Study On Optimization Of Fermentation Conditions And Biochemical Properties Of The Fermentation Broths For Two Nematode-trapping Fungi

It is generally believed that the killing effect of nematode-trapping fungi on nematodes is due to the degradation of the nematode cuticle by the various proteases excreted by the fungi,so that the hyphae can penetrate the body and kill the nematodes.Therefore,it is of a great significance to study the biochemical substances produced by the nematode-trapping fungi in the process of trapping and killing nematodes,which will be of great help for the use of the nematode-trapping fungi to control parasitic nematode disease in future.Based on the preparation of spore suspension of two nematode-trapping fungi,different dispersants and media were used to screen the best medium for each the nematode-trapping fungus.Then,the optimization of the culture medium was prepared.The components of the fermentation broths from nematode-trapping fungi-Duddingtonia flagrans and Arthrobotrys oligospora under different conditions were analyzed and compared.The results of the relevant studies were as follows:(1)Through the comparison of the amounts of spores and appearance of the two kinds of spore suspension of different fungus,the PDA medium with high sporulation ability,easy collecting and fewer impurities was screened out to be the initial culture medium for the two kinds of liquid fermentation.(2)In two kinds of basic liquid fermentation media,different concentrations of pectin,gelatin and sodium carboxymethyl cellulose were added,and the optimal combination of the ingredients was obtained to produce extracellular protease by changing the growth state of mycelial during liquid fermentation.Finally,2 g/L gelatin LMZ liquid medium was selected as the optimized medium for two kinds of fungi.(3)The 3rd larvae of Strongylus and complex amino acids(DL-valine:L-phenylalanine =1:1)were used to stimulate the nematode-trapping fungi in the fermentation broth with fermentation broth treated by distilled water as the control;Then analysis and comparison was carried out to the biochemical components of three corresponding fermentation liquid of two kinds of fungi.The results showed that the total amounts of amino acids in the amino acid-induced and distilled water groups were higher than that in the Strongylus larvae group;the crude protein and total sugar content were the highest in the distilled water group and the lowest in the larvae group.Among three different treatments,the amounts of amino acids of D.flagrans treatments were lower than those of A.oligospora,but the total sugar contents were higher than that of A.oligospora.The content of crude protein in D.flagrans fermentation broth was higher than that of A.oligospora with the same treatment,while the results of two fungi were opposite in the groups induced by complex amino acid and distilled water.(4)Strongylus(L3),Haemonchus contortus(L3)and Caenorhabditis elegans were used to test the effects by the fermented broths of two kinds of fungi in all six goroups,and the killing rates were higher in D.flagrans than in A.oligospora under the same treatment.Considering the different inductors,the activity in the larvae group was the highest in the same fungus treatment,followed by complex amino acids group and distilled water group,respectively.(5)Using the mass spectrometry-based non-labeled quantitaty technique,the proteogics of the fermentation broths of D.flagrans with different inductors were studied.The results indicated that the total number of proteins identified was 325,and protein-number in AA group(amino acid-induced),DF group(distilled water treated)and NE group(Strongylus(L3)-induced)was 200,232 and 209,respectively.Among all the proteins,there were up-regulated 21,down-regulated 25 in AA/NE,up-regulated 1,down 6 in AA/DF and up-regulated 24,down-regulated 22 in NE/DF,correspondingly.(6)In addition to joining the processes of biological reactions,such as carbon metabolism,amino acid biosynthesis,differential proteins also involved in methane metabolism,biological regulation and other processes.Seen from the molecular function analysis results,compared with the distilled water group,the proteins in the rested groups of D.flagrans,mainly had catalytic and binding molecular function.KEGG pathways for analysis showed that differential proteins were mainly involved in carbon metabolism and amino acid biosynthesis.All these indicated that D.flagrans can respond to nematode larvae and complex amino acid stimuli,and stayed in stress state.Therefore,further analysis of the differential proteome is helpful to reveal the molecular mechanism of killing nematodes by D.flagrans.