Biological Activity and Phenolic Content of Kombucha Beverages under the Influence of Different Tea Extract Substrates

Abstract

In this study, the influence of different tea extract substrates on the biological activities of kombucha beverages was investigated. The variations in bioactive compounds such as polyphenols and flavonoids and their potential health-promoting properties represented by antioxidant activity were analyzed. Our findings shed light on the diverse effects of tea substrates on the production of bioactive compounds and their subsequent impact on the biological activities of kombucha, providing valuable insights for optimizing kombucha production and its potential health benefits. The new tea substrate for kombucha, called horchata, an Ecuadorian tea, shows a similar trend but with a low content of phenolics (4.511 ± 0.111 mg gallic acid equivalent (GAE)/g dry weight (DW)) and flavonoids (1.902 ± 0.0455 mg quercetin equivalent (QE)/g DW), and antioxidant activity (DPPH—33.569 ± 1.377 µmol TROLOX/g DW, ABTS—20.898 ± 2.709 µmol TROLOX/g DW, FRAP—34.456 ± 2.0618 Fe²⁺ mM/100 g DW compared to black and green tea as substrates for kombucha. Through HPLC-DAD, several polyphenols were registered, and homovanillic acid showed the highest concentration (74.45 mg/100 g). Horchata kombucha scored the highest in sweetness and smell, reflecting its popularity among the tasters, making it a valuable candidate as a kombucha substrate.

1. Introduction

Functional beverages, a subset of the functional food industry and the most rapidly expanding segment of the functional food market, have gained popularity among health-conscious consumers for their perceived health advantages [1]. Beverages that contain ingredients recognized to provide specific health benefits, beyond basic nutrition, such as improved energy, enhanced hydration, or support for overall well-being, are known as functional beverages. Kombucha is an example of a functional beverage, known for its probiotic, antioxidant, and potential digestive health benefits. Typically, kombucha is made with tea (of various origins), sugar, and a symbiotic culture of acetic acid bacteria and yeast, fermented for 7–21 days [2].

The predominant bacterium in this symbiotic culture is Acetobacter xylinum, while the yeasts belong to the genera Zygo-saccharomyces, Schizosaccharomyces, Saccharomyces, Saccharomycodes, Candida, Pichia, Brettanomyces, and Torulopsis [3]. Under aerobic conditions, Kombucha symbiosis can convert the substrate (sucrose and tea), over 7–10 days, into a slightly carbonated, mildly sour, and refreshing beverage [4].

Due to the presence of bioactive compounds in kombucha beverages, they are recognized as having beneficial functions for humans, such as treatment and prevention of diabetes, lowering of cholesterol and triglyceride levels, and oxidative stress control [5]. The increased popularity of kombucha has recently led to a rise in research on its biological effects. The active compounds found in kombucha can originate from the tea used as substrate in its production, as well as from the metabolic processes of the microorganisms involved in the fermentation process [6]. Traditionally, teas from the Camellia sinensis plant, such as black and green tea, are used as substrates for kombucha [7]. Black tea sweetened with sucrose is considered to be the ideal substrate for kombucha beverage fermentation, and green tea is a popular alternative to black tea [8]. Recently, there has been a shift towards using a variety of raw materials as substrates for kombucha production, including herbs, fruits, and by-products from the food industry. Several studies have explored the use of these alternative ingredients to prepare kombucha, expanding the traditional use of Camellia sinensis teas [9]. The bioactive compounds in kombucha beverages made from non-Camelia sinensis teas could offer significant health benefits due to the high content of secondary metabolites characteristic of the chosen plant species.

Few studies have explored the factors that impact the levels of bioactive compounds in the final product. Therefore, this article focuses on the impact of different substrates that influence the production of these bioactive compounds and the antioxidant activity of kombucha, going beyond the traditional use of green and black teas.

In our study, horchata was used as a non-Camellia sinensis tea substrate. The term ‘horchata’ (a generic Spanish word) is used to describe a variety of sweet drinks that share similar production processes and different ingredients (different combinations of grains, ground nuts, spices, medicinal plants, etc.) depending on the country of origin [10]. In our case, horchata is a traditional beverage originating from Loja City in southern Ecuador. It is well known for having notable therapeutic and nutritional qualities. Historically known as “healing water due to its therapeutic benefits”, Loja’s horchata is crafted from flowers, fruits, and medicinal herbs. Among the plants used in horchata, the following stand out: Aerva sanguinolenta (L.) Blume (escancel), Aloysia triphylla Royle (cedron), Aloysia citrodora Paláu (lemon verbena), Ocimum basilicum L. (basil), Melissa officinalis L. (lemon balm), Mentha × piperita L. (mint), Borago officinalis L. (borage), Malva sylvestris L. (mallow flowers), Matricaria chamomilla (chamomile), plantain, Nasturtium officinale W.T. Aiton (watercress), Equisetum arvense L. (horsetail), Soliva sessilis Ruiz & Pav. (burweed), Sanguisorba minor (burnet), Linum usitatissimum L. (flaxseed), and Amaranthus quitensis Kunth (ataco), among others [10].

The medicinal herbs used in the preparation of horchata are organically grown in the northwestern parishes of the Loja canton, and due to its rich history and therapeutic qualities, this traditional beverage has become an integral part of the region’s cultural and gastronomic heritage.

The study novelty consists of investigating horchata tea as a kombucha substrate, even if horchata properties have been recognized since ancient times. Black tea, oolong tea, and green tea are the main teas used in Kombucha fermentation; bioactive compounds depend on the tea type [6]. Our study is a pilot study conducted to check if the content of bioactive compounds and antioxidant activity follow the same trend as the well-known substrates represented by black and green teas. Studying the bioactive compounds in kombucha based on horchata may be a valuable starting point for understanding their importance in antioxidant activity potentiation. Taking into account that nearly half of Camellia species are at risk of extinction in the wild, while tea (Camellia sinensis) itself is assessed as Data Deficient, due to a lack of available information [11], the use of other sources as a substrate for kombucha preparation can contribute to the sustainable use of plant resources, by reducing the impact on established species.

The current research aims to understand and develop new functional beverages that promote health and wellness. This involves cultivating kombucha on different substrates such as black, green, and horchata teas, and assessing their biological (antioxidant activity) and chemical characteristics. The goal is to evaluate the use of the biological activity and phenolic content of kombucha beverages under the influence of different tea extract substrates, to characterize horchata tea as a new substrate for a nutraceutical beverage like kombucha.

2. Materials and Methods

2.1. Chemicals

Ultrapure water was obtained from a Milli-Q water purification system (Millipore Corp., Bedford, MA, USA). ABTS (2,2-azinobis-3-ethyl-benzothiazoline-6-sulfonic acid), DPPH (1,1-diphenyl-2-picrylhydrazyl), TPTZ (2,4,6-tris(2-pyridyl)-(S)-triazine), Folin–Ciocalteau’s reagent, and ferrous chloride were also purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA), as well as the Liquid Chromatography-Mass Spectrometry (LC-MS) grade solvents (ethanol, formic acid, and acetonitrile). All chemicals were of analytical and High Performance Liquid Chromatography (HPLC) grade and reagents were prepared according to standard analytical procedures.

2.2. Plant Material

Three types of tea were used in the kombucha production: two were based on Camellia sinensis (black and green tea), and the third was a non-Camellia sinensis tea substrate based on a mix of flowers, fruits, and medicinal herbs. The teas were obtained using tea bags from a tea shop located in Quito, Ecuador. The flowchart of the kombucha tea research methodology was presented in the Figure 1.

2.3. Kombucha Tea Preparation

Kombucha tea was prepared in a sterile environment, avoiding cross-contamination. Every surface (crystal vases) was sterilized in an autoclave at 121 °C for 15 min prior to working with Symbiotic Culture of Bacteria and Yeast (SCOBY) and tea extracts.

SCOBY film was obtained from store-bought kombucha SCOBY. Previous steps to fermentation were carried out, following the methodology proposed by Sevilla (2023) [12]. SCOBY film was propagated using a standard liquid culture broth that contained 2 L of water, 150 g/L of white cane sugar, and 10 g/L of black tea (from 3 tea bags). Three consecutive fermentations of 7 days each were carried out, producing a total of 6 SCOBY films. The experimental culture broth was then prepared for each batch, following the previous steps, using 10 g/L of black tea, 10 g/L of green tea, and 10 g/L of horchata tea. Crystal vases of 2 L of capacity were used for the experimental batches. Based on the methodology proposed by Greenwalt et al. (2000) [13], a SCOBY stabilization step was carried out to adapt it to the new substrate (based on a mix of escancel, lemon verbena, basil, lemon balm, mint, borage, mallow flowers, chamomile, congona, horsetail, violet, shullo, and ataco [14]) in the horchata infusion.

A test of five replicates was carried out with 100 mL of sugared horchata infusion, 50 mL of starter tea, and a thin biofilm of SCOBY covered with a cotton cloth, at room temperature for 10 days. When a 2 cm thick biofilm was obtained, the fermentation proceeded. The fermentation was carried out following the method described by Buzia et al. (2018) [15] by adding 850 mL of the three tea samples, in infusions, 50 g of sugar, 150 mL of the previously fermented infusion, and the SCOBY previously formed, under similar conditions to those described by González Tellez et al. (2018) [16] for 10 days at room temperature. Controlled sampling was conducted every 48 h, with caution, under strict sterile conditions, and avoiding exposing the vases to direct light. Each time, a sample of 50 mL was tested for pH until the beverages reached a pH of 3.5–4.0. No contamination was visible, and new SCOBY films grew in each batch. Kombucha involves two processes: fermentation, which takes place under anaerobic conditions, a process in which yeasts break down sugars into carbon dioxide and ethanol, and respiration, which takes place under aerobic conditions that convert sugars into carbon dioxide and water [17]; thus, after carbonation, the kombucha produced from the three types of tea was filtered with medium-pore filter paper to eliminate any remains of SCOBY, medicinal plants, or solids in suspension, to produce a translucent drink. SCOBY films were stored in the same broth that was prepared for the previous steps of experimental fermentation.

2.4. Extract Preparation

Dried material from the black, green, and horchata tea bags was macerated with ethanol 99.5% for 24 h at room temperature in triplets and gathered for further analysis.

2.5. Phytochemical Analysis

2.5.1. Quantification of Total Phenolic Content (TPC)

For TPC quantification, the method used was based on the methodology described by Thaweesang (2019) [18] with modifications. The Folin–Ciocalteu colorimetric method was used for the assay, whereby an aliquot of each extract was added to 1 mL of Folin–Ciocalteu reagent for 5 min for a reaction at room temperature, followed by the addition of 2 mL of a 100 g/L solution of Na₂CO₃ and 1.5 mL of water and kept at dark for 60 min. Absorbance at 765 nm was measured in a spectrophotometer against a blank, while a calibration curve was constructed using standard solutions within the range of 0–500 mg/L using gallic acid, obtaining a correlation coefficient of R² = 0.9941 (y = 0.0061x + 0.1393, n = 7, p = 9.26504 × 10⁻⁷, detection limit (DL) = 7.9118 mg/L, quantitation limit (QL) = 26.3726 mg/L). The results were expressed in mg GAE/g DW.

2.5.2. Quantification of Total Flavonoid Content (TFC)

An aluminum chloride colorimetric method was used as described by Pękal and Pyrzynska (2014) [19] with modifications. A volume of 1 mL of the tea extracts was added to 0.3 mL of 10% (v/v) AlCl₃ solution, 0.2 mL of 1 M sodium acetate, and 5.6 mL of distilled water for 40 min. The assay was carried out in triplets for each sample at room temperature (25 °C). A calibration curve was obtained using quercetin (QE) standard solutions at a range of 1–100 mg/L, with a coefficient of R² = 0.9915 (y = 0.0149x + 0.0983, n = 8, p = 1.9012 × 10⁻⁷, DL = 0.3519 mg/L, QL = 1.1732 mg/L). The spectrophotometric measure was carried out at an absorbance of 435 nm. The TFC results were expressed as mg QE/g DW.

2.6. Antioxidant Analysis

2.6.1. Ferric Ion Reducing Antioxidant Power (FRAP) Assay

For the ferric ion reducing antioxidant power (FRAP) assay, the method used was described by Rajurkar and Hande (2012) [20]. The FRAP reagent was obtained by mixing acetate buffer (300 mM, pH 3.6), a solution of 10 mM of TPTZ in 40 mM of HCl, and 20 mM of FeCl₃ at 10:1:1 (v/v/v). An aliquot of 1 mL of each extract was added to 3 mL of the FRAP reagent and left in the dark for 5 min at room temperature. Absorbance was measured at 593 nm. Ferric sulfate (Fe₂SO₃) standard solutions in the range of 0.10–1.00 mmol/L were used for the calibration curve (y = 0.5981x − 0.0082, n = 7, R² = 0.9989, p = 1.3166 × 10⁻⁸, DL = 0.01546 mg/L, QL = 0.05153 mg/L).

2.6.2. Determination of ABTS Free Radical Scavenging Activity

For the determination of ABTS free radical scavenging activity, the method described by Kuskoski et al. (2005) [21] was used. The working solution was obtained by mixing ABTS (2 mM) and potassium persulfate (70 mM) and calibrated to an absorbance of 0.700 ± 0.005 at 734 nm by diluting the solution with 99.5% absolute ethanol. The assay was carried out in triplets by adding 100 μL of each tea sample to 2 mL of ABTS solution. TROLOX standard solutions within the range of 10.0–150.0 mM was used for the calibration curve, obtaining an R² value of 0.9622 (y = 0.5981x − 0.0082, n = 7, p = 6.01 × 10⁻⁵, DL = 0.001681 mM, QL = 0.005604 mM). The absorbance was measured at 734 nm.

2.6.3. Determination of the 2,2-Diphenyl-1-picrylhydrazyl (DPPH) Radical Scavenging Activity

To determine the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, the method described by Guo et al. (2008) [22] with modifications. An aliquot of 50 µL of each extract was added to 2 mL of fresh 0.15 mM DPPH solution diluted in ethanol for 30 min at dark at room temperature. The scavenging activity was calculated with the formula (A control − A sample/A control) × 100. For the calibration curve, TROLOX standard solutions within the range of 0–2.5 mM were used to obtain the regression equation y = 18.073x + 1.2252 with a correlation coefficient of R² = 0.9788 (n = 7, p = 1.4045 × 10⁻⁵, DL = 0.004831 mM, QL = 0.01610 mM).

2.7. High-Performance Liquid Chromatography with Diode-Array Detection (HPLC-DAD)

High-performance liquid chromatography with diode-array detection (Agilent Technologies 1260, Santa Clara, CA, USA) accompanied by a C18 (150 × 4.6 mm, particle size of 5 μm) column was used for polyphenol detection in the horchata samples. The samples were frozen at −80 °C and lyophilized for 1 week. The samples and methanolic extract were made with methanol 80:20 with 1 g of sample. It was filtered and centrifuged to be able to be analyzed by HPLC. The mobile phase used for chromatographic analysis [23], consisted of a two-solvent system, used in gradient elution, using a linear gradient elution program for separation with trifluoroacetic acid (0.1% TCA, solvent A) and 5–100% acetonitrile (solvent B) for 40 min. The gradient elution schedule consisted of 5% B at the start of the process, followed by 15% B at 16.5 min, 33% B at 22.5 min, 100% B at 30.5 min, and 5% B every 35 to 40 min. The elution flow was set at 1 mL/min, the column temperature at 5 °C, and the injection volume at 30 μL. The diode array detection was set to collect data in the 200 ÷ 400 nm range. Calibration curves of the HPLC-DAD method were obtained by plotting the peak area (counts) against the concentration of standards 10, 50, 100, 200, and 400 μg/mL⁻¹. The reference standards used (100 μg mL⁻¹), were chosen based on their widespread representation in the plant world and strong antioxidant capacity. They were prepared from stock solutions by dissolving in a 30% ethanol–water solution, filtered through a 0.45 μm syringe filter, and stored at −18 °C in 2 mL vials. Identification and quantification were achieved by comparison with standard spectra at each retention time.

2.8. Sensory Evaluation

Three varieties of kombucha were evaluated: horchata, green tea, and black tea prepared with standardized procedures to ensure consistency in fermentation and flavor profile. A panel of 15 trained tasters rated the samples on five organoleptic attributes: appearance, color, odor, acidity, and sweetness, using a scale of 0 to 5. The scores were averaged and statistically analyzed using ANOVA to identify significant differences between samples, followed by Tukey’s test for multiple comparisons. The results are presented in a table for a clear comparison of the organoleptic profiles of the kombuchas (

Table 1. Average scores obtained in the sensory evaluation of the organoleptic attributes of the three kombucha varieties.

Attribute	Horchata Kombucha	Green Tea Kombucha	Black Tea Kombucha
Appearance	4.3	3.9	4.1
Color	4.1	3.7	4.0
Odor	4.5	4.0	3.8
Acidity	3.7	4.2	4.3
Sweetness	4.6	3.5	3.9

).

2.9. Statistical Analysis

Three replications were used; the results are expressed as means ± standard deviation (SD). Pearson’s correlations were used to determine the relationship between secondary metabolite content and antioxidant capacities. A nested ANOVA test was applied to determine the differences. The statistical analysis was performed with RStudio [24].

3. Results

3.1. Quantification of Total Phenolic (TPC) and Flavonoid Content (TFC) in Kombuchas with Different Tea Substrates

A similar trend was found for both assays, resulting in a higher value for green tea (TPC: 13.029 ± 0.627 mg GAE/g DW, 5.444 ± 0.257 mg QE/g DW), followed closely by black tea and horchata tea. The nested ANOVA showed significant differences (F_5,12 = 214.6, p < 0.0001) in the content of total phenolic content as well as total flavonoid content (Figure 2).

3.2. Antioxidant Analysis

Antioxidant properties highlighted by ferric ion reducing antioxidant power (FRAP), ABTS free radical scavenging activity (ABTS), and DPPH• radical scavenging activity (DPPH) assays were evaluated in triplets. For the ABTS assay, green tea and black tea samples showed similar values, with green tea having the highest antioxidant activity, while horchata showed a lower value compared to the other samples. Regarding the DPPH, similar activities were found between green tea and horchata, while black tea showed the highest value. In the case of FRAP, similar trends were found to those described for the total phenolic content, with green tea having the highest score, followed by black tea and horchata with the lowest values. Regarding the kombucha tea sample analysis, the three beverages showed a similar antioxidant capacity in the DPPH scavenging assay, while for ABTS scavenging assay, a similar trend as for the tea samples was found. In the FRAP assay, the green tea kombucha showed a higher value than the other two kombucha types (Figure 3). The nested ANOVA test showed significant differences (p < 0.0001) between the three sample types.

Pearson coefficient correlation was carried out for the secondary metabolites and antioxidant capacity data. For total phenolic content (TPC), a high positive correlation was found for ABTS (r = 0.872) and FRAP (r = 0.903) for the general data, and for total flavonoid content (TFC), similar coefficient values were generated in ABTS (r = 0.880) and FRAP (r = 0.825) assays. The DPPH scavenging assay showed an opposite low negative correlation for both tested metabolites (Figure 4).

3.3. HPLC—DAD Analysis of Horchata Tea

Several polyphenols were registered in the horchata sample. Homovanillic acid showed the highest concentration (74.45 mg/100 g), followed by gallic acid (1.89 mg/100 g), 3,4 dimethoxyphenyl acetic acid (1.78 mg/100 g), vanillic acid (1.02 mg/100 g), trans-cinnamic acid (0.98 mg/100 g), and 2,5 dihydroxyphenyl acetic acid (0.92 mg/100 g), while the lowest (<0.78 mg/100 g) was for p-cumaric acid (Figure 5).

3.4. Sensory Evaluation

The sensory evaluation of the different varieties of kombucha revealed distinctive characteristics in terms of appearance, color, smell, acidity, and sweetness. Using a panel of tasters in a laboratory, it was determined that horchata kombucha was the most generally accepted due to its flavor, smell, and color, followed by green tea kombucha, and finally, black tea kombucha. To visualize these differences, the results of the comparative analysis are summarized in Table 1.

The results showed that horchata kombucha scored the highest in sweetness and smell, reflecting its popularity among the tasters. Green tea kombucha attracted attention in appearance and color, while black tea kombucha, although less preferred in flavor and smell, exhibited a balanced profile in acidity and sweetness.

In detail, the horchata kombucha presented a high sweetness with a score of 3.9 and a smell of 3.7, making it particularly attractive to the tasters. Green tea kombucha, on the other hand, received notable scores in appearance (4.0) and color (4.2), indicating its visual appeal. Black tea kombucha, although with lower scores in sweetness and smell (3.9 and 3.8 respectively), showed a moderate acidity profile (2.8), suggesting a more robust and distinctive flavor.

4. Discussion

This study discusses the biological activity and phenolic content of kombucha beverages under the influence of different tea extract substrates, represented by horchata and black and green teas. Kombucha is a fermented beverage that contains polyphenols and other compounds with antioxidant power and relevant biological activities that would help in chronic disease treatment caused by oxidative stress [25], and it has anti-microbial, anti-carcinogenic, and anti-diabetic properties [26]. Due to its scientifically proven properties, the interest in kombucha is growing in the world market. While numerous varieties of kombucha are produced using different types of tea, primarily derived from Camellia sinensis, particularly black tea, there is a growing availability of other kombucha varieties made from diverse tea types such as green, white, and red tea in the market [27]. Despite extensive research on the microbiological content and antibacterial properties of kombucha, there remains a scarcity of studies focusing on the distinct health benefits associated with various types of tea. Even though the chemical composition of the tea leaves was thoroughly studied, in the case of kombucha these data are few [2].

The high antioxidant activity of horchata was underlined previously [28,29,30]. The chemical composition of kombucha is influenced by various factors, with the type of tea used being a significant determinant [31]. The abundance of compounds in the tea substrate directly impacts the antioxidant activity of kombucha tea [32]. Our study highlights the potential of horchata as a unique tea substrate for kombucha, showcasing distinct phenolic and flavonoid profiles compared to traditional black and green teas. The lower phenolic and flavonoid content in horchata may impact the antioxidant activity of the resulting kombucha, as evidenced by reduced DPPH, ABTS, and FRAP values. These differences in bioactive compounds could contribute to variations in the health-promoting properties of the kombucha beverages produced from different tea substrates. Our findings align with those of Jakubczyk et al. (2020) [27], highlighting the high antioxidant capacity of green tea, which is associated with elevated levels of phenolic and flavonoid content. Specifically, our study revealed a higher reductive potential measured by the FRAP assay compared to other antioxidant capacity tests, indicating the presence of antioxidants in the sample. The FRAP assay is recognized as a suitable method for assessing total antioxidants in plants consumed by humans [33].

Kombucha fermentation can vary between 7 and 60 days, although experts recommend an optimal time of 15 days, as explained by Benítez & Pavone (2023) [34]. Increasing the fermentation time will result in the production of more antioxidants, but it can also increase the toxic level due to organic acids, with an unpleasant taste similar to vinegar; therefore, an optimal time of 10 days was established in our study to terminate the fermentation process in the three tea substrates and carry out antioxidant assays.

Likewise, a more acidic pH between 3 and 4 was taken into consideration to standardize the fermentation time, which is achieved thanks to the production of acids such as acetic acid, gluconic acid, and glucuronic acid [35]. Vildozo (2022) [36] indicates that in this acidic environment, a favorable condition is established for the growth of the organisms that make up the SCOBY (acid-tolerant yeasts such as Dekkera and Brettanomyces and acetic acid bacteria such as Komagataeibacter sp.), which contribute to the selection of microorganisms, preventing contamination of the beverage, as was achieved in the present study.

Fermentation substrates influence the variation in the production of bioactive components and the chemical composition of kombucha [37]. In recent years, new analogous fermentation substrates (fruits, vegetables, herbal infusions, etc.) have been studied. These substrates may require additional or specific steps in fermentation, which may achieve/potentiate additional health properties. In the case of herbal infusions (used for many years as health-promoting beverages), the chemical composition and bioactive properties promote various physiological effects and can be improved after the SCOBY fermentation process [38].

In Ecuador, the demand for tea consumption is low, although it has shown a slight growth due to greater recognition of its nutraceutical properties, which is why the market for products based on aromatic and medicinal herbs has grown in the country. Black tea and green tea in Ecuador can be obtained at a price that can reach 4.00 USD per 100 g, increasing the production cost of the drinks that are prepared from the infusions of these substrates [39,40]. Meanwhile, horchata in Ecuador is a traditional drink based on medicinal herbs, widely consumed in the Ecuadorian Highlands. Horchata can even replace the consumption of coffee and sugary drinks, with a cost that can reach a maximum of 2.00 USD [41], which has contributed to reducing the production costs of kombucha produced with horchata compared to those produced with black and green tea, despite sugar and water use being the same.

In our case, there is a lack of correlation between the TPC, TFC, and DPPH radical scavenging activities, which can be explained by the fact that the main phenolic acids show a weak anti-radical effect in experiments with the DPPH radical [42] or by the limitations of the Folin–Ciocalteu method caused by other reducing agents (carotenoids, chlorophyll, L-ascorbic acid, and sulfur dioxide) that are abundant in cells and can react with the Folin–Ciocalteu agent [43].

The current study utilized the HPLC-DAD method to identify important phenolic compounds and secondary metabolites. In Ecuador, “horchata” tea, characterized by a reddish color, is an infusion of several aromatic plants such as escancel, lemon verbena, mint, borage, mallow flowers, lemon balm, and chamomile, among others. This tea is known for its medicinal properties, including aiding digestion, and is considered energizing, diuretic, and hydrating [15]. Of all secondary metabolites identified in kombucha based on horchata tea, only gallic acid was found by others in horchata based on black and green teas [44]. Homovanillic acid has a good antioxidant and antiradical capacity [45], showing promising therapeutic potential for depression in combination with a mixed probiotic cocktail [46] and for autism spectrum disorders [47]. Gallic acid, one of the most abundant phenolic acids, used as a flavoring agent and food preservative, has numerous biological and pharmacological activities (antioxidant, antimicrobial, anti-inflammatory, anticancer, cardioprotective, neuroprotective, and gastroprotective effects) [48]. Vanillic acid, also used as a flavoring agent, is characterized by anticancer, antidiabetic, anti-obesity, antibacterial, anti-inflammatory, and antioxidant effects [49]. Interestingly, horchata has been used as a medicinal tea and has a similar composition to green or black tea in terms of secondary metabolites. This suggests a potential overlap in their health-promoting properties and chemical constituents.

5. Conclusions

The chemical composition and biological activity of kombucha vary significantly depending on the type of tea used and its specific chemical composition. We found that the substrates used for kombucha, including horchata, black, and green tea, exhibited similar phenolic and flavonoid content and antioxidant activity, likely due to their comparable profiles of phenolic compounds. Furthermore, our analysis showed that the kombucha beverages obtained using these substrates maintained this trend, with even higher levels of total phenolics, flavonoids, and antioxidant capacity compared to the teas themselves. This suggests that complex phenolic compounds may undergo degradation to smaller molecules during fermentation. Our results support the potential use of horchata tea as a substrate for obtaining kombucha nutraceutical beverages to sustain the use of plant resources. Further studies are needed to discern possible synergistic or antagonistic interactions between the species used in horchata production.

Our HPLC-DAD analysis revealed a notable presence of polyphenols in kombucha prepared with horchata, with homovanillic acid emerging as a significant compound with a high concentration. This finding suggests that horchata-based kombucha may offer specific health benefits associated with homovanillic acid, known for its antioxidant and potential therapeutic properties in conditions like depression and autism spectrum disorders.

In addition, the cost-effectiveness of producing kombucha with horchata compared to black and green teas in Ecuador provides insights into the economic feasibility and accessibility of utilizing alternative tea substrates. The affordability and availability of horchata as a traditional medicinal tea could potentially influence the market competitiveness and consumer appeal of horchata-based kombucha products.