Antifungal Activity of Kombucha Tea against Human Pathogenic Fungi (Accepted)

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Vol 9, Issue 5, 2016
Online - 2455-3891
Print - 0974-2441
ANTIFUNGAL ACTIVITY OF KOMBUCHA TEA AGAINST HUMAN PATHOGENIC FUNGI
ARI YUNIARTO1*, KUSNANDAR ANGGADIREDJA2, RIANA ANNISA NUR AQIDAH1
1Department of Pharmacology Research Group, Pharmacology Research Group, Bandung School of Pharmacy, Jl. Soekarno – Hatta No. 754,
Bandung, West Java, Indonesia. 2Department of Pharmacology Research Group, Pharmacology and Clinical Pharmacy Research Group,
School of Pharmacy, Bandung Institute of Technology (ITB), Jl. Ganeca 10, Bandung, West Java, Indonesia. Email: ariyuniarto@yahoo.co.id
Received: 11 June 2016, Revised and Accepted: 16 June 2016
ABSTRACT
Objective: In general, infectious diseases still known as a major contributor to the number of morbidity and mortality in the worldwide beside
other diseases. Infections are ordinarily caused by viruses, bacteria, and fungi. Several human pathogenic fungi involving Aspergillus flavus, Candida
albicans, and Microsporum gypseum have responsible against infectious diseases. The objective of the research was to evaluate the antifungal activity
of kombucha tea against human pathogenic fungi such as A. flavus, C. albicans, and M. gypseum.
Methods: The antifungal activity of the kombucha tea against A. flavus, C. albicans, and M. gypseum were tested by disc diffusion method with duration
of fermentation in 6, 12, and 18 days.
Results: Based on the present study, inhibitory diameter of A. flavus, C. albicans, and M. gypseum are 16.83, 15.36, and 25.06 mm, respectively. The
inhibitory diameter was obtained from kombucha tea activity with duration of fermentation in 6 days.
Conclusion: The results provide support for the use of kombucha tea as a potential antifungal source against human pathogenic fungi involving
A. flavus, C. albicans, and M. gypseum.
Keywords: Antifungal, Kombucha tea, Pathogenic, Fungi.
INTRODUCTION
Infectious diseases still known as a leading problem occurred in
several countries. Infections are the invasion mechanism of the host
by the microorganism. Infections are ordinarily caused by fungi,
bacteria, and viruses [1]. In fact, infectious diseases still ranked as
the major contributor to the number of morbidity and mortality in
the worldwide including in Indonesia. Typical of fungal infections
which take place in Indonesia are triggered by various factors. They
are tropical climate conditions, which maintain the fungal growth,
poor personal sanitation, and hygiene, and inadequate immunity
(immunocompromised patients such as AIDS, cancer diseases, or
organ transplantation) [2-4].
Pathogenic fungi, which predominantly attack humans, are
Microsporum gypseum that provokes skin disease; Candida albicans that
induce candidiasis, and Aspergillus flavus that leads to Aspergillosis.
The value of incident of fungal infections triggered by Microsporum has
reached 1.5 billion cases per year as well as the sickness which caused
by C. albicans and A. flavus that count 9.5 million and 1 million cases per
year, respectively [5].
Meanwhile, fungal infections management can be executed in both non-
pharmacological and pharmacological treatment. Non-pharmacological
treatment can be completed by maintaining personal hygiene and
protect the immune system. At the same time, pharmacological
treatment can be executed by administering proper antifungal drugs
based on the cause of the infecting fungi. The use of antifungal drugs
which incorrectly give contributions for the resistance of the fungi.
Therefore, it is required to develop potential antifungal that effectively
eradicate the fungi such as kombucha tea.
Kombucha is a beverage of a fermented tea consumed traditionally
in many countries and have several benefits. Kombucha obtained by
fermenting sweetened tea and tea fungus. It is recognized as symbiotic
culture of bacteria and yeast which forms as gelatin [6-8]. Tea fungus
is a symbiotic culture of acetic acid bacteria (Acetobacter aceti,
Acetobacter pasteurianus, and Gluconobacter oxydans) and yeasts
(Saccharomyces sp., Zygosaccharomyces kombuchaensis, and
Torulopsis sp.) [9]. This jelly form has brownish-white, springy
texture, and immense water content. This symbiosis results acid and
alcohol that interfere the advancement of other microorganisms that
are inhibited from the kombucha tea. The objective of the present
study was to evaluate the antifungal activity of kombucha tea
against human pathogenic fungi such as A. flavus, C. albicans, and
M. gypseum.
METHODS
Fungal specimens and preparation of inoculum
Fungal specimens that used in this study such as A. flavus, C. albicans,
and M. gypseum. The fungal specimens were grown at 25°C on
sabouraud dextrose agar (SDA) medium for 24-72 hrs.
Preparation of fungal suspension
Fungal specimens suspended in liquid media of 0.9% sodium
chloride. It is incubated for 18-24 hrs at 25°C. Furthermore,
shake the suspension and control the turbidity to get close to the
standard of turbidity, 0.5 McFarland. The standard of turbidity,
0.5 McFarland, is equal to the fungal content which counts 108 colony
forming unit/ml. It has absorbance in the value of 0.08-0.10 in
wavelength of 530 nm.
Kombucha culture rejuvenation
The aim of this procedure is to establish kombucha culture which will
be inoculated into fermentation media. Kombucha culture rejuvenation
was started with preparing the fermentation media by heating of
1000 ml aquadest in a sterilized beaker glass until temperature of
100°C for ±15 minutes, followed by the addition of 0.5% black tea.
Then, the mixture was filtered. Results of black tea involving residue
and filtrate which contributes as the growth media of kombucha.
The filtrate was measured to 600 ml, added with 10% sucrose,
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Yuniarto et al.
and diluted until homogeneous. The solution is packed into a glass
bottle. Furthermore, the media is cooled down to room temperature
(25°C±2°C). Progressing into the next stage, inoculation stage, the
prepared media is combined with kombucha culture 10%. Hereafter,
the bottle is covered with porous cloth and sealed with rubber. Then, it
is located in a stable area, which is protected from sunrays. The result
of this rejuvenation of kombucha tea culture is ready for the final step
which is the fermentation for 18 days.
Measurement of antifungal activity
Antifungal activity of kombucha tea was tested by the paper disc method.
First, the materials and tools are prepared into laminar air flow. Heat
the SDA until it melts. Cool it down to 40-50°C. Then, add 1 ml of fungal
suspension specimen. Add about 20 ml of cooled SDA to the Petri plates.
Let the mixture of mold suspension and the media is solidified. Prepare
the paper disc and the liquid which is originated from the fermentation
of kombucha tea. On the paper disc, using sterilized tweezers, place 3
drops of 20 µl of the liquid aseptically (1.5 cm from the side of Petri
plates). This medium is then incubated at room temperature for 3 × 24
hrs. The antifungal activity is displayed by the inhibition zone of the
specimen around the paper disc.
Measurement of the equivalence of kombucha tea solution and
miconazole
The test of equivalence of kombucha tea and antifungal agent,
miconazole is executed by paper disc method. From the primary
solution of 20,000 µg/ml, they were created a series of solution with
distinctive concentration from 18,000, 16,000, 14,000, 12,000, 10,000,
8000, 6000, and 5000 µg/ml. 1 ml of fungal suspension is added into
sterilized Petri plates. Add ±20 ml of liquid sterilized SDA. Furthermore,
shake the Petri plates to homogenize the mixture of the media and the
suspension. When it solidifies, aseptically add three paper disc by
distance of 1.5 cm from the side of Petri plates. Ensure that the paper
disc is added with 20 µl of diluted miconazole in dimethyl-sulfoxide.
Hereafter, it is incubated in 25°C for 24-72 hrs. Subsequently, it
measures the inhibitory diameter.
Statistical analysis
Statistical analysis of the result was performed using the one-way
analysis of variance, followed with the post-hoc test Tukey honestly
significant difference. A value of p<0.05 was used to explain data
significance.
RESULTS AND DISCUSSION
Kombucha tea is a traditional beverage originated from the fermentation
of tea and sugar. It has unique aromatic, sweet, and sour taste and
contains diversified type of vitamin, minerals, and organic acid of
fermented tea leaves. Kombucha is the results of fermentation of yeast
culture and acid bacteria. The fermentation process of kombucha is
affected by several factors of surroundings such as amount of inoculum,
incubation temperature, pH, and initial sugar concentration [10,11].
In kombucha fermentation, tea is used as the fermentation media.
Tea has functioned as mineral supply for the reaction of yeast and
bacteria. Besides, it takes a role in giving the flavor and specific aroma
of the kombucha. In this research, it used black tea which commonly
consumed because its polyphenol oxidation helps conveying the strong
color and unique taste. Black tea produces more metabolic material
compared to other media [12].
Antifungal activity test of kombucha tea was performed by diffusion
method using paper disc method against human pathogenic fungi
such as A. flavus, C. albicans, and M. gypseum. In this research, the fungi
were performed as it represents the three mycosis infections consist
of cutaneous mycosis (M. gypseum), mucosal mycosis (C. albicans), and
systemic mycosis (A. flavus). In the test of the medium, SDA is applied
due to its objective as a specific media for the fungal growth.
Test of inhibitory activity of the fungi aims to determine the ability of
the product (the result of kombucha tea fermentation) in inhibiting the
growth of pathogenic fungi such as A. flavus, C. albicans, and M. gypseum.
The activity test is accomplished in 6, 12, and 18 days.
According to Table 1, test of inhibitory activity of kombucha tea against
fungal specimen in the time of 6, 12, and 18 days exhibit different
inhibitions regarding the obtained inhibition zone. This phenomenon
indicates that, in those fermentation conditions, kombucha tea
genuinely has antifungal activity. As the duration of fermentation is
increased, the condition of the media is getting acid.
In the present study, it reveals that the increase of fermentation period
will diminish the antifungal activity. It is supported with the data that
there is a declining trend of inhibitory diameter during 6, 12, and 18 days
of fermentation. It is expected that in the 6 days of fermentation, the
yield of metabolic compound is greater than the yield of 12 and 18 days
fermentation, as well as having higher ability in impeding the growth
of fungi. The probable compounds that perform as the antifungal agent
are acetic acid, lactic acid, ethanol, glucuronic acid, which originated
from the fermentation of kombucha.
Acetic acid or CH3COOH is a clear fluid that has sour taste and pungent
odor. Acetic acid is weakly ionized in aqueous solution and historically
used as preservative due to its antimicrobial effect [13]. Organic acid
(acetic acid) can be applied as antimicrobial compound as it suppresses
the growth of pathogenic microbes by deactivating the work system
of the cell: Cell wall, cell membrane, metabolic enzymes, and protein
synthesis system.
In positive control test, miconazole has a role as the antifungal agent.
Miconazole is derived from synthetic imidazole which relatively
stable and has broad antifungal spectrum against dermatophyte fungi.
Miconazole inhibits the fungal activity of Trichophyton, Epidermophyton,
Microsporum, Candida, and Malassezia furfur [14]. Moreover, dimethyl-
sulfoxide is used as the solvent of miconazole which acts as a negative
control. As tea is known for its antimicrobial activity, however, the 5%
concentration of black tea which is used as the negative control results
no inhibition of the fungal growth.
From the Fig. 1, it showed that inhibitory diameter of kombucha tea in
6 days of fermentation, gives the equivalence value of 20 µl kombucha
tea and miconazole: 5696 µg/ml for A. flavus, 5487.38 µg/ml for
C. albicans, and 9174.44 µg/ml for M. gypseum, respectively.
Table 1: The average of inhibitory diameter (mm) of kombucha
tea against A. flavus, C. albicans, and M. gypseum
Duration (days) Average of inhibitory diameter (mm)
A. flavus C. albicans M. gypseum
6 16.83±0.05 15.36±0.87 25.06±0.32
12 15.43±0.25 14.56±0.98 22.50±0.75
18 11.06±0.35* 11.00±0.81* 21.16±0.90*
*Data represent the mean±SD of observations, significant different during
fermentation period p<0.05. SD: Standard deviation, A. flavus: Aspergillus flavus,
C. albicans: Candida albicans, M. gypseum: Microsporum gypseum
Fig. 1: Antifungal activity of miconazole against inhibitory
diameter of fungi

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Asian J Pharm Clin Res, Vol 9, Issue 5, 2016, 253-255
Yuniarto et al.
CONCLUSION
The results from the present study provide support for the use
of kombucha tea as a potential antifungal source against human
pathogenic fungi involving A. flavus, C. albicans, and M. gypseum.
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