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,

40100 Shah Alam, 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

310

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: 10.5220/0008361903100315

In Proceedings of BROMO Conference (BROMO 2018), pages 310-315

ISBN: 978-989-758-347-6

thought to be responsible for many of the

mushroom's beneficial effects.(Barrie, 2011).Shitake

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

Antimicrobial Activity of Selected Malaysian Mushrooms Against Staphylococci and Streptococci

311

Streptococcus. The plates were incubated at 37°C

for 24 h for bacterial strains. After incubation, the

inoculated plates were observed for zones of

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.

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).

Table 1 Antimicrobial effect of different types of mushrooms, in different types of solvents against Staphylococci and

Streptococci

Zone of

inhibition

Types of

solvents

Zone Of Inhibition After 24 hours

(

)

Shiitake Mushroom O

ster Mushroom White Button Mushroom

Staphylococci Streptococci Staphylococci Streptococci Staphylococci Streptococci

Acetone 0 12 0 0 0 0

Chloroform 0 14 0 0 0 0

Ethanol 0 0 0 0 0 0

Amoxycillin

(

ositive)

17 20 17 20 17 20

Distilled

Water

(ne

ative)

0 0 0 0 0 0

BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity

312

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

Zone of Inhibition

Types of Mushrooms

Zone of inhibition of extracts against

Streptococci

Acetone

Extract

Chloroform

Extract

Ethanol

Extract

Amoxycillin

Distilled

water

Zone of inbition

Types of Mushrooms

Zone of inhibition of extracts against

Staphylococci

Acetone

Chloroform

Ethanol

Amoxycillin

Distilled

water

Antimicrobial Activity of Selected Malaysian Mushrooms Against Staphylococci and Streptococci

313

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

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.

REFERENCES

Anne Hart (2009, October 31). Ground Report. Retrieved

May 2, 2016, from Health and Science:

http://www.groundreport.com/Health_and_Science/Sh

ould-you-grow-your-own-anti-viral-mushrooms-

Whos/2911171.

Barrie Cassileth(2011). Lentinula edodes Complementary

Therapies, Herbs, and Other OTC

Agents.Retrieved from

http://www.cancernetwork.com/integrative-

oncology/content/article/10165/1859511 on 3rd May

2016.

Bock-Gie Jung, Jin-A Lee, Bong-Joo Lee (2012).

Immunoprophylactic effects of shiitake mushroom

(Lentinula edodes) against Bordetella bronchiseptica in

mice. Journal of Microbiology , 1003-1008.

DayongWu,MunkyongPae,ZhihongRen,ZhuyanGuo,Dona

l Smith and SiminNikbinMeydani(2011). Dietary

Supplementation With White Mushroom Enhances

Natural Killer Cell Activity In C57bl Mice. JN The

Journal Of Nutrition. 1472-7. Retrieved

fromwww.ncbi.nlm.nih.gov.

Julie Cerrato (November 30, 2012). Mushroom Magic.

Food and Health. Retrieved on 10

June 2016 from

http://www.examiner.com/article/mushroom-magic.

KuznetsovOlu,Mil’kovaEV,SosninaAE,SotnikovaNlu(200

5).Antimicrobial action of Lentinusedodes juice on

human microflora.80-2. Retrived from

http://pubget.com/paper/15773410/Antimicrobial

action of Lentinusedodes juice on human microflora.

Lena Ciric,AnnaTymon, EgijaZaura, Peter

Lingström, Monica Stauder, Adele Papetti, Caterina

Signoretto, Jonathan Pratten, Michael

Wilson and David Spratt(2011). In Vitro Assessment

of Shiitake Mushroom (Lentinula edodes) Extract for

Its Antigingivitis Activity. Journal of Biomedicine and

Biotechnology, 7.

M. Ashrafuzzaman, A. K.M Kamruzzaman, M. Razi

Ismail, S.M. Shahidullah, S.A. Fakir (2009).

Substrate affects growth and yield of shiitake. African

Journal of Biotechnology, 2999.

MasatomoHirasawa,NaotoShauji,TomotakeNeta,Kazuo

Fukushima and Kazuko Takada (1999). Three kinds

of antibacterial substances from Lentinusedodes.

International Journal of Antimicrobial Agents .Volume

11, Issue 2 . 151-157.

Murphy,Bakaletz,Smeesters(2009).Microbial interaction

in the respiratory tract.US National Library of

Medicine. Retrieved from

http://www.ncbi.nlm.nih.gov/pubmed/19918134 on

April 2016.

Narasimha , Praveen, Mallikarjuna, Deva Prasad

Raju(2011). Mushrooms (Agaricusbisporus) mediated

biosynthesis of sliver nanoparticles, characterization

and theirantimicrobial activity. International Journal

of Nano Dimension.

National Institute of Allergy and Infectious

Disease.Retrieved from

http://www.niaid.nih.gov/topics/microbes/Pages/defaul

t.aspx on 4

April 2016.

BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity

314

Pushpa Sharma (2012). Antioxidant Mushroom.

International journal research of pharmacy , 6.

Regina Hiroko Hassegawa,Maria Catarina Megumi

Kasuya,Maria Cristina DantasVanetti(2004).Growth

and antibacterial activity of Lentinula edodes in liquid

media supplemented with agricultural

wastes.Electronic Journal of Biotechnology.Vol 8.

Taufiqur Rahman (2012). Shiitake Mushroom: A Tool of

Medicine. Bangladesh J Medicinal Biochemical , 24.

Traci Vandermark(2011).The Health Benefits of Button

Mushrooms.Retrived from

http://www.livestrong.com/article/415319-the-health-

benefits-of-button-mushrooms/ on 1

May 2016.

Antimicrobial Activity of Selected Malaysian Mushrooms Against Staphylococci and Streptococci

315