Phycological Research 2010; 58: 151–156
Agar from the red seaweed, Laurencia flexilis (Ceramiales,
Rhodophyta) from northern Philippines
pre_573
151..156
Ronald D. Villanueva,1* Jumelita B. Romero,2 Anita Linda R. Ragasa3† and Marco Nemesio E. Montaño1
1
The Marine Science Institute, College of Science, University of the Philippines, Diliman, 1101 Quezon City, 2Mindanao
State University, Tawi-Tawi, Bongao, 1702 Tawi-Tawi, and 3College of Fisheries, Mariano Marcos State University,
Currimao, 2903 Ilocos Norte, Philippines
SUMMARY
The worldwide production of the gelling agent agar
mainly rely on the red algae of the order Gracilariales
and Gelidiales for raw material. We investigate here
the potential of a species from another red algal order,
Ceramiales as an agar source. The agar from Laurencia
flexilis collected in northern Philippines was extracted
using native and alkali treatment procedures and
the properties of the extracts were determined using
chemical, spectroscopic and physical methods. The
native agar, 26% dry weight basis, forms a gel with
moderate gel strength (200 g cm-2). Alkali-treatment
did not enhance the gel strength, indicating insignificant amounts of galactose-6-sulfate residue, the precursor of the gel-forming 3,6-anhydrogalactose (3,6AG) moieties. Furthermore, the Fourier transform
infrared and chemical analysis showed low sulfate and
high 3,6-AG levels, not affected significantly by the
alkali treatment. Nuclear magnetic resonance spectroscopic analysis revealed 3-linked 6-O-methyl-Dgalactose and 4-linked 3,6-anhydro-L-galactose as the
major repeating unit of the native extract, with minor
sulfation at 4-position of the 3-linked galactose residues. The native and alkali treated agars have comparably high gelling and melting temperatures, whereas
the former exhibits higher gel syneresis. Laurencia
flexilis could be a good source of agar that possesses
physico-chemical and rheological qualities appropriate
for food applications. Due to the inability of alkali
treatment to enhance the key gel qualities of the
native extract, it is recommended that commercial
agar extraction from this seaweed would be done
without pursuing this widely-used industrial procedure.
Key words: agar, alkali treatment, Ceramiales, Laurencia flexilis.
INTRODUCTION
Agar is a biopolymer with a wide variety of applications
in food, biotechnology and medicine, primarily as a
© 2010 Japanese Society of Phycology
gelling agent. Chemically, agar is an unbranched
polysaccharide built on a disaccharide repeating unit of
3-linked b-D-galactopyranosyl (G) and 4-linked 3,6anhydro-a-L-galactopyranosyl (LA) residues (Araki
1966). Heterogeneity in this structure comes from substitution with sulfate hemiesters, methyl ethers and
pyruvate ketals at various positions. The variation in
position and degree of substitution influence the property of agar gels. For example, higher degree of substitution with sulfate groups produces lesser agar gel
strength, that is, softer gels.
Agar is synthesized by a number of seaweeds under
the class Rhodophyceae (Craigie 1990). However, only
those from the order Gelidiales (Gelidium, Pterocladia
and Gelidiella) and Gracilariales (Gracilaria, Gracilariopsis and Hydropuntia) are being commercially
exploited as raw materials for its production as gelling
agent in various applications (McHugh 1991; Armisen
1995). In the order Ceramiales, despite the elucidation
of the chemical structure of the agaroid polysaccharide
in the cell-wall of several species (e.g. Campylaephora
hypnaeoides J. Agardh (Usov et al. 1983), Griffithsia
antarctica J.D. Hooker & Harvey and Ceramium uncinatum Harvey (Miller 2003)), the evaluation of the gel
quality of their agar extract is generally lacking. Recent
advances in the agaroid polysaccharides from some
Ceramialean representatives involve screening for pharmacologic activities (Duarte et al. 2004; Grünewald
et al. 2009).
We investigate here the potential of the seaweed,
Laurencia flexilis Setchell (Rhodomelaceae, Ceramiales) for the production of agar. Several species of the
genus Laurencia have been previously studied chemically to primarily contain highly substituted agar
(Bowker & Turvey 1968a,b; Usov et al. 1989; Usov &
Elashvili 1991, 1997; Miller et al. 1993; Siddhanta
*To whom correspondence should be addressed.
Email: [email protected]
†Deceased.
Communicating editor: T. Motomura.
Received 1 October 2008; accepted 24 November 2009.
doi: 10.1111/j.1440-1835.2010.00573.x
152
et al. 2002) and possess poor gelling properties (Miller
et al. 1993). Recent molecular phylogenetic analysis
revealed L. flexilis to be an atypical species in the
genus Laurencia and is an intermediate species
between Laurencia and Chondrophycus (Abe et al.
2006). We further explore here the implication of this
phylogenetic distinctness to agar chemistry and quality.
MATERIALS AND METHODS
Algal material
Laurencia flexilis were collected at Currimao, Ilocos
Norte, northern Philippines (18°03′N, 120°59′E) in
June 1997. Samples were cleaned of epiphyte, calcareous debris, and extraneous seaweeds and then ovendried at 60°C. Voucher specimen of the alga was lodged
at the G.T. Velasquez Phycological Herbarium (University of the Philippines – Marine Science Institute) with
accession number T19321.
Agar extraction
Extraction of native agar was done by boiling 20 g dried
seaweed in 800 mL water for 1 h. Diatomaceous earth
was added and the mixture was homogenized then
pressure-filtered while hot. The polysaccharide was
recovered from the filtrate by the freeze-thawing
method, followed by drying at 60°C.
Alkali modification of the seaweed sample was
carried out by heating 20 g dried seaweed in 750 mL of
5% NaOH solution at 90°C for 1 h. The sample was
washed thoroughly in running water and then soaked in
600 mL 0.5% HOAc at room temperature (28–30°C)
for 1 h, to neutralize excess alkali. The alkali-modified
seaweed was again washed and processed further as
described in native extraction. Extractions were done in
three replicate samples.
R. D. Villanueva et al.
500 mg) was set on the gel surface. The set-up was
then placed in a water bath and heated slowly. The
temperature at which the lead shot dropped to the
bottom of the test tube was recorded as the melting
temperature.
To measure gel syneresis, approximately 10 g of hot
1.5% (w/w) agar solutions were poured into test tubes
(21 mm diameter) and allowed to gel at room temperature (28–30°C) for 24 h. The initial weights of the gels
were recorded before placing them on dry Whatman
(No. 1) filter papers. Loss of exudates from the gels was
monitored by weighing the gels after 2 h. The syneresis
index values of the gel samples were taken as the
percent of gel weight loss (over the initial gel weight).
Chemical analyses
The sulfate content was quantified by the barium chloride precipitation technique of Jackson and McCandless (1978), after sulfate hydrolysis by 1N HCl at
110°C for 4 h. The 3,6-anhydrogalactose content of the
agar samples was determined colorimetrically using the
resorcinol method of Yaphe and Arsenault (1965).
Spectroscopic analyses
The Fourier transform infrared (FT-IR) spectra of
samples were recorded on films using a Perkin-Elmer
Series 2000 FT-IR Spectrometer in transmittance mode.
Films were prepared by drying 5 mL of 0.5% agar
solution on a Teflon-coated pan (3 cm diameter) at 60°C.
13
C-Nuclear magnetic resonance (NMR) spectroscopy was carried out using a Jeol 400 Spectrometer
operating at 100.40 MHz. Spectra of about 5%
polysaccharide solutions in D2O were obtained at 80°C,
using a 5 mm nuclear probe, with acquisition time of
0.3 s and a 15 ms delay. Chemical shifts were measured in parts per million relative to internal
3-(trimethyl)-silyl proprionic acid-d4-sodium salt, TSP
(d = 2.4 ppm) standard.
Gel analyses
Gel analyses were conducted according to Montaño
et al. (1999). Gel strengths of 1.5% (w/w) agar solutions were measured using a Marine Colloids Gel Tester
(Model GT-1, Marine Colloids Inc., Springfield, NJ,
USA). Gelling temperature was determined using a hot
solution of 1.5% agar in a test tube. A thermometer was
inserted, with its bulb situated just below the solution
surface, through a stopper. The solution was allowed to
cool and glass beads (diameter: 2.85 mm; weight:
30 mg) were dropped at intervals of 0.5°C. The temperature at which a bead failed to drop through the agar
solution was recorded as the gelling temperature. To
determine the melting temperature, the gel set-up used
in the gelling temperature determination was refrigerated and a lead shot (diameter 4.30 mm; weight
Statistical analyses
Data were tested for normality (Shapiro-Wilk’s test) and
for homogeneity of variance (Bartlett’s test) with the aid
of SAS statistical software (SAS Institute Inc., Cary,
NC, USA). Gel strength data were square transformed to
meet the latter parametric assumption. Analysis of variance (ANOVA) was carried out to detect significant difference in agar yield and physico-chemical parameters
between native and alkali-treated agars (P < 0.05).
RESULTS AND DISCUSSION
The native and alkali-treated polysaccharide extracts
of Laurencia flexilis were obtained in 26% and 19%
yields, respectively. The lower yield in the latter sug© 2010 Japanese Society of Phycology
Agar from Laurencia flexilis
153
gests that agar could have leached out to the alkali
solution in the pretreatment step, as earlier observed in
agar extraction from Gracilaria spp. (Freile-Pelegrín &
Murano 2005).
The FT-IR spectra (Fig. 1) of both extracts are similar
which include a weak band at 1250/cm, indicative of a
low level of sulfate ester (Stancioff & Stanley 1969). No
remarkable variation in the intensity of this band was
apparent between the spectra of the two preparations,
implying the absence of alkali-labile sulfate groups in
Fig. 1. Fourier transform infrared spectra of native (N) and
alkali-treated (AT) agar extracted from Laurencia flexilis. Arrows
indicate peaks at 1250, 930 and 845/cm, attributable to total
sulfate, 3,6-anhydrogalactose and sulfation at the 4-position of
galactose moieties, respectively.
the algal polysaccharide. This was corroborated by the
absence of a peak at 820/cm, the diagnostic band for a
sulfate ester at the C-6 position of the galactosyl backbone in the native extract. Alkali treatment could eliminate such sulfate substitution to effect the formation of
3,6-anhydrogalactose among 4-linked residues. Further
evidence of the absence of alkali labile sulfation is that
there is no reduction in the sulfate content of the
polysaccharide upon alkali modification, despite the
slight but not significant increase, as seen in the chemical analysis (Table 1). The sulfation pattern in the
polysaccharide is mainly an alkali-stable axial sulfate
ester at C-4 of 3-linked galactosyl moieties, as indicated by a persistent weak signal at 845/cm (Stancioff
& Stanley 1969) which is particularly discernible in the
native extract (Fig. 1). The strong signal at 930/cm
demonstrated the presence of high content of anhydrogalactose residues in both preparations that is consistent with the results of the chemical analysis (Table 1).
The lack of amyloglucosidase treatment in the
extraction procedure afforded polysaccharide extracts
with considerable amounts of floridean starch as
detected through C-NMR spectroscopy. Signals attributed to the starch (Table 2, Fig. 2) were assigned in
reference to Lahaye et al. (1986). It has been demonstrated that the freeze-thaw process in the recovery of
agar during the extraction process can eliminate the
starch as it dissolves in the thaw water (Chiovitti et al.
2004). Despite the use of the same process in the
present study, still the agar extracts contain appreciable
amounts of the starch. The presence of starch in the
product can have an effect on the quality of gel preparations, as the gelling agent (agar) concentration in the
gel is in effect lowered and there could be some
physico-chemical interactions between the two polymers. A repeated freeze-thaw process or the use of
amylase treatment may remove such a contaminant,
but the added cost of such a protocol should be taken
into consideration when used on an industrial scale.
Amylase treatment is seldom used in the commercial
production of food-grade agar.
Table 1. Yield and physico-chemical properties of agars from Laurencia flexilis using native
and alkali-treated extraction (mean ⫾ SE, n = 3)
Native agar
Agar yield (%)
Gel strength (g/cm2)
Gelling temperature (°C)
Melting temperature (°C)
Syneresis index (%)
Sulfate (%)
3,6-anhydrogalactose (%)
25.7
200
46.2
92.3
16.0
1.9
31.4
⫾
⫾
⫾
⫾
⫾
⫾
⫾
0.5
5
1.0
1.2
1.1
0.2
1.2
Alkali-treated agar
19.3
175
47.7
93.3
12.3
2.2
29.5
⫾
⫾
⫾
⫾
⫾
⫾
⫾
0.4
24
0.9
1.9
1.1
0.1
1.5
F-value
P-value
168.02
2.23
2.31
0.45
11.01
4.55
2.07
0.0002*
0.2096ns
0.2028ns
0.5391ns
0.0294*
0.1000ns
0.2239ns
Results of ANOVA are shown as probability (P) values, with <0.05 indicating significant
difference in the parameter between native and alkali-treated agars (df = 1). *Significant. ns,
not significant.
© 2010 Japanese Society of Phycology
154
R. D. Villanueva et al.
Table 2.
Assignments of major
13
C-nuclear magnetic resonance (NMR) resonances of the
native polysaccharide from Laurencia flexilis
Repeating unit
G6MLA
FS
Sugar
3-linked (G6M)
4-linked (LA)
Glucose
Carbon atom
C-1
C-2
C-3
C-4
C-5
C-6
Methyl
102.3
98.3
100.1
82.1
80.1
73.8
69.0
77.4
77.9
73.5
75.6
71.8
71.8
69.3
61.2
59.1
70.1
69.8
72.1
The major repeating unit is G6MLA: 3-linked 6-O-methyl-D-galactopyranosyl and
4-linked 3,6-anhydro-L-galactopyranosyl units. Chemical nomenclature is adopted from
Knutsen et al. (1994). FS, floridean starch.
Fig. 2 13C-Nuclear magnetic resonance (NMR) spectrum of the
native polysaccharide from Laurencia flexilis. Signals at 96.3 and
59.1 ppm are attributed to C-1 of anhydro-L-galactose (LA) vicinal
to a sulfated C-4 of D-galactose (G) or 6-O-methyl-D-galactose
(G6M) and methyl carbon, respectively. Asterisks indicate signals
attributable to floridean starch (see Table 2 for assignments).
In the native polysaccharide 13C-NMR spectrum
(Fig. 2), the assignment of chemical shifts to a 3-linked
6-methyl-D-galactose and 4-linked 3,6-anhydro-Lgalactose (G6MLA) repeating unit (Table 2) were done in
reference to Craigie and Jurgens (1989). Methylation at
G6 is of widespread occurrence among the galactans
synthesized by Laurencia species; however, it is not
detected in several species and other agar-producing
algae (Craigie 1990; Miller 1997). Another methylation
pattern of diverse distribution among agarophytes is
methyl ether at LA2 (Craigie 1990); however, it was not
detected in the alga studied here. The occurrence of
sulfate ester at G-4/G6M-4 produced a resonance at
96.3 ppm, which is attributed to the upfield shift of LA1
(Usov et al. 1980). Sulfation pattern of this type has not
been reported in Laurencia, but was observed in several
genera of the family Rhodomelaceae (Craigie 1990) and
occurs in high levels in Dascylonium incisum (J. Agardh)
Kylin (Miller et al. 1993). On the other hand, sulfate
ester at G2 was reported in L. pinnatifida (Hudson) J.V.
Lamouroux (Bowker & Turvey 1968a), L. nipponica
Yamada (Usov & Elashvili 1991), and L. thyrsifera J.
Agardh (Miller et al. 1993). Whereas, L6 sulfation,
generic among rhodophycean agars (Craigie 1990;
Miller 1997) as it is considered the precursor of the gel
forming residue 3,6-anhydrogalactose, is absent in the
L. flexilis native extract.
Alkali modification did not enhance gel strength
and other commercially relevant properties, except
syneresis, of the agar extract from L. flexilis (Table 1).
This observation is in accordance with the fact that no
substantial chemical transformation (desulfation) in
the native polysaccharide was effected by the procedure, as there is no significant difference in sulfate
and 3,6-AG contents between the two extracts
(Table 1). Such a procedure is hence not recommended during commercial use of the seaweed as agar
source. Boiling (~100°C) is an appropriate method of
extraction on an industrial scale, at lower production
cost and more eco-friendly. Native extraction at this
temperature produces agar with better gel quality than
extraction at lower temperatures (Pereira-Pacheco
et al. 2007).
One peculiar rheological aspect of the agar obtained
from this Laurencia species is its moderate gelling
property, which is not in unity with the very poor gelling
properties (<50 g cm-2 gel strength) of agars reported for
several species of the Rhodomelaceae (Miller et al.
1993). The native extract has a gel strength that is
within the range of values for agars from species of the
Gracilariales and is slightly lower than those from the
Gelidiales (Table 3). It is noteworthy that the gel strength
of the agar from L. flexilis may be further improved with
the elimination of floridean starch during extraction.
The high gelling temperature of the native and alkalitreated agar preparations (46–48°C, Table 1) can be due
to the high level of methyl ether substitution of the
samples (Guiseley 1970), which is a typical feature of
Gracilaria agars (Murano 1995). This specific agar characteristic disqualifies such agars from being used in
microbiological media as agars with lower gelling temperatures (<40°C) are required (Armisen & Galatas
1987).
© 2010 Japanese Society of Phycology
Agar from Laurencia flexilis
Table 3.
155
Characteristics of native agars from several species in Gracilariales and Gelidiales
Species
Yield (%)
Gel strength
(g/cm2)
Source
Gracilaria edulis
Gracilaria crassa
Gracilaria foliifera
Gracilaria corticata
Gracilaria dura
Gracilaria bursa-pastoris
Gracilaria gracilis
Gracilaria cervicornis
Gracilaria crassissima
Gracilaria blodgettii
Gelidiella acerosa
Gelidiella acerosa†
Gelidiella acerosa†
Gelidium madagascariense
25
23
22
16
33
35
30
39
30
37
~10–37
22–25
20–34
19
100
250
100
100
318
22
630
<50
180
~750
~50–449
400–845
360–800
807
Meena et al. 2008
Marinho-Soriano 2001
Freile-Pelegrín & Murano 2005
Ganesan et al. 2008
Prasad et al. 2007
Roleda et al. 1997
Mollion et al. 1990
†Though acetic acid treated, no alkali treatment was used in the extraction.
CONCLUSION
Structural analyses of the polysaccharide from Laurencia flexilis reveal a 6-methylated D-galactose alternating
with 3,6-anhydro-L-galactose as a major repeating unit.
Its agar makes gel with moderate strength that is appropriate for food applications. Alkali treatment during
extraction did not enhance the gel properties and is not
recommended for commercial processing.
Further investigations regarding other aspects in the
use of the resource should be pursued, such as the
effect of season to agar quantity and quality (e.g. FreilePelegrín et al. 1996; Villanueva et al. 1999; MarinhoSoriano & Bourret 2003; Romero et al. 2007; Ganesan
et al. 2008) and refinements in the technique of extraction (e.g. Li et al. 2008).
ACKNOWLEDGMENTS
We are grateful to J.T. Aguilan for the NMR experiments. This work was supported by DOST-PCAMRD and
UP. This is Marine Science Institute (University of the
Philippines) contribution no. 388 and MSU-Tawi-Tawi
contribution no. 004.
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