Application of a fermented lactobacillus muciaginousus with high bacteriostatic and biofilm inhibition capacity and its product in preparation of health products

Abstract

The application belongs to the technical field of microorganisms, and particularly relates to a fermented Lactobacillus muci genus with high efficient bacteriostatic and biofilm inhibiting capacity and application of a product of the fermented Lactobacillus muci genus in preparation of health products. Limosilactobacillus fermentum ​ ) H57-AN-Na1-4 is preserved as CGMCC No. 35329. The fermented Lactobacillus muci genus of the application has a significant inhibiting effect on Gardnerella, Streptococcus group B, Escherichia coli, Staphylococcus aureus and Candida albicans infection and also has a good inhibiting capacity on biofilms of the pathogenic bacteria, thereby reducing the infection risk. The fermented Lactobacillus muci genus can be used for preventing or treating diseases related to the infection of the pathogenic bacteria. The fermented Lactobacillus muci genus of the application has a high lactic acid production, promotes a healthy microecological environment, has superior cell colonization capacity and enhances the bacteriostatic effect. In addition, the safety of the fermented Lactobacillus muci genus of the application is jointly proved by hemolytic test, antibiotic resistance gene detection and functional verification.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to the application of a fermenting *Lactobacillus mucinus* with highly efficient antibacterial and biofilm-inhibiting capabilities and its products in the preparation of health products. Background Technology

[0002] Gardnerella vaginalis and Group B Streptococcus (Gardnerella vaginalis) are present in the human micro-ecosystem. Group B Streptococcus GBS), Escherichia coli ( Escherichia coli Staphylococcus aureus ( Staphylococcus aureus ) and Candida albicans ( Candida albicans As common opportunistic pathogens, microorganisms are widely colonized on the skin and mucous membranes. When the host's immune function is normal and the microecological environment is at homeostasis, these microorganisms maintain a harmonious symbiotic relationship with the host and do not cause disease. However, when the body's immunity declines, the mucosal barrier is damaged, or the microecological balance is disrupted, they can breach their colonization sites and invade different tissues and organs of the body, thereby causing a variety of infectious diseases.

[0003] Staphylococcus aureus can cause a full spectrum of purulent diseases, ranging from skin and soft tissue infections (such as boils and carbuncles) to deep organ infections (such as pneumonia and osteomyelitis) and even bloodstream infections. In recent years, the emergence and prevalence of multidrug-resistant strains such as methicillin-resistant Staphylococcus aureus (MRSA) have posed significant challenges to clinical treatment, severely limiting the choice of therapeutic agents. Escherichia coli is a core member of the intestinal commensal flora and is also the most common pathogen causing urinary tract infections. Furthermore, it can translocate through the intestines, causing invasive diseases such as abdominal infections and neonatal purulent meningitis. Candida albicans, a common colonizing fungus in human mucous membranes, primarily infects mucous membranes such as the oral cavity and vagina, as well as skin folds. In immunocompromised individuals, it can cause disseminated candidiasis, a disease with an extremely high mortality rate. However, when the vaginal pH level rises and the number of lactobacilli, the dominant flora in the vaginal microecology, decreases significantly, Gardnerella vaginalis will overproliferate, leading to bacterial vaginosis (BV). GBS is one of the leading pathogens causing neonatal sepsis and purulent meningitis, and it can also cause adverse pregnancy outcomes such as puerperal infection and premature rupture of membranes in pregnant women.

[0004] Currently, antibiotics are the primary means of clinical treatment for the aforementioned opportunistic microbial infections. However, this treatment method has many limitations. First, while antibiotics target and eliminate pathogens, they indiscriminately destroy the host's symbiotic flora, leading to a sharp decline in the number of beneficial bacteria such as lactobacilli. This can not only induce secondary infections such as fungal vaginitis but also weaken the immune barrier function of the local mucosa, significantly increasing the risk of infection recurrence. Second, antibiotic treatment can cause various adverse reactions, such as gastrointestinal disorders, rashes, and liver and kidney damage; severe drug allergic reactions can even be life-threatening. Third, long-term or improper use of antibiotics can accelerate the development and spread of drug resistance in pathogens, limiting the choice of drugs for subsequent treatment and posing a serious public health threat. Finally, antibiotics are unable to completely eliminate pathogens that have formed biofilms; the residual biofilms can become a "reservoir" for infection recurrence, eventually developing into chronic persistent infections.

[0005] Microecological therapy offers a novel strategy for the prevention and control of opportunistic microbial infections. This therapy, by targeting and regulating the host's microecological environment, not only disrupts the biofilm structure of pathogens, inhibiting their colonization and proliferation, but also restores the acidic microenvironment in areas such as the vagina through immunomodulation, thus restoring flora homeostasis and fundamentally reducing the risk of infection recurrence. Simultaneously, microecological therapy avoids the drug resistance problems caused by antibiotic overuse, exhibiting significant advantages such as high safety and sustainability. However, current clinical applications and commercially available probiotic preparations still face numerous technical bottlenecks and safety concerns. Some lactobacillus strains (such as certain...) Lactobacillus rhamnosus The strain may be hemolytic or carry a tetracycline resistance gene. tetM Macrolide resistance genes ( ermB The presence of mobile resistance elements, such as serotonin, severely limits their clinical application. Existing probiotic strains have a narrow antibacterial spectrum, exhibit weak inhibitory activity against fungi such as Candida albicans, and research on their antagonistic effects against GBS is also very limited. Furthermore, commercially available probiotic preparations generally suffer from insufficient colonization capacity, making it difficult to form a stable colonization barrier on the host mucosal surface and maintain their microecological regulatory effects over the long term.

[0006] In summary, existing probiotic preparations are not ideal in treating urogenital tract infections, inhibiting the proliferation of opportunistic pathogens, and preventing biofilm formation. Furthermore, they lack stable in vivo colonization capabilities, making it difficult to meet clinical needs for the prevention and control of opportunistic microbial infections. Therefore, developing a novel probiotic preparation that combines broad-spectrum antibacterial activity, biofilm inhibition, and safe colonization characteristics has become a critical scientific problem urgently needing to be solved in the fields of clinical microbiology and translational medicine. Summary of the Invention

[0007] In view of the defects and deficiencies in the existing technology, the present invention provides a fermented Lactobacillus mucinus with highly efficient antibacterial and biofilm inhibition capabilities and its application in the preparation of health products.

[0008] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a fermenting *Lactobacillus mucilaginosus* with highly efficient antibacterial and biofilm-inhibiting capabilities, characterized in that: the fermenting *Lactobacillus mucilaginosus* ( Limosilactobacillus fermentum H57-AN-Na1-4 was deposited on July 22, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 35329.

[0009] In a second aspect, the present invention provides a composition comprising live bacteria of *Lactobacillus fermentans* as described in the first aspect and its products, inactivated bacterial cells, fermentation broth, or cell-free supernatant.

[0010] In a third aspect, the present invention provides the use of the fermented Lactobacillus mucinus described in the first aspect or the composition described in the second aspect in the preparation of health products.

[0011] Alternatively, in the above applications, the health product can inhibit pathogens.

[0012] Alternatively, in the above applications, the health product can inhibit pathogens, eliminate biofilms of pathogens, and / or reduce the load of pathogens.

[0013] Alternatively, in the above applications, the pathogenic bacteria include Gardnerella vaginalis, Group B Streptococcus, Escherichia coli, Staphylococcus aureus, or Candida albicans.

[0014] Alternatively, in the above applications, the health product can be used to prevent or treat diseases associated with the aforementioned pathogenic bacteria infection.

[0015] Preferably, the diseases include bacterial vaginosis, vaginal flora imbalance, recurrent vaginitis, pelvic inflammatory disease, as well as premature birth, premature rupture of membranes, maternal urinary tract infection, chorioamnionitis, postpartum endometritis, puerperal infection, neonatal sepsis, meningitis, pneumonia, urinary tract infection, skin and soft tissue infection, bacteremia, acute cystitis, pyelonephritis, recurrent urinary tract infection, gastroenteritis, diarrhea, sepsis, abdominal infection, wound infection, bacteremia, infective endocarditis, osteomyelitis, suppurative arthritis, food poisoning, toxic shock syndrome, vulvovaginal candidiasis, recurrent candidal vaginitis, oral candidiasis, esophageal candidiasis, urinary tract candidiasis, candidemia, or invasive candidiasis.

[0016] Alternatively, in the above applications, the product may be a drug, health product, functional food, food supplement, food for special medical purposes, medical device, or hygiene product.

[0017] Alternatively, in the above applications, the product may also contain a pharmaceutically, health-promoting, or food-grade carrier.

[0018] Preferably, the carrier includes fillers, binders, wetting agents, disintegrants, lubricants, or flavoring agents known in the art.

[0019] Alternatively, in the above applications, the dosage form of the product includes pills, tablets, lozenges, lyophilized powders, granules, capsules, aqueous solutions, alcoholic solutions, oil solutions, syrups, emulsions, suspensions, suppositories, solutions for injection or infusion, ointments, gels, tinctures, creams, patches, lotions, sprays, aerosols, powder sprays, effervescent tablets, transdermal therapy systems, microcapsules, or implants.

[0020] Preferably, the drug is for oral, topical, or vaginal administration. The carrier in the drug is a conventionally used carrier suitable for preparing oral, topical, or vaginal forms.

[0021] Preferably, the term "external use" refers to application to the skin, mucous membranes, or vulva.

[0022] More preferably, the daily dose of the drug depends on the method of administration (oral, topical, or vaginal) and the type of treatment (therapeutic or prophylactic).

[0023] Preferably, the health products, functional foods, food supplements, and foods for special medical purposes are for oral administration.

[0024] Alternatively, in the above applications, the sanitary products include sanitary wipes, sanitary napkins, panty liners, sanitary tampons, sanitary napkins, vaginal washes, and feminine antibacterial / bacteriostatic washes.

[0025] Alternatively, in the above applications, the product may also contain a second component.

[0026] Preferably, the second component includes live bacterial secretions, probiotics, postbiotics, prebiotics, antibacterial agents, or immunomodulators.

[0027] Compared with the prior art, the present invention has the following advantages: (1) The fermented mucinous lactobacillus of the present invention has a significant inhibitory effect on Escherichia coli, Staphylococcus aureus, Candida albicans, Gardnerella vaginalis, and Group B streptococci, which is significantly better than market products and can effectively protect the human body from pathogens.

[0028] (2) The fermented mucinous lactobacillus of the present invention can inhibit the growth and biofilm formation of Escherichia coli, Staphylococcus aureus, Group B Streptococcus, Candida albicans, Gardnerella vaginalis, etc., and greatly reduce the survival and reproduction of pathogens.

[0029] (3) In terms of cell colonization, the colonization rate of the fermented mucoid lactobacillus of the present invention on vaginal epithelial cells, etc. is much higher than that of products on the market, which can better regulate the balance of flora and maintain the stability of human microecology. Attached Figure Description

[0030] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings: Figure 1 MALDI-TOF mass spectrum of strain 35329.

[0031] Figure 2 Blood agar hemolysis test for strain number 35329.

[0032] Figure 3 Long-read whole-genome sequencing results of strain number 35329.

[0033] Figure 4 Tracking the inhibitory effect of strain No. 35329 and its market products on Escherichia coli over 24 hours (77 cycles).

[0034] Figure 5 The inhibitory effect of strain No. 35329 and its market products on Escherichia coli within 24 hours.

[0035] Figure 6 Tracking the inhibitory effect of strain No. 35329 and its market products on Staphylococcus aureus over 24 hours (77 cycles).

[0036] Figure 7The inhibitory effect of strain No. 35329 and its market products on Staphylococcus aureus over 24 hours.

[0037] Figure 8 Tracking the inhibitory effect of strain No. 35329 and its market products on Group B Streptococcus for 24 hours (77 cycles).

[0038] Figure 9 The inhibitory effect of strain No. 35329 and its market products on Group B Streptococcus within 24 hours.

[0039] Figure 10 Tracking the inhibitory effect of strain No. 35329 and its market products on Candida albicans over 24 hours (77 cycles).

[0040] Figure 11 The inhibitory effect of strain No. 35329 and its market products on Candida albicans within 24 hours.

[0041] Figure 12 Tracking the inhibitory effect of strain 35329 supernatant on Gardnerella vaginalis over 48 hours (145 cycles).

[0042] Figure 13 The inhibitory effect of the supernatant of strain No. 35329 on Gardnerella vaginalis after 48 hours.

[0043] Figure 14 The inhibitory effect of strain No. 35329 and its market products on the biofilm of Staphylococcus aureus.

[0044] Figure 15 The biofilm inhibitory effect of strain No. 35329 and its market products on group B streptococci.

[0045] Figure 16 The biofilm inhibition effect of strain No. 35329 and its market products on Candida albicans.

[0046] Figure 17 Adsorption of strain 35329 and its marketed products on epithelial VK2 / E6E7 cells. Detailed Implementation

[0047] The present invention will be further described in detail below with reference to embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. The following are the experimental methods and results used in the implementation examples.

[0048] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0049] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0050] Example: This invention isolates a novel fermenting mucinous lactobacillus with highly efficient antibacterial and biofilm-inhibiting capabilities from the vaginal secretions of healthy women.

[0051] The fermented mucinous lactobacillus ( Limosilactobacillus fermentum H57-AN-Na1-4 was deposited on July 22, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 35329.

[0052] 1. Strains Isolation and Culture Sample source: Vaginal secretions from healthy women (informed consent was obtained from the subjects, and pregnant women and recent antibiotic users were excluded).

[0053] 1.1 Separation and Purification Steps Spread culture: Anaerobic culture was performed on NYCIII plates at 37°C for 24 hours. Morphologically typical and well-isolated single colonies were picked from the plates and purified on appropriate media to ensure the acquisition of a single strain. Colony morphology was then observed, and preliminary judgment was made based on basic phenotypic characteristics such as Gram staining. After obtaining pure cultures, an appropriate amount of fresh bacterial cells was used for MALDI-TOF MS identification. After matrix treatment, the samples were analyzed using the MALDI-TOF MS, and their protein mass spectrometry fingerprints were acquired and compared with reference spectra in the database. Based on the matching score and identification results provided by the system, it was confirmed whether the isolate was the target species.

[0054] The mass spectrometry results of this bacterium, as identified by MALDI-TOF MS, are as follows: Figure 1 As shown, the fingerprint pattern matches that of fermenting *Lactobacillus mucinus*.

[0055] 1.2 Blood agar hemolysis test This experiment aims to determine whether a bacterial strain can lyse red blood cells, thus indicating its virulence or cytotoxicity. The *Lactobacillus mucilaginosus* culture of this invention was streaked onto blood agar plates containing 5% defibrinated sheep blood. After 24 hours of anaerobic incubation, the hemolytic activity of the strain was observed visually. The type of hemolysis was determined based on the phenomena surrounding the colonies: a clear zone indicated β-hemolysis, a green halo indicated α-hemolysis, and no change indicated γ-hemolysis. *Staphylococcus aureus* was used as a positive control.

[0056] 1.2.1 Experimental Methods (1) Inoculate the bacterial strain onto a blood agar plate.

[0057] (2) Anaerobic culture at 37°C for 24 hours.

[0058] (3) Observe the type of hemolysis around the colony.

[0059] 1.2.2 Experimental Results Figure 2 The results showed that after 24 hours of culture on blood agar plates, there was no hemolysis, proving the safety of fermented Lactobacillus mucinus for human use.

[0060] 1.3 Long-read sequencing of the entire genome and antibiotic resistance genes 1.3.1 Experimental Methods This invention utilizes whole-genome long-read sequencing to detect antimicrobial resistance genes. The specific steps are as follows: (1) Sample preparation and sequencing: Collect genomic DNA from the samples to be tested and obtain their genomic sequences using long-chain nanopore sequencing. Ensure that the genomic sequences of the samples are saved in FASTA format to obtain complete genomic data for subsequent analysis.

[0061] (2) Data processing: Perform quality control and filtering on the raw data obtained from sequencing to remove low-quality sequences.

[0062] (3) Genome alignment: The query genome is aligned with the reference genome of Lactobacillus fermentum.

[0063] (4) Circular plot display: A circular plot is used to display the comparison results between the query genome and the reference genome. The query genome is marked in orange and the reference genome is marked in green.

[0064] (5) Annotation: Annotate the functions of the queried genome, and identify genes, functional regions, etc.

[0065] 1.3.2 Experimental Results Long-read sequencing whole-genome sequencing results are as follows Figure 3 As shown, no known antibiotic resistance genes were detected in the potential plasmids identified by sequencing. The safety of its use was verified at the gene level.

[0066] 1.4 Antibiotic susceptibility testing 1.4.1 Experimental Methods Pure cultures of identified strains were used for antibiotic susceptibility testing. First, standardized bacterial suspensions were prepared and evenly spread on the surface of NYCIII medium. Then, susceptibility testing discs containing different antibiotics were placed on the inoculated plates and incubated anaerobically at 37°C. After incubation, the diameter of the inhibition zone around each antibiotic disc was measured.

[0067] 1.4.2 Experimental Results As shown in Table 1, the results indicate that the fermented *Lactobacillus mucilaginosus* is sensitive to common antibiotics.

[0068] Table 1: Size of the inhibition zone 2. Inhibition of pathogenic bacteria Pathogenic strains included: *Escherichia coli* ST131 (associated with urethritis and other inflammations), *Staphylococcus aureus* (ATCC 29213), *Group B Streptococcus* (CCUG 47293), and *Candida albicans* (a fungus, CCUG44135), and *Gardnerella vaginalis* (CCUG44012). A commercially available vaginal lactobacillus active capsule (Dingjunsheng DJS) was purchased as a product control.

[0069] 2.1 Inhibitory effect of fermented Lactobacillus mucin supernatant on various pathogenic bacteria 2.1.1 Experimental Methods Escherichia coli ST131, Staphylococcus aureus (ATCC 29213), Group B Streptococcus (CCUG47293), and Candida albicans (a mold, CCUG 44135) were cultured overnight in LB aerobic dishes. The strains were then cultured overnight in liquid LB medium at 37°C under aerobic conditions. The final cultures of the above strains were centrifuged and resuspended in fresh NYCIII medium, and the OD was adjusted. 600 To a concentration of 0.2, add 100 μL of cell-free supernatant of the tested *Lactobacillus* (OD=1) to 100 μL of the strain with adjusted OD value. Use 100 μL of sterile culture medium as a control. Place the 96-well plate in an aerobic workstation's microplate reader and incubate at 37°C for 24 hours, monitoring the results. Read the OD value every 20 minutes. 600 After cultivation, bacterial growth was quantified by measuring the absorbance at 600 nm in each well. Each experiment had three replicates, and the experiment was repeated three times. The following figures illustrate the common trends across the three experiments. Statistical analysis was performed using one-way ANOVA. p<0.1, p<0.001, p<0.0001.

[0070] 2.1.2 Experimental Results like Figure 4 and Figure 5As shown, Escherichia coli infection, especially ST131, is very common in urethritis, vaginitis, and other inflammatory infections. A comparison was made using the fermented *Lactobacillus mucosa* of this invention and a commercially available product (vaginal lactobacillus active capsules (Dingjunsheng DJS)). Compared to the control group which had no lactobacillus supernatant, the fermented *Lactobacillus mucosa* supernatant showed significant inhibition of *E. coli* upon addition. This inhibitory effect persisted for 24 hours after the end of the experiment. The inhibitory effect of this bacterium on *E. coli* was also significantly superior to the commercially available product over 77 cycles.

[0071] like Figure 6 and Figure 7 As shown, Staphylococcus aureus is a common opportunistic pathogen in clinical practice, associated with various diseases such as skin and soft tissue infections, vaginal and intestinal infections, wound infections, bacteremia, and neonatal infections. It can cause serious infections in newborns, premature infants, and immunocompromised individuals. To evaluate the antibacterial effect of the fermented *Lactobacillus mucinus* of this invention, the supernatant of the fermented *Lactobacillus mucinus* of this invention was compared with commercially available products. The results showed that, compared with the control group without any added *Lactobacillus* supernatant, the supernatant of the fermented *Lactobacillus mucinus* of this invention showed a significant inhibitory effect on *S. aureus* immediately after addition, and this inhibitory effect lasted for 24 hours until the end of the experiment. During the experiment, the growth of *S. aureus* was significantly inhibited. The inhibitory effect of the fermented *Lactobacillus mucinus* of this invention on *S. aureus* remained significantly better than that of commercially available products even after 77 cycles.

[0072] like Figure 8 and Figure 9 As shown, Group B Streptococci are common pathogens causing skin, vaginal, and intestinal infections, especially dangerous in premature infants. A comparison was made between the fermented *Lactobacillus mucinus* of this invention and commercial products. Compared to the control group which contained no *Lactobacillus* supernatant, the fermented *Lactobacillus mucinus* supernatant showed significant inhibition of Group B Streptococci upon addition. This inhibition persisted for 24 hours after the end of the experiment. No Group B Streptococci grew. The inhibitory effect of the fermented *Lactobacillus mucinus* of this invention on Group B Streptococci was also significantly superior to commercial products over 77 cycles.

[0073] like Figure 10 and Figure 11 As shown, Candida albicans is a common skin, vaginal, and nosocomial infection, and is a WHO key pathogen of antibiotic resistance concern. A comparison was made between the fermented *Lactobacillus mucosa* of this invention and commercial products. Compared to the control group which contained no *Lactobacillus* supernatant, the fermented *Lactobacillus mucosa* supernatant showed inhibitory effects on Candida albicans upon addition. This inhibitory effect persisted for 24 hours after the end of the experiment. The inhibitory effect of this bacterium on Candida albicans was also significantly superior to commercial products over 77 cycles.

[0074] 2.2 Inhibitory effect of fermenting *Lactobacillus mucinus* on *Gardnerella vaginalis* 2.2.1 Experimental Methods Inhibition assays against *Gardnerella vaginalis* were performed in 96-well plates, with a final volume of 200 μL per well. In short, *Gardnerella vaginalis* was streaked from cryopreserved tubes onto NYCIII plates and anaerobically incubated at 37°C for 24 hours. Colonies were scraped from the plates using a 1 μL inoculation loop and resuspended in 2 mL of NYCIII broth, and anaerobically incubated at 37°C for 24 hours. All culture steps were performed in an anaerobic workstation. The final culture of *Gardnerella vaginalis* was centrifuged (10,000 g, 10 min), resuspended in fresh NYCIII medium, and the OD was adjusted. 600 To a concentration of 0.2, add 100 μL of *Gardnerella vaginalis* suspension (adjusted OD value) to 100 μL of the supernatant of *Lactobacillus mucinus* to be tested. Use 100 μL of sterile culture medium as a control. Place the 96-well plate in an anaerobic workstation's microplate reader and incubate at 37°C for 800 minutes, monitoring the results. Read the OD value every 20 minutes. 600 After incubation, bacterial growth was quantified by measuring the absorbance at 600 nm in each well. Each experiment had three replicates, and was repeated twice. The following figures illustrate the common trends from the two experiments. Statistical analysis was performed using one-way ANOVA. p<0.1.

[0075] 2.2.2 Experimental Results like Figure 12 and Figure 13 As shown, compared to the control group which had no fermented Lactobacillus supernatant, the fermented Lactobacillus supernatant showed significant inhibition of Gardnerella vaginalis at approximately 24 hours, and this inhibitory effect persisted until 48 hours after the end of the experiment.

[0076] 3. Anti-biofilm test (crystal violet staining method) 3.1 Biofilm culture Inoculation with pathogenic bacteria: Take the bacterial suspension of Staphylococcus aureus, Group B Streptococcus, and Candida albicans in the logarithmic phase, dilute it with LB medium to OD=0.1, and incubate at 37℃ for 24 hours.

[0077] 3.2 Quantitative analysis of biofilm Washing: Discard the supernatant and wash 3 times with PBS buffer; Staining: Add 50 μL of crystal violet staining solution to each well and incubate at room temperature for 30 minutes; Decolorization: Discard the staining solution, wash 3 times with distilled water, add 100 μL of 33% glacial acetic acid for 20 minutes to decolorize; Detection: OD measured using an enzyme-linked immunosorbent assay (ELISA) reader. 570 The biofilm inhibition rate is calculated as follows: (OD value of control group - OD value of experimental group) / OD value of control group × 100%.

[0078] The following figures illustrate the common trends across the three experiments. The statistical method used was one-way ANOVA. p<0.01, p<0.001, p<0.0001.

[0079] 3.3 Experimental Results Experimental results are as follows Figures 14 to 16 As shown, the results indicate that the formation of biofilms in Staphylococcus aureus, Group B Streptococcus, and Candida albicans was significantly reduced when the supernatant of the fermented Lactobacillus mucinus of the present invention was added, indicating that the supernatant of the fermented Lactobacillus mucinus played a significant inhibitory role in the biofilm formation of these bacteria.

[0080] 4. Adhesion experiment on vaginal cells 4.1 Experimental Methods: VK2 / E6E7 cell line was planted at 5 × 10⁶ cells per well. 4 Cells were seeded at a density of 100% in 24-well plates and cultured in a CO2 incubator until 80%-90% confluence. 24 hours before the adhesion assay, the old culture medium was discarded, and the cells were gently washed twice with pre-warmed PBS. 1 mL of antibiotic-free cell culture medium was added to each well. Simultaneously, the target *Lactobacillus* strain was cultured anaerobically at 37°C for 24 hours. For the adhesion assay, cells from 3 wells were resuspended in 1 mL of fresh, pre-warmed antibiotic-free culture medium, and cell counts were performed to determine the multiple of infection. When the cells reached 100% confluence, the number of cells per well was approximately 5 × 10⁶. 5 Each lactobacillus was washed with 1 mL PBS, resuspended in antibiotic-free cell culture medium, and OD was adjusted. 600 Up to 1.0 (at which point the bacterial concentration is approximately 10). 9 CFU / mL). The bacterial concentration was adjusted according to the multiplicity of infection (cells:bacteria = 1:100), and added to the cell wells. Each sample was prepared in triplicate, with a negative control (no bacteria added). After inoculation, the 24-well plate was centrifuged at 1000 rpm for 10 minutes, and then incubated in a CO2 incubator for 4 hours. After incubation, the cells were washed twice with pre-warmed PBS to remove unattached bacteria. 300 μL of TrypLE Express digestion solution was added to each well, and digestion was performed at 37°C for 5 minutes, followed by the addition of 700 μL of PBS to terminate the digestion. 1 mL of cell suspension was collected from each well and serially diluted with PBS (10-10). - ¹ to 10 -610 μL of each dilution was used to seed MRS plates for viable bacterial count. MRS plates were incubated anaerobically at 37°C for 24-48 hours. Appropriate dilutions with colony counts between 30-300 were selected for counting to determine the number of adherent lactobacilli. The figure below shows the common trend across the three experiments. One-way ANOVA was used for statistical analysis. p<0.0001.

[0081] 4.2 Experimental Results: like Figure 17 As shown, the experimental results indicate that the fermented *Lactobacillus mucilaginosus* of the present invention has an extremely strong adsorption capacity for cells, which is significantly higher than that of commercial products, demonstrating its superior colonization ability.

[0082] Obviously, the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make various modifications and variations to the present invention without departing from the spirit and scope of the invention. Therefore, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention also intends to include these modifications and variations.

Claims

1. A fermenting *Lactobacillus mucinus* with highly efficient antibacterial and biofilm-inhibiting capabilities, characterized in that: The fermented L. muci- de (Lactobacillus muci- de) (Lactobacillus muci- de) Limosilactobacillus fermentum ) H57-AN-Na1-4, which was preserved in the China General Microbiological Culture Collection Center on July 22, 2025, at 1st North Chenxi Road, No. 3, Chaoyang District, Beijing, with a preservation number of CGMCC No. 35329.

2. A composition, characterized in that: It comprises live bacteria of the fermenting Lactobacillus mucinus and its products as described in claim 1, inactivated bacterial cells, fermentation broth or cell-free supernatant.

3. The use of the fermented Lactobacillus mucinus according to claim 1 or the composition according to claim 2 in the preparation of health products.

4. The application according to claim 3, characterized in that: The health product can inhibit pathogens, eliminate biofilms of pathogens, and / or reduce the load of pathogens.

5. The application according to claim 4, characterized in that: The pathogens include Gardnerella vaginalis, Group B Streptococcus, Escherichia coli, Staphylococcus aureus, or Candida albicans.

6. The application according to claim 5, characterized in that: The health product is capable of preventing or treating diseases associated with pathogenic bacterial infections as described in claim 5. Preferably, the diseases include bacterial vaginosis, vaginal flora imbalance, recurrent vaginitis, pelvic inflammatory disease, as well as premature birth, premature rupture of membranes, maternal urinary tract infection, chorioamnionitis, postpartum endometritis, puerperal infection, neonatal sepsis, meningitis, pneumonia, urinary tract infection, skin and soft tissue infection, bacteremia, acute cystitis, pyelonephritis, recurrent urinary tract infection, gastroenteritis, diarrhea, sepsis, abdominal infection, wound infection, bacteremia, infective endocarditis, osteomyelitis, suppurative arthritis, food poisoning, toxic shock syndrome, vulvovaginal candidiasis, recurrent candidal vaginitis, oral candidiasis, esophageal candidiasis, urinary tract candidiasis, candidemia, or invasive candidiasis.

7. The application according to claim 3, characterized in that: The product is a drug, health product, functional food, food supplement, food for special medical purposes, medical device or hygiene product. Preferably, the hygiene product includes sanitary wipes, sanitary napkins, panty liners, sanitary tampons, sanitary cotton, vaginal wash, and feminine antibacterial / bacteriostatic wash.

8. The application according to claim 7, characterized in that: The product also contains pharmaceutically, health-promoting, or food-grade carriers.

9. The application according to claim 7, characterized in that: The dosage forms of the products include pills, tablets, lozenges, lyophilized powders, granules, capsules, aqueous solutions, alcoholic solutions, oil solutions, syrups, emulsions, suspensions, suppositories, solutions for injection or infusion, ointments, gels, tinctures, creams, patches, lotions, sprays, aerosols, powder sprays, effervescent tablets, transdermal therapy systems, microcapsules, or implants.

10. The application according to claim 4, characterized in that: The product also contains a second component, which includes probiotics, postbiotics, prebiotics, antimicrobial agents, or immunomodulators.

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