**4. Fractionation and its application in the extraction of polysaccharides from the mushroom species,** *G. lucidum*

environment and the ingredient of medium have also been reported to strongly impact the growth of mycelia and the production of exopolysaccharides [44]. A report by Yang and Liau [45] noted that production of *G. lucidum* polysaccharides by fermenter-grown mycelia cultivar was optimum at pH of 4–4.5 and temperature 30–35°C. Acceleration of mycelial growth and bioactive components could be produced by addition of supplements such as fatty acids.

54 Fractionation

Three types of dissimilar pre-treat ways were applied on the different life stages of mushroom before the hot water extraction. An investigation of the non-volatile composition of *G. lucidum* [46] reported that the mushroom contains 59% crude fiber, 26–28% carbohydrate, 7–8% crude protein, 3–5% crude fat and 1.8% ash. Nonetheless, a wide variety of bioactive molecules are also found in *G. lucidum* which include terpenoids, phenols, nucleotides and their derivatives, steroids, glycoproteins and polysaccharides. *G. lucidum* proteins contain most of the essential amino acids and are especially affluent in leucine and lysine. The fermentation broth contained media, mycelia and spores. After concentration, the volume of liquid was reduced. Addition of Sevag's reagent for the purpose of deproteinization could eliminate the protein ingredient in the mixture. Xia Li *et al.* [47] proposed an explanatory theory of the advantage of using Sevag's reagent. With regards to deproteinization rate and polysaccharide residual rate, the process of optimization, was based on the following parameters: deproteinization frequency (example: 4 times), time of oscillating (example: 11 min), the volume ratio of sample to Sevag's reagent (2:1), and the volume ratio of chloroform to n-butanol (5:1), The resulting deproteinization rate was 64.2%, the polysaccharide loss rate was 34.3%, and the purity of polysaccharide was raised by about 2.8 times. Compared to other stage of cultivation, mycelia of *G. lucidum* were treated simply and without any step of deproteination and removal of fatty substances. One of the reasons for these could be that at the initial stage of mycelial mat forming, little protein was available. Excluding the step of deproteination allowed the hot water extraction at 70°C to be conducted directly and shorten the time for processing polysaccharides. Similarly, deproteination for fruiting body powder is also not required. Instead, petroleum ether is used to remove fatty and fat-soluble substances. The high proportion of polyunsaturated fatty acids and low total fat content relative to the total fatty acids of *G. lucidum* are considered significant contributors to the heath value of *G. lucidum*. [48]. The residues accumulated after the defatted step would allow the hot water extraction to proceed. In this case, the main concern at the end of experiment was isolation of polysaccharides. As in other studies, from the accumulated filtrates of several replicates of hot water extraction, the precipitate treated by addition of ethanol and freeze-dried would contain crude polysaccharides, indicating that several types of polysaccharides are found in the crude. The subsequent step in this study was the application of DEAE-Sephadex A-25 column as anion exchange chromatography intended for the further separation of each type of crude polysaccharides. According to the data file of ion exchange [49], Sephadex ion exchangers are derived from either Sephadex G-50 or Sephadex G-25. The G-50 matrix is less highly cross-linked than the G-25. Ion-exchangers based on Sephadex-50 have less rigidity and thus swell more than those based on G-25, which are more rigid. These properties mean that A-50 and C-50 types are better suited to larger biomolecules, such as polysaccharides or proteins in the molecular weight range of 30,000–100,000 MW, whereas A-25 and C-25 type ion exchangers are a better choice

for small molecules up to about 30,000 MW.

The discovery of new polysaccharides from *G. lucidum* has long been a research of great interest in a wide range of field. Bao *et al.* [50] reported six different functionalized derivatives of the (1→3)–α-D-glucan which were isolated from the spores of *G. lucidum*. The process included aminopropylated, sulfated, carboxymethylated, carboxymethylated and sulfated, and benzylamidated-carboxymethylated with varying degrees of substitution synthesized. These modified derivatives have shown potent stimulating effects on the lymphocyte proliferation and antibody production. On the other hand, the induction of carboxymethyl group with low degree of substitution was the best choice for the improvement of the immunostimulating activity. A one-step to prepared selenium nanoparticles (SeNPs) decorated by the water–soluble derivative of *G. lucidum* polysaccharides (SPS) was tested on their anti-inflammatory activity against murine RAW264.7 macrophage cells induced by lipopolysaccharides [51]. The results suggested that seNPs-SPS complexes possessed anti-inflammatory potential modulating pro/anti-inflammation cytokine secretion profiles. The isolated polysaccharide (molecular weight 1×10<sup>6</sup> Da) isolated from the sporocarps of *G. lucidum* was used to determined radioprotective property *in vivo* and *in vitro* by survival studies [52]. The findings indicated *Ganoderma* polysaccharides were significantly protective against radiation-induced damages. Another study showed positive effect of polysaccharides extracted from *G. lucidum* on blood glucose, serum level, lipid peroxidation, nonenzymaic and enzymic antioxidants in the plasma and liver of streptozotocin-induced diabetic rats [53]. The neutral crude polysaccharides extracted from *G. lucidum* fruiting body claimed by Chen *et al.* [54] exhibited the higher DPPH-, O- and OH- free radical scavenging activities. It could significantly enhance the antioxidant enzyme activities (SOD, CAT and GPx) and reduce levels of IL-1β, IL-6 and TNF-α in rats with cervical cancer. Two fractions purified from the fruiting body of *G. lucidum* were tested on activation of macrophage cell (RAW 264.7) and antitumor activities on the human breast cancer cell (MDA-MB-231) [55]. The results indicated that both fractions increased the proliferation and pinocytic activity of macrophage significantly and played an inhibiting effect on the cancer cell.
