The increasing demand of carrageenan led to intensive cultivation in developing high quality seaweeds. The increasing worldwide demand and development of new applications of carrageenan caused a rapid expansion and growth to occur in the seaweed farming industry. Due to the increasing demand of raw material for the production of carrageenans, carrageenan processors were forced to source out for new or additional raw material sources (Freile-Pelegrín and Robledo, 2007). The discovery of Eucheuma cottonii made a large contribution to the seaweed industry since it is the fastest growing seaweed discovered with an ability to double its biomass within 15 to 30 days (Doty, 1996). The extraction of E. cottonii produces kappa carrageenan. Each carrageenan manufacturer carefully controls the raw materials and process parameters to produce a high amount of carrageenan extracts with well-defined properties (Imeson, 2000).
Nevertheless, in spite of the high commercial value of E. cottonii and of its polysaccharide, detailed studies on the properties contributing to the quality of carrageenan is limited. Since there is only little information on carrageenan properties, the present study is an attempt to process E. cottonii for the extraction and purification of carrageenan, and to then identify the influences of alkali treatment in isolating carrageenan from E. cottonii. Another aim of this study is to investigate the carrageenan properties, such as yield, gel strength, gelling and melting temperatures, of native and treated carrageenan from E. cottonii. Lastly, the final aim of this study is to identify the type of carrageenan extracted from E. cottonii. Potentially the results can be used in processing higher grade carrageenan.
5.2 CARRAGEENAN ISOLATION
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In this project, studies were extended to what was identified as a kappa type carrageenan that was extracted from E. cottonii. Alkaline extraction of E. cottonii is commonly used for the industrial production of the gel forming polysaccharide, kappa carrageenan. However, even though E. cottonii and its carrageenan have high commercial value, not many detailed studies have been carried out on the characteristics of the carrageenan and on the best conditions for the alkaline extraction to produce a 'nearly pure' kappa carrageenan.
As for this study, a total of twelve samples of E. cottonii weighing at 30 grams each were extracted to obtain the gelling polysaccharide, carrageenan. The results show that carrageenan extracted using alkali solutions are of better quality and quantity compared to carrageenan extracted using distilled water. Potassium hydroxide and sodium hydroxide were used as extracting agents since these solutions are known to aid in extracting high yield carrageenan. The mechanism of extraction requires a high temperature condition where the seaweed is boiled in order to release carrageenan out into the extracting solution. An extraction period of two hours was used as a standard to compare the effects of each extracting agent. Ethanol and isopropanol were used as precipitating agents because their function is to coagulate carrageenan. In non-alkali treated carrageenan, isopropanol proved to be a better precipitating agent compared to ethanol since it resulted in higher yield carrageenan.
5.3 GELLING TEMPERATURE AND MELTING TEMPERATURE
The gelling and melting temperatures of the samples resembled that of a pure carrageenan system. Gelling and melting temperatures increase with cation concentration as reflected when E. cottonii was extracted using KOH and NaOH. Carrageenan molecules are neutralized more rapidly and extensively with the increment of cation levels which results in the formation of additional double helices that creates a more extensive 3-dimensional network at higher temperatures (Norziah et al., 2006). Gelation occurs on cooling at a critical temperature and it has been attributed to a two-stage reaction involving a coil-helix transition followed by aggregation of helices (Morris et al., 1980; Morris, 1998).
Based on the observations made, the gelation process is highly influenced by certain factors such as the type and concentration of salts in solution, cooling and heating rates as well as the concentration of the hydrocolloid. In this study, the gelling temperatures of carrageenan samples were within the range of 21.5°C to 41°C. Melting temperatures are significantly higher than the gelling temperatures. Hysteresis is the difference in temperature between the transition temperature measured upon heating and cooling (van de Velde et al., 2005). Hysteresis in this case is attributed to the extra energy required to overcome the helical associations to melt the carrageenan network (Bubnis, 2000). The melting temperatures were within the range of 53.0°C to 61.0°C.
5.4 GEL STRENGTH
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Gelation occurs due to the formation of intermolecular bridges between carrageenans. Kappa carrageenan has an ability to gel by means of the formation of a double helix structure and alignment of helices into junction zones. The carrageenan-carrageenan interaction occurs at the junction zones which results in a three-dimensional network with the capability of the immobilization of water molecules in its interstices (Arnott et al., 1974). Gelation takes place by hydrogen bonding which occurs on cooling below the glass transition temperature leading to the double helix formation (Viebke and Piculell, 1996).
Carrageenan gelation not only depends on the amount and type of carrageenan molecules, but also on the presence of cations in solution (Smith and Cook, 1953; Bayley, 1955; Smith et al., 1955; Pernas et al., 1967; Towle, 1973, Reid, 1978; Morris et al., 1980, Dickinson, 1998; Syrbe et al., 1998). Gelation depends on the presence of cations like K+ in a kappa carrageenan system (Glicksman, 1969; Towle, 1973; Syrbe et al., 1998; Chen et al., 2002; Sedlmeyer et al., 2003). Kappa carrageenan has the ability to form a gel in the presence of specific cations such as potassium and sodium. The action of cations has been attributed to controlling the solubility of the carrageenan and hence its tendency to precipitate from solution (Percival and McDowell, 1967). The gel is heat stable above boiling point after heating and cooling cycle (Thomas, 1992).
FIGURE 5.1 General gelling mechanism of carrageenans. [Adapted from Millán et al., 2001]
The gel strength of carrageenans varies with the species, location, season and extraction method of the seaweed (Hoyle, 1978). In this study, the gel strength of carrageenans from E. cottonii was exclusively species specific, collected from the same location and hence shared the same environmental conditions. High contents of 3,6-anhydrogalactose are generally associated with strong gels, whereas a high content of sulphate is associated to weak gels (Armisen, 1995). The gel strength of the carrageenan polysaccharides extracted from E. cottonii was markedly improved by alkali treatment. The increase in gel strength was induced by the formation of 3,6-anhydro-L-galactose. Based on the information presented in this study, KOH-treated carrageenan turned out to be the best source in terms of gel strength of carrageenans with gel strength of 225 g/cm2. Whereas, non-alkali treated carrageenan gave the lowest gel strength of 86.3 g/cm2.
The gelling mechanism indicates that kappa gels are thermoreversible gels. The gels become fluid when heated above the melting temperature and will gel upon cooling with minimal to no loss of their original gel strength (Bubnis, 2000). Kappa gels are strong and brittle. The potassium and sodium ions in the alkali solutions neutralize the sulphate group in the kappa carrageenan structure which then promotes hydrogen bonding leading to the formation of a double helix (Campo et al., 2009). These helices contribute to the strength and brittleness of the kappa carrageenan. However, due to the brittleness of the gel, when the gel is subjected to shear it is unable to reheal itself.
FIGURE 5.2 Gelation mechanism of kappa carrageenan. [Adapted from Bubnis, 2000]
The solubility of carrageenans is fundamental to their application in the food industry, as the required product is an evenly dispersed mixture to promote a consistent food system composition (Lamond, 2004). All carrageenan samples were found to be soluble in water at high temperatures of 80°C and above, developing low fluid processing viscosities. The carrageenan samples formed cloudy viscous suspensions in water. Turbidity was higher in alkali-treated samples due to pigment transfer from the alkaline solution. The dissolution temperature of depends on cation concentration and levels of sulphate groups (Campo et al., 2009). The increased salt levels hinder solubility because there is a reduced amount of free water available in the system to interact with the carrageenan (Bubnis, 2000). Therefore, higher temperatures are required to free enough water from the solvated cations to dissolve the carrageenan. As a result, non-alkali-treated samples were found to disperse more readily compared to that of alkali-treated samples. Other than that, all carrageenans were found to be insoluble in ethanol. This is due to the function of ethanol as a coagulating agent causing the carrageenan molecules to coagulate and hence preventing dispersion.
FIGURE 5.3 Effect of cations on carrageenan dispersion. [Adapted from Bubnis, 2000]
5.6 IDENTIFICATION OF HYDROCOLLOID AND PREDOMINANT TYPE OF COPOLYMER
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Kappa carrageenan is soluble in hot water. The addition of potassium ions induces the formation of a double helix which causes the gel to be short textured, rigid but brittle. Kappa carrageenan exhibits high levels of syneresis which is the elimination of water from a gel as the gel structure tightens and contracts (Imeson, 2010). Kappa carrageenan is a slightly opaque gel due to the transfer of pigments from the addition of potassium ions.
Iota carrageenan is soluble in hot water. The addition of calcium ions induces the formation of a compliant and elastic gel. Iota carrageenan does not show any signs of syneresis. Iota carrageenan displays a clear gel and is freeze/thaw stable. This gel also exhibits thixotropic characteristics which mean that they thin under shear stress but is able to regain its original form once the stress is removed (Caldwell, 2009).
Lambda carrageenan is a free flowing, non-gelling pseudo-plastic solution in water. This form of carrageenan is fully soluble in hot water. However, lambda carrageenans are unable to form gels which results in random distribution of polymer chains. The addition of cations has slight effect on viscosity (Prajapati et al., 2007).
In this study, observations on the carrageenan characteristics were made throughout the experiment. The carrageenan was soluble in hot water. The gel was short textured, rigid and brittle. The addition of potassium ions contributed to higher gel strength and yield. The carrageenan also showed signs of syneresis and is slightly opaque. The gel is not freeze/thaw stable and does not exhibit thixotropic characteristics. Therefore, based on these observations, the carrageenan isolated from E. cottonii was identified as kappa carrageenan.
5.7 pH VALUE
Generally all carrageenans are stable at neutral and alkaline pH's. However, a decrease in pH showed a reduction in gelling and melting temperatures. Basically, reduction in pH caused the gel to be weaker and more brittle due to an increment in the number of shorter chains which prevented the formation of junction zones (Norziah et al., 2006). The pH of alkali-treated carrageenans was higher than that of non-alkali treated carrageenans. The pH of non-alkali-treated carrageenans ranged from 7.32 to 7.84 whereas, the pH of alkali-treated carrageenans ranged from 10.28 to 11.00. Hence, gelling and melting temperatures as well as gel strengths of alkali-treated carrageenans were higher compared to non-alkali-treated carrageenans.
The total yield of carrageenan extracted from alkali treated E. cottonii was higher than that of the non-alkali-treated samples. These findings are in agreement with previous observations (Tuvikene et al., 2006) regarding the higher yield in alkali-treated samples due to the alkali solution which promotes desulphation at the 6-position of the galactose units of the carrageenan, resulting in recurring 3,6 anhydrous galactose polymers by dehydration and reorientation causing coagulation of carrageenan and hence giving a higher carrageenan yield (Kalinowski, 2007). This shows that the yield and the quality of carrageenan depend strongly on extraction and purification techniques. KOH-treated carrageenan resulted in the highest yield of 80.3% while non-alkali treated carrageenan resulted in the lowest yield of 20.0%.
5.9 COMPARISON ON THE EFFECTS OF EXTRACTION METHODS
In this study, it was found that the gelling and melting temperatures, gel strength and yield were higher in carrageenans isolated through freeze-thaw method compared to hot extraction method in non-alkali treated carrageenan. However, the results could be attributed to the precipitating agents as well. In this case, isopropanol was a better precipitating agent compared to ethanol in terms of carrageenan yield in non-alkali-treated carrageenan. Overall, alkali-treated carrageenan had the best quality. Potassium hydroxide proved to be the best extracting agent in the hot extraction method. KOH-treated carrageenan had the highest gelling and melting temperatures, gel strength and yield.