Journal of Applied Food Technology 9 (1) 5–10

Journal of Applied Food Technology

Hom e page : https://e journal2.undip.ac .id/index.php/j af t

Effect of Fermentation Time on Chemical, Microbiological and Hedonic

Quality of Kombucha of Arabica Coffee Cascara (Coffea arabica L.)
Adrina Eka Shafira*, Antonius Hintono, Siti Susanti

Department of Food Technology, Faculty of Animal and Agricultural Sciences, Diponegoro University, Semarang,
Indonesia

*Corresponding author ([email protected])

Article information:
Abstrac Received: 1 July 2020
This research aims to determine the effect of different fermentation times Accepted: 17 June 2022
on the chemical, microbiological and hedonic quality of Kombucha of Arabica Available online: 30 June 2022
Coffee Cascara. The material used in this study is Arabica Coffee Cascara as
Keywords:
a raw material for making kombucha with different fermentation treatment. The Bacteria
research method used was a Completely Randomized Design (CRD) with 4 Cascara
treatments and 5 repetitions. Fermentation time variations carried out are 2, 4, Fermentation
6 and 8 days. Data from the testing of chemical and microbiological properties Kombucha
were analyzed using Analysis of Variance (ANOVA) with a 95% confidence Yeast
level and if there is a real influence then proceed with the Duncan Multiple
Range Test (DMRT). Data from the hedonic quality test results were analyzed © 2022
using Kruskal Wallis nonparametric test with a confidence level of 95% and if Indonesian Food Technologists
All rights reserved.
there was a real influence, then proceed with the Mann-Whitney test. The
results showed that the different fermentation time of Kombucha of Arabica This is an open access article
Coffee Cascara had a significant effect (p <0.05) on total acid, total sugar, total under the CC BY-NC-ND
alcohol, total microbes, and hedonic quality of the product. The longer the license.
fermentation lasts, the total acid and total alcohol will increase while the total
sugar, total microbes and antioxidant activity will decrease. Kombucha of doi: 10.17728/jaft.8399
Arabica Coffee Cascara with 8 days fermentation time is the best treatment
product because it meets the quality standards and is preferred by panelists.

Introduction al., 2018). SCOBY can grow in environments that contain

Kombucha is a traditional beverage made by a lot of sugar as a carbon source (Puspitasari et al.,
fermenting a tea solution with sugar using a Kombucha 2017). The type of sugar that is consumed by many
starter for several days (Wistiana and Zubaidah, 2015). people is High Fructose Syrup (HFS). HFS is a liquid
The length of time of fermentation can affect the quality sweetener derived from cassava with an enzymatic
of Kombucha (Puspitasari et al., 2017). Kombucha process that has a high sweetness level, does not form
fermentation can last for 7 to 14 days (Susilowati, 2013). crystals, and has a high level of solubility (Prastiwi et al.,
Chemical content contained in Kombucha, including 2018). HFS has a sweetness level 1.8 times higher than
vitamins B1, B2, B3, B6, B12, B15, vitamin C, folic acid, sucrose and has a low calorie of 3.9 calories/gram
glucuronic acid, acetic acid, lactic acid, amino acids, (Qonitah et al., 2016).
enzymes, and antibiotic compounds (Naland, 2004). Kombucha can be made from various types of tea,
Kombucha is very beneficial for the human body one of them is the Arabica cascara tea derived from the
because it can facilitate digestion, as an antibiotic, Arabica coffee cherry skin. Cascara is a product derived
antioxidant and antibacterial. The various benefits of from the outer skin of coffee cherries which is dried and
Kombucha make Kombucha known as a health drink and processed in such a way that it can be brewed as it is
popular in China, Russia, Germany, and the United brewing tea in general (Sastra and Bawono, 2018).
States (Dipti et al., 2003). Cascara which has been brewed into tea has a
SCOBY or Symbiotic Culture of Bacteria and distinctive sour taste and aroma like fruits (Nafisah and
Yeast is a symbiotic starter between bacteria and yeast Widyaningsih, 2019). The distinctive sour taste is the
used in the fermentation of kombucha tea (Mousavi et effect of the content of chlorogenic acid and caffeic acid

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 Journal of Applied Food Technology 9 (1) 5–10

in cascara (Rohdiana, 1999). Fresh cascara generally total microbes and hedonic tests.
contains crude protein, crude fiber, tannin, caffeine,
lignin, fat, ash, calcium, phosphorus, and several Arabica Coffee Cascara Tea Making
secondary metabolite compounds, such as the The making of The cascara tea method referred to
polyphenol group (Sumihati et al., 2011). Polyphenol the method of Yuwanti et al. (2018) with modification.
compounds contained in Cascara are flavan-3-ol, 32.5 grams (5%) of Arabica Coffee Cascara and 650 mL
hydroxynamic acid, flavanols, anthocyanin, catechins, of water was heated at 90°C for 5 minutes until it forms
tannins and ferulic acids (Esquivel and Jimenez, 2012). an Arabica Coffee Cascara tea. The tea was filtered to
Cascara also contains vitamins C and E which are good separate the pulp and then put it into a container/bottle
for human skin (Nuraini et al., 2019). This research aims glass. The Arabica Coffee Cascara tea was cooled to
to determine the effect of different fermentation times room temperature.
against the chemical, microbiological and hedonic quality
of Kombucha of Arabica Coffee Cascara. Kombucha of Arabica Coffee Cascara Making
The making of Kombucha of Arabica Coffee
Materials and methods Cascara method referred to the method of Nurhayati et
Materials al. (2018) with modification. Arabica Coffee Cascara tea
The ingredients used are Arabica Coffee Cascara, was mixed with 5.5% (v/v) of High Fructose Syrup and
starter SCOBY (Symbiotic Culture of Bacteria and put into a sterile glass container, then added a starter
Yeast), High Fructose Syrup, NaOH 0.1 N, PP indicators, SCOBY and Kombucha vinegar until the solution
PCA (Plate Count Agar), distilled water, physiological reached pH 4. The glass container was closed using
NaCl 0.85%, methanol and DPPH reagents. The tools plastic and sealed using a rubber band. The tea was
used are beaker, measuring cup, analytical scales, drop incubated at room temperature with a treatment duration
pipettes, volume pipettes, test tubes, thermometers, of fermentation for 2, 4, 6 and 8 days. The fermented tea
vortices, Erlenmeyer, burettes, Kjeldahl distillation was filtered to separate Nata (secondary product) from
flasks, pycnometers, scales, autoclaves, petri dishes, the Kombucha of Arabica Coffee Cascara. Kombucha of
stoves, thermometers, refractometers, and Arabica Coffee Cascara was pasteurized at 70°C for 10
spectrophotometers. minutes.

Methods Total Acid Analysis

The study was conducted in December 2019 to The total acetic acid analysis was carried out using
January 2020 in the Laboratory of Food and Agricultural a method that refers to Leasa and Matdoan (2015) using
Products Engineering, and the Laboratory of Chemistry titration. 10 mL of Kombucha samples were put into
and Food Nutrition, Faculty of Animal and Agricultural Erlenmeyer, then diluted to 250 mL and added 2 drops
Sciences and Integrated Laboratory of Diponegoro of the phenolphthalein (PP) indicator. Samples were
University, Semarang. The analysis included total acid titrated using 0.1 N NaOH to form a constant pink color.
analysis, total sugar, total alcohol, antioxidant activity, The total acid was calculated using Equation
ml NaOH 𝑥𝑥 N NaOH 𝑥𝑥 BM Acetic Acid
Total Acid =
mL sample
𝑥𝑥 100% Equation 1
Analysis of Total Alcohol put into a pycnometer that has been weighed before. The
The total alcohol analysis was carried out using a pycnometer contained distillate was weighed and their
method that refers to AOAC (2000) using the distillation weight is recorded. The same procedure was carried out
method. 100 ml of Kombucha samples were put into the on aquadest as a comparison. The results of the alcohol
Kjeldahl distillation flask and then added to distilled water density calculation converted using the alcohol
as much as 100 mL, then distilled at 80°C. The distillate conversion table. The alcohol density was calculated
was stored in an Erlenmeyer to a volume of 50 mL, then using Equation 2.
(picnometer weight + distillate)− empty picnometer weight
Alcohol Density = (picnometer
weight+aquadest)− empty picnometer weight
Equation 2

Analysis of Total Sugar media. As much as 1 mL of Kombucha sample was put

Total sugar analysis was carried out using a into a test tube containing 9 mL of physiological NaCl
method that refers to Surtinah (2007) using a Hand 0.85% (dilution 10-1). Next, 1 mL of dilution 10-1 was put
Refractometer. A hand refractometer must be cleaned into a test tube containing 9 mL physiological NaCl
using distilled water and dry tissue before use. As much 0.85% (dilution 10-2). The same step was repeated until
as 1 drop of the Kombucha sample was dropped to the dilution 10-6. At the last 3 dilutions, as much as 1 ml of
lens then tightly closed (make sure there are no air the last 3 dilutions was put into a petri dish that has been
bubbles was formed) and then looked at the device. A sterilized, then added with sterile PCA media and mixed
dark and light boundary scale was set so the total sugar well. The plates were incubated at 37°C for 2 x 24 hours.
of the product could be seen. The total sugar figure was
right on the boundary line of the hand refractometer. Analysis of Antioxidant Activity
The antioxidant activity analysis was carried out
Analysis Total Microbe using a method that refers to Yuwanti et al. (2018) using
The total microbial analysis was carried out using DPPH. As much as 0.1 mL of Kombucha sample was
a method that refers to Wati (2018) using the Total Plate prepared in a test tube then added 2.95 mL of methanol
Count (TPC) method with PCA (Plate Count Agar) and 3 mL of 300 µm DPPH solution and then

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homogenized using a vortex. The homogeneous wavelength of 517 nm. Blanks were made using
samples were leavened for 30 minutes. The absorbance methanol. The antioxidant levels were calculated using
value was measured using a spectrophotometer at a Equation 3.

Absorbance blanks − Absorbance samples

𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 (%) =
Absorbance blanks
𝑥𝑥 100% Equation 3

Hedonic Quality Test Range Test (DMRT) to determine the differences

The hedonic quality test was carried out using a between the treatments given. The hedonic test data
method that refers to Permata and Sayuti (2016) which were analyzed using the Kruskal Wallis nonparametric
includes aroma, color, sour taste, and overall preference. test with a 95% confidence level, then followed by the
The Kombucha samples were tested on 25 semi-trained Mann-Whitney Test. The entire analysis process was
panelists. The hedonic quality analysis was carried out carried out using SPSS 22.0 for Windows application.
that samples prepared in plastic cups and then given a
three-digit code. Each plastic cup was filled with 15 mL Results and Discussion
samples of Kombucha of Arabica Coffee Cascara. Total Acid
Panelists were asked to give a favorite score using 5 Based on the test results presented in Table 1, the
likeness scales, namely a score of 1 (Dislike), a score of treatment of different fermentation time on making
2 (Somewhat Dislike), a score of 3 (Ordinary), a score of Kombucha of Arabica Coffee Cascara had a significant
4 (Somewhat Like) and a score of 5 (Like). effect (p<0.05) on the total acid value of the product. The
longer fermentation of Kombucha made an increase in
Data Analysis total acid value. This is consistent with the opinion of
The data of total acid, total alcohol, total sugar, Simanjuntak et al. (2016) which states that the long
total microbes, and antioxidant activity were analyzed period of Kombucha fermentation can cause a decrease
using Analysis of Variance (ANOVA) with a 95% in acidity and an increase in the total acid of the product.
confidence level, then followed by Duncan Multiple

Table 1. Total Acid, Total Sugar, Total Alcohol, Total Microbes and Antioxidant Activity of
Kombucha of Arabica Coffee Cascara
Treatments
Parameters
2 days 4 days 6 days 8 days
Total Acid (%) 0.20 ± 0.02a 0.21 ± 0.01a 0.24 ± 0.03a 0.29 ± 0.04b
Total Sugar (°brix) 6.80 ± 0.28b 6.52 ± 0.48b 6.28 ± 0.33ab 5.92 ± 0.43a
Total Alcohol (%) 0.74 ± 0.37a 0.91 ± 0.39a 1.21 ± 0.43a 2.59 ± 0.36b
Total Microbes (104 CFU/ml) 12.00 ± 7.58b 5.80 ± 3.65a 3.60 ± 2.10a 2.22 ± 1.66a
Antioxidant Activity (%) 89.55 ± 0.43 89.00± 0.63 88.63 ± 0.61 87.01 ± 2.67
Notes: Numbers followed by different superscripts show significant effects (p<0.05).

The increase of total acid value in Kombucha of the increase in fermentation time. The decrease in the
Arabica Coffee Cascara was caused by an increase in total sugar of Kombucha of Arabica Coffee Cascara was
the number of organic acids due to the metabolic process caused by using sugar as the main nutrient for microbes.
of the starter SCOBY consisting of various bacteria and This is supported by the opinion of Puspitasari et al.
yeast with sugar. This is supported by the opinion of (2017) which states that the sugar content in Kombucha
Ayuratri and Kusnadi (2018) which states that the serves as a carbon source of microbes that can be used
sucrose contained in Kombucha will run into a recast to grow and metabolism. Sugar used by bacteria and
carried out by bacteria and yeast and will then be yeast in the SCOBY starter to metabolize and convert to
changed to organic acids such as acetic acid and organic acids and alcohol. This is supported by the
gluconic acid. The formed acetic acid would be used to opinion of Suhardini and Zubaidah (2016) which states
stimulate the growth of yeast Saccharomyces cerevisiae that the level of sugar in Kombucha which is shrinking is
which could metabolize and produce alcohol. This is caused by the activity of yeast which remodels sugar to
consistent with the opinion of Nur et al. (2018) which be converted into alcohol as well as the activity of
states that Saccharomyces cerevisiae can be stimulated bacteria which remodels glucose into gluconic acid.
by growth due to the intake of acetic acid produced by
the bacterium Acetobacter xylinum during aerobic Total Alcohol
fermentation. Based on the test results presented in Table 1, the
different fermentation length treatment in making
Total Sugar Kombucha of Arabica Coffee Cascara had a significant
Based on the test results presented in Table 1, the effect (p<0.05) on the total alcohol value of the product.
different fermentation length treatment in making The longer fermentation of Kombucha made an increase
Kombucha of Arabica Coffee Cascara had a significant in total alcohol value. This is consistent with the opinion
effect (p<0.05) on the total sugar value of the product. of Simanjuntak et al. (2016) which states that the total
The longer fermentation of Kombucha caused a value of alcohol contained in Kombucha products will
decrease in total sugar value. This is consistent with the increase with increasing fermentation time. The increase
opinion of Simanjuntak et al. (2016) which states that the in the total alcohol of Kombucha of Arabica Coffee
total sugar content of Kombucha will decrease along with Cascara was caused by the metabolic processes carried

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out by yeast on the SCOBY starter which converts sugar to release and the antioxidant activity of kombucha
into alcohol. This is in accordance with the opinion of decreases.
Insani et al. (2018) which states that yeast run in a
fermentation process will metabolize and remodels Hedonic Test of Aroma
glucose into alcohol. Based on the test results presented in Table 2, the
difference in fermentation time treatment had no
Total Microbes significant effect (p> 0.05) on the aroma of Kombucha of
Based on the test results presented in Table 1, the Arabica Coffee Cascara. The results of the panelist score
different fermentation length treatments in the making of on the aroma attributes ranged from 3.20 to 3.32, which
Kombucha of Arabica Coffee Cascara had a significant was included in the Normal (neutral) category. The
effect (p<0.05) on the total product microbes. The longer longer the fermentation lasts, the stronger the aroma
fermentation of Kombucha caused a decrease in the total becomes. This is in accordance with the opinion of
microbes. This is consistent with the opinion of Wistiana Mahadi et al. (2016) which states that the fermentation
and Zubaidah (2015) which states that the longer the process that lasts longer can affect the aroma of
fermentation time in Kombucha, the rate of microbial Kombucha becoming more acidic. The aroma of the acid
growth inhibited, and the amount decreased. A decrease was thought to cause the panelists to score "normal"
in the rate of microbial growth in Kombucha of Arabica because the panelists are not yet familiar with the scent
Coffee Cascara was caused by a decrease in the amount of Kombucha. The aroma of acid in Kombucha of Arabica
of sugar which is the main nutrient for microbes. This is Coffee Cascara was thought to originate from the content
supported by the opinion of Ayuratri and Kusnadi (2017) of organic acids and volatile alcohol. This is consistent
which states that along with the increase in fermentation with the opinion of Purnami et al (2018) which states that
time, sugar as a source of carbon for microbes will Kombucha has a distinctive sour aroma derived from the
decrease in number due to the recast process by yeast content of volatile compounds, such as acetic acid,
to become organic acids so that microbes lose their alcohol, and other organic acids. The aroma of cascara
source of nutrients to grow and the amount decreases. as a raw material for Kombucha also influenced the
Some sugar, which was the main nutrient for bacteria result of the Kombucha aroma. Guntoro et al. (2019)
and yeast during the fermentation process can also be expressed the opinion that the coffee cherry or cascara
turned into an inhibitor because it has been overhauled skin has a strong and distinctive aroma of fruit.
into organic acids and alcohol which contribute to the
decrease in total microbes. This is consistent with the Color Hedonic
opinion of Goh et al. (2012) which states that the sugar Based on the test results presented in Table 2, the
content in Kombucha has the potential to be an inhibitor difference in fermentation time treatment had a
for microorganisms because sugar that has been turned significant effect (p<0.05) on the color preference of
into organic acids and alcohol can reduce the ability of Kombucha of Arabica Coffee Cascara. Panelists
microorganisms to metabolize. commented on the brown color seen in Kombucha. The
color of Kombucha of Arabica Coffee Cascara was
Antioxidant Activity thought to come up due to the content of the polyphenol
Based on the test results presented in Table 1, the compound in cascara which was the raw material for the
different fermentation length treatments in making product. This is in accordance with the opinion of
Kombucha of Arabica Coffee Cascara did not show any Yuwanti et al. (2018) which states that cascara contains
significant effect (p>0.05) on the antioxidant activity of several polyphenol compounds, one of them was
the product. The longer fermentation of Kombucha anthocyanin which can give a color profile to the results
caused a decrease in the antioxidant activity. This is of steeping. The brown color in Kombucha was thought
supported by the opinion of Kusuma and Fibrianto (2019) to be due to the content of catechin compounds in
which states that the antioxidant activity in Kombucha will cascara which is easily oxidized to produce theaflavin
tend to decrease with increasing fermentation time. A and thearubigin compounds as a determinant of steeping
decrease in the antioxidant activity of Kombucha of color. This is supported by the opinion of Towaha (2013)
Arabica Coffee Cascara was caused by a decrease in which states that catechin compounds are compounds
acidity (pH) so that the solution has a more acidic that are easily oxidized and produce theaflavin and
atmosphere. Hassmy et al. (2017) states that acidic pH thearubigin compounds which can affect the color yield
values can cause phenolic compounds to become more of steeping tea products.
stable so that protons that can bind to DPPH are difficult
Table 2. Scores of Hedonic Properties of Kombucha from Arabica Coffee Cascara
Treatments
Hedonic Attributes
2 days 4 days 6 days 8 days
Aroma 3.28 ± 0.84 3.20 ± 0.64 3.24 ± 1.16 3.32 ± 0.98
Color 3.72 ± 0.73ab 3.56 ± 0.58b 4.08 ± 0.81a 3.36 ± 0.70b
Sour Taste 2.96 ± 1.01 a 3.88 ± 0.92 b 3.60 ± 1.11 b 3.36 ± 0.99ab
Overall 3.24 ± 0.83ac 3.68 ± 0.55a 3.68 ± 0.90ab 3.00 ± 0.64c
Notes: Numbers followed by different superscripts show significant effects (p<0.05).

Sour Taste Hedonic difference in fermentation time treatment had a

Based on the test results presented in Table 2, the significant effect (p<0.05) on the taste of Kombucha of
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Arabica Coffee Cascara. Kombucha of Arabica Coffee (kombucha): effects of sucrose concentration and
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