Evaluating the Health Implications of Kombucha Fermented with Gardenia jasminoides Teas: A Comprehensive Analysis of Antioxidant, Antimicrobial, and Cytotoxic Properties

Abstract

Background/Objectives: Plant-derived compounds are increasingly valued in drug discovery for their therapeutic potential. This study aims to examine the antimicrobial, antioxidant, and anticancer properties of kombucha beverages fermented with Gardenia jasminoides (GJ) and various types of Camellia sinensis teas: matcha green tea (MGT), organic green tea (OGT), and decaffeinated green tea (DGT). Methods: Two experimental designs were employed: (1) using black tea as a base substrate, infusing the four teas post-fermentation over 0–14 days, and (2) directly fermenting tea–herb combinations over 0–21 days. Antioxidant activity was assessed via the DPPH assay. Microbial dynamics were analyzed through total mesophilic bacteria and Lactobacillus counts. Antimicrobial potential was evaluated against E. coli, S. aureus, and S. enteritidis over 24 h. Cytotoxicity assays were conducted on Caco-2 and U251 cell lines to assess anticancer effects, with pH-adjusted controls used to differentiate bioactivity from acidity. Results: In the first experiment, GJ kombucha displayed the highest antioxidant potential (IC50: 14.04 µg/mL), followed by MGT (IC₅₀: 32.85 µg/mL) and OGT (IC₅₀: 98.21 µg/mL). In the second setup, unfermented GJ kombucha initially showed high antioxidant activity (IC₅₀: 12.94 µg/mL), improving during fermentation to reach an IC₅₀ of 18.26 µg/mL by day 21. Microbial analysis indicated moderate increases in total mesophilic bacteria and Lactobacillus in GJ kombucha after 14 days, while MGT, OGT, and DGT exhibited higher increments. GJ kombucha consistently demonstrated the highest antimicrobial activity against E. coli, S. aureus, and S. enteritidis, with significant inhibitory effects observed by 24 h. Cytotoxicity assays showed that GJ kombucha reduced Caco-2 cell viability to 20% at 800 µg/mL after 14 days, while U251 cells maintained 50% viability at the same concentration. Conclusions: This study highlights the antimicrobial, antioxidant, and anticancer potential of GJ kombucha, with fermentation enhancing bioactive metabolite production. Optimizing fermentation conditions, identifying specific bioactive compounds, expanding cytotoxicity testing, and exploring broader therapeutic applications of kombucha could maximize its health benefits and establish it as a natural antimicrobial and anticancer agent.

1. Introduction

Plant-derived compounds have garnered significant scientific interest due to their potent bioactive properties, notably antimicrobial, antioxidant, and anticancer activities, making them promising candidates for drug discovery [1,2]. These bioactives offer potential solutions for addressing issues like antimicrobial resistance, oxidative stress, and cancer progression. Specifically, plant-derived antimicrobials exhibit broad-spectrum efficacy against bacteria, fungi, and viruses, positioning them as valuable alternatives in developing new antimicrobial agents [3]. The antioxidant effects of these compounds are largely attributed to their phenolic content, which plays a crucial role in neutralizing reactive oxygen species (ROS) and reducing oxidative stress, thereby protecting cellular structures from free radical-induced damage [4]. Rich in various antioxidants, such as vitamins E, A, and C, as well as polyphenolic compounds, plants significantly contribute to health promotion and the prevention of age-related diseases [5,6,7,8].

Beyond their antimicrobial and antioxidant effects, certain plant-derived bioactives, including flavonoids, terpenoids, and alkaloids, show promising anticancer potential. These compounds can inhibit cancer cell proliferation, induce apoptosis, and prevent metastasis by modulating the key pathways associated with tumorigenesis, such as those involved in cell cycle regulation, inflammation, and oxidative stress [9]. While the bioactive properties of various well-known plant-based compounds are extensively studied [5,9,10], the specific antimicrobial, antioxidant, and anticancer potential of alternative plant sources, such as those used in kombucha production, remains underexplored. There is a notable gap in research regarding how these compounds contribute to the bioactive properties of kombucha, especially when derived from less conventional tea bases and plants like Gardenia jasminoides (GJ). Systematic exploration of these novel plant sources could reveal unique health-promoting properties and applications in combating modern health challenges.

Kombucha, a widely enjoyed fermented tea beverage, is made by fermenting tea and sugar with a symbiotic culture of bacteria and yeasts (SCOBY) [11]. This process not only gives kombucha its characteristic tangy flavor but also enriches it with potential health benefits, including antioxidant, antimicrobial, and anticancer effects. The SCOBY used in fermentation contains a complex array of microorganisms essential to the process [12,13]. Key bacteria like Acetobacter and Gluconacetobacter convert alcohol to acetic acid, which gives kombucha its tang, while Lactobacillus bacteria produce lactic acid, enhancing both acidity and potential probiotic benefits. Yeasts, including Saccharomyces cerevisiae, Brettanomyces, and Zygosaccharomyces, help convert sugars into alcohol and produce aromatic compounds that deepen kombucha’s flavor profile [14,15]. The resulting beverage is rich in organic acids, minerals, vitamins, amino acids, and bioactive compounds derived from the tea base [11,12,13,14,15].

Driven by consumer demand for non-alcoholic and healthier beverage choices, kombucha has rapidly become a major player in the functional beverages market [16]. Its popularity has led to a diverse range of offerings, including both natural and flavored varieties, with flavored kombucha particularly appealing to consumers seeking innovative options [17]. Reflecting this growth, the kombucha market was valued at EUR 1.84 billion in 2019 and is projected to reach EUR 10.45 billion by 2027 [17].

In response to this growing interest, scientific research has focused on optimizing kombucha’s sensory appeal and health benefits. Historically, black tea has been the primary substrate for kombucha fermentation, but recent interest in other teas is expanding the beverage’s flavor and health potential [11]. Variables like tea type, fermentation duration, SCOBY density, and temperature all influence kombucha’s final properties [13,18,19,20]. Systematically investigating kombucha made with different tea bases will provide valuable insights into optimizing its health benefits and applications [21,22].

Studies have explored alternative ingredients in kombucha production, substituting the traditional tea base with options like herbal teas [23], fruit juices [24,25], or even coffee [26]. Research has also evaluated various sugar sources [27] and alternative microbial cultures to replace the standard SCOBY [28]. These efforts aim to diversify kombucha’s flavor and boost its health-promoting properties. However, despite these innovations, there is still a significant research gap in understanding how the selection of tea bases and the timing of herbal infusions during fermentation specifically impact kombucha’s bioactive properties. While research has investigated various sugars and microbial cultures as potential modifiers of kombucha’s characteristics, the role of different tea varieties in enhancing the antimicrobial, antioxidant, and anticancer potentials has not been systematically addressed.

This study addresses these gaps by comparing the bioactive properties of kombucha fermented with different teas and GJ, focusing on enhancing its health-promoting potential. Specifically, it explores the antimicrobial, antioxidant, and anticancer properties of kombucha made from MGT, organic green tea (OGT), decaffeinated green tea (DGT), and GJ. Two experimental approaches were employed: one in which the teas and GJ were infused post-fermentation using black tea as a substrate and another where the tea and herbs were directly fermented over a specific period. Through these experimental designs, this study provides a comprehensive understanding of how the choice of tea base and timing of herbal infusion during fermentation can optimize kombucha’s bioactive properties. This research is especially timely, given the growing consumer demand for functional foods with enhanced health benefits, and may pave the way for innovative approaches to kombucha production.

GJ, a plant from the Rubiaceae family, is traditionally used in Chinese medicine for its antidiabetic, anti-inflammatory, antidepressant, and antioxidant effects [29,30,31,32,33]. GJ is rich in bioactive compounds like geniposide, genipin, gardenoside, crocin, and iridoid glycosides, which transfer into kombucha during fermentation, enhancing its therapeutic properties [34,35]. Geniposide has anti-inflammatory, antidepressant, and antidiabetic effects, while genipin improves insulin resistance and alleviates liver oxidative stress and mitochondrial dysfunction [36,37,38,39,40,41]. Crocetin contributes to GJ’s antidiabetic and antihyperlipidemic properties, offering protection against retinal damage and renal dysfunction [42,43].

Particularly significant are crocetin, crocin-1, and crocin-2, which have shown potent anticancer effects by inducing apoptosis and inhibiting cancer cell proliferation [44]. Crocetin stands out due to its superior gastrointestinal absorption and antiproliferative effects on cancer cell lines such as MKN-28 (stomach), MCF-7 (breast), and Caco-2 (colon) [44]. Additionally, crocetin downregulates pro-inflammatory cytokines like IL-1β and TNF-α, further supporting its anticancer and anti-inflammatory properties. These effects highlight GJ’s potential in both cancer treatment and prevention [44].

GJ’s bioactive components also modulate the AKT/mTOR pathway, which is crucial for cancer progression, and have shown effectiveness in targeting aggressive cancers like glioblastoma multiforme (GBM). When combined with chemotherapy agents like cisplatin, GJ enhances cytotoxicity in cancer cells while reducing toxicity to normal cells, offering a promising strategy to minimize the side effects of conventional cancer treatments [45]. Furthermore, chlorogenic acids, particularly 3,5-dicaffeoylquinic acid, present in GJ, contribute to its anticancer, anti-inflammatory, antimicrobial, and antioxidant effects, supporting its potential in health promotion and cancer prevention [46,47,48].

Originating in East Asia, C. sinensis is widely cultivated in tropical and sub-tropical regions, producing various types of green tea (GT), including OGT, MGT, and DGT [49]. OGT is distinguished by its lack of chemicals, herbicides, and pesticides throughout the entire production process [19]. MGT, which consists of finely ground leaves of C. sinensis grown in the shade, is notable for its high chlorophyll content, caffeine, and theanine [50]. With notably high concentrations of phenolic acids, quercetin, rutin, theanine, and chlorophyll, it surpasses other green tea varieties in terms of its nutritional profile [50]. The infusion and extracts of MGT show promise in combating lifestyle diseases caused by free radicals and inflammation, as well as in mitigating premature aging processes [50,51,52,53]. OGT, which contains various compounds with both antioxidant and antimicrobial effects, is particularly rich in polyphenols, including catechins like catechin, gallocatechin, epicatechin, and epigallocatechin, which are known for their potent antioxidant properties [54]. These compounds neutralize free radicals, reduce oxidative stress, and contribute to overall health. Furthermore, the antimicrobial effects of OGT are attributed to catechins, specifically epigallocatechin gallate (EGCG), which damages bacterial cell membranes and impairs barrier function in microorganisms [54].

In summary, this study aims to systematically evaluate the bioactive properties of kombucha produced from various tea bases, with a particular emphasis on enhancing its antimicrobial, antioxidant, and anticancer potentials through distinct fermentation strategies. By examining the impact of different tea types and infusion techniques on kombucha’s bioactive profile, this research seeks to identify the optimal conditions for maximizing the therapeutic properties of the beverage. The results of this investigation may contribute to the advancement of functional kombucha, offering a tailored approach to developing beverages that deliver specific health benefits. Such findings are especially relevant in response to the increasing consumer demand for innovative, health-enhancing functional foods and could potentially provide new insights into kombucha production for the broader health and wellness industry.

2. Materials and Methods

2.1. Tea Selection and Kombucha Fermentation

In this study, Gardenia jasminoides (GJ), Barry’s OGT, Pukka Supreme MGT, and Twinings DGT were procured from Dunnes Stores, Dublin 1, Dublin, Ireland. Two sets of experiments were conducted to investigate the antioxidant and microbial properties of four types of tea [55].

The first experiment involved the preparation of kombucha beverages infused with the four types of teas, such as GJ, OGT, MGT, and DGT, after 14 days of fermentation. A black tea infusion, denoted as black tea, served as the starting kombucha beverage (Figure 1). Following the protocol outlined by Marsh et al. (2014), with minor modifications, 5% dry black tea leaves (m/v) were steeped in 1 L of boiling water for 3 min. Subsequently, 9% sucrose (m/v) was added and boiled for an additional 1 min. After cooling to room temperature and filtration, 100 mL of the resulting black tea infusion, containing 2% (m/v) SCOBY and a starter culture (φ = 10%), was inoculated into each glass jar for kombucha tea fermentation. The sample flasks were then incubated at room temperature for 14 days. Different herb teas were added for antioxidant activity and antimicrobial testing [56].

The second experiment involved the incubation of the four herb teas at room temperature for 0, 7, 14, and 21 days. Herb-fermented kombucha beverages were prepared by adding 5 g of each herb (GJ, OGT, MGT, and DGT) to 1 L of boiled water containing 9% sucrose. After this incubation period, the liquid portions of the kombucha beverages were collected and centrifuged at 1370× g for 10 min for subsequent experimentation (Figure 2). The kombucha tea samples were sterilized by filtration through a sterile microfilter (0.22 μm pore size) under aseptic conditions to ensure the integrity of the samples [57].

2.2. Antioxidant Assay

Antioxidant assays were performed on the kombucha beverages derived from GJ, MGT, OGT, and DGT using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay [58]. This assay utilizes the synthetic free radical DPPH (0.04 mg/mL) to evaluate the ability of antioxidants to scavenge free radicals. A color change was observed from red to yellow, indicating the effectiveness of the antioxidants. The IC₅₀ parameter, which represents the concentration of antioxidants needed to achieve a 50% reduction in the initial radical concentration, was determined. These IC₅₀ values serve as quantitative indicators of the efficacy of the antioxidants present in the extracts and ferments from different tea sources. The assay was conducted using vitamin C/Ascorbic acid as a standard to establish a standard curve. Each sample (1 mL) was combined with 3 mL of the DPPH solution in test tubes and kept in the dark for 30 min. After incubation, the contents were transferred to plastic cuvettes, with absolute methanol serving as the reference sample. The absorbance was measured at 520 nm using a colorimeter, with the sample concentrations ranging from 0.1 to 1000 μg/mL. The percentage inhibition, indicative of the DPPH scavenging effect, was calculated using the formula:

DPPH scavenging effect (%) = {(A₀ − A₁)/A₀) × 100}

(1)

where A₀ represents the absorbance of the control reaction, and A₁ is the absorbance in the presence of the extract samples and blank [59].

2.3. Quantification of 3,5-Dicaffeoylquinic Acid in GJ Kombucha Ferments

The quantification of 3,5-Dicaffeoylquinic acid in the GJ kombucha ferments was conducted using high-performance liquid chromatography (HPLC). The objective was to determine the concentration of 3,5-Dicaffeoylquinic acid, a bioactive compound renowned for its potent antioxidant properties, in kombucha samples obtained over a 14-day fermentation period in experimental setting one. Kombucha ferments were systematically collected at distinct time points (0, 7, and 14 days) during the fermentation process. Following sample collection, a process of filtration and extraction was implemented to obtain a concentrated solution enriched with 3,5-Dicaffeoylquinic acid. The HPLC system, featuring a suitable column and detector, was deployed for comprehensive analysis. To facilitate accurate quantification, a standard solution comprising pure 3,5-Dicaffeoylquinic acid was prepared to establish a calibration curve. HPLC analysis was performed using the Alliance Waters 2998 PDA instrument (Waters Corporation, Milford, MA, USA). Separation was achieved using an Agilent Eclipse Plus C18 column (Agilent Technologies, Santa Clara, CA, USA) (4.6 × 150 mm) with a particle size of 5 µm. The mobile phase consisted of a mixture of acetonitrile with 0.05% formic acid (solvent A) and water with 0.05% formic acid (solvent B). Gradient elution was employed according to the following program: 0–20 min from 10% A to 20% A (and from 90% B to 80% B). The thermostat temperature was set at 20 °C, and the flow rate was maintained at 1 mL/min. Chromatograms were recorded at 280 nm, with the retention time peak at 14 min, indicating the presence of 3,5-Dicaffeoylquinic acid. Both the kombucha extract and standard solutions were injected into the HPLC system. Each procedure was performed in triplicate to enhance the statistical validity of the findings.

2.4. Microbiological Profile

Microbiological profiling of the samples was conducted on days 0 and 14 of fermentation. A volume of 1 mL of the samples was uniformly extracted from the fermentation vessels, and serial dilutions of the samples were prepared as described by the protocol [60]. Selective media were inoculated with 200 µL of the 10⁻⁵ and 10⁻⁶ diluted samples, and this procedure was repeated three times for each sample. Yeast extract glucose chloramphenicol (YGC) agar was utilized for yeast quantification, while the glucose yeast extract calcium carbonate (GYC) agar was employed for counting acetic acid bacteria. Mesophilic bacterial colonies were enumerated using the plate count agar, while the De Man Rogasa Sharp (MRS) agar was utilized for Lactobacillus quantification. After incubation for five days at 30 °C for the MRS, PCA, and GYC plates and at 25 °C for the YGC plates, colonies were enumerated, and the colony-forming units (CFU) per milliliter were calculated using the following equation:

$$N={\displaystyle \frac{C}{[V\times \left(N1+\left(0.1.N2\right)\right)]}}$$

(2)

where N represents the total amount of microorganisms in one milliliter, C is the total number of colonies counted in a sample, V is the volume of serial dilutions transferred to the samples (mL), N1 is the number of replicates of the samples prepared with the first of the serial dilutions, N2 is the number of replicates of the samples prepared with the second of the serial dilutions, and d is the most concentrated of the successive serial dilutions (16).

2.5. Antimicrobial Assay

The antimicrobial efficacy of the kombucha beverages, which were infused with four distinct tea varieties, was evaluated against three bacterial strains: E. coli (ATCC 25922), S. aureus (ATCC 25923), and S. enteritidis (ATCC 14028). Tryptone Soy Agar (TSA) plates were prepared by dissolving 37 g of TSA in 1 L of deionized water, followed by heating, stirring, and autoclaving at 120 °C for 20 min. After cooling to 80 °C, the molten agar was poured into sterile Petri dishes under sterile conditions and stored at 4 °C until required. Bacterial suspensions were prepared by dissolving 5 g of nutrient broth in 250 mL of deionized water, followed by autoclaving, cooling, and inoculation. Bacterial samples were collected from the agar plates and incubated at 37 °C for 24 h, and isolated colonies were obtained through streaking on TSA plates, followed by further incubation at 35 °C for 24 h. The inoculum preparation involved picking up 1–2 colonies of each bacterial strain using sterile loops. Time–kill studies were conducted by adjusting the bacterial cell densities to approximately 10 CFU/mL and exposing them to four groups with concentrations of minimum inhibitory concentration (MIC). The bacterial reduction rates were measured at various time points (0, 1, 2, 4, 6, and 24 h). Antimicrobial activity was assessed using the broth dilution method, with the serial dilutions ranging from 16 to 0.015 mg/mL prepared in 100 mL volumes in a 96-well microtiter tray over 24 h. The experiments were performed in duplicate, and the data were analyzed using the minimum inhibitory concentration (MIC) against the three bacterial strains [61,62].

2.6. Anticancer Assays

The anticancer potential of the GJ kombucha was assessed using the human epithelial cell line Caco-2 (ATCC HTB-37™, Manassas, VA, USA) and the U-251 MG (product no.09063001, Sigma-Aldrich, St. Louis, MO, USA.) human brain glioblastoma astrocytoma cells. The cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with L-glutamine, 10% fetal bovine serum (FBS), and 1% penicillin–streptomycin. The cultures were maintained at 37 °C with 95% humidity and 5% CO₂ in 75 cm² culture flasks. For the dose–response evaluation, the Caco-2 and U-251 MG cells were seeded into 96-well plates at a density of 1 × 10⁴ cells per well in 100 µL of DMEM. The cells were allowed to adhere for 24 h. The GJ kombucha tea was filled by Whatman^® GD/X Syringe Filters (Pore Size 0.2 μm) from Merck before it was mixed in a cell culture medium. The medium was prepared with various concentrations of GJ kombucha (12.5, 25, 50, 100, 200, 400, and 800 µg/mL). The cells were incubated for 48 h under the same incubation conditions. To assess cell viability, the MTT assay was performed. After the treatment period, the medium was aspirated and replaced with 100 µL of a 5 mg/mL MTT solution (Amresco, Solon, OH, USA). The cells were incubated with MTT for 4 h at 37 °C. Following incubation, the MTT solution was removed, and 100 µL of Dimethyl Sulfoxide (DMSO) was added to each well to dissolve the formazan crystals. The plates were mixed for 30 min, and absorbance was measured at 570 nm using a Spectromax M3 Multi-Mode Microplate Reader. Cell viability was calculated based on the absorbance readings, comparing the treated cells to untreated controls. This approach enabled the evaluation of the cytotoxic effects of GJ kombucha on the Caco-2 and U-251 MG cell lines.

3. Results

3.1. DPPH Assay

The results obtained from the DPPH assay conducted in experimental settings 1 and 2 provide valuable insights into the antioxidant capacity of kombucha beverages (Figure 3). In the first experimental setting, the IC₅₀ values for GJ, OGT, MGT, and DGT were determined to be 14.04 µg/mL, 98.21 µg/mL, 32.85 µg/mL, and 43.68 µg/mL, respectively. In the second experimental setting, the antioxidant capacity of GJ was assessed on days 0, 7, 14, and 21. The IC₅₀ values obtained were 36 µg/mL on day 0, 29.75 µg/mL on day 7, 26.97 µg/mL on day 14, and 18.26 µg/mL on day 21. The IC₅₀ value for unfermented GJ was 12.94 µg/mL.

3.2. Quantification of 3,5-Dicaffeoylquinic Acid in GJ Kombucha Ferments

The quantification of 3,5-Dicaffeoylquinic acid in the GJ kombucha ferments involved three replicates per sampling day from 0 to 14, revealing a pronounced increase in concentration as the fermentation progressed (Figure 4). Initially, at day 0, the average concentration was 1.9 mg/100 mL, representing a baseline before substantial fermentation activity commenced. Over time, this concentration steadily rose to 3.1 mg/100 mL by day 7 and further to 3.7 mg/100 mL by day 14. Statistical analysis, using a significance level of 0.05, underscored significant differences in 3,5-Dicaffeoylquinic acid production across time points: from day 0 to day 7 (p < 0.0001), from day 0 to day 14 (p < 0.0001), and from day 7 to day 14 (p = 0.008). These findings highlight a dynamic shift in the content of 3,5-Dicaffeoylquinic acid during fermentation, emphasizing its temporal accumulation in GJ kombucha.

3.3. Microbiological Profile

Table 1. Microbiological characteristics of kombucha cultures on days 0 and 14 of fermentation.

Beverage	t (Fermentation)/day
	0	14	0	14	0	14	0	14
	N (Microorganism)/(CFU/mL)
	ACC		TMB		Yeast		Lactobacillus
GJ	(2.3 ± 0.2) × 103	(3.2 ± 0.1) × 106	(6.7 ± 0.2) × 103	(9.2 ± 0.4) × 105	(1.7 ± 0.1) × 103	(6.7 ± 0.7) × 106	(7.4 ± 0.3) × 103	(5.8 ± 0.2) × 106
MGT	(2.3 ± 0.4) × 103	(1.0 ± 0.2) × 107	(6.7 ± 0.2) × 103	(3.2 ± 0.4) × 107	(1.7 ± 0.3) × 103	(2.7 ± 0.7) × 107	(7.4 ± 0.3) × 103	(1.8 ± 0.1) × 107
OGT	(2.3 ± 0.1) × 103	(3.0 ± 0.3) × 107	(6.7 ± 0.2) × 103	(3.7 ± 0.4) × 107	(1.7 ± 0.1) × 103	(2.9 ± 0.6) × 107	(7.4 ± 0.2) × 103	(2.1 ± 0.1) × 107
DGT	(2.3 ± 0.4) × 103	(3.0 ± 0.3) × 107	(6.7 ± 0.2) × 103	(4.2 ± 0.4) × 107	(1.7 ± 0.1) × 103	(3.7 ± 0.4) × 107	(7.4 ± 0.4) × 103	(2.8 ± 0.1) × 107

presents the microbiological characteristics of kombucha cultures on days 0 and 14 of fermentation, focusing on the abundance of different microorganisms, including acetic acid bacteria (ACC), total mesophilic bacteria (TMB), yeast, and Lactobacillus. Initially, all beverages displayed similar initial microbial counts for ACC, yeast, and Lactobacillus, ranging from (2.3 ± 0.1) × 10³ to (2.3 ± 0.4) × 10³ CFU/mL for ACC, (6.7 ± 0.2) × 10³ CFU/mL for yeast, and (7.4 ± 0.2) × 10³ to (7.4 ± 0.4) × 10³ CFU/mL for Lactobacillus, respectively.

After 14 days of fermentation, significant changes in the microbial counts were observed across all beverages. Specifically, the GJ exhibited a moderate increase in the TMB and Lactobacillus counts compared to the MGT, OGT, and DGT. GJ showed a rise in the TMB counts from (3.2 ± 0.1) × 10⁶ CFU/mL to (6.7 ± 0.7) × 10⁶ CFU/mL, while the MGT, OGT, and DGT displayed higher increases, reaching (2.7 ± 0.7) × 10⁷ CFU/mL, (2.9 ± 0.6) × 10⁷ CFU/mL, and (3.7 ± 0.4) × 10⁷ CFU/mL, respectively. Similarly, the Lactobacillus counts in the GJ increased from (9.2 ± 0.4) × 10⁵ CFU/mL to (5.8 ± 0.2) × 10⁶ CFU/mL, whereas the MGT, OGT, and DGT exhibited higher increments, reaching (1.8 ± 0.1) × 10⁷ CFU/mL, (2.1 ± 0.1) × 10⁷ CFU/mL, and (2.8 ± 0.1) × 10⁷ CFU/mL, respectively. These results suggest differential microbial proliferation dynamics influenced by the specific characteristics of each tea substrate and fermentation conditions, necessitating further investigation to comprehend their implications for kombucha fermentation outcomes and product quality.

3.4. Kill Time

Kill time refers to the duration required for a substance or treatment to effectively inhibit or kill a particular microorganism. In the context of the presented study, the kill time indicates the inhibitory effect of four types of tea-infused kombucha, GJ, OGT, MGT, and DGT, on the growth of three bacterial strains: E. coli, S. aureus, and S. enteritidis over a period of 24 h (Figure 5). Each graph presents the colony-forming units per milliliter (CFU/mL) as a function of time, demonstrating the potential antimicrobial effects of kombucha infusions with various tea types against different bacteria, with the control group indicating the natural growth rate of the bacteria without any kombucha influence. The y-axis varies between the graphs to illustrate the bacterial growth range specific to each microorganism tested.

The growth inhibitory effects of the GJ, OGT, MGT, and DGT tea-infused kombucha on E. coli, S. aureus, and S. enteritidis were investigated over a 24 h period. Initially, all kombucha variants and the control showed comparable CFUs/mL, suggesting no immediate impact on bacterial growth. However, over time, all kombucha types exhibited decreased CFUs/mL compared to the control, indicating inhibition of bacterial growth. Notably, at the 24 h mark, the GJ kombucha consistently demonstrated the most pronounced inhibitory effect on all tested bacterial strains, followed by the OGT, MGT, and DGT, respectively. These variations likely stem from differences in tea composition, fermentation processes, and specific antimicrobial properties. Overall, the findings highlight the potential of tea-infused kombucha as a natural antimicrobial agent, with differential effectiveness against various bacterial strains.

3.5. Differential Cytotoxic Effects of GJ Kombucha on Caco-2 and U251 Cancer Cell Lines

The cytotoxic properties of GJ kombucha were systematically assessed across two distinct human cancer cell lines: Caco-2, a colorectal carcinoma line, and U251, a glioblastoma astrocytoma line. The study employed a dose–response analysis, wherein cell viability was measured following exposure to a gradient of GJ concentrations, specifically at 12.5, 25, 50, 100, 200, 400, and 800 µg/mL. The relationship between the cell viability and concentration of GJ is shown in Figure 6.

The left panel (Figure 6a) shows the significant cytotoxic effects of GJ kombucha on the Caco-2 cell line. As the concentration of GJ kombucha increased, cell viability decreased dramatically, especially at the 800 µg/mL concentration, where viability dropped to 20%. This suggests that the 14-day fermented GJ kombucha has a potent cytotoxic effect on colorectal carcinoma cells, likely attributed to higher levels of bioactive compounds generated during prolonged fermentation. At lower concentrations, such as 12.5 µg/mL to 200 µg/mL, cell viability remained relatively stable for both the 7-day fermented and unfermented GJ powder samples (>70–90%), indicating that the 14-day fermentation process is crucial in enhancing cytotoxicity. In contrast, the 7-day fermented kombucha and GJ powder exhibited minimal cytotoxic effects, with cell viability higher than 70%, even at 800 µg/mL. This suggests that the 7-day fermentation period and unfermented GJ powder contain lower concentrations of bioactive compounds or less potent bioactivity compared to the 14-day fermented kombucha. The pH-adjusted control maintained near 100% cell viability, confirming that the pH variations in the GJ kombucha samples were not solely responsible for the observed cytotoxicity.

The right panel (Figure 6b) presents the response of U251 glioblastoma cells to the same GJ kombucha samples. While a dose-dependent reduction in cell viability was observed, the effects were less pronounced than in the Caco-2 cells. At the highest concentration of 800 µg/mL, the 14-day fermented GJ kombucha reduced U251 cell viability to around 50%. Although this reduction is substantial, it is much milder compared to the Caco-2 cells, indicating that U251 cells are less susceptible to the cytotoxic effects of the bioactive compounds in GJ kombucha. The 7-day fermented kombucha and unfermented GJ powder showed minimal cytotoxicity, with viability remaining consistently above 80% across most concentrations. These findings suggest that glioblastoma cells might have adaptive resistance mechanisms, or they may be less responsive to the bioactive components in GJ kombucha. The differential responses between U251 and Caco-2 cells highlight the potential selective cytotoxic effects of GJ kombucha, with colorectal carcinoma cells appearing more sensitive to prolonged fermentation products.

4. Discussion

The findings of the current study offer comprehensive insights into the antioxidant, antimicrobial, and anticancerous properties of tea-infused kombucha, with a particular focus on how different tea varieties influence fermentation dynamics and the resulting bioactivity of the beverage. By evaluating two experimental settings, we identified distinct patterns in antioxidant capacity, microbial dynamics, and anticancer activity, highlighting the complex interplay between tea substrates, fermentation processes, and post-fermentation infusion. This study underscores the potential of kombucha as a functional beverage with enhanced bioactive properties.

The key distinction between the two experimental settings lies in their approach to investigating the effects of tea varieties on kombucha fermentation. In the first experiment, the study examined the influence of tea infusion post-fermentation, where kombucha beverages fermented with black tea served as the base substrate. Following this initial fermentation period, the kombucha was infused with four distinct tea types, namely GJ, OGT, MGT, and DGT. This approach allowed for the assessment of how the infusion of different teas impacts the microbiological profile and antioxidant activity of the fermented beverage. In contrast, the second experiment focused on directly fermenting four herb teas—GJ, OGT, MGT, and DGT—without utilizing pre-fermented kombucha. By incubating the herb teas over a specified period, this study aimed to observe the fermentation dynamics and resultant properties of the kombucha beverages. This experimental setup provided insights into how various herb tea substrates interact with the fermentation process and contribute to the final characteristics of the beverage.

The antioxidant capacity of kombucha beverages was evaluated using the DPPH assay, with vitamin C/Ascorbic acid as the reference standard (IC₅₀: 10.03 µg/mL). The results demonstrated varying antioxidant potential across the kombucha variants. In the first experimental setting (Figure 3a), kombucha beverages infused with four types of tea showed significant differences in their antioxidant activities. GJ kombucha exhibited the highest antioxidant potential, with an IC₅₀ value of 14.04 µg/mL, closely approximating the standard antioxidant capacity of vitamin C. MGT kombucha displayed the second-highest activity (IC₅₀: 32.85 µg/mL), followed by DGT kombucha (IC₅₀: 43.68 µg/mL), while OGT kombucha demonstrated the lowest antioxidant potential (IC₅₀: 98.21 µg/mL).

The dose–response curves presented in Figure 3a clearly illustrate these trends, showing a steeper inhibition curve for GJ kombucha compared to the other variants. This steepness corresponds to its lower IC₅₀ value, indicating higher antioxidant efficacy. The gradual curves observed for MGT, DGT, and especially OGT reflect their comparatively lower antioxidant capacities. Integrating these visual data with the numerical IC₅₀ values provides a comprehensive understanding of the antioxidant activity differences among the kombucha variants.

The potent antioxidant activity observed in GJ kombucha can be attributed to its unique bioactive composition. Compounds such as geniposide, genipin, crocin, and crocetin, abundant in GJ tea, are well-documented for their strong antioxidant properties, acting through mechanisms like free radical scavenging and inhibition of oxidative stress pathways [63,64]. Furthermore, phenolic compounds, including rutin hydrate, gallic acid, quercetin, and (+)-catechin, significantly enhance its antioxidant capacity, as these molecules are known to neutralize free radicals and chelate transition metals effectively [65,66].

Similarly, MGT kombucha exhibited significant antioxidant activity, likely due to its rich concentration of phenolic acids, quercetin, rutin, theanine, and chlorophyll. Matcha green tea’s unique cultivation under shade increases its chlorophyll and amino acid contents, contributing to its antioxidant profile [67,68]. These compounds are known to exert antioxidant effects through various mechanisms, including direct radical scavenging and enhancement of endogenous antioxidant enzyme activities [69].

DGT kombucha retained notable antioxidant capacity (IC₅₀: 43.68 µg/mL) despite the decaffeination process, which removes caffeine, a minor contributor to antioxidant activity. The preservation of key phenolic compounds, such as catechins and flavonoids, underscores the importance of these compounds in the antioxidant mechanisms of kombucha [70]. However, the slightly lower activity compared to GJ and MGT may be attributed to the loss of the synergistic effects involving caffeine.

OGT kombucha demonstrated the least antioxidant potential (IC₅₀: 98.21 µg/mL), as evidenced by its dose–response curve in Figure 3a, which shows a significantly flatter gradient compared to other variants. This diminished activity could result from differences in cultivation practices, processing methods, or a reduced concentration of key antioxidant compounds [15,52]. While green tea is generally rich in catechins and flavonoids, the observed lower efficacy in OGT may stem from a reduced presence of these bioactives due to oxidative degradation or thermal processing.

Several studies related to herbal teas have shown that fermentation increases the bioavailability of compounds linked to antioxidant capacity. For instance, the oxidation of catechins into theaflavins and thearubigins is catalyzed by polyphenol oxidase (PPO). Theaflavins, including theaflavin-3-gallate and thearubigins, exhibit stronger antioxidant activity compared to their precursor catechins. Research has demonstrated that these compounds increase during fermentation, contributing to the superior antioxidant capacity of black tea when compared to non-fermented teas such as green tea [71,72,73].

Flavonoids, including flavonols and flavones, are also affected by fermentation. Specific processes, such as the fermentation of sun-dried green tea with tea fungi, result in an increase in compounds like kahenol and myricetin. This rise in flavonoid content is associated with enhanced antioxidant activity. For example, fermentation with Starmerella davenportii has been shown to maximize the total flavonoid content in black tea extracts within 36 h. Acidic fermentation conditions further facilitate the release of bound flavonoids, improving their bioavailability and antioxidant efficacy [74,75,76,77,78,79].

Non-flavonoid polyphenols, including phenolic acids such as gallic acid, are also transformed during fermentation. In some cases, such as when fermented with Aspergillus niger, gallic acid levels increase due to tannase-mediated metabolism, further enhancing the antioxidant activity of the tea. However, extended fermentation periods, which are typical in black tea production, may limit the accumulation of certain phenolic acids [80,81,82].

Different types of tea used as substrates for fermentation can introduce varying levels of antioxidants into the kombucha. For instance, in this first experimental setting, black tea served as the base substrate, known for its polyphenolic compounds, such as catechins and theaflavins, which contribute to its antioxidant properties. Therefore, kombucha fermented with black tea as the base would inherently possess a certain antioxidant capacity. During the infusion process with different types of teas post-fermentation, additional antioxidants are introduced into the kombucha. This infusion adds to the existing antioxidant content derived from the fermentation of black tea. Each type of infused tea brings its unique profile of antioxidants, thereby influencing the overall antioxidant capacity of the final product. The antioxidant capacity of the final kombucha product can vary depending on the composition and concentration of antioxidants in the infused teas. The fermentation process itself can influence the release and modification of antioxidants present in the tea substrate. Factors such as fermentation duration, temperature, and microbial activity can affect the bioavailability and potency of antioxidants in the final product. The combination of antioxidants from different tea sources may exhibit synergistic effects, enhancing the overall antioxidant capacity of the kombucha. Certain antioxidants may interact with each other to potentiate their individual antioxidant activities, thereby amplifying the overall protective effect against oxidative stress. Conversely, certain conditions during fermentation or infusion may lead to the degradation or loss of antioxidants, potentially reducing the overall antioxidant capacity of the kombucha. Overall, the first experimental setting highlights the complex interplay of tea substrates, fermentation processes, and post-fermentation infusion on the antioxidant capacity of kombucha beverages. It underscores the importance of tea selection and fermentation conditions in modulating the antioxidant profile of the final product [83,84].

In the second experimental setting, the antioxidant capacity of GJ was assessed at multiple time points throughout a 21-day fermentation process: days 0, 7, 14, and 21. The IC₅₀ values recorded for GJ were 36 µg/mL on day 0, 29.75 µg/mL on day 7, 26.97 µg/mL on day 14, and 18.26 µg/mL on day 21. For unfermented GJ, the IC₅₀ value was measured at 12.94 µg/mL. The progressive decline in IC₅₀ values over the 21-day period (as shown in Figure 3b) clearly demonstrates the increasing antioxidant potential of GJ, with a marked enhancement by day 21. As the IC₅₀ values decreased over time, the dose–response curves shifted, reflecting the enhanced antioxidant activity of GJ during fermentation. The steepening of the curve, particularly by day 21, signifies a more potent antioxidant effect, which is consistent with the observed reduction in IC₅₀ values. These graphical representations underscore the progressive nature of the fermentation-induced enhancement in antioxidant capacity. This trend indicates that the fermentation process played a significant role in boosting the antioxidant activity of GJ, likely due to the microbial conversion of compounds and the production of bioactive metabolites.

The herb teas utilized GJ, OGT, MGT, and DGT, each contributing its unique antioxidant profile to the fermentation process. In addition to the direct antioxidant content from the herb teas, the observed enhancement in antioxidant potential during fermentation can be attributed to various factors, including the production of postbiotics by microbial fermentation processes within the SCOBY. The metabolic activities of the microorganisms SCOBY during fermentation result in the generation of bioactive compounds such as organic acids, peptides, and phenolic compounds. These microbial-derived compounds, known as postbiotics, contribute significantly to the overall antioxidant capacity of the fermented beverage.

Chlorogenic acids and their derivatives (CQAs), categorized as depsides of trans-cinnamic acids and quinic acid, represent significant bioactive components in GJ. These compounds exhibit diverse biological activities, including anti-inflammatory, hypolipidemic, antimicrobial, and antioxidant properties [47,48,85,86,87]. Among these, 3,5-dicaffeoylquinic acid is a prominent bioactive compound found in GJ, known for its potent antioxidant properties. It belongs to the group of dicaffeoylquinic acids, which are derivatives of chlorogenic acid. This compound has been identified as a significant free radical scavenger, particularly effective in neutralizing ABTS•+ radicals. Its antioxidant capacity, measured by Trolox equivalent antioxidant capacity (TEAC), has been reported at 1.1 mM, indicating high antioxidant activity compared to other compounds analyzed in gardenia extracts [48,85,86,87,88,89]. The presence of 3,5-dicaffeoylquinic acid in GJ highlights its potential therapeutic and health-promoting benefits, making it a valuable target for further research into natural antioxidants and their applications in medicine and functional foods [47,48,85,86,87,88,89].

The quantification of 3,5-DCQA in GJ kombucha in the post-fermentation tea infusion in the current experiment revealed a significant increase in concentration during fermentation [Figure 4]. Initial levels of 3,5-DCQA were 1.9 mg/100 mL on day 0, rising to 3.1 mg/100 mL by day 7 and further to 3.7 mg/100 mL by day 14. This increase indicates that the metabolic activities of the microbial consortium within the SCOBY contribute to the enhanced presence of this compound. These results are statistically significant, indicating that the metabolic activities of the microbial consortium within the SCOBY contribute to the enhanced presence of this compound. The significant increase in 3,5-DCQA concentration during fermentation has several important implications, including enhanced antioxidant capacity, improved bioavailability and efficacy, and optimized fermentation parameters to maximize the production of this and other beneficial compounds. Understanding the dynamics of 3,5-DCQA accumulation can help optimize fermentation conditions, such as duration, temperature, and microbial composition, to maximize the production of this and other beneficial compounds. The insights gained from this study can inform the development of new kombucha products with targeted health benefits tailored to deliver higher concentrations of specific bioactive compounds. Additionally, these data provide a benchmark for comparing the antioxidant properties of kombucha made from different tea substrates, aiding in the selection of optimal ingredients for desired health outcomes.

During kombucha fermentation, microbial dynamics are pivotal in refining fermentation methodologies and determining the quality attributes of the beverage. The primary bacterial players, ACC, are responsible for converting alcohol into acetic acid, while dominant yeast species such as Zygosaccharomyces further contribute to the fermentation process [13,90,91,92,93,94]. The intricate interaction between these microbial species significantly influences the chemical composition of kombucha, including its taste, acidity, and sensory profiles. Balancing the activities of ACC and yeast is essential for controlling the production of organic acids and ethanol, thereby shaping the overall characteristics of the beverage. Changes in the bacterial and yeast communities can lead to variations in the production of organic acids, metabolites, and flavor profiles, highlighting the importance of understanding and managing these microbial dynamics [13,90,91,92,93]. ACC concentrations play a crucial role in influencing kombucha’s antioxidant properties, with higher concentrations correlating with elevated levels of antioxidants such as polyphenols and vitamin C. Similarly, yeast concentrations impact kombucha’s antioxidant and antimicrobial properties by contributing to the synthesis of organic acids, vitamins, and enzymes, as well as possessing antimicrobial properties crucial for inhibiting harmful microorganisms. While specific information regarding the direct influence of TMB concentrations on kombucha’s antimicrobial properties remains limited, their indispensable role in shaping the microbial population during fermentation underscores their importance. Concentrations of Lactobacillus sp. also significantly contribute to both the antioxidant and antimicrobial properties of kombucha, influencing the production of bioactive compounds such as polyphenols and organic acids. Variations in Lactobacillus sp. concentrations across different kombucha variants result in differences in properties and potential health benefits. Understanding and managing these microbial dynamics are crucial for refining fermentation methodologies to achieve the desired flavor, acidity, and overall quality attributes of kombucha. By comprehending the complexities underlying kombucha fermentation, producers can optimize production processes and enhance the beverage’s nutritional and sensory characteristics [13,90,91,92,93].

This study conducted a microbiological characterization of kombucha cultures over a 14-day fermentation period, with a specific focus on quantifying populations of ACC, TMB, yeast, and Lactobacillus species concentrations. Initial microbial counts at day 0 ranged from 1.7 × 10³ to 7.4 × 10³ CFU/mL across various kombucha variants, indicating a baseline colonization level. These microorganisms play pivotal roles in mediating kombucha fermentation, impacting its sensory attributes and purported health benefits. Following the 14-day fermentation period, a substantial escalation in microbial counts was observed, with counts surpassing 10⁷ CFU/mL in the majority of samples like the OGT, MGT, and DGT. This pronounced proliferation underscores the fermentative vigor intrinsic to kombucha production, driven by the metabolic activities of acetic acid bacteria, yeast, and Lactobacillus species. Notably, the kombucha variant fermented with GJ exhibited lower counts [(3.2 ± 0.1) ×10⁶ for ACC, (6.7 ± 0.7) ×10⁶ for TMB, (9.2 ± 0.4) ×10⁵ for yeast, and (5.8 ± 0.2) ×10⁶ for Lactobacillus] compared to other variants. The observed variation in microbial dynamics, particularly the lower counts in the kombucha variant fermented with GJ, may stem from several intertwined factors. Firstly, differences in the substrate composition between GJ and other tea substrates used in kombucha fermentation could influence nutrient availability, thereby impacting microbial growth and metabolic activity. Additionally, the distinct microbial community associated with GJ may engage in unique interactions compared to other substrates, potentially leading to altered growth patterns and population dynamics. Furthermore, pH levels and acidity—pivotal factors in kombucha fermentation—might also fluctuate between substrates, influencing microbial proliferation differently. Furthermore, GJ may possess inherent antimicrobial properties that could inhibit the growth of specific microbial species, contributing to lower overall microbial counts in the fermented product. Polyphenols, such as catechins and flavonoids found in GJ, exhibit antimicrobial activity by disrupting microbial growth through interactions with cell membranes, enzyme activities, and essential cellular processes. Modulating the growth dynamics of specific microbial species during kombucha fermentation potentially inhibits the proliferation of certain bacteria and contributes to reduced overall microbial counts compared to other tea varieties. Moreover, variations in fermentation conditions such as temperature, oxygen availability, and fermentation duration across different batches may further contribute to the observed variation in microbial populations. A comprehensive investigation into these factors, through targeted experiments and microbial community analysis, holds promise for elucidating the underlying mechanisms and refining fermentation protocols to achieve desired product characteristics and consistency. Accordingly, the observed microbial dynamics elucidate the intricate interplay between microbial consortia and fermentation parameters during kombucha elaboration. A profound understanding of these dynamics is imperative for refining fermentation methodologies, optimizing product quality attributes, and harnessing the full potential of this esteemed fermented beverage.

The investigation of kill time, defined as the duration necessary for teas infused in kombucha to effectively inhibit or eliminate the growth of E. coli, S. aureus, and S. enteritidis bacteria, serves as a pivotal metric in assessing the antimicrobial efficacy of various tea-infused kombuchas. By tracking the kill time, researchers can distinguish the effectiveness of kombucha as a natural antimicrobial agent, shedding light on how different tea types may influence its antimicrobial properties. This parameter not only elucidates the potential applications of kombucha in food safety and preservation but also underscores its significance in microbial control strategies. In this study, four types of tea-infused kombuchas—GJ, MGT, OGT, and DGT—were evaluated for their antimicrobial effects against E. coli, S. aureus, and S. enteritidis over a 24 h period (Figure 5). Monitoring the CFU/mL of bacteria over time allowed for the characterization of distinct growth patterns and antimicrobial activity among the different kombucha infusions. Initially, all kombucha variants and the control group showed comparable CFUs/mL, indicating that there was no immediate impact on bacterial growth upon exposure to the kombucha. This suggests that any observed differences in bacterial growth over time are likely attributable to the kombucha treatments rather than an inherent variability in initial bacterial populations. As time progressed, all kombucha variants exhibited a decrease in CFU/mL compared to the control group. This indicates that the kombucha treatments inhibited the growth of the tested bacterial strains over the 24 h period. The decrease in CFU/mL suggests that the antimicrobial properties of the kombucha were effective in limiting bacterial proliferation. At the 24 h mark, the GJ kombucha consistently demonstrated the most pronounced inhibitory effect on all tested bacterial strains, followed by the OGT, MGT, and DGT, respectively. This variation in inhibitory effects among the kombucha variants suggests that differences in tea composition, fermentation processes, and specific antimicrobial properties contribute to their effectiveness against the tested bacterial strains. Each type of tea used in the kombucha fermentation process (GJ, OGT, MGT, and DGT) possesses unique biochemical compounds that can influence microbial growth. For instance, green teas contain polyphenolic compounds such as catechins, which have known antimicrobial properties. The presence and concentration of these compounds vary among tea types, thus affecting their antimicrobial efficacy. The fermentation of kombucha involves the action of SCOBY on the tea substrate. During fermentation, the microorganisms in the SCOBY metabolize the sugars in the tea, producing organic acids, carbon dioxide, and trace amounts of alcohol. These metabolic byproducts contribute to the acidity and flavor of the final product and can also exhibit antimicrobial properties. The duration and conditions of fermentation can influence the production and concentration of these compounds, thereby affecting the antimicrobial activity of the kombucha. The antimicrobial activity of kombucha can be attributed to multiple factors, including the presence of organic acids (e.g., acetic acid, lactic acid), polyphenols, and other bioactive compounds produced during fermentation. These substances can disrupt bacterial cell membranes, inhibit enzyme activity, and interfere with the cellular processes essential for growth and survival. The effectiveness of kombucha against different bacterial strains may vary depending on their susceptibility to these antimicrobial agents. The variation in inhibitory effects observed among the tested bacterial strains (E. coli, S. aureus, and S. enteritidis) likely reflects differences in their susceptibility to the antimicrobial compounds present in tea-infused kombucha. Certain bacterial species may possess inherent resistance mechanisms or exhibit varying degrees of sensitivity to specific antimicrobial agents. Additionally, the composition and structure of their cell walls may influence their susceptibility to penetration and disruption by the active compounds in kombucha. Overall, the findings highlight the potential of tea-infused kombucha as a natural antimicrobial agent against a range of bacterial strains. The observed inhibitory effects underscore the importance of tea composition and fermentation processes in determining the antimicrobial efficacy of kombucha. By leveraging the natural antimicrobial properties of kombucha, food preservation strategies can employ a synergistic combination of hurdles, including refrigeration, modified atmosphere packaging, and pH control, to effectively inhibit microbial growth without relying solely on intensive thermal treatments. This approach not only enhances food safety but also preserves product quality by minimizing the impact of excessive heat exposure on sensory attributes and nutritional content. Incorporating tea-infused kombucha into hurdle technology offers a versatile and sustainable solution applicable to a wide range of food products, aligning with consumer preferences for minimally processed and naturally preserved foods. Further research into the specific mechanisms underlying kombucha’s antimicrobial effects will provide valuable insights for optimizing its application in food preservation strategies. Further research into the specific mechanisms underlying these effects could provide valuable insights into the development of kombucha-based antimicrobial products.

The cytotoxic effects of the GJ kombucha on Caco-2 and U251 cell lines observed in this study suggest selective anticancer potential. Caco-2 cells, representing colorectal carcinoma, were more sensitive to kombucha, while the U251 cells, a model for glioblastoma, showed resistance. These cell lines were selected to explore the effects of kombucha on cancers with distinct origins and biological characteristics, providing an initial understanding of its therapeutic potential. Colorectal cancer (Caco-2) is among the most common cancers, and its gastrointestinal origin aligns with the natural route of kombucha consumption, making it a relevant model. Glioblastoma (U251), in contrast, represents an aggressive brain tumor with a poor prognosis and serves as a challenging target to evaluate the limitations of kombucha’s cytotoxic effects.

This differential response may indicate that kombucha targets the specific pathways more critical in colorectal cancer cells, suggesting its potential for targeted therapies that minimize damage to healthy cells. However, the resistance observed in the U251 cells highlights the need for alternative strategies, such as combination therapies, to overcome glioblastoma’s inherent resistance.

While these findings provide valuable insights into the selective cytotoxic effects of GJ kombucha, we acknowledge that they may not be directly generalized to other cancer types. Different cancer cells have distinct molecular and metabolic profiles, potentially leading to variable responses to kombucha. Future studies should expand the evaluation to a broader panel of cancer cell lines, including those representing breast, lung, and pancreatic cancers, to better assess the therapeutic potential and broader applicability of GJ kombucha.

To further explore the therapeutic potential of GJ kombucha, future research should focus on isolating and characterizing the bioactive compounds responsible for these cytotoxic effects. Molecular studies, including gene expression analysis and proteomics, will provide deeper insights into the pathways influenced by kombucha. Additionally, investigating the bioavailability of key metabolites through future in vivo studies will clarify their absorption, distribution, metabolism, and excretion, which is essential for understanding their therapeutic effectiveness. Exploring kombucha’s effects across a wider range of cancer types and its interaction with the tumor microenvironment will be crucial for understanding its broader application in cancer therapy.

The impact of prolonged fermentation on the bioactive properties of kombucha should also be explored. While the current study focused on fermentation up to 21 days, extending the fermentation period could provide valuable insights into how fermentation time affects the production and stability of bioactive compounds, optimizing antioxidant, antimicrobial, and anticancer properties.

Nanoparticle-based delivery systems, such as liposomes, gold nanoparticles, and nanoemulsions, could enhance the bioavailability and stability of kombucha’s bioactive compounds. These systems may also offer targeted delivery, amplifying the therapeutic impact, particularly for cancer and inflammatory conditions. Future research should focus on optimizing these nanoparticle formulations to improve delivery efficiency, stability, and bioavailability, as well as evaluating their safety profiles and potential interactions with the human microbiome [94,95].

In conclusion, this study highlights the promising antimicrobial, antioxidant, and anticancer properties of GJ kombucha, particularly in the context of fermentation. The fermentation process was found to significantly enhance the production of bioactive compounds, contributing to GJ kombucha’s potent antioxidant and antimicrobial activities, as well as its selective cytotoxic effects on cancer cell lines. Among the kombucha variants tested, the GJ kombucha demonstrated the most robust activity across all assays, underscoring its therapeutic potential. The findings suggest that the choice of tea substrate and fermentation time are crucial factors influencing the bioactivity of kombucha. Further research is needed to optimize fermentation conditions, identify the specific bioactive compounds responsible for these effects, and evaluate the broader therapeutic applications of GJ kombucha. Expanding the scope of cytotoxicity testing to include additional cancer cell lines and animal models will be vital in determining its potential as a natural adjunct therapeutic for cancer treatment and microbial infections. This study lays a strong foundation for the future exploration of kombucha as a multifaceted, plant-based remedy with significant health benefits.