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Optimization of Carrageenan Extraction from Eucheuma Spinosum Using Pilot Scale Ohmic Technology
Salengke Salengkea,*, Andi Hasizahb, Supratomoa, Abdul Warisa, Meta Mahendradattac, Amran Lagac, Metusalachd
aDept. of Agricultural Engineering, Hasanuddin University, Makassar, INDONESIA bAgricultural Systems Study Program, Graduate School, Hasanuddin University, Makassar, INDONESIA
cDept. of Food Science, Hasanuddin University, Makassar, INDONESIA dDept. of Fisheries, Hasanuddin University, Makassar, INDONESIA
*Corresponding author e-mail address: salengke@unhas.ac.id
Abstract:
Carrageenans are important hydrocolloids extracted from red seaweeds (Rhodophyceae) of genus Eucheuma, Iridiae,
Gigartina, and Chondrus. Carrageenan extraction technologies currently applied in modern industries are based on
conventional heating using double-jacketed tanks supplied with steam as heating medium. This technology is inefficient in
energy use and poses a complicated processing setup since it requires a dedicated steam generator and steam delivery system.
The objectives of this study were to test the performance of ohmic-based technology for carrageenan extraction and to find
optimum conditions for extraction of carrageenan from Eucheuma spinosum using ohmic heating technology. The experiment
was conducted using a 7.5 liter ohmic heating chamber. The experiment was designed using central composite design (CCD)
of Response Surface Methodology (RSM) with four parameters (heating temperature, extraction time, KOH concentration, and
seaweed to solution ratio) and the responses measured were carrageenan yield, viscosity, and gel strength. The experimental
results showed that extraction yields and gel strength generally increased as both temperature and extraction time was
increased. Optimum extraction condition was obtained at 88.1oC, extraction duration at 163.5 min, KOH concentration at
0.4M, and seaweed to solution ratio (w/v) at 1:36.1. The responses at the optimum conditions were carrageenan yield 52.27%,
gel strength 62.46 Bloom, and viscosity 274.5 cP.
Keywords: carrageenan extraction, Eucheuma spinosum, ohmic technology
1. Introduction
Carrageenans are hydrocolloids extracted from red seaweeds (Rhodophyceae) of genus Eucheuma, Iridiae,
Gigartina, and Chondrus. Carrageenans are versatile ingredients which are used in food and non-food industries as
stabilizing, thickening, and gelling agents. Global production of cultivated seaweeds exceeded 23 million metric
tons in 2012 where approximately 99% of the total production was contributed by Asian countries (FAO 2014).
Carrageenan productions are mostly done by heating carrageenan producing seaweeds in alkali solutions such
as KOH and NaOH. Temperature and duration of heating are determined based on the desired final product. For
production of semi-refined carrageenan, heating temperature is mostly kept under 80oC to avoid carrageenan loss
due to leaching. Heating of seaweeds in alkali solution convert non-gelling carrageenan precursor (mu and nu
carrageenans) into gelling carrageenans (kappa and iota respectively) (Falshaw et al., 2001). The concentration of
alkali used and the length of heating are based on the degree of conversion (desulfation) desired but these two
factors affect yield and properties of carrageenan produced. Other processing factors that have been reported to
affect the yield and the properties of carrageenan are heating temperature and ratio between the volumes of alkali
solution used in the heating process and the weight of the seaweed processed.
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Studies on modification and extraction of carrageenan from various seaweeds have been reported by many
researchers and the aforementioned processing factors have been identified to affect yield and properties of
carrageenan produced (Hoffmann et al., 1995; Normah and Nazarifah, 2003; Hilliou et al., 2005; Tuvikene et al.,
2006; Distantina et al., 2011; Webber et al., 2012; Villanueva and Montano, 2014). Many of the studies reported in
literature have shown that processing conditions such as temperature and length of heating as well as concentration
of alkali used as extracting solution have reciprocal effects on yield and quality (gel strength and viscosity). For
instance, increasing KOH concentration reduced extraction yield and intrinsic viscosity but increased gel strength
significantly (Tuvikene et al., 2006; Distantina et al., 2011; Villanueva and Montano, 2014). Due to the
contradictory effects of processing factors on yield and quality of carrageenan produced, it is important to optimize
the processing conditions in order to obtain acceptable quality at optimum yield.
Optimizations of processing conditions for carrageenan production from various types of seaweeds have been
reported by several researchers. However, all studies on carrageenan extraction that have been reported in
literatures were based on small scale experiments using conventional heating. In a recent study reported by Hasizah
et al. (2017), extraction of carrageenan from Eucheuma spinosum using pilot scale ohmic heating technology
showed promising results. The same technology was used in this study to find an optimum condition for extraction
of carrageenan from Eucheuma spinosum.
Optimization of extraction of carrageenans using ohmic based technology is important in order to obtain
optimum yield and quality and improve overall efficiency of the extraction process and economic viability of the
technology. Processing factors affecting yield and carrageenan quality have been identified by several researchers
(Suryaningrum et al., 2003; Tuvikene et al., 2006; Freile-Pelegrin et al., 2006; Mustapha et al., 2011; Webber et
al., 2012; and Bono et al., 2014) and optimum conditions for carrageenan extraction using conventional heating
technology have been identified. In this study, optimum condition for extraction of carrageenan from Eucheuma
spinosum using pilot scale ohmic heating technology was determined using Central Composite Design (CCD) of
response surface methodology (RSM). The RSM has been used extensively for improving and optimizing
processes (Myers et al., 2009; Kumar et al., 2009; Sobukola et al., 2009; El-Sersy et al., 2010; Elumalai and
Thangavelu, 2010; Fan et al., 2011; Bono et al., 2014).
2. Materials and method
Extraction of carrageenan from Eucheuma spinosum was done using a pilot scale ohmic heating chamber. The
chamber was made of PVC pipe (5 inch inside diameter) with internal volume of 7.5 liters. The details of the
ohmic heating setup were described in Hasizah et al. (2017). Extraction experiment was designed based on Central
Composite Design (CCD) with four variables (temperature, X1; extraction duration, X2; KOH concentration, X3;
and ratio between KOH volumes to seaweed weight, X4). Each variable was varied at three levels (coded -1, 0, +1)
such that the total number of experimental runs were 31 (16 at factorial points, 8 at axial points, and 7 at center
point for estimation of pure error and curvature of response surface).
The values of each variable at factorial points (levels -1, 0, and +1) were determined based on the range of
values reported by various researchers in literatures. Extraction temperatures commonly used by researchers are in
the range of 85–100oC and extraction durations were in the range of 3–6 hours. Therefore, the -1 level for
extraction temperature and duration were set at 85oC and 2 hours respectively, the 0 level were at 90oC and 3
hours, and the +1 level were at 95oC and 4 hours. The –α and +α levels for temperature and duration were at 80oC
and 1 hour and 100oC and 5 hours respectively. KOH concentrations used in the experiments were 0.2 M (level –
α), 0.4 M (level -1), 0.6 M (level 0), 0.8 M (level +1) and 1.0 M (level +α) and the ratios of KOH solution volume
to seaweed mass were set at 15:1 (level –α), 25:1 (level -1), 35:1 (level 0), 45:1 (level +1), and 55:1 (level +α).
Following each extraction process, the KOH solution containing the carrageenan extracted from the seaweed was
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separated from solid residues by filtration using cheese clothes and the residues were washed three times with 2
liters of fresh KOH solution. The carrageenan contained in the KOH solution was recovered by precipitation using
90% methanol with the ratio between methanol and KOH solution was 2:1 (v/v). The carrageenan obtained was
sun dried to about 12% moisture content. The responses measured were extraction yield, gel strength (Bloom value
of carrageenan gel at 1.5% carrageenan), and viscosity at standard condition (1.5% solution at 75oC). The gel
strength was measured using texture analyzer (Stable Micro Systems, Surrey, UK) equipped with Bloom test
apparatus and solution viscosity was measured using Brookfield viscometer (Brookfield Engineering, Middleboro,
Massachusetts, US). The data obtained were analyzed using Design Expert software. The main effects of each
variable as well as their interactions were analyzed using analysis of variance (ANOVA). Regression analyses were
also performed to obtain models for estimation of responses. Adequacy of each model was evaluated using two
criteria, i.e. R2 value and F value from lack of fit test.
3. Results and discussion
The choice of the independent variables in the optimization of extraction conditions was based on previous
research reported by various researchers. Several research on carrageenan extraction from various types of
seaweeds reported that temperature and duration of heating, alkali concentration, and ratio between the volumes of
alkali solution and the weight of seaweed affected extraction yield and carrageenan quality (Suryaningrum et al.,
2003; Tuvikene et al., 2006; Freile-Pelegrin et al., 2006; Mustapha et al., 2011; Webber et al., 2012; and Bono et
al., 2014). All of the aforementioned studies used conventional heating such as heating using hot plate during
extraction process. Therefore, in this study, we employed the four variables in an effort to find an optimum
condition for extraction of carrageenan from Eucheuma spinosum using ohmic heating technology. The middle
values (zero level) used in experiment were temperature (X1) 90oC, extraction duration (X2) 180 menit, KOH
concentration (X3) 0.6 M, and ratio of solution volume to seaweed weight (X4) 35:1. Recovery of extracted
carrageenan from KOH solution was done using 90% methanol with wolume ratio of 2 parts methanol to 1 part of
solution. The responses measured were extraction yield, gel strength, and viscosity.
Extraction yield, gel strength, and carrageenan viscosity obtained from 31 experimental runs were shown in
Table 1. Extraction yield varied from 42.1% - 74.2% (12% moisture content) or equivalent to 37% - 65% dry basis.
These values are in accordance with average yield of 57.9% reported by Dawes et al. (1977), 44-59% reported by
Buriyo et al. (2001), 53 and 56% reported by Vairappan et al. (2003), 56.6% reported by Wakibia et al. (2006),
and 43.5 – 57.2% reported by Freile-Pelegrin and Robledo (2008). The wide range of extraction yields obtained in
this study indicates that extraction yield was affected by extraction condition.
The results of the sequential regression analysis performed by Design Expert software (Table 2) show that the
recommended model for extraction yield was a linear model. The addition of two factor interaction (X1X2, X1X3,
X1X4, X2X3, X2X4, and X3X4) into the linear model did not improve the model prediction significantly (p>0.05). The
addition of quadratic factors (X12, X2
2, X32, and X4
2) and cubic factors (X13, X2
3, X33, and X4
3) also did not give
significant improvement to model prediction (p>0.05). The lack of fit test in Table 2 indicate that the F value for
the linear model is 0.54 with p value = 0.86. These values show that the lack of fit is insignificant and the linear
model can provide adequate prediction of the extraction yield. The result of analysis of variance for the linear
model is given in Table 3. Based on the p values of each factor in Table 3, it can be seen that the significant factors
affecting extraction yield were extraction duration (p=0.046) and ratio between solution volumes to seaweed
weight (p=0.0075). Extraction temperature only provides marginal effect (p=0.1192) while the effect of KOH
concentration was insignificant (p=0.5527). It can also be seen from Table 3 that the F and the p values for the
linear model (3.94 and 0.0125 respectively) suggest that all the factors in the model can significantly predict
extraction yield. The linear model for the prediction of extraction yield is presented in Equation 1 (for coded
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factors) and Equation 2 (for actual factors) and indicates that extraction duration and ratio of KOH solution
volumes to seaweed mass significantly affect extraction yield. Response surface plot at the zero level of
temperature (90oC) and KOH concentration (0.6M) show that extraction yield increases liniearly as extraction
duration and the ratio of KOH solution volumes to seaweed mass increases.
Table 1. Extraction yield, gel strength, and viscosity of carrageenan from E. spinosum at various extraction
conditions.
Run # Temp, C Duration, t
KOH Conc., M
Ratio Yield, % Gel strength, Bloom
Viscosity, cP
1 95 240 0.8 45:1 65.8236 56.8348 258
2 90 180 0.6 35:1 60.094 58.6147 259
3 85 240 0.8 25:1 46.606 89.7947 283
4 85 240 0.8 45:1 74.051 66.4528 267
5 80 180 0.6 35:1 44.3531 85.6976 280
6 90 180 0.6 35:1 43.775 64.2331 265
7 90 180 1 35:1 56.5422 68.33 268
8 95 240 0.4 45:1 69.8345 71.6528 272
9 90 180 0.6 55:1 61.5603 59.5262 260
10 95 240 0.8 25:1 68.5349 62.8278 262
11 90 300 0.6 35:1 51.9665 64.6532 251
12 95 240 0.4 25:1 69.5205 60.6466 261
13 85 120 0.4 25:1 42.1015 78.5045 277
14 95 120 0.8 45:1 62.3359 63.3395 264
15 90 180 0.6 35:1 56.1189 75.3221 273
16 90 180 0.2 35:1 59.1648 63.5889 264
17 85 240 0.4 45:1 53.6213 91.4047 285
18 90 180 0.6 35:1 60.9063 90.9929 284
19 90 60 0.6 35:1 47.509 47.3098 268
20 90 180 0.6 35:1 74.2074 85.3453 280
21 95 120 0.4 45:1 58.3038 67.1754 268
22 85 120 0.8 45:1 64.5904 57.4026 258
23 90 180 0.6 15:1 44.5054 86.6547 282
24 85 120 0.4 45:1 59.595 62.6989 262
25 85 120 0.8 25:1 47.2264 72.236 271
26 90 180 0.6 35:1 71.9847 77.9081 276
27 90 180 0.6 35:1 62.2888 66.7691 267
28 95 120 0.4 25:1 43.03 57.4593 258
29 85 240 0.4 25:1 56.6032 70.3171 271
30 95 120 0.8 25:1 52.7163 69.3193 270
31 100 180 0.6 35:1 53.6902 79.39 284
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Table 2. Results of analysis of variance (ANOVA) for extraction yield.
Sequential Model Sum of Squares
Source Sum of Squares
DF Mean Square
F Value Prob > F
Mean 1.03E+05 1 1.03E+05
Linear 1047.99 4 262 3.94 0.0125 Suggested
2FI 332.43 6 55.41 0.79 0.5859
Quadratic 229.08 4 57.27 0.78 0.5515
Cubic 309.87 8 38.73 0.36 0.9144 Aliased
Residual 857.7 8 107.21
Total 1.05E+05 31 3398.29
Lack of Fit Tests Source Sum of
Squares DF Mean
Square F Value Prob > F
Linear 1111.77 20 55.59 0.54 0.8601 Suggested
2FI 779.34 14 55.67 0.54 0.8389
Quadratic 550.26 10 55.03 0.53 0.8178
Cubic 240.39 2 120.2 1.17 0.3728 Aliased
Pure Error 617.31 6 102.88
Table 3. Analysis of variance table for linear model.
Source Sum Squares DF Mean Square F Value Prob > F
Model 1047.99 4 262 3.94 0.0125 significant
Temperature (X1) 172.69 1 172.69 2.6 0.1192
Duration (X2) 291.28 1 291.28 4.38 0.0463
KOH conc (X3) 24.06 1 24.06 0.36 0.5527
Ratio (X4) 559.96 1 559.96 8.42 0.0075
Residual 1729.08 26 66.5
Lack of Fit 1111.77 20 55.59 0.54 0.8601 not significant
Pure Error 617.31 6 102.88
Cor Total 2777.07 30
42 83.448.352.57 XXYield (1)
RtYield 483.005806.0164.30 (2)
Figure 1. Response surface plot for the effect of extraction
Results of measurement for gel strength show that the gel strength ranged from 47.31 to 91.4 Bloom
equivalent to 37.365 to 72.186 g/cm2 with an average of 55.341 g/c
the less than 50 g/cm2 gel strength reported by
strength of 158 g/cm2 reported by Istini et al. (1994)
viscosity of the carrageenan obtained in this study ranged from
higher than the values reported from the study by Vairappan
– 254 cP. These values are also higher than the maximum
Pelegrin et al. (2006) and Freile-Pelegrin and Robledo (200
Eucheuma isiforme.
Results of analysis of variance (Table 4)
model but model prediction is significant only at 10% level (
that the lack of fit is insignificant (p=0.8712) which indicate that the quadratic model can adequately fit the
experimental results. The quadratic equation for gel strength is given in Eq. 3 (for coded factors) and Eq. 4 (for
actual factors). It is important to note that the factor
17.74strengthGel
33.707strengthGel
Response plots for gel strength at various conditions are given in Figure 2.
two variables while the other two variables are kept at the zero level. It can be seen from the figure that gel strength
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Figure 1. Response surface plot for the effect of extraction conditions on yield.
Results of measurement for gel strength show that the gel strength ranged from 47.31 to 91.4 Bloom
with an average of 55.341 g/cm2. This average value is slightly higher than
gel strength reported by Freile-Pelegrin et al. (2006, 2007) but lower than the average gel
reported by Istini et al. (1994) and 100.8 g/cm2 reported by Wakibia
viscosity of the carrageenan obtained in this study ranged from 251 to 285 cP (average 269.29).
higher than the values reported from the study by Vairappan et al. (2003) which indicated average viscosity of 250
ese values are also higher than the maximum viscosity of 161.3 cP and 144.6 cP reported by Freile
Pelegrin and Robledo (2007) respectively for iota carrageenan extracted from
(Table 4) indicate that the gel strength can be best modeled using quadratic
model but model prediction is significant only at 10% level (p=0.0816). Nonetheles, result of lack of fit test
=0.8712) which indicate that the quadratic model can adequately fit the
The quadratic equation for gel strength is given in Eq. 3 (for coded factors) and Eq. 4 (for
It is important to note that the factors included in the model are only those with p values < 0.1.
43221 76.467.484.3 XXXX
)*(3798.2)(001298.0)(245.15 2 RCtT
Response plots for gel strength at various conditions are given in Figure 2. Each plot represents the effect of
other two variables are kept at the zero level. It can be seen from the figure that gel strength
on yield.
Results of measurement for gel strength show that the gel strength ranged from 47.31 to 91.4 Bloom value or
. This average value is slightly higher than
but lower than the average gel
reported by Wakibia et al. (2006). The
251 to 285 cP (average 269.29). These values are
(2003) which indicated average viscosity of 250
viscosity of 161.3 cP and 144.6 cP reported by Freile-
for iota carrageenan extracted from
indicate that the gel strength can be best modeled using quadratic
lack of fit test indicate
=0.8712) which indicate that the quadratic model can adequately fit the
The quadratic equation for gel strength is given in Eq. 3 (for coded factors) and Eq. 4 (for
s included in the model are only those with p values < 0.1.
(3)
(4)
Each plot represents the effect of
other two variables are kept at the zero level. It can be seen from the figure that gel strength
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was slightly affected by extraction temperature and significantly affected by extraction duration. The slight effect
of the interaction between KOH concentration and solution to seaweed ratio is also apparent from the plot.
Results of analysis of variance for viscosity indicate that there no adequate model for predicting viscosity. The
lowest p value obtained (p=0.1238) was for quadratic model which indicate that all models are insignificant at 10%
level. Anova result for response surface quadratic model also show an F value of 1.43 with p value of 0.2447 which
imply that the model is insignificant relative to the noise. Result of analysis of variance for response surface
quadratic model shows that the only significant factor in the model is extraction duration (p=0.0464) and the
interaction between KOH concentration and ratio of KOH solution to seaweed weight. Response plots for
carrageenan viscosity are given in Figure 3.
Table 4. Results of analysis of variance (ANOVA) for gel strength.
Sequential Model Sum of Squares
Source Sum of Squares DF Mean Square F Value Prob>F
Mean 1.52E+05 1 1.52E+05 Suggested
Linear 859.99 4 215 1.82 0.1547
2FI 720.74 6 120.12 1.02 0.4383
Quadratic 907.93 4 226.98 2.53 0.0816 Suggested
Cubic 494.22 8 61.78 0.52 0.8103 Aliased
Residual 943.34 8 117.92
Total 1.56E+05 31 5037.51
Lack of Fit Tests
Source Sum of Squares DF Mean Square F Value Prob > F
Linear 2247.55 20 112.38 0.82 0.6601
2FI 1526.81 14 109.06 0.8 0.6605
Quadratic 618.88 10 61.89 0.45 0.8712 Suggested
Cubic 124.66 2 62.33 0.46 0.6536 Aliased
Pure Error 818.68 6 136.45
Figure 2. Response surface plot for the effect of
Figure 3. Response surface plot for the effect of processing conditions on viscosity.
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Figure 2. Response surface plot for the effect of processing conditions on gel strength.
Figure 3. Response surface plot for the effect of processing conditions on viscosity.
processing conditions on gel strength.
Figure 3. Response surface plot for the effect of processing conditions on viscosity.
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The optimum conditions for extraction of carrageenan from Eucheuma spinosum were obtained from Design
Expert software (Stat. Ease Minneapolis, USA). It can be seen from the solutions given by the software that
extraction condition with highest desirability (0.754) was extraction at 85oC for 238.91 minutes using KOH
concentration of 0.4 M and solution volume to seaweed weight ratio of 44.99:1. Under this condition, extraction
yield is expected at 65.77% with gel strength of 82.129 Bloom and viscosity of 276.022 cP. If gel strength and
viscosity are to be maximized, the optimum extraction condition is at temperature 85oC, heating duration 225.8
minutes, KOH concentration 0.68 M, and solution volume to seaweed weight ratio 27.69. Under this condition, gel
strength is 85.481 Bloom value (67.512 g/cm2) and viscosity 278.96 cP.
4. Conclusions
Overall results indicate that ohmic heating technology is effective for extraction of carrageenan from Eucheuma
spinosum. Yield of extraction was comparable to the yields reported in literatures from laboratory experiments.
The most significant factors affecting extraction yield were extraction temperature and solution volume to seaweed
weight ratio. Extraction yield can be modeled adequately using linier model. Optimum condition for extraction of
carrageenan from Eucheuma spinosum was at temperature 85oC, heating duration 238.91 minutes, KOH
concentration 0.4 M, and solution volume to seaweed weight ratio of 44.99:1.
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