# A Radish Root Ferment Filtrate for Cosmetic Preservation: A Study of Efficacy of Kopraphinol
## 2 Materials and Methods
### Antifungal Activity of Kopraphinol and Methylparaben  
The antifungal activity of Kopraphinol was assessed on _C. albicans_, _P. rubens_, and _A. brasiliensis_ standard strains and on four _Candida_ spp. clinical isolates. Fungi were grown on SDA plates at 35 \({}^{\circ}\)C (_Candida_ strains) or 25 \({}^{\circ}\)C (other fungi).  
The M.I.C.s of Kopraphinol for _Candida_ strains were determined by using a 96-well plate microdilution method; twofold dilutions of the preservative, ranging from 50% to 0.39% vol/vol, were prepared in SLM. All assays were performed at least in triplicate. The microplates were inoculated with about 1 \(\times\) 10\({}^{4}\) yeasts/well and aerobically incubated at 35 \({}^{\circ}\)C for 24 h. After the incubation, the plates were visually checked for fungal growth, and the M.I.C. was defined as the lowest concentration at which no growth was observed. To determine the M.F.C. (Minimum Fungicidal Concentration), aliquots of 2 \(\mu\)L of the medium from each well with no visible growth were subcultured onto SDA plates, which were then incubated at 35 C for 24 h; the M.F.C. was defined as the lowest concentration at which no growth was detectable. The results are reported in Table 1.  
The M.I.C.s of Kopraphinol against _A. brasiliensis_ and _P. ovale_ were determined using an agar macrodilution method [12]. Twofold serial dilutions of the product in SDA were made in 5 mm Petri dishes (final volume 10 mL), obtaining final concentrations of 10%, 5%, 2.5%, and 1.25% vol/vol. The experiments were all performed in triplicate. Control plates containing only SDA were run simultaneously. The agar surface of the plates was then inoculated into the center with 1-3 \(\mu\)L of a conidial or yeast cell suspension prepared in sterile distilled water +0.05% Tween-80, containing 10\({}^{3}\)-10\({}^{4}\) conidia or cells. The plates, wrapped with Parafilm to maintain the correct water activity in the medium, were inverted and incubated at room temperature (about 25 \({}^{\circ}\)C). The plates were visually checked for fungal growth; M.I.C.s were defined as the lowest concentrations of Kopraphinol preventing the growth of macroscopically visible colonies on Kopraphinol-containing plates when there was visible growth on the Kopraphinol-free control plates. The results are reported in Table 1.  
\begin{table}
\begin{tabular}{l c c} \hline
**Microorganisms** & **Kopraphinol (vol/vol)** & **Methylparaben (wt/vol)** \\ \hline _Escherichia coli_ (ATCC 8739) & 0.78 \(\pm\) 0.00\% (6.25 \(\pm\) 0.00\%) & 0.2 \(\pm\) 0.00\% (0.2 \(\pm\) 0.00\%) \\ \hline _Staphylococcus aureus_ (ATCC 6538) & 1.56 \(\pm\) 0.00\% (12.5 \(\pm\) 0.00\%) & 0.2 \(\pm\) 0.00\% (0.2 \(\pm\) 0.00\%) \\ \hline _Pseudomonas aeruginosa_ (ATCC 9027) & 6.25 \(\pm\) 0.00\% (6.25 \(\pm\) 0.00\%) & 0.2 \(\pm\) 0.00\% (0.2 \(\pm\) 0.00\%) \\ \hline _Candida albicans_ (ATCC 10231) & 1.56 \(\pm\) 0.00\% (3.13 \(\pm\) 0.00\%) & 0.05 \(\pm\) 0.00\% (0.05 \(\pm\) 0.00\%) \\ \hline _Candida_ spp. (from rectal swab) & 3.13 \(\pm\) 0.00\% (6.25 \(\pm\) 0.00\%) & n.d. \\ \hline _Candida_ spp. (from throat swab) & 1.56 \(\pm\) 0.00\% (1.56 \(\pm\) 0.00\%) & n.d \\ \hline _Candida_ spp. (from vaginal swab) & 1.56 \(\pm\) 0.00\% (6.25 \(\pm\) 0.00\%) & n.d. \\ \hline _Candida_ spp. (from mucosa of the cheek) & 1.56 \(\pm\) 0.00\% (3.13 \(\pm\) 0.00\%) & n.d. \\ \hline _Aspergillus brasiliensis_ (ATCC 16404) & 5.00 \(\pm\) 0.00\% & 0.1 \(\pm\) 0.00\% \\ \hline _Penicillium rubens_ (ATCC 9179) & 5.00 \(\pm\) 0.00\% & n.d. \\ \hline \end{tabular}
\end{table}
Table 1: Evaluation of the antimicrobial activity of Kopraphinol and methylparaben. The values (\(\pm\)SD) represent the minimum inhibitory concentrations (MIC) (in brackets, the minimum bactericidal/ fungicidal concentrations, MBC/MFC, are shown) (n.d. = not determined). MIC and MBC/MFC values are expressed as % vol/vol for Kopraphinol and as wt/vol for methylparaben.