This work aimed to evaluate the fungicide activity of chitosan-silver nanocomposites (Ag-Chit-NCs) against Penicillium expansum from feed samples. The physicochemical properties of nanocomposites were characterized by X-ray fluorescence analysis (XRF), small-angle X-ray scattering (SAXS), X-ray photoelectron spectroscopy (XPS), and transmission electron microscopy (TEM). The morphological integrity of the nanohybrid was confirmed by electron transmission. By the data of RFA (X-ray fluorescent analysis), the contents of Ag in Ag-chitosan composite were 5.9 w/w%. The size distribution of the Ag nanoparticles incorporated in the chitosan matrix was investigated by SAXS. The main part of the size heterogeneity distribution in the chitosan matrix corresponds to the portion of small particles (3-4 nm). TEM analysis revealed a spherical morphology in the form of non-agglomerated caps, and 72% of the nanoparticles measured up to 4 nm. The minimum inhibitory concentration of NCs was evaluated in petri dishes. Three different concentrations were tested for antifungal activity against the mycotoxigenic P. expansum strain. Changes in the mycelium structure of P. expansum fungi by scanning electron microscopy (SEM) were observed to obtain information about the mode of action of Ag-Chit-NCs. It was shown that NC-Chit-NCs with sizes in the range from 4 to 10 nm have internalized sizes in cells, form agglomerates in the cytoplasm, and bind to cell organelles. Besides, their ability to influence protein and DNA fragmentation was examined in P. expansum. SDS-PAGE explains the apparent cellular protein response to the presence of various Ag-Chit-NCs. The intensity of P. expansum hyphal cell protein lines treated with Ag-Chit-NCs was very thin, indicating that high molecular weight proteins are largely prevented from entering the electrophoretic gel, which reflects cellular protein modification and possible damage caused by the binding of several protein fragments to Ag-Chit-NCs. The current results indicate that Ag-Chit-NCs <10 nm in size have significant antifungal activity against P. expansum, the causative agent of blue mold-contaminated dairy cattle feed.

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