## 3 Results and Discussion
### Comparative Analysis of COS and \(SO_{2}\) Antimicrobial Effects  
\(SO_{2}\) is a traditional food additive that has been permitted for use in wine. The total limited concentration of \(SO_{2}\) in wine can vary, such as 200 mg/L or 300 mg/L, because laws restricting \(SO_{2}\) use in wine vary among different countries. However, winemakers tend to supplement wine with as little \(SO_{2}\) as possible owing to negative health effects on consumers with special sensitivity. To simulate the real environment of contamination during winemaking, nine types of wine spoilage organism were mixed together and inoculated into a pasteurized must. Free \(SO_{2}\), rather than its bound form, can inhibit microbes, but the amount of free \(SO_{2}\) is closely related to the temperature and pH of a must. \begin{table}
\begin{tabular}{c c c c c} \hline \hline \multirow{2}{*}{**Wine spoilage Organism**} & \multicolumn{4}{c}{**Colony Count (CFU/mL)**} \\ \cline{2-5}  & **No COS** & **100 mg/L COS** & **500 mg/L COS** & **1000 mg/L COS** \\ \hline _Enterococcus_ & \((1.46\pm 0.59)\times 10^{8}\) & \((1.15\pm 0.54)\times 10^{8}\) & \((1.81\pm 0.45)\times 10^{4}\) & \((5.30\pm 2.10)\times 10^{2}\) \\ _Bacillus cereus_ & \((2.39\pm 1.42)\times 10^{8}\) & \((6.45\pm 2.12)\times 10^{7}\) & \((1.61\pm 0.93)\times 10^{3}\) & \((1.99\pm 0.30)\times 10^{2}\) \\ _Paenibacillus_ & \((1.71\pm 1.03)\times 10^{8}\) & \((6.16\pm 2.58)\times 10^{7}\) & \((8.47\pm 3.12)\times 10^{3}\) & \((1.73\pm 0.65)\times 10^{2}\) \\ _Alkaliphilus_ & \((1.83\pm 0.77)\times 10^{7}\) & \((4.24\pm 1.31)\times 10^{6}\) & \((4.92\pm 1.63)\times 10^{2}\) & 0 \\ _Pantoa_ sp. & \((2.20\pm 1.25)\times 10^{8}\) & \((1.10\pm 0.20)\times 10^{7}\) & \((1.85\pm 0.51)\times 10^{5}\) & \((9.33\pm 6.66)\times 10^{2}\) \\ _Lactococcus_ & \((1.77\pm 0.12)\times 10^{7}\) & \((1.55\pm 0.31)\times 10^{7}\) & \((5.67\pm 2.73)\times 10^{2}\) & 0 \\ _Aspergillus flavus_ & \((1.61\pm 0.76)\times 10^{8}\) & \((1.93\pm 0.66)\times 10^{7}\) & \((1.89\pm 0.74)\times 10^{3}\) & \((1.23\pm 0.45)\times 10^{2}\) \\ _Aurebosidium pullulans_ & \((2.10\pm 0.41)\times 10^{6}\) & \((5.88\pm 1.81)\times 10^{5}\) & \((6.43\pm 3.41)\times 10^{3}\) & \((7.33\pm 3.66)\times 10^{1}\) \\ _Penicillium_ & \((1.52\pm 0.15)\times 10^{7}\) & \((8.51\pm 3.17)\times 10^{6}\) & \((6.87\pm 2.54)\times 10^{4}\) & \((2.55\pm 0.67)\times 10^{3}\) \\ \hline \hline \end{tabular}
\end{table} 
Table 1: The colony counts of nine types of spoilage organisms when applying 100, 500, and 1000 mg/L COS in the must. Therefore, the fermentation temperature was kept at 25 \({}^{\circ}\)C, and the pH values of all musts before fermentation were determined to be around 3.5. Finally, SO\({}_{2}\) (up to 200 mg/L) was added to investigate its antimicrobial effect. Surviving colonies were only reduced by 15% when supplemented with 50 mg/L SO\({}_{2}\) compared to that without additives. Therefore, 50 mg/L SO\({}_{2}\) was clearly insufficient for large-scale antimicrobial action. Increasing the SO\({}_{2}\) concentration to 100 or 200 mg/L significantly reduced the amount of surviving spoilage organisms, which finally reached less than 0.01%. The amount of surviving colonies with supplementation of 500 mg/L COS was close to that achieved by adding 100 mg/L SO\({}_{2}\) (Table 2). Furthermore, comparative analysis of the antimicrobial effects of COS (500 mg/L) and SO\({}_{2}\) (100 mg/L) as individual additives on nine different types of wine spoilage organism was conducted. As shown in Table 3, 500 mg/L COS showed better antibacterial effects than 100 mg/L SO\({}_{2}\) on all the nine wine spoilage organisms, except _Enterococcus_ and _Alkaliphilus_. For three types of fungi, COSM showed greater antifungal effects than SO\({}_{2}\). This might be due to COS originating from chitin, which composes the fungal cell wall.