Combination of lactobacillus rhamnosus LRa05, lactobacillus acidophilus La88 and lactobacillus plantarum Lp90 and application of combination in regulation of vaginal mucosal barrier protein

Abstract

The invention relates to the field of prevention and treatment of vaginitis, in particular to a composition of lactobacillus rhamnosus LRa05, lactobacillus acidophilus La88 and lactobacillus plantarum Lp90 and application of the composition to regulation of vaginal mucosa barrier protein. The lactobacillus rhamnosus LRa05 is a lactobacillus rhamnosus LRa05 with the preservation number of CGMCC (China General Microbiological Culture Collection Center) NO.24377, and the lactobacillus rhamnosus LRa05 is a lactobacillus rhamnosus LRa05 with the preservation number of CGMCC NO.24377. The lactobacillus acidophilus La88 is the lactobacillus acidophilus La88 of which the preservation number is CGMCC (China General Microbiological Culture Collection Center) NO.24109, and the lactobacillus acidophilus La88 is a lactobacillus acidophilus La88 of which the preservation number is CGMCC NO.24109. The lactobacillus plantarum Lp90 is the lactobacillus plantarum Lp90 with the preservation number of CGMCC (China General Microbiological Culture Collection Center) NO.10453, and the lactobacillus plantarum Lp90 has the preservation number of CGMCC NO.10453. The lactobacillus rhamnosus LRa05, the lactobacillus acidophilus La88, the lactobacillus plantarum Lp90 and the combination thereof disclosed by the invention can regulate the expression of the vagina mucosal barrier protein.

Description

Technical Field

[0001] This application relates to the field of regulating the mechanical barrier function of the vaginal mucosa, specifically to the combination of Lactobacillus rhamnosus LRa05, Lactobacillus acidophilus La88 and Lactobacillus plantarum Lp90 and their application in regulating vaginal mucosal barrier proteins. Background Technology

[0002] The mechanical barrier function of the vaginal mucosa is a core foundation for maintaining the health of the female reproductive system. Its structural integrity and functional stability directly depend on the normal expression of tight junction proteins between epithelial cells. For example, three key proteins—Zonula Occludens-1 (ZO-1), Occludin (OCLN), and Claudin-1 (CLDN1)—reduce vaginal epithelial permeability and prevent the invasion of harmful external substances by forming tight junction complexes between cells. Their expression levels and functional status directly determine the defensive capability of the vaginal mucosal barrier.

[0003] In existing technologies, *Lactobacillus reuteri* and *Lactobacillus acidophilus* have been shown to participate in reproductive health protection by regulating vaginal flora structure and maintaining an acidic vaginal environment. However, neither *Lactobacillus reuteri* nor *Lactobacillus acidophilus* has been found to directly regulate vaginal mucosal tight junction proteins ZO-1, OCLN, and CLDN1, and their regulatory role and mechanism on the vaginal mucosal mechanical barrier remain unclear. Furthermore, current probiotic products primarily focus on "flora balance regulation" or "mucosal adhesion and colonization," lacking precise design targeting "vaginal epithelial tight junction protein expression regulation," thus failing to achieve targeted enhancement of the vaginal mucosal mechanical barrier function. Summary of the Invention

[0004] This application provides a probiotic preparation comprising at least one of the following: a) to c)

[0005] a): A mixture of at least one or more of the following: dead cells, live cells, inactivated cells, and metabiotics of Lactobacillus rhamnosus LRa05 with accession number CGMCC NO.24377;

[0006] b): A mixture of at least one or more of the following: dead cells, live cells, inactivated cells, and metabiotics of *Lactobacillus acidophilus* La88 with accession number CGMCC NO.24109; and

[0007] c): A mixture of at least one or more of the following: dead cells, live cells, inactivated cells, and metabiotics of Lactobacillus plantarum Lp90 with accession number CGMCC NO.10453.

[0008] In some embodiments, the probiotic preparation is a regulator of vaginal mucosal barrier proteins, wherein the vaginal mucosal barrier proteins are at least one of ZO-1, OCLN, and CLDN1.

[0009] In some embodiments, the probiotic preparation is an antibacterial agent containing Alcaligenes bacteria.

[0010] In some embodiments, the probiotic preparation is an antibacterial preparation containing Gardnerella vaginalis.

[0011] In some embodiments, the probiotic preparation is a preparation that reduces the abundance of Proteobacteria in the gut and / or increases the abundance of Firmicutes.

[0012] In some embodiments, the probiotic preparation is an inactivated bacterial agent used to regulate the Proteobacteria / Firmwallia ratio in an in vitro gut microbiota simulation system.

[0013] In some embodiments, the probiotic preparation is one that reduces the abundance of Alcaligenes in the gut.

[0014] In some embodiments, the probiotic preparation is one that increases the abundance of Streptococcus spp. in the gut.

[0015] In some embodiments, the probiotic preparation is a preparation that reduces inflammatory factors and MPO activity in vaginal tissue lysate.

[0016] This application also provides applications of at least one of the following: *Lactobacillus rhamnosus* LRa05 (CGMCC NO. 24377), *Lactobacillus acidophilus* La88 (CGMCC NO. 24109), and *Lactobacillus plantarum* Lp90 (CGMCC NO. 10453), the applications including:

[0017] Preparation of a modulatory agent for vaginal mucosal barrier proteins;

[0018] Prepare an antibacterial agent containing an inhibitory agent against Alcaligenes spp.;

[0019] Prepare an antibacterial agent containing Gardnerella vaginalis;

[0020] Preparations of formulations that reduce the abundance of Proteobacteria in the gut and / or increase the abundance of Firmicutes;

[0021] Prepare inactivated bacterial cell reagents for regulating the Proteobacterium / Firmwallia ratio in an in vitro gut microbiota simulation system;

[0022] Preparation of formulations to reduce the abundance of Alcaligenes in the gut;

[0023] Preparation of formulations that increase the abundance of Streptococcus spp. in the gut;

[0024] Preparations were made to reduce the activity of inflammatory factors and MPO in vaginal tissue lysate. Attached Figure Description

[0025] Figure 1 The statistical results of vaginal pH in mice from the NC, MC, Lp90 and FH groups provided for the test cases are shown in the figure.

[0026] Figure 2 Images of the vaginal opening and vaginal tissue of mice in the NC, MC, Lp90 and FH groups provided for the test cases.

[0027] Figure 3 HE staining images of vaginal tissues from mice in the NC, MC, Lp90, and FH groups provided for the test cases.

[0028] Figure 4 A statistical chart showing the copy number of Gardnerella vaginalis in mice from the MC, Lp90, and FH groups provided for the test cases.

[0029] Figure 5 β-diversity analysis of the microbial community in vaginal secretions from MC, Lp90, and FH mice provided for the test cases.

[0030] Figure 6 Phylogenetic analysis of the microbial community in vaginal secretions from MC, Lp90, and FH mice provided as test cases.

[0031] Figure 7 Genus-level analysis of the microbial community in vaginal secretions from MC, Lp90, and FH mice provided for the test cases.

[0032] Figure 8 Statistical chart of expression levels of anti-inflammatory factors (TNF-α, IL-1β, IL-6, IL-17) in MC, Lp90 and FH group mice provided for the test cases.

[0033] Figure 9 Statistical graphs showing the expression levels of IL-10, MPO, and SIgA in mice from the MC, Lp90, and FH groups provided for the test cases.

[0034] Figure 10 Analysis of vaginal mucosal barrier protein factors (ZO-1, OCLN, CLDN1) in MC, Lp90 and FH mice provided for the test cases.

[0035] In the figure, different numbers of "*" marks indicate statistically significant differences. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application. Reagents not specifically described in detail herein are all conventional reagents and are commercially available; methods not specifically described in detail are all conventional experimental methods and can be learned from the prior art.

[0037] Lawson PA, et al., “Moldovan Medical Journal. March 2021;64(1):35-40”, pointed out that the combined use of L. reuteri and L. rhamnosus (usually in combination with estrogen) can significantly improve the vaginal environment, restore healthy flora, and indirectly enhance the mucosal barrier function. However, it cannot directly change the expression trend of vaginal mucosal barrier proteins.

[0038] Based on this, the embodiment provides a probiotic preparation that can alter the expression patterns of vaginal mucosal barrier proteins, particularly ZO-1, OCLN, and CLDN1, specifically promoting the expression of these three proteins. Specifically, the probiotic preparation contains an effective amount of at least one of the following: *Lactobacillus rhamnosus* LRa05 (CGMCC NO. 24377), *Lactobacillus acidophilus* La88 (CGMCC NO. 24109), and *Lactobacillus plantarum* Lp90 (CGMCC NO. 10453).

[0039] Among them, the *Lactobacillus plantarum* strain Lp90 was classified and named *Lactobacillus plantarum*, deposited on January 27, 2015, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.10453. The *Lactobacillus rhamnosus* strain LRa05 was classified and named *Lactobacillus rhamnosus*, deposited on January 24, 2022. The *Lactobacillus acidophilus* strain La88 was classified and named *Lactobacillus acidophilus*, deposited on December 15, 2021, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.10453.

[0040] The embodiment also provides a probiotic preparation comprising at least one or more of the following: dead cells, live cells, and inactivated cells of Lactobacillus rhamnosus LRa05 with accession number CGMCC NO.24377.

[0041] The embodiment also provides a probiotic preparation comprising at least one or more of the following: dead cells, live cells, and inactivated cells of Lactobacillus acidophilus La88 with accession number CGMCC NO.24109.

[0042] The embodiment also provides a probiotic preparation comprising at least one or more of the following: dead, live, and inactivated cells of Lactobacillus rhamnosus LRa05 with accession number CGMCC NO.24377; at least one or more of the following: dead, live, and inactivated cells of Lactobacillus acidophilus La88 with accession number CGMCC NO.24109; and a mixture of at least one or more of the following: dead, live, and inactivated cells of Lactobacillus plantarum Lp90 with accession number CGMCC NO.10453.

[0043] In some embodiments, the Lactobacillus rhamnosus LRa05 strain according to this application may be an isolated bacterial strain or a pure culture colony.

[0044] In some embodiments, the Lactobacillus acidophilus La88 strain according to this application may be an isolated bacterial strain or a pure culture colony.

[0045] In some embodiments, the Lactobacillus plantarum Lp90 strain according to this application may be an isolated bacterial strain or a pure culture colony.

[0046] In some of the formulations provided in the embodiments, the concentration of at least one or more of the dead bacterial cells, live bacterial cells, and inactivated bacterial cells is 10. 2 Up to 10 17 Within the range of colony-forming units per gram or per milliliter (CFU / g or CFU / mL), for example, in 10 5 -10 17 Within the range of CFU / g or CFU / mL, for example, in the range of 10 6 -10 17 Within the range of CFU / g or CFU / mL, for example, in the range of 10 7 -10 17 Within the range of CFU / g or CFU / mL, for example, in the range of 10 8 -10 17 Within the range of CFU / g or CFU / mL, for example, in the range of 10 9 -10 17 Within the range of CFU / g or CFU / mL, for example, in the range of 10 10 -10 17 Within the range of CFU / g or CFU / mL, for example, in the range of 10 11 -10 16 Within the range of CFU / g or CFU / mL, for example, in the range of 10 12 -1016 Within the range of CFU / g or CFU / mL, for example, in the range of 10 13 -10 16 Within the range of CFU / g or CFU / mL, for example, in the range of 10 7 -10 16 Within the range of CFU / g or CFU / mL, for example, in the range of 10 8 -10 15 Within the range of CFU / g or CFU / mL, for example, in the range of 10 9 -10 15 Within the range of CFU / g or CFU / mL, for example, in the range of 10 10 -10 15 Within the range of CFU / g or CFU / mL, for example, in the range of 10 11 -10 15 Within the range of CFU / g or CFU / mL, for example, in the range of 10 12 -10 15 Within the range of CFU / g or CFU / mL.

[0047] In some of the compositions provided in the embodiments, the spores of the *Lactobacillus acidophilus* La88 strain are in a concentration range determined by a spore activation-selective culture binding count method, with a concentration in the range of 10. 3 Up to 10 17 Within the range of colony-forming units per gram or per milliliter (CFU / g or CFU / mL), for example, in 10 5 -10 17 Within the range of CFU / g or CFU / mL, for example, in the range of 10 6 -10 17 Within the range of CFU / g or CFU / mL, for example, in the range of 10 7 -10 17 Within the range of CFU / g or CFU / mL, for example, in the range of 10 8 -10 17 Within the range of CFU / g or CFU / mL, for example, in the range of 10 9 -10 17 Within the range of CFU / g or CFU / mL, for example, in the range of 10 10 -10 17 Within the range of CFU / g or CFU / mL, for example, in the range of 10 11 -10 16 Within the range of CFU / g or CFU / mL, for example, in the range of 10 12 -10 16 Within the range of CFU / g or CFU / mL, for example, in the range of 10 13 -10 16 Within the range of CFU / g or CFU / mL, for example, in the range of 107 -10 16 Within the range of CFU / g or CFU / mL, for example, in the range of 10 8 -10 15 Within the range of CFU / g or CFU / mL, for example, in the range of 10 9 -10 15 Within the range of CFU / g or CFU / mL, for example, in the range of 10 10 -10 15 Within the range of CFU / g or CFU / mL.

[0048] In the context of this application, *Lactobacillus rhamnosus* LRa05, *Lactobacillus acidophilus* La88, or *Lactobacillus plantarum* Lp90, as defined herein, can be provided in the composition according to this application in the form of at least one or more of dead, live, and inactivated bacterial cells. Live bacterial cells refer to live *Lactobacillus reuteri* bacteria with intact cell structure, capable of normal metabolism and reproduction, for example, cultured in a culture medium (such as MRS medium), centrifuged, washed to retain viability, and usually preserved in lyophilized form (such as lyophilized bacterial powder). For example, live bacterial cells refer to the bacterial solid obtained by fermenting the strain in MRS medium, centrifuging, and washing with physiological saline. Dead bacterial cells refer to bacterial cells that have died naturally or lost activity through physical / chemical treatment (such as high temperature or ultraviolet light), and whose cell structure may be intact or partially destroyed. For example, dead bacterial cells are obtained by freeze-drying live bacterial cells, grinding and crushing them, and passing them through a 200-mesh sieve to obtain bacterial cell fragment powder (protein content ≥30%). Inactivated bacteria specifically refer to bacteria that have been killed through controlled methods (such as heat inactivation, formaldehyde treatment, or autoclaving) while retaining their cell surface structures (such as cell walls and capsules). Inactivated bacteria emphasize "structural preservation," while dead bacteria may suffer structural damage due to the treatment method. For example, inactivated bacteria are produced by sterilizing live bacteria with moist heat at 60°C for 30 minutes (or treating with 0.5% formaldehyde solution for 1 hour) to ensure no live bacteria are detected (live bacteria count <10 CFU / mL using plate counting method), thus preserving the bacterial structure and metabolic products.

[0049] In some of the formulations provided in the embodiments, Lactobacillus rhamnosus LRa05, Lactobacillus acidophilus La88, or Lactobacillus plantarum Lp90 can be used as the active ingredient in at least one or more of the following: dead cells, live cells, and inactivated cells, and the active ingredient accounts for 0.0001% (w / w) to 99% (w / w) of the total mass of the formulation.

[0050] In some embodiments, the probiotic preparation is a metabiotic. "Metabiotic" refers to the collective term for physiologically active bacterial components and metabolites produced by probiotics (such as *Lactobacillus rhamnosus*, *Lactobacillus acidophilus*, and *Lactobacillus plantarum* in this text) after specific processing. Its core characteristic is that it can exert its functions without relying on the live bacterial state. Specifically, "metabiotic" includes at least one of the following: "inactivated bacterial cells," a solution containing "inactivated bacterial cells," a fermentation broth containing "inactivated bacterial cells," or an inactivated fermentation solution.

[0051] In some embodiments, the formulation may be provided in solid, liquid, viscous, emulsion, or dry form. The formulations provided in some examples are preferably formulated as pastes, soft gelatin capsules, hard gelatin capsules, powders, talc, granules, beads, lozenges, effervescent tablets, lozenges, chewable tablets, sublingual tablets, oils, liquids, solutions, tinctures, emulsions, fruit juices, concentrates, syrups, sprays, mists, drinking ampoules, gels, gums, tablets, or coated pills.

[0052] Some of the embodiments provide formulations that are powders, tablets, ointments, emulsions, oils, suspensions, lotions, gels, pastes, foams, dairy products, gels, mists, sprays, or fermented preparations.

[0053] Some embodiments provide probiotic preparations as vaginal gels, which have been tested and found to promote the expression of vaginal mucosal barrier protein factors ZO-1, OCLN, and CLDN1. For example, *Lactobacillus rhamnosus* LRa05, *Lactobacillus acidophilus* La88, or *Lactobacillus plantarum* Lp90 can be formulated into vaginal gels using at least one or more of the following bacterial forms: dead, live, and inactivated. Taking 100g as an example, this gel contains *Lactobacillus rhamnosus* LRa05, *Lactobacillus acidophilus* La88, or *Lactobacillus plantarum* Lp90, all in a mixture of at least one or more of the following bacterial forms: dead, live, and inactivated (1×10⁻⁶). 9 Preparation process: Carbomer (CFU live cells or 2g inactivated cells or 2g dead cells), Carbomer 940 (1%-2%), Sodium carboxymethyl cellulose (0.5%-1%), Trehalose (5%-8%), Glycerin (3%-5%), Ethylparaben (0.05%-0.1%), Citric acid (0.1%-0.3%), and purified water to a final volume of 100g. Preparation process: Carbomer swells in purified water for 24 hours. Add mucosal adhesive, osmotic pressure regulator, preservative, and pH adjuster, and stir until homogeneous. Finally, add the bacterial strain combination (live cells require stirring at a low temperature <30℃). After homogenization, dispense into 5g / vial delivery units.

[0054] Some embodiments provide probiotic preparations for vaginal use, which have been tested and found to promote the expression of vaginal mucosal barrier protein factors ZO-1, OCLN, and CLDN1. For example, *Lactobacillus rhamnosus* LRa05, *Lactobacillus acidophilus* La88, or *Lactobacillus plantarum* Lp90 can be formulated into vaginal suppositories using at least one or more of the following: dead cells, live cells, and inactivated cells. Taking each 2g suppository as an example, this vaginal suppository contains *Lactobacillus rhamnosus* LRa05, *Lactobacillus acidophilus* La88, or *Lactobacillus plantarum* Lp90, which can be formulated using at least one or more of the following: dead cells, live cells, and inactivated cells (5 × 10⁻⁶). 8 The ingredients are: CFU live bacteria or 0.5g inactivated bacteria / 0.5g dead bacteria or a mixture thereof; polyethylene glycol 4000 (40%-60%); polyethylene glycol 6000 (20%-30%); lactose (5%-10%); chitosan (0.5%-1%); and ε-polylysine (0.1%-0.3%). Preparation process: The polyethylene glycol matrix is ​​heated to 60℃ to melt, excipients are added and stirred to dissolve, the temperature is lowered to 40℃, the bacterial strain combination is added, mixed thoroughly, poured into suppository molds (pre-cooled to 4℃), cooled and solidified, and then packaged.

[0055] Some embodiments provide probiotic preparations for vaginal washes, which have been tested and found to promote the expression of vaginal mucosal barrier protein factors ZO-1, OCLN, and CLDN1. For example, *Lactobacillus rhamnosus* LRa05, *Lactobacillus acidophilus* La88, or *Lactobacillus plantarum* Lp90 can be formulated into a vaginal wash using at least one or more of the following: dead cells, live cells, and inactivated cells. Taking 100 mL of this vaginal wash as an example, it contains *Lactobacillus rhamnosus* LRa05, *Lactobacillus acidophilus* La88, or *Lactobacillus plantarum* Lp90, which can be formulated using at least one or more of the following: dead cells, live cells, and inactivated cells (0.1%-0.5% inactivated cells, or 0.1%-0.5% dead cells or 1×10⁻⁶ live cells). 7 Ingredients: CFU or a mixture thereof), xanthan gum (0.1%-0.3%), polysorbate 80 (1%-2%), glycerol (2%-3%), benzalkonium chloride (0.02%-0.05%), lactic acid (0.2%-0.5%), and purified water to a final volume of 100 mL. Preparation process: Disperse and swell xanthan gum in purified water, add other excipients and stir to dissolve, adjust pH to 4.0-4.5, add the bacterial strain combination and mix well, filter through a 0.45 μm filter membrane (filtration is omitted for live bacteria), and dispense into 100 mL / bottle.

[0056] Some embodiments provide probiotic preparations as vaginal sprays, which have been tested and found to promote the expression of vaginal mucosal barrier protein factors ZO-1, OCLN, and CLDN1. For example, *Lactobacillus rhamnosus* LRa05, *Lactobacillus acidophilus* La88, or *Lactobacillus plantarum* Lp90 can be formulated into a vaginal spray using at least one or more of the following: dead cells, live cells, and inactivated cells. Taking 100 mL of this vaginal spray as an example, it contains *Lactobacillus rhamnosus* LRa05, *Lactobacillus acidophilus* La88, or *Lactobacillus plantarum* Lp90, which can be formulated using at least one or more of the following: dead cells, live cells, and inactivated cells (0.1%-0.5% inactivated cells, or 0.1%-0.5% dead cells or 1×10⁻⁶ live cells). 7 Ingredients: CFU or a mixture thereof), xanthan gum (0.05%-0.1%), polysorbate 80 (1%-2%), glycerol (2%-3%), benzalkonium chloride (0.02%-0.05%), lactic acid (0.2%-0.5%), and purified water to a final volume of 100 mL. Pour into a spray bottle (press-down type, each 0.1 mL spray contains ≥10³ CFU or 0.1 mg of bacteria).

[0057] Some embodiments provide probiotic preparations in the form of vaginal effervescent tablets, which have been tested and found to promote the expression of vaginal mucosal barrier protein factors ZO-1, OCLN, and CLDN1. For example, *Lactobacillus rhamnosus* LRa05, *Lactobacillus acidophilus* La88, or *Lactobacillus plantarum* Lp90 can be formulated into vaginal effervescent tablets using at least one or more of the following: dead cells, live cells, and inactivated cells. Taking 1g as an example, this vaginal effervescent tablet contains *Lactobacillus rhamnosus* LRa05, *Lactobacillus acidophilus* La88, or *Lactobacillus plantarum* Lp90, which can be prepared by direct compression of powder into tablets using at least one or more of the following: dead cells and inactivated cells (0.2g inactivated, 0.2g dead cells, or a mixture thereof), sodium bicarbonate (10%-15%), citric acid (8%-12%), microcrystalline cellulose (20%-30%), and hydroxypropyl methylcellulose (2%-5%).

[0058] Some of the formulations provided in the examples contain pharmaceutically, health-promoting, or food-grade excipients.

[0059] In some embodiments, the excipients include lyophilization protectants, mucosal adhesives, osmotic pressure regulators, and preservatives.

[0060] In some embodiments, the lyophilization protectant is selected from at least one type of protectant chosen from sugars, proteins, polymers, or amino acids, and there is no limitation on the specific protectant substance. Examples of sugars include trehalose, sucrose, lactose, and glucose; examples of proteins include skim milk powder, casein, and gelatin; examples of polymers include polyethylene glycol and PVP; and examples of amino acids include glutamic acid and proline. More specifically, the lyophilization protectant accounts for 5%-20% of the total weight of the probiotic preparation provided in the embodiments. In some embodiments, the probiotic preparation contains 5%-15% by weight of a sugar lyophilization protectant. In some embodiments, the probiotic preparation contains 10%-20% by weight of a protein lyophilization protectant. In some embodiments, the probiotic preparation contains 3%-10% by weight of sugars and 5%-10% by weight of a protein lyophilization protectant.

[0061] In some embodiments, the mucosal adhesive is selected from natural or synthetic polymers, and there is no limitation on the specific mucosal adhesive. Examples of natural polymers include sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, gum arabic, xanthan gum, and chitosan; examples of synthetic polymers include carbomer 940 / 980 and sodium polyacrylate. More specifically, the mucosal adhesive accounts for 0.5%-5% of the total weight of the probiotic preparation provided in the embodiments. In some embodiments, the probiotic preparation contains 0.5%-3% by weight of a natural polymer compound. In some embodiments, the probiotic preparation contains 1%-5% by weight of a synthetic polymer compound.

[0062] In some embodiments, the osmotic pressure regulator is selected from at least one of glycerol, propylene glycol, mannitol, sorbitol, sodium chloride, and potassium chloride. More specifically, the osmotic pressure regulator accounts for 1%-10% of the total weight of the probiotic preparation provided in the embodiments. In some embodiments, the probiotic preparation contains at least one of glycerol, propylene glycol, mannitol, and sorbitol at a weight ratio of 1%-8%. In some embodiments, the probiotic preparation contains at least one of sodium chloride and potassium chloride at a weight ratio of 0.5%-2%.

[0063] In some embodiments, the preservative is selected from at least one of ethylparaben, propylparaben, benzalkonium chloride, chlorhexidine, tea polyphenols, and ε-polylysine. More specifically, the preservative accounts for 0.01%-0.5% of the total weight of the probiotic preparation provided in the embodiments. In some embodiments, the probiotic preparation contains 0.01%-0.1% by weight of a chemical preservative. In some embodiments, the probiotic preparation contains 0.1%-0.5% by weight of a natural preservative.

[0064] Furthermore, the excipients also include a pH adjuster, such as at least one of citric acid, lactic acid, and sodium bicarbonate. More specifically, the pH adjuster comprises 0.05%-1% of the total weight of the probiotic preparation provided in the examples, specifically to control the pH to 3.8-4.5.

[0065] Furthermore, the excipients also include a cosolvent, such as at least one of ethanol and polysorbate 80. More specifically, the cosolvent accounts for 0.5%-5% of the total weight of the probiotic formulation provided in the examples.

[0066] Furthermore, the excipients also include fragrances, such as at least one of menthol and rose essential oil. More specifically, the fragrances comprise 0.01%-0.1% of the total weight of the probiotic preparation provided in the examples.

[0067] Some embodiments provide formulations that may contain one or more thickeners, and / or one or more sweeteners and / or one or more artificial sweeteners, wherein the thickener is preferably selected from cellulose ethers, polysaccharides, and selected from the group consisting of xanthan gum, gelatin, highly dispersed silica, starch, carrageenan, alginate, astragalus gum, agar, gum arabic, pectin and polyvinyl ester, and the sweetener is selected from the group consisting of glucose, fructose, sucrose, glucose syrup, sorbitol, mannitol, xylitol, maltitol, steviol glycosides, saccharin, cyclamate, acesulfame K and / or aspartame.

[0068] Preferred foods and nutritional supplements in the sense of this application may include effervescent tablets, vitamin tablets, dietary supplements, mineral tablets, trace element tablets, beverage powders, beverages, fruit juices, dairy beverages, yogurt, mineral water, non-carbonated water, filled gummies, chewable tablets, fruit juices or syrups, coated pills and tablets, and aerosols.

[0069] In addition, the formulation may also contain detergents, enzymes, electrolytes, pH adjusters, thickeners, prebiotics, fluorescent whitening agents, ashing inhibitors, dye transfer inhibitors, foam modifiers and / or colorants.

[0070] Furthermore, the compositions or formulations provided in this application may also contain prebiotics. Prebiotics are chemical products that induce the growth and / or activity of symbiotic microorganisms (e.g., bacteria and fungi) that contribute to the health of the host. Prebiotics are indigestible carbohydrates that pass through the upper gastrointestinal tract undigested and stimulate the growth and / or activity of beneficial bacteria that colonize the gut or skin microbiota.

[0071] Prebiotics are food ingredients that are not easily digested but promote the growth of specific microorganisms. Synbiotics are compositions containing at least one probiotic and at least one prebiotic. Such compositions are understood to promote the growth of probiotics. Therefore, powerful synbiotics are based on a combination of specific strains of probiotics and carefully selected prebiotics. They can provide important health benefits to mammals.

[0072] Some oligosaccharides used as prebiotics are fructooligosaccharides (FOS), xylooligosaccharides (XOS), polydextrose, pectin, galactooligosaccharides (GOS), or human milk oligosaccharides (HMOs). In addition, disaccharides such as lactulose or some monosaccharides such as lactose or tagatose can also be used as prebiotics.

[0073] In a very broad sense, prebiotics are all compounds that can be metabolized by probiotics. Preferably, prebiotics are indigestible or poorly digestible by mammals. Therefore, after being ingested by mammals, indigestible prebiotics can pass through the small intestine and into the large intestine to stimulate the growth of probiotics in that compartment. Thus, prebiotics can serve as a food source for probiotics. It is believed that prebiotics (many of which are poorly digestible carbohydrates) promote the growth of probiotics. Prebiotics are naturally found in, for example, cabbage, onions, whole grains, bananas, garlic, honey, leeks, artichokes, fortified foods and beverages, and dietary supplements. Prebiotics are well known in the art, and there are no particular limitations on prebiotics themselves when used in this application.

[0074] In some embodiments, at least one prebiotic product in the composition is selected from the following compounds and compositions: indigestible carbohydrates, β-glucan, mannooligosaccharides, inulin, fructooligosaccharides, human milk oligosaccharides (HMO), galactooligosaccharides (GOS), lactulose, lactulose oligosaccharides, galactotriose, fructooligosaccharides (FOS), cellobiose, cellodextrin, cyclodextrin, maltitol, lactitol, glycosilsucrose, betaine, vitamin E, or variants thereof (wherein the variants are selected from α, β, γ, δ tocopherols, tocotrienols, and tocomonenophenols). Optionally, mannooligosaccharides and / or inulin may be preferred. HMOs may include lact-N-tetrasaccharide, lact-N-fucopentose, lact-N-triose, 3'-sialyllactose, lact-N-neofucopentose, sialic acid, L-fucose, 2-fucosyllactose, 6'-sialyllactose, lact-N-neotetrasaccharide, and 3-fucosyllactose.

[0075] In some embodiments, the prebiotic may also be: lactose, β-glucan, mannooligosaccharide, inulin, fructooligosaccharide, galactooligosaccharide (GOS), lactulose, lactulose oligosaccharide, galactotriose, fructooligosaccharide (FOS), cellobiose, cellodextrin, cyclodextrin, maltitol, lactitol, glucosylsucrose, betaine, vitamin E or its variants (wherein the variants are selected from α, β, γ, δ tocopherols, tocotrienols and tocomonenophenols), lact-N-tetrasaccharide, lact-N-fucopentose, lact-N-propose, 3'-sialyllactose, lact-N-neofucopentose, sialic acid, 2-fucosyllactose, 6'-sialyllactose, lact-N-neotetrasaccharide and 3-fucosyllactose. Optionally, lactose and / or mannooligosaccharide and / or inulin may be preferred.

[0076] D- and L-fucose enhance the natural defenses of the skin or mucous membranes, stimulate epidermal immune defenses, and / or prevent and / or treat autoimmune diseases of the skin. In some embodiments of this application, the composition comprises D- or L-fucose.

[0077] In some embodiments of this application, the composition or formulation further comprises L-fucose at a concentration of 10 mM to 500 mM in the composition.

[0078] In the context of this application, additives and / or excipients include auxiliary substances known to those skilled in the art for preparing solid, semi-solid, or liquid forms, such as diluents, solvents (including water, glycerol, and ethanol), solubilizers, acidifiers, thickeners, sweeteners, flavor enhancers, colorants, lubricants, surfactants, preservatives, pH-stabilizing buffers, and mixtures thereof.

[0079] According to the test examples in this application, both *Lactobacillus acidophilus* La88 and *Lactobacillus rhamnosus* LRa05 provided in this application can upregulate the expression levels of ZO-1, OCLN, and CLDN1 in the vaginal mucosa of mice infected with *Gardnerella vaginalis*. Specifically, probiotic preparations obtained by using dead cells of *Lactobacillus acidophilus* La88, dead cells of *Lactobacillus rhamnosus* LRa05, and dead cells of *Lactobacillus plantarum* Lp90, or combinations of the three, can all upregulate the expression levels of ZO-1, OCLN, and CLDN1 in the vaginal mucosa of mice infected with *Gardnerella vaginalis*.

[0080] According to the test examples in this application, the Lactobacillus acidophilus La88, Lactobacillus rhamnosus LRa05 and their combinations provided in this application can reduce serum cellular inflammatory factors in mice infected with Clostridium difficile.

[0081] According to the test examples in this application, the Lactobacillus acidophilus La88 provided in this application can inhibit harmful bacteria such as Shigella spp., Alcaligenes spp., and Enterococcus spp.

[0082] According to the test examples in this application, the Lactobacillus acidophilus La88, Lactobacillus rhamnosus LRa05 and their combinations provided in this application can reduce the vaginal Gardnerella vaginalis load in infected mice.

[0083] According to the test examples of this application, the Lactobacillus acidophilus La88, Lactobacillus rhamnosus LRa05 and their combination provided in this application reduce the expression of vaginal pro-inflammatory factors in mice infected with Gardnerella vaginalis and enhance the secretion of immunoglobulin SIgA.

[0084] To aid understanding, the technical effects of this application are explained in detail below with several test examples.

[0085] 1. Test sample

[0086] Lactobacillus acidophilus La88, Lactobacillus rhamnosus LRa05, and Lactobacillus plantarum Lp90, stored at -80℃, were activated three times in MRS liquid medium. The seed culture was then inoculated into MRS liquid medium at a 2% (v / v) inoculum, and subsequently cultured anaerobically at 37℃ for 24 h, yielding a viable count of 5.0 × 10⁻⁶. 9 CFU / mL bacterial suspension (containing bacterial cells and their metabolites) was prepared. The bacterial suspension was inactivated (70℃, 30 min), concentrated, and freeze-dried to prepare *Lactobacillus acidophilus* La88 postbiotic inoculant, *Lactobacillus rhamnosus* LRa05 postbiotic inoculant, and *Lactobacillus plantarum* Lp90 postbiotic inoculant, respectively. The La88, LRa05, and Lp90 postbiotic inoculants were mixed in a 1:1:1 weight ratio to obtain a compound postbiotic inoculant (FH).

[0087] 2. Experimental animals

[0088] Forty-eight SPF-grade female BALB / c mice (8 weeks old, weighing 18-20g) were housed in a controlled environment with a room temperature maintained at 22±2℃ and humidity at 55%±5%, following a 12-hour light / dark cycle. They had free access to food and water. All experimental procedures involving the mice complied with the animal care and use ethics guidelines stipulated by the Hubei Provincial Animal Experiment Evaluation Center.

[0089] 3. Animal grouping

[0090] After one week of adaptive feeding, 48 mice were randomly divided into a healthy group (NC group), a model group (MC group), an Lp90 group, an LRa05 group, a La88 group, and a compound group (FH), with 8 mice in each group.

[0091] Except for the healthy group, mice in all other groups were induced to enter estrus by subcutaneous injection of 100 μL of estradiol benzoate solution, with injections repeated every 4 days. After estrus, 20 μL of a 1×10⁻⁶ bacterial solution was inoculated intravaginally into the vagina of mice in the model group, Lp90 group, LRa05 group, La88 group, and the combined group. 10 Gardnerella vaginalis (Gv) at CFU / mL was inoculated once daily for 5 consecutive days until the model was established. Healthy individuals received an intravaginal injection of 20 μL of physiological saline. Successful model establishment was defined as: redness and swelling of the vaginal opening accompanied by abundant vaginal discharge; observation of vaginal tissue lesions using HE staining; and Gv copy number calculation via qPCR in the secretions confirming significant proliferation of the target bacteria.

[0092] In addition to intravaginal administration of Gv to mice, the Lp90 group also received intravaginal administration of 1×10⁻⁶ g of Gv. 10 Lp90 probiotic agent was administered at a dose of CFU / mouse; the LRa05 group also simultaneously inoculated mice vaginally with 1×10 10CFU / / mouse dose of LRa05 probiotic agent; La88 group also simultaneously inoculated mice vaginally with 1×10 10 CFU / / mouse dose of La88 postbiotic agent; the compound group was also simultaneously inoculated vaginally with 1×10 10 The compound probiotic agent was administered at a dose of CFU / mouse. Mice in the Lp90 group, LRa05 group, La88 group, and compound group were inoculated for 17 days, once a day.

[0093] All mice were fed with regular feed and sterile water from the start to the end of the experiment (a total of 28 days).

[0094] 4. Vaginal pH measurement of mice in each group

[0095] Twenty-four hours after the last administration, pH test strips were inserted into the vaginal opening of mice, ensuring full contact with vaginal secretions before being quickly removed. The vaginal pH value was then compared with a standard colorimetric card to record the value.

[0096] The results are as follows Figure 1 As shown, compared with the NC group, the vaginal pH value of mice in the MC group was abnormally elevated. Elevated vaginal pH is a key indicator of bacterial vaginosis, reflecting an imbalance in the vaginal microbiota. After intervention with various metabiotics, especially the FH group, the vaginal pH value of mice with bacterial vaginosis significantly decreased. This result indicates that the metabiotic intervention involved in this invention indicates a shift towards a healthy vaginal environment.

[0097] 5. Observation of vaginal opening and vaginal tissue in each group of mice

[0098] After completing the pH measurement, the redness and swelling of the vaginal opening of each group of mice were observed and photographed. After vaginal irrigation, the mice were euthanized by cervical dislocation, and the vaginal tissue was dissected and photographed to observe the redness and swelling of the vaginal tissue.

[0099] The results are as follows Figure 2 As shown, compared with the NC group, the MC group showed slight redness and swelling of the vaginal tissue, accompanied by congestion, indicating successful model establishment. Furthermore, the pathological symptoms in the Lp90 and its compound probiotic groups were all reduced compared to the MC group.

[0100] 6. Pathological analysis of vaginal tissue sections from each group of mice

[0101] Mice were euthanized by cervical dislocation and vaginal tissue was collected from each group of mice. Some tissue was fixed by soaking in 10% paraformaldehyde solution (some tissue was directly stored at -80℃ for later use). Hematoxylin-eosin (HE) staining was then performed, and the vaginal tissue structure was observed under an optical microscope at 400x magnification.

[0102] like Figure 3As shown, HE staining of vaginal tissue from mice in the NC group revealed that the vaginal mucosal epithelium was continuous, intact, and smooth, without necrosis or sloughing, and no obvious inflammatory cell infiltration was observed. Furthermore, the intercellular connections between the spinous cells of the vaginal tissue were tight, and no obvious intercellular bridge structures were found. Compared to the NC group, the vaginal mucosal epithelium in the MC group was severely damaged, with extensive sloughing of vaginal mucosal epithelial tissue extending to the lamina propria, accompanied by inflammatory cell infiltration. Compared to the MC group, mice in the Lp90 and its compound postbiotic agent groups showed less damage to the vaginal mucosal epithelium, with the vaginal mucosal epithelium gradually becoming continuous, less inflammatory cell infiltration, and a relatively tight lamina propria structure.

[0103] Based on this, the embodiment also provides a vaginal topical daily cleansing / care preparation for daily vaginal cleansing, maintaining the integrity of the vaginal mucosal barrier, and relieving vaginal mucosal discomfort related to GV. Specifically, it can be prepared as a wash, gel, wipe, or care solution, containing at least one of the following: *Lactobacillus rhamnosus* LRa05 (CGMCC NO. 24377), *Lactobacillus acidophilus* La88 (CGMCC NO. 24109), and *Lactobacillus plantarum* Lp90 (CGMCC NO. 10453). The preparation method is specifically the same as that used in the probiotic preparation provided in the above embodiment for the preparation of the active ingredients, and the excipients are also prepared in the same way as those used in the probiotic preparation, and will not be elaborated here.

[0104] Based on this, the embodiments also provide a vaginal microecological daily regulation raw material / composition for regulating the local microecological balance of the female vagina and maintaining the normal state of the vaginal mucosa. It contains at least one of the following: *Lactobacillus rhamnosus* LRa05 (CGMCC NO. 24377), *Lactobacillus acidophilus* La88 (CGMCC NO. 24109), and *Lactobacillus plantarum* Lp90 (CGMCC NO. 10453). The specific preparation method is the same as that used in the probiotic preparations provided in the above embodiments, and the excipients are also prepared in the same way as those used in the probiotic preparations, which will not be elaborated here.

[0105] Based on this, the embodiments also provide a Gv-induced vaginal mucosal injury model experimental reagent, used in in vitro / animal experiments, as an inactivated strain postbiotic experimental reagent to slow down Gv-induced vaginal mucosal epithelial damage in mice, for research institutions and laboratories to conduct mechanism studies and efficacy screening. Specifically, it contains at least one of the following: *Lactobacillus rhamnosus* LRa05 with accession number CGMCC NO.24377, *Lactobacillus acidophilus* La88 with accession number CGMCC NO.24109, and *Lactobacillus plantarum* Lp90 with accession number CGMCC NO.10453. The preparation method is specifically the same as the preparation method of the active ingredient in the probiotic preparation provided in the above embodiments, and the excipients are also prepared in the same way as the excipients in the above probiotic preparations, which will not be repeated here.

[0106] Based on this, the embodiment also provides an inactivated strain postbiotic raw material for general food additives, which can be added to general foods (such as yogurt, beverages, solid beverages, and candies) to improve food flavor, extend shelf life, and enhance food quality (without claiming any health / wellness benefits). Specifically, it contains at least one of the following: *Lactobacillus rhamnosus* LRa05 with accession number CGMCC NO.24377, and *Lactobacillus plantarum* Lp90 with accession number CGMCC NO.10453. The preparation method is specifically the same as the preparation method of the active ingredients in the probiotic preparations provided in the above embodiments, and the excipients are also prepared in accordance with the above-described preparation method of the excipients in the probiotic preparations, which will not be repeated here.

[0107] Based on this, the embodiments also provide a food-grade postbiotic antibacterial / preservative composition, which is an inactivated strain postbiotic antibacterial and preservative composition used in food processing and storage to inhibit harmful microorganisms (such as Gv-related bacteria) in food and extend the shelf life of food. This is for food industry application only and does not involve claims regarding human health. Specifically, it contains at least one of the following: *Lactobacillus rhamnosus* LRa05 with accession number CGMCC NO. 24377, and *Lactobacillus plantarum* Lp90 with accession number CGMCC NO. 10453. The preparation method is specifically the same as the preparation method of the active ingredients in the probiotic preparations provided in the above embodiments, and the excipients are also prepared in the same way as the excipients in the above probiotic preparations, which will not be repeated here.

[0108] 7. Analysis of Gv load in vaginal secretions of mice in each group

[0109] Mice were vaginally cleansed three times with 300 μL of physiological saline, and the vaginal lavage fluid was collected. The number of Gardnerella vaginalis in the lavage fluid was quantified using qPCR. The upstream and downstream primer sequences used are shown in Table 1. The qPCR reaction volume (10 μL) is shown in Table 2. The reaction program was set to 95℃ pre-denaturation for 30 s, 95℃ denaturation for 5 s, and 60℃ annealing for 30 s, for 40 cycles. In another step, the temperature was increased from 65℃ to 95℃ in 0.5℃ increments every 5 s to establish a melting curve. The fluorescence threshold cycle value (CT) was determined, and the copy number was calculated based on the standard curve (log copy number / / μL vs. CT value). Each sample was tested in triplicate.

[0110] Table 1

[0111]

[0112] Table 2

[0113]

[0114] The results are as follows Figure 4 As shown, Gardnerella vaginalis was not detected in the vaginal irrigation fluid of the NC group, while the Gardnerella vaginalis load in the model group was significantly higher than that in the healthy group, indicating that Gardnerella vaginalis successfully colonized the vagina. After intervention in each postbiotic group, the Gardnerella vaginalis load in each postbiotic group was significantly reduced. At the same time, compared with the Lp90 group, the FH compound postbiotic agent group was significantly more effective than the single postbiotic group in reducing the Gardnerella vaginalis load in the vagina.

[0115] 8. Analysis of vaginal flora in each group of mice

[0116] Total microbial DNA was extracted from the homogenate lysate of mouse vaginal tissue. The V3-V4 region of the 16S rRNA gene was amplified by PCR, followed by paired-end sequencing. The sequencing data underwent quality control and filtering, and cluster analysis was performed to determine phylogenetic relationships. Finally, changes in the mouse vaginal flora were assessed.

[0117] To visualize the overall vaginal microbiota structure, this test case used PCA to analyze the β-diversity of vaginal flora in each group of mice, such as... Figure 5 As shown, the model group was completely separated from the healthy group, and the community structure of Lp90 and its compound postbiotic agent group was close to that of the healthy group, indicating that Lp90 and its compound postbiotic agent group can effectively regulate the vaginal microbial community structure of BV mice.

[0118] Figure 6The study revealed the phylum-level microbial community structure characteristics of the gut microbiota in each group of mice. For Proteobacteria, the NC group had the lowest proportion, while the MC group saw a sharp increase to 92%, the LP90 group decreased to 70%, and the FH group decreased to 62%. For Firmicutes, the NC group had approximately 48% proportion, the MC group almost eliminated it, the LP90 group recovered to approximately 20%, and the FH group recovered to approximately 35%. Other phyla (such as Campylobacteria and Bacteroidetes) were almost suppressed in the MC group, but showed varying degrees of recovery after LP90 and FH interventions.

[0119] Depend on Figure 6 It was found that compared with the NC group, the MC group showed drastic changes in its microbial community structure, with excessive proliferation of Proteobacteria and suppression of other beneficial phyla, indicating that the vaginitis model was successfully established, leading to an imbalance in the gut / vaginal microbiome. After intervention with the LP90 strain, the proportion of Proteobacteria decreased significantly, while the proportion of beneficial phyla such as Firmicutes increased, indicating that the LP90 strain can improve the microbial community imbalance in the vaginitis model mice to some extent. Compared with the LP90 group, the FH group (compound probiotics) had a lower proportion of Proteobacteria and a higher proportion of Firmicutes, with community diversity closer to that of the NC group, indicating that the intervention effect of the compound probiotics was better than that of the single LP90 strain.

[0120] Based on this, the embodiments also provide postbiotic reagents for reducing the abundance of Proteobacteria in the mouse gut and increasing the abundance of Firmicutes, for universities and research institutions to conduct research on the "interaction mechanism between Gv and gut microbiota". These reagents can be used to construct a Gv-induced mouse gut microbiota dysbiosis model, and to verify the gut microbiota regulation pathway by detecting changes in microbiota abundance after intervention; or as control reagents for screening "active substances related to microbiota balance regulation". Specifically, it contains at least one of the following: *Lactobacillus rhamnosus* LRa05 (CGMCC NO. 24377), *Lactobacillus acidophilus* La88 (CGMCC NO. 24109), and *Lactobacillus plantarum* Lp90 (CGMCC NO. 10453). The preparation method is specifically the same as that of the active ingredients in the probiotic preparations provided in the above embodiments, and the excipients are also prepared in the same way as those in the probiotic preparations, which will not be elaborated here.

[0121] Based on this, the embodiments also provide inactivated bacterial reagents for regulating the Proteobacteria / Firmwallia ratio in in vitro gut microbiota simulation systems. These reagents are used in in vitro gut simulators (such as the SHIME model and dynamic gut fermentation systems) to simulate the human / animal gut microecological environment, regulating the ratio of Proteobacteria (related to harmful bacteria) to Firmicutes (related to beneficial bacteria) within the system. This provides in vitro experimental support for industrial research on gut microbiota and the development of novel microbiota regulation substances. Specifically, it contains at least one of the following: *Lactobacillus rhamnosus* LRa05 (CGMCC NO. 24377), *Lactobacillus acidophilus* La88 (CGMCC NO. 24109), and *Lactobacillus plantarum* Lp90 (CGMCC NO. 10453). The preparation method is specifically the same as that of the active ingredients in the probiotic preparations provided in the above embodiments, and the excipients are also prepared in the same way as those in the probiotic preparations described above; therefore, they will not be elaborated here.

[0122] Based on this, the embodiments also provide inactivated strain postbiotic preparations for inhibiting the enrichment of Proteobacteria in artificial micro-ecosystems. These preparations are used in artificial micro-ecosystems such as wastewater treatment plants and industrial micro-ecosystem reactors to inhibit excessive enrichment of Proteobacteria, prevent the accumulation of their metabolites (such as harmful substances), and ensure the stable operation of the artificial micro-ecosystem (e.g., improving wastewater treatment efficiency and optimizing the industrial fermentation environment). Specifically, it contains at least one of the following: *Lactobacillus rhamnosus* LRa05 (CGMCC NO. 24377), *Lactobacillus acidophilus* La88 (CGMCC NO. 24109), and *Lactobacillus plantarum* Lp90 (CGMCC NO. 10453). The preparation method is specifically the same as that of the active ingredients in the probiotic preparations provided in the above embodiments, and the excipients are also prepared in the same way as those in the probiotic preparations, which will not be elaborated here.

[0123] Based on this, the embodiments also provide postbiotic industrial raw materials for enhancing the abundance of Firmicutes in fermentation systems. These materials are used in industrial fermentation systems (such as biofuel, biopharmaceutical, and organic acid fermentation) to increase the abundance of Firmicutes within the fermentation system, enhance the synthesis efficiency of the target fermentation product, and optimize industrial fermentation process parameters. Specifically, they include at least one of the following: *Lactobacillus rhamnosus* LRa05 (CGMCC NO. 24377), *Lactobacillus acidophilus* La88 (CGMCC NO. 24109), and *Lactobacillus plantarum* Lp90 (CGMCC NO. 10453). The preparation method is specifically the same as that of the active ingredients in the probiotic preparations provided in the above embodiments, and the excipients are also prepared in the same way as those in the probiotic preparations, which will not be elaborated here.

[0124] Figure 7 The results of species taxonomic analysis of the gut microbiota of each group of mice at the genus level are shown. Figure 7 As shown, compared with the NC group, the abundance of potentially harmful bacteria such as *Escherichia Shigella*, *Alcaligenes*, and *Enterococcus* was significantly increased in the MC group, while the abundance of beneficial bacteria such as *Streptococcus* was significantly decreased, indicating that vaginitis led to an imbalance in the gut microbiota at the genus level in mice. After intervention with LP90 strain, the abundance of *Escherichia Shigella*, *Alcaligenes*, and *Enterococcus* decreased significantly, while the abundance of *Streptococcus* recovered to near normal levels, indicating that LP90 can effectively improve the imbalance of gut microbiota at the genus level in mice with vaginitis. Furthermore, compared with the LP90 group, the abundance of *Escherichia Shigella* in the FH group was further decreased, and the abundance of *Streptococcus* was further increased, with the overall microbiota structure being closer to that of the NC group, suggesting that the intervention effect of compound probiotics is better than that of single LP90 strain.

[0125] Based on this, the embodiment also provides a metabiotic reagent that reduces the abundance of *Escherichia coli*, *Alcaligenes*, and *Enterococcus* in the mouse gut induced by Gv, and increases the abundance of *Streptococcus*. This reagent is intended for use by universities, research institutes, and pharmaceutical R&D centers in basic animal model research, for constructing Gv-induced mouse gut microbiota dysbiosis models, and as a standardized experimental reagent for intervening in the mouse gut. It provides experimental materials for studying the genus-level regulatory mechanisms of gut microbiota and the mechanisms of microbial interactions, and is used for laboratory detection and data analysis. Specifically, it contains at least one of the following metabiotics: *Lactobacillus rhamnosus* LRa05 (CGMCC NO. 24377), *Lactobacillus acidophilus* La88 (CGMCC NO. 24109), and *Lactobacillus plantarum* Lp90 (CGMCC NO. 10453). The specific preparation method is the same as that for the preparation of active ingredients in the probiotic preparations provided in the above embodiments. The preparation method for excipients is also the same as that for excipients in the above probiotic preparations, and will not be repeated here.

[0126] Based on this, the embodiments also provide inactivated bacterial preparations for regulating the abundance of *Escherichia coli*, *Alcaligenes*, *Enterococcus*, and *Streptococcus* in in vitro intestinal simulation systems. These preparations are applied to in vitro intestinal fermentation simulators and artificial intestinal microecological systems to precisely regulate the in vitro microbial community structure. They are used to screen microbial regulatory factors and verify the targeted regulatory capabilities of postbiotic flora, providing in vitro testing tools for microecological industrial research and development. Specifically, they contain at least one of the following postbiotics: *Lactobacillus rhamnosus* LRa05 (CGMCC NO. 24377), *Lactobacillus acidophilus* La88 (CGMCC NO. 24109), and *Lactobacillus plantarum* Lp90 (CGMCC NO. 10453). The preparation method is specifically the same as that of the active ingredients in the probiotic preparations provided in the above embodiments, and the excipients are also prepared in the same way as those in the probiotic preparations, which will not be elaborated here.

[0127] Based on this, the embodiments also provide a strain postbiotic tool formulation for studying the correlation of abundance at the genus level in intestinal flora. This formulation serves as a research tool reagent for analyzing the abundance correlation patterns of *Escherichia coli* / *Alcaligenes* / *Enterococcus* / *Streptococcus*. Specifically, it contains at least one of the following postbiotics: *Lactobacillus rhamnosus* LRa05 (CGMCC NO. 24377), *Lactobacillus acidophilus* La88 (CGMCC NO. 24109), and *Lactobacillus plantarum* Lp90 (CGMCC NO. 10453). The preparation method is specifically the same as that for the active ingredients in the probiotic formulations provided in the above embodiments, and the excipients are also prepared in accordance with the methods described for the excipients in the above probiotic formulations; therefore, they will not be elaborated upon here.

[0128] Based on this, the embodiments also provide a postbiotic food preservative preparation that reduces the abundance of *Shigella*, *Alcaligenes*, and *Enterococcus* in a food system. As a food industry preservation aid, it is added to prepared vegetables, meat products, ready-to-eat foods, and fermented dairy products to inhibit the proliferation of spoilage bacteria such as *Shigella*, *Alcaligenes*, and *Enterococcus* in the food matrix, extending the shelf life of food and improving the microbial safety of food processing. It is used only in food production processes and does not claim any health benefits for humans. Specifically, it contains at least one of the postbiotics *Lactobacillus rhamnosus* LRa05 (CGMCC NO. 24377) and *Lactobacillus plantarum* Lp90 (CGMCC NO. 10453). The preparation method is specifically the same as the preparation method of the active ingredients in the probiotic preparations provided in the above embodiments, and the excipients are also prepared in the same way as the excipients in the above probiotic preparations, which will not be repeated here.

[0129] Based on this, the embodiment also provides an inactivated strain postbiotic fermentation aid for enhancing the abundance of Streptococcus in a food fermentation system. This aid is applied to the production of fermented foods such as yogurt, kimchi, fermented beverages, and brewed foods. It directionally enhances the abundance of Streptococcus within the fermentation system, optimizes the fermentation microbial community structure, and improves the flavor, texture, and mouthfeel of the fermented food. It is a common food processing auxiliary material and is not included in the category of health foods. Specifically, it contains at least one of the following: *Lactobacillus rhamnosus* LRa05 (CGMCC NO. 24377) and *Lactobacillus plantarum* Lp90 (CGMCC NO. 10453). The preparation method is specifically the same as that of the active ingredient in the probiotic preparation provided in the above embodiment, and the excipients are also prepared in the same way as those in the probiotic preparation, which will not be elaborated here.

[0130] Based on this, the embodiment also provides a postbiotic compound preparation for regulating the abundance of microbial flora at the genus level in food, used in food enterprise formulation research and development, compounded in ordinary foods (pastries, solid beverages, cereal foods) to maintain the stability of the internal microecology of food, reduce the growth of harmful contaminating bacteria, and is used only as a raw material for food industry formulations. Specifically, it contains at least one of the postbiotics Lactobacillus rhamnosus LRa05 with preservation number CGMCC NO.24377 and Lactobacillus plantarum Lp90 with preservation number CGMCC NO.10453. The specific preparation method is the same as the preparation method of the active ingredients in the probiotic preparation provided in the above embodiment, and the excipients are also prepared in the same way as the excipients in the above probiotic preparation, which will not be repeated here.

[0131] 9. Analysis of local immune factors in the vagina of mice in each group

[0132] Vaginal tissue samples stored at -80℃ were processed strictly according to the instructions of the ELISA kit. The levels of cellular inflammatory factors TNF-α, IL-1β, IL-6, IL-17, IL-10, MPO and SIgA in the vaginal tissue lysate of each group of mice were detected.

[0133] The results are as follows Figure 8 , Figure 9As shown, the levels of pro-inflammatory factors TNF-α, IL-1β, IL-6, and IL-17, as well as MPO activity, were at relatively low levels in the NC group. In the MC group, infection with Gardnerella vaginalis stimulated the host's innate immune system, resulting in an approximately three-fold increase in the concentrations of pro-inflammatory factors TNF-α, IL-1β, IL-6, and IL-17, and MPO activity. This result indicates the successful establishment of a bacterial vaginosis model, simulating the local immune response in the vaginal environment under bacterial vaginosis conditions, reflecting the inflammatory state and immune dysfunction of the vaginal microenvironment. After intervention in each postbiotic group, the concentrations of pro-inflammatory factors TNF-α, IL-6, and IL-17, as well as MPO activity, all decreased to varying degrees. Among them, the FH compound postbiotic group showed a better reduction in the concentrations of pro-inflammatory factors TNF-α, IL-6, and IL-17, and MPO activity than the Lp90 and other single-bacterial postbiotic groups. Simultaneously, it significantly enhanced the secretion levels of anti-inflammatory factor IL-10 and immunoglobulin SIgA, and the FH compound postbiotic preparation group showed better results than Lp90 and other single-strain postbiotic groups. These changes indicate that whether it is La88, LRa05, Lp90, or other single-strain postbiotics, or the FH compound postbiotic preparation, they can effectively inhibit the local vaginal immune response, reduce the production and expression of inflammation-related cytokines and enzyme activities, promote the expression of anti-inflammatory cytokines and immunoglobulins, help enhance the local immune protection of the vaginal mucosa, and improve the defense against pathogens. Moreover, the FH postbiotic preparation is more effective than the Lp90 postbiotic preparation and other single-strain postbiotic preparations.

[0134] Based on this, the embodiment also provides an experimental reagent for reducing the activity of inflammatory factors and MPO in vaginal tissue lysate in a Gv-induced mouse vaginitis model. For example, it can be used as a core experimental reagent for constructing animal models related to bacterial vaginosis and studying the mechanisms of vaginal mucosal inflammation; for example, it can be used to establish a standardized Gv-induced mouse vaginal inflammation pathological model for basic verification of inflammatory regulation mechanisms; for example, it can be used as a dedicated modeling reagent for vaginal inflammation animal models, providing standardized experimental consumables for scientific research projects; for example, it can be used for basic scientific research experiments on vaginal microbial imbalance and local immune inflammation. Specifically, it contains at least one of the following: *Lactobacillus rhamnosus* LRa05 (CGMCC NO. 24377), *Lactobacillus acidophilus* La88 (CGMCC NO. 24109), and *Lactobacillus plantarum* Lp90 (CGMCC NO. 10453). The specific preparation method is the same as that for the preparation of active ingredients in the probiotic preparations provided in the above embodiments. The preparation method for excipients is also the same as that for excipients in the above probiotic preparations, and will not be repeated here.

[0135] Based on this, the embodiments also provide experimental postbiotic preparations for downregulating pro-inflammatory factors and MPO activity in in vitro vaginal inflammatory cell models. For example, an in vitro inflammatory model co-cultured with vaginal epithelial cells and Gv can be constructed to study the regulatory role of postbiotics on mucosal cell inflammatory responses; as a cellular-level inflammatory intervention reagent, it can be used for in vitro screening of microbial anti-inflammatory activity; and to study the interaction between vaginal pathogens and host cells, exploring the in vitro intervention patterns of inflammatory factor release. Specifically, it contains at least one of the following: *Lactobacillus rhamnosus* LRa05 (CGMCC NO. 24377), *Lactobacillus acidophilus* La88 (CGMCC NO. 24109), and *Lactobacillus plantarum* Lp90 (CGMCC NO. 10453). The preparation method is specifically the same as that of the active ingredients in the probiotic preparations provided in the above embodiments, and the excipients are also prepared in the same way as those in the probiotic preparations, which will not be elaborated here.

[0136] Based on this, the embodiments also provide control reagents for screening / evaluating candidate substances for anti-Gv-induced vaginal inflammation. For example, they can be used as positive control reagents to compare the inflammatory intervention effects of new compounds and novel microbial preparations; for example, they can be used to conduct comparative studies of similar anti-inflammatory microbial preparations to ensure the standardization and comparability of experimental data; and as standard reference reagents when providing technical services for evaluating the anti-inflammatory activity of microorganisms. Specifically, it contains at least one of the following: *Lactobacillus rhamnosus* LRa05 (CGMCC NO. 24377), *Lactobacillus acidophilus* La88 (CGMCC NO. 24109), and *Lactobacillus plantarum* Lp90 (CGMCC NO. 10453). The preparation method is specifically the same as the preparation method of the active ingredients in the probiotic preparations provided in the above embodiments, and the excipients are also prepared in the same way as the excipients in the above probiotic preparations, which will not be repeated here.

[0137] Based on this, the embodiments also provide experimental strains of postbiotic materials for studying Gv-induced vaginal inflammation signaling pathways. For example, using this preparation as an intervention material, the regulatory mechanisms of inflammatory signaling pathways such as NF-κB and MAPK can be studied. Specifically, it contains at least one of the following: *Lactobacillus rhamnosus* LRa05 (CGMCC NO. 24377), *Lactobacillus acidophilus* La88 (CGMCC NO. 24109), and *Lactobacillus plantarum* Lp90 (CGMCC NO. 10453). The preparation method is specifically the same as that used in the probiotic preparations provided in the above embodiments, and the excipients are also prepared in accordance with the methods described above for the excipients in the probiotic preparations; therefore, they will not be elaborated upon here.

[0138] Based on this, the embodiments also provide inactivated bacterial raw materials for preparing non-medical external cleaning / care matrix, as biological formulation raw materials for ordinary external cleaning products (such as feminine hygiene wipes, external cleaning solutions), which are only added as ingredients and do not claim any anti-inflammatory, antibacterial, or health-promoting effects. Specifically, it contains at least one of the following: *Lactobacillus rhamnosus* LRa05 with accession number CGMCC NO.24377, *Lactobacillus acidophilus* La88 with accession number CGMCC NO.24109, and *Lactobacillus plantarum* Lp90 with accession number CGMCC NO.10453. The preparation method is specifically the same as the preparation method of the active ingredients in the probiotic preparations provided in the above embodiments, and the excipients are also prepared in the same way as the excipients in the above probiotic preparations, which will not be repeated here.

[0139] Based on this, the embodiments also provide microbial raw materials for in vitro antibacterial / anti-inflammatory evaluation, serving as standard microbial raw materials to establish an in vitro anti-inflammatory activity evaluation system within the laboratory; and for internal strain characteristic testing and process development within the enterprise, serving only as laboratory testing raw materials. Specifically, it includes at least one of the following: *Lactobacillus rhamnosus* LRa05 with accession number CGMCC NO.24377, *Lactobacillus acidophilus* La88 with accession number CGMCC NO.24109, and *Lactobacillus plantarum* Lp90 with accession number CGMCC NO.10453. The preparation method is specifically the same as the preparation method of the active ingredients in the probiotic preparations provided in the above embodiments, and the excipients are also prepared in accordance with the above excipient preparation method for probiotic preparations, which will not be elaborated here.

[0140] 10. Analysis of vaginal mucosal barrier protein factors in each group of mice

[0141] Vaginal tissue samples stored at -80℃ were processed strictly according to the instructions of the ELISA kit, and the contents of vaginal mucosal barrier protein factors ZO-1, OCLN, and CLDN1 in the vaginal tissues of mice in each group were detected.

[0142] ZO-1, CLDN1, and OCLN proteins are standard markers for assessing barrier integrity. The results of the detection of the levels of vaginal mucosal barrier protein factors ZO-1, OCLN, and CLDN1 in vaginal tissues of mice in each group are shown below. Figure 10 As shown, compared with the NC group, the levels of vaginal mucosal barrier protein factors ZO-1, OCLN, and CLDN1 in the MC group were significantly reduced, indicating severe damage to the vaginal mucosal barrier, which is consistent with the results observed in HE-stained pathological sections. However, after intervention with postbiotics in each group, the levels of vaginal mucosal barrier protein factors in each postbiotic group were effectively increased, indicating that Lp90 and its compound postbiotic agent can effectively repair the damaged vaginal mucosal barrier, and the compound postbiotic agent is more effective than the Lp90 single postbiotic agent.

[0143] Based on this, the embodiments also provide experimental postbiotic reagents for reducing the expression of ZO-1, OCLN, and CLDN1 in a Gv-induced mouse vaginal tissue model. Specifically, these reagents are used to construct a Gv-induced mouse vaginal mucosa experimental model, serving as a core intervention reagent for basic scientific research experiments on the regulation of vaginal mucosal tight junction protein expression; to establish animal models related to vaginal mucosal protein expression for verification studies on the interaction mechanism between pathogenic bacteria and mucosal proteins, supporting research grants and academic papers; as standardized experimental consumables for vaginal mucosal animal models, providing dedicated experimental reagents for research projects; or to study the regulatory mechanism of Gv on vaginal mucosal tight junction proteins. Specifically, the reagents include at least one of the following: *Lactobacillus rhamnosus* LRa05 (CGMCC NO. 24377), *Lactobacillus acidophilus* La88 (CGMCC NO. 24109), and *Lactobacillus plantarum* Lp90 (CGMCC NO. 10453). The specific preparation method is the same as that for the preparation of active ingredients in the probiotic preparations provided in the above embodiments. The preparation method for excipients is also the same as that for excipients in the above probiotic preparations, and will not be repeated here.

[0144] Based on this, the embodiments also provide experimental inactivated bacterial preparations for downregulating the expression of ZO-1, OCLN, and CLDN1 in an in vitro vaginal mucosal epithelial cell model. Specifically, these preparations are used to construct an in vitro cell model co-cultured with vaginal epithelial cells and Gv, serving as an intervention reagent to study the regulatory effect of bacterial metagenes on the expression of tight junction proteins in mucosal cells under in vitro conditions; for cellular-level mucosal protein expression testing, serving as in vitro screening material for the effects of microbial preparations on mucosal cells; and as teaching and training material for cell culture and protein detection experiments. Specifically, they contain at least one of the following: *Lactobacillus rhamnosus* LRa05 (CGMCC NO. 24377), *Lactobacillus acidophilus* La88 (CGMCC NO. 24109), and *Lactobacillus plantarum* Lp90 (CGMCC NO. 10453). The specific preparation method is the same as that for the preparation of active ingredients in the probiotic preparations provided in the above embodiments. The preparation method for excipients is also the same as that for excipients in the above probiotic preparations, and will not be repeated here.

[0145] Based on this, the embodiments also provide control reagents for screening candidate substances that regulate the expression of vaginal mucosal tight junction proteins. For example, they can be used as experimental control reagents to compare the intervention effects of novel compounds and microbial preparations on Gv-induced changes in mucosal protein expression; or as standard reference reagents when conducting in vitro functional evaluation services for microbial preparations to ensure the standardization and comparability of experimental data. Specifically, it contains at least one of the following: *Lactobacillus rhamnosus* LRa05 (CGMCC NO. 24377), *Lactobacillus acidophilus* La88 (CGMCC NO. 24109), and *Lactobacillus plantarum* Lp90 (CGMCC NO. 10453). The preparation method is specifically the same as the preparation method of the active ingredients in the probiotic preparations provided in the above embodiments, and the excipients are also prepared in the same way as the excipients in the above probiotic preparations, which will not be elaborated here.

[0146] Based on this, the embodiments also provide experimental postbiotic materials for studying the signaling pathway of Gv-induced vaginal mucosal protein expression. For example, this preparation can be used as an intervention material to study the cell signaling pathway mechanism of Gv regulating vaginal mucosal tight junction proteins; or to analyze the regulatory rules of inactivated bacterial postbiotics on vaginal mucosal cell protein expression; or as a shared experimental material for studying the interaction mechanism between vaginal microorganisms and host mucosal cells. Specifically, it contains at least one of the following: *Lactobacillus rhamnosus* LRa05 with accession number CGMCC NO.24377, *Lactobacillus acidophilus* La88 with accession number CGMCC NO.24109, and *Lactobacillus plantarum* Lp90 with accession number CGMCC NO.10453. The preparation method is specifically the same as the preparation method of the active ingredients in the probiotic preparations provided in the above embodiments, and the excipients are also prepared in accordance with the above-mentioned excipient preparation method for probiotic preparations, which will not be repeated here.

[0147] Based on this, the embodiments also provide a fermentation preparation of a strain of bacteria for preparing a metabiotic that can regulate the expression of Gv-induced mouse vaginal mucosal proteins. For example, it can be used for small-scale and pilot-scale production of experimental metabiotic reagents, optimizing the fermentation and inactivation processes of the strain, and ensuring batch stability of experimental materials; or it can be used to develop a high-density fermentation and inactivation process for the strain, providing technical support for the large-scale production of scientific reagents; or it can be used for the standardized culture of functional strains, providing standard fermentation raw materials for scientific research collaboration. Specifically, it contains at least one of the following: *Lactobacillus rhamnosus* LRa05 (CGMCC NO. 24377), *Lactobacillus acidophilus* La88 (CGMCC NO. 24109), and *Lactobacillus plantarum* Lp90 (CGMCC NO. 10453). The preparation method is specifically the same as the preparation method of the active ingredients in the probiotic preparations provided in the above embodiments, and the excipients are also prepared in the same way as the excipients in the above probiotic preparations, which will not be repeated here.

[0148] Based on this, the embodiments also provide intermediate formulations for developing inactivated bacterial reagents for vaginal mucosal experimental models. For example, as intermediates in scientific reagent development, they can optimize postbiotic formulations and activity retention processes; or be used for research on probiotic inactivation and postbiotic formulation technologies, providing stable experimental materials for basic scientific research; and provide process optimization testing raw materials for scientific reagent manufacturers, without involving the production of end-use medical / health products. Specifically, it contains at least one of the following: *Lactobacillus rhamnosus* LRa05 (CGMCC NO. 24377), *Lactobacillus acidophilus* La88 (CGMCC NO. 24109), and *Lactobacillus plantarum* Lp90 (CGMCC NO. 10453). The preparation method is specifically the same as the preparation method of the active ingredients in the probiotic formulations provided in the above embodiments, and the excipients are also prepared in accordance with the above-mentioned excipient preparation method for probiotic formulations, which will not be elaborated here.

[0149] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. A probiotic preparation comprising at least one of the following: a) to c) a): A mixture of at least one or more of the following: dead cells, live cells, inactivated cells, and metabiotics of Lactobacillus rhamnosus LRa05 with accession number CGMCC NO.24377; b): A mixture of at least one or more of the following: dead cells, live cells, inactivated cells, and metabiotics of *Lactobacillus acidophilus* La88 with accession number CGMCC NO.24109; and c): A mixture of at least one or more of the following: dead cells, live cells, inactivated cells, and metabiotics of Lactobacillus plantarum Lp90 with accession number CGMCC NO.10453.

2. The probiotic preparation according to claim 1 is a regulatory preparation for vaginal mucosal barrier proteins, wherein the vaginal mucosal barrier protein is at least one of ZO-1, OCLN, and CLDN1.

3. The probiotic preparation according to claim 1 is a preparation containing an antibacterial agent against Alcaligenes spp.

4. The probiotic preparation according to claim 1 is a preparation containing an antibacterial agent against Gardnerella vaginalis.

5. The probiotic preparation according to claim 1 is a preparation for reducing the abundance of Proteobacteria in the gut and / or increasing the abundance of Firmicutes.

6. The probiotic preparation according to claim 1 is an inactivated bacterial agent for regulating the ratio of Proteobacteria / Firmwallia in an in vitro intestinal flora simulation system.

7. The probiotic preparation according to claim 1 is a preparation for reducing the abundance of Alcaligenes in the intestine.

8. The probiotic preparation according to claim 1 is a preparation that increases the abundance of Streptococcus spp. in the intestine.

9. The probiotic preparation according to claim 1 is a preparation for reducing the activity of inflammatory factors and MPO in vaginal tissue lysate.

10. The application of at least one of the following: *Lactobacillus rhamnosus* LRa05 with accession number CGMCC NO.24377, *Lactobacillus acidophilus* La88 with accession number CGMCC NO.24109, and *Lactobacillus plantarum* Lp90 with accession number CGMCC NO.10453, wherein the application includes: Preparation of a modulatory agent for vaginal mucosal barrier proteins; Prepare an antibacterial agent containing an inhibitory agent against Alcaligenes spp.; Prepare an antibacterial agent containing Gardnerella vaginalis; Preparations of formulations that reduce the abundance of Proteobacteria in the gut and / or increase the abundance of Firmicutes; Prepare inactivated bacterial cell reagents for regulating the Proteobacterium / Firmwallia ratio in an in vitro gut microbiota simulation system; Preparation of formulations to reduce the abundance of Alcaligenes in the gut; Preparation of formulations that increase the abundance of Streptococcus spp. in the gut; Preparations were made to reduce the activity of inflammatory factors and MPO in vaginal tissue lysate.

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