303

•k J. Appl. Glycosci., Vol. 47, No. 3 & 4, p. 303-310 (2000)•l

Isolation and Characterization of l -Carrageenan from

Eucheuma serra (Togekirinsai)*

Li-hwa Lin, Masakuni Tako** and Fujiya Hongo

Department of Bioscience and Biotechnology, Faculty of Agriculture , University of the Ryukyus

(1, Senbaru, Nishihara, Okinawa 903-0123, Japan)

A gelling polysaccharide was extracted from Togekirinsai (Eucheuma serra), which was col

lected from Miyako Island, Okinawa Prefecture, Japan, and purified by gelation with calcium chlo

ride. The purified polysaccharide obtained from Togekirinsai was colorles fibrous powder; yield
,
38.3% (w/w) based on dried seaweed and 4.6% (wet seaweed) . The total carbohydrate, ash and

moisture contents of the polysaccharide were 71.4, 21.2 and 7.1%, respectively . The content of to
tal sulfate was estimated to be 23.8%. The polysaccharide was composed of D-galactose
, 3,6-anhy-
dro-D-galactose and ester sulfate at a molar ratio of 1.2: 1.0: 1.5. Molecular mass of the polysac

charide was estimated to be about 2.8•~105. The infrared spectrum and values of optical rotation of

the polysaccharide at different temperatures were in agreement with those of standard c -car-

rageenan. The 13C- and 1H-NMR spectra showed the polysaccharide isolated from Togekirinsai was

composed of D-galactopyranosyl-4-sulfate and 3,6-anhydro-D-galactopyranosyl-2-sulfate . These re

sults indicate that the gelling polysaccharide is a Ă-carrageenan.

Ă-Carrageenan is found in certain species of red Togekirinsai (Eucheuma serra), which also be-
seaweed (Rhodophyceae) and is prepared by selec longs to red seaweed (Rhodophyceae), is grown on
tive precipitation with calcium chloride. The com Miyako Island of Okinawa Prefecture and has
position and properties of c Ă-carrageenan is very been used as a gelling additive of "Urusu" since
close to ƒÈ-carrageenan. It has the same carbohy long ago. Recently, because this seaweed is also
drate chain built with ƒÈ-carrageenan, that is the used as salad dressing, its utilization in the food
position of sulfate located at C-4 of 3-O-linked ƒÀ- industry is on the increase. The polysaccharide
D-galactopyranose residues, but differs from Ă-car from Togekirinsai (Eucheuma serra) has not been
rageenan in the position of sulfate groups located well studied yet.
at C-2 of 4-O-linked 3,6-anhydro-a-D-galactopy- We report herewith the isolation and characteri-
ranose residues.1,2) Polysaccharides, such as agar, zation of the gelling polysaccharide from Toge-
fucoidan and K-carrageenan, were isolated from kirinsai.
Kubireogonori (Gracilaria blodgettii),3) Yumigata-

ogonori (Gracilaria arcuata),4) Okinawamozuku MATERIALS AND METHODS

(Cladosiphon okamuranus),5) Itomozuku (Nemacy-

stus decipiens)6) and Ibaranori (Hypnea charoi- Materials. Togekirinsai (Eucheuma serra )

des ),7) which were harvested from the Okinawa Is- used in this study was collected in July 1999 from

lands. However, c -carrageenan has never been iso- Miyako Island, Okinawa. The collected seaweed

lated from any Okinawa seaweeds. was washed with tap water and then dried by an

*presented at th air-dried oven at 40•Ž for 24 h. c -Carrageenan,

e Annual Meeting of the Japanese Soci-
used as the standard, was extracted from Euc-
ety for Food Science and Technology, Fukuoka, Ja-
heuma spinosum supplied by Taiyo Kagaku Co.,
pan, September 6-8, 1999.
**Conesponding author
. Ltd., Japan.
 304 T Annl 17R,nncri Vnl 47 No 3 & 4 (2000)

Extraction and purification of polysaccharide. treated with 0.5 M hydrogen chloride in methanol

An air-dried seaweed sample (5 g) was suspended at 105•Ž for 12 h in a sealed tube. The reaction

in water and heated on a heater with distilled mixture was neutralized with silver carbonate at

water (500 mL) at 100•Ž for 2 h to extract the 60•Ž, and then filtered and evaporated.12)

polysaccharide. The extract was then centrifuged at Paper chromatography, liquid chromatography

5000 rpm for 20 min and the supernatant was fil- and thin-layer chromatography. Paper chroma-

tered through a suction filter (Celite 545). The pre- tography was performed on Advantec Filter Paper

cipitate was then dialyzed against distilled water No. 50 using a solvent of butanol-ethanol-water

overnight to complete the dissolution. The solution (4: 1: 5). Chromatograms were sprayed with ani-
was diluted with distilled water to 500 mL, and fil- line hydrogen phthalate in butanol saturated with

tered through a suction filter (Celite 545) again. water and heated at 105•Ž for 15 min.

The extract was concentrated in an evaporator to Two milliliters of the hydrolyzate was applied

about half of the original volume. In the presence to a liquid chromatograph, DM 500 (Column;

of calcium chloride (0.2%, 20 mL), ethanol (2 Carbo. PacPAL 4•~250mm, Dionex Co., Ltd.),

vols) was added to the solution. The precipitate equilibrated with 5 mM NaOH. The chromatogra-

was dried in vacuo. The crude polysaccharide (2.3 phy was carried out at a flow rate of 1 mL/min at

g) was dissolved in 300 mL of distilled water and 35•Ž.

filtered through a suction filter (Celite 545). The Thin-layer chromatography was carried out on

filtrate was dialyzed against distilled water until glass plate (20 cm in length) treated with silica gel
free from chloride, and then ethanol (2 vols) was containing calcium sulfate as the binder and using

added to the dialyzate. The precipitate was dried in a solvent of butanol-ethanol-water (4: 1: 5). Chro-

vacuo. matograms were sprayed with 10% sulfuric acid in

Acid hydrolysis. The purified polysaccharide water and heated at 110•Ž for 15 min.

(50 mg) was dissolved in distilled water (20 mL) Molecular mass determination. The molecular

at 80•Ž and sulfuric acid was added to a final con- mass of the polysaccharide was determined by

centration of 0.4 M. The mixture was then heated high-performance liquid chromatography (HPLC)

at 100•Ž for 3 h. The hydrolyzate was neutralized (Shimadzu SCL-6 B; Shimadzu Seisakusho, Co.

with BaCO3. Ltd., Japan) on a Superdex 200 column (TSK gel

Determination of total carbohydrate, D- galac- G 4000 PW•~L) with a sample loop of 200 p L.

tose and 3, 6-anhydro-D- galactose. The total The HPLC operation was performed at room tem-

carbohydrate was determined by the phenol-sulfu- perature. The column was developed with 50 mM
ric acid method.8' D-Galactose and 3,6-anhydro-D- phosphoric acid buffer, and the same buffer sup-

galactose were determined by the methods of cys- plemented with 150 mM sodium chloride and frac-
teine-hydrochloric acid9' and resorcinol,10,11)
respec- tions were collected at a flow rate of 0.2 mL/min.

tively. The fractions were treated by the phenol-sulfuric

Estimation of ester sulfate. Purified polysac- acid reagent, and color development was measured

charide (40 mg) was dissolved in distilled water at 490 nm. Standard pullulan (P-82), P-400 (mo-

(20 mL) at 80•Ž and hydrochloric acid was added lecular mass, 4.0•~105), P-100 (1.0•~105), P-20

to the solution to a final concentration of 1.0 M (2.0•~104), and P-5 (0.5•~104), Pharmacia Chemi-
HCI. The mixture was then heated at 110•Ž for 3 cals Co., Ltd., Sweden, having definite molecular

h. Two milliliters of the hydrolyzate was applied mass were used for calibration.

to a liquid chromatograph, DM 500 (Dionex Co., The molecular mass of the polysaccharide was

Ltd., USA), on a column (AS 4 A-SC 4•~250 mm) also determined from the intrinsic viscosity at

equilibrated with 1.7 mM NaHCO3•~1.8 mM Na2 25•Ž in 0.1 M NaCI according to the relationship

CO3. The chromatography was carried out at a [ƒÅ]= 3.1•~10-3• M0.95, determined for ƒÇ-car-

flow rate of 1.5mL/min at 35•Ž. rageenan.13) The intrinsic viscosity [ƒÅ] was deter-

Methanolysis. The polysaccharide (10mg) was mined by measuring the specific viscosity with an
 Isolation of c-Carrageenan from Togekirinsai 305

Ostwald-type viscometer. the shape of a thorny branch, the thick part of

Measurements of infrared spectra and optical which was about 5.0 mm and its tip was sharp.
rotation. Infrared spectra were recorded with an The collected seaweed (2000 g) was washed

infrared spectrophotometer (IR-8200, Japan Spec- with tap water and then dried by an air-dried oven

troscopic Co., Ltd., Japan) for samples dispersed at 40•Ž for 24 h. The weight of the air-dried sea-

in KBr discs. weed decreased to 240 g. The gelling polysaccha-

Optical rotation was measured at 589 nm on a ride was prepar ed and purified as described in MA-

polarimeter (DIP-180, Japan Spectroscopic Co., TERIALS AND METHODS. The purified polysacch aride

Ltd., Japan) for a 0.1% (w/v) solution in distilled obtained from Togekirinsai was a colorless, fibrous

water. powder, with a yield of 38.3% (w/w) based on

13 C-and1H-nuclear magnetic resonance (NMR) dried seaweed and 4.6% (wet seaweed).

spectroscopy. The 13C-and 1H-NMR were recorded

at 80•Ž on a FT-NMR spectrometer (JNM ƒ¿500, Chemical components of the polysaccharide.

Nihon Denshi, Co., Ltd., Japan). The chemical The total carbohydrate and ash of the purified
shifts were expressed in parts per million (ppm) polysaccharide were estimated to be 71.4 and
relative to internal dimethyl sulphoxide (DMSO), 21.2%, respectively. As shown in Table 1, the
39.6 ppm for 13C-NMR and 2.697 ppm for 1H- moisture of the purified polysaccharide was esti-
NMR, respectively. The polysaccharide isolated mated to be 7.1%. The sulfuric acid was estimated
from Togekirinsai (80 mg) and standard Ă-car- to be 23.8% by HPLC after hydrolysis of the poly-
rageenan were dissolved in 4 mL of D2O at 80•Ž saccharide. The 3,6-anhydro-D-galactose and D-ga-
for 30 min and recorded at 80•Ž.14-17) lactose were estimated to be 30.5 and 39.1%, re-
spectively, by colorimetric determination with cys-
RESULTS

Preparation of polysaccharide from Table 1. Chemical components of purified polysaccharide

from Eucheuma serra.
Togekirinsai.
Figure 1 shows a photograph of Togekirinsai
(Eucheuma serra) which was collected in July
from Miyako Island of Okinawa Prefecture, Japan .
The Togekirinsai reached 15-20 cm long having

Fig. 1. Photograph of Eucheuma serra (Togekirinsai).

 306 J. Appl. Glycosci., Vol. 47, No. 3 & 4 (2000)

Fig. 2. Thin-layer charomatogram

of methanolyzate of the po
lysaccharide isolated from
Eucheuma serra.
1. methyl-/3-D-galactoside; 2, me-

thyl-a-D-galactoside; 3, methanoly-

zate of the polysaccharide from

Eucheuma serra ; 4, methanolyzate

Fig. 3. Infrared spectra of the polysaccharide from Eucheuma serra and
of standard c -carrageenan. (1), 3,6-
Ă -carrageenan standard .
anhydro-methyl-ƒÀ-D-galactoside; (2),
(1), polysaccharide from Eucheuma serra; (2), Ă-carrageenan standard.
3,6-anhydro-methyl-a-D-galactoside;
Experimental conditions are described in the text.
(3), methyl-f3- and -a-D-galactose.

Experimental conditions are de-

scribed in the text.

Table 2. Optical rotation of the polysaccharide extracted from Eucheuma serra.

teine-hydrochloric acid-resorcinol reagents. Determination of molecular mass.

The molecular mass of purified polysaccharide

Identification of sugar components of the poly- isolated from Togekirinsai was measured by the

saccharide. gel chromatography method on a superdex 200

A paper chromatogram (data not shown) of the colunm. According to the standard calibration

hydrolyzate of the purified polysaccharide and c- curve obtained from the definite molecular mass

carrageenan used as the standard showed spots pullulan, the molecular weight of this purified
identical to D-galactose. Only one peak was also polysaccharide was calculated to be approximately
shown on a liquid chromatogram, and it was iden- 2.8•~105. The molecular mass was also calculated

tical to D-galactose. As shown in Fig. 2, examina- to be about 2.8•~105 by the viscometric method

tion of the methanolysis product of the polysac- with an Ostwald-type viscometer.

charide by thin-layer chromatography indicated the

presence of 3,6-anhydro methyl ƒÀ-D-galactoside at Infrared spectra and optical rotation of the
the position of spot 1, 3,6-anhydro methyl-a-D-ga- polysaccharide.
lactoside at the position of spot 2,18) and a mixture The infrared spectrum of the polysaccharide
of methyl-a- and ƒÀ-D-galactoside at the position (Fig. 3) showed a broad band at 1240-1250 cm-1,
of spot 3. and was common to all of the sulfated polysaccha-
rides due to sulfate absorption.19,20)The peak at 805
cm-1 was assigned to be a ester sulfate on C 2 of
 Isolation of Ă-Carrageenan from Togekirinsai 307

Fig. 5. 1H-NMR spectrum of the polysaccharide from

Eucheuma serra and standard Eucheuma spino-

sum at 80•Ž.

Details are described in the text.

Fig. 4. 13C-NMR spectrum of the polysaccharide from

Eucheuma serra and standard Eucheuma spino-

sum at 80•Ž.

Details are described in the text.

Table 3. Twelve chemical shifts of the 13C-NMR spectrum of the purified poly-
saccharide isolated from Togekirinsai and the standard Eucheuma spinosum.
 308 J. Appl. Glycosci., Vol. 47, No. 3 & 4 (2000)

Table 4. Twelve chemical shifts of the 'H-NMR spectrum of the purified poly-
saccharide isolated from Togekirinsai and the standard Eucheuma spino-
sum (supplied by Taiyo Kagaku Co., Ltd.) at a concentration of 2.0%
(w/v).

the 4-linked 3,6-anhydro-D-galactose and the peak fate), 69.7 (C-2), 77.2 (C-3), 75.2 (C-5) and 61.7
at 845 cm-1 to be ester sulfate on C 4 of the 3- ppm (C-6) of D-galactose, and 77.4 (C-2) 80.2 (C-
linked-D-galactose unit, and 930 cm 1 showed 3,6- 3) 80.7 (C-4) 79.4 (C-5) and 72.3 ppm (C-6) of
anhydro-D-galactose.19-25) These data were consis- 3,6-anhydro - galactose, respectively (Table 3).
tent with those of standard c -carrageenan prepared These chemical shifts are assigned to the repeating
from Eucheuma spinosum. unit of c-carrageenan.2,14-17,23,26,27)
No other chemical
The optical rotation of the purified polysaccha- shift assigned to the carbon atom of the sulfate
ride at various temperatures is summarized in groups substituted at any carbon atoms in the D-
Table 2. It showed a value of+0.092•‹at 60•Ž galactose or 3,6-anhydro-D-galactose was esti-
and then increased gradually with decreasing tem- mated. This means that the polysaccharide isolated
perature. The values were also in agreement with from Togekirinsai was composed of D-galactose-4-
those of standard c -carrageenan at various tem- sulfate and 3,6-anhydro-D-galactose-2-sulfate.
peratures. The proton chemical shift of the polysaccharide

isolated from Togekirinsai is shown in Fig. 5. The

13C- and 1H-nuclear magnetic resonance (NMR) 1H -NMR spectrum of the Ca salt of the polysac-

spectra analysis. charide and commerical Ca salt of the Ă-car-

As shown in Fig. 4, at 80°C, in D2O, the 13C-nu- rageenan prepared from Eucheuma spinosum sup-

clear magnetic resonance (13C-NMR) spectrum of plied by Taiyo Kagaku Co., Ltd, show nine major
the Ca salt of the polysaccharide isolated from To- peaks, with the chemical shifts at 4.649, 3.640,

gekirinsai at a concentration of 2.0% (w/v) and 3.999, 4.910, 3.805, and 3.828 ppm corresponding

commercial Ca salt of c -carrageenan prepared to G 1, G 2, G 3, G 4, G 5, and G 6, respectively.

from Eucheuma spinosum supplied by Taiyo Ka- The chemical shifts values of 5.314, 4.849, 4.677,

gaku Co., Ltd. show 12 major peaks. The chemical 4.70 12, 4.648, and 4.116 ppm correspond to A 1,

shifts were estimated at G 1, 104.6; G 2, 69.7; G 3, A 2, A 3, A 4, A 5, and A 6, respectively. The

77.2; G 4, 72.5; G 5, 75.2; G 6, 61.7; A 1, 94.4; A coalescing signals of A 3, almost together with

2, 77.4; A3, 80.2; A4, 80.7; AS, 79.4; and A6, A 5, appear as one peak with a maximum at 3.805

72.3 ppm, respectively; here, G refers to D-galac- ppm (Table 4). These data were in agreement with
tose and AG is the 3,6-anhydro-D-galactose resi- those of standard c -carrageenan. As shown in

due. These data were consistent to those of stan- Fig. 5 and Table 4, these chemical shifts of the

dard c -carrageenan which showed the chemical polysaccharide isolated from Togekirinsai are as-
shifts at 104.6 ppm (C 1 of 3-linked ƒÀ-D-galac- signed to the repeating unit of c -carrageenan.17,24)

topyransoyl-4-sulfate), 94.4 ppm (C 1 of 4-linked This means that the polysaccharide isolated from

3,6-anhydro-ƒ¿-D-galactopyransoyl-2-sulfate), 72.5 Togekirinsai is composed predominantly of ƒÇ-car-

ppm (C 4 of 3-linked ƒÀ-D-galactopyransoyl-4-sul- rageenan.

 DISCUSSION REFERENCES

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ロ ーD一
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