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Indonesian Journal of Chemical Research
http://ojs3.unpatti.ac.id/index.php/ijcr
Indo. J. Chem. Res., 11(1), 29-36, 2023
The Effect of Fermentation on the Characteristics and Antioxidant Activity of Wuluh Starfruit
Leaf Kombucha Tea (Avverhoa bilimbi Linn.)
Fanny Fajrin Aulia Rosyada, Eva Agustina*, Hanik Faizah
Biology Deparment, Faculty Sains and Technology, UIN Sunan Ampel Surabaya, Dr. Ir, H. Seokarno Street Number 682,
Surabaya, Indonesia.
*Corresponding Author: eva_agustina@uinsby.ac.id
Received: December 2022
Received in revised: February 2023
Accepted: April 2023
Available online: May
Abstract
Kombucha is a fermented drink that provides health effects. Wuluh starfruit leaves are
one of the ingredients that can be used in making kombucha. This study was conducted
to determine the effect of fermentation time on the physical, chemical, and antioxidant
characteristics of wuluh starfruit leaf kombucha tea (Avverhoa bilimbi Linn.). This
research is an experimental study with a completely randomized design (CRD)
consisting of 4 treatments. Kombucha wuluh starfruit leaves are made with a
fermentation time of 0, 4 8, and 12 days. The physical characteristics tested include an
organoleptic test of scent, color, and taste. The chemical characteristics tested include
pH, levels of titrated acids (tta), phenolic, and alcohols. The antioxidant activity is
known by determining the value of IC50. Organoleptic, pH, tat, and phenolic assessment
data were statistically analyzed using Kruskal Wallis and Mann Whitney. Alcohol
content and antioxidant activity were analyzed descriptively. The best result of physical,
and chemical characteristics and antioxidant activity are on the 12th day of fermentation
with a pH of 3, TTA 0.110.070%, phenolic 87.331.140 mg/ml GAE and alcohol of
0.41% with an IC50 value of 3.65 ppm.
Keywords: Antioxidant, fermentation, Kombucha, total titrated acids, phenolic
INTRODUCTION
Health is a very valuable thing for a human that
is generally often overlooked. The emergence of
various health problems makes people aware of the
importance of maintaining health to implement a
healthy lifestyle by consuming healthy foods and
drinks (Cahanar & Suhanda, 2006). One of the drinks
that can improve body health is kombucha.
Kombucha is one of the traditional drinks fermented
from black tea containing sugar with tea mushrooms
commonly known as Scoby (Symbiotic culture of
bacteria and yeast). Kombucha has a taste like
sparkling apple cider. During the fermentation period,
kombucha will undergo a change in taste from
refreshing sour to sour like vinegar (Goh et al., 2012;
Sun, Li, & Chen, 2015). This happens because the
bacteria and yeast in kombucha will convert sugar
into several major compounds, namely acetic acid,
ethanol, and glucuronic acid as well as minor
compounds, namely lactic origin, phenolic acid, B
vitamins, and enzymes. Organic acid compounds will
increase during the fermentation process. This will
affect the decrease in pH and increase in total acid in
kombucha (Villarreal-Soto, Beaufort, Bouajila,
Souchard, & Taillandier, 2018; Wistiana & Zubaidah,
2015).
Organic acids produced from fermentation are
the main metabolites produced and can act as active
ingredients to provide health effects such as
antioxidants (Muhialdin et al., 2019; Purnami, Anom
Jambe, & Wayan Wisaniyasa, 2018; Suhardini &
Zubaidah, 2016). An antioxidant compound is defined
as a compound that can delay, slow down, and
prevent oxidation processes because it has the
property of being easily oxidized or is a strong
reductor compared to other molecules (Mahardika &
Roanisca, 2018; Simanjutak, 2012). Kombucha tea is
a drink that is known to have antioxidant activity, so
consuming kombucha can have a fairly good effect on
the activity of the intestines, stomach, and glands. The
antioxidant effect on kombucha can also overcome
diabetes, and aging problems, act as a laxative, and is
known to relieve joint rheumatism, gout, and
hemorrhoids (Firdaus, C, Isnaini, & Aminah, 2020;
Goh et al., 2012).
In kombucha to produce a good taste, generally
use black tea because black tea is a type of tea that
has a fragrant scent compared to other types of tea,
but for health purposes, the manufacturer of
kombucha can use other herbal plants that contain
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active substances in the form of natural antioxidants
with higher levels (Naland, 2008). According to Nasir
& Rahmani (2015), the scent and taste quality of a dry
tea is influenced by the content of chemical
compounds in it such as caffeine, tannins, and
essential oils. The protection of several chemical
compounds found in plants so that they have the
potential to become a source of taste, aromatic
compounds, and drugs that are very beneficial for
human health (Fadeyi, Adeniran, & Akiode, 2022).
Kombucha can be made from leaves that have a high
content of polyphenols such as tannins and
flavonoids. One of the plants that have the potential to
be used as a material for making kombucha is star
fruit leaves. The use of wuluh starfruit as a material
for making kombucha has never been reported. Even
star fruit leaves have several chemical compounds
that have the potential to be utilized such as saponins,
tannins, sulfur, formic acid, and phenol compounds,
namely flavonoids. The total flavonoid content in star
fruit leaves is not less than 0,7% (Misfadhila,
Chandra, & Wahyuni, 2020). In addition to
flavonoids, wuluh starfruit leaves also have a high
tannin content of 10,92% compared to green tea of
1,44% and lime leaves of 1,8% (Andriani, Gde
Mayun Permana, & Rai Widarta, 2019).
One of the factors that can affect when making
kombucha is the fermentation time. Generally, the
fermentation time of kombucha is 6-12 days. Based
on research (Nurhayati, Yuwanti, & Urbahillah,
2020), the best treatment with the highest phenolic
levels was obtained at a fermentation time of
Kombucha 8 days. Research (Falahuddin, Apriani, &
Nurfadilah, 2017) about kombucha soursop leaves are
known to be the best treatment by producing the
highest vitamin C value, namely at a fermentation
time of 12 days.
Research about the effect of fermentation time on
kombucha using wuluh starfruit leaves (Avverhoa
bilimbi Linn.) to determine the physical, chemical,
and antioxidant activity characteristics has never been
carried out. Thus, it is necessary to research the effect
of the treatment of long fermentation time on the
kombucha of star fruit leaves (Avverhoa bilimbi
Linn.).
METHODOLOGY
This study used an experimental method with a
completely randomized design (CRD) consisting of 4
treatments with 3 replicates, that is 0, 4, 8, and 12
days of fermentation time at 10% sugar concentration.
Materials and Instrumentals
The ingredients used in this study were wuluh
starfruit leaves (Avverhoa bilimbi Linn.), kombucha
culture starter, black tea, granulated sugar, aquades,
Na2CO3, gallic acid, methanol, ethanol 96%,
Indicators pp, Folin-ciocalteu, DPPH, and NaOH.
The instruments used in this study are glass
containers, clean cloths, rubber bands, autoclaves,
vortexes, ovens, UV-vis spectrophotometers, cameras,
and label paper.
Methods
Tool sterilization
Sterilization of glass containers that have been
covered with aluminum foil is carried out using an
autoclave with a temperature of 121 and a pressure
of 1 atm for 15 minutes.
Preparation of the kombucha starter
Water amount 500 ml boiled then 50 grams of
10% sugar (w/v) is added. After that, 2.5 grams of
0.5% (w/v) black tea is added. Then filtering is
carried out and poured into sterile glass containers.
After the tea has a temperature equal to room
temperature, 50 ml (10% (w/v)) kombucha culture is
added to the brewed black tea. Then cover the
container with a clean cloth then tie it with a rubber
band. Starter propagation of kombucha culture
fermented for 14 days.
Preparation of the wuluh starfruit tea
The wuluh starfruit leaves separated by twigs.
The part used for making tea is young leaves of wuluh
starfruit. After that, the leaves are thoroughly washed
under running water. Then, cut the leaves into small
pieces and drying using the oven at a temperature of
55
Preparation of the wuluh starfruit leaves
kombucha
Wuluh starfruit leaves tea of as much as 12
grams 0.5% (w/v) is brewed using 2400 ml boiling
water. After that, poured 200 ml into each container
and added sugar with as much as 10% (w/v). Then 20
ml of the kombucha liquid starter added, the glass
container is covered with a clean cloth. After that, the
brewed wuluh starfruit leaves tea is fermented for 0,
4, 8, and 12 days.
The test pH analysis of starfruit leaves kombucha
tea
The measurement of pH in kombucha wuluh
starfruit leaves tea samples is carried out by taking
about 50 ml of the kombucha solution and then
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putting it into a glass beaker. Then, the pH is
measured using a universal pH, and the color changes
on the pH paper are matched to the color standard
listed in packaging.
Total levels of titrated acid
Total titrated acid was tested using the principle
of acid titration by base referring to Cahyaningtyas
(2018). The test was taking 10 ml of the sample into a
100 ml volumetric flask and adding distilled water
until the boundary mark. After that, a sample was
filtered. 10 ml of filtrate is taken and put into
erlenmeyer and added indicator pp. After that,
titration is carried out with a solution of NaOH 0.1 N.
Titration is carried out until the solution changes color
to pink. The calculation is carried out by the
following Equation 1.
Total Acid (%)
(1)
V NaOH is volume NaOH for titration, N NaOH is
NaOH standard concentration, Vsample is the volume
of the sample for titrate, and MW is the Molecular
weight of acetic acid
The Phenolic Level Test
Measurement of phenolic levels in kombucha
using the folin ciocalteu method refers to Pourmorad
(2006). The test was carried out by making a standard
solution of gallic acid with 5 concentration variations,
namely 10 ppm, 20 ppm, 30 ppm 40 ppm, and 50
ppm. Then each concentration is taken 1 ml and put
into a test tube. 0.5 ml of folin ciocalteu inserted.
After that, 4 ml of Na2CO3 7% solution was added
and homogenized using a vortex and allowed to stand
for 8 minutes and calculated absorbance with a
wavelength of 760 nm.
Measurements on the sample were carried out by
taking 1 ml of the sample and putting it into a test
tube and giving 0.5 ml of folin ciocalteu. Then 4 ml
of Na2CO3 7% solution is added. It is homogenized
using a vortex and allowed to stand for 8 minutes.
Absorbance is calculated with a wavelength of 760
nm. Phenolic content can be calculated using equality
and regression and using the following Equation 2.
TPC =
(2)
TPC is total phenolic content (mg/g GAE), c is
concentration (value x) (ppm), v is extract volume
(ml), fp is Dilution factor, g is sample weight (grams)
Alcohol test
The measurement of alcohol content using the
Skoog (1985) by taking 25 ml of wuluh starfruit
leaves kombucha solution. Then, the neutralization of
the solution is carried out using sodium hydroxide
(NaOH) 3 N. followed by the distillation process.
Then the distillation results of 25 ml are put into a 25
ml pycnometer equipped with a thermometer. Before
the treatment of pycnometer and thermometer tools
are weighed first. After that, the pycnometer is put in
cold water until the temperature has reached 28.
Then, the pycnometer weighed the weight again, and
the calculation of specific gravity can be done by the
following Equation 3 (Bp is pycnometer weight, and)
BM is molecular weight).
.
( 3)
Antioxidant activity test
Antioxidant activity was tested using the DPPH
method referring to Hassmy et al. (2017). In testing
the antioxidant activity added 1.1 ml of DPPH
solution (10 mg/L) and 2 ml of Ethanol. Then 4 ml of
kombucha sample solution was added with variations
in concentrations of 50 ppm, 70 ppm, and 110 ppm.
Then incubation is carried out for 30 minutes. Then
the absorbance was measured at a wavelength of 516
nm. DPPH absorption inhibition calculation is as
follows Equation 4 (A0 is Absorbance control (DPPH
+Aquades), and As and Sample absorbance and
DPPH).
% Inhibition
(4)
The determination of IC50 is determined by
plotting the percentage of inhibition obtained into the
regression equation y=ax + b where x is the sample
volume and y is the value of % inhibition.
Organoleptic test
The process of collecting data on organoleptic
tests was carried out using hedonic tests of scent,
color, and taste conducted by 15 panelists. The
hedonic scale ranges from 1-4 where 1: dislike, 2:
somewhat like, 3: like, and 4: very like. Testing was
carried out by pouring kombucha wuluh starfruit
leaves as many as 3 samples on a container that has
been coded to mark the identity of the kombucha tea
according to the fermentation time and sugar
concentration.
This section must also be able to provide
complete procedural information so that it can be
carried out by other researchers who will be returning.
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All procedures that have been used must use
international standard units and terms that are
commonly used in scientific research.
Data Analysis
Research data including pH, total levels of
titrated acid (tat), phenolic and organoleptic
assessment were carried out non-parametric tests with
a value of α <0.05. If there are differences, continue
with the Mann Whitney test to find out the differences
between treatments. Qualitative data, including
alcohol content and antioxidant activity were
analyzed descriptively.
RESULTS AND DISCUSSION
Characteristics of Physics: Organoleptic Test
The physical characteristics test on kombucha tea
was known by conducting hedonic tests, including
scent, color, and taste on 15 panelists. Hedonic tests
are carried out to determine the acceptance of
products in the community as drinks (Nainggolan,
2009). The results of organoleptic test research can be
seen in Table 1
Table 1. The kombucha organoleptic test results
Note: A (fermentation time 4 days), B (fermentation time 8
days), C (fermentation time 12 days). Similar letter
notation means there is no real difference in Mann
Whitney's test
The results of the scent test statistically with the
kruskal wallis test were found to have no significant
differences. The results of the hedonic scent test in
Table 1 the longer the fermentation time will also
experience an increase in the hedonic value of the
scent. Wuluh starfruit leaf kombucha tea has a strong
sour scent and is quite pungent. The sour scent
formed during the fermentation period is caused by
the fermentation process of alcohol and acetic acid
which causes the formation of volatile compounds
that can be felt by the human sense of smell
(Nurhidayah, 2018). Color is one of the factors of
consumer acceptance of a product because it uses the
sense of sight (Leal, Suárez, Jayabalan, Oros, &
Escalante-Aburto, 2018; Rosita, Handito, & Amaro,
2021). The results color of wuluh starfruit leaf
kombucha can be seen in Figure 1.
The results of the color test statistically with the
kruskal wallis test were found to have no significant
differences. The results of the color hedonic test in
Table 1 the longer the fermentation time will make
the hedonic value of the color increase. Wuluh
starfruit leaf kombucha tea has a faded color from
brownish yellow to clear yellow. According to
(Pratiwi & Aryawati, 2012) the color fading in
kombucha tea is caused by a change in pH to acid so
that more fermentation time results in the
decomposition of the components in the solution,
namely tannins which are included in phenol
compounds are damaged due to the presence of acids
so that the concentration is reduced.
Figure 1. The result of wuluh starfruit leaves
kombucha tea with fermentation time treatment A
(4 days), B (8 days), and C (12 days)
The results of the taste test statistically with the
Kruskal wallis test are known to have significant
differences. The results of the color hedonic test in
Table 1 the longer the fermentation time will also
experience an increase in the hedonic value of the
taste. The hedonic value of the most preferred taste is
treatment C (fermentation time 12 days). Kombucha
wuluh starfruit leaves wuluh treatment C has a
slightly sweet and alcoholic sour taste. According to
Nasir and Rahmani (2015) fermentation that is carried
out long enough will make the sweetness of
kombucha decrease because the existing sugar has
been fermented. An overhaul of sugar during
fermentation by fermentative microorganisms occurs
by changing its chemical composition to acids,
alcohols, and CO2.
Chemical characteristics
This research was conducted to determine the
effect of fermentation time on the chemical
characteristics of kombucha wuluh starfruit leaves tea
(Avverhoa bilimbi Linn.). Kombucha wuluh starfruit
Treatme
nt
Organoleptic Test and Standard deviation
Scent
Color
Taste
A
1.870.834
2.470.8
34
2.330.816
B
2.201.014
2.670.9
76
3.071.033
C
2.271.100
2.600.9
86
3.130.990
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tea has been observed parameters include pH value,
total levels of total titrated acid (TTA), phenolic, and
alcohol during the fermentation period of 0 days, 4
days, 8 days, and 12 days. The results of the kruskall
wallis test on chemical characteristics are known to
show differences in pH values, TTA levels, and
phenolic levels of kombucha tea of star fruit leaves
(Avverhoa bilimbi Linn.). The results of the chemical
characteristics test can be seen in the following Table
2 (Note: P1 (fermentation time 0 days), P2
(fermentation time 4 days), P3 (fermentation time 8
days), P4 (fermentation time 12 days)).
Table 2. The kombucha chemical characteristics test
results
*Similar letter notation means there is no real
difference in Mann Whitney's test
The results of the Mann Whitney test at pH in
Table 2 are shown by assigning different letters. The
letter (a) notation indicates that the treatment of P1 is
significantly different from P2, P3 and P4. The letter
(b) notation indicates that P2 treatment is significantly
different from P1, P3 and P4 treatments. The letter c
notation indicates that the P3 treatment is
significantly different from P1 and P2 but not
significantly different from the P4 treatment.
The pH test results in Table 2 can be seen that
the longer the fermentation time, the more the pH
value of kombucha decreases. The decrease in pH
occurs because during fermentation there are growth
and metabolic processes of acetic acid bacteria, lactic
acid, and yeast which can increase organic acids
(Jayabalan, Malbaša, Lončar, Vitas, & Sathishkumar,
2014). According to Hapsari et al. (2021), the pH
value in the fermentation process of kombucha is one
of the most important environmental parameters
because it is formed from several acid compounds
such as acetic acid and gluconic. The acidity of
kombucha drinks that are safe for consumption ranges
from a pH value of 2.5-4.6 (Naland, 2008).
The effect of acidity estimation on taste and
aroma in addition to using pH can be done with a
TTA test. While the pH value only measure the total
acid under dissociated conditions. It can be concluded
that the measurement of TTA is better than pH
(Angelia, 2017). The results of the Mann Whitney test
at TTA in Table 2 shown by letter notation are known
that the letter a notation shows that the treatment on
P1 is significantly different from P2, P3 and P4. The
letter (bc) notation indicates that P2 is significantly
different from P1 and not significantly different from
P3 and P4.
The results of the tat test in Table 2 are known
that there is an increase in Total Titrated Acid (TTA)
during the fermentation time take place. The highest
total titrated acid in wuluh starfruit leaf kombucha
was in the P4 treatment (fermentation time of 12
days) of 0.1100,070%. This is comparable to the
research that has been carried out by Pratiwi and
Aryawati (2012) that there is an increase in total acid
levels during the fermentation time in the seaweed
kombucha Saggarsum sp. The increase in total acid
levels is suspected to occur due to sugar in kombucha
which will be overhauled in the fermentation process
by bacteria and yeast into organic acids.
The results of the Mann Whitney test on phenolic
levels in Table 2 shown by letter notation are known
that the letter (a) notation indicates that the P1
treatment is significantly different from P3 and P4
treatment, but not significantly different from P2
treatment. The letter (ab) notation indicates that the
treatment of P2 is significantly different from P4 but
not significantly different from P1 and P3. The (bc)
notation indicates that the treatment of P3
significantly different from P1 but not significantly
different from P2 and P4. The letter (c) notation
indicates that the P4 treatment is significantly
different from the P1 and P2 treatments, but not
significantly different from the P3 treatment.
Based on the phenolic test result in Table 2, it is
known that with the longer fermentation time in
kombucha wuluh starfruit leaves tea, it is known that
phenolic levels increase until the 8th day of
fermentation and will decrease on the 12th day.
According to Suhardini and Zubaidah (2016), the
increase in phenolic levels is suspected to be due to
microbes of the bacterial and yeast groups that can
metabolize to produce flavonoid compounds through
enzymatic reactions, these conditions affect the total
amount of phenols in kombucha tea. According to
Ardheniati et al. (2009), the fermentation process
causes polyphenol content to decrease due to the
oxidation reaction. Decreased phenolic levels are also
Treat
ment
pH
SD
TTA (%)
SD
Phenolic
Levels (mg/ml
GAE) SD
Alcohol
(%)
P1
6a
0.010
0.001a
58.67
2.663a
0.11
P2
4b
0.018
0.025bc
61
4.52106ab
0.21
P3
3c
0.05
0.025bc
92.53
1.363bc
0.82
P4
3c
0.11
0.070bc
87.33
1.140c
0.41
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associated with a decrease in the number of microbial
cells because reduced sugar as an energy source in
microbes also decreases.
The fermentation process of kombucha is also
subject to oxidative chemical changes by
microorganisms within the substrate. As a result, the
microorganisms dissolve in the form of a more
complex compound and enzymes convert the sugars
into alcohols (Herwin, Kosman, & Siami, 2013). The
results of the alcohol content test of wuluh starfruit
leaf kombucha tea in Table 2 the longer fermentation
time causes alcohol to increase. However, the alcohol
content will decrease on the 12th day of fermentation.
The increase in alcohol content is caused during the
fermentation process of the yeast Saccharomyces
cerevisiae produces alcohol anaerobically, and then
alcohol stimulates the growth of Acetobacter xylinum
to produce acetic acid aerobically, while acetic acid
will stimulate the growth of Saccharomyces
cerevisiae. Then alcohol will be used by Acetobacter
bacteria for the formation of acetic acid. So kombucha
tea has decreased alcohol levels (Pratiwi & Aryawati,
2012).
Antioxidant Activity
Antioxidant activity can be determined by a
value of IC50. The results of measuring the IC50 are
known that all treatments produced kombucha tea
with very strong antioxidants. In the antioxidant
activity test, wuluh starfruit leaves kombucha tea was
analyzed descriptively. The results of the antioxidant
activity of wuluh starfruit leave kombucha tea are
presented in Table 3.
Table 3. IC50 Value Result
Treatment
IC50 (ppm)
Information
P1
2.68
Very Strong
P2
4.26
Very Strong
P3
3.77
Very Strong
P4
3.65
Very Strong
Antioxidants are defined as compounds that have
the role of delaying, slowing down, and preventing
oxidation processes (Simanjutak, 2012). This study
was tested for antioxidant activity by determining the
IC50 value. In this study, it was known that there was a
relationship between phenolic and Tat levels to
antioxidant activity in wuluh starfruit kombucha. It is
known that higher the phenolic and Tat levels make
the IC50 values low which indicates that the activity
on kombucha is getting stronger during the
fermentation time. According to (Rustiah & Umriani,
2018) phenol content has a positive linear relationship
with the content of antioxidant activity. Because
phenol compounds such as phenolic acid are natural
antioxidants in a plant. This is related to the
Kombucha starter which produces several enzymes
such as proteases, lipases, and polyphenol oxidase
which can catalyze the biodegradation of the flavin
and hydroxylates which are powerful antioxidants
(Chu & Chen, 2006).
The IC50 value is the concentration of the extract
needed to dampen 50% of the total DPPH. The results
of the IC50 value test in Table 3 are known that the
antioxidant activity in wuluh starfruit leaf kombucha
tea has an IC50 value <50 so it can be said that in all
treatments it produces strong antioxidant compounds.
According to Tristantini et al. (2016), a compound is
said to be a very strong antioxidant if the IC50 value is
less than 50, strong (50-100), and weak (151-200). So
it can be concluded that the smaller the IC50 value, the
higher the antioxidant activity. In the figure, it is
known that at the fermentation time of the 4th to the
8th day, it is known that there is a decrease in
antioxidant activity marked by an increase in the
value of IC50. According to Hunandar (2017), the
decrease in antioxidant activity can be caused
because, during the fermentation process, new phenol
compounds have been formed, but these compounds
cannot inhibit free radicals. This happens because
polyphenol compounds such as catechins are lost and
further polymerized into higher molecular weight
molecules leading to the detection of lower
polyphenol content. At the time of fermentation on
the 12th day, it is known that the antioxidant activity
gradually increases again. The increase in antioxidant
activity is due to the biotransformation process of
several phenol compounds due to the presence of
enzymes produced during the kombucha fermentation
process and the release of catechins from
microorganisms that are sensitive to acids (Hunandar,
2017). It can be concluded that the longer the
fermentation of the IC50 value will reach the optimum
point then decrease.
CONCLUSION
Fermentation time affects the physical, and
chemical characteristics and antioxidant activity of
wuluh starfruit leaf kombucha tea. Wuluh starfruit
leaf kombucha tea which has the best physical, and
chemical characteristics and antioxidant activity is in
the P4 treatment (fermentation time 12 days) with a
hedonic value of scent is 2.27 with color
value is 2.60, taste value is 3.13, pH value is 3, the
total titrated acid level is 0.1100.070%, the phenolic
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level is 87.331.140 mg/ml GAE, alcohol is 0.41%,
and IC50 value is 3.65 ppm.
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