Data-Independent\nAcquisition Proteome Technology for\nAnalysis of Antifungal and Anti-aflatoxigenic Properties of Eugenol\nto Aspergillus flavus

Several toxicogenic <i>Aspergilli</i>, such\nas Aspergillus flavus and A. parasiticus, could biosynthesize aflatoxin B<sub>1</sub> (AFB<sub>1</sub>) and\nother mycotoxins. Chemical fungicides are commonly used to control\nfungal contamination, but chemical residues may pose significant risks\nto human health and environmental stability. Consequently, natural\nantifungal and aflatoxin-inhibiting agents could be sustainable alternatives.\nEugenol has been used as an inhibitor of aflatoxins (AFs), which is\na common essential oil. Nevertheless, the definite mechanism by which\neugenol exerts its inhibitory effect on <i>Aspergillus</i> remains unclear. This research demonstrates that eugenol significantly\nsuppressed fungi growth and AF production as the dose increased (40.9\nto 100%). With the proteomics approach, the inhibition pathway of\neugenol was investigated. The production of proteins involved in cell\nwall integrity was notably reduced under eugenol treatment, indicating\nthat eugenol destroys the cell wall integrity of A.\nflavus. Furthermore, exposure to eugenol downregulated\nseveral fungal developmental regulators and subsequently inhibited A. flavus development. Energy metabolism in A. flavus is closely related to its secondary metabolism.\nUnder eugenol treatment, the synthesis of proteins relevant to the\npentose phosphate pathway was significantly enhanced, leading to a\ndecrease in the availability of acetyl-CoA, a precursor for AF biosynthesis.\nSimultaneously, the valine, leucine, and isoleucine biosynthesis pathways\nwere enhanced, further reducing the content of acetyl-CoA. This might\nbe the primary factor in the inhibition of AF biosynthesis by eugenol.\nRibosome biogenesis was the most dysregulated pathway based on KEGG\ndata, indicating that eugenol disturbed ribosome biogenesis and affected\nits normal function in A. flavus. In\nconclusion, eugenol inhibits the cellular integrity, energy metabolism,\nand protein synthesis and then suppresses A. flavus development and AF biosynthesis, which provides a clearer grasp\nof the inhibitory mechanism meaningful for A. flavus and AF contamination control.