Next Article in Journal
Optimization of Fermentation and Transcriptomic Analysis: The Impact of Aspartic Acid on the Antioxidant Activity of Termitomyces
Previous Article in Journal
Eco-Friendly Biosurfactant: Tackling Oil Pollution in Terrestrial and Aquatic Ecosystems
Previous Article in Special Issue
Functional Component Production Capabilities in Milk Fermentation of Some Featured Lactic Acid Bacteria Species for Use in Different Food Processes
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Fermentation
Volume 11
Issue 4
10.3390/fermentation11040201
fermentation-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Evaluation of the Impact of Fermentation Conditions, Scale Up and Stirring on Physicochemical Parameters, Antioxidant Capacity and Volatile Compounds of Green Tea Kombucha

by
Raniele Oliveira Alves
1,
Rodrigo Lira de Oliveira
2,
Marcílio Martins de Moraes
3,
Wallysson Wagner Vilela Santos
4,
Cláudio Augusto Gomes da Câmara
3,
Suzana Pedroza da Silva
4,
Camila Souza Porto
5,* and
Tatiana Souza Porto
1,6,*
1
Northeast Biotechnology Network, Federal Rural University of Pernambuco (UFRPE), Av. Dom Manoel de Medeiros, s/n, Dois Irmãos, Recife 52171-900, PE, Brazil
2
Multi-User Food Science and Technology Laboratory, Federal University of the Agreste of Pernambuco (UFAPE), Av. Bom Pastor, Boa Vista, s/n, Garanhuns 55292-270, PE, Brazil
3
Department of Chemistry, Federal Rural University of Pernambuco, Av. Dom Manoel de Medeiros, s/n, Dois Irmãos, Recife 52171-900, PE, Brazil
4
Postgraduate Program in Environmental Sciences, Federal University of the Agreste of Pernambuco (UFAPE), Av. Bom Pastor, Boa Vista, s/n, Garanhuns 55292-270, PE, Brazil
5
Laboratory of Bioproducts and Bioprocesses Development, Education Unit of Penedo, Federal University of Alagoas, Penedo 57200-000, AL, Brazil
6
Department of Morphology and Animal Physiology, Federal University of Pernambuco, Recife 52171-900, PE, Brazil
*
Authors to whom correspondence should be addressed.
Fermentation 2025, 11(4), 201; https://doi.org/10.3390/fermentation11040201
Submission received: 27 February 2025 / Revised: 28 March 2025 / Accepted: 2 April 2025 / Published: 9 April 2025
(This article belongs to the Special Issue Precision Fermentation: Applications in the Food and Beverage Industry)
Browse Figures
Versions Notes

Abstract

:
This study evaluated the influence of tea, sucrose, and inoculum concentrations on green tea kombucha’s physicochemical properties and antioxidant capacity to optimize its production. The highest total phenolic content (98.61 mg GAE/100 mL) and radical scavenging activity for ABTS (9647.14 μmol/mL) and DPPH (6640.00 μmol/mL) were observed with 8 g/L of tea, 80 g/L of sucrose, and 30% inoculum. Principal Component Analysis highlighted inoculum as the key factor influencing these parameters. Following this, fermentation was scaled up in 6.5 L bioreactors operating under static and stirred conditions. Monitoring physicochemical properties, antioxidant capacity, and volatile compounds revealed the impact of agitation on fermentation, with the kombucha obtained by static cultivation presenting higher biological activity. Eleven volatile compounds were identified, including carboxylic acids, terpenes, esters, alcohols, and phenols. Notably, α-terpinolene, dodecanoic acid, and 2,4-di-tert-butylphenol, found in kombucha, exhibit antioxidant properties linked to health benefits. Differences in volatile compound profiles were observed between static and stirred processes. This study concluded that kombucha maintains its physicochemical characteristics and bioactivity during scale-up, contributing to a better understanding of large-scale production. It also suggests stirred cultivation as an alternative for kombucha production.

1. Introduction

Kombucha is a beverage obtained from the fermentation of sweetened Camellia sinensis tea (mainly green or black tea) with the addition of a Symbiotic Culture of Bacteria and Yeast (SCOBY). The first reports of kombucha production and consumption date back to China during the Chin dynasty. The beverage later spread to Japan, Russia, and Eastern Europe, gaining worldwide popularity during World War II and more recently becoming widely consumed in Western societies, particularly in Europe and the United States [1].
During the fermentation process, the biochemical composition of kombucha continuously changes. The process occurs at room temperature for 7–10 days and occurs with the participation of yeast and bacteria, accompanied by the formation of a cellulose-thick biofilm at the liquid-air interface [2]. Due to ethanol fermentation, the sucrose concentration decreases [3]. The duration of the process could affect not only the sensorial features of the product but also the stability and, therefore, the biological activities of its components [4].
Kombucha has a slightly acidic and carbonated taste, providing greater consumer acceptance [5]. After fermentation, the beverage contains different organic acids like gluconic and glucuronic acids, various sugars, vitamins, amino acids, proteins, enzymes, ethanol, phenolic compounds, and metabolic products of yeasts and bacteria [6]. Studies have highlighted kombucha’s beneficial effects, as it is a vast source of bioactive compounds that are digested, absorbed, and metabolized by the body, exerting cellular-level effects. Some of these benefits include improved immune responses and liver detoxification [7].
Several factors can influence kombucha fermentation, including temperature, pH, oxygen, substrate, sucrose concentration, the origin of the SCOBY, container geometry, and fermentation time. The amount of substrate, sucrose, and a symbiotic culture of kombucha used in fermentation, as well as specific preparation conditions, have already been reported in the literature. Any changes in these parameters impact on the final product quality, as well as its nutritional, biological, and sensory properties [8]. Scale-up is another factor that affects the characteristics of the kombucha fermentation process. The industrial-scale fermentation process is challenging due to the difficulty in evaluating the factors that affect the scale-up process during cultivation [9,10].
Different methods can be used for kombucha production, ranging from traditional low-tech static fermentation to high-tech bioreactors in a controlled environment. Various types of bioreactors are employed depending on the scale, process control requirements, and available resources [11,12]. Scaling up kombucha production poses challenges, particularly in maintaining product quality and consistency. Proper control of the fermentation process is crucial to avoid variations in flavor and quality. Fermentation kinetics can be influenced by temperature, pH, dissolved oxygen, type of sugars, fermentation time, and vessel geometry [13].
Since kombucha also is an aerobic fermentation process, oxygen is essential for the metabolism of the microorganisms involved. Agitation plays a significant role, as it improves oxygen dispersion in the medium, influences the release of phenolic compounds and antioxidants, and affects the beverage’s sensory complexity. However, excessive agitation can hinder SCOBY formation, which typically occurs at the liquid surface under static conditions [9].
This work aimed to assess the influence of the variable’s tea, sucrose concentration, and inoculum on the laboratory-scale kombucha production. Based on these findings, we propose the scale-up production of kombucha in a static and stirred bioreactor. We also evaluated the physicochemical properties, phenolic profile, antioxidant capacity, and volatile compounds of the beverages produced.

2. Materials and Methods

2.1. Preparation of Green Tea Infusion and Kombucha Fermentation

The starter culture was obtained from Indupropil Indústria e Comércio Ltd.a (Ijuí, Brazil). The inoculum was taken from a previous kombucha fermentation, and the cellulosic biofilm (SCOBY) was not used in this fermentation process, which was carried out as described by Alves et al. [14]. The tea infusion was prepared by the addition of green tea (C. sinensis) in 400 mL of boiling water for 10 min, after which sucrose was added. The cooled tea was transferred to a 750 mL cylindrical glass container, and the fermented liquid broth (inoculum) was added to prepare the kombucha. The glass container was covered with cellulose paper and secured with a rubber band, and fermentation occurred for a maximum of 10 days at 28 °C, as recommended by the FDA [15], with samples being taken every two days.
The investigation of the experimental conditions of kombucha fermentation followed a 23-full factorial design plus three central points. The independent variables were sucrose concentration (40, 60, and 80 g/L), green tea concentration (4, 6, and 8 g/L), and inoculum concentration (10, 20, and 30%). The responses were the physicochemical parameters (pH, total titratable acidity, and total soluble solids), total phenolic content, and antioxidant activities against the free radicals ABTS and DPPH. The experimental procedures for the determination of each variable are described in Section 2.3, Section 2.4, and Section 2.5, respectively.
The statistical analysis of the experimental design was performed using the software Statistica 7.0 (Statsoft Inc., Tulsa, OK, USA). Principal Component Analysis (PCA) was performed using the FactoMineR and Factoshiny packages in R software (version 4.3.1) and Rstudio (version 2023.06.2 + 561), reducing the dimensionality of the data and identifying the main patterns of variability. The obtained dataset was evaluated through multivariate statistical analysis, where the PCA was conducted with the concentration of tea, sucrose, inoculum, and fermentation time (FT) as supplementary variables. The eigenvalues, percentage of variance explained, and cumulative percentage of variance were calculated for each principal component. Additionally, to assess the accuracy of the eigenvalue estimates, 95% confidence intervals were computed based on the bootstrap sample distribution using the BCa (Bias-Corrected and Accelerated) method, which corrects for bias and adjusts for variability.

2.2. Evaluation of Scale-Up and Effect of Stirring on Green Tea Kombucha Fermentation

After defining the most suitable fermentative conditions based on the 23-full factorial design, the process was scaled up 10-fold (0.5 L to 5.0 L). Kombucha tea was fermented in a laboratory bioreactor model BIOTEC-C (TECNAL, Piracicaba, Brazil), with an inner diameter of 18.5 cm and a total capacity of 6.5 L, operating with static and stirred (50 rpm) processes. Both cultivations were performed at 28 °C, and samples were collected on different days (2, 4, 6, 8, and 10) and stored at −22 °C for further analysis of the physicochemical parameters, total phenolic content, antioxidant capacity, and the volatile compounds profile as described in Section 2.3, Section 2.4, Section 2.5 and Section 2.6, respectively.

2.3. Physicochemical Parameters Determination

The pH of kombucha beverages was measured using a Marconi/PA-200 electronic pH meter (Piracicaba, São Paulo, Brazil). Total titratable acidity (TTA) was determined by titrating 100 mL of samples against 0.01 N NaOH, following the method described by Bhattacharya et al. [16]. Total Soluble Solids (TSS) were measured using a portable digital refractometer (Megabrix/REDI-P-101, São Paulo, Brazil) and expressed in °Brix. The total reducing sugar released from kombucha samples of bioreactor conducted processes was determined by reaction with 3,5-dinitrosalicylic acid, as described by Miller et al. [17].

2.4. Total Phenolic Content

The total phenolic content (TPC) of each sample was determined using the method described by Singleton et al. [18]. A 1 mL aliquot of Folin–Ciocalteu reagent (diluted 1:10) was added to 0.2 mL of kombucha (diluted 1:10). After 1 min, 0.8 mL of a solution of Na2CO3 (75 g/L) was added, and the mixture was homogenized. Following a 2 h incubation, the absorbance was measured at 765 nm using a spectrophotometer (Bel/Wuv-M51, São Paulo, Brazil). The phenolic compound content was expressed as gallic acid equivalent in mg/100 mL.

2.5. Antioxidant Capacity and Total Phenolic Content of Kombucha Fermented Tea

The free radical scavenging ability of 2,2-diphenyl-1-picrylhydrazyl (DPPH) in fermented kombucha solutions was determined using the method described by Brand-Williams et al. [19] and adapted by Mizuta et al. [20]. A 0.1 mL aliquot of fermented kombucha solution was mixed with 3.9 mL of 0.1 mmol/L ethanolic DPPH solution. The mixture was shaken vigorously and allowed to stand under the protection of light for 30 min, after which the absorbance was measured at 515 nm using a spectrophotometer. Ethyl alcohol was used as a blank, and a standard curve was constructed with Trolox solution. Results were expressed in μmol Trolox/mL of kombucha.
The total antioxidant activity was assessed using the ABTS (2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic)) free radical scavenging, according to Mizuta et al. [20]. In a dark environment, a 50 μL aliquot of the kombucha sample was combined with 950 μL of ABTS radical for 10 min. Absorbance was measured at 734 nm using a spectrophotometer, with ethanol used as a blank. Quantitation was performed using a Trolox standard curve, and the result was expressed as μmol Trolox/mL of Kombucha.

2.6. Identification of Volatile Compounds

Volatile compounds in kombucha samples obtained by static and stirred processes in bioreactors were analyzed using gas chromatography-mass spectrometry (GC-MS) to determine their volatile constituents. The analysis was performed on a Shimadzu GC-MS QP2010 SE equipment (Shimadzu Company, Kyoto, Japan) with a mass selective detector, operating at an electron impact of 70 eV with a scan interval of 0.5 s and fragments of m/z 40 to 550 Da. The equipment was equipped with a Shimadzu OAC 6000 Headspace automatic injector (Shimadzu Company, Kyoto, Japan) (with direct injection of the headspace) and with a nonpolar DB-5 fused silica capillary column (30 m × 0.25 mm × 0.25 mm) (J&W Scientific, Folsom, CA, USA). The mobile phase used was helium gas with a flow rate of 1.0 mL min−1. The injection system settings were set as follows: incubation time 5 min, 80 °C, 250 rpm, syringe pre-purge time 60 s, injection flow rate 25 mL/min, and post-injection residence time 60 s. The column temperature was initially set to 50 °C (hold for 4 min), increased to 290 °C (held for 5 min) at 6 °C min−1, and then further increased to 310 °C at 10 °C min−1 (held for 5 min). The injector and detector temperatures were 250 °C. The amount of each compound was calculated from the GC peak areas in the order of elution from the DB-5 column and was expressed as a relative percentage of the total chromatogram area.
The volatile components were identified based on the comparison of the linear retention indices calculated with reference to a homologous series of C8–C40 n-alkanes, using the equation by van Den Dool [21] with the mass spectra available in the literature according to Sparkman [22] and by a computerized comparison of the mass spectra obtained with those contained in the mass spectral library of the GC-MS data system (WILEY 21). Results were expressed as a percentage of the area of the volatile compound.

3. Results

3.1. Evaluation of the Influence of Fermentation Conditions on Kombucha Production from Green Tea from Camellia Sinensis

The experimental conditions and results of green tea kombucha production after 10 days of fermentation, performed according to a 23-factorial design, are presented in Table 1. It is important to mention that during the kombucha fermentation assay, samples were withdrawn every two days. The results of each parameter determined in the 2, 4, 6, and 8 days of fermentation can be visualized in the Supplementary Materials Table S1. The statistical evaluation of the effects of the independent variables (sucrose and green tea concentrations and inoculum) and their interactions on the physicochemical parameters (pH, total titratable acidity, and total soluble solids), total phenolic content, and antioxidant capacity against the DPPH and ABTS free radicals obtained at the tenth day of fermentation can be observed in Table 2.

3.1.1. Effects on pH, Total Titratable Acidity and Total Soluble Solids

The results obtained for pH indicate a progressive reduction throughout fermentation, as illustrated in Table S1 (Supplementary Materials). In the present study, conducted for 10 days, the final pH values ranged from 2.92 to 3.41 (Table 1). This trend is associated with an increase in acidity, as evidenced by the inverse relationship between total titratable acidity (TTA) and pH during the fermentation process (Supplementary Materials Table S1). At the end of fermentation (day 10), titratable acidity varied according to the experimental conditions, reaching a maximum value of 2.27 g/L of acetic acid.
Total soluble solids (TSS) varied throughout the fermentation, influenced by the specific composition of each test evaluated. TSS values ranged from 3.2 to 7.8 °Brix. At the end of fermentation, the lowest TSS value was observed for the sample with the lowest initial sucrose concentration (3.2 °Brix), while the highest value recorded was 7.8 °Brix for the test with the highest initial sucrose concentration.

3.1.2. Total Phenolic Compounds and Antioxidant Capacity

The amount of phenolic content in kombucha beverage samples at the end of fermentation can be observed in Table 1. Throughout fermentation progress, TPC production varied as a function of time; the highest production was observed on the tenth day (98.61 mg GAE/100 mL).
Tea concentration, as an independent variable, presented a significant positive effect (Table 2). Sucrose and inoculum concentrations exhibited negative and positive effects, respectively. The interaction between all variables studied (1 × 2 × 3) was statistically significant, evidencing the synergistic interaction, with the maximum response observed at the highest levels of each variable.
All independent variables, tea, sucrose, and inoculum concentration, were statistically significant for the antioxidant capacity of the kombucha beverage against the DPPH free radical, showing positive effects. The interaction between the inoculum and tea concentrations (2 × 3) had a synergistic effect on the DPPH response, as illustrated in the geometric interpretation diagram (Figure 1A). It can be observed that for the DPPH free radical, the highest antioxidant capacity was obtained in the run with the highest tea concentration (8 g/L) and the highest inoculum (30%), corresponding to run 8, in which both independent variables were at their highest levels.
Figure 2B presents the three-dimensional graphic representation of the 23-factorial design, allowing the visualization of all the trials studied. Our results confirm that run 8 obtained the highest antioxidant capacity, corroborating the trends observed in Figure 1A.
The ABTS radical scavenging activity when analyzing the antioxidant potential of the kombucha beverage at different fermentation times (See Table S1) showed a progressive increase with advancing fermentation days. The ABTS value on the tenth day varied between 4661.42 μmol/mL and 9647.14 μmol/mL in the runs studied (Table 1).

3.2. Principal Component Analysis (PCA) of Green Tea Kombucha Fermentation Process

A multivariate statistical analysis was performed from the dataset (Table 1 and Supplementary Materials Table S1) of the results obtained during all days of fermentation (2, 4, 6, 8, and 10 days). The PCA results demonstrated that the first two principal components represented 71.15% of the total data variability, with 47.96% for Dim1 and 23.19% for Dim2 (Figure 2). The PCA results showed how the variables used in the factorial design influence the increase or decrease of responses for the physicochemical parameters, antioxidant activity, and phenolic compounds (Figure 2).
The PCA factor loadings showed that the parameters DPPH (0.70), ABTS (0.84), TPC (0.80), and TTA (0.72) had a positive correlation with the first principal component (Dim1), which can be associated with the antioxidant capacity and phenolic compounds. On the other hand, pH showed a negative correlation (−0.59), indicating that higher pH values are associated with lower concentrations of antioxidant compounds. Regarding the second principal component (Dim2), an inverse relationship was observed between the pH and TTA parameters, with pH (0.76) showing a positive correlation and TTA (−0.62) showing a negative correlation. This behavior is related to the acid-base profile during the kombucha fermentation process. The factor loadings of the other parameters evaluated in the kombucha fermentation experiments can be verified in Supplementary Table S2.

3.3. Scale-Up of and Evaluation of Stirring Influence on Green Tea Kombucha Fermentation

The scale-up for kombucha production was based on the assay that showed the highest values for DPPH, ABTS, and phenolic compounds, specifically in run 8 (tea concentration 8 g/L, sucrose concentration 80 g/L, and inoculum concentration 30%). These fermentation conditions were defined in a previous study by our research group involving the fermentation of kombucha green tea [14].
Kombucha production in the static and stirred bioreactor provided values for the physicochemical and biological parameters studied and were used as metrics to verify the feasibility of scaling up the process. Regarding pH, as shown in Figure 3A, from the second day of fermentation, the pH decay rate remains similar for both fermentation processes. On the sixth day, the pH values were 2.67 and 2.66 for static and stirred, respectively, and on the last day of fermentation, they were 2.67 and 2.54, respectively, for a 10-day fermentation.
The TTA variation during kombucha fermentation in static and stirred systems can be observed in Figure 3B. It increased throughout fermentation, reaching peak values on the 6th day: 2.05 g/L for the static system and 3.12 g/L for the stirred system. At the end of fermentation, TTA values were 3.03 g/L for the stirred system and 2.1 g/L for the static system. The TSS results in kombucha beverages are presented in Figure 3C. At the end of kombucha fermentation, the samples obtained values of 6.7% for fermentation in the stirred system and 6.1% in the same period for the static system.
In terms of sucrose consumption, the production of reduced sugar can be observed in Figure 3D. At the end of fermentation, the reducing sugar values were 24.22 mg/mL for the static system and 20.77 mg/mL for the stirred system.
Figure 4A shows changes in the results for TPC, DPPH, and ABTS free radical scavenging capacity in kombucha production in static and stirred systems. At the end of fermentation, the phenolic compounds for the static and stirred systems presented values of 32.33 mg GAE/100 mL and 37.22 mg GAE/100 mL at the end of fermentation, respectively, and the values for antioxidant activity against DPPH 4144.28 μmol/mL and ABTS 5633.33 μmol/mL for the static system were higher than for the stirred one (DPPH: 842.73 μmol/mL and ABTS: 2689.74 μmol/mL).

3.4. Volatile Compounds of Green Tea Kombucha Produced in Static and Stirred Processes

The kinetics of volatile organic compounds (VOCs) were determined during the fermentation, and the identified compounds can be observed in Table 3. A total of 11 compounds were identified, including 4 carboxylic acids, 2 terpenes, 1 ester, 1 alcohol, 1 aromatic hydrocarbon, 1 phenol, 1 ketone. Table 3 shows the changes in these volatiles throughout the fermentation.
During the progress of kombucha fermentation in a static and stirred process, some compounds emerge, such as 1,3-Di-tert-butylbenzene, which appears from the fourth day of fermentation in the stirred system and remains until the end of fermentation (tenth day), whereas in the static process, this compound is present at the beginning of fermentation, but from the fourth day onwards it is no longer detected. This behavior can also be observed for Decanoic acid and Dodecanoic acid.

4. Discussion

The acidity of kombucha, generated mainly from organic acids such as acetic, lactic, gluconic, and glucuronic through microbial metabolism, is generally affected by the carbon source, nitrogen source, and microbial community [23]. This decrease in pH occurs due to the production of organic acids during fermentation. There are three stages during fermentation: alcoholic fermentation, lactic fermentation, and acetic acid fermentation [24]. During the alcoholic fermentation phase, yeasts play an important role in the conversion of glucose and fructose into ethanol. Subsequently, acid and lactic bacteria use the alcohol produced by the yeasts to produce various organic acids such as lactic, gluconic, and glucuronic acids, with acetic acid being the most predominant, leading to a reduction in pH [2].
Studies developed by Amarasinghe [4] show that the pH of kombucha made with black tea decreases from about 4.7 to 2.92 over 10 days of fermentation. The authors report a decrease in pH decreased from 5.6 to 3.6 in the first seven days of fermentation. Other authors observed a similar trend when also evaluating the production of kombucha beverages with green tea, where after 10 days of fermentation, the pH of the beverage reached 2.63 [25]. The statistical analysis (Table 2) indicates that sucrose concentration and inoculum presented a negative significant effect in contrast with tea concentration.
This can be explained by the increase in the concentration of organic acids, mainly acetic and succinic acids. As the fermentation progressed, the microorganisms present produced new acids. Lactic and acetic bacteria are responsible for the production of acids. That is, the samples with higher TTA had better growth conditions for the bacteria to grow and produce acids, thus reflecting the increase in TTA in the samples studied. Notably, low pH and high titratable acidity allow only microorganisms capable of sustaining such conditions to thrive, which may offer some protection against invasive contaminants [16]. Studies indicate that sucrose levels decrease linearly at the beginning of fermentation and then decline more gradually, correlating with the TSS values of the samples evaluated [7].
A positive correlation was demonstrated between phenolic compound content and reducing potential, considering that phenolic compounds are primarily responsible for antioxidant activity. This study confirms the observations made by Chakravorthy et al. [26], who reported that the amounts of total phenolic compounds in kombucha increased by approximately 54% over the fermentation period. Similar qualitative results were obtained by Lobo et al. [27], who found that the metabolic conversion of tea constituents during fermentation by microbial enzymes may contribute to the increased antioxidant activity of kombucha.
The analysis of the antioxidant potential of the samples studied revealed the potential to produce antioxidant compounds during the fermentation of the kombucha fermented tea. Due to hydrogen-donating antioxidants, tea constituents scavenge radicals from the aqueous phase and act as chain-breaking antioxidants to eliminate lipid peroxyl radicals [28]. The free radicals ABTS and DPPH were chosen to study the antioxidant capacity of the kombucha beverage, considering that they are very stable radicals.
For the dependent variables DPPH and ABTS, the effect of tea concentration was positive, showing that the highest concentration of tea resulted in an increase in antioxidant capacity. This may be attributed to the modification of bioactive composition by microbial activity during fermentation. The decrease in pH observed during fermentation creates conditions for the bound phenolic constituents to be released by enzymatic processes, which modifies the content and structure of secondary metabolites and results in increased antioxidant capacity.
Regarding the inoculum, which also had a positive effect on DPPH, ABTS, and total phenolics, the presence and amount of certain chemical compounds depend on the microorganisms found with the SCOBY, fermentation parameters (time and temperature), sucrose concentration, and tea type. Other authors have reported that the antioxidant capacity of the kombucha beverage was significantly affected by the composition of the microorganisms present in the fermentation broth. In this study, differences in antioxidant capacities between kombucha samples can be explained by the abundance of phenolics and TPC diversity in the tea leaves used [29]. The molecular state, free and bound form of phenolics also contribute to these differences, as mentioned by several authors [4,11].
Phenolic compounds are primarily responsible for antioxidant activities; similar results were also reported by Zou et al. [30], showing a good correlation between catechin concentrations and ABTS scavenging abilities. In a previous study, the phenolic profile of kombucha green tea was identified by the presence of catechin, epicatechin, and their derivatives: (−)-epicatechin 3-O-gallate, (ECG), (+)-catechin 3-O-gallate (CG) and (−)-epigallocatechin 3-O-gallate (EGCG) [14]. The increase in antioxidant activity in kombucha depends on factors such as fermentation time, type of substrate, and the normal microbiota of the kombucha culture, which determines the nature of the secondary metabolites that develop during the fermentation process [31].
PCA demonstrated that samples located close to a specific vector (represented by a physicochemical parameter, antioxidant activity, and phenolic compounds) are characterized by these parameters. The responses of pH, acidity, DPPH, ABTS, and phenolic compounds (Figure 2A) can be correlated with the concentration parameters of tea, sucrose, and inoculum (supplementary variables).
As fermentation progresses over the days, the pH decreases, which is inversely proportional to the increase in acidity. An interesting point is that the tea vector aligns with the pH vector, indicating that higher tea concentrations promote a decrease in pH. Thus, in Figure 2B, it can be seen that Run 3 has similar characteristics and exhibits lower pH values. The pH is also closely related to microbial growth and structural changes in phytochemical compounds, which can influence antioxidant activity and phenolic compounds [32].
Runs 7 (R7) and 8 (R8), located in the first quadrant (Figure 2B), can be correlated with the parameters DPPH, ABTS, and phenolic compounds. The variable inoculum is the main component influencing the increase in these parameters. Consequently, assays with higher initial inoculum concentrations exhibit higher values for DPPH, ABTS, and phenolic compounds. Additionally, a higher inoculum concentration resulted in a shorter process duration for the same beverage volume while maintaining similar values of pH, DPPH, ABTS, and phenolic compounds [33].
The scale-up process is influenced by several factors, including temperature, pH, oxygen levels, dissolved CO2, operational system, precursor supply, shear rate in the fermenter, as well as the nature and composition of the medium [34]. The greater pH drop at the end of fermentation in the stirred process can be explained by the action of acetic acid bacteria in kombucha fermentation, which converts ethanol into acetic acid under aerobic environmental conditions. The surface of the container is the only place where oxygen from the air is transmitted to the fermentation liquid and is necessary for the bacteria to express their metabolic activities [10].
Without the agitation factor, there is less possibility of oxygen transmission and, at the same time, of acetic acid production. Therefore, it can be observed that in the kombucha produced in the stirred bioreactor, oxygen becomes more available to both bacteria and yeast; with this, the production and conversion of ethanol into acetic acid becomes greater, and thus, the pH of the kombucha at the end of fermentation becomes lower than in the static process. The agitation process also affects the structure of the biofilm due to the loss of mechanical resistance [35]. Mainly in the absence of agitation, where microbial disintegration can occur between the aerobic acetic bacteria that will tend to occupy the surface layer and the yeast that can precipitate at the bottom of the vessel, and this can have negative effects on the fermentation process [36]. The effect of the process variables and their variation on a selected controlled characteristic of the product (pH = 3.2) is determined, and a decision is made on the scale-up of the process [33].
Kombucha production in a stirred system demonstrated lower conversion of sucrose into its fermentation byproducts when compared to the static system. This conversion process occurs mainly by yeast, which produces invertase and breaks down sucrose into glucose and fructose. The lower conversion observed can be attributed to the influence of agitation on the yeast, possibly affecting the adaptation of the microorganisms present, which did not demonstrate a good relationship with the agitation process, making it less effective under these conditions. In addition, the decrease in sucrose concentration over time can be used as an indicator of the fermentation rate [9,25]
The synthesis of volatile compounds (VOCs) in kombucha varies according to the microorganisms in the environment since the culture used has different groups of yeasts and bacteria, and these lead to the formation of specific VOC profiles. Therefore, controlling the fermentation kinetics is crucial, as well as adjusting factors such as time, temperature, and aeration, allowing the VOC profile to be modulated and the sensory complexity of the kombucha to be customized [37].
The agitation factor in this study affected both the concentration of VOCs during the fermentation process and revealed the absence of some of these compounds when compared to kombucha production in a static system. Given that agitation influenced the oxygen available to the microorganisms present, it reflected in the rate of VOC production, which is directly related to the speed of sugar consumption and the growth rate of yeast.
Saccharomyces yeasts mainly metabolize the alcohols found in kombucha; however, mixed fermentation produces VOCs by yeasts other than Saccharomyces, in this case, Zygosaccharomyces sp., Dekkera sp., Candida sp., and Pichia sp., which contribute to the final aroma of the product, but also to the formation of organic acids through their oxidation. Together with aldehydes, they make up the main substrates for the formation of organic acids and esters by the metabolic pathways of the bacteria present in SCOBY. Thus, alcohols are used by microorganisms to synthesize volatile aromatic acids that acidify the medium [38,39].
Compounds such as terpenes that have been identified are also present mainly in tea, with main compounds such as α-Terpinolene. Terpenes have several properties involved in the chemical resistance of the tea plant against biotic and abiotic stresses. More precisely, α-Terpinolene is a compound that has antioxidant and antimicrobial activity [40]. In this study, it was observed that this compound was formed both in the kombucha fermentation process in the stirred and static bioreactors. However, at the end of fermentation on day 10, these compounds disappear.
Furthermore, dodecanoic acid and 1,3-Di-tert-butylbenzene were present in all kombucha samples; however, at the end of fermentation for the stirred system, this compound was absent. Microbial degradation during fermentation is one of the factors that contribute to the disappearance of some volatile compounds in kombucha fermentation, as observed for α-Terpinolene. The families of molecules are mainly metabolites synthesized by the microbiome based on organic substrates present in the environment. The metabolic pathways related to kombucha fermentation are adapted to the nutrients present in the tea (mainly nitrogenous compounds) [41].
In kombucha, VOCs have several important biological functions, both in the microbial ecosystem and in the sensory and functional properties of the beverage. Thus, the production of VOCs can influence the fermentation rate, regulating enzymatic activity and the metabolism of sugars and other substrates [42]. In addition to contributing to the aroma and flavor of kombucha, VOCs are responsible for the fruity, floral, and acidic aromas [41], as well as some volatile compounds, such as phenols, which can contribute to beneficial biological activities, helping to combat oxidative stress [43].
Monitoring the pH, acidity, and concentration of bioactive compounds throughout fermentation is essential to ensure product stability and quality on a large scale. The static system has proven to be more effective in preserving the integrity of bioactive compounds, in addition to offering the advantage of reducing operating costs. In addition, excessive agitation influences the system, affecting the quality of the kombucha drink produced and may result in undesirable variations in the final product, compromising standardization for sale.

5. Conclusions and Perspectives Futures

The combination of statistical approaches allowed us to assess the impact of process conditions on kombucha fermentation. It was observed that the higher concentration of green tea, sucrose, and inoculum resulted in a significant increase in the antioxidant capacity for DPPH and ABTS radicals, as well as in the concentration of phenolic compounds, reaching values of 6640.00 μmol/mL, 9647.14 μmol/mL and 98.61 mg GAE/100 mL, respectively. PCA analysis showed that increasing the inoculum concentration can reduce fermentation time without compromising important parameters such as pH and antioxidant activity. The scale-up of the process proved to be effective, preserving the physicochemical quality, bioactive compounds, and biological activity of the fermented beverages, with 11 volatile compounds (VOCs) being identified. Therefore, monitoring the pH, acidity, and concentration of bioactive compounds throughout the fermentation is essential to ensure the stability and quality of the product on a large scale. The static system proved to be more effective in preserving the integrity of the bioactive compounds, in addition to offering the advantage of reducing operating costs. Future studies may further investigate the antioxidant, anti-inflammatory, and antimicrobial effects of kombucha in different populations, expanding our knowledge of its functional properties.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/fermentation11040201/s1: Table S1: Experimental conditions and average results of green tea kombucha fermentation according to 23-full factorial design performed at 28 °C for 2 to 8 days. Table S2: Factor loadings of the parameters in the principal components represented by dimensions (Dim1 and Dim2) for the variables pH, Total Titratable acidity (TTA), Total soluble solids (TSS), Total phenolic content (TPC), Antioxidant activity against the free radical DPPH and ABTS.

Author Contributions

Conceptualization, R.O.A., and T.S.P.; methodology, R.O.A., M.M.d.M., W.W.V.S., and C.A.G.d.C.; software, R.O.A., M.M.d.M., W.W.V.S., and C.A.G.d.C.; validation, T.S.P., C.S.P., S.P.d.S., and R.O.A.; formal analysis, R.O.A., M.M.d.M., R.L.d.O., W.W.V.S., and C.A.G.d.C.; investigation, R.O.A., R.L.d.O., W.W.V.S., and M.M.d.M.; resources, T.S.P.; data curation, R.O.A., R.L.d.O., and W.W.V.S.; writing—original draft preparation, R.O.A.; writing—reviewing and editing, T.S.P., C.S.P., S.P.d.S., and R.L.d.O.; visualization, T.S.P., C.S.P., S.P.d.S., and R.L.d.O.; supervision, T.S.P.; project administration, T.S.P.; funding acquisition, T.S.P. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Coordination for the Improvement of Higher Education. Personnel (CAPES: Brazil) under grant number 001, the National Council for Scientific and Technological Development (CNPq, Brazil) under grant number 315249/2021-8, and the Foundation for Science and Technology of the State of Pernambuco (FACEPE, Brazil) under grant number APQ-0726-5.07/21 and grant IBPG-0385-5.07/21.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article and Supplementary Materials. Further inquiries can be directed to the corresponding authors.

Acknowledgments

The first author is grateful to the FACEPE (Foundation for Science and Technology of the State of Pernambuco, Brazil) for the PhD scholarship and the financial support that made this research possible. All the authors are grateful to the Federal University of Agreste of Pernambuco (UFAPE) for the research infrastructure.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
VOCsVolatile organic compounds
PCAPrincipal component analysis
TTATotal titratable acidity
TSSTotal soluble solids
SCOBYSymbiotic culture of bacteria and yeast

References

  1. Batista, P.; Penas, M.R.; Pintado, M.; Oliveira-Silva, P. Review Kombucha: Perceptions and Future Prospects. Foods 2022, 11, 1977. [Google Scholar] [CrossRef] [PubMed]
  2. Jakubczyk, K.J.; Piotrowska, G.; Janda, K. Characteristics and Biochemical Composition of Kombucha—Fermented Tea. Med. Ogólna i Nauk. o Zdrowiu 2020, 26, 94–96. [Google Scholar] [CrossRef]
  3. Bertsch, P.; Etter, D.; Fischer, P. Transient: In Situ Measurement of Kombucha Biofilm Growth and Mechanical Properties. Food Funct. 2021, 12, 4015–4020. [Google Scholar] [CrossRef] [PubMed]
  4. Amarasinghe, H.; Weerakkody, N.S.; Waisundara, V.Y. Evaluation of Physicochemical Properties and Antioxidant Activities of Kombucha “Tea Fungus” during Extended Periods of Fermentation. Food Sci. Nutr. 2018, 6, 659–665. [Google Scholar] [CrossRef]
  5. Leal, J.M.; Suárez, L.V.; Jayabalan, R.; Oros, J.H.; Escalante-Aburto, A. A Review on Health Benefits of Kombucha Nutritional Compounds and Metabolites. CYTA-J. Food 2018, 16, 390–399. [Google Scholar] [CrossRef]
  6. Laavanya, D.; Shirkole, S.; Balasubramanian, P. Current Challenges, Applications and Future Perspectives of SCOBY Cellulose of Kombucha Fermentation. J. Clean. Prod. 2021, 295, 126454. [Google Scholar] [CrossRef]
  7. Kayisoglu, S.; Coskun, F. Determination of Physical and Chemical Properties of Kombucha Teas Prepared with Different Herbal Teas. Food Sci. Technol. 2021, 41, 393–397. [Google Scholar] [CrossRef]
  8. Emiljanowicz, K.E.; Malinowska-Pańczyk, E. Kombucha from Alternative Raw Materials–The Review. Crit. Rev. Food Sci. Nutr. 2019, 60, 3185–3194. [Google Scholar] [CrossRef]
  9. Sanwal, N.; Gupta, A.; Bareen, M.A.; Sharma, N.; Sahu, J.K. Kombucha Fermentation: Recent Trends in Process Dynamics, Functional Bioactivities, Toxicity Management, and Potential Applications. Food Chem. Adv. 2023, 3, 100421. [Google Scholar] [CrossRef]
  10. Cvetković, D.; Markov, S.; Djurić, M.; Savić, D.; Velićanski, A. Specific Interfacial Area as a Key Variable in Scaling-up Kombucha Fermentation. J. Food Eng. 2008, 85, 387–392. [Google Scholar] [CrossRef]
  11. Villarreal-Soto, S.A.; Beaufort, S.; Bouajila, J.; Souchard, J.P.; Renard, T.; Rollan, S.; Taillandier, P. Impact of Fermentation Conditions on the Production of Bioactive Compounds with Anticancer, Anti-Inflammatory and Antioxidant Properties in Kombucha Tea Extracts. Process Biochem. 2019, 83, 44–54. [Google Scholar] [CrossRef]
  12. Bortolomedi, B.M.; Paglarini, C.S.; Brod, F.C.A. Bioactive Compounds in Kombucha: A Review of Substrate Effect and Fermentation Conditions. Food Chem. 2022, 385, 132719. [Google Scholar] [CrossRef] [PubMed]
  13. Tran, T.; Grandvalet, C.; Verdier, F.; Martin, A.; Alexandre, H.; Tourdot-Maréchal, R. Microbiological and Technological Parameters Impacting the Chemical Composition and Sensory Quality of Kombucha. Compr. Rev. Food Sci. Food Saf. 2020, 19, 2050–2070. [Google Scholar] [CrossRef]
  14. Alves, R.O.; Monteiro, A.L.B.; da Silva, C.M.; da Silva, T.M.S.; de Oliveira, R.L.; Porto, C.S.; Porto, T.S. Investigation of the Influence of Different Camellia Sinensis Teas on Kombucha Fermentation and Development of Flavored Kombucha with Brazilian Fruits. Beverages 2025, 11, 13. [Google Scholar] [CrossRef]
  15. Kitwetcharoen, H.; Phung, L.T.; Klanrit, P.; Thanonkeo, S.; Tippayawat, P.; Yamada, M.; Thanonkeo, P. Kombucha Healthy Drink—Recent Advances in Production, Chemical Composition and Health Benefits. Fermentation 2023, 9, 48. [Google Scholar] [CrossRef]
  16. Bhattacharya, D.; Bhattacharya, S.; Patra, M.M.; Chakravorty, S.; Sarkar, S.; Chakraborty, W.; Koley, H.; Gachhui, R. Antibacterial Activity of Polyphenolic Fraction of Kombucha Against Enteric Bacterial Pathogens. Curr. Microbiol. 2016, 73, 885–896. [Google Scholar] [CrossRef]
  17. Miller, G.L. Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar. Anal. Chem. 1959, 31, 426–428. [Google Scholar] [CrossRef]
  18. Singleton, V.L.; Orthofer, R.; Lamuela-Raventós, R.M. [14] Analysis of Total Phenols and Other Oxidation Substrates and Antioxidants by Means of Folin-Ciocalteu Reagent. Methods Enzymol. 1999, 299, 152–178. [Google Scholar]
  19. Brand-Williams, W.; Cuvelier, M.E.; Berset, C. Use of a Free Radical Method to Evaluate Antioxidant Activity. LWT-Food Sci. Technol. 1995, 28, 25–30. [Google Scholar] [CrossRef]
  20. Mizuta, A.G.; De Menezes, J.L.; Dutra, T.V.; Ferreira, V.; Castro, J.C.; Antonia, C.; Jorge, E.; Junior, M.M.; Alves, B.; Filho, D.A. Evaluation of Antimicrobial Activity of Green Tea Kombucha at Two Fermentation Time Points against Alicyclobacillus spp. LWT-Food Sci. Technol. 2020, 130, 109641. [Google Scholar] [CrossRef]
  21. van Den Dool, H.; Kratz, P.D. A Generalization of the Retention Index System Including Linear Temperature Programmed Gas—Liquid Partition Chromatography. J. Chromatogr. A 1963, 11, 463–471. [Google Scholar] [CrossRef] [PubMed]
  22. Sparkman, O.D. Identification of Essential Oil Components by Gas Chromatography/Mass Spectroscopy Robert P. Adams. J. Am. Soc. Mass Spectrom. 1997, 8, 671–672. [Google Scholar] [CrossRef]
  23. Zhang, J.; Van Mullem, J.; Dias, D.R.; Schwan, R.F. The Chemistry and Sensory Characteristics of New Herbal Tea-Based Kombuchas. J. Food Sci. 2021, 86, 740–748. [Google Scholar] [CrossRef]
  24. Ayed, L.; Ben Abid, S.; Hamdi, M. Development of a Beverage from Red Grape Juice Fermented with the Kombucha Consortium. Ann. Microbiol. 2017, 67, 111–121. [Google Scholar] [CrossRef]
  25. Ahmed, R.F.; Hikal, M.S.; Abou-Taleb, K.A. Biological, Chemical and Antioxidant Activities of Different Types Kombucha. Ann. Agric. Sci. 2020, 65, 35–41. [Google Scholar] [CrossRef]
  26. Chakravorty, S.; Bhattacharya, S.; Chatzinotas, A.; Chakraborty, W.; Bhattacharya, D.; Gachhui, R. Kombucha Tea Fermentation: Microbial and Biochemical Dynamics. Int. J. Food Microbiol. 2016, 220, 63–72. [Google Scholar] [CrossRef]
  27. Lobo, R.O.; Dias, F.O.; Shenoy, C.K. Kombucha for Healthy Living: Evaluation of Antioxidant Potential and Bioactive Compounds. Int. Food Res. J. 2017, 24, 541–546. [Google Scholar]
  28. Malbaša, R.V.; Lončar, E.S.; Vitas, J.S.; Čanadanović-Brunet, J.M. Influence of Starter Cultures on the Antioxidant Activity of Kombucha Beverage. Food Chem. 2011, 127, 1727–1731. [Google Scholar] [CrossRef]
  29. Jayabalan, R.; Subathradevi, P.; Marimuthu, S.; Sathishkumar, M.; Swaminathan, K. Changes in Free-Radical Scavenging Ability of Kombucha Tea during Fermentation. Food Chem. 2008, 109, 227–234. [Google Scholar] [CrossRef]
  30. Zou, C.; Li, R.Y.; Chen, J.X.; Wang, F.; Gao, Y.; Fu, Y.Q.; Xu, Y.Q.; Yin, J.F. Zijuan Tea- Based Kombucha: Physicochemical, Sensorial, and Antioxidant Profile. Food Chem. 2021, 363, 3–10. [Google Scholar] [CrossRef]
  31. Jayabalan, R.; Malbaša, R.V.; Lončar, E.S.; Vitas, J.S.; Sathishkumar, M. A Review on Kombucha Tea-Microbiology, Composition, Fermentation, Beneficial Effects, Toxicity, and Tea Fungus. Compr. Rev. Food Sci. Food Saf. 2014, 13, 538–550. [Google Scholar] [CrossRef] [PubMed]
  32. Villarreal, S.A.; Beaufort, S.; Bouajila, J.; Souchard, J.; Taillandier, P. Understanding Kombucha Tea Fermentation: A Review. J. Food Sci. 2018, 83, 580–588. [Google Scholar]
  33. Lonǎr, E.; Djurić, M.; Malbaša, R.; Kolarov, L.J.; Klašnja, M. Influence of Working Conditions upon Kombucha Conducted Fermentation of Black Tea. Food Bioprod. Process. 2006, 84, 186–192. [Google Scholar] [CrossRef]
  34. Marsh, A.J.; O’Sullivan, O.; Hill, C.; Ross, R.P.; Cotter, P.D. Sequence-Based Analysis of the Bacterial and Fungal Compositions of Multiple Kombucha (Tea Fungus) Samples. Food Microbiol. 2014, 38, 171–178. [Google Scholar] [CrossRef] [PubMed]
  35. Cvetkovic, D.; Markov, S. Preparation of Kombucha from Winter Savory (Satureja montana L.) in the Laboratory Bioreactor. Acta Period. Technol. 2005, 266, 187–196. [Google Scholar] [CrossRef]
  36. Malbaša, R.; Lončar, E.; Djurić, M.; Došenović, I. Effect of Sucrose Concentration on the Products of Kombucha Fermentation on Molasses. Food Chem. 2008, 108, 926–932. [Google Scholar] [CrossRef]
  37. Coelho, R.M.D.; de Almeida, A.L.; do Amaral, R.Q.G.; da Mota, R.N.; Sousa, P.H.M.d. Kombucha: Review. Int. J. Gastron. Food Sci. 2020, 22, 100272. [Google Scholar] [CrossRef]
  38. Antolak, H.; Piechota, D.; Kucharska, A. Kombucha Tea—A Double Power of Bioactive Compounds from Tea and Symbiotic Culture of Bacteria and Yeasts (SCOBY). Antioxidants 2021, 10, 1541. [Google Scholar] [CrossRef]
  39. Zailani, N.S.; Adnan, A. Substrates and metabolic pathways in symbiotic culture of bacteria and yeast (SCOBY) fermentation: A mini review. J. Teknol. 2022, 84, 155–165. [Google Scholar] [CrossRef]
  40. Suffys, S.; Richard, G.; Burgeon, C.; Werrie, P.Y.; Haubruge, E.; Fauconnier, M.L.; Goffin, D. Characterization of Aroma Active Compound Production during Kombucha Fermentation: Towards the Control of Sensory Profiles. Foods 2023, 12, 1657. [Google Scholar] [CrossRef]
  41. Wang, Z.; Ahmad, W.; Zhu, A.; Geng, W.; Kang, W.; Ouyang, Q.; Chen, Q. Identification of Volatile Compounds and Metabolic Pathway during Ultrasound-Assisted Kombucha Fermentation by HS-SPME-GC/MS Combined with Metabolomic Analysis. Ultrason. Sonochem. 2023, 94, 106339. [Google Scholar] [CrossRef] [PubMed]
  42. Meng, Y.; Wang, X.; Li, Y.; Chen, J.; Chen, X. Microbial Interactions and Dynamic Changes of Volatile Flavor Compounds during the Fermentation of Traditional Kombucha. Food Chem. 2024, 430, 137060. [Google Scholar] [CrossRef] [PubMed]
  43. Zhang, J.; Ma, H.; Wang, H.; Sun, M.; Yu, C.; Liu, Q.; He, Z.; Song, S.; Feng, T.; Yao, L. Flavor and Sensory Profile of Kombucha Fermented with Raw Pu-Erh Tea and Evaluation of the Antioxidant Properties. LWT 2024, 200, 116220. [Google Scholar] [CrossRef]
Fermentation 11 00201 g001
Figure 1. (A) Diagram for the geometric interpretation of the effects of inoculum concentration and green tea concentration on the antioxidant capacity of the kombucha beverage against the DPPH free radical. (B) Three-dimensional graphical representation of the 23-factorial design for the kombucha beverage (Table 1), illustrating the influence of the variable’s tea concentration and inoculum concentration on the antioxidant capacity against the DPPH free radical.
Figure 1. (A) Diagram for the geometric interpretation of the effects of inoculum concentration and green tea concentration on the antioxidant capacity of the kombucha beverage against the DPPH free radical. (B) Three-dimensional graphical representation of the 23-factorial design for the kombucha beverage (Table 1), illustrating the influence of the variable’s tea concentration and inoculum concentration on the antioxidant capacity against the DPPH free radical.
Fermentation 11 00201 g001
Fermentation 11 00201 g002
Figure 2. Principal component analysis of physicochemical parameters, phenolic compounds, and antioxidant capacity of green tea kombucha beverage. (A) PCA graph for the independent variables pH, Total Titratable acidity (TTA), Total soluble solids (TSS), Total phenolic content (TPC), Antioxidant activity against the free radical DPPH and ABTS, (B) PCA graph for the trained runs performed using 23-factorial design.
Figure 2. Principal component analysis of physicochemical parameters, phenolic compounds, and antioxidant capacity of green tea kombucha beverage. (A) PCA graph for the independent variables pH, Total Titratable acidity (TTA), Total soluble solids (TSS), Total phenolic content (TPC), Antioxidant activity against the free radical DPPH and ABTS, (B) PCA graph for the trained runs performed using 23-factorial design.
Fermentation 11 00201 g002
Fermentation 11 00201 g003
Figure 3. Changes in pH (A), total titratable acidity (B), total soluble solids (C), and (D) reducing sugars during green tea kombucha fermentation in static and stirred bioreactors performed at 28 °C. Different letters (a–d or A–E) indicate a statistically significant difference among values (p < 0.05).
Figure 3. Changes in pH (A), total titratable acidity (B), total soluble solids (C), and (D) reducing sugars during green tea kombucha fermentation in static and stirred bioreactors performed at 28 °C. Different letters (a–d or A–E) indicate a statistically significant difference among values (p < 0.05).
Fermentation 11 00201 g003
Fermentation 11 00201 g004
Figure 4. Changes in total phenolic content (A) and antioxidant capacity against DPPH (B) and ABTS (C) free radicals during green tea kombucha fermentation in static and stirred bioreactors performed at 28 °C. Different letters (a–c or A–E) indicate a statistically significant difference among values (p < 0.05).
Figure 4. Changes in total phenolic content (A) and antioxidant capacity against DPPH (B) and ABTS (C) free radicals during green tea kombucha fermentation in static and stirred bioreactors performed at 28 °C. Different letters (a–c or A–E) indicate a statistically significant difference among values (p < 0.05).
Fermentation 11 00201 g004
Table 1. Experimental conditions and results of green tea kombucha fermentation according to 23-full factorial design performed at 28 °C for 10 days.
Table 1. Experimental conditions and results of green tea kombucha fermentation according to 23-full factorial design performed at 28 °C for 10 days.
RunIndependent VariablesDependent Variables
Sucrose
(g/L)		Green Tea
(g/L)		Inoculum
(%)		pHTTA a
(g/L)		TSS b
(°Brix)		TPC c
(mg GAE/100 mL)		DPPH d
(µmol/mL)		ABTS e
(µmol/mL)
1404103.201.70 ± 0.02 c3.60 ± 0.05 g37.87 ± 0.44 h2590.00 ± 41.48 b4661.42 ± 80.81 e
2804103.061.83 ± 0.03 b,c6.80 ± 0.10 b,c49.93 ± 0.08 f3031.67 ± 188.56 b5654.28 ± 20.30 d,e
3408103.411.34 ± 0.05 d,e3.20 ± 0.05 h79.01 ± 0.84 c2681.67 ± 205.61 b8540.00 ± 232.33 a,b
4808103.092.09 ± 0.06 a,b6.80 ± 0.05 b,c47.13 ± 0.52 g3190.00 ± 129.64 b5368.57 ± 30.30 d,e
5404303.031.64 ± 0.08 c,d5.80 ± 0.03 e84.73 ± 0.36 b4590.00 ± 388.91 a,b8068.57 ± 338.60 b,c
6804302.941.88 ± 0.01 b,c6.50 ± 0.05 d64.96 ± 1.09 d4690.00 ± 35.35 a,b5911.42 ± 10.10 d
7408303.131.19 ± 0.06 e4.10 ± 0.02 f78.44 ± 1.17 c6156.67 ± 11.78 a7618.57 ± 363.65 b,c
8808303.122.27 ± 0.09 a7.80 ± 0.01 a98.61 ± 0.24 a6640.00 ± 341.77 a9647.14 ± 40.41 a
9606203.321.83 ± 0.10 b,c7.00 ± 0.05 b58.68 ± 0.52 e3305.00 ± 188.56 b7525.71 ± 313.14 c
10606203.341.91 ± 0.08 b,c6.90 ± 0.02 b,c58.51 ± 0.32 e3380.00 ± 129.64 b8018.57 ± 303.04 b,c
11606203.271.79 ± 0.09 b,c6.70 ± 0.04 c,d58.51 ± 1.41 e3305.80 ± 153.21 b8332.85 ± 101.01 b,c
a Total Titratable acidity, b Total soluble solids, c Total phenolic content, d Antioxidant activity against the free radical DPPH; e Antioxidant activity against the free radical ABTS. Different letters (a–h) indicate statistically significant differences among values (p < 0.05).
Table 2. Estimated effects of the independent variables and their interactions on green tea kombucha fermentation carried out according to a 23-full factorial design after 10 days of fermentation at 28 °C.
Table 2. Estimated effects of the independent variables and their interactions on green tea kombucha fermentation carried out according to a 23-full factorial design after 10 days of fermentation at 28 °C.
Variable or InteractionpHTTA aTSS bTPC cDPPH dABTS e
(1) Sucrose concentration−5.491 *13.594 *25.922 *−14.540 *12.519 *−2.004
(2) Tea concentration5.099 *−1.102−1.85148.8887 *30.754 *5.977 *
(3) Inoculum−5.295 *−1.1028.795 *83.935 *86.412 *6.101 *
1 × 2−0.9809.675 *7.869 *−2.9763.6740.186
1 × 33.5302.327−5.554 *15.045 *−2.9931.781
2 × 30.392−0.1220.001−8.170 *26.672 *−0.266
1 × 2 × 32.5491.3476.017 *62.415 *2.5857.256 *
* Statistically significant at 95% confidence level (p < 0.05). a Total Titratable acidity, b Total soluble solids, c Total phenolic content, d Antioxidant activity against the free radical DPPH; e Antioxidant activity against the free radical ABTS.
Table 3. Relative change in peak area (%) of volatile components in green tea kombucha during fermentation in static and stirred bioreactors.
Table 3. Relative change in peak area (%) of volatile components in green tea kombucha during fermentation in static and stirred bioreactors.
RT (Min)CompoundsClassStatic System (Days)Stirred System (Days)
246810246810
7.408Cyclopentyl 4-ethylbenzoateEster2.532.453.551.852.3117.782.453.551.852.31
10.7662-ethyl-1-Hexanol Alcohol11.846.8310.869.967.625.736.8310.869.967.62
12.833α-TerpinoleneTerpenesnd0.992.011.29nd7.080.992.011.29nd
15.490Octanoic acidCarboxylic acids8.605.453.014.143.8710.705.453.014.143.87
16.9961,3-Di-tert-butylbenzeneHydrocarbon2.67ndndndndndndndndnd
17.714IononeKetone9.868.597.998.038.97nd8.597.998.038.97
20.341Decanoic acidCarboxylic acidsndndndndnd6.64ndndndnd
21.100β-CaryophylleneTerpenes0.160.451.972.162.920.730.451.972.162.92
22.536PentadecaneCarboxylic acids15.3616.1214.2615.6016.44nd16.1214.2615.6016.44
22.9772,4-Di-tert-butylphenolPhenols26.7122.3720.7421.1423.2629.0622.3720.7421.1423.26
24.618Dodecanoic acidcarboxylic acidsndndndndndndndndndnd
Total (%)77.7363.2564.3964.1765.3977.7290.8774.4375.0580.55
RT: Retention time; nd: not detected.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Alves, R.O.; de Oliveira, R.L.; de Moraes, M.M.; Santos, W.W.V.; Gomes da Câmara, C.A.; da Silva, S.P.; Porto, C.S.; Porto, T.S. Evaluation of the Impact of Fermentation Conditions, Scale Up and Stirring on Physicochemical Parameters, Antioxidant Capacity and Volatile Compounds of Green Tea Kombucha. Fermentation 2025, 11, 201. https://doi.org/10.3390/fermentation11040201

AMA Style

Alves RO, de Oliveira RL, de Moraes MM, Santos WWV, Gomes da Câmara CA, da Silva SP, Porto CS, Porto TS. Evaluation of the Impact of Fermentation Conditions, Scale Up and Stirring on Physicochemical Parameters, Antioxidant Capacity and Volatile Compounds of Green Tea Kombucha. Fermentation. 2025; 11(4):201. https://doi.org/10.3390/fermentation11040201

Chicago/Turabian Style

Alves, Raniele Oliveira, Rodrigo Lira de Oliveira, Marcílio Martins de Moraes, Wallysson Wagner Vilela Santos, Cláudio Augusto Gomes da Câmara, Suzana Pedroza da Silva, Camila Souza Porto, and Tatiana Souza Porto. 2025. "Evaluation of the Impact of Fermentation Conditions, Scale Up and Stirring on Physicochemical Parameters, Antioxidant Capacity and Volatile Compounds of Green Tea Kombucha" Fermentation 11, no. 4: 201. https://doi.org/10.3390/fermentation11040201

APA Style

Alves, R. O., de Oliveira, R. L., de Moraes, M. M., Santos, W. W. V., Gomes da Câmara, C. A., da Silva, S. P., Porto, C. S., & Porto, T. S. (2025). Evaluation of the Impact of Fermentation Conditions, Scale Up and Stirring on Physicochemical Parameters, Antioxidant Capacity and Volatile Compounds of Green Tea Kombucha. Fermentation, 11(4), 201. https://doi.org/10.3390/fermentation11040201

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop