Lactobacillus johnsonii and uses thereof

Abstract

The present application relates to a kind of lactobacillus johnsonii and application, belong to microbial technology field.Lactobacillus johnsonii JoA9-1 of the present application is preserved in China typical culture preservation center, and the preservation number is CCTCCM2025369.Lactobacillus johnsonii JoA9-1 of the present application has no virulence factor, does not hemolyze, and has good safety, and its beneficial effect is: can produce multiple organic acids and has tolerance to acidic environment, can adapt and maintain the acidic environment of female genital tract;Can inhibit the propagation of pathogenic bacteria;Can inhibit the formation of Candida albicans hypha;It has immune regulation function, and the inhibitory effect on inflammatory factor TNF alpha is superior to clotrimazole treatment, can significantly enhance mouse vaginitis treatment;Treatment occurs in the site of vagina infection, can repair the local tissue of Candida albicans vaginitis mouse;Regulate the relative abundance of vaginal microbial flora after Candida albicans vaginitis infection of mouse, restore vaginal microbial flora environment.

Description

TECHNICAL FIELD

[0001] The present application relates to Lactobacillus johnsonii and application, and belongs to the field of microbial technology. BACKGROUND

[0002] Female reproductive tract diseases cover a variety of types, such as vaginitis, cervicitis, pelvic inflammatory disease, etc., and its incidence is on the rise worldwide. Traditional treatment methods are mostly focused on the antibacterial or anti-inflammatory treatment against pathogens, but often have high recurrence rate and easily lead to vaginal microecological imbalance. Female reproductive tract microecological disorder is characterized by the increase of microbial diversity and the excessive growth of anaerobic bacteria, and when the number of lactic acid bacteria decreases, the inhibitory effect on other microorganisms will also decrease, the number of pathogenic bacteria in the reproductive tract increases, and the diversity of flora increases. Vaginal lactobacillus, as the dominant bacteria in the normal vaginal flora, plays a key role in maintaining the balance of vaginal microecology.

[0003] Lactobacillus inhibits the growth of pathogenic and opportunistic pathogenic microorganisms through the mechanisms of producing lactic acid, hydrogen peroxide, bacteriocin and regulating immunity, which is very important for maintaining the reproductive health of women. Lactobacillus has long-term positive effects on vaginal health, including maintaining vaginal microecological balance, enhancing local immune function, reducing inflammatory response, improving vaginal epithelial barrier integrity, reducing disease recurrence rate and anticancer effect, etc. Lactobacillus johnsonii is a kind of vaginal lactobacillus, which belongs to gram-positive bacteria and is facultative anaerobic. Under suitable culture conditions, Lactobacillus johnsonii can ferment various sugars to produce lactic acid, which is one of its important metabolic characteristics, which is crucial for maintaining the acidic environment of vagina.

[0004] Vaginitis, also known as vaginosis, is a common gynecological outpatient disease. Patients often have abnormal vaginal discharge, odor, and a series of symptoms such as itching, burning, and irritating pain. Vaginitis is mainly caused by internal or external factors leading to pathogenic bacteria invasion, which destroys the microecological balance of the healthy reproductive tract and causes reproductive tract diseases. According to the different causes of the disease, there are more than ten types of vaginitis, including bacterial vaginosis, vulvovaginal candida vaginosis, trichomonas vaginosis, senile vaginosis, viral vaginosis, and aerobic bacterial vaginosis. Vulvovaginal candidiasis (VVC) is mainly caused by overgrowth of Candida albicans, often occurs in patients with long-term use of broad-spectrum antibiotics, poor blood sugar control in diabetic patients, pregnancy, and other conditions of low immune function or local microenvironment changes. The typical symptoms are pruritus and burning pain of the vulva, and the leukorrhea is like tofu residue. The Candida associated with VVC includes C. albicans, C. glabrata, C. parapsilosis, and C. tropicalis, while the common infection is caused by C. albicans as the pathogenic bacteria. Bacterial vaginosis (BV) is a common type of vaginitis in women of childbearing age, mainly caused by the imbalance of normal flora in the vagina, the overgrowth of Gardnerella, anaerobic bacteria, and the decrease of lactobacilli. Its incidence is closely related to factors such as frequent sexual intercourse, vaginal irrigation, and the use of antibiotics.

[0005] The current treatment for vaginitis is mainly the use of antibiotics. Although antibiotics have a positive effect on disease control, with the widespread use of antibiotics, antibiotic resistance in the treatment of vaginitis is becoming increasingly serious. In candidal vaginitis, the sensitivity of Candida to fluconazole and clotrimazole and other drugs has decreased, increasing the risk of recurrence and treatment failure. Antibiotics not only kill pathogenic bacteria, but also destroy the normal flora in the vagina. Long-term use may increase the number of certain bacteria in the vagina, break the mutual restraint relationship between the flora, lead to vaginal flora imbalance, and even induce or aggravate vaginitis. The development of new antibiotics and alternative therapies, such as probiotic therapy, antimicrobial peptides, and immunotherapy, is needed. Probiotics can inhibit the growth of pathogenic bacteria and promote the recovery of beneficial flora, which is helpful for the prevention and treatment of vaginitis.

[0006] In the vaginal flora of healthy women, Lactobacillus jensenii is usually one of the dominant flora, and the stability of its quantity and activity is crucial for maintaining the balance and health of the vaginal microecology. There are some published patents and documents reporting the role of Lactobacillus jensenii, for example, Chinese patent CN118185812A discloses Lactobacillus jensenii and its application. However, considering that so far, few studies have evaluated the anti-Candida activity and probiotic properties of Lactobacillus jensenii strains in VVC and the performance differences between different strains of the same species, therefore, screening vaginal lactobacilli with better probiotic ability is an unmet need, and supplementing probiotics such as Lactobacillus jensenii in the state of female genital tract disease helps to restore the balance of vaginal microecology and assist in the treatment of vaginal diseases. SUMMARY

[0007] The purpose of the present application is to overcome the defects in the prior art, and provide a Lactobacillus jensenii and 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 application solves the technical problem by the following technical scheme: first, a Lactobacillus jensenii (Lactobacillus jensenii) is provided, Lactobacillus johnsonii JoA9-1, which is isolated from the vaginal secretions of healthy women of childbearing age, is deposited in the China Center for Type Culture Collection, and the deposit number is CCTCCM2025369.

[0009] The present application further provides a method for identifying and selecting the Lactobacillus jensenii strain, which comprises identifying by appearance characteristics, genome identification and metabolic group organic acid quantitative determination to obtain the Lactobacillus jensenii strain with the highest acid-producing capacity.

[0010] The Lactobacillus jensenii JoA9-1 of the present application has the following morphological characteristics:

[0011] The colony edge is regular, opaque, white and round, with a full middle and a smooth and moist surface in MRS solid medium.

[0012] (2) Gram-positive, rod-shaped, often single, paired or short chain.

[0013] The Lactobacillus jensenii JoA9-1 of the present application has no virulence factor and does not hemolyze, has good safety, strong lactic acid-producing capacity, can reduce the pH of the female vagina, and can produce antibacterial substance hydrogen peroxide, which can effectively inhibit the reproduction of pathogenic bacteria.

[0014] The present application further provides genome analysis of Lactobacillus jensenii.

[0015] The application further provides a culture method of the Lactobacillus johnsonii strain, which comprises inoculating the Lactobacillus johnsonii into a specific culture medium for proliferation culture, and finally obtaining the proliferated Lactobacillus johnsonii strain. The culture medium is MRS culture medium.

[0016] The application further provides an application of the Lactobacillus johnsonii strain in female reproductive tract diseases, which comprises improving the vaginal microecological environment, improving the pathogenic bacteria load, the expression level of inflammatory factors (TNF alpha), inflammatory cell infiltration and vaginal mucosa integrity of vaginal secretions.

[0017] When used for improving the vaginal microecological environment, the product is used as a bacteriostatic agent or a bactericide for inhibiting or killing pathogenic bacteria.

[0018] The inhibition of pathogenic bacteria in the above application comprises inhibiting the growth of one or two or more of Escherichia coli, Gardnerella vaginalis, Candida albicans, Staphylococcus aureus and Salmonella.

[0019] It is confirmed by observation of HE staining sections of complete tissues such as heart, liver, spleen, lung, kidney, vagina and ovary of mice that the aforementioned Lactobacillus johnsonii JoA9-1 has good safety and probiotic properties. The Lactobacillus johnsonii JoA9-1 improves the relative abundance of vaginal microbial flora of a mouse vaginitis model. Lactobacillus johnsonii JoA9-1 (CCTCC M 2025369), has significant innovative significance in the field of microbial technology, especially in the direction of female reproductive tract health maintenance. In addition to the high safety (no virulence factor, no hemolysis), the ability to adapt to the acidic environment of the reproductive tract, the inhibition of pathogenic bacteria and Candida albicans hyphae formation, the immune regulation and local tissue repair function, the key to the regulation and repair effect of the Lactobacillus johnsonii JoA9-1 on the vaginal microbial flora of mice infected with Candida albicans vaginitis is:

[0020] I. Precise improvement of the relative abundance imbalance of post-infection vaginal microbial flora

[0021] The occurrence of Candida albicans vaginitis is closely related to the disorder of vaginal microbial community structure. Under normal conditions, Lactobacillus is the dominant bacteria in the vagina, which inhibits the overgrowth of harmful bacteria through the production of organic acids and competition for nutrients. After infection, Candida albicans proliferates rapidly, and the abundance of other harmful microorganisms (such as Proteus bacteria) increases, while the abundance of beneficial bacteria such as Lactobacillus decreases significantly, forming an imbalance state of harmful bacteria dominance and insufficient beneficial bacteria. Lactobacillus jensenii JoA9-1 can specifically improve this imbalance: in the intervention of a mouse model infected with 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 colonization number and relative proportion of Candida albicans. In addition, for other conditional pathogenic bacteria with abnormal abundance increase during infection, this strain can inhibit their reproduction through competition for nutrients, secretion of antibacterial metabolites (such as various organic acids, H2O2, etc.), and other ways, thereby promoting the transformation of the vaginal microbial community from an imbalance state to a beneficial bacteria-dominated state, and building a stable bacterial community foundation for vaginal health.

[0022] II. Effectively restore the microecological environment of vaginal microbial community

[0023] The health of vaginal microbial community not only depends on the reasonable proportion of bacterial abundance, but also depends on the symbiotic relationship between bacteria, metabolic balance, and the synergistic maintenance of vaginal microenvironment. Candida albicans infection can disrupt this homeostasis: on the one hand, the massive growth of Candida albicans can occupy the living space of other microorganisms, disrupting the competition and symbiotic balance between bacteria; on the other hand, its metabolic products can change the microenvironment parameters such as pH and oxidation-reduction potential in the vagina, further inhibiting the growth of beneficial bacteria (such as Lactobacillus), forming a vicious cycle of "infection-bacterial imbalance-microenvironment deterioration". Lactobacillus jensenii JoA9-1 has a dual role in restoring the microecological environment of vaginal microbial community:

[0024] 1. Rebuilding symbiotic balance: After colonization, this strain can form a synergistic effect with other normal microorganisms in the vagina (such as other Lactobacillus species and a small amount of beneficial anaerobes) as a dominant beneficial bacteria, inhibiting the overgrowth of Candida albicans and other harmful bacteria, restoring the species diversity and mutual restraint and interdependence of symbiotic relationship between bacteria, and reducing the risk of single harmful bacteria-dominated community structure.

[0025] 2. Synergistic optimization of vaginal microenvironment: On the one hand, the strain can produce various organic acids (such as lactic acid, acetic acid, etc.), maintain the acidic environment (pH 3.8-4.5) in the vagina, which 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 metabolic products 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 the health of the flora, thereby achieving the overall recovery of the vaginal microbial flora microecological environment and reducing the recurrence risk of Candida albicans vaginitis.

[0026] In summary, Lactobacillus johnsonii JoA9-1 provides a new path for targeted 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, which is different from the traditional drug treatment method that only targets pathogenic bacteria. It focuses more on improving the vaginal health status from the root of the microecology, and has higher safety and long-term maintenance value.

[0027] The Lactobacillus johnsonii JoA9-1 of the present application has no virulence factor and does not hemolyze, and has good safety. Its beneficial effects are: it can produce various organic acids and has tolerance to acidic environment, can adapt to and maintain the acidic environment of female genital tract; it can produce a certain amount of hydrogen peroxide, has effective bacteriostatic ability to Escherichia coli, Staphylococcus aureus, Candida albicans and other bacteria, and can inhibit the reproduction of pathogenic bacteria; it can inhibit the formation of Candida albicans hyphae, indicating that it can greatly improve vaginitis; it has immune regulation function, can reduce the expression level of pro-inflammatory factor TNFα, and the inhibition effect on inflammatory factor TNFα is better than that of clotrimazole treatment, can significantly enhance the treatment of mouse vaginitis; it has no effect on the main organs such as heart, liver, spleen, lung and kidney of mice during treatment, has good safety, the treatment site is at the infected site of vagina, can repair the local tissue of Candida albicans vaginitis mice; 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 DRAWINGS

[0028] Figure 1 It is a front view of Lactobacillus johnsonii JoA9-1 strain.

[0029] Figure 2 It is a gram staining photo of Lactobacillus johnsonii JoA9-1 strain.

[0030] Figure 3 It is KEGG gene function analysis of Lactobacillus johnsonii JoA9-1 strain.

[0031] Figure 4 It is genome circle analysis of Lactobacillus johnsonii JoA9-1 strain.

[0032] Figure 5 This is a low pH growth tolerance curve for Lactobacillus joA9-1 strain.

[0033] Figure 6 The acid-producing capacity and growth curve of Lactobacillus joA9-1.

[0034] Figure 7 The diagram shows the formation and changes of Candida albicans and its hyphae (400x magnification) in mouse vaginal irrigation fluid.

[0035] Figure 8 HE staining of mouse heart, liver, lung, kidney and ovary tissues (200x), HE staining of spleen tissue (400x), and pathological changes in mouse vaginal tissue (HE staining 400x).

[0036] Figure 9 The expression of the inflammatory factor TNFα after treatment with Lactobacillus joA9-1 and clotrimazole.

[0037] Figure 10 A phylum-level species abundance map.

[0038] Figure 11 Shannon index and Chao plot after treatment with Lactobacillus joA9-1 and clotrimazole.

[0039] Figure 12 Heatmap of species clustering at the hierarchical level after treatment with Lactobacillus joA9-1 and clotrimazole.

[0040] Figure 13 Abundance of Proteobacteria and Firmicutes after treatment with Lactobacillus joA9-1 and clotrimazole.

[0041] Figure 14 Abundance of Lactobacillus species after treatment with Lactobacillus joA9-1 and clotrimazole.

[0042] The strain preservation information is as follows: Strain name: JoA9-1; Preservation number: CCTCCM2025369;

[0043] Category Naming: Lactobacillus johnsonii JoA9-1; Depository: China Center for Type Culture Collection; Address: Wuhan University, Wuhan, Hubei Province; Date of deposit: March 4, 2025. Detailed Implementation

[0044] 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.

[0045] Lactobacillus johnsonii JoA9-1, which is derived from vaginal secretion of a healthy woman of childbearing age, is identified as Lactobacillus johnsonii by appearance characteristics and 16S rRNA Lactobacillus johnsonii and finally the subtype of the strain through metabolomics and genome sequencing.

[0046] Example 1

[0047] Isolation of the strain

[0048] A vaginal swab was used to collect vaginal secretion sample from the posterior 1 / 3 of the vagina of a healthy woman of childbearing age by scraping in a clockwise direction at one time; the swab was immediately placed into a 10 mL centrifuge tube after being taken out, and the number was recorded. 5 mL of MRS liquid medium and 2 mL of liquid paraffin were added into the centrifuge tube, and the tube was obliquely inserted into a shaker for 48 h with full shaking. A small amount of liquid was taken from the liquid paraffin surface with a 10 μL inoculation loop and coated on MRS solid medium with added calcium carbonate, the number was recorded, and the medium was placed in an anaerobic incubator at 37°C for culture. After 48 h of culture, colonies with obvious calcium dissolution ring on the calcium-containing medium were picked for Gram staining and microscopic examination. The colony morphology and bacterial morphology were observed and recorded, and the suspected bacteria that met the conditions were preliminarily determined as Lactobacillus. Then the remaining colonies of the suspected Lactobacillus after microscopic examination were inoculated in liquid medium and continued to be streaked on solid plates until the pure bacteria were determined (streak culture at least 3 times). According to the colony morphology on the MRS solid medium, 1 μL of the inoculation loop was used to select different single colonies and re-streaked on new solid medium and the number was recorded, and the incubation was continued at 37°C for 48 h under anaerobic conditions. The strain was subcultured until it was completely purified. A single colony was inoculated in a large solid medium and incubated at 37°C for 48 h under anaerobic conditions to expand the culture of the bacteria. A certain number of cryotubes and 2 mL EP tubes were prepared, 200 μL of sterilized glycerol and 800 μL of broth were added to the cryotubes, and 1 mL of sterilized PBS was added to the EP tubes, and the numbers were recorded. The expanded strain was scraped with an inoculation loop and placed in a 2 mL EP tube, and stirred, and stored at -20°C; the remaining colonies were scraped with the same inoculation loop and placed in a cryotube, and stirred, and stored at -80°C.

[0049] Example 2

[0050] Identification of the strain

[0051] The purified single colony was picked and placed in liquid medium for 24 h to prepare a bacterial suspension, part of which was continued to be cultured, and part of which was subjected to DNA extraction, PCR amplification of 16S rRNA gene, and sent to Shanghai Sangon Biological Engineering Co., Ltd. for sequencing of the purified PCR product. The PCR results were compared by BLAST, and finally identified as Lactobacillus johnsonii.

[0052] After PCR verification of Lactobacillus johnsonii strain JoA9-1, the bacterial solution was streaked to MRS solid medium, and cultured at 37°C for 24-48h in anaerobic condition. The colony edge was regular, opaque, white on the front, raised in the middle, smooth and moist on the surface. The front view is shown in Figure 1 .

[0053] A small amount of colony was taken and diluted with 200μL sterile ddH2O, and then evenly coated on a glass slide. After fixing under a alcohol lamp, the sample was dyed by Gram staining method, and observed under a microscope for its morphology, size, arrangement and whether there was spore, etc. Lactobacillus johnsonii is a gram-positive bacterium, rod-shaped, often single, paired or short chain, no spore, see Figure 2 .

[0054] Example 3

[0055] Strain genome sequencing

[0056] Lactobacillus johnsonii strain JoA9-1 was inoculated into 5mL of anaerobic configuration (liquid paraffin sealing) MRS broth, and cultured to the late logarithmic growth phase. The whole genome DNA of the strain was extracted, and three generations of whole genome sequencing were performed. After assembly and annotation, the nucleic acid sequence was input into KEGG database for gene function analysis, genome circle diagram analysis and virulence factor analysis by Virulence Factor Databases (VFDB). The results are as follows: the strain does not have virulence factors, and Table 1 shows that the whole genome of the strain is 2039243bp, there is 1 contigs in the genome, the GC content is 34.75%, and the N50 value is 2039243bp. In addition, KEGG database integrates data of genome, chemical molecules and biochemical systems, etc. Among them, the core metabolic pathway database divides biological metabolic pathways into six categories: metabolism, genetic information processing, environmental information processing, cellular processes, organismal systems and human diseases, see Figure 3 KEGG gene function analysis of Lactobacillus johnsonii strain JoA9-1, Figure 4Genome circle analysis of Lactobacillus johnsonii strain JoA9-1. The analysis showed that the gene function annotation of Lactobacillus johnsonii JoA9-1 was mainly concentrated in three aspects of metabolism, environmental information processing and genetic information processing. There were 639 annotated genes in the metabolic pathway, of which the number of genes related to carbohydrate metabolism was the largest, with 193, indicating that Lactobacillus johnsonii JoA9-1 may have strong sugar metabolism capacity. There were 225 annotated genes in genetic information processing, of which the number of genes related to replication and repair was the largest, with 83. There were 175 annotated genes in environmental information processing, of which the number of genes related to membrane transport was the largest, with 104, mainly including genes related to various types of transmembrane transport proteins such as ABC transporters and PTS transporters. It is speculated that the strain has strong adaptability to the external environment and can resist the pressure of environmental changes to a certain extent.

[0057] Table 1 QUAST analysis results of Lactobacillus johnsonii JoA9-1

[0058] Example 4

[0059] Acid resistance experiment

[0060] Lactobacillus johnsonii was activated in MRS broth with pH value of 5.7 and cultured at 37°C overnight for 12 hours; an appropriate amount of bacterial suspension was centrifuged at 10,000 r / min for 10 min, and the supernatant was discarded. The bacterial slurry was resuspended with sterilized PBS buffer and calibrated to OD600 of 1 at 600 nm wavelength in a microplate reader. Inoculate at an inoculation amount of 1% into MRS broth with pH value of 4.5 prepared in advance and sterilized and divided into 10 mL centrifuge tubes, isolate oxygen with sterilized liquid paraffin, and incubate at 37°C under anaerobic conditions. Measure the OD600 concentration of the bacterial solution every 2 hours thereafter, and mix and wait for stratification before each time the bacterial solution is aspirated to prevent the fermentation broth from sinking to the bottom. At the same time, use MRS broth with pH value of 4.5 without inoculation as a blank control. Continuously measure for 24 hours, record the data, and draw the pH growth tolerance curve. The results show that Lactobacillus johnsonii JoA9-1 can grow in a low pH environment (pH value of 4.5) and has acid resistance characteristics, as shown in Figure 5 .

[0061] Example 5

[0062] Acid production capacity determination and growth curve experiment

[0063] Lactobacillus johnsonii JoA9-1 was resuscitated and inoculated on MRS solid medium, and incubated in an anaerobic box at 37°C for 48 h. A single colony was picked up with a loop and inoculated into 40 mL of sterilized broth medium, and sealed with liquid paraffin. The culture was shaken for 8 h. The supernatant was collected in a centrifuge tube, and centrifuged at 400-1000 g for 10 min to remove cells. The supernatant was centrifuged at 2000 g to remove cell debris and precipitate. The supernatant was then sent to Suzhou Paimin Biomedical Co., Ltd. for organic acid targeting detection analysis. The results showed that Lactobacillus johnsonii JoA9-1 had strong lactic acid production characteristics, 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 results of Lactobacillus johnsonii JoA9-1 organic acid quantification.

[0064] Table 2 Lactobacillus johnsonii JoA9-1 organic acid quantification results

[0065] Lactobacillus johnsonii JoA9-1 was inoculated in MRS medium and cultured for two generations. Single colonies with regular edges, opaque, white round shape, full in the middle, smooth and moist surface were selected and inoculated in MRS liquid medium. After overnight culture at 37°C in a shaker, an appropriate amount of bacterial suspension was centrifuged at 10000 r / min for 10 min, and the supernatant was discarded. The bacteria were resuspended with sterilized PBS buffer and the OD600 was adjusted to 1 using a microplate reader. The bacteria were inoculated in MRS broth medium at a concentration of 1% and sterilized. The medium was sealed with sterilized liquid paraffin to prevent oxygen absorption. Uninoculated MRS broth medium was used as a blank control. The samples were placed in a 37°C incubator. 100 μL of bacterial suspension was taken from below the liquid paraffin surface using a pipette and transferred to a 96-well plate. This process was repeated three times. The OD600 value was measured using a microplate reader, and the average value was calculated and recorded with a system error reduction. The initial OD600 value of the bacterial suspension at 0 h was recorded. The OD600 value of the bacterial suspension was measured every 2 h up to 24 h, and the bacterial suspension was mixed before each measurement to prevent sedimentation. At the same time, the pH was measured every 2 h using a handheld pH meter, and the average value was calculated by measuring each sample and blank control three times to construct the acid production curve. The results showed that Figure 6 Lactobacillus johnsonii JoA9-1 was in the lag phase at 0-2 h, the logarithmic growth phase at 2-22 h, and the stationary phase at 22 h. The pH of the MRS culture medium was 5.7 when Lactobacillus johnsonii JoA9-1 was inoculated. The pH of the culture medium decreased significantly at 2 h, and rapidly decreased at 4-16 h. The pH tended to be stable at 16-22 h, and reached 3.8 at 24 h. This indicated that Lactobacillus johnsonii JoA9-1 had good acid production performance.

[0066] Example 6

[0067] Drug sensitivity test

[0068] The drug sensitivity of L. johnsonii was determined by the paper disc agar diffusion method, with E. coli and S. aureus as control quality strain. Before the test, the three strains were activated and cultured in advance, and the strains were evenly stirred in PBS buffer using a disposable inoculation ring. The above three bacterial solutions were adjusted to 0.5 using a McFarland turbidimeter. A sterile cotton swab was used to pick up an appropriate amount of bacterial solution and evenly spread it on MRS solid medium, MacConkey medium and MH solid medium. After the water on the plate was completely absorbed by the agar, sterile forceps were used to evenly disperse the drug sensitivity discs (ampicillin, penicillin, streptomycin, gentamicin, erythromycin, tetracycline, chloramphenicol, and ciprofloxacin) on the surface of the medium. Four were pasted on each plate, and the inhibition zone position was reserved. Then, incubate at 37°C in an anaerobic box for 24h, and measure the diameter of the inhibition zone with a vernier caliper. The results showed that L. johnsonii JoA9-1 was sensitive to tetracycline, chloramphenicol, erythromycin, penicillin and ampicillin; resistant to ciprofloxacin, streptomycin and gentamicin, as shown in Table 3. Average size of inhibition zone diameter of L. johnsonii JoA9-1 drug sensitivity test.

[0069] Table 3 Average size of inhibition zone diameter of L. johnsonii JoA9-1 drug sensitivity test

[0070] Example 7

[0071] Hemolysis test

[0072] L. johnsonii JoA9-1 single colony was inoculated on Columbia blood plates, with 3 parallel groups set for each group. After incubation at 37°C for 48h, observation was carried out, with Enterococcus faecalis (β-hemolytic, ATCC29212, purchased from Ningbo Mingzhou Biotechnology Co., Ltd.) as the positive control and blank medium as the negative control. The bacterial hemolytic activity was determined according to the following rules: if 1-2mm of grass-green translucent hemolytic ring was observed around the colony, the strain was identified as α-hemolytic and pathogenic; if 2-4mm of 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, i.e. non-hemolytic and non-pathogenic. The results showed that no change was observed around the colony of L. johnsonii JoA9-1, which was γ-hemolytic, i.e. non-hemolytic and non-pathogenic.

[0073] Example 8

[0074] Bacteriostatic test

[0075] The inhibition ability of the isolated probiotic bacteria on pathogenic bacteria was determined by the punch method. Lactobacillus johnsonii JoA9-1, Staphylococcus aureus and Escherichia coli were inoculated in liquid medium and incubated in a shaker at 37°C overnight. An appropriate amount of Lactobacillus johnsonii liquid was taken and centrifuged at 6000 rpm for 5 min to obtain the supernatant. 10 μL of Staphylococcus aureus and Escherichia coli were respectively inoculated on LB plates.

[0076] 200 μL of the supernatant of the Lactobacillus johnsonii culture was added to the punched holes (three replicates were made for each group, and MRS medium was used as a blank control). The plates were incubated at 37°C overnight, and the size of the inhibition zone on each plate was observed and measured. The results showed that Lactobacillus johnsonii JoA9-1 had an inhibitory effect on Staphylococcus aureus and Escherichia coli. The average diameter of the inhibition zone of Lactobacillus johnsonii JoA9-1 is shown in Table 4.

[0077] Table 4 Average diameter of the inhibition zone of Lactobacillus johnsonii JoA9-1 in the inhibition test

[0078] The inhibition rate of the isolated probiotic bacteria on pathogenic bacteria was determined by the micro-hole plate method. 190 μL of the supernatant of the Lactobacillus johnsonii JoA9-1 culture and 10 μL of the suspension of the indicator bacteria (10 6 CFU / mL of Staphylococcus aureus, Escherichia coli and Candida albicans) were added to each hole of the 96-hole plate, and MRS liquid medium was used instead of the supernatant of the Lactobacillus johnsonii JoA9-1 culture as a control (three replicates were made for each group). After incubation at 37°C for 48 h for Staphylococcus aureus and Escherichia coli and at 28°C for Candida albicans, the absorbance was measured at 580 nm using a microplate reader. The inhibitory activity of the Lactobacillus was calculated according to formula (1) based on the inhibition rate of the Lactobacillus on the indicator fungi.

[0079] Inhibition rate (%) = (1 - OD A / OD control ) x 100 (1)

[0080] In the formula, OD A is the absorbance of the sample group, and OD control is the absorbance of the control group. The results showed that Lactobacillus johnsonii JoA9-1 had a certain inhibitory ability on Staphylococcus aureus, Escherichia coli and Candida albicans. The inhibition rate of Lactobacillus johnsonii JoA9-1 is shown in Table 5.

[0081] Table 5 Inhibition rate of Lactobacillus johnsonii JoA9-1

[0082] Example 9

[0083] Determination of hydrogen peroxide production ability

[0084] The hydrogen peroxide (H2O2) content detection kit was used for determination, and the specific steps were as follows: Lactobacillus johnsonii JoA9-1 was inoculated in MRS liquid medium, and was placed in a 37℃ shaker for 48h, then was centrifuged at 10000r / min for 10min, and the supernatant was discarded; 1mL of acetone was added per 50 million bacteria, and after ultrasonic crushing, 8000g of 4℃ centrifugation was performed for 10min, and the supernatant was taken and placed on ice for determination; the reagents two (3mL of concentrated hydrochloric acid was added before use to dissolve thoroughly for standby use), three and four prepared by the kit were placed in a 25℃ water bath for 10min or more; the standard concentration was 2μmol / mL; according to the kit, sample (250μL), standard (250μL), acetone (250μL) and reagents two (25μL), three (50μL) were added in different EP tubes, 4000g, centrifuged at room temperature for 10min, the supernatant was discarded, and the flow sediment was obtained; then reagent four (250μL) was added, the sediment was dissolved thoroughly after shaking, and was placed at room temperature for 5min, 200μL was taken and added to a 96-well plate to determine the absorbance at 415nm, and the H2O2 content was calculated (three repeats were made for each group), the minimum detection limit of the kit was 0.0027μmol / mL, and the linear range was 0.0195-3μmol / mL. The results showed that Lactobacillus johnsonii JoA9-1 produced 1.2μmol / mL of H2O2 under the kit, and had strong H2O2 production ability.

[0085] Example 10

[0086] Mouse vaginitis model experiment

[0087] 32 mice (SPF grade female ICR, 20-22g, mouse age 6-8 weeks, fed in an environment with temperature 25℃, humidity 60%, light / dark 12h cycle alternation, and free access to water and food) were randomly divided into 4 groups, 8 mice in each group, A healthy blank group, B untreated group as control group, C Lactobacillus johnsonii treatment group, D drug (clotrimazole) control group, adaptive feeding for 1 week.

[0088] The mice in the other groups except the healthy blank group were subcutaneously injected with benzoic acid estradiol (2mg / mL) (to adjust the physiological state of the mouse reproductive tract, proliferate and keratinize the vaginal epithelial cells, simulate the vaginal environment in the female reproductive cycle, and enhance the susceptibility of mice to Candida albicans), every two days (injection in the morning every day), 0.05mL each time, after 3 times of benzoic acid estradiol injection, vaginal swabs were taken, and 20μL (10 6CFU / mL) C. albicans suspension was inoculated into the vagina of mice for 4 consecutive days, and the inoculated mice were hung upside down for 3-5 min. After inoculation, the vulva and secretions of the mice were observed every day (every day after inoculation, the vaginal mucosa was observed for inflammation such as hyperemia, edema, bleeding, erosion, and increased secretion). On the 5th day after inoculation, 100 μL of sterile saline was used to flush the vagina twice or three times to obtain the lavage fluid, which was subjected to Gram staining, plate coating (inoculated on Sabouraud glucose agar medium, cultured at 28°C for 72 h, then observed for colony count), and 16S rRNA sequencing of the vaginal swab to determine whether the modeling was successful. The results showed that a large number of C. albicans hyphae were detected by Gram staining, and a large number of C. albicans were observed by plate coating, indicating that the modeling was successful.

[0089] After successful modeling, the treatment group C was treated with 20 μL (10 8 CFU / mL) of L. johnsonii JoA9-1 bacterial suspension, the drug control group D was treated with the drug clotrimazole (100 mg / mL), the healthy blank group A and the control group B were given the same amount of normal saline, and the treatment route was vaginal irrigation, which lasted for 6 days. After inoculation, the vaginal mucosa was observed every day for inflammation such as hyperemia, edema, bleeding, erosion, and increased secretion. On the last day of treatment, the mouse vagina was irrigated with 100 μL of normal saline, and the lavage fluid was placed in a clean sterile tube for microscopic examination and coating to observe whether C. albicans was present. The remaining vaginal lavage fluid was stored at 4°C for future use, and a vaginal swab was taken, then the mouse was sacrificed by cervical dislocation, and the mouse vaginal tissue, heart, liver, spleen, kidney, lung, and ovary were removed and stored in paraformaldehyde.

[0090] Detection of vaginal inflammation-related indicators before and after treatment:

[0091] (1) C. albicans hyphae formation and C. albicans coating in vaginal lavage fluid: After 6 days of treatment, 100 μL of normal saline was used to flush the mouse vagina, and the lavage fluid was subjected to Gram staining and coating on Sabouraud solid medium, which was cultured at 28°C for 72 h, and the number of C. albicans was observed. The results showed that compared with the model group, the number of C. albicans hyphae detected by Gram staining in the vaginal lavage fluid of the mice treated with L. johnsonii JoA9-1 was less, and the number of C. albicans observed by plate coating was significantly reduced, as shown in Figure 7 .

[0092] (2) Mouse vulva and secretion condition evaluation and mouse tissue inflammation condition evaluation: During the treatment, the mouse vaginal mucosa was observed daily for hyperemia, edema, erosion, increased secretion, etc., and comparative analysis was performed. The vaginal symptom scoring standard was as follows: normal, no abnormality in the external genitalia, no secretion or only a small amount of transparent secretion (0 points), red but not swollen or swollen but not red or increased secretion (1 point), red with swelling, red with increased secretion or swelling with increased secretion (2 points), red and swollen with increased secretion, red and swollen with erosion, red and swollen with increased secretion and erosion (3 points). The results showed that after treatment with Lactobacillus johnsonii JoA9-1 for 6 days, the VVC model control group mice had hyperemia, edema, and increased secretion at the vaginal orifice; the Lactobacillus johnsonii JoA9-1 treatment group and the clotrimazole group significantly reduced the symptoms of hyperemia and edema at the vaginal orifice and reduced the secretion of vaginal secretion, as shown in Table 6.

[0093] Table 6 Lactobacillus johnsonii JoA9-1 treatment vaginal symptom score table

[0094] (3) Mouse local histopathological examination: After 6 days of treatment, the complete tissue of the vagina, heart, liver, spleen, kidney, lung, and ovary were taken and preserved in paraformaldehyde. After the tissue fixation was completed, the steps of dehydration, embedding, sectioning, etc. were performed to prepare pathological sections, and then HE staining was performed. Finally, the pathological changes of each tissue were observed under a microscope.

[0095] HE staining: ① Embedding: After the tissue samples fixed with 4% paraformaldehyde were rinsed with running water, tissue blocks were prepared, placed in a pathological embedding plastic basket, and subjected to gradient alcohol dehydration, xylene transparency, wax immersion, and embedding. ② Sectioning: The tissues were cut into 5 pm thick slices using a Leica RM2235 microtome, spread in warm water, and fixed on glass slides. ③ Staining: The sections were deparaffinized to water, stained with hematoxylin-eosin, dehydrated with gradient alcohol, and transparentized with xylene, and then sealed with neutral resin glue. ④ After checking the sample numbers, the appearance of the sections was observed with the naked eye to determine whether the coverslips were properly positioned, and then the sections were examined under a microscope to observe the integrity and coloration of the tissue sections. The results showed that after 6 days of continuous vaginal administration in all treatment groups, the main organs including the heart, liver, spleen, lung, and kidney were not affected. In addition, after Candida albicans infection, the ovaries of all treatment groups and the control group treated with normal saline were also not affected. This is specifically reflected in:

[0096] Heart: The myocardial fibers were short and cylindrical, arranged in order, and the structure was complete. The myocardial fibers had obvious cross striations. The myocardial tissue of each group showed no obvious pathological changes and exhibited normal histological characteristics.

[0097] Liver: The liver cells were arranged radially around the central vein, with large and round nuclei and abundant eosinophilic cytoplasm. The structures of interlobular arterys, interlobular veins and interlobular bile ducts in portal area were clear. The "feather-like" appearance of liver cells was related to the content of glycogen, which caused the difference in the size of the cavities in the liver tissue sections of each group. No obvious pathological changes were observed in the liver of each group, and the normal histological features were presented.

[0098] Spleen: The boundary between white pulp and red pulp was clear. The structures of lymph nodule and periarterial lymph sheath in white pulp were complete, and the cells were dense. The structures of splenic cord and splenic sinus in red pulp were clear, and the splenic cord contained abundant macrophages and lymphocytes, and the splenic sinus contained abundant red blood cells. The spleen of each group presented normal histological morphology.

[0099] Lung: The alveolar structure was complete, and the morphological structure of each level of bronchus, lung chamber and alveolar wall was clear. The air content in the airway was moderate, and no obvious pathological changes were observed in the lung of each group.

[0100] Kidney: The kidney tissue structure of mice was complete, the glomerulus was round or oval structure, located in the renal cortex, the size was uniform, the capillary loop distribution was clear, and the mesangial area was not obviously widened. The renal tubular epithelial cells were arranged in order, the brush border was clear, the lumen was unobstructed, and the interstitial structure was normal. The kidney of each group presented normal histological morphology.

[0101] Ovary: The ovarian tissue morphology was normal, and primary follicle, secondary follicle and corpus luteum at each level could be clearly identified. The follicular granulosa cell layering was clear, and the follicle grew actively and developed well. No obvious pathological changes were observed in the ovary of each group.

[0102] At the same time, the vaginal tissue was stained by HE. The vaginal mucosa epithelium of the healthy blank group was smooth, and no congestion and edema, inflammatory cell infiltration, or necrotic tissue shedding was observed. Compared with the blank group mice, the squamous epithelial cells of the mucosa layer of the control group mice proliferated, a small area of epithelial cells degenerated, necrosed and shed, and small abscesses were scattered. There were a large number of inflammatory cells mainly composed of neutrophils in the submucosal layer. The thickness of the vaginal mucosa epithelial layer of the Lactobacillus jensenii JoA9-1 treatment group was close to that of the healthy group, the epithelial cells were arranged in order, the number and range of small abscesses in the surface layer were reduced, and the inflammatory cell infiltration in the submucosal layer was significantly reduced or disappeared, achieving a similar therapeutic effect as the clotrimazole group, as shown in Figure 8 .

[0103] (4) Determination of cellular inflammatory factor TNFα: Six days after treatment in mice, vaginal lavage fluid was collected from mice, and the average level of cellular inflammatory factor TNFα in the vaginal lavage fluid of each group of mice was detected strictly according to the instructions of the ELISA kit. The results showed that after 6 days of treatment, the expression of pro-inflammatory factor TNFα was significantly increased in the control group compared with the healthy control group. After treatment with Lactobacillus joeeri JoA9-1 and clotrimazole, the expression of TNFα was reduced to varying degrees compared with the control group; among them, the difference between the treatment with Lactobacillus joeeri JoA9-1 and clotrimazole and the control group was significant, and the therapeutic effect of Lactobacillus joeeri JoA9-1 was better than that of the positive control drug clotrimazole. P <0.05). These results indicate that *Lactobacillus joyes* JoA9-1 possesses immunomodulatory functions and may exert a therapeutic effect on vaginitis caused by *Candida albicans* by reducing TNFα expression, with a therapeutic effect comparable to clotrimazole. While the antifungal drug clotrimazole significantly inhibits the growth and mycelial formation of *Candida albicans*, its inhibitory effect on the inflammatory factor TNFα is not as strong as that of *Lactobacillus joyes* JoA9-1. (See [link to relevant documentation]). Figure 9 .

[0104] (5) The effect of Lactobacillus joA9-1 treatment on the relative abundance of vaginal microbiota in a mouse vaginitis model was detected by 16S rRNA sequencing. Analysis of vaginal swab samples showed that mice infected with Candida albicans exhibited dysbiosis, reduced biodiversity, a decreased proportion of Firmicutes, and an increased proportion of Proteobacteria. Simultaneously, compared with the control group, Lactobacillus joA9-1 treatment significantly improved bacterial microbiota diversity in mice with Candida vaginitis. (See [reference needed]). Figures 10-12 At the phylum level, Lactobacillus joA9-1 treatment significantly reduced the relative abundance of Proteobacteria and increased the relative abundance of Firmicutes, reshaping a healthier vaginal microbiome. (See [link to relevant documentation]). Figure 13 At the genus level, the relative abundance of lactic acid bacteria increased significantly, see [reference needed]. Figure 14 Compared to clotrimazole, Lactobacillus joA9-1 can increase the richness and diversity of the vaginal microbiome, especially lactobacilli, thus effectively treating candidal vaginitis.

[0105] In addition to the above-described embodiments, the present invention may have other implementations. All technical solutions formed by equivalent substitution or equivalent transformation fall within the protection scope claimed by the present invention.

Claims

1. A Lactobacillus johnsonii (Lj) strain, characterized in that: Lactobacillus johnsonii The Lactobacillus johnsonii is preserved in China Center for Type Culture Collection, and the preservation number is CCTCC No: M 2025369. ​ 2. The use of the Lactobacillus johnsonii according to claim 1 in the preparation of a medicine for treating vaginitis caused by Candida albicans.

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