Isolation And Antagonism Mechanism Of Components In Aspergillus Niger Spores

In this paper,secondary metabolites of Aspergillus niger?xj?spores were separated by method of natural product separation which got five monomeric compounds ANS 1-5.Through the screening of antagonistic activity,ANS-4 has significant antagonistic activity against Rastonia solanacearum Q11-2 and Erwina carotovora EC-1.The antagonistic mechanism is preliminarily studied.The main experimental conclusions are as follows:1.Reflux extraction of 20kg Aspergillus niger spores using 95%ethanol which got1345g crude extract after low pressure concentration,the yield is 6.7%.After the extraction of petroleum ether and ethyl acetate,the antagonistic activity of three parts was screened and result is:petroleum ether segment>ethyl acetate segment>water segment.The petroleum ether segment had the best antagonistic activity against Q11-2,EC-1 and T-37 with the antagonistic activity respectively up to 83.26%±3.62%,92.15%±2.14%and 84.45%±3.15%.2.The petroleum ether segment A1-A11 segment was further subdivided by chromatographic column chromatography,Sephadex LH-20 gel column separation,decompression chromatographic column chromatography and recrystallization,and five monomer compounds were obtained,which were named as ANS-1 to ANS-5.Monomer compounds were identified by nuclear magnetic resonance ¹H-NMR,¹³C-NMR and EI-MS.Ergosterol?ANS-1?,tetra-sitosterol?ANS-2?,5-pentacylresorcinol?ANS-3?,5-hydroxymethyl-furan acid?ANS-4?,and succinimide?ANS-5?were isolated from Aspergillus niger for the first time.3.The results of the antagonistic activity screening of monomer ANS 1-5 of the compound showed that the antagonistic activity of ANS-4 on Q11-2 and EC-1 was the strongest,which were 94.26±1.92%,96.31±2.83%and 19.18±4.51%,respectively.The EC₅₀of compound ANS-4 on Q11-2 and EC-1 were 0.5611mg/m L and 0.5605mg/m L.The results of cell permeability and integrity tests showed that ANS-4 had a certain effect on the cell permeability of Q11-2 at the concentration of EC₅₀,which increased the extracellular conductivity of Q11-2,but had no significant effect on the cell permeability of EC-1.The nucleic acid and protein measurements showed no significant intracellular material leakage,such as proteins and nucleic acids,while the observation of cell morphology of Q11-2 and EC-1 by scanning electron microscopy indicated that ANS-4 had no serious effect on the bacterial membrane,but only caused partial malformation and enlargement of cell surface.Through the determination of total protein content of the two types of bacteria,it was shown that the normal growth and metabolic function of the bacteria were affected by ANS-4,and the protein expression was significantly decreased.The results of determination of intracellular ROS content showed that ANS-4 rapidly broke the intracellular ROS level in a short time and caused its abnormal accumulation.In addition to ANS-4 against extracellular polysaccharide of Q11-2 and EC-1,make its extracellular polysaccharide content significantly decreased,content determination results combined with biological membrane,significantly inhibited the biofilm formation of Q11-2 and EC-1.Q11-2 and EC-1 swimming ability also by inhibition of ANS-4,ANS-4 affecting the swimming ability of the bacteria by inhibition of extracellular polysaccharide and the function of biological membrane structure.The activity of succinate dehydrogenase and malate dehydrogenase showed that ANS-4 had a significant effect on the activity of these two key tricarboxylic acid enzymes.