A strain of paracasei that simultaneously improves oxidative damage, skin aging, and female intimate health

Abstract

The application discloses a paracasei lactis strain for simultaneously improving oxidative damage, skin aging and female private health, and belongs to the technical field of microorganisms. The application screens a paracasei lactis strain CCFM1317, wherein the paracasei lactis strain CCFM1317 has the effects of repairing oxidative damage related symptoms caused by vaginitis and improving intestinal health and skin health. The effects are as follows: improving the Nrf2 content of cell supernatant, improving oxidative stress of a mouse vagina, enhancing the anti-aging ability of a mouse skin, enhancing the antioxidant capacity of a mouse intestine, improving pathological characteristics of a mouse vagina, reducing the pathogenic bacteria load of a mouse vagina, reducing the gardnerella load of a mouse vagina, and improving pathological characteristics of a mouse vagina. Therefore, the paracasei lactis strain CCFM1317 has great application prospects in products for improving and repairing oxidative damage caused by vaginitis and skin health and intestinal health problems caused by vaginitis.

Description

Technical Field

[0001] This invention relates to a strain of *Lactobacillus paracasei* that simultaneously improves oxidative damage, skin aging, and women's intimate health, belonging to the field of microbial technology. Background Technology

[0002] Facultative anaerobic bacteria such as Gardnerella vaginalis invade the vagina and proliferate rapidly, leading to bacterial vaginosis (BV), a common vaginal infection in women of reproductive age. Gardnerella vaginalis, invading the vaginal environment, adheres to reproductive tract epithelial cells and secretes toxins, causing damage to mitochondria. The mitochondrial respiratory chain (ETC) is a major site of reactive oxygen species (ROS) production. Electrons are leaked during ETC transport, directly transferring to oxygen and generating large amounts of ROS, causing an imbalance in the body's redox system and resulting in oxidative damage. Nuclear factor-erythrocyte-associated factor 2 (Nrf2) is an important antioxidant transcription factor. Under normal circumstances, Nrf2 is maintained at low levels in the cytoplasm by binding to KELCH-like ECH-associated protein 1 (Keap1). Studies have shown that probiotics, by activating the Nrf2 signaling pathway, can increase the expression levels of the Nrf2 gene and protein, thereby increasing the anti-inflammatory and antioxidant capacity of cells. MAPK is an intracellular threonine protein kinase that can be activated extracellularly by ROS, thereby further regulating the transcription of MMPs, especially the MMP family of interstitial collagenase (MMP-1), matrix-degrading enzyme-1 (MMP-3), and gelatinase (MMP-9), leading to the degradation of collagen and elastin.

[0003] Currently, the treatment for BV vaginitis mainly involves oral or topical application of anti-anaerobic antibiotics such as nitroimidazoles (metronidazole, tinidazole) and lincomycins (clindamycin) to inhibit their overgrowth. However, while antibiotics kill the pathogens, they also eliminate all the inherent vaginal microorganisms, failing to repair the vaginal environment damaged by oxidative stress and increasing the risk of vaginitis recurrence.

[0004] Studies have shown that the addition of live lactobacillus capsules to the treatment of recurrent bacterial vaginosis can significantly reduce the recurrence rate (Observation on the effectiveness of live lactobacillus capsules in the treatment of recurrent vaginitis, Mingyi, 2020). Live lactobacillus preparations can effectively improve the local microecological environment, achieving the goal of treating BV vaginitis with significant efficacy (Comprehensive clinical evaluation of live lactobacillus capsules for the treatment of bacterial vaginosis, Chinese Journal of New Drugs, 2022).

[0005] In 1989, the Institute of Microecology at Dalian Medical University isolated and screened a strain of Lactobacillus DM8909 from vaginal secretions of healthy women. Preliminary experiments ("Study on the Inhibition of Vaginal Infection in Mice by Lactobacillus DM8909," *Chinese Journal of Microecology*, 2003; "Study on the Inhibitory Effect of Lactobacillus DM8909 on Escherichia coli Adhesion to Vaginal Epithelial Cells," *Chinese Journal of Microecology*, 1997; "Preliminary Study on the Adhesion Phenomenon of Lactobacillus DM8909 to Vaginal Epithelial Cells," *Chinese Journal of Microecology*, 1993) and clinical data ("Phase II Clinical Trial Study of Lactobacillus delbrueckii DM8909 in the Treatment of Bacterial Vaginosis," *Chinese Journal of Microecology*, 2001) showed that Lactobacillus delbrueckii DM8909 possesses good probiotic properties, such as a certain adhesion advantage to vaginal epithelial cells, while simultaneously inhibiting the growth and reproduction of harmful pathogenic bacteria and regulating the vaginal flora balance, thus effectively treating bacterial vaginosis. However, the specific effects of this Lactobacillus on the oxidative damage that causes vaginitis were not reported.

[0006] Some patent applications have involved the ability of *Lactobacillus paracasei* to inhibit the growth of pathogenic bacteria (CN116426435A and CN116179413A); however, the oxidative damage caused by vaginitis by the protected strains has not been studied. Furthermore, there is still no *Lactobacillus paracasei* strain with an elucidated mechanism that can be clearly used to alleviate bacterial vaginosis and promote gut and skin health. Summary of the Invention

[0007] This invention provides a strain of *Lactobacillus paracasei* CCFM1317 that can alleviate bacterial vaginosis, improve oxidative damage caused by vaginitis, and address skin and intestinal health problems caused by vaginitis. *Lactobacillus paracasei* CCFM1317 exhibits good ability to activate the Keap1 / Nrf2 pathway in vitro, and has the potential to alleviate bacterial vaginosis. SPF-grade BALB / c mice, female, 7 weeks old, weighing 18-20g, were selected. After the adaptation period, mice were injected with estradiol valerate for three consecutive days to induce estrus. Gardnerella vaginalis was then used to infect the mice for 5 consecutive days, once daily. The specific infection procedure involved preparing a 10... 10 CFU / mL Gardnerella vaginalis suspension was administered at a dose of 20 μL per mouse. 20 μL of the suspension was slowly injected into the vagina of the mouse using a pipette tip. The mouse was then inverted and held for 1-2 minutes before being placed in a cage. Mice with a bacterial vaginosis model were divided into a model group and an intervention group. The intervention group received 100 μL of a CFU / mL Gardnerella vaginalis suspension via gavage. 9The model group underwent simple Gardnerella vaginalis infection with a CFU / mL probiotic suspension, without any subsequent intervention. The intervention group underwent Gardnerella vaginalis infection and received probiotics as a subsequent intervention. A blank control group was also included, which did not undergo Gardnerella vaginalis infection and did not receive any subsequent probiotic intervention. During the experiment, the efficacy of treatment for bacterial vaginosis in mice was evaluated by analyzing Gardnerella vaginalis colonization, vaginal epithelial cell shedding, secretion of antioxidant factor (Nrf2), expression of the antioxidant pathway (Nrf2), and histopathological findings. Simultaneously, the expression of MAPK / AP-1 / MMP-1 mRNA in mouse skin tissue and the expression of Keap1 / Nrf2 mRNA and Nrf2 levels in colon tissue were detected to evaluate the intervention effect on skin aging and intestinal health problems caused by vaginitis in mice.

[0008] This invention provides a strain of Lacticaseibacillus paracasei CCFM1317; the Lacticaseibacillus paracasei CCFM1317 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on August 4, 2023, with the accession number GDMCC No: 63713.

[0009] The present invention provides a microbial inoculant containing the above-mentioned Lactobacillus paracasei CCFM1317 or its fermentation broth or its lysate.

[0010] In one embodiment of the present invention, the microbial agent is a liquid preparation, a powder preparation, or a granular preparation.

[0011] In one embodiment of the present invention, the microbial agent is freeze-dried powder of Lactobacillus paracasei CCFM1317.

[0012] In one embodiment of the present invention, the amount of *Lactobacillus paracasei* CCFM1317 added to the above-mentioned microbial agent is at least 10. 6 CFU / mL or 10 6 CFU / g.

[0013] The present invention provides a product containing the above-mentioned Lactobacillus paracasei CCFM1317 or containing the above-mentioned microbial agent.

[0014] In one embodiment of the present invention, the amount of *Lactobacillus paracasei* CCFM1317 added to the above-mentioned product is at least 10. 6 CFU / mL or 10 6 CFU / g.

[0015] In one embodiment of the present invention, the product is a pharmaceutical or hygiene product.

[0016] In one embodiment of the present invention, the sanitary products include sanitary wipes, sanitary napkins, panty liners, sanitary tampons, sanitary cotton pads, vaginal washes, and feminine antibacterial / bacteriostatic washes.

[0017] In one embodiment of the invention, the pharmaceutical product comprises the above-described composition and a pharmaceutically permissible carrier.

[0018] In one embodiment of the present invention, the carrier includes one or more of the following commonly used in medicine: fillers, adhesives, wetting agents, disintegrants, lubricants, and flavoring agents.

[0019] In one embodiment of the present invention, the dosage form of the medicine includes granules, capsules, tablets, pills, suppositories, or oral liquids.

[0020] In one embodiment of the present invention, the pharmaceutical product includes enteric-coated tablets and capsules, oral liquids, vaginal suppositories, tablets, gelatin capsules, sprays, creams, and gels.

[0021] The present invention also provides the use of the above-mentioned Lactobacillus paracasei CCFM1317 or the above-mentioned microbial agent in the preparation of products for relieving and / or treating bacterial vaginosis or anti-aging.

[0022] In one embodiment of the present invention, the amount of *Lactobacillus paracasei* CCFM1317 added to the above-mentioned product is at least 10. 6 CFU / mL or 10 6 CFU / g.

[0023] In one embodiment of the present invention, the product is a pharmaceutical or hygiene product.

[0024] In one embodiment of the present invention, the sanitary products include sanitary wipes, sanitary napkins, panty liners, sanitary tampons, sanitary cotton pads, vaginal washes, and feminine antibacterial / bacteriostatic washes.

[0025] In one embodiment of the invention, the pharmaceutical product comprises the above-described composition and a pharmaceutically permissible carrier.

[0026] In one embodiment of the present invention, the carrier includes one or more of the following commonly used in medicine: fillers, adhesives, wetting agents, disintegrants, lubricants, and flavoring agents.

[0027] In one embodiment of the present invention, the dosage form of the medicine includes granules, capsules, tablets, pills, suppositories, or oral liquids.

[0028] In one embodiment of the present invention, the pharmaceutical product includes enteric-coated tablets and capsules, oral liquids, vaginal suppositories, tablets, gelatin capsules, sprays, creams, and gels.

[0029] The present invention also provides the use of the above-mentioned *Lactobacillus paracasei* CCFM1317 or the above-mentioned microbial inoculant in the preparation of products or microbial inoculants having any of the following uses:

[0030] (1) Increase the Nrf2 content in cell supernatant;

[0031] (2) Improves vaginal oxidative stress in mice: Increases Nrf2 levels in vaginal tissue;

[0032] (3) Enhance the anti-aging ability of mouse skin: Downregulate the expression of MAPK / AP-1 / MMP-1 mRNA;

[0033] (4) Enhance the antioxidant capacity of mouse intestines: activate the Keap1 / Nrf2 pathway and increase Nrf2 levels in colon tissue;

[0034] (5) Improved vaginal pathological features and reduced pathogenic bacterial load in mice by reducing Gardnerella vaginalis load in mice.

[0035] (6) Improves the pathological characteristics of the mouse vagina.

[0036] In one embodiment of the present invention, the product is a pharmaceutical or hygiene product.

[0037] In one embodiment of the present invention, the sanitary products include sanitary wipes, sanitary napkins, panty liners, sanitary tampons, sanitary cotton pads, vaginal washes, and feminine antibacterial / bacteriostatic washes.

[0038] Beneficial effects

[0039] This invention screened and obtained a strain of *Lactobacillus paracasei* CCFM1317. This *Lactobacillus paracasei* CCFM1317 has the effect of repairing symptoms related to oxidative damage caused by vaginitis and promoting intestinal and skin health, specifically manifested in the following ways:

[0040] (1) Increase the Nrf2 content in cell supernatant;

[0041] (2) Improves vaginal oxidative stress in mice: Increases Nrf2 levels in vaginal tissue;

[0042] (3) Enhance the anti-aging ability of mouse skin: Downregulate the expression of MAPK / AP-1 / MMP-1 mRNA;

[0043] (4) Enhance the antioxidant capacity of mouse intestines: activate the Keap1 / Nrf2 pathway and increase Nrf2 levels in colon tissue;

[0044] (5) Improved vaginal pathological features and reduced pathogenic bacterial load in mice by reducing Gardnerella vaginalis load in mice.

[0045] (6) Improved pathological features of the mouse vagina;

[0046] Therefore, Lacticaseibacillus paracasei CCFM1317 has great application potential in products that improve and repair oxidative damage caused by vaginitis, as well as skin and intestinal health problems caused by vaginitis.

[0047] Preservation of biological materials

[0048] A strain of Lacticaseibacillus paracasei (CCFM1317), taxonomically named Lacticaseibacillus paracasei, was deposited on August 4, 2023, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No:63713), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0049] Figure 1 Effect of different bacterial lysates on Nrf2 content in the supernatant of vaginal epithelial cell VK2 / E6E7 culture; compared with the model group, *p<0.05, **p<0.01, ***p<0.001.

[0050] Figure 2 Animal experiment design flowchart.

[0051] Figure 3 Effect of Lactobacillus paracasei on vaginal Nrf2 mRNA expression; **p<0.01, ****p<0.0001.

[0052] Figure 4 Figure: Effect of Lactobacillus paracasei on colonic oxidation; where a represents the expression of antioxidant Nrf2; b represents the effect of Keap1 mRNA expression; compared with the model group, ****p<0.0001.

[0053] Figure 5Figure: Effect of Lactobacillus paracasei on skin aging; where a represents the effect on MAPK mRNA expression; b represents the effect on AP-1 mRNA expression; c represents the effect on MMP-1 mRNA expression; compared with the model group, **p<0.01, ***p<0.001, ****p<0.0001.

[0054] Figure 6 : Colonization of Gardnerella vaginalis in the vagina; Gardnerella vaginalis load in the vagina of BV mice on day 7 and day 17; a and b represent significant differences between different groups on day 17 (p<0.01).

[0055] Figure 7 : Histopathological evaluation of mouse vaginal tissue. Detailed Implementation

[0056] The vaginal epithelial cells VK2 / E6E7 involved in the following examples were kindly provided by the Department of Obstetrics and Gynecology of People's Hospital.

[0057] Gardnerella vaginalis ATCC 14018 was purchased from the Guangdong Institute of Microbiology Culture Collection Center (GDMCC). The *Lactobacillus paracasei* FCQNA15L3 and *Lactobacillus gasseri* QJSWX195M1 mentioned in the following examples were deposited at the Jiangnan University Food Microbiology Culture Collection Center.

[0058] The Lactobacillus delbrueckii DM8909 was purchased from Dingjunsheng (Lactobacillus live bacteria capsules for vaginal use).

[0059] The culture media involved in the following examples are as follows:

[0060] MRS medium: yeast extract 5.0 g / L, beef extract 10.0 g / L, peptone 10.0 g / L, glucose 20.0 g / L, anhydrous sodium acetate 2.0 g / L, diammonium citrate 2.0 g / L, dipotassium hydrogen phosphate 2.6 g / L, manganese sulfate monohydrate 0.25 g / L, magnesium sulfate heptahydrate 0.5 g / L, Tween-80 1 mL, pH 6.2–6.4.

[0061] BHI medium: tryptone 10.0 g / L, bovine heart extract 17.5 g / L, sodium chloride 5.0 g / L, glucose 3.0 g / L, disodium hydrogen phosphate dodecahydrate 2.5 g / L, yeast extract 10.0 g / L, maltose 1.0 g / L, pH 7.2–7.4; after cooling to about 55°C, add 10% sterile fetal bovine serum.

[0062] Cell culture medium: 89% (v / v) DMEM medium + 10% (v / v) fetal bovine serum + 1% (v / v) 100× penicillin

[0063] A mixed solution of penicillin and streptomycin (the mixed solution contains 10,000 U / mL of penicillin and 10 mg / mL of streptomycin).

[0064] Lactobacillus paracasei CCFM1317 bacterial suspension: Lactobacillus paracasei CCFM1317 was inoculated into MRS liquid medium at a 2% inoculum size and incubated at 37°C for 24 hours. The bacterial suspension concentration was then adjusted to 1×10⁻⁶. 9 CFU / mL.

[0065] Gardnerella vaginalis suspension: Gardnerella vaginalis strain ATCC 14018 was cultured in BHI medium at 37°C for 24 hours, and the bacterial suspension concentration was adjusted to 10. 10 CFU / mL.

[0066] The detection methods involved in the following embodiments are as follows:

[0067] Detection of Nrf2 content in VK2 / E6E7 cell culture supernatant

[0068] Two mL of culture medium containing lysates of *Lactobacillus paracasei* CCFM1317, *Lactobacillus paracasei* FCQNA15L3, *Lactobacillus delbrueckii* DM8909, or *Lactobacillus gasseri* QJSWX195M1 were added to each well of a 12-well plate and incubated for 24 h. Each sample was tested in triplicate. After co-culture, the culture supernatant was collected, and the Nrf2 content in the vaginal epithelial cell culture supernatant was detected by ELISA.

[0069] Secretion of the antioxidant factor Nrf2 and expression of Keap1 / Nrf2 mRNA in mouse vaginal tissue

[0070] Total RNA was extracted from mouse vaginal tissue using the Trizol method, and OD was detected using Nanodrop. 260 / OD 280 The values ​​were determined by reverse transcription. 1 μg of total RNA was reverse transcribed into 20 μL of cDNA using a reverse transcription kit. The reaction mixture was prepared using ChamQ Universal SYBR qPCR Master Mix for quantitative real-time PCR. GAPDH was used as an internal control gene, based on 2... -ΔΔCt The expression level of the target gene was calculated using the following methods; the primers are shown in Table 1.

[0071] Table 1: Primers and Sequences

[0072]

[0073] Secretion of the antioxidant factor Nrf2 and expression of Keap1 / Nrf2 mRNA in mouse colon tissue

[0074] 20 mg of mouse colon tissue was used to measure cytokines. Vaginal tissue was homogenized with 180 μL of pre-chilled PBS. The sample was centrifuged at 3000 rpm for 15 min at 4°C. The supernatant from the vaginal tissue was used to determine Nrf2 concentration according to the kit instructions (Nanjing Senbeijia Biotechnology Co., Ltd.). Total RNA was extracted from mouse colon tissue using the Trizol method, and OD was detected using Nanodrop. 260 / OD 280 The values ​​were determined by reverse transcription. 1 μg of total RNA was reverse transcribed into 20 μL of cDNA using a reverse transcription kit. The reaction mixture was prepared using ChamQ Universal SYBR qPCR Master Mix for quantitative real-time PCR. GAPDH was used as an internal control gene, based on 2... -ΔΔCt The expression level of the target gene was calculated using the method described in Table 2.

[0075] Table 2: Primers and Sequences

[0076]

[0077] MAPK / AP-1 / MMP-1 mRNA expression in mouse skin tissue

[0078] 20 mg of mouse dorsal skin tissue was collected for cytokine determination. Vaginal tissue was homogenized with 180 μL of pre-chilled PBS. The sample was centrifuged at 3000 rpm for 15 min at 4°C. The supernatant from the vaginal tissue was used to determine Nrf2 concentration according to the kit instructions (Nanjing Senbeijia Biotechnology Co., Ltd.). Total RNA was extracted from mouse vaginal tissue using the Trizol method, and OD was detected using Nanodrop. 260 / OD 280 The values ​​were calculated, and 1 μg of total RNA was reverse transcribed into 20 μL of cDNA using a reverse transcription kit. The reaction mixture was prepared using ChamQ Universal SYBR qPCR Master Mix for quantitative real-time PCR. GAPDH was used as an internal control gene, based on 2... -ΔΔCt The expression level of the target gene was calculated using the method described in Table 3.

[0079] Table 3: Primers and Sequences

[0080]

[0081] Gardnerella vaginalis colonization in mouse vagina

[0082] Sampling was performed by using a pipette tip to aspirate a certain amount of phosphate buffered solution from the mouse vagina to obtain 300 μL of vaginal lavage fluid sample. Gardnerella vaginalis was counted using qPCR. The entire experimental period was 17 days, with two samplings. The first sampling was on day 7 (the first day after infection ended) as a colonization test for Gardnerella vaginalis, and the second sampling was on day 17 after the intervention to quantify the load of Gardnerella vaginalis and Lactobacillus. First, DNA was extracted from the vaginal lavage fluid using a soil rapid DNA rotation kit (MP Biomedical, USA) and a QIAQuick Gel extraction kit (Qiagen, Germany) according to the manufacturer's instructions. Subsequently, Gardnerella vaginalis was quantitatively detected using qPCR. Primers were selected based on the bacterial 16S rRNA sequence. The reaction mixture (10 μL) consisted of 5 μL of 2×ChamQ Universal SYBRqPCR Master Mix (Nanjing Novizan Biotechnology Co., Ltd.), 1 μL of template DNA (10 ng / μL), 0.5 μL of forward and reverse primers (10 μM each), and 3 μL of double-distilled water. The thermal cycling conditions were: initial denaturation at 95 °C for 30 s; followed by 95 °C for 5 s and 60 °C for 30 s, repeated 40 times. In another step, melting was established by increasing the temperature from 65 °C to 95 °C in 0.5 °C increments every 5 s. The threshold cycle value (CT) was determined, and the copy number was calculated based on the standard curve (Log copies / μL vs. CT value). Each sample was tested in triplicate.

[0083] Table 4: Species-specific primers for real-time quantitative PCR detection of Gardnerella vaginalis

[0084]

[0085] Histopathological analysis of mouse vaginal tissue

[0086] At the end of the experiment, mice were euthanized and their vaginas were removed. A portion of the vaginal tissue was used for histopathological examination. The vaginal tissue was fixed in 4% paraformaldehyde, embedded in paraffin, sectioned into 5 mm thick sections, and stained with hematoxylin and eosin (H&E). The vaginal tissue samples were observed at 40x magnification using a pathological slide scanner (Panoramic MIDI, 3DHistech Ltd, Budapest, Hungary).

[0087] Example 1: Isolation and identification method of Lacticaseibacillus paracasei (CCFM1317)

[0088] The specific steps are as follows:

[0089] 1. Screening

[0090] The samples were obtained from feces of healthy adults. After pretreatment, the samples were stored in 30% glycerol at -80°C. After thawing, the samples were mixed and 0.5 mL of the sample was added to 4.5 mL of physiological saline. The samples were then serially diluted with physiological saline containing 9 g / L. The appropriate serial dilutions were plated on MRS solid medium and incubated at 37°C for 48 h. Typical colonies of *Lactobacillus paracasei* were picked and streaked onto MRS solid medium for purification. Single colonies were then transferred to MRS liquid medium for enrichment and preserved in 30% glycerol to obtain *Lactobacillus paracasei* CCFM1317. The typical colonies of *Lactobacillus paracasei* are round, milky white, smooth, and raised.

[0091] 2. Identification

[0092] The genome of strain CCFM1317 was extracted, and the 16S rDNA of the strain was amplified and sequenced (performed by Shanghai Meiji Biomedical Technology Co., Ltd., and the nucleotide sequence of the 16S rDNA obtained by amplification of CCFM1317 is shown in SEQ ID NO.1). The nucleotide sequence of the amplified 16S rDNA of the strain was then compared with the nucleic acid sequence in NCBI. The results showed that the strain was Lactobacillus gasseri, and named Lactobacillus paracasei CCFM1317.

[0093] Example 2: The ability of Lactobacillus to regulate cellular oxidative stress in vitro

[0094] The specific steps are as follows:

[0095] (1) Preparation of bacterial suspension

[0096] Lactobacillus paracasei CCFM1317, Lactobacillus delbrueckii DM8909, and Lactobacillus gasseri QJSWX195M1 were inoculated into MRS solid medium and cultured in a water-jacketed incubator at 37°C for 24-48 hours to obtain single colonies; single colonies were picked and inoculated into MRS liquid medium and cultured at 37°C for 12-18 hours to obtain culture solution;

[0097] The culture medium was inoculated into MRS liquid medium at a 2% (v / v) inoculum and cultured at 37℃ for 12 h to obtain the seed culture. The seed culture was then inoculated into MRS liquid medium at a 3-5% (v / v) inoculum for expansion culture and cultured at 37℃ for 18-24 h to obtain a concentration of 3.4 × 10⁻⁶. 8 CFU / mL bacterial suspension; the bacterial suspension was homogenized under high pressure (800-1200 MPa, 5 times) to obtain bacterial lysate.

[0098] Vaginal epithelial cells VK2 / E6E7 were used at a concentration of 2.5 × 10⁻⁶. 4 Cells were seeded at a concentration of 1 / mL, with 2 mL inoculated per well in a 12-well plate. After culturing for 24 h, control and treatment groups were set up. The treatment group was supplemented with serum-free medium containing 1.25 μg / mL LPS, while the control group was supplemented with complete medium. After 24 h, the old medium was discarded, and the cells were washed three times with PBS. The control group was supplemented with 5% MRS medium. The treatment group was supplemented with an equal proportion of bacterial lysate. 2 mL of medium containing lysates of *Lactobacillus paracasei* CCFM1317, *Lactobacillus delbrueckii* DM8909, or *Lactobacillus gasseri* QJSWX195M1 was added to each 12-well plate and cultured for 24 h. Each sample was tested in triplicate. After co-culture, the culture supernatant was collected, and the Nrf2 content in the vaginal epithelial cell culture supernatant was detected by ELISA. The results are as follows: Figure 1 As shown.

[0099] The results show:

[0100] Figure 1 The results showed that, compared with the control group (Nrf2 content of 106.10 ng / L), the model group had a significantly lower Nrf2 content of 89.60 ng / L. The Nrf2 content after intervention with the bacterial lysates prepared from *Lactobacillus delbrueckii* DM8909, *Lactobacillus paracasei* CCFM1317, and *Lactobacillus paracasei* FCQNA15L3 were 111.10 ng / L, 119.10 ng / L, and 88.92 ng / L, respectively. Compared with *Lactobacillus delbrueckii* DM8909, the group intervened with *Lactobacillus paracasei* CCFM1317 significantly increased the Nrf2 content of vaginal epithelial cells (by 32.92% compared with the model group), while *Lactobacillus paracasei* FCQNA15L3 did not have this ability.

[0101] Example 3: Application of Lactobacillus paracasei CCFM1317 in alleviating bacterial vaginosis in mice

[0102] The specific steps are as follows:

[0103] 1. Animal experiments

[0104] (1) Preparation of bacterial suspension

[0105] Lactobacillus was inoculated into MRS solid medium and cultured in a water-jacketed incubator at 37°C for 24-48 hours to obtain single colonies; single colonies were picked and inoculated into MRS liquid medium and cultured at 37°C for 12-18 hours to obtain culture medium 1.

[0106] Inoculate the culture medium 1 into the MRS liquid medium at an inoculation amount of 2% (v / v), and culture it at 37 °C for 12 h to obtain a seed solution; inoculate the seed solution into the MRS liquid medium at 3 - 5% (v / v) for subculture, and culture it at a temperature of 37 °C for 18 - 24 h to obtain a bacterial solution with a concentration of ~10 9 CFU / mL.

[0107] (2) Animal experiment. The specific experimental procedure is shown in Figure 2 and Table 5:

[0108] Table 5: Experimental scheme and grouping:

[0109]

[0110]

[0111] SPF - level BALB / c mice, female, 7 - week - old, weighing 18 - 20 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Production License Number: SCXK(Beijing)2012 - 0001). The mice were randomly divided into 5 groups according to their body weights. Referring to Table 5, all groups of mice were normally raised throughout the experiment.

[0112] Both the bacterial vaginitis model group and the intervention group needed to undergo subcutaneous injection of 100 μL of estradiol valerate solution (0.5 mg of estradiol valerate dissolved in 100 μL of filtered and sterilized sesame oil) into the neck for 3 consecutive days (days 1 - 3) to induce estrus. The vagina was infected with Gardnerella vaginalis for 5 consecutive days (days 3 - 7). (The specific operation method for infection was to aspirate 20 μL of the bacterial suspension of Gardnerella vaginalis with a pipette tip, slowly inject it into the vagina of the mouse, invert the mouse, keep it for 1 - 2 minutes, and then put it into the cage).

[0113] The experimental period was 18 days (days 1 - 18):

[0114] Inducing estrus: Subcutaneous injection of 100 μL of estradiol valerate solution (0.5 mg of estradiol valerate dissolved in 100 μL of filtered and sterilized sesame oil) into the neck for 1 - 3 days to induce estrus.

[0115] Infection experiment (model - building period): On the 3rd day, the mice in the blank control group were inoculated with 20 μL of PBS into the vagina, and the other mice were inoculated with 20 μL of the Gardnerella vaginalis bacterial suspension with a viable bacteria count of 10 10 CFU / mL. After inoculation, the mice were inverted for 1 - 2 minutes to prevent the bacteria from flowing out, and this was done for 5 consecutive days until the inoculation of Gardnerella vaginalis ended on the 7th day.

[0116] Intervention experiment: Starting from the 8th day, the details are as follows:

[0117] Blank control group: Gavage with 100 μL of PBS once a day;

[0118] Model group mice: 100 μL PBS was administered by gavage once daily;

[0119] Lactobacillus delbrueckii DM8909 group: Mice were administered 100 μL once daily by gavage. 9 CFU / mL Lactobacillus delbrueckii DM8909 bacterial suspension;

[0120] Lactobacillus paracasei CCFM1317 group: Mice were administered 100 μL once daily by gavage. 9 CFU / mL Lactobacillus paracasei CCFM1317 bacterial suspension;

[0121] Lactobacillus gasseri QJSWX195M1 group: Mice were administered 100 μL once daily by gavage. 9 CFU / mL Lactobacillus gasseri QJSWX195M1 bacterial suspension;

[0122] At the end of infection (day 7) and the end of intervention (day 17), 50 μL of phosphate buffer solution was aspirated from the vagina of mice using a pipette tip each time. Finally, 300 μL of vaginal irrigation fluid was collected to determine the vaginal Gardnerella vaginalis and lactobacillus load.

[0123] Meanwhile, on day 18, all experimental mice were euthanized and vaginal, colon, and skin tissues were dissected for subsequent histopathological analysis. The expression of the antioxidant pathway (Nrf2) in vaginal tissue, the expression of the aging pathway (MAPK / AP-1 / MMP-1) in skin tissue, and the expression of the antioxidant pathway (Keap / Nrf2) in colon tissue were detected.

[0124] 2. Experimental Results:

[0125] (1) Secretion of antioxidant factor Nrf2 and expression of Keap1 / Nrf2 mRNA in mouse vaginal tissue

[0126] Total RNA was extracted from mouse vaginal tissue using the Trizol method, with GAPDH as an internal reference gene, based on 2 -ΔΔCt The method calculates the expression level of the target gene; the results are as follows: Figure 3 As shown.

[0127] The results showed that the expression level of Nrf2 mRNA in the control group was 1, while the expression level in the model group was 0.10, which was significantly lower than that in the control group. The Nrf2 mRNA expression levels after intervention with Lactobacillus delbrueckii DM8909, Lactobacillus paracasei CCFM1317, and Lactobacillus gasseri QJSWX195M1 were 0.48, 2.87, and 2.94, respectively. Compared with Lactobacillus delbrueckii DM8909, the Lactobacillus paracasei CCFM1317 intervention group significantly increased the expression of Nrf2 mRNA.

[0128] (2) Secretion of antioxidant factor Nrf2 and expression of Keap1 / Nrf2 mRNA in mouse colon tissue

[0129] 20 mg of mouse colon tissue was collected to measure cytokines. Simultaneously, total RNA was extracted from mouse colon tissue using the Trizol method, with GAPDH as an internal reference gene. Based on 2... -ΔΔCt The method calculates the expression level of the target gene; the results are as follows: Figure 4 As shown.

[0130] 1) By Figure 4 As shown in 'a', compared with the control group, there was no significant difference in Nrf2 secreted by the colonic tissue of the model group mice. After probiotic intervention, Lactobacillus paracasei CCFM1317 (23.35 ng / L) significantly increased the concentration of the antioxidant Nrf2 in the colonic tissue compared with other groups (Lactobacillus delbrueckii DM8909: 19.27 ng / L, Lactobacillus gasseri QJSWX195M1: 20.26 ng / L).

[0131] 2) By Figure 4 As shown in b, with the Keap1 mRNA expression level in the control group as 1, the expression level in the model group was 3.33, significantly higher than the control group. The Keap1 mRNA expression levels after intervention with *Lactobacillus delbrueckii* DM8909, *Lactobacillus paracasei* CCFM1317, and *Lactobacillus gasseri* QJSWX195M1 were 1.86, 1.41, and 1.94, respectively. Compared to other groups (*Lactobacillus delbrueckii* DM8909 decreased by 44.14%, and *Lactobacillus gasseri* QJSWX195M1 decreased by 41.74% compared to the model group), the *Lactobacillus paracasei* CCFM1317 intervention group significantly reduced Keap1 mRNA expression (by 57.66% compared to the model group). In summary, *Lactobacillus paracasei* CCFM1317 can significantly promote the antioxidant capacity of colonic tissue.

[0132] (3) Expression of MAPK / AP-1 / MMP-1 mRNA in mouse skin tissue

[0133] Total RNA was extracted from mouse dorsal skin tissue using the Trizol method, with GAPDH as an internal reference gene, based on 2 -ΔΔCt The method calculates the expression level of the target gene; the results are as follows: Figure 5 As shown.

[0134] 1) By Figure 5As shown in 'a', with the expression level of MAPK mRNA in the control group being 1, the expression level in the model group was 5.61, which was significantly higher than that in the control group. The MAPK mRNA expression levels after intervention with Lactobacillus delbrueckii DM8909, Lactobacillus paracasei CCFM1317, and Lactobacillus gasseri QJSWX195M1 were 2.14, 3.40, and 0.99, respectively, proving that mice with vaginitis do indeed have skin health risks, and oral probiotics can help resist this risk.

[0135] 2) By Figure 5 As shown in b, with the expression level of AP-1 mRNA in the control group as 1, the expression level in the model group was 1.17, which was higher than that in the control group. The expression levels of AP-1 mRNA after intervention by Lactobacillus delbrueckii DM8909, Lactobacillus paracasei CCFM1317, and Lactobacillus gasseri QJSWX195M1 were 0.51, 0.19, and 1.41, respectively. Lactobacillus gasseri QJSWX195M1 did not show a significant regulatory effect. Compared with Lactobacillus delbrueckii DM8909 (p<0.01), the Lactobacillus paracasei CCFM1317 intervention group (p<0.001) reduced the expression of MAPK mRNA more significantly.

[0136] 3) By Figure 5 As shown in c, with the expression level of MMP-1 mRNA in the control group as 1, the expression level in the model group was 1.34, which was significantly higher than that in the control group. The MMP-1 mRNA expression levels after intervention with Lactobacillus delbrueckii DM8909, Lactobacillus paracasei CCFM1317, and Lactobacillus gasseri QJSWX195M1 were 0.73, 0.41, and 0.86, respectively. Compared with other groups (compared to the model group, Lactobacillus delbrueckii DM8909 decreased by 45.52%, and Lactobacillus gasseri QJSWX195M1 decreased by 35.82%), the Lactobacillus paracasei CCFM1317 intervention group reduced the expression of MMP-1 mRNA more significantly (69.40% lower than that in the model group).

[0137] In summary, Lactobacillus paracasei CCFM1317 can significantly improve skin aging problems caused by vaginitis.

[0138] (4) Gardnerella vaginalis colonization in mouse vagina

[0139] Sampling was performed by using a pipette tip to aspirate a certain amount of phosphate buffered solution from the mouse vagina to obtain 300 μL of vaginal lavage fluid. Gardnerella vaginalis and Lactobacillus were counted using qPCR. The entire experimental period was 17 days, with two samplings. The first sampling was performed on day 7 (the first day after infection ended) to assess Gardnerella vaginalis colonization. The second sampling was performed on day 17 after the intervention to quantify the load of Gardnerella vaginalis and Lactobacillus.

[0140] from Figure 6 The results showed that, except for the blank control group, approximately 12.42 lg copies / μL of Gardnerella vaginalis could be detected in mice on day 7, indicating successful colonization of Gardnerella vaginalis. In the blank control group, Gardnerella vaginalis was not detected throughout the experiment. Several days after probiotic intervention, the colonization level of Gardnerella vaginalis in the CCFM1317 group was significantly reduced to 9.41 lg copies / μL. Compared with the model group, neither Lactobacillus delbrueckii DM8909 nor Lactobacillus gasseri QJSWX195M1 significantly reduced Gardnerella vaginalis colonization (p>0.05).

[0141] (5) Histopathological analysis of mouse vaginal tissue

[0142] HE staining of mouse vaginal tissue can effectively assess the inflammation status of vaginal tissue in different groups of mice.

[0143] like Figure 7 As shown, the vaginal epithelium in the blank control group was smooth and continuous, with an intact tissue structure and no obvious inflammatory cell infiltration. In the model group, the vaginal mucosal epithelium showed poor continuity, with superficial cell erosion forming pores, submucosal stroma congestion, and a large number of inflammatory cells infiltrating the epithelium and stroma. After appropriate probiotic intervention for a period of time, the *Lactobacillus delbrueckii* DM8909 group showed less inflammatory cell infiltration than the model group, but significant squamous epithelial hyperplasia was observed. In the other two probiotic intervention groups, *Lactobacillus gasseri* QJSWX195M1 did not significantly improve the recovery of mouse vaginal tissue compared to the model group; compared to the control group, different parts of the vaginal epithelium still showed discontinuity of the vaginal epithelium and inflammatory cell infiltration. *Lactobacillus paracasei* CCFM1317 showed the opposite effect to *Lactobacillus gasseri* QJSWX195M1, for example, restoring epithelial continuity and reducing inflammatory cell infiltration. The recovery of mouse vaginal tissue after *Lactobacillus paracasei* CCFM1317 intervention was superior to that of the *Lactobacillus delbrueckii* DM8909 group.

[0144] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A strain of Lactobacillus paracasei ( Lacticaseibacillus paracasei CCFM1317 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on August 4, 2023, with accession number GDMCC No: 63713.

2. A microbial inoculant, characterized in that, The microbial agent contains Lactobacillus paracasei CCFM1317 or its fermentation broth as described in claim 1.

3. A product characterized in that, The product contains Lactobacillus paracasei CCFM1317 as described in claim 1 or contains the microbial agent as described in claim 2, and the product is a pharmaceutical or hygiene product.

4. The product according to claim 3, characterized in that, The hygiene products include sanitary wipes, panty liners, tampons, sanitary napkins, or feminine antibacterial / bacteriostatic washes.

5. The product according to claim 3, characterized in that, The hygiene product in question is a sanitary napkin.

6. The product according to claim 3, characterized in that, The hygiene product in question is a vaginal wash.

7. The product according to claim 3, characterized in that, The dosage form of the medicine is granules, capsules, tablets, pills, suppositories, or oral liquid.

8. The use of *Lactobacillus paracasei* CCFM1317 as described in claim 1 or the microbial agent as described in claim 2 in the preparation of products for relieving and / or treating bacterial vaginosis, or for anti-aging or women's intimate health, characterized in that... The product in question is a pharmaceutical or hygiene product.

9. The application according to claim 8, wherein the sanitary product includes sanitary wipes, panty liners, sanitary tampons, sanitary napkins, or feminine antibacterial / bacteriostatic wash.

10. The application according to claim 8, wherein the sanitary product is a sanitary napkin.

11. The application according to claim 8, wherein the sanitary product is a vaginal wash.

12. The use of Lactobacillus paracasei CCFM1317 as described in claim 1 or the microbial agent as described in claim 2 in the preparation of a pharmaceutical product having any of the following uses: (1) Increase the Nrf2 content in cell supernatant; (2) Improve individual vaginal oxidative stress: increase Nrf2 levels in vaginal tissue; (3) Enhance individual skin anti-aging ability: Downregulate MAPK / AP-1 / MMP-1 mRNA expression; (4) Enhance the individual's intestinal antioxidant capacity: Activate the Keap1 / Nrf2 pathway and increase the Nrf2 level in colon tissue; (5) Improved individual vaginal pathological features and reduced Gardnerella vaginalis load in mice; (6) Improve individual vaginal pathological features.

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