Montmorillonite-probiotic microsphere preparation as well as preparation method and application thereof

Abstract

The invention relates to a montmorillonite-probiotic microsphere preparation as well as a preparation method and application thereof, the microsphere preparation comprises montmorillonite, a probiotic biofilm and functional small molecules, and microspheres of the microsphere preparation are prepared by crosslinking sodium alginate in a collection solution based on a microfluidic technology. The microsphere preparation is mainly used for oral delivery of montmorillonite-probiotic biofilms and functional small molecules, so as to treat intestinal diseases. Compared with the prior art, the microsphere preparation provided by the invention has the advantages of gastric acid invasion resistance, intestinal responsive release and the like, and can effectively treat intestinal inflammation. In addition, the probiotic biofilm wrapped by the microspheres can enhance the interaction with the intestinal mucosa and prolong the residence time in the intestinal tract. Meanwhile, the used functional small molecules have excellent anti-inflammatory and intestinal barrier repairing effects, and can cooperate with the probiotics to regulate and control intestinal flora, so that the intestinal health is further improved.

Description

Technical Field

[0001] The present invention relates to the technical field of oral probiotic preparations, and in particular to a montmorillonite-probiotic microsphere preparation and a preparation method and application thereof. Background Art

[0002] Inflammatory bowel disease (IBD) is a type of chronic inflammatory gastrointestinal disease, including Crohn's disease and ulcerative colitis. These diseases often lead to intestinal mucosal inflammation, impaired intestinal barrier function, and intestinal flora imbalance. Montmorillonite, as a natural layered silicate mineral, has shown significant efficacy in the treatment of diarrhea caused by IBD due to its excellent pathogen adsorption capacity and intestinal mucosal protection. In addition, montmorillonite is mainly mined from natural bentonite mines and has high safety. The harmful substances it adsorbs can be excreted from the body with gastrointestinal peristalsis, thereby reducing the risk of systemic side effects. However, the mechanism of action of montmorillonite is mainly limited to symptom relief and local protection, and the root cause of IBD has not yet been solved.

[0003] Oral probiotics can provide the possibility of systemic treatment of IBD by regulating host immune response, producing anti-inflammatory substances, strengthening intestinal barrier and regulating intestinal flora. In order to overcome the therapeutic limitations of montmorillonite, immobilizing probiotics on the surface of montmorillonite is a potential solution. Montmorillonite has excellent cation exchange properties. The cations between its layers (such as Ca 2+ , Na + ) easily reacts with hydrogen ions (H + ) exchange, which changes the charge on the surface of montmorillonite from negative to positive, helping to enhance the electrostatic interaction with probiotics, thereby promoting the formation of biofilms on the surface of probiotics. The biofilm of probiotics is composed of probiotics and their secreted extracellular polymers, which provides a good attachment surface for probiotics and enhances their adhesion ability in the intestine. Therefore, this method can also effectively overcome the problems of insufficient adhesion ability and short retention time of probiotics caused by rapid gastrointestinal transit and mucosal damage at the site of inflammation.

[0004] Chinese patent CN 112972503 A discloses a preparation method and application of a probiotic system, which combines probiotics in a living organism with an inorganic material system in vitro, and forms a composite microsphere of "bacteria-inorganic material-biofilm" by mixed culture, wherein the inorganic material includes montmorillonite. Although the prepared microspheres can be well adsorbed on the inner surface of the host's intestine and provide a safe and reliable environment for the stable growth and reproduction of probiotics, the biofilm has limited gastric acid resistance, and a large number of bacteria will still be inactivated when they are orally taken to the stomach.

[0005] Chinese patent CN 117462513 A discloses a colon-targeted polymer microsphere and its preparation method and application, wherein the colon-targeted polymer microsphere includes a microsphere wall material and a microsphere core material; wherein the raw materials of the wall material include methacrylated dextran and methacrylated tannic acid, and the raw materials of the microsphere core material include probiotics and postbiotics. The method is based on droplet microfluidics technology, and generates monodisperse polymer microspheres with uniform particle size encapsulating probiotics and postbiotics through a simple, rapid and spatiotemporally controllable photocrosslinking reaction. The polymer microsphere has the functions of resisting gastrointestinal environmental invasion, colon-targeted degradation and intestinal mucosal adhesion, and can efficiently deliver probiotics and postbiotics for the treatment of intestinal diseases. However, the colon-targeted polymer microsphere is prepared by crosslinking with oil-in-water droplets as templates. The oil phase is not conducive to the maintenance of bacterial activity, and after the preparation is completed, it needs to be repeatedly washed to ensure the effective removal of the oil phase, resulting in a more complicated overall process. Summary of the invention

[0006] The purpose of the present invention is to provide a montmorillonite-probiotic microsphere preparation and a preparation method and application thereof, so as to systematically and efficiently treat IBD.

[0007] The purpose of the present invention can be achieved by the following technical solutions:

[0008] The invention provides a montmorillonite-probiotic microsphere preparation, which comprises montmorillonite, a probiotic biofilm and functional small molecules.

[0009] Preferably, the microsphere preparation is a microsphere composed of montmorillonite, probiotic biofilm, functional small molecules and microsphere wall material, wherein the probiotic biofilm is attached to the surface of montmorillonite, and the functional small molecules are dispersed between montmorillonite and the microsphere wall material.

[0010] Further preferably, the microsphere wall material is prepared by cross-linking sodium alginate in a collection solution, and the montmorillonite, probiotic biofilm and functional small molecules are encapsulated in the microsphere wall material prepared from sodium alginate.

[0011] Preferably, the probiotics are selected from one or more of lactobacillus, bifidobacterium, and Clostridium butyricum.

[0012] Preferably, the functional small molecule is selected from one or more of indole-3-propionic acid, propionic acid, butyric acid, lipoteichoic acid, and peptidoglycan.

[0013] The montmorillonite-probiotic microsphere preparation provided by the present invention can overcome the limitations of the action of montmorillonite and realistic factors such as low intestinal adhesion of probiotics, susceptibility to environmental attacks and poor single efficacy, and develops a microsphere preparation that simultaneously encapsulates montmorillonite-probiotic biofilm and functional small molecules, and endows it with the characteristics of resistance to gastric acid and intestinal responsive release.

[0014] In a second aspect, the present invention further provides a method for preparing the above-mentioned montmorillonite-probiotic microsphere preparation, comprising the following steps:

[0015] S1, dispersing the sterilized montmorillonite particles in a probiotic culture solution in a logarithmic growth phase, and culturing at 28-40° C. for 36-60 hours to obtain a montmorillonite-probiotic biofilm;

[0016] S2, dispersing montmorillonite-probiotic biofilm and functional small molecules in sodium alginate solution, mixing them evenly and injecting them into the microfluidic chip channel, generating spray droplets under the action of static voltage, and cross-linking into microsphere preparations in the collected liquid.

[0017] Preferably, in step S1, the concentration of the montmorillonite particles dispersed in the probiotic culture solution is 5 to 20 mg / mL.

[0018] Preferably, in step S1, the probiotic culture solution in the logarithmic growth phase refers to OD 600 Probiotic culture solution with a value of 0.4 to 0.5.

[0019] Preferably, in step S1, the culturing refers to anaerobic culturing.

[0020] Preferably, in step S1, the montmorillonite-probiotic biofilm is further washed three times with a sterile PBS solution.

[0021] Preferably, in step S1, the sterilized montmorillonite particles refer to montmorillonite particles sterilized by ultraviolet irradiation for 0.5 to 1 h.

[0022] Preferably, in step S2, the concentration of the montmorillonite-probiotic biofilm dispersed in the sodium alginate solution is 10-40 mg / mL; the concentration of the functional small molecules dispersed in the sodium alginate solution is 0.5-2 mg / mL.

[0023] Preferably, in step S2, the concentration of the sodium alginate solution is 1-3% (W / V).

[0024] Further preferably, in step S2, the solvent of the sodium alginate solution is PBS buffer.

[0025] Preferably, in step S2, the static voltage is 4-8 kV.

[0026] Preferably, in step S2, the collection solution is selected from one or more of calcium chloride solution, barium chloride solution and aluminum chloride solution.

[0027] Further preferably, the solvent of the calcium chloride solution, barium chloride solution and aluminum chloride solution is sterile water, and sterile water refers to water that has been treated to remove all forms of microorganisms such as bacteria, viruses, and microbial spores.

[0028] Preferably, in step S2, the concentration of the collected solution is 1-3% (W / V).

[0029] Preferably, in step S2, the cross-linking time is 1 to 5 minutes.

[0030] Preferably, the liquid flow rate in the microfluidic chip channel is 3 to 6 mL / h.

[0031] In a third aspect, the present invention further provides a use of the above-mentioned montmorillonite-probiotic microsphere preparation in the preparation of an oral live probiotic preparation for treating intestinal diseases.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] (1) The present invention directly uses montmorillonite as a carrier of probiotics to form a montmorillonite-probiotic biofilm complex, and then disperses the montmorillonite-probiotic biofilm complex and functional small molecules in a sodium alginate solution, and cross-links in the collected solution to prepare a microsphere preparation. The microsphere preparation is mainly used for oral delivery of montmorillonite-probiotic biofilm and functional small molecules, has the advantages of resisting gastric acid invasion and intestinal responsive release, and can effectively treat intestinal inflammation.

[0034] (2) The montmorillonite-probiotic microsphere preparation of the present invention uses montmorillonite with cation exchange capacity, which can promote the formation of biofilm by acid-producing probiotics.

[0035] (3) The montmorillonite-probiotic microsphere preparation of the present invention, the montmorillonite-probiotic biofilm used can enhance the adhesion of the intestinal mucosa, thereby prolonging the retention time of probiotics in the intestine, and to a certain extent enhance the ability of probiotics to resist the adverse environment in the body.

[0036] (4) The montmorillonite-probiotic microsphere preparation of the present invention is prepared by cross-linking sodium alginate in the collection solution. The sodium alginate used has good biocompatibility, can protect the probiotics from gastric acid invasion, and is responsively released under the intestinal environment.

[0037] (5) The montmorillonite-probiotic microsphere preparation of the present invention is prepared by combining sodium alginate and Ca 2+ The preparation is obtained by cross-linking in an aqueous phase, the preparation process is simple, and it is beneficial to maintaining bacterial activity.

[0038] (6) The montmorillonite-probiotic microsphere preparation of the present invention can synergistically encapsulate functional small molecules to solve the problem of poor therapeutic efficacy of a single probiotic.

[0039] (7) The preparation method of the montmorillonite-probiotic microsphere preparation of the present invention is based on microfluidic electrospray technology, which has a simple preparation process, high flux, fast cross-linking speed, and the microsphere size is uniform and controllable with excellent monodispersity. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The figure is a flow chart of the preparation of montmorillonite-probiotic microsphere preparation;

[0041] Figure 2 This is a scanning electron microscope photo of the biofilm formed by Lactobacillus acidophilus on the surface of montmorillonite (B in the figure is a partial enlarged view);

[0042] Figure 3 Optical microscope photos of montmorillonite-Lactobacillus acidophilus biofilm and indole-3-propionic acid microsphere preparation;

[0043] Figure 4 Scanning electron micrographs of montmorillonite-Lactobacillus acidophilus biofilm and indole-3-propionic acid microsphere preparation. DETAILED DESCRIPTION

[0044] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0045] Unless otherwise specified, the reagents, methods, instruments and equipment used in the present invention are conventional reagents, methods, instruments and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0046] The invention provides a montmorillonite-probiotic microsphere preparation, which is a microsphere composed of montmorillonite, a probiotic biofilm, a functional small molecule and a microsphere wall material, wherein the probiotic biofilm is attached to the surface of the montmorillonite, the functional small molecule is dispersed between the montmorillonite and the microsphere wall material, the microsphere wall material is prepared by cross-linking sodium alginate in a collection liquid, the montmorillonite, the probiotic biofilm and the functional small molecule are encapsulated in the microsphere wall material prepared by sodium alginate; the probiotic is selected from one or more of lactobacillus, bifidobacterium and clostridium butyricum, and the functional small molecule is selected from one or more of indole-3-propionic acid, propionic acid, butyric acid, lipoteichoic acid and peptidoglycan.

[0047] like Figure 1 As shown, the above-mentioned montmorillonite-probiotic microsphere preparation is obtained by the following preparation method:

[0048] S1, dispersing the montmorillonite particles sterilized by ultraviolet irradiation in a probiotic culture solution in a logarithmic growth phase, anaerobic culture at 28-40°C for 36-60 hours to obtain a montmorillonite-probiotic biofilm, and washing it three times with a sterile PBS solution. The concentration of the montmorillonite particles dispersed in the probiotic culture solution is 5-20 mg / mL.

[0049] S2, dispersing montmorillonite-probiotic biofilm and functional small molecules in sodium alginate solution, mixing them evenly and injecting them into the microfluidic chip channel, generating spray droplets under the action of 4-8kV static voltage, and cross-linking them in the collecting liquid for 1-5 minutes to prepare a microsphere preparation. Among them, the concentration of montmorillonite-probiotic biofilm dispersed in sodium alginate solution is 10-40mg / mL; the concentration of functional small molecules dispersed in sodium alginate solution is 0.5-2mg / mL; the concentration of sodium alginate solution is 1-3% (W / V); the collecting liquid is selected from one or more of calcium chloride solution, barium chloride solution, and aluminum chloride solution with a concentration of 1-3% (W / V).

[0050] The glass capillaries in the microfluidic chip channels used in the following embodiments are from SUTTER INSTRUMENTAL CO. Model: B100-58-15.

[0051] Example 1

[0052] A montmorillonite-Lactobacillus acidophilus-indole-3-propionic acid microsphere preparation, the preparation method comprising the following steps:

[0053] (1) Bacterial culture and sample preparation:

[0054] Thaw the glycerol-frozen Lactobacillus acidophilus (purchased from Beijing Biobo Biotechnology Co., Ltd.) strain in a 37°C water bath. Then, take 100 μL of the bacterial suspension and add it to 5 mL of sterile MRS liquid culture medium, and culture it anaerobically in a 37°C constant temperature incubator for 24 hours. After the bacteria are revived, transfer 100 μL of the revived bacterial solution to 5 mL of sterile MRS liquid culture medium, and culture it in a 37°C constant temperature incubator for 24 hours. Follow the above steps for 3 consecutive activations to increase the activity of Lactobacillus acidophilus. After activation, inoculate the Lactobacillus acidophilus bacterial solution into the MRS liquid culture medium at a ratio of 1:50 (V / V), and culture it anaerobically in a 37°C constant temperature incubator until OD 600 The value is 0.4~0.5, for backup.

[0055] (2) Preparation of Lactobacillus acidophilus biofilm:

[0056] First, montmorillonite was sieved through a 500-mesh sieve to obtain particles with a particle size of about 20 μm. Then, these particles were sterilized by ultraviolet irradiation for 1 hour. Next, the sterilized montmorillonite particles were dispersed in an OD 600 The Lactobacillus acidophilus liquid with a pH value of 0.4 to 0.5 was cultured anaerobically in a 37°C constant temperature incubator for 48 hours. Finally, the montmorillonite-Lactobacillus acidophilus biofilm was obtained by centrifugation (1000 rpm, 2 min) and washed three times with PBS buffer.

[0057] (3) Preparation of Lactobacillus acidophilus biofilm and indole-3-propionic acid microsphere preparation

[0058] Montmorillonite-Lactobacillus acidophilus biofilm (30 mg / mL) and indole-3-propionic acid (1.5 mg / mL) were dispersed in 1.5% (W / V) sodium alginate solution, mixed thoroughly and injected into the channel of a microfluidic chip via a syringe. Monodisperse spray droplets were generated under the conditions of 6 kV voltage and 4 mL / h flow rate, and were quickly cross-linked in 2.0% (W / V) calcium chloride collection solution to form a microsphere preparation.

[0059] Figure 2 This is a scanning electron microscope photo of the biofilm formed by Lactobacillus acidophilus on the surface of montmorillonite in this example. Figure 3 The optical microscope photographs of the montmorillonite-Lactobacillus acidophilus biofilm and the indole-3-propionic acid microsphere preparation of this example show that the prepared microsphere preparation has good monodispersity and uniform size.

[0060] Figure 4 The scanning electron microscope photographs of the montmorillonite-Lactobacillus acidophilus biofilm and indole-3-propionic acid microsphere preparation of this example are shown. It can be seen from the figure that the particle size of the prepared microspheres is 250 μm.

[0061] Example 2

[0062] A montmorillonite-Bifidobacterium-butyric acid microsphere preparation, the preparation method comprising the following steps:

[0063] (1) Bacterial culture and sample preparation:

[0064] Thaw the glycerol-frozen Bifidobacterium strain (purchased from Beijing Biobo Biotechnology Co., Ltd.) in a 37°C water bath. Then, take 50 μL of the bacterial suspension and add it to 5 mL of sterile PYG liquid culture medium, and culture it anaerobically in a 37°C constant temperature incubator for 24 hours. After the bacteria are revived, transfer 50 μL of the revived bacterial solution to 5 mL of sterile PYG liquid culture medium, and culture it in a 37°C constant temperature incubator for 24 hours. Follow the above steps for 3 consecutive activations to increase the activity of the Bifidobacterium. After activation, inoculate the Bifidobacterium bacterial solution into the PYG liquid culture medium at a ratio of 1:100 (V / V), and culture it anaerobically in a 37°C constant temperature incubator until OD 600 The value is 0.4~0.5, for backup.

[0065] (2) Preparation of Bifidobacterium Biofilm:

[0066] First, montmorillonite was sieved through a 500-mesh sieve to obtain particles with a particle size of about 20 μm. Then, these particles were sterilized by ultraviolet irradiation for 1 hour. Next, the sterilized montmorillonite particles were dispersed in an OD 600 The bifidobacterium liquid with a value of 0.4 to 0.5 was further cultured in a 37°C constant temperature incubator for 48 hours. Finally, the montmorillonite-bifidobacterium biofilm was obtained by centrifugation (1000 rpm, 2 min), and then washed three times with PBS buffer.

[0067] (3) Preparation of Bifidobacterium biofilm and butyrate microsphere preparations

[0068] Montmorillonite-Bifidobacterium biofilm (30 mg / mL) and butyric acid (1.0 mg / mL) were dispersed in 1.5% (W / V) sodium alginate solution, mixed thoroughly and placed in the channel of a microfluidic chip. Monodisperse spray droplets were generated under the conditions of 6 kV voltage and 4 mL / h flow rate, and quickly cross-linked in 2.0% (W / V) calcium chloride collection solution to form a microsphere preparation.

[0069] Example 3

[0070] The application of montmorillonite-Lactobacillus acidophilus-indole-3-propionic acid microsphere preparation in the treatment of colitis, the experimental method comprises the following steps:

[0071] (1) Construction of a dextran sulfate sodium (DSS)-induced colitis mouse model

[0072] BALB / C female mice were randomly divided into healthy control group, DSS group and microsphere preparation group, with 5 mice in each group. The mice in DSS group and microsphere preparation group were fed with 3% (W / V) DSS aqueous solution for 1 week, and the mice in healthy control group were fed normally.

[0073] (2) Treatment of colitis using the montmorillonite-Lactobacillus acidophilus-indole-3-propionic acid microsphere preparation prepared in Example 1

[0074] The prepared montmorillonite-Lactobacillus acidophilus-indole-3-propionic acid microsphere preparation (containing 5 mg / 10 g body weight of montmorillonite-Lactobacillus acidophilus biofilm and 0.2 mg / 10 g body weight of indole-3-propionic acid) was dispersed in a 1.0% (W / V) carboxymethyl cellulose aqueous solution and intragastrically administered once a day for 5 days. The DSS group mice and the healthy control group mice were intragastrically administered with the same volume of 1.0% (W / V) carboxymethyl cellulose aqueous solution.

[0075] (3) Evaluation of the anti-colitis efficacy of montmorillonite-Lactobacillus acidophilus-indole-3-propionic acid microsphere preparation

[0076] After the administration, blood and colon samples were collected from each group of mice. The colon length of mice in the microsphere preparation group was significantly longer than that in the DSS group. The collected blood samples were centrifuged to obtain serum, and the ELISA test showed that compared with the DSS group, the concentration of pro-inflammatory factors in the serum of mice in the microsphere preparation group was significantly reduced, and the concentration of anti-inflammatory factors was significantly increased.

[0077] In summary, the present invention can provide a montmorillonite-probiotic microsphere preparation, which can be used for oral delivery of montmorillonite-probiotic biofilm and functional small molecules to treat intestinal diseases. The prepared microsphere preparation has the advantages of resistance to gastric acid invasion and intestinal responsive release. The microsphere preparation still maintains high bacterial activity when it reaches the stomach through oral administration, so that IBD can be treated more systematically and efficiently.

[0078] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A montmorillonite-probiotic microsphere preparation, characterized in that: The microsphere preparation comprises montmorillonite, probiotic biofilm and functional small molecules.

2. The montmorillonite-probiotic microsphere preparation according to claim 1, characterized in that: The microsphere preparation is a microsphere composed of montmorillonite, probiotic biofilm, functional small molecules and microsphere wall material, wherein the probiotic biofilm is attached to the surface of montmorillonite, and the functional small molecules are dispersed between montmorillonite and the microsphere wall material.

3. The montmorillonite-probiotic microsphere preparation according to claim 2, characterized in that: The microsphere wall material is prepared by cross-linking sodium alginate in a collection solution, and the montmorillonite, probiotic biofilm and functional small molecules are encapsulated in the microsphere wall material prepared from sodium alginate.

4. The montmorillonite-probiotic microsphere preparation according to claim 1, characterized in that: The probiotics are selected from one or more of lactobacillus, bifidobacterium and clostridium butyricum.

5. The montmorillonite-probiotic microsphere preparation according to claim 1, characterized in that: The functional small molecule is selected from one or more of indole-3-propionic acid, propionic acid, butyric acid, lipoteichoic acid, and peptidoglycan.

6. A method for preparing the montmorillonite-probiotic microsphere preparation according to any one of claims 1 to 5, characterized in that: The following steps are involved: S1, dispersing the sterilized montmorillonite particles in a probiotic culture solution in a logarithmic growth phase, and culturing at 28-40° C. for 36-60 hours to obtain a montmorillonite-probiotic biofilm; S2, dispersing montmorillonite-probiotic biofilm and functional small molecules in sodium alginate solution, mixing them evenly and injecting them into the microfluidic chip channel, generating spray droplets under the action of static voltage, and cross-linking into microsphere preparations in the collected liquid.

7. The method for preparing the montmorillonite-probiotic microsphere preparation according to claim 6, characterized in that: In step S1, the concentration of the montmorillonite particles dispersed in the probiotic culture solution is 5 to 20 mg / mL.

8. The method for preparing the montmorillonite-probiotic microsphere preparation according to claim 6, characterized in that: In step S2, the concentration of the montmorillonite-probiotic biofilm dispersed in the sodium alginate solution is 10-40 mg / mL; the concentration of the functional small molecules dispersed in the sodium alginate solution is 0.5-2 mg / mL.

9. The method for preparing the montmorillonite-probiotic microsphere preparation according to claim 6, characterized in that: In step S2, the concentration of the sodium alginate solution is 1-3% (W / V); the static voltage is 4-8 kV; the collecting solution is selected from one or more of calcium chloride solution, barium chloride solution, and aluminum chloride solution; the concentration of the collecting solution is 1-3% (W / V), and the cross-linking time is 1-5 min.

10. Use of the montmorillonite-probiotic microsphere preparation according to any one of claims 1 to 5 in the preparation of an oral live probiotic preparation for treating intestinal diseases.

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