Jurnal Kelautan Tropis Juni 2023 Vol. 26(2):238-244
P-ISSN : 1410-8852 E-ISSN : 2528-3111
Carrageenan Extraction of Kappaphycus alvarezii Seaweed from Nusa
Lembongan Waters Using Different Alkaline Treatments
Ichoun Brawici Dhewang1, Ervia Yudiati1*, Subagiyo1, Rabia Alghazeer2
1Department
of Marine Science, Faculty of Fisheries and Marine Sciences, Universitas Diponegoro
Jl. Prof Jacub Rais, Tembalang, Semarang, Central Java 50275 Indonesia
3Department of Chemistry, Faculty of Sciences, University of Tripoli
Tariq Sayyidi al Misri Road, Tripoli, Libya
Email : eyudiati@gmail.com
Abstract
Kappaphycus alvarezii is a carrageenan-producing red seaweed that is widely cultivated in Nusa Lembongan waters, Bali,
Indonesia. Carrageenan is generally extracted using an alkaline base,. iIn this study three different types of alkali were used
to extract carrageenan originating from Nusa Lembongan Waters. This study aims to determine the quality of the extraction.
The three alkalis used were KOH, NaOH, Ca(OH)2, and distilled water as a control. The 20 g dried seaweed was weighed,
washed with the tap running water, and immersed in 0.15% alkaline solution (KOH, NaOH, Ca(OH)2) as well as distilled water
for 24 hrs. Followed by rinsing with running water until pH was neutral. The solution was soaked and heated at 100ºC for 2 hours
with a ratio of seaweed and water 1 kg in 20 L, and filtered. The extract was mixed with 1.25% KCI. The carrageenan precipitate
was put in the oven at 60-80ºC until dry for 48 hours. The yield test results showed that the use of alkaline Ca(OH)2 resulted in a
yield of 29.28% better than alkaline KOH (28.18%) and NaOH (27.7%). Based on the analysis of Fourier Transform Infrared
Spectroscopy (FTIR), extraction using alkaline Ca(OH)2 showed functional groups characteristic of iota-carrageenan, whereas
using alkaline KOH and NaOH showed functional groups characteristic of kappa-carrageenan.
Keywords: Alkaline, Carrageenan, FTIR, Kappaphycus alvarezii
INTRODUCTION
Globally, seaweed is one of the most important marine resource sectors either biologically or
economically. Seaweed cultivation is one of the fast growing aquaculture sectors (Mariot et al.,
2021). Indonesia is one of the largest superior seaweed producers in the world with a production
value of up to 9.9 million tons (FAO, 2021). The Province of Bali contributed 149 tons of seaweed
production (KKP, 2020), most of which was produced from the waters of Nusa Lembongan.
Kappaphycus alvarezii seaweed cultivation has developed very rapidly since the demand
for carrageenan from industry has increased in many countries, including Indonesia (Kasim &
Mustafa, 2017). K. alvarezii is one of the seaweeds that is widely cultivated by the people of Nusa
Lembongan because it has a fast growth rate (about 4.5% per day) (Rudke et al., 2022). In spite of
that, K. alvarezii also has a high amount of polysaccharides in the cell wall so this can be a potential
source of carrageenan (Manuhara et al., 2016; Riyaz et al., 2022). Carrageenan is a hydrocolloid
compound extracted from red seaweed species namely K. alvarezii, Chondrus crispus, and
Eucheuma spinosum (Azevedo et al., 2015; Bouanati et al., 2020; Arun et al., 2021). Carrageenan can
be used as a stabilizer, emulsifier, thickener, food, cosmetics, biostimulants for plants and animals, as
well as immunostimulant (Mariot et al., 2021; Vaghela et al., 2022). Carrageenan acts as a stabilizer
because it contains sulfate groups which are negatively charged along the polymer chain and are
hydrophilic which can bind water and other hydroxyl groups (Supriyantini et al., 2017).
Carrageenan extraction from K. alvarezii is generally carried out using an alkaline base (KOH)
(Bono et al., 2014; Solorzano et al., 2019). The function of KOH is to help the extraction of
polysaccharides effectively and to accelerate the elimination process of 6-sulfate to 3,6-anhydro-Dgalactose thereby increasing the quality of the carrageenan produced (Azevedo et al., 2015).
However, several other researchers used different alkaline bases such as NaOH and Ca(OH) 2 as
carrageenan extracting compounds (Das et al., 2021; Ganesan et al., 2018). According to research
*) Corresponding author
www.ejournal2.undip.ac.id/index.php/jkt
Diterima/Received : 07-02-2023, Disetujui/Accepted : 10-04-2023
DOI: https://doi.org/10.14710/jkt.v26i2.17389
Jurnal Kelautan Tropis Juni 2023 Vol. 26(2):238-244
conducted by Panggabean, et al in (2018), it is known that differences in concentration of alkali can
affect yield results. The higher of alkali concentration during the alkalization process takes place,
resulting the higher pH, and this will enhance the ability of alkali to extract.
Research on the extraction of carrageenan using different alkaline methods from seaweed
cultivation in Nusa Lembongan has never been explored before. There is still a lack of information,
so, it is important to provide information about the chemical-based carrageenan data of K. alvarezii
and the potency utilization of K. alvarezii seaweed, especially in Nusa Lembongan, Bali.
MATERIALS AND METHODS
The seaweed used in the study was K. alvarezii which was obtained from a seaweed
cultivation area in Nusa Lembongan Waters, Bali. Dried seaweed was extracted at the Diponegoro
University, Biology Laboratory, Central Java. The research method used in this study was laboratory
experimental, to investigate cause-and-effect relationships by giving one or several treatments
(Sunaryo et al., 2015). The obtained data were analyzed using descriptive methods.
The process of extracting carrageenan from K. alvarezii was carried out according to the
protocol of Erjanan et al. (2017) with a few modifications. The dried seaweed was weighed as much
as 20 g, then the seaweed was cut into small pieces (± 1 cm), then the seaweed was washed with
running water until clean. Immersion in 0.15% alkaline solution (KOH, NaOH, Ca(OH)2) and distilled
water was done. After 24 hours the seaweed was then washed again with running water until a
neutral pH was obtained, then the seaweed was soaked and heated at 100ºC for 2 hours with a ratio
of seaweed and water 1 kg in 20 L, so the water needed was 400 ml. After the heating process,
filtering is carried out with a filter cloth in a hot state. The filtered filtrate is then mixed with 1.25% KCI
in each treatment, and let stand for 30 mins. KCl solution is used for the carrageenan precipitation
process. The carrageenan precipitate is put in the oven at 60-80ºC until dry for 2 days (48 hours). After
finishing in the oven, the carrageenan is crushed into carrageenan powder and then weighed for
the analysis process. Yield analysis was carried out by comparing the weight of carrageenan with
the weight of the dried seaweed used. Yield is calculated based on the formula:
Yield (%) = (Carrageenan weight)/(Dry seaweed weight) x 100% (Leksono et al., 2018)
Carrageenan Composition Test (FT-IR)
The functional group produced by carrageenan was tested using an infrared
spectrophotometer. The infrared spectrum is used to determine the presence of several chemical
bonds in organic compounds. The functional group test followed the method of Silverstein & Bassler
(1962). The sample is weighed as much as 1 mg, then mixed with 100 mg KBr and pressed for 10
minutes at a pressure of 8-10 psi until a thin pellet is obtained, then the pellet is inserted into the cell
holder and the spectra are made.
RESULTS AND DISCUSSION
Yield is a comparison of the dry weight of the extract with the amount of raw material before
extraction process. Yield is one of the important parameters to determine the effectiveness of the
extraction process (Sinurat, 2017). The percentage yield of carrageenan resulting from K. alvarezii
extraction with three different alkaline treatments is shown in Figure 1. It shows that the extraction
using alkaline Ca(OH)2 resulted in the highest average percentage of 29.28% compared to other
alkaline treatments. Whereas extraction using distilled water produces the lowest average yield
percentage, which is equal to 23%. The average yield percentage from the KOH alkaline treatment
was 28.18% and the NaOH alkaline treatment was 27.7%. Giving alkali in the extraction process can
accelerate the process of cell wall plasmolysis thereby facilitating the separation process between
the cell wall and cytoplasm of seaweed cells so that the ability to extract is greater than using
aqueous water (Wulandari et al., 2019).
Carrageenan Extraction of Kappaphycus alvarezii (I.B. Dhewang et al.)
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Jurnal Kelautan Tropis Juni 2023 Vol. 26(2):238-244
35,00
30,00
Yield
25,00
20,00
15,00
10,00
5,00
0,00
% Yield
% Yield
% Yield
% Yield
KOH
NaOH
Ca(OH)2
Aquades
Alkaline
KOH % Yield
NaOH % Yield
Ca(OH)2 % Yield
Aquades % Yield
Figure 1. Yield from carrageenan extraction using 4 different alkaline treatments namely (KOH, NaOH,
Ca(OH)2, and distilled water.
The yield from the study showed that alkaline treatment could increase the yield ie. 25%. This
results is in accordance with FAO standards (2007). On the other hand, treatment without alkali
(distilled water) produced a yield value of less than 25%. Alkali treatment resulted in a higher yield
value allegedly because the alkaline solution was able to accelerate the process of forming 3,6
anhydro-galactose. Later, most of the sodium, magnesium and calcium content in carrageenan will
be bound to the ester group and the 3,6 anhydro-galactose capopolymer so that the extraction
process can increase the value of the yield (Wulandari et al., 2019). This statement is supported by
(Gerung et. al, 2019) that the administration of an alkaline/base solution can break down the cell
wall thereby increasing the solubility of carrageenan in water and preventing the hydrolysis reaction
of the glycosidic bond in the carrageenan molecule.
The yield values of the three types of alkaline treatment, namely the treatment with Ca(OH) 2,
had the highest yield value of 29.28% compared to other alkaline treatments, namely KOH of (28.18%)
and NaOH (27.7%). This difference occurs because in the extraction stage besides dissolving
carrageenan, the reaction between carrageenan and alkaline solvents also takes place. It is
estimated that the reaction that took place involved the exchange of ions, that is, cations in
dissolution replaced sulfate ions in carrageenan (Distantina et al., 2013). The calcium cation in
Ca(OH)2 has the largest molecular weight of 74.093 g/mol (Winaya et al., 2020) compared to the
molecular weight of the potassium cation in KOH of 56.11 g/mol (Park et al., 201) and the sodium
cation in NaOH of 39.997 g/mol (Winaya et al., 2020). The Ca(OH)2 yield result was the highest than
KOH, NaOH, and distilled water extraction results.
FT-IR Spectrum Analysis of Carrageenan in Three Different Treatments
Samples suspected of carrageenan compounds can be proven by identifying them using an
infrared spectrophotometer or FTIR. FTIR analysis is a technique used to determine the functional
group of a compound. Based on Figure 2 and Table 1, the results of carrageenan extraction using
different alkalis, namely KOH, NaOH, and Ca(OH) 2, show that the functional groups of glycosidic
bonds and sulfate esters are present in all carrageenan with four different alkaline treatments. The
ester sulfate and 3,6-anhydrogalactose functional groups from the use of alkali Ca(OH) 2 have a
wave number that is almost close to the wave number of commercial carrageenan (Sham, 2022),
while the use of alkali Ca(OH)2 also produces functional group 3, 6 anhydrogalactose 2 sulfates. On
the other side, the use of alkaline KOH, NaOH, and distilled water did not produce this functional
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Jurnal Kelautan Tropis Juni 2023 Vol. 26(2):238-244
group. The four alkaline treatments produced the O-H hydroxyl functional groups with the resulting
wave numbers not approaching the commercial carrageenan wave numbers. Furthermore the
glycosidic linkage produced in each treatment was pointed from different wave numbers. KOH is
pointed from 1073.270 cm-1, NaOH (1071.31cm-1), Ca(OH)2 (1075.81cm-1), and distilled water
(1072.74cm-1), respectively.
FTIR test results are used to identify the functional groups and compounds that have been
extracted. The FTIR spectra of the extracted carrageenans were compared to one another, based
on the differences in the alkali used. The wave range observed in this FTIR test is 4000-400 cm-1. FTIR
test results of K. alvarezii extraction using 3 different alkaline treatments showed several important
absorption peaks (wavelengths) indicating functional groups that are generally found in
carrageenan.
Figure 2. The FTIR test results analysis of carrageenan extracted using alkali (A) NaOH, (B) KOH, (C)
Ca(OH)2, and (D) distilled water.
Table 1. Wavelength analysis on carrageenan bonds based on four alkaline treatments (NaOH, KOH,
Ca(OH)2, and distilled water)
Wave number
(cm-1)
3200-3600
1220-1265
928-933
840-870
800-805
1010-1080
Functional groups
O-H
Ester sulfate
3,6anhydrogalactose
3,6anhidrogalactose-4sulfate
3,6
anhydrogalactose2sulfate
Glikosidic linkage
Commercial
Carrageenan
KOH
(Syam, 2022)
3410,15
3427,22
1261,45
1258,22
Alkaline Treatments
NaOH Ca(OH)2 Distilled Water
3435,46 3454,49
1241,81 1262,26
3433,87
1257,34
931,62
929,5
929,45
930,52
929,77
864,11
847,98
847,9
849,38
848,84
408,46
1071,31 1075,81
1072,74
Carrageenan Extraction of Kappaphycus alvarezii (I.B. Dhewang et al.)
1073,27
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FTIR test results of carrageenan extraction with alkaline KOH treatment had a wavelength peak
in the 3427 cm-1 region which indicated the O-H functional group due to the presence of hydrogen
bonds in the carrageenan structure which caused a shift towards shorter wave numbers. The peak
wavelength in the area around 1258 cm-1 indicates absorption of sulfate esters in the carrageenan
extraction results using alkaline KOH, this is supported by Balqis et al., 2017) indicating that the wave
number of sulfate ester absorption obtained is approx. at 1228.928 cm-1. The results of other studies
on carrageenan stated that sulfate esters were shown at a wave number of 1240-1260 cm-1 (Chopin
and Whalen, 1993), as well as research by Tanusorn et al. (2018) noted that sulfated esters were
shown at a wave number of 1210 and 1260 cm-1.
The FTIR test value of the alkaline NaOH treatment showed the same functional group as O-H
at a wave number of 3435 cm-1 and the S=O ester sulfate functional group a wave number of
1241cm-1. It is shown that the sulfate ester content in the alkaline NaOH treatment was compared to
the alkaline KOH treatment. Meanwhile, the wave number at 929.25 cm-1 indicates the presence of
the C-O-C functional group. In the 3,6-anhydrogalactose structure, the C-O-C content of 3,6anhydrogalactose from the alkaline NaOH treatment is slightly higher than the alkaline KOH
treatment. The peak at a wave number of 847.9 indicates the presence of the 3,6-anhydrogalactose4-sulfate functional group which has a strong intensity, the same thing was also found in the KOH and
distilled water treatment.
Based on the results of the FTIR test for the Ca(OH) 2 treatment, it was shown that there were 2
functional groups 3,6-anhydrogalactose-4-sulfate and 3,5-anhydrogalactose-2-sulfate with
respective wave number of 849.38 cm-1 and 408.48 cm-1. The functional group 3,5anhydrogalactose-2-sulfate was only found in the alkaline Ca(OH) 2 treatment and was not found in
other treatments. The alkaline treatment Ca(OH) 2 also found several other groups such as O-H with
a wave number of 3454.49 cm-1, a sulfate ester group with a wave number of 1262.26 cm-1 as the
same as the other treatments.
From the results of identification with infrared spectroscopy and the description of the
wavelength, it can be concluded that the carrageenan produced by Ca(OH)2 is of the Iota type.
Iota type is characterized by the presence of galactose 2-sulfate and 4-sulfate, 3,6anhydrogalactose, and sulfate ester groups (Villanueva et al., 2004; Campo et al., 2009). Other
treatments using alkaline KOH, NaOH, and distilled water shows the chemical structure of
carrageenan from kappa-carrageenan type, this is based on the functional groups found such as
(ester sulfate), (3,6-anhydrogalactose), and (galactose-4-sulfate) (Campo et al ., 2009;Verma et al.,
2014).
CONCLUSION
The application of alkaline Ca(OH)2 in extraction methods produces the highest amount of
carrageenan yield compared to the application of alkaline KOH, NaOH, or distilled water. Based on
the FTIR analysis, Ca(OH)2 produced iota-carrageenan type. KOH, NaOH, and distilled water
extraction methods produced kappa-carrageenan type.
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