Study On Physical Mutagenesis And Ultrasound Assisted Fermentation Of Phellinus Igniarius

Phellinus igniarius(P.igniarius)generally called Sanghuangjun in Chinsese,is a rare medicinal fungi,which has a unique medicinal value and a wide range of biological activity.Polysaccharide is the main active component of P.igniarius.To improve the yield of polysaccharides by Phellinus igniarius,a method of rational screening of low power He-Ne laser and Ultraviolet(UV)-induced mutants was employed in this work.Preliminary properties,chemical compositions,biological activity and SEM images of P.igniarius mycelia polysaccharides were studied.Meanwhile,we studied the mutation mechanism using two-dimensional electrophoresis technology.The effect of ultrasound on P.igniarius mycelia fermentation was examined and the mechanism was discussed preliminarily.The main research work and conclusions are as follow:(1)The optimization of protoplast preparation condition.The optimal preparation conditions of protoplast from P.igniarius mycelia as follows:mycelial age of P.igniarius mycelia was 10d,1%lywallzyme and 0.25%driselase as enzymatic reaction system,enzymolysis temperature 30℃,enzymolysis time 3h.(2)The selection of mutagenesis method and mutation.The positive mutation rate of composite irradiation of He-Ne laser and UV and only UV irradiation were compared.The result showed that the positive mutation rate of protoplast from P.igniarius by composite irradiation of He-Ne laser and UV was over 2 times higher than by UV only.P.igniarius protoplasts were irradiated by low energy He-Ne laser combined with UV and 9 mutant strains were selected after five generations.The mutant strain JZx possessed the best producing ability and genetic stability.In shake flasks,dry mycelia weight derived from mutant JZx fermentation was 1.40-fold from control and endo-polysaccharides production 1.57-fold,respectively.(3)Identification of dominant mutant strain.The antagonistic experiment and isozyme electropheresis spectrogram analysis were employed to identify mutants.The results showed that genetic material of the screened mutants changed.The analysis of isozyme electropheresis spectrogram indicated that this novel mutation method might expand the scope of the mutational range for P.igniarius.(4)Comparison of the mycelium polysaccharide physicochemical properties,molecular structure and biological activity form JZx and CK strains.Compared with CK,JZx contains higher content of total sugar and uronic acid.The polysaccharide from CK and JZx basically composed of L-rhamnose,D-glucose and a small amount of D-Mannose,in a molar ratio of 9:1:3 and 2:1:16,respectively.Molecular weight distribution results showed that the polysaccharide from JZx contained more polysaccharide fragment with low molecular weight(1.5kDa,61%)compared to control.Infrared spectroscopy indicated that the laser and UV mutation caused the fracture of glycosidic bond but did not affect the main chemical structure of polysaccharide.Polysaccharide from JZx exhibited higher scavenging ability of hydroxyl free radical compared with the control.It was obvious that polysaccharide from JZx showed the higher antioxidant activities with TEAC value of 195.43μmol Trolox/g sample and FRAP value of 20.57μmol Fe2+/g sample,compared with the control increased 32.44%and 22.8%,respectively.The result of determination of anti HepG2 in vitro activity of polysaccharide showed that polysaccharide from JZx had more obvious inhibitory effect on HepG2 than control.(5)The comparison of solid polysaccharides morphology from JZx and CK mycelium was investigated.The solid state morphology of polysaccharide was also observed by scanning electron microscopy(SEM).The result showed that the morphology of polysaccharide changed between JZx and the control,polysaccharide polymer sheet and the aperture size of JZx were smaller than CK.(6)The separation and purification of endo-polysaccharides from JZx.We got two polysaccharide fraction PIPS1 and PIPS2 from JZx mycelium polysaccharide using DEAE-52—Sepharose Fast Flow ion exchange chromatography.The polysaccharide contents of PIPS1 and PIPS2 were high,about 99%,and the content of protein and uronic acid were very small,indicating that PIPS1 and PIPS2 in high purity.PIPS1 and PIPS2 mainly composed of D-glucose,L-rhamnose,and a small amount of D-Mannose,in the mole ratio of 4:9:1 and 3:7:1,respectively.PIPS 1 contained more polysaccharide fragments with low molecular weight(3.1 KDa,93%).(7)Study on the mutation mechanism.With total protein of mycelia from JZx and CK strains as the research object,20 differential protein spots were obtained using two-dimensional electrophoresis and TOF-MS/MS.The results revealed that there were 4 protein spots related with sugar metabolism,3 protein spots might involved in synthetic macromolecules in cell,2 protein spots were identified as molecular chaperone,1 protein spot might involved in spore germination.The results suggested that external stress factors such as laser and UV could induce the metabolism of P.igniarius cell changed and high expression of molecular chaperones and the protein involve in spore germination.(8)Ultrasonic assisted P.igniarius liquid fermentation.In this study,a novel flat-plate ultrasound technology was developed to stimulate polysaccharides production from Phellinus igniarius mycelial fermentation.Three-factor-three-level Box-Behnken design was used to optimize the fermentation time applying ultrasound(culture time),ultrasonic treatment time,duty cycle time and culture time for a high yield of P.igniarius polysaccharides(PIPS).Optimal conditions were found to be culture time 3.8 d,ultrasound treatment time 65 min and duty cycle time 25 s,and that gave a maximum PIPS yield of 1.8002 g/L,which increased～22.64%compared with the control(without any ultrasound).(9)Comparison of the mycelium polysaccharide physicochemical properties,molecular structure and biological activity before and after ultrasonic reaction.PIPS mainly contained low-molecular weight(MW)polysaccharides(3.1 kDa,80%)composed of glucose,rhamnose and mannose in a molar ratio of 11.0:14.0:1.0.PIPS with higher carbohydrate and uronic acid contents exhibited strong antioxidant activities in vitro.(10)The effect of ultrasound on P.igniarius mycelia morphology.Laser scanning confocal microscope(LSCM)was used to observe the changes of mycelia morphology before and after the ultrasound treatment.The result suggested that ultrasound could change the morphology and structure of P.igniarius mycelium,and accelerate the transfer of nutrients and metabolites.