Study On Physical Mutation And Fermentation From Phellinus Igniarius

In order to exploit and utilize Phellinus igniarius as national resources of Chinese medicinal materials farther and wider,the research status of biological characteristics, composition and pharmacological action of Phellinus igniarius was reviewed in this paper.Based on analysis of literatures and recent researches,selective breeding of fermentation strains,fermentation technology,polysaccharide extraction and activities of the polysaccharide were studied.Phellinus igniarius growed slowly in the process of culture,Conidium was difficult to induce,so protoplast only can be separated from mycelium.The suitable conditions of protoplast separation and regeneration were as follows:mycelial age 10d, mannitol as osmotic pressure stabilizer on separation and regeneration,1.5% lywallzyme and 0.5%driselase as reactive synthase,enzymatic temperature 30℃, enzymatic time 3h,improved PDA as regeneration culture medium.With LA and LA-UV mutation from four mutant modes,average fermentation yields of screened mutant strains were 11.57±1.86mg/mL and 8.52±1.52mg/mL respectively,which were better than others,further the positive mutation rates with LA and LA-UV mutation were 6.08%and 5.52%respectively.The strains with LA-UV mutation produced larger variation and had great increasing potential,so LA-UV mutant mode was applied to large-scale screening.The S2 strain with UV mutation was selected,which had faster growth rate,then the protoplast of S2 strain was mutated with LA-UV,through five generation screening,five mutant strains were selected,Results showed that the five mutant strains had good genetic stability,they also had antagonism to the original strains,and the electrophoretograms of esterase and peroxidase were changed.The analytical methods of fermentation yield on intracellular and extracellular polysaccharide from Phellinus igniarius were established.The fermentation yields of polysaccharide from all selected strains were determined,then results showed that the fermentation yield of polysaccharide from SJZ2 strain,which were highest,increased by 36.88%than original strain.The culture medium of SJZ2 was screened and optimized.The results showed that the best carbon source was wheat flour,the best nitrogen source was rice bran,the optimum conditions were as follows:wheat flour 5.16%,rice bran 1.38%,KH₂PO₄ 0.094%,MgSO₄ 0.054%,under these conditons,the factors influencing fermentation yield were in the order as follows:rice bran content＞wheat flour content＞KH₂PO₄ content＞MgSO₄ content,and regression model was well predictable,which had been confirmed by verification experiment.The single factor experiments of shaking flask that had an effect on fermentation yield were studied.The results showed that the optimum conditions were as follows: media amount 120/250（mL/mL）,the inoculum size 17mL,temperature 26℃,rotate speed 135r/min,under these conditions,the factors influencing fermentation yield were in the order as follows:temperature＞inoculum size＞rotate speed＞media amount, and the regression model was well predictable,which had been confirmed by verification experiment.The kinetic changes of mycelial yield,soluble protein content,polysaccharide content,viscosity,LiP,MnP,Lac,carboxymethyl cellulase and filter paper cellulase were studied in the process of shaking flask fermentation,then michaelis constant and maximum reaction speed of LiP,MnP,Lac were determined.Base on shaking flask fermentation,fermentor experiments were studied.The results showed that the optimum conditions of fermentation were as follows:media amount 70/100（mL/mL）,inoculation volume 10%,stirring speed 180r/min, temperature 29℃,air flow 1:0.6v/v.m,under these conditions,3.5L fermentation culture medium could produce mycelium 68.23±2.51g（n=3）,and extracellular polysaccharide 10.23±0.35g（n=3）.Polysaccharides from Phellinus igniarius were extracted with compound enzymes. Different enzymes were screened and compound,The screened enzymes for extraction were cellulase,helicase,lywallzyme and neutrase（3:1:0.5:4）.Base on single factor tests,extraction conditions were optimized.The results showed that the optimum conditions of polysaccharide extracting rate were as follows:pH 6.5,temperature 40.9℃,extraction time 3.78h,enzyme quantity 3.37%,the optimum conditions of polysaccharide purity were as follows:pH 6.7,temperature 34.5℃,extraction time 2.35h, enzyme quantity 2.57%,under these conditions,the factors influencing polysaccharide extracting rate were in the order as follows:pH＞enzyme quantity＞temperature＞extraction time,factors influencing polysaccharide purity were in the order as follows: extraction time＞pH＞temperature＞enzyme quantity,and the regression model was well predictable,which had been confirmed by verification experiment.The part physicochemical properties of PIPS were studied.The results showed that the reaction of I-KI reagent was negative,that ofα-naphthol reagent was positive,that of Folin’s reagent was negative,inherent viscosity of EPS at 25℃was 0.85dL/g,and inherent viscosity of IPS at 25℃was 1.2dL/g.Through fractionation of PIPS,EPS_0.1and EPS_0.2were obtained from EPS,eight fractions were obtained from IPS such as IPS_0.01,IPS_0.02,IPS_0.1and so on,and the monosaccharide composition of PIPS and part fractions were determined.The results showed that the monosaccharide composition and molar ratio of IPS were 1（Xyl）:50（Rha）:33（Ara）:225（Man）:375（Glu）,those of IPS_0.1were 10（Rha）:33（Man）:70 （Glu）,those of IPS_0.01were 9（Man）:5（Glu）,those of EPS were 1（Gal）:35（Man）:35（Glu）, those of EPS_0.1were 7（Man）:5（Glu）,and those of EPS_0.2were 13（Man）:5（Glu）.Antioxidative activities of polysaccharide from Phellinus igniarius showed better dose-effect relationship.Through the curvilinear regression computation,the result showed that the EC₅₀of IPS on DPPH radical scavenging capacity was 1.09mg/mL,EPS was 1.52mg/mL,the EC₅₀of IPS on hydroxyl radical scavenging capacity was 0.78mg/mL,EPS was 0.23mg/mL,the EC₅₀of IPS on Superoxide Anions scavenging capacity was 29.97μg/mL,EPS was 20.31μg/mL,the EC₅₀of IPS on Fe²⁺ Chelation was 1.36mg/mL,EPS was 1.66mg/mL,under these conditions,the reduction activity of IPS was y=1.3023x+0.0178（R²=0.9991）,EPS was y=1.8004x+0.0185 （R2=0.9992）.