Preparation method and application of coprophilous fungus transplantation microcapsule

Abstract

The invention relates to the technical field of oral microbial preparations, and particularly discloses a coprophilous fungus transplantation microcapsule preparation method and application thereof, and the preparation method comprises the following steps: coprophilous fungus suspension acquisition, layer-by-layer embedding, calcium / zinc reinforcement, coprophilous fungus microcapsule freeze-drying and the like. The carboxymethyl chitosan, the sodium alginate / sulfated polysaccharide, the calcium ions and the like are used as raw materials, the hydrogel with a double-network structure is prepared through specific ionic crosslinking and electrostatic adsorption reaction, and the hydrogel has excellent mechanical performance and stability and can effectively protect coprophilous fungi from being damaged by the external environment. The prepared double-network hydrogel is used as a wall material, coprophilous fungi are wrapped in the double-network hydrogel to form coprophilous fungi microcapsules, and the variety of receptor intestinal flora communities after coprophilous fungi transplantation is effectively enhanced.

Description

Technical Field

[0001] The present invention belongs to the technical field of oral microbial preparations, and specifically relates to a method for preparing fecal microbiota transplantation microcapsules and applications thereof. Background Art

[0002] The intestinal microecology plays a vital role in various physiological and pathological processes in the human body. Intestinal microbiome disorders, whether changes in flora composition or flora diversity, may become a key pathogenic factor in intestinal and extraintestinal diseases, such as inflammatory bowel disease, Clostridium difficile infection, functional constipation, autism and Parkinson's disease. Fecal microbiota transplantation reshapes the structure and function of the recipient's intestinal flora by transplanting the flora in the donor's feces into the recipient's intestine, maintaining the homeostasis of the human intestinal microecology, and achieving the treatment of intestinal and extraintestinal diseases.

[0003] Fecal microbiota transplantation is a direct and efficient emerging therapy for reshaping and optimizing intestinal microbial communities. Its administration route is mainly divided into upper, middle and lower gastrointestinal tract administration according to different parts of the digestive tract, covering a variety of administration methods, such as colonoscopy infusion, enema, nasogastric tube infusion and oral fecal microbiota capsules. Among them, colonoscopy infusion, enema and nasogastric tube infusion are effective, but their invasive characteristics and operational complexity greatly limit their widespread popularization and application.

[0004] In contrast, oral capsules have become the most ideal method of administration due to their economy and convenience. However, oral administration faces the challenge of maintaining the activity of the flora. Most fecal bacteria are strictly anaerobic, so it is difficult to ensure their activity during the extraction and freeze-drying of fecal bacteria. At the same time, oral capsules encounter harsh physical and chemical conditions (gastric acid, digestive enzymes, etc.) when passing through the gastrointestinal tract, which will significantly reduce the vitality and quantity of the flora, thereby affecting the effective treatment effect. Therefore, ensuring the activity and quantity of fecal bacteria during oral administration is a key issue that needs to be solved urgently. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing fecal microbiota transplantation microcapsules and its application to solve the problems raised in the above-mentioned background technology.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A method for preparing fecal microbiota transplantation microcapsules and its application, comprising the following steps:

[0008] S1. Obtaining fecal suspension: Collect fresh fecal samples from the donor, add the fecal samples to sterile saline and stir evenly until there are no obvious large particles, filter step by step to remove food residues and large particles, and collect the supernatant by centrifugation to obtain fecal suspension;

[0009] S2. Embedding layer by layer: Add the fecal bacteria suspension into the carboxymethyl chitosan solution and sodium alginate solution / sulfated polysaccharide successively and incubate on a shaker, then centrifuge and wash to remove the residual wall materials, finally forming a fecal bacteria suspension embedded in a bilayer cross-linked network;

[0010] S3. Calcium / zinc strengthening: Drop the stabilizing solution into the embedded fecal bacteria suspension, stir evenly and then wash with the washing solution, and centrifuge to obtain the embedded fecal bacteria microcapsules;

[0011] S4. Freeze-drying of fecal bacteria microcapsules: Pre-freeze the fecal bacteria microcapsules at -80 °C, and then vacuum-dry to obtain the fecal bacteria microcapsule preparation, which is stored anaerobically and frozen.

[0012] Preferably, the treatment of the donor fecal sample in S1 is carried out in an anaerobic environment, the ratio of the fecal sample to sterile physiological saline is 100 mg: 0.5 - 2 mL, when filtering step by step, take 1.0 mm and 0.1 mm molecular sieves to filter successively, and the centrifugal force during centrifugation is 300 g and the centrifugation time is 5 min.

[0013] Preferably, S2 is carried out in an anaerobic environment, the concentrations of the carboxymethyl chitosan solution, sodium alginate solution, and sulfated polysaccharide solution are 1 - 4 mg / mL, the shaker speed is 100 rpm, the centrifugal force during centrifugation is 500 g, the centrifugation time is 5 min, and wash twice with sterile physiological saline.

[0014] Preferably, S3 is carried out in an anaerobic environment, the stabilizing solution is calcium chloride or zinc chloride solution, the concentration is 0.05 - 0.15 mol / L, the washing solution is sterile physiological saline or PBS washing solution, wash 2 - 4 times, and centrifuge to obtain the final fecal bacteria microcapsules, the centrifugal force is 1000 g and the centrifugation time is 5 min.

[0015] Preferably, in S4, the fecal bacteria microcapsules are pre-frozen for 48 h and vacuum-dried for 24 h, and the pressure is 0.300 mbar.

[0016] Compared with the prior art, the beneficial effects of the present invention are:

[0017] Based on carboxymethyl chitosan, sodium alginate / sulfated polysaccharide, and calcium ions as raw materials, the present invention prepares a hydrogel with a double-network structure through specific ion cross-linking and electrostatic adsorption reactions. This hydrogel has excellent mechanical properties and stability, and can effectively protect fecal bacteria from being damaged by the external environment. Using the prepared double-network hydrogel as the wall material to encapsulate fecal bacteria therein to form fecal bacteria microcapsules effectively enhances the species richness of the recipient intestinal flora community after fecal microbiota transplantation. Description of the Drawings

[0018] Figure 1SEM image of the fecal microcapsules of the embodiment of the present invention magnified 1000 times;

[0019] Figure 2 SEM image of the fecal bacteria suspension of the embodiment of the present invention magnified 1000 times;

[0020] Figure 3 Simpson index (left) and Shannon index (right) schematic diagrams in the experimental examples of the present invention. Detailed implementation manners

[0021] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0022] Example 1:

[0023] A fecal microcapsule based on donor feces, comprising the following components in parts by weight: 10 g of a fresh fecal sample from the donor, 0.2 g of sodium alginate, 0.2 g of carboxymethyl chitosan, 200 g of a calcium chloride solution with a concentration of 0.1 mol / L, and 500 g of sterile physiological saline.

[0024] The preparation method of the above fecal microcapsule based on donor feces comprises the following steps, and the whole reaction is carried out in an anaerobic environment:

[0025] (1) Collect fresh fecal samples from SPF-grade C57BL / 6 mice, mix the fecal samples from the same group of mice, add 10 g of the fecal sample to 100 mL of sterile physiological saline, stir evenly until there are no obvious large particles, perform vortex homogenization treatment, filter through 1 mm and 0.1 mm molecular sieves successively to further remove food residues and large particle substances in the feces, and centrifuge at 300 g for 5 min to collect the supernatant to obtain a fecal bacteria suspension.

[0026] (2) Take 2 mL of the fecal bacteria suspension and add it to 10 mL of a carboxymethyl chitosan solution with a concentration of 2 mg / mL, stir evenly, incubate on a shaker at 100 rpm for 10 min to make the positively charged carboxymethyl chitosan evenly adsorb on the negatively charged cell surface. Then centrifuge at 500 g for 5 min, resuspend and wash with 10 mL of physiological saline and centrifuge 2 times.

[0027] Add 10 mL of sodium alginate solution with a concentration of 2 mg / mL to the obtained fecal bacteria-carboxymethyl chitosan precipitate, stir evenly, incubate at 100 rpm on a shaker for 10 min to form fecal bacteria cells encapsulated by a carboxymethyl chitosan-sodium alginate double-network cross-linked wall material. Subsequently, centrifuge at 500 g for 5 min and resuspend and wash twice with 10 mL of normal saline to remove the residual sodium alginate.

[0028] (3) Take 0.1 mol / L calcium chloride solution and dropwise add it to 10 ml of the finally obtained embedded fecal bacteria suspension in the above step, stir at a speed of 1000 r / min for 10 min. Subsequently, centrifuge at 1000 g for 5 min and wash once with 10 ml of normal saline to obtain fecal bacteria microcapsules.

[0029] (4) Pre-freeze the fecal bacteria microcapsules at -80 °C for 48 hours, and then vacuum dry at a pressure of 0.300 mbar for 24 h to obtain a fecal bacteria microcapsule preparation, which is stored anaerobically at -40 °C.

[0030] The obtained fecal bacteria microcapsule preparation is subjected to microscopic morphology observation and germ-free colonization test.

[0031] Example 2:

[0032] A fecal bacteria microcapsule based on donor feces, comprising the following components in parts by weight: 10 g of a fresh fecal sample from a donor, 0.2 g of sodium alginate, 0.2 g of carboxymethyl chitosan, 200 g of a zinc chloride solution with a concentration of 0.1 mol / L, and 500 g of PBS washing solution.

[0033] The preparation method of the above fecal bacteria microcapsule based on donor feces comprises the following steps, and the whole reaction process is carried out in an anaerobic environment:

[0034] (1) Collect fresh fecal samples from SPF-grade C57BL / 6 mice, mix the fecal samples from the same group of mice, add 10 g of the fecal sample to 100 mL of sterile physiological saline, stir evenly until there are no obvious large particles, perform vortex homogenization treatment, filter through 1 mm and 0.1 mm molecular sieves successively to further remove food residues and large particle substances in the feces, and centrifuge at a centrifugal force of 300 g for 5 min to collect the supernatant to obtain a fecal bacteria suspension.

[0035] (2) Take 2 mL of the fecal bacteria suspension and add it to 10 mL of a carboxymethyl chitosan solution with a concentration of 2 mg / mL, stir evenly, incubate at 100 rpm on a shaker for 10 min to make the positively charged carboxymethyl chitosan evenly adsorb on the negatively charged cell surface of the bacteria. Subsequently, centrifuge at 500 g for 5 min, resuspend and wash and centrifuge twice with 10 mL of normal saline.

[0036] Add 10 mL of sodium alginate solution with a concentration of 2 mg / mL to the obtained fecal bacteria - carboxymethyl chitosan precipitate, stir evenly, incubate at 100 rpm on a shaker for 10 min to form fecal bacteria cells encapsulated by a carboxymethyl chitosan - sodium alginate double - network cross - linked wall material. Subsequently, centrifuge at 500 g for 5 min, resuspend and wash twice with 10 mL of normal saline to remove the residual sodium alginate.

[0037] (3) Take 0.1 mol / L zinc chloride solution and add it dropwise to 10 mL of the finally obtained embedded fecal bacteria suspension in the above - mentioned steps, stir at a speed of 1000 r / min for 10 min. Subsequently, centrifuge at 1000 g for 5 min and wash once with 10 mL of PBS washing solution to obtain fecal bacteria microcapsules.

[0038] (4) Pre - freeze the fecal bacteria microcapsules at - 80 °C for 48 hours, and then vacuum - dry at 0.300 mbar for 24 h to obtain a fecal bacteria microcapsule preparation, which is stored anaerobically at - 40 °C.

[0039] Comparative Example 1:

[0040] This comparative example presents a fecal bacteria suspension based on donor feces, including the following components in parts by weight: 10 g of fresh donor feces sample and 200 g of sterile normal saline.

[0041] The preparation method of the above - mentioned fecal bacteria suspension based on donor feces includes the following steps. The whole reaction process is carried out in an anaerobic environment: collect fresh feces samples from SPF - level C57BL / 6 mice, mix the feces samples from the same group of mice, add 10 g of feces samples to 100 mL of sterile normal saline, stir evenly until there are no obvious large particles, perform vortex homogenization treatment, filter through 1 mm and 0.1 mm molecular sieves successively to further remove food residues and large - particle substances in the feces, centrifuge at 300 g for 5 min, and collect the supernatant to obtain a fecal bacteria suspension. The obtained fecal bacteria suspension is subjected to microscopic morphology observation and germ - free mouse colonization test.

[0042] Microscopic morphology observation

[0043] Perform scanning electron microscopy observation on the fecal bacteria suspension and fecal bacteria microcapsules prepared in Example 1 and Comparative Example 1. Figure 1 It can be seen that for the fecal bacteria microcapsules obtained in Example 1, a regular network structure covers the surface of the fecal bacteria, which is a carboxymethyl chitosan - sodium alginate double - ion calcium cross - linked network. Figure 2 Visible un - encapsulated fecal bacteria.

[0044] Germ - free mouse colonization test

[0045] Twelve 8-week-old ICR germ-free mice were randomly divided into two groups: the fecal microbiota suspension transplantation group (FMT) and the fecal microbiota microcapsule transplantation group (eFMT). The two groups of germ-free mice were separately housed in individual germ-free isolators. The prepared fecal microbiota suspension and fecal microbiota microcapsules were transferred into the isolators according to the requirements of aseptic transfer. On the 1st and 3rd days of the experiment, the germ-free mice were gavaged with the transplantation materials, and each mouse was gavaged with 200 μL of fecal microbiota suspension or fecal microbiota microcapsules once.

[0046] On the 4th day of the experiment, fresh mouse fecal samples were collected and transferred to -80 °C for storage. The fecal samples were subjected to 16S rRNA sequencing, and the specific steps were as follows: The total fecal bacterial DNA was extracted according to the kit operation and its concentration was measured. The V3-V4 variable region of the 16S rRNA gene was amplified by PCR, and equal amounts of purified amplicons were combined for 16S amplicon sequencing.

[0047] The α-diversity of the intestinal microbiota in the samples was analyzed. As Figure 3 shown, both the Shannon index and the Simpson index of the eFMT group were higher than those of the FMT group, indicating that the method in Example 1 enhanced the species richness of the recipient intestinal microbiota community after fecal microbiota transplantation, and confirmed that the fecal microbiota microcapsules obtained in Example 1 could promote the colonization of fecal microbiota in the intestine and promote the early colonization and growth of fecal microbiota.

[0048] It should be understood that in the development of any actual implementation, such as in any engineering or design project, a large number of specific implementation decisions can be made. Such development efforts may be complex and time-consuming, but for those of ordinary skill in the art who benefit from this disclosure, without excessive experimentation, such development efforts will be a routine task of design, manufacture, and production.

[0049] It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and are not restrictive. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered by the scope of the claims of the present invention.

Claims

1. A method for preparing a fecal microbiota transplantation microcapsule, characterized in that, It includes the following steps: S1. Obtaining fecal microbiota suspension: Collect fresh fecal samples from donors, add the fecal samples into sterile physiological saline and stir evenly until there are no obvious large particles, filter step by step to remove food residues and large particle substances, and centrifuge to collect the supernatant to obtain the fecal microbiota suspension; S2. Embedding layer by layer: Add the fecal microbiota suspension into carboxymethyl chitosan solution, sodium alginate solution / sulfated polysaccharide in sequence and incubate on a shaker, centrifuge and wash to remove the residual wall materials, and finally form a fecal microbiota suspension embedded with a bilayer cross-linked network; S3. Calcium / zinc strengthening: Drop the stabilizing solution into the embedded fecal microbiota suspension, stir evenly and then wash with the washing solution, and centrifuge to obtain the embedded fecal microbiota microcapsules; S4. Freeze-drying the fecal microbiota microcapsules: Pre-freeze the fecal microbiota microcapsules at -80 °C, and then conduct vacuum drying to obtain the fecal microbiota microcapsule preparation, and store it anaerobically and frozen.

2. The preparation method of the fecal microbiota transplantation microcapsule according to claim 1, wherein: In S1, the treatment of the donor fecal samples is carried out in an anaerobic environment. The ratio of the fecal samples to sterile physiological saline is 100 mg: 0.5 - 2 mL. When filtering step by step, 1.0 mm and 0.1 mm molecular sieves are taken for filtration in sequence. When centrifuging, the centrifugal force is 300 g and the centrifugation time is 5 min.

3. A method for preparing a fecal microbiota transplantation microcapsule according to claim 1, characterized in that: In S2, it is carried out in an anaerobic environment. The concentrations of the carboxymethyl chitosan solution, sodium alginate solution, and sulfated polysaccharide solution are 1 - 4 mg / mL. The shaker speed is 100 rpm. When centrifuging, the centrifugal force is 500 g and the centrifugation time is 5 min, and it is washed 2 times with sterile physiological saline.

4. A method for preparing a fecal microbiota transplantation microcapsule according to claim 1, characterized in that: In S3, it is carried out in an anaerobic environment. The stabilizing solution is calcium chloride or zinc chloride solution, and the concentration is 0.05 - 0.15 mol / L. The washing solution is sterile physiological saline or PBS washing solution, for 2 - 4 times, and the final fecal microbiota microcapsules are obtained by centrifugation, with a centrifugal force of 1000 g and a centrifugation time of 5 min.

5. A method for preparing a fecal microbiota transplantation microcapsule according to claim 1, characterized in that: In S4, the fecal microbiota microcapsules are pre-frozen for 48 h and vacuum dried for 24 h, and the pressure is 0.300 mbar.

6. Application of the method for preparing a fecal microbiota transplantation microcapsule according to claim 1 in oral microbial preparations.

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