Cheese lactobacillus casei strain JYLC-132 for improving vaginitis, postbiotic preparation and application thereof

Abstract

The present application relates to the technical field of probiotics, in particular to a strain of Lactobacillus casei JYLC-132 for improving vaginitis, a postbiotic preparation and application thereof. Lacticaseibacillus casei The Lactobacillus casei JYLC-132 for improving vaginitis has been preserved in the China General Microbiological Culture Collection Center on February 26, 2024, and the address is No. 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC NO. 29894. The postbiotic preparation prepared from the Lactobacillus casei JYLC-132 can improve vaginitis, in particular, non-gonococcal vaginitis and Candida albicans vaginitis.

Description

Technical Field

[0001] The present invention relates to the technical field of probiotics, and in particular to a Lactobacillus casei JYLC-132 strain for improving vaginitis, and a postbiotic preparation and application thereof. Background Art

[0002] Vaginitis is a common infectious disease of the female reproductive system, primarily caused by an imbalance in the vaginal microbiome. Invading pathogens trigger an inflammatory response in the vaginal mucosa, manifesting clinically with symptoms such as vulvar itching, abnormal vaginal discharge, and burning pain. Long-term unresolved vaginitis not only impacts patients' quality of life but can also lead to serious complications such as pelvic inflammatory disease and infertility. In recent years, the incidence of non-gonococcal vaginitis (NGV) and diabetic vaginitis complicated by Candida albicans has been increasing.

[0003] Non-gonococcal vaginitis is a vaginal inflammation caused by infection with mycoplasma, chlamydia, or other non-gonococcal pathogens. Chlamydia trachomatis is the most common, accounting for approximately 40%-50% of cases, and Ureaplasma urealyticum accounts for approximately 20%-30%. Immune cell dysfunction plays a key role in the pathogenesis of non-gonococcal vaginitis. Candida albicans vaginitis is a fungal vaginitis caused by an overgrowth of Candida albicans. When the vaginal microecology is out of balance, Candida albicans multiplies and invades the vaginal mucosa, causing typical symptoms such as vulvar itching, burning pain, and increased vaginal discharge with a dreg-like appearance. Because diabetic women are in a state of high blood sugar for a long time, the glycogen content in the vaginal epithelial cells increases, providing rich nutrients for the growth and reproduction of Candida albicans; at the same time, high blood sugar can also damage the body's immune defense function and weaken the white blood cells' ability to phagocytize and kill pathogens, making diabetic women a high-risk group for Candida albicans vaginitis. The condition of these patients is often more stubborn, difficult to cure, and prone to relapse, and they need safe and effective targeted treatments.

[0004] Currently, the treatment of vaginitis relies primarily on antibiotics, antifungal drugs, and topical cleansers. While antibiotics can rapidly inhibit the growth of pathogens, they can disrupt the normal vaginal flora, leading to the proliferation of drug-resistant bacteria and recurrent disease. Antifungal drugs have limited efficacy in treating diabetic vaginitis caused by Candida albicans and are prone to toxic side effects with long-term use. Topical cleansers only temporarily relieve symptoms and fail to fundamentally regulate the vaginal microecological balance. Chinese patent application CN117778221A discloses a strain of Lactobacillus rhamnosus that is effective in preventing and treating female vaginitis. The strain significantly inhibits the growth of pathogens such as Gardnerella vaginalis, Candida albicans, and Staphylococcus aureus. However, its efficacy in preventing vaginitis is explored solely based on its ability to inhibit pathogens, adhere to genital tract cells, and its inherent probiotic properties. Animal studies have not been conducted to verify its efficacy, and therefore it is unclear whether Lactobacillus rhamnosus can improve non-gonococcal vaginitis or diabetic vaginitis caused by Candida albicans. Summary of the Invention

[0005] To address the technical problems in existing vaginitis treatments, such as the destruction of normal vaginal flora, limited efficacy, and significant toxic and side effects, especially the lack of effective treatments for non-gonococcal vaginitis and diabetic vaginitis complicated by Candida albicans, the present invention provides a strain of Lactobacillus casei JYLC-132 for improving vaginitis, as well as its postbiotic preparation and application.

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

[0007] In a second aspect, the present invention provides a postbiotic preparation of the above-mentioned Lactobacillus casei JYLC-132. The preparation method is as follows: activating Lactobacillus casei JYLC-132 on an MRS plate culture medium, picking up the activated single colony and inoculating it into an MRS liquid culture medium, and culturing it at a constant temperature of 37°C under anaerobic conditions for 12 hours to obtain a seed liquid; inoculating the seed liquid into the MRS liquid culture medium at a 1% inoculum amount, and culturing it at a constant temperature of 37°C under anaerobic conditions for 24 hours. The obtained bacterial liquid is heat-inactivated, concentrated, and freeze-dried to obtain the postbiotic preparation of Lactobacillus casei JYLC-132.

[0008] Furthermore, the number of bacteria in the postbiotic preparation was 1.0×10 10 CFU / g.

[0009] Furthermore, the postbiotic preparation also includes pharmaceutically acceptable excipients.

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

[0011] In a third aspect, the present invention further provides a use of the above-mentioned postbiotic preparation in the preparation of a medicament for improving vaginitis, wherein the 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 CD3+, CD4+ and / or CD8+ cell levels.

[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 induced by diabetes by inhibiting the expression of TGF-β1.

[0016] The beneficial effects of the present invention are:

[0017] The present invention provides a strain of Lactobacillus casei JYLC-132. A postbiotic preparation prepared from this strain can improve vaginitis, particularly non-gonococcal vaginitis and Candida albicans vaginitis. Experiments have shown that the postbiotic preparation of Lactobacillus casei JYLC-132 can improve vaginitis caused by Candida albicans infection caused by diabetes by inhibiting TGF-β1 expression. The postbiotic preparation of Lactobacillus casei JYLC-132 can also significantly improve vaginitis caused by Ureaplasma urealyticum infection by increasing CD3+ and CD4+ cell levels, stabilizing the immune response, and reducing CD8+ cell levels, slowing immune damage and restoring cellular immunity to normal. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0019] Figure 1 It is a bar graph of TGF-β1 levels in vaginal tissues of rats in the blank control group and the diabetic group.

[0020] Figure 2 The effect of a postbiotic preparation of Lactobacillus casei JYLC-132 on TGF-β1 levels in rat vaginal tissue.

[0021] Figure 3 The effect of a postbiotic preparation of Lactobacillus casei JYLC-132 on the peripheral blood CD3+ level in mice with non-gonococcal vaginitis.

[0022] Figure 4 The effect of a postbiotic preparation of Lactobacillus casei JYLC-132 on the peripheral blood CD4+ level in mice with non-gonococcal vaginitis.

[0023] Figure 5 The effect of a postbiotic preparation of Lactobacillus casei JYLC-132 on the peripheral blood CD8+ level in mice with non-gonococcal vaginitis. DETAILED DESCRIPTION

[0024] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0025] Those skilled in the art will appreciate that the leavener described in Example 1, also known as starter, yeast fertilizer, old fertilizer, or dough starter, is a mixed fermentation system of multiple strains. It primarily relies on the combined effects of the respiration of microorganisms such as yeast, mold, and lactic acid bacteria to produce changes in the texture and flavor of steamed buns.

[0026] Example 1 Isolation, screening and identification of bacterial strains

[0027] 1. Strain screening and purification

[0028] (1) Sampling: Yeast was collected in Jinzhuang Town, Sheqi County, Nanyang City, Henan Province in July 2023.

[0029] (2) Strain separation: Take 1g of fermenter and add it into a sterile Erlenmeyer flask containing 10mL of sterile saline (0.85%), shake it to obtain a sample solution; dilute the sample solution with sterile saline to prepare dilutions of different concentration gradients, which are 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 ; Then, 100 μL of 7 dilutions with different concentration gradients were spread onto MRS plate culture medium using a spreader and cultured at 37°C under anaerobic conditions for 48 h;

[0030] The preparation method of MRS plate culture medium is as follows:

[0031] 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 triammonium 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 in deionized water, make up to 1 L, adjust the pH to 6.3, and sterilize with high-pressure steam at 115°C for 30 min. Then, under sterile conditions, pour the sterilized culture medium into a sterilized plate and let it cool for use.

[0032] (3) Colony selection: Select colonies based on their characteristics of white, round, moist, opaque, and neat edges.

[0033] (4) Isolation and purification: The single colony was inoculated onto an MRS plate using the three-zone streak method and cultured at 37°C under anaerobic conditions for 48 h. Repeat the above operation for a total of three streaking steps to purify 8 pure strains. The pure strains were placed in glycerol tubes and stored at -80°C.

[0034] 2. Screening of postbiotic preparations with inhibitory effects on Candida albicans

[0035] (1) Preparation of postbiotic preparations

[0036] Eight strains stored at -80°C were activated on MRS plate medium. One single colony was picked and transferred to 100 mL of MRS liquid medium. The culture was kept at 37°C for 12 h to prepare seed solution. The seed solution was inoculated into MRS liquid medium at a 1% (v / v) inoculum. The culture was then kept at 37°C for 24 h under anaerobic conditions to obtain a viable bacterial count of 1.0 × 10 10 The bacterial solution (containing bacteria and their metabolites) was heat-inactivated (115°C, 30 min) to obtain a heat-inactivated bacterial solution. The heat-inactivated bacterial solution was concentrated and freeze-dried to obtain 8 kinds of bacteria containing 1.0×10 10 cfu / g of the postbiotic preparations were named Postbiotic Preparation 1, Postbiotic Preparation 2, Postbiotic Preparation 3, Postbiotic Preparation 4, Postbiotic Preparation 5, Postbiotic Preparation 6, Postbiotic Preparation 7 and Postbiotic Preparation 8 respectively.

[0037] Wherein, the preparation method of MRS liquid culture medium is as follows:

[0038] 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 triammonium citrate, 2 g of dipotassium hydrogen phosphate, 0.58 g of magnesium sulfate, 0.25 g of manganese sulfate, and 1 mL of Tween-80, dissolve them in deionized water, make up to 1 L, adjust the pH to 6.3, and sterilize by high-pressure steam at 115°C for 30 min. After cooling, divide the mixture into aseptic packages for later use.

[0039] (2) Inhibition test of Candida albicans

[0040] The standard strain of Candida albicans ATCC10231 (purchased from the Microbiology Laboratory of the Hubei Provincial Center for Disease Control and Prevention) was inoculated into Sabouraud broth (purchased from Qingdao Haibo Biotechnology Co., Ltd.) and cultured with shaking at 35°C for 18-24 hours. 200 μL of the culture was evenly spread on a Sabouraud broth plate (purchased from Qingdao Haibo Biotechnology Co., Ltd.) and allowed to stand for 30 minutes. The test solution was prepared by mixing 1 g of the postbiotic preparation with 1 mL of sterile water. Using an Oxford cup, wells were punched onto the surface of the Sabouraud broth plate coated with C. albicans. 200 μL of the test solution was added to each well. Fluconazole was used as a positive control, and saline was used as a negative control. After incubation at 37°C for 24 hours, the diameter of the inhibition zone was observed and measured. The test results are shown in Table 1.

[0041] Table 1 Antibacterial results of postbiotic preparations

[0042]

[0043] As shown in Table 1, all eight 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.

[0044] 3. Identification and preservation

[0045] The strain used to prepare the postbiotic preparation 2 was sent for identification by Jinan Tianyi Biotechnology Co., Ltd. The primers used during the identification process were as follows:

[0046] 27F (sequence 1): 5′-AGAGTTTGATCCTGGCTCAG-3′;

[0047] 1492R (sequence 2): 5'-CTACGGCTACCTTGTTACGA-3'.

[0048] The strain was identified as Lactobacillus casei ( Lacticaseibacillus casei ), named Lactobacillus casei JYLC-132.

[0049] The Lactobacillus casei ( Lacticaseibacillus casei ) The 16S rDNA gene sequence of JYLC-132 (SEQ ID 3) is:

[0050]

[0051] Lactobacillus casei ( Lacticaseibacillus casei ) JYLC-132 was sent to the General Microbiology Center of China Culture Collection Administration for Microorganisms for preservation. The preservation date was February 26, 2024, and the preservation number was CGMCCNO.29894.

[0052] Example 2 Preparation of a postbiotic preparation of Lactobacillus casei JYLC-132

[0053] Lactobacillus casei JYLC-132 stored at -80°C was activated on an MRS plate medium (preparation method see Example 1), and a single colony after activation was picked and transferred to 100 mL of MRS liquid medium (preparation method see Example 1), and cultured at a constant temperature of 37°C under anaerobic conditions for 12 h to prepare a seed solution, which was inoculated into the MRS liquid medium at a 1% (v / v) inoculum amount, and then cultured at a constant temperature of 37°C under anaerobic conditions for 24 h to obtain a viable cell count of 1.0×10 10 The bacterial solution (containing bacteria and their metabolites) was heat-inactivated (115°C, 30 min), concentrated, and freeze-dried to prepare a postbiotic preparation of Lactobacillus casei JYLC-132.

[0054] The number of bacteria in the postbiotic preparation is 1.0×10 10 CFU / g.

[0055] Example 3 Preparation of a postbiotic preparation of Lactobacillus casei JYLC-132

[0056] Lactobacillus casei JYLC-132 stored at -80°C was activated on an MRS plate medium (preparation method see Example 1), and a single colony after activation was picked and transferred to 100 mL of MRS liquid medium (preparation method see Example 1), and cultured at a constant temperature of 37°C under anaerobic conditions for 12 h to prepare a seed solution, which was inoculated into the MRS liquid medium at a 1% (v / v) inoculum amount, and then cultured at a constant temperature of 37°C under anaerobic conditions for 24 h to obtain a viable cell count of 1.0×10 10 The bacterial solution (containing bacteria and their metabolites) was heat-inactivated (115°C, 30 min), concentrated, freeze-dried, and then mixed with glucose to prepare a postbiotic preparation of Lactobacillus casei JYLC-132.

[0057] The number of bacteria in the postbiotic preparation is 1.0×10 10 CFU / g.

[0058] Example 4 Preparation of a postbiotic preparation of Lactobacillus casei JYLC-132

[0059] Lactobacillus casei JYLC-132 stored at -80°C was activated on an MRS plate medium (preparation method see Example 1), and a single colony after activation was picked and transferred to 100 mL of MRS liquid medium (preparation method see Example 1), and cultured at a constant temperature of 37°C under anaerobic conditions for 12 h to prepare a seed solution, which was inoculated into the MRS liquid medium at a 1% (v / v) inoculum amount, and then cultured at a constant temperature of 37°C under anaerobic conditions for 24 h to obtain a viable cell count of 1.0×10 10 The bacterial solution (containing bacteria and their metabolites) 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.

[0060] The number of bacteria in the postbiotic preparation is 1.0×10 10 CFU / g.

[0061] Those skilled in the art will appreciate that the glucose and erythritol added to the postbiotic preparations of Examples 3 and 4 are merely auxiliary ingredients for adjusting the taste. If the postbiotic preparations are used to improve diabetes combined with Candida albicans vaginitis (vaginitis caused by Candida albicans infection caused by diabetes), the postbiotic preparations of Lactobacillus casei JYLC-132 of Examples 2 and 4 are preferred to avoid the negative impact of glucose on diabetes.

[0062] Experimental Example 1 Effect of a postbiotic preparation of Lactobacillus casei JYLC-132 on diabetic Candida albicans vaginitis

[0063] 1. Preparation

[0064] Experimental animals: 60 female Sprague-Dawley rats (weighing approximately 200 g) were purchased from the Experimental Animal Center of Tongji Medical College, Huazhong University of Science and Technology. Inoculated strain: Candida albicans standard strain ATCC10231, sixth generation, purchased from the Microbiology Laboratory of Hubei Provincial Center for Disease Control and Prevention.

[0065] 2. Grouping and modeling

[0066] After one week of adaptive feeding, 60 female Sprague-Dawley rats were randomly divided into a diabetic group (n=40) and a blank control group (n=20). After blood glucose levels were normalized in all groups, rats were fasted but not watered. Twelve hours later, the diabetic group received a single intraperitoneal injection of 65 mg / kg of 10 mg / mL streptozotocin (STZ, pH 4.5). Simultaneously, the blank control group received a single intraperitoneal injection of an equal volume of citric acid-sodium citrate buffer solution. One week after the intraperitoneal injection of STZ, blood glucose levels were measured in all groups. Successful establishment of a diabetic rat model was determined to be successful if the blood glucose level was ≥16.7 mmol / L.

[0067] Ten rats from the blank control group and ten rats from the model diabetic group were randomly selected. After vaginal lavage, they were killed by cervical dislocation and then fixed on a sterile operating table. The hair on both sides of the upper edge of the pubic symphysis and the lower midline of the abdomen were trimmed and disinfected. The abdominal wall and peritoneum were incised along the midline of the abdomen, the Y-shaped uterus was found and the surrounding connective tissue was peeled off. The vagina was found along the uterus downward, and the vaginal tissue was quickly removed and stored in a -70℃ refrigerator for later use.

[0068] The remaining 30 rats in the diabetic group were randomly divided into a diabetic Candida albicans vaginitis group (n=10), a low-dose group (n=10), and a high-dose group (n=10). The remaining blank control group served as a non-diabetic Candida albicans infection group (n=10). The vaginas of the rats in the four groups were inoculated with a sterile saline suspension of Candida albicans spores (containing 5×10 Candida albicans spores). 8 One day after inoculation, rats in the low-dose group were orally administered with 100 million CFU / d of the postbiotic preparation (prepared in Example 2), and rats in the high-dose group were orally administered with 1 billion CFU / d of the postbiotic preparation (prepared in Example 2). Administration was continued for 6 consecutive days. Rats in the remaining two groups (diabetic Candida albicans vaginitis group and non-diabetic combined Candida albicans infection group) were orally administered with an equal volume of sterile saline. A normal diet was maintained during modeling.

[0069] After the administration, the rats in the four groups were sacrificed by cervical dislocation after vaginal lavage, and then fixed on a sterile operating table. The hair on both sides of the upper edge of the pubic symphysis and the lower midline of the abdomen were trimmed and disinfected. The abdominal wall and peritoneum were incised along the midline of the abdomen, the Y-shaped uterus was found, and the surrounding connective tissue was peeled off. The vagina was found along the uterus, and the vaginal tissue was quickly removed and stored in a -70℃ refrigerator for later use.

[0070] 3. TGF-β1 detection

[0071] Homogenate Preparation: Vaginal tissues from the six groups stored at -70°C were minced and added with a physiological saline homogenate diluent (containing 0.05% Triton X-100, 5 mM CaCl2, and 10 μg / mL indomethacin). The homogenates were ground on ice to prepare homogenates. The homogenates were centrifuged at 3000 g for 15 minutes at 4°C. The supernatant was collected and stored at -70°C until testing.

[0072] ELISA: TGF-β1 levels in vaginal tissue from each group were determined using an enzyme-linked immunosorbent assay (ELISA) sandwich assay. Both the pre-coated and detection antibodies are affinity-purified polyclonal antibodies. The detection antibody is biotinylated. Samples and the biotinylated antibody (detection antibody) are added to the ELISA plate for reaction, followed by washing with PBS or TBS. Peroxidase-labeled avidin is then added for reaction. After thorough washing with PBS or TBS, the plate is developed with the substrate TMB. TMB converts to blue under the catalysis of peroxidase and to yellow under the action of acid. The intensity of the color is positively correlated with the concentration of rat TGF-β1 in the sample.

[0073] Determine the number of antibody-coated ELISA plate wells required for this assay and add one blank TMB well. Add 0.1 mL of each standard at concentrations of 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 a row of seven wells. One well, containing only the sample diluent, serves as the zero well. Add 100 μL of the supernatant from each group to be tested to each well of the ELISA plate. Cover the plate and incubate at 37°C for 90 minutes. After the reaction, shake off the liquid in the ELISA plate and tap it against absorbent paper several times without rinsing. Add 0.1 mL of the prepared biotinylated anti-rat TGF-β1 antibody working solution to each well. Incubate at 37°C for 60 minutes. After the reaction, shake off the liquid in the ELISA plate and wash three times with 0.01 M PBS, soaking for approximately 1 minute each time. Add 0.1 mL of the prepared ABC working solution to each well. Incubate at 37°C for 30 minutes. Discard the liquid from the plate and wash five times with 0.01M PBS, soaking for approximately 1-2 minutes each time. Add 90 μL of TMB colorimetric solution to each well and incubate at 37°C in the dark for 20-25 minutes. Add 0.1 mL of TMB stop solution to each well, which should immediately turn the blue color to yellow. Measure the OD value at 450 nm using a microplate reader. Set well zero as the control.

[0074] After subtracting the absorbance of the zero well from the absorbance of the standard and supernatant samples, the resulting data can be directly plotted on graph paper. Based on the absorbance of the sample, the corresponding TGF-β1 concentration in the test sample is calculated according to the standard curve.

[0075] Data are expressed as mean ± standard deviation. All results were analyzed using SPSS 16.0 statistical analysis software. Student's t-test was used to compare the blank control group and the diabetic group. One-way analysis of variance was used to compare the diabetic C. albicans vaginitis group, the low-dose group, the high-dose group, and the non-diabetic combined C. albicans infection group. Intergroup comparisons were performed using the LSD method. P < 0.05 was considered statistically significant.

[0076] 4. Test results

[0077] Depend on Figure 1 The TGF-β1 test results showed that the TGF-β1 level in the vaginal tissue of rats in the diabetic group was significantly higher than that in the blank control group (P < 0.05). The difference was statistically significant, indicating that the vaginitis model caused by Candida albicans infection induced by diabetes was successfully established.

[0078] like Figure 2 As shown, one week after inoculation with Candida albicans, TGF-β1 levels were measured in the vaginal tissues of rats in the non-diabetic combined Candida albicans infection group, the diabetic Candida albicans vaginitis group, the low-dose group, and the high-dose group. TGF-β1 levels were significantly increased in the diabetic Candida albicans vaginitis group compared with the non-diabetic combined Candida albicans infection group (P < 0.05), indicating a statistically significant difference. Following treatment with a postbiotic preparation of Lactobacillus casei JYLC-132, TGF-β1 levels in the vaginal tissues of rats in the low-dose and high-dose groups were significantly altered compared with the diabetic Candida albicans vaginitis group (P < 0.05), indicating a statistically significant difference. These results indicate that the postbiotic preparation of Lactobacillus casei JYLC-132 significantly improves vaginitis caused by diabetic Candida albicans infection.

[0079] Experimental Example 2 Effects of a postbiotic 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)

[0080] 1. Preparation

[0081] Experimental Animals: 70 SPF-grade healthy female BALB / c mice, 4-6 weeks old, weighing 18-24 g, were purchased from the Laboratory Animal Research Center of Inner Mongolia Medical University. Mice were housed under standard conditions: room temperature of 23-25°C, humidity of 45%-55%, and a 12-hour light-dark cycle (light cycle from 7:00 AM to 7:00 PM). Mice were provided with ample food and water, with free access to food and water. Experiments were conducted between 8:00 AM and 6:00 PM. Ureaplasma urealyticum serotype 8 (ATCC 27816) was purchased from the American Type Culture Collection (ATCC) and maintained after resuscitation and passage.

[0082] 2. Grouping, modeling and drug administration

[0083] Fifty mice were randomly selected and divided into five groups, namely normal control group, model group, roxithromycin group, low-dose group, and high-dose group, with 10 mice in each group.

[0084] After one week of adaptive feeding, the mice in the other four groups, except the normal control group, were pretreated with estrogen and injected subcutaneously with estradiol benzoate injection at 0.4 mg / 20 g body weight in the neck for seven consecutive days. Then, a sterile dry cotton swab was rotated several times at the vaginal opening of the mice to expand the vaginal opening. The mice in the model group, roxithromycin group, low-dose group, and high-dose group were inoculated with 50 μL of Ureaplasma urealyticum serotype 8 bacterial solution (inoculation concentration of 1×10 4 A vaginal Ureaplasma urealyticum infection model was established by vaginal inoculation of 100 mg / mL (CCU / mL). Mice in each group were placed in an inverted position for 15 minutes to prevent vaginal fluid from leaking. This inoculation was repeated daily for three consecutive days, with a booster inoculation on day 7 of modeling (the date of the first inoculation with Ureaplasma urealyticum serotype 8 was designated as day 1 of modeling). The normal control group showed no significant changes in hair color, behavior, or mental state before and after modeling. Seven days after modeling, mice in the remaining four Ureaplasma urealyticum-infected groups generally developed dry, split hair, poor mental state, and exhibited irritability and aggressiveness. The Ureaplasma-infected mice also displayed significant redness and swelling of the vulva, along with profuse discharge.

[0085] On the 11th day of modeling, mice were gavaged with 100 million CFU / d of the postbiotic preparation (the postbiotic preparation prepared in Example 2). The high-dose group was gavaged with 1 billion CFU / d of the postbiotic preparation (the postbiotic preparation prepared in Example 2). The dosage of roxithromycin in the mice was calculated based on the body surface area ratio of a 60 kg human (human: mouse = 1:9.1), and the gavage dose of roxithromycin for mice was 0.05 g / kg. The normal control group and the model group were given the same volume of normal saline, and the gavage volume in each group was controlled at 0.3 mL / mouse.

[0086] On the 7th day of continuous gavage administration, blood was collected from the orbital venous plexus using a capillary tube. On the 21st day of continuous gavage administration, blood was collected from the eyeballs of each group of mice. 1 mL of blood was placed in an enzyme-free centrifuge tube, allowed to stand at 4°C for 30 minutes, and then centrifuged for 20 minutes (4°C, 3000 rpm). The pale yellow liquid on the upper layer was serum. Three 80 μL aliquots of serum were added to 10 μL of CD3-PerCP, 10 μL of CD4-FITC, and 10 μL of CD8-PE monoclonal antibodies, respectively, and incubated in the dark for 20 minutes. 2 mL of ACK red blood cell lysis buffer was added to each tube, gently tapped to mix, and lysed for 2 minutes. The tubes were centrifuged at 4°C, 1500 rpm, for 5 minutes. The red supernatant was discarded, and the pellet was gently resuspended in 2 mL of PBS with gentle mixing. The pellet was then centrifuged again at 4°C, 1500 rpm, for 5 minutes. The supernatant was discarded, and 0.5 mL of PBS was added. Fluorescence percentage was immediately determined by flow cytometry. Flow cytometry was used to detect the changes in the levels of peripheral blood lymphocyte subsets in each group of mice.

[0087] 3. Test results

[0088] T lymphocyte subsets CD3+, CD4+, and CD8+ cells are the main indicators of cellular immune function. Figure 3 、 Figure 4 and Figure 5 As shown, during the first week of treatment, the roxithromycin group showed a more pronounced immune response regulation than the other groups. However, as treatment progressed, and dosing gradually increased to day 21, the high-dose postbiotic preparation significantly outperformed the roxithromycin group. The results showed that the postbiotic preparation derived from Lactobacillus casei JYLC-132 significantly increased serum CD3+ and CD4+ cell levels, stabilizing the immune response, while reducing CD8+ cell levels, mitigating immune damage, and restoring normal cellular immunity. The postbiotic preparation derived from Lactobacillus casei JYLC-132 improves vaginitis caused by Ureaplasma urealyticum infection by regulating peripheral blood T lymphocyte subsets CD3+, CD4+, and CD8+.

[0089] Although the present invention has been described in detail with reference to the accompanying drawings and in conjunction with preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, persons of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and such modifications or substitutions shall be within the scope of the present invention. Any changes or substitutions that can be easily conceived by persons skilled in the art within the technical scope disclosed in the present invention shall be within the scope of protection of the present invention.

Claims

1. An application of a postbiotic preparation of Lactobacillus casei JYLC-132 in the preparation of a medicament for improving vaginitis, characterized in that: Vaginitis is caused by ureaplasma urealyticum infection; Lactobacillus casei ( Lacticaseibacillus casei JYLC-132 was deposited on February 26, 2024, at the General Microbiology Center of the China Culture Collection Administration (CGCCC), located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the accession number CGMCC NO. 29894. The preparation method of the postbiotic preparation of Lactobacillus casei JYLC-132 comprises the following steps: activating Lactobacillus casei JYLC-132 on an MRS plate culture medium, picking a single colony after activation and inoculating it into an MRS liquid culture medium, and culturing the culture medium at a constant temperature of 37° C. for 12 hours under anaerobic conditions to obtain a seed solution; The seed liquid was inoculated into MRS liquid culture medium at a 1% inoculum rate and cultured at a constant temperature of 37°C under anaerobic conditions for 24 hours. The obtained bacterial liquid was heat-inactivated, concentrated, and freeze-dried to obtain a postbiotic preparation of Lactobacillus casei JYLC-132.

2. The use according to claim 1, characterized in that The number of bacteria in the postbiotic preparation is 1.0×10 10 CFU / g.

3. The use according to claim 1, characterized in that Postbiotic preparations also include pharmaceutically acceptable excipients.

4. The use according to claim 3, characterized in that Pharmaceutically acceptable excipients include glucose and erythritol.

5. The use according to claim 1, characterized in that Improving vaginitis includes improving vaginitis caused by ureaplasma urealyticum infection by regulating CD3+, CD4+ and / or CD8+ cell levels.

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