Electrochemical Alternative for Evaluating Antioxidant Capacity in Kombuchas

Abstract

Kombucha is a millennia-old beverage crafted from green or black tea and saccharides and fermented with a symbiotic culture of bacteria and yeast (SCOBY). This functional drink boasts health benefits, such as improved intestinal flora function, hepatoprotection and inhibition of amyloid fibers. It contains bioactive antioxidants, such as catechins, ascorbic acid, vitamins and other polyphenolic compounds. With kombucha’s rising popularity, the Food and Drug Administration (FDA) has implemented control procedures to ensure the quality and safety of this food product. Due to the antioxidant properties of the major bioactive compounds in kombucha, feasible and low-cost electroanalytical methods emerge as promising alternatives. The objective of this study was to evaluate the voltammetric behavior of kombucha samples to establish and compare their redox profiles and antioxidant activities. Thus, 18 kombucha samples were used, comprising commercial samples and samples prepared in the laboratory from different SCOBYs purchased from different countries, and analyzed by differential pulse voltammetry (DPV) and square wave voltammetry (SWV) on a carbon paste electrode (CPE). The electrochemical index (EI) values determined from the samples were used to establish their antioxidant activities. The EI values were also compared with spectrophotometric data from Folin–Ciocalteu (FC) and Ferric Reducing Antioxidant Power (FRAP) assays.

1. Introduction

Kombucha is a probiotic drink easily prepared by the infusion or extraction of “Camellia sinensis”, followed by the fermentation of sugars. The fermentation is carried out using a symbiotic culture of bacteria and yeast, called symbiotic culture of bacteria and yeast (SCOBY), and this stage usually takes 7 to 14 days. Its production is not yet a well-established process, and parameters such as the tea concentration, infusion temperature, SCOBY type and fermentation time have not yet been standardized [1,2,3,4,5].

Kombucha is already a commercial beverage with increasing popularity due to its benefits to human health. Most of the reports emphasize its antioxidant capacity, due to its high concentrations of polyphenols, and other benefits, due to the presence of vitamins and minerals [4,5,6,7,8,9,10].

Experimental factors are essential for the quality of kombucha. The temperature must be controlled at 28 ± 2 °C and the pH between 2.5 and 4.2, since these parameters influence its metabolic profile and its antioxidant activity. The fermentation time is directly associated with an increase in the acidity of the beverage; the concentrations of ethanol, organic acids, polyphenols and bioactive compounds; and the sensory characteristics desired for the final product [4,5,6,7]. Carbonation and the amount of dissolved oxygen influence the activity of aerobic acetic acid bacteria, responsible for the conversion of monosaccharides and ethanol into organic acids. The yeasts are responsible for the production of ethanol, conferring alcoholic characteristics that may interfere with the antioxidant capacity of the beverage, while the acetic bacteria contribute to the development of the acidic flavor [1].

Several methods have been used to evaluate the total antioxidant capacity in fruits, vegetables, beverages and other foodstuffs [7,8,9,10,11,12,13]. Usually, spectrophotometric methods such as Ferric Reducing Antioxidant Power (FRAP) and Folin–Ciocalteu (FC) assays are used [10,11].

Antioxidant activity has been measured by means of the electrochemical index (EI), which considers thermodynamic, kinetic and quantitative parameters from DP voltametric assays. The voltammetric techniques cyclic voltammetry (CV), differential pulse voltammetry (DPV) and square wave voltammetry (SWV) have been used to evaluate the redox profile of electroactive molecules, including those present in natural products and foods [12,13].

Concerning the evaluation of natural products, foods and beverages, the spectrophotometric methods are usually hampered by chromogenic interfering agents. Electroanalysis allows the tentative identification of antioxidants [12,13].

The aim of this study was to characterize and compare the voltammetric behavior of kombuchas, to establish their electrochemical profiles and antioxidant activities. Thus, 18 kombucha samples were used, either commercial or prepared in the laboratory from different symbiotic cultures acquired from different countries, and analyzed by DPV and SWV, on a carbon paste electrode (CPE). This work established and compared the electrochemical behavior of 18 kombuchas. In addition, the EI values determined for the samples were used to establish their antioxidant activities. The EI values were also compared here with the spectrophotometric data of FRAP and FC.

2. Materials and Methods

2.1. Chemicals and Reagents

The commercial kombuchas, all flavored, were acquired from local stores in Goiânia and Anápolis, Goiás, Brazil (

Table 1. Commercial (^®) and laboratory-made (֍) kombucha samples.

Sample	®	֍	SCOBY Procedence	Fermentation Time	Added Flavoring	SCOBY Additives
A	🗹		China	14 d	Passion Fruit	-
B	🗹		China	14 d	Vanilla	-
C	🗹		China	14 d	Cajuína	-
D	🗹		China	14 d	Hibiscus 1	-
E	🗹		China	14 d	Sicilian Lemon	-
F	🗹		China	14 d	Guava	-
G		🗹	Germany	14 d	-	-
H		🗹	China	14 d	-	-
I		🗹	USA	14 d	-	-
J		🗹	Qatar	14 d	-	-
K		🗹	China	14 d	-	Jum *
L		🗹	Canada	14 d	-	-
M		🗹	Japan	14 d	Kombu **	-
N		🗹	Iceland	14 d	Oolong tea	-
O		🗹	Australia	14 d	-	-
P		🗹	China	14 d	-	Hibiscus
Q		🗹	China	14 d	-	-
R		🗹	India	14 d	-	Coffee

* Honey instead of sugar; ** salty seaweed; 🗹 confirmation if the sample is either commercial or laboratory-made.

, samples A to F). Kombuchas prepared in the laboratory used SCOBYs from different countries (Table 1, samples G to S). The SCOBYs related to the samples were acquired from Tao kombuchas^®, Porto Alegre, RS, Brazil (A–F) and Naturall^® probiotics, Shah Alam, Selangor, Malasia, (G–S). P and R kombucha samples were prepared following secondary fermentations in the presence of hibiscus and coffee, respectively.

The sodium acetate trihydrate, acetone, glacial acetic acid, hydrochloric acid, methanol, ferric chloride hexahydrate, ferrous sulfate heptahydrate, 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ), gallic acid, sodium carbonate ≥ 99.0% purity, ethyl alcohol (HPLC grade), Folin–Ciocalteu (FC) reagent, graphite powder and mineral oil for carbon paste were sourced from Sigma-Aldrich, St. Louis, MO, USA. All solutions were prepared using analytical grade reagents and deionized water (conductivity 0.1 µS cm⁻¹) (Millipore S.A., Molsheim, France). All solutions were prepared at a concentration of 100 µM from dilution of stock solutions (1 mM).

2.2. Preparation of Kombucha

The preparation of kombucha was based on the methodology proposed by Greenwalt et al. [1]. In brief, the green tea leaves were infused with hot water containing 20% sucrose and free of carbon dioxide. The resulting tea was cooled to room temperature, transferred to a sterile, gauze-covered container and finally fermented with SCOBY for 14 days. To reduce the risk of contamination by pathogenic and spoilage microorganisms, sanitary measures were adopted; thus, the previously fermented culture broth was acidified and kept in an oven at room temperature. After four to seven days, a new SCOBY film was formed, and the resulting drink was filtered to remove the suspended microorganisms [1,6,10].

For secondary fermentation and flavor addition, SCOBYs were added for 5 days to the culture broth previously fermented with C. sinensis and 20% (v/v) of the aqueous extract of the flavoring fruit or spice.

The main features of the resulting samples are presented in Table 1.

2.3. Antioxidant Activity Determinations

The spectrophotometric determination of antioxidant activity was carried out in triplicate by two traditional methods [11,12], namely Folin–Ciocalteu (FC) expressed in mg L⁻¹ of gallic acid equivalents (GAE) of sample, and the Ferric Reducing Antioxidant Power (FRAP) expressed in mg L⁻¹ of ferrous sulfate.

From the fresh kombucha aliquot, at least three different dilutions were prepared in triplicate in test tubes. The Folin–Ciocalteu (FC) spectrophotometric method was used to determine total phenolic compounds in kombucha. Each aliquot of 50 µL of the kombucha sample at a concentration of 1% was placed in a test tube containing 1 mL of distilled water and 250 µL of the FC reagent [11]. After 5 min, 750 µL of a 20% Na₂CO₃ solution and 2950 µL of distilled water were added. The mixture was incubated in the absence of light for 60 min; afterwards, the absorbance was measured in a spectrophotometer at 765 nm, using the blank solution as a reference. The quantification of phenolic compounds in kombucha samples was carried out in triplicate, and the average result was reported. The results were expressed in gallic acid equivalents in µg mL⁻¹, from a calibration curve obtained under the same conditions for sample analysis. The percentage of the remaining radicals after 5 min of reaction between the sample and FC^• was calculated using the following Equation (1):

Remaining percentage (%) = (AbsC − AbsS)/(AbsC) × 100

(1)

where AbsC and AbsS are the absorbance of the control (FC^• solution) and of the sample, respectively.

The FRAP assay procedure is based on the Fe³⁺ reduction to Fe²⁺. The FRAP reagent, prepared at the beginning of the analysis, consisted of a mixture of 2,4,6-Tripyridyl-s-Triazine (TPTZ) (10 mmol L⁻¹), HCl (40 mmol L⁻¹), FeCl₃ (20 mmol L⁻¹) and sodium acetate–acetic acid buffer solution (pH 3.6) in the following proportion, 1:1:1:10, respectively. An amount of 100 µL aliquots of the kombucha sample were injected into 1.8 mL FRAP reagent and left in a water bath at 37 °C for 10 min. Then, absorbance was measured at 593 nm. FeSO₄·7H₂O aqueous solutions (500, 1000, 1500 and 2000 µM) were used in the calibration curve, and the results were expressed as µg Fe II equivalents per mL of the kombucha sample [12].

A calibration curve for ferrous sulfate was plotted at concentrations of 500, 1000, 1500 and 2000 µM. The evaluation (595 nm) was performed after 30 min of mixing, and the FRAP reagent was used as a blank to calibrate the spectrophotometer. Ferrous sulfate concentrations (mM) were placed on the X axis and the respective absorbances on the Y axis in a spreadsheet, where the linear regression equation was calculated. The absorbance was calculated for 1000 mM of ferrous sulfate, according to Equation (1).

Absorption spectra were recorded using an Agilent 8453 UV-Vis spectrophotometer with a photodiode array detector and UV-Visible ChemStation software G1115AA (Agilent Technologies, Inc., Santa Clara, CA, USA).

2.4. Voltammetric Assays

Electrochemical assays were carried out with an Autolab III^® potentiostat/galvanostat (Santa Clara, CA, USA) integrated with NOVA 2.1 software (Metrohm, Herisau, Switzerland). Measurements were made in a single-compartment 5 mL electrochemical cell with a 3-electrode system, consisting of a carbon paste electrode (CPE), an Ag/AgCl/KCl_sat electrode and a platinum wire (purchased from Lab Solutions, São Paulo, Brazil), representing the working, reference and auxiliary electrode, respectively. The experimental conditions for differential pulse voltammetry (DPV) were pulse amplitude of 50 mV, pulse width of 0.5 s and scan rate of 10 mV s⁻¹. DP voltammograms were baseline corrected using a step window of 5.00 mV.

The experimental conditions for square wave voltammetry (SWV) were a pulse amplitude of 50 mV with a frequency of 50 Hz and a potential rise of 2 mV, corresponding to a scan rate of 100 mV s⁻¹. The experimental conditions for cyclic voltammetry (CV) were a scanning range of 0.0 to 1.0 V and a scan rate of 100 mV s⁻¹.

All experiments were carried out in triplicate, at room temperature, and the main electrolyte used was 0.1 M phosphate-buffer solution (PBS), pH = 7.0.

Electrochemical Index

The sum of the ratios I_pa/E_pa for all peaks observed in DPV was used to obtain the EI values.

$$EI=\sum _{i=1}^n{\displaystyle \frac{I_{pa}}{E_{pa}}}$$

(2)

For the identified anodic peaks, the respective currents (I_pa) and potentials (E_pa) were associated with the electrooxidation of phenolic compounds (antioxidants). The I_pa is directly related to the concentration and electron transfer kinetics of the identified electrooxidation reaction. E_pa, in turn, expresses the thermodynamic reducing character of the antioxidant [13,14,15,16,17,18,19,20].

2.5. Statistical Analysis

Analysis of variance (ANOVA) and Tukey’s test were used to identify significant differences between the analytical data sets obtained from the analyzed samples. The analysis was conducted using Origin 8.0 software, an application for analysis, statistical processing and graphical visualization of experimental data. The ANOVA test allowed us to verify the existence of statistically significant variations between the group means, assuming homogeneity of variances and normality of the data.

Subsequently, Tukey’s multiple comparison test was applied as a post hoc method to identify which pairs of samples presented significant differences. The use of these statistical tests provides greater robustness to the analysis of the experimental results, ensuring the validity of the conclusions based on rigorous quantitative criteria. The ANOVA and Tukey tests evaluated significant differences in the analytical data between samples. For each assay (FC, FRAP and EI), we ranked the 18 samples (A–R) within that assay only, using the raw assay values. Because rank is a relative measure, no scaling or normalization was applied; ranking is invariant to any monotonic transformation of the data. We assumed that higher values indicate better antioxidant performance in all four assays (as is standard for these readouts).

Thus, within each assay, we sorted samples in descending order and assigned integer positions starting at 1 (best), 2, 3, …, 18 (worst). Ties were handled with average (mid) ranks. If k samples tie and would occupy rank positions p through p + k − 1, each of those samples receives the average of those positions. As an example, if three samples tie for ranks 2, 3 and 4, each obtains (2 + 4)/2 = 3. These 18 samples (A–R) were analyzed using four assays related to antioxidant activity: FC, FRAP and EI. Because each assay is reported in different units and scales, comparisons between samples were made within each assay to avoid biased interpretations due to scale differences. To this end, we assigned ranks per assay, ordering the values in descending order (highest value = best antioxidant performance in that assay). A “rank average” was used in the event of ties. Thus, in the “Ranks (1 = best by method)” table, rank 1 indicates the best sample for that specific method. To summarize overall performance, a composite rank was also calculated per sample, defined as the arithmetic mean of the four ranks (one for each assay). The lower the composite rank, the better the overall performance, considering simultaneously phenolic/related content (FC), reducing power (FRAP), and EI.

This summary appears in the table “Composite ranking (average of ranks).” We quantified the association among four antioxidant readouts, FC, FRAP and EI, using two complementary measures: Pearson’s product–moment correlation and Spearman’s rank correlation. Pearson’s correlation assesses linear relationships between raw values, assuming interval-scale comparability; Spearman’s correlation assesses monotonic relationships by correlating ranks, making it more robust to outliers and nonlinear but monotonic trends. Because correlation coefficients are invariant to linear rescaling (e.g., min–max or z-score), all analyses were performed directly on the raw assay values; scaling was not required for correlation estimation. For reporting and visualization, we built two 3 × 3 correlation matrices—one for Pearson’s and one for Spearman’s—using only the short variable names (FC, FRAP, EI).

We quantified the association among four antioxidant variables, that is, FC, FRAP and EI, using two complementary measures: Pearson’s product–moment correlation and Spearman’s rank correlation. Pearson’s correlation assesses linear relationships between raw values, assuming interval-scale comparability; Spearman’s correlation assesses monotonic relationships by correlating ranks, making it more robust to outliers and nonlinear but monotonic trends.

3. Results

3.1. Voltammetric Profile

For each Kombucha sample, an aliquot was added to PBS (pH = 7.0), with a dilution of 1:10, and a DP voltammogram was recorded on CPE, as shown in Figure 1. In general, the DP voltammograms of the samples presented two profiles, one with three anodic peaks, the first at E_p1a ~ 0.35 V, the second at E_p2a ~ 0.50 V and the third at E_p3a ~ 0.85 V (samples A–F), as shown in Figure 1A. The other samples (G–R) presented a voltammetric profile with two anodic peaks, at E_p1a ~ 0.20 V and at E_p3a ~ 0.75 V, as shown in Figure 1B. However, the voltammograms of samples M and R showed intermediate profiles between the groups, since for sample M, a peak at ~ 0.45 V characteristic of group 1 was identified, and in sample R, in addition to the peak around 0.20 V, anodic peaks at ~0.40 V and 0.60 V were identified. These variations are justified considering the complexity of the samples, as well as the variety of flavorings and additives used, as shown in Table 1.

The above experiments were repeated by SWV, as shown in Figure 2. For example, the SW voltammogram of kombucha sample B indicated two reversible anodic processes, at E_p1a = 0.15 V and E_p2a = 0.23 V, and one irreversible oxidation peak at E_p3a = 0.65 V, as shown in Figure 2A. In the SW voltammogram of sample G, only two peaks were detected, the first reversible at E_p2a = 0.21 V and the second irreversible at E_p3a = 0.75 V. The overall SW results were in agreement with the DP data, thus exhibiting reversibility only for potential peaks, E_pa < 0.4 V.

To investigate the electrochemical profile and identify the anodic processes of kombucha samples, DP voltammograms were recorded in a 1.0 mM catechin solution and in a 1.0 mM gallic acid solution in PBS (pH = 7.0), on a carbon paste electrode (CPE), as shown in Figure 3.

3.2. Antioxidant Activity Determinations and Electrochemical Index

FC and FRAP were two spectrophotometric methods used here to establish the antioxidant activity of kombucha samples, as shown in

Table 2. Data calculated using FC, FRAP and the values of electrochemical indexes (EIs) of kombucha samples analyzed by DPV.

Sample	FC (GAE µg mL−1)	FRAP (Ferrous Sulfate µg mL−1)	EI (µA V−1)
A	80.6200	708.3740	39.5622
B	83.3200	468.6980	31.8808
C	78.9533	512.0980	22.9271
D	44.8200	433.7780	35.6435
E	63.3200	668.0330	20.6107
F	64.5200	674.0480	31.3077
G	44.7200	476.2140	19.9652
H	84.1200	555.6310	17.8947
I	67.9533	594.9480	23.3381
J	59.8200	318.1590	27.0224
K	53.2867	561.4890	15.1649
L	53.1200	474.1330	26.0263
M	74.8700	400.0040	15.0421
N	22.4200	361.8620	20.5263
O	18.3533	315.4870	17.4333
P	48.4533	610.9470	17.8580
Q	60.9200	594.7180	28.3636
R	51.4867	445.0740	20.8357

. Both methods are based on a simple electron transfer redox reaction. Using the FRAP method, in the presence of the phenolic compound (antioxidant), Fe³⁺ is reduced to Fe²⁺, resulting in a color change in the solution, and by FC, the reduction occurs from a Mo⁶⁺ solution to Mo⁵⁺. The antioxidant activities of kombucha samples were established from the inhibition percentages of the Mo⁶⁺ concentration of 63.4 µM. The FC assay is more commonly used to establish the antioxidant activity of phenolics. The calibration curves for the FC and FRAP analyses were linear with values of correlation coefficient (r) = 0.997 and r = 0.996, respectively. These assays were performed in triplicate. Table 2 shows the antioxidant capacity of kombucha samples by means of Total Phenolic Content spectrometric assays FC and FRAP [11,12]. The absorbance of each kombucha sample was recorded after 30 min of reaction with FC and FRAP.

Table 2 and Figure 4 show the antioxidant capacity of kombucha samples by FC and FRAP spectrometric assays. Samples A, B, C, I and N showed the highest antioxidant capacity, whereas the lowest FC values were detected in samples O and P, below 50%.

The DP voltammograms of the kombucha samples (A–R) were used to establish the EI values, as shown in Table 2 and Figure 5. The data presented indicated a variation in the EI values of the samples between 39.5622 and 15.1649 µA V⁻¹. The calculated coefficient of variation (CV, %) was 29.56%. This CV% value is expected, considering that it refers to the EI values obtained for different kombucha samples. Here, we are not evaluating and comparing the precision of the method, but the antioxidant activity of different kombucha samples. Overall, the results also indicated high EI values, suggesting that the kombucha samples exhibited high antioxidant activity, primarily associated with the presence of catechins.

Spectrophotometric and electrochemical assays clearly demonstrated that all kombucha samples exhibited antioxidant activity. Using the FC method, which predominantly reflects phenolic contributions, sample H led, followed by B and A, suggesting a higher load of reducing species compatible with the FC reagent. Using FRAP, A was the best, with F and E close behind; this pattern highlights samples with the greatest capacity to reduce the ferric/ferrous pair under the assay conditions. Finally, in EI, sample A showed the best electrochemical response, followed by D and B, signaling favorable redox characteristics in this instrumental setup. The integrated reading via composite rank favors samples with consistently good performance across multiple mechanisms. By this metric, H performed best overall (average rank ≈ 3.75), placing 1st in FC, with intermediate positions in the remaining tests. Next came A (≈ 5.50; 1st by FRAP and 3rd by FC), F (≈7.25; 2nd by FRAP) and I (≈8.00; balanced performance). At the opposite extreme, the worst averages were observed for R (≈14.00), O (≈13.25), D (≈11.75) and a group with weaker overall performance formed by N and J (both ≈ 11.25), confirming the mechanism-dependent nature of the readings.

The results of the antioxidant activity of the analyzed kombucha samples obtained by spectrophotometric methods were partly convergent (Table 2); however, variations between them are expected considering several possibilities, such as the time interval between studies, and the variety and complexity of the samples.

Thus, N (leading in EI) or F (strong in FRAP/EI) exemplify specific performances by mechanism, while H and A stand out for their cross-sectional robustness.

From a practical standpoint, when a single “general” candidate is required, H and A emerge as the most defensible choices, as they exhibit high and consistent performance across multiple fronts. However, if the target application prioritizes a specific mechanism, the method-specific ranking tables should guide the decision: H/B/A for phenolics (FC), A/F/E for reducing power (FRAP) and N/H/F when the electrochemical response is more relevant. Finally, for statistical inference between samples (e.g., ANOVA + Tukey’s within-method), replicates per sample and method will be required, allowing for estimation of variability and testing for mean differences with appropriate rigor. Until then, the ranks and the composite rank should be interpreted as comparative summaries sensitive to the mechanism measured by each assay.

Spearman’s correlation (based on ranking) highlighted a moderate positive association between the FC and FRAP spectrophotometric methods (≈0.44) (

Table 3. Correlation matrices with Pearson’s and Spearman’s methodologies for FC, FRAP and EI.

Spearman’s Correlation	FC	FRAP	EI
FC (GAE µg mL−1)	1	0.44066	0.294118
FRAP (Ferrous sulfate µg mL−1)	0.44066	1	0.153767
EI (µA V−1)	0.294118	0.153767	1
Pearson’s Correlation	FC	FRAP	EI
FC (GAE µg mL−1)	1	0.501018	−0.03165
FRAP (Ferrous sulfate µg mL−1)	0.501018	1	−0.0494
EI (µA V−1)	−0.03165	−0.0494	1

). Spearman’s correlation also showed positive associations of EI with FC (≈0.29) and FRAP (≈0.15), suggesting a slight monotonic covariance that was not captured as linear by Pearson’s correlation, as shown in Table 3. Overall, the statistical data indicated a good correlation between FC and FRAP data and a moderate correlation between FC and FRAP data with respect to EI.

4. Discussion

Camellia sinensis tea, as well as its kombucha products, is a rich source of polyphenolic antioxidants, such as catechins and gallic acid [1,2,3,4,5,6]. Therefore, the redox profile and the determination of antioxidant capacity are undeniable quality parameters for this functional foodstuff.

In this study, the electrochemical profile of 18 kombucha samples (A to R), commercially acquired or made in the laboratory (Table 1), was evaluated.

The electrochemical behavior of kombucha samples was investigated using DPV and SWV to verify the reducing power and reversibility of electroactive species identified. These data are closely related to pro-oxidant and antioxidant behavior, and were used to estimate EI values. The EI values were subsequently compared with classical spectrometric assays for antioxidant evaluation [13,14,15,16,17,18,19,20].

The results presented in Figure 1 and Figure 2 showed two main patterns, which can be explained by the flexibility of production protocols [3,4,5,6,7,8,9,10].

Thus, many production procedures, i.e., SCOBY source, flavoring additives and operational parameters, such as pH and temperature control, can exert a relevant impact on redox profile. For instance, higher pH can lead to low redox stability, as well as to the development of unwanted microorganisms [2,3,5,6,7,8,9,10]. Yet, the use of additives such as fruits, coffee and other natural sources of polyphenols will increase the antioxidant power of this functional probiotic beverage. Furthermore, enzymes, including glucosidase, pectinase, xylanase, cellulase and glucanase, produced during fermentation degrade the oligomeric and polymeric polyphenolic compounds in tea into smaller polyphenols, such as catechin and gallic acid, thereby increasing the total phenol and flavonoid content in the kombucha samples [4,5,6,7,8,9].

Although catechin and its derivatives are the main kombucha antioxidants, many other electroactive compounds can be found, i.e., ascorbic and phenolic acids [1,2,3,4,5,6,7,8,9], thus impacting the DPV profile. In fact, the samples herein evaluated have exhibited two patterns, one with three anodic peaks, with the first at E_p1a c.a. 0.35 V, the second at E_p2a c.a. 0.50 V, and the third at E_p3a c.a. 0.80 V (Figure 1A); and the other with two anodic peaks, with the first at E_p1a c.a. 0.2 V and the second at E_p2a c.a. 0.75 V (Figure 1B).

The anodic peaks between 0.20 and 0.40 V are typical for catechin and other “catechol-like flavonoids” (Figure 1), yet the anodic peaks at E_pa ≥ 0.6 V are consistent with the presence of resorcinol- and monophenol-like moieties [13,14,15,16,17,18,19,20].

The oxidation of catechin occurs first reversibly, at the electron-donating 3′,4′-dihydroxyl groups of catechol, at very low positive potentials (Figure 1, Figure 2 and Figure 3). In turn, the hydroxyl groups of the resorcinol moiety are oxidized at more positive potentials and from an irreversible anodic process (Figure 1 and Figure 3) [14,15,16,19,20].

Nevertheless, the anodic peaks between 0.2 and 0.3 V are typical for any catechol and gallic acid-like polyphenols, Figure 3, yet the anodic peaks at E_pa ≥ 0.6 V are consistent with the presence of resorcinol and monophenol-like moieties.

Moreover, the oxidation of polyphenols may precede electron transfer via the formation of a phenoxyl radical (semiquinone) intermediate. Semiquinone is unstable and decays via dimerization or polycondensation reactions [13,14,15,16,17,18,19,20], with other present electroactive and non-electroactive compounds, thus leading to split and shoulder DPV peaks (Figure 1). Polysaccharides can also exert an indirect effect on diffusional processes by changing the medium viscosity and through binding properties [5,6,7,8,9,10,13,14,15,16,17,18,19,20].

It is worth noting that the P and K samples produced in the lab used a SCOBY modified with hibiscus and honey, two potential sources of polyphenols, as shown in Table 1 and Figure 1B. Furthermore, bioactive compounds such as epicatechin, catechin, caffeic acid and chlorogenic acid are produced or released during the metabolism of microorganisms involved in fermentation, affecting the antioxidant and redox profile [3,4,5,6,7,8,9,10].

When comparing the spectrophotometric assays performed on the kombucha samples, the FC method data indicated the same trend as those of FRAP, with a slightly more significant difference only in samples K and N. However, especially for samples A-F, there are large differences with the other assays, FC and FRAP, as shown in Table 2.

The data indicated that all samples presented significant antioxidant activities, since the EI values were high. Samples A, B, D and F presented an EI above 30 µA V⁻¹, while the others presented values above 15 µA V⁻¹. However, unlike the FC, FRAP and DPPH tests, the antioxidant activity is related to the presence and high concentration of phenolics, since the other components of the samples are not electroactive on CPE, such as sucrose, glucose, fructose, acetic acid, gluconic acid and glucuronic acid and ethanol, or are in low concentrations, such as vitamins and minerals. The results also indicated a CV of 29.56% for the EI values. This value is expected considering the variety and complexity of kombucha samples.

Moreover, some divergence between the three methods is scientifically plausible, since FRAP and FC are related to the reducing capacity of the samples on Fe³ and Mo⁶⁺ chemical reduction, respectively. Yet, EI reflects the general electrochemical properties of the sample. In general, electron transfer in transition-metal states is faster than in phenolic compounds, which involve simultaneous hydrogen transfer [13,14,15,16,17,18,19,20].

However, overall, all methods indicated that these kombucha samples all presented antioxidant properties, regardless of the method used. Furthermore, these results indicated the advantages of the voltammetric EI method, since it is simpler and faster.

Previous studies have used comparisons of the antioxidant activity of foods from EI and traditional methods such as FC, FRAP and DPPH, in wines [13] and walnut kernel extracts [16]; however, EI has not been used for kombucha samples.

Statistical evaluation of EI, FRAP and FC using ANOVA and Tukey’s test indicated no significant differences among the methods (p > 0.05). The difference between the spectrophotometric assays and the electroanalytical EI parameter was expected due to the presence of chromogenic interfering substances in natural samples, which decreased the overall correlation. The other groups were similar on average. Additionally, an ANOVA test was performed with the normalized data, in which all data averages had no significant difference (pH0 = 0.31795). Thus, the findings from each antioxidant capacity variable seem to be in agreement.

Besides the well-established use of EI values for antioxidant evaluation, voltammetric methods can provide complete electrochemical characterization of molecules and food samples, revealing information on redox stability, redox reversibility and pro-oxidant/antioxidant character, and allowing tentative identification of electroactive species [13,14,15,16,17,18,19,20].

5. Conclusions

The results highlight the advantages of electroanalytical approaches in antioxidant evaluation, when compared to traditional spectrophotometric methods.

The highest antioxidant capacity observed for commercial samples can be attributed to the use of additives. In fact, the highest antioxidant capacity of laboratory-made kombuchas was also observed for samples in which the SCOBYs were modified with some potential source of natural antioxidants, i.e., coffee, honey and hibiscus.

These findings not only highlight the relevance of kombucha as a potential source of antioxidants but also underscore the need to continue investigating and optimizing production conditions to maximize its health benefits.