EFFICACY OF CARICA PAPAYA ENZYME AS ANTIBACTERIAL HAND

SANITIZER

By:

Penaflor, Nuela
Perez Kenneth
Pintor, Jalene
Reyes, Charrisa Jiezel
Sandot, Kimberly
Santos, Randeline
Susa, Valerie
Tabones, Edward
Tamano, Joey Ann
Valdez, Airah
Villamor, Oressah Freya

2020
 ABSTRACT

Every year there is a significant increase in the mortality rate of people due to lack of
sanitation, poor hand-washing practices or simple because of little no access to water. The
current water shortage in the Philippines intensifies this problem. Based on numerous studies,
Papaya contains antiseptic and antibacterial properties. An enzyme from papaya leaves yields
antibacterial and antifungal behavior called papain enzyme which will help in reducing the
number of bacteria where it is no longer possess a threat. The researchers want to formulate an
alternative antibacterial sanitizing product that can provide protection against Staphylococcus
aureus at half of the cost. The researchers will make use of the Maceration Method for the
extraction wherein they will leave ground papaya leaves in liquid for a period of 24 to 48 hours.
Then apply the Agar Dilution Process for the testing of the antibacterial efficacy of the
aforementioned product.
 CHAPTER 1
INTRODUCTION

1. Background of the Study

Papaya belongs to Caricaceae family under kingdom Plantae. It originates from Mexico
and South America. The botanical name of papaya is Carica papaya. It is a small tropical tree
with a straight stem marked by scars where leaves have fallen from it directly. There are
many varieties of papaya, but the main varieties grown in the United State are Red Lady,
Maradol, and various Solo types. Christopher Columbus (2012) reputedly referred to the
tropical fruit papaya as ‘fruit of angels.’ It is rich in vitamin A, E, K, and B, fiber, calcium,
magnesium, phosphorus and zinc, as well as the essential nutrients lycopene, folate, lutein
and enzymes. According to the book Healing Foods by DK Publishing House, papaya is
known to have antibacterial properties and promotes good digestion and almost every part of
the plant can be used.

Carica papaya plants produced natural compounds (Annonaceous acetegenins) in leaf

bark and wing tissues. The leaves are large, usually are 50-70cm in diameter, deeply
palmately lobed, with seven lobes. Several scientific investigations on the biological
activities had been done through the leaves. It was suggested that a potential lucrative
industry based simply on production of plant biomass could develop for production of anti-
cancer drugs, pending Food and Drug Agency approval, and natural botanical pesticides
(McLanghlin, 1992). The high level of natural self-defense compounds in the tree makes it
high resistant to insect and disease infestations. Fresh and green papaya leaf has a therapeutic
value due to its antiseptic quality. It cleans the intestines from bacteria. In French Guiana,
both leaf and root are prepared in combination with other ingredients for the treatment of
malaria fever (Vigneron et al., 2005). Dried and pulverized leaves are sold for making tea;
 also, the leaf decoction is administered as purgative for horses and used for the treatment of
genetic urinary system.

Papaya fruits are widely consumed in food industry, while the processing of papaya
fruits, papaya leaf is not consumed in any industry. In order to solve such problem, the study
on the papaya leaf had been done. From the study done by Simmonne (2005), papain enzyme
from the papaya leaf yields the antibacterial behavior. As referring to the research title,
production of antibacterial hand sanitizer by the natural enzyme are important for the bio
green product which is preferable than the chemical made product of hand sanitizer. It is also
environmental-friendly and non-toxic product which solve the garbage formation by papaya
tree.

Papain enzyme can be obtained from papaya fruit, latex or roots. The researchers used
papaya leaf extract to obtain papain enzyme which have phenolic compounds such as
protocatechuic acid, p-coumarie acid, dimethoxymarin, caffeic acid, kaempferol, quereetin,
chlorogenic acid. These compounds have antimicrobial activity and have been proven to be
able to inhibit the growth of Rhizupos stolonifer. Papaya leaf has been chosen for this
research because it provides the best potential for further commercial form and most
preferable material used for the papain enzyme extraction. Then, Maceration Method was
chosen for the papain extraction to be followed by the Agar Dilution Method to test its
susceptibility against the bacteria Staphylococcus aureus.

2. Statement of the Problem

The current water shortage in the Philippines intensifies the significant increase in the
mortality rate of people due to lack of sanitation, poor hand-washing practices or simple no
access to water. That being said the researchers from Our Lady of Fatima University
Antipolo Campus come up with a solution of conducting an experiment regarding the
Efficacy of Carica Papaya Enzyme as Antibacterial Hand Sanitizer.

3. Objectives
 Listed below are the expected outcomes at the end this research.

1.

1. Significance of the Study

4. Scope and Limitation

 CHAPTER 3
METHODOLOGY

3.1 Procedure

The papaya leaves used in this research were 20-25 cm in length. The papaya was
washed, dried with oven, blended to obtain leaf powder. The leaf powder was then
macerated with three kinds of solvent: ethanol, ethyl acetate, and hexane. After the
maceration procedure is done, it will be tested if it’s proven effective against three
pathogenic bacteria: Staphylococcus aureus, Bacillus subtilis, and Escherichia coli.

3.2 Preparation of the sample

· Collect fresh leaves of the plant sample.

· Remove the extraneous matter by washing the sample with water.

· Wipe the leaves with cloth.

· Dry the leaves using an oven not exceeding 60℃ temperature

· Grind the dried leaves using an osterizer.

3.3 Maceration

Maceration is a procedure done to extract the active components of a medicinal

plant, leaving it in liquid for a period of six hours or several weeks. It involves soaking
plant materials (coarse or powdered) in a stoppered container with a solvent and allowed
to stand at room temperature for a period of minimum 3 days with frequent agitation. The
processed intended to soften and break the plant’s cell wall to release the soluble
phytochemicals. After 3 days, the mixture is pressed or strained by filtration Azwanida
(2015). This technique has been adopted from winemaking and has now been used
effectively in getting the extracts from various medicinal plants. Botanical Online (2019),
states that macerations are made in proportion of 20 parts of liquid to 1 part of plant.
Zhang et., al (2018), outlines that maceration requires the use of organic solvents and a
long extraction time. It must be done at room temperature.

3.3.1 Materials:

· Weighing scale
· Solvent: ethanol, ethyl, acetate, hexane
· Beaker
· 3 glass jars with seal
· 3 stoppered bottles
· Coffee filters

3.3.2 Extraction Procedure:

1. Measure about 10g of ground papaya leaves each and place in three tightly sealed
jars or vessel.

2. Add 20g of ethanol in one jar. Ethyl acetate in the second jar. Hexane in the third
jar.

3. Tightly seal the jars and allow it to stand for 24 to 48 hours. Note the amount of
alcohol volume used. Occasionally shake the jars to prevent molds from forming.
Label the jars with the date it was prepared and the date it will be opened.

4. Strain the extracts off into three containers using a coffee filter.

5. Make sure the substance is clean and clear from solid materials or else it will rot
over time.

6. Measure the exact volume of each concentrated extracts. Observe and write down
the different effects the solvents did to the mixture.
 7. Store the extract in a tightly stoppered bottle. Properly label the container with the
name of the plant, concentration of the extract, and compute the grams plant
material/mL extract, concentration of the extract in gram-extract/mL plant, and
date of extraction.

3.4 Kirby Bauer Disc Diffusion Method

The Kirby-Bauer Test, also known as disk diffusion method or agar diffusion
method, uses wafers or disks saturated with antibiotics to test the susceptibility of specific
bacteria to certain antibiotics. In this research, the papaya leaf extracts will be tested
against Staphylococcus aureus, Bacillus subtilis, and Escherichia coli. Reynolds (2019),
states that the presence or absence of zones is crucial in identifying if the bacteria is
sensitive to the antibiotic. If there is no growth around the area of the disk, then that
means the bacteria is killed or inhibited by the papaya extract. The size of the zones
determines how effective the papain at preventing the growth of the different bacteria.

3.4.1 Materials:

· Petri dishes with Mueller-Hinton agar

· Test tube of bacterial culture
· Sterile swabs
· Forceps
· Cellulose discs with test chemicals
· Bench top sanitizer
· Alcohol burner
· Ethanol for flame sterilizing

3.4.2 Procedure:

1. Properly label the outside rim of the petri dishes with organism name, extract (with
the type of solvent used), and treatment information.
 2. Use a sterile swab to inoculate the plates. Briefly flame sterilize the opening of the
test tube then dip the swab into the bacterial broth. Remove swab and again gently
flame sterilize the test tube.

3. Pick up the petri dish and in a side-to-side motion, swab the plate gently. To create a
uniform layer of bacteria, slightly rotate the dish and continue swabbing. Properly
dispose the swab in a biohazard container. Allow it to dry for about 5 minutes.

4. Using a marker, divide the dish into four quadrants.

5. Quickly dip the forceps in ethanol and gently flame sterilize it. Make sure the ethanol
has completely burned off and allowed to briefly cool.

6. Pick up a cellulose disk, place it on one quadrant, and tap lightly. Flame sterilize the
forceps between placing disks. Do this until all quadrants are filled.

7. Plates should be incubated overnight at a temperature of 37 C

REFERENCES

Zhang, Q. W., Lin, L. G., & Ye, W. C. (2018). Techniques for extraction and isolation of natural
products: a comprehensive review. Chinese medicine, 13, 20. doi:10.1186/s13020-018-0177-x
Courts D. (2013, April 30). How To Make A Simple Herbal Extract. Retrieved from https://home-
remedies.wonderhowto.com/how-to/make-simple-herbal-extract-0146585/

Reynolds J. (2019, June 3). Kirby-Bauer (Antibiotic Sensitivity). Retrieved from

https://bio.libretexts.org/Bookshelves/Ancillary_Materials/Laboratory_Experiments/Microbiology_Labs
/Microbiology_Labs_I/09%3A_Kirby-Bauer_(Antibiotic_Sensitivity)

(2013, July 31) Standardization of the Kirby-Bauer Method. Retrieved from

https://web.archive.org/web/20130731092046/http://aminj.myweb.uga.edu/KIRBY-BAUER.html