# A Radish Root Ferment Filtrate for Cosmetic Preservation: A Study of Efficacy of Kopraphinol
## 2 Materials and Methods
### Challenge Test  
The challenge test consists in artificially contaminating the cosmetic formulations with a standard inoculum of suitable microorganisms and evaluating the number of surviving microorganisms at predetermined intervals of time. Different challenge test protocols are commonly available in evaluating the microbiological stability of cosmetics [14]. In this investigation, to test the preservative efficacy of Kopraphinol compared to methylparaben, the method of the European Pharmacopoeia 7 th Edition [15] was chosen, because it has the strictest acceptance criteria, which are difficult to achieve with many preservative systems [14]. Preliminary challenge tests were performed on several simple formulations to choose the most suitable one; more precisely, Carbopol 940- and hydroxyethylcellulose-based gels, emulsions formulated with Sepigel(r) as an emulsifier, and Cetomacrogol aqueous cream were prepared and tested. However, hydroxyethylcellulose-based gels have proven to be unsuitable because they already exhibit antimicrobial activity due to the presence of 10% glycerin, whereas Carbopol- and Sepigel-containing formulations show a sharp decrease in viscosity at the time of the addition of Kopraphinol. Therefore, challenge tests were performed on Cetomacrogol aqueous cream, which does not undergo alterations after the addition of the preservative; moreover, it is a formulation rich in free water, and for this reason, it represents a good substrate for microbial growth. Cetomacrogol cream was prepared as described by [PERSON] et al. [16]. Its composition is as follows: 15 g of lipophilic phase petrolatum, 6 g of mineral oil, 7.2 g of cetostearyl alcohol, 1.8 g of hydrophilic phase Cetomacrogol 1000, and 70 g of purified water. Kopraphinol was added to the creams at 4% vol/vol, as suggested by the manufacturer, while methylparaben was used at 0.2% wt/vol. The addition of Kopraphinol did not cause appreciable changes in the appearance and texture of the cream. The following bacterial strains were employed in the challenge tests: _E. coli_ (ATCC 8739), _P. aeruginosa_ (ATCC 9027), _S. aureus_ (ATCC 6538), _C. albicans_ (ATCC 10231), and _A. brasiliensis_ (ATCC 16404). For each microorganism, fifty grams of cream were contaminated with a standard inoculum to obtain a concentration of about \(10^{6}\) colony-forming units (cfu)/g, and the preparations were stored at room temperature. At time 0 and after 1, 7, 14, and 28 days, one gram of each product was aseptically sampled and suspended in 10 mL of peptone water; the resulting suspension was subjected to decimal serial dilutions in sterile saline and analyzed for the enumeration of viable microorganisms onto TSA or SDA plates. The challenge tests were carried out in parallel with the corresponding controls (cream without preservative). The antimicrobial activity at each time was expressed in terms of the logarithmic reduction in the number of viable microorganisms compared to that at time 0. The reduction in the log number in each sample should comply with criterion A in the European Pharmacopoeia: 2 log reduction after 24 h and 3 log reduction after 7 days for bacteria and 2 log reduction at 14 days for fungi (with no increase at 28 days in both cases). The results are reported in Tables 4 and 5. \begin{table}
\begin{tabular}{c c c c c c c c c c} \hline \multirow{2}{*}{**TIME**} & **Eur. Ph.** & \multicolumn{3}{c}{_Escherichia coli_} & \multicolumn{3}{c}{_Staphylococcus aureus_} & \multicolumn{3}{c}{_Pseudomonas aeruginosa_} \\  & **Criteria** & **W** & **K** & **M** & **W** & **K** & **M** & **W** & **K** & **M** \\ \hline Day 2 & 2 & 0.01 & \textgreater{}5 & \textgreater{}5 & 0.209 & \textgreater{}5 & 0.017 & \(-1.438\) & 4.828 & 1.216 \\ \hline Day 7 & 3 & \(-0.138\) & \textgreater{}5 & \textgreater{}5 & \textgreater{}5 & \textgreater{}5 & \textgreater{}5 & \(-2.885\) & \textgreater{}5 & \(-1.924\) \\ \hline \end{tabular}
\end{table} 
Table 4: Results of the challenge test performed on bacterial strains.