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Characterization of refined carrageenan from Kappaphycus alvarezii

extracted using marine fungi
To cite this article: S Sulistiawati et al 2020 IOP Conf. Ser.: Earth Environ. Sci. 404 012071

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EMBRIO 2019 IOP Publishing
IOP Conf. Series: Earth and Environmental Science 404 (2020) 012071 doi:10.1088/1755-1315/404/1/012071

Characterization of refined carrageenan from Kappaphycus

alvarezii extracted using marine fungi

S Sulistiawati1, N H Ain1, Uju1, K Tarman1,2*

1
Department of Aquatic Product Technology, Bogor Agricultural University (IPB
University), Bogor, Indonesia
2
Center of Coastal and Marine Resources Studies, Bogor Agricultural University (IPB
University), Bogor, Indonesia

*E-mail: kustiaz@apps.ipb.ac.id

Abstract. Carrageenan is a polysaccharide that widely used in food, pharmaceutical, cosmetic,

textile and printing industries as a coagulate agent, stabilizer, and gelling agent. The extraction
of carrageenan usually is carried out by alkali treatment, but it may have toxic effect for the
environment. Therefore, another extraction method is required to reduce the toxic effect from
carrageenan. Carrageenan can also be extracted by using a cellulolytic enzyme. A natural
cellulolytic enzyme can be produced from endophytic fungi. The objective of this research was
to extract carrageenan using endophytic fungal cellulase and to evaluate the effect of fungal
cellulase concentration and extraction time on the quality of carrageenan. The materials used in
this research were marine endophytic fungus (EN) and dried seaweed Kappaphycus alvarezii.
This research was conducted in three steps. The first step was cultivating of endophytic fungus
(EN) in cellulose basal medium. The second step was cellulase extraction from endophytic
fungus EN. The last step was extraction of carrageenan by cellulase endophytic fungus. The
highest yield was obtained by added 1% of EN cellulase enzyme and extraction period for 3
hours. Characteristics of carrageenan in generally met the standard, but the gel strength and
whiteness was still below the standard.

Keywords: algae, cellulose, endophyte, marine fungi, natural

1. Introduction

Carrageenan is a hydrocolloid consisting of the ammonium, calcium, magnesium, potassium, and

sodium sulfate ester from galactose and polysaccharide 3.6-anhidrogalaktose (FAO 2007). In the food
industry, carrageenan has been widely used because has the functional ability as thickening, gelling, and
stabilizing agent, improving cheese texture, controlling the viscosity and texture of pudding, also as a
filler and stabilizer in meat processing (Campo et al 2009). Carrageenan is classified into several types
such as λ, κ, ι, ε, and μ carrageenan which all contain sulfate 22-35 percent (Necas 2013). Kappa
carrageenan (κ-carrageenan) is the most used type of carrageenan in various industrial fields. Kappa
carrageenan commonly obtained from seaweed Kappaphycus alvarezii (Webber et al 2012).

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EMBRIO 2019 IOP Publishing
IOP Conf. Series: Earth and Environmental Science 404 (2020) 012071 doi:10.1088/1755-1315/404/1/012071

Cellulolytic enzymes produced by various types of microorganisms, such as fungi. There have been
many explorations of cellulolytic enzymes produced by fungi. Aspergillus sp. and Trichoderma sp. are
known have ability to produce cellulolytic enzymes with high activity (Sivaramanan 2014, Reddy et al
2014). Carrageenan extraction can be done by utilizing fungi as a cellulolytic enzyme producer. The
ability of fungi to produce cellulolytic enzymes is very important in the extraction process, but it is also
necessary to use cellulolytic fungi that safe to use. Varadarajan et al (2009) reported that enzymatic
carrageenan extraction using Aspergillus niger produced more carrageenan yield than the conventional
extraction. Muthezilan et al (2014) has also performed carrageenan extraction using pure enzyme
produced from coastal plant endophytic shoots and obtained a higher yield until 52%.

The endophytic fungus strain (EN) isolated from seagrass has been known to produce extracellular
cellulase enzyme (Oktavia et al 2014). In previous research, EN fungus produced cellulase with high
cellulolytic activity. Irma (2018) has reported that EN fungus could be used in carrageenan extraction
from Kappaphycus alvarezii by utilizing EN fungal culture directly. Based on information about EN
capability from previous research, further research is needed to know the ability of cellulase enzyme
isolated from EN fungus in the carrageenan extraction process. The aims of this research were to obtain
carrageenan extract from Kappaphycus alvarezii using EN fungal cellulase and to determine the effect
of extraction period, EN cellulase enzyme concentration as well as the interaction between extraction
period and concentration of EN cellulase enzyme on carrageenan extract yield.

2. Materials and methods

2.1. Cultivation of endophytic fungus producer of cellulolytic enzyme

The cultivation was carried out by inoculation 10% of EN fungus on Basal Medium without carbon
source (NaNO3 2.0 g/L; KH2PO4 1.0 g/L; MgSO4.7H2O 0.5 g/L; FeSO4 10.0 mg/L). EN endophytic
fungus was cultivated on 500 mL Erlenmeyer flask with 200 mL of media and added with carbon source
of dried Kappaphycus alvarezii seaweed (1%). The culture was incubated for seven days at room
temperature and shaked with 120 rpm. Furthermore, for cellulase enzyme production, inoculation of
10% EN culture in 500 mL of the same production media (basal medium and seaweed) was done. The
culture was incubated at room temperature and shaked with 120 rpm for 9 days (Oktavia et al 2014).
.
2.2. Cellulase enzyme extraction from en fungus
The fungus culture was filtered using filter paper. The filtrate was centrifuged at a rate of 3,000 rpm for
20 min. Centrifugation has done to obtained crude cellulase extract. The crude cellulase extract was
purified by precipitation using ammonium sulfate (Muthezilan et al 2014).

2.3. Carrageenan extraction from Kappaphycus alvarezii using cellulase enzyme

The carrageenan extraction process was carried out by suspending 12.5 g of flour Kappaphycus alvarezii
with 250 mL of distilled water (Varadarajan et al 2009) and added with three different concentrations
(0 %, 1 % and 2%) of cellulase enzymes from EN endophytic fungus. Sample suspension was heated
on shaking incubator with two different extraction times (2 h and 3 h). Centrifugation of suspension
with speed at 7,000 rpm and 4°C for 15 min (Varadarajan et al 2009). The supernatant was mixed with
a 2-propanol solution with a ratio of 1:1. To remove the liquid part, the sample was centrifuged with
7,000 rpm at a temperature of 4°C for 20 min. The sample was then dried using an oven.

2.4. Analysis of carrageenan extract

2.4.1. Yield. The yield of carrageenan extraction was calculated based on the ratio between
carrageenan weight produced and weight of dried seaweed that was processed respectively.

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EMBRIO 2019 IOP Publishing
IOP Conf. Series: Earth and Environmental Science 404 (2020) 012071 doi:10.1088/1755-1315/404/1/012071

2.4.2 Sulfate content. The carrageenan was weighed 1 g and put into the Erlenmeyer flask 50 mL of
HCl 0.2 N then refluxed for 6 h until the solvent became clear. This solvent was transferred into a cup
glass and heated to boiling. Furthermore, 10 mL of BaCl2 solution was added over the water bath for 2
h. The precipitate formed was filtered with a non-gray filter paper and washed with boiling water to
chloride free. The filter paper was dried into the dryer ovenat 1,000°C until white ash was obtained. Ash
was cooled in a desiccator and then weighed (FMC Corp 1977).

2.4.3 Viscosity. Viscosity measurement was done by dissolving 1.5% carrageenan water then heated
in a boiling water bath until the solvent temperature reached 75°C. Viscosity was measured using
viscometer. The hot solvent was adjusted to a precise, viscometer was turned on and the temperature of
the solvent was measured. When the solvent temperature reached 75°C and the viscosity value was
measured on a scale of 1 to 100. The measurement was done after one minute of a full rotation for
spindle number three (FMC Corp 1977).

2.4.4 Gel strength. The 1.6% carrageenan solution and 0.16% KCl were heated in a bath of boiling
water with regular stirring to 80°C. The volume of the solution was 50 mL. The hot solution was then
put into the 4 cm diameter mould and kept at 10°C for two hours. The gel in the mould was inserted into
the measuring instrument (Rheoner RE-3305). The plunger that will be in contact with the gel was in
the middle. The plunger was activated and the samples were measured. Evaluation of measurement
results was done by reading the resulting graph. The maximum force (gel strength) can be read on the
recorder (FMC Corp 1977).

2.4.5 Acid-insoluble ash content (FMC Corp. 1977). The carrageenan was boiled with 25 mL of 10%
HCl for 5 minutes. The unsolved ingredients were filtered using a non-gray filter paper, then cooled in
a desiccator for further weight.

2.5 Functional group test by FTIR

Potassium bromide (KBr) powder was finely grounded by 0.1 g, then weighed carrageenan samples of
0.01 g were added and crushed together until smooth and homogeneous. The water content was removed
by heating in an oven 60°C for 48 h. Further FTIR spectrophotometric analysis was performed to
determine the functional group (Smith 2011).

3. Results and discussion

3.1. Cellulase enzyme production

The cellulase enzyme in this study was obtained through the cultivation of EN fungus in the basal
medium. Cultivation was done by growing EN fungus in the basal medium with dried Kappaphycus
alvarezii as its substrate source. The cellulase enzyme activity of the crude extract was measured after
obtaining enzyme on fungal culture broth after nine days cultivation. The crude extracts enzyme was
obtained by the filtration process and the centrifugation of culture broth of EN fungus. The supernatant
from the centrifugation was the crude extract enzyme that has been produced. The value of enzyme
activity of crude extract obtained was 0.0015 U/mL.

The semi-pure enzyme was obtained by precipitation using 80% of ammonium sulfate. The value of
semi-pure cellulase enzyme activity was 0.014 U/mL. This semi-pure cellulase enzyme was used in the
carrageenan extraction. This result was still low if compared to the cellulolytic activity of the best known
producer of cellulases Trichoderma reesei. Zhao et al (2018) reported the cellulase production of T.
reesei cultured in different media and pretreatment. The cellulase production by T. reesei cultured in
steam-treated willow was 0.7 U/mL and increased to 1.6 U/mL when the culture medium was induced
with acetic acid.

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EMBRIO 2019 IOP Publishing
IOP Conf. Series: Earth and Environmental Science 404 (2020) 012071 doi:10.1088/1755-1315/404/1/012071

3.2. Yield of carrageenan

Yield is the ratio of the amount of produced carrageenan by the amount of seaweed extracted. The
seaweeds used as the raw material and carrageenan were weighed in dried form. The results of
carrageenan extraction can be seen in figure 1.
35 d
e f
30 b c
a
25
Yield (%)

20
15
10
5
0
2h 3h 2h 3h 2h 3h
0% 1% 2%
Treatment

Figure 1. Effect of cellulase enzyme concentration and time of extraction on carrageenan yield.

Based on the results of yield analysis, it is known that the enzyme concentration factor, extraction time
and interaction between the two factors show the significant effect with p-value <0.01. The highest yield
produced in this study was carrageenan obtained from 1% enzyme concentration and 3 h of extraction
time. The highest yield value was 30.96%.

The yield of this treatment had a value of 6.44% higher than the treatment without enzyme addition.
However, when compared to some previous studies on enzymatic extraction of carrageenan, the yield
value in this study was still lower than other studies. Varadarajan et al (2009) yielded 45% of
carrageenan, while Muthezilan et al (2014) obtained carrageenan yield of 52% by extraction using
purified enzyme with dialysis. This is allegedly due to differences in the value of cellulase enzyme
activity used in carrageenan extraction. The research of Varadarajan et al (2009) used a cellulase enzyme
with an activity value of ≥700 U/g in the carrageenan extraction process.

The value of yield with 1% enzyme concentration was the highest accounting for 28.42%. There was
4.51% difference if compared to the treatment with no enzyme addition. This means that the addition of
1% cellulase enzyme from EN fungus increased the productivity of carrageenan extraction by 4.51%.
The highest value of yield based on the extraction period was obtained after 3 hours extraction with
27.89%. This result shows that the extension of extraction period increased the carrageenan by 2.72%.

3.3. Sulfate content

Sulfate content is a parameter used for various types of polysaccharides contained in red algae (Winarno
1996). The sulfate content affects the gel strength of carrageenan. The value of carrageenan sulfate
content in this study can be seen in figure 2.

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EMBRIO 2019 IOP Publishing
IOP Conf. Series: Earth and Environmental Science 404 (2020) 012071 doi:10.1088/1755-1315/404/1/012071

18 a a a a a a
16

Sulfate content (%)

14
12
10
8
6
4
2
0
2h 3h 2h 3h 2h 3h
0% 1% 2%
Treatment

Figure 2. Effect of cellulase enzyme concentration and time of extraction on carrageenan sulfate
content.

Based on the result of sulfate content analysis using ANOVA, it is known that the interaction between
enzyme concentration and time of extraction did not have a significant effect on the sulfate content with
p-value>0.01. The sulfate content of carrageenan in this study ranged from 16.05% to 16.95%, the value
still meets the quality standard of carrageenan according to FAO (2007) with sulfate content ranged
from 15-50%. Although the sulfate content in this study still meets the standards, this value remains
very low when compared to the sulfate content of the carrageenan produced using alkali compounds.
Wenno et al (2012) in his research obtained the value of sulfate content in carrageenan was 27.43%.

3.4. Viscosity
Viscosity is the power of molecular flow in the solution system. Viscosity test was done to know the
degree of viscosity of carrageenan solution at a certain concentration and temperature. In this study,
viscosity measurements were performed using 1.5% carrageenan solution at 75°C and measured by a
Brookfield viscometer. The result of viscosity analysis can be seen in figure 3.
35 b
30 d d
c
Viscosity (cP)

25 a a
20
15
10
5
0
2h 3h 2h 3h 2h 3h
0% 1% 2%
Treatment

Figure 3. Effect of cellulase enzyme concentration and time of extraction on carrageenan viscosity.

Based on the results of viscosity analysis using ANOVA, it was found that enzyme concentration factor,
extraction time and interaction between two factors showed a significant effect with a p-value<0.01.
Viscosity of carrageenan extracted for 2 h was higher than that of 3 h extraction with a difference of
2.67 cP. This result shows that the extension of extraction time decreased the viscosity value by 2.67
cP.

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EMBRIO 2019 IOP Publishing
IOP Conf. Series: Earth and Environmental Science 404 (2020) 012071 doi:10.1088/1755-1315/404/1/012071

The enzyme concentration had a significant effect on the viscosity value, with the highest value at the
enzyme concentration level of one percent and the lowest for the treatment with no enzyme addition.
The results of this analysis indicate that the addition of enzyme concentration increased carrageenan
viscosity with a concentration limit of 1%. The highest viscosity value 29.5 cP was found in the
interaction of treatment level of enzyme concentration of 1% and 2 h of extraction time. Overall, the
carrageenan viscosity value still follows the quality standard of carrageenan according to FAO that is
above 5 cP.

3.5. Gel strength

Gel strength is one of the characteristics to determine the use of carrageenan. The gel strength of
carrageenan is in correlation with its sulfate content. The gel strength value of carrageenan gel produced
in this study can be seen in figure 4.
30
24.75
25 20.2 21.75 22.2
Gel strength (gF)

20.04
20
14.6
15
10
5
0
2h 3h 2h 3h 2h 3h
0% 1% 2%
Treatment

Figure 4. Effect of cellulase enzyme concentration and time of extraction on carrageenan gel strength.

The gel strength values generated in this study ranged from 14.6 gF to 24.75 gF, with the highest value
in the SB carrageenan with treatment 3 h of extraction time and 1% of enzyme concentration. SB
carrageenan has the lowest sulfate content compared with other treatments. This result is similar to that
of Wenno et al (2012) that the smaller of sulfate content increases the consistency of carrageenan gel
strength.

3.6. Acid-insoluble ash content

The acid-insoluble ash content is an indicator of the hygiene of carrageenan. In this study it was known
that the acid-insoluble ash value was in the range of 0.02 to 0.21% of each treatment, this value is still
under the maximum limit according to FAO (2007) that is 1%. This low acid-insoluble ash content
shows that the carrageenan was produced in this study have a high level of hygiene.

3.7. Functional group test by FTIR

The identification using FTIR aims to show the functional groups present in the carrageenan samples.
The FTIR data of carrageenan from this study were compared to commercial carrageenan. FTIR analysis
showed that the results of this research have a number of waves that are similar to the commercial
carrageenan, hence it can be stated that the carrageenan produced in this research was confirmed. The
FTIR spectrum of commercial carrageenan and the results of this study can be seen in figure 5.

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EMBRIO 2019 IOP Publishing
IOP Conf. Series: Earth and Environmental Science 404 (2020) 012071 doi:10.1088/1755-1315/404/1/012071

d
c
b
a

1300 1000 700 400

Wavenumber (cm-1)

Figure 5. Functional groups of each hydrolysis product with different enzyme concentration, FTIR
spectrum (a) commercial carrageenan, (b) carrageenan with 0% enzyme, (c) carrageenan with enzyme
1% (d) carrageenan with 2% enzyme.

4. Conclusion

The enzyme of marine fungus EN can be used to extract carrageenan. Enzyme concentration, extraction
period and interaction of extraction time and enzyme concentration are affect the yield value, viscosity
and gel strength of the carrageenan.

Acknowledgements

Acknowledgements are given to Ministry of Research, Technology and Higher Education

of the Republic of Indonesia who has funded this research through Competency-Based Research Scheme
to Dr. Kustiariyah Tarman, SPi, MSi with contract number 1442 / IT3.11 / PN / 2017 and 1608 / IT3.11
/ PN / 2018.

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IOP Conf. Series: Earth and Environmental Science 404 (2020) 012071 doi:10.1088/1755-1315/404/1/012071

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