OPTIMATION OF GLUCOSE CONCENTRATION AND INCUBATION TIME ON GROWTH AND

SECONDARY METABOLITES PRODUCED BY Schizophyllum commune

RESEARCH PROPOSAL SEMINAR

YASMINE AURELLIA
B1B017019
Supervisor Co-supervisor
Dr. Nuraeni Ekowati, M.S Drs. Aris Mumpuni, M.Phil.
NIP 196111291986032002 NIP 196403291988031002

THE MINISTRY OF EDUCATION AND CULTURE

JENDERAL SOEDIRMAN UNIVERSITY
FACULTY OF BIOLOGY
PURWOKERTO
2021
 Background
Schizophyllum commune
Macroscopic medicinal mushroom from Basidiomycota phylum, Schizophyllaceae family, Agaricomycetes
class and Agaricales order. One of the most important microorganisms in the biotechnological industries
because of their biosynthetic capabilities of metabolites such as enzymes, polysaccharides, organic acids
and antibiotics.

Secondary Metabolites
Compounds that are synthesized by a microbe, not to fulfill their primary needs (to grow and develop)
but to maintain their existence in interacting with their environment.

Glucose
Important carbon source which is used as a source of energy which can influence the production of
fungal mycelium biomass and production of secondary metabolites of S. commune mushroom.
 Background (Con’t)
Incubation Time
The growth of fungal mycelia is also influenced by the length of incubation time. The longer the
incubation time, the number of fungal mycelia will increase to some extent, as well as the production of
secondary metabolites.

Liquid Medium
Has the potential for high mycelium biomass production and in a shorter time with less risk of
contamination.

Extraction Method
Secondary metabolites can be isolated from mycelium and culture filtrate using the extraction method.
Research Problems
● How do the different glucose concentrations
and incubation times affect the growth and
production of secondary metabolites of S.
commune.
Objectives
● What is the optimum glucose concentration ● To determine the effect of different glucose
and incubation time for the production of concentrations and incubation times on the
secondary metabolites of S. commune. growth and production of secondary
metabolites of S. commune.
● To determine the optimum glucose
concentration and incubation time for the
growth and production of secondary
metabolites of S. commune.
 Benefits
This research is expected to be useful to provide scientific information regarding glucose concentration
in the medium and optimal incubation time for growth and secondary metabolites produced by S.
commune mushrooms and can be a reference for further research on the utilization of secondary
metabolites of S. commune mushroom.

Hypothesis
1. A concentration of 30 g / L glucose in the medium and an incubation time of 30 days are optimal
for the growth of S. commune.
2. A concentration of 30 g / L glucose in the medium and an incubation time of 30 days are optimal
for the production of secondary metabolites from S. commune.
 Materials Tools●● Mortar
Pestle
● S. commune ● Laminar air flow ● Petri dish
isolates (LAF)
● Instant potato ● Glacial acetic acid ● Autoclave
dextrose agar (PDA) ● Methylated spirits ● Oven ● Test tube
● Distilled water ● Matches ● Water bath ● Loop needle
● Glucose ● Cotton ● Vacuum pump ● Spatula
● Malt extract ● Plastic wrap ● Hot plate ● Cork drill
● Peptone ● Filter paper ● pH meter ● Vial bottle
● Yeast extract ● 70% alcohol ● Sprayer ● Stationery
● KH 2 PO 4 ● TLC silica gel 60 F254 ● Beaker glass ● Camera
● MgSO 4 .7H 2O ● Dragendorff ● Erlenmeyer flask
● NaOH reagent ● Methylated
● HCl ● Sulfuric acid spirits
● Chloroform vanillin reagent ● Digital scales
● Ethyl acetate ● AlCl3 reagent ● Buchner funnel
● Methanol ● Tissue
● Hexane ● Heat resistant
● N-hexane plastics
● Label paper
● Aluminum foil
 Location and Time of Research
The research will conduct at Laboratory of Mycology and Phytopathology, Faculty of Biology, Jenderal
Soedirman University, from May until August 2021.
 Research Method
1. Experimental Design

This research will be carry out experimentally with a completely randomized design (CRD) with
factorial pattern consisting of 9 treatments with 3 replications. The treatment combinations to be
test are:

G1I1: glucose concentration 10 g / L, G2I1: glucose concentration 20 g / L, G3I1: glucose concentration 30 g / L,

incubation time of 20 days incubation time of 20 days incubation time of 20 days
G1I2: glucose concentration 10 g / L, G2I2: glucose concentration 20 g / L, G3I2: glucose concentration 30 g / L,
incubation time of 25 days incubation time of 25 days incubation time of 25 days
G1I3: glucose concentration 10 g / L, G2I3: glucose concentration 20 g / L, G3I3: glucose concentration 30 g / L,
incubation time of 30 days incubation time of 30 days incubation time of 30 days

1 22 3
2. Research Variables and Parameters

Variables
• Independent Variables: Glucose concentration and incubation time
• Dependent Variables: Production of secondary metabolites and growth of S. commune mycelium

Parameters
• Main Parameters: Types of secondary metabolites and dry weight of mycelium from S. commune
• Supporting Parameter: Final pH of the medium
3. Research Procedure

1) Sterilization Tools

Preparation of petri Autoclave all tools Sterilize ose needle

dishes, Erlenmeyer Wash all tools and with a temperature of using 70% alcohol
flasks, test tubes and allow to dry 121℃, 2 atm pressure and then burn above
cork drills for 20 minutes bunsen

2) Preparation Instant PDA

Sterilize using an
Put 39 g instant PDA Heat on a hot plate Pour the media into
autoclave with a
into 1 L distilled and homogenize an Erlenmeyer flask
temperature of 121℃, 2
water in a beaker using a magnetic and close using a
atm pressure for 15
glass stirrer cotton plug
minutes
3) Rejuvenation of S. commune
Inoculate on
Pour medium
Heat the PDA Take 1 plug of S. PDA medium
into the petri Wrap in wrapper
medium until it commune into petri dishes
dish until it is and incubate
melts culture at LAF
solid
aseptically

4) Preparation of Mushroom Complete Medium (MCM)

Dissolve 10 g, 20 g and 30 g Homogeniz The pH is measure Pour 100 Autoclave
glucose, 10 g malt extract , 2 g e with a hot to be 6. If it is too mL of sterilization
peptone , 2 g yeast extract , 2 g plate and acidic, add 10% medium 121, 2 atm
KH22PO44, 1 g MgSO44.7H22O in 1 L magnetic NaOH, too alkaline into for 15
distilled water stirrer add 5% HCl Erlenmeyer minutes
5) Cultivation of S. commune

Inoculate 5 plugs into Erlenmeyer

Cut mushroom Incubate with the
containing 100 mL of liquid Measure final pH
mycelium on PDA help of a shaker at
medium with glucose of the medium
medium with a 5 mm room temperature for
concentrations of 10 g, 20 g and with a pH meter
cork drill 20, 25 and 30 days
30 g

6) Harvesting and Weighing of S. commune Mycelium

Filter the mycelium in the

Weighed the dry
Erlenmeyer flask using filter Separate mycelium from Dry in an oven at
weight of mycelium
paper with a Buchner funnel and filtrate and weighed in 60 until a constant
and record the
the filtration is accelerate using a wet weight weight is obtain
results
vacuum pump
7) Final pH Measurement of Medium

Wash electrodes with

Turn on the pH Insert electrode into Record pH value as the
meter distilled water and dry with a the media final pH of the media
tissue

8) Preparation of S. commune Mycelium Extract

Crush dry After 24 Separate

Extraction is carry
mycelium, add hours, the The The results of
out using Evaporate
with 25 mL of separate the mycelium mycelium is the first and
chloroform solvent until a thick
chloroform solvent mycelium from the extract using second
for the second extract is
and macerate for 1 from the solvent ethyl acetate extraction are
time and macerate obtain
x 24 hours solvent using using filter as a solvent mixed
for 1 x 24 hours
filter paper paper
9) Preparation of S. commune Culture Filtrate Extract
Allow to Separate the The
Put 50 mL
Add 25 mL of Attach the stand for 5 extraction results separated The extraction
culture filtrate
chloroform separating minutes until and filtrate is is evaporate
into a
and shake for funnel to solvent accommodate extract using until a thick
separating
10 minutes the stative separate from with an ethyl acetate extract is obtain
funnel
the filtrate Erlenmeyer flask solvent

10) Identification of Secondary Metabolite Compounds

Draw top and bottom
Cut silica gel F254
254 outlines with distance Dotted the S. Put into a
thin layer
between the tip of the F254 commune extract chamber which
chromatography 254 in the center of the Allow to dry is saturated
silica gel plate and the upper lower border as with the
plates into 10 x
and lower boundary lines is 1 much as 8 µl mobile phase
10 cm sizes
cm each
10) Identification of Secondary Metabolite Compounds (Cont’d)

a. Alkaloid Identification

The The mobile phase use for

Heat at a temperature of 100
stationary chloroform extract is methanol:
Spray Dragendorff for 5 minutes. The presence
phase use hexane: glacial acetic acid (8: 2:
reagent to detect of alkaloids is indicated by
is silica gel 0,5) and for ethyl acetate extract
eluted spots the formation of yellow or
is methanol: hexane: glacial acetic
F254 orange patches
254 acid (8: 1: 0,5)

b. Terpenoid Identification
The mobile phase use for
The Heat at a temperature of 100
chloroform extract is n-hexane:
stationary Spray sulfuric acid for 5 minutes. The presence of
ethyl acetate: glacial acetic acid (8:
phase use is vanillin reagent to terpenoids is indicate by the
2: 0,5) and for ethyl acetate
detect eluted spots formation of blackish purple
silica gel F254 extract is ethyl acetate: n-hexane:
254 spots
glacial acetic acid (8: 2: 0,5)
 10) Identification of Secondary Metabolite Compounds (Cont’d)

c. Flavonoid Identification

The mobile phase use for

chloroform extract is n-
View at 366 nm UV
The stationary hexane: ethyl acetate:
Spray AlCl33 light. The presence of
phase use is silica glacial acetic acid (8: 2:
reagent to detect flavonoids is indicated
0.5) and for ethyl acetate
gel F254 eluted spots by the formation of
254 extract is ethyl acetate: n-
blue green spots
hexane: glacial acetic acid
(8: 2: 0, 5)

For each eluted spot the retardation factor (Rf) value is calculate with the following formula.
Rf =
 Data Analysis
The data obtain are analyze using analysis of variance (ANOVA) at the 95% confidence level. The results
of the ANOVA test that are significantly different are followed by the duncan multiple range test (DMRT).
Research
Framework
Research Schedule
Month
No. Type of activity March April May June July August
1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4
1. Preparation                                              
  - Proposal seminar                                                
  - Permit arrangement and site survey                                                
  - Preparation of tools and materials                                                
2. Analysis of samples in the laboratory                                              
3. Data analysis                                                
4. Literature review                                                
5. Thesis drafting                                                
6. Research results seminar                                                

7. Thesis revision and thesis ratification                                                

Data Tabulation
Table. 1 Data Tabulation of Secondary Metabolites Characterization
Sulfuric Acid Vanillin AlCl3 Reagent
Dragendorff Reagent
Reagent
No. Treatment Dark Purple Color Blue Green Flavonoid
Yellow Alkaloid Group
Terpenoid Group Group
A B A B A B
1. G1I1            
2. G1I2            
3. G1I3            
4. G2I1            
5. G2I2            
6. G2I3            
7. G3I1            
8. G3I2            
9. G3I3            
Data Tabulation (Cont’d)
Table. 2 Data Tabulation of Mycelium Dry Weight
Repetition
Glucose (Dry Weight of Mycelium)
Incubation Time Total
Concentration
1 2 3
10 20
10 25
10 30
20 20
20 25
20 30
30 20
30 25
30 30
Total        
Data Tabulation (Cont’d)
Table. 3 Anova Table

Source of
df SS MS F Sig.
Variation

Glucose
3        
Concetration

Incubation Time 3        

Glucose
Concentration* 9        
Incubation Time

Error          
Total          
 References
Chen, Z., Yin, C., Fan, X., Ma, K., Yao, F., Zhou, R., Shi, D., Cheng, W. & Gao, H., 2019. Characterization of Physicochemical and Biological Properties of Schizophyllum
commune Polysaccharide Extracted with Different Methods. International Journal of Biological Macromolecules, 156, pp.1425-1434.

Ekowati, N., Ina R, N. & Mumpuni, A., 2016. Potensi Jamur Trametes Versicolor dan Russula Sp. dalam Menghasilhan β-glukan Melalui Proses Fermentasi. Seminar
Nasional Pendidikan dan Saintek. Fakultas Biologi, Universitas Jenderal Soedirman.

Harborne, A., 1987. Metode Fitokimia. Bandung: Penerbit ITB.

Hutabarat, F. V., Diba, F. & Sisillia, L., 2019. Daya Hambat Ekstrak Kulit Jati (Tectona grandis Linn F) terhadap Pertumbuhan Jamur Pelapuk Kayu Schizophyllum
commune Fries. Jurnal Hutan Lestari, 7(3), pp.176-180.

Indrawati, I. & Fakhrudin, S. D., 2016. Isolasi dan Identifikasi Jamur Patogen pada Air Sumur dan Air Sungai di Pemukiman Warga Desa Karangwangi, Cianjur, Jawa
Barat. Jurnal biodjati, 1(1), pp.27-38.

Jones, M., Bhat, T., Kandare, E., Thomas, A., Joseph, P., Dekiwadia, C., Yuen, R., John, S., Ma, J. & Wang, C.H., 2018. Thermal Degradation and Fire Properties of
Fungal Mycelium and Mycelium-Biomass Composite Materials. Scientific reports, 8(1), pp.1-10.
 References (Cont’d)
Kim, S. W., Hwang, H. J., Park, J. P., Cho, Y. J., Song, C. H. & Yun, J. W., 2002. Mycelial Growth and Eko-biopolymer Production by Submarged Culture of Various
Edible Mushroom Under Different Medium. Letters in Applied Microbiology, 34, pp. 55-61.

Maharani, M. M., Ratnaningtyas, N. I. & Priyanto, S., 2014. Penggunaan Beberapa Medium Semisintetik untuk Produksi Miselium Jamur Maitake (Grifola frondosa
(Dickson: Fr.) SF Gray) Isolat Cianjur dan Ekstrak Kasarnya. Scripta Biologica, 1(1), pp.22-27.

Margino, S., 2008. Produksi Metabolit Sekunder (Antibiotik) oleh Isolat Jamur Endofit Indonesia. Majalah Farmasi Indonesia, 19(2), pp.86-94.

Sumaryono, W., 1999. Produksi Metabolit Sekunder Tanaman Secara Bioteknologi. Seminar Nasional Kimia Bahan Alam. Unesco, Universitas Indonesia.

Vamanu, E., 2013. Antioxidant Properties and Chemical Compositions of Various Extracts of the Edible Commercial Mushroom, Pleurotus ostreatus, in Romanian
Markets. Revista de Chimie, 64, pp.49-54.

Yap, T. & Ng, M. I. M., 2001. An Improved Methods for the Isolation of Lentinan from Lentinula edodes (Berk) Sing (Agaricomycetidae). International Journal of
Medical Mushroom, 3, pp. 6-19.
 Thank you
CREDITS: This presentation template was created by Slidesgo,
including icons by Flaticon, infographics & images by Freepik