## 3 Results and Discussion
### Effects of COS on Saccharomyces Cerevisiae and PichiaPastoris Cell Growths  
To investigate the effects of COS on the cell growths of _Saccharomyces cerevisiae_ and _PichiaPastoris_, a pasteurized must with 500 mg/L COS as an additive was inoculated with only _Saccharomyces cerevisiae_ or _PichiaPastoris_, without any wine spoilage organism. A pasteurized must without any additive and inoculated with only _Saccharomyces cerevisiae_ or _PichiaPastoris_ was used as a control group. The bacterial concentration was evaluated using the OD\({}_{600}\) value. The logarithmic phase of the cell growth of _Saccharomyces cerevisiae_ was slightly extended, when 500 mg/L COS was added, because bacterial growth was slightly slowed down. When the stationary phase was reached, the bacterial concentrations (OD\({}_{600}\) values) with or without COS were not significantly different. However, the growth of _PichiaPastoris_, a type of wild yeast that can cause wine spoilage [1], was significantly inhibited by 500 mg/L COS (Figure 2). Therefore, 500 mg/L COS supplemented into the must inhibited _PichiaPastoris_ cell growth but did not influence _Saccharomyces cerevisiae_ cell growth. Therefore, using 500 mg/L COS as an additive during winemaking would not affect the fermentation process. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Colony Count (CFU/mJ)** & \multicolumn{2}{c}{**24 h**} & **48 h** & **72 h** & **96 h** & **120 h** \\ \hline \multirow{3}{*}{_Lactobacillus plantarum_} & / & \((1.54\pm 0.52)\times 10^{6}\) & \((2.74\pm 0.58)\times 10^{7}\) & 0 & – & – \\  & COS & \((1.77\pm 0.41)\times 10^{5}\) & \((5.08\pm 1.50)\times 10^{5}\) & 0 & – & – \\  & SO\({}_{2}\) & \((1.98\pm 0.23)\times 10^{4}\) & \((5.93\pm 2.23)\times 10^{5}\) & 0 & – & – \\ \hline \multirow{3}{*}{_Acetobacter aceti_} & / & \((5.83\pm 2.67)\times 10^{6}\) & \((9.10\pm 1.73)\times 10^{7}\) & \((4.33\pm 1.96)\times 10^{8}\) & \((1.60\pm 0.16)\times 10^{2}\) & 0 \\  & COS & \((1.95\pm 0.18)\times 10^{4}\) & \((1.24\pm 0.10)\times 10^{5}\) & \((5.57\pm 0.97)\times 10^{6}\) & \((6.83\pm 1.76)\times 10^{1}\) & 0 \\  & SO\({}_{2}\) & \((1.61\pm 0.45)\times 10^{5}\) & \((3.13\pm 1.35)\times 10^{6}\) & \((2.69\pm 0.45)\times 10^{7}\) & \((6.96\pm 1.91)\times 10^{1}\) & 0 \\ \hline \multirow{3}{*}{_Peliococcus damnosus_} & / & \((1.83\pm 0.23)\times 10^{6}\) & \((9.23\pm 3.95)\times 10^{6}\) & \((2.85\pm 0.24)\times 10^{7}\) & \((7.70\pm 1.25)\times 10^{1}\) & 0 \\  & COS & \((6.47\pm 3.88)\times 10^{4}\) & \((2.23\pm 0.51)\times 10^{5}\) & \((7.50\pm 1.27)\times 10^{5}\) & \((1.80\pm 0.43)\times 10^{1}\) & 0 \\  & SO\({}_{2}\) & \((1.16\pm 0.47)\times 10^{4}\) & \((2.72\pm 0.62)\times 10^{5}\) & \((9.00\pm 1.92)\times 10^{5}\) & \((4.23\pm 1.02)\times 10^{1}\) & 0 \\ \hline \multirow{3}{*}{_Lactobacillus brevis_} & / & \((9.73\pm 1.19)\times 10^{5}\) & \((9.10\pm 2.46)\times 10^{6}\) & \((1.04\pm 0.59)\times 10^{2}\) & 0 & – \\  & COS & \((2.75\pm 0.37)\times 10^{4}\) & \((1.78\pm 0.58)\times 10^{5}\) & \((2.33\pm 0.77)\times 10^{1}\) & 0 & – \\ \cline{1-1}  & SO\({}_{2}\) & \((5.37\pm 2.94)\times 10^{3}\) & \((2.43\pm 0.59)\times 10^{5}\) & \((2.93\pm 1.38)\times 10^{1}\) & 0 & – \\ \hline \end{tabular}
\end{table} 
Table 4: The colony counts of four known wine spoilage organisms at each time point (24, 48, 72, 96, and 120h) after using 500 mg/L COS or 100 mg/L SO\({}_{2}\) as an additive. As summarized in Table 5, compared with previously reported multiple additions and physical methods, COS showed a more universal antimicrobial effect without any adverse effects. Using 500 mg/L COS as an additive during winemaking did not influence _Saccharomyces cerevisiae_ cell growth, meaning that COS did not influence the fermentation process. Furthermore, the radical scavenging activity of wine could be increased by adding COS, as confirmed by analysis of the DPPH and hydroxyl radical scavenging activities (data not shown). Therefore, COS shows potential for applications in the winemaking industry.