S. C. Lindstrom & D. J. Chapman (eds), Fifteenth International Seaweed ... a School of Pharmacy and Biomedical Sciences, University of Portsmouth, King Henry ...
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Hydrobiologia 326/327 : 497-500, 1996.
S. C. Lindstrom & D. J. Chapman (eds), Fifteenth International Seaweed Symposium . ©1996 Kluwer Academic Publishers. Printed in Belgium.
Structure and properties of agar from two unexploited agarophytes from Venezuela . Erminio Murano' ,2 *, Renato Toffanin'° 2 , Cristiano Pedersini 2 , Alfredo Carabot-Cuervo 3 , Gerald Blunden 4 & Roberto Rizzo' ,5 'POLY-bibs Research Center, AREA di Ricerca, Padriciano 99, 1-34012 Trieste, Italy, Fax : +39 40 7797091 ; e-mail address : murano @ P OLY01 . tbs. trieste. i t 2 POLY-tech, AREA di Ricerca, Padriciano 99, 1-34012 Trieste, Italy 3 Faculty of Pharmacy, University of Los Andes, Merida, Venezuela a School of Pharmacy and Biomedical Sciences, University of Portsmouth, King Henry I Street, Portsmouth POI 2DZ, UK 5 Department of Biochemistry, Biophysics and Macromolecular Chemistry, University of Trieste, Via L . Giorgieri 1, 1-34127, Trieste, Italy * Author for correspondence
Key words: agar, Gelidiella acerosa, Gracilaria mammillaris, red algae, red seaweed, sulphate
Abstract The red seaweeds Gelidiella acerosa and Gracilaria mammillaris growing along the coast of Venezuela were investigated as potential economic sources of agar . Agar extracted from Gracilaria mammillaris accounted for 27% of the algal dry weight but had quite a poor gelling ability because of the presence of alkali-stable sulphate groups on the D-galactose residue . However, the gel strength of its aqueous solutions was considerably enhanced by the addition of potassium, sodium and calcium ions (up to 1N) . On the contrary, the galactan from Gelidiella acerosa (yield of about 20% w/w) exhibited quite good properties . In particular, the gel strength was comparable to that of commercial agaroses . Sulphate esters were not detectable by chemical methods and NMR spectroscopy revealed an agarose backbone with a high degree of methylation on both D and L-galactose residues . Introduction Agarocolloids are synthesised as cell wall constituents in a number of Rhodophyta (Craigie, 1990) and they are one of the most important and widely used groups of algal polysaccharides (Painter, 1983) . Recently, the rodophycean genus Gracilaria has grown in importance worldwide as an agar source owing to its abundance in biomass and successful cultivation (Critchley, 1993) . For example, Gracilaria lemaneiformis (Bory) Dawson is being grown commercially in Venezuela as a source of agar (Rincones, 1994) . However, several other species of Gracilaria are available in the country, as well as other agar-yielding genera, such as Gelidium, Pterocladia (Lemus et al., 1991) and GelidielIa . A programme to study the properties of the agars
extracted from different Venezuelan species of marine algae is underway. In this communication we report the properties of the agars extracted from Gracilaris mammillaris (Mont.) Howe and Gelidiella acerosa (Forsskal) J . Feldman & Hamel.
Materials and methods Gracilaria mammillaris and Gelidiella acerosa were collected at San Juan de los Cayos, Falcon State, Venezuela in January 1994 . The algae samples were first air dried and then oven dried at 40 °C . Agar extractions were performed on 7 grams of each dried algae at 95 °C in 0 .05 M phosphate-buffer (Murano et al., 1993) . The crude agar extracts were treat-
498 ed with amylase (Termamyl® , Novo Enzymes) and recovered after isopropanol precipitation as previously described (Murano et al., 1992). Alkali treatment of agar extracted from Gracilaria mammillaris was performed according to the method proposed by Craigie & Leigh (1978) . The sulphate content of the agar samples was quantified in triplicate with sodium rhodizonate according to the method of Terho & Hartiala (1971), after sulphate hydrolysis by 1N HCl at 110 °C for 4 h . Nuclear magnetic resonance (NMR) experiments were performed at 80 °C using a Bruker AC 200 NMR spectrometer equipped with a 5 mm multinuclear probe . The ' H-NMR (200.13 MHz) spectra of 0 .5% (w/v) agar solutions in D20 (99 .9%) were recorded with presaturation of the HOD residual signal . The ' H chemical shifts were measured in parts per million (ppm) from internal sodium 2,2,3,3-tetradeuterio3-(trimethylsilyl)-propionate (TSP) . Proton composite pulse decoupled 13 C-NMR spectra (50 .33 MHz) of 2 .5% (w/v) agar solutions in D 2 0 were acquired overnight. The ' 3 C chemical shifts were referred to tetramethylsilane (TMS) by setting the internal dimethylsulphoxide resonance to 39 .6 ppm . The 2-0-methyl and 6-0-methyl content was estimated by the ratio between 1/3 of the area of the methyl resonances at 3 .52 and 3 .43 ppm, respectively, and the area of the H- Is of 4-linked-L-galactose in the region 5 .29-5 .15 ppm (Lahaye et al ., 1986), assuming a perfect alternating agar backbone . Gel strength, melting and dynamic gelling temperatures were determined in triplicate (Murano et al ., 1992) . A Stevens-LFRA texture analyser was used for the gel strength determination on 1 .5% (w/w) agar cylinders (15 mm diameter, 15 mm height) . In the case of Gracilaria mammillaris, the gel strength was measured on gel cylinders obtained from agar solutions at different concentration of potassium, sodium and calcium chloride, up to 1N . Fourier-Transform Infrared (FT-IR) spectra were recorded on films using a PerkinElmer 1750 FTIR spectrometer. Small amplitude oscillatory theological tests were carried out on gels at 25 °C by introducing the warm solutions (1% w/w) between the plates (50 mm diameter, 1 mm gap) of a Rheometrics fluid spectrometer RFS 8500 allowing gelation to occur in situ . The determination of the molecular weight averages and molecular weight distributions was carried out by means of a high performance size exclusion chromatography system previously described (Martinsen et al ., 1991 ; Murano et al., 1992) . Agar samples were dissolved at
60 °C in dimethyl sulphoxide/water (0 .8 :0 .2) in order to avoid association of agar macromolecules .
Results and discussion Agar extracted from Gracilaria mammillaris accounted for 27% of the algal dry weight and the weightaverage molecular weight (Table 1) of its native form was higher than that of agars isolated from other Gracilaria species (Murano, 1995) . FT IR and NMR spectroscopy investigation showed that the substitution pattern of this agarocolloid was characterised by a high degree of complexity owing to the presence of differently sulphated, methylated and pyruvated residues . The degree of substitution (DS) by methyl groups, estimated by ' H-NMR, was quite high, especially for the D-galactose residue (Table 1) . The large degree of methylation of this residue was also indicated by the presence in the typical agarose 13 C-NMR spectrum (Usov et al ., 1980), of additional peaks at 73 .6 and 71 .8 ppm (Figure 1), attributable to C-5 and C6 of 6-O-methyl-o-D-galactose residues, respectively (Nicolaisen et al ., 1980) . Despite the high degree of methylation and the high weight-average molecular weight, a 1 .5% (w/w) aqueous solution of agar from G . mammillaris had low gelling and melting temperatures and low gel strength (Table 1) . Such a poor gelling ability of the native form was attributed to the presence of alkali-stable sulphate hemiesters located at C-6 and, contrary to previously reported data on G. mammillaris (Valiente et al ., 1992), C-4 of the D-galactose residues . The occurrence of D-galactose 4-sulphate and D-galactose 6-sulphate was revealed, in the FT IR spectrum by the presence of peaks at 850 and 820 cm -1 , respectively (Armisen & Galatas, 1987) (Figure 2) . Furthermore, in the ' 33 C-NMR spectrum, the peaks at 96 .7 ppm and those at 67 .4, 68 .4 and 73 .0 ppm were attributed to the C-1 of 3,6-anhydro-L-galactose adjacent to a D-galactose 4-sulphate and to C-6, C4 and C-5 of a D-galactose 6-sulphate, respectively (Lahaye & Yaphe, 1988 ; Usov et al ., 1983) (Figure 1) . Although a little reduction of the peak at 820 cm - ' in the IR spectrum was observed and a small increase of gel strength was measured after alkali treatment (data not shown), nearly 5% (w/w) of sulphate was still present in the agar from G . mammillaris . The presence of alkali-stable sulphated residues, in particular D-galactose 4-sulphate, confers to the agar extracted from G. mammillaris structural and rheological similarity to rc-carrageenan . In fact, the storage
499 Table 1 . Chemical and physical parameters of agars from Gracilaria mammillaris and Gelidiella acerosa .
Gracilaria mammillaris Gelidiella acerosa
Total yield (% w/w)
Mm
27 21
469 350
x 10-3
Sulphate ester (mol/C6H1f105)
Gel strength( 2) (gxcm -2)
Gelling Temp . (° C)
Melting Temp . (° C)
2-O-Me-LGal (DS)( 3)
6-O-Me-D-
0.19
12 561
42 45
75 93
0.15 0 .30
0 .60 0 .45
Gal (DS) ( ' )
( 1 ) Weight-average molecular weight . ( 2 ) Measured on 1 .5% (w/w) gel cylinders . ( 3 ) Degree of substitution expressed as 2-O-methyl-3,6-anhydro-L-galactose to 3,6-anhydro-L-galactose and 6-O-methyl-D- galactose to Dgalactose, respectively, assuming a perfectly alternating agar backbone . (-) Below the detection limit of the method .
Geliidiella acemsa
0 0
0 iuc
oe
io
P
0.5 N (eq/L)
1
PPM ~
Figure 1 . 13 C-NMR spectra of native agar from Gracilaria mummillaris and Gelidiella acerosa . The peak marked with (V) is due to the anomeric carbon of the a-L-galactopyranose residue adjacent to a D-galactose 4-sulphate . Arrows indicate the carbons of the Dgalactose 6-sulphate residues . Signals marked with (0) and (0) are attributed to carbons of the 2-O-methyl-3,6-anhydro-a-L-galactose and 6-O-methyl-/3-D-galactose residues, respectively .
z Q m 0
Gelidlalla acamsa
f
a
Gracilaria mammillarls
1300
1100
900
cm'
Figure 2 . FT-lR spectra of native agar from Gracilaria mammillaris and Gelidiella acerosa . Arrows indicate the peaks due to ester sulphate in C-6 (820 cm -1 ), ester sulphate in C-4 (850 cm -1 ) and total ester sulphate (1250 cm -1 ) link vibration, respectively .
Figure 3 . Gel strength of native agar from Gracilaria mammillarisc as a function of KCI (-U-), NaCl (-0-) and CaC12 (-A-) concentration .
modulus (G') of its aqueous solutions is considerably increased by the addition of potassium ions, up to 1N (data not shown) . Interestingly, the gel strength showed a strong dependence not only on the potassium but also on sodium and, to a less extent, calcium ions concentration (Figure 3) . These findings suggest a complex mechanism for the gelation of this sulphated galactan . However, further investigation will be conducted to better understand the rheological behaviour of such a peculiar agar. Agar isolated from Gelidiella acerosa exhibited quite good gelling properties as a result of its regular chemical structure . Pyruvate ketals and sulphate hemiesters could not be detected by chemical methods and only very weak signals at 103 .7 and 101 .3 ppm, ascribed to the presence of L-galactose 6-sulphate residues (Lahaye et al., 1985), were observed in the 13 C-NMR spectrum . Similarly, the FT IR spectrum showed very low intensity absorbances at 1250 and
500, 820 cm -1 due to total sulphate and the ester sulphate in C-6 link vibrations, respectively . Moreover, NMR experiments showed the sample to have an agarose backbone with a high degree of methylation on both D (6-0-methyl) and L (2-0-methyl)-galactose residues . Signals at 98 .7, 78 .9 and 78 .5 ppm were attributed to C-1, C-2 and C-3, respectively in the 2-O-methyl-3,6anhydro-L-galactose residues (Shashkov et al ., 1978) . A 1 .5% (w/w) agar gel exhibited a high breaking force, comparable with both that of commercial bacteriological agars and that previously reported for the same Hawaiian species (Santos & Doty, 1983) . As a result of the remarkable DS with methoxyl the gelling temperature was rather high but still in the range of those of agars from Gracilaria (Table 1) .
Conclusions The Venezuelan red seaweed Gelidiella acerosa can be considered as a potential economic source of high gel strength agar with a high degree of methoxylation in its native form . On the contrary, agar extracted from Gracilaria mammillaris has quite a poor gelling ability owing to the presence of alkali-stable sulphate hemiesters . However, its gel strength was considerably enhanced by the addition of NaCl and KCI, suggesting a mixed gelation mechanism between that of agarose and that one of rc-carrageenan .
Acknowledgements The authors wish to thank E . D'Agnolo for the molecular weight determinations . The Consiglio Nazionale delle Ricerche ('Progetto Finalizzato Chimica Fine IF) is gratefully acknowledged for financial support .
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