Lactobacillus gasseri and compositions thereof for simultaneously improving female intimate health and skin aging

Abstract

The application discloses Lactobacillus gasseri and a composition thereof for simultaneously improving female private health and skin aging, and belongs to the technical field of microorganisms.The application screens a Lactobacillus gasseri CCFM1313 from vaginal secretions of healthy women, the Lactobacillus gasseri CCFM1313 and the composition containing the Lactobacillus gasseri CCFM1313 and Paracaseiclovor CCFM1316 or a fermentation liquor thereof have the effects of repairing oxidative damage related symptoms caused by vaginitis and relieving skin aging.Therefore, the Lactobacillus gasseri CCFM1313 and the composition containing the Lactobacillus gasseri CCFM1313 and Paracaseiclovor CCFM1316 or the fermentation liquor thereof have great application prospects in products for improving and repairing oxidative damage caused by vaginitis and skin health problems caused by vaginitis.

Description

Technical Field

[0001] This invention relates to Lactobacillus gasseri and its compositions that simultaneously improve women's intimate health and skin aging, belonging to the field of microbial technology. Background Technology

[0002] Vaginal diseases are common vaginal infections among women of reproductive age, characterized by high recurrence rates and atypical clinical symptoms, making them easy to miss in clinical practice.

[0003] Pathogenic bacteria invading the vaginal environment adhere to reproductive tract epithelial cells, releasing toxins that damage mitochondria and produce large amounts of reactive oxygen species (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 remains at low levels in the cytoplasm. Studies have shown that probiotics can increase the expression levels of the Nrf2 gene and protein by activating the Nrf2 signaling pathway, thereby increasing the anti-inflammatory and antioxidant capacity of cells. Simultaneously, Nrf2 can synergistically enhance superoxide dismutase (SOD) to induce oxidative stress responses. MAPK is an intracellular threonine protein kinase that can be activated extracellularly by ROS, activating the transcription factor AP-1 to further regulate the transcription of MMPs, especially the MMP family members interstitial collagenase (MMP-1), matrix hydrolysin-1 (MMP-3), and gelatinase (MMP-9), leading to the degradation of collagen and elastin.

[0004] Currently, the main medications used to treat vaginitis include nitroimidazoles (metronidazole, tinidazole) and lincomycins (clindamycin). However, antibiotics cannot repair the vaginal environment damaged by oxidative stress, resulting in a persistently high risk of vaginitis recurrence.

[0005] Studies have shown that using live lactobacillus capsules as an adjunct to the treatment of recurrent vaginitis can significantly reduce the recurrence rate. Furthermore, a comparison of the efficacy of three single-strain probiotics and compound probiotic formulations containing the corresponding single strains revealed that both single and compound probiotics effectively promoted immune function in mice, with the compound probiotics showing a more significant therapeutic effect. Therefore, the combined use of multiple strains may produce a synergistic effect, enhancing the survival ability of the strains and providing continuous, stable, and comprehensive regulation of the body.

[0006] Currently, the number of domestically produced probiotic products for treating vaginitis on the market is very limited. In 1989, Kang Bai et al. isolated and screened a strain of *Lactobacillus delbrueckii* DM8909 from vaginal secretions of healthy women. Preliminary experiments ("Study on the Inhibition of Vaginal Infection in Mice by *Lactobacillus delbrueckii* DM8909 Strain," *Chinese Journal of Microecology*, 2003) and clinical data ("Phase II Clinical Trial Study on the Treatment of Bacterial Vaginosis by *Lactobacillus delbrueckii* DM8909 Strain," *Chinese Journal of Microecology*, 2001) showed that this bacterium has good probiotic properties and is effective in treating bacterial vaginosis. In addition, some patents involve probiotic compositions for vaginitis, mainly focusing on regulating vaginal inflammatory factors (CN110339216A, CN111647525A). However, there are no reports evaluating the effects of probiotic compositions on antioxidant capacity, and no studies have been conducted on their effects on oxidative damage caused by vaginitis. Therefore, there is still no *Lactobacillus delbrueckii* or its compositions with elucidated mechanisms and proven efficacy in alleviating bacterial vaginosis and vaginal oxidative damage and skin health caused by vaginitis. Summary of the Invention

[0007] This invention provides a strain of *Lactobacillus gasseri* capable of alleviating vaginitis, improving oxidative damage caused by vaginitis, and alleviating skin health problems caused by vaginitis. *Lactobacillus gasseri* CCFM1313 exhibits a good ability to activate Nrf2 expression and increase Nrf2 concentration in vaginal epithelial cell culture supernatant in vitro, and has the potential to alleviate oxidative stress caused by vaginitis. SPF-grade BALB / c mice, female, 7 weeks old, weighing 18-20g, were selected. After the adaptation period, the 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 the vaginitis 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 probiotic suspension at CFU / mL was prepared by combining CCFM1313 and CCFM1316 in a 1:1 ratio. The model group underwent only Gardnerella vaginalis infection without any subsequent intervention, while 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 colonization of Gardnerella vaginalis, vaginal epithelial cell shedding, secretion of antioxidant factor (Nrf2), expression of antioxidant pathway (Nrf2 / SOD1), and histopathological findings were analyzed to evaluate the therapeutic effect on bacterial vaginosis in mice. At the same time, the expression of MAPK / AP-1 / MMP-1 mRNA in mouse skin tissue was detected to evaluate the intervention effect on skin aging caused by vaginitis in mice.

[0008] This invention provides a strain of Lactobacillus gasseri CCFM1313, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on August 4, 2023, with accession number GDMCC No: 63709.

[0009] The present invention provides a microbial inoculant containing Lactobacillus gasseri CCFM1313 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 Lactobacillus gasseri CCFM1313 lyophilized powder.

[0012] In one embodiment of the present invention, the amount of Lactobacillus gasseri CCFM1313 added to the above-mentioned microbial agent is at least 10. 6 CFU / mL or 10 6 CFU / g.

[0013] This invention provides a microbial inoculant containing Lactobacillus gasseri CCFM1313 or its fermentation broth or lysate; or the microbial inoculant simultaneously contains Lactobacillus gasseri CCFM1313 or its fermentation broth or lysate, and Lactobacillus paracasei CCFM1316 or its fermentation broth or lysate.

[0014] The Lactobacillus paracasei CCFM1316 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on August 4, 2023, with accession number GDMCC No: 63712.

[0015] In one embodiment of the present invention, the microbial agent is a liquid preparation, a powder preparation, or a granular preparation.

[0016] In one embodiment of the present invention, the microbial agent is a freeze-dried powder containing a mixture of Lactobacillus gasseri CCFM1313 and Lactobacillus paracasei CCFM1316.

[0017] In one embodiment of the present invention, the microbial agent contains Lactobacillus gasseri CCFM1313 and Lactobacillus paracasei CCFM1316 mixed in a 1:1 ratio, and the viable count in the microbial agent is at least 10. 6 CFU / mL or 10 6 CFU / g.

[0018] The present invention provides a product containing the above-mentioned Lactobacillus gasseri CCFM1313 or containing the above-mentioned microbial agent.

[0019] In one embodiment of the present invention, the amount of Lactobacillus gasseri CCFM1313 added to the above product is at least 10. 6 CFU / mL or 10 6 CFU / g.

[0020] In one embodiment of the present invention, the product is food, medicine, health product or hygiene product.

[0021] In one embodiment of the present invention, the food includes dietary supplements, ordinary food, and health products; the hygiene products include sanitary wipes, sanitary napkins, panty liners, sanitary tampons, sanitary cotton pads, vaginal washes, and antibacterial / bacteriostatic feminine washes.

[0022] In one embodiment of the invention, the pharmaceutical product comprises the above-described composition and a pharmaceutically permissible carrier.

[0023] 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.

[0024] In one embodiment of the present invention, the dosage form of the medicine includes granules, capsules, tablets, pills, suppositories, or oral liquids.

[0025] 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.

[0026] This invention provides a product containing Lactobacillus gasseri CCFM1313 or its fermentation broth or its lysate, and Lactobacillus paracasei CCFM1316 or its fermentation broth or its lysate; the Lactobacillus paracasei CCFM1316 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on August 4, 2023, with accession number GDMCC No: 63712.

[0027] In one embodiment of the present invention, the product contains Lactobacillus gasseri CCFM1313 and Lactobacillus paracasei CCFM1316 mixed in a 1:1 ratio, and the viable count in the microbial agent is at least 10. 6 CFU / mL or 10 6 CFU / g.

[0028] In one embodiment of the present invention, the product is food, medicine, health product or hygiene product.

[0029] In one embodiment of the present invention, the food includes dietary supplements, ordinary food, and health products; the hygiene products include sanitary wipes, sanitary napkins, panty liners, sanitary tampons, sanitary cotton pads, vaginal washes, and antibacterial / bacteriostatic feminine washes.

[0030] In one embodiment of the invention, the pharmaceutical product comprises the above-described composition and a pharmaceutically permissible carrier.

[0031] 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.

[0032] In one embodiment of the present invention, the dosage form of the medicine includes granules, capsules, tablets, pills, suppositories, or oral liquids.

[0033] 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.

[0034] The present invention also provides the use of the above-mentioned Lactobacillus gasseri CCFM1313 or an agent containing the above-mentioned microorganisms in the preparation of products for relieving and / or treating bacterial vaginosis or for anti-aging or ovarian maintenance.

[0035] In one embodiment of the present invention, the product is food, medicine, health product or hygiene product.

[0036] In one embodiment of the present invention, the food includes dietary supplements, ordinary food, and health products; the hygiene products include sanitary wipes, sanitary napkins, panty liners, sanitary tampons, sanitary cotton pads, vaginal washes, and antibacterial / bacteriostatic feminine washes.

[0037] In one embodiment of the invention, the pharmaceutical product comprises the above-described composition and a pharmaceutically permissible carrier.

[0038] 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.

[0039] In one embodiment of the present invention, the dosage form of the medicine includes granules, capsules, tablets, pills, suppositories, or oral liquids.

[0040] 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.

[0041] The present invention also provides the use of compositions containing Lactobacillus gasseri CCFM1313, Lactobacillus paracasei CCFM1316 or their fermentation broth in the preparation of products for relieving and / or treating bacterial vaginosis or for anti-aging.

[0042] In one embodiment of the present invention, the composition contains Lactobacillus gasseri CCFM1313 and Lactobacillus paracasei CCFM1316 mixed in a 1:1 ratio, and the viable count of the microbial agent is at least 10. 6 CFU / mL or 10 6 CFU / g.

[0043] In one embodiment of the present invention, the product is food, medicine, health product or hygiene product.

[0044] In one embodiment of the present invention, the food includes dietary supplements, ordinary food, and health products; the hygiene products include sanitary wipes, sanitary napkins, panty liners, sanitary tampons, sanitary cotton pads, vaginal washes, and antibacterial / bacteriostatic feminine washes.

[0045] In one embodiment of the invention, the pharmaceutical product comprises the above-described composition and a pharmaceutically permissible carrier.

[0046] 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.

[0047] In one embodiment of the present invention, the dosage form of the medicine includes granules, capsules, tablets, pills, suppositories, or oral liquids.

[0048] 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.

[0049] The present invention also provides the use of the above-mentioned Lactobacillus gasseri CCFM1313 or a composition containing Lactobacillus gasseri CCFM1313, Lactobacillus paracasei CCFM1316 or their fermentation broth in the preparation of microbial preparations having any of the following uses:

[0050] (1) It upregulates the expression of Nrf2 mRNA in cells and increases the content of Nrf2 in cell supernatant;

[0051] (2) Reduced vaginal epithelial cell shedding in mice with vaginitis;

[0052] (3) Improved pathological features of the mouse vagina;

[0053] (4) Improves vaginal oxidative stress in mice: Activates the Nrf2 / SOD1 pathway and increases the levels of Nrf2 and SOD in vaginal tissue;

[0054] (5) Downregulates the level of TNF-α, a inflammatory factor in the vagina of mice;

[0055] (6) Enhance the anti-aging ability of mouse skin: downregulate the expression of MAPK / AP-1 / MMP-1 mRNA.

[0056] Beneficial effects

[0057] This invention screened a strain of Lactobacillus gasseri CCFM1313 from the vaginal secretions of healthy women. This Lactobacillus gasseri CCFM1313, and compositions containing Lactobacillus gasseri CCFM1313, Lactobacillus paracasei CCFM1316, or their fermentation broth, have the effect of repairing symptoms related to oxidative damage caused by vaginitis and alleviating skin aging, specifically manifested in:

[0058] (1) Upregulates Nrf2 mRNA expression in cells and increases Nrf2 content in cell supernatant;

[0059] (2) Reduced vaginal epithelial cell shedding in mice with vaginitis;

[0060] (3) Improved pathological features of the mouse vagina;

[0061] (4) Improves vaginal oxidative stress in mice: Activates the Nrf2 / SOD1 pathway and increases the levels of Nrf2 and SOD in vaginal tissue;

[0062] (5) Reduces the content of TNF-α, an inflammatory factor in the vagina of mice;

[0063] (6) Enhance the anti-aging ability of mouse skin: Downregulate MAPK / AP-1 / MMP-1 mRNA expression;

[0064] Therefore, Lactobacillus gasseri CCFM1313, and compositions containing Lactobacillus gasseri CCFM1313, Lactobacillus paracasei CCFM1316, or their fermentation broth, have great application potential in products that improve and repair oxidative damage caused by vaginitis, as well as skin health problems caused by vaginitis.

[0065] Preservation of biological materials

[0066] A strain of Lactobacillus gasseri (CCFM1313), taxonomically named Lactobacillus gasseri, was deposited on August 4, 2023, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No: 63709), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, Institute of Microbiology.

[0067] A strain of *Lactaseibacillus paracasei* (CCFM1316), taxonomically named *Lactaseibacillus paracasei*, was deposited on August 4, 2023, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No: 63712), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, Institute of Microbiology. Attached Figure Description

[0068] Figure 1 Effects of different bacterial cell lysates on the oxidation response of VK2 / E6E7 vaginal epithelial cells: a represents the Nrf2 content in the culture supernatant; b represents the effect on Nrf2 mRNA expression. Compared with the model group, *p<0.05, **p<0.01, ***p<0.001.

[0069] Figure 2 Animal experiment design flowchart.

[0070] Figure 3 Image showing vaginal epithelial cell shedding; p<0.0001.

[0071] Figure 4 : Histopathological evaluation of mouse vaginal tissue.

[0072] Figure 5 Figure: Effect of Lactobacillus gasseri on vaginal antioxidant targets; where a represents the effect on Nrf2 mRNA expression; b represents the effect on SOD1 mRNA expression; compared with the model group, *p<0.05, ***p<0.001, ****p<0.0001.

[0073] Figure 6 : Effect of Lactobacillus gasseri on vaginal antioxidant response; where a represents the effect of Nrf2 concentration on antioxidant factor; b represents the effect of SOD concentration on antioxidant enzyme; compared with the model group, *p<0.05, ****p<0.0001.

[0074] Figure 7Effect of Lactobacillus gasseri on the level of vaginal inflammatory factor TNF-α; compared with the model group, *p<0.05, **p<0.1.

[0075] Figure 8 Figure: Effect of Lactobacillus gasseri 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<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Detailed Implementation

[0076] The vaginal epithelial cells VK2 / E6E7 used in the following examples were kindly provided by the Department of Obstetrics and Gynecology, People's Hospital. Gardnerella vaginalis ATCC 14018 was purchased from the Guangdong Institute of Microbiology Culture Collection Center (GDMCC).

[0077] The culture media involved in the following examples are as follows:

[0078] 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.

[0079] 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.

[0080] Cell culture medium: 89% (v / v) DMEM medium + 10% (v / v) fetal bovine serum + 1% (v / v) 100× penicillin

[0081] A mixed solution of penicillin and streptomycin (the mixed solution contains 10,000 U / mL of penicillin and 10 mg / mL of streptomycin).

[0082] Lactobacillus gasseri CCFM1313 bacterial suspension: Lactobacillus gasseri CCFM1313 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.

[0083] 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.

[0084] The detection methods involved in the following embodiments are as follows:

[0085] Detection of Nrf2 content:

[0086] The Nrf2 content in the supernatant was detected by ELISA. Simultaneously, each well was rapidly washed three times with PBS, and 500 μL of Trizol was added to each well. RNA was extracted by repeated pipetting and collection, and then reverse transcribed into cDNA using an RT-PCR reverse transcription kit. Gene expression in VK2 / E6E7 cells was detected by real-time quantitative PCR. -ΔΔCt The formula was used to calculate the expression level of Nrf2 mRNA, with GAPDH as the internal reference. Primers are described in Table 1.

[0087] Table 1 Primers and Sequences

[0088]

[0089] Formula for calculating the combined effect of probiotics:

[0090] The formula for calculating the combined effect of probiotics uses the Jin Zhengjun Q-value method: Q = E a+b / (E a +E b -E a ×E b ), where E a+b E represents the rate of change of the measured index value when A and B are combined. a and E b These are the rate of change of the detected index values ​​when A and B act alone, respectively. In the formula, the numerator represents the "measured combined effect", the denominator represents the "expected combined effect", and Q is the ratio of the two. Q < 0.85 indicates an antagonistic effect, 0.85 ≤ Q < 1.15 indicates an additive effect, and Q ≥ 1.15 indicates a synergistic effect ("Evaluation of the Synergistic Effect of Matrine Combined with Doxorubicin on Human Breast Cancer Cells by Jin Zhengjun Q-value Method", Shizhen Guoyi Guoyao, 2018).

[0091] Vaginal epithelial cell shedding in mouse vagina

[0092] Ten μL of sample solution was taken from the vaginal irrigation fluid of mice on day 17, transferred to a glass slide, and gently smeared using the outer wall of a pipette tip. The smear specimens were preserved with a fixative and stained using the Diff-Quik staining method (a rapid staining method modified from Wright's staining). Under a microscope, five fields of view were captured from each sample (one mouse), and epithelial cells were counted from each image to determine the mean. Epithelial cell shedding was evaluated under a 400x optical microscope.

[0093] Histopathological analysis of mouse vaginal tissue

[0094] 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).

[0095] Expression of Nrf2 / SOD1 mRNA, an antioxidant-related gene, in mouse vaginal tissue

[0096] 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 method described in Table 2.

[0097] Table 2 Primers and Sequences

[0098]

[0099] Secretion of antioxidant factor Nrf2 and antioxidant enzyme SOD in mouse vaginal tissue

[0100] 20 mg of mouse vaginal tissue was collected for cytokine determination. The vaginal tissue was homogenized with 180 μL of pre-cooled PBS. The sample was centrifuged at 3000 r / min for 15 min at 4℃. The supernatant of the vaginal tissue was collected for Nrf2 and SOD concentration determination according to the kit instructions (Nanjing Senbeijia Biotechnology Co., Ltd.).

[0101] MAPK / AP-1 / MMP-1 mRNA expression in mouse skin tissue

[0102] Total RNA was extracted from mouse dorsal skin 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 qPCRMaster Mix for quantitative real-time PCR. GAPDH was used as an internal control gene, based on 2... -ΔΔCt Methods were used to calculate the expression levels of MAPK / AP-1 / MMP-1 genes. Primers are shown in Table 3.

[0103] Table 3 Primers and Sequences

[0104]

[0105] Example 1: Isolation and identification method of Lactobacillus gasseri CCFM1313

[0106] The specific steps are as follows:

[0107] 1. Screening

[0108] The samples were obtained from vaginal secretions of healthy adult women. 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 gasseri* CCFM1313. The typical colonies of *Lactobacillus gasseri* are round and milky white.

[0109] 2. Identification

[0110] The genome of strain CCFM1313 was extracted, and the 16S rDNA of the strain was amplified and sequenced (performed by Shanghai Meiji Biopharmaceutical Technology Co., Ltd., and the nucleotide sequence of the 16S rDNA obtained from CCFM1313 amplification is shown in SEQ ID NO.1):

[0111] The nucleotide sequence of the amplified 16S rDNA of the strain was then compared with the nucleic acid sequence in NCBI, and the results showed that the strain was Lactobacillus gasseri, named Lactobacillus gasseri CCFM1313.

[0112] Example 2: The ability of Lactobacillus to regulate cellular oxidative stress in vitro

[0113] The specific steps are as follows:

[0114] (1) Preparation of bacterial suspension

[0115] Lactobacillus delbrueckii DM8909 and Lactobacillus gasseri CCFM1313 were inoculated into MRS solid medium and cultured in a water-jacketed incubator at 37°C for 2448 h to obtain single colonies; single colonies were picked and inoculated into MRS liquid medium and cultured at 37°C for 1218 h to obtain culture solution.

[0116] 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 35% (v / v) inoculum for expansion and cultured at 37℃ for 1824 h to obtain the culture with a concentration of 1.58 × 10⁻⁶. 9 CFU / mL Lactobacillus delbrueckii culture and 1.01×10 7 CFU / mL Lactobacillus gasseri bacterial suspension. The bacterial suspension was homogenized under high pressure (800-1200 MPa, 5 times) to obtain bacterial cell lysates.

[0117] Vaginal epithelial cells VK2 / E6E7 were used at a concentration of 2.5 × 10⁻⁶. 4 Cells were inoculated at a density of 2 mL / well in 12-well plates. After culturing for 24 hours, control and treatment groups were established. 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 hours, 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 volume of bacterial lysate: 2 mL of medium containing lysates of *Lactobacillus gasseri* CCFM1313 and *Lactobacillus delbrueckii* DM8909 were added to 12-well plates and cultured for 24 hours. Each sample was tested in triplicate. After the incubation period, the supernatant was collected, and the Nrf2 content in the supernatant was detected by ELISA. The results are as follows: Figure 1 As shown.

[0118] The results show:

[0119] (1) Figure 1 As shown in a, 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 of Lactobacillus delbrueckii DM8909 and Lactobacillus gasseri CCFM1313 after intervention were 111.10 ng / L and 116.10 ng / L, respectively. Compared with Lactobacillus delbrueckii DM8909, the group intervened with Lactobacillus gasseri CCFM1313 significantly increased the Nrf2 content of vaginal epithelial cells (p<0.001).

[0120] (2) Figure 1 As shown in b, the expression level of Nrf2 mRNA in the control group was 1, and the expression level in the model group was 0.59, which was significantly higher than that in the control group. The Nrf2 mRNA expression levels after intervention with Lactobacillus delbrueckii DM8909 cell lysate and Lactobacillus gasseri CCFM1313 cell lysate were 0.43 and 1.92, respectively. Compared with other groups, the Lactobacillus gasseri CCFM1313 intervention group significantly upregulated the expression of Nrf2 mRNA (p<0.01).

[0121] Cellular experiments showed that Lactobacillus gasseri CCFM1313 can increase Nrf2 content and upregulate Nrf2 expression, thus improving the ability of vaginal tissue to reduce oxidative stress.

[0122] Example 3: Application of Lactobacillus gasseri CCFM1313 in alleviating vaginitis in mice

[0123] The specific steps are as follows:

[0124] (1) Preparation of bacterial suspension

[0125] 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 solution;

[0126] The culture medium was inoculated into MRS liquid medium at a 2% (v / v) inoculum and cultured at 37°C for 12 h to obtain the seed culture. The seed culture was then inoculated into MRS liquid medium at a 35% (v / v) inoculum for expansion and cultured at 37°C for 18-24 h to obtain a concentration of ~10. 9 CFU / mL bacterial suspension.

[0127] Preparation of the composition:

[0128] Lactobacillus gasseri CCFM1313 and Lactobacillus paracasei CCFM1316 were compounded in a volume ratio of 1:1 to obtain a composition, and the viable count in the composition was 1×10⁻⁶. 9 CFU / mL.

[0129] (2) Animal experiments, see the specific experimental procedures. Figure 2 and Table 4:

[0130] Table 4: Experimental Design and Grouping:

[0131]

[0132] SPF-level BALB / c mice, female, 7 weeks old, weighing 18 - 20 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Production License No. SCXK(Beijing)2012 - 0001).

[0133] The mice were randomly divided into 5 groups according to their body weights. Referring to Table 2, all groups of mice were normally raised throughout the experiment.

[0134] 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. And they needed to undergo vaginal infection 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 and slowly inject it into the vagina of the mice. Then, hold the mice upside down for 1 - 2 minutes and place them in the cage).

[0135] The experimental period was 18 days (days 1 - 18):

[0136] 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.

[0137] Infection experiment (model establishment period): Starting from day 3, 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 bacterial suspension of Gardnerella vaginalis 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, for 5 consecutive days until the end of Gardnerella vaginalis inoculation on day 7.

[0138] Intervention experiment: Starting from day 8, the details were as follows:

[0139] Blank control group: Administer 100 μL of PBS by gavage once a day;

[0140] Mice in the model group: Administer 100 μL of PBS by gavage once a day;

[0141] Lactobacillus gasseri CCFM1313 group: Mice were administered 100 μL of the bacterial suspension of Lactobacillus gasseri CCFM1313 at 10 9 CFU / mL by gavage once a day;

[0142] Lactobacillus paracasei CCFM1316 group: Mice were administered 100 μL of the bacterial suspension of Lactobacillus paracasei CCFM1316 at 10 9 CFU / mL by gavage once a day;

[0143] Composition group: Mice were administered 100 μL of the composition at 10 9CFU / mL bacterial suspension;

[0144] At the end of the intervention (day 17), 50 μL of phosphate buffer solution was aspirated from the mouse vagina each time using a pipette tip to collect a sample. Finally, 300 μL of vaginal irrigation fluid was collected for subsequent analysis of vaginal epithelial cell shedding in the mouse vagina.

[0145] Meanwhile, on day 18, all experimental mice were euthanized and their vaginal and back skin tissues were dissected for subsequent histopathological analysis. The analysis included the detection of antioxidant factor (Nrf2) secretion, antioxidant enzyme (SOD) content, and antioxidant pathway (Nrf2 / SOD1) expression in the vaginal tissue, as well as the expression of collagen degradation-related pathways (MAPK / AP-1 / MMP-1) in the skin tissue.

[0146] (3) Experimental results:

[0147] 1) Shedding of vaginal epithelial cells in mouse vagina

[0148] Epithelial cell shedding was evaluated under a 400x optical microscope; results are as follows: Figure 3 As shown.

[0149] The results showed that the model group had severe epithelial cell shedding (39.33±2.67 cells / field), while the blank control group had approximately 10.67 cells per field. Compared with the model group, Lactobacillus gasseri CCFM1313, Lactobacillus paracasei CCFM1316, and the combination significantly alleviated vaginal epithelial cell shedding (p<0.0001), with approximately 29.17, 21.25, and 28.00 cells per field in each group, respectively.

[0150] 2) Histopathological analysis of mouse vaginal tissue

[0151] HE staining of mouse vaginal tissue can effectively assess the inflammation status of vaginal tissue in different groups of mice. Results are as follows... Figure 4 As shown.

[0152] The results showed that 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 into the epithelium and stroma. Connective tissue proliferation and repair were observed in the dermis, with abundant fibroblasts and collagen fibers interwoven. After a period of appropriate probiotic intervention, submucosal stroma congestion improved, especially in the group treated with the combined probiotics, where epithelial continuity was restored and inflammatory cell infiltration was reduced. Staining results of CCFM1313 and its combination showed that the dermal fibroblasts were tightly arranged, and the connective tissue morphology returned to normal.

[0153] 3) Expression of Nrf2 / SOD1 mRNA, an antioxidant-related gene, in mouse vaginal tissue

[0154] The expression levels of Nrf2 / SOD1 mRNA were detected, and the results are as follows: Figure 5 As shown.

[0155] The results show:

[0156] Depend on Figure 5 As shown in 'a', the expression level of Nrf2 mRNA in the control group was 1, and the expression level in the model group was 0.10, which was significantly lower than that in the control group. The expression levels of Nrf2 mRNA in Lactobacillus gasseri CCFM1313, Lactobacillus paracasei CCFM1316, and after the combination intervention were 2.94, 1.35, and 3.85, respectively. The combination intervention group upregulated Nrf2 mRNA expression more significantly (upregulated by 3.75 times compared to the model group), while other groups upregulated by at most 2.85 times. Among them, the Q value of the combination was 11.99, indicating that the combination achieved a synergistic effect.

[0157] Depend on Figure 5 As shown in b, with the SOD1 mRNA expression level of the control group as 1, the expression level of the model group was 0.20, which was significantly lower than that of the control group. The SOD1 mRNA expression levels of Lactobacillus gasseri CCFM1313, Lactobacillus paracasei CCFM1316, and the combined intervention group were 1.20, 0.58, and 0.89, respectively, and the probiotic intervention group significantly increased the expression of SOD1 mRNA.

[0158] In summary, Lactobacillus gasseri CCFM1313 and its composition can significantly promote the antioxidant capacity of vaginal tissue, and the composition can achieve a synergistic effect.

[0159] 4) Secretion of antioxidant factor Nrf2 and antioxidant enzyme SOD in mouse vaginal tissue

[0160] Vaginal tissue supernatant was collected and Nrf2 and SOD concentrations were determined according to the kit instructions (Nanjing Senbeijia Biotechnology Co., Ltd.). Results are as follows: Figure 6 As shown.

[0161] The results show:

[0162] Depend on Figure 6As shown in 'a', this indicates the concentration of the antioxidant Nrf2 in vaginal tissue. The results showed that, compared with the control group, the Nrf2 secreted by the vaginal tissue of the model group mice was significantly decreased (15.95 ng / L) (p<0.05). After probiotic intervention, the Nrf2 content significantly increased. The Nrf2 concentrations of Lactobacillus gasseri CCFM1313, Lactobacillus paracasei CCFM1316, and the combination after intervention were 24.79, 30.29, and 55.67 ng / L, respectively, and the Q value of the combination was 2.61, demonstrating a synergistic effect.

[0163] Depend on Figure 6 As shown in b, this indicates the concentration of the antioxidant enzyme SOD in vaginal tissue. The results showed that compared to the control group (76.08 pg / mL), the SOD secreted by the vaginal tissue of the model group mice (68.49 pg / mL) was significantly decreased (p<0.05). After probiotic intervention, only the composition showed a significant increase in SOD content (p<0.0001). The SOD concentrations of Lactobacillus gasseri CCFM1313, Lactobacillus paracasei CCFM1316, and the composition after intervention were 76.54, 71.85, and 92.59 pg / mL, respectively, and the composition's Q value was 2.13, demonstrating a synergistic effect.

[0164] 5) TNF-α content in mouse vaginal tissue

[0165] Vaginal tissue supernatant was collected and TNF-α concentration was determined according to the kit instructions (Nanjing Senbeijia Biotechnology Co., Ltd.). Results are as follows: Figure 7 As shown.

[0166] The results show:

[0167] Depend on Figure 7 The results showed the concentration of the inflammatory factor TNF-α in vaginal tissue. Compared with the control group (167.18 ng / mL), the TNF-α secreted by the vaginal tissue of the model group mice (243.35 ng / mL) was significantly increased (p<0.1). After probiotic intervention, the tissue TNF-α content decreased significantly. The SOD concentrations of *Lactobacillus gasseri* CCFM1313, *Lactobacillus paracasei* CCFM1316, and the combined intervention were 165.33, 186.43, and 173.43 ng / mL, respectively. The *Lactobacillus gasseri* CCFM1313 intervention group showed the greatest decrease in TNF-α content (32.06% lower than the model group).

[0168] 6) Expression of MAPK / AP-1 / MMP-1 mRNA in mouse skin tissue

[0169] Back skin tissue was collected to detect the expression of MAPK / AP-1 / MMP-1 mRNA. Results are as follows: Figure 8As shown.

[0170] The results show:

[0171] Depend on Figure 8 As 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 (p<0.0001). The expression levels of MAPK mRNA in Lactobacillus gasseri CCFM1313, Lactobacillus paracasei CCFM1316, and after the combined intervention were 0.98, 1.41, and 1.61, respectively, all of which significantly downregulated the expression of the target gene (p<0.0001), proving that mice with vaginitis do indeed have a risk to skin health, and oral probiotics can help resist this risk.

[0172] Depend on Figure 8 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 in Lactobacillus gasseri CCFM1313, Lactobacillus paracasei CCFM1316, and the combined treatment were 1.30, 0.48, and 0.31, respectively. Although the expression level of Lactobacillus gasseri CCFM1313 was higher than that in the model group, it did not cause a significant change, indicating that Lactobacillus gasseri alone did not have a regulatory effect on downregulating AP-1 mRNA expression. However, Lactobacillus paracasei CCFM1316 (p<0.01) and the combined treatment (p<0.001) both significantly reduced the expression of MAPK mRNA. The Q value of the combined treatment was 1.18, which shows that the combination showed a synergistic effect in downregulating AP-1 mRNA expression.

[0173] Depend on Figure 8 As shown in c, the expression level of MMP-1 mRNA in the control group was 1, and the expression level in the model group was 1.41, which was significantly higher than that in the control group (p<0.05). The expression levels of MMP-1 mRNA in Lactobacillus gasseri CCFM1313, Lactobacillus paracasei CCFM1316, and the probiotic intervention group were 1.06, 0.99, and 1.27, respectively. Compared with the model group, the probiotic intervention group showed a decrease in MMP-1 mRNA expression.

[0174] In summary, Lactobacillus gasseri CCFM1313 and its composition, and the synergistic effect of the composition, can significantly improve skin aging problems caused by vaginitis.

[0175] 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 gasseri CCFM1313 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on August 4, 2023, with accession number GDMCC No: 63709.

2. A microbial inoculant, characterized in that, The microbial agent contains *Lactobacillus gasseri* CCFM1313 or its fermentation broth as described in claim 1; or the microbial agent simultaneously contains *Lactobacillus gasseri* CCFM1313 or its fermentation broth as described in claim 1, and *Lactobacillus paracasei* CCFM1316 or its fermentation broth; the *Lactobacillus paracasei* CCFM1316 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on August 4, 2023, with accession number GDMCC No: 63712.

3. A product characterized in that, The product contains Lactobacillus gasseri CCFM1313 as described in claim 1 or contains the microbial agent as described in claim 2, and the product is food, medicine or health product.

4. The product according to claim 3, characterized in that, The food products include dietary supplements.

5. The use of Lactobacillus gasseri CCFM1313 or its fermentation broth as described in claim 1, or the microbial agent as described in claim 2, in the preparation of a medicine for relieving and / or treating bacterial vaginosis.

6. The application according to claim 5, characterized in that, The applications include: (1) Upregulates Nrf2 mRNA expression in cells and increases Nrf2 content in cell supernatant; (2) Reduced vaginal epithelial cell shedding in mice with vaginitis; (3) Improved pathological features of the mouse vagina; (4) Improves vaginal oxidative stress in mice: Activates the Nrf2 / SOD1 pathway and increases the levels of Nrf2 and SOD in vaginal tissue; (5) Enhances the anti-aging ability of mouse skin and downregulates the expression of MAPK / AP-1 / MMP-1 mRNA.

7. The use of a composition comprising Lactobacillus gasseri CCFM1313 or its fermentation broth as described in claim 1, and Lactobacillus paracasei CCFM1316 or its fermentation broth, in the preparation of a medicament for relieving and / or treating bacterial vaginosis; wherein Lactobacillus paracasei CCFM1316 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on August 4, 2023, with accession number GDMCC No: 63712.

8. The application according to claim 7, characterized in that, The applications include: (1) Upregulates Nrf2 mRNA expression in cells and increases Nrf2 content in cell supernatant; (2) Reduced vaginal epithelial cell shedding in mice with vaginitis; (3) Improved pathological features of the mouse vagina; (4) Improves vaginal oxidative stress in mice: Activates the Nrf2 / SOD1 pathway and increases the levels of Nrf2 and SOD in vaginal tissue; (5) Enhances the anti-aging ability of mouse skin and downregulates the expression of MAPK / AP-1 / MMP-1 mRNA.

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