Antimicrobial Activity of Selected Malaysian Mushrooms Against
Staphylococci and Streptococci
Al Dhalli S.
1
, Pavithira P. R.
1
, Jiyauddin K.
1
, Kaleemullah M.
1
,Mohd Fadli A.
1
Eddy Y.
1
1
School of Pharmacy, Management & Science University, Selangor Darul Ehsan, Malaysia
Keywords: Lentinula edodes, Pleurotus ostreatus, Agaricus bisporus, Staphylococci and Streptococci.
Abstract : One of the biggest problems in the developing world is the proliferation of treatable bacterial infections. The
use of mushrooms as nutritional food as well as medicine is gaining popularity. The aim of this research is
to evaluate the antimicrobial activity in Lentinula edodes, Pleurotus ostreatus and Agaricus bisporus against
Staphylococcus and Streptococcus microorganism. The mushroom was dried and grounded into powder and
extracted by cold maceration. The solvent was extracted through evaporation and then the extract was tested
for its antimicrobial activity against Staphyloccoci and Streptoccoci microorganism by disc Diffusion
Method. The chloroform extract of Shiitake mushroom exhibited maximum zone of inhibition against
Streptococci species was 14 mm. Moderate activity of acetone extracts against Streptococci species was
observed as 12 mm. The chloroform extracts showed broad spectrum of antibacterial activity against the
tested bacterial pathogens than other solvents. It was found that the shiitake mushroom has antibacterial
activity and it gives reliable indication of the concentration of drug required to inhibit the growth of
microorganism compared with to the other two types of mushrooms. Shiitake mushrooms cultivated in
Malaysia are potential sources of bioactive compound against streptococcus species and should be
investigated for natural antibiotics.
1 INTRODUCTION
Microbes are tiny organisms which cannot be seen
without a microscope, yet they are abundant on
Earth. They live everywhere such as in air, soil,
rock, and water (NIAID,2010). Some microbes need
oxygen to live, but others do not. These microscopic
organisms are found in plants and animals as well as
in the human body. Some microbes cause disease in
humans, plants, and animals. Others are essential for
a healthy life, and we could not exist without them.
Indeed, the relationship between microbes and
humans is delicate and complex. Microbes make up
more than 60 % of the Earth’s living matter and
scientists estimate that 2-3 billion species share the
planet with us.
Most microbes belong to one of four major
groups: bacteria, viruses, fungi, or protozoa. Since
the 19
th
century, we have known microbes cause
infectious diseases. Near the end of the 20
th
century,
researchers began to learn that microbes also
contribute to many chronic diseases and conditions.
Mounting scientific evidence strongly links
microbes to some forms of cancer, coronary artery
disease, diabetes, multiple sclerosis, and chronic
lung diseases (NIAID,2010).
One of the biggest problems in the developing
world is the proliferation of treatable bacterial
infections which run unchecked through populations
due to lack of access to medications and medical
treatment. Therefore, suitable action must be taken
at the right time to combat the problem. For
example, developing new drugs either synthetic or
natural rather than using of antibiotic only for
treating infectious disease. The use of mushrooms as
nutritional food as well as medicine is gaining
popularity in recent times. The nutritive and
medicinal properties of many mushrooms have been
documented. Few examples of mushrooms which
are known for its antimicrobial activities are
Letinula edodes , Pleurotus ostreatus and Agaricus
bisporus.
Lentinula edodes (shiitake) an edible mushroom
indigenous to East Asia, is cultivated worldwide for
its purported health benefits. Lentinan ([1,3] beta-D-
glucan), a polysaccharide isolated from shiitake, is
thought to be responsible for many of the
mushroom's beneficial effects.(Barrie, 2011).Shitake
S., A., P. R., P., K., J., M., K., A., M. and Y., E.
Antimicrobial Activity of Selected Malaysian Mushrooms Against Staphylococci and Streptococci.
DOI: 10.5220/0009846500002406
In Proceedings of BROMO Conference (BROMO 2018) - Symposium on Natural Product and Biodiversity, page 1
ISBN: 978-989-758-347-6
Copyright
c
2022 by SCITEPRESS Science and Technology Publications, Lda. All rights reserved
1
mushrooms have both medicinal and culinary
properties which encourages the plantation of it
throughout the world. Lentinula edodesposses
antibacterial effects against bacteria such as
Streptococcus spp., Actinomyces spp., Lactobacillus
spp and also against cancer. Apart from it L.edodes
also has antitumour, antiviral, hypocholesterolemic
and hypoglycemic properties which plays an
important role in the consumption of these
mushrooms.
Apart from it, Pleurotus ostreatus (oyster) are
also another common type of mushroom which has
been extensively used in traditional Chinese
medicine from as early as 3,000 years ago. These
mushrooms commonly used due to huge advantages
which include its nutritional value and other
medicinal benefits which presents in it. In detailed,
oyster mushrooms posses an antioxidant property as
well as antibacterial activities which against various
species of Gram positive and Gram negative
bacteria. Besides, Pleurotus ostreatus present with
an antitumor activities which may be effective for
improving antioxidant capacity and preventing
tumor.
In addition, Agaricus bisporus (button
mushrooms ) are also included in the category of
medicinal mushrooms due to some evidence such as
the results of a study, published in the July 2010
issue of "Nutrition Journal," show that button
mushrooms reduce inflammation in arterial cells
and prevent white blood cells from sticking to
arterial walls.(Traci, 2011) The researchers conclude
that consuming button mushrooms may be a means
to prevent heart disease and also released a report
stating that button mushrooms have been found
effective at treating breast, colon and prostate
cancers.
2 MATERIALS AND METHODS
2.1 Materials
2.1.1 Mushrooms
Lentinula edodes, Pleurotus ostreatus and Agaricus
bisporus Bacteria strain Staphylococci and
Streptococci Antibiotic Amoxycillin
2.1.2 Other Materials
70% Ethanol , distilled water , Acetone , Chloroform
, Whatsman filter paper No 60 , beaker , blender ,
incubator , Mueller Hinton Agar , Sterile Petri dish
,test tubes , nutrient broth , aluminium foil , sterile
cotton swab , Bunsen burner , weighing balance ,
spatula ,filter funnel , conical flask , measuring
cylinder , micropipette and ruler.
2.2 Methods
2.2.1 Collection of Samples
The Lentinula edodes, Pleurotusostreatus and
Agaricusbisporus mushrooms was collected from a
supermarket.
2.2.2 Preparation of Powder
The collected mushrooms were washed with running
tap water to remove adhering materials. Then, the
mushrooms were sliced and dried at temperature not
exceeding 50
o
C.
These dried materials were pulverized
mechanically into coarse powder. The fine powder
was separated by passing through sieve No. 60.
The coarse powder obtained was used for the
extraction process.
2.2.3 Preparation of Extracts
The coarse powdered of mushrooms such as
shiitake, oyster and white button mushrooms
(250gm) were taken in an aspirator bottle separately
and extracted successively by cold maceration
technique with solvents like aqueous, chloroform,
acetone and ethanol respectively for six days.At the
end of each extraction they were filtered through
filter paper. Except aqueous extract all other extracts
were distilled over water bath to remove 80% of the
solvent. The aqueous extract was concentrated by
distilling on a mantle. The remaining portion of all
the solvents from the extracts was removed under
vacuum.
2.2.4 In-vitro Antibacterial Screening for
Extracts by Disc Diffusion Method
The antibacterial activity of the extract determined
by streaking bacterial cultures with a nutrient agar
medium in petri plates. Sterilized filter paper discs
(Whatman No 1) soaked in different beakers
containing the dissolved extracts of different
mushrooms were taken out with sterilized forceps
and air-dried and placed on plates with the different
organisms such as Staphylococcus and
Streptococcus. The plates were incubated at
37°C for 24 h for bacterial strains. After incubation,
the inoculated plates were observed for zones of
BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity
2
inhibition in millimeter diameter using a transparent
ruler. The sensitivity or susceptibility of the test
bacteria to the standard drug was tested using an
inoculated agar plate and Amoxicillin 250mg. The
zones of inhibition were measured and compared
with those of the plant extract.
Table 1: Antimicrobial effect of different types of mushrooms, in different types of solvents against Staphylococci and
Streptococci.
2.2.5 Determination of Minimal Inhibitory
Concentration(MIC)
The determination of MIC extracts will be measured
by using a tube dilution method with a slight
modification. Briefly, extracts will be subjected to a
series of serial dilution. Extracts with different
concentration will be added aseptically into different
labeled test tube containing sterile Muller Hinton
broth. Then,the bacterial suspension and fungal
suspension will be inoculated into respective test
tubes. The test tubes will be incubated bacteria at
37
0
c for 21-24hours and fungus at 28
0
c for 24-
28hrs.The MIC value will be measured by
comparing turbidity of the whole series of test tubes
with a negative control and positive controls.MIC
value will be stated as the highest concentration that
shows no turbidity which indicates no growth of
bacteria. Each test will be performed in triplicate.
The tube dilution test is the standard method for
determining levels of resistance to an antibiotic.
Serial dilutions of the antibiotic are made in a liquid
medium which is inoculated with a standardized
number of streptococcus organisms and incubated
for 72 hours time. The lowest concentration (highest
dilution) of antibiotic preventing appearance of
turbidity is considered to be the minimal inhibitory
concentration (MIC). At this dilution the antibiotic
is bacteriostatic. First of all, test tubes numbered
from 1 to 4. Then,1.0 ml of sterile broth added to all
test tubes.2.0 ml of shiitake acetone extraction and
1ml of streptococcus organism suspension added to
the first test tube(100 mg/ml).1.0 ml of the contents
from the first tube transferred to the second tube.
Using a separate pipette, the contents of this tube
mixed and transferred 1.0 ml to the third tube.
Continue dilutions in this manner to tube number 4,
being certain to change pipettes between tubes to
prevent carryover of antibiotic on the external
surface of the pipette. Finally 1.0 ml from tube 4 is
removed and discarded. Same process followed for
shiitake chloroform extraction and all test tubes
incubated at 35
o
C for 72 hours. After the
determined period , tubes examined for visible signs
of bacterial growth. The highest dilution without
growth is said to be the minimal inhibitory
concentration (MIC).
Zone of
inhibition
/
Types of
solvents
Zone Of Inhibition After 24 hours (mm)
Shiitake Mushroom
Oyster Mushroom
White Button Mushroom
Staphylococci
Staphylococci
Streptococci
Staphylococci
Streptococci
Acetone
0
0
0
0
0
Chloroform
0
0
0
0
0
Ethanol
0
0
0
0
0
Amoxycillin
(positive)
17
17
20
17
20
Distilled
Water
(negative)
0
0
0
0
0
Antimicrobial Activity of Selected Malaysian Mushrooms Against Staphylococci and Streptococci
3
3 RESULTS
Figure 1 Antimicrobial effect of extracts and controls
against Streptococci
Figure 2 Antimicrobial effect of extracts and controls
against Staphylococci
4 DISCUSSION
In the present investigation, the antibacterial
properties of chloroform,acetone,alcohol and
aqueous extracts of medicinal mushrooms such as
shiitake,oyster and white button mushrooms were
tested against human pathogenic bacteria. The
antibacterial properties of the extracts were also
comparatively analysed against standard antibiotics
by antibiotic sensitivity test.
The chloroform extract of shiitake mushroom
exhibited maximum zone of inhibition against
streptococcus species was 14mm. The moderate
activity of acetone extracts against streptococcus
species was observed as 12mm. The chloroform
extracts showed broad spectrum of antibacterial
activity against the tested bacterial pathogens than
other solvents.
The medicinal mushrooms such as white button
and oyster against staphylococcus and streptococcus
species resistance against solvent extraction of
chloroform, acetone, ethanol and aqueous. Similarly,
shiitake against staphylococcus species also
resistance against all the solvent extractions.
The antibiotic sensitivity test using standard
antibiotic which is amoxycillin were tested against
pathogenic bacteria. The antibiotic used was
exhibited antibacterial activity. The results
confirmed that the solvent extracts such as
chloroform and ethanol of shiitake mushroom
exhibited a higher antibacterial activity against
streptcoccus pathogenic bacteria. The result of
antibacterial effect of chloroform, acetone, ethanol
and aqueous solvents of white button and oyster
mushrooms revealed no activity against pathogenic
bacterial strains.
Thus, MIC assay are capable of verifying that the
shiitake mushroom has antibacterial activity and that
it gives reliable indication of the concentration of
drug required to inhibit the growth of
microorganism. Acetone and chloroform extract of
shiitake mushroom was subjected to get the MIC
against strpetococcus species and it was found to be
50mg/ml for acetone extract and 12.5mg/ml for
chloroform extract.
5 CONCLUSION
The in- vitro comparative study of antimicrobial
activity of selected Malaysian mushrooms against
Streptococci and Staphylococci in different solvent
0
5
10
15
20
25
Zone of Inhibition
Types of Mushrooms
Zone of inhibition of extracts
against Streptococci
Acetone
Extract
Chlorofor
m Extract
Ethanol
Extract
Amoxycilli
n
Distilled
water
0
2
4
6
8
10
12
14
16
18
Zone of inbition
Types of Mushrooms
Zone of inhibition of extracts against
Staphylococci
Acetone
Chloroform
Ethanol
Amoxycillin
Distilled
water
BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity
4
extract concludes that the chloroform extract of
shiitake mushroom exhibited maximum zone of
inhibition against Streptococcus species was 14mm.
The moderate activity of acetone extracts against
Streptococcus species was observed as 12mm. The
chloroform extracts showed broad spectrum of
antibacterial activity against the tested bacterial
pathogens than other solvents.
ACKNOWLEDGEMENT
The author is very thankful and grateful towards the
research committee and lecturers of Management
and Science University, Malaysia for providing all
the needed materials, equipment as well continuous
guidance and support throughout this research
project.
CONFLICT OF INTEREST
Author declared there is no conflict of interest.
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Antimicrobial Activity of Selected Malaysian Mushrooms Against Staphylococci and Streptococci
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