Biology, Medicine, & Natural Product Chemistry ISSN 2089-6514 (paper) Volume 14, Number 1, April 2025 | Pages: 453-463 | DOI: 10.14421/biomedich.2025.141.453-463 ISSN 2540-9328 (online) Potential of Kedabu (Sonneratia ovata Backer) Fruit Juice Supplemented in Kombucha Beverage as Antibacterial Agent Musyirna Rahmah Nasution*, Clara Nabilah, Meiriza Djohari, Nurhapipah Departement of Pharmacy, Sekolah Tinggi Ilmu Farmasi Riau, Pekanbaru, Riau, Indonesia. Corresponding author* musyirnarahmah@stifar-riau.ac.id Manuscript received: 03 May, 2025. Revision accepted: 15 July, 2025. Published: 18 July, 2025. Abstract Riau Province has abundant natural resources of mangrove forests almost along the coast of Riau. Mangrove fruit can be developed into various processed foods, but processed foods derived from mangroves have not been widely developed and are of interest to coastal communities. Kombucha tea is a probiotic beverage produced through the fermentation of tea and sugar using a kombucha starter known as SCOBY (Symbiotic Culture of Bacteria and Yeast). The presence of sugar in kombucha colonies is a vital source of nutrition. Additionally, fruit juice can be incorporated to create variations of kombucha tea. Kedabu fruit (Sonneratia ovata B.), a native fruit of Riau Province, is recognized for its high carbohydrate content and secondary metabolites, which exhibit potential antibacterial properties. This research developed four formulations of kombucha tea, each with different concentrations of kedabu fruit juice (0%, 10%, 20%, and 30%). The study’s objectives were to formulate, evaluate the physicochemical properties and antibacterial activity of these formulations against Escherichia coli and Staphylococcus aureus. The organoleptic properties, alcohol content, pH, %TTA (Total Acidity Titratable), total LAB (Lactic Acid Bacteria), and yeast count were assessed during a 14-day fermentation period. The assay results indicated that the kombucha fruit kedabu tea formulation exhibited favorable organoleptic characteristics, a decrease in pH, an increase in %TTA, total LAB, and yeast count within the acceptable range defined by standards. Moreover, it displayed inhibitory activity against Escherichia coli and Staphylococcus aureus bacteria, falling within the weak to moderate categories. Keywords: Kombucha; Kedabu; Antibacterial; Escherichia coli; Staphylococcus aureus. INTRODUCTION Riau Province, located in Indonesia, is known for its abundant natural resources including land, mining, agriculture, forestry, marine, and industry. With a coastline stretching over 2,078 kilometers, the province boasts extensive mangrove forests along its shores. Mangroves offer a wide range of valuable resources that have been extensively utilized by coastal communities, both in the form of timber forest products and non-timber forest products. However, the development of processed foods derived from mangroves remains limited, despite the potential demand from these communities. Mangroves are a type of plant that thrives in tidal areas, particularly along protected tropical and sub-tropical coasts as well as sheltered sub-tropical regions. The mangrove plant encompasses various tree species, including Avicennia, Sonneratia, Rhizophora, Bruguiera, Ceriops, Lumnitzera, Excoecaria, Xylocarpus, Aegiceras, Scyphyphora, and Nypai (Kathiresan et al, 2001). One of the mangrove plants is Kedabu (Sonneratia ovata Backer) which is utilized in food and health. Gastrointestinal tract infections pose a significant health concern due to the digestive system’s vulnerability to bacterial invasion. As the primary entry point for food and drinks, the gastrointestinal tract risks contamination by pathogenic bacteria. Traditionally, antibiotics have been utilized to treat bacterial infections. However, the indiscriminate use of antibiotics can result in bacterial resistance, necessitating the exploration of alternative treatment options (Aminov, 2010). One such approach to prevent gastrointestinal tract infections is using kombucha tea. Kombucha tea is a fermented beverage produced by the fermentation of black tea and sugar using a symbiotic culture of bacteria and yeast (SCOBY). The fermentation process typically takes place over 7-14 days. SCOBY is composed of various microorganisms, including acetic acid bacteria (such as Acetobacter xylinum, Acetobacter acetic, and Gluconobacter oxydans), yeast (such as Saccharomyces cerevisiae, Zygosaccharomyces sp, and Torulopsis sp), and lactic acid bacteria (such as Lactobacillus sp and Lactococcus sp) (Laureys et al., 2020). Kombucha is known to contain a diverse array of chemical compounds, including acetic acid, gluconic acid, lactic acid, vitamins (B1, B2, B6, B12, C), ethanol, carbon dioxide, polyphenols, and others (Herwin, 2022). These compounds contribute to the pharmacological https://doi.org/10.14421/biomedich.2025.141.453-463 454 Biology, Medicine, & Natural Product Chemistry 14 (1), 2025: 453-463 effects of kombucha tea, which include improved digestion, antibacterial properties, antioxidant activity, and more. The presence of secondary metabolite compounds such as triterpenoids, alkaloids, phenolics, saponins, and flavonoids. These compounds are known for their potential health benefits and contribute to the overall bioactive profile (Chandraekara et al., 2018). Furthermore, a study revealed that kombucha tea exhibited inhibitory effects on Escherichia coli and Staphylococcus aureus, with inhibition diameters of 20.5 mm and 20.15 mm, respectively, after a 14-day fermentation period (Bhattacharya et al., 2016). Escherichia coli and Staphylococcus aureus were the most susceptible bacteria to the inhibitory action of kombucha tea, resulting in inhibition diameters of 19 mm and 18 mm, respectively. Increased fermentation time of kombucha tea led to an enhanced antibacterial activity (Al-Mohammadi et al., 2021). One of the crucial factors influencing kombucha production is the presence of sugar. Sugar plays a vital role as an energy source for yeast in the SCOBY (Miranda et al., 2016). Within the SCOBY, yeast metabolizes sugar by converting it into glucose and fructose, which are subsequently fermented into ethanol. Acetobacter sp. then utilizes ethanol to produce acetic acid (Laureys et al., 2020). In kombucha tea, it is possible to substitute sugar with fruit juice as an alternative sweetener. Sonneratia ovata Backer, commonly known as kedabu or pedada plant, is a mangrove species frequently found along the coastlines of Riau Province, Indonesia. The fruit of the kedabu plant is a significant source of nutrients. It can be processed into various products, including candy, jam, syrup, and dodol. Importantly, the kedabu fruit is non-toxic and can be consumed directly. Analysis conducted by Astuti et al. revealed that Sonneratia ovata Backer fruit contains approximately 2.19% carbohydrates. Furthermore, phytochemical screening demonstrated the presence of flavonoids, phenolics, and saponins and triterpenoids (such as oleanolic acid and maslinic acid) in the extracts and fractions of Sonneratia ovata fruit (Wu et al., 2009). Research exploring the utilization of kedabu fruit in functional food products remains limited. This is notable given the fruit's potential as a source of secondary metabolite compounds with antibacterial properties, as well as its high nutritional content. Moreover, no existing publications specifically focused on kombucha tea made from kedabu fruit, which presents an interesting opportunity for further investigation. Thus, this study aimed to formulate kombucha tea by incorporating kedabu fruit juice and evaluate its antibacterial activity against Escherichia coli and Staphylococcus aureus bacteria. By developing kombucha tea using kedabu fruit juice, researchers aim to highlight the potential of this fruit in Riau Province, Indonesia, and promote its utilization as an alternative antibacterial remedy MATERIALS AND METHODS Sampling and Sample Identification The samples were collected from West Dumai District, Dumai City, Riau, Indonesia. The samples were then taken to the Botanical Laboratory, Department of Biology, Faculty of Mathematics and Natural Sciences, University of Riau, Pekanbaru, for identification. The sample identification was conducted under the reference number 611/UN19.5.1.1.3-4.1/EP/2021 (Table 2). Kedabu Fruit Juice Preparation Fresh Kedabu fruit weighing 500 grams was thoroughly washed. The fruit flesh was then finely chopped into small pieces and mixed with water at 1:2 (w/v). The mixture was blended using a blender to obtain a homogeneous fruit pulp. Subsequently, the kedabu fruit pulp was filtered to obtain pure kedabu fruit juice. The kedabu fruit juice was further prepared in concentrations of 10%, 20%, and 30% by dilution (Chan et al., 2021). Formulation of Kombucha Formulation details can be found in Table 1. 250 mL of distilled water was boiled in a stainless steel pot to prepare the kombucha tea. Black tea, sugar, and kedabu juice were added to the boiling water. The mixture was then filtered and transferred to a glass jar. The kombucha tea mixture was allowed to cool down to 25°C. Next, 10% (v/v) of kombucha starter culture was added to the mixture, and distilled water was added to bring the total brew volume to 500 mL. The mixture was homogenized thoroughly. The glass jar was covered with a clean cloth or tissue and securely tied. Fermentation was conducted at a temperature of 25°C for 14 days. Samples were taken for antibacterial and chemical analysis during the fermentation process. Table 1. Formulation of kombucha tea with kedabu fruit juice. No Composition *F1 F2 F3 F4 1 Black Tea 1,6% 1,6% 1,6% 1,6% 2 Sugar 10% 10% 10% 10% 3 Kombucha Starter 10% 10% 10% 10% 4 Kedabu Fruit Juice 0% 10% 20% 30% 5 Aquadest ad 500 mL ad 500 mL ad 500 mL ad 500 mL Note : Formula (*F) Nasution et al. – Potential of Kedabu (Sonneratia ovata Backer) Fruit Juice Supplemented … 455 Evaluation of chemical changes and antibacterial activity Some parameters used in evaluating the quality of kombuca tea supplemented with kedabu fruit juice as a fermented beverage were organoleptic test, phytochemical screening test, determination of pH with pH meter, alcohol content with alcohol meter. The total tritabel acidity (TTA) was determined using the alkalimetric method. Determination of total lactic acid bacteria (LAB) and total yeast in kombucha tea is done by using the standar plate count/SPC method (Pour plate method). As a functional beverage, one pharmacological effect test was antibacterial activity against E coli and S aureus bacteria using the disc diffusion method. Evaluation of all parameters was made before fermentation and after fermentation on the 7th and 14th day. Data analysis The data obtained from the antibacterial activity assay of kombucha tea supplemented with kedabu fruit juice against Escherichia coli and Staphylococcus aureus bacteria, collected at different time points (before fermentation, 7th day, and 14th day of fermentation), were analyzed using the One Way ANOVA statistical test. The analysis was performed using the SPSS application to determine if there were any significant differences among the groups. To further determine which formula group exhibited the best antibacterial activity, a post-hoc analysis was conducted using the Tukey test. This test allows for a pairwise comparison between groups to identify significant differences. Additionally, the data obtained from the evaluation of the kedabu fruit kombucha tea formula, which included organoleptic test, pH test, total lactic acid bacteria (LAB) test, total titratable acid (TTA) test, total yeast test, and alcohol test, were analyzed descriptively. Descriptive analysis provides a comprehensive summary of the data, including measures of central tendency and variability, to describe the characteristics of the kedabu fruit kombucha tea formula. These data analyses provide valuable insights into the antibacterial activity and overall quality of the kedabu fruit kombucha tea formula, allowing for an objective assessment of its effectiveness as a functional beverage. RESULTS AND DISCUSSION Sample Identification The sample identification was conducted under the reference number 611/UN19.5.1.1.3-4.1/EP/2021 (Table 2). The results of sample identification showed that the sample had the regional name Kedabu and the species name Sonneratia ovata Backer. Table 2. The result of the identification sampel Sample Identification Sample Kingdom Plantae Phylum Magnoliophyta Classis Magnoliosida Ordo Myrtales Family Lythraceae Genus Sonneratia Species Sonneratia ovata Backer Regional Name Kedabu Figure 1. Kedabu Plant (a); Kedabu Fruit (a and c). Kedabu or pedada plants (Sonneratia sp.) are a species of mangrove plants commonly found along the coastlines of Riau Province, Indonesia. The fruit of the kedabu plant has gained attention due to its nutritional composition and bioactive compounds (Halifah et al, 2019). Exploiting natural resources to develop functional food products is an important aspect of utilizing traditional medicinal plants. In this study, the focus was on the production of kombucha tea using kedabu fruit juice as an ingredient. Kombucha tea is a fermented beverage known for its potential health benefits and is typically prepared using black tea and sugar fermented by a symbiotic culture of 456 Biology, Medicine, & Natural Product Chemistry 14 (1), 2025: 453-463 FI FIV bacteria and yeast (SCOBY) (Chakravorty et al, 2016). By incorporating kedabu fruit into the kombucha tea formulation, it is possible to enhance the nutritional and bioactive properties of the final product. The results of the sample identification confirmed that it was Sonneratia ovata Backer, commonly known as kedabu fruit. The phytochemical screening test indicated that the fermentation time and addition of kedabu fruit juice significantly effected the content of secondary metabolites, increasing their levels. Kombucha tea enriched with kedabu fruit juice exhibited higher secondary metabolites, including flavonoids, phenolics, terpenoids, and saponins (Table 3). These secondary metabolites are well-known for their pharmacological effects, such as antimicrobial, antioxidant, and antidiabetic activities (Herwin, 2022). Table 3. The results of screening phytochemical of kombuca tea with or without kedabu fruit juice. Secondary Metabolite Reagent Before Fermentation After fermentation on 14 days Kombucha Tea Kombucha Tea + Kedabu Juice Kombucha Tea Kombucha Tea + Kedabu Juice Alkaloid Mayer - - - - Fenolik Logam Mg + HCl(P) + + + + Flavonoid FeCl3 1 % + + + + Terpenoid Lieberman-Bouchard - + + + Steroid Lieberman-Bouchard - - - - Saponin Air - + - + Evaluation of chemical changes and antibacterial activity. The evaluation of kombucha tea's quality was conducted based on the standard parameters outlined in SNI 7552- 2018 for fermented beverages. The organoleptic test was performed after a fermentation period of 14-days. The results demonstrated that an extended fermentation time led to a lighter color of kedabu fruit kombucha tea (Table 4). This color change can be attributed to the activity of microorganisms responsible for color degradation. Bacteria utilize total soluble solids as an energy source, resulting in a depletion of solvents in the media and a clearer liquid (Jayabalan et al., 2014). Regarding the aroma, the observations of kombucha tea indicated that a longer fermentation time resulted in a more distinct aroma in the kedabu fruit kombucha. Furthermore, increasing the concentration of kedabu fruit led to a more pronounced sour taste. The sour taste in kedabu fruit kombucha tea is attributed to the production of lactic acid and acetic acid during the fermentation process (Wang et al., 2022) Table 4. Observation of kedabu fruit kombucha tea before and after fermentation for 14 days Description The Color of Kedabu Fruit Kombucha Tea Before Fermentation (0-day) After Fermentation on 14- day Note : F1: Kombucha tea without kedabu juice ; F2,F3, F4 : Kombucha tea+kedabu juice (10%, 20% ,30%) FII FIII FIV FI FII FIII FIV Nasution et al. – Potential of Kedabu (Sonneratia ovata Backer) Fruit Juice Supplemented … 457 The subsequent test involved measuring the alcohol content using an alcohol meter. Alcohol measurement is a crucial aspect in assessing the quality and safety of kombucha tea. As fermented beverages, kombucha is known to contain alcohol. In Indonesia, where the majority of consumers are Muslims, it is essential for these beverages to comply with the halal food standards established by the Indonesian Ulama Council (MUI). According to MUI (Fatwa No. 10 of 2018) (FMUIN, 2018). fermented beverage products containing less than 0.5% alcohol/ethanol are considered halal if they are medically harmless (Pauzi et al., 2019) In this study, the obtained results revealed that the alcohol content in all formulations of kombucha tea was 0% throughout the fermentation process from day-0 to day-14 (Table 5). It is worth noting that previous research has indicated that the alcohol content in kombucha generally ranges from 0.1% to 1%. Table 5. Evaluation of organoleptic test, alcohol content, %tta, total lab, total yeast and antibacterial activity of kedabu fruit kombucha tea formulations Formula Parameters Observation Results 0-day 7-day 14- day FI Taste Sweet Sour Sour Flavor typical kombucha falvor typical kombucha falvor typical kombucha falvor Color dark brown dark brown brown pH 3,37 2,94 2,92 Alcohol 0% 0% 0% %TTA 0,086% 0,33% 0,51% Total LAB 9,9 x 107 cfu/mL 18,1 x 107 cfu/mL 16 x 107 cfu/mL Total Yeast 2,32 x 106 cfu/mL 1,56 x 106 cfu/mL 2,47 x 106 cfu/mL Antibacterial Activity Escherichia coli 6,8 mm 7,8 mm 8,2 mm Antibacterial Activity Staphylococcus aureus 7,2 mm 8,6 mm 8,3 mm FII Taste Sweet Sour Sour Flavor typical kombucha falvor typical kombucha falvor typical kombucha falvor Color Dark brown light brown golden brown pH 3,25 2,86 2,72 Alcohol 0% 0% 0% %TTA 0,16% 0,35% 0,75% Total LAB 6,5 x 107 cfu/mL 16,2 x 107 cfu/mL 13,4 x 107 cfu/mL Total Yeast 1,98 x 106 cfu/mL 1,3 x 106 cfu/mL 2,29 x 106 cfu/mL Antibacterial Activity Escherichia coli 7,2 mm 8,03 mm 9 mm Antibacterial Activity Staphylococcus aureus 7,6 mm 8,8 mm 10,2 mm FIII Taste Sweet Sour Sour Flavor Kedabu flavor typical kombucha falvor typical kombucha falvor Color brown light brown golden brown pH 3,25 2,92 2,61 Alcohol 0% 0% 0% %TTA 0,16% 0,58% 0,99% Total LAB 5,1 x 107 cfu/mL 9,95 x 107 cfu/mL 9,9 x 107 cfu/mL Total Yeast 1,53 x 106 cfu/mL 1,19 x 106 cfu/mL 1,9 x 106 cfu/mL Antibacterial Activity Escherichia coli 7,4 mm 8,6 mm 9,96 mm Antibacterial Activity Staphylococcus aureus 7,9 mm 9,2 mm 10,93 mm FIV Taste Sour Sour Sour Flavor Kedabu flavor typical kombucha falvor typical kombucha falvor Color brown light brown golden brown pH 3,18 2,8 2,5 Alcohol 0% 0% 0% %TTA 0,17% 0,58% 1,02% Total LAB 6,15 x 107 cfu/mL 6,95 x 107 cfu/mL 8,55 x 107 cfu/mL Total Yeast 1,3 x 106 cfu/mL 0,92 x 106 cfu/mL 1,68 x 106 cfu/mL Antibacterial Activity Escherichia coli 7,8 mm 10,5 mm 11,8 mm Antibacterial Activity Staphylococcus aureus 7,93 mm 10,3 mm 15 mm 458 Biology, Medicine, & Natural Product Chemistry 14 (1), 2025: 453-463 The pH measurements of all formulations revealed a decrease in pH values ranging from 3.37 to 2.5 (Table 5). As the concentration of kedabu juice in kombucha tea increased, the pH value decreased. The duration of fermentation also influenced the pH value. The highest pH value was observed on the 0th day. The fermentation time increases, the pH value decreases (Nurikasari et al., 2017). The decrease in pH value in kedabu fruit kombucha tea is attributed to the conversion of glucose into organic acids by microorganisms. The concentration of organic acids increases with prolonged fermentation (Laureys et al., 2020). The pH of kombucha tea typically ranges from 2.5 to 4.2 (Murphy et al., 2018). Overall, the pH of the produced kedabu fruit kombucha tea meets the general pH requirements for kombucha tea. The total titratable acid (TTA) values were measured using the alkalimetric titration method. All formulas exhibited changes in TTA values ranging from 0.086% to 1.02% (Table 5). The maximum % total acid in kombucha tea is 3% (w/v) (Greenwalt et al., 2000). Thus, the overall results obtained in this study meet the standard % total acid requirement for kombucha tea. The %TTA increases with the addition of kedabu juice in kombucha tea and the duration of fermentation. The increase in %TTA is associated with the enhanced activity of lactic acid bacteria in metabolizing sucrose into organic acids (Nguyen et al., 2015). The relationship between total titratable acid and pH is inversely proportional, meaning that as the %TTA value increases, the pH value decreases and vice versa. This observation is consistent with the findings of previous research on kombucha tea (Laureys et al., 2020). The acidic nature of kombucha tea, attributed to the presence of organic acids such as acetic acid and lactic acid, contributes to its characteristic taste and potential health benefits. Acetic acid is the primary organic acid produced during kombucha fermentation. It plays a crucial role in inhibiting the growth of pathogenic bacteria. The formation of acetic acid in kombucha fermentation has been found to have potential antibacterial properties (Herwin, 2022). As acetic acid is produced in kombucha, it undergoes decomposition, releasing free protons that lead to a decrease in pH (Hamed et al., 2012). Acetic acid in kombucha can disrupt the lipid bilayer structure of bacterial cells by introducing protons into the cytoplasm. This accumulation of intracellular protons results in acidic conditions, leading to protein denaturation and energy loss within the bacterial cells. The higher the content of organic acids, particularly acetic acid, the greater the potential for inhibiting the growth of pathogenic bacteria. The ideal formation of organic acids in kombucha reduces pH, creating highly acidic conditions that significantly affect bacterial growth. This acidic environment inactivates the cytoplasm of pathogenic bacteria and causes severe damage to their cells (Herwin, 2022). These findings highlight the antimicrobial potential of acetic acid in kombucha and its ability to create an unfavorable environment for pathogenic bacteria. The presence of acetic acid, along with other organic acids formed during kombucha fermentation, contributes to its overall antimicrobial activity and potential health benefits. The results of the total lactic acid bacteria (LAB) testing indicate that all treatments kedabu fruit kombucha tea contained a sufficient amount of LAB, meeting the minimum requirements for probiotic beverage quality standards, which is at least 107 cfu/mL (Nyanzi et al., 2021). The total LAB count increased from the 0th day to the 7th day of fermentation. This increase can be attributed to the availability of an adequate nutrient source, promoting the growth of LAB. However, from the 7th day until the 14th day, there was a decrease in the total LAB count (Table 5). This decline may be attributed to the depletion of nutrients in the kedabu fruit kombucha tea medium. At the initial stages of fermentation, there is an abundance of nutrients that actively support the division and growth of bacteria. However, as fermentation progresses, the available nutrients decrease, reducing in bacterial activity and reproduction. This decline in LAB count indicates that the bacteria have surpassed their logarithmic growth phase and are entering a phase of decreased reproduction (Villarreal et al., 2018). These findings highlight the dynamic nature of LAB growth during the fermentation of kedabu fruit kombucha tea. The initial increase in LAB count indicates active fermentation and bacterial proliferation, while the subsequent decline can be attributed to nutrient depletion. It is essential to carefully monitor the fermentation process to ensure optimal LAB growth and maintain the probiotic qualities of kombucha tea. The minimum requirement for total yeast in kombucha tea is at least 104 cfu/mL (Nyhan et al., 2022). The study results indicate that all kedabu juice kombucha tea treatments met this requirement, with yeast counts exceeding 104 cfu/mL. The findings demonstrate that the length of fermentation time has an impact on the total yeast count. A decrease in yeast count was observed from the 0th day to the 7th day of fermentation, followed by an increase from the 7th day to the 14th day. The initial decrease in yeast count during the early stages of fermentation can be attributed to the yeast undergoing a lag phase or an adaptation phase with its environment. During this phase, some enzymes may not have been synthesized yet, and metabolic processes are still slow, resulting in a lack of cell division. As a result, the yeast count may remain stable or even decrease during this phase. However, as fermentation progresses, the yeast adapts to its environment, and the growth rate increases. The growth rate of yeast is influenced by the availability of substrates and the pH of the kombucha medium, which Nasution et al. – Potential of Kedabu (Sonneratia ovata Backer) Fruit Juice Supplemented … 459 FI FII FIII FIII provides an ideal environment for yeast growth and the conversion of glucose into alcohol and organic acids (De Miranda et al., 2022). In the case of kedabu juice kombucha tea, the substrate for yeast growth is obtained from the kedabu juice. These findings emphasize the dynamic nature of yeast growth during kombucha fermentation. The initial decrease in yeast count is followed by an increase as the yeast adapts to the fermentation environment and utilizes the available substrates. Monitoring and understanding the yeast population dynamics during fermentation is crucial to ensure the desired quality and characteristics of kombucha tea. Table 6. The result of antibacterial activity of kedabu juice kombucha tea before fermentation. Formula Diameter of Inhibition (mm) → Eschericia coli Average ± SD I II III K (-) 6 6 6 6 ± 0a K (+) 21 24 22 22,3 ± 1,53b F1 7,1 6,2 7,0 6,8 ± 0,49 a F2 7,2 6,9 7,4 7,2 ± 0,25 a F3 7,5 6,8 8,0 7,4 ± 0,6 a F4 7,8 7,0 8,5 7,8 ± 0,75 a Formula Diameter of Inhibition (mm)→ Staphylococcus aureus Average ± SD I II III K (-) 6 6 6 6 ± 0a K (+) 23,3 25,1 26 24,8 ± 1,37b F1 7,8 7,1 6,8 7,2 ± 0,51a F2 8,5 7,1 7,4 7,6 ± 0,74 a F3 8,8 7,4 7,5 7,9 ± 0,78 a F4 8,7 7,6 7,5 7,93 ± 0,66 a Figure 2. Antibacterial Activity of Kedabu Fruit Kombucha Tea against bacteria Escherichia coli and Staphylococcus aureus before fermentation (on 0th day). Table 7. The result of antibacterial activity of kedabu juice kombucha tea after fermentation 7-day. Formula Diameter of Inhibition (mm) → Eschericia coli Average ± SD I II III K (-) 6 6 6 6 ± 0a K (+) 22,1 21,7 21 21,6 ± 0,56c F1 9,2 7,2 7,0 7,8 ± 1,22ab F2 9,5 7,4 7,2 8,03 ± 1,27 ab F3 10 7,6 8,1 8,6 ± 1,26 ab F4 11 7.8 10 10,5 ± 0,7b Formula Diameter of Inhibition (mm) → Staphylococcus aureus Average ± SD I II III K (-) 6 6 6 6 ± 0a K (+) 21,6 22,1 23,5 22,4 ± 0,98c F1 8,7 9,1 8,0 8,6 ± 0,56 b F2 9 9,4 7,9 8,8 ± 0,78 b F3 9,1 9,6 9 9,2 ± 0,32 b F4 9,6 11 10,2 10,3 ± 0,7 b 460 Biology, Medicine, & Natural Product Chemistry 14 (1), 2025: 453-463 FI K(-) FII FIV FI FIII FI FIV FIII FI FII K(-) FIV FIII Figure 3. Antibacterial Activity of Kedabu Fruit Kombucha Tea against Escherichia coli and Staphylococcus aureus on 7-day. Table 8. The result of antibacterial activity of kedabu juice kombucha tea after fermentation day-14. Formula Diameter of Inhibition (mm) → Eschericia coli Average ± SD I II III K (-) 6 6 6 6 ± 0a K (+) 21,5 22,3 24 22,6 ± 1,27d F1 7,8 8,3 8,4 8,2 ± 0,32ab F2 8,2 9,9 8,9 9 ± 0,85b F3 10,5 9 10,4 9,96 ± 0,84bc F4 11 12 12,5 11,8 ± 0,7c Formula Diameter of Inhibition (mm) → Staphylococcus aureus Average ± SD I II III K (-) 6 6 6 6 ± 0 a K (+) 22,4 23,4 23 22,93 ± 0,5e F1 8,7 8,3 7,9 8,3 ± 0,4b F2 11,3 10,2 9 10,2 ± 1,15bc F3 12,4 10,5 9,9 10,93 ± 1,3c F4 15,5 15,2 14,4 15 ± 0,56d Figure 4. Antibacterial Activity of Kedabu Fruit Kombucha Tea against Escherichia coli and Staphylococcus aureus on 14th day. The antibacterial activity test was conducted to evaluate the effectiveness of kombucha tea with and without the addition of kedabu fruit juice against Escherichia coli and Staphylococcus aureus bacteria. Four different formulations of kedabu fruit juice kombucha tea (designated as F1, F2, F3, and F4) were tested, and their inhibitory effects were categorized as weak to moderate. Statistical analysis using the One Way ANOVA test revealed a significant difference in the diameter of the inhibition zones among the various kombucha tea formulas containing kedabu fruit juice. Further analysis using the Tukey test indicated that the formula labeled F4 exhibited a significant difference in inhibiting Escherichia coli and Staphylococcus aureus bacteria. These results suggest that increasing the concentration of kedabu fruit juice in kombucha tea leads to a greater diameter of inhibition zone formation. The observed antibacterial activity can be attributed to bioactive compounds in kombucha tea, such as organic Nasution et al. – Potential of Kedabu (Sonneratia ovata Backer) Fruit Juice Supplemented … 461 acids, phenolic compounds, and other secondary metabolites. The concentration of these bioactive compounds is known to increase during the fermentation process, especially with prolonged fermentation time. The elevated levels of organic acids, including lactic acid and acetic acid, contribute to the stronger antibacterial activity of the kombucha tea. This finding is consistent with previous research demonstrating that organic acid content increases with prolonged fermentation time (Jayabalan et al., 2007). It is important to note that the antibacterial activity of kombucha tea is influenced by various factors, including the type and concentration of bioactive compounds, fermentation conditions, and the targeted bacteria. Further studies are needed to elucidate the specific mechanisms underlying the antibacterial effects of kombucha tea and to identify the key bioactive components responsible for its activity against Escherichia coli and Staphylococcus aureus. The inhibitory effects of kedabu fruit kombucha tea against Escherichia coli and Staphylococcus aureus bacteria can be attributed to the presence of lactic acid and acetic acid, which are produced by lactic acid bacteria (LAB) and acetic acid bacteria (AAB). These organic acids have been recognized as significant antibacterial agents in kombucha tea. The antibacterial activity of organic acids is closely associated with the pH of the medium. As the pH decreases, the antibacterial activity of the organic acids increases. Lactic acid and acetic acid have the ability to disrupt the metabolic processes of pathogenic bacteria by damaging enzyme activity, as well as the structure and function of cell walls and membranes. This disruption inhibits nutrient absorption and protein synthesis in the bacteria. Furthermore, LAB-derived bacteriocin compounds can also contribute to the antibacterial activity by disrupting the membranes of pathogenic bacteria. These compounds damage essential components such as potassium ions and ATP, leading to intracellular pH imbalance and ultimately cell failure. The findings mentioned above are supported previous research who investigated the antibacterial mechanisms of organic acids and bacteriocins produced by LAB. Their study demonstrated the damaging effects of these compounds on the cell membranes and intracellular components of bacteria, leading to the inhibition of growth and survival (Hou et al., 2021). It is important to note that the antibacterial activity of kedabu fruit kombucha tea is a complex interplay of various factors, including the specific composition of organic acids, the presence of other bioactive compounds, and the targeted bacteria strains. Further research is needed to explore the detailed mechanisms of action and the specific interactions between the organic acids and pathogenic bacteria. The kedabu fruit kombucha tea has been found to contain various secondary metabolites as indicated by the results of phytochemical screening, including flavonoids, phenolics, terpenoids, and saponins (Table 3). Phenolic compounds identified in kombucha tea include epicatechin gallate, epigallocatechin, catechin, epicatechin, and epigallocatechin gallate. These compounds have been associated with pharmacological activities (Kaewkod et al., 2019). Flavonoid compounds present in kedabu fruit kombucha tea have been shown to exhibit inhibitory effects on nucleic acid synthesis during DNA and RNA formation. Additionally, flavonoids have the potential to disrupt bacterial cell wall permeability, microsomes, and lysosomes through interactions with bacterial DNA (Cushnie et al., 2005). The findings mentioned above highlight the presence of various bioactive compounds in kedabu fruit kombucha tea, particularly phenolic and flavonoid compounds. These compounds have been extensively studied for their potential therapeutic properties and antimicrobial activities. Further investigations are necessary to explore the specific mechanisms by which these secondary metabolites interact with bacterial DNA and exert their pharmacological effects. The inhibitory effect of phenolic compounds on bacterial cell walls is achieved by interfering with forming the cell wall peptidoglycan layer, disrupting it (González et al., 2009). Terpenoids, on the other hand, act as antibacterial agents by damaging transmembrane proteins, which serve as pathways for the entry and exit of compounds, thereby reducing the permeability of bacterial cell walls. Consequently, the lack of nutrients in the bacterial cells leads to their death (Cowan, 1999). Saponins exert their antibacterial activity through a mechanism involving the diffusion of saponins through the outer membrane and cell wall. Once inside the bacterial cell, saponins bind to the cytoplasmic membrane, destabilizing it and causing the release of cytoplasmic contents. Ultimately, this disruption leads to cell death (Pandey et al., 2011). The mechanisms described above illustrate how phenolic compounds, terpenoids, and saponins exhibit antibacterial effects by targeting different components of bacterial cells. These mechanisms contribute to the understanding of the potential antimicrobial activities of these secondary metabolites found in kedabu fruit kombucha tea. CONCLUSIONS Kombucha tea supplemented with kedabu fruit juice complies with the quality standards of fermented beverages and exhibits antibacterial activity against Escherichia coli and Staphylococcus aureus. The Kombucha tea made from kedabu fruit juice demonstrates favorable results in various quality parameters, including organoleptic evaluation, pH value, total titratable acidity, and the counts of lactic acid bacteria (LAB) and yeast, all of which meet the 462 Biology, Medicine, & Natural Product Chemistry 14 (1), 2025: 453-463 established standards. Additionally, the kombucha tea exhibits inhibitory effects against Escherichia coli and Staphylococcus aureus bacteria, falling within the weak to moderate categories. Statistical analysis using One Way ANOVA reveals a significant variation in the diameter of the inhibition zone among the different formulas of kedabu fruit kombucha tea. Further analysis using the Tukey test demonstrates that formula 4 significantly differs in its inhibitory effects on Escherichia coli and Staphylococcus aureus bacteria. 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