**3. Review of available** *G. lucidum* **polysaccharides 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 [23]. 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 [24]. 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 watersoluble 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 [20], acidic precipitation with acetic acid [21], ion-exchange chromatography [22], gel filtration [23] and affinity chromatography [24].

with a molecular weight of 584,900 Da was isolated from a hot water extract of wood-cultured *G. lucidum* by Cao *et al.* [13]. It was found to contain more than 40% β-D-glucan. Another report by You *et al.* showed a type of polysaccharide peptide extracted from the same species with an average molecular weight of 513,000 Da. It contains 16 kinds of amino acids [14]. Most of the polysaccharides extracted and bonded with protein had high molecular weight and played a specific function related to immune stimulation system. It was reported by Giavasis [15] and Ho *et al.* [16] that bioactive polysaccharides which are higher in molecular weight have been shown to exhibit significant antitumor properties, immunomodulatory activity, antioxidant activity, and neuroprotection. A series of fractionation has been conducted with the purpose of studying this little known fungus using advanced technology. A pure *G. lucidum* polysaccharide labeled as GLP-1-1 was isolated from a culture broth with molecular weight of 22,014 Da [17]. Its monosaccharide content contained glucose, mannose, and galactose with molar percentages of 92.33, 7.55, and 0.22%, respectively. A water-soluble polysaccharide [18] extracted from the spores of *G. lucidum*, subsequently went through water extraction and sequential alcohol precipitating method and purified by anion-exchange and gel filtration chromatography. It was eluted as a single and symmetrical sharp peak corresponding to an average molecular weight of 8000 Da as determined by high performance gel permeation chromatography. Sugar compositional analysis showed that it is only composed of D-glucose. Wang *et al.* [19] used a native *G. lucidum* polysaccharide in a linear, water-insoluble β-D-(1→3)-glucan to prepare sulfated polysaccharides. The molecular weight of *G. lucidum* polysaccharide was 133,000 Da. In addition, another water-soluble neutral polysaccharide was isolated from the fruiting bodies of *G. lucidum* by DEAE Sepharose Fast Flow and Sephacryl S-500 High Resolution Chromatography [20]. GC analysis showed that this polysaccharide was mainly composed of glucose and galactose in the molar ratio of 34:1 and its average molecular weight was approximately 2,500,000 Da. Contradictorily, there are two lowmolecular weight of glucan purified from a crude *G. lucidum* polysaccharide preparation [21]. Their physiochemical properties marked a glucan with 5200 Da and another glucan with molecular weight 15,400 Da. The later was composed of glucose, galactose and mannose in a ratio of 29:1.8:1.0. This wide range of molecular weight of *G. lucidum* polysaccharides apparently clearly

indicated that they are still abundant polysaccharides of *G. lucidum* yet to be discovered.

β-l→3 and side chains with β-1→6 linkages and a D-galactopyranosyl β-1→6 linkage.

**3. Review of available** *G. lucidum* **polysaccharides extraction and** 

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

**fractionation methodology**

42 Fractionation

The molecular structure of *G. lucidum* polysaccharides which is associated closely with glycosidase linkage is another factor vitally influencing the bioactivities. The types of monosaccharide used to be the backbone play an important role as skeleton to support the other types monosaccharide as branches. Actually, it is a complex polymer upon which the different types of monosaccharides were involved plus the different position of carbon binding. For example, two peptidoglycans named Ganoderan B and C isolated from the fruiting body of *G. lucidum*, were shown to exhibit hypoglycemic efficacy with molecular weight 7400 and 5800 Da [22]. Chemical and physicochemical studies demonstrated that the backbone of ganoderan B contain D-glucopyranosyl β-1→3 and side chains with β-1→6 linkages. The backbone of Ganoderan C contains glucopyranosyl Comparing to the ancient era, definitely today's new technology is enhancing the yields of the extraction. A lot of new polysaccharides of *G. lucidum* have been discovered and reported to possess certain level of therapeutic efficacy [25, 26]. However, the method of polysaccharides extractions of this mushroom including the fractionation performed by the researchers are not standardized. Numerous methods resulted in the extraction of incorrect polysaccharides especially when using the hot water extraction method. The objective of this review paper is to discuss several polysaccharides extraction methods and verify these methods which include fractionation on the selected mushroom, *G. lucidum*.

#### **3.1. Extraction and fractionation of a novel water-soluble β-D-glucan from the spores of** *G. lucidum* **using ethanol precipitation**

Analysis was based on the conceptual framework proposed in five papers authored by Bao *et al.* from 2000 to 2002 [27] (please refer to **Figure 4**). The subject model was the spore of *G. lucidum* which was sporoderm-broken. With reference to the paper by Bao *et al.* [28], a novel water-soluble β-D-glucan was discovered after 10 years of study. Most of the steps of the extraction and fractionation were similar except that several procedures were modified in order to obtain better results. This illustrated that older methods could still work well and these methods could give rise to more effective methods that could lead to new discoveries.

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 [29]. 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

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For this study, 15% of trichloroacetic acid (TCA) was used to procure protein precipitation reaction [30]. 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 *et al.* [28] reported using 3 days of running water followed by 1 day of distilled water to remove components with molecular weight of 3000 Da. Later the concentrated retentate was stirred slowly and 95% ethanol was added at low temperature to promote polysaccharides precipitation. After centrifugation the precipitate was washed with two solvents before being dried in vacuum. Compared to previous study [28], the precipitate obtained was dissolved in water at room temperature, 0.5 L of ethanol was added and another round of centrifugation was conducted. The resulting brownish crude product was the key component used for further fractionation. The packed column with DEAE-cellulose (diethylaminoethyl) was used in anion-exchange chromatography. It achieves separation by using positively charged ion exchange matrix with an affinity for molecules having net negative surface charges [31]. The elution by using water and various affinity salts would be adequate to dissolve the polysaccharide components which are retained in the matrix. The fractions collected in tube were colorless, and the best determination method was the phenol-sulfuric acid method [32], since the yellowish end product could be used for the calibration graph based on absorbance at 490 nm versus number of fractions. The peak determined from the graph could be used to determine combination of fractions for further purification. Thus sephacyl-S-200HR [33] is used as size exclusion ordinal isolating each different polysaccharides based on their molecular weight. In this case, the compound labeled as GLSA50-1B was chosen for the

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 proteinprecipitation, 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

processing.

characterization as a novel β-D-glucan.

from mushroom spores.

**Figure 4.** Flowchart of extraction, fractionation and isolation methodology of polysaccharides from *Ganoderma lucidum* spores.

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 [30]. 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 *et al.* [28] reported using 3 days of running water followed by 1 day of distilled water to remove components with molecular weight of 3000 Da. Later the concentrated retentate was stirred slowly and 95% ethanol was added at low temperature to promote polysaccharides precipitation. After centrifugation the precipitate was washed with two solvents before being dried in vacuum. Compared to previous study [28], the precipitate obtained was dissolved in water at room temperature, 0.5 L of ethanol was added and another round of centrifugation was conducted. The resulting brownish crude product was the key component used for further fractionation. The packed column with DEAE-cellulose (diethylaminoethyl) was used in anion-exchange chromatography. It achieves separation by using positively charged ion exchange matrix with an affinity for molecules having net negative surface charges [31]. The elution by using water and various affinity salts would be adequate to dissolve the polysaccharide components which are retained in the matrix. The fractions collected in tube were colorless, and the best determination method was the phenol-sulfuric acid method [32], since the yellowish end product could be used for the calibration graph based on absorbance at 490 nm versus number of fractions. The peak determined from the graph could be used to determine combination of fractions for further purification. Thus sephacyl-S-200HR [33] is used as size exclusion ordinal isolating each different polysaccharides based on their molecular weight. In this case, the compound labeled as GLSA50-1B was chosen for the characterization as a novel β-D-glucan.

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 proteinprecipitation, 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.

**Figure 4.** Flowchart of extraction, fractionation and isolation methodology of polysaccharides from *Ganoderma lucidum*

spores.

44 Fractionation

### **3.2. Extraction and fractionation of** *G. lucidum* **polysaccharide from the mycelia of**  *G. lucidum* **using 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 [34] (please refer to **Figure 5**). The methodology described seems short and insufficient to determine the major characteristics of polysaccharides. To date, studies investigating *G. lucidum* polysaccharides have produced equivocal results that contributed to misunderstanding of polysaccharides. Firstly, when the extraction steps were carried out using hot water extraction and terminated after precipitation with 95% ethanol, most of the claimed polysaccharides (labeled as GLP) comprised total carbohydrates. In addition, majority of the polar compounds with plenty of –OH groups could possibly have been evacuated from the matrix. The experiment was unable to encompass the entire full scope of detailed molecular level of specific polysaccharides. Secondly the composition of GLP included alduronic acid (19.27%) and protein (5.39%). This scenario directly indicated the GLP content was not purely polysaccharides. The objectives of this study focused on efficacy of GLP treatment in plasma insulin concentration and gut microbiota composition in mice. There is notable paucity of empirical research focusing especially on polysaccharide.

The extraction started with pulverized *G. lucidum* mycelia with water. The process was repeated twice and the mixture was agitated for 4 h at 70–80°C. Comparing the common hot water extraction which provided 100°C of boiling water, it can be seen that the reduced temperature used was to avoid the formation of bubbles. The agitation was accomplished by continued shaking for 4 h. These mechanical activities are adjustable to ensure the extraction would be maximum. However, there is no mention on the speed of shaking in the experiment and the condition of the mycelia.

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. lucidum* **polysaccharides F31 from the fruiting body of** *G. lucidum* **using absolute ethanol at 4°C**

Comparing with the common hot water extraction, this study applied 80°C instead of 100°C water [35] (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

**Figure 5.** Flowchart of extraction, fractionation methodology of polysaccharides from *Ganoderma lucidum* mycelia.

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Extraction and Fractionation of Polysaccharides from a Selected Mushroom Species... http://dx.doi.org/10.5772/intechopen.78047 47

**3.2. Extraction and fractionation of** *G. lucidum* **polysaccharide from the mycelia of** 

The topic has mentioned "polysaccharides" which implied many types of polysaccharides. and this was frequently attributed to hot water extraction of mushroom resources [34] (please refer to **Figure 5**). The methodology described seems short and insufficient to determine the major characteristics of polysaccharides. To date, studies investigating *G. lucidum* polysaccharides have produced equivocal results that contributed to misunderstanding of polysaccharides. Firstly, when the extraction steps were carried out using hot water extraction and terminated after precipitation with 95% ethanol, most of the claimed polysaccharides (labeled as GLP) comprised total carbohydrates. In addition, majority of the polar compounds with plenty of –OH groups could possibly have been evacuated from the matrix. The experiment was unable to encompass the entire full scope of detailed molecular level of specific polysaccharides. Secondly the composition of GLP included alduronic acid (19.27%) and protein (5.39%). This scenario directly indicated the GLP content was not purely polysaccharides. The objectives of this study focused on efficacy of GLP treatment in plasma insulin concentration and gut microbiota composition in mice. There is notable paucity of empirical research focus-

The extraction started with pulverized *G. lucidum* mycelia with water. The process was repeated twice and the mixture was agitated for 4 h at 70–80°C. Comparing the common hot water extraction which provided 100°C of boiling water, it can be seen that the reduced temperature used was to avoid the formation of bubbles. The agitation was accomplished by continued shaking for 4 h. These mechanical activities are adjustable to ensure the extraction would be maximum. However, there is no mention on the speed of shaking in the experiment

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.

Comparing with the common hot water extraction, this study applied 80°C instead of 100°C water [35] (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

**3.3. Extraction and fractionation of** *G. lucidum* **polysaccharides F31 from the fruiting** 

*G. lucidum* **using agitation and filtration**

46 Fractionation

ing especially on polysaccharide.

and the condition of the mycelia.

**body of** *G. lucidum* **using absolute ethanol at 4°C**

**Figure 5.** Flowchart of extraction, fractionation methodology of polysaccharides from *Ganoderma lucidum* mycelia.

**3.4. Extraction and fractionation of high molecular weight bioactive-β-glucan from the fruiting body of** *G. lucidum* **using centrifugation and high performance anion** 

demonstrated a strong and consistent association with immune system of host.

needed 2 mg of GLP20, a total of 2775 times of analysis can be conducted.

increasing precipitation of substrates including polysaccharides.

Previous studies have reported that the fractionation for polysaccharides from mushroom sample normally started with hot water extraction [37] (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

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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

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 *G. lucidum*. If each batch of analysis

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

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 [40]. It is possible that these reports were influenced by lack of information on glycosidic linkages. The final analysis

**exchange chromatography**

larger sample of volume being used.

step included total protein test.

**Figure 6.** Flowchart of extraction, fractionation and isolation methodology of polysaccharides from *Ganoderma lucidum* fruiting body. The gel DEAE Sepharose Fast Flow and Sephacryl S-300 were the main tools to obtain the pure polysaccharides.

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 [36] 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 × 10<sup>6</sup> 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. lucidum* **using 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 [37] (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 *G. lucidum*. If each batch of analysis needed 2 mg of GLP20, a total of 2775 times of analysis can be conducted.

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.

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

**Figure 6.** Flowchart of extraction, fractionation and isolation methodology of polysaccharides from *Ganoderma lucidum* fruiting body. The gel DEAE Sepharose Fast Flow and Sephacryl S-300 were the main tools to obtain the pure polysaccharides.

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 [36] 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

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

to

determine of protein or peptide content.

polysaccharides for bioassay guided test.

1.5 × 10<sup>6</sup> Da.

48 Fractionation

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 [40]. 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. lucidum* **and** *Poria cocos* **using soxhlet extractor and** 

color complex which can be measured colorimetrically at 625 nm.

**DEAE-Sephadex A-25 and Sepharose 6B**

Two types of mushroom have been used [41] (please refer to **Figure 8**). They were *G. lucidum* and *Poria cocos*. The methodology used was similarly applied on both mushrooms. Sample mushrooms were in the form of mycelia and fruiting body, respectively. In other words, the method aimed to produce total polysaccharide as mentioned in the article and there was no specific technique to produce targeted polysaccharide from either mycelia or fruiting body. There was no indication whether the samples were in fresh or dried condition, and it was assumed that 2 g of dried powder sample plus 90 mL of distilled water processed in soxhlet extractor. The final concentrated extract after distillation for few hours was diluted with distilled water. This step is important because the final reading of absorbance at 625 nm is limited at 3. When 10% volume of diluted soxhlet extract was added with ethanol, it was possible that the micro-molecules that existed during the soxhlet heating were eliminated. Hence the next step of chilling at 4°C provided ample time for the suspension of the unwanted debris. The speed and duration of centrifugation were commonly applied to most of precipitate. After separation, the precipitate still needs to be diluted for determination of carbohydrate by the anthrone method. The principle of anthrone method is that dehydrated carbohydrates in various form included monosaccharides, disaccharides, starch, gums, glycosides, dextrins and definite polysaccharides during the process will condense with anthrone to form a green

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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. lucidum* **polysaccharides from the fermentation broth, mycelia, fruiting body using open column with resin** 

Three different life stages of *G. lucidum* were involved in this study [42] (please refer to **Figure 9**). Since it takes around 3 months for cultivation of *G. lucidum* fruiting body, the mycelia-based and broth-based products have assumed extraordinary importance because of the requirement for heightened quality-control and year-round production [43]. The processes and the different types of growth parameters including temperature and pH concerned in submerged mycelial medium, are simple to be standardized under controlled circumstances. In addition, the purification and other succeeding processing of active components such as polysaccharides excreted into the culture medium normally involve relatively simple procedures. The cultivation

**anthrone sulfate)**

**Figure 7.** Flowchart of extraction, fractionation and isolation methodology of polysaccharides from *Ganoderma lucidum* fruiting body.

### **3.5. Extraction and fractionation of mushroom polysaccharides from the mycelia and fruiting body (dried of** *G. lucidum* **and** *Poria cocos* **using soxhlet extractor and anthrone sulfate)**

Two types of mushroom have been used [41] (please refer to **Figure 8**). They were *G. lucidum* and *Poria cocos*. The methodology used was similarly applied on both mushrooms. Sample mushrooms were in the form of mycelia and fruiting body, respectively. In other words, the method aimed to produce total polysaccharide as mentioned in the article and there was no specific technique to produce targeted polysaccharide from either mycelia or fruiting body. There was no indication whether the samples were in fresh or dried condition, and it was assumed that 2 g of dried powder sample plus 90 mL of distilled water processed in soxhlet extractor. The final concentrated extract after distillation for few hours was diluted with distilled water. This step is important because the final reading of absorbance at 625 nm is limited at 3. When 10% volume of diluted soxhlet extract was added with ethanol, it was possible that the micro-molecules that existed during the soxhlet heating were eliminated. Hence the next step of chilling at 4°C provided ample time for the suspension of the unwanted debris. The speed and duration of centrifugation were commonly applied to most of precipitate. After separation, the precipitate still needs to be diluted for determination of carbohydrate by the anthrone method. The principle of anthrone method is that dehydrated carbohydrates in various form included monosaccharides, disaccharides, starch, gums, glycosides, dextrins and definite polysaccharides during the process will condense with anthrone to form a green color complex which can be measured colorimetrically at 625 nm.

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. lucidum* **polysaccharides from the fermentation broth, mycelia, fruiting body using open column with resin DEAE-Sephadex A-25 and Sepharose 6B**

**Figure 7.** Flowchart of extraction, fractionation and isolation methodology of polysaccharides from *Ganoderma lucidum*

fruiting body.

50 Fractionation

Three different life stages of *G. lucidum* were involved in this study [42] (please refer to **Figure 9**). Since it takes around 3 months for cultivation of *G. lucidum* fruiting body, the mycelia-based and broth-based products have assumed extraordinary importance because of the requirement for heightened quality-control and year-round production [43]. The processes and the different types of growth parameters including temperature and pH concerned in submerged mycelial medium, are simple to be standardized under controlled circumstances. In addition, the purification and other succeeding processing of active components such as polysaccharides excreted into the culture medium normally involve relatively simple procedures. The cultivation

**Figure 8.** Flowchart of extraction, fractionation methodology of polysaccharides from *Ganoderma lucidum* and *Poria cocos*, fruiting body and mycelia.

**Figure 9.** Flowchart of extraction, fractionation and isolation methodologies of polysaccharides from *Ganoderma lucidum*

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fermentation broth, mycelia and fruiting body.

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**Figure 9.** Flowchart of extraction, fractionation and isolation methodologies of polysaccharides from *Ganoderma lucidum* fermentation broth, mycelia and fruiting body.

**Figure 8.** Flowchart of extraction, fractionation methodology of polysaccharides from *Ganoderma lucidum* and *Poria cocos*,

fruiting body and mycelia.

52 Fractionation

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.

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

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The excellent results presented in this study illustrated the effective methodology for purification of polysaccharides from *G. lucidum* using three different life stages. The dissimilar way of pre-treating the sample in the beginning has potentially separated all the polysaccharides. The results clearly showed each main isolated polysaccharide contained different total sugar content, monosaccharides composition, molecular weight and the bioactivity. This study had successfully isolated the main polysaccharides from fermentation broth, mycelia and fruiting

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

mined 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

Da) isolated from the sporocarps of *G. lucidum* was used to deter-

each stage of *G. lucidum* was successfully eluted with 50 mM Tris-HCl buffer.

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

body of *G. lucidum*.

(molecular weight 1×10<sup>6</sup>

inhibiting effect on the cancer cell.

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.

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 *G. lucidum* was successfully eluted with 50 mM Tris-HCl buffer.

The excellent results presented in this study illustrated the effective methodology for purification of polysaccharides from *G. lucidum* using three different life stages. The dissimilar way of pre-treating the sample in the beginning has potentially separated all the polysaccharides. The results clearly showed each main isolated polysaccharide contained different total sugar content, monosaccharides composition, molecular weight and the bioactivity. This study had successfully isolated the main polysaccharides from fermentation broth, mycelia and fruiting body of *G. lucidum*.
