of the present invention, in step (3), the weight-to-volume ratio of the bacterial cells to the first excipient solution is 1 g: (5-40) mL.

[0028] According to an embodiment of the present invention, in step (3), the mass fraction of the excipient in the first excipient solution is 4 to 30 wt%, and the excipient has the meaning as described above.

[0029] According to an embodiment of the present invention, in step (3), the solvent of the first excipient solution is selected from an aqueous sodium chloride solution, wherein the aqueous sodium chloride solution has the meaning described above. More preferably, the solvent of the first excipient solution is selected from physiological saline, for example, a 0.9 wt% aqueous sodium chloride solution.

[0030] According to an embodiment of the present invention, in step (3), the inactivation treatment method is selected from at least one of thermal inactivation, cryogenic inactivation or chemical inactivation, preferably thermal inactivation.

[0031] For example, the heat inactivation temperature is 60-100°C, and the heat inactivation time is 10-60 min.

[0032] According to an embodiment of the present invention, in step (4), a second excipient solution is added to make the total weight of the inactivated bacterial stock solution the same as the weight of the bacterial cell solution before inactivation in step (3). Preferably, the second excipient solution is the same as or different from the first excipient solution.

[0033] According to an embodiment of the present invention, the excipient in the second excipient solution has a mass fraction of 4 to 30 wt%, and the excipient has the meaning described above.

[0034] Preferably, the solvent of the second excipient solution is selected from an aqueous sodium chloride solution, wherein the aqueous sodium chloride solution has the meaning described above. More preferably, the solvent of the first excipient solution is selected from physiological saline, such as a 0.9 wt% aqueous sodium chloride solution.

[0035] For example, the second excipient solution is the same as the first excipient solution.

[0036] According to an embodiment of the present invention, in step (5), the drying method is selected from vacuum freeze drying and / or spray drying, preferably vacuum freeze drying.

[0037] For example, the conditions for vacuum freeze drying include: a freezing temperature of -20 to -40°C, a freezing time of 1 to 3 hours, and a vacuum degree of 0.20 to 0.25 mbar.

[0038] For example, the vacuum freeze-drying process includes: pre-freezing at -40±2℃ for 1 to 3 hours, pre-freezing at -20±2℃ for 0.5 to 1 hour, and finally pre-freezing at -40±2℃ for 0.5 to 2 hours, followed by drying and desorption drying under a vacuum of 0.25 mbar to prepare inactivated bacterial powder.

[0039] According to an embodiment of the present invention, the centrifugation conditions are not specifically limited in the preparation method, as long as the desired centrifugation effect can be achieved, for example, the centrifugation speed is 10000-20000 rpm.

[0040] According to embodiments of the present invention, in the above applications and / or uses, the composition may be a pharmaceutical composition.

[0041] Preferably, in the above applications and / or uses, the pharmaceutical composition contains an inactivated Bacteroides fragilis ZY-312 bacterial powder with a pharmaceutically effective dose and the preservation number CGMCC No. 10685.

[0042] In the above-mentioned pharmaceutical composition, the dosage form of the pharmaceutical composition is pills, tablets, granules, capsules, powders, suspensions, oral liquids or enemas, etc.

[0043] Preferably, the pharmaceutical composition is administered orally or via enema.

[0044] The above-described pharmaceutical composition is wherein the administration cycle of the pharmaceutical composition is intermittent administration, periodic administration, continuous administration, or long-term administration.

[0045] Compared with the prior art, the beneficial effects of the present invention are:

[0046] This invention demonstrates through various animal diarrhea model experiments that *Bacteroides fragilis*, particularly *Bacteroides fragilis* ZY-312 (CGMCC No. 10685), including live, lysate, and inactivated strains, has the effect of improving and treating both infectious and non-infectious diarrhea. The morphologically intact *Bacteroides fragilis* inactivated powder provided by this invention, at different concentration formulations, shows even better efficacy in improving both infectious and non-infectious diarrhea without side effects on the body, demonstrating excellent application prospects. Attached Figure Description

[0047] Figure 1 This is a colony morphology diagram of Bacteroides fragilis ZY-312 after anaerobic culture according to the present invention;

[0048] Figure 2 Gram-stained microscopic image of Bacteroides fragilis ZY-312 of this invention;

[0049] Figure 3 This is a transmission electron microscope image of the inactivated Bacteroides fragilis ZY-312 powder of the present invention;

[0050] Figure 4A -E represents the expression level of interferon-stimulated genes (ISGs) in the small intestine tissue of suckling mice in each group of Examples 10 of this invention;

[0051] Note: lfit1, lfit2, lfit3, Oasl2, and Rsad2 are interferon-stimulated genes (ISGs). Compared with the model group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; compared with the NCTC 9343 live bacterial culture group, # P<0.05, ## P<0.01, ### P<0.001; compared with the NCTC 9343 inactivated bacterial solution group, + P<0.05, + P<0.01, +++ P < 0.001, T tests;

[0052] Figure 5 This is a bar chart showing the total number of oocysts in a single New Zealand rabbit in each group during the treatment period in Example 12 of the present invention.

[0053] The microbial strain used in the implementation of this invention was deposited on April 2, 2015, at the China General Microbiological Culture Collection Center (CGMCC) (No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing). Classification and naming: *Bacteroides fragilis* ZY-312, accession number CGMCC No. 10685. *Bacteroides fragilis* ZY-312 was isolated and obtained by the applicant and is already protected by an authorized patent (patent number 201510459408.X). According to the patent examination guidelines, it can be purchased by the public through commercial channels or has already been granted, so it does not need to be deposited, i.e., no deposit certificate is required. Detailed Implementation

[0054] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0055] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0056] Unless otherwise specified, the following examples and comparative examples all use methods known in the art to ferment and culture Bacteroides fragilis, wherein the Bacteroides fragilis with accession number CGMCC NO.10685 is Bacteroides fragilis ZY-312.

[0057] Example 1: Fermentation culture of Bacteroides fragilis

[0058] Bacteroides fragilis ZY-312 was streaked onto blood agar plates and anaerobic incubated for 48 hours. Colony morphology, staining characteristics, size, coccoid shape, and distribution were observed.

[0059] Colony characteristics: After 48 hours of culture on blood agar plates, Bacteroides fragilis ZY-312 colonies are round, slightly convex, translucent, white, with a smooth surface and no hemolysis. The colony diameter is between 1 and 3 mm. See [link to relevant documentation]. Figure 1 .

[0060] Microscopic morphology: Gram-stained Bacteroides fragilis ZY-312 is a Gram-negative bacterium, exhibiting a typical rod shape with blunt, rounded, and deeply stained ends. The unstained portion in the middle of the cell resembles a vacuole. (See also...) Figure 2 .

[0061] Select a single colony and inoculate it into the culture medium for fermentation culture for 8 hours (temperature is 37℃). Centrifuge the obtained bacterial solution at 3000 r / min for 15 min, remove the supernatant, and collect the precipitate to obtain Bacteroides fragilis ZY-312 bacterial sludge.

[0062] Example 2 Sample Preparation

[0063] I. Preparation of live Bacteroides fragilis culture, lysis buffer, and inactivated culture solution

[0064] (1) Enrichment: Select a single colony from the anaerobic culture in Example 1 and inoculate it into TSB (tryptone soybean broth containing 5% fetal bovine serum) for enrichment and fermentation culture. The resulting bacterial solution is stored for later use.

[0065] (2) Bacteroides fragilis live bacterial suspension: The bacterial suspension prepared in step (1) was used to determine the bacterial count using a McFarland turbidity tube, and then diluted to 10 with physiological saline. 6 CFU / mL, 10 7 CFU / mL, 10 8 CFU / mL, 10 9 CFU / mL and 10 10 CFU / mL, store for later use.

[0066] (3) Bacteroides fragilis lysate: Bacteroides fragilis lysate was prepared by ultrasonic disruption. Specific steps: 5 mL of the Bacteroides fragilis live bacterial solution prepared in step (2) was ultrasonically disrupted for 30 min, with an on cycle of 10 seconds and an off cycle of 10 seconds. The lysis efficiency of this system reached 99%. After lysis, the bacterial solution was centrifuged at 6000 rpm for 10 min at 4℃ and then filtered for later use.

[0067] (4) Bacteroides fragilis inactivated bacterial solution: The Bacteroides fragilis inactivated bacterial solution was prepared by high temperature or ultraviolet irradiation. Specific steps: Take 10g of the solution prepared in step (2) and 10g of the solution. 7 Cells / mL and 10 9 Place 5 mL of live Bacteroides fragilis culture (cells / mL) into a beaker, and then place the beaker containing the culture in a constant temperature water bath at 100℃ for 20–30 min or in a UV environment for 30–60 min to prepare an inactivated Bacteroides fragilis culture.

[0068] II. Preparation of Bacteroides fragilis inactivated powder

[0069] (1) Take the fermentation broth of Bacteroides fragilis prepared in Example 1, centrifuge the fermentation broth, collect the wet cells, add physiological saline at a ratio of cells:physiological saline = 1:(10-30)(m:v) to resuspend and wash the bacterial sludge, and centrifuge again to collect the washed cells. m represents mass and v represents volume.

[0070] (2) Add 5% maltodextrin + 0.9% sodium chloride mixture to the bacterial cells obtained in step (1) according to the ratio of bacterial cells: excipient = 1:(5~15)(m:m). After stirring and dispersing, heat inactivate at (70~100)±5℃ for (20~40)±5 minutes and then centrifuge to collect the bacterial sludge.

[0071] (3) Add excipients to the inactivated bacterial mud collected in step (2) so that the total weight is consistent with the weight of the bacterial liquid before inactivation, stir until completely dissolved, and obtain the original solution of inactivated bacterial powder.

[0072] (4) The inactivated bacterial powder stock solution obtained in step (3) is subjected to vacuum freeze-drying. After pre-freezing at -40±2℃ for 1 to 3 hours, it is pre-freezed at -20±2℃ for 0.5 to 1 hour, and finally pre-freezed at -40±2℃ for another 0.5 to 2 hours. The inactivated bacterial powder is prepared by drying once (at -5±2℃ and 0±2℃) and desorption drying (at 35±2℃) under a vacuum of 0.25 mbar. The bacterial count of the powder reaches 1×10⁻⁶. 11 Cells / g or higher. (From) Figure 3 Microscopic examination revealed that the bacterial cells in the prepared inactivated bacterial powder were intact.

[0073] NCTC 9343 inactivated bacterial powder was prepared using the same method described above.

[0074] The sample prepared in this embodiment is used in the following embodiments.

[0075] Example 3: Effects of inactivated Bacteroides fragilis powder on senna-induced diarrhea in mice.

[0076] I. Experimental Methods

[0077] This embodiment uses 60 SPF-grade Kunming mice, half male and half female, weighing 18-22g, for the experiment. Each experimental mouse is assigned a unique number. Before grouping the animals, the project number, species / strain, sex, cage number, and animal number should be labeled on the mouse cages. Using BioBook software, the mice are randomly grouped according to their initial weight and sex into 6 groups: blank group (group 1), model group (group 2), positive control group (group 3, atropine sulfate solution 0.0030g / mL), and low (group 4), medium (group 5), and high (group 6) doses of Bacteroides fragilis ZY-312 inactivated powder obtained in Example 2, with 10 mice in each group.

[0078] Preparation of senna leaf solution: Weigh senna leaves, add water and boil for about 10 minutes, filter, and concentrate the filtrate under reduced pressure to obtain a solution of 1.33 g / mL.

[0079] Establishment of a senna leaf diarrhea model: Four hours prior to senna leaf administration, mice were fasted but allowed free access to water. Ten mice (blank group) were administered an equal volume of physiological saline by gavage, while the remaining 50 mice were administered 1.33 g / mL senna leaf solution by gavage at a dose of 15 μL / g body weight, twice daily for four consecutive days. After model establishment, the 50 mice were randomly divided into corresponding groups.

[0080] Administration regimen: Except for the control group (group 1) and the model group (group 2), which were administered an equal volume of physiological saline by gavage, the other groups were given the corresponding drug (approximately 0.2 mL) by single gavage at a dose of 10 μl / g body weight for 7 consecutive days. After treatment, mice were placed in cages lined with filter paper, one mouse per cage. The filter paper was changed every 1 hour, and the mice were observed for 5 consecutive hours. The number of loose stools and the total number of stools per hour for each mouse were recorded, and the loose stool rate, loose stool grade, and diarrhea index were calculated.

[0081] Mouse stool can be classified into five types: normal stool, normal-looking but high-moisture stool, abnormally shaped soft stool, watery stool, and mucous stool. The first two types are considered normal stool, and the latter three are considered diarrheal stool. The difference between dry and loose stool is determined by the presence or absence of stains on filter paper. The frequency of defecation is defined as one pellet or clump (if the number of pellets cannot be determined).

[0082] Diarrhea Index = Loose Stool Rate × Loose Stool Grade. The loose stool rate is the ratio of the number of loose stools excreted by each mouse to the total number of stools. The loose stool grade is determined by the size of the stain formed on filter paper by the loose stool, and is divided into four grades: Grade 1 (less than 1 cm), Grade 2 (1–1.9 cm), Grade 3 (2–3 cm), and Grade 4 (greater than 3 cm).

[0083] During the statistical analysis, first count the grade of each pile of wet feces, then add up all the grades of the rat's loose feces and divide by the number of loose feces to obtain the average grade of the loose feces, which is the loose feces grade. Measurement of grade diameter: If the feces are round, measure the diameter directly; if the feces are oval, measure the diameter of the longest and approximately round parts, add the two together and divide by 2.

[0084] The specific experimental groups and dosing regimens are shown in Table 1.

[0085] Table 1. Effects of inactivated Bacteroides fragilis powder on senna-induced diarrhea in mice; experimental groups and administration regimens.

[0086]

[0087] II. Experimental Results

[0088] Table 2. Effects of inactivated Bacteroides fragilis powder on senna-induced diarrhea in mice.

[0089]

[0090]

[0091] Note: Compared with the model group, **P<0.01, *P<0.05.

[0092] The results are shown in Table 2. The diarrhea index of the positive control group and each dose group of the inactivated Bacteroides fragilis powder was significantly lower than that of the model group (P < 0.01). This indicates that the inactivated Bacteroides fragilis powder provided by this invention can effectively improve diarrhea symptoms in mice and exhibits a dose-response effect.

[0093] Example 4: Effects of Bacteroides fragilis on Senna-induced Diarrhea in Mice

[0094] I. Experimental Methods

[0095] Same as Example 3. The difference is that 70 SPF-grade Kunming mice were randomly divided into 7 groups: a blank control group (group 1), a model group (group 2), a positive control group (group 3, Imodium 4 mg / kg), and groups receiving *Bacteroides fragilis* live bacterial solution (group 4), lysis buffer (group 5), inactivated bacterial solution (group 6), and inactivated bacterial powder (group 7), with 10 mice in each group. After 4 days of continuous gavage administration of senna leaves to establish the model, *Bacteroides fragilis* treatment was administered twice daily, once in the morning and once in the afternoon, for 4 consecutive days. The mice's behavioral characteristics and clinical symptoms were observed daily, with particular attention to diarrhea, which was recorded. After treatment, the mice were dissected for pathological observation.

[0096] The specific experimental groups and dosing regimens are shown in Table 3.

[0097] Table 3. Effects of Bacteroides fragilis on senna-induced diarrhea in mice: experimental groups and administration regimens.

[0098]

[0099]

[0100] II. Experimental Results

[0101] Table 4. Changes in the number of animals with soft stools / total number of animals in each group after treatment with Bacteroides fragilis.

[0102]

[0103] Note: Compared with the model group, *P<0.05.

[0104] As shown in Table 4, the positive control group (Imodium) showed significant efficacy on day 2 of treatment (P < 0.05) and was completely cured on day 3; while the groups treated with Bacteroides fragilis live bacterial solution, lysate, inactivated bacterial solution, and inactivated bacterial powder showed significant efficacy on day 3 (P < 0.05) and were essentially completely cured by day 5. This indicates that the Bacteroides fragilis live bacterial solution, lysate, inactivated bacterial solution, and inactivated bacterial powder provided by this invention all have a significant effect on improving diarrhea symptoms in mice.

[0105] Example 5: Effects of Bacteroides fragilis on magnesium sulfate-induced diarrhea in mice

[0106] I. Experimental Methods

[0107] This embodiment uses 70 SPF-grade Kunming mice, half male and half female, weighing 18-22g, for the experiment. Each experimental mouse is assigned a unique number. Before grouping the animals, the project number, species / strain, sex, cage number, and animal number should be labeled on the mouse cages. Using BioBook software, the mice are randomly grouped according to their initial weight and sex into 6 groups: blank group (group 1), model group (group 2), positive control group (group 3, 0.8g / mL magnesium sulfate solution), low (group 4), medium (group 5), and high (group 6) doses of Bacteroides fragilis ZY-312 inactivated powder obtained in Example 2, and live bacteria group obtained in Example 2 (group 7), with 10 mice in each group.

[0108] Magnesium sulfate diarrhea model establishment: Four hours prior to magnesium sulfate administration, mice were fasted but allowed free access to water. Ten mice (blank group) were administered an equal volume of physiological saline via gavage, while the remaining 60 mice were administered 0.8 g / mL magnesium sulfate solution via gavage at a dose of 10 μl / g body weight, twice daily for four consecutive days. After model establishment, the 60 model mice were randomly divided into corresponding groups.

[0109] Administration regimen: Except for the control group (group 1) and the model group (group 2), which were administered an equal volume of physiological saline by gavage, the other groups were given the corresponding drug (approximately 0.2 mL) by single gavage at a dose of 10 μl / g body weight for 7 consecutive days. After treatment, mice were placed in cages lined with filter paper, one mouse per cage. The filter paper was changed every 1 hour, and the mice were observed for 5 consecutive hours. The number of loose stools and the total number of stools per hour for each mouse were recorded, and the loose stool rate, loose stool grade, and diarrhea index were calculated.

[0110] Mouse stool can be classified into five types: normal stool, normal-looking but high-moisture stool, abnormally shaped soft stool, watery stool, and mucous stool. The first two types are considered normal stool, and the latter three are considered diarrheal stool. The difference between dry and loose stool is determined by the presence or absence of stains on filter paper. The frequency of defecation is defined as one pellet or clump (if the number of pellets cannot be determined).

[0111] Diarrhea Index = Loose Stool Rate × Loose Stool Grade. The loose stool rate is the ratio of the number of loose stools excreted by each mouse to the total number of stools. The loose stool grade is determined by the size of the stain formed on filter paper by the loose stool, and is divided into four grades: Grade 1 (less than 1 cm), Grade 2 (1–1.9 cm), Grade 3 (2–3 cm), and Grade 4 (greater than 3 cm).

[0112] During the statistical analysis, the grade of each pile of wet stool was first counted. Then, the grades of all loose stools of the mouse were added together and divided by the number of loose stools to obtain the average grade of the loose stool, which is the loose stool grade. For measuring the diameter of the grade: if the stool was round, the diameter was measured directly; if the stool was elliptical, the diameter of the longest and approximately round section was measured, and the two were added together and then divided by 2. Specific experimental groups and drug administration protocols are shown in Table 5.

[0113] Table 5. Effects of Bacteroides fragilis on magnesium sulfate-induced diarrhea in mice: experimental groups and administration regimens.

[0114]

[0115] II. Experimental Results

[0116] Table 6. Effects of Bacteroides fragilis on magnesium sulfate-induced diarrhea in mice.

[0117]

[0118] Note: Compared with the model group, **P<0.01, *P<0.05; compared with the live bacteria group, ## P<0.01.

[0119] Experimental results showed that the diarrhea index of the positive control group, all dose groups of inactivated Bacteroides fragilis powder, and the live bacteria group were significantly lower than that of the model group (P < 0.01); however, the diarrhea index of the live Bacteroides fragilis group was significantly higher than that of the inactivated Bacteroides fragilis group at the same dose. These results indicate that the inactivated Bacteroides fragilis powder provided by this invention can effectively improve diarrhea symptoms in mice, exhibits a dose-response effect, and is more effective than the live Bacteroides fragilis.

[0120] Example 6: Effects of Bacteroides fragilis on Escherichia coli-induced diarrhea in mice

[0121] I. Experimental Methods

[0122] (1) Preparation of bacterial culture

[0123] Escherichia coli preserved on slant agar plates were inoculated onto nutrient agar plates and incubated at 37°C for 24 h. Several colonies were then picked and inoculated onto MH broth and incubated at 37°C for 24 h. The broth was diluted with sterile physiological saline and turbided using a 0.5 McFarland turbidimetric tube to adjust the bacterial count to 3 × 10⁻⁶. 8 CFU / mL.

[0124] (2) Treatment trial on mice with Escherichia coli diarrhea model

[0125] Select mice weighing approximately 20g and administer 0.2mL of a bacterial culture containing 3×10⁻⁶ cells via intraperitoneal injection. 8 Mice were induced to have diarrhea by CFU / mL Escherichia coli bacterial solution. The positive control group was administered 0.01 g / mL berberine hydrochloride aqueous solution by gavage. The experimental grouping and methods were the same as in Example 3. The mice were observed continuously for 5 hours, and the diarrhea index was recorded. The specific experimental grouping and administration regimen are shown in Table 7.

[0126] Table 7. Effects of Bacteroides fragilis on Escherichia coli-induced diarrhea in mice: experimental groups and administration regimens.

[0127]

[0128] II. Experimental Results

[0129] Table 8. Effects of Bacteroides fragilis on Escherichia coli-induced diarrhea in mice.

[0130]

[0131] Note: Compared with the model group, **P<0.01, *P<0.05.

[0132] Table 8 shows that the diarrhea index of the positive control group, all dose groups of the inactivated Bacteroides fragilis powder, and the live bacteria group were significantly lower than those of the model group (P < 0.01). However, the diarrhea index of the live Bacteroides fragilis group was significantly higher than that of the inactivated Bacteroides fragilis group at the same dose. These results indicate that the inactivated Bacteroides fragilis powder provided by this invention can effectively improve diarrhea symptoms and exhibits a dose-response effect.

[0133] Example 7: Effects of Bacteroides fragilis on antibiotic-induced diarrhea in rats

[0134] An antibiotic-associated diarrhea model was constructed by combining three antibiotics (clindamycin, ampicillin, and streptomycin) in a 1:1:1 mass ratio, followed by administration of 1×10... 9 NCTC 9343 inactivated bacterial powder suspension at 1×10⁻⁶ cells / mL 9 Cells / mL of inactivated Bacteroides fragilis powder suspension and 1×10 9 Treatment was performed using a CFU / mL suspension of live Bacteroides fragilis (prepared in Example 2), and the efficacy of inactivated Bacteroides fragilis powder was observed.

[0135] I. Experimental Methods

[0136] Sixty female SD rats aged 7-8 weeks and weighing 200-250g were randomly divided into 6 groups: blank control group (group 1), model group (group 2), positive control group (group 3, Imodium 1.5mg / kg), and NCTC 9343 inactivated bacterial powder group (group 4, 1×10⁻⁶). 9 Cells / mL), Bacteroides fragilis inactivated powder group (group 5, 1×10) 9 Cells / mL) and Bacteroides fragilis live bacteria group (group 6, 1×10) 9 CFU / mL), 10 animals per group.

[0137] Animals in each group were intraperitoneally injected with 0.2 mL of a triple antibiotic solution at a concentration of 180 mg / mL, while the control group was injected with an equal volume of physiological saline. This was administered once daily for 7 consecutive days. On day 8, animals in the corresponding groups were administered physiological saline, Imodium suspension, or 1×10 mg / mL triple antibiotic solution via gavage, respectively. 9 NCTC 9343 inactivated bacterial powder (cells / mL), 1×10 9 Cells / mL Bacteroides fragilis inactivated bacterial powder suspension and 1×10 9 CFU / mL Bacteroides fragilis live bacterial suspension was administered twice daily, once in the morning and once in the afternoon, for 7 consecutive days. Rats were observed daily for behavioral and clinical symptoms. After treatment, rats were dissected for pathological examination.

[0138] On the afternoon of day 7 (after modeling) and the afternoon of day 14 (after the experiment), feces from each rat were collected in a single cage within 4 hours, and the wet weight was measured. The feces were then dried in an 80℃ oven for 3 hours, and the dry weight was measured and recorded. The fecal moisture content (%) was calculated using the formula: (fecal wet weight - fecal dry weight) / fecal wet weight × 100.

[0139] The specific experimental groups and dosing regimens are shown in Table 9.

[0140] Table 9. Effects of Bacteroides fragilis on antibiotic-induced diarrhea in rats: experimental groups and administration regimens.

[0141]

[0142]

[0143] II. Experimental Results

[0144] Table 10 Effects of Bacteroides fragilis on antibiotic-induced diarrhea in rats

[0145] Note: Compared with the model group, ***P<0.001, **P<0.01, *P<0.05; compared with the NCTC9343 inactivated bacterial powder group,## P<0.01; compared with the Bacteroides fragilis live powder group, @ P<0.05.

[0146] Table 10 shows that the fecal water content in the *Bacteroides fragilis* inactivated powder group was comparable to that in the positive control group, and was significantly lower than that in the model group (P<0.001). The fecal water content in the NCTC 9343 inactivated powder group was higher, but still significantly lower than that in the model group (P<0.05). The fecal water content in the *Bacteroides fragilis* live powder group was significantly lower than that in the model group (P<0.001), but statistically significant compared to the *Bacteroides fragilis* inactivated powder group (P<0.05). These results indicate that the *Bacteroides fragilis* inactivated powder provided by this invention can effectively improve antibiotic-associated diarrhea symptoms, and its therapeutic efficacy is comparable to that of Imodium, and superior to NCTC 9343 inactivated powder (P<0.01) and the *Bacteroides fragilis* live powder group (P<0.05).

[0147] Example 8: Effect of Bacteroides fragilis on Clostridium difficile-induced diarrhea in mice

[0148] A Clostridium difficile diarrhea model was established by intraperitoneal injection of clindamycin combined with gavage of Clostridium difficile, followed by administration of 1×10 9 NCTC 9343 inactivated bacterial powder suspension (cells / ml), 1×10 9 Cells / ml of inactivated Bacteroides fragilis powder suspension and 1×10 9 Treatment was performed using a CFU / ml suspension of live Bacteroides fragilis powder (prepared in Example 2), and the efficacy of inactivated Bacteroides fragilis powder was observed.

[0149] I. Experimental Methods

[0150] 1. Preparation of bacterial culture

[0151] Clostridium difficile, preserved on slant culture, was inoculated onto CCFA agar plates and incubated at 37°C in an anaerobic gas mixture (80% N2-10% CO2-10% H2) incubator for 72 h. Several colonies were then picked and inoculated into CCFA broth, incubated at 37°C for 72 h, diluted with sterile physiological saline, and turbided using a 0.5 McFarland turbidimetric tube to adjust the bacterial count to 3 × 10⁻⁶. 8 CFU / mL.

[0152] 2. Treatment trial on mice with Clostridium difficile diarrhea model

[0153] Sixty female C57BL / 6 mice aged 6-8 weeks and weighing 18-22g were randomly divided into 6 groups: blank control group (group 1), model group (group 2), positive control group (group 3, Imodium 4mg / kg), and NCTC 9343 inactivated bacterial powder group (group 4, 1×10⁻⁶).9 Cells / mL), Bacteroides fragilis inactivated powder group (group 5, 1×10) 9 Cells / mL) and Bacteroides fragilis live bacteria group (group 6, 1×10) 9 CFU / mL), 10 animals per group.

[0154] Animals in each group were intraperitoneally injected with 100 mg / kg clindamycin solution, while the control group was injected with an equal volume (0.1 mL / 10 g body weight) of physiological saline, once daily for 3 consecutive days; on the 4th day, they were administered 3 × 10 g clindamycin via gavage. 8 CFU / mL Clostridium difficile bacterial suspension, once daily for 7 consecutive days; on day 11, the corresponding groups of animals were administered physiological saline (0.1 mL / 10 g body weight), Imodium suspension, or 1×10⁻⁶ CFU / mL Clostridium difficile bacterial suspension by gavage. 9 NCTC 9343 inactivated bacterial powder suspension and Bacteroides fragilis inactivated bacterial powder suspension (Cells / mL) were administered twice daily, once in the morning and once in the afternoon, for 7 consecutive days.

[0155] Observe the behavioral characteristics and clinical symptoms of mice daily. Record the diarrhea status of each mouse daily. The diarrhea scoring criteria refer to the diarrhea scoring method in the study of Kurita A et al. 0 points: normal or no stool; 1 point: mild diarrhea, stool is slightly wet and soft; 2 points: moderate diarrhea, stool is relatively wet and unformed, and there is mild perianal staining; 3 points: severe diarrhea, watery stool with severe perianal staining (refer to Kurita A, Kado S, Kaneda N, et al. Modified irinotecanhydrochloride (CPT-11) administration schedule improves induetion of delayed-onset diarrhea in rats[J]. Cancer Chem other Pharmacol,2000,46(3):211-220)).

[0156] After treatment, the mice were euthanized on the morning of the 18th day and then 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.):

[0157] (1) Edema

[0158] 0 points: No edema.

[0159] 1 point: Only a small amount (<2X) of mild edema with multiple submucosal dilatations.

[0160] 2 points: Moderate (2-3X) multiple submucosal dilatations with moderate edema.

[0161] 3 points: Severe edema with numerous (>3X) multiple submucosal dilatations.

[0162] 4 points: Severe edema with diffuse submucosal dilatation.

[0163] (2) Inflammatory cell infiltration

[0164] 0 points: No inflammation.

[0165] 1 point: A small number of multifocal neutrophil infiltrations.

[0166] 2 points: Moderate multifocal neutrophil infiltration (involving more of the submucosa).

[0167] 3 points: Extensive multifocal or even clustered neutrophil infiltration (involving more of the submucosa and muscle layer).

[0168] 4 points: The lesion involves the same area as in 3 points, but an abscess or more extensive muscle layer involvement has occurred.

[0169] (3) Intestinal epithelial injury

[0170] 0 points: No changes in intestinal epithelial damage.

[0171] 1 point: Small number of multifocal superficial epithelial lesions (vacuolation, apoptosis of individual cells, attenuation / necrosis of villous tips).

[0172] 2 points: Moderate multifocal superficial epithelial damage (vacuolation, apoptosis of individual cells, attenuation / necrosis of villous tips).

[0173] 3 points: Numerous multifocal epithelial lesions with vacuoles, apoptosis of individual cells, and attenuation / necrosis of villous tips ± pseudomembrane formation (fibrinous exudate containing neutrophils and desquamated epithelium within the lumen).

[0174] 4 points: More obvious pseudomembrane or epithelial ulceration (complete epithelial shedding at the lesion site) appears on the basis of 3 points.

[0175] II. Experimental Results

[0176] Table 11 Effects of Bacteroides fragilis on Clostridium difficile-induced diarrhea scores in mice

[0177]

[0178]

[0179] Note: Compared with the model group, **P<0.01, *P<0.05.

[0180] Table 12 Effects of Bacteroides fragilis on pathological scores in mice with Clostridium difficile-induced diarrhea

[0181]

[0182] Note: Compared with the model group, **P<0.01, *P<0.05; compared with the NCTC 9343 inactivated bacterial powder group, # P<0.05; compared with the live Bacteroides fragilis group, @ P<0.05.

[0183] The results in Tables 11 and 12 show that the diarrhea and pathological scores of the positive control group and the *Bacteroides fragilis* inactivated powder group were significantly lower than those of the model group (P<0.01). The diarrhea and pathological scores of the NCTC 9343 inactivated powder group were higher, but still significantly lower than those of the model group (P<0.05). The diarrhea and pathological scores of the *Bacteroides fragilis* live powder group were significantly lower than those of the model group (P<0.01), but the pathological score was statistically significant compared with the *Bacteroides fragilis* inactivated powder group (P<0.05). These results indicate that the *Bacteroides fragilis* inactivated powder provided by this invention can effectively improve the symptoms of Clostridium difficile infectious diarrhea, and its therapeutic effect is stronger than that of NCTC 9343 inactivated powder (P<0.01) and *Bacteroides fragilis* live powder (P<0.05).

[0184] Example 9: Effects of Bacteroides fragilis on chronic diarrhea induced by high lactose in mice

[0185] Wistar rats were fed a high-lactose diet for 21 days to establish a chronic diarrhea model. They were then treated with live Bacteroides fragilis solution, lysate, inactivated solution and inactivated powder prepared in Example 2 to observe the efficacy of Bacteroides fragilis in treating chronic diarrhea.

[0186] I. Experimental Methods

[0187] 1. Feed preparation

[0188] The feed was formulated according to the AOAC recommended formula (reference: The Official Mehods of Analysis of the AOAC. 14th ed. US, 1984, 877), as shown in Table 13.

[0189] Table 13. Content of various feed components (%)

[0190] Note: 'a' indicates that 1 kg of basic feed contains: Vitamin A (dry, stable) 20000 IU, Vitamin D (dry, stable) 2000 IU, Vitamin E (dry, stable) 100 IU, Vitamin K 5 mg, Choline 2000 mg, Inositol 100 mg, Para-aminobenzoic acid 100 mg, Nicotinic acid 40 mg, Calcium pantothenate 40 mg, Vitamin B1 5 mg, Vitamin B2 8 mg, Vitamin B6 5 mg, Vitamin B... 12 0.03mg, folic acid 2mg, biotin 0.4mg.

[0191] b indicates that 1 kg of basic feed contains: KH2PO4 194.5g, CaCO3 190.7g, NaCl 69.6g, MgSO4 28.65g, FeSO4·7H2O 13.5g, MnSO4·H2O 2.05g, KI 0.395g, ZnSO4·7H2O 0.274g, CuSO4·5H2O 0.239g, CoCl2·6H2O 0.012g.

[0192] 2. Treatment trial on mice with high lactose-induced chronic diarrhea

[0193] Eighty SPF-grade Wistar rats aged 3-4 weeks (half male and half female) were selected and acclimatized for one week. The rats were then housed individually in cages lined with filter paper and iron frames. They were randomly divided into two groups: a control group (group 1, n=10) and a model group (n=70). The control group was fed a basal diet, while the model group was fed a high-lactose diet. A chronic diarrhea model was established after 21 days. Sixty rats with chronic diarrhea were randomly divided into four groups: a model group (group 2), a recovery group (group 3), a positive control group (group 4, probiotic 300 mg / kg), and groups receiving live Bacteroides fragilis solution (group 5), lysate (group 6), inactivated solution (group 7), and inactivated powder (group 8), with 10 rats in each group. On day 22, the rats were administered the drug via gavage (0.1 mL / 10 g body weight) twice daily, once in the morning and once in the afternoon, for 14 consecutive days. Observe the mice's behavioral characteristics and clinical symptoms daily, paying particular attention to diarrhea and recording the findings. On day 35, after anesthesia, blood was drawn from the abdominal aorta and the rats were dissected for pathological observation.

[0194] The specific experimental groups and dosing regimens are shown in Table 14.

[0195] Table 14 Effects of Bacteroides fragilis on rats with high-lactose-induced chronic diarrhea: Experimental groups and administration regimens

[0196]

[0197]

[0198] Similar to Example 3, the behavioral characteristics and clinical symptoms of the mice were observed daily, and the diarrhea status and scores of each Wistar rat were recorded. Serum IL-6 levels were measured after the experiment.

[0199] II. Experimental Results

[0200] Table 15 Effects of Bacteroides fragilis on body weight and diarrhea score in Wistar rats with high lactose-induced chronic diarrhea.

[0201] Note: Compared with the model group, *P<0.05, **P<0.01, ***P<0.001.

[0202] Table 16 Effects of Bacteroides fragilis on serum IL-6 in Wistar rats with lactose-induced chronic diarrhea.

[0203]

[0204]

[0205] Note: Compared with the model group, *P<0.05, **P<0.01.

[0206] The results in Tables 15 and 16 show that after the experiment, the body weight of rats in the positive control group and each of the Bacteroides fragilis groups was significantly higher than that in the model group, while the diarrhea score and serum IL-6 level were significantly lower than those in the model group (P < 0.01). This indicates that the Bacteroides fragilis provided by this invention can effectively improve the symptoms of chronic diarrhea, and the mechanism may be related to the reduction of the expression of the pro-inflammatory factor IL-6. At the same time, it was found that there were no significant differences in body weight, diarrhea score, and IL-6 level between the Bacteroides fragilis groups and the positive group, indicating that the efficacy of Bacteroides fragilis provided by this invention in treating chronic diarrhea is comparable to that of Enterogermina (Bacillus licheniformis live bacteria capsules).

[0207] Example 10: Effect of Bacteroides fragilis on rotavirus-induced diarrhea in mice

[0208] Rotavirus is a major cause of severe dehydrating gastroenteritis in 5-year-old children, with a high incidence in autumn and winter. Its host cell receptor binding sites are VP4 and VP7 proteins. A viral diarrhea model was established in mice by gavage with the Wa strain of rotavirus. Subsequently, mice were treated with live / inactivated NCTC 9343 bacterial suspension, live / inactivated Bacteroides fragilis ZY-312 bacterial suspension, inactivated Bacteroides fragilis ZY-312 powder, and lysis buffer, respectively. The efficacy of these treatments in treating rotavirus-induced diarrhea in mice was observed.

[0209] I. Experimental Methods

[0210] Three-day-old SPF-grade Kunming suckling mice, half male and half female, were randomly divided into 10 groups of 8 mice each: control group (group 1), model group (group 2), and NCTC 9343 live bacteria solution group (group 3, 1×10⁻⁶). 9 CFU / mL), NCTC 9343 inactivated bacterial solution group (group 4, 1×10) 9 Cells / mL), low-dose group of Bacteroides fragilis live bacterial suspension (group 5, 1×10⁻⁶ cells / mL), 5 CFU / mL), Bacteroides fragilis live bacterial suspension medium dose group (group 6, 1×10 7 CFU / mL), high-dose group of Bacteroides fragilis live bacterial solution (group 7, 1×10) 9 CFU / mL), low-dose group of Bacteroides fragilis inactivated bacterial solution (group 8, 1×10⁻⁶ CFU / mL), 5 Cells / mL), Bacteroides fragilis inactivated bacterial solution medium dose group (group 9, 1×10 7 Cells / mL), high-dose group of Bacteroides fragilis inactivated bacterial solution (group 10, 1×10⁻⁶ cells / mL), 9 Cells / mL), Bacteroides fragilis inactivated powder group (group 11, 1×10) 9 Cells / mL) and Bacteroides fragilis lysate group (group 12, 1×10) 9 Cells / mL). After 1 hour of constant-temperature starvation, suckling mice in groups 2-12 were orally inoculated and filtered through a 0.22 μm filter to obtain a titer of 1 × 10⁻⁶. 7 50 μL of PFU-containing Wa strain rotavirus solution was administered to suckling mice in group 1 via gavage, while 50 μL of PBS was administered as a control. 24 hours after gavage challenge, the treated group mice developed yellow, watery stools. Fecal samples were collected and a fecal suspension was prepared. 100 μL of the supernatant was pipetted vertically into the wells of a rotavirus colloidal gold detection kit (Hangzhou Abbott Pharmaceutical Co., Ltd.). The appearance of red bands in the control and detection areas within 10-20 minutes indicated a positive rotavirus test, successful model establishment, and the ability to proceed with treatment. Treatment was administered twice daily for 7 consecutive days. Mouse behavior and clinical symptoms were observed daily, with particular attention to diarrhea, which was recorded. Fecal samples were collected at the end of model establishment and treatment. The supernatant was centrifuged and analyzed using ELISA (Millipore) to detect the amount of rotavirus antigen in the feces. At the experimental endpoint, mice were euthanized by cervical dislocation, and RNA was extracted from the small intestine for qPCR detection of interferon-stimulated genes (ISGs) expression. Specific experimental groupings and administration regimens are shown in Table 17.

[0211] Table 17 Experimental Groups and Dosing Regimens

[0212]

[0213]

[0214] II. Experimental Results

[0215] The diarrhea scores of the suckling mice at the start of treatment and 7 days after treatment are shown in Table 18, and the amount of rotavirus antigen in the feces is shown in Table 19.

[0216] Table 18 Effects of Bacteroides fragilis on rotavirus-induced diarrhea scores in mice

[0217] Note: Compared with the model group, ***P<0.001, ****P<0.0001; compared with the NCTC 9343 live bacteria solution group, # P<0.05, ### P<0.001, #### P<0.0001; compared with the NCTC 9343 inactivated bacterial solution group, + P<0.05, +++ P < 0.001, T tests.

[0218] Table 19 Fecal antigen levels

[0219]

[0220]

[0221] Note: Compared with the model group, ***P<0.001; compared with the NCTC 9343 live bacteria group, # P<0.05, ## P<0.01; compared with the NCTC 9343 inactivated bacterial solution group, +++ P < 0.001, T tests.

[0222] According to the data in Tables 18 and 19, after the experiment, the diarrhea scores of rats in the NCTC 9343 live bacteria and inactivated bacteria groups (P<0.05) and the various groups of Bacteroides fragilis ZY-312 (P<0.0001) were significantly lower than those in the model group, indicating that Bacteroides fragilis ZY-312 provided by this invention can effectively improve the symptoms of virus-induced diarrhea. At the same time, it was found that the amount of rotavirus antigen in the feces of the Bacteroides fragilis live bacteria solution, inactivated bacteria solution, inactivated bacteria powder, and lysate groups was significantly reduced compared with the model group (P<0.001), while there was no significant difference between the NCTC 9343 live bacteria and inactivated bacteria groups and the model group. This suggests that the improvement of diarrhea by Bacteroides fragilis ZY-312 may be related to reducing the viral antigen load in the body, thereby reducing the severity of diarrhea by reducing the viral antigen load in the host. Meanwhile, Figure 4 shows that compared to the model group, the expression levels of interferon-stimulated genes (ISGs) in each group of *Bacteroides fragilis* ZY-312 were significantly increased (P<0.05). ISG expression drives the antiviral state of infected and uninfected neighboring cells, directly interfering with viral replication and transmission. This indicates that *Bacteroides fragilis* can upregulate ISG expression, initiate dendritic cell-mediated connection between innate and adaptive immunity through the interferon-related pathway, induce a restrictive antiviral state in cells, induce apoptosis in infected cells, and regulate immune cell subsets crucial for antiviral responses, coordinating the host's immune response to the virus, producing potent antiviral activity, and improving diarrhea. The effects of each group of *Bacteroides fragilis* ZY-312 were superior to those of live and inactivated NCTC 9343 bacteria (P<0.05). The improvement effects of low, medium, and high doses of *Bacteroides fragilis* ZY-312 bacterial suspension on viral diarrhea were: high dose > medium dose ≈ low dose; the effect of live bacteria was comparable to that of inactivated bacteria.

[0223] Example 11: Resistance of Bacteroides fragilis to norovirus infection

[0224] Human norovirus causes over 90% of nonbacterial gastroenteritis cases worldwide, with peak incidence in spring and autumn. It causes severe morbidity and mortality globally, with the two main genotypes infecting humans being GI and GII. Transmission occurs via the fecal-oral route after the ingestion of packaged viral particles. After an incubation period of 24-48 hours, the virus causes symptomatic diarrhea and vomiting within the next 12-60 hours. Norovirus primarily infects individuals aged 5-17 years and adults, causing diarrhea along with upper respiratory symptoms. Its binding site is the P structure of the VP1 protein, responsible for recognizing host histoblood group antigen (HBGA) receptors. Blocking the binding of norovirus to HBGAs reduces viral attachment to host cells, weakens viral infectivity, and alleviates the diarrhea it causes.

[0225] I. Experimental Methods

[0226] The GII.17 norovirus (NoV) P protein (GenBank ID: KU557839) was expressed in Escherichia coli BL21 competent cells, and the protein size and concentration were determined by SDS-PAGE electrophoresis. Boiled human type A / B saliva was diluted 1:1000 with PBS and coated onto a 96-well ELISA plate overnight (100 μL / well), then blocked with 5% skim milk. P protein (GII.17) (1 μg / mL) was pre-incubated with serially diluted Bacteroides fragilis at 37°C for 1 hour, then added to each well of the 96-well plate. 100 μL of mouse anti-NoV polyclonal antibody serum (1:3000, homemade) was added to each well, and the plate was incubated at 37°C for 1 hour. HRP-goat anti-mouse IgG antibody (1:6000, Abcam) was added and incubated at 37°C for 1 hour. All samples were washed five times with 0.05% PBS-Tween 20. A substrate buffer containing TMB (freshly prepared, 100 μL / well) was added, and the plate was incubated at room temperature in the dark for 10 minutes for color development. The reaction was stopped by adding 50 μL of 2 mol / L H2SO4, and the OD value was measured at 450 nm using a microplate reader. The experimental groups and administration regimens are shown in Table 20, and the bacterial concentrations of Bacteroides fragilis ZY-312 are shown in Table 21.

[0227] Table 20 Experimental groupings of Bacteroides fragilis against norovirus

[0228]

[0229]

[0230] Table 21 Dosage concentrations of live and inactivated Bacteroides fragilis solutions

[0231]

[0232] II. Experimental Results

[0233] The blocking ability of Bacteroides fragilis to bind to NoV-HBGAs was evaluated by the blocking rate, where the blocking rate = (1-OD) / ( ... 450 Experimental group / OD 450 (Mean value of positive control group) × 100%. The results are shown in Table 22.

[0234] Table 22. Blocking rate of Bacteroides fragilis binding to NoV-HBGAs

[0235]

[0236]

[0237] Note: Compared with the positive control group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, T tests.

[0238] As shown in Table 22, compared with the positive control group, all groups of Bacteroides fragilis were able to block the binding of NoV-HBGAs (P<0.05), and OD 450 The value decreased significantly (P<0.05), 1×10 9 The blocking rate of Bacteroides fragilis at a concentration of Cells / mL was nearly 50% (P<0.0001). Furthermore, with increasing drug concentration of Bacteroides fragilis, the OD... 450 The lower the value, the stronger the ability to block NoV-HBGAs binding, indicating that Bacteroides fragilis can dose-dependently weaken viral infectivity, has strong antiviral activity against norovirus, and improves and resists norovirus-induced diseases such as diarrhea. The improvement effect of low, medium, and high doses of Bacteroides fragilis bacterial suspension on viral diarrhea is: high dose > medium dose > low dose; the effect of live bacteria is comparable to that of inactivated bacteria.

[0239] Example 12 Effect of Bacteroides fragilis on immunogenicity in rabbits with Cryptosporidium infection and diarrhea

[0240] A parasite-associated diarrhea model was established in New Zealand white rabbits by gavage with Cryptosporidium cuniculus oocysts (extracted from a single positive fecal sample from a rabbit farm in Jilin). Subsequently, the rabbits were treated with Bacteroides fragilis live bacterial solution, inactivated bacterial powder, and lysis buffer (prepared in Example 2), and the efficacy of these three treatments was observed.

[0241] I. Experimental Methods

[0242] New Zealand white rabbits aged 35 days were randomly divided into 6 groups of 8 rabbits each: control group (group 1), model group (group 2), positive control group (nitrazepam-methyl) (group 3), Bacteroides fragilis inactivated powder group (group 4), Bacteroides fragilis lysate group (group 5), and Bacteroides fragilis live bacterial solution group (group 6). New Zealand white rabbits in groups 2-6 were inoculated by gavage with 1.5 × 10⁻⁶ ozonated bacteria. 4 Cryptosporidium oocysts were collected, and the control group was administered physiological saline by gavage. Three days after gavage, New Zealand rabbits developed yellow, watery stools. Fecal samples were collected and a fecal suspension was prepared. Oocyst structures were observed under a microscope, indicating successful modeling and readiness for treatment. The control group received physiological saline, while the treatment group received the medication twice daily for seven consecutive days. The diarrhea status of the New Zealand rabbits was observed daily, and fecal samples were collected on the first day of treatment and at the end of treatment. The number of oocysts was observed under a microscope. Specific experimental groupings and administration protocols are shown in Table 23.

[0243] Table 23 Experimental groupings and administration regimens of Bacteroides fragilis live bacteria, lysate, and inactivated powder against Cryptosporidium-induced diarrhea in New Zealand rabbits.

[0244]

[0245] II. Experimental Results

[0246] The number of oocysts in the feces of New Zealand rabbits daily during the treatment period is shown in Table 24. The total number of oocysts per New Zealand rabbit during the treatment period is shown in Table 24. Figure 5 As shown.

[0247] Table 24. Number of oocysts in daily feces of New Zealand rabbits during drug administration.

[0248]

[0249]

[0250] Note: Compared with the model group (T tests), *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0251] According to the data in Table 24, after the experiment, the number of oocysts in the feces of New Zealand rabbits in both the positive control group (nitrothiazide) and the Bacteroides fragilis groups was significantly lower than that in the model group (P<0.01), indicating that the Bacteroides fragilis provided by this invention can effectively improve diarrhea symptoms caused by Cryptosporidium. Specifically, in the Bacteroides fragilis live bacterial solution group, the number of oocysts in the feces of New Zealand rabbits was zero at the endpoint, indicating that the Bacteroides fragilis live bacterial solution can significantly improve parasite-related diarrhea (P<0.0001). Figure 5 It was found that during the treatment period, the total number of oocysts in the feces of each New Zealand rabbit in the positive control group (nitrothiacetylcholine) and each of the Bacteroides fragilis groups was significantly lower than that in the model group (P<0.0001). Furthermore, the total number of oocysts in the feces of each New Zealand rabbit in the Bacteroides fragilis lysate group and the Bacteroides fragilis live bacterial solution and inactivated bacterial powder groups was significantly lower than that in the positive control group (nitrothiacetylcholine) (P<0.0001). This indicates that Bacteroides fragilis is more effective than the existing drug nitrothiacetylcholine in treating diarrhea caused by Cryptosporidium.

[0252] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. The use of Bacteroides fragilis in the preparation of compositions for improving and / or treating viral infectious diarrhea, characterized in that, The *Bacteroides fragilis* is a morphologically intact inactivated bacterium selected from *Bacteroides fragilis* ZY-312 with accession number CGMCC No. 10685. The viral infectious diarrhea is diarrhea caused by norovirus infection.

2. The application according to claim 1, characterized in that, The dosage form of the composition is pills, tablets, granules, capsules, powders, suspensions, oral liquids, or enemas.

3. The application according to claim 2, characterized in that, The composition is administered orally or via enema.

4. The application according to claim 1, characterized in that, The dosing cycle of the composition can be intermittent, periodic, continuous, or long-term.

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