Jurnal Akademika Kimia, 14(2): 70-76, May 2025
ISSN (online) 2477-5185 | ISSN (print) 2302-6030
http://jurnal.fkip.untad.ac.id/index.php/jak/

OPEN ACCESS

1

Soybean Tempeh: Effect of Yeast Concentration and Fermentation
Duration on Organoleptic Test as a Learning Resource
Mufida, *Tri Santoso, Sri H. V. Pulukadang, & Dewi S. Ahmar

Chemistry Education Study Program/Faculty of Teacher Training and Education – Universitas Tadulako, Palu –
Indonesia 94119
Received 28 February 2025, Revised 07 March 2025, Accepted 14 March 2025
doi: 10.22487/j24775185.2025.v14.i2.pp70-76

Abstract
Tempeh is a traditional Indonesian fermented food known for its high nutritional value. This study sought to assess the impact
of yeast concentration and fermentation length on moisture content, protein content, and organoleptic evaluation of yellow soybean
tempeh sourced from Tojo Una-Una, Poso, and Palu. The research included a 3 × 3 factorial design incorporating yeast concentrations
of 1, 2, and 3 grams, alongside fermentation durations of 30, 48, and 72 hours. The moisture content was assessed using the
gravimetric method, the protein content was determined via the Kjeldahl method, and 20 panelists performed an organoleptic
evaluation. The findings indicated that the moisture content of tempeh generally rose with prolonged fermentation time and elevated
yeast concentration. The maximum moisture content, 63.22%, was observed in tempeh from Tojo Una-Una, which used 2 grams of
yeast and had a fermentation duration of 72 hours. The minimum value, 50.18%, was also from Tojo Una-Una with 1 gram of
yeast and 30 hours of fermentation. Protein levels peaked at 48 hours of fermentation with 3 grams of yeast, reaching a maximum
of 26.88% in the Palu area. In contrast, the minimum was recorded at 18.55% in tempeh from Tojo Una-Una with 2 grams of
yeast and 30 hours of fermentation. The organoleptic test results indicated that panelists favored Poso tempeh in terms of flavor,
aroma, and texture. The concentration of yeast and the time of fermentation influence the moisture content, protein content, and
organoleptic quality of tempeh. The methodologies and findings of this study serve as a resource for chemistry education in the format
of LKPD.
Keywords: Fermentation, organoleptic, yeast, yellow soybean tempeh

utilizing the fungi Rhizopus oligosporus and
Rhizopus orizae.
In Indonesia, a wide variety of tempeh,
including tempe gembus, tempe mangel kacang,
tempe menjes, tempeh benguku, and tempe
romtoro. Despite the diversity of tempeh, yellow
soybean-based tempeh is more recognized and
favored. Yellow soybeans are a superior source of
plant-derived protein and are rich in calcium,
magnesium, and iron (Jafarzadeh et al., 2024).
One advantage of yellow soybeans is their
aesthetic appeal, aligning with customer
preferences. The Center for Agricultural Data and
Information Systems of the Ministry of Agriculture
reported that yellow soybean production in 2015
was 954,997 tons. Consequently, to satisfy the
demand, Indonesia imported 2,256,931.68 tons of
soybeans. The protein content in yellow soybeans
ranged from 31% to 48%. The fat content ranged
from 11% to 21%. Soybeans serve as the primary
raw materials in the production of tempeh, tofu, and
soy sauce due to their high protein content. Yellow
soybeans (Glycine max) are typically utilized for

Introduction
Soybeans constitute a significant source of
nourishment. The nutritional makeup of soybeans
changes based on the developed variety and the
coloration of the epidermis and cotyledons (Prativi
et al., 2023). Yellow soybeans have a substantial
nutritional composition of 38% protein, 18% fat,
15% ash, and 29% carbs per 100 grams, along with
vitamins and minerals (Afzaal et al., 2022; Moroșan
et al., 2023).
Tempeh is a traditional Indonesian dish,
originating in Central Java in the 1700s and now
recognized globally (Romulo & Surya, 2021).
Tempeh is recognized as a fermented product
originating from soybean components (Aziez et al.,
2022). Tempeh possesses a superior protein content
of 18.3% in comparison to other processed
soybeans, including tauco at 10.5%, tofu at 7.9%,
soy sauce at 5.5%, and soy milk at 2.8% (Subali et
al., 2023). Tempeh is a food product derived from
the fermentation of soybeans or other legumes

⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯

*Correspondence:
Tri Santoso
e-mail: trisantoso@untad.ac.id
© 2025 the Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons
Attribution-NonCommercial-ShareAlike 4.0 International, which permits unrestricted non-commercial use, distribution,
and reproduction in any medium, provided the original work is properly cited.

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Jurnal Akademika Kimia
Methods

tempeh production; however, individuals often
innovate by substituting soybeans with legumes
such as kidney beans (Phaseolus vulgaris) (Safitry et
al., 2022).
To date, soybeans have been recognized as a
fundamental ingredient for the production of
tempeh. The limited storage duration of tempeh
fosters innovation in the further processing of
yellow soybeans. The procedure for producing
tempeh from yellow soybeans and other soybean
varieties is identical, encompassing washing, boiling,
peeling, soaking, and fermentation. Fermentation is
a crucial phase in tempeh production, when yeast is
introduced to facilitate the transformation of
soybean seeds into tempeh. The fermentation of
soybeans for tempeh production necessitates the
involvement of Rhizopus sp. mold. The molds
commonly utilized for tempeh production are
Rhizopus oryzae and Rhizopus oligosporus (Ellent
et al., 2022).
Several previous studies have explored the
effects of yeast concentration and fermentation
time on tempeh quality. For example, Anita (2018)
studied black soybean tempeh, while Andika et al.
(2023), Hanafi et al. (2023), and Zhao et al. (2024)
examined the impacts of fermentation on sensory
and chemical properties. However, these studies did
not focus on yellow soybean tempeh sourced from
diverse regions, nor on its potential as a contextual
chemistry learning resource. This study seeks to fill
that research gap by analyzing the effects of yeast
and fermentation on yellow soybean tempeh from
Central Sulawesi (Tojo Una-Una, Poso, Palu) and by
developing an LKPD (Student Worksheet) based
on the results.
Multiple factors influence the quality of
tempeh production, specifically yeast concentration
and fermentation duration. Tempeh yeast
comprises the fungus Rhizopus sp, commonly
referred to as tempeh mushroom. Rhizopus fungus
is the predominant species in tempeh yeast,
characterized by its white coloration and mycelia
that link soybean seeds to tempeh (Teoh et al.,
2024). Tempeh production involves two
fermentation processes. The primary fermentation
process begins when the soybeans are soaked in
water. The second fermentation, also known as
primary fermentation, occurs when yeast is
introduced to soybeans that are prepared for
fermentation. The study by Diez-Simon et al. (2020)
examines the influence of yeast concentration and
fermentation duration on the chemical and sensory
characteristics of black soybean tempeh (Glycine
soja), sharing similarities in variables and
methodologies. The author will highlight the
contrasting aspects of the yellow soybean variable
utilized as a chemistry learning resource, and will
also employ a distinct research location from other
studies, specifically three sites in Central Sulawesi.

Tools and Materials

The tools used are a porcelain cup, a
desiccator (Schott Duran brand), an analytical balance
(Denver Instrument M-310), and an oven. For protein
content analysis, the following equipment is
required: Kjeldahl heater, 100 mL Kjeldahl flask
(Iwaki), distillation device (Iwaki), stopper and
clamp (Pyrex), Erlenmeyer (Herma), 50 mL burette
(Pyrex), and analytical balance (Shimadzu AUX220).
The materials required for analyzing the moisture
and protein content of yellow soybean tempeh
samples include Kjeldahl tablets, concentrated
H2SO4, aquades, 30% NaOH, 0.1 N HCl, and
methyl red indicators.

Sample preparation

The initial phase involves assembling all
necessary tools and ingredients. Begin with 1 kg of
yellow soybeans, then sort and wash them. After
cleaning, boil the soybeans for 30 minutes to
facilitate skin removal. Subsequently, soak the
soybeans for 12 hours, then boil them again for 30
minutes. Allow them to cool before adding 1 gram
of yeast, along with 2 and 3 grams. Finally, wrap the
soybeans in plastic, puncture holes, and ferment for
30, 48, and 72 hours (Anita, 2018).

Tempeh water content analysis

A 10-gram sample of mashed yellow soybean
tempeh is placed into a porcelain cup. The mixture
is thereafter cooked in the oven at 100°C for
approximately three hours. The substance is
subsequently cooled with a desiccant for one hour
and measured with an analytical balance (Anita,
2018).

Analysis of tempeh protein content

The initial phase is the destruction step,
wherein a 1-gram sample of yellow soybean tempeh
is placed into a 100 mL Kjeldahl flask, followed by
the addition of one Kjeldahl tablet and 10 mL of
concentrated H2SO4. Subsequently, all the
constituents in the pumpkin are denatured (heated)
until a transparent solution is achieved. The
subsequent phase is distillation, wherein the
products of the destruction phase are diluted with
100/10 mL of distilled water and agitated until
homogeneous. Subsequently, 10 mL is extracted
and transferred into a distillation flask. Introduce 10
mL of 30% NaOH solution into the pumpkin's wall
until two distinct layers are established. The flask is
subsequently attached to a condenser. Steam from
the boiling liquid passes through the condenser. It
is collected in an Erlenmeyer flask containing 10 mL
of 0.1 N HCl solution, with a red methyl indicator
added. The distillate is subsequently titrated with a
standardized NaOH solution (0.1 N) using oxalic
acid as the titrant. The titration is concluded after
the distillate exhibits a pink hue. The latter creates a
blank solution, achieved by substituting the sample
with distilled water (Nurfadila, 2024).

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Mufida et al.

Data analysis techniques

1 - poor. LKPD is deemed viable if the percentage
score of 61% of the Student Worksheet eligibility
percentage is classified within the eligibility category
outlined in Table 1.

Determination of moisture content

(𝑊𝑜 + 𝑊𝑠 )
× 100% (1)
𝑊𝑠
(Badan Standarisasi Nasional, 2009)

𝑤𝑎𝑡𝑒𝑟 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 (%) =

Table 1. Percentage of student worksheet
eligibility

Information:
Ws = sample weight before diovened (grams)
Wi = sample weight + cup after ovening (grams)
Wo = weight of empty cups (grams)

Skor percentage (%)
81− 100
61− 80
41− 60
21 − 40
20

Determination of protein levels
(𝑡𝑠 + 𝑡𝑏 ) × 𝐻𝐶𝑙 × 14 × 10
× 100% (2)
𝑚𝑔 𝑠𝑎𝑚𝑝𝑒𝑙
% 𝑃𝑟𝑜𝑡𝑒𝑖𝑛 = % 𝑁 × 5.71

Source: (Riduwan, 2012).

%𝑁 =

Results and Discussion
The results of the moisture content analysis
in soybean samples from Tojo Una-Una, Poso, and
Palu City are presented in Table 2 after being
analyzed and calculated.

(Badan Standarisasi Nasional, 2009)

Keterangan:
VS
= HCl volume of 0.1 N for the titration of
the sample, expressed in milliliters (mL);
Vb
= HCl volume of 0.1 N for blank titration,
expressed in milliliters (mL);
N
= normality of HCl solution;
W
=sample weight, expressed in
milligrams (mg);
14
= atomic weight Nitrogen;
5.71
= protein factor for Soybeans

Table 2. Average moisture content
Region
Tojo una-una
Poso
Palu

Moisture content
56.58
56.29
58.08

The determination of moisture content in
this study uses the gravimetric method, which
involves drying the sample at 105°C until it reaches
a constant weight. Moisture content is calculated
from the difference in weight before and after
drying and expressed in percent (Zambrano et al.,
2019).
In this study, tempeh moisture content was
influenced by soybean origin, yeast concentration,
and fermentation duration. Longer fermentation
generally increased moisture content, especially in
soybeans from Tojo Una-Una and Palu, due to the
enzymatic activity of Rhizopus sp., which softened
the substrate. In contrast, soybeans from Poso
showed relatively stable moisture content,
presumably due to a denser seed structure.
Increasing yeast concentration also tended to
increase moisture content due to the higher
intensity of mold metabolism.
In theory, prolonged fermentation can lead
to enzymatic activity from Rhizopus sp. molds,
which hydrolyze soybean components like proteins
into smaller compounds. This process can draw
water into the tempeh tissue, leading to an increase
in moisture content (Hanafi et al., 2023). However,
several technical and biological factors can cause the
moisture content to decrease as fermentation time
increases. This was observed in the results, where an
increase in mold respiration activity led to denser
mold growth with longer fermentation. The molds
perform respiration that produces heat, which can
cause greater water evaporation. This causes the
water content of tempeh to decrease.
Sulistiyawati & Lusiani (2022) explained that
the increased fermentation temperature due to mold

Organoleptic test

This study engaged 20 untrained panelists
from the Chemistry Education Study Program at
Tadulako University. Samples of yellow pumpkin
seed tempeh were produced in containers
designated P1, P2, and P3. Panelists were thereafter
provided with samples and questionnaires to
provide hedonic evaluations based on aroma, color,
taste, texture, and overall product appearance
criteria. The panelists' preferences were assessed
using a 5-point hedonic scale: 1 (extremely dislike),
2 (dislike), 3 (somewhat like), 4 (like), and 5 (very
like). Subsequently, the panelists were requested to
complete a questionnaire and engage in a discussion
regarding the provided sample (Tanone & Prasetya,
2019).

Creation and validation of student worksheets
(LKPD)

The initial Student Worksheet (LKPD) was
developed to encompass work practices and
research findings shown in the accompanying draft,
intended as a resource for chemistry education. The
LKPD's validity was evaluated by two specialists: a
content expert and a design expert. The validity
criteria are deemed practical if they receive a
minimum score of 2.75.
𝑃𝑒𝑟𝑠𝑒𝑛𝑡𝑎𝑠𝑒 =

Category
Highly Valid
Valid
Quite Valid
Less Valid
Very Invalid

𝑠𝑢𝑚 𝑜𝑓 𝑣𝑎𝑙𝑖𝑑𝑎𝑡𝑖𝑜𝑛 𝑟𝑒𝑠𝑢𝑙𝑡 𝑠𝑐𝑜𝑟𝑒𝑠
× 100%
ℎ𝑖𝑔ℎ𝑒𝑠𝑡 𝑠𝑐𝑜𝑟𝑒

The assessment results will be evaluated using
a Likert scale with the following classifications: 5 very good; 4 - good; 3 - moderate; 2 - adequate; and

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metabolism can accelerate water evaporation.
Besides the temperature and air circulation during
fermentation, whether carried out in the open or
with good air circulation, the water released during
metabolism will evaporate, especially from the
second day onward. The longer the fermentation
lasts under these conditions, the more water is lost,
and as the texture of tempeh becomes denser, the
yeast concentration increases. The fermentation
time is extended. The faster the mold forms thick
mycelium, the denser the tempeh becomes, which
binds water more strongly or even causes it to lose
water as the texture hardens and dries.
Thus, in general theory, the water content
increases with fermentation time. However, the
results of this study show that the process does not
always occur linearly and can be influenced by
environmental conditions and local raw materials.
The microbial metabolic process produces water as
a by-product of aerobic fermentation (Mori et al.,
2023).
The results of the moisture content analysis
in soybean samples from Tojo Una-Una, Poso, and
Palu City are presented in Table 3 after being
analyzed and calculated.

environmental
conditions,
and
physical
characteristics of the seeds. This finding aligns with
Andika et al. (2023), who stated that high yeast
concentration and optimal fermentation can
increase protein content. The best treatment was
obtained at 3 grams of yeast and 48 hours of
fermentation, while Tojo Una-Una soybean with 2
grams of yeast also showed optimal protein levels.
In theory, fermentation by Rhizopus sp. increases
protein levels through the synthesis of mold
biomass, the breakdown of complex proteins, and
the reduction of antinutritional compounds.
The high protein yield in tempeh from Tojo
Una-Una supports the theory of Widodo et al.
(2021) that moderate yeast concentration, when
combined with good soybean quality, can optimize
enzymatic activity. Tojo Una-Una soybeans are
thought to have high initial protein, ideal seed size,
and stable moisture, resulting in efficient
fermentation even with only 2 grams of yeast. This
finding demonstrates the efficient use of yeast
without decreasing yield, relevant for an economical
biochemical learning example.
In contrast, tempeh from Palu and Poso
showed lower protein levels despite using more
yeast. This indicates that increasing yeast and
fermentation duration does not always increase
protein content, as the initial soybean quality and
fermentation conditions are also influential.
Soybeans from Palu and Poso had lower initial
protein levels, with Poso experiencing excess
moisture that inhibited mold growth. In addition,
the use of excessive yeast in some treatments
actually decreased fermentation efficiency.
According to Hanafi et al. (2023), too high
yeast concentration can trigger microorganism
competition and disrupt fermentation stability,
thereby reducing tempeh quality. Inappropriate
fermentation time, especially if it is too long, can
also prevent significant increases in protein levels
due to protein degradation or microbial
contamination.
This study shows that the protein content of
tempeh meets the quality standards, with the best
results obtained at a yeast concentration of 3 grams
and optimal fermentation conditions. The less yeast
and the faster the fermentation, the higher the
protein content tends to be. However, adding yeast
to the optimum point also increases the activity of
the proteolytic enzyme Rhizopus oligosporus,
which breaks down complex proteins into simple
compounds (Perdani & Utama, 2020; Zhao et al.,
2024).
The increase in protein is also supported by
an increase in soluble nitrogen and free amino acids.
However, excess yeast can increase the
fermentation temperature and accelerate protein
degradation, thus reducing protein levels (Surbakti
et al., 2022). Therefore, the balance between yeast
concentration and fermentation time is essential to
produce tempeh with the best chemical and sensory
quality (Lusiana et al., 2023).

Table 3. Average protein content
Region
Tojo una-una
Poso
Palu

Protein content
21.75
24.34
22.18

The results showed that the protein content
in yellow soybean tempeh increased with both the
yeast concentration and the length of fermentation
time. Tempeh fermented for 48 hours with a yeast
concentration of 3 grams had the highest protein
content compared to other treatments. This aligns
with research by Widodo et al. (2021), which states
that the increase in protein levels in tempeh results
from the activity of protease enzymes produced by
Rhizopus sp. These enzymes can hydrolyze
complex proteins into simpler, easily measurable
forms. The increase in protein levels can also be
caused by the growth of mold biomass during
fermentation. Rhizopus sp. grows and develops on
the surface of soybean seeds. This mold mycelium
itself contains protein, so it contributes to the total
protein content of tempeh. During fermentation,
the process of breaking down complex compounds
degrades antinutritional compounds, such as trypsin
inhibitors, which inhibit protein digestion. This
increases the availability of protein that can be
measured chemically. Besides that, it can be seen
from the Optimal Fermentation conditions that
during the 48-hour fermentation with 3 grams of
yeast, temperature and humidity tend to approach
optimal conditions for mold growth, allowing the
fermentation process to occur more completely.
The protein content of tempeh varied among
soybean origins (Palu, Poso, and Tojo Una-Una)
due to differences in varieties, fermentation

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Mufida et al.

Organoleptic test

low aroma score could result from rapid or
uncontrolled fermentation due to higher ambient
temperatures, which lead to the production of sharp
or undesirable odors.
Although the texture of Palu tempeh (3.45) is
still acceptable, it lags behind the other regions. This
may be related to fluctuating humidity or
suboptimal environmental conditions that disrupt
consistent mold growth during fermentation.
Implications for chemistry learning beyond
sensory analysis, these findings provide meaningful
insights for contextualized chemistry education.
The differences in organoleptic scores across
regions can be used to design student worksheets
(LKPD) that incorporate real data, encouraging
students to explore the connection between
fermentation, biomolecules, enzyme activity, and
sensory perception.
By analyzing data from local food products,
students can relate chemistry concepts to everyday
experiences. This not only improves scientific
understanding but also fosters appreciation for local
wisdom and traditional food biotechnology. The
integration of tempeh into chemistry learning
enhances engagement and relevance in the
classroom. According to Table 4 of the results, it
was determined that learning resources in the form
of LKPD are very suitable for use as a learning
medium. The results achieved were 80% for
material experts and 90% for design experts.
Consequently, the LKPD (Student Worksheet) is
deemed legitimate as a learning resource, since it
satisfies the percentage criteria, achieving a range of
80% to 61% (Riduwan, 2012).

The organoleptic test used in this study is the
Hedonic Scale Scoring method, as one of the
consumer acceptance tests. This test used 20
untrained panelists. Organoleptic analysis was
carried out by preparing test samples and
questionnaires. For the hedonic test, the test
parameters contained smell, color, taste, and texture
of the product. The results are shown in Figure 1.
5
4

3
2
1
0
Smell

Flavor
Tojo

Texture
Poso

Color

Palu

Figure 1. Organoleptic test
The results show that tempeh from Tojo
Una-Una received the highest scores in texture
(3.95) and taste (3.73). The superior texture
indicates a well-developed mycelium network of
Rhizopus sp., resulting in a firm but tender tempeh
structure. This aligns with the findings of
Nirmalasari et al. (2024), who stated that optimal
fermentation yields a compact, fully bound structure
with white, dense mycelium covering the surface.
The high taste score also reflects the balance
of moisture and protein levels, leading to favorable
fermentation by-products such as free amino acids
and volatile compounds that enhance the flavor
profile. These sensory characteristics suggest that
fermentation in Tojo Una-Una proceeded under
stable and favorable conditions.
Poso-produced tempeh achieved the highest
aroma score (3.91), slightly surpassing Tojo UnaUna (3.90). This suggests that the fermentation
process in Poso produces a more appealing tempeh
aroma, potentially due to environmental factors or
higher-quality soybean substrates that promote
optimal microbial metabolism. According to
Sulistiyawati & Lusiani (2022), aroma formation
during fermentation is highly influenced by
temperature, humidity, yeast concentration, and the
generation of volatile compounds such as esters,
alcohols, and aldehydes. Poso also showed
consistent scores in taste and texture (both 3.70),
indicating overall uniform sensory quality. This
implies that soybeans from Poso are highly
responsive to fermentation, resulting in wellaccepted sensory characteristics.
In contrast, tempeh from Palu consistently
received the lowest scores across all parameters,
particularly in aroma (3.30) and color (3.02). The
lower color rating may be due to uneven fungal
growth, which results in patches, discoloration, or
black spots on the tempeh surface. The relatively

Table 4. Average validation assessment of the
LKPD Aspect Assessment Rating Scale
Aspect Assessment
Material Expert
Design Expert
Total
Average

Rating Scale (%)
80
90
170
85

Source: Data from Material Expert and Design Expert
Validation Results

The LKPD developed contains research
data, including water content, protein content, and
organoleptic scores. Students are directed to analyze
the data and relate it to chemical concepts such as
biomolecules, enzymes, and changes in compounds
during fermentation. By using tempeh as a learning
medium, students can develop critical thinking skills
and understand the application of chemistry in
everyday life.
Conclusions
The study indicates that the optimal yeast
concentration for producing yellow soybean
tempeh meets desirable chemical parameters
(moisture and protein content) and offers superior
organoleptic quality. This concentration produces

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elevated protein levels and suitable moisture
content, and is organoleptically favored. The
optimal fermentation duration for producing yellow
soybean tempeh with satisfactory chemical and
organoleptic quality is 48 to 72 hours, contingent
upon the specific qualities of the soybeans from
each location. Fermentation for 48 hours yields
ideal protein levels, whereas 72-hour fermentation
markedly elevates moisture content and alters the
texture of tempeh. The validation test results of the
LKPD (Student Worksheet) indicate its
appropriateness as a learning resource.

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Conflict of Interest
The authors declare that there are no
conflicts of interest associated with the publication
of this article. Each author contributed equally and
without pressure from other parties.
Acknowledgment
The author expresses his deepest gratitude to
all individuals and entities who have contributed to
this research. Special thanks go to the Chemistry
Laboratory FKIP of Tadulako University for the
assistance and facilities provided throughout the
research process.
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