An intestinal flora preparation, preparation method and application, and spore content detection method

Abstract

The present invention belongs to the technical field of intestinal flora transplantation, and discloses an intestinal flora preparation, preparation method and application, and a method for detecting spore content. In the preparation method provided by the present invention, a human bacterial suspension obtained by purifying a fecal sample is treated by combining a slow heating pretreatment, a high temperature incubation treatment with a volatile ethanol solution, and a density gradient centrifugation method, which can effectively increase the spore yield and the germination rate of the obtained spores, while effectively removing harmful metabolites, viruses and other substances, thereby obtaining an intestinal flora preparation with high purity and high activity.

Description

Technical Field

[0001] The invention belongs to the technical field of intestinal flora transplantation, and in particular relates to an intestinal flora preparation, a preparation method and application thereof, and a method for detecting spore content. Background Art

[0002] Intestinal flora transplantation technology, also known as fecal microbiota transplantation (FMT), refers to the technology of extracting intestinal flora from the feces of a specific healthy individual and transplanting it into the patient's intestines to reshape the patient's unbalanced intestinal flora to achieve the treatment of related diseases. In recent years, many clinical trials have confirmed the safety of this technology and shown that it has significant effects in treating certain intestinal diseases, such as refractory Crohn's disease and recurrent Clostridium difficile infection.

[0003] The most critical step in FMT technology is to extract intestinal flora from the feces of specific healthy individuals. The treatment of the extracted intestinal flora greatly affects the effect of the treatment. However, the existing fecal intestinal flora extraction process is difficult to completely remove pathogenic Gram-negative bacteria, harmful metabolites and viruses, resulting in the presence of the above-mentioned impurities and potential harmful substances in the extracted intestinal flora. These substances may inhibit the colonization of beneficial bacteria, reduce the success rate of flora transplantation, and even cause adverse reactions, which poses certain risks. Therefore, obtaining a method that can effectively separate intestinal flora from harmful substances in flora transplantation in fecal samples is of great significance for improving the success rate of intestinal flora transplantation and biosafety.

[0004] Most of the bacteria on the domestic probiotic catalog are Gram-positive bacteria, and are Firmicutes bacteria, while common pathogenic bacteria such as Escherichia coli and Shigella are mostly Gram-negative bacteria. Therefore, based on the differences in cell wall structure between Firmicutes and pathogenic bacteria and the spore dormant bodies unique to some species of Firmicutes, the probiotic vegetative body can be converted into a spore dormant body by a pretreatment process to achieve effective removal of pathogenic bacteria. In the Chinese patent application with publication number CN110917220A, a method for preparing a therapeutic composition is disclosed, which involves stimulating probiotics to produce spores through low-temperature freezing technology, and purifying the obtained spores through heat treatment or alcohol treatment methods. In the Chinese patent application with publication number CN116478820A, a process for separating and screening spore flora from intestinal flora is disclosed, which first uses high-temperature treatment technology to induce probiotics to form spores, and then purifies the produced spores through heat treatment technology.

[0005] However, the above-mentioned spore treatment process has problems such as low spore yield, low spore germination rate and inability to effectively remove harmful metabolites, viruses and other substances. This limits the application of intestinal flora preparations in the form of spores in intestinal flora transplantation and has certain limitations. Summary of the invention

[0006] The first purpose of the present invention is to provide a method for preparing an intestinal flora preparation. In the preparation method, a human bacterial suspension obtained by purifying a stool sample is treated by combining slow heating pretreatment, high-temperature incubation treatment with a volatile ethanol solution, and density gradient centrifugation, which can effectively improve the spore yield and spore germination rate of the intestinal flora preparation while effectively removing harmful metabolites, viruses and other substances, thereby obtaining an intestinal flora preparation with high purity and high activity.

[0007] The second purpose of the present invention is to provide an intestinal flora preparation.

[0008] The third purpose of the present invention is to provide the application of the above-mentioned intestinal flora preparation in intestinal flora transplantation technology.

[0009] The fourth object of the present invention is to provide a method for testing the spore content of intestinal flora preparations.

[0010] Specifically, the preparation method of the intestinal flora preparation provided by the present invention includes: S1, taking a fecal sample for purification treatment to obtain a human bacterial suspension; S2, taking the human bacterial suspension for slow heating pretreatment and centrifugation treatment to obtain a pretreated bacterial mud; S3, taking the pretreated bacterial mud and mixing it with an ethanol solution with a concentration of 70-75%, performing a high-temperature incubation and sealing treatment to obtain an incubated bacterial solution; S4, taking the incubated bacterial solution for density gradient centrifugation treatment to obtain the intestinal flora preparation.

[0011] Furthermore, in step S1, the purification treatment is selected from one or more of centrifugation treatment, filtration treatment and dialysis treatment.

[0012] Furthermore, in step S2, the starting temperature of the slow heating treatment is 0-30°C, the heating rate is 0.5-2°C / min, the final temperature is 40-70°C, and the insulation time is 2-10h.

[0013] Furthermore, in step S3, based on the total volume of the ethanol solution, the added concentration of the pretreated bacterial sludge is 0.05-2 g / mL.

[0014] Furthermore, in step S3, the temperature of the high temperature incubation treatment is 60-100° C., and the time is 1-5 hours.

[0015] Furthermore, in step S4, the medium for density gradient centrifugation treatment is a 20-40 wt% sodium chloride solution, the temperature is 2-8° C., the rotation speed is 6000-16000 rpm, and the time is 10-40 min.

[0016] Furthermore, the preparation method includes step S5; step S5 includes: mixing the intestinal flora preparation with a germination agent, inducing incubation, and obtaining a spore suspension; and performing germination culture on the spore suspension to obtain an activated intestinal flora preparation.

[0017] Furthermore, the germination agent is selected from one or more of sodium taurocholate, ox bile powder and ox bile salt.

[0018] Furthermore, the intestinal flora preparation is added at a concentration of 0.05 to 2 g / mL; and the germination agent is added at a concentration of 0.001 to 2 wt%.

[0019] Furthermore, the induction incubation temperature is 20-60°C and the time is 5-40 minutes.

[0020] Furthermore, the culture medium for germination culture comprises 0.01-0.08 wt % of ox bile powder and 0.01-0.08 wt % of taurocholic acid.

[0021] Furthermore, the germination culture temperature is 30-40°C and the time is 48-96h.

[0022] The intestinal flora preparation provided by the present invention is prepared by the above-mentioned intestinal flora preparation preparation method.

[0023] The present invention also provides application of the intestinal flora preparation in intestinal flora transplantation technology.

[0024] The test method for the spore content of an intestinal flora preparation provided by the present invention comprises: mixing the intestinal flora preparation with a fluorescent reaction solution, performing fluorescence detection to obtain fluorescence intensity; and calculating the spore content in the preparation based on the fluorescence intensity; wherein the fluorescent reaction solution comprises 0.5 to 2 mM EuCl2, 0.5 to 2 mM TbCl3 and 0.5 to 2 mM aminocarboxylic acid complexing agent.

[0025] Furthermore, based on the total volume of the fluorescent reaction solution, the added amount of the intestinal flora preparation is 0.0005-0.05 g / mL.

[0026] Furthermore, the formula for calculating the spore content based on the fluorescence intensity is shown in formula (1):

[0027] Y = 7.225X + 56.36 Formula (1);

[0028] Among them, Y is the spore content, the unit is CFU / g; X is the fluorescence intensity, the unit is au.

[0029] Beneficial effects:

[0030] The preparation method of the intestinal flora preparation provided by the present invention has an excellent spore yield and the obtained intestinal flora preparation has a relatively ideal spore germination rate. A large number of live bacteria can be obtained by culturing it. At the same time, the preparation method can also effectively remove alcohol-soluble and / or fat-soluble harmful metabolites that are difficult to remove in the existing intestinal flora preparation process. It has the advantages of high purity and high activity, can be well applied to intestinal flora transplantation technology, and has broad application prospects.

[0031] It is speculated that the reason why the preparation method provided by the present invention has the above-mentioned technical effects is that: in the preparation process, the slow heating pretreatment method can gradually increase the thermal stimulation given, optimize the process of activating the nutrient cells of Firmicutes bacteria to transform into spores, and at the same time can better destroy or change the proteins and nucleic acid substances of common pathogenic bacteria such as Escherichia coli and Shigella, so as to obtain pretreated bacterial mud; and the obtained pretreated bacterial mud is subjected to high-temperature incubation treatment with an ethanol solution having a concentration of 70-75%, and the high-concentration ethanol in the early stage of the high-temperature incubation treatment is combined with the high-temperature treatment to achieve the Escherichia coli and Shigella in the pretreated bacterial mud. The common pathogenic bacteria such as fungi are completely killed and the incompletely transformed vegetative cells are further stimulated to transform into spores. During the high-temperature incubation process, the ethanol in the solution is in a dynamic process of volatilization and dissolution. At this time, the dynamic changes of ethanol in the solution can play a role in initially stimulating the activation of spores, which is beneficial to improving the spore germination rate. At the same time, the pretreated bacterial sludge is subjected to high-temperature incubation treatment with a volatile ethanol solution, and the density gradient centrifugation treatment is used to effectively remove the alcohol-soluble and / or fat-soluble harmful metabolites contained therein, so as to ultimately achieve the effect of improving the spore yield and spore germination rate and effectively removing harmful impurities. DETAILED DESCRIPTION

[0032] Based on the problems of low spore yield and spore germination rate in existing intestinal flora spore treatment processes and the inability to effectively remove harmful metabolites, viruses and other substances, the inventors of the present invention conducted extensive and in-depth research and a large number of experiments to obtain the technical solution of the present invention.

[0033] In the present invention, a preparation method of an intestinal flora preparation is first provided. The preparation method specifically comprises: taking a stool sample for purification treatment to obtain a human bacterial suspension; S2, taking the human bacterial suspension for slow heating pretreatment to obtain a pretreated bacterial mud; S3, taking the pretreated bacterial mud and mixing it with an ethanol solution, performing a high-temperature incubation sealing treatment, and obtaining an incubated bacterial solution; S4, taking the incubated bacterial solution for density gradient centrifugation treatment to obtain the intestinal flora preparation.

[0034] In the present invention, the purification treatment in step S1 is a method for purifying stool samples conventionally used in the field of intestinal flora transplantation technology, which is limited to the removal of large solid impurities such as food residues and digestive tract secretions. Those skilled in the art can make adaptive choices according to actual needs, and the present invention does not impose any special restrictions on it. Among them, specific examples of the purification treatment include but are not limited to: one or more of centrifugal treatment, filtration treatment and dialysis treatment.

[0035] In the present invention, the slow heating pretreatment in step S2 refers to the process of treating the human bacterial suspension by slowly heating the temperature, so as to activate the transformation of the vegetative cells of the probiotic bacteria of the genus Firmicutes into spores while destroying or changing the proteins and nucleic acid substances of common pathogenic bacteria such as Escherichia coli and Shigella.

[0036] In some specific embodiments, the conditions for the slow heating pretreatment in step S2 include: the starting temperature is preferably 0-30°C, such as 0°C, 4°C, 8°C, 10°C, 25°C, 30°C or any value therebetween; the heating rate is preferably 0.5-2°C / min, such as 0.5°C / min, 0.8°C / min, 1°C / min, 1.1°C / min, 1.2°C / min, 1.5°C / min, 1.8°C / min, 2°C / min or any value therebetween; the final temperature is preferably 40-70°C, such as 40°C, 40.3°C, 42°C, 45°C, 48°C, 50°C, 55°C, 60°C, 70°C or any value therebetween; the holding time is preferably 2-10h, such as 2h, 2.5h, 3h, 4h, 5h, 7h, 9h, 10h or any value therebetween.

[0037] In the present invention, the high temperature incubation closed treatment in step S3 refers to the process of treating the pretreated bacterial sludge using high temperature conditions, volatile ethanol and its concentration changes, so as to completely kill common pathogenic bacteria such as Escherichia coli and Shigella, further stimulate the nutrient cells that have not been completely converted into spores to be converted into spores and perform preliminary activation of the spores, and effectively remove alcohol-soluble and / or fat-soluble harmful metabolites using the similar compatibility principle.

[0038] In the present invention, the concentration of the ethanol solution in step S3 is specifically 70-75% (v / v), such as 70% (v / v), 71% (v / v), 72% (v / v), 74% (v / v), 75% (v / v) or any value therebetween.

[0039] In some specific embodiments, in step S3, based on the total volume of the ethanol solution, the added concentration of the pretreated bacterial sludge is preferably 0.05-2 g / mL, such as 0.05 g / mL, 0.1 g / mL, 0.2 g / mL, 0.3 g / mL, 0.4 g / mL, 0.5 g / mL, 1.0 g / mL, 1.5 g / mL, 2.0 g / mL or any value therebetween.

[0040] In some specific embodiments, the conditions for the high temperature incubation sealing treatment in step S3 include: the temperature is preferably 60-100°C, such as 60°C, 63°C, 68°C, 70°C, 75°C, 80°C, 85°C, 90°C, 100°C or any value therebetween; the time is preferably 1-5h, such as 1h, 1.3h, 1.8h, 2h, 3h, 4h, 5h or any value therebetween; and it should be noted that the container solvent used in the high temperature incubation sealing treatment is larger than or much larger than the volume of the solution being treated, so that the high temperature incubation sealing treatment is always in a safe state and leaves enough space to promote the dynamic changes in the concentration of ethanol in the solution.

[0041] In the present invention, the density gradient centrifugation treatment described in step S4 refers to a purification method in which a continuous or discontinuous density gradient is formed in a centrifuge tube using a certain medium, a cell suspension or homogenate is placed on the top of the medium, and the cells are stratified and separated by the action of gravity or a centrifugal field, so as to achieve the separation of spores and other impurities in the incubated bacterial liquid. Those skilled in the art can make adaptive choices according to actual needs, and the present invention does not impose any special limitation on it.

[0042] In some specific embodiments, the medium for the density gradient centrifugation treatment in step S4 is preferably a 20-40wt% sodium chloride solution; and the conditions for the density gradient centrifugation treatment include a temperature of preferably 2-8°C; such as 2°C, 4°C, 6°C, 8°C or any value therebetween; a rotation speed of preferably 6000-16000rpm, such as 6000rpm, 8000rpm, 10000rpm, 12000rpm, 14000rpm, 16000rpm or any value therebetween; and a time of 10-40min, such as 10min, 20min, 30min, 40min or any value therebetween.

[0043] In the present invention, based on the existence form of microorganisms in the obtained intestinal flora preparation, the preparation method preferably also includes step S5; the step S5 specifically includes: mixing the intestinal flora preparation with a germination agent, inducing incubation to obtain a spore suspension; taking the spore suspension for germination culture to obtain an activated intestinal flora preparation.

[0044] In the present invention, the germination agent in step S5 refers to a substance that can germinate spores to form vegetative cells. Specific examples of the germination agent include but are not limited to: one or more of pig bile powder, pig bile salt, ox bile powder and ox bile salt. In some specific embodiments, the germination agent preferably includes one or more of sodium taurocholate, ox bile powder and ox bile salt.

[0045] In some specific embodiments, the added concentration of the intestinal flora preparation in step S5 is preferably 0.05-2 g / mL; such as 0.05 g / mL, 0.1 g / mL, 0.2 g / mL, 0.3 g / mL, 0.4 g / mL, 0.5 g / mL, 1.0 g / mL, 1.5 g / mL, 2.0 g / mL or any value therebetween; the added concentration of the germination agent is preferably 0.001-2 wt%, such as 0.001 wt%, 0.005 wt%, 0.01 wt%, 0.1 wt%, 0.5 wt%, 1 wt%, 1.2 wt%, 2 wt% or any value therebetween.

[0046] In some specific embodiments, the conditions for inducing incubation in step S5 include: the temperature is preferably 20-60°C, such as 20, 23, 25, 30, 35, 40, 45, 50, 60 or any value therebetween; the time is preferably 5-40 min, such as 5 min, 8 min, 10 min, 15 min, 20 min, 25 min, 30 min, 40 min or any value therebetween.

[0047] In the present invention, the germination culture in step S5 refers to the process of culturing spores to germinate and form nutrient cells; wherein the culture medium used in the germination culture is a type of culture medium conventionally used in intestinal flora culture, and those skilled in the art can make adaptive choices according to actual needs, and the present invention does not impose any particular limitation on it. In some specific embodiments, specific examples of the culture medium used in the germination culture include but are not limited to: BHIS culture medium and / or YCFA culture medium.

[0048] In some specific embodiments, the culture medium for germination culture preferably includes ox bile powder and taurocholic acid. The ox bile powder is preferably added at a concentration of 0.01 to 0.08 wt%, such as 0.01 wt%, 0.03 wt%, 0.05 wt%, 0.07 wt%, 0.08 wt% or any value therebetween; the taurocholic acid is preferably added at a concentration of 0.01 to 0.08 wt%, such as 0.01 wt%, 0.02 wt%, 0.03 wt%, 0.04 wt%, 0.05 wt%, 0.06 wt%, 0.07 wt%, 0.08 wt% or any value therebetween.

[0049] In some specific embodiments, the conditions for the germination culture in step S5 include: the temperature is preferably 30-40°C, such as 30°C, 32°C, 34°C, 36°C, 38°C, 40°C or any value therebetween; the time is 48-96h, such as 48h, 72h, 96h or any value therebetween.

[0050] The present invention also provides an intestinal flora preparation, which is prepared by the above-mentioned intestinal flora preparation preparation method and has the advantages of high purity and high activity.

[0051] Based on the advantages of high purity and high activity of the intestinal flora preparation prepared by the preparation method of the intestinal flora preparation, the present invention also provides the application of the intestinal flora preparation in intestinal flora transplantation technology.

[0052] In order to further improve the quality of the intestinal flora preparation obtained by the preparation method of the intestinal flora preparation, the present invention also provides a method for testing the spore content of the intestinal flora preparation. The method utilizes the fluorescence reaction of DPA specifically contained in the spores with EuCl2, TbCl3 and aminocarboxylic acid complexing agent, measures the spore content in the intestinal flora preparation, and calculates based on the measured spore content, so as to finally realize the rapid detection of the spore content of the intestinal flora preparation.

[0053] In the present invention, the testing method specifically includes: mixing the intestinal flora preparation with a fluorescent reaction solution, performing fluorescence detection to obtain fluorescence intensity; and calculating based on the fluorescence intensity to infer the spore content in the preparation.

[0054] In the present invention, the fluorescent reaction solution includes EuCl2, TbCl3 and aminocarboxyl complexing agent; and the concentration of EuCl2 is preferably 0.5-2mM, such as 0.5mM, 0.8mM, 1mM, 1.5mM, 2mM or any value therebetween; the concentration of TbCl3 is preferably 0.5-2mM, such as 0.5mM, 0.6mM, 0.9mM, 1.3mM, 1.8mM, 2mM or any value therebetween; the concentration of aminocarboxyl complexing agent is preferably 0.5-2mM, such as 0.5mM, 0.9mM, 1.3mM, 1.8mM, 2mM or any value therebetween. Wherein, the aminocarboxyl complexing agent refers to a class of organic substances containing amino and carboxyl groups in the molecule and capable of forming stable chelates with most metal ions, and specific examples thereof include but are not limited to: ethylenediaminetetraacetic acid and / or cyclohexanediaminetetraacetic acid.

[0055] In some specific embodiments, based on the total volume of the fluorescent reaction solution, the added amount of the intestinal flora preparation is preferably 0.0005-0.05 g / mL, such as 0.0005 g / mL, 0.001 g / mL, 0.005 g / mL, 0.01 g / mL, 0.05 g / mL or any value therebetween.

[0056] In the present invention, the formula for calculating the spore content in the preparation based on the fluorescence intensity is shown in formula (1):

[0057] Y = 7.225X + 56.36 Formula (1);

[0058] Wherein, Y is the estimated spore content in the preparation, in CFU / g; X is the fluorescence intensity, in au.

[0059] Embodiments of the present invention are described in detail below, and the examples of the embodiments are intended to be used to explain the present invention, but should not be construed as limiting the present invention. In the embodiments, if specific techniques or conditions are not specified, the techniques or conditions described in the literature in this area or the product specifications are used. If the manufacturer is not specified for reagents or instruments used, they are all conventional products that can be obtained commercially.

[0060] It should be noted that the human bacterial suspensions used in the examples and comparative examples shown below are all from the same stool sample, and the preparation specifically includes: taking 200 g of stool samples from healthy donors that have passed the examination and mixing them with 2 L of normal saline, using a bacterial flora separation and extraction instrument (Chengge Pharmaceutical) and referring to the instructions for homogenization and multi-stage filtration, collecting the human bacterial suspension, and dividing the human bacterial suspension into 12 equal portions, and refrigerating at 4 ° C for later use.

[0061] The reagents and sources involved in the following examples include:

[0062] BHIS medium (Top Biotechnology, catalog number MD115);

[0063] Sodium taurocholate (West Asia Reagents, catalog number A20565);

[0064] Ox bile powder (Solarbio, catalog number B8210);

[0065] Ox bile salt (Solarbio, catalog number G8281);

[0066] Example 1

[0067] This embodiment provides a method for preparing an intestinal flora preparation, which specifically comprises:

[0068] (1) Take 200 mL of human bacterial suspension, slowly heat it at an initial temperature of 4°C and a heating rate of 2°C / min, heat it to 60°C and keep it warm for 3.5 hours, centrifuge it at 4°C and 8000 rpm for 10 minutes, and collect the pretreated bacterial sludge.

[0069] (2) The pretreated bacterial sludge was mixed with 75% ethanol solution at a dosage of 0.1 g / mL and added to a 1 L sterile container. The mixture was incubated at 80°C for 2 h in a closed environment. The mixture was centrifuged at 4°C and 8000 rpm for 10 min. The supernatant was removed and the precipitate was washed three times with saline. The precipitate was resuspended with saline to obtain a 0.5 g / mL incubation bacterial solution.

[0070] (3) Using 20-40 wt % sodium chloride solution as a medium, the incubated bacterial solution was subjected to density gradient centrifugation at 4° C. and 8000 rpm for 20 min to obtain the intestinal flora preparation.

[0071] Example 2

[0072] This example adopts the method provided in Example 1 to prepare an intestinal flora preparation, except that in step (1), the starting temperature is 30°C, the heating rate is 1°C / min, the final temperature is 60°C, the insulation time is 3.5h, and other conditions are the same to obtain an intestinal flora preparation.

[0073] Example 3

[0074] This example adopts the method provided in Example 1 to prepare an intestinal flora preparation, except that in step (1), the starting temperature is 0°C, the heating rate is 1.5°C / min, the final temperature is 40°C, the insulation time is 10 h, and other conditions are the same to obtain an intestinal flora preparation.

[0075] Example 4

[0076] This example adopts the method provided in Example 1 to prepare an intestinal flora preparation, except that in step (1), the starting temperature is 25°C, the heating rate is 1.5°C / min, the final temperature is 70°C, the insulation time is 10 h, and other conditions are the same to obtain an intestinal flora preparation.

[0077] Example 5

[0078] In this example, the intestinal flora preparation is prepared by the method provided in Example 1, except that in step (2), an equal volume of 70% ethanol solution is used instead of 75% ethanol solution, and the high temperature incubation temperature is 100°C and the time is 1 h. Other conditions are the same to obtain an intestinal flora preparation.

[0079] Comparative Example 1

[0080] This comparative example adopts the method provided in Example 1 to prepare the intestinal flora preparation, except that in step (2), an equal volume of 50% ethanol solution is used instead of 75% ethanol solution, and the temperature of high temperature incubation treatment is 100°C and the time is 1 hour. Other conditions are the same to obtain the intestinal flora preparation.

[0081] Comparative Example 2

[0082] In this example, the intestinal flora preparation is prepared by the method provided in Example 1, except that in step (2), an equal volume of 90% ethanol solution is used instead of 75% ethanol solution, and the high temperature incubation temperature is 60°C and the time is 5 hours. Other conditions are the same to obtain an intestinal flora preparation.

[0083] Comparative Example 3

[0084] This example adopts the method provided in Example 1 to prepare an intestinal flora preparation, except that in step (2), an equal volume of 75% isopropanol solution is used instead of 75% ethanol solution, and other conditions are the same to obtain an intestinal flora preparation.

[0085] Comparative Example 4

[0086] The comparative example provides a method for preparing an intestinal flora preparation, which specifically includes: (1) taking 200 mL of human bacterial suspension and centrifuging it at 4°C and 8000 rpm for 10 minutes, collecting bacterial mud, taking the pre-treated bacterial mud and mixing it with 75% ethanol solution according to the addition amount of 0.1 g / mL, adding it to a 1L sterile container, incubating it at 80°C for 2 hours in a closed environment, centrifuging it at 4°C and 8000 rpm for 10 minutes, removing the supernatant, and washing the precipitate with saline for 3 times, and resuspending the precipitate with saline to obtain 0.1 g / mL incubation bacterial solution. (2) using 20-40wt% sodium chloride solution as a medium, performing density gradient centrifugation on the incubation bacterial solution at 4°C and 8000 rpm for 20 minutes to obtain the intestinal flora preparation.

[0087] Comparative Example 5

[0088] The comparative example provides a method for preparing an intestinal flora preparation, which specifically includes: (1) taking 200 mL of human bacterial suspension, freezing it at -80°C for 4 hours, taking it out and thawing it at room temperature, and centrifuging the thawed bacterial suspension at 4°C and 8000 rpm for 10 minutes to obtain a pretreated bacterial sludge. (2) taking the pretreated bacterial sludge and mixing it evenly with a 75% ethanol solution according to an addition amount of 0.1 g / mL, adding it to a 1L sterile container, incubating it at 80°C for 2 hours in a closed environment, centrifuging it at 4°C and 8000 rpm for 10 minutes, removing the supernatant, and washing the precipitate with saline for 3 times, and resuspending the precipitate with saline to obtain a 0.1 g / mL incubation bacterial solution. (3) using a 20-40wt% sodium chloride solution as a medium, performing density gradient centrifugation on the incubation bacterial solution at 4°C and 8000 rpm for 20 minutes to obtain the intestinal flora preparation.

[0089] Comparative Example 6

[0090] The comparative example provides a method for preparing an intestinal flora preparation, specifically comprising: (1) taking 200 mL of a human bacterial suspension, placing it at 4°C for 4 hours, centrifuging it at 4°C and 8000 rpm for 10 minutes, removing the supernatant, and obtaining a pretreated bacterial sludge. (2) taking the pretreated bacterial sludge and mixing it evenly with a 75% ethanol solution according to an addition amount of 0.1 g / mL, adding it to a 1L sterile container, incubating it at 80°C in a closed environment for 2 hours, centrifuging it at 4°C and 8000 rpm for 10 minutes, removing the supernatant, and washing the precipitate with saline for 3 times, and resuspending the precipitate with saline to obtain a 0.1 g / mL incubation bacterial solution. (3) using a 20-40wt% sodium chloride solution as a medium, subjecting the incubation bacterial solution to a density gradient centrifugation treatment at 4°C and 8000 rpm for 20 minutes to obtain the intestinal flora preparation.

[0091] Comparative Example 7

[0092] This comparative example adopts the method provided in Example 1 to prepare the intestinal flora preparation, except that in step (2), the pretreated bacterial sludge and 75% ethanol solution are incubated at room temperature for 2 hours, and other conditions are the same to obtain the intestinal flora preparation.

[0093] Test Example 1

[0094] This test example is used to illustrate the number of germinated spores of the intestinal flora preparations provided in the above embodiments and comparative examples. The test specifically includes:

[0095] (1) Take 0.1 g of intestinal flora preparation and mix it with 0.9 mL of normal saline, incubate at 37°C for 30 min to obtain the test solution; take 100 μL of the test solution and spread it on a BHIS agar plate, incubate it at 37°C for 24 h, pick a single colony and send it for 16S rDNA testing, and judge whether the intestinal flora preparation contains Gram-negative pathogens based on the 16S rDNA sequencing results;

[0096] (2) Take 0.1g of intestinal flora preparation and mix with 0.9mL of normal saline, use 10-fold gradient dilution method to prepare test solution of appropriate dilution, take 100μL of test solution and spread on BHIS agar plate, spread on 3 plates in parallel, incubate at 37℃ for 24h, count and take the average value to obtain the number of viable germinated bacteria of intestinal flora preparation. The test results are shown in Table 1.

[0097] Table 1.

[0098]

[0099] It can be seen from the test results shown in Table 1 that, compared with comparative examples 1 to 7, the method provided by embodiments 1 to 5 of the present invention can effectively remove Gram-negative bacteria in fecal samples, and the number of live bacteria obtained after germination treatment of the obtained intestinal flora preparations is significantly higher than that of comparative examples 1 to 7, indicating that the intestinal flora preparations provided by embodiments 1 to 5 of the present invention have a higher spore yield and the obtained spores have good germination ability.

[0100] Test Example 2

[0101] This test example is used to illustrate the concentrations of four alcohol-soluble metabolites, namely, prostaglandin F1a, prostaglandin H2 2-glyceryl Ester, calcitriol, and 24-hydroxycalcitriol, in the intestinal flora preparations provided in the above embodiments and comparative examples, and sent to Shanghai Baiqu Biomedical Technology Co., Ltd. for metabolomics targeted detection. The test results are shown in Table 2.

[0102] Table 2.

[0103]

[0104] It can be seen from the test results shown in Table 2 that, compared with Comparative Example 1, the method provided by Examples 1 to 5 or Comparative Examples 2 to 7 of the present invention can effectively remove alcohol-soluble metabolites in the human bacterial suspension, and the content of alcohol-soluble metabolites in the obtained intestinal flora preparation is significantly reduced, which has higher biosafety.

[0105] Example 6

[0106] This embodiment provides a method for germination treatment of an intestinal flora preparation, taking the intestinal flora preparation provided in Example 1 as a treatment object, specifically comprising:

[0107] (1) 0.1 g of the intestinal flora preparation was mixed with 0.9 mL of the germination agent shown in Table 3, and the mixture was incubated at 37° C. for 30 min to obtain an activated intestinal flora preparation.

[0108] Table 3.

[0109]

[0110]

[0111] (2) A test solution with an appropriate dilution was prepared by a 10-fold gradient dilution method, 100 μL of the activated intestinal flora preparation was taken on a BHIS agar plate containing 0.05 wt% ox bile powder and 0.05 wt% sodium taurocholate, three plates were spread in parallel, anaerobically cultured at 37°C for 72 h, and the plate count was performed. The results are shown in Table 4.

[0112] Table 4.

[0113] Germination Agent <![CDATA[Number of viable germinated bacteria (×10 7 CFU / g)]]> 1 0.8 2 7.0 3 10.0 4 7.0 5 10.7 6 12.3 7 11.0 8 16.7

[0114] From the test results shown in Table 4, it can be seen that compared with other germination agents, the use of physiological saline including 0.04wt% sodium taurocholate, 0.04wt% ox bile powder and 0.02wt% ox bile salt as a germination agent to treat the same intestinal flora preparation can germinate more live bacteria and has an excellent effect on promoting spore germination.

[0115] Example 10

[0116] This embodiment provides a method for detecting the spore content in an intestinal flora preparation. The intestinal flora preparation provided in the above embodiment is used as a sample. The testing method specifically includes:

[0117] Take 0.1 g of the intestinal flora preparation and resuspend it with a small amount of physiological saline. After heating at 121°C for 30 minutes, centrifuge and collect the supernatant, filter it through a 0.22 μm filter membrane to obtain a test solution. Take 10 μL of the test solution and mix it evenly with 90 μL of the fluorescent reaction solution. Use a fluorescence detector to detect the fluorescence signal at an excitation wavelength of 275 nm and an emission wavelength of 543 nm, and calculate the spore content of the preparation based on the following formula.

[0118] Y = 7.225X + 56.36 Formula (1);

[0119] Among them, Y is the estimated spore content of the preparation, the unit is CFU / g; X is the fluorescence intensity, the unit is au, R 2 =0.9067. The test results are shown in Table 5.

[0120] Table 5.

[0121]

[0122] From the comparison of the test results shown in Table 1 and Table 5, it can be seen that the intestinal flora preparation is tested using the detection method provided in this embodiment, and the trend ratio between the inferred preparation spore content obtained and the results of each group obtained by the plate count method used in Test Example 1 is highly consistent, and this test method can make up for the limitation of the plate count method that cannot detect uncultivable bacteria, thereby obtaining an inferred spore content that is closer to the actual situation of the intestinal flora preparation.

[0123] That is, this detection method can effectively replace the plate count method and be applied in the production process of intestinal flora preparations to realize rapid detection of the activity of intestinal flora preparations. It has higher real-time and authenticity, allowing producers to adjust the production process in time according to real-time detection results, thereby preparing intestinal flora preparations with better activity.

[0124] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and purpose of the present invention.

Claims

1. A method for preparing an intestinal flora preparation, characterized in that: The preparation method comprises: S1, taking a stool sample for purification treatment to obtain a human bacterial suspension; S2, taking the human bacterial suspension for slow heating pretreatment to obtain a pretreated bacterial mud; S3, taking the pretreated bacterial mud and mixing it with an ethanol solution with a concentration of 70-75% (v / v), performing a high-temperature incubation and sealing treatment to obtain an incubated bacterial solution; S4, taking the incubated bacterial solution for density gradient centrifugation treatment to obtain the intestinal flora preparation.

2. The method for preparing the intestinal flora preparation according to claim 1, characterized in that: In step S1, the purification treatment is selected from one or more of centrifugation treatment, filtration treatment and dialysis treatment.

3. The method for preparing the intestinal flora preparation according to claim 1, characterized in that: In step S2, the starting temperature of the slow heating treatment is 0-30°C, the heating rate is 0.5-2°C / min, the final temperature is 40-70°C, and the insulation time is 2-10h.

4. The method for preparing the intestinal flora preparation according to claim 1, characterized in that: In step S3, based on the total volume of the ethanol solution, the concentration of the pretreated bacterial sludge added is 0.05-2 g / mL; Optionally, the high temperature incubation treatment is performed at a temperature of 60 to 100° C. and a time of 1 to 5 hours.

5. The method for preparing the intestinal flora preparation according to claim 1, characterized in that: In step S4, the medium for density gradient centrifugation treatment is a 20-40 wt% sodium chloride solution, the temperature is 2-8° C., the rotation speed is 6000-16000 rpm, and the time is 10-40 min.

6. The method for preparing the intestinal flora preparation according to claim 1, characterized in that: The preparation method comprises step S5; step S5 comprises: mixing the intestinal flora preparation with a germination agent, inducing incubation, and obtaining a spore suspension; performing germination culture on the spore suspension, and obtaining an activated intestinal flora preparation; Optionally, the germination agent includes one or more of sodium taurocholate, ox bile powder and ox bile salt; Optionally, the intestinal flora preparation is added at a concentration of 0.05 to 2 g / mL; the germination agent is added at a concentration of 0.001 to 2 wt%; Optionally, the induction incubation temperature is 20 to 60°C and the time is 5 to 40 minutes; Optionally, the culture medium for germination culture comprises 0.01-0.08 wt % of ox bile powder and 0.01-0.08 wt % of taurocholic acid; Optionally, the germination culture is carried out at a temperature of 30 to 40° C. and for a time of 48 to 96 hours.

7. An intestinal flora preparation, characterized in that: The intestinal flora preparation is prepared by the preparation method of any one of claims 1 to 6.

8. Use of the intestinal flora preparation according to claim 7 in intestinal flora transplantation technology.

9. A method for testing the spore content of an intestinal flora preparation, characterized in that: The testing method comprises: mixing an intestinal flora preparation with a fluorescent reaction solution, performing fluorescence detection to obtain fluorescence intensity; and calculating the spore content in the preparation based on the fluorescence intensity; wherein the fluorescent reaction solution comprises 0.5 to 2 mM EuCl2, 0.5 to 2 mM TbCl3 and 0.5 to 2 mM aminocarboxylic acid complexing agent.

10. The method for testing the spore content of the intestinal flora preparation according to claim 9, characterized in that: Based on the total volume of the fluorescent reaction solution, the added amount of the intestinal flora preparation is 0.0005-0.05 g / mL; Optionally, the formula for calculating the estimated spore content based on the fluorescence intensity is as shown in formula (1): Y = 7.225X + 56.36 Formula (1); Among them, Y is the estimated spore content, the unit is CFU / g; X is the fluorescence intensity, the unit is au.

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