Antibacterial Activity of Unripe Chico (Manilkara zapota) Fruit Extract Against Escherichia coli

and Staphylococcus aureus

In partial fulfillment for the requirements of the

subject
SCIENCE RESEARCH II

ALVAREZ, KIRZTEN AVRIL R. | X-Mendel

BORJA, CARL JOSEPH N. | X-Fleming
CORPUS, NORMAN BERNARD PATRICK M. | X-Pasteur
Researchers

S.Y. 2022-2023

John Angelo B. Almazar

Teacher
ABSTRACT

Organic antibiotics are in demand even until now. The search for organic antibiotics that
are “budget-friendly”, effective, has less rate of risk, and environmental friendly has been too
challenging. According to Bano M., and Bilal A., the leaves of chico (Manilkara zapota L.) is
rich in antioxidant properties and was also found that it exhibits anti diabetic property. In this
investigative project, the antibacterial activity of immature chico (Manilkara zapota) fruit extract
against Escherichia coli and Staphylococcus aureus was studied.

Plant extract concentrations 25%, 50%, 75%, and 100% were prepared and their
antibacterial activity on gram-negative bacteria E. coli and gram-positive bacteria S. aureus were
assessed through the process of Kirby Bauer Disk Diffusion Assay in which 4 plates with three
replicates. Each disks contained 4 paper filters with the extracts respectively. The antibacterial
activities were determined by measuring the zone of inhibition diameter using a ruler as the
instrument in measuring the zone. Ethanol and Streptomycin were used as the negative and
positive control respectively.

The results showed that Chico (M. zapota) fruit extracts with 100% concentration have
the highest activity on S. aureus amongst all of the concentrations. However, the positive control
exhibited the highest activity amongst all of the treatments with 25mm diameter. All of the
concentrations did not utilize any activity on E. coli, providing us with the hypothesis that it is
not significant (p<0.05)

INTRODUCTION

Due to its pleasant taste and aroma, Manilkara zapota or most commonly known as chico
here in the Philippines is widely used as a dessert. Frequently, it is even chilled to emphasize its
flavors. In India, the fruit is said to be used as an ingredient to make a sherbet, ice cream, or is
eaten as a dried fruit (Mickelbart, 1996). Some even made vinegar and syrup out of the chico
fruit. (García, 1988).

For many years, the latex in from the sapodilla tree which is called ‘chicle’ is used as the
main ingredient for making chewing gums. Its contents are 15% rubber and 38% resin which are
both tasteless.

A methanolic extract of sapota fruit was recently discovered to suppress the growth of
tumors (Srivastava et al., 2014). There are roughly 40 different kinds of sapota grown throughout
the world, with "Kalipatti" being the most popular variety in India. In addition to creating a
probiotic sapota juice, (Vijay Kumar et al. 2015) researched the growth kinetics of LAB,
antioxidant, antibacterial, and sensory qualities. According to the results of this study, sapota
juice can also be used to make probiotic drinks with a high cell density (8 108 CFU/mL).
 One of the main fruit harvests in India, Mexico, and Venezuela is the sapota (Achras
sapota L. or Manilkara zapota L.). Significant amounts of fermentable sugars, protein, phenolics,
carotenoids, ascorbic acid, minerals (potassium, copper, and iron), and vitamins are all present in
sapota fruit (A, C, folate, and pantothenic acid). Sapota is regarded as a healthy fruit to treat
micronutrient deficiencies as a result of these qualities. The fruits and leaves of the plant are rich
in antioxidants and phytochemicals which are good in suppressing tumors. (Tulloch, A.,
Goldson- Barnaby, A., Bailey, D., & Gupte, S., 2019)

Escherichia coli, or most commonly known as E. coli has been abundant and growing
continuously. It is found in the lower intestinal tract. Some of its variety has mild effects while
some could lead to serious food poisoning. The person infected by this bacteria will experience
extreme dehydration and fatigue. (Cleveland Clinic, 2020).

Staphylococcus aureus is a gram-positive bacteria that causes a wide range of clinical

infections. Infections caused by this pathogen are common in both community and hospital
settings. The emergence of multi-drug resistant strains such as MRSA (Methicillin-Resistant
Staphylococcus aureus) makes treatment challenging. S. Although S. aureus does not normally
cause infection on healthy skin, once it is allowed to enter the internal tissues or bloodstream, it
can cause a number of potentially serious infections. (Taylor T., Unakal C., 2022).
STATEMENT OF THE PROBLEM

This study aims to know the effectiveness of Manilkara zapota fruit extracts as an
antibacterial against E. coli and S. aureus to lessen the risk of getting affected by it. Hence, the
study enables us to answer the following questions:

1. Is there a significant difference in the antibacterial activity of the immature

Manilkara zapota fruit extracts and the positive control Streptomycin?

2. Do the immature Manilkara zapota fruit extracts exhibit an increase in effectiveness

in inhibiting the bacterial growth of Escherichia coli and Staphylococcus aureus?

HYPOTHESIS

Based on the stated problems, the following hypotheses was formulated, it is in null
form:

1. There is no significant difference in the antibacterial activity of immature chico fruit

extracts and positive control Streptomycin against Escherichia coli and Staphylococcus
aureus.

ENGINEERING GOALS

This research study aims to find a local fruit from the Philippines which can inhibit
antibacterial activity against the viral bacteria–E. coli and S. aureus.

EXPECTED OUTPUT

Manilkara zapota fruit extract is expected to be feasible and can surpass the effectiveness
of the positive control Streptomycin in terminating Escherichia coli and Staphylococcus aureus.
MATERIALS AND METHODS

I. Verification and Authentication of Plants Sample

The plant samples of Immature chico (Manilkara zapota) were presented to the Department
of Biology, Angeles University-Foundation, Angeles City, Pampanga for proper identification
and authentication.

II. Collection of the Plant Material

The collection of fresh immature Manilkara zapota are picked at a local garden in F.K
Ilagan St., Sinipit Pob. Bongabon, Nueva Ecija and prepared for step-by-step extraction. A total
of a kilogram of Manilkara zapota was handpicked.

III. Ethanolic Extraction of Fruit Extracts

After the Manilkara zapota were harvested and collected, they were first coarsely
chopped, then air-dried for three days, then were percolated for an additional three days in 95%
analytical-grade ethanol. Thereafter, the ethanol underwent Rotary Vacuum Evaporation at the
Pharmacy Laboratory at Araullo University in Cabanatuan.

IV. Culturalization of Bacteria

The test microorganisms were collected at the Department of Biological Sciences,

College of Science in CLSU. The tests were conducted on the gram-negative E. coli and gram-
positive S. aureus. After being injected into a tube of Mueller Hinton Agar, they were left to
incubate for 24 hours at 37°C. (Lozano, Cajucom, & Lumague, 2019)
V. Anti-Bacterial Testing (Kirby Bauer Disc Diffusion Assay)

The experiment was laid out in a petri dish with six (6) treatments: four (4) experimental
setups and two (2) control setups. Treatment 1 (application of 100% concentration of M. zapota
extract), Treatment 2 (application of 75% concentration of M. zapota extract), Treatment 3
(application of 50% concentration of M. zapota extract), Treatment 4 (application of 25%
concentration of M. zapota extract), Treatment 5 (application of Streptomycin as the positive
control), Treatment 6 (application of 95% ethanol as negative control).

In conclusion, a total of twelve (12) petri dishes were used for conducting this
experimental and control set-ups with 2 microbes (E. coli and S. aureus), 3 replicates (replicate
1, replicate 2, and replicate 3) with 6 treatments in each plates (100%, 75%, 50%, 25%,
Streptomycin, and Ethanol).

VI. Risk Assessment

The hazards and risk factors (hazardous chemicals, harmful bacteria, microorganisms,
devices) that can potentially harm in this experiment are the components of the antibiotic such as
the hazardous chemicals on it, (E. coli and S. aureus), and the possibilities of getting a cut from
chopping the Manilkara zapota. Possibility of getting diarrhea from the E. coli and skin infection
from S. aureus could also add up to the courses of risks that could be attained by conducting this
experiment.

VII. Safety Precautions

In this experiment, precautions were planned and also performed accordingly with the
supervision of a qualified scientist. The qualified scientist was used to ensure that every step in
our experiment is aided by them, also to make sure that no experimenter is in the line of danger
during and after the experiment.

Utilizing personal protective equipment (PPE) such as gloves, goggles, and lab coats
were used to protect the individuals from any substances that may contain harmful elements from
the bacteria E.coli and S. aureus and to ensure the safety of every individual that will conduct the
experiment. Safety precautions of level BSL-2 were used to protect the individuals from getting
the viral E. coli and S. aureus.

VIII. Disposal of materials

This study followed the protocols of Central Luzon State University in disposal of the
materials and the usage of autoclave to eliminate the bacteria.
IX. Statistical Analysis

For hypothesis testing, ANOVA was used using MS Excel Version 2013. In order for the
treatment to be significant, p-values should be less than 0.05.

Results and Discussion

Table I. Results of Kirby Bauer Disc Diffusion Assay on Staphylococcus

aureus

Zone of Inhibition

Treatments R1 R2 (mm) R3 (mm) Mean (%) p-value Verbal

(mm) Description

100% 10 9 9 9.33 <0.001 Significant

75% 9 9 9 9 <0.001 Significant
50% 8 8 8 8 <0.001 Significant

25% 8 8 8 8 <0.001 Significant

Positive Control 25 23 24 24 <0.001 Significant
(Streptomycin)

Negative Control 0 0 0 0 <0.001 Significant

(Ethanol)

The table shows the zone of inhibition of Staphylococcus Aureus which are treated with
different concentrations. The ethanolic extract of the M. zapota exhibited poor antibiotic activity
against S. aureus within 48 hours with 3 replicates. The concentration of the ethanolic plant
extract was 1 milliliter (mL) of extract decreasing until 0.25 milliliters (mL) of extract.
According to Bano, M., and Bilal, A (2017)., the chico leaves are high in antioxidants. The high
antioxidant capacity detected for acetone extracts of sapodilla indicates that this plant can be
used as a supplement in the foods. However, there are no published findings that the fruit has an
antibacterial property.
 Table II. Results of Kirby Bauer Disc Diffusion Assay on Escherichia coli

Zone of Inhibition

Treatments R1 R2 (mm) R3 (mm) Mean (%) p-value Verbal

(mm) Description

100% 5 5 5 5 <0.001 Significant

75% 5 5 5 5 <0.001 Significant
50% 5 5 5 5 <0.001 Significant
25% 5 5 5 5 <0.001 Significant
Positive Control 25 23 25 24.5 <0.001 Significant
(Streptomycin)

Negative Control 0 0 0 0 <0.001 Significant

(Ethanol)

The table shows the zone of inhibition of Escherichia coli which are treated with different
concentrations. The ethanolic extract of the M. zapota exhibited little to no antibiotic activity
against Escherichia coli within 48 hours with 3 replicates. The concentration of the ethanolic
plant extract was 1 milliliter (mL) of extract decreasing until 0.25 milliliters (mL) of extract. A.
Murnisyazwani (2019, February) claims that Chico leaves are rich in antioxidants, according to
Rabeta, MS. The strong antioxidant potential of sapodilla acetone extracts suggests that this plant
can be employed as a dietary supplement. The leaves of the plant do not, however, appear to
have any antibacterial properties according to published research.
 Figure I. Graphical representation of the antibacterial activity of Manilkara zapota against
E. coli as indicated by the zone of inhibition (mm).

As indicated in Figure I, all concentrations of 100%, 75%, 50%, and 25% showed activity
which wasn’t the same as the positive control Streptomycin. It induced an activity of 5mm
however it was not close to the inhibited zone of Streptomycin which averaged 24.5 mm.
However, it can be claimed that although the plant sample possesses antibacterial capabilities, it
is not as strong as the positive control, Streptomycin.

Figure II. Graphical representation of the antibacterial activity of Manilkara zapota

against S. aureus as indicated by the zone of inhibition (mm).

As shown in Figure II, all concentrations of 100%, 75%, 50%, and 25% demonstrated
activity that were different from the positive control Streptomycin. It induced an activity
averaging of 8.58 mm on all concentrations and replicates; however, it was not close to the
inhibited zone of Streptomycin which averaged 24 mm. Thus, it can be said that although the
plant sample has antibacterial properties, they are not as effective as those of the positive control,
Streptomycin.
CONCLUSION

This study highlighted that the extracts of the M. zapota fruit exhibited minimal to no
antibiotic activity against E. coli and S. aureus. As the results show, we can conclude that the M.
zapota fruit extract is not as effective as the positive control Streptomycin.

Although the extracts of M. zapota fruit showed that it is not effective on the statistical
analysis, the data showed that the M. zapota fruit contained antibiotic properties and managed to
make a zone of inhibition.

Therefore, it is concluded that even so that the M. zapota fruit shows little antibiotic
properties it still managed to kill some bacteria and clear a zone of inhibition. So in conclusion,
M. zapota fruit is still effective against the bacteria S. aureus and E. Coli just as not as effective
as our positive control Streptomycin.

RECOMMENDATIONS

After conducting the study and predicating on the obtained results and findings, the
following measures are recommended:

The main recommendation is further study. The study showed that the fruit extracts
exhibited minimal activity against the bacteria. However, it did not have the same outcome with
the positive control Streptomycin.

Using more than one plant sample as an additional would also be recommended.
Preferably, from the same plant family for it to be easily determined which property it carries
that exhibited the antibacterial property.

Another part of the plant could be used as a sample to determine the full capabilities of a
chico fruit and another property it carries.

Changing the solvent could also help in determining in which component of the extract
had the antibacterial property.
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Appendices
Figure 1. Preparation of the fruit extracts

Figure 2. Rotary Vacuum Evaporation at Phinma Araullo University

Figure 3. Preparation of agar medium for the bacteria culturization

Figure 4. Preparation of the concentrations

Figure 5. Culturalization of bacteria

Figure 6. Zone of Inhibition by 12 hours on S. aureus

Figure 7. Zone of Inhibition by 12 hours on E. coli