**2. Separation and characterization**

The cell wall of fungi is composed mainly of polysaccharides such as lichenan, isolichenan, and galactomannan (fig.1) (Elix and Stocker-Wörgötter, 2008). Bernard and Latgé (2001)

described that the fungal wall is a complex structure composed typically of chitin, 1,3-- and 1,6--glucan, mannan and proteins, although wall composition frequently varies markedly between the species of fungi.

**Figure 1.** Structures of some polysaccharides contained in fungal cell wall

For the separation and isolation of lichen polysaccharides, traditional methods and modern techniques are used. Traditional methods basically involved freezing and thawing of the material originally extracted with hot water. Dialysis and ethanol precipitation has been employed for further purification. Also, alkali solutions have been used to extract these compounds. The polysaccharides present in the thalli of *Umbilicaria mammulata* were isolated by an exhaustive method starting with successive extraction with hot water at 100oC followed by extraction of the residue with hot 2% KOH at 100 oC. The alkaline extract was further treated with Fehlings solution to yield the 1,3-β- glucan from the supernantant and a 1,6-β- glucan was obtained from the precipitated Fehlings complex (Carbonero *et al*., 2006). In our laboratory, we have used a similar alkaline extraction procedure to isolate a polysaccharide from *Usnea* cf. *cornuta* Körb. Chromatographic techniques and filtration devices were utilized to separate lichen polysaccharides (Paulsen *et al*., 2002). However, different column chromatographic methods, including GP-HPLC, HPLC and ion exchange chromatography have been used recently to separate polysaccharides from lichens. Olafsdottir *et.al* (1999), fractionated the polysaccharides from *Cetraria islandica* on DEAE Sepharose CL-6B anion exchange columns with a 0 -1 M NaCl gradient. Further purification was performed by Sephacryl S-400 gel filtration. To determine homogeneity and *M*r of these compounds, electrophoretic methods and gel permeation chromatography are used. For the determination of monosaccharides, TLC and GC methods are used. The monosaccharides could be derivatized into alditol acetates or into trimethylsilyl (TMS) ethers for analysis by GC. The linkage analysis is performed by using NMR spectroscopy (1D proton, 2D-COSY, NOESY, 2D-TOCSY, 1H 13C-HSQC, HMBC, H2BC and HSQC-NOESY) (Paulsen *et al*., 2002 and Jensen *et al*., 2010) and methylation analysis (Omarsdottir *et.al*., 2005; Olafsdottir *et. al*., 1999; Ruthes *et al*. 2010).
