Lactobacillus casei JYLC-132 for improving vaginitis as well as postbiotic preparation and application of lactobacillus casei JYLC-132

Abstract

The invention relates to the technical field of probiotics, in particular to a lactobacillus casei JYLC-132 strain for improving vaginitis as well as a postbiotic preparation and application of the lactobacillus casei JYLC-132 strain. The invention relates to a lactobacillus casei JYLC-132 for improving vaginitis, the lactobacillus casei JYLC-132 is preserved in China General Microbiological Culture Collection Center on February 26, 2024, the preservation address is No.3, No.1 yard, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC NO.29894. The invention further discloses a preparation method of the lactobacillus casei JYLC-132 for improving vaginitis. The metagen preparation prepared from the casei casei JYLC-132 can be used for improving vaginitis, especially improving non-gonococcal vaginitis and candida albicans vaginitis.

Description

Technical Field

[0001] The present invention relates to the technical field of probiotics, and particularly relates to a Lactobacillus casei JYLC-132 for improving vaginitis, its postbiotics preparation and application. Background Art

[0002] Vaginitis is a common infectious disease of the female reproductive system, mainly caused by the imbalance of the vaginal microecology. The invasion of pathogens causes an inflammatory reaction in the vaginal mucosa, and the clinical manifestations are symptoms such as vulvar pruritus, abnormal leucorrhea and burning pain. If it is not cured for a long time, it will not only affect the quality of life of patients, but also may cause serious complications such as pelvic inflammatory disease and infertility. In recent years, the incidence rates of non-gonococcal vaginitis (NGV) and vulvovaginal candidiasis complicated with diabetes have shown an upward trend.

[0003] Non-gonococcal vaginitis is a vaginal inflammation caused by the infection of mycoplasma, chlamydia or other non-gonococcal pathogens. Among them, Chlamydia trachomatis infection is the most common, accounting for about 40%-50% of the causes of the disease, and the proportion of Ureaplasma urealyticum infection is about 20%-30%. In the pathological process of non-gonococcal vaginitis, the abnormal function of immune cells plays a key role. Vulvovaginal candidiasis is a fungal vaginal inflammation caused by the overgrowth of Candida albicans. When the vaginal microecology is imbalanced, Candida albicans will multiply in large numbers and invade the vaginal mucosa, resulting in typical symptoms such as vulvar pruritus, burning pain, and increased leucorrhea with a curd-like appearance in patients. Due to the long-term hyperglycemic state of diabetic women, the glycogen content in vaginal epithelial cells increases, providing rich nutrients for the growth and reproduction of Candida albicans; at the same time, hyperglycemia also damages the body's immune defense function and weakens the phagocytosis and killing ability of white blood cells against pathogens, making diabetic women a high-risk group for vulvovaginal candidiasis. Moreover, the condition of such patients is often more stubborn, with the characteristics of being difficult to cure and easy to relapse, and more targeted and effective treatment means are needed.

[0004] At present, the treatment of vaginitis mainly relies on antibiotics, antifungal drugs, and local cleaning agents. Although antibiotics can quickly inhibit the growth of pathogenic bacteria, they can disrupt the normal vaginal flora, leading to the growth of drug-resistant bacteria and recurrence of diseases; antifungal drugs have limited efficacy in the treatment of diabetic vulvovaginal candidiasis, and long-term use is prone to cause toxic and side effects; local cleaning agents can only temporarily relieve symptoms and cannot fundamentally regulate the vaginal microecological balance. Chinese Patent Application CN117778221A discloses a Lactobacillus casei rhamnosus with the efficacy of preventing and treating female vaginitis, which can significantly inhibit the growth of pathogenic bacteria such as Gardnerella vaginalis, Candida albicans, and Staphylococcus aureus. However, the exploration of its intervention effect on vaginitis only considers aspects such as the ability to inhibit pathogenic bacteria, genital tract cell adhesion, and the probiotic characteristics of the strain itself, without conducting animal experiment efficacy verification, and it is impossible to know whether this Lactobacillus casei rhamnosus can improve non-gonococcal vaginitis and diabetic vulvovaginal candidiasis. Summary of the Invention

[0005] Aiming at the technical problems existing in the current treatment of vaginitis, such as disrupting the normal vaginal flora, limited efficacy, obvious toxic and side effects, especially the lack of effective treatment means for non-gonococcal vaginitis and diabetic vulvovaginal candidiasis, the present invention provides a Lactobacillus casei JYLC-132 for improving vaginitis, its postbiotics preparation, and applications.

[0006] In the first aspect, the present invention provides a Lactobacillus casei JYLC-132 for improving vaginitis. The Lactobacillus casei ( Lacticaseibacillus casei ) JYLC-132 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on February 26, 2024. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO. 29894.

[0007] In the second aspect, the present invention provides a postbiotics preparation of the above-mentioned Lactobacillus casei JYLC-132. The preparation method is to activate Lactobacillus casei JYLC-132 on an MRS plate medium, pick the activated single colony and inoculate it into an MRS liquid medium, and incubate it statically at 37°C under anaerobic conditions for 12 h to obtain a seed solution; inoculate the seed solution into an MRS liquid medium at an inoculation amount of 1%, and incubate it statically at 37°C under anaerobic conditions for 24 h. The obtained bacterial solution is subjected to heat inactivation treatment, concentrated, and freeze-dried to obtain the postbiotics preparation of Lactobacillus casei JYLC-132.

[0008] Further, the number of bacteria in the postbiotics preparation is 1.0×10 10 CFU / g.

[0009] Further, the postbiotics preparation also includes pharmaceutically acceptable excipients.

[0010] Furthermore, pharmaceutically acceptable excipients include glucose and erythritol.

[0011] In a third aspect, the present invention also provides an application of the above-mentioned postbiotic preparation in the preparation of a drug for improving vaginitis, where vaginitis includes non-gonococcal vaginitis and Candida albicans vaginitis.

[0012] Furthermore, non-gonococcal vaginitis includes vaginitis caused by Ureaplasma urealyticum infection.

[0013] Furthermore, improving vaginitis includes improving vaginitis caused by Ureaplasma urealyticum infection by regulating the levels of CD3+, CD4+ and / or CD8+ cells.

[0014] Furthermore, Candida albicans vaginitis includes vaginitis caused by Candida albicans infection triggered by diabetes.

[0015] Furthermore, improving vaginitis includes improving vaginitis caused by Candida albicans infection triggered by diabetes by inhibiting the expression of TGF-β1.

[0016] The beneficial effects of the present invention are as follows: The present invention provides a strain of Lactobacillus casei JYLC-132, and the postbiotic preparation prepared from this strain can improve vaginitis, especially non-gonococcal vaginitis and Candida albicans vaginitis. Experiments have shown that the postbiotic preparation of Lactobacillus casei JYLC-132 can achieve the effect of improving vaginitis caused by Candida albicans infection triggered by diabetes by inhibiting the expression of TGF-β1; the postbiotic preparation of Lactobacillus casei JYLC-132 can also make the immune response tend to be stable by increasing the levels of CD3+ and CD4+ cells, and slow down immune damage by reducing the level of CD8+ cells, so that cellular immunity returns to normal, and significantly improve vaginitis caused by Ureaplasma urealyticum infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, for those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0018] Figure 1 It is a bar graph of the TGF-β1 levels in the vaginal tissues of rats in the blank control group and the diabetes group.

[0019] Figure 2 It is the effect of the postbiotic preparation of Lactobacillus casei JYLC-132 on the TGF-β1 level in the vaginal tissues of rats.

[0020] Figure 3 Effect of postbiotics preparation of Lactobacillus casei JYLC-132 on the level of CD3+ in peripheral blood of mice with non-gonococcal vaginitis.

[0021] Figure 4 Effect of postbiotics preparation of Lactobacillus casei JYLC-132 on the level of CD4+ in peripheral blood of mice with non-gonococcal vaginitis.

[0022] Figure 5 Effect of postbiotics preparation of Lactobacillus casei JYLC-132 on the level of CD8+ in peripheral blood of mice with non-gonococcal vaginitis. Detailed implementation manners

[0023] To enable those skilled in the art to better understand the technical solutions in the present invention, the following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0024] Those skilled in the art can understand that the ferment starter described in Embodiment 1 is also called ferment head, dough starter, old dough, dough leavener, etc. It is a multi-strain mixed fermentation system, and the texture and flavor of steamed buns are mainly affected by the combined effects of the respiration of microorganisms such as yeasts, molds, and lactic acid bacteria.

[0025] Embodiment 1 Isolation, screening and identification of strains 1. Strain screening and purification (1) Sampling: The ferment starter was collected from Jinzhuang Town, Sheqi County, Nanyang City, Henan Province in July 2023.

[0026] (2) Strain isolation: Take 1 g of the ferment starter and add it to a sterile Erlenmeyer flask containing 10 mL of sterilized normal saline (0.85%), shake it to obtain a sample solution; dilute the sample solution with sterilized normal saline to prepare dilution solutions with different concentration gradients, which are 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 ; then use a spreader to spread 100 μL of the dilution solutions with 7 different concentration gradients onto MRS plate medium, and culture them at 37 °C under anaerobic conditions for 48 h; Among them, the preparation method of MRS plate medium is as follows: Take 10 g of peptone, 10 g of beef extract, 5 g of yeast extract powder, 20 g of glucose, 5 g of sodium acetate, 2 g of ammonium citrate, 2 g of dipotassium hydrogen phosphate, 0.58 g of magnesium sulfate, 0.25 g of manganese sulfate, 1 mL of Tween-80, and 15 g of agar powder. Dissolve them with deionized water, make up the volume to 1 L, adjust the pH to 6.3, sterilize at 115 °C under high-pressure steam for 30 min, and then pour the sterilized medium into sterilized petri dishes under sterile conditions and let it cool for later use.

[0027] (3)Select colonies: Select colonies according to the colony characteristics of white, round, moist surface, opaque, and neat edges.

[0028] (4)Isolation and purification: Inoculate the selected single colony onto the MRS plate medium by the three-zone streaking method, and culture it at 37 °C under anaerobic conditions for 48 h. Repeat the above operation, and after a total of 3 streaks, 8 pure strains are obtained by purification. Add the pure strains to glycerol tubes and store them at -80 °C.

[0029] 2. Screening of postbiotics preparations with inhibitory effect on Candida albicans (1)Preparation of postbiotics preparations Activate the 8 strains stored at -80 °C on the MRS plate medium respectively. Pick 1 activated single colony and transfer it to 100 mL of MRS liquid medium, and culture it statically at a constant temperature of 37 °C for 12 h to obtain a seed solution. Inoculate the seed solution into the MRS liquid medium at an inoculation amount of 1% (v / v), and then culture it statically at a constant temperature of 37 °C under anaerobic conditions for 24 h to obtain a bacterial solution with a viable count of 1.0×10 10 CFU / mL (containing bacteria and their metabolites). After heat-inactivating the bacterial solution (115 °C, 30 min), a heat-inactivated bacterial solution is obtained. Concentrate and freeze-dry the heat-inactivated bacterial solution to obtain 8 postbiotics preparations with a bacterial cell count of 1.0×10 10 cfu / g, named postbiotics preparation 1, postbiotics preparation 2, postbiotics preparation 3, postbiotics preparation 4, postbiotics preparation 5, postbiotics preparation 6, postbiotics preparation 7, and postbiotics preparation 8 respectively.

[0030] Among them, the preparation method of MRS liquid medium is as follows: Take 10 g of peptone, 10 g of beef extract, 5 g of yeast extract powder, 20 g of glucose, 5 g of sodium acetate, 2 g of ammonium citrate, 2 g of dipotassium hydrogen phosphate, 0.58 g of magnesium sulfate, 0.25 g of manganese sulfate, 1 mL of Tween-80. Dissolve them with deionized water, make up the volume to 1 L, adjust the pH to 6.3, sterilize at 115 °C under high-pressure steam for 30 min, and after cooling, dispense it under sterile conditions for later use.

[0031] (2)Inhibitory test on Candida albicans The Candida albicans standard strain ATCC10231 (purchased from the Microbiology Laboratory of Hubei Provincial Center for Disease Control) was inoculated on Sabouraud liquid medium (purchased from Qingdao Haibo Biotechnology Co., Ltd.). It was cultured with shaking at 35°C for 18 - 24 hours. Then, 200 μL of the bacterial solution was evenly spread on a Sabouraud medium plate (purchased from Qingdao Haibo Biotechnology Co., Ltd.) and left for 30 min. 1 g of the postbiotic preparation was mixed with 1 mL of sterile water to prepare the test solution. Oxford cups were used to punch holes on the surface of the Sabouraud medium plate coated with Candida albicans. 200 μL of the test solution was added to each hole, with fluconazole as the positive control and normal saline as the negative control. After culturing at 37°C for 24 h, the diameter of the inhibition zone was observed and measured. The test results are shown in Table 1.

[0032] Table 1 Inhibitory results of the postbiotic preparation

[0033] As can be seen from Table 1, all 8 postbiotic preparations can inhibit Candida albicans, but the inhibitory effect of postbiotic preparation 2 on Candida albicans is the most obvious. Therefore, the strain used to prepare postbiotic preparation 2 was selected for identification.

[0034] 3. Identification and preservation The strain used to prepare postbiotic preparation 2 above was sent for identification. The identification unit: Jinan Tianyi Biotechnology Co., Ltd. During the identification process, the following primers were used: 27F (Sequence 1): 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R (Sequence 2): 5'-CTACGGCTACCTTGTTACGA-3'.

[0035] The strain was identified as Lactobacillus casei ( Lacticaseibacillus casei ), named Lactobacillus casei JYLC-132.

[0036] The 16S rDNA gene sequence (Sequence 3) of this Lactobacillus casei ( Lacticaseibacillus casei ) JYLC-132 is:

[0037] Lactobacillus casei ( Lacticaseibacillus casei ) JYLC-132 was deposited with the China General Microbiological Culture Collection Center on February 26, 2024, and the deposit number is CGMCC NO. 29894.

[0038] Example 2 Preparation of postbiotics of Lactobacillus casei JYLC-132 Lactobacillus casei JYLC-132 stored at -80°C was activated on MRS plate medium (preparation method is shown in Example 1). One activated single colony was picked and transferred into 100 mL of MRS liquid medium (preparation method is shown in Example 1). It was statically cultured at 37°C under anaerobic conditions for 12 h to obtain a seed solution. The seed solution was inoculated into MRS liquid medium at an inoculation amount of 1% (v / v), and then statically cultured at 37°C under anaerobic conditions for 24 h to obtain a bacterial solution with a viable count of 1.0×10 10 CFU / mL (containing bacteria and their metabolites). The bacterial solution was heat-inactivated (115°C, 30 min), concentrated, and freeze-dried to prepare the postbiotics of Lactobacillus casei JYLC-132.

[0039] The number of bacteria in the postbiotics is 1.0×10 10 CFU / g.

[0040] Example 3 Preparation of postbiotics of Lactobacillus casei JYLC-132 Lactobacillus casei JYLC-132 stored at -80°C was activated on MRS plate medium (preparation method is shown in Example 1). One activated single colony was picked and transferred into 100 mL of MRS liquid medium (preparation method is shown in Example 1). It was statically cultured at 37°C under anaerobic conditions for 12 h to obtain a seed solution. The seed solution was inoculated into MRS liquid medium at an inoculation amount of 1% (v / v), and then statically cultured at 37°C under anaerobic conditions for 24 h to obtain a bacterial solution with a viable count of 1.0×10 10 CFU / mL (containing bacteria and their metabolites). The bacterial solution was heat-inactivated (115°C, 30 min), concentrated, freeze-dried and mixed with glucose to prepare the postbiotics of Lactobacillus casei JYLC-132.

[0041] The number of bacteria in the postbiotics is 1.0×10 10 CFU / g.

[0042] Example 4 Preparation of postbiotics of Lactobacillus casei JYLC-132 The Lactobacillus casei JYLC-132 stored at -80°C was activated on an MRS plate medium (the preparation method is shown in Example 1). One single colony after activation was picked and transferred into 100 mL of MRS liquid medium (the preparation method is shown in Example 1). It was cultured statically at a constant temperature of 37°C for 12 h under anaerobic conditions to obtain a seed solution. The seed solution was inoculated into the MRS liquid medium at an inoculation amount of 1% (v / v), and then cultured statically at a constant temperature of 37°C for 24 h under anaerobic conditions to obtain a bacterial solution with a viable count of 1.0×10 10 CFU / mL (containing bacteria and their metabolites). The bacterial solution was heat-inactivated (115°C, 30 min), concentrated, freeze-dried and then mixed with erythritol to prepare a postbiotic preparation of Lactobacillus casei JYLC-132.

[0043] The number of bacteria in the postbiotic preparation is 1.0×10 10 CFU / g.

[0044] As known to those skilled in the art, the glucose and erythritol added to the postbiotic preparations in Example 3 and Example 4 are only excipients for adjusting the taste. If the postbiotic preparation is used for the improvement of diabetes complicated with vulvovaginal candidiasis (vaginitis caused by Candida albicans infection induced by diabetes), the postbiotic preparations of Lactobacillus casei JYLC-132 in Example 2 and Example 4 are preferably selected to avoid the negative impact of glucose on diabetes.

[0045] Experimental Example 1 Effect of the postbiotic preparation of Lactobacillus casei JYLC-132 on diabetes vulvovaginal candidiasis 1. Preparation work Experimental animals: 60 female SD rats (weighing about 200 g), purchased from the Experimental Animal Center of Tongji Medical College, Huazhong University of Science and Technology. Inoculated strain: The sixth generation of Candida albicans standard strain ATCC10231, purchased from the Microbiology Laboratory of Hubei Provincial Center for Disease Control and Prevention.

[0046] 2. Grouping and model establishment After one week of adaptive feeding, 60 female SD rats were randomly divided into a diabetes group (n = 40) and a blank control group (n = 20). After measuring that the blood glucose values of each group of rats were normal, they were given water treatment without food. After 12 h, the rats in the diabetes group were intraperitoneally injected with 10 mg / mL of streptozotocin (STZ, pH 4.5) at a dose of 65 mg / kg. At the same time, the blank control group was intraperitoneally injected with an equal amount of citric acid-sodium citrate buffer solution. One week after the intraperitoneal injection of streptozotocin, the blood glucose values of each group of rats were measured, and the diabetes rat model was determined to be successfully constructed when the blood glucose value ≥ 16.7 mmol / L.

[0047] Randomly select 10 rats from the blank control group and 10 rats from the established diabetic group. After vaginal lavage, decapitate the rats, then fix them on the sterile operating table, cut off the hair on both sides of the upper edge of the pubic symphysis in the lower abdomen and the midline of the lower abdomen and disinfect. Incise the abdominal wall and peritoneum along the midline of the abdomen, find the Y-shaped uterus and dissect the surrounding connective tissue, find the vagina along the uterus downward, quickly remove the vaginal tissue, and store it in a -70°C refrigerator for later use.

[0048] Randomly divide the remaining 30 rats in the diabetic group into a diabetic vulvovaginal candidiasis group (n = 10), a low-dose group (n = 10), and a high-dose group (n = 10). The remaining blank control group serves as a non-diabetic combined Candida albicans infection group (n = 10). The vaginas of the four groups of rats are inoculated with a sterile normal saline suspension of Candida albicans spores (containing 5×10 8 spores). One day after inoculation, intragastrically administer the postbiotic preparation (the postbiotic preparation prepared in Example 2) at a dose of 100 million CFU / d to the rats in the low-dose group and 1 billion CFU / d to the rats in the high-dose group, and continuously administer the drug for 6 days. The rats in the other two groups (the diabetic vulvovaginal candidiasis group and the non-diabetic combined Candida albicans infection group) are intragastrically administered an equal volume of sterile normal saline. Feed the rats a normal diet during the modeling period.

[0049] After the drug administration is completed, decapitate the four groups of rats after vaginal lavage, then fix them on the sterile operating table, cut off the hair on both sides of the upper edge of the pubic symphysis in the lower abdomen and the midline of the lower abdomen and disinfect. Incise the abdominal wall and peritoneum along the midline of the abdomen, find the Y-shaped uterus and dissect the surrounding connective tissue, find the vagina along the uterus downward, quickly remove the vaginal tissue, and store it in a -70°C refrigerator for later use.

[0050] 3. Detection of TGF-β1 Preparation of homogenate: Cut the vaginal tissues of the above six groups stored at -70°C into small pieces, add physiological saline homogenate diluent (containing 0.05% Triton X-100, 5 mM CaCl2, and 10 μg / mL indomethacin), and grind them on ice to make a homogenate. Centrifuge the homogenate at 3000 g at 4°C for 15 min, take the supernatant, and store it in a -70°C refrigerator for later measurement.

[0051] ELISA assay: The level of TGF-β1 in vaginal tissues of each group was determined by the enzyme-linked immunosorbent antibody sandwich method. The pre-coated antibody and the detection-phase antibody were both affinity-purified polyclonal antibodies. The detection-phase antibody was biotin-labeled. After the sample and the biotin-labeled antibody (detection-phase antibody) were added to the wells of the enzyme-linked immunosorbent assay (ELISA) plate successively for reaction, they were washed with PBS or TBS. Subsequently, peroxidase-labeled avidin was added for reaction; after thorough washing with PBS or TBS, the substrate TMB was used for color development. TMB was converted into blue under the catalysis of peroxidase and into the final yellow under the action of acid. The intensity of the color was positively correlated with the concentration of rat TGF-β1 in the sample.

[0052] Determine the number of wells of the enzyme-linked immunosorbent assay (ELISA) plate coated with the antibody required for this test, and add 1 well for TMB blank color development. Add 0.1 mL of the standard products at 1000 pg / mL, 500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5 pg / mL, 31.3 pg / mL, and 15.6 pg / mL to one row of 7 wells one by one, and add only the sample diluent to 1 well as the zero well. Add 100 μL of the supernatant samples of each group to be tested to the wells of the ELISA plate one by one. Cover the ELISA plate and react at 37 °C for 90 min. After the reaction, discard the liquid in the ELISA plate, and then pat it a few times against the blotting paper without washing. Add 0.1 mL of the prepared biotinylated anti-rat TGF-β1 antibody working solution to each well in turn. React at 37 °C for 60 min. After the reaction, discard the liquid in the ELISA plate and wash it 3 times with 0.01 M PBS, soaking for about 1 min each time. Add 0.1 mL of the prepared ABC working solution to each well in turn. React at 37 °C for 30 min. Discard the liquid in the ELISA plate and wash it 5 times with 0.01 M PBS, soaking for about 1 - 2 min each time. Add 90 μL of the TMB color development solution to each well in turn and react at 37 °C in the dark for 20 - 25 min. Add 0.1 mL of the TMB stop solution to each well in turn, and at this time the blue color immediately turns yellow. Measure the OD value at 450 nm with an ELISA reader. Set the zero well as the control.

[0053] After subtracting the absorbance value of the zero well from the absorbance values of the standard products and supernatant samples used, the obtained data can be directly used to draw a curve on the coordinate paper. According to the absorbance value of the sample, calculate the corresponding TGF-β1 concentration in the test sample according to the standard curve.

[0054] The data are expressed as mean ± standard deviation. All results were analyzed using the SPSS 16.0 statistical analysis software package. The t-test was used between the blank control group and the diabetes group. One-way analysis of variance was used for comparison among the diabetic vulvovaginal candidiasis group, the low-dose group, the high-dose group, and the non-diabetic combined with Candida albicans infection group, and the LSD method was used for comparison between groups. The results were considered to have statistical differences when P < 0.05.

[0055] 4. Test results From Figure 1 The TGF-β1 detection results show that the level of TGF-β1 in the vaginal tissues of rats in the diabetes group is significantly higher than that in the blank control group, with P < 0.05, indicating a statistically significant difference. This shows that the vaginitis model induced by Candida albicans infection caused by diabetes was successfully established.

[0056] As Figure 2 Shown, one week after inoculation with Candida albicans, the TGF-β1 levels in the vaginal tissues of rats in four groups, namely the non-diabetic combined Candida albicans infection group, the diabetic Candida albicans vaginitis group, the low-dose group, and the high-dose group, were detected. The TGF-β1 level in the vaginal tissues of rats in the diabetic Candida albicans vaginitis group was significantly higher than that in the non-diabetic combined Candida albicans infection group, with P < 0.05, indicating a statistically significant difference. After treatment with the postbiotics preparation of Lactobacillus casei JYLC-132, the TGF-β1 level in the vaginal tissues of rats in the low-dose group and the high-dose group changed significantly compared with that in the diabetic Candida albicans vaginitis group, with P < 0.05, indicating a statistically significant difference. The results show that the postbiotics preparation of Lactobacillus casei JYLC-132 has a significant improvement effect on vaginitis caused by Candida albicans infection induced by diabetes.

[0057] Experimental Example 2 Effects of the postbiotics preparation of Lactobacillus casei JYLC-132 on the levels of CD3+, CD4+, and CD8+ in the peripheral blood of mice with non-gonococcal vaginitis (NGV) 1. Preparation work Experimental animals: 70 SPF-grade healthy female BALB / c mice, 4 - 6 weeks old, weighing 18 - 24 g, were purchased from the Experimental Animal Research Center of Inner Mongolia Medical University. The mice were all raised under standard conditions, with a room temperature of 23 - 25°C, a humidity of 45% - 55%, and a light-dark cycle of 12 hours (7:00 - 19:00 for light time). Sufficient feed and water were provided, and the mice could freely eat and drink. Animal experiments were carried out between 8 am and 6 pm. Ureaplasma urealyticum serotype 8 (ATCC27816) was purchased from the American Type Culture Collection (ATCC) and stored after resuscitation and subculture.

[0058] 2. Grouping, model establishment, and administration Randomly select 50 mice and divide them into 5 groups, namely the normal control group, the model group, the roxithromycin group, the low-dose group, and the high-dose group, with 10 mice in each group.

[0059] After 1 week of adaptive feeding of mice, except for the normal control group, the remaining 4 groups of mice were pretreated with estrogen. Estradiol benzoate injection was subcutaneously injected into the neck at a dose of 0.4 mg / 20 g body weight for 7 consecutive days. Then, a sterile dry cotton swab was rotated several times at the vaginal orifice of the mice to dilate the vaginal orifice. Mice in the model group, roxithromycin group, low-dose group, and high-dose group were vaginally inoculated with 50 μL of the bacterial solution of Ureaplasma urealyticum serotype 8 (the inoculation concentration was 1×10 4 CCU / mL) to establish a vaginal Ureaplasma urealyticum infection model. Each group of mice was placed in an inverted position for 15 minutes to prevent the liquid from flowing out of the vagina. This was done once a day for 3 consecutive days, and on the 7th day of model establishment (taking the time of the first inoculation with Ureaplasma urealyticum serotype 8 as the 1st day of model establishment), a booster inoculation was carried out once. Before and after model establishment, there were no obvious changes in the hair color, movement behavior, and mental state of the mice in the normal control group; after 7 days of model establishment in the remaining 4 groups of mice infected with Ureaplasma urealyticum, the mice generally had dry and split hair, and a poor mental state, showing irritable symptoms and aggressiveness. In addition, the vulva of the mice infected with Ureaplasma urealyticum had obvious red and swollen symptoms and more secretions.

[0060] On the 11th day of model establishment, intragastric administration was carried out. The low-dose group was intragastrically administered with 100 million CFU / d of the postbiotic preparation (the postbiotic preparation prepared in Example 2); the high-dose group was intragastrically administered with 1 billion CFU / d of the postbiotic preparation (the postbiotic preparation prepared in Example 2); the dosing dose of roxithromycin in the roxithromycin group of mice was converted according to the body surface area ratio of a person with a body weight of 60 kg (human:mouse = 1:9.1), and the dose of roxithromycin intragastrically administered to mice was 0.05 g / kg; the normal control group and the model group were given the same volume of normal saline, and the intragastric administration volume per group was controlled at 0.3 mL / animal.

[0061] On the 7th day of continuous intragastric administration, orbital venous plexus blood collection was performed using a capillary tube. On the 21st day of continuous intragastric administration, blood was collected by enucleating the eyeballs of each group of mice. 1 mL of blood was placed in a non-enzymatic centrifuge tube, allowed to stand at 4°C for 30 minutes, and then centrifuged for 20 minutes (4°C, 3000 r / min). The upper light yellow liquid was serum. 80 μL of serum from each of the 3 aliquots was added with 10 μL of CD3-PerCP, 10 μL of CD4-FITC, and 10 μL of CD8-PE monoclonal antibodies, and incubated in the dark for 20 minutes. 2 mL of ACK red blood cell lysate was added to each of the above tubes, gently tapped and mixed evenly, and lysed for 2 minutes. Centrifuged at 4°C and 1500 r / min for 5 minutes, the red supernatant was discarded, 2 mL of PBS was added, gently mixed evenly to resuspend the precipitate, and then centrifuged again at 4°C and 1500 r / min for 5 minutes, the supernatant was discarded, 0.5 mL of PBS was added, and flow cytometry was immediately used to detect the fluorescence percentage. The changes in the levels of peripheral blood lymphocyte subsets in each group of mice were detected by flow cytometry.

[0062] 3. Test results T lymphocyte subsets CD3+, CD4+, and CD8+ cells are the main indicators reflecting cellular immune function. As can be seen from Figure 3 , Figure 4 and Figure 5 , in the first week of drug administration, the regulation of immune response in the roxithromycin group was significantly more effective than that in other groups. However, as the treatment time extended and the drug administration gradually increased to 21 days, the high-dose group of the postbiotic preparation was significantly more effective than the roxithromycin group. The results showed that the postbiotic preparation of Lactobacillus casei JYLC-132 could significantly increase the cell levels of CD3+ and CD4+ in the serum, make the immune response tend to be stable, reduce the CD8+ cell level, slow down immune damage, and restore normal cellular immunity. The postbiotic preparation of Lactobacillus casei JYLC-132 improved vaginitis caused by Ureaplasma urealyticum infection by regulating peripheral blood T lymphocyte subsets CD3+, CD4+, and CD8+.

[0063] Although the present invention has been described in detail by referring to the accompanying drawings and in combination with the preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, those of ordinary skill in the art can make various equivalent modifications or substitutions to the embodiments of the present invention, and these modifications or substitutions should all be within the scope of the present invention. / Any person skilled in the art within the technical scope disclosed by the present invention can easily think of changes or substitutions, which should all be covered by the protection scope of the present invention.

Claims

1. A Lactobacillus casei JYLC-132 for improving vaginitis, characterized in that, Lactobacillus casei Lacticaseibacillus casei JYLC-132 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on February 26, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO. 29894.

2. A postbiotic preparation of Lactobacillus casei JYLC-132 as described in claim 1, characterized in that, The preparation method is to activate Lacticaseibacillus casei JYLC-132 on MRS plate medium, pick a single colony after activation and inoculate it into MRS liquid medium, and statically culture it at a constant temperature of 37°C for 12 h under anaerobic conditions to obtain a seed solution; Inoculate the seed solution into MRS liquid medium according to an inoculation amount of 1%, and statically culture it at a constant temperature of 37°C for 24 h under anaerobic conditions. The obtained bacterial solution is subjected to heat inactivation treatment, concentrated, and freeze-dried to obtain a postbiotic preparation of Lacticaseibacillus casei JYLC-132.

3. The postbiotic preparation according to claim 2, characterized in that, The viable count of the postbiotic preparation is 1.0×10 10 CFU / g.

4. The postbiotic preparation according to claim 2, wherein, The postbiotic preparation also includes pharmaceutically acceptable excipients.

5. The postbiotic preparation according to claim 4, wherein, Pharmaceutically acceptable excipients include glucose and erythritol.

6. Use of a postbiotic preparation according to any one of claims 2-5 in the preparation of a medicament for improving vaginitis, characterized in that, Vaginitis includes non-gonococcal vaginitis and vulvovaginal candidiasis.

7. The application according to claim 6, wherein Non-gonococcal vaginitis includes vaginitis caused by Ureaplasma urealyticum infection.

8. The application according to claim 7, characterized in that, Improving vaginitis includes improving vaginitis caused by Ureaplasma urealyticum infection by regulating the levels of CD3+, CD4+ and / or CD8+ cells.

9. The application according to claim 6, characterized in that, Vulvovaginal candidiasis includes vaginitis caused by Candida albicans infection triggered by diabetes.

10. The application according to claim 9, wherein Improving vaginitis includes improving vaginitis caused by Candida albicans infection triggered by diabetes by inhibiting the expression of TGF-β1.

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