| Cordyceps guangdongensis has been approved as the second novel food of Cordyceps species in China.Its fruiting body has a variety of significant active effects,such as antioxidation,immunity enhancement,renal failure treatment,life spans prolongation.Studies have shown that polysaccharide is an important active ingredient in Cordyceps fungi,but the polysaccharide of C.guangdongensis has not been systematically studied.Therefore,in this study,the preparation methods,composition,immunity and antioxidative activities of polysaccharide from C.guangdongensis fruiting bodies were systematically studied.And at the same time,a preliminary exploration of the key enzyme gene Pgm during polysaccharide biosynthesis was conducted,in order to lay the foundation for the further study of polysaccharide and its related gene functions on synthesis of C.guangdongensis.The contents and the results of this study are as follows:(1)Screening method of polysaccharides from C.guangdongensis and the preparation of crude polysaccharide-1 CPS-1(Crude Polysccharide-1): By comparing the four extraction methods,hot water extraction method was adopted here to extract polysaccharide from C.guangdongensis for the next studies,duing to its simplicity of operation and low cost.And first of all,using hot water extraction method to separate the polysaccharide from its fruit body,and following by the subsequently purification steps as ethanol precipitation,sevage method for primary purification to obtain CPS,then through dialysis bag for dialyzing,finally,freeze-dried to obtain the polysaccharide powder CPS-1.(2)Isolation and characteristics analysis of crude polysaccharide: The crude polysaccharide sample CPS was separated into two components PPS-W and PPS-S byDEAE-52 cellulose gel.The monosaccharide composition was mainly composed of glucose,mannose and galactose of both two groups PPS with HPLC and GC analysis;Fourier infrared spectrometer and SEM analysis results showed that the main types of the glycosidic bonds are all ?-type and the surface microstructure of both two groups PPS were shown a sponge shape,respectively.(3)Evaluation of antioxidant activity: The hydroxyl radical scavenging ability,ferrous ion chelating ability,ABTS scavenging ability,DPPH scavenging and reducing ability of CPS-1,PPS-W and PPS-S were measured by spectrophotometry.The results showed that all of the three components of the polysaccharide have significant antioxidant activity,and the antioxidant activity of CPS-1 is the most effective one(for the value of p < 0.05).(4)Improving immunity activity evaluation: in this part,the effect of CPS-1 on the humoral immune function of normal mice was examined.The results showed that CPS-1 in the high-dose group can obviously enhance the function of humoral immunity in mice.(5)Preliminary analysis of phosphoglucomutase gene Pgm on polysaccharide synthesis: in this section,the gene sequence and the encoded protein sequence were analyzed,a Pgm overexpression vector was constructed,and a protoplast transformation method was initially explored.This study established a method for the preparation of polysaccharides from C.guangdongensis and its composition and characteristics have been identified.It also confirmed that the polysaccharide of C.guangdongensis have good activities on anti-oxidation and immunity enhancement,furthermore,the overexpression vector of Pgm was obtained.The study here lays an important foundation for the further study of the effectiveness and its development and utilization of polysaccharide of C.guangdongensis. |
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