Intestinal flora enrichment microsphere, preparation method and application thereof, and enrichment method of intestinal flora

Abstract

The invention belongs to the technical field of intestinal flora transplantation, and discloses an intestinal flora enrichment microsphere as well as a preparation method and application thereof and an enrichment method of intestinal flora. The intestinal flora enrichment microsphere provided by the invention specifically comprises a calcium alginate gel substrate and a porous adsorbent doped on the calcium alginate gel substrate, and the intestinal flora-enriched microspheres have a special pore size distribution situation, namely the pore size is gradually increased along the direction from the center to the outer layer. When the intestinal flora enrichment microspheres are applied to enrichment of intestinal flora, active flora cells can be effectively separated from small impurities such as intestinal contents and flora metabolites while the proportion of the high-activity flora cells is kept, and the intestinal flora enrichment microspheres have an excellent intestinal flora enrichment effect.

Description

Technical Field

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

[0002] Microbiota transplantation is a new medical technology that transplants the intestinal flora from the feces of healthy donors to the recipients to regulate or rebuild the balance of the recipients' intestinal flora. However, the intestinal flora isolated from the feces of healthy donors will still contain various intestinal contents, microbiota metabolites and other tiny impurities, which will not only affect the transplantation effect, but may also cause adverse reactions such as diarrhea, constipation, fever, nausea and vomiting.

[0003] At present, centrifugal washing, suction filtration, physical screening and filtration, and other biochemical treatments such as cellulase and protease pretreatment are the main methods for impurity removal and bacterial cell enrichment. Centrifugal washing refers to the use of centrifugal force to effectively separate bacterial cells from impurity molecules, thereby enriching bacterial cells; however, due to the large number and proportion of impurities in the bacterial solution, it is impossible to precipitate bacterial cells through simple centrifugation, so multiple centrifugation treatments are required, which will easily lead to a decrease in the yield of bacterial cells and the destruction of the bacterial structure, thereby reducing the proportion of active bacteria in the obtained intestinal flora, and there are problems such as low bacterial sludge yield and low proportion of live bacteria. The suction filtration method and physical screening filtration method are based on the difference in particle size between intestinal flora and other impurities. The flora cells are sorted through holes or membranes with specific mesh sizes to achieve enrichment of flora cells; however, due to the high diversity of flora cells and the wide distribution of particle sizes, it is difficult to effectively remove impurities through a single mesh treatment, and the impurity removal effect is poor. Multi-level mesh treatment will lead to a significant decrease in the yield of flora cells, and the process is complicated and cumbersome, which is easy to cause blockage and other situations, and the actual production application effect is poor. Other biochemical treatment methods such as cellulase and protease pretreatment are easy to damage the flora cell structure and destroy the flora vitality. The impurities after treatment will exist in the sample in other forms, and effective flora cell enrichment effect cannot be achieved.

[0004] Therefore, obtaining a method that can effectively enrich intestinal flora cells while maintaining a high proportion of active flora cells is of great significance for the research and development of flora transplantation technology. Summary of the invention

[0005] The first purpose of the present invention is to provide an intestinal flora enrichment microsphere. When the intestinal flora enrichment microsphere is used for the enrichment of intestinal flora, it can achieve the enrichment of intestinal flora cells well while maintaining the proportion of highly active flora cells, and effectively increase the total bacterial amount, total live bacteria amount and live bacteria proportion in the final obtained bacterial sludge, and has good application prospects.

[0006] The second purpose of the present invention is to provide a method for preparing the above-mentioned intestinal flora-enriched microspheres.

[0007] The third purpose of the present invention is to provide the application of the above-mentioned intestinal flora enriched microspheres in intestinal flora transplantation technology.

[0008] The fourth purpose of the present invention is to provide a method for enriching intestinal flora.

[0009] Specifically, the intestinal flora enriched microspheres provided by the present invention include a calcium alginate gel substrate and a porous adsorbent doped on the calcium alginate gel substrate; and the pore size of the intestinal flora enriched microspheres increases from the center to the outer layer.

[0010] Furthermore, the porous adsorbent is selected from one or more of porous starch, biochar, activated carbon, attapulgite, activated clay, diatomaceous earth, talc, kaolin and zeolite.

[0011] Furthermore, the porous adsorbent is diatomaceous earth and / or attapulgite.

[0012] Furthermore, the average particle size of the porous adsorbent is 100-300 μm, and the average pore size is 20-80 μm.

[0013] Furthermore, based on the total mass of the intestinal flora-enriched microspheres, the loading amount of the porous adsorbent is 0.001 to 10 wt %.

[0014] Furthermore, the average particle size of the intestinal flora enriched microspheres is 1 to 10 mm.

[0015] The preparation method of intestinal flora enriched microspheres provided by the present invention comprises: S1, mixing sodium alginate, a porous adsorbent and a solvent to obtain a mixed solution; S2, dropping the mixed solution dropwise into a calcium chloride solution in a static state, stirring and incubating after the dropwise addition is completed to obtain the intestinal flora enriched microspheres.

[0016] Furthermore, in step S1, the solvent is water and / or physiological saline.

[0017] Furthermore, in step S1, in the mixed solution, the concentration of sodium alginate is 0.2-2% (w / v), and the concentration of the porous adsorbent is 0.2-2% (w / v).

[0018] Furthermore, in step S1, the mixing treatment is mechanical stirring, the rotation speed of the mechanical stirring is 200-1500 r / min, the temperature is 35-40° C., and the time is 2-10 min.

[0019] Furthermore, in step S2, the concentration of the calcium chloride solution is 0.4-1.5% (w / v).

[0020] Furthermore, in step S2, the volume ratio of the mixed solution to the calcium chloride solution is 1:(1-4).

[0021] Furthermore, in step S2, the dropping speed of the mixed solution is 2 to 10 mL / min.

[0022] Furthermore, in step S2, the stirring incubation treatment is carried out at a rotation speed of 100 to 600 rpm, a temperature of 35 to 40° C., and a time of 10 to 30 min.

[0023] The present invention further provides application of the intestinal flora enriched microspheres in intestinal flora transplantation technology.

[0024] The intestinal flora enrichment method provided by the present invention comprises: (1) mixing a treatment solution and a stool sample, removing impurities, and obtaining a bacterial suspension; (2) mixing the intestinal flora enrichment microspheres with the bacterial suspension, adsorbing, filtering, and centrifuging to enrich the intestinal flora.

[0025] Furthermore, in step (1), the treatment solution is selected from one or more of a betaine solution, a laver extract solution, a sargassum extract solution, a phosphate buffer solution and a physiological saline solution.

[0026] Furthermore, in step (1), the addition ratio of the treatment solution to the stool sample is 5:1 to 20:1 (mL:g).

[0027] Furthermore, in step (1), the speed of mixing the treatment solution and the stool sample is 1000-6000 r / min, the temperature is 0-5° C., and the time is 2-20 min.

[0028] Furthermore, in step (1), the impurity removal treatment is a suction filtration treatment, and the mesh size of the filter for the suction filtration treatment is 10 to 400 meshes.

[0029] Furthermore, in step (2), based on the volume of the bacterial suspension, the added amount of the intestinal flora enrichment microspheres is 5-20% (w / v).

[0030] Furthermore, in step (2), the rotation speed of the adsorption treatment is 50 to 150 r / min, the temperature is 0 to 5° C., and the time is 10 to 25 min.

[0031] Beneficial effects:

[0032] First, the intestinal flora enrichment microspheres provided by the present invention specifically include a calcium alginate gel substrate and a porous adsorbent doped thereon; and the intestinal flora enrichment microspheres have a special pore size distribution trend - the pore size increases from the center to the outer layer. When the intestinal flora enrichment microspheres are applied to the enrichment of intestinal flora, it can effectively separate active flora cells from tiny impurities such as intestinal contents and flora metabolites while maintaining a high proportion of active flora cells, and has an excellent intestinal flora enrichment effect.

[0033] It is speculated that the reasons why the intestinal flora enrichment microspheres have the above-mentioned excellent intestinal flora enrichment effect include: using negatively charged calcium alginate hydrogel as the substrate, the incorporated porous adsorbent and the substrate together form a nested, multi-scale composite pore structure, and at a more macroscopic level, the pore size has a trend of increasing from the center to the outer layer. The intestinal flora enrichment microspheres formed have an ideal multi-level composite pore structure that can effectively adsorb tiny impurities and dead bacterial cells and intercept live bacterial cells, so as to achieve effective adsorption of tiny impurities and dead bacterial cells, and the intestinal flora enrichment microspheres have a certain elasticity. Through the microspheres and the pore structure thereon, the mutually bound live cells, dead cells and impurities can be effectively peeled off without destroying the activity of the live bacterial cells, and the tiny impurities and dead bacterial cells in the solution can be synergistically adsorbed and fixed to optimize the bacterial solution state, so that the live bacterial cells therein can be precipitated and collected and enriched by the bacterial community (present in the supernatant) after simple centrifugation, thereby achieving the effect of improving the bacterial sludge yield, the total bacterial amount of the bacterial sludge and the proportion of bacterial sludge activity.

[0034] Secondly, the present invention also provides an example of a method for preparing the above-mentioned intestinal flora enriched microspheres. In the preparation method, the gelation of sodium alginate into microspheres is achieved by combining static dripping and dynamic incubation. In the static dripping process, the sodium alginate and Ca 2+ The porous adsorbent is quickly combined with gelation and coated to form a dense three-dimensional gel network core; then, during the dynamic incubation process, the free sodium alginate and Ca in the solution 2+ And the porous adsorbent continues to grow on the three-dimensional gel network core to form a relatively loose three-dimensional gel network outer layer, so as to obtain intestinal flora-enriched microspheres with a special pore size distribution. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1This is a microscopic observation picture of the cross section of the intestinal flora-enriched microspheres provided in Preparation Example 1 of the present invention (magnification is ×80);

[0036] Figure 2 A microscopic observation of the cross section of the intestinal flora-enriched microspheres provided in Comparative Preparation Example 1 (magnification is ×80);

[0037] Figure 3 This is a microscopic observation of the cross-section of the intestinal flora-enriched microspheres provided in Comparative Preparation Example 2 (magnification is ×80). DETAILED DESCRIPTION

[0038] The intestinal flora enriched microspheres provided by the present invention comprise a calcium alginate gel substrate and a porous adsorbent doped on the calcium alginate gel substrate; and the pore size of the intestinal flora enriched microspheres increases along the direction from the center to the outer layer.

[0039] In the present invention, the porous adsorbent refers to a type of substance having many tiny pores and a certain adsorption capacity, and specific examples thereof include but are not limited to: one or more of porous starch, biochar, activated carbon, attapulgite, activated white clay, diatomaceous earth, talc, kaolin and zeolite.

[0040] In some specific embodiments, the porous adsorbent is preferably diatomaceous earth and / or attapulgite. In this case, the porous adsorbent has a relatively good cation exchange capacity, and cooperates with the negatively charged calcium alginate gel substrate, and the prepared intestinal flora enrichment microspheres are applied to the intestinal flora enrichment treatment and have a better enrichment effect.

[0041] In the present invention, the average particle size of the porous adsorbent is preferably 100-300 μm, such as 100 μm, 130 μm, 150 μm, 180 μm, 200 μm, 250 μm, 300 μm or any value therebetween; more preferably 150-200 μm. The average pore size of the porous adsorbent is preferably 20-80 μm, such as 20 μm, 25 μm, 30 μm, 45 μm, 60 μm, 70 μm, 80 μm or any value therebetween; more preferably 30-60 μm. At this time, the porous adsorbent can form a more ideal nested, multi-scale composite pore structure with the calcium alginate substrate, so as to give the obtained intestinal flora enrichment microspheres a better enrichment effect when used in intestinal flora enrichment treatment.

[0042] In the present invention, based on the total mass of the intestinal flora enriched microspheres, the loading amount of the porous adsorbent is preferably 0.001-10wt%, such as 0.001wt%, 0.02wt%, 0.13wt%, 1wt%, 2wt%, 2.5wt%, 3wt%, 5wt%, 8wt%, 10wt% or any value therebetween. At this time, the obtained intestinal flora enriched microspheres have a more ideal multi-level composite pore structure that can effectively adsorb tiny impurities and dead bacterial cells and intercept live bacterial cells.

[0043] In the present invention, the average particle size of the intestinal flora enriched microspheres is preferably 1 to 10 mm, such as 1 mm, 1.5 mm, 2 mm, 2.8 mm, 3 mm, 5 mm, 7 mm, 9 mm, 10 mm or any value therebetween, more preferably 3 to 6 mm. At this time, the intestinal flora enriched microspheres have excellent porosity and specific surface area, and are applied to intestinal flora enrichment treatment to have a better enrichment effect.

[0044] Based on the purpose of obtaining the above-mentioned intestinal flora enriched microspheres, the present invention also provides a preparation method of intestinal flora enriched microspheres. The preparation method specifically comprises: S1, mixing sodium alginate, a porous adsorbent and a solvent to obtain a mixed solution; S2, adding the mixed solution dropwise to a calcium chloride solution, stirring and incubating after the addition is completed, to obtain the intestinal flora enriched microspheres.

[0045] It should be noted that the above-mentioned intestinal flora-enriched microspheres can be prepared by this preparation method or by other technical means.

[0046] In the present invention, in step S1, the solvent is a type of reagent conventionally used in the preparation of existing gel microspheres. Those skilled in the art can make adaptive choices according to actual needs. The present invention does not impose any special limitations on it. Specific examples include but are not limited to: water and / or physiological saline.

[0047] In the present invention, in step S1, the mixing treatment is a type of technical means commonly used in the prior art to achieve uniform mixing of materials. Those skilled in the art can make adaptive choices based on actual needs. The present invention does not impose any particular limitation on it. Specific examples include but are not limited to: one or more of magnetic stirring, ultrasonic mixing and mechanical stirring.

[0048] In some specific embodiments, in step S1, the concentration of sodium alginate in the mixed solution is preferably 0.2-2% (w / v), such as 0.2% (w / v), 0.3% (w / v), 0.5% (w / v), 0.9% (w / v), 1% (w / v), 1.4% (w / v), 1.8% (w / v), 2% (w / v) or any value therebetween, more preferably 1-1.3% (w / v); the concentration of the porous adsorbent is preferably 0.2-2% (w / v), such as 0.2% (w / v), 0.4% (w / v), 0.6% (w / v), 0.8% (w / v), 1.4% (w / v), 1.6% (w / v), 2% (w / v) or any value therebetween, more preferably 1-1.3% (w / v).

[0049] In some specific embodiments, in step S1, the mixing treatment is preferably mechanical stirring, and the conditions of the mixing treatment include: the rotation speed is preferably 200-1500r / min, such as 200r / min, 250r / min, 300r / min, 380r / min, 500r / min, 850r / min, 1000r / min, 1350r / min, 1500r / min or any value therebetween, more preferably 500-1000r / min; the temperature is preferably 35-40°C, such as 35°C, 37°C, 38°C, 40°C or any value therebetween; the time is preferably 2-10min, such as 2min, 2.8min, 3min, 3.6min, 4min, 6min, 9min, 10min or any value therebetween.

[0050] In the present invention, in step S2, the method of adding the mixed solution dropwise into the calcium chloride solution is a type of feeding method commonly used in the prior art. Those skilled in the art can make an adaptive choice according to actual needs. The present invention does not impose any particular limitation on it. Specific examples include but are not limited to: one or more of the pouring method, the dropper dropping method and the automatic burette dropping method.

[0051] In some specific embodiments, in step S2, the concentration of the calcium chloride solution is preferably 0.4-1.5% (w / v), such as 0.4% (w / v), 0.5% (w / v), 0.8% (w / v), 1.1% (w / v), 1.3% (w / v), 1.5% (w / v) or any value therebetween, more preferably 0.8-1.1% (w / v).

[0052] In some specific embodiments, in step S2, the volume ratio of the mixed solution to the calcium chloride solution is preferably 1:(1-4), such as 1:1, 1:1.3, 1:1.5, 1:2, 1:2.8, 1:3, 1:4 or any value therebetween.

[0053] In some specific embodiments, in step S2, the dropping speed of the mixed solution is preferably 2-10 mL / min, such as 2 mL / min, 2.5 mL / min, 2.8 mL / min, 3 mL / min, 3.6 mL / min, 4 mL / min, 5 mL / min, 8 mL / min, 10 mL / min or any value therebetween, more preferably 5-7 mL / min.

[0054] In some specific embodiments, in step S2, the conditions for the stirring incubation treatment include a rotation speed of preferably 100-600 rpm, such as 100 rpm, 180 rpm, 200 rpm, 250 rpm, 300 rpm, 500 rpm, 600 rpm or any value therebetween, more preferably 200-300 r / min; a temperature of preferably 35-40°C, such as 35°C, 36°C, 37°C, 38°C, 39°C, 40°C or any value therebetween; a time of preferably 10-30 min, such as 10 min, 12.8 min, 15 min, 18 min, 20 min, 25 min, 30 min or any value therebetween, more preferably 15-20 min.

[0055] Based on the excellent effect of the above-mentioned intestinal flora enrichment microspheres in achieving intestinal flora enrichment, the present invention also provides the use of the above-mentioned intestinal flora enrichment microspheres in intestinal flora transplantation technology.

[0056] Based on the application of the above-mentioned intestinal flora enrichment microspheres in intestinal flora transplantation technology, the present invention also provides an intestinal flora enrichment method. The enrichment method specifically comprises: (1) mixing the treatment solution and the fecal sample, removing impurities, and obtaining a bacterial suspension; (2) mixing the above-mentioned intestinal flora enrichment microspheres with the bacterial suspension, adsorbing, filtering, and centrifuging to enrich the intestinal flora.

[0057] In the present invention, in step (1), the treatment solution is used to fully dissolve the intestinal flora in the stool sample and protect the intestinal flora in the subsequent treatment steps. Specific examples include: one or more of betaine solution, laver extract solution, seaweed extract solution, phosphate buffer solution and physiological saline.

[0058] In some specific embodiments, in step (1), the addition ratio of the treatment solution and the stool sample is preferably 5:1 to 20:1 (mL:g), such as 5:1 (mL:g), 5.5:1 (mL:g), 6:1 (mL:g), 8:1 (mL:g), 10:1 (mL:g), 16.5:1 (mL:g), 20:1 (mL:g) or any value therebetween, more preferably 8:1 to 10:1 (mL:g).

[0059] In some specific embodiments, in step (1), the conditions for mixing the treatment solution and the stool sample include a rotation speed of preferably 1000-6000 r / min, such as 1000 r / min, 2000 r / min, 4000 r / min, 5000 r / min, 6000 r / min or any value therebetween, more preferably 2000-4000 rpm; a temperature of preferably 0-5°C, such as 0°C, 0.1°C, 0.5°C, 1°C, 3°C, 5°C or any value therebetween; and a time of preferably 2-20 min, such as 2 min, 3 min, 5 min, 7 min, 10 min, 15 min, 20 min or any value therebetween, more preferably 8-15 min.

[0060] In the present invention, in step (1), the impurity removal treatment is a method conventionally used in the preparation of existing intestinal flora preparations to remove large particle impurities such as intestinal contents, undigested food residues and flora metabolites. Those skilled in the art can make adaptive choices based on actual needs. The present invention does not impose any particular limitation on it. Specific examples include but are not limited to: filtration and / or suction filtration.

[0061] In some specific embodiments, in step (1), the impurity removal treatment is preferably a vacuum filtration treatment, and the filter mesh number of the vacuum filtration treatment is preferably 10 to 400 mesh, such as 10 mesh, 15 mesh, 20 mesh, 30 mesh, 50 mesh, 80 mesh, 100 mesh, 300 mesh, 400 mesh or any value therebetween.

[0062] In some more preferred embodiments, in step (1), the impurity removal treatment is preferably a multi-stage filtration treatment, and the number of filter layers in the multi-stage filtration treatment is 1-6 layers, and the number of filter meshes increases with the increase of the number of layers.

[0063] In the present invention, in step (2), the adsorption treatment is a process of utilizing intestinal flora enriched microspheres to achieve sufficient adsorption and fixation of tiny impurities and dead bacterial cells in the solution. Those skilled in the art may adopt stirring, shaking and other technical means according to actual needs to achieve a sufficient structure between the intestinal flora enriched microspheres and the bacterial suspension, and the present invention does not impose any particular limitation thereto.

[0064] In some specific embodiments, in step (2), based on the volume of the bacterial suspension, the added amount of the intestinal flora enriched microspheres is preferably 5-20% (w / v), such as 5% (w / v), 7% (w / v), 9% (w / v), 10% (w / v), 12.3% (w / v), 15% (w / v), 18% (w / v), 20% (w / v) or any value therebetween, more preferably 10-15% (w / v).

[0065] In some specific embodiments, in step (2), the conditions for the adsorption treatment include a rotation speed of preferably 50 to 150 r / min, such as 50 r / min, 58 r / min, 60 r / min, 75 r / min, 100 r / min, 125 r / min, 150 r / min or any value therebetween; a temperature of preferably 0 to 5°C, such as 0°C, 1°C, 2°C, 3°C, 4°C, 5°C or any value therebetween; and a time of preferably 10 to 25 min, such as 10 min, 11 min, 12 min, 14 min, 18 min, 20 min, 25 min or any value therebetween.

[0066] 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.

[0067] The reagents and their sources used in the following preparation examples and embodiments are specifically:

[0068] Sodium alginate (Anhui Zhonghong Bioengineering Co., Ltd., catalog number D52003216);

[0069] Porous starch (Shaanxi Shengyuantai Biotechnology Development Co., Ltd., product number SYTDKDF824);

[0070] Diatomaceous earth (Shijiazhuang Tourmaline Mineral Products Co., Ltd., product number sjz131);

[0071] Attapulgite (Xuyi Qihong Attapulgite Powder Co., Ltd., product number 0189);

[0072] Zeolite (manufacturer: Henan Jiufeng Water Treatment Co., Ltd., product number: JFFS-3518);

[0073] Herba Lysimachiae Extract (Shaanxi Gubenyuan Biotechnology Co., Ltd., product number gby984);

[0074] LIVE / DEAD TM BacLightTM Bacterial Viability Kit (Thermo Fisher Scientific, catalog number 2714429).

[0075] Preparation Example 1

[0076] This preparation example is used to illustrate the preparation of intestinal flora enriched microspheres, which specifically includes:

[0077] S1. Mix sodium alginate, diatomaceous earth and 0.85% physiological saline, and vortex mix them at 37° C. and 800 r / min for 8 min to prepare a mixed solution, wherein the concentration of sodium alginate in the mixed solution is 1.3% (w / v), and the concentration of diatomaceous earth is 1.3% (w / v);

[0078] S2. Under static condition, take 200 mL of the mixed solution and add it dropwise into 200 mL of 1.1% (w / v) calcium chloride solution at a titration rate of 6 mL / min. After the addition is completed, incubate it at 37°C and 200 r / min for 15 min, filter it with a 30-mesh filter to obtain intestinal flora-enriched microspheres.

[0079] Figure 1 This is a microscopic observation picture of the cross section of the intestinal flora enrichment microspheres prepared in this preparation example (magnification is ×80), in which the black part is the adsorbent matrix and the bright white part is the pore structure inside the microspheres. Figure 1 It can be seen that the pore size of the intestinal flora enriched microspheres increases from the center to the outer layer, and the loading amount of diatomaceous earth in the intestinal flora enriched microspheres is about 0.13wt%.

[0080] Preparation Example 2

[0081] This preparation example adopts the method provided in Preparation Example 1 to prepare intestinal flora enriched microspheres, except that in step S1, an equal mass of porous starch is used instead of diatomaceous earth to prepare a mixed solution, and other conditions are the same to obtain intestinal flora enriched microspheres, and the loading amount of porous starch in the intestinal flora enriched microspheres is about 0.13wt%.

[0082] Preparation Example 3

[0083] This preparation example adopts the method provided in Preparation Example 1 to prepare intestinal flora enriched microspheres, except that in step S1, an equal mass of attapulgite is used instead of porous starch to prepare a mixed solution, and other conditions are the same to obtain intestinal flora enriched microspheres, and the loading amount of attapulgite in the intestinal flora enriched microspheres is about 0.13wt%.

[0084] Preparation Example 4

[0085] This preparation example adopts the method provided in Preparation Example 1 to prepare intestinal flora enriched microspheres, except that in step S1, an equal mass of zeolite is used instead of porous starch to prepare a mixed solution, and other conditions are the same to obtain intestinal flora enriched microspheres, and the loading amount of zeolite in the intestinal flora enriched microspheres is about 0.13wt%.

[0086] Preparation Example 5

[0087] This preparation example adopts the method provided in Preparation Example 3 to prepare intestinal flora enriched microspheres, except that in step S1, the concentration of sodium alginate in the mixed solution is 2% (w / v), the concentration of attapulgite is 0.2% (w / v), and other conditions are the same to obtain intestinal flora enriched microspheres, and the loading amount of attapulgite in the intestinal flora enriched microspheres is about 0.02wt%.

[0088] Preparation Example 6

[0089] This preparation example adopts the method provided in Preparation Example 3 to prepare intestinal flora enriched microspheres, except that, in step S1, the concentration of sodium alginate in the mixed solution is 0.5% (w / v), the concentration of attapulgite is 2% (w / v), and other conditions are the same to obtain intestinal flora enriched microspheres, and the loading amount of attapulgite in the intestinal flora enriched microspheres is about 0.2wt%.

[0090] Preparation Example 7

[0091] This preparation example adopts the method provided in Preparation Example 3 to prepare intestinal flora enriched microspheres, except that in step S2, the concentration of the calcium chloride solution is 1.5% (w / v), and other conditions are the same to obtain intestinal flora enriched microspheres, and the loading amount of attapulgite in the intestinal flora enriched microspheres is about 0.13wt%.

[0092] Preparation Example 8

[0093] This preparation example adopts the method provided in Preparation Example 3 to prepare intestinal flora enriched microspheres, except that in step S2, the concentration of the calcium chloride solution is 0.4% (w / v), and other conditions are the same to obtain intestinal flora enriched microspheres, and the loading amount of attapulgite in the intestinal flora enriched microspheres is about 0.13wt%.

[0094] Preparation Example 9

[0095] This preparation example adopts the method provided in Preparation Example 3 to prepare intestinal flora enriched microspheres, except that, in step S2, after the mixed solution is added dropwise, the solution is incubated at a temperature of 37°C, a rotation speed of 100 r / min, and a time of 30 min. Other conditions are the same to obtain intestinal flora enriched microspheres, and the loading amount of attapulgite in the intestinal flora enriched microspheres is about 0.13 wt%.

[0096] Preparation Example 10

[0097] This preparation example adopts the method provided in Preparation Example 3 to prepare intestinal flora enriched microspheres, except that, in step S2, after the mixed solution is added dropwise, the solution is incubated at a temperature of 37°C, a rotation speed of 600 r / min, and a time of 30 min. Other conditions are the same to obtain intestinal flora enriched microspheres, and the loading amount of attapulgite in the intestinal flora enriched microspheres is about 0.13wtt%.

[0098] Comparative Preparation Example 1

[0099] This comparative preparation example provides a method for preparing intestinal flora-enriched microspheres, which specifically includes:

[0100] S1. Mix sodium alginate with 0.85% physiological saline, and vortex mix at 37°C and 800 rpm for 8 min to prepare a sodium alginate solution with a concentration of 1.3% (w / v);

[0101] S2. Take 200 mL of sodium alginate solution and add it dropwise into 200 mL of 1.1% (w / v) calcium chloride solution at a titration rate of 6 mL / min. After the addition is completed, incubate at 37°C and 200 r / min for 15 min, filter, and obtain intestinal flora-enriched microspheres.

[0102] Figure 2 This is a microscope picture of the intestinal flora-enriched microspheres obtained in this comparative preparation example (magnification is ×80).

[0103] Comparative Preparation Example 2

[0104] This comparative example is used to illustrate the preparation of an intestinal flora enriched microsphere, which specifically includes:

[0105] S1. Sodium alginate, porous starch and 0.85% physiological saline were mixed, and vortexed for 8 min at 37° C. and 800 rpm to prepare a mixed solution, wherein the concentration of sodium alginate in the mixed solution was 1.3% (w / v), and the concentration of porous starch was 1.3% (w / v);

[0106] S2. At 37°C and 200 r / min, 200 mL of the mixed solution was added dropwise to 400 mL of 1.1% (w / v) calcium chloride solution at a titration rate of 6 mL / min. After the addition was completed, the mixture was incubated for 15 min and filtered to obtain intestinal flora-enriched microspheres, and the loading amount of porous starch in the intestinal flora-enriched microspheres was about 0.13%.

[0107] Figure 3 This is a microscope picture of the intestinal flora-enriched microspheres obtained in this comparative preparation example (magnification is ×80).

[0108] It should be noted that the fresh stool samples from healthy donors used in the following examples and comparative examples are the same.

[0109] Example 1

[0110] This example is used to illustrate a method for enriching intestinal flora, which specifically includes:

[0111] (1) 16 g of fresh fecal sample from a healthy donor was mixed with 128 mL of 0.85% saline, and vortexed at 3000 r / min and 4°C for 10 min to obtain a fecal sample solution; the fecal sample solution was filtered through 100-mesh and 200-mesh sieves in turn, each filtration time was 1 min, and the filtrate was collected to obtain a bacterial suspension.

[0112] (2) The intestinal flora-enriched microspheres provided in Preparation Example 1 were added to the bacterial suspension at an addition amount of 10% (w:v). After incubation at 100 rpm and 4°C for 15 min, the intestinal flora-enriched microspheres were removed by filtration using a 30-mesh filter, the filtrate was collected, and the filtrate was centrifuged at 14000 r / min and 4°C for 20 min. The supernatant was removed and the bacterial sludge was collected.

[0113] Example 2

[0114] This example uses the method provided in Example 1 to enrich intestinal flora, except that in step (1), an equal volume of 10% (w / v) betaine solution is used instead of 0.85% saline, and other conditions are the same to collect bacterial sludge.

[0115] Example 3

[0116] This example uses the method provided in Example 1 to enrich intestinal flora, except that in step (1), an equal volume of 10% (w / v) lycopodiella extract solution is used instead of 0.85% saline, and other conditions are the same to collect bacterial sludge.

[0117] Embodiments 4 to 12

[0118] Examples 4 to 12 use the method provided in Example 1 to enrich the intestinal flora, except that in step (2), the intestinal flora enrichment microspheres provided in Preparation Example 1 are replaced by different intestinal flora enrichment microspheres of equal mass, as shown in Table 1. Other conditions are the same, and bacterial sludge is collected.

[0119] Table 1.

[0120] Group Intestinal flora enrichment microspheres Group Intestinal flora enrichment microspheres Group Intestinal flora enrichment microspheres Example 4 Preparation Example 2 Example 5 Preparation Example 3 Example 6 Preparation Example 4 Example 7 Preparation Example 5 Example 8 Preparation Example 6 Example 9 Preparation Example 7 Example 10 Preparation Example 8 Embodiment 11 Preparation Example 9 Example 12 Preparation Example 10

[0121] Comparative Example 1

[0122] This comparative example provides a method for enriching intestinal flora, and the enrichment method specifically comprises:

[0123] (1) 16 g of fresh fecal sample from a healthy donor was mixed with 128 mL of 0.85% saline, and vortexed at 3000 r / min and 4°C for 10 min to obtain a fecal sample solution; the fecal sample solution was filtered through 100-mesh and 200-mesh sieves in turn, each filtration time was 1 min, and the filtrate was collected to obtain a bacterial suspension.

[0124] (2) The bacterial suspension was centrifuged at 14000 r / min and 4°C for 20 min, the supernatant was removed, and the bacterial sludge was collected.

[0125] Comparative Example 2

[0126] This comparative example adopts the method provided in Example 1 to enrich the intestinal flora, except that in step (2), the intestinal flora enrichment microspheres provided in Comparative Preparation Example 1 are used instead of the intestinal flora enrichment microspheres provided in Preparation Example 1 with an equal mass, and other conditions are the same, and bacterial sludge is collected.

[0127] Comparative Example 3

[0128] This comparative example adopts the method provided in Example 1 to enrich the intestinal flora, except that in step (2), the intestinal flora enrichment microspheres provided in Comparative Preparation Example 2 of equal mass are used to replace the intestinal flora enrichment microspheres provided in Preparation Example 1, and other conditions are the same, and bacterial sludge is collected.

[0129] Test Case

[0130] This test is used to illustrate the enrichment effect of the intestinal flora enrichment method provided in the above embodiments and comparative examples. The test specifically includes: weighing and recording the mass of the bacterial sludge collected in each embodiment and comparative example, and diluting the bacterial sludge 1000 times with 0.85% saline, and then LIVE / DEAD TM BacLight TM The Bacterial Viability Kit was used for cell viability detection and the staining was performed according to the instructions. The test was performed by flow cytometry. The test results are shown in Table 2.

[0131] Table 2.

[0132]

[0133]

[0134] It can be seen from the test results shown in Table 2 that, compared with Comparative Examples 1 to 3, when the method provided by Examples 1 to 12 of the present invention is used to process the same stool sample, there is a significant improvement in the weight, unit bacterial count and activity of the bacterial sludge, and the active bacterial community in the stool sample is effectively enriched, so that the finally collected bacterial sludge shows a higher total bacterial count and total live bacterial count.

[0135] 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 microsphere enriched with intestinal flora, characterized in that: The intestinal flora enriched microspheres include a calcium alginate gel substrate and a porous adsorbent doped on the calcium alginate gel substrate; and the pore size of the intestinal flora enriched microspheres increases from the center to the outer layer.

2. The intestinal flora enriched microspheres according to claim 1, characterized in that: The porous adsorbent is selected from one or more of porous starch, biochar, activated carbon, attapulgite, activated clay, diatomaceous earth, talc, white clay and zeolite; Optionally, the porous adsorbent is diatomaceous earth and / or attapulgite; Optionally, the porous adsorbent has an average particle size of 100 to 300 μm and an average pore size of 20 to 80 μm; Optionally, based on the total mass of the intestinal flora-enriched microspheres, the loading amount of the porous adsorbent is 0.001 to 10 wt %.

3. The intestinal flora enriched microspheres according to claim 1, characterized in that: The average particle size of the intestinal flora enriched microspheres is 1 to 10 mm.

4. The method for preparing intestinal flora-enriched microspheres according to any one of claims 1 to 3, characterized in that: The preparation method comprises: S1, mixing sodium alginate, a porous adsorbent and a solvent to obtain a mixed solution; S2, dropping the mixed solution dropwise into a calcium chloride solution in a static state, stirring and incubating after the dropping is completed to obtain the intestinal flora enriched microspheres.

5. The method for preparing intestinal flora-enriched microspheres according to claim 4, characterized in that: In step S1, the solvent is water and / or physiological saline; Optionally, the concentration of sodium alginate in the mixed solution is 0.2-2% (w / v), and the concentration of the porous adsorbent is 0.2-2% (w / v); Optionally, the mixing treatment is mechanical stirring, the rotation speed of the mechanical stirring is 200 to 1500 r / min, the temperature is 35 to 40° C., and the time is 2 to 10 min.

6. The method for preparing intestinal flora-enriched microspheres according to claim 4, characterized in that: In step S2, the concentration of the calcium chloride solution is 0.4-1.5% (w / v); Optionally, the volume ratio of the mixed solution to the calcium chloride solution is 1:(1-4); Optionally, the dropwise addition speed of the mixed solution is 2 to 10 mL / min; Optionally, the stirring incubation treatment is performed at a rotation speed of 100 to 600 rpm, a temperature of 35 to 40° C., and a time of 10 to 30 min.

7. Use of the intestinal flora-enriched microspheres according to any one of claims 1 to 3 in intestinal flora transplantation technology.

8. A method for enriching intestinal flora, characterized in that: The enrichment method comprises: (1) mixing the treatment solution and the stool sample, removing impurities, and obtaining a bacterial suspension; (2) The intestinal flora enrichment microspheres according to any one of claims 1 to 3 are mixed with a bacterial suspension, subjected to adsorption treatment, filtration, and centrifugation to enrich the intestinal flora.

9. The method for enriching intestinal flora according to claim 8, characterized in that: In step (1), the treatment solution is selected from one or more of a betaine solution, a laver extract solution, a sargassum extract solution, a phosphate buffer solution and a physiological saline solution; Optionally, the treatment solution and the stool sample are added in a ratio of 5:1 to 20:1 (mL:g); Optionally, the treatment solution and the stool sample are mixed at a speed of 1000-6000 r / min, a temperature of 0-5°C, and a time of 2-20 min; Optionally, the impurity removal treatment is a suction filtration treatment, and the mesh size of the filter in the suction filtration treatment is 10 to 400 meshes.

10. The method for enriching intestinal flora according to claim 8, characterized in that: In step (2), the amount of the intestinal flora enrichment microspheres added is 5-20% (w / v) based on the volume of the bacterial suspension; Optionally, the rotation speed of the adsorption treatment is 50-150 r / min, the temperature is 0-5° C., and the time is 10-25 min.

Images