An intestinal flora freeze-drying embedding method based on an o / w emulsion stabilizing system and application thereof

Abstract

The application discloses an intestinal flora freeze-drying embedding method based on an O / W emulsion stable system and application thereof, and belongs to the technical field of biological medicine and intestinal microorganism delivery. The method comprises the following steps: preparing a medium-chain glyceride and a natural protein solution with emulsification activity, and performing emulsification treatment to construct an O / W emulsion; mixing screened intestinal flora, a freeze-drying protective agent and the O / W emulsion to form a composite system; and finally, freeze-drying is performed to obtain a freeze-dried powder of the intestinal flora. The application can protect the number of live bacteria and maintain the community structure of the complex mixed flora.

Description

Technical Field

[0001] This invention relates to a freeze-drying and embedding method for intestinal flora based on an O / W emulsion stabilization system and its application, belonging to the field of biomedical technology. Background Technology

[0002] Currently, fecal microbiota transplantation (FMT) is a novel biotherapy technique that transplants functional gut microbiota from the feces of a healthy donor into the intestines of a patient, showing significant efficacy in the treatment of diseases such as Clostridium difficile infection (CDI) and inflammatory bowel disease (IBD). However, traditional FMT formulations face serious challenges: Low efficiency of live bacteria delivery: Exogenous bacteria need to be taken orally and pass through the "oral-stomach-small intestine-colon" route. The strong acid in the stomach and the digestive enzymes in the small intestine form multiple physiological barriers, resulting in a significant decrease in the number of live bacteria. The proportion that finally reaches the colon is only 1% to 10%.

[0003] Lack of targeting capability: Existing vectors are difficult to achieve precise release into the colon, resulting in large fluctuations in clinical dosing doses.

[0004] Existing encapsulation technologies, such as spray drying, are prone to inactivation at high temperatures; sol-gel methods have long production cycles; and ionogel methods have weak acid resistance. Therefore, developing a gut microbiota encapsulation technology that can efficiently withstand the stresses of the gastrointestinal environment and is cost-effective is a pressing technical problem that the industry needs to solve.

[0005] In recent years, technical routes for enhancing the gastrointestinal tolerance of probiotics by encapsulating them in protein-stabilized emulsions have been reported. Existing routes typically require pre-preparing probiotics as lyophilized powders and dispersing them in an oil phase, then constructing a protein-polysaccharide dual-network emulsion gel using cross-linking agents such as gluconate-δ-lactone, gellan gum, and calcium ions to resist gastric acid and bile salt stress and achieve colonic delivery. However, these routes have the following inherent drawbacks: First, the bacterial cells must undergo a freeze-drying process, which can damage the initial activity due to secondary drying. Furthermore, the freeze-dried bacterial powder comes into direct, large-area contact with oils, easily causing biofilm damage. Second, the gelation process requires the introduction of multiple exogenous cross-linking agents and prolonged static setting, resulting in a lengthy and costly process. Residual cross-linking agents also pose biosafety risks. Third, and most importantly, existing emulsion embedding studies primarily target single purebred probiotic strains, with evaluation indicators only considering the survival rate of individual strains. However, the complex mixed gut microbiota from feces contains a large number of strictly anaerobic bacteria with varying tolerances and high sensitivity to oxygen and dehydration. Different species exhibit vastly different tolerances to acid, bile salts, freezing, and drying stress. Conventional embedding and processing methods can easily lead to selective death of sensitive species, resulting in a significant shift in the community structure of the delivered microbiota compared to the original sample, thus weakening the ecological efficacy of fecal microbiota transplantation (FMT) formulations. Therefore, how to achieve full-spectrum protection and community structure fidelity of complex mixed gut microbiota with a simplified process, without omitting the pre-freeze-drying and gelation steps, has become a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0006] This invention provides a method for freeze-drying and embedding intestinal flora based on an O / W emulsion stabilization system and its application, which can effectively solve the above-mentioned problems.

[0007] This invention provides a method for freeze-drying and embedding gut microbiota based on an O / W emulsion stabilization system, comprising the following steps: (1) Raw material preparation: medium chain triglycerides (MCT), natural protein aqueous solution with emulsifying activity, water-containing intestinal flora sludge and freeze-drying protectant aqueous solution are prepared; the water-containing intestinal flora sludge is intestinal flora sludge derived from feces of healthy donors and has not been freeze-dried; (2) Preparation of O / W emulsion: The natural protein aqueous solution and the medium chain triglyceride are mixed at an oil-water volume ratio of 1:8-10 and emulsified at 25-35°C to obtain an O / W emulsion without layering and without oil droplets floating. (3) Construction of composite system: First, mix the fungal mud with the protective agent aqueous solution at a ratio of 1g:4-10mL, and then mix it with the O / W emulsion at a ratio of 1g:8-10mL to form a "fungal mud-protectant-O / W emulsion" composite system; no cross-linking agent or gelling agent is added to the system, and no cross-linking curing treatment is performed; (4) Freeze-drying: The composite system is directly subjected to vacuum freeze-drying to obtain freeze-dried intestinal flora powder; the vacuum freeze-drying includes a pre-freezing stage, a sublimation drying stage and a desorption drying stage in sequence, the pre-freezing temperature of the pre-freezing stage is -60℃ to -40℃, and the final drying temperature of the desorption drying stage is 25℃ to 35℃.

[0008] In this invention, the intestinal flora is provided in the form of undried aqueous bacterial sludge, and is directly mixed in aqueous phase with an aqueous solution of a freeze-drying protectant, and then blended with a pre-prepared O / W emulsion; there is no need to pre-freeze the bacterial cells into powder, nor is there a need to disperse the bacterial cells in the oil phase; no cross-linking agent or gelling agent is added to the composite system, and no cross-linking and curing treatment is performed; the composite system is directly subjected to one-step vacuum freeze-drying after uniform mixing; and a non-layered structure is formed by first emulsifying an aqueous solution of natural protein with emulsifying activity with medium-chain triglycerides. An O / W emulsion without oil droplets is prepared and then blended with a "bacterial sludge-freeze-drying protectant" system in a specific ratio. During freeze-drying, a composite protective layer of "protein continuous phase + lipid dispersed phase" is formed in situ on the surface of the bacteria. The lipid dispersed phase forms a hydrophobic protective barrier, while the protein continuous phase maintains interfacial stability. The freeze-drying process employs a mild procedure with a pre-freezing temperature of -60℃ to -40℃ and a final desorption drying temperature of 25℃ to 35℃, which maintains the activity of the bacterial community while avoiding damage to the bacteria from severe ice crystals.

[0009] Through the synergistic effect of the above-mentioned technical features, the present invention enables the method to achieve highly efficient protection of complex intestinal flora using only a simplified process of "pre-prepared O / W emulsion + water-containing bacterial mud blending + one-step freeze drying" without the need for cross-linking agents and gelation treatment.

[0010] In some embodiments, the preparation of the aqueous intestinal flora sludge in step (1) includes: taking a fresh fecal sample from a healthy donor, adding physiological saline, stirring and mixing at 4°C, filtering through 100-mesh, 150-mesh and 200-mesh filters in sequence, and collecting the filtrate; centrifuging the filtrate at 4°C and 5000×g for 10 min, and discarding the supernatant; resuspending and washing the sludge at a ratio of 1g:9mL (sludge weight to physiological saline volume), centrifuging again at 4°C and 8000×g for 10 min, discarding the supernatant, and collecting the aqueous intestinal flora sludge.

[0011] In some embodiments, the preparation of medium-chain triglycerides in step (1) includes: heating medium-chain triglycerides to 45-55°C and maintaining the temperature, removing crystalline particles, and filtering through an organic phase filter membrane for sterilization; the preparation method of the natural protein aqueous solution with emulsifying activity is as follows: dissolving natural protein powder with emulsifying activity in pre-cooled deionized water at 4°C at a mass-volume concentration of 3%-5%, adjusting the pH to 6.9-7.1, letting it stand overnight to allow the protein to expand, and filtering through an aqueous filter membrane for sterilization.

[0012] In some embodiments, the preparation process of the O / W emulsion in step (2) is as follows: placing an aqueous solution of natural protein with emulsifying activity in a water bath at 25-35°C, and slowly adding medium-chain triglycerides at a volume ratio of 1:8-10 for oil and water while stirring, and continuing to stir until an O / W emulsion without layering, without obvious oil droplets floating, and without clumping on the wall when the container is inverted is formed.

[0013] In some embodiments, the emulsifying natural protein is selected from one or more of whey protein, soy protein isolate, and casein; the freeze-drying protectant includes one or more of lactose, maltose, trehalose, maltotriose, fructooligosaccharides, sodium caseinate, and gelatin.

[0014] In some embodiments, the freeze-drying protectant is a mixture of lactose, maltose and trehalose, or a mixture of trehalose, maltotriose, fructooligosaccharides, sodium caseinate and gelatin.

[0015] In some embodiments, the pre-freezing temperature of the pre-freezing stage is -60℃ to -40℃, and the holding time is 3 to 6 hours; the sublimation drying stage is carried out under vacuum conditions with the temperature gradually increased from -45℃ to -35℃, then to -35℃ to -25℃, and then to -25℃ to -15℃, until it reaches -15℃ to 0℃; the desorption drying stage is carried out with the temperature gradually increased from 0℃ to 15℃, then to 25℃ to 35℃, until it reaches 15℃ to 25℃.

[0016] This invention provides a freeze-dried intestinal flora powder prepared by the above method. The freeze-dried powder contains intestinal flora, a freeze-drying protectant, and an O / W emulsion freeze-dried product formed by natural proteins with emulsifying activity and medium-chain triglycerides. The natural proteins with emulsifying activity and medium-chain triglycerides are coated on the surface of the bacteria to form a composite protective layer.

[0017] In some embodiments, the composite protective layer comprises a continuous phase composed of the natural protein with emulsifying activity and a dispersed phase composed of the medium-chain triglycerides, and the composite protective layer continuously coats the surface of the bacterial cells.

[0018] This invention provides the application of the above-mentioned lyophilized intestinal flora powder in the preparation of oral intestinal flora transplantation formulations or intestinal microbial delivery drugs, wherein the oral intestinal flora transplantation formulations or intestinal microbial delivery drugs are used to treat Clostridium difficile infection (CDI) or inflammatory bowel disease (IBD), or to maintain the diversity of the intestinal flora community structure.

[0019] The beneficial effects of this invention are: Existing emulsion encapsulation technologies (including those delivering probiotics in emulsion gel form) all target single pure bacterial strains, and their effects are limited to improving the survival rate of individual bacteria. However, in the complex mixed gut microbiota derived from feces, different bacterial species exhibit vastly different tolerances to acid, bile salts, freezing, and dehydration stress. Processing can easily cause selective death of sensitive anaerobic bacteria and induce community shift. In this invention, the lyophilized gut microbiota powder, after reconstitution, is treated with simulated gastric fluid for 2 hours and then with simulated intestinal fluid for 3 hours. Characterization using 16S rRNA high-throughput sequencing shows that the β-diversity clustering distance between the lyophilized community structure and the original gut microbiota sample is smaller than the β-diversity clustering distance between the control lyophilized powder (without the O / W emulsion) treated under the same conditions and the original gut microbiota sample. In other words, compared to the control, the lyophilized powder obtained in this invention, after simulated gastrointestinal fluid stress, more closely resembles the original microbiota sample, demonstrating that this invention preserves the number of live bacteria while completely maintaining the community structure of the complex mixed microbiota. The fidelity of the microbial community structure indicates that the present invention can achieve non-selective and proportional protection for microbial species with different tolerances. This is a technical effect that existing encapsulation techniques for single pure strains cannot achieve or expect.

[0020] This invention forms a composite protective layer of "continuous protein phase + dispersed lipid phase" on the surface of bacteria during freeze-drying (the formation mechanism is speculated to be the in-situ self-assembly of protein and oil droplets into a film induced by ice crystal growth and compression during freezing). The hydrophobic layer formed by the MCT effectively resists gastric acid erosion, and the natural protein with emulsifying activity enhances the stability of the system. Experimental data show that the average survival rate of bacteria treated by the method of this invention after treatment with simulated gastrointestinal fluid can reach more than 42%, which is much higher than that of the control group (about 2%).

[0021] After being subjected to combined stress treatment with artificial gastric and intestinal fluids, the survival rate of live bacteria in the freeze-dried powder of this invention is significantly higher than that of the blank control and the existing conventional sodium alginate embedding control. This provides a structural basis for the preparation of oral intestinal microbiota transplantation (FMT) preparations, which is beneficial to reduce clinical dosage fluctuations and reduce adverse reactions such as abdominal distension and diarrhea caused by overdose.

[0022] This invention uses natural, edible MCT and natural proteins with emulsifying activity, with no chemical residues, good biocompatibility, low raw material costs, and a process suitable for large-scale production. Attached Figure Description

[0023] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0024] Figure 1 Image of the lyophilized powder from the blank control group of Example 1. Shows the appearance of the lyophilized powder without added O / W emulsion.

[0025] Figure 2 Image of the control group (conventional sodium alginate embedding) lyophilized powder of Example 1. Shows the appearance of the lyophilized powder after conventional sodium alginate embedding.

[0026] Figure 3 Images of the lyophilized powder from the experimental group in Example 1. Showing the appearance of the lyophilized powder prepared by adding the O / W emulsion system of this invention.

[0027] Figure 4 Microscopic image of bacterial sludge from the blank control group in Example 1 (before passing through artificial gastrointestinal fluid). Showing the original bacterial cell state.

[0028] Figure 5 Microscopic image of bacterial sludge from the control group (conventional sodium alginate embedding) in Example 1 (before exposure to artificial gastrointestinal fluid). This shows the conventional sodium alginate embedding state.

[0029] Figure 6 Microscopic image of bacterial sludge from the experimental group in Example 1 after treatment with the O / W emulsion stabilization system (before exposure to artificial gastrointestinal fluid). This shows the bacterial cells being encapsulated by the emulsion system.

[0030] Figure 7 Microscopic images of the blank control group in Example 1 (after treatment with artificial gastrointestinal fluid). The images show a significant reduction in bacterial count and sparse field of view after treatment with gastrointestinal fluid.

[0031] Figure 8 Microscopic images of the control group (conventional sodium alginate embedding) in Example 1 (after treatment with artificial gastrointestinal fluid). The images show a reduction in bacterial count and sparser field of view after treatment with gastrointestinal fluid.

[0032] Figure 9 Microscopic images of bacterial sludge from the experimental group in Example 1 after treatment with the O / W emulsion stabilization system (after treatment with artificial gastrointestinal fluid). The images show that after treatment with gastrointestinal fluid, the bacterial cells were still effectively encapsulated and protected, maintaining their morphology and quantity significantly.

[0033] Figure 10: 16S rRNA high-throughput sequencing phylum-level diversity results, C is the original bacterial sludge sample, B-1 is the freeze-dried sample of Example 1, A-1 is the freeze-dried sample of Example 1 after treatment with gastrointestinal fluid, B-2 is the freeze-dried sample of the blank control group, A-2 is the freeze-dried sample of the blank control group after treatment with gastrointestinal fluid, and the horizontal axis is the relative abundance. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention.

[0035] Example 1 Preparation of freeze-dried intestinal flora powder Preparation of medium-chain triglycerides (MCT): MCT oil with a purity of ≥98% was placed in a 50℃ water bath and heated for 30 min to remove trace crystal particles to ensure the homogeneity of the system. After being filtered through a 0.22 μm organic phase filter membrane for sterilization, it was dispensed into sterile glass containers and stored at 4℃ in the dark for later use.

[0036] Preparation of whey protein (WPI) solution: Accurately weigh WPI powder with a protein purity ≥92%, and slowly add it to pre-cooled deionized water at 4℃ at a ratio of 5% (w / v). Stir magnetically at 300 rpm for 60 min until completely dissolved. Adjust the pH of the solution to 7.0±0.1 with 0.1 mol / L NaOH, and let it stand overnight at 4℃ to allow the protein molecules to fully expand. After filtration through a 0.22 μm aqueous filter membrane for sterilization, allow it to return to room temperature (25℃) for later use.

[0037] O / W emulsion preparation: Pour the prepared WPI aqueous solution into a 500 mL beaker and place it in a 30℃ constant temperature water bath; turn on the digital display constant speed stirrer and set the speed to 600 rpm. After the solution forms a stable vortex, slowly pour in MCT oil at an oil-to-water volume ratio of 1:9 and continue stirring for 15 min. During this time, gently scrape the beaker wall with a glass rod to ensure that the oil droplets at the edge are fully dispersed, forming a uniform milky white crude emulsion that is free of layering, has no obvious oil droplets floating on the surface, and does not clump when the container is inverted. This is the O / W emulsion.

[0038] Preparation of unemulsified MCT-protein mixture (control): Pour the prepared WPI aqueous solution into a 500mL beaker, and slowly pour in MCT oil at an oil-to-water volume ratio of 1:9 to obtain the unemulsified MCT-protein mixture (control).

[0039] Preparation of bacterial suspension: 150 g of fresh fecal samples from three different healthy donors (donors A, B, and C) were selected and added to 1000 mL of physiological saline. The samples were stirred at 4000 rpm for 10 min at 4℃. The mixture was then filtered sequentially through 100-mesh, 150-mesh, and 200-mesh filters. The filtrate was collected as the bacterial suspension. The bacterial suspension was centrifuged at 5000×g for 10 min at 4℃, and the supernatant was removed. The bacterial sludge was resuspended and washed with physiological saline at a ratio of 1 g:9 mL, and then centrifuged again at 8000×g for 10 min at 4℃. The supernatant was removed, and the purified bacterial sludge was collected.

[0040] Preparation of sodium alginate-embedded bacterial sludge: Weigh 5.0 g of food-grade sodium alginate powder, add 100 mL of deionized water, and stir magnetically in a 60℃ water bath until completely dissolved. After cooling to room temperature, filter through a 0.22 μm filter membrane to obtain a 5% (w / v) sodium alginate aqueous solution, which is stored for later use. Take the pure bacterial sludge prepared above and add it to the 5% sodium alginate aqueous solution at a ratio of 1 g: 9 mL of bacterial sludge weight. Vortex mix to form a bacterial-sodium alginate mixture. Drop the mixture into a 0.2 mol / L sterile CaCl2 solution at a rate of 1 mL / min using a syringe to form microspheres with a diameter of 2-3 mm. Crosslink and solidify at room temperature for 30 min, wash three times with sterile physiological saline, and collect the sodium alginate-embedded bacterial microspheres, which are the sodium alginate-embedded samples to be freeze-dried.

[0041] Preparation of intestinal flora freeze-drying protectant: Take 40 g of food-grade lactose, 30 g of food-grade maltose, and 10 g of food-grade trehalose, add 1000 mL of physiological saline to dissolve and mix well; after high temperature and high pressure sterilization, let stand and cool at room temperature, and store in a 4℃ refrigerator for later use.

[0042] Preparation of the composite system: Add the intestinal flora freeze-drying protectant to the bacterial sludge at a ratio of 1g:6mL and vortex mix well; then add the O / W emulsion prepared above at a ratio of 1:9 and continue to vortex mix well to form a "bacterial sludge-freeze-drying protectant-O / W emulsion" composite system.

[0043] Activity detection and freeze-drying: A portion of the composite system sample was diluted 100-fold with physiological saline and stained with SYTO-9 and propidium iodide (PI) fluorescent dye (commercially available live / dead bacteria detection kit). After a dark reaction of 15 min, the activity before freeze-drying was detected by flow cytometry. The remaining sample was placed in a vacuum freeze dryer for freeze-drying, and the drying program was carried out according to the freeze-drying process parameters in Table 1 (pre-freezing to -55℃, and finally warming to 35℃). After freeze-drying, the freeze-dried intestinal flora powder was collected, ground evenly, and the activity after freeze-drying was detected using the same flow cytometry method. The freeze-drying survival rate was calculated according to the formula "freeze-drying survival rate (%) = activity of sample after freeze-drying (%) / activity of sample before freeze-drying (%)".

[0044] Table 1. Freeze-drying procedure for gut microbiota

[0045] The results showed that, in this embodiment, the activity of the sample from donor A before freeze-drying was 61.66%±0.78%, and the activity of the sample after freeze-drying was 51.01%±1.25%, with a calculated freeze-drying survival rate of 82.73%. For donor B, the activity of the sample before freeze-drying was 66.81%±1.69%, and the activity of the sample after freeze-drying was 48.06%±2.40%, with a calculated freeze-drying survival rate of 71.94%. For donor C, the activity of the sample before freeze-drying was 64.71%±1.72%, and the activity of the sample after freeze-drying was 55.68%±1.23%, with a calculated freeze-drying survival rate of 86.05%. These results indicate that the embedding method of this invention can effectively preserve the activity of intestinal flora during freeze-drying and reduce damage to the flora during the freeze-drying process.

[0046] Gastrointestinal fluid resistance test Artificial gastric fluid: Accurately measure 16.4 mL of dilute HCl, add 800 mL of ultrapure water, then add 10 g of pepsin, stir until completely dissolved, and bring the volume to 1000 mL with ultrapure water. Adjust the pH to 2.5 with 1 mol / L HCl, and after complete dissolution, filter through a 0.22 μm microporous membrane for sterilization. Store at 4°C for later use.

[0047] Artificial intestinal fluid: Weigh 6.8g of potassium dihydrogen phosphate, dissolve it in ultrapure water, and bring the volume to 500 mL. Adjust the pH to 6.8 with 0.1 mol / L sodium hydroxide solution. Separately weigh 10g of pancreatic enzyme, dissolve it in an appropriate amount of ultrapure water, mix the two solutions, and dilute them with ultrapure water to 1000 mL. Store at 4℃ for later use.

[0048] A blank control group, a control group (sodium alginate embedding), and an experimental group were set up. Experimental group 1 consisted of the lyophilized intestinal flora powder prepared in Example 1 with the addition of an O / W emulsion, and its image is shown below. Figure 3As shown in the image. Experimental group 2 consisted of lyophilized intestinal flora powder prepared with unemulsified MCT-protein mixture (control). The blank control group consisted of lyophilized intestinal flora powder directly lyophilized without the addition of O / W emulsion (other preparation steps were the same as the experimental group), and its image is shown in the image below. Figure 1 As shown, the control group (sodium alginate embedding) consisted of lyophilized intestinal flora powder conventionally embedded with sodium alginate of the same specifications as the experimental group (other preparation steps were the same as the experimental group), and its image is shown below. Figure 2 As shown.

[0049] Experimental group: 1 g of the lyophilized intestinal flora prepared in Example 1 was used, and its activity before gastrointestinal fluid treatment was detected by flow cytometry; another 10 g of the lyophilized intestinal flora was added to artificial gastric fluid at a ratio of 1:9, vortexed, and incubated on a shaker at 37°C for 2 h; then centrifuged at 6000 ×g for 10 min to remove the gastric fluid, and artificial intestinal fluid was added at a ratio of 1:9, vortexed, and incubated on a shaker at 37°C for 3 h; centrifuged again at 6000 ×g for 10 min to remove the intestinal fluid, and the precipitated flora was detected by flow cytometry. The gastrointestinal fluid survival rate was calculated using the formula "Gastrointestinal fluid survival rate (%) = Sample activity after gastrointestinal fluid treatment (%) / Sample activity before gastrointestinal fluid treatment (%)". Microscopic images of donor A in this group before artificial gastrointestinal fluid treatment are shown below. Figure 6 As shown, the microscopic images after processing with artificial gastrointestinal fluid are as follows. Figure 9 As shown.

[0050] Blank control group: Intestinal flora lyophilized powder of the same specifications as the experimental group, without added O / W emulsion, was used. Artificial gastric and intestinal fluids were processed and activity was tested following the same steps as the experimental group. The survival rate of the gastrointestinal fluids was calculated for comparative analysis. Microscopic images of donor A in this group before artificial gastrointestinal fluid treatment are shown below. Figure 4 As shown, the microscopic images after processing with artificial gastrointestinal fluid are as follows. Figure 7 As shown.

[0051] Control group (sodium alginate embedding): Lyophilized intestinal flora powder with the same specifications as the experimental group, routinely embedded with sodium alginate, was processed using artificial gastric and intestinal fluids, and its activity was tested following the same steps as the experimental group. The survival rate of the gastrointestinal fluids was calculated for comparative analysis. Microscopic images of donor A in this group before artificial gastrointestinal fluid treatment are shown below. Figure 5 As shown, the microscopic images after processing with artificial gastrointestinal fluid are as follows. Figure 8 As shown.

[0052] Activity assay results showed: After treatment with artificial gastrointestinal fluid, the intestinal flora activity of experimental group 1 of donor A was 25.03%±1.32%, which was 3028.75% higher than that of the blank control group (the activity of the blank control group was 0.80%±0.08%), and the survival rate was 49.07%, which was 2061.67% higher than that of the blank control group (the survival rate of the blank control group was 2.27%). The survival rate of the control group (sodium alginate embedding) was 6.36%, which was 180.18% higher than that of the blank control group. The survival rate of experimental group 2 (unemulsified MCT-protein mixture (control)) was 34.60%, which was 1424.23% higher than that of the blank control group.

[0053] After treatment with artificial gastrointestinal fluid, the intestinal flora activity of donor B in experimental group 1 was 23.38%±0.56%, which was 3192.96% higher than that of the blank control group (the activity of the blank control group was 0.71%±0.06%). The survival rate was 48.65%, which was 2216.67% higher than that of the blank control group (the survival rate of the blank control group was 2.10%). The survival rate of the control group (sodium alginate embedding) was 6.12%, which was 191.43% higher than that of the blank control group. The survival rate of experimental group 2 (unemulsified MCT-protein mixture (control)) was 31.33%, which was 1391.90% higher than that of the blank control group.

[0054] After treatment with artificial gastrointestinal fluid, the intestinal flora activity of experimental group 1 of donor C was 24.97%±0.57%, which was 4061.67% higher than that of the blank control group (the activity of the blank control group was 0.60%±0.10%). The survival rate was 44.85%, which was 2364.29% higher than that of the blank control group (the survival rate of the blank control group was 1.82%). The survival rate of the control group (sodium alginate embedding) was 6.17%, which was 239.01% higher than that of the blank control group. The survival rate of experimental group 2 (unemulsified MCT-protein mixture (control)) was 29.52%, which was 1521.98% higher than that of the blank control group. For detailed data, please refer to Tables 2, 3 and 4.

[0055] In summary, the average survival rate of gut microbiota without O / W emulsion stabilization system treatment after artificial gastrointestinal fluid treatment was only 2.06%, while the average survival rate of gut microbiota with sodium alginate encapsulation treatment after artificial gastrointestinal fluid treatment was 6.22%. The average survival rate of experimental group 2 (unemulsified MCT-protein mixture (control)) after artificial gastrointestinal fluid treatment was 31.82%, while the average survival rate of gut microbiota after O / W emulsion stabilization system treatment (experimental group 1) reached 47.52%, which was much higher than that of the untreated group, the sodium alginate encapsulation group, and the unemulsified MCT-protein mixture (control) experimental group 2, with an average survival rate increase of 2214.21%. For detailed data, please refer to Table 5.

[0056] Table 2. Relevant data on the artificial gastrointestinal fluid test of gut microbiota after treatment with O / W emulsion stabilization system (donor A)

[0057] Table 3. Relevant data on the artificial gastrointestinal fluid test of gut microbiota after treatment with O / W emulsion stabilization system (donor B)

[0058] Table 4. Relevant data on the artificial gastrointestinal fluid test of gut microbiota after treatment with O / W emulsion stabilization system (donor C)

[0059] Table 5. Comprehensive data of intestinal flora and artificial gastrointestinal fluid after O / W emulsion stabilization system treatment.

[0060] The lyophilized intestinal flora powder prepared in Example 1 of this invention exhibited excellent tolerance in a simulated gastrointestinal environment, and the survival rate of gastrointestinal fluid was significantly improved compared with the control group. This indicates that the dense hydrophobic protective layer formed by the O / W emulsion stabilization system can effectively resist the stress of gastric acid and intestinal fluid and protect the activity of intestinal flora.

[0061] 16S rRNA high-throughput sequencing results (see appendix) Figure 10 The results showed significant differences between the original bacterial sludge and the freeze-dried samples of Example 1, the freeze-dried samples of Example 1 treated with gastrointestinal fluid, the freeze-dried samples of the blank control group, and the freeze-dried samples of the blank control group treated with gastrointestinal fluid. However, the microbial differentiation gradient among these five groups was: original bacterial sludge sample > freeze-dried samples of Example 1 treated with gastrointestinal fluid > freeze-dried samples of the blank control group treated with gastrointestinal fluid / freeze-dried samples of Example 1 / freeze-dried samples of the blank control group. This trend aligns with the β-diversity clustering distance trend, indicating that the microbial structure of the freeze-dried samples of Example 1 treated with gastrointestinal fluid is closest to that of the original bacterial sludge sample, while the distance between the freeze-dried samples of the blank control group treated with gastrointestinal fluid and the original bacterial sludge sample is the greatest. These results demonstrate that the present invention not only significantly improves the survival rate of live bacteria in gastrointestinal fluid but also achieves full-spectrum, non-selective protection of mixed microbial communities, ensuring that the delivered microbial community structure remains highly consistent with the original microbial community. Since the prior art only discloses the known effect of "improving the survival rate of a single pure strain" which is unrelated to community structure, those skilled in the art cannot expect "the complete maintenance of the mixed microbial community structure" from this known effect. Therefore, the present invention achieves a new effect that the prior art cannot expect.

[0062] Example 2 Preparation of freeze-dried intestinal flora powder The raw material preparation was the same as in Example 1, but the WPI concentration was adjusted to 3% (3,000 g dissolved in 100 mL of water). The protective agent formulation was adjusted to: 5 g trehalose, 4 g maltotriose, 3 g fructooligosaccharides, 2 g sodium caseinate, and 0.5 g gelatin dissolved in 1000 mL of physiological saline.

[0063] O / W emulsion preparation: Same as in Example 1.

[0064] Treatment of bacterial suspension: Same as in Example 1.

[0065] Preparation of sodium alginate-encapsulated bacterial sludge: Same as in Example 1.

[0066] The freeze-drying program was adjusted to the parameters in Table 6 (pre-freeze to -45℃, then finally heat up to 25℃).

[0067] Table 6. Freeze-drying procedure for gut microbiota

[0068] The results showed that, in this embodiment, the activity of the sample from donor A before freeze-drying was 61.66%±0.78%, and the activity of the sample after freeze-drying was 52.90%±0.06%, with a calculated freeze-drying survival rate of 85.79%. For donor B, the activity of the sample before freeze-drying was 66.81%±1.69%, and the activity of the sample after freeze-drying was 53.20%±0.41%, with a calculated freeze-drying survival rate of 79.63%. For donor C, the activity of the sample before freeze-drying was 64.71%±1.72%, and the activity of the sample after freeze-drying was 53.06%±0.94%, with a calculated freeze-drying survival rate of 82.00%. These results indicate that the embedding method of this invention can effectively preserve the activity of intestinal flora during freeze-drying and reduce damage to the flora during the freeze-drying process.

[0069] Gastrointestinal fluid resistance test The testing method is the same as in Example 1.

[0070] A blank control group, a control group (sodium alginate embedding), and an experimental group were set up. Experimental group 1 was the intestinal flora lyophilized powder prepared by adding O / W emulsion as described in Example 2. Experimental group 2 was the intestinal flora lyophilized powder prepared by adding unemulsified MCT-protein mixture (control). The blank control group was the intestinal flora lyophilized powder directly lyophilized without adding O / W emulsion (other preparation steps were the same as the experimental group). The control group (sodium alginate embedding) was the intestinal flora lyophilized powder conventionally embedded with sodium alginate of the same specifications as the experimental group (other preparation steps were the same as the experimental group).

[0071] Activity assay results showed: After treatment with artificial gastrointestinal fluid, the intestinal flora activity of experimental group 1 of donor A was 21.86%±1.62%, which was 2632.50% higher than that of the blank control group (the activity of the blank control group was 0.80%±0.08%). The survival rate was 41.32%, which was 1720.26% higher than that of the blank control group (the survival rate of the blank control group was 2.27%). The survival rate of the control group (sodium alginate embedding) was 6.36%, which was 180.18% higher than that of the blank control group. The survival rate of experimental group 2 (unemulsified MCT-protein mixture (control)) was 29.63%, which was 1205.29% higher than that of the blank control group.

[0072] After treatment with artificial gastrointestinal fluid, the intestinal flora activity of experimental group 1 of donor B was 21.84%±2.02%, which was 2976.06% higher than that of the blank control group (the activity of the blank control group was 0.71%±0.06%), and the survival rate was 41.05%, which was 1854.76% higher than that of the blank control group (the survival rate of the blank control group was 2.10%). The survival rate of the control group (sodium alginate embedding) was 6.12%, which was 191.43% higher than that of the blank control group. The survival rate of experimental group 2 (unemulsified MCT-protein mixture (control)) was 31.24%, which was 1387.62% higher than that of the blank control group.

[0073] After treatment with artificial gastrointestinal fluid, the intestinal flora activity of experimental group 1 of donor C was 23.90%±1.00%, which was 3883.33% higher than that of the blank control group (the activity of the blank control group was 0.60%±0.10%). The survival rate was 45.04%, which was 2374.73% higher than that of the blank control group (the survival rate of the blank control group was 1.82%). The survival rate of the control group (sodium alginate embedding) was 6.17%, which was 239.01% higher than that of the blank control group. The survival rate of experimental group 2 (unemulsified MCT-protein mixture (control)) was 30.71%, which was 1587.36% higher than that of the blank control group. For detailed data, please refer to Tables 7, 8 and 9.

[0074] In summary, the average survival rate of gut microbiota without O / W emulsion stabilization system treatment after artificial gastrointestinal fluid treatment was only 2.06%, while the average survival rate of gut microbiota with sodium alginate encapsulation treatment after artificial gastrointestinal fluid treatment was 6.22%. The average survival rate of experimental group 2 (unemulsified MCT-protein mixture (control)) after artificial gastrointestinal fluid treatment was 30.53%, while the average survival rate of gut microbiota after O / W emulsion stabilization system treatment (experimental group 1) reached 42.47%, which was much higher than that of the untreated group, the sodium alginate encapsulation group, and the unemulsified MCT-protein mixture (control) experimental group 2, with an average survival rate increase of 1983.25%. For detailed data, please refer to Table 10.

[0075] The above experimental results demonstrate that, under the premise that all components and all other process conditions are completely consistent, the survival rate can be improved by nearly 40% to 50% simply by "pre-emulsifying to form a stable O / W emulsion". That is, "pre-made O / W emulsion" has an independent and irreplaceable contribution to the technical effect of the present invention, and this contribution is verified by the dual indicators of live bacteria survival rate and bacterial community structure fidelity, rather than a simple summation of the effects of each component.

[0076] Table 7. Relevant data on the artificial gastrointestinal fluid test of gut microbiota after O / W emulsion stabilization system treatment (donor A)

[0077] Table 8. Relevant data on the artificial gastrointestinal fluid test of gut microbiota after O / W emulsion stabilization system treatment (donor B)

[0078] Table 9. Relevant data on the artificial gastrointestinal fluid test of gut microbiota after O / W emulsion stabilization system treatment (donor C)

[0079] Table 10. Comprehensive data from the artificial gastrointestinal fluid test of gut microbiota after O / W emulsion stabilization system treatment.

[0080] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the invention should be included within the scope of protection of the invention.

Claims

1. A method for freeze-drying and embedding intestinal flora based on an O / W emulsion stabilization system, characterized in that, Includes the following steps: (1) Raw material preparation: medium-chain triglycerides, natural protein aqueous solution with emulsifying activity, water-containing intestinal flora sludge and freeze-drying protectant aqueous solution are prepared; the water-containing intestinal flora sludge is intestinal flora sludge derived from feces of healthy donors and has not been freeze-dried; (2) Preparation of O / W emulsion: The natural protein aqueous solution and the medium chain triglyceride are mixed at an oil-water volume ratio of 1:8-10 and emulsified at 25-35°C to obtain an O / W emulsion without layering and without oil droplets floating. (3) Construction of composite system: First, mix the fungal mud with the protective agent aqueous solution at a ratio of 1g:4-10mL, and then mix it with the O / W emulsion at a ratio of 1g:8-10mL to form a "fungal mud-protectant-O / W emulsion" composite system; no cross-linking agent or gelling agent is added to the system, and no cross-linking curing treatment is performed; (4) Freeze-drying: The composite system is directly subjected to vacuum freeze-drying to obtain freeze-dried intestinal flora powder; the vacuum freeze-drying includes a pre-freezing stage, a sublimation drying stage and a desorption drying stage in sequence, the pre-freezing temperature of the pre-freezing stage is -60℃ to -40℃, and the final drying temperature of the desorption drying stage is 25℃ to 35℃.

2. The method according to claim 1, characterized in that, The preparation of the aqueous intestinal flora sludge in step (1) includes: taking a fresh fecal sample from a healthy donor, adding physiological saline, stirring and mixing at 4°C, filtering through 100-mesh, 150-mesh and 200-mesh filters in sequence, and collecting the filtrate; centrifuging the filtrate at 4°C and 5000×g for 10 min, and discarding the supernatant; resuspending and washing the sludge at a ratio of 1g:9mL (sludge weight to physiological saline volume), centrifuging again at 4°C and 8000×g for 10 min, discarding the supernatant, and collecting the aqueous intestinal flora sludge.

3. The method according to claim 1, characterized in that, The preparation of medium-chain triglycerides in step (1) includes: heating medium-chain triglycerides to 45-55°C and maintaining the temperature, removing crystalline particles, and filtering through an organic phase filter membrane for sterilization; the preparation method of the natural protein aqueous solution with emulsifying activity is as follows: dissolving natural protein powder with emulsifying activity in pre-cooled deionized water at 4°C at a mass-volume concentration of 3%-5%, adjusting the pH to 6.9-7.1, letting it stand overnight to allow the protein to expand, and filtering through an aqueous filter membrane for sterilization.

4. The method according to claim 1, characterized in that, The preparation process of the O / W emulsion in step (2) is as follows: place the natural protein aqueous solution with emulsifying activity in a water bath at 25-35℃, and slowly add medium chain triglycerides at a volume ratio of 1:8-10 for oil and water while stirring. Continue stirring until an O / W emulsion without layering, without obvious oil droplets floating, and without clumping on the wall when the container is inverted is formed.

5. The method according to claim 1, characterized in that, The emulsifying natural protein is selected from one or more of whey protein, soy protein isolate, and casein; the freeze-drying protectant includes one or more of lactose, maltose, trehalose, maltotriose, fructooligosaccharides, sodium caseinate, and gelatin.

6. The method according to claim 1, characterized in that, The freeze-drying protectant is a mixture of lactose, maltose and trehalose, or a mixture of trehalose, maltotriose, fructooligosaccharides, sodium caseinate and gelatin.

7. The method according to claim 1, characterized in that, The pre-freezing temperature in the pre-freezing stage is -60℃ to -40℃, and the holding time is 3 to 6 hours; the sublimation drying stage is carried out under vacuum conditions, with the temperature gradually increased from -45℃ to -35℃, then to -35℃ to -25℃, and then to -25℃ to -15℃, until it reaches -15℃ to 0℃; the desorption drying stage is carried out with the temperature gradually increased from 0℃ to 15℃, then to 15℃ to 25℃ to 25℃, until it reaches 25℃ to 35℃.

8. A freeze-dried powder of intestinal flora prepared by the method according to any one of claims 1 to 7, characterized in that, The freeze-dried powder contains intestinal flora, freeze-drying protectant, and an O / W emulsion freeze-dried product formed from emulsifying natural proteins and medium-chain triglycerides, wherein the emulsifying natural proteins and medium-chain triglycerides coat the surface of the bacteria to form a composite protective layer.

9. The lyophilized intestinal flora powder according to claim 8, characterized in that, The composite protective layer comprises a continuous phase composed of the natural protein with emulsifying activity and a dispersed phase composed of the medium-chain triglycerides, and the composite protective layer continuously coats the surface of the bacterial cells.

10. The use of the lyophilized intestinal flora powder of claim 8 in the preparation of oral intestinal flora transplantation formulations or intestinal microbial delivery drugs, characterized in that, The oral intestinal flora transplantation preparation or intestinal microbial delivery drug is used to treat Clostridium difficile infection or inflammatory bowel disease, or to maintain the diversity of the intestinal flora community structure.

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