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CHAPTER 2

LITERATURE REVIEW

2.1

General characteristics of Ganoderma Lucidum


Ganoderma lucidum, the most famous species in the group of
ganoderma is a legendary mushroom in China with a long
fascinating history since two thousand years ago. Not only it is a
sparkling beautiful woody mushroom, but more importantly,
Ganoderma lucidum is known as the mushroom of immortality and
is the number one medicinal mushroom in China. It is also known as
"A Special One Medicine" because no side effects occurred even it
have been used for a long time plus it can improve one's health,
raising its body heal and prolong the life of a person as well. In fact,
Dr. B.K. Kim who is a world leader in research on Ganoderma in
Korea showed that Ganoderma lucidum has also had an anti-AIDS
property (Kim, Shim, Choi and Kim,1997).
Had been introduced into culture more than 2000 year ago,
Ganoderma lucidum is widely used in oriental folk medicine for
treating various diseases. In the past few years, this fungus has
been

actively

studied

as

producer

of

biologically

active

compounds.

Individual

compounds

isolated

from

Ganoderma

lucidum exhibit hypolipidemic and bactericidal activities, stimulate


immunity,

and

are

effective

in

treating

diseases

of

the

cardiovascular and nervous systems (Stamets, 2000). Remarkably,


these include very important diseases such as hypertension,
diabetes, hepatitis, cancers and HIV (Paterson, 2006). It is believed
that therapeutic properties of this fungus are determined by its
unique

chemical

composition.

It

contains

proteins,

lipids,

polysaccharides, phenolic compounds, steroids, terpenoids, and


microelements including germanium, which ensures intensive
oxygen consumption by cells (Bao et al., 2002). Polysaccharides (glucans) constitute the main active component of this complex.
Instead of its medicinal value, a lignocellulose-degrading
fungus such as Ganoderma lucidum is well known as enzymes
producer. It produces cellulases, hemicellulases, pectinases and
ligninases for plant cell wall hydrolysis. These enzymes are
increasingly employed in the bioprocessing of plant materials for
fuel, feed and chemical feedstocks. They are also employed in
agriculture as silage additives, feed additives and retting additives.
The textile and paper industries also use such enzymes. The high
demand

for

these

enzymes

has

intensified

the

search

for

microorganisms producing high levels of enzyme activity and for


improved fermentation processes for their production.

2.2

Cultivation of Ganoderma lucidum


Generally, the cultivation of Ganoderma lucidum can be done
in both solid-state cultivation and submerged cultivation. The solidstate

cultivation

of

Ganoderma

lucidum

on

lignocelluloses

substrates is commonly used to obtain the fruiting bodies of this


mushroom. However, it usually takes several months to cultivate
the fruiting body of Ganoderma lucidum and it is also difficult to
control the product quality (Tang and Zhong, 2002). Thus,
submerged cultivation is more preferable rather than solid-state
cultivation when the nutritive requirements of a mushroom are to
be studied since it provides a better growth of the mushroom and
allows the addition of many substrates without worsening the mass
transfer conditions in the culture.
In

recent

years,

submerged

cultivation

of

Ganoderma

lucidum has been developed to obtain mycelial biomass, ganoderic


acid and polysaccharides which can be used to produce medicinal
products (Yang and Liau, 1998). Recently, the major concerns were
to find environmentally good and economically feasible compounds
that stimulate the acceleration of mycelial growth and metabolite
production of Ganoderma lucidum (Yang et al., 2000). Therefore,
the submerged culture of Ganoderma lucidum has received great
attention as a promising alternative for efficient production of its
valuable metabolites, especially polysaccharide (Lee, 1999a; Yang

and Liau, 1998) and ganoderic acid. Plus, submerged cultivation is


widely used in order to determine the cellulase activity of
Ganoderma lucidum since it was a lignocellulose-degrading fungi.

2.3

Temperature and pH Effects on Ganoderma lucidum Growth


The temperature and pH is one of the most important factors
in order to ensure that the Ganoderma lucidum culture could grow
well. The ideal temperature and pH for the growth of Ganoderma
lucidum is 26

and 6.0-6.5 respectively. Unsuitable temperature

and pH may affect cell membrane function, cell morphology and


structure, the ionic state of substrates, the uptake of various
nutrients, and product biosynthesis (Yang and Liau, 1998). In
general, cells can only grow within a certain temperature and pH
range,

and

metabolite

formation

is

also

often

affected

by

temperature and pH.


2.4

Ganoderma

lucidum

Active

and

Pharmacological

Components
Lin Zhi-Bin and his group embarked on separate studies to
prove the efficacy of this famous herb. Their work has resulted not
only in the improvement of its cultivation and extraction method
but more importantly it surfaced the science behind this wonder

herb. They had identified the major bio-active components of the


Ganoderma lucidum and its specific action to its revered use. In
general, individual compounds isolated from Ganoderma lucidum
exhibit

hypolipidemic

immunity,

and

are

and

bactericidal

effective

in

activities,

treating

diseases

cardiovascular and nervous systems (Stamets, 2000).

stimulate
of

the

2.5

Polysaccharides of Ganoderma lucidum


Polysaccharides represent a structurally diverse class of
biological macromolecules with a wide-range of physicochemical
properties.

The

major

bioactive

Ganoderma

lucidum

polysaccharides species are -1-3 and -1-6-D glucans. As fungal


wall constituent, bioactive polyglycan (-glucans) in Ganoderma
lucidum are found in all parts of mushroom, including the
mycelium. Fungal polysaccharides can also be secreted into the
growth media and become extracellular polysaccharides. Bioactive
polyglycans in Ganoderma lucidum include neutral polysaccharides
(-1-3, -1-6 homo D-glucan), acidic glucan and polyglycan,
protein-bond heteroglucan, arabinoxyoglucan, a highly branched
heteroglucan, a heteroglycan with -1-4 core and peptidoglycan,
ganoderans A, B and C (in the fruiting body), -D-glucan and
Lucidan which is a protein-bond heteroglycan, as well as other
polyglycans in the mycelium which have not been characterized
(Mizuno, 1992).
In recent studies, bioactive polysaccharides were isolated
from the basidiocarps (Bao et al., 2002; Zhang and Lin, 2004) and
from the mycelial biomass that cultivated in liquid culture (Kim et
al., 1993).In fact, there are few bioactive polysaccharides were
isolated from the culture medium of Ganoderma lucidum culture

(Kim

et al., 2003). Statistically, there are more than 200

polysaccharides have been isolated from the fruiting bodies,


spores, mycelia and cultivation broth Ganoderma lucidum (Huie
and Di, 2004).

Polysaccharides of Ganoderma lucidum are believed to exert


anti-tumour activities through an enhancement of host-mediated
immunity rather than a direct cytotoxicity to the tumour cells. They
have high molecular weights as a common feature which tends to
increase water solubility and result in more effective anti-tumour
activity. However, some water insoluble polysaccharides also
possess anti-tumour activity (Wang et al., 1993) and polysaccharide
branching affects activity. In fact, it should be noted that the
amount of bioactive water-insoluble polysaccharides was found to
be higher than that of water-soluble polysaccharides.
The antitumor activity of polysaccharides (-D-glucan) that
isolated from Ganoderma lucidum is more effective than those
produced by other mushrooms because Ganoderma lucidum
polysaccharides

are

better

absorbed

orally

(Chang,

1995).

Experimentally, when the edible mushroom, such as Lentinula


edodus were fed to laboratory animals, tumor regression could be
induced at a significant level only when they were supplemented as
20% - 30% of the diet. In contrast, only 5% (w/w) powdered
Ganoderma lucidum dry fruiting body as a diet supplement
produced a significant retardation in the formation and growth
micro adenomas in colon.

2.6

Cellulase Activity of Ganoderma lucidum

2.61

Cellulase Enzyme

Cellulolytic enzymes or cellulases are generally induced as


multienzyme systems, composed of three or more different
enzymes. It produced by microorganisms such as plant pathogens
and fuction to degrade (hydrolyse) the plant polysaccharide which
is called as cellulose. All cellulases cleave the same chemical bond
which is the -l, 4-glycosidic bond, but there is variation in the
microenvironment of these bonds in natural substrates.
Cellulase enzymes have been traditionally divided into three
classes,

C1

(endoglucanases

(cellobiohydrolases)

and

&

exoglucanases),

-glucosidases

(cellobioses)

CX
which

combined as cellulase complex (Sadhu et al., 2011). The vital


characteristics of this cellulase complex are

the system is

multienzymatic, it contains at least three enzyme components


which are both physically and chemically distinct, and all those
three components must play essential roles in the hydrolysis of
cellulose to glucose. At least two steps in cellulose degradation by
microorganisms begin with the preparatory prehydrolytic step that
involving

an

enzyme

(C1)

which

swells

and/or

hydrate

anhydroglucose chains. While the second step uses hydrolytic


enzymes (Cx) and beta glucosidase (cellobiase).

Recently, there are several types of fungi have been


extensively studied about the cellulase enzyme complex including
Ganoderma lucidum. This complex converts crystalline, amorphous,
and chemically derived celluloses quantitatively to glucose. This
complex converts cellulose to beta-dextrins and ultimately to Dglucose. It is widely accepted that cellulases secreted by fungi
consist of three major components which are endoglucanases,
(EGs) cellobiohydrolases (exocellulases, CBHs) and -glucosidases
(Zhou, Chen and Li, 2004). The fungal cellulases are available in
large amounts and do not seem to form physical complexes with
each other, but they act in strong synergy. For this reason the
purity of the enzymes preparations is of utmost importance for the
elucidation of their enzymatic activities, since even traces of
another type of activity can seriously bias the results.

2.62

Cellulase Assay

The whole cellulase activity is often represented by FPA


including synergistic actions of the three parts (Zhou, Chen and Li,
2004).

Normally,

represented

by

single

endoglucanase

carboxymethyl

cellulase

activity
(CMCase)

is

often

activity.

Endoglucanase (CMCase) attacks randomly in the interior of the


cellulose structure and not very active against crystalline cellulose,

but they are capable of hydrolyzing substituted cellulases such as


carboxymethylcellulose

(CMC)

to

produce

cellulodextrins.

Its

activity can be assayed with a water-soluble cellulose derivate,


such as carboxymethylcellulose (CMC) or phosphoric acid-swollen
cellulose.
Besides that, exocellulase (FPase) activity can only be
estimated by measuring the release of glucose from crystalline
cellulose such as Avicel, filter paper or cotton by using the
International Union of pure and Applied Chemists (IUPAC) standard
filter paper assay (FPA). This assay uses the dinitrosalicylic acid
(DNS) method to determine reducing sugars released from 50 mg
of Whatman #1 filter paper by a complex cellulase mixture.
Generally, -glucosidase activity can also be assayed by
using DNS method since it completes the hydrolysis by converting
cellobiose and cellooligosaccharides into glucose (Parry et al.,
2001). In fact, -glucosidase is also considered to be part of a
cellulase system, since it stimulates the rate and extent of cellulose
hydrolysis by relieving cellobiose-induced inhibition of endo- and
exo-cellulases (Bhat, Gaikwad and Maheshwari, 1993). However, in
some cases, -glucosidase activity was assayed by measuring the
rate of hydrolysing P-nitrophenyl- -glucopyrosidase (pNPG) into Pnitrophenol.