Study On Grifola Frondosa’s Physical Mutagenesis And Liquid Fermentation For Polysaccharide In Rice And Wheat Bran

Grifola frondosas are edible and medicinal fungus which have attracted extensive researches in recent years. Polysaccharides from Grifola frondosas have the function of adjuvant therapy of tumour and immunity enhancement. The Grifola frondosas which could grow fast in the culture medium that mostly involved rice and wheat bran have been chosen and we have investigated its optimum component proportion of culture medium and the best fermentation conditions. The new technological system will be established and be the basis of extensive usage for rice and wheat bran.(1) In order to enhance the successful odds, we have investigated the best conditions of mutation as follows:The best UV-mutation time is 30s, lethality rate is 94.79%. The best microwave mutation time is 10s, lethality rate is 82.76%. After the stability experiment for five generations, fermentation for three generations, we choose Grifola frondosas UVW-1 which is fast grow and high yield of polysaccharide. The result accords with the grey relational grade analysis. Quickly distinguishes by the special medium, we get the Grifola frondosas UVW-5 which can make use of cellulose fast. It is helpful to research the solid state fermentation for the second use of rice and wheat bran. Identifies and analyzes by the isozyme electrophoresis, we have proved that there are some genetic material different between the old Grifola frondosas and UVW-1, UVW-5.(2) After single factor experimentations of enzyme degradation on cellulose, the Uniform Experimental U12*(1210) were designed to examine the relationship between rice bran and wheat bran. Finally we choose two optimum component proportion of culture medium as follows:rice bran 100g/L, wheat bran 110g/L, with intracellular polysaccharide 146.48mg/100mL, extracellular polysaccharide 124mg/100mL and the dry weight of mycelium 0.992g/100mL; rice bran 30g/L, wheat bran 20g/L, with intracellular polysaccharide 118.79mg/100mL, extracellular polysaccharide 16mg/100mL and the dry weight of mycelium 0.843g/100mL. The fermentation production in the second culture medium is similar to the glucose (3%) culture medium, it is more close to the goal which rice and wheat bran replaces glucose. On the basis of it, we establish the growth dynamic model of UVW-1.(3) In order to meet the requirement of research, we have investigated other culture mediums and treatment methods for rice and wheat bran. With the dry weight of mycelium and yield of polysaccharides as criteria, single factor experimentations were designed to carbon source, nitrogen source, different addition level of glucose and peptone. Then, the Uniform Experimental U12*(1210) were designed to examine the relationship between glucose (0-5%), rice bran (1-12%), peptone (0-0.5%), wheat bran (1-12%), KH2PO4 (0.02-0.35%) and MgSO4 (0.01-0.23%) After intuitionistic and SPSS analysis, the optimum culture medium were established as follows:rice bran 20g/L, glucose 30g/L, peptone 1g/L, wheat bran 110g/L, KH2PO4 1.1 g/L, MgSO4 1.5g/L,with intracellular polysaccharide 59mg/100mL, extracellular polysaccharide 119mg/100mL and the dry weight of mycelium 0.9225g/100mL; rice bran 100g/L,wheat bran 120g/L,KH2PO4 2.3g/L, MgSO4 1.1g/L,with intracellular polysaccharide 61mg/100mL, extracellular polysaccharide 235mg/100mL and the dry weight of mycelium 0.704g/100mL.It is indicated that the fermentation of UVW-1 in rice and wheat bran culture medium without glucose is better. Taking all these factors into consideration, we presume that glucose can be replaced by rice and wheat bran.(4) Exterior conditions of liquid fermentation to produce polysaccharide on the optimized culture medium with rice and wheat bran as the main component was studied. The effects of loading volume (50-150mL), pH (4-8), temperature (20-36℃) and rotation speed (90-210r/min) on dry weight of mycelium and yield of polysaccharides were studied in single factor experimentations. The optimum culture external conditions were established as follows:fermentation temperature 24-28℃, pH 6-7, rotation speed 90-210r/min and loading volume 50%(250mL conical flask).(5) Whether the grown of UVW-1 in the culture medium of rice and wheat bran is better, Scale-up experiment was performed in 20L fermenter to test, with loading volume (18L), fermentation temperature (28±1℃), ventilation volume (0.8v/v/min), stirring speed (150r/min), gage pressure (0.05MPa) and fermentation time (96h). Compare the different index change of UVW-1 with the old Grifola frondosas. The results of fermentation experiment showed that after 56h, the max contents of polysaccharides and dry weight of mycelium can be obtained as 81.27mg/100mL and 744 mg/100mL. After 96h fermentation, the dry weight of mycelium and intracellular polysaccharide has enhanced 39.24% and 42.58% individually. It is indicated that the UVW-1 is good at growing in the culture medium of rice and wheat bran. However.The metabolic process of polysaccharide expanded production by Grifola frondosas was gained, which laid a foundation for the establishment of production parameters and control steps.(6)The fermentation liquids which acquired in different fermentation time of UVW-1 has been scanned by NIR spectra. Take different indexes as chemical value, we establish the PLS model of reducing Sugar, the fitting coefficient is 96.98% with the calibration set is ten, the prediction set is five. It laid a foundation for the establishment of automatic control and fast forecast the process of fermentation.