Synergistic Bioactive Potential of Combined Fermented Kombucha and Water Kefir

Abstract

The rising interest in functional fermented beverages, such as kombucha and water kefir, has stimulated research into their health benefits. This study aimed to investigate the combined bioactive potential of kombucha and water kefir by fermenting a novel medium prepared by mixing them in a 1:1 v/v ratio. The fermentation process involved using both SCOBY and water kefir grains (WKGs) separately, as well as co-cultivation, to explore the bioactive properties of the three fermented beverages. Samples were analyzed at 24, 48, and 72 h for changes in pH, microorganism growth, and concentrations of flavonoids and phenolics. Antioxidant activity was assessed using DPPH, ABTS, and FRAP tests, alongside colorimetric analysis and enzyme inhibition assays against α-amylase, α-glucosidase, and lipase. The results demonstrated that longer fermentation times increased both bioactive compound content and antioxidant capacity. The highest phenolic concentration was found in the WKG-fermented mixture (47.58 ± 2.13 mg GAE/100 mL), while the highest iron-reducing capacity was observed in the product fermented with both WKGs and the co-culture of SCOBY-WKGs. Additionally, SCOBY fermentation showed significant inhibitory activity (over 70%) against digestive enzymes. These findings suggest that co-fermenting kombucha and water kefir represents a promising alternative to traditional water kefir, with improved bioactive compound profiles.

1. Introduction

Over the past decade, there has been a growing awareness of minimally processed food products that offer high nutritional value and health benefits. As a result, fermented foods have been receiving increasing attention for their potential advantages [1]. Lavefve et al. investigated the microbial ecology of fermented vegetables and non-alcoholic beverages, highlighting the rich diversity in and the dynamic nature of the microbial communities involved in these fermentations [1]. Their review also examined current evidence regarding the potential health benefits of these foods, with a particular focus on gut health and immune system support. In ancient times, fermentation was primarily used as a method for food preservation. Today, however, our understanding has expanded to encompass the complex biochemical processes underlying fermentation. These transformations not only improve the nutritional profile and digestibility of foods but also result in the production of bioactive metabolites with potential health-promoting properties, as noted by Azcárate-Peril et al. [2]. Their study explored the role of probiotics and prebiotics present in fermented foods in modulating the gut microbiota. It highlighted their ability to enhance microbial diversity, strengthen the gut barrier, and support overall gastrointestinal health. Additionally, the authors emphasized how specific microbial strains and fermentation substrates can influence immune function and metabolic pathways. The health benefits associated with the consumption of fermented foods have been recognized for generations and continue to be supported by emerging scientific evidence. Although many of the health claims surrounding fermented products were once largely anecdotal, recent research in the fields of food and nutrition science has started to offer more robust evidence. Studies now suggest that fermented foods may have beneficial effects on chronic conditions such as cardiovascular disease, obesity, and diabetes—primarily through their ability to modulate the gut microbiota and exert anti-inflammatory effects [3]. These benefits are often attributed to the microorganisms in the food and the metabolites they produce [1]. Additionally, increasing attention has been given to the role of the human microbiota in the host health and how it interacts with the microbiota, particularly through gut microbes. As highlighted by Marco et al. [4], fermented foods can significantly influence the gut microbiota and, in turn, host physiology. Their study emphasized that the health benefits associated with fermented foods are largely attributed to the presence of live microorganisms and the bioactive metabolites generated during the fermentation process [4]. The growing interest in functional beverages, particularly fermented ones, has led to an increase in research on the health benefits and bioactive properties of drinks such as kombucha and water kefir.

Kombucha is a centuries-old, fermented beverage of probable Manchurian origins, traditionally made by fermenting sweetened tea with a symbiotic culture of bacteria and yeast (SCOBY). Diez-Ozaeta and Astiazaran investigated kombucha’s capacity to produce bacterial cellulose, highlighting its promising potential for applications in both the food and biomedical fields [5]. The symbiotic culture is embedded in a biocellulose biofilm that is described as a gelatinous mass. Thus, when brewing a new batch of kombucha tea, SCOBY is introduced into the sugared tea infusion, and two different phases can be perceived: a floating cellulose biofilm and the liquid phase (tea infusion). Villarreal-Soto et al. conducted a comprehensive analysis of kombucha fermentation, emphasizing the crucial roles played by bacteria and yeasts in the production of organic acids, ethanol, and various bioactive compounds. Their study also explored how factors such as fermentation conditions, the type of tea used, and sugar concentration affect the chemical composition and microbial dynamics of the final product [6]. Kombucha is traditionally brewed using teas from the Camellia sinensis plant, such as black and green tea. Gaggìa et al. explored the fermentation of kombucha using green, black, and rooibos teas, uncovering distinct microbial communities associated with each tea type that significantly influenced fermentation dynamics. Their study found that kombucha brewed with green tea exhibited the highest antioxidant activity, likely due to its rich polyphenol content [7]. Complementing these findings, Dutta and Paul emphasized that black tea sweetened with sucrose serves as an optimal substrate for kombucha production, as its polyphenol content supports microbial growth and enhances antioxidant properties. While green tea remains a popular alternative, it tends to produce different fermentation patterns and bioactive compound profiles [8]. During fermentation, the yeast converts the sugar into alcohol, while the bacteria metabolize the alcohol, producing various organic acids, vitamins, and other bioactive compounds. Antolak et al. reported that kombucha exhibits dual bioactive potential derived from both the tea base and the symbiotic culture of bacteria and yeasts (SCOBY). The SCOBY contributes additional metabolites such as organic acids and enzymes that further amplify the health-promoting properties of the beverage [9]. Kombucha is especially valued for its rich content of polyphenolic compounds, including catechins, phenolic acids such as gallic and chlorogenic acid, and flavonoids like rutin and quercetin. These bioactive compounds, originating from the tea and enhanced through fermentation, are believed to contribute to kombucha’s potential health benefits, such as supporting gut health, strengthening immune function, aiding detoxification, and reducing oxidative stress. Miranda et al. emphasized that the fermentation process transforms polyphenols in a way that increases their bioavailability and antioxidant capacity, reinforcing kombucha’s classification as a promising functional beverage [10]. Today, kombucha is widely available in diverse flavors across global markets, with the use of non-traditional substrates such as herbal infusions and fruit extracts expanding its functional potential even further [5,11].

Sugar water kefir, commonly known as water kefir or sugary kefir, is a fermented beverage made by fermenting a sugary liquid (such as sugar water or fruit juice) with water kefir grains, which contain a symbiotic culture of beneficial yeasts, LAB, and acetic acid bacteria (AAB). Randazzo et al. explored the creation of innovative non-dairy probiotic beverages by fermenting Mediterranean fruit juices with water kefir microorganisms. The study assessed fermentation performance, microbial dynamics, and chemical changes across different fruit substrates. The researchers found that water kefir grains effectively adapted to the juices, sustaining viable populations of lactic acid bacteria and yeasts throughout the fermentation process [12]. Unlike kombucha, which is tea-based, sugar water kefir offers a more versatile, often caffeine-free alternative. The fermentation process of water kefir results in the production of various organic acids, such as lactic acid, acetic acid, and succinic acid, along with vitamins (particularly B vitamins) and minerals, all of which support overall health. Fiorda et al. investigated the complex microbial community within water kefir grains, exploring how these microorganisms metabolize sucrose and other nutrients to produce organic acids (lactic and acetic), ethanol, carbon dioxide, and exopolysaccharides. These compounds contribute to the functional properties of the beverage [13]. Sugar water kefir is known for its high probiotic content, particularly strains of Lactobacillus and Saccharomyces, which support gut health, enhance digestion, and may have positive effects on immune function. Papadopoulou et al. examined the antioxidant, antithrombotic, and anti-inflammatory properties of bioactive compounds derived from water kefir grains (WKGs) and a fermented beverage made with apple pomace. The study aimed to evaluate the functional potential of WKGs, not only as a fermentation starter but also as a source of health-promoting molecules. The researchers conducted a series of in vitro biochemical and cellular assays to assess the effects. Both the WKG extract and the fermented beverage demonstrated strong antioxidant activity, inhibited platelet aggregation, and reduced inflammatory mediators. These findings suggest that WKG and its fermented beverage have antithrombotic and anti-inflammatory potential [14,15].

The majority of global scientific studies primarily focus on examining kefir and kombucha independently, often overlooking the exploration of the potential symbiotic relationships and synergistic effects that may arise from their combined use. To the best of the authors’ knowledge, there are limited studies on the combination of kombucha and water kefir beverages, as well as their co-cultured products involving SCOBY and WKGs. Pihurov et al. investigated the synergistic microbial interactions between SCOBY and WKGs, as lyophilized cultures, during co-fermentation, with a focus on their combined contribution to postbiotic compound production. Using metagenomic and metabolomic approaches, the authors characterized the microbiota dynamics and metabolic outputs of co-fermentation. The complementary interaction between SCOBY and WKGs microbiomes resulted in increased postbiotic diversity and concentration, enhancing antioxidant, antimicrobial, and immunomodulatory properties, thus improving the co-fermented beverage’s functionality [16]. The study by Filho et al. examined the fermentation dynamics of kombucha and kefir using a cashew nut beverage (Anacardium occidentale L.) as a substrate. They tested three formulations: 100% kefir, 50/50% kefir and kombucha, and 100% kombucha, all fermented for 72 h at 28 °C. The results showed that mixed cultures of kefir and kombucha did not affect the glucuronic acid production but were efficient at consuming oligosaccharides compared to individual cultures. The fermentation of the cashew nut beverage with kefir and kombucha improved the flavor and texture of the beverage, making it a potential healthy and innovative alternative for new culinary recipes [17]. However, there are no data in the literature on a new formulated medium created by combining kombucha and sugar water kefir. With a view of enhancing the nutritional and biological profiles of the two beverages by exploiting their combined bioactive potential, the focus of the present study was to formulate a new fermentation medium by mixing kombucha and water kefir in a 1:1 v/v ratio and to investigate the fermented products resulting from the use of both SCOBY and WKGs separately, as well as from the co-cultivation of SCOBY and WKGs.

A detailed understanding of the dynamics occurring during fermentation, such as changes in pH, grain growth, and SCOBY, and the concentration of bioactive compounds, will provide valuable insights into potentially enhancing the benefits of the beverage. Furthermore, the evaluation of the inhibition of enzymatic activities, such as lipase and amylase, could further elucidate the functional potential of these fermented beverages.

2. Materials and Methods

2.1. Chemicals and Reagents

The symbiotic culture of bacteria and yeast (SCOBY) and water kefir grains that were used for the preparation of the fermented beverages were purchased from Kefiralia (Burumart Commerce S.L, Arrasate, Spain). In addition, water (Leo, Sila, Carlopoli, Italy), black tea (TeAti, Rotterdam, The Netherlands), and white sugar (Eridania, San Quirico, Italy) were from a local market.

Dimethylsulphoxide (DMSO), absolute ethanol and methanol, and HPLC-grade formic acid and acetonitrile were acquired from Carlo Erba (Milan, Italy). Folin–Ciocâlteu reagents, sodium carbonate, gallic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azinobis-(3-ethylbenzthiazolin-6-sulfonic acid, ABTS), sodium carbonate (Na₂CO₃), potassium persulfate (K₂S₂O₈), aluminum chloride (AlCl₃), 2,4, 6-Tris(2-pyridyl)-s-triazine (TPTZ), FeCl₃, sodium acetate, FeSO₄, and butylhydroxytoluene (BHT) were obtained from Sigma Aldrich (Milan, Italy). Lipase (EC 3.1.1.3), α-amylase (EC 3.2.1.1), α-glucosidase (EC 3.2.1.20), dihydro chloride, maltose, potassium chlorate, aluminum chloride, sodium acetate, o-dianisidine solution (DIAN), peroxidase/glucose oxidase system color reagent (PGO), p- nitrophenyl octanoate (NPC), and TRIZIMA were from Sigma-Aldrich s.r.l. (Milan, Italy).

2.2. Fermentation Conditions

For the kombucha preparation, boiling water (1 L) was added with a commercial black tea (7 g, 0.7% w/v) in a sterile glass vessel previously sterilized at 121 °C, 15 psi for 60 min. The tea infusion was allowed to steep for a few minutes, after which sucrose (100 g, 10% w/v) was added and stirred until fully dissolved. Once the solution cooled to room temperature, it was inoculated with a symbiotic culture of bacteria and yeast (SCOBY, S) and incubated at room temperature for 24 h.

After fermentation, the resulting infusion was divided equally into two portions (500 mL each) and transferred into sterile amber glass containers. One portion was designated as the kombucha control (Ko), while the other was reserved for mixing with water kefir.

Water kefir was prepared by dissolving sucrose (100 g, 10% w/v) in 1 L of natural water, to which four dates were added as a natural nitrogen source. The solution was inoculated with WKGs (100 g, 10% w/v) and fermented at room temperature for 24 h. After fermentation, the beverage was filtered and divided into two 500 mL portions. One portion served as the water kefir control (Ke), and the other was mixed with the kombucha sample as described above.

A new fermentation medium was created by mixing 500 mL of kombucha with 500 mL of water kefir (KoKe). This mixture was then partitioned into four 250 mL portions: 250 mL was kept as a control (KoKe), 250 mL was inoculated with 25 g of WKGs (KoKeG), 250 mL was inoculated with 40 g of SCOBY culture (KoKeS), and the remaining 250 mL was cultivated with both WKGs and SCOBY (S) (25 g and 40 g, respectively, KeKoGS). Each portion was stored in a sterile glass container, covered by a breathable cloth and kept at room temperature for 72 h. During the fermentation period, 50 mL aliquots were collected from each container at 24, 48, and 72 h. An overview of the fermentation process is shown in Figure 1. All samples were stored at −20 °C until further analysis. All analyses were performed in triplicate, and the results are given as mean ± standard deviation.

2.3. pH Evaluation

The pH values of all samples were evaluated using an electronic pH meter (Hanna Instruments, Woonsocket, George Washington Hwy, Smithfield, RI, USA) [18].

2.4. Total Phenolic Content (TPC) Determination

A Folin–Ciocâlteu assay was carried out to evaluate the total phenolic content (TPC) [19]. Briefly, 100 µL of the sample solubilized in 2 mL of H₂O was added to the Folin–Ciocâlteu reagent (500 µL) and a 7.5% Na₂CO₃ solution (400 µL). After incubation at room temperature for 30 min, the absorbance was measured (λ = 765 nm) by a UV–vis spectrophotometer (model V-10 plus, Onda, Europe) against a blank. The TPC was determined by a seven-point gallic acid curve that was made by analyzing stock solutions at concentrations ranging from 0.0001 to 0.5 mg/mL. The results, reported as the mean value of three measurements ± standard deviation, were expressed as mg of gallic acid equivalents per 100 mL of sample (GAE/100 mL).

2.5. Total Flavonoid Content (TFC) Determination

The total flavonoid content (TFC) was evaluated by applying a colorimetric assay [20]. The TFC was evaluated by a quercetin calibration curve that was obtained by analyzing seven stock solutions at concentration in the range from 0.01 to 1 mg/mL. The results, reported as a mean value of three replicates ± standard deviation, were expressed as mg of QE/100 mL of sample.

The mathematical kinetic model was a very useful method to analyze the impact of fermentation on bioactive compounds.

The first-order degradation kinetic was applied following the equation:

−ln (Ct/C0) = kt

where C0 was the initial TPC or TFC concentration, and Ct was the concentration of both TPC and TFC after 48 and 72 h of fermentation.

2.6. Antioxidant Activity Evaluation

The DPPH, ABTS, and FRAP assays were performed to evaluate the antioxidant activities of all samples. The inhibition capacity of both 2,2-diphenyl-1-picrylhydrazyl (DPPH) and azino-bis(3-ethylbenzothiazoline-6-sulfonate (ABTS•⁺) radicals was expressed as mg of Trolox equivalents per mL of samples (mg TE/ 100 mL) and the ferric-reducing antioxidant power (FRAP) values were reported as mM of FeSO₄ per 100 mL of sample.

2.6.1. DPPH Assay

A working solution of DPPH at a concentration of 0.1 mM was prepared from the commercial DPPH solution (1 mM) by dilution with methanol [21]. In total, 100 µL of the DPPH solution was added to different volumes of each sample (10, 25, 50, and 100 µL) and the volume went up to 3 mL with methanol. The solutions were incubated for 30 min in the dark, covered with aluminum foil, under magnetic stirring. A decrease in absorbance was monitored at 517 nm. Similarly, the calibration curve was prepared using 100 μL of Trolox at a concentration range of 0.25–0.01 mg mL⁻¹ in methanol (2.8 mL) and 100 µL of the DPPH working solution. The total antioxidant capacity of samples was expressed as mg of Trolox equivalents (TEAC) per 100 mL of sample. Each sample was measured in triplicate. Mean and standard deviation (n = 3) were calculated. Also, EC50 values were calculated using GraphPad Prism 8 software (GraphPad Inc., San Diego, CA, USA).

2.6.2. ABTS Assay

A solution of cation-radical ABTS•⁺ was prepared by mixing a 7 mM ABTS solution and 2.45 mM of potassium persulfate (K₂S₂O₈) and allowing the mixture to stand for 16 h in the dark. The final working solution of ABTS•⁺ was obtained, allowing the solution to stand for 16 h under the laboratory temperature in the dark. For the spectrophotometric measurement of the samples, 1 mL of diluted ABTS•⁺ (3 mL) and different volumes of each sample (10, 25, 50, and 100 µL) were mixed. A six-point calibration curve was prepared using Trolox at the concentration range of 0.50–0.01 mg mL⁻¹ in methanol, and the absorbance was acquired at 734 nm after 5 min of incubation [22]. The ABTS•⁺ scavenging activity of the samples was expressed in 100 mL samples as mg of Trolox equivalents (mg of TE 100 mL⁻¹). Each sample was measured in triplicate. Mean and standard deviation (n = 3) were calculated. Also, the radical scavenging activity was given as EC₅₀ values (µL ± SD).

2.6.3. FRAP Test

The ferric-reducing antioxidant power (FRAP) assay is a widely used method that measures the antioxidant potential in samples through the reduction of ferric iron (Fe³⁺) to ferrous iron (Fe²⁺) by the antioxidants present in the samples in a redox-linked colorimetric reaction. Ferric (Fe³⁺) to ferrous (Fe²⁺) ion reduction at a low pH causes the formation of a blue-colored ferrous complex (ferrous-tripyridyltriazine (Fe²⁺-TPTZ)) from a colorless ferric complex (ferric-2,4,6-tripyridyl-s-triazine (Fe³⁺-TPTZ)) in the presence of antioxidant compounds. The assay was performed according to a previously reported protocol [23]. A working FRAP reagent was prepared as required by mixing 0.25 M of sodium acetate buffer (pH 3.6), 10 mM of tri-pyridyl-triazine (TPTZ) solution in 40 mM of HCl, and 20 mM of FeCl₃·6H₂O solution in a ratio of 10:1:1. FRAP solution reagent (2 mL) was mixed with 0.9 mL of distilled water and 0.1 mL samples (1 mg/mL_DMSO). The mixtures were allowed to stand under a magnetic stirrer for 30 min in the dark. Then, the absorbance changes in the Fe²⁺ complex, due to the action of an antioxidant compound in the samples, were measured at 593 nm by the spectrophotometer (model V-550, Jasco, Milano, Italy, Europe). The standard curve of an FeSO₄ solution was linear between 10 and 0.001 mg/mL. The positive control was BHT. The results were expressed as mM of FeSO₄ per 100 mL of samples.

2.7. Determination of the Phenolic Acids, Flavonoids, and Caffeine Content by High-Performance Liquid Chromatography (HPLC-DAD) Analysis

The phenolic compounds of the samples were separated by high-performance liquid chromatography (HPLC, Shimadzu, Kyoto, Japan) equipped with an auto sampler (SIL 20A), two pumps (LC 20AD), and a system controller CMB-20A. The separation was performed on a Mediterranea SEA C18 column (4.6 mm × 25 cm, 5 μm, Terrassa, Spain) using a mobile phase that consisted of formic acid (0.1%) in double-distilled water (A) and acetonitrile, ACN (B). All samples and commercial standards were analyzed utilizing the following gradient over a total run time of 50 min: 0.01 min, 10% B; 20 min, 22% B; 40 min, 40% B; 45 min, 10% B; 50 min, 10% B. The flow rate of the mobile phase was 0.8 mL/min. Polyphenolic compounds in the eluent were detected with a diode array detector (SPD M20A, Shimadzu, Kyoto, Japan), at different wavelengths. The compounds in the samples were identified by overlapping their chromatograms and standard ones: chlorogenic acid (λ = 327 nm), ellagic acid, gallic acid, rutin and vanillic acid (λ = 254 nm), caffeine (λ = 273 nm), epigallocatechinagallato (λ = 254 nm), ferulic acid (λ = 325 nm), p-coumaric acid (λ = 310 nm), and catechin (λ = 280 nm). The individual phenols were quantified by the external standard method using the respective calibration curves in the range of 0.1–0.5 mg/mL for all standards.

2.8. Colorimetric Analysis

Colorimetric analysis was made by using a colorimeter PCE CSM-4 (PCE, Lucca, Italy), capable of detecting CIELab parameters (a*, b*, and L*). The chroma C* and hue angle (h*) were also calculated following Equations (1) and (2).

C* = (a*2 + b*2)1/2

(1)

Hue angle (h*) = tan−1 (b*/a*)

(2)

The evaluation of the overall color change was assessed by the calculation of ∆E (3):

∆E = [(L0∗ − L∗)2 + (a0∗ − a∗)2(b0∗ − b∗)2]1/2

(3)

where the index “0” indicates the sample at time 0, and the letters without the index correspond to the parameters after different fermentation times.

2.9. Enzyme Inhibitory Activities

The porcine pancreatic lipase inhibitory activity of the samples was determined following the previously published method [24]. Briefly, a solution of 4-nitrophenyl octanoate (NPC) in dimethyl sulfoxide (DMSO) (5 mM), lipase (1 mg/mL), and Tris-HCl buffer (pH 8.5) was prepared. Briefly, a sample (25 µL) was mixed with the enzyme, NPC solution, and Tris-HCl buffer (pH 8.5). The mixture was left to react at 37 °C for half an hour; after that, the absorbance was read at λ= 405 nm using a UV-Vis Jenway 6003 spectrophotometer (Carlo Erba, Milan, Italy). Orlistat was used as a positive control. The α-amylase and α-glucosidase inhibitory activity was assessed as previously reported [24]. In the α-amylase inhibitory assay, a starch solution of the enzyme (EC 3.2.1.1) and colorimetric reagent (CRS) was prepared. Both the control and sample (40 µL) were added to the starch solution and left to react with the enzyme at 25 °C for 5 min. The absorbance was read at 540 nm. After that, the absorbance was read at λ = 540 nm. In the α-glucosidase inhibitory assay, a maltose solution, α-glucosidase (EC 3.2.1.20) solution, O-dianisidine (DIAN) solution, and peroxidase/glucose oxidase (PGO) system-color reagent solution were prepared. The sample or control (5 µL) was mixed with the maltose solution (250 µL), and enzyme solution (5 µL), and the obtained mixture was left to incubate at 37 °C for 30 min. Then, perchloric acid solution (50 µL) was added, and the mixture was centrifuged. The supernatant was collected and mixed with DIAN (5 µL) and PGO (300 µL), and left to incubate at 37 °C for 30 min. The absorbance was read at λ = 492 nm with UV-Vis Jenway 6003 spectrophotometer (Carlo Erba, Milan, Italy). Acarbose was used as a positive control in both tests.

2.10. Statistical Analysis

The experiments were conducted in three replications, and the averages were subjected to variance analysis using GraphPad Prism 10.3.1 software (GraphPad Software, San Diego, CA, USA). The study opted for the two-way ANOVA followed by the Tukey test to determine the differences between the means ± standard deviations of pH, TPC, TFC antioxidant activities, phenolic acids, flavonoids, caffeine content, and CIELab parameters. Significance was established at p values < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****).

3. Results and Discussion

3.1. pH Values

During fermentation, a decrease in pH was observed in all samples as early as 24 h (Figure 2). After one day, separately fermented kombucha (Ko) and water kefir (Ke) showed pH values of 4.1 ± 0.2 and 4.5 ± 0.4, respectively. The pH of Ko remained relatively stable throughout the fermentation, reaching 3.8 ± 0.1 by 72 h, while Ke showed a gradual decrease, reaching 4.0 ± 0.1 (p < 0.05). In contrast, the mixed beverage (KoKe) exhibited a more pronounced acidification, with pH values dropping from 3.5 ± 0.6 at 24 h to 2.8 ± 0.1 at 48 h and 2.7 ± 0.1 at 72 h. The incorporation of water kefir grains (KoKeG), SCOBY (KoKeS), or both (KoKeGS) into the KoKe mixture further accelerated the acidification process. For instance, KoKeG and KoKeS already reached a pH of 3.0 ± 0.1 after 24 h, which was lower than KoKe alone at the same time point. After 72 h, KoKeG and KoKeS showed significantly lower pH values compared to their 24 h measurements (p < 0.05), while KoKeGS displayed no significant change between 48 and 72 h.

The results obtained for the separately fermented Ko and Ke beverages were comparable with the pH values reported in the literature, which ranged from pH 4.0 to pH 3.0 and pH 4.5 to pH 3.5, respectively, over the first three days of fermentation [20,25,26,27,28]. The pH significantly decreased within the first 24 h of the process from an initial value of 6.5–6.2, due to the metabolic activity of the microorganisms of SCOBY and WKGs that converted the sugars, resulting in higher acid production. The co-fermentation of kombucha and water kefir introduces a complex microbial ecosystem involving yeasts and bacteria from both cultures. While our study did not employ metagenomic or culture-dependent analyses, the existing literature provides insights into potential interactions [16,29,30]. In water kefir, the coexistence of yeasts and LAB is characterized by mutualistic interactions. Yeasts, such as Zygotorulaspora florentina, produce essential nutrients like amino acids and vitamins (e.g., B6) through their metabolic activities. These compounds are utilized by LAB species, including Lactobacillus hordei and Lactobacillus nagelii, promoting their growth and lactic acid production. Conversely, LAB acidify the medium, creating an environment conducive to yeast growth and enhancing the overall fermentation process [31,32]. Studies have demonstrated that the co-cultivation of yeasts and lactobacilli in water kefir medium significantly enhances the growth of all interaction partners, indicating a mutualistic relationship. For instance, Z. florentina benefits from the acidification of the medium by lactobacilli, while lactobacilli receive essential nutrients produced by yeasts, such as amino acids and vitamins [31].

Kombucha fermentation involves a symbiotic culture of bacteria and yeasts (SCOBY), where acetic acid bacteria (e.g., Gluconacetobacter sp.) and yeasts (e.g., Saccharomyces cerevisiae) collaborate. The acetic acid bacteria oxidize ethanol produced by yeasts into acetic acid, contributing to the characteristic tangy flavor of kombucha.

When SCOBY and WKGs are co-cultured, their microbial communities may engage in synergistic interactions that enhance the fermentation process. Yeast-driven sugar fermentation produces ethanol that acetic acid bacteria can oxidize, contributing to organic acid production. In parallel, lactic acid bacteria from kefir grains may benefit from the mild acidic environment generated by acetic acid bacteria from SCOBY, further supporting their growth and metabolite production.

After the fast initial decrease, the pH values showed a drop between 0.6 and 0.4 units in the next few days, along with the increase in the organic acid concentrations in the medium due to the metabolism of the acetic acid bacteria (AAB) that used ethanol generated by yeasts for acetic acid production [33] which, in turn, stimulated yeast activity in ethanol production, highlighting the synergistic growth of the microorganisms.

From 24 h onward, a stabilization occurred with the pH values, which may be related to the buffer effect arising from the reactions between synthesized organic acids and minerals from the substrate [34]. In addition, acetic acid may have less interference in pH because it is a weak acid with a low ionization rate in the medium [17]. In the newly formulated medium KoKe, prepared by mixing the two fermented beverages Ko and Ke in a 1:1 v/v ratio, the pH resulted in lower values compared to the starting Ko and Ke at all times investigated. This decrease in pH values can be attributed to the metabolic activity of the consortium of microorganisms, involving a wide variety of lactic and acetic acid bacteria and yeasts, which worked in mutualistic symbiosis in the fermented beverages even after mixing. The addition of the starters S, G, and SG led to a lowering of the pH value only in the first 48 h. The same trend of pH values was reported by Lima Araujo Filho et al. [17]. They prepared a cashew nut beverage (CNB) by blending dried and raw cashew nuts (Anacardium occidentale L.) and inoculated 100 mL of CBN with 5 mL of kombucha and 5 mL of kefir (10% v/v). After 36 h of fermentation, the beverage had reached a pH value of 4.23, which decreased to 3.96 after 48 h and remained unchanged after 72 h [17]. Similar values were found by Shori et al. [35] while producing cashew nut yogurt. The authors fermented cashew nut milk with lactic acid bacteria, strained at 42 °C for 3 h, and obtained a product with a pH of 4.06, with little variation after refrigeration and storage for 21 days.

3.2. TPC

The Folin–Ciocâlteu assay revealed that tea kombucha (Ko) exhibited the highest absolute phenolic content among all samples analyzed (Figure 3). Phenolic compounds are widely recognized as key contributors to the bioactive properties of fermented beverages, with their composition varying depending on the source material. Zhao et al. analyzed 30 tea infusions from green, black, oolong, white, yellow, and dark teas, profiling their phenolic compounds and assessing antioxidant activities. The study found that green and yellow teas had the highest total phenolic content and strongest antioxidant capacity, highlighting the significant role of tea type in determining bioactive potential [36]. In kombucha (Ko), the presence of symbiotic microorganisms within the SCOBY has been shown to enhance phenolic content during fermentation [37,38]. Across all fermentation times, Ko exhibited relatively stable total phenolic content (TPC), ranging from 56 to 58 mg GAE/100 mL. In contrast, water kefir (Ke) initially displayed a low phenolic content (0.21 ± 0.03 mg GAE/100 mL at 24 h), which relied on date fruits as a source of sugar. However, this value increased progressively over the three-day fermentation period, reaching 0.55 ± 0.01 mg GAE/100 mL. The TPC values observed in our kombucha samples did not align with those reported by Laureys and De Vuyst, who found total phenolic content levels of approximately 0.30 mg GAE/mL at 24 h and 0.45 mg GAE/mL at 48 h of fermentation [27]. Similarly, Hsieh et al. reported a TPC of 0.7 mg GAE/mL after 72 h of fermentation, indicating higher phenolic content under their experimental conditions [39]. The findings highlighted how the phenolic content of the newly fermented medium KoKe markedly improved compared to Ke (31.93 ± 0.19 mg GAE/100 mL, **** p < 0.0001), although the content significantly reduced over 72 h of fermentation (**** p < 0.0001), (24.24 ± 0.11 mg GAE/100 mL at 48 h, 22.33 ± 0.05 mg GAE/100 mL at 72 h). The TPC trend was likely caused by the initial metabolic activity of microorganisms in the mixed medium, which continued to hydrolyze and transform kombucha polyphenols from bound to freeform during the first 24 h. Microbial activity significantly decreased in the following two days, as observed by Sarkara et al. [40]. This reduction was likely due to nutrient depletion, the buildup of inhibitory metabolites, or environmental changes. Their findings highlight the dynamic nature of microbial processes in polyphenol biotransformation and emphasize the importance of optimizing fermentation conditions to sustain microbial activity and improve polyphenol production efficiency. The addition of the cultures G, S, and SG to KoKe improved the phenolic amount after 48 h, ranging between 29.31 ± 0.04 and 34.00 ± 0.08 mg GAE/100 mL, and especially after 72 h, reaching values between 34.93 ± 0.59 and 47.58 ± 2.13 mg GAE/100 mL. The greatest increase in phenolic concentration was observed in KoKeG, likely because kefir grains exhibited higher LAB β-glucosidase activity, which is responsible for breaking down phenolic compounds into simpler forms compared to SCOBY [41,42]. Michlmayr and Kneifel provided an in-depth analysis of the β-glucosidase activities of lactic acid bacteria (LAB) that hydrolyze plant metabolite glucoconjugates, leading to the release of bioactive compounds and enhancing the sensory attributes of fermented foods. The review discusses the biochemical and genomic mechanisms critical for the hydrolysis of β-glucosides by food-fermenting LAB. It highlights the role of the phosphotransferase system (PTS) in the uptake of β-glucosides and the subsequent action of β-glucosidases in their hydrolysis.

A key factor in this process was the speed at which metabolites were produced during fermentation by WKGs and SCOBY; for WKGs, the average time was typically 24 h, whereas for SCOBY, it took up to 20 days [20,43,44]. The similarity of TPC values between KoKeS and KoKeGS suggested that combining kefir and kombucha cultures did not enhance the efficiency of metabolizing bound phenolic or releasing free phenolic into the medium. The symbiotic culture of bacteria and yeast (SCOBY) in kombucha primarily comprises acetic acid bacteria (AAB) and yeasts, which may not possess the same level of β-glucosidase activity. The co-culture of SCOBY-WKGs introduces additional microbial dynamics. While AAB can metabolize ethanol to acetic acid, potentially enhancing the medium acidity and stability, the presence of SCOBY may not significantly augment the β-glucosidase-mediated hydrolysis of bound phenolics. This could explain why the TPC in KoKeGS did not surpass that of KoKeG, despite the added complexity of the SCOBY. Furthermore, the co-culture of SCOBY and WKGs may introduce competitive interactions for nutrients or the production of inhibitory metabolites, such as bacteriocins or organic acids, potentially modulating the growth of specific taxa or metabolite profiles. These complex interactions may partly explain the differences observed in phenolic content across fermentation conditions in this study. These findings underscore the pivotal role of LAB β-glucosidase activity in the fermentation process, highlighting the importance of selecting appropriate microbial consortia to optimize the release and bioavailability of phenolic compounds in fermented beverages.

3.3. TFC

The total flavonoid content of all samples is shown in Figure 4. Ko showed the highest concentration at all fermentation times (57.57 ± 0.31, 61.48 ± 1.13, and 75.63 ± 0.59 mg QE/100 mL at 24, 48, and 72h, respectively). Several studies reported that the flavonoid content in kombucha changes during fermentation, often increasing in the early stages and decreasing over time [39]. This trend can be attributed to the degradation of flavonoids caused by microbial metabolism, oxidation, and the acidic conditions of the fermentation process [45]. Unlike kombucha, which had tea-derived flavonoids [46], sugar water kefir lacked a significant source of flavonoids as no fruits were used. Any trace amounts of flavonoids from the dates used as the sugar source [47] were unlikely to undergo significant degradation or transformation during the 24, 48, or 72 h of fermentation. Essentially, sugar water kefir did not contain measurable amounts of flavonoids. However, no studies have specifically examined the flavonoid content in sugar water kefir without fruits. KoKe exhibited a significant improvement in flavonoid content compared to Ke (**** p < 0.0001), having reached concentrations of 39.02 ± 1.15 mg QE/100 mL at 24 h, 40.69 ± 0.45 mg QE/100 mL at 48 h, and 50.02 ± 0.70 mg QE/100 mL at 72 h of fermentation. Unlike the phenolic content, the addition of the WKGs and/or SCOBY did not have a positive impact on the TFC values. The reduced flavonoid concentrations observed in KoKeG, KoKeS, and KoKeGS compared to KoKe can be attributed to the increased microbial diversity resulting from the addition of starter cultures (G, S, or GS) to the existing microbial community in the KoKe beverage. This expansion likely promoted antagonistic microbial interactions, where microorganisms compete for available nutrients, including flavonoids, which serve as carbon and energy sources for microbial growth and metabolic processes. During fermentation, microorganisms such as lactic acid bacteria (LAB) and acetic acid bacteria (AAB) produce various enzymes, including β-glucosidases, which can hydrolyze flavonoid glycosides into aglycones [48]. While this enzymatic activity can enhance the bioavailability of flavonoids, it may also lead to the depletion of free flavonoid concentrations in the medium. Additionally, the presence of multiple microbial strains can introduce complex interactions, such as competition for substrates and the production of antimicrobial compounds, further influencing flavonoid levels. Therefore, the addition of starter cultures (G, S, or GS) to the KoKe beverage likely increased the microbial diversity, intensifying the competition for flavonoids and leading to their reduced concentrations in the final product. This underscores the importance of carefully managing microbial consortia in fermentation processes to balance the enhancement of bioactive compounds with the preservation of desired nutrient levels.

Tu et al. identified Pichia, Aspergillus, Zygosaccharomyces, Acetobacter, and Ralstonia as the key microorganisms responsible for metabolizing flavonoids and producing major volatile flavor compounds in the kombucha beverage [49]. The best result was found to be the TFC value of KokeG at 72 h (34.56 ± 0.69 mg QE/100 mL).

A kinetic model was applied to assess the changes in TPC and TFC during the fermentation process. The results indicated that the samples followed a first-order reaction, as shown in

Table 1. Kinetics analysis of TPC and TFC evolution in kombucha samples during fermentation.

Bioactives	Treatment	Reaction Order	K-Value	Equation	R2
TPC
	Ko	1	0.0009	y = 0.0009x + 3.9922	0.871
	Ke *	-	-	-	-
	KoKe	1	0.1065	y = −0.1065x + 32.763	0.462
	KoKeG	1	0.004	y = 0.004x + 3.2384	0.817
	KoKeS	1	0.007	y = 0.0071x + 3.2989	0.756
	KoKeGS	1	0.0028	y = 0.0028x + 3.3425	0.882
TFC
	Ko	1	0.0057	y = 0.0057x + 3.8928	0.917
	Ke *	-	-	-	-
	KoKe	1	0.0052	y = 0.0052x + 3.5122	0.874
	KoKeG	1	0.0016	y = 0.0016x + 3.271	0.992
	KoKeS	1	0.0123	y = 0.0123x + 2.7128	0.910
	KoKeGS	1	0.0122	y = 0.0122x + 2.4345	0.919

* not calculable. Ko: kombucha; Ke: kefir; KoKe: kombucha and water kefir (1:1 v/v) (b); KoKeG: KoKe fermented by WKGs; KoKeS: KoKe fermented by SCOBY; KoKeGS: KoKe co-fermented by SCOBY and WKGs.

.

Data analysis revealed an increase in polyphenolic compounds in all samples, both in terms of TPC and TFC, except for the TPC of KoKe. This could be attributed to two distinct mechanisms. First, polyphenols with large molecular structures, such as theaflavins, thearubigins, and thearubins present in tea [50], are broken down during fermentation by cellulases, glucanases, xylanases, pectinases, and microbial glucosidases, resulting in smaller polyphenolic monomers and an increase in TPC and TFC [51]. Secondly, the cell walls of tea leaves contain a high amount of insoluble bound phenols that are difficult to release into the beverage [52]. In this case, microbial fermentation likely aids in the release of these insoluble bound phenols, as observed in other matrices. Kim et al. observed that microbial fermentation significantly enhanced the bioaccessibility of phenolic compounds in rice spent water (RSW). Specifically, methanol-extractable free phenolic contents were seven times higher in fermented RSW compared to fresh RSW [53].

3.4. Content of Phenolic Acids, Flavonoids, and Caffeine Determined by HPLC Analysis

The polyphenolic profiles of kombucha (Ko), water kefir (Ke), and their mixtures (KoKe), fermented with different microbial cultures, were analyzed using HPLC-DAD. The targeted compounds included ellagic acid, rutin, gallic acid, catechin, EGCG (epigallocatechin gallate), ferulic acid, chlorogenic acid, and caffeine. The results, presented in

Table 2. Content of phenolic acids, flavonoids, and caffeine in kombucha samples expressed as µg/100 mL.

STD	Ellagic Acid	Rutin	Gallic Acid	Caffeine	Catechin	EGCG	Ferulic Acid	Chlorogenic Acid
λ (nm)	254	254	270	273	280	280	325	327
RT (min)	47.7	46.5	11.7	30	31	12.2	42.2	32.2
Samples
Ko24h	0.54 ± 0.01 a	2.75 ± 0.01 a	163.87 ± 1.03 a	13.65 ± 0.35 c	n.d. b	12.06 ± 0.02 c	5.05 ± 0.01 a	n.d. b
Ko48h	0.55 ± 0.01 a	2.73 ± 0.01 a	112.70 ± 0.08 b	15.32 ± 0.06 b	n.d. b	7.94 ± 0.15 d	5.55 ± 0.01 a	54.75 ± 0.22 a
Ko72h	0.57 ± 0.01 a	3.17 ± 0.01 a	111.28 ± 0.46 b	15.69 ± 0.28 b	n.d. b	6.54 ± 0.40 d	6.02 ± 0.01 a	55.23 ± 0.02 a
Ke24h	0.15 ± 0.01a	n.d. b	12.17 ± 0.01 g	n.d. e	n.d. b	n.d. f	1.52± 0.03 f	n.d. b
Ke48h	0.35 ± 0.02 a	n.d. b	20.65 ± 0.04 f	n.d. e	n.d. b	n.d. f	2.08 ± 0.05 e	n.d. b
Ke72h	0.45 ± 0.03 a	n.d. b	33.23 ± 0.03 e	n.d. e	n.d. b	n.d. f	3.12 ± 0.01 d	n.d. b
KoKe24h	0.55 ± 0.01 a	2.78 ± 0.09 a	113.12 ± 0.74 b	15.24 ± 0.56 b	n.d. b	6.33 ± 0.08 d	4.02 ± 0.01 c	54.99 ± 0.13 a
KoKe48h	0.55 ± 0.01 a	2.84 ± 0.01 a	110.32 ± 0.26 c	13.17 ± 0.13 c	n.d. b	5.94 ± 0.41 e	4.44 ± 0.01 b	54.50 ± 0.05 a
KoKe72h	0.55 ± 0.01 a	2.93 ± 0.01 a	111.22 ± 0.36 b	13.45 ± 0.17 c	8.88 ± 0.03 a	7.21 ± 0.22 d	5.03 ± 0.01 b	54.50 ± 0.11 a
KoKeG24h	0.55 ± 0.01 a	2.44 ± 0.01 a	111.15 ± 0.16 b	11.11 ± 0.01 d	8.97 ± 0.01 a	6.21 ± 0.05 e	5.50 ± 0.01 a	54.22 ± 1.71 a
KoKeG48h	0.56 ± 0.01 a	2.63 ± 0.02 a	112.18 ± 0.32 b	14.24 ± 0.50 c	8.91 ± 0.02 a	6.72 ± 0.09 e	5.61 ± 0.01 a	54.75 ± 0.01 a
KoKeG72h	0.71 ± 0.01 a	2.64 ± 0.03 a	127.40 ± 0.01 d	13.42 ± 0.05 c	8.93 ± 0.01 a	15.68 ± 0.01 a	5.73 ± 0.01 a	54.67 ± 0.06 a
KoKeS24h	0.55 ± 0.01 a	2.59 ± 0.05 a	113.35 ± 2.08 b	14.86 ± 1.42 b	8.90 ± 0.01 a	7.18 ± 1.21 d	4.89 ± 0.01 b	54.98 ± 0.34 a
KoKeS48h	0.56 ± 0.01 a	2.58 ± 0.01 a	111.89 ± 0.43 b	13.72 ± 0.24 c	8.92 ± 0.02 a	6.54 ± 0.31 d	5.10 ± 0.01 a	54.82 ± 0.01 a
KoKeS72h	0.59 ± 0.01 a	3.01 ± 0.15 a	124.72 ± 0.83 d	21.43 ± 0.40 a	8.97 ± 0.02 a	12.87 ± 0.55 b	5.30 ± 0.01 a	57.01 ± 0.13 a
KoKeGS24h	0.55 ± 0.01 a	2.58 ± 0.05 a	111.42 ± 0.02 b	14.15 ± 0.45 c	8.91 ± 0.01 a	6.49 ± 0.26 d	5.04 ± 0.01 b	54.69 ± 0.10 a
KoKeGS48h	0.55 ± 0.01 a	2.56 ± 0.01 a	112.01 ± 0.05 b	13.91 ± 0.02 c	8.92 ± 0.02 a	6.66 ± 0.03 d	5.04 ± 0.01 b	54.81 ± 0.04 a
KoKeGS72h	0.56 ± 0.01 a	2.57 ± 0.01 a	112.15 ± 0.40 b	13.42 ± 0.01 c	8.92 ± 0.01 a	6.42 ± 0.07 d	5.02 ± 0.01 b	54.68 ± 0.05 a

n.d. not detected. The results are expressed as the mean of three replicates ± standard deviation in traditional kombucha (Ko), water kefir (Ke), kombucha and water kefir mixed in a 1/1 v/v ratio (KoKe), KoKe fermented by WKGs (KoKeG), KoKe fermented by SCOBY (KoKeS), and KoKe co-fermented by WKGs and SCOBY (KoKeGS). Statistical significance is indicated by different superscript letters.

, reported the concentrations of these compounds (µg/100 mL) across different fermentation times (24 h, 48 h, and 72 h) for each sample type. This study revealed that various phenolic compounds respond differently to the fermentation process, highlighting the complex interactions between microbial activity and phenolic metabolism. The observed fluctuations in compound concentrations over the fermentation periods (24 h, 48 h, and 72 h) underscore the dynamic and time-dependent nature of fermentation. For example, gallic acid and chlorogenic acid levels changed with fermentation time, and this variation indicates that fermentation not only influences the types of compounds but also their relative abundance.

Gallic acid and chlorogenic acid were the predominant phenolic compounds detected across all samples. The highest concentration of gallic acid was observed in kombucha after 24 h of fermentation (163.87 ± 1.03 µg/100 mL), while the peak chlorogenic acid content was recorded in KoKeS at 72 h (57.01 ± 0.13 µg/100 mL). In kombucha, gallic acid levels declined over time, decreasing to 112.70 ± 0.08 µg/100 mL at 48 h and 111.28 ± 0.46 µg/100 mL at 72 h, suggesting possible degradation or microbial transformation. In contrast, gallic acid concentrations increased during fermentation in KoKeG (from 111.15 ± 0.16 µg/100 mL at 24 h to 127.40 ± 0.01 µg/100 mL at 72 h) and in KoKeS (from 113.35 ± 2.08 µg/100 mL to 124.72 ± 0.83 µg/100 mL) over the same period. While chlorogenic acid levels remained relatively stable throughout fermentation in most samples, a noticeable increase was observed after 72 h in Ko and KoKeS. Water kefir (Ke) exhibited significantly lower concentrations of phenolic compounds compared to kombucha, with only ellagic acid, gallic acid, and ferulic acid detected in small amounts. This finding supports previous research, indicating that water kefir typically has a less complex polyphenolic profile than kombucha [54,55]. Additionally, EGCG (epigallocatechin gallate), a major polyphenol found in green tea, was detected in kombucha but at lower concentrations relative to other phenolic compounds.

The concentration of EGCG (epigallocatechin gallate) in kombucha progressively declined over the course of fermentation, decreasing from 12.06 ± 0.02 µg/100 mL at 24 h to 6.54 ± 0.40 µg/100 mL at 72 h. This reduction is consistent with the known sensitivity of EGCG to oxidative degradation during fermentation. In contrast, EGCG levels increased in the mixed samples—KoKe, KoKeS, and most notably in KoKeG—where they reached 15.68 ± 0.01 µg/100 mL at 72 h, up from 6.21 ± 0.05 µg/100 mL at 24 h. This trend suggests that the presence of water kefir grains and their interaction with kombucha tea components may help stabilize or even enhance EGCG content during fermentation. Ellagic acid and rutin were detected in the lowest concentrations across all samples. The variability observed in the quantitative data was not entirely consistent with previous studies, underscoring the complexity and variability of polyphenolic profiles in fermented beverages. For instance, Pihurov et al. reported that the predominant bioactive compounds identified in the fermented product were epicatechin (1135.69 μg/mL), rutin trihydrate (568.93 μg/mL), and caffeic acid (255.64 μg/mL). In contrast, gallic acid, ferulic acid, and chlorogenic acid were detected in much lower concentrations, 39.68 ± 2.56 μg/mL, 0.25 ± 0.02 μg/mL, and 0.36 ± 0.00 μg/mL, respectively [16]. However, the findings showed how the fermentation process and culture type influenced the content and diversity of phenolic compounds. For consumers interested in the health benefits associated with these beverages, it was crucial to note that the bioactive compounds in kombucha (especially gallic acid and chlorogenic acid) may be most concentrated at early fermentation stages, while other compounds like EGCG might degrade or fluctuate over time. The potential synergistic effects of kombucha and water kefir fermentation also offer an interesting avenue for future research and product development.

3.5. Antioxidant Activity

Antioxidant activity was evaluated using three different assays: DPPH, ABTS, and FRAP, across four concentrations (100, 50, 25, and 10 µL). The percentage of inhibition was assessed, allowing for the calculation of EC₅₀ (µL). The DPPH and ABTS results were expressed as milligrams of Trolox equivalents per 100 milliliters of the beverage (mg TE/100 mL), while the FRAP test results were reported as FeSO₄ mM per 100 mL.

3.5.1. DPPH

Table 3. TEAC and EC₅₀ values expressed as mg/ 100 mL and µL, respectively, against DPPH radicals.

Samples	100 µL	50 µL	25 µL	10 µL	EC50 (µL ± SD)
Ko24h	34.8 ± 0.2	33.9 ± 0.1	31.3 ± 0.1	28.0 ± 0.1	4.3 ± 0.5 a,A
Ko48h	35.6 ± 0.1	33.9 ± 0.1	32.5 ± 0.2	29.0 ± 0.1	3.6 ± 0.6 a,A
Ko72h	36.8 ± 0.1	34.7 ± 0.1	33.1 ± 0.1	29.5 ± 0.1	3.2 ± 0.5 a,A
Ke24h	0	0	0	0	- *c,E
Ke48h	0.5 ± 0.3	0	0	0	26751 ± 4.4 b,E
Ke72h	4.7 ± 0.2	0	0	0	2749 ± 3.4 a,E
KoKe24h	34.7 ± 0.3	33.2 ± 0.4	28.0 ± 0.2	13.5 ± 0.7	11.1 ± 1.0 a,B
KoKe48h	36.1 ± 0.1	34.4 ± 0.2	28.9 ± 0.1	13.9 ± 0.1	10.1 ± 1.0 a,B
KoKe72h	36.3 ± 0.2	34.4 ± 0.2	28.9 ± 0.1	13.9 ± 0.1	10.1 ± 1.0 a,B
KoKeG24h	35.5 ± 0.1	33.5 ± 0.3	16.1 ± 0.1	0.6 ± 0.2	23.3 ± 1.4 a,C
KoKeG48h	35.9 ± 0.2	35.1 ± 0.4	17.6 ± 0.1	0	22.1 ± 1.3 a,C
KoKeG72h	31.6 ± 1.2	27.1 ± 2.7	27.0 ± 2.7	5.4 ± 3.9	18.6 ± 1.3 a,C
KoKeS24h	33.8 ± 0.2	32.6 ± 0.1	18.7 ± 0.1	9.1 ± 1.2	16.8 ± 1.3 b,D
KoKeS48h	36.6 ± 0.1	34.8 ± 0.6	31.8 ± 0.1	11.2 ± 0.6	9.9 ± 1.2 a,D
KoKeS72h	36.7 ± 0.1	34.9 ± 4.6	31.9 ± 1.1	12.2 ± 0.9	9.7 ± 1.2 a,D
KoKeGS24h	36.6 ± 0.1	34.9 ± 0.6	27.8 ± 0.1	1.9 ± 0.1	15.4 ± 1.2 a,B
KoKeGS48h	36.0 ± 1.1	33.1 ± 0.1	25.7 ± 1.2	8.3 ± 1.9	14.8 ± 1.2 a,B
KoKeGS72h	37.1 ± 0.1	36.5 ± 0.3	30.2 ± 0.1	2.6 ± 0.6	13.6 ± 1.1 a,B

* not calculable. TEAC values against DPPH of traditional kombucha (Ko), water kefir (Ke), kombucha and water kefir (1:1 v/v) (KoKe), KoKe fermented by WKGs (KoKeG), KoKe fermented by SCOBY (KoKeS), and KoKe co-fermented by WKGs and SCOBY (KoKeGS). Statistical significance of EC₅₀ is indicated by different letters in the superscript. Small letters indicate statistical analysis within the sample at different fermentation times; capital letters indicate statistical analysis between different samples but at the same fermentation times.

reports the total antioxidant capacity (TEAC) values expressed as mg/100 mL for all samples, measured against the DPPH radical. The EC₅₀ values, which indicated the concentration at which the sample achieves 50% of its maximal antioxidant activity, were also provided for each sample. The findings clearly showed the effects of fermentation time and microbial involvement on the DPPH radical scavenging of Ko, Ke, and their mixtures KoKe. Kombucha and its mixtures exhibited significant antioxidant properties, while water kefir contributed less to the antioxidant activity, especially at the early stages of fermentation. The enhanced phenolic and flavonoid profiles developed during the fermentation process were reflected in the strong antioxidant activity of the samples against DPPH. The antioxidant activity of kombucha (Ko) increased slightly over time, as reflected in the TEAC values (from 34.8 ± 0.2 mg/100 mL at 24 h to 36.8 ± 0.1 mg/100 mL at 72 h), indicating that the antioxidant capacity strengthened as fermentation progressed. The EC₅₀ values showed a consistent decrease from 4.3 ± 0.5 µL at 24 h to 3.2 ± 0.5 µL at 72 h, suggesting that kombucha became more effective in scavenging free radicals as the fermentation time increased. Ke showed no antioxidant activity at 24 h, and this improved only after 48 h of fermentation (0.5 ± 0.3 mg/100 mL). By 72 h, Ko reached a TEAC of 4.7 ± 0.2 mg/100 mL, but the EC₅₀ remained very high (2749 ± 3.4 µL), indicating limited antioxidant efficiency at this stage. The mixed beverage (KoKe) showed significant antioxidant activity right from 24 h (34.7 ± 0.3 mg/100 mL at the concentration of 100 µL) and sustained high levels over time, stabilizing at 36.3 ± 0.2 mg/100 mL after 72 h. The EC₅₀ values remained relatively stable, around 10.1 ± 1.0 µL, indicating consistent antioxidant activity throughout the fermentation process. The combination of kombucha and water kefir appeared to enhance the antioxidant capacity as compared to the water kefir alone.

The mixed beverage fermented with WKGs (KoKeG) showed an initial strong antioxidant activity at 24 h (35.5 ± 0.1 mg/100 mL), which slightly decreased over time to 31.6 ± 1.2 mg/100 mL at 72 h, showing a lack of correlation with the increasing trend in polyphenolic content during the 72 h fermentation. In contrast, the SCOBY-fermented mixed beverage (KoKeS) exhibited antioxidant activity steadily increasing from 33.8 ± 0.2 mg/100 mL at 24 h to 36.7 ± 0.1 mg/100 mL at 72 h, comparable to that of kombucha at the same time and concentration, but the corresponding EC₅₀ value at 72 h was 3-fold that of kombucha (9.7 ± 1.2 µL) reflecting a lower antioxidant activity compared to kombucha alone. The mixed beverage fermented by both WKGs and SCOBY (KoKeGS) showed consistent antioxidant activity at all days, reaching the highest value of all samples (37.1 ± 0.1 mg/100 mL) at the highest concentration. Nevertheless, the EC₅₀ value was significantly lower than the corresponding Ko one, since Ko had an almost unchanged TEAC value even at the lowest concentration of 10 µL. Kombucha (Ko) not only consistently outperformed water kefir (Ke) but also demonstrated the highest effectiveness in scavenging free radicals among all samples tested. However, the mixed kombucha and water kefir beverage (KoKe) consistently exhibited high radical scavenging properties, and its fermentation by both WKGs and SCOBY slightly enhanced the antioxidant properties compared to KoKe alone, suggesting a possible synergy between the two fermentation methods.

To the best of the authors’ knowledge, there are limited results on the DPPH radical scavenging activity of mixed fermented kombucha and sugar water kefir, as well as their combined fermentation. Pihurov et al. [16] examined the antioxidant properties of beverages fermented with various lyophilized SCOBY and WKG combinations, finding that DPPH radical scavenging increased during fermentation, peaking at 72 h with a value of 2.412 µM TE/mL.

3.5.2. ABTS

The data presented in

Table 4. TEAC and EC₅₀ values, expressed as mg/100 mL and µL, against ABTS radicals.

Samples	100 µL	50 µL	25 µL	10 µL	EC50 (µL ± SD)
Ko24h	52.6 ± 0.1	52.1 ± 0.1	51.5 ± 0.1	41.1 ± 0.4	2.3 ± 0.4 c,A
Ko48h	52.3 ± 0.1	51.5 ± 0.1	49.7 ± 0.2	48.4 ± 0.2	1.1 ± 0.1 b,A
Ko72h	53.3 ± 0.2	53.3 ± 0.2	53.3 ± 0.2	50.7 ± 0.3	0.4 ± 0.1a,A
Ke24h	46.4 ± 0.2	46.1 ± 0.1	0	0	39.8 ± 2.6 a,E
Ke48h	48.1 ± 2.2	46.7 ± 0.4	3.1 ± 0.8	0	36.3 ± 1.5 a,E
Ke72h	49.1 ± 0.4	48.5 ± 0.2	2.1 ± 0.2	1.2 ± 1.8	34.3 ± 1.4 a,C
KoKe24h	51.9 ± 0.2	49.9 ± 0.3	22.1 ± 0.2	7.6 ± 0.1	20.8 ± 1.2 b,D
KoKe48h	50.1 ± 0.3	46.9 ± 0.4	25.2 ± 0.1	13.6 ± 2.6	18.3 ± 1.3 b,D
KoKe72h	53.1 ± 0.2	53.1 ± 0.2	53.1 ± 0.2	41.2 ± 0.1	2.1 ± 0.3 a,B
KoKeG24h	50.7 ± 0.1	49.3 ± 0.3	36.4 ± 0.1	24.9 ± 0.1	9.4 ± 0.9 b,C
KoKeG48h	51.2 ± 1.7	50.5 ± 1.8	37.5 ± 1.9	23.8 ± 1.1	9.2 ± 0.8 b,C
KoKeG72h	53.1 ± 0.2	53.1 ± 0.2	53.1 ± 0.2	40.1 ± 0.3	2.2 ± 0.4 a,B
KoKeS24h	51.7 ± 0.1	50.4 ± 3.4	48.1 ± 0.8	21.0 ± 0.3	7.8 ± 1.0 b,B
KoKeS48h	51.8 ± 0.1	49.8 ± 0.4	48.1 ± 0.1	23.3 ± 0.2	7.1 ± 3.4 b,B
KoKeS72h	53.1 ± 0.2	53.1 ± 0.2	53.1 ± 0.2	34.6 ± 1.1	3.4 ± 0.4 a,B
KoKeGS24h	51.6 ± 0.1	50.3 ± 0.5	45.1 ± 0.1	25.7 ± 0.1	6.8 ± 0.7 a,B
KoKeGS48h	51.9 ± 0.1	51.5 ± 0.2	46.8 ± 0.1	26.5 ± 0.4	6.2 ± 0.8 a,B
KoKeGS72h	53.2 ± 1.1	53.2 ± 1.1	45.4 ± 0.2	30.5 ± 0.3	5.1 ± 0.6 a,B

TEAC values against ABTS of traditional kombucha (Ko), water kefir (Ke), kombucha and water kefir (1:1 v/v) (KoKe), KoKe fermented by WKGs (KoKeG), KoKe fermented by SCOBY (KoKeS), and KoKe co-fermented by WKGs and SCOBY (KoKeGS). Statistical significance of EC₅₀ is indicated by different letters in the superscript. Small letters indicate statistical analysis within the sample at different fermentation times; capital letters indicate statistical analysis between different samples but at the same fermentation times.

confirmed the DPPH results, as all samples demonstrated high efficiency in scavenging ABTS radicals. Ko, Ke, KoKe, KoKeG, KoKeS, and KoKeGS exhibited increased antioxidant activity over time, as indicated by the rise in TEAC values and the corresponding decrease in EC₅₀ values after 72 h of fermentation. Ko showed clear and consistent antioxidant activity exhibiting the highest activity at 72 h, with TEAC values ranging from 53.3 ± 0.2 mg/100 mL at 100 µL to 50.7 ± 0.3 mg/100 mL at 10 µL. These findings are consistent with the results reported by Kim et al. [56] who observed a DPPH value of 140.01 ± 3.0 μmol TE/mL for kombucha.

In contrast, water kefir showed significantly weaker antioxidant activity compared to kombucha. For instance, at 24 h, the antioxidant activity was minimal, with TEAC values near zero for some concentrations, and the EC₅₀ value was quite high at 39.8 ± 2.6 µL. However, as fermentation progressed, the antioxidant activity improved, reaching more measurable values at 72 h (49.1 ± 0.4 mg/100 mL at 100 µL) and a lower EC₅₀ of 34.3 ± 1.4 µL. Despite this improvement, water kefir remained significantly less potent than kombucha. KoKe demonstrated a moderate enhancement in antioxidant activity compared to water kefir (Ke) alone. At the highest tested concentration (100 µL), KoKe reached a TEAC value of 53.3 ± 0.2 mg/100 mL after 72 h, a level comparable to that of kombucha. However, at the lowest concentration (10 µL), its TEAC value was approximately 0.79 times lower than that of kombucha, which corresponded to a higher EC₅₀ value for KoKe (2.1 µL) compared to kombucha (0.4 µL). These results suggest that while KoKe displays improved antioxidant potential relative to Ke, kombucha retains greater efficiency at lower concentrations. The observed antioxidant enhancement in KoKe supports the idea of a potential synergistic interaction between kombucha and water kefir components. TEAC values were consistently higher than the corresponding DPPH radical inhibition values across all samples. The type of starter culture used influenced the antioxidant capacity of the beverages in distinct ways. Fermentation with water kefir grains (KoKeG) generally resulted in enhanced antioxidant properties compared to fermentation with SCOBY alone or the co-culture of WKGs and SCOBY (KoKeGS). Nevertheless, the antioxidant efficiency of KoKeG remained lower than that of kombucha alone, particularly when evaluated based on EC₅₀ values.

3.5.3. FRAP

The FRAP test also confirmed the findings from the DPPH and ABTS assays (Figure 5). Specifically, Ko demonstrated strong ferric-reducing power, which remained nearly constant across all concentrations (659.44 ± 1.35 − 637.92 ± 0.35 mM FeSO₄/100 mL). In contrast, the Ke sample exhibited moderate antioxidant activity of 88.25 ± 0.49 mM FeSO₄/100 mL at 24 h, which significantly increased to 301.55 ± 1.42 mM FeSO₄/100 mL after 72 h. The combination of the two fermented drinks resulted in a significant enhancement of the ferric-reducing power of Ke, with a mean value of 474.75 ± 0.37 mM FeSO₄ /100 mL. Inoculation with G and GS boosted the FRAP values of the KoKe mixed drink, while the presence of SCOBY did not cause any change.

The data from the antioxidant assays provided valuable insights into how different samples behaved when assessed for their antioxidant capacities using various testing methods. All the samples tested exhibited a better response to the ABTS assay compared to the DPPH one. Specifically, the EC₅₀ values, which denote the concentration of a sample required to achieve 50% of its maximum antioxidant activity, were found to be significantly lower in the ABTS test than in the DPPH one. The lower EC₅₀ values for ABTS suggest that the samples were more effective at scavenging ABTS radicals than DPPH ones, indicating a stronger antioxidant capability in neutralizing the ABTS free radicals. While the DPPH test targets a stable free radical (DPPH•), the ABTS assay uses a more reactive radical (ABTS•⁺), which could explain the enhanced performance observed in the ABTS test. This difference in reactivity may be a key factor in why the samples exhibited better overall antioxidant activity when assessed with the ABTS method compared to the DPPH method. Chou et al. mentioned that kombucha typically shows lower DPPH scavenging activity compared to ABTS [57]. Specifically, the optimized kombucha exhibited a DPPH scavenging activity of 12.44%, which was significantly lower than its ABTS scavenging activity of 78.75%. This pattern highlights the difference in the antioxidant capabilities of kombucha when assessed using different radical scavenging assays, with ABTS showing stronger antioxidant activity compared to DPPH. Similar results were reported by Ziemlewska [58], who evaluated kombucha fermented with different additives (Ribes nigrum, Aronia melanocarpa, and Vaccinium myrtillus). They observed that the EC₅₀ values for ABTS•⁺ radical scavenging were consistently lower than those for the DPPH assay, indicating stronger antioxidant activity in the ABTS test. Specifically, ABTS EC₅₀ values were 122 ± 1.9 µg/mL for Ribes nigrum, 128 ± 2.6 µg/mL for Aronia melanocarpa, and 131 ± 1.3 µg/mL for Vaccinium myrtillus, while the corresponding DPPH EC₅₀ values were significantly higher: 753 ± 2.5 µg/mL, 791 ± 7.4 µg/mL, and 1375 ± 8.5 µg/mL, respectively. Comparable findings were reported by Jakubczyk et al., who evaluated the antioxidant potential of eight commercial green tea-based kombuchas using DPPH, ABTS, and FRAP assays. [59]. The ABTS radical scavenging activity ranged from 95.52% to 98.26%, while the DPPH radical inhibition varied between 87.51% and 94.99%. Papadopoulou et al. evaluated the antioxidant capacity of an apple pomace-based water kefir beverage (WKB) and reported that the ABTS assay produced significantly stronger results than the DPPH test [14]. Specifically, the ABTS value for amphiphilic lipids extracted from the beverage reached 44.62 μmol TE/g extract, while the DPPH value was considerably lower at only 0.0507 μmol TE/g extract. A clear and significant difference was observed in the results of the FRAP (ferric-reducing antioxidant power) assay. The FRAP assay measures a sample’s ability to reduce ferric ions (Fe³⁺) to ferrous ions (Fe²⁺), which serves as a key indicator of the sample’s electron-donating capacity, an aspect of antioxidant activity that differs from radical scavenging. The results from the FRAP test demonstrated that the KoKe samples fermented with water kefir grains (KoKeG) and those co-fermented with both WKGs and SCOBY (KoKeGS) exhibited a marked improvement in their iron-reducing capacities compared to the KoKe sample. This enhancement suggests that the fermentation processes involving either WKGs alone or both WKG-SCOBY increased the samples’ abilities to donate electrons, thereby improving their capacity to reduce ferric ions. This improvement indicates a higher antioxidant potential through the electron transfer mechanism.

The differences observed in the results of the various assays can be attributed to the distinct mechanisms of action that each test measures [60]. The ABTS and DPPH assays focus primarily on free radical scavenging, assessing a sample’s ability to neutralize reactive radicals. In contrast, the FRAP assay evaluates the sample electron-donating ability and its capacity to reduce ferric ions, which is a different but equally important aspect of antioxidant activity. The combination of Ko and Ke appeared to confer a strong free radical scavenging ability, as indicated by the ABTS and DPPH assays, while the fermentation with G or GS enhanced the iron-reducing capacity, as demonstrated by the FRAP test.

Across all three antioxidant assays (DPPH, ABTS, and FRAP), kombucha demonstrated the highest antioxidant potential when compared to water kefir (Ke) and to various mixed fermentations, including KoKe, KoKeG, KoKeS, and KoKeGS. This superior performance is attributed to kombucha’s distinct microbial composition, optimal fermentation substrate, and stable metabolic pathways [61]. Several interconnected factors contribute to this enhanced functional outcome.

1.

Microbial synergy in kombucha fermentation

Kombucha is produced through fermentation by a SCOBY, a symbiotic culture of acetic acid bacteria (e.g., Acetobacter and Gluconobacter) and yeasts (e.g., Saccharomyces and Zygosaccharomyces) that grow well in a tea-based, acidic environment. This microbial consortium enables coordinated metabolic processes [62].

During fermentation, yeasts break down sucrose into glucose and fructose, which are then converted into ethanol and carbon dioxide. Acetic acid bacteria subsequently oxidize ethanol into acetic acid and produce additional bioactive compounds such as gluconic and glucuronic acids, both known for their antioxidant and detoxifying properties. Simultaneously, the microbial community facilitates the breakdown and transformation of complex tea polyphenols such as catechins and theaflavins into more bioavailable and bioactive antioxidant molecules. This process results in the production of a diverse range of antioxidant metabolites, including organic acids, vitamins, and bioactive peptides, which together contribute to the potent antioxidant activity observed in kombucha [59,63,64].

2.

Alteration of microbial synergy in mixed fermentations

When kombucha and water kefir are combined in a 1:1 (v/v) ratio and fermented using a SCOBY, water kefir grains (WKGs), or both, the fermentation process and antioxidant production become less effective [59,65]. This reduced performance is likely due to several factors:

(a)

Dilution of substrate and microbial preferences

Kombucha microbes are adapted to a polyphenol-rich, acidic tea environment. Mixing with water kefir dilutes these substrates and introduces a sweeter, less complex nutrient profile. This change makes the environment less suitable for SCOBY microbes, which may lead to a reduced ability to transform polyphenols into antioxidant compounds.

(b)

Microbial Competition and Interference

Water kefir grains introduce lactic acid bacteria (LAB) and sugar-tolerant yeasts that prefer simple, carbohydrate-rich environments with low tannin content. LAB rapidly consume sugars and lower the pH by producing acids. This acidification can inhibit the activity of acetic acid bacteria, which are essential for ethanol oxidation and the production of key antioxidant compounds like acetic and gluconic acids [66]. In addition, yeasts from both cultures may compete or co-ferment, but the resulting byproducts such as ethanol and CO₂ are not efficiently utilized. This loss of microbial synergy can further reduce the antioxidant potential of the fermentation.

(c)

Reduced activity of the oxidative pathway

Kombucha’s strong antioxidant effects mainly come from oxidative fermentation, especially through pathways driven by Acetobacter bacteria. However, in mixed fermentations, LAB become more dominant, shifting the process toward lactic acid fermentation. While lactic acid helps with taste and preservation, it does not significantly contribute to antioxidant production. As a result, the transformation of polyphenols into more active antioxidant compounds is less efficient. This is likely due to lower enzyme activity related to oxidative metabolism. In these mixed systems, antioxidant levels tend to stabilize or even decrease. Experimental results show that although total polyphenol levels may remain high, they do not match the antioxidant activity, indicating that the polyphenols are not being converted into their active forms. This points to a loss of coordination between the microbes involved in fermentation.

3.

Stability and efficiency of the natural kombucha system

The natural fermentation system of kombucha is both stable and well suited for producing antioxidant compounds over time. This system has been improved through repeated fermentation cycles, ensuring consistent results and high yields. In contrast, adding new microbial populations from water kefir or changing the fermentation conditions can disrupt this balance, often leading to less efficient antioxidant production. The SCOBY-based system, however, ensures a reliable conversion of tea polyphenols into active antioxidant compounds.

4.

Functional efficiency and biochemical output

In functional fermentation, efficiency should be measured not just by sugar consumption or acid production but also by the quality and strength of the bioactive compounds created. Kombucha, with its specialized and cooperative microbial community, effectively converts the limited substrates (tea and sugar) into a wide range of beneficial antioxidant compounds. On the other hand, the mixed kombucha–kefir fermentation is less effective at producing these bioactive compounds due to microbial conflict, incompatible substrates, and interference in metabolic pathways [67].

Despite offering potential benefits such as novel flavors or probiotic diversity, the mixed system sacrifices functional efficiency, particularly in generating antioxidants due to the loss of oxidative specialization inherent to kombucha’s original microbial network. Even when polyphenol levels are similar or higher, the antioxidant activity is lower in mixed systems, emphasizing the importance of microbial compatibility and pathway optimization in fermentation design.

3.6. Effect of Fermentation on CIELab Parameters of Investigated Samples

Food color is a key quality parameter, as it influences consumer purchasing choices. To assess the impact of color on all samples, the CIELab parameters after 24, 48, and 72 h were measured. As can be seen in

Table 5. Evolution of CIELab parameters in fermentation samples.

	24	48	72	24	48	72	24	48	72
Samples	L *			C *			Hue
Ke	43.15 ± 3.52 a	43.95 ± 3.91 a	45.49 ± 3.82 a	4.70 ± 1.28 d	4.93 ± 1.33 e	5.11 ± 1.28 e	1.56 ± 0.30 b	1.57 ± 0.36 a	1.55 ± 0.32 b
Ko	36.91 ± 2.63 d	36.54 ± 2.46 e	38.21 ± 2.86 e	24.80 ± 2.18 a	23.05 ± 1.92 a	24.35 ± 1.85 a	1.35 ± 0.25 c	1.35 ± 0.25 c	1.38 ± 0.25 c
KoKe	36.72 ± 2.76 d	38.86 ± 2.85 c	39.59 ± 2.94 c	24.25 ± 2.17 b	17.48 ± 1.64 c	17.24 ± 1.69 c	1.32 ± 0.23 d	1.37 ± 0.28 b	1.38 ± 0.28 c
KoKeGS	37.25 ± 2.85 b	39.03 ± 2.98 b	40.15 ± 3.11 b	7.18 ± 1.26 e	8.85 ± 1.75 b	8.64 ± 1.37 d	0.71 ± 0.15 e	1.37 ± 0.21 e	1.06 ± 0.18 e
KoKeG	35.53 ± 2.14 e	38.62 ± 2.87 c	40.05 ± 3.02 b	24.86 ± 1.97 c	15.35 ± 1.58 d	22.82 ± 1.74 b	1.33 ± 0.28 c	1.38 ± 0.22 d	1.44 ± 0.39 a
KoKeS	38.27 ± 2.78 c	38.14 ± 2.55 d	38.87 ± 2.90 d	23.86 ± 1.87 c	18.78 ± 1.72 c	17.62 ± 1.66 c	1.40 ± 0.39 a	1.36 ± 0.22 d	1.34 ± 0.22 d

Ko: kombucha; Ke: kefir; KoKe: kombucha and water kefir (1:1 v/v) (b); KoKeG: KoKe fermented by WKGs; KoKeS: KoKe fermented by SCOBY; KoKeGS: KoKe co-fermented by SCOBY and WKGs. Data are expressed as means ± SD (n = 3). Differences between samples were assessed by one-way ANOVA followed by Tukey’s post hoc test. Results followed by different letters are significant. * p < 0.05.

, there were several significant differences. Comparing the data relating to the L* parameter, it emerged that during the fermentation of Kombucha and related formulations, several modifications of brightness occurred. The L* parameter underwent an increase in all samples. After 72 h, the maximum L* value was reached by Ke samples (45.49). An increase in the Chroma C* parameter was observed during fermentation, with C* values from 5.11 to 24.35 after 72 h. No significant changes were observed in the hue angle.

The total color difference (ΔE) was also calculated after 48 and 72 h of fermentation. As shown in Figure 6a,b, the KoKe and KoKeS samples exhibited the most notable colorimetric variations as a result of the fermentation processes.

3.7. Enzyme Inhibition Capacity

The results of the enzyme inhibition assay, which evaluates the inhibitory capacity of the sample against the digestive enzymes, were expressed as the percentage of inhibition. An analysis of the data evidenced that kombucha (Ko) showed the highest inhibitory activity, and its inhibitory activity increased over the fermentation time with maximum values of 91.28, 85.77, and 71.28% for α-glucosidase, α-amylase, and lipase, respectively, after 72 h of fermentation (Figure 7a–c).

A promising enzyme inhibitory activity was observed also with KoKeS, where 77.58% was observed against α-amylase, whereas 75.48% was observed against the α-glucosidase enzyme. It is interesting to note that the KoKe sample showed a lower activity with a percentage of inhibition of 24.72–26.72% against α-glucosidase and lipase, respectively. The comparison of kefir (Ke) activity on the three enzymes reveals that, although marginally, lipase was the most sensitive enzyme, showing inhibition percentages ranging from 35.75% at 24 h to 25.22% at 72 h of fermentation. The Pearson correlation coefficient highlighted a strong positive correlation between total flavonoid content (TFC) and α-glucosidase (r² = 1) for the Ko sample, which also showed a positive correlation between α-amylase and both total phenolic content (TPC) and TFC (r² values of 0.96 and 0.97, respectively). A perfect correlation (r² = 1) was observed between TFC and α-amylase in the KoKeG sample. Various studies have demonstrated the hypoglycemic and antihyperlipidemic effects of different kombucha tea formulations. Teixeira Oliveira et al. also confirmed the inhibitory effect of kombucha on α-glucosidase [37]. In this work, authors found percentages of inhibition of 78.17 and 73.64% for green tea and kombucha, respectively. The α-glucosidase exerts a prominent role in carbohydrate digestion, catalyzing the hydrolysis of disaccharides and oligosaccharides in the small intestine [68]. As a result, this leads to improved glycemic control and enhanced insulin sensitivity. In a previous study, Seehusen et al. [69] demonstrated that kombucha tea inhibited pancreatic α-amylase, with an EC₅₀ value of 0.16 ± 0.06% (mL/U), while unfermented tea showed no effect on the carbohydrate hydrolysis enzyme. Although the exact mechanism by which kombucha tea induces hypoglycemia has not been fully characterized in vivo, the authors suggested that catechins are primarily responsible. However, due to the complex nature of kombucha, which contains both known and unknown compounds, alternative explanations should also be considered. These could include pH-dependent enzyme inactivation, the biotransformation of tea catechins, or the production of an inhibitory factor during fermentation. More recently, Puspitasari et al. [70] confirmed the α-glucosidase inhibitory activity of tea and Kombucha tea from Rhizophora mucronata leaves with EC₅₀ values of 0.12 and 0.09 mg/mL for unfermented tea and the kombucha tea, respectively. Kallel et al. [71] evidenced that kombucha tea could inhibit α-amylase and that the inhibition potency increases during the progression of fermentation. The effective inhibitors are likely the phenolic compounds, with their concentration in the one-thousand-fold diluted sample being approximately 1 mg/L. Among these, the gallate-containing monomers are the primary contributors to the observed bioactivity. The inhibitory effect of kombucha on carbohydrate-hydrolyzing enzymes was also confirmed by Xiudong et al. [72], who demonstrated that kombucha fermentation significantly enhances the inhibitory properties of a soymilk beverage, especially when the fermentation temperature reaches 37 °C. Under these conditions, the beverage exhibited strong enzyme inhibition, with α-glucosidase and α-amylase inhibitory activities reaching 84% and 66%, respectively. The hypoglycemic and hypolipidemic effects of kombucha and black tea were validated in vivo. Both kombucha and black tea were administered to Alloxan-induced diabetic rats (5 mL/kg per day) for 30 days. Results evidenced that kombucha tea exerts a higher inhibitory activity against α-amylase and lipase. Interestingly, administering kombucha to surviving diabetic rats restored lipase activity in the plasma and pancreas to 68  ±  10% and 62  ±  10% (p  <  0.05), respectively. This was accompanied by a marked delay in the absorption of LDL-cholesterol and triglycerides, along with a significant increase in HDL-cholesterol levels, reaching 157  ±  30% [73]. Pramono et al. [74] examined the bioactive components of a strawberry kombucha drink (SKD) and their effects on metabolic disorder markers and found that the fermented drink exerted promising α-amylase, α-glucosidase, and lipase inhibitory activities with potency greater than the positive control. Specifically, the EC₅₀ value for α-amylase inhibition by the strawberry kombucha drink was 5.39 µg/mg, demonstrating greater potency than Acarbose, which showed an EC₅₀ of 10.38 µg/mg. In contrast, the EC₅₀ for α-glucosidase inhibition was slightly higher for the kombucha (15.03 µg/mg) compared to Acarbose (10.62 µg/mg). For lipase inhibition, the kombucha exhibited an EC₅₀ of 39.70 µg/mg, while Orlistat showed stronger activity with an EC₅₀ of 16.14 µg/mg. In another study, kombucha was used for the fermentation of dried and fresh mulberry leaves (ML-DS and ML-FS, respectively), with the aim of increasing the content of bioactive compounds [75]. The results showed that fresh mulberry leaves were more favorable for kombucha fermentation than dried leaves. At a concentration of 45.6 mg/L, the inhibition rate of ML-D12-FS on α-glucosidase reached 85.88%. This enhanced activity may be attributed to the ability of the organisms in kombucha to generate new flavonoids with potent α-glucosidase inhibitory properties. A similar effect was observed with kombucha tea made from seagrapes (Caulerpa racemosa), where EC₅₀ values of 80.44% and 90.42% were recorded for α-amylase and α-glucosidase inhibition, respectively. Additionally, this kombucha tea improved blood glucose and total cholesterol levels in mice fed a cholesterol- and fat-enriched diet [76]. The ability of kombucha to exert an antidiabetic effect was studied and compared with the drug metformin. Zubaidah et al. [77] administered snake fruit (Salacca zalacca) kombucha, black tea kombucha, and metformin orally to diabetic rats over a 28-day period. All kombucha formulations led to a reduction in fasting plasma glucose levels. Notably, black tea kombucha demonstrated more pronounced hypoglycemic and lipid-lowering effects than black tea alone. The lipase inhibitory activity of various kombucha formulations was also observed. Aji et al. [78] found that butterfly pea flower kombucha was more effective than butterfly pea infusion in inhibiting pancreatic lipase, with EC₅₀ values of 162.83 μg/mL and 239.39 μg/mL, respectively.

4. Conclusions

This study explored the fermentation of a kombucha and water kefir mixture (1:1 v/v), inoculated with SCOBY, WKGs, or both. Key parameters such as pH, microbial growth, and concentrations of phenolic compounds and flavonoids were monitored over 72 h. Results showed that longer fermentation increased the levels of bioactive compounds and antioxidant activity, as demonstrated by DPPH, ABTS, and FRAP assays.

The WKG-fermented sample showed the highest phenolic content, while the co-fermented product (KoKeGS) had the greatest iron-reducing capacity. The KoKeG sample exhibited the strongest ABTS radical scavenging activity, and SCOBY-fermented products showed the highest enzyme inhibition, particularly against α-glucosidase and α-amylase. Color analysis revealed significant changes during fermentation, indicating bioactive compound development.

While kombucha was found to contain a higher concentration of bioactive compounds and demonstrated superior antioxidant activity, the findings highlighted the potential to develop a newly fermented beverage that improved the quality of water kefir. These findings support the potential of co-fermentation as a method to enhance the bioactive profile of fermented beverages.

Future studies should include extended fermentation trials, sensory evaluation for consumer acceptance, and advanced metagenomic and metabolomic analyses to better understand microbial interactions and their impacts on functional compound formation.