Characterization and thermal behavior of six sulphated polysaccharides from seaweeds

Food Hydrocolloids Vol.8 no.3-4 pp.215-232, 1994

Characterization and thermal behavior of six sulphated polysaccharides from seaweeds Min-feng Lai I, Chin-Fung Li 1 and Cheng-yi Li 1.2,3

Graduate Institute of Food Science and Technology, National Taiwan University, Taipei, Taiwan and 2Institute of Chemistry, Academic Sinica, Nankang, Taipei, Taiwan, ROC

I

3To whom correspondence should be addressed at Institute of Chemistry. Academia, Sinica, Nankang, Taipei, Taiwan, ROC Abstract. The composition of water soluble polysaccharides from Halymenia ceylanica, Grateloupia [ilicina, Pterocladia capillacea, Viva arasakii, Helminthocladia australis and Liagoropsis schramni were analyzed by high performance anion exchange chromatography. The IR and "C-NMR spectra of these polysaccharides were also investigated. The Pterocladia capillacea agar was a regular, alternating and nearly 'absolute' structure. The G.filicina polysaccharide possessed the 'intermediate' structure between agar and carrageenan. And H.cey/anica was not well resolved because of its high heterogeneity. In a diluted salt solution the three Rhodophytaceae and the two Helminthocladiaceae polysaccharide molecules showed Gaussian coil behavior with different properties of the polymer-solvent pairs, while Ulvaceae polysaccharide exhibited very different behavior. The thermal behaviors of the polysaccharides were examined by DSC. The thermogram of Pterocladia polysaccharide gel showed three melting-transition endotherms at 40. 75 and lJ5°C. which might be attributed to the double helix-coil conformation change, the melting of junction zone and the disentangling of the tightest cross-links respectively. There were two endotherrns detected on the thermograms of Grateloupia (20-40 and 70°C) and Halymenia (25-50 and Boac) polysaccharides corresponded to the conformation change and to the aggregation of the random coils. The similar viscometric and thermal behaviors among the polysaccharides in the same family were observed, which could result from their acidic groups, structures and/or conformations. and were useful for the algal taxonomy and applications.

Introduction

Seaweed is one of the most important sources of the polysaccharide gum. Recent studies about the physicochemical properties and structure of the sulphated polysaccharides of new or economic seaweeds have added considerably to knowledge in the field (1). It has been attempted to establish the molecular taxonomy of polysaccharides in seaweeds from the physicochemical properties (2-8), from the distribution of functional group in IR spectra (3,4,8,9-12) or from the structure or the conformation elucidated by 13C-NMR (13-24). Furthermore, there are still many seaweed species which have not yet been identified. The nature of the water-soluble, sulfated polysaccharide in the marine alga was known by the characteristics of its biosynthesis (12,24,25,26). The conformation was ribbon-like, helical, or a completely disordered form with linear or branched structure (25) which could be changed with different environmental conditions (27). The chemical component of the polysaccharide was affected by the cultivation environment (24) and extraction conditions (6,15,18,22). Consequently, the different physicochemical properties of the 215

M.-f.Lai, C.-F.Li and C.-y.Li

polysaccharides from identical species of seaweed could be observed. Such variations would influence their functions and applications. Significant information was gained of the highly cooperative structure of the seaweed polysaccharide as it underwent conformational or phase transition, and/ or any chemical reaction upon being heated or cooled could be obtained by DSC (27). There were a few studies concerning the phase transition of the gellingseaweed polysaccharide with DSC (28-31). However, very few investigations on the non-gelling polysaccharide were made under different conditions. The purposes of this study were the investigation of the physicochemical characteristics, the thermal behavior of the seaweed sulfated polysaccharides and the masking structure of galactan from red seaweed. All varieties of seaweed were grown in Taiwan. The study might be useful not only for collecting the preliminary data for taxonomy, but also the development of new industrial sources of the polysaccharide gum. Six varieties of tropical algae, including red and green seaweeds, were applied during the investigation. Among them, Helminthocladia sp. and Liagoropsis sp. were studied for the first time, and Halymenia sp. had been only seldom investigated. Materials and methods

Seaweeds

Six varieties of the tropical seaweed were used in this study. There were Pterocladia capillacea (Gmelin) Bornet et Thuret (Gelidiaceae, Gelidiales), Grateloupia filicina (Wulf) C. Ag. (Halymeniaceae, Cryptonemiales), Halymenia ceylanica Weber-van Bosse (Halymeniaceae, Cryptonemiales), VIva arasakii (Ulvaceae, Ulvales), Helminthocladia australis Harvey (Helminthocladiaceae, Nemaliales), and Liagoropsis schrammi (Maze et Schramm) Doty et Abbott. (Heliminthocladiaceae, Nemaliales) were grown in the southern part of Taiwan and were harvested in the spring of 1991 or 1992. The seaweed was washed, dried in an air oven at 45°C, and milled. Sample preparation DC

The seaweed was extracted with fifty times its weight of water at 80 for 5 h. The extract was cooled to room temperature and then centrifuged. The supernatant was filtered through the Whatman No. 1 filter paper. Treatment with chloroform and amyl alcohol by the Sevag method (32) was applied for the removal of proteins. The purified polysaccharide was precipitated with three volumes of 95% ethanol and washed with ethanol and acetone, followed by drying in vacuo at ambient temperature. The P.capillacea agar was prepared by the freeze-thawing method (33). Analysis of chemical constituents

The polysaccharide was hydrolysed with 2 mol/dm ' trifluoroacetic acid (TFA) at 95°C for 16 h (34,35). The hydrolyzate was dried in vacuo with a centrifugal evaporator (Savant Speed-Vac 100, Savant Instruments, Inc., Farmingdale, NY) 216

Sulphated polysaccharides from seaweeds

at ambient temperature, then redissolved in 18 Mfl deionized water. The solution was purified with Amberlite IRA-400 resin (CI- form), and filtered through a membrane of pore size 0.45 urn. The neutral monosaccharides of the filtrate were determined with high-performance anion-exchange chromatography (HPAEC). HPAEC was conducted with a Oionex OX-300 Bio-LC Model 4000i system with a CarboPac PA 1 Guard and CarboPac PAl column (250 x 4 mm IO), and a Model PAD II (Oionex, Sunnyvale, CA, USA). Three pulse potentials at range 2 were E, = 0.10 V, £2 = 0.60 V, and E, = -0.80 V, and the durations were t, = 120 ms, t 2 = 60 ms, and t J = 180 ms respectively (36). The output range was 300 or 100 nA. The sample loop size was 25 ul. Because the dissociation constant of monosaccharide to its alcoxide ion was regarded as the alkali concentration, different monosaccharides have characteristic retention times with differnt concentration and flow rate of the alkali eluant. In order to characterize the monosaccharides adequately, eluent 1(%) (0.2 mol/dm' NaOH) and eluent 2(%)) (0.1. water), and flow rate (ml/mn) of three analytical programs by Oionex gradient pump were (1) 20:80, 0.75; (2) 10:90,1.00; and (3) 0: 100, 1.50. Program (1) was used for the quantitative analysis of the monosaccharide with o-fructose used as the internal standard. All data were triplicate and calculated with a Oatajet integrator (Spectra Physica Inc., San Jose, CA). The total carbohydrate content of the polysaccharide fraction was measured by the phenol-sulfuric acid method (37). Because of the significant colorimetric differences between different sugars, it was calibrated by the standard curve of one or two composites of the major monosaccharide compositions (referred to the results of HPAEC analysis). The pyruvate content was determined from the acid hydrolysate by HPLC (ModeI1081B, Hewlett Packard, USA). The column was packed with the REZEX organic acid (H+ form) and was eluted with 1% H JP0 4 at 35°C, with a RI detector. The sulphate ester content was examined by the sodium rhodizonate method, with sodium sulphate as standard (38). 3,6Anhydrogalactose was assayed by the improved resorcinol method (39), 0fructose as standard. The hexauronic acid was analyzed by the harmine-sulfuric acid method (40), o-galacturonic acid as standard. All the chemical compositions, except for total carbohydrate, were shown as molar percentages and detailed in Table I.

Determination of the structure The proton-decoupled, IJC-NMR was carried out using a Bruker ACP~300 and AMX-500 NMR spectrometer operating at 75.48 and 125.77 MHz respectively, and with a 5 mm dual probe. The spectra of 5-6% polysaccharide solution in 0 20 were recorded at 80°C with a spectral width of 6-21 KHz and relaxation delay of 0.4-1.3 s. The carbon chemical shifts were measured in parts per million (p.p.m.) relative to internal dimethyl sulfoxide and converted to values related to tetramethyl silane. The conversion constant was 39.5. Films of polysaccharides for infrared analysis were obtained from a 2 ml aqueous solution containing 5 mg polysaccharide by drying in Teflon molds 217

N >-' 00

f

:"" I:""

~.

r

~

r.

Table I. Chemical components of the polysaccharides from six varieties of seaweed

Total carbohydrate (%)" Monosaccharide (%)b D-galactose 3,6-anhydro-galactose L-rhamnose n-mannose methyl-galactose 2-deoxY-D-ribose n-xylose n-ribose t.-fucose n-arabinose D-galactosamine D-glucosamine monic acid Sulfate ester" Pyruvic acid" G:A:S d

Halymenia ceylanica

Grateloupia filicina

Pterocladia capillacea

Viva arasakii

Helminthocladia australis

Liagoropsis schramni

45.0

73.7

83.9

54.9

74.7

80.8

52.8 7.0 nd" nd 4.6 15.2 7.8 0.5 nd 5.3 trace nd 6.8 0.735 0.417 1:0.13:0.65

38.6 26.0 1.3 nd 4.6 8.6 3.3 0.9 0.9 nd trace nd 15.7 0.256 0.076 1:0.67:0.16

41.9 39.9 nd nd 9.3 4.2 2.9 nd nd nd trace nd 1.8 0.Q15 0.039 1:0.95:0.008

nd nd 33.2 4.3 nd 2.8 11.0 0.4 0.4 1.2 trace nd 46.7 0.430 nct

nd nd nd 62.1 nd 1.4 28.6 nd 0.5 1.4 nd trace 6.1 0.208 nd

nd nd nct 72.2 nd nd 23.6 nd 0.7 nd nd nd 3.6 0.617 nd

"Represented as weight % of the product. "Shown as molar % of total carbohydrate. "Expressed with molar ratio of acidic group to the monosaccharide unit. dThe molar ratio of galactose:3,6-anhydro-galactose:sulfate. e Not detectable.

oo ::l

0-

r ..:,

r.

Sulphated polysaccharides from seaweeds

(2.7 em I.D. X 2.0 em H) at ambient temperature. The spectra were recorded on a Perkin Elmer 882 Infrared spectrophotometer with the standard condition for slit program and time drive. Measurements of physical properties

Intrinsic viscosity, [1)], was determined by capillary viscometer, Carmon-Fenske No. 50, 75 or 100, at 25 ± 0.05°C and 50 ± 0.1°C. The Huggins plot [1)Sp/C = [1)] + k' x [1)f x C) (41) and the Fuoss-Strauss plot [1)Sp/C = A/(1 + B x YC)] (5,6) of the viscosity data in aqueous 0.1 mol/drn ' NaCI and distilled water respectively, gave good linear behaviours in the range of polymer concentration, C, from 0.01 to 0.25 g/d\. The expansion factor, a'll' is given by [1)]w/[1)] = a'll3 (6,41), where [1)]w is the intrinsic viscosity in distilled water, [1)] is the intrinsic viscosity in aqueous 0.1 mol/drn ' NaCl. The apparent viscosity, 1) and 1)w' of the polysaccharide solution (1 g/dl in aqueous 0.1 mol/drrr' NaCl and distilled water respectively) was measured by a cone/plate viscometer model DV-1I + with a 1.565° cone spindle No. cp-42 (Brookfield Engineering Laboratories, Inc., USA) and 100 r.p.m. at 25 ± 0.1°C. The sample size for determination was 1.0 m\. The temperature dependence of the degree of optical rotation was measured with 1 or 0.12 g/dl (in distilled water) of the viscous or gelling polysaccharide solution respectively, by the polarimeter (Polartronic Universal, Schmidt & Haensch Gmbh & Co., Germany) with 100 mm quartz plate tube and sodium Dline (589 or 546 nm) (4). Because the specific optical rotation degree, [d], was the representation of whole product, it was reasonably calculated by basing on dry polysaccharide mass, not on total carbohydrate content. The gel strength of 1 g/dl (in distilled water) gel of Pterocladia agar was determined by compression test with a Sun Rheometer model CR-200D (Sun Scientific Co., Ltd, Japan) with 1 kg mode and 10 mm cylindrical probe, and analysed with Rheo Data Analyzer software v. 2.5. The compressing speed and depth were 60 mm/min and 4 mm respectively. The sample was prepared by cooling the hot solution in a Teflon mold (27 mm, I.D. x 17 mm H), then infused in silicone oil immediately, and tested after aging at 5°C for two days (42). The gel strength was presented as maximum stress (g/cm") of a generalized texture profile analysis (42, 43). The thermal behavior of the polysaccharide was examined with the Setaram DSC121 system (Setaram Co., France). The polysacchride was dissolved completely in distilled water to a concentration of 4 g/dl (dry basis) (30,31), and weighed 100.5 ± 0.5 mg of the freshly-prepared, hot polysaccahride solution into a stainless steel crucible and sealed with a stainless steel cap and aluminium 0ring. The sample was aged at 5°C for two days before testing (30). During the determination the sample was equilibrated at 5°C for 10 min first, then heated to 95°C at a rate of 1°C/min. The reference crucible was filled with an equivalent amount of distilled water.

219

M.-r.Lai, C.-F.U and c.-y.U

Results and discussion Chemical properties

The chemical components of extracts from the six varieties of seaweed, after purifying with Sevag method, are listed in Table I. The total amount of carbohydrate content was in inverse proportion to the amount of the acidic group content, due to the existence of residue salt in the polysaccharides. The polysaccharide from Hiceylanica contained the least of total carbohydrate content with a small amount of 3,6-anhydro-galactose and the largest amount of sulfate ester (~O. 735 mole per monosaccharide molar unit) and pyruvate (~0.417 mole per monosaccharide molar unit) among the six polysaccharides. The molar ratio of galactose, 3,6-anhydro-galactose and sulfate ester (G:A:S) in the Halymenia polysaccharide was 1:0.13:0.65. The total carbohydrate content of G.filicina polysaccharide was high, with larger amounts of 3,6-anhydrogalactose and uronic acid and smaller amounts of sulfate ester and pyruvic acid than those of the Halymenia polysaccharide. The G:A:S ratio of Grateloupia polysaccharide was 1:0.67: 0.16. The compositions of these two polysaccharides of the family Halymeniaceae (sometimes classified into Grateloupiaceae) (3,4,8) were close to pi-carrageenan (one kind of lambda-type carrageenan) (8). The P.capillacea agar contained very low charged substitution and the ratio of G:A:S was 1:0.95:0.008. In addition to galactose and its derivatives (i.e. 3,6-anhydrogalactose and methylated galactose), 2-deoxy-o-ribose, xylose, uronic acid, 0arabinose, L-rhamnose, o-ribose or L-fucose, etc. were presented in the red algal polysaccharides (Table I). The polysaccharides from the remaining three, non red algal varieties were unpyruvated. The Ulva arasakii composed of high contents of L-rhamnose (33.2 molar %) and uronic acid (46.7 molar %), which was identified as o-glucuronic acid (44), fairly large amount of n-xylose; and small amounts of n-mannose , 2-deoxy-o-ribose, n-arabinose, n-ribose, and Lfucose. The compositions of the polysaccharides from Hiaustralis and L.schramni were similar. Both major constituents were o-mannose (62.1 and 72.2 molar % respectively) and n-xylose (28.6 and 23.6 molar %), and the minor components were uronic acid and L-fucose. The data indicated that those polysaccharides from red algae (i.e. Hiceylanica, Gi[ilicina, and P.capillacea) had different degrees of pyruvation. As DiNinno et al. (1979) described (45), the occurrence of pyruvate ketal bound to carrageenan and agar might have taxonomic significance, and was further proved. Six algal polysaccharides analysed had their characteristic compositions specified by seaweed family and related to the cell-wall structure and biological activity in seaweed (25,26). These were somewhat heterogeneous sugar substances existing in the algal polysaccharide. During the investigation the HPAEC, with the three elution programmes, was found very effective for the determination of minor sugar components. IR spectroscopy determination

The IR spectra of P.capillacea agar with G:A:S = 1:0.95:0.008 (Table I) showed 220

Sulphated polvsaccharides from seaweeds

the peaks of the total sulfate ester at 1240 ern-I (Fig. 1) which corresponded to the 5-0 stretching vibration and the sharp peak at 925 cm -I (3,6-anhydrogalactose). The above characteristic absorption bands were also observed in the agars from other varieties (10,24). The Gfilicina polysaccharide had the characteristic sulfate ester absorption band at 1210 em -I and a small shoulder at 930 cm -I related to 3,6-anhydro-galactose. The band from 780 to 870 em- 1 indicated the presence of several types of sulfate esters (8). Two peaks at 830 and 800 cm -I were assigned to the secondary equatorial sulfate at C 2 of the 1,3linked galactose unit and a primary axial sulfate at C 2 of 1,4-linked 3,6-anhydrogalactose respectively (3,4,8,10,12). The 1375 cm" peak seemed to be the asymmetric 5-0 stretch peak described by Rochas et al. (10). The sulfate ester

p

I

2000

(

•

!

!

1600

,

1200

800

400

WAVE NUMBER, em-I

Fig. I. IR spectra of the polysaccharides from Pterocladia capillacea (P). Grateloupia [ilicina (G) and Halymeina ceylanica (H).

221

M.-f.Lai, C.-F.Li and C.-y.Li

of the polysaccharide was more diverse than those of the polysaccharide from Hawaiian Gfilicina (8). The IR spectra of the polysaccharide from Hiceylanica showed peaks at 1198 ern -1 for the large amount of sulfate ester, and 820 cm- 1 for galactose 6-sulfate. The small 3,6-anhydro-galactose content was not detected on the spectra (H) because of the broad 1300-900 em -I band. There was no signal about galactose-4-sulfate observed on the IR spectra of the three red algal polysaccharides. The wave number of the peak for sulfate ester would shift to the lower value with any increment in the sulfate content. It was implied that the amount of the functional group played an important role during the elucidation of IR spectra by the absorption peak. There was a sharp peak at 886 em -I in the spectra of P. capillacea and Gfilicina seldom reported in

,

2000

I

1600

,

,

,

I

1200

,

,

,

J,

800

,I

400

WAVE NUMBER, em-I

Fig. 2. IR spectra of the polysaccharides from Viva arasakii (0), Helminthocladia australis (H) and Liagoropsis schramni (L).

222

Sulphated polysaccharides from seaweeds

the literature. The size of this peak was increased with the increase in 3,6anhydrogalactose content. All IR spectra of the polysaccharides from LLarasakii, Hiaustralis, and L.schramni had the peak at 1200 em -I for sulfate esters, and the peaks 870800 cm- I for various kinds of sulfate esters (Fig. 2). The type of sulfate ester could not be identified from the spectra. However. two types of sulfate esters (822 and 780 cm- I ) were detected in the Uiarasakii and three types (850, 810 and 760 em -1) in the other two varieties of seaweed. There were the characteristic bands in the IR spectra of the polysaccharides from different families. NMR spectra characterization

The 13C-NMR spectroscopy at 75.48 MHz was applied to analyse the polysaccharides from three varieties of red seaweed. The spectrum of P.capillacea agar showed a regular, alternating structure of 3-0-D-galactopyranosyl (G) [Gl, 102.4; G2, 70.2; G3, 82.8; G4, 68.7; G5, 75.3; and G6, 61.4 p.p.m.] and 40-3,6-anhydro-L-galactopyranosyl (A) [AI, 98.2; A2, 69.9; A3, 80.1; A4, 77.3; A5, 75.6; and A6, 69.4 p.p.m.] (13, 19,21,22) (Fig. 3). The small split signals (indicated by arrows) were probably attributed to the presence of different conformers (46) or to the non-uniform distribution of the sulfated or methylated units in the polymer. There was no methoxy group signal (59.1 p.p.m.) or pyruvate methyl group signal (25.7 p.p.m.) observed in the spectrum. due to insufficeint amounts of pyruvate and methoxy groups detected. A2 A5 A1

A3

A6 G5

G1

G3

"

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100

G6

1

90

G4 G2

A4

1t,

~"'r

I

I

80

70

\~,!I"'I'

I

60

PPM

Fig. 3. 75.48 MHz DC NMR spectrum of Pterocladia capillacea agar (recorded at 80°C, 6872 scans, spectral width 17241 Hz and acquisition time 0.47 s). G, 3-0-D-galactopyranosyl; A, 4-0-3,6anhydro-L-galactopyranosyl.

223

M.-r.Lai, C.-F.Li and C.-y.Li

A6 86

C6

xe

06

I

I

100

90

I

I

I

80

70

50

PPM

Fig. 4. 75.48 MHz 13C-NMR spectrum of Grateloupia filicina galactan (recorded at 80aC, 11 648 scans, spectral width 6024 Hz and acquisition time 1.36 s). A, B, C, D, X, and Yare as in Table II footnotes.

Table II. Assignment of resonances (p.p.m.) in 13C-NMR spectrum of sulfated galactan from Grateloupia filicina

-O~oHf:ro0J~}O~otr OH

~H

OH

OH

HO

CH,oH

OH

D

ABC CH2

HY-0 0'-

-o~ X

OH

Y

Residue

Cl

C2

C3

C4

C5

C6

A

104.7 96.1 103.7 101.1 102.7 94.6

70.4 69.8 71.8 70.4 70.2 69.8

79.0 69.3 79.0 69.8 80.4 78.0

65.5 78.4 65.5 78.4 66.4 79.4

75.3 69.3 75.3 70.4 75.3 76.9

61.3 61.3 61.3 61.3 61.3 6D

B

C D X

Y

224

Sulphated polysaccharides from seaweeds

The spectrum of G.filicina galactan (Fig. 4) consisted of more than three series of resonances as described by Usov (13). It is not only analogous to the spectra of the desulfated lambda-carrageenan and the porphyran, but also to the desulfated kappa-carrageenan. The assignment of resonances in llC-NMR and the possible structures of G.filicina galactan are listed in Table II. A small methoxy group signal (59.1 p.p.m.) was observed. The unknown peaks of 76.1, 73.6,72.3 and 68.2 p.p.m. might be attributed to the carbons with (i) galactose2-sulfate, 3,6-anhydro-galactose-2-sulfate and/or pyruvate ketal, and (ii) uronic acid, and/or 2-deoxy-o-ribose residues. The splitting peaks were the complicated results of the different conformers with random locations of the functional groups and the residues. Further studies of the series of different structures corresponding to the different molecular fractions or incorporated in the same polysaccharide chain are required. The polysaccharide of G.filicina which might be classified into Grateloupiaceae or Halymeniaceae, showed an 'intermediate' structure between agar and carrageenan (13). The backbone structure was built of the alternating 3- and 4-linked galactose constituents, the latter one contained both 0- and L-forms (e.g. G.divaricata) (13). The polysaccharide of H.ceylanica was also in the same family as Gi[ilicina but with a more complicated structure. Hence, the NMR spectrum of H.ceylanica was carried out with a proton-decoupled 13C-NMR spectrometer at 125.77 MHz. There was one Cl signal (101.3 p.p.m.) at the anomeric region and one C6 signal (61.3 p.p.m.) (Fig. 5). The spectrum provided some data about the molecular structure and the conformation, although the C2-C5 resonance peaks were not well resolved. It suggested that the structure of H.ceylanica polysaccharide which had a high sulfate ester content (0.735 mole per monoaccharide mole) and pyruvate (0.417 mole per monosaccharide mole) and low uronic acid, 3,6anhydro-galactose and 2-deoxy-o-ribose, was more randomly sulfated and pyruvated than that of G.filicina polysaccharide. The complicated splitting peaks were the result of high heterogeneity and many different conformers 61.3

I

100

I

90

I

80

I 70

I

60

PPM

Fig. 5. 125.77 MHz uC-NMR spectrum of Halytnenia ceylanica galactan (recorded at 80°C, 15 024 scans, spectral width 20 833 Hz and acquisition time 0.78 s).

225

M.-f.Lai, C.-F.U and C.-y.U

existed. The methoxyl group signal (58.9 p.p.m.) and methyl group signal (25.4 p.p.m.) were also observed. Physical properties

The degree of specific optical rotation, [d]D, is attributed primarily to the sugar composition and its glycosidic linkage. The polysaccharide solutions of P.capillacea and Uiarasakii showed levorotatory, [d]D = -18 and -54° respectively (Table III), and the other four polysaccharides were dextrotatory. The [df5 of Hiceylanica, +12°, was much lower than that of Hivenusta Boergesen, +118° (4); and G.filicina, + 16°, was different from G.lanceola, +54.9° (3). The polysaccharide of Uiarasakii was close to those of Uilactuca (47) and the higher molecular weight fraction of Uconglobata (5,6), but differed from that of Upertusa (5,6,7). The [df5 of Hiaustralis and L.schramni were + 14 and 32° respectively. From the result, the polysaccharides belonging to the same family showed the same dextrotatory or levorotatory behaviour. However, the degrees of optical rotation of polysaccharides, (e.g. VIva sp.) varied with different seaweed species (6,7), growth environments, and polysaccharide fractions which were derived from different extraction conditions, DEAE fractions, etc. (5,48). Consequently, it would be difficult to apply the optical property of the polysaccharide as an index for the taxonomy of algae. The intrinsic viscosities of the polysaccharide were measured both in distilled water and 0.1 mol/drrr' NaCI solution. In general, the intrinsic viscosities, [1]], in 0.1 mol/drrr' NaCI solution were much lower than those in water among the polysaccharide studied (Table III). Grateloupia sp. polysaccharide had the highest [1]] among the three red algae, which could be the consequence of the highest expansion properties, all = 2.93, caused by the electrostatic repulsion of the sulfate ester residues. However, Halymenia sp. polysaccharide molecule possessed the highest sulfate ester conterit and pyruvate, but with a lower expansion factor, all = 2.31, than that of Grateloupia. This could be explained by the heterogeneous structure of the molecule which might diminish the electrostatic force of the sulfate ester. Pterocladia sp. agar with small sulfate and pyruvate contents, gave a very low expansion effect, a'l = 1.09. The VIva sp. polysaccharide showed the lowest [TI] value, 1.09 dl/g at 25°C and an expansion factor, all = 2.75, which was higher than those of Helminthocladia and Liagoropsis polysaccharides (Table III). It could be attributed to the large amounts of uronic acid and sulfate ester existing in the Ulva polysaccharide (Table I). The Huggins constant, k', of all polysaccharides, except that of VIva sp., was proportional to the [TI] under the same determining conditions (Table III). The k' constants of the sulfated galactans measured were in the range of 0.44-0.59, and Helminthocladia and Liagoropsis polysaccharides were close, 0.26 and 0.27 respectively. The concentration used during the determination of the intrinsic viscosity was very dilute and beyond the critical concentration for intermolecular entanglement. So, the frictional property contributing to viscosity was related to the 226

Table III. Some physical properties of the purified polysaccharides from six varieties of seaweed Source

Halymenia ceylanica Grateloupia [ilicina Pterocladia capillacea Viva arasaki Helminthocladia australis Liagoropsis schramni

[dj25 (water)

+12 +16 -18 -54 +14 +32

25°C [111" (dllg)

[11V (dllg)

a

5.58 5.90 5.32 1.09 3.44 4.91

68.98 148.49 6.83 22.69 23.12 23.95

2.31 2.93 1.09 2.75 1.89 1.70

"The data of [11] and 11 were determined in aqueous 0.1 mol/drn' NaCI. bThe data of [11]w and 11w were determined in distilled water. "a; = (h]w/[11])l/3. dThe k' value was derived from the Huggins plot (11,p/C = [11] + k'*[11f*C). "Not determined.

C

"

k'd

0.57 0.59 0.44 13.90 0.26 0.27

50°C [11]a (dllg)

k'd

1 g/dl solution 11" 11w b (cps) (cps)

5.60 5.85

0.42 0.51

29.1 42.6

34.2 53.7

5.3 7.3 13.2

6.3 9.5 13.8

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M.-f.Lai, C.-F.Li and C.-y.Li

interactions between the polymer-polymer and the polymer-solvent, and to the size and shape of the molecule (41), and could be deduced from the k' of Huggins equation (11'r/C = [11] + k' x [11f x C). The k' values of three sulfated galactans were close to 0.5, which revealed a poor polymer-solvent pair. Because the molecular conformation of the polysaccharide in dilute solution was linear, it showed a partially ordered helix at ambient temperature which transformed to random coil when heated (49,50). Both k' values of Helminthocladia and Liagoropsis sp. polysaccharides were smaller and showed the good polymer-solvent pair. These four polysaccharides showed Gaussian coil behaviour. It might be concluded that the polysaccharides from the same family had similar viscometric parameters relating to their molecular structure. The peculiar property of Ulva sp. polysaccharide in 0.1 mol/drrr' NaCI solution (k' = 13.90) required further study to explain. The [11] and k' values at 25 and 50° (Table III) corresponded to the temperatures of the phase transition from the DSC thermograms (Fig. 7) of Halymenia and Grateloupia polysaccharides. The temperature only affected [11] very slightly. It implied that the temperature would not influence the value which strongly suggested the influence of molecular hydrodynamic volume. However, the k' value became smaller and followed the phase transition which might be due to the effect of a better polymer-solvent pair and the slight change of molecular conformation between 25 and 50°C (Fig. 7). The apparent viscosities of the six polysaccharides measured are listed in Table III, G.filicina had the highest viscosities both in water and the salt solution, followed by Hiceylanica, then the other three varieties of non-gelling seaweed. The apparent viscosity was proportional to [11] or [11]w' The gel strength of the Taiwan P.capillacea agar extracted with hot water was 1034.0 ± 56.4 g/cm", and was higher than that from Venezuela (735.3 g/crrr') (51). Thermal properties

The thermal behavior of the polysaccharides was also investigated. the P.capillacea agar had three endotherms with peak temperatures, T p , 'at 38.9, 72.6, and 93.3°C, and the enthalpies of transitions were -50.5, -12.0 and -1.9 mJ/mg respectively (Fig. 6). The results of DSC thermogram and optical rotation versus temperature coincided closely. The exotherms shown at low temperature (20-30°C) might be attributed to the interaction between the agar and the water molecules (31). The first large endothermic peak of the gel-sol transition was related to the double helix-random coil conformational change, and was similar to the thermogram of the kappa-carrageenan (Sigma Chemical Co., USA) (Tp 38.1°C, and enthalpy change, -67.18 mJ/mg) (31). The temperature ofthe second endotherm was close to disentangling the multiple junction zone with no conformation change in agarose gel (31). The third one was seldom observed, and might correspond to the unfolding of the most tightly cross-linked chains of polysaccharides (52). It might be due to the fact that the gel of P.capillacea agar after curing at 5°C for two days contained a mixed network structure. 228

Sulphated polysaccharides from seaweeds

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Fig. 7. DSC thermogram s and optical rotatio n of Gratelo up ia filicina (0) and Halym enia ceylanica (H) polysaccharides. T he 4% concent rat io n (w/v) was for th e DSC at heating ra te of 1°C/min ; the 1.0% concentratio n was for the det ection of optical rotation at 589 nm .

229

M.-f.Lai, C.-F.Li and C.-y.Li

During the thermal analysis of the non-gelling polysaccharide, the thermograms of G.fi/icina and Hiceylanica were very similar (Fig. 7). There were two endotherms at 25-45 and 65-76°C, and -3.159 and -0.083 mJ/mg (enthalpies of transition) respectively for G.filicina, and 30-50 and 77-90°C, and - 3.56 and -0.32 mJ/mg respectively for Hiceylanica. The first endothermic transition corresponded to intramolecular reorganization with the change of molecular conformation, from ordered conformer to random coil (29,30). This was further proved by the shift of the optical rotation. The second endothermic peak might be due to the dissociation of the randomly coiled molecule (53). It was peculiar that the G.filicina with the high intrinsic viscosity showed less enthalpy at transition, which occurred at lower temperature. The reorganization peak (at low temperature) of kappa-carrageenan gel with the large amount of acidic substitutes, reported by Watase and Nishinari (29,31), was also observed in the non-gelling polysaccharide solution (Figs 7,8). In addition, a sharp, reorganized endotherm at 29.7°C was detected from the Hiceylanica which also contained great amounts of charged substitutes. Further study would be required for the elucidation of such results. There were three endothermic peaks at 26.4,38.6 and 52.9°C which appeared on the thermograms of U.arasakii (Fig. 8), which might result in intermolecular interaction but not conformation change. The total enthalpy change of the transition of U.arasakii polysaccharide solution was -6.62 mJ/mg. And enthalpy and the peak temperature of Uiarasakii were lower than those of Haustra/is and L.schramni (Fig. 8), and higher than those of Gi[ilicina and Hiceylanica (Fig. 7). Both Hiaustralis and L.schramni had one small exothermic peak at 27.5°C (0.50

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Fig. 8. DSC thermograms and optical rotation of Ulva arasakii (U), Helminthocladia australis (H), and Liagoropsis schramni (L) polysaccharides. The 4% concentration (w/v) was for theDSC at heating rate of I''Czmin; the 1.0% concentration was for the detection of optical rotation at 589 nm.

230

Sulphated polysaccharides from seaweeds

mJ/mg) and 24.3°C (0.71 mJ/mg) respectively, and one large endotherm at a higher temperature, 55.3°C (-66.42 mJ/mg) and 48.9°C (-23.81 mJ/mg). The enthalpy of the transition for the former was significantly higher than the latter, although they had similar monosaccharide constituents and charged groups. Conclusion From the physicochemical properties of the seaweed polysaccharides investigated, they could be divided into four groups. (1) Hceylanica and G.filicina were sulfated and pyruvated galactans with high viscosity and expansion factor, and were similar to lambda-type carrageenan. (2) The polysaccharide from P.capillacea was a kind of agar with the low expansion factor and high gel strength. (3) Uronic acid, rhamnose and xylose were the major components of U.arasakii polysaccharide with less viscosity. (4) Mannose and xylose existed mainly in the polysaccharides of Hiaustralis and L.schrammi. The structures of three polysaccharides from red seaweed had been elucidated with IR and 13C-NMR spectroscopy. The P.capillacea agar was composed of Dgalactose and 3,6-anhydro-L-galactose residues to a regular, alternating and nearly 'absolute' structure. The G.filicina polysaccharide possessed the 'intermediate' structure between agar and carrageenan. The structure of the sulfated galactan from H ceylanica was high heterogeneity with randomly sulfated, pyruvated and heterogeneous residues. In a dilute salt solution the three Rhdophytaceae and the two Helminthoc1adiaceae polysaccharide molecules exhibited Gaussian coil behavior with different properties of polymer-solvent pairs, but, the Ulvaceae polysaccharide behavior differed from the Gaussian coil. The DSC thermogram of gel-sol transition and the optical rotation change during heating might imply that the hybrid, gel-network structure existed in the cured gel of the P.capillacea polysaccharide. The thermal behaviors of the five non-gelling polysaccharides could be used to elucidate the conformational changes, the mechanisms of the intermolecular aggregations and the intramolecular reorganizations of molecules. The molecular properties derived from the viscosity and the thermal behavior of the algal polysaccharide might be useful for the algal taxonomy and applications. Acknowledgements We would like to express our great appreciation to Professor Young- Meng Chiang, Graduate Institute of Oceanography, National Taiwan University for supplying all seaweed samples. The authors were also grateful to Ms Su-Ching Lin for the NMR technical assistance. This study was supported by grant (NSC81-0418-B-001-530-BG) from the National Science Council, Executive Yuan, Taiwan, ROC. References 1. Usov,A.1. and Elashvili,M.Y. (1991) Bot. Mar .. 34, 553-560.

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2. 3. 4. 5. 6. 7. 8. 9. 10. [ 1. [2. [3. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

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