Synergistic Antimicrobial Effect of Ging

University of San Agustin
COLLEGE OF HEALTH AND ALLIED MEDICAL PROFESSION

MEDICAL LABORATORY SCIENCE PRORAM
SYNERGISTIC ANTIMICROBIAL EFFECT OF GINGER (Zingiber officinale)
ETHANOLIC EXTRACTS AND PROBIOTIC (Lactobacillus casei) AGAINST
ENTERIC PATHOGENIC BACTERIA
A Research Presented to the Faculty of the
College of Health and Allied Medical Professions
MLS Program
In Partial Fulfillment of the Requirements for the Degree of
Bachelor of Science in Medical Laboratory Science
BESAS, Analiza
CASADOR, Rochelle Dominique
CASTILLO, Armeline
CERCADO, Mayz
CHAVEZ, Euna Grace
DEL CASTILLO, Ma. Angelica
DITALO, James Dominic
November, 2020
Table of Contents
Title Page
CHAPTER
1. INTRODUCTION
Background and Rationale 1
Objectives of the Study 2
Hypothesis 4
Research Framework 4
Significance of the Study 5
Scope and Delimitations 6
Definition of terms 7
2. REVIEW OF RELATED LITERATURE
Gastrointestinal Infection 13
Antibiotics and Antimicrobial Resistance 14
Zingiber officinale 16
Antimicrobial Activities of Ginger 17
Probiotics and Lactobacillus casei 19
Antimicrobial Activities of Probiotics and L. casei 22
Escherichia coli Pathotype 23
Staphylococcus aureus Pathotype 25
Synthesis 27
3. METHODOLOGY
Research Design 29
ii
Social Preparations and Ethical Considerations 29
Sampling Design and Sample Size Determination 30
Procedures/Methods 31
Data Processing 35
Data Analysis 36
REFERENCES 37
APPENDICES
A. Draft of Letter to the Research Laboratory In-charge 42
B. Schematic Diagram of Research Procedures 44
TIMETABLE FOR RESEARCH PROPOSAL 50
BUDGETARY REQUIREMENTS 51
DUMMY TABLE 52
CURRICULUM VITAE 54
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Chapter 1
INTRODUCTION
Background and rationale
A significant burden of acute and chronic illness is induced by gastrointestinal
infections, with diarrhea being the most frequent manifestation. According to the
Epidemiology Bureau Department of Health, there were 61, 281 cases or 59.1 rate of
acute bloody diarrhea in the Philippines in 2016, while there was 109.1 rate of acute watery
diarrhea, mostly were caused by bacterial and parasitic infections (DOH, 2016).
Conversely, use of antibiotics could cause adverse complications and bacterial resistance
to antimicrobial drugs is increasing worldwide (Rahmani et al., 2014) such that rate of
resistance to ciprofloxacin varied from 8.4% to 92.9% for E. coli and from 4.1% to 79.4%
for K. pneumoniae according to Global Antimicrobial Resistance and Use Surveillance
System (GLASS) (WHO, 2020).
Zingiber officinale (ginger) is a plant used in traditional medicine against different
diseases because of its various properties (antimicrobial, antioxidant, anti-inflammatory,
etc.) and recognized as “safe” by the US Food and Drug Administration. Ginger contains
monoterpenoids, sesquiterpenoids, phenolic compounds, and its derivatives which
provide a broad antimicrobial spectrum against different microorganisms (Beristain-Bauza
et al., 2019). Ethanol extracts of this rhizome showed remarkable activity against various
microorganisms with the highest activity on E. coli (Yusha’u et al., 2008) and also have
shown a wide range of influence on gram-positive aerobic bacteria especially in the area
of influence on tested staphylococci (Santo-Grace et al., 2017).

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The probiotics are first described by Lilley and Stillwell as substances secreted by
one microorganism that stimulated another's growth and were later used to describe tissue
extracts that stimulated microbial growth (Nazir et al., 2018). Being enteric in nature,
probiotics do not have any parasitic or pathogenic effect on humans as they retard the
growth of harmful microorganisms and are very potent against pathogens protecting the
enteric flora (Shipradeep et al., 2012). Probiotics help the infantile immune system by
lowering the pH of the gut, rendering it unsuitable for pathogenic bacteria to
survive. Lactobacillus is one of a number of probiotics considered to be biological
therapeutics and host immune-modulating biologicals that are generally recognized as
safe (Chen et al., 2019).
Prakasita et al. (2019) reported that the tested combinations of herbs and
probiotics can adhere to the intestinal tract. Studying the collective antimicrobial activities
of ginger and probiotics may help find alternative solutions in improving antibiotic formulas
without any harmful side effects to the immune system and to the body’s microbiota and
it will also help reduce morbidity of gastrointestinal diseases.
Objectives of the study
The main purpose of this study is to determine the synergistic antimicrobial effect
of ginger ethanolic extract and the probiotic Lactobacillus casei Shirota strain against
enteric pathogenic bacteria such as Escherichia coli and Staphylococcus aureus.
Specifically, it aims to:

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1. determine the minimum inhibitory concentration of ginger ethanolic extracts by
disk diffusion method to be used in the succeeding assay;
2. determine the zone of inhibition of the following preparations on Escherichia coli
and Staphylococcus aureus using disk diffusion method:
a. 1 ml ginger ethanolic extracts (1mg/ml)
b. 1 ml Lactobacillus casei Shirota strain cell-free supernatant
c. 1:1 proportion of ginger ethanolic extracts and Lactobacillus casei Shirota
strain
d. 2:1 proportion of ginger ethanolic extracts and Lactobacillus casei Shirota
strain
e. 1:2 proportion of ginger ethanolic extracts and Lactobacillus casei Shirota
3. determine if there is a significant difference in the zones of inhibition produced by
the following preparations on Escherichia coli and Staphylococcus aureus:
a. 1 ml ginger ethanolic extracts (1mg/ml)
b. 1 ml Lactobacillus casei Shirota strain cell-free supernatant
c. 1:1 proportion of ginger ethanolic extracts and Lactobacillus casei Shirota
strain
d. 2:1 proportion of ginger ethanolic extracts and Lactobacillus casei Shirota
strain
e. 1:2 proportion of ginger ethanolic extracts and Lactobacillus casei Shirot

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Hypothesis
There is no significant difference in the zones of inhibition produced by ginger
ethanolic extracts, Lactobacillus casei Shirota strain, and their combinations in different
proportions on Escherichia coli and Staphylococcus aureus.
Research framework
Dependent Variables
Independent Variables
serial dilutions of ginger ethanolic Minimum inhibitory

extracts concentration (MIC) and
zone of inhibition on
1000 μg/ml - 100μg/ml
a. Escherichia coli
b. Staphylococcus
Treatment 1: aureus
1mL Ginger ethanolic extract (1mg/ml
conc.)
Treatment 2:
1mL Lactobacillus casei Shirota strain
cell free supernatant
Treatment 3: zone of inhibition produced

1:1 proportion of ginger ethanolic on
extracts and Lactobacillus casei Shirota
a. Escherichia coli
Treatment 4:
b. Staphylococcus
2:1 proportion of ginger ethanolic
aureus
Treatment 5:
1:2 proportion of ginger ethanolic
Figure 1. Research Framework

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Based on the theory of Debalke et al. (2018), aqueous-ethanolic plant extracts
solutions have been used to determine the zone of inhibition to cultures of E. coli and
S. aureus. It showed that the zone of inhibition and susceptibility of the microorganisms to
the solutions varies depending on the concentration of the extracts added to each plate
media.
Generally, zones of inhibition mean of the antibacterial activity of the plant extract
depends on its concentrations and on the strain of tested bacteria (Hasan et al., 2012).
Lactobacillus can produce lactic acid, acetic acid, formic acid and other acids to reduce
intestinal pH and demonstrated its ability to inhibit several bacterial pathogens,
including Escherichia coli and Staphylococcus aureus (Chen et al., 2019).
The research paradigm presented above depicts the independent and dependent
variables of the study. Ginger ethanolic extract and Lactobacillus casei Shirota strain and
their combinations as listed are considered as the independent variables. On the other
hand, the minimum inhibitory concentration and zone of inhibition produced by these
preparations on Escherichia coli and Staphylococcus aureus are considered as the
dependent variables. This research paradigm is based on the report of Shipradeep et al.
(2012) that probiotics and essential extracts both have a great potential in terms of their
synergistic effect against microbial gut which is normally higher than any known drug due
to their complementary actions.
Significance of the study
The results of the study may be beneficial to the following:

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Community. The study will help the community of both adults and children find
alternative remedy in treating certain illness especially those with gastrointestinal
diseases. This will also benefit them in lowering the risk of being susceptible against
enteric pathogenic bacteria.
Health professionals. Studying the collective antimicrobial activities of ginger and
probiotics may enhance the knowledge to help find alternative solutions in improving
antibiotic formulas without any harmful side effects to the immune system and to the
body’s microbiota and to help reduce morbidity of gastrointestinal diseases.
Future researchers. This study may serve as related literature for future
researchers.
Scope and delimitations
This study aims to determine the synergistic antimicrobial effect of ethanolic
extracts of Zingiber officinale and Lactobacillus casei Shirota strain against Escherichia
coli and Staphylococcus aureus, in vitro.
This study will be conducted by the researchers at the University of San Agustin
Medial Technology Laboratory in the 2nd semester of Academic year 2020-2021. Only the
ethanolic extract of ginger and commercially available Lactobacillus casei Shirota strain
will be tested in this study.
Tests organisms to reflect the synergistic antimicrobial effect of ginger and
commercially available Lactobacillus casei Shirota strain and their different combinations
will be limited only to Escherichia coli and Staphylococcus aureus.

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Minimum inhibitory concentration of ginger ethanolic extracts will be determined
using disk diffusion by series of dilutions to prepare different extract concentrations.
Similarly, synergistic antimicrobial effect of ginger and Latobacillus casei Shirota strain will
also be indicated by the zone of inhibition produced on the test organisms using the disk
diffusion method. This study will be performed in three trials with five replicates per trial.
Definition of terms
To promote understanding between the researchers and the readers, the following
terms related to the study are defined as follows:
Synergistic implies the combined effect of two or more agents that is greater than
the sum of their individual effects (Forbes, 2016).
In this study, it refers to the collective or collaborative antimicrobial effect of ginger
ethanolic extract and Lactobacillus casei Shirota strain to the test bacteria, Escherichia
coli and Staphylococcus aureus.
Antimicrobial is a chemical substance produced either by a microorganism or by
synthetic means that is capable of killing or suppressing growth of microorganism (Forbes,
2016).
In this study, antimicrobial refers to ability of the ginger ethanolic extract and
Lactobacillus casei Shirota strain and their combinations to inhibit the growth of the test
bacteria, Escherichia coli and Staphylococcus aureus as indicated by the zone of inhibition
produced using the disk diffusion method.

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Effect refers to a cause, a result or consequence or a bring about of an action
(Oates & Enquist, 2013)
In this study, effect refers to the influence of ginger ethanolic extract and
Lactobacillus casei Shirota strain and their combinations to the test bacteria, Escherichia
coli and Staphylococcus aureus in terms of zone of inhibition produced using the disk
diffusion method.
Ginger is also known as Zingiber officinale belongs to Zingiberaceae family that
have strong aromatic and medicinal properties, and are characterized by their tuberous or
non-tuberous rhizomes (Santo Grace et al., 2017).
In this study, ginger is a sample used as a source of ethanolic extracts for
investigation of its antimicrobial properties in combination with Lactobacillus casei Shirota
strain against the test bacteria, Escherichia coli and Staphylococcus aureus by measuring
the zone of inhibition produced using the disk diffusion method.
Ginger ethanolic extract refers to the extract obtained when ginger powder is
mixed with 95% ethanol incubated at room temperature for 72 hours then filtered with
Whatman filter paper and concentrated using a rotary evaporator at 78°C (Yadufashije et
al., 2020).
In this study, ginger ethanolic extract refers to the isolates separated from ginger
rhizome that will be tested for its antimicrobial effect in synergism with Lactobacillus casei
Shirota strain to the test bacteria, Escherichia coli and Staphylococcus aureus.
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Lactobacillus casei Shirota strain is a strain discovered by Dr. Minoru Shirota
and considered to belong in the genus Lactobacillus producing lactic acid that has been
scientifically proven to reach the intestine alive (Lactobacillus casei, 2020).
In this study, Lactobacillus casei Shirota strain will be used to investigate its
antimicrobial effect in combination with ginger ethanolic extracts to the test bacteria,
Escherichia coli and Staphylococcus aureus by measuring the zone of inhibition produced
using the agar well diffusion method.
Probiotic refers to the substances secreted by one microorganism that stimulated
another's growth (Nazir et al. 2018).
In this study, probiotic refers to the Lactobacillus casei Shirota strain which can be
obtained from a commercial probiotic product and tested for their antimicrobial effect in
combination with ginger ethanolic extracts to Escherichia coli and Staphylococcus aureus.
Escherichia coli is a Gram-negative bacillus commonly found as a commensal in
the human microbiota (Rojas-Lopez et al., 2018).
In this study, Escherichia coli, specifically it’s pathogenic strain, is used as the test
bacteria to determine the synergistic antimicrobial effect of ginger ethanolic extracts and
Lactobacillus casei Shirota strain which will be measured using disk diffusion method.
Staphylococcus aureus is a gram-positive bacterium and one of the most
common causative agents of wide range infective diseases such as skin infections,
bacteremia, endocarditis, pneumonia and food poisoning (Enany et al., 2017).
In this study, Staphylococcus aureus, specifically it’s pathogenic strain, is used as
the test bacteria to determine the synergistic antimicrobial effect of ginger ethanolic
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extracts and Lactobacillus casei Shirota strain which will be measured using disk diffusion
method.
Minimum Inhibitory Concentration is defined the concentration giving the least
inhibitory activity and below which there is no further inhibition (Rahman et al., 2020).
In this study, this will be used to determine the lowest concentration of ginger
ethanolic extract that could inhibit growth of Escherichia coli and Staphylococcus aureus
by serial dilutions to prepare different extract concentration.
Zone of Inhibition is a circular area around the spot of the antibiotic in which
the bacteria colonies do not grow and can be used to measure the susceptibility of the
bacteria towards antibiotic (Bhargav et al., 2016)
In this study, it refers to the method used to determine the antimicrobial effect of
ethanolic extracts and Lactobacillus casei Shirota strain and their combination to the test
bacteria, Escherichia coli and Staphylococcus aureus.
In vitro refers to the technique of performing a given procedure in a controlled
environment outside of a living organism (Seladi-Schulman, 2019)
In this study, it pertains to the means used to determine the effectivity of ginger
ethanolic extracts, Lactobacillus casei Shirota strain and their combination to the test
bacteria, Escherichia coli and Staphylococcus aureus by measuring the zone of inhibition
through disk diffusion method.
Disk diffusion method is classified as an agar diffusion method and uses a filter
paper disk saturated with the plant extract, which is placed on top of an agar surface to
measure the inhibition zone (Horváth et al., 2016).

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In this study, disk diffusion method refers to the technique which will be used to
measure the zone of inhibition produced by ginger ethanolic extract, Lactobacillus casei
Shirota strain and their combinations to the test bacteria, Escherichia coli and
Staphylococcus aureus.
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Chapter 2
REVIEW OF RELATED LITERATURE
Gastrointestinal disorders with presentation of diarrhea has been a relevant health
problem in the Philippines which are usually common among children. It was known to be
acquired from commercially and homely formulated foods without the observance of
cleanliness upon preparation which eventually makes food become a vehicle for
pathogens.
Ninety five percent (95%) of the disease manifestations that were described for the
food-borne disease outbreaks (FBDOs) since 2005 as reported by Azanza et. al (2018)
associated with multiple food vehicles with unknown etiological agents of illnesses were
gastrointestinal symptoms including vomiting, diarrhea, and stomach ache. In relation to
this, it was also stated that eleven (11) morbidity cases linked to FBDO were caused by
the pathogenic strain of Escherichia coli manifesting the same symptoms with
gastrointestinal disorders while 1.24% were due to staphylococcal enterotoxins.
As the setback with gastrointestinal diseases associated with enteric pathogens
and the evolution of these pathogens into a more resistant specie continue to arise,
studies for the provision of adequate treatment has also been constantly developed with
more focus on the antibacterial properties of plants to produce antibiotics with no harmful
side effects. Having all the prevalent problems in different places, this study primarily aims
to determine the synergistic effect of the antimicrobial properties of Zingiber officinale
(Ginger), specifically its ethanolic extract, and probiotic supplement specifically
Lactobacillus casei against certain enteric bacteria.

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So, this chapter provides discussions on the variables and their relationship in
relevance with the study and the explanations regarding the standpoint on why the study
exists to answer the objectives at hand. Furthermore, this portion also includes the
methodologies used by the related studies to arrive at a specific method that could be
appropriately used by this research.
The chosen references included recent research reports, journals, evaluative
reports, reviews of literature, a dissertation, essays and articles pertaining to Zingiber
officinale, probiotics, and enteric pathogenic bacteria.
Gastrointestinal Infections
Gastrointestinal (GIT) diseases contribute greatly to the worldwide burden of
infectious diseases with diarrhea as the second leading cause of preventable illness in
children under the age of five (Fletcher, McLaws, & Ellis, 2013).
As revealed in the study of Karimi et al. (2018), approximately one billion cases of
diarrhea occur in children younger than 5 years, annually, of which around 4.5 million lose
their lives. Due to this, many studies have been conducted to evaluate pathogens causing
diarrhea, as well as innovative and effective treatment methods worldwide. It was
demonstrated that after viruses, bacteria are the second causative agents in diarrhea with
almost 30% cases with high treatment costs (Karimi et al., 2018).
Moreover, a review of the literature world-wide indicates that a causative organism
is identified in about 50% of symptomatic cases and the majority of gastrointestinal
illnesses can be transmitted through the fecal-oral route. A significant proportion, about
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36%, of gastrointestinal illnesses is attributable to food borne transmission. Among all
illnesses attributable to food borne transmission, 30% are caused by bacteria, 3% by
parasites, and 67% by viruses (Fletcher, McLaws, & Ellis, 2013).
In Australia about 32% of all gastroenteritis are food-borne,
and Campylobacter, non-typhoid Salmonella, pathogenic E. coli and norovirus are
responsible for over 80% of foodborne illness from known pathogens (Fletcher, McLaws,
& Ellis, 2013).
Meanwhile, in the Philippines, 95% of the disease manifestations with food-borne
disease outbreak since 2005, were mostly associated with bacterial pathogens. The
highest incidences involving multiple food vehicles were institutionally prepared, followed
by home prepared foods as indicated in the study of Azanza et. al., 2018. For example,
poisoning caused by the durian candy was the third multiple household outbreak that was
distributed in the entire region of CARAGA (southern part of the Philippines), and was
found out to be contaminated with S. aureus reflecting 1.24% of the FBDOs cases due to
staphylococcal toxin.
Gastrointestinal diseases affect both resource-rich and less developed countries,
despite the clear correlation between gastrointestinal diseases and factors such as poor
sanitation, insufficient access to clean drinking water, and other risk factors.
Antibiotics and antimicrobial resistance
The increase usage of antibiotics has induced microorganisms to acquire
resistance factor which have become a burning predicament. In general, bacteria present
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in the biofilm culture are more resistant to antimicrobials than planktonic extracts (Santo
Grace et al., 2017). To compensate with this, there is an urgent need to find alternative
chemotherapeutic drugs in diseases treatment particularly those of plant origin, which are
easily available and have considerably less side effect.
According to Belda Junior et al. (2007), the emergence of new infectious diseases,
the revival of many seemingly controlled infections and the rise in bacterial resistance
have created the need for studies aimed at developing new antimicrobials. Given the
inability of microorganisms to develop new molecules with antimicrobial properties,
refining the screening methods used to distinguish antimicrobials from other natural
sources is of great importance.
For the past decades, many researches have established the side effect of
overused and misused antibiotic which can harm vital organs as well as their impact on
the immune system and in the normal flora of the body. Due to this, the known success of
traditional medicine has guided the search for new chemotherapeutic alternatives to
eliminate the infections caused by drug-resistant microbes and to reduce the harm caused
by antibiotic (Bocanegra-Garcia et al., 2009).
Antibiotics can selectively decrease tissue invasion and eliminate aggressive
bacterial species or decrease luminal and mucosal bacterial concentrations, depending
on their spectrum of activity. So, rational use of antibiotic is the key approach to improve
the antibiotic performance and tackling of the antimicrobial resistance. The efficacy of
antimicrobials is influenced by many factors: (1) bacterial status (susceptibility and
resistance, tolerance, persistence, biofilm) and inoculum size; (2) antimicrobial

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concentrations (mutant selection window and sub-inhibitory concentration); (3) host
factors (serum effect and impact on gut micro-biota) (Li et al., 2017).
Therefore, further understanding of the link between antimicrobial uses, bacterial
status and host reaction provides new insights and stimulates the analysis to evolving
antimicrobial treatment regimens that produce improved clinical results and reduce
resistance at the same time.
Zingiber officinale
Due to their significant antimicrobial principles and phytoconstituents and broader
therapeutic abilities, traditional herbs have been a valuable source of medicine in virtually
all cultures and societies worldwide.
One of the traditional medicines widely used to treat various illnesses is the ginger
rhizome. Zingiber officinale or simply ginger belongs to Zingiberaceae family that have
strong aromatic and medicinal properties, and are characterized by their tuberous or non-
tuberous rhizomes. The rhizome is rich in secondary metabolites such as phenolic
compounds (gingerol, paradol and shogaoal), volatile sesquiterpenes (zingiberene and
bisabolene) and monoterpenoids (curcumene and citral) (Santo Grace et al., 2017).
Zingiber officinale has been widely used all over the world since antiquity for a wide
array of unrelated ailments including arthritis, cramps, rheumatism, sprains, sore throats,
muscular aches, pains, constipation, vomiting, hypertension, indigestion, dementia, fever,
and infectious diseases (Ali, 2008 as stated in Santo Grace et al., 2017).
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Because of the extensive use of this rhizome, studies confirmed that ginger have
anti-inflammatory, anti-thrombotic, cholesterol-lowering, blood pressure-lowering, anti-
microbial, anti-oxidant, anti-tumor, and hypoglycemic properties (Shahrajabian et al.,
2019), but the most relevant property attributed to this study is its antimicrobial properties.
Due this property as well as its easy availability, it was used in several medicinal plant
extraction researches that have shown direct antimicrobial properties against pathogenic
agents (Al-Juraifani, 2011, as cited in Abd El-Khalek, et al., 2016), thus can be used in
treatment of bacterial infection and generally recognized as safe (GRAS) by the US FDA
(Kamrul et al. 2014, as cited in Yadufashije et al., 2020) as well as by the Aliment and
Drug Administration of Egypt for utilization as pabulum supplement (Abd El-Khalek et al.,
2016).
Antimicrobial activities of ginger
The antibacterial and inhibition activities of ginger extracts could be attributed to
its chemical properties (Kamrul et al., 2014, as cited in Yadufashije et al., 2020), with
zingiberene sesquiterpenoids as the main component. Furthermore, according to the
study of Garvey et al. (2009), the extracts are active at high concentration and inactive at
very low concentrations based on the results of the bacterial growth inhibition (Yadufashije
et al., 2020) suggesting that inhibition activities were dose-dependent (Foustine et al.,
2019).
In vitro experiments have shown that active ginger constituents prevent colon
bacteria from multiplying. These bacteria, which cause flatulence, ferment undigested
carbohydrates which could be retorted by ginger (Yadufashije et al., 2020). Ginger

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essential oil and ethanolic extract showed different degree of antibacterial activity against
food-borne bacteria due to the compound contained within respective extracts and these
demonstrate that there may be different antibacterial activity in different ginger extracts
(Foustine et al., 2019).
In relation to this, some studies, as revealed in the study of Abd El-Khalek et al.
(2016), investigated the antimicrobial property of the volatile oil extracted from ginger
rhizomes and showed inhibition activity against Aspergillus niger, Saccharomyces
cerevisiae, and Bacillus cereus, which were determined using disc diffusion method.
In the same study conducted by Yadufashije et al., (2020) using the soybean oil
extract of ginger, it has shown that at boiling temperature, this extract has potential
antimicrobial activities with highest zone of inhibition (11.67±1.53mm) against Salmonella
spp. and lowest zones of inhibition (8.0±1.73mm, 8.67±2.52mm) against Escherichia coli
and Staphylococcus aureus.
Moreover, Z. officinale ethanolic extract has also displayed a wide range of
influence on gram-positive aerobic bacteria and as experimented, has confirmed its
significance, especially in its area of influence on tested staphylococci (Santo Grace et al.,
2017) as well as on Bacillus. spp. E. coli, and Salmonella spp. (Onyeagba et al. 2004, as
cited in Abd El-Khalek et al., 2016). Meanwhile, ginger methanolic extracts presented an
extent of inhibition sensitivity in terms of zones of inhibition against different gram-negative
bacteria using paper disk method (Malik, 2015, as cited in Abd El-Khalek et al., 2016).
In the study conducted by Foustine et al. (2019), in which ginger essential oil and
ginger ethanolic extract antimicrobial activities were compared using disk diffusion method
against four types of food-borne bacteria, it was revealed that ginger essential oil denoted
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a much wider activity against E. coli, B. subtilis, S. typhi, and S. aureus than ginger
ethanolic extracts. However, Yassen & Esmaeel (2016), indicated that ethanolic,
methanolic, and hexanoic extracts of ginger produced marked inhibitory effect on S.
aureus and E. coli than aqueous and acetonic extracts on microorganism test.
Therefore, as stated by Foustine et al., (2019), different ginger extraction methods
can result in various antimicrobial properties due to the different extracted substances.
Probiotics and Lactobacillus casei
Lilley and Stillwell first used the term “probiotics” to describe substances secreted
by one microorganism that stimulated another's growth and were later used to describe
tissue extracts that stimulated microbial growth and supplements of animal feed that have
a beneficial impact on animals by adding to their balance of intestinal flora (Nazir et al.
2018). Since then, the definition of probiotic had evolved over time.
In a scientific sense, Metchnikoff (1907, as cited in George Kerry et al., 2018)
expressed probiotics as an alteration of floral or microbial diversity in the human body and
as a replacement useful against harmful microbes. In addition, Tissier (1906, as cited in
George Kerry et al., 2018) suggested the oral administration of live organisms
(bifidobacteria) to patients with diarrhea especially in infants to help restore a healthy gut
flora.
According to the latest report of the Food and Agriculture Organization (FAO) and
World Health Organization (WHO) as stated by Karimi et al. (2018), probiotics are live
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microorganisms whose adequate intake causes beneficial effects on health and several
studies have demonstrated their effects on prevention and treatment of many diseases.
Probiotics also have many health benefits for digestion, metabolism, innate
epithelial immunity, and even in exclusion of pathogenic microorganisms (Azad et al
2019). George Kerry et al. (2018), defined the variety of health-related properties of
probiotics namely anti-pathogenicity, anti-diabetic, anti-obesity, anti-inflammatory, anti-
cancer, anti-allergic, and angiogenic activities as wells as a significant effect on the brain
and central nervous system (CNS).
Furthermore, these also stimulate, modulate, and regulate the host's immune
response and can also control the release of gastrointestinal hormone via initiating the
activation of a specific genes of a localized host cells, and influence brain behavior through
neural signaling, as part of the gut-brain axis (George Kerry et al., 2018). Therefore, based
on the above-mentioned properties, probiotics would perhaps exhibit antimicrobial activity
which is an important aspect in the study.
The beneficial effects of viable probiotic bacteria as dietary supplements have
gained huge research interest (Prabhurajeshwar & Chandrakanth, 2019), and probiotic-
containing products have long been studied and appreciated for their positive effect on
gastrointestinal (GI) health (Sutula et al., 2013). The most common genera with probiotic
characteristics are the Lactobacillus spp. and L casei as the most commonly utilized
probiotic species (Aktas et al., 2016) which are now widely used to prepare fermented
dairy products such as yoghurts, milk-shakes and etc. (Prabhurajeshwar & Chandrakanth,
2019).
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However, bacterial growth can drastically be affected by pH, so in order to be
used as a probiotic, these microorganisms must adapt to the pH of the human gut which
varies from 1.5 to 4.5 depending on the type of foods consumed and digested
(Prabhurajeshwar & Chandrakanth, 2019).
Lactobacillus casei Shirota (LcS), a species in the genus Lactobacillus
encompassing most of the lactic acid bacteria in the body (Lactobacillus casei, 2020),
found in the probiotic-fermented milk drink Yakult at a reported concentration of
1.2 × 1010 cfu/100ml (Hu et al., 2019), has over 75 years’ history of safe consumption and
proven health benefits, backed by comprehensive clinical studies focused primarily on the
reduction and immune-modulating effects of functional and infectious gut diseases
(Macmillan Publishers Limited, part of Springer Nature, 2019).
Taking the daily probiotic products such as Yakult was associated with a lower rate
of clinical presentation with diverticulitis episodes and improvement in gastrointestinal
symptoms (Nichols et al., 2020). Conversely, probiotic therapy has also been introduced
for the maintenance of oral health and a study by Sutula et al., (2013, as cited in Hu et al.,
2019), revealed that four weeks of consumption of 6.5×109 viable LcS per day by healthy
denture wearers showed a transient colonization of the oral cavity and denture surfaces
by this strain during the consumption period and for up to 7 weeks of washout, posed no
significant effect on acidogenic populations such as levels of lactobacilli and streptococci.
In addition, Yakult intake changed the abundance of some bacteria related to
dental caries, suggesting that the change of composition may be beneficial to oral health,
while the overall microbiota structure remained unaffected (Hu et al., 2019).
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Antimicrobial activities of probiotics and L. casei
Antimicrobial behavior is known to be one of the most beneficial results of
probiotics, as disruption or alteration in the composition of the diverse gut microbiota
community is prevented, unlike conventional antibiotics (George Kerry et al., 2018).
Tejero-Sarinena et al. (2013, as cited in George Kerry et al., 2018) investigated
the influence of probiotics on the survival of Salmonella enterica, Serovar typhimurium
and Clostridium difficile in an in vitro model and claimed that probiotics inhibit pathogens
by the production of short-chain fatty acids (SCFAs), such as acetic, propionic, butyric and
lactic acids. Whereas, the most common bacteriocins include lacticin, lactocin, pediocin,
pisciolin, enterocin, reuterin, plantaricin, enterolysin and nisin (Fijan, 2016).
In the study conducted by Karimi et at. (2018) revealed that a study from Martyr
Chamran University of Ahvaz was carried out from 2013 to 2014 involving a total of thirteen
(13) probiotics colonies isolated from 20 samples of common dairy products and
vegetables to evaluate the antimicrobial effect of each against two E. coli pathotype using
disk diffusion method. It was pointed that five out of thirteen probiotic strains have
demonstrated growth inhibitory activity against Escherichia coli pathotypes specifically on
Escherichia coli O157:H7 strain.
Additionally, it has also been reported in the review study conducted by Azad et
al., (2018) where in homogenates prepared from several probiotics,
including Lactobacillus rhamnosus GG, Lactobacillus acidophilus, Lactobacillus
delbrueckii bulgaricus, Bifidobacterium lactis, and Streptococcus thermophiles, have the
ability to suppress the proliferation of mononuclear cells. Moreover, the same study
asserted that Bifidobacterium bifidum has a significant effect in enhancing antibody

23
responses to ovalbumin, whereas B. breve has an increased humoral immune response
after stimulation with IgA (Azad et al., 2018).
Typically, according to Fijan (2016), probiotic strains usually develop more than
one antimicrobial agent that can function synergistically, expanding the scope of
microorganisms that are attacked. As long as this antimicrobial spectrum is limited to
pathogenic microorganisms, this property can be desirable, but it cannot be ruled out that
it would not affect the normal gut microbiota or other microbiotas as well.
Further, Fijan (2016) also suggested that probiotic properties are strain based and
that identification of strains is imperative. Hence, it should be remembered that the
beneficial effect of one probiotic preparation does not imply efficacy of other preparations
containing different bacterial strains, because each individual probiotic strain has its
unique biological properties.
Escherichia coli pathotype
Escherichia coli is a Gram-negative bacterium commonly found as a commensal
in the human microbiota. The plasticity of its genome, however, has contributed to the
evolution of this organism into pathogenic strains capable of causing human and animal
diseases and syndromes of public health significance (Rojas-Lopez et al., 2018).
Recently, unprecedented outbreaks of Escherichia coli O157:H7 infection have
occurred in Japan, Scotland and America, and in Europe and Australia with other
Enterohemorrhagic Escherichia coli (EHEC) serotypes (Karimi et al., 2018). Based on this
24
evidence, further research into the application of new methods to prevent and regulate
these bacteria appears to be appropriate.
Based on the review study of Rojas-Lopez et al. (2018), pathogenic E. coli are
mainly divided into two groups depending on the disease location namely extraintestinal
pathogenic E. coli (ExPEC) and intestinal pathogenic E. coli (InPEC). ExPEC strains are
chiefly associated with neonatal meningitis (NMEC) and urinary tract infections (UPEC) in
adults, conversely, InPEC strains are usually related to diarrheal disease which are further
subdivided into at least 6 well-known pathotypes: enteropathogenic E. coli (EPEC),
enterohemorrhagic E. coli (EHEC), enteroinvasive E. coli (EIEC), enteroaggregative E.
coli (EAEC), diffusely adherent E. coli (DAEC) and enterotoxigenic E. coli (ETEC).
The Enterohemorrhagic Escherichia coli (EHEC) strains were discussed as
causative agents of hemorrhagic colitis and hemolytic uremic syndrome and the shiga-like
toxin of this strain has verotoxic effects. Additionally, there are over 700 antigenic types
(serotypes) producing shiga toxin and these serotypes are necessary to distinguish strains
that actually cause disease (Todar, 2020). However, the most common serotypes are
0157:H7 wherein the infective dose of this strain is low with up to 100 bacteria causing
disease (Karimi et al., 2018).
Pathogenic E. coli infection usually causes severe diarrhea (Yang et al., 2017) with
8–10% of cases in children (Karimi et al., 2018). Diarrhea is the result of the reversal of
the normal net absorptive status of water and electrolyte absorption to secretion.
Fortunately, diarrheal disease caused by pathogenic E. coli is preventable by improved
environmental sanitation and is treatable by antibiotics. The treatment of diarrheal disease
is generally effective with oral rehydration and maintaining electrolyte balance through the
25
diet (Yang et al., 2017) and nowadays, probiotics are consumed to inhibit pathogens and
increase shelf life of foods as they release antimicrobial to compete with pathogens that
lead to an increase in the immune response of the host (Karimi et al., 2018).
Nevertheless, novel therapeutic methods are also required for the incidence of
antibiotic resistance, loss of normal intestinal flora and verotoxin caused by E. coli strains,
particularly as a result of taking antibiotics.
Staphylococcus aureus pathotype
Staphylococcus aureus is a gram-positive bacterium and one of the most common
causative agents of wide range infective diseases such as skin infections, bacteremia,
endocarditis, pneumonia and food poisoning (Enany et al., 2017).
It is a facultative anaerobe that belongs to the Staphylococcus genus within the
Staphylococcae family and is often classified in a routine microbiology laboratory as a
clinically significant bacterium. It appears as irregular tiny clusters of gram-positive cocci
and is cultivated for 18-24 hours in a blood agar plate. (Bagnoli et al., 2018).
Furthermore, S. aureus can exist commensally with humans as a colonizer but can
also exist as a pathogen. It is a major pathogen in bacteremia whether community
acquired or hospital acquired. It has proven its versatility by continuing to be an important
infectious pathogen that has contributed to increasing morbidity and mortality of patients
over the years (Youssef & Molony, 2017).
According to Enany et al. (2017), it was indicated that the process of S. aureus
infections involves five stages: (1) colonization, (2) local infection, (3) systemic
26
dissemination and/or sepsis, (4) metastatic infections, (5) toxicosis. Consequently, this
microorganism is in carrier state in the anterior nares and may appear asymptomatic for
weeks or months and its colonization may proceed to infection once exposed to prolonged
hospitalization, immune suppression, surgeries, use of medical invasive medical devices
and chronic metabolic diseases.
Commonly, S. aureus when in contact with skin can cause skin abscess and can
further spread and result to localized infections such as carbuncle, cellulitis, and impetigo
bullosa or wound infection. They may also enter the blood and spread systematically to
different organs resulting to sepsis and their spread may result to endocarditis,
osteomyelitis, renal carbuncle, septic arthritis and epidural abscess (Enany et al., 2017).
Statistics showed that approximately 20-30% of human population carries
S.aureus and about 40-70% of associated nosocomial infections worldwide were caused
by Methicillin-Resisted Staphyloccocus aureus (MRSA) which was first reported in United
Kingdom after the introduction of methicillin to treat patients with penicillin-resistant
staphylococcus infection (Enany et al., 2017).
Drug resistance developed in human pathogenic bacteria is emerging and has
become a global problem. Due to this, there’s a difficulty in looking for the best drug that
could treat S. aureus infection. According to David and Daum (2017), the history of S.
aureus is characterized by the emergence of this resistance to each new class of
antimicrobial anti-staphylococcal drugs, including penicillin, sulfonamides, tetracyclines,
glycopeptides, and others, resulting in complications in treatment.
During 1960s, it was identified that S.aureus became resistant to Methicilin, β-
lactam antimicrobial active initially against a majority of S.aureus strains. Moreover, a

27
particular mobile genetic element (MGE) called staphylococcal chromosome mec
(SCCmec) was responsible for this methicillin resistance in staphylococci, which brings
many virulence factors with it (Enany et al., 2017).
Additionally, it was also stated by Enany et al. that back in 1920s, Penicillin was
discovered as the first beta-lactam antibiotic to become an effective weapon against
S.aureus infections, but during 1940s, resistant to this drug also arise after its introduction
into clinics due to the production of plasmid-encode beta-lactamase enzyme
(penicillinase) which enzymatically cleaved the beta-lactam ring of penicillin rendering the
antibiotic inactive. Daptomycin, Linezolid, Ceftaroline, can be used as alternatives to
vancomycin for infections caused by methicillin-resistant Staphylococcus aureus (MRSA)
as suggested by Hemeg et al. (2019).
Despite the rapid advancement of modern medicine, S. aureus infections still
remain as highly prevalent in human populations as transmission can be of direct contact.
As these bacteria are growing around human environment, therefore it is important to be
aware of its pathogenesis as it is known to be commensal at the same time opportunistic
pathogen.
Synthesis
Despite the clear correlation between gastrointestinal diseases and factors such
as poor sanitation, insufficient access to clean drinking water, and other risk factors, as
well as the rapid advancement of modern medicine, pathogenic bacteria still continue to
evolve into much more resistant strain and will constantly remain as a risk to human
28
gastrointestinal tract. Further understanding of the connection between antimicrobial uses,
bacterial status, and host reaction provides new insights and stimulates analysis into
emerging regimens of antimicrobial therapy that deliver improved clinical outcomes while
reducing resistance. Hence, there is a need to conduct studies that investigate properties
of bacteria and the possible treatment to their infection from natural resources to decrease
the gap in knowledge in treatment formulation and to reduce further harm.
This study will contribute into reaching this knowledge as this research aims to
determine the synergistic effect of the antimicrobial properties of Zingiber officinale
(Ginger), specifically its ethanolic extract, and Lactobacillus casei strain Shirota from
commercial probiotic product against pathogenic strains of Escherichia coli and
Staphylococcus aureus, guided by the principles stated in this review.

29
Chapter 3
METHODOLOGY
Research design
The research design that will be employed in this study is the quasi-experimental
research design. This will involve manipulation of the test variables in the study,
observation and determination of the synergistic antimicrobial effect of ethanolic extracts
of ginger and Lactobacillus casei Shirota strain which are purposely selected as
experimental groups of this study. Determination of the minimum inhibitory concentration
ethanolic extracts will also be done. The antimicrobial activity of ginger ethanolic extracts,
Lactobacillus casei Shirota strain and their combination will be measured through the zone
of inhibition produced in cultures of Escherichia coli and Staphylococcus aureus using disk
diffusion method.
Social preparations and ethical considerations
Prior to the conduct of the study, the researchers must first obtain approval from
the Ethics Committee of the University of San Agustin to be given an ethical clearance
form indicating that the study is ethical and can be performed. Upon approval by the Ethics
Committee, the researchers will prepare a letter of permission which will be addressed to
the Mendel Research Laboratory of the University of San Agustin where the experiment
will be conducted. Upon approval by the Mendel Research Laboratory in-charge,

30
researchers must then prepare the instruments that are needed in the course of the
experiment to be requested from the laboratory.
In the whole duration of the experiment, researchers must observe and adhere to
the proper handling of biochemical hazards and other hazards inside the laboratory. The
researchers must treat all microorganisms as potential pathogens and all materials that
will be used for culturing microorganisms should be sterilized properly to ensure the safety
of all equipment. Disinfecting the working areas before and after use is needed as well as
proper handwashing. All cultures, chemicals, disinfectant, and media as well as hazardous
substances should be clearly and securely labeled to avoid acquiring infections and any
injury during experimental work in the laboratory (Sarosh et al., 2015).
The researchers must also observe proper waste disposal during the course of the
experiment. All of the equipment and supplies used in experiments involving bacterial
cultures should be sterilized. This includes the tools used for transferring media or
bacteria, such as the inoculating instruments (loops and needles) and pipettes for liquid
transfer. Decontamination should be carried out by autoclaving at 121°C for 60 minutes.
After autoclaving, media may be disposed of through normal waste disposal or incineration
procedures.
Sampling design and sample size determination
Purposive non-probability sampling design is used in selecting the samples for the
study guided by the principles presented in the literature review. Ginger ethanolic extracts
and Lactobacillus casei Shirota strain are purposely chosen as the main samples and
31
independent variables in the study and their antimicrobial effect will be measured by the
zone of inhibition produced on Escherichia coli and Staphylococcus aureus, purposely
selected as the test bacteria, using disk diffusion method. These samples will be most
useful in achieving the main objective of the study which is to determine the synergistic
antimicrobial effect of ginger ethanolic extracts and Lactobacillus casei Shirota strain
against Escherichia coli and Staphylococcus aureus.
Procedures/methods
Preparation of ginger ethanolic extracts
The preparation of ethanolic extracts from ginger will be based from the study of
Nguyen et al. (2019) with some modifications. Fresh ginger rhizome will be acquired from
the local market and 100g will be cut into smaller pieces and be utilized for extraction by
maceration. It will be soaked in 500ml 95% ethanol (Yadufashije et al., 2020) in a Becher
for 72 hours at room temperature. After 72 hours, the whole mixture will be filtered using
cheese cloth and secondary filtration will be done using Whatman No.1 to remove
residues. The filtrate that will be obtained will be concentrated at 50°C by rotary evaporator
and the dried extract will be dissolved in ethanol to a final concentration of 100mg/ml (Gull
et al., 2012). The solution will then be stored at 4°C in a refrigerator to be used for
subsequent procedures (Yassen & Esmaeel, 2016).
Minimum Inhibitory Concentration (MIC) of ginger ethanolic extracts

32
Minimum inhibitory concentration of ginger ethanolic extract will be determined by
the method described by Rahman et al., 2020 with some modifications. The stock solution
of 100mg/ml of ginger extract will be used. It will be prepared by diluting 1g of the dried
extract in 10ml ethanol so 1ml of working solution contains 100mg of ethanolic ginger
extracts. This will be labeled as the Stock Solution-I. To prepare more diluted working
solution, 1:100 dilution will be done to the Stock Solution-I by adding 99 ml of ethanol. So,
1ml of the working solution will contain 1mg of ginger extract; this will be labeled as the
Stock Solution-II and will be used to observe minimum inhibitory concentration against E.
coli and S. aureus by making different working solutions of different concentrations (Table
1).
Sterile disks with a diameter of 5 mm will be dipped in different dilutions of ethanol
extracts of ginger for 1 hour and will be placed over MHA agar plates seeded with 107
CFU of each bacterial culture. Plates will then be incubated at 37°C for 24 hours. The
zone of inhibition in each case will be measured as diameter of the clearing zones and
results will be recorded.
Table 1: Composition and different concentrations of working solutions with
control
No. of Test Stock Solution-II Distilled Water Concentration of

(ml) (ml) Ethanolic Ginger
extract (μg/ml)
Test 1 9 ml 1 ml 900μg/ml
33
Test 11 - 10 ml -
Preparation of Lactobacillus casei Shirota strain cell-free supernatant (CFS)
Lactobacillus casei Shirota strain will be collected from a commercial probiotic
drink containing Lactobacillus casei Shirota strain and isolation will be based from the
study of Rafieian-Kopaei et al. (2017) with some modifications. 2ml of the commercial
probiotic drink will be transferred in a flask containing de Man, Rogosa & Sharpe (MRS)
Broth as enrichment media. 100 mL of distilled water will be added to the media and will
be incubated at 37°C for 24 hours. For identification and confirmation of the bacteria,
Gram-staining will be done and subsequent cultures will be performed.
Preparation of cell-free supernatant (CFS) will be based from the study of
Koohestani et al. (2018) with minor modifications. Fresh culture of the isolate will be
centrifuged at 8000 rpm for 15 minutes then CFS will be decanted aseptically and will be
sterilized using a 0.20 µm pore size filter. The CFS will be used freshly for the subsequent
assay. Antimicrobial effect of the isolate will be evaluated by disk diffusion test on MHA
medium plated with the test bacteria. Blank sterile disks (5mm in diameter) will be dipped
in 1000μL supernatant 1 hour before placing on separate MHA media inoculated with E.
34
coli and S. aureus. The plates will then be incubated at 37°C for 24 hours and zone of
inhibition will be measured using a ruler.
Preparation of the test bacteria
The test bacteria which will be used in the study are Escherichia coli and
Staphylococcus aureus and they will be commercially acquired from a laboratory and 107
CFU will be evenly suspended in Mueller Hinton Agar (MHA) poured into the Petri dishes.
The bacteria will be incubated for 24 hours at 37°C to get the active strains and will serve
as inoculums for the subsequent studies (Foustine et al., 2019).
Antimicrobial assay (Disk Diffusion Method)
Antimicrobial activity of ginger ethanolic extract and Lactobacillus casei Shirota
strain will be assessed using disk diffusion method based on the study of Foustine et al.
(2019) and it will be performed under aseptic conditions. Sterile disks will be prepared
from Whatman filter paper no. 1 with a diameter of 5 mm and will be dipped into each
working solution of the samples 1 hour before testing. Different proportions of working
solutions will be prepared for each treatment. Treatments to be done are demonstrated in
Table 2.
35
Table 2: Preparation of treatments
Treatment 100mg/ml of Lactobacillus

Ginger Ethanolic casei Shirota
Extract (ml) strain CFS (ml)
1 1 mL -
2 - 1 mL
3 1 mL 1 mL
(1:1 proportion)
4 2 mL 1 mL
(2:1 proportion)
5 1 mL 2 mL
(1:2 proportion)
Thereafter, the disks will be placed on top of the media that have been inoculated
with the test bacteria such as Escherichia coli and Staphylococcus aureus in a manner of
three trials with five replicates. All of the inoculated Petri dishes will be incubated for 24
hours at 37°C. After 24 hours, the media will be observed for the area of inhibition and
diameter of the area in millimeters will be measured using a ruler.
Data processing
Study findings will be explained in words, tables, and figures. Specifically, results
from testing minimum inhibitory concentration of ginger ethanolic extracts will be
processed in tabular from. Tabulation and graphical representations will be utilized in
presenting the findings that will be obtained from measuring the zone of inhibition
produced by the three trials in five replicates. To show if there will be significant difference
in the three trials performed in five replicates, tabulation will also be used to present the
findings that will be obtained.

36
Data analysis
To address the first objective which is to measure the minimum inhibitory
concentration of ginger ethanolic extract, data will be analyzed by determining the highest
and lowest values of concentration to which visible growth is inhibited. To answer second
objective which is to measure the zone of inhibition produced by ginger ethanolic extracts,
Lactobacillus casei Shirota strain and their combinations on Escherichia coli and
Staphylococcus aureus, data will be analyzed by getting the mean and standard deviation
from the mean of the zone of inhibition in three trials performed which will be presented in
tabular and graphical forms. To answer the third objective which is to determine the
significant difference in the zones of inhibition produced by ginger ethanolic extracts,
Lactobacillus casei Shirota strain and their combination on Escherichia coli and
Staphylococcus aureus, means of the zone of inhibition produced in three trials with five
replicates will be analyzed using one-way analysis of variance (ANOVA) with 0.05 level of
significance.
37
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42
APPENDIX A
DRAFT OF LETTER TO THE MENDEL RESEARCH LABORATORY IN-CHARGE
October 30, 2020
DR. DORALYN S. DALISAY

In-charge, Mendel Research Laboratories
General Luna St., Iloilo City
Dear Dr. Dalisay,
A warm Agustinian greetings!
We are the students from MLS 3H Group 2 of the College of Health and Allied Medical
Professions. We would like to ask permission from your office to allow us to work in Mendel
Research Laboratories of the University of San Agustin on______________ to perform
experimental study on the antimicrobial activity of ethanolic extracts and Lactobacillus
casei Shirota strain involving the incubation of the test bacteria such as Escherichia coli
and Staphylococcus aureus for our research entitled Synergistic Antimicrobial Effect
of Ginger (Zingiber officinale) Ethanolic Extracts and Probiotic (Lactobacillus casei)
Against Enteric Pathogenic Bacteria
We will take full responsibility and accountability for any damage that may occur to the
building, laboratory, and equipment during the conduct of our experiment. The University
of San Agustin and its personnel will not be held liable for any damage or loss of our
personal belongings, personal injury or death.
We are hoping for your generous approval.
Thank you and God Bless.
Respectfully yours,
43
ROCHELLE DOMINIQUE CASADOR MAYZ CERCADO
MA. ANGELICA DEL CASTILLO ANA LIZA R. BESAS
ARMELINE CASTILLO JAMES DOMINIC DITALO
EUNA GRACE N. CHAVEZ
Conforme:
Research Adviser: ZESIL GAY E. GELLE, RMT, MSMT
Email address: zgelle@usa.edu.ph
JOSE G. PEREZ, JR., RMT, MSMT

Academic Supervisor, MLS Program
SOFIA COSETTE P. MONTEBLANCO, RN, MAN

Dean, CHAMP
44
APPENDIX B
Schematic Diagram of Research Procedures
Preparation of Ginger Ethanolic Extracts
Obtain 100g Ginger Soak the ginger rhizome

rhizome and slice by 500 mL 95% Ethanol
into small pieces. for 72 hours at room
temperature.
Filter the mixture with

Dissolve the dried extract in
cheese cloth and refilter
ethanol to a final
using Whatman no.1.
concentration of 100mg/ml
then store at 4°C
Concentrate the filtrate to
dryness at 50°C using
rotary evaporator to obtain
pure extract
Minimum inhibitory concentration (MIC) of ginger ethanolic extracts
A. Preparation of stock solution
Dissolve 1 g of dried ginger

ethanolic extract in 10ml
ethanol to yield a
concentration of 100mg/ml
Label this solution as

Stock Solution-I
Add 99 ml Ethanol to Stock

Solution-I to make 1:100
dilution then label as Stock
Solution-II
45
B. Preparation of different working solutions with different concentrations
Prepare 10 ml of different concentration of ginger ethanolic extract using distilled
water.
No. of Test Stock Solution-II Distilled Water Concentration of

(ml) (ml) Ethanolic Ginger
extract (μg/ml)
Test 11 - 10 ml
46
C. Determining minimum inhibitory concentration
Dip 2 sterile disks to each Remove the sterile

Prepare sterile
working solution of ginger disks from each
disks with
ethanolic extracts solution by a forcep;
diameter of 5mm
prepared in different place on a sterile petri
concentration for 1 hour. dish and allow to dry.
While waiting for the

Place the remaining disks to dry, prepare plate
sterile disk from Place one of the two
sterile disks from each media inoculated with E.
each solution to coli and S. aureus. Label
MHA agar plate solution to MHA agar
plate inoculated with and section the plates
inoculated with according to the
Staphylococcus Escherichia coli
concentration of each
aureus working solution.
Place blank disk as control Measure the zone of inhibition

to each plate. Place the produced and identify the
plates in an incubator for minimum inhibitory
24 hours at 37°C concentration from each
bacterial plate.
Preparation of Lactobacillus casei Shirota strain cell-free supernatant

A. Enrichment of the bacteria
Transfer 2ml of Yakult in

a flask with MRS broth
Add 100 ml of distilled

water in the flask
Incubate the MRS broth

for 24 hours at 37°C
47
B. Gram staining
Perform smear Gently shake the MRS Using the inoculating

preparation using broth tube containing loop, fish out a
aseptic method. Lactobacillus casei loopful of specimen
Shirota strain avoiding to touch the
side from the tube
Heat fix the smear Leave smears in the Apply evenly the
by passing it Biosafety Cabinet for smear at the center
through the flame 3 a few minutes and of the slide in a
times. allow to air dry. circular motion from
inner to outer.
Flood the smear with Add 3-8 drops of Add 3-8 drops of
crystal violet for 30 sodium bicarbonate sodium bicarbonate
seconds and let it stand for and let it stand for
5-10 seconds 5-10 seconds
Gently flood the

Once dry counterstain Decolorize smear smear with Gram’s
the smear using 1:1 ethanol-acetone iodine and let it
Safranin and wash drop by drop until stand for 5-10
gently with tap water. only a blue tinge seconds. Drain
Allow to dry then appears excess Gram’s
examine under OIO. iodine
48
C. Preparation of CFS and antimicrobial assay
Centrifuge fresh culture of Decant supernatant

the isolate at 8000 rpm for aseptically and sterilized
15 minutes using 0.20 µm pore size filter
After 1 hour, remove the Prepare 1000μL supernatant

disk using a forcep and and dip 5mm blank sterile
allow to dry in a petri dish. disks on the supernatant for
1 hour.
Place the disks over MHA Incubate the plates for 24

media inoculated with E. coli hours at 37°C and measure
and S. aureus. the zone of inhibition using a
ruler
Antimicrobial assay (disk diffusion method)

A. Preparation of treatment
Treatment 100mg/ml of Lactobacillus

Ginger Ethanolic casei Shirota
Extract (ml) strain CFS (ml)
1 1 mL -
2 - 1 mL
3 1 mL 1 mL
(1:1 proportion)
4 2 mL 1 mL
(2:1 proportion)
5 1 mL 2 mL
(1:2 proportion)
49
B. Disk diffusion method
Prepare sterile disks from

Whatman filter paper no. 1
with 5mm diameter
Dip sterile disks into each

treatment prepared 1 hour
before testing and allow to
dry.
Place the disks on top of

each MHA media
inoculated with E. coli and
S. aureus
Incubate the inoculated

petri dishes for 24 hours at
37°C.
After incubation, measure

the zone of inhibition
produced.
50
TIMETABLE OF RESEARCH PROPOSAL

51
BUDGETARY REQUIREMENTS
BUDGET
DETAILS AMOUNT
I. PERSONAL SERVICES
A. Honoraria
1. Panel Members (Proposal & Final Php 3,000.00
Defense) Php 2,000.00
2. Statistician
SUBTOTAL Php 5,000.00
II. MAINTENANCE AND OPERATING

EXPENSES
A. Materials and Laboratory Supplies Php 11,000.00
B. Office Supplies and Printing Php 1,000.00
SUBTOTAL Php 12, 000.00
III. CONTINGENCY Php 3,000.00
TOTAL Php 20, 000.00

52
DUMMY TABLES
Table 3. Minimum Inhibitory Concentration
No. of Stock Distilled Concentration of Zone of Inhibition

Test Solution-II Water (ml) Ethanolic Ginger (mm)
(ml) extract (μg/ml) E. coli S. aureus
Test 11 - 10 ml -
Table 4. Zone of inhibition produced on Escherichia coli
ZONE OF INHIBITION (mm)
Escherichia coli Ethanolic Lactobacillus casei 1:1 1:2 2:1

extracts Shirota strain proportion proportion proportion
TRIAL 1
TRIAL 2
TRIAL 3
MEAN
53
Table 5. Zone of inhibition produced on Staphylococcus aureus
ZONE OF INHIBITION (mm)
Staphylococcus Ethanolic Lactobacillus casei 1:1 1:2 2:1

aureus extracts Shirota strain proportion proportion proportion
TRIAL 1
TRIAL 2
TRIAL 3
MEAN
Table 6. Mean of the Zone of inhibition produced in three trials

MEAN OF THE ZONE OF INHIBITION (mm)
Test Bacteria Ethanolic Lactobacillus casei 1:1 1:2 2:1

extracts Shirota strain proportion proportion proportion
Escherichia coli
Staphylococcus aureus
Mean
Grand Mean
Table 7. Analysis of Variance
Source of Variance SS df MS Interpretation

Between Trials
Within Trials
Total
F Critical
P Value
54
CURRICULUM VITAE
NAME: ANA LIZA BESAS
ADDRESS: BRGY. SAN FERNANDO, SAN JOSE, ANTIQUE
CONTACT NUMBER: 09365425946
E-MAIL ADDRESS: albesas@usa.edu.ph
MOTHER’S NAME: MARILYN BESAS
FATHER’S NAME: ARTHUR BESAS
EDUCATIONAL BACKGROUND:
PRIMARY: ST. ANTHONY’S COLLEGE GRADE SCHOOL
SECONDARY: ST. ANTHONY’S COLLEGE HIGH SCHOOL
TERTIARY: UNIVERSITY OF SAN AGUSTIN

55
CURRICULUM VITAE
NAME: ROCHELLE DOMINIQUE T. CASADOR
ADDRESS: BRGY. NEW ISABELA, TACURONG CITY,
SULTAN KUDARAT
E-MAIL ADDRESS: rdcasador@usa.edu.ph
MOTHER’S NAME: ROCELIA JUDITH T. CASADOR
FATHER’S NAME: ROGER T. CASADOR
PRIMARY: NEW ISABELA CENTRAL ELEMENTARY SCHOOL
SECONDARY: NOTRE DAME-SIENA COLLEGE OF TACURONG

56
CURRICULUM VITAE
NAME: ARMELINE A. CASTILLO
ADDRESS: BRGY. CABAYOGAN BADIANGAN, ILOILO CITY
E-MAIL ADDRESS: Arcastillo@usa.edu.ph
MOTHER’S NAME: JENELYN CASTILLO
FATHER’S NAME: ARTURO CASTILLO
PRIMARY: ST. JULIAN ACADEMY
SECONDARY: ST. JULIAN ACADEMY

57
CURRICULUM VITAE
NAME: MAYZ P. CERCADO
ADDRESS: DISTRICT IV, FORNIER ST. SIBALOM, ANTIQUE
E-MAIL ADDRESS: mpcercado@usa.edu.ph
MOTHER’S NAME: MONICA MAY CERCADO
FATHER’S NAME: VICENTE CERCADO
PRIMARY: ANTIQUE SPED CENTER
SECONDARY: ANTIQUE NATIONAL SCHOOL

58
CURRICULUM VITAE
NAME: EUNA GRACE N. CHAVEZ
ADDRESS: BRGY, SOOC AREVALO PROJECT 3 ILOILO CITY
E-MAIL ADDRESS: egchavez@usa.edu.ph
MOTHER'S NAME: FAITH CHAVEZ
FATHER'S NAME: LEOVIN CHAVEZ
PRIMARY: MANDURRIAO ELEMENTARY SCHOOL
SECONDARY: ILOILO CITY NATIONAL HIGH SCHOOL

59
CURRICULUM VITAE
NAME: MA. ANGELICA A. DEL CASTILLO

ADDRESS: LOT 1, BLOCK 21, PHASE 4
GRAN PLAINS SUBDIVISION, JARO, ILOILO CITY

E-MAIL ADDRESS: mdelcastillo@usa.edu.ph
MOTHER’S NAME: THELMA A. DEL CASTILLO

FATHER’S NAME: ROBERTO P. DEL CASTILLO
PRIMARY: ILOILO SACRED HEART SCHOOL, INC.

SECONDARY: ST. PAUL’S UNIVERSITY ILOILO
60
CURRICULUM VITAE
NAME: JAMES DOMINIC S DITALO
ADDRESS: ILAYA BUGASONG ANTIQUE
E-MAIL ADDRESS: jdboyditalo07@gmail.com
MOTHER’S NAME: ROSELYN SUAREZ DITALO
FATHER’S NAME: DOMINGO SUNGCAYAWON DITALO
PRIMARY: SAINT JOSEPH ACADEMY
SECONDARY: CENTRAL PHILIPPINES UNIVERSITY

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University of San Agustin

COLLEGE OF HEALTH AND ALLIED MEDICAL PROFESSION

SYNERGISTIC ANTIMICROBIAL EFFECT OF GINGER (Zingiber officinale)

ETHANOLIC EXTRACTS AND PROBIOTIC (Lactobacillus casei) AGAINST

ENTERIC PATHOGENIC BACTERIA

A Research Presented to the Faculty of the

College of Health and Allied Medical Professions

In Partial Fulfillment of the Requirements for the Degree of

Bachelor of Science in Medical Laboratory Science

Background and Rationale 1

Objectives of the Study 2

Significance of the Study 5

Scope and Delimitations 6

2. REVIEW OF RELATED LITERATURE

Antibiotics and Antimicrobial Resistance 14

Antimicrobial Activities of Ginger 17

Probiotics and Lactobacillus casei 19

Antimicrobial Activities of Probiotics and L. casei 22

Escherichia coli Pathotype 23

Staphylococcus aureus Pathotype 25

Social Preparations and Ethical Considerations 29

Sampling Design and Sample Size Determination 30

A. Draft of Letter to the Research Laboratory In-charge 42

B. Schematic Diagram of Research Procedures 44

TIMETABLE FOR RESEARCH PROPOSAL 50

Background and rationale

A significant burden of acute and chronic illness is induced by gastrointestinal

for K. pneumoniae according to Global Antimicrobial Resistance and Use Surveillance

System (GLASS) (WHO, 2020).

Zingiber officinale (ginger) is a plant used in traditional medicine against different

diseases because of its various properties (antimicrobial, antioxidant, anti-inflammatory,

monoterpenoids, sesquiterpenoids, phenolic compounds, and its derivatives which

provide a broad antimicrobial spectrum against different microorganisms (Beristain-Bauza

of influence on tested staphylococci (Santo-Grace et al., 2017).

lowering the pH of the gut, rendering it unsuitable for pathogenic bacteria to

survive. Lactobacillus is one of a number of probiotics considered to be biological

therapeutics and host immune-modulating biologicals that are generally recognized as

safe (Chen et al., 2019).

it will also help reduce morbidity of gastrointestinal diseases.

Objectives of the study

enteric pathogenic bacteria such as Escherichia coli and Staphylococcus aureus.

Specifically, it aims to:

1. determine the minimum inhibitory concentration of ginger ethanolic extracts by

disk diffusion method to be used in the succeeding assay;

2. determine the zone of inhibition of the following preparations on Escherichia coli

and Staphylococcus aureus using disk diffusion method:

a. 1 ml ginger ethanolic extracts (1mg/ml)

b. 1 ml Lactobacillus casei Shirota strain cell-free supernatant

c. 1:1 proportion of ginger ethanolic extracts and Lactobacillus casei Shirota

d. 2:1 proportion of ginger ethanolic extracts and Lactobacillus casei Shirota

e. 1:2 proportion of ginger ethanolic extracts and Lactobacillus casei Shirota

3. determine if there is a significant difference in the zones of inhibition produced by

the following preparations on Escherichia coli and Staphylococcus aureus:

a. 1 ml ginger ethanolic extracts (1mg/ml)

b. 1 ml Lactobacillus casei Shirota strain cell-free supernatant

c. 1:1 proportion of ginger ethanolic extracts and Lactobacillus casei Shirota

d. 2:1 proportion of ginger ethanolic extracts and Lactobacillus casei Shirota

e. 1:2 proportion of ginger ethanolic extracts and Lactobacillus casei Shirot