Study On The Growth And Nutrition Characteristics And The Extracellular Enzyme Activity Of Cordyceps Militaris

Cordyceps militaris,known as "Oriental holy grass",is a kind of valuable edible and medicinal fungus with medicinal and nourishing effect.Many studies show that C.militaris contains cordycepin,cordycepic acid,cordyceps polysaccharide,superoxide dismutase(SOD),vitamins,mineral elements and other active ingredients,which are far more than the famous and precious traditional Chinese medicine Cordyceps sinensis,C.militari,as a succedaneum for Cordyceps sinensis,is often used as medicine in clinical,therefore,the development prospects of the c.militaris is very promising.In this study,6 strains of C.militaris were used as the object for the study of molecular identification,mycelial culture characteristics respectively,screened out the dominant strains.The optimum liquid strain medium formula of C.militaris was optimized by orthogonal experimental design.Compared the growth difference of each strain during cultivation process and fruit body active ingredients,also studied the changes of extracellular enzyme activity of 6 strains in different growth periods.The aim of this study was to provide theoretical and technical basis for the selection of excellent C.militaris strains,optimization of culture conditions and large-scale cultivation.The main results were as follows:(1)In the screening test of 6 strains of C.militaris,It was found there were antagonisms between the two different strains and the same strain.The identification of ITS sequence and phylogenetic tree analysis of rDNA showed that all the 6 strains were C.Militaris,and the genetic relationship between them were close.Under the optimum culture condition,the mycelial growth rate of CM3 was the fastest,and the growth rate of CM8 was the slowest,but the growth potential of CM5 was the best.The mycelial Color-changed culture showed that CM4 and CM5 were faster and darker in colouring,CM8 couldn't turn color.Under the condition of liquid culture,the dehydrogenase activity of CM5 was the highest,and the lowest was CM8,CM5 mycelial biomass was the highest,pellet size uniform,density.In the liquid culture experiment for culturing mushroom,CM5,CM4 were orange,stroma was long and dense,fruiting tidiness consistent,CM8 didn't turn color and no fruiting.The comprehensive analysis indicated that CM5 and CM4 were quality strains of the 6 tested strains,CM1 was not as good as them,CM3 and CM2 were poor,and CM8 was a degenerate strain which couldn't produce mushrooms.(2)The dominant species CM5 was selected as the research object,the mycelium biomass was takenas the index to optimize the liquid culture medium.The results showed that the optimum carbon source was sucrose,and the optimum nitrogen source was yeast powder,the optimum medium formula was sucrose3%,yeast powder1%,KH2PO4 0.2% and MgSO4 0.2%.(3)The solid cultivation of the 6 strains and the detection of the active ingredients of the fruiting bodies showed that CM5,CM4 mycelium growed fast,white,thick,growing well,turned color fast,orange,primordium differentiation early,Polystromatic formation,their fruiting body is uniform and dense,the top of the fruiting body with papillary perithecia,orderly musheroom,no pollution,short growth cycle,good commercial character and high yield,CM8 was unable to turn color and produce fruiting body.The highest content of cordycepin in the fruiting bodies of 5 strains was CM4,the lowest was CM2,the highest content of both Cordyceps acid and Cordyceps polysaccharide was CM5,the lowest was CM1,in addition,a variety of other active ingredients were detected in the fruiting bodies of the 5 strains.(4)Among the three kinds of extracellular enzymes of the 6 tested strains,the highest activity was amylase,followed by protease,and the lowest was cellulase.The activity of cellulase and amylase in the strains with good colouring ability reached the peak value in the veraison period,the cellulose enzyme activity was positively correlated with the formation of stroma,the high amylase activity inhibited the normal differentiation of the stroma,but was positively correlated with the growth of the fruiting body.In the formative period of the stroma,strains with high protease activity,stroma differentiation was better,however,the strains with lower protease activity in the growth period of the fruiting body had higher yield,appears to be negatively correlated with it.The changes of cellulase,amylase and protease activity of CM4 and CM5 with good morphology and high yield were all exactly the same.Without color change and mushroom,activity of extracellular enzyme of CM8 changed little,and no apparent peak value occurred.