than freezing method. It is 20% higher than the freezing–drying method which was 11%. Which means agar, produced by freezing method, has more impurities like water and other soluble impurities, than the syneresis method.
Treatment: alkaline solution is used to extract polysaccharide from the cell wall of seaweed. For example: for Gracilaria any stronger base is required to extract agar from its cell wall. Strong base helps in alkaline hydrolysis of the sulfates resulting stronger agar gel strength.
Extraction: Extracted agar is later dissolved into boiling water above atmospheric pressure and on a high pH to obtain a high yield.
Filtration: Most standard filtration techniques can be used in this step.
5.4 Structure of Agar
The agar was originally thought to be made up of simple sulfated poly galatose structure wise. After the development of electron microscopy in 1937, it was shown that agar can be separated into two different fractions—agarose and agaropectin. The agarose works as gelling component of agar. The percentage of agarose and agaropectin may differ in different species. A higher ratio of agarose to agaropectin gives higher gel strength. By the late fifties more research was done on the structure of agar and agarose has been described as linear polymer, consisting of alternate D-galactose and 3,6 anhydro-Lgalactose. However its structure is a complex polysaccharide that varies from source to source. A study in 1991 showed that agar has at least 11 different agarobiose structure [12]. The variation in structure depends upon the gender of the species, the environment it grows in, the time of year, agar got extracted, etc. In summary agar is a complex of interchanging of β-(1–3)-D and α-(1–4)-L linked galactose residues [13]. Out of which α-(1–4)-L residues are converted by the 3,6 anhydro bridge as shown in Figure 5.1. Or we can say it is a composition of heterogeneous molecules that differ in their physico-chemical properties. However sulfates, pyruvates, urinate or methoxyl groups are also some of the modifications in the agarose complexes. Agarose is typically a molecule of higher molecular weight and low in sulfates. Agaropectin is just opposite to agarose, it has lower molecular weight compared to agarose and higher sulfates at about 5–8%. Some agars also have xylose in it.
Figure 5.1 Chemical structure of agarobiose (the fundamental repeating unit of agar). Source: Europian Food Cunsumption Database.
Modern alkaline treatment method helps in increasing the anhydrous bridges within molecule, thus increasing gel strength. Methoxy levels in agar determine the gel setting temperature. The level of methoxy content in gel is directly proportion to its strength. Thus higher level of methoxy in agar leads to the higher gel setting temperature and vice versa.
5.5 Heterogeneity of Agar
The classical ‘freeze–thaw’ example of fractionating the agar is the oldest known fractionation method of agar. The gelling counterparts of polysaccharides (agarose) were broken up from their non gelling counterparts (agaropectin), purely by freezing, thawing and then pressing the agar gel. In 1979 Usov [14] and his team recognised this non-gelling counterpart as agaropectin. In old times, to exhibit the heterogeneity of agar and separate agarose (Polysaccharide fraction with highest gelling capacity) from agaropectin (the charged polysaccharide counterpart of poor or non-gelling quality), the differential solubility of agar polysaccharide was used. In 1946 Yanagawa [15] extracted polysaccharide fractions with sulfate content by mixing agar of G. amansii at 3, 50–55 and at 75–80 °C in water. As well as he acquired different yields of agar from Gloiopeltis, Gracilaria, Ceramium, Gelidium and Acanthopeltis species of agarophytes, simply by boiling them at 70, 60, 50% of alcohol. In 1970 Guiseley [16] developed a method to precipitate polysaccharides. Since methylated agar polysaccharide is soluble in hot ethanol, in 1970 Guiseley used this property of agar to precipitate polysaccharide. In 1986 Lahaye developed a procedure to extract agar. In this method water is added to the different boiling ethanol-water solutions at different temperatures to get a sequential solvent of agar. Izumi [17] and Yaphe’s group studied the differences in charges densities of agar polysaccharide by using anion exchange chromatographic techniques.
5.6 Physico-Chemical Characteristics of Agar
The ability to gel and solubilize for agar polysaccharides solely depends upon the comparative hydrophobicity of structural repeating units of agarobiose. Gelling property of agar also alters when the 1,4-linked 3,6-anhydro-α-L-galactopyranoses gets substituted with hydrophobic groups like methoxyl and polar groups like sulfates and pyruvates.
In 1977 Rees and Welsh [18] stated that agar gels are created only when agar polysaccharide reaches it helical conformation and these helical conformations accumulate at one place. According to Arnott et al. 1974, the hydrogen bond between water molecule and the O2 of galactose and O5 anhydrogalactose maintain this structure. The detailed structure of agar was explained by X-ray diffraction studies, which have revealed that extended single helices with a lead of 0.888–0.973 nm are created predominantly during the gelling or drying of the agar. These structures were verified later with the help of molecular modelling of agaro-oligosaccharides, showing a triple folded left handed single helix with a lead of 0.95 nm and 2.85 nm pitch. This confirms that clustering of single helices is also a possible step during agar gelation. X-ray diffraction study of agarose by Chandrasekaran in 1998 revealed that it is a double helical structure which is stabilized by the hydrogen bonds between agarose and water molecules at O-2 and O-5 of galactose unit. The helix is left handed with 1.9 nm pitch. The inner and outer dimensions are 0.42 and 1.36 nm. The length of repeating unit is 0.633 nm built only with sugar in 4C1 conformation. However it consists of three-fold symmetry. Therefore we can say agarose is a crystallized, oriented, double helical left handed compound with repeating units of interconnecting galactose units. Since only O-2 of galactose unit is needed in gelling process, anything that changes its conformation, abrupt the gelling process.
Polymerization of agar gel takes place when hexagonal fibbers of 6 double helices club together to make bigger clusters as shown in Figure 5.2. L-galactose 6-sulfate and L-galactose both having 1C4 conformation, tend to replace their biological precursor 3,6-anhydrogalactose, which has 4C1 conformation. This switching between precursors, prevent the helix formation in agar or introduce kinks or interrupt it otherwise. These kinks would help in the formation of 3-D structure of the gel. Moreover, when replacement of these chemicals on O2 of anhydrogalactose and O6 and O4 galactose does not disturb the helical conformation of agar, they help in lessen the clustering of helices hence stops the gel formation. It can be prevented by increasing the temperature so that ordered conformation can take place. In 1970, Guiseley [19] stated that methoxyl content of agar partially controls the gelling temperature of agar. Agar with higher methoxyl content needs higher temperature to form gel. However, an opposite association was obtained with synthetically methylated agar and revealing that synthetic methylation takes place at random sites in agar compared to its counterparts which naturally occurs at O6 of galactose and/or O2 of 3,6anhydrogalactose in agar.
Figure 5.2 Chemical construct of different agarose units [20].
Commercially prepared agar seems to have molecular weight ranging from 35.7 to 144 kD. In sequential solvent extraction method, the molecular weight of agar doesn’t appear to determine its differential solubility. Like the other polymers, the solubility of agar depends upon its nature of solvent, like if solvent is capable of distorting its helices and conformations,
