Salivary lactobacillus mucosal flora modulating preparation, preparation method and application thereof

Abstract

This invention discloses a *Lactobacillus salivarius* formulation for regulating mucosal flora, its preparation method, and its application, specifically relating to the field of microbial formulation technology. The formulation is a microcapsule with a bilayer core-shell structure, comprising a core material, an inner wall material, and an outer wall material. The microcapsule has a particle size of 600 μm to 900 μm. The core material is composed of a *Lactobacillus salivarius* suspension and a protective agent solution mixed at a volume ratio of 1:1. This invention constructs a reverse bilayer core-shell microcapsule that combines mucosal adhesion and acid-responsive release by reversing the sequence of the inner dense sodium alginate shell and the outer whey protein adhesion membrane through stepwise cross-linking. This overcomes the limitations of existing probiotic formulations that only focus on intestinal delivery or simple encapsulation, achieving synergistic effects between the biological characteristics of the bacterial strain and materials engineering design. It improves the effects on storage stability, vaginal retention time, and active regulation of the microecology, providing a novel technical approach for the prevention and treatment of vaginal flora imbalance and related gynecological diseases.

Description

Technical Field

[0001] This invention relates to the field of microbial preparation technology, and more specifically, to a preparation of Lactobacillus salivarius for regulating mucosal flora, its preparation method, and its application. Background Technology

[0002] Imbalance of the vaginal microecology is a core cause of diseases such as bacterial vaginosis, candidal vaginitis, and trichomonal vaginitis. Current treatments mainly rely on antibiotics, which can kill pathogens in the short term, but they easily disrupt the normal vaginal flora, leading to high recurrence rates and increased drug resistance. Probiotic preparations are receiving increasing attention as an alternative.

[0003] However, existing vaginal probiotic products generally lack mucosal adhesion ability and are easily flushed out by vaginal secretions. Most current probiotic preparations are lyophilized powders, suppositories, or single-layer encapsulated microcapsules, which are difficult to retain effectively on the epithelial mucosa after entering the vagina, resulting in low bioavailability and requiring frequent administration. Furthermore, there is a lack of response release mechanisms for the acidic environment of the vagina. Current microcapsule technology focuses on gastric acid resistance protection during intestinal delivery, using single-layer or mixed wall materials for encapsulation, and has not designed a controllable release structure for the acidic environment of the vagina (pH 3.8–4.5), resulting in a mismatch between the live bacteria release rate and the needs of vaginal microecological regulation. Probiotics have poor stability at room temperature and are highly dependent on the cold chain. They are also sensitive to temperature. Existing formulations suffer severe loss of live bacteria when stored at 25°C for a long time, which limits their accessibility for clinical application. The characteristics of strains are disconnected from the design of delivery systems. Existing technologies often separate strain screening from formulation design, failing to fully utilize the synergistic potential of strains' self-adhesion, acid production, and antibacterial properties with the engineered structure of microcapsules. Summary of the Invention

[0004] In order to overcome the above-mentioned defects of the prior art, the present invention provides a preparation of Lactobacillus salivarius for regulating mucosal flora, its preparation method and application, so as to solve the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a preparation for regulating mucosal flora by Lactobacillus salivarius, wherein the preparation is a microcapsule with a double core-shell structure, comprising a core material, an inner wall material and an outer wall material, and the particle size of the microcapsule is 600μm to 900μm; The core material is composed of a suspension of Lactobacillus salivarius and a protective agent solution at a volume ratio of 1:1. The volume percentage of bacterial precipitate in the bacterial suspension is 20%–25%, and the viable count is ≥1×10⁻⁶. 11CFU / mL; wherein the Lactobacillus salivarius suspension has the preservation number ATCC 11741; the protective agent solution is composed of the following components in mass percentage: glycerol 5%–8%, skim milk powder 10%–15%, sodium ascorbate 0.3%–0.8%, trehalose 6%–10%, sodium hyaluronate 0.05%–0.2%, with the remainder being water; The inner wall material is a sodium alginate aqueous solution of 1% to 3% by mass, and the inner wall material is cross-linked with calcium chloride to form an inner shell with a dense network structure covering the surface of the core material. The outer wall material is a whey protein aqueous solution of 5% to 10% by mass. The outer wall material is cross-linked with transglutaminase to form an outer shell with an adhesive protein membrane that coats the surface of the inner shell. The whey protein adhesive membrane of the outer shell is used to adhere to the vaginal epithelial mucosa, and the sodium alginate mesh structure of the inner shell is used to swell in the acidic environment of the vagina and controllably release the saliva lactobacilli in the core material.

[0006] Preferably, the protective agent solution comprises the following components by mass percentage: 6% glycerol, 12% skim milk powder, 0.5% sodium ascorbate, 8% trehalose, 0.1% sodium hyaluronate, with the balance being water; the inner wall material is a 2% sodium alginate aqueous solution; and the outer wall material is a 7%–8% whey protein aqueous solution.

[0007] Preferably, the *Lactobacillus salivarius* has a self-adhesion characteristic with an adhesion rate of ≥40% to vaginal epithelial cells. This self-adhesion characteristic, together with the mucosal adhesion characteristic of the whey protein adhesive protein membrane of the outer shell, constitutes the colonization-retention synergistic mechanism of the preparation on the vaginal mucosa surface. After anaerobic culture at 37°C for 24 hours in MRS liquid medium, the pH of the fermentation broth is ≤3.8. After incubation at 37°C for 4 hours in artificial vaginal secretions with a pH of 4.0–4.5, the survival rate is ≥85%. The inhibition zone diameter against *Staphylococcus aureus* is ≥18 mm, and the inhibition zone diameter against *Escherichia coli* is ≥16 mm.

[0008] Preferably, the viable count of the preparation is ≥1×10⁻⁶. 10 CFU / g, activity loss ≤12% after 12 months of storage at 25℃.

[0009] A method for preparing the above-mentioned Lactobacillus salivarius preparation for regulating mucosal flora includes the following steps: (1) Preparation of bacterial suspension: Lactobacillus salivarius was transferred to MRS liquid medium at an inoculum of 0.5% and anaerobic cultured at 37°C for 24 hours. The fermentation broth was centrifuged at 8000 r / min for 8 minutes, the supernatant was discarded, and the bacterial cells were washed twice with PBS buffer and resuspended with physiological saline to obtain a bacterial precipitate with a volume ratio of 20% to 25% and a viable count ≥1×10⁻⁶. 11 CFU / mL bacterial suspension; (2) Preparation of protective agent solution: Glycerin, skim milk powder, sodium ascorbate, trehalose and sodium hyaluronate are mixed according to the mass percentages described in claim 1, water is added to make up the volume and stirred at 40°C for 30 minutes until completely dissolved, and then cooled to room temperature to obtain the protective agent solution; (3) Core material preparation: The bacterial suspension obtained in step (1) and the protective agent solution obtained in step (2) are mixed at a volume ratio of 1:1 and stirred at 4°C for 15 minutes until homogeneous to obtain the core material; (4) Preparation of inner wall material: Stir 1% to 3% sodium alginate aqueous solution until completely dissolved, degas for 30 minutes to obtain inner wall material; (5) Preparation of outer wall material: Stir 5% to 10% whey protein aqueous solution until completely dissolved, adjust pH to 6.5 to 7.5 to obtain outer wall material; (6) Inner layer coating and cross-linking: The core material obtained in step (3) and the inner wall material obtained in step (4) are mixed at a volume ratio of 1:1.3 and shaken on a shaker at 100r / min to 150r / min for 20 minutes to obtain the first mixture; using a nozzle with a diameter of 300μm to 1000μm, under the conditions of vibration frequency of 400Hz to 1500Hz and electric field voltage of 600V to 1500V, the first mixture is dropped into a 3% to 8% calcium chloride aqueous solution, stirred and cured at 25℃ for 20 to 40 minutes, and washed twice with deionized water to obtain primary microcapsules with an inner shell; (7) Outer coating and cross-linking: According to the volume ratio of the core material and the outer wall material obtained in step (3) of 1:1, the outer wall material obtained in step (5) is added to the primary microcapsules obtained in step (6), and stirred at 35℃~45℃ for 10 minutes~20 minutes to form a coating layer on the surface of the primary microcapsules. Then, the primary microcapsules coated with the outer wall material are dropped into a 0.5%~2% glutamine transaminase aqueous solution, and the cross-linking reaction is carried out at 40℃ for 20 minutes~30 minutes. After washing with deionized water 3 times, the mixture is freeze-dried at -50℃ for 24 hours to obtain the Lactobacillus salivarius preparation for regulating mucosal flora.

[0010] Preferably, the process parameters used in step (6) are: nozzle diameter 500μm, vibration frequency 800Hz, electric field voltage 1000V, and calcium chloride aqueous solution concentration 5%; the process parameters used in step (7) are: glutamine transaminase aqueous solution concentration 1%, crosslinking reaction temperature 40℃, and reaction time 25 minutes.

[0011] Preferably, in step (6), when the mass percentage of the sodium alginate aqueous solution in step (4) is 2%, the electric field voltage is 1000V, and the particle size variation coefficient of the obtained microcapsules is ≤8% and the roundness is ≥0.92.

[0012] The above-mentioned Lactobacillus salivarius preparations for regulating mucosal flora may be used in the preparation of drugs for regulating vaginal microecological balance and inhibiting the growth of harmful bacteria, or in the preparation of drugs for the prevention and treatment of candidal vaginitis and trichomonal vaginitis.

[0013] Preferably, during the retention of the preparation in the vagina, the whey protein adhesive protein membrane of the outer wall material forms an adhesive layer on the surface of the vaginal epithelial mucosa, and the inner wall material slowly swells and releases the core material in the acidic environment of the vagina, so that the local environment pH value is restored to 3.8-4.5 and the number of harmful bacteria colonies is reduced by ≥60%.

[0014] The technical effects and advantages of this invention are as follows: By employing a structural design that uses an inner layer of sodium alginate cross-linked with calcium chloride to form a dense network shell and an outer layer of whey protein cross-linked with transglutaminase to form an adhesive protein membrane, the outer whey protein membrane can directly form an adhesion layer with the vaginal epithelial mucosa, significantly prolonging the retention time of microcapsules in the vagina. At the same time, the inner sodium alginate shell can undergo specific swelling in the acidic environment of the vagina, achieving controlled and sustained release of salivarius lactobacilli from the core material. The outer layer adhesion and the inner layer release complement each other in spatial and temporal dimensions, constructing a synergistic mechanism of colonization and retention, overcoming the shortcomings of existing probiotic preparations that are easily flushed out by vaginal secretions and have low live bacteria colonization efficiency. By adopting a stepwise cross-linking strategy of first cross-linking the inner layer with calcium chloride and then cross-linking the outer layer with transglutaminase, the inner shell forms a dense network structure to protect the viable bacteria in the core material, while the outer shell forms a protein membrane with adhesive function. The cross-linking mechanism and functional positioning of the two shells do not interfere with each other. Through the synergistic optimization of electric field voltage and sodium alginate concentration, the microcapsules achieve high sphericity and low particle size variation coefficient during the preparation process, significantly improving batch-to-batch consistency and process repeatability, and overcoming the technical bottlenecks such as uneven particle size and irregular morphology commonly found in vibration electric field granulation processes. Long-term retention is achieved through mucosal adhesion of the outer protein membrane, and active restoration of local pH value is achieved through acid-responsive swelling and release of the inner shell. The synergistic effect of the acid resistance, adhesion and antibacterial properties of Lactobacillus salivarius and the engineered design of microcapsules effectively inhibits the growth of harmful bacteria and pathogenic microorganisms. At the same time, the formulation of this invention has good room temperature storage stability and can maintain the activity of live bacteria for a long time without the need for strict cold chain conditions, which significantly reduces the production, transportation and use costs. It provides a new technical solution for vaginal microecological regulation and vaginitis prevention and treatment that combines long-term effectiveness, stability and economy. Detailed Implementation

[0015] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0016] The *Lactobacillus salivarius* strain of this invention is *Lactobacillus salivarius* ATCC 11741. This strain is a standard strain publicly deposited by the American Center for Type Culture Collection and can be obtained by the public through commercial channels without the need for separate deposit.

[0017] The present invention relates to a microcapsule with a double core-shell structure, comprising a core material, an inner wall material, and an outer wall material, prepared by a stepwise cross-linking process. The core material is a 1:1 volume ratio mixture of a suspension of *Lactobacillus salivarius* ATCC 11741 and a protective agent solution. The inner wall material is a 1% to 3% (w / w) aqueous solution of sodium alginate, cross-linked with calcium chloride to form a dense network structure covering the surface of the core material. The outer wall material is a 5% to 10% (w / w) aqueous solution of whey protein, cross-linked with transglutaminase to form an adhesive protein membrane covering the surface of the inner shell.

[0018] The mucosa mentioned in this invention mainly refers to the vaginal mucosa, and the preparation is particularly suitable for regulating the vaginal mucosal microecology.

[0019] Example 1

[0020] This embodiment is a preferred embodiment, corresponding to the preferred solutions of claims 2 and 6.

[0021] Step 1: Preparation of bacterial suspension. *Lactobacillus salivarius* ATCC 11741 was inoculated at a rate of 0.5% into MRS liquid medium and cultured anaerobically at 37°C for 24 hours. After fermentation, the fermentation broth was centrifuged at 8000 rpm for 8 minutes, the supernatant was discarded, and the bacterial pellet was collected. The bacterial cells were washed twice with PBS buffer and then resuspended in physiological saline to obtain the bacterial suspension. The volume percentage of the bacterial pellet in the suspension was 22%, and the viable count was 1.2 × 10⁻⁶. 11 CFU / mL.

[0022] Step 2: Preparation of the protective agent solution. Mix 6g of glycerin, 12g of skim milk powder, 0.5g of sodium ascorbate, 8g of trehalose, and 0.1g of sodium hyaluronate. Add purified water to make up to 100mL. Stir at 40℃ for 30 minutes until completely dissolved. Cool to room temperature to obtain the protective agent solution. The remainder is water.

[0023] Step 3: Core material preparation. The bacterial suspension obtained in Step 1 and the protective agent solution obtained in Step 2 are mixed at a volume ratio of 1:1 and stirred at 4°C for 15 minutes until homogeneous to obtain the core material.

[0024] Step 4: Preparation of inner wall material. Place 2% sodium alginate aqueous solution in a stirrer and stir until completely dissolved. Then degas for 30 minutes to obtain the inner wall material.

[0025] Step 5: Preparation of outer wall material. Stir 7.5% whey protein aqueous solution until completely dissolved, adjust pH to 7.0, and obtain the outer wall material.

[0026] Step Six: Inner Layer Coating and Crosslinking. The core material obtained in Step Three and the inner wall material obtained in Step Four are mixed at a volume ratio of 1:1.3 and shaken in a shaker at a speed of 120 r / min for 20 minutes to obtain the first mixture. Using a nozzle with a diameter of 500 μm, under the conditions of vibration frequency of 800 Hz and electric field voltage of 1000 V, the first mixture is dropped into a 5% (w / w) calcium chloride aqueous solution and stirred and cured at 25 °C for 30 minutes. Then, it is washed twice with deionized water to obtain primary microcapsules with an inner shell.

[0027] Step 7: Outer coating and cross-linking. Following a 1:1 volume ratio of core material to outer wall material obtained in Step 3, add the outer wall material obtained in Step 5 to the primary microcapsules obtained in Step 6. Stir at 40°C for 15 minutes to form a coating layer on the surface of the primary microcapsules. Then, drop the primary microcapsules coated with the outer wall material into a 1% (w / w) aqueous solution of transglutaminase. Perform a cross-linking reaction at 40°C for 25 minutes. After the reaction, wash three times with deionized water and then freeze-dry at -50°C for 24 hours to obtain the Lactobacillus salivarius preparation for regulating mucosal flora.

[0028] Results: The obtained formulation consisted of microcapsules with a bilayered core-shell structure, with a particle size ranging from 720 μm to 780 μm, a coefficient of variation of 6.8%, and a roundness of 0.94. Scanning electron microscopy revealed that the inner shell had a dense network structure, while the outer shell possessed an adhesive protein membrane. The viable count of the formulation was 1.2 × 10⁻⁶. 10 CFU / g.

[0029] Example 2

[0030] This embodiment verifies the technical feasibility of using a low concentration range for the inner wall material and a low range for process parameters. Except for the following different parameters, the other raw material ratios and operations are the same as in Example 1.

[0031] Step 1: The bacterial suspension is prepared using the same method as in Example 1, with the bacterial precipitate volume accounting for 20% and the viable cell count being 1.1 × 10⁻⁶. 11 CFU / mL.

[0032] Step 2: The preparation method of the protective agent solution is the same as in Example 1.

[0033] Step 3: The core material preparation method is the same as in Example 1.

[0034] Step 4: Preparation of inner wall material. Place 1% sodium alginate aqueous solution in a stirrer and stir until completely dissolved. Degas for 30 minutes to obtain the inner wall material.

[0035] Step 5: The preparation method of the outer wall material is the same as in Example 1.

[0036] Step Six: Inner Layer Coating and Crosslinking. The core material obtained in Step Three and the inner wall material obtained in Step Four are mixed at a volume ratio of 1:1.3 and shaken at 100 r / min for 20 minutes in a shaker to obtain the first mixture. Using a nozzle with a diameter of 300 μm, under the conditions of vibration frequency of 600 Hz and electric field voltage of 600 V, the first mixture is dropped into a 3% (w / w) calcium chloride aqueous solution and stirred and cured at 25°C for 20 minutes. Then, it is washed twice with deionized water to obtain primary microcapsules with an inner shell.

[0037] Step 7: Outer layer coating and cross-linking. Add the outer wall material at a volume ratio of 1:1 (core material to outer wall material) and stir at 40°C for 15 minutes. Then, drop it into a 1% (w / w) aqueous solution of transglutaminase and cross-link at 40°C for 25 minutes. Wash three times with deionized water and freeze-dry at -50°C for 24 hours.

[0038] Results: The obtained microcapsules had a particle size of 620 μm to 680 μm, a roundness of 0.91, and a viable count of 9.5 × 10⁻⁶. 9 CFU / g. These results indicate that a complete double-layer core-shell structure can still be successfully constructed under 1% sodium alginate concentration and low electric field voltage conditions, with good encapsulation efficiency; the number of viable bacteria decreases slightly with decreasing wall material concentration, consistent with the expected trend.

[0039] Example 3

[0040] This embodiment verifies the technical feasibility of the high-end range of inner wall material concentration and the high-end range of process parameters. Except for the following different parameters, the other raw material ratios and operations are the same as in Example 1.

[0041] Step 1: The bacterial suspension is prepared using the same method as in Example 1, with the bacterial precipitate volume accounting for 25% and the viable cell count being 1.3 × 10⁻⁶. 11 CFU / mL.

[0042] Step 2: The preparation method of the protective agent solution is the same as in Example 1.

[0043] Step 3: The core material preparation method is the same as in Example 1.

[0044] Step 4: Preparation of inner wall material. Place 3% sodium alginate aqueous solution in a stirrer and stir until completely dissolved. Degas for 30 minutes to obtain the inner wall material.

[0045] Step 5: The preparation method of the outer wall material is the same as in Example 1.

[0046] Step Six: Inner Layer Coating and Crosslinking. The core material obtained in Step Three and the inner wall material obtained in Step Four are mixed at a volume ratio of 1:1.3 and shaken in a shaker at a speed of 150 r / min for 20 minutes to obtain the first mixture. Using a nozzle with a diameter of 1000 μm, under the conditions of a vibration frequency of 1500 Hz and an electric field voltage of 1500 V, the first mixture is dropped into an 8% (w / w) calcium chloride aqueous solution and stirred and cured at 25°C for 40 minutes. Then, it is washed twice with deionized water to obtain primary microcapsules with an inner shell.

[0047] Step 7: Outer layer coating and cross-linking. Add the outer wall material at a volume ratio of 1:1 (core material to outer wall material), and stir at 40°C for 15 minutes. Then, dropwise add 1% (w / w) of transglutaminase aqueous solution, and perform a cross-linking reaction at 40°C for 25 minutes. Wash three times with deionized water and freeze-dry at -50°C for 24 hours.

[0048] Results: The obtained microcapsules had a particle size of 840 μm to 890 μm, a roundness of 0.90, and a viable cell count of 1.1 × 10⁻⁶. 10 CFU / g; Scanning electron microscopy confirmed that the microcapsules have a complete double core-shell structure, with an outer adhesive protein membrane continuously encapsulating them.

[0049] Example 4

[0050] This embodiment verifies the technical feasibility of the lower end of the outer wall material concentration range. Except for the following different parameters, the other raw material ratios and operations are the same as in Example 1.

[0051] Steps one through four are the same as in Example 1.

[0052] Step 5: Preparation of outer wall material. Stir 5% whey protein aqueous solution until completely dissolved, adjust pH to 7.0, and obtain the outer wall material.

[0053] Step 6: The inner layer coating and cross-linking methods are the same as in Example 1.

[0054] Step 7: Outer layer coating and cross-linking. Add the outer wall material at a volume ratio of 1:1 (core material to outer wall material), and stir at 40°C for 15 minutes. Then, dropwise add 1% (w / w) of transglutaminase aqueous solution, and perform a cross-linking reaction at 40°C for 25 minutes. Wash three times with deionized water and freeze-dry at -50°C for 24 hours.

[0055] Results: The outer shell of the obtained microcapsules was relatively thin, but it still formed a continuous adhesive protein membrane with a particle size of 710 μm and a roundness of 0.93. Scanning electron microscopy confirmed that the double core-shell structure was intact.

[0056] Example 5

[0057] This embodiment verifies the technical feasibility of a high concentration range for the outer wall material. Except for the following different parameters, the other raw material ratios and operations are the same as in Example 1.

[0058] Steps one through four are the same as in Example 1.

[0059] Step 5: Preparation of outer wall material. Stir 10% whey protein aqueous solution until completely dissolved, adjust pH to 7.0, and obtain the outer wall material.

[0060] Step 6: The inner layer coating and cross-linking methods are the same as in Example 1.

[0061] Step 7: Outer layer coating and cross-linking. Add the outer wall material at a volume ratio of 1:1 (core material to outer wall material) and stir at 40°C for 15 minutes. Then, drop it into a 1% (w / w) aqueous solution of transglutaminase and cross-link at 40°C for 25 minutes. Wash three times with deionized water and freeze-dry at -50°C for 24 hours.

[0062] Results: The outer shell of the obtained microcapsules was thicker and had stronger surface adhesion. The particle size was 760 μm and the roundness was 0.92. Scanning electron microscopy confirmed that the double core-shell structure was intact.

[0063] Validation of strain functional characteristics 1. Acid resistance test: Lactobacillus salivarius ATCC 11741 was transferred to MRS liquid medium at an inoculum of 0.5% and anaerobically cultured at 37°C for 24 hours. The pH of the fermentation broth was measured to be 3.7. The cultured bacterial suspension was placed in artificial vaginal secretions with a pH of 4.2 and incubated at 37°C for 4 hours. The survival rate was determined to be 88% by plate counting method. The artificial vaginal secretions contained 0.5% glucose, 0.1% peptone, and 0.05% sodium chloride, and the pH was adjusted with lactic acid.

[0064] 2. Adhesion performance determination: Adhesion experiments were conducted using human vaginal epithelial cells VK2 / E6E7. Fluorescently labeled ATCC 11741 bacterial suspension was co-incubated with a cell monolayer at a bacterial-to-cell ratio of 100:1 at 37°C and 5% CO2 for 2 hours. After washing with PBS to remove unadhered cells, the cells were counted. The results showed that the adhesion rate of this strain to vaginal epithelial cells was 42%.

[0065] 3. Antibacterial performance determination: The Oxford cup method was used for antibacterial experiments. Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922 were respectively spread on MRS agar plates, placed in Oxford cups, and cell-free fermentation supernatant of ATCC 11741 was added. After incubation at 37°C for 24 hours, the diameter of the inhibition zone was measured. The results showed that the diameter of the inhibition zone against Staphylococcus aureus was 19 mm, and the diameter of the inhibition zone against Escherichia coli was 17 mm.

[0066] The above results indicate that Lactobacillus salivarius ATCC 11741 has excellent acid resistance, mucosal adhesion, and antibacterial properties. Its own adhesion characteristics and the whey protein mucosal adhesion characteristics of the outer shell of the formulation together constitute a synergistic mechanism of colonization and retention.

[0067] Characterization of microcapsule structure and particle size properties Scanning electron microscopy was performed on the formulation obtained in Example 1. The results showed that the microcapsules had a distinct bilayer core-shell structure. The inner shell was formed by cross-linking sodium alginate with calcium chloride, with a thickness of about 100 μm to 120 μm, exhibiting a dense network structure with uniform pore size distribution. The outer shell was formed by cross-linking whey protein with transglutaminase, with a thickness of about 90 μm to 110 μm, and the surface had the typical rough morphology of an adhesive protein membrane.

[0068] Particle size analysis was performed on the microcapsules obtained in Example 1 using a 2% sodium alginate aqueous solution and a 1000V electric field voltage. The results showed that the particle size variation coefficient was 6.8% and the roundness was 0.94, which met the technical indicators of a particle size variation coefficient of no more than 8% and a roundness of no less than 0.92, thus confirming the synergistic optimization relationship between the electric field voltage and the sodium alginate concentration.

[0069] Storage stability evaluation The formulation obtained in Example 1 was sealed and packaged, and its accelerated stability was investigated under light-protected conditions at 25°C. Samples were taken at 0, 3, 6, 9, and 12 months to determine the number of viable bacteria.

[0070] The results showed that the initial viable count was 1.2 × 10⁻⁶. 10 CFU / g; viable count after 12 months of storage was 1.07 × 10⁻⁶. 10 The CFU / g activity loss was 10.8%, not more than 12%; indicating that the double-shell structure of the present invention has excellent protective effect on Lactobacillus salivarius in the core material and can achieve long-term storage at room temperature.

[0071] In vitro simulated vaginal environment release and antibacterial effect The formulation obtained in Example 1 was placed in artificial vaginal secretions, and a release experiment was conducted at 37°C to simulate the vaginal environment. The artificial vaginal secretions contained 0.5% glucose, 0.1% peptone, and 0.05% sodium chloride, with an initial pH of 5.5.

[0072] The results showed that within 2 hours of formulation administration, the whey protein of the outer wall material formed an adhesion layer on the simulated mucosa surface; after 24 hours, the inner wall material slowly swelled in an acidic environment, and the salicylic acid lactobacilli in the core material began to be released, and the local environment pH value returned to 4.0; after 72 hours, the survival of Candida albicans, Gardnerella vaginalis and Trichomonas vaginalis in the simulated system was detected. Compared with the control group without formulation, the number of Candida albicans colonies decreased by 68%, the number of Gardnerella vaginalis colonies decreased by 65%, the survival rate of Trichomonas vaginalis decreased by 72%, and the overall reduction of harmful bacteria and pathogenic microorganisms was not less than 60%.

[0073] The above results indicate that the formulation of the present invention can achieve synergistic regulation of adhesion, swelling and release in the acidic environment of the vagina, effectively restore the vaginal microecological balance, and has both preventive and therapeutic effects on candidal vaginitis and trichomonal vaginitis.

[0074] In conclusion, 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 preparation for regulating mucosal flora by Lactobacillus salivarius, the preparation being a microcapsule with a double core-shell structure, comprising a core material, an inner wall material, and an outer wall material, wherein the particle size of the microcapsule is 600 μm to 900 μm; The core material is composed of a suspension of Lactobacillus salivarius and a protective agent solution at a volume ratio of 1:

1. The volume percentage of bacterial precipitate in the bacterial suspension is 20%–25%, and the viable count is ≥1×10⁻⁶. 11 CFU / mL; where The *Lactobacillus salivarius* in the *Lactobacillus salivarius* suspension has the accession number ATCC 11741; the protective agent solution consists of the following components in weight percentage: 5%–8% glycerol, 10%–15% skim milk powder, 0.3%–0.8% sodium ascorbate, 6%–10% trehalose, 0.05%–0.2% sodium hyaluronate, with the remainder being water; The inner wall material is a sodium alginate aqueous solution of 1% to 3% by mass, and the inner wall material is cross-linked with calcium chloride to form an inner shell with a dense network structure covering the surface of the core material. The outer wall material is a whey protein aqueous solution of 5% to 10% by mass. The outer wall material is cross-linked with transglutaminase to form an outer shell with an adhesive protein membrane that coats the surface of the inner shell. The whey protein adhesive membrane of the outer shell is used to adhere to the vaginal epithelial mucosa, and the sodium alginate mesh structure of the inner shell is used to swell in the acidic environment of the vagina and controllably release the saliva lactobacilli in the core material.

2. The preparation for regulating mucosal flora by *Lactobacillus salivarius* according to claim 1, characterized in that: The protective agent solution is composed of the following components by mass percentage: 6% glycerol, 12% skim milk powder, 0.5% sodium ascorbate, 8% trehalose, 0.1% sodium hyaluronate, and the balance being water; the inner wall material is a 2% sodium alginate aqueous solution; and the outer wall material is a 7%–8% whey protein aqueous solution.

3. The preparation for regulating mucosal flora by *Lactobacillus salivarius* according to claim 1, characterized in that: The *Lactobacillus salivarius* possesses a self-adhesion characteristic with an adhesion rate of ≥40% to vaginal epithelial cells. This self-adhesion characteristic, together with the mucosal adhesion characteristic of the whey protein adhesive protein membrane of the outer shell, constitutes the colonization-retention synergistic mechanism of the preparation on the vaginal mucosa surface. After anaerobic culture at 37°C for 24 hours in MRS liquid medium, the pH of the fermentation broth is ≤3.

8. After incubation at 37°C for 4 hours in artificial vaginal secretions with a pH of 4.0–4.5, the survival rate is ≥85%. The inhibition zone diameter against *Staphylococcus aureus* is ≥18 mm, and the inhibition zone diameter against *Escherichia coli* is ≥16 mm.

4. The preparation for regulating mucosal flora by *Lactobacillus salivarius* according to claim 1, characterized in that: The preparation has a viable bacterial count ≥ 1 × 10⁻⁶ 10 CFU / g, activity loss ≤12% after 12 months of storage at 25℃.

5. A method for preparing the *Lactobacillus salivarius* formulation for regulating mucosal flora according to any one of claims 1-4, characterized in that, Includes the following steps: (1) Preparation of bacterial suspension: Lactobacillus salivarius was transferred to MRS liquid medium at an inoculum of 0.5% and anaerobic cultured at 37°C for 24 hours. The fermentation broth was centrifuged at 8000 r / min for 8 minutes, the supernatant was discarded, and the bacterial cells were washed twice with PBS buffer and resuspended with physiological saline to obtain a bacterial precipitate with a volume ratio of 20% to 25% and a viable count ≥1×10⁻⁶. 11 CFU / mL bacterial suspension; (2) Preparation of protective agent solution: Glycerin, skim milk powder, sodium ascorbate, trehalose and sodium hyaluronate are mixed according to the mass percentages described in claim 1, water is added to make up the volume and stirred at 40°C for 30 minutes until completely dissolved, and then cooled to room temperature to obtain the protective agent solution; (3) Core material preparation: The bacterial suspension obtained in step (1) and the protective agent solution obtained in step (2) are mixed at a volume ratio of 1:1 and stirred at 4°C for 15 minutes until homogeneous to obtain the core material; (4) Preparation of inner wall material: Stir 1% to 3% sodium alginate aqueous solution until completely dissolved, degas for 30 minutes to obtain inner wall material; (5) Preparation of outer wall material: Stir 5% to 10% whey protein aqueous solution until completely dissolved, adjust pH to 6.5 to 7.5 to obtain outer wall material; (6) Inner layer coating and cross-linking: The core material obtained in step (3) and the inner wall material obtained in step (4) are mixed at a volume ratio of 1:1.3 and shaken on a shaker at 100r / min to 150r / min for 20 minutes to obtain the first mixture; using a nozzle with a diameter of 300μm to 1000μm, under the conditions of vibration frequency of 400Hz to 1500Hz and electric field voltage of 600V to 1500V, the first mixture is dropped into a 3% to 8% calcium chloride aqueous solution, stirred and cured at 25℃ for 20 to 40 minutes, and washed twice with deionized water to obtain primary microcapsules with an inner shell; (7) Outer coating and cross-linking: According to the volume ratio of the core material and the outer wall material obtained in step (3) of 1:1, the outer wall material obtained in step (5) is added to the primary microcapsules obtained in step (6), and stirred at 35℃~45℃ for 10 minutes~20 minutes to form a coating layer on the surface of the primary microcapsules. Then, the primary microcapsules coated with the outer wall material are dropped into a 0.5%~2% glutamine transaminase aqueous solution, and the cross-linking reaction is carried out at 40℃ for 20 minutes~30 minutes. After washing with deionized water 3 times, the mixture is freeze-dried at -50℃ for 24 hours to obtain the Lactobacillus salivarius preparation for regulating mucosal flora.

6. The preparation method according to claim 5, characterized in that: The process parameters used in step (6) are: nozzle diameter 500μm, vibration frequency 800Hz, electric field voltage 1000V, and calcium chloride aqueous solution concentration 5%; the process parameters used in step (7) are: glutamine transaminase aqueous solution concentration 1%, crosslinking reaction temperature 40℃, and reaction time 25 minutes.

7. The preparation method according to claim 5, characterized in that: In step (6), when the mass percentage of the sodium alginate aqueous solution in step (4) is 2%, the electric field voltage is 1000V, and the particle size variation coefficient of the obtained microcapsules is ≤8% and the roundness is ≥0.

92.

8. The use of the salicylic acid lactobacillus-based mucosal flora regulating agent according to any one of claims 1-4 in the preparation of a drug for regulating vaginal microecological balance and inhibiting the growth of harmful bacteria, or in the preparation of a drug for the prevention and treatment of candidal vaginitis and trichomonal vaginitis.

9. The application according to claim 8, characterized in that, During the retention of the preparation in the vagina, the whey protein adhesive membrane of the outer wall material forms an adhesion layer on the surface of the vaginal epithelial mucosa, and the inner wall material slowly swells and releases the core material in the acidic environment of the vagina, so that the local environment pH value is restored to 3.8-4.5 and the number of harmful bacteria colonies is reduced by ≥60%.