STRUCTURAL CHARACTERIZATION OF AGAR EXTRACTED FROM SIX RED SEAWEED SPECIES GROWING IN THE COAST OF VIETNAM

Polysaccharides extracted from six red seaweed species growing in Vietnam have been studied. Characterisation of their structure by chemical and spectroscopic methods showed that all of them have a basic repeating structure of alternating 3-linked -D-galactopyranosyl and 4-linked 3,6-anhydro -L-galactopyranosyl units with substituted methyl ether groups. The native agar offers only weak gelling abilities owing to the 4-linked -L-galactopyranosyl 6-sulfate as its precursor. Conversion of this unit into the corresponding 3,6-anhydride by treating with hot alkali generally led to gel strength increasing. The agars from G. fisheri and G. firma are poorly substituted, while those from G.asiatica, G.tenustipitata and G. heteroclada are partly methylated on position 6 of the 3-linked -Dgalactose. Agar from Gelidiella acerosa is partlly methylated on both positions 6 and 2 of the 4linked 3,6-anhydro -L-galactose. The alkalimodified agars have been obtained in acceptable quantities with gel strength of 300932 g/cm 2 . The obtained results showed that all the six algal species are suitable as raw material for commercial agar production and worthy of further cultivation.


INTRODUCTION
Agar is a well known polymer originating from two main algal families ( Gracilariaceae and Gelidiaceae ).It has a linear backbone made of an alternating 3-linked -D-galactose (G) and 4linked 3,6-anhydro L-galactose (LA) units, i.e. agarose, although the latter may be present as a biochemical precursor, i.e. 4-linked -L-galactopyranosyl 6-sulfate (Fig. 1) which can be converted into the corresponding 3,6-anhydride by alkali treatment.The hydroxyl groups in this basic structure are often substituted, mostly with methyl groups on position O-6 of G-or O-2 of LA units.G-unit may contain a pyruvic group 4,6-O (R)-1-carboxyethylidene acetal, O-4 or O-6 sulfate esters, and a branching xylopyranose or 4-O methyl L-galactose1,2.The gelling properties of agar closely depend on the degree and type of substitution 3, 4, while sulfated residues strongly influence the gel strength and methyl eter groups impact the elasticity and gelling temperature.
In Vietnam five algae among 13 Gracilaria species and Gelidiella acerosa have been found to be a major souce for agar production, that ' s why they have been widely studied.
The aim of this study was to elucidate chemical structure of the agars extracted from the above mentioned seaweeds to estimate their potential as raw material for commercial agar production as well as for their further cultivation

Extraction of native polysaccharides
Samples of air-dried seaweed (40g) were acid treated in 2l of solution 0,11 -0,18 % H 2 SO 4 for 1h, and washed in running tap water for another 2h.The samples were then boiled for 1h with 1,5 l of distilled water (pH 7 -8) using a Bunsen burner in a 2l Erlenmeyer flask equipped with a reflux condenser.The agar extracts were dehydrated by freezing and pressing.

Preparation and extraction of alkali-modified agar
Samples of air-dried seaweed (40 g) were treated in 2l of solution 6% NaOH at 70 -80 0 C for 3 h, and washed in runing tap water for 30 min.The seaweed samples were then extracted as described above for native polysaccharides.

Gel strength measurements
dried agar (1 g) was placed in a 250 ml beaker, dissolved in distilled water (100 ml) and was pressure cooked for 15 min.The weight of the solution was then adjusted to 100g with additional water.After stirring to ensure that it was homogeneous, the solution was left at room temperature overnight to gel.The beaker was then placed on a top-pan balance, a circular stainless steel rod (crosssection area of 1cm 2 ) was pressed by hand into the gel until it collapsed, and the maximum balance reading was noted.The calculated gel strength was an average of four determnations on the same sample.

Gel-melting temperature measurements
Agar (30mg), distilled water (3 ml) and a small piece of lead as a weight were placed in a screw-capped culture tube (13 ×100 mm) and presure -cooked for 20 min ( twice if not properly disolved).After shaking, the solution was allowed to set overnight in a vertical position.A 5 mm glass bead was placed on the gel surface in a glycol bath and heated on hotplate with a temperature rising rate approximately 2 0 C/min, The gel-melting range was determined as the temperatures of the glycol bath at which the bead started to sink and reached the bottom of the tube.The heating was then stopped, and the tube was allowed to cool.Every minute the tube was removed briefly from the bath and turned horizontally.The gel-setting range was determined as the temperatures between which the solution showed sign of gelling and at which it would no longer flow.All determinations were performed in doublicate.

Constitutent sugar analysis
Alditol acetate derivatives were prepared by a double hydrolysis-reduction procedue 6 and analysed by gas liquid chromatography (GLC) according to the method of Falshaw and Furneux 7.

Glycosyl linkage analysis
Samples were converted to a form of triethylammonium salt by dialysis against triethylammonium hydrochloride ( 0,1 M, pH7, X2), then methylated by the procedure of Stevenson and Furneux 6 and converted to alditol acetate derivatives for GLC analysis as described above.

13 C-Nuclear magnetic resonance spectroscopy
The 13 C-NMR spectra were recorded on 5% W/V agar solution, in D 2 O-H 2 O (1:1) at 80 0 C on a Bruker AVANCE 500 MHZ.

Agar yield and physical properties
The yield of agar and its gel strength, gelling and melting temperatures from the six red seaweed species are shown in table 1.The yield of native agar (N) from six species of red seaweed varied from 15,5 to 40,0% and the yield of alkali-modified agar (A) from these ones varied at an interval 12,4 -28,5%.
Treatment of the seaweed samples with hot alkali prior to extraction resulted in a considerable positive effect on gel strength of the agars produced.Gels made from 1% concentration of the alkali-modified agar (A) yielded rather modest strength value of 180 g/cm 2 for G. fisheri and to very strong value 932 g/cm 2 for G. tenuispititata, meanwhile gel strength of native agar (N), with exception of Gelidiella acerosa, ranged 33 -102,9 g/cm 2 .As mentioned, the presence of the precursor units in the corresponding native agar significantly weakens their ability to form gel, that is generally a common observation for agar and carrageenan.Gelling temperature of agar extracted from the Gracilaria species varied in an interval of 34 -44 0 C, while that of Gelidiella varied between 42 -46 0 C. On the other hand, melting temperature of agar from Gracilaria -between 74,5 -94,5 0 C, and that from Gelidiella ranged from 95 -98 0 C.This agar melted near boiling point of water, about 8 0 C higher than other commercial agars.
Gracilaria materials imported from Japan usually provide an agar yield of 14 -25% which at 1,5% concentration forms a gel strength of 600 -800 g/cm 2 and gelling temperature (39-44 0 C) 8.The yield of agar(A) from Vietnamese G. fisheri and G. firma was not higher than 14% but the yield from the other red seaweeds ranged 17 -28,2 %.The gel strength value 180-932 g/cm 2 of the Vietnamese Gracilaria agars was lower than that of the Japanese ones.Japanese species, such as G. chorda, showed gel strength as high as 1139 g/cm 2 at 1,5 % concentration 8, while Gracilaria species from Taiwan, Philippnes and Thailand gave the values of 660,45 g/cm 2 , 622,44 g/cm 2 and 716,14 g/cm 2 , respectively 9.

Chemical composition
The structure of the alkali-modified agars from six red seaweed species was studied by constituent sugar analysis (table 2), glycosyl linkage analysis (Table 3) and 13 C NMR spectroscopy ( Fig. 2  has less than 10 % methylation on any position.Methylation is not unique to Gracilaria, but the presence of substantial methylation (at whatever position) can be used as taxonomic marker for Gracilaria species in combination with other characteristic features such as the gross morphology and the presence of tetrasporangial and spermatangical nemathecia 12 .This had already led to the reassessment of the identity of one mislabelled sample.Precursor residue.In idealized agar structure, the total number of 4-linked 3,6anhydrogalactopyranosyl unit (determined by constituent sugar analysis as alditol acetate species Angal + 2-MeAngal) should equal the total number of 3-linked galactopyranosyl unit (determined as Gal + 6-MeGal ).A lower level of total 3,6 anhydrogalactopyranosyl unit may indicate the presence of precursor (L6S/L2M6S) unit.Constituent sugar analysis would generate alditol acetate patterns Gal and 2-MeGal from L6S and L2M,6S units, respectly.The level of these precursor unit in the native agar can be estimated from either the decrease in the level of the alditol acetate patterns Gal and/or 6-MeGal (Gal) or the increase in Angal and/or 2-Me Angal (Angal) value in the constituent sugar analyses by going from native to alkali-modified agars once normalised to exclude the variations in level of other sugars 13.From these values it can be seen that levels of precursor (L6S/L2M,6S) units ranged up to 14,8 mol % (table2).

Species
Deduced substitution/linkage 13 C NMR spectroscopy. 13C NMR spectroscopy of hot aqueous solutions of alkali-treat agar samples enabled the major structures identified from constituent sugar analysis to be confirmed by comparing the observed chemical shifts (table 4) with literature value for the agars methylated at G6 or LA2 and at both G6 and LA2 positions 14.
The alkali-modified agar from G. fisheri and G. firma were poorly methyl substituted.Its 13 C NMR spectrum (fig 2A) revealed 12 major signals corresponding to those of unsubstituted agarose.The constuent analysis of the alkali-modified agar from G. fisheri ( table 2 ), however, indicated that the structure may not be as simple as that deduced from 13 C NMR spectra.As agar was alkali-modified, the total number of 4-linked 3,6-anhydrogalactosyl units should be equal to the total number of 3-linked galactosyl units, hence the expected 1:1 ratio of 3-linked to 4-linked units should be found.However, the calculated ratio was 1,52 :1.The presence of T-gal in the glycosil linkage analysis data indicated that the galactosyl unit occurs as a single branch of the main agar chain.4).About 21,7% for G. tenuistipitata, 31,2 % for G. asiatica and 18,5 % for G. heteroclada of the 3-linked units were found to be methylated on O-6 position from relative peak area of G6M-5/G5+G6M-5.Constituent sugar analysis of alkali-modified agar revealed nearly equal amount of (Gal+6MeGal) and (Angal) as expected.Glycosyl linkage analysis data of agar from the three Gracilaria species confirmed the predominance of simple agar of alternating 3-linked -Dgalactopyranosyl and 4-linked 3,6-anhydro -L-galactopyranosyl units.In addition, the presence 4-galactose confirmed the existence of linked galactopyranosyl units as a main agar chain.

CONCLUSION
Polysaccharides extracted from six red seaweed species growing in Vietnam were structurally investigated using constituent sugar and glycosyl linkage analyses, and 13 C-NMR spectroscopy.Characterisation of their structure by chemical and spectroscopic methods showed that all of them have a basic repeating structure of alternating 3-linked -D-galactopyranosyl and 4-linked 3,6-anhydro -L-galactopyranosyl units with substituted methyl ether groups.
The experimental data showed that agars from Gracilaria fisheri and G. firma were poorly substituted, while for G. asiatica, G. tenuistipitata, G. heteroclada and Gelidiella acerosa the methylation (on G6) was considerable.
For the alkali-modified agars from G. asiatica, G. heteroclada and G. tenuistipitata the 13 C NMR spectra demonstrated major characteristic resonnaces of agarose and minor ones of agarose methylated at O-6 of the 3-linked galactose.The glycosyl linkage analysis data of agar from the three Gracilaria species confirmed the predominance of simple agar with alternating 3linked -D-galactopyranosyl and 4-linked 3,6-anhydro -L-galactopyranosyl units.
The obtained results indicated that all the six algal species are suitable as raw material for commercial agar production and worthy of further cultivation.

Fig 1 :
Fig 1: Structure of idealized agar (A) and agar precursor (B) with common positions of methyl ether substitution.

Table 1 :
Extraction yield and physical properties of the agar speciments.
and table 4).ether substitution of agar.Sugar constituent of agar from the red seaweed species are shown in table 2. Three substitution patterns were observed, reflecting the sites of methylation in various species.The agars from G. fisheri and G. firma were poorly subtituted (2 -4,2 % of 6-O-methyl-D-galactose; 0 -2,4% of 2-O-methyl 3,6 anhydro-L-galactose), while methylation on G6 (>12%) and little on LA2 were observed for G. asiatica, G. tenuispititata and G. heteroclada.The agar from Gelidiella acerosa was methylated on both G6 and LA2.Levels of methylation have also been observed in gel-forming agars from few red algal species, for example, hightly 6-O methylated agar from Euptilota formosissima ( in order Ceramiales) 10, 2-O methylated agar from Chinese sample of G.eucheumoides 6 and agar methylated on both G6 and LA2 from Japanese G. eucheumoides 11.In general, however, levels of methylation in agar are much lower.So agar from Gelidium and Pteroclada (as well as few other Gracilaria)