Fractionation plays a big role in most of the sample processing especially in mushroom polysaccharides extraction. This intermediate step will determine further purification process which will lead to the type of polysaccharides that will be obtained. Four types of Ganoderma lucidum cultured medium used in the research papers were randomly chosen. They are spores, mycelia, fruiting body and fermentation broth. For water soluble polysaccharides, hot water extraction is typically applied. The following ethanol precipitation could be appropriate used to sediment the component with OH-group including polysaccharide. The next step of fractionation consist of anion exchange chromatography or gel filtration enhance the purity of polysaccharides. Using these extraction and fractionation techniques, high quality polysaccharides could be successfully obtained from the mushroom that are useful for further studies. This review examined the various extraction and fractionation techniques used in the study of polysaccharides from G. lucidum.
- Ganoderma lucidum
- anion exchange chromatography
1. Mushroom polysaccharides
Mushroom polysaccharides have been studied intensively since the therapeutic effects of hot water extract from several fungi were evaluated on Swiss albino mice . The fungi,
The long chain of main β-glucan molecules is capable of having branching side-chains with other monosaccharides in the position of C1 and C3 or C1 and C6 through condensation. Likewise, other types of molecules such as proteins are found in polysaccharide-K (Figure 2) .
Basically D-glucose units are the common forms of β-glucans and mostly they are binding with β-1,3 links. The β-glucans of mushroom, yeast or other fungi contains 1–6 side branches (Figure 3), while cereal β-glucans contain β-1,4 and β-1,3 backbone bonds.
In short, there are many possible branches of different types of monosaccharides with glycosidic linkages. This scenario is closely associated with molecular weight of the polysaccharide.
Presently the effects of mushroom polysaccharides have been studied intensively which consist of antioxidant , anti-inflammation , anti-hyperglycemia , anti-tumor  and immunostimulation . Nevertheless the process of discovery of new polysaccharides is still on-going. Moreover, research revealed that the chemical characteristics of the constituents of the mushroom polysaccharides, such as molecular weight, molecular structure, solubility in water, the location, length and frequency of the side-chains have a correlative relationship with the function of polysaccharides . The issue of types of isolated polysaccharides have received considerable critical attention on the step of the process of extraction especially fractionation. Fractionation has a pivotal role in the intermediate stage between extraction and isolation. Polysaccharides fractionation is essential for a wide range of technologies for separation process including certain quantity of a crude polysaccharides. The crude will be continuously separated into smaller quantities with diverse compositions.
2. Polysaccharides of selected species,
The molecular structure of
3. Review of available
G. lucidumpolysaccharides extraction and fractionation methodology
Mushroom polysaccharides are present as structural components of fungal cell wall which is composed of two major types of polysaccharides. They are divided into a rigid fibrillar of cellulose and a matrix-like glycoprotein, α-glucan or β-glucan . Selection of mushroom polysaccharides extraction method normally relies on the cell wall structure. A reliable procedure for successful extraction of polysaccharides from either cultivar mycelia or fruiting body has been developed . The extraction method commonly involves 80% ethanol for elimination of low molecular substances from mushroom material, followed by 3–5 successive repeating extractions with water (100°C, 2–4 h). The alternative choice is 5% sodium hydroxide (80°C, 6 h) or 2% ammonium oxalate (100°C, 6 h). In fact, the hot water extraction yields water-soluble polysaccharides, while the extraction with alkali solution is the best method to produce water-insoluble polysaccharides. Although various types of extraction method can be applied depending on the structure and water-solubility of polysaccharides, however, it is important to rupture the hard cell wall from the outer layer to the inner layer with weak-to-strong extraction conditions (pH and temperature). A combination of techniques should be chosen for further extraction of polysaccharides, normally ethanol precipitation will be the first choice as it excludes the impurities from the extracted polysaccharides. Other techniques included fractionation precipitation , acidic precipitation with acetic acid , ion-exchange chromatography , gel filtration  and affinity chromatography .
Comparing to the ancient era, definitely today’s new technology is enhancing the yields of the extraction. A lot of new polysaccharides of
3.1. Extraction and fractionation of a novel water-soluble β-D-glucan from the spores of
G. lucidumusing ethanol precipitation
Analysis was based on the conceptual framework proposed in five papers authored by Bao
Normally 95% ethanol is added at the beginning of extraction to remove the lipids especially after the sporoderm of the spores were broken. The exposure to the environment actually escalated the oxidation of the content of spores. Two types of spores breaking mechanisms are applicable nowadays. The traditional way, i.e., via mechanical vibration grinding resulted in the bioactive ingredients and unsaturated oil being directly exposed to the air, while the Supersonic Airflow Pulverizer would only break the outside hard chitinous layer and keep the inner covering layer complete . The methodology in this study did not mention the types of spores breaking mechanism and storage period of sporoderm-broken spores before starting the extraction.
The defatted step, conducted twice, 5 days duration in each, would remove maximum lipid contents. After centrifugation, the residues, with most of the ethanol content being removed, have to be air-dried to ensure complete ethanol free. The hot water extraction is particularly useful to extract water-soluble polysaccharides in the mushroom. Six replicates of extraction with boiling water with interval of 4 h each would allow the changing of new batch of hot water. This suggested that the bigger spin probe provided at the center of the container with appropriate speed would reduce the bubbles and increase the volume of spores with water. The extraction of substances with OH group under power of polarity of water and temperature could ensure the effective extraction of reishi spores polysaccharides. Therefore the accumulated 60 L of hot water filtrate needs to be centrifuged until 1 L of concentrated filtrate is obtained for further processing.
For this study, 15% of trichloroacetic acid (TCA) was used to procure protein precipitation reaction . In fact, most of the protein components have been denatured during boiling. Continuously added TCA eliminated components that interrupted the polysaccharide purification process. The next step was dialysis of the supernatant with 1 M of sodium hydroxide aimed to neutralize the TCA. The dialysis was completed after 2 days under running water. However, Bao
The key aspect of this experiment was isolation of the pure polysaccharides and not total carbohydrates. Hence the definition of polysaccharide needs to be fully understood. There was confusion on the nature of polysaccharides when the experiment was terminated at the time when only accumulated filtrates were obtained after a few rounds of hot water extractions. The claim that polysaccharide could not be substantiated, when steps included protein-precipitation, dialysis, ion-exchange chromatography and size exclusion, were ignored. The center concept of polysaccharide is that it consists of the skeleton of the sugar backbone structure, the glycosidase linkage of branches, and specific rotation to indicate the configuration. The result of this study provided further support of new and significant new polysaccharides from mushroom spores.
3.2. Extraction and fractionation of
G. lucidumpolysaccharide from the mycelia of G. lucidumusing agitation and filtration
The topic has mentioned “polysaccharides” which implied many types of polysaccharides. and this was frequently attributed to hot water extraction of mushroom resources  (please refer to Figure 5). The methodology described seems short and insufficient to determine the major characteristics of polysaccharides. To date, studies investigating
The extraction started with pulverized
A variety of perspectives were expressed at the end of study. The striking result to emerge from the data was the positive and significance of GLP treatment. However, this finding cannot be extrapolated to determine the effectiveness of polysaccharides due to the impurity of GLP.
3.3. Extraction and fractionation of
G. lucidumpolysaccharides F31 from the fruiting body of G. lucidumusing absolute ethanol at 4°C
Comparing with the common hot water extraction, this study applied 80°C instead of 100°C water  (please refer to Figure 6). Recent trends using temperature lower than 100°C have led to the preservation of protein content and elimination of bubble forming. One of the greatest challenges is the extraction of the polysaccharides with hot water, concurrently preventing the denaturation of the protein content. Typically, when the temperature is above 41°C, the protein will probably be melted but most of the peptides maintain their primary structures. Questions have been raised about the degree of denaturation of protein at temperature below 80°C compared to 100°C. Eventually the secondary and tertiary structures of protein will be destroyed under higher temperature. Heat could be a contributing factor for the production of end products during the process of water extraction. In this case, protein analysis had been carried on during the process of extraction. Despite the fact that the main objective of the study was to accentuate the polysaccharides extraction, certain conditions were required to determine of protein or peptide content.
After the precipitation of GLPs by DEAE-sepharose Fast Flow, fractionation was conducted using affinity chromatography. The fractionation has been applied intensively with the purpose of obtaining single pure compound. The subsequent usage of Sephacryl-S-300 resin  as high resolution size exclusion chromatography allows rapid and reproducible purification of polysaccharides, proteins and other macromolecules. In this study, this resin purified the polysaccharides with fractionation range of globular polysaccharides size 1 × 104 to 1.5 × 106 Da.
The results showed that P31 has been successfully fractionated and characterized with subsequent determination of molecular weight. The different techniques of analytical results indicated that P31 is a pure compound. Thus, this study showed an example of obtaining pure polysaccharides for bioassay guided test.
3.4. Extraction and fractionation of high molecular weight bioactive-β-glucan from the fruiting body of
G. lucidumusing centrifugation and high performance anion exchange chromatography
Previous studies have reported that the fractionation for polysaccharides from mushroom sample normally started with hot water extraction  (please refer to Figure 7). This initial step, albeit old, ensures the polysaccharides are the main compounds obtained at the end of the extraction. The function of polysaccharide is largely based upon empirical studies that demonstrated a strong and consistent association with immune system of host.
Similar procedure of hot water extraction was used on dried dark brownish powder form of fruiting body and freeze-dried white powder (labeled GLP20 in this paper), compared to procedure for mycelia and spores. The minor differences of the temperature, duration of hot water extraction, speed and time of centrifugation and volume of ethanol could be due to the larger sample of volume being used.
The fractionation of GLP20 started when 2 M trifluoroacetic acid (TFA) was added at 110°C for 3 h. This colorless strong acid has a higher acid ionization constant compared to acetic acid and trichloroacetic acid, due to the fluorine atom exerting the inductive effect that strategically stabilizes the CO2− charge of the carboxylic anion more than the other corresponding acids [38, 39]. Therefore the glycosidic linkage of GLP20 was broken down and hydrolyzed to monosaccharides composition. One possible implication of this is the separation of the sugar units of the polysaccharide as its large molecular structure, and later the determination of the monosaccharides which can be done with analytical column. However, another issue has to be considered. GLP20 was the product of the precipitation by ethanol. In this study, open column for further purification of GLP20 was not used, despite the fact that references were cited in the section of results on the purification by columns such as DEAE cellulose chromatography and Sephadex series size exclusion chromatography. The authors commented that this process was tedious and the yield of purified polysaccharides was low. The present studies yielded 0.37% (w/w) GLP20 on the basis of the dry weight of fruiting bodies. The mean 5.55 g of GLP20 was obtained by using 1500 g fruiting body of
The present study was over-reliance on self-initiated methodology. The advantages of using a series of chromatography for isolation at the final stage of fractionation are for the purpose of maximizing the purification. The use of 20% ethanol precipitation was supposed to precipitate all substrates with ▬OH group. Even at the higher % of ethanol applied, there are increasing precipitation of substrates including polysaccharides.
The degree of purification of GLP20 was uncertainly proven. Thus the content of monosaccharides reported later did not appear to support the assumption of single polysaccharide obtained from this extraction, since the process of hydrolysis was not specific to determine the bonding of each sugar component. Generally, hydrolysis or saccharification is a step in the degradation of a polysaccharide substance. The cleavage of H▬O bond reaction of cation and anion or both with water molecule takes place due to pH conditions . It is possible that these reports were influenced by lack of information on glycosidic linkages. The final analysis step included total protein test.
3.5. Extraction and fractionation of mushroom polysaccharides from the mycelia and fruiting body (dried of
G. lucidumand Poria cocosusing soxhlet extractor and anthrone sulfate)
Two types of mushroom have been used  (please refer to Figure 8). They were
This soxhlet extraction method is one of the practical ways to exclude types of carbohydrate from the mushroom. Therefore, the polysaccharides mentioned in the chapter would refer to various forms of carbohydrates which included polysaccharides as one of them. The result and conclusion has positively quoted the significance of said production of polysaccharides potentially acting as prebiotics by manipulating gut microbiota composition. It was decided that the best method to adopt for this investigation was to provide a platform for beneficial bacteria associated with polysaccharides treatment, making these polysaccharides candidate prebiotics. However, there was a lack of result specifically on types of polysaccharides from both basidiomycetes.
3.6. Extraction and fractionation of three kinds of
G. lucidumpolysaccharides from the fermentation broth, mycelia, fruiting body using open column with resin DEAE-Sephadex A-25 and Sepharose 6B
Three different life stages of
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
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 , 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.
In this stage of fractionation, Sephadex A-25 was used instead of A-50 and followed by another stage of fractionation with Sepharose 6B. Hence, most of the molecules more than 30,000 MW could pass through. In order to completely isolate the polysaccharides, the main fraction of each stage of
The excellent results presented in this study illustrated the effective methodology for purification of polysaccharides from
4. Fractionation and its application in the extraction of polysaccharides from the mushroom species,
The discovery of new polysaccharides from
The method of
The authors thank the Director General of Health, Malaysia for the permission to publish and the Director of Institute for Medical Research (IMR), Kuala Lumpur for the support. This work was financially supported by the Ministry of Health Research Grant Scheme (Grant ID: NMRR-11-953-10736). The authors also thank Prof. Vikineswary (University of Malaya), Dr. Wan Abd Al Qadr Imad Wan mothar (UM) and Dr. Rahayu Ahmad (MIP) for critically reviewing this chapter.