Lactobacillus rhamnosus for preventing and/or treating diseases caused by reproductive tract flora disorder and/or bone loss
A technology for Lactobacillus rhamnosus and diseases, applied in the direction of Lactobacillus, bone diseases, and microbial-based methods, can solve the problems of drug resistance of pathogenic bacteria, unfavorable reproductive tract maintenance, and reduced abundance of probiotics, etc.
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Embodiment 1
[0053] Example 1: Isolation and identification of Lactobacillus rhamnosus OF44-15Ph10T
[0054] 1. Sample collection
[0055] The isolated sample comes from the feces of a healthy female. The feces are collected into sterile sample tubes and brought back to the laboratory for sorting within 1 hour.
[0056] 2. Separation and purification
[0057] Immediately transfer the collected fresh samples to an anaerobic operation box, take 0.2g sample in 1mL sterile PBS (phosphate buffer), shake and mix well, and then carry out gradient dilution coating, the medium adopts the modified version of PYG medium (purchased from Huankai Microbial Technology Co., Ltd.), the specific formula is (1L): tryptone 8g, soybean peptone 2g, polyprotein 1g, casein 1g, yeast powder 10g, beef extract 5g, glucose 5g, K 2 HPO 4 2g, maltose 0.5g, cellobiose 0.5g, soluble starch 0.5g, sodium sulfide 0.25g, Tween 80 0.5mL, Cysteine-HCl·H 2 O 0.5g, glycerol 0.5mL, sodium acetate 5g, heme 5mg, vitamin K 11μL...
Embodiment 2
[0072] Example 2: Genome sequencing, species classification and functional gene analysis of Lactobacillus rhamnosus OF44-15Ph10T
[0073] 1. Genome sequencing
[0074] Centrifuge the overnight cultured OF44-15Ph10T bacterial solution at 7,227 g at 4°C for 10 minutes, resuspend the obtained pellet in 1 mL of Tris-EDTA, add 50 μL of 10% SDS and 10 μL of proteinase K (20 mg / mL) at 55°C The cells were lysed in a water bath for 2 hours, and the DNA was extracted by the phenol-chloroform method. And use the Illumina Hiseq 2000 platform to sequence it, the sequencing length is 500bp in both directions, and use SOAPdenovo to assemble the reads. After evaluation, use GCskew to analyze the GC content of the whole genome and visualize the whole genome sequence and functional distribution ( figure 1 ).
[0075] 2. Classification of strain genome species
[0076] Use Checkm software to compare and analyze the whole genome sequence, and get the species information closest to the genome ...
Embodiment 3
[0079] Example 3: Biologically active substances of Lactobacillus rhamnosus OF44-15Ph10T
[0080] The bioactive substances of OF44-15Ph10T mainly examine the content of L-lactic acid and D-lactic acid in the metabolites, and the production of hydrogen peroxide.
[0081] 1. Sample pretreatment
[0082] The strain OF44-15Ph10T was inoculated into MRS medium, and cultured at 37°C for 24h under aerobic and anaerobic conditions, respectively.
[0083] Take 1mL of the bacterial solution and centrifuge at 8000r / min for 5min, and take the supernatant to detect the content of L-lactic acid and D-lactic acid.
[0084] Then take 1 mL of the bacterial solution and add lysozyme (final concentration: 1 mg / mL), let it stand at 37°C for 15 min, centrifuge at 8000 r / min for 5 min, take the supernatant, and test the concentration of hydrogen peroxide.
[0085] 2. Measurement method
[0086] The contents of L-lactic acid and D-lactic acid were measured using L-Lactic Acid (L-Lactate) Assay Ki...
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