# Influence of Ozone on the Biochemical Composition of Birch Sap
## 4 Discussion  
The use of ozone in the food industry is not new, but its effects on the quality of food and drink have not yet been thoroughly researched. One such well-known drink \begin{table}
\begin{tabular}{l c c c} \hline \hline  & \multicolumn{3}{c}{Correlation Coefficient} \\ \hline  & **Tittatable Acidity,** & **pH** & **Electrical Conductivity, \(\mu\)S cm\({}^{-1}\)** \\ \hline
**Total soluble solids, \({}^{*}\)Brix** & \(-0.308\) * & \(0.283\) * & \(-0.164\) \\ \hline
**Tittatable Acidity, mg 100 mL\({}^{-1}\)** & - & \(-0.900\) ** & \(0.616\) ** \\ \hline
**pH** & - & - & \(-0.576\) ** \\ \hline \hline \end{tabular}  
* Correlation is significant at the 0.05 level (\(n=126\)). ** Correlation is significant at the 0.01 level (\(n=126\)).  
\end{table} 
Table 1: Correlation analysis data between components in the sap and acidity values. Figure 10: Comparison of components of fresh and ozonated for 30 min birch sap: 1 (control)—non-ozonated sap (initial); 7—gap ozonated for 30 min. Data are presented as means \(\pm\) standard deviations (\(p<0.05\), \(n=6\)). is birch sap. A review of the studies reveals that there is still no specificity in terms of ozone concentrations and their use intervals. The aim of this study was to determine the effectiveness of ozone in the reduction of microorganisms in the sap while assessing the quality of the sap, monitoring its changes immediately after ozonation and after storing the sap in different environments. The parameters of fresh sap were compared to the parameters of ozone-treated and stored sap. At the beginning of the study, an observation was made that ozone saturation in sap is not constant compared to ozone saturation in water. During the ozonation of the sap, the average ozone concentration reached 0.99 \(\pm\) 0.09 mg L\({}^{-1}\) after 10 min. After 5 min of ozonation, only 0.087 \(\pm\) 0.009 mg L\({}^{-1}\) was recorded in the sap. However, between 5 and 10 min, the ozone concentration was not studied, so it is not clear exactly at which point it rose. Compared with [PERSON] and [PERSON]'s study [42], which used similar ozone concentrations in map sap, saturated ozone concentrations (about 1 mg L\({}^{-1}\)) were reached within 5 min and persisted throughout the ozonation period (40 min). The obtained results show that the number of colony-forming units of microorganisms in the ozonated sap significantly decreased due to ozone treatment. The total reduction in the number of colony-forming units of microorganisms in the sap right after ozonation was 6.32-6.48 log CFU mL\({}^{-1}\), the reduction in lactic acid bacteria was 2.46-3.15 log CFU mL\({}^{-1}\), and the reduction of yeast was 3.16-3.76 log CFU mL\({}^{-1}\). During the storage, ozonated sap also retained lower levels of microorganisms. Ozone's impact on microorganisms has long been known from studies with drinking water [30; 43; 44; 45], various juices [18; 46; 47; 48; 49], and berries, fruit, and vegetables [50; 51; 52; 53; 54; 55]. However, there were no such studies where the main objective was to reduce the number of microorganisms in birch sap using ozone treatment. One study was found on map sap by researchers [PERSON] and [PERSON] [42], in which the sap was treated with ozone at a concentration of approximately 1 mg L\({}^{-1}\). The object of the study was to make an aerobic plate count and yeast count. The researchers found that there was a significant decrease in the number of aerobic plates after 40 min, where less than 3 log were recorded, and after 20 min, the aerobic plate count decreased by only 1 log. This contradicts our study, where at 0.99 \(\pm\) 0.09 mg L\({}^{-1}\) ozone concentration after 20 min of treatment there was a significant (\(p\) < 0.05) effect of ozone on microorganisms. As [PERSON] and [PERSON] [42] conclude, this may have been influenced by the presence of sucrose at a concentration of 3%. In the case of the present study, the sucrose content in fresh silver birch (_Betula pendula_ Roth) sap was significantly lower at 0.12 \(\pm\) 0.01 mg 100 mL\({}^{-1}\).