Polysaccharide Biosynthesis In Ganoderma Lucidum Under Nitrogen Availability And Application Of Its Nanoparticles

The elucidation of the Ganoderma lucidum genome makes this organism a potential model system in medicinal fungi study.The main bioactive component of G.lucidum is expolysaccharide from its submerged culture?GLP?.To date,the research mainly focused on the following aspects about GLP,including extracting,purification,structure identification,biological activities and optimization of fermentation conditions,addition of inducers,development of new bioprocessing strategies to increase GLP production.However,few work on biosynthesis and bioactivity enhancement of GLP have been done.In this thesis,effects of nitrogen availability on GLP biosythesis in G.lucidum was studied.Then structure of GLP was analyzed and G.lucidum exopolysaccharide nanoparticles?GLPN?were prepared and characterized.Furthermore,the effects of GLPN on immune regulation were investigated.The main results are as followings:?1?A high yield of GLP of 0.4 g/L was achieved in a submerged culture of G.lucidum under low nitrogen concentration?5 mM of C₄H₇NO₄?,which was 23.21%higher yield than that obtained under high nitrogen concentration?20 mM of C₄H₇NO₄?.qRT-PCR further confermed that the expression levels of key genes of glucokinase,?-phosphoglucomutase,UDP-glucose pyrop-hosphorylase and 1,3-?-glucan synthase involved in polysaccharide biosynthesis were upregulated by 30%,35%,16.67%and 17.85%respectivly due to the nitrogen limitation.?2?A G.lucidum polysaccharide GLP-1-1 was isolated from a culture broth with Mw of 22014 Da.Monosaccharide contained glucose,mannose,and galactose with mole percentages of 92.33%,7.55%,and 0.22%,respectively.Moreover,FTIR and methylation analysis showed that Mannose had only one linkage pattern of Man(1-and was the terminal sugar in GLP-1-1 and glucose was the backbone and branching residue sugar in GLP-1-1.The results of antioxidant activity analysis showed that GLP-1-1 had a great DPPH and ABTS radical scavenging activity.Meanwhile,GLP-1-1 also exhibited anti-tumor activity to A431 and MDA-MB-231 cells,and inhibitory rates were dose-dependent.During culturing with GLP-1-1,the G₁/G₀ cell percentage of A431 cells and MDA-MB-231 cells were increased by 73.77%and 28.6%respectively.Therefore,the anti-tumor activity of GLP-1-1may be caused by inducing the G₁/G₀ arrest of tumor cells.?3?Ganoderma lucidum polysaccharide nanoparticles?GLPN?were prepared by ball milling method.The physicochemical properities of GLPN were characterized by AFM and Zetasizer in detail.It showed that nanoparticles exhibited surface morphology and arrays forms.GLPN were characteristic of little particle size,narrow size distribution and high zeta potential.Average size was 465nm.?4?Incorporation of GLPN significantly influenced the pasting properties and farinograph characteristics of the dough,as well as texture,sensory properties and flavor of buns.In contrast to the control group,supplementation with 5%GLPN makes the values of the water absorption,the development time,the stability time and FQN?FU?increased by5.49%,30.27%,57.69%and 78.38%,while the degree of softening decreased by 27.27%;The values of peak viscosity,trough viscosity,breakdown,final viscosity,setback and peak time decreased by 17.09%,10.12%,8.45%,16.88%,19.31%and 8.24%,while the pasting temperature increased by 4.94%.The addition of 5%GLPN significantly increased the hardness,chewiness and gumminess by 9.63%,23.38%,36.19%,while the adhesiveness and springiness decreased by 19.51%and 5.43%.Sensory evaluation showed that the addition of GLPN increased the concentrations of flavor compounds,which enhanced the smells and tastes of the resulting buns.According to the result of mice feeding experiment,blood glucose of the mice feeding with GLPN would attain peak 30 mins later than that of control group and average peak value decreased by 14.03%,14.25%,12.04%.The results indicate that GLPN buns could delay the onset of blood glucose peak and effectively control the blood glucose.