Effect Of Ultrasound-assisted Ganoderma Lucidum Liquid Fermentation On Its Growth And Metabolism And Its Mechanism Research

Ganoderma lucidum(G.lucidum) is a well-known fungus with rich active ingredients.In recent years,with the completion of the whole genome sequencing of G.lucidum,the establishment of transgenic system,the improvement of reverse genetic operation system,and its own unique secondary metabolism,it has become a potential model basidiomycete.G.lucidum’s liquid fermentation technology has the advantages of short cycle time,low cost,high yield,and is conducive to industrialization.However,the contents and types of fermentation mycelium important active ingredient,triterpenoids,are lower than those of fruiting bodies.This research intended to perform ultrasonic treatment in the G.lucidum fermentation process,and improved its fermentation efficiency through the method of low-intensity physics.In this study,the use of divergent ultrasound equipment to assist the fermentation of G.lucidum,and to compare the cell levels and physical and chemical differences of mycelia before and after ultrasonic treatment,then analyzed the effect mechanism of ultrasonic treatment on the fermentation efficiency of G.lucidum.This study used metabolomics analysis to clarify the effect of ultrasound on the final metabolites of mycelium,and transcriptomics to analyze the effect of ultrasound on global gene expression of G.lucidum.The involved molecular mechanisms of ultrasound promoting the growth and metabolism of G.lucidum were investigated for the first time using transcriptome and metabolome in this study.The main research contents and results of this research were as follows:(1)Research on low-intensity ultrasound to promote the fermentation of G.lucidum.Using mycelium biomass and triterpenoids production as indicators,the effects of different ultrasonic parameters on G.lucidum liquid fermentation were studied.Raman spectroscopy,scanning electron microscopy and intracellular Fluo-4/AM probe method were used to study the effect of ultrasonic treatment on the cell structure of G.lucidum.The results showed that the best ultrasonic treatment conditions were: treatment in the logarithmic growth phase(96 h and 120 h after inoculation)for 15 min respectively,ultrasonic frequency of 28±2 k Hz,and power density of 15 W/L.Compared with the control group,the mycelial biomass and triterpenoids production increased by 26.99% and 33.62%(p<0.05),and the intracellular and extracellular polysaccharide contents increased by 18.48% and 35.90%(p<0.05),respectively.The contents of flavonoids inside and outside the cells increased by 30.27% and 12.00%,respectively(p<0.05).The results of the study showed that ultrasonic treatment promoted the growth and metabolism of G.lucidum.The macro-structure results of G.lucidum cells showed that ultrasonic treatment can change the content or structure of polysaccharides,fatty acids and amino acids in the mycelium.Microscopic results showed that after the ultrasonic treatment,the mycelium was partially broken and the contents leaked at recovery time of 0.5 h,and at recovery time of 3 h,the mycelial part was recovered,and the mycelium branches increased and the winding was tighter.Fluo-4/AM probe method observed that the intracellular calcium ion concentration in the mycelium increased after treatment,indicating that ultrasonic treatment changed the cell membrane permeability of G.lucidum.(2)Study the effect of ultrasonic treatment on the metabolites of G.lucidum fermentation through metabolomics.A total of 857 different metabolites(578up-regulated and 279 down-regulated)were identified by LC-MS non-targeted metabolomics.Up-regulated metabolites were more than down-regulated,indicating that ultrasonic treatment induced the synthesis of a large number of metabolites.These differential metabolites were enriched in multiple KEGG metabolic pathways such as amino acid metabolism,sugar metabolism,terpenoid biosynthesis,phospholipid and sphingolipid metabolism,and transporter pathways,etc,indicating that the complex metabolic pathways of cells would undergo complex changes in response to external stress after sonication.To sum up,ultrasonic treatment can significantly change the metabolic level of G.lucidum.(3)Analyze the effect of ultrasonic treatment on the gene expression profile of G.lucidum by transcriptomics.Compared with the control group,569 and 932 differentially expressed genes(DEGs)were identified after 0.5 h and 3 h of recovery after sonication,respectively,and 334 DEGs continued to change during the recovery time of 3 h.The results showed that ultrasonic treatment can significantly change the G.lucidum at the transcription level,and had a certain lasting effect.The identified DEGs were enriched into the corresponding GO function analysis and KEGG metabolic pathways.The results showed that the ultrasonic treatment group and the control group had significant differences in biological processes,cell composition and molecular function.Cytochrome P450,sulfur,carbon,amino acids,fatty acid metabolism,terpenoid biosynthesis,and sphingolipid signaling pathways were all involved in the response of G.lucidum to ultrasound.(4)Combined analysis of transcriptome and metabolome showed that the identified differential genes and metabolites mainly involve the following pathways:carbohydrate metabolism,energy metabolism,biosynthesis and metabolism of amino acids and terpenoids,membrane transport,membrane composition composition,cell wall,signal transmission,ROS removal and heat shock protein expression,etc.The results showed that ultrasonic treatment can promote the induction of primary metabolism,secondary metabolism,membrane structure and membrane properties,cell wall,antioxidant mechanism regulation and differential expression of thermal effects of G.lucidum,thereby synergistically affecting the growth and metabolism of G.lucidum.In summary,ultrasonic treatment can be used as an effective method to promote the liquid fermentation of G.lucidum.