# A Radish Root Ferment Filtrate for Cosmetic Preservation: A Study of Efficacy of Kopraphinol
## 2 Materials and Methods
### Contact Time (Killing Time)  
The antimicrobial activity of Kopraphinol was also characterized by a \"killing time\" test, performed on _E. coli, P. aeruginosa, S. aureus_, and _C. albicans_; the purpose of this assay is to evaluate the reduction in the viable count when microorganisms are incubated with the product in a liquid medium that does not support cell growth. The tests, carried out in triplicate, were performed following the protocol developed by [PERSON] et al. [13]. Microorganisms in the logarithmic phase of growth were suspended at a density of 5 \(\times\) 10\({}^{5}\)-1 \(\times\) 10\({}^{6}\) cfu/mL in a suitable volume of PBS (pH 7.3) containing 4% vol/vol of Kopraphinol (concentration suggested by the producer for practical use). A control tube (microorganisms suspended in PBS only) was included in each experiment. At time zero and after 1, 2, and 3 h of incubation at 35 \({}^{\circ}\)C, 0.5 mL of the suspensions was removed, subjected to serial tenfold dilutions in PBS, and seeded onto TSA (_E. coli, S. aureus_, and _P. aeruginosa_) or SDA (_Candida albicans_) plates. The number of surviving microorganisms at each time was evaluated by counting the colonies after incubation for 24 h at 35 \({}^{\circ}\)C. The results are reported in Table 3.  
\begin{table}
\begin{tabular}{l c c c c} \hline  & _E. coli_ **(ATCC 8739)** & _P. aeruginosa_ **(ATCC 9027)** & _S. aureus_ **(ATCC 6538)** & _C. albicans_ **(ATCC 10231)** \\ \hline MIC K & 0.78 \(\pm\) 0.00\% & 6.25 \(\pm\) 0.00\% & 1.56 \(\pm\) 0.00\% & 1.56 \(\pm\) 0.00\% \\ \hline MIC K50 & 0.78 \(\pm\) 0.00\% & 6.25 \(\pm\) 0.00\% & 1.56 \(\pm\) 0.00\% & 1.56 \(\pm\) 0.00\% \\ \hline MIC K100 & 0.78 \(\pm\) 0.00\% & 6.25 \(\pm\) 0.00\% & 1.56 \(\pm\) 0.00\% & 1.56 \(\pm\) 0.00\% \\ \hline MIC K\({}^{\star}\) & 0.78 \(\pm\) 0.00\% & 6.25 \(\pm\) 0.00\% & 1.56 \(\pm\) 0.00\% & 1.56 \(\pm\) 0.00\% \\ \hline \end{tabular}
\end{table}
Table 2: Antimicrobial activity of Kopraphinol subjected to thermal treatment (K = no heat treatment: K50 = heating to 50 \({}^{\circ}\)C for 1 h; K100 = heating to 100 \({}^{\circ}\)C for 1 h; K\({}^{\star}\) = freezing at \(-80\)\({}^{\circ}\)C for 24 h and subsequent thawing). The values (\(\pm\)SD) represent the minimum inhibitory concentrations (MIC) expressed as % vol/vol.  
\begin{table}
\begin{tabular}{l c c c c} \hline  & **STRAIN** & **Time 0** & **1 h** & **2 h** & **3 h** \\ \hline _E. coli_ (ATCC 8739) & 100\% & 30 \(\pm\) 6.6\% & 0.46 \(\pm\) 0.25\% & 0.17 \(\pm\) 0.09\% \\ \hline _P. aeruginosa_ (ATCC 9027) & 100\% & 91.7 \(\pm\) 7.6\% & 33.3 \(\pm\) 5.6\% & 9.8 \(\pm\) 4.5\% \\ \hline _S. aureus_ (ATCC 6538) & 100\% & 120.8 \(\pm\) 7.7\% & 141.7 \(\pm\) 9.9\% & 83.3 \(\pm\) 7.5\% \\ \hline _C. albicans_ (ATCC 10231) & 100\% & 98.3 \(\pm\) 8.7\% & 84.5 \(\pm\) 5.0\% & 70.7 \(\pm\) 10.5\% \\ \hline \end{tabular}
\end{table}
Table 3: Killing time test results. The values (\(\pm\)SD), the average of three experiments, represent the percentage of surviving bacteria after different times of contact with Kopraphinol 4% vol/vol.