Research On Extraction, Purification And Cell Immune Activity Of Polysaccharides From The Base Of Flammulina Velutipes Stipe

Flammulina velutipes is rich in nutrients which has become the world’s third largest edible fungus. With more and more people understand the Flammulina Velutipes nutritional value, it will have great broad prospect for development. At the same time, there will be more and more the base of Flammulina velutipes stipe bring out. In the present reports, studies on Flammulina velutipes mostly focus on the fruit body and mycelia, but there have no research on the base of Flammulina velutipes stipe until now. So the purpose of this study is fully utilization of the base of Flammulina velutipes stipe. It’s to reduce environment pollution and what’s more to improve its value.In this study, response surface methodology, base on Box-Behnken design was used to optimize the ultrasonic power, ultrasonic time and ratio of water to raw material. The results show that the optimum extraction parameters were ultrasonic power680W, ultrasonic time19.8min and the ratio of water to raw material28mL/g, the yield of polysaccharides was16.20%which there was no significant difference with the predicted values. Compared with the hot water extraction, the yield was highly increased by70%and extraction time shortened by92%.FSP was further purified by DEAE-cellulose-52to obtain three fractions of FSP-1, FSP-2and FSP-3. Basic physicochemical characterization of crude FSP, FSP-1, FSP-2and FSP-3were determined by using the methods of chemistry and physics. As to crude FSP, FSP-1, FSP-2and FSP-3, polysaccharides content was64.19%,99.19%,96.42%and65.17%respectively. Protein content was3.74%, not detected,2.07%and3.00%. Uronic acid content was2.94%,4.63%,3.40%and1.72%respectively. Sulfuric radical content was0.55%,0.49%,1.37%and0.32%respectively. In the FT-IR spectrum, characteristic absorptions of polysaccharides, carboxyl group and pyranose ring were observed in crude FSP, FSP-1, FSP-2and FSP-3.Immunoregulation activity in vitro of crude polysaccharides, FSP-1, FSP-2and FSP-3were evaluated by using the cell model experiments. Results displayed remarkable immune activity which could improve the proliferation rate and enhance ability of devouring neutral red of macrophages in vitro. Crude FSP showed much higher immune enhancing activities than FSP-1, FSP-2and FSP-3. At5μg/mL of crude FSP, the proliferation rate of crude FSP in macrophages was121.52%.By the culture of the B16F10and L929, results exhibited that FSP and macrophages co-culture medium had the significant growth inhibition on B16F10and L929. The highest inhibition rate of B16F10was23.71%. And the highest inhibition rate of L929was44.61%at the50μg/mL of FSP-1, as six times high as directly with FSP. At the same time, the inhibition rate of L929was much higher than B16F10’s, because L929is more sensitive to TNF than B16F10.