The Metabolic Characteristics Of Temperature-driven Fungal Growth And Aflatoxin Biosynthesis In Aspergillus Flavus

Aspergillus flavus is a kind of saprophytic fungus,which is mainly found on mildewed grain,grain and oil products,and is the main factor causing food mildew.As one of the secondary metabolites of Aspergillus flavus,aflatoxin is a highly toxic carcinogen,which will lead to liver cancer and even death if the food contaminated by aflatoxin is ingested.The biosynthesis of aflatoxin is affected by many factors,including temperature,water activity,p H and CO₂,among which temperature is one of the most important factors affecting the production of aflatoxin.In this study,Aspergillus flavus A3.2890 strain was used as material,and physiological and biochemical analysis,ultramorphological analysis and matabolomic analysis were used to compare the differences in intracellular ultramorphology and small molecule metabolites of Aspergillus flavus growing at toxic temperature 28?and non-toxic temperature37?.The main research results were summarized as follows:1.The morphology and internal ultrastructure of the mycelium of Aspergillus flavus during different growth stages at 28?and 37?were observed and compared by scanning electron microscopy(SEM)and transmission electron microscopy(TEM)from the perspective of cell morphology for the first time.The results showed that at 28?,the content of aflatoxin increased and the content of glucose and nitrogen in the medium decreased gradually during 24h to 44h.There were filamentous sticky secretions on the mycelium surface and large vesicles in the mycelium cells.During 48h to 72h,the content of afflatoxin continued to increase,the content of glucose and nitrogen in the medium also decreased,and the mycelium gradually appeared shrinkage,collapse and distortion,and the internal organelles of the mycelium were blurred.Comparing with 28?,accumulation of aflatoxin was not detected at 37?for 24 h to 72 h,but dry mycelium on this period have been higher than 28?,the consumption of glucose and nitrogen was also accelerated at 37?,the mycelium was robust and full,the number of mitochondria and liposomes peaked at 44h and 48h,and the membrane structure was observed after 72 h.Subsequently,the ultrastructure of Aspergillus flavus mycelium cultured at 30?and40?was compared to verify the above findings.Taken together,28?and 30?promoted the aflatoxins biosynthesis,but induced earlier and faster senescence of Aspergillus flavus mycelia.On the contrast,37?and 40?were not conducive to the biosynthesis of aflatoxin,but were beneficial to the growth of mycelia,and the senescence process of mycelia was relatively slow.2.The non-targeted metabolome of Aspergillus flavus at 28?and 37?was analyzed by gas chromatography-mass spectrometry(GC-MS).A total of 140 peaks were detected by GC-MS,of which 75 peaks were identified by standards.Further statistical analysis showed that there were significant differences in mycelium metabolome between the two temperatures.There were significant differences in the intracellular metabolism of mycelia during different developmental stages at the two temperatures,especially in the first two days after inoculation,and then the metabolic differences gradually decreased.These differential metabolites were organic acid,amino acid and sugar,differential metabolic pathway mainly concentrated in the tricarboxylic acid cycle,glucose metabolism and amino acid metabolism.The tricarboxylic acid cycle pathway,glucose metabolism pathway and amino acid metabolism pathway were all promoted at 37?,which was consistent with the observation that hypha growth was more vigorous at this temperature.3.The lipid profiles of Aspergillus flavus at 28?and 37?were analyzed by liquid chromatography-mass spectrometry(LC-MS).A total of 155 lipid metabolites were identified in positive ion mode and 113 lipid molecules were identified in negative ion mode.Glycerol phospholipid and glycerol ester were the main lipids in Aspergillus flavus.Glycerol phospholipid content was very high in the early stage(2d and 3d)of Aspergillus flavus growth and development,and gradually decreased with the prolongation of inoculation time.Glycerides were present throughout the growth and development of Aspergillus flavus.At the same time,the glycerol phospholipids and glycerides bound to the saturated fatty acid chain were mostly upregulated at 37?,while the glycerol phospholipids and glycerides bound to the unsaturated fatty acid chain were mostly down-regulated at 37?.In order to further verify the results,we conducted targeted fatty acid analysis based on GC-MS,and quantitatively analyzed10 kinds of saturated fatty acids and 7 kinds of unsaturated fatty acids.The results showed that compared with 28?,the content of saturated fatty acids was significantly up-regulated at 37?,while the content of unsaturated fatty acids was significantly down-regulated at 37?.The results were in good agreement with those of lipidome analysis.