A probiotic compound and its use in the prevention and repair of damage to the mucosa of the gynecological reproductive tract

Abstract

The present application provides a kind of probiotic compound and its application in preventing and repairing gynecological reproductive tract mucosa damage.The compound is composed of core probiotic, mucosa repair auxiliary component and sustained-release carrier, which are collectively encapsulated in the sustained-release carrier to form drug-loaded microspheres, with total viable count ≥1×10 10 CFU / g; the mucosa repair auxiliary component is a specific ratio combination of snail mucus active peptide, L-arginine and phytosterol; the sustained-release carrier is γ-ray modified chitosan and sodium alginate composite microspheres, which are matched with prebiotics for directional synergism, realize bacteriostasis, mucosa repair and microecological regulation functions through multi-component synergy, solve the problem of easy inactivation and weak repair ability of traditional products, provide a safe and effective new solution for gynecological health, and can be prepared into various dosage forms such as suppositories and gels, with wide application prospect.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and gynecological care technology, and in particular to a probiotic complex and its application in the prevention and repair of gynecological reproductive tract mucosal damage. Background Technology

[0002] The gynecological reproductive tract mucosa is a natural barrier of the female reproductive system, and its integrity is crucial for resisting pathogen invasion and maintaining the balance of the vaginal microecology. However, in clinical practice, the causes of gynecological reproductive tract mucosal damage are diverse, and women experience varying degrees of vaginal mucosal damage. Common causes include improper sexual activity, iatrogenic procedures, inflammatory stimulation, and hormonal changes. Damaged mucosa can lead to vaginal pH imbalance and a reduction in beneficial bacteria, increasing the risk of HPV infection and making women more susceptible to recurrent infections such as vaginitis and cervicitis, seriously affecting women's health.

[0003] Current clinical treatments for mucosal damage have significant limitations: traditional antibiotics, while inhibiting harmful bacteria, easily disrupt the vaginal flora balance, exacerbating the weakening of the mucosal barrier function, and cannot achieve mucosal tissue repair and regeneration; single probiotic preparations struggle to maintain activity in the complex vaginal environment, exhibiting low colonization efficiency and short duration of action, only partially regulating flora balance and failing to address the needs of mucosal damage repair; while repair drugs containing growth factors and polysaccharides can only promote local tissue regeneration, lacking long-term inhibitory capabilities against pathogens, unable to prevent secondary infections at the damaged site, and some components have poor biocompatibility, posing a risk of local irritation. Furthermore, existing related products have significant shortcomings in dosage form design; ordinary formulations struggle to achieve targeted release and long-term retention of active ingredients, and probiotics and repair components are prone to rapid leakage or inactivation, leading to fluctuations in efficacy; traditional carrier materials have limited encapsulation and protection capabilities, failing to address the technical challenge of simultaneous and sustained action of antibacterial and repair components.

[0004] Therefore, developing novel formulations that combine antibacterial, repair, and microecological regulation functions has become a key need for solving the problem of gynecological reproductive tract mucosal damage. Summary of the Invention

[0005] In view of this, the present invention proposes a probiotic complex and its application in the prevention and repair of gynecological reproductive tract mucosal damage, thereby solving the above problems.

[0006] The technical solution of this invention is implemented as follows: a probiotic complex includes core probiotics, mucosal repair auxiliary components, and a sustained-release carrier. The core probiotics and mucosal repair auxiliary components are co-encapsulated within the sustained-release carrier to form drug-loaded microspheres. The core probiotics include *Lactobacillus rhamnosus*, *Lactobacillus reuteri*, *Lactobacillus helveticus*, *Lactobacillus curvatureii*, and *Lactobacillus gasseri*, with a viable count ratio of (1-4):(1-3):(0.5-2):(0.5-2.5):(0.5-2). The total viable count of the core probiotics in the drug-loaded microspheres is ≥1×10⁻⁶. 10 CFU / g.

[0007] Preferably, it comprises the following components in parts by weight: 10-20 parts of core probiotics, 2-8 parts of prebiotics, 3-5 parts of mucosal repair auxiliary ingredients, and a sustained-release carrier.

[0008] Preferably, the mucosal repair auxiliary component comprises snail mucus active peptides, L-arginine, and phytosterols in a mass ratio of 1:(1.2-2.3):(2.5-3.5), wherein the molecular weight of the snail mucus active peptides is 300-1000 Da, and the phytosterols are selected from one or more combinations of β-sitosterol, stigmasterol, campesterol, and sitosterol.

[0009] Preferably, the method for extracting the snail mucus active peptides includes the following steps: (1) Select live white jade snails, clean their body surface with sterile physiological saline, collect fresh mucus, add 3-5 times the volume of pH 6.5-7.2 phosphate buffer, stir evenly to obtain mucus suspension; (2) Extract the mucus suspension by magnetic stirring at 4-10℃ for 1-2 hours, then centrifuge at 8000-10000 rpm for 15-20 minutes and take the supernatant. (3) The supernatant was initially filtered through an ultrafiltration membrane with a molecular weight cutoff of 1 kDa. The filtrate was collected, purified by gel filtration chromatography, and the eluent was collected. The eluent was then freeze-dried at -40°C to -50°C to obtain snail mucus active peptide powder.

[0010] Preferably, the sustained-release carrier is modified chitosan and sodium alginate, wherein the modified chitosan is modified by γ-ray irradiation with an irradiation dose of 10-30 kGy for 20-40 min.

[0011] Preferably, the prebiotic is selected from one or more combinations of fructooligosaccharides, galactooligosaccharides, inulin, and resistant dextrin.

[0012] This invention also provides a method for preparing a probiotic complex, comprising the following steps: S1. Cultivation and pretreatment of core probiotics: Core probiotics were inoculated into MRS medium and cultured under anaerobic conditions at 35-37℃ for 18-24 hours. Then, the bacterial sludge was collected by centrifugation at 8000-10000rpm for 15-20 minutes. The bacterial sludge of each strain was mixed evenly according to the live cell count ratio and pre-frozen at -40℃ to -60℃ for 2-4 hours. After that, the compound freeze-dried bacterial powder was obtained by freeze drying. The sublimation drying temperature was 0-10℃, the desorption drying temperature was 20-30℃, and the total drying time was 12-18 hours. Then, prebiotics were added and mixed evenly to obtain probiotic-prebiotic mixed powder. S2. Preparation of mucosal repair auxiliary ingredients: Take snail mucus active peptide powder, mix it with L-arginine and phytosterol, and stir at 100-200 rpm for 15-30 minutes until uniform to obtain mucosal repair auxiliary powder. S3. Preparation of sustained-release carrier: S31. Mix the probiotic-prebiotic mixed powder obtained in S1 with the mucosal repair auxiliary powder obtained in S2 evenly to obtain the total mixed powder; S32. Dissolve the γ-ray modified chitosan in a 0.5-1.5% (v / v) aqueous solution of glacial acetic acid to prepare a 0.5-2.0% (w / v) modified chitosan acid solution. Dissolve sodium alginate in phosphate buffer to prepare a 0.5-2.0% (w / v) sodium alginate solution for later use. S33. Disperse the total mixed powder in a modified chitosan solution and stir until homogeneous to form a suspension; add it dropwise to a sodium alginate solution, crosslink it with calcium ions, filter it, and dry it to obtain probiotic complex microspheres with a particle size of 50-200 μm.

[0013] Preferably, the suspension described in step S33 is stirred at 25-35°C and 200-300 rpm for 30-60 minutes, and the final concentration of the suspension is 10-20% (w / v). In step S33, the dropwise flow rate is 0.5-2 mL / min, and the suspension is added dropwise to the sodium alginate solution at a volume ratio of 1:8-12. After the dropwise addition is completed, a calcium chloride solution with a concentration of 0.1-0.3 mol / L is immediately added to the mixed solution, with a volume ratio of calcium chloride solution to sodium alginate solution of 1:3-5. The reaction is continued to be stirred for 1-2 hours.

[0014] The present invention relates to the application of probiotic complex in the preparation of products for preventing and repairing gynecological reproductive tract mucosal damage.

[0015] Preferably, the product is a suppository, gel, effervescent tablet, or vaginal douche solution.

[0016] Compared with the prior art, the beneficial effects of the present invention are: This invention utilizes a scientifically formulated blend of core probiotics such as *Lactobacillus rhamnosus* and *Lactobacillus reuteri* to achieve potent inhibition of pathogenic bacteria in the gynecological reproductive tract. For example, the inhibition zone diameter for *Candida albicans* and *Gardnerella vaginalis* can reach 17.8 ± 1.0 mm, significantly superior to single-strain or traditional probiotic preparations. By adding prebiotics such as fructooligosaccharides and galactooligosaccharides, the proliferation and metabolic activity of the core probiotics can be targeted to enhance their colonization stability and numerical dominance in the vagina. The mucosal repair auxiliary ingredients utilize a specific combination of snail mucus active peptides, L-arginine, and phytosterols. These three components work synergistically to exert a repairing effect, exhibiting excellent biocompatibility and mucosal penetration. They can directly promote the migration and proliferation of vaginal epithelial cells, accelerate the healing of damaged mucosa, improve local blood circulation and nutrient supply, and enhance the mucosal repair and regeneration capacity. They can also regulate the mucosal inflammatory response, reduce irritation at the damaged site, and strengthen the physical defense function of the mucosal barrier. Combined with probiotics, they inhibit pathogenic bacteria while promoting mucosal tissue regeneration, shortening the healing time and solving the technical pain point of traditional products that only inhibit bacteria but do not repair.

[0017] This invention uses γ-ray irradiated modified chitosan and sodium alginate to construct a composite sustained-release carrier. The modified chitosan has increased cross-linking density, and the three-dimensional network structure formed with sodium alginate has excellent encapsulation protection and controlled-release performance, effectively protecting the activity of probiotics, prolonging the duration of action, and ensuring that probiotics maintain a high number of viable bacteria in the complex environment of the vagina, overcoming the defects of ordinary probiotics that are easy to inactivate and have a short colonization time. Detailed Implementation

[0018] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.

[0019] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0020] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0021] In this invention, Lactobacillus rhamnosus, derived from the China Center for Type Culture Collection, Latin name: Lacticaseibacillus rhamnosus Accession number: CCTCC AB 2015377; Lactobacillus reuteri, also known as *Lactobacillus reuteri*, is derived from the China General Microbiological Culture Collection Center. Limosilactobacillus reuteri Accession number: CGMCC 1.12733; Lactobacillus helveticus was obtained from the China Center for Type Culture and Preservation. Latin name: Lactobacillus helveticus Accession number: CCTCC S2022088; Lactobacillus curvatureii was obtained from the China General Microbiological Culture Collection Center. Its Latin name is: Lactobacillus crispatus Accession number: CGMCC 1.2743; Lactobacillus gasseri was obtained from the China General Microbiological Culture Collection Center. Its Latin name is: Lactobacillus gasseri Accession number: CGMCC 1.3396.

[0022] Example 1 1. Probiotic complex formula: 10 servings of core probiotics: Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus helveticus, Lactobacillus brevis, and Lactobacillus gasseri live bacteria count: 1:1:0.5:0.5:0.5; total live bacteria count ≥1×10⁻⁶ 10 CFU / g; 2 servings of prebiotics: fructooligosaccharides; Three portions of mucosal repair auxiliary ingredients: snail mucus active peptide, L-arginine and phytosterol in a mass ratio of 1:1.2:2.5. The molecular weight of snail mucus active peptide is 300 Da, and the phytosterol is β-sitosterol. Sustained-release carriers: modified chitosan and sodium alginate; 2. Preparation of key components: Snail mucus active peptides (1) Select live white jade snails, clean their body surface with sterile physiological saline, collect fresh mucus, add 4 times the volume of pH=7 phosphate buffer, stir evenly to obtain mucus suspension. (2) Extract the mucus suspension by magnetic stirring at 7°C for 1.5 h, then centrifuge at 10000 rpm for 18 min and take the supernatant; (3) The supernatant was initially filtered through an ultrafiltration membrane with a molecular weight cutoff of 1 kDa. The filtrate was collected, purified by gel filtration chromatography, and the eluent was collected and freeze-dried at -45°C to obtain snail mucus active peptide powder.

[0023] Modified chitosan was treated with γ-ray irradiation at a dose of 20 kGy for 30 min.

[0024] Example 2 1. Probiotic complex formula: 20 servings of core probiotics: Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus helveticus, Lactobacillus brevis, and Lactobacillus gasseri live bacteria count = 4:3:2:2.5:2; total live bacteria count ≥ 1×10⁻⁶ 10 CFU / g; 8 servings of prebiotics: stachyose; Five parts of mucosal repair auxiliary ingredients: snail mucus active peptide, L-arginine and phytosterol in a mass ratio of 1:2.3:3.5. The molecular weight of snail mucus active peptide is 1000 Da, and the phytosterol is stigmasterol. Sustained-release carriers: modified chitosan and sodium alginate; 2. The preparation of key components is the same as in Example 1.

[0025] Example 3 1. Probiotic complex formula: 15 portions of core probiotics: Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus helveticus, Lactobacillus brevis, and Lactobacillus gasseri live bacteria count = 3:2:1.5:1.5:1; total live bacteria count ≥ 1×10⁻⁶ 10 CFU / g; 5 servings of prebiotics: galactooligosaccharides; Four portions of mucosal repair auxiliary ingredients: snail mucus active peptide, L-arginine and phytosterol in a mass ratio of 1:1.8:3. The molecular weight of snail mucus active peptide is 800 Da, and the phytosterol is sitosterol. Sustained-release carriers: modified chitosan and sodium alginate; 2. The preparation of key components is the same as in Example 1.

[0026] 3. The probiotic complexes of Examples 1-3 above were prepared according to the following method: S1. Cultivation and pretreatment of core probiotics: Core probiotics were inoculated into MRS medium and cultured at 36℃ under anaerobic conditions for 20h. Then, the bacterial sludge was collected by centrifugation at 10000rpm for 20min. The bacterial sludge of each strain was mixed evenly according to the live cell count ratio. After pre-freezing at -50℃ for 3h, the compound freeze-dried bacterial powder was obtained by freeze drying. The sublimation drying temperature was 5℃, the desorption drying temperature was 25℃, and the total drying time was 15h. Then, prebiotics were added and mixed evenly to obtain probiotic-prebiotic mixed powder. S2. Preparation of mucosal repair auxiliary ingredients: Snail mucus active peptide powder is mixed with L-arginine and phytosterol, and stirred at 150 rpm for 20 minutes until uniform to obtain mucosal repair auxiliary powder. S3. Preparation of sustained-release carrier: S31. Mix the probiotic-prebiotic mixed powder obtained in S1 with the mucosal repair auxiliary powder obtained in S2 evenly to obtain the total mixed powder; S32. Dissolve the γ-ray modified chitosan in a 1% (v / v) aqueous solution of glacial acetic acid to prepare a 1.5% (w / v) modified chitosan acid solution. Dissolve sodium alginate in phosphate buffer to prepare a 1.5% (w / v) sodium alginate solution for later use. S33. Disperse the total mixed powder in a modified chitosan solution and stir for 50 minutes at 30℃ and 250 rpm to prepare a 15% (w / v) suspension. Add the suspension dropwise to a sodium alginate solution at a volume ratio of 1:10 and a flow rate of 1 mL / min. After the addition is complete, immediately add a 0.2 mol / L calcium chloride solution to the mixed solution. The volume ratio of calcium chloride solution to sodium alginate solution is 1:4. Continue stirring for 2 hours, filter, and dry to obtain probiotic complex microspheres with a particle size of 100 μm.

[0027] The total viable count of probiotics in Examples 1-3 was measured to be ≥1×10⁻⁶. 10 CFU / g.

[0028] Comparative Example 1 The difference between this comparative example and Example 3 is that Lactobacillus curvaturei and Lactobacillus gasseri are not added, and Lactobacillus rhamnosus, Lactobacillus reuteri, and Lactobacillus helveticus are configured into 15 portions of core probiotics in a live count ratio of 3:2:1.

[0029] The other components and preparation methods are the same as in Example 3.

[0030] Comparative Example 2 The difference between this comparative example and Example 3 is that the ratio of live core probiotics is 5:4:3:3:3.

[0031] The other components and preparation methods are the same as in Example 3.

[0032] Comparative Example 3 The difference between this comparative example and Example 3 is that the probiotic complex does not contain prebiotics.

[0033] The other components and preparation methods are the same as in Example 3.

[0034] Comparative Example 4 The difference between this comparative example and Example 3 is that snail mucus active peptides are not added, and the mucosal repair auxiliary ingredients only contain L-arginine and β-sitosterol in a mass ratio of 1.8:3.

[0035] The other components and preparation methods are the same as in Example 3.

[0036] Comparative Example 5 The difference between this comparative example and Example 3 is that the sustained-release carrier is ordinary chitosan, while the other components and preparation methods are the same as in Example 3.

[0037] Comparative Example 6 The difference between this comparative example and Example 3 is that the modified chitosan-sodium alginate composite carrier is not used. Instead, the probiotic-prebiotic mixed powder and the mucosal repair auxiliary powder are directly mechanically mixed and stirred at 300 rpm for 30 minutes until uniform, thus forming a powdered composite.

[0038] I. Functionality Test 1. In vitro mucosal repair rate test Methods: A scratch assay was performed using human vaginal epithelial cells (VK2 / E6E7). After the cells had grown into a confluent monolayer, scratches were created using a sterile pipette tip, and the culture medium was replaced with extracts of the samples from Examples 1-3 and Comparative Examples 1-5 (concentration 1 mg / mL). The cells were incubated at 37°C and 5% CO2 for 24 hours, and the scratch width was measured by photographing under a microscope at 0 and 24 hours.

[0039] Calculation: Mucosal repair rate (%) = [(0-hour scratch width - 24-hour scratch width) / 0-hour scratch width] × 100%.

[0040] 2. Probiotic colonization rate test Methods: An in vitro model of human vaginal epithelial cells (VK2 / E6E7) was constructed, simulating the vaginal fluid environment (pH 4.2, containing 0.1% mucin). Cultured vaginal epithelial cells were cultured at 1×10⁶ cells per well. 5 Cells were seeded at a density of 1,000 cells per well in 24-well cell culture plates and cultured for 24 hours until the cells were fully adhered.

[0041] Add 1 mL of simulated vaginal fluid to each well, then add 100 μL of a solution with a concentration of 1×10⁻⁶. 8 The probiotic culture at CFU / mL was co-cultured at 37℃ in a 5% CO2 incubator for 48 hours. 0.5 mL of 0.25% trypsin digestion solution was added to each well, and the cells were digested at 37℃ for 5 minutes. The cells were resuspended in PBS, and 100 μL of the cell suspension was used for colony counting. After appropriate dilution of the resuspended solution, 100 μL was spread on the corresponding probiotic culture medium plate and incubated upside down at 37℃ for 24-48 hours. The number of colonies on the plate was counted.

[0042] Colony adhesion rate (%) = (number of adhered probiotic colonies / number of added probiotic colonies) × 100%.

[0043] The test results are as follows: Table 1

[0044] Results analysis: This invention employs a combined design of synergistic colonization of five probiotics, prebiotic enhancement, direct repair via snail mucus active peptides, and long-term protection via a modified carrier. This results in a mucosal repair rate of ≥83% and a simulated vaginal colonization rate of ≥81.5% in Examples 1-3, significantly outperforming existing technologies. Example 3 demonstrates the best performance, enabling rapid reconstruction of vaginal microecological balance in clinical applications while efficiently repairing damaged mucosa. It provides a scientifically sound and practical technical solution for the prevention and treatment of gynecological reproductive tract mucosal damage and related infections. The synergistic effect of the core probiotics, mucosal repair auxiliary components, and sustained-release carrier is key to this invention. The absence of any core component or adjustment of the ratio in the comparative examples significantly reduced the repair effect, confirming the indispensability of each component in promoting mucosal repair. II. Sustained-release effect test 1. Simulated release medium: vaginal fluid simulation solution (pH 4.2, containing 0.1% mucin, 0.2% lactic acid, 0.3% sodium chloride, 0.05% calcium chloride, osmotic pressure 300±10mOsm / kg), which conforms to the physiological environment of the female reproductive tract.

[0045] 2. Test temperature and oscillation conditions: 37℃ constant temperature oscillation oven, 100rpm; molecular weight cutoff 10kDa (allowing probiotics and snail mucus active peptides to permeate, while retaining microspheres and carrier fragments). 3. Sample dosage: 0.5g of each sample (comparative example 6 is powder), at time points of 12h, 24h, 48h, 72h, and 96h, with 3 parallel tests.

[0046] 4. Methods for detecting active ingredients 4.1 Probiotic viable count: dilution plating method (MRS medium, anaerobic culture at 37℃ for 48h, CFU / mL count). 4.2 Snail mucus active peptides: HPLC method (C18 column, mobile phase methanol-water = 60:40, detection wavelength 220nm, external standard method for quantification) Cumulative release rate (%) = (release amount at a certain time point / total content of the component in the sample) × 100%, Total content detection: After ultrasonic crushing and enzymatic hydrolysis, the total number of live probiotics and the total mass of snail mucus active peptides were measured.

[0047] Table 2: Cumulative Release Rate of Live Probiotics (%)

[0048] Table 3: Cumulative Release Rate of Snail Mucus Active Peptides (%)

[0049] Results analysis: The release curves of the probiotics and snail mucus active peptides in Examples 1-3 showed a high degree of agreement, with a cumulative release rate of ≥88% over 96 hours. Furthermore, the initial burst release rates were ≤34.4% for probiotics and ≤31.2% for active peptides, demonstrating that the modified chitosan-sodium alginate microspheres achieved simultaneous sustained release of both active ingredients. This indicates that after γ-ray irradiation, the cross-linking density of the modified chitosan increased, and the resulting three-dimensional network structure effectively prevented rapid leakage of the active ingredients while allowing for continuous release through slow swelling in vaginal fluid. Example 3 exhibited the best sustained-release effect, with a 96-hour release rate of ≥94% for both components and the lowest burst release rate. This indicates optimal matching between the molecular size of the active peptides and the pore size of the microspheres, and that the shell thickness formed by the carrier concentration balanced the release rate and protective effect. Compared with Comparative Examples 5 and 6, the application of the sustained-release carrier effectively protected the activity of the probiotics, maintaining a high viable count in the complex vaginal environment and prolonging the duration of action. This not only ensures that probiotics can continue to play a role in regulating the balance of the gut microbiota, but also provides a stable microenvironment for mucosal repair, further enhancing the overall repair effect.

[0050] III. Antibacterial Test The Oxford cup method was used, with Candida albicans (ATCC10231) and Gardnerella vaginalis (ATCC14018) as indicator bacteria, to determine the diameter (mm) of the inhibition zone. A diameter ≥10mm was considered to indicate antibacterial activity.

[0051] Test results: Table 4: Diameter of inhibition zone test (mm)

[0052] Analysis of the results of the inhibition zone test: Examples 1-3 all showed inhibition zones greater than 15 mm against both Candida albicans and Gardnerella vaginalis, with Example 3 showing the best effect. The inhibition zone diameter against Candida albicans reached 17.8 ± 1.0 mm, and against Gardnerella vaginalis, it was 17.3 ± 1.1 mm, indicating that the core probiotic complex has a strong inhibitory effect on both target pathogens. This demonstrates that the present invention achieves highly efficient inhibition of pathogens through the synergistic effect of core probiotics, mucosal repair auxiliary components, and sustained-release carriers, providing strong support for the prevention of gynecological reproductive tract infections.

[0053] IV. Combining safety and effectiveness No significant adverse reactions were observed during the experiment, indicating that the probiotic complex of this invention has good biocompatibility and safety. Its dual efficacy in preventing and repairing mucosal damage provides a new solution for gynecological reproductive tract health care and has broad application prospects.

[0054] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A probiotic complex, characterized in that, The product comprises core probiotics, mucosal repair auxiliary components, and a sustained-release carrier. The core probiotics, prebiotics, and mucosal repair auxiliary components are encapsulated within the sustained-release carrier to form drug-loaded microspheres. The core probiotics include *Lactobacillus rhamnosus*, *Lactobacillus reuteri*, *Lactobacillus helveticus*, *Lactobacillus brevis*, and *Lactobacillus gasseri*, with a viable count ratio of (1-4):(1-3):(0.5-2):(0.5-2.5):(0.5-2). The total viable count of the core probiotics in the drug-loaded microspheres is ≥1×10⁻⁶. 10 CFU / g.

2. The probiotic complex as described in claim 1, characterized in that, It includes the following components by weight: 10-20 parts core probiotics, 2-8 parts prebiotics, 3-5 parts mucosal repair auxiliary ingredients, and a sustained-release carrier.

3. The probiotic complex as described in claim 1, characterized in that, The mucosal repair auxiliary component includes snail mucus active peptides, L-arginine, and phytosterols in a mass ratio of 1:(1.2-2.3):(2.5-3.5). The molecular weight of the snail mucus active peptides is 300-1000 Da, and the phytosterols are selected from one or more combinations of β-sitosterol, stigmasterol, campesterol, and sitosterol.

4. The probiotic complex as described in claim 3, characterized in that, The method for extracting the active peptides from snail mucus includes the following steps: (1) Select live white jade snails, clean their body surface, collect fresh mucus, add 3-5 times the volume of pH 6.5-7.2 phosphate buffer, stir evenly to obtain mucus suspension; (2) Extract the mucus suspension by magnetic stirring at 4-10℃ for 1-2 hours, then centrifuge at 8000-10000 rpm for 15-20 minutes and take the supernatant. (3) The supernatant was initially filtered through an ultrafiltration membrane with a molecular weight cutoff of 1 kDa, the filtrate was collected, purified, the eluent was collected, and freeze-dried to obtain snail mucus active peptide powder.

5. The probiotic complex as described in claim 1, characterized in that, The sustained-release carrier is modified chitosan and sodium alginate. The modified chitosan is treated with γ-ray irradiation at a dose of 10-30 kGy for 20-40 min.

6. The probiotic complex as described in claim 1, characterized in that, The prebiotics are selected from one or more combinations of fructooligosaccharides, stachyose, galactooligosaccharides, inulin, and resistant dextrin.

7. The method for preparing the probiotic complex according to any one of claims 1-6, characterized in that, Includes the following steps: S1. Cultivation and pretreatment of core probiotics: Core probiotics were inoculated into MRS medium and cultured at 35-37℃ under anaerobic conditions for 18-24 hours. Then, the bacterial sludge was collected by centrifugation at 8000-10000 rpm for 15-20 minutes. The bacterial sludge of each strain was mixed evenly according to the live bacteria ratio, and then pre-frozen at -40℃ to -60℃ for 2-4 hours. After freeze-drying, a compound freeze-dried bacterial powder was obtained. Prebiotics were then added and mixed evenly to obtain a probiotic-prebiotic mixed powder. S2. Preparation of mucosal repair auxiliary ingredients: Take snail mucus active peptide powder, mix it with L-arginine and phytosterol, and stir at 100-200 rpm for 15-30 minutes until uniform to obtain mucosal repair auxiliary powder. S3. Preparation of sustained-release carrier: S31. Mix the probiotic-prebiotic mixed powder obtained in S1 with the mucosal repair auxiliary powder obtained in S2 evenly to obtain the total mixed powder; S32. Dissolve the γ-ray modified chitosan in a 0.5-1.5% (v / v) aqueous solution of glacial acetic acid to prepare a 0.5-2.0% (w / v) modified chitosan acid solution. Dissolve sodium alginate in phosphate buffer to prepare a 0.5-2.0% (w / v) sodium alginate solution for later use. S33. Disperse the total mixed powder in a modified chitosan solution and stir until homogeneous to form a suspension; add it dropwise to a sodium alginate solution, crosslink it with calcium ions, filter it, and dry it to obtain probiotic complex microspheres with a particle size of 50-200 μm.

8. The preparation method according to claim 7, characterized in that, The suspension described in step S33 is stirred at 25-35°C and 200-300 rpm for 30-60 minutes, and the final concentration of the suspension is 10-20% (w / v). In step S33, the dropwise flow rate is 0.5-2 mL / min, and the suspension is added dropwise to the sodium alginate solution at a volume ratio of 1:8-12. After the dropwise addition is completed, a calcium chloride solution with a concentration of 0.1-0.3 mol / L is immediately added to the mixed solution, with a volume ratio of calcium chloride solution to sodium alginate solution of 1:3-5. The reaction is continued to be stirred for 1-2 hours.

9. The use of the probiotic complex as described in any one of claims 1-7 or the probiotic complex prepared by the preparation method of claim 8 in the preparation of products for preventing and repairing gynecological reproductive tract mucosal damage.

10. The application as described in claim 9, characterized in that, The product is a suppository, gel, effervescent tablet, or vaginal douche solution.

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