processes

Article
Production and Characterization of Kombucha Tea from
Different Sources of Tea and Its Kinetic Modeling
Kubra Tarhan Kuzu 1,2 , Gamze Aykut 1 , Serap Tek 1 , Ercan Yatmaz 1,3 , Mustafa Germec 1 , Ibrahim Yavuz 1
and Irfan Turhan 1, *

1 Department of Food Engineering, Faculty of Engineering, Akdeniz University, 07058 Antalya, Turkey;
kubratarhan@comu.edu.tr (K.T.K.); gamze_3507@hotmail.com (G.A.); seraptek91@hotmail.com (S.T.);
ercanyatmaz@akdeniz.edu.tr (E.Y.); afatsumcemreg@gmail.com (M.G.); ibrahimyavuz07@gmail.com (I.Y.)
2 Department of Food Processing, Vocational School of Technical Sciences, Çanakkale Onsekiz Mart University,
Terzioglu Campus, 17100 Çanakkale, Turkey
3 Göynük Culinary Arts Vocational School, Akdeniz University, 07980 Antalya, Turkey
* Correspondence: iturhan@akdeniz.edu.tr; Tel.: +90-(242)-310-6573; Fax: +90-(242)-310-6306

Abstract: This study aimed to investigate the fermentation performance, sugar consumption, pH
changes, total phenolic compounds, and antioxidant activity produced using different tea extracts and
sugar concentrations and the kinetic characteristics of Kombucha fermentation. Three independent
sugar concentrations (10 g/L, 40 g/L, and 70 g/L) were used in the fermentation process. The results
showed that the Kombucha culture consumed all sugar in the fermentation medium when the sugar
concentration was below a certain threshold, but when the sugar concentration was high, not all
substrate was consumed. Sugar consumption values ranged from 48.39 to 55.40 g/L and affected
biomass formation, with higher sugar consumption resulting in increased biomass production. The
pH decreased during fermentation due to the production of organic acids and microbial by-products,
while total acidity increased. Total phenolic compounds increased during fermentation, with the
highest concentrations observed in herbal Kombucha teas. Antioxidant activity varied, with some
samples showing a decrease in DPPH scavenging ability. Kinetic characterization revealed the
relationship between substrate depletion, sugar consumption, total acidity, and phenolic compound
production. The results showed that sugar concentration influenced the fermentation kinetics and
Citation: Tarhan Kuzu, K.; Aykut, G.;
end-product characteristics of Kombucha tea. Overall, this study provides valuable insights into the
Tek, S.; Yatmaz, E.; Germec, M.;
Yavuz, I.; Turhan, I. Production and
fermentation process of Kombucha tea and its impact on various parameters, contributing to the
Characterization of Kombucha Tea understanding of the factors affecting its quality and health benefits.
from Different Sources of Tea and Its
Kinetic Modeling. Processes 2023, 11, Keywords: Kombucha fermentation; bioactive component; proximate composition; kinetic parameters;
2100. https://doi.org/10.3390/ kinetic modeling
pr11072100

Academic Editor: Chi-Fai Chau

Received: 11 June 2023 1. Introduction

Revised: 8 July 2023
Kombucha has been consumed all over the world but historically in China, Rus-
Accepted: 11 July 2023
sia, and Eastern European countries. Kombucha is a fermented sugared black tea by
Published: 14 July 2023
yeasts and Acetobacter species [1,2]. The various yeast species of Kombucha tea are
Brettanomyces bruxellensis [3], Candida stellata [3], Schizosaccharomyces pombe [3], Torulaspora
delbrueckii [3], Zygosaccharomyces bailii [3], Saccharomycodes ludwigii [4], Kloeckera apiculata [5],
Copyright: © 2023 by the authors.
Saccharomyces cerevisiae [4], Brettanomyces lambicus [6], Brettanomyces custersii [6], Candida
Licensee MDPI, Basel, Switzerland. krusei [5], and Pichia species [7]. This means that Kombucha culture differs from place to
This article is an open access article place and could easily be understood from different research about Kombucha cultures [2].
distributed under the terms and Namely, the kombucha culture is a symbiotic culture of bacteria and yeast (SCOBY) that is
conditions of the Creative Commons essential for the fermentation process of Kombucha [2]. The role of yeasts in the Kombucha
Attribution (CC BY) license (https:// fermentation is to hydrolyze sucrose from the cultivation medium to glucose and fructose
creativecommons.org/licenses/by/ and metabolize these monosaccharides to ethanol, which is further oxidized to acetic acid
4.0/). by acetic acid bacteria (AAB). AAB cannot uptake sucrose alone because of the lack of

Processes 2023, 11, 2100. https://doi.org/10.3390/pr11072100 https://www.mdpi.com/journal/processes

Processes 2023, 11, 2100 2 of 16

enzymes for the extracellular hydrolysis of sucrose or its transport into the cell. AAB uses
yeast-derived glucose to synthesize gluconic acid and bacterial cellulose in the form of a
pellicle, which is commonly described as the “fungus” [8–10]. Microbial community type
and composition play an important role in the biochemistry dynamics of Kombucha. These
associations help to decrease pH and reduce microbial growth of other microorganisms
with antimicrobial metabolites [11]. The time of Kombucha fermentation is between 7 and
60 days. During this time, biological activities increase. On the other hand, it was reported
that the best results were yielded in an average of 10 days [12]. According to the Food and
Drug Administration Model Food Code for Kombucha Brewing, more than 10 days of fer-
mentation are not suggested if produced for human consumption [13]. Therefore, 8–10 days
can be enough to obtain the best beverage specifications, and microorganisms use sugar to
produce value-added acids and antimicrobial metabolites [14]. The kombucha tea yielded
after fermentation consists of sugars (glucose, fructose), gluconic, glucuronic, L-lactic,
acetic, malic, tartaric, malonic, citric, and oxalic acids, as well as ethanol, 14 amino acids,
water-soluble vitamins, antibiotically active matters, and some hydrolytic enzymes [15].
Kombucha tea has beneficial features on human health, such as improving the im-
mune system, detoxifying harmful substances, lowering blood pressure, treating gastritis
and cholesterol, and exhibiting antioxidant, antibacterial, anticancer, and antidiabetic
activities [2]. The research about the antimicrobial activity of Kombucha tea showed that
the antimicrobial agent was acetic acid content, and it inhibited Agrobacterium tumefaciens,
Bacillus cereus, Salmonella choleraesuisserotypetyphimurium, Staphylococcus aureus, and
Escherichia coli. However, due to the fermented samples including 33 g/L total acid
(7 g/L acetic acid), these values indicated the yielded beverage samples were not suit-
able for drinkable levels, but Kombucha had antimicrobial activity against pathogenic
bacteria [16]. The other research demonstrated that Kombucha tea had an antimicrobial
effect against a range of pathogenic bacteria, several clinical Candida species, fermented
L. citriodora, and F. vulgare [17]. Kombucha could also be used against enteropathogenic
bacterial infections due to its polyphenolic content [18]. Various Kombucha cultures
also showed different antioxidant activity under the same fermentation conditions (10%
starter addition to the fresh medium prepared, 30 ◦ C, and 15 days fermentation time),
mostly indicating time-dependent properties [19]. The conformable research showed
the difference between antioxidant activity values from different starter cultures and tea
extracts [15]. The Kombucha fermentation with different initial sucrose concentrations
(ISCs) (70 g/L, 50 g/L, and 35 g/L of sucrose) was studied, and the highest sugar concen-
tration value was found to be an optimal concentration of carbon source, providing high
pH, low acetic acid, and high L-lactic acid content and highest sucrose consumption [20].
In the literature, there are some similar studies regarding the production of Kombucha
tea from different types of herbal and fruit teas. For instance, Zubaidah et al. [21] examined
the physical, chemical, and microbiological features of Kombucha from different varieties
of apples (Anna, Manalagi, Fuji, Granny Smith, Red Delicious, Rome Beauty, and Royal
Gala). Based on the results, it was reported that the best treatment was yielded on Fuji
varieties of Kombucha apple (total acid 1.33%, pH 2.95, total phenol 268.57 µg/mL GAE,
total sugar 6.74%, antibacterial activity against Staphylococcus aureus 21.30 mm, antibacterial
activity Escherichia coli 21.20 mm, antioxidant activity 35.62%, organoleptic aroma 3.55,
taste 3.3, and color 3.4 (on a scale of 1–5)) [21]. In another study, where the different carbon
sources (glucose, fructose, xylose, lactose, sucrose (70 g/L)), types of teas (black tea, green
tea, sage tea, pomegranate (hibiscus) tea, blueberries tea, and rosehip tea), and coffee were
used as resources to produce Kombucha [22], the pH, acidity, antioxidant activity, phenolic
substance, biomass development, color change, organic acid profile, ethanol, and sensory
analysis were examined. The results indicated that the value of pH decreased during
fermentation, and the Kombucha from fruit teas were greater acidity than herbal teas and
coffee extract. The phenolic substance content and antioxidant activity of the Kombucha
produced have been found to have the potential to be an important product. Regarding
biomass growth, it was determined most in glucose and sucrose (tea samples) and lactose
Processes 2023, 11, 2100 3 of 16

(coffee extract) and at the least in fructose (tea samples) and lactose (coffee extract). When
color changes were examined, it was detected that the L, a, and b values of herbal tea
changed in a fermentation medium supplemented with glucose, xylose, or fructose. During
the fermentation, most of the organic acids, including oxalic acid, tartaric acid, malic acid,
lactic acid, citric acid, succinic acid, and fumaric acid, were measured. On the other hand,
it was reported that no ethanol production was observed at the end of the fermentation.
Based on the sensory analysis, the most and least preferred Kombucha teas were produced
from pomegranate and sage teas, respectively [22]. In a different study, Tamer et al. [23]
evaluated the bio-accessibility and functional features of Kombucha teas fortified with
different medicinal plant extracts (linden, lemon balm, sage, Echinacea, mint, and cinnamon).
Based on the results, the antioxidant capacity (AC), ferric-reducing antioxidant power, and
cupric-reducing AC were 13.96%, 48.90%, and 55.54%, respectively. It was also found that
during 9-day storage, the bio-accessibility of total phenolic and AC dramatically increased
after gastric and intestinal digestion [23]. Additionally, the changes in the content of
organic acids and polyphenols during the Kombucha fermentation from green tea, black
tea, and tea manufacturing waste [24] and the antibacterial and antifungal activities of
black and green Kombucha teas [25] were also examined. Moreover, the kinetics of sucrose
fermentation by Kombucha culture was also studied by using Boltzmann’s functions [26].
The fermentation conditions were performed on 1.5 g/L of black tea, with 67 g/L of sucrose,
and using 10% or 15% of Kombucha culture (v/v). The model was described as a sigmoid
function at two different temperatures (22 ◦ C and 30 ◦ C). Based on the results, it was
determined that the rate of fermentation was maximum on days 4–5, and after reaching
the maximal rate, it dramatically decreased. It was reported that as the temperature
and inoculum concentration increased, the rate of the fermentation increased, the optimal
fermentation time was 3.5–5 days under the implemented circumstances, and the saturation
curves indicated the sigmoid kinetics at the selected sucrose concentration [26]. When
considering this information, this study has novelty in terms of the use of some different
types of teas in the production of Kombucha teas, kinetic characterization of Kombucha
fermentations performed at different substrate concentrations, and kinetic modeling of
Kombucha fermentations in terms of substrate consumption and total acidity. Therefore,
this study is filled the significant gap in the literature.
Kombucha tea is generally produced from black and green tea, but commercial firms’
market started to produce new Kombucha teas with lemon, apples, peach, blackberries, and
rosehip. Therefore, the objective of this study is to investigate the production of Kombucha
teas with diverse chemical compositions by utilizing various substrates. Additionally, the
study seeks to analyze the kinetic properties of Kombucha fermentation and develop a
kinetic model for fermentations involving different tea sources.

2. Materials and Methods

2.1. Kombucha Culture and Media
Kombucha culture was obtained from the commercial firm “Comboutea” (Tema Phar-
maceutical Vitamin Cosmetics Limited Company, Samsun, Turkey). The media components
for stock and pre-culture were 10 g/L yeast extract, 20 g/L glucose, and 20 g/L peptone [27].
After the medium composition was prepared, the medium pH was adjusted to 4 using
10 N HCl. The prepared medium was sterilized at 121.1 ◦ C for 15 min. Subsequently, the
medium was cooled to room temperature. It was inoculated with 10% (v/v) of Kombucha
culture. The stock and pre-cultures were incubated at 24 ◦ C for 10 days, and the stock
cultures were stored at 4 ◦ C. Stock cultures were renewed for one month to have viability
and productivity.

2.2. Experimental Design

In this study, different fruit (bilberry, rosehip, apple, and pomegranate tea) and herbal
(green, sage, linden, and black tea) teas were used for tea extraction. On the other hand,
the limited sugar value to produce Kombucha from different types of tea was determined
Processes 2023, 11, 2100 4 of 16

from a previous study [15]. After determining the maximum sugar limit, sucrose as the
sole carbon source was added to the fermentation medium by decreasing it to 30 g/L
to instigate the effect of the initial sucrose concentration. Thus, the ISCs were 10 g/L,
40 g/L, and 70 g/L in the present study. Each of the extracts was prepared with three
different ISCs (10 g/L, 40 g/L, and 70 g/L), and a coded system for the samples is given
in Table 1. All production and analyses were replicated two times. Kinetic parameters of
Kombucha fermentation were also calculated. Fermentations were kinetically modeled
using the logistic model (LM) and the Luedeking–Piret model (LPM) [28]. The LM was
used to predict the experimental substrate consumption values, and LPM was employed to
estimate the experimental total acidity values of fermentation.

Table 1. Sample code system.

Initial Sugar Concentration (g/L)

Tea Origin Tea
10 40 70
Sage tea (ST) ST-10 ST-40 ST-70
Linden tea (LT) LT-10 LT-40 LT-70
Herbal tea
Green tea (GT) GT-10 GT-40 GT-70
Black tea (BT) BT-10 BT-40 BT-70
Apple tea (AT) AT-10 AT-40 AT-70
Rosehip tea (RT) RT-10 RT-40 RT-70
Fruit tea
Pomegranate tea (PT) PT-10 PT-40 PT-70
Bilberry tea (BBT) BBT-10 BBT-40 BBT-70

2.3. Preparation of Tea Extracts, Inoculation, and Fermentation

Four different herbal teas (green (GT), sage (ST), linden (LT), and black tea (BT)) and
four various fruit teas (bilberry (BBT), rosehip (RT), apple (AT), and pomegranate tea (PT))
were used for Kombucha production. All the tea samples were provided by the Unilever
Company in Konya, Turkey.
The extraction process was realized by mixing 1 L of boiled pure water with 10 g tea
and waiting for 15 min to obtain tea extracts [29]. The mix was filtered by using roughing
filter paper (cellulosic filter paper) to separate the insoluble materials. After filtration,
different amounts of sucrose (10 g/L, 40 g/L, or 70 g/L) were immediately added. After the
sugar was completely dissolved, the mixture was transferred into 250 mL flasks (100 mL
working volume) and cooled to room temperature. It was stored in appropriate conditions
until inoculation.
After pre-culture and fermentation media were prepared, the flasks were inoculated
with 10 mL of pre-culture. Inoculated sugared tea mixtures were incubated at 24 ◦ C for
10 days with no agitation, and samples were taken daily under aseptic conditions and
stored at 4 ◦ C [30].

2.4. Analysis
The total acidity was determined by adding 0.1 N NaOH to samples until the pH
was 8.2 [31]. The pH values of fermented teas were measured with an electronic pH
meter (Thermo Scientific Orion 4 Star, Singapore). The total biomass of fermented sam-
ples was gravimetrically determined. The collected samples during fermentation were
filtered by using pre-weighed filter paper (Whatman No.: 1), and the fermented broth
was removed. The filter cake (biomass) was then dried at 60 ◦ C in the oven until constant
weight [32]. The residual sugar concentration was spectrophotometrically determined
using the 3,5-dinitrosalicylic acid method [33]. The Folin–Ciocalteu method was used to
determine the total phenolic substance concentration in samples. The results were given as
milligrams of gallic acid equivalents per liter (mg GAE/L) of Kombucha [34]. The antioxi-
dant analysis was determined with the α, α-diphenyl-β-picrylhydrazyl (DPPH) free radical
scavenging method, and the antioxidant capacity was determined as % inhibition [19].
Processes 2023, 11, 2100 5 of 16

2.5. Kinetic Parameters

Kinetic parameters including substrate consumption (∆S, g/L), maximum substrate
consumption rate (QS , g/L/d), substrate utilization yield (η, %), biomass production (∆X,
g/L), maximum biomass production rate (QX , g/L/d), biomass yield (YX/S , g biomass/g
substrate), total acidity (TA, %), maximum total acidity production rate (QTA , %/d), pheno-
lics production (∆PH, mg/L), maximum phenolics production rate (QPH , mg/L/d), and
phenolics yield (YPH/S , mg phenolics/g substrate) were calculated. The details regarding
how kinetic parameters are calculated can be found in previous similar studies [28].

2.6. Kinetic Modeling

The LM (Equation (1)) and LPM (Equation (2)) were utilized to estimate the experi-
mental substrate consumption and total acidity data of Kombucha fermentation. Microsoft
Office Excel 2013 was used. Traditionally, LM is used to describe cell growth. However,
in this work, the model was modified to define sugar consumption and independently
employed cell growth data because there is no sigmoid growth of biomass because of high
acidity or low pH values.
−dS
 
S
= µm,S 1 − S (1)
dt Sm
where −dS/dt is the substrate consumption rate (g/L/d), µm, S is the specific sugar con-
sumption rate (1/d), S is the residual substrate concentration at the time “t” (g/L), and Sm
is the maximum substrate concentration (g/L).
The LPM is utilized to define the metabolite production rate (dP/dt) related to cell
growth. However, dP/dt is also thought to be contingent on both momentary S and
dS/dt linearly [35].
dP dS
=α + βS (2)
dt dt
where dP/dt is the total acidity rate (%/d) and α and β are the empirical constants that
vary based on fermentation conditions and are determined with the best appropriate real
data. Moreover, the determination of coefficient (R2 ) was utilized to comprehend whether
modeling is accomplished or not [28].

2.7. Statistical Analysis

The SAS version 7 program (Statistical Analysis System, TS P1, Cary, NC, USA) was
used for the statistical evaluation of the data obtained from the study, and variance analysis
was performed. Significant differences were evaluated by the Duncan Multiple Comparison
Test at a confidence level of 95%.

3. Results and Discussion

3.1. Sugar Consumption and Biomass Production
Three ISCs were used to determine the fermentation performance of the commercial
Kombucha culture. It was determined that all the sugar in the fermentation medium is
consumed by the Kombucha culture in fermentations performed with AT-10, RT-10, PT-10,
and BT-10. However, when the fermentation medium contains high sugar concentration,
all the substrate in the medium is not consumed by the Kombucha culture at the end of the
fermentation (Figure 1A and Supplementary Materials). The highest sugar consumption
value is 55.40 g/L for LT-70 herbal Kombucha tea, whereas its highest value is 48.39 g/L
for AT-70 fruit Kombucha tea (Figure 1A and Table 2). These sugar consumption values
also affect biomass formation. In Figure 1B and the Supplementary Materials, biomass
production curves from different medium compositions are given. The difference in the
medium used, amount of sugar, the composition of culture, fermentation conditions, and
applied period are effective in the chemical composition of Kombucha [36]. Moreover, ISC
affects total sugar consumption. High sugar consumption is observed in fermentations
with high ISCs (Table 2). It is determined that residual sugar concentration decreases
 values also affect biomass formation. In Figure 1B and the Supplementary Materials, bio-
mass production curves from different medium compositions are given. The difference in
the medium used, amount of sugar, the composition of culture, fermentation conditions,
and applied period are effective in the chemical composition of Kombucha [36]. Moreover,
Processes 2023, 11, 2100 ISC affects total sugar consumption. High sugar consumption is observed in fermenta- 6 of 16
tions with high ISCs (Table 2). It is determined that residual sugar concentration decreases
during fermentation. Furthermore, the highest biomass productions for herbal and fruit
Kombucha teas are determined as 6.46 g/L for GT-70 and 8.77 g/L for PT-70 (Figure 1B).
during fermentation. Furthermore, the highest biomass productions for herbal and fruit
These resultsteas
Kombucha show
arethat biomassas
determined formation
6.46 g/L increases
for GT-70 with an increase
and 8.77 in sugar
g/L for PT-70 consump-
(Figure 1B).
tion (Table 2). In work conducted by Muhialdin et al. [37], the amount and yield of
These results show that biomass formation increases with an increase in sugar consumptionbiomass
were associated
(Table 2). In workwith the sugarby
conducted source.
Muhialdin et al. [37], the amount and yield of biomass
were associated with the sugar source.

Figure 1. Change of substrate concentration, biomass production, pH, total acidity, phenolic, and
Figure 1. Change
antioxidant of during
activity substrate concentration,
Kombucha biomass
fermentation production,
performed pH, containing
on media total acidity, phenolic,
different initialand
antioxidant activity during Kombucha fermentation performed on media containing different initial
sugar concentrations. (A) Substrate consumption vs. time with sage tea (ST-70) and linden tea (LT-70).
sugar concentrations. (A) Substrate consumption vs. time with sage tea (ST-70) and linden
(B) Biomass production vs. time with green tea (GT-70) and pomegranate tea (PT-70). (C) pH changetea (LT-
vs. time with sage tea (ST-40) and blueberry tea (BBT–40). (D) Total acidity vs. time with sage tea
(ST-40) and rosehip tea (RT-70). (E) Phenolic change vs. time with green tea (GT-40) and blueberry
tea (BBT-40). (F) Antioxidant activity vs. time with green tea (GT-40) and rosehip tea (RT-70).
Processes 2023, 11, 2100 7 of 16

Table 2. Kinetic parameters of Kombucha fermentation performed with extracts from different types
of tea with various initial sugar concentrations.

Kinetic Parameters
Tea [Substrate] ∆S QS ∆X QX YX/S ∆TA QTA ∆PH QPH YPH/S
η (%)
(g/L) (g/L/d) (g/L) (g/L/d) (g/g) (%) (%/d) (mg/L) (mg/L/d) (mg/g)
10 g/L 8.52 1.29 79.48 0.72 0.12 0.08 0.17 0.08 445.96 97.18 52.34
ST 40 g/L 13.10 3.71 29.39 1.74 0.51 0.13 4.10 0.60 883.17 169.75 67.42
70 g/L 26.62 4.05 32.71 3.96 0.94 0.15 1.51 0.28 900.31 189.07 33.82
10 g/L 11.66 2.72 97.25 1.02 0.22 0.09 0.59 0.17 242.12 26.80 20.77
LT 40 g/L 19.85 4.00 47.40 2.72 1.20 0.14 1.04 0.27 399.65 49.54 20.13
70 g/L 55.40 11.28 72.78 3.80 0.14 0.07 1.63 0.31 227.06 48.14 4.10
10 g/L 12.13 2.40 88.80 0.74 0.26 0.06 0.35 0.15 1217.60 410.68 100.38
GT 40 g/L 32.43 4.90 56.65 2.52 0.78 0.08 0.87 0.33 1215.90 345.69 37.49
70 g/L 44.07 6.34 53.72 6.46 1.22 0.15 1.42 0.37 1268.40 264.16 28.78
10 g/L 15.75 2.82 100.00 1.09 0.34 0.07 0.34 0.15 321.98 64.14 20.44
AT 40 g/L 18.61 3.07 39.89 3.76 1.39 0.20 1.18 0.27 785.45 159.90 42.21
70 g/L 48.39 5.28 63.15 4.28 1.37 0.09 1.64 0.41 466.38 74.71 9.64
10 g/L 15.43 3.65 100.00 2.01 0.23 0.13 1.07 0.36 130.66 21.39 8.47
RT 40 g/L 28.74 6.37 54.33 4.71 2.17 0.16 1.61 0.49 295.36 76.28 10.28
70 g/L 45.66 8.32 51.38 5.60 1.31 0.12 2.77 0.82 255.86 56.83 5.60
10 g/L 10.33 2.47 100.00 2.20 0.91 0.21 0.39 0.20 549.89 95.36 53.23
PT 40 g/L 21.04 3.67 44.69 3.25 1.43 0.15 1.30 0.30 800.04 101.66 38.02
70 g/L 42.92 6.36 50.61 8.77 1.66 0.20 2.18 0.34 930.94 166.20 21.69
10 g/L 12.67 2.54 95.62 0.81 0.31 0.06 0.44 0.18 655.20 95.51 51.71
BBT 40 g/L 16.05 3.07 32.56 2.07 0.70 0.13 1.52 0.39 1311.92 187.92 81.74
70 g/L 21.41 3.24 26.02 3.55 1.40 0.17 1.73 0.41 782.90 178.13 36.57
∆S, substrate consumption (g/L); QS , maximum substrate consumption rate (g/L/d); η, substrate utilization
yield (%); ∆X, biomass production (g/L); QX , maximum biomass production rate (g/L/d); YX/S , biomass yield
(g biomass/g substrate); ∆TA, total acidity (%); QTA , maximum total acidity production rate (%/d); ∆PH,
phenolic production (mg/L); QPH , maximum phenolic production rate (mg/L/d); and YPH/S , phenolic yield (mg
phenolic/g substrate).

3.2. pH and Total Acidity

Kombucha fermentation was performed with herbal and fruit tea extracts for 10 days,
and pH changes are shown in Figure 1C and the Supplementary Materials. Herbal and
fruit tea extracts’ initial pH values range from 4.86 to 5.90 and from 3.12 to 3.53, respectively.
Differences between initial and final values of pH are higher for herbal tea samples than
for fruit tea samples (Figure 1C and Supplementary Materials). The lowest final pH
values of herbal and fruit teas are measured to be 2.53 for ST-40 and 2.46 for BBT-40
(Figure 1C), and their highest values are 3.73 for ST-10 and 3.57 for AT-10 (Supplementary
Materials). It is seen that a slight pH increase at the end of Kombucha fermentation in AT
supplemented with 10 g/L sucrose (Supplementary Materials). This may be due to the
breakdown of dead cells in the fermentation medium [38]. All pH values, except for that
of AT-10, decrease during fermentation because microorganisms metabolize sugar into
different metabolites, such as organic acids and by-products (Figure 1C and Supplementary
Materials). For all samples, a significant decrease in pH is noticed between days 0 and 5,
and changes in pH are statistically insignificant after day 5 (p < 0.05). Total acidity values in
all herbal and fruit tea samples are below 0.30% acetic acid at the beginning of fermentation.
Initial total acidity values differ from 0.04% to 0.22% for herbal tea extracts and 0.15% to
0.27% for fruit tea extracts (Figure 1D and Supplementary Materials). The highest and
lowest total acidity values for herbal and fruit teas are determined to be 4.21% (ST-40)
(Figure 1D), 0.15% (GT-10) (Supplementary Materials), and 2.55% (RT-70) (Figure 1D),
0.23% (AT-10) (Supplementary Materials). In general, when examining Figure 1D and
the Supplementary Materials, an increase in total acidity values is observed as a result
of organic acid production and dead cell fragmentation during fermentation [26]. These
results show that organic acid production and microbial by-products, which occur in
Processes 2023, 11, 2100 8 of 16

parallel with sugar consumption during fermentation, decrease pH and increase total
acidity (Figure 1D and Supplementary Materials).

3.3. Total Phenolic Compounds and Antioxidant Activity

The changes in total phenolic compounds for herbal and fruit Kombucha teas are given
in Figure 1E and the Supplementary Materials. The phenolic concentration increases in
almost all herbal (except for those of BT-40 and BT-70) and fruit Kombucha tea experiments
at the end of fermentation. The highest total phenolic compound values are determined to
be 1522.90 mg GAE/L in ST-40, 1061.55 mg GAE/L in LT-10, and 3266.05 mg GAE/L in
GT-40 (Figure 1E), and 1025.67 mg GAE/L in BT-10 for herbal Kombucha teas after 10 days
fermentation (Supplementary Materials). For fruit Kombucha teas, the highest total pheno-
lic compound values are calculated to be 1041.21 mg GAE/L in AT-40, 1016.99 mg GAE/L
in RT-40, 1420.44 mg GAE/L in PT-40 (Supplementary Materials), and 1907.53 mg GAE/L
in BBT-40 (Figure 1E) after 10 days fermentation. When considering all fermentations,
the highest increase in total phenolic substance concentration is yielded as 215.44% with
BBT-40. An increase in phenolic content with fermentation may be related to the enzymes
of mixed Kombucha culture, the acidic environment of Kombucha tea, the synergistic effect
of different components in tea, and the breakdown of complex phenolic compounds [39].
Moreover, the fact that phenolic components are more stable at acidic pH may cause dif-
ferences in the total amount of phenolic substances during fermentation [40]. The decline
in total phenolic concentration in black Kombucha tea samples (BT-40 and BT-70) might
be due to the characteristics of black tea of that season. The total amount of phenolic
substances in green tea is higher than in black tea [40]. Moreover, the percentage increase
in total phenolic concentration in fruit Kombucha teas (average 112.49%) is higher than
in herbal Kombucha teas (average 49.14%) (Figure 1). In summary, the total amount of
phenolic compounds increased.
The antioxidant activity results are given in Figure 1F and the Supplementary Ma-
terials. The final DPPH scavenging ability decreases except for GT-70 (+4.46%), AT-10
(+5.99%), and AT-40 (+38.08%) assays. Decline values change from 0.86% to 31.97% for
herbal Kombucha teas and 2.92% to 17.50% for fruit Kombucha teas. The maximal decrease
is calculated to be 31.97% for LT-40. This decline could be about substrate and starter
culture types. Because of research about the influence of starter culture on Kombucha
fermentation, results show that DPPH scavenging ability is slightly increased in the first 3
days and decreased after day 3 of fermentation [15]. The final antioxidant values are lower
than the initial values of this research. The highest final DPPH scavenging ability value is
66.59% for the GT-40 at the end of the fermentation (Figure 1F). In a study [19], half of eight
different Kombucha samples showed a regular increase in antioxidant activity, while the
rest of them had irregular and variable results. It was predicted that Kombucha is affected
by different environments, sugar quantity, fermentation conditions, and ionization change
that occurs during fermentation may cause this variability. The highest initial and final
DPPH scavenging ability results are calculated in green tea samples (Figure 1F). All other
herbal and fruit tea Kombucha samples are lower than green tea samples (Figure 1F and
Supplementary Materials). It is also reported that the highest DPPH scavenging ability
is generally obtained from green tea samples [41]. Moreover, the change in antioxidant
activity is affected by tea type and fermentation temperature. DPPH scavenging abil-
ity, despite decreases and increases during fermentation, generally increases at the end
of fermentation [41].

3.4. Kinetic Characterization

Based on the kinetic results given in Table 2, the minimum and maximum ∆S are
determined as 8.52 g/L in ST-10 and 55.40 g/L in LT-70, respectively. As the sugar concen-
tration in the fermentation medium increases, ∆S increases. However, this is not alone as
an indicator that indicates the success of fermentation. Therefore, other kinetics regarding
substrate depletion, QS , and η, were estimated. The results indicate that the lowest and
Processes 2023, 11, 2100 9 of 16

highest values of QS are 1.29 g/L/d in ST-10 and 11.28 g/L in LT-70, which are the same
as ∆S. Moreover, as the substrate concentration increases, QS increases. The η was also
determined, and the values of η range from 26.02 in BBT-70 to 100% in AT-10, RT-10, and
PT-10. When the sugar amount added into the fermentation medium was minimum, almost
all the sugar was consumed by the Kombucha culture. However, when the substrate con-
centrations in the fermentation medium are 40 g/L and 70 g/L, the η varies from 29.39% in
ST-40 to 56.65% in GT-40 and 26.02% in BBT-70 to 72.78% in LT-70, respectively. Therefore,
we can say that as the substrate concentration in the fermentation environment increases, η
decreases in general.
Similar to the kinetics regarding substrate consumption, when the fermentation
medium is enriched with 10 g/L sucrose, the minimum ∆X is 0.72 g/L for ST-10, whereas its
maximum value is 2.20 g/L for PT-10. When 40 g/L sucrose is added into the fermentation
medium, the lowest and highest values of ∆X are 1.74 g/L and 4.71 g/L in ST-10 and RT-40,
respectively. Similarly, when the substrate concentration in the medium is 70 g/L, the ∆X
varies from 3.55 to 8.77 g/L in BBT-70 and PT-70. Therefore, as the substrate concentration
increases, ∆X increases. As for the QX , when the fermentation medium is supplemented
with 10 g/L, 40 g/L, and 70 g/L of sucrose, the lowest and highest values of QX are calcu-
lated as 0.12 g/L/d and 0.91 g/L/d (ST-10 and PT-10), 0.51 g/L/d and 2.17 g/L/d (ST-40
and RT-40), and 0.14 g/L/d and 1.66 g/L/d (LT-70 and PT-70), respectively. Between both
the minimum and maximum QX values, the highest QX values are yielded when 40 g/L
substrate is added into the medium. Moreover, when the fermentation medium is supple-
mented with 10 g/L, 40 g/L, and 70 g/L, the lowest values of YX/S are 0.06 g/g, 0.08 g/g,
and 0.07 g/g, whereas their highest values are 0.21 g/g, 0.20 g/g, and 0.20 g/g, respectively.
The minimum and maximum values of YX/S at different substrate concentrations are highly
close to each other. Although ∆X increases depending on the substrate concentration, this
situation is not valid for the YX/S .
Regarding the kinetic results related to the total acidity, the minimum and maximum
∆TA values are determined as 0.17% and 1.07%, 0.87% and 4.10%, and 1.42% and 2.77%
of ST-10 and RT-10, GT-40 and ST-40, and GT-70 and RT-70 when 10 g/L, 40 g/L, and
70 g/L sucrose are inserted into the medium, respectively. Except for the ∆TA values of
the Kombucha fermentation of ST, as the substrate concentration increases, ∆TA values
increase (Table 2). The lowest and highest QTA values are found as 0.08%/d and 0.36%/d
(ST-10 and RT-10), 0.27%/d and 0.60%/d (LT-40 and ST-40), and 0.28%/d and 0.82%/d
(ST-70 and RT-70) with 10 g/L, 40 g/L, and 70 g/L of sucrose concentration added into the
medium. As it is in the values of ∆TA, except for QTA values of ST, QTA values increase
with an increase in substrate concentration (Table 2).
The lowest values of ∆PH are 130.66 mg/L, 295.36 mg/L, and 227.06 mg/L with
10 g/L, 40 g/L, and 70 g/L sucrose concentrations inserted into the RT-10, RT-40, and
LT-70 media, respectively. Contrarily, its maximum values are yielded as 1217.60 mg/L
from GT-10, 1311.92 mg/L from BBT-40, and 1268.40 mg/L from GT-70. Except for the
∆PH values of ST, GT, and BT, the highest ∆PH values are obtained when 40 g/L sucrose
is used in the medium (Table 2). Additionally, the minimum and maximum values of
QPH are 21.39 mg/L/d and 410.68 mg/L/d, 49.54 mg/L/d and 245.69 mg/L/d, and
48.14 mg/L/d and 264.16 mg/L/d for RT-10 and GT-10, LT-40 and GT-40, and LT-70 and GT-
70, respectively. It is realized that the Kombucha teas from GT supplemented with 10 g/L,
40 g/L, and 70 g/L sucrose give the highest phenolic substance amounts. As the substrate
concentration in the GT-based medium increase, the values of QPH decrease. Conversely, the
QPH increase with an increase in substrate concentration added into the ST- and PT-based
media. For the rest of QPH , the maximum peak values of QPH are yielded when 40 g/L
substrate concentration is added into the fermentation medium. Concerning the YPH/S ,
its lowest values are obtained to be 8.47 mg PH/g, 10.28 mg PH/g, and 4.10 mg PH/g
substrate when the fermentation media are RT-10, RT-40, and LT-70, respectively. Maximum
YPH/S values are also calculated as 100.38 mg PH/g, 81.74 mg PH/g, and 36.57 mg PH/g
substrate for GT-10, BBT-40, and BBT-70, respectively. As the sugar concentration in the
Processes 2023, 11, 2100 10 of 16

medium increases, the maximum YPH/S value decreases. Moreover, it is determined that
YPH/S values decrease with an increase in the substrate levels of GT- and PT-based media.
For the remaining media, except for that of the LT, the highest YPH/S peak values are
yielded when the media are enriched with 40 g/L sucrose (Table 2).

3.5. Kinetic Modeling

The observed values of total acidity were estimated by the LPM, while the actual
values related to sugar depletion were predicted by the LM (Figure 2). Concerning
the prediction of sugar depletion values, the observed and estimated sugar depletion
curves are plotted vs. time in Figure 2. As indicated in Figure 2, ST, LT, GT, AT, RT,
PT, and BBT, the experimental and estimated sugar depletion values are generally in
good agreement, except for that of the Kombucha fermentation performed with ST
supplemented with 40 g/L sucrose because its R2 value (0.6038) is lower than 0.75
(Table 3). Indeed, the experimental data on days 4–7 of fermentation are overestimated
by the LM (Figure 2, ST). Additionally, if the R2 value is higher than 0.75, meaning that
the model can be used to estimate the fermentation experimental data [42]. Therefore,
the yielded R2 values are found between 0.7731 and 0.9750, except for R2 = 0.6038,
demonstrating that the proposed model for substrate depletion adequately fits the actual
data of substrate depletion. Additionally, the values of µm, S, and Sf are between 0.17
and 1.14 d−1 and 0.34 and 9.64 g/L, respectively. The minimum and maximum values
of µm, S are obtained when the BBT supplemented with 40 g/L and 10 g/L sucrose is
used in the production of Kombucha tea, respectively. The highest µm, S values are
achieved when 10 g/L sucrose is added to the fermentation media (Table 3). The lowest
and highest Sf values are also yielded with ST supplemented with 10 g/L sucrose and
RT supplemented with 70 g/L sucrose, respectively. Nevertheless, the values of Sf
increase with an increase in sucrose concentration added into the fermentation media
in general (Table 3). Mahdinia et al. [35] predicted the observed substrate depletion
data of Menaquinoe-7 fermentation in the biofilm reactor with glucose- or glycerol-
based medium using the LM. Based on the modeling results, it was declared that the
R2 values of the process were determined between 0.953 and 0.991, indicating that
the suggested models fit well with the experimental substrate consumption data. The
values of µm, S for glucose- and glycerol-based media were 0.059 h−1 and 0.054 h−1 ,
respectively [35], which are all higher than those of this study (Table 3). Ilgın et al. [43]
estimated the substrate consumption data of Aspergillus niger inulinase fermentation
from carob extract under shake flask fermentation circumstances using the LM. From the
calculations, it was reported that the values of µm, S, and Sf were found to be 0.062 h−1
and 0.93 g/L, respectively, showing that the µm, S value is greater than the results yielded
from the current study while the Sf value is compatible with those of this study (Table 3).
In another study in which A. niger inulinase fermentation of sugar beet molasses in
the large-scale stirred tank bioreactor was taken place [44], the experimental data of
substrate consumption were forecasted by using the LM; thus, the µm, S , and Sf values
are computed as 0.042 h−1 and 1.18 g/L, respectively. It is determined that the computed
µm, S, and Sf values are highly consistent with the present study (Table 3). It was reported
that the proposed model successfully fitted the experimental data of substrate depletion
with a high R2 value (R2 = 0.9778) [44].
Processes
Processes2023,
2023, 11,
11, x2100
FOR PEER REVIEW 11 of 16
11 of 16

Figure 2.
Figure Substrate consumption
2. Substrate consumption and
and total
total acidity
acidity curves
curves fitted
fitted by
by the
the LM
LM and
and LPM.
LPM.
Processes 2023, 11, 2100 12 of 16

Table 3. The model parameters calculated for kinetically modeling the Kombucha fermentation.

Kinetics for Substrate Consumption Kinetics for Acidity

Tea [Substrate]
µm, S (1/d) Sm (g/L) Sf (g/L) R2 β (%/gS.d) α (%/gS) A > β Fold R2
10 (g/L) 0.4738 10.24 0.34 0.9438 0.0001 0.0201 360.19 0.5675
ST 40 (g/L) 0.2752 44.58 1.87 0.6038 0.0076 0.2584 34.00 0.9064
70 (g/L) 0.2864 77.45 2.54 0.8572 0.0013 0.0662 49.85 0.8481
10 (g/L) 0.7821 11.99 1.19 0.9672 −0.0058 0.0631 10.91 0.8060
LT 40 (g/L) 0.3462 41.42 3.89 0.8055 −0.0006 0.0343 60.27 0.5337
70 (g/L) 0.5947 73.16 0.83 0.9655 0.0004 0.0211 55.41 0.4809
10 (g/L) 0.7168 13.62 0.84 0.9487 −0.0024 0.0142 5.87 0.7586
GT 40 (g/L) 0.2909 52.03 4.44 0.9185 0.0005 0.0315 63.17 0.7220
70 (g/L) 0.2503 82.03 9.18 0.9413 0.0001 0.0394 282.80 0.8259
10 (g/L) 0.7798 15.34 1.24 0.9636 −0.0075 0.0468 6.24 0.9228
AT 40 (g/L) 0.2757 46.12 2.46 0.9012 0.0019 0.0863 45.75 0.8479
70 (g/L) 0.3324 71.27 5.07 0.9563 0.0015 0.0365 23.65 0.8251
10 (g/L) 0.7978 14.64 2.49 0.9671 −0.0030 0.0876 29.15 0.8465
RT 40 (g/L) 0.2765 51.46 9.47 0.7731 −0.0003 0.0475 145.00 0.6153
70 (g/L) 0.2459 87.58 9.64 0.8645 −0.0006 0.0671 110.88 0.7225
10 (g/L) 1.0969 8.94 1.39 0.9750 −0.0039 0.0554 14.30 0.9526
PT 40 (g/L) 0.2011 46.40 6.97 0.8137 0.0008 0.0568 75.30 0.8141
70 (g/L) 0.2589 79.37 6.13 0.9332 0.0013 0.0493 39.13 0.8193
10 (g/L) 1.1422 12.71 0.52 0.9166 −0.0023 0.0371 16.34 0.7676
BBT 40 (g/L) 0.1671 46.97 4.90 0.8257 0.0010 0.1349 129.31 0.7937
70 (g/L) 0.3043 79.67 1.49 0.9088 0.0008 0.1122 135.44 0.6790
µm, S , specific sugar consumption rate (1/d); Sm , maximum substrate concentration (g/L); Sf , final substrate
concentration (g/L); R2 , determination of coefficient; β, empirical constant (%/gS.d); α, empirical constant
(%/gS); ST, sage tea; LT, linden tea; GT, green tea; AT, apple tea; RT, rosehip tea; PT, pomegranate tea; and BBT,
bilberry tea.

The actual and estimated total acidity values are also plotted vs. time and shown
in Figure 2. It is detected that the values of R2 range from 0.4809 to 0.9526. It can be
said that those with R2 values higher than 0.75 (in this case, they are ST-40, ST-70, LT-10,
GT-10, GT-70, AT-10, AT-40, AT-70, RT-10, PT-10, PT-40, PT-70, BBT-10, and BBT-40) are
adequately fitted by the LPM (Table 3). Therefore, most of the Kombucha fermentation from
different tea extracts supplemented with different concentrations of sucrose is satisfactorily
fitted by the LPM with the R2 value greater than 0.75. Moreover, α and β values, which
can change based on the fermentation circumstances, were estimated (Table 3). If α 6= 0
and β = 0, then the total acidity is associated with substrate consumption. If α = 0 and
β 6= 0, then the total acidity is non-associated with substrate consumption. The values of
β vary from −0.0075 to 0.0076%/gS.d, which are so close to zero, while the values of α
range from 0.0142 and 0.2584%/gS. The values of α are 5.87 to 360.19 times greater than
those of β (Table 3). Therefore, we can say that the total acidity is associated with substrate
consumption. Mahdinia et al. [35] studied the kinetic modeling of Menaquinoe-7 fabrication
from glucose and glycerol in the biofilm reactor using the LPM. The model parameters
of the LPM, which are α and β, were calculated to be −0.138 mg/g and 0.00010 mg/g/h
for the production in the glucose-based medium and −0.089 mg/g and 0.00301 mg/g/h
for the production in glycerol-based medium, respectively. Therefore, the values of α
were 1380- and 29.57-fold higher than those of β, respectively, showing that Menaquinoe-7
fabrication was associated with substrate consumption [35], as it is in the current study. To
the best of our knowledge, there is no study regarding the kinetic modeling of Kombucha
fermentation using different tea extracts enriched with different concentrations of sucrose
as a carbon source. Therefore, this study is important in terms of contributing to science
because of the information it contains.
Processes 2023, 11, 2100 13 of 16

4. Conclusions
In conclusion, the study focused on the fermentation performance of a commercial
Kombucha culture using different herbal and fruit tea extracts with varying sugar concen-
trations. The results showed that the Kombucha culture effectively consumed all sugar
in the fermentation medium when supplemented with AT-10, RT-10, PT-10, and BT-10,
but when higher sugar concentrations were present, not all substrate was consumed. The
highest sugar consumption value was 55.40 g/L for LT-70 herbal Kombucha tea, while
AT-70 fruit Kombucha tea had the highest value at 48.39 g/L. These sugar consumption
values were correlated with biomass production, showing an increase with higher sugar
consumption. The pH and total acidity of the Kombucha teas changed during fermentation,
with herbal teas showing more significant fluctuations than fruit teas. The organic acid
production and microbial by-products during fermentation decreased pH and increased
total acidity. Additionally, the total phenolic compounds and antioxidant activity increased
during fermentation, with higher increases observed in fruit kombucha teas compared to
herbal teas. Kinetic characterization revealed the relationships between sugar depletion,
biomass production, total acidity, and phenolic compounds during fermentation. The
values of different kinetic parameters varied depending on the sugar concentration in
the fermentation medium. However, this study had some limitations, such as the lack
of detailed analysis of the specific enzymes involved in the breakdown of complex phe-
nolic compounds and the influence of environmental factors on fermentation variability.
Furthermore, only a specific commercial Kombucha culture was used, which may not
fully represent the diversity of Kombucha cultures available. Despite these limitations,
this study provides valuable insights into the fermentation performance of Kombucha
cultures under different conditions, which could be beneficial for further research and
industrial applications.

Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/pr11072100/s1, Figure S1: Change of substrate concentration,
biomass production, pH, total acidity, phenolic, and antioxidant activity during Kombucha fer-
mentation performed on media containing different initial sugar concentrations. (A, H, and O):
Sage tea (ST); (B, I, and Q): Linden tea (LT); (C, J, and P): Green tea (GT); (D, K, and R): Apple
tea (AT); (E, L, and S): Rosehip tea (RT); (F, M, and T): Pomegranate tea (PT); and (G, N, and U):
Bilberry tea (BBT).
Author Contributions: K.T.K. conceptualization, investigation, methodology, project administra-
tion, and writing—original draft preparation; G.A. conceptualization, investigation, methodology,
project administration, and writing—original draft preparation; S.T. conceptualization, investigation,
methodology, project administration, and writing—original draft preparation; E.Y. writing—review
and editing, project administration, supervision, and formal analysis; M.G. writing—review and
editing, formal analysis, software, and modeling; I.Y. writing—review and editing, formal analysis,
software, and modeling; I.T. writing—review and editing, resources, supervision, conceptualization,
and methodology. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Data Availability Statement: Not applicable.
Acknowledgments: This study was supported by the TUBITAK 2209-University Student Research
Project Support Program.
Conflicts of Interest: The authors declare no conflict of interest.
Processes 2023, 11, 2100 14 of 16

Abbreviations

Abbreviations Full Name

ISC Initial sucrose concentration
AAB Acetic acid bacteria
R2 Determination of coefficient
α and β Empirical constants
N Normal
HCl Hydrochloric acid
LPM Luedeking–Piret model
LM Logistic model
GT Green tea
ST Sage tea
LT Linden tea
BT Black tea
BBT Bilberry tea
RT Rosehip tea
AT Apple tea
PT Pomegranate tea
NaOH Sodium hydroxide
GAE Gallic acid equivalents
DPPH α, α-diphenyl-β-picrylhydrazyl
∆S Substrate consumption, g/L
QS Maximum substrate consumption rate, g/L/d
η Substrate utilization yield, %
∆X Biomass production, g/L
QX Maximum biomass production rate, g/L/d
YX/S Biomass yield, g biomass/g substrate
TA Total acidity, %
QTA Maximum total acidity production rate, %/d
∆PH Phenolic production, mg/L
QPH Maximum phenolic production rate, mg/L/d
YPH/S Phenolic yield, mg phenolic/g substrate
−dS/dt Substrate consumption rate, g/L/d
µm, S Specific sugar consumption rate, 1/d
S Residual substrate concentration at the time “t”, g/L
Sm Maximum substrate concentration, g/L
Sf Final substrate concentration, g/L
dP/dt Total acidity rate, %/d
SAS Statistical Analysis System

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