Extraction of Kappa-Carrageenan from Red Seaweed

Carbohydrates are macromolecules whose basic units are called monosaccharides (or simple sugars). These simple sugars are polyhydroxy aldehydes (aldoses) or ketones (ketoses), depending on the chemical nature of their carbonyl group, containing at least three carbon atoms. The smallest monosaccharides with only three carbon atoms are collectively known as trioses; while those with four, five, six, and so on carbon atoms are called tetroses, pentoses, hexoses and so on, respectively. Carbohydrates may also be classified according to the number of combining monosaccharide units, forming an oligosaccharide or polysaccharide.

In the experiment, the sulfated polysaccharide, κ-carrageenan is extracted. Carrageenans, in general, have high molecular weights polysaccharides made up of repeating galactose units and 3,6 anhydrogalactose (3,6-AG), joined by alternating alpha 1-3 and beta 1-4 glycosidic linkages. There are various types of the polysaccharide which include alpha, beta, lamba, kappa, mu, and iota carrageenan (figure 1). However, iota, kappa and lambda are the three carrageenans of commercial interest. The primary factors influencing the properties of these three are the number and position of the ester sulfate groups on the repeating galactose units. Nevertheless, all carrageenans are extracted from red seaweed and assume a gelatinous structure.

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Figure 1.Different types of carrageenan. They differ in structure, properties and uses.

Production of carrageenan, particularly κ-carrageenan can be done in either of two ways. The first method, which is considered as the original one, involves the extraction of carrageenan into an aqueous solution, filtration to remove the seaweed residue and finally, the recovery of the carrageenan from the solution. Hence, product of this extraction is called refined or filtered carrageenan. In the second method, carrageenan is not actually extracted but rather, everything soluble in alkali and water is washed out from the seaweed, leaving the insoluble parts which are largely consisted of carrageenan and cellulose. The produced carrageenan is then dried and sold as semi-refined carrageean (SRC). For the experiment, the first processing method is employed.

The Euchemia species are the red seaweeds indigenous of the Philippine waters. In particular, Eucheuma Cottonii (figure 2) is the raw material used for κ-carrageenan extraction. Sun-dried Eucheuma Cottonii is rinsed with running water to remove salts, sands, and other foreign materials. The seaweed is then oven dried at 60oC overnight. This temperature, however, was adjusted to 90oC to enhance drying since the extraction proper is to be done early in the morning of the following day, and therefore, time for drying is not enough.

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Figure 2.Eucheuma cottonii

From the oven-dried seaweed, 5.00g was weighed. This was placed in a 1-L beaker containing 500 mL of 5% NaOH solution and was heated at 100oC for 1 hour. An alkali solution, NaOH in this case, is used because it removes some of the sulfate groups from the molecules and increases the formation of 3,6-AG, thereby increasing the gel strength of the final product. The resulting solution is the filtered using suction filtration to remove seaweeds that were not dissolved, and increase carrageenan concentration in the solution from 1-2% to 2-3%.

To the filtrate, isopropyl alcohol was added until precipitation; this was followed by freeze drying of the precipitate for 24 hours. This process is the alcohol method wherein carrageenan of any type is recovered as a fibrous coagulum. Alcohol serves as a dehydrating agent, removing solvent from the carrageenan. Unlike this method, however, the gel method, which relies on the ability of kappa carrageenan to form a gel with potassium salts, can only be used for the recovery of kappa-carrageenan. In this method, dehydration of the gel can be done by either subjecting it to a freeze-thaw process or squeezing it (pressure application). The former method involves the carrageenan (usually as fine spaghetti-like pieces) being washed with potassium chloride to remove solvent, pressed to remove surplus liquid and then frozen. The schematic diagram of the refined carrageenan process appears in figure 3 below.

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Figure 3.Schematic diagram for the production of refined or filtered carrageenan.

Characterization of the extracted kappa-carrageenan can be carried out using Ostwald viscometer and IR spectroscopy. The former is for the determination of the average molecular weight of the carrageenan through viscosity measurement. On the other hand, the latter can provide information about the structure of the carrageenan, especially since sulfated polysaccharides have strong, broad absorption bands in the 1000-1100 cm-1 region. To further characterize the carrageenan, qualitative tests for carbohydrates may also be employed. Tests such as Molisch’s, Bial’s, Seliwanoff’s, Benedict’s, Barfoed’s, Mucic acid and, hydrolysis of polysaccharides and iodine test also provide information on the nature and properties of the polysaccharide; with the first three being based on the production of furfural and furfural derivatives and the other four based on the reactivity of the aldehyde or potential aldehyde group. In the experiment, however, characterization of the extracted κ-carrageenan was not done.