Inhibition Mechanism Of Aspergillus Flavus By Pamamycin Produced By Streptomyces Alboflavus TD-1 And Its Application In Corn Storage

In China,as a large grain producing country,it is inevitable that cereal food raw materials are contaminated by Aspergillus flavus and other molds in the field,storage and transportation.At present,it is safer,more efficient and environmentally friendly to use biological methods to prevent and control mold contamination such as Aspergillus flavus.In this paper,Streptomyces alboflavus TD-1 isolated from soil was used as the research object to isolate the active substance produced by metabolism,to study the inhibitory effect and mechanism of pamamycin on the growth of Aspergillus flavus and other fungi,and to study the anti-mildew effect of Streptomyces alboflavus TD-1 on corn during storage,and to explore the potential of Streptomyces alboflavus TD-1 as biocontrol bacteria.This study is of great significance to the development of new biological anti-mildew agents with independent intellectual property rights,to reduce grain loss in China,to prevent food raw materials from being contaminated by mold and toxins,and to ensure food safety.The main results are as follows:（1）The inhibitory effect of Streptomyces alboflavus TD-1 on the growth of Aspergillus flavus 2219,Botrytis cinerea,Trichoderma harzianum,Aspergillus oryzae,Penicillium citrus and Aspergillus Niger was studied by filter paper method.Streptomyces alboflavus TD-1 had good inhibitory effect on Aspergillus flavus 2219,Botrytis cinerea,Trichoderma harzianum,Aspergillus oryzae,Penicillium citrus and Aspergillus Niger,especially on Aspergillus flavus and Aspergillus niger,the inhibition rate was higher than50%.Its inhibitory effect on Botrytis cinerea,Trichoderma harzianum,Aspergillus oryzae and Penicillium citrus was good,and the inhibition rate was between 25%and 50%.（2）The active substances in Streptomyces alboflavus TD-1 were isolated and identified by thin layer chromatography（TLC）,silica gel column chromatography,high performance liquid chromatography（HPLC）and liquid chromatography-mass spectrometry（LC-MS）.The molecular formula was predicted to be C₃₅H₆₁NO₇,pamamycin and its homologue,and the relative molecular weight was 593,607,621,635,649.The silica gel column chromatography eluent for the separation of pamamycin was optimized.And it was determined that the eluent ratio of ethyl acetate:n-hexane:diisopropylamine=1:80:0.5,which could elute 86.17%of pamamycin with a purity of 84.43%,When the dosage was 30m L.At the same time,it was also judged that pamamycin was a slightly acidic substance.（3）The stability of pamamycin was determined.Firstly,the stability of pamamycin in different storage time,temperature and state was determined by HPLC.Pamamycin was suitable to be stored at-20℃in dry state,degraded with time,decreased by 37.03%on the7th day,and then tended to be stable.In addition,after heat treatment,p H treatment and UV treatment,the inhibition experiment of Aspergillus flavus 2219 was carried out to determine the stability of pamamycin activity.Pamamycin has good thermal stability and still has strong antibacterial activity after sterilization on 121℃at 30 min.The bacteriostatic activity of pamamycin decreased after acid-alkali treatment.The stability of ultraviolet radiation was very good,and the antibacterial activity of ultraviolet lamp irradiation for 5 hours was not weakened.（4）The minimal inhibitory concentration（MIC）of pamamycin against Aspergillus flavus 2219 and other fungi was determined by 96-well plate microdilution method.The MIC of pamamycin was 0.125mg/m L to Penicillium citrus,0.5mg/m L to Botrytis cinerea,and 1mg/m L to Aspergillus flavus 2219,Aspergillus niger,Trichoderma harzianum and Aspergillus oryzae.The morphology of mold cells treated with pamamycin was observed by scanning electron microscope,and the spores and hyphae were distorted in different degrees.The inhibitory mechanism of pamamycin on Aspergillus flavus 2219 and other molds was studied by measuring the integrity of mold cell wall,cell membrane and mitochondria and the content of ergosterol.And it was found that the target of action of pamamycin on mold was ergosterol,the structural component of fungal cell membrane.The inhibition mechanism was to inhibit the growth and reproduction of fungi by destroying the integrity of cell wall,changing the permeability of cell membrane,affecting the energy metabolism of mitochondria and inhibiting the biosynthesis of ergosterol in mycelial cell membrane.And it could inhibit 98.24%the synthesis of AFB₁.（5）The infection experiment of Aspergillus flavus 2219 was carried out by using the fermentation broth of Streptomyces alboflavus TD-1.It was found that Aspergillus flavus2219 began to be inhibited when the concentration of Streptomyces alboflavus TD-1（the proportion of mycelium in the total volume of fermentation broth）was 35%.In addition,it had no effect on the change of water content in corn within 10 days of storage,and the fatty acid value increased slowly,and the germination rate decreased not obviously.