Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC) Determination

To estimate the minimal inhibitory concentration (MIC) of the investigated products, the broth microdilution method was used against M. circinelloides, A. flavus, A. fumigatus, A. niger, and P. notatum isolates used in the biological assays [15]. One hundred microliters (100 μL) of SDB A solution were dispensed into each well of a microdilution plate with 96 "U"-shaped wells. Next, 100 μL of the nanomaterials emulsion was put to the first horizontal row of wells on the plate. In double serial dilutions, a 100 μL aliquot was taken from the well with the highest concentration and transferred to the next well, resulting in concentrations ranging from 1000 to 1.9 μg/mL. In the end, 10 μL of an inoculum solution comprising various strains examined was put to each well of the plate, where each column represented a fungal strain. In the presence of the standard antifungal cyclohexamide with 500 mg/L media for prevention of rapid growing molds, a positive control (media with the materials but without fungal strains) and negative control (media without the materials and fungal strains) were grown (media with the fungi but without nanomaterials).

All CSNPs prepared at various pH levels were dissolved in acetic acid (1%). Consequently, 100 µL of SDB containing varied concentrations of tested chitosan nanoparticles in two fold serial dilutions was added to each well (1000, 500, 250, 125, 62.5, 31.25, 15.62, 7.81, 3.90, and 1.95 µg/mL) After diluting the NPs, 10 µL suspensions containing 1.5 × 10⁸⁷ fungal strains/mL were inoculated; acetic acid served as the negative control.

At 25 °C, the plates were incubated for 72 h. The presence (or absence) of growth was visually observed after the proper incubation period. The MIC was established as the lowest concentration that visibly inhibited fungal growth while considering the formation of cell clusters or "buttons" in the plate wells. The studied nanoparticles' antibacterial activity was explained (considered active or not) based on the criteria outlined by Morales et al. [16]: strong/good activity (MIC: lower than 1000 μg/mL).

On SDA-coated Petri dishes, 1 μL aliquots of MIC, MIC × 2, and MIC × 4 of the tested nanomaterials, Cyclohexamide (500 mg/L), and the negative control for fungal growth were subcultured to determine the MFC. After 24–48 h of incubation at 35 °C, the MFC was evaluated based on its growth relative to the controls. The minimal fungicidal concentration (MFC) was then established as the lowest product concentration that inhibited the development of various fungal species and produced either 50% or 99.9% fungicidal activity [17]. Biological activity assays were performed in triplicate, and the results were expressed as the arithmetic means of the MIC and MFC concentrations. Subsequently, it was possible to determine whether the substance was active using both dilution methods. The MFC test was carried out since it was unclear if it would actually kill the fungus or just inhibit its growing. Depending on the tested fungus species, small aliquots of each broth dilution test were sub cultured on a rich solid medium and incubated for a predefined duration of time and temperature. According to Chemical and Laboratory Standard Institute-standardized publications. The MFC is recognized as the lowest concentration of the chemical at which observable subculture growth is not seen. Moreover, MFC might offer information on fungicide or fungistatic action. If MFC and MIC are same, the material is a compound fungicide; if MFC is greater than MIC, the substance is fungistatic [18].