A kind of polysaccharide rpp1 prepared by Bacillus natto fermented shellfish and its purification method and application
A technology of Bacillus natto and polysaccharides, which is applied in the field of polysaccharide RPP1 prepared by Bacillus natto fermented shellfish and its purification, which can solve the problems of not getting good results, not very prominent effects, etc.
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Embodiment 1
[0035] (1) Extraction of crude polysaccharide
[0036] The crude polysaccharide used in the examples is the active polysaccharide mixture obtained by the inventors using Bacillus natto to ferment clam philippines. The specific steps are: take natto by weighing, screen and cultivate to obtain Bacillus subtilis natto subspecies PS-1-HepG2 seed bacterial suspension, take fresh and alive Philippine clams, take meat, homogenate, add distilled water for high-temperature sterilization, and then insert The above-mentioned seed bacteria suspension is fermented, adding distilled water to the fermented product, centrifuging to obtain the supernatant, adding ethanol for alcohol precipitation, and adopting the Sevag method to remove protein after obtaining the precipitate; ultrafiltration and freeze-drying to obtain crude polysaccharide powder.
[0037] (2) Purification of polysaccharide RPP1
[0038] Step 1: Ion Exchange Column Chromatography
[0039] Connect a DEAE Sepharose Fast Flow ...
Embodiment 2
[0045] (1) Physicochemical properties analysis of polysaccharide RPP1
[0046] Coomassie brilliant blue method was used to detect protein content, and the standard curve was as follows image 3 As shown, the regression equation is y=0.0037x+0.0101, R 2 =0.99531, the linear fitting is better in the range of 0-100μg / mL.
[0047] The sulfate content was determined by gelatin turbidimetry, Figure 4 Be the standard curve of sulfate radical content, regression equation is y=0.0037x+0.0101, R 2 =0.99531, the linear fit is good within the range of 0-600 μg / mL.
[0048] Adopt m-hydroxybiphenyl method to measure uronic acid content, the standard curve of uronic acid content is as follows Figure 5 As shown, the regression equation is y=0.0219x-0.0046, R 2 =0.99558, the linear relationship is good in the range of 0-15 μg / mL.
[0049] According to the above detection and analysis, the total sugar content of RPP1 is 90.01%, the protein content is 0.24%, the sulfate content is 0.54%,...
Embodiment 3
[0063] Study on Antitumor Activity of Polysaccharide RPP1
[0064] 1. Main materials
[0065] Human colon cancer cell line HT29 (Shanghai Cell Bank, Chinese Academy of Sciences) BALB / C, four-week-old 18-22g male nude mice (Shanghai Slack Experimental Animal Co., Ltd.), polysaccharide RPP1 isolated and purified from fermented clam philippines.
[0066] 2. The effect of polysaccharide RPP1 on apoptosis of HT-29 cells
[0067] 1. Experimental method
[0068] The HT-29 cells were taken out from the liquid nitrogen tank and cultured with McCoy's 5A medium containing 10% fetal calf serum. Mix the resuspended cells in the medium and inoculate them into a 96-well culture plate. Each cell can be inoculated into 2 wells. The culture plate is placed at 37°C and 5% CO 2 cultivated in an incubator. After 24 hours, the state of the cells was observed, the medium was changed, and the culture was continued. The cells were grown to a density of about 90%, and the cells were collected in a...
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