Isolation,Purification And Bioactivity Analysis Of Polysaccharide From Beiqi Mushroom

Pleurotus ostreatus（P⁰）is a common edible fungus,which is widely cultivated in China.With the deepening of the research on Pleurotus ostreatus,Astragalus as an ingredient to the Pleurotus ostreatus culture medium,and developed a new type of mushroom with richer nutritional value--Beiqi Mushroom(P¹⁵).In this experiment,we selected Beiqi mushroom and Pleurotus ostreatus grown in the same environment for polysaccharide extraction,and then make a preliminary comparative study on the physical and chemical properties,structure and biological activity of the extracts.The main contents and results are as follows:1、The polysaccharide of P⁰/P¹⁵ was extracted by ultrasonic assisted hot water extraction.Combined with the experimental results and the feasibility of practical operation,the extraction conditions were determined.The final extraction conditions were as follows:ultrasonic treatment for 20 minutes,ultrasonic power of 100W,hot water extraction at 85℃for 2 h,solid-liquid ratio of 1:10.Under the above conditions,the extraction rate of polysaccharide P⁰ was 22.70±0.14%,the extraction rate of polysaccharide P¹⁵ was 25.45±0.16%.2、Separating and purifing the crude polysaccharides from mushroom extracted above.The results showed that the protein removal rates were 84.49±0.63%and 83.96±0.29%,the polysaccharide retention rates were 72.16±0.86%and 68.29±0.69%,the pigment removal rates were 68.74±0.06%and67.69±1.20%,and the polysaccharide retention rates were 86.05±0.80%and84.30±1.53%,respectively.Then the obtained polysaccharides were purified by column,and the pure product of polysaccharides P⁰OP-I,P⁰OP-II,P¹⁵OP-I and P¹⁵OP-II were obtained,and their sugar contents were 93.17±0.13%、90.56±0.48%、94.21±0.35%and 90.16±0.17%,respectively.3、We selected the P⁰OP-I and P¹⁵OP-I to explore their primary structure.By the UV scanning and freeze thaw experiments we can find that the purification effect of polysaccharide was very good;the results of infrared sca-ning showed that P⁰OP-I and P¹⁵OP-I contained mannose and were linked by pyranoside bond;the molecular weight analysis results showed that the relative molecular weight of P⁰OP-I was 21706.96 Da,P¹⁵OP-I was 20172.65 Da.The results of monosaccharide composition showed that P⁰OP-I and P¹⁵OP-I were mainly composed of Man,Glc UA,Glc,Gal and Ara,and their molar ratios were 22.61:40.94:33.70:2.49:0.26 and 33.6:14.48:47.23:4.17:0.52.4、The immunomodulatory activities of P⁰OP-I and P¹⁵OP-I were studied by constructing the mouse macrophage RAW264.7 cell model in vitro.The results showed that the polysaccharide components P⁰OP-I and P¹⁵OP-I could induce the activation and release of NO and TNF-αin RAW264.7 cells,and showed a strong concentration dependent effect on polysaccharide.5、Selecting appropriate inhibitors of MAPK and NF-κB signaling pathways to conduct a preliminary study on the mechanism of the polysaccharide components P⁰OP-I and P¹⁵OP-I in inducing activation of RAW264.7 cells to release NO and secrete TNF-α.It was found that they induced NO release in RAW264.7 cells by activating NF-κB,JNK MAPK and Erk MAPK signaling pathway,while stimulating RAW264.7 cells to secrete TNF-αwas through JNK and Erk MAPK signaling pathway,and the induction activity of P¹⁵OP-I was higher than that of polysaccharide component P⁰OP-I.The research results of this paper provide a theoretical basis for other scholars to further study and develop in many fields.