## 4 Discussion  
Molds secrete large amounts of enzymes into the environment. These substances are used extensively in various fields, such as the food, beverages, and pharmaceutical industries [5; 22]. However, extracellular enzymes enable the growth of molds under various conditions and contribute to the biodegradation of materials such as paper or textiles [10]. This can lead to substantial material and economic losses. The gemini surfactants used in this study inhibited the growth of molds. All of the compounds, both neutral and cationic, decreased the concentration of enzymes produced by _A. brasiliensis_ strain ATCC 16404. This contradicts results reported by [PERSON] et al. [23], who investigated the influence of colloidal silver nanoparticles (Ag NPs) on the activity of extracellular enzyme. Nanosilver at 22.5 ppm caused the activity of all the enzymes to increase. In our work, after the addition of gemini surfactants, some enzymes, namely alkaline phosphatase, esterase, esterase lipase, and \(\alpha\)-glucosidase, were not detected in the mycelium. Minor changes were noted for acid phosphatase and naphthol-AS-BI-phosphohydrolase. The former is found in the mycelium cell wall [24], which was not destroyed by the action of gemini surfactants. This may explain why the concentration of this enzyme was the same as in the untreated sample. Acid phosphatase and naphthol-AS-BI-phosphohydrolase are often detected in microscopic fungi that cause infections, such as _Candida_ spp. [25] or _Malassezia_ spp. [26]. They are also found in elevated concentrations in the mycelia of _Aspergillus niger_, _Cidosporium cladosporoides_, or _Stachybotrys chartarum_ inhabiting building materials such as wallpaper, gypsum, and carton-gypsum board [10; 27]. Since extracellular enzymes play a key role in biodegradation and infections, it is important to limit their synthesis. Like most filamentous fungi, strains of _A. niger_ have the ability to secrete high levels of both proteins and metabolites [28]. The intra- and extracellular proteome of _Aspergillus_ spp. has been analyzed extensively using different approaches such as SDS-PAGE, two-dimensional (2D)-PAGE gel electrophoresis, and MALDI-ToF (Matrix-Assisted Laser Desorption/ Ionization- Time of Flight) [29; 30; 31]. However, little is known about the influence of gemini surfactants on fungal proteins, and there have been no studies addressing this issue. The neutral and cationic gemini surfactants tested in our research caused several changes in the extracellular protein profiles of _A. brasiliensis_ ATCC 16404. No significant differences were observed between the types of biocide and the changes in the protein patterns. This is in agreement with results reported by [PERSON] et al. [18], who observed that the number of patterns in the protein profiles of _Candida albicans_ were noticeably lower after treatment with essential oils (peppermint and clove oils). Changes in electrophoretic profiles depend on the concentrations of gemini surfactants used. Surfactants produce larger changes in the protein fraction profiles at 2 MIC than at MIC. [PERSON] et al. [32] suggested that proteins can interact with surfactants both _in vitro_ and _in vivo_. Ionic surfactants can lead to denaturation and conformational changes in proteins [33]. The changes that occurred after treatment with gemini surfactants were clearly visible using an electron microscope (TEM). These observations supported those described previously using scanning microscopy (SEM) [16]. There are only a few publications describing the impact of antifungal biocides (not antibiotics) on the ultrastructure of mold cells. The action of quaternary ammonium compounds (QAC) on yeast _Saccharomyces cerevisiae_ was described by [PERSON] et al. [14; 34]. Monomeric surfactants, especially n-dodecyl-N,\(N\)-dimethylalaninum methyl bromide (DMALM-12), were found to cause cell wall and cell membrane deformation. In such cases, the cell interior fills with electronically dense granules while single lipid drops form in the cytoplasm.