Antibacterial Activity of Lemon (Citrus limon) Peel

Extract against Staphylococcus aureus

and Escherichia coli

A Research presented to the

Senior High School Department
(Science, Technology, Engineering and Mathematics)

JAM CHLOE Z. RIVAS

Researcher

AGUSAN DEL SUR NATIONAL SCIENCE HIGH SCHOOL

Gov. D.O. Plaza Gov’t Center, Prosperidad, ADS

March 2021

i.
 ABSTRACT

WHO (2020) stated that antibiotic resistance is a growing concern globally. In the
Philippines, it was reported that several bacteria such as Staphylococcus aureus and
Escherichia coli have high rates of resistance to conventional antibiotics. As a result,
attempts to find and develop new antimicrobial agent including from plants are
significant in today's world. This study aims to expand the range of antibacterial agents
from natural resources so, crude extracts from lemon peels undergone antibacterial
screening through disc diffusion assay to identify the average zone of inhibition of the
plant sample against S. aureus and E. coli. Results indicated antibacterial property
present in the lemon peel extract with an average diameter of 24 mm zone of inhibition
against S. aureus and 26mm against E. coli. Results further showed that there is
significant difference between the treatments since the p-value is less than 0.05.
Clindamycin, the positive control exhibited the highest inhibition zone with 37.5 mm
against S. aureus; whilst the lemon peel extract against S. aureus showed the least
inhibitory performance with 24mm. Zones of inhibition that are greater than 19mm is
analyzed as a very active antibacterial performance, therefore, this implies that both
treatment groups exhibit a very active antibacterial activity against S. aureus and E. coli;
yet different concentrations of the plant samples has to be tested for a more thorough
investigation of the antibacterial potential of the extract.

Keywords: anti-bacterial, lemon peel, inhibition

ii.
 ACKNOWLEDGMENT

Immeasurable appreciation and deepest gratitude for the help and support are

extended to the following persons who in one way or another have contributed in making

this study possible.

Mr. Ace Michael B. Magalso, her research adviser, for sharing his expertise and

giving multiple considerations for the researcher upon conducting the study. Your

enthusiasm, guidance and exacting attention to detail had been an inspiration from the

beginning to the final draft of this paper.

Mrs. Jine L. Havana, her Principal, for also granting all researchers into several

considerations upon conducting their studies.

Ma’am Willame Paracale and Mr. Algerd Moscosa, her teachers, for giving

comments and suggestions that gave benefits in the completion and success of this study.

Mrs. Rowena Rivas and Mr. Moises Rivas, her parents, for the full support

especially for providing financial aid despite the pandemic.

Above all, to GOD ALMIGHTY, for establishing her plans and making all things

work together. He has been the researcher’s greatest strength since day one. All the glory

belongs to Him alone.

The Researcher

iii.
 TABLE OF CONTENTS

Title Page i
Abstract ii
Acknowledgement iii
Table of Contents iv
List of Tables vi
List of Figures vi
List of Appendices vii

Chapter 1 - INTRODUCTION 1-16

1.1 Background of the Study 1-2
1.2 Research Objectives 3
1.3 Research Problem 3
1.4 Research Hypotheses 4
1.5 Conceptual Framework 4
1.6 Scope and Delimitations 5
1.7 Significance of the Study 5-6
1.8 Definition of Terms 6-9
1.9 Review of Related Literature 10-16

Chapter 2 - METHODOLOGY 17-23

2.1 List of Materials 17
2.2 Research Design 18
2.3 Procedure/Methods 19-23
2.3.3 Antibacterial Screening 20-21

Chapter 3 – RESULTS AND DISCUSSIONS 24-27

3.1 Antibacterial Screening 24-25
3.2 Statistical Analysis 26-27

v
Chapter 4 – SUMMARY, CONCLUSIONS AND RECOMMENDATIONS 28-29
4.1 Summary and Findings 28
4.2 Conclusions 29
4.3 Recommendations 29

Bibliography 30-34
Appendix 35-39
Curriculum Vitae 40

v
 LIST OF TABLES

Table Table Title Page

1 The Research Design 18

2 ZOI of the treatment groups against S. aureus and E. coli 24

3 Univariate Analysis of Variance of the Zone of Inhibition of the Plant

Samples and the Positive Control against S. aureus and E. coli 26

4 Post Hoc Analysis for the ANOVA Result (Scheffe) 27

LIST OF FIGURES

Figure Figure Title Page

1 Conceptual Paradigm of Study 4

2 Diameter of ZOI of the two treatments against S. aureus 25

3 Diameter of ZOI of the two treatments against E. coli 25

vi
 LIST OF APPENDICES

Appendix Appendix Title Page

A Flow Chart 35

B Raw Data 35

C Report of Microbiological Analysis 36-37

D Plagiarism Test 38

E Documentation 39

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 Antibacterial Activity of
Lemon Peel Extract
Chapter 1

INTRODUCTION

1.1 Background of the Study

Antibiotic resistance is a growing concern globally. It is a major threat to global

health and food security. A growing list of infections such as pneumonia, tuberculosis,

blood poisoning, gonorrhea, and foodborne diseases are becoming harder, and

sometimes impossible, to treat as antibiotics become less effective (WHO, 2020). The

global health care system is struggling to find effective and affordable drugs to treat

microbial infections, which account for about half of all infections (Elisha et al., 2017).

According to Bhalodia and Shukla (2011), it is important to investigate newer

drugs that have lesser resistance. Drugs derived from natural sources play a significant

role in the prevention and treatment of human diseases. In many developing countries,

traditional medicine is one of the primary healthcare systems. Natural products such as

herbs and spices are widely used in traditional medicine to treat human diseases. Over

90% of new drugs developed during the past three decades have been based on natural

sources. However, the discovery rate of new antimicrobial agents is declining.

Antimicrobial properties of medicinal plants are being increasingly reported from

different parts of the world. Plants are rich in a wide variety of secondary metabolites

such as tannins, terpenoids, alkaloids, flavonoids, glycosides and many more which have

been found in vitro to have antimicrobial properties. It is also already known to man for

centuries about the therapeutic efficacy of herbal medicine or many indigenous plants for

1
 Antibacterial Activity of
Lemon Peel Extract
various of disorders has been acknowledged by practitioners of traditional medicine

(Bhalodia & Shukla,2011).

According to Shaik et al.,(2009), plant extracts or their active ingredients are

estimated to be utilized as folk medicine by 80% of the world's population in traditional

therapies. It is expected that plant extracts showing target sites other than those used by

antibiotics will be active against drug resistant microbial pathogens. However, very little

information is available on such activity of medicinal plants. (Valle et. Al, 2015)

The Philippines is one of the Asian countries with a diverse flora, and many

species are said to have medicinal characteristics. (Quisumbing, 2010) Hence, numerous

studies have used this advantage to investigate and identify the plants with potential

bioactive molecules of pharmaceutical importance in the management of MDR bacteria,

in vitro methods of evaluation on test organisms were performed using the plant extracts;

this is due to the alarming increase in antibiotic resistance among bacteria.

Traditional healers have used plant preparations as antibacterial, as well as

treatments for pneumonia, diarrhea, and other bacterial illnesses. Plants create

metabolites to protect themselves against bacteria in the environment; therefore this may

be a logical method (Lazarte et. Al., 2020)

In an effort to expand the range of antibacterial agents from natural resources, this

study is designed to determine the antibacterial activity of Lemon (Citrus limon) peel

extract against Staphylococcus aureus and Escherichia coli- a bacteria which is a leading

cause of disease among humans.

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 Antibacterial Activity of
Lemon Peel Extract

1.2 Research Objectives

The general objective of the study is to assess the antibacterial activity of Lemon

peel extract (LPE) against Staphylococcus aureus and Escherichia coli.

Specifically, this study aims to:

1. Assess the antibacterial activity of the Lemon peel extract against S. aureus and E. coli.

2. Determine the mean diameter of the average zone of inhibition (ZOI) of the lemon peel

eztract in S. aureus and E. coli.

3. Determine if there is a significant difference between the average ZOI of the lemon peel

extract and the positive control in S. aureus and E. coli.

1.3 Research Problem

The study endeavors to identify the antibacterial efficacy of the Lemon peel

extract against Staphylococcus aureus and Escherichia coli. Specifically, this aims to

answer the following:

1. Does the lemon peel extract have anti-bacterial property against S. aureus and E.

coli?

2. What is the mean diameter of the average ZOI of the lemon peel extract against

S. aureus and E. coli?

3. Is there a significant difference between the average ZOI of the lemon peel extract

and the positive control in S. aureus and E. coli?

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 Antibacterial Activity of
Lemon Peel Extract
1.4 Hypotheses

Based on the foregoing research problem, the following null hypotheses are

formulated:

1. The lemon peel extract contains no anti-bacterial property against S. aureus and E.

coli.

2. The mean diameter of the average ZOI of the lemon peel extract is zero against S.

aureus and E.coli.

3. There is no significant difference between the average ZOI of the lemon peel extract

and the positive control in S. aureus and E. coli.

1.5 Conceptual Framework

Input Process Output

Lemon Peel Antibacterial Antibacterial

Extract Screening- Disc Agent
Diffusion Assay

Figure 1. Conceptual Paradigm of the Study

Figure 1 shows the main concept of this study which is to find an alternative and

effective antibacterial agent that is less costly, has increased potency and a natural source.

This indicates the framework of this investigation wherein the antibacterial activity of

Lemon Peel Extract provides a feasible solution of the main concept of the research

problem.

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 Antibacterial Activity of
Lemon Peel Extract
1.6 Scope and Limitations

This study entitled, “Antibacterial Activity of Lemon (Citrus limon) Peel Extract

against Staphylococcus aureus and Escherichia coli” focused on the development of an

antibacterial agent using lemon peels. The study is limited in determining its antibacterial

activity on S. aureus and E. coli only as the bacteria isolates. This study includes

collecting of samples within Prosperidad, Agusan del Sur, extraction of the lemon peels

through crude ethanol extraction, testing its antibacterial activity using disc diffusion

assay in 3 trials each bacteria only, determining the mean diameter of the average zone of

inhibition of the different concentrations, gathering of data and arriving at the

conclusions. This was conducted at the Department of Science and Technology, Butuan

City; whilst the diameter of zone of inhibition of the positive control was based on an

existing result of a published antibacterial research paper by Muchtaromah et. Al, (2020).

1.7 Significance of the Study

This study can contribute in preventing health care-associated infections and the

spread of antimicrobial resistance. It is the most vital thing to avoid the transmission of

harmful germs and prevent the infections.

The result of the study also benefit the following:

Community. The result of the study will help to deter the spread of germs and

illness-causing bacteria, particularly in busy environments like schools and offices. S.

aureus and E. coli is a common bacteria that exists anywhere, hence this study could

provide an alternative antimicrobial agent so communities could have an alternative

antibacterial agent, which is cheaper, efficient and effective against S. areus and E. coli.

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 Antibacterial Activity of
Lemon Peel Extract
Science and Public Health Organizations. These organizations will also benefit

from this investigation. This study may be able to contribute to the development of

products that can serve as antimicrobial agents and by having this new data and

knowledge regarding the anti-bacterial activity of Lemon Peel extract against E. coli and

S. aureus.

Future Researchers. This study will be able to contribute to the existing body of

knowledge that will be beneficial for future researchers who aim to investigate and widen

the scope of their study with regards to the efficiency of medicinal plants.

1.8 Definition of Terms

Agar Plates - An agar plate is a petri dish with a growth media

solidified with agar for microorganism culture.

Antibiotics and other selective chemicals are

sometimes added to impact development.

Antibacterial - It is directed or effective against bacteria such as

an antibacterial soap and antibacterial drug or it

could be an agent that is able to kill bacteria.

Broth Culture - Broth cultures are liquid cultures used in

laboratories to cultivate bacteria. A scientist starts

with a sterile liquid growing medium to make a

broth culture. The bacteria are inoculated into the

medium, which is then placed in an incubator at the

proper temperature.

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 Antibacterial Activity of
Lemon Peel Extract
Clindamycin - Clindamycin is an antibiotic that is used to treat

bone and joint infections, pelvic inflammatory

disease, strep throat, pneumonia, middle ear

infections, and endocarditis, among other bacterial

illnesses.

Crude Extract - A concentrated preparation of a drug obtained by

removing the active constituents of the drug with

suitable solvents, evaporating all or nearly all of the

solvent, and adjusting the residual mass or powder

to the prescribed standard

Disc Diffusion Assay - The disk diffusion test is a culture-based

microbiology assay used in diagnostic and drug

discovery laboratories. In diagnostic labs, the assay

is used to determine the susceptibility of bacteria

isolated from a patient’s infection to clinically

approved antibiotics.

Escherichia Coli - These are bacteria found in the environment,

foods, and intestines of people and animals. It is a

large and diverse group of bacteria. Some kinds of

E. coli can cause diarrhea, while others cause

urinary tract infections, respiratory illness and

pneumonia, and other illnesses.

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 Antibacterial Activity of
Lemon Peel Extract
Gram Negative - Gram-negative bacteria are bacteria that do not

retain the crystal violet stain used in the gram-

staining method of bacterial differentiation. Gram-

negative bacteria cause infections including

pneumonia, bloodstream infections, wound or

surgical site infections, and meningitis in healthcare

settings.

Gram Positive - Gram-positive bacteria are bacteria that test

positive in the Gram stain test, which has

traditionally been used to swiftly divide bacteria

into two major categories based on their cell wall

type.

Infectious Agents - These are organisms that are capable of producing

infection or infectious disease. Infectious diseases

are caused by diverse living agents that replicate in

their hosts.

Inoculum - It is a substance used for inoculation. It is a small

amount of material containing bacteria, viruses, or

other microorganisms that is used to start a culture.

McFarland Standard - The 0.5 McFarland standard is used to adjust the

turbidity of the inoculum prior to the cotton

swabbing of the agar plates for the antimicrobial

8
 Antibacterial Activity of
Lemon Peel Extract
assays. The standard should be prepared and

subjected to quality control prior to use.

Pathogens -Pathogens are any organism that can produce

disease. A pathogen may also be referred to as an

infectious agent, or simply a germ.

Staphylococcus aureus - Staphylococcus aureus is a Gram-positive, round-

shaped bacterium that belongs to the Firmicutes

family. It is a common member of the body's

microbiota, commonly found in the upper

respiratory tract and on the skin.

Turbidity - Turbidity is a measurement of a liquid's relative

clarity. When a light is shined through a water

sample, it is a measurement of the amount of light

dispersed by material in the water.

Zone on Inhibition (ZOI) - This is an area of media where bacteria are unable

to grow, due to presence of a drug that impedes

their growth.

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 Antibacterial Activity of
Lemon Peel Extract
1.9 Review of Related Literature

Antibiotic resistance is a problem that continues to threaten the healthcare

industry in both developing and developed countries around the world. The emergence of

multidrug-resistant (MDR) bacteria in hospitals and communities is a major unsolved

issue that places a significant strain on health-care systems (Cosgrove & Carmeli, 2003).

Infectious complications remain a major source of mortality and morbidity among

hospitalized patients, despite breakthroughs in antibiotic treatments. Although medical

practitioners can treat these individuals with second- or third-choice medications, the

usage of synthetic medications may put the patient at more danger due to the chance of

the drugs creating more dangerous side effects.

To address this problem, steps must be done to diminish it, such as controlling

antibiotic use, understanding the genetic mechanisms of resistance, and creating new

antibiotics and therapeutic techniques. Chemical leads for new drugs are emerging as a

result of advances in identifying novel sources of natural compounds with antibacterial

properties and expanding antibiotic chemical diversity. (Silva, 2013)

Cowan (2010) discussed that the majority of modern medicines have their roots in

old herbal traditions. Plant-based traditional medicine is based on hundreds of years of

observation and belief that predates the emergence of modern medicine. Medicinal plants

have been utilized as cures for human diseases for generations because they contain

medicinal components. There are a variety of plant-based natural remedies with

antifungal, antibacterial, and antiprotozoal properties that can be used systemically or

locally. Several plants with active ingredients such as volatile oils, polyphenols, and

alkaloids are used as folk remedies, while others have acquired appeal as completed

10
 Antibacterial Activity of
Lemon Peel Extract
products known as phytomedicines (Goyal, 2008). The adoption of traditional medicine

as an alternative form of health treatment, as well as the development of microbe

resistance to standard antibiotics, motivated researchers to examine the antibacterial

activity of medicinal plants in the second half of the twentieth century. Plant-derived

antimicrobials have significant therapeutic promise. They are effective in the treatment of

infectious diseases while simultaneously mitigating many of the side effects that are often

associated with synthetic antimicrobials.

Medicinal plants produce a wide range of chemicals with proven therapeutic

benefits. Antimicrobial activity is one of the most important properties of these

therapeutic herbs. Infectious infections are rare in plants, which indicates that they have

developed effective defense mechanisms (Bolla, 2011).Candidates for creating novel

antimicrobial medications are chemicals that can either restrict the growth of bacteria or

kill them with little or minimal toxicity to host cells (Lee et. Al., 2014). Some bioactive

substances, alone or in conjunction with other therapeutic treatments, may inhibit the life

activities of disease-causing bacteria (Sivananthan, 2013).

Antimicrobial capabilities of medicinal plants have been increasingly documented

from various parts of the world in recent years. Plant extracts with target sites other than

those employed by antibiotics are likely to be effective against drug-resistant microbial

infections. However, there is a scarcity of knowledge on medicinal plants' activity in this

regard (Abu-Shanab, 2016).

The importance of traditional medicinal herbs as alternatives to modern

antibacterial and antifungal drugs has been documented multiple times in the medical

literature. For practical and economic reasons, the majority of these published

11
 Antibacterial Activity of
Lemon Peel Extract
publications originate from several countries that continue to use herbal medicine for the

treatment of various disorders (Akhtar et. Al, 2015). These studies are important

resources for local medical researchers who want to learn more about the antibacterial

and antifungal properties of Philippine medicinal plants, especially against MDR

bacteria. For the design of future investigations, knowledge of various antimicrobial tests

and plant bioactive substances is essential.

In the Philippines, Infections caused by bacteria are still a major source of

morbidity, with respiratory, gastrointestinal, and urinary tract infections all being

common. Antibiotics are one of the most important discoveries of the modern age, having

been utilized to cure bacterial diseases.

Unfortunately, because of extensive abuse and misuse of antibiotics both in

humans and animals, rates of antimicrobial resistance have been increasing. According to

a WHO Report in 2014, the top 7 common bacteria that cause serious diseases with

widespread resistance include E.coli, S. aureus, H. influenzae, Shigella, P. aeruginosa,

non typhoidal Salmonella, Klebsiella, and N. gonorrhea. In the Philippines,

Staphylococcus aureus, Pseudomonas aeruginosa. and Acinetobacter baumannii were

reported to have high rates of resistance to conventional antibiotics. Abreu, et. Al (2012)

stated that what is being feared now is an occurrence of a post-antibiotic era, wherein

present antibiotics are ineffective against common diseases. As a result, attempts to find

and develop new antimicrobials are still crucial in today's world. To make matters worse,

few pharmaceutical companies have invested in the development of novel antibiotic

classes in the recent three decades.

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 Antibacterial Activity of
Lemon Peel Extract
Plants as sources of antimicrobials are an old concept which dates back to

traditional medical sytems in numerous cultures (Lewis, 2006). Herbal preparations have

been used by traditional healers as antiseptics, as well as cures for pneumonia, diarrheas,

and other bacterial infections. This may be a logical strategy since plants produce

metabolites to protect themselves against microbes in the environment (DOH, 2017)

Many modern day medicines and even antibiotics are also nature-derived or plant-

based or plant-derived. Quinine and artemisinin are two success stories of antimalarial

agents derived from the cinchona bark and Artemisia annua, respectively (Willcox &

Bodeker, 2004). Traditional antibacterials, inhibitors of multidrug resistance pumps, and

chemicals that target bacterial pathogenicity are three categories of antibacterials that can

be created from plants (Quintana & Saraos, 2007)

Staphylococcus aureus

Staphylococcus aureus is a Gram-positive, aerobic bacterium that is an important

cause of infections in humans. Approximately 30% of the healthy human population are

carriers of S. aureus. Common carriage sites are the anterior nares, pharynx, perineum

and skin. S. aureus can cause invasive infections in the community and healthcare setting

and has a broad spectrum of clinical syndromes, ranging from rather benign infections

(e.g. folliculitis) to potentially life-threatening infections (e.g. bloodstream infection).

Common healthcare-associated infections (HAIs) caused by S. aureus include surgical

site infections (SSIs), hospital-acquired bloodstream infections (HA-BSIs) and

pneumonia. These are important causes of morbidity, mortality and increased healthcare

expenditure (Troeman, Hout and Kluytmans, 2019).

13
 Antibacterial Activity of
Lemon Peel Extract
Steven et. al, (2015) also discussed that Staphylococcus aureus is a major human

pathogen that causes a wide range of clinical infections. It is a leading cause of

bacteremia and infective endocarditis as well as osteoarticular, skin and soft tissue,

pleuropulmonary, and device-related infections. The past 2 decades have witnessed two

clear shifts in the epidemiology of S. aureus infections: first, a growing number of health

care-associated infections, particularly seen in infective endocarditis and prosthetic

device infections, and second, an epidemic of community-associated skin and soft tissue

infections driven by strains with certain virulence factors and resistance to β-lactam

antibiotics.

Many resistant strains such as Pseudomonas, Klebsiella, Enterobacter,

Staphylococcus and Enterococcus has caused problems in treating infections caused by

these bacteria. Studies on extracts prepared from collected plants with antimicrobial

compounds having different mechanisms of action against resistant microbial strains are

of clinical importance as discussed by Jafari et al., (2012).

Escherichia coli

Escherichia coli (E. coli) is a Gram-negative, rod-shaped, facultative anaerobic

bacterium. This microorganism was first described by Theodor Escherich in 1885. Most

E. coli strains harmlessly colonize the gastrointestinal tract of humans and animals as a

normal flora. However, there are some strains that have evolved into pathogenic E. coli

by acquiring virulence factors through plasmids, transposons, bacteriophages, and/or

pathogenicity islands. These pathogenic E. coli can be categorized based on serogroups,

pathogenicity mechanisms, clinical symptoms, or virulence factors. Among them,

14
 Antibacterial Activity of
Lemon Peel Extract
enterohemorrhagic E. coli (EHEC) is defined as pathogenic E. coli strains that produce

Shiga toxins and cause hemorrhagic colitis and the life-threatening sequelae hemolytic

uremic syndrome (HUS) in humans (Lim et.al, 2013)

E. coli's hardiness, versatility, broad palate and ease of handling have made it the

most intensively studied and best understood organism on the planet. However, research

on E.coli has primarily examined it as a model organism, one that is abstracted from any

natural history. But E. coli is far more than just a microbial lab rat. Rather, it is a highly

diverse organism with a complex, multi-faceted niche in the wild. Recent studies of

‘wild’ E. coli have, for example, revealed a great deal about its presence in the

environment, its diversity and genomic evolution, as well as its role in the human

microbiome and disease (Blount, 2015)

Citrus Limon Peels

The peel of citrus fruits is a rich source of flavanones and many polymethoxylated

flavones, which are very rare in other plants. These compounds, not only play an

important physiological and ecological role, but are also of commercial interest because

of their multitude of applications in the food and pharmaceutical industries. The citrus

peel oils show strong antimicrobial activity (Dhanavade et. al, 2011)

It is also an important medicinal plant, used mainly for its alkaloids, which are

having anticancer activities, and the antibacterial potential of the crude extracts against

clinically significant bacterial strains has been reported. The plant is a potential source of

vitamin C, and the oil is used in various preparations to reduce skin itching, for skin

nourishment, and the pulp left after extraction of the juice is reported to be used for the

15
 Antibacterial Activity of
Lemon Peel Extract
treatment of pimples and wrinkles and to soften facial skin (Orang et al., 2015). These

facts have inspired the screening of C. limon for its antioxidant potential and

antimicrobial efficacy agents against a panel of microbes implicated in skin diseases.

The essential oil can disrupt the bacterial membrane and damage the lipids and

proteins layer. The flavonoid can inhibit specific enzymes and scavenge free radical. It

concluded that lemon (Citrus limon) peel extract have a high potential on antimicrobial

activity against the Escherichia coli (Saikia et. al, 2017)

Citrus limon Peels Bioactive Compounds

Although we often throw them away, lemon peels are packed with bioactive

compounds. First, lemon peels are a good source of nutrition; it is an excellent source of

vitamin C, as well as calcium, vitamin B6 and thiamine. It is also an amazing source of

antioxidants, anti-inflammatory compounds and dietary fibre in addition to its many

bioactive compounds.

Lemon peels contain many flavonoid antioxidants, as well as a unique antioxidant

compound known as D-limonene, which increases enzymes in the body to reduce

oxidative stress, illness and chronic diseases. Furthermore, lemon peels can also help to

promote heart health in many ways and reduce the risk of developing heart disease.

Studies have shown that the high amounts of flavonoid antioxidants, along with vitamin

C and fibre, can boost heart health and reduce risk factors associated with heart disease.

As well, studies have shown that lemon peels can help to lower “bad” LDL cholesterol

and triglyceride levels, while at the same time, increase “good” HDL cholesterol (Mejia,

2019).

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 Antibacterial Activity of
Lemon Peel Extract
Chapter 2

METHODOLOGY

2.1 Materials

A. Reagents

1L of 80% Ethanol

B. Organic Material

462g of ripe lemon peels

C. Apparatus

Bottles

Beaker

Erlenmeyer flask

Knife

Petri Dish

Reagent Bottle

Sterile cotton swab

D. Equipments

Rotary evaporator

17
 Antibacterial Activity of
Lemon Peel Extract

2.2 Research Design

Table 1. The Research Design

Diameter of Inhibition (mm)

After 24 hrs of incubation

Treatments
Staphylococcus aureus Escherichia coli

Plant Sample X1 X2 X3 X1 X2 X2

(Lemon Peel
Extract)

Positive X1 X2 X3 X1 X2 X3
Control

(Clindamycin)

(Let x= Diameter of Zone of Inhibition in millimeters)

The research design that is used in this study is a two-group experimental design

where two groups were contrasted with each other. The research is descriptively circling

on the investigation of the antibacterial activity of the two treatments which is lemon peel

extract to be compared to the positive control which is clindamycin against S. aureus and

E. coli. Both treatments underwent three trials of disc diffusion assay to determine the

diameter of zone of inhibition. The significant differences of the two are desired.

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 Antibacterial Activity of
Lemon Peel Extract
2. 3 Procedures

2.3.1 Collection and Preparation of Samples

The materials that are used in this study are lemon fruit peelings of a ripe lemon,

1L of 80% ethanol, and containers. The lemon fruits were excoriated, leaving the peels to

be washed and air-dried. 462g of lemon peels was collected. It was assured that the

obtained fruit peels did not manifest any signs of decay or discoloration

2.3.2 Plant Extraction

Air dried-chopped samples were soaked in 80% ethyl alcohol for 48 hours.

The mixture of each plant sample was filtered to get the ethanol solution. The

solutions were then subjected to ROTAVAP in order to obtain the crude extracts

of the plant samples.

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 Antibacterial Activity of
Lemon Peel Extract
2.3.3 Antibacterial Screening

The treatments including the positive control underwent Disc Diffusion Assay as

the method for antibacterial screening and compared the results in this procedure to

determine the antibacterial activity of Lemon peel extract. This method was conducted by

a micro analyst at the testing site and it includes the following procedures:

2.3.3.1 Preparation of 0.5 Mcfarland Standard

The test organisms used are S.areus and E. coli. This standard contains

approximately 1.5 x 10^8 CFU/ml (colony-forming units per milliliter) of the test

organism. Preparation of 0.5 Mcfarland Standard began by mixing 0.5ml of 0.048M

BaCl2 to 99.5 ml of 0.36 N H2SO4; and 5 ml was distributed into screw-cap tubes of the

same dimension as those to be used in preparing the culture suspension. The tubes were

tightly sealed and was stored in the dark at room temperature.

2.3.3.2 Preparation of the Nutrient Broth

A loopful of bacteria (S. aureus-gram positive & E.coli- gram negative) was taken

from the culture slant and was inoculated in 50ml nutrient broth. The culture broth was

incubated for 18-24 hours at 35°C. The culture broth was observed for turbidity,

indicative of microbial growth.

2.3.3.3 Adjusting the turbidity of the inoculum

Aseptically, 5ml of culture broth was transferred in sterile screw-capped tubes.

The bacterial suspension was agitated on a vortex mixer and immediately compared

against the 0.5 Mcfarland standard prepared. The adjusted turbidity serves as the

inoculum for the microbial assay. This also serves as the inoculum to be swabbed onto

agar plates.

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 Antibacterial Activity of
Lemon Peel Extract
2.3.3.4 Preparation of Plates

15 ml of melted nutrient agar was poured into dry and sterile petri dishes and

letting the medium solidify. A sterile cotton swab was moistened into the test organism

(inoculum) suspension. A cotton swab with wooden applicator handles was used. The

sterile cotton swab was then dipped into the suspension of the test organism/inoculum.

The moistened swab was pressed and rotated firmly against the inside wall of the tube

just above the fluid level to remove the excess liquid.

2.3.3.5 Cotton Swabbing on the Agar Plate

After preparing the agar plates, the test organism was swabbed aseptically into the

solidified nutrient agar by streaking the swab over the entire surface of the agar plate

three times; the plate was rotated 60 degrees after each application to ensure an even

distribution of the inoculum.

2.3.3.6 Paper Disc Diffusion Method

A 13 mm absorbent filter disc was used to absorb the crude extract of the lemon

peel. One paper disc at a time was immersed into the plant extract using forceps. The

moistened filter disc was then laid on the seeded agar plate. The discs were tapped with

forceps to ensure maximum full contact with the agar medium. The plates were incubated

by storing them inverted at 37°C for 24 hours.

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 Antibacterial Activity of
Lemon Peel Extract
2.3.4 Measurement of Zone of Inhibition

After incubation, it was followed by reading of the assay plates. The “halo” or

“clearing” around the discs are known as the zone of inhibition (ZOI). The plates were

read inversely with a reflected light against a dark background. And with a ruler, the

diameter of each inhibition zone was measured in millimeters.

The antimicrobial activity is analyzed as:

<10 mm, expressed as inactive

10-13 mm, partially active

14-19mm, active

>19 mm, very active

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 Antibacterial Activity of
Lemon Peel Extract
2.3.6 Disposal

The samples were disposed immediately after the experimentation to avoid

hazardous effect. Before organizing all the reusable apparatus in their own storages, it

was properly cleansed and disinfected. While the other important equipment from the

laboratory was properly be taken care of the laboratory aide.

2.3.7 Statistical Analysis

The result of the disc diffusion assay went through a thorough analysis.

Univariate Analysis of Variance with Post Hoc Analysis was used for the scrutiny of the

results. The study chose to manage and organize the results with SPSS 16.0.

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 Antibacterial Activity of
Lemon Peel Extract
Chapter 3

RESULTS AND DISCUSSION

3.1 Antibacterial Screening

Table 2. Zone of inhibition of the treatment groups against S. aureus and E. coli

Diameter of Inhibition (mm)

Treatments After 24 hrs of incubation

Staphylococcus aureus Escherichia coli

Plant Sample 24mm 24mm 25mm 27mm 26mm 26mm

(Lemon Peel
Extract)

Positive 37mm 38mm 37.5mm 30mm 30mm 31mm

Control

(Clindamycin)

The table above shows the diameter of the zone of inhibition of the two treatments

which is the lemon peel extract and the positive control (Clindamycin) after 24 hours of

incubation. The lemon peel extract demonstrated an average diameter of 24mm against S.

aureus and 26mm against E. coli. While the positive control (Clindamycin) yielded an

average diameter of 37.5mm against S. aureus and 30.3mm against E. coli. Since the

minimum value to determine a very active antibacterial activity is 19mm, therefore, this

implies that both treatment groups exhibit a very active antibacterial performance against

S. aureus. and E. coli.

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 Antibacterial Activity of
Lemon Peel Extract

Figure 2. Diameter of zone of Inhibition of the two treatments against S. aureus

40 37 38 37.5
35
30 25
24 24
25
ZOI in mm

20
15
10
5
0
Trial 1 Trial 2 Trial 3

Lemon Peel Extract Positive Control

The chart shown above shows the different zone of inhibition of the 3 trial

performances of the two treatments against S. aureus only. The positive control,

Clindamycin, showed the best performance in inhibition in all trials.

Figure 3. Diameter of zone of Inhibition of the two treatments against E. coli

40
35 30 30 31
30 27 26 26
25
ZOI in mm

20
15
10
5
0
Trial 1 Trial 2 Trial 3

Lemon Peel Extract Positive Control

The chart shown above demonstrates the inhibition zones of the 3 trial

performances of both treatments against E. coli only. The positive control, Clindamycin,

showed the best performance in inhibition in all trials.

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 Antibacterial Activity of
Lemon Peel Extract

3.2 Statistical Analysis

Table 3. Univariate Analysis of Variance of the Zone of Inhibition of the Plant

Samples and the Positive Control against S. aureus and E. coli

Type III Sum of

Source Squares df Mean Square F Sig.

Corrected Model 302.667a 3 100.889 385.212 .000

Intercept 9308.907 1 9308.907 3.554E4 .000

Treatment 302.667 3 100.889 385.212 .000

Error 1.833 7 .262

Total 9877.250 11

Corrected Total 304.500 10

Table 3 shows the result of the analysis on the zone of inhibition of the lemon
peel extract (plant sample) together with the positive control (clindamycin) against S.
aureus and E. coli. The result shows that there is significant difference between the
treatments since the p-value is 0.00 which is less than 0.05 – the level of significance.
Thus, the null hypothesis was rejected.

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 Antibacterial Activity of
Lemon Peel Extract

Table 4. Post Hoc Analysis for the ANOVA Result (Scheffe)

Subset
Treatment N 1 2 3 4
Lemon Peel Extract against
3 24.3333
S. aureus
Lemon Peel Extract against
3 26.3333
E.coli
Positive control against E.
2 30.0000
coli
Positive Control against S.
3 37.5000
aureus
Sig. 1.000 1.000 1.000 1.000

Table 5 shows the result of the post hoc analysis of the mean absorbance reading on

the zone of inhibition of all treatments. This was employed to identify which treatments

are different from the others as ANOVA showed significant difference of the treatments.

Result shows that there is significant difference between all of the treatments

against S. aureus and E. coli since all of them fall on different subsets. It can also be

deduced from the data that the lemon peel extract against S. aureus showed the least

inhibitory performance or antibacterial activity among all other treatments; while the

positive control (clindamycin) against S. aureus showed the best inhibitory performance

or antibacterial activity.

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 Antibacterial Activity of
Lemon Peel Extract

Chapter 4

SUMMARY OF FINDINGS, CONCLUSIONS AND RECOMMENDATIONS

4.1 Summary of Findings

This research study investigated the antibacterial activity of Lemon Peel extract

against Staphylococcus aureus and Escherichia coli. Through antibacterial screening-

disc diffusion assay, the zone of inhibition of the plant sample was identified: the lemon

peel extract demonstrated an average diameter of 24 mm against S. aureus and 26mm

against E. coli. While the positive control (Clindamycin) yielded an average diameter of

37.5mm against S. aureus and 30.3mm against E. coli. Zones of inhibition that are greater

than 19mm is analyzed as a very active antibacterial activity, therefore, this implies that

both treatment groups exhibit a very active antibacterial performance against S. aureus

and E. coli.

The antibacterial screening also showed significant difference between the

treatments since the p-value is 0.00 which is less than 0.05 – the level of significance.

Meanwhile, results of the post hoc analysis of the mean absorbance reading on the zone

of inhibition of all treatments indicate that the lemon peel extract against S. aureus

showed the least inhibitory performance with 24mm zone of inhibition; while the positive

control (clindamycin) against S. aureus with 37.5 mm exhibited the highest inhibition

zone.

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 Antibacterial Activity of
Lemon Peel Extract

4.2 Conclusions

After the thorough analysis of the results, the following conclusions were formulated:

1. The lemon peel extract contains anti-bacterial property against S. aureus and E.

coli.

2. The mean diameter of the average ZOI of the lemon peel extract is 24mm against

S. aureus and 26mm against E.coli.

3. There is a significant difference between the average ZOI of the lemon peel

extract and the positive control in S. aureus and E. coli.

4.3 Recommendations

After the conclusions, the following are recommended for future studies:

1. Utilize different concentrations of plant samples for a more thorough investigation

of the potential effectiveness of the extract.

2. Incorporate different extraction processes in obtaining the extract to analyze the

most effective procedure.

3. Compare the plant sample with a control that is not based on an existing result but

with a current antibacterial screening- the same conducting date with all of the

treatments.

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 Antibacterial Activity of
Lemon Peel Extract

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 Antibacterial Activity of
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APPENDICES

Appendix A. Flow Chart

Collection of Plant Disc Diffusion

the Materials Extraction Assay

Statistical Measurement of
Disposal
Analysis Zone of Inhibition

Appendix B. Raw Data

Treatment Result of Inhibition Result of Inhibition (mm)
(mm) in S. aureus in E. coli

Lemon Peel Extract

Trial 1 24mm 27mm

Trial 2 24 mm 26 mm

Trial 3 25 mm 26 mm

Average 24 mm 26 mm
Positive Control

Trial 1 37mm 30mm

Trial 2 38mm 30mm

Trial 3 37.5 mm 31mm

Average 37.5 mm 30.3 mm

35
 Antibacterial Activity of
Lemon Peel Extract
Appendix C. Report of Microbiological Analysis

36
Antibacterial Activity of
Lemon Peel Extract

37
 Antibacterial Activity of
Lemon Peel Extract
Appendix D. Plagiarism Test

A. Plagiarism test- Introduction

A. Plagiarism test- RRL

38
 Antibacterial Activity of
Lemon Peel Extract
Appendix E. Documentation

Collection of Lemon Peels Air-drying of Lemon Peels

Obtained Lemon Peel Extract Absorbent Disc soaked in

concentrated lemon peel

S.areus and E. coli plates with S.areus and E. coli plates with
discs containing lemon peel discs containing lemon peel
extract before incubation extract after incubation
39
 Antibacterial Activity of
Lemon Peel Extract

CURRICULUM VITAE

NAME : Jam Chloe Zerrudo Rivas

GENDER : Female

HOME ADDRESS : Purok-6, Azpetia,

Prosperidad, Agusan del Sur

CIVIL STATUS : Single

PHONE NUMBER : 09350945907

DATE OF BIRTH : November 18, 2002

PLACE OF BIRTH : Butuan City

RELIGION : Born Again Christian

EMAIL ADDRESS : [email protected]

PARENTS : Moises C. Rivas

: Rowena Z. Rivas

EDUCATIONAL ATTAINMENT :

SCHOOL YEAR

ELEMENTARY : Philippine Normal University (CTL) 2009-2015

JUNIOR HIGH SCHOOL : Agusan del Sur National Science High School 2015-2019

SENIOR HIGH SCHOOL : Agusan del Sur National Science High School 2019-2021

40