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Article

Development of Low-Caffeine Kombucha Using Lotus Root Tea and an Evaluation of Its Functional Properties

by
Jin Seon Baek
1,
Younhee Nam
1,
Sunghee Kim
2,
Hee Song Kim
3,
Eun Jin Lee
3,
Mee-Ryung Lee
3,* and
Soo Rin Kim
1,2,4,*
1
Department of Advanced Bioconversionce, Kyungpook National University, Daegu 41566, Republic of Korea
2
School of Food Science and Biotechnology, Kyungpook National University, Daegu 41566, Republic of Korea
3
Department of Food and Nutrition, Daegu University, Gyeongsan 38453, Republic of Korea
4
Research Institute of Tailored Food Technology, Kyungpook National University, Daegu 41566, Republic of Korea
*
Authors to whom correspondence should be addressed.
Beverages 2025, 11(2), 55; https://doi.org/10.3390/beverages11020055
Submission received: 12 March 2025 / Revised: 9 April 2025 / Accepted: 17 April 2025 / Published: 18 April 2025
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Abstract

:
Kombucha, traditionally fermented from black or green tea, is well known for its potential health benefits. However, its high caffeine content may limit consumption for certain individuals. Therefore, this study aimed to develop a low-caffeine kombucha using lotus root tea as an alternative to black or green tea. Lotus root was roasted and brewed to prepare the tea base, to which sugar and a SCOBY were added for primary fermentation. Subsequently, Lactobacillus plantarum (1.0 × 109 and 3.0 × 109 CFU/mL) was inoculated to carry out secondary fermentation. The kombucha samples were assessed for their organic acid composition, antioxidant activity, antimicrobial effects, β-glucuronidase inhibition, and protective effects against Salmonella infection in a Caenorhabditis elegans model. The caffeine concentration of lotus root tea kombucha was significantly lower than that of conventional kombucha. L. plantarum fermentation increased the lactic acid concentration and enhanced antimicrobial activity, particularly against Escherichia coli OP50 and Salmonella typhimurium. Additionally, β-glucuronidase inhibition significantly improved, suggesting potential gut health benefits. In C. elegans, kombucha consumption improved survival rates following Salmonella infection, indicating a protective effect. This study demonstrates that fermentation using Lactobacillus plantarum can enhance the bioactivity of lotus root kombucha, highlighting its potential as a low-caffeine functional beverage.

Graphical Abstract

1. Introduction

Kombucha is one of the most popular low-alcohol fermented drinks worldwide and is among the fastest-growing products in the functional beverage market [1,2]. It is traditionally produced by fermenting green or black tea with added sugar using a symbiotic culture of bacteria and yeast (SCOBY) [3,4]. During fermentation, various organic acids and bioactive compounds, including acetic acid, glucuronic acid, and lactic acid, are produced [5]. These compounds help to regulate the gut environment, inhibit the growth of pathogenic bacteria, and protect the intestinal barrier [6]. Additionally, the bacteriocins and microbial metabolites generated during fermentation exhibit antimicrobial activity and anti-inflammatory effects, further supporting gut health [7,8]. As a result, kombucha has gained recognition as a functional fermented beverage that promotes a healthy gut microbiome [3,9]. Beyond its gut-related benefits, kombucha has been reported to offer various health advantages, including anti-inflammatory properties, antioxidant activity, and reductions in cholesterol levels, blood pressure, and cancer cell proliferation. Additionally, it has been associated with improvements in liver function, immune system response, and overall gastrointestinal health [10,11].
Traditional kombucha is fermented using black or green tea as a base, resulting in a relatively high caffeine content [12]. Caffeine is a naturally occurring xanthine alkaloid found in various plants, including coffee, tea, and cocoa [13]. It is widely consumed due to its stimulatory effects on the central nervous system and its ability to enhance energy levels [13]. For the average adult, a daily intake of up to 400 mg is considered safe [14]; however, excessive caffeine consumption has been associated with adverse effects, including adrenal stimulation, increased blood pressure, anxiety, and cardiac arrhythmia [15]. As awareness of the potential health risks associated with caffeine consumption increases, the demand for low-caffeine foods and beverages is growing. These products may serve as more suitable alternatives for caffeine-sensitive individuals, such as pregnant women and children [16,17].
To meet this demand, lotus root (Nelumbo nucifera) has been considered a potential low-caffeine substrate for fermentation. It is a rich source of polyphenols and dietary fiber, serving as a prebiotic that supports gut microbiota balance [18]. Its high insoluble dietary fiber concentration promotes intestinal motility and helps prevent constipation by facilitating bowel movements [19,20]. Recent studies suggest that the bioactive properties of lotus root contribute to gut microbiota homeostasis and improved digestive health [21,22]. Beyond its digestive benefits, lotus root exhibits various biological activities, including antioxidant [23], immunomodulatory [24], anti-obesity [25], hypoglycemic [26], neuropharmacological [27], and memory-enhancing effects [28]. These functional properties are attributed to its phenolic compounds, such as catechol, gallic acid, catechin, epicatechin, gallocatechin, rutin, quercetin, and chlorogenic acid [29]. Additionally, lotus root is rich in flavonoids, vitamin C, and inulin, a prebiotic compound known to support beneficial gut bacteria [30,31,32]. However, its distinct aroma and unique texture limit consumer preference. To enhance its palatability and broaden its applications, it has been processed into various food products, such as lotus root tea, powder, and snacks [33,34]. Given its health-promoting properties, combining lotus root with microbial fermentation presents a promising strategy to further enhance its functional potential. In particular, fermentation using lactic acid bacteria (LAB) can improve both its bioactivity and sensory qualities.
Lactic acid bacteria (LAB) have been widely used in both traditional and modern food fermentation due to their ability to enhance flavor and preserve food. Fermented foods containing LAB are not only valued for their taste, but are also associated with various health benefits [35]. Kombucha provides a favorable environment for the growth of Lactobacillus plantarum due to its fermentation conditions, which support the survival and activity of LAB. The incorporation of LAB in kombucha fermentation has been reported to enhance its functional properties, including the increased production of organic acids, antimicrobial activity, and improved gut microbiota modulation [36,37]. Previous studies have suggested that LAB strains, particularly L. plantarum, enhance the functionality of fermented foods by increasing organic acid production, modulating gut microbiota, and producing bioactive compounds, such as D-saccharic acid 1,4-lactone (SAL), which contributes to detoxification and β-glucuronidase inhibition [38,39,40,41,42]. In particular, LAB stimulates the production of D-saccharic acid 1,4-lactone (SAL), a bioactive compound known for its detoxification effects and β-glucuronidase inhibition, while also promoting the survival of acetic acid bacteria, which play a key role in kombucha fermentation [37,43]. Moreover, L. plantarum displays broad substrate flexibility, genome plasticity, and the efficient production of bioactive compounds—traits that make it more suitable than many other LAB strains for the fermentation of plant-based matrices such as tea and vegetable substrates [44]. Therefore, L. plantarum was selected for this study based on its proven ability to enhance the biofunctional properties of fermented beverages, including kombucha.
Currently available decaffeinated or low-caffeine kombucha products are limited in variety, and often lack the bioactive complexity found in traditional tea-based kombucha. In particular, little research has focused on the use of naturally low-caffeine substrates like lotus root tea as a fermentation base. Furthermore, the enhancement of their functional properties through probiotic co-fermentation, such as with L. plantarum, remains underexplored. To address these gaps, this study aimed to develop a low-caffeine kombucha using lotus root tea and to investigate whether its functional properties could be enhanced through secondary fermentation with L. plantarum. To this end, kombucha samples with and without L. plantarum treatment were compared in terms of organic acid composition, antioxidant capacity, antimicrobial activity, β-glucuronidase inhibition, and protective effects against Salmonella infection using a Caenorhabditis elegans model.

2. Materials and Methods

2.1. Materials

The roasted lotus root used in this study was provided by Daebon Agricultural Corporation (Gyeongsan, Republic of Korea). The symbiotic culture of bacteria and yeast (SCOBY) and SCOBY culture liquid were purchased from Slowoon (Seoul, Republic of Korea), while the sugar was obtained from CJ CheilJedang Co., Ltd. (Incheon, Republic of Korea) through a commercial source.

2.2. Preparation and Fermentation of Lotus Root Tea Kombucha

The processing flow chart for the preparation of lotus root tea kombucha and its Lactobacillus plantarum-fermented variants is presented in Figure 1.

2.2.1. Preparation of Lotus Root Tea Kombucha

To prepare lotus root tea kombucha, 100 g of roasted lotus root was steeped in 1 L of water at a temperature above 85 °C for 15 min. After removing the lotus root, 100 g of sugar was added and completely dissolved. Then, 20 g of SCOBY and 70 mL of SCOBY culture liquid were introduced. The mixture underwent primary fermentation under aerobic and static conditions at 25 °C until the pH reached 3.8. Although the fermentation period varied depending on the condition of the SCOBY, primary fermentation was typically completed within 72 h (3 days).
The fermentation method was developed based on a previously registered patent, with modifications to optimize the final product. Specifically, the fermentation time and temperature were referenced from a domestic patent entitled “Method for manufacturing fermented tea using lotus root and fermented tea produced thereby” (Korean Patent No. 10-2459439) [45].
Following primary fermentation, byproducts were removed, and the liquid was transferred to a pressure-resistant container for secondary fermentation under anaerobic and static conditions for two days. After completion of secondary fermentation, the mixture was centrifuged at 12,000× g for 5 min, and only the supernatant was collected. The resulting kombucha was designated as LK (Lotus root tea kombucha) and used for further fermentation with Lactobacillus plantarum. The kombucha (LK) was prepared in three independent batches.

2.2.2. Additional Fermentation with Lactobacillus plantarum

To introduce Lactobacillus plantarum into the kombucha, the previously prepared LK underwent an additional fermentation step with L. plantarum ATCC14917. L. plantarum was first cultured in MRS broth at 37 °C for 24 h, harvested via centrifugation at 13,000 rpm for 1 min, and washed three times with sterile distilled water. The washed cells were then inoculated in 10 mL of LK at two concentrations: 1.0 × 109 CFU/mL (LK-P1) and 3.0 × 109 CFU/mL (LK-P3). These final concentrations were selected based on their superior β-glucuronidase inhibitory activity observed in preliminary screening experiments (Table S1). Fermentation was carried out under static conditions at 25 °C for six days.

2.3. HPLC Analysis of Caffeine and Organic Acids (SCFAs and Lactic Acid)

The concentrations of caffeine in kombucha were analyzed using high-performance liquid chromatography (HPLC) (Alliance 2996, Waters, Milford, MA, USA) equipped with a photodiode array (PDA) detector. A Kinetex C18 column (5 µm, 100 Å, 250 mm × 4.6 mm; Phenomenex, Torrance, CA, USA) was used with methanol, acetic acid, and water (20:1:79) as the mobile phase. The analysis was conducted at a flow rate of 1.0 mL/min, with the column maintained at 30 °C. Kombucha samples were filtered through a 0.45 µm syringe filter and subsequently analyzed at 280 nm [46].
The organic acid concentration, including acetic acid, propionic acid, butyric acid, and lactic acid in kombucha was analyzed using high-performance liquid chromatography (HPLC) (1260 Infinity, Agilent, Santa Clara, CA, USA) equipped with a refractive index (RI) detector. A Rezex ROA-Organic Acid H+ column (8%, 150 mm × 4.6 mm; Phenomenex) was used, with 0.005 N H2SO4 as the mobile phase. The analysis was conducted at a flow rate of 0.6 mL/min, with the column maintained at 50 °C. Kombucha samples were diluted 10-fold, filtered through a 0.45 µm syringe filter, and subsequently analyzed.

2.4. The Antioxidant Activity

2.4.1. DPPH Radical Scavenging Activity

The antioxidant activity of the samples was evaluated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay [47]. Before analysis, samples were filtered through a 0.22 µm membrane filter. The assay was performed in a 96-well plate, with each well containing 20 µL of the sample and 180 µL of 0.1 mM DPPH solution. A blank control was prepared using ethanol instead of the sample under the same conditions. Ascorbic acid (100 µg/mL) was used as a positive control. The reaction mixtures were incubated in the dark for 30 min to allow sufficient interaction between the samples and DPPH radicals. After incubation, absorbance was measured at 517 nm using a spectrophotometer (SpectraMax iD3, Molecular Devices, San Jose, CA, USA). The DPPH radical scavenging activity was calculated as the percentage of inhibition relative to the blank control using the following equation:
% DPPH radical scavening ability = {(Absblank − Abssample)/Absblank} × 100

2.4.2. ABTS Radical Scavenging Activity

Then, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS) radical cations were generated by mixing 7.0 mM ABTS with 2.6 mM potassium persulfate in distilled water, followed by incubation in the dark at room temperature for 24 h [48]. Before use, the solution was diluted with distilled water to an absorbance of 0.70 ± 0.02 at 732 nm. In a 96-well microplate, 180 µL of the diluted ABTS solution and 20 µL of the sample were added to each well. A control well was prepared by adding 20 µL of distilled water instead of the sample. Ascorbic acid (100 µg/mL) was used as the positive control. The plate was incubated in the dark at room temperature (25 °C) for 30 min. Absorbance was measured at 732 nm using a microplate reader. The ABTS radical scavenging activity was calculated as the percentage of inhibition compared to the control.
% ABTS radical scavening ability = {(Absblank − Abssample)/Absblank} × 100

2.5. Antimicrobial Activity Assay

The antimicrobial activity of kombucha was evaluated against Escherichia coli OP50 and Salmonella typhimurium ATCC 13311 [49]. For preculture, bacterial strains were inoculated into LB and incubated at 37 °C for 20 h. For the main culture, a 200 µL bacterial suspension was mixed with 1800 µL of LB as the control, whereas the test groups contained 200 µL of bacterial suspension, 200 µL of LB, and 1600 µL of kombucha (undiluted or 10-fold diluted) resulting in a final volume of 2 mL. Cultures were incubated statically at 37 °C, and bacterial growth was measured at 0 and 24 h via spectrophotometry at 620 nm. Growth inhibition was determined by comparing the optical density (OD) values of the test samples with those of the control group. The percentage of bacterial growth inhibition (% inhibition) was calculated using the following equation:
% inhibition = {1 − (ODsample 24 h − ODsample 0 h)/(ODcontrol 24 h − ODcontrol 0 h)} × 100

2.6. β-Glucuronidase Inhibition Assay

The β-glucuronidase inhibition assay was performed to evaluate the inhibitory effect of kombucha samples on β-glucuronidase activity [50]. The enzyme solution (10 units/mL) was prepared in 0.1 M phosphate buffer (pH 7.5), and 70 µL was used per reaction. A 30 µL aliquot of filtered sample was added to the enzyme solution and incubated at 37 °C for 15 min. Lotus root tea kombucha samples (LK, LK-P1, and LK-P3) were prepared by diluting them at a 1:100 ratio in distilled water (DW). As a control, 30 µL of distilled water was used instead of the sample. After incubation, 100 µL of p-nitrophenyl-β-glucuronide (0.1 mM, 0.1 M phosphate buffer, pH 7.5) was added as the substrate, and the reaction proceeded at 37 °C. Absorbance was measured at 405 nm at 30 min to quantify the enzymatic activity based on p-nitrophenol release. Saccharide acid 1, 4-lactone (SAL) (IC50 = 0.05 mM) was used as the positive control [51]. The β-glucuronidase inhibition activity (%) was calculated using the following equation:
% inhibition = {(Abscontrol − Abssample)/Abscontrol} × 100

2.7. C. elegans Assays

2.7.1. Maintenance and Strains

Caenorhabditis elegans strains were obtained from the Caenorhabditis Genetics Center (CGC) (Minneapolis, MN, USA). The wild-type strain Bristol N2 was used and maintained on nematode growth medium (NGM) agar plates seeded with Escherichia coli OP50, provided by CGC. The nematodes were maintained at 15 °C under standard laboratory conditions.
For all experiments, synchronized populations of C. elegans at the L4 larval stage were used. Synchronization was achieved using a bleaching solution consisting of 400 µL sodium hypochlorite (NaOCl) and 100 µL of 5 N sodium hydroxide (NaOH). After bleaching, the embryos were incubated in M9 buffer (3 g KH2PO4, 1 M MgSO4, 5 g NaCl, and 6 g Na2HPO4) without food until the L1 larval stage [52]. The synchronized L1 larvae were then transferred to NGM plates seeded with E. coli OP50 and incubated at 20 °C until they reached the L4 stage.

2.7.2. Pharyngeal Pumping Assay

The effects of LK, LK-P1, and LK-P3 on the pharyngeal pumping rate of C. elegans were evaluated [53]. Kombucha medium was prepared by spreading 33 µL of lotus root tea kombucha and 0.2 mL of E. coli OP50 (108 CFU/mL in M9 buffer) onto 60 mm NGM plates. The plates were left at room temperature for 2 h to allow for absorption.
Adult C. elegans feeding on E. coli OP50 were randomly transferred to NGM plates containing E. coli OP50 and kombucha. The pharyngeal pumping activity was recorded using a stereomicroscope (SZMN45TR-ST2, SUNNY KOREA, Jeju, Republic of Korea) at 180× magnification for 1 min. The number of pharyngeal pumps was visually examined and manually counted. Each test was performed twice to ensure reproducibility.

2.7.3. Pathogen Resistance Assays of Salmonella enterica Infection

For infection experiments, Salmonella serovar Typhimurium strain ATCC 13311 was used. The bacteria were cultured in Luria Broth (LB) at 37 °C for 24 h and then resuspended in M9 buffer at a concentration of 108 CFU/mL. A 30 mm NGM plate was seeded with 0.1 mL of the prepared S. typhimurium suspension and allowed to dry before use.
For the pathogen resistance assay, C. elegans were pre-conditioned on kombucha medium at 25 °C for 36 h, where they were fed both E. coli OP50 and kombucha. Subsequently, the worms were transferred to NGM plates seeded only with S. typhimurium to initiate infection. The control group consisted of worms pre-conditioned on E. coli OP50 without kombucha [54].
The pathogen resistance assay was conducted at 30 °C, and the worms were transferred to fresh S. typhimurium-seeded plates every 12 h. The worms were considered dead if they failed to respond to gentle touch. Individuals that died due to internal hatching or vulval rupture were excluded from statistical analysis. All pathogen resistance assays were performed in triplicate, with each plate containing 25 worms.

2.8. Statistical Analysis

All experiments were performed in triplicate, and the results were expressed as mean ± standard deviation (SD). Statistical analysis was conducted using SPSS (Windows version 19.0) software. One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was used to determine statistical significance, with p < 0.05 considered significant.
The survival curves were analyzed using the Kaplan–Meier method, and the statistical significance between groups was determined using the log-rank test.

3. Results

3.1. Caffeine Concentration of Lotus Root Tea Kombucha

The caffeine concentration of lotus root tea kombucha (LK) and Lactobacillus plantarum-fermented lotus root tea kombucha (LK-P1, LK-P3) is presented in Table 1. The caffeine concentration in LK was 2.75 µg/mL, which was significantly higher than in the L. plantarum-fermented groups (p < 0.05). The caffeine concentrations in LK-P1 and LK-P3 were 2.29 µg/mL and 2.32 µg/mL, respectively, both of which were significantly lower than LK.
Previous studies have investigated caffeine degradation during black tea fermentation, reporting that caffeine levels decreased from 1060 µg/mL on day 0 to 948 µg/mL on day 7 [55]. This indicates that black tea retains significantly higher caffeine levels compared to lotus root tea kombucha fermented for five days. Additionally, an analysis of nine commercially available kombucha products in the United States reported caffeine concentrations ranging from 15.1 µg/mL to 227.4 µg/mL [56]. These findings indicate that the caffeine concentration in lotus root tea kombucha, both with and without L. plantarum fermentation, is considerably lower than in conventional kombucha. During the fermentation process with L. plantarum, the caffeine concentration tended to decrease further; however, no significant difference was observed between LK-P1 and LK-P3.

3.2. Effect of L. plantarum Fermentation on Organic Acids in Lotus Root Tea Kombucha

Short-chain fatty acids (SCFAs) are key metabolites produced by gut microbiota, with acetic acid, propionic acid, and butyric acid being the most representative types [57]. The concentration of acetic acid, propionic acid, butyric acid, and lactic acid in lotus root tea kombucha (LK) and L. plantarum-fermented lotus root tea kombucha (LK-P1, LK-P3) are shown in Figure 2.
The acetic acid concentration increased in the L. plantarum-fermented groups compared to LK. Specifically, LK had an acetic acid concentration of 2.15 g/L, whereas LK-P1 and LK-P3 contained 3.24 g/L and 3.45 g/L, respectively. Although this increase was not statistically significant, it suggests that L. plantarum fermentation may enhance acetic acid production.
The concentration of propionic acid also increased following fermentation. LK contained 0.25 g/L of propionic acid, while LK-P1 and LK-P3 had 0.41 g/L and 0.50 g/L, respectively. These differences between LK and both L. plantarum-fermented groups were statistically significant (p < 0.05), while no significant difference was observed between LK-P1 and LK-P3. Butyric acid was not detected (ND) in any of the samples.
The most pronounced change was observed in the lactic acid concentration. While LK contained no detectable lactic acid, its concentration significantly increased in the L. plantarum-fermented groups. LK-P1 exhibited a lactic acid concentration of 4.07 g/L, whereas LK-P3 reached 4.58 g/L. A significant difference (p < 0.05) was observed between LK-P1 and LK-P3, indicating that a higher inoculation level of L. plantarum enhances lactic acid production. A significant decrease in pH was observed in the L. plantarum-fermented groups, consistent with the increased production of organic acids, particularly lactic acid (Table S2).

3.3. Antioxidant Activity of Lotus Root Tea Kombucha

Antioxidant activity plays a crucial role in various physiological functions, including anti-aging, anti-inflammatory effects, and immune enhancement. The antioxidant activity of lotus root tea kombucha (LK) and L. plantarum-fermented lotus root tea kombucha (LK-P1, LK-P3) was evaluated using the DPPH radical scavenging assay (Figure 3A). Ascorbic acid (100 µg/mL) was used as a positive control and exhibited a high antioxidant activity of 90.79%. The antioxidant activity of LK at 1× concentration was 61.37%. Similarly, LK-P1 exhibited a scavenging activity of 61.36%, whereas LK-P3 showed a slightly lower scavenging activity of 53.99%.
Although no statistically significant differences were observed among the samples, LK and LK-P1 exhibited similar antioxidant activities (~61%), while LK-P3 showed a slightly lower scavenging activity (53.99%). Previous studies have reported antioxidant activities ranging from 35.4% to 60.3% for kombucha samples fermented under similar conditions [58], suggesting that lotus root tea kombucha possesses relatively high antioxidant activity.
In addition to DPPH analysis, the ABTS radical scavenging activity of lotus root tea kombucha and L. plantarum-fermented variants was also evaluated (Figure 3B). Ascorbic acid (100 µg/mL), used as the positive control, exhibited a high ABTS radical scavenging activity of 93.05%. The ABTS scavenging activities of LK, LK-P1, and LK-P3 were 92.99%, 93.08%, and 92.89%, respectively, showing uniformly high antioxidant potential across all treatments.
No statistically significant differences were observed among the samples, indicating that the addition of L. plantarum did not significantly influence the ABTS radical scavenging capacity under the tested conditions. These results suggest that lotus root tea kombucha itself possesses strong antioxidant activity, as measured using the ABTS assay.

3.4. Antimicrobial Activity of Lotus Root Tea Kombucha

The antimicrobial activity of lotus root tea kombucha (LK) and Lactobacillus plantarum-fermented lotus root tea kombucha (LK-P1, LK-P3) was evaluated against Escherichia coli OP50 and Salmonella typhimurium (Figure 4).
Against E. coli OP50, the inhibition rates were 57.82% for LK, 78.33% for LK-P1, and 66.74% for LK-P3 (Figure 4A). The inhibitory activity of LK-P1 was approximately 1.35 times higher than that of LK, while LK-P3 exhibited a 1.15-fold increase. Although these differences were not statistically significant, L. plantarum-fermented lotus root tea kombucha (LK-P1 and LK-P3) exhibited a trend toward higher inhibitory activity compared to LK.
Similarly, against S. typhimurium, the inhibition rates were 47.88% for LK, 49.55% for LK-P1, and 61.88% for LK-P3 (Figure 4B). The inhibitory activity of LK-P1 was slightly higher than that of LK (1.04-fold increase), whereas LK-P3 exhibited a 1.29-fold increase, which was statistically significant (p < 0.05). Overall, lotus root tea kombucha exhibited approximately 50% antimicrobial activity against E. coli OP50 and S. typhimurium, and this activity was further enhanced by L. plantarum fermentation.

3.5. Inhibitory Effect of Lotus Root Tea Kombucha on β-Glucuronidase Activity

β-Glucuronidase activity is considered an important indicator of gut health, as its excessive levels are often associated with gut dysbiosis, inflammation, and an increased risk of colorectal diseases. Inhibiting β-glucuronidase activity can help maintain a healthier gut microbiota composition, reduce toxin reabsorption, and support overall gastrointestinal health.
The β-glucuronidase inhibitory activity of lotus root tea kombucha (LK) and Lactobacillus plantarum-fermented lotus root tea kombucha (LK-P1 and LK-P3) was evaluated (Figure 5). The inhibition rates were 28.79% for LK, 47.84% for LK-P1, and 60.51% for LK-P3. Statistical analysis revealed no significant difference between LK and the positive control, Saccharic acid 1,4-lactone (SAL) (IC50 0.05 mM). However, LK-P3 exhibited significantly higher β-glucuronidase inhibition than LK (p < 0.05). No significant difference was observed between LK-P1 and LK-P3, suggesting that β-glucuronidase inhibition reached a plateau at higher L. plantarum inoculation levels. Overall, lotus root tea kombucha exhibited approximately 29% β-glucuronidase inhibitory activity, and this effect was further enhanced by L. plantarum fermentation, reaching up to 60% inhibition.

3.6. Protective Effect of Lotus Root Tea Kombucha Against Salmonella Infection Resistance in C. elegans

The protective effect of lotus root tea kombucha (LK) and Lactobacillus plantarum-fermented kombucha (LK-P1, LK-P3) against Salmonella enterica serovar Typhimurium infection in C. elegans was evaluated by assessing pharyngeal pumping activity and worm survival (Figure 6).
The pharyngeal pumping activity was measured to assess the feeding rate and ingestion ability of C. elegans [59]. The control group exhibited a pharyngeal pumping rate of 227.4 pumps/min, whereas worms exposed to LK showed a significant reduction (146.9 pumps/min) (Figure 6A). In contrast, worms treated with L. plantarum-fermented kombucha exhibited pumping rates of 250.6 pumps/min (LK-P1) and 227.3 pumps/min (LK-P3), showing no significant difference compared to the control group.
The effect of kombucha consumption on survival following S. typhimurium infection was evaluated by monitoring worm viability over time. The control group exhibited the shortest lifespan, with complete mortality observed by 132 h, after which survival monitoring was discontinued (Figure 6). In contrast, worms pre-fed with LK, LK-P1, or LK-P3 demonstrated prolonged survival. Statistical analysis confirmed that survival rates were significantly higher in all kombucha-treated groups compared to the control group (LK: p = 0.007, LK-P1: p = 0.003, and LK-P3: p < 0.001). L. plantarum fermentation further enhanced the protective effect, as lifespan was longer in LK-P1 and LK-P3 compared to LK.
At 12 h post infection, mortality in the control group was 17.6%, whereas LK-, LK-P1-, and LK-P3-treated worms exhibited mortality rates of 5.4%, 8.1%, and 1.4%, respectively (Figure 6C). This trend persisted throughout the experiment, with the control group reaching a peak mortality rate of 40.5% at 24 h. Similarly, LK and LK-P1 groups also exhibited peak mortality at 24 h, but at a lower rate of 29.7%. In contrast, the LK-P3 group exhibited a delayed peak mortality at 36 h, with a rate of 18.9%. These findings suggest that lotus root tea kombucha consumption may delay C. elegans mortality following S. typhimurium infection.

4. Discussion

In this study, lotus root tea kombucha (LK) exhibited inherent antimicrobial activity against Escherichia coli OP50 and Salmonella typhimurium, with enhanced inhibitory effects observed in LK-P1 and LK-P3. This suggests that lotus root tea kombucha itself possesses antimicrobial properties, likely due to the production of organic acids and bioactive metabolites during fermentation. The enhanced inhibition in LK-P1 and LK-P3 correlated with increased acetic acid, propionic acid, and lactic acid levels, with LK-P3 showing the highest lactic acid concentration (approximately 5 g/L). Organic acids such as short-chain fatty acids (SCFAs) and lactic acid exhibit strong antimicrobial activity, playing a crucial role in inhibiting harmful bacteria and improving the intestinal environment [60]. SCFAs also contribute to gut health by strengthening the intestinal mucosa and regulating immune function [61]. This increase in organic acids was accompanied by a significant pH reduction in LK-P1 (3.43) and LK-P3 (3.33) compared to LK (3.74), reinforcing their contribution to pathogen inhibition. These findings suggest that the addition of L. plantarum further amplified the antimicrobial potential of lotus root tea kombucha, likely by enhancing organic acid production and acidification.
In addition to its antimicrobial activity, lotus root tea kombucha exhibited significant β-glucuronidase inhibitory activity, with LK-P1 and LK-P3 showing enhanced effects. β-Glucuronidase is associated with the reactivation of carcinogens and other harmful substances, and its inhibition is linked to a reduced risk of colorectal cancer and intestinal inflammation. While this study did not directly confirm the presence of D-saccharic acid 1,4-lactone (SAL) in lotus root tea kombucha, previous research has identified SAL as a key β-glucuronidase inhibitor in traditional kombucha [62,63,64]. It is plausible that SAL may also be produced during lotus root tea kombucha fermentation, contributing to the observed enzyme-inhibitory activity. Additionally, kombucha has been reported to suppress the growth of β-glucuronidase-producing bacteria (Escherichia coli, Streptococcus spp., Clostridium spp.) [38,65,66,67,68], thereby reducing the overall enzyme levels in the gut. Polyphenols such as catechins, theaflavins, and thearubigins, which are commonly found in kombucha [69,70], may further support β-glucuronidase inhibition by suppressing β-glucuronidase-producing bacteria and protecting intestinal barrier integrity through antimicrobial and antioxidant properties.
Furthermore, the addition of L. plantarum to the kombucha fermentation process enhanced β-glucuronidase inhibition, suggesting a synergistic effect between kombucha-derived bioactive compounds and L. plantarum-mediated fermentation. This bacterium is known for its diverse physiological functions, including antimicrobial [71], anti-inflammatory [72], and enzyme-modulating activities [73]. Previous studies have reported its β-glucuronidase inhibitory effects, and the findings of this study align with these previous results [74]. The precise molecular mechanism underlying β-glucuronidase inhibition in lotus root tea kombucha remain unclear, but the results suggest that multiple factors, including SAL, polyphenols, organic acids, and microbiota modulation, may contribute to its inhibitory activity.
C. elegans has been widely utilized as a model organism to investigate the impact of gut microbiota on host physiology [75,76,77]. The ability to culture C. elegans under germ-free conditions allows for precise experimental control over microbial exposure, making it a valuable model for gut health research [77,78,79]. While previous studies have evaluated the activity of kombucha-derived microorganisms in C. elegans [80], research directly examining the effect of kombucha consumption in this model remains limited. In this study, we assessed the pharyngeal pumping activity to confirm whether kombucha consumption affects C. elegans feeding behavior. Our findings indicated that L. plantarum-fermented kombucha did not negatively impact feeding, as the pharyngeal pumping rate was comparable to that of the control group. This suggests that kombucha ingestion is well tolerated in C. elegans, supporting its suitability as a model for investigating the biological effects of kombucha.
The pathogenicity of S. typhimurium in C. elegans is characterized by its proliferation within the intestine, leading to increased mortality. Infection disrupts pharyngeal peristalsis, induces gut distension due to bacterial overgrowth, and damages the intestinal barrier [81]. In this study, worms pre-exposed to kombucha for 36 h were transferred to S. typhimurium-containing plates, and survival was monitored every 12 h. The results indicated that kombucha significantly extended lifespan by delaying mortality, suggesting a protective effect against S. typhimurium-induced lethality. This protective effect was particularly pronounced in worms exposed to L. plantarum-fermented lotus root tea kombucha, suggesting the potential synergistic effect of kombucha-derived bioactive compounds and L. plantarum fermentation.
Despite a three-fold difference in the L. plantarum inoculum levels, most in vitro functional parameters—including antimicrobial, antioxidant, and β-glucuronidase inhibitory activities—did not show statistically significant differences between LK-P1 and LK-P3. This may be due to a plateau effect, in which functional benefits saturate beyond a certain microbial threshold [82]. However, in the C. elegans infection model, LK-P3 exhibited a more pronounced protective effect, suggesting that higher probiotic concentrations can still exert biologically meaningful outcomes in vivo, even when the in vitro differences are minimal, as supported by previous studies [83].
The protective mechanism of kombucha may be attributed to its organic acid and bioactive compounds. Previous research has shown that SCFAs produced during kombucha fermentation promote the growth of beneficial gut bacteria, protect the intestinal mucosa, and reduce inflammation [84,85]. Additionally, in this study, we directly confirmed the antioxidant activity of lotus root tea kombucha, which may have contributed to mitigating S. typhimurium-induced damage [86]. While our findings provide valuable insights into the potential protective effects of kombucha against S. typhimurium infection, further research is required to elucidate the precise mechanisms underlying its lifespan-extending properties. Future studies should focus on identifying the specific kombucha-derived metabolites responsible for these effects and exploring their interactions with host immune responses and gut microbiota.

5. Conclusions

This study demonstrated that lotus root tea can be used as a low-caffeine base for kombucha production. When further fermented with Lactobacillus plantarum, the resulting kombucha exhibited enhanced functional properties, including increased levels of organic acids, improved antimicrobial activity, and greater inhibition of β-glucuronidase. Moreover, consumption of the fermented kombucha improved survival in Caenorhabditis elegans following Salmonella infection, suggesting protective effects on host health.
These results highlight the potential of lotus root tea kombucha as a functional, non-caffeinated beverage with gut-health-promoting properties. However, further in vivo or clinical studies are required to confirm its health benefits in humans. Future research could also explore its underlying mechanisms of action and assess its sensory acceptability for consumer applications.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/beverages11020055/s1. Table S1: β-glucuronidase inhibitory activity of lotus root kombucha fermented with varying concentrations of Lactobacillus plantarum. Table S2: pH of lotus root tea kombucha and Lactobacillus-fermented lotus root tea kombucha.

Author Contributions

Investigation, J.S.B. and Y.N.; project administration, S.K.; resources, H.S.K. and E.J.L.; supervision, S.R.K. and M.-R.L.; visualization, J.S.B.; writing—original draft preparation, J.S.B. and S.K.; writing—review and editing, S.R.K. All authors have read and agreed to the published version of the manuscript.

Funding

This research was financially supported by the Ministry of Small and Medium-sized Enterprises (SMEs) and Startups (MSS), Republic of Korea, under the “Regional Specialized Industry Devel-opment Plus Program (R&D, S3401227)” supervised by the Korea Technology and Information Promotion Agency for SMEs.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article/Supplementary Materials; further inquiries can be directed to the corresponding authors.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
CFUColony-forming unit
DPPH2,2-Diphenyl-1-Picrylhydrazyl
HPLCHigh-performance liquid chromatography
LABLactic acid bacteria
LKLotus root tea kombucha
LK-P1Lotus root tea kombucha supplemented with 1.0 × 109 CFU/mL of L. plantarum
LK-P3Lotus root tea kombucha supplemented with 3.0 × 109 CFU/mL of L. plantarum
NGMNematode growth medium
ODOptical density
SALD-saccharic acid 1,4-lactone
SCOBYSymbiotic culture of bacteria and yeast
SCFAsShort-chain fatty acids

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Figure 1. Processing flow chart for the preparation of lotus root tea kombucha (LK) and Lactobacillus plantarum-fermented variants.
Figure 1. Processing flow chart for the preparation of lotus root tea kombucha (LK) and Lactobacillus plantarum-fermented variants.
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Figure 2. Organic acid profiles of lotus root tea kombucha with and without Lactobacillus plantarum fermentation. LK represents kombucha without L. plantarum, while LK-P1 and LK-P3 represent kombucha supplemented with 1.0 × 109 CFU/mL and 3.0 × 109 CFU/mL of L. plantarum, respectively. The concentrations of acetic acid, propionic acid, butyric acid, and lactic acid were quantified using calibration curves for each compound and expressed as g/L. ND indicates that the compound was not detected. Statistical significance for each organic acid was determined using Tukey’s test (p < 0.05), and different letters indicate significant differences among them. When no significant differences were found, no letters were shown. Error bars represent standard deviations.
Figure 2. Organic acid profiles of lotus root tea kombucha with and without Lactobacillus plantarum fermentation. LK represents kombucha without L. plantarum, while LK-P1 and LK-P3 represent kombucha supplemented with 1.0 × 109 CFU/mL and 3.0 × 109 CFU/mL of L. plantarum, respectively. The concentrations of acetic acid, propionic acid, butyric acid, and lactic acid were quantified using calibration curves for each compound and expressed as g/L. ND indicates that the compound was not detected. Statistical significance for each organic acid was determined using Tukey’s test (p < 0.05), and different letters indicate significant differences among them. When no significant differences were found, no letters were shown. Error bars represent standard deviations.
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Figure 3. Antioxidant activity of lotus root tea kombucha measured via (A) DPPH and (B) ABTS radical scavenging assays. LK represents kombucha without Lactobacillus plantarum, while LK-P1 and LK-P3 represent kombucha supplemented with 1.0 × 109 CFU/mL and 3.0 × 109 CFU/mL of L. plantarum, respectively. Statistical significance was determined using Tukey’s test (SPSS, n = 3), and different letters indicate significant differences (p < 0.05). When no significant differences were found, no letters were shown. Error bars represent standard deviations.
Figure 3. Antioxidant activity of lotus root tea kombucha measured via (A) DPPH and (B) ABTS radical scavenging assays. LK represents kombucha without Lactobacillus plantarum, while LK-P1 and LK-P3 represent kombucha supplemented with 1.0 × 109 CFU/mL and 3.0 × 109 CFU/mL of L. plantarum, respectively. Statistical significance was determined using Tukey’s test (SPSS, n = 3), and different letters indicate significant differences (p < 0.05). When no significant differences were found, no letters were shown. Error bars represent standard deviations.
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Figure 4. Antimicrobial activity of lotus root tea kombucha against Escherichia coli and Salmonella typhimurium. Antimicrobial activity of lotus root tea kombucha (LK) and Lactobacillus-fermented LK against E. coli OP50 (A) and S. typhimurium ATCC 13311 (B). LK represents kombucha without L. plantarum, while LK-P1 and LK-P3 represent kombucha supplemented with 1.0 × 109 CFU/mL and 3.0 × 109 CFU/mL of L. plantarum, respectively. Statistical significance was determined using Tukey’s test (SPSS, n = 3), and different letters indicate significant differences (p < 0.05). When no significant differences were found, no letters were shown. Error bars represent standard deviations.
Figure 4. Antimicrobial activity of lotus root tea kombucha against Escherichia coli and Salmonella typhimurium. Antimicrobial activity of lotus root tea kombucha (LK) and Lactobacillus-fermented LK against E. coli OP50 (A) and S. typhimurium ATCC 13311 (B). LK represents kombucha without L. plantarum, while LK-P1 and LK-P3 represent kombucha supplemented with 1.0 × 109 CFU/mL and 3.0 × 109 CFU/mL of L. plantarum, respectively. Statistical significance was determined using Tukey’s test (SPSS, n = 3), and different letters indicate significant differences (p < 0.05). When no significant differences were found, no letters were shown. Error bars represent standard deviations.
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Figure 5. β-Glucuronidase inhibitory activity of lotus root tea kombucha. LK represents kombucha without L. plantarum, while LK-P1 and LK-P3 represent kombucha supplemented with 1.0 × 109 CFU/mL and 3.0 × 109 CFU/mL of L. plantarum, respectively. Saccharic acid 1,4-lactone (SAL) (IC50 = 0.05 mM) was used as the positive control. Kombucha samples were diluted to 10 µL/mL before measurement. Statistical significance was determined using Tukey’s test (SPSS, n = 3), and different letters indicate significant differences (p < 0.05). Error bars represent standard deviations.
Figure 5. β-Glucuronidase inhibitory activity of lotus root tea kombucha. LK represents kombucha without L. plantarum, while LK-P1 and LK-P3 represent kombucha supplemented with 1.0 × 109 CFU/mL and 3.0 × 109 CFU/mL of L. plantarum, respectively. Saccharic acid 1,4-lactone (SAL) (IC50 = 0.05 mM) was used as the positive control. Kombucha samples were diluted to 10 µL/mL before measurement. Statistical significance was determined using Tukey’s test (SPSS, n = 3), and different letters indicate significant differences (p < 0.05). Error bars represent standard deviations.
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Figure 6. Effects of lotus root tea kombucha and Lactobacillus plantarum-fermented kombucha on pharyngeal pumping and survival of C. elegans exposed to Salmonella typhimurium. (A) The effects of kombucha samples on the pharyngeal pumping rate of C. elegans. (B) Survival curve of C. elegans exposed to Salmonella typhimurium after supplementation with kombucha. (C) The effects of kombucha samples on the time-dependent mortality rates of C. elegans following S. typhimurium infection. LK represents kombucha without L. plantarum, while LK-P1 and LK-P3 represent kombucha fermented with L. plantarum at 1.0 × 109 and 3.0 × 109 CFU/mL, respectively. Pharyngeal activity was assessed via manual counting under 180× magnification. Survival analysis was performed using the Kaplan–Meier method and log-rank test (n = 74 worms per group, across three plates). Higher mortality in the heatmap is shown in red; right-censored data indicate worms alive at the end of the experiment. ns, **, and *** indicate levels of statistical significance. ns: not significant; **: p < 0.01; ***: p < 0.001.
Figure 6. Effects of lotus root tea kombucha and Lactobacillus plantarum-fermented kombucha on pharyngeal pumping and survival of C. elegans exposed to Salmonella typhimurium. (A) The effects of kombucha samples on the pharyngeal pumping rate of C. elegans. (B) Survival curve of C. elegans exposed to Salmonella typhimurium after supplementation with kombucha. (C) The effects of kombucha samples on the time-dependent mortality rates of C. elegans following S. typhimurium infection. LK represents kombucha without L. plantarum, while LK-P1 and LK-P3 represent kombucha fermented with L. plantarum at 1.0 × 109 and 3.0 × 109 CFU/mL, respectively. Pharyngeal activity was assessed via manual counting under 180× magnification. Survival analysis was performed using the Kaplan–Meier method and log-rank test (n = 74 worms per group, across three plates). Higher mortality in the heatmap is shown in red; right-censored data indicate worms alive at the end of the experiment. ns, **, and *** indicate levels of statistical significance. ns: not significant; **: p < 0.01; ***: p < 0.001.
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Table 1. Caffeine concentration of lotus root tea kombucha.
Table 1. Caffeine concentration of lotus root tea kombucha.
LKLK-P1LK-P3
Caffeine
(µg/mL)		2.75 ± 0.02 a2.29 ± 0.09 b2.32 ± 0.10 b
LK: kombucha without L. plantarum, LK-P1: kombucha supplemented with 1.0 × 109 CFU/mL of L. plantarum, and LK-P3: kombucha supplemented with 3.0 × 109 CFU/mL of L. plantarum. Different letters indicate significant differences (p < 0.05) as determined by Tukey’s test (SPSS, n = 3).
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MDPI and ACS Style

Baek, J.S.; Nam, Y.; Kim, S.; Kim, H.S.; Lee, E.J.; Lee, M.-R.; Kim, S.R. Development of Low-Caffeine Kombucha Using Lotus Root Tea and an Evaluation of Its Functional Properties. Beverages 2025, 11, 55. https://doi.org/10.3390/beverages11020055

AMA Style

Baek JS, Nam Y, Kim S, Kim HS, Lee EJ, Lee M-R, Kim SR. Development of Low-Caffeine Kombucha Using Lotus Root Tea and an Evaluation of Its Functional Properties. Beverages. 2025; 11(2):55. https://doi.org/10.3390/beverages11020055

Chicago/Turabian Style

Baek, Jin Seon, Younhee Nam, Sunghee Kim, Hee Song Kim, Eun Jin Lee, Mee-Ryung Lee, and Soo Rin Kim. 2025. "Development of Low-Caffeine Kombucha Using Lotus Root Tea and an Evaluation of Its Functional Properties" Beverages 11, no. 2: 55. https://doi.org/10.3390/beverages11020055

APA Style

Baek, J. S., Nam, Y., Kim, S., Kim, H. S., Lee, E. J., Lee, M.-R., & Kim, S. R. (2025). Development of Low-Caffeine Kombucha Using Lotus Root Tea and an Evaluation of Its Functional Properties. Beverages, 11(2), 55. https://doi.org/10.3390/beverages11020055

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