Jurnal Ilmu Farmasi dan Farmasi Klinik
(Journal of Pharmaceutical Science and Clinical Pharmacy)
ISSN 1693-7899 (print) - 2716-3814 (online)
DOI: 10.31942/jiffk.v21i1.9529

Sustainable Antibiofilm Strategy Using Fruit Waste: EcoEnzyme from Pineapple Core and Lemon Peel Against
Streptococcus mutans and Candida albicans
Harris Antonius 1, Eldiza Puji Rahmi1*, Rika Revina1, Dhigna Luthfiyani Citra Pradana1,
Abdi Wira Septama2
1 Pharmacy Program, Faculty of Medicine, Universitas Pembangunan Nasional “Veteran” Jakarta, South

Jakarta, DKI Jakarta, Indonesia
2 Research Centre for Pharmaceutical Ingredients and Traditional Medicine, National Research and

Innovation Agency (BRIN), Kawasan Sains Dan Teknologi (KST) Soekarno, Cibinong, Indonesia

ABSTRACT: Eco-enzyme is a versatile liquid produced by fermenting organic material in
molasses solution and has some potential antimicrobial compounds. In this study, the organic
materials utilized were pineapple cores and lemon peels, which are typically discarded. Oral
microbes, particularly Streptococcus mutans and Candida albicans, often develop antimicrobial
resistance through their defense mechanisms by forming a polymeric layer known as biofilm.
This biofilm, composed of polysaccharides, protects microbes from exposure to antimicrobial
agents, allowing them to adapt and survive. The objective of this study was to evaluate the
antibiofilm properties of eco-enzymes derived from various organic materials. The assessment
involved determining the minimum inhibitory concentration (MIC) and minimum bactericidal
concentration (MBC)/minimum fungicidal concentration (MFC) and conducting an antibiofilm
assay. The eco-enzyme formulated with pineapple cores exhibited higher antimicrobial
activity, with an MIC of 25% and an MBC of 50% against Streptococcus mutans. Additionally,
the highest antibiofilm activity was observed in the eco-enzyme containing a specific organic
matter combination, with values of 68526.23 ppm against Streptococcus mutans and 2235.30
ppm against Candida albicans. The IC50 value for all three eco-enzyme samples was analyzed
using a one-way ANOVA statistical test, revealing significant inhibitory activity against biofilm
formation by Streptococcus mutans (p=0.023) and Candida albicans (p=0.002).
Keywords: Biofilm; Candida albicans; Eco-Enzyme; Streptococcus mutans

*Corresponding author:
Name
:Eldiza Puji Rahmi
Email
: eldizapr@upnvj.ac.id
Address : Faculty of Medicine, Universitas Pembangunan Nasional “Veteran” Jakarta, South Jakarta, DKI
Jakarta, Indonesia

127
Submitted: 19-07-2024.; Received in Revised Form: 19-09-2024; Accepted: 17-05-2025

Antonius et al.

INTRODUCTION
The oral cavity is known for having the second highest diversity of microbiota,
following the gastrointestinal tract, forming a complex ecosystem that includes various
opportunistic pathogens capable of causing significant oral and dental diseases. Dental
plaque, a biofilm that develops on both soft and hard oral tissues, is central to the
pathogenesis of these conditions (Salehi et al., 2020). Statistics from Riset Kesehatan Dasar
2013 (Kementerian Kesehatan RI, 2013) and Survei Kesehatan Indonesia 2023 (Badan
Kebijakan Pembangunan Kesehatan, 2023) reveal a dramatic rise in the prevalence of oral
and dental problems in Indonesia, from 25.9% in 2013 to 56.9% in 2023. The most frequent
complaints included toothaches, damaged teeth, and dental caries, which made up 86% of all
cases.
Among the microbes found in the oral cavity, Streptococcus mutans and Candida
albicans play a crucial role by forming biofilms and demineralizing teeth, ultimately leading
to the development of dental caries (Kim et al., 2021). Biofilms serve as a vital defense
mechanism for these microbes, allowing them to withstand antimicrobial treatments (Zhou
et al., 2015). Microbial adherence to tooth surfaces produces extracellular polymeric
substances (EPS) that provide protection against antimicrobial agents (Rather et al., 2021).
While antimicrobial mouthwash is commonly used to prevent biofilm thickening by
inhibiting microbial adhesion during its formation, it may inadvertently contribute to the
emergence of resistant strains when used on mature biofilms (Takenaka et al., 2022).
In light of these challenges, alternative strategies that are both effective and
environmentally sustainable are urgently needed. One promising approach involves the use
of eco-enzymes—bioactive liquids produced through the fermentation of organic kitchen
waste, including fruit and vegetable scraps (Gu et al., 2021). These eco-enzymes contain
hydrolytic enzymes such as proteases, amylases, and lipases, which can degrade biofilm
matrices by targeting their protein and polysaccharide components (Soleha et al., 2023)
(Shree et al., 2023). Furthermore, the acidic environment generated during fermentation
enhances their antimicrobial activity (Gu et al., 2021). Research by Lestariningsih &
Moordiani (2022) demonstrated that an eco-enzyme formulated as a mouthwash can inhibit
the growth of Candida albicans, achieving a zone of inhibition of 4.83±0.00 mm. Another
study by Welfalini et al. (2022) found that eco-enzymes inhibited the growth of Streptococcus
spp. taken from the ectodermal tissue of a dog's skin, with a zone of inhibition of 8.30±0.25
mm. Controlling the growth of Streptococcus mutans and Candida albicans can effectively
reduce biofilm formation.
The production of eco-enzymes from household waste, such as pineapple cores and
lemon peels, not only offers a bioactive alternative for biofilm control but also aligns with
sustainable waste management practices. Fruit waste represents a significant portion of
domestic organic refuse (Athia et al., 2021). In Indonesia, per capita consumption of
pineapple increased from 0.209 kg in 2013 to 0.469 kg in 2017, while lemon consumption
reached 12.57 grams per person per day in 2021 (Badan Pusat Statistik, 2021; Fauzi et al.,
2021). Globally, lemon consumption in Western Europe also rose from 1.6 kg to 1.9 kg per
person between 2013 and 2017 (Confederation of British Industry, 2023). This increase in
consumption results in significant waste accumulated in landfills, posing environmental
challenges (Aili Hamzah et al., 2021). Left untreated, carbohydrates and proteins from this
waste can produce unpleasant odors (Asendy et al., 2018).
Addressing this issue supports the achievement of the United Nations Sustainable
Development Goal (SDG) 12, which emphasizes responsible consumption and production
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(BAPPENAS, 2023). The dual benefit of eco-enzymes, as biofilm inhibitors and as a means of
valorizing food waste, presents a sustainable and innovative solution in the development of
natural antimicrobial agents. Maximizing community-level eco-enzyme production may
contribute not only to oral health improvement but also to environmental sustainability and
antimicrobial stewardship.
METHODS
Tools and Materials
The laboratory instruments and equipment employed in this study included closed
plastic containers, universal pH indicators, an incubator, a vortex mixer, micropipette with
sterile pipette tips, inoculating loops, 96-well flat-bottom microplates, and a microplate
reader. Reagents and materials utilized comprised pineapple core, lemon peel, molasses,
distilled water, chlorhexidine (0.2%), Mayer’s reagent, Dragendorff’s reagent, Bouchardat’s
reagent, Liebermann-Burchard’s reagent, magnesium powder (Mg), hydrochloric acid (HCl),
ferric chloride (FeCl₃), n-hexane, Benedict’s reagent, Lugol's iodine solution, Mueller Hinton
Agar (MHA, Himedia®), Mueller Hinton Broth (MHB, Himedia®), glucose, sodium chloride
(NaCl, Merck®), McFarland standard 0.5 (Himedia®), crystal violet solution, and ethanol
(Merck®). Microbial strains of Streptococcus mutans and Candida albicans were obtained
from the Research Centre for Pharmaceutical Ingredients and Traditional Medicine.
Preparation of Eco-Enzymes
Three eco-enzymes were prepared from pineapple core, lemon peel, and a 1:1
combination of both, using a fermentation process at the Pharmacology Laboratory, UPN
Veteran Jakarta. Fresh pineapple cores and lemon peels were collected from a citrus juice
vendor at the Angke Fruit Market, West Jakarta, and subsequently washed under running
water, cut into small pieces, and weighed. Each organic material was combined with molasses
and distilled water in a ratio of 1:3:10. The mixture was placed in closed plastic containers,
shielded from direct sunlight, and allowed to ferment at an ambient temperature for a period
of three months. After fermentation, the resulting eco-enzyme liquid was filtered to separate
it from the residual solids (Gumilar, 2023). Three variants were produced: (1) pineapple core
eco-enzyme, (2) lemon peel eco-enzyme, and (3) a 1:1 combination of both.
Phytochemical Screening
Qualitative phytochemical screening was conducted to detect the presence of major
secondary metabolites in each eco-enzyme variant. Standard procedures were employed to
test for alkaloids, flavonoids, tannins/polyphenols, saponins, steroids/triterpenoids, and
carbohydrates using appropriate reagents.
pH Determination
The universal pH indicator was employed to measure the pH of pineapple core, lemon
peel, and a combination of both eco-enzymes.
Antimicrobial Activity Test
Minimum Inhibitory Concentration (MIC) Determination
We conducted the MIC determination using the broth microdilution method, following
the guidelines of Weinstein et al., 2020. Microbial suspensions were prepared to match a 0.5
McFarland standard and diluted in MHB to obtain a final concentration of 5 × 10⁵ CFU/mL.
MIC determination was performed using the microdilution method with a 96-well flatbottom microplate, which has twelve columns and eight rows. The first column (A1-H1)
served as the negative control, filled with 100 μl of MHB and 100 μl of the bacterial
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suspension. The second column (A2-H2) acted as the positive control, containing 100 μl of
0.2% chlorhexidine and 100 μl of the bacterial suspension. The empty wells in rows A to C,
with the exception of column 12, were filled with 100 μl MHB. In well A12, 200 μl of pineapple
core eco-enzyme was added, and 100 μl of this solution was transferred to well A11. This
step was repeated down to well A3, which received a 1/512 dilution of the solution from well
A12. The same procedure was followed for the lemon peel eco-enzyme in row B and the 1:1
ratio combination of eco-enzymes in row C. After filling the wells, each well was
supplemented with 100 μl of bacterial suspension, resulting in a total volume of 200 μl per
well. The microplate was incubated at 35±2°C for 18-24 hours for Streptococcus mutans and
at 37°C for 24-48 hours for Candida albicans. The MIC was identified as the lowest
concentration in which the wells remained non-turbid, indicating no microbial growth
(Marak & Dhanashree, 2018; Sukandar et al., 2014).
Minimum Bactericidal and Fungicidal Concentration (MBC and MFC) Determination
For each non-turbid well, 5 μl of the content was transferred to a petri dish containing
MHA and inoculated with an inoculating loop. The petri dishes were subsequently incubated
at 35±2°C for 18-24 hours. The MBC and MFC values were defined as the lowest
concentrations of each sample that demonstrated no microbial growth (Afroz et al., 2020;
Hendiani et al., 2020; Sukandar et al., 2014).
Antibiofilm Activity Assay
Biofilm Formation Assay
Biofilm formation of Streptococcus mutans and Candida albicans was done using the
microtiter plate method. A total of 100 μL of microbial suspension was mixed with 100 μL of
MHB supplemented with 2% glucose in each well. Microplate incubated at 37°C for 24 hours
for Streptococcus mutans and 48 hours for Candida albicans.
Wells were then gently washed with sterile distilled water, stained with 0.1% crystal
violet for 15 minutes, washed again, and dried at room temperature. The presence of violetstained biofilms was confirmed visually. Each well was then solubilised with 200 μL ethanol,
and the absorbance was measured at 595 nm. Biofilm formation was classified based on
OD595 results that were interpreted by range of specific OD595 values as follows: >0.5
(strong), 0.3–0.5 (moderate), <0.3 (weak), <0.15 (very weak), and 0 (non-adherent) (AlBayati & Samarasinghe, 2022; Arjuna et al., 2018; Gunardi et al., 2022; Hejazinia et al., 2020;
Neglo et al., 2022; O’Toole, 2011).
Biofilm Growth Inhibition Activity (Antibiofilm Assay)
Antibiofilm activity testing of eco-enzymes was done using the same biofilm formation
procedure, with the addition of eco-enzyme treatments. Each eco-enzyme variant was tested
at concentrations of 100%, 50%, 25%, 12.5%, and 6.25%. Diluted microbial suspensions of
as much as 100 μL were added to 100 μL of MHB supplemented with 2% glucose and
considered a negative control in this experiment. The positive control used was 200 μl
chlorhexidine 2%, then 100 μl was transferred to the adjacent well, which already contained
100 μl MHB supplemented with 2% glucose. Dilution was repeated until the fifth well with a
concentration of chlorhexidine of 0,125%. Each well was then added with 100 μl diluted
microbial suspension. Each eco-enzyme sample of 100 μl was added to the well, and then 100
μl microbial suspension was added. Microplates were incubated at 37°C for 24 jam for
Streptococcus mutans and 48 jam for Candida albicans. The incubated microplate was then
washed in a sterile aquadest. Each well in the microplate was stained with 200 μl of 0,1%
crystal violet for 15 minutes, then washed with sterile distilled water and dried at room
temperature. A violet stain inside the well showed biofilm formation. After biofilm formation
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was confirmed, each well was reconstituted with 200 μl of ethanol, and its absorbance value
(Optical Density or OD) was measured at 595 nm. Experiments were done in triplicate, and
then the average and standard deviation values were calculated. Lastly, after the OD value
was obtained, the inhibition rate was calculated using these formulas (Alenazy, 2023; Marak
& Dhanashree, 2018; Shinde et al., 2021).
Inhibition Rate (%) = [(ODcontrol– ODTreatment)/ODcontrol] x 100
Data Analysis
All experimental procedures were performed in triplicate. The inhibition rates for each
concentration and eco-enzyme variant were plotted against the logarithmic concentration to
generate regression curves and determine the IC₅₀ values, which then was averaged and
counted as its standard deviation. Data homogeneity was assessed using Levene’s test (p >
0.05 indicating homogeneity), and normality was tested using the Shapiro–Wilk test (p > 0.05
indicating normal distribution) due to a sample size <50 (Usmadi, 2020) (Quraisy, 2022).
Statistical significance was evaluated using one-way ANOVA followed by post-hoc Tukey’s
test for multiple comparisons. Analyses were conducted using IBM SPSS Statistics software,
with significance set at α = 0.05 (Shinde et al., 2021).
RESULTS AND DISCUSSION
Eco-Enzyme Production
Three variations of eco-enzyme were produced based on the type of organic material
used, each yielding 3 liters of final product. A white to yellowish surface film developed
during fermentation, identified as a pitera layer, a fungal biofilm rich in amino acids, peptides,
proteins, organic acids, vitamins, and minerals (Natasya et al., 2023). Lactic acid bacteria
were reported to be found in eco-enzymes. During fermentation, carbohydrates present in
the organic materials are first broken down into glucose, which is then converted into pyruvic
acid. Under aerobic conditions, pyruvic acid is further transformed into acetaldehyde by
pyruvate decarboxylase. Acetaldehyde can either be reduced to ethanol by alcohol
dehydrogenase or oxidised into acetic acid via acetaldehyde dehydrogenase (Rukmini &
Astuti Herawati, 2023) (He et al., 2022). Concurrently, lactic acid is produced by lactic acid
bacteria through lactate dehydrogenase (Wang et al., 2021). Additionally, organic acids
derived from the feedstock are bioconverted into enzymes such as protease, lipase, and
amylase during fermentation (Gumilar, 2023).
Phytochemical Screening
All three eco-enzyme samples demonstrated similar phytochemical profiles (Table 1),
containing alkaloids, flavonoids, tannins/polyphenols, saponins, and carbohydrates. Steroid
in pineapple eco-enzyme was previously detected by research conducted by (Sai et al.
(2023); the results aligned with the steroid compound contained in pineapple. However, in
this research, steroids were not detected despite the organic materials used containing
steroids. The absence of steroids could be attributed to microbial biotransformation through
processes such as isomerization, hydroxylation, oxidation, and condensation (Cano-Flores et
al., 2020), which may have degraded the original steroidal compounds.
pH Measurement
The pH values of the eco-enzyme samples (Table 2) ranged between 4 and 7,
consistent with previous studies on fermented eco-enzymes. The increase in pH level was
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expected because of the ammonification process of enzymes and protein contained in ecoenzymes. Protein, with the help of microbes, undergoes decomposition into amino acids, its
most simple form, and subsequently deaminated, resulting in the production of ammonia, a
base that increases the pH of eco-enzyme (Pashaei et al., 2022). This process also altered the
odor of eco-enzymes, particularly those made with mixed organic materials, resulting in a
strong and unpleasant smell. These changes indicate potential degradation in eco-enzyme
quality, including reduced antimicrobial activity.
Table 1. Qualitative Phytochemical Screening Results of Eco-Enzyme
Organic Matters of Eco-Enzyme
Phytochemical
Description
Pineapple
Combination
Compound
Lemon Peel
Core
1:1
Alkaloid
- Mayer
No precipitate formed
- Dragendorff
+
+
+
Precipitate formed
- Bouchardat
+
+
+
Precipitate formed
Flavonoid
+
+
+
Orange color change
Tanin/polyphenol +
+
+
Blackish color change
Saponin
+
+
+
Stable bubble formed
Steroid/
No color change
triterpenoid
Carbohydrate
- Benedict
+
+
+
Precipitate formed
- Lugol
No color change

Table 2. pH Determination of Eco-Enzyme Sample
Sample
Pineapple Core Eco-Enzyme
Lemon Peel Eco-Enzyme
Combination 1:1 Eco-enzyme

pH
4–5
4–5
6–7

Antimicrobial Activity
The MIC, MBC, and MFC values of the eco-enzymes and chlorhexidine against
Streptococcus mutans and Candida albicans are shown in Table 3. The antimicrobial activity
of eco-enzymes was suspected because of the presence of acetic acid in them (Vidalia et al.,
2023). Based on previous research by Salma and Ratni in 2022, the acetic acid content of ecoenzymes that fermented for 90 days was as much as 9.69%. Acetic acid can interfere with the
structure of the microbial cell wall, resulting in a loss of energy (in the form of ATP) for the
microbe. The acetic acid contained in the eco-enzyme was enough to inhibit the growth of
Candida albicans, which requires an acetic acid concentration of 10% to inhibit its growth
(Zinn & Bockmühl, 2020). That concentration was also enough to kill Streptococcus mutans
entirely because it only needed 3% acetic acid with an exposure time of 30 minutes (Halstead
et al., 2015).
Antibiofilm Activity
The antibiofilm activity of the three eco-enzyme samples was evaluated against
Streptococcus mutans and Candida albicans, with appropriate controls included. The
concentrations tested were selected based on MIC results, under the assumption that
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concentrations capable of inhibiting microbial growth might also impede biofilm formation
(Table 4). Biofilm development is a complex process regulated by quorum sensing, a
mechanism that facilitates intercellular communication among microbial cells. This
signalling system coordinates various physiological activities, including the transition from
planktonic to biofilm-associated lifestyles, thereby enhancing microbial survival and
virulence (Preda & Săndulescu, 2019). Quorum sensing mechanism produces protease, an
extracellular enzyme that plays a crucial role in biofilm maturation and dispersion
(Dewatisari et al., 2023). Protease produced from quorum sensing hydrolyzes biofilm
structure, resulting in the dispersion of microbe cells from the biofilm, leading to the spread
of microbes (Ramírez-Larrota & Eckhard, 2022).
Table 3. Minimum inhibitory value and minimum bactericidal/fungicidal concentration of ecoenzyme and chlorhexidine against Streptococcus mutans and Candida albicans
MBC and MFC Value + Standard
MIC Value + Standard Deviation
Deviation
Sample
Streptococcus
Streptococcus
Candida albicans
Candida albicans
mutans
mutans
Chlorhexidine
< 0.00625% + 0% < 0.00625% + 0% < 0.00625% + 0% < 0.00625% + 0%
Pineapple Core Eco25% + 0%
50% + 0%
50% + 0%
No inhibition
Enzyme
Lemon Peel Eco-Enzyme 50% + 0%
50% + 0%
No inhibition
No inhibition
Combination 1:1 Eco50% + 0%
50% + 0%
No inhibition
No inhibition
enzyme

Table 4. Results of Antibiofilm Activity Testing of Eco-Enzyme and Chlorhexidine Samples
Sample
Chlorhexidine
Pineapple core eco-enzyme
Lemon peel eco-enzyme
Combination 1:1 eco-enzyme

IC50 Value + Standard Deviation
Streptococcus mutans
Candida albicans
2936.28 + 1756.30 ppm
1865.69 + 433.59 ppm
84229.72 + 68909.47 ppm 145026.38 + 56157.65 ppm
557162.94 + 252732.39
185613.82 + 66102.54 ppm
ppm
68526.23 + 54994.44 ppm 2235.30 + 3122.52 ppm

Eco-enzymes are known to contain protease enzyme, which may contribute to destroy
and prevent the forming of a biofilm matrix (Soleha et al., 2023). Referring to the organic
material used for the production of the eco-enzyme, specifically, eco-enzymes derived from
pineapple core are likely to retain bromelain, a proteolytic enzyme naturally present in
pineapple, even after fermentation (Ilyas, 2020). The IC50 values for the antibiofilm activity
of eco-enzyme with single organic materials shows that pineapple core eco-enzyme was
more active than lemon peel eco-enzyme, likely due to bromelain's dual role in degrading the
microbial cell wall and inhibiting biofilm formation (Muhsinin et al., 2021). Furthermore, the
eco-enzyme made with mixed organic materials demonstrated the most effective antibiofilm
activity, evidenced by the lowest IC₅₀ values. This benefit could be attributed to its higher pH,
which aligns with the optimal activity range of protease enzymes (~pH 6) (Soleha et al.,
2023). Optimal protease activity contributes significantly to microbial cell death and
prevents biofilm development (Liaqat et al., 2021). The theory was in line with the research
results, where the eco-enzyme with the highest pH value, eco-enzyme with The combination
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of organic materials exhibited better antibiofilm activity against both Streptococcus mutans
and Candida albicans.
CONCLUSION
This study demonstrated that all three eco-enzymes from the pineapple core, lemon
peel, and organic material combinations have antimicrobial activity against Candida albicans
with an MIC value of 50% ± 0% and Streptococcus mutans with an MIC value of 50% ± 0%,
except for the pineapple core eco-enzyme, which got an MIC value of 25% ± 0% and an MBC
value of 50% ± 0%. All three eco-enzymes were beneficial for inhibiting biofilm formation, as
seen from the one-way ANOVA statistical significance of 0.023 for Streptococcus mutans and
0.002 for Candida albicans. The effective concentrations of pineapple core, lemon peel, and
organic material combination eco-enzyme that inhibit 50% of Streptococcus mutans growth
are 84229.72 ± 68909.47 ppm, 185613.82 ± 66102.54 ppm, and 68526.23 ± 54994.44 ppm.
At the same time, for Candida albicans, they are 145026.38 + 56157.65 ppm, 557162.94 ±
252732.39 ppm, and 2235.30 ± 3122.52 ppm.
ACKNOWLEDGMENT
This research is supported by the Pharmacy Study Program, Faculty of Medicine, UPN
“Veteran” Jakarta, and the National Research and Innovation Agency.
AUTHOR CONTRIBUTION
HA: literature search; experimental studies; data analysis; manuscript preparation.
EPR: Concepts or ideas; design; definition of intellectual content; manuscript editing;
manuscript review.
RR: Design; manuscript editing; manuscript review.
DLCP: Manuscript review.
AWS: Manuscript editing; manuscript review.
CONFLICT OF INTEREST
None to declare
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