Study On Mutation Of Grifola Frondosa And Liquid Fermentation Of New Strain With Rice Bran And Wheat Bran Added Without Or With Selenium

China is an agricultural power, there are 10 million tons of rice bran and 30 million tons of wheat bran produced per year. It is important to develop a highly efficient and valuable new deep-processing technology of rice bran and wheat bran for raising competitive ability of Chinese agriculture and related enterprises.Grifola frondosa is a kind of edible and medicinal fungus, which has rich nutritional substances and has many physiological active functions, such as immunity adjustment, antitumor, liver protection. Grifola frondosa contained various enzymes to degrade rice bran and wheat bran. Liquid fermentation is a rapid and industrial scale production technology. For efficiently and valuably transforming rice bran and wheat bran, the present dissertation aimed to using the mutated Grifola frondosa strain to ferment whole rice bran and wheat bran, added without or with selenium, to obtain target materials such as mycelia, mycelial polysaccharides, mycelia rich with selenium, and mycelial polysaccharides rich with selenium. Hence, mutation of Grifola frondosa strain, fermentation technologies, fermentation dynamics, fermentation technology for enriching selenium and dynamics of enriching selenium were well investigated. The main results and conclusions are as follows.(1) For obtaining new better Grifola frondosa strain to degrade rice bran and wheat bran, ultraviolet and microwave were used to mutate Grifola frondosa CCTCC M2011113. An UV mutated new strain Grifola frondosa JSULIU-Dl and a microwave mutated new strain Grifola frondosa JSULIU-D2 was obtained respectively by 10-time UV mutation or microwave mutation and preliminary screening, secondary screening and shaker fermentation screening. When fermenting in 100mL medium with rice bran and wheat bran, the mycelial dry weight of Grifola frondosa JSULIU-Dl is 1.7g, and mycelial polysaccharides is 110mg, while the mycelial dry weight of Grifola frondosa JSULIU-D2 is 1.6g, and mycelial polysaccharides is 135mg.The new two strains has rapid growth rate of 1.02mm/h, and mycelial polysaccharide is 20% or 50% more than that of original strain respectively, but UV mutation strain’s fermentation is thoroughgoing and better.(2) Fermentation parameters of Grifola frondosa JSULIU-Dl were investigated including ten single-factor experiments, optimization of medium components, and fermentation of 10L,50L,500L. The ten factors with their level ranges were set as rotation speed of 60-210r/min, temperature of 16-36℃,rice bran of 20-100g/L,wheat bran 20-100g/L, KH2PO4 of 0-3.6g/L, MgSO4.7H2O of 0-3.6 g/L, inoculation volume ratio of 4-20%, pH of 3-11, loading volume of 50-200 mL in 250 mL conical flask, fermentation time of 2-12 d. Six criteria by 100mL fermentation volume were employed for every single-factor experiment, they were mycelial dry weight, residue dry weight, loss weight of dry materials of substrates, mycelial polysaccharides weight, exo-polysaccharides weight, total sugar weight. Sixty curves were obtained to guide selecting fermentation parameters.With U15 (54) uniform design, rice bran of 10-50g/L, wheat bran of 10-30g/L, KH2PO4 of 0-3.6g/L, MgSO4.7H2O of 0-1.6 g/L were optimized by determining the mycelia weight, loss weight of substrates, mycelial polysaccharide weight, exo-polysaccharide weight and total sugar weight. Five model equations of five criteria were obtained. The optimum media consists of rice bran of 50g/L, wheat bran of 30g/L, MgSO4.7H2O of 0.8 g/L. Other fermentation parameters were selected as inoculation volume ratio of 10%, temperature of 28℃, rotation speed of 150 r/min, natural pH, loading volume of 100 mL in 250 mL conical flask, fermentation time of 9 d after considering the reasonable economy and technology. Result showed that the weight concentrations of dry mycelium, loss of substrates, mycelial polysaccharide, exo-polysaccharide and total sugar were obtained as 30.9 g/L,25.5g/L,0.90g/L,2.914g/L, and 3.815g/L, respectively.The summary tables of input and output in the fermentor of 10L,50L and 500L were obtained. For the fermentation of 500L scale, the media compositions included rice bran of 74.34g/L, wheat bran of 9.66g/L, KH2PO4 of 3.6g/L, MgSO4.7H2O of 3.6 g/L. The output is dry mycelia of 17.0kg and exocell material of 11.0kg, by 1m3 liquid volume of media.(3) The uniform design U24(245) was used to optimize the weight of rice bran, wheat bran,KH2PO4,MgSO4.7H2O in original media of 100mL and time based on the mycelial dry weight of Grifola frondosa JSULIU-D1. The empirical dynamic equation was also obtained. The optimum parameters were as follows:rice bran of 48.0g/L, wheat bran of 34.4g/L, KH2PO4 of 3.6 g/L, MgSO4.7H2O of 1.8g/L, inoculation volume ratio of 10%, temperature of 28C, rotation speed of 150r/min, natural pH, loading volume of 100 mL in 250 mL flask, fermentation time of 5.44 d. The maximal mycelial dry weight concentration was 23.47±1.32g/L.(4) With U2o(2O4) design table, based on the mycelium empirical dynamic equation of (3), mycelial dry weight in original media of 100mL at different time were predicted for 20 experiments of uniform design.The theoretical dynamic equation of Logistic model equation and the regression equations of its parameters of μm and Cmax with the weight of rice bran,wheat bran,KH2PO4,MgSO4.7H2O were obtained.(5) With U24(245) design table, uniform design experiments were done with factors of weight of rice bran (quantity of wheat bran per liter decided by 86.25 minus the quantity of rice bran), selenium, KH2PO4,MgSO4.7H2O in original media of 100mL and time. The weight of dry mycelium, residue, loss of substrates, mycelial polysaccharide, exo-polysaccharides, total sugar, mycelial selenium, and selenium weight per kg mycelium and transformation ratio of selenium were determined. The weight of mycelium, mycelial polysaccharide, exo-polysaccharide, and mycelial selenium were used to build four model equations of Grifola frondasa JSULIU-D1 of fermenting liquid whole rice bran and wheat bran with selenium. The suitable parameters were obtained as:rice bran of 86.25g/L(may be replaced by wheat bran less than 86.25g of 25%),wheat bran of Og/L,selenium of 0.319mg/L, KH2PO4 of 0.15 g/L, MgSO47H2O of 0.15g/L, inoculation volume ratio of 10%, temperature of 28C,rotation speed of 150r/min, natural pH, loading volume of 100 mL in 250 mL conical flask, fermentation time of 9 d, and corresponding mycelial dry weight concentration of 35.44 g/L, mycelial polysaccharides weight concentration of 1.931g/L, mycelial selenium weight concentration of 1.546mg/L, exo-polysaccharides weight concentration of 2.43mg/L were obtained.(6) Four criteria including weight of dry mycelium, mycelial polysaccharide, exo-polysaccharide, mycelial selenium were used to estimate four optimum factors of Grifola frondosa JSULIU-Dl to ferment liquid whole rice bran and wheat bran with selenium. Under the conditions of inoculation volume ratio of 10%, temperature of 28C,rotation ratio of 150r/min, natural pH, loading volume of 100 mL in 250 mL conical flask, four optimum technologies were as follows:maximum mycelial dry weight concentration of 42.48 g/L,mycelial selenium concentration of 1.54mg/L,rice bran of 86.25g/L,wheat bran of 0g/L,selenium of 0.319mg/L, KH2PO4 of 3.6 g/L, MgSO4.7H2O of 3.6g/L, fermentation time of 8.93 d; maximum mycelial polysaccharides concentration of 2.655g/L, rice bran of 86.25g/L,wheat bran of Og/L, selenium of 5.182mg/L, KH2PO4 of 0.15 g/L, MgSO4.7H2O of 0.15g/L, fermentation time of 6.11 d; maximum exo-polysaccharides concentration of 9.231 g/L, rice bran of 0g/L,wheat bran of 86.25g/L,selenium of 7.65mg/L, KH2PO4 of 0.15 g/L, MgSO4.7H2O of 3.6g/L, fermentation time of 12 d; maximum mycelial selenium concentration of 3.995mg/L, rice bran of 86.25g/L,wheat bran of 0g/L,selenium of 7.65mg/L, KH2PO4 of 0.15 g/L, MgSO4.7H20 of 0.15g/L, fermentation time of 12 d.(7) With U20(2O4) design table, based on the mycelial dry weight empirical dynamic equation of (5), mycelial dry weight in original media of 100mL at different time were predicted for twenty experiments of uniform design. The theoretical dynamic equations of Logistic model equation and the regression equations of its parameters of μm and Cmax with weight of rice bran, wheat bran, selenium, KH2PO4, MgSO4.7H2O were obtained.(8) Freeze-drying mycelia were pulverized with nano-machinery and analyzed by scanning electrical microscope (SEM) to check the total broken cell wall, which provided basis of taking mycelial powder directly. The selenium and residual heavy metals of Pb, Hg, Cd in mycelium were analyzed with inductive couple plasm mass spectrometer (ICP-MS). Residual heavy metals are basically close to the requirements of National Standards, which indicated that raw rice bran and wheat bran must be selected strictly. Free amino acids were analyzed with amino acid analyzer. The contain of free amino acids is 80-100g per kg dry mycelia, and ratio of 8 essential amino acids is about 50% of total amino acid.