Application of bacteroides fragilis in improving and treating functional gastrointestinal diseases

Abstract

Different animal model tests prove that the bacteroides fragilis, especially viable bacteria, lysate, inactivated bacteria, extracts and the like of the bacteroides fragilis ZY-312 with the preservation number of CGMCC No.10685 and the bacteroides fragilis NCTC 9343 have the effects of improving and treating the functional gastrointestinal diseases, especially functional dyspepsia and irritable bowel syndrome. For functional dyspepsia, bacteroides fragilis can effectively improve weight loss, anorexia, visceral hypersensitivity and gastric motility disorder caused by functional dyspepsia. For irritable bowel syndrome, bacteroides fragilis can effectively improve diarrhea symptoms and reduce intestinal sensitivity.

Description

Technical Field

[0001] The invention relates to the technical field of application of Bacteroides fragilis, and in particular to application of inhibiting Bacteroides fragilis in preparing medicines for preventing and treating functional gastrointestinal diseases. Background Art

[0002] Functional gastrointestinal disorders (FGIDs), also known as abnormal gut-brain interaction, are a group of non-organic digestive tract disorders characterized by abdominal pain, nausea and vomiting, diarrhea and constipation, and difficulty in excreting food or feces due to factors such as motility disorders, visceral hypersensitivity, changes in mucosal immune function, changes in intestinal flora, and abnormal central nervous system processing functions. Clinically, functional dyspepsia and irritable bowel syndrome are more common. Among them, functional dyspepsia (FD) refers to a group of clinical syndromes caused by symptoms caused by gastric and duodenal dysfunction without organic diseases. The main symptoms include postprandial fullness, early satiety, upper abdominal distension and pain, belching, anorexia, nausea, etc. The pathogenesis is not yet clear, and it may be related to gastrointestinal motility disorders, visceral hypersensitivity, decreased relaxation function of the gastric fundus to food, mental and social factors, etc. Irritable bowel syndrome (IBS) is a common functional intestinal disease characterized by abdominal pain or discomfort accompanied by changes in bowel habits without organic lesions. Clinically, it can be divided into diarrhea, constipation and mixed types according to the characteristics of defecation and the properties of stool. The pathogenesis is not yet clear, but it is currently believed to be the result of multiple factors and multiple pathogenic mechanisms, including abnormal gastrointestinal motility, visceral sensory abnormalities, intestinal infection, gastrointestinal hormones, and mental and psychological disorders.

[0003] More and more studies have confirmed that intestinal microbiota plays an important role in FGIDs and is one of the important factors causing the disease. The incidence of small intestinal bacterial overgrowth (SIBO) in IBS patients and FD patients is significantly increased compared with healthy controls. In about 10% of IBS patients, symptoms can be traced back to episodes of infectious gastroenteritis that lead to persistent changes in the structure of the microbiota. Other studies have shown that by transplanting feces from IBS patients into feces from germ-free (GF) mice, the gastrointestinal transit, visceral hypersensitivity, and intestinal permeability of animals can be changed.

[0004] The intestinal microbiota is a potential target for the treatment of FGIDs. Most current microbiota-oriented treatment strategies (including probiotics and synbiotics, prebiotics and dietary interventions, antibiotics, and the recent fecal microbiota transplantation (FMT)) have not shown consistent results. Among them, the strains used in clinical studies related to probiotics are mostly first-generation probiotics, so it is necessary to develop second-generation probiotics with better effects, better safety, and stronger tolerance. Summary of the invention

[0005] In view of the above problems, the present invention provides use of Bacteroides fragilis in improving and / or treating functional gastrointestinal diseases.

[0006] In order to better solve the above problems, the present invention adopts the following technical solutions:

[0007] First aspect

[0008] The present invention provides an application of Bacteroides fragilis in preparing a product for improving and / or treating a functional gastrointestinal disease, wherein the Bacteroides fragilis is selected from Bacteroides fragilis ZY-312 or Bacteroides fragilis NCTC 9343, and the active ingredient is a live bacterium, an inactivated bacterium or an extract of Bacteroides fragilis; and the functional gastrointestinal disease is functional dyspepsia.

[0009] According to an embodiment of the present invention, the Bacteroides fragilis is selected from live bacteria of Bacteroides fragilis ZY-312, inactivated bacteria of Bacteroides fragilis ZY-312, live bacteria of Bacteroides fragilis NCTC 9343, inactivated bacteria of Bacteroides fragilis NCTC 9343, and an extract of Bacteroides fragilis ZY-312.

[0010] According to an embodiment of the present invention, the Bacteroides fragilis is selected from the inactivated bacteria of Bacteroides fragilis ZY-312 and the inactivated bacteria of Bacteroides fragilis NCTC 9343; preferably, the Bacteroides fragilis is selected from the inactivated bacteria of Bacteroides fragilis ZY-312.

[0011] According to an embodiment of the present invention, the inactivated bacteria are morphologically intact inactivated bacteria and / or morphologically incomplete inactivated bacteria.

[0012] According to an embodiment of the present invention, the inactivated bacteria are inactivated by any one or more of dry heat, moist heat, filtration, organic solvents, chemical reagents, ultraviolet or infrared rays, fermentation, freeze drying, genetic recombination, genetic modification or transformation.

[0013] According to an embodiment of the present invention, the inactivated bacteria is inactivated Bacteroides fragilis bacteria powder.

[0014] Preferably, the preparation method of the inactivated Bacteroides fragilis powder is as follows: fermenting and culturing Bacteroides fragilis and centrifuging to obtain bacterial cells, washing the bacterial cells with a sodium chloride aqueous solution and centrifuging to obtain washed bacterial cells, adding an excipient to resuspend, inactivating and centrifuging to obtain inactivated bacterial sludge, and drying.

[0015] Further preferably, the preparation method of the inactivated Bacteroides fragilis powder is as follows:

[0016] (1) fermenting Bacteroides fragilis, centrifuging the fermentation broth after the fermentation is completed, and collecting the bacterial cells;

[0017] (2) adding sodium chloride aqueous solution at a weight volume ratio of 1 g of bacterial cells to sodium chloride aqueous solution (10-30) mL) for washing, and centrifuging to obtain washed bacterial cells;

[0018] (3) adding the first excipient solution to the washed bacterial cells, mixing and resuspending to obtain a bacterial cell solution, then performing an inactivation treatment, centrifuging, and collecting the inactivated bacterial sludge;

[0019] (4) adding a second excipient solution to the inactivated bacteria sludge obtained in step (3) to obtain an inactivated bacteria stock solution;

[0020] (5) Drying the inactivated bacteria stock solution obtained in step (4) until the residual moisture is less than 5 wt %, thereby obtaining inactivated Bacteroides fragilis bacteria powder.

[0021] According to an embodiment of the present invention, in step (1), the bacterial count of the fermentation liquid reaches 10 8 CFU / mL and above.

[0022] According to an embodiment of the present invention, in step (2), the mass concentration of the sodium chloride aqueous solution is 0.6-1.5wt%, preferably 0.65-1.2wt%, more preferably 0.8-1.0wt%, most preferably 0.85-0.95wt%, for example 0.9wt% sodium chloride aqueous solution.

[0023] According to an embodiment of the present invention, the first excipient in step (3) is the same as or different from the second excipient in step (4), and is independently selected from at least one of mannitol, sorbitol, maltodextrin, lactose, sodium chloride, maltose, sucrose, glucose, trehalose, dextran, proline, lysine, alanine, casein, and skim milk.

[0024] According to an embodiment of the present invention, in step (3), the weight-to-volume ratio of the bacteria to the first excipient solution is 1 g:(5-40) mL.

[0025] According to an embodiment of the present invention, in step (3) and / or step (4), the mass fraction of the excipient is 4 to 30 wt%.

[0026] According to an embodiment of the present invention, in step (3) and / or step (4), the excipient solution is selected from a sodium chloride aqueous solution; preferably, the excipient solution is selected from physiological saline, such as a 0.9 wt % sodium chloride aqueous solution.

[0027] According to an embodiment of the present invention, in step (3), the inactivation method is selected from at least one of heat inactivation, freeze inactivation or chemical inactivation, preferably heat inactivation.

[0028] Exemplarily, the temperature of the heat inactivation is 60 to 100° C., and the time of the heat inactivation is 10 to 60 minutes.

[0029] 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 consistent with the weight of the bacterial solution before inactivation in step (3).

[0030] Illustratively, the second excipient solution is the same as the first excipient solution.

[0031] According to an embodiment of the present invention, in step (5), the drying method is selected from vacuum freeze drying and spray drying, preferably vacuum freeze drying.

[0032] According to an embodiment of the present invention, 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.

[0033] Exemplarily, the vacuum freeze-drying process includes: pre-freezing at -40±2°C for 1 to 3 hours, pre-freezing at -20±2°C for 0.5 to 1 hour, and finally freezing at -40±2°C for 0.5 to 2 hours, and preparing the inactivated bacterial powder through primary drying and analytical drying under a vacuum degree of 0.25 mbar.

[0034] According to an embodiment of the present invention, in the preparation method, the centrifugal conditions are not specifically limited, as long as the desired centrifugal effect can be achieved, for example, the centrifugal speed is 10000-20000 rpm.

[0035] According to an embodiment of the present invention, the product is selected from any one of a pharmaceutical composition, a food, a health product or a food additive.

[0036] Preferably, the pharmaceutical composition contains a pharmaceutically effective dose of inactivated Bacteroides fragilis ZY-312 powder with a preservation number of CGMCC No.10685.

[0037] 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.; preferably, the pharmaceutical composition is administered orally or by enema.

[0038] The administration cycle of the above-mentioned pharmaceutical composition is intermittent administration, periodic administration, continuous administration or long-term administration.

[0039] Preferably, the extract of Bacteroides fragilis ZY-312 contains Bacteroides fragilis capsular polysaccharide A.

[0040] According to an embodiment of the present invention, the method for preparing an extract of Bacteroides fragilis ZY-312 comprises the following steps:

[0041] (i) centrifuging the fermented Bacteroides fragilis culture, resuspending the culture in purified water, adjusting the pH to 3.5, extracting, centrifuging, and collecting the supernatant to obtain a crude sugar solution;

[0042] (ii) the crude sugar solution is ultrafiltered and concentrated through a 10KD ultrafiltration membrane to remove small molecular impurities until the conductivity is stable, and the reflux liquid is collected;

[0043] (iii) adding an equal volume of buffer to the reflux liquid for salt conversion; performing DEAE Sepharose Fast Flow ion exchange column chromatography at a flow rate of 20 mL / min, gradient elution for 25 column volumes, collecting fractions in a 100 mL / bottle (component), tracking and monitoring by SEC-HPLC, combining fractions with a single, symmetrical absorption peak at 206 nm, ultrafiltration through a 10KD ultrafiltration membrane, adding purified water for repeated ultrafiltration until the conductivity is stable, collecting the reflux liquid, and lyophilizing to obtain a Bacteroides fragilis extract.

[0044] According to an embodiment of the present invention, in step (i), purified water is added to resuspend the bacterial cell pellet at a weight ratio of 1 g:6 g of purified water;

[0045] And / or, extraction at 100°C for 1.5h.

[0046] According to an embodiment of the present invention, in step (iii), the buffer is 40 mmol / L Tris-HCl (pH 8.5);

[0047] And / or, the eluent is 20mmol / L Tris-HCl (pH8.5, containing 0.2mol / L NaCl).

[0048] According to an embodiment of the present invention, the method for preparing an extract of Bacteroides fragilis ZY-312 comprises the following steps:

[0049] (i) centrifuging the fermented Bacteroides fragilis culture, resuspending the culture by adding purified water at a weight ratio of 1 g:6 g, adjusting the pH to 3.5 with 1 mol / L hydrochloric acid solution, extracting at 100° C. for 1.5 h, cooling to room temperature, centrifuging at 12000 g for 10 min at room temperature, and collecting the supernatant to obtain a crude sugar solution;

[0050] (ii) the crude sugar solution is ultrafiltered and concentrated through a 10KD ultrafiltration membrane to remove small molecular impurities until the conductivity is stable, and the reflux liquid is collected;

[0051] (iii) adding an equal volume of 40 mmol / L Tris-HCl (pH 8.5) to the reflux liquid for salt conversion; performing DEAE Sepharose Fast Flow ion exchange column chromatography (16 mm × 200 mm) at a flow rate of 20 mL / min, gradient eluting with 20 mmol / L Tris-HCl (pH 8.5, containing 0.2 mol / L NaCl) for 25 column volumes, collecting fractions in a 100 mL / bottle (component), monitoring by SEC-HPLC, combining fractions with a single, symmetrical absorption peak at 206 nm, ultrafiltration using a 10KD ultrafiltration membrane, adding purified water for repeated ultrafiltration until the conductivity is stable, collecting the reflux liquid, and lyophilizing to obtain a Bacteroides fragilis extract.

[0052] Second aspect

[0053] The present invention provides an application of Bacteroides fragilis in preparing a product for improving and / or treating a functional gastrointestinal disease, wherein the Bacteroides fragilis is selected from Bacteroides fragilis ZY-312 or Bacteroides fragilis NCTC 9343, and the active ingredient is an inactivated bacterium or a lysate of Bacteroides fragilis; and the functional gastrointestinal disease is irritable bowel syndrome.

[0054] According to an embodiment of the present invention, irritable bowel syndrome includes diarrhea-type irritable bowel syndrome and post-inflammatory irritable bowel syndrome.

[0055] According to an embodiment of the present invention, the Bacteroides fragilis is selected from the inactivated bacteria of Bacteroides fragilis ZY-312, the inactivated bacteria of Bacteroides fragilis NCTC 9343, and the lysate of Bacteroides fragilis ZY-312; preferably, the Bacteroides fragilis is selected from the inactivated bacteria of Bacteroides fragilis ZY-312.

[0056] According to an embodiment of the present invention, the inactivated bacteria are morphologically intact inactivated bacteria and / or morphologically incomplete inactivated bacteria.

[0057] According to an embodiment of the present invention, the inactivated bacteria are inactivated by any one or more of dry heat, moist heat, filtration, organic solvents, chemical reagents, ultraviolet or infrared rays, fermentation, freeze drying, genetic recombination, genetic modification or transformation.

[0058] According to an embodiment of the present invention, the inactivated bacteria is inactivated Bacteroides fragilis bacteria powder.

[0059] Preferably, the preparation method of the inactivated Bacteroides fragilis powder is as follows: fermenting and culturing Bacteroides fragilis and centrifuging to obtain bacterial cells, washing the bacterial cells with a sodium chloride aqueous solution and centrifuging to obtain washed bacterial cells, adding an excipient to resuspend, inactivating and centrifuging to obtain inactivated bacterial sludge, and drying.

[0060] Further preferably, the preparation method of the inactivated Bacteroides fragilis powder is as follows:

[0061] (1) fermenting Bacteroides fragilis, centrifuging the fermentation broth after the fermentation is completed, and collecting the bacterial cells;

[0062] (2) adding sodium chloride aqueous solution at a weight volume ratio of 1 g of bacterial cells to sodium chloride aqueous solution (10-30) mL) for washing, and centrifuging to obtain washed bacterial cells;

[0063] (3) adding the first excipient solution to the washed bacterial cells, mixing and resuspending to obtain a bacterial cell solution, then performing an inactivation treatment, centrifuging, and collecting the inactivated bacterial sludge;

[0064] (4) adding a second excipient solution to the inactivated bacteria sludge obtained in step (3) to obtain an inactivated bacteria stock solution;

[0065] (5) Drying the inactivated bacteria stock solution obtained in step (4) until the residual moisture is less than 5 wt %, thereby obtaining inactivated Bacteroides fragilis bacteria powder.

[0066] According to an embodiment of the present invention, in step (1), the bacterial count of the fermentation liquid reaches 10 8 CFU / mL and above.

[0067] According to an embodiment of the present invention, in step (2), the mass concentration of the sodium chloride aqueous solution is 0.6-1.5wt%, preferably 0.65-1.2wt%, more preferably 0.8-1.0wt%, most preferably 0.85-0.95wt%, for example 0.9wt% sodium chloride aqueous solution.

[0068] According to an embodiment of the present invention, the first excipient in step (3) is the same as or different from the second excipient in step (4), and is independently selected from at least one of mannitol, sorbitol, maltodextrin, lactose, sodium chloride, maltose, sucrose, glucose, trehalose, dextran, proline, lysine, alanine, casein, and skim milk.

[0069] According to an embodiment of the present invention, in step (3), the weight-to-volume ratio of the bacteria to the first excipient solution is 1 g:(5-40) mL.

[0070] According to an embodiment of the present invention, in step (3) and / or step (4), the mass fraction of the excipient is 4 to 30 wt%.

[0071] According to an embodiment of the present invention, in step (3) and / or step (4), the excipient solution is selected from a sodium chloride aqueous solution; preferably, the excipient solution is selected from physiological saline, such as a 0.9 wt % sodium chloride aqueous solution.

[0072] According to an embodiment of the present invention, in step (3), the inactivation method is selected from at least one of heat inactivation, freeze inactivation or chemical inactivation, preferably heat inactivation.

[0073] Exemplarily, the temperature of the heat inactivation is 60 to 100° C., and the time of the heat inactivation is 10 to 60 minutes.

[0074] 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 consistent with the weight of the bacterial solution before inactivation in step (3).

[0075] Illustratively, the second excipient solution is the same as the first excipient solution.

[0076] According to an embodiment of the present invention, in step (5), the drying method is selected from vacuum freeze drying and spray drying, preferably vacuum freeze drying.

[0077] According to an embodiment of the present invention, 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.

[0078] Exemplarily, the vacuum freeze-drying process includes: pre-freezing at -40±2°C for 1 to 3 hours, pre-freezing at -20±2°C for 0.5 to 1 hour, and finally freezing at -40±2°C for 0.5 to 2 hours, and preparing the inactivated bacterial powder through primary drying and analytical drying under a vacuum degree of 0.25 mbar.

[0079] According to an embodiment of the present invention, in the preparation method, the centrifugal conditions are not specifically limited, as long as the desired centrifugal effect can be achieved, for example, the centrifugal speed is 10000-20000 rpm. According to an embodiment of the present invention, the product can be any one of a pharmaceutical composition, a food, a health product or a food additive.

[0080] Preferably, the pharmaceutical composition contains a pharmaceutically effective dose of inactivated Bacteroides fragilis ZY-312 powder with a preservation number of CGMCC No.10685.

[0081] 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.; preferably, the pharmaceutical composition is administered orally or by enema.

[0082] The above-mentioned pharmaceutical composition, the administration cycle of the pharmaceutical composition is intermittent administration, periodic administration, continuous administration or long-term administration.

[0083] According to an embodiment of the present invention, the lysate of Bacteroides fragilis ZY-312 is prepared by ultrasonic disruption.

[0084] Preferably, the method for preparing the lysate of Bacteroides fragilis ZY-312 comprises the following steps:

[0085] (1) fermenting Bacteroides fragilis, centrifuging the fermentation liquid after the fermentation is completed, and collecting bacterial sludge;

[0086] (2) adding physiological saline in a weight ratio of bacterial sludge to physiological saline of 1:(5-15) and stirring to resuspend;

[0087] (3) The resuspended bacterial solution is lysed by ultrasonication;

[0088] (4) After lysis, the cells are centrifuged and filtered to obtain a lysate.

[0089] According to an embodiment of the present invention, in step (3), the lysis conditions are lysis for 30 min, On for 10 seconds, Off for 10 seconds;

[0090] And / or, the centrifugation conditions in step (4) are 6000 rpm, 10 min, and 4°C.

[0091] Compared with the prior art, the present invention has the following beneficial effects:

[0092] The present invention proves through different animal model experiments that Bacteroides fragilis, especially the live bacteria, lysate, inactivated bacteria, extracts, etc. of Bacteroides fragilis ZY-312 with a preservation number of CGMCC No. 10685 and Bacteroides fragilis NCTC 9343 have the effect of improving and treating functional gastrointestinal diseases, especially functional dyspepsia and irritable bowel syndrome. For functional dyspepsia, Bacteroides fragilis can effectively improve weight loss, anorexia, visceral hypersensitivity and gastric motility disorders of functional dyspepsia. For irritable bowel syndrome, Bacteroides fragilis can effectively improve diarrhea symptoms and reduce intestinal sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0093] Figure 1 This is a colony morphology diagram of Bacteroides fragilis ZY-312 after anaerobic culture of the present invention;

[0094] Figure 2This is a Gram staining microscopic image of Bacteroides fragilis ZY-312 of the present invention;

[0095] Figure 3 This is a transmission electron microscope image of the inactivated Bacteroides fragilis ZY-312 powder of the present invention;

[0096] Figure 4 It is the chemical structural formula of the structural unit of capsular polysaccharide A in the Bacteroides fragilis extract of the present invention.

[0097] Strain deposit information

[0098] The microbial strains used in the implementation of the present invention have been deposited in the General Microbiological Center (CGMCC) of the China Microbiological Culture Collection Committee (No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing) on ​​April 2, 2015. Classification name: Bacteroides fragilis ZY-312, deposit number CGMCC No.10685. Bacteroides fragilis ZY-312 was isolated and obtained by the applicant of the present invention, and has been protected by a patent (patent number 201510459408.X). According to the provisions of the Patent Examination Guidelines, the public can buy it from commercial channels or it has been authorized, and no deposit is required, that is, no deposit certificate is required. DETAILED DESCRIPTION

[0099] The technical scheme of the present invention will be further described in detail in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary illustrations and explanations 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 included in the scope that the present invention is intended to protect.

[0100] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.

[0101] Unless defined otherwise or clearly indicated by the context, all technical and scientific terms in the present disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs.

[0102] Example 1 Fermentation culture of Bacteroides fragilis

[0103] The Bacteroides fragilis ZY-312 strain was streaked onto a blood plate and cultured anaerobically for 48 hours. The colony morphology, staining characteristics, size, club shape, and distribution were observed.

[0104] Colony characteristics: After 48 hours of culture on a blood plate, Bacteroides fragilis ZY-312 is round, slightly convex, translucent, white, smooth, and non-hemolytic. The colony diameter is between 1 and 3 mm. Figure 1 .

[0105] Microscopic morphology: Gram staining of Bacteroides fragilis ZY-312 showed that it was a Gram-negative bacterium with a typical rod shape, blunt and darkly stained ends, and a non-stained part in the middle of the bacteria that looked like a vacuole. Figure 2 .

[0106] A single colony was selected and inoculated into the culture medium for fermentation culture for 8 hours (temperature was 37°C). The obtained bacterial solution was centrifuged at a speed of 3000 r / min for 15 min. The supernatant was removed and the precipitate was collected to obtain Bacteroides fragilis ZY-312 bacterial sludge.

[0107] Example 2 Sample preparation

[0108] 1. Live Bacteroides fragilis

[0109] The bacterial sludge of Bacteroides fragilis prepared in Example 1 was taken, and physiological saline was added to the bacterial sludge at a ratio of bacterial sludge: physiological saline = 1: (5-15) (m:m), and the mixture was stirred and resuspended to prepare a live bacterial solution of Bacteroides fragilis.

[0110] 2. Preparation of Bacteroides fragilis lysate

[0111] The lysate of Bacteroides fragilis was prepared by ultrasonic disruption. The bacterial sludge of Bacteroides fragilis prepared in Example 1 was taken, and physiological saline was added to the bacterial sludge at a ratio of bacterial sludge: physiological saline = 1: (5-15) (m:m), and stirred and resuspended. The resuspended bacterial solution was lysed by an ultrasonic instrument for 30 minutes, On for 10 seconds, Off for 10 seconds, and the bacterial cell lysis efficiency of the system reached 99%. After lysis, it was centrifuged at 6000 rpm, 10 minutes, and 4°C, and filtered for use.

[0112] 3. Preparation of inactivated Bacteroides fragilis

[0113] (1) Take the Bacteroides fragilis bacterial sludge prepared in Example 1, add physiological saline at a ratio of bacterial sludge: physiological saline = 1: (10-30) (m:v) to resuspend and wash the bacterial sludge, and centrifuge again to collect the washed bacterial cells.

[0114] (2) Add an excipient prepared by mixing 5% maltodextrin and 0.9% sodium chloride to the bacterial cells obtained in step (1) at a ratio of bacterial cells: excipient = 1: (5-15) (m:m), stir and disperse, heat inactivate at (70-100) ± 5°C for (20-40) ± 5 minutes, and then centrifuge to collect the bacterial sludge.

[0115] (3) adding excipients to the inactivated bacteria sludge collected in step (2) to make the total weight consistent with the weight of the bacteria solution before inactivation, stirring to completely dissolve, and obtaining an inactivated bacteria powder stock solution.

[0116] (4) The inactivated bacterial powder stock solution obtained in step (3) is subjected to vacuum freeze drying, pre-frozen at -40±2°C for 1 to 3 hours, pre-frozen at -20±2°C for 0.5 to 1 hour, and finally pre-frozen at -40±2°C for 0.5 to 2 hours, and then dried once (at -5±2°C and 0±2°C) and dried by desorption (at 35±2°C) under a vacuum degree of 0.25 mbar to prepare inactivated bacterial powder, wherein the bacterial count of the bacterial powder reaches 1×10 11 Cells / g or more. Figure 3 It can be seen from the microscopic examination that the bacterial morphology in the prepared inactivated bacterial powder is complete.

[0117] The same method as above was used to prepare the inactivated bacteria of NCTC 9343.

[0118] 4. Preparation of Bacteroides fragilis extract

[0119] (1) Take the bacterial sludge precipitate obtained in Example 1, add purified water at a weight ratio of bacterial precipitate to purified water of 1 g:6 g to resuspend the bacterial sludge, adjust the pH to 3.5 with 1 mol / L hydrochloric acid solution, extract at 100° C. for 1.5 h, cool to room temperature, centrifuge at 12000 g for 10 min at room temperature, and take the supernatant to obtain a crude sugar solution;

[0120] (2) the crude sugar solution is ultrafiltered and concentrated through a 10KD ultrafiltration membrane to remove small molecular impurities until the conductivity is stable, and the reflux liquid is collected;

[0121] (3) Add an equal volume of 40mmol / L Tris-HCl (pH8.5) to the reflux liquid for salt conversion; DEAE Sepharose Fast Flow ion exchange column chromatography (16mm×200mm), flow rate 20mL / min, 20mmol / L Tris-HCl (pH8.5, containing 0.2mol / L NaCl) gradient elution for 25 column volumes, segmented collection, 100mL / bottle (component), SEC-HPLC tracking monitoring, merge the components with a single, symmetrical peak at 206nm absorption peak, ultrafilter with 10KD ultrafiltration membrane, add purified water and repeat ultrafiltration until the conductivity is stable, collect the reflux liquid, freeze-dry, and obtain the Bacteroides fragilis ZY-312 extract. According to nuclear magnetic resonance spectrometer analysis, the extract is capsular polysaccharide A, and the chemical structure diagram is shown in the attached Figure 4 .

[0122] This Example The samples were prepared for use in the following examples.

[0123] Example 3 Effect of Bacteroides fragilis on the functional dyspepsia rat model induced by iodoacetamide plus platform standing method

[0124] 1. Experimental Methods

[0125] 1. Animal Modeling and Grouping and Drug Administration

[0126] 84 10-day-old young SD rats were randomly divided into 7 groups: normal control group, model group, ZY-312 live bacteria group, NCTC 9343 live bacteria group, ZY-312 inactivated bacteria group, NCTC 9343 inactivated bacteria group, and ZY-312 extract group, with 12 rats in each group. After grouping, the normal control group animals were gavaged with 0.2mL 2% sucrose solution, and the other experimental groups were gavaged with 0.2mL 0.1% iodoacetamide sucrose solution for 6 consecutive days. The animals were kept until 28 days old, and the experimental group animals except the normal control group were placed in a water-filled platform box to stand for 6 hours every day for modeling, for 28 consecutive days.

[0127] When the animals were 28 days old, the corresponding test substances were given by gavage twice a day according to Table 1 for 28 consecutive days.

[0128] Table 1 Experimental groups and drug administration methods

[0129]

[0130]

[0131] 2. Index detection

[0132] (1) Body weight and food intake

[0133] The body weight and food intake of the rats were recorded on the 14th and 28th days after drug administration / platform standing.

[0134] (2) Gastric sensitivity test

[0135] After the administration, half of the animals in each group were randomly selected, anesthetized after fasting, and an intragastric balloon was placed through the esophagus under anesthesia. After placement in the stomach, the balloon was inflated with about 10 mL of air to ensure that it was located in the stomach and could be smoothly inflated and expanded. The balloon was connected to an electronic barostat through an airway. A pair of silver needle electrodes were inserted into the trapezius muscle of the rat and connected to an electrophysiological recorder. The experiment was started after the rats woke up and adapted for 2 hours. The myoelectricity of the trapezius muscle of the rat in the stable state was recorded, and 30 seconds were intercepted as the baseline to calculate the basic root mean square. The intragastric balloon of the rat was then pressurized in stages, with pressures of 20, 40, and 60 mmHg, respectively. The pressurization time was 30 seconds each time, and the myoelectric activity of the trapezius muscle of the neck was recorded every 180 seconds between pressurizations. Gastric sensitivity was evaluated by the root mean square change rate of the trapezius muscle of the neck as an indicator. The root mean square change rate was calculated as follows: root mean square change rate = (root mean square during pressurization - basic root mean square) / basic root mean square × 100%.

[0136] (3) Gastric emptying test

[0137] After the administration, half of the animals in each group were randomly selected. Before the experiment, the animals were fasted but not watered. Each group of animals was given nutritional semisolid paste by gavage. After 30 minutes, the animals were dissected and the stomachs were taken for gastric emptying detection. The gastric emptying rate (%) was calculated according to the formula [1-(total stomach weight-net stomach weight) / semisolid paste gavage amount] × 100%

[0138] 2. Experimental Results

[0139] The body weight and food intake of rats on the 14th and 28th days of drug administration / platform standing are shown in Table 2. The results showed that compared with the normal control, the model group animals had reduced body weight and reduced food intake, which was statistically significant (P < 0.05 or P < 0.01). Compared with the model group, the body weight and food intake of live bacteria, inactivated bacteria and extracts of ZY-312 on the 14th and 28th days were significantly increased (P < 0.05 or P < 0.01), and the body weight of live bacteria and inactivated bacteria of NCTC9343 increased significantly on the 14th and 28th days, and the food intake increased significantly on the 28th day (P < 0.05 or P < 0.01). Moreover, the body weight and food intake were significantly increased when the live bacteria of ZY-312 were compared with the live bacteria of NCTC9343, and when the inactivated bacteria of ZY-312 were compared with the inactivated bacteria of NCTC9343 (P < 0.05 or P < 0.01), indicating that the live bacteria / inactivated bacteria of ZY-312 are more effective than NCTC 9343 in improving the slow weight gain and anorexia caused by the functional dyspepsia model.

[0140] Table 2 Animal body weight and food intake

[0141]

[0142] Note: Compared with the model group, * indicates P < 0.05, ** indicates P < 0.01; compared with the NCTC 9343 live bacteria group, # indicates P < 0.05, ## indicates P < 0.01; compared with the NCTC 9343 inactivated bacteria group, ■ indicates P < 0.05, ■■ indicates P < 0.01.

[0143] After the administration, the results of the gastric sensitivity test are shown in Table 3. Compared with the normal control group, the root mean square change rate of the cervical trapezius muscle electromyography induced by gastric distension stimulation at 20, 40, and 60 mmHg pressure in the model group was significantly increased (P < 0.01). Compared with the model group, the root mean square change rate of electromyography in the live bacteria, inactivated bacteria, and extract groups of ZY-312 and the live bacteria and inactivated bacteria groups of NCTC9343 at each pressure was significantly decreased (P < 0.05 or P < 0.01). Moreover, the root mean square change rate of electromyography in the live bacteria of ZY-312 was significantly lower than that in the live bacteria of NCTC9343, and the root mean square change rate of electromyography in the inactivated bacteria of ZY-312 was significantly lower than that in the inactivated bacteria of NCTC9343 at each pressure (P < 0.01), indicating that ZY-312 is better than NCTC9343 in improving the increased gastric sensitivity of functional dyspepsia.

[0144] Table 3 RMS change rate of rat neck electromyography in gastric sensitivity test

[0145]

[0146]

[0147] Note: Compared with the model group, * indicates P < 0.05, ** indicates P < 0.01; compared with the NCTC 9343 live bacteria group, # indicates P < 0.05, ## indicates P < 0.01; compared with the NCTC 9343 inactivated bacteria group, ■ indicates P < 0.05, ■■ indicates P < 0.01.

[0148] After the administration, the results of gastric emptying rate are shown in Table 4. Compared with the normal control group, the gastric emptying rate of the model group animals was significantly reduced (P < 0.01), indicating that gastric motility was impaired. Compared with the model group, the live bacteria, inactivated bacteria and extract groups of ZY-312 and the live bacteria and inactivated bacteria groups of NCTC9343 could significantly increase the gastric emptying ratio (P < 0.01). Moreover, the gastric emptying rate of ZY-312 was significantly higher than that of NCTC9343 live bacteria, and that of ZY-312 inactivated bacteria was significantly higher than that of NCTC9343 inactivated bacteria (P < 0.05 or P < 0.01), indicating that ZY-312 is better than NCTC9343 in improving the motility disorder of the functional dyspepsia model.

[0149] Table 4 Gastric emptying rate (%)

[0150] Group Gastric emptying rate (%) Normal control group <![CDATA[86.2±3.4 ** ]]> Model Group 53.4±4.2 ZY-312 live bacteria group <![CDATA[73.8±3.2 **# ]]> NCTC 9343 live bacteria group <![CDATA[64.3±5.9 ** ]]> ZY-312 Inactivated Bacteria Group <![CDATA[78.8±6.3 **■■ ]]> NCTC 9343 inactivated bacteria group <![CDATA[63.6±4.1 ** ]]> ZY-312 extract group <![CDATA[71.5±2.9 ** ]]>

[0151] Note: Compared with the model group, * indicates P < 0.05, ** indicates P < 0.01; compared with the NCTC 9343 live bacteria group, # indicates P < 0.05, ## indicates P < 0.01; compared with the NCTC 9343 inactivated bacteria group, ■ indicates P < 0.05, ■■ indicates P < 0.01.

[0152] Example 4 Effect of Bacteroides fragilis on TNBS-induced irritable bowel syndrome (PI-IBS) model in rats

[0153] PI-IBS (post-infectious / post-inflammatory irritable bowel syndrome) is a common type of IBS, also known as post-inflammatory irritable bowel syndrome. The TNBS model is the most commonly used PI-IBS model. About 40% of PI-IBS animal models use trinitrobenzene sulfonic acid (TNBS), and this model can reflect the characteristics of irritable bowel syndrome (i.e., visceral hypersensitivity, intestinal motility dysfunction, permeability and secretion abnormalities).

[0154] 1. Experimental Methods

[0155] 1. Animal Modeling and Grouping and Drug Administration

[0156] A total of 84 SD rats were randomly divided into 7 groups according to their body weight: normal control group, model group, positive drug group, ZY-312 live bacteria group, ZY-312 inactivated bacteria group, ZY-312 lysate group, and ZY-312 extract group, with 12 rats in each group.

[0157] Rats in each group were fasted for 18 h before modeling (without water deprivation), and the animals were anesthetized (1% sodium pentobarbital solution, 45 mg / kg) after fasting. The normal control group animals were enema with 0.8 mL of normal saline solution as a control, and the other groups were enema with 0.8 mL of TNBS ethanol solution for modeling.

[0158] After modeling, each group was given the test drugs by gavage every day according to Table 5 for 28 consecutive days.

[0159] Table 5 Experimental groups and drug administration methods

[0160] Group animal Test substance Dosage Dosage Normal control group Normal animals Normal saline / PO, BID, day 1~28 Model Group Model Animals Normal saline / PO, BID, day 1~28 Positive drug group Model Animals Pinaverium Bromide 20mg / kg PO, BID, day 1~28 ZY-312 live bacteria group Model Animals ZY-312 live bacteria <![CDATA[1×10 9 CFU per animal]]> PO, BID, day 1~28 ZY-312 Inactivated Bacteria Group Model Animals ZY-312 inactivated bacteria <![CDATA[1×10 9 cells / cell]]> PO, BID, day 1~28 ZY-312 Lysate Group Model Animals ZY-312 Lysate <![CDATA[1×10 9 cells / cell]]> PO, BID, day 1~28 ZY-312 extract group Model Animals ZY-312 Extract 5mg / kg PO, BID, day 1~28

[0161] 2. Index detection

[0162] (1) Stool scoring

[0163] Animal feces were collected weekly and the feces characteristics were scored, with hard feces scored as 1 point, soft feces scored as 2 points, and unformed feces scored as 3 points.

[0164] (2) Rectal balloon dilation (CRD) test and pain threshold test

[0165] After 28 days of administration, the rats in each group were subjected to rectal balloon dilation (CRD) test and pain threshold test.

[0166] The paraffin-oil-lubricated F8 catheter with a balloon was inserted through the anus for about 6 cm, and the catheter was fixed to the base of the rat's tail with tape. The catheter was connected to a 60 mL syringe and a mercury sphygmomanometer via a three-way tube. The balloon pressure was quickly inflated to 20, 40, and 60 mmHg, respectively. Each pressure was maintained for 20 seconds, and the switching interval was 4 minutes. The abdominal reflex (AWR) score of each pressure was recorded. The AWR score was defined as follows: 0 points, no behavioral response to expansion; 1 point, only slight head movement and body stillness; 2 points, slight contraction of abdominal muscles; 3 points, abdomen raised; 4 points, body arched with pelvic lift.

[0167] Inflate the balloon slowly to increase the pressure (not exceeding 80 mmHg) until the AWR score reaches 3 points. The pressure value at this time is the pain threshold. If the pressure reaches 80 mmHg and the AWR score still does not reach 3 points, it will be counted as 80 mmHg. Repeat 3 times and take the average value, with an interval of 2 minutes between each time.

[0168] 2. Experimental Results

[0169] The results of the fecal status scores of rats during the experiment are shown in Table 6. The results showed that compared with the normal control, the feces of the model animals were soft or unformed, and the fecal status scores were significantly increased (P < 0.01). Compared with the model group, the live bacteria, inactivated bacteria, lysate and extract of ZY-312 could reduce the fecal status scores to varying degrees (P < 0.05 or P < 0.01). The scores of the ZY-312 inactivated bacteria group were significantly lower than those of the live bacteria, lysate or extract groups of ZY-312 (P < 0.05 or P < 0.01), indicating that the inactivated bacteria of ZY-312 had a better therapeutic effect on the diarrhea symptoms of the PI-IBS model.

[0170] Table 6 Fecal status scores of TNBS-induced PI-IBS model rats (Mean±SEM)

[0171] Group Day 7 of medication Day 14 of medication Day 21 of medication 28th day of medication Normal control group <![CDATA[1.00±0.00 ** ]]> <![CDATA[1.10±0.10 ** ]]> <![CDATA[1.00±0.00 ** ]]> <![CDATA[1.14±0.10 ** ]]> Model Group 2.83±0.16 2.64±0.24 2.29±0.15 1.97±0.17 Positive drug group 2.78±0.11 2.52±0.13 2.31±0.18 1.98±0.19 ZY-312 live bacteria group <![CDATA[2.59±0.12 *▲▲ ]]> <![CDATA[2.19±0.12 *▲ ]]> <![CDATA[1.93±0.17 *▲ ]]> <![CDATA[1.51±0.13 *▲ ]]> ZY-312 Inactivated Bacteria Group <![CDATA[2.12±0.28 ** ]]> <![CDATA[1.71±0.23 ** ]]> <![CDATA[1.45±0.13 ** ]]> <![CDATA[1.14±0.19 ** ]]> ZY-312 Lysate Group <![CDATA[2.47±0.14 *▲ ]]> <![CDATA[2.22±0.15 *▲▲ ]]> <![CDATA[1.97±0.16 *▲ ]]> <![CDATA[1.48±0.14 *▲ ]]> ZY-312 extract group <![CDATA[2.39±0.11 **▲ ]]> <![CDATA[2.08±0.21* *▲ ]]> <![CDATA[1.84±0.19 *▲ ]]> <![CDATA[1.43±0.16 *▲ ]]>

[0172] Compared with the model group, * indicates P < 0.05, ** indicates P < 0.01;

[0173] Compared with the ZY-312 inactivated bacteria group, ▲ indicates P < 0.05, ▲▲ indicates P < 0.01.

[0174] The results of rectal balloon dilation (CRD) test and pain threshold test of animals in each group after 28 days of administration are shown in Table 7. The results showed that compared with the normal control, the AWR scores of 20mmHg, 40mmHg, and 60mmHg in the CRD test of the model animals were significantly increased (P < 0.01), and the pain threshold was significantly reduced (P < 0.01). Compared with the model group, the positive drug, live bacteria, inactivated bacteria, lysate and extract of ZY-312 can significantly reduce the AWR scores of 20mmHg, 40mmHg, and 60mmHg in the CRD test (P < 0.01), and the pain threshold was significantly increased (P < 0.01). Compared with the live bacteria, lysate or extract groups of ZY-312, the AWR scores at 40 mmHg and 60 mmHg in the inactivated bacteria group of ZY-312 were significantly reduced, and the pain threshold was increased (P < 0.05 or P < 0.01), indicating that the inactivated bacteria of ZY-312 is more effective in improving the increased intestinal sensitivity and abdominal pain symptoms of the PI-IBS model.

[0175] Table 7 CRD test and pain threshold test results of TNBS-induced PI-IBS model rats (Mean±SEM)

[0176]

[0177]

[0178] Compared with the model group, * indicates P < 0.05, ** indicates P < 0.01;

[0179] Compared with the ZY-312 inactivated bacteria group, ▲ indicates P < 0.05, ▲▲ indicates P < 0.01.

[0180] Example 5 Effect of inactivated Bacteroides fragilis on the diarrhea-type irritable bowel syndrome model induced by acetic acid and restraint

[0181] 1. Experimental Methods

[0182] 1. Animal Modeling and Grouping and Drug Administration

[0183] A total of 96 SD rats were randomly divided into 8 groups according to their body weight: normal control group, model group, positive drug group, ZY-312 live bacteria group, ZY-312 inactivated bacteria low-dose group, ZY-312 inactivated bacteria medium-dose group, ZY-312 inactivated bacteria high-dose group and NCTC 9343 inactivated bacteria group, with 12 rats in each group.

[0184] On Day 1, the animals were anesthetized (1% sodium pentobarbital solution, 45 mg / kg) after fasting. After anesthesia, a silicone tube connected to a syringe was inserted through the anus (8 cm from the anus). The normal control group animals were given an enema with 1 mL of normal saline as a control, and the other groups were given an enema with 1 mL of acetic acid for modeling. After slowly pulling out the silicone tube, the anus was pressed by hand and the rat's tail was lifted for 30 seconds, and then the colon was flushed with 1 mL of PBS buffer.

[0185] After enema, rats in each group rested for 6 days (Day 1 to Day 6). On Day 7-Day 9, rats in the experimental groups except the normal control group were placed in a special transparent cylindrical tube that restricted their limbs but did not affect their breathing for restraint stress, once a day, 30 minutes each time. Rats in the normal control group were placed in a quiet, warm (18-20℃), light-proof environment for feeding, free access to water and food.

[0186] Starting from Day 1, the test drugs were administered by gavage every day according to Table 8 for 9 consecutive days.

[0187] Table 8 Experimental groups and administration methods

[0188]

[0189]

[0190] 2. Index detection

[0191] (1) Stool scoring

[0192] Animal feces were collected on Day 4 and Day 8, and the feces characteristics were scored, with hard feces scored as 1 point, soft feces scored as 2 points, and unformed feces scored as 3 points.

[0193] (2) Rectal balloon dilatation (CRD) test

[0194] After 9 days of administration, rats in each group were subjected to rectal balloon dilation (CRD) test. The F8 catheter with a balloon lubricated with paraffin oil was inserted through the anus for about 6 cm, and the catheter was fixed to the base of the rat's tail with tape. The catheter was connected to a 60mL syringe and a mercury sphygmomanometer through a three-way tube. Rapid gas injection was performed to make the balloon pressure reach 20, 40, and 60 mmHg, respectively. Each pressure was maintained for 20 seconds, and the switching interval was 4 minutes. The abdominal reflex (AWR) score of each pressure was recorded. The AWR score was defined as follows: 0 points, no behavioral response to dilation; 1 point, only slight head movement and body stillness; 2 points, slight contraction of abdominal muscles; 3 points, abdomen raised; 4 points, body arched with pelvic lifting.

[0195] 2. Experimental Results

[0196] The results of the fecal status scores of rats on the 4th and 8th days of the experiment are shown in Table 9. The results showed that compared with the normal control, the feces of the model animals were soft or unformed, and the fecal status scores were significantly increased (P < 0.01). Compared with the model group, the ZY-312 live bacteria group, the high, medium and low dose groups of the inactivated bacteria of ZY-312, and the NCTC 9343 inactivated bacteria group all significantly reduced the fecal status scores (P < 0.01). At the same dose level, the score of the high dose group of the inactivated bacteria of ZY-312 was significantly lower than that of the live bacteria group of ZY-312 and the inactivated bacteria group of NCTC 9343 (P < 0.05 or P < 0.01), indicating that the therapeutic effect of the inactivated bacteria of ZY-312 on the diarrhea symptoms of the irritable bowel syndrome model caused by acetic acid and restraint is better than that of the live bacteria of ZY-312 and the inactivated bacteria of NCTC 9343.

[0197] Table 9 Stool status scores of the diarrhea-type irritable bowel syndrome model induced by acetic acid plus restraint (Mean±SEM)

[0198]

[0199]

[0200] Compared with the model group, * indicates P < 0.05, ** indicates P < 0.01;

[0201] Compared with the high-dose ZY-312 inactivated bacteria group, ● indicates P < 0.05, ●● indicates P < 0.01.

[0202] After 9 days of administration, the results of rectal balloon dilation (CRD) test of animals in each group are shown in Table 10. The results showed that compared with the normal control, the AWR scores of 20 mmHg, 40 mmHg, and 60 mmHg in the CRD test of the model animals were significantly increased (P < 0.01). Compared with the model group, the positive drug, ZY-312 live bacteria group, ZY-312 inactivated bacteria high, medium and low dose groups, and NCTC 9343 inactivated bacteria group could significantly reduce the AWR scores of 20 mmHg, 40 mmHg, and 60 mmHg in the CRD test (P < 0.01). Moreover, at the same dose level, the AWR scores of 20 mmHg, 40 mmHg and 60 mmHg in the high-dose ZY-312 inactivated bacteria group were significantly lower than those in the ZY-312 live bacteria group and the NCTC 9343 inactivated bacteria group (P < 0.01), indicating that the effect of ZY-312 inactivated bacteria on improving the increased intestinal sensitivity and abdominal pain symptoms in the irritable bowel syndrome model caused by acetic acid plus restraint is better than that of ZY-312 live bacteria and NCTC 9343 inactivated bacteria.

[0203] Table 10 CRD test results of acetic acid plus restraint-induced diarrhea-type irritable bowel syndrome model

[0204]

[0205] Compared with the model group, * indicates P < 0.05, ** indicates P < 0.01;

[0206] Compared with the high-dose ZY-312 inactivated bacteria group, ● indicates P < 0.05, ●● indicates P < 0.01.

[0207] The above is an explanation of the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of Bacteroides fragilis in preparing a product for improving and / or treating functional gastrointestinal diseases, wherein the Bacteroides fragilis is selected from Bacteroides fragilis ZY-312 or Bacteroides fragilis NCTC 9343, and the active ingredient is live bacteria, inactivated bacteria or extracts of Bacteroides fragilis; and the functional gastrointestinal disease is functional dyspepsia; Preferably, the Bacteroides fragilis is selected from the group consisting of live bacteria of Bacteroides fragilis ZY-312, inactivated bacteria of Bacteroides fragilis ZY-312, live bacteria of Bacteroides fragilis NCTC 9343, inactivated bacteria of Bacteroides fragilis NCTC 9343, and an extract of Bacteroides fragilis ZY-312.

2. The use according to claim 1, characterized in that: The inactivated bacteria are morphologically intact inactivated bacteria and / or morphologically incomplete inactivated bacteria; Preferably, the inactivated bacteria are inactivated by any one or more of dry heat, moist heat, filtration, organic solvents, chemical reagents, ultraviolet or infrared rays, fermentation, freeze drying, genetic recombination, genetic modification or transformation.

3. The use according to claim 1 or 2, characterized in that: The inactivated bacteria is inactivated Bacteroides fragilis powder; Preferably, the preparation method of the inactivated Bacteroides fragilis powder is as follows: fermenting and culturing Bacteroides fragilis and centrifuging to obtain bacterial cells, washing the bacterial cells with a sodium chloride aqueous solution and centrifuging to obtain washed bacterial cells, adding an excipient to resuspend, inactivating and centrifuging to obtain inactivated bacterial sludge, and drying; Further preferably, the preparation method of the inactivated Bacteroides fragilis powder is as follows: (1) fermenting Bacteroides fragilis, centrifuging the fermentation broth after the fermentation is completed, and collecting the bacterial cells; (2) adding sodium chloride aqueous solution at a weight volume ratio of 1 g of bacterial cells to sodium chloride aqueous solution (10-30) mL) for washing, and centrifuging to obtain washed bacterial cells; (3) adding the first excipient solution to the washed bacterial cells, mixing and resuspending to obtain a bacterial cell solution, then performing an inactivation treatment, centrifuging, and collecting the inactivated bacterial sludge; (4) adding a second excipient solution to the inactivated bacteria sludge obtained in step (3) to obtain an inactivated bacteria stock solution; (5) Drying the inactivated bacteria stock solution obtained in step (4) until the residual moisture is less than 5 wt %, thereby obtaining inactivated Bacteroides fragilis bacteria powder.

4. The use according to any one of claims 1 to 3, characterized in that: The product is selected from any one of a pharmaceutical composition, a food, a health product or a food additive; Preferably, the pharmaceutical composition contains a pharmaceutically effective dose of inactivated Bacteroides fragilis ZY-312 powder with a preservation number of CGMCC No.10685.

5. The use according to any one of claims 1 to 4, characterized in that: The extract of Bacteroides fragilis ZY-312 contains Bacteroides fragilis capsular polysaccharide A; Preferably, the method for preparing the extract of Bacteroides fragilis ZY-312 comprises the following steps: (i) centrifuging the fermented Bacteroides fragilis culture, resuspending the culture in purified water, adjusting the pH to 3.5, extracting, centrifuging, and collecting the supernatant to obtain a crude sugar solution; (ii) the crude sugar solution is ultrafiltered and concentrated through a 10KD ultrafiltration membrane to remove small molecular impurities until the conductivity is stable, and the reflux liquid is collected; (iii) adding an equal volume of buffer to the reflux liquid for salt conversion; performing DEAE Sepharose Fast Flow ion exchange column chromatography at a flow rate of 20 mL / min, gradient elution for 25 column volumes, collecting fractions in a 100 mL / bottle (component), tracking and monitoring by SEC-HPLC, combining fractions with a single, symmetrical absorption peak at 206 nm, ultrafiltration through a 10KD ultrafiltration membrane, adding purified water for repeated ultrafiltration until the conductivity is stable, collecting the reflux liquid, and lyophilizing to obtain a Bacteroides fragilis extract.

6. Use of Bacteroides fragilis in preparing a product for improving and / or treating functional gastrointestinal diseases, wherein the Bacteroides fragilis is selected from Bacteroides fragilis ZY-312 or Bacteroides fragilis NCTC 9343, and the active ingredient is an inactivated bacterium or a lysate of Bacteroides fragilis; and the functional gastrointestinal disease is irritable bowel syndrome; Preferably, the irritable bowel syndrome includes diarrhea-predominant irritable bowel syndrome and post-inflammatory irritable bowel syndrome. Preferably, the Bacteroides fragilis is selected from the group consisting of inactivated bacteria of Bacteroides fragilis ZY-312, inactivated bacteria of Bacteroides fragilis NCTC 9343, and lysate of Bacteroides fragilis ZY-312.

7. The use according to claim 6, characterized in that: The inactivated bacteria are morphologically complete inactivated bacteria and / or morphologically incomplete inactivated bacteria. Preferably, the inactivated bacteria are inactivated by any one or more of dry heat, moist heat, filtration, organic solvents, chemical reagents, ultraviolet or infrared rays, fermentation, freeze drying, genetic recombination, genetic modification or transformation.

8. The use according to claim 6 or 7, characterized in that: The inactivated bacteria is inactivated Bacteroides fragilis powder; Preferably, the preparation method of the inactivated Bacteroides fragilis powder is as follows: fermenting and culturing Bacteroides fragilis and centrifuging to obtain bacterial cells, washing the bacterial cells with a sodium chloride aqueous solution and centrifuging to obtain washed bacterial cells, adding an excipient to resuspend, inactivating and centrifuging to obtain inactivated bacterial sludge, and drying; Further preferably, the preparation method of the inactivated Bacteroides fragilis powder is as follows: (1) fermenting Bacteroides fragilis, centrifuging the fermentation broth after the fermentation is completed, and collecting the bacterial cells; (2) adding sodium chloride aqueous solution at a weight volume ratio of 1 g of bacterial cells to sodium chloride aqueous solution (10-30) mL) for washing, and centrifuging to obtain washed bacterial cells; (3) adding the first excipient solution to the washed bacterial cells, mixing and resuspending to obtain a bacterial cell solution, then performing an inactivation treatment, centrifuging, and collecting the inactivated bacterial sludge; (4) adding a second excipient solution to the inactivated bacteria sludge obtained in step (3) to obtain an inactivated bacteria stock solution; (5) Drying the inactivated bacteria stock solution obtained in step (4) until the residual moisture is less than 5 wt %, thereby obtaining inactivated Bacteroides fragilis bacteria powder.

9. The use according to any one of claims 6 to 8, characterized in that: The product is selected from any one of a pharmaceutical composition, a food, a health product or a food additive; Preferably, the pharmaceutical composition contains a pharmaceutically effective dose of inactivated Bacteroides fragilis ZY-312 powder with a preservation number of CGMCC No.10685.

10. The use according to any one of claims 6 to 9, characterized in that: The lysate of Bacteroides fragilis ZY-312 was prepared by ultrasonic disruption; Preferably, the method for preparing the lysate of Bacteroides fragilis ZY-312 comprises the following steps: (1) fermenting Bacteroides fragilis, centrifuging the fermentation liquid after the fermentation is completed, and collecting bacterial sludge; (2) adding physiological saline in a weight ratio of bacterial sludge to physiological saline of 1:(5-15) and stirring to resuspend; (3) The resuspended bacterial solution is lysed by ultrasonication; (4) After lysis, the cells are centrifuged and filtered to obtain a lysate.

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