Intestinal bacteria capsule and preparation process thereof

Abstract

The invention relates to the technical field of coprophilous fungus transplantation, in particular to an intestinal fungus capsule and a preparation process thereof. The preparation process comprises the following steps: preparing the fecal fungi; adding a special freeze-drying protective agent and then carrying out freeze-drying treatment; and filling the freeze-dried bacterial powder into a capsule to prepare the intestinal bacterial capsule. The freeze-drying protective agent comprises the following raw materials: cane sugar, whey protein, mannitol, trehalose, lecithin, vitamin C and water. By optimizing a freeze-drying protective agent formula and an accurate freeze-drying process, the freeze-drying survival rate of thalli is remarkably improved, and a test result shows that the survival rate of probiotics such as bifidobacterium and lactic acid bacteria exceeds 90%. The capsule form is adopted to ensure that thalli are accurately released in the intestinal tract, discomfort and thalli loss caused by a traditional transplantation mode are avoided, and the storage stability is effectively improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of fecal microbiota transplantation, and particularly relates to an intestinal bacteria capsule and a preparation process thereof. BACKGROUND

[0002] In recent years, with the deepening of intestinal microbial research, the important role of intestinal flora in the occurrence, development and treatment of various diseases has been gradually discovered. As a treatment method, fecal microbiota transplantation (FMT) can transplant the fecal flora of a healthy donor into the intestinal tract of a patient, thereby reconstructing the intestinal micro-ecosystem and improving intestinal function and restoring intestinal health. At present, fecal microbiota transplantation has been used to treat various diseases, including Clostridium difficile infection, inflammatory bowel disease (such as Crohn's disease and ulcerative colitis), irritable bowel syndrome and metabolic disorder diseases, and has shown good therapeutic effect in clinical application.

[0003] Although the efficacy of fecal microbiota transplantation has been widely recognized, its promotion and application still face many practical problems. On the one hand, the traditional fecal microbiota transplantation method usually needs to use a stomach tube, a gastroscope or a colonoscope to directly introduce the fecal microbiota liquid into the intestinal tract of the patient. This method is complicated to operate, has low patient acceptance, and may even cause psychological resistance. On the other hand, the preparation and storage of the fecal microbiota sample also have technical bottlenecks. At present, the fecal microbiota sample is usually stored at-80℃, but this method has strict storage conditions, high logistics transportation cost, and the activity of the bacteria is easily damaged. In addition, the traditional fecal microbiota freeze-drying technology often causes a high loss rate of bacteria during the freeze-drying process, and the survival rate of the bacterial powder is low, which seriously affects the quality and effectiveness thereof.

[0004] As a new form of fecal microbiota transplantation, the fecal microbiota capsule provides a new idea for solving the problems in the traditional method. The fecal microbiota capsule replaces the traditional stomach tube, gastroscope and colonoscope transplantation operation by using an oral administration method, greatly reduces the discomfort of the patient, simplifies the operation process of the doctor, and improves the convenience of clinical application. At present, the fecal microbiota capsule usually uses an enteric capsule technology to enable the bacteria to avoid inactivation in the stomach acid environment and to be accurately released in the intestinal tract, thereby achieving effective intestinal flora transplantation. However, the existing fecal microbiota capsule technology still has some deficiencies, such as the insufficient optimization of the formula of the freeze-drying protective agent, which causes a low survival rate of the bacteria during the freeze-drying and long-term storage, and the effective storage time of the bacterial powder is relatively short, which is difficult to meet the clinical and commercialization needs. SUMMARY

[0005] The present application aims to provide an intestinal bacteria capsule and a preparation process thereof to solve the problems in the background.

[0006] The present application discloses a preparation process of an intestinal bacteria capsule, which comprises the following steps:

[0007] S1: preparing fecal bacteria;

[0008] S2: adding fecal bacteria transplant special freeze-drying protectant and freeze-drying;

[0009] S3: loading into capsules to obtain the intestinal bacteria capsules;

[0010] The fecal bacteria transplant special freeze-drying protectant in the step S2 includes the following raw materials in percentage by mass:

[0011] The vitamin C effectively provides antioxidant protection to reduce oxidative damage to the cells during freeze-drying; the lecithin serves as a cell membrane protectant to enhance membrane stability and reduce membrane rupture during freeze-drying; the mannitol reduces mechanical damage to the cell membrane during freeze-drying by inhibiting ice crystal formation; the trehalose enhances the stability of the cell membrane and the anti-damage ability of the cells during resuscitation after freeze-drying; the whey protein provides protein support for the cells to further enhance the protective effect of the membrane and reduce damage during freeze-drying; the sucrose provides osmotic pressure protection to prevent water loss from the cell membrane during freeze-drying, thereby stabilizing the cell structure; and the water serves as a solvent to ensure uniform dispersion of the other components and the effectiveness of the freeze-drying protectant. These components synergistically maximize the freeze-drying protection effect and ensure the stability and resuscitation ability of the cells.

[0012] Preferably, the fecal bacteria transplant special freeze-drying protectant includes the following raw materials in percentage by mass:

[0013] 0.05-0.9% vitamin C;

[0014] 0.2-2.0% lecithin;

[0015] 1.0-3.0% mannitol;

[0016] 1.0-5.0% trehalose;

[0017] 2.0-8.0% whey protein;

[0018] 3.0-10.0% sucrose;

[0019] and the balance is water.

[0020] Preferably, the fecal bacteria transplant special freeze-drying protectant includes the following raw materials in percentage by mass:

[0021] 0.1-0.7% vitamin C;

[0022] 0.3-1.7% lecithin;

[0023] 1.2-2.8% mannitol;

[0024] 1.4-4.5% trehalose;

[0025] 2.0-8.0% whey protein;

[0026] 4.0-10.0% sucrose;

[0027] the balance being water.

[0028] Preferably, the freeze-drying protective agent for fecal bacteria transplantation comprises the following raw materials in percentage by mass:

[0029] 0.2-0.5% vitamin C;

[0030] 0.5-1.5% lecithin;

[0031] 1.5-2.5% mannitol;

[0032] 2.0-4.0% trehalose;

[0033] 3.5-6.5% whey protein;

[0034] 5.0-9.0% sucrose;

[0035] the balance being water.

[0036] Further preferably, another membrane phospholipid, phosphatidylserine, can be added in an appropriate amount to the freeze-drying protective agent for fecal bacteria transplantation to further improve its performance. Specifically, the freeze-drying protective agent for fecal bacteria transplantation comprises the following raw materials in percentage by mass:

[0037] 0.2-0.5% vitamin C;

[0038] 0.3-0.7% phosphatidylserine;

[0039] 0.5-1.5% lecithin;

[0040] 1.5-2.5% mannitol;

[0041] 2.0-4.0% trehalose;

[0042] 3.5-6.5% whey protein;

[0043] 5.0-9.0% sucrose;

[0044] the balance being water.

[0045] Preferably, the mass ratio of the fresh fecal sample to the freeze-drying protective agent for fecal bacteria transplantation is 100:(80-120).

[0046] The freeze-drying protective agent for fecal bacteria transplantation can be prepared by stirring and mixing the above raw materials uniformly.

[0047] Preferably, the capsule in the step S3 is an HPMC capsule.

[0048] The present application also discloses an intestinal bacteria capsule prepared by the above method. The intestinal bacteria capsule prepared by the present application can be widely applied to intestinal diseases such as inflammatory bowel disease (IBD), Clostridium difficile infection, irritable bowel syndrome (IBS), chronic constipation or diarrhea, etc., and effectively improves the symptoms of patients by reestablishing the balance of intestinal flora. In the treatment of metabolic diseases such as obesity, type 2 diabetes and non-alcoholic fatty liver disease (NAFLD), the capsule promotes metabolic balance by regulating the intestinal microbiome. In addition, it can also be used for adjuvant therapy of immune-related diseases, including allergic diseases, certain autoimmune diseases, and nervous system diseases such as depression, autism spectrum disorder (ASD) and Parkinson's disease, and shows significant intervention potential.

[0049] The intestinal bacteria capsule and its preparation process provided by the present application significantly improve the survival rate of bacterial cells during freeze-drying by optimizing the formulation of freeze-drying protectants and precise freeze-drying process. The test results show that the survival rate of probiotics such as Bifidobacterium and Lactobacillus is more than 90%, which is much higher than the traditional technical level. The capsule form not only ensures the accurate release of bacterial cells in the intestinal tract, avoids the discomfort and loss of bacterial cells caused by traditional transplantation methods for patients, but also effectively improves the storage stability, prolongs the storage time of bacterial powder to more than one year, and significantly reduces the psychological and operational burden of patients and operators. DETAILED DESCRIPTION

[0050] Vitamin C, product number: L65550, purchased from Shanghai Jizisheng Biotechnology Co., Ltd.

[0051] Lecithin, product number: 1043895, purchased from Shanghai Haohong Biomedical Technology Co., Ltd.

[0052] Mannitol, product number: 1105019, purchased from Shanghai Haohong Biomedical Technology Co., Ltd.

[0053] Trehalose, product number: D14630, purchased from Shanghai Jizisheng Biotechnology Co., Ltd.

[0054] Whey protein, product number: YB124936, purchased from Hunan Yunbang Biotechnology Co., Ltd.

[0055] Sucrose, product number: S-71320, purchased from Tianjin Xinsisheng Biotechnology Co., Ltd.

[0056] Phosphatidylserine, product number: LMM03115, purchased from Nanjing Luomai Biotechnology Co., Ltd.

[0057] Example 1:

[0058] A preparation process of an intestinal bacteria capsule, comprising the following steps:

[0059] S1: 100 grams of donor fresh fecal sample is uniformly mixed with 500 grams of 0.9wt% NaCl aqueous solution, filtered with a 0.4mm filter screen, and the filtrate is collected; the filtrate is centrifuged at 3000rpm for 15 minutes, and the precipitate is collected to obtain a fecal bacteria material;

[0060] S2: 100 grams of freeze-drying protective agent for fecal bacteria transplantation is added to the fecal bacteria material, stirred at 300rpm for 10 minutes to uniformly disperse the fecal bacteria material in the freeze-drying protective agent for fecal bacteria transplantation, and vacuum drying is performed at a vacuum degree of 10Pa and a temperature of-50℃ for 36 hours by using a Christ Epsilon 2-6D LSC plus freeze dryer to obtain a freeze-dried powder;

[0061] The formula of the freeze-drying protective agent for fecal bacteria transplantation is: 0.3 grams of vitamin C, 1.0 gram of lecithin, 2.0 grams of mannitol, 3.0 grams of trehalose, 5.0 grams of whey protein, 7.0 grams of sucrose, and the balance is water; and the total amount of the freeze-drying protective agent for fecal bacteria transplantation is ensured to be 100 grams. The above raw materials are uniformly stirred and mixed at room temperature to obtain the freeze-drying protective agent for fecal bacteria transplantation.

[0062] S3: The freeze-dried powder is loaded into DRcaps TM The acid-resistant HPMC capsule contains 350mg of freeze-dried powder per capsule, and the intestinal bacteria capsule is finally obtained and stored at-80℃.

[0063] Example 2:

[0064] The difference between Example 1 and Example 2 is that the formula of the freeze-drying protective agent for fecal bacteria transplantation is different. Specifically, the formula of the freeze-drying protective agent for fecal bacteria transplantation is: 0.3 grams of vitamin C, 1.5 grams of lecithin, 2.0 grams of mannitol, 3.0 grams of trehalose, 5.0 grams of whey protein, 7.0 grams of sucrose, and the balance is water; and the total amount of the freeze-drying protective agent for fecal bacteria transplantation is ensured to be 100 grams.

[0065] Example 3:

[0066] The difference between Example 1 and Example 3 is that the formula of the freeze-drying protective agent for fecal bacteria transplantation is different. Specifically, the formula of the freeze-drying protective agent for fecal bacteria transplantation is: 0.3 grams of vitamin C, 0.5 grams of phosphatidylserine, 1.0 gram of lecithin, 2.0 grams of mannitol, 3.0 grams of trehalose, 5.0 grams of whey protein, 7.0 grams of sucrose, and the balance is water; and the total amount of the freeze-drying protective agent for fecal bacteria transplantation is ensured to be 100 grams.

[0067] Comparative Example 1:

[0068] The difference between Example 1 and the present application is that the freeze-drying protective agent used in the present application is different. Specifically, the freeze-drying protective agent used in the present application is prepared by mixing 20 g of skimmed milk powder, 1 g of sucrose, 3 g of gelatin, and 76 g of water at room temperature.

[0069] Comparative Example 2:

[0070] The difference between Example 1 and the present application is that the freeze-drying protective agent used in the present application is different. Specifically, the freeze-drying protective agent used in the present application is prepared by mixing 20 g of skimmed milk powder, 1 g of sucrose, 3 g of gelatin, and 76 g of water at room temperature.

[0071] Test Example:

[0072] Method for determining the survival rate of freeze-drying:

[0073] (1) Detection of viable bacteria before freeze-drying:

[0074] An appropriate amount of the sample to be tested is diluted step by step according to the gradient dilution method, and 1 ml of sample solution is taken at each dilution gradient. The sample is evenly distributed in the culture medium using the pour plate count method. After incubating the plate at 37°C for 24 hours, the number of colonies on the plate is counted. According to the dilution factor and the number of colonies, the total number of viable bacteria before freeze-drying (CFU / g) is calculated.

[0075] (2) Detection of viable bacteria after freeze-drying:

[0076] The freeze-dried sample is added with an appropriate amount of sterile 0.9wt% NaCl aqueous solution, and the original volume before freeze-drying is made up to restore the sample to the state before freeze-drying. The same gradient dilution treatment is performed on the supplemented sample solution, and the viable bacteria count (CFU / g) of the recovered sample is detected using the pour plate count method. Ensure that the culture conditions are consistent with those before freeze-drying (37°C, incubation for 24 hours).

[0077] Calculation of freeze-drying survival rate:

[0078]

[0079] Table 1: Bacterial survival rate test table

[0080] Survival rate of lactic acid bacteria, % Survival rate of bifidobacteria, % Example 1 96.2 90.9 Example 2 97.1 91.6 Example 3 98.5 94.7 Comparative Example 1 92.3 80.7 Comparative Example 2 91.4 80.5

[0081] The freeze-drying protective agent for fecal bacteria transplantation used in embodiment 1 of the present application significantly improves the survival rate of bacteria in the freeze-drying process through the precise combination and synergistic effect of multiple components. Vitamin C, as a high-efficiency antioxidant, reduces the oxidative damage to the cell membrane in the freeze-drying process and protects the activity of bacteria; lecithin prevents the membrane from rupturing under the condition of dehydration by enhancing the stability of the cell membrane; and mannitol reduces the mechanical damage of ice crystals to the cell membrane by virtue of its ability to inhibit the formation of ice crystals. These core components jointly act from antioxidant, membrane protection to physical damage protection, and multiple levels ensure the integrity and stability of bacteria in the freeze-drying process. In addition, trehalose plays an important role in improving the stability of the cell membrane and enhancing the anti-damage ability of recovery; whey protein provides necessary protein support for cells and cooperates with membrane phospholipid to further strengthen the protection effect of the membrane; and sucrose, as an osmotic pressure regulator, effectively prevents the loss of water in the cell membrane during the freeze-drying process and maintains the structural stability of the membrane.

[0082] Compared with the simple formula in Comparative Example 1 which depends on skimmed milk powder, sucrose and gelatin alone, the protective agent of embodiment 1 of the present application exhibits stronger multi-dimensional protection ability and significantly improves the survival rate of bacteria, especially the survival rate of Bifidobacterium and Lactobacillus, which are both more than 90%. Through the comprehensive optimization of antioxidant, membrane stabilization and rehydration protection, the protective agent of the present application effectively makes up for the shortcomings of traditional technology and has significant improvement in technical performance and practical application.

[0083] The freeze-drying survival rate of embodiment 3 is significantly better than that of embodiment 2 and Comparative Example 2, and the experimental results show that phosphatidylserine (PS) and lecithin (PC) may have a synergistic effect, and the mechanism may be derived from the functional complementation of the two in membrane protection. PS may reduce the mechanical damage of dehydration and ice crystal formation to the membrane by enhancing the stability of the cell membrane and maintaining charge balance, while providing certain antioxidant protection; PC may ensure the structural integrity and functional recovery of the cell membrane in the dehydration and rehydration process by improving the fluidity and flexibility of the membrane. The two components may ensure the stability of the membrane while enhancing its flexibility at a moderate ratio, avoiding the membrane hardening that may be caused by the use of PS alone and making up for the lack of antioxidant protection when PC is used alone, thereby forming a possible optimal protection effect. The above speculation needs to be verified through further experiments to clarify the specific synergistic mechanism of the two.

Claims

1. A preparation process of enterobacteria capsule, characterized in that: The steps include: S1: Preparation of fecal microbiota; S2: Add a freeze-drying protective agent specifically for fecal microbiota transplantation and freeze-dry; S3: filling the capsule to obtain the enterobacteria capsule; The raw materials of the freeze-dried protective agent for fecal microbiota transplantation in step S2 include sucrose, whey protein, mannitol, trehalose, lecithin, vitamin C and water.

2. The preparation process of the enterobacteria capsule according to claim 1, characterized in that: The fecal microbiota transplantation-specific freeze-dried protective agent comprises the following raw materials, calculated by mass percentage: 0.05-0.9% vitamin C; 0.2-2.0% lecithin; 1.0-3.0% mannitol; 1.0-5.0% trehalose; 2.0-8.0% whey protein; 3.0-10.0% sucrose; The balance is water.

3. The preparation process of the enterobacteria capsule according to claim 2, characterized in that: The fecal microbiota transplantation-specific freeze-dried protective agent comprises the following raw materials, calculated by mass percentage: 0.1-0.7% vitamin C; 0.3-1.7% lecithin; 1.2-2.8% mannitol; 1.4-4.5% trehalose; 2.0-8.0% whey protein; 4.0-10.0% sucrose; The balance is water.

4. The preparation process of the enterobacteria capsule according to claim 2, characterized in that: The fecal microbiota transplantation-specific freeze-dried protective agent comprises the following raw materials, calculated by mass percentage: 0.2-0.5% vitamin C; 0.5-1.5% lecithin; 1.5-2.5% mannitol; 2.0-4.0% trehalose; 3.5-6.5% whey protein; 5.0-9.0% sucrose; The balance is water.

5. The preparation process of the enterobacteria capsule according to any one of claims 1 to 4, characterized in that: The mass ratio of the fresh stool sample to the freeze-dried protective agent for fecal microbiota transplantation is 100:(80-120).

6. The process for preparing the enterobacteria capsule according to any one of claims 1 to 4, characterized in that: Sucrose, whey protein, mannitol, trehalose, lecithin, vitamin C and water are stirred and mixed evenly to obtain the freeze-dried protective agent specially used for fecal microbiota transplantation.

7. The process for preparing the enterobacteria capsule according to any one of claims 1 to 4, characterized in that: The capsule in step S3 is an HPMC capsule.

8. An enterobacteria capsule, characterized in that: The invention is prepared by the method according to any one of claims 1 to 7.

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