Production of kombucha from Muntingia calabura L. leaves and evaluation of its antibacterial activity and total flavonoid content

Plant material

Muntingia calabura leaves were provided commercially and authenticated by the Division of Botany, Laboratory of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Universitas Muslim Indonesia.

Starter culture

The culture of kombucha was obtained from the Laboratory of Microbiology, Faculty of Pharmacy, Universitas Muslim Indonesia. The production of kombucha consisted of 1% of M. calabura leaves and 10% of sucrose. The starter culture of kombucha was used as a symbiotic culture of acidic bacteria and yeast (Kaewkod et al., 2019).

Preparation of kombucha

The M. calabura leaves were obtained in Makassar city. The leaves were picked at 10:00 a.m. and sorted. The leaves were dried in an oven at 105°C for 60 minutes to reduce the moisture content. The leaves were then mashed to facilitate the extraction process. As much as 5 g of M. calabura leaves was added to 500 ml of distilled water and boiled for 15 minutes. Into the hot tea, sucrose 10% was added. The mixture was cooled at room temperature. The starter culture 10% was inoculated into a mixture and left at fermentation temperature for 12 days (Kaewkod et al., 2019). After the fermentation process, the kombucha layer was removed and then washed with clean water. The kombucha product was sterilized.

Determination of pH and total acidity

The pH of kombucha was determined using a digital pH meter. The pH meter was calibrated with a phosphate buffer pH 4 and pH 7 before being used. The total acidity of kombucha was evaluated according to a previous study (Kaewkod et al., 2019). Kombucha was titrated with 0.1 M NaOH. Phenolphthalein 1% was used as an indicator of the reaction. The volume of the NaOH solution was calculated in terms of grams of acetic acid per liter of the sample.

Determination of alcohol content

Analysis of alcohol content was carried out using an ultraviolet-visible (UV-Vis) spectrophotometer (Thermo Scientific Evolution 201). The kombucha was prepared in various concentrations, namely, 1%, 4%, 7%, 10%, and 13%, and then analyzed at a maximum wavelength of 263 nm. In this analysis, the absorbance value of the sample whose concentration is known will be tested in percent. The linearity of the standard series was carried out through analysis. The results of the linearity equation were used to determine the amount of alcohol in the sample (Rahmi et al., 2013).

To support the data of the UV-Vis spectrophotometer, the alcohol content was determined also by high-performance liquid chromatography (HPLC). The HPLC analysis was performed by the standard addition method. A standard solution of ethanol was made with the concentration series. The sample of 2.5 ml of kombucha was diluted in 10 ml of distilled water. The standard ethanol addition is carried out by adding a standard solution of 10%, 15%, and 20% ethanol of as much as 1.5 ml to a volumetric flask containing 2.5 ml of the sample. The samples were injected into the HPLC system using column C18 reverse phase. A mixture of acetonitrile and phosphate buffer at a ratio of 5:9 at pH buffer 7 was used as the mobile phase. The HPLC data were analyzed to determine the amount of ethanol in the sample (Fawwaz and Baits, 2016; Rahmi et al., 2013).

Determination of microbiological contaminants

Total microbial analysis (bacteria and fungi) was carried out by taking 1 ml of each of the dilution samples and putting it in a sterile Petri dish. Furthermore, 15 ml of NA and potato dextrose agar was poured into the Petri dish, respectively. The Petri dishes were carefully rotated and moved horizontally or in parallel to homogenize the mixture and then allowed to solidify. The Petri dishes were incubated at a temperature of 36°C for 24 hours and 25°C for 48–72 hours for bacteria and fungi, respectively.

In the most probable number (MPN) test, nine test tubes each contained Durham tubes and lactose broth (LB) medium. A sample of 1 ml was inserted into each test tube based on the sample concentration series, namely, 100, 101, and 102, in three series of tubes and then incubated at 37°C for 24 hours.

Determination of antibacterial activity

The antibacterial activity was carried out using the agar diffusion method as in a previous study described with slight modification (Kaewkod et al., 2019). A single colony of bacteria Escherichia coli, Shigella dysenteriae, Salmonella typhi, Bacillus subtilis, and Vibrio cholerae was prepared in the form of a suspension in a 0.85% NaCl solution. The bacteria suspension was inserted of as much as 1 ml into a test tube of semisolid nutrient agar (NA) media, which was still liquid and homogenized, and then poured into a Petri dish that had been placed with a buffer. After the semisolid medium solidified, the kombucha with various concentrations of 0.1%, 1%, 10%, and 100% was put into the reservoir according to the concentration of each sample. The plates were further incubated at 37°C for 24 hours. Vernier calipers measured the zones of bacterial growth inhibition.

Determination of total flavonoid content

The total flavonoid content of kombucha was assessed by the aluminum chloride colorimetric method (Baba and Malik, 2015). Kombucha 100 µg/ml was prepared for 1 ml, and then 0.3 ml of a 5% NaNO₂ solution and 0.3 ml of a 10% AlCl₃ solution were added after 5 minutes of incubation. After the mixture was allowed to stand for 6 minutes, 2 ml of a 1 mol/L NaOH solution was added. The final volume of the mixture was brought to 10 ml with distilled water. The mixture was allowed to stand for 15 minues, and the absorbance was measured at 417 nm by a UV-Vis spectrophotometer. The total flavonoid content was calculated from a calibration curve, and the result was expressed as mg quercetin equivalent per g dry weight (mgQE/g).

pH and total acidity

The pH of kombucha (4.78) is safe for consumption because the acidity is higher than 2.5. As is known, the pH level of kombucha will drop dramatically during the fermentation process, from 6.71 to pH 2.91, and the lowest it can reach is 2.5 (Zhao et al., 2018). The acidic level, 0.22% as acetic acid, is in the range of the acidic level requirement from the Indonesia National Standard, which is 0.2–0.9%. During fermentation, there is a breakdown of sucrose into alcohol and also the formation of other organic acids by bacteria. The acid formed will release protons so that the pH value becomes low (Ganigué et al., 2016).

The fermentation time of kombucha affects its acidity. The longer fermentation time correlates to the total acid increases. This is because during the fermentation process yeast and bacteria metabolize sucrose and produce a number of organic acids such as acetic acid, gluconic acid, and glucuronic acid; therefore, an increase in the levels of organic acids occurs. The higher organic acids contained in kombucha contribute to the higher total acid (Martínez-Leal et al., 2018). It is known that the sour taste that occurs in kombucha is largely a contribution of the emergence of organic acids, antioxidants, and vitamin C in kombucha (Martínez-Leal et al., 2018).

Alcohol content

The quantitative analysis of alcohol was performed by UV-Vis spectrophotometry at a wavelength of 263 nm as the maximum wavelength of ethanol. The concentration series of the sample was analyzed by a UV-Vis spectrophotometer at the maximum wavelength. The absorbance of the sample can be seen in Table 1, and from these data, a linear curve was obtained with the R² value 0.996, as shown in Figure 1. Linear regression analysis was performed to prove the existence of a relationship between actual concentration and method response. The determination of the alcohol content in the kombucha was analyzed based on the calculation of the obtained linear regression equation y = 0.0658x + 0.1118. The alcohol content determined by the UV-Vis spectrophotometry method exhibited that the higher concentration of kombucha was in line with the absorbance, as shown in Table 1.

The chromatogram data of kombucha based on the ethanol addition standard using sample concentrations of 10%, 15%, and 20% were shown in Table 2. The determination of the alcohol content in the fermented kombucha leaves was performed by the HPLC method (Fig. 2). The analysis was carried out by the measurement of the maximum wavelength of pure alcohol using a UV-Vis spectrophotometer because the HPLC for beverage analysis uses a UV-Vis detector. The maximum wavelength (λ_max) of ethanol was 263 nm. Determination of alcohol content using HPLC using a phosphate buffer pH 7 has provided the maximum peak area. The pH 7 of the buffer solution was the optimum condition for analyzing the ethanol content of beverages using a mobile phase mixture of acetonitrile: phosphate buffer (5:9). The linear regression, y = 0.13x + 1.6167, was obtained by a linear curve equation based on the area of the peak with the concentration of the sample. The results of measuring the alcohol content by HPLC showed that the alcohol content of the sample with a concentration of 10% kombucha drink was 0.726%. The formation of alcohol in kombucha was the result of the fermentation process (Kaewkod et al., 2019).

Table 1. Alcohol content of kombucha from M. calabura leaves by UV-Vis spectrophotometry. [Click here to view]

Figure 1. Linear curve of kombucha from M. calabura leaves by a UV-Vis spectrophotometer. [Click here to view]

Table 2. Alcohol content of kombucha from M. calabura leaves by the HPLC [Click here to view]

Figure 2. The chromatogram of kombucha from M. calabura leaves. [Click here to view]

Microbiological contaminants

Microbiological analysis based on the total plate count (TPC) value of bacteria and fungi exhibited that the kombucha from M. calabura has no microbial contamination. The values of TPC-bacteria and fungi were zero colony forming unit (CFU)/ml and 2 × 100 CFU/ml, respectively. The value of the MPN of coliform and pathogenic bacteria exhibited the absence of bacterial growth on the LB medium and eosin methylene blue agar medium. Both microbiological contaminant assessments conclude that kombucha is safe as a daily beverage because the values were in the range of the standard of the Indonesian Food and Drug Authority (BPOM).

Table 3. Antibacterial activity of kombucha from M. calabura leaves by agar diffusion. [Click here to view]

Antibacterial activity

Kombucha showed a remarkable potency of antimicrobial activity against a pathogenic bacterium, which has demonstrated the inhibition of the growth of E. coli, S. dysenteriae, S. typhi, B. subtilis, and V. cholerae. The largest inhibition zone diameter was obtained at a concentration of 100% (32 mm) against S. typhi, as shown in Table 3.

These results showed that the fermented kombucha has antibacterial activity based on the formation of an inhibition zone. The activity was due to the presence of organic acids in the M. calabura leaves kombucha which can inhibit the growth of bacteria such as gluconic acid, glucuronic acid, ascorbic acid, and acetic acid (Kaewkod et al., 2019).

The diameter of the inhibition zone of kombucha with concentrations of 0.1%, 1%, and 10% indicates activity in the weak category while at a concentration of 100% the diameter of the inhibition zone toward bacteria E. coli (22 mm), S. typhi (32 mm), and B. subtilis (30 mm) was classified as strong activity, S. dysenteriae (19 mm) was classified as moderate activity, and V. cholerae (13 mm) was classified as weak activity. The interpretation of the antibacterial activity of an agent based on the magnitude of the clear zone is divided into three categories, namely, weak if the resulting clear zone diameter is ≤ 11 mm, moderate if the resulting inhibitory zone diameter ranges from 12 to 21 mm, and strong if the resulting clear zone diameter is ≥ 22 mm (Harmita and Radji, 2008).

The organic acids formed in the kombucha of M. calabura leaves during the fermentation process provide antibacterial activity. One of these compounds is acetic acid, and the formation of acetic acid in kombucha can inhibit the growth of Gram-negative bacteria (Sreeramulu et al., 2000). Undissociated acetic acid can damage the lipid bilayer structure of bacteria and introduce protons into the cytoplasm. The large number of intracellular protons makes the cytoplasm in acidic conditions, thus causing protein denaturation and energy loss. Therefore, the high acetic acid content can inhibit bacterial growth (Park et al., 2021). In addition, the organic acids that are formed in the kombucha will form the pH of the drink at an acidic level. The low pH of kombucha affects bacterial growth.

Total flavonoid content

The determination of total flavonoid content was performed by UV-Vis spectrophotometry at a wavelength of 417 nm as the maximum wavelength of quercetin. The concentration series of quercetin was measured by a UV-Vis spectrophotometer. The absorbances of the quercetin series were used to obtain the linear regression. The R² value was 0.996. Linear regression was performed to prove the existence of a relationship between the actual concentration and method response. The determination of the total flavonoid content in the kombucha was analyzed based on the calculation of the obtained linear regression equation y = 0.0081x − 0.0725.

The absorbance of the sample was measured in triplicate. The total flavonoid content in kombucha was expressed in QE, which is the amount of quercetin milligram equivalence in milliliters of samples. The calculation of total flavonoid levels based on the absorbance data exhibited that the kombucha contains 35 mg/ml QE. This result indicates that the kombucha has a potential to be an antioxidant due to its total flavonoid content. The polyphenol and the carotenoid compound contribute to the antioxidant activity of natural resources (Fawwaz., 2021). In addition, the total flavonoid content was positively correlated with the antibacterial activities. Although in this study we did not prove that, a previous study exhibited that the level of total phenolic and flavonoid content in the extract contributed to the antibacterial activities against E. coli and S. aureus (Mahboubi et al., 2015).

The total flavonoid content was determined using aluminum chloride reagents. Aluminum chloride will form a stable complex with the carbonyl groups in C4 and hydroxyl in C3 (flavanols) and C5 in flavanols and flavones. The principle of the aluminum chloride method is the formation of a stable complex with the C4 keto group, as well as in the C3 or C5 hydroxyl groups of flavones and flavanols. In addition, aluminum chloride forms a stable acidic complex with the orthohiroxyl groups in the A- or B- rings of flavonoid compounds. Quercetin was chosen as a comparison solution because it is one of the flavonoid group compounds that can react with aluminum chloride to form complexes (P?kal and Pyrzynska, 2014).