Lactobacillus rhamnosus for preventing and treating mastitis and application thereof
A technology of Lactobacillus rhamnosus, mastitis, applied in the direction of Lactobacillus, application, bacteria used in food preparation, etc., can solve problems such as drug dependence and drug resistance
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Embodiment 1
[0018] Example 1: Isolation, screening and identification of Lactobacillus rhamnosus Probio-M9.
[0019] 1. Materials and methods
[0020] 1.1 Source, isolation and identification of strains
[0021] From a healthy woman's breast milk sample, weigh 10 g of the sample and dilute it with 90 g of sterile normal saline, then take 0.1 mL of it and spread it on a selective MRS solid medium, and culture it anaerobically at 37°C for 72 hours. Pick typical colonies for Gram-staining microscopic examination, select Gram-positive strains and streak them on MRS solid medium for 2-3 times to obtain pure colonies. The morphology of this strain is Gram-like bacteria observed under the microscope, which is straight rod-shaped, single, sometimes in pairs or in chains; the diameter of colonies on MRS solid medium is 1-3mm, milky white, raised, round, The surface is smooth and has no kinematic properties.
[0022] Physiological and biochemical identification and 16s rRNA molecular identifica...
Embodiment 2
[0025] Example 2 Lactobacillus rhamnosus Probio-M9 (Lactobacillus rhamnosus Probio-M9) gastrointestinal juice Tolerance performance.
[0026] 1. Gastrointestinal fluid tolerance test method:
[0027] 1.1. Add 3.5 g / L pepsin to sterilized PBS at pH 2.5 (adjusted with 1 mol / L HCl), filter and sterilize with a 0.22 μm microporous membrane to prepare simulated gastric juice.
[0028] 1.2. Select the strains that have been screened for acid resistance for cultivation. After two generations of cultivation, centrifuge and wash the bacteria twice, collect the bacteria, add the same amount of pH 2.5 simulated gastric juice as the culture medium, culture at 37°C for 3 hours, and use at 0h and 3h MRS agar medium pouring method was used to determine the number of viable bacteria.
[0029] 1.3. In sterilized PBS with pH 8.0 (adjusted with 0.1 mol / L NaOH), add 0.1% trypsin and 1.8% ox bile salt and filter with a 0.22 μm microporous membrane to prepare simulated intestinal fluid.
[00...
Embodiment 3
[0042] Example 3: Prevention and treatment effect of Lactobacillus rhamnosus Probio-M9 on mouse mastitis.
[0043] 1 Experimental method
[0044] 1.1 Experimental strain preparation
[0045] Lactobacillus rhamnosus Probio-M9 powder preparation, live bacteria content 2x10 11 CFU / g, provided by Beijing Ketuo Hengtong Biotechnology Co., Ltd.
[0046] 1.2 Experimental animals
[0047] The experiment used 4-month-old C57BL / 6J sterile pregnant mice, provided by the Third Military Medical University, for the establishment of animal models of mastitis.
[0048] 1.3 Experimental main kits
[0049] Abcam MPO (Myeloperoxidase), IL-10 (Interleukin-10) mouse ELISA kit; Biolegend company IFN-y, IL-17, IL-1-β, TNF-α, IL-6 mouse ELISA kit; Shanghai Sangon mouse endotoxin, IL-4, lysozyme ELISA kit; BD Anti-NF-kB P65 antibody, Anti-IkB beta antibody, Anti-P38 antibody, Anti-JNK1+JNK2+JNK3 antibody, Anti- ERK1+ERK2 antibody, Anti-CD45 antibody, Anti-CLC4 antibody, Anti-STAT3 antibody, An...
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