Evaluation of physicochemical properties and antioxidant activities of kombucha “Tea Fungus” during extended periods of fermentation

Abstract Kombucha fermentation is traditionally carried out by inoculating a previously grown tea fungal mat into a freshly prepared tea broth and incubating under aerobic conditions for 7–10 days. In this study, four kombucha beverages were prepared by placing the tea fungal mats in sugared Sri Lankan black tea at varying concentrations for a period of 8 weeks. The antioxidant activities, physicochemical, and qualitative properties were monitored prior to the commencement of the fermentation process, one day after the inoculation with the microorganisms and subsequently on a weekly basis. All samples displayed a statistically significant decrease (p < .05) in the antioxidant activity at the end of 8 weeks, which was indicative of the decreasing functional properties of the beverage. The physicochemical properties indicated increased acidity and turbidity, which might decrease consumer appeal of the fermented beverage. Further studies are necessary to test the accumulation of organic acids, nucleic acids, and toxicity of kombucha on human organs following the extended period of fermentation.

It is believed that kombucha originated in China over 2,000 years ago (Martin et al. 1995;Liu et al. 1996;Srihari & Satyanarayana, 2012), while many other historical reports indicate that this beverage was consumed in countries such as Russia, Germany, and the Middle East as well (Dipti, Yogesh, & Kain, 2003). The kombucha fermentation is traditionally carried out for 7-10 days in household preparation conditions.
As the fermentation progresses, the taste of kombucha tea changes from a pleasurably fruity, sour, and sparkling flavor to a mild vinegarlike taste, thus increasing the consumer acceptability of the flavor and other sensory aspects of the beverage (Goh et al., 2012;Marsh, Sullivan, Hill, Ross, & Cotter, 2014;Watawana et al., 2015). Ultimately, a pleasantly sour, slightly sparkling, apple cider-like beverage is produced. It can be produced at home by fermentation using mail order fungus or a tea fungal mat which is domestically prepared. Although green tea can be used for the preparation of this beverage, the combination of black tea and white sugar is considered the finest substrates.
The beverage is well-known to possess many prophylactic and therapeutic benefits, where it is believed to help with digestion, give relief free radicals, in particular reactive oxygen species (ROS), which are considered to possess high levels of wide-ranging antioxidant properties (Srihari & Satyanarayana, 2012). The presence of tea polyphenols is known to impart the antioxidant activity to the kombucha as well (Jayabalan et al., 2014). Studies examining kombucha tea prepared using substrates such as green tea, black tea, and tea waste material have been shown to have a high radical scavenging activity (Jayabalan, Subathradevi, Marimuthu, Sathishkumar, & Swaminathan, 2008).
However, the total antioxidant activity depends on the fermentation time, type of the tea material, and the microbiota present in the tea fungal mat (Jayabalan et al., 2014;Sievers et al., 1995;Sreeramulu et al., 2000). Although the tea fungus has the ability to enhance the radical scavenging activity by the fermentation process, whether these beneficial effects of the beverage are enhanced through extended periods of fermentation remains a question. To address this issue, this study focused on monitoring the changes to the antioxidant activities and physicochemical effects of kombucha "Tea Fungus" during an extended fermentation period of 8 weeks. The fermentation process has not been monitored for such a lengthy duration, and as such, the antioxidant properties of the beverage have not been evaluated for such a period as well. It is of interest to see whether the functional properties of the beverage as well as other physicochemical aspects are altered beyond the typical 7-10 days of fermentation. Most of the physicochemical properties evaluated in this study are related to the mouthfeel and appearance of the beverage. These sensory aspects are important above all as being a fermented beverage, consumers may tend to judge the drink mostly based on the color, clarity, and flow properties.

| MATERIALS AND METHODS
The kombucha tea fungal mat was originally obtained from the Yunnan Province of China, following which it was domestically grown in Singapore until three "daughter" mats were developed. The third "daughter" mat was used for this experiment. The dominant acetic acid bacterial species found in the tea fungal mat were Acetobacter xylinum, A. xylinoides, A. aceti, A. pausterianus, and Bacterium gluconicum. Kloeckera spp., Schizosaccharomyces pombe, Saccharomyces ludwigii, S. cerevisiae, Torulaspora spp., Zygosaccharomyces bailii, and Pichia spp., were the dominant fungal species. Sugared black tea was prepared by addition of Sri Lankan black tea into boiling water and allowed to infuse for 5 mins. The black tea used for this experiment was obtained from Kenilworth Estate in Ginigathena, which is one of the tea estates of Watawala Plantations in Sri Lanka. Different tea concentrations were prepared by changing the amount of Sri Lankan black tea. The concentrations of tea in the experimental samples which were prepared are shown in Table 1. The infusion was filtered using a sterile sieve. To this, brown sugar as amounts displayed in Table 1 was added and dissolved. The extracts were left to cool to room temperature. The tea was poured into a sterile glass jar, and the tea fun-

| pH value and total soluble solids (TSS) of Kombucha
The pH values were measured using an electronic handheld pH meter (Testo 206 PH1, Keison, London, UK). The TSS were measured with a hand refractometer (ERMA Inc. A-Contrast II-520 -0, Tokyo, Japan).

| Change in weight of kombucha mat and clarity
Observations for mat formation and changes in clarity (clear/turbid) were recorded on a weekly basis. In addition, the percentage of weight gains or loss of the tea fungus mat (mother culture + daughter cultures) between day 0 (before the initiation of the fermentation process) and the day of data collection were calculated based on the differences in the wet weights of the total tea fungal mat in the broth.

| Oxygen radical absorbance capacity (ORAC) assay
The ORAC assay was carried out according to the method by Prior et al. (2003)

| DPPH radical scavenging assay
The DPPH radical scavenging activity assay was carried out using the

| Statistical analysis
All data were presented as means (± standard deviation) of at least three independent experiments (n ≥ 3), with each experiment having a minimum of three replicates of each sample. For comparisons between samples, data were analyzed by ANOVA using the software Minitab version 17. A probability of 5% or less was considered as statistically significant.

| pH and total soluble solids
A statistically significant decrease (p < .05) was observed in the overall pH of all kombucha samples with the fermentation time as compared to Tea 1-F. There were no statistically significant differences (p < .05) of pH between control and fermented samples on day 0, week 2, and week 3, but a statistically significant difference in pH (p < .05) between kombucha samples was observed in week 5. The initial pH of the tea samples was 5.3, and it dropped to 4.3 during the fermentation period. It showed a rapid decrease until 2 weeks of fermentation and continued to decrease to a certain extent up to 8 weeks. Studies show that the pH of kombucha at drinkable level decreases from around 5.0 to 2.5 within 6 days of fermentation (Liu et al. 1996;Greenwalt, Ledford, & Steinkraus, 1998). Similarly, in this study, the pH decreased from 5.6 to 3.6 within the first week (7 days) of fermentation in the control sample. According to the study of Chen and Liu (2000), the final pH value of the liquid broth after 30 days was 2.5, which was much lower than the pH for optimum growth (pH 5.4-6.3) of yeasts.
The decrease in pH value could be due to the increased concentration of organic acids produced during the fermentation process by bacteria and yeasts in the tea fungal mat, which then seeped into the broth.
It was also observed that the pH and TSS of the broths of the con- which the kombucha fungal mats have not been added. Additionally, the changes in the pH and TSS in the control samples may not have necessarily been due to contamination, as given the total period of analysis of 2 weeks, it is possible that the beverage itself underwent changes in viscosity due to exposure to oxygen, and therefore as a result of oxidation. Attempts were made, nevertheless, to minimize any such exposure or contamination, despite bearing in mind the inevitable.

| Weight of the kombucha mat and clarity
Changes to the weight of the kombucha mat are presented in Figure 2c. Similar to the parameters of pH and TSS, it was observed that the clarity of the control samples also changed during the period of analysis from "very clear" to "turbid." As in the instance of the previous parameters, although it may be interpreted as a evidence of contamination, this may not have necessarily been the case for the same reason as oxidation and other biochemical changes take place within the tea itself, through exposure to air-although attempts were made as much as possible to minimize such activity.

| TPC assay, ORAC assay, and DPPH radical scavenging assay
TPC assay, ORAC assay, and DPPH radical scavenging assay data of the broths are presented in Figure 3. The TPC of kombucha samples did not statistically significantly increase (p < .05) with the fermentation time compared with the unfermented samples. This was being observed for the first time, as many of the previous studies focused on lesser (i.e., 7-10 days) of fermentation. TPC of kombucha samples shows a statistically significant difference (p < .05) compared with the control samples, except in weeks 2, 3, 4, and 6. Tea 3-F and 4-F showed statistically significantly higher (p < .05) TPC in week 3 when compared with the initial values. Two outliers were observed as displayed in Figure 3a. Tea 3-F displayed a higher TPC value in week 3. The reduction in TPC may be due to the utilization of phenolic compounds by the tea fungus. The sudden increases in the TPCs could have coincided with the increases in the microbiological activities which was not an aspect investigated in this study. Nevertheless, the quantity of the TPC did not always determine the antioxidant activities of Kombucha, whereas the types of metabolites produced might have had the key effect instead (Chu & Chen, 2006;Greenwalt et al. 2000).
The ORAC values of kombucha samples had not statistically significantly (p < .05) increased with the fermentation time compared with the unfermented samples. The ORAC value of the kombucha beverages prepared using a higher concentration of tea dust (Tea 3-F, Tea 4-F) displayed a statistically significantly higher (p < .05) antioxidant activity compared with the other samples of lower tea concentrations. According to the mean separation, Tea 4-F had a statistically significantly higher (p < .05) ORAC value than other samples.
Tea 3-F shows the highest antioxidant activity in week 5. However, the increase in the ORAC values as the fermentation progressed was gradual, when compared with previous studies such as that by Chu and Chen (2006) [study of 15 days] and Jayabalan et al. (2008) [8 days]. This may possibly be because the antioxidants had lost their antioxidant properties due to prolonged exposure to oxidation.
T A B L E 2 Changes to the clarity of kombucha samples and controls T1-F  T1-C  T2-F  T2-C  T3-F  T3-C  T4-F  T4- indicated that the antioxidant activity in terms of the DPPH radical scavenging potential was statistically significantly decreasing (p < .05). Reiss (1994) and Blanc (1996) have shown that the composition of different kombucha beverages is greatly affected by the individual tea fungal mats being used. This probably results from the variability of the normal microflora found in different tea fungus samples (Chen & Liu, 2000;J'unior et al., 2009;Jayasekera et al., 2011). Moreover, Malbaˇsa et al. (2011) reported that the application of different kombucha starters causes a development of different antioxidant activities on both substrates.

| CONCLUSIONS
Overall, the kombucha samples displayed a decrease in the anti-

ACKNOWLEDGMENTS
The authors are grateful to the financial and analytical support rendered by the National Institute of Fundamental Studies, Hantane Road, Kandy, Sri Lanka.