i

INVESTIGATION OF THE ANTIMICROBIAL

POTENCY OF GARBAGE ENZYMES PRODUCED FROM
BANANA PEELINGS AND CABBAGE PEELINGS

A research paper submitted to the

Faculty of the Philippine Science High School-
Cordillera Administrative Region Campus
in fulfillment of the course requirements in
Science, Technology, Engineering and Mathematics Research

ALEXANDRA S. SANDAGA
ARNEE FILIA B. SORIANO

10 May 2019
 ii

APPROVAL SHEET

In fulfillment of the requirements in Science, Technology, Engineering and Mathemati

cs Research 2 (STR2), this research entitled, “INVESTIGATION OF THE ANTIMICROBIA
L POTENCY OF GARBAGE ENZYMES PRODUCED FROM BANANA PEELINGS AND
CABBAGE LEAVES” is submitted by Alexandra S. Sandaga and Arnee Filia B. Soriano on 1
0 May 2019.

FIONA U. PAREDES
Adviser

This research paper is hereby accepted by the Research Council.

CONRADO C. ROTOR, Jr., Ph.D.

Chair

MELBA C. PATACSIL
Co-chair

JAY JAY F. MANUEL MARITES P. RIVERA

Member Member

RICARIDO M. SATURAY, JR. FREDA M. WONG

Member Member

ACKNOWLEDGEMENT
 ii

This research was supported by Philippine Science High School - CARC. First and foremost,

we would to express our gratitude to our research adviser, Sir Ricarido Saturay for accepting our resea

rch proposal and Ma’am Fiona Paredes for guiding us on our research study. Thank you for accompan

ying us and providing us one-on-one consultations on improving our study. You both have provided in

sight and expertise that greatly assisted the research. We would also like to thank Ma’am Freda Wong

for aiding us in the antimicrobial potency test. Thank you for showering us with knowledge and for th

e consultations regarding the disk diffusion test. We want to express our gratitude to Sir Jayjay Manu

el for acknowledging us in our part of environmental action. We would like to thank the SRA Unit co

mprising Sir Orlando, Sir Francis, and Sir Kyrie for helping us throughout the conduction of our study

in the biology and chemistry laboratory. We could not have finished the experiment without their guid

ance and laboratory equipment availability. We would like to thank all the people who believed in us, t

eachers, students, and our families, thank you so much. We may conduct a different study next school

year, our final year in Pisay that still revolves around Biology and Chemistry but we will surely not fo

rget all the principles and additional knowledge we gained from all the lectures, consultations, and res

earch. This year has been full of ups and downs and we could not have finished this without believing

in our capabilitues.
 ii

ABSTRACT

Alexandra S. Sandaga and Arnee Filia B. Soriano. Philippine Science High School – Cordi
llera Administrative Region Campus, 10 May 2019. “INVESTIGATION OF THE ANTIMIC
ROBIAL POTENCY OF GARBAGE ENZYMES PRODUCED FROM BANANA PEELIN
GS AND CABBAGE LEAVES”

Adviser: Fiona U. Paredes

Abstract:

Biodegradable wastes eventually rot and cause a lot of inconveniences, among

which is the unpleasant odor emitted that attracts flies. However, garbage enzym

es produced from the fermented fruit and/or vegetable peelings lessen biodegrad

able waste generated in local households while simultaneously serving as an alte

rnative cleaning agent. In this study, the antimicrobial potential of eco-enzymes

extracted from fermented cabbage leaves and from banana peelings is evaluated.

The garbage enzyme solution was filtered from a fermented mixture of sugar,ve

getable/fruit peelings, and water combined in a ratio of 1:3:5 (sugar: vegetable/f

ruit peelings: water). The mixture was set aside to ferment for three months prio

r to filtration. An antimicrobial assay against Staphylococcus aureus and Escher

ichia coli was conducted through the Kirby-Bauer Disk Diffusion Susceptibility

test. The positive and negative controls were Gentamicin (antibiotic) and distille

d water, respectively. Gentamicin exhibited antimicrobial activity on both patho

gens with a zone of inhibition of 21.07 mm. However, relative to this, the observ

ed antimicrobial activity of the solutions filtered from both the fermented banan

a leaves and cabbage peels is negligible.In a similar study, it was found that the f

erment from lakatan banana flesh does not inhibit growth in E. coli and S. aureu

s.
 ii

TABLE OF CONTENTS

APPROVAL SHEET i
ACKNOWLEDGEMENT ii
ABSTRACT iii
List of Figures iv
List of Tables v
CHAPTER I: INTRODUCTION

Background of the Study 1

Objectives of the Study 2
2
Significance of the Study 3
Scope and Limitations of the Study
DEFINITION OF TERMS 5

CH CHAPTER II: REVIEW OF RELATED LITERATURE 6

xCHAPTER III: MATERIALS AND METHODS 10

Research Design 10
Locale of the Study 11
Materials and Research Instruments 11
Procedures 12
Treatment of Data 15

CH CHAPTERR IV: RESULTS AND DISCUSSION 17

C CHAPTER V: CONCLUSION AND RECOMMENDATION 22

S

LI LITERATURE CITED 23

A APPENDICES

Appendix A: Raw Data 25

Appendix B: Formulas/Statistical Tables 28
Appendix C: Documentation 29
 ii

LIST OF FIGURES

Figure Title Page

1 Flowchart of activities 12
2 Zone of Inhibition against E. coli 19
3 Zone of Inhibition against S. aureu 20
4 Fermentation Process 29
5 Fermented Solutions 29
6 MHA Preparation 29
7 Vacuum Filtration 29
8 Fermentation Process 29
 ii

LIST OF TABLES

Table Title Page

1 List of variables 10
2 Materials/instruments 11
3 Corresponding zone of inhibition inferences 14
4 pH of each fruit/vegetable batch 17
5 Presence of Biofilm 17
6 Mean Zone of Inhibition of Banana Peelings 18
and Cabbage Leaves against E. coli and S. aureus
7 Acceptable inhibitory zone diameter (mm) limit of 25
control strains
8 Zone diameter interpretative standard for veterinary 26
pathogens
9 Continuation: Zone diameter interpretative standard 27
for veterinary pathogens
10 Zone of Inhibition Means per Batch 28
 CHAPTER I
INTRODUCTION

Background of the Study

Biodegradable waste such as fruit and vegetable peelings are produced by food processing ind

ustries, food markets, restaurants, and households. As observed in the public market and local househ

olds, these are disposed carelessly, and are found clogged in sewers or scattered on the ground, causin

g an inconvenience, or simply being the source of foul odor (How effective are garbage enzymes, 201

2).

Around 20,500 tons of biodegradable waste is produced all over the Philippines (Senate of the

Philippines, 2017). The increase of biodegradable was contributed by population increase and high de

mand for fruits and vegetables. According to the National Solid Waste Management Commission (NS

WMC), a total of 664.75 tons of biodegradable waste were generated in Cordillera Administrative Re

gion in 2016.

Biodegradable waste could be utilized to produce garbage enzymes (GE). It is also known as

eco enzyme, which is a solution produced from the fermentation of fresh fruit or vegetable dregs, such

as peelings, cuttings and bits, molasses or brown sugar, and water, much like how wine is produced. T

he fermentation process creates a vinegar-like solution with containing natural proteins, mineral salts,

and enzymes, containing acidic properties that are claimed suitable as a non-toxic cleaner which benef

its the environment, including households. It requires waste easily accessible in the kitchen, which ma

kes it a suitable alternative for chemical cleaning agents, such as dishwashing liquid. (Prakash, 2011).

The production of garbage enzymes will lessen biodegradable waste production in local house

holds. Despite the problem on biodegradable waste, there is limited affordable cleaning agents found i

n the market. Through the production of garbage enzymes, this will be able to provide the local comm

unity a non-toxic cleaner that does not utilize chemicals that will harm the environment and the comm

on people. Moreover, it is an affordable and suitable alternative for chemical cleaning agents that is en

vironmentally-friendly because it utilized fruit and vegetable dregs and also, the ingredients are readil

y available and could be acquired at a negligible cost.

 This would also act as a guide for future researchers since there are not many studies on the sa

id topic. Most of the research studies found mainly focus on combined biodegradable waste and not o

n specific kinds of fruits and vegetables that contain the properties.

Statement of the Objectives

General Objective/s

To evaluate the antimicrobial potency of garbage enzymes produced from fermented banana p

eelings and cabbage leaves.

Specific Objective/s

1. To determine the antimicrobial potency of garbage enzymes from fermented banana peels t

hrough disk diffusion against E. Coli and S. Aureus by measuring the zone of inhibition.

2. To determine the antimicrobial potency of garbage enzymes from fermented cabbage leave

s through disk diffusion against E. Coli and S. Aureus by measuring the zone of inhibition.

3. To compare the antimicrobial potency of garbage enzymes produced from fermented banan

a peelings and cabbage leaves through disk diffusion by measuring the zone of inhibition.

Significance of the Study

The antimicrobial properties of eco enzymes have been in the media for years. The production

of eco-enzymes from the fermentation of fruit and vegetable peelings are beneficial, first and foremost,

to the common people and the local households. They are beneficial since Garbage enzymes are prod

uced through fruit and vegetable dregs, which are easily accessible in kitchens of the common people.

Fruit and vegetable waste are locally available because of the large food consumption of the communi

ty. If proven to be successful, the production, along with the investigation of the anti-bacterial properti

es of the eco-enzymes will provide a cheaper and more accessible alternative to chemical cleaning age

nts such as dishwashing liquid.

 Moreover, the people who produce and capitalize from the production and retail of fruits and

vegetables, in this case bananas and cabbages, obtain the opportunity to lessen the waste produced, in

small scale and also, lessen the risk. Piling of biodegradable waste in homes or production areas is a n

uisance at best, and a health hazard at worst. Asides from the common people, the investigation of the

antimicrobial potency of eco-enzymes will also benefit the scientific community, as it provides the dat

a that is most commonly absent in the media. The research fills in knowledge gaps of the antimicrobia

l potency of the enzyme.

Greenhouse gases are gases that absorb infrared radiation in the atmosphere. Methane and nitr

ous oxides are examples of greenhouse gases, which are produced by the disposal of these decomposa

ble wastes, either in landfill or by composting. When thrown in the environment, this decomposable w

aste can be utilized in order to produce value added bio-product, which in turn, reduces the production

of greenhouse gas created from it (Saramanda, 2017). Therefore, garbage enzymes will have a long te

rm benefit to the community.

Scope of Limitations of the Study

The research is focused on the investigation of the antimicrobial potency of garbage enzymes produce

d from banana peelings and cabbage leaves. The bacterial strains used are Staphylococcus aureus and

Escherichia coli. There were three batches for each fruit/vegetable and three replicates per batch. It w

as conducted at the Philippine Science High School - Cordillera Administrative Region Campus - Che

mistry Laboratory and Biology Laboratory.

The controls used for the disk diffusion test were distilled water and gentamicin, an antibiotic.

Distilled water was used as the negative control since it does not affect the results. It is pure and has a

pH of level of 7 of neutral. In addition, it does not contain ions that may interfere with the chemical re

actions involved. The antibiotic that was used as the positive control is Gentamicin. It is readily availa

ble in the school laboratory and is an acceptable antimicrobial agent for E. coli and S. aureus accordi

ng to the disk diffusion protocol by Tendencia (2004)

 DEFINITION OF TERMS

Disk Diffusion - a test of the antibiotic sensitivity of bacteria. It uses antibiotic discs to test th

e extent to which bacteria are affected by those antibiotics.

Enzyme - a substance produced by a living organism that acts as a catalyst to bring about a sp

ecific biochemical reaction. They speed up chemical reactions in the body, but do not get used up in th

e process.

Fermentation - an anaerobic cellular process in which an organic substance converts a carbo

hydrate, such as starch or a sugar, into an alcohol or an acid in the absence of oxygen.

Garbage Enzyme - also known as eco enzyme, which is a solution produced from the fermen

tation of fresh fruit or vegetable dregs, such as peelings, cuttings and bits, molasses or brown sugar, a

nd water, much like how wine is produced.

Zone of Inhibition - the clear region around a disc saturated with an antimicrobial agent on t

he agar surface. The clear region is an indication of the absence, or the effective inhibition, of microbi

al growth by the antimicrobial agent.

 CHAPTER II
REVIEW OF RELATED LITERATURE

A. Garbage Enzyme
Garbage Enzymes (GEs), also termed Eco- Enzyme according to Prakash, is a solution that is organic

in nature, produced by the fermentation of fresh vegetable or fruit waste, brown sugar or molasses, an

d water. Bacteria and microorganisms metabolize molasses, the key ingredient in the solution, into al

cohol.

This is reduced in its final form to acetic acid or vinegar. Vinegar with its acidic properties is

well known as an all-round non-toxic cleaner. It has a strong sweet and sour fermented scent and a dar

k-brown color. Through the process, it creates vinegar composed of natural proteins, mineral salts, an

d enzymes that make it multipurpose for agriculture, household, etc. (How effective are garbage enzy

mes,2012)

Dr. Rosukon Poompanvong, a researcher based in Thailand, developed garbage enzymes. She

has been actively involved in enzyme research for more than 30 years. She encourages people to make

eco enzymes at home since it can be easily made. This will be able to ease global warming and lessen

greenhouse gases in our atmosphere. (Prakash, 2017).

The enzymes are obtained through the fermentation of fresh vegetable dregs and/or fresh fruit

waste, along with brown sugar or molasses, and water. Fermentation is defined as the anaerobic proce

ss whereby organic compounds such as carbohydrates are broken down to form products such as lacti

c acid or alcohol.

The production of eco enzymes has been published and promoted by the media dating back fro

m its discovery, thirty years ago. Claims and results of its effectiveness as a substitute for cleaning age

nts can be found on the internet, and all these sources state similar processes on how to produce the en

zymes.

A research by Arun states that the enzymes were produced from the molasses taken from suga

rcane industries, and the fruit waste and vegetable dregs provided by shops, specifically, pineapple, or

ange, and mango dregs, respectively. Three parts of the waste, containing equal grams of each of the d

regs, were mixed with one part of molasses and ten parts of water in air tight containers, which were t
hen placed in a cool, dry, and well ventilated area in order for the complete degradation of organic mat

ter to occur Three months were dedicated for the fermentation of the mixture, before the filtering of t

he residue to acquire the enzyme is done (Arun, 2015).

To know if the fermentation process is working, the researcher must be able to see a white lay

er of biofilm on the surface of the enzyme. This indicates the presence of microorganisms.

The process for preparing the mixture is known to take about 5 minutes. However, it states that

the producer must open the lid at least once a week to release the gases to avoid the build up of gasses

that may cause the container to explode (How effective are garbage enzymes, 2012).

As for the pH, pure garbage enzymes are acidic, with a pH of 3.6. Enzymes are affected by ch

anges in pH. The shape of the enzyme may be affected therefore, either the substrate cannot bind to th

e active site or it cannot undergo catalysis. Within a narrow pH range, changes in the structural shapes

of the enzymes and substrates may be reversible. But for a significant change in pH levels, the enzym

e and the substrate may undergo denaturation.

In this study, garbage enzymes will be prepared from banana peels and cabbage leaves.

B. Disk Diffusion: Anti-Microbial Testing

Escherichia coli and Staphylococcus aureus are a serious cause of a variety of community- an

d hospital-acquired infections. E. coli is one of the most common nosocomial pathogens that cause uri

nary tract infections (UTIs) and enterocolitis. S. aureus is also an etiological infection agent responsib

le for significant levels of morbidity and mortality. According to Broad Institute. In 2010, Escherichia

coli accounts for 17.3% of clinical infections requiring hospitalization and is the second most commo

n source of infection behind Staphylococcus aureus (18.8%). In recent years, the emergence of resista

nt Staphylococcus aureus and resistant Escherichia coli strains to many antibiotics has been observed

worldwide. These have become a major concern in global public health invigorating the need for new

antimicrobial compounds. (Bachir raho, G., 2015).

As previously mentioned, garbage enzymes, in its reduced and final form of vinegar, has acidi

c properties that is well known as an all-around non-toxic cleaner. The antimicrobial activity of the ga
rbage banana peeling ferment and the cabbage leaves will be evaluated through the disk diffusion met

hod.

The antibiotic saturated disk will be placed on the agar that was previously inoculated with th

e test bacterium, wherein it will pick up moisture, and the antibiotic will diffuse radially outward thro

ugh the agar medium producing an antibiotic concentration gradient. This will be the basis for the prin

ciple of the disk diffusion method (Tendencia, 2004).

Susceptibility testing is used to determine which antimicrobials will inhibit the growth of the ba

cteria or fungi causing a specific infection. The results from this test will help a healthcare practitioner

determine which drugs are likely to be most effective in treating a person's infection. (American Asso

ciation of Clinical Chemistry, 2018)

The best medium for routine susceptibility tests due to its good reproducibility, low amount in

sulfonamide, trimethoprim, and tetracycline inhibitors, is the Mueller-Hinton Agar. These properties

mean it gives satisfactory growth results of most bacterial pathogens, which is why the disk diffusion

method is performed using MHA (Tendencia, 2004). In this particular study, Nutrient Agar will be util

ized. microorganisms since it grows the largest number of different types of microbes - fungi and bact

eria. Yet, not all bacteria can grow on these. Some find it too rich, and others find it deficient (Liu &

Usinger, 2019).

The diameter of the zone of inhibition in the test is compared with those in the chart in interpretative s

tandard for veterinary pathogens. The result is reported as Resistant, Intermediate, or Susceptible (Ten

dencia, 2004).

Susceptibility test results using agents other than those listed in the chart is interpreted on the

basis of the absence or presence of a definite zone of inhibition and is considered only as qualitative u

ntil such time as interpretative zones have been established.

Once the fermentation process is complete, the solution or vinegar will be filtered using filter

paper. It will then be transferred into another container and sealed to be used in the antimicrobial anal

ysis (Arun, 2015)

 CHAPTER III

MATERIALS AND METHODS

Research Design

The garbage enzymes were obtained through the fermentation of banana peelings and cabbage leaves

with brown sugar. There were three batches for each fruit/vegetable.

The measurements were based on a ratio of 1:3:5 (Saramanda, 2017), therefore, 40g brown s

ugar, 120g fresh vegetable/fruit peelings, and 200g distilled water was used. The mixture was placed i

n an airtight glass jar in a cool, dry, and ventilated area for complete degradation of the organic matter.

The fermentation process was conducted for three months.

 The potency of the garbage enzymes was analyzed through the disk diffusion method. The ant

imicrobial activity of the enzyme to bacteria, specifically Escherichia coli and Staphylococcus aureus

was measured.

The controls that were used in the assay are listed in the table below,

Table 1. List of Variables

Constant Variable/s distilled water

type of antibiotic: gentamicin

type of ferment: banana solution

type of ferment: cabbage solution

Independent Variable/s duration of the disk diffusion method

amount of bacteria for disk diffusion method

Dependent Variable/s zone of inhibition

Locale of the Study

The fermentation process and testing of the antimicrobial potency of garbage enzymes produced from

banana peelings and cabbage leaves will be conducted at the Philippine Science High School - Cordill

era Administrative Region Campus - Chemistry Laboratory and Biology Laboratory, located at Purok

12 Lime Kln, Baguio City, Benguet, Philippines. The banana peelings and cabbage leaves will be brou

ght fresh from the Baguio City Public Market.

Materials and Research Instruments

Table 2. Materials/Instruments
Material/Instrument Specifications

Ohaus: Pioneer Plus Analytical Bal Model: Ohaus Pioneer Plus Analytical Balance and Precisio
ance and Precision Series n Series
Description: It is designed to accurately weigh materials in t
he sub-milligram range.

Magnetic Hotplate stirrer Model: Success Technique Industries Magnetic Hotplate Sti
rrer
Description: t is generally used to heat glassware or its cont
 ents.

Biosafety Cabinet Model: Esco, Streamline Class II Biosafety Cabinet (BSC)

Description: It is an enclosed, ventilated laboratory worksp
ace for materials requiring a defined biosafety level. It prev
ents further contamination to the environment.

Autoclave Model: Jinan Hanon Instruments Vertical Autoclave, YXQ -

5O
Maximum Working/Design Temperature: 135°C/139°C
Maximum Working/Design Pressure: 0.22MPa/0.25MPa
Volume: 60 L
Power: 3.1 KW
Description: It provides a physical method for disinfection
and sterilization through high temperature and pressure to e
liminate microorganisms and spores.

Incubator Model: Digisystems Laboratory Instruments Inc. Incubator

Description: It provides a controlled, contaminant-free envi
ronment for safe, reliable work of plate cultures by regulati
ng conditions such as temperature, humidity, and CO2.

Nutrient Broth Model: HiMedia Laboratories Nutrient Broth, M002-500G

Description: It is used for general cultivation of less fastidio
us microorganisms, can be enriched with blood or biologica
l fluids.

Nutrient Agar Model: HiMedia Laboratories Nutrient Agar, M001-500G

Description: It is used for general cultivation of less fastidio
us microorganisms, can be enriched with blood or biologica
l fluids.

Electronic pH Indicator It is used to measure hydrogen-ion activity (alkalinity or aci

dity)

Petri Dish Model: Glassco Pyrex Petro Dish

It is a shallow cylindrical glass or plastic lidded dish that bi
ologists use to culture cells such as bacteria, enzymes, etc.

Whatman FIlter Paper Name/Model: Whatman Filter Paper

Cat No.: 1001 125
Size: 125 mm
Description: It is a semi-permeable paper barrier placed per
pendicular to a liquid or air flow. It is used to separate fine s
ubstances from liquids or air.

Procedures

Acquisition Collection o
Fermentation
of Materials f Dregs
 Medium Pr Inoculation of
Filtration pH Analysis
eparation DIsks

Figure 1. Flow Chart of Activities

The garbage enzymes extracted were based on the ratio, 1:3:5 (Saramanda, 2017), following 4

0g brown sugar, 120g fresh vegetable/fruit peelings, and 200g distilled water. The mixture was placed

in an airtight glass jar in a cool, dry, and ventilated area for complete degradation of the organic matter

The fermentation process was conducted for a minimum of three months.

. The antimicrobial potential of the garbage enzyme was investigated to characterize its proper

ties. The antimicrobial assay was based from the Kirby-Bauer disk diffusion method (Hudzicki, 2009)

and the The Philippine Standard Disk Diffusion method (Tendencia, 2004). A suspension was prepare

d by dissolving of 15.12 grams of Mueller-Hinton Agar powder in 540 mL of purified/distilled water.

The resulting solution was heated and stirred continuously until it boils. The medium was then steriliz

ed by autoclaving it at 15 lbs pressure (121 °C) for 45 minutes and was cooled to room temperature.

Twelve Petri dishes were autoclaved using the same parameters, 15 lbs pressure (121 °C) in 4

5 minutes. The medium was transferred onto these Petri dishes under the Biosafety Cabinet.

Inoculum and Bacteria Preparation

The bacterial strains, Escherichia coli (Gram negative) and Staphylococcus aureus (Gram positive) w

ere obtained from the Science and Research Assistant (SRA) unit of Philippine Science High School -

CAR.

Gram Negative Bacteria are bacteria which do not retain the crystal violet stain because of its

physical makeup and will stain pink while Gram positive bacteria has a very thick cell wall made of p

rotein called peptidoglycan. These bacteria retain the crystal violet dye.

The inoculum is a small amount of substance containing bacteria from a pure culture which is
used to start a new culture or to infect an experimental animal. For preparation of the sub-culture (Ten

dencia, 2004), 0.52 g of nutrient broth powder and nutrient agar powder was weighed and put into a 2

50 mL Erlenmeyer flask. A graduated cylinder was used in measuring 40 mL of purified/distilled wate

r which was then added into the erlenmeyer flask. It was dissolved by stirring and heating the solution

then were autoclaved using the same parameters stated. The medium was then transferred into 2 airtig

ht screw-capped test tubes in a slanted position. After hardening, E. coli and S. aureus were inoculated

in each test tube and incubated for at least 24 hours.

From the sub-culture made, four or five colonies were taken with a wired inoculating loop. Th

e bacterial colonies were then transferred into the test tube containing the nutrient broth solution. This

method was done separately for both bacterial strains. The turbidity of the test bacterial suspension wi

th that of 0.5 McFarland (vigorously shaken before use) was compared against a white background wi

th contrasting black line under adequate light.

Disk Diffusion

The Kirby-Bauer Disk Diffusion Assay was conducted in accordance to Tendencia (2004). A sterile co

tton swab was dipped into the standardized bacterial suspension. Excess inoculum was removed by lig

htly pressing the swab against the tube wall at a level above that of the liquid. The agar was inoculate

d by streaking the cotton swab containing the inoculum. The plate was rotated by 60° and this was rep

eated multiple times until the plate was fully covered using the rubbing procedure. This ensured an ev

en distribution of the inoculum. The surface of the medium was allowed to dry for 3-5 minutes but not

longer than 15 minutes to allow for absorption of excess moisture. The diameter of the zone of inhibiti

on were observed for both cabbage leaves and banana peelings against S.aureus and E. coli in accorda

nce to the corresponding inferences (Table 3).

 Table 3. Corresponding zone of inhibition inferences (Tendencia 2004)

< 10 mm may be expressed as in active

10 - 13 mm partially active

14 - 19 mm active

> 19 mm greatly active

Antibiotic Disks

Using Whatman filter paper, more than 72 disks of 6 mm diameter were made to be utilized f

or paper disk diffusion. There were four disks, each disk was dispensed each solution: the first sampl

e, second sample, positive control, and negative control.

To measure the diameter of the zones of inhibition, use a ruler graduated to 0.5mm and round

up the zone measurement to the nearest millimeter (Tendencia 2004).Susceptibility test results using a

gents other than those listed in the chart was interpreted on the basis of the presence or absence of a d

efinite zone of inhibition and it was considered only as qualitative until such time as interpretative zon

es have been established (Tendencia, 2004)

Treatment of Data

Statement of the Hypotheses

There is no recognizable zone of inhibition for the fermented banana peelings solution agains

t the bacterial strains, E. coli and S. aureus.

There is no recognizable zone of inhibition for the fermented banana peelings solution against

the bacterial strains, E. coli and S. aureus.

There is no significant difference in the mean zone of inhibition between the fermented banan

a peelings solution and the fermented cabbage leaves solution.

Analysis of Data
The diameter of the zone of inhibition was based in accordance to the inferences of the disk diffusion

protocol by Tendencia (2004).

CHAPTER IV
RESULTS AND DISCUSSION

pH Test

Table 4. pH of each fruit/vegetable batch

1 2 3

Banana Peelings 4.20 9.00 9.20

 Cabbage Leaves 3.20 3.00 invalid

The ferment generally has acidic pH since the average pH of fermented garbage “enzyme” solution is

3.6. As shown above (Table 4), the second and third banana ferment should be disregarded as it is not

relevant to the study since the solution became basic or alkaline, making its contamination apparent.

Preliminary Data
Table 5. Presence of Biofilm
BANANA CABBAGE
WEEKS
I II III I II III
WEEK 1
WEEK 2
WEEK 3
WEEK 4
WEEK 5
WEEK 6
WEEK 7
WEEK 8
WEEK 9
WEEK 11
WEEK 12

Legend: orange - unknown, red - none, green - present

The fermented solution were observed over a 12-week duration for the formation of biofilm, ther

efore, indicating there is the presence of microbes (How effective are garbage enzymes, 2012).

Antimicrobial Susceptibility of Fermented Cabbage Leaves

The Disk Diffusion test was performed on the bacterial strains Staphylococcus aureus and Es

cherichia coli with the fermented samples in accordance with the Kirby-Bauer protocol used (Hudzick

i, 2009). The zone of inhibition of the fermented solution of banana peelings and cabbage leaves with

the two bacterial strains, S. aureus and E. coli were compared.

 Table 6. Mean Zone of Inhibition for Banana Peelings and Cabbage Leaves against E. coli and S. aur
eus
Treatment Mean Zone of Inhibition (in mm)

Escherichia coli Staphylococcus aureus

Mean 土 standard devia Mean 土 standard devia

tion tion

Solution from Banana peelings 6 mm 6 mm

Solution from Cabbage leaves 6 mm 6 mm

Gentamicin 20.6 mm 20.6 mm

Sterilized Drinking Water 6 mm 6 mm

Four disks were placed in each petri plate: the positive control (Gentamicin, A), negative cont

rol (Distilled Water, DW), banana peelings ferment (B), and the cabbage leaves ferment (C).
 R1 R2

R3 R4

R5 R6

Figure 2. Zone of Inhibition against E. coli (R: Replicates, A: Antibiotic, B: Banana, C: Cabbage, DW
Distilled Water)
R1 R2

R3 R4

R5 R6

Figure 3. Zone of Inhibition against S. aureus (R: Replicates, A: Antibiotic, B: Banana, C: Cabbage,
DW: Distilled Water)

The zone of inhibition of the fermented solutions against the bacterial strains Escherichia coli an

d Staphylococcus aureus are measured in millimeters. Compared to Gentamicin, both the banana peel

ing and cabbage leaves ferment did not inhibit the growth of both bacteria, E. coli and S. aureus, as sh

own by the six replicates for each fruit/vegetable. There was no inhibition because it only covered the

size of the disk which is 6 mm. Gentamicin, the positive control, exhibited great activity (Table 3) aga

inst S.aureus and E.coli on all six replicates with a mean zone of inhibition of 20.6 mm (Table 6). The

sterilized drinking water, the negative control, showed no zones of inhibition in all trials, as expected.
However, relative to this, the observed antimicrobial activity of the solutions filtered from both the fer

mented banana leaves and cabbage peels is negligible.

In a similar study entitled, “The Antimicrobial Property of Lakatan Banana” conducted by Joseph

Gil in 2013, parallel results were obtained when it was found that lakatan banana flesh ferment did no

t inhibit growth in E. coli and S. aureus (Gil,2013).

 CHAPTER V
CONCLUSION AND RECOMMENDATIONS

The study evaluated the antimicrobial potency against the bacterial strains, E. coli and S. aureus, of ga

rbage enzymes produced from fermented banana peelings and cabbage leaves through disk diffusion.

The zone of inhibition from each ferment was compared to evaluate the antimicrobial potency of the p

roduced enzymes. These were also compared with the positive control, Gentamicin.

Compared with Gentamicin (zone of inhibition: 20.6 mm), the observed antimicrobial activity

of the fermented banana peelings and cabbage leaves solutions are negligible. Hence, both the banana

peelings ferment and cabbage leaves ferment did not inhibit the growth of E. coli and S. aureus, as ob

served from the six replicates of the ferment.

It is recommended to conduct the antimicrobial assay and disk diffusion immediately after the

fermentation and filtration process as this may have affected the antimicrobial and chemical properties

of the enzyme solution. As observed in the fermentation process, the presence of protein should be co

nfirmed. A protein assay such as the Biuret test and the Lowry protein assay test must be conducted. T

he Lowry protein assay is a biochemical assay used to measure and determine the total concentration

of protein present in a solution. It uses copper which bonds with peptide bonds under alkaline solution

s while the biuret test is a chemical test used for detecting the presence of peptide bonds. In the presen

ce of peptides, a copper(II) ion forms mauve-colored coordination complexes in an alkaline solution.

LITERATURE CITED

American Association for Clinical Chemistry. (22 July 2018). Antibiotic susceptibility testing.
 Retrieved on 16 November 2018 and from https://labtestsonline.org/ tests/antibiotic-susceptib
ility-testing
Arun, C. (2015). Process Safety and Environmental Protection. Investigation of biocatalytic pote
ntial of garbage and its influence on stabilization of industrial waste activated sludge, 94, 471-478. D
oi:10.1016/j.psep.2014.10.008
Aryal, S. (15, April 2015). Nutrient Agar: Composition, Preparation, and Uses. Retrieved on 4
February 2019 from https://microbiologyinfo.com/nutrient-agar-composition- preparation-an
d-uses/
Aryal, S. (7, July 2015). Potato Dextrose Agar (PDA) – Principle, Uses, Composition, Procedure, and
Colony Characteristics. Retrieved on 4 February 2019 from https://microbiologyinfo.com/potat
o-dextrose-agar-pda-principle-uses-composition-procedu re-and-colony-characteristics/
Bahir raho, G. and Abouni, B. (2015). Escherichia coli and Staphylococcus aureus most common
source of infection; The Battle Against Microbial Pathogens: Basic Science, Technological Adv
ances and Educational Programs (A. Méndez-Vilas, Ed.). Laboratory of Molecular Microbiolog
y, Health and proteomics, University of Sidi Bel abbes, Algeria
Gil, J. (2013). The Antimicrobial Property of Lakatan Banana. Retrieved on 01 April 2019 and fro
m https://prezi.com/the-antibacterial-properties-of-lakatan-banana
Hartree-Lowry and Modified Lowry Protein Assays. (2019). Retrieved 05 February, 2019 from
https://www.ruf.rice.edu/~bioslabs/methods/protein/lowry.html
HiMedia Laboratories (n.d.) Mueller Hinton Agar [PDF File]. Retrieved on 4 February 2019 fro
m http://himedialabs.com/TD/M173.pdf
How effective are garbage enzymes. (6 December 2012). Retrieved on 28 August 2018 and from
http://www.ecowalkthetalk.com
Hudzicki, J. (08 December 2009). Kirby-Bauer DIsk DIffusion Susceptibility Test Protocol. Retr
ieved on 07 January 2019 from http://www.asmscience.org.content/education/protocol/
protocol.3189
Liu, Shijun & Usinger, Laurie. (2019). Science buddies: All about agar. Retrieved on 22 March, 201
9 from https://www.sciencebuddies.org/science-fair-projects/references/
grow-microbes-agar
Nazim, F. and Meera, V., 2013. Treatment of synthetic grey water using 5% and 10% garbage
enzyme solution. Bonfring Int. J. Ind. Eng. Manage. Sci. 3 (4), 111–117.
NCCLS. 2002. Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for
Bacteria Isolated from Animals; Approved Standard-Second Edition. NCCLS document M31-A2 (IS
BN 1-56238-461-9). NCCLS, 940 West Valle Road, Suite 1400, Wayne, Pennsylvania 19087-1898,
USA
Prakash, B. (27 February 2011). How to make and use garbage enzymes. Retrieved on 31 Aug
ust 2018 and from http://www.ecowalkthetalk.com/blog/2011/02/27/how-to- make-and-use- garb
age- enzymes/
Saramanda, G. (2017). Antimicrobial Activity of Fermented Citrus Fruit Peel Extract. Antimicrobi
al Activity of Fermented Citrus Fruit Peel Extract, 7(11), 7th ser., 25-28. doi:10.9790/9622-07110725
28
Smitha, R.B. (2010). Dual production of endotoxin and amylase from Bacillus thuringiensis sub
sp. Kurtsaki by fermentation and efficacy studies of endotoxin against eriophyid mite. Retrieved o
n 15 September 2018 and from http://shodhganga.inflibnet.as.in/handle/10603/4017
Tang, F. and Tong, C.W.. (2011). “A Study of the Garbage Enzyme’s Effects in Domestic Wastewater’
Digital Open Science Index, Environmental and Ecological Engineering Vol:5, No:12, 2011. waset.or
g/Publication/6989
Senate of the Philippines. (November 2017). Philippine solid wastes. Retrieved on November 15,
2018 and from https://www.senate.gov.ph/publications/SEPO/ AAG_Philippine %2520Solid
%2520Wastes_Nov2017.pdf
Tendencia, E. A. (2004). Disk diffusion method. In Laboratory manual of standardized methods for
antimicrobial sensitivity tests for bacteria isolated from aquatic animals and environment (pp. 1
3-29). Tigbauan, Iloilo, Philippines: Aquaculture Department, Southeast Asian Fisheries Developmen
t Center.
What is eco enzyme. (2018). Retrieved on 28 August 2018 and from http://www.enzyesos.co/
 what-is-eco-enzyme
Worthington Biochemical Corporation. (n.d.). Effects of pH. Retrieved on 17 November 2018 and
from http://www.worthington-biochem.com/introbiochem/effectsph.html
 APPENDIX A
RAW DATA

Table 7. Acceptable inhibitory zone diameter (mm) limit of control strains

Table 8. Zone diameter interpretative standard for veterinary pathogens
Table 9. Continuation: Zone diameter interpretative standard for veterinary pathogens
 APPENDIX B
STATISTICAL TABLES

Table 10. Zone of Inhibition meas per Batch

Treatment Mean Zone of Inhibition (in mm)

Escherichia coli Staphylococcus aureus

Mean 土 standard devia Mean 土 standard devia

tion tion

Mean 土 standard devia Mean 土 standard devia

Solution from Banana peelings
tion tion

Mean 土 standard devia Mean 土 standard devia

Solution from Cabbage leaves
tion tion

Mean 土 standard devia Mean 土 standard devia

Gentamicin
tion tion

Mean 土 standard devia Mean 土 standard devia

Sterilized Drinking Water tion tion
 APPENDIX C
DOCUMENTATION

Figure 4. Fermentation Process Figure 5. Fermented Solutions

Figure 6. MHA Preparation Figure 7. Vacuum filtration

Figure 8. E. coli and S. aureus subculture