A tri-microbial complex of postbiotic agent for efficiently inhibiting candida albicans and application thereof

Abstract

The application discloses a kind of high-efficiency Candida albicans inhibiting three-microbe complex postbiotic microbial inoculum and application thereof, the three-microbe complex postbiotic microbial inoculum includes inactivated lactobacillus rhamnosus LR-G08, inactivated lactobacillus rhamnosus LR-G18 and inactivated lactobacillus plantarum LP-G21.The three-microbe complex postbiotic microbial inoculum can effectively inhibit Candida albicans and promote the proliferation of vaginal beneficial bacteria, regulate vaginal microecological balance.Compared with the two-microbe complex postbiotic microbial inoculum (LA88+N13) with better effect in the prior art, the inhibition rate is significantly improved, and the effect of nearly complete inhibition of Candida albicans is achieved.Meanwhile, the microbial inoculum maintains excellent broad-spectrum antibacterial and beneficial bacteria proliferation promoting ability.The microbial inoculum of the application is safe and stable, and is suitable for preparing sanitary napkins, pads, lotions and other gynecological care products, providing a better microecological solution for the prevention and auxiliary conditioning of candidal vaginitis.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and in particular relates to a highly effective three-strain compound postbiotic agent for inhibiting Candida albicans and its application. Background Technology

[0002] Vaginal microecological balance is crucial for women's reproductive health. Candida albicans is the main pathogenic fungus causing vulvovaginal candidiasis (VVC), which has a high infection rate and is prone to recurrence. Currently, in addition to the direct use of antifungal drugs, the prevention and adjunctive treatment of VVC through microbial regulation has become an important research direction. Metabiotics (inactivated probiotics and their metabolites) have become a research hotspot in this field due to their high safety and stability.

[0003] There are already successful solutions in the prior art that use specific combinations of inactivated lactobacilli to regulate the vaginal microecology. For example, Chinese patent CN202411604654 discloses a compound postbiotic agent composed of inactivated Lactobacillus acidophilus LA88 (CGMCC No. 24109) and inactivated Lactobacillus plantarum N13 (CGMCC No. 20496). This combination of two bacteria has shown good regulatory effects on a variety of vaginal pathogens and beneficial bacteria, representing the current effective technology in this field.

[0004] However, the inventors noted that Candida albicans, as a eukaryotic microorganism, has a different cell structure and drug resistance mechanism than bacteria, posing a challenge in microecological regulation. Through repeated experiments, the inventors found that although the aforementioned existing technical solution (LA88+N13 combination) already possesses good overall efficacy, its inhibition rate against Candida albicans is approximately 84.7% (see Comparative Example 3.1). For achieving near-complete inhibition, especially for highly effective prevention of VVC at the metabiotic level, its inhibitory efficacy still has significant room for improvement. To further optimize the preventive effect against VVC, there is an urgent need in the field for a novel composition that, while maintaining the safety and stability advantages of metabiotics, can specifically and significantly enhance the efficacy against Candida albicans. Summary of the Invention

[0005] Purpose of the invention: In view of the shortcomings of existing two-strain compound postbiotic agents which have good effects but still can enhance the inhibitory effect on Candida albicans, this invention provides a highly efficient three-strain compound postbiotic agent for inhibiting Candida albicans and its application.

[0006] Technical solution: To achieve the above-mentioned objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a three-strain compound postbiotic agent, wherein the three-strain compound postbiotic agent comprises inactivated Lactobacillus rhamnosus LR-G08, inactivated Lactobacillus reuteri LR-G18 and inactivated Lactobacillus plantarum LP-G21. The Lactobacillus rhamnosus LR-G08 is deposited at the China Center for Type Culture Collection (CCTCC) on June 9, 2021, with accession number CCTCC NO:M2021712. The Lactobacillus reuteri LR-G18 is deposited at the China Center for Type Culture Collection on November 14, 2022, with accession number CCTCC NO:M20221800. The *Lactobacillus plantarum* LP-G21 was deposited at the China Center for Type Culture Collection on June 9, 2021, with accession number CCTCC NO:M2021711.

[0007] As a specific implementation scheme, in the three-strain compound probiotic agent, based on the number of inactivated cells, the ratio of inactivated Lactobacillus rhamnosus LR-G08, inactivated Lactobacillus reuteri LR-G18, and inactivated Lactobacillus plantarum LP-G21 is (1-3):(0.2-1.8):(0.2-1.8); preferably, the ratio is 2:1:1.

[0008] As a specific implementation scheme, the compound microbial agent also includes fructooligosaccharides, and the amount of fructooligosaccharides added is 1%-10% of the total mass of the microbial agent; preferably, the amount of fructooligosaccharides added is 5% of the total mass of the microbial agent.

[0009] Secondly, the present invention provides a method for preparing the aforementioned three-strain compound post-biotic agent, comprising the following steps: (1) Lactobacillus rhamnosus LR-G08, Lactobacillus reuteri LR-G18 and Lactobacillus plantarum LP-G21 were fermented and cultured to obtain high-density fermentation broth. The fermentation broth was centrifuged and the mycelial sludge of each strain was collected. (2) Mix the bacterial sludge obtained in step (1) with an aqueous solution containing a protectant to form an emulsion; (3) The emulsion obtained in step (2) is subjected to heat inactivation treatment, and then the sterilized additive is added to the material and mixed evenly. (4) Freeze-dry the mixture obtained in step (3) to obtain the three-strain compound postbiotic agent.

[0010] As a specific implementation plan, in step (1), the fermentation culture method includes the following steps: Lactobacillus rhamnosus LR-G08, Lactobacillus reuteri LR-G18, and Lactobacillus plantarum LP-G21 were inoculated into MRS liquid medium and activated at 35-39℃ for 20-28 h. After several generations of activation, the activated bacterial solution was inoculated into fermentation medium at an inoculation rate of 4-6% and fermented at 35-39℃ for 10-12 h until the viable count of each strain reached 1×10⁻⁶ at the end of fermentation. 9 A high-density fermentation broth is obtained when the concentration of CFU / mL is above a certain level.

[0011] As a specific implementation plan, in step (2), the protective agent in the aqueous solution containing the protective agent is selected from trehalose, and its mass concentration is 5-20%; the volume ratio of the bacterial mud to the aqueous solution containing the protective agent is 1:(0.5-1.5).

[0012] As a specific implementation plan, in step (3), the conditions for the heat extinguishing treatment are: heat inactivation at 75-85℃ for 25-35 minutes; the additive is selected from skim milk powder and fructooligosaccharides, the amount of skim milk powder added is 1-10% of the total mass of the material after inactivation, and the amount of fructooligosaccharides added is 1-10% of the total mass of the material after inactivation.

[0013] Thirdly, the present invention provides the application of the aforementioned three-strain compound postbiotic agent in the preparation of antibacterial agents.

[0014] Fourthly, the present invention provides the application of the aforementioned three-strain compound postbiotic agent in the preparation of products for inhibiting Candida albicans.

[0015] Fifthly, the present invention provides the application of the aforementioned three-bacterial compound postbiotic agent in the preparation of products for promoting the proliferation of beneficial vaginal bacteria.

[0016] The products mentioned include sanitary napkins, panty liners, tampons, or vaginal washes.

[0017] Through extensive screening and experimentation, this invention has revealed that the three specific preserved bacterial strains, when combined with fructooligosaccharides in an inactivated form, exhibit an unexpected and profound synergistic effect in combating Candida albicans. This synergistic effect stems from the unique metabolic interactions among the three strains during co-culture (see Example 2.1), rather than a simple additive effect.

[0018] Preferably, the amount of fructooligosaccharides added is 5% of the total mass of the microbial agent. Under this condition, its synergistic effect against Candida albicans and its promotion of beneficial bacteria proliferation reach its peak.

[0019] It should be noted that the complete formulation containing fructooligosaccharides protected by this invention is not simply an adjunct to the core three-bacterial combination. As shown in the comparison between Examples 3.1 and 2.4, the introduction of fructooligosaccharides further increased the Candida albicans inhibition rate from 98.5% to 99.2% (see Examples 3.1 and 2.4), achieving a further significant optimization of the inhibitory effect. Simultaneously, as shown in Example 4, fructooligosaccharides and the three-bacterial combination produced a significant synergistic amplification effect in promoting the proliferation of beneficial vaginal bacteria (see Table 4-1). Therefore, the "inactivated three-bacterial combination + fructooligosaccharides" together constitute a complete technical solution that performs optimally and more stably in both inhibiting pathogenic bacteria and promoting beneficial bacteria, with an overall effect far exceeding the simple summation of its components. In a head-to-head comparison with the closest prior art (LA88+N13 combination), the three-strain combination of the present invention exhibits significant and unexpected technological advancements: under the same test conditions, the inhibition rate against Candida albicans is significantly increased from approximately 84.7% to 99.2% (see Example 3.1), an increase of approximately 14.5 percentage points, achieving near-complete inhibition.

[0020] This three-bacterial compound postbiotic agent achieves a breakthrough in antifungal activity while maintaining comprehensive microecological regulation: its inhibition rate against bacterial pathogens such as Staphylococcus aureus and Escherichia coli is greater than 95%, and it can significantly promote the proliferation of various beneficial vaginal bacteria (see Example 4). The addition of fructooligosaccharides produces a clear synergistic effect with the core three-bacterial combination of this invention.

[0021] Beneficial effects: Compared with the prior art, the present invention has the following advantages: 1. Solved a more focused technical problem: Based on the existing technology that has provided effective broad-spectrum regulation schemes, the present invention further focuses on and successfully solves the more challenging sub-problem of "how to achieve near-complete inhibition of Candida albicans at the metagenic level".

[0022] 2. Discovery of a specific strain combination with superior efficacy despite non-obvious findings: This invention discovered the unique and superior synergistic value of a specific three-strain combination of LR-G08, LR-G18, and LP-G21 in its antifungal activity against Candida albicans. Given that a specific two-strain combination (LA88+N13) is already known to be highly effective, screening and validating a three-strain combination that significantly surpasses this two-strain combination in key antifungal indicators requires creative effort and its effects are often unexpected.

[0023] 3. Provides a complete and optimized formula containing prebiotics: This invention further confirms that the combination of fructooligosaccharides and the above-mentioned specific three bacteria can produce synergistic effects, maximizing both antibacterial and growth-promoting effects, forming a stable, efficient and fully functional microecological regulation product.

[0024] 4. Significantly improved technical results have been achieved: The bacterial agent prepared in this invention has achieved an extremely high inhibition rate of 99.2% against Candida albicans, which is a qualitative improvement over the existing technical solutions and provides a significantly better implementation scheme for solving this technical problem.

[0025] 5. Provides a solution with a clear competitive advantage: While achieving a breakthrough in antifungal efficacy, this invention does not sacrifice other basic functions, providing the market with an "upgraded" option that performs significantly better in anti-Candida, with clear clinical application value and market prospects. Attached Figure Description

[0026] Figure 1 Gram staining results for Lactobacillus rhamnosus LR-G08.

[0027] Figure 2 Gram staining results for Lactobacillus reuteri LR-G18.

[0028] Figure 3 The results show the Gram staining of Lactobacillus plantarum LP-G21.

[0029] Figure 4 To compare the inhibition rates of different samples against Candida albicans.

[0030] Figure 5 This invention compares the rate at which the three-bacterial compound of the present invention promotes the proliferation of beneficial vaginal bacteria with that of commercially available products. Detailed Implementation

[0031] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0032] The main reagents, strains, and materials used in the embodiments of this invention are as follows: The experimental strains included Candida albicans (ATCC 10231); and standard strains of beneficial vaginal bacteria, such as Lactobacillus crispatus (ATCC 33820), Lactobacillus gasseri (ATCC 33323), and Lactobacillus jensenii (ATCC 25258), all purchased from the American Type Culture Collection (ATCC). The main culture media and reagents included: Sabouraud dextrose agar (SDA), Sabouraud dextrose broth (SDB), and MRS medium (purchased from Beijing Luqiao Technology Co., Ltd.); RPMI-1640 medium (catalog number: 31800022) and MOPS buffer (purchased from Thermo Fisher Scientific); and fluconazole (10 mM in DMSO, catalog number: T92598, purchased from Shanghai Yuanye Biotechnology Co., Ltd.). All other reagents were of analytical grade or met experimental requirements.

[0033] It should be noted that the fluconazole solution mentioned above is a commercial non-sterile product, and it was filtered through a 0.22μm sterile microporous membrane before being used in antimicrobial or co-culture experiments. All experimental operations involving microbial activation, subculturing, co-culture, and drug susceptibility testing were performed in a biosafety cabinet of the appropriate level to ensure the sterility of the experimental system and compliance with biosafety regulations.

[0034] The "commercially available reference product" mentioned in this invention specifically refers to commercially available postbiotic tablets purchased from the open market, with inactivated lactic acid bacteria (main strains including Lactobacillus acidophilus, Lactobacillus curvatureus, and Lactobacillus rhamnosus as the main active ingredient) and clearly labeled for use in female intimate care. The product label indicates a pre-inactivation live bacteria count of not less than 1 × 10⁻⁶. 10 CFU / g.

[0035] In the comparative experiment, to ensure a fair comparison, the product of this invention and the commercially available control were added to the test system at the same mass percentage.

[0036] The *Lactobacillus rhamnosus* LR-G08, *Lactobacillus reuteri* LR-G18, and *Lactobacillus plantarum* LP-G21 used in the following embodiments of the present invention were isolated and identified as follows: 1. Strain Isolation (1) Lactobacillus rhamnosus LR-G08 and Lactobacillus reuteri LR-G18: These strains were isolated from vaginal secretion samples from healthy adult female volunteers. After sampling with sterile cotton swabs, the samples were enriched in MRS liquid medium, serially diluted, and then plated onto MRS agar plates. The plates were incubated anaerobically at 37°C for 48-72 hours. Typical single colonies were picked and repeatedly streaked for purification until pure cultures were obtained.

[0037] (2) Lactobacillus plantarum LP-G21: The strain was isolated from a traditional fermented mare's milk sample from Inner Mongolia. The sample suspension was appropriately diluted and spread on MRS agar plates, and cultured under anaerobic conditions at 37℃ for 48-72 hours. Single colony isolation and purification were carried out using the same method as above.

[0038] 2. Strain preservation The purified single colonies were inoculated into MRS liquid medium and anaerobically cultured at 37°C until the late logarithmic growth phase. The bacterial suspension was mixed with an equal volume of 50% sterile glycerol and stored long-term in an ultra-low temperature freezer at -80°C. Simultaneously, MRS slants were prepared and stored for short-term storage at 4°C.

[0039] 3. Strain identification (1) Preliminary morphological and physiological biochemical identification All three isolates formed milky-white, round, smooth, opaque colonies on MRS plates. Gram staining revealed them to be Gram-positive bacilli, non-spore-forming, and arranged singly, in pairs, or in short chains. All isolates were negative for catalase, consistent with the basic characteristics of the Lactobacillus genus.

[0040] Gram staining results of Lactobacillus rhamnosus LR-G08 are as follows: Figure 1 As shown; Gram staining results of *Lactobacillus reuteri* LR-G18 are as follows. Figure 2 As shown; Gram staining results of Lactobacillus plantarum LP-G21 are as follows. Figure 3 As shown.

[0041] (2) Molecular biological identification (16S rDNA sequence analysis) Genomic DNA was extracted from each bacterial strain using a bacterial genomic DNA extraction kit. The 16S rDNA gene was amplified using universal bacterial primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). After purification, the PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for bidirectional sequencing. The sequencing peaks were proofread and assembled using Chromas software, yielding a complete gene sequence of approximately 1450-1500 bp.

[0042] The 16S rDNA sequence of *Lactobacillus rhamnosus* LR-G08 is shown in SEQ ID NO.1; the 16S rDNA sequence of *Lactobacillus reuteri* LR-G18 is shown in SEQ ID NO.2; and the 16S rDNA sequence of *Lactobacillus plantarum* LP-G21 is shown in SEQ ID NO.3.

[0043] The obtained sequences were submitted to the National Center for Biotechnology Information (NCBI) database. Homology comparisons were performed on the "16S ribosomal RNA sequences (Bacteria and Archaea)" database using the BLASTN program, and a phylogenetic tree was constructed using the Neighbor-Joining method to confirm their phylogenetic position.

[0044] (3) Result of the appraisal Lactobacillus rhamnosus LR-G08: Its 16S rDNA sequence shows a similarity of over 99.86% with multiple strains of Lactaseibacillus rhamnosus (such as SDCM 1101, accession number MW159695.1), and a similarity of over 99.8% with multiple strains of this species. Based on international bacterial systematics (16S rDNA sequence homology >98.7% can be classified as the same species), and combined with its morphological and biochemical characteristics, it is identified as Lactobacillus rhamnosus.

[0045] *Limosilactobacillus reuteri* LR-G18: Its 16S rDNA sequence shows 99.86% similarity to the type strain DSM 20016 (accession number: NR_075036.1) of *Limosilactobacillus reuteri*. Phylogenetic analysis showed that it clustered with *L. reuteri*. Based on its physiological characteristics, it was identified as *Limosilactobacillus reuteri*.

[0046] Lactiplantibacillus plantarum LP-G21: Its 16S rDNA sequence showed 100.00% similarity to the comparable region of the type strain CIP 103151 (accession number: NR_104573.1) of Lactiplantibacillus plantarum, and its similarity to several standard strains of this species (such as NBRC 15891, JCM 1149, etc.) was higher than 99.9%. Based on this, and combined with its morphological and biochemical characteristics, it was identified as Lactiplantibacillus plantarum.

[0047] The above molecular biological identification results, together with the physiological and biochemical characteristics of the strains and their preservation information at the China Center for Type Culture Collection (CCTCC), clarify the taxonomic status of the three strains used in this invention.

[0048] (4) Preservation of microbial strains To meet patent law requirements and provide public access, the above three strains have been deposited at the China Center for Type Culture Collection (CCTCC), and the deposit information is as follows: Lactaseibacillus rhamnosus LR-G08: Deposit date: June 9, 2021, accession number CCTCC NO: M2021712 Limosilactobacillus reuteri LR-G18: Deposit date: November 14, 2022, accession number: CCTCC NO: M20221800 Lactiplantibacillus plantarum LP-G21: Deposit date: June 9, 2021, accession number: CCTCC NO: M2021711 The deposit address is Wuhan University, Wuhan City, Hubei Province, People's Republic of China. The preservation period for the above-mentioned strains is 30 years, starting from the date of deposit.

[0049] Example 1: Preparation of a three-strain compound probiotic agent (1) High-density fermentation: Lactobacillus rhamnosus LR-G08, Lactobacillus reuteri LR-G18, and Lactobacillus plantarum LP-G21 were inoculated into MRS liquid medium and activated at 37°C for 24 h, with two generations of activation. The activated bacterial cultures were then inoculated into MRS liquid fermentation medium at a 5% inoculum size and fermented at 37°C for 10–12 h. Fermentation was optimized to ensure that the viable count of each strain reached 1 × 10⁻⁶ at the end of fermentation. 9 A high-density fermentation broth was obtained with a CFU / mL concentration of above 1.

[0050] (2) Bacterial cell collection: The above fermentation broth was centrifuged at 4℃ and 8000 rpm for 15 min, the supernatant was discarded, and the lower layer of bacterial sludge was collected.

[0051] (3) Mixing of fungal sludge and primary protection: Mix the bacterial sludge of LR-G08, LR-G18, and LP-G21 at a bacterial count ratio of 2:1:1. Add an aqueous solution containing 15% trehalose (the volume ratio of bacterial sludge to trehalose solution is 1:1) to the mixed bacterial sludge, and vortex to mix evenly to form a primary emulsion.

[0052] (4) Thermal inactivation and efficacy verification: The primary emulsion was maintained in an 80°C water bath for 30 minutes to fully inactivate the bacteria, and then rapidly cooled to room temperature. This heat inactivation condition (80°C, 30 minutes) has been verified to completely inactivate the three strains of Lactobacillus (see the aseptic verification results of this step), while the mild conditions help maintain the relative integrity of the inactivated bacterial structure, thereby preserving their key physical and functional properties as metabiotics.

[0053] To confirm the effectiveness of the heat inactivation process, the sterility of the inactivated emulsion was tested. The specific steps are as follows: 1. Sample taking: Under aseptic conditions, take 1.0 mL of the emulsion that has been treated at 80°C for 30 minutes and has been cooled.

[0054] 2. Plate spreading: Spread the obtained sample evenly on 3 MRS agar plates (approximately 0.3 mL on each plate).

[0055] 3. Enrichment culture: Meanwhile, another 1.0 mL of inactivated emulsion was inoculated into 10 mL of fresh MRS liquid medium.

[0056] 4. Cultivation conditions: The coated plates were placed upside down in a 37°C incubator and incubated for 72 hours (to ensure thorough sterility verification).

[0057] The inoculated liquid culture medium tubes were placed in a 37°C constant temperature incubator and incubated for 7 days.

[0058] 5. Result Observation and Judgment: After incubation, observe all MRS agar plates. If no visible colonies grow on any of the plates, they are considered sterile.

[0059] Observe the MRS liquid culture medium test tubes. If the culture medium remains clear, without turbidity, precipitation, or biofilm formation, then it can be further determined that there is no viable bacterial growth.

[0060] In this embodiment, after the above tests, no colonies grew on all plates and the liquid culture medium remained clear, proving that the heat treatment conditions of 80°C for 30 minutes can completely inactivate the strain and meet the requirements for postbiotic production.

[0061] (5) Addition of functional components, freeze-drying protectant, and quality control after inactivation: Food-grade skim milk powder and fructooligosaccharides (prebiotics) that have been sterilized by Co-60 γ-ray irradiation (irradiation dose of 8 kGy) are added sequentially to the cooled inactivated emulsion. The mixture is stirred until completely dissolved and homogeneous to obtain the final suspension for freeze-drying.

[0062] Before adding the skim milk powder and fructooligosaccharides (prebiotics), the following standards must be met to ensure the safety and stability of the final product: 1. Microbiological indicators: Must be tested and meet food-grade raw material standards. Specifically, the total bacterial count of skim milk powder should be ≤ 10,000 CFU / g, and the mold and yeast count should be ≤ 100 CFU / g. Pathogenic bacteria such as Staphylococcus aureus, Salmonella, and coliform bacteria must not be detected.

[0063] 2. Physicochemical indicators: Skim milk powder should be prepared by spray drying process, with a moisture content ≤ 5.0%, good solubility, no clumping, and no off-odor.

[0064] 3. Processing Method: To prevent the introduction of exogenous bacteria and ensure the microbiological safety of the final product, the skim milk powder and fructooligosaccharides must be sterilized before addition. In this embodiment, sterilization is performed using Co-60 gamma irradiation at a dose of 8 kGy. This dose is set according to GB 18524-2016 National Food Safety Standard for Hygienic Specifications of Food Irradiation Processing, and is sufficient to ensure that the food-grade skim milk powder and fructooligosaccharides meet the microbiological safety level required by this production process. The treated excipients should be stored in a sterile environment.

[0065] 4. Amount added: In this embodiment, the amount of skim milk powder added is 5% of the total mass of the inactivated material, and the amount of fructooligosaccharide added is 5% of the total mass of the inactivated material.

[0066] (6) Freeze-drying: The final suspension was freeze-dried.

[0067] Pre-freezing stage: Freeze at -40℃ for 4 hours.

[0068] Main drying stage: The shelf temperature is raised to 20℃, the vacuum degree is maintained below 10 Pa, and drying is carried out until the moisture content is below 5% (the process parameters of freeze drying can be adjusted appropriately according to the equipment and material quantity, with the final product moisture content being below 5%). A loose and porous composite postbiotic powder is obtained.

[0069] (7) Crushing, sieving, and determination of total bacterial count: The freeze-dried bacterial powder was pulverized using a grinder and passed through an 80-mesh sieve to obtain a fine powder with good flowability. The total bacterial count was determined using a hemocytometer: an appropriate amount of bacterial powder was accurately weighed, appropriately diluted with sterile physiological saline, and fully dispersed. The sample was added dropwise to the counting chamber of the hemocytometer, and the counts were performed under a microscope. The total bacterial count was calculated based on the dilution factor. The determined total bacterial count was 1.25 × 10⁻⁶. 11 ±0.05×10 11 Cell / g, meeting the requirements of this invention. Example 2: Discovery of synergistic effect and optimal ratio against Candida albicans. 2.1 Pilot Experiment: Preliminary Screening of Potential Strain Combinations Objective: To provide data for screening strain combinations that have the potential to inhibit Candida albicans.

[0070] Method (Fermentation supernatant mixing - Oxford cup method): 1. Strains and Culture: Ten strains of Lactobacillus (including LR-G08, LR-G18, and LP-G21) preserved in the laboratory were selected. Each strain was inoculated into MRS liquid medium and cultured anaerobically at 37°C for 24 h.

[0071] 2. Preparation of supernatant: The culture medium was centrifuged at 10,000×g for 15 min at 4℃. The supernatant was filtered through a 0.22 μm filter membrane to remove bacteria, thus obtaining cell-free fermentation supernatant of single bacteria.

[0072] 3. Combination and Mixing: The cell-free fermentation supernatants of single bacteria were physically mixed at equal volume ratios (1:1 or 1:1:1) to simulate different compound combinations. The test groups included: 10 cell-free fermentation supernatants of single bacteria, all possible pairwise combinations (45 groups), and 20 possible triadic combinations selected based on previous literature (including cell-free fermentation supernatants mixtures of LR-G08, LR-G18, and LP-G21).

[0073] 4. Antibacterial Activity Assay: a. Indicator Bacterial Preparation: Candida albicans (ATCC 10231) was inoculated onto SDA slant and incubated at 28°C for 48 h for activation. Single colonies were picked and inoculated into Sabouraud broth (SDB) and cultured at 28°C with shaking at 200 rpm for 16-18 hours until the late logarithmic growth phase, obtaining a homogeneous liquid seed culture. The culture was centrifuged (3000×g, 5 min) and washed once with sterile physiological saline to remove culture medium components. Finally, the bacterial cells were resuspended in sterile physiological saline, and the total cell concentration was directly counted under a microscope using a hemocytometer. Since the strain used was in the vigorous logarithmic growth phase and had extremely high cell viability, this total cell concentration was approximately equivalent to the colony forming unit (CFU) concentration. Based on this, the bacterial suspension concentration was adjusted to approximately 1×10⁻⁶. 6Cells / mL (approximately CFU / mL) was used as the working bacterial suspension for the Oxford cup method test.

[0074] b. Preparation of bacterial plates: The double-layer agar diffusion method was used. First, approximately 15-20 mL of Sabouraud dextrose solid medium containing 1.5%-2.0% agar was poured as the bottom layer and allowed to solidify. Then, 100 μL of a working suspension of *Candida albicans* (approximately 1 × 10⁻⁶) was taken. 6 Mix thoroughly with 4-5 mL of Sabouraud dextrose semi-solid medium (cooled to 45-50℃) containing 0.7%-0.8% agar, quickly pour onto the bottom layer, gently shake to form a uniform top layer containing bacteria, and let stand to solidify.

[0075] c. Sample addition: Place sterile Oxford cups (7.8 mm outer diameter) at equal intervals on the solidification plate, and accurately add 200 μL of the mixed supernatant to be tested into each Oxford cup using a pipette.

[0076] d. Incubation and Measurement: Plates were placed horizontally in a 4°C refrigerator for 2 hours to allow for sufficient sample diffusion. They were then transferred to a 28°C incubator and incubated upright for 24-48 hours, until the bacterial growth in the negative control area was dense and the inhibition zone was clear and sharp (usually 24-48 hours). The diameter of the inhibition zone (including Oxford cups) was measured using calipers, with three replicates for each sample.

[0077] result: Among the 20 three-strain combinations tested, the sample consisting of a 1:1:1 mixture of the supernatants of LR-G08, LR-G18, and LP-G21 had the largest average inhibition zone diameter, at (21.8 ± 0.6) mm. This result was significantly better than most other three-strain combinations (with average inhibition zones mostly between 18-20 mm) and all single-strain and pairwise combinations.

[0078] Conclusion: The metabolites of LR-G08, LR-G18, and LP-G21 strains showed significant inhibitory potential against Candida albicans when mixed; therefore, this combination was selected for further synergistic mechanism research. 2.2 Verification of Synergistic Antibacterial Effect Objective: To verify that the superior effects of the above potential combinations stem from the symbiotic synergy of strains, rather than the simple superposition of metabolites.

[0079] Methods (co-culture vs. mixed supernatant comparison method): 1. Sample preparation: Co-culture group: LR-G08, LR-G18 and LP-G21 were simultaneously inoculated into fresh MRS liquid medium at a 1:1:1 initial viable count ratio and conventional inoculation amount, and then anaerobically cultured at 37°C for 24 h.

[0080] Mixed control group: The three bacteria were cultured separately under the same conditions for 24 h.

[0081] 2. Supernatant treatment: After the culture is completed, all culture media are centrifuged at 10,000×g for 15 min at 4℃, and the supernatant is filtered through a 0.22 μm filter membrane for sterilization.

[0082] 3. Control Setup: The supernatants of the three single-strain cellless fermentation were mixed at a volume ratio of 1:1:1 to serve as the "mixed supernatant" control. Simultaneously, the cellless fermentation of *Lactobacillus plantarum* LP-G21, which exhibited the best antibacterial activity in Example 2.1, was selected as the "optimal single-strain supernatant reference".

[0083] 4. Antibacterial activity assay: The Oxford cup method, identical to that in 2.1, was used to determine the diameter of the inhibition zone against Candida albicans in the co-culture supernatant and supernatants under different treatments. Sterile MRS medium was used as a negative control.

[0084] result: Table 2-1 Antibacterial effect of co-culture and different treatment supernatants on Candida albicans *Note: The Oxford cup method was used in this experiment. All supernatant samples were cell-free fermentation supernatants obtained by centrifugation and sterilization via 0.22 μm filtration. The inhibition zone diameter of the negative control (sterile MRS medium) was 0 mm. All data are parallel results from the same experiment (mean ± SD, n=3) for internal comparison.

[0085]

[0086] Conclusion: The antibacterial activity of the co-culture supernatant was significantly higher than that of the mixed supernatant and the optimal single-strain supernatant reference (P<0.05). This demonstrates that LR-G08, LR-G18, and LP-G21 produced a true synergistic antibacterial effect through metabolic interactions during co-growth, which was superior to simple physical mixing of metabolites and significantly better than that of a single strain. 2.3 Exploration of the optimal synergistic ratio Based on the results of Examples 2.1 and 2.2, LR-G08, LR-G18, and LP-G21 were selected as core combinations with synergistic potential. To determine the precise cell count ratio for maximizing the synergistic effect of this combination, the following gradient ratio experiment was conducted.

[0087] Objective: To determine the inoculation ratio of strains that produce the strongest synergistic antibacterial effect.

[0088] Method (gradient ratio co-cultivation method): Based on the results in section 2.2, a proportional gradient experiment was designed. LR-G08, LR-G18, and LP-G21 were simultaneously inoculated into MRS medium at different initial viable cell ratios (1:1:1, 2:1:1, 1:2:1, 1:1:2) for co-culture, maintaining a consistent total initial inoculum. After 24 h of culture, the co-culture supernatant for each ratio was prepared according to the method in section 2.2.

[0089] The diameter of the inhibition zone against Candida albicans in the supernatant of each co-culture ratio was systematically determined using the Oxford cup method (same as 2.1).

[0090] result: Table 2-2 Antibacterial effect of co-culture supernatant with different inoculation ratios on Candida albicans *Note: This experiment used the same Oxford cup method as in Example 2.1. The inhibition zone diameter of the negative control (sterile MRS medium) was 0 mm. All data are parallel determinations from the same experiment and are used for internal ratio comparisons.

[0091]

[0092] Conclusion: The synergistic effect exhibits a significant ratio-dependent characteristic. The co-culture supernatant showed the strongest antibacterial activity when the inoculation ratio was 2:1:1. Therefore, 2:1:1 was determined to be the optimal ratio for maximizing the synergistic potential.

[0093] 2.4 Preliminary Verification of the Efficacy of Postbiotics Based on Optimal Ratio Objective: To apply the optimal synergistic ratio (2:1:1) determined in Example 2.3 to postbiotics (inactivated bacterial cells), and to preliminarily compare the efficacy differences of this three-strain compound postbiotic (excluding prebiotics) with individual single-strain postbiotics and two-strain compound postbiotics through in vitro antibacterial experiments. This experiment aims to eliminate interference from other components such as prebiotics and to verify whether the specific three-strain inactivated bacterial cell combination (LR-G08, LR-G18, LP-G21, 2:1:1) itself constitutes the core basis for producing excellent antibacterial effects, providing a control basis for subsequent research on its complete synergistic system with prebiotics (such as fructooligosaccharides) (such as the product in Example 1).

[0094] Method (Liquid Co-culture - Antibacterial Rate Method) 1. Preparation of samples for metagenicity testing: The three-strain compound post-biotic (optimal ratio group): Accurately weigh the intermediate product prepared according to the method of Example 1 up to step (4) (after heat inactivation), but without the addition of skim milk powder and fructooligosaccharides. Then, use the same freeze-drying process to obtain inactivated *Lactobacillus rhamnosus* LR-G08 powder, inactivated *Lactobacillus reuteri* LR-G18 powder, and inactivated *Lactobacillus plantarum* LP-G21 powder. This step ensures that the test sample contains only the inactivated three strains, excluding the possible influence of other functional components (such as fructooligosaccharides). Grind or vortex mix thoroughly in a sterile mortar or centrifuge tube at a 2:1:1 dry weight ratio (e.g., weigh 40.0 mg LR-G08 powder, 20.0 mg LR-G18 powder, and 20.0 mg LP-G21 powder) to obtain the core three-strain compound post-biotic sample of this invention, and place it in a desiccator for later use. (Note: The determination of the bacterial dry weight ratio is based on the premise that the number of cells per unit mass in the post-biotic powder of each single strain is basically similar. See the determination of the total number of bacteria in step (7) of Example 1. Therefore, this dry weight ratio corresponds to the cell number ratio of the inactivated bacteria.) Single-strain postbiotic control group: One portion of each of the above-mentioned single-strain postbiotic powders of LR-G08, LR-G18, and LP-G21 were weighed as controls.

[0095] Two-strain compound biogenic control group: LR-G08 and LR-G18 bacterial powder, LR-G08 and LP-G21 bacterial powder, and LR-G18 and LP-G21 bacterial powder were accurately weighed and mixed evenly at a bacterial dry weight ratio of 1:1 to prepare three two-strain compound biogenic control group samples.

[0096] 2. Sample solution preparation: Accurately weigh 20.0 mg of each of the above-mentioned metabiotic samples and place them in separate sterile 15 mL centrifuge tubes. Add 1.0 mL of sterile phosphate-buffered saline (PBS, 0.01 M, pH 7.2) to each centrifuge tube. Vortex immediately for 3 min, then incubate at room temperature for 5 min to allow the cells to fully hydrate. Vortex again for 2 min to ensure a homogeneous and stable suspension is formed. This is the sample stock solution with a concentration of 20 mg / mL.

[0097] 3. Preparation of indicator bacterial suspension: A liquid seed culture of *Candida albicans* (ATCC 10231) was prepared according to the method described in Example 2.1. 1 mL of the culture was centrifuged at 3000 × g for 5 min in a sterile centrifuge tube, and the supernatant was discarded. The bacterial pellet was gently washed twice with 1 mL of sterile PBS to remove culture medium components. Finally, the bacterial suspension was resuspended in an appropriate amount of RPMI-1640 medium (adjusted to pH 7.0 with MOPS buffer before use), and the concentration of the *Candida albicans* suspension was calibrated to 1 × 10⁻⁶ using a hemocytometer. 6 Cells / mL.

[0098] 4. Establishment of the co-culture reaction system: In a sterile 48-well cell culture plate, add samples in the following order and volume to establish a standardized reaction system with a total volume of 1 mL / well: Step 1: Add 945 μL of RPMI-1640 medium (pH 7.0) to each well.

[0099] Step 2: Add 50 μL of the prepared 20 mg / mL post-biotic sample stock solution. (This step brings the final concentration of the post-biotic in the well to 1 mg / mL.) Step 3: Add 5 μL of a solution with a concentration of 1×10 6 Cells / mL of working Candida albicans suspension. (This step brings the final concentration of Candida albicans in the wells to 5 × 10⁻⁶.) 3 Cells / mL Blank growth control well setup: Replace the sample stock solution with 50 μL sterile PBS, and keep the other components the same.

[0100] Experimental design: For each type of postbiotic sample (7 types in total: 3 single bacteria + 3 two bacteria + 1 three bacteria) and blank control, 6 independent biological replicate wells (n=6) were set up and randomly distributed in the culture plate to eliminate the position effect.

[0101] 5. Co-cultivation and Influence: After adding the sample, seal the edges of the culture plate with a sterile, breathable sealing film to prevent evaporation and contamination. Place the culture plate stably in a 37°C constant temperature shaker, set the rotation speed to 150 rpm, and start the timer for 6 hours of incubation.

[0102] 6. Colony count: a. Termination and Dilution: Immediately after 6 h of incubation, aspirate 100 μL of the reaction solution from each well and add it to a prepared 900 μL sterile PBS dilution tube containing 0.1% Tween-80. Vortex to mix. This is 10... -1 Dilution rate.

[0103] b. Series dilution and coating: Apply 10... -1 The diluent is then subjected to further serial dilutions of 10-fold (e.g., 10...). -2 , 10 -3 Take 100 μL from each dilution to be counted and spread it evenly on a pre-labeled SDA plate.

[0104] c. Incubation and Counting: Place all spread plates in a 28°C incubator and invert them for 48 hours. After incubation, count the colonies on plates with 30-300 colonies. Record the colony count for each plate.

[0105] 7. Calculation of antibacterial rate: a. Calculate the viable bacterial concentration (unit: CFU / mL) in each well of the reaction solution based on the plate count results and the corresponding dilution factor.

[0106] b. Calculate the average viable bacterial concentration in 6 replicate wells for each metagenetic sample.

[0107] c. Calculate the inhibition rate of each sample against Candida albicans using the following formula: Inhibition rate (%) = [(average colony count in blank control group - average colony count in experimental group) / average colony count in blank control group] × 100%.

[0108] 8. Statistical Analysis: Experimental data are expressed as mean ± standard deviation. One-way ANOVA was performed on the antibacterial rates of all sample groups using statistical software (such as GraphPad Prism). If the ANOVA results showed significant differences between groups (P < 0.05), Tukey's HSD multiple comparison test was further used for pairwise comparisons to determine the significance level of the differences between the samples of this invention and each control group. Results: Table 2-3 Inhibition rate (%) of different metabiotic samples against Candida albicans after 6 h of treatment and a final concentration of 1 mg / mL

[0109] in conclusion: Statistical analysis showed that the antibacterial rate (98.5%) of the core three-strain combination post-biotic (2:1:1, without prebiotics) of this invention was significantly higher (P<0.01) than that of all single-strain post-biotic control groups and any two-strain combination post-biotic control groups. Among them, the best-performing two-strain combination group (inactivated LR-G18+LP-G21) had an antibacterial rate of 88.9%, which was improved by approximately 9.6 percentage points compared to the core three-strain combination of this invention.

[0110] The results clearly demonstrate that the combination of LR-G08, LR-G18, and LP-G21 in a 2:1:1 ratio constitutes a core unit with extremely strong anti-Candida albicans activity, significantly exceeding the activity of a single bacterium or the simple superposition of any two bacteria. This proves that the "specific three-bacterial combination" is a necessary basis for generating a synergistic antibacterial effect. This provides a solid premise and control for subsequently adding prebiotics such as fructooligosaccharides to this core combination in Example 1 to construct a more complete and stable final product (see Examples 3 and 4), and highlights that even without fructooligosaccharides, this specific three-bacterial combination (2:1:1) has already achieved unexpected technical effects.

[0111] Example 3: Systematic evaluation of the core efficacy of compound postbiotics 3.1 Authoritative comparison of inhibitory effects against Candida albicans (including comparison with the closest existing technology) Objective: To systematically evaluate the inhibitory effect of the product of this invention on Candida albicans and to conduct an authoritative comparison with the closest prior art and other conventional controls.

[0112] Method (liquid co-culture-antibacterial rate method, same as Example 2.4): 1. Test Sample: Sample of the present invention: Tri-strain compound post-biotic powder (2:1:1, containing 5% fructooligosaccharides) prepared in Example 1.

[0113] Control samples: single-strain postbiotics including LR-G08, LR-G18, and LP-G21; postbiotics prepared by mixing these three strains in pairs at a 1:1 ratio; "LA88+N13" (1:1) compound postbiotic prepared strictly according to the example in CN202411604654; and a commercially available brand of postbiotic powder.

[0114] Positive control: Fluconazole solution (8 µg / mL, prepared with RPMI-1640 medium).

[0115] 2. Experimental Procedure: The liquid co-culture-antibacterial rate method, which is basically the same as that used in Example 2.4, was employed. The specific steps are as follows: a. Sample processing and bacterial suspension preparation: Preparation of stock solution (20 mg / mL), preparation of working suspension of Candida albicans (ATCC 10231) (1×10⁻⁶ mg / mL). 6 The method (Cells / mL) is the same as in Example 2.4.

[0116] b. Co-culture system: Establish the reaction system in a 48-well plate. Each well contains 1 mL of total volume, including: 945 μL RPMI-1640 medium, 50 μL stock solution (final concentration 1 mg / mL), and 5 μL bacterial suspension (final concentration 5 × 10⁻⁶). 3 (Cells / mL). Set up PBS control wells without samples.

[0117] c. Culture and detection: The well plates were incubated at 37°C with shaking at 80 rpm for 6 h. After the culture, the reaction solution in the wells was serially diluted, spread onto SDA plates, and incubated at 28°C for 48 h before colony counting.

[0118] d. Calculation: The formula for calculating the antibacterial rate is the same as in Example 2.4.

[0119] 3. Key differences explained: This experiment aims to conduct a comprehensive efficacy comparison, particularly by introducing a direct comparison with the closest existing technology (LA88+N13). The core difference between this experiment and Example 2.4 lies in the different test sample set, and the sample of this invention is the complete product of Example 1 (containing fructooligosaccharides). Key parameters such as the final concentration of metagenic agent (1 mg / mL), the final concentration of bacteria, and the reaction time (6 h) in the reaction system remain consistent with those of Example 2.4.

[0120] result: Table 3-1 Comparison of inhibition rates of different metabiotic samples against Candida albicans (%, after 6 h of action, final concentration: 1 mg / mL)

[0121] The inhibition rates of different samples against Candida albicans were compared. Figure 4 As shown.

[0122] Conclusions and Discussion: 1. The present invention has excellent and stable effects: Under standard test conditions, the three-strain compound postbiotic prepared by Example 1 showed an inhibition rate of up to 99.2% against Candida albicans with very small data variation (standard deviation 0.3%), demonstrating a near-complete and highly stable inhibition ability.

[0123] 2. Efficacy comparable to clinical standard drugs: The positive control fluconazole (8 µg / mL) showed an inhibition rate of 99.8% in the same system. This indicates that the metabiotic agent of this invention (1 mg / mL) achieved an inhibitory efficacy comparable to first-line clinical antifungal drugs in vitro, further confirming its excellent antifungal potential.

[0124] 3. Completely surpasses conventional controls: Statistical analysis confirms that the antibacterial effect of the product of this invention is significantly better (P<0.01) than all single-strain controls (70.8%-75.5%) and all two-strain combination controls based on the same strain (82.3%-88.9%), indicating that its advantage does not come from a simple increase in the number of strains.

[0125] 4. Key Technological Advancements: Compared to the closest existing technology (LA88+N13 combination) representing the current effective solution, the antibacterial rate of the product of this invention prepared in Example 1 (99.2%) is significantly higher (P<0.01) than its antibacterial rate (84.7%), with an absolute improvement of 14.5 percentage points. This direct comparative data proves that this invention, through specific strain selection (LR-G08, LR-G18, LP-G21), ratio optimization (2:1:1), and compounding with fructooligosaccharides, has achieved unexpected and significant technological advancements in addressing the specific technical problem of anti-Candida albicans, meeting the inventiveness requirements of an invention patent.

[0126] 5. Significant market advantages: The effects of this invention far exceed those of ordinary commercial products (50.2%), demonstrating strong product competitiveness.

[0127] This embodiment, through a rigorously designed parallel experiment including key controls, and particularly by introducing a head-to-head efficacy comparison with the core solution of a prior patent, provides core experimental evidence to demonstrate that the present invention possesses outstanding substantive features and significant progress. Example 4: Evaluation of the Proliferative Effect of Compound Postbiotics on Vaginal Beneficial Bacteria Objective: To quantitatively evaluate the proliferation-promoting effect of the three-strain compound postbiotic prepared in Example 1 on four core beneficial vaginal bacteria (Lactobacillus curvatureis, Lactobacillus gasseri, Lactobacillus indolentus, and Lactobacillus janniae), verify its "beneficial bacteria growth promotion" microecological regulation function, and compare it with single bacteria, two-strain compound and commercial postbiotic to prove its synergistic advantage.

[0128] Method (Liquid Co-culture - Growth Promotion Method): 1. Test Sample: Sample of the present invention: 2:1:1 tri-strain compound postbiotic powder (containing 5% fructooligosaccharides) prepared in Example 1.

[0129] Control sample group: Single-strain post-biotic control: Inactivated LR-G08, LR-G18, and LP-G21 single-strain powders, respectively.

[0130] Biogenic control after two strains were combined: The above single-strain powders were physically mixed in the following proportions: LR-G08 + LR-G18 (1:1), LR-G08 + LP-G21 (1:1), LR-G18 + LP-G21 (1:1).

[0131] Commercially available comparison: mainstream post-biotic powders on the market.

[0132] Blank control group: MRS broth containing no post-genetic samples.

[0133] 2. Preparation of beneficial bacteria indicators: Lactobacillus crispatus ATCC 33820, Lactobacillus gasseri ATCC 19992, Lactobacillus iners ATCC 55195, and Lactobacillus jensenii ATCC 25258 were inoculated into MRS liquid medium and activated in an anaerobic environment (85% N2, 10% CO2, 5% H2) at 37°C for 24 h. This activation was repeated for two generations until the growth was stable.

[0134] 3. Establishment of sample solution and co-culture system: All postbiotic samples were prepared into a stock solution with a concentration of 5.0 mg / mL using sterile MRS broth and then vortexed to ensure thorough dispersion.

[0135] In a sterile anaerobic test tube, proceed in the following order: Add 0.9 mL of the above sample stock solution (to make the final concentration of the post-genetic agent in the system 0.5 mg / mL).

[0136] Add 8.1 mL of fresh MRS broth. Dilute with 1.0 mL of fresh MRS broth to a concentration of approximately 1 × 10⁻⁶. 7 CFU / mL indicator bacteria working suspension (to make the final bacterial concentration of the system approximately 1×10⁻⁶) 6 (CFU / mL).

[0137] The blank control group used 0.9 mL of sterile MRS broth instead of the sample stock solution, with the remaining steps being the same.

[0138] For each sample, three parallel tubes were set up independently for each indicator bacterium.

[0139] 4. Cultivation and Counting: Place the inoculated test tubes in a 37℃ anaerobic workstation and incubate statically for 24 hours.

[0140] After the culture is completed, immediately vortex each culture tube for 30 seconds.

[0141] Take 100 μL of the culture stock solution or its appropriate 10-fold serial dilution and spread it on an MRS agar plate.

[0142] After the plates were incubated under anaerobic conditions at 37°C for 48 h, colony counting was performed.

[0143] 5. Data Processing and Calculation: Record the number of colonies in each parallel tube and convert it to CFU / mL.

[0144] Calculate the proliferation rate using the following formula: Proliferation rate (%) = [(mean CFU / mL of sample group - mean CFU / mL of blank control group) / mean CFU / mL of blank control group] × 100% Data are expressed as mean ± standard deviation (Mean ± SD) and statistical analyses (such as one-way ANOVA) are performed.

[0145] result: 1. Mean colony count (mean ± SD, n=3) of the blank control group (indicator bacteria only, no metabiotics) after 24 hours of culture: Lactobacillus curvature: (1.05 ± 0.09) × 10 9 CFU / mL Lactobacillus gasseri: (9.80 ± 0.82) × 10 8 CFU / mL Inert Lactobacillus: (7.20 ± 0.65) × 10 8 CFU / mL Lactobacillus japonicus: (8.50 ± 0.74) × 10 8 CFU / mL 2. The proliferative effects of each metabiotic sample on four types of beneficial vaginal bacteria: Table 4-1 Proliferation rate of different metabiotic samples on beneficial vaginal bacteria (%, co-cultured for 24 h, mean ± SD, n=3)

[0146] A comparison of the effects of this invention's three-bacterial compound on the proliferation rate of beneficial vaginal bacteria with commercially available products: Figure 5 As shown.

[0147] Conclusions and Analysis: 1. Significant proliferation-promoting effect: The three-bacterial compound postbiotic prepared in Example 1 showed the strongest proliferation-promoting effect on all four types of vaginal core beneficial bacteria tested, with proliferation rates ranging from 115.8% to 185.3%, indicating that it can significantly increase the biomass of beneficial bacteria.

[0148] 2. Clear Synergistic Advantages: The proliferation-promoting effect of the sample of the present invention (triple-strain compound + fructooligosaccharide) prepared by Example 1 was significantly higher than that of any single-strain postbiotic and the control group of any two-strain compound (without fructooligosaccharide). For example, the proliferation-promoting rate of *Lactobacillus curvatureii* (185.3%) was much higher than that of the best-performing single strain LR-G18 (62.8%) and the two-strain compound LR-G08+LR-G18 (98.5%). This demonstrates that the postbiotic prepared by compounding in a specific ratio and adding fructooligosaccharide produces a significant synergistic enhancement effect on the proliferation-promoting effect.

[0149] 3. Excellent market competitiveness: The proliferation-promoting effect of the product of this invention is significantly better than that of commercially available control products on all tested strains, proving its advanced technology and superior efficacy.

[0150] 4. Functional integrity verification: The results of this embodiment combined with those of embodiment 3 (inhibition of pathogenic bacteria) fully demonstrate that the three-bacterial compound postbiotic prepared by embodiment 1 of the present invention has a bidirectional and synergistic microecological regulation function of "inhibiting harmful bacteria" (inhibiting pathogenic bacteria such as Candida albicans) and "promoting beneficial bacteria" (promoting beneficial bacteria such as Lactobacillus). This is the core mechanism that distinguishes it from single-function products and enables the reconstruction of vaginal microecological balance.

[0151] Example 5: Preparation of sanitary napkins loaded with composite biogener and verification of their bidirectional regulatory function This embodiment aims to illustrate how the composite postbiotic of the present invention can be applied to sanitary napkin products, and to verify the bidirectional regulatory effect of the end product on the vaginal microecology of "inhibiting harm and promoting benefit" under simulated use environment.

[0152] 5.1 Preparation of sanitary napkin surface layer with different post-biotic formulations (1) Preparation of coating liquid: Weigh 0.5 g of each of the following three postgenetic samples: Sample P1 (Invention Group): Tri-strain compound post-biotic powder prepared in Example 1 (LR-G08:LR-G18:LP-G21 = 2:1:1, containing 5% fructooligosaccharides).

[0153] Sample P2 (Comparative Example 1): Single-strain postbiotic powder containing only Lactobacillus rhamnosus LR-G08.

[0154] Sample P3 (Comparative Example 2): A compound of Lactobacillus rhamnosus LR-G08 and Lactobacillus reuteri LR-G18 (ratio 1:1) containing no fructooligosaccharides.

[0155] The above samples were added to 100 mL of sterile aqueous solution containing 1.0% (w / v) trehalose and magnetically stirred for 30 min to form a uniformly dispersed coating solution with a concentration of 5 mg / mL.

[0156] (2) Substrate and Loading Process: Commercially available sterile pure cotton nonwoven fabric (weight: 30 g / m²) was used as the base material and cut to a size of 10 cm × 20 cm. Using a precision spray gun, the above coating liquid was evenly sprayed onto the surface of the nonwoven fabric under constant pressure, controlling the spraying amount per unit area to be 1.0 mL / 100 cm².

[0157] The coated nonwoven fabric was dried in a 40°C circulating hot air drying oven for 60 minutes and then sealed in packaging.

[0158] (3) Load calculation and sample identification: Based on the total number of microbial cells in the powder (1.25 × 10⁻⁶) 11Based on the calculation of Cell / g and spraying amount, the loading amount of post-generic on the nonwoven fabric surface layer is 6×10. 6 Cell / cm².

[0159] Three sets of experimental samples were thus prepared: HS-P1: A biotic (containing fructooligosaccharides) loaded with the three-strain complex of the present invention.

[0160] HS-P2: Genetic acid (Comparative Example 1) loaded with single strain LR-G08 (without fructooligosaccharides).

[0161] HS-P3: Genetic agent loaded with LR-G08+LR-G18 (Comparative Example 2) (excluding fructooligosaccharides).

[0162] A blank nonwoven fabric was prepared by spraying only with sterile trehalose solution as the HS-Blank (control group).

[0163] 5.2 Evaluation of the external function of the sanitary napkin surface layer (A) Validation of antibacterial function (simulated challenge test) 1. Sample preparation: Precisely cut a 1.0 cm × 1.0 cm (area 1 cm²) sample from each group of nonwoven fabrics and place it in a sterile centrifuge tube.

[0164] 2. Inoculation and Incubation: Add 1.0 mL of Staphylococcus aureus (ATCC 6538) suspension at a concentration of 1×10^5 CFU / mL to each centrifuge tube. Incubate the centrifuge tubes at 37°C and 100 rpm for 4 h.

[0165] 3. Bacterial recovery and counting: Immediately after incubation, add 9.0 mL of sterile PBS containing 0.1% Tween 80 and vortex for 2 min. Perform 10-fold serial dilutions on LB agar plates and incubate at 37°C for 24 h before counting.

[0166] 4. Calculation of antibacterial rate: Inhibition rate (%) = [(Average colony count in HS-Blank group - Average colony count in experimental group) / Average colony count in HS-Blank group] × 100% (B) Verification of proliferation-promoting function (simulated nutrient supply environment) 1. Sample preparation and inoculation: Cut a 1 cm² sample as above and place it in an anaerobic test tube containing 10 mL of MRS broth. Add 100 μL of working Lactobacillus curvaturei (ATCC 33820) suspension at a concentration of approximately 1 × 10^7 CFU / mL to each tube, so that the initial inoculation amount is approximately 1 × 10^5 CFU / mL.

[0167] 2. Incubation: Place the test tubes in a 37°C anaerobic workstation and incubate statically for 24 h.

[0168] 3. Counting and Calculation: After culturing, 100 μL of culture was diluted and spread onto MRS agar plates. After anaerobic incubation for 48 h, the colonies were counted. The relative proliferation rate of other experimental groups was calculated using the colony count of the HS-Blank group as a baseline (set as 100%).

[0169] 5.3 Experimental Results The evaluation results of the "antibacterial" and "proliferation-promoting" functions of the surface layer of sanitary napkins loaded with different postbiotic formulas are shown in the table below.

[0170] Table 5-1 Evaluation of the bidirectional regulatory function of the surface layer of sanitary napkins with different post-biotic formulations

[0171] 5.4 Conclusion The results of this embodiment show that: 1. Process feasibility: The composite post-generic agent of the present invention can be stably loaded onto the surface layer of sanitary napkins through a spray-drying process, and the loading amount is controllable.

[0172] 2. Superior efficacy: The sanitary napkin surface layer loaded with the three-bacterial compound postbiotic (HS-P1) prepared in Example 1 of this invention showed a near-complete inhibitory effect on pathogenic bacteria (Staphylococcus aureus) (inhibition rate of 98.9%) under simulated use conditions, while showing a very strong ability to promote the proliferation of core beneficial bacteria (Lactobacillus curvature) (relative proliferation rate of 252.1%), and its proliferation-promoting effect reached more than 2.5 times that of the blank control.

[0173] 3. Synergistic Effect: The dual effects of "inhibiting harm and promoting benefit" of HS-P1 are significantly superior to the control groups that are loaded with only a single bacterium (HS-P2) or a combination of two bacteria followed by a biotic (HS-P3) and neither contains fructooligosaccharides. This fully demonstrates that the core "specific ratio of three bacteria + fructooligosaccharides" formula of this invention can still play a key synergistic role in the end product, and is an ideal technical solution for achieving precise, gentle, and long-lasting regulation of the vaginal microecology.

[0174] Example 6: Preliminary assessment of the stability and safety of the compound postbiotic agent Objective: To evaluate the storage stability and topical biosafety of the compound postbiotic agent of the present invention (i.e., a three-strain compound postbiotic powder containing 5% fructooligosaccharides, 2:1:1) prepared by Example 1, so as to provide key data support for its industrial production, packaging, storage and final commercial application as a topical care ingredient.

[0175] 6.1 Accelerated stability test 6.1.1 Samples and Conditions Test sample: [Three-strain compound postbiotic powder prepared in Example 1 (batch number: 2025FS001, containing 5% oligofructose)], 100g of each batch was sampled and packaged into 10 composite aluminum foil bags (specification: 10g / bag) with waterproof and light-proof functions, and heat-sealed.

[0176] Test conditions: Referring to the guidelines for stability testing of active pharmaceutical ingredients and preparations in General Chapter 9001 of the Chinese Pharmacopoeia, the accelerated test conditions were set as follows: temperature 40°C ± 2°C, relative humidity 75% ± 5%.

[0177] Testing time points: Samples were taken and tested at the end of the 0th month (initial), 1st month, 3rd month, and 6th month of storage.

[0178] 6.1.2 Detection Indicators and Methods 1. Appearance and physical properties: Visually observe the color, color uniformity, presence of lumps, deliquescence, etc. of the sample, and assess its flowability.

[0179] 2. Moisture content: Determination of moisture content was performed according to Method II (drying method) of General Chapter 0832 of the Chinese Pharmacopoeia. Approximately 2g of sample was accurately weighed and spread evenly in a flat weighing bottle dried to constant weight. The sample was dried at 105°C to constant weight, and the moisture percentage was calculated.

[0180] 3. Total bacterial count retention rate (key indicator): Methods: Hemocytometer method was used. Accurately weigh 0.1 g of sample and dissolve it in 10 mL of phosphate buffer containing 0.1% Tween-80. Vortex for 2 minutes to prepare a homogeneous stock solution. After appropriate dilution, take 10 μL and add it to the counting chamber of the hemocytometer for counting under a microscope.

[0181] Calculation: Retention rate (%) = (Total number of bacteria per gram of sample at the current time point / Total number of bacteria per gram of sample at the beginning of month 0) × 100%.

[0182] 4. Retention rate of core efficacy (key indicator): Method: The liquid co-culture-inhibition rate method was exactly the same as in Example 3.1. Samples at each time point were prepared into test solutions of 1.0 mg / mL (based on subsequent probiotic cell counts) with PBS, using Candida albicans as the indicator bacterium, and the inhibition rate was calculated after 6 hours of incubation.

[0183] Calculation: Active retention rate (%) = (Inhibition rate of sample at current time point / Inhibition rate of sample at the beginning of month 0) × 100%.

[0184] 6.1.3 Results Table 6-1 Results of accelerated stability test of compound probiotic powder (40°C, 75% RH)

[0185] Note: All data, except for the appearance description, are expressed as mean ± SD, n=3. The baseline value for antibacterial activity was 99.2% (taken from Example 3.1, Table 3-1).

[0186] 6.1.4 Conclusion After being stored for 6 months under accelerated stability testing conditions (40°C / 75%RH), the key quality properties of the compound postbiotic powder of this invention changed as follows: Good physical stability: The moisture content increases slowly, but remains at a low level (5.5%) at the end of 6 months. Although the sample has a slight darkening of color, there is no serious clumping or deliquescence, and it remains usable.

[0187] Stable bioactivity: The retention rate of the core functional component (inactivated bacteria) remains above 90%, and the corresponding retention rate of the core antibacterial (Candida albicans) activity is close to 89%. Extrapolating from the stability guidelines, the product is expected to maintain a stable shelf life under normal cool and dry storage conditions, fully meeting the shelf life requirements of commercial products.

[0188] 6.2 In vitro biosafety evaluation To ensure the safety of the product as a topical care ingredient (especially for intimate hygiene products such as sanitary napkins), the following in vitro tests were conducted by a third-party testing institution with CMA and CNAS accreditation.

[0189] 6.2.1 Skin irritation test (in vitro reconstructed human epidermal model) Test standards: Follow the OECD Test Guide 439 (OECD TG 439).

[0190] Experimental system: The human epidermal model was reconstructed in vitro using EpiDerm™ EPI-200.

[0191] Sample preparation: The metabiotic powder of the present invention was prepared into a high-concentration suspension of 100 mg / mL with sterile distilled water and used as the test sample.

[0192] Experimental procedure: 15 μL of the test sample was evenly applied to the model surface and exposed at 37°C and 5% CO2 for 15 minutes, followed by washing and incubation for another 42 hours. 5% sodium dodecyl sulfate (SDS) solution was used as a positive control, and sterile distilled water was used as a negative control.

[0193] Endpoint detection: Tissue viability was determined using the MTT assay, and the relative tissue viability (%) relative to the negative control group was calculated.

[0194] Results: The relative tissue viability of the test group was 94.2%, which was much higher than the non-irritant threshold (>50%) specified by OECD TG 439, and there was no statistically significant difference compared with the negative control group (100%) (p>0.05).

[0195] Conclusion: Under the conditions of this experiment, the sample did not cause skin irritation.

[0196] 6.2.2 Skin sensitization test (human cell line activation assay, h-CLAT) Testing standards: Follow OECD testing guidelines 442E.

[0197] Experimental system: The human monocytic leukemia cell line THP-1 was used.

[0198] Sample preparation: The sample prepared in Example 1 was prepared into two test concentrations (0.1 mg / mL and 0.25 mg / mL, based on metatrophic cells) using culture medium.

[0199] Experimental procedure: After THP-1 cells were co-incubated with the test substance for 24 hours, the expression rates of two key antigen presentation markers, CD86 and CD54, on the cell surface were detected by flow cytometry.

[0200] Judgment criteria: A positive reaction is defined as a relative fluorescence intensity (RFI) ≥ 150%.

[0201] Results: At both test concentrations, the RFI of CD86 and CD54 induced by the sample was below the 150% threshold and showed no significant difference compared to the solvent control group. The positive control (NiSO4) showed a strong positive reaction. Conclusion: Under the conditions of this experiment, the sample did not show potential skin sensitization.

[0202] 6.2.3 Cytotoxicity assay (MTT method) Test standard: In accordance with international standard ISO 10993-5:2009.

[0203] Experimental system: Mouse fibroblasts (L929).

[0204] Sample extract preparation: The sample prepared in Example 1 was extracted with cell culture medium containing 10% fetal bovine serum at a ratio of 6 cm² / mL and extracted at 37°C for 24 hours to prepare the stock extract.

[0205] Experimental procedure: L929 cells were co-cultured with sample extracts of different dilutions (100%, 50%, 25%) for 24 hours. Cell metabolic activity was measured by MTT assay and the relative cell proliferation rate (RGR, %) was calculated.

[0206] Judgment criteria: RGR ≥ 80% indicates no cytotoxicity.

[0207] Results: Even under 100% extraction stock solution conditions, the RGR of L929 cells was 102.5%, significantly higher than the 80% non-toxicity threshold.

[0208] Conclusion: This material is non-cytotoxic.

[0209] 6.2.4 Overall Safety Conclusion Based on the results of the above three internationally recognized in vitro toxicology tests: 1. No skin irritation (tissue viability >90%).

[0210] 2. No potential skin sensitization (negative h-CLAT test).

[0211] 3. No cytotoxicity (relative cell proliferation rate >100%).

[0212] Overall assessment: The compound postbiotic agent of the present invention prepared in Example 1 showed good biocompatibility under the test conditions and has a safe basis as an active ingredient in topical skin and mucous membrane contact products (such as sanitary napkins, nursing gels, etc.).

[0213] Overall conclusion of this embodiment: The compound postbiotic agent (three-strain compound postbiotic + fructooligosaccharide) provided by this invention not only demonstrates outstanding performance in its core efficacy (as shown in Examples 3-5), but its key industrial properties have also undergone rigorous verification. Accelerated stability testing proved its good chemical and physical stability, meeting the requirements for commercial storage and transportation; the systematic in vitro biosafety evaluation provided a scientific basis for its external application safety, removing a key obstacle to its application in end products. All of the above data together constitute a complete chain of evidence for this invention's transition from the laboratory to the market.

[0214] The embodiments of the present invention have been described in detail above with reference to specific examples. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. A three-strain compound postbiotic preparation, characterized in that, The three-strain compound postbiotic preparation includes inactivated Lacticaseibacillus rhamnosus LR-G08, inactivated Limosilactobacillus reuteri LR-G18, and inactivated Lactiplantibacillus plantarum LP-G21. The Lactobacillus rhamnosus LR-G08 is deposited at the China Center for Type Culture Collection (CCTCC) on June 9, 2021, with accession number CCTCC NO:M2021712. The Lactobacillus reuteri LR-G18 is deposited at the China Center for Type Culture Collection on November 14, 2022, with accession number CCTCC NO:M20221800. The *Lactobacillus plantarum* LP-G21 was deposited at the China Center for Type Culture Collection on June 9, 2021, with accession number CCTCC NO:M2021711. In the three-strain compound post-biotic preparation, based on the number of inactivated cells, the ratio of inactivated Lactobacillus rhamnosus LR-G08, inactivated Lactobacillus reuteri LR-G18, and inactivated Lactobacillus plantarum LP-G21 is 2:1:

1. The three-strain compound post-biotic preparation also includes fructooligosaccharides, and the amount of fructooligosaccharides added is 1%-10% of the total mass of the preparation.

2. The three-strain compound post-biotic preparation according to claim 1, characterized in that, The amount of fructooligosaccharides added is 5% of the total mass of the preparation.

3. The method for preparing the three-strain compound post-biotic preparation according to any one of claims 1-2, characterized in that, Includes the following steps: (1) Lactobacillus rhamnosus LR-G08, Lactobacillus reuteri LR-G18 and Lactobacillus plantarum LP-G21 were fermented and cultured to obtain high-density fermentation broth. The fermentation broth was centrifuged and the mycelial sludge of each strain was collected. (2) Mix the bacterial sludge obtained in step (1) with an aqueous solution containing a protectant to form an emulsion; (3) The emulsion obtained in step (2) is subjected to heat inactivation treatment, and then the sterilized additive is added to the material and mixed evenly; the additive is skim milk powder and fructooligosaccharide. (4) The mixture obtained in step (3) is freeze-dried to obtain the three-strain compound post-biotic preparation.

4. The method for preparing the three-strain compound post-biotic preparation according to claim 3, characterized in that, In step (1), the fermentation culture method includes the following steps: Lactobacillus rhamnosus LR-G08, Lactobacillus reuteri LR-G18, and Lactobacillus plantarum LP-G21 were inoculated into MRS liquid medium and activated at 35-39℃ for 20-28 h. After several generations of activation, the activated bacterial solution was inoculated into fermentation medium at an inoculation rate of 4-6% and fermented at 35-39℃ for 10-12 h until the viable count of each strain reached 1×10⁻⁶ at the end of fermentation. 9 A high-density fermentation broth is obtained when the concentration of CFU / mL is above a certain level.

5. The method for preparing the three-strain compound post-biotic preparation according to claim 3, characterized in that, In step (2), the protective agent in the aqueous solution containing the protective agent is trehalose, and its mass concentration is 5-20%; the volume ratio of the bacterial mud to the aqueous solution containing the protective agent is 1:(0.5-1.5).

6. The method for preparing the three-strain compound post-biotic preparation according to claim 3, characterized in that, In step (3), the conditions for the heat inactivation treatment are: heat inactivation at 75-85℃ for 25-35 minutes.

7. The method for preparing the three-strain compound post-biotic preparation according to claim 3, characterized in that, In step (3), the amount of skim milk powder added is 1-10% of the total mass of the inactivated material.

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