# Influence of Ozone on the Biochemical Composition of Birch Sap
## 4 Discussion
It can be concluded that the presence of organic matter affects the ozone dispersion and uniformity in the sap. It has previously been shown that the presence of organic matter, pH, or temperature can affect the effects of ozone on microorganisms [56; 57]. The amount of organic matter can inhibit the effectiveness of microorganisms' inactivation. This was also confirmed by [PERSON] et al. [18], in whose study the ozonation (75-78 \(\upmu\)g mL\({}^{-1}\)) effect on reducing _Escherichia coli_ in orange juice started after 60 s and got deactivated in low-pulp juice after 6 min, and in unfiltered juice after 15-18 min of treatment. More research has been carried out monitoring the changes of microbiota in the sap during storage and identifying the microorganisms as well as treating the sap with different methods by inactivating microorganisms [3; 9; 58; 59]. In our studies, it was found that the temperature increment resulted in the growth of microorganisms in the sap. Researchers [PERSON] and [PERSON] [9] found that the number of microorganisms CFU in birch sap increased rapidly during the first week of storage but the difference was not significant, regardless of the temperature (4 \({}^{\circ}\)C and 20 \({}^{\circ}\)C). Physical, chemical, and microbiological changes occur in birch sap during storage. The rate of these changes depends on the initial parameters, storage conditions, and technology of processing. The technologies must not damage the internal parameters of the product. The composition of birch sap has been well studied. The physicochemical and rheological properties and composition of minerals, organic acids, amino acids, and sugar have been described in numerous studies [3; 4; 5; 6; 10; 58; 60; 61]. Most studies on birch sap have focusedon yield and composition, while, to our knowledge, no studies have reported the effects of ozone on birch sap constituents, pH, and color. Birch sap is colorless and, according to the results of this work, ozone had an effect on both chroma and hue angle parameters. It can be concluded that ozone is a factor that reduces the color of the sap. However, a visual difference in the color of birch sap is difficult to discern. There are no exact data on how ozone affects the color of birch sap. Some researchers have reported that the ozonation of fruit and berry juices--orange [62; 63], melon [64], grape [65], apple [66], and blackberry [67]--changes the color. There are several studies in which ozone did not induce a color change: by treating prebiotic orange juice with ozone at a concentration of 0.057, 0.128, or 0.230 mgO\({}_{3}\) mL\({}^{-1}\) of juice (15, 30, 45, or 60 s) [31] or by treating apple juice with 0.08-0.31 mg L\({}^{-1}\) ozone at different temperatures [68]. According to [PERSON] et al. [68], the color change of juice in different studies may be due to different treatment methods and control parameters (concentration, ozone gas flow, and treatment interval). During the study, it was found that the values of the active acidity of fresh birch sap and immediately after ozonation ranged from 6.10 to 6.49. During storage, the values of pH decreased after seven days at 2 \({}^{\circ}\)C and after five days at 20 \({}^{\circ}\)C. A larger decrease was seen after seven days when the sap was stored at 20 \({}^{\circ}\)C. The pH values obtained for fresh sap are in most cases consistent with other studies. [PERSON] and [PERSON] [61] found that the pH of birch sap can vary from 7.5 to 5.5 in the spring. The study data showed that the pH dropped faster at 20 \({}^{\circ}\)C. It was confirmed by [PERSON] and [PERSON] et al. [3] that pH drops during storage, and [PERSON] and [PERSON] [9], by studying the physicochemical properties of the sap, found that the pH decreased faster at 20 \({}^{\circ}\)C (pH: 3.41) compared to 4 \({}^{\circ}\)C (pH: 3.79) after 58 days. The pH of fresh birch sap studied by [PERSON] et al. [60] ranged between 6.0 and 6.6 and dropped only 0.2-0.3 in pH units during storage. According to [PERSON] et al.