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Extraction and characterisation of alginate from brown seaweeds (Fucales,

Phaeophyceae) collected from Port Dickson, Peninsular Malaysia

Article  in  Journal of Applied Phycology · April 2011

DOI: 10.1007/s10811-010-9533-7

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Extraction and characterisation of
alginate from brown seaweeds (Fucales,
Phaeophyceae) collected from Port
Dickson, Peninsular Malaysia

Journal of Applied Phycology

ISSN 0921-8971
Volume 23
Number 2

J Appl Phycol (2011)

23:191-196
DOI 10.1007/
s10811-010-9533-7

1 23
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1 23
 Author's personal copy
J Appl Phycol (2011) 23:191–196
DOI 10.1007/s10811-010-9533-7

Extraction and characterisation of alginate from brown

seaweeds (Fucales, Phaeophyceae) collected from Port
Dickson, Peninsular Malaysia
Swee-Yong Chee & Ping-Keong Wong & Ching-Lee Wong

Received: 10 December 2009 / Revised and accepted: 12 May 2010 / Published online: 3 June 2010
# Springer Science+Business Media B.V. 2010

Abstract Four species of brown seaweeds, namely Sar- temperature. Alginate extracted from S. baccularia was
gassum baccularia, Sargassum binderi, Sargassum found to be very heat-sensitive. Its Mw has dropped more
siliquosum and Turbinaria conoides, harvested from Port than 83%, from 7.52×105 to 1.23×105 g mol−1, when the
Dickson, Negeri Sembilan, Malaysia were analysed extraction temperature was raised. The effect of heat on
for ash content, alginate yield and alginate properties. the extent of depolymerisation of the alginate molecule
Seaweeds calcined at 450°C were found to have low of the other three brown seaweed species was less
amount of non-combustible residue as these were not significant, with decrease in molecular weight ranging
contaminated by calcareous animals. Alginate was between 13% and 16%.
extracted from these seaweeds by two methods: hot and
cold. In the hot method, the storing time was 3 h and the Keywords Alginate yield . Ash content . Brown seaweed .
processing temperature was 50°C, whilst in the cold Intrinsic viscosity . Molecular weight
method, the sample was stored overnight at room
temperature. Higher yield of alginate was obtained by
the hot method compared to the cold method, but alginate Introduction
extracted by the cold method gave higher molecular
weight. In the hot method, 49.9% of alginate was Seaweeds are a potential renewable resource in the marine
extracted from S. siliquosum, followed by T. conoides environment where about 6,000 species have been identi-
(41.4%), S. binderi (38.9%) and S. baccularia (26.7%). fied and grouped into different classes: green (Chlorophy-
Alginate extracted from T. conoides has an average ceae), brown (Phaeophyceae) and red (Rhodophyceae)
molecular weight, Mw, of 8.06×105 g mol−1, whereas algae. Total global seaweed production in 2004 was more
alginate from S. siliquosum was the lowest in Mw (4.81× than 15 million t, of which nearly 15–20% is contributed by
105 g mol−1) when the extraction was done at room the Indian Ocean region. About 100,000 t (wet weight) of
seaweed are harvested across the Indian coast (Chandini et
Paper presented at the 7th Asia Pacific Congress on Algal al. 2008). Seaweeds are extensively used as food by coastal
Biotechnology, New Delhi, 2009. peoples, particularly in East Asia such as China and
S.-Y. Chee (*) : P.-K. Wong Vietnam, but also in some parts of Europe including
Faculty of Science, Universiti Tunku Abdul Rahman, Scotland. This is because seaweeds are known to provide
Jalan Universiti, an excellent source of bioactive compounds such as
Bandar Barat,
carotenoids, dietary fibre, essential fatty acids, vitamins
31900 Kampar, Perak, Malaysia
e-mail: csy@utar.edu.my and minerals (Chandini et al. 2008). Malaysia has a high
diversity of marine algae, with 261 taxa, although only few
C.-L. Wong studies on the distribution and ecology of algae have been
Department of Science, Faculty of Engineering and Science,
reported in Peninsular Malaysia (Wong and Phang 2004).
Universiti Tunku Abdul Rahman,
Jalan Genting Kelang, Sargassum and Turbinaria are widespread tropical
53300 Kuala Lumpur, Malaysia genera, and more than 400 species of Sargassum exist
 Author's personal copy
192 J Appl Phycol (2011) 23:191–196

worldwide and 25 species are found in Malaysia (Wong and study on the effect of temperature is vital in avoiding
Phang 2004). Turbinaria is relatively species-poor, with extra cost of heating. The control of alginate viscosity is
only 17 species described (Rohfritsch et al. 2007). Both a vital factor for the industry as the use of alginate
Sargassum and Turbinaria are found abundantly at Port depends mainly on its viscosity. Low viscosity is used in
Dickson, Negeri Sembilan, west coast of Peninsular paper making and fruit industry. High viscosity alginate
Malaysia. is normally used in food and cosmetics (Hernandez-
Alginate, or algin, is present in the cell walls of brown Carmona et al. 2000). The ability of sodium alginate to
seaweeds, and it is partly responsible for the flexibility of form viscous solutions and gels in aqueous media is
the seaweed (McHugh 2003). Alginate is a linear glycuronan important in the pharmaceutical industry (Sriamornsak et
of (1,4)-linked α-L-guluronate (G) and β-D-mannuronate al. 2007). Thus, the main focus of the present study was to
(M) residues arranged in a non-regular blockwise pattern determine the ash content, the alginate yield, alginate
along the chain (Hernandez-Carmona et al. 2000; Larsen et viscosity and molecular weight, and the effect of temper-
al. 2003). The actual chemical structure of the alginate varies ature on the alginate properties of four species of brown
between genera, and a similar variability is found in the seaweeds commonly found at Port Dickson, Malaysia.
properties of the alginate that is extracted from the seaweed
(McHugh 2003). The molecular weight of alginate ranges
generally between 500 and 1,000 kDa. Its solubility is Materials and methods
influenced by factors such as pH, concentration, ions in
solution and the presence of divalent ions (Rioux et al. Four species of brown seaweeds, Sargassum baccularia,
2007). The properties of the alginate vary between species, Sargassum binderi, Sargassum siliquosum and Turbinaria
so the choice of which seaweeds to harvest is based on conoides were collected from Port Dickson, Negeri
both the availability of particular species and the Sembilan, west coast of Peninsular Malaysia. The seaweed
properties of the alginate that they contain. The main samples were washed with water to remove impurities
commercial sources are species of Ascophyllum, Durvil- such as sand and air-dried for at least 2 days. Then, the
laea, Ecklonia, Laminaria, Lessonia, Macrocystis, Sar- seaweed samples were cut into small pieces and subse-
gassum and Turbinaria (McHugh 1987). Alginates are quently kept in bottles and stored in the refrigerator.
widely used in the pharmaceutical, cosmetic, food and To determine the ash content, three sets of dry samples
biotechnology industries (Hernandez-Carmona et al. 2000) (5 g each) were placed in crucibles and dried in the oven at
to produce products such as paper coatings, adhesives, 105°C for 30 min. The dry samples were weighed again
dyes, gels and explosives. The quality of alginate is and then calcined in the muffle furnace at 450°C for 3 h.
determined by how well it performs in thickening aqueous Calcined samples were cooled in a desiccator and weighed
solutions and forming gels (McHugh 2003). again to determine the ash content according to the
Alginic acid is present in brown seaweeds mainly in the following equation:
form of calcium salt of alginic acid, although other salts
Weight of ash
such as sodium might be present. The purpose of the Ash content ð%Þ ¼  100:
Weight of dry seaweed
extraction step is to convert the alginate to the soluble form
of sodium alginate and remove it from the algae (Hernandez-
Carmona et al. 2000). In principle, the isolating process of Alginate was extracted by two methods. In the cold
alginates includes stages of pre-extraction with hydrochloric method, two sets of 20 g of air-dried seaweed samples
acid (HCl), washing, filtration and neutralisation with were soaked in 300 mL of 1% CaCl2 solution at room
alkali. Lastly, sodium alginate is precipitated from the temperature (27°C) overnight (around 18 h). Then, the
solution by alcohol and re-precipitated in the same way. seaweed was washed with distilled water (3×300 mL),
Larsen et al. (2003) also treated the seaweed with stored in 5% HCl solution for 1 h and washed again with
formaldehyde before extracting with HCl. However, this distilled water (3×300 mL). After that, the samples were
step was not carried out in this study. For the pre-extraction stored in 300 mL of 3% Na2CO3 solution for 1 h and
step, some researchers suggest that acid treatment is not 250 mL of water was added into it before it was left to
essential for the extraction of sodium alginate, the reason stand overnight. The viscous mixture was separated from its
being that alginic acid present in seaweed is mostly in residue by centrifuging at 14,000×g. Sodium alginate
the form of free acid (Hernandez-Carmona et al. 2000). The extracted was precipitated from the solution by adding an
extraction step can be used to control the viscosity of the ethanol/water mixture (1:1, v/v). The precipitate was
final product. Higher temperature during extraction can lead filtered, washed with ethanol again and dried in air,
to the breakdown of uronic acid chains and consequently followed by drying in a vacuum oven. In the hot method,
lower the viscosity of the sodium alginate extracted. The samples were treated similarly as the cold method, except
 Author's personal copy
J Appl Phycol (2011) 23:191–196 193

Table 1 Ash content and percentage yield of alginate extracted using cold and hot extraction methods

Species Ash content of algae (%) Cold method Hot method

Mean weight of alginate (g) Mean yield (%) Mean weight of alginate (g) Mean yield (%)

S. baccularia 11.6 4.78 23.9 5.34 26.7

S. binderi 14.3 5.75 28.7 7.77 38.7
S. siliquosum 14.6 7.78 38.9 9.97 49.9
T. conoides 16.7 8.10 40.5 8.28 41.4

that the storing time was 3 h at 50°C. Yield of alginate was Houwink–Sakurada equation: ½h ¼ kMva where the k used
calculated as follows: was 0.023 and the a was 0.984, as proposed by Clementi et
al. (1998) as quoted by Torres et al. (2007) to empirically
Weight of alginate
Yield of alginate ð%Þ ¼  100: relate [η] and the weight average molecular weight, Mw.
Weight of dry seaweed
The a value in the Mark–Houwink–Sakurada equation
Viscosity measurements for alginate samples were carried ranges from 0.73 to 1.31 depending on the ionic strength
out at 25°C for 0.4% alginate solutions with a Ubbelohde and alginate composition (Torres et al. 2007).
glass viscometer (Cannon, USA) which has a capillary
diameter of 0.56 mm. The flow time of the solution, t, was
measured relative to the flow time of distilled water, t0. The Results
water bath was set to 25.0±0.1°C. After 10 mL of dissolved
sample was measured into the viscometer, it was left in the There average ash content was 11.6% in S. baccularia,
water bath for about 10 min to attain thermal equilibrium. 14.3% in S. binderi, 14.6% in S. siliquosum and 16.7% in
The solution was pumped above the upper indicator line T. conoides (Table 1).
and allowed to flow under the influence of gravity and From Table 1, it was found that the hot method extracted
timed. This was repeated three times, and the average slightly more sodium alginate as compared to the cold
reading was taken as the flow time, t. Concentration of the method. The hot method extracted 26.7% of alginate from
sample was reduced with the addition of 1 mL of distilled S. baccularia, whilst 23.9% of alginate was extracted by
water subsequently, and dilution was repeated at least six the cold method. A similar trend was also observed in T.
times. The intrinsic viscosity was determined by plotting a conoides, with 41.4% and 40.5% of alginate extracted by
graph of reduced viscosity, ηsp/c (Eq. 3), and inherent the hot and cold methods, respectively. About 28.7% and
viscosity (Eq. 4), ln ηr/c, against the concentration of the 38.9% of alginate was extracted from S. binderi and S.
sample solution, c. siliquosum respectively by the cold method as compared to
Relative viscosity, 38.9% and 49.9% of alginate extracted by the hot method.
The intrinsic viscosity of alginate from S. baccularia
t
hr ¼ ð1Þ dropped drastically (83.3%) when it was extracted through
t0
the hot method, whereas the intrinsic viscosities of
Specific viscosity, alginate from both S. binderi and T. conoides were the
least affected (13–14%) by heat, followed by S. siliquosum
hsp ¼ hr  1 ð2Þ
(16.0%, Table 2).
Reduced viscosity,
hsp hr  1
¼ ð3Þ Table 2 Intrinsic viscosity of the alginate samples
c c
Inherent viscosity, Alginate samples Intrinsic viscosity, [η] (dLg−1)

ln hr Cold method Hot method

hinh ¼ ð4Þ
c
S. baccularia 15.6 2.6
where t is the flow time of solution and t0 is the flow time S. binderi 11.0 9.5
of solvent. S. siliquosum 10.0 8.4
Based on the intrinsic viscosity, viscosity average
T. conoides 16.7 14.5
molecular weight, Mv, was calculated by the Mark–
 Author's personal copy
194 J Appl Phycol (2011) 23:191–196

Table 3 Molecular weight, Mw,

of alginate samples extracted Alginate samples Molecular weight, Mw ×105 (gmol−1) Mw reduction upon heating (%)
from S. baccularia, S. binderi,
S. siliquosum and T. conoides Cold method Hot method

S. baccularia 7.52 1.23 83.6

S. binderi 5.27 4.55 13.7
S. siliquosum 4.81 4.04 16.0
T. conoides 8.06 7.01 13.0

The calculated Mw was in kDa before it was converted to Characterisation of alginate

g mol−1 (Table 3). Among the four species, alginate
extracted from T. conoides has the highest molecular Before extraction, most authors suggest a pre-extraction
weight (8.06×105 g mol−1), followed by S. baccularia. step using acid (Truus et al. 2001), alkali (Baranov et al.
However, alginate from S. baccularia was found to be very 1967) or formalin (Hernandez-Carmona et al. 1999).
heat-sensitive. Its Mw dropped from 7.52×105 to 1.23× Hernandez-Carmona et al. (1999) proved that formaldehyde
105 g mol−1, which was equal to an 83.6% Mw reduction, solution can fix some of the phenolic compounds and
when higher temperature was used in the extraction encourage discolouration. However, as addition of acid may
process. The extent of depolymerisation of the alginate lead to loss of alginate viscosity (Hernandez-Carmona et al.
molecules for the other three species was less severe. For 1999) and discolouration was not necessary in the early
S. binderi, the Mw of the alginate extracted were 5.27×105 stage of extraction, pre-extraction step was not adopted in
and 4.55×105 g mol−1 for the cold and hot methods, this study.
respectively. The magnitude of change in the Mw values At the beginning of extraction, calcium chloride
for S. siliquosum and T. conoides were quite similar to S. solution was used to soak the air-dried sample at 27°C
binderi, which ranged between 13% and 16% in Mw (room temperature) in the cold method and 50°C for the
reduction (Table 3). hot method. Different temperatures in extraction are
known to affect the yield and viscosity of sodium
alginate (Hernandez-Carmona et al. 1999) and the
Discussion rheological properties (Truus et al. 2001). Even a slight
rise in treatment temperature can lead to a rise in
The purpose of keeping the dry seaweed in the refrigerator extraction yield and a drastic fall in viscosity. Hence, the
was to preserve the freshness of the samples. Algae must be treatment temperature normally ranges between room
collected, transported and dried as quickly as possible to temperature and 50°C, and little degradation of alginate
avoid destruction of alginates (Truus et al. 2001). Dry is found to occur with most species of seaweed at
seaweeds were chopped and cut into small pieces before temperatures up to 50°C (McHugh 1987).
storing in the refrigerator. Reduction to small pieces, Calcium chloride is important for the formation of
preferably 5–10 mm2, has two advantages. The first is that calcium alginate with fibrous texture that does not dissolve
in the treatment with acid and alkali, these reagents can in water. HCl was added to convert it back into alginic acid
penetrate the seaweed more thoroughly and more rapidly, (McHugh 2003). Water used before and after the addition
whilst the second advantage is that the seaweed can be of acid washes away excessive calcium chloride and HCl.
transported much more readily (McHugh 1987). Sodium carbonate was added to convert alginic acid to
The low ash content (Table 1) showed that there was sodium alginate (McHugh 2003; McHugh et al. 2001;
little non-combustible residue such as minerals in the Hernandez-Carmona et al. 1999). McHugh (1987) also
seaweed samples. The low percentage of ash also indicated
that the samples were not contaminated by any calcareous Table 4 Average molecular weight of alginates from different sources
organisms which could not be removed by extensive (Torres et al. 2007)
washing (Larsen et al. 2003). The other factor that might
Species Origin Molecular weight, Mw ×105 (gmol−1)
affect the ash content in the sample is the harvesting distance
from land. The collected species grow in the intertidal area of S. fluitans Florida 3.00
coral reefs (<0.4 m above sea level); therefore, frequent S. dentifolium Egypt 6.06
exposure to the sun during the lowest tide might contribute S. asperifolium Egypt 7.34
to the reduction in mass to keep afloat and not being S. latifolium Egypt 4.16
detached from the holdfast when the tide comes in, thus the S. vulgare Brazil 3.30
lower ash content of the seaweeds studied.
 Author's personal copy
J Appl Phycol (2011) 23:191–196 195

states that less soda ash is needed if seaweeds had lowest from S. siliquosum extracted by the cold method.
undergone acid pretreatment. Alginate extracted from S. baccularia was found to be very
To recover sodium alginate, the mixture was centrifuged heat-sensitive. The effect of heat on the extent of
instead of being filtered as it was too viscous for filtration. depolymerisation of the alginate molecular weight from
As sodium alginate does not dissolve in the mixture of the other three brown seaweed species was less significant.
alcohol and water (McHugh 2003), the solution mixture of The molecular weight of the alginate obtained from this
ethanol and water (50:50, v/v) was used to precipitate out work is generally comparable to those obtained from Egypt
the solid form of sodium alginate. The disadvantage of this and Brazil (Torres et al. 2007). Thus, the alginate extracted
step is that alcohol is quite expensive. Instead of sodium from Malaysian species has the potential to be used in
alginate, alginate could also be recovered as alginic acid. relevant industries according to its viscosity range.
However, one of the disadvantages in precipitating it with
acid is that gelatinous precipitate formed is difficult to Acknowledgments The authors express their gratitude to the
dewater (McHugh et al. 2001). The solid sodium alginate government of Malaysia, represented by the Ministry of Science,
Technology and Innovation (MOSTI) for funding.
was then cut into smaller pieces and dried in an oven at
50°C. This was to avoid breakage of bonds in alginate due
to heating. Both the samples that were extracted by the cold
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