A kind of extracellular polysaccharide of Parabacteroides dineri and its extraction method and application
A technology of parabacteroides and exopolysaccharides, applied in the field of exopolysaccharides of parabacteroides distirii and its extraction, can solve problems such as bacteremia and infection, achieve enhanced immunity, low preparation cost, and simple and easy extraction process line effect
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Embodiment 1
[0051] The method for extracting the extracellular polysaccharide of Parabacteroides dineri (named PEP 1-A) includes the following steps:
[0052] (1) Extraction and decolorization of crude polysaccharide
[0053]Using Parabacteroides diselia ATCC 8503 (can be purchased from Shanghai Kewei Chemical Technology Co., Ltd.) as the experimental strain, the parabacteroides diselia ATCC 8503 (Pd bacteria) stored at -80 ℃ was inoculated into the brain heart leachate liquid culture The base was cultured at 37°C for 24h, and after activation for two generations, it was inoculated into the brain-heart extract liquid medium with 3% inoculum for expansion, and cultured for 48h under the conditions of anaerobic, 37°C, and 100 rpm shaker shaking.
[0054] After the cultivation is completed, the fermentation broth is centrifuged (4°C, 4000rpm, 30min) to remove the bacterial cells, the supernatant is collected, boiled at 100°C for 30min, centrifuged (4°C, 4000rpm, 30min), and the supernatant i...
Embodiment 2
[0059] The exopolysaccharide PEP 1-A of Parabacteroides dineri obtained in Example 1 was used as a raw material, and its composition and structure were analyzed.
[0060] 1. Determination of total sugar content
[0061] Using D-mannose (Man) as the reference substance, the total sugar content of PEP 1-A was determined by the sulfuric acid phenol method. Accurately and accurately weigh an appropriate amount of D-mannose reference substance, add pure water to prepare a 0.5mg / mL solution, take 0, 20, 40, 60, 80, 120, 160, 200 μL in turn and place them in a 2mL EP tube, add pure Make up to 400 μL with water. Accurately weigh an appropriate amount of PEP 1-A, add pure water to prepare 0.25 mg / mL, and take 400 μL into a 2 mL EP tube. Then add 200 μL of 5% redistilled phenol and 1 mL of concentrated sulfuric acid to each EP tube, shake well, stand at room temperature for 10 min, keep at 40 °C for 15 min, and take an ice-water bath for 10 min. In the secondary hole, the absorbance ...
Embodiment 3
[0078]Effects of PEP 1-A administration on the proliferation activity of macrophage RAW264.7 cells in vitro
[0079] Take the RAW264.7 cells in the logarithmic growth phase by pipetting and mixing, and then divide the cells into 2 × 10 cells. 5 Cells / mL were seeded in 96-well plates, 100 μL per well. After the cells were fully adherent, the old medium was discarded, and 100 μL of different concentrations of PEP 1-A (2 μg / mL, 5 μg / mL, 10 μg / mL) were added. , 25 μg / mL, 50 μg / mL, 100 μg / mL), the control group only added medium, and 1 μg / mL lipopolysaccharide (LPS) was used as a positive control. After culturing for 24 hours, observe and take pictures under an inverted microscope. Add 20 μL of 5 mg / mL MTT solution to the wells, continue to incubate for 4 h, remove the supernatant, add 200 μL of DMSO to each well, shake gently on a shaker at room temperature for 10 min, and measure the absorbance at 490 nm of each well with a microplate reader.
[0080] Depend on Figure 15 The r...
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