A freeze-dried powder biological preparation of intestinal flora transplantation and a preparation method thereof

Abstract

The application discloses a kind of intestinal flora transplantation freeze-dried powder biological agent and preparation method thereof, freeze-dried powder biological agent includes the mixed powder prepared mainly with inulin, sweet orange fruit powder, cydonia freeze-dried powder, day day flower freeze-dried powder and intestinal bacteria freeze-dried powder;Mixed powder is according to mass fraction, ratio is as follows:inulin 75 parts, sweet orange fruit powder 10 parts, cydonia freeze-dried powder 12 parts, day day flower freeze-dried powder 3 parts;Intestinal bacteria freeze-dried powder and mixed powder are according to mass ratio 7:3;In the application, construct the four-element composite protection system of inulin+sweet orange fruit powder+cydonia freeze-dried powder+day day flower freeze-dried powder, realize "active protection+flavor blending+medicinal auxiliary+synergistic taste masking" fourfold efficacy through accurate component allocation ratio design;The selection of sweet orange fruit powder is in line with the dispersibility requirement of freeze-dried powder, the rich aroma of cydonia freeze-dried powder is synergized with day day flower freeze-dried powder, accurately covers up the fishy smell of freeze-dried powder, and formula ratio adapts to the mixing process of freeze-dried powder, ensure that there is no particle aggregation, improve preparation uniformity.

Description

Technical Field

[0001] This invention relates to the field of intestinal flora transplantation biological agents, and particularly to an intestinal flora transplantation lyophilized powder biological agent and its preparation method. Background Technology

[0002] Gut microbiota transplantation (GMT) is an important treatment for diseases related to gut microbiota imbalance (such as inflammatory bowel disease and irritable bowel syndrome). Its core principle is to transfer the gut microbiota of a healthy donor to the patient's gut, restoring the patient's gut microbiota balance. The key to successful GMT formulation lies in maintaining the activity of the microbiota during preparation, storage, and transport, while simultaneously optimizing the palatability of the formulation to improve patient compliance.

[0003] Currently, intestinal flora transplantation preparations are mainly divided into two mainstream dosage forms: lyophilized powder preparations and bacterial sludge capsule preparations. The corresponding protectants mostly use single ingredients such as trehalose, mannitol, glycerol, and sucrose or simple compound systems. Their main function is to maintain the activity of the flora, but they have obvious functional limitations and cannot meet the diversified needs of clinical applications.

[0004] Existing protective agents are mostly simple mixtures of components, without forming functional synergy, and cannot simultaneously achieve multiple functions such as activity protection, flavor blending, pharmaceutical assistance, and long-lasting taste masking. Moreover, existing freeze-dried powder formulation systems have poor compatibility and low dispersibility.

[0005] Therefore, existing technologies still need to be improved and enhanced. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the purpose of this invention is to provide a lyophilized powder biological agent for intestinal flora transplantation and its preparation method, aiming to solve the problems of poor compatibility, low dispersibility and lack of functional synergy in existing lyophilized powder formulation systems.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: Firstly, a lyophilized biological agent for intestinal flora transplantation, comprising inulin, sweet orange fruit powder, citron lyophilized powder, bitter orange lyophilized powder, and intestinal bacteria lyophilized powder.

[0008] Furthermore, the freeze-dried biological agents, by mass parts, are as follows: inulin 72-78 parts, sweet orange fruit powder 9-11 parts, citron freeze-dried powder 11-13 parts, and bitter orange freeze-dried powder 2.5-3.5 parts.

[0009] The second aspect is a method for preparing a lyophilized powder biological agent for intestinal flora transplantation, the preparation method comprising: Step 1: Prepare citron freeze-dried powder and bitter orange freeze-dried powder separately, and mix inulin and sweet orange fruit powder with bergamot freeze-dried powder and bitter orange freeze-dried powder in proportion to prepare mixed powder; Step 2: Take intestinal flora and perform routine freeze-drying to prepare intestinal flora freeze-dried powder; then mix the intestinal flora freeze-dried powder with the mixed powder in a mass ratio, and stir under sterile conditions to obtain intestinal flora transplantation freeze-dried powder preparation.

[0010] Furthermore, in step 1, the citron freeze-dried powder is prepared using a low-temperature freeze-drying process, with a freeze-drying temperature of 40℃-50℃ and a freeze-drying time of 12-24h, preserving its natural aromatic substances and active ingredients, and a particle size of 100-200 mesh.

[0011] Furthermore, in step 1, the freeze-dried powder of Bitter Orange Flower is prepared using a low-temperature freeze-drying process, with a particle size of 100-200 mesh, retaining natural volatile oil components, and the volatile oil content is above 0.1%.

[0012] Furthermore, in step 1, the mass ratio of citron freeze-dried powder to bitter orange freeze-dried powder is 11:3.5-13:2.5 (the other two types of inulin and sweet orange fruit powder are added and mixed according to the required ratio of 7.5:1).

[0013] Furthermore, in step 2, the enterovirus freeze-dried powder and the mixed powder are mixed at a mass ratio of 7:3.

[0014] Furthermore, in step 2, the aseptic environment humidity is below 35%, the ambient temperature is 2-8℃, and the finished intestinal flora transplantation lyophilized powder preparation is sealed and stored in a refrigerated environment at 2-8℃.

[0015] Furthermore, the stirring rate is 50-100 r / min and the stirring time is 5-10 min.

[0016] The technical solution adopted in this invention has the following beneficial effects: In this invention, citron and bitter orange, two medicinal and edible raw materials, are combined and applied to the preservative of lyophilized intestinal flora transplantation powder. Neither raw material causes gastrointestinal irritation or inhibits intestinal flora activity, and they achieve synergistic medicinal value, overcoming the limitations of traditional preservatives that only focus on activity protection while neglecting auxiliary medicinal value. Citron and bitter orange, both medicinal and edible raw materials, possess natural aromatic scents, and when combined, they form a synergistic taste-masking effect, effectively masking the fishy and unpleasant odors of the lyophilized intestinal flora powder itself. Combined with the flavor-harmonizing effect of sweet orange fruit powder, this further optimizes the palatability of the formulation, addressing the industry pain point of low patient compliance due to unpleasant odor in lyophilized intestinal flora transplantation powder formulations. Furthermore, the aromatic substances are stable and do not easily volatilize during storage, ensuring a long-lasting taste-masking effect without affecting flora activity. A quaternary composite protective system of inulin + sweet orange fruit powder + citron lyophilized powder + bitter orange lyophilized powder is constructed. Through precise component ratio design, it achieves "activity protection + flavor harmonization + medicinal assistance + synergistic taste masking". Four-fold efficacy, precisely adapted to the characteristics and application scenarios of intestinal flora transplantation freeze-dried powder formulations; specifically adapted to intestinal flora freeze-dried powder formulations, the selection of sweet orange fruit powder meets the dispersibility requirements of freeze-dried powder, the rich aroma of citron freeze-dried powder works synergistically with bitter orange freeze-dried powder to precisely mask the fishy smell of freeze-dried powder, and the formula ratio is adapted to the mixing process of freeze-dried powder to ensure no particle agglomeration and improve the uniformity of the formulation. Detailed Implementation

[0017] To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0018] A lyophilized biological agent for intestinal flora transplantation, comprising a mixed powder mainly composed of inulin, sweet orange fruit powder, citron lyophilized powder, and bitter orange lyophilized powder, and intestinal flora lyophilized powder, wherein the above components are in any proportion.

[0019] The preferred formulation of the above components is as follows: the mixed powder is in the following proportions by mass: 72-78 parts inulin, 9-11 parts sweet orange fruit powder, 11-13 parts citron freeze-dried powder, and 2.5-3.5 parts bitter orange freeze-dried powder.

[0020] A method for preparing a lyophilized powder biological agent for intestinal flora transplantation, the preparation method comprising: Step 1: Prepare citron freeze-dried powder and bitter orange freeze-dried powder separately, and mix inulin and sweet orange fruit powder with bergamot freeze-dried powder and bitter orange freeze-dried powder in proportion to prepare mixed powder; Citron freeze-dried powder is prepared using a low-temperature freeze-drying process at a temperature of 40℃-50℃ for 12-24 hours, preserving its natural aromatic substances and active ingredients, with a particle size of 100-200 mesh. Bitter orange freeze-dried powder is also prepared using a low-temperature freeze-drying process, with a particle size of 100-200 mesh, preserving natural volatile oil components, and a volatile oil content of over 0.1%. The mass ratio of citron freeze-dried powder to bitter orange freeze-dried powder is 11:3.5-13:2.5 (the other two powders, inulin and sweet orange fruit powder, are added and mixed according to a ratio of 7.5:1 as required). Both citron and bitter orange freeze-dried powders must be stored away from light to prevent the volatilization of aromatic substances.

[0021] Inulin, as the main protective substrate, also possesses prebiotic activity, maintaining the activity and stability of intestinal flora after freeze-drying and during storage. Food-grade inulin with a purity of ≥95% is selected. Sweet orange fruit powder, as an auxiliary protective agent, enhances the dispersibility and stability of the protective agent system while harmonizing the basic flavor. Natural freeze-dried sweet orange fruit powder is selected, with no added preservatives or sucrose.

[0022] Step 2: Collect intestinal flora and perform routine freeze-drying to prepare intestinal flora freeze-dried powder. Then, mix the intestinal flora freeze-dried powder with the mixed powder in a specific mass ratio under sterile conditions to obtain the intestinal flora transplantation freeze-dried powder preparation. The intestinal flora freeze-dried powder and the mixed powder are mixed in a mass ratio of 7:3. The sterile environment humidity is below 35%, and the ambient temperature is 2-8℃. The finished intestinal flora transplantation freeze-dried powder preparation is sealed and stored in a refrigerated environment at 2-8℃, with a shelf life of over 12 months. The stirring rate is 50-100 r / min, and the stirring time is 5-10 min.

[0023] Example 1: Protectant for lyophilized intestinal flora transplantation powder: Take 75g of inulin, 10g of sweet orange fruit powder, 12g of citron freeze-dried powder, and 3g of bitter orange freeze-dried powder. The citron freeze-dried powder and bitter orange freeze-dried powder are prepared by a low-temperature freeze-drying process at -45℃ for 18 hours, with a particle size of 150 mesh. Mix the above components evenly in a sterile environment at 2-8℃ and humidity ≤30% to obtain the intestinal flora transplantation freeze-dried powder protectant of the present invention. Take 70g of intestinal flora freeze-dried powder and mix it with 30g of the protectant. Stir for 8 minutes at a temperature of 5℃, humidity of 25%, and stirring speed of 80r / min, with sterile operation throughout. After stirring, aseptically package to obtain the final intestinal flora transplantation freeze-dried powder preparation. The preparation is sealed and stored in a refrigerated environment at 2-8℃, with a shelf life of more than 12 months.

[0024] To verify the technical effectiveness of the protective agent of this invention, multiple sets of comparative experiments were conducted, strictly following the "Microbial Limit Test Method" and the "Food Safety Toxicology Evaluation Procedure". The core test data are as follows (all are the average values ​​of 3 parallel tests, RSD≤3.5%, to ensure data reliability): Microbial activity test Test subjects: freeze-dried powder protectant of this invention, traditional protectant (trehalose + mannitol compound), and single citron / bitter orange flower protectant.

[0025] Experimental method: Each protectant was mixed with the enteric bacteria lyophilized powder and stored under specified conditions. The survival rate of the bacterial community was tested after 1 month, 3 months, 6 months and 12 months of storage.

[0026] Experimental results: The freeze-dried powder formulation treated with the protective agent of this invention achieved a bacterial survival rate of 89.6% after 6 months of storage and 81.2% after 12 months of storage, both far exceeding the industrial storage requirements (the industry standard requirement is ≥75%), and perfectly matching the description of the 12-month shelf life of freeze-dried powder. The freeze-dried powder treated with the traditional protective agent (trehalose:mannitol = 1:1) had a survival rate of only 74.2% after 6 months, failing to meet the 12-month qualified storage standard. The freeze-dried powder treated with a single citron protective agent had a survival rate of 78.5% after 6 months, and the freeze-dried powder treated with a single Fructus davidiana had a survival rate of 77.8% after 6 months. Without synergistic effects, the protective effect was significantly weaker than the compound system of this invention.

[0027] Gastrointestinal irritation test Test subjects: the freeze-dried powder protectant of this invention (diluted according to the usage ratio) and physiological saline (blank control).

[0028] Experimental methods: Animal experiments (mice) were conducted. The drugs were administered by gavage for 7 consecutive days. The condition of the gastrointestinal mucosa, feeding and defecation of mice were observed, and the pathological sections of gastrointestinal tissue were examined.

[0029] Experimental Results: Forty SPF-grade Kunming mice were randomly divided into four groups (n=10 per group, half male and half female, weighing 20±2g). Each group was administered the following by gavage: a diluted protective agent of this invention (10-fold diluted to the clinical concentration), physiological saline (blank control), or a positive control (0.1mol / L hydrochloric acid). The administration was repeated for 7 consecutive days, with a daily gavage volume of 0.2mL / 10g body weight. After gavage, the gastric and intestinal mucosa of the mice were dissected and observed. The gastric and intestinal mucosa of the blank control group and the protective agent group were smooth, without congestion or edema, with a gastric mucosal damage index of 0.0±0.0. The gastric mucosa of the positive control group showed significant congestion and edema, with a damage index of 3.8±0.5. Histopathological examination of the gastrointestinal tissue showed that the gastrointestinal cells of the protective agent group were neatly arranged, without inflammatory cell infiltration, and there was no significant difference compared to the blank control group (P>0.05). This is consistent with the description in the component instructions that "citron and bitter orange have no gastrointestinal irritation," proving that the protective agent of this invention has no gastrointestinal irritation and good safety.

[0030] Odor evaluation test Test subjects: lyophilized powder formulations treated with the protective agent of this invention, lyophilized powder formulations treated with traditional protective agents, and lyophilized powder formulations without added protective agents.

[0031] Experimental method: A sensory evaluation group of 10 people was formed to evaluate the odor of each preparation using a 1-10 scoring method (1 point: extremely poor odor, strong fishy smell; 10 points: good odor, no unpleasant odor, faint fragrance). The average value was taken.

[0032] Experimental Results: The freeze-dried powder formulation treated with the preservative of this invention initially scored 9.0 points in odor, effectively masking the unpleasant odor of the enteric bacteria freeze-dried powder to the greatest extent. After 6 months of storage, the score remained at 8.7 points, decreasing by only 0.3 points, demonstrating excellent stability of aromatic substances. The freeze-dried powder treated with the traditional preservative initially scored 5.8 points, decreasing to 4.8 points after 6 months of storage, indicating poor odor masking and significant attenuation. The freeze-dried powder without the preservative initially scored only 2.5 points, showing no odor masking effect and extremely poor odor. The experiment proves that the synergistic odor masking effect of the combination of two medicinal and edible homologous raw materials in this invention is significant and long-lasting, while not affecting the activity of the bacterial flora.

[0033] Adaptability testing Test subject: The mixed system of the freeze-dried powder protectant of this invention and enteric bacteria freeze-dried powder.

[0034] Test method: The dispersion uniformity, particle agglomeration and flowability (angle of repose) of the mixed system were tested, and the judgment was made in accordance with the industry quality standards for lyophilized powder preparations.

[0035] Test results: After the protective agent of this invention is mixed with the enteric bacteria lyophilized powder, the dispersion uniformity reaches 98.7%, and there is no particle agglomeration (the number of particles with a particle size ≥100μm is 0). In the flowability test, the angle of repose is 31.2°, which is far lower than the judgment standard of "good flowability" (≤35°) and the industry standard (≤38°), fully meeting the quality requirements of lyophilized powder formulations. It is consistent with the description of "uniform stirring and no particle agglomeration" in the usage method and is suitable for the industrial mixing process of lyophilized powder.

[0036] The above test parameters are configured as follows: Basic test parameters: Number of parallel experimental groups: 3 groups, with an average RSD ≤ 3.5%.

[0037] Mouse experiment: 40 SPF-grade Kunming mice were used, with 10 mice in each group (half male and half female), weighing 20±2g. The gavage cycle was 7 days, and the daily gavage volume was 0.2mL / 10g body weight.

[0038] Odor evaluation: A sensory evaluation panel of 10 people, using a 1-10 scoring method.

[0039] Industry standard reference: microbial survival rate ≥75% (industrialization requirement); freeze-dried powder angle of repose ≤38° (industry standard).

[0040] The values ​​for bacterial community activity testing (core verification items) are shown in Table 1: Table 1 Gastrointestinal irritation test values Positive control (0.1 mol / L hydrochloric acid): gastric mucosal damage index 3.8±0.5, with obvious congestion and edema. Blank control (physiological saline): gastric mucosal damage index 0.0±0.0, mucosa smooth and without abnormalities.

[0041] The protective agent group of this invention: gastric mucosal damage index 0.0±0.0, no congestion and edema, and no inflammatory infiltration in pathological sections (P>0.05).

[0042] Related description: Consistent with the ingredient description that "citron and bitter orange have no gastrointestinal irritation".

[0043] The odor evaluation test values ​​are shown in Table 2: Table 2 Compatibility test values ​​(lyophilized powder compatibility) Dispersion uniformity: 98.7%.

[0044] Particle aggregation: The number of particles with a diameter ≥100μm is 0, indicating no aggregation.

[0045] Angle of repose: 31.2° (≤35°, good fluidity, meets industry standard ≤38°).

[0046] Related description: Consistent with the description of "uniform stirring and no particle agglomeration" in the preparation method.

[0047] The novel formulation of the lyophilized powder protectant for intestinal flora transplantation provided by this invention has the following significant advantages compared to existing technologies: Stable and long-lasting activity protection: Through the core protective effect of inulin, combined with the synergistic assistance of citron and bitter orange, two medicinal and edible components, the activity of intestinal flora can be effectively maintained during preparation, storage and transportation. The freeze-dried powder preparation can have a shelf life of more than 12 months under refrigeration conditions of 2-8℃, which is far superior to traditional protective agents and meets the long-term storage needs of industrialization and clinical application.

[0048] High safety and wide applicability: All components are food-grade or pharmaceutical-grade. The combination of citron and bitter orange, two medicinal and edible raw materials, does not cause gastrointestinal irritation. Animal experiments have verified its good safety profile, making it suitable for long-term use by different groups of people. Its safety in clinical applications is guaranteed.

[0049] Synergistic medicinal value enhances clinical value: The combination of citron and bitter orange, two medicinal and edible raw materials, achieves complementary medicinal value, which can help improve intestinal function and provide additional support for patients' intestinal health. This breaks through the single function of traditional protectants that only have active protection and enhances the clinical application value of the preparation.

[0050] Significant and long-lasting flavor masking effect: The synergistic flavor masking system formed by citron and bitter orange can completely mask the fishy smell of freeze-dried intestinal flora powder. Combined with the flavor harmonizing effect of sweet orange fruit powder, it greatly optimizes the palatability of the formulation. Moreover, the aromatic substances are stable and do not volatilize significantly during storage. They can maintain a good flavor masking effect throughout the entire shelf life, significantly improve patient compliance, and solve a core pain point in the industry.

[0051] Highly adaptable and easy to industrialize: Specifically designed for lyophilized powder formulations, the formulation ratio is highly compatible with the preparation and mixing process of lyophilized powders. The mixed formulation has excellent dispersibility and flowability, with no particle agglomeration. No adjustment is required to existing production equipment, making it highly practical and directly applicable to large-scale industrial production.

[0052] With outstanding innovation, it is the first time that the compound of citron and bitter orange, two medicinal and edible aromatic raw materials, has been applied to the intestinal flora transplantation freeze-dried powder protectant, forming a unique four-element composite protection system technical solution, which is of great technological innovation.

Claims

1. A lyophilized biological agent for intestinal flora transplantation, characterized in that, Freeze-dried biological agents include inulin, sweet orange fruit powder, citron freeze-dried powder, bitter orange freeze-dried powder, and enterobacteria freeze-dried powder.

2. The lyophilized biological agent for intestinal flora transplantation according to claim 1, characterized in that, The freeze-dried biological agents are distributed in the following proportions by weight: inulin 72-78 parts, sweet orange fruit powder 9-11 parts, citron freeze-dried powder 11-13 parts, and bitter orange freeze-dried powder 2.5-3.5 parts.

3. A method for preparing the lyophilized biological agent for intestinal flora transplantation as described in claim 1, characterized in that, Preparation methods include: Step 1: Prepare citron freeze-dried powder and bitter orange freeze-dried powder separately, and mix inulin and sweet orange fruit powder with bergamot freeze-dried powder and bitter orange freeze-dried powder in proportion to prepare mixed powder; Step 2: Take intestinal flora and perform routine freeze-drying to prepare intestinal flora freeze-dried powder; then mix the intestinal flora freeze-dried powder with the mixed powder in a mass ratio, and stir under sterile conditions to obtain intestinal flora transplantation freeze-dried powder preparation.

4. The method for preparing the lyophilized biological agent for intestinal flora transplantation according to claim 3, characterized in that, In step 1, the citron freeze-dried powder is prepared using a low-temperature freeze-drying process. The freeze-drying temperature is 40℃-50℃, and the freeze-drying time is 12-24h, which preserves its natural aromatic substances and active ingredients. The particle size is 100-200 mesh.

5. The method for preparing the lyophilized biological agent for intestinal flora transplantation according to claim 3, characterized in that, In step 1, the freeze-dried powder of bitter orange is prepared by low-temperature freeze-drying process, with a particle size of 100-200 mesh, retaining natural volatile oil components, and the volatile oil content is above 0.1%.

6. The method for preparing the lyophilized biological agent for intestinal flora transplantation according to claim 3, characterized in that, In step 1, the mass ratio of citron freeze-dried powder to bitter orange freeze-dried powder is 11:3.5-13:2.5, and inulin and sweet orange fruit powder are added and mixed in a ratio of 7.5:1 as required.

7. The method for preparing the lyophilized biological agent for intestinal flora transplantation according to claim 3, characterized in that, In step 2, the enterovirus freeze-dried powder and the mixed powder are mixed at a mass ratio of 7:

3.

8. The method for preparing the lyophilized biological agent for intestinal flora transplantation according to claim 3, characterized in that, In step 2, the aseptic environment humidity is below 35% and the ambient temperature is 2-8℃. The finished intestinal flora transplantation lyophilized powder preparation is sealed and stored in a refrigerated environment at 2-8℃.

9. The method for preparing the lyophilized biological agent for intestinal flora transplantation according to claim 3, characterized in that, The stirring rate is 50-100 r / min and the stirring time is 5-10 min.

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