Porous fiber composite microbial microsphere and application thereof

Abstract

The invention provides porous fiber composite microbial microspheres and application thereof, and belongs to the technical field of probiotic preparations. The porous fiber composite microbial microsphere has a porous structure skeleton composed of A-CMC and ADH-HA, and probiotics loaded by the skeleton, and an ADH-HA film wrapping the probiotics is attached to the surface of the skeleton of the microsphere. The microspheres are obtained by shearing a precursor solution of the composite fiber microspheres into liquid drops with the diameter of 200-600 microns and freeze-drying the liquid drops. Wherein the composite fiber microsphere precursor solution is prepared by mixing an A-CMC suspension solution with the concentration of 0.9 to 1.3 percent, an ADH-HA solution with the concentration of 0.1 to 0.9 percent and probiotics treated by glycerol. The microsphere is good in cell compatibility, actively adheres to the wall in intestinal juice, has specific adhesiveness to a colitis disease part, can realize targeted therapy, and can ensure that enough active probiotics reach the colitis disease part.

Description

Technical Field

[0001] The present invention relates to the technical field of probiotic preparations, and in particular to porous fiber composite microbial microspheres and applications thereof in treating inflammatory bowel disease. Background Art

[0002] Inflammatory bowel disease (IBD) is a chronic and difficult-to-relieve inflammatory disease of the gastrointestinal tract. Standard clinical treatments for IBD often have serious side effects, such as headaches, rashes, nausea, abdominal pain, liver dysfunction, pancreatitis, pneumonia, etc. Moreover, a large proportion of patients do not see significant results after treatment, or develop drug resistance over time, making it difficult to cure. Ingesting probiotics to regulate the balance of intestinal microbiota is an effective and safe new therapy. Through reasonable colonization of intestinal microbiota, it can induce the activation of cells with systemic anti-inflammatory functions in the intestine, effectively improve intestinal efficiency, and restore the health of patients. It has great application prospects in the treatment of IBD. However, ingested probiotics are easily deactivated by the influence of digestive tract fluids, resulting in a significant reduction in efficacy. Encapsulation technology is needed to protect probiotics to ensure that sufficient active probiotics can pass through gastric and intestinal fluids to reach the colon and exert their effects.

[0003] In the existing technology, common encapsulation technologies include spray drying, extrusion, electrospinning, and emulsion. Among them, the spray drying method requires preparation under high temperature conditions, which will cause a large number of probiotics to be inactivated, affecting the efficacy of the medicine; the probiotic medicine prepared by the extrusion method is too large in size, which limits the application of the medicine; the electrospinning method requires the use of a high-voltage power supply and has low production efficiency, resulting in increased medicine costs; the emulsion method requires the use of toxic reagents such as emulsifiers, resulting in poor biocompatibility of the prepared probiotic medicine. In addition, existing probiotic medicines also have technical problems such as insufficient amount of probiotics reaching the colon, affecting efficacy, and inability to achieve targeted treatment.

[0004] In view of this, it is necessary to design a porous fiber composite microbial microsphere and its application to solve the above problems. Summary of the Invention

[0005] In view of the technical problems existing in the background technology, the present application provides a porous fiber composite microbial microsphere and its application, using aldehyde-modified carboxymethyl cellulose (A-CMC) and hydrazide functionalized hyaluronic acid (ADH-HA) with good biocompatibility as microsphere skeletons to load probiotics. First, the present application utilizes the porous structure of the microspheres and the pH response characteristics of A-CMC to enable the microspheres to shrink in gastric juice and expand in intestinal fluid, thereby making it easier for the microspheres to actively adhere to the intestinal wall. Secondly, the present application utilizes the high hydrophilicity of the material and the porous structure of the microspheres, as well as the characteristics of the microspheres expanding to increase in volume and decrease in density, making it easier for the microspheres to actively adhere to the intestinal wall without adhering to the stomach wall. Finally, the present application also utilizes the large number of carboxylic acid groups (-COOH) carried on A-CMC and ADH-HA to make the microspheres carry a negative charge in the intestinal fluid, thereby achieving specific adhesion to the site of colon inflammation for targeted therapy.

[0006] In the first aspect, an embodiment of the present application provides a porous fiber composite microbial microsphere, which has a porous structure skeleton composed of aldehyded carboxymethyl cellulose and hydrazide functionalized hyaluronic acid, and probiotics loaded by the skeleton, and the skeleton surface of the porous fiber composite microbial microsphere is also attached with an ADH-HA film wrapping the probiotics; the porous fiber composite microbial microsphere is obtained by shearing the composite fiber microsphere precursor into droplets with a diameter in the range of 200-600 μm and freeze-drying the droplets; the composite fiber microsphere precursor is prepared by mixing an A-CMC suspension with a concentration of 0.9-1.3%, an ADH-HA solution with a concentration of 0.1-0.9%, and probiotics treated with glycerol.

[0007] Furthermore, the porous fiber composite microbial microspheres are hydrophilic and carry a negative charge; when the porous fiber composite microbial microspheres are dispersed in an acidic solution, the microspheres shrink and their diameters decrease; when the microspheres are dispersed in an alkaline solution, the microspheres expand and their diameters increase.

[0008] Furthermore, the porous fiber composite microbial microspheres are used to treat inflammatory bowel disease; the porous fiber composite microbial microspheres do not adhere to the wall in gastric juice, but do adhere to the wall in intestinal juice or colonic juice; the porous fiber composite microbial microspheres have specific adhesion properties to the site of colon inflammation.

[0009] Furthermore, the optimal storage temperature of the porous fiber composite microbial microspheres is -20°C; the porous fiber composite microbial microspheres are spherical or drop-shaped.

[0010] Furthermore, the porous fiber composite microbial microspheres are prepared by the following steps:

[0011] S1, adding sodium periodate to the CMC aqueous suspension, stirring at room temperature in the dark, then adding ethylene glycol to quench the unreacted periodate. After the reaction is completed, dialyzing with deionized water to obtain an A-CMC suspension, which is freeze-dried to obtain A-CMC;

[0012] S2, adding oxalic acid dihydrazide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide to a hyaluronic acid solution to obtain a mixed solution, followed by reacting at room temperature for a period of time, adding hydrochloric acid solution dropwise during the reaction to maintain the pH of the mixed solution at 4.7-4.8, and when the pH value of the system no longer increases, adding sodium hydroxide solution dropwise to adjust the pH of the solution to 7 to terminate the reaction, followed by dialysis with deionized water and freeze-drying to obtain ADH-HA;

[0013] S3, using the A-CMC obtained in step S1 to prepare an A-CMC suspension with a concentration of 0.9-1.3%, and using the ADH-HA obtained in step S2 to prepare an ADH-HA solution with a concentration of 0.1-0.9%; then, the A-CMC suspension and the ADH-HA solution are mixed and stirred for 30 minutes, and the probiotics treated with glycerol are added and uniformly dispersed to obtain a composite fiber microsphere precursor solution;

[0014] S4, the composite fiber microsphere precursor obtained in step S4 is transferred to the raw material warehouse, the microfluidic device and the nitrogen-assisted device cooperate with each other, the microfluidic device controls the precursor to flow out of the fluid needle, and the nitrogen-assisted device shears the outflowing precursor into small droplets, and the small droplets are dripped into the liquid nitrogen of the molding device, and the porous fiber composite microbial microspheres are obtained after freeze-drying; the small droplets pass through a temperature control device before being dripped into the liquid nitrogen, and the temperature of the temperature control device is set to -50°C to 10°C.

[0015] Furthermore, the preparation steps of the porous fiber composite microbial microspheres also include:

[0016] In step S1, the stirring time at room temperature in the dark is 6-8 hours, the molar ratio of NaIO4 to CMC is (0.5-1):1, and the concentration of the CMC aqueous suspension is 1-2 wt.%; and,

[0017] In step S2, the mass ratio of ADH to HA and EDC is 1:(2-4):(0.1-0.2), the concentration of the HA solution is 0.5-2 wt.%, the concentration of the hydrochloric acid solution is 0.4-0.6 mol / L, and the concentration of the sodium hydroxide solution is 0.05-0.1 mol / L; and,

[0018] In step S3, the volume ratio of the A-CMC suspension to the ADH-HA solution is (8-12):1; and

[0019] In step S4, the microfluidic device and the nitrogen assist device cooperate with each other to shear the precursor liquid at a speed of 9-10 mL / h, and the nitrogen pressure is 35-65 KPa.

[0020] Furthermore, in step S4, treating the probiotics with glycerol specifically refers to dispersing the probiotics in glycerol, treating at 4° C. for 30-60 minutes, and then centrifuging to obtain glycerol-treated probiotics, wherein the glycerol concentration is 10-20%.

[0021] Furthermore, the method for preparing spherical porous fiber composite microbial microspheres also includes setting the temperature of the temperature control device to -10°C to 10°C in step S4; the method for preparing droplet-shaped porous fiber composite microbial microspheres also includes placing the composite fiber microsphere precursor obtained in step S3 in a refrigerator until the viscosity of the precursor reaches 3-300 Pa.s, and then transferring it to the raw material warehouse, as well as setting the microfluidic rate to 1mm / min-3mm / min and the temperature of the temperature control device to -50°C to -40°C.

[0022] Furthermore, step S4 uses the following preparation device: including a microfluidic device for controlling the outflow rate of the precursor liquid, a nitrogen-assisted device that cooperates with the microfluidic device to blow off the precursor liquid, and a molding device for freeze-drying the blown-off precursor droplets; the microfluidic device includes a raw material warehouse and a liquid flow needle, and the inner diameter of the liquid flow needle is 250-350 μm; the nitrogen-assisted device is arranged below the raw material warehouse, including a hollow tube 1 connected to the nitrogen input end, a hollow tube 2 connected to the nitrogen output end, a hollow tube 3 arranged below the liquid flow needle and connected to the liquid flow needle, a hollow tube 4 connected to the molding device, and a four-way connection connecting hollow tubes 1, 2, 3, and 4; the molding device is arranged below the nitrogen-assisted device, and the molding device includes a temperature control device and liquid nitrogen.

[0023] In a second aspect, an embodiment of the present application provides an application of the porous fiber composite microbial microspheres described in any one of the aforementioned technical solutions, wherein the porous fiber composite microbial microspheres are used to treat inflammatory bowel disease.

[0024] The beneficial effects of this application are as follows:

[0025] (1) This application uses aldehyde-modified carboxymethyl cellulose (A-CMC) and hydrazide-functionalized hyaluronic acid (ADH-HA) with good biocompatibility as the microsphere skeleton to load probiotics, and the surface of the skeleton is attached with an ADH-HA film wrapped with probiotics. The porous fiber composite microbial microspheres provided by this application are porous structures with complex pore sizes. They are not through-holes. Invasion of the inner layer of the microspheres requires passing through multiple layers of hyaluronic acid, which can better protect the inner layer probiotics. At the same time, the porous fiber composite microbial microspheres provided by this application will shrink in acidic gastric juice, which can prevent harmful substances from entering the interior of the microspheres and further protect the inner layer probiotics.

[0026] (2) The present application utilizes the porous structure of the microspheres and the pH response characteristics of A-CMC to enable the microspheres to shrink in gastric juice and expand in intestinal juice. At the same time, since the material constituting the microspheres is highly hydrophilic and the microspheres are porous, there is a lot of air inside. After the microspheres come into contact with the aqueous solution, the water that quickly infiltrates will lock the air inside the microspheres, thereby forming tiny bubbles. In an alkaline environment, the microspheres expand, causing the volume to increase and the density to decrease. Under the synergistic effect of buoyancy and capillary action, the microspheres containing bubbles are pulled toward the hydrophilic glass wall (simulating the stomach wall and intestinal wall that are also hydrophilic), causing the microspheres to adhere to the wall. The microspheres are more likely to actively adhere to the intestinal wall rather than the stomach wall. This is because the volume of the microspheres decreases and the density increases after they shrink, and the buoyancy and capillary action are not enough to pull the microspheres.

[0027] (3) The present application also utilizes the large number of carboxylic acid groups (-COOH) carried on A-CMC and ADH-HA to make the microspheres carry a negative charge in the intestinal fluid, thereby achieving specific adhesion to the site of colon inflammation (the site of colon inflammation is usually positively charged) for targeted treatment.

[0028] The above description is only an overview of the technical solution of the present application. In order to more clearly understand the technical means of the present application, it can be implemented in accordance with the contents of the specification. In order to make the above and other purposes, features and advantages of the present application more obvious and easy to understand, the specific implementation methods of the present application are listed below. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] To more clearly illustrate the technical solution of this application, the following is a brief introduction to the drawings used in this application. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be derived from these drawings without inventive effort.

[0030] Figure 1 This is a morphology diagram of the porous fiber composite microbial microspheres of Example 1 of the present application; wherein, Figure 1 (a) is the morphology of multiple porous fiber composite microbial microspheres observed using an optical microscope. Figure 1 (b) is a morphology of a single microsphere observed by scanning electron microscopy (SEM), and (c) is a local magnified view of a single microsphere observed by scanning electron microscopy (SEM);

[0031] Figure 2 This is a schematic diagram of probiotic activity obtained by staining the probiotics in the porous fiber composite microbial microspheres of Example 1 using acridine orange and ethidium bromide and photographing them using laser confocal microscopy;

[0032] Figure 3 This is a comparison diagram of the adhesion phenomenon of porous fiber composite microbial microspheres in an in vitro model in the examples of this application;

[0033] Figure 4 This is a morphology diagram of the porous fiber composite microbial microspheres obtained in Example 7 of the present application;

[0034] Figure 5 This is a comparison chart of the MPO activity of mice in Application Example 1 of this application;

[0035] Figure 6 This is the in vivo fluorescence imaging of mice in Application Example 2 of this application, wherein: Figure 6 (a) is an inflammation mouse, Figure 6 (b) normal mice;

[0036] Figure 7 This is a device for preparing porous fiber composite microbial microspheres in the embodiments of this application. DETAILED DESCRIPTION

[0037] The following embodiments of the technical solution of the present application will be described in detail with reference to the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present application and are therefore only examples and are not intended to limit the scope of protection of the present application.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this application belongs; the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit this application; the terms "including" and "having" and any variations thereof in the specification and claims of this application and the above-mentioned figure descriptions are intended to cover non-exclusive inclusions.

[0039] In the description of the embodiments of this application, the technical terms "first" and "second" are used only to distinguish different objects and should not be understood to indicate or imply relative importance or implicitly specify the quantity, specific order, or primary and secondary relationship of the indicated technical features. In the description of the embodiments of this application, the meaning of "plurality" is more than two, unless otherwise clearly and specifically defined.

[0040] References herein to "embodiments" mean that a particular feature, structure, or characteristic described in connection with the embodiments may be included in at least one embodiment of the present application. The appearance of this phrase in various places in the specification does not necessarily refer to the same embodiment, nor does it constitute an independent or alternative embodiment that is mutually exclusive of other embodiments. It is understood, both explicitly and implicitly, by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0041] In the description of the embodiments of this application, the term "and / or" is simply a description of the association relationship between associated objects, indicating that three relationships can exist. For example, A and / or B can represent the following three situations: A exists alone, A and B exist simultaneously, and B exists alone. In addition, the character " / " in this document generally indicates that the associated objects are in an "or" relationship.

[0042] In the description of the embodiments of the present application, the term "plurality" refers to two or more (including two). Similarly, "multiple groups" refers to two or more (including two groups), and "multiple sheets" refers to two or more (including two sheets). For those skilled in the art, the specific meanings of the above terms in the embodiments of the present application can be understood according to specific circumstances.

[0043] Existing probiotic preparations have technical problems such as insufficient number of active bacteria, difficult to control the size of the preparation, poor biocompatibility, high production cost, and insufficient survival rate in gastrointestinal fluid, which affects the efficacy and cannot accurately identify the site of colon inflammation for targeted treatment.

[0044] In order to solve the above technical problems, the present application provides a porous fiber composite microbial microspheres and applications thereof, wherein aldehyde-modified carboxymethyl cellulose (A-CMC) and hydrazide-functionalized hyaluronic acid (ADH-HA) with good biocompatibility are used as microsphere skeletons to load probiotics. First, the present application utilizes the porous structure of the microspheres and the pH response characteristics of A-CMC to enable the microspheres to shrink in gastric juice and swell in intestinal fluid, thereby making it easier for the microspheres to actively adhere to the intestinal wall. Secondly, the present application utilizes the high hydrophilicity of the material and the porous structure of the microspheres, as well as the characteristics of the microspheres expanding to increase in volume and decrease in density, making it easier for the microspheres to actively adhere to the intestinal wall instead of adhering to the stomach wall. Finally, the present application also utilizes the large number of carboxylic acid groups (-COOH) carried on A-CMC and ADH-HA to make the microspheres carry a negative charge in the intestinal fluid, thereby achieving specific adhesion to the site of colon inflammation for targeted therapy.

[0045] In the first aspect, an embodiment of the present application provides a porous fiber composite microbial microsphere having a skeleton of a porous structure composed of aldehyded carboxymethyl cellulose (A-CMC) and hydrazide functionalized hyaluronic acid (ADH-HA), and probiotics loaded by the skeleton, and an ADH-HA film wrapped with probiotics is also attached to the surface of the skeleton of the microsphere. The microsphere is obtained by shearing the composite fiber microsphere precursor into droplets with a diameter in the range of 200-600 μm and freeze-drying the droplets. The composite fiber microsphere precursor is prepared by mixing an A-CMC suspension with a concentration of 0.9-1.3%, an ADH-HA solution with a concentration of 0.1-0.9%, and probiotics treated with glycerol.

[0046] In the present application embodiment, porous fiber composite microbial microspheres have hydrophilicity and carry negative charge.Wherein, hydrophilicity is mainly related to the hydrophilicity of material A-CMC and ADH-HA and the porous structure of porous fiber composite microbial microspheres, and negative charge is mainly related to the large amount of carboxylic acid groups (-COOH) carried on A-CMC and ADH-HA. Microspheres are dispersed in acidic solution and protonation occurs. Now, the molecular chain of microspheres is neutral, and the hydrogen bond between the molecular chains is enhanced, causing the microspheres to shrink and the diameter to become smaller; Microspheres are dispersed in alkaline solution and deprotonation occurs. Now, the molecular chain of microspheres is negatively charged and there is electrostatic repulsion, causing the molecular chain to stretch, and the microspheres expand and the diameter becomes larger.

[0047] In the examples of this application, porous fiber-composite microbial microspheres are used to treat inflammatory bowel disease. These microspheres do not adhere to the wall of the gastric fluid, but do adhere to the wall of the intestinal or colonic fluid. Furthermore, since inflamed areas of the colon are generally positively charged, the negatively charged porous fiber-composite microbial microspheres exhibit specific adhesion to these areas, enabling targeted treatment.

[0048] In the embodiment of the present application, the optimal storage temperature of the porous fiber composite microbial microspheres is -20°C.

[0049] The porous fiber composite microbial microspheres provided in this application are prepared by the following steps:

[0050] S1, sodium periodate (NaIO4) is added to a CMC aqueous suspension (solvent is water), stirred at room temperature in the dark, and then ethylene glycol is added to quench the unreacted periodate. After the reaction is completed, the mixture is dialyzed with deionized water to obtain an A-CMC suspension, which is then freeze-dried to obtain A-CMC.

[0051] In the embodiment of the present application, the stirring time at room temperature in the dark is 6-8 hours, the molar ratio of NaIO4 to CMC is (0.5-1):1, and the concentration of the CMC aqueous suspension is 1-2 wt.%.

[0052] In the embodiment of the present application, the dialysis time is 3 days, and the deionized water is replaced 3 times a day during the dialysis process.

[0053] S2, adding oxalic acid dihydrazide (ADH) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) to a hyaluronic acid (HA) solution to obtain a mixed solution, which is then reacted at room temperature for a period of time. During the reaction, hydrochloric acid solution is added dropwise to maintain the pH of the mixed solution at 4.7-4.8. When the pH value of the system no longer increases, sodium hydroxide solution is added dropwise to adjust the pH of the solution to 7 to terminate the reaction, which is then dialyzed with deionized water and freeze-dried to obtain ADH-HA.

[0054] In the examples of the present application, the mass ratio of ADH to HA and EDC is 1:(2-4):(0.1-0.2).

[0055] In the examples of the present application, the concentration of the HA solution is 0.5-2 wt.%, the concentration of the hydrochloric acid solution is 0.4-0.6 mol / L, and the concentration of the sodium hydroxide solution is 0.05-0.1 mol / L.

[0056] Preferably, hydrochloric acid solution is added dropwise during the reaction to maintain the pH of the mixed solution at 4.75.

[0057] In the embodiment of the present application, the dialysis time is 3 days, and the deionized water is replaced 3 times a day during the dialysis process.

[0058] S3, using the A-CMC obtained in step S1 to prepare an A-CMC suspension with a concentration of 0.9-1.3%, and using the ADH-HA obtained in step S2 to prepare an ADH-HA solution with a concentration of 0.1-0.9%. Next, the A-CMC suspension and the ADH-HA solution are mixed and stirred for 30 minutes, and the glycerol-treated probiotics are added and uniformly dispersed to obtain a composite fiber microsphere precursor solution. The A-CMC and ADH-HA undergo chemical crosslinking upon mixing, which stabilizes the prepared microsphere structure and prevents premature release of the probiotics.

[0059] In the examples of the present application, treating the probiotics with glycerol specifically involves dispersing the probiotics in glycerol, treating at 4°C for 30-60 minutes, and then centrifuging to obtain the glycerol-treated probiotics. The glycerol concentration is 10-20%, preferably 10%. The centrifugation temperature is 4°C and the speed is 7000 rpm.

[0060] In the examples of the present application, the volume ratio of the A-CMC suspension to the ADH-HA solution is (8-12):1.

[0061] In the embodiment of the present application, the concentration of probiotics in the composite fiber microsphere precursor solution is 2×10 9 CFU / mL.

[0062] S4, the composite fiber microsphere precursor obtained in step S4 is transferred to the raw material warehouse, the microfluidic device and the nitrogen-assisted device cooperate with each other, the microfluidic device controls the precursor to flow out of the fluid needle, and the nitrogen-assisted device shears the outflowing precursor into small droplets, which are then poured into the liquid nitrogen of the molding device, and the porous fiber composite microbial microspheres are obtained after freeze-drying.

[0063] In the embodiment of the present application, the microfluidic device and the nitrogen-assisted device cooperate with each other to shear the precursor liquid at a speed of 9-10 mL / h.

[0064] In the examples of this application, the nitrogen pressure is 35-65 kPa. During the gas-assisted preparation of microspheres, nitrogen destroys the surface tension of the microsphere precursor, facilitating its dripping. Therefore, the gas pressure has the most direct effect on the diameter of the microspheres, and the diameter of the microspheres can be adjusted by controlling the gas pressure.

[0065] In the embodiment of the present application, the small liquid droplets pass through a temperature control device before being injected into the liquid nitrogen, and the temperature of the temperature control device is set to -50°C to 10°C.

[0066] The apparatus for preparing porous fiber composite microbial microspheres in step S4 is shown in FIG. Figure 7 The preparation device includes a microfluidic device for controlling the outflow rate of the precursor liquid, a nitrogen assist device that cooperates with the microfluidic device to blow off the precursor liquid, and a molding device for freeze-drying the blown-off precursor droplets.

[0067] The microfluidic device includes a raw material bin and a fluid flow needle, and the inner diameter of the fluid flow needle is 250-350 μm.

[0068] The nitrogen auxiliary device is arranged below the raw material bin, and includes a hollow tube 1 connected to the nitrogen input end, a hollow tube 2 connected to the nitrogen output end, a hollow tube 3 arranged below the liquid flow needle and connected to the liquid flow needle, a hollow tube 4 connected to the forming device, and a four-way connection connecting hollow tubes 1, 2, 3, and 4.

[0069] The molding device is arranged below the nitrogen assist device, and the molding device includes temperature control equipment and liquid nitrogen.

[0070] In the embodiment of the present application, the porous fiber composite microbial microspheres are spherical or teardrop-shaped, and the porous fiber composite microbial microspheres are porous structures. The preparation method of spherical porous fiber composite microbial microspheres also includes setting the temperature of the temperature control device to -10°C to 10°C in step S4. The preparation method of teardrop-shaped porous fiber composite microbial microspheres also includes placing the composite fiber microsphere precursor obtained in step S3 in a refrigerator (0-5°C) until the viscosity of the precursor reaches 3-300Pa.s, and then transferring it to the raw material warehouse, and setting the microfluidic rate to 1mm / min-3mm / min, and setting the temperature of the temperature control device to -50°C to -40°C.

[0071] Among them, placing the precursor liquid in a low-temperature environment (0-5°C) can make the internal network structure tighter and increase the number of cross-linkable points per unit volume, thereby promoting cross-linking and increasing the viscosity of the precursor liquid.

[0072] In a second aspect, an embodiment of the present application provides an application of porous fiber composite microbial microspheres, which are used to treat inflammatory bowel disease and are taken orally.

[0073] In this embodiment, the microspheres sequentially enter the stomach, intestines, and colon. They do not adhere to the stomach wall and release almost no probiotics; they quickly adhere to the intestines, releasing only a small amount of probiotics; and they slowly adhere to the colon wall, releasing a large amount of probiotics. When the A-CMC concentration is 1.3% and the ADH-HA concentration is 0.9%, the amount of probiotics released by the microspheres in the colon exceeds the total amount released in the stomach and intestines by 2,100 times.

[0074] Some specific examples are listed below. It should be noted that the examples described below are exemplary and are only used to explain the present application, and should not be construed as limiting the present application. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in this area or the product specifications are used. Reagents or instruments used without manufacturer's indication are all commercially available conventional products.

[0075] Example 1

[0076] Example 1 proposes a porous fiber composite microbial microsphere, which is prepared by the following steps:

[0077] S1, NaIO4 was added to the CMC aqueous suspension with a concentration of 1.5wt.% (molar ratio n NaIO4 :n CMC =0.5:1), stirred in the dark at room temperature for 6 h, then added ethylene glycol to quench unreacted periodate, and after the reaction was completed, dialyzed with deionized water for 3 days, changing the water 3 times a day during the dialysis process to obtain an A-CMC suspension, which was then freeze-dried to obtain A-CMC.

[0078] S2, 0.5g ADH and 0.07g EDC were added to 100mL of 1wt.% hyaluronic acid (HA) solution, and the reaction was carried out at room temperature for a period of time. During the reaction, the pH value of the reaction system was maintained at 4.75 by adding 0.5mol / L hydrochloric acid solution. When the pH value of the system no longer increased, 0.05mol / L sodium hydroxide solution was added to adjust the pH of the solution to 7, and then dialyzed with deionized water for 3 days and freeze-dried to obtain ADH-HA.

[0079] S3, the probiotics were dispersed in 10% glycerol, treated at 4°C for 30 min, and then centrifuged at 7000 r / min at 4°C to obtain the glycerol-treated probiotics; then, 0.9% ADH-HA solution and 0.9% A-CMC solution were prepared at a volume ratio of V A-CMC :V ADH-HA =10:1 and stirred for 30 min, then the probiotics treated with glycerol were added and evenly dispersed to obtain a composite fiber microsphere precursor solution. The probiotic concentration in the precursor solution was 2×10 9 CFU / mL.

[0080] S4, the probiotic composite fiber microsphere precursor obtained in step S3 is transferred to the raw material warehouse, and is matched with a nitrogen auxiliary device (nitrogen pressure is 35KPa) by a microfluidic device (microfluidic rate is 0.4mm / min), and the microfluidic device controls the precursor to flow out from the liquid needle, and the nitrogen auxiliary device shears the precursor into small droplets. The microfluidic device and the nitrogen auxiliary device cooperate with each other to shear the precursor at a speed of 9.6mL / h. The small droplets obtained by shearing are dripped into the liquid nitrogen of the molding device, and porous fiber composite microbial microspheres are obtained through freeze-drying. Wherein, the small droplets will pass through a temperature control device before being dripped into the liquid nitrogen. The temperature of the temperature control device is set to 0°C, and the time of freeze-drying is 2 days.

[0081] The diameter of the porous fiber composite microbial microspheres obtained in Example 1 is 600 μm.

[0082] See also Figure 1 , Figure 1 (a) is the morphology of porous fiber composite microbial microspheres observed using an optical microscope. Figure 1 (b) and (c) are the morphology and local magnification of a single microsphere observed by scanning electron microscopy (SEM). It can be seen that the microsphere has a porous structure, and a layer of hyaluronic acid membrane is attached to the non-porous area, and many probiotics are wrapped in the hyaluronic acid.

[0083] The probiotics in the microspheres were stained with acridine orange and ethidium bromide and photographed by laser confocal microscopy to observe the activity of the probiotics. Figure 2As shown, strong green fluorescence and weak red fluorescence can be seen in the microspheres, indicating that the production process of the present application can preserve the activity of most probiotics.

[0084] Comparative Example 1

[0085] Comparative Example 1 differs from Example 1 in that, in step S3, the probiotics were not treated with glycerol; instead, the probiotics were directly mixed with the A-CMC solution and the ADH-HA solution. All other steps were the same as in Example 1 and are not further described here. The diameter of the microspheres obtained in Comparative Example 1 was 600 μm.

[0086] The survival rate of probiotics was tested by the plate coating method, and the results are shown in Table 1.

[0087] Table 1. Probiotic concentrations in porous fiber composite microbial microspheres treated with and without glycerol

[0088] project Before lyophilization (CFU / mL) After lyophilization (CFU / mL) Glycerin treatment <![CDATA[8.27×10 8 ]]> <![CDATA[1.21×10 8 ]]> Glycerin-free treatment <![CDATA[6.22×10 8 ]]> <![CDATA[0.46×10 8 ]]>

[0089] The encapsulation activity obtained by the plate coating method was approximately 14.63%, which is lower than the results obtained by acridine orange and ethidium bromide staining tests. This is because the colonies grown on the plate coating are not necessarily individual probiotics, but may be aggregates of many probiotics, resulting in a lower number. However, comparative data still shows that the survival rate of probiotics is significantly improved after glycerol treatment, especially after freeze-drying. After glycerol treatment, the concentration of probiotics increased by 2.63 times. This shows that glycerol plays an important protective role in the preparation of microspheres. This is because glycerol reduces the formation of ice crystals in the probiotic cell membrane, effectively improving the survival rate of probiotics during liquid nitrogen quick freezing.

[0090] Examples 2-3 and Comparative Example 2

[0091] The difference between Examples 2-3 and Comparative Example 2 and Example 1 is that the concentrations of the ADH-HA solution in step S3 are changed to 0.1%, 0.5%, and 1.3%, respectively. The remaining concentrations are the same as in Example 1 and are not further described here. The diameter of the microspheres obtained in Examples 2-3 and Comparative Example 2 was 600 μm.

[0092] The porous fiber composite microbial microspheres obtained in Examples 2-3 and Comparative Example 1 were sequentially immersed in gastric simulated fluid, intestinal simulated fluid, and colon simulated fluid (i.e., first immersed in gastric simulated fluid, then immersed in intestinal simulated fluid, and finally immersed in colon simulated fluid), and the survival rate of probiotics in the microspheres was tested (the initial bacterial content in the microspheres was 1.21×10 8 CFU / mL), and the results are shown in Table 2.

[0093] Table 2. Probiotic survival of microspheres prepared with different concentrations of ADH-HA in an in vitro model

[0094]

[0095]

[0096] It can be seen that the probiotics were completely inactivated after the microspheres were prepared using a 1.3% ADH-HA concentration and immersed in gastric simulated fluid for 2 hours. This is because an excessively high HA concentration will increase the shrinkage of the microspheres in gastric fluid, and the A-CMC will be unable to support the microsphere structure and will be torn, causing the probiotics to be exposed to gastric fluid and inactivated. Therefore, the concentration of ADH-HA is preferably 0.1-0.9%, and is further preferably 0.9% based on the survival rate of probiotics in colon simulated fluid.

[0097] Example 4 and Comparative Example 3

[0098] The difference between Example 4 and Comparative Example 3 and Example 1 is that the concentration of the A-CMC solution in step S3 is changed to 1.3% and 0.5%, respectively. The other processes are the same as in Example 1 and are not described here. The diameter of the microspheres obtained in Example 4 and Comparative Example 3 is 600 μm.

[0099] The survival rate of probiotics in the porous fiber composite microbial microspheres obtained in Example 4 and Comparative Example 3 in the gastric simulated fluid, intestinal simulated fluid and colon simulated fluid (the initial bacterial content was 1.21×10 8 CFU / mL), and the results are shown in Table 3.

[0100] Table 3. Probiotic survival of microspheres prepared with different concentrations of A-CMC in an in vitro model

[0101]

[0102] It can be seen that the microspheres prepared with a concentration of 0.5% A-CMC were immersed in the gastric / intestinal / colon simulated fluid for 1-2 hours, and the probiotics were completely inactivated, while the microspheres prepared with a concentration of 1.3% A-CMC had a better probiotic survival rate. When the diameter of the flow needle is 300μm, an A-CMC concentration higher than 1.3% will cause needle clogging. If the A-CMC concentration needs to be further increased, the diameter of the flow needle needs to be adjusted. Therefore, the concentration of A-CMC is preferably 0.9-1.3%, and is further preferably 1.3% based on the survival rate of probiotics in the colon simulated fluid.

[0103] Examples 5-6 and Comparative Example 4

[0104] The difference between Examples 5-6 and Example 4 is that the nitrogen pressure in step S4 is 45KPa and 65KPa respectively, so as to prepare porous fiber composite microbial microspheres with different diameters. The diameter distribution of the microspheres is shown in Table 4. The rest is the same as Example 1 and will not be repeated here. Comparative Example 4 is a method with a concentration of 8×10 9 CFU / mL of pure bacterial solution.

[0105] Table 4. Average diameter of porous fiber composite microbial microspheres obtained in Examples 4-6

[0106] project Average diameter (μm) Example 4 (35KPa) 600μm Example 5 (45KPa) 400μm Example 6 (65KPa) 200μm

[0107] According to the above table, the greater the air pressure, the smaller the average diameter of the porous fiber composite microbial microspheres, which shows that the size of the microspheres can be adjusted by controlling the nitrogen pressure.

[0108] The survival rate of the probiotics in the microspheres prepared in Examples 4-6 and Comparative Example 4 was tested in an in vitro model (stomach / intestinal / colon simulation fluid) (the initial bacterial content in the porous fiber composite microbial microspheres was 1.21×10 8 CFU / mL), and the results are shown in Table 5.

[0109] Table 5. Survival rates of probiotics in porous fiber composite microbial microspheres with different diameters in an in vitro model

[0110]

[0111]

[0112] It can be seen that the survival rate of probiotics in the simulated fluid of the intestine and colon increases with the increase of the diameter of the microspheres, while the mortality rate of the pure bacterial solution in the simulated gastric fluid is 99.9994%. This is because, on the one hand, the porous fiber composite microbial microspheres provided by this application are porous structures, and the pore size is intricate and not through-holes. Invasion of the inner layer of the microspheres requires passing through multiple layers of hyaluronic acid. On the other hand, the microspheres will shrink in acidic gastric juice, which can prevent harmful substances from entering the interior of the microspheres and further protect the inner layer of probiotics. In addition, the porous fiber composite microbial microspheres provided by this application have reached the minimum viable count of 10 recommended by the World Gastroenterology Organization. 6 -10 7 The microsphere diameter of 200 μm is easier to use in real life, so Example 6 is the best example.

[0113] The potential of the porous fiber composite microbial precursor solution prepared in Example 6 was tested and was -83 mV, indicating that the porous fiber composite microbial microspheres provided in this application are negatively charged, while the inflamed colon site is generally positively charged. This is conducive to the specific adhesion of the microspheres to the inflamed colon site and better targeted treatment.

[0114] In an in vitro model, when the porous fiber-composite microbial microspheres were transferred from a PBS buffer with a pH of 7.4 to a gastric fluid with a pH of 2, the microspheres shrank and their diameters decreased. This is because A-CMC contains a large number of -COOH groups, which are protonated in an acidic environment, making the molecular chain neutral and strengthening the hydrogen bonds between the molecular chains, causing the microspheres to shrink. When the porous fiber-composite microbial microspheres were transferred from gastric fluid to intestinal fluid with a pH of 6.8, the microspheres continued to expand and their diameters increased. This is because the -COONa groups on A-CMC are deprotonated in an alkaline environment, making the molecular chain negatively charged. Under the action of electrostatic repulsion, the molecular chain stretches and the preparation expands.

[0115] In addition, because the material that makes up the microspheres is highly hydrophilic and the microspheres are porous, there is a lot of air inside. When the microspheres come into contact with an aqueous solution, the rapidly infiltrating water will lock the air inside the microspheres, forming tiny bubbles. In an alkaline environment, the microspheres expand, causing their volume to increase and their density to decrease. Under the synergistic effect of buoyancy and capillary action, the microspheres containing bubbles are pulled toward the hydrophilic glass wall (simulating the stomach wall and intestinal wall, which are also hydrophilic), causing the microspheres to adhere to the wall. However, the microspheres do not adhere to the wall in a low pH (acidic) environment. This is because the volume of the microspheres decreases and their density increases after shrinkage, and the buoyancy and capillary action are not enough to pull the microspheres.

[0116] See Figure 3 The figure below compares the adhesion behavior of porous fiber-composite microbial microspheres in an in vitro model. It can be seen that porous fiber-composite microbial microspheres do not readily adhere to the stomach wall but rather actively adhere to the intestinal wall. Testing showed that the microspheres adhered to the intestinal wall after immersion in colon-simulating fluid for 35 seconds, after immersion in intestinal-simulating fluid for 15 seconds, and did not adhere to the intestinal wall in gastric fluid.

[0117] The storage performance of the porous fiber composite microbial microspheres prepared in Example 6 was tested at -20°C, 4°C and room temperature, respectively. The results are shown in Table 6.

[0118] Table 6. Storage performance of porous fiber composite microbial microspheres at -20℃, 4℃ and room temperature

[0119] project -20℃ 4℃ normal temperature Storage 0 days 120000000 120000000 120000000 7-day storage 105000000 70000000 22300000 14-day storage 112000000 52000000 14000000

[0120] It can be seen that the activity of the porous fiber composite microbial microspheres was slightly reduced when stored in a -20°C environment for 14 days, but was significantly reduced in an environment of 4°C or room temperature.

[0121] Endothelial cells were used as test cells, and the cell compatibility of the porous fiber composite microbial microspheres prepared in Example 6 was tested using the CCK-8 staining method. The obtained data are shown in Table 7.

[0122] Table 7. Cytocompatibility of porous fiber composite microbial microspheres

[0123] Sample concentration (%) 0 25 50 100 Cell survival rate (%) 100 103 93.5 104

[0124] It can be seen that the cell activities are all greater than 90%, indicating that the porous fiber composite microbial microspheres have no cytotoxicity.

[0125] Comparative Example 5

[0126] Comparative Example 5 differs from Example 6 in that no probiotics were added in step S3, and the composite fiber microsphere precursor solution was prepared directly using the A-CMC solution and the ADH-HA solution. Other steps were the same as in Example 6 and are not described here. The porous fiber composite microspheres prepared in Comparative Example 5 had a diameter of 200 μm.

[0127] Example 7

[0128] The difference between Example 7 and Example 1 is that step S4 is specifically, first placing the probiotic composite fiber microsphere precursor obtained in step S3 in a refrigerator at 4°C, and waiting for the viscosity of the precursor to reach 10Pa.s, and then transferring the precursor to the raw material warehouse, through the microfluidic device (microfluidic rate is 1.5mm / min) and the nitrogen auxiliary device (nitrogen pressure is 35KPa), the microfluidic device controls the precursor to flow out of the liquid needle, and the nitrogen auxiliary device shears the precursor into small droplets. The microfluidic device and the nitrogen auxiliary device cooperate with each other to shear the precursor at a speed of 9.6mL / h. The small droplets obtained by shearing are poured into the liquid nitrogen of the molding device, and porous fiber composite microbial microspheres are obtained through freeze-drying. Among them, the small droplets will pass through a temperature control device before being poured into the liquid nitrogen. The temperature of the temperature control device is set to -45°C, and the freeze-drying time is 2 days. The morphology of the porous fiber composite microbial microspheres obtained in Example 32 is drop-shaped. The morphology obtained by optical microscope observation is shown in FIG. Figure 4 shown.

[0129] Application Example 1

[0130] Mice were divided into six groups (n=6 per group): a blank group, a model group, a probiotic group, a sterile microsphere group, a 5-ASA group (5-aminosalicylic acid, an anti-inflammatory drug), and a porous fiber composite microsphere group. The efficacy of the porous fiber composite microspheres in treating inflammatory bowel disease was tested using an acute IBD model. The experimental conditions are shown in Table 8. MPO activity was measured after the experiment.

[0131] Table 8. Experimental conditions of each group of mice

[0132]

[0133] See Figure 5 The figure below shows a comparison of MPO activity in mice. MPO can measure the activity of neutrophils and assist in determining the severity of inflammation; the lower the intensity, the weaker the inflammation. It can be seen that the MPO activity in the model group was significantly higher than that in the blank group. The MPO activity in the 5-ASA group and the probiotic group was similar, indicating a certain anti-inflammatory effect. However, the MPO activity in the porous fiber composite microbial microsphere group was closest to that of the blank group, demonstrating the best anti-inflammatory effect.

[0134] Application Example 2

[0135] Two groups of mice were given free access to 3.5% DSS solution (inflammation mice) and normal water (normal mice) for 7 days. On the 8th day, both groups of mice were fed 0.0139 g of porous fiber composite microbial microspheres labeled with Cy5.5. Six hours later, the hearts, lungs, livers, kidneys, spleens, stomachs, small intestines, and colons of the mice were harvested for in vivo fluorescence imaging. Figure 6 As shown, Figure 6 (a) is an inflammation mouse, Figure 6 (b) shows a normal mouse. A comparison shows that the stomach and colon of the inflamed mouse exhibit strong fluorescence, while only the stomach of the normal mouse exhibits fluorescence. This suggests that the hyaluronic acid in the porous fiber-composite microbial microspheres does not spread systemically, demonstrating good safety. Furthermore, the microspheres possess inflammation-specific adhesion, enabling targeted therapy.

[0136] In summary, the present application provides a porous fiber composite microbial microsphere and its application. The porous fiber composite microbial microsphere uses aldehyde-modified carboxymethyl cellulose (A-CMC) and hydrazide-functionalized hyaluronic acid (ADH-HA) with good biocompatibility as the microsphere skeleton to load probiotics. It not only has good cell compatibility, but also actively adheres to the wall in the intestinal fluid and has specific adhesion to the site of colon inflammation, which can achieve targeted treatment. Moreover, the number of active probiotics reaching the colon fluid has reached the minimum viable count recommended by the World Gastroenterology Organization (10 6 -10 7 The porous fiber composite microbial microspheres provided in this application are more effective than 5-ASA and ordinary probiotic solutions in treating inflammatory bowel disease.

[0137] It should be noted that the present application is not limited to the above-mentioned embodiments. The above-mentioned embodiments are merely examples, and any embodiments having substantially the same structure and effect as the technical concept within the scope of the present application are all included in the technical scope of the present application. In addition, without departing from the scope of the present application, any other embodiments that can be conceived by those skilled in the art and that combine some of the constituent elements in the embodiments are also included in the scope of the present application.

Claims

1. A porous fiber composite microbial microsphere, characterized in that: The porous fiber composite microbial microspheres have a porous structure skeleton composed of aldehyde-modified carboxymethyl cellulose and hydrazide-functionalized hyaluronic acid, and probiotics loaded on the skeleton. The surface of the skeleton of the porous fiber composite microbial microspheres is also attached with an ADH-HA film encapsulating the probiotics. The porous fiber composite microbial microspheres are obtained by shearing a composite fiber microsphere precursor liquid into droplets with a diameter in the range of 200-600 μm and freeze-drying the droplets. The composite fiber microsphere precursor solution is prepared by mixing an A-CMC suspension with a concentration of 0.9-1.3%, an ADH-HA solution with a concentration of 0.1-0.9%, and probiotics treated with glycerol.

2. The porous fiber composite microbial microspheres according to claim 1, characterized in that: The porous fiber composite microbial microspheres are hydrophilic and carry negative charges. When the porous fiber composite microbial microspheres are dispersed in an acidic solution, the microspheres shrink and their diameters decrease. When the microspheres are dispersed in an alkaline solution, the microspheres expand and their diameters increase.

3. The porous fiber composite microbial microspheres according to claim 1, characterized in that: The porous fiber composite microbial microspheres are used to treat inflammatory bowel disease; the porous fiber composite microbial microspheres do not adhere to the wall in gastric juice, but do adhere to the wall in intestinal juice or colonic juice; the porous fiber composite microbial microspheres have specific adhesion properties to the site of colon inflammation.

4. The porous fiber composite microbial microspheres according to claim 1, characterized in that: The optimal storage temperature of the porous fiber composite microbial microspheres is -20°C; the porous fiber composite microbial microspheres are spherical or drop-shaped.

5. The porous fiber composite microbial microspheres according to claim 1, characterized in that: The porous fiber composite microbial microspheres are prepared by the following steps: S1, adding sodium periodate to the CMC aqueous suspension, stirring at room temperature in the dark, then adding ethylene glycol to quench the unreacted periodate. After the reaction is completed, dialyzing with deionized water to obtain an A-CMC suspension, which is freeze-dried to obtain A-CMC; S2, adding oxalic acid dihydrazide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide to a hyaluronic acid solution to obtain a mixed solution, followed by reacting at room temperature for a period of time, adding hydrochloric acid solution dropwise during the reaction to maintain the pH of the mixed solution at 4.7-4.8, and when the pH value of the system no longer increases, adding sodium hydroxide solution dropwise to adjust the pH of the solution to 7 to terminate the reaction, followed by dialysis with deionized water and freeze-drying to obtain ADH-HA; S3, using the A-CMC obtained in step S1 to prepare an A-CMC suspension with a concentration of 0.9-1.3%, and using the ADH-HA obtained in step S2 to prepare an ADH-HA solution with a concentration of 0.1-0.9%; then, the A-CMC suspension and the ADH-HA solution are mixed and stirred for 30 minutes, and the probiotics treated with glycerol are added and uniformly dispersed to obtain a composite fiber microsphere precursor solution; S4, the composite fiber microsphere precursor obtained in step S4 is transferred to the raw material warehouse, the microfluidic device and the nitrogen-assisted device cooperate with each other, the microfluidic device controls the precursor to flow out of the fluid needle, and the nitrogen-assisted device shears the outflowing precursor into small droplets, and the small droplets are dripped into the liquid nitrogen of the molding device, and the porous fiber composite microbial microspheres are obtained after freeze-drying; the small droplets pass through a temperature control device before being dripped into the liquid nitrogen, and the temperature of the temperature control device is set to -50°C to 10°C.

6. The porous fiber composite microbial microspheres according to claim 5, characterized in that: The preparation steps of the porous fiber composite microbial microspheres also include: In step S1, the stirring time at room temperature in the dark is 6-8 hours, the molar ratio of NaIO4 to CMC is (0.5-1):1, and the concentration of the CMC aqueous suspension is 1-2 wt.%; and, In step S2, the mass ratio of ADH to HA and EDC is 1:(2-4):(0.1-0.2), the concentration of the HA solution is 0.5-2 wt.%, the concentration of the hydrochloric acid solution is 0.4-0.6 mol / L, and the concentration of the sodium hydroxide solution is 0.05-0.1 mol / L; and, In step S3, the volume ratio of the A-CMC suspension to the ADH-HA solution is (8-12):1; and In step S4, the microfluidic device and the nitrogen assist device cooperate with each other to shear the precursor liquid at a speed of 9-10 mL / h, and the nitrogen pressure is 35-65 KPa.

7. The porous fiber composite microbial microspheres according to claim 5, characterized in that: In step S4, treating the probiotics with glycerol specifically refers to dispersing the probiotics in glycerol, treating at 4° C. for 30-60 minutes, and then centrifuging to obtain glycerol-treated probiotics, wherein the glycerol concentration is 10-20%.

8. The porous fiber composite microbial microspheres according to claim 5, characterized in that: The method for preparing spherical porous fiber composite microbial microspheres also includes setting the temperature of the temperature control device to -10°C to 10°C in step S4; the method for preparing droplet-shaped porous fiber composite microbial microspheres also includes placing the composite fiber microsphere precursor liquid obtained in step S3 in a refrigerator until the viscosity of the precursor liquid reaches 3-300 Pa.s, and then transferring it to the raw material warehouse, as well as setting the microfluidic rate to 1mm / min-3mm / min and the temperature of the temperature control device to -50°C to -40°C.

9. The porous fiber composite microbial microspheres according to claim 5, characterized in that: Step S4 uses the following preparation device: including a microfluidic device for controlling the outflow rate of the precursor liquid, a nitrogen-assisted device that cooperates with the microfluidic device to blow off the precursor liquid, and a molding device for freeze-drying the blown-off precursor droplets; the microfluidic device includes a raw material warehouse and a liquid flow needle, and the inner diameter of the liquid flow needle is 250-350 μm; the nitrogen-assisted device is arranged below the raw material warehouse, including a hollow tube 1 connected to the nitrogen input end, a hollow tube 2 connected to the nitrogen output end, a hollow tube 3 arranged below the liquid flow needle and connected to the liquid flow needle, a hollow tube 4 connected to the molding device, and a four-way connection connecting hollow tubes 1, 2, 3, and 4; the molding device is arranged below the nitrogen-assisted device, and the molding device includes a temperature control device and liquid nitrogen.

10. A use of the porous fiber composite microbial microspheres according to any one of claims 1 to 9, characterized in that: The porous fiber composite microbial microspheres are used for treating inflammatory bowel disease.

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