Lactobacillus paracasei VCare-360 related to female private care and application thereof

Abstract

The present invention relates to a Lactobacillus paracasei VCare‑360 related to female private care and its application. Specifically, the present invention discloses a strain of Lactobacillus paracasei VCare‑360 related to female private care, and its preservation number is CGMCC No.31095. The Lactobacillus paracasei of the present invention has the characteristics of being resistant to gastric acid, resistant to bile salts, high acid production, producing hydrogen peroxide, strong adhesion to vaginal epithelial cells VK2 / E6E7, no resistance to antibiotics, and being able to inhibit fungal or bacterial vaginitis, and can be used to prepare a private care product that relieves vaginitis symptoms and improves female vaginal health.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and relates to Lactobacillus paracasei VCare-360 and applications thereof related to female private care, and more specifically to Lactobacillus paracasei VCare-360 and applications thereof for relieving symptoms of vaginitis. Background Art

[0002] Vaginitis is a common gynecological disease caused by infection, characterized by an imbalance of vaginal microecology. The main symptoms are abnormal vaginal discharge, itching or burning sensation, etc. Among them, bacterial vaginitis, candidal vaginitis, Trichomonas vaginitis and aerobic vaginitis are the most common, accounting for 70% to 90% of all cases. At present, oral or topical antibiotics are mainly used for clinical treatment. For bacterial vaginitis, oral metronidazole or topical clindamycin can be used, and for fungal vaginitis, oral fluconazole or topical miconazole can be used. Antibiotic treatment can usually relieve patients' adverse symptoms in the short term, but continued use can lead to increased pathogen resistance, destruction of vaginal flora and high recurrence rates.

[0003] Currently, microbiota transplantation, oral plant extracts and probiotics have become alternative and auxiliary therapies for antibiotic treatment. [1] In 2014, the vaginal flora of healthy women was used to treat patients with refractory and recurrent bacterial vaginitis, and clinical symptoms and bacterial flora indicators were improved. [2-4] The benefits of herbal therapy and probiotic therapy for vaginitis were studied. Herbal therapy is the use of natural herbs such as carvacrol, limonene, thymol and essential oils to treat vaginitis. [2] Probiotics can benefit the host's health when consumed in sufficient amounts. A large number of studies have shown that topical or oral probiotics can prevent and relieve vaginitis by restoring vaginal flora homeostasis and host immune homeostasis. [3,5] .

[0004] Therefore, it is of great value to screen probiotics that have the function of preventing and alleviating vaginitis. Summary of the invention

[0005] To solve the above problems, the present invention provides a Lactobacillus paracasei VCare-360, which can colonize well in the vagina, inhibit the growth of Gardnerella and Candida albicans, and reduce inflammatory reactions. The Lactobacillus paracasei prepared into a bacterial agent or product will help alleviate the symptoms of vaginitis, improve women's vaginal health, and can be used in women's private care products.

[0006] Specifically, the present invention provides the following technical solutions:

[0007] On the one hand, the present invention provides Lactobacillus paracasei VCare-360, which was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on June 27, 2024, with a deposit number of CGMCC No. 31095.

[0008] In another aspect, the present invention provides a probiotic comprising the Lactobacillus paracasei VCare-360 as described above.

[0009] In some embodiments, the dosage form of the probiotic is selected from one of lyophilized powder, liquid, ointment, suppository, pill, capsule, tablet, oil drop and granule.

[0010] In another aspect, the present invention provides a biological preparation comprising the Lactobacillus paracasei VCare-360 as described above.

[0011] In some embodiments, the probiotics or biological preparations further comprise one or more excipients, wherein the excipients are selected from one or more of prebiotics, plant extracts and sugar substitutes.

[0012] In some embodiments, the prebiotic is selected from one or more of fructooligosaccharides, galacto-oligosaccharides, xylo-oligosaccharides, isomaltooligosaccharides, soybean oligosaccharides, inulin, spirulina, versicolor polysaccharides, stachyose, manno-oligosaccharides and polydextrose.

[0013] In some embodiments, the plant extract is selected from one or more of Curcuma zedoaria extract, eucalyptus extract, Sophora flavescens extract, Cnidium monnieri extract, Phellodendron amurense extract, honeysuckle extract, Portulaca oleracea extract, carvacrol, limonene and thymol.

[0014] In some embodiments, the sugar substitute is selected from one or more of neotame, acesulfame potassium, mannitol, sorbitol, aspartame, erythritol, xylitol and sucralose.

[0015] On the other hand, the present invention provides use of the Lactobacillus paracasei VCare-360 as described above in preparing a product for preventing or alleviating vaginal inflammation or regulating the balance of vaginal flora.

[0016] In some embodiments, the vaginitis is bacterial vaginitis or fungal vaginitis.

[0017] In another aspect, the present invention provides use of the Lactobacillus paracasei VCare-360 as described above in the preparation of a product for inhibiting the growth of Gardnerella and Candida albicans.

[0018] In another aspect, the present invention provides use of the Lactobacillus paracasei VCare-360 as described above in the preparation of a product for inhibiting the growth of Atopobium.

[0019] In some embodiments, the product is a probiotic, a biologic, or a pharmaceutical product.

[0020] In some embodiments, the drug is selected from one of a vaginal wash and an antibacterial / antibacterial wash for women.

[0021] In some embodiments, the product is resistant to gastric acid, resistant to bile salts, capable of producing acid, capable of producing hydrogen peroxide, and has the ability to adhere to intestinal cells.

[0022] In some embodiments, the dosage form of the probiotic, biological agent or drug is selected from one of liquid, ointment, suppository, pill, capsule, tablet, oil drop and granule, but is not limited thereto.

[0023] In another aspect, the present invention provides a medicine comprising the Lactobacillus paracasei VCare-360 as described above.

[0024] In some embodiments, the pharmaceutical product further comprises one or more pharmaceutically acceptable carriers.

[0025] In some embodiments, the drug is selected from one of a vaginal wash and an antibacterial / antibacterial wash for women.

[0026] Lactobacillus paracasei is a common candidate probiotic strain and can be used in the preparation of probiotics and medicines.

[0027] Candida albicans is a fungus that is widely found in nature. When the glycogen content of women's vaginal epithelial cells increases and the acidity increases, the growth of Candida albicans can cause vaginitis.

[0028] Gardnerella is a Gram-negative small rod and a common pathogen of female reproductive tract infection. When it grows excessively in the vagina, it causes an imbalance in the normal microecological balance of the vaginal flora, causing female reproductive tract infections such as bacterial vaginitis and perineal infection.

[0029] Atopobium is one of the vaginal flora, and its overgrowth can lead to an imbalance in the normal microecological balance of the vaginal flora.

[0030] Beneficial Effects

[0031] As a probiotic, the Lactobacillus paracasei VCare-360 provided by the present invention is safe and has no obvious drug resistance to antibiotics. In addition, the Lactobacillus paracasei VCare-360 provided by the present invention can also be used to develop probiotics, biological preparations or medicines.

[0032] The VCare-360 of the present invention is Lacticaseibacillus paracasei, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Committee on June 27, 2024, at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCC No. 31095. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The acid production results of 6 bacterial strains are shown.

[0034] Figure 2 A hydrogen peroxide standard curve is shown.

[0035] Figure 3 The results of hydrogen peroxide production by the strains are shown.

[0036] Figure 4 The colony morphology of VCare-360 is shown.

[0037] Figure 5 A microscopic morphology of VCare-360 is shown.

[0038] Figure 6 The results of self-aggregation of the strains are shown.

[0039] Figure 7 The hydrophobicity results of the strains are shown.

[0040] Figure 8 The anti-inflammatory effect of the strain in a fungal inflammation model was shown.

[0041] Fig. 9 The anti-inflammatory effect of the strains in a bacterial inflammation model is shown. DETAILED DESCRIPTION

[0042] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0043] Example 1. Strain Isolation and Screening

[0044] 1. Isolation of strains

[0045] The strain VCare-360 was isolated from a sample from the saccharification process of a winery in Hunan. 10.0 g of the saccharification process sample was accurately weighed and added to 90 mL of sterile water. After shaking at 180 r / min for 20 min, it was diluted step by step and the appropriate gradient was selected and coated on an MRS solid plate (10.0 g of peptone, 10.0 g of beef extract powder, 5.0 g of yeast extract powder, 2.0 g of diammonium hydrogen citrate, 20.0 g of glucose, 1.0 mL of Tween-80, 5.0 g of sodium acetate, 2.0 g of dipotassium hydrogen phosphate, 0.58 g of magnesium sulfate, 0.25 g of manganese sulfate, 18.0 g of agar, fixed to 1 L, pH 6.5, autoclaved), and cultured at 37°C for 36-48 h. A single colony with a smooth surface and a milky white round or oval shape was picked with an inoculation loop, and then streaked on an MRS solid medium and cultured at 37°C for 36-48 h. This was repeated for 5-6 generations. Randomly select 10 single colonies on the solid plate that are milky white, smooth, round or oval in shape, use an inoculation loop to pick up the 10 purified single colonies, inoculate them into MRS liquid culture medium respectively, culture them at 37°C for 18-24 h, and subculture them 2-3 times with a 2% inoculum size before freezing.

[0046] 2. Evaluation of strains’ resistance to gastric acid and bile salts

[0047] Refer to T / CNHFA435-2024 "Gastric Juice Tolerance Test Method for Probiotics" to evaluate the gastric acid resistance of the candidate strains; refer to this method to evaluate the bile salt resistance of the candidate strains.

[0048] The evaluation results of the candidate strains' resistance to gastric acid and bile salts are as follows:

[0049] Table 1 Survival rate results of 10 strains resistant to gastric acid and bile salt

[0050]

[0051] Combined with the above gastric acid resistance and bile salt resistance results, BC 30091 (named VCare-360), BC 30097, BC30102, and BC 30103 were screened out for subsequent performance evaluation; commercially available Lactobacillus rhamnosus GR-1 (hereinafter referred to as GR-1) and commercially available Lactobacillus reuteri RC-14 (hereinafter referred to as RC-14) were selected as control strains.

[0052] In the present invention, "BC 30091" or "VCare-360" is the number given by the inventor to the Lactobacillus paracasei provided by the present invention during the research process, and "CGMCC No.31095" is the preservation number of the strain. These three represent the same strain, and their numbers can be used interchangeably in the following text.

[0053] 3. Evaluation of strain growth performance

[0054] The strains BC 30091 (named VCare-360), BC 30097, BC 30102, BC 30103, GR-1, and RC-14 were inoculated into fresh MRS liquid medium and cultured overnight at 37°C for 18-24 h. The OD 600 The normalized samples were inoculated into fresh MRS liquid medium at a 2% inoculum and incubated at 37 °C for 18 h before measuring the OD 600 The competitive strains were tested with Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14.

[0055] Table 2 Growth performance test results of 6 strains

[0056]

[0057] As can be seen from Table 2, strains BC 30091 (named VCare-360), BC 30097, BC 30102, and BC 30103 grew rapidly, and their growth performance was better than that of GR-1 and RC-14, especially strain BC 30091 (named VCare-360) had very good growth performance.

[0058] 4. Evaluation of the acid production performance of the strain

[0059] The strains BC 30091 (named VCare-360), BC 30097, BC 30102, BC 30103, GR-1, and RC-14 were inoculated into fresh MRS liquid medium and cultured overnight at 37°C for 18 h. OD 600 Normalization, the normalized bacterial liquid was inoculated into fresh MRS liquid medium at a 2% inoculation amount, and the supernatant was centrifuged and filtered after culturing at 37 °C for 18 h. The lactic acid production was detected by HPLC. The competitive strain (Novonesis) was tested with Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14, respectively.

[0060] from Figure 1 It can be seen that strain BC 30091 (named VCare-360) has the strongest acid production performance, which is 18.75 g / L, strain RC-14 has the worst acid production performance, which is only 4.54 g / L, and the acid production capacity of the other four strains is above 15 g / L. This result shows that strain BC 30091 (named VCare-360) has strong acid production performance and has potential probiotic properties.

[0061] 5. Evaluation of the hydrogen peroxide production performance of the strain

[0062] The ability to produce hydrogen peroxide was tested by the 4-aminoantipyrine method. The strains BC 30091 (named VCare-360), BC 30097, BC 30102, BC 30103, GR-1, and RC-14 were inoculated into fresh sterile MRS liquid medium and cultured overnight at 37°C for 18-24 h. The OD 600 The samples were normalized and inoculated into fresh MRS liquid medium at 2% inoculation volume. The supernatant was collected after centrifugation at 37°C and 200 rpm for 18-24 h. The competitive strain (Novonesis) was tested with Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14, respectively.

[0063] Hydrogen peroxide standard solution: Weigh an appropriate amount of hydrogen peroxide standard, add the corresponding volume of water, and prepare 0, 0.5, 1, 1.5, 2, 5, 8, 10, 25, and 50 μg / mL hydrogen peroxide standard solutions for drawing a standard curve, such as Figure 2 shown.

[0064] from Figure 3 It can be seen that the strain BC 30091 (named VCare-360) has the best hydrogen peroxide production performance, with a production of 4.98 μg / mL. The commercial strain GR-1 does not produce hydrogen peroxide. The strains BC 30103 and RC-14 produce hydrogen peroxide in an amount of 4.78 μg / mL and 4.87 μg / mL, respectively. Based on the above test results of gastric acid resistance, bile salt resistance, acid production and hydrogen peroxide production, the strain BC 30091 (named VCare-360) with good performance was selected for further research and evaluated together with the commercial strains GR-1 and RC-14. GR-1 TM Lactobacillus rhamnosus strains and RC-14 TM The Lactobacillus reuteri strain has been included in the "New Dietary Ingredient Notification" list by the U.S. Food and Drug Administration (FDA) and is a reliable edible probiotic strain.

[0065] Example 2 Identification and preservation of VCare-360 strain

[0066] The strain was identified as Lacticaseibacillus paracasei, which was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on June 27, 2024, with the deposit number CGMCC No.31095. Figure 4 This is the colony morphology of VCare-360 on the culture medium plate. Figure 5 This is a microscopic morphology diagram of VCare-360.

[0067] The 16S rDNA sequence is shown below, and the similarity with the 16S rDNA sequence of Lactobacillus paracasei JCM 1171 is 100.0%.

[0068] 16S rDNA sequence information:

[0069] CAAGTCGAACGAGTTCTCGTTGATGATCGGTGCTTGCACCGAGATTCAACATGGAACGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCTTAAGTGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAGATCCAAGAACCGCATGGTTCTTGGCTGAAAGATGGCGTAAGCTATCGCTTTTGGATGGACCCGCGGCGTATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGATGATACGTAGCCGAACTGAGAGGTTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTGGAGAAGAATGGTCGGCAGAGTAACTGTTGTCGGCGTGACGGTATCCAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCCTCGGCTTAACCGAGGAAGCGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAATGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAAT (SEQ ID NO:1)

[0070] Example 3 Evaluation of the antibacterial performance of VCare-360

[0071] The indicator strains Escherichia coli, Staphylococcus aureus, and Salmonella were inoculated into LB liquid medium, Candida albicans was inoculated into YPD liquid medium (purchased from Beijing Aoboxing, 02-107), Gardnerella and Atopobium were inoculated into BHI liquid medium (purchased from Beijing Luqiao, CM917B) + 10% sterile defibrinated sheep blood, and placed in an anaerobic workstation at 37°C for culture activation; after the indicator bacteria were cultured overnight, they were transferred to the corresponding fresh culture medium and cultured until OD 600 =1 or viable count 10 8 CFU / mL, dilute to 10 5 CFU / mL was used as the indicator bacterial solution.

[0072] The strain BC 30091 (named VCare-360), GR-1, and RC-14 glycerol tubes were inoculated into fresh sterile MRS liquid medium and cultured overnight at 37°C for 18-24 h. The OD 600 Normalized, inoculated with 2% inoculation in fresh MRS liquid medium, incubated at 37°C for 18-24 h, centrifuged to obtain the supernatant, sterilized by membrane, and used as the test sample. MRS liquid medium was used as a negative control, and ampicillin or kanamycin was used as a positive control. Reaction system: S1: 100 μL fermentation broth supernatant: 100 μL indicator bacteria dilution; S2: 50 μL fermentation broth supernatant: 150 μL indicator bacteria dilution; S3: 25 μL fermentation broth supernatant: 175 μL indicator bacteria dilution, inhibition rate (%) = ×100%, where: A0: negative control sample OD 600 Value; A: sample OD 600 Values ​​(where A0 and A are the OD 600 -OD before culture 600 The competitor strain (Novonesis) was tested using Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14.

[0073] Table 3 The inhibitory effects of the three strains on pathogenic bacteria under different conditions

[0074]

[0075] Table 3 continued

[0076]

[0077] Table 3 continued

[0078]

[0079] As can be seen from Table 3, the inhibition rate of strain BC 30091 (named VCare-360) against two strains of Escherichia coli was slightly lower than that of RC-14; the inhibition ability against Staphylococcus aureus, Salmonella, Candida albicans, Gardnerella vaginalis, and Atopobium was better than that of GR-1 and RC-14. This shows that strain BC 30091 (named VCare-360) has strong antibacterial ability and has the potential to improve fungal vaginitis and bacterial vaginitis.

[0080] Example 4 Evaluation of the adhesion performance of strains in vitro

[0081] 1. Evaluation of self-aggregation performance

[0082] The strain BC 30091 (named VCare-360), GR-1 and RC-1 glycerol tubes were inoculated into fresh MRS liquid medium at 2‰ and cultured overnight at 37°C for 18-24 h. The OD 600 Normalized, inoculated with 2% inoculum in fresh MRS liquid medium, cultured at 37°C, 200 rpm for 18-24 h, and then measured the OD 600 After washing twice with saline, resuspend the 600 =1, pipette 100 μL of bacterial solution into the ELISA plate and detect the initial OD 600 The bacterial solution was then allowed to stand for 4 h, and 100 μL of the upper bacterial solution was taken to the ELISA plate to detect the OD value after standing. 600 The value is recorded as A1, and the aggregation is calculated using the following formula: strain self-aggregation rate (%) = (1- ) × 100%. The competitor strain (Novonesis) was tested with Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14, respectively.

[0083] The strain will self-aggregate during the rapid reproduction period, and it is positively correlated with the adhesion ability of the strain. The self-aggregation rate of strain BC30091 (named VCare-360) is the highest, reaching 67.99% ( Figure 6 ).

[0084] 2. Hydrophobic performance evaluation

[0085] Pipette the above OD 600 =1, add 100 μL of bacterial solution to the ELISA plate and detect the initial OD 600 Then, 900 μL of bacterial solution was aspirated, 180 μL of dodecane was added, vortexed, and allowed to stand for 20 min to separate layers. The lower aqueous phase was taken to measure its OD 600 Value, recorded as A, use the following formula to calculate hydrophobicity, hydrophobicity (%) = × 100%. The competitive strain (Novonesis) was tested with Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14.

[0086] High bacterial hydrophobicity can help bacteria adhere to the intestinal mucosa. Therefore, the higher the bacterial hydrophobicity, the stronger its adhesion ability. The commercially available strain RC-14 has the highest hydrophobicity of 34.23%. The hydrophobicity of strain BC 30091 (named VCare-360) is 15.53%, which is lower than RC-14 but higher than strain GR-1 ( Figure 7 ).

[0087] Example 5 Safety evaluation of strains

[0088] 1. Hemolytic evaluation

[0089] The overnight cultured VCare-360 bacterial solution was inoculated on Columbia blood agar plates (according to the instructions, weigh an appropriate amount of Columbia blood agar medium powder, add the corresponding volume of water, sterilize at 121°C for 15 min and set aside; add 10% sterile defibrinated sheep blood before use), with Escherichia coli and Staphylococcus aureus as positive control strains.

[0090] The results showed that strain BC 30091 (named VCare-360) had no hemolysis. The positive control strain Escherichia coli ATCC25922 showed no hemolysis, while Staphylococcus aureus CMCC26001 had β-hemolysis zones around its white colonies.

[0091] 2. Evaluation of antibiotic sensitivity

[0092] Antibiotic information: ampicillin: 16 μg / mL; vancomycin: 128 μg / mL; gentamicin: 256 μg / mL; kanamycin: 1024 μg / mL; streptomycin: 256 μg / mL; erythromycin: 8 μg / mL; clindamycin: 16 μg / mL; tetracycline: 64 μg / mL; chloramphenicol: 64 μg / mL. Antibiotic samples of different concentrations were prepared using the two-fold dilution method.

[0093] The strain BC 30091 (named VCare-360) was inoculated in a glycerol tube into fresh MRS liquid medium and cultured at 37°C overnight for 18-24 h. It was then inoculated into fresh MRS liquid medium at a 2% inoculum and cultured at 37°C, 200 rpm for 4 h until the OD 600 =1, the number of viable bacteria is about 1.5×10 8 CFU / mL.

[0094] The activated bacterial solution was diluted 1000 times to a viable bacterial count of about 1.5×10 5CFU / mL, and 100 μL of antibiotic solutions of different concentrations were respectively added to a 96-well plate and cultured at 37°C for 24 h. The OD before and after culture was detected. 600 MRS liquid culture medium was used as a blank control, and aqueous solution was used as a negative control. The concentration at which the strain in the well plate is completely inhibited is called the minimum inhibitory concentration (MIC). The MIC value is compared with the breakpoint value in the "General Rules for Probiotics for Food Use" (T / CIFST 009-2022) to determine its antibiotic sensitivity.

[0095] The results of antibiotic sensitivity tests are as follows:

[0096] Table 4 Results of antibiotic sensitivity test of BC 30091

[0097]

[0098] As can be seen from Table 4, BC 30091 (named VCare-360) is highly sensitive to antibiotics and has no obvious resistance to the above antibiotics. This result shows that strain BC 30091 (named VCare-360) is safe and can be used to develop edible products such as probiotic preparations.

[0099] Example 6 Effect of VCare-360 on the Vaginal Epithelial Cell VK2 / E6E7 Inflammation Model

[0100] 1. Vaginal epithelial cell culture VK2 / E6E7 and determination of probiotic application concentration

[0101] VK2 / E6E7 cells (purchased from ATCC) were resuspended in VK2 / E6E7 culture medium containing 10% fetal bovine serum and 1% double antibody (penicillin-streptomycin mixture), the cell density was adjusted, and the cells were seeded into 6 cm culture dishes and cultured at 5% CO2 and 37°C. The cells were passaged every 3 days. VCare-360 and positive control strains (RC-14:GR-1 = 1:1 mixture) were diluted with cell KSMF complete medium (Gibco) to a final treatment concentration of 1×10 8 , 1×10 7 , 1×10 6 CFU / mL, and 3 replicates were set for each concentration. The group without probiotics was used as the blank control group. After incubation with cells for 24 h under 5% CO2 and 37 °C, the CCK8 kit was used to detect cell viability and the effective concentration of probiotics was determined to be 1×10 7 CFU / mL.

[0102] 2. Determination of the adhesion of strains to VK2 / E6E7 cells

[0103] VK2 / E6E7 cells were grown at 106 10 cells / mL were inoculated in a six-well plate with a cover glass. After 24 h of adherence, the cell culture medium containing the double antibody was removed and 2 mL of 10 8 CFU / mL of probiotic suspension was co-cultured with cells under 5% CO2 and 37 ℃ for 2 h. The non-adherent probiotics were washed with PBS buffer, fixed with methanol at room temperature for 20 min, washed with ddH2O and dried. The Gram staining kit was used for staining according to the operating instructions. The adhesion of 20 random probiotic cells was observed under a microscope and the adhesion index was calculated. The VCare-360 adhesion index was 19.85±5.85 CFU / cell, and the positive control adhesion index was 8.25±3.16 CFU / cell. The positive control strain was a 1:1 mixture of Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 (Novonesis, Urex ).

[0104] 3. Construction of cellular inflammation model and evaluation of the anti-inflammatory effect of probiotics

[0105] 3.1 Construction of Candida albicans cell inflammation model (fungal vaginitis model)

[0106] VK2 / E6E7 cells were grown at 10 5 The cells / mL were inoculated in a 12-well plate and grown for 24 h. The Candida albicans culture was diluted to 10 with KSMF complete medium. 7 CFU / mL. Each well was incubated with 10 5 CFU / mL of Candida albicans solution was used to stimulate cells for 2 h under 5% CO2 and 37°C.

[0107] 3.2 Construction of Gardnerella cell inflammation model (bacterial vaginosis model)

[0108] Same as above, add 10 6 The cells were stimulated with a Gardnerella bacterial solution of 1.3 CFU / mL and in the presence of 5% CO2 and 37°C for 2 h.

[0109] 3.3 Screening of anti-inflammatory activity of strains in vitro

[0110] In the VK2 / E6E7 cell fungal and bacterial vaginitis validation model, the final incubation concentration was 10 7 CFU / mL of probiotics were incubated for 6 h, with the group without pathogenic bacteria and probiotics as the blank control group (control group), the group with pathogenic bacteria alone as the model group (the fungal cell vaginitis validation model was the Ca group, and the bacterial cell vaginitis validation model was the Gv group), and a 1:1 mixture of competitor strains GR-1 and RC-14 (Novonesis, Urex ) incubation group was the positive bacteria control group (Pb group). After removing the cell culture medium and washing with PBS three times, the cell RNA samples of each group were extracted using a cell RNA extraction kit to detect the expression levels of inflammatory factors TNF-α, IL-8, IL-6, and IL-1β in different groups. BC 30091 (named VCare-360) has a certain anti-inflammatory effect on both fungal and bacterial cell inflammation models. In the fungal cell inflammation model, the level of inflammatory factors in the Ca group was significantly higher than that in the control group, proving that the cell inflammation model caused by fungal infection was successfully established. Among them, the expression levels of 3 inflammatory factors in BC 30091 (named VCare-360) were lower than those in the Ca group, and the expression level of IL-6 (0.61±0.15) was comparable to that of the Pb group (0.57±0.47), proving that strain BC 30091 (named VCare-360) can alleviate the inflammatory response of VK2 / E6E7 cells caused by fungal infection; in the bacterial cell inflammation model, the level of inflammatory factors in the Gv group was significantly higher than that in the control group, proving that the cell inflammation model caused by bacterial infection was successfully established. Among them, the expression levels of TNF-α and IL-1β in BC 30091 (named VCare-360) were lower than those in the Gv group, and the expression level of IL-1β (0.82±0.61) was better than that of the Pb group (1.10±0.78), proving that strain BC 30091 (named VCare-360) can alleviate the inflammatory response of VK2 / E6E7 cells caused by bacterial infection

[0111] 3.4 Competitive resistance of strains to the colonization ability of Candida albicans cells

[0112] Same as above steps, VK2 / E6E7 cells were cultured at 10 6 10 cells / mL were inoculated into a six-well plate with a cover glass, and the probiotic solution and Candida albicans solution were diluted with KSMF complete medium, and 2 mL of 10 8 CFU / mL of probiotics and a concentration of 10 7CFU / mL suspension of Candida albicans, in which no probiotics were added as blank control. It was co-cultured with cells for 2 h under 5% CO2 and 37 ℃. After the incubation, the excess non-adherent probiotics were washed with PBS buffer, fixed with methanol at room temperature for 20 min, washed with ddH2O and dried, stained with Gram staining kit, and the adhesion of Candida albicans to cells was observed under a microscope. 20 cells were randomly selected and the number of Candida albicans adhered to the cells was counted. The adhesion index of Candida albicans was 41.75±13.14 CFU / cell, the adhesion index of the 1:1 mixture of positive control GR-1 and RC-14 was 13.45±3.71 CFU / cell, and the adhesion index of VCare-360 was 10.95±4.46 CFU / cell. Therefore, VCare-360 has the best competitive adhesion performance with Candida albicans on VK2 / E6E7 cells.

[0113] References

[0114] 1. Bisanz JE, et al. A Systems Biology Approach Investigating the Effect of Probiotics on the Vaginal Microbiome and Host Responses in a DoubleBlind, Placebo-Controlled Clinical Trial of Post-Menopausal Women. PLoS One. 2014;9(8):e104511. (PubMed)

[0115] 2. Ho M, et al. Oral Lactobacillus rhamnosus GR-1 and Lactobacillusreuteri RC-14 to reduce Group B Streptococcus colonization in pregnant women: A randomized controlled trial. Taiwan, China J Obstet Gynecol. 2016;55(4):515-8. (PubMed)

[0116] 3. Hummelen R, et al. Lactobacillus rhamnosus GR-1 and L. reuteri RC-14 to prevent or cure bacterial vaginosis among women with HIV. Int JGynaecol Obstet. 2010;111(3):245-8. (PubMed)

[0117] 4. Martinez RC, et al. Improved treatment of vulvovaginal candidiasis with fluconazole plus probiotic Lactobacillus rhamnosus GR-1 andLactobacillus reuteri RC-14. Lett Appl Microbiol. 2009;48(3):269-74. (PubMed)

[0118] 5. Reid G, et al. Oral use of Lactobacillus rhamnosus GR-1 and L.fermentum RC-14 significantly alters vaginal flora: randomized, placebo-controlled trial in 64 healthy women. FEMS Immunol Med Microbiol. 2003;35(2):131-4. (PubMed)

[0119] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Lactobacillus paracasei VCare-360, characterized in that The Lactobacillus paracasei VCare-360 was deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration on June 27, 2024, with the deposit number CGMCC No.31095.

2. A probiotic, characterized in that: The probiotic comprises the Lactobacillus paracasei VCare-360 according to claim 1.

3. The probiotic according to claim 2, characterized in that The dosage form of the probiotic is selected from one of powder, liquid, ointment, suppository, pill, capsule, tablet, oil drop and granule.

4. A biological agent, characterized in that The biological preparation comprises the Lactobacillus paracasei VCare-360 according to claim 1.

5. The probiotic according to claim 2 or 3 or the biological preparation according to claim 4, characterized in that: The invention also comprises one or more auxiliary materials, wherein the auxiliary materials are selected from one or more of prebiotics, plant extracts and sugar substitutes.

6. The probiotic agent or biological preparation according to claim 5, characterized in that: The prebiotics are selected from one or more of fructooligosaccharides, galacto-oligosaccharides, xylooligosaccharides, isomaltooligosaccharides, soybean oligosaccharides, inulin, spirulina, versicolor polysaccharides, stachyose, manno-oligosaccharides and polydextrose.

7. The probiotic agent or biological preparation according to claim 5, characterized in that: The plant extract is selected from one or more of Curcuma zedoaria extract, eucalyptus extract, Sophora flavescens extract, Cnidium monnieri extract, Phellodendron amurense extract, honeysuckle extract, Portulaca oleracea extract, carvacrol, limonene and thymol.

8. The probiotic agent or biological preparation according to claim 5, characterized in that: The sugar substitute is selected from one or more of neotame, acesulfame potassium, mannitol, sorbitol, aspartame, erythritol, xylitol and sucralose.

9. Use of Lactobacillus paracasei VCare-360 according to claim 1 in preparing a product for preventing and alleviating vaginitis or regulating the balance of vaginal flora, inhibiting the growth of Gardnerella and Candida albicans, wherein the product is a probiotic, a biological preparation or a medicine.

10. The use according to claim 9, characterized in that The vaginitis is bacterial vaginitis or fungal vaginitis.

11. The use according to claim 9, characterized in that The dosage form of the probiotic, biological preparation or medicine is selected from one of powder, liquid, ointment, suppository, pill, capsule, tablet, oil drop and granule.

12. A medicine, characterized in that The medicine comprises the Lactobacillus paracasei VCare-360 according to claim 1.

13. The drug according to claim 12, characterized in that The medicine is selected from one of vaginal wash and women's antibacterial / antibacterial wash.

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