Purification of agar from Gracilaria - heteroclada alga by means of ion-exchange chromatography

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1 Journal of Chemistry, Vol. 41, No. 3, P , 2003 Purification of agar from Gracilaria - heteroclada alga by means of ion-exchange chromatography Received Le Dinh Hung, Ngo Quoc Buu, Bui Minh Ly, Huynh Quang Nang Institute of Materials Science Nha Trang branch Summary Agarocolloids from Gracilaria heteroclada growing in Vietnam have a potential to be a good food-grade agar. The genus Gracilaria of Vietnam including G. heteroclada as a rule contains more sulfate groups and divalent metal ions than other genera of the world, which would degrade agar quality. Purification procedure of the algae extraction juices from G. heteroclada using ionexchange resins was implemented by percolating the extraction juices successively through a cationic ion-exchange column in the Na + form, anion-exchange column in the Cl - 2- and /or SO 4 and OH - forms. The experimental data showed that such a purification process allows producing food-grade agar of high quality with a high recovery coefficient due to the conversion of 6-sulfate L- galactose into 3,6-anhydrous galactose. The treatment of agar extraction juices with ionexchange resins enabled to make agar with increased gel strength, transparency of agar solution, as well as agar solubility. I - Introduction Agar is a mixture of polysaccharides: agarose and agaropectin of high molecular weight from 30,000 to 250,000. Agarose constitutes neutral polysaccharides, while agaropectin is composed essentially of the agarose skeleton to which may be linked sulfate and other substantial groups and represents a charged fraction of the agar molecule. The presence of sulfate esters is a common feature of agars from Vietnamese Gracilaria species and usually impairs the gel strength and other qualities of agars. There are many treatment types for improving agar qualities. A common method is treatment of algae with an alkali solution at o C to convert the 6-sulfate L- galactose into 3,6-anhydrous L-galactose from (Murano, 1995), but alkaline may cause chain breaking of agar molecule. Sulfate ester groups in the agaropectin fraction of agar may also be eliminated by precipitating agaropectin by means of complexion agents (ethylene glycol, cetylpiridinium chloride etc...), but these agents are costly and cause the loss of entire agaropectin fraction which amounts to 10 to 50% by weight of the entirety, consequently decrease the agar yield and rend the obtained product more expensive (Lebbar et al, 1988). The object of this study is application of ion-exchange chromatographic method to produce desulfated food-grade agar from Vietnamese Gracilaria heteroclada without losing a fraction of the agar and consequently without significantly increase in cost. 111

2 II - Materials and method 1. Materials Gracilaria heteroclada samples were harvested at Song Cau, Phu Yen province. Algal samples were cleaned, rinses with sea water, dried by natural sunlight and stored at 2 o C - 4 o C for subsequent extraction and chemical analysis. Cation-exchange resin in Na + from (Amberlite IR-120), anion-exchange resin in Cl -, SO 2-4 and OH - forms (Amberlite IRA and IRA - 120). Ion-exchange columns were of 10 mm in diameter and 500 mm in height, containing about 30 ml of resin. Flow rate through cation-exchange column in the Na + form and anion-exchange columns in the Cl - and SO 2-4 forms is 6 ml/min and 3.5 ml/min for that in the OH - form. 2. Chemical D-galactose, D-fructose, resorcinol reagent, anthrone reagent, rhodizonate - sodium and benzoic acid, DEAE-sephadex A-50 (Cl - ) were received from Nacalai Tesque Inc, Japan. 3. Method for agar extraction Extraction of agar was performed according to Craigie and Leigh (Craigie &Leigh, 1978). Seaweed samples were washed with distilled water and salt, epiphyte and contaminants were removed, then the samples were treated with HCl 10-3 N at room temperature for 1 h and washed to remove excess acid before extraction. The algae were extracted with boiling water at 121 o C in autoclave for 2 h, filtered through Whatman paper and subsequently diatomite filter-aid, then the extraction juices were passed through ion-exchange resin columns, maintained at 55 o C by thermostat, collected and poured into shallow trays and allowed to gel at room temperature, then frozen. The gel was thawed, washed with distilled water and dried at 60 o C overnight. A process for purifying the agar juices from G. heteroclada by ion-exchange chromatography comprises: (a) placing the extraction juice in the presence of a cation-exchange resin conditioned into the Na + form, then putting it (b) through an anion-exchange column conditioned into the Cl - and/or SO 4 2- form, and finally (c) passing the juice through an anionexchange column conditioned into the OH - form. Determination of total galactose (Dubois, 1956); 3-6 anhydrous galactose (Yaphe, 1965); Sulfate content (Terho & Hartiala, 1971). The purified agar products were fractionated on DAEA-sephadex A-50 (Cl - ) column for agarose and agaropectin separation and the sugar content for each fraction was determined (Duckworth et al., 1971; Whyte et al.,1981; Stevenson & Furneaux, 1991). Determination of average molecular weight was carried out following Martinsen (Martinsen et al.,1991). Concentration of Na, Ca, Mg and Fe were analyzed by AES and AAS. III - Results and discussion Infrared spectroscopy is a convenient method to follow sulfate, 3,6-anhydrous L- galactose and L-galactose- 6-sulfate concentrations in agars (Lahaye, Rochas & Yaphe, 1986; Lahaye & Yaphe,1988; Rochas et al., 1986). The spectra of agar from Gracilaria and a strong absorbance at 930 cm -1 attributed to 3,6- anhydro L-galactose (Stanley, 1963) and a weak absorbance for total sulfate at 1250 cm -1 (Lloyd et al., 1961). Furthermore, the absorbance at 820 cm -1 was attributed to L- galactose-6-sulfate and the absorbance at 2920 cm -1 for total sugar (Stancioff & Stanley, 1969). IR spectra of the agar samples were obtained on the IR spectrometer of the Institute of Chemistry, NCST of Vietnam. The absorbance ratio relating 3,6-anhydro L-galactose and total sulfate to sugar content of agars after passing though ion-exchange columns were reported in table 1. The infrared absorbance ratio relating total sulfate to sugar content (1250/2920) is highest in native agar (Agar 1) (1.65) and greatly decreased in sample Agar 4 (1.14), mean while 112

3 the highest ratio relating 3,6-anhydro galactose to total sugar was found in Agar 4 (2.57) and the lowest one in Agar 1 (1.37). This means that percolating the agar extraction juice through an anion-exchange resin in OH - and/or Cl - forms enable to convert L-galactose 6- sulfate into the 3,6-anhydro form, consequently enhance the gelling ability of agar (gel strength of Agar 1 is 100 g/cm 2, while that of Agar 4 amounts to as much as 180 g/cm 2 ). Table 1: Infrared absorbance ratio of 3,6-anhydro galactose (3,6-AG) and total sulfate to total sugar content of agar samples from G. heteroclada and gel strength after percolating the agar extraction juice (AEJ) successively through ion-exchange columns (IEC) conditioned into different counter-ion forms Sample Treatment method for agar juices 3,6-AG/Total sugar (932/2920) Sulfate/Total sugar (1250/2920) Gel strength (1%), g/cm 2 Agar 1 Native agar (non-treated) Agar 2 AEJ passed through IEC in Cl - form Agar 3 AEJ passed through IEC in OH - form Agar 4 AEJ passed through IEC (Cl - ) and then IEC (OH - ) The experimental data in table 2 show that there are considerable variations in 3,6-anhydro galactose, agarose, agaropectin and sulfate contents and gel strength, when agar extraction juice were successively passed through ionexchange resin columns conditioned into different forms as the counter-ions. The contents of 3,6-anhydro galactose (30.4%) and gel strength (270 g/cm 2 ) were highest, while those of agaropectin (55%) and sulfate (2.58%) were lowest in the sample S9. On the other hand, these variations slightly depend upon the sequence of percolation of agar juices through the ion-exchange columns (compare to the samples S6 and S9, table 2). The purification process by means of ionexchange resins allows producing a desulfatized agar at minimal cost compared to the existing procedures. It suffices to percolate the extraction juice obtained from step (b) through an IEC in OH - form step (c) at a temperature exceeding the gelling threshold of said juice (55 o C). By this means the enrgetically unstable axial sulfate at C-6 of the L-galactose will be eliminated, giving rise to the more stable 3,6-anhydro galactose without losing an agaropectin fraction in the agar. For the same gelling strength (270 G/cm 2 ) the IECtreated agar sample gave a yield of 21.4%, compared with 16.8% for the 1% alkali alga sample (table 2, last column). The coloring agents from the algae which represent a significantly depreciation factor of the agar quality are fixed onto the resin essentially by anion-exchange with salting-out of the chloride or sulfate ions in the juice. This fixation is remarkably effective, that it can be seen from the optical density data reported in table 3. For the native agar sample (S1) and the samples passed through an IEC in Na + form (S 2 - S9) the OD values are of order , while for the algae extraction juices passed through an IEC in Cl - 2- or SO 4 form these values considerably decreased (0.105 and for S3 and S5, respectively). The native agar extraction juice contains an amount of metal elements, including Mg, Fe and especially Ca, that might be attributed to the formation of intra- and inter-molecular Ca 2+ -bridges and rend the agar poorly soluble (Tako &Nakamura, 1988). Table 4 showed that the extraction juices of agar were passed through IEC conditioned into Na + form, the contents of 113

4 Ca 2+, Mg 2+ and Fe n+ decreased mg/g order up to traces that implies the enhancement of the agar solubility in water. The data also indicated that molecular weight of agar did not change during treatment of agar juices by means of ionexchange resin. 114 Sample Table 2: Properties of the agars from Vietnamese G. heteroclada after purification by ion-exchange chromatography (in % of agar) Total sugar 3-6 AG Agarose Agaropectin Sulfate Gel strength Agar yield, (1,5%), g/cm 2 % (dry alga) S S S S S S S S S S S1: native agar; S2: AEJ passed through IEC (Na + ); S3: AEJ passed sequentially through IEC (Na + ) and IEC (Cl - ); S4: AEJ passed sequentially through IEC (Na + ) and IEC (OH - ); S5: AEJ passed sequentially through IEC (Na + ) and IEC (SO 4 2- ); S6: AEJ passed sequentially through IEC (Na + ), IEC (OH - ) and IEC (Cl - ); S7: AEJ passed sequentially through IEC (Na + ), IEC (OH - ) and IEC (SO 4 2- ); S8: AEJ passed sequentially through IEC (Na + ), IEC (SO 4 2- ) and IEC (OH - ); S9: AEJ passed sequentially through IEC (Na + ), IEC (Cl - ) and IEC (OH - ); S10: AEJ after pre-treatment of the alga with alkali (1%) and non-treated by IE chromatography (The remarks are also meant for table 3 and 4). Table 3: Optical densities (OD) and ph of agar juices from G. heteroclada at 0,5-0,6% concentration. (OD measurements at 340 nm in a 1 cm cell at 60 o C) S * Native agar IEC (Na + ) IEC (Cl - ) IEC (SO - 4 ) IEC (OH - ) IEC (SO - 4 ) IEC (Cl - ) OD ph OD ph OD ph OD ph OD ph OD ph OD ph S S S S S S S S S * See remarks in table 2.

5 Table 4: The concentration of some important elements in the agars from G. heteroclada treated by means of IE resins (mg/g Agar) Sample Na + Ca ++ Mg ++ Fe ++ Cl - Average molecular weight S ,600 S trace trace ,200 S ,600 S ,800 S ,000 S ,200 S ,000 S ,200 S ,000 * See remarks in table 2. III - Conclusion The agarocolloids from Gracilaria heteroclada growing along the coast of Southern Vietnam appear to be a food-grade agar of good quality. Treatment of the agar extraction juices with ion-exchange resins enabled to eliminate heavy metal ions, which affect the solubility of the agar product, remove the coloring agents, which depreciator agar quality and convert the 6-sulfate L-galactose into 3,6-anhydro galactose from without losing a agaropectin fraction in the agar. This purification procedure qualitatively enhanced the agar properties. It was shown that the agarose content in native agar is 26%, but for the agar juice successively passed through IEC in the Na +, Cl - and OH - forms that value amounts to 40%, while the gel strength increases from 140 to 270 g/cm 2. On the other hand, for the same gelling strength (270g/cm 2 ) the IEC-treated agar sample gave an yield of 21.4%, in contrast to 16.8% for the 1% alkalitreated alga sample. References 1. E. Murano. Chemical structure and quality of Agar from Gracilaria. J. Appl. Phycol, 7, (1995). 2. R. Lebbar, M. Delmas and A. Gaset. Process for producing agar - agar from an algae extraction iuice; Patent number 4, 780, 534, Oct. 25. (1988). 3. J. S. Craigie, C. Leigh. Carrageenas and Agar. In: Handbook of Phycological Methods, Physiological and Biochemical methods - Cambridge Univ. Press (1978). 4. W. S. Dubois, O. C. Wilton, J. M. C. Caskilll, H. J. Humm, F. A. Wolf. Colorimetric method for determination of sugars and related substances. Anal. Chem, Vol. 28, P (1956). 5. W. Yaphe, G. P. Arsenault. Improved resorcinol reagent for the determination of fructose and 3,6-anhydrogalactose in polysaccharides. Anal. Biochem, Vol. 3, P (1965). 6. T. T. Terho, K. Kartiala. Method for determination of the sulphate content of glycosaminoglycans - Anal. Biochem, 41, P (1971). 7. M. Duckworth, W. Yaphe. The structure of agar, Part I, Fractionation of a complex mixture of polysaccharides. Carbohyd. Res, 16, (1971). 8. J. N. C. Whyte, J. R. Englar, R. G. Saunders, J. C. Lindsay. Biomass and agar 115

6 from reproductive and vegetative Gracilaria; Botanica Marina, Vol. XXIV, P (1981). 9. T. T. Stevenson, R. H. Furneaux. Chemical methods for the analysis of sulphated galactans from red algae. Carbohydr. Res, Vol. 210, P (1991). 10. A. Martinsen, G. Skjak-Brak, O. Smidsrod, F. Zanetti, S. Paoletti. Compar. of different methods for determin. of molecular weight and molecular weight distrib. of alginates. Carbohydr. Polym, Vol. 15, P (1991). 11. M. Lahaye, C. Rochas, W. Yaphe. A new procedure for determining the hetergeneity of agar polymers in the cell wall of Gracilaria spp. (Gracilariaceae). Can. J. Bot, Vol. 64, P (1986). 12. M. Lahaye, W. Yaphe. Effect of seasons on the Chemi. Struc. and gel strength of Gracilaria psedoverrucosa agar (Gracilariaceae, Rhodophyta). Carbohydr. Polym., Vol. 8, P (1988). 13. C. Rochas, M. Lahaye, W. Yaphe. Sulfate content of carrageenan and agar determined by infrared spectroscopy. Bot. Mar, 29, P (1986). 14. D. J. Stancioff, N. F. Stanley. Infrared and chemical studies on algal polysaccharides. Proc. Int. Seaweed Symp., Vol. 6, P (1969). 15. M. Tako, S. Nakamura. Gelation mechanism of agarose. Carbohydr. Res., Vol. 180, P (1988). 116

The Cell Wall Galactan of Catenella nipae Zanardini from Southern Australia

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