Antimicrobial Activity of Selected Malaysian Mushrooms Against

Staphylococci and Streptococci

Al Dhalli S.

, Pavithira P. R.

, Jiyauddin K.

, Kaleemullah M.

,Mohd Fadli A.

Eddy Y.

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

century, we have known microbes cause

infectious diseases. Near the end of the 20

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

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

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

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

c for 21-24hours and fungus at 28

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

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

Streptococci

Staphylococci

Streptococci

Staphylococci

Streptococci

Acetone

Chloroform

Ethanol

Amoxycillin

(positive)

Distilled

Water

(negative)

Antimicrobial Activity of Selected Malaysian Mushrooms Against Staphylococci and Streptococci

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

Zone of Inhibition

Types of Mushrooms

Zone of inhibition of extracts

against Streptococci

Acetone

Extract

Chlorofor

m Extract

Ethanol

Extract

Amoxycilli

Distilled

water

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

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