## 6 Methods for Obtaining Selenium-Containing Polysaccharides
### Extraction from Natural Products  
As stated in the previous paragraph, chemical synthesis of high molecular-weight polysaccharides is practically impossible. Therefore, these compounds are either isolated from natural sources or obtained by biotechnological methods and, in certain cases, chemically modified. Depending on the biological material from which the polysaccharides are extracted, slightly different extraction conditions have to be chosen [318] due to the different cell wall structure of bacteria, fungi, and plants. Polysaccharides are mostly soluble in water, but insoluble in organic solvents; therefore, water is the most common extraction medium [319] and the base solvent in the acid or alkali extraction and the enzymolysis method [320]. Basically, the Se-polysaccharides are isolated and purified using the same methods as non-selenium-enriched polysaccharides. Certain differences, observed in our research on the biosynthesis, extraction, and purification of these compounds, consist in different solubilities and limitations in the use of aggressive reagents in derivatization processes (strong acids and bases) due to the instability of some selenium compounds in such an environment [252; 320]. As it has been observed, selenium polysaccharides have lower water solubility than non-selenated analogues, which thus affects their extraction efficiency [39]. In plants, breaking the cell wall with ultrasound or microwave methods is required prior to the polysaccharide extraction [321; 322]. In mushrooms, on the other hand, the biologically active polysaccharides (mainly \(\beta\)-glucans) are components of the outer layer of the cell wall [323]. In this case the extraction of cell wall polysaccharides commonly involves a pre-extraction with 80% ethanol for elimination of low molecular substances [324]. However, apart from those slight differences, the basic extraction methods are similar and include hot water extraction, precipitation with alcohol (ethanol or methanol), deproteinization, and fractionation, utilizing different methods. Taking into account the differences in the physical and chemical properties of the various polysaccharides (Se-polysaccharides), an appropriate extraction method should be chosen based on the structure and solubility (in water or alkali) of the target compounds. During the extraction, a transition from gentle to aggressive extraction conditions (pH, temperature, pressure) is essential. This way the cell wall is gradually destroyed, resulting in the release of Se-polysaccharides from deeper cell wall layers. Procedures are often time-consuming and multiphasic [321]. The example procedure for the extraction of the active polysaccharides from mushroom cultures may involve the following steps. The initial water extraction is carried out with organic solvents, mainly ethanol, acetone, or a mixture of chloroform and methanol in order to remove non-polar compounds such as lipids, phenols, and terpen. The next step is centrifugation, which produces a supernatant called cold water extract. The residue is then extracted in boiling water, and the biomass is isolated from the hot water extract by another centrifugation. The extraction is repeated to increase the yield [325]. The aqueous extraction residue is extracted with an aqueous base solution (with 2% NaOH or KOH solutions) at the boiling point [326]. The precipitate is separated by a centrifugation using NaBH\({}_{4}\) to protect against degradation of the end units of the polysaccharide chains. Exactly the same procedures were used to extract Se-polysaccharides from selenium enriched mycelium of _L. edodes_ and _Hericium erinaceus_[11; 13; 39; 249]. After the extraction, the polysaccharides are purified from proteins, amino acids, monosaccharides, phenolic derivatives, and other molecules. Protein contaminants are removed using a 20% trichloroacetic acid solution by an enzymatic process using a protease enzyme, by the Sevage method, or by extraction with a phenolic reagent [326].