Lactobacillus johnsonii and application thereof

Abstract

The invention relates to lactobacillus johnsonii and application thereof, and belongs to the technical field of microorganisms. The lactobacillus johnsonii JoA9-1 disclosed by the invention is preserved in the China Center for Type Culture Collection, and the preservation number of the lactobacillus johnsonii JoA9-1 is CCTCCM2025369. The lactobacillus johnsonii JoA9-1 is free of toxic factors, free of hemolysis and good in safety, and has the beneficial effects that the lactobacillus johnsonii JoA9-1 can produce various organic acids, has tolerance to an acid environment and can adapt to and maintain the acid environment of a female genital tract; the reproduction of pathogenic bacteria can be inhibited; the formation of candida albicans hyphae can be inhibited; the composition has an immunoregulation function, has an inhibitory effect on inflammatory factor TNF alpha superior to that of clotrimazole treatment, and can significantly enhance treatment of mouse vaginitis; the treatment occurrence part is at the vaginal infection part, and local tissues of mice with candida albicans vaginitis can be repaired; the relative abundance of the vagina microbial flora after the mouse is infected by the candida albicans vaginitis is regulated, and the vagina microbial flora environment is recovered.

Description

Technical Field

[0001] The invention relates to Lactobacillus johnsonii and application thereof, belonging to the technical field of microorganisms. Background Art

[0002] Female reproductive tract diseases encompass a wide range of types, such as vaginitis, cervicitis, and pelvic inflammatory disease, and their incidence is on the rise worldwide. Traditional treatments often focus on antibacterial or anti-inflammatory treatments targeting pathogens, but these often result in high recurrence rates and an imbalance in the vaginal microbiome. Disturbances in the female reproductive tract microbiome are characterized by increased microbial diversity and overgrowth of anaerobic bacteria. When the number of lactic acid bacteria decreases, the inhibitory effect on other microorganisms also decreases, leading to an increase in pathogenic bacteria and greater diversity in the reproductive tract. Vaginal Lactobacillus, as the dominant bacteria in the normal vaginal flora, plays a key role in maintaining vaginal microecological balance.

[0003] Lactobacillus inhibits the growth of pathogenic and opportunistic pathogens by producing lactic acid, hydrogen peroxide, antibacterial peptides, and regulating immunity, which is very important for maintaining women's reproductive health. Lactobacillus has long-term positive effects on vaginal health, including maintaining vaginal microecological balance, enhancing local immune function, reducing inflammatory responses, improving vaginal epithelial barrier integrity, reducing disease recurrence rates, and having anti-cancer effects. Lactobacillus johnsonii is a type of vaginal lactobacillus, a Gram-positive bacterium that is facultatively anaerobic. Under appropriate culture conditions, Lactobacillus johnsonii can ferment a variety of sugars to produce lactic acid, which is one of its important metabolic characteristics and is crucial for maintaining an acidic vaginal environment.

[0004] Vaginitis, also known as vaginosis, is a common condition seen in gynecological clinics. Patients often experience abnormal vaginal discharge and odor, along with a range of symptoms such as itching, burning, and irritation. Vaginitis is primarily a reproductive tract disease caused by the invasion of pathogens due to internal or external factors, disrupting the microecological balance within the healthy reproductive tract. Depending on the cause, there are over ten types of vaginitis, including bacterial vaginosis, vulvovaginal candidiasis, Trichomonas vaginosis, senile vaginosis, viral vaginosis, and aerobic vaginosis. Vulvovaginal candidiasis (VVC) is often caused by an overgrowth of Candida albicans and often occurs in conditions such as long-term use of broad-spectrum antibiotics, poor blood sugar control in diabetic patients, and pregnancy, which can lead to compromised immune function or altered local microenvironment. Typical symptoms include vulvar itching and burning, and a dreg-like vaginal discharge. Candida species associated with VVC include Candida albicans, Candida glabrata, Candida parapsilosis, and Candida tropicalis, with Candida albicans being the most common pathogen. Bacterial vaginosis (BV) is a common type of vaginitis in women of childbearing age, primarily caused by an imbalance in the normal vaginal flora, with overgrowth of Gardnerella and anaerobic bacteria and a decrease in Lactobacilli. Its onset is closely associated with factors such as frequent sexual activity, vaginal douching, and antibiotic use.

[0005] Current treatment for vaginitis primarily relies on antibiotics. While antibiotics have a positive effect on disease control, their widespread use has led to an increasing problem of antibiotic resistance in vaginitis treatment. In candidal vaginitis, Candida species have decreased sensitivity to drugs such as fluconazole and clotrimazole, increasing the risk of recurrence and treatment failure. Antibiotics not only kill pathogens but also disrupt the normal vaginal flora. Long-term use may increase the number of certain bacteria in the vagina, disrupting the mutual control between these flora, leading to an imbalance in the vaginal flora and potentially inducing or exacerbating vaginitis. The development of new antibiotics and alternative therapies, such as probiotics, antimicrobial peptides, and immunotherapy, is being promoted. Probiotics can inhibit the growth of pathogens and promote the recovery of beneficial flora, potentially helping to prevent and treat vaginitis.

[0006] In the vaginal flora of healthy women, Lactobacillus johnsonii is usually one of the dominant bacteria, and the stability of its number and activity is crucial for maintaining the balance and health of the vaginal microecology. There are some published patents and literature reports on the role of Lactobacillus johnsonii. For example, Chinese patent CN118185812A discloses a Lactobacillus johnsonii and its application. However, considering that to date, there are few studies evaluating the anti-candida activity and probiotic properties of Lactobacillus johnsonii strains in VVC, as well as the differences in performance between different strains of the same genus, screening vaginal lactobacilli with better probiotic ability is an unmet need. In the case of female reproductive tract diseases, supplementing with probiotics such as Lactobacillus johnsonii can help restore the balance of the vaginal microecology and assist in the treatment of vaginal diseases. Summary of the Invention

[0007] The purpose of the present invention is to address the defects of the existing technology and propose a Lactobacillus johnsonii and its application, which has good safety and probiotic properties, can produce lactic acid and hydrogen peroxide, and effectively inhibit the growth of pathogenic bacteria.

[0008] The present invention solves the technical problem through the following technical solutions: First, a Lactobacillus johnsonii ( Lactobacillus johnsonii ) JoA9-1 was obtained independently from the vaginal secretions of healthy women of childbearing age and deposited in the China Center for Type Culture Collection with the accession number CCTCCM2025369, Lactobacillus johnsonii JoA9-1.

[0009] The present invention further provides a method for identifying and selecting the Lactobacillus johnsonii strain, comprising obtaining the Lactobacillus johnsonii strain with the highest acid production capacity through appearance feature identification, genome identification, and quantitative determination of organic acids by the metabolome.

[0010] The Lactobacillus johnsonii JoA9-1 of the present invention has the following morphological characteristics: In MRS solid medium, the colonies have regular edges, are opaque, white and round, full in the middle, and have a smooth and moist surface; (2) They are Gram-positive, rod-shaped, and often exist singly, in pairs, or in short chains.

[0011] The Lactobacillus johnsonii JoA9-1 of the present invention has no virulence factors, is non-hemolytic, has good safety, has a strong lactic acid production ability, can reduce the pH of the female vagina, and can produce the antibacterial substance hydrogen peroxide, which can effectively inhibit the reproduction of pathogenic bacteria.

[0012] The present invention further provides genome analysis of Lactobacillus johnsonii.

[0013] The present invention further provides a method for culturing a Lactobacillus johnsonii strain, the method comprising inoculating the Lactobacillus johnsonii into a specific culture medium for proliferation and culturing, and ultimately obtaining the proliferated Lactobacillus johnsonii strain. The culture medium is an MRS culture medium.

[0014] The present invention further provides the use of Lactobacillus johnsonii strains in female reproductive tract diseases, including improving the vaginal microecological environment, improving the pathogenic bacteria load in vaginal secretions, the expression level of inflammatory factors (TNFα), inflammatory cell infiltration and vaginal mucosal integrity.

[0015] When used to improve the vaginal microecological environment, the product acts as an antibacterial agent or bactericide to inhibit or kill pathogenic bacteria.

[0016] The inhibition of pathogens in the above application includes inhibiting the growth of one or more of Escherichia coli, Gardnerella vaginalis, Candida albicans, Staphylococcus aureus and Salmonella.

[0017] By HE staining sections of intact tissues such as mouse heart, liver, spleen, lung, kidney, vagina and ovary, it was confirmed that the aforementioned Lactobacillus johnsonii JoA9-1 has good safety and probiotic properties. The effect of Lactobacillus johnsonii on improving the relative abundance of vaginal microbial flora in a mouse vaginitis model. Lactobacillus johnsonii JoA9-1 (Accession No.: CCTCC M 2025369) has significant innovative significance in the field of microbial technology, especially in maintaining female reproductive tract health. Its core advantages include high safety (no virulence factors, no hemolysis), adaptability to the acidic environment of the reproductive tract, inhibition of pathogenic bacteria and Candida albicans hyphae formation, immune regulation, and local tissue repair. Its key advantages include its ability to regulate and repair the vaginal microbiome in mice following Candida albicans vaginitis infection. Specifically, 1. Accurately improve the imbalance of relative abundance of vaginal microbial flora after infection The occurrence of Candida albicans vaginitis is closely related to the disorder of the vaginal microbial flora structure. Under normal conditions, Lactobacillus is the dominant flora in the vagina, which inhibits the excessive reproduction of harmful bacteria by producing organic acids and competing for nutrients. After infection, Candida albicans proliferates rapidly, while the abundance of other harmful microorganisms (such as certain conditional pathogens such as Proteus) increases, while the abundance of beneficial bacteria such as Lactobacillus decreases significantly, forming an unbalanced state in which harmful bacteria dominate and beneficial bacteria are insufficient. Lactobacillus johnsonii JoA9-1 can specifically improve this imbalance problem: in the intervention of a mouse model of Candida albicans vaginitis, this strain can gradually increase the relative abundance of Lactobacillus microorganisms in the vagina through its own colonization ability in the vaginal mucosa, while significantly reducing the number and relative proportion of Candida albicans colonization; in addition, for other conditional pathogens whose abundance increases abnormally during the infection process, this strain can also inhibit their reproduction by competing for nutrients and secreting antibacterial metabolites (such as various organic acids, H2O2, etc.), thereby promoting the vaginal microbial flora from an unbalanced state to a state dominated by beneficial bacteria, and building a stable flora foundation for vaginal health.

[0018] 2. Effectively restore the vaginal microbial flora microecological environment The health of the vaginal microbiome depends not only on the proper balance of bacterial abundance, but also on the symbiotic relationship between the microbiome, metabolic balance, and synergistic maintenance of the vaginal microenvironment. Candida albicans infection can disrupt this homeostasis: on the one hand, the massive growth of Candida albicans will crowd out the living space of other microorganisms, disrupting the competition and symbiotic balance between the microbiome; on the other hand, its metabolites may change microenvironmental parameters such as the pH value and redox potential in the vagina, further inhibiting the growth of beneficial bacteria (such as Lactobacillus), forming a vicious cycle of "infection-microbial imbalance-microenvironmental deterioration." Lactobacillus johnsonii JoA9-1 has a dual role in restoring the microecological environment of the vaginal microbiome: 1. Rebuilding the symbiotic balance of the bacterial flora: After colonization, this strain can act as a dominant beneficial bacterium to form a synergistic effect with other normal microorganisms in the vagina (such as other Lactobacillus species and a small amount of beneficial anaerobic bacteria), inhibiting the excessive expansion of Candida albicans and other harmful bacteria, restoring the species diversity and the mutually restrictive and interdependent symbiotic relationship among the bacterial flora, and reducing the risk of a bacterial flora structure dominated by a single harmful bacterium.

[0019] 2. Synergistically optimize the vaginal microenvironment: On the one hand, this strain can produce a variety of organic acids (such as lactic acid, acetic acid, etc.), maintaining an acidic environment in the vagina (pH 3.8-4.5). This environment is not only conducive to the growth of itself and other lactobacilli, but also directly inhibits the reproduction of pathogenic bacteria such as Candida albicans. On the other hand, its metabolic activity can regulate the composition of metabolites in the vagina, reduce the accumulation of harmful metabolites, and increase the content of beneficial metabolites (such as lactic acid, H2O2, etc.), providing a suitable metabolic environment for bacterial health, thereby achieving a comprehensive restoration of the vaginal microbial flora microecological environment and reducing the risk of recurrence of Candida albicans vaginitis.

[0020] In summary, Lactobacillus johnsonii JoA9-1 provides a new path for targeted microbial flora intervention in the treatment of Candida albicans vaginitis by precisely regulating the relative abundance of vaginal microbial flora after infection and effectively restoring the microecological environment. Different from traditional drugs that only target pathogenic bacteria, it focuses more on improving vaginal health from the root of the microecology, and has higher safety and long-term maintenance value.

[0021] The Lactobacillus johnsonii JoA9-1 of the present invention has no virulence factors, is non-hemolytic, and has good safety. Its beneficial effects are: it can produce multiple organic acids and has tolerance to acidic environments, and can adapt to and maintain the acidic environment of the female reproductive tract; the bacterium can produce a certain amount of hydrogen peroxide, has effective antibacterial ability against Escherichia coli, Staphylococcus aureus, Candida albicans, etc., and can inhibit the reproduction of pathogenic bacteria; it can inhibit the formation of Candida albicans hyphae, indicating that it can improve vaginal inflammation to a great extent; it has an immunomodulatory function, can reduce the expression level of the pro-inflammatory factor TNFα, and has an inhibitory effect on the inflammatory factor TNFα that is better than clotrimazole treatment, and can significantly enhance the treatment of vaginitis in mice; during the treatment process, it has no effect on the main organs of the mouse, such as the heart, liver, spleen, lungs, and kidneys, and has good safety. The treatment occurs at the site of vaginal infection, and can repair the local tissue of mice with Candida albicans vaginitis; it regulates the relative abundance of vaginal microbial flora after Candida albicans vaginitis infection in mice, and restores the vaginal microbial flora environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a front view photo of Lactobacillus johnsonii JoA9-1 strain.

[0023] Figure 2 This is a Gram-stained photograph of Lactobacillus johnsonii JoA9-1 strain.

[0024] Figure 3 KEGG gene function analysis of Lactobacillus johnsonii JoA9-1 strain.

[0025] Figure 4 This is the genome circle analysis of Lactobacillus johnsonii JoA9-1 strain.

[0026] Figure 5 This is the low pH growth tolerance curve of Lactobacillus johnsonii JoA9-1 strain.

[0027] Figure 6 This is the acid production capacity and growth curve of Lactobacillus johnsonii JoA9-1.

[0028] Figure 7 This is a diagram showing the formation and changes of Candida albicans and its hyphae (400 times) in the vaginal lavage fluid of mice.

[0029] Figure 8 HE staining of mouse heart, liver, lung, kidney and ovary tissues (200 times), HE staining of spleen tissue (400 times), and pathological changes of mouse vaginal tissue (HE staining 400 times).

[0030] Figure 9 The expression of inflammatory factor TNFα after treatment with Lactobacillus johnsonii JoA9-1 and clotrimazole.

[0031] Figure 10 Species abundance map at the phylum level.

[0032] Figure 11 Shannon index and Chao graph after treatment with Lactobacillus johnsonii JoA9-1 and clotrimazole.

[0033] Figure 12 Heat map of species clustering at the gate level for Lactobacillus johnsonii JoA9-1 and clotrimazole treatment.

[0034] Figure 13 Figure 3. Abundance of Proteobacteria and Firmicutes after treatment with Lactobacillus johnsonii JoA9-1 and clotrimazole.

[0035] Figure 14 Figure 2 shows the abundance of Lactobacillus genus after treatment with Lactobacillus johnsonii JoA9-1 and clotrimazole.

[0036] The strain deposit information is as follows: strain name: JoA9-1; deposit number: CCTCCM2025369; Category naming: Lactobacillus johnsonii JoA9-1; Depository: China Center for Type Culture Collection; Depository address: Wuhan University, Wuhan City, Hubei Province; Deposit date: March 4, 2025. DETAILED DESCRIPTION

[0037] The methods used in the following examples are all conventional methods. Unless otherwise specified, the raw materials, reagents and equipment used can be purchased through commercial channels and will not be described in detail.

[0038] Lactobacillus johnsonii JoA9-1, derived from vaginal secretions of healthy women of childbearing age, was identified as Lactobacillus johnsonii based on appearance characteristics and 16S rRNA. Lactobacillus johnsonii ), and then the subtype of the strain was finally determined through metabolomics and genomic sequencing.

[0039] Example 1 Isolation of strains Vaginal secretion samples were collected from healthy women of childbearing age by scraping the posterior third of the vaginal canal with a vaginal swab in a clockwise motion. Immediately after removal, the sample was placed into a 10mL centrifuge tube and numbered. 5mL of MRS liquid culture medium and 2mL of liquid paraffin were added to the centrifuge tube, which was then placed on a shaker at an angle for 48 hours and thoroughly shaken. A 10μL inoculating loop was inserted below the liquid paraffin level, and a small amount of liquid was spread onto MRS solid culture medium supplemented with calcium carbonate. The sample was numbered and incubated in an anaerobic incubator at 37°C. After 48 hours of incubation, colonies with a distinct calcium-soluble zone on the calcium-containing culture medium were selected for Gram staining and microscopic examination. Colony morphology and bacterial body size were carefully observed and recorded. Suspected bacteria that met the criteria were preliminarily identified as Lactobacilli. The remaining suspected Lactobacilli colonies were then inoculated into liquid culture medium and streaked onto solid plates until pure bacteria were confirmed (streaking at least three times). Based on the morphology of the colonies on the MRS solid medium, use a 1μL inoculating loop to select individual colonies and re-streak them onto new solid medium. Record the number and continue incubating under anaerobic conditions at 37°C for 48 hours. Subculture until the strain is completely purified. Select individual colonies and inoculate them onto a larger solid medium. Incubate at 37°C under anaerobic conditions for 48 hours to expand the bacterial culture. Prepare a number of cryovials and 2mL polystyrene prepolymer tubes. Add 200μL of sterilized glycerol and 800μL of broth to the cryovials. Add 1mL of sterilized PBS to the polystyrene prepolymer tubes and record the number. Use an inoculating loop to scrape a small amount of the amplified strain into a 2mL polystyrene prepolymer tube, stir, and store at -20°C. Use the same inoculating loop to scrape the remaining colonies into a cryovial, stir, and store at -80°C.

[0040] Example 2 Identification of strains A purified single colony was picked and placed in liquid culture medium for 24 hours to prepare a bacterial suspension. Part of the suspension was cultured, and part of the suspension was used for DNA extraction and PCR amplification of the 16SrRNA gene. The purified PCR product was then sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. The PCR results were compared by BLAST and finally confirmed to be Lactobacillus johnsonii.

[0041] Take the PCR-verified strain of Lactobacillus johnsonii JoA9-1 and streak the bacterial liquid onto MRS solid medium. Incubate anaerobically at 37℃ for 24-48h. The colony has regular edges, is opaque, white on the front, convex in the middle, and has a smooth and moist surface. Figure 1 .

[0042] Using the smear method, take a small amount of colonies, dilute with 200μL sterile ddH2O, evenly spread on a slide, fix with a slight heat of an alcohol lamp, stain with Gram staining, and observe its morphology, size, arrangement, and whether there are spores under a microscope. Lactobacillus johnsonii is a Gram-positive bacterium, rod-shaped, often existing alone, in pairs or short chains, without spores.Figure 2 .

[0043] Example 3 Strain genome sequencing Lactobacillus johnsonii JoA9-1 was inoculated into 5 mL of anaerobic MRS broth (sealed with liquid paraffin) and cultured until late logarithmic growth. Whole-genome DNA was extracted and subjected to third-generation whole-genome sequencing. After assembly and annotation, the nucleotide sequence was entered into the KEGG database for gene function analysis and genome circle mapping analysis, and into the Virulence Factor Databases (VFDB) for virulence factor analysis. The results showed that this strain does not possess virulence factors. Table 1 shows that the entire genome is 2,039,243 bp in length, contains one contig, has a GC content of 34.75%, and an N50 value of 2,039,243 bp. In addition, the KEGG database integrates data on genomes, chemical molecules, and biochemical systems. The core metabolic pathway database divides biological metabolic pathways into six major categories: metabolism, genetic information processing, environmental information processing, cellular processes, organismal systems, and human diseases. Figure 3 KEGG gene function analysis of Lactobacillus johnsonii JoA9-1 strain, Figure 4 This is a genome map analysis of Lactobacillus johnsonii JoA9-1. The analysis revealed that functional annotation of Lactobacillus johnsonii JoA9-1 genes focused on three key areas: metabolism, environmental information processing, and genetic information processing. A total of 639 genes were annotated in the metabolic pathway, with the largest number (193) associated with carbohydrate metabolism, suggesting that Lactobacillus johnsonii JoA9-1 may possess strong carbohydrate metabolism capabilities. A total of 225 genes were annotated in the genetic information processing pathway, with 83 genes associated with replication and repair being the most numerous. A total of 175 genes were annotated in the environmental information processing pathway, with 104 genes associated with membrane transport being the most numerous, primarily genes related to various transmembrane transport proteins, such as ABC transporters and PTS transporters. This suggests that this bacterium has a strong ability to adapt to its external environment and can withstand the stress of environmental changes to a certain extent.

[0044] Table 1 Analysis results of Lactobacillus johnsonii JoA9-1QUAST

[0045] Example 4 Acid resistance test Lactobacillus johnsonii was activated in MRS broth at pH 5.7 and cultured overnight at 37°C for 12 hours. An appropriate amount of the bacterial suspension was centrifuged at 10,000 rpm for 10 minutes, and the supernatant discarded. The bacterial slurry was resuspended in sterile PBS buffer and calibrated to an OD600 of 1 at 600 nm using a microplate reader. A 1% inoculum was inoculated into MRS broth at pH 4.5, prepared and sterilized in 10 mL centrifuge tubes. Oxygen was isolated using sterile liquid paraffin and incubated anaerobically at 37°C. The OD600 concentration of the culture was measured every 2 hours. Before each aspiration, the culture was mixed with a turbine and allowed to stand for stratification to prevent sedimentation. Uninoculated MRS broth at pH 4.5 was used as a blank control. Measurements were taken continuously for 24 hours, and the data were recorded to plot a pH-tolerance growth curve. The results showed that Lactobacillus johnsonii JoA9-1 could grow in a low pH environment (pH 4.5) and had acid resistance. Figure 5 .

[0046] Example 5 Acid production capacity determination and growth curve experiment Lactobacillus johnsonii JoA9-1 was revived and inoculated onto MRS solid medium in an anaerobic chamber at 37°C for 48 hours. A single colony was picked with an inoculating loop and transferred to 40 mL of sterilized broth, sealed with liquid paraffin, and shaken for 8 hours. The culture supernatant was collected into a centrifuge tube and centrifuged at 400-1000 g for 10 minutes to remove cells. The supernatant was then centrifuged at 2000 g to remove cell debris and precipitate. The supernatant was then sent to Suzhou Pamino Biopharmaceutical Co., Ltd. for targeted organic acid analysis. The results showed that Lactobacillus johnsonii JoA9-1 exhibited strong lactic acid production, with a lactic acid content of 312.6732 μg / mL. In addition, the citric acid content was as high as 2300.792 μg / mL. See Table 2 for the quantification of organic acids in Lactobacillus johnsonii JoA9-1.

[0047] Table 2 Quantitative results of organic acids of Lactobacillus johnsonii JoA9-1

[0048] After two generations of inoculation of Lactobacillus johnsonii JoA9-1 in MRS medium, single colonies with regular, opaque, white, rounded edges, a plump center, and a smooth, moist surface were selected and transferred to MRS liquid medium. The colonies were incubated at an angle in a shaker at 37°C overnight. An appropriate amount of the bacterial suspension was centrifuged at 10,000 rpm for 10 minutes, and the supernatant discarded. The suspension was resuspended in sterile PBS buffer and calibrated to an OD600 of 1 on a microplate reader. A 1% inoculum of the suspension was inoculated into previously prepared, sterilized MRS broth aliquoted in 10 mL centrifuge tubes. Oxygen was isolated using sterile liquid paraffin. Uninoculated MRS broth was used as a blank control and inoculated at an angle at 37°C for static incubation. Using a pipette, 100 µL of the bacterial suspension was transferred to a 96-well plate, repeated three times. The OD600 values ​​were measured using a microplate reader, and the average value was calculated and recorded to minimize systematic error. The OD600 value of the initial bacterial solution at 0h was recorded. Thereafter, the OD600 value of the bacterial solution was measured every 2h and recorded until 24h. The bacterial solution needed to be mixed before each aspiration to prevent the fermentation liquid from sinking to the bottom. At the same time, the pH change was measured every 2h with a handheld pH meter. Each sample and blank control were measured three times and the average value was calculated to construct the acid production curve. The results showed that Figure 6 , Lactobacillus johnsonii JoA9-1 is in the lag phase at 0-2h, in the logarithmic growth phase at 2-22h, and reaches the stable phase at 22h; when Lactobacillus johnsonii JoA9-1 is just inoculated, the pH of the MRS culture medium is 5.7, the pH of the culture medium drops significantly at 2h, drops rapidly during 4-16h, tends to be stable at 16-22h, and reaches 3.8 at 24h, which indicates that Lactobacillus johnsonii JoA9-1 has good acid production performance.

[0049] Example 6 Drug sensitivity testing Antimicrobial susceptibility testing of Lactobacillus johnsonii was performed using the disc agar diffusion method, with Escherichia coli and Staphylococcus aureus serving as control strains. All three strains were pre-activated and cultured. The strains were evenly stirred in PBS buffer using a disposable inoculating loop. The concentration of the bacterial suspension was adjusted to 0.5 using a McFadden turbidimeter. A sterile cotton swab was used to apply an appropriate amount of the bacterial suspension to MRS solid medium, MacConkey medium, and MH solid medium. After the agar had completely absorbed the moisture from the plates, drug susceptibility strips (ampicillin, penicillin, streptomycin, gentamicin, erythromycin, tetracycline, chloramphenicol, and ciprofloxacin) were evenly distributed on the surface of the culture medium using sterile tweezers. Four strips were applied to each plate, with the inhibition zone marked. The plates were then incubated in an anaerobic chamber at 37°C for 24 hours, and the diameters of the inhibition zones were measured using a vernier caliper. The results showed that Lactobacillus johnsonii JoA9-1 was sensitive to five antibiotics, including tetracycline, chloramphenicol, erythromycin, penicillin and ampicillin; and was resistant to three antibiotics, including ciprofloxacin, streptomycin and gentamicin. See Table 3 for the average diameter of the inhibition zone in the drug sensitivity test of Lactobacillus johnsonii JoA9-1.

[0050] Table 3 Average values ​​of the diameter of the inhibition zone in the drug sensitivity test of Lactobacillus johnsonii JoA9-1

[0051] Example 7 Hemolysis test Single colonies of Lactobacillus johnsonii JoA9-1 were inoculated onto Columbia blood agar plates in triplicate and incubated anaerobically at 37°C for 48 hours before observation. Enterococcus faecalis (β-hemolytic, ATCC29212, purchased from Ningbo Mingzhou Biotechnology Co., Ltd.) served as a positive control, and blank culture medium served as a negative control. Bacterial hemolytic activity was assessed according to the following criteria: if a 1-2 mm, grass-green, translucent hemolytic ring was observed around the colony, the strain was identified as α-hemolytic and pathogenic; if a 2-4 mm, transparent hemolytic ring was observed around the colony, the strain was identified as β-hemolytic and highly pathogenic; if no change was observed, the strain was identified as γ-hemolytic, meaning it was non-hemolytic and non-pathogenic. The results showed that no change was observed around the Lactobacillus johnsonii JoA9-1 colony, indicating it was γ-hemolytic, meaning it was non-hemolytic and non-pathogenic.

[0052] Example 8 Antibacterial experiment The antibacterial activity of isolated probiotics against pathogens was determined using the punch method. Lactobacillus johnsonii JoA9-1, Staphylococcus aureus, and Escherichia coli were revived and inoculated into liquid culture medium. The culture was then placed in a shaker and incubated overnight at 37°C. An appropriate amount of the Lactobacillus johnsonii culture was aspirated and placed in a centrifuge. The culture was centrifuged at 6000 rpm for 5 minutes, and the supernatant was collected. 10 μL of each of Staphylococcus aureus and Escherichia coli were spread onto LB plates.

[0053] 200 μL of vaginal Lactobacillus johnsonii culture supernatant was added to the wells (three replicates per group, with MRS medium serving as a blank control). Incubate overnight at 37°C. Observe the size of the inhibition zone on each plate, and measure and record the diameter of the zone. The results showed that L. johnsonii JoA9-1 exhibited antibacterial activity against Staphylococcus aureus and Escherichia coli. Table 4 shows the average diameters of the inhibition zones for L. johnsonii JoA9-1.

[0054] Table 4 Average values ​​of the diameter of the inhibition zone in the antibacterial test of Lactobacillus johnsonii JoA9-1

[0055] The antibacterial rate of isolated probiotics against pathogens was determined by microtiter plate. 190 μL of Lactobacillus johnsonii JoA9-1 culture supernatant and 10 μL of indicator bacteria suspension (10 6 CFU / mL of Staphylococcus aureus, Escherichia coli, and Candida albicans suspensions were used. MRS liquid medium was used instead of the culture supernatant of Lactobacillus johnsonii JoA9-1 as a control (three replicates per group). Staphylococcus aureus and Escherichia coli were cultured at 37°C, and Candida albicans was cultured at 28°C for 48 h. The absorbance was measured at 580 nm on a microplate reader. The antibacterial activity of Lactobacilli was calculated as the inhibition rate against the indicator fungus according to formula (1).

[0056] Inhibition rate (%) = (1-OD A ) / OD control ×100(1) Where: OD A is the absorbance of the sample group, OD control The results showed that Lactobacillus johnsonii JoA9-1 had a certain antibacterial ability against Staphylococcus aureus, Escherichia coli and Candida albicans, as shown in Table 5.

[0057] Table 5 Antibacterial rate of Lactobacillus johnsonii JoA9-1

[0058] Example 9 Determination of hydrogen peroxide production capacity The hydrogen peroxide (H2O2) content detection kit was used for determination. The specific steps were as follows: Lactobacillus johnsonii JoA9-1 was inoculated into MRS liquid culture medium, placed obliquely in a 37°C shaker for 48 hours, centrifuged at 10,000 r / min for 10 minutes, and the supernatant was discarded; 1 mL of acetone was added for every 5 million bacteria, and after ultrasonic disruption, the supernatant was centrifuged at 8,000 g at 4°C for 10 minutes, and the supernatant was taken and placed on ice for testing; reagents 2 (add 3 mL of concentrated hydrochloric acid to fully dissolve before use), 3, and 4 prepared in the kit were water-bathed at 25°C for more than 10 minutes; the concentration of the standard was 2 μmol / mL; The kit adds the sample (250 μL), standard (250 μL), acetone (250 μL), and reagents 2 (25 μL) and 3 (50 μL) to separate EP tubes. Centrifuge at 4000 g for 10 minutes at room temperature, discard the supernatant, and remove the precipitate. Then, add reagent 4 (250 μL), shake thoroughly to dissolve the precipitate, let it stand at room temperature for 5 minutes, and a 200 μL aliquot of the sample is added to a 96-well plate. The absorbance at 415 nm is measured and the H2O2 content is calculated (triplicates are performed for each group). The kit has a minimum detection limit of 0.0027 μmol / mL and a linear range of 0.0195-3 μmol / mL. Results showed that L. johnsonii JoA9-1 produced 1.2 μmol / mL of H2O2 using this kit, demonstrating strong H2O2 production.

[0059] Example 10 Mouse vaginitis model experiment Thirty-two mice (SPF female ICR, 20-22 g, 6-8 weeks old, housed in an environment with a temperature of 25°C, 60% humidity, and a 12-h light / dark cycle, with free access to water and food) were randomly divided into four groups, with 8 mice in each group: A healthy blank group, B untreated group as control group, C Lactobacillus johnsonii treatment group, and D drug (clotrimazole) control group, and adaptive feeding was performed for 1 week.

[0060] Except for the healthy blank group, mice in other groups were injected subcutaneously with estradiol benzoate (2 mg / mL) (which regulates the physiological state of the mouse reproductive tract, causes the proliferation and keratinization of vaginal epithelial cells, simulates the vaginal environment during the female reproductive cycle, and enhances the susceptibility of mice to Candida albicans) once every two days (injected every morning), with each injection of 0.05 mL. After 3 injections of estradiol benzoate, vaginal swabs were taken, and 20 μL (10 6CFU / mL) suspension of Candida albicans was inoculated into the mouse vagina for four consecutive days. The inoculated mice were hung upside down for 3-5 minutes. The vulva and secretions of the mice were observed daily after inoculation (the vaginal mucosa was visually inspected for inflammatory changes such as congestion, edema, bleeding, erosion, and increased secretions). On the fifth day after inoculation, 100 μL of sterile saline was aspirated two to three times to obtain vaginal lavage fluid. This was then subjected to Gram staining, plate plating (inoculation on Sabouraud dextrose agar, incubation at 28°C for 72 hours, and then observation and counting of colonies), and 16S rRNA sequencing of vaginal swabs to verify the success of the model. The results showed that Gram staining detected a large number of Candida albicans mycelium, and plate plating also observed a large number of Candida albicans, indicating that the model was successful.

[0061] After the model was successfully established, the treatment group C was given 20 μL (10 8 The mice were treated with a suspension of Lactobacillus johnsonii JoA9-1 containing 100 CFU / mL (400 μg / mL). The drug control group, group D, was treated with clotrimazole (100 mg / mL). The healthy control groups, groups A and B, were given an equal volume of normal saline. Treatment was via vaginal instillation for 6 days. The vaginal mucosa was visually observed daily after inoculation for inflammatory changes such as congestion, edema, bleeding, erosion, and increased secretions. On the last day of treatment, the mice were vaginally lavaged with 100 μL of normal saline. The lavage fluid was placed in a clean, sterile tube and examined microscopically and smeared for the presence of Candida albicans. The remaining vaginal lavage fluid was stored at 4°C until further use. Vaginal swabs were then obtained, and the mice were sacrificed by cervical dislocation. The intact vaginal tissue, heart, liver, spleen, kidney, lung, and ovary tissues were removed and stored in paraformaldehyde.

[0062] Vaginitis related indicators before and after treatment: (1) Vaginal lavage fluid Candida albicans mycelium formation and Candida albicans smear: After 6 days of treatment, 100 μL of normal saline was taken with a pipette to flush the mouse vagina, and the lavage fluid was Gram-stained and smeared on Sabouraud solid culture medium. It was cultured at 28°C for 72 hours, and the Candida albicans count was observed. The results showed that compared with the model group, the vaginal lavage fluid of mice treated with Lactobacillus johnsonii JoA9-1 had fewer Candida albicans mycelium detected by Gram staining, and the plate smear also observed a significant reduction in Candida albicans. Figure 7 .

[0063] (2) Evaluation of the vulva and secretions of mice and the evaluation of the inflammation of mouse tissues: During the treatment process, the vaginal mucosa of mice was observed with the naked eye every day for congestion, edema, erosion, increased secretions, etc., and a comparative analysis was performed. The vaginal symptom scoring criteria were as follows: normal, no abnormality in the vulva, no secretions or only a small amount of transparent secretions (0 points), redness without swelling or swelling without redness or increased secretions (1 point), redness with swelling, redness with increased secretions, or swelling with increased secretions (2 points), redness and swelling with increased secretions, redness and swelling with erosion, redness and swelling with increased secretions and erosion (3 points). The results showed that after 6 days of treatment with Lactobacillus johnsonii JoA9-1, the vaginal opening of mice in the VVC model control group was congested, edematous, and secretions increased; the Lactobacillus johnsonii JoA9-1 treatment group and the clotrimazole group significantly reduced the symptoms of vaginal congestion and edema, and reduced the amount of vaginal secretions, as shown in Table 6.

[0064] Table 6 Vaginal symptom score table for Lactobacillus johnsonii JoA9-1 treatment

[0065] (3) Local histopathological examination of mice: After 6 days of treatment, intact vaginal tissue, heart, liver, spleen, kidney, lung, and ovary tissues were collected and stored in paraformaldehyde. After tissue fixation, dehydration, embedding, and slicing were performed in sequence to prepare pathological sections, which were then stained with HE. Finally, the pathological changes of each tissue were observed under a microscope.

[0066] HE staining: ① Embedding: The tissue samples were fixed with 4% paraformaldehyde, rinsed with running water, and then the tissue blocks were trimmed and placed in a pathological embedding plastic basket for gradient alcohol dehydration, xylene transparency, wax immersion and embedding. ② Sectioning: The tissue was cut into 5µm thick slices using a Leica RM2235 slicer, spread in warm water, and fixed on a glass slide. ③ Staining: The slices were dewaxed to water, stained with hematoxylin-eosin, dehydrated with gradient alcohol, transparentized with xylene, and sealed with neutral resin glue. ④ After checking the sample number, the appearance of the slices was observed with the naked eye to see if the coverslip was in the appropriate position. The quality inspection was then performed under a microscope to observe the integrity and staining of the tissue slices. The results showed that after 6 days of continuous vaginal administration in all treatment groups, major organs including the heart, liver, spleen, lungs and kidneys were not affected. In addition, after infection with Rosa albicans, the ovaries of all treatment groups and the control group treated with normal saline were also not affected. This is specifically reflected in: Heart: Myocardial fibers were neatly arranged in a short cylindrical shape, with intact structure. Longitudinal sections showed distinct transverse striations. No significant pathological changes were observed in the myocardial tissue of any group, exhibiting normal histological features.

[0067] Liver: Hepatocytes were arranged radially around the central vein, with large, round nuclei and abundant eosinophilic cytoplasm. Interlobular arteries, veins, and bile ducts were clearly visible in the portal area. The "feathery" appearance of hepatocytes is related to glycogen content, resulting in differences in the size of the cavities in liver tissue sections between groups, which is normal. No significant pathological changes were observed in the livers of any group, displaying normal histological features.

[0068] Spleen: The demarcation between the white and red pulp was clear. The lymphoid nodules and periarterial lymphoid sheaths in the white pulp were intact and densely cellular. The splenic cords and sinusoids in the red pulp were clearly defined, with abundant macrophages and lymphocytes within the cords and abundant red blood cells within the sinusoids. All spleens in the groups exhibited normal histological morphology.

[0069] Lungs: The alveolar structure was intact, the morphology and structure of the bronchi, atria and alveolar walls at all levels were clear, the air volume in the airways was moderate, and no obvious pathological changes were found in the lungs of all groups.

[0070] Kidney: Mouse kidney tissue was structurally intact, with round or oval glomeruli located within the renal cortex and uniform in size. Capillary loops were clearly distributed, and the mesangial region showed no significant widening. Tubular epithelial cells were neatly arranged, with distinct brush borders, unobstructed tubular lumens, and normal interstitial structure. All kidneys exhibited normal histological morphology.

[0071] Ovaries: Ovarian tissue showed normal morphology and structure, with primary and secondary follicles and the corpus luteum clearly identifiable. Granulosa cells were clearly stratified, and follicles were actively growing and well-developed. No significant pathological changes were observed in the ovaries of any group.

[0072] At the same time, HE staining of vaginal tissue showed that the vaginal mucosal epithelium of the healthy blank group was smooth, without congestion and edema, inflammatory cell infiltration, and necrotic tissue shedding. Compared with the blank group mice, the squamous epithelial cells in the mucosal layer of the control group mice proliferated, and a small number of epithelial cells were degenerated, necrotic and shedding in small areas, with scattered small abscesses formed, and a large number of inflammatory cells, mainly neutrophils, infiltrated in the submucosal layer. The thickness of the vaginal mucosal epithelial layer in the Lactobacillus johnsonii JoA9-1 treatment group was close to that of the healthy group, the epithelial cells were arranged neatly, the number of small abscesses on the surface was reduced, the range was reduced, and the infiltration of inflammatory cells in the submucosal layer was significantly reduced or disappeared, achieving a therapeutic effect similar to that of the clotrimazole group. Figure 8 .

[0073] (4) Determination of TNFα: After 6 days of treatment, vaginal lavage fluid was collected from mice and the ELISA kit was operated strictly according to the operating instructions to detect the average level of TNFα in the vaginal lavage fluid of each group of mice. The results showed that after 6 days of treatment, the expression of the pro-inflammatory factor TNFα in the control group was significantly increased compared with the healthy blank group. After treatment with Lactobacillus johnsonii JoA9-1 and clotrimazole, the expression of TNFα decreased to varying degrees compared with the control group. Among them, the difference between the treatment with Lactobacillus johnsonii JoA9-1 and clotrimazole and the control group was significant, and the therapeutic effect of Lactobacillus johnsonii JoA9-1 was better than that of the positive control drug clotrimazole ( P <0.05). The above results indicate that Lactobacillus johnsonii JoA9-1 has immunomodulatory function and may play a therapeutic role in vaginitis caused by Candida albicans by reducing the expression of TNFα, and its therapeutic effect is comparable to that of clotrimazole. Although the antifungal drug clotrimazole significantly inhibits the growth and mycelium formation of Candida albicans, its inhibitory effect on the inflammatory factor TNFα is not as good as that of Lactobacillus johnsonii JoA9-1. Figure 9 .

[0074] (5) 16S rRNA sequencing was used to detect the effect of Lactobacillus johnsonii JoA9-1 on the relative abundance of vaginal microbial flora in the mouse vaginitis model. Vaginal swab sample analysis showed that the bacterial flora of mice infected with Candida albicans was dysbiotic, with reduced biodiversity, a decreased proportion of Firmicutes, and an increased proportion of Proteobacteria. At the same time, compared with the control group, treatment with Lactobacillus johnsonii JoA9-1 significantly improved the bacterial microbial diversity of mice with Candida vaginitis, as shown in Figure 2. Figures 10-12 At the phylum level, Lactobacillus johnsonii JoA9-1 treatment significantly reduced the relative abundance of Proteobacteria and increased the relative abundance of Firmicutes, reshaping a healthier vaginal microbiome. Figure 13 At the genus level, the relative abundance of lactic acid bacteria increased significantly, as shown in Figure 14 Compared with clotrimazole, Lactobacillus johnsonii JoA9-1 can increase the richness and diversity of the vaginal microbiome, especially lactic acid bacteria, thereby effectively treating candidal vaginitis.

[0075] In addition to the above embodiments, the present invention may also have other implementations. Any technical solution formed by equivalent replacement or equivalent transformation falls within the scope of protection required by the present invention.

Claims

1. A Lactobacillus johnsonii, characterized in that: The Lactobacillus johnsonii JoA9-1 is preserved in the China Center for Type Culture Collection with the preservation number CCTCCM2025369.

2. The use of Lactobacillus johnsonii in the preparation of a medicament for vaginitis according to claim 1, characterized in that: It includes the use in the preparation of drugs for improving the vaginal microecological environment, improving the pathogenic bacteria load in vaginal secretions, the expression level of the inflammatory factor TNFα, inflammatory cell infiltration and vaginal mucosal integrity.

3. The use of Lactobacillus johnsonii in the preparation of a medicament for vaginitis according to claim 2, characterized in that: The improvement of vaginal microecological environment includes inhibiting the growth and reproduction of pathogenic bacteria.

4. The use of Lactobacillus johnsonii in the preparation of a medicament for vaginitis according to claim 3, characterized in that: The pathogenic bacteria inhibited is at least one of Escherichia coli, Gardnerella vaginalis, Candida albicans, Staphylococcus aureus and Salmonella.

5. Use of the Lactobacillus johnsonii according to claim 1 in the preparation of a medicament for inhibiting or eliminating vaginal Candida albicans.

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