Application of lactobacillus jensenii with high bacteriostatic and biofilm inhibition capacity and product thereof in preparation of health products

Abstract

The application belongs to the technical field of microorganisms, and particularly relates to application of Lactobacillus jensenii with high efficient bacteriostatic and biofilm inhibiting capacity and a product thereof in preparation of health products. Lactobacillus jensenii ) H21-AN-Na1-1 with a preservation number of CGMCC No. 35328. The Lactobacillus jensenii has a significant inhibiting effect on Gardnerella, Streptococcus group B, Escherichia coli, Staphylococcus aureus and Candida albicans infection, and has a good inhibiting capacity on biofilms of the pathogenic bacteria, thereby reducing the infection risk. The Lactobacillus jensenii can be used for preventing or treating diseases related to the infection of the pathogenic bacteria. The Lactobacillus jensenii has high lactic acid production, promotes formation of a healthy microecological environment, has superior cell colonization capacity, and enhances the bacteriostatic effect. In addition, the Lactobacillus jensenii has good safety, which is 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 Lactobacillus japonicus and its products, which have highly efficient antibacterial and biofilm inhibition capabilities, in the preparation of health products. Background Technology

[0002] Gardnerella vaginalis, Group B Streptococci, Escherichia coli, Staphylococcus aureus, and Candida albicans are common opportunistic pathogens in clinical practice, widely colonizing human skin and mucous membranes. These microorganisms typically maintain a symbiotic relationship with the host, but if the body's immunity is weakened, the mucosal barrier is damaged, or the microecological environment is imbalanced, they can breach their colonization sites and invade different tissues and organs, causing various infectious diseases. For example, when vaginal pH increases and the number of lactobacilli, the dominant flora in the vaginal microecology, sharply decreases, Gardnerella vaginalis can proliferate excessively and cause bacterial vaginosis. Group B Streptococci are one of the leading pathogens of neonatal sepsis and purulent meningitis, and can also lead to adverse pregnancy outcomes such as puerperal infection and premature rupture of membranes in pregnant women. Staphylococcus aureus can cause infections from the skin and soft tissues (such as boils, etc.). From carbuncles to deep organ infections (such as pneumonia and osteomyelitis) and even bloodstream infections, the prevalence of methicillin-resistant strains has become a serious challenge for clinical treatment. Escherichia coli is a core member of the intestinal commensal flora and the most common pathogen of urinary tract infections. It can also cause invasive diseases such as abdominal infections and neonatal purulent meningitis through intestinal translocation. Candida albicans is a common colonizing fungus in human mucous membranes, mainly affecting mucous membranes such as the oral cavity and vagina, as well as skin folds. Disseminated candidiasis can occur in immunocompromised individuals, with an extremely high mortality rate.

[0003] Currently, antibiotics are the main 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 induce secondary infections such as fungal vaginitis and weaken the local mucosal immune barrier function, significantly increasing the risk of infection recurrence. Second, antibiotic treatment can cause various adverse reactions, including gastrointestinal disorders, rashes, and liver and kidney damage. Severe drug allergic reactions can even be life-threatening. Third, long-term or irregular use of antibiotics can accelerate the development and spread of drug resistance in pathogens, leading to limited choices of subsequent treatment drugs and posing a serious public health threat. Fourth, antibiotics are difficult to completely eliminate pathogens that have formed biofilms. The residual biofilms can become a "reservoir" for infection recurrence, eventually developing into chronic persistent infections.

[0004] Microecological therapy offers a novel strategy for the prevention and control of opportunistic microbial infections: this therapy, through targeted regulation of the host's microecological environment, not only disrupts the biofilm structure of pathogens and inhibits their colonization and proliferation, but also rebuilds the acidic microenvironment in areas such as the vagina through immunomodulation, restoring flora homeostasis and fundamentally reducing the risk of infection recurrence. Simultaneously, it avoids the drug resistance problems caused by antibiotic overuse, exhibiting significant advantages such as high safety and strong sustainability. However, current clinical applications and commercially available probiotic preparations still face numerous technical bottlenecks and safety risks: 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 α, β, and γ, severely limits their clinical application; existing probiotic strains have a narrow antibacterial spectrum, weak inhibitory activity against fungi such as Candida albicans, and research on their antagonistic effects against Group B Streptococcus is also very limited; in addition, commercially available probiotic preparations generally have insufficient colonization capacity, making it difficult to form a stable colonization barrier on the host mucosa surface and maintain their microecological regulatory effects in the long term.

[0005] In summary, current 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, failing 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 issue urgently needing to be addressed in the fields of clinical microbiology and translational medicine. Summary of the Invention

[0006] In view of the defects and deficiencies in the existing technology, the present invention provides a Lactobacillus japonicus with highly efficient antibacterial and biofilm inhibition capabilities and its application in the preparation of health products.

[0007] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a *Lactobacillus japonicus* with highly efficient antibacterial and biofilm-inhibiting capabilities, characterized in that: the *Lactobacillus japonicus* ( Lactobacillus jensenii H21-AN-Na1-1 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. 35328.

[0008] In a second aspect, the present invention provides a composition comprising live Lactobacillus japonicus and its products as described in the first aspect, inactivated bacterial cells, fermentation broth, or cell-free supernatant.

[0009] In a third aspect, the present invention provides the use of Lactobacillus japonicus described in the first aspect or the composition described in the second aspect in the preparation of health products.

[0010] Alternatively, in the above applications, the health product can inhibit pathogens.

[0011] Alternatively, in the above applications, the health product can inhibit pathogens, eliminate biofilms of pathogens, and / or reduce the load of pathogens.

[0012] Alternatively, in the above applications, the pathogenic bacteria include Gardnerella vaginalis, Group B Streptococcus, Escherichia coli, Staphylococcus aureus, or Candida albicans.

[0013] Alternatively, in the above applications, the health product can be used to prevent or treat diseases associated with the aforementioned pathogenic bacteria infection.

[0014] 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.

[0015] 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.

[0016] Alternatively, in the above applications, the product may also contain a pharmaceutically, health-promoting, or food-grade carrier.

[0017] Preferably, the carrier includes fillers, binders, wetting agents, disintegrants, lubricants, or flavoring agents known in the art.

[0018] 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.

[0019] 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.

[0020] Preferably, the term "external use" refers to application to the skin, mucous membranes, or vulva.

[0021] 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).

[0022] Preferably, the health products, functional foods, food supplements, and foods for special medical purposes are for oral administration.

[0023] 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.

[0024] Alternatively, in the above applications, the product may also contain a second component.

[0025] Preferably, the second component includes probiotics, postbiotics, prebiotics, antibacterial agents, or immunomodulators.

[0026] Compared with the prior art, the present invention has the following advantages: This invention isolates a novel *Lactobacillus japonicus* species with highly efficient antibacterial and biofilm-inhibiting capabilities from vaginal secretions of healthy women. The *Lactobacillus japonicus* species of this invention exhibits significant inhibitory effects against *Gardnerella vaginalis*, Group B Streptococcus, *Escherichia coli*, *Staphylococcus aureus*, and *Candida albicans* infections, and also demonstrates good inhibitory ability against the biofilms of these pathogenic bacteria, reducing the risk of infection. It can be used for the prevention or treatment of diseases related to infections caused by these pathogenic bacteria. The *Lactobacillus japonicus* species of this invention has high lactic acid production, promotes a healthy microecological environment, has excellent cell colonization ability, and enhances the probiotic effect. Furthermore, this invention demonstrates the good safety profile of *Lactobacillus japonicus* species of this invention through hemolysis tests, antibiotic resistance gene detection, and functional verification. Attached Figure Description

[0027] 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 35328.

[0028] Figure 2 Blood agar hemolysis test for strain number 35328.

[0029] Figure 3 Long-read whole-genome sequencing results of strain number 35328.

[0030] Figure 4 Comparison of lactic acid concentrations secreted by strain 35328, its market products, and the control.

[0031] Figure 5 The direct inhibitory effect of strain 35328 and its marketed products on Group B Streptococcus is shown in the figure above. The top image compares colonies after serial dilutions in BHI culture dishes, with each column representing a concentration from right to left, decreasing 10-fold to the left. The bottom image shows a statistical comparison of CFU readings.

[0032] Figure 6 Tracking the inhibitory effect of strain 35328 supernatant on Gardnerella vaginalis over 48 hours (145 cycles).

[0033] Figure 7 Tracking the inhibitory effect of strain No. 35328 and its market products on Escherichia coli over 24 hours (77 cycles).

[0034] Figure 8 The inhibitory effect of strain No. 35328 and its market products on Escherichia coli within 24 hours.

[0035] Figure 9 Tracking the inhibitory effect of strain No. 35328 and its market products on Staphylococcus aureus over 24 hours (77 cycles).

[0036] Figure 10 The inhibitory effect of strain No. 35328 and its market products on Staphylococcus aureus over 24 hours.

[0037] Figure 11 Tracking the inhibitory effect of strain No. 35328 and its market products on Group B Streptococcus for 24 hours (77 cycles).

[0038] Figure 12 The inhibitory effect of strain No. 35328 and its market products on Group B Streptococcus within 24 hours.

[0039] Figure 13 Tracking the inhibitory effect of strain No. 35328 and its market products on Candida albicans over 24 hours (77 cycles).

[0040] Figure 14 The inhibitory effect of strain No. 35328 and its market products on Candida albicans within 24 hours.

[0041] Figure 15 The inhibitory effect of strain No. 35328 and its market products on the biofilm of Staphylococcus aureus.

[0042] Figure 16 The biofilm inhibitory effect of strain No. 35328 and its market products on Group B Streptococcus.

[0043] Figure 17 The biofilm inhibition effect of strain No. 35328 and its market products on Candida albicans.

[0044] Figure 18 Adsorption of strain 35328 and its marketed products on epithelial VK2 / E6E7 cells. Detailed Implementation

[0045] 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.

[0046] 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.

[0047] 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.

[0048] Example: This invention isolates a novel Lactobacillus janniae with highly efficient antibacterial and biofilm-inhibiting capabilities from vaginal secretions of healthy women.

[0049] The Lactobacillus janniae ( Lactobacillus jensenii H21-AN-Na1-1 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. 35328.

[0050] 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).

[0051] 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. After obtaining the pure culture, an appropriate amount of fresh bacterial cells was used for MALDI-TOF MS identification. The samples were then processed with a matrix before being analyzed by the MALDI-TOF MS system. 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.

[0052] The mass spectrometry results of this bacterium, as identified by MALDI-TOF MS, are as follows: Figure 1 As shown, the fingerprint pattern conforms to that of Lactobacillus janniae.

[0053] 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 japonicus* 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.

[0054] 1.2.1 Experimental Methods (1) Inoculate the bacterial strain onto a blood agar plate.

[0055] (2) Anaerobic culture at 37°C for 24 hours.

[0056] (3) Observe the type of hemolysis around the colony.

[0057] 1.2.2 Experimental Results Figure 2 The results showed that after 24 hours of culture on blood agar plates, no α- or β-hemolytic loops were observed, demonstrating the safety of *Lactobacillus japonicus* for human use.

[0058] 1.3 Long-read sequencing of the entire genome and antibiotic resistance genes 1.3.1 Experimental Methods This invention utilizes whole-genome 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.

[0059] (2) Data processing: Perform quality control and filtering on the raw data obtained from sequencing to remove low-quality sequences.

[0060] (3) Genome alignment: The query genome is aligned with the Lactobacillus janniae reference genome.

[0061] (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.

[0062] (5) Annotation: Annotate the functions of the queried genome, and identify genes, functional regions, etc.

[0063] 1.3.2 Experimental Results Long-read sequencing whole-genome sequencing results are as follows Figure 3 As shown, no known mobile antibiotic resistance genes were detected in the potential plasmids identified by sequencing. The safety of its use was verified at the genetic level.

[0064] 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.

[0065] 1.4.2 Experimental Results As shown in Table 1, the results indicate that this Lactobacillus janniae is sensitive to common antibiotics.

[0066] Table 1: Size of the inhibition zone 2. Lactic acid production 2.1 Experimental Methods D-Lactate Production Assay: The D-lactate colorimetric assay kit was used, strictly following the manufacturer's instructions. The supernatant consisted of *Lactobacillus japonicus* 35328 (as described in this invention) and the purchased standard *Lactobacillus japonicus* CCUG 44499 from a bacterial bank as controls. Since there were no commercially available *Lactobacillus japonicus* products, *Lactobacillus* live bacteria capsules (Dingjunsheng DJS) were purchased as a control. After anaerobic culture in fresh NYCIII medium for 24 hours, the OD value was adjusted to 1, and the culture was centrifuged to obtain cell-free supernatant. To ensure that the sample readings were within the detection range of the standard curve, the culture supernatant was diluted 500-fold with ultrapure water. Each experiment had three replicates, and the experiment was repeated five times. The following figures show the common trends of the three experiments. The statistical method was one-way ANOVA. p<0.1, p<0.0001.

[0067] 2.2 Experimental Results Experimental results are as follows Figure 4 As shown, under the same culture conditions, strain 35328 of this test strain secretes a high concentration of D-lactic acid, exceeding 340,000 nmol / mL, which is significantly superior to the Lactobacillus janniae strain in the bacterial bank and commercial products. This demonstrates that this strain has a better function in controlling the acidic environment and inhibiting the growth of other bacteria.

[0068] 3. Inhibition of pathogenic bacteria 3.1 Direct inhibitory effect of Lactobacillus japonicus culture on Group B Streptococcus. Activation: Group B Streptococcus (CCUG 29376) was transferred into the resistance plasmid pMSP3535 and inoculated into BHI medium (8 μg / mL erythromycin) and cultured overnight at 37°C. Then, 1 mL of Lactobacillus suspension (OD=1) and 1 mL of Group B Streptococcus suspension (OD=0.2) were mixed in 2 mL of NYCIII medium and anaerobically cultured for 24 hours. The co-culture was removed, serial dilutions were performed, and 10 µL of each dilution was plated onto BHI plates containing erythromycin (8 µg / mL). The plates were incubated aerobically at 37°C overnight. Colony counting was performed within the appropriate dilution range (20–200 colonies).

[0069] The results are as follows Figure 5 As shown, the results indicate that *Lactobacillus japonicus* of the present invention has a direct inhibitory effect on Group B Streptococcus. Compared with the control group without the addition of *Lactobacillus japonicus* of the present invention and the control group with the addition of commercial products, *Lactobacillus japonicus* of the present invention significantly inhibited the growth of Group B Streptococcus.

[0070] 3.2 Inhibitory effect of Lactobacillus japonicus fermentation supernatant on 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.

[0071] 3.2.1 Inhibitory effect of Lactobacillus japonicus fermentation supernatant on Gardnerella vaginalis 3.2.1.1 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 supernatant of *Lactobacillus japonicus* (OD=1) for testing. 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 figure below shows the common trend between the two experiments. One-way ANOVA was used for statistical analysis.

[0072] 3.2.1.2 Experimental Results like Figure 6 As shown, after 24 hours, the supernatant of *Lactobacillus japonicus* exhibited an increasingly stronger inhibitory effect on *Gardnerella vaginalis*, reaching its peak at the end of the 48-hour experiment. This experiment demonstrates the inhibitory effect of *Lactobacillus japonicus* supernatant on the growth of *Gardnerella vaginalis*.

[0073] 3.2.2 Inhibitory effect of Lactobacillus jenny fermentation supernatant on various other pathogenic bacteria 3.2.2.1 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. 600After 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<0.01, p<0.001, p<0.0001.

[0074] 3.2.2.2 Experimental Results like Figure 7 and Figure 8 As shown, Escherichia coli infection, especially ST131, is very common in urethritis, vaginitis, and other inflammatory infections. A comparison was made using the *Lactobacillus japonicus* of this invention and a commercially available product (vaginal lactobacillus active capsules (Dingjunsheng DJS)). The experimental results showed that *Lactobacillus japonicus* 35328 had a significant inhibitory effect on *Escherichia coli* from the moment of addition. This inhibitory effect lasted for 24 hours until the end of the experiment. At the end of the experiment, *Lactobacillus japonicus* 35328 statistically significantly reduced the number of *Escherichia coli*, and also showed a significantly better effect compared to commercially available products.

[0075] like Figure 9 and Figure 10 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 *Lactobacillus japonicus* of this invention, the supernatant of *Lactobacillus japonicus* of this invention was compared with commercially available products. The results showed that *Lactobacillus japonicus* 35328 had a significant inhibitory effect on *Staphylococcus aureus*. At the end of the experiment, *Lactobacillus japonicus* 35328 statistically significantly reduced the growth of *Staphylococcus aureus*, especially compared to commercially available products, *Lactobacillus japonicus* 35328 had a more significant inhibitory effect on *Staphylococcus aureus*.

[0076] like Figure 11 and Figure 12As shown, Group B Streptococci are common pathogens causing skin, vaginal, and intestinal infections, especially dangerous in premature infants. A comparison was made between the *Lactobacillus japonicus* 35328 of this invention and commercially available products. The experimental results showed that *Lactobacillus japonicus* 35328 exhibited a significant inhibitory effect on Group B Streptococci from the moment of its introduction. This significant inhibitory effect became more pronounced as the experiment progressed. At the end of the experiment (24 hours), *Lactobacillus japonicus* 35328 statistically significantly reduced the growth of Group B Streptococci, and also showed a statistically significantly greater inhibitory effect compared to commercially available products.

[0077] like Figure 13 and Figure 14 As shown, Candida albicans is a common cause of cutaneous, vaginal, and nosocomial infections, and is a key pathogen of concern for WHO antibiotic resistance. A comparison was made between the *Lactobacillus japonicus* 35328 of this invention and commercially available products. The results showed that *Lactobacillus japonicus* 35328 had a significant inhibitory effect on Candida albicans. This inhibitory effect improved as the experiment progressed. At the end of the 24-hour experiment, *Lactobacillus japonicus* 35328 statistically significantly reduced the growth of Candida albicans. Compared to commercially available products, *Lactobacillus japonicus* 35328 also showed a significantly more pronounced inhibitory effect on Candida albicans.

[0078] 4. Anti-biofilm test (crystal violet staining method) 4.1 Biofilm culture Inoculation with pathogenic bacteria: Take the bacterial suspension of Staphylococcus aureus, Group B Streptococcus, and Candida albicans in the logarithmic phase, dilute with LB medium to OD=0.1 and incubate at 37°C for 24 hours.

[0079] 4.2 Quantitative analysis of biofilms Washing: Discard the supernatant and wash 3 times with PBS buffer; air dry at room temperature for 15-25 minutes.

[0080] Staining: Add 50 μL of crystal violet staining solution (1% w / w) to each well and incubate at room temperature for 25 minutes; Destaining: Discard the staining solution, wash 3 times with PBS, add 100 μL of 30% glacial acetic acid for 20 minutes to destain; Detection: OD measured by ELISA reader 595 value.

[0081] The following figures illustrate the common trends across the three experiments. The statistical method used was one-way ANOVA. p<0.01, p<0.0001.

[0082] 4.3 Experimental Results Experimental results are as follows Figures 15 to 17As 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 *Lactobacillus japonicus* of this invention was added, indicating that the supernatant of *Lactobacillus japonicus* significantly inhibited biofilm formation in these bacteria. Compared to the control, the biofilm concentration of different pathogenic bacteria with the supernatant of *Lactobacillus japonicus* 35328 was reduced by 2-3 times compared to the control without the supernatant of *Lactobacillus japonicus*.

[0083] 5. Adhesion experiment on vaginal cells 5.1 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 -6 10 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<0.1, p<0.0001.

[0084] 5.2 Experimental Results like Figure 18 As shown, the experimental results indicate that the Lactobacillus japonicus of the present invention has an extremely strong adsorption capacity for cells, which is significantly higher than that of commercial products, proving its superior colonization ability.

[0085] 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 strain of *Lactobacillus japonicus* with highly efficient antibacterial and biofilm-inhibiting capabilities, characterized in that: The Lactobacillus janniae ( Lactobacillus jensenii H21-AN-Na1-1 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. 35328.

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

3. The use of Lactobacillus japonicus of claim 1 or the composition of 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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