Happy Birthday JP!!!!

University of San Agustin
COLLEGE OF HEALTH AND ALLIED MEDICAL PROFESSIONS

Medical Laboratory Science Program
CHAPTER 1
INTRODUCTION
Background of the Study

Staphylococcus aureus is derived from the Greek word staphyle which
means bunch of grapes and Latin word aurum which means gold. It is grampositive cocci that appear in clusters. This type of bacteria is a normal flora of the
skin and commonly found on hair of people and animals. Staphylococcus aureus
can cause several serious diseases which are sometimes hard to treat. Both
community-associated and hospital-acquired infections with Staphylococcus
aureus have increased in the past 20 years and the rise in incidence has been
accompanied by a rise in antibiotic-resistant strains. However, the continuous
use of synthetic drugs encourages the development of potent strains.
Hence,many people resort to alternative drugs from local plants (Edelstein et al.,
2011).
Escherichia coli are a large and diverse group of bacteria that is normally
present in environment, foods, and intestines of people and animals. Most strains
of E. coli are harmless but others can make you sick. Some strains of E. coli can
cause diarrhea, while others cause urinary tract infections, respiratory illness and
pneumonia, and other illnesses (CDC, 2013).
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Over the years, plant and vegetable extracts have been used as antibacterial agent for different bacteria. In the Philippines, the use of plant extracts
as medication has been passed on from one generation to another, and has
established its importance in health delivery, considering the expensive Western
treatment that most Filipinos cannot afford or cannot easily access. (Holgado
et.al, 2015)
Banana is used widely because of its nutritional values. In the past years,
there are studies that show that banana plant fruit and its parts can be used to
treat human diseases. Banana peel is a part of banana fruit that has antibacterial
and antifungal activity but has not been studied extensively. There are some
studies that relate the antibacterial activity of banana peel against periodontal
pathogens, but the varieties of banana that are commonly found here in the
Philippines are not yet tested and compared. Hence, the aim of this study is to
determine the antibacterial activity of banana peel ethanolic extract from Lakatan
(Musa sapientum Linnaeus), Sab-a (Musa acuminata balbisiana) and Seorita
(Musa acuminata paradisiaca) on Staphylococcus aureus (Kapadia et.al,
2015).

Objectives of the Study

This study aims to determine the antibacterial activity of Lakatan (Musa
sapietum Linnaeus), Sab-a (Musa acuminata balbisiana) and Seorita (Musa
acuminata paradisiaca) peel ethanolic extracts against Staphylococcus aureus
and Escherichia coli.
Specifically, this study aims;
1. to determine the phytochemical properties of
Lakatan (Musa
sapientum Linnaeus), Sab-a (Musa acuminata paradisiaca) and

Seorita (Musa acuminata paradisiaca) ethanolic peel extract
2. to determine the zone of inhibition of following treatments, positive and
negative control against Staphylococcus aureus and Escherichia coli:
Treatment LEE Lakatan Ethanolic Peel Extract
Treatment SEE Saba Ethanolic Peel Extract
Treatment SEE- Seorita Ethanolic Peel Extract
Treatment LSS- Lakatan, Saba and Seorita Ethanolic Peel Extract
Positive Control Penicillin
Negative Control Distilled Water
3. to determine if there is significant difference in the mean zones of
inhibitions produced by the different treatments, using penicillin as
positive control and distilled water as negative control.
4. to determine which among the treatments showed the widest zone of
inhibition against Staphylococcus aureus and Escherichia coli.
Hypothesis:

1. There is no significant difference between the mean zones of inhibition

produced by the treatments: LEE, SEE, SEE, LSSE, penicillin and
distilled water .
Conceptual framework
The paradigm of the study (Figure 1) shows the relationship between the
treatments and their effects on the diameter of zones of inhibition against
Staphylococcus aureus and Escherichia coli. This is based on the concept of
Andrada et al., (unpublished thesis 2014), cited in Buenafe et al., (unpublished
thesis 2013), that there are plant bioactive chemicals which are proven to have
antibacterial activity. Furthermore, phytochemicals can be combined to produce
antibacterial
activity.Variables
Thus, it possesses antibacterial activity based on the zone
Independent
of treaments
inhibition produced.
Different
LEE- Lakatan Ethanolic Peel Extract
SEE- Saba Ethanolic Peel Extract
SEE- Seorita Ethanolic Peel Extract
LSSEE Lakatan, Saba and
Seorita Ethanolic Peel Extract
SLSEE- Saba, Lakatan and
Seorita
Extract
Ethanolic Peel
Dependent Variable
SLSEE- Seorita,Lakatan and Saba

Ethanolic Peel Extract
Positive Control Penicillin
Negative Control Distilled water
Diameter of the zone of

inhibition produced against
Staphylococcus aureus and
Escherichia coli.

Figure 1
Scope and Delimitation of the Study

This study will be limited to identifying the antibacterial activity of Lakatan
(Musa sapientum Linnaeus), Sab-a (Musa acuminata paradisiaca) and
Seorita (Musa acuminata paradisiaca) banana peel ethanolic extract against
Staphylococcus aureus and Escherichia coli.
The control organism, Staphylococcus aureus (ATCC 25923) and
Escherichia coli (ATCC 25922) will be obtained from the University of the
Philippines Visayas, Miagao.
On the other hand, Lakatan (Musa sapientum Linnaeus) fruit will be
obtained from West Visayas State University, Lambunao campus, Saba (Musa

acuminata balbisiana) from Garin Farm and Seorita (Musa acuminata

paradisiaca) from Hibaoan, Pavia.
Extraction process will be conducted in the Mendel Hall Research
Laboratory of the University of San Agustin, Iloilo City.
The treatments will be made by weighing and diluting the crude extracts
and the positive control, Penicillin, which can be commercially purchased in the
capsule form.
The treatment did not include ethanol as a negative control since it already
has an antibacterial effect which can interfere with the zones of inhibition of other
treatments.
Furthermore, the statistical tool to be used in the analysis of data will be
the Mean, Standard Deviation, One-Way Analysis of Variance, followed by the
Post Hoc (Least Significant Difference) analysis with a confidence level of 0.05.
The phytochemical screening test will include only alkaloids, flavonoids,
saponins, tannins and terpennoids.
The researchers will no longer go beyond the things that are said above.
Definition of Terms
For the purpose of clarity and better understanding, the following terms
are defined conceptually and operationally.
Agar Well Diffusion Method is one method for quantifying the ability of
antibiotics to inhibit bacterial growth. It is based on the assumption that
antibiotics diffuse freely in the solid nutrient medium (Bonav et al., 2008)
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In this study, it refers to the method used to test the antibacterial activity of
Lakatan (Musa sapientum Linnaeus) , Saba (Musa acuminata balbisiana) and
Seorita (Musa acuminata paradisiaca) peel ethanolic extracts against
Antibacterial Activity Assay is used for drug discovery, epidemiology and

prediction of therapeutic outcome. Standard and clinically isolated bacterial
strains were used for antibacterial assays (Journal of Pharmaceutical Analysis,
2016).
In this study, the antibacterial activity assay is the method used to
determine the effectiveness of the treatments.
Antibiotics are chemicals that kill or inhibit the growth of bacteria and are
used to treat bacterial infections (Microbiology Society, 2016).
In this study, the antibiotic used is Penicillin.
Bacteria are a group of single-celled organisms that can cause infection

and can also be beneficial (Merriam-Webster, 2011).
In this study, the test organism to be used will be Staphylococcus aureus
and Escherichia coli.

Banana is an edible fruit, botanically a berry, produced by several kinds of

large herbaceous flowering plants in the genus Musa. (Wikipedia, 2016)
In this study, the peel of the banana varieties to be used are Lakatan,
Saba and Senorita .
Ethanolic extract is the material that is obtained from an object (plant

matter, coral samples, sponges, and aqueous mixtures, etc) by extracting that
item with ethanol .
In this study, it is the solvent to be used for extraction of the antibacterial
constituents .
Escherichia coli is conceptually defined as a facultative anaerobe gramnegative rod that lives I the intestinal tracts of living organisms in the health and
disease (Todar, 2012).
In this study, it is defined as the microorganism which will be tested for the
antibacterial activity.
Extract is a preparation containing the active ingredient of a substance in

concentrated form (Oxford Dictionary, 2016).
In this study, ethanolic extract of banana peel will be used.

Lakatan (Musa sapientum Linnaeus) are diploid banana cultivars from the
Philippines. It is one of the most common banana cultivars in the Philippines
(Wikipedia, 2016).
In this study, Lakatan (Musa sapientum Linnaeus) peel ethanolic extract
will be used as one of the treatments.
Peel is the outer covering of the banana fruit. Banana peels are used as
food for animals, in water purification, for manufacturing of several biochemical
products as well as for jokes and comical situations (The Free Dictionary, 2009).
In this study, peel extracts will be used as another treatment for
Penicillin is an antibiotic of low toxicity, produced naturally by molds of the

genus Penicilium and also semisynthetically, having a bactericidal action on
many susceptible Gram-positive and Gram-negative cocci and bacilli, some also
effective against certain spirochetes (The Free Dictionary, 2009).
In this study , it is the antibiotic to be used as positive control.
Sab-a (Musa acuminata balbisiana) is a triploid hybrid (ABB) banana

cultivar originating from the Philippines. It is primarily a cooking banana though it

can also be eaten raw. It is one of the most important banana varieties in
Philippine cuisine (Wikipedia, 2016).
In this study, Saba (Musa acuminata balbisiana) ethanolic peel extract
will be used as one of the treatments.
Seorita (Musa acuminata paradisiaca) (also known as Monkoy,

Cariosa, or Cuarenta Dias) are diploid cultivars of the banana Musa acuminata
originating from the Philippines. They are very small stout bananas which, like all
bananas belonging to the AA cultivar group, and are known for being
extraordinarily sweet. (Wikipedia, 2016)
In this study, Senorita (Musa acuminata paradisiaca) ethanolic peel
extract will be used as one of the treatments.
Staphylococcus aureus is a type of bacteria which stains Gram positive, is

non-motile cocci and appears as grape-like (staphylo-) clusters. (NewsMedical.net, 2016)
In this study, Staphylococcus aureus is the pathogenic bacterium that will
be utilized as the test organism for the anti-bacterial activity assay of Lakatan
(Musa sapientum Linneus) peel extract, Sab-a (Musa acuminata balbisiana)
peel extract and Seorita (Musa acuminata paradisiaca) peel extract.
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Zone of inhibition it is an area that surrounds the wafer where bacteria are
not capable of growing (Rao, 2012) and it determines microbial sensitivity
(Sustento, et al., 2013)
In this study, it will be used to determine the resistance or susceptibility of
the test organisms to the different treatments.
Significance of the Study

The results of the study will yield valuable information to the following:
Patients. To provide an alternative way to prevent and treat infections
caused by Staphylococcus aureus. The plant sample can be an abundant and
effective alternative antibacterial source for boils, pimples and other bacterial
infections.
Doctors. They may have alternative synthetic drug to give to the patients
suffering from the diseases caused by Staphylococcus aureus and other health
benefits given by banana.
Pharmaceutical Companies. They may have an alternative source in
producing antibiotics to treat patients who have acquired a disease caused by
Staphylococcus aureus.
Future researchers and Students. They may be able to use the results of
their study to supplement their own studies on their subject.
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CHAPTER 2
REVIEW OF RELATED LITERATURE
Staphylococcus aureus
Staphylococcus aureus is facultative anaerobic gram-positive cocci which
occur singly, in pairs, and irregular clusters.
It is a non-motile, non-spore
forming, and has both catalase and coagulase. Typical colonies are yellow to
golden yellow, smooth, circular, slightly raised, and hemolytic on 5% sheep blood
agar. However, many strains may appear dirty white and are non-hemolytic. It
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also gives a positive mannitol fermentation and deoxyribonuclease test.

(Turnidge, J., Rao, N., et al. 2008)
Staphylococcus aureus is both a commensal organism, which colonizes
the nares, axillae, vagina, pharynx, or damaged skin surfaces, and an extremely
versatile pathogen. It causes supercial lesions (boils, furunculosis), deep-seated
and systemic infections (endocarditis, osteomyelitis), and toxemic syndromes
(food poisoning by releasing enterotoxin into food, toxic shock syndrome by
release of superantigens into the blood stream, localized and generalized
exfoliation by the production of exfoliative toxins). In pyogenic infections the
pathogenesis results from the combined action of a variety of factors. Infection is
initiated when a break of the skin or mucosal barrier allows Staphylococci access
to adjacent tissues or to the bloodstream. Its also leading cause of food
poisoning,
resulting
from
the
consumption
of
food
contaminated
with
enterotoxins. Staphylococcal food intoxication involves rapid onset of nausea,

vomiting, abdominal pain, cramps, and diarrhea. Symptoms usually resolve after
24 hours. (Turnidge, J., Rao, N., et al. 2008)
Animal bite infected with Staphylococcus aureus can result in local
infections, cellulitis, erythema, tenderness, mild fever, adenopathy, and
lymphangitis (rarely). Scalded skin syndrome is caused by exfoliative toxins
secreted on the epidermis and mostly affects neonates and young children.
Other skin conditions caused by Staphylococcal exfoliative toxins include blisters,
13

skin loss, pimples, furuncles, impetigo, folliculitis, abscesses, poor temperature

control, fluid loss, and secondary infection. Staphylococcus aureus which can
also cause necrotizing fasciitis in immunocompromised individuals, although this
is very rare. Necrotizing fasciitis is life-threatening and causes severe morbidity.
Toxic shock syndrome is associated with vaginal colonization with toxinproducing Staphylococcus aureus during menstruation, complications with
staphylococcal infection at other sites, or complications of surgical procedures.
Deep infections include endocarditis, peritonitis, necrotizing pneumonia,
bacteremia, meningitis, osteomyelitis, septic arthritis, and infections of bones,
joints and organs. (Public Health Agency of Canada, 2011)
Banana
Banana is an elongated usually tapering tropical fruit with soft pulpy fresh
enclosed in a soft usually yellow rind. It is a widely cultivated perennial herb, of
the family Musaceae and Genus Musa, bearing fruit in compact dependent
(Merriam-Webster Dictionary,Inc., 2016).
Medicinal Properties of Banana

A study entitled Dietary Fiber, Phytonutrients and Antioxidant Activity of
Common Fruit Peels as Potential Functional Food Ingredient by Samonte and
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Trinidad (2012) shows that plantain banana peel is rich in dietary fiber and
phytonutrients and is a potential functional food/ingredients. Also the study
concluded that it has potential in preventing for risk of chronic diseases such as
diabetes mellitus, cardiovascular diseases and some cancers.
According to Quisumbing, E. (1978) in his book entitled Medicinal Plants
of the Philippines asserts that the growing plant parts contain tannic and gallic
acids which are used in medicine. It also contains phosphorus, calcium, iron,
chlorine, vitamin a, vitamin b, vitamin c, magnesia, soda, lime and potash.
Medicinally its leaves are used to dress inflamed and blistered surfaces and are
used as cooling application for headaches. Its also stated that the root extract is
antifebrile and are restorative. Moreover, the sap from the roots and juice from
the trunks were used to treat diarrhea by Cambodians and Malays. The flowers
were used to treat dysmenorrhea, menorrhagia, earache and heartburn.. Lastly
the fruit is used for treat anemia, diarrhea and scurvy by drinking its extracts and
eating the fruit itself.
Musa acuminata balbisiana also known as Sab-a banana is one of the

most important banana cultivars in the Philippines. It is eaten raw or cooked into
various desserts or dishes. It has the highest nutrient content because it has the
deepest roots among other banana plants, thus absorb more nutrients from the
soil (Safepac Corporation Davao, 2015).
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Lakatan (Musa sapientum Linnaeus) is one of the most distinct banana

cultivars widely grown in the Philippines today. It grows in many soil types and
land classification. It tolerates a wide range of soil pH from strong acidity of pH
3.4 to medium alkalinity of pH 7.8. It can thrive at an elevation of 1.5 to 1,000
meters above sea level and was found to produce sweeter fruits at elevation of
600 meters and more above sea level (PCARRD, 2004).
Among the banana varieties, lakatan is the most popular table fruit
preferred by most consumers due to its physical characteristics like sweetness
and sturdiness that makes its ripened state last longer without affecting its taste
(Castro, 2005).
Latakan (Musa sapietnum L.) is found all over Philippines. The male
inflorescence is extensively used as a vegetable. Its fibers are made into clothes
while the leaves are used for polishing floors and lining pots for cooking. The
bananas are good sources of vitamin A, B and C (Quisumbing, 1978)
Seorita/
Monkoy/
are diploid cultivars of
the
Cariosa/
Musa
banana Musa
acuminata
acuminata originating
paradisiacas
from
the
Philippines. They are small stout bananas which, like all bananas belonging to
the cultivar group and are known for being extraordinarily sweet. Seorita
bananas are some of the shortest banana cultivars, growing to a height of only
2.44 m (8.0 ft) with a pseudostem girth of 42 cm (17 in) at 1 m (3.3 ft) height. The
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pseudostem is green and shiny with a pink-purple underlying color. The leaves
are very waxy with petioles that are sometimes bordered with pink-purple to red.
The inflorescence hangs vertically with red-purple bracts which are yellow or
green on the inside surface. Male flowers are colored yellow and are persistent.
The plant begins to flower at about 231 days after planting. The time period from
flowering to harvesting is 54 days, hence the name Cuarenta dias (Revolvy,
2016).
A study performed by Jain, P., et al. (2011) entitled "Antibacterial and
antioxidant activities of local seeded banana fruits" resulted to a conclusion that
the ethanol extract of Musa Sapientum L. showed antibacterial activity in disk
diffusion method. Also the study found out that the seeds had the highest
phenolic content (19.46mg GAE/g of extract) followed by the banana pulp extract
and banana peel extract.
Another study conducted by Mokbel, M. and Hashinaga F. (2005) entitled
"Antibacterial and Antioxidant Activities of Banana (Musa, AAA cv. Cavendish)
Fruits Peel" revealed that the components of the Fruit Peels were active against
Gram-negative and Gram-positive bacteria. The most active components were
D-malic acid and 12-hydroxystearic acid. These extracts and its isolated
components were evaluated using disk diffusion methods and Minimum Inhibition
Concentration.
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Another study Antibacterial activity of extracts of twelve common

medicinal plants from the Philippines done by Penicilla and Magno (2011)
conducted an experiment on antibacterial activity of Musa Sapientum extract had
slight activity on S.aureus and B. subtilis only. Also it is stated that the plants
have very potent antibacterial activity.
The study Antibacterial and phytochemical analysis of Banana fruit peel
of Ehiowemwenguan, Emoghene and Inetianbor (2014) resulted as Musa
sapientum
have
antibacterial
potency
against
Klebsiella
pnuemoniae,
Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus in the

Antibacterial Activity Assay.
Moreover, a study entitled "Microbial Effect of Aqueous Banana Peel
Extract, Iraq" by Chabuck Z., et al. (2013), found out that Musa sapientum had
inhibitory effects on the microbial isolates. Among the microbial isolates the
highest inhibitory effect was against M. catarrhalis and Staphylococcus aureus
with 30mm inhibition zones. Thus, making Musa sapientum a good antibacterial
agent against Staphylococcus aureus.
The study of Alsula K. L., et al. (2013) entitled "Antibacterial Properties of
Cavendish Banana Peel (Yellow and Green) on Pathogens Escherichia coli and
Staphylococcus aureus", the results of the disk diffusion method the peel extract
on yellow Cavendish banana does not inhibit the growth of Staphylococcus
aureus but inhibits E. coli growth. Other the other hand the green cavendish
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banana extract could inhibit growth of both bacteria. Minimum Inhibitory

Concentration was performed to determine if the extracts were bacteriostatic.
The test resulted to a very turbid solution, and arrived to conclude that the
extracts were neither bacteriostatic nor bactericidal.
In the study of Fagbemi, J., et al. (2009) entitled "Evaluation of the
antimicrobial properties of unripe banana (Musa sapientum L.), lemon grass
(Cymbopogon citratus S.) and turmeric (Curcuma longa L.) on pathogens",
concluded that ethanolic extracts had greater microbicidal activity than aqueous
extracts from the same species of banana. All of the pathogens tested were
susceptible to the ethanolic extracts of the unripe banana, lemon grass and
turmeric. Also unripe banana had less that 2-hour killing time for Staphylococcus
aureus ATCC 25921.
A study entitled Anti-bacterial Potentials of Banana Peel in Ethanolic and
Water Extracts of Bahadar, N., et al. (2014) showed that the banana extracts
produced zones of inhibition on Staphylococcus aureus and E. coli culture, thus
banana extracts have antibacterial properties.
Phytochemicals
Phytochemicals are non-nutritive chemicals, produce by plants, that have
protective or disease preventive properties. Though it is produced by plants to
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protect themselves, these also are non-essential nutrients for humans and
protects them against diseases.
Alkaloids are any class of naturally occurring nitrogen containing bases.
These have diverse and important physiological effects on humans. Some of the
well-known alkaloids are morphine, strychnine, quinine, ephedrine and nicotine.
Most of the time a given specie contains a few kinds of alkaloids (Encyclopdia
Britannica, Inc., 2016). Alkaloids have antibiotic-enhancing activity and directantibacterial activity. It can inhibit bacterial growth or make bacteria more
susceptible to antibiotics like ciprofloxacin (Cushnie, 2014).
Flavonoids is a phytochemical famous as an antioxidant and antiinflammatory agent. It contributes as an additive and additive in food (The
George Mateljan Foundation, 2016). Flavonoids are also found to have
physiologically active constituents have been used to treat human diseases. Also
Flavonoids possess antifungal, antiviral and antibacterial potential. Quercetin, for
example, inhibits of DNA gyrase, cytoplasmic membrane function, and energy
metabolism. (Cushnie, 2006)
Saponins are glycosides with a distinctive foaming characteristic.
Consisting of a polycyclic aglycone that is either a choline steroid or triterpenoid.
It has the ability to foam is due to the combination of the nonpolar sapogenin and
the water soluble side chain. Some saponins are able to assist in digestion by
accelerating the body's ability to absorb calcium and silicon (Cornell University,
20

2015). Saponin is known to have antibacterial activity against Gram-positive

bacteria and discrete antifungal activity (Avato, 2006).
Tannins also called polyphenols are naturally occurring compounds,
commonly found in grapes (Vinepair, Inc., 2016). Tannin is known to have
antimicrobial activity by inhibition of extracellular microbial enzymes, iron
deprivation, deprivation of the substrates required for microbial growth or
inhibition of oxidative phosphorylation (Scalbert, 1991).
Terpenoids or isoprenoids is a subclass of the prenyllipids. It represents
the oldest group of small molecular products synthesized by plants. They are
largely found in essential oils and were used in the Ancient Egypt for various
religious aims (Leray, 2016). Terpenoids can increase the permeability of living
cells thus inhibit the growth of bacteria, yeast and molds (Griffin, 2000).
Floxacillin
Floxacillin is a type of penicillin that works by interfering with bacterial cell
wall formation. Bacteria need cell walls for their survival. The cell wall regulates
everything that goes in and out of the bacterium. Floxacillin interferes by
weakening the bonds that hold the bacterial cell wall together.
A study conducted by Jennifer M Andrews (2006) entitled Determination
of Minimum Inhibitory Concentrations. The Minimum Inhibitory Concentration of
Floxacillin is against Staphylococcus aureus ATCC 25923 is 0.6mg/L.
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Summary of Related Literature

In conclusion, Staphylococcus aureus is facultative anaerobic grampositive cocci which occur singly, in pairs, and irregular clusters. Its non-motile,
non-spore forming, and has a positive test result for mannitol fermentation,
deoxyribonuclease, catalase and coagulase. It causes supercial lesions (boils,
furunculosis), deep-seated and systemic infections (endocarditis, osteomyelitis),
and toxemic syndromes (food poisoning by releasing enterotoxin into food, toxic
shock syndrome by release of superantigens into the blood stream, localized and
generalized exfoliation by the production of exfoliative toxins.
Moreover, Musa acuminata balbisiana also known as Sab-a banana is
an ingredient in Filipino cooking and known for its high nutrient content. Lacatan
or Musa sapientum L. on the other hand, is widely grown and due to its versatility
in growing in different conditions. Lastly Seorita also known as Monkoy,
Cariosa and Musa acuminata paradisiacas are very small stout bananas. Due
to the tannic and gallic acid contained in the plant itself it has viable use in
medicine. It has been used for treating blister, inflammation, diarrhea,
dysmenorrhea, menorrhagia, earache and heartburn. Lastly the fruit is used for
supportive treatment for anemia, diarrhea and scurvy.
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Floxacillin is an antibiotic belonging to the penicillin group. It kills bacteria

by interfering with cell wall formation. And without the cell wall the bacteria would
die.
Phytochemicals are non-nutritive plant-produced chemicals that have
protective or disease preventive potentials.
CHAPTER 3
METHODOLOGY
Research Design and Purpose of the Study

The purpose of the study is to identify the antibacterial activity of Lakatan
(Musa sapientum Linnaeus), Saba (Musa acuminata balbisiana) and Seorita
23

(Musa acuminata paradisiaca) peel ethanolic extract against Staphylococcus

aureus and Escherichia coli.
This research is Post-Test Only Control Group Experimental Research
Design, Quantitative data will be collected and analyzed to answer the
objectives of the study. The results obtained in the data gathering
procedure will be validated by a registered medical technologist and will
be standardized based on CLSI Standards (2012).
Study Locale
This study will be conducted at the Gregor Mendel Research Laboratory of
the University of San Agustin.
Data Gathering Procedure

Plant Collection and Identification
Two kilograms of each ripe Lakatan (Musa sapientum Linnaeus) fruit from
West Visayas State University, Lambunao campus, Saba (Musa acuminata
balbisiana) from Garin Farm and Seorita (Musa acuminata paradisiaca) from
Hibaoan, Pavia will be used for the study in order to ensure that the bananas ore
24

of the correct variety ,the plants used will be identified and verified by the Bureau
of Plant Industries Iloilo City Office.
Processing of Plant Samples

The Lakatan (Musa sapientum Linnaeus), Saba (Musa acuminata
balbisiana) and Seorita (Musa acuminata paradisiaca) peels will be washed
thoroughly with tap water to remove unwanted materials and allowed to dry. They
will be cut into smaller pieces and weighed to approximately 200 grams for each
extraction (IOSR, 2014).
Preparation Ethanol Extract

For the plant extraction with ethanol, Claustros procedure will be adapted.
The Banana peel cuts, weighing approximately 200 grams will be placed
separately in flasks containing 400 mL of 95% ethyl alcohol and soaked for 48
hours, with the flask stoppered tightly, and mixed constantly with the aid of
mechanical shaker. After 48 hours of soaking, it will be filtered using filter paper
leaving the filtrates to be concentrated to 20 mL with the use of a Rotary
Evaporator. The concentrated extracts are to be kept in 60 mL amber bottles in
temperature below 5C. The remaining crude extracts reserved for phytochemical
analysis will be kept in the same way (IOSR, 2014).
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Subculture of Bacterial Isolates

The pure cultures of test microoganisms, Staphylococcus aureus (ATCC
25923) and Escherichia coli (ATCC 25922) will be obtained from the University of
the Philippines Visayas, Miagao Campus. To confirm that bacteria received are
pure culture, the test organisms will be subcultured in Eosin Methylene Blue agar
and Mannitol Salt Agar for Escherichia coli and Staphylococcus aureus
respectively (CLSI, 2012).
Screening for presence of Phytochemicals.

Alkaloids
One milliliter of freshly prepared crude ethanolic extracts of Sab-a (Musa
acuminata balbisiana ), Lakatan (Musa sapientum Linnaeus) and Seorita
(Musa acuminata paradisiaca) peel extracts will be transferred in separate test
tubes added with Hagers Reagent. The appearance of creamy white precipitate
confirms the presence of alkaloids (Sofowora, 1993).
Flavonoids
Three milliliters of freshly prepared ethanolic extracts of Sab-a (Musa
(Musa acuminata paradisiaca) peel extracts will be transferred in three test
26

tubes. Three pieces of magnesium ribbons will be added to each tube, the
reaction is then enhanced with addition of three drops of concentrated
hydrochloric acid. An orange to pink discoloration is indicative of the presence of
flavonoids (Sofowora, 1993).
Saponins
acuminata balbisiana ), Lakatan (Musasapientum Linnaeus) and Seorita
(Musa acuminata paradisiaca) peel extracts will be transferred in clean test
tubes. Three fourths of the tubes will be filled with water and sealed with cork
stopper. The tubes will be shaken vigorously to froth and let it stand for 3-5
minutes. Continued frothing indicates the presence of saponins (Sofowora,
1993).
Tannins
tubes. A drop of 5% ferric chloride will be added in each test tube. A green to
black precipitation indicates the presence of tannins (Sofowora, 1993).
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Terpennoids
tubes. A drop of concentrated sulfuric acid will be added to each tube.The
appearance of a dark brown to black color is indicative of terpennoids
(Sofowora, 1993).
Preparation of Treatment
Antibacterial Susceptibility Testing (Agar-Well Diffusion Method)

In this method, the antibacterial constituents present in the plant extracts
are allowed to diffuse into the medium and allowed to interact in a plate freshly
seeded with the test organisms. The zones of inhibition will appear uniformly
circular as there will be a confluent lawn of growth. The diameter of the zone of
inhibition will be measured in millimeters (CLSI, 2012). The reagents used will be
28

Mueller-Hinton Agar Medium, Nutrient Broth, and Penicillin solution as the

positive control.
This procedure will start with the preparation of 140-mm petri plates
containing Mueller-Hinton medium with a height of 4mm. The procedure for
medium preparation can be found in Appendix C and the seeding of agar plates
can be found in Appendix B. The volume of the treatments pipetted into each well
(40L) will be based on the pretesting of the allowable content of each well by
the researchers, and also the study of Taye et al. (2011) where 50L of the plant
extract will be introduced into 6mm diameter well.
The treatments are as follows:
Treatment LEE- Lakatan (Musa sapientum Linnaeus) Ethanolic Extract (100%)
Treatment SEE- Saba (Musa acuminata x balbiciana) Ethanolic Extract (100%)
Treatment SEE- Seorita (Musa acuminata x paradisiaca) Ethanolic Extract
(100%)
Treatment LSSEE- Lakatan (Musa sapientum Linnaeus) (50%), Saba (Musa
acuminata x balbiciana) (25% )and Seorita (Musa acuminata x paradisiaca)
Ethanolic Extract (25%)
29

Treatment SLSEE- Saba (Musa acuminata x balbiciana) (50%), Lakatan (Musa

sapientum Linnaeus) (25%) and Seorita (Musa acuminata x paradisiaca)
Ethanolic Extract (25%)
Treatment SLSEE- Seorita (Musa acuminata x paradisiaca)(50%), Saba
(Musa acuminata x balbiciana) (25%) and Lakatan (Musa sapientum Linnaeus)
(25%)
Positive Control Penicillin (100g)
Negative Control Distilled water
Preparation of the Inoculum

For the preparation of the inoculum the procedure is based on Appendix C
. The researchers will make direct broth suspensions of the colonies from the
pure cultures of Staphylococcus aureus and Escherichia coli. The suspensions
will be adjusted to achieve a turbidity equivalent to a 0.5 McFarland turbidity
standard against a white background with contrasting black lines resulting to a
suspension containing approximately 1.25 x 107 cfu/ml of Staphylococcus aureus
ATCC 25923 and Escherichia coli ATCC 25922.
All procedures will be performed in a type IIA biosafety cabinet with
complete Personal Protective Equipment.
30

Disposal of Biological and Chemical wastes

Laboratories are mandated to follow specific rules regarding industry
specific waste disposal according to Occupational Safety and Health
Administration (OSHA), as seen in Appendix B, because several hundred
thousand people are working in laboratories, which are places where myriad
chemicals and biological agents are handled everyday some of which are
hazardous to health or even life-threatening, therefore has formulated guidelines
specifically for laboratories.
Waste products, test organisms, apparatus and other special wastes (e.g
gloves, masks, and news papers, used culture media and inoculum) will be
segregated, disinfected (10% hypochlorite solution) and sterilized according to
the protocols of the USA Research Laboratory. Properly trained laboratory
personnel will assist in the performance of the study. Protective measures
inherent in the laboratory will be applied (Buenafe et al. 2013)
Statistical Data and Analysis Procedure
31

To identify the mean zone of inhibition of the treatments against

Staphylococcus aureus and Escherichia coli, the mean and standard deviation
will be obtained.
To identify if there is a significant difference in the mean zones of inhibition
among the different treatments against Staphylococcus aureus and Escherichia
coli, One-Way ANOVA with = 0.05 will be used.
The Post Hoc (Least Significant Difference) will be used to identify which
among treatments of the Lakatan, Saba and Seorita peel ethanolic extract is
the most effective.
References
Acharya, T. (2013). Minimum Inhibitory concentration (MIC): Broth dilution
method-procedure and interpretation. Retrieved from
http://microbeonline.com/minimum-inhibitory-concentration-mic-brothdilution-method-procedure-interpretation/
Alkaloid. (2016). Retrieved from https://www.britannica.com/science/alkaloid
32

Ananya M, MD, (2016). News-Medical.net: What is Staphylococcus Aureus?.

http://www.news-medical.net/health/What-is-StaphylococcusAureus.aspx
Andrada, CM et al., (2014). Synergistic Antibacterial Activity of Ampicillin and
Calamansi (Citrus microcarpa) Crude Rind Methanolic and Aqueous
Extracts against Escherichia coli and Staphylococcus aureus.
Avato P., Bucci R., Tava A., Vitali C., Rosato A., Bialy Z. and Jurzysta M. (2006).
Antimicrobial activity of saponins from Medicago sp.: structure-activity
relationship. Retrieved from
https://www.ncbi.nlm.nih.gov/pubmed/16619355
Benefits of Cardava (Cardaba) Banana that You Might Not Know. (2015).
Retrieved from https://safepaccorporationdavao. wordpress
.com/2015/06/02/benefits-of-cardava-cardaba-banana-that-you-might-notknow/
Bonav, B. et al (2008). Principles of Assessing Bacterial Susceptibility to
Antibiotics sing the Agar diffusion Method. Retrieved from
http://m.jac.oxfordjournals.org/content/61/6/1295.full
Buenafe, et al. (2013). Synergistic Antibacterial Activity of Amoxicillin and
Bakhaw (Rhizophora mucronata) brk crudenmethanolic and aqueous
extract against Staphylococcus aureus and Escherichia coli . Unpublished
thesis.University of San Agustin, Iloilo.
Castro, D. P. (2005). Castro Lakatan Farm. A Case Study. Iloilo City, Philippines:
College of Agriculture, Central Philippine University, Iloilo City
Causes and Symptoms of Staphylococcus aureus. (2014). Retrieved from
http://www.health.state.mn.us/divs/idepc/diseases/staph/basics.html
Clinical Laboratory Standards Institute (2003). M100-S23 Performance
Standards for Antimicrobial Susceptibility Testing. Twenty Third
Informational Supplement, Pennsylvania.
Cushnie, T., Cushnie, B., Lamb, A. (2014). Alkaloids: an overview of their
antibacterial, antibiotic-enhancing and antivirulence activities. Retrieved
from http://www.sciencedirect.com/science/article/pii/S0924857914001885
Cushnie, T., Lamb, A. (2005). Antimicrobial activity of flavonoids. Retrieved from
https://www.ncbi.nlm.nih.gov/pubmed/16323269
33

Designs for Experiments. (2016). Retrieved from

http://web.csulb.edu/~msaintg/ppa696/696exper.htm#Post-Test Only
Control Group
Edelstein et al., (2011). The Jama Network: Antibiotics, Acne,
and Staphylococcus aureus Colonization, 147(8), 917-921.
Ehiowemwenguan, G. (2014). IOSR Journal of Pharmacy: Antibacterial and
phytochemical analysis of Banan a fruit peel, 4(8), 18-25.
Flavonoids. (2016). Retrieved from http://www.whfoods.com/genpage.php?
tname=nutrient&dbid=119
Griffin S.G. (2000). Aspects of antimicrobial activity of terpenoids and the
relationship to their molecular structure. Retrieved from
http://researchdirect.uws.edu.au/islandora/object/uws%3A243
Holgado et.al, (2015). Journal of ethnopathology: Antimicrobial activity of
selected Peruvian medicinal plants, 88, 199204.
Kapadia et.al, (2015). PMC: Detection of antimicrobial activity of banana peel
(Musa paradisiaca L.) on Porphyromonas
gingivalis and Aggregatibacter
actinomycetemcomitans: An in
vitrostudy, 6(4): 496499.
Leray, Claud. (2016). Terpenes, terpenoids. Retrieved from
http://www.cyberlipid.org/simple/simp0004.htm
Mandal, A. MD, (2016). News-Medical.net: What is Staphylococcus Aureus?
http://www.news-medical.net/health/What-is-StaphylococcusAureus.aspx
McJohnson, R. (2013). Waste disposal. Preferred waste disposal retrieved
from http://www.preferredsepticanddisposal.com/
Merriam-Webster, (2011). Definition of bacteria. Retrieved from
http://www.merriam webster.com/dictionary/bacteria
Oxford Dictionary, (2016). Definition of extract in English. Retrieved from
https://en.oxforddictionaries.com/definition/extract
Philippine Council for Agriculture, Forestry and Natural Resources Research and
Development. The Philippines Recommends for Banana (1992). Los
Baos, Laguna, Philippines: PCARRD.
34

Public Health Agency of Canada, (2011). Public health Agency of Canada:

Pathogen Safety Data Sheet - Infectious Substances
Quisumbing, E. (1978). Medicinal Plants of the Philippines. Iloilo City,
Philippines: Katha Publishing Co., Inc
Rao, K.B.V % Kumar, S.R.S, (2012). In vitro antagonistic and hemolytic activity of
marine Actinobacteria Retrieved from http://ijals.com/wpcontent/uploads/201/08/2.In-vitro-antagonistic-and -hemolyticactivity-ofmarine-Actinobacteria.pdf
Safepac Corporation Davao, (2015). Fruit Concentration. Retrieved from
https://safepaccorporationdavao.wordpress.com/
Saponins. (2015). Retrieved from
http://poisonousplants.ansci.cornell.edu/toxicagents/saponin.html
Seorita banana. Revolvy. (2016, June 5). Retrieved from
http://www.revolvy.com/main/index.php?s=Se%C3%B1orita
%20banana&uid=1575
Sofowora, J. (1993). Phytochemical constituents of some selected medicinal
plants. African Journal of Pure and Applied Chemistry V, 3, 228-233.
Retrieved from http://www.academicjournals.org/article/article1.pdf
Staphylococcus aureus-Pathogen Safety Data Sheets-Public Health Agency of
Canada (2012).Retrieved from http://www.phac-aspc.gc.ca/lab
bio/res/psdsftss/staphylococcus-aureus-eng.php
Sumpay, Jr., R. C. (2007). Growth Performance of Lacatan Banana (Musa
sapientum L.) fertilized with different brands of Commercial Organic
Fertilizer at Ilong Bukid Sumpay, Jr., R. C. (2007). Growth Performance of
Lacatan Banana (Musa sapientum L.) fertilized with different brands of
Commercial Organic Fertilizer at Ilong Bukid, San Rafael, Iloilo Conditions:
A Community based Participatory Research
Sustento, V.A (2013). Laboratory Activities in Bacteriolgy 2013 Edition. Iloilo:
University of San Agustin Publishing House.
Taye, B. et al. (2011). Antibacterial activities of selected medicinal plants in
traditional treatment of human wounds in Ethopia. Rettrieved from
http://www.ncbi.nml.nih.gov/omc/articles/PMc3614199/
35

The Free Dictionary, (2009). Peeling. Retrieved from

http://www.thefreedictionary.com/peeling
Thomas, J & Veda, B. et al. (2008). SCREENING OF TEN INDIAN MEDICINAL
PLANTS FOR THEIR
ANTIBACTERIAL ACTIVITY AGAINST SHIGELLA SPECIES
AND ESCHERICHIA COLI. Retrieved from
http://www.google.com.ph/?gfe_rd=cr
%ei=zbC7U_KMEcqE8QeYqYGwAg#q=agar+well+contains+40+microliter+of
+extract+or+treatment.
Todar, K. (2008). Staphylococcus auereus and Staphylococcal Disease.
Rerieved from:
http://kishnoy.med.harvard.edu/pdf/Torella_2010.pdf
Turnidge, J., Rao, N., et al. (2008). Libyan Journal of Medicine: Isolation of
antibiotic-resistant pathogenic and potentially pathogenic bacteria from
carpets of mosques in Tripoli, Libya, v5i0.5536.
Xi'an Jiaotong University, (2016). Journal of Pharmaceutical Analysis: Methods
for in vitro evaluating antimicrobial activity, 6, 7179.
36

Appendix A
37

Steps in preparing the Lakatan,Saba and Seorita Peel:

1. Choose a fruit free from pesticides, dyes, and wax so there are no ther
components to interfere.
2. Wash and dry the fruit thoroughly, whether it is oganic or not.. Do not
include any colorations or bad spots.
3. Peel off the banana, use a knf, orscissors for cutting peels uniformly.
4. Put in a plastic zip or zip lock at room temperature in preparation for
extraction.
Plant Extraction:
1. Weigh 200g of pell for each extraction method.
2. Soak weight peel in 400mL ethanol in an Erlenmeyer flask for 48 hours
at room temperature with constant stirrig using the magnetic shaker.
3. Filter the extracts using a vcuum filtering system.
4. Subject the filtered extracts in rotary evapratio for further
concentration.
5. Store in air-tight vials below 5C until use.
38

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University of San Agustin

COLLEGE OF HEALTH AND ALLIED MEDICAL PROFESSIONS

Background of the Study

University of San Agustin

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Objectives of the Study

sapientum Linnaeus), Sab-a (Musa acuminata paradisiaca) and

University of San Agustin

1. There is no significant difference between the mean zones of inhibition

SLSEE- Seorita,Lakatan and Saba

Diameter of the zone of

University of San Agustin

Scope and Delimitation of the Study

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acuminata balbisiana) from Garin Farm and Seorita (Musa acuminata

University of San Agustin

Antibacterial Activity Assay is used for drug discovery, epidemiology and

Bacteria are a group of single-celled organisms that can cause infection

University of San Agustin

Banana is an edible fruit, botanically a berry, produced by several kinds of

Ethanolic extract is the material that is obtained from an object (plant

Extract is a preparation containing the active ingredient of a substance in

University of San Agustin

Penicillin is an antibiotic of low toxicity, produced naturally by molds of the

Sab-a (Musa acuminata balbisiana) is a triploid hybrid (ABB) banana

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Seorita (Musa acuminata paradisiaca) (also known as Monkoy,

Staphylococcus aureus is a type of bacteria which stains Gram positive, is

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Significance of the Study

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University of San Agustin

also gives a positive mannitol fermentation and deoxyribonuclease test.

enterotoxins. Staphylococcal food intoxication involves rapid onset of nausea,

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skin loss, pimples, furuncles, impetigo, folliculitis, abscesses, poor temperature

Medicinal Properties of Banana

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Musa acuminata balbisiana also known as Sab-a banana is one of the

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Lakatan (Musa sapientum Linnaeus) is one of the most distinct banana

are diploid cultivars of

University of San Agustin

University of San Agustin

Another study Antibacterial activity of extracts of twelve common

Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus in the

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banana extract could inhibit growth of both bacteria. Minimum Inhibitory

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University of San Agustin

2015). Saponin is known to have antibacterial activity against Gram-positive

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Summary of Related Literature

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Floxacillin is an antibiotic belonging to the penicillin group. It kills bacteria

Research Design and Purpose of the Study

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(Musa acuminata paradisiaca) peel ethanolic extract against Staphylococcus

Data Gathering Procedure

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Processing of Plant Samples

Preparation Ethanol Extract