2. important difference that in agar the 3,6-anhydro-a-galactopyr-
anose units are in the L-configuration and not in the D-configura-
tion as is the case for carrageenans (Usov, 2011).
Both carrageenans and agar form gels in aqueous environments
via helix formation and aggregation of the polysaccharide chains
through inter-molecular hydrogen bonds (Morris, 1986; Schafer
Stevens, 1995) - for carrageenans the gelation is supported by the
presence of cations that induce the formation of a stable three-
dimensional gel-network (usually with potassium for k-carra-
geenan and with calcium for i-carrageenan) (Montero Pe, 2002;
Rhein-Knudsen et al., 2015). However, being biologically synthe-
sized in nature, natural carrageenans and agar are inherently het-
erogeneous. Extracted k- and i-carrageenans may thus contain
traces of their biosynthetic precursors, i.e. m- and n-carrageenan,
respectively, whereas agar may hold porphyran structures, i.e. the
precursor for agar, having a-L-galactose-6-sulfate instead of 3,6-
anhydro-a-L-galactopyranose, along with other hybrid structures
(Rhein-Knudsen et al., 2015). The level of these different precursors
and the extent of structural differences vary in different red
seaweed species, and this variation obviously affects the rheological
properties of the hydrocolloids.
Tanzania has long been a producer of carrageenan from seaweed
farming (Hurtado et al., 2015), but neither seaweed collection nor
farming are currently practiced in Ghana, even though the 540 km
long Atlantic coastline in the south is a habitat for different
seaweed species with potential for local hydrocolloid production.
Some of the wild red seaweed species such as Hypnea spp., Cryp-
tonemia crenulata and Hydropuntia spp. found along the coast of
Ghana are known, however, to contain hydrocolloids, notably
carrageenan and agar (Mtolera Buriyo, 2004; Pereira-Pacheco,
Robledo, Rodríguez-Carvajal, Freile-Pelegrín, 2007; Saito de
Oliveira, 1990). Cultivation of e.g. Hypnea musciformis for extraction
of k-carrageenan has been studied in India and Brazil, but has not
reached a commercial stage (Berchez, Pereira, Kamiya, 1993;
Ganesan, Thiruppathi, Jha, 2006). Only few studies have been
conducted on Ghanaian seaweeds, and these have mainly focused
on elemental analysis and assessments of iodine levels (e.g. Serfor-
Armah, Nyarko, Osae, Carboo, Seku, 1999; Serfor-Armah et al.,
2000). In 1975, John and Asare (John Asare, 1975) published a
preliminary assessment study of the yields and properties of hy-
drocolloids extracted from certain Ghanaian red seaweeds, but to
our knowledge, no recent data are available on the rheological
characteristics or hydrocolloid levels in wild red seaweed species
from Ghana. The hypothesis of the present work was that red
seaweed species native to Ghana hold galactose-rich hydrocolloids
of the carrageenan or agar type, and that the rheological properties
of hydrocolloids extracted from these seaweed species could be on
par with commercially used carrageenans and agar. The present
study was conducted to assess the carbohydrate composition of
local red seaweed species found along the coast in Ghana, and to
characterize the rheological properties of the hydrocolloids
extracted as a base for considering local carrageenan and agar
production from red seaweed resources in the region.
2. Materials and methods
2.1. Chemicals
All chemicals were purchased from Sigma-Aldrich Chemical Co.
(St. Louis, MO, USA) unless stated otherwise. Guluronic acid was
purchased from Chemos GmbH (Regenstauf, Germany).
2.2. Seaweed sampling and sample preparation
Wild red seaweed samples were collected in the coastal areas of
Ghana, except the Kappaphycus alvarezii (Table 1). All samples ob-
tained from Ghana were immediately frozen in aliquout portions
at À20 C after collection. Before use, the seaweed samples were
gently thawn, and then rinsed to remove epiphytes, entangled
materials, and sand. The washed seaweed samples were then
freeze-dried and milled, then passed through a 1 mm mesh sieve
(MF 10 basic Microfine grinder drive, IKA) to obtain uniform par-
ticle sizes. The milled seaweed samples were stored in sealed
plastic bags at À20 C. Cultured Kappaphycus alvarezii was received
in dried form from Vietnam (Nhatrang Institute of Technology
Research and Application) and used as a benchmark for carra-
geenan extraction and rheological properties of carrageenan.
2.3. Composition analysis
The amount of dry matter and ash in the seaweed samples were
determined according to the National Renewable Energy Labora-
tory (NREL) procedure and the weight of biomass used in the ex-
periments was mathematically corrected for the amount of
moisture present in the samples (Sluiter et al., 2004, 2008). For
carbohydrate composition analysis, the collected seaweed samples
were hydrolyzed according to a modification of the NREL two-step
sulfuric acid hydrolysis procedure, as described by Manns et al.
(Manns, Deutschle, Saake, Meyer, 2014). In brief, 100 mg dry
matter seaweed material per mL was mixed with 72% H2SO4
(weight/volume, w/v) and left to react at 30 C for 1 h. The reaction
mixture was then diluted to 4% w/v H2SO4 and hydrolyzed in an
autoclave at 120 C for 40 min (Manns et al., 2014). The acid hy-
drolysate and seaweed residuals were then separated by vacuum
filtration and the supernatants filtered through a 0.22 mm nylon
syringe tip filter (Frisenette Aps, Knepel, DK) and diluted in 500 mM
NaOH prior to injection for high performance anion exchange
chromatography (HPAEC) analysis. HPAEC separation of the
seaweed polysaccharides was performed using a HPAEC-PAD,
ICS3000 system (Dionex Corp. Sunnyvale, CA) equipped with a
CarboPac™ PA20 column by a method principally as described by
Arnous and Meyer (2008). L-fucose, L-arabinose, L-rhamnose, D-
galactose, D-glucose, D-xylose, D-mannose, D-galacturonic acid, D-
guluronic acid, and D-glucuronic acid were used as mono-
saccharide standards for quantification; quantification was done
using Chromeleon software (Dionex Corp. Sunnyvale, CA). Recovery
values for the monosaccharides were estimated from parallel runs
of monosaccharide standards (Arnous Meyer, 2008).
2.4. Hydrocolloid extraction and characterization
2.4.1. Carrageenan extraction
Water extraction of carrageenan was performed using 1.5 g (dry
matter) samples from H. musciformis, C. crenulata and K. alvarezii
that were hydrated overnight in 30 mL milli-Q water before direct
extraction at 99 C for 1.5 h. The pH following overnight soaking
was between 7.5 and 8.5 and based on previous results, this pH was
high enough to prevent depolymerization of the seaweed galactans
(Capron, Yvon, Muller, 1996; John Asare, 1975). Alkali-
treatment was carried out using a slightly modified procedure of
the method described by Istinii, Ohno, and Kusunose (1994). Briefly,
60 mL 6% w/v KOH was added to 1.5 g of the seaweed samples and
reaction was carried out at 80 C for 3 h. KOH was removed by
washing the seaweed samples and soaking in water overnight.
Carrageenan extraction was then performed on the alkali-treated,
washed, seaweed samples (1.5 g dry matter) in 30 mL milli-Q wa-
ter at 99 C for 1.5 h. The extracts were pressure filtered (filter
paper, PP filter cloth, Sigma-Aldrich) after being mixed with dia-
tomaceous earth (Celite, Sigma-Aldrich), precipitated in 80% iso-
propanol, filtrated, and recovered by freeze-drying. Yields were
N. Rhein-Knudsen et al. / Food Hydrocolloids 63 (2017) 50e58 51
3. determined by weighing and confirmed by HPAEC analysis of HCl
hydrolyzed carrageenans (0.5% by weight of carrageenan in 1M HCl,
105 C, 3 h) (Istinii et al., 1994). Standard grade carrageenans ob-
tained from Sigma-Aldrich Chemical Co. (k-carrageenan: 22048,
Lot# BCBK1080V, i-carrageenan: C1138, Lot# SLBJ7874V) (St. Louis,
MO, USA) were used for comparison. 3,6-anhydro-galactose con-
tents of the hydrocolloids were determined, where practically
possible, following the reducing acid hydrolysis procedure
described by Jol, Neiss, Penninkhof, Rudolph, and De Ruiter (1999).
(the third addition of the reducing agent methylmorpholine-
borane complex in the procedure was left out to improve the
subsequent analytical HPAEC quantitation, however). Quantifica-
tion was done using HPAEC-PAD with 3,6-anhydro-galactose as a
standard (purity ! 95% as informed by the manufacturer) (Dextra
Laboratories Ltd., UK).
2.4.2. Agar extraction
Direct water extraction of agar and extraction of agar after
alkali-treatment were performed according to the methods
described by Freile-Pelegrín and Murano (2005). For the water
extraction, 1.5 g (dry matter) of Hydropuntia dentata was hydrated
in 30 mL milli-Q water overnight. The pH of the suspensions was
adjusted to 6e6.5 and extraction was performed at 99 C for 1.5 h.
The procedure was selected from the literature where it is also
stated that agar is stable at pH 6 when extracting at boiling point
(without pressure) (Armisen Galatas, 1987; Freile-Pelegrín
Murano, 2005; Marinho-Soriano Bourret, 2003). For extraction
of agar after alkali-treatment, the seaweeds were soaked in 30 mL
5% w/v NaOH overnight and modification of the seaweed carbo-
hydrates was done at 90 C for 3 h. The alkali-treated biomasses
were then washed and soaked in water overnight to remove
remaining alkali and extraction was performed in 45 mL milli-Q
water, pH 6e6.5 at 99 C for 1.5 h. The extracts were filtered as
described above for carrageenans. Agar was recovered by freeze-
drying. Yields were determined by weighing and confirmed by
HPAEC analysis of HCl hydrolyzed agar (0.5% agar in 1M HCl, 105 C,
3 h) (Freile-Pelegrín Murano, 2005). Standard grade agar ob-
tained from Sigma-Aldrich Chemical Co. (A1296, Lot# BCBQ5740V)
(St. Louis, MO, USA) was used for comparison. 3,6-anhydro-galac-
tose contents were determined as described in section 2.4.1.
2.4.3. Sulfate content analysis
The inorganic sulfate content was determined by turbidity
analysis as described by Jackson and McCandless (1978) after hy-
drolysis of the hydrocolloids with 1M HCl at 105 C for 3 h (Jackson
McCandless, 1978).
2.4.4. Fourier transform infrared spectroscopy
Attenuated total reflectance Fourier transform infrared spec-
troscopy (ATR-FTIR) was carried out on a Nicolet iS50 FTIR spec-
trometer (Thermo Fisher Scientific Inc., USA) with ATR module. The
spectra were recorded in the range of 4000e400 cmÀ1
by acquiring
32 scans with 4 cmÀ1
resolution.
2.4.5. Oscillatory rheology
Samples for oscillatory rheology analysis were prepared by a
modified procedure from Thrimawithana, Young, Dunstan, and
Alany (2010). In brief, the carrageenans were prepared by dissolv-
ing at 80 C for the k-carrageenans and at 95 C for the i-carra-
geenans. Solutions were diluted with 4% KCl to obtain final
concentrations of 1.5% w/v carrageenan and 1% of KCl. After addi-
tion of KCl, the solutions were heated for an additional time of
20 min. Agar at 1.5% w/v was dissolved in milli Q water at 95 C and
analyzed without further addition of ions (Thrimawithana et al.,
2010). The volume used for each analysis was 3 mL. Rheological
properties of the hydrocolloids were assessed by small angle
oscillatory rheological measurements on a HAAKE MARS rotational
rheometer (Thermo Scientific Inc., Germany) equipped with a
serrated parallel-plate (Reologica Instruments AB) with a diameter
of 60 mm and a gap of 1.0 mm. Temperature sweep tests were
conducted at 0.1 Hz to evaluate the viscoelastic properties during
gelation by in situ cooling (80/95e20 C) and heating (20e80/95 C)
of the hydrocolloid mixture at a rate of 1 C/min. To avoid dehy-
dration during experiment, the plate was covered with silicone oil
(Hall Miba A/S, Kgs. Lyngby, Denmark). The storage modulus (G0),
the loss modulus (G00), and the thermal hysteresis behavior of the
gels were determined as a function of temperature.
2.5. Statistics
Carbohydrate composition analyses, hydrocolloid extractions,
and sulfate content determinations were performed in triplicates
and the data are presented as mean ± standard deviations (SD).
Analyses of variances (ANOVA) were used to determine significance
differences in yields and compositions with the Tukey-Kramer test
from pooled standard deviations (JMP 11 Statistical Software, SAS).
Values of P 0.05 were considered statistically significant.
3. Results and discussion
3.1. Seaweed composition
All the seaweed samples were found to contain significant
amounts of galactose, namely from 21 to 26% by weight (w/w) of
the dry matter for the Hypnea musciformis and Cryptonemia cren-
ulata samples, and 15% w/w for the Hydropuntia dentata sample
(Table 2), which supports the expectation that the seaweed species
could contain carrageenan and agar, respectively (Table 1). For the
compositional analysis of the whole seaweed samples the quanti-
fied galactose was assumed to represent both the galactose moi-
eties and the 3,6-anhydro-galactose, though it is known that the
3,6-anhydro bridges are acid labile (Jol et al., 1999). The mono-
saccharide composition showed that the C. crenulata and
H. musciformis samples had almost as high galactose levels
(constituting 21e26% by weight of the seaweed dry matter) as that
found in the carrageenan benchmark red seaweed sample from
K. alvarezii (30% % by weight of the seaweed dry matter) (Table 2) e
the latter was used as benchmark since K. alvarezii is currently the
most significant commercial k-carrageenan source (Hurtado et al.,
Table 1
Overview of seaweed samples, expected hydrocolloids, and origin of the red seaweed samples used in the present study.
Red seaweed samples Expected hydrocolloid type Origin
Hypnea musciformis(I) Carrageenan Old Ningo, Greater Accra, Ghana/Jan. 2015
Hypnea musciformis(II) Carrageenan Prampram, Greater Accra, Ghana/Aug. 2015
Cryptonemia crenulata Carrageenan Prampram, Greater Accra, Ghana/ Jan. 2015
Kappaphycus alvarezii Carrageenan (benchmark) Nha Trang, Vietnam/2015
Hydropuntia dentata Agar Prampram, Greater Accra, Ghana/Jan. 2015
N. Rhein-Knudsen et al. / Food Hydrocolloids 63 (2017) 50e5852
4. 2015). Comparison of the polysaccharide contents in the Ghanaian
seaweeds and K. alvarezii was done even though the post-harvest
handling differed. The ash contents and glucose levels varied
slightly in the two H. musciformis samples, which were collected
from two different sites along the Ghanaian coast (Table 1), but the
galactose levels were similar (Table 2).
Several studies involve estimation of carbohydrate content in
K. alvarezii and Meinita et al. (2012) estimated the carbohydrate
content to range from 35 to 78% by weight in different K. alvarezii
tissues collected from various places in Indonesia (Meinita et al.,
2012). By extraction of crude carbohydrates, Arunkumar,
Palanivelu, and Darsis (2014) found a carbohydrate content of
40% for a H. musciformis sample collected in India (Arunkumar et al.,
2014). When adding the monosaccharide levels the carbohydrate
levels found in the H. musciformis samples collected in Ghana of
37e39% w/w (Table 2) were thus in complete accordance with the
total level of crude carbohydrates found in the Indian sample by
Arunkumar et al. (2014). Glucose was found in levels of 11e18% w/w
(Table 2). Since red seaweeds are generally believed to contain less
than 10% w/w cellulose, some of the glucose may derive from flo-
ridean starch, the storage carbohydrate of red algae that is built of
1,4-linked a-D-glucopyranose chains with branches at position 6
(Usov, 2011). Other minor monosaccharide constituents were
identified in the red seaweed hydrolysates, namely: mannose,
rhamnose, arabinose, xylose, galacturonic acid, guluronic acid and
glucuronic acid. These minor saccharides have also been identified
in red seaweeds earlier (Usov, 2011). For the C. crenulata, minor
monosaccharides were mainly rhamnose, while the other seaweed
samples contained mainly xylose and some traces of uronic acids as
minor monosaccharides. These findings are in agreement with
previous findings reporting the presence of rhamnose in the red
algae Rhodella maculata (Fareed Percival, 1977), while xylose
substitutions on agar and carrageenans from red seaweed species
have been described by Araki, Arai, and Hirase (1967) and Estevez,
Ciancia, and Cerezo (2000) respectively. The ash contents in sea-
weeds are generally higher than those of terrestrial plants, and
ranged from 19 to 36% w/w (Table 2), which is in agreement with
previous findings for these seaweed species (Arunkumar et al.,
2014).
The observed differences in the composition of these red
seaweed species may be attributed to the differences in the algal
source and the growth environment (Table 1). It is well known that
wild seaweeds show significant variation in nutrient contents at
different environmental conditions such as water temperature,
water salinity, nutrients, and light (Marinho-Soriano, Fonseca,
Carneiro, Moreira, 2006).
Once the exact impact of the different factors is understood, it
may be possible to optimize the monosaccharide composition and
hydrocolloid levels.
3.2. Hydrocolloid yield and characteristics
3.2.1. Hydrocolloid yield
To validate whether the galactose units found in the red sea-
weeds, section 3.1, were actually constituents of hydrocolloid
polysaccharides, the seaweeds were subjected to direct water-
extraction and alkali-treatment methods as described in Section
2.4.
Evidently, most of the extracted material contained galactose
and sulfate, which are the main components of agar and carra-
geenan. Extraction yields for the putative carrageenans ranged
from 19 to 27% by weight while yields were slightly lower for the
extracted agar hydrocolloids, but generally with no profound dif-
ferences in yields between water- and alkali-treatment (Table 3).
The lower (agar) hydrocolloid extraction yield for H. dentata is in
agreement with the lower level of galactose-moieties found in
H. dentata than in the other seaweed samples expected to contain
carrageenan (Table 2). The carrageenan and agar yields from
H. musciformis and H. dentata were in accordance with the results
published by John and Asare (1975) showing Ghanaian
H. musciformis carrageenan extraction yields of 25e45% by weight
and Ghanaian H. dentata (referred to as Gracilaria dentata in their
study) agar extraction yields of approximately 10e30% dry weight,
depending on season of harvest (John Asare, 1975). A comparison
of the total content of galactose-monomers found in the seaweed
samples (Table 2) and the hydrocolloid extraction yields and the
hydrocolloids compositions (Table 3) showed that 48e89% by
weight of the galactose residues present in the different seaweeds
were extracted as hydrocolloids. Generally, the alkali-treatment
was most effective for obtaining more galactose-rich hydrocol-
loids, resulting in high extraction of galactose in the hydrocolloids,
e.g. reaching a yield of 89% of the galactose-moieties present in
H. musciformis(II) in the extracted hydrocolloid (calculated from the
Data in Tables 2 and 3). In comparison, 81% of the galactose-
moieties present in the H. musciformis (II) were released as hy-
drocolloid by water-extraction. Although we did not observe a
significant difference in hydrocolloid extraction yield between
direct water-extraction and alkali-treated the modification of the
seaweed hydrocolloids was successfully accomplished by the
alkali-treatment, since the water-extracted hydrocolloid contained
significantly higher amounts of sulfate compared to the ones
extracted via alkali-treatment (Table 3). The analysis of the sulfate
content supported the expectation that the hydrocolloid poly-
saccharides from the two Ghanaian Hypnea musciformis samples
could be k-carrageenans. The water-extracted hydrocolloids from
C. crenulata had sulfate contents that were comparable with the
standard grade i-carrageenan, whereas the sulfate level in the
alkali-extracted hydrocolloids was more at the level of sulfate in k-
carrageenan (Table 3). Literature regarding C. crenulata is limited
but has been described as producing both i-carrageenan (Saito de
Table 2
Monomeric carbohydrate yields [mean ± SD]1
determined by HPAEC-PAD analysis after two-step sulfuric acid hydrolysis and ash content determined gravimetrically after
igniting at 550 C. Different roman superscript letters column-wise indicate significant differences (P 0.05) by one-way ANOVA.
Seaweed samples Monosaccharides and ash levels
Galactose
[% dry material]
Glucose
[% dry material]
Others2
[% dry material]
Ash content
[% dry material]
Hypnea musciformis(I) 26a,b
±0.9 11d
± 0.7 2c
±0.1 22d
± 0.8
Hypnea musciformis(II) 21b
± 0.9 13c
±0.6 3c
±0.5 30b
± 0.2
Cryptonemia crenulata 25a,b
±1.7 18a
±0.9 4c
±0.6 19e
±0.6
Kappaphycus alvarezii 30a
±4.5 11d
± 0.3 14a
±4.8 23c
±0.1
Hydropuntia dentata 15c
±0.8 15b
± 0.7 9b
± 0.8 36a
±0.2
1
All monosaccharide values are given on per weight basis as dehydrated monomers.
2
Others: mannose, rhamnose, arabinose, xylose, galacturonic acid, guluronic acid, and glucuronic acid.
N. Rhein-Knudsen et al. / Food Hydrocolloids 63 (2017) 50e58 53
5. Oliveira, 1990) and a hybrid galactan composed of l-carrageenan
and agar (Zibetti, Noseda, Cerezo, Duarte, 2005). The 3,6-
anhydro-galactose contents were quantified by HPAEC-PAD anal-
ysis following reductive hydrolysis (Table 3). The results supported
that the alkali-treatment was effective as evident from the lower
3,6-anhydro-galactose content in the water-extracted samples
(Table 3). The standard grade i-carrageenan was shown to contain
only 8% 3,6-anhydro-galactose, while the k-carrageenan contained
higher amounts (Table 3). The commercial i-carrageenan was
commercial grade Type II: predominantly i-carrageenan and may
contain higher amounts of its precursor n-carrageenan that pro-
duce flexible gels thus has low content of 3,6-anhydro-galactose.
Small amounts of glucose, fucose and xylose were also identified
in the extracted hydrocolloids (Table 3). Several types of mono-
saccharide substitutions on the galactose-rich hydrocolloids,
including xylose substitutions on the galactose-moieties in agar,
have been described in the literature, e.g. by Araki et al. (1967).
Estevez et al. (2000) have similarly described xylose-substitutions
on carrageenan derived from the red seaweed Kappaphycus alvar-
ezii (Estevez et al., 2000). Surprisingly high amounts of glucose
were found in the standard grade k-carrageenan sample (Table 3).
With reductive acid hydrolysis the level of glucose remained the
same (data not shown), which verified that the detection of glucose
was not a result of decomposition of anhydro-galactose moieties.
Unfortunately, the manufacturing method for this commercial k-
carrageenan sample was not specified by the manufacturer. The
presence of glucose could be a result of insufficient separation of
the carrageenan during extraction.
3.2.2. Fourier transform infrared (FTIR) spectroscopy
The FTIR spectra of carrageenan products from the
H. musciformis, K. alvarezii, and C. crenulata seaweed samples ob-
tained from two different extraction procedures, namely water-
and extractions and alkali-treatments, were compared with those
of commercial k-carrageenan and i-carrageenan (Fig. 1A). The
spectra of the carrageenans (Fig.1A) showed the main features of k-
and i-carrageenan, since notably the moderately strong intensity at
approximately 845 cmÀ1
is assigned to CeOeSO4 on C4 of D-
galactose-4-sulfate (Pereira, Amado, Critchley, van de Velde,
Ribeiro-Claro, 2009; Pereira, Sousa, Coelho, Amado, Ribeiro-
Claro, 2003). The weak intensity band at approximately 867 cmÀ1
(Fig.1A) also indicates CeOeSO4, but on C6 of galactose. This sulfate
substitution is present on m and n-carrageenans, the precursors for
k- and i-carrageenan, respectively, as well as on l-carrageenan. As
we see a strong intensity band at 845 cmÀ1
from D-galactose-4-
sulfate, which is not part of l-carrageenan, the weak band at
867 cmÀ1
is appointed to m and n-carrageenan (Pereira et al., 2009).
The findings are in accord with the chemical composition data
(Table 3) showing that although sulfate contents were decreased in
the alkali-extracted samples, the results indicated that only partial
removal of sulfate substitutions from the galactose-moieties in the
carrageenan took place during the alkali-treatment. The presence
of a strong band at approximately 930 cmÀ1
indicated the presence
of 3,6-anhydro-D-galactose and was thus assigned as CeO bonds
(Matsuhiro, 1996). In Fig. 1Ahej, an absorption band around
805 cmÀ1
was also visible, indicating the presence of a sulfate ester
at C2 of the anhydro-D-galactose, i.e. CeOeSO4 on C2 of the 3,6-
anhydro-D-galactose-unit, which is a profound band characteristic
for i-carrageenan and q-carrageenan (Matsuhiro, 1996; Pereira
et al., 2009). No clear bands were observed between 820 cmÀ1
and 830 cmÀ1
to verify the presence of l-carrageenan as obtained
from the CeOeSO4 on C2 and C6 on galactose (Pereira et al., 2009).
These findings suggest that the main type of hydrocolloid present
in the Ghanaian H. musciformis was indeed k-carrageenan (and as
expected, the K. alvarezii hydrocolloid was also confirmed to be k-
carrageenan), whereas C. crenulata is expected to contain i-carra-
geenan, based on the presence of a band at strong intensity band at
845 cmÀ1
, from CeOeSO3 on C4 of galactose, and a weaker band at
805 cmÀ1
, assigned to the CeOeSO3 of C2 on 3,6-anhydro-galactose.
The glucose, especially the high amount of glucose detected in
the commercial k-carrageenan, having characteristic bands be-
tween 990 and 1150 cmÀ1
, was not clearly revealed in the FTIR
spectrum. As the samples for FTIR analysis were not weighed for
accurate estimation of contents, it was difficult to make further
conclusions about this issue; this conclusion is in accord with a
previous FTIR report (Adina, Florinela, Adbelmoumen, Carmen,
2010).
Although the FTIR spectra of the agar samples were generally
significantly different from the FTIR spectra of the carrageenan
samples, some similarities between the two sets of spectra were
evident (Fig. 1B). For example, as in the carrageenan hydrocolloid
spectra, the presence of a strong band in the region around
Table 3
Overview of seaweed type (hydrocolloid source), hydrocolloid extraction method (direct water-extraction or after alkali treatment), hydrocolloid and monomer1
yields, and
sulfate levels [data given as means ± SD]. Different roman superscript letters indicate significant differences (P 0.05) column-wise for carrageenans and agar yields,
monosaccharides, and sulfate content by one-way ANOVA.
Hydrocolloid
source
Hydrocolloid
extraction method
Hydrocolloid extraction yield
[% dry material]
Hydrocolloid composition
Galactose (3,6-anhydrogalactose3
)
[% hydrocolloid]
Glucose
[% hydrocolloid]
Others4
[% hydrocolloid]
Sulfate content
[% hydrocolloid]
H. musciformis(I) Direct water-extraction 24a
±1.7 72b,c
±1.0 (24) 4d,e
±0.8 1d
± 0.01 20c,d
±1.2
Alkali-treated 26a
±1.6 84a
±0.9 (48) 2f
±0.3 2b
± 0.2 16d
± 0.4
H. musciformis (II) Direct water-extraction 24a
±1.9 71b,c
±1.5 (n.a.) 8b,c
±0.7 1.45c,d
21c,d
±2.4
Alkali-treated 27a
±4.5 70c
±1.5 (n.a.) 4d,e,f
±0.6 1c,d
±0.5 16d
± 1.2
C. crenulata Direct water-extraction 21a
±1.9 61d
± 2.7 (n.a.) 4d,e
±0.6 1d
± 0.1 32b
± 4.3
Alkali-treated 19a
±0.1.8 68c
±1.6 (n.a.) 3e,f
±0.2 3b
± 0.2 25c
±2.5
K. alvarezii Direct water-extraction 21a
±2.8 68c,d
±5.3 (23) 3e,f
±0.3 2b,c
±0.1 20c,d
±0.7
Alkali-treated 23a
±5.9 77b
± 2.4 (52) 6c,d
±0.5 2b,c
±0.1 16d
± 0.6
k-carrageenan2
47e
±1.5 (17) 30a
±1.5 n.d 22c,d
±0.6
i-carrageenan2
40e
±2.0 (8) 9b
± 0.6 4a
± 0.1 39a
±3.8
H. dentata Direct water-extraction 15b
± 1.7 79b
± 2.5 (34) 1b
± 0.1 6a
±0.4 8a
±0.2
Alkali-treated 13b
± 0.02 79b
± 2.6 (46) 2a
±0.6 6a
±0.2 5b
± 0.2
Agar2
89a
±1.1 (48) 2a,b
±0.3 3b
± 0.6 7b
± 0.6
1
All monosaccharides values are given as dehydrated monomers,
2
purchased from Sigma-Aldrich,
3
data in parenthesis are amounts of 3,6 anhydrogalactose out of total galactose quantified by reducing TFA hydrolysis, n.a. not analyzed (i.e. not done),
4
Fucose, xylose, mannose; n.d: not detected.
N. Rhein-Knudsen et al. / Food Hydrocolloids 63 (2017) 50e5854
6. 930 cmÀ1
for the agar samples (Fig. 1B), could be attributed to the
CeO bonds of 3,6-anhydro-a-L-galactopyranose. The characteristic
broad band of a sulfate ester between 1210 and 1260 cmÀ1
was
much stronger in carrageenan than in agar (Fig. 1A and B) indi-
cating that the amount of sulfates was higher in the extracted
carrageenan hydrocolloids than in the agar, in accord with the
compositional data (Table 3). The bands at 740 and 770 cmÀ1
in the
spectra of the commercial agar and the water-extracted H. dentata
hydrocolloids (Fig. 1B) were assigned to the skeletal bending of the
galactose ring and especially in the anomeric region
(700e950 cmÀ1
), where agar and carrageenan have previously
been reported to show several similar bands (Pereira, Gheda,
Ribeiro-claro, 2013).
The spectral feature of agar at approximately 890 cmÀ1
(Fig. 1B)
is interpreted to be mainly associated with the CeH bending at the
anomeric carbon in b-galactose residues (Matsuhiro, 1996). The
spectra of the agar hydrocolloids extracted from H. dentata revealed
that effective modification had occurred during the alkali
treatment, as the band at 867 cmÀ1
, indicating CeOeSO4, in the
water-extracted sample (Fig. 1Beb), was not present in the equiv-
alent alkali-extracted sample (Fig. 1Bec) (Guerrero, Etxabide,
Leceta, Pe~nalba, De La Caba, 2014). These findings confirmed
that the alkali-treatment used modified the sulfates in the hydro-
colloids, in agreement with the sulfate content analysis, that
showed that the sulfate content was consistently lower in the
alkali-extracted than in the water-extracted samples (Table 3).
3.2.3. Rheological properties
Oscillatory rheological measurements were performed to assess
the gelling characteristics of the extracted hydrocolloids from the
Ghanaian seaweed samples. 1.5% carrageenans with 1% KCl added
were used for the carrageenan gel analysis, while the gelling
properties of the agar samples were determined using 1.5% agar
dissolved in milli-Q water. The storage modulus (G0) and the loss
modulus (G00) were measured over temperature ranges from
80 Ce20 C for k-carrageenan and 95 Ce20 C for i-carrageenan
and agar. (Rheological analysis from 80 Ce20 C was performed for
i-carrageenan and agar as well, and the same rheological pattern
was observed (Results not shown). The temperature was increased
as no cross-over of G0 and G00 was observed). To evaluate the
reversible gelling properties, the parameters were determined by
heating back to 80 C and 95 C, respectively. For all the tested
hydrocolloids, G0 increased as a result of gel formation as the
temperature decreased (Fig. 2, and Supplementary material
Figures S1-S3). The gel strengths were estimated at 25 C as the
average of the G’ values measured at the three temperatures closest
to 25 C and are summarized in Table 4. Clear differences in the gel
strengths were evident between the hydrocolloids obtained by
alkali-treatment and those obtained by water extraction (Table 4).
These differences are most likely attributable to the formation of
anhydro-galactose by the alkali-treatment for extraction, as
corroborated by the sulfate content differences described in section
3.2. The alkali-treated carrageenans are expected to have higher gel
strengths after removal of sulfate in the b-1,4-linked galactose units
forming higher amounts of the gel-inducing 3,6-anhydro-galactose
units. Statistical analysis revealed that the water-extracted carra-
geenans from the Ghanaian seaweeds had similar gel strengths to
the k-carrageenan extracted by from the Vietnamese K. alvarezii
sample (Table 4). No significant difference in gel strength was
observed between the alkali-extracted carrageenans from the
H. musciformis samples collected at different sites along the coast
and at different times, indicating that both samples contain hy-
drocolloids with remarkable gelling potential and that the collec-
tion site and time had no influence in this particular study.
The extracted k-carrageenans from the seaweed samples
showed significantly higher gel strengths at 25 C than the com-
mercial k-carrageenan obtained from Sigma-Aldrich (Fig. 2A and
Table 4). It is difficult to make any conclusive statements when
comparing the k-carrageenan in the present study to the standard
grade k-carrageenan, as the origin and extraction technique of the
standard grade compound is undisclosed. Nevertheless, it is
evident from the composition analysis that the lower gel strength
obtained from the commercial k-carrageenan is likely related to the
higher glucose to galactose ratio as the sulfate levels were the same
(Table 3). In addition, several other factors have an impact on the
gelling properties, such as molecular weight and ionic content, and
further analyses are thus required for a full understanding on the
origin of the differences in the rheological behaviors of the hy-
drocolloid gels (Chen, Liao, Dunstan, 2002). As was the case for
the k-carrageenans (Fig. 2A), the storage modulus (G0) values for
the i-carrageenan from C. crenulata also increased upon cooling
(Fig. 2B). The increase in G’ for i-carrageenan occurred earlier than
for k-carrageenan, indicating that sol-gel transition occurred at
Fig. 1. A) FTIR analysis of carrageenans from a) Sigma-Aldrich (k-carrageenan), b)
H. musciformis(I) (water-extracted), c) H. musciformis (II) (water extracted), d)
H. musciformis(II) (alkali-treated), e) H. musciformis(I) (alkali-treated), f) K. alvarezii
(alkali-treated), g) K. alvarezii (water-treatedh) C. crenulata (water-extracted), i)
C. crenulata (alkali-treatedand j) Sigma-Aldrich (i-carrageenan. B) FTIR analysis of agar
from a) Sigma-Aldrich, b) H. dentata (water-extracted), c) H. dentata (alkali-treated).
N. Rhein-Knudsen et al. / Food Hydrocolloids 63 (2017) 50e58 55
7. higher temperatures in the i-carrageenan hydrocolloids than in the
k-carrageenan. The gel strengths for the i-carrageenans were esti-
mated as described above and the data are summarized in Table 4.
The lower gel strengths observed for the i-carrageenan samples are
in accord with theory, and the characteristics of k-carrageenan to
produce stronger and more rigid gels than i-carrageenan is also
well established (Rhein-Knudsen et al., 2015).
The estimated gel strengths were based on gel formation by
potassium ions. Calcium ions are far more effective at increasing
the gel strength for i-carrageenan than potassium, which is best for
optimization of gel strength in k-carrageenan samples
(Thrimawithana et al., 2010). We observed a significant difference
in gel strength between the water e and alkali-extracted i-carra-
geenan samples, corroborating that an effective alkali-modification
procedure was accomplished (Table 3). For the i-carrageenan
samples, a change in slope occurred close to 30e40 C which was
evident only in a non-logarithmic plot (Fig. 2B). This change in
slope may be attributable to the co-existence of k-carrageenan in
the sample as occurrence of hybrid carrageenans has been
described previously (Blanco-Pascual, Aleman, Gomez-Guillen,
Montero, 2014; van de Velde, Peppelman, Rollema, Hans,
2001). The observed type of gelling behavior is believed to be a
result of a two-step gelation process (Parker, Brigand, Miniou,
Trespoey, 1993). The presence of k-carrageenan should ideally in-
crease the gel strength, since k-carrageenan makes stronger gels
than i-carrageenan, but the abrupt drop in gel strength appears to
contradict this expectation. Nevertheless, the sudden drop in slope
(Fig. 2B) was not evident when examining the logarithmic plot for
i-carrageenan more closely (Figure S2). Serrated plates were used
to avoid risk of sliding of the samples. Thus the observed drop in gel
strength may be due to minor syneresis, which may influence the
measurements due to the formation of a solvent layer (Chen et al.,
2002; Parker et al., 1993; Richardson Goycoolea, 1994).
The water-extracted agar from H. dentata had gel strength at
25 C of around 10 Pa, much lower than the one obtained from the
standard grade agar (Fig. 2C). The alkali-extracted agar from
H. dentata on the other hand, showed a gel strength which was
statistically similar to the commercial sample (Table 4). The gel
strength of commercial agar kept increasing when further cooled,
while the one extracted from H. dentata seemingly reached its
maximum around 20e25 C (Fig. 2C). The gel strength measured
for the agar samples were generally much lower than those for k-
Fig. 2. Storage modulus, G’ [Pa], measured from 80 C to 20 C at a rate of 1 C/min for
A) 1.5% k-carrageenan with 1% added KCl, and the storage modulus, G’ [Pa], measured
from 95 C to 20 C at a rate of 1 C/min for B) 1.5% i-carrageenan with 1% added KCl
and C) 1.5% agar in milli-Q water.
Table 4
Overview of the parameters determined by oscillatory rheology for 1.5% carrageenan with 1% added KCl and 1.5% agar in milli-Q water. Gel strength at 25 C was determined as
the averages of the three values determined closest to 25 C ± SD; different roman superscript letters indicate significantly different values (p 0.05) by ANOVA (carrageenan
samples assessed separately from agar samples).
Hydrocolloid source Extraction method Gel strength at z25
C [Pa]2
(G0
at z25
C) Tgel [
C] (G' G00
) Tmelt [
C] (G' G00
)
H. musciformis(I) Direct water-extraction 3126d
± 209.7 33 56
Alkali-treated 6409b
± 147.0 36 60
H. musciformis(II) Direct water-extraction 3075d
± 218.4 33 56
Alkali-treated 6538a,b
±79.8 36 60
C. crenulata Direct water-extraction 1590e
±38.00 74 e
Alkali-treated 4035c
±135.3 71 e
K. alvarezii Direct water-extraction 3126d
± 209.8 32 53
Alkali-treated 6905a
±241.7 35 55
k-carrageenan1
1263e
±11.0 41 57
i-carrageenan1
790f
±10.3 70 80
H. dentata Direct water-extraction 10b
± 1.1 38 74
Alkali-treated 287a
±13.7 52 e
Agar1
238a
±71.2 48 e
1
Purchased from Sigma-Aldrich,
2
Average values of the three measurements closest to 25
C, - could not be determined.
N. Rhein-Knudsen et al. / Food Hydrocolloids 63 (2017) 50e5856
8. and i-carrageenan (Fig. 2). The low gel strength of the putative agar
hydrocolloids, is likely due to the agar being measured in absence of
ionic interaction (i.e. no ions added for gelation), whereas the gel
strengths for the carrageenans were measured according to the
standard procedure in the presence of potassium ions to promote
the gel formation.
The gelling temperatures Tgel and melting temperatures Tmelt
were estimated for all the hydrocolloid samples and were defined
as the point in which the storage modulus G0 exceeded the value of
the loss modulus G’’ (G’ G00), during cooling and re-heating
respectively (Table 4) (Hossain, Miyanaga, Maeda, Nemoto,
2001). Comparing the water-extracted with the alkali-extracted
hydrocolloids, we observed that both the gelling and melting
temperatures were slightly lower for the water-extracted hydro-
colloids. The alkali- and water-extracted hydrocolloid from
C. crenulata, assumed to contain mainly i-carrageenan poly-
saccharides, had higher gelling temperature compared to that of k-
carrageenan, which was in agreement to standard grade sample i-
carrageenan. This result highly indicated that the gelling charac-
teristics of C. crenulata carrageenan were similar to those of iota
carrageenan as exemplified by the benchmark sample. The deter-
mined gelling and melting temperatures for H. dentata and
H. musciformis was similar to those reported earlier by John and
Asare (John Asare, 1975). The gelling temperature is dependent
on the concentration of the negatively charged sulfate groups, as
they inhibit gel formation due to repulsion of the carrageenan
chains. The potassium ions promotes the gel formation as they
stabilize the “junction zones” between the hydrocolloid helices by
binding to the sulfate groups without hindering cross-linking of the
two helices. The higher sulfate content and the absence of counter
ions in the water-extracted hydrocolloids will hence decrease the
gelling temperature due to the repulsion effects (Rhein-Knudsen
et al., 2015). The gelling temperature for the commercial k-carra-
geenan was different from the carrageenans extracted from the
Ghanaian seaweed used in the present study. As shown in Table 3,
the k-carrageenan sample had significantly higher amounts of
sulfate than the other k-carrageenan samples. In addition it also
contained high amounts of glucose that promotes the increase of
the gelling temperature. The glucose thus appears to have a higher
effect than the charged provided by the sulfate esters.
The gels obtained from seaweed sources are thermo-reversible
and the melting temperatures and corresponding G0 values were
estimated as described above for the gelling temperatures. The
profiles of sulfate dependences of Tmelt were similar to those
determined of Tgel (Table 4). The melting temperatures for the
carrageenan extracted from C. crenulata and the agar derived from
H. dentata could not be determined, as the cross-over between G0
and G00 was not observed within the chosen temperature range
(Supplementary material Figures S2, S3).
Carrageenans and agar are commercially extracted by either hot
water- or alkali-treatment techniques. Hot water extraction is used
for the extraction of native agar and carrageenan, whereas the
alkali-treatment is a combined chemical modification and extrac-
tion method. The alkali removes the sulfate esters from the pre-
cursors, i.e. from porphyran, m-carrageenan, and n-carrageenan,
and moreover causes formation of the 3,6-anhydro-bridge on the
galactopyranose moieties, which in turn promotes gel formation as
the de-sulfatation and 3,6-anhydro-bridge formation cause
conformational transitions of the polysaccharides (Genicot-Joncour
et al., 2009). Hence, both the chemistry and the rheological prop-
erties of the red seaweed hydrocolloids are influenced by several
factors, notably the algal source, life-stage, growth environment,
and the final rheological properties are moreover affected by the
hydrocolloid extraction method (Rhein-Knudsen et al., 2015).
4. Conclusion
Selected wild red seaweed samples collected along the coastal
areas in Ghana were assessed for their potential as new source for
hydrocolloid production. The assessments were based on compar-
ison of monosaccharides composition, yields and characteristics of
the extracted hydrocolloids with carrageenan from Kappaphycus
alvarezii, the most important source of commercial carrageenan
(Hurtado et al., 2015) Rheological characteristics and gelling
properties were also compared with benchmark samples of
standard-grade carrageenan and agar. Two-step sulfuric acid hy-
drolysis and HPAEC-PAD analysis showed that the Ghanaian red
seaweeds contained high and similar amounts of galactose, the
main component of carrageenan and agar. The extraction tech-
nology adopted in this study i.e. water e and alkali-treatment re-
quires overnight treatment, high temperature and long extraction
hours which can be optimized using milder extraction technology
which is crucial to preserving the integrity of the hydrocolloids.
Nevertheless the yield for carrageenan (21e27%) was very satis-
factory similar to the carrageenan yield of K. alvarezii. Moreover, the
composition analysis of the extracted hydrocolloids indicated suc-
cessful modification during alkali-treatment, which was supported
by FTIR analysis and oscillatory rheological measurements. Esti-
mation of hydrocolloids rheological properties showed only minor
differences between the hydrocolloids extracted from the Ghanaian
red seaweed (H. musciformis) and from K. alvarezii from Vietnam. A
comparison with commercial hydrocolloids revealed that the car-
rageenans from the Ghanaian H. musciformis produced gels of
higher strength due to the absence of glucose and the formation of
anhydro-bridge in the 4-linked galactose unit resulting in lower
sulfate content. The C. crenulata collected in Ghana was found to
contain a hybrid carrageenan having the characteristics similar to
commercial i-carrageenan but with k-carrageenan co-existing in
the i-carrageenan matrix. The modification during extraction was
able to promote gelation, resulting in a gel strength exceeding the
one for the commercial i-carrageenan. The same trend was
observed from agar extracted from H. dentata, which showed poor
gelling abilities when extracted with water, but was improved
during alkali-treatment. Analysis of rheological parameters indi-
cated that the alkali-treated agar from H. dentata did not produce
good gel as the commercially used agar sample, if used at lower
temperatures. Based on the presented analysis of the seaweed
hydrocolloids and its characteristics, it is evident that the Ghanaian
seaweed samples have potential to be used for extraction of func-
tional hydrocolloids. In particular, the carrageenans from the
Ghanaian H. musciformis showed pronounced similarities with the
carrageenan derived from K. alvarezii and even surpassed the gel-
ling abilities of the standard grade k-carrageenan. In addition, the
results clearly showed that the seaweed hydrocolloids composition
and rheological parameters could be enhanced by modification
during extraction, which is worth noting when considering appli-
cation for these hydrocolloid polysaccharides.
Conflict of interest
All authors declare no conflict of interest.
Acknowledgements
This work was funded via the Seaweed Biorefinery Research
Project in Ghana (SeaBioGha) supported by Denmark's develop-
ment cooperation (Grant DANIDA-14-01DTU), Ministry of Foreign
Affairs of Denmark. We will also like to thank the Water Research
Institute, Council for Scientific Research, Accra, Ghana for their
assistance in collecting the seaweed samples.
N. Rhein-Knudsen et al. / Food Hydrocolloids 63 (2017) 50e58 57
9. Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.foodhyd.2016.08.023.
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