Intestinal content separation and collection device, method and extract

Abstract

The present invention provides an apparatus and method for separating and recovering intestinal contents, which includes an impurity removal module and a target content collection module. The impurity removal module has a cross-flow filtration structure and removes impurities from the intestinal contents. The target content collection module is mainly composed of a microporous membrane cross-flow filter. The method removes impurities from the intestinal contents through rough filtration and cross-flow fine filtration, and then obtains a concentrate through the microporous membrane cross-flow filter to collect the target contents. The microporous membrane has a sponge-like porous structure, which, combined with cross-flow filtration, can quickly and effectively separate and recover the bacterial flora from the intestinal contents. The separated and recovered bacterial flora powder accounts for at least 10% to 20% of the initial wet intestinal contents, and the bacterial content is at least 10%. 11 Each piece is 1g of powder.

Description

[Technical field]

[0001] The present invention relates to the field of medical application technology, and in particular to an apparatus for separating and recovering intestinal contents, a method for separating and recovering intestinal contents, and an extract thereof. [Background technology]

[0002] The intestinal flora of mammals is an important metabolic and immune "organ" in the mammalian body, and affects the host's overall metabolism. The host's metabolism is influenced by its own genes and the genes of the intestinal flora, and there is a "cometabolic" process between the host and the flora. Intestinal bacteria can participate in the metabolism of the host and establish a cometabolic relationship with the host. Under normal circumstances, the intestinal flora in a mammal's body is relatively stable, but once it becomes unbalanced, it can cause the host's immune system to malfunction, leading to disease.

[0003] The human body is home to a huge number of symbiotic microorganisms, most of which reside in the human intestinal tract and number approximately 1000 trillion (10 14 The number of intestinal microorganisms exceeds 100,000, which is about 10 times the total number of cells in the human body. More than 99% of intestinal microorganisms are bacteria, including the majority of anaerobic bacteria and a small number of aerobic bacteria. Intestinal microorganisms are important participants in human metabolism, providing substrates, enzymes, and energy to human metabolic processes, and fatty acids obtained by the metabolism of intestinal microorganisms promote the growth and differentiation of human epithelial cells, and are involved in the synthesis of vitamins and the absorption of various ions. The intestine is the largest immune organ in the human body, and the cooperation between intestinal microorganisms and the host on the surface of the intestinal mucosa promotes the establishment and development of the immune system, becoming an important immune barrier for the human body. In addition, intestinal microorganisms provide a protective function for the human body by forming a "bacterial membrane barrier."

[0004] The intestinal microorganisms of the human body not only help digestion of food, but also act as a barrier to effectively resist pathogens that invade the human body from the outside, and have always grown with mankind. The balanced and stable intestinal microecological environment formed in the long evolutionary process plays an important role in the nutrition, metabolism and immunity of the human body. In the long evolutionary process, intestinal microorganisms cooperate with mankind and play a very important role in the nutrition, metabolism and immunity of the human body.

[0005] The number of various bacteria in the intestinal tract plays a role only when an equilibrium is maintained. This balance is very fragile, and most cases of abdominal pain and diarrhea are caused by an imbalance in the intestinal flora. Malnutrition, obesity, diabetes, and other conditions may all be related to an imbalance in the intestinal flora. Recent research has shown that senile dementia and autism may also be related to an imbalance in the intestinal flora.

[0006] In December 2013, the top ten scientific advances published by Science magazine included research on the relationship between intestinal microbiota and human health. The intestinal microbial flora is very complex, and the intestinal microbiota, which are combined in a certain ratio and dependent on each other while restricting each other, form an ecological balance in terms of quantity that is symbiotic with the host and benefits both. Food, drink, and probiotics can have a certain degree of action or influence on the intestinal microbial flora. However, although improving diet can improve the intestinal microecological balance, the process is slow and takes a relatively long time. In addition, there are many types of intestinal microbiota, and even if probiotics, which are limited in quantity and type, are supplemented, it is difficult to restore the overall balance of the intestinal microecology, and the therapeutic effect is limited. In recent years, fecal microbiota transplantation (FMT) has become an important new technology in the medical field, and the reconstruction of the intestinal microbiota has gradually been recognized by people and has attracted clinical attention.

[0007] The feces of mammals, such as humans, mainly contain undigested residues (e.g., dietary fiber), intestinal secretions (including mucus), proteins, white blood cells, epithelial cells, bacteria, inorganic matter (mainly calcium and phosphate), indigestible or unabsorbable food, and water. Most healthy adults defecate once a day, and the amount of feces excreted varies according to the type of food, the amount of food eaten, and the functional state of the digestive organs. For example, those who mainly consume rice, wheat flour, and meat produce delicate and small amounts of feces, while those who consume a lot of miscellaneous grains and especially a lot of vegetables produce coarse and large amounts of feces due to the high fiber content.

[0008] The number of bacteria in feces is very large, accounting for 1 / 3 of the dry weight, and most of them are normal bacterial flora. In healthy infant feces, there are mainly bifidobacteria, bacteroids, enterobacter, enterococci, staphylococci, etc. In adult feces, Escherichia coli, anaerobic bacteria, and enterococci are the main bacterial flora, accounting for about 80%, while most of the bacteria such as aerogenes, Proteus, and Pseudomonas aeruginosa are transient bacteria, accounting for no more than 10%. The total amount of spore-forming bacteria (such as Clostridium) and yeast does not exceed 10%. The number of bacteria and bacterial family tree in feces are usually in a relatively stable state, and maintain an ecological balance with the host ("Clinical Laboratory Diagnosis and Clinical Application" edited by Wang Ruihui, Changchun, China: Jilin Science and Technology Press, March 2017).

[0009] Fecal microbial transplantation transfers the bacterial flora (intestinal flora) in the feces of healthy individuals into the patient's body, allowing the patient to have the intestinal flora of healthy individuals, regulating the imbalance of the intestinal flora, and reconstructing a normally functional intestinal microecosystem, thereby providing help in the treatment of intestinal and extraintestinal diseases. In modern times, during treatment using fecal microbial transplantation, the fecal bacterial state used is relatively often fresh fecal bacterial fluid, or fecal juice, fermented fecal juice, or child fecal juice. The "Huanglong Tang" of ancient Chinese doctors is a prescription that contains human fecal juice. Fecal microbial transplantation using fecal bacterial fluid requires medical personnel and patients to endure a great mental challenge, but a more important issue is how to complete the isolation and preservation of fecal bacteria within a time period that does not affect the function of the bacterial flora.

[0010] There are at least 1000-1150 kinds of bacteria in the human intestine, and on average, each host contains about 160 dominant species. The main constituent groups of the human intestinal microorganisms are very similar, but the relative content and strain types of each microbial group are significantly different between different host individuals. Factors that affect the differences in microbial flora include the host's region, age, physiological state, dietary habits, and other factors. Human fecal bacteria are currently the only organ that humans can essentially share, and there is no need to consider the problem of immune rejection. The intestinal flora of each individual has a different ecological balance and can have large differences. This also increases the difficulty of fecal microbial transplantation to a certain extent.

[0011] One of the important points of fecal fluid transplantation is the preparation of fecal bacteria for transplantation. Currently, fecal bacterial fluid is relatively common. However, there is still no consensus on the preparation of fecal bacterial fluid, and there are many factors that affect the prepared fecal bacterial fluid. First, fecal volume: The weight of feces is not proportional to the number of bacterial flora, and there is a relatively large individual difference, so there is no unified standard for determining the amount of feces collected when preparing fecal bacterial fluid. Second, preparation time: Fecal bacterial flora contains many anaerobic bacteria, and the metabolic time of each bacterial species is different. Therefore, it is necessary to complete the separation and extraction within the shortest possible time, otherwise the death of anaerobic bacteria and the relative abundance of bacterial flora species will occur, which will affect the clinical treatment effect. Third, conventional preparation flow and method: mainly using manual coarse filtration plus centrifugal concentration method, or fine filtration plus centrifugal concentration method. Fecal bacteria obtained by conventional preparation methods have a large difference in the type and abundance of bacterial flora compared to the bacterial flora distribution in the initial intestinal contents, and the reproducibility is poor. Fourth, the state of fecal bacterial liquid: there are fresh fecal bacterial liquid and freeze-dried fecal bacterial liquid. Freeze-dried fecal bacterial liquid is prepared by adding glycerin to fresh fecal bacterial liquid and then freezing it at a low temperature, or it can be prepared into frozen fecal bacterial capsules.

[0012] Currently, automatic fecal bacteria separation and recovery systems often use multi-stage filtration to remove other impurities, and some documents in China have been published, such as CN103330961B, CN105624024B, and CN204097475U. However, all of these fecal bacteria separation and recovery systems are obtained by filtering and separating large particle impurities in fecal solutions, such as undigested food residues, and the bacterial solution should contain a large amount of food residues, bacterial metabolic products, and water. Currently, the commonly used method is to centrifuge the mixed liquid from which the large particle impurities have been filtered out by a centrifuge to separate it into layers, and discard the supernatant or sediment to obtain useful bacterial flora. Most intestinal bacteria are between 0.1 and 50 μm in size, and discarding the supernatant will result in the loss of a large amount of small-sized microorganisms. When the supernatant is left and the sediment is discarded, the large-sized microorganisms will be lost. Furthermore, due to the diversity and complexity of stool sample components and the differences in operation between different operators, the method varies greatly and is therefore uncontrollable, making it difficult to achieve standardized reproducible operation, and the operation is inconvenient.

[0013] However, these methods have poor controllability, cannot selectively screen specific intestinal microorganisms, and cannot meet the needs of precision treatment of intestinal diseases in the future. At the same time, such devices use conventional filter filtration methods to perform microfiltration and remove impurities with relatively small sizes. The microfiltration filter has an aperture size of 0.01mm to 1mm and is very prone to clogging by impurities, which greatly reduces the efficiency of filtration. At the same time, feces contains a large amount of viscous components, which greatly reduces the efficiency of the conventional filter filtration method and makes it impossible to achieve separation and extraction of bacterial flora in an effective time. Due to the high bacterial content in fecal bacteria, the conventional multi-stage filtration patent technology has a low bacterial content per unit volume obtained, and the reproducibility of the operation and the consistency of the bacterial flora and the sample are relatively poor, which increases the difficulty of bacterial flora transplantation.

[0014] WO2012122478A1 discloses a composition of human fecal extract. The mixed (blended) sample is passed through a filter of less than 2.0 mm, and the filtrate is collected in one centrifuge tube and centrifuged for 10 minutes. The supernatant is removed, the cells are resuspended in a diluent, and centrifuged again for 10 minutes. The final supernatant is discarded, and the cells are resuspended in an aqueous solution to obtain a composition. In the method of the document, a large amount of bacteria is discarded, and the operation in air causes the death of a large amount of anaerobic bacteria.

[0015] WO2016065777A1 discloses the following: A microorganism separation device for luminal contents, in which the outlet of a current tank is connected to the inlet of a stage filtration device, the stage filtration device is composed of one or more stage filtration flasks, the liquid outlet of the storage tank is connected to the liquid inlet of the current tank and at least one stage filtration flask, the material supply port of a sealed separated material tank is connected to the outlet of the stage filtration device, the dispensing device is composed of at least one dispensing bottle, the bacteria liquid inlet of the first dispensing bottle is connected to the bacteria liquid outlet of the separated material tank, the odor inlet of the deodorizing device is connected to the odor outlet of the current tank and the odor outlet of the separated material tank via an odor pipe, and at least one air pump is provided in the odor pipe to introduce the odors of the current tank and the separated material tank into the deodorizing device, and the separation device assists filtration by performing vibrations such as rocking, swinging, or waving on the liquid in the stage filtration flask.

[0016] WO2016201114A1 discloses a method for producing an oral microbiota restoration therapy (MRT) composition, the method comprising: collecting a fecal sample and purifying the fecal sample to obtain a purification intermediate, wherein the purification of the fecal sample includes adding a diluent to the fecal sample, mixing the fecal sample with the diluent to form a mixture, filtering the mixture, transferring the filtrate from the filtration step to a centrifuge tube, centrifuging the filtrate to obtain a purification intermediate, freeze-drying the purification intermediate to obtain a plurality of freeze-dried pellets, and further encapsulating the plurality of freeze-dried pellets in one or more capsules.

[0017] CN201810852561.2 discloses the following: An intestinal content separation and recovery device, method and product thereof, which adopts a microporous membrane separation module combining sidewall filtration and rotary separation technology to extract intestinal microorganisms instead of the traditional centrifugal separation technology. In the application of intestinal content separation, the filtration characteristics of the nuclear pore membrane are utilized to collect various types of intestinal contents through filtration of each nuclear pore membrane with different pore sizes, and what is captured on the nuclear pore membrane is the desired product. This invention is an early invention by the inventor, and has made a relatively large improvement in obtaining intestinal microorganisms by centrifugation, but still uses traditional filtration means in the impurity removal stage, and the final collection of intestinal microorganisms uses a nuclear pore membrane, which is a membrane structure with a nearly uniform pore size, and it is necessary to apply negative pressure to the lower end to perform cross-flow filtration, so there is still room for improvement in the overall filtration efficiency and the quality of the obtained intestinal microorganisms.

[0018] Traditional fecal microbiota extraction processes often use a centrifugation process. However, there are differences between intestinal contents in the centrifugation process, including the composition and abundance of various food residues and bacteria in the feces, and the results of each separation are different, and the species of bacteria that are eliminated are also different. Even if the parameters of the centrifugation process, such as the rotation speed and solid-liquid ratio, are the same, the product quality of the final microbiota is uncontrollable and effective reproducibility cannot be achieved. Moreover, manual operations are involved in the operation process, and the reproducibility of the entire process faces a great challenge.

[0019] Secondly, the extraction efficiency is generally low, and the loss of intestinal bacteria during the extraction process is serious, limiting the number of bacteria extracted, which reduces the therapeutic effect of FMT and increases the cost of FMT. At the same time, fecal microbiota needs to be processed by multiple flows, and because fecal microbiota is mainly anaerobic, it needs to be centrifuged in a nitrogen gas-substituted biomechanical chamber (glove box), which makes the operation difficult. In addition, not all operations can be performed in a nitrogen gas-substituted biomechanical chamber (glove box), so it is difficult to guarantee the quality and the treatment effect is unstable.

[0020] Therefore, the lack of standardized fecal bacterial isolation and transplantation technology in the prior art is one of the important reasons restricting the development of fecal microbial transplantation. How to quickly and effectively perform fecal bacterial isolation, while ensuring its reproducibility, standardization, and ensuring the consistency of the obtained fecal bacteria with the initial sample are of great significance to fecal bacterial isolation. The technical field is required to effectively solve the above-mentioned problems of centrifugation by an intestinal content isolation and recovery device and an intestinal content isolation and recovery method, and to eliminate or alleviate all or part of the shortcomings in the prior art. Summary of the Invention

[0021] In response to the problems with the prior art described above, an object of the present invention is, first, to provide an intestinal content separation and recovery device which realizes efficient, precise and controllable collection of specific intestinal bacterial flora by microporous membrane ultrafine module cross-flow filtration.

[0022] At the same time, the present invention further provides a method for separating and recovering intestinal contents, and an extract of the intestinal contents obtained by the separation and recovery.

[0023] It should be noted here that unless otherwise specified, the terms used in this specification have the same meaning as the general meaning of each scientific and technical term in the relevant technical field, and the technical terms defined in various technical dictionaries, textbooks, etc.

[0024] The present invention first provides an intestinal content separation and recovery device, wherein the content separation and recovery device includes an impurity removal module and a target content collection module, the impurity removal module has a cross-flow filtration structure and removes impurities from the intestinal content, the target content collection module is mainly composed of a microporous membrane cross-flow filter, the microporous membrane has a sponge-like porous structure, the pore size range of the microporous membrane is 0.01 to 20 μm, preferably the pore size range of the microporous membrane is 0.1 to 10 μm, more preferably the pore size range of the microporous membrane is 0.2 to 5 μm, the aperture ratio (opening rate) of the microporous membrane is 30% or more, preferably 50%, 60% or more, more preferably 70%, 80%, 85%, 90% or more, and the microporous membrane is a hydrophilic membrane.

[0025] Furthermore, the microporous membrane crossflow filter has a disk-shaped structure, and the ratio of the effective filtration area of ​​the crossflow disk to the total area of ​​the crossflow disk is 0.3 to 0.9, preferably 0.4 to 0.8, and more preferably 0.5 to 0.6. The ratio of these two values ​​indicates the effective utilization rate of the microporous membrane in crossflow filtration. In the present invention, when the total area of ​​the microporous membrane crossflow disk is about 270 cm, 2 , with an effective filtration area of ​​approximately 150 cm 2 It can be said that: Furthermore, the flow path of the microporous membrane crossflow filter is a spiral flow path, which provides effective support for the flow of liquid and allows crossflow filtration to be performed using as much of the microporous membrane as possible. Furthermore, the cross-sectional area of ​​the microporous membrane cross-flow filter is smaller than the cross-sectional area of ​​the inlet pipe, and the ratio of the former to the latter is 0.8, 0.6, or 0.5 or less. For example, when the cross-sectional area of ​​the inlet pipe is about 35 mm 2 In this case, the cross-sectional area of ​​the flow passage of the microporous membrane cross-flow filter is about 15 to 20 mm 2 In the present invention, cross-flow filtration can be performed without using separate pressurized or negative pressure equipment, and although this increases additional costs, it does not mean that the corresponding equipment cannot be used.

[0026] Furthermore, the separation and recovery device includes a pump, which transports the intestinal content solution from the impurity removal module to the target content collection module through a pipe, and the pump is preferably a peristaltic pump to avoid cross-infection of the intestinal content solution and meet the requirements of medical consumables.

[0027] Furthermore, the impurity removal module includes a multi-stage filtration module, for example, a coarse filtration module and a fine filtration module. The fine filtration module preferably uses cross-flow filtration.

[0028] Furthermore, the concentrate from the cross-flow filtration mixes with the permeate from the previous stage of filtration to form a circulation circuit. The microporous membrane may be one or more of polyethersulfone membrane, cellulose membrane, polymethacrylate membrane, modified or unmodified. Preferably, the microporous membrane, polyethersulfone membrane, is a wet casting phase inversion membrane. The polyethersulfone membrane may be an improved polyethersulfone filtration membrane, and may be a disk filtration membrane 003 (0.03 μm), 010 (0.1 μm), 020 (0.2 μm), 045 (0.45 μm), 065 (0.65 μm), 080 (0.80 μm), 120 (1.2 μm), 500 (5.0 μm), or 1000 (10 μm). The microporous membrane of the present invention has a network structure constructed by connecting particles with belt-like fibers, the non-uniform pore structure shows a three-dimensional or three-dimensional change in the pores, and the sponge-like porous structure has a high porosity. Such a membrane structure exhibits superior performance to the relatively uniform Nuclepore membrane during cross-flow filtration, and can achieve cross-flow filtration quickly and effectively without the need for a separate pressurizing or negative pressure device. At the same time, a spacer may be provided on the back side of the microporous membrane to prevent it from being damaged during use.

[0029] Further, one embodiment of the present invention provides an intestinal content separation and recovery apparatus, the intestinal content separation and recovery apparatus including an impurity removal module and a target content collection module, the impurity removal module including a stirring coarse filtration module and a fine filtration module; The stirring / coarse filtration module is a vessel having an stirring device at an upper portion, a coarse filtration filter at a lower portion, and an outlet and a reflux port at a bottom portion, The microfiltration module includes a microfiltration cross-flow disk filter; The intestinal contents (feces) are mixed with a solvent (e.g., physiological saline), and then first thoroughly stirred by the above-mentioned stirring device so as to thoroughly disperse the intestinal contents in the solvent. Next, relatively large impurities are removed by a coarse filtration filter. The contents are then transported by a first transport pump to the above-mentioned precision filtration cross-flow disk type filter, and passed through the above precision filtration cross-flow disk type filter to remove relatively fine impurities. The target content collection module includes a microporous membrane cross-flow disk filter using a microporous membrane filter, and the permeate after filtration by the precision filtration module is transported to the microporous membrane cross-flow disk filter by a second transport pump, and the target content is collected from the concentrate of the microporous membrane cross-flow disk filter after cross-flow filtration by the microporous membrane filter.

[0030] Furthermore, the above-mentioned microfiltration cross-flow disc-type filter has a filter pore size of 5 to 100 μm, preferably 5 to 50 μm, and more preferably 10 μm, 20 μm, 25 μm or 30 μm. Furthermore, the filter (microporous membrane) of the microporous membrane crossflow disk type filter has a pore size of 0.01 to 20 μm, preferably 0.1 to 10 μm, and more preferably 0.2 μm, 0.5 μm, 1 μm, 2 μm, 5 μm or 7 μm.

[0031] In addition, the microfiltration module further comprises a storage tank, and the permeate filtered by the microfiltration cross-flow disk filter enters the storage tank and then enters the target content collection module. The microfiltered concentrate is returned to the vessel through the return port via a pipe to mix with the coarsely filtered permeate and form a circulation circuit.

[0032] In addition, the target content collection module further includes a permeate effluent tank, the permeate filtered by the microporous membrane cross-flow disk filter enters the permeate effluent tank, and the concentrated liquid flows back to the storage tank of the precision filtration module via a pipeline. After circulating and filtering for 10 to 60 minutes, preferably 50 minutes, the target content is collected from the concentrated liquid of the microporous membrane cross-flow disk filter.

[0033] Further, the target content collection module includes one microporous membrane cross-flow disk type filter, or the target content collection module includes a plurality of microporous membrane cross-flow disk type filters with different pore sizes, which are arranged in sequence from larger pore size to smaller pore size, and are used to collect target contents of different sizes.

[0034] Furthermore, the target contents are intestinal microorganisms (intestinal bacterial flora), including bifidobacteria, lactobacilli, Escherichia coli, enterococci, etc., and the size of the intestinal microorganisms is 0.1 μm to 50 μm.

[0035] Furthermore, a cover is installed on the outside of the container of the stirring coarse filtration module, and an exhaust port is provided in the ceiling of the cover, and smelly gas from feces is exhausted from the exhaust port and deodorized by a deodorizing device.

[0036] Furthermore, the separation and recovery apparatus is equipped with an oxygen removal module to ensure and maintain the intestinal contents in a low-oxygen or oxygen-free atmosphere during the separation and recovery process.

[0037] The present invention also provides a method for separating and recovering intestinal contents using the device of the present invention, said method being carried out using the device of the present invention, Step 1) Dispersion step: mixing the intestinal contents with a liquid dispersion medium, the ratio of the intestinal contents to the liquid dispersion medium is 100g:200-5000ml, preferably 100g:300-1000ml, or 100g:500-800ml, and mechanically dispersing the resulting mixture to form a uniform dispersion mixture; Step 2) impurity removal step: passing the dispersion mixture through a filtration filter to remove impurities, the filtration including cross-flow filtration, to obtain a permeate (filtrate); Step 3) a collection step, the permeate of step 2) is filtered through a microporous membrane cross-flow filter to obtain a concentrate and a filtrate waste, the concentrate is for collecting the target content; The microporous membrane of the microporous membrane crossflow filter has a sponge-like porous structure, the pore size range of the microporous membrane is 0.01 to 20 μm, preferably the pore size range of the microporous membrane is 0.1 to 10 μm, more preferably the pore size range of the microporous membrane is 0.2 to 5 μm, the opening ratio of the microporous membrane is 30% or more, preferably 50%, 60% or more, more preferably 70%, 80%, 85%, 90% or more, and the microporous membrane is a hydrophilic film.

[0038] Furthermore, in the dispersion mixture of step 1), the content of bacteria is at least 10 8 pieces / ml, or 10 9 pieces / ml, or 10 10 pieces / ml. Furthermore, the concentrated solution from step 3) is refluxed and mixed with the permeate from step 2) to form a circulation circuit.

[0039] Furthermore, the filtration and impurity removal in step 2) is divided into coarse filtration and fine filtration, and the filter pore size of the coarse filtration is greater than 10 μm, or greater than 50 μm, or greater than 100 μm, or greater than 200 μm, or greater than 500 μm, and a crude permeate (filtrate) is obtained, and then the crude permeate is subjected to fine filtration.

[0040] Furthermore, the microfiltration in step 2) is cross-flow filtration, and the filter pore size of the microfiltration is 5 to 100 μm, preferably 5 to 50 μm, more preferably 10 μm, 20 μm, 25 μm or 30 μm, and a micropermeate (filtrate) and a concentrated liquid are obtained, of which the micropermeate enters the microporous membrane cross-flow filter in step 3) and is filtered to obtain a concentrated liquid and a filtrate effluent.

[0041] Furthermore, the concentrated liquid from step 3) returns to the previous step and mixes with the permeate, thereby forming a circulation circuit.

[0042] Furthermore, the mechanical means in step 1) above may be stirring or other similar means for effectively dispersing a solid-liquid mixture, such as ball milling, shaking, and the like.

[0043] Furthermore, the concentrated bacterial liquid from step 3) is freeze-dried to prepare a powder. A composition comprising an intestinal contents extract, the intestinal contents extract being prepared by any of the intestinal contents separation and recovery devices or any of the intestinal contents separation and recovery methods described above, and the size of the obtained intestinal contents extract is 0.01 to 50 μm, 0.1 to 40 μm, 0.2 to 30 μm, or 0.5 to 20 μm.

[0044] Furthermore, the intestinal content extract is a concentrated bacterial liquid, which can be freeze-dried to produce a powder with a bacterial content of at least 10 11 Pieces / 1g powder, or 2×10 11 Pieces / 1g powder, or 5×10 11 1g powder or 10 12 Each piece is 1g of powder.

[0045] The distribution (type and abundance) of the bacterial flora in the intestinal content extracts is very close to that of the intestinal contents. Such specific substances in the intestinal contents that are waiting to be processed have characteristics such as high stickiness and large individual differences, as well as individual differences in the intestinal contents and differences in the operation of the operators. Therefore, centrifugation is often used in conventional bacterial flora extraction, but a large amount of bacteria is separated and discarded through centrifugation, which results in poor quality control, a large amount of bacterial loss, low biodiversity, poor reproducibility, inconvenient operation, poor environmental control, and low bacterial survival rate (large amount of anaerobic bacteria in the intestinal tract), which leads to poor or unstable therapeutic effects.

[0046] Since the proliferation rates of the bacterial species contained in the intestinal contents are different, if the separation and extraction of the intestinal contents is not completed within an effective time, for example within 1.5 hours, preferably within 1 hour, the distribution of bacterial species in the obtained extract will differ from the distribution of bacterial species in the initial (initial) intestinal contents.

[0047] The intestinal contents separation and recovery device of the present invention does not use any centrifugal parts, does not need to obtain the target contents by centrifugal means, and obtains the target contents by combining cross-flow filtration and microporous membrane. While ensuring filtration efficiency, extraction is completed within an effective time, thus solving problems such as blockage and efficiency reduction, and effectively extracting all bacterial species while maintaining the relative abundance of the original bacterial species.

[0048] The present invention uses microporous membrane cross-flow filtration to concentrate the target intestinal contents instead of the conventional centrifugal separation technique, which allows for more precise control of the extraction step, higher separation efficiency, and at the same time simplifies the operation and allows for a higher level of automation. In addition, by using microporous membranes with different pore sizes, the size effect can be exploited to selectively separate the desired intestinal contents.

[0049] In the present invention, filtration is divided into coarse filtration and fine filtration, and fine filtration is performed by cross-flow filtration. Conventionally, fine filtration is performed using a filter method, but the pore size of the filter for fine filtration is 0.01 to 1 mm, which is very prone to clogging (blockage) by impurities, greatly reducing the efficiency of filtration, and since there are relatively many viscous components in feces, the efficiency of conventional filter filtration is reduced. Since such a large amount of target microorganisms cannot pass smoothly due to clogging of the fine filtration filter, the content of the target microorganisms finally collected is greatly reduced. By using the cross-flow filtration method, relatively small impurities in the fecal sample are removed, and the residues (captured materials) on the membrane surface are washed away by the tangential force (shear force) parallel to the membrane surface generated by the water flow, which prevents clogging of the pores, improves separation efficiency, greatly improves the probability of microorganisms passing through the filter, and extends the life of the filter.

[0050] Conventionally, microfiltration modules are easily clogged, so it is difficult to filter with a filter having a pore size of 20 μm or less. The filtered material also contains a high proportion of small-sized impurities. The microfiltration module of the present invention uses a cross-flow filtration method, so that the microfiltration module uses a cross-flow filter with a pore size of 15 μm or even smaller, which greatly reduces the content of smaller-sized impurities passing through the cross-flow filter, and can increase the purity of the target content collected by the target content collection module.

[0051] After the microfiltration step, the present invention uses microporous membrane cross-flow filtration to concentrate the target intestinal contents, achieving the cooperation of the microfiltration module and the target contents collection module. Target contents of different sizes can be concentrated by designing the pore size of the filter of the microfiltration module and the target contents collection module. For example, a cross-flow filter with a pore size of 5 μm can be used as the microfiltration module, and a cross-flow filter with a pore size of 1 μm can be used as the target contents collection module to selectively collect target contents with a size between 1 and 5 μm.

[0052] The cross-flow filtration of the microfiltration module and the target content collection module of the present invention preferably uses a cross-flow disk filter, and cross-flow filtration is performed through a spirally arranged pipe inside the micro cross-flow disk filter, thereby greatly increasing the effective filtration area and reducing the volume of the filter.

[0053] The method for separating and recovering intestinal contents according to the present invention has a higher extraction rate and a higher extraction efficiency, and the percentage of the separated and recovered bacterial flora powder is at least 10% to 20% of the initial (initial) wet intestinal contents, and the bacterial content is at least 10%. 11 The bacterial flora powder obtained from 100 g of initial wet feces is at least 10 g to 20 g, and the extraction efficiency of the number of bacteria according to the present invention is at least 6.7 × 10 9 pieces / (cm 2 h)~1.34×10 10 pieces / (cm 2 h), that is, for 100 g of initial wet feces, it is about 150 cm 2 With an effective filtration area of ​​100 mm, separation and recovery can be completed in a maximum of 60 min, and the resulting bacterial flora powder is at least 10 g to 20 g, with a bacterial count of 1 × 10 12 pieces~2×10 12 There are 10 pieces. [Brief description of the drawings]

[0054] The drawings described herein are for the purpose of providing a better understanding of the embodiments of the present invention and constitute a part of the present invention, and the schematic embodiments of the present invention and the description thereof are not intended to unduly limit the present invention but are used to interpret the present invention. Please refer to the drawings.

[0055] [Figure 1] FIG. 1 shows an intestinal content separation and extraction system according to the first embodiment. [Diagram 2] FIG. 2 is a schematic diagram of an intestinal content separation and extraction system using a conventional centrifugal separation method in Comparative Example 1. [Diagram 3]FIG. 3 is a 200x microscope photograph of the product separated by the intestinal content separation and recovery apparatus described in Example 1 and collected from the concentrate on the microporous membrane cross-flow disk filter (12). [Figure 4] FIG. 4 is a 200x microscope photograph of 2 g of solid precipitate in the lower sediment obtained by the conventional centrifugal separation method in Comparative Example 1, dissolved in 25 ml of physiological saline. [Diagram 5] FIG. 5 is a 200x microscope photograph of the product collected from the concentrate of the microporous membrane cross-flow disk filter after separation by the intestinal content separation and recovery device in Comparative Example 2. [Figure 6] FIG. 6 is a structural schematic diagram of the cross-flow disc filter of the first embodiment. [Figure 7] FIG. 7 shows photographs of samples after centrifugation in Comparative Example 1 (left panel, Fig. 7a: photograph of the sample after the first centrifugation; middle panel, Fig. 7b: photograph of the sample after the second centrifugation; right panel, Fig. 7c: photograph of the sample after the third centrifugation). [Figure 8] FIG. 8 shows electron microscope photographs of the supernatant after centrifugation in Comparative Example 1 (left panel, Fig. a: supernatant after the first centrifugation; middle panel, Fig. b: supernatant after the second centrifugation; right panel, Fig. c: supernatant after the third centrifugation). [Figure 9] FIG. 9 is a comparison diagram of the alpha diversity of the target contents separated and recovered in Example 6 and Comparative Example 3. [Figure 10] FIG. 10 is a comparison diagram of the beta diversity of the target contents and initial (initial) feces separated and collected in Example 6 and Comparative Example 3. [Figure 11] FIG. 11 is a comparison diagram of the beta diversity of the target contents and initial (initial) feces separated and collected in Example 6 and Comparative Example 3. [Figure 12] FIG. 12 is a comparative diagram of the bacterial diversity of the target contents and initial (initial) feces separated and collected in Example 6 and Comparative Example 3. [Figure 13]13 is a comparison diagram of the log value of the average number of copies / g powder of the target contents separated and collected by Example 7 and Comparative Examples 4 to 5. [Explanation of symbols] 1: impurity removal module, 2: target contents collection module, 3: stirring coarse filtration module, 4: microfiltration module, 5: stirring device, 6: container, 7: coarse filtration filter, 8: outlet, 9: reflux port, 10: microfiltration cross-flow disk type filter, 11: first transport pump, 12: microporous membrane cross-flow disk type filter, 13: second transport pump, 14: storage tank, 15: effluent tank, 16: cover, 17: exhaust port, 18: deodorization device, 201: stirring coarse filtration module, 202: microfiltration module, 203: stirring device, 204: container, 205: coarse filtration filter, 206: microfiltration filter, 501: cross-flow inlet, 502: concentrated liquid outlet, 503: cross-flow filtration pipe arranged in a spiral shape. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0056] In order to make the technical configuration and advantages of the present invention clearer, the technical configuration of the present invention is described below in a clear and comprehensive manner with the aid of specific embodiments and corresponding drawings. It goes without saying that the described embodiments are only a part of the embodiments of the present invention, and not all of them. Any other embodiments that a person skilled in the art can obtain based on the embodiments of the present invention without inventive efforts are within the scope of protection of the present invention.

[0057] In the present invention, the bacterial flora of the intestinal contents is mainly measured by 16S rRNA gene amplicon sequencing. Hereinafter, technical configurations of embodiments of the present invention will be described in detail with reference to the drawings. EXAMPLES

[0058] FIG. 1 shows an intestinal content separation and recovery apparatus according to one embodiment of the present invention, in which the intestinal content separation and recovery apparatus includes an impurity removal module 1 and a target content collection module 2, in which the impurity removal module 1 includes a stirring coarse filtration module 3 and a fine filtration module 4.

[0059] The stirring coarse filtration module 3 includes a vessel 6 having a stirring device 5 disposed therein, the stirring device 5 being located at the upper part of the vessel 6, a coarse filtration filter 7 being disposed at the lower part of the vessel 6, the stirring device 5 being located above the coarse filtration filter 7, and a liquid outlet 8 and a reflux port 9 being disposed at the bottom of the vessel 6, which are separated from each other.

[0060] During operation, for example, 130 g of initial wet intestinal contents (feces) and 750 ml of physiological saline are mixed in a container 6, then thoroughly stirred and dispersed using a stirrer 5, filtered through a coarse filtration filter 7, and discharged through an outlet 8, thereby removing relatively large particle impurities in the feces.

[0061] The microfiltration module 4 includes a microfiltration cross-flow disk-type filter 10, in which the liquid filtered by the coarse filtration is discharged from the outlet 8, and then transported from a first transport pump 11 to the microfiltration cross-flow disk-type filter 10, where relatively fine impurities are removed. The microfiltration module 4 further includes a storage tank 14 that communicates with the fluid of the microfiltration cross-flow disk-type filter 10, and the permeate filtered by the microfiltration cross-flow disk-type filter 10 flows into the storage tank 14. The concentrated liquid can be returned to the bottom of the container 6 via the return port 9 through a pipe and mixed with the coarsely filtered permeate. The microfiltration cross-flow disk-type filter 10 has a filter pore size of 10 μm.

[0062] The target content collection module 2 includes a microporous membrane cross-flow disk-type filter 12 using a microporous membrane filter, and the permeate filtered by the precision filtration module 4 is transported to the microporous membrane cross-flow disk-type filter 12 by a second transport pump 13, filtered by the microporous membrane filter, and the target content is collected from the concentrated liquid in the microporous membrane cross-flow disk-type filter 12.

[0063] The microporous membrane crossflow disk filter 12 has a filter pore size of 0.1 μm. The microporous membrane is a hydrophilic polyethersulfone membrane with an opening rate of 85%. The crossflow disk has an effective filtration area of ​​155 cm. 2 The ratio of the cross-flow disc area to the total area is 0.5. The cross-sectional area of ​​the inlet pipe is approximately 35 mm 2 The cross-sectional area of ​​the cross-flow disc passage is approximately 15 mm 2 It is.

[0064] The permeate filtered by the microporous membrane cross-flow disk filter 12 entered the effluent tank 15, and the concentrated liquid was returned to the storage tank 14 of the microfiltration module via a pipeline. During operation, after circulating filtration for, for example, 5 minutes, the target contents were collected from the concentrated liquid of the microporous membrane cross-flow disk filter 12. All samples could be filtered and collected in about 60 minutes. After the samples were freeze-dried, 15 g of powder was obtained, and the bacterial content in the powder was 10 11 Each piece is 1g of powder.

[0065] The collected concentrate from the microporous membrane cross-flow disk filter 12 was observed under a 200x microscope, as shown in FIG.

[0066] The microporous membrane cross-flow disk filter 12 has a spiral structure, as shown in Figure 7. The spiral structure design is relatively compact, which contributes to reducing the overall volume of the equipment, and also provides a relatively good filtering effect.

[0067] The container 6 of the stirring coarse filtration module 3 can be provided with an external cover 16, and an exhaust port 17 is provided at the top of the cover 16. The smelly gas generated from the feces is discharged from the exhaust port 17 and deodorized by a deodorizing device 18.

[0068] [Comparative Example 1] The intestinal content separation and recovery device includes only an impurity removal module, as shown in FIG. 2, in comparison with the intestinal content separation and recovery device in Example 1. The impurity removal module includes an agitation coarse filtration module 201 and a microfiltration module 202. The agitation coarse filtration module 201 is a container 204 with an agitator 203 at the top and a coarse filtration filter 205 at the bottom, as in the agitation coarse filtration module 3 in Example 1. After the feces and water are mixed in the container 204, they are thoroughly dispersed by the agitator 203 and filtered by the coarse filtration filter 205, so that impurities with relatively large particles in the feces are removed. The microfiltration module 202 includes a microfiltration filter 206 and is used to remove impurities with relatively small particles in the feces by a normal filtration method (non-cross-flow filtration). The pore size of the microfiltration filter is relatively small, so that in the filtration step, the permeation speed of the filtrate is slow, and the microfiltration filter is clogged even with a small amount of sample, and needs to be frequently replaced or cleaned, otherwise it is difficult for the filtrate to permeate through.

[0069] After removing impurities with the microfiltration filter 206, the fecal suspension was centrifuged to separate intestinal microorganisms at 1500 rpm for 3 minutes, and repeated three times. The sample photographs after each centrifugation are shown in FIG. 8. The upper supernatant liquid after each centrifugation was subjected to electron microscope analysis, and the results are shown in FIG. 9. As can be seen from FIG. 8 and FIG. 9, when the obtained sample is centrifuged, a large amount of bacterial species is contained in the supernatant liquid obtained by centrifugation, and the effective amount of the finally obtained intestinal contents extract is reduced due to the loss of these bacterial species, and the distribution of bacterial species is different from that in the initial (original) intestinal contents.

[0070] The final bottom sediment after three centrifugations is about 6g in total, and 2g of solid sediment is dissolved in 25ml of physiological saline, and the 200x microscope photograph is shown in Figure 4. As can be seen from Figure 4, the collected bottom sediment has a low microbial content. Therefore, the effect of collecting microorganisms by centrifugation is relatively poor, and the microbial content in the product is low, which results in an undesirable therapeutic effect.

[0071] In Example 1, compared with Comparative Example 1, microfiltration is performed by cross-flow filtration to remove relatively small impurities in the fecal sample, and the residue (captured material) on the membrane surface is washed away by the tangential force parallel to the membrane surface generated by the water flow, thereby preventing clogging of the pores, improving separation efficiency, and extending the life of the filter. After the microfiltration step, instead of the conventional centrifugal separation technology, microporous membrane cross-flow filtration is used to concentrate the target intestinal contents, which allows the extraction step to be more precisely controlled, increasing separation efficiency, and at the same time simplifying the operation and increasing the automation level.

[0072] [Comparative Example 2] The intestinal content separation and recovery device includes an impurity removal module and a target content collection module. The impurity removal module includes a stirring coarse filtration module and a fine filtration module. The stirring coarse filtration module is a container with a stirring device at the top, a coarse filtration filter at the bottom, and an outflow port and a reflux port at the bottom. 130 g of feces and 750 ml of saline were mixed in the container, then thoroughly dispersed by the stirring device and filtered through the coarse filtration filter to remove relatively large particle impurities in the feces.

[0073] The microfiltration module includes two layers of microfiltration filters with different pore sizes, the pore sizes of which are 0.5 mm and 10 μm on the upper and lower sides, respectively. The liquid filtered by coarse filtration is transported to the microfiltration filter, which removes relatively fine impurities. The microfiltration filter further includes a storage tank, and the permeate after filtration by the microfiltration filter flows into the storage tank.

[0074] The target content collection module is the same as the target content collection module in Example 1. However, the microfiltration module used a conventional multi-stage filtration method, and the filtration efficiency was low. All samples required about 150 minutes for filtration and collection, which was significantly longer than Example 1, and the target product obtained was about 7 g. The collected concentrate of the microporous membrane cross-flow disk filter was observed under a 200x microscope, as shown in Figure 5. The collected microbial content was increased compared to Comparative Example 1, but was significantly lower than Example 1. In addition, the impurity content of Comparative Example 2 was obviously higher than that of Example 1.

[0075] As can be seen, in Example 1, compared to Comparative Example 2, the microfiltration module and the target collection module simultaneously use a cross-flow filtration method, and the tangential force parallel to the membrane surface generated by the water flow washes off the residue (captured material) on the membrane surface, preventing clogging of the pores. In addition, washing off the residue (captured material) on the membrane surface also contributes to the separation of microorganisms and captured impurities, increasing the filtration efficiency and greatly improving the probability that microorganisms pass through the filter. In Comparative Example 2, the entire recovery and extraction process took a long time, the filter was easily clogged in the filtration step, and the amount of target product obtained was small.

[0076] [Examples 2 to 5] In another embodiment 2 of the present invention, an intestinal contents separation and recovery device is provided, the overall structure of which is the same as that of embodiment 1. The filter pore size of the microfiltration cross-flow disk filter 10 is 5 μm, and the filter pore size of the microporous membrane cross-flow disk filter (12) is 1 μm. The particle size distribution of the concentrated liquid separated in embodiment 1 and embodiment 2 is compared as shown in FIG. 6. Depending on the design of the pore size of the microfiltration module and the target contents collection module filter, target contents of different sizes can be concentrated, and the particle size distribution of the target contents in the concentrated liquid in embodiment 2 is narrower.

[0077] Another embodiment 3 of the present invention provides an intestinal content separation and recovery device, the overall structure of which is the same as that of embodiment 1. The filter pore size of the microfiltration cross-flow disk-type filter 10 is 100 μm, and the filter pore size of the microporous membrane cross-flow disk-type filter 12 is 0.05 μm.

[0078] Another embodiment 4 of the present invention provides an intestinal content separation and recovery device, the overall structure of which is the same as that of embodiment 1. The filter pore size of the microfiltration cross-flow disk-type filter 10 is 50 μm, and the filter pore size of the microporous membrane cross-flow disk-type filter 12 is 20 μm.

[0079] Another embodiment 5 of the present invention provides an intestinal content separation and recovery device, the overall structure of which is the same as that of embodiment 1. The filter pore size of the microfiltration cross-flow disk-type filter 10 is 5 μm, and the filter pore size of the microporous membrane cross-flow disk-type filter 12 is 0.05 μm.

[0080] [Example 6 and Comparative Example 3] The same intestinal contents (feces) were divided into three evenly based on weight and labeled DY, MY, and F, respectively. Example 6 was labeled DY, Example 3 was labeled MY, and the initial (initial) feces was labeled F.

[0081] In Example 6, the separation and collection of intestinal contents was performed using the same separation and collection device as in Example 1, including: Step 1) Dispersion: The intestinal contents are mixed with the liquid dispersion medium so that the ratio of the intestinal contents to the liquid dispersion medium (physiological saline) is 100 g:500 ml, and the resulting mixture is dispersed by mechanical means while being thoroughly stirred with a stirring device 5 to form a dispersion mixture. Step 2) impurity removal: the dispersion mixture is passed through a filter structure to remove impurities, the filtration including coarse filtration and cross-flow fine filtration, the dispersion mixture in the container 6 is filtered through the coarse filtration filter 7 and discharged from the outlet 8, and the relatively large impurities of the particles in the feces are removed; The coarsely filtered permeate enters a precision filtration cross-flow disk filter 10 with a filter pore size of 50 μm to remove relatively fine impurities. After stirring for 5 minutes with the stirring device 5, sufficient permeate is obtained and the microfiltration operation can be started. After performing microfiltration for 10 minutes, sufficient permeate (filtrate) is obtained and the collection step of the target content can be started. Step 3) collection: the permeate of step 2) is filtered through a microporous membrane cross-flow filter to obtain a concentrate and a filtrate waste, and the concentrate is used to collect the target content; The microporous membrane of the microporous membrane crossflow filter has a non-uniform pore structure, the pore size range of the microporous membrane is 0.2 μm, the aperture ratio is 80%, the microporous membrane is a hydrophilic membrane, and the effective filtration area of ​​the crossflow disk is 155 cm 2 , the cross-sectional area of ​​the inlet pipe is about 35 mm 2 , the cross-sectional area of ​​the cross-flow disc passage is about 15 mm 2 and Step 4) Freeze-drying: The target content obtained in step 3) was freeze-dried to form 12 g of powder, and the bacterial content in the powder was 1.5 × 10 11 Each piece is 1g of powder. It is made into capsules. Among them, the concentrate obtained by microfiltration in the impurity removal step is returned and mixed with the permeate obtained by coarse filtration to form a circulation circuit, and the concentrate from the microporous membrane crossflow filter is returned and mixed with the permeate obtained by microfiltration in the impurity removal step to form a circulation circuit. The steps 1) to 3), i.e., the separation and recovery steps, take a total of 60 min.

[0082] [Comparative Example 3] Using an intestinal content separation and recovery device as shown in Figure 2, 100 g of intestinal content was mixed with 550 ml of physiological saline, stirred for 5 minutes with a stirrer, and then filtered through a coarse filtration filter and a microfiltration filter with a pore size of 0.25 mm. The permeate obtained by the microfiltration was centrifuged at a centrifuge speed of 2000 rpm for 5 minutes, after which the supernatant was discarded and physiological saline was added. Centrifugation was repeated three times in this manner, the supernatant was discarded, and the remaining precipitate, i.e., the target content, was obtained. Example 6 (DY) and Comparative Example 3 (MY) were repeated four times (four experiments were performed on the stool of the same donor within one week), and the types and abundances of the bacterial species in the obtained target contents and initial (initial) stool (F) were determined using the 16sR DNA sequencing method.

[0083] Microbial diversity is considered from the perspective of community ecology, and the diversity analysis of a single sample (Alpha diversity) can reflect the abundance and diversity of the microbial community, including the estimation of the abundance and diversity of species in environmental communities through a series of statistical analysis indexes. Shannon: Used to estimate one of the microbial diversity indexes in a sample. The higher the Shannon value, the higher the community diversity.

[0084] FIG. 9 shows that the bacterial flora extracted by the present invention has a higher biodiversity than that obtained by the centrifugation method, and the difference from the centrifugation method is clear.

[0085] PCA (Principal Component Analysis) uses variance decomposition to reflect the differences in multi-group data on a two-dimensional coordinate diagram, and the coordinate axes take the two eigenvalues ​​that can reflect the variance value to the maximum. The closer the composition of the samples, the closer the distance reflected in the PCA diagram.

[0086] 10 to 12 show that the bacterial flora extracted by the present invention is closer to the initial feces in terms of species and abundance (abundance), the similarity is higher, and the statistical difference is significant.

[0087] The target contents obtained in Example 6 (DY) and Comparative Example 3 (MY) are significantly statistically different from the initial feces (F).

[0088] The R-value is between (-1, 1), and an R-value greater than 0 indicates a significant difference between groups. The confidence level of statistical analysis is expressed by the P-value, with P<0.05 indicating a statistically significant difference.

[0089] Table 1 shows the statistical values ​​of the target contents and initial feces (F) obtained in Example 6 (DY) and Comparative Example 3 (MY). [Table 1]

[0090] As can be seen, the target contents (bacterial flora) obtained by the present invention are closer in type and distribution to the type and distribution of the bacterial flora in the initial (initial) intestinal contents (feces) than those obtained by the centrifugation method. At the same time, the present invention has been found to have better reproducibility.

[0091] [Example 7 and Comparative Examples 4 to 5] The thoroughly and uniformly mixed intestinal contents (feces) were divided into nine portions according to weight, with each portion weighing 100 g, and three portions were used in Example 7, three portions were used in Comparative Example 4, and three portions were used in Comparative Example 5.

[0092] In Example 7, the intestinal contents were separated and collected using the same separation and collection device and method as in Example 1, with the only difference being as follows. The amount of saline added to the vessel 6 is 500 ml, the filter pore size of the coarse filtration is 2 mm, the filter pore size of the precision filtration module 4 is 40 μm, the filter pore size of the microporous membrane cross-flow disk filter 12 is 0.5 μm, and the aperture ratio is 85%.

[0093] Separation and recovery were completed over a period of 60 minutes, and the resulting concentrated bacterial liquid was freeze-dried to obtain bacterial powder. This was repeated three times, yielding 12 g, 11 g, and 12 g, respectively.

[0094] Comparative Example 4 was carried out with reference to Example 3 in Chinese patent application CN201810852561.2, with the only difference being that the saline solution was 500 ml, and the pore sizes of the microfiltration filters were 0.5 mm and 40 μm from top to bottom, respectively.

[0095] The bacteria were separated and collected over a period of 120 minutes, and the resulting concentrated bacterial liquid was freeze-dried to obtain bacterial powder. This was repeated three times, yielding 8g, 8g, and 7g, respectively.

[0096] Comparative Example 5 was carried out with reference to the method of Comparative Example 1, with the only difference being that the amount of saline added to the container 6 was 550 ml, the filter pore size of the coarse filtration was 2 mm, and the filter pore size of the fine filtration module 4 was 40 μm.

[0097] The bacteria were separated and collected over a period of 150 minutes, and the resulting concentrated bacterial liquid was freeze-dried to obtain bacterial powder. This was repeated three times, yielding 3g, 2g, and 2g, respectively.

[0098] The average number of replicates was measured for the samples obtained in Example 7, Comparative Example 4, and Comparative Example 5. The average number of replicates of the sample reflects the number of biological samples in the sample.

[0099] The results of the average number of replicas / g powder for the samples of Example 7, Comparative Example 4, and Comparative Example 5 are shown in Table 2.

[0100] [Table 2]

[0101] The logarithm of the average number of replicates / g powder values ​​in Table 2 was taken and is shown in FIG. From the results of Example 7, Comparative Example 4 and Comparative Figure 5, it can be seen that the results obtained by the method of the present invention are consistent in order of magnitude, have good reproducibility, have high separation and recovery efficiency, can be completed within 1 h, and the quality of the obtained bacterial flora powder is higher. Comparative Example 4 using the conventional technology has the results obtained in order of magnitude and has good reproducibility, but in the microfiltration step, the conventional filtration and the nuclepore membrane are still used when collecting by crossflow, the overall separation and recovery time is longer, the quality of the obtained bacterial flora powder is inferior to that of the present invention, and the average copy number / g powder value is three orders of magnitude lower than that of the present invention. Comparative Example 5 using conventional multi-stage normal filtration has obvious differences in the results obtained, have poor reproducibility, the overall separation and recovery time is longer, the mass of the obtained bacterial flora powder is smaller, and the average copy number / g powder value is more than eight orders of magnitude lower than that of the present invention.

[0102] Although the above describes the embodiments of the present invention in detail, they are merely examples for the convenience of understanding and explanation, and are not intended to limit the present invention. Although the above-mentioned embodiments describe the present invention in detail, those skilled in the art can modify the technical configurations described in the above-mentioned embodiments or equivalently change the technical features therein, and it should be understood that these modifications or changes fall within the spirit and scope of the technical configurations described in the embodiments of the present invention.

Claims

1. An intestinal content separation and collection device, comprising: an impurity removal module and a target content collection module; The impurity removal module removes impurities from the intestinal contents, The impurity removal module includes a stirring coarse filtration module and a fine filtration module; The microfiltration module includes a microfiltration cross-flow disk filter; The microfiltration cross-flow disk filter has a filter pore size of 5 to 100 μm, the target content collection module comprises a microporous membrane crossflow filter; The microporous membrane has a sponge-like porous structure, The pore size range of the microporous membrane is 0.01 to 20 μm; The opening rate of the microporous membrane is 30% or more, the microporous membrane is a hydrophilic membrane; The intestinal contents separation and recovery device, wherein the microporous membrane crossflow filter has a disk-shaped structure and has a spiral fluid flow passage therein, and the ratio of the effective filtration area of ​​the crossflow disk to the total area of ​​the crossflow disk is 0.3 to 0.9:

1.

2. 2. The device according to claim 1, wherein the ratio of the effective filtration area of ​​the cross-flow disc to the total area of ​​the cross-flow disc is 0.4-0.8:

1.

3. 3. The device according to claim 1 or 2, characterized in that the flow passage of the microporous membrane cross-flow filter is a spiral cross-flow passage.

4. The device according to any one of claims 1 to 3, characterized in that the flow path cross-sectional area of ​​the microporous membrane cross-flow filter is smaller than the cross-sectional area of ​​the inlet pipe.

5. The device according to any one of claims 1 to 4, characterized in that the microporous membrane comprises one or more of the following membranes: polyethersulfone membrane, cellulose membrane, polymethacrylate membrane, modified or unmodified.

6. A method for separating and recovering intestinal contents, comprising the steps of: Step 1) Dispersion step: mixing the intestinal contents with a liquid dispersion medium, the ratio of the intestinal contents to the liquid dispersion medium is 100 g: 200-5000 ml, and dispersing the resulting mixture by mechanical means to form a uniform dispersion mixture; Step 2) impurity removal step: passing the dispersion mixture through a filtration filter to remove impurities and obtain a permeate (filtrate); The filtration includes coarse filtration and cross-flow microfiltration, and the filter pore size of the cross-flow microfiltration is 5 to 100 μm; Step 3) a collecting step, the permeate of step 2) is filtered through a microporous membrane cross-flow filter to obtain a concentrate and a filtrate waste, the concentrate is for collecting the target content; The method for separating and recovering intestinal contents, wherein the microporous membrane of the microporous membrane crossflow filter has a sponge-like porous structure, the pore size range of the microporous membrane is 0.01 to 20 μm, the opening rate of the microporous membrane is 30% or more, and the microporous membrane is a hydrophilic membrane.

7. The concentration of the microorganisms in the dispersion mixture of step 1) is at least 10 ８ The method according to claim 6, characterized in that the concentration is 1 / ml.

8. The method according to claim 6 or 7, characterized in that the concentrate of step 3) is refluxed and mixed with the permeate of step 2) to form a circulation circuit.

9. The method according to any one of claims 6 to 8, characterized in that the concentrated liquid of step 3) is returned to the previous stage and mixed with the permeate to form a circulation circuit.

10. A composition comprising an intestinal contents extract, The intestinal content extract is obtained by the intestinal content separation and recovery device according to any one of claims 1 to 5. or obtained by preparing the intestinal contents separation and recovery method according to any one of claims 6 to 9, The size of the obtained intestinal content extract is characterized by being 0.01 to 50 μm. A composition comprising the intestinal contents extract.

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