TALISAY CITY SCIENCE HIGH SCHOOL

Poblacion, Talisay City, Cebu

The Antibacterial Efficacy of Tomato (Solanum lycopersicum) Fruit Extract on Bacillus

cereus Using Varied Solvents for Extraction

REYES, MARYAH KOREEN B.

A Science Investigatory Project Entry from Talisay City Science High School to the Regional
Science and Technology Fair 2017

Research Adviser:
MR. CARL LAWRENCE LERAY

Date of Submission:
October 20, 2017
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Abstract

This study aims to assess the antibacterial efficacy of Tomato (Solanum lycopersicum)
fruit extract on Bacillus cereus using different solvents – Ethanol, Methanol, and distilled water –
to determine if Tomatoes can be a good substitute for pharmaceutical antibiotics. The tomatoes
were blended and the juices (1000 mL) were soaked each in 1000 ml of 95% Ethanol, Methanol,
and distilled water. A Rotary evaporator was used to separate the solvents after extracts have
undergone of filtration. The researchers prepared 6 setups for each extract: 25% concentration,
50%, 75%, 100%, Positive (Amoxicillin) and Solvent control. Each treatment contains 20 mL
concentration of extract and distilled water mixture. The researchers then performed the Kirby-
Bauer Diffusion test and compared the setups in 3 replicates and 3 trials of each extract from
different solvents.
The results obtained shows that the 75% and 100% concentration obtained with the use of
Ethanol and 100% concentration from distilled water have successfully and effectively inhibited
bacterial growth. On the other hand, setups obtained with the use of Methanol showed
inconsistent results.
The data shows that tomato extracts can be a good antibiotic and a less risky substitute
for Amoxicillin.
The researchers recommend that when studying a microorganism, it is safer to choose
non-pathogenic bacteria. It is best to ask for assistance from a supervisor or a professional. This
is important to ensure the safety of the researchers specially when studying bacteria.
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Acknowledgement

The researcher conveys his deepest appreciation and sincerest gratitude to the following

people for their most valued assistance, succor and moral support for the completion of this

scientifically inclined research project.

First of all, the researcher would like to solemnly thank his co-researchers, Rosyl Niña A.

Deiparine and Andy Brian G. Tagpuno, for their unwavering support and assistance to fulfill the

completion of our study.

To the researcher’s Science and Research Advisers, Sir Carl Lawrence Leray and Sir

Glendale Niadas of Talisay City Science High School (TCSHS) for their support and guidance

through their teaching that led the researchers into the making of a relevant research topic and

efficient research paper. To Ms. Mary Hannah Rose Padayao, University of San Carlos

Researcher, the researcher’s very sedulous Research Supervisor, for considerately helping us

conduct our study, giving discounts, sharing her professional teaching and most of all for her

assiduous efforts and patient tutelage thus making this scientifically inclined research study a

success.

To the University of San Carlos – Talamban Campus (USC-TC) for allowing the

researchers to get access of their Biology Laboratory. Without their approval, they wouldn’t be

able to conduct the study at all. To the entire University of San Carlos Biology Department Staff

for sharing them reliable information and their expert consultation.

For the unwavering moral, financial support and insights of our parents who never ceased

to support us. And above all, utmost thanks to The Almighty God for the divine intervention and

guidance through this academic endeavor.

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Table of Contents

A. Title Page……………………………………………………………………………………………….i.
B. Abstract………………………………………………………………………………………....2
C. Acknowledgement……………………………………………………………………………...3

1 INTRODUCTION……………………………………………………………………5-13

Background of the Study………………………………………………………………..5-6

Statement of the Problem……………………………………………………………….6-7
Significance of the Study………………………………………………………………..7-8
Scope and Limitations…………………………………………………………………….8
Review of Related Literature…………………………………………………………..9-12
Research Flow…………………………………………………………………………....13
Hypothesis………………………………………………………………………...……..14
Definition of Terms………………………………………………………………......14-15

2 METHODOLOGY………………………………………………………………….16-18

Preparation of Materials………………………………………………………………….14
Preparation of Ethanolic extract………………………………………………………....14
Preparation of Inoculum…………………………………………………………………14
Kirby-Bauer Diffusion Method………………………………………………………….15
Gathering of Data and Results…………………………………………………………...15

3 RESULTS AND DISCUSSION………………………………………………….....19-26

4 CONCLUSION AND RECOMMENDATIONS……………………………………...27

Conclusions…………………………………………………………………………..….27
Recommendations……………………………………………………………………….27

Bibliography……………………………………………………………………………….....28-29

Appendices…………………………………………………………………………………...30-35
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INTRODUCTION

Background of the Study

In our modern era, people learned to innovate not only in the field of technology but also

in the concoction of comestibles – which is the primary source of energy and nourishment. There

have been a variety of food choices, however; because of this, people have become unwary of

the possible risks to human health. In this fast-paced world, people tend to neglect befitting food

preparations which may lead to diseases and complications.

Food-borne diseases, or known as food poisoning, are conditions resulting from the food

spoilage catalyzed from pathogenic bacteria, viruses, toxins, chemicals or parasites (US Centers

for Disease Control and Prevention, 2011). According to the World Health Organization (2017),

death rates every year reached 125,000 globally and 40% of people under 5 years old endure

food-related conditions. The WHO South-East Asia Region has the second highest burden of

food-borne diseases per population, after the African Region, having more than 175,000

alarming death cases every year (Geneva, 2015).

The Bacillus cereus is one of the potential causes of food poisoning. According to a study

in Analytical and Bioanalytical Chemistry, the pathogen yields 19 variants that cause nausea and

vomiting to human. In 2015, a food poisoning case in Germany was reported at a number of

daycare centers induced by Bacillus cereus. Around 100 children and staff was infected by the

said bacteria found in rice (Technische Universitaet Muenchen (TUM), 2015). The types of food

most likely to harbor the bacteria are starchy staples, increasing the risks of food poisoning cases

here in Philippines since the country is known to have rice as its staple food. (TUM, 2015).

Tomato (Solanum lycopersicum) is a fruit under the Solanaceae family and genus
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Solanum (International Plant Names Index, 1768). Tomatoes grow in places with either warm or

temperate climate, like Central and South America – where its species originated – which

explains why Tomatoes are abundant here in Philippines. The researchers come up with the idea

of testing the antibacterial effectivity of tomato (Solanum lycopersicum) not only because of its

availability but also because they possess nutrients or antioxidants, such as flavonoids and

Lycopene that were proven to inhibit bacterial growth.

It is with this thought that the researchers conceptualize the idea of determining the

antibacterial property of Tomato (Solanum lycopersicum) fruit on Bacillus cereus.

Statement of the Problem

The study was aimed to determine the efficacy of Tomato (Solanum lycopersicum) fruit

extract as an antibacterial component to Bacillus cereus. Tomatoes contain antioxidant nutrients

– the main components in the antibacterial testing of the study.

The research also sought to answer these following questions:

1.) What are the mean diameters of the Zone of Inhibition of Tomato (Solanum

lycopersicum) fruit ethanolic, methanolic and distilled water extracts on Bacillus

cereus with the following concentrations in comparison with the positive and negative

controls:

a. 25%,

b. 50%,

c. 75%,

d. 100%,

e. Amoxicillin Trihydrate (positive control), and

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f. Ethanol, methanol and distilled water (negative control)?

2.) Is the mean diameter of the Zone of Inhibitions of varied concentrations of Tomato

(Solanum lycopersicum) fruit ethanolic extracts significantly different on Bacillus

cereus with the positive and negative controls?

3.) Is the mean diameter of the Zone of Inhibitions of varied concentrations of Tomato

(Solanum lycopersicum) fruit methanolic extracts significantly different on Bacillus

cereus with the positive and negative controls?

4.) Is the mean diameter of the Zone of Inhibitions of varied concentrations of Tomato

(Solanum lycopersicum) fruit extracted using distilled water, significantly different on

Bacillus cereus with the positive and negative controls?

5.) Which among the varied solvents for extraction and concentrations manifested the

highest inhibition of Bacillus cereus?

6.) Will the Tomato (Solanum lycopersicum) fruit extract inhibit the growth of Bacillus

cereus?

Significance of the Study

Nowadays, people tend to forget to ensure the safety of the food they consume. Cases like

these may lead to food-borne diseases. Reports of food poisoning internationally and locally

intensify by time. Further escalation of these cases and negligence of the proper measures in

cooking may lead to worse intestinal complications and life-threatening conditions like

gastrointestinal infection and circulatory shock. Thus, the researchers decided to use a specific

fruit to test its effectivity in killing the bacteria that associates with this dilemma.

The study to be conducted would be very beneficial to the people suffering from financial
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problem affected by the food-borne diseases, associated by Bacillus cereus. If successful, the

tomato fruit extract can be of use as a natural antibiotic to prevent the spread or inhabiting of

bacteria in comestibles.

The researchers believe that if the study is successful, they could reduce the increasing

risk of food-borne diseases or possibly, deplete the cases of outbreaks in the Philippines.

Scope and Limitations

The materials used were obtained from the Cebu Southern Pharmacy / Global Scientific

Pharmacy. The researchers used 2.5 kilograms of Tomatoes for each solvent, obtained from a

local market in Carbon Market, Cebu City and solvents, 1000 mL each – 95% Ethanol, Methanol

and distilled water.

The study was conducted in The University of San Carlos – Talamban Campus in the

Biology Department, assisted and guided by Ms. Mary Hannah Rose Padayao, a professional

Research Technician, to ensure the safety of the researchers. The researchers conducted nine (9)

trials, with six (6) setups – consisting of four (4) sets of concentrations (25%, 50%, 75%, 100%),

positive (amoxicillin) and solvent control – on each type of extract. The cultured Bacillus cereus

bacteria were provided by the university. The university’s Laboratory Bio-Safety Rules prohibit

non-professionals, such as students, to culture pathogenic bacteria thus assistance from a

professional Technician is necessary in conducting the study.

The results and data were gathered after 24 hours of incubation. The researchers gathered

the data by measuring the diameter of zone of inhibition (in mm) of each setup.

The study is limited only to its antibacterial activity and will not involve the factors

associating oral consumption of human beings.

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Review of Related Literature

Tomatoes (Solanum lycopersicum)

Tomatoes are commonly mistaken as vegetables; however they are classified as fruits. It

is under the genus Solanum, but was once part of Lycopersicon; and the family Solanaceae

(International Plant Names Index, 1768). Its species first originated in the Central and Southern

America. In Philippines, Pangasinan, Bukidnon, Ilocos Norte, Ilocos Sur, Iloilo and Nueva Ecija

are the major providers in tomato production. Tomatoes are accessible because of their versatile

nature, it has no particular season of planting, and it can be planted at anytime of the year

(Institute of Plant Breeding, 2008).

Tomatoes contribute to several nutritional benefits. They contain beneficial antioxidant

components such as vitamin C, Lycopene, beta-carotene, chlorogenic acid and naringin (T.

Watanabe, 2006). Aside from improving skin health, tomatoes help reduce the risk of cancer –

prostate, breast and pancreatic cancer – and heart diseases (WebMD, 2005). However, the study’s

focal point is the component present in the Tomato that aids in the antibacterial activity against

Bacillus cereus.

Since tomatoes possess antioxidant components such as Flavonoids and Lycopene which

are good antibacterial components, thus making it a potential antibiotic.

Bacillus cereus

Bacillus cereus is a gram-positive bacteria, frequently have strains which causes harm to

humans and results to food-borne illnesses, it is also beneficial in some strains for it can be as

probiotics to the animals. (Ryan et al., 2004) It is one of the vital causes in food poisoning,

commonly the cause of “Fried Rice Syndrome”.

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Bacillus cereus is also responsible for a minority of foodborne illnesses are facultative

anaerobes and the same as the other genus, it produces protective endospore endospores. Its

virulence factors include cereolysin and phospholipase C. The endospores appear when the food

is improperly cooked.

Flavonoids

Flavonoids are a diverse group of phytonutrients (plant chemicals) found in almost all

fruits and vegetables. Along with carotenoids, they are responsible for the vivid colors in fruits

and vegetables. There are several significant groups of flavonoids, including anthocyanidins,

flavanols, flavones, flavonols, flavonones and isoflavones. (Szalay, 2015)

Many researchers have set a goal to explain the antibacterial mechanisms of action of

selected flavonoids. The activity of quercetin, for example, has been at least partially attributed

to inhibition of DNA gyrase (Cushnie et al., 2005).

The mechanism of antibacterial action of the tomato extracts used in this research relies

on the flavonoid content of the tomato, but it is still unknown how most bacteria are sucsceptible

to flavonoids found in most fruits and plants. However, results of recent studies suggested that

the most probable cause of the antibacterial characteristic of some of these compounds is due to

the inhibition of nucleic acid synthesis which is the process in which nucleic acids (DNA and

RNA) are synthesized and degraded (Ulanowska et al., 2006).

Lycopene

Lycopene, an extract of tomatoes, has been reported to have an anti-inflammatory effect

via an antioxidative function. In recent studies, researchers evaluated the synergistic effect of

lycopene and ciprofloxacin antibiotic to treat rats with chronic bacterial prostatis and compared it

to using ciprofloxacin alone. The lycopene/ciprofloxacin group showed a statistically significant

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decrease in bacterial growth and improvement in prostatic inflammation compared with the

ciprofloxacin group. (Han, Chang Hee, et al., 2008)

According to the Nutrition research of Rao and Agarwal (2012), Lycopene exhibit

biological and antibacterial activity with the use of animal subjects like mice. Results have

shown that the mice’s resistance to bacterial infections has successfully increased.

Ethanol as Solvent for Extraction

Ethanol, also known as ethyl alcohol, is a compound with a formula C2H5OH and

abbreviated as EtOH. It is a colorless liquid with a quite distinctive scent and is a major

ingredient in alcohol beverages. Among all the alcohols, Ethanol is the least toxic and is only

poisonous in large amounts. This makes it suitable in consumer products. It is also used as a

preservative in pharmaceutical measures; antiseptic; burning fuel, characterized from its volatile

and flammable nature; and widely used as a chemical solvent in synthesis of other non-polar or

polar organic compounds (Human Metabolome Database (HMD), 2004) with a boiling point of

78.37⁰C which is lower than that of water (Haynes, 2011).

Ethanol is said to be the second best chemical solvent, after water itself. Due to its

hydroxyl group, Ethanol is a very polar molecule with a polarity index of 5.2; however, the ethyl

group – ethanol – is non-polar. This explains why ethanol dissolves both polar and non-polar

substances.

Methanol as Solvent for Extraction

Methanol, also known as methyl alcohol, is a the simplest alcohol with the formula

CH3OH (often abbreviated MeOH). Like ethanol, it is a clear, volatile and flammable liquid that

also has a similar odor that of Ethanol. However, Methanol, unlike ethanol, is extremely toxic

and is not suitable for human consumption (National Institute for Occupational Safety and
 12

Health, 2008). It is mostly used as fuel and waste water treatment and for producing biodiesel

(Methanex, 2006).

Methanol is also a known chemical solvent with a polarity index of 5.1, thus only used to

extract polar compounds. It also more preferable than that of Ethanol because it is less costly and

easier to evaporate (with a boiling point of 64.7⁰C).

Distilled Water as Solvent for Extraction

Distilled water is simply water that has undergone the process of distillation, a process

that removes impurities and dirt from water through decocotion and condensation. Its chemical

formula is H2O, meaning that its molecule contains one oxygen and two hydrogen atoms that are

connected by covalent bonds.

In chemical and biological laboratories, cheaper alternatives such as deionized water are

preferred to distilled water. But if these alternatives are not pure enough, distilled water is used.

Distilled water has several usage aside from consumption and hydration. It is also used to top off

lead acid batteries in vehicular engines. This can also be used as an effective chemical solvent.

Water is an excellent solvent for many substances. It is the ‘universal solvent’ which

makes it a good chemical solvent. It is a polar compound with a polarity index of 10.2 and

boiling point of 100⁰C.

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Research Flow

PREPARATION OF MATERIALS

EXTRACTION PROCESS OF TOMATO

(Solanum lycopersicum) FRUIT

PREPARATION OF BACTERIAL
INOCULUM

KIRBY-BAUER DISK METHOD

GATHERING OF RESULTS AND DATA

Figure 1. Research Flow of the Study

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Hypothesis

The Tomato (Solanum lycopersicum) fruit extract cannot inhibit the growth of Bacillus

cereus.

Ho1: There is no significant difference between the mean diameters of the Zone of

Inhibitions of varied concentrations of Tomato (Solanum lycopersicum) fruit

ethanolic extract on the bacteria Bacillus cereus in comparison with the positive and

negative controls.

Ho2: There is no significant difference between the mean diameters of the Zone of

Inhibitions of varied concentrations of Tomato (Solanum lycopersicum) fruit

methanolic extract on the bacteria Bacillus cereus in comparison with the positive

and negative controls.

Ho3: There is no significant difference between the mean diameters of the Zone of

Inhibitions of varied concentrations of Tomato (Solanum lycopersicum) fruit extract

using distilled water on the bacteria Bacillus cereus in comparison with the positive

and negative controls.

Ho4: There is no significant interaction between the concentrations of Tomato (Solanum

lycopersicum) fruit extract and the varied solvents used for extraction.

Definition of Terms

Antioxidant – a substance that inhibits oxidation, especially one used to counteract the

deterioration of stored food products

DNA Gyrase (Gyrase) - is an essential bacterial enzyme that catalyzes the ATP-

dependent negative super-coiling of double-stranded closed-

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circular DNA. This is essential for most microorganisms to

replicate DNA.

Flavonoids – is a phenolic compound present in almost all fruits and plants and is known

for its antioxidant and anti-inflammatory effect.

Kirby-Bauer method - is a standardized test for microbiologic susceptibility performed

by transferring a standardized pure culture of the organism of

interest onto a sensitivity plate and observing growth in the

presence of disks containing antibiotics.

Lycopene – a red carotene pigment and phytochemical found in tomatoes.

Mueller-Hinton agar – is a microbiological growth medium that is commonly used

for antibiotic susceptibility testing.

Normal Saline Solution - is used for preparing microbial suspensions when it is

necessary to deliver a set number of microbes to an

identification test battery, to antimicrobial agents, or to growth

media used for disk susceptibility testing; recommended for

procedures that require the use of an isotonic diluent.

Rotary Evaporator – is a device used in chemical laboratories for the efficient and

gentle removal of solvents from samples by evaporation.

Solvent - a liquid used for dissolving other substances.

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METHODOLOGY

Preparation of Materials

The researchers used 10 kilograms of Tomatoes which were obtained from a local market

in Magallanes, Cebu City, Philippines.

The culture medium was prepared by dissolving 23g of Mueller-Hinton agar into 1000 ml

of distilled water with pH level of constant (7.0 – 7.2). 100-ml beakers were prepared and were

labeled according to its concentration, Positive and Negative control. Normal Saline Solution

(NSS) was also prepared for the inoculum. All prepared materials were sterilized at 121⁰C for 15

minutes with the use of autoclave. Petri dishes were dried oven at 160⁰C for 2 hours to ensure

these were sterilized.

Preparation of extracts

In preparing the extract, the researchers, first, cleaned the tomatoes with deionized water

before blending them using a cloth strainer. After filtration, 4 L of fruit juice was obtained and

from then, was separated in different containers, labeled according to the solvent used. By then,

the fruit juice was soaked each in 1000 mL of corresponding solvent - 95% ethyl alcohol,

methanol and distilled water – for 24 hours in room temperature. Afterwards, using Whatman

filter paper no. 1, the juices were filtered to obtain the filtrate. The filtrate was then evaporated

under reduced pressure using rotary evaporator in 60⁰C; 150 rpm. Subsequently, the researchers

decocted the obtained extract after solvent evaporation, in order to remove excess amount of

water within the aqueous tomato extract.

The researchers obtained 200 mL each of the final extracts after solvent evaporation and

placed it to each sterile beaker according to designated concentration – 25%, 50%, 75%, 100%.

Each treatment will contain 20 mL of concentration or mixture. The Positive control

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(Amoxicillin) was mixed with 20 ml distilled water and the Negative containing merely 20 mL

of corresponding solvent.

Preparation of inoculum

In preparation of the bacterial inoculum, 24-hour cultured Bacillus cereus bacteria were

obtained, provided by the University of San Carlos. The microorganisms were inoculated to the

sterile Saline Solution on tubes using inoculating loop until comparable to the 0.5 McFarland

turbidity standard. The bacterial suspension was performed using aseptic techniques inside the

Biological Safety Cabinet level 2 conditioned in room temperature (25⁰-26⁰C).

Kirby-Bauer Diffusion Method / Agar Diffusion Test

Sterile Muller-Hinton agar was cooled to 50-55⁰C. The cooled medium was poured to the

individual sterile petri dishes and set aside to dry for about 5 minutes. Inoculum of test bacteria

which is comparable to the standard turbidity were swabbed uniformly on solidified sterile

Muller-Hinton agar plates using sterile cotton swabs in one direction, plate rotated in 90⁰ then

streaked in the same direction. This process is repeated thrice until the entire scope of agar plate

is swabbed. The sterile disks of 6 mm diameter (Whatman no. 1) were soaked to the prepared

treatments. The soaked disks were placed on the swabbed plates aseptically using sterile forceps.

Forcep was sterilized by soaking it in 95% ethanol and was passed unto flame.

The inoculated plates were incubated at 30°C for 24 hours in upright position and the

zones of inhibition that formed around the well were measured using a ruler. Inoculation was

done in the Biosafety cabinet. Diameters of the inhibition zone by the extract and the known

drug were compared.

All procedures were done in three trials with three replicates at the Microbiology and

Microtechnique Laboratory, University of San Carlos, Talamban, Cebu City, Philippines

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Gathering of Data and Results

The researchers measured the diameters of zone of inhibition on each treatment. Results

were compared and tabulated quantitatively. A table is shown which contain the results from

each concentration including the positive and negative controls.

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RESULTS AND DISCUSSION

A. Tomato (Solanum lycopersicum) Fruit Ethanolic Extract

The Table below shows the diameter of zone of inhibition of varied concentrations (A =

50% concentration, B = 75% concentration, C = 100% concentration) of Tomato (Solanum

lycopersicum) fruit ethanolic extract and the positive control (Amoxicillin) and negative control

(Ethanol).

Table 1. Diameter (in mm) of zone of inhibition (using Ethanol as solvent)

Setups Replicate 1 Replicate 2 Replicate 3

Trial (diameter in mm) Trial (diameter in mm) Trial (diameter in mm)
1 2 3 1 2 3 1 2 3
A 0 0 7 7 6 7 0 0 0
B 9 8 11 11 8 8 8 9 8
C 11 10 13 15 12 15 13 10 11
D 17 15 17 14 17 16 17 15 16
E (ethanol) ND ND ND ND ND ND ND ND ND
*ND = not determined

The data above were gathered results after incubation of bacteria. This shows the

diameter of inhibition on each treatment obtained after the study. Results were measured in

millimeters with the use of ruler. Table shows that treatment D which is the positive control

(Amoxicillin) has the widest diameters compared to the other treatments. Treatment C (100%

concentration) has the second widest inhibition and highest between the experimental set-ups.

While results obtained in treatment E has shown negative results. Data from treatment A has

shown inconsistent results, having both negative and positive results.

The results of the study implies that the higher the concentration of Tomato (Solanum

lycopersicum) fruit ethanolic extract, the higher the inhibition of Bacillus cereus. The 100 %

concentration or set-up C has successfully inhibited bacterial growth because of its high
 20

concentration of flavonoids and lycopene which has high antibacterial property. The result of the

research affirms the study of Rao and Agarwal (2012) which lycopene exhibits antibacterial

property. Flavonoid is also a main component in the inhibition of the growth of Bacillus cereus

found in tomato (Solanum lycopersicum) fruit. According to Ulanowska et al. (2006), flavonoids

inhibits the synthesis of nucleic acid those inhibits the growth of the bacteria.

Table 2. Mean Diameter (in mm) of Zone of Inhibition for Tomato Fruit Ethanolic Extract.
Replicates
Treatments Row Total (Row Total)2
1 2 3
A 2.33 6.67 0 9.00 81.00
B 9.33 9.00 8.33 26.66 710.76
C 11.33 14.00 11.33 36.66 1343.96
D 16.33 15.67 16.00 48.00 2304.00
E 0 0 0 0 0
Total 120.32 14, 476.90

The table above shows the mean diameter of trials in each replicate. The total and of each

row were also calculated. Each treatment has shown quantitative results, except for treatment E,

having constant negative results.

Table 3. Summary of One – Way ANOVA (using Ethanolic Extract)

Sources of
df SS MS f p-value
Variation
Before Treatment 4 514.777 128.694 45.3030 0.00001
After Treatment 10 28.407 2.841
Total 14 543.184

The data were further statistically analyzed to determine whether the extract had

significant difference. Based from the table above, the computed p-value is 0.00001 which

shows a significance difference as indicated that P-value < α = 0.05 thus, rejecting the first null

hypothesis. Therefore, the difference of means of the five treatments is statistically significant.
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B. Tomato (Solanum lycopersicum) Fruit Methanolic Extract

The table below shows the diameter (in mm) of zone of inhibitions of Bacillus cereus

using tomato (Solanum lycopersicum) fruit methanolic extract.

Table 4. Diameter (in mm) of Zone of Inhibition (using Methanolic as solvent)

Setups Replicate 1 Replicate 2 Replicate 3

Trial (diameter in mm) Trial (diameter in mm) Trial (diameter in mm)
1 2 3 1 2 3 1 2 3
A ND ND ND ND ND ND ND ND ND
B ND ND ND ND ND ND ND ND ND
C 8 ND ND ND ND 7 7 ND 7
D 17.5 18 18.5 17 15.1 18.2 20 19.2 19
E (Methanol) ND ND ND ND ND ND ND ND ND
*ND = not determined

Based from the data above, only set-ups C and D showed inhibition of Bacillus cereus.

Set-ups A (50%) and B (75%) manifested a constant ND (not determined) result which means

there were no inhibition of the growth of the bacteria. Among the experimental set-ups, only set-

up C which is the 100% concentration of tomato fruit methanolic extract displayed antibacterial

activity.

The results of the study implies that the higher the concentration of Tomato (Solanum

lycopersicum) fruit methanolic extract, the higher the inhibition of Bacillus cereus. The 100 %

concentration or set-up C has successfully inhibited bacterial growth because of its high

concentration of flavonoids and lycopene which has high antibacterial property. The result of the

research affirms the study of Rao and Agarwal (2012) which lycopene exhibits antibacterial

property. Flavonoid is also a main component in the inhibition of the growth of Bacillus cereus

found in tomato (Solanum lycopersicum) fruit. According to Ulanowska et al. (2006), flavonoids

inhibits the synthesis of nucleic acid those inhibits the growth of the bacteria.
 22

Table 5. Mean Diameter (in mm) of Zone of Inhibition (using Methanolic Extract)

Replicates
Treatments Row Total (Row Total)2
1 2 3
A 0 0 0 0 0
B 0 0 0 0 0
C 2.67 2.33 4.67 9.67 93.51
D 18.00 16.77 19.40 54.17 2934.39
E (Methanol) 0 0 0 0 0
Total 63.84 4075.55

The table above shows the mean diameter of trials in each replicate. The total and of each

row were also calculated. Each treatment has shown quantitative results, except for treatment E,

having constant negative results.

Table 6. Summary of One – Way ANOVA (using Methanolic Extract)

Sources of
df SS MS f p-value
Variation
Before Treatment 4 737.596 184.399 276.8616 0.00001
After Treatment 10 6.660 0.666
Total 14 744.257

The data were further statistically analyzed to determine whether the extract had

significant difference. Based from the table above, the computed p-value is 0.00001 which

shows a significance difference as indicated that P-value < α = 0.05 thus, rejecting the second

null hypothesis. Therefore, the difference of means of the five treatments is statistically

significant.

C. Tomato (Solanum lycopersicum) Fruit Extract using Distilled Water

The table below shows the diameter (in mm) of zone of inhibitions of Bacillus cereus

using tomato (Solanum lycopersicum) fruit extract obtained by means of distilled water.
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Table 7. Diameter (in mm) of Zone of Inhibition (Tomato Fruit Extract using Distilled
Water).

Setups Replicate 1 Replicate 2 Replicate 3

Trial (diameter in mm) Trial (diameter in mm) Trial (diameter in mm)
1 2 3 1 2 3 1 2 3
A ND ND ND ND ND ND ND ND ND
B ND ND ND ND ND ND ND ND ND
C 9 7 7.2 ND 7 7.4 7 9 8
D 17.5 18.0 18.0 17.5 15.5 18.0 19.5 19.5 19.0
E (Distilled ND ND ND ND ND ND ND ND ND
Water)

According to Table 7, only set-ups C (100% concentration) and D (positive control)

showed inhibition of Bacillus cereus. Among the experimental set-ups, only set-up C which is

the 100% concentration of tomato fruit extract using distilled water exhibited antibacterial

activity. Set-ups A (50% concentration) and B (75% concentration) revealed a constant ND (not

determined) result which means there were no inhibition of the growth of the bacteria.

The results of the study implies that the higher the concentration of Tomato (Solanum

lycopersicum) fruit extract using distilled water, the higher the inhibition of Bacillus cereus. The

100 % concentration or set-up C has successfully inhibited bacterial growth because of its high

concentration of flavonoids and lycopene which has high antibacterial property. The result of the

research affirms the study of Rao and Agarwal (2012) which lycopene exhibits antibacterial

property. Flavonoid is also a main component in the inhibition of the growth of Bacillus cereus

found in tomato (Solanum lycopersicum) fruit. According to Ulanowska et al. (2006), flavonoids

inhibits the synthesis of nucleic acid those inhibits the growth of the bacteria.
 24

Table 8. Mean Diameter (in mm) of Zone of Inhibition (Tomato Fruit Extract using
Distilled Water).

Replicates
Treatments Row Total (Row Total)2
1 2 3
A 0 0 0 0 0
B 0 0 0 0 0
C 7.73 4.80 8.00 20.53 421.4809
D 17.83 17.00 19.33 54.16 2933.3056
E (Distilled 0 0 0
Water)
Total 74.69 5578.5961

Table 8 shows the mean diameter of trials in each replicate. The total and of each row

were also calculated. Each treatment has shown quantitative results, except for treatment E,

having constant negative results.

Table 9. Summary of One – Way ANOVA (using Distilled Water)

Sources of
df SS MS f p-value
Variation
Before Treatment 4 746.356 186.589 205.3015 0.00001
After Treatment 10 9.089 0.909
Total 14 755.444

The data were further statistically analyzed to determine whether the extract had

significant difference. Based from the table above, the computed p-value is 0.00001 which

shows a significance difference as indicated that P-value < α = 0.05 thus, rejecting the second

null hypothesis. Therefore, the difference of means of the five treatments is statistically

significant.

D. Interaction Between the Concentration of Tomato (Solanum lycopersicum) Fruit Extract

and the Varied Solvents Used for Extraction.

 25

Table 10 reveals the results of diameter (in mm) of inhibition of Bacillus cereus of the

100% concentration of the varied chemical solvents (ethanol, methanol and distilled water).

Based on the tables 1, 4 and 7, the 100% concentration of Tomato (Solanum lycopersicum) fruit

extract in all the chemical solvents (ethanol, methanol and distilled water) is the only

experimental set-up that shows highest inhibition of the growth of Bacillus cereus.

Table 10. Summary of Results for Diameter (in mm) of Zone of Inhibition of 100%
Concentrations of Tomato (Solanum lycopersicum) Fruit Extract of Varied
Chemical Solvents.
Diameter (in mm) of Inhibition of 100% Concentration of Tomato
Chemical Solvents Fruit Extract
Replicate 1 Replicate 2 Replicate 3
Ethanol 11.33 14.00 11.33
Methanol 2.67 2.33 4.67
Distilled Water 7.73 4.80 8.00

Based from the table above, ethanol used as a chemical solvent in obtaining Tomato

(Solanum lycopersicum) fruit extract showed the highest inhibition of Bacillus cereus in

comparison to methanol and distilled water. This is because Ethanol can dissolve both polar and

non-polar compounds, unlike Methanol and distilled water which are both polar molecules.

Table 11. Summary of Two – Way ANOVA

Df SS MS f p-value
Chemical Solvent 2 249.08 124.54 59.48 <.0001
Concentration 2 195.4 97.7 46.66 <.0001
Chemical Solvents 49.81 12.45 5.95 0.0031
4
x Concentration
Error 18 37.69
Total 26 531.98

The data were further statistically analyzed to determine whether the extract had

significant difference. Based from the table above, the computed p-value of chemical solvent

and the concentration of tomato fruit extract is less than 0.0001 which shows a significance

difference as indicated that P-value < α = 0.05. The p-value of the interaction between the two
 26

factors is 0.0031. Since the p-value of the interaction is less than α = 0.05, then it shows a

significant interaction between the type of chemical solvent and the concentration of the tomato

fruit extract. Thus, rejecting the fourth null hypothesis.

 27

CONCLUSION AND RECOMMENDATIONS

Conclusion

The researcher concludes that there is a significant difference between the treatments of

Tomato (Solanum lycopersicum) on Bacillus cereus using all chemical solvents. According to the

gathered data, among the experimental set ups, the 100% concentration has consistently inhibited

bacterial growth in all chemical solvents – Ethanol, Methanol and distilled water. However,

among the solvents used in extraction, Ethanol had the widest Zone of inhibition of Bacillus

cereus.

Therefore, the Tomato (Solanum lycopersicum) fruit extract which contains flavonoids,

and lycopene is an effective antibacterial component in inhibiting the growth of Bacillus cereus.

Recommendations

The researchers recommend that when studying a microorganism, it is safer to choose

over non-pathogenic bacteria. It is best to ask for assistance from a supervisor or a professional.

This is important to ensure the safety of the researchers specially when studying bacteria. It is

also recommended to undergo phytochemical analysis after the solvent evaporation to make sure

the composition, chemicals and compounds present in the extract. Make certain that water from

the extract is evaporated before preparing the concentration with distilled water. Such situation

may lead your experiment to negative results because of excessive amount of water.
 28

BIBLIOGRAPHY

Haynes, William M., ed. (2011). CRC Handbook of Chemistry and Physics (92nd ed.). Boca
Raton, FL: CRC Press. p. 3.246. ISBN 1439855110.

Cushnie, TP Tim, and Andrew J. Lamb. "Antimicrobial activity of flavonoids." International

journal of antimicrobial agents 26.5 (2005): 343-356.

Magee, Elaine.( 2017). Health Properties of Tomatoes. Available at: www.webmd.com/food-

recipes/features/health-properties-tomatoes#1 (Accessed: 9/19/2017)

Marxen, Sandra, et al. "Chemodiversity of cereulide, the emetic toxin of Bacillus cereus."
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McNaught, Alan D; Wilkinson, Andrew; IUPAC (1997), "IUPAC Compendium of Chemical

Terminology", IUPAC Compendium of Chemical Terminology (2 ed.), Oxford:
Blackwell

Methanex (2006). “Technical Information & Safe Handling Guide for Methanol”. Retrieved
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And-Safety/Safety-Resources/Health---Safety/Methanex-TISH-Guide.aspx

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Retrieved from: https://pubchem.ncbi.nlm.nih.gov/compound/702/

Peralta, I. E.; Spooner, D. M. (2001). "Granule-bound starch synthase (GBSSI) gene phylogeny
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PMID 21669622. doi:10.2307/3558365.

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8385-8529-9.

Technische Universitaet Muenchen (2015). “The Number of Food Poisoning Cases Caused by
Bacillus cereus is on the Rise.” Retrieved from:
http://www.infectioncontroltoday.com/news/2015/04/the-number-of-food-poisoning-
cases-caused-by-bacillus-cereus-is-on-the-rise.aspx/

Ulanowska, Katarzyna, et al. "Assessment of antibacterial effects of flavonoids by estimation of

generation times in liquid bacterial cultures." Biologia 62.2 (2007): 132-135.

World Health Organization (2015). “WHO’s first ever global estimates of foodborne diseases

find children under 5 account for almost one third of deaths.” Retrieved from:
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World Health Organization (2017). "Food Safety." Retrieved from:

http://www.who.int/mediacentre/factsheets/fs399/en/
 30

APPENDICES

The following picture were taken while conducting the study

The materials essential in the study: Tomatoes, Normal Saline Solution (NSS), agar (Mueller-
Hinton agar), distilled water, Ethanol, Methanol, n-Hexane
 31

Extraction of Tomatoes Soaking the fruit juice in solvents

Filtration Process Separating solvent from extract

 32

Preparation of Bacterial inoculum

Preparation of Concentrations
 33

Results (Ethanol)
Replicate 1
Trial 1 Trial 2 Trial 3

Replicate 2
Trial 1 Trial 2 Trial 3

Replicate 3
Trial 1 Trial 2 Trial 3
 34

(Methanol & Distilled water)

Replicate 1
Trial 1 Trial 2 Trial 3

Replicate 2
Trial 1 Trial 2 Trial 3

Replicate 3
Trial 1 Trial 2 Trial 3
 35

SUMMARY OF TWO-WAY ANOVA