Lactobacillus acidophilus respiratory health formulation to boost mucosal barrier

Abstract

This invention discloses a respiratory health preparation of *Lactobacillus acidophilus* that enhances the mucosal barrier, relating to the field of microbial preparation technology. The preparation includes probiotic active ingredients and a pharmaceutically or food-grade acceptable carrier. The probiotic active ingredients consist of *Lactobacillus acidophilus* LH-001, *Lactobacillus rhamnosus* LR-002, and *Bifidobacterium bifidum* BB-003, which have been selectively adapted for respiratory use. It also includes synergistic functional components. By weight, the synergistic functional components include 1-5 parts of 2'-fucosylated lactose, 0.5-2 parts of low-molecular-weight sodium hyaluronate, and 0.1-0.5 parts of baicalin. The weight-average molecular weight of the low-molecular-weight sodium hyaluronate is 10-30 kDa. This invention obtains three functionally complementary exclusive strains through selective respiratory adaptation, and, combined with the synergistic effect of the synergistic components and targeted formulation technology, solves the problems of poor compatibility and difficult live bacteria delivery of conventional intestinal strains, achieving in-situ efficacy in the respiratory tract and meeting the needs of multiple commercial dosage forms.

Description

Technical Field

[0001] This invention relates to the field of microbial preparation technology, specifically to a Lactobacillus acidophilus respiratory health preparation that enhances the mucosal barrier. Background Technology

[0002] The respiratory mucosal barrier is the body's first and core line of defense against external pathogens and allergens. It is composed of a physical barrier made up of epithelial cells, a microbial barrier made up of mucosal flora, and an immune barrier made up of secretory antibodies. In recent years, increased air pollution, increased exposure to allergens, recurrent respiratory infections, and antibiotic overuse leading to dysbiosis have all damaged the respiratory mucosal barrier, which is a core cause of allergic rhinitis, recurrent respiratory discomfort, chronic cough, and other problems. As research on probiotics and respiratory health deepens, probiotic formulations specifically formulated for the respiratory physiological environment have become a core demand in the industry.

[0003] Current respiratory-related probiotic regimens primarily use commercially available, gut-compatible strains as their core components. These strains haven't been specifically adapted to the slightly acidic mucus environment, microaerobic conditions, and ciliary clearance mechanisms of the respiratory tract. Consequently, they exhibit extremely weak adhesion to human respiratory epithelial cells, are easily and rapidly eliminated from the body, and mostly exert their effects indirectly through the gut-lung axis, resulting in slow onset and limited efficacy. Furthermore, existing regimens often involve simple combinations of common strains without synergistic functional design. The accompanying formulations also fail to address the issues of short survival time, low survival rate, and insufficient targeting of live bacteria in the respiratory tract, making it difficult to achieve the desired results in practical use. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a Lactobacillus acidophilus respiratory health preparation that enhances the mucosal barrier. This invention involves the targeted acclimatization of Lactobacillus acidophilus, Lactobacillus rhamnosus, and Bifidobacterium bifidum into the respiratory tract, combined with synergistic components such as 2'-fucosylated lactose and targeted formulation technology, to create a probiotic preparation specifically for respiratory health. The acclimatized strains are adapted to the micro-oxygen and mucus environment of the respiratory tract, exhibiting significantly improved adhesion and survival rates, enabling them to colonize and exert their effects in situ within the respiratory tract, synergistically strengthening the mucosal barrier repair. The accompanying microencapsulation technology and multi-dosage form design, including nasal sprays and enteric-coated oral formulations, solve the problems of difficult delivery and weak efficacy of traditional intestinal probiotics, while also providing excellent storage stability, precisely meeting the needs of various scenarios such as acute respiratory discomfort and daily conditioning.

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solution: On the one hand, a Lactobacillus acidophilus respiratory health preparation that enhances the mucosal barrier, the preparation comprising probiotic active ingredients and a pharmaceutically or food-grade acceptable carrier; The probiotic active ingredients consist of Lactobacillus acidophilus LH-001, Lactobacillus rhamnosus LR-002, and Bifidobacterium bifidum BB-003, which have been induced to develop respiratory tract-directed domestication. The Lactobacillus acidophilus LH-001 strain was obtained through atmospheric pressure, room temperature plasma mutagenesis combined with continuous passage and adaptive domestication of human respiratory epithelial cells, and its 24-hour adhesion rate to human respiratory epithelial cells was ≥60%. The Lactobacillus rhamnosus LR-002 strain was obtained through targeted screening for antagonism against respiratory pathogens combined with adaptive domestication of the inflammatory microenvironment. It has an inhibition zone diameter of ≥12mm against common respiratory pathogens. The Bifidobacterium bifidum BB-003 strain was obtained through microaerobic environment adaptation and targeted screening combined with co-culture of mucosal immune cells. Its survival rate is ≥80% in a microaerobic environment with a volume fraction of 5% carbon dioxide + 5% oxygen for 72 hours.

[0006] Furthermore, the viable count ratio of Lactobacillus acidophilus LH-001, Lactobacillus rhamnosus LR-002, and Bifidobacterium bifidum BB-003 is (1-3):(1-2):(2-4), and the total viable count in the finished product is ≥1×10⁻⁶. 9 CFU / g.

[0007] Furthermore, the formulation also includes synergistic functional components; by mass parts, the synergistic functional components include 1-5 parts of 2'-fucosylated lactose, 0.5-2 parts of low molecular weight sodium hyaluronate, and 0.1-0.5 parts of baicalin; the weight-average molecular weight of low molecular weight sodium hyaluronate is 10-30 kDa; wherein 2'-fucosylated lactose can serve as a dedicated carbon source for probiotic active ingredients, low molecular weight sodium hyaluronate can enhance the physical adhesion of the formulation to the respiratory mucosa, and baicalin can help reduce local inflammatory responses in the respiratory tract.

[0008] Furthermore, the formulation is a respiratory tract-targeted adhesion pH-responsive live bacteria microcapsule formulation; the wall material of the live bacteria microcapsule formulation is a chitosan-hyaluronic acid-sodium alginate ternary composite carrier, and the core material is probiotic active ingredients and optional synergistic functional components; the pH-responsive characteristics are that it can rapidly swell in the respiratory tract inflammatory microenvironment of pH 6.0-7.5, and maintain structural stability in an acidic environment of pH 1.0-3.0.

[0009] Furthermore, the live bacteria microcapsule formulation has a particle size of 5-10 μm, a live bacteria encapsulation rate of ≥90%, a live bacteria survival rate of ≥90% after 6 months of sealed storage at 25°C, and a live bacteria release rate of ≥85% within 1-2 hours in the respiratory tract inflammatory microenvironment.

[0010] Furthermore, the dosage form of the preparation is a nasal spray, a nebulized inhalation liquid, a dry powder inhaler, or an oral enteric-coated lyophilized preparation; wherein the nasal spray, nebulized inhalation liquid, and dry powder inhaler can act directly on the upper respiratory tract mucosa, and the oral enteric-coated lyophilized preparation can exert its effect through the gut-lung axis after being absorbed by the intestine.

[0011] On the other hand, a Lactobacillus acidophilus respiratory health preparation that enhances the mucosal barrier, the preparation method of which includes the following steps: S1. Strain culture and preparation of bacterial sludge: Lactobacillus acidophilus LH-001, Lactobacillus rhamnosus LR-002, and Bifidobacterium bifidum BB-003 were activated and cultured separately. After fermentation, the bacterial cells were collected by low-temperature centrifugation and washed with sterile physiological saline to prepare single-strain wet bacterial sludge. The mixture was then mixed evenly according to the target ratio to obtain mixed bacterial sludge. S2. Preparation of basic materials: Mix the mixed bacterial sludge with optional synergistic functional components and sterile physiological saline, and stir evenly in a low-temperature, light-proof, anaerobic environment to prepare a basic material applicable to all dosage forms. S3. Optional microencapsulation: This step is performed only when preparing a microencapsulation dosage form. An emulsification-ionic crosslinking-gradient composite coating process is used to encapsulate the base material obtained in step S2 with a chitosan-hyaluronic acid-sodium alginate ternary composite carrier to prepare live bacteria microcapsules. If a microencapsulation dosage form is not prepared, this step is skipped. S4. Formulation: Based on the target dosage form, select the basic material obtained in step S2 or the live bacteria microcapsules prepared in step S3, mix them with a pharmaceutically or food-grade acceptable carrier for the corresponding dosage form, and prepare the finished product of the target dosage form under low temperature, low oxygen, and sterile conditions.

[0012] Furthermore, in step S3, the specific operation of the emulsification-ionic crosslinking-gradient composite coating process is as follows: Aqueous phase preparation: Sodium alginate was dissolved in sterile physiological saline to prepare a sodium alginate solution with a mass-to-volume ratio of 1.5%-2.5%, which was then uniformly mixed with the base material at a volume ratio of 1:1 to obtain the aqueous phase. Emulsification: The aqueous phase was slowly added dropwise to the liquid paraffin oil phase containing Span 80, and emulsified by high-speed stirring to prepare a stable W / O type emulsion; Cross-linking and curing: Calcium chloride solution was added dropwise to the emulsion, and cross-linking and curing were completed by stirring at low speed. After centrifugation and washing, primary microcapsules were obtained. Gradient composite coating: The primary microcapsules are redispersed in sterile water. Hyaluronic acid solution is added and stirred to complete the first layer of coating. Chitosan acetic acid solution is added and stirred to complete the second layer of coating. After centrifugation and washing, coated microcapsules are obtained. Gradient composite coating can enhance the adhesion and retention ability of microcapsules on the respiratory mucosa.

[0013] Furthermore, in step S1, the temperature for low-temperature centrifugation is 2-8℃, and the centrifugation speed is 4000-6000 rpm; the coated microcapsules prepared in step S3 are added with a freeze-drying protectant and pre-frozen at -70℃ to -80℃ for 3-5 hours, and then freeze-dried under vacuum for 20-24 hours to prepare freeze-dried live bacteria microcapsule powder; the freeze-drying protectant can reduce the loss of probiotic activity during the freeze-drying process and ensure the stability of live bacteria during the storage period of the preparation.

[0014] Compared with existing technologies, this Lactobacillus acidophilus respiratory health preparation formula for enhancing the mucosal barrier has the following beneficial effects: I. This invention utilizes a targeted domestication process tailored to the respiratory physiological environment to obtain three functionally complementary probiotic strains, precisely addressing the core issues of poor compatibility and inability of existing general-purpose intestinal strains to exert their effects in situ within the respiratory tract. After domestication, *Lactobacillus acidophilus* significantly enhances its adhesion to human respiratory epithelial cells, directly repairing damaged epithelial junctions and strengthening the physical barrier; *Lactobacillus rhamnosus* can selectively inhibit common respiratory pathogens without interfering with normal mucosal flora; *Bifidobacterium bifidum* adapts to the microaerobic environment of the respiratory tract, promoting the production of secretory antibodies and strengthening local immunity. After optimized formulation, these three strains can colonize and exert their effects in situ within the respiratory tract, eliminating the need to rely solely on the indirect effects of the gut-lung axis.

[0015] II. This invention addresses the core issues of existing solutions, such as lack of synergy between components and poor live bacteria delivery, through the design of synergistic components that match the functions of bacterial strains and a targeted formulation process. The synergistic components form a precise synergy with the core bacterial strains. 2'-Fucose-based lactose can provide targeted energy to the target strains without nourishing pathogenic bacteria, and can also help block the binding of allergens, pathogenic bacteria, and mucosa. Low-molecular-weight hyaluronic acid and baicalin can help soothe mucosal discomfort and amplify the barrier repair effect. The accompanying targeted microcapsule formulation protects the live bacteria from degradation, prolongs mucosal retention time, and achieves precise release at inflamed sites. The preparation process is adaptable to the production needs of various commercial dosage forms.

[0016] Other advantages, objectives and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination or study, or may be learned from the practice of the invention. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0018] Figure 1 This is a flowchart illustrating the preparation method of a Lactobacillus acidophilus respiratory health preparation for enhancing the mucosal barrier according to the present invention. Figure 2 This is a schematic diagram of the directed domestication process of the strains of this invention; Figure 3 This is a flowchart illustrating the key process of microencapsulation in this invention. Detailed Implementation

[0019] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided below.

[0020] Example 1 This invention provides a Lactobacillus acidophilus respiratory health preparation that enhances the mucosal barrier. The preparation has the following components: The total live bacteria count of the probiotic active ingredients is 1×10⁻⁶. 10 The concentration is CFU / g, with Lactobacillus acidophilus LH-001 accounting for 60% of the live bacteria, Lactobacillus rhamnosus LR-002 accounting for 20% of the live bacteria, and Bifidobacterium bifidum BB-003 accounting for 20% of the live bacteria. Based on 100 parts by weight of probiotic active ingredients, the synergistic functional components include 3 parts of 2'-fucosylated lactose, 1 part of low molecular weight sodium hyaluronate, and 0.3 parts of baicalin. The wall material is a ternary composite carrier of chitosan, hyaluronic acid, and sodium alginate, with the remainder being pharmaceutically acceptable carriers such as composite lyophilization protectants. The composite lyophilization protectant consists of skim milk powder, trehalose, and mannitol.

[0021] The preparation method of this formulation is as follows: Figure 1 As shown, the specific preparation method is as follows: Strains culture and preparation of mycelial sludge: *Lactobacillus acidophilus* LH-001, *Lactobacillus rhamnosus* LR-002, and *Bifidobacterium bifidum* BB-003, preserved in glycerol, were used. The domestication process of these three strains is as follows: Figure 2As shown, two generations of activation were performed separately. For the first generation activation, the preserved strains were inoculated into MRS liquid medium and anaerobically cultured at 37°C for 18 hours to complete the first generation activation. For the second generation activation, the activated bacterial solution from the first generation was transferred to fresh MRS liquid medium at a 2% inoculum and anaerobically cultured at 37°C for 12 hours to complete the second generation activation, obtaining seed culture suitable for large-scale cultivation. The second-generation activated seed cultures of the three strains were inoculated into their respective optimized media at a 3% inoculum. *Lactobacillus acidophilus* and *Lactobacillus rhamnosus* were cultured in modified MRS medium at 37°C under a strictly anaerobic environment, while *Bifidobacterium bifidum* was cultured in a dedicated *Bifidobacterium* MRS medium supplemented with 0.5 g / L cysteine ​​hydrochloride at 37°C under a microaerobic environment with 5% CO2 + 5% Oxygen. The bacterial concentration was monitored in real-time during cultivation, and the concentration was maintained at a level not lower than 1 × 10⁻⁶. 9 When the concentration of CFU / mL was reached, the culture was terminated, and fermentation broths of three strains were obtained. The fermentation broths of the three strains were placed in a low-temperature centrifuge, and the centrifugation temperature was controlled at 4℃, the centrifugation speed was 5000 rpm, and the centrifugation time was 15 min. After centrifugation, the supernatant was discarded and the bacterial precipitate was collected. Sterile physiological saline pre-cooled to 4℃ was added to the bacterial precipitate, the bacterial cells were resuspended, and centrifuged again. The washing operation was repeated twice to finally prepare single-strain wet bacterial sludge. According to the live cell count ratio set in the formula, the wet bacterial sludge of the three strains was added to a sterile mixing container. The ambient temperature was controlled at 2℃-8℃ throughout the process, and the mixture was mixed evenly under anaerobic and light-proof conditions to obtain mixed bacterial sludge.

[0022] Preparation of basic materials: The mixed bacterial sludge, the formula-produced synergistic functional components, and pre-cooled sterile saline were added to a sterile mixing tank. The ambient temperature was controlled at 4°C, and nitrogen was introduced throughout the process to maintain an anaerobic environment. The mixture was stirred at a low speed of 80 rpm for 30 minutes under dark conditions until the system was completely mixed and homogeneous, thus obtaining the basic materials.

[0023] Microencapsulation: such as Figure 3As shown, food-grade sodium alginate was added to sterile physiological saline pre-cooled to 4°C and stirred at low speed until completely dissolved to prepare a sodium alginate solution with a mass-to-volume ratio of 2%. The sodium alginate solution and the base material were added to a sterile container at a volume ratio of 1:1 and stirred at 100 rpm for 20 minutes under anaerobic and light-protected conditions at 4°C until homogeneous to obtain an aqueous phase. The oil phase was prepared in advance by adding food-grade Span 80 to liquid paraffin and stirring until homogeneous to obtain a solution containing 2% Span 80 by mass-to-volume ratio. The liquid paraffin oil phase was pre-cooled at 4°C for later use. A crosslinking agent dispersion was prepared in advance by dissolving anhydrous calcium chloride in a small amount of sterile water, then adding liquid paraffin and Span 80, and dispersing at high speed to obtain a calcium chloride oil phase dispersion, which was then pre-cooled to 4°C for later use. The aqueous phase was slowly added dropwise to the oil phase at a uniform rate, with a volume ratio of 1:4. The temperature was maintained at 4°C throughout the dropwise addition, and the mixture was stirred at 8000 rpm. After the dropwise addition was completed, high-speed stirring was continued for 20 minutes to prepare the desired product. A stable W / O emulsion was prepared. Calcium chloride oil phase dispersion was slowly added dropwise to the emulsion at a uniform rate, with the temperature controlled at 4°C during the addition. The mixture was stirred at a low speed of 300 rpm. After the addition was complete, stirring continued for 30 min to achieve ionic cross-linking and curing through interfacial diffusion. After cross-linking, the system was centrifuged at 3000 rpm for 10 min at 4°C. The supernatant was discarded, and the precipitate was collected. The precipitate was washed twice with pre-cooled sterile physiological saline to obtain primary microcapsules. The primary microcapsules were redispersed in sterile water pre-cooled to 4°C to prepare a microcapsule suspension. A 1% (w / v) hyaluronic acid solution was added to the suspension, and the mixture was stirred at 200 rpm for 20 min at 4°C in the dark to complete the first hydrophilic coating. After centrifugation and washing, the first-coated microcapsules were collected. The first-coated microcapsules were then redispersed in pre-cooled sterile water, and a 1.5% (w / v) chitosan acetic acid solution was added. The mixture was stirred for 20 min under the same conditions to complete the second positively charged coating. After centrifugation and washing, complete coated microcapsules were obtained.

[0024] Freeze-drying of the formulation: Add pre-cooled composite freeze-drying protectant solution to the coated microcapsules, stir evenly, and spread evenly in a freeze-drying tray, with a material thickness not exceeding 8 mm; transfer the freeze-drying tray into a vacuum freeze dryer, first cool to -75℃, and pre-freeze at a constant temperature for 4 hours to ensure complete freezing of the material; after pre-freezing, turn on the vacuum system, control the vacuum degree to within 10 Pa, raise the temperature to -20℃ for sublimation drying, and maintain for 20 hours; after sublimation drying, raise the temperature to 25℃ for desorption drying, and maintain for 2 hours. The freeze-drying process is carried out in the dark, and finally freeze-dried live bacteria microcapsule powder, i.e., the target formulation, is obtained.

[0025] Testing revealed that the microcapsule formulation prepared in this embodiment had a particle size of 6μm-8μm, a viable bacterial encapsulation rate of 94.2%, a viable bacterial survival rate of 93.5% after 6 months of sealed storage at 25℃, and a viable bacterial survival rate of 95.1% after 3 months of refrigerated storage at 2℃-8℃. The adhesion rate to human respiratory epithelial cells was 86.7% after 24 hours, and the viable bacterial survival rate was 91.3% after 72 hours under microaerobic conditions. The survival time in isolated respiratory mucosa could reach over 12 hours. In vitro respiratory epithelial model testing showed that it could increase the expression level of tight junction proteins by 42.6% and the production of secretory IgA in the mucosa by 38.9%.

[0026] The targeted microcapsule formulation prepared in this embodiment fully realizes the functional advantages brought about by the targeted domestication of bacterial strains. The ternary composite carrier wall material of chitosan, hyaluronic acid, and sodium alginate achieves effective protection of live bacteria and precise release at the site of inflammation, with all core performance indicators meeting the expected targets. The formulation can significantly enhance the adhesion and retention time of bacterial strains in the respiratory tract, achieving in-situ efficacy in the respiratory tract, while also exhibiting excellent stability under both room temperature and refrigerated storage, making it suitable for precise intervention scenarios involving respiratory mucosal damage.

[0027] Example 2 This invention provides a Lactobacillus acidophilus respiratory health preparation that enhances the mucosal barrier. The preparation has the following components: The total live bacteria count of the probiotic active ingredients is 2×10⁻⁶. 9 The concentration is CFU / mL, with 50% viable Lactobacillus acidophilus LH-001, 25% viable Lactobacillus rhamnosus LR-002, and 25% viable Bifidobacterium bifidum BB-003. Based on 100 parts by weight of probiotic active ingredients, the synergistic functional components include 2 parts 2'-fucosylated lactose, 1.5 parts low molecular weight sodium hyaluronate, and 0.2 parts baicalin. The remainder consists of sterile isotonic saline, antibacterial stabilizers, and other pharmaceutically acceptable carriers.

[0028] The specific preparation method of this formulation is as follows: Strain Cultivation and Mycotic Sludge Preparation: *Lactobacillus acidophilus* LH-001, *Lactobacillus rhamnosus* LR-002, and *Bifidobacterium bifidum* BB-003, preserved in glycerol, were individually activated for two generations. For the first generation activation, the preserved strains were inoculated into MRS liquid medium and anaerobically cultured at 37°C for 18 hours to complete the first generation activation. For the second generation activation, the activated bacterial solution from the first generation was transferred to fresh MRS liquid medium at a 2% inoculation rate and anaerobically cultured at 37°C for 12 hours to complete the second generation activation, yielding a microbial sludge suitable for use in mycotic sludge preparation. Expanded seed culture: Second-generation activated seed cultures of the three strains were inoculated at 3% onto their respective optimized culture media. *Lactobacillus acidophilus* and *Lactobacillus rhamnosus* were cultured in a modified MRS medium at 37°C under a strictly anaerobic environment. *Bifidobacterium bifidum* was cultured in a dedicated Bifidobacterium MRS medium supplemented with 0.5 g / L cysteine ​​hydrochloride at 37°C under a microaerobic environment of 5% CO2 + 5% Oxygen. Cell concentration was monitored in real-time during culture, and cultured until the cell concentration stabilized at no less than 1 × 10⁻⁶. 9 When the concentration of CFU / mL was reached, the culture was terminated, and fermentation broths of three strains were obtained. The fermentation broths of the three strains were placed in a low-temperature centrifuge, and the centrifugation temperature was controlled at 4℃, the centrifugation speed was 5000 rpm, and the centrifugation time was 15 min. After centrifugation, the supernatant was discarded and the bacterial precipitate was collected. Sterile physiological saline pre-cooled to 4℃ was added to the bacterial precipitate, the bacterial cells were resuspended, and centrifuged again. The washing operation was repeated twice to finally prepare single-strain wet bacterial sludge. According to the live cell count ratio set in the formula, the wet bacterial sludge of the three strains was added to a sterile mixing container. The ambient temperature was controlled at 2℃-8℃ throughout the process, and the mixture was mixed evenly under anaerobic and light-proof conditions to obtain mixed bacterial sludge.

[0029] Preparation of basic feed solution: The synergistic functional components and antibacterial stabilizers were dissolved in sterile isotonic physiological saline in advance, and then filtered through a 0.22μm filter membrane for sterilization and pre-cooled to 4℃ for later use. Under a Class 100 sterile environment, the mixed bacterial sludge, sterilized excipient solution, and sterile isotonic physiological saline were added to a sterile mixing tank. The ambient temperature was controlled at 2℃-6℃, and nitrogen gas was introduced throughout the process to maintain an anaerobic and light-protected environment. The mixture was stirred at a low speed of 60rpm for 40min to ensure that all components were completely and evenly dispersed. After stirring, the pH value of the system was measured, and the pH value was adjusted to the physiologically suitable range of 6.5-7.0 with sterile buffer solution. Finally, sterile isotonic physiological saline was used to bring the volume to the target volume, and the mixture was stirred evenly to obtain the feed solution to be dispensed.

[0030] Aseptic filling and molding: Under a Class 100 sterile environment, at a low temperature of 2℃-6℃ and in an anaerobic and light-protected environment, the liquid to be filled is filled into a pharmaceutical nebulizer bottle that has been treated with moist heat sterilization, and the bottle is sealed and capped. Throughout the process, live bacteria are prevented from coming into contact with high temperature and oxygen to prepare the finished nebulized inhalation liquid.

[0031] Testing showed that the viable bacterial survival rate of the nebulized inhalation solution prepared in this embodiment was 90.1% after refrigeration at 2℃-8℃ for 3 months; the adhesion rate of human respiratory epithelial cells was 78.3% after 24 hours, and the viable bacterial survival rate was 87.6% after 72 hours under microaerobic conditions; the survival time of the solution on isolated respiratory mucosa could reach more than 8 hours; in vitro respiratory epithelial model testing showed that it could increase the expression level of tight junction protein by 35.7% and the production of mucosal secretory IgA by 32.4%; the droplet size after nebulization met the requirements for respiratory tract deposition and could effectively reach the bronchial and pulmonary mucosa.

[0032] The nebulized inhalation solution prepared in this embodiment can directly deliver the bacterial strain to the respiratory mucosa via nebulization, avoiding the influence of the digestive tract environment on the live bacteria and enabling rapid local action. The targeted-acclimated bacterial strains possess excellent respiratory mucosal adhesion and microaerobic tolerance. Combined with synergistic components, they can achieve rapid repair of the mucosal barrier and exhibit good stability during refrigerated storage, making them suitable for intervention scenarios involving acute respiratory discomfort and local mucosal damage.

[0033] Example 3 This invention provides a Lactobacillus acidophilus respiratory health preparation that enhances the mucosal barrier. The preparation has the following components: The total live bacteria count of the probiotic active ingredients is 5×10⁻⁶. 9 The concentration is CFU / g, with 40% viable Lactobacillus acidophilus LH-001, 30% viable Lactobacillus rhamnosus LR-002, and 30% viable Bifidobacterium bifidum BB-003. Based on 100 parts by weight of probiotic active ingredients, the synergistic functional components include 4 parts 2'-fucosylated lactose, 0.8 parts low molecular weight sodium hyaluronate, and 0.4 parts baicalin. The remainder consists of food-grade acceptable carriers such as compound freeze-drying protectants, fillers, lubricants, and enteric coating materials. The compound freeze-drying protectant is composed of skim milk powder, trehalose, and mannitol.

[0034] The specific preparation method of this formulation is as follows: Strain Cultivation and Mycotic Sludge Preparation: *Lactobacillus acidophilus* LH-001, *Lactobacillus rhamnosus* LR-002, and *Bifidobacterium bifidum* BB-003, preserved in glycerol, were individually activated for two generations. For the first generation activation, the preserved strains were inoculated into MRS liquid medium and anaerobically cultured at 37°C for 18 hours to complete the first generation activation. For the second generation activation, the activated bacterial solution from the first generation was transferred to fresh MRS liquid medium at a 2% inoculation rate and anaerobically cultured at 37°C for 12 hours to complete the second generation activation, yielding a microbial sludge suitable for use in mycotic sludge preparation. Expanded seed culture: Second-generation activated seed cultures of the three strains were inoculated at 3% onto their respective optimized culture media. *Lactobacillus acidophilus* and *Lactobacillus rhamnosus* were cultured in a modified MRS medium at 37°C under a strictly anaerobic environment. *Bifidobacterium bifidum* was cultured in a dedicated Bifidobacterium MRS medium supplemented with 0.5 g / L cysteine ​​hydrochloride at 37°C under a microaerobic environment of 5% CO2 + 5% Oxygen. Cell concentration was monitored in real-time during culture, and cultured until the cell concentration stabilized at no less than 1 × 10⁻⁶. 9 When the concentration of CFU / mL was reached, the culture was terminated, and fermentation broths of three strains were obtained. The fermentation broths of the three strains were placed in a low-temperature centrifuge, and the centrifugation temperature was controlled at 4℃, the centrifugation speed was 5000 rpm, and the centrifugation time was 15 min. After centrifugation, the supernatant was discarded and the bacterial precipitate was collected. Sterile physiological saline pre-cooled to 4℃ was added to the bacterial precipitate, the bacterial cells were resuspended, and centrifuged again. The washing operation was repeated twice to finally prepare single-strain wet bacterial sludge. According to the live cell count ratio set in the formula, the wet bacterial sludge of the three strains was added to a sterile mixing container. The ambient temperature was controlled at 2℃-8℃ throughout the process, and the mixture was mixed evenly under anaerobic and light-proof conditions to obtain mixed bacterial sludge.

[0035] Preparation of basic materials: The mixed bacterial sludge, the formula-produced synergistic functional components, the compound freeze-drying protectant, and the pre-cooled sterile saline were added to a sterile mixing tank. The ambient temperature was controlled at 2℃-8℃. Nitrogen gas was introduced throughout the process to maintain an anaerobic and light-proof environment. The mixture was stirred at a low speed of 80 rpm for 30 minutes until the system was completely mixed and homogeneous, thus obtaining the basic materials.

[0036] Vacuum freeze drying: The base material is spread evenly in a freeze drying tray, with a material thickness not exceeding 10 mm, and then transferred into a vacuum freeze dryer; it is first rapidly cooled to -80℃ and pre-frozen at a constant temperature for 5 hours to ensure that the material is completely frozen; after pre-freezing, the vacuum system is turned on and the vacuum degree is controlled within 8 Pa, and the temperature is raised to -15℃ for sublimation drying, which is maintained for 18 hours; after sublimation drying, the temperature is gradually raised to 30℃ for desorption drying, which is maintained for 3 hours. The freeze drying process is carried out in the dark and anaerobic environment, and finally, the compound probiotic freeze-dried bacterial powder is prepared.

[0037] Formulation: The compound probiotic freeze-dried powder and the prescribed amount of filler and lubricant are pretreated, and all materials are passed through an 80-mesh sieve to ensure uniform particle size. All sieved materials are added to a three-dimensional motion mixer, and the speed is controlled at 15 rpm for 30 minutes to obtain a uniform mixed powder. Using a dry granulation process, the mixed powder is fed into a dry granulator, and the pressure of the pressure roller is controlled at 2 MPa and the granulation speed is controlled at 10 rpm to prepare uniform granules of 20-40 mesh. The prepared granules are added to a tableting machine and tableted in a low-temperature, low-humidity, light-proof, and anaerobic environment, with the tableting pressure controlled at 5 kN-8 kN, to produce tablets with uniform weight. Pharmaceutical enteric coating solution is prepared, and the tablets are coated with an enteric film in a high-efficiency coating machine, with the coating weight gain controlled at 8%-10%. After coating, the tablets are dried to obtain the oral enteric freeze-dried tablets.

[0038] Testing showed that the oral enteric-coated lyophilized formulation prepared in this embodiment had a viable bacterial survival rate of 91.7% after being sealed and stored at 25°C for 6 months, and a viable bacterial survival rate of 93.2% after being refrigerated at 2°C-8°C for 3 months. After standing in a specified simulated gastric fluid for 2 hours, the viable bacterial survival rate was 92.4%, which can achieve targeted release into the intestine. The adhesion rate of human respiratory epithelial cells was 72.5% after 24 hours, and the viable bacterial survival rate was 85.2% after 72 hours under microaerobic conditions. Through the gut-lung axis, it can increase the production of secretory IgA in the respiratory mucosa by 29.6%, and the intestinal flora regulation effect is significant, which can indirectly improve the immune status of the respiratory mucosa.

[0039] The oral enteric-coated lyophilized formulation prepared in this embodiment solves the core problem of oral probiotic inactivation in gastric juice through enteric film coating, enabling precise release of live bacteria into the gut. It exhibits excellent stability under both room temperature and refrigerated storage. By regulating the gut microbiota, the formulation exerts a respiratory health intervention effect through the gut-lung axis. Simultaneously, the targeted-acclimated strains retain the potential for respiratory mucosal adhesion and repair, thus addressing both gut and respiratory health needs and making it suitable for daily long-term conditioning and intervention scenarios for chronic respiratory discomfort.

[0040] Comparative Example 1 The comparative formulation has the following components: Commercially available, universally compatible gut microbiota strains were used, with a total viable count of 1×10⁻⁶. 10 The product contains CFU / g, of which Lactobacillus acidophilus NCFM accounts for 60%, Lactobacillus rhamnosus GG accounts for 20%, and Bifidobacterium animalis Bb-12 accounts for 20%. No synergistic functional components are added, and the remainder consists of conventional excipients such as compound freeze-drying protectants.

[0041] The specific preparation method of this formulation is as follows: Strain Cultivation and Mycotic Sludge Preparation: *Lactobacillus acidophilus* NCFM, *Lactobacillus rhamnosus* GG, and *Bifidobacterium animalis* Bb-12, preserved in glycerol, were individually activated for two generations. For the first generation activation, the preserved strains were inoculated into MRS liquid medium and anaerobically cultured at 37°C for 18 hours. For the second generation activation, the activated bacterial solution from the first generation was transferred to fresh MRS liquid medium at a 2% inoculation rate and anaerobically cultured at 37°C for 12 hours, yielding a seed culture suitable for large-scale cultivation. The second-generation activated seed cultures of the three strains were inoculated into MRS expansion medium at a 3% inoculation rate and anaerobically cultured at 37°C. The bacterial concentration was monitored in real-time during cultivation, and cultivation continued until the bacterial concentration stabilized at no less than 1 × 10⁻⁶. 9 When the concentration of CFU / mL was reached, the culture was terminated, and fermentation broths of the three strains were obtained. The fermentation broths of the three strains were placed in a low-temperature centrifuge, and the centrifugation temperature was controlled at 4℃, the centrifugation speed was 5000 rpm, and the centrifugation time was 15 min. After centrifugation, the supernatant was discarded and the bacterial precipitate was collected. Sterile physiological saline pre-cooled to 4℃ was added to the bacterial precipitate, the bacterial cells were resuspended, and centrifuged again. The washing operation was repeated twice to finally prepare single-strain wet bacterial sludge. The wet bacterial sludges of the three strains were mixed evenly according to the live cell count ratio set in the formula to obtain mixed bacterial sludge.

[0042] Preparation of freeze-dried material: Add compound freeze-drying protectant and pre-cooled sterile saline to the mixed bacterial sludge, stir evenly at 4°C and in the dark to prepare freeze-dried material.

[0043] Formulation: The freeze-dried material is spread evenly in a freeze-drying tray, transferred to a vacuum freeze dryer, pre-frozen at -75℃ for 4 hours, with the vacuum degree controlled within 10Pa, sublimation drying for 20 hours, and desorption drying for 2 hours to finally obtain a conventional probiotic powder formulation.

[0044] Testing revealed that the viable bacterial survival rate of the formulation prepared in this comparative example was 72.3% after 6 months of sealed storage at 25°C and 76.8% after 3 months of refrigerated storage at 2°C-8°C. The adhesion rate of human respiratory epithelial cells was 21.6% after 24 hours, and the viable bacterial survival rate was 34.7% after 72 hours under microaerobic conditions. The survival time of the formulation on isolated respiratory mucosa was less than 2 hours. After standing in simulated gastric fluid for 2 hours, the viable bacterial survival rate was 31.5%. In vitro respiratory epithelial model testing showed that the expression level of tight junction protein increased by 8.2%, and the production of mucosal secretory IgA increased by 7.5%. The core performance indicators were far lower than those of the embodiments of this invention.

[0045] This comparative example uses commonly used gut-adapted probiotic strains in existing technologies. These strains have not undergone targeted adaptation to the respiratory environment, resulting in extremely poor adhesion to respiratory epithelial cells, low survival rate in the micro-oxygen environment of the respiratory tract, extremely short mucosal retention time, and poor storage stability and resistance to gastric juice. They cannot achieve in situ efficacy in the respiratory tract and can only exert a weak indirect effect through the gut-lung axis, with limited barrier repair effects. This fully reflects the core defects of existing technologies.

[0046] Performance Comparison Table of Examples and Comparative Examples:

[0047] Example 2 is a liquid formulation, and only its refrigeration stability was tested, not its long-term stability at 25°C; Example 3 is an oral enteric-coated formulation, with the core pathway of action being the gut-lung axis, and no data was directly retained on the respiratory mucosa; Examples 1 and 2 are topical formulations, and there is no need to test the survival rate of the artificial gastric fluid.

[0048] As shown in the table above, the three embodiments of the present invention are significantly superior to Comparative Example 1, which uses conventional intestinal strains, in terms of strain adhesion, mucosal retention, immune enhancement, micro-oxygen tolerance, and storage stability. The microencapsulated formulation of Example 1 exhibits the most prominent in-situ effect in the respiratory tract, with optimal stability at both room temperature and refrigerated storage. The nebulized liquid formulation of Example 2 demonstrates good refrigerated stability and can quickly reach the respiratory tract to exert its effect. The enteric-coated formulation of Example 3 effectively resists the gastric juice environment, ensuring live bacteria reach the intestines, thus protecting both intestinal and respiratory health. Overall, the data indicates that the present invention, through targeted strain domestication and matching formulation processes, truly solves the problems of conventional probiotics being difficult to take effect in the respiratory tract, having insufficient stability, and weak efficacy.

[0049] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A Lactobacillus acidophilus respiratory health preparation that enhances the mucosal barrier, characterized in that, The formulation includes probiotic active ingredients, as well as a pharmaceutically or food-grade acceptable carrier; The probiotic active ingredients consist of Lactobacillus acidophilus LH-001, Lactobacillus rhamnosus LR-002, and Bifidobacterium bifidum BB-003, which have been induced to develop respiratory tract-directed domestication. The Lactobacillus acidophilus LH-001 strain was obtained through adaptive domestication by atmospheric pressure room temperature plasma mutagenesis combined with continuous passage of human respiratory epithelial cells. The Lactobacillus rhamnosus LR-002 strain was obtained through targeted screening against respiratory pathogens combined with adaptive domestication to the inflammatory microenvironment. The Bifidobacterium bifidum BB-003 strain was obtained through microaerobic environment adaptation and targeted screening combined with co-culture of mucosal immune cells.

2. The Lactobacillus acidophilus respiratory health preparation for enhancing the mucosal barrier according to claim 1, characterized in that, The viable count ratio of Lactobacillus acidophilus LH-001, Lactobacillus rhamnosus LR-002, and Bifidobacterium bifidum BB-003 is (1-3):(1-2):(2-4), and the total viable count of the preparation is ≥1×10⁻⁶. 9 CFU / g.

3. The Lactobacillus acidophilus respiratory health preparation for enhancing the mucosal barrier according to claim 1, characterized in that, The formulation also includes synergistic functional components; by mass parts, the synergistic functional components include 1-5 parts of 2'-fucosylated lactose, 0.5-2 parts of low molecular weight sodium hyaluronate, and 0.1-0.5 parts of baicalin; the weight average molecular weight of low molecular weight sodium hyaluronate is 10-30 kDa.

4. The Lactobacillus acidophilus respiratory health preparation for enhancing the mucosal barrier according to claim 1, characterized in that, The formulation is a respiratory tract-targeted adhesion pH-responsive live bacteria microcapsule formulation; the wall material of the live bacteria microcapsule formulation is a chitosan-hyaluronic acid-sodium alginate ternary composite carrier, and the core material is the probiotic active ingredient and optional synergistic functional components.

5. The Lactobacillus acidophilus respiratory health preparation for enhancing the mucosal barrier according to claim 4, characterized in that, The live bacteria microcapsule formulation has a particle size of 5-10 μm, a live bacteria encapsulation rate of ≥90%, and a live bacteria survival rate of ≥90% after being sealed and stored at 25°C for 6 months.

6. The Lactobacillus acidophilus respiratory health preparation for enhancing the mucosal barrier according to claim 1, characterized in that, The formulation is in the form of a nasal spray, a nebulized inhalation solution, a dry powder inhaler, or an oral enteric-coated lyophilized preparation.

7. The Lactobacillus acidophilus respiratory health preparation for enhancing the mucosal barrier according to claim 1, characterized in that, The preparation method of this formulation includes the following steps: S1. Strain culture and preparation of bacterial sludge: Lactobacillus acidophilus LH-001, Lactobacillus rhamnosus LR-002, and Bifidobacterium bifidum BB-003 were activated and cultured separately. After fermentation, the bacterial cells were collected by low-temperature centrifugation and washed with sterile physiological saline to prepare single-strain wet bacterial sludge. The mixture was then mixed evenly according to the target ratio to obtain mixed bacterial sludge. S2. Preparation of basic materials: Mix the mixed bacterial sludge with optional synergistic functional components and sterile physiological saline, and stir evenly in a low-temperature, light-proof, anaerobic environment to prepare a basic material applicable to all dosage forms. S3. Optional microencapsulation: This step is performed only when preparing a microencapsulation dosage form. An emulsification-ionic crosslinking-gradient composite coating process is used to encapsulate the base material obtained in step S2 with a chitosan-hyaluronic acid-sodium alginate ternary composite carrier to prepare live bacteria microcapsules. If a microencapsulation dosage form is not prepared, this step is skipped. S4. Formulation: Based on the target dosage form, select the basic material obtained in step S2 or the live bacteria microcapsules prepared in step S3, mix them with a pharmaceutically or food-grade acceptable carrier for the corresponding dosage form, and prepare the finished product of the target dosage form under low temperature, low oxygen, and sterile conditions.

8. The Lactobacillus acidophilus respiratory health preparation for enhancing the mucosal barrier according to claim 7, characterized in that, In step S3, the specific operation of the emulsification-ionic crosslinking-gradient composite coating process is as follows: Aqueous phase preparation: Sodium alginate was dissolved in sterile physiological saline to prepare a sodium alginate solution with a mass-to-volume ratio of 1.5%-2.5%, which was then uniformly mixed with the base material at a volume ratio of 1:1 to obtain the aqueous phase. Emulsification: The aqueous phase was slowly added dropwise to the liquid paraffin oil phase containing Span 80, and emulsified by high-speed stirring to prepare a stable W / O type emulsion; Cross-linking and curing: Calcium chloride solution was added dropwise to the emulsion, and cross-linking and curing were completed by stirring at low speed. After centrifugation and washing, primary microcapsules were obtained. Gradient composite coating: The primary microcapsules are redispersed in sterile water. Hyaluronic acid solution is added and stirred to complete the first layer of coating. Chitosan acetic acid solution is added and stirred to complete the second layer of coating. After centrifugation and washing, coated microcapsules are obtained.

9. A Lactobacillus acidophilus respiratory health preparation for enhancing the mucosal barrier according to claim 7, characterized in that, In step S1, the temperature of low-temperature centrifugation is 2-8℃ and the centrifugation speed is 4000-6000 rpm; the coated microcapsules prepared in step S3 are added with a freeze-drying protectant and pre-frozen at -70℃~-80℃ for 3-5 hours, and then freeze-dried under vacuum for 20-24 hours to prepare freeze-dried live bacteria microcapsule powder.

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