**4. Discussion**

Kombucha tea was prepared from green, oolong, and black teas. The microbial community and chemical properties presented during kombucha fermentation were demonstrated in this study. In this study, the starter culture of kombucha tea was used as a symbiosis culture of acetic acid bacteria including *Acetobacter xylinum* and yeast cells. Therefore, cellulose production from *A. xylinum* was present on the surface of the kombucha tea during 3–15 days of fermentation. The production of the cellulose of this species is important for kombucha tea fermentation since a floating cellulose pellicle provides benefits to microorganisms by enhancement of the association between the bacteria and yeast cells and allows for the exposure of the microorganisms to atmospheric oxygen [20]. Moreover, caffeine and other related xanthines that are found in tea showed the ability to stimulate cellulose production by way of the bacteria that is present [3].

Normally, kombucha tea was completely fermented when fermentation time was 12–21 days and the inoculation size of starter culture was 5–10%. Thus, a period of kombucha fermentation of 15 days was selected in this study since the highest level of acidity was demonstrated during this fermentation process [11,21–23]. Moreover, yeast cells are known to convert sucrose into glucose and fructose by invertase enzymes. Additionally, ethanol was also produced. Glucose in kombucha was further utilized by acetic acid bacteria to produce gluconic acid, whereas ethanol was used to produce acetic acid. 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. After increasing the fermentation time, the total counts of acetic acid bacterial and yeast cells were found to be significantly higher than were observed at the beginning of the fermentation period. After 15 days of fermentation, the total counts of acetic acid bacteria in the kombucha prepared from green tea and black tea were significantly higher than in the kombucha prepared from oolong tea. Additionally, the total counts of yeast cells in each kombucha tea were not significantly different. Therefore, the increasing amounts of bacteria present in kombucha during the fermentation process is dependent upon the different types of tea that are used as a substrate [3].

The pH values of the kombucha prepared from green, oolong, and black teas decreased due to the production of organic acids during the fermentation process. The lowest pH value was recorded in the kombucha that had been prepared from black tea, followed by that of oolong tea and green tea. The total acidity of kombucha prepared from black tea showed the highest total amount of acids, which related to the low pH value of the kombucha. Moreover, microorganisms were able to use sucrose as the major substrate to produce many organic acids that resulted in the decrease of total soluble solids during fermentation of each kombucha tea. These different values of pH and total acidity correspond to the different types of tea used and affected the degree of organic acid production. Notably, green tea is considered a non-fermented type of tea, whereas black tea and oolong tea are classified as fully fermented tea and semi-fermented tea, respectively. The process of tea preparation resulted in different oxidation levels of catechins. Green tea was found to possess the highest total phenolic content, and black tea showed the highest content of polyphenol oxidation products after the fermentation process [24].

Moreover, this study has enabled researches to optimize new conditions of HPLC for detection of several organic acids including glucuronic, gluconic, DSL, ascorbic, acetic, and succinic acid in kombucha tea. All organic acids were clearly separated by isocratic elution buffer using the C18 conventional column. Moreover, an elution buffer of 20 mM KH2PO4, pH 2.4, and a UV detector of 210 nm were suitable for the separation of each organic acid. Low molecular weight organic acids with high levels of polarity produced the greatest level of separation using mobile phase 20 mM KH2PO4 as an elution buffer. In addition, a low pH buffer was used to ensure that all acidic groups were protonated, which allowed for protection of the change from organic acids to the neutral form, thus allowing the best interaction between the organic acids and the C18 stationary phase [25]. The HPLC result revealed that gluconic acid and acetic acid were identified as the major organic acids in the kombucha tea that was prepared via the fermentation of green, oolong, and black tea for a period of 15 days.

In addition, kombucha prepared from black tea presented higher values of organic acid content in terms of glucuronic, gluconic, DSL, ascorbic, acetic, and succinic acid, when compared to oolong tea and green tea. The highest level of organic acid content in kombucha tea was found to be gluconic acid. The glucuronic acid present in kombucha has been associated with a number of benefits to the liver. Glucuronic acid plays an important role in liver detoxification and in the process associated with the excretion of exogenous chemicals known as glucuronidation [26].

The DPPH scavenging abilities of the kombucha prepared from green, oolong, and black teas significantly increased from the beginning of fermentation. During kombucha fermentation, many compounds with radical scavenging properties were obtained from the tea leaves and were considered by-products of the metabolic pathway of microorganisms. Catechins belong to polyphenols in green tea and they display high levels of antioxidant properties. Catechins also have the ability to scavenge free radicals and reactive oxygen species [13]. Notably, the increase of antioxidant potential against DPPH radicals from kombucha tea significantly reduced oxidative injuries in rats [27]. Moreover, kombucha tea prepared from green, oolong, and black teas showed significantly high amounts of total phenolic contents on day 3 of fermentation. After 3–6 days of kombucha tea fermentation, the amounts of phenolic compounds could maintain stability and then continued to be stable during 15 days of the fermentation process. The kinetics of microorganisms in kombucha fermentation increased around 3 days after beginning inoculation, which might be the reason for the enhancement of the phenolic compounds [28]. Many enzymes are produced during kombucha fermentation, such as phytase, α-galactosidase, and tannase, which are all related to the degradation of complex polyphenols to small molecules and are known to cause an increase in total phenolic compounds [29].

The study of kombucha prepared from Chinese black tea, Chinese green tea, Chinese oolong tea and Sri Lankan black tea also yielded the highest amounts of phenolic compounds on day 1 of fermentation. Notably, the phenolic compounds maintained a level of stability throughout 7 days of the kombucha fermentation period. Additionally, individual polyphenol contents yielded variations in terms of quantity in each type of tea [30]. The enzymes that were liberated from the bacteria and yeast in the tea fungus consortium degraded the complex of phenolic compounds in the tea, and the degradation was increased in the acidic environment of the fermentation process [21].

Moreover, kombucha contains other antioxidant substances, such as ascorbic acid and DSL, which were shown to be present in high levels in the kombucha that had been prepared from black tea. DSL, a derivative of D-glucaric acid, demonstrated detoxification, anticarcinogenic, and cholesterol-reduction properties [5,31–33]. DSL has been found to reduce ameliorate alloxan-induced type 1 diabetes by inhibiting the apoptotic death of pancreatic β-cells [5]. Moreover, DSL also revealed the greatest benefit in terms of anti-oxidative properties and displayed the ability to decrease oxidative damage to certain cellular biomolecules, such as on the lipids and proteins found in human blood platelets [34]. In addition, kombucha prepared from green tea revealed the highest level of phenolic content. An increase in the total phenolic content during kombucha fermentation that occurred from the ability of the bacteria and yeast was found to liberate enzymes, such as phytase, which could break down the cellulosic backbone of the tea leaves to release polyphenol compounds [21].

In this study, the kombucha prepared from green, oolong and black teas efficiently inhibited all tested pathogenic enteric bacteria: *Escherichia coli*, *E. coli* O157:H7 DMST 12743, *Shigella dysenteriae* DMST 1511, *Salmonella* Typhi DMST 22842, and *Vibrio cholerae*. The strongest antibacterial activity of kombucha tea was related to the presence of organic acid, such as the acetic acid found in kombucha tea. This antibacterial activity displayed a significant level of inhibitory activity against all tested bacteria. The studies by Dibner and Buttin [35] reported that organic acids displayed antimicrobial activity and also displayed an inhibitory effect against acid-intolerant species such as *E. coli*, *Salmonella* sp., and *Campylobacter* sp. that had been obtained from the guts of piglets. Weak organic acids, such as acetic acid and benzoic acid, were reported to show antimicrobial activity since the organic acid molecules could induce cytoplasmic acidification and destroy bacterial cells [36].

Kombucha tea displayed a remarkable level of antimicrobial activity against a broad range of microorganisms, which have demonstrated an ability to inhibit the growth of pathogens such as *Helicobacter pylori*, *Escherichia coli*, *Entamoeba cloacae*, *Pseudomonas aeruginosa*, *Staphylococcus aureus*, *S. epidermidis*, *Agrobacterium tumefaciens*, *Bacillus cereus*, *Aeromonas hydrophila*, *Salmonella typhimurium*, *S. enteritidis*, *Shigella sonnei*, *Leuconostoc monocytogenes*, *Yersinia enterocolitica*, *Campylobacter jejuni*, and *Candida albicans* [3,37]. Both acetic acid and catechins are known to inhibit a range of Gram-positive and Gram-negative microorganisms [2]. Therefore, kombucha tea has been recognized as containing active compound substances that could inhibit bacterial pathogens [23,38]. Battikh et al. [39] reported that kombucha prepared from green tea could inhibit *S. epidermidis*, *S. aureus*, *Micrococcus luteus*, *S. typhimurium*, *E. coli*, *Listeria monocytogenes*, and *P. aeruginosa* with diameters ranging from 12 to 22 mm, while kombucha prepared from black tea showed inhibition zones against these bacteria ranging from 10.5 to 19 mm. Furthermore, other studies showed that antibacterial activities did not exclusively from acetic acid or other organic acids such as citric acid, lactic acid, malic acid, and pyruvic acid [40] but possibly from other biologically active components such as bacteriocins, proteins, enzymes, and tea-derived phenolic compounds as well as tannins originally present in the tea broth that could have been involved as antimicrobial substances [37,38].

In contrast, unfermented tea did not display any antimicrobial activity against the tested microorganisms. This probably occurred because of the low concentrations of tea broth (1%, *w*/*v*) and polyphenol levels. Thus, unfermented tea did not display any inhibitory effects against the tested microorganisms [41]. In this study, neutralized kombucha did not display any inhibitory activity on all tested bacteria. Therefore, the use of the kombucha tea will provide natural organic acids for health benefits. The antibacterial activity of kombucha occurred from the acidity present in the kombucha tea. The same result was obtained from the previous studies of Cetojevic-Simin et al. [42], all of whom reported that neutralized kombucha prepared from black tea could not inhibit pathogenic bacteria.

In addition, heat-denatured kombucha tea prepared after boiling (100 ◦C, 20 min) and autoclaving (121.5 ◦C for 15 min) inhibited all tested pathogenic enteric bacteria. These results confirm that the antimicrobial components in the kombucha are thermostable. Moreover, heat-denatured kombucha tea that was achieved by boiling could significantly inhibit bacteria more effectively than the heat-denatured kombucha tea that had been autoclaved. The acidity of kombucha tea treated by boiling was maintained and the amount of total acidity was significantly higher than in the kombucha tea that was treated by autoclave. Moreover, the amount of total acidity present between heat-denatured kombucha tea by boiling and kombucha tea without any treatment was not significantly different. In contrast, the total acidity of kombucha tea that was treated by autoclave significantly decreased the amount of acidity when compared to kombucha tea without any treatment. This phenomenon indicated that the acidity activity of kombucha tea was reduced at high temperature by being autoclaved. This is particularly noteworthy with regard to the pasteurization of kombucha tea beverages that were prepared to preserve the potential antibacterial agents that are present in kombucha tea. Moreover, other components, such as catechins, and the antioxidant activities recorded after the autoclaving of these tea drinks at 120 ◦C for 20 min also decreased [43].

Kombucha prepared from green, oolong, and black tea were found to display effective toxicity against Caco-2 colorectal cancer cells. Interestingly, kombucha prepared from green tea and black tea showed the specific toxicity on cancer cells. Therefore, this is a new report about the cytotoxicity effect of kombucha prepared from different kinds of tea leaves on Caco-2 colorectal cancer cells. However, toxicity test of kombucha tea on different types of cancer and normal cell lines should be further studied.

The major beneficial components of green tea, such as EGCG, restrained carcinogenesis in a variety of tissues through the inhibition of mitogen-activated protein kinases, growth factor-related cell signaling, activation of activator protein 1 and nuclear factor-B, topoisomerase I, and matrix metalloproteinases along with other potential targets [44]. Previous reports have shown that kombucha prepared from black tea contained dimethyl 2-(2-hydroxy-2-methoxypropylidene) malonate and vitexin that caused certain cytotoxic effects on 786-O (human renal carcinoma) and U2OS (human osteosarcoma) cells by the reduction of cell invasion, cell motility, and matrix metalloproteinase activity [45,46]. Moreover, kombucha tea was found to significantly decrease the survival rate of prostate cancer cells by downregulating the expression of angiogenesis stimulators like matrix metalloproteinase, cyclooxygenase-2, interleukin-8, endothelial growth factor, and human inducible factor-1α [47]. The active substances of kombucha are associated with many of the compounds found in each type of the tea leaves and are related to the acid production that occurs from the microorganisms.

Tea polyphenols in kombucha that are present in the tea leaves and during kombucha fermentation were identified as anticancer substances. Tea polyphenols could inhibit the mechanisms of cancer formation such as gene mutation and cancer cell proliferation. Notably, tea polyphenols also induced the apoptosis of cancer cells and terminated cancer cell metastasis [48–50]. Both types of tea leaves that had been infused (1%, *w*/*v*) and acidified in the kombucha tea showed the ability to inhibit Caco-2 colorectal cancer cells. In 2013, a study by Zhao et al. [51] showed that fermented and unfermented specimens of Pu-erh tea and green tea could inhibit HT-29 colon cancer. Additionally, kombucha prepared from green tea and black tea could inhibit A549 human lung carcinoma cells and Hep-2 epidermoid carcinoma, while kombucha prepared from black tea could inhibit Hep-2 cells [23].

In this study, acetic acid in kombucha displayed toxicity against Caco-2 cancer cells. However, toxicity activities against cancer cells were also found to have occurred as a result of the presence of other organic acids such as glucuronic, gluconic, DSL, ascorbic, acetic, and succinic acid. DSL had the ability to inhibit the activity of glucuronidase enzymes, such as hydrolyzed glucuronides and produced aglycones. Aglycones are known to be toxic substances that are able to induce normal cells to become cancer cells [52]. Gluconic acid, glucuronic acid, lactic acid, and ascorbic acid are known to have the ability to reduce the occurrence of stomach cancer [53]. Notably, kombucha prepared from black tea was found to contain several organic acids. In this study, black tea revealed the lowest level of toxicity on Caco-2 cells because of the presence of other components found in the black tea, such as thearubigins and theaflavin, which were consistently degraded during kombucha fermentation. In contrast, catechins in green tea and oolong tea were not degraded during kombucha tea fermentation [22]. Theaflavins in black tea have been reported to possess activity against carcinogenesis by interfering with the signaling pathways and suppressing the transcription of certain oncoproteins [54]. Anticancer and antibacterial activity decreased when kombucha tea was neutralized at pH 7.0 by adjustments with NaOH. However, the anticancer components of kombucha tea were found to be thermostable after the tea was heated by either boiling or by being autoclaved. Especially, kombucha tea prepared from green and black tea after treatment by boiling at 100 ◦C for 20 min that also retained the cytotoxicity effect to cancer cells. However, kombucha tea at 15 days of fermentation revealed low pH values (2.70–2.94). Thus, kombucha tea should be prepared at a pH value of around 4.2 and should not be consume in amounts of more than 4 oz per day [55]. This scientific research study clarified that kombucha tea demonstrated the health benefits of effectively treating pathogenic enteric bacterial infection, anti-oxidation, and toxicity to colorectal cancer cells, which might help to promote the consumption of kombucha beverages among consumers.

#### **5. Conclusions**

In the present study, kombucha tea was fermented with a symbiotic culture of acetic acid bacteria and yeasts that produced a significant amount of organic acids. The kombucha tea prepared from different types of tea, namely green, oolong, and black tea, displayed significantly different values of pH, total acidity, total soluble solids, and organic acid content. Moreover, the HPLC system for the detection of several organic acids in kombucha tea was optimized in this study using the C18 conventional column. Isocratic elution buffer of 20 mM KH2PO4, pH 2.4 with 210 nm UV detector was used as a new adaptive HPLC condition for organic acid detection. Kombucha prepared from black tea at 15 days of fermentation showed the highest degree of organic acid content, such as with glucuronic, gluconic, DSL, ascorbic, acetic, and succinic acids. These organic acids were found to be effective against pathogenic enteric bacteria *Escherichia coli*, *E. coli* O157:H7, *Shigella dysenteriae*, *Salmonella* Typhi, and *Vibrio cholera*. They attributed to a large extent of the antibacterial effect observed to organic acids because when neutralizing the kombucha samples, antibacterial effect was not observed. Moreover, kombucha prepared from green tea and black tea demonstrated toxicity on Caco-2 colorectal cancer cells. These findings indicate the greatest potential health benefits of kombucha tea with regard to inhibiting pathogenic enteric bacteria and by promoting healthy function of the digestive system in the gastrointestinal tract. In addition, kombucha displayed antioxidant activity against DPPH radicals. Therefore, kombucha tea could be considered as a potential source of compounds presenting antioxidant activity, inhibitory activity against pathogenic enteric bacteria and selective toxicity on colorectal cancer cells.

**Author Contributions:** Conceptualization and Investigation, T.K., S.B., and Y.T.; Methodology, T.K., S.B., and Y.T.; Formal analysis, T.K. and Y.T.; Writing—original draft preparation, T.K. and Y.T.; Writing—review and editing, T.K. and Y.T.; Funding acquisition, Y.T.

**Funding:** This research study was supported by the Royal Golden Jubilee (RGJ) Ph.D. Program Scholarship, the Thailand Research Fund, grant no. PHD/0061/2558; and was partially supported by the Center of Excellence in Bioresources for Agriculture, Industry, and Medicine, Department of Biology, Faculty of Science, and The Graduate School, Chiang Mai University, Chiang Mai, Thailand.

**Acknowledgments:** This research work was partially supported by Chiang Mai University. The Tea Gallery Group (Thailand) Co., Ltd. and Chatchai Kitipornchai are also thoughtfully acknowledged. Department of Medical Technology, Faculty of Associated Medical Sciences, Department of Biology, Faculty of Science, and The Graduate School, Chiang Mai University, Chiang Mai, Thailand are also acknowledged.

**Conflicts of Interest:** The authors declare no conflict of interest.
