Fermented Lactobacillus mucilaginosus and its use in inhibiting the production of Gardnerella biofilm

Abstract

The present invention belongs to the technical field of probiotics, and particularly relates to a fermented mucus lactobacillus and its use in inhibiting the production of vaginal Gardnerella biofilm. The strain is a fermented mucus lactobacillus fermertum LF50 with a deposit number of CCTCC NO: M 2024382. The strain has an inhibitory ability of up to 96% on the production of vaginal Gardnerella biofilm, and also has extremely high hydrophobicity and self-aggregation ability. At the same time, after vaginal epithelial cells are infected with vaginal Gardnerella, the fermented mucus lactobacillus of the present invention can intervene to alleviate the secretion of cell pro-inflammatory factors and alleviate the condition. This fermented mucus lactobacillus can be used as a new option for microecological treatment of bacterial vaginosis.

Description

Technical Field

[0001] The invention belongs to the technical field of probiotics, and particularly relates to a fermented mucus lactobacillus strain and application thereof in inhibiting the production of Gardnerella biofilm. Background Art

[0002] Bacterial vaginosis is a common lower genital tract infection in women of childbearing age, accounting for 36-60% of patients with vaginal inflammation in gynecological clinics. It is mainly related to the increase of anaerobic bacteria such as Gardnerella vaginalis and the decrease of Lactobacillus, the dominant bacteria that maintains the normal acidic environment of the vagina. Due to the presence of biofilm dominated by Gardnerella vaginalis, the clinical recurrence rate of bacterial vaginosis within 1 year is as high as 60%.

[0003] There are three main approaches to the treatment of vaginal inflammation: the use of antibiotics, vaginal acidifiers, or probiotic supplementation. The first two treatment methods have a short course of treatment and quick results, but are not conducive to the reconstruction and stabilization of the vaginal microecology after treatment, and are often accompanied by problems such as high recurrence rates. Supplementing the vagina with probiotics or probiotic preparations can also have a therapeutic effect. Lactobacillus, which has a good inhibitory effect on vaginal Gardnerella, can not only produce acid to maintain a low pH value for vaginal health, but also compete for space, secrete antibacterial substances, and destroy biofilms. Microecological therapy has attracted much attention due to its few side effects, high efficacy, and low recurrence rate.

[0004] The strain that has been most thoroughly studied and has been put into clinical treatment for bacterial vaginosis is Lactobacillus rhamnosus GR-1 (L. rhamnosus GR-1), which has excellent performance in microecological regulation and immune regulation. Commercial microecological preparations have obvious species, strain and population specificity. Lactobacillus rhamnosus GR-1 can improve the symptoms of Chinese BV patients but cannot colonize in the vagina of Chinese women. Therefore, it is of great clinical significance to find lactobacilli that are suitable for the Chinese population and can effectively reduce the recurrence and persistent infection of BV. Summary of the invention

[0005] In order to overcome the problems of the prior art, the present invention discloses a fermented mucus lactobacillus and its use in inhibiting the production of Gardnerella biofilm. The present invention takes the inhibition of vaginal Gardnerella biofilm production as the core indicator, screens out a fermented mucus lactobacillus LF50 with extremely high biofilm inhibition ability, and the effect is better than the currently developed and marketed Lactobacillus rhamnosus GR-1.

[0006] The object of the present invention is achieved in that:

[0007] The first aspect of the present invention provides a strain of fermented mucus Lactobacillus fermertum LF50, which was deposited in the China Center for Type Culture Collection on March 4, 2024, address: Wuhan University, Wuhan, China, with a deposit number of CCTCC NO: M 2024382.

[0008] The second aspect of the present invention provides a method for culturing the fermented Lactobacillus mucilaginosus LF50, comprising the following steps:

[0009] (1) transferring the fermented Lactobacillus mucilaginosus LF50 into MRS liquid culture medium, placing it at 37° C. and culturing it for 24 hours to obtain an activated bacterial solution;

[0010] (2) The activated bacterial solution in step (1) is inoculated into a liquid MRS culture medium at an inoculum amount of 1% to 3%, and the culture is continued to obtain a bacterial solution of fermented Lactobacillus mucosa LF50.

[0011] The third aspect of the present invention provides a probiotic agent prepared by the fermented Lactobacillus mucilaginosus LF50.

[0012] The fourth aspect of the present invention provides a probiotic fermentation product prepared by the fermented Lactobacillus mucilaginosus LF50.

[0013] The fifth aspect of the present invention provides a product prepared from the fermented Lactobacillus mucilaginosus LF50, the probiotic agent or the probiotic fermentation product.

[0014] Preferably, the product is a female vaginal care product, a food or a medicine for treating vaginitis.

[0015] Preferably, the content of fermented mucus Lactobacillus LF50 in the female vagina care product or the medicine for treating vaginitis is 1×10 9 ~2×10 9 CFU / mL.

[0016] Preferably, the vaginitis is vaginitis caused by Gardnerella vaginalis.

[0017] The sixth aspect of the present invention provides the application of the fermented Lactobacillus mucilaginosus LF50, comprising:

[0018] (1) It is used to inhibit the production of vaginal Gardnerella biofilm;

[0019] (2) Used in the preparation of female vaginal care products, foods or medicines for the treatment of vaginitis.

[0020] Information on deposited strains:

[0021] Fermented mucus Lactobacillus LF50, depository institution: China Center for Type Culture Collection (CCTCC); address: Wuhan University, Wuhan, China; deposit date: March 4, 2024; deposit number: CCTCC NO: M 2024382; classification name: fermented mucus Lactobacillus fermertum.

[0022] The control strain used in the present invention is:

[0023] Lactobacillus rhamnosus GR-1 was isolated from the Youleshi brand probiotic capsules, produced by Shanghai Puwei Health Food Co., Ltd., approval number: National Food and Health G20130762.

[0024] Other strains used in the present invention:

[0025] G. vaginalis S23 was isolated from a female patient with clinical bacterial vaginosis.

[0026] The advantages and beneficial effects of the present invention are:

[0027] 1. The present invention provides a new strain of fermented mucus lactobacillus Lactobacillus fermertum LF50, which has better effect than the currently developed and marketed Lactobacillus rhamnosus L.rhamnosus GR-1. This strain has a good ability to inhibit the production of vaginal Gardnerella biofilm. Intervention using the fermented mucus lactobacillus LF50 of the present invention can reduce the production of vaginal Gardnerella biofilm by about 96%, with almost no biofilm residue.

[0028] 2. The fermented Lactobacillus mucosa LF50 of the present invention has extremely high hydrophobicity and strong self-aggregation ability, with a measured value of up to 90%, which is much higher than that of L. rhamnosus GR-1, and is very likely to have an advantage in the process of adhering to vaginal epithelial cells.

[0029] 3. The use of the fermented mucus Lactobacillus LF50 to treat female vaginal inflammation caused by vaginal Gardnerella can significantly reduce the secretion of pro-inflammatory cytokines and alleviate the disease progression, indicating that the strain described in the present application has a certain ability to alleviate vaginal inflammation strongly associated with vaginal Gardnerella, and can be used as a new option for microecological treatment of bacterial vaginosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0031] Figure 1 The inhibition rate of each lactic acid bacteria strain on the biofilm production of G. vaginalis S23 is shown.

[0032] Figure 2 The results of the comparison between the effects of fermented mucus lactobacillus LF50 and L.rhamnosus GR-1 under laser confocal microscopy; A: The production of G.vaginalis S23 biofilm, the biofilm is uniform and dense, thick, and mostly live bacteria; B: After the intervention of L.rhamnosus GR-1, the amount of G.vaginalis S23 biofilm generation was significantly reduced, but there were still obvious dead G.vaginalis S23 bacteria remaining; C: After the intervention of fermented mucus lactobacillus LF50, no obvious G.vaginalis S23 and its biofilm residues were found.

[0033] Figure 3 The figure shows the comparison of hydrophobicity and self-aggregation ability between fermented Lactobacillus mucus LF50 and L. rhamnosus GR-1; wherein, A: the hydrophobicity of fermented Lactobacillus mucus LF50 bacteria is significantly higher than that of L. rhamnosus GR-1; B: the self-aggregation ability of fermented Lactobacillus mucus LF50 bacteria is significantly higher than that of L. rhamnosus GR-1, and its lower aqueous solution is almost clear; C: after the hydrophobicity experiment of fermented Lactobacillus mucus LF50 is completed, the lower aqueous solution is almost clear and transparent, while the lower aqueous solution of L. rhamnosus GR-1 is still turbid; D: after the self-aggregation experiment of fermented Lactobacillus mucus LF50 is completed, almost all bacteria are settled at the bottom of the test tube, while L. rhamnosus GR-1 bacteria are evenly suspended in PBS.

[0034] Figure 4 The results show the alleviating effect of fermented Lactobacillus mucinus LF50 on inflammation caused by G. vaginalis S23; A: changes in IL-6 secretion, after the intervention of fermented Lactobacillus mucinus LF50, the secretion of this immune factor was significantly reduced; B: changes in IL-8 secretion, after the intervention of fermented Lactobacillus mucinus LF50, the secretion of this immune factor was significantly reduced. DETAILED DESCRIPTION

[0035] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0036] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0037] The present invention will be described in detail below by way of examples. It should be understood that the following examples are only used to further explain and illustrate the content of the present invention in detail, and are not used to limit the present invention.

[0038] Example 1

[0039] This example uses the crystal violet staining method to evaluate the amount of biofilm formed after lactobacillus antagonism, and the specific method is as follows:

[0040] 1. Inoculation of each strain

[0041] After three generations of activation, fermented Lactobacillus mucilaginosus LF50 and L. rhamnosus GR-1 were inoculated in liquid MRS medium at a 1% inoculum and cultured to the middle of the stable phase for use in experiments. After two generations of anaerobically activated Gardnerella vaginalis S23 in double-resistance chocolate agar medium, single colonies were inoculated in modified BHI broth (BHI, 3 g / L soluble starch, 3 g / L glucose) and cultured to the middle of the stable phase for use in experiments.

[0042] The concentrations of vaginal Gardnerella and Lactobacillus were adjusted to a turbidity of 0.5 using a McFarland turbidimeter, and 2 μL of the bacterial solution was inoculated into 196 μL of modified BHI broth, and cultured anaerobically at 37°C in a 96-well plate for 48 h. A single well culture of G. vaginalis S23 was set as a negative control, and 2 μL of the bacterial solution was inoculated into 198 μL of modified BHI broth in the same culture method. Each treatment was repeated at least three times, and each repeat contained three technical parallel wells.

[0043] 2. Crystal Violet Staining

[0044] Discard the bacterial solution and rinse slowly with PBS three times to remove floating bacteria in the wells. Air dry for 60 minutes and stain the biofilm with 200 μL 0.4% (w / v) gentian violet for 30 minutes. Then wash gently with 200 μL PBS to remove excess stains, air dry for 5 minutes, and dissolve the gentian violet with 200 μL 33% (v / v) acetic acid solution. Use an enzyme-labeled instrument to measure the absorbance of gentian violet at a wavelength of 595 nm.

[0045] 3. Data processing

[0046] The measured values ​​were averaged and the inhibition rate was calculated using the following formula:

[0047]

[0048] It has been calculated that L. rhamnosus GR-1 can reduce the amount of biofilm produced by about 90%, but using this strain for intervention can reduce the amount of vaginal Gardnerella biofilm produced by about 96%, with almost no biofilm remaining.

[0049] Example 2: Observation of the action mode of the strain using laser confocal microscopy

[0050] 1. Inoculation of each strain

[0051] The cultivation of each strain is the same as 1. Inoculation of each strain in Example 1.

[0052] Use a 24-well plate and place a 14 mm × 14 mm cell slide at the bottom of each well. Use a McFarland turbidimeter to adjust the concentration of Gardnerella vaginalis and Lactobacillus fermentans LF50 to a turbidity of 0.5, take 10 μL of the bacterial solution and inoculate it in 980 μL of modified BHI broth, and culture it anaerobically at 37°C for 48 hours. Set up a single-well culture of G. vaginalis S23 as a negative control, and take 10 μL of the bacterial solution from this well and inoculate it in 990 μL of modified BHI broth, and culture it in the same way.

[0053] 2. Fluorescent dye staining and photography

[0054] After incubation, remove the cell slides and wash them three times with BHI. Add 3 μL of propidium iodide staining solution and 3 μL 9. Add 1 mL of BHI to prepare the staining solution (prepare immediately before use), take 300 μL of fluorescent staining solution and incubate at 25°C in the dark for 25 minutes. After the sample is incubated in the staining solution, gently wash it three times with BHI to remove excess staining.

[0055] Biofilms were observed at 63× magnification with a 0.75 zoom oil lens (LSM510, Carl Zeiss, Thornwood, NY), and spectral lasers (green, 488 nm; red, 561 nm) were used for excitation light observation, and the images were retained, with the same manipulation time for each sample. Tomographic scanning was performed at 1 μm intervals in the z-axis direction to obtain a series of images of each layer, and the three-dimensional images were combined.

[0056] like Figure 2 As shown, laser confocal microscopy observation and verification showed that when L. rhamnosus GR-1 was used for intervention, some dead Gardnerella and the biofilm produced during their growth process remained in the culture system; however, after intervention with fermented mucus Lactobacillus LF50, no obvious residual vaginal Gardnerella was found in the system.

[0057] Example 3: Hydrophobicity and self-aggregation experiments

[0058] 1. Hydrophobicity experiment

[0059] Inoculate the activated fermented Lactobacillus mucilaginosus LF50 in MRS medium at 1% inoculum and culture at 37° for 20-24h. Centrifuge at 6000×g for 5min to collect the bacteria and resuspend them in sterile PBS. Adjust the bacterial suspension to OD600nm =0.8, take 1mL of bacterial solution and mix with 1mL of xylene, shake thoroughly for 2min, and let stand at room temperature for 1h. Discard the upper solution and take the lower bacterial solution to measure OD.

[0060] The hydrophobicity of the strain was calculated using the formula:

[0061]

[0062] 2. Self-aggregation experiment

[0063] Inoculate the activated fermentative Lactobacillus mucilaginosus LF50 at 1% inoculum into MRS medium and culture at 37°C for 14-16 hours. Collect the cells by centrifugation at 6000×g for 5 minutes and resuspend in sterile PBS. Adjust the bacterial suspension to OD 600nm =0.8, take 2 mL and dispense into 5 mL EP tubes, let stand at 37 °C for 8 h, and aspirate the upper bacterial suspension to determine OD.

[0064] The self-aggregation ability of the strain was calculated using the formula:

[0065]

[0066] like Figure 3 As shown, the fermented mucus Lactobacillus LF50 bacteria have extremely high hydrophobicity and self-aggregation ability, with a measured value of up to 90%, which is much higher than L. rhamnosus GR-1, and is very likely to have an advantage in the process of adhering to vaginal epithelial cells.

[0067] Example 4: Evaluation of the effect of fermented Lactobacillus mucilaginosus LF50 on relieving inflammation caused by G. vaginalis S23

[0068] 1. Intervention Cell Culture

[0069] Vaginal epithelial cell line VK2 / E6E7 (ATCC-CRL2616) was cultured and activated with endothelial cell culture medium (ECM). The activated cells were adjusted to a concentration of 5×10 5 Each well of a 6-well plate was inoculated with 1 mL of culture medium and cultured in a 37°C, 5% CO2 incubator for 24 h to allow the epithelial cells to fully adhere to the wall.

[0070] 2. Cell modeling and strain intervention

[0071] The activated fermented Lactobacillus mucilaginosus LF50 was adjusted to a bacterial concentration of 1×10 4 / mL, G.vaginalis S23 adjusted the bacterial concentration to 1×10 5 The cells were resuspended in cell culture medium without antibiotics.

[0072] Discard the spent cell culture medium, gently rinse three times with sterile PBS to remove residual antibiotics, inoculate each well with 1 mL of cell culture medium containing G. vaginalis S23, and culture in a 37°C, 5% CO2 incubator for 24 hours. Use blank culture medium ECM without bacteria as a negative control.

[0073] After 24 hours of modeling, discard the waste cell fluid, rinse gently three times with sterile PBS to remove residual vaginal Gardnerella and necrotic cells, inoculate each well with 1 mL of cell culture medium containing fermented Lactobacillus mucosa LF50, and culture in a 37°C, 5% CO2 incubator for 24 hours. Use blank culture medium ECM without bacteria as negative control.

[0074] After the intervention, the cell culture supernatant was collected and centrifuged at 1000 × g for 15 min at 4°C to remove bacteria and cell debris as much as possible, then aliquoted as needed and stored at -80°C.

[0075] The contents of human immune factors IL-6 and IL-8 in the cell culture supernatant were determined according to the kit protocol.

[0076] like Figure 4 As shown, treatment with fermented Lactobacillus mucosa LF50 can significantly reduce the secretion of pro-inflammatory cytokines and alleviate the disease progression. This strain can be used as a new option for microecological treatment of bacterial vaginosis.

[0077] Finally, it should be noted that the above is only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred arrangement scheme, a person skilled in the art should understand that the technical solution of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A strain of Lactobacillus mucilaginosus ( Lactobacillus fermertum ) LF50, characterized in that, It was deposited in the China Center for Type Culture Collection on March 4, 2024, address: Wuhan University, Wuhan, China, with the deposit number being CCTCC NO: M2024382.

2. The method for culturing the fermented Lactobacillus mucilaginosus LF50 according to claim 1, wherein: The steps include: (1) The fermented Lactobacillus mucilaginosus LF50 was transferred to MRS liquid culture medium and cultured at 37°C for 24 h to obtain an activated bacterial solution; (2) The activated bacterial solution in step (1) is inoculated into a liquid MRS culture medium at an inoculum amount of 1% to 3%, and the culture is continued to obtain a bacterial solution of fermented Lactobacillus mucosa LF50.

3. A probiotic agent prepared by fermenting Lactobacillus mucilaginosus LF50 according to claim 1.

4. A probiotic fermentation product prepared by fermenting Lactobacillus mucilaginosus LF50 according to claim 1.

5. A medicine prepared from the fermented Lactobacillus mucilaginosus LF50 according to claim 1, the probiotic agent according to claim 3 or the probiotic fermentation product according to claim 4.

6. The drug according to claim 5, characterized in that: The medicine is a medicine for treating vaginitis.

7. The medicine according to claim 6, characterized in that: The content of fermented mucus lactobacillus LF50 in the medicine is 1×10 9 ~2 × 10 9 CFU / mL.

8. The drug according to claim 6, characterized in that: The vaginitis is vaginitis caused by Gardnerella vaginalis.

9. The use of fermented Lactobacillus mucilaginosus LF50 as claimed in claim 1, characterized in that: The applications include: (1) Used in the preparation of drugs that inhibit the production of vaginal Gardnerella biofilm; (2) Used in the preparation of medicines for treating vaginitis, wherein the vaginitis is caused by Gardnerella vaginalis.

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