A strain of Lactobacillus crispatus and its pharmaceutical use
A technology of Lactobacillus flexus and strains, applied in the direction of antibacterial drugs, bacteria, drug combinations, etc., can solve the problems of poor effect, toxic and side effects, etc.
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Embodiment 1
[0038] Isolation and identification of embodiment 1 Lactobacillus flexus strain
[0039] 1. Isolation of Lactobacillus flexus strains
[0040] MRS medium: peptone 10.0g; beef extract 10.0g; yeast extract 5.0g; diammonium hydrogen citrate 2.0g; glucose 20.0g; Tween 80, 1.0mL; sodium acetate (CH 3 COONa·3H 2 O) 5.0g; Dipotassium hydrogen phosphate (K 2 HPO4·3H 2 O) 2.0g; Magnesium sulfate (MgSO 4 ·7H 2 O) 0.58g; manganese sulfate (MnSO 4 ·H 2 O) 0.25g; distilled water 1000mL; adjust to pH 6.2-6.6.
[0041] More than 200 samples of vaginal secretions from healthy women without symptoms and signs of vaginitis were collected. The samples were numbered sequentially, inoculated in MRS liquid medium, and incubated anaerobically at 37°C for 24 hours. For each sample, 5 single colonies were randomly selected and cultured in 2 mL MRS liquid medium for 24 hours, and the colonies were numbered sequentially. Bacteria are amplified for bacterial conservation and DNA preparation. Hund...
Embodiment 2
[0051] Embodiment 2 Lactobacillus flexus IMC-105 produces H 2 o 2 detection
[0052] h 2 o 2 Preparation of standard curve: firstly, piperazine-N, N'-di-ethanesulfonic acid (Piperazine-N, N-Bis 2-ethanesulfonic acid, PIPES) with a concentration of 100mmol / L was used to dissolve 30% (v / v) H 2 o 2 (equivalent to 9.128mol / L) stock solution diluted to 1mol / L, and then 1mol / L H with 100mmol / L PIPES 2 o 2 Dilute to 0μmol / L, 20μmol / L, 40μmol / L, 60μmol / L, 80μmol / L, 100μmol / L working solution; then take 100μL of the above H 2 o 2 Mix the working solution with 100 μL of TMB (tetramethylbenzidine) at a concentration of 20 mmol / L and 2 μL of horseradish peroxidase (1 mg / mL), incubate at 16°C for 10 min, and measure the OD 630 value. end up with H 2 o 2 Concentration standard curve: y=0.0033x-0.0496, R 2 = 0.992.
[0053] Lactobacillus flexure IMC-105 liquid culture H 2 o 2 Concentration detection: Pick the colony of Lactobacillus flexus IMC-105, inoculate 1mL of the bacteri...
Embodiment 3
[0057] Example 3 Adhesion of Lactobacillus flexus IMC-105 to vaginal epithelial cells
[0058] Vaginal epithelial exfoliated cells were obtained from more than 20 healthy women of childbearing age (clinical tests showed no reproductive system infection). Each cell sample was labeled and added to MEM medium (pH 4.0), centrifuged at 120g for 10 minutes, and then repeated centrifuged twice. Wash off the surface bacteria, and adjust the concentration of exfoliated vaginal epithelial cells to 105 cells / mL with MEM medium; activate the overnight culture of Lactobacillus flexus IMC-105 to make the concentration (108CFU / mL). The vaginal epithelial exfoliated cells and the Lactobacillus flexus IMC-105 were mixed in equal volumes of 500 μL each, and then placed on a shaker at 37°C (rotating speed: 100r / min, radius of rotation: 2.54cm) and incubated for 1h, and then filtered through a 0.8μm filter membrane , remove the free Lactobacillus flexure IMC-105, and lightly press the filter memb...
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