*3.2. Microbial and Chemical Changes that Occur During Kombucha Tea Fermentation*

Microorganisms in kombucha tea utilize a carbon source and begin to produce cellulose which appears as a thin layer on top of the culture medium. The different types of tea; green, oolong, and black tea, that were used in this study did not affect the growth of the tea fungus. The total counts of bacterial and yeast cells during kombucha tea fermentation are shown in Figure 2. After inoculation of the 10% (*v*/*v*) starter culture, total counts of acetic acid bacterial colonies at the beginning of the kombucha tea fermentation process for green, oolong, and black teas were 5.72, 5.81, and 5.54 log CFU/mL, respectively. Moreover, the total counts of yeast cells at the beginning of kombucha tea fermentation were 5.68, 5.89, and 5.79 log CFU/mL in the kombucha tea prepared from green, oolong, and black teas, respectively. After increasing the fermentation time, the number of bacterial and yeast cells were found to be significantly higher than when observed at the beginning of the fermentation period (Figure 2A,B). After 15 days of fermentation, a total count of 7.80 log CFU/mL was found in the kombucha that had been prepared from green tea and black tea. The acetic acid bacterial colonies were significantly higher in the kombucha prepared from green and black teas than in the kombucha prepared from oolong tea (7.54 log CFU/mL). In addition, the total counts of the yeast cells in the kombucha tea prepared from green tea (7.64 log CFU/mL), oolong tea (7.74 log CFU/mL) and black tea (7.78 log CFU/mL) were not significantly different in each type of kombucha tea.

**Figure 2.** Alteration of the total count of acetic acid bacteria (**A**), total count of yeast cells (**B**), pH (**C**), total acidity (**D**), and total soluble solids (**E**) during fermentation of kombucha prepared from green tea (ᶺ), oolong tea (-), and black tea (). \* The values were significantly different between the beginning of the fermentation and the end of 15 days of the fermentation period (*p* < 0.05). \*\* The values were significantly different for each type of kombucha tea at the end of 15 days of fermentation (*p* < 0.05). The results are presented as mean ± SD of three independent experiments.

The blank control without acetic acid and yeast was performed. However, after incubation for 2–3 days the contamination from other microorganisms was presented because the blank control showed pH around 4.57–5.25. However, pH of 3.73–3.92 was determined after inoculation of starter culture at 0 day of fermentation time (Figure 2C). This acidic condition of kombucha tea inhibited other contaminated microorganisms in kombucha tea.

The alteration of pH values during kombucha tea fermentation with significantly different initial pH values is shown in Figure 2C. At the end of the 15-day fermentation period, the pH value of kombucha tea that had been prepared from black tea was the lowest at a pH value of 2.70. Kombucha that was prepared from green tea and oolong tea revealed pH values of 2.94 and 2.89, respectively. On the other hand, changes in titratable acidity that occurred during the fermentation process were significantly increased, which indicated a concentration of these organic acids (Figure 2D). The total acidity of the kombucha prepared from black tea was significantly higher (16.75 g/L) than that of the kombucha prepared from oolong (12.24 g/L) and green teas (11.72 g/L). In contrast, total soluble solids of kombucha prepared from green, oolong and black tea were significantly decreased from 10 ◦*Brix* to 6 ◦*Brix* at 15 days of fermentation (Figure 2E). In addition, no alcohol content was detected during the process of kombucha tea fermentation.

#### *3.3. Organic Acids in Kombucha Tea*

The organic acids in kombucha tea were analyzed by HPLC assay. The HPLC system was optimized for the detection of several organic acids in kombucha tea with a conventional C18 column. HPLC conditions were optimized and 20 mM KH2PO4 with a pH value of 2.4 in the isocratic elution buffer was used with a 210 nm UV detector. Six organic acids including glucuronic acid, gluconic acid, D-saccharic acid 1,4-lactone (DSL), acetic acid, ascorbic acid, and succinic acid were clearly separated. Chromatograms of six standard organic acids, including glucuronic acid, gluconic acid, DSL, acetic acid, ascorbic acid, and succinic acid, were eluted at different retention times (Figure 3).

**Figure 3.** HPLC chromatograms of running time 40 min (**A**) and the six organic acids (**B**) are presented as standards; glucuronic acid (Peak 1), gluconic acid (Peak 2), D-saccharic acid 1,4-lactone (DSL) (Peak 3), acetic acid (Peak 4), ascorbic acid (Peak 5), and succinic acid (Peak 6).

After 15 days of kombucha fermentation, the amounts of each type of organic acid that were present in the kombucha prepared from green, oolong, and black teas were analyzed by HPLC. The chromatograms and production values of organic acids at 15 days of fermentation are shown

in Figure 4 and Table 1. The highest content of gluconic acid was found in the kombucha that was prepared from green, oolong, and black teas, followed by acetic acid, DSL, ascorbic acid, and glucuronic acid. In contrast, succinic acid was detected in the kombucha that had been prepared from black tea, but it was not detected in the kombucha prepared from green tea and oolong tea. Therefore, kombucha prepared from black tea revealed the highest amounts of organic acid content: glucuronic acid (1.58 g/L), gluconic acid (70.11 g/L), DSL (5.23 g/L), ascorbic acid (0.70 g/L), acetic acid (11.15 g/L), and succinic acid (3.05 g/L), when compared to both oolong tea and green tea.

**Figure 4.** HPLC chromatograms of kombucha prepared from green tea (**A**), oolong tea (**B**), and black tea (**C**) at 15 days of fermentation. Peak (**1**)—glucuronic acid; Peak (**2**)—gluconic acid; Peak (**3**)—D-saccharic acid 1,4-lactone (DSL); Peak (**4**)—acetic acid; Peak (**5**)—ascorbic acid; Peak (**6**)—succinic acid, respectively.


**Table 1.** Organic acid content of kombucha tea prepared from green tea, oolong tea, and black tea at 15 days of the fermentation process.

a, b, c The data of different superscript letters (a, b, c) are presented as mean ± SD of three independent experiments and revealed significantly different values for each type of kombucha tea (*p* < 0.05). <sup>d</sup> ND: organic acid in kombucha tea was not detected.
