Extrusion type multi-flux coprophilous fungus preparation method and application thereof

Abstract

The invention discloses an extrusion type multi-flux coprophilous fungus preparation method and application thereof, and belongs to the technical field of medicine. The method comprises the following steps: (1) homogenizing fresh excrement, namely culturing and counting viable bacteria of a donor excrement sample and preparing a coprophilous bacteria homogenizing solution; (2) step-by-step extrusion type filtration: a rotary extrusion device is used for filtration, the diluent is firstly sieved by a 50-250-mesh standard sieve and then sieved by a 1100-1300-mesh standard sieve, and filtrate is collected; and (3) centrifuging to prepare a suspension. According to the multi-flux extrusion type method and the arrangement of the filtering tool, food residues are effectively filtered, the activity of microorganisms in coprophilous fungi can be kept, and the viable count is increased. Experiments show that the method is easy and effective to operate, harmful food residues can be removed, and the coprophilous fungi extraction rate, survival rate, yield and production efficiency are greatly improved.

Description

Technical Field

[0001] The present invention relates to a method for preparing excrement bacteria with extrusion multi-flux and its application, belonging to the field of medical technology. Background Art

[0002] Fecal Microbiota Transplantation (FMT) is to transplant the functional flora in the feces of specific healthy people into the patient's intestine in a certain way to reshape the intestinal homeostasis, restore the diversity of intestinal flora, and achieve the effect of treating intestinal flora disorders and improving disease symptoms. The "Technical Specifications for National Medical Service Items (2023 Edition)" provides a reference standard for the application of intestinal flora transplantation, making it more standardized and regularized. The above expert consensus and technical specifications have greatly promoted the development of FMT treatment, ensured the safety of its clinical application, and promoted the healthy development of FMT.

[0003] In the prior art, Chinese Patent CN 107513515 A discloses a process for preparing fecal bacteria liquid. By passing the fecal dilution through an 80-mesh standard sieve and a 300-mesh standard sieve, and then subjecting the collected filtrate to suction filtration through 20-mm, 5-mm, and 0.8-μm filter membranes, the extraction rate of viable bacteria is finally 76%. This process flow is complex. The fecal dilution needs to be filtered first and then suction filtered, which significantly increases the processing time, reduces the processing efficiency, and is not conducive to high-throughput production. Moreover, the viable bacteria extraction rate of this process is still relatively low. Therefore, in the preparation of fecal bacteria, there is still an urgent need to further simplify the process, improve the processing efficiency, and increase the viable bacteria recovery rate. Summary of the Invention

[0004] [Technical Problem]

[0005] The technical problem to be solved by the present invention is to provide a method for preparing fecal bacteria liquid with high production flux, high efficiency, short time, simple process, few impurities in the bacterial sludge, increased viable bacteria amount and viable bacteria volume ratio.

[0006] [Technical Solution]

[0007] In order to achieve the above object, the following technical solutions are provided:

[0008] The present invention first provides a method for preparing fecal bacteria, characterized in that the method comprises the following steps:

[0009] (1) Pretreatment: Mix feces with water and stir evenly to prepare a fecal dilution;

[0010] (2) Double-layer filter screen extrusion filtration: Collect the fecal dilution obtained in step (1) and perform extrusion filtration through a double-layer filter screen to obtain a fecal filtrate;

[0011] (3) Fecal bacteria preparation: Collect the fecal filtrate obtained in step (2), perform solid-liquid separation, and collect the solid bacterial sludge, which is the fecal bacteria.

[0012] In one embodiment, step (1) includes: Mix feces with 6 to 10 times the weight of physiological saline, and stir evenly to obtain a fecal dilution.

[0013] In one embodiment, the viscosity of the fecal dilution is 45 to 65 mPa·s.

[0014] In one embodiment, step (2) includes: Collect the fecal dilution obtained in step (1), and perform extrusion filtration through a double-filter layer net, so that the fecal dilution passes through the inner sieve and the outer sieve in sequence to obtain a fecal filtrate: the inner sieve is 50 to 250 mesh, and the outer sieve is 1100 to 1300 mesh.

[0015] In one embodiment, the inner sieve is 150 mesh and the outer sieve is 1200 mesh.

[0016] In one embodiment, the extrusion speed is 20 to 80 mm / s.

[0017] In one embodiment, the extrusion speed is 50 mm / s.

[0018] In one embodiment, the sieve used in step (2) is a nylon sieve.

[0019] In one embodiment, in step (2), the fecal dilution is extruded until the filter residue becomes lumpy, and then the extrusion is stopped.

[0020] In one embodiment, the solid-liquid separation in step (3) is carried out at 2 to 10 °C.

[0021] In one embodiment, step (3) further includes: Resuspending the bacterial sludge to obtain a fecal bacteria liquid.

[0022] In one embodiment, the method does not involve the diagnosis or treatment of diseases.

[0023] The present invention also provides the fecal bacteria prepared by the method.

[0024] The present invention also provides a product containing the fecal bacteria.

[0025] In one embodiment, the product contains a bacterial suspension, bacterial cells, or freeze-dried bacterial cells of the fecal bacteria.

[0026] The present invention also provides the application of the method in increasing the viable count or extraction rate of viable fecal bacteria.

[0027] The present invention also provides an application of the method in improving the reduction degree of the flora in fecal bacteria.

[0028] The present invention also provides an application of the method, or the fecal bacteria, or the product in the preparation of a product for regulating the intestinal flora.

[0029] The present invention has the following beneficial effects:

[0030] (1) The present invention adopts double - filter integrated extrusion filtration, and the viable bacteria recovery rate of the obtained fecal bacteria liquid is 84.8%, which is significantly higher than the processes using gauze filtration or negative pressure filtration.

[0031] (2) When preparing fecal bacteria by the method of the present invention, the production time of the bacterial sludge is 20 - 30 minutes, which is significantly lower than that of gauze filtration or negative pressure filtration. The shortened production time can significantly improve the preparation efficiency and reduce the pollution risk.

[0032] (4) Before the extrusion - type filtration treatment of the present invention, the precipitation amount of the bacterial sludge is the same as that of the processes using gauze filtration or negative pressure filtration, but the viable bacteria count is significantly higher than that of gauze filtration or negative pressure filtration. At the same time, the quality of the bacterial sludge is significantly lower than that of gauze filtration or negative pressure filtration, indicating that the method provided by the present invention results in fecal bacteria containing less impurities.

[0033] (5) The method of the present invention not only simplifies the process steps, but also reduces the loss of the flora during the production process. The reduction degree of the flora structure of the obtained bacterial sludge is higher compared with the original feces of healthy donors, basically reaching 100%.

[0034] (6) The method of the present invention has low cost, high efficiency, large output and no cross - contamination. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 : Schematic diagram of the rotary extrusion device used in the multi - flux extrusion preparation process, wherein, 1: screw, 2: pressing plate, 3: liquid outlet, 4: outer sieve, 5: inner sieve.

[0036] Figure 2 : Venn diagram of the reduction degree of the flora structure. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0037] The present invention will be further described in detail below in conjunction with the specific embodiments. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0038] In the experimental methods of the following examples, unless otherwise specified, they are all conventional methods, carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0039] Calculation of viable bacteria recovery rate: Total viable bacteria count after treatment / Total viable bacteria count before treatment × 100%.

[0040] Example 1: Optimization of the extrusion preparation process

[0041] A multi-flux extrusion preparation process for standard bacterial liquid in fecal microbiota transplantation includes the following steps:

[0042] Homogenization treatment of fresh feces: Take 210 g of feces from 3 healthy donors respectively. Each fecal sample is injected into 6 - 7 times the sample volume of sterile normal saline and diluted to a sample viscosity of 60 mPa·s. Place it in a nitrogen biological operation cabinet and use a blade stirrer at a low speed of 300 w for 30 s to stir the feces until it becomes a suspension without obvious large particles and impurities.

[0043] 1. Single-filter integrated extrusion filtration

[0044] Determination of the mesh number of the inner filter: Divide the original fecal suspension after sample pretreatment into three equal parts, record the weight and detect the viable bacteria amount by PMA-qPCR method. Pass the fecal bacteria homogenate through standard nylon sieve meshes of 50, 150, and 250 meshes respectively. Rotate the screw of the extrusion device, with an extrusion stroke of 300 mm and an extrusion speed of 50 mm / s, to perform extrusion filtration on the sample. Stop extrusion until the filter residue becomes lumpy. Open the liquid outlet, collect all the filtrate, weigh it, record the filtrate weight and filtration time, and detect the viable bacteria amount in the filtrate by PMA-qPCR method. The percentage of the ratio of the viable bacteria amount in the filtrate to the viable bacteria amount in the original fecal suspension is the total viable bacteria recovery rate of the filtrate. Centrifuge the filtrate at 4°C and 6500 g for 5 min, remove the supernatant to obtain a bacterial sludge precipitate, weigh and record it, and compare the impurity content in the filtrate.

[0045] The results are shown in Table 1. When the inner filter is selected with 250 meshes, the filtration is difficult, and the filtrate recovery rate (weight after filtration / weight before filtration × 100%), the total viable bacteria recovery rate of the filtrate, and the quality of the bacterial sludge are all low, and the filtration time is long. Therefore, it is discarded; there is no obvious difference in the filtrate recovery rate and extrusion filtration time between 50 and 150 meshes, but the total viable bacteria recovery rate of the filtrate with 150 meshes is high, and the quality of the bacterial sludge is less than that of the 50-mesh bacterial sludge, indicating that its impurity retention amount is lower. Therefore, the inner filter is preferably 150 meshes.

[0046] Table 1 Filtrate recovery rate, total viable bacteria recovery rate of the filtrate, filtration time, and quality of the bacterial sludge before and after extrusion filtration of the inner filter

[0047]

[0048] 2. Double - filter integrated extrusion filtration:

[0049] Add an outer filter under the lower layer of the inner filter to further filter the filtrate collected in Step 1. Determination of the mesh number of the outer sieve: According to the mesh number of the inner sieve determined in the above steps, determine the combination method of the mesh number with the outer sieve. The mesh numbers of the outer sieve are selected as 1100 mesh, 1200 mesh, 1300 mesh, and 1400 mesh respectively. Record the weight of the original fecal suspension after sample pretreatment, and detect the viable bacteria count by PMA - qPCR method. Divide the fecal bacteria homogenate into four equal parts, and the four equal parts are subjected to extrusion filtration using double - filter combinations with different mesh numbers of the outer filter, namely 150 / 1100, 150 / 1200, 150 / 1300, and 150 / 1400 mesh. Collect the filtrate, record the weight and filtration time, and detect the viable bacteria count in the filtrate by PMA - qPCR method. Centrifuge the filtrate at 4°C and 6500g for 5 min, remove the supernatant to obtain a bacterial sludge precipitate, weigh and record it, and compare the impurity content in the filtrate.

[0050] The filtration results of the combined double - filters of 150 / 1100, 150 / 1200, 150 / 1300, and 150 / 1400 mesh are shown in Table 2. The results show that when the outer filter is selected with 1300 mesh, the maximum total viable bacteria recovery rate of the filtrate is 84.5%. When the outer filter is selected with 1400 mesh, the filtrate recovery rate, the total viable bacteria recovery rate of the filtrate, and the quality of the bacterial sludge all decrease significantly (p < 0.01). Among them, the 150 / 1300 - mesh combination has a relatively high filtrate recovery rate and total viable bacteria recovery rate of the filtrate, and the quality of the bacterial sludge is relatively low, indicating that the impurities in the bacterial liquid obtained by this combination filtration are reduced, the unit viable bacteria amount is further increased, and the bacterial sludge is purer. Therefore, the 150 / 1300 - mesh combination nylon net is selected. The total viable bacteria recovery rate of the double - filter and single - filter schemes is comparable. However, compared with the single - filter, the quality of the bacterial sludge of the double - filter is significantly reduced on the premise of ensuring the filtrate recovery rate, indicating that the unit viable bacteria amount is increased and more impurities are filtered out. Therefore, considering the filtrate recovery rate, the total viable bacteria recovery rate of the filtrate, the filtration time, and the quality of the bacterial sludge, the 150 / 1300 - mesh combination double - filter is finally selected.

[0051] Table 2 Filtrate recovery rate, total viable bacteria recovery rate, filtration time, and bacterial sludge quality before and after extrusion filtration of combined filters with different mesh numbers

[0052]

[0053] 3. Optimize the extrusion speed according to the sample viscosity

[0054] The homogenization treatment steps of fresh feces are the same as above. Measure the viscosity of the diluted sample, filter it through a combined double-screen nylon sieve of 150 / 1300 mesh, rotate the screw of the extrusion device, with an extrusion stroke of 300 mm. Set the extrusion speeds of the same sample to 20, 50, and 80 mm / s respectively, and conduct extrusion filtration on the sample until the filter residue becomes lumpy and stop extrusion. Open the liquid outlet, weigh all the collected filtrate, record the filtrate weight and filtration time, and detect the viable bacteria count in the filtrate by PMA-qPCR method. Centrifuge the filtrate at 4°C and 6500g for 5 min, remove the supernatant to obtain the bacterial sludge precipitate, weigh and record it, and compare the impurity content in the filtrate.

[0055] The results show that the maximum viscosity of the sample that can be tolerated by one-time extrusion should not exceed 85 mPa·s. As shown in Table 3, when the viscosity value of the diluted sample < 45 mPa·s, the extrusion speed of 80 mm / s is preferred to obtain the maximum filtrate recovery rate and total viable bacteria recovery rate of the filtrate; when the viscosity value of the diluted sample is 45 - 65 mPa·s, the extrusion speed of 50 mm / s is preferred to obtain the maximum filtrate recovery rate and total viable bacteria recovery rate of the filtrate; when the viscosity value of the diluted sample is 65 - 85 mPa·s, the extrusion speed of 20 mm / s is preferred to obtain the maximum filtrate recovery rate and total viable bacteria recovery rate of the filtrate.

[0056] Table 3 Effects of extrusion speed on filtrate recovery rate and total viable bacteria recovery rate of diluted samples in different viscosity value ranges during extrusion

[0057]

[0058] Example 2: Multi-flux extrusion preparation process of standard bacterial solution for fecal microbiota transplantation

[0059] A multi-flux extrusion preparation process of standard bacterial solution in fecal microbiota transplantation includes the following steps:

[0060] Homogenization treatment of fresh feces: Take 200 g of feces from 5 healthy donors, divide each sample into four parts A, B, C, and D and place them in sterile beakers, 50 g for each. Inject 6 - 7 times the sample volume of sterile physiological saline into each fecal sample to dilute the sample viscosity to 45 - 65 mPa·s, place it in a nitrogen biological safety cabinet, use a blade stirrer at a low speed gear with a power of 300 w and stir for 30 s to stir the feces into a suspension without obvious large particles and impurities. After suspension, take 1 mL for viable bacteria counting by PMA-qPCR method, and further calculate the total viable bacteria count of the suspension.

[0061] Double - filter integrated extrusion filtration: Pass the fecal bacteria homogenate through a 150 - mesh standard nylon sieve and a 1300 - mesh standard nylon sieve successively. Rotate the screw of the extrusion device to extrude and filter the sample. The extrusion speed is 50 mm / s. Stop extrusion when the filter residue becomes lumpy. Open the liquid outlet, collect all the filtrate, take 1 mL for viable bacteria counting by PMA - qPCR method, and further calculate the total viable bacteria recovery rate of the filtrate.

[0062] Centrifugation to prepare suspension: Place the filtrate collected by extrusion filtration in a sterile centrifuge tube, centrifuge at 4°C, 6000 - 7000g for 5 minutes, discard the supernatant to obtain a bacterial sludge precipitate, weigh it, and perform metagenomic sequencing to analyze the bacterial community structure.

[0063] Comparative Example 1: Gauze filtration preparation process of standard bacterial solution for fecal microbiota transplantation

[0064] A gauze filtration preparation process of standard bacterial solution for fecal microbiota transplantation includes the following steps:

[0065] From part B of the feces collected in Example 2, take each fecal sample and inject it into sterile physiological saline at 6 - 7 times the sample volume to dilute the sample viscosity to 45 - 65 mPa·s. Place it in a nitrogen biological operation cabinet, fix the stirring paddle and stirrer at a low speed of 80 w for 5 minutes. After suspension, take 1 mL for viable bacteria counting by PMA - qPCR method, and further calculate the total viable bacteria number of the suspension. Then first filter the large - particle residues with a single - layer sterile gauze, and then filter with a four - layer sterile gauze. Collect the filtrate, take 1 mL for viable bacteria counting by PMA - qPCR method, and further calculate the total viable bacteria recovery rate of the filtrate. Centrifuge at 4°C, 6500g for 5 minutes, discard the supernatant to obtain a bacterial sludge precipitate, weigh it, and perform metagenomic sequencing to analyze the bacterial community structure.

[0066] Comparative Example 2: Negative - pressure suction filtration preparation process of standard bacterial solution for fecal microbiota transplantation

[0067] A negative - pressure suction filtration preparation process of standard bacterial solution for fecal microbiota transplantation includes the following steps:

[0068] From the fecal C part collected in Example 2, each fecal sample was injected into sterile normal saline at 6-7 times the sample volume and diluted until the sample viscosity was 45-65 mPa·s. It was placed in a nitrogen biological operation cabinet, the stirring paddle and stirrer were fixed, and stirred at a low rate of 80 w for 5 min. After suspension, 1 mL was taken for viable bacteria counting by PMA-qPCR method, and the total viable bacteria count of the suspension was further calculated. For negative pressure filtration, the sample solution was pumped into the filter screen using a peristaltic pump. When the sample was completely pumped into the filter screen, the vacuum pump was turned on and turned off when the filtration was complete. Since negative pressure suction filtration easily causes the filter membrane to clog, a filter screen with a mesh size greater than 300 will clog and be damaged to a certain extent during negative pressure suction filtration. Therefore, it is impossible to use the 1000-mesh filter screen in Example 2 for operation. Therefore, filter screens with mesh sizes of 10, 30, 80, 150, 200, and 300 were used for filtration in sequence. After filtration was completed, the filtrate was collected using a peristaltic pump, 1 mL was taken for viable bacteria counting by PMA-qPCR method, and the total viable bacteria recovery rate of the filtrate was further calculated. Centrifuge at 4°C and 6500 g for 5 min, remove the supernatant to obtain a bacterial sludge precipitate, weigh it, and perform metagenomic sequencing to analyze the bacterial community structure.

[0069] Comparative Example 3: Stepwise filtration preparation process of standard bacterial solution for fecal microbiota transplantation

[0070] A stepwise filtration preparation process of standard bacterial solution in fecal microbiota transplantation includes the following steps:

[0071] From the fecal D part collected in Example 2, each fecal sample was injected into sterile normal saline at 6-7 times the sample volume and diluted until the sample viscosity was 45-65 mPa·s. It was placed in a nitrogen biological operation cabinet, the stirring paddle and stirrer were fixed, and stirred at a low rate of 80 w for 5 min. After suspension, 1 mL was taken for viable bacteria counting by PMA-qPCR method, and the total viable bacteria count of the suspension was further calculated. The stirred fecal diluent was successively passed through 80-mesh and 300-mesh standard sieves, filtered while stirring, the filtrate was collected, 1 mL was taken for viable bacteria counting by PMA-qPCR method, and the total viable bacteria recovery rate of the filtrate was further calculated. Centrifuge at 4°C and 6500 g for 5 min, remove the supernatant to obtain a bacterial sludge precipitate, weigh it, and perform metagenomic sequencing to analyze the bacterial community structure.

[0072] Table 4 Total viable bacteria recovery rate, bacterial sludge quality, and production preparation time (including stirring, filtration, and centrifugation steps) of filtrate before and after fecal bacteria extrusion method, gauze filtration method, negative pressure suction filtration method, and stepwise filtration through a sieve

[0073]

[0074] For Example 2 and Comparative Examples 1, 2, and 3, the total viable bacteria recovery rate of the filtrate, the quality of the bacterial sludge, and the production preparation time before and after fecal bacteria treatment are shown in Table 4. The results show that the total viable bacteria recovery rate of the filtrate in the extrusion method of Example 2 is 84.47%, which is significantly higher than that in each comparative example (p < 0.01). Moreover, the quality of the bacterial sludge obtained by the extrusion method is the lowest, which is 23.14 g. Considering comprehensively, the quality of the bacterial sludge prepared in Example 2 is the least among the three groups, but the viable bacteria amount is greatly increased, and the production preparation time is greatly shortened. The preparation method of the present invention not only increases the viable bacteria number of fecal bacteria but also greatly reduces the food digestion residues, and can improve the clinical efficacy of fecal microbiota transplantation.

[0075] Comparative Example 4: Effects of Different Preparation Methods and Mesh Numbers of Screens on the Reduction Degree of Bacterial Community Structure

[0076] Take 120 g of feces from 5 healthy donors, and divide each sample into 6 portions and place them in a sterile beaker, with one portion being 20 g. Prepare fecal bacteria liquid according to the grouping in Table 4. The filtrate is centrifuged at 6500 g for 5 - 10 min at 4°C, and the supernatant is removed to obtain a bacterial sludge precipitate, and the reduction degree of the bacterial community structure is analyzed by metagenomic sequencing. The preparation methods include 150 / 1300 - mesh extrusion method, 80 / 300 - mesh extrusion method, 150 / 1100 - mesh extrusion method, gauze filtration method of Comparative Example 1, negative pressure filtration method of Comparative Example 2, and step - by - step filtration of screens of Comparative Example 3. Among them, the extrusion method preparation process with an 80 / 300 - mesh combined filter screen refers to the same operation steps as in Example 2, and the mesh number combination is changed. The homogenate is filtered successively through an 80 - mesh standard nylon screen and a 300 - mesh standard nylon screen, and other steps are the same as those in Example 2. The extrusion method preparation process with a 150 / 1100 - mesh combined filter screen refers to the same operation steps as in Example 2, and the mesh number combination is changed. The homogenate is filtered successively through a 150 - mesh standard nylon screen and an 1100 - mesh standard nylon screen, and other steps are the same as those in Example 2.

[0077] The above - mentioned reduction degree of the bacterial community structure = the number of common bacterial species with the original feces / the number of bacterial species in the original feces × 100%.

[0078] Table 5 Effects of Different Preparation Methods and Mesh Numbers of Screens on the Reduction Degree of Bacterial Community Structure

[0079]

[0080]

[0081] The results are as Figure 2As shown in Table 5, the sludge precipitate obtained by the extrusion method with 150 / 1300 mesh is almost the same as the original fecal flora structure, and the reduction degree of the flora structure is 98.9%, which is significantly higher than that of other preparation methods and the extrusion method with other combinations of sieve mesh numbers. The preparation method of the present invention retains the flora structure of the original feces of healthy donors, is closer to the in-situ transplantation of flora, can maximize the advantages of donor-recipient matching in the early stage of fecal microbiota transplantation, and greatly improves the clinical efficacy of fecal microbiota transplantation.

[0082] Comparative Example 5: Effect of different centrifugal forces on the total viable bacteria recovery rate of sludge precipitate

[0083] The specific implementation manner refers to Example 2, with the difference that after collecting the filtrate, the centrifugal force is changed to 3500g, 5000g, 6500g, 8000g, 9500g to prepare the sludge precipitate. After centrifugation, 0.1g of the sludge precipitate is taken for viable bacteria counting by PMA-qPCR method, and the total viable bacteria number of the sludge precipitate is further calculated and compared with the total viable bacteria number in the filtrate to calculate the total viable bacteria recovery rate. The results show that the total viable bacteria recovery rate of the sludge precipitate is the highest at 6500g, reaching 90%. If the centrifugal force is too low, some bacteria still remain in the supernatant and cannot be enriched. If the centrifugal force is too high, some bacteria will die. Therefore, 6500g is preferably used as the centrifugation process parameter.

[0084] Table 6 Effect of different centrifugal forces on the total viable bacteria recovery rate of sludge precipitate

[0085]

[0086] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various modifications and decorations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A method for preparing fecal bacteria, characterized in that: The method comprises the following steps: (1) Pretreatment: diluting the feces to prepare a feces dilution solution; (2) double-layer filter extrusion filtration: collecting the fecal dilution obtained in step (1), extruding and filtering through a double-layer filter until the filter residue becomes agglomerated, and then stopping the extrusion to obtain a fecal filtrate; (3) Preparation of fecal bacteria: Collect the fecal filtrate obtained in step (2), separate the solid and liquid, and collect the solid bacterial sludge, which is the fecal bacteria.

2. The method according to claim 1, characterized in that The viscosity of the feces dilution in step (1) is 45 to 65 mPa·s.

3. The method according to claim 1, characterized in that The double filter layer mesh in step (2) comprises an inner sieve and an outer sieve; the inner sieve is 400-600 mesh, and the outer sieve is 1100-1300 mesh.

4. The method according to any one of claims 1 to 3, characterized in that: The feces dilution liquid first passes through the inner screen and then passes through the outer screen; it is squeezed while passing through the screen.

5. The method according to claim 4, characterized in that The extrusion speed is 20-80 mm / s.

6. The method according to claim 5, characterized in that The screen is a nylon screen.

7. The method according to claim 6, characterized in that The solid-liquid separation in step (3) is performed by centrifugation at 2-10°C and 6000-7000g for at least 5 minutes.

8. Fecal bacteria prepared by the method according to any one of claims 1 to 7.

9. Use of the method of claims 1 to 7 in increasing the number of live fecal bacteria or increasing the degree of reduction of fecal bacteria.

10. Use of the method according to claims 1 to 7 or the fecal bacteria according to claim 8 in the preparation of a product for regulating intestinal flora.

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