A fecal microbial freeze-drying preservation solution and its application

Abstract

This invention provides a fecal microbial freeze-drying protective solution. Through the synergistic effect of components such as phosphorylcholine polymer, glycerol, trehalose, ergothioneine, L-cysteine, melibiose, isomaltooligosaccharide, and stachydrine, it effectively reduces the damage to bacterial activity during the freeze-drying process, improves the stability of freeze-drying preservation, and enhances the ability to maintain bacterial metabolic activity. It plays a significant role in maintaining the diversity and functional activity of the bacterial community.

Description

Technical Field

[0001] This invention relates to the field of microbial preservation technology, and in particular to a fecal microbial freeze-drying preservation solution and its application. Background Technology

[0002] Fecal microbiota transplantation (FMT) is a treatment method that transplants functional microbiota from the feces of healthy individuals into the gastrointestinal tract of patients to reshape the recipient's gut microbiota structure and restore normal gut microbial function. Since being included in US guidelines for the treatment of recurrent Clostridium difficile infection in 2013, FMT has been widely used in the treatment and exploratory research of various microbiota-related diseases.

[0003] The preservation method of fecal microbiota preparations is a key factor affecting the clinical efficacy of fecal microbiota therapy (FMT). Currently, the main preservation methods for fecal microbiota include cryopreservation, freeze-drying, and refrigeration. Compared with cryopreservation, freeze-drying has significant advantages: it can be stored for a long time at 4°C or room temperature, greatly reducing transportation and storage costs and extending the product's shelf life.

[0004] However, both the low-temperature freezing and dehydration drying stages of the freeze-drying process damage bacterial cells. During the freezing stage, the formation of ice crystals inside and outside the cell can lead to mechanical damage to the cell membrane; during the drying stage, the phase transition and dehydration of the cell membrane phospholipid bilayer can cause membrane structure disruption, and the conformation of biomolecules such as proteins may undergo irreversible changes. For complex microbial communities like fecal microbiota, which consist of hundreds of bacterial species, different species exhibit significant differences in their tolerance to the freeze-drying process. Some sensitive strains are easily inactivated during freeze-drying, leading to changes in the microbial community structure and reduced diversity.

[0005] Therefore, developing a freeze-drying protective solution designed specifically for the characteristics of fecal microbiota is of great significance for the preservation and transplantation of fecal microbiota, as it can systematically protect microbiota with different physiological characteristics and maintain microbiota diversity and functional activity. Summary of the Invention

[0006] To address the problems existing in the prior art, the present invention provides a fecal microorganism freeze-drying protective solution and its application.

[0007] The first aspect of this invention provides a fecal microbiota freeze-drying protective solution, comprising:

[0008] 5%-20% of ice crystal damage inhibitory components;

[0009] 0.1%-0.5% of oxidative stress protective components;

[0010] 6%-12% of resuscitation-active components;

[0011] 0.1%-2% phosphorylcholine polymer.

[0012] Preferably, the ice crystal damage inhibiting component includes glycerol and trehalose. More preferably, the ice crystal damage inhibiting component includes 1%-5% glycerol and 5%-15% trehalose.

[0013] Preferably, the oxidative stress protective component includes ergothioneine and L-cysteine. More preferably, the oxidative stress protective component includes 0.02%-0.2% ergothioneine and 0.005%-0.05% L-cysteine.

[0014] Preferably, the revival synergistic component includes melibiose and isomaltooligosaccharide. More preferably, the revival synergistic component includes 3%-6% melibiose and 3%-6% isomaltooligosaccharide.

[0015] Preferably, the fecal microorganism freeze-drying protective solution further includes 0.1%-1% stachydrine.

[0016] A second aspect of the present invention provides a method for freeze-drying and preserving fecal microorganisms, comprising:

[0017] Add the fecal microorganism freeze-drying protective solution as described in the first aspect of the present invention to the fecal microorganism sample, place it in a freeze dryer for freeze drying, prepare fecal microorganism freeze-dried powder, and seal and store it.

[0018] A third aspect of the present invention provides a fecal microbiota freeze-drying protection kit, comprising the fecal microbiota freeze-drying protection solution provided in the first aspect of the present invention and the fecal microbiota freeze-drying preservation method provided in the second aspect of the present invention.

[0019] The fourth aspect of the present invention provides the application of the fecal microorganism freeze-drying protection solution provided in the first aspect of the present invention, the fecal microorganism freeze-drying protection method described in the second aspect of the present invention, or the fecal microorganism freeze-drying protection kit described in the third aspect of the present invention in the freeze-drying preservation of bacteria.

[0020] Compared with the prior art, the advantages of the present invention are:

[0021] The fecal microorganism freeze-drying protective solution provided by this invention utilizes glycerol, trehalose, and isomaltooligosaccharide (MOSO) to synergistically maintain osmotic pressure balance, forming a glassy matrix that inhibits ice crystal damage during the freeze-drying process. The oxidative stress resistance combination of ergothioneine and L-cysteine ​​provides long-lasting "precise removal" of strong oxidative damage by clearing ROS across the entire spectrum inside and outside the cell membrane. Simultaneously, ergothioneine protects the sulfhydryl groups of L-cysteine ​​from excessive oxidation, prolonging its intracellular antioxidant activity, while L-cysteine ​​metabolism... The glutathione generated by the enzyme can help regenerate ergothioneine; phosphorylcholine polymers can form a membrane-like hydration protective layer, reducing protein deposition and cell adhesion, reducing non-specific aggregation / adhesion of the microbial community during freezing, synergistically stabilizing membrane proteins with trehalose, and synergistically stabilizing osmotic pressure balance with glycerol; melibiose and isomaltooligosaccharide serve as specific carbon sources to maintain metabolic pathways and can improve the survival rate of fecal bacteria during recovery; stachydrine's biocompatibility, osmotic pressure regulation ability, and membrane stabilization properties provide additional protection for fecal bacteria during freeze-drying stress. The fecal bacteria freeze-drying protectant provided by this invention can effectively reduce the damage to bacterial activity during freeze-drying, improve freeze-drying stability, enhance the ability to maintain bacterial metabolic activity, and inhibit opportunistic pathogens while increasing the proportion of healthy functional bacteria, playing a significant role in maintaining microbial diversity and functional activity. Attached Figure Description

[0022] Figure 1 The image shows the plate colony results of the fecal microbial solution after 6 months of freeze-drying and rehydration provided in the embodiments of the present invention. A, F represent Comparative Example 1, Comparative Example 2, Comparative Example 3, Example 1, Example 2, and Example 3, respectively. The results show that the number of colonies in Examples 1-3 is significantly higher than that in Comparative Examples 1-3.

[0023] Figure 2 This is a flow cytometry chromatogram for detecting live and dead bacteria after 6 months of rehydration following freeze-drying of fecal microbiota solution, provided in an embodiment of the present invention. Figure 2 A is the flow cytometry plot of Comparative Example 3. Figure 2 B is the flow cytometry chromatogram of Example 2, where P2 represents viable bacteria and P5 represents dead bacteria. The results show that the number of viable bacteria in Example 2 is significantly higher than that in Comparative Example 3.

[0024] Figure 3 The graph shows the results of relative abundance detection of fecal bacteria communities under different freeze-dried protective solutions provided in the embodiments of the present invention. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0026] A fecal microbiota freeze-drying protective solution, comprising:

[0027] 5%-20% of ice crystal damage inhibitory components;

[0028] 0.1%-0.5% of oxidative stress protective components;

[0029] 6%-12% of resuscitation-active components;

[0030] 0.1%-2% phosphorylcholine polymer.

[0031] In some embodiments, the ice crystal damage inhibiting component includes glycerol and trehalose. In some preferred embodiments, the ice crystal damage inhibiting component includes 1%-5% glycerol and 5%-15% trehalose.

[0032] In some embodiments, the oxidative stress protective component includes ergothioneine and L-cysteine. In some preferred embodiments, the oxidative stress protective component includes 0.02%-0.2% ergothioneine and 0.005%-0.05% L-cysteine.

[0033] In some embodiments, the revival synergistic component includes melibiose and isomaltooligosaccharide. In some preferred embodiments, the revival synergistic component includes 3%-6% melibiose and 3%-6% isomaltooligosaccharide.

[0034] In some embodiments, the fecal microorganism freeze-drying protective solution further includes 0.1%-1% stachydrine.

[0035] Example

[0036]

[0037] Application Example 1: Validation of bacterial count detection during freeze-drying recovery of fecal microbial freeze-dried preservation solution

[0038] 1. Experimental Objective

[0039] The number of bacteria in the lyophilized fecal microorganisms after lyophilization and revival was determined by plate counting method, thus verifying the lyophilization preservation effect of the lyophilized fecal microorganisms provided by this invention on fecal microorganisms.

[0040] 2. Experimental Materials

[0041] 1) Fecal microbial solution;

[0042] 2) The fecal microbial freeze-drying protective solution provided in Examples 1-3 of this invention;

[0043] 3) Fecal microbial solution without any added freeze-drying protectant (Comparative Example 1), 5% glycerol (Comparative Example 2), 5% glycerol and 10% trehalose (Comparative Example 3).

[0044] 3. Experimental Methods

[0045] 3.1 Mix the fecal bacteria solution with the fecal bacteria freeze-dried preservation solution provided in Examples 1-3 and Comparative Examples 2-3 of this invention in equal proportions, pour the mixture into a freeze-drying tray, let it stand under anaerobic conditions for 20 minutes, seal and transfer it to a -80℃ refrigerator for pre-freezing for 4 hours.

[0046] 3.2 Turn on the freeze dryer and set the following freeze-drying parameters:

[0047]

[0048] After pre-freezing, the frozen bacterial solution is quickly put into a freeze dryer to prepare fecal bacteria freeze-dried powder.

[0049] 3.3 Samples were taken on the day the fecal microbiota lyophilized powder was prepared and again 6 months after lyophilization (stored in a sealed container at 4°C) for reconstitution and revival. The lyophilization-rehydration-reconstitution-revival process was as follows: Weigh 0.1g of fecal microbiota lyophilized powder into a centrifuge tube. Using a sterile pipette, an appropriate amount of preheated (37°C) and deoxygenated physiological saline was drawn. The pipette tip was placed firmly against the inner wall of the centrifuge tube, and the medium was slowly added, allowing the liquid to flow down the wall to avoid directly impacting the lyophilized powder. Volume is crucial; typically, 0.5mL of physiological saline was added first, and the mixture was incubated at room temperature for 3-5 minutes. After incubation, another 0.5mL of physiological saline was added to bring the total reconstitution volume to 1.0mL. After adding the medium, the mixture was gently blown up and down or vortexed to completely dissolve the lyophilized powder and form a homogeneous suspension. Vigorous shaking was strictly prohibited. The resulting reconstitution stock solution could be diluted 10 times (e.g., 0.1mL of stock solution plus 0.9mL of physiological saline) before subsequent testing for plate coating or flow cytometry.

[0050] 3.4 Place the above uniform suspension in an anaerobic workstation and incubate at 37°C for 30 minutes to obtain the reconstituted and revived fecal bacteria solution.

[0051] 3.5 Plate count method for survival rate detection: Take 1 mL of the reconstituted fecal microbiota solution and add it to a centrifuge tube containing 9 mL of physiological saline. Dilute it serially in physiological saline (select the most suitable dilution gradient according to the sample). Take 10... -6 10 -7 10 -8100 μL of each culture medium was spread evenly on blood agar plates, with 3 plates (replicated) for each concentration. The plates were inverted and incubated in an anaerobic environment at 37°C for 72 h. After incubation, the CFU of each test group was calculated based on the appearance of 30-300 colonies on the plate.

[0052] 4. Experimental Results

[0053] The plate colony results after 6 months of freeze-drying and rehydration of fecal microbial solution are shown in the figure below. Figure 1 As shown, A, F represent Comparative Example 1, Comparative Example 2, Comparative Example 3, Example 1, Example 2, and Example 3, respectively. The results show that the colony counts in Examples 1-3 are significantly higher than those in Comparative Examples 1-3. The specific plate count results are as follows:

[0054]

[0055] 5. Results Analysis

[0056] The results showed that the bacterial count on the colony plate of Comparative Example 1 on the day of freeze-drying was only 20.7% of that before freeze-drying. This result indicates that the fecal microbial solution lacking freeze-drying protectant had a poor preservation effect on fecal microorganisms during freeze-drying and could not provide protection against the low-temperature environment during freeze-drying. The bacterial counts on the colony plates of Comparative Examples 2 and 3 on the day after freeze-drying were 38.2% and 47.3% of those before freeze-drying, respectively. This result indicates that existing protectants such as glycerol and trehalose can reduce the damage to bacterial activity caused by the freeze-drying environment to some extent and improve the survival rate of bacteria in the low-temperature environment during freeze-drying, but still cannot prevent more than half of the bacteria from dying in the freeze-drying environment. In contrast, the bacterial counts on the colony plates of Examples 1, 2, and 3 on the day after freeze-drying were 58.8%, 69.4%, and 65.3% of those before freeze-drying, respectively. Compared with Comparative Examples 1, 2, and 3, the fecal microbial freeze-drying protectant provided by this invention can enable more than half of the bacteria to survive during freeze-drying, greatly reducing the damage to bacterial activity caused by the freeze-drying environment and further improving the survival rate of bacteria in the low-temperature environment during freeze-drying.

[0057] The experiment also compared the number of bacteria on colony plates on the day of freeze-drying with those after 6 months of freeze-drying. The results showed that the number of bacteria on colony plates of Comparative Example 1 after 6 months of freeze-drying was only 65.0% of that on the day of freeze-drying, indicating that the fecal microorganism freeze-dried powder lacking freeze-drying protectant was not well preserved in a sealed container at 4 degrees Celsius, and the bacteria in the freeze-dried powder continued to die over time. The number of bacteria on colony plates of Comparative Examples 2 and 3 after 6 months of freeze-drying was 69.7% and 81.1% of that on the day of freeze-drying, respectively. This result indicates that existing protectants such as glycerol and trehalose can improve the bacterial activity and survival rate of the freeze-dried powder to some extent, but 20-30% of the bacteria still die in the freeze-drying state. In contrast, the number of bacteria on colony plates of Examples 1, 2, and 3 after 6 months of freeze-drying all exceeded 95% of that before freeze-drying, indicating that the fecal microorganism freeze-dried powder prepared with the fecal microorganism freeze-drying protectant provided by this invention is more stable and can maintain the bacterial activity and survival rate of the fecal microorganism freeze-dried powder more steadily, which is beneficial for the preservation of the fecal microorganism freeze-dried powder.

[0058] Application Example 2: Verification of the number of viable and dead bacteria during the freeze-drying and thawing of fecal microbial freeze-dried preservation solution.

[0059] 1. Experimental Objective

[0060] The number of live and dead bacteria after freeze-drying and revival was detected by SYTO9 / PI fluorescence staining using the fecal microbial freeze-drying protectant solution, thus verifying the freeze-drying preservation effect of the fecal microbial freeze-drying protectant solution provided by the present invention on fecal microbial.

[0061] 2. Experimental Materials

[0062] 1) Fecal microbial solution;

[0063] 2) The fecal microbial freeze-drying protective solution provided in Examples 1-3 of this invention;

[0064] 3) Fecal microbial solution without any added freeze-drying protectant (Comparative Example 1), 5% glycerol (Comparative Example 2), 5% glycerol and 10% trehalose (Comparative Example 3).

[0065] 3. Experimental Methods

[0066] 3.1 Prepare the freeze-dried fecal bacteria powder according to the steps in Application Example 1 above, and then reconstitute and revive it to obtain the reconstituted and revised fecal bacteria solution.

[0067] 3.2 Dilute the reconstituted fecal microbiota solution 100-fold. Add 10 μL of the diluted fecal microbiota solution to a counting microsphere; add 980 μL of PBS buffer; add 5 μL of SYTO9 cell dye, and gently vortex to mix; incubate at room temperature in the dark for 20 min; add 5 μL of PI dye, and vortex to mix.

[0068] 3.3 On-machine testing: Taking the BD FACSCanto II flow cytometer as an example, configure three lasers and eight-color fluorescence channels (488nm, 633nm, 405nm lasers) to detect the number of live and dead bacteria.

[0069] 4. Experimental Results

[0070] The flow cytometry chromatogram for detecting live and dead bacteria after 6 months of freeze-drying and rehydration of fecal microbiota solution is shown below. Figure 2 As shown, where Figure 2 A is the flow cytometry plot of Comparative Example 3. Figure 2 B is the flow cytometry chromatogram of Example 2, where P2 represents viable bacteria and P5 represents dead bacteria. The results show that the number of viable bacteria in Example 2 is significantly higher than that in Comparative Example 3. The specific count results of viable and dead bacteria after thawing and incubation of the frozen fecal microbial solution are as follows:

[0071]

[0072] 5. Results Analysis

[0073] The results showed that Comparative Example 1 had only 20.8% of the viable bacteria count on the day of freeze-drying, and only 62.1% of the count on the day of freeze-drying after 6 months. This indicates that the fecal microbial solution lacking freeze-drying preservatives had poor preservation effects on fecal microorganisms during freeze-drying and could not provide protection against the low-temperature environment during freeze-drying. Similarly, the freeze-dried fecal microbial powder lacking freeze-drying preservatives did not perform well when sealed in a 4°C refrigerator, and the bacteria in the freeze-dried powder continued to die over time. Comparative Examples 2 and 3 had 38.0% and 47.8% of the viable bacteria count on the day of freeze-drying, respectively, but only 62.1% and 69.0% of the count on the day of freeze-drying after 6 months. This indicates that existing preservatives such as glycerol and trehalose can, to some extent, reduce the damage to bacterial activity caused by the freeze-drying environment and improve the survival rate of bacteria in the low-temperature environment during freeze-drying, but still... It is unavoidable that more than half of the bacteria will die during the freeze-drying process. While existing preservatives such as glycerol and trehalose can maintain bacterial activity in the freeze-dried powder to some extent and improve its survival rate, 20%-30% of the bacteria still die during freeze-drying. In contrast, the viable bacterial counts on the day of freeze-drying in Examples 1, 2, and 3 were 56.3%, 65.7%, and 63.6% of those before freeze-drying, respectively. Even after 6 months of freeze-drying, the viable bacterial count still reached more than 95% of that on the day of freeze-drying. Compared with Comparative Examples 1, 2, and 3, the fecal microorganism freeze-drying preservative solution provided by this invention can ensure that more than half of the bacteria survive during the freeze-drying process, greatly reducing the damage to bacterial activity caused by the freeze-drying environment. This can further improve the survival rate of bacteria in the low-temperature environment during the freeze-drying process, while maintaining good bacterial activity in the freeze-dried powder. This allows for a more stable maintenance of bacterial activity and survival rate in the fecal microorganism freeze-dried powder, which is beneficial for the preservation of the fecal microorganism freeze-dried powder.

[0074] Application Example 3: Detection of metabolic activity of fecal microorganisms after freeze-dried preservation and thawing

[0075] 1. Experimental Objective

[0076] The metabolic activity of lyophilized fecal microorganisms preserved in the lyophilized preservation solution after rehydration and revival was detected by CTC dye, verifying the lyophilized preservation effect of the lyophilized preservation solution provided by the present invention on fecal microorganisms.

[0077] To further enhance the freeze-dried fecal microorganism powder's resistance to freeze-drying stress and improve its metabolic activity after reconstitution and recovery, stachydrine was added to the formula used in Example 2. Stachydrine is a quaternary ammonium alkaloid, a plant-derived compatible solute that can accumulate in plant cells under extreme environmental stress, regulating osmotic pressure, stabilizing proteins and membrane structures, and enhancing stress resistance. Its biocompatibility, osmotic pressure regulation, and membrane stabilization properties may provide additional protection against freeze-drying stress. Details are as follows:

[0078]

[0079] 2. Experimental Materials

[0080] 1) Fecal microbial solution;

[0081] 2) The fecal microbial freeze-drying protective solution provided in Examples 2 and 4-6 of this invention;

[0082] 3) Fecal microbial solution without any added freeze-drying protectant (Comparative Example 1), 5% glycerol (Comparative Example 2), 5% glycerol and 10% trehalose (Comparative Example 3).

[0083] 3. Experimental Methods

[0084] 3.1 Prepare the freeze-dried fecal bacteria powder according to the steps in Application Example 1 above, and then reconstitute and revive it to obtain the reconstituted and revised fecal bacteria solution.

[0085] 3.2 CTC dye detection of metabolic activity: The reconstituted fecal bacteria solution was placed at room temperature for 10 minutes and thoroughly vortexed to mix; the bacterial solution was diluted 100 times with PBS buffer and set aside.

[0086] 3.4 Take 10 μL of diluted bacterial solution and add it to a counting microsphere tube; add 980 μL of PBS buffer and gently vortex to mix; add 10 μL of 50 mM CTC working solution to a final concentration of 5 mM; gently vortex to mix, and incubate at 37°C for 30 min in the dark throughout the process; after incubation, let it stand at room temperature for 5 min to equilibrate the temperature.

[0087] 3.5. Add 1 μL of 10 μM SYTO 24 Green working solution to a final concentration of 10 mM. Incubate at room temperature in the dark for 15 min without washing. Add 5 μL of DAPI dye, vortex to mix, and let stand at room temperature in the dark for 5 min.

[0088] 3.6. On-machine testing: Taking the BD FACSCanto II flow cytometer as an example, configure three lasers and eight-color fluorescence channels (488nm, 633nm, 405nm lasers).

[0089] 3.7 Result Calculation:

[0090] CTC+DAPI double staining analysis: live and metabolically active bacteria (CTC) + DAPI - ); dead bacteria (CTC) - DAPI + ); Metabolically weakened live bacteria (CTC) - DAPI - ).

[0091] Metabolic activity reduction capacity (%) = (CTC) + (Bacterial count / viable count) × 100%

[0092] 4. Experimental Results

[0093]

[0094] 5. Results Analysis

[0095] Comparative Example 1 showed a metabolic activity reduction capacity of only 20.7% on the day of freeze-drying and only 15.4% after 6 months. This indicates that the metabolic activity reduction capacity of fecal microbial solution lacking freeze-drying preservatives is poor, and the metabolic activity reduction capacity continues to decrease over time in the freeze-dried state. Comparative Examples 2 and 3 showed metabolic activity reduction capacities of 37.9% and 47.8% on the day of freeze-drying, respectively, and 29.8% and 40.3% after 6 months. This suggests that existing freeze-drying preservatives such as glycerol and trehalose can mitigate the decline in metabolic activity reduction capacity caused by freeze-drying to some extent, but more than 50% of the live bacteria still experience metabolic degradation. The metabolic activity reduction capacity of the bacteria in the freeze-dried state was weak, and the rate of decline of metabolic activity reduction capacity over time was slowed down. However, the metabolic activity reduction capacity of Example 2 of the present invention was 68.8% and 62.4% on the day of freeze-drying and 6 months of freeze-drying, respectively, which was significantly higher than that of Comparative Examples 1-3. In Examples 4-6, stachydrine was added to the basis of Example 2, and the metabolic activity reduction capacity on the day of freeze-drying was 79.5%, 82.1%, and 81.1%, respectively, and the metabolic activity reduction capacity after 6 months of freeze-drying was 77.6%, 81.7%, and 79.9%, respectively. The metabolic activity reduction capacity was further improved and stabilized, indicating that a certain concentration of stachydrine can further enhance the metabolic activity of bacteria.

[0096] Application Example 4: Detection of relative abundance of fecal microbiota under different fecal microbiota freeze-drying preservation solutions

[0097] 1. Experimental Objective

[0098] The relative abundance of fecal microbiota preserved in different fecal microbiota freeze-drying preservation solutions was detected by 16S rRNA sequencing to verify the freeze-drying preservation effect of the fecal microbiota freeze-drying preservation solution provided by the present invention on fecal microbiota.

[0099] 2. Experimental Materials

[0100] 1) Fecal microbial solution;

[0101] 2) The fecal microbial freeze-drying protective solution provided in Example 5 of this invention;

[0102] 3) Fecal microbial solution without any added freeze-drying protective solution (Comparative Example 1), 5% glycerol, 10% trehalose (Comparative Example 3).

[0103] 3. Experimental Methods

[0104] 3.1 Prepare fecal bacteria freeze-dried powder according to the steps in Application Example 1 above, and store it in a sealed refrigerator at 4 degrees Celsius for 6 months.

[0105] 3.2. Take samples for reconstitution and recovery to obtain reconstituted fecal microbial solution, and extract total DNA from the samples.

[0106] 3.3 Library Construction and Sequencing: Primers were designed based on conserved regions. Sequencing adapters were added to the primer ends, and PCR amplification was performed. The products were then purified, quantified, and homogenized to form a sequencing library. The constructed library underwent quality control. Libraries that passed quality control were sequenced using Illumina NovaSeq 6000. The raw image data files obtained from high-throughput sequencing (such as sequencing platforms like Illumina NovaSeq) were converted into raw sequencing reads through base calling analysis. The results were stored in FASTQ (fq) file format, which included the sequence information of the sequencing reads and their corresponding sequencing quality information.

[0107] 4. Experimental Results

[0108] This embodiment uses 16S rRNA sequencing technology to compare the relative abundance changes of fecal microbiota in Comparative Example 1, Comparative Example 3 and Example 5, and focuses on analyzing the differences in key functional genera.

[0109] The results are as follows Figure 3 As shown in Comparative Example 1, no Bifidobacterium or Lactobacillus were detected. However, the total proportion of probiotics such as Broutbacterium, Collins, Bacteroides, and Eubacterium hopterii was only 20.58%, while the total proportion of opportunistic pathogens such as Citrobacter, Escherichia coli / Shigella, and Cronobacter was as high as 49.64%. This indicates that the freeze-drying preservation effect of fecal microbiota without the addition of freeze-drying protectant is poor, with a high proportion of opportunistic pathogens, a low proportion of probiotics, a severely unbalanced microbiota structure, and significantly disordered function, making it difficult to exert immune regulation and colonization protection effects.

[0110] In Comparative Example 3, Bifidobacterium and Lactobacillus accounted for 0.42% and 0.24%, respectively; while Broutella increased to 13.68%, Collins to 12.69%, Bacteroides to 5.12%, and Eubacterium hominis to 5.37%; the combined proportion of conditionally pathogenic bacteria such as Citrobacter, Escherichia coli / Shigella, and Cronobacter to 33.87% indicates that the addition of glycerol and trehalose reduced the proportion of conditionally pathogenic bacteria in fecal microbiota, and healthy functional bacteria began to gradually accumulate, with the microbiota structure transitioning towards a healthier direction, thus improving the freeze-drying protection effect of fecal microbiota to a certain extent.

[0111] In Example 5, the proportions of Bifidobacterium and Lactobacillus were 3.58% and 2.40%, respectively, showing a further increase. The proportions of Broutbacterium increased to 19.72%, Collins to 18.11%, Bacteroides to 5.77%, and Eubacterium hominis to 6.25%. The combined proportions of conditionally pathogenic bacteria such as Citrobacterium, Escherichia coli / Shigella, and Cronobacter were reduced to 19.21%, showing significant inhibition. This indicates that the fecal microbiota freeze-dried protective solution provided by this invention has a good protective effect on the fecal microbiota. It can increase the proportion of healthy functional bacteria, inhibit conditionally pathogenic bacteria, significantly optimize the fecal microbiota structure, and help exert immune regulation and colonization protection effects, thereby facilitating subsequent fecal microbiota transplantation.

[0112] The above results indicate that the fecal microbiota freeze-drying protectant provided by the present invention can effectively reduce the damage to bacterial activity during the fecal microbiota freeze-drying process, improve the freeze-drying preservation stability, enhance the ability to maintain bacterial metabolic activity, and inhibit conditionally pathogenic bacteria while increasing the proportion of healthy functional bacteria, thereby optimizing and reconstructing the intestinal flora structure. It is an ideal choice for fecal microbiota freeze-drying protection.

[0113] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A fecal microbial freeze-drying protective solution, characterized in that, include: 5%-20% of ice crystal damage inhibitory components; 0.1%-0.5% of oxidative stress protective components; 6%-12% of resuscitation-active components; 0.1%-2% phosphorylcholine polymer.

2. The fecal microbial freeze-drying protective solution as described in claim 1, characterized in that, The ice crystal damage inhibitory components include glycerol and trehalose.

3. The fecal microbial freeze-drying protective solution as described in claim 2, characterized in that, The ice crystal damage inhibitory component includes 1%-5% glycerol and 5%-15% trehalose.

4. The fecal microbial freeze-drying protective solution as described in claim 1, characterized in that, The oxidative stress protection components include ergothioneine and L-cysteine.

5. The fecal microbial freeze-drying protective solution as described in claim 4, characterized in that, The oxidative stress protection component includes 0.02%-0.2% ergothioneine and 0.005%-0.05% L-cysteine.

6. The fecal microbial freeze-drying protective solution as described in claim 1, characterized in that, The revival-enhancing components include melibiose and isomaltooligosaccharide.

7. The fecal microbial freeze-drying protective solution as described in claim 1, characterized in that, It also includes 0.1%-1% stachydrine.

8. A method for freeze-drying and preserving fecal microorganisms, characterized in that, include: Add the fecal microorganism freeze-drying protective solution as described in any one of claims 1-7 to the fecal microorganism sample, place it in a freeze dryer to freeze dry, prepare fecal microorganism freeze-dried powder, and seal and store it.

9. A fecal microbiota freeze-dried preservation kit, characterized in that, It includes the fecal microorganism freeze-drying protective solution as described in claim 1 and the fecal microorganism freeze-drying preservation method as described in claim 8.

10. The application of the fecal microorganism freeze-drying preservation solution of claim 1, the fecal microorganism freeze-drying preservation method of claim 8, or the fecal microorganism freeze-drying preservation kit of claim 9 in bacterial freeze-drying preservation.

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