Freeze-drying protective agent, preparation method thereof, intestinal fecal bacteria freeze-drying process and application of intestinal fecal bacteria freeze-drying process

Abstract

The invention discloses a freeze-drying protective agent which comprises glycerol, skimmed milk powder, mannitol, glutathione, inulin and sterile water. By optimizing the components of the freeze-drying protective agent, the activity of the coprophilous fungi can be effectively protected, the survival rate of the intestinal coprophilous fungi in the freeze-drying process is remarkably improved, meanwhile, the coprophilous fungi can be kept stable for a long time after being freeze-dried, long-term storage and transportation are facilitated, the functional stability of the intestinal coprophilous fungi in the freeze-drying process is remarkably improved, and the survival rate of the intestinal coprophilous fungi in the freeze-drying process is improved. The invention further discloses a freeze-drying process of the coprophilous fungus freeze-drying protective agent, by accurately controlling parameters of all stages, damage to coprophilous fungi is reduced to the maximum extent, the activity of the coprophilous fungi is effectively protected, the quality and stability of freeze-dried products are ensured, and meanwhile the freeze-drying process is simple, convenient, easy to operate and suitable for large-scale production.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and more specifically, to a freeze-drying protectant and a preparation method thereof, as well as a freeze-drying process and application of intestinal fecal bacteria. Background Art

[0002] In the field of modern medicine, intestinal microbiota transplantation (FMT) has attracted widespread attention as an innovative treatment method. By transplanting fecal microbiota from healthy donors into the recipient's intestines and regulating the balance of the recipient's intestinal flora, it can have a therapeutic effect on a variety of intestinal and extraintestinal diseases, such as inflammatory bowel disease, metabolic syndrome, Clostridium difficile infection, etc. A large number of clinical studies and basic research have confirmed the important role of intestinal flora in human health and the occurrence and development of diseases, as well as the potential application value of FMT. However, to achieve effective intestinal microbiota transplantation, it is key to ensure that the fecal bacteria can survive, colonize and function well in the recipient's intestines after transplantation, and the survival rate and functionality of fecal bacteria during collection, preparation and transplantation face many challenges.

[0003] Freeze-drying technology is a commonly used method for preserving microorganisms and is widely used for long-term storage and transportation of microorganisms. However, in the treatment of intestinal fecal bacteria, the existing freeze-drying technology has many problems:

[0004] 1. The traditional freeze-drying process lacks targeted fecal bacteria protection measures, which makes the fecal bacteria susceptible to damage during the freeze-drying process, reduces the survival rate, and thus affects the effect after transplantation.

[0005] 2. Existing freeze-dried protective agents have poor stability during long-term storage or under different storage conditions, which limits their operability in clinical applications. In addition, the components of the protective agents are often complex, and the synergistic mechanism between different substances is not yet clear, resulting in uncertainty in the protective effect. Therefore, there is a lack of a freeze-dried protective agent for intestinal fecal bacteria with clear ingredients, significant protective effect and suitable for clinical application.

[0006] 3. Although the traditional freeze-drying process can maintain the activity of fecal bacteria to a certain extent, due to the high requirements of fecal bacteria for low temperature and low oxygen environment, complex and expensive equipment is often required in actual operation, which increases production costs.

[0007] 4. Although some studies have attempted to improve the traditional freeze-drying process, such as optimizing freeze-drying parameters and adding different protective agents, the results are still not ideal, and it is difficult to take into account multiple factors such as cost, operational complexity and fecal bacteria survival rate at the same time.

[0008] In order to improve the survival rate and functional stability of intestinal fecal bacteria during freeze-drying and transplantation, it is of great significance to develop suitable freeze-drying protectants and freeze-drying processes. Summary of the Invention

[0009] In order to solve at least one of the above technical problems, the present invention provides a freeze-drying protectant and a preparation method thereof, as well as a freeze-drying process for intestinal fecal bacteria using the freeze-drying protectant and a preparation method of a freeze-dried preparation of intestinal fecal bacteria.

[0010] The first aspect of the present invention provides a freeze-drying protective agent, comprising glycerol, skim milk powder, mannitol, glutathione, inulin, and sterile water.

[0011] In some embodiments, by mass percentage, glycerol is 10%-20%, skim milk powder is 2%-8%, mannitol is 1%-6%, glutathione is 0.1%-0.2%, inulin is 5%-7.5%, and the balance is sterile water.

[0012] In some embodiments, by mass percentage, glycerol is 15%, skim milk powder is 8%, mannitol is 3%, glutathione is 0.15%, inulin is 6%, and the balance is sterile water.

[0013] The second aspect of the present invention provides a method for preparing the above-mentioned lyoprotectant, comprising the following steps:

[0014] S1: Dissolve skim milk powder in sterile water and stir until completely dissolved;

[0015] S2: Add glycerol, mannitol, glutathione and inulin in sequence and continue stirring until mixed evenly.

[0016] The third aspect of the present invention provides the use of the above-mentioned lyoprotectant in the preparation of a biomedical product for intestinal flora transplantation.

[0017] A fourth aspect of the present invention provides a freeze-drying process for intestinal fecal bacteria, using the above-mentioned freeze-drying protectant for freeze-drying.

[0018] In some embodiments, a freeze-drying process for intestinal fecal bacteria specifically comprises the following steps:

[0019] S1: Pre-freezing stage: Freeze the mixture of lyophilized protective agent and fecal microbial solution at -50℃ for 3.5-4.5 hours; fecal microbial solution is prepared by the following method:

[0020] A1: Mix stool samples from screened healthy donors with sterile saline at a mass-to-volume ratio of 1:5, homogenize with stirring, and perform graded filtration.

[0021] A2: Centrifuge the filtered bacterial solution at 3500 rpm for 5 minutes, remove the supernatant, wash with sterile saline, and resuspend. Repeat the centrifugation and washing steps 2-3 times to obtain the fecal bacterial solution.

[0022] S2: Primary drying stage: maintain vacuum degree ≤10Pa, raise temperature to -30℃, and last for 9.5-10.5 hours;

[0023] S3: Secondary drying stage: maintain vacuum degree ≤10Pa, raise temperature to 25℃, and continue for 7.5-8.5 hours.

[0024] In some embodiments, a freeze-drying process for intestinal fecal bacteria specifically comprises the following steps:

[0025] S1: Pre-freezing stage: The mixture of lyophilized protective agent and fecal microbial solution was frozen at -50°C for 4 hours; the fecal microbial solution was prepared by the following method:

[0026] A1: Mix stool samples from screened healthy donors with sterile saline at a mass-to-volume ratio of 1:5, homogenize with stirring, and perform graded filtration.

[0027] A2: Centrifuge the filtered bacterial solution at 3500 rpm for 5 minutes, remove the supernatant, wash with sterile saline, and resuspend. Repeat the centrifugation and washing steps 2-3 times to obtain the fecal bacterial solution.

[0028] S2: Primary drying stage: maintain vacuum degree ≤10Pa, raise temperature to -30℃, and continue for 10 hours;

[0029] S3: Secondary drying stage: maintain vacuum degree ≤10Pa, raise temperature to 25℃, and continue for 8 hours.

[0030] A fifth aspect of the present invention provides the use of the above-mentioned freeze-drying process in the preparation of biomedical products for intestinal flora transplantation.

[0031] A sixth aspect of the present invention provides a method for preparing a freeze-dried preparation of intestinal fecal bacteria, comprising the following steps:

[0032] S1: Preparing a lyoprotectant: The preparation of the lyoprotectant comprises the following steps: dissolving 5% skim milk powder in 50% sterile water, by mass percentage, and stirring until completely dissolved; adding 15% glycerol, 3% mannitol, 0.15% glutathione, and 6% inulin in sequence, and continuously stirring until uniformly mixed, and making up the balance with sterile water;

[0033] S2: Mix the fecal bacteria liquid and the freeze-dried protective agent in a volume ratio of 1:1 to obtain a fecal bacteria-protective agent mixed solution; the fecal bacteria liquid is prepared by the following method:

[0034] A1: Mix stool samples from screened healthy donors with sterile saline at a mass-to-volume ratio of 1:5, homogenize with stirring, and perform graded filtration.

[0035] A2: Centrifuge the filtered bacterial solution at 3500 rpm for 5 minutes, remove the supernatant, wash with sterile saline, and resuspend. Repeat the centrifugation and washing steps 2-3 times to obtain the fecal bacterial solution.

[0036] S3: Freeze-drying: Pre-freezing stage: Freeze the fecal bacteria-protectant mixture at -50℃ for 4 hours; primary drying stage: maintain the vacuum degree ≤10Pa, raise the temperature to -30℃, and continue for 10 hours; secondary drying stage: maintain the vacuum degree ≤10Pa, raise the temperature to 25℃, and continue for 8 hours to obtain the intestinal fecal bacteria freeze-dried preparation.

[0037] Repeated centrifugation and washing during the preparation of the fecal bacteria in the present invention can ensure that the obtained fecal bacteria liquid is clear and transparent, thereby reducing later side effects.

[0038] The components of the freeze-drying protectant in the present invention have the following synergistic effects: glycerol and skim milk powder synergistically form a composite protective layer, glycerol reduces the formation of ice crystals in cells, and skim milk powder stabilizes the extracellular matrix; among glutathione and inulin, the former neutralizes oxidative stress, and the latter provides a carbon source, accelerating the metabolic recovery of the flora during the recovery period. Without affecting the activity of fecal bacteria, it can effectively resist the damage to fecal bacteria caused by adverse environmental factors such as low temperature and hypoxia during the freeze-drying process, and significantly improve the survival rate of fecal bacteria after the freeze-drying operation, thereby ensuring the colonization and function of fecal bacteria in the recipient's intestine after transplantation.

[0039] The freeze-drying process of the present invention has the following synergistic effects: first, the pre-freezing temperature is set at -50°C, and the temperature is rapidly lowered to form fine ice crystals (DSC analysis shows that the ice crystal diameter is <5μm), thereby reducing cell damage. After the pre-freezing is completed, a sublimation drying stage is carried out, in which free water is slowly sublimated under low vacuum (sublimation rate ≤0.5g / h) to avoid collapse; and bound water is desorbed by heating to ensure that the final water content is ≤2.5%.

[0040] Unless otherwise specified, the experimental methods used in the present invention are all conventional methods.

[0041] Unless otherwise specified, the raw materials and materials used in the present invention can be obtained through commercial channels.

[0042] Compared with the prior art, the beneficial effects of the present invention are embodied in:

[0043] 1. The present invention can effectively protect the activity of fecal bacteria and significantly improve the survival rate of intestinal fecal bacteria during the freeze-drying process by optimizing the freeze-drying protectant components.

[0044] 2. The freeze-drying protectant of the present invention enables fecal bacteria to remain stable for a long time after freeze-drying, facilitating long-term storage and transportation, and significantly improving the functional stability of intestinal fecal bacteria during the freeze-drying process.

[0045] 3. The freeze-drying process of the fecal bacteria freeze-drying protectant provided by the present invention minimizes fecal bacteria damage by precisely controlling the parameters at each stage, effectively protects the activity of fecal bacteria, ensures the quality and stability of the freeze-dried product, and has a short freeze-drying time, making it suitable for industrial production.

[0046] 4. The preparation method of the lyoprotectant, lyophilization process and preparation method of the freeze-dried preparation of intestinal fecal bacteria provided by the present invention are simple and easy to operate, and are suitable for large-scale production. The technical solution provided by the present invention is particularly suitable for the treatment of diseases such as recurrent Clostridium difficile infection, inflammatory bowel disease, metabolic syndrome, etc., and has important clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 These are fluorescent graphs of bacterial viability distribution obtained by analyzing the fecal bacteria in each group of Example 5 after freeze-drying using a bacterial viability meter, wherein Figure A is a traditional protective agent, Figure B is an 8% glycerol group, Figure C is a 10% glycerol group, Figure D is a 15% glycerol group, Figure E is a 20% glycerol group, and Figure F is a 22% glycerol group.

[0048] Figure 2 This is a fluorescent graph of the bacterial viability distribution obtained by analyzing the fecal bacteria-lyophilized protective agent mixture in Example 7 with a bacterial viability meter after freeze-drying, wherein Figure A is experimental group 1, Figure B is experimental group 2, Figure C is experimental group 3, and Figure D is the control group.

[0049] Figure 3 This is a fluorescent graph of bacterial viability distribution obtained by analyzing the fecal bacteria-lyophilized protective agent mixture in Example 8 using a bacterial viability meter after freeze-drying, wherein Figure A is Group A and Figure B is Group B. DETAILED DESCRIPTION

[0050] In order to make the technical means, creative features, objectives and effects of the invention easier to understand, the present invention is further described in conjunction with specific implementation methods and drawings, but the present invention is not limited to the following implementation cases.

[0051] Example 1 Preparation of Lyoprotectant

[0052] This embodiment provides a lyoprotectant, and the preparation of the lyoprotectant comprises the following steps:

[0053] Weighing of raw materials: Weigh the following components by mass percentage: 15% glycerol, 8% skim milk powder, 3% mannitol, 0.15% glutathione, and 6% inulin.

[0054] Dissolving and mixing: Add skim milk powder to 50% of the total amount of sterile water and stir in a 40°C constant temperature water bath until completely dissolved (approximately 30 minutes). Add glycerol, mannitol, glutathione, and inulin in sequence, stirring continuously until well mixed. Add sterile water to 100% and stir for 10 minutes.

[0055] Example 2 Standardized preparation of fecal bacteria liquid

[0056] Stool sample processing

[0057] Take an appropriate amount of fecal sample from a healthy donor and mix it with 5 volumes of sterile saline (1:5, w / v). Homogenize it using a blender for 10 minutes.

[0058] Hierarchical filtration

[0059] Filter through four levels of pore size filters (pore size decreasing: 500 μm → 200 μm → 100 μm → 50 μm) to remove large particles of impurities. Collect the filtrate and store at 4°C for later use.

[0060] Centrifugal washing

[0061] The filtrate was centrifuged at 3500 rpm and 4°C for 5 minutes, and the supernatant was discarded. The pellet was resuspended in sterile saline and centrifuged and washed three times to obtain a clarified fecal bacterial solution (viable bacterial count ≥ 1 × 10^10 CFU / mL).

[0062] Example 3 Freeze-drying process

[0063] S1: Pre-freezing stage: Freeze the mixture of the lyophilized protective agent and the fecal bacteria solution obtained in Example 2 at -50°C for 4 hours;

[0064] S2: Primary drying stage: maintain vacuum degree ≤10Pa, raise temperature to -30℃, and continue for 10 hours;

[0065] S3: Secondary drying stage: maintain vacuum degree ≤10Pa, raise temperature to 25℃, and continue for 8 hours.

[0066] Example 4 Preparation Method of Intestinal Faecium Freeze-dried Preparation

[0067] S1: Preparation of lyoprotectant: The preparation of lyoprotectant comprises the following steps: dissolving 8% skim milk powder in 50% sterile water by mass percentage and stirring until completely dissolved; adding 15% glycerol, 3% mannitol, 0.15% glutathione and 6% inulin in sequence, stirring continuously until uniformly mixed, and making up the remainder with sterile water.

[0068] S2: The fecal bacteria solution obtained in Example 2 was mixed with a freeze-dried protective agent in a volume ratio of 1:1 to obtain a fecal bacteria-protective agent mixed solution;

[0069] S3: Freeze-drying: Pre-freezing stage: Freeze the fecal bacteria-protectant mixture at -50℃ for 4 hours; primary drying stage: maintain the vacuum degree ≤10Pa, raise the temperature to -30℃, and continue for 10 hours; secondary drying stage: maintain the vacuum degree ≤10Pa, raise the temperature to 25℃, and continue for 8 hours to obtain the intestinal fecal bacteria freeze-dried preparation.

[0070] Example 5 Effect of different glycerol concentrations on fecal bacteria survival rate

[0071] Experimental design

[0072] Control group: traditional protective agent group (containing trehalose 8%, sucrose 5%, and the balance being sterile water).

[0073] Experimental group: divided into 5 groups, with glycerol concentrations of 8%, 10%, 15%, 20%, and 22% respectively. The remaining components and concentrations were the same as in Example 1, and the balance was sterile water.

[0074] Freeze-drying process: The parameters described in Example 3 were followed (pre-freezing at -50°C / 4h, primary drying at -30°C / 10h, and secondary drying at 25°C / 8h).

[0075] Experimental methods

[0076] Fecal bacteria liquid from the same source (preparation method see Example 2) was mixed with each protective agent at a volume ratio of 1:1. After freeze-drying, it was redissolved in sterile physiological saline and the survival rate was determined using a bacteria counter (fluorescence method).

[0077] The experimental results are shown in Table 1 and Figure 1 ( Figure 1 Figure 3 is a fluorescent graph of bacterial viability distribution obtained by analysis with a bacterial viability meter, wherein Figure A is the traditional protectant group, Figure B is the 8% glycerol group, Figure C is the 10% glycerol group, Figure D is the 15% glycerol group, Figure E is the 20% glycerol group, and Figure F is the 22% glycerol group, where green represents surviving bacteria and red represents dead bacteria).

[0078] Table 1

[0079]

[0080]

[0081] Conclusion: As can be seen from Table 1, the 15% glycerol group had the highest survival rate after freeze-drying, and the experimental groups (8% glycerol group, 10% glycerol group, 15% glycerol group, 20% glycerol group, 22% glycerol group) had significantly better survival rates after freeze-drying than the control group (traditional protectant group). It can be seen that the freeze-dried protectant component of the present invention does have the effect of significantly improving the survival rate of fecal bacteria after freeze-drying. In comparison within the experimental group with added glycerol, the protective effect of glycerol at a concentration of 10-20% was above 70%. When the glycerol concentration exceeded 20% and was lower than 10%, the survival rate after freeze-drying showed a downward trend (survival rate was lower than 70%), so the glycerol concentration between 10-20% is considered to be the optimal concentration range.

[0082] Example 6 Effect of different skim milk powder concentrations on fecal bacteria survival rate

[0083] Control group: traditional protective agent group (containing trehalose 8%, sucrose 5%, and the balance being sterile water).

[0084] Experimental group: divided into 5 groups, with skim milk powder concentrations of 0%, 2%, 5%, 8%, and 10% respectively. The remaining components and concentrations were the same as those in Example 1, and the balance was sterile water.

[0085] Freeze-drying process: The parameters described in Example 3 were followed (pre-freezing at -50°C / 4h, primary drying at -30°C / 10h, and secondary drying at 25°C / 8h).

[0086] Experimental methods

[0087] Fecal bacteria liquid from the same source (preparation method see Example 2) was mixed with each protective agent at a volume ratio of 1:1. After freeze-drying, it was redissolved in sterile physiological saline and the survival rate was determined using a bacteria counter (fluorescence method).

[0088] The experimental results are shown in Table 2.

[0089] Table 2

[0090]

[0091]

[0092] Conclusion: As can be seen from Table 1, the 8% skim milk powder group had the highest survival rate after freeze-drying, and the experimental groups (0% skim milk powder group, 2% skim milk powder group, 5% skim milk powder group, 8% skim milk powder group, 10% skim milk powder group) had significantly better survival rates after freeze-drying than the control group (traditional protectant group). It can be seen that the freeze-dried protectant component of the present invention does have the effect of significantly improving the survival rate of fecal bacteria after freeze-drying. In comparison with the experimental groups to which skim milk powder was added, the protective effect of skim milk powder at a concentration of 2-8% was above 75%. When the concentration of skim milk powder exceeded 8% and was lower than 2%, the survival rate after freeze-drying showed a decreasing trend (survival rate was lower than 75%), so the skim milk powder concentration between 2-8% was considered to be the optimal concentration range.

[0093] Example 7 Comparative Experiment on Freeze-dried Protectant Formula

[0094] Experimental design

[0095] Control group: Contains only skim milk powder (8%) and glycerol (15%), with the remainder being sterile water.

[0096] Experimental group 1: the formulation of Example 1.

[0097] Experimental Group 2: The glycerin component was removed from the formulation of Example 1.

[0098] Experimental group 3: The skim milk powder component was removed from the formula of Example 1.

[0099] Experimental method: Fecal bacteria liquid from the same source (preparation method see Example 2) was mixed with each group of lyophilization protectants at a volume ratio of 1:1. After freeze-drying, it was re-dissolved in sterile saline and the survival rate was determined using a bacteria counter (fluorescence method).

[0100] The test results are shown in Table 3 and Figure 2 ( Figure 2 Figure 1 is a fluorescent graph of bacterial viability distribution obtained by analysis with a bacterial viability meter. Figure A is experimental group 1, Figure B is experimental group 2, Figure C is experimental group 3, and Figure D is the control group. Green represents surviving bacteria, and red represents dead bacteria.

[0101] Table 3

[0102]

[0103] Conclusion: According to Table 3 and Figure 2 The survival rate of fecal bacteria after freeze-drying in the freeze-dried lyoprotectant formulation of experimental group 1 (86.4%) was significantly higher than that in the control group (54.5%). This indicates that the freeze-dried lyoprotectant components of the present invention have a synergistic effect and can indeed improve the survival rate of fecal bacteria. Furthermore, the freeze-dried survival rate of experimental group 1 when glycerol and skim milk powder were absent was far lower than when both were present, demonstrating the importance of the combined effect of the two components.

[0104] Example 8 Freeze-drying Process Optimization Verification

[0105] Experimental design: The protective agent formula (Example 1) was fixed, and the fecal bacteria-protectant mixture was prepared and divided into two portions. The freeze-drying parameters were adjusted:

[0106] Group A: pre-freezing at -50°C / 4h → primary drying at -30°C / 10h → secondary drying at 25°C / 8h (same as Example 2).

[0107] Group B: pre-freezing at -40℃ / 3h→primary drying at -25℃ / 9h→secondary drying at 30℃ / 6h (traditional process).

[0108] The experimental results are shown in Table 4 and Figure 3 ( Figure 3 Figure 1 is a fluorescent graph of bacterial viability distribution obtained by analysis with a bacterial viability meter. Figure A is group A, and Figure B is group B. Green represents surviving bacteria, and red represents dead bacteria.

[0109] Table 4

[0110]

[0111] Conclusion: According to Table 4 and Figure 3 It can be seen that the survival rate of group A (Example 2) after freeze-drying is significantly higher than that of group B (traditional process). Therefore, it can be concluded that the freeze-drying process of the present invention does have the effect of significantly improving the survival rate of bacteria after freeze-drying.

[0112] Example 9 Effects of different freeze-dried protective agent combinations on the survival rate of freeze-dried fecal bacteria

[0113] Experimental design:

[0114] Group 1: 15% glycerol + 8% skim milk powder, the balance is sterile water;

[0115] Group 2: 15% glycerol + 8% skim milk powder + 3% mannitol, the balance is sterile water;

[0116] Group 3: 15% glycerol + 8% skim milk powder + 3% mannitol + 0.15% glutathione, the balance is sterile water;

[0117] Group 4: 15% glycerol + 8% skim milk powder + 3% mannitol + 0.15% glutathione + 6% inulin, the balance being sterile water (same as Example 1).

[0118] Experimental method: Fecal bacteria liquid from the same source (preparation method see Example 2) was mixed with the freeze-dried protective agent of each group at a volume ratio of 1:1. After freeze-drying, it was re-dissolved in sterile saline and the survival rate was determined using a bacterial counter (fluorescence method).

[0119] The experimental results are shown in Table 5.

[0120] Table 5

[0121]

[0122] Conclusion: It can be seen from Table 5 that as the components of the lyoprotectant tend to the components of Example 1, the survival rate of fecal bacteria after freeze-drying is significantly improved, and the effect of Group 4 (Example 1) is the best (86.4%). It can be concluded that the lyoprotectant components of the present invention do have the effect of significantly improving the survival rate of fecal bacteria after freeze-drying.

[0123] Example 10 Lyoprotectant and Lyophilization Process Stability

[0124] Experimental design:

[0125] The optimal protective agent formula of the present invention (Example 1) was mixed with fecal bacteria samples and divided into five groups on average. After freeze-drying using the freeze-drying process of Example 2 (pre-freezing -50°C / 4h→primary drying -30°C / 10h→secondary drying 25°C / 8h), the survival rate of fecal bacteria after freeze-drying was measured.

[0126] The experimental results are shown in Table 6.

[0127] Table 6

[0128]

[0129] Conclusion: It can be seen from Table 6 that the lyophilized preparation of fecal bacteria prepared by the lyophilization protectant provided in Example 1 and the lyophilization process of Example 2 has a high survival rate and strong stability. It can be concluded that the lyophilized protectant and lyophilization process of the present invention effectively improve the activity of fecal bacteria and do have the effect of improving the survival rate of fecal bacteria after lyophilization and maintaining detection stability.

[0130] Example 11 Long-term storage stability of lyoprotectants

[0131] Experimental design:

[0132] The freeze-dried preparation of intestinal fecal bacteria was stored at -80°C, and the number of viable bacteria was measured at 0 days, 15 days, 30 days, and 60 days.

[0133] Experimental method: (same as Example 4)

[0134] S1: Preparation of lyoprotectant: The preparation of lyoprotectant comprises the following steps: dissolving 8% skim milk powder in 50% sterile water by mass percentage and stirring until completely dissolved; adding 15% glycerol, 3% mannitol, 0.15% glutathione and 6% inulin in sequence, stirring continuously until mixed evenly, and making up the balance with sterile water.

[0135] S2: Mixing the fecal bacteria solution and the freeze-dried protective agent in a volume ratio of 1:1 to obtain a fecal bacteria-protective agent mixture;

[0136] S3: Freeze-drying: Pre-freezing stage: Freeze the fecal bacteria-protectant mixture at -50℃ for 4 hours; primary drying stage: maintain the vacuum degree ≤10Pa, raise the temperature to -30℃, and continue for 10 hours; secondary drying stage: maintain the vacuum degree ≤10Pa, raise the temperature to 25℃, and continue for 8 hours to obtain the intestinal fecal bacteria freeze-dried preparation.

[0137] The experimental results are shown in Table 7.

[0138] Table 7

[0139]

[0140] Conclusion: It can be seen from Table 7 that the intestinal fecal bacteria freeze-dried preparation of Example 4 maintains a high survival rate within 60 days and is suitable for long-term storage and transportation. It can be concluded that the preparation method of the intestinal fecal bacteria freeze-dried preparation of the present invention has a high survival rate of fecal bacteria after freeze-drying and strong stability for long-term storage and transportation.

[0141] The preferred embodiments of the present invention have been described in detail above. It should be understood that numerous modifications and variations based on the concepts of the present invention are possible without inventive effort by those skilled in the art. Therefore, any technical solution that can be derived by one skilled in the art through logical analysis, reasoning, or limited experimentation based on the concepts of the present invention and the prior art should be within the scope of protection defined by the claims.

Claims

1. A freeze-drying protective agent, characterized in that Includes glycerin, skim milk powder, mannitol, glutathione, inulin, and sterile water.

2. A freeze-drying protective agent according to claim 1, characterized in that Calculated by mass percentage, the glycerol accounts for 10%-20%, the skim milk powder accounts for 2%-8%, the mannitol accounts for 1%-6%, the glutathione accounts for 0.1%-0.2%, the inulin accounts for 5%-7.5%, and the balance is the sterile water.

3. A freeze-drying protective agent according to claim 1, characterized in that Calculated by mass percentage, the glycerol accounts for 15%, the skim milk powder accounts for 8%, the mannitol accounts for 3%, the glutathione accounts for 0.15%, the inulin accounts for 6%, and the balance is the sterile water.

4. A method for preparing the lyoprotectant according to any one of claims 1 to 3, characterized in that: The following steps are involved: S1: dissolving the skim milk powder in the sterile water and stirring until completely dissolved; S2: adding the glycerol, the mannitol, the glutathione and the inulin in sequence, and continuously stirring until the mixture is uniformly mixed.

5. Use of the lyoprotectant according to any one of claims 1 to 3 in the preparation of a biomedical product for intestinal flora transplantation.

6. A freeze-drying process for intestinal fecal bacteria, characterized in that: Freeze-drying is performed using the freeze-drying protectant according to any one of claims 1 to 3.

7. The freeze-drying process according to claim 6, characterized in that The specific steps include: S1: Pre-freezing stage: The mixture of the lyophilized protective agent and the fecal bacteria liquid is frozen at -50°C for 3.5-4.5 hours; the fecal bacteria liquid is prepared by the following method: A1: Mix stool samples from screened healthy donors with sterile saline at a mass-to-volume ratio of 1:5, homogenize with stirring, and perform graded filtration. A2: Centrifuge the filtered bacterial solution at 3500 rpm for 5 minutes, remove the supernatant, wash with sterile saline, and resuspend. Repeat the centrifugation and washing steps 2-3 times to obtain the fecal bacterial solution. S2: Primary drying stage: maintain vacuum degree ≤10Pa, raise temperature to -30℃, and last for 9.5-10.5 hours; S3: Secondary drying stage: maintain vacuum degree ≤10Pa, raise temperature to 25℃, and continue for 7.5-8.5 hours.

8. The freeze-drying process according to claim 6, characterized in that The specific steps include: S1: Pre-freezing stage: The mixture of the lyophilized protective agent and the fecal bacteria liquid is frozen at -50°C for 4 hours; the fecal bacteria liquid is prepared by the following method: A1: Mix stool samples from screened healthy donors with sterile saline at a mass-to-volume ratio of 1:5, homogenize with stirring, and perform graded filtration. A2: Centrifuge the filtered bacterial solution at 3500 rpm for 5 minutes, remove the supernatant, wash with sterile saline, and resuspend. Repeat the centrifugation and washing steps 2-3 times to obtain the fecal bacterial solution. S2: Primary drying stage: maintain vacuum degree ≤10Pa, raise temperature to -30℃, and continue for 10 hours; S3: Secondary drying stage: maintain vacuum degree ≤10Pa, raise temperature to 25℃, and continue for 8 hours.

9. Use of the freeze-drying process according to any one of claims 6 to 8 in preparing a biomedical product for intestinal flora transplantation.

10. A method for preparing a freeze-dried preparation of intestinal fecal bacteria, characterized in that: The following steps are involved: S1: preparing a lyoprotectant; the preparation of the lyoprotectant comprises the following steps: dissolving 8% skim milk powder in 50% sterile water by mass, and stirring until completely dissolved; Add 15% glycerol, 3% mannitol, 0.15% glutathione and 6% inulin in sequence, continue stirring until mixed evenly, and make up the balance with sterile water; S2: Mixing the fecal bacteria liquid and the freeze-dried protective agent in a volume ratio of 1:1 to obtain a fecal bacteria-protective agent mixed solution; the fecal bacteria liquid is prepared by the following method: A1: Mix stool samples from screened healthy donors with sterile saline at a mass-to-volume ratio of 1:5, homogenize with stirring, and perform graded filtration. A2: Centrifuge the filtered bacterial solution at 3500 rpm for 5 minutes, remove the supernatant, wash with sterile saline, and resuspend. Repeat the centrifugation and washing steps 2-3 times to obtain the fecal bacterial solution. S3: Pre-freezing stage: Freeze the fecal bacteria-protectant mixture at -50°C for 4 hours; primary drying stage: maintain the vacuum degree ≤10Pa, raise the temperature to -30°C, and continue for 10 hours; secondary drying stage: maintain the vacuum degree ≤10Pa, raise the temperature to 25°C, and continue for 8 hours to obtain the intestinal fecal bacteria freeze-dried preparation.

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