Targeted enteric bacteria-loaded pellets based on selective permeable membrane and 3D-printed porous carrier

Abstract

The present application relates to a kind of based on selective permeable membrane and 3D printing porous carrier large intestine targeted bacteria-loaded pellets, belong to oral large intestine targeted drug delivery technical field, and large intestine targeted bacteria-loaded pellets are obtained by mixed bacteria body being wrapped by semi-permeable shell membrane as shell, the semi-permeable shell membrane is obtained by selective permeable membrane covering 3D printing porous carrier, and the 3D printing porous carrier is used as support skeleton.By mixed bacteria body being wrapped by semi-permeable shell membrane as shell, the semi-permeable shell membrane is obtained by selective permeable membrane covering 3D printing porous carrier, selective permeable membrane is evenly coated on the outer surface and pore inner wall of 3D printing porous carrier, and together form the shell with mechanical strength and large intestine targeted permeability, so that large intestine targeted bacteria-loaded pellets, release rate 3-4% in simulated gastric acid 2 hours, can effectively protect the safety of internal embedded flora through stomach, after entering intestine, release rate 94-96% in 4 hours in large intestine simulation liquid, and cumulative total release rate is as high as 97-99%.

Description

Technical Field

[0001] This invention belongs to the field of oral colon-targeted drug delivery technology, specifically relating to a colon-targeted bacterial microsphere based on a selectively permeable membrane and a 3D-printed porous carrier. Background Technology

[0002] Oral colon-targeted drug delivery refers to the use of colon-targeted bacterial pellets to enable oral drug formulations to resist the digestive environment of the stomach and small intestine, and to be released or activated only after reaching the large intestine, thereby exerting a therapeutic effect locally or systemically.

[0003] However, in diseases such as ulcerative colitis, the gastrointestinal environment of patients is completely different from that of healthy individuals. Their intestines are often accompanied by mucosal congestion and edema, increased inflammatory exudate, rapid intestinal transit, or physical narrowing. These pathological changes can seriously interfere with the normal passage of colon-targeted agents, their disintegration at the lesion site, and subsequent drug absorption. More importantly, in this disordered process, free bacterial cells in the human body, such as pathogenic or opportunistic pathogens, can easily invade the microspheres loaded with bacteria. The colonization of these exogenous bacteria will competitively occupy the microecological niche, change the microenvironment, and may produce harmful metabolites, thereby seriously affecting the survival rate and function of the therapeutic flora loaded inside the microspheres, ultimately leading to a significant reduction in the therapeutic effect. Summary of the Invention

[0004] The purpose of this invention is to provide a targeted E. coli microsphere based on a selectively permeable membrane and a 3D-printed porous carrier in order to solve the above-mentioned problems.

[0005] The present invention achieves the above objectives through the following technical solutions: This invention provides a coli-targeted bacterial microsphere based on a selectively permeable membrane and a 3D-printed porous carrier. The coli-targeted bacterial microsphere is obtained by encapsulating a mixed bacterial culture with a semi-permeable membrane as the shell. The semi-permeable membrane is obtained by coating a 3D-printed porous carrier with a selectively permeable membrane.

[0006] As a further optimization of the present invention, a 3D printed porous carrier is used as a support skeleton, which is made by 3D printing of printing paste.

[0007] As a further optimization of the present invention, the raw materials for preparing the printing paste, by weight, include: 15-20 parts HPMC E5, 2-4 parts microcrystalline cellulose, 3-5 parts 50% glycerol aqueous solution, 65-75 parts purified water, 0.5-1 parts titanium dioxide and 0.1 parts sodium benzoate.

[0008] As a further optimization of the present invention, the preparation process of the printing paste is as follows: (a) Add HPMC E5 and microcrystalline cellulose to purified water, stir evenly and let stand for 1-3 hours to obtain a pre-mixed solution. (ii) Add glycerol aqueous solution, titanium dioxide and sodium benzoate to the pre-mixed solution in sequence, heat to 40°C, stir at 1000-1200r / min for 1-2h, degas under vacuum for 20-45min, and let stand at 25°C for 12-16h to obtain printing paste.

[0009] As a further optimization of the present invention, the selectively permeable membrane is prepared by a casting solution, and the preparation process of the casting solution is as follows: (a) Add cellulose powder to an 18% sodium hydroxide aqueous solution and stir at 80-120 r / min for 10-25 min at 25℃ to obtain an alkali cellulose mixture; (ii) Add urea to the alkali cellulose mixture, cool to -10℃ and stir at 1000-1200r / min for 45-60min to obtain the casting solution.

[0010] As a further optimization of the present invention, the solid-liquid ratio of cellulose powder to 18% sodium hydroxide aqueous solution is 1:10 (g / mL), and the mass ratio of the alkali cellulose mixture to urea is 1:0.08.

[0011] As a further optimization of the present invention, the mixed bacterial culture is prepared by mixing Bifidobacterium longum, Lactobacillus acidophilus, Clostridium plasmidonum and Lactobacillus casei, and the mass ratio of each strain in the mixed bacterial culture is (2-3):(1-2):(1-2):(1-2).

[0012] This invention also provides a method for preparing E. coli-targeted bacterial microspheres based on selectively permeable membranes and 3D-printed porous carriers, comprising the following steps: S1, Printing the support skeleton: Add the printing paste to the 3D printer, the printing extrusion speed is 2-5 mm / s, the nozzle moving speed is 3-6 mm / s, the nozzle diameter is 0.3-0.5 mm, and print the cap frame and the body frame in sequence. After printing, dry them to a moisture content of 5-8% and use the cap frame and the body frame as the support skeleton. S2, Preparation of selectively permeable membrane: Immerse the support framework in the casting solution, and after 2-3 minutes, vertically lift the support framework at a constant speed of 1-2 mm / s. Hang it vertically for 30-60 seconds, and then transfer it to an environment of 30-35℃ and vacuum degree -0.06MPa to dry for 2-4 hours to obtain a pre-formed shell membrane. Immerse the pre-formed shell membrane in a 3% dilute sulfuric acid regeneration bath, let it stand at room temperature for 30 minutes, and then rinse it with deionized water until the pH of the effluent is 6.5-7.0. S3, Glycerylation treatment: Immerse the pre-made shell membrane after washing with water in step S3 in a 20% glycerol aqueous solution for 10-15 minutes. After taking it out, use filter paper to absorb the excess liquid on the surface and place it in an environment of 35-40℃ and vacuum degree -0.05MPa to dry for 4-6 hours to obtain a semi-permeable shell membrane. S4, Preparation of E. coli-targeted bacterial microspheres: Mix the mixed bacterial cells with the bacterial cell protectant evenly, and then freeze-dry them to prepare bacterial powder. Under aseptic conditions, the bacterial powder is filled into a semi-permeable membrane using a vacuum filling method to obtain E. coli-targeted bacterial microspheres.

[0013] As a further optimization of the present invention, the diameter of the cap frame is 1.0-1.5mm and the depth is 0.2-0.3mm; the diameter of the body frame is 0.9-1.4mm and the depth is 0.5-0.8mm.

[0014] As a further optimization of the present invention, the freeze-drying protectant is trehalose and skim milk, wherein the amount of trehalose added is 8-12% of the wet weight of the mixed bacterial cells, and the amount of skim milk added is 5-8% of the wet weight of the mixed bacterial cells.

[0015] The beneficial effects of this invention are as follows: This invention uses a semi-permeable membrane as a shell to encapsulate mixed bacterial cells. The semi-permeable membrane is obtained by coating a 3D-printed porous carrier with a selectively permeable membrane. The selectively permeable membrane is uniformly coated on the outer surface and inner wall of the pores of the 3D-printed porous carrier, together forming a shell that combines mechanical strength and colon-targeted permeability. This allows the colon-targeted bacterial pellets to release 3-4% of their contents in simulated gastric acid for 2 hours, effectively protecting the embedded bacteria as they safely pass through the stomach. After entering the intestines, the release rate in simulated colonic fluid is 94-96% in 4 hours, with a cumulative total release rate as high as 97-99%. When the colon-targeted bacterial microspheres enter the stomach, the gastric acid cannot dissolve them. Upon entering the small intestine, they resist degradation by digestive enzymes such as trypsin and lipase, and the bacterial powder is firmly locked in the lumen. When they enter the large intestine, the abundant symbiotic microorganisms in the colon, such as Bacteroides and Clostridium, secrete cellulase, which destroys the integrity of the colon-targeted bacterial microspheres, increasing the pore size until the structure collapses. At the same time, the micropore size of the colon-targeted bacterial microspheres is in the nanometer range, while the size of bacterial cells is in the micrometer range. The size of bacterial cells is much larger than the pore size of the membrane, so bacterial cells cannot enter. This design allows small molecules, such as oxygen, water, nutrients, metabolites, and intestinal enzymes, to slowly permeate, thereby maintaining the microenvironment required by the internal flora while achieving effective encapsulation and protection of the bacterial cells. Detailed Implementation

[0016] The present application will now be described in further detail. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0017] Unless otherwise specified, all methods used in this invention are conventional methods known to those skilled in the art, and all reagents and materials used are commercially available products.

[0018] Example 1 The preparation process of the printing paste is as follows: 15 parts HPMC E5 and 2 parts microcrystalline cellulose are added to 65 parts purified water, stirred evenly and allowed to stand for 1 hour to obtain a pre-mixed solution; 3 parts glycerol aqueous solution, 0.5 parts titanium dioxide and 0.1 parts sodium benzoate are added to the pre-mixed solution in sequence, the temperature is raised to 40℃, stirred at 1000 r / min for 1 hour, vacuum degassing for 20 minutes, and allowed to stand at 25℃ for 12 hours to obtain the printing paste; The selectively permeable membrane was prepared by casting solution, which was prepared as follows: cellulose powder was added to an 18% sodium hydroxide aqueous solution (the solid-liquid ratio of cellulose powder to 18% sodium hydroxide aqueous solution was 1:10 (g / mL)), and stirred at 80 r / min for 10 min at 25°C to obtain an alkali cellulose mixture; urea was added to the alkali cellulose mixture (the mass ratio of alkali cellulose mixture to urea was 1:0.08), and the mixture was cooled to -10°C and stirred at 1000 r / min for 45 min to obtain the casting solution. Printing the support skeleton: Add the printing paste to the 3D printer, print at an extrusion speed of 2 mm / s, a nozzle movement speed of 3 mm / s, and a nozzle diameter of 0.3 mm. Print the cap frame and the body frame in sequence (cap frame diameter: 1.0 mm, depth: 0.2 mm; body frame diameter: 0.9 mm, depth: 0.5 mm). After printing, dry them to a moisture content of 5%. Use the cap frame and body frame as the support skeleton. Selective permeability membrane preparation: The support framework was immersed in the casting solution, and after 2 minutes, it was vertically lifted out at a constant speed of 1 mm / s. It was then suspended vertically for 30 seconds and then transferred to an environment of 30°C and vacuum degree of -0.06 MPa to dry for 2 hours to obtain a pre-formed membrane. The pre-formed membrane was immersed in a 3% dilute sulfuric acid regeneration bath and allowed to stand at room temperature for 30 minutes. After removal, it was rinsed with deionized water until the pH of the effluent was 6.5. Glycerylation treatment: The pre-made shell membrane after washing with water in step S3 is immersed in a 20% glycerol aqueous solution for 10 min. After taking it out, excess liquid on the surface is absorbed with filter paper, and it is dried in an environment of 35℃ and vacuum degree -0.05MPa for 4 h to obtain a semi-permeable shell membrane. Preparation of coli-targeted bacterial microspheres: Bifidobacterium longum, Lactobacillus acidophilus, Clostridium plasmidonum, and Lactobacillus casei were mixed to obtain a mixed bacterial cell (the mass ratio of Bifidobacterium longum, Lactobacillus acidophilus, Clostridium plasmidonum, and Lactobacillus casei was 2:1:1:1). The mixed bacterial cell was mixed evenly with a cell protectant (the freeze-drying protectant was trehalose and skim milk, wherein the amount of trehalose added was 8% of the wet weight of the mixed bacterial cell, and the amount of skim milk added was 5% of the wet weight of the mixed bacterial cell). After freeze-drying protection, the bacterial powder was prepared. Under aseptic conditions, the bacterial powder was filled into a semi-permeable shell membrane using a vacuum filling method to obtain coli-targeted bacterial microspheres.

[0019] Example 2 The preparation process of the printing paste is as follows: 18 parts HPMC E5 and 3 parts microcrystalline cellulose are added to 70 parts purified water, stirred evenly and allowed to stand for 2 hours to obtain a pre-mixed solution; 4 parts glycerol aqueous solution, 0.8 parts titanium dioxide and 0.1 parts sodium benzoate are added to the pre-mixed solution in sequence, the temperature is raised to 40℃, stirred at 1100 r / min for 1.5 hours, vacuum degassing is performed for 30 minutes, and the mixture is allowed to stand at 25℃ for 14 hours to obtain the printing paste; The selectively permeable membrane was prepared by casting solution, which was prepared as follows: cellulose powder was added to an 18% sodium hydroxide aqueous solution (the solid-liquid ratio of cellulose powder to 18% sodium hydroxide aqueous solution was 1:10 (g / mL)), and stirred at 100 r / min for 18 min at 25°C to obtain an alkali cellulose mixture; urea was added to the alkali cellulose mixture (the mass ratio of alkali cellulose mixture to urea was 1:0.08), and the mixture was cooled to -10°C and stirred at 1100 r / min for 55 min to obtain the casting solution. Printing the support skeleton: Add the printing paste to the 3D printer, print at an extrusion speed of 3 mm / s, a nozzle movement speed of 5 mm / s, and a nozzle diameter of 0.4 mm. Print the cap frame and the body frame in sequence (cap frame diameter: 1.2 mm, depth: 0.2 mm; body frame diameter: 1.1 mm, depth: 0.5 mm). After printing, dry them to a moisture content of 7%. Use the cap frame and body frame as the support skeleton. Preparation of selectively permeable membrane: The support framework was immersed in the casting solution. After 2 minutes, the support framework was vertically lifted out at a constant speed of 1 mm / s and suspended vertically for 45 seconds. Then, it was transferred to an environment of 32°C and vacuum degree of -0.06 MPa to dry for 3 hours to obtain a pre-formed shell membrane. The pre-formed shell membrane was immersed in a 3% dilute sulfuric acid regeneration bath and allowed to stand at room temperature for 30 minutes. After removal, it was rinsed with deionized water until the pH of the effluent was 6.5. Glycerylation treatment: The pre-made shell membrane after washing with water in step S3 is immersed in a 20% glycerol aqueous solution for 12 minutes. After taking it out, excess liquid on the surface is absorbed with filter paper, and it is dried in an environment of 37°C and vacuum degree -0.05MPa for 5 hours to obtain a semi-permeable shell membrane. Preparation of coli-targeted bacterial microspheres: Bifidobacterium longum, Lactobacillus acidophilus, Clostridium plasmidonum, and Lactobacillus casei were mixed to obtain mixed bacterial cells (the mass ratio of Bifidobacterium longum, Lactobacillus acidophilus, Clostridium plasmidonum, and Lactobacillus casei was 2:2:1:1). The mixed cells were then mixed evenly with a cell protectant (the freeze-drying protectant was trehalose and skim milk, wherein the amount of trehalose added was 10% of the wet weight of the mixed bacterial cells, and the amount of skim milk added was 6% of the wet weight of the mixed bacterial cells). After freeze-drying protection, the bacterial powder was prepared. Under aseptic conditions, the bacterial powder was filled into a semi-permeable shell membrane using a vacuum filling method to obtain coli-targeted bacterial microspheres.

[0020] Example 3 The preparation process of the printing paste is as follows: 20 parts of HPMC E5 and 4 parts of microcrystalline cellulose are added to 75 parts of purified water, stirred evenly and allowed to stand for 3 hours to obtain a pre-mixed solution; 5 parts of glycerol aqueous solution, 1 part of titanium dioxide and 0.1 parts of sodium benzoate are added to the pre-mixed solution in sequence, the temperature is raised to 40℃, stirred at 1200r / min for 2 hours, vacuum degassing is performed for 45 minutes, and the mixture is allowed to stand at 25℃ for 16 hours to obtain the printing paste; The selectively permeable membrane was prepared by casting solution, which was prepared as follows: cellulose powder was added to an 18% sodium hydroxide aqueous solution (the solid-liquid ratio of cellulose powder to 18% sodium hydroxide aqueous solution was 1:10 (g / mL)), and stirred at 120 r / min for 25 min at 25℃ to obtain an alkali cellulose mixture; urea was added to the alkali cellulose mixture (the mass ratio of alkali cellulose mixture to urea was 1:0.08), and the mixture was cooled to -10℃ and stirred at 1200 r / min for 60 min to obtain the casting solution. Printing the support skeleton: Add the printing paste to the 3D printer, print at an extrusion speed of 5 mm / s, a nozzle movement speed of 6 mm / s, and a nozzle diameter of 0.5 mm. Print the cap frame and the body frame in sequence (cap frame diameter: 1.5 mm, depth: 0.3 mm; body frame diameter: 1.4 mm, depth: 0.8 mm). After printing, dry them to a moisture content of 8%, and use the cap frame and body frame as the support skeleton. Selective permeability membrane preparation: The support framework was immersed in the casting solution, and after 3 min, it was vertically lifted out at a constant speed of 2 mm / s and suspended vertically for 60 s. Then, it was transferred to an environment of 35℃ and vacuum degree -0.06 MPa to dry for 4 h to obtain a pre-formed shell membrane. The pre-formed shell membrane was immersed in a 3% dilute sulfuric acid regeneration bath and allowed to stand at room temperature for 30 min. After removal, it was rinsed with deionized water until the pH of the effluent was 7.0. Glycerylation treatment: The pre-made shell membrane after washing with water in step S3 is immersed in a 20% glycerol aqueous solution for 15 minutes. After taking it out, excess liquid on the surface is absorbed with filter paper, and it is dried in an environment of 40°C and vacuum degree -0.05MPa for 6 hours to obtain a semi-permeable shell membrane. Preparation of coli-targeted bacterial microspheres: Bifidobacterium longum, Lactobacillus acidophilus, Clostridium plasmidonum, and Lactobacillus casei were mixed to obtain a mixed bacterial cell (the mass ratio of Bifidobacterium longum, Lactobacillus acidophilus, Clostridium plasmidonum, and Lactobacillus casei was 3:2:2:2). The mixed bacterial cell was mixed evenly with a cell protectant (the freeze-drying protectant was trehalose and skim milk, wherein the amount of trehalose added was 12% of the wet weight of the mixed bacterial cell, and the amount of skim milk added was 8% of the wet weight of the mixed bacterial cell). After freeze-drying protection, the bacterial powder was prepared. Under aseptic conditions, the bacterial powder was filled into a semi-permeable shell membrane using a vacuum filling method to obtain coli-targeted bacterial microspheres.

[0021] Comparative Example 1 The preparation process of the printing paste is as follows: 18 parts HPMC E5 and 3 parts microcrystalline cellulose are added to 70 parts purified water, stirred evenly and allowed to stand for 2 hours to obtain a pre-mixed solution; 4 parts glycerol aqueous solution, 0.8 parts titanium dioxide and 0.1 parts sodium benzoate are added to the pre-mixed solution in sequence, the temperature is raised to 40℃, stirred at 1100 r / min for 1.5 hours, vacuum degassing is performed for 30 minutes, and the mixture is allowed to stand at 25℃ for 14 hours to obtain the printing paste; The selectively permeable membrane was prepared by casting solution, which was prepared as follows: cellulose powder was added to an 18% sodium hydroxide aqueous solution (the solid-liquid ratio of cellulose powder to 18% sodium hydroxide aqueous solution was 1:10 (g / mL)), and stirred at 100 r / min for 18 min at 25°C to obtain an alkali cellulose mixture; urea was added to the alkali cellulose mixture (the mass ratio of alkali cellulose mixture to urea was 1:0.08), and the mixture was cooled to -10°C and stirred at 1100 r / min for 55 min to obtain the casting solution. Printing the support skeleton: Add the printing paste to the 3D printer, print at an extrusion speed of 3 mm / s, a nozzle movement speed of 5 mm / s, and a nozzle diameter of 0.4 mm. Print the cap frame and the body frame in sequence (cap frame diameter: 1.2 mm, depth: 0.2 mm; body frame diameter: 1.1 mm, depth: 0.5 mm). After printing, dry them to a moisture content of 7%. Use the cap frame and body frame as the support skeleton. Preparation of selectively permeable membrane: The support framework was immersed in the casting solution. After 2 minutes, the support framework was vertically lifted out at a constant speed of 1 mm / s and suspended vertically for 45 seconds. Then, it was transferred to an environment of 32°C and vacuum degree of -0.06 MPa to dry for 3 hours to obtain a pre-formed shell membrane. The pre-formed shell membrane was immersed in a 3% dilute sulfuric acid regeneration bath and allowed to stand at room temperature for 30 minutes. After removal, it was rinsed with deionized water until the pH of the effluent was 6.5. Glycerylation treatment: The pre-made shell membrane after washing with water in step S3 is immersed in a 20% glycerol aqueous solution for 12 minutes. After taking it out, excess liquid on the surface is absorbed with filter paper, and it is dried in an environment of 37°C and vacuum degree -0.05MPa for 5 hours to obtain a semi-permeable shell membrane. Preparation of coli-targeted bacterial microspheres: Bifidobacterium longum, Lactobacillus acidophilus, Clostridium plasmidonum, and Lactobacillus casei were mixed to obtain a mixed bacterial cell (the mass ratio of Bifidobacterium longum, Lactobacillus acidophilus, Clostridium plasmidonum, and Lactobacillus casei was 2:2:1:1). The mixed bacterial cell was then mixed evenly with a cell protectant (the freeze-drying protectant was sucrose and skim milk, wherein the amount of sucrose added was 10% of the wet weight of the mixed bacterial cell, and the amount of skim milk added was 6% of the wet weight of the mixed bacterial cell). After freeze-drying protection, the bacterial powder was prepared. Under aseptic conditions, the bacterial powder was filled into a semi-permeable shell membrane using a vacuum filling method to obtain coli-targeted bacterial microspheres.

[0022] Comparative Example 2 The preparation process of the printing paste is as follows: 18 parts HPMC E5 and 3 parts microcrystalline cellulose are added to 70 parts purified water, stirred evenly and allowed to stand for 2 hours to obtain a pre-mixed solution; 4 parts glycerol aqueous solution, 0.8 parts titanium dioxide and 0.1 parts sodium benzoate are added to the pre-mixed solution in sequence, the temperature is raised to 40℃, stirred at 1100 r / min for 1.5 hours, vacuum degassing is performed for 30 minutes, and the mixture is allowed to stand at 25℃ for 14 hours to obtain the printing paste; The selectively permeable membrane was prepared by casting solution, which was prepared as follows: cellulose powder was added to an 18% sodium hydroxide aqueous solution (the solid-liquid ratio of cellulose powder to 18% sodium hydroxide aqueous solution was 1:10 (g / mL)), and stirred at 100 r / min for 18 min at 25°C to obtain an alkali cellulose mixture; urea was added to the alkali cellulose mixture (the mass ratio of alkali cellulose mixture to urea was 1:0.08), and the mixture was cooled to -10°C and stirred at 1100 r / min for 55 min to obtain the casting solution. Printing the support skeleton: Add the printing paste to the 3D printer, print at an extrusion speed of 3 mm / s, a nozzle movement speed of 5 mm / s, and a nozzle diameter of 0.4 mm. Print the cap frame and the body frame in sequence (cap frame diameter: 1.2 mm, depth: 0.2 mm; body frame diameter: 1.1 mm, depth: 0.5 mm). After printing, dry them to a moisture content of 7%. Use the cap frame and body frame as the support skeleton. Preparation of selectively permeable membrane: The support framework was immersed in the casting solution. After 2 minutes, the support framework was vertically lifted out at a constant speed of 1 mm / s and suspended vertically for 45 seconds. Then, it was transferred to an environment of 32°C and vacuum degree of -0.06 MPa to dry for 3 hours to obtain a pre-formed shell membrane. The pre-formed shell membrane was immersed in a 3% dilute sulfuric acid regeneration bath and allowed to stand at room temperature for 30 minutes. After removal, it was rinsed with deionized water until the pH of the effluent was 6.5. Glycerylation treatment: The pre-made shell membrane after washing with water in step S3 is immersed in a 20% glycerol aqueous solution for 12 minutes. After taking it out, excess liquid on the surface is absorbed with filter paper, and it is dried in an environment of 37°C and vacuum degree -0.05MPa for 5 hours to obtain a semi-permeable shell membrane. Preparation of coli-targeted bacterial microspheres: Bifidobacterium longum, Lactobacillus acidophilus, Clostridium plasmidonum, and Lactobacillus casei were mixed to obtain a mixed bacterial cell (the mass ratio of Bifidobacterium longum, Lactobacillus acidophilus, Clostridium plasmidonum, and Lactobacillus casei was 2:2:1:1). The mixed bacterial cell was mixed evenly with a bacterial cell protectant (the freeze-drying protectant was trehalose and gelatin, wherein the amount of trehalose added was 10% of the wet weight of the mixed bacterial cell, and the amount of gelatin added was 6% of the wet weight of the mixed bacterial cell). After freeze-drying protection, the bacterial powder was prepared. Under aseptic conditions, the bacterial powder was filled into a semi-permeable shell membrane using a vacuum filling method to obtain coli-targeted bacterial microspheres.

[0023] Comparative Example 3 The preparation process of the printing paste is as follows: 18 parts HPMC E5 and 3 parts microcrystalline cellulose are added to 70 parts purified water, stirred evenly and allowed to stand for 2 hours to obtain a pre-mixed solution; 4 parts glycerol aqueous solution, 0.8 parts titanium dioxide and 0.1 parts sodium benzoate are added to the pre-mixed solution in sequence, the temperature is raised to 40℃, stirred at 1100 r / min for 1.5 hours, vacuum degassing is performed for 30 minutes, and the mixture is allowed to stand at 25℃ for 14 hours to obtain the printing paste; The selectively permeable membrane was prepared by casting solution, which was prepared as follows: cellulose powder was added to an 18% sodium hydroxide aqueous solution (the solid-liquid ratio of cellulose powder to 18% sodium hydroxide aqueous solution was 1:10 (g / mL)), and stirred at 100 r / min for 18 min at 25°C to obtain an alkali cellulose mixture; urea was added to the alkali cellulose mixture (the mass ratio of alkali cellulose mixture to urea was 1:0.08), and the mixture was cooled to -10°C and stirred at 1100 r / min for 55 min to obtain the casting solution. Printing the support skeleton: Add the printing paste to the 3D printer, print at an extrusion speed of 3 mm / s, a nozzle movement speed of 5 mm / s, and a nozzle diameter of 0.4 mm. Print the cap frame and the body frame in sequence (cap frame diameter: 1.2 mm, depth: 0.2 mm; body frame diameter: 1.1 mm, depth: 0.5 mm). After printing, dry them to a moisture content of 7%. Use the cap frame and body frame as the support skeleton. Selective permeable membrane preparation: The support skeleton was immersed in the casting solution. After 2 minutes, the support skeleton was vertically lifted out at a constant speed of 1 mm / s and suspended vertically for 45 seconds. Then, it was transferred to an environment of 32°C and vacuum degree -0.06 MPa to dry for 3 hours to obtain the pre-made shell membrane. Glycerylation treatment: The pre-made shell membrane after washing with water in step S3 is immersed in a 20% glycerol aqueous solution for 12 minutes. After taking it out, excess liquid on the surface is absorbed with filter paper, and it is dried in an environment of 37°C and vacuum degree -0.05MPa for 5 hours to obtain a semi-permeable shell membrane. Preparation of coli-targeted bacterial microspheres: Bifidobacterium longum, Lactobacillus acidophilus, Clostridium plasmidonum, and Lactobacillus casei were mixed to obtain mixed bacterial cells (the mass ratio of Bifidobacterium longum, Lactobacillus acidophilus, Clostridium plasmidonum, and Lactobacillus casei was 2:2:1:1). The mixed cells were then mixed evenly with a cell protectant (the freeze-drying protectant was trehalose and skim milk, wherein the amount of trehalose added was 10% of the wet weight of the mixed bacterial cells, and the amount of skim milk added was 6% of the wet weight of the mixed bacterial cells). After freeze-drying protection, the bacterial powder was prepared. Under aseptic conditions, the bacterial powder was filled into a semi-permeable shell membrane using a vacuum filling method to obtain coli-targeted bacterial microspheres.

[0024] Performance testing (i) The release performance of the Escherichia coli targeted bacterial microspheres prepared by the methods of Examples 1-3 and Comparative Example 3 was tested according to the dissolution and release determination method of the General Chapter 0931 of Part IV of the Chinese Pharmacopoeia 2025. The test results are shown in Table 1.

[0025] Table 1

[0026] As shown in Table 1, the coli-targeted microspheres prepared using the methods of Examples 1-3 had a release rate of 3-4% in simulated gastric acid for 2 hours, effectively protecting the internally embedded bacteria as they safely passed through the stomach. After entering the intestine, the release rate in simulated coliform fluid was 94-96% in 4 hours, with a cumulative total release rate as high as 97-99%. In contrast, although the coli-targeted microspheres prepared using the method of Comparative Example 3 also had a low release rate in the stomach, their release rate in the coliform environment was only 56%, with a cumulative release of 58%. This indicates that their encapsulation system did not completely disintegrate and released slowly in the intestine, failing to achieve effective delivery of the bacteria.

[0027] (ii) The coli-targeted bacterial microspheres prepared by the methods of Example 2 and Comparative Examples 1-2 were stored, and the survival rate of mixed bacteria in the coli-targeted bacterial microspheres during storage was calculated. The calculation results are shown in Table 2.

[0028] Table 2

[0029] As can be seen from Table 2, the cell survival rate of Example 2 was the highest regardless of the storage temperature of 4℃ or 37℃. In particular, after 6 months of storage, the survival rate of Example 2 was still 76% at 4℃ and 68% at 37℃, which was significantly better than that of Comparative Examples 1-2.

[0030] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A type of E. coli-targeted bacterial microsphere based on a selectively permeable membrane and a 3D-printed porous carrier, characterized in that, The coli-targeted bacterial microspheres are prepared by encapsulating a mixed bacterial culture with a semi-permeable membrane as the shell, and the semi-permeable membrane is prepared by coating a 3D-printed porous carrier with a selectively permeable membrane.

2. The *E. coli*-targeted bacterial microspheres based on a selectively permeable membrane and a 3D-printed porous carrier according to claim 1, characterized in that... The 3D-printed porous carrier serves as a support skeleton, which is made by 3D printing printing paste.

3. The *E. coli*-targeted bacterial microspheres based on a selectively permeable membrane and a 3D-printed porous carrier according to claim 2, characterized in that... The raw materials for preparing the printing paste, by weight, include: 15-20 parts HPMC E5, 2-4 parts microcrystalline cellulose, 3-5 parts 50% glycerol aqueous solution, 65-75 parts purified water, 0.5-1 parts titanium dioxide and 0.1 parts sodium benzoate.

4. The *E. coli*-targeted bacterial microspheres based on a selectively permeable membrane and a 3D-printed porous carrier according to claim 3, characterized in that... The preparation process of the printing paste is as follows: (a) Add HPMC E5 and microcrystalline cellulose to purified water, stir evenly and let stand for 1-3 hours to obtain a pre-mixed solution. (ii) Add glycerol aqueous solution, titanium dioxide and sodium benzoate to the pre-mixed solution in sequence, heat to 40°C, stir at 1000-1200r / min for 1-2h, degas under vacuum for 20-45min, and let stand at 25°C for 12-16h to obtain printing paste.

5. The *E. coli*-targeted bacterial microspheres based on a selectively permeable membrane and a 3D-printed porous carrier according to claim 1, characterized in that... The selectively permeable membrane is prepared by a casting solution, and the preparation process of the casting solution is as follows: (a) Add cellulose powder to an 18% sodium hydroxide aqueous solution and stir at 80-120 r / min for 10-25 min at 25℃ to obtain an alkali cellulose mixture; (ii) Add urea to the alkali cellulose mixture, cool to -10℃ and stir at 1000-1200r / min for 45-60min to obtain the casting solution.

6. The *E. coli*-targeted bacterial microspheres based on a selectively permeable membrane and a 3D-printed porous carrier according to claim 5, characterized in that... The solid-liquid ratio of the cellulose powder to the 18% sodium hydroxide aqueous solution is 1:10 (g / mL), and the mass ratio of the alkali cellulose mixture to urea is 1:0.

08.

7. The *E. coli*-targeted bacterial microspheres based on a selectively permeable membrane and a 3D-printed porous carrier according to claim 1, characterized in that... The mixed bacterial culture is prepared by mixing Bifidobacterium longum, Lactobacillus acidophilus, Clostridium plasmidonum and Lactobacillus casei, and the mass ratio of each strain in the mixed bacterial culture is (2-3):(1-2):(1-2):(1-2).

8. A method for preparing *E. coli*-targeted bacterial microspheres based on a selectively permeable membrane and a 3D-printed porous carrier as described in any one of claims 1-7, characterized in that... Includes the following steps: S1, Printing the support skeleton: Add the printing paste to the 3D printer, the printing extrusion speed is 2-5 mm / s, the nozzle moving speed is 3-6 mm / s, the nozzle diameter is 0.3-0.5 mm, and print the cap frame and the body frame in sequence. After printing, dry them to a moisture content of 5-8% and use the cap frame and the body frame as the support skeleton. S2, Preparation of selectively permeable membrane: Immerse the support framework in the casting solution, and after 2-3 minutes, vertically lift the support framework at a constant speed of 1-2 mm / s. Hang it vertically for 30-60 seconds, and then transfer it to an environment of 30-35℃ and vacuum degree -0.06MPa to dry for 2-4 hours to obtain a pre-formed shell membrane. Immerse the pre-formed shell membrane in a 3% dilute sulfuric acid regeneration bath, let it stand at room temperature for 30 minutes, and then rinse it with deionized water until the pH of the effluent is 6.5-7.

0. S3, Glycerylation treatment: Immerse the pre-made shell membrane after washing with water in step S3 in a 20% glycerol aqueous solution for 10-15 minutes. After taking it out, use filter paper to absorb the excess liquid on the surface and place it in an environment of 35-40℃ and vacuum degree -0.05MPa to dry for 4-6 hours to obtain a semi-permeable shell membrane. S4, Preparation of E. coli-targeted bacterial microspheres: Mix the mixed bacterial cells with the bacterial cell protectant evenly, and then freeze-dry them to prepare bacterial powder. Under aseptic conditions, the bacterial powder is filled into a semi-permeable membrane using a vacuum filling method to obtain E. coli-targeted bacterial microspheres.

9. The method for preparing E. coli-targeted bacterial microspheres based on a selectively permeable membrane and a 3D-printed porous carrier according to claim 8, characterized in that, The diameter of the cap frame is 1.0-1.5 mm and the depth is 0.2-0.3 mm; the diameter of the body frame is 0.9-1.4 mm and the depth is 0.5-0.8 mm.

10. The method for preparing E. coli-targeted bacterial microspheres based on a selectively permeable membrane and a 3D-printed porous carrier according to claim 8, characterized in that, The freeze-drying protectant is trehalose and skim milk, wherein the amount of trehalose added is 8-12% of the wet weight of the mixed bacterial cells, and the amount of skim milk added is 5-8% of the wet weight of the mixed bacterial cells.

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