Lactobacillus rhamnosus and application thereof

Abstract

The invention provides lactobacillus rhamnosus and application thereof. The lactobacillus rhamnosus is an original strain separated and purified from vaginal secretion of healthy women. The lactobacillus rhamnosus is identified by the China General Microbiological Culture Collection Center (CGMCC) and is classified and named as lactobacillus rhamnosus, and the preservation number is CGMCC No.33918. According to the lactobacillus rhamnosus disclosed by the invention, through optimization of growth conditions, a large amount of efficient antibacterial substances such as L-lactic acid and H2O2 can be generated in a fermentation culture solution of the lactobacillus rhamnosus, and the growth of gardneria vaginalis can be effectively inhibited. The lactobacillus rhamnosus disclosed by the invention is a microorganism capable of efficiently inhibiting gardneria vaginalis and relieving bacterial vaginosis of mice, and has a relatively high application value.

Description

Technical Field

[0001] This invention relates to the application of probiotics, specifically to a type of Lactobacillus rhamnosus and its applications. Background Technology

[0002] Bacterial vaginosis (BV), caused by vaginal microecological imbalance, is one of the most common gynecological inflammations, characterized by a decrease in dominant vaginal lactobacilli and an overgrowth of anaerobic bacteria, especially Gardnerella vaginalis. BV is associated with adverse outcomes such as pelvic inflammatory disease and endometritis, and increases the risk of miscarriage, premature birth, and premature rupture of membranes in pregnant women. Currently, antibiotics are the main drugs used to treat BV clinically, but their use leads to high recurrence rates, drug resistance, and disruption of the normal vaginal flora. In recent years, probiotics have gained attention as an alternative and complementary therapy to antibiotic treatment for BV. Lactobacilli are important probiotics in a healthy vaginal environment; their metabolites and potential antibacterial abilities play a crucial role in maintaining vaginal cleanliness and fighting infection. Numerous studies have shown that oral or vaginal administration of lactobacilli can alleviate BV symptoms and reduce recurrence rates. However, there are currently few probiotic preparations available for clinical use, and most studies focus on non-vaginal lactobacillus strains, lacking research on dominant vaginal lactobacilli and their functional roles. Lactobacillus rhamnosus is one of the most common bacteria in the vagina of healthy women, and its highly efficient production of antibacterial substances and its bacteriostatic ability are expected to be further studied. Summary of the Invention

[0003] To address the shortcomings of existing technologies, the present invention aims to provide a *Lactobacillus rhamnosus* and its applications. This *Lactobacillus rhamnosus* is suitable for alleviating bacterial vaginosis caused by *Gardnerella vaginalis* and has promising application prospects.

[0004] To address the problems in the existing technology, the technical solution adopted by this invention is as follows:

[0005] This invention provides a self-selected Lactobacillus rhamnosus strain with high inhibitory activity against Gardnerella vaginalis, classified and named Lactobacillus rhamnosus. This strain was deposited on March 21, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33918. The address of the depository is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0006] The present invention also discloses the application of the above-mentioned Lactobacillus rhamnosus in alleviating bacterial vaginosis.

[0007] Furthermore, the Lactobacillus rhamnosus includes the Lactobacillus rhamnosus described above or a suspension of the Lactobacillus rhamnosus described above.

[0008] Furthermore, the method for preparing the Lactobacillus rhamnosus bacterial suspension includes the following steps: inoculating Lactobacillus rhamnosus into a solid slant culture medium for activation, performing seed culture on the activated strain, and fermenting the seed culture solution to obtain the Lactobacillus rhamnosus bacterial suspension.

[0009] Furthermore, the culture temperature of the Lactobacillus rhamnosus is 37°C.

[0010] Furthermore, the Lactobacillus rhamnosus culture time is 48 hours.

[0011] Furthermore, the fermentation culture is carried out at 37°C and statically for 24 hours.

[0012] The colonies of the *Lactobacillus rhamnosus* are milky white, raised and opaque, with varying edge morphology. The fermentation culture of this strain produces large amounts of highly effective antibacterial substances such as L-lactic acid and H2O2, effectively inhibiting the growth of *Gardnerella vaginalis*. Its growth temperature range is 35-37℃, and its optimal growth pH range is 5-7.

[0013] The preparation method of the above-mentioned Lactobacillus rhamnosus bacterial suspension includes:

[0014] Lactobacillus rhamnosus was activated by inoculating solid slant culture medium at 37℃ for 48 h; the activated solid plate strain was then inoculated into primary seed culture medium for primary seed expansion culture at 37℃ for 24 h; the primary seed culture was then inoculated into fermentation medium at an inoculation rate of 2% for expansion culture at 37℃ for 24 h.

[0015] The present invention also provides a study on the antibacterial efficacy of the above-mentioned Lactobacillus rhamnosus or the above-mentioned Lactobacillus rhamnosus bacterial suspension against Gardnerella vaginalis.

[0016] The present invention also provides the application of the above-mentioned Lactobacillus rhamnosus or the above-mentioned Lactobacillus rhamnosus bacterial suspension in relieving bacterial vaginosis.

[0017] The present invention also provides a method for the above-mentioned Lactobacillus rhamnosus to alleviate bacterial vaginosis, comprising the following steps: taking bacterial culture for 24 hours, centrifuging at 5000 rpm for 10 minutes to obtain bacterial cells, resuspending with sterile physiological saline, washing 2-3 times, inoculating into a mouse model of bacterial vaginosis infected with Gardnerella vaginalis, and detecting its alleviating effect.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1. Low cost and clear background: The Lactobacillus rhamnosus strain of this invention is a probiotic strain that is listed in the "List of Strains that can be Used in Food" issued by the National Health Commission of the People's Republic of China. The raw materials are readily available and inexpensive.

[0020] 2. Excellent probiotic characteristics: This vaginal lactobacillus exhibits typical characteristics of vaginal lactobacilli, with a high ability to produce antibacterial substances such as L-lactic acid and H2O2. It also shows good inhibitory effects against Gardnerella vaginalis. The selected Lactobacillus rhamnosus produced lactic acid at a level as high as 85.07 mmol / L and H2O2 at a level as high as 21.02 mmol / L in 24 hours. The inhibition diameter against Gardnerella vaginalis was 12.04 ± 0.08 mm.

[0021] 3. Effectively alleviates bacterial vaginosis in mice: Continuous administration of Lactobacillus rhamnosus bacterial suspension to the vulva for 5 days effectively alleviates inflammation levels in mice with bacterial vaginosis, lowers vaginal pH, and to some extent restores the normal dominant vaginal flora. This has significant practical application value, providing a valuable bacterial strain resource for the use of vaginal lactobacilli in treating bacterial vaginosis. Attached Figure Description

[0022] Figure 1 This is the morphology of Lactobacillus rhamnosus (YD-S-66) of the present invention on a solid culture medium.

[0023] Figure 2 The image shows the morphology of Lactobacillus rhamnosus (YD-S-66) of this invention under a microscope.

[0024] Figure 3 This is a comparative diagram showing the L-lactic acid production of multiple strains of Lactobacillus in this invention.

[0025] Figure 4 This is a comparative diagram showing the H2O2 production of multiple strains of Lactobacillus in this invention.

[0026] Figure 5 The antibacterial effect of fermentation broth of multiple strains of lactobacillus on Gardnerella vaginalis.

[0027] Figure 6 Example 6: Verification score for bacterial vaginosis in mice.

[0028] Figure 7 Comparison of IL-6 expression levels in Example 6.

[0029] Figure 8 Comparison of IL-10 expression levels in Example 6.

[0030] Figure 9 Graph showing changes in vaginal pH in mice in Example 6.

[0031] Figure 10The effects of different interventions on the vaginal flora of mice in Example 6. Detailed Implementation

[0032] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. After reading this invention, any modifications of the present invention in various equivalent forms, or direct or indirect applications in other related technical fields, made by those skilled in the art, shall fall within the scope defined by the appended claims.

[0033] Example 1:

[0034] 1. Screening of strains

[0035] (1) Collect vaginal secretion samples from healthy women, rinse thoroughly into sterile saline solution, and mix well. Use sterile saline solution to perform serial dilutions of the vaginal swab rinse solution: 10... 1 10 2 10 3 Take 0.2 mL of each serially diluted solution and spread it onto solid culture medium (culture medium formula: glucose 20 g, peptone 10 g, beef extract 10 g, yeast extract 5 g, anhydrous sodium acetate 5 g, dipotassium hydrogen phosphate 2 g, diammonium hydrogen citrate 2 g, Tween 80 1.0 g, magnesium sulfate heptahydrate 0.2 g, manganese sulfate monohydrate 0.05 g, agar 15 g, purified water 1000 mL). Incubate at 37℃ for 48 h.

[0036] (2) Select different colonies and streak them onto a solid culture medium (20g glucose, 10g peptone, 10g beef extract, 5g yeast powder, 5g anhydrous sodium acetate, 2g dipotassium hydrogen phosphate, 2g diammonium hydrogen citrate, 1.0mL Tween 80, 0.2g magnesium sulfate heptahydrate, 0.05g manganese sulfate monohydrate, 15g agar, 1000mL purified water), and incubate at 37℃ for 48h. Observe whether the isolated colonies are purified. If there are contaminating bacteria, repeat the step until a pure culture is obtained.

[0037] (3) The purified strain was inoculated into liquid culture medium (20g glucose, 10g peptone, 10g beef extract, 5g yeast powder, 5g anhydrous sodium acetate, 2g dipotassium hydrogen phosphate, 2g diammonium hydrogen citrate, 1mL Tween 80, 0.2g magnesium sulfate heptahydrate, 0.05g manganese sulfate monohydrate, 1000mL purified water) and incubated at 37℃ for 24h.

[0038] (4) Take 0.2 mL of the above bacterial solution and inoculate it into 10 mL of fermentation culture medium. Incubate at 37℃ for 24 h, centrifuge at 8000 rpm for 5 min, detect the content of L-lactic acid and H2O2 in the supernatant, and inoculate the strain on a bacterial plate containing Gardnerella vaginalis. Measure the diameter of the inhibition zone and screen out the strain Lactobacillus rhamnosus (YD-S-66) with strong acid production and H2O2 production. This strain is a highly effective antibacterial strain and is identified.

[0039] The aforementioned Lactobacillus rhamnosus was deposited on March 21, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33918.

[0040] Example 2: Identification of bacterial strains

[0041] Lactobacillus rhamnosus (YD-S-66) was cultured on MRS solid medium at 37°C for 48 hours for bacterial identification. The results are as follows: the colonies are milky white, small, raised, opaque, and have irregular edge morphology (see...). Figure 1 The 16S rRNA gene sequence of the strain was determined by the Institute of Microbiology, Chinese Academy of Sciences. Based on the comprehensive analysis of experimental data such as the culture characteristics, microscopic characteristics, and 16S rRNA gene sequence, the strain was identified as Lactobacillus gasseri.

[0042] The 16S rRNA gene sequence of *Lactobacillus rhamnosus* (YD-S-66) is as follows:

[0043] GCTTGCATCTTGATTTGATTTTGAACGAGTGGCGGACGGGTGAGTAACACGTG

[0044] GGTAACCTGCCCTTAAGTGGGGGATAACATTTGGAAACAGATGCTAATACCGCATA

[0045] AATCCAAGAACCGCATGGTTCTTGGCTGAAAGATGGCGTAAGCTATCGCTTTTGGA

[0046] TGGACCCGCGGCGTATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAATGATA

[0047] CGTAGCCGAACTGAGAGGTTGATCGGCCACATTGGGACTGAGACACGGCCCAAAC

[0048] TCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAG

[0049] CAACGCCGCGTGAGTGAAGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTGGAGAA

[0050] GAATGGTCGGCAGAGTAACTGTTGTCGGCGTGACGGTATCCAACCAGAAAGCCAC

[0051] GGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAT

[0052] TTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCCTCG

[0053] GCTTAACCGAGGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACA

[0054] GTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGC

[0055] GAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGA

[0056] ACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAATGCTAGGTGTTGGA

[0057] GGGTTTCCGCCCTTCAGTGCCGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTA

[0058] CGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGA

[0059] GCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCTTTT

[0060] GATCACCTGAGAGATCAGGTTTCCCCTTCGGGGGCAAAATGACAGGTGGTGCATG

[0061] GTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAAC

[0062] CCTTATGACTAGTTGCCAGCATTTAGTTGGGCACTCTAGTAAGACTGCCGGTGACA

[0063] AACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTA

[0064] CACACGTGCTACAATGGATGGTACAACGAGTTGCGAGACCGCGAGGTCAAGCTAA

[0065] TCTCTTAAAGCCATTCTCAGTTCGGACTGTAGGCTGCAACTCGCCTACACGAAGTC

[0066] GGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTG

[0067] TACACACC

[0068] Example 3 Gram staining microscopic examination

[0069] (1) Picking colonies: Generally, fresh plates are selected to pick colonies.

[0070] (2) Smear: The smear should not be too thick and should be distributed as evenly as possible. Before smearing, add a small amount of sterile water or physiological saline to the slide to facilitate the even distribution of bacteria.

[0071] (3) Fixation: After the smear dries, quickly pass the specimen over an alcohol lamp three times, for a total of about 2 to 3 seconds.

[0072] (4) Initial staining: Stain with crystal violet for 1 min.

[0073] (5) Mordant: Stain with iodine solution for 1 min.

[0074] (6) Decolorization: Add a few drops of 95% ethanol. Once the purple color just stops appearing, stop the decolorization process immediately.

[0075] (7) Re-dyeing: Before re-dyeing, use a paper towel to absorb excess water, then re-dye with phenol red for 30-60 seconds, and rinse with a small stream of water.

[0076] (8) Microscopic examination: First observe with a low-power or high-power microscope. After the target is found, observe with an oil immersion lens. After observing with an oil immersion lens, clean the objective lens with xylene in time.

[0077] The results are as follows Figure 2 As shown, Lactobacillus rhamnosus (YD-S-66) is a Gram-positive bacillus under microscopic examination. It does not produce spores and is often arranged in chains.

[0078] Example 4: Detection of L-lactic acid production capacity of Lactobacillus rhamnosus

[0079] The activated bacterial strains were uniformly concentrated and inoculated into MRS broth tubes at a 2% inoculum rate, and cultured statically at 37°C for 24 hours. 1 mL of the culture was centrifuged at 8000 rpm for 5 minutes, the supernatant was collected, the precipitate was discarded, and the culture was placed on ice for testing. The L-lactic acid production capacity of the four bacterial strains after 24 hours of culture was detected using a colorimetric assay kit. The results are as follows: Figure 3 As shown, Lactobacillus rhamnosus (YD-S-66) is a high-yielding L-lactic acid strain with a yield of 85.07 mmol / L.

[0080] Example 5: Detection of H2O2 production capacity of Lactobacillus rhamnosus

[0081] The activated bacterial culture concentration was adjusted to a uniform concentration, and 2% inoculum was added to MRS broth tubes. The tubes were then incubated statically at 37°C for 24 hours. 1 mL of the culture was centrifuged at 8000 rpm for 5 minutes, the supernatant was collected, the precipitate was discarded, and the culture was placed on ice for testing. The H₂O₂ production capacity of the four bacterial strains after 24 hours of incubation was detected using a colorimetric assay kit. The results are as follows: Figure 4 As shown, Lactobacillus rhamnosus (YD-S-66) is a high-yield H2O2 strain with a yield of 21.02 mmol / L.

[0082] Example 6: Detection of the ability of Lactobacillus rhamnosus to inhibit Gardnerella vaginalis.

[0083] Take 100 μL of Gardnerella vaginalis culture (concentration approximately 1 × 10⁻⁶). 7 CFU / mL), evenly spread onto Columbia blood agar plates, with 4 Oxford cups, 3 test wells, and 1 blank well (with sterile medium) placed on each plate. Add 100 μL of supernatant from each strain to each well sequentially. Incubate the plates upright in a 37℃ anaerobic gas-generating container for 48 h. Observe the inhibition zone after incubation and measure its diameter. The appearance of an inhibition zone indicates that the presence of the strain inhibits the growth of Gardnerella vaginalis; the larger the diameter of the inhibition zone, the stronger the inhibitory effect. Results are as follows: Figure 5As shown, the supernatant of Lactobacillus rhamnosus (YD-S-66) produced the largest inhibition zone, which was 12.04±0.08 mm.

[0084] Example 7: Evaluation of the efficacy of Lactobacillus rhamnosus in alleviating bacterial vaginosis in mice.

[0085] (1) Construction of a vaginitis model. Activated Gardnerella vaginalis was inoculated into a modified BHI broth medium and cultured statically at 37°C. The Gardnerella vaginalis suspension was transferred to a sterile centrifuge tube and centrifuged at 5000 rpm for 10 min to collect the bacterial cells. The bacterial cells were washed with sterile physiological saline and centrifuged again, repeated 2-3 times, and then resuspended in sterile physiological saline. The bacterial concentration was adjusted to 1×10⁻⁶. 8 CFU / mL was used to establish the BV model.

[0086] (2) Preparation of probiotic suspension. Activated probiotics were inoculated into MRS broth and incubated statically at 37°C. The probiotic suspension was then transferred to a sterile centrifuge tube and centrifuged at 5000 rpm for 10 min to collect the bacterial cells. The bacterial cells were washed with sterile physiological saline and centrifuged again. This process was repeated 2-3 times before resuspending the cells in sterile physiological saline. The concentration of each bacterial suspension was adjusted to approximately 1 × 10⁻⁶. 9 CFU / mL was administered vaginally to mice. Metronidazole was used as a positive control, and experiments were conducted in an infection group, a Lactobacillus rhamnosus group, and a saline group.

[0087] (3) Method for alleviating bacterial vaginosis in mice using Lactobacillus rhamnosus. After successful modeling, the Lactobacillus rhamnosus group was treated with vaginal injection of 100 μL of bacterial suspension (1×10⁻⁶). 9 The treatment group received CFU / mL; the saline group received 100μL of saline vaginally; the metronidazole group received metronidazole vaginal effervescent tablets; and the model group received no treatment. The treatment was administered once a day for one week.

[0088] (4) Evaluation of the effect of Lactobacillus rhamnosus in alleviating bacterial vaginosis in mice. The effect was initially verified by inflammation score (vaginal appearance evaluation, grade 1-5, ≤2 can be judged as normal), inflammatory factors (pro-inflammatory factor IL-6, anti-inflammatory factor IL-10), pH change, and flora change. Figures 6-9 This study evaluates the alleviating effect of Lactobacillus rhamnosus (YD-S-66) suspension on bacterial vaginosis in mice. Figure 10 This study demonstrates the effects of different interventions on the vaginal flora of mice. The overall results show that intervention with *Lactobacillus rhamnosus* YD-S-66 effectively improved vaginal inflammation, reduced inflammation levels, and restored the vaginal pH environment in mice compared to the model group. Figure 10It can be seen that intervention with Lactobacillus rhamnosus YD-S-66 can significantly reduce the number of Gardnerella vaginalis pathogens in the vagina, and to some extent restore the normal dominant flora in the mouse vagina.

Claims

1. A type of Lactobacillus rhamnosus, characterized in that: The strain was classified and named Lactobacillus rhamnosus. It was deposited at the China General Microbiological Culture Collection Center (CGMCC) on March 21, 2025, with accession number CGMCC No. 33918.

2. The use of Lactobacillus rhamnosus as described in claim 1 in alleviating bacterial vaginosis.

3. The application according to claim 2, characterized in that, The pathogen causing the bacterial vaginosis is Gardnerella vaginalis.

4. The application according to claim 2, characterized in that, The Lactobacillus rhamnosus includes the Lactobacillus rhamnosus or a suspension of Lactobacillus rhamnosus as described in claim 1.

5. The application according to claim 2, characterized in that, The method for preparing the bacterial suspension of Lactobacillus rhamnosus includes the following steps: Lactobacillus rhamnosus is inoculated into a solid slant culture medium for activation, the activated strain is seed cultured to obtain a primary seed liquid, and the primary seed liquid is inoculated into a broth culture medium for fermentation culture to obtain a bacterial suspension of Lactobacillus rhamnosus.

6. The application according to claim 5, characterized in that, The culture temperature of Lactobacillus rhamnosus was 37℃.

7. The application according to claim 5, characterized in that, The Lactobacillus rhamnosus culture time was 48 hours.

8. The application according to claim 5, characterized in that, The fermentation culture temperature was 37°C.

9. The application according to claim 5, characterized in that, The fermentation culture time is 24 hours of static culture.

10. The application according to claim 5, characterized in that, The primary seed culture was inoculated into shake flask culture medium at an inoculation rate of 2% for fermentation.

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