Chapter I

INTRODUCTION

Background of the Study

The continuous spreading of infections caused by different microorganisms in the

country and elsewhere is one of the serious problems that man faced. This trend is

reflected through the progressive increase in number of medicinal drugs throughout the

market. Often the prices of manufactured drugs are high and the availability is not

always assured. The high cost of recommended drugs especially those antibiotics

besets the treatment of human diseases. This is the reason why researches have been

conducted in almost all parts of nature in search of medicinal properties. The

continuous inflation in prices of medicinal drugs motivated consumers to seek ways and

means by which medicinal drugs can be substituted. A positive and an economical

approach to this problem is the utilization of cheap but highly effective available

indigenous sources of drugs for curing skin diseases.

Diseases spread quickly. This is because microbes-bacterium or fungus are

everywhere and it is difficult to move away from them. When people catch disease,

normally they buy antibiotic to fight it off and feel well again.

As the price of major necessities continuously increases, some cannot afford to

buy antibiotic to fight their sickness. That is why antibacterial and antifungal medium

with lower prices than those of commercially produced can help those people who find

commercial drugs costly.

Honey is one of nature’s exceptional health offerings. Bees make it for

themselves and their brood, to live on through the winter – as such, it’s packed with

1
everything they need, including vitamins, minerals, antioxidants, and enzymes that are

beneficial for us. Honey is a sweet food made by bees using nectar from flowers. The

variety produced by honey bees is the one most commonly referred to, as it is the type

of honey collected by most beekeepers and consumed by people. Honey produced by

other bees (bumblebees, stingless bees) and other hymenoptera insects (e. g. honey

wasps) have different properties, and they are not discussed in this article. Honey bees

transform nectar into honey by a process of regurgitation and evaporation. They store it

as a primary food source in wax honey combs inside the beehive.

This study wants to determine the efficacy of honey extract to resist and kill

bacteria and fungi that cause diseases to animals.

Statement of the Problem

This study entitled “HONEY EXTRACT AS GROWTH INHIBITORS AGAINST

Escherichia coli, Staphylococcus aureus, and Aspergillus fumigatus” was

primarily conducted to assess the efficiency and effectiveness of honey extract as

growth inhibitors of common pathogens Escherichia coli, Staphylococcus aureus, and

Aspergillus fumigatus.

Specifically, this study sought answers to the following questions:

1. Can Honey Extract be used as an antimicrobial agent?

2. What percentage concentration of extracts (100%, 50%, and 25%) significantly

inhibited the growth of (bacteria) Staphylococcus aureus and Escherichia coli as

well as (fungus) Aspergillus fumigates as compared with the positive control?

2
 3. Is there a significant difference on the zone of inhibition of the pathogens

(Staphylococcus aureus, Eschericia coli, and Aspergillus fumigatus) subjected to

different treatments?

Hypothesis of the Study

This study made use of the following:

 Honey extract is a good antimicrobial agent.

 There is no significant difference on the effect of using honey extract in inhibiting

the growth of (bacteria) Staphylococcus aureus and Eschericia coli as well as

(fungus) Aspergillus fumigatus.

 There is no significant difference on the zone of inhibition of the different

pathogens namely Staphylococcus aureus, Eschericia coli as well as (fungus)

Aspergillus fumigates subjected to different treatments.

Significance of the Study

The aim of this study is to produce and prove the capability of Honey extract in

treating bacterial and fungal infections as compared to the commercially available

drugs. The success of this study would provide a very wide and safe alternative

medication and represent a potential natural source for pharmacological and

pharmaceutical application. The study provides concrete information regarding the

antimicrobial property of Honey Extract against common pathogens.

Furthermore, the discovery of honey as a medicine for bacterial and fungal

infections will give information to the public to watch, protect and preserve it.

3
 This can be a possible start for another innovation in enriching the knowledge

about honey achieving a greater purpose for this organism to improve the lives of

humans for government and non-government organizations. Enrichment of the aspects

of the study can unlock new potentials of ants that can help people especially those in

the field of medicine.

Specifically, this study will be significant and helpful to the following persons:

CONSUMERS: This study will be beneficial to them for they can save a lot of money.

Instead of buying high priced commercial drugs, they can cure their skin diseases as

well as diarrhea with these locally available medicinal drugs.

DRUG MANUFACTURERS: This study can serve as eye opener for them to be

interested in organic and non-conventional materials to manufacture cheap and readily

available medicinal drugs.

RESEARCHERS: The study can serve as a basis for further researchers and

development in search for effective and economically feasible solutions to industrial,

commercial, medicinal and environmental waste problem. This study will enhance their

skill and knowledge in conducting experiments which they can use for personal

development and even for income generation.

STUDENTS: This study may serve as eye opener and encourage them to undertake

product development.

4
Scope and Delimitations of the Study

This study entitled “HONEY EXTRACT AS GROWTH INHIBITORS AGAINST

Escherichia coli, Staphylococcus aureus, and Aspergillus fumigatus” was focused

on the determination of the antibacterial and anti-fungal properties of honey extract. It

was also conducted to produce an antimicrobial formulation for skin diseases and to use

as a drug for diarrhea out of cheap, easy to produce, and readily available materials like

honey.

The study was also limited in testing the experimental samples on one type of

fungus and two types of bacterial pathogens.

The preparation of this research study was conducted from June to August 2014.

Collection honey as well as preparation of the extract was done at Brgy. Tugatog,

Bongabon, Nueva Ecija. Preparation and culturing of bacteria was done at Veterinary

Microbiology Laboratory at the College of Veterinary Medicine, Central Luzon State

University.

The measurement of the zone of Inhibition and testing of the inhibitory effect of

the experimental product on the microorganisms was also done at Veterinary

Microbiology Laboratory, College of Veterinary Medicine, Central Luzon State

University.

5
 Chapter II

REVIEW OF RELATED LITERATURE AND STUDIES

The researchers have conducted a survey of related literature and studies.

Certain books, journals, encyclopedias, and studies, which have direct and indirect

connection with the study, were carefully reviewed. These studies helped the

researchers in conducting scientific studies on the same fields.

Medicinal Drugs

The widely held belief that modern medicine is all the product of complex

chemical syntheses is a fallacy. More than 120 current prescription drugs are still

obtained from higher plants and about 25% of all prescriptions contain one or more

active ingredients from plants.

According to Odugbemi (2006), the continuous evolution of bacterial resistance

to currently available antibiotics has necessitated the search for novel and effective

antimicrobial compounds. Globally, plant extracts are employed for their antibacterial,

antifungal and antiviral activities. It is known that more than 400,000 spp. of tropical

flowering plants have medicinal properties and this has made traditional medicine

cheaper than modern medicine. Some plant decoctions are of great value in the

treatment of diarrhea or gastrointestinal disorder, urinary tract infections, skin infections,

infertility, wound and cutaneous abscesses (Meyer et al., 1996; Dimayuga and Gracia,

1991).

6
 Black (1996) mentioned that there is a growing recognition of the economic value

of traditional medicines and their importance to local people in many developing

countries. These factors have been important in promoting the establishment of

protected areas for medicinal plants.

Honey

According to National Honey Board (2011), honey is a sweet food made by bees

using nectar from flowers. It is also stated to cure some allergies; particularly localized

honey to an area could help minimize seasonal allergies as bees feed on pollen from

local plants which eventually find its way to form honey. Its use for centuries is as a

treatment for sore throats and coughs, and according to recent research, may in fact be

as effective as many common cough medicines. Scientists have revealed that honey

has powerful anti-bacterial properties on at least sixty species of bacteria, and unlike

antibiotics, which are often useless against certain types of bacteria; honey is non-toxic

and has strong effects. The pH of honey is commonly between 3.2 and 4.5. This

relatively acidic pH level prevents the growth of many bacteria.

Escherichia coli

Escherichia coli (commonly abbreviated E. coli; named after it is discovered), is a

gram negative rod-shaped bacterium that is commonly found in the lower intestine of

warm-blooded organisms (endotherms). Most E. coli strains are harmless, but some

such as serotype O157:H7 can cause serious food poisoning in humans, and are

occasionally responsible for product recalls ( Vogt and Dippold 2005).

7
 E. coli are not always confined to the intestine, their ability to survive for brief

periods outside the body makes them an ideal indicator organism to test environmental

samples for fecal contamination(Feng, Weagant, Grant 2002).

The bacteria can also grown easily and its genetics are comparatively simple

and easily-manipulated or duplicated through a process of metagenics, making it one of

the best–studied prokaryotic model organisms, and an important species in

biotechnology and microbiology. (Thompson 2007)

Staphylococcus aureus

Staphylococcus aureus (literally the “golden cluster seed” or “the seed gold” and

also known as golden staph and Oro staphira) is the most common cause of staph

infections. It is a spherical bacterium, frequently part of the skin flora found in the nose

and on skin. About 20% of the populations are long-term carriers of S. aureus.

S. aureus can cause a range of illnesses from minor skin infections, such as

pimples ,impetigo(may also be caused by Streptococcus pyogenes), boils(furuncles),

cellulitis folliculitis, carbuncles, scalded skin syndrome and abscesses to life–

threatening diseases such as pneumonia, meningitis, osteomyelitis, endocarditis, toxic

shock syndrome (TSS) and bacteremia sepsis. Its incidence is from skin, soft tissue,

respiratory, bone, joint, endovascular to wound infections. It is still one of the five most

common causes of nosocomial infections, often causing postsurgical wound infections.

Abbreviated to S. aureus or Staph aureus in medicinal literature, S. aureus should not

be confused with the similarly dangerous (and also medically relevant) species of the

genus Streptococcus (Kluytmans, vanBelkum, Verbrugh 1997).

8
Aspergillus fumigatus

Aspergillus fumigates is a fungus of the genus Aspergillus, and is one of the most

common Aspergillus species which cause disease in immuno-compromised individuals.

A.fumigatus is a saprotroph that is widespread in nature and typically found in

soil and decaying organic matter such as compost heaps where it plays an essential

role in carbon and nitrogen recycling. Colonies of the fungus are produced from

conidiophores’ thousands of minute grey-green conidia (2-3µm) that readily become

airborne. For many years A.fumigatus was thought to only reproduce asexually, as

neither mating nor meiosis had ever been observed. However, in 2008 it was shown tha

A.fumigatus possesses a fully functional sexual reproductive cycle,145 years after its

original description by Fresenius (O’Gorman CM 2008).

RELATED STUDIES

Honey as an Antibiotic: Scientists Identify a Secret Ingredient in Honey That Kills

Bacteria

A new research published in the July 2010 print edition of the FASEB

Journal explains for the first time how honey kills bacteria. Specifically, the research

shows that bees make a protein that they add to the honey, called defensin-1, which

could one day be used to treat burns and skin infections and to develop new drugs that

could combat antibiotic-resistant infections.

"We have completely elucidated the molecular basis of the antibacterial activity of

a single medical-grade honey, which contributes to the applicability of honey in

medicine," said Sebastian A.J. Zaat, Ph.D., a researcher involved in the work from the

9
Department of Medical Microbiology at the Academic Medical Center in Amsterdam.

"Honey or isolated honey-derived components might be of great value for prevention

and treatment of infections caused by antibiotic-resistant bacteria."

To make the discovery, Zaat and colleagues investigated the antibacterial activity

of medical-grade honey in test tubes against a panel of antibiotic-resistant, disease-

causing bacteria. They developed a method to selectively neutralize the known

antibacterial factors in honey and determine their individual antibacterial contributions.

Ultimately, researchers isolated the defensin-1 protein, which is part of the honey bee

immune system and is added by bees to honey. After analysis, the scientists concluded

that the vast majority of honey's antibacterial properties come from that protein. This

information also sheds light on the inner workings of honey bee immune systems, which

may one day help breeders create healthier and heartier honey bees.

"We've known for millennia that honey can be good for what ails us, but we

haven't known how it works," said Gerald Weissmann, M.D., Editor-in-Chief of

the FASEB Journal, "Now that we've extracted a potent antibacterial ingredient from

honey, we can make it still more effective and take the sting out of bacterial infections."

Antifungal Activity of Four Honeys of Different Types from Algeria against

Pathogenic Yeast: Candida albicans and Rhodotorula sp.

A study by Asian Pacific Journal of Tropical Biomedicine(Volume 2, Issue 7;

Pages 554–557) on July 2012 evaluated the antifungal activity of four honeys of

different types from Algeria against pathogenic yeast i.e.Candida albicans (C. albicans)

and Rhodotorula sp.

10
 Four Algeria honeys of different botanical origin were analyzed to test antifungal

effect against C. albicans, and Rhodotorula sp. Different concentrations (undiluted,

10%, 30%, 50% and 70% w/v) of honey were studied in vitro for their antifungal activity

using C. albicans and Rhodotorula sp. as fungal strains.

The range of the diameter of zone of inhibition of various concentrations of tested

honey was (7–23 mm) for Rhodotorula sp., while C. albicans showed clearly resistance

towards all concentrations used. The MICs of tested honey concentrations against C.

albicans and Rhodotorula sp. were (70.09–93.48) % and (4.90–99.70) % v/v,

respectively. This study demonstrates that, in vitro, these natural products have clearly

an antifungal activity against Rhodotorula sp. and C. albicans.

Antimicrobial Activity of Honey from Five Species of Brazilian Stingless Bees

A study conducted by Universidade Estadual de Santa Cruz (UESC) Laboratório

de Química de Produtos Naturais e Bioativos, Campus Soane Nazaré de Andradeand

Universidade Estadual de Feira de Santana (UEFS) in Brazil (Apr. 2013) states the

antimicrobial activity of honey produced by Melipona asilvai, Melipona

quadrifasciataanthidioides, Friseomelita doederleinei, Tetragonisca angustula and

Plebeia sp. were investigated. The agar well diffusion assay demonstrated that all

honeys had antibacterial activity against Staphylococcus aureus, but only the samples

from M. quadrifasciata anthidioides and F. doederleinei inhibited the growth of

Escherichia coli. In the Minimum Inhibitory Concentration determination assay, M.

asilvai, M. quadrifasciata anthidioides, F. doederleinei and T. angustula honeys were

more active than that from Plebeia sp. for S. aureus and E. coli. The microorganisms

11
Pseudomonas aeruginosa and Candida albicans were resistant to the all native

stingless bee honey in both assays. Honey was more effective against bacteria than a

sugar solution, suggesting that the mechanism for bacterial growth inhibition is not only

related to the osmotic effect. The results of antimicrobial activity may explain the

popular medicinal use of these honeys in bacterial diseases.

Ethno Botany and Antimicrobial Perspective of Spices and Honey Against

Candida albicans

A study by J Intercult Ethno pharmacol (2013) identified the spices used in

Ethiopian food through ethno botanical survey and studies their antifungal activity

against Candida albicans. Ethno botanical survey of the selected Kebeles of Jimma,

Ethiopia was conducted using a semi structured questionnaire from October 2006 to

November 2007. Antifungal nature of the spices and combination of spices and honey

were evaluated by agar well diffusion assay from September 2008 to July 2010. Ethno

botanical survey indicated fourteen species of spices and honey play a major role in

Ethiopian food & beverages. Single plant extract of Trachyspermum copticum showed

highest activity against C. albicans. The same plant showed antagonistic effect when

combined with brown and white honey. Cinamomum zeylanicum showed highest

synergistic effect with both brown & white honey when compared to Allium ursenum,

Cuminum cyminum, Nigella sativa, Rosemarinus officinalis and Occimum

hodiense. Thus spices used in Ethiopian food could be a preventive as well as a cure

for oral candidiasis caused by C.albicans.

12
 Relevance of Review of Related Literature and Studies

To the Present Study

The related literature and readings provide significant information that can help

the researchers develop the study better; providing clear and reliable processes and

concept involved; like the description of the different bacterial pathogens that can be

used in the study.

The related studies provide the researchers the knowledge about the past

experiments and studies to avoid duplication of variables and descriptive information

regarding the past studies about the ability and characteristics of honey.

13
 Chapter III

METHODOLOGY

The chapter presents the method of research utilized in this study, the

techniques adopted in gathering data, and the procedures followed in the

experimentation.

Materials

The main material used in this research is the honey. Honey was bought in the

public market. The material was selected because of their relatively low cost, nutrient

content, and availability in the locality. Materials such as graduated cylinder, petri dish,

spatula, vials, alcohol lamp, weighing scale, test tube, transferpette, vernier caliper,

beaker, incubator, electric hot plate, cheese cloth and auto clave were also utilized in

the study. Lysol, Penicillin and water is also used as the positive and negative control.

Experimental Design

Three species of pathogen, Escherichia coli, Staphylococcus aureus, Aspergilus

fumigatus were used in the study. Each microorganism was subjected to varying

percent of honey extract at 100%, 50%, and 10%. There was also positive and negative

control.

14
 Table 1.Treatment Assignment using Honey Extract Against
Staphylococcus aureus, E.coli (bacteria) and Aspergillus fumigatus(fungus)

Honey
PERCENTAGE Distilled
TREATMENTS Extract
CONCENTRATION Water(ml)
(ml)
Treatment 1 100% 0 5
Treatment 2 50% 2.5 2.5
Treatment 3 10% 4.5 0.5
Penicillin
Positive Control 0 0
(bacteria)

Positive Control Lysol (fungus) 0 0

Negative
Water(bacteria &fungus) 2 0
Control

The positive control (Penicillin) for the Echerichia coli and Staphylococcus aureus

was different with the one used for the Aspergillus fumigatus, since Aspergillus

fumigatus (Lysol 50mg/ml) is a fungus not a bacteria.

 Preparation of Test Organism

Pure culture of Escherichia coli, Staphylococcus aureus, and Aspergillus

fumigates was obtained from the Veterinary Microbiology College of Veterinary

Medicine, Central Luzon State University. The organism was inoculated into separate

test tubes with sterile distilled water and incubated at 37°C for 24 hours. Suitable culture

medium was prepared to serve as the growing environment of the bacteria.

The microbial species used were cultured. Pure bacteria (Escherichia coli and

Staphylococcus aureus) were placed in a petri dish with nutrient agar and incubated to

grow 24 hours while the fungus (Aspergillus fumigatus) used was cultured for three

days.

15
  Preparation of Growth Agar for the Test Organism

Nutrient agar is the type of medium that was used in this study. It was

prepared by weighing 7.2 grams of Soyabean Digest Agar for the E. coli and 5.2 grams

for the Staphylococcus aureus and dissolves it in a sterile 180 ml of water. For the

Aspergillus fumigatus 11.7 grams of Sabouraud Dextrose Agar was dissolved in 180 ml

of sterile water. After preparing the solution, it will be covered with clean cotton with

gauze and covered with foil. The prepared medium will be cooked using electric hot

plate at 25°C. After cooking, approximately 20 ml were poured sterile onto sterile petri

dish and allowed to harden. These were incubated at room temperature in a level

position to avoid atmospheric contamination.

 In-vitro Bioassay

In the eradicate test against the pathogen, the crude extracts were tested against

existing infections and measured its zones of inhibitions; presence of inhibition only

meant that the extract can kill the pathogen from an infected host. While protectant

against the pathogen meant that the extracts can stop the pathogen from attacking the

organism.

 Inoculation of Pathogen

The prepared nutrient agar was sterilized for 30 minutes at 15 psi. Upon cooling,

20 ml of nutrient agar will aseptically transfer evenly in the whole plate. It is allowed to

solidify at room temperature. When the medium had already solidified, the bacterial

organisms were incorporated and incubated for a maximum 24 hours to show their

growth on an agar at 37°C.

16
 The zone of inhibitions were determined and measured at the nearest

(mm) using a vernier caliper after 24 hours of inoculation. If the zone of inhibition

appeared in the plate, it signified the effectivity of the extracts against the pathogen.

Extracts that showed no zone of inhibition means that the extract had no inhibiting

property against the test organism. Three replicates were prepared for each treatment.

Data to be Gathered

The following data were gathered and analyzed:

1. Diameter of zone of inhibition effected by the treatment with anti-bacterial and

antifungal properties in in-vitro bioassay.

2. Diameter of zone of inhibition exhibited by the positive control.

17
 Chapter IV

RESULTS AND DISCUSSION

This part presents, analyses, and interprets the data gathered in an organized

manner regarding the use of the treatments from honey extract inhibiting the growth of

human pathogenic fungi and bacteria as compared to commercial drug.

 In vitro test result

Table 2.0 Sensitivity of Escherichia coli to Honey Extract

Measurement of the Zone of Inhibition (mm)
Treatments Replication 1 Replication 2 Replication 3 Mean SD
T1=100 % - - - - -
T2=50% - - - - -
T3=10% - - - - -
T4=Positive Control
21.70 21.85 22.30 21.95 0.31
T5=Negative Control - - - - -

Note:(-) No zone of Inhibition

Sensitivity of Escherichia coli to Honey Extract

Measurement of the Zone of Inhibition (mm)

M 40

E
20
A
N 0

Experimental Treatment

Fig. 1 Sensitivity of Escherichia coli to Honey Extract Measurement of the Zone of

Inhibition

18
 The table shows the sensitivity of Escherichia coli to the experimental honey

extract against the positive control (Penicillin) and the negative control (distilled water)

after 24 hours of incubation. Disc diffusion method was the procedure done on the

experiment. Table 2.0 shows the mean measurements of the zone of inhibition of E. coli

subjected to different treatments. It shows that Treatment 4, Penicillin was significantly

the most effective treatment with a mean of 21.95 mm. The treatment 1(100%

concentration of Honey extract) was not effective as well as treatment 2,3 and 5. This

implied that only that positive control (penicillin) has antibacterial property against E.

coli.

Analysis of Variance (ANOVA) table shows that there is no significant difference

among the mean zone of inhibitions of the treatments 100%, 50% and 10%

concentration of honey extract as compared to the positive control (Penicillin).

The result of the investigation showed that honey extract does not inhibit the

growth of Escherichia coli.

Table 3.0 Sensitivity of Staphylococcus aureus to Honey Extract

Measurement of the Zone of Inhibition (mm)

Treatments Replication 1 Replication 2 Replication 3 Mean SD

T1=100 % 16.60 17.40 14.70 16.23 1.39
T2=50% - - - - -
T3=10% - - - - -
T4=Positive Control 46.90 43.30 46.10 45.43 1.89

T5=Negative Control - - - - -

Note:(-) No zone of Inhibition

19
 Sensitivity of Staphylococcus aureus to Honey Extract
Measurement of the Zone of Inhibition (mm)

M 60

E 40

A 20
N
0

Experimental Treatment

Fig. 2 Sensitivity of Staphylococcus aureus to Honey Extract Measurement of the

Zone of Inhibition

Table 3 presents the result of the antibacterial test of the experimental treatment

against the positive control (Penicillin), and the negative control (distilled water) after 24

hours of incubation against Staphylococcus aureus. The table shows the mean

measurements of the zone of inhibition of Staphylococcus aureus subjected to different

treatments. It shows that Treatment 4, Penicillin was significantly the most effective

treatment with a mean of 45.53mm. The 100% concentration of Honey extract was next

most effective treatment with a mean measurement of 16.23 mm. This implied that

100% honey extract has antibacterial property against E. coli. The effectivity of the

antibacterial property of Honey extract lessens as the concentration of the extract

lessens.

20
 Treatment 2 (50% honey extract), treatment 3 (10% honey extract) and

Treatment 5 (negative control) showed no zone of inhibition against the test organism.

(ANOVA) Appendix Table 3.0 shows that there is no significant difference

among the mean zone of inhibitions of the treatments 100%, 50% and 10%

concentration of honey extract as compared to the positive control (Penicillin).

The result of the investigation showed that different concentration of

honey extract especially 100% is the best concentration to show the Antibacterial

property of the honey against Staphylococcus aureus.

Table 4.0 Sensitivity of Aspergillus fumigatus to Honey Extract

Measurement of the Zone of Inhibition (mm)

Treatments Replication 1 Replication 2 Replication 3 Mean SD

T1=100 % 51.85 34.60 45.60 44.02 8.73
T2=50% - - - - -
T3=10% - - - - -
T4=Positive Control 26.35 23.80 24.80 24.98 1.28
T5=Negative Control - - - - -

Note:(-) No zone of Inhibition

21
 Sensitivity of Aspergillus fumigatus to Honey Extract
Measurement of the Zone of Inhibition (mm)
50
M
40
E
30
A
20
N
10

0
T1=100 % T2=50% T3=10% T4=Positive T5=Negative
Control Control
Experimental Treatment

Fig. 1 Sensitivity of Aspergillus fumigatus to Honey Extract Measurement of the

Zone of Inhibition

Table 4.0 showed the mean of the measurement of the zone of inhibition of

Aspergillus fumigates subjected to different treatments. Treatment 1 (100% honey

extract) was significantly the most effective with the mean in the zone of inhibition of

43.92 mm. This was followed by treatment 4(Positive control) with an average zone of

inhibition of 24.98 mm. This implied that the 100% honey extract has antifungal

properties against Aspergillus fumigatus. Treatment 2 (50% honey extract) Treatment 3

(10% honey extract) and treatment 5 (negative control) were significantly no zone of

inhibition.

Analysis of Variance (ANOVA) table shows that there is no significant difference

among the mean zone of inhibitions of the treatments 100%, 50% and 10%

concentration of honey extract as compared to the positive control(Lysol).

22
 The result of the investigation showed that variable concentration of honey

extract has no effect on the microorganisms. The pure extract greatly inhibits the

growth of the organisms more effective than the positive control.

23
 Chapter V

SUMMARY, CONCLUSIONS, AND RECOMMENDATIONS

Summary

The different treatments were placed in the wells of different culture media and

the zones of inhibition were measured after the incubation period.

Honey extract concentrations 100%, 50% and 10% were found that there is no

antibacterial property for E. coli. In the case of Staphylococcus aureus, honey extract

concentration 100% was found to have antibacterial properties. Of all the treatments,

the positive control Penicillin for E. coli and Staphylococcus aureus showed the highest

zone of inhibition still.

100% Honey concentration were found to have anti-fungal properties against

Aspergillus fumigatus. Of all the treatments, the 100% concentration of honey extract for

Aspergillus fumigatus showed the highest zone of inhibition.

Distilled water, negative control for the three organisms showed no effects by

giving zero zone of inhibition.

Conclusion

The results of the study showed that honey extract have anti-fungal properties

better than the positive control Lysol. Antibacterial properties of the honey extract, on

the other hand, are only showed in Staphylococcus aureus and not on the E.coli

bacteria.

24
Recommendations

The honey extract used in this study is bought in the market, so it is

recommended to get honey extract directly from their den.

It is also recommended to compare the zone of inhibition of the treatments to the

standard zone of inhibition that could qualify to be an anti-bacterial and anti-fungal

active ingredient for ointments or drugs.

The researcher recommends conducting study on the shelf-life of the

experimentally made honey drug. Further research must be conducted to test the anti-

fungal property and antibacterial property of the extract to other test organisms. The

period and method of application should also be modified to attain better results.

Chemical analysis of the product must also be conducted.

25
 BIBLIOGRAPHY

Black, J.K. (1996) Microbiology: Principles and Application. 3rd ed. Prentice Hall,

Incorporated, New Jersey

MANZANO,RALPH ALDEN B. PASIGUE,ANN JILLIAN P. and SERINILLA,JUSTIN

JESSE L. Central Luzon State University Science High School, March 2010.Common

FENG P,WEAGANT S,GRANT,M (2002-09-01),Enumeration of Escherichia coli and

ColifornBacteria, Bacteriological Analytical Manual (8thed.).FDA/Center for Food Safety

& Applied Nutrition.Retrieved 2007-01-25.

GORMAN CM et al. (2008).Discovery of a sexual cycle in the opportunistic fungal

pathogen Aspergillusfumigatus.Nature
.

KLUYTMANS J,VAN BELKUM A,VERBRUGH H (July 1997).Nasal carriage of

Staphylococcus aureus:epidemiology,underlying mechanisms,and associated

risks.Clin.Microbiol.Rev.10 (3): 505-20.

THOMPSON,ANDREA (2007-06-04).”E.coliThrives in Beach Sands”. Live Science.

VOGT RL,DIPPOLD L (2005). Escherichia coli 0157:H7 outbreak associated with

consumptionof ground beef, June-July 2002.Public Health Rep.

WADE,NICHOLAS (15 July 2008).”Taking a Cue From Ants on Evolution of

Humans”.New York Times

Meyer et al., 1996; Dimayuga and Gracia, 1991

Van Mele&Cuc (2000)

Peng & Christian 2005)

Integrated Pest Management (IPM)

26
Sebastian A.J. ZaatPh.D Department of Medical Microbiology at the Academic Medical

Center in Amsterdam FASEB JOURNAL

Gerald WeissmannMD,Editor-in-chief of the FASEB JOURNAL

Banaynal (1999)

Black (1996)

National Honey Board

(Offenberg&Wiwatwitaya 2010)

Asian Pacific Journal of Tropical Biomedicine (July 2012) Antifungal activity of four

honeys of different types from Algeria against pathogenic yeast: Candida albicans and

Rhodotorula sp.; Volume 2, Issue 7, Pages 554–557

Laboratório de Química de Produtos Naturais e Bioativos, Universidade Estadual

de Feira de Santana (UEFS), Universidade Estadual de Santa Cruz (UESC),

Campus Soane Nazaré de AndradeAntimicrobial activity of honey from five species of

Brazilian stingless bees,(Apr. 2013), Ilhéus, BA, Brasil

J Intercult Ethnopharmacol (2013)Ethno botany and antimicrobial perspective of

Spices and Honey against Candida albicans

27
Appendices

28
 Materials

Erlenmeyer Flask¸Beaker and

Vials Graduated Cylinder

Petri Dish Lysol

Test tube Syringe 29

 Materials

Vernier Caliper Weighing Scale

Alcohol Lamp Electric Hot Plate

Auto-clave Transferpette 30
 Materials

Honey Extract Sabouraud Digest Agar

Soyabean Casein Digest Agar

31
 APPENDIX FIGURES

Appendix Figure 1.Media Preparation

Appendix Figure 2. Sterilization of the materials needed in the study

32
Appendix Figure 3. Measurement of the Zone of Inhibtion of

Aspergillus fumigatus

Appendix Figure 4. Measurement of the Zone of Inhibtion of

Staphylococcus aureus

33
Appendix Figure 5. Measurement of the Zone of Inhibtion of

Escherichia coli

Appendix Figure 6. Incubation of Petri dish

34
 Appendix Figure 7.0 Weighing of Nutrient Agar used in the study

Appendix Figure 8.Trasferring the Treatments in different concentration into the

wells.

35
 Appendix Figure 9. Microscopic view of Escherichia coli

Appendix Figure 10. Microscopic view of Staphylococcus aureus

36
 APPENDIX TABLES

Appendix Table 1.0 Analysis of Variance on the zone of inhibition of Escherichia

coli to Honey Extract

ANOVA
Source of
Variation SS df MS F P-value F crit
Between Groups 0.039 2 0.0195 0.000202 0.999798 3.885294
Within Groups 1156.482 12 96.3735

Total 1156.521 14

Appendix Table 2.0 Analysis of Variance on the zone of inhibition of

Staphylococcus aureus to Honey Extract

ANOVA
Source of
Variation SS df MS F P-value F crit
Between Groups 1.009333 2 0.504667 0.001285 0.998716 3.885294
Within Groups 4711.444 12 392.6203

Total 4712.453 14

Appendix Table 3.0 Analysis of Variance of the zone on inhibition of Aspergillus

fumigatus to Honey Extract

ANOVA
Source of
Variation SS df MS F P-value F crit
Between Groups 39.792 2 19.896 0.048288 0.953044 3.885294
Within Groups 4944.353 12 412.0294

Total 4984.145 14

37