1

INTRODUCTION

Background of the Study

Coconut (Cocos nucifera) is a well distributed fruit tree all

around the world, providing food, especially in the tropical and

subtropical regions and for its many uses it is often called the “tree of

life”. Almost all parts of the tree have benefits, from the fruits, to the

leaves, to the roots, even the husk. The constituents of C. nucifera

husks have some biological effects, such as anthelminthic, anti-

inflammatory, antinociceptive, antimicrobial, and antitumor activities.

Chromatographic and spectrometric procedures were also performed

to isolate and identify the components present in the extract

(Wilkinson, 2009).

Throughout history, humans have used medicinal plants

therapeutically, and minerals, plants, and animals have traditionally

been the main sources of drugs. Further, S. aureus is a gram-

positive coccal bacterium that is a member of the Firmicutes, and is

frequently found in the nose, respiratory tract, and on the skin. It is

often positive for catalase and nitrate reduction and is a facultative

aerobe that can grow without the need for oxygen. Although S.

aureus is not always pathogenic, it is a common cause of skin

infections such as abscesses, respiratory infections such as sinusitis,

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and food poisoning (Wilkinson, 2009). Moreover, Escherichia coli is a

gram-negative, facultatively anaerobic, rod-shaped, coliform bacterium

of the genus Escherichia that is commonly found in lower intestine of

warm-blooded organisms (Wilkinson, 2009).

According to Silva, et al (2013), on their study entitled “Chemical

and antimicrobial analysis of husk fiber aqueous extract from Cocos

nucifera ” The observed antimicrobial profile of Cocos nucifera husk

fiber extracts against S. aureus strains supports the use of the plant

on folk medicine. Two unreported substances present in the C.

nucifera husk fiber aqueous crude extract, gallic and ellagic acids

besides procyanidins already reported were isolated and identified. In

this regard, the elucidation of the composition of the extracts

contributes to the knowledge on the mechanisms involved in the

activity displayed by them, as already described by our research

group. The results presented here provide a motivation for further

investigation on the subject, since this plant has several medicinal

popular uses.

However, research studies reported the use of Coconut husk

fiber extract by different countries all over the globe. Recent studies

focuses on the Coconut husk fiber aqueous extract against S. aureus

and E. coli as a sort of inhibitory property. As the beneficial effects of

this plant material specifically on the plant extract, with respect to

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antimicrobial properties against common Pathogenic bacteria, are not

scientifically proven, thus, present study will be conducted (Esquanazi

et al., 2002).

This study will determine the inhibitory property of Coconut

(Cocos nucifera) husk fiber extract in S. aureus and E. coli by following

responsive processes. The husk of the coconut will be collected and

fiber extract will be prepared and antimicrobial properties against

common pathogenic bacteria like S. aureus and E. coli.

Objectives of the Study

The overall aim of this future study is to determine the

Inhibitory property of Coconut (Cocos nucifera) Husk Fiber Extract.

Specifically, the study aims to:

1. determine the degree of inhibition of C. nucifera husk fiber

extract on the growth of S. aureus and E. coli;

2. evaluate the difference on the degree of inhibition of C. nucifera

husk fiber extract on the growth of S. aureus and E. coli;

3. develop laboratory activity in microbiology from the study.

Significance of the Study

The result of the study will be beneficial to the following:

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Teachers. They can gain more information about the importance of

phytochemicals, especially in the Coconut (C. nucifera) Husk

Fiber Extract.

Students. They will be able to acquire knowledge about the functions

of phytochemicals specifically in Coconut (C. nucifera) Husk

Fiber Extract which can help in their studies.

Community. They will be able to know the significance of Coconut (C.

nucifera) Husk Fiber Extract.

Future Researchers. This study will be able to help the future

researchers who are eager to continue discovering new remedies

to combat diseases. Specifically, anti-microbial product.

Scope and Delimitation of the Study

This study focused on phytochemicals analysis and inhibitory

property of matured Coconut (C. nucifera) Husk Fiber Extract against

S. aureus and E.coli only as the test species. DNA analysis and

elemental analysis will not be included in the study.

Time and Place of the Study

This study was conducted and performed at Department of

Science and Technology (DOST) Laboratory, Butuan City on May to

September 2017.
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Operational Definition of Terms

For better understanding of this study, several relevant terms

are defined as follows:

Agar plate Method refers to the medium of experiment with the use of

agar plates.

Alkaloids refers to the phytochemical present in Coconut husk fiber.

Coconut refers to the plant being studied specifically it’s Husk Fiber

Extract.

Degree of inhibition refers to the amount or level present in Coconut

husk fiber extract that can inhibit the development of bacteria

specifically, S. aureus and E. coli;

Inhibitory property it refers to the amount of chemical present in

Coconut Husk Fiber Extract to decrease, limit, or block the

action or function in the development of S. aureus and E. coli.

Phytochemicals refers to the plant chemical which will be determined

in Coconut Husk Fiber Extract.

Phytochemical property refers to the chemical compounds that

occur in Coconut husk fiber extract

The phytochemical analysis is the analysis used to the presence of

phytochemicals in Coconut Husk Fiber Extract.

Test species refers to species being studied, specifically S. aureus and

E. coli.
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Conceptual Framework

The Biological constituents of Coconut (C. nucifera) depend on

the method and the efficacy of the inhibitory property of Coconut Husk

Fiber Extract.

Input Process Output

Agar Plate Method

R1 - Agar growth media (15ml)

+ (8ml) of coconut husk Zone of
Coconut (C. fiber extract. Inhibition
nucifera) R2 - Agar growth media (15ml) a. S. aureus
Husk Fiber + (8ml) of coconut husk
Extract fiber extract. b. E. coli
R3 - Agar growth media (15ml)
+ (8ml) of coconut husk
fiber extract.

Figure 1. Paradigm of Study

The Figure 1 above shows the research paradigm of the study

Specifically, the relationship between the input Coconut (C. nucifera)

Husk Fiber extract, the process (Preparetion of Agar Plate Method)

with Coconut (C. nucifera) Husk Fiber Extract, and the output Result

of Inhibitory Property evaluation.

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REVIEW OF RELATED LITERATURE

Phytochemical content of Coconut (C. nucifera)

Plants contain chemicals which serve as its secondary

metabolites (Yao et al., 2004) these chemicals are not necessary

nutrients but it can affect health (Temple, 2000). Flavonols,

isoflavones, flavones, phenolic acid, and glucosinolates are common

type of phytochemical. The formation of phytochemicals structure is

due to the process of conjugation of sugar and aglycones (Luthria and

Natarajan, 2009). The study of natural product is called

phytochemistry. These phytochemical are present in vegetables, fruits,

drinks and preservatives (Doughari and Obida, 2008). In plants,

phytochemicals serve as protection and defense mechanism of plants

against pest and microbial organism (Krishnaiah et al., 2007).

In almost plants, 10-15% of concentrations are alkaloids. They

are water soluble and heterocyclic compound because it contains

nitrogen in a negative oxidation state that can form salt with acids.

Most of the alkaloids are having a definite melting point, a crystalline

solid, colorless, and bitter in taste. It activated and inhabited the

central process at cellular and organ level in animals. Moreover,

alkaloids possess allergic and antimitotic effects at cellular level.

Alkaloids play a vital role in chemical ecological perspectives life plant-

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microbial interactions, plant-herbivore interaction, and plant-plant

interaction. Though poisonous, many alkaloids have a physiological

effect that has medicinal value that can cure diseases including

diabetics, cardiac dysfunction, cancer, malaria, etc. In addition,

alkaloids are also used as anaesthesia to relieve (Mahajan et al.,

2014).

Furthermore, Phytochemical studies of the coconut fiber

(mesocarp) ethanolic extract revealed that the presence of phenols,

tannins, leucoanthocyanidins, flavonoids, triterpenes, steroids, and

alkaloids, while a butanol extract recovered triterpenes, saponins, and

condensed tannins. Notably, compounds like flavonoids having

antioxidant action are widely distributed in edible vegetables, fruits,

and many herbs. The lyophilized extract and fractions, as well as ethyl

acetate extracts, from the C. nucifera fiber are rich in polyphenols,

compounds such as catechins, epicatechins, tannins, and flavonoids.

The constituents of the liquid albumen were identified as vitamin B,

nicotinic acid (B3, 0.64 mg/mL), pantothenic acid (B5, 0.52 mg/mL),

biotin (0.02 mg/mL), riboflavin (B2, o0.01 ng/mL), folic acid (0.003

mg/mL), with trace quantities of vitamins B1, B6, and C, pyridoxine,

thiamine, folic acid, amino acids, L-arginine, plant hormones (auxin,

1,3-diphenylurea, cytokinin), enzymes (acid phosphatase, catalase,

dehydrogenase, diastase, peroxidase, RNA polymerases), and growth-

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promoting factors. Furthermore, oil extracted from the solid albumen

is primarily lauric acid and alpha tocopherol. Root phenolic

compounds were identified as flavonoids and saponins. Other

compounds identified in leaf epicuticularwax were lupeol methylether,

skimmiwallin, [3b-methoxy-25-ethyl-9,19-cyclolanost-24(241)-ene],

and isoskimmiwallin [3b-methoxy-24-ethyl-9,19-cyclolanost-25 (251)-

ene]. These studies were performed using coconut husk fiber extracts,

suggesting that this part of the plant is a highly potent analgesic.

Cocos nucifera may enable the production of new low-cost medicines

for several ailments and may provide a very inexpensive source of new

analgesic drugs. Further investigations are warranted. Further

bioassay-guided fractionation and isolation of specific molecules are

highly recommended so that the chemical moiety responsible for the

activity can be identified and its mechanism of action established

(Bano, 2007).

Economic Importance of Coconut

The coconut palm is one of the most beautiful and useful trees

in the world, grown in more than 80 tropical countries. It supplies

food, drink and shelter and also supplies raw material to number of

industries intimately connected with domestic as well as economic life.

All the parts-of the Wonder palm are useful to making in one way or
 10

other. On account of this, the palm has been regarded as Kalpvriksh

(Tree of heaven) (Kobayashi et al., 2015).

C. nucifera L. (Arecaceae) is a widely distributed species around

the tropical areas. Popular uses have been reported in the treatment of

arthritis and diarrhea. This study evaluates the antimicrobial activity

of husk fiber aqueous extract from C. nucifera and performed the

identification of some biological active substances. The minimal

inhibitory concentration (MIC) against human pathogen

microorganisms was determined. Chromatographic and spectrometric

procedures were also performed to isolate and identify the components

present in the extract. In the MIC assay of crude aqueous extract, only

the methicillin sensible and the resistant (MRSA) S. aureus strains

were susceptible at 156 μg/mL. The ethyl acetate partition taken from

crude extract was more promising (MIC of 78 μg/mL). No fungal

growth inhibition was observed. Catechin, epicatechin, two

procyanidin dimers and condensed tannins were found in the organic

phase. In addition, gallic and ellagic acids were detected for the first

time in C. nucifera husk fiber. Gallic acid showed MIC of 39 μg/mL

and minimal bactericidal concentration (MBC) at 78 μg/mL. Ellagic

acid was not active against the tested strains, as well as catechin and

epicatechin. Additionally catechin, epicatechin, two procyanidin

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dimers and condensed tannins were also detected. The antimicrobial

activity observed was selective to S. aureus strains (Silva, et al., 2013).

Common disease caused by Staphylococcus aureus

S. aureus (“staph”) facts, including how S. aureus is spread,

common symptoms and complications. S. aureus has long been

recognized as one of the most important bacteria that cause disease in

humans. It is the leading cause of skin and soft tissue infections such

as abscesses (boils), furuncles, and cellulitis. Although most staph

infections are not serious, S. aureus can cause serious infections such

as bloodstream infections, pneumonia, or bone and joint infections .

Most infections caused by S. aureus are skin and soft tissue infections

such as abscesses or cellulitis (Chan, et al., 1998).

S. aureus causes the vast majority of skin and soft tissue

infections (SSTIs) in humans. S. aureus has become increasingly

resistant to antibiotics and there is an urgent need for new strategies

to tackle S. aureus infections. Vaccines offer a potential solution to

this epidemic of antimicrobial resistance. However, the development of

next generation efficacious anti S. aureus vaccines necessitates a

greater understanding of the protective immune response against S.

aureus infection. In particular, it will be important to ascertain if

distinct immune mechanisms are required to confer protection at

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distinct anatomical sites. Moreover, recent discoveries have

highlighted that interleukin-17-producing T cells play a particularly

important role in the immune response to S. aureus skin infection and

suggest that vaccine strategies to specifically target these types of T

cells may be beneficial in the treatment of S. aureus SSTIs. S. aureus

expresses a large number of cell wall-anchored (CWA) proteins, which

are covalently attached to the cell wall peptidoglycan. The virulence

potential of many CWA proteins has been demonstrated in infection

models; however, there is a paucity of information regarding their roles

during SSTIs. In this review, we highlight potential candidate antigens

for vaccines targeted at protection against SSTIs (Adebayo, 2013).

S. aureus is responsible for numerous food poisonings due to

the production of enterotoxins by strains contaminating foodstuffs,

especially dairy products. Several parameters, including interaction

with antagonistic flora such as Lactococcus lactis, a lactic acid

bacterium widely used in the dairy industry, can modulate S. aureus

proliferation and virulence expression. We developed a dedicated S.

aureus microarray to investigate the effect of L. lactis on

staphylococcal gene expression in mixed cultures. This microarray was

used to establish the transcriptomic profile of S. aureus in mixed

cultures with L. lactis in a chemically defined medium held at a

constant pH (6.6). Under these conditions, L. lactis hardly affected S.

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aureus growth. The expression of most genes involved in the cellular

machinery, carbohydrate and nitrogen metabolism, and stress

responses was only slightly modulated: a short time lag in mixed

compared to pure cultures was observed. Interestingly, the induction

of several virulence factors and regulators, including the agr locus,

sarA, and some enterotoxins, was strongly affected. This work clearly

underlines the complexity of L. lactis antagonistic potential for S.

aureus and yields promising leads for investigations into nonantibiotic

biocontrol of this major pathogen (Akinpelu, et al., 2015).

S. aureus is a major human pathogen that causes a wide range

of diseases, such as septicemia, meningitis, endocarditis,

osteomyelitis, and toxic shock syndrome. S. aureus is also a major

causative agent of food poisonings. Staphylococcal food poisonings

(SFP) are due to the production of staphylococcal enterotoxins (SEs) by

S. aureus strains contaminating foodstuffs. Milk and dairy products

are often incriminated in SFP, especially in France. During the cheese-

making process, contamination by S. aureus may come from several

sources. Raw milk (notably milk from mastitic cows) may be a source

of contamination; however, strains endemic in the processing plant

environment (present on fomites and in biofilms), as well as strains

present in healthy human carriers, may also contaminate dairy

products. A complex and balanced ecosystem is required throughout

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the cheese-making process for the development of the organoleptic

properties of the final product. The inhibition of S. aureus growth and

of the production of SE in cheeses must thus be achieved by

nonantibiotic means so that the biodiversity and correct microbial

ecology of the environment may be maintained, yielding a satisfactory

final product. To this end, the appropriate and rational use of lactic

acid bacteria (LAB) appears to be a promising strategy in the fight

against S. aureus (Dua, et al., 2013).

Interactions between S. aureus and LAB in several ecosystems,

including fermented foodstuffs but also nasal and vaginal

environments, have been explored for years. However, apart from

offering general observations, studies have not yet comprehensively

described the mechanism of inhibition of S. aureus by LAB. LAB have

frequently been considered to be a factor that influences S. aureus

growth by physically and chemically changing the environment.

Several possibilities have been proposed to explain the inhibition of S.

aureus by LAB, including the production of bacteriocins and hydrogen

peroxide, competition for nutrients, and needless to say, acidification,

even if the impact of the latter mechanism has been questioned.

Studies of the inhibition of S. aureus virulence expression by LAB,

including the inhibition of SE production, are quite scarce. Few

studies have described the inhibition of enterotoxin production in the

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presence of LAB, and none have unraveled the mechanisms involved

in such antagonism (Renjith, et al., 2013).

Virulence expression in S. aureus is controlled by complex

regulatory networks that include two-component systems (e.g., AgrAC,

SrrAB, SaeRS, and ArlRS) and transcription factors (e.g., SarA and its

homologs and Rot) but also the Clp proteolytic complex and the

alternative sigma factor σB. Among these regulatory systems, the

accessory gene regulator (agr) is a key modulater of virulence

expression. It combines a two-component system and a quorum-

sensing system. The agr system comprises two transcripts, RNAII and

RNAIII, which are divergently transcribed. RNAII encodes AgrBDCA,

the structural components of the quorum-sensing system. RNAIII is

the effector molecule of the agr system but also encodes the delta-

hemolysin. The accessory gene regulator is responsible for the post-

exponential-phase induction of several virulence factors, including

exoproteins). The activity of agar itself can be modulated by other

regulatory systems and in response to environmental variations

(Alviano et al., 2004).

Exploring the interactions between positive microbiota and S.

aureus and understanding the mechanisms involved in the inhibition

of S. aureus growth or virulence will enable the setting of rational

screening criteria for positive microbiota to be used in food

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preservation and will also yield new strategies for fighting against this

major human pathogen. Such an endeavor is, however, complex and

requires the employment of new approaches. DNA microarrays are

powerful tools for the investigation of these multifactorial interactions.

The microarray technique has been widely used to investigate the

global responses of S. aureus to several stimuli. Nonetheless, a

transcriptomic analysis of S. aureus in interaction with other bacterial

species has, as of yet, not been reported. In this study, we have

developed a dedicated S. aureus-specific microarray, usable for the

study of mixed cultures of S. aureus and LAB. This microarray was

used to investigate the transcriptomic response of S. aureus to the

presence of Lactococcus lactis in a chemically defined medium (CDM)

at a constant pH. Surprisingly, while L. lactis hardly affected S. aureus

growth and the expression of genes involved in central metabolism

processes under these conditions, it was able to dramatically impair

the expression of several virulence genes (Easmon, 1983).

Common diseases caused by Escherichia coli

E. coli is one of the most frequent causes of many common

bacterial infections, including choelecystitis, bacteremia, cholangitis,

urinary tracth infection(UTI), and traveler’s diarrhea, and other clinical

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infections such as neonatal meningitis and pneumonia (Amor, et al.,

2006).

Morphology of Staphylococcus aureus

Staphylococci are Gram-positive cocci about 0.5 – 1.0 μm in

diameter. They grow in clusters, pairs and occasionally in short

chains. The clusters arise because staphylococci divide in two planes.

The configuration of the cocci helps to distinguish micrococci and

staphylococci from streptococci, which usually grow in chains.

Observations must be made on cultures grown in broth, because

streptococci grown on solid medium may appear as clumps. Several

fields should be examined before deciding whether clumps or chains

are present (DebMandal, et al., 2011)

S. aureus is one of the main causes of hospital-and community-

acquired infection which can results in serious consequences

(solangih, et al., 2001). Nosocomial S. aureus infections affect the

blood stream, skin, soft tissues and lower respiratory tracts. S. aureus

can be a cause of central venous catheter-associated bacteremia and

ventilator-assisted pneumonia. It also causes serious deep-seated

infections, such as endocarditis and osteomyelitis (Alviano, et al.,

2004). In addition to the infections listed above, S. aureus is often

responsible for toxins-mediated diseases, such as toxic shock

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syndrome, scolded skin syndrome and staphylococcal foodborne

disease (SFD). Hospitalized patients are particularly exposed to S.

aureus infections due to their compromised immune system and

frequent catheter and insertions and injections (koschek, et al., 2008).

Penicillin was used in order to combat this organism in the year

1940, and all isolates were sensitive to penicillin during that period.

With this antibiotic, the mortality rate of bacteremia caused by S.

aureus decreased from striking 80% to 25% (Le Loir, et al., 2003).

While in 1944, the first penicillin-resistant strange of S. aureus was

discovered. As clinicians used more and more penicillins as the

primary treatment of S. aureus infections, the prevalence of penicillin-

resistant strains increased in the 1960’s. It was during this time that a

new class of antibiotic was developed to target this pathogen

specifically. This class of antibiotics is known as the penicillinase-

resistant penicillins, which includes nafcillin, dicloxacillin, and

oxacillin. One year after this class of antibiotic was developed, a

resistant strain known as methicillin-resistant S. aureus (MRSA) was

discovered in both the United States and the United Kingdom (Zaoutis,

2006).

Methicillin-resistant S. aureus (MRSA) became increasingly

prevalent, especially in large hospitals, in the 1970’s. From the late

1970’s to the early 1990’s, methicillin resistant S. aureus (MRSA) was

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usually a hospital-acquired pathogen. It was not until 1998 that the

first case of CA-MRSA in children was identified, since then CA-MRSA

has become more prevalent in children throughout the United States

(Zaoutis, 2006).

Morphology of Escherichia coli

E. coli is a gram negative, rod-shaped bacterium that is

commonly found in the lower intestine of warm blooded organism

(endotherms). Most E. coli strains are harmless, but some stereotypes

can cause serious food poisoning in humans. The harmless strains are

part of the normal flora of the gut, and can benefit their host by

producing vitamin k2, and by preventing the establishment of

pathogenic bacteria within the intestine. E. coli and related bacteria

constitute about 0.1 % of gut flora, and fecal-oral transmission is the

major route through which pathogenic strains of the bacterium cause

disease (Deb Mandal, 2011).

Staphylococcus Aureus Treatment

S. aureus causes a variety of manifestations and diseases. The

treatment of choice for S. aureus infection is penicillin. In most

countries, S. aureus strains have developed a resistance to penicillin

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due to production of an enzyme by the bacteria called penicillinase.

The first line therapy is penicillinase-resistant penicillins like oxacillin

or flucloxacillin. Therapy is often given in combination with

aminoglycosides like gentamicin for more serious infections. The

duration of treatment depends on the site of infection and on severity

(Deb Mandal, 2011).

Genetic mutation and modification

Genetic mutation and modification is said to be the mechanisms

that makes S. aureus resistant to methicillin to produce Methicillin

Resistant S. aureus or MRSA. The modification in the mecA gene of the

bacteria which codes for an altered penicillin-binding protein leads to

a lower affinity for binding β-lactams (penicillins, cephalosporins and

carbapenems). This allows for resistance to all β-lactam antibiotics.

MRSA infections in both the hospital and community setting are

commonly treated with non-β-lactam antibiotics such as clindamycin

(a lincosamine) and co-trimoxazole (also commonly known as

trimethoprim/sulfamethoxazole). In severe cases vancomycin is used

(Zaoutis, 2006).
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Enzyme modification and other methods of resistance

Aminoglycosides like gentamicin, amikacin, streptomycin and

Kanamycin were once effective against Staphylococcal infections. They

have developed resistance by modifying enzymes, changing the

ribosomal attachment sites and by actively pushing out the drug from

the bacteria (Deb Mandal, 2011).

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METHODOLOGY

A. Research Design

The experiment conducted used Completely Randomized Design

(CRD). Completely randomized designs are the simplest in which the

treatments are assigned to the experimental units completely at

random. It allows every experimental unit, i.e. plot, animal, soil

sample, etc. to have equal probability of receiving a treatment that test

subjects are applied to treatment levels of the primary factor at

random. Levels of significance between treatments measured using T-

test on CRD. The study on Inhibitory property of Coconut (C. nucifera)

Husk Ash Fiber Extract on growth of S. aureus and E. coli was

conducted using CRD as follows;

B. Treatments

Agar growth media (15ml) + 8ml cocos nucifera husk fiber

extract.

C. Collection and drying of Cocos nucifera Husk Fiber Extract

The fibrous husk of matured Coconut (C. nucifera) were

collected from the local coconut growers at Trento Agusan del Sur and
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was weighed in 500g-1000g. It was washed with distilled water to

remove dirt, cut into smaller pieces and air dried for 10 days using the

traditional drier of Calamansi which was performed at Trento Agusan

del Sur.

D. Preparing the Coconut (C. nucifera) Husk material

This procedure underwent the standardized procedure by the

Department of Science and Technology DOST-Caraga, Ethanolic

Extraction Laboratory.

a. Materials

Weighing Scale

80% ethyl alcohol

Buchner funnel

Procedure:

1000g of Coconut (C. nucifera) husk was weighed using weighing

scale. The husk was treated with sufficient 80% ethyl alcohol to

completely submerge the material in a flask. The C. nucifera husk was

soaked for 24-28 hours. It was filtered through Buchner funnel with

gentle suction. The flask and Coconut husk materials was rinsed with

fresh portions of alcohol. The used water and coconut husk material
 24

was transferred to the funnel, combining the washings with the

filtrate. It was applied with gentle suction to complete collection of the

coconut husk. The husk’s residue was discarded and concentrates the

filtrate under vacou at temperature below 50-0°C to about 20 ml. the

husk volume was measured of the concentrated husk. The test

materials were completely dry, and syrupy in consistency. Water

soluble husk were dissolved in normal saline solution, oily and

resinous extracts were suspended in the saline solution with 0.2 %

between 80.

Normal saline solution, prepared by dissolving 0.85g of A.R

sodium chloride in 100ml distilled water.

E. Preparation of the test organisms

The researchers acquired S. aureus and E. coli culture slants

from Department Of Science and Technology -Microbiology Laboratory.

F. Preparation of treatment agar

It was poured with 15ml of melted nutrient agar into dry and

sterile petri dishes. Let the medium solidify. Moisten a sterile cotton

swab into the S. aureus and E. coli (inoculum) suspension. Use cotton

swab with wooden applicator handles. It was dip a sterile cotton swab
 25

into a suspension of the S. aureus / inoculum. Press and rotate the

moistened swab firmly against the inside wall of the tube just above

the fluid to remove the excess liquid.

Agar content:

0.5% Peptone

It is an enzymatic digest of animal protein. Peptone is the

principal source of organic nitrogen for the growing bacteria.

0.3% beef extract/yeast extract

It is the water-soluble substances which aid in bacterial growth,

such as vitamins, carbohydrates, organic nitrogen compounds and

salts.

1.5% agar

It is the solidifying agent.

0.5% NaCl

The presence of sodium chloride in nutrient agar maintains a

salt concentration in the medium that is similar to the cytoplasm of

the microorganisms.

Distilled water

Water is essential for the growth of and reproduction of micro-

organisms and also provides the medium through which various

nutrients can be transported.

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pH is adjusted to neutral (7.4) at 25 °C.

Procedure:

1. Suspend 28 g of nutrient agar powder in 1 litre of distilled water.

2. Heat this mixture while stirring to fully dissolve all components.

3. Autoclave the dissolved mixture at 121 degrees Celsius for 15

minutes.

4. Once the nutrient agar has been autoclaved, allow it to cool but

not solidify.

5. Pour nutrient agar into each plate and leave plates on the sterile

surface until the agar has solidified.

F.1. Preparation of different Experimental Treatments

Agar growth media (15ml) + 8ml C. nucifera husk fiber extract

G. Inoculation of Test Organism using Aseptic Technique

a. Label a Petri dish

1. Petri dishes will be labeled on the bottom rather than on

the lid.

2. Write close to the edge of the bottom of the plate to

preserve area to observe the plate after it has incubated.

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Labels usually include the organism name, type of agar,

date, and the plater's name or initials.

3. Using sterile cotton swabs, remove any visible water on

the agar in the plate or around the inner rim of the petri

plate.

4. Observe the plate and mentally divide it into three sectors.

The plate will then be turned clockwise (if you are right

handed) with the agar side up.

5. The second sector will then be at the top for streaking and

then the plate is turned again so that the third sector can

be streaked.

b. Sterilize the Transfer Loop before Obtaining a Specimen

1. To streak a specimen from a culture tube, metal transfer

loops will be sterilized by flaming the wire loop held in the

light blue area of a Bunsen burner just above the tip of

inner flame of the flame until it is red-hot. If a hot

incinerator is available, the loop may be sterilized by

holding it inside the incinerator for 5 to 7 seconds.

2. Once sterile, the loop is allowed to cool by holding it still.

 28

3. Do not wave it around to cool it or blow on it. When

manipulating bacteria, transfer loops are usually held like

a pencil.

4. If plastic disposable loops are being utilized, they are

removed from the packaging to avoid contamination and

after being used, are discarded into an appropriate

container.

5. A new loop is recommended for each sector of an isolation

streak plate.

c. Open the culture tube and collect a sample of specimen

using the sterile loop

1. Isolation will be obtained from any of a variety of

specimens. This protocol describes the use of a mixed

broth culture, where the culture contains several different

bacterial species or strains.

2. The specimen will be streaked on a plate could come in a

variety of forms, such as solid samples, liquid samples,

and cotton or foam swabs.

3. Material containing possibly infectious agents should be

handled appropriately in the lab using bio safety

procedure.
 29

4. Remove the test tube cap. It is recommended that the cap

be kept in your right hand (the hand holding the sterile

loop).

5. Curl the little finger of your right hand around the cap to

hold it or hold it between the little finger and third finger

from the back.

6. Modern test tube caps extend over the top of the test

tube, keeping the rim of the test tube sterile while the rim

of the cap has not been exposed to the bacteria.

7. The cap can also be placed on the disinfected table, if the

test tube is held at an angle so that air contamination

does not fall down into the tube.

8. Insert the loop into the culture tube and remove a loopful

of broth.

9. Replace the cap of the test tube and put it back into the

test tube rack.

d. Streak the Plate

The lid of the agar plate was opened just sufficiently enough to

streak the plate with the inoculation loop. Minimize the amount of
 30

agar and the length of time the agar is exposed to the environment

during the streak process.

e. Three Sector Streaks (t streak):

1. Sterilize the wire loop.

2. Cool the loop by touching it on the edge of the sterile agar

plate.

3. Dip the loop into the broth culture containing the mixture

of bacteria.

4. Lift the lid of the plate just enough to insert the loop. Drag

the loop over the surface of the top one-third of the plate

back and forth in a "zig-zag" formation.

5. The loop has picked up thousands of bacteria which are

spread out over the surface of the agar.

6. Sterilize the loop in the flame.

7. Turn the plate 90 degrees and drag the loop through the

area you have just streaked two to three times and

continue to drag the loop in a "zig-zag" formation in the

remaining half of the plate without touching that area

again.

8. Sterilize the loop in the flame.

 31

9. Turn the plate 90 degrees. Repeat the procedure. Drag the

loop two to three times through the area you just

streaked, and fill in the remaining area of the plate (zig-

zag formation), being very careful not to touch any of the

areas you previously streaked.

10. Incubate the plate for 24 hours. If you streaked correctly,

you will see isolated colonies in the third sector. The

heaviest growth will be in the first sector. There will be less

growth and some isolated colonies in the second sector.

The third area should have the least growth with isolated

colonies.

This process was performed at Department of Science and

Technology Microbiological Laboratory.

H. Incubation of the test organism

The test organisms were incubated for 18-24 hours at 35 °C.

 32

Data Gathering

Zone of Inhibition

A. The plates

1. Look for “HALO” or “Clearing” around the discs. This

known as the zone of inhibition.

2. Invert the plates and with a ruler measures the

diameter of each inhibition zone in millimeters.

3. Express result as mm diameter zone of inhibition.

4. Record the diameter of the paper disc used in the assay

in mm.

B. Analyzing the results

This procedure was taken from the Department of science and

Technology, Regional Standards and Testing Laboratory.

Statistical Treatment

T-test was used to determine the significant difference on the

zone of inhibition of the different treatments.

 33

Proper Waste Disposal

Wastes that accumulated during the experiment were properly

disposed according to standard laboratory waste disposal procedures.

C. Laboratory Activity in Microbiology

A Laboratory Activity in Microbiology was produced after the

study being conducted.

 34

Procedural Flow

Collection of
matured Coconut S. aureus
(C. nucifera) Husk
And
Fiber extracts
E. coli

Drying of Coconut
Husk Fiber Extract

Ethanolic
Extraction

Agar Plate Method

Inoculation of test
organism using
aseptic technique

Incubation

Zone of Inhibition

Statistical Analysis
using T-test

Proper waste
disposal

Laboratory Activity
in Microbiology

Fig. 1 Procedural flow of the study

 35

RESULTS AND DISCUSSION

Phytochemicals present in Coconut (C. nucifera) Husk Fiber

The Coconut (C. nucifera) Husk Fiber has the presence of

phytochemical properties such as of phenols, tannins,

leucoanthocyanidins, flavonoids, triterpenes, steroids and alkaloids.

This implies that Coconut husk fiber extract has the ability to inhibit

such bacteria and can be used for antimicrobial testing (Bano, S.

2007).

Degree of Inhibition

Table 1 shows the degree of inhibition of Coconut (C. nucifera)

husk fiber extract against S. aureus. Wherein, replicate 1 indicates the

result of 9 mm as the result of inhibition. The replicate 2 indicates the

result of 10 mm as the result of inhibition. The replicate 3 indicates

the result of 8 mm as the result of inhibition and 7 mm as the total

number of inhibition. This implies that the degree of inhibition of

Coconut (C. nucifera) husk fiber extract on the growth of S. aureus is

active and has the potential to inhibit S. aureus provided that 0 mm is

inactive, 1 mm – 5 mm is quite active, 6 mm – 10 mm is active, 11 mm

 36

– 15 mm is intermediately active and 16 mm – 20 mm is very active

(Bauer, et.al. 2007).

Table 1. The degree of inhibition of Coconut (C. nucifera) Husk

Fiber Extract on S. aureus.

Sample number Sample code Parameter Result of zone

of inhibition
Micro-608 Coconut Husk Staphylococcus
Extract aureus
R1 9mm
R2 10mm
R3 8mm

Average 9mm

Table 2 shows the degree of inhibition of Coconut (C. nucifera)

husk fiber extract against E. coli. Wherein, replicate 1 indicates the

result of 6 mm as the result of inhibition. The replicate 2 indicates the

result of 8 mm as the result of inhibition. The replicate 3 indicates the

result of 7 mm as the result of inhibition and 9 mm as the total

number of inhibition. This implies that the degree of inhibition of

Coconut (C. nucifera) husk fiber extract on the growth of E. coli is

active and has the potential to inhibit E. coli provided that 0 mm is

inactive, 1 mm – 5 mm is quite active, 6 mm – 10 mm is active, 10 mm

– 15 mm is intermediately active and 16 mm – 20 mm is very active

(Bauer, et.al)
 37

Table 2 The degree of inhibition of Coconut (C. nucifera) Husk

Fiber Extract on E. coli.

Sample number Sample code Parameter Result of zone

of inhibition
Micro-608 Coconut Husk Escherichia
Extract coli
R1 6mm
R2 8mm
R3 7mm

Average 7mm

Significant difference between the E. coli and S. aureus

Table 3 presents the significant difference between E. coli and S.

aureus. It shows that there was no significant difference between the

E. coli and S. aureus provided with p-value 0.070 which is greater than

the level of significant (atα =¿ 0.050). This implies that Coconut Husk

Extract can inhibit the growth of E. coli and S. aureus with almost the

same inhibition (Bauer, et.al. 2007)

Table 3. The significant difference between E. coli and S. aureus.

Bacteria Standard Mean T-value P-value Remarks

deviation
Escherichia coli 1.00 7.00
-2.449 0.070 Not
Staphylococcus 1.00 9.00 Significan
aureus t
 38

SUMMARY, CONCLUSION AND RECOMMENDATION

Summary

This research study focused on the inhibitory property of

Coconut (C. nucifera) husk fiber extract against S. aureus and

Escherichia coli: Basis for Developing Laboratory Activity in

Microbiology. This was conducted at Department of Science and

Technology (DOST) Ampayon, Butuan City. The experiment used the

Completely Randomized Design (CRD). The Coconut (C. nucifera) Husk

fiber was extracted through the process of ethanolic extraction at

DOST Caraga. The result of inhibition of Coconut (C. nucifera) on S.

aureus with three (3) replicates has the weighted mean of 9 mm and

the E. coli with three (3) replicates which has the weighted mean of

7mm, provided that 0 mm is inactive, 1 mm – 5 mm is quite active, 6

mm – 10 mm is active, 10 mm – 15 mm is intermediately active and 16

mm – 20 mm is very active.

This study was conducted to find out the degree of inhibition of

Coconut (C. nucifera) husk fiber extract against S. aureus and E. coli

and determine the significant difference and the phytochemical

present in Coconut (C. nucifera) husk fiber. Also, develop laboratory

activity in Microbiology.
 39

The result revealed further that the phytochemical present in

Coconut Husk Fiber were phenols, tannins, leucoanthocyanidins,

flavonoids, triterpenes, steroids and alkaloids. which has the ability to

inhibit such bacteria. The degree of inhibition of Coconut (C.

nucifera) husk fiber extract against S. aureus was active provided that

the mean is 9 mm and the E. coli which have the mean of 7mm which

is active also. However, there was no significant difference between E.

coli and S. aureus provided with p-value 0.070 which is greater than

the level of significant (atα =¿ 0.050). This happens because the

Standard deviation resulted into 1.00 based on the results of the zone

of inhibition of each bacterium.

Conclusions

Base on the result of the study the following conclusion are

drawn:

1. The Coconut (C. nucifera) Husk Fiber has the presence of

phytochemical properties such as of phenols, tannins,

leucoanthocyanidins, flavonoids, triterpenes, steroids and

alkaloids.

2. The degree of inhibition of Coconut (C. nucifera) Husk Fiber

Extract against S. aureus and E. coli were both active provided

 40

that the mean of inhibition of S. aureus was 9 mm and the mean

of E. coli was 7mm.

3. There was no Significant Difference between between E.coli and

S. aureus provided with p-value 0.070 which is greater than the

level of significant (atα =¿ 0.050).

4. A laboratory activity was developed based on the research

methodology.

Recommendations

Based on the results of the study, the researchers recommended

and suggested the following:

1. Other researchers may conduct other study about Coconut

Husk fiber extract because it is not just only essential for

antimicrobial property but could be also effective in anti-

helminthic property and other diseases due to the presence of

other phytochemical properties such as flavonoids and tannins.

2. Teachers can create other laboratory activity out from the study

that are related to biology.

3. Teachers from science courses can conduct orientation or

symposium about the importance of plants.

 41

LITERATURE CITED

Adebayo JO, Balogun EA, Malomo SO, Soladoye AO, Olatunji LA,
Kolawole OM, et al. (2013) Antimalarial activity of Cocos
nucifera husk fibre: further studies. Evid Based Complement
Alternat Med 2013; 2013: 742476, doi: 10.1155/2013/742476.

Akinpelu DA, Alayande KA, Aiyegoro OA, Akinpelu OF, Okoh AI. (2015)
Probable mechanisms of biocidal action of Cocos nucifera Husk
extract and fractions on bacteria isolates. BMC Complement
Altern Med.2015;15:116. doi: 10.1186/s12906-015-0634-3.

Alviano DS, Rodrigues KF, Leitão SG, Rodrigues ML, Matheus ME,
Fernandes PD, Antoniolli AR, Alviano CS (2004).
Antinociceptive and free radical scavenging activities of Cocos
nucifera L. (Palmae) husk fiber aqueous extract. J.
Ethnopharmacol. 92:269-273.

Ammor, S., G. Tauveron, E. Dufour, and I. Chevallier. (2006).

Antibacterial activity of lactic acid bacteria against spoilage and
pathogenic bacteria isolated from the same meat small-scale
facility. 2. Behaviour of pathogenic and spoilage bacteria in dual
species biofilms including a bacteriocin-like-producing lactic
acid bacteria. Food Control 17:462-468.

Bano, S. (2007). Chemistry of Natural Products. Department of

Chemistry, Faculty of Science, JamiaHamdard, New delhi-
110062.

Chan PF, Foster SJ (1998) Role of SarA in virulence determinant

production and environmental signal transduction in
Staphylococcus aureus. J Bacteriol 180: 62326241.

DebMandal M, Mandal S (2011). Coconut (Cocos nucifera L.:

Arecaceae): In health promotion and disease prevention. Asian
Pac. J. Trop. Med. 241-247.

Doughari, J.H. & Obida, J.S. (2008). Antibacterial potentials of stem

bark extracts of Leptadenialacifoli against some pathogenic

bacteria. Pharmacologyonline 3(11): 839-848

 42

Dua K, Sheshala R, Ying LT, Hui LS, Gorajana A (2013).

Antiinflammatory, antibacterial and analgesic potential of Cocos
nucifera L: A review. Antiinflamm. Antiallergy Agents Med.
Chem. In press.

Easmon CSF, Adlam C (1983): Staphylococci and staphylococcal

infections. Vols 1 and 2. Academic Press, London, 1983 .

Esquenazi D, Wigg MD, Miranda MM, Rodrigues HM, Tostes JB,

Rozental S, Da silva, AJ, Alviano CS (2002). Antimicrobial and
antiviral activities of polyphenols from Cocos nucifera Linn
(Palmae) husk fiber extract. Res. Microbiol. 153:647-652.

Koschek PR, Alviano, Gattass CR (2008). The husk fiber of Cocos

nucifera (Palmae) is a source of antineoplastic activity. Braz J
Med Biol Res. 2007;40:1339–43.

Kobayashi, S.D.; Malachowa, N.; DeLeo, F.R. (2015). Pathogenesis of

Staphylococcusaureus abscesses. Am. J.Pathol. 2015, 185,
1518–1527.

Krishnaiah D, Sarbatly R, Bono A, (2007). Phytochemical antioxidants

for health and medicine: A move towards nature.
BiotechnolMolBio rev 1: 97-104.

Le Loir Y, Baron F, Gautier M (2003). Staphylococcus aureus and food

poisoning. Genetics and Molecular Research 2(1):63–76

Luthria, D. L., & Natarajan, S. S. (2009). Influnce of sample

preparation on the assay of isoflavones. Planta Medical, 75,
704-710.

Mahajan, M. Kumar, V and Yadav S.V. (2014). Alkaloids: Properties,

application and pharmacological effects pp. 1-36.

Moran, G.J.; Krishnadasan, A.; Gorwitz, R.J.; Fosheim, G.E.;

McDougal, L.K.; Carey, R.B.; Talan, D.A. (2006). Methicillin-
resistant S. aureus infections among patients in the emergency
department. N. Engl. J. Med. 2006, 355, 666–674.

Renjith RS, Chikku AM, Rajamohan T. (2013). Cytoprotective,

antihyperglycemic and phytochemical properties of Cocos
 43

nucifera (L.) inflorescence. Asian Pac J Trop Med. 2013;6:804–

810.

Silva E, Deora R, Misra TK (2013) Characterization of the primary

sigma factor of Staphylococcus aureus. J Biol Chem 271:
21828-21834.

Solangih A, Iqbal ZA (2001). Chemical composition of meat (kernel)

and nut water of major coconut (Cocos nucifera L.) cultivars at
coastal area of Pakistan. Pak. Am J Bot. 2011;43:357–363.

Temple, N. J. (2000). Antioxidant and disease: More questions than

Treasure and Evans Pharmacognosy. (2002). 15th edition. W.B
Saunders. London

Wilkinson BJ (1997) Biology. In: Crossley KB, Archer GL, eds. The
Staphylococci in Human Diseases. Churchill Livingston,
London. pp 1-38.

Yao, L. H., Jiang, Y. M., Shi, J., Tomas-Barberan, F. A., Datta, N.,
Singanusong, R., et al. (2004). Flavanoids in food and their
Health Benefits. Plant Foods for Human Nutrition, 59, 113-112.
 44

APPENDICES
45
 46

Name: Yr. & Sec. Date:

Anti-microbial Test of Coconut (Cocos nucifera) Husk Fiber

Extract on Staphylococcus aureus and Escherichia coli

Microbiology Laboratory Activity Sheet

Introduction

Coconut (Cocos nucifera) is a well distributed fruit tree all

around the world, providing food, especially in the tropical and
subtropical regions and for its many uses it is often called the “tree of
life”. Almost all parts of the tree have benefits, from the fruits, to the
leaves, to the roots, even the husk. The constituents of C. nucifera
husks have some biological effects, such as anthelminthic, anti-
inflammatory, antinociceptive, antimicrobial, and antitumor activities.

Staphylococcus aureus is a gram-positive coccal bacterium that

is a member of the Firmicutes, and is frequently found in
the nose, respiratory tract, and on the skin. It is often positive
for catalase and nitrate reduction and is a facultative aerobe that can
grow without the need for oxygen. Although S. aureus is not
always pathogenic, it is a common cause of skin infections such
as abscesses, respiratory infections such as sinusitis, and food
poisoning (Wilkinson, 2009). Moreover, Escherichia coli are a gram-
negative, facultatively anaerobic, rod-shaped, coliform bacterium of
the genus Escherichia that is commonly found in lower intestine of
warm-blooded organisms (Wilkinson, 2009).

Objectives:

 To describe the significant difference of Staphylococcus aureus

and Escherichia coli.
 To determine the biological effects of Coconut Husk Fiber
Extract.
 To perform anti-microbial testing using Coconut Husk Fiber
Extract on Staphylococcus aureus and Escherichia coli.

Materials:
*Coconut Husk Fiber Extract *Cotton Swab
 47

*Staphylococcus aureus *wooden applicator handles

*Escherichia coli *Sterile Loop
*Agar *Bunsen burner
*Agar plate *Test tube
*Petri Dish *Incubator
*Laboratory Gown *Distilled water
*Laboratory Gloves and Masks

Procedures:
1. Prepare the Coconut Husk Fiber Extract
2. Prepare the test organisms.
Staphylococcus aureus – gram-positive cocci
Escherichia coli – gram-negative rods

Bacteria: Gram positive or Gram negative

The 0.5 M McFarland standard is used to adjust the turbidity of the inoculum
prior to the cotton swabbing of the agar plates for the antimicrobial assays.The
standard should be prepared and subjected to quality control prior to use.

This standard contains approximately 1.5x108 CFU/ml of the test organisms.

3. Preparation of the nutrient broth

Refer to label for the preparation of 1000ml solution.

Take a loopful of bacteria, gram-positive or gram negative, from the

culture slant and inoculate in 50ml nutrient broth;

Incubate the culture broth for 18-24 hours at 35°C;

Observe the culture broth for turbidity, indicative microbial growth.

4. Adjusting the turbidity of the inoculum

Adjust turbidity serves as the inoculum for the microbial assay. This will serve
as the inoculum which will be swabbed onto agar plates.

Use within 15 minutes after adjusting the turbidity.

 48

Aseptically transfer 5ml of the culture broth is sterile screw-capped

tubes.

Agitate on a vortex mixer the bacterial suspension and immediately

compare against the 0.5 Mcfarland standard prepared.

If the bacterial suspension does not appear to be of the same density

as the McFarland standard, adjust the turbidity by adding sterile
saline solution or culture broth and subsequently compare the
resulting turbidity to the standard.

5. Preparation of plates
Prepare plates depending on the number of test organisms and
replications required. Common practice requires 2 replicates for each
extract and for each test organisms.

Pour approximately 15ml of melted nutrient agar into dry and sterile
petri dishes. Let the medium solidify

Moisten a sterile cotton swab into the test organism (inoculum)

suspension. Use cotton swab with wooden applicator handles. Dip a
sterile cotton swab into a suspension of the test organism/inoculum.
Press and rotate the moistened swab firmly against the inside wall of
the tube just above the fluid level to remove the excess liquid.

6. Cotton swabbing
Aseptically swab the test organism into a solidified nutrient agar by
streaking the swab over the entire surface of the agar plate 3X,
rotating the plate 60 degrees after each application to ensure an even
distribution of the inoculum on the surface of the medium.

Let the swabbed plates stand for 5 minutes.

 49

7. Paper disc diffusion method

Materials:
Screw-capped tube of test organism
Nutrient agar
Forcep
10mm paper disc, sterilized in petri dish (Pack disc in sets of 10)

Using the forceps pick out one paper disc and immerse the paper
disc into the plant extract.

Lay the moistened filter disc gently on the seeded agar plate.

Gently tap the disc forcep to ensure maximum full contact of the
disc with the agar medium.

Incubate the plates inverted.

Technique for handling the paper disc and moistening with the plant extract;

With the forcep on one hand and the petri dish containing the sterilized filter
discs on the other hand, open the dish and pick out one paper disc with the
forcep.

Immerse the disc into the assay solution.

Remove excess liquid by letting the moist paper disc to touch the inside wall of
the container of the assay solution.

8. Reading the assay plates

Look for “HALO” or “Clearing” around the disc. This is known as
the zone of inhibition.

Invert the plates and with a ruler measure the diameter of each
inhibition zone in millimeters.

Express results as mm diameter zone of inhibition.

Record the diameter of the paper disc used in the assay in mm..
 50

Result

(Guide Questions)

Answer the following based on the experiment being conducted:

1. What are the biological effects of Coconut Husk Fiber Extract?

2. What are the significant difference between Staphylococcus

aureus and Escherichia coli ?

3. Which test organism has the strong inhibition? Explain this

phenomenon?
 51

4. Draw the zone of inhibition of the test organism

Conclusion:

References:
Adebayo JO, Balogun EA, Malomo SO, Soladoye AO, Olatunji LA,
Kolawole OM, et al. (2013) Antimalarial activity of Cocos
nucifera husk fibre: further studies. Evid Based Complement
Alternat Med 2013; 2013: 742476, doi: 10.1155/2013/742476.

DebMandal M, Mandal S (2011). Coconut (Cocos nucifera L.:

Arecaceae): In health promotion and disease prevention. Asian
Pac. J. Trop. Med. 241-247.

Esquenazi D, Wigg MD, Miranda MM, Rodrigues HM, Tostes JB,

Rozental S, Da silva, AJ, Alviano CS (2002). Antimicrobial and
antiviral activities of polyphenols from Cocos nucifera Linn
(Palmae) husk fiber extract. Res. Microbiol. 153:647-652.
 52

Staphylococcus aureus

Replicate 1: Replicate 2

Replicate 3:
 53

Escherichia coli

Replicate 1: Replicate 2:

Replicate 3:
54
 55

APPENDIX A

LETTER OF PERMISSION

Republic of the Philippines

Agusan Del Sur State College of Agriculture and Technology
Bunawan, Agusan Del Sur

JENNIFER J. DEJARME
Chief Laboratory Chemist
DOST-Caraga

Maam:

We the researchers are having the study entitled INHIBITORY

PROPERTY OF COCONUT (Cocos nucifera) HUSK ASH FIBER
EXTRACT AGAINST Staphylococcus aureus.

In this connection, we would like to ask permission from your good

office for the conduct of phytochemical analysis and zone inhibition on
our plants against Staphylococcus aureus.

We are hoping for your approval. Thank you and God bless!

Respectfully yours,

RICO S. FERNANDEZ

CHRISTIAN M. TAYAB
Researchers

Noted:

VIRMALYN D. ENOBIO
Adviser

Approved:

ALVIN O. CAYOGYOG, Ph.D

COLLEGE DEAN
 56

Appendix B

CURRICULUM VITAE

PERSONAL BACKGROUND:

Name : Rico Serato Fernandez

N-Name : “Budo”
Age : 19 years old
Gender : Male
Civil Status : Single
Home Address : P-1B, Villa Ondayon, Bayugan City
Birthdate : May 30, 1997
Birthplace : Villa Ondayon, Bayugan City
Tribe : Cebuano
Religion : Roman Catholic
Mother’s Name : Marilyn Serato Fernandez
Father’s Name : Luis Bellera Fernadez Sr.

EDUCATIONAL BACKGROUND:

Elementary : Villa Ondayon Elementary School

Villa Ondayon, Bayugan City

High School : Bayugan National Comprehensive High School

Bayugan City

College : Agusan del Sur State University

Bunawan, Agusan Del Sur

Course : Bachelor of Secondary Education

Major in Biology
 57

CURRICULUM VITAE

PERSONAL BACKGROUND:

Name : Christian Manceras Tayab

N-Name : “Ken-ken”
Age : 18 years old
Gender : Male
Civil Status : Single
Home Address : P-1 Bliss, Pulanglupa, Trento, Agusan Del Sur
Birthdate : April 10, 1998
Birthplace : Trento, Agusan Del Sur
Tribe : Cebuano
Religion : Roman Catholic
Mother’s Name : Virginia Manceras Tayab
Father’s Name : Barle Dioric Tayab

EDUCATIONAL BACKGROUND:

Elementary : Pulanglupa Elementary School

Pulanglupa, Trento, Agusan Del Sur

High School : Trento National High School

Trento, Agusan Del Sur

College : Agusan Del Sur State College of Agriculture and

Technology
Bunawan, Agusan Del Sur

Course : Bachelor of Secondary Education

Major in Biology
58
 59

ACKNOWLEDGEMENT

The researchers were gratefully expresses their sincerest and

heartfelt appreciation to those who extended their help, assistance,

guidance, encouragement and pieces of advice during the conduct of

this study.

First and foremost: to our Heavenly Father, for his goodness,

guidance, protection, immeasurable blessings and grace. The

researchers were also thankful for the wisdom and strength bestowed

upon him;

Virmalyn D. Enobio, Chairperson of the Advisory Committee for

her suggestions and patience in checking the manuscript and guiding

the researchers’ throughout the duration of the study;

Ronald R. Baldo, Roselita A. Renoblas and Vivian C. Peligro,

examining committee members, for their valuable suggestion and

comments;

Marvin S. Pizon, statistician, for his patience in the statistical

analysis of data;

To Sheryl T. Ytoc, for editing the manuscript;

 60

To our family Mr. and Mrs. Fernandez and Mr. and Mrs. Tayab

for always there to support morally and financially especially to the

success of our study;

To our friends and classmates who share their ideas to make

this research even more successful.

To all of you, thank you so much.

Budo and Ken-ken