Studies On Grifolan And Enriched Organic Selenium From Grifola Frondosa Liquid Cultured In Rice Bran Substrate

Grifola frondosa is a edible and medicinal fungus which has attracted extensive researches in recent years.Grifolan from Grifola frondosa have the function of adjuvant therapy of antitumer and immunity enhancement.Selenium biotransformed by Grifola frondosa has the advantage of low toxicity,and also can enhance the immunity.As the by-product of the rice milling process,rice bran is in a large yield and need more application to improve its value.This research adopted liquid fermentation of Grifola frondosa to produce grifolan and enrich organic Selenium,as a novel way and a new technology,which solved problems of grifolan production, organic Selenium biotransformation and the further application of rice bran together. The results provided the foundation for the industrial production of special health food by a large-scale use of cheap raw material.Thus,this research has a definite value on theory and practice.The research contented:(1) The feasiblity of liquid fermentation to produce grifolan on the mediem with rice bran as the main component was studied,as well as the application mode of rice bran and the composition of rice bran medium.Grifola frondosa induced by Ultraviolet irradiation which could grow well on rice bran mediems was applied as the strain.Based on the basic medium,which contained potato 200g,KH₂PO₄ 1.5g/L, MgSO₄ 0.75g/L,V_B1 10mg/L,single factor experimentations were made on carbon source,nitrogen source,addition level of glucose(0.5-2.5%) and rice bran(1-5%,wt). Then,L₉(3⁴) orthogonal experiments,with the dry weight of mycelium and yield of polysaccharides as criteria,were performed among the addition level of glucose (1-2%),rice bran(4-6%) and peptone(0.3-0.5%).The optimum culture medium were established as follows:rice bran 40-60g/L,glucose 15g/L,peptone 3-4g/L,with intracellular polysaccharide 281.3mg/100mL,the dry weight of mycelium 1246.7mg/100mL.Results showed that rice bran could be an alternative of glucose to decrease the production cost.Basic medium could be made without potato,and the different application style played a relatively unimportant role on the yield of intracellular polysaccharide.(2) Exterior conditions of liquid fermentation to produce grifolan on the optimized mediem with rice bran as the main component was studied.The effects of inoculation volume(4-12mL),loading volume(50-150mL),culture time(3-8d), temperature(22-34℃) 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 28℃,inoculation volume 8%,rotation speed 120-150r/min,fermentation time 5d and loading volume 20%(250mL conical flask).(3) The Selenium enrichment and grifolan production together in the liquid fermentation of Grifola frondosa on the mediem with rice bran as the main component was studied.Single factor experiments were performed with different Selenium addition level,and the Uniform Experimental U₁₂^*(12¹⁰) were designed to examine the relationship between Selenium,rice bran,glucose and treatment methods for rice bran,which got an analysis of regression model made by SPSS.Respectively, the regression equations of multiple linear regression and binary logistic regression are:Y₁=0.070 X₂+0.234 X₃+0.209;Y₂=0.024 X₂+0.087X₃+0.046; Y₃=0.034X₁X₃-0.009X₄+0.118;Y₄=0.003X₂+0.559X₁X₃-0.006X₂X₃+0.022. (Selenium X₁,rice bran X₂,glucose X₃,treatment methods for rice bran X₄,mycelium dry weight Y₁,polysaccharides contents Y₂,Selenium contents Y₃,Selenium transformation rate Y₄).(4) Scale-up experiment was performed in 18L fermenter,with loading volume (15L),fermentation temperature(28±1℃),ventilation volume(0.8v/v/min),stirring speed(150r/min),gage pressure(0.05MPa) and fermentation time(1-5d).The results of fermentation experiment showed that after 2 days,the max contents of crude polysaccharides in the mycelium can be obtained as 631mg/100mL.The metabolic process of Selenium enrichment and grifolan expanded production by Grifola frondosa was gained,which laid a foundation for the establishment of production parameters and control steps.(5) The optimum extraction technique of mycelium grifolan in Grifola frondosa had been studied.By means of single factor tests,L₁₆(2¹⁵) orthogonal experiments wwre performed.By the variance analysis and range analysis,the optimum of mycelial grifolan in Grifola frondosa was determined as follows:extracting temperature 75℃,extracting time 90min,extracting number 3,ethanol content 67v/v.(6) Isolation by DEAE ion-exchange cellulose(DEAE-52) and structure identification of crude grifolan were studied.Three refined grifolans were gained.The analysis of physicochemical property proved that the gained samples were polysaccharides without starch,amino acid,peptide,and protein.Infrared spectrum was scaned in the range of 4000-400cm^-1,and the analysis showed that grifolans refined have the characteristic peak of polysaccharides.