Fecal fungi capsule freeze-drying protective agent for intestinal flora transplantation and preparation method of fecal fungi capsule freeze-drying protective agent

Abstract

The invention discloses a coprophilous fungus capsule freeze-drying protective agent for intestinal flora transplantation and a preparation method of the coprophilous fungus capsule freeze-drying protective agent for intestinal flora transplantation and belongs to the technical field related to pharmaceutical preparations, and the coprophilous fungus capsule freeze-drying protective agent for intestinal flora transplantation comprises nano-liposome; polydextrose; plant selenoprotein; tea polyphenol; vitamin E; chitosan oligosaccharide; an Omega-3 fatty acid; 3-5% of inulin; the nano-liposome forms a phospholipid bilayer to form a physical barrier on the surface of a cell, and polydextrose and inulin regulate the extracellular medium viscosity and inhibit the growth rate of ice crystals; omega-3 fatty acid is inserted into a phospholipid bilayer to maintain the fluidity of a membrane at a low temperature, vitamin E and tea polyphenol reduce membrane lipid peroxidation products in the freeze-drying process, and the stability of an oxidant cell membrane is protected through damage of ice crystals, so that cells are protected, and the survival of the cells is improved.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical preparation technology, and particularly relates to a fecal microbiota capsule freeze-dried protectant for intestinal microbiota transplantation and its preparation method. Background Technology

[0002] Fecal microbiota transplantation (FMT) is defined as the transplantation of functional microbiota from the feces of healthy individuals into the gastrointestinal tract of a patient, thereby rebuilding a new gut microbiota and achieving the treatment of intestinal and extraintestinal diseases. The freeze-drying process for microorganisms, most commonly used for lactic acid bacteria and probiotics, typically requires the addition of preservatives in addition to common fillers and excipients.

[0003] Traditional freeze-drying protectants protect cells only through osmotic pressure regulation (such as mannitol) or simple encapsulation (such as skim milk), which cannot effectively inhibit the growth rate of ice crystals. They also have limited antioxidant components, and the membrane lipid peroxidation problem is significant during freeze-drying, which cannot promote the colonization of probiotics. Summary of the Invention

[0004] In view of the above situation and to overcome the defects of the prior art, the present invention provides a fecal microbiota capsule freeze-drying protectant for intestinal microbiota transplantation and its preparation method. The freeze-drying protectant components can protect cells and improve cell survival in terms of ice crystal damage protection and cell membrane stability, thereby improving colonization efficiency and microbiota survival rate.

[0005] The technical solution adopted in this invention is as follows:

[0006] In a first aspect, the present invention provides a fecal microbiota capsule freeze-drying protectant for intestinal microbiota transplantation, comprising the following components:

[0007] Nanoliposomes 1-3%;

[0008] Polydextrose 3-5%

[0009] Plant selenoprotein 0.1-0.3%

[0010] Tea polyphenols 0.5-1.5%

[0011] Vitamin E 0.5-1.5%

[0012] Chitosan oligosaccharides 0.2-0.5%

[0013] Omega-3 fatty acids 0.5-1.5%;

[0014] Inulin 3-5%.

[0015] Preferably, the fecal microbiota capsule freeze-drying protectant for intestinal microbiota transplantation comprises the following components:

[0016] Nanoliposomes 2%;

[0017] Polydextrose 4%

[0018] Plant selenium protein 0.2%

[0019] 1% tea polyphenols

[0020] Vitamin E 1%

[0021] Chitosan oligosaccharide 0.35%

[0022] 1% Omega-3 fatty acids;

[0023] Inulin 4%.

[0024] In a second aspect, this application provides a method for preparing a fecal microbiota capsule freeze-drying protectant for intestinal microbiota transplantation, comprising the following steps:

[0025] (1) Preparation of nanoliposomes: Phosphatidylcholine and cholesterol were dissolved in chloroform at a mass ratio of 3:1; organic solvent was removed by rotary evaporation at 40°C to form a film; PBS buffer (pH 7.4) was added for hydration and incubated overnight at 4°C; liposome suspension with a particle size of <200 nm was obtained by high pressure homogenization (1000 bar, 3 cycles);

[0026] (2) Pretreatment of water-soluble components: Polydextrose, plant selenoprotein, tea polyphenols and chitosan oligosaccharide were dissolved in sterile water; sterilized by filtration through a 0.22μm filter membrane and stored at 4℃ in the dark;

[0027] (3) Pretreatment of fat-soluble components: Vitamin E and Omega-3 fatty acids were dissolved in a small amount of ethanol; 1% Tween 80 was added for emulsification, and the mixture was magnetically stirred at 300 rpm for 30 minutes.

[0028] (4) Mixing: Add 3%-5% polydextrose to PBS buffer to a final volume of 500ml;

[0029] Add 0.1-0.3% plant selenoprotein and 0.5-1.5% tea polyphenols, stir at 200 rpm for 15 minutes, and adjust the pH to 6.8±0.2;

[0030] Slowly add 0.5-1.5% vitamin E and 0.5-1.5% Omega-3 fatty acid emulsion, reduce the stirring speed to 100 rpm, and maintain for 30 minutes;

[0031] Add 0.2-0.5% chitosan oligosaccharide and 3-5% inulin; add 1-3% nanoliposome suspension; stir at 200 rpm for 15 minutes, and filter through a 0.22 μm filter membrane;

[0032] (5) Freeze-drying: Pre-freeze by step cooling; dry once; dry twice to obtain a fecal microbiota capsule freeze protectant for intestinal microbiota transplantation.

[0033] Furthermore, the step (5) of the stepped cooling process is as follows: maintain 4°C for 15 minutes;

[0034] Keep at -20℃ for 1 hour;

[0035] Keep at -40℃ for 8 hours: Ensure the material is completely cured; the temperature must be 5-10℃ below the eutectic point.

[0036] Cooling rate: 1℃ / minute.

[0037] Furthermore, the drying parameters for step (5) are as follows:

[0038] Temperature control: -33℃, the temperature of the heating plate must be lower than the eutectic point of the material;

[0039] Vacuum level: 0.2 mbar (20 Pa);

[0040] Duration: 36 hours, ensuring over 95% sublimation of free water.

[0041] Furthermore, the secondary drying parameters in step (5) are as follows:

[0042] Temperature: 18℃;

[0043] Time: 54 hours, ensuring residual moisture content is less than 3%;

[0044] Endpoint determination: Pressure rise test ΔP < 0.01 mbar / 10 min.

[0045] After adopting the above solution, the beneficial effects of the present invention are as follows:

[0046] (1) A fecal microbiota capsule freeze-drying protectant for intestinal microbiota transplantation, the components of which are nanoliposomes, polydextrose, plant selenoproteins, tea polyphenols, vitamin E, chitosan oligosaccharides, Omega-3 fatty acids, and inulin. Among them, nanoliposomes form a physical barrier on the cell surface by forming a phospholipid bilayer; polydextrose and inulin regulate the viscosity of the extracellular medium and inhibit the growth rate of ice crystals; Omega-3 fatty acids maintain membrane fluidity at low temperatures by inserting into the phospholipid bilayer; vitamin E and tea polyphenols reduce membrane lipid peroxidation products during freeze-drying, and protect the stability of the cell membrane by protecting against oxidants caused by ice crystal damage, thereby protecting cells and improving cell survival.

[0047] (2) Plant selenoproteins, chitosan oligosaccharides and vitamin E scavenge free radicals, inulin and polydextrose can be utilized by bifidobacteria, increasing the abundance of bifidobacteria; nanoliposomes achieve continuous release under intestinal pH conditions, enhancing bacterial colonization; chitosan oligosaccharides form a complex with intestinal mucosal mucin, which can prolong the retention time of the protectant; polydextrose and inulin form a three-dimensional network structure during freeze-drying, shortening the product reconstitution time.

[0048] (3) A method for preparing a freeze-dried protectant for fecal microbiota transplantation, wherein the step-down cooling method is a gradual cooling process of 4℃→-20℃→-40℃, which can reduce the mechanical damage of ice crystals to microbial cells compared with the traditional direct cooling method (such as direct pre-freezing at -40℃). Detailed Implementation

[0049] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0050] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0051] All methods not specifically described in this invention are conventional methods in the art, and all reagents not specifically described are conventional reagents in the art.

[0052] Example 1: A fecal microbiota capsule freeze-drying protectant for intestinal microbiota transplantation, comprising the following components:

[0053] Nanoliposomes 3%; polydextrose 5%; plant selenoprotein 0.3%; tea polyphenols 1.5%; vitamin E 1.5%; chitosan oligosaccharide 0.5%; Omega-3 fatty acids 1.5%; inulin 5%.

[0054] Example 2: A fecal microbiota capsule freeze-drying protectant for intestinal microbiota transplantation, comprising the following components:

[0055] Nanoliposomes 2%; polydextrose 4%; plant selenium protein 0.2%; tea polyphenols 1%; vitamin E 1%; chitosan oligosaccharide 0.35%; Omega-3 fatty acids 1%; inulin 4%.

[0056] Example 3: A fecal microbiota capsule freeze-drying protectant for intestinal microbiota transplantation, comprising the following components:

[0057] Nanoliposomes 1%; polydextrose 3%; plant selenoprotein 0.1%; tea polyphenols 0.5%; vitamin E 0.5%; chitosan oligosaccharide 0.2%;

[0058] Omega-3 fatty acids 0.5%; inulin 3%.

[0059] Comparative Example 1: A lyophilized preservative for fecal microbiota transplantation capsules, comprising the following components:

[0060] Polydextrose 4%; plant selenium protein 0.2%; tea polyphenols 1%; vitamin E 1%; chitosan oligosaccharide 0.35%; Omega-3 fatty acids 1%; inulin 4%.

[0061] Comparative Example 2: A fecal microbiota capsule lyophilization protectant for intestinal microbiota transplantation, comprising the following components:

[0062] Nanoliposomes 2%; polydextrose 4%; plant selenoprotein 0.2%; chitosan oligosaccharide 0.35%; Omega-3 fatty acids 1%; inulin 4%.

[0063] Comparative Example 3: A fecal microbiota capsule lyophilization protectant for intestinal microbiota transplantation, comprising the following components:

[0064] Nanoliposomes 2%; plant selenium protein 0.2%; tea polyphenols 1%; vitamin E 1%; chitosan oligosaccharide 0.35%; Omega-3 fatty acids 1%;

[0065] Comparative Example 4: A fecal microbiota capsule lyophilization protectant for intestinal microbiota transplantation, comprising the following components:

[0066] Nanoliposomes 2%; polydextrose 4%; plant selenium protein 0.2%; tea polyphenols 1%; vitamin E 1%; Omega-3 fatty acids 1%; inulin 4%.

[0067] Comparative Example 5: A freeze-dried protectant for fecal microbiota transplantation capsules, comprising the following components: 10% skim milk and 5% mannitol.

[0068] Example 4

[0069] The preparation method of the lyophilized protectant for fecal microbiota transplantation capsules includes the following steps:

[0070] (1) Preparation of nanoliposomes: Phosphatidylcholine and cholesterol were dissolved in chloroform at a mass ratio of 3:1; organic solvent was removed by rotary evaporation at 40°C to form a film; PBS buffer (pH 7.4) was added for hydration and incubated overnight at 4°C; liposome suspension with a particle size of <200 nm was obtained by high pressure homogenization (1000 bar, 3 cycles);

[0071] (2) Pretreatment of water-soluble components: Polydextrose, plant selenoprotein, tea polyphenols and chitosan oligosaccharide were dissolved in sterile water; sterilized by filtration through a 0.22μm filter membrane and stored at 4℃ in the dark;

[0072] (3) Pretreatment of fat-soluble components: Vitamin E and Omega-3 fatty acids were dissolved in a small amount of ethanol; 1% Tween 80 was added for emulsification, and the mixture was magnetically stirred at 300 rpm for 30 minutes.

[0073] (4) Mixing: Add 3%-5% polydextrose to PBS buffer to a final volume of 500ml;

[0074] Add 0.1-0.3% plant selenoprotein and 0.5-1.5% tea polyphenols, stir at 200 rpm for 15 minutes, and adjust the pH to 6.8±0.2;

[0075] Slowly add 0.5-1.5% vitamin E and 0.5-1.5% Omega-3 fatty acid emulsion, reduce the stirring speed to 100 rpm, and maintain for 30 minutes;

[0076] Add 0.2-0.5% chitosan oligosaccharide and 3-5% inulin; add 1-3% nanoliposome suspension; stir at 200 rpm for 15 minutes, and filter through a 0.22 μm filter membrane;

[0077] (5) Freeze-drying: Pre-freeze by step cooling; dry once; dry twice to obtain a fecal microbiota capsule freeze protectant for intestinal microbiota transplantation.

[0078] The stepped cooling steps are as follows: 4℃ for 15 minutes; -20℃ for 1 hour; -40℃ for 8 hours: to ensure complete solidification of the material, the temperature must be 5-10℃ below the eutectic point; cooling rate: 1℃ / minute.

[0079] The parameters for one drying cycle are:

[0080] Temperature control: -33℃, heating plate temperature must be below the eutectic point of the material; Vacuum degree: 0.2mbar (20Pa);

[0081] Duration: 36 hours, ensuring over 95% sublimation of free water.

[0082] The secondary drying parameters are:

[0083] Temperature: 18℃; Time: 54 hours, ensuring residual moisture <3%; Endpoint determination: pressure rise test ΔP < 0.01mbar / 10min.

[0084] Experimental Example 1

[0085] Fecal microbiota capsule freeze-drying protectants for intestinal microbiota transplantation, as described in Example 4, were prepared for Examples 1-3 and Comparative Examples 1-4, respectively. Fecal microbiota suspension was uniformly inoculated (1×10^8 CFU / ml); the following indicators were detected:

[0086] Plate counting method for detecting survival rate;

[0087] Flow cytometry was used to detect membrane integrity.

[0088] MDA content detection to assess oxidative damage;

[0089] qPCR was used to detect the colonization efficiency of fecal microbiota 72 hours after drug administration;

[0090] The test results are shown in Tables 1 and 2.

[0091] Table 1: Comparison of freeze-drying performance of each group.

[0092] Group Survival rate (%) Reconstitution time (min) Membrane integrity rate (%) MDA content (nmol / mg) Example 1 92.5 1.6 94.2 1.5 Example 2 90.7 1.8 94.8 1.3 Example 3 88.3 2.1 92.4 1.2 Comparative Example 1 63.7 5.8 71.5 3.8 Comparative Example 2 72.4 2.5 83.6 5.1 Comparative Example 3 79.3 2.3 90.2 1.5 Comparative Example 4 82.6 2.2 91.8 1.3 Comparative Example 5 42.3 8.5 53.7 7.9

[0093] Table 2 shows the results of customized testing for each group of microbial communities.

[0094] Group Bifidobacteria (×10 CFU / g) Escherichia coli (×10 CFU / g) B / E ratio Intestinal retention time (h) Example 2 5.9 7.5 0.79 13.7 Comparative Example 1 3.1 12 0.26 9.8 Comparative Example 2 4.2 8.5 0.49 10.3 Comparative Example 3 2.8 8.9 0.31 11.2 Comparative Example 4 4.9 7.8 0.63 8.2 Comparative Example 5 1.5 4.3 0.35 6.5

[0095] Based on the above experimental data, in terms of survival rate, Examples 1-3 were significantly higher than Comparative Examples 1-5; Comparative Example 1 lacked nanoliposomes, and its survival rate was lower than that of Examples 1-3, confirming its role in physical barrier function; Comparative Example 2 lacked tea polyphenols and vitamin E, and its survival rate was lower than that of Examples 1-3, showing the synergistic protective effect of tea polyphenols and vitamin E.

[0096] Regarding reconstitution, Examples 1-3 showed significantly better results than Comparative Examples 1-5, while the reconstitution times of Comparative Examples 1-5 showed no significant difference. This indicates that the overall formulation determines the reconstitution characteristics.

[0097] Regarding membrane integrity, Comparative Example 1 (lacking nanoliposomes) was significantly lower than Examples 1-3 (reduced by 20+ percentage points), and Comparative Example 2 (lacking tea polyphenols and vitamin E) was lower than Examples 1-3 (reduced by 8 percentage points), indicating that oxidative damage can disrupt cell membrane structure.

[0098] Regarding microbial colonization, Comparative Example 3 (lacking inulin and glucan) had a lower number of Bifidobacteria, verifying the growth-promoting effects of inulin and glucan. Comparative Example 4 (lacking chitosan) had a lower intestinal retention time, indicating that chitosan enhances the colonization effect by prolonging the retention time. The B / E ratio of Example 2 reached 0.79, which was significantly higher than other control groups.

[0099] Through the above-mentioned comparative experiments, the components of the fecal microbiota capsule freeze-dried protectant for intestinal microbiota transplantation in this application enhance mucosal adhesion, provide physical protection, reduce oxidative damage, and promote the proliferation of beneficial bacteria through different mechanisms, which is significantly better than the traditional formula (Comparative Example 5).

[0100] Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents. In short, if those skilled in the art, inspired by this description, design similar structural methods and embodiments without departing from the spirit of the invention, such designs should fall within the protection scope of the present invention. Each component of this application can be driven by a corresponding external motor; this is prior art and will not be elaborated upon here.

Claims

1. A fecal microbiota capsule freeze-drying protectant for intestinal microbiota transplantation, characterized in that, Includes the following components: Nanoliposomes 1-3%; Polydextrose 3-5% Plant selenoprotein 0.1-0.3% Tea polyphenols 0.5-1.5% Vitamin E 0.5-1.5% Chitosan oligosaccharides 0.2-0.5% Omega-3 fatty acids 0.5-1.5%; Inulin 3-5%.

2. The fecal microbiota capsule freeze-drying protectant for intestinal microbiota transplantation according to claim 1, characterized in that, Includes the following components: Nanoliposomes 2%; Polydextrose 4% Plant selenium protein 0.2% 1% tea polyphenols Vitamin E 1% Chitosan oligosaccharide 0.35% 1% Omega-3 fatty acids; Inulin 4%.

3. The method for preparing the fecal microbiota capsule freeze-drying protectant for intestinal microbiota transplantation according to claim 1, characterized in that: Includes the following steps: (1) Preparation of nanoliposomes: Phosphatidylcholine and cholesterol were dissolved in chloroform at a mass ratio of 3:1; organic solvent was removed by rotary evaporation at 40°C to form a film; PBS buffer (pH 7.4) was added for hydration and incubated overnight at 4°C; liposome suspension with a particle size of <200 nm was obtained by high pressure homogenization (1000 bar, 3 cycles); (2) Pretreatment of water-soluble components: Polydextrose, plant selenoprotein, tea polyphenols and chitosan oligosaccharide were dissolved in sterile water; sterilized by filtration through a 0.22μm filter membrane and stored at 4℃ in the dark; (3) Pretreatment of fat-soluble components: Vitamin E and Omega-3 fatty acids were dissolved in a small amount of ethanol; 1% Tween 80 was added for emulsification, and the mixture was magnetically stirred at 300 rpm for 30 minutes. (4) Mixing: Add 3%-5% polydextrose to PBS buffer to a final volume of 500ml; Add 0.1-0.3% plant selenoprotein and 0.5-1.5% tea polyphenols, stir at 200 rpm for 15 minutes, and adjust the pH to 6.8±0.2; Slowly add 0.5-1.5% vitamin E and 0.5-1.5% Omega-3 fatty acid emulsion, reduce the stirring speed to 100 rpm, and maintain for 30 minutes; Add 0.2-0.5% chitosan oligosaccharide and 3-5% inulin; add 1-3% nanoliposome suspension; stir at 200 rpm for 15 minutes, and filter through a 0.22 μm filter membrane; (5) Freeze-drying: Pre-freeze by step cooling; dry once; dry twice to obtain a fecal microbiota capsule freeze protectant for intestinal microbiota transplantation.

4. The method for preparing the fecal microbiota capsule freeze-drying protectant for intestinal microbiota transplantation according to claim 3, characterized in that: The step (5) cooling process is as follows: maintain 4℃ for 15 minutes; Keep at -20℃ for 1 hour; Keep at -40℃ for 8 hours: Ensure the material is completely cured; the temperature must be 5-10℃ below the eutectic point. Cooling rate: 1℃ / minute.

5. The method for preparing the fecal microbiota capsule freeze-drying protectant for intestinal microbiota transplantation according to claim 3, characterized in that: The drying parameters for step (5) are as follows: Temperature control: -33℃, the temperature of the heating plate must be lower than the eutectic point of the material; Vacuum level: 0.2 mbar (20 Pa); Duration: 36 hours, ensuring over 95% sublimation of free water.

6. The method for preparing the fecal microbiota capsule freeze-drying protectant for intestinal microbiota transplantation according to claim 3, characterized in that: The secondary drying parameters for step (5) are as follows: Temperature: 18℃; Time: 54 hours, ensuring residual moisture content is less than 3%; Endpoint determination: Pressure rise test ΔP < 0.01 mbar / 10 min.