CHAPTER 3

MATERIALS AND METHODS

3.1

Strain
The culture of Ganoderma lucidum was obtained from Dr.
Mohd Nor Bin Abdul Wahab, the professor from the Department of
Biochemistry

and

Microbiology,

Faculty

Biotechnology

and

Biomoleculear Science, University Putra Malaysia. Then, the culture

of Ganoderma lucidum had been sub-cultured into the Potato
Dextrose Agar (PDA), Semi Ganoderma Complete Medium (SGCM)
and Meat Extract Agar (MEA) and was kept at 24C before
proceeding into the next step of the project.
3.2

Media Agar Preparation

In order to determine the best media for the mycelial growth
of Ganoderma lucidum, three types of media agar was used which
were PDA, SGCM and MEA media. In preparing the PDA media,
19.5g of PDA powder was weighed and put in the 1L autoclave
bottle. Then, 500ml of distilled water was added and the media
solution was stirred and heated until all the PDA powder was fully
dissolved. After that, the media was autoclaved at 121

for 15

minutes. The media was cooled first before being poured into the
agar plate. The media must be poured into the agar plate
aseptically quickly in order to avoid the media from becoming
solidified. Then, the prepared media agar was kept in 4 and
could be used for the mycelial growth of Ganoderma lucidum.
For the SGCM media, the preparation of the media was quite
complicated compared to PDA and MEA. The media was consisted
of 20g of glucose, 2.5g of peptone, 2.5g of yeast extract, 0.5g of
KH2PO4, 0.5g K2HPO4, 0.2 MgSO4.7H2O and 9g of agar powder that
had been fully dissolved in the 500ml of distilled water by stirring
and heating the mixture. Then, the next steps of autoclaving,
cooling and storing the media were just similar like the preparation
of the PDA media.
The preparation of MEA media is almost similar with PDA and
SGCM media but only differed in term of the media composition.
This media were made from the mixture of 12.75g of maltose,
2.25g of dextrose, 2.35g of glycerol, 0.78g of peptone and 15g of
agar powder that had been dissolved in 1000ml of distilled water.
The next step was just as same as the preparation step of PDA and
SGCM agar.
3.3

Growth of Ganoderma lucidum on Media Agar

The strain was grown on the three different agar media which
were PDA, MEA and SGCM media. The growth of the strain was
determined by measuring the area of the mycelial growth. The
measurement of the mycelia had been taken every day until the
mycelia had fully covered the plate. The graph of area of the
mycelial growth versus time (day) was plotted. Based on the graph,
the growth rate of the Ganoderma lucidum was determined by
using this calculation: Growth rate of Ganoderma lucidum= (Area of
mycelia that fully cover the plate) / (Time needed by mycelia to
fully cover the plate)

3.4

Inoculum Preparation of Ganoderma lucidum

Inoculum of Ganoderma lucidum was prepared by growing
Ganoderma lucidum culture on a rotary shaker at 150 rpm and 26
C in 250-ml flasks containing 100 ml of following standard medium
which consisted glucose, 10.0 gl-1; NH4NO3, 1.0 gl-1; KH2PO4, 0.8 g l-1;
K2HPO4, 0.8 g l-1; Peptone, 2.5 g l-1 Na2HPO4, 0.2 g l-1; MgSO47H2O,
0.5 g l-1 and yeast extract, 2.0 g l-1. The standard medium was
prepared by dissolving all the materials above in 1000ml of distilled
water. Then, the medium was autoclaved at 121 , 15psi for 15
minutes due to sterilization purpose. The medium then was
adjusted to pH 5.5-6.0 with 2 M NaOH. The inoculums then were
used for the submerged fermentation (SF) of the Ganoderma
lucidum.

3.5

Cultivation Conditions of Ganoderma lucidum

For the production of cell biomass and polysaccharides of

Ganoderma

lucidum,

the

submerged

fermentation

(SF)

of

Ganoderma lucidum was carried out in the above-mentioned

medium with same cultivation condition. However, there are 3
different types of carbon source that had been used for the

submerged

fermentation

for

comparison

glucose, sucrose and lactose.

purpose

which

are

By using the same media and

conditions, the effects of substrates on the cellulase enzyme

activity of Ganoderma lucidum was analysed by using various types
of substrates instead of glucose as a carbon source for the
submerged cultivation of Ganoderma lucidum.
3.6

Estimation of Mycelial Growth

The mycelial growth of Ganoderma lucidum was determined

by measuring the dry cell weight of mycelia at different time

interval which is at 10th day, 20th day and 30th day. In order to
determine the dry cell weight of mycelia, the fermentation culture
was filtered by using Sinta Glass Filter No 1 and the mycelia were
separated from the fermentation culture filtrate. Then, the mycelia
were dried at 60C for sufficient time until constant dry weight of
mycelia was achieved. The graph of dry cell weight of mycelia
versus cultivation time was plotted in order to see the mycelial
growth of Ganoderma lucidum.

3.7

Polysaccharides Analysis
For this analysis, two types of polysaccharides were analysed,

extracellular

polysaccharides

(EPS)

and

intracellular

polysaccharides (IPS). As to the determination of Ganoderma

lucidum

polysaccharides,

the

EPS

was

extracted

from

the

fermentation culture filtrate of Ganoderma lucidum while the IPS

was extracted from the mycelia of the Ganoderma lucidum. The
method used for the EPS and IPS extraction is a modified method of
Kim et al. (2005). The total polysaccharides of the EPS and IPS then
were measured by using the standard of Phenol Sulphuric Method.

3.71

Extracellular Polysaccharides Extraction (EPS)

The EPS of Ganoderma lucidum was extracted from the

fermentation culture filtrate of Ganoderma lucidum at different time
interval. The crude EPS was extracted at 10 th day, 20th day and 30th
day in order to see the production of EPS during the mycelial
growth.

For the EPS extraction, the crude EPS was mixed with four

volumes of absolute ethanol, stirred vigorously before being kept at

4C for overnight for EPS precipitation. Then, the precipitated EPS
was centrifuged at 3000 x g for 20 minutes. The supernatant was
removed and the pellet which is the EPS was dissolved in 1M NaOH
and centrifuged again.

3.72

Intracellular Polysaccharides Extraction (IPS)

For the analysis of IPS, the mycelia were separated from the
fermentation culture by using Sinta Glass Filter No. 1. Then, the
mycelia were dried at 60C for sufficient time until constant dry
weight of mycelia was achieved. To extract the IPS, the dried
mycelia was washed with 80% ethanol and submerged in the
distilled water. After that, the samples were autoclaved at 121C for
1 hour and cooled down before being centrifuged at 2400 x g for 5
minutes in order to separate supernatant from the pellet.

3.73

Total Polysaccharides Determination

For the analysis, the total polysaccharides were measured by

using standard method of Phenol Sulphuric Analysis.

The Phenol

Sulphuric method is an example of a colorimetric method that is

widely

used

to

determine

the

total

concentration

of

polysaccharides. In this analysis, all non-reducing sugars were

converted to reducing sugars by the sulphuric acid, so that this
method would determine the total polysaccharides present.

3.8

Cellulase Enzyme Activity Analysis

3.81

Crude Enzyme Extraction

For this study, the crude enzyme was extracted from

the fermentation culture filtrate of Ganoderma lucidum that

made of different types of substrates. About 1.5ml of
fermentation culture filtrate was taken from the flask at
different time interval in order to study the enzymatic activity
of cellulase enzyme which is Filter Paper-ase (Fpase),
Carboxymethylcellulase (CMCase) and -Glucosidase.

The

culture filtrate was collected for each 10 days starting from

0th day. Then, the collected filtrate was centrifuged at 13000
rpm for 10 minutes at room temperature. The pellet was
removed and supernatant was kept at 4C before being used

in further enzymatic analysis. The crude enzyme must be

kept at low temperature in order to avoid the reduction of
enzyme activity as well as avoiding enzyme degradation.
3.82
Filter paper-ase (Fpase) Assay
For this analysis, the method that was used is DNS method
that was commonly used to assay for the reducing end products.
Reactions were carried out by mixing and incubating the enzyme
preparation with a known amount of substrate. This enzyme was
assayed by measuring the rate at which they produce reducing
sugars from their respective substrates. A Whatman #1 filter paper
with 1x 3cm size was put in the test tube and then, 1.8ml of citrate
buffer and 0.2ml of crude enzyme solution was added. The test
tube was incubated in water bath at 40C for 60 minutes in order to
allow the reaction of the enzyme and substrates. Then, 3ml DNS
reagent was added and the mixture was vortex before being boiled
in the water bath for 15 minutes. After boiling, 1ml of Rochelle salt
was added into the tube. Then, the test tube was vortex and the
absorbance reading of the solution was read at 575nm to
determine the activity of the FPase enzyme.

3.82 Carboxymethylcellulase (CMCase) Assay

For the CMCase enzyme assay, the method used was quite
similar with the FPAse enzyme assay which is DNS method. The

only different is in term of the substrate used. The reactions were

carried out by mixing and incubating the enzyme solution with the
CMC (substrate). This enzyme was assayed by measuring the rate
at which they produce reducing sugars from their respective
substrates. During the analysis, 1.8ml of CMC buffer and 0.2ml of
crude enzyme solution was added into the test tube. The test tube
was incubated in water bath at 40C for 30 minutes in order to
allow the reaction of the enzyme and substrates. Then, 3ml DNS
reagent was added and the mixture was vortex before being boiled
in the water bath for 15 minutes. After boiling, 1ml of Rochelle salt
was added into the tube. Then, the test tube was vortex and the
absorbance reading of the solution was read at 575nm to
determine the activity of CMCase enzyme.

3.83

-glucosidase Assay

For the analysis of -glucosidaseassay, the method that was

used is quite simpler compared to FPase and CMCase enzyme
assay. The analysis was carried out by mixing and incubating the
enzyme solution with the P-nitrophenyl- -glucopyrosidase (pNPG)
which used as a substrate. This enzyme was assayed by measuring
the rate at which they produce p-nitrophenols from their respective
substrates. During the analysis, 1.8ml of pNPG and 0.2ml of crude
enzyme solution was added into the test tube. After that, the test

tube was incubated in water bath at 40C for 30 minutes in order to

allow the reaction of the enzyme and substrates.

Then, 2ml of

Sodium Carbonate (Na2CO3) was added into the test tube and the
mixture was vortex to homogenize it. The function of Sodium
Carbonate is to stop the reaction of the enzyme. Then, the
absorbance reading of the solution was read at 400nm to
determine the existence of the -glucosidaseenzyme in the sample.
The absorbance reading data was used to calculate the enzymatic
activity of -glucosidasein the Ganoderma lucidum culture.