# Influence of Ozone on the Biochemical Composition of Birch Sap
## 3 Results
### Microbiological Research
An essential difference (Tukey HSD test and Dunnett's test, \(p<0.05\)) was found between the raw sap and the treatment intervals of 25 min (No. 6) and 30 min (No. 7), where the total bacterial count was 89.4% and 87.5% lower, respectively, than in the untreated sap. The highest total bacterial count was recorded in the untreated sap stored at 20 \({}^{\circ}\)C for five days. This led to storing the sap at a temperature of 20 \({}^{\circ}\)C for only five days, unlike the 2 \({}^{\circ}\)C sap that was stored for seven days. After five days of storage, the mean total bacterial count in the untreated sap was 8.54 log CFU mL\({}^{-1}\) (No. 1, Day 5, 20 \({}^{\circ}\)C). No significant difference was observed in total bacterial count in the sap (Tukey HSD test, \(p<0.05\)). However, a comparison of the control group and the ozone treatment groups showed a significant difference between treatment groups 6 and 7 ([PERSON]'s tests, \(p<0.05\)). The total bacterial count detected in fresh sap varies depending on storage temperature (Figure 3). After seven days of storage at 2 \({}^{\circ}\)C, the total bacterial count increased by 13.3%, and after five days of storage at 20 \({}^{\circ}\)C, the bacterial count increased by 24%. In experiments on the lactic acid bacteria count (Figure 4), it was found that the untreated sap (No. 1) contained 3.20 log CFU mL\({}^{-1}\) (day 1), 3.72 log CFU mL\({}^{-1}\) (day 7, 2 \({}^{\circ}\)C), or 6.61 log CFU mL\({}^{-1}\) (day 5, 20 \({}^{\circ}\)C). The lactic acid bacteria count decreased with each longer treatment interval. However, compared to the untreated control group ([PERSON]'s tests, \(p<0.05\)), significant differences were recorded immediately after treatment in groups (No. 3, 4, 5, 6, and 7). Sap ozonation for 30 min (No. 7) had a statistically significant effect after five days when the sap was stored at 20 \({}^{\circ}\)C.  
When comparing sap treatment methods, the most significant effect on lactic acid bacteria (Tukey HSD test, \(p<0.05\)) was achieved on day 1 after ozonation of sap for 15 min, 20 min, 25 min, or 30 min, and after five days' (20 \({}^{\circ}\)C) storage after ozone treatment for 30 min. The lactic acid bacteria count recorded in the non-ozonated sap (Figure 4) increased by 13.8% after seven days of storage (2 \({}^{\circ}\)C) and by 51.5% after five days (20 \({}^{\circ}\)C). The most effective reduction of lactic acid bacteria count was when the sap was ozonated for 30 min. Figure 5: Changes in the yeast colonies count in birth sap: 1 (control)—not ozonated sap (initial); 2—gap ozonated for 5 min; 3—gap ozonated for 10 min; 4—gap ozonated for 15 min; 5—gap ozonated for 20 min; 6—gap ozonated for 25 min; 7—gap ozonated for 30 min. Significant, substantial differences are distinguished by different letters (Tukey HSD test). Single letters indicate that there is no significant difference. Differences were considered significant when \(p\) values were less than 0.05 (\(n=18\)). In the Dunnett test, significant differences between the control group (No. 1) and the treatment group are marked with an asterisk “**”. No mold was found in the sap; only yeast was detected. Figure 5 shows the change in the number of yeasts in birch sap when the sap was tested on the first day (immediately after ozonation), after seven days of being stored at 2 \({}^{\circ}\)C, and after five days when the sap was stored at 20 \({}^{\circ}\)C.  
The highest count of yeast colonies was found in untreated sap, both fresh and stored for five or seven days. The yeast colonies count in the sap decreased immediately after ozonation (day 1) from 3.77 log CFU mL\({}^{-1}\) (No. 1) to 1.98 log CFU mL\({}^{-1}\) (No. 7); after 30 min ozonation, the yeast colonies count decreased by 98.4%, while after seven days the count of yeast colonies in sap ozonated for 30 min was 88.9% lower than that in non-ozonated sap. After five days, the yeast colony count in the sap ozonated for 30 min was 97.1% lower than in the non-ozonated.